TEL-AVIV UNIVERSITY GEORGE S.

WISE FACULTY OF LIFE SCIENCES GRADUATE SCHOOL

Morphological and molecular aspects of sea urchins (genus Echinometra) from Okinawa, Zanzibar and Eilat

Thesis submitted towards the M.Sc. degree in Ecology and Environmental Quality At Tel-Aviv University

By

Omri Bronstein

The research was performed in the Department of Zoology Under the supervision of Professor Yossi Loya

March 2009

To my grandparents,

"If we knew what it was we were doing, it would not be called research, would it?"

Albert Einstein

Acknowledgments
First, I would like to thank my supervisor, Prof. Yossi Loya for his endless support, vision, and inspiration. It has been a privilege taking this gurney with you! I would also like to stretch my deepest thanks to my friends and collogues who walked along with me

Dr. Noa Shenkar for guiding me through the maze of science, for her advice and for her care but above all for her friendship. Dr. Esti Winter for her perspectives and ideas in science and life, and for her editorial assistance. To Prof. Micha Ilan and his lab, for sharing their space and their cakes, and for always keeping their door open. To the Benayahu lab, past and present, and especially to Chen Yoffe, whos been my compass when the sea was rough. To my colleagues at the Loya lab, Meir, Maya, and Itzik and especially to Roee Segal a dear friend and an absolute must for working at sea. Above all to Ada Alamaru, for setting me an example in scientific perfection and for unconditional help and friendship. To the stuff at the IMS in Zanzibar, TBRC in Okinawa, and IUI in Eilat, for their help and support in the lab and at sea, and especially Dr. Naoko Isomura for introducing me to the world of molecular biology. To the Beer family for making me feel at home, on the other side of the globe. Amir Szitenberg, for his patience and dedication in hours spent exploring urchin phylogeny. To Rina Yeger and Yona Lichtenfeld, from the electron microscopy unit at BGU, for their professionalism, enthusiasm, and interest in this urchins tale. To my friends, and especially Ami, Tal, and Sharoni, for always being there, keeping the pieces together!

Finally, I would like to thank my family for their love and encouragement and especially my grandparents whom this work is dedicated to.

And to several anonymous sea urchins

Table of contents
List of figures.......................................................................................................................... 2 List of tables ........................................................................................................................... 2 1. Abstract............................................................................................................................... 4 2. Introduction ....................................................................................................................... 6
2.1 Morphologically inferred taxonomy......................................................................................8 2.2 Molecular taxonomy...............................................................................................................9 2.3 Morphological vs. molecular tools ......................................................................................11 2.3.1 The problems associated with morphological techniques..................................................11 2.3.2 The problems associated with molecular techniques..........................................................13 2.4 The genus Echinometra (Gray 1825) ..................................................................................15

3. Materials and methods ................................................................................................... 19

3.1 Collection of samples ...........................................................................................................19 3.2 Morphological measurements ..............................................................................................21 3.3 Species identification based on morphological characters.................................................23 3.4 Echinometra nomenclature ..................................................................................................24 3.5 Sperm morphology using SEM............................................................................................24 3.6 Molecular assays...................................................................................................................25 3.6.1 DNA extraction .....................................................................................................................26 3.6.2 PCR amplification and sequencing......................................................................................27 3.6.3 Alignment and analysis of sequences..................................................................................28 3.7 Statistical analysis .................................................................................................................30

4. Results ............................................................................................................................... 30
4.1 Morphological diversity in Echinometra ............................................................................30 4.2 External morphological characteristics ...............................................................................32 4.2.1 Color of spines ......................................................................................................................32 4.2.2 Milled rings ...........................................................................................................................33 4.2.3 Skin of peristome ..................................................................................................................34 4.3 Internal morphological characteristics.................................................................................35 4.3.1 Spicules..................................................................................................................................35 4.3.2 Tubefeet spicules ..................................................................................................................36 4.3.3 Gonad spicules ......................................................................................................................37 4.3.4 Sperm morphology ...............................................................................................................38 4.4 Sex, length and morphology.................................................................................................41 4.5 Phylogenetic relationship among sequences .......................................................................41 4.6 Comparing morphological keys and molecular taxonomy.................................................45

5. Discussion ......................................................................................................................... 46
5.1 5.2 5.3 5.4 The effect of sex on the morphological characters .............................................................54 Phylogenetic relationships and comparison to morphological data...................................55 Biogeography and cladogenic events ..................................................................................56 Morphological keys and molecular attribution ...................................................................61

6. Summary........................................................................................................................... 63 7. Reference .......................................................................................................................... 65 8. 67 ..................................................................................................................................

List of figures Map of the Indian Ocean and Eastern Pacific .................................................. 19 Study sites ........................................................................................................... 21 External anatomy of a regular urchin in lateral view....................................... 22 Sperm and eggs in gonads of Echinometra spp. from Okinawa ..................... 24 Echinometra species from Okinawa, Eilat and Zanzibar................................. 31 Spine color occurrence in Echinometra from Okinawa, Zanzibar, and Eilat. 33 Occurrence and state of milled rings in Echinometra from Okinawa, Zanzibar, and Eilat ............................................................................................. 34 Figure 9. Color of the skin around the peristome in Echinometra from Okinawa, Zanzibar, and Eilat............................................................................................. 35 Figure 10. Spicule types from Echinometra ...................................................................... 36 Figure 11. Shape of the spicules in the tubefeet of Echinometra from Okinawa, Zanzibar, and Eilat............................................................................................. 37 Figure 12. Shape of the spicules in the gonads of Echinometra from Okinawa, Zanzibar, and Eilat............................................................................................. 38 Figure 13. Sperm of Okinawa, Mauritius, and Eilat Echinometra................................... 39 Figure 14. Neighbor Joining tree of a 104 COI haplotypes.............................................. 44 Figure 15. Neighbor-Joining trees showing the phylogenetic relationships among clades of CO sequences of Indo-Pacific Echinometra species...................... 54 Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 7. Figure 8.

List of tables Table 1. Sperm head size (length and width) and shape (length/width) of Echinometra from Okinawa, Mauritius, Eilat, Bonin, Guam, and Hawaii. ............................ 40 Table 2. Percentage sequence divergence within and between populations.................... 42

Abbreviations
TBRC Tropical Biosphere Research Center IUI Inter University Institute IMS Institute of Marine Science CO Cytochrome c oxidase subunit 1 ML Maximum Likelihood NG Neighbor Joining MP Maximum Parsimony SN Supernatant PCR Polymerase Chain Reaction mtDNA mitochondrial DNA WIO Western Indian Ocean IWP Indo-West Pacific

1. Abstract
Accurate assessment of species diversity is essential to nearly all areas of biology: studies of biodiversity, ecology, conservation, and policy-making all necessitate correct species identification. Although considerable attention is devoted to species concepts in the primary evolution literature, far less consideration is dedicated to developing methods for empirically delimiting and assessing species boundaries. Moreover, even when species boundaries have been hypothesized, little thought has been given to how these constructs can be assessed in a rigorous manner. Sea urchins from the genus Echinometra (Gray 1825) currently comprise eight known species, with cosmopolitan distribution. Where they occur, these species are often among the most prevalent urchins in the reef. One of these species, Echinometra mathaei, has been called the most common urchin in the world. The ecological impact of these urchins is substantial, through grazing and bioerosion on benthic habitats in general and on coral reefs in particular. Among these urchins, four closely-related species are reported from Okinawa, and one of them, E. mathaei, was also reported from the western Indian Ocean (WIO) and the Gulf of Aqaba. However, whereas in Okinawa the four Echinometra species are well studied, investigations into the nature of Echinometra from the WIO and the Gulf of Aqaba have been superficial and based on mere observations. The first objective of the current study was to evaluate the general concordance between morphological and molecular tools used for identifying species. The four closely-related Okinawan Echinometra are used here to investigate this relationship. Ratifying the delineation of these four species based on mitochondrial cytochrome c oxidase subunit I molecular phylogeny, allows scrutinization of the morphological keys used for their identification. The second

objective of this work was to investigate the nature of Echinometra found in the WIO (Zanzibar) and the Gulf of Aqaba (Eilat). This investigation was based on the insights gained from the Okinawan urchins and presents, for the first time, molecular data regarding Echinometra species from these rejoins. The results revealed that the morphological keys available for identifying Okinawan Echinometra are highly compatible with molecularly inferred identification (~80% concordance). Furthermore, the color of the spines was found to be the least shared character-state among species. Consequently applying a hierarchical classification with color of spine predominance, improved concordance levels to ~91%. However, despite their significance in ecological studies, external morphological features alone are in marked discordance with molecular data and could not be used to infer species attribution in these urchins. Sex of an individual had no effect on its species ascription throughout the characters examined. Results based on both morphological and molecular examinations of Echinometra from Zanzibar and Eilat refute the claims that these urchins are E. mathaei. Furthermore, molecular taxonomy strongly supports these urchins as an entire new species of the genus Echinometra, based on their considerable molecular divergence from other Echinometra species, and their well supported monophily. Moreover, the urchins from Eilat and Zanzibar appear to be the same species, and a rigorous sampling carried out around the Island of Zanzibar suggests that only one species of Echinometra exist there. A clear biogeographic pattern is discernible between the Pacific and Indian Ocean with populations, with the Eilat and Zanzibar populations being allopatric to those of the Pacific. However, the nature of the barrier that split these populations some 1-2 million years ago is still unknown.

2. Introduction
Species level is recognized as the major unit of biodiversity (Hull 1977; Claridge et al. 1997; Mayden 1997; Ereshefsky 2001). Almost all studies in biology, whether at the level of molecules, cells, individuals or populations, are typically referenced to the level of the species. Prior to Darwin, naturalists viewed species as ideal or general types, which could be exemplified by an ideal specimen bearing all the morphological traits general to the species. In order to do so, these specimens had to be differentiated into species. After Darwin, whose theories shifted attention from uniformity to variation and from the general to the particular (Darwin 1859), scientific interest shifted towards the relation of an individual to the other individuals with which it interacts. These two basic goals of attributing a newly-discovered specimen to a species and determining the relationship among groups of organisms is called taxonomy, the science of classification. The process of allocating individuals to a given species obviously depends on the criteria by which species are defined and delimited, which are in turn determined by the concept of what a species is. Classical taxonomists classified individuals as members of a species based on a suite of shared morphological characters that were diagnostic and differentiated them from other such morphologically defined groups. Numerical taxonomy shares the concept of a morphological species; however it differs from classical taxonomy mainly in its methodology: the use of a large number of phenotypic characters, rather than only diagnostic ones, with equal a priori weighting, and in its emphasis on species as clusters derived from measures of overall similarity (Sneath & Sokal 1973). The biological species concept (Dobzhansky 1937; Mayr 1942) defines species based on their ability to interbreed: species are considered as natural entities

distinguishable from other species by the criterion of reproductive isolation and not overall phenotypic similarity. However, the biological species concept has been criticized for several reasons, mainly for its lack of universality (for example, inapplicability to asexual taxa: Mishler & Theriot 2000), and the difficulty of applying it to allopatric populations (Mallet 1995). Phylogenetic species concepts are increasingly being proposed as alternatives to the biological species concept. In recent years there have been several attempts to delimit species boundaries based on phylogenetic reconstructions, mostly using mitochondrial DNA haplotypes (e.g. Doan & Castone 2003; Hendrixon & Bond 2005; Flot et al. 2008). Although some of these studies have revealed concordance with species defined by classical taxonomy, others have not (Wiens and Penkrot 2002). In their colloquium paper: Phylogenetics and the origin of species Avise and Wollenberg (1997) succeeded in connecting population genetics, gene genealogies and reproductive isolation. They pointed out that reproductive isolation is an important component of all lineage-based phylogenetic species concepts as it provides the restriction or elimination of gene flow between populations, resulting in independently evolving lineages. One may then argue that, for sexually-reproducing taxa, reproductive isolation does provide a sound criterion for delimiting species. Reproductive isolation may thus be looked upon as a sufficient but not necessary condition for delimiting species boundaries: where it does exist, it is likely to unambiguously delimit species. The debate over the validity of different species concepts is unlikely to be resolved in the near future. However, concordance between boundaries obtained using different species concepts or different data sets within a particular taxon, would

significantly increase our level of certainty in defining a group of individuals as a new species and enable the construction of viable identification keys.

2.1 Morphologically inferred taxonomy

Morphology refers to the form and configuration of an organism (MerriamWebster dictionary). This includes aspects of external appearance such as shape, structure and color, as well as the form and structure of internal parts like bones, spicules, and organs. Most taxa differ morphologically from other taxa. Typically, closely-related taxa differ much less than more distantly related ones, but there are exceptions. Such a exceptions are often seen through convergent evolution where unrelated taxa acquire similar appearance (Niemi 1985). The classification of living organisms, i.e. Alpha taxonomy, and associated biodiversity assessments, are still mainly based on morphological characters (Ereshefsky 2001; Will & Rubinoff 2004). Once the morphological boundaries of an operational species are determined, identification either results in the inclusion of the organism under study in a group, or not. If the individual does not have the diagnostic combination of characters of a named taxon then redefinition is necessary, i.e. its placement in a new or different group, or expansion of the existing taxon definition (Will & Rubinoff 2004). Since morphology is a complex and non-neutral marker that requires a great deal of expertise, taxonomical assessments solely based on morphology could lead to under- or over-estimations of biodiversity. One such example could be found in the European genus Niphargus, which is believed to be in gross underestimation of the true level of its biodiversity (Sket 1999). Will and Rubinoff (2004) however, state that morphological assessments are still the best alternative for

understanding and studying whole organisms when determining identities or systematic relationships. Only in specific cases, they add, if we are to address questions that defy resolution using traditional character systems, are the use of molecular techniques required.

2.2 Molecular taxonomy

With a greater need for biological inventories and studies of biodiversity than ever before, and with fewer researchers to do the job (McAllister 2000; Hopkins & Freckleton 2002), it has been suggested to delaminate organisms by using parts of the genome as a marker in a sort of molecular typification. Genomic approaches to taxon diagnosis exploit diversity among DNA sequences to identify organisms (Kurtzman 1994; Wilson 1995, Hebert et al. 2003a). In a very real sense, these sequences can be viewed as genetic barcodes that are embedded in every cell. By this molecular approach, a small fraction of an organisms total genome, its barcode, is used as an identifying tag for the organism (Blaxter 2003, 2004; Hebert et al. 2003a,b; Stoeckle 2003). Two key concepts drive the use of molecular markers: species identification and species delimitation. Discerning the two concepts and associated terminology is fundamental to understanding the development and applicability of these markers. Molecular species identification, also referred to as DNA barcoding, deals with the assignment of specific molecular markers to species. In other words, this approach attempts to produce a molecular entity, shared by all taxa, that differs in its character state between species. Such a tool would be extremely beneficial in determining the existence of new species and invaluable in inferring cryptic species. Molecular species delimitation

is the attempt to draw the boundaries between species. This approach tries to incorporate molecular level population comparisons, such as molecular divergence indices, to traditional phylogenetics. It differs from species identification in that it attempts to also infer relations among taxa. Using molecular markers for phylogenetic reconstructions is thought to be a pragmatic approach and an objective tool to help taxonomy both in difficult situations (e.g. absence of specialists, morphological convergences, poorly preserved samples, etc.), and in verifying otherwise obtained relationships between taxa (namely morphologically inferred). The Cytochrome c oxidase subunit 1 (COI) gene has been proposed as the main barcoding gene for metazoans (Hebert et al. 2003a,b). Based on initial screening and theoretical considerations, Hebert et al. (2003a,b) suggested that an approximately 650 bp stretch of this gene may be sufficient to obtain resolution on all levels between species and phylum for the majority of groups. Barcoding is already being employed for complex groups such as nematodes and mosquitoes (Floyd et al. 2002; Floyd & Abebe 2002; Besansky et al. 2003), to name but a few. In Crustacea, for example, a COI threshold of 0.16 subst./site was found to be a decisive criterion in species delimitation (Lefbure et al. 2006). Other potential markers such as the mitochondrial genes encoding ribosomal (12S, 16S) DNA and several nuclear genes were also proposed as candidates for molecular barcoding and phylogenetic reconstruction (Hebert et al. 2003a,b). However, their use in broad taxonomic analyses is constrained by the prevalence of insertions and deletions (indels) that greatly complicate sequence alignments (Doyle and Gaut 2000). Moreover, many of the other markers suggested tend to evolve more slowly than COI and are therefore more suitable for ranking higher taxonomic groups than species (Lefbure et

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al. 2006). The relative ease of amplifying COI with standard primers (Folmer et al. 1994), and the accumulating data from many research groups, is turning this DNA fragment into an attractive candidate gene for organism diversity screening.

2.3 Morphological vs. molecular tools 2.3.1 The problems associated with morphological techniques
Hebert et al. (2003a) claim that the approach of routine species identification based on morphology has four significant limitations. First, both phenotypic plasticity and genetic variability in the characters employed for species recognition can lead to incorrect identifications. Second, morphologically cryptic taxa, which are common in many groups (Knowlton 1993; Jarman & Elliott 2000), are likely to be overlooked in this approach. Third, since morphological keys are often effective only for a particular life stage or sex, individuals may happen to be in a life history stage or of a sex that does not display the diagnostic morphological characters that separate it from other species. Finally, the use of morphology often demands such a high level of expertise that misdiagnoses are common. This final shortcoming mentioned by Hebert et al. (2003a) becomes ever more severe in light of the dwindling numbers of taxonomists in the current biodiversity crisis (Daly 1995; Buyck 1999; Lammers 1999; McAllister 2000; Hopkins & Freckleton 2002). Another problem is that of the geographically limited sample collection in the process of establishing the key criteria for a particular species definition. It is not uncommon for authors to present morphologically defining criteria based on a small number of samples collected on a limited spatial scale (Palumbi & Metz 1991; Chen et al.

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2008). In such a sampling design, only a part of the character states for a given morphological character will be included in the species identification key. These gaps in information may lead to future misidentifications of species, as specimens containing the missing character states are encountered and evaluated based on the existing keys. Subjective and imprecise character definition used to convey the exact nature of the defined characters is yet another common shortcoming of many morphologicallybased species identifications encountered in the literature. Further, in many cases, species descriptions are not exhaustive and often suffer from an inconsistent inclusion of characters, even among descriptions of very similar species. Arakaki et al. (1998) for instance, uses the terms dark and bright to describe the state of the skin around the peristome in Indo-West Pacific populations of sea urchins. Color is also frequently used as a character state with no quantitative support or reference (Arakaki et al. 1998; McClanahan & Muthiga 2001). Using such ambiguous descriptions renders the associated morphological characters useless not just in delaminating extremely plastic characters but also relatively distinct ones. For example, Brumfield et al. (2001) found such a color-based classification system to be misleading in the case of a small passerine which breeds in tropical South America. Finally, samples damaged during collection, prolonged or inadequate storing, and fixation using different chemicals (e.g. Formaldehyd, EtOH) is likely to cause loss of diagnostically important characters (such as color or body structure in soft-bodied animals), rendering the samples useless for morphological assessments (Schander & Willassen 2005).

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2.3.2 The problems associated with molecular techniques

COI is frequently used as a species delimitation tool for phylogenetic and evolutionary inference. Although COI is considered as a very good gene for that purpose by some researchers (Miya & Nishida 2000; Hebert et al. 2003a,b), others contend that molecular barcoding may not be the most effective tool for forming robust phylogenetic hypotheses (Schander & Willassen 2005). Furthermore, exclusive promotion of COI (or any other marker for that matter) as an all-purpose species diagnostic gene could be potentially deceptive. The Cnidaria, for instance, seem to have a unique DNA repair system that results in low levels of variability and few distinctive features in COI (Hebert et al. 2003a,b). When using COI for either delimitation or identification, it is also seen as a major issue that this molecule may not be able to distinguish recent species (e.g. Mathews et al. 2002). However, this constitutes a challenge for any taxonomy and not only for DNA-based ones. Will and Rubinoff (2004) argue that molecular identification of species is fraught with the same constraints and inconsistencies that plague morphological judgments of species boundaries. They claim that as most morphological assessments rely on a suite of complex morphological characters upon which to base their conclusions, molecular techniques rely on parts of a single gene. A fundamental problem associated with applying molecular tools for species identification is that as robust and reliable as these tools may be, they rely on the very system they claim to replace. In order for a sequence to have a meaning it must be compared to an existing sequence that has been previously assigned to a particular species.

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Field identification and non-invasive sampling, two increasingly important requirements in biodiversity surveys, are not always possible using molecular methods. Moreover, if molecular techniques are to improve the rate by which we identify new species, then the fact that they rely on traditional slow methodologies to attach the organism to the DNA sequence, hinders that goal. Limited or biased sample collection is yet another major problem not to be overlooked. Although this shortcoming is not directly linked to the molecular techniques perse, its consequences are far-reaching. Just as establishing morphological keys based on limited sample size could result in underestimating the existing variability and natural proportions of character-states, so too could assigning the possible molecular entities to a species based on a limited number of samples be misleading. Many authors draw their conclusions on vast geographical scales after having examined only a mere few samples (Flot et al. 2008). Another bias in sample collection is that of choosing the right or morphologically defined specimens, while neglecting individuals that do not fall into the typical species description (Flot et al. 2008). The outcome of such biased sampling will result in failure to ascertain the entire species range. If one aims at deducing results for entire populations, avoiding such bias can only be achieved by obtaining a large number of samples of randomly collected individuals of the species of interest. Finally, assigning the wrong molecular barcodes based on insufficient morphological keys can lead to erroneous species identifications and wrongly inferred phylogenies. Currently, no screening system has been devised to validate sequences entering the data bases. As a result numerous errors are frequently discovered in the

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submissions without any possibility of checking the original material (e.g. Harris 2003). Such errors will lead to misidentifications, with no real way to verify the findings. The debate between supporters and critics of using molecular techniques as taxonomic tools (e.g., Tautz et al. 2002, 2003; Hebert et al. 2003a; Schander & Willassen 2005) and (e.g., Lipscomb et al. 2003; Mallet & Willmott 2003; Seberg et al. 2003; Will & Rubinoff 2004) is fierce. However the mutualism between molecular systematics and traditional taxonomy is clearly an obligate relationship. DNA sequence data are an important and powerful part of taxonomy and systematics, and molecular data have an indisputable role in the analysis of biodiversity. A good example of this can be seen in the barcoding of life project (www.dnabarcodes.org). In this initiative molecular barcoding is utilized for species identification, while the organisms true identity is guaranteed by attaching taxonomically identified vouchers (a reference specimen for each reported taxon) to all the sequences submitted to the project. However, DNA-based data should not be seen as a substitute for understanding and studying whole organisms when determining identities or systematic relationships. This is therefore the reason why I chose to implement and compare both techniques in my study of Echinometra spp.

2.4 The genus Echinometra (Gray 1825)

Eight common species of genus Echinometra (Echinodermata: Echinoidea) are known to exist globally (McClanahan & Muthiga 2001). Ecological comparisons of the eight species show differences in morphology, diet, tolerance to environmental stress, behavior, and reproduction between and within species (Kelso 1970; Russo 1977; Tsuchiya & Nishihira 1984, 1985, 1986; Neill 1988; Arakaki & Uehara 1991). These sea

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urchins have variable roles in coral reef ecosystems. As grazers of benthic algae, they reduce algal cover and break down reef substratum, which creates topographic complexity and may enhance coral recruitment (Dart 1972; Sammarco et al. 1974; Birkeland & Randall 1981). On the other hand, high sea urchin abundance is associated with a high erosion rate of coral reef substratum and low topographic complexity of reefs (McClanahan & Shafir 1990; Eakin 1996); therefore, high sea urchin abundance may have long-term detrimental effects on the structure and ecology of these reefs (McClanahan & Obura 1995; Jennings & Polunin 1996). Echinometra are also known to feed on living corals, thereby reducing coral survival and calcium carbonate deposition (Glynn et al. 1979; Bak & van Eys 1975; Lawrence 1975). Since Echinometra is known to be the dominant urchin species in many reefs around the world (McClanahan & Muthiga 2001), and as reports of their growing numbers are mounting (Muthiga & McClanahan 1987; McClanahan & Kaunda-Arara 1996; McClanahan 1994), the need to thoroughly study this group becomes crucial. Four genetically and morphologically divergent species of tropical sea urchins belonging to the Echinometra mathaei sensu lato species complex are currently recognized in the literature (Palumbi 1996; Arakaki et al. 1998). These species are among the most ubiquitous and abundant shallow-water echinoids in the warm Indo-Pacific region (Palumbi 1996; Palumbi et al. 1997; McClanahan & Muthiga 2001). Their wide distribution stretches from central Japan in the north, to south-east Australia in the south, Mexico in the east, and to the Gulf of Suez in the west (Mortensen 1943; Kelso 1970; Clark & Rowe 1971; Russo 1977). Originally, although two species were described from the Indo-Pacific (de Blainville 1825), E. mathaei from Mauritius and E. oblonga from an

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undisclosed locality, subsequent morphological studies of adults and larvae (Mortensen 1943) concluded that only one species existed. The same conclusion was reached by Clark and Rowe (1971). Thus, Echinometra mathaei became to be known as the worlds most abundant sea urchin. However, extensive investigations into ecological distribution, test morphology, spicules in gonads, and gamete compatibility proved that E. mathaei and E. oblonga are entirely separate species (Kelso 1970). Later morphological, behavioral, and embryological studies (Tsuchiya & Nishihira 1984, 1985; Uehara & Shingaki 1984, 1985; Uehara et al. 1986, 1990) established the existence of four Echinometra species in Okinawa. These findings were also supported by enzyme electrophoresis (Matsuoka & Hatakana 1991) and mitochondrial DNA studies (Palumbi & Metz 1991), which concluded that the four types of urchins found in Okinawa are distinct but closely-related species. Across their huge range of geographical distribution many morphological variants have been described for these species (Russo 1977), including some from the Gulf of Aqaba at the northern tip of the Red Sea (Fishelson 1971; Lawrence 1983). Although much work was done in the Pacific, taxonomical studies on these species of both morphological and molecular nature are scarce for the WIO. However, despite this lack of sufficient taxonomical background, urchins from many parts of the WIO and the Gulf of Aqaba have been referred to only as E. mathaei in numerous ecological studies from that region (e.g. Lawrence 1983; McClanahan & Shafir 1990; McClanahan & Muthiga 2001). By combining both morphological keys and molecular analysis, the current study

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will ascertain the occurrence of E. mathaei in the WIO and the Gulf of Aqaba, and will provide an up-to-date revision of the geographical range of this species. The objectives of this research were: (1) To test the reliability of current morphological species identification keys for Okinawa Echinometra spp., and to define, if possible, the critical identifying features. (2) To compare morphological identification to molecular barcoding in these closely-related Okinawa species. (3) To identify the species of Echinometra in Eilat (Gulf of Aqaba) and Zanzibar using both morphological and molecular tools. (4) To determine the phylogenetic relationships between the WIO and Red Sea species and the IWP species.

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3. Materials and methods

3.1 Collection of samples
Samples of Echinometra spp. were collected between June 2007 and November 2008 from three locations worldwide, Okinawa, Zanzibar and Eilat (Gulf of Aqaba, northern red sea) (Fig. 1 & 2). Okinawa samples were collected during June-July 2007 at Sesoko Island (263803.44N, 1275151.24E), off the coast of Okinawa, from a depth of 1 m at low tide. Urchins were collected separately from 9 randomly placed 1 m2 quadrats.

Figure 1. Map of the Indian Ocean and eastern Pacific. Dots mark the three sampling sites:
Okinawa in the Pacific, Zanzibar in the Indian Ocean, and Eilat in the Red Sea, connecting to the Indian Ocean.

The Zanzibar samples were collected in November 2007, March-April 2008 and November 2008 from eight sites around the Island: Changu, Bawe, Chumbe, Kizimkazi, Jambiani, Pongwe, Metemwe and Nungwi (060703.10S, 391006.89E;

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060816.36S, 392906.70E;

390757.82E; 061957.32S,

061654.51S, 393332.16E;

391032.85E; 060120.99S,

062812.81S, 392530.21E;

054851.17S, 392133.47E; 054347.44S, 391727.75E, respectively). These eight sites were selected to represent possibly different environments surrounding the Island of Zanzibar where Echinometra were expected to inhabit. Urchins from all sites were randomly collected at low tide from depths of 1-3 meters. The Eilat samples were collected in October 2007 and March 2008 from Tur-Yam (293059.10N, 345534.63E), south of the oil jetty terminal (KATZAA). Urchins were collected from depths of 1-3 meters using SCUBA. Throughout this study, sea urchins were sampled randomly in the field and the sampling site was recorded for each individual. Following collection, the samples were brought to the lab (IMS in Zanzibar, IUI in Eilat and TBRC in Okinawa) and maintained in running seawater. Further analysis was completed within 24 hours of collection. A total of 118, 86 and 42 individuals were collected from Zanzibar, Okinawa and Eilat respectively.

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Figure 2. Study sites; Zanzibar (A), Gulf of Aqaba (B) and Okinawa (C). Sampling sites are
indicated by arrows: Tur-Yam in the Gulf of Aqaba; Changu, Bawe, Chumbe, Kizimkazi, Jambiani, Pongwe, Mnemba and Nungwi in Zanzibar; and adjacent to the TBRC (Tropical Biosphere Research Center) on Sesoko island, Okinawa.

3.2 Morphological measurements

The morphological characteristics used for describing the differences among Echinometra spp. were: color of the spines, brightness of milled ring, brightness of the skin around the peristome, and spicule shape in the tubefeet and gonads (Fig. 3). An array of morphometric measurements was taken, including length, width and height (measured along the oral-aboral axis). Measurements were done on tests only (i.e. not including the spines) to the nearest 0.5 mm using Vernier calipers. Thin blade calipers were used to prevent the spines from interfering with the measurements. Proportions of length/width, length/height, and width/height were calculated (denoted as L/W, L/H, and W/H,

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respectively). Appearance of milled ring and skin around the peristome was determined as being bright or dark for the skin around the peristome, and bright, faded or dark for the milled rings. Color of the spines was described in words. Following external examination, urchins were dissected and the gonads were exposed. Gonad samples were then used for spicule analysis, determination of the specimens sex and for DNA extraction. To determine a specimens sex, a small piece of gonad was detached using a forceps and observed under a light microscope at a 20X magnification. The presence of sperm or eggs was used to determine the sex (Fig. 4). If no gonads were found in an individual, it was noted as NG (no gonads). In several male samples originating from Eilat, the shape and dimensions of the sperm were recorded using electron microscopy in addition to the measurements noted above.

Figure 3. External anatomy of a

regular urchin in lateral view. Left side: test only;

right side: spines, tube feet and intact.

epidermis

(after Hendler et al. 1995).

22

In order to obtain tubefeet spicules, several tubefeet were detached with a forceps while gonad spicules were obtained from a small portion of the gonad. Tissue samples from both tubefeet and gonads were photographed using a light microscope (a 10X magnification for gonad spicules and a 20X magnification for tubefeet spicules) fitted with a Ken-a-vision PupilCAM, digital camera. In order to determine spicule morphology and dimensions, pictures were analyzed using the software ImageJ (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA,

http://rsb.info.nih.gov/ij/, 1997-2008).

3.3 Species identification based on morphological characters

In order to evaluate the reliability of currently used morphological keys, I chose the Okinawa Echinometra as a model group. A matrix including the entire morphological data set was constructed. For each character-state, the names of all possible species exhibiting that state were recorded. Following analysis of all morphological characters, a score was calculated by summing up the number of times each species name appeared for every individual (referred to as total score). The species name with the highest score was determined as the species of that individual. If a similar score was achieved for two (or more) species, no decision could be made, and the individual was assigned as unidentified. For those individuals, an arbitrary priority, achieved by multiplying the score of a character by a factor of two, was given to different characters. Species identification was performed once again according to the revised scores. Subsequently, the new proportion of unidentified individuals was calculated. This method of assigning

23

species names based on morphology was later tested by the molecular attributions imposed by the phylogenetic reconstruction of the CO sequences.

Figure 4. Sperm (A) and eggs (B) in gonads of Echinometra spp. from Okinawa. Scale bar indicates 100 m.

3.4 Echinometra nomenclature

The nomenclature used to describe the four Echinometra species found in Okinawa is Echinometra spp. A, B, C, and D, where Echinometra spp. B and D are considered as E. mathaei and E. oblonga respectively (Edmondson 1935; Motokawa 1991; Nishihira et al. 1991). Zanzibar Echinometra are hereby named E. spp. ZE (ZE=Zanzibar Echinometra), while Eilat Echinometra are named E. spp. EE (EE=Eilat Echinometra).

3.5 Sperm morphology using SEM

The examination of sperm was carried out using a scanning electron microscope (JEOL JSM5610LV scanning electron microscope at Ben-Gurion University). Sperm

24

samples were obtained as undiluted semen, i.e., dry sperm by removing the testes and a modified protocol from Arakaki et al. (1998) was followed. Specimens were fixed in 2.5% glutaraldehyde in filtered (0.2m) seawater (FSW) and stored for four months at 4C. Fixed samples were washed twice in FSW for a total of three hours and centrifuged at 5000 rpm in order to concentrate the rinsed sperm. Subsequently, samples were postfixed in 1% osmium tetroxide fixative (OsO4) in FSW for one hour at low temperature (about 4C). Following post-fixation, the samples were spinned down, washed twice with ddH2O and, finally, 50l of ddH2O were added to each sample. Several drops of the rinsed diluted samples were mounted on poly-l-lysine covered cover-glass (renders the cover-glass sticky) and left to stand for 10 minutes to spread and adhere the sperm to the glass. The glass was put in a balance glass vial. The process of dehydration in a graded series of ethanol (30% - 5 min, 50% - 5 min, 70% - 2.5 days, 90% - 3 min, 100% - 3 min, 100% - 3 min) was carried out in the glass vial. Following complete dehydration, samples were placed in the hood and treated with a graded series of ethanol:hexamethyldisilazane (HMDS) (3:1, 2:1, 1:1, 1:2, 1:3 all briefly) to replace the ethanol, and finally washed twice with 100% HMDS. Samples were left to dry, then coated with palladium gold (about 12 nm) using an Ion Coater (Emitech K575X).

3.6 Molecular assays

A ~600 bp long portion of Cytochrome C oxidase subunit (CO), a mitochondrial gene, was sequenced to confirm species identification based on morphology and to reconstruct the phylogenetic trees (Geyer & Palumbi 2003). The phylogenetic tree reconstruction approach was used to test the presence of cryptic sibling

25

species. Throughout this study, DNA extractions were performed at the collection sites i.e. the TBRC in Okinawa, the IMS in Zanzibar and the IUI in Eilat. Extracted DNA was transported to Tel-Aviv University for completion of the analysis.

3.6.1 DNA extraction

DNA was extracted from the gonads based on a protocol modified from Xavier Pochon (Pochon et al. 2001). To make 212 ml of extraction solution, a mix of 100 ml ddH2O, 100 g Guanidinium isothiocyanate, 10.6 ml Tris (1M pH 7.6) and 4.25 ml EDTA (0.5M) were dissolved by stirring for 10 min at 60-70C. Then, 4.24 g Sarkosyl and 2.1 ml -mercaptoethanol were added to the mixture and the total volume adjusted to 212 ml by adding ddH2O. Extraction solution was maintained in the dark at 4C. A small piece of gonad was clipped off using forceps, rinsed with ddH2O, dried and placed in a 1.5 ml microcentrifuge tube filled with 300 l extraction solution. Tubes were vortexed and stored at 4C for 3 hr to enable tissue dissolving. Samples were then vortexed again, quickly centrifuged and incubated at 72C for 10 min during which they were vortexed 34 times. Tubes were then centrifuged at maximum speed for 5 min and a volume of 200 l of supernatant (SN) was placed in new 1.5 ml microcentrifuge tubes. DNA was precipitated by adding 200 l of isopropanol, vortexing and incubating overnight at 20C. Following incubation, the tubes were centrifuged at maximum speed for 15 min. All remaining SN around the pellet was discarded using a slim pipette. The extracted DNA was rinsed with 100 l of 70% EtOH, vortexed and centrifuged again at maximum speed for 10 min. SN was again discarded and the tubes inverted open and allowed to dry. Finally DNA was resuspended in 50 l of super distilled water while placed on ice

26

and gently vortexed every 15 min. Resuspended DNA was stored at -20C until use. Following extraction, DNA purity was assessed using a NanoDrop ND-1000 spectrophotometer and the concentration of each sample was recorded. Extractions revealed as impure were discarded and the extraction was repeated. Otherwise, DNA was diluted to ca. 2.5 ng/l with ddH2O.

3.6.2 PCR amplification and sequencing

CO was amplified using the primers CO-f (5-

CCTGCAGGAGGAGGAGAYCC-3, Geyer & Palumbi 2003) and CO-d (5GAACATGATGAAGAAGTGCACCTTCCC-3, Geyer & Palumbi 2003), which correspond to positions 6439-7039 in the Strongylocentrotus purpuratus mitochondrial genome. The polymerase chain reaction (PCR) was performed in 40 l total volume and its chemistry as fallows: 4 l 10X reaction buffer, 3.2 l dNTP (2.5mM), 2.4 l MgCl (25mM), 0.8 l (8 pmol) of each primer, 0.4 l of GoTaq Flexi DNA Polymerase (Promega), 26.4 l ddH2O and 2 l of DNA template (ca. 5 ng/l). Amplifications were conducted in a Peqlab Primus 96 machine and used a standard amplification profile of an initial denaturation step at 94C for 4 min followed by 35 cycles of 94C for 30 sec, 55C for 30 sec and 72 C for 30 sec (Geyer & Palumbi 2003). PCR products (ca. 600bp) were checked on an ethidium-bromide-stained 1% Tris-Acetate-EDTA agarose gel. PCR products purification and sequencing were preformed by Macrogen Inc., Korea. PCR products were purified using a Montage Millipore column kit. Sequencing reactions were performed in a MJ Research PTC-225 Peltier Thermal Cycler using a ABI PRISM BigDyeTM Terminator Cycle Sequencing Kits with AmpliTaq DNA

27

polymerase (FS enzyme) (Applied Biosystems), according to protocols supplied by the manufacturer. Single-pass sequencing was performed on each template twice, sequencing both directions using primers CO-f and CO-d. The fluorescent-labeled fragments were purified from the unincorporated terminators with an ethanol precipitation protocol. The samples were resuspended in distilled water and subjected to electrophoresis in an ABI 3730xl sequencer (Applied Biosystems).

3.6.3 Alignment and analysis of sequences

Chromatograms were checked and edited manually using ChromasPro version 1.42 (Technelysium Pty Ltd) and aligned using Clustal X version 2.0.9 (Larkin et al. 2007). The sequences were then translated to amino acids sequences using MEGA version 4.0 (Tamura et al. 2007) and the reading frame was located by comparison to Echinodermata CO amino acid sequences from the GenBank. Eight additional Echinometra CO sequences, were obtained from GenBank aligned and cut at the same positions as the Echinometra sampled (accession numbers: AY262861, AY262876, AY262880, AY262886, AY262930; AF255507, AF255522; AF018884, from Landry et al. 2003, McCartney et al. 2000 and Palumbi et al. 1997, respectively). Three species were chosen as outgroups, Echinometra insularis, Echinometra vanbrynti and Echinometra viridis, as their mitochondrial genomes were consistently recovered as the most closely related to the Echinometra sampled using BLAST in GenBank (accession numbers: AY262907, AY262883; AF255522, from Landry et al. 2003 and McCartney et al. 2000, respectively). Eventually only E. insularis was used as an outgroup as this species was found to be the closest to the Echinometra species examined, and gave the

28

best resolution in the phylogenetic reconstruction. The CO fragments were screened for individual haplotypic variation using the program DnaSP version 4.90 softwear (Rozas et al. 2003). Phylogenetic reconstruction was performed on all haplotypes found using three independent phylogenetic methods, Maximum Likelihood (ML), Neighbor Joining (NJ), and Maximum Parsimony (MP). Neighbor Joining clustering analyses (NJ; nucleotide model: maximum composite likelihood, homogeneous pattern among lineages and uniform rates among sites, pairwise deletion) were conducted using MEGA version 4.0 with 1,000 non-parametric bootstrap replicates (Felsensteis 1985). The monophyly of the groups obtained was checked by performing Maximum Likelihood (ML; GTR model with gamma law, invariable sites), and Maximum Parsimony (MP; search option: CNI level = 1, initial tree by random addition with 10 replications, use all sites) phylogenetic analyses (1,000 bootstrap replicates) in Treefinder version Oct 2008

(http://www.treefinder.de) and MEGA version 4.0, respectively. For maximum likelihood analysis, the best probabilistic model of sequence evolution was determined using Modeltest 3.04 (Posada & Crandall 1998). The phylogenetic tree reconstruction approach was used to infer species boundaries and to test for the presence of cryptic sibling species. Within and among-species polymorphism of COI sequences were estimated using ARLEQUIN software ver. 3.11 (Laval & Schneider 2005) with Tamura model (Tamura & Nei 1993).

29

3.7 Statistical analysis

Morphological data were analyzed using STATAISTICA 7 software. To perform analysis of variance, normal distribution and homogeneity of variances assumptions were verified as follows: normal distribution was tested using the Kolmogorov-Smirnov test and homogeneity of variances was tested using Cochrans test (Zar 1999). Since homogeneity of variances did not exist, a non-parametric analysis (i.e. Kruskal-Wallis) was applied. Multiple comparisons of mean ranks tests were then used as post-hoc comparisons when significant differences were detected. Separate students t-tests were performed in order to test differences in sperm size between samples from Eilat and the Pacific. Results are presented as mean SE throughout the text unless denoted otherwise.

4. Results
4.1 Morphological diversity in Echinometra
Echinometra from Okinawa, Zanzibar, and Eilat are shown in figure 5. In Okinawa, four morphologically separated groups of Echinometra were identified (corresponding to the four established Echinometra species, E. spp. A, mathaei, C, and oblonga) (pictures 1-8 in figure 5). In Eilat and Zanzibar, a single morphological Echinometra group was encountered in each of the locations, and these were assigned the names E. sp. EE in Eilat (pictures 9-10 in figure 5) and E. sp. ZE in Zanzibar (pictures 11-12 in figure 5). A summary of the morphological characteristics of Echinometra from Okinawa, Zanzibar, and Eilat is presented in appendix A.

30

Figure 5.

Echinometra species

from Okinawa, Eilat and Zanzibar. Oral (right column) and aboral (left column) views of the same individual are sown. E. sp. A, E. mathaei, E. sp. C and E. oblonga from Okinawa are shown in pictures 1-2, 3-4, 5-6 and 7-8, respectively. Eilat Echinometra (E. sp. EE) is shown in pictures 910 and Zanzibarian (E. sp. Black ZE) in

Echinometra

pictures 11-12. The bars indicate 1 cm and applies for both pictures in that row.

31

4.2 External morphological characteristics 4.2.1 Color of spines

Spine colors varied greatly from light brown to violet, green, and black. In total eight character-states were defined based on color. In one occasion (E. sp. C), spine colors varied substantially and were indiscriminate. As a result, in this species colors were pooled to a single category (various colors, no white tips). Adding information on spine tip color for this category was used in order to discriminate them from E. sp. A. E. sp. A is distinctively characterized by white-tipped spines (100%). E. mathaei is characterized by a dominant light brown spine morph (76.92%), and a less prominent brown spine morph (23.08%). The spine colors exhibited by E. sp. C are diverse, but none of the individuals in this category were found to be black or bear white tips. E. oblonga, previously characterized as displaying only black spines, was found to have a dark brown spine morph (37.5%) as well as the dominant black spine morph (62.5%). E. sp. ZE displayed three spine color morphs, black (62.75%), brown (23.53%), and violet (13.72%). E. sp. ZE also displayed three spine color morphs, brown (47.5%), dark brown (22.5%), and dark brown/green (30%) (Fig. 6).

32

100%

80%

60%

40%

20%

0% A B C D ZE EE

Figure 6. Spine color occurrence in Echinometra from Okinawa, Zanzibar, and Eilat. X axis
represents species; Y axis represents percentage of occurrence. Sample size for E. sp. A, E. mathaei, E. sp. C, E. oblonga from Okinawa, E. sp. ZE from Zanzibar and E. sp. EE from Eilat is, n=4, n=13, n=56, n=8, n=51, and n=40 respectively. Color legend: White tips, Light brown, Brown, Various colors no white tips, Black, Dark brown, Violet, Dark brown/green.

4.2.2 Milled rings

Milled rings displayed three character-states: bright, dark, and faded. The first two appeared in Okinawa individuals only. The faded morph was strictly confined to the Zanzibar and Eilat individuals. The presence of a bright milled ring was found in all samples (100%) of E. sp. A. In E. sp. C and E. mathaei, most of the individuals exhibited bright milled rings (61.54% and 80.36%, respectively) while the rest of the population presented dark rings (38.46% and 19.64%, respectively). In E. oblonga all the samples (100%) were characterized by dark milled rings. In E. sp. ZE an intermediate milled ring state was found and denoted as faded (35.29%). The majority (62.75%) of E. sp. ZE however displayed dark milled rings and only a small minority (1.96%) showed

33

bright rings. E. sp. EE had mostly bright (70%) milled rings and the rest were faded (30%) (Fig. 7).
100%

80%

60%

40%

20%

0% A B C D ZE EE

Figure 7. Occurrence and state of milled rings in Echinometra from Okinawa, Zanzibar, and
Eilat. X axis represents species; Y axis represents percentage of occurrence. Sample size for E. sp. A, E. mathaei, E. sp. C, E. oblonga from Okinawa, E. sp. ZE from Zanzibar and E. sp. EE from Eilat is, n=4, n=13, n=56, n=8, n=51, and n=40 respectively. Color legend: Bright, Faded, Dark

4.2.3 Skin of peristome

The nature of the skin around the peristome was described as either dark or bright. However these two character-states were not evenly distributed as the dark morph predominated all but one species. E. sp. A had a 50% - 50% ratio between dark and bright. E. mathaei had 84.62% dark-skinned individuals and 15.38% of bright ones. In E. sp. C still the majority of individuals had dark skin (55.36%), but the proportions of bright-skinned individuals (44.64%), was greater than in E. mathaei. E. oblonga was entirely dark-skinned around the peristome (100%). E. spp. ZE and EE had highly similar milled ring proportions. For both, proportions of the bright skinned individuals were very

34

low (1.96% and 2.5%, respectively) in comparison to the dark skinned individuals (98.04% and 97.5%, respectively) (Fig. 8).
100%

80%

60%

40%

20%

0% A B C D ZE EE

Figure 8.

Color of the skin around the peristome in Echinometra from Okinawa,

Zanzibar, and Eilat. X axis represents species; Y axis represents percentage of occurrence. Sample size for E. sp. A, E. mathaei, E. sp. C, E. oblonga from Okinawa, E. sp. ZE from Zanzibar and E. sp. EE from Eilat is, n=4, n=13, n=56, n=8, n=51, and n=40 respectively. Color legend: Bright, Dark.

4.3 Internal morphological characteristics 4.3.1 Spicules

A total of four different spicule types (Fig. 9) were encountered in gonads and tubefeet during this work, in eleven different spicule combinations. Two additional spicule arrangements were defined as none in having no spicules, and as multiple in having a mixed array of spicules.

35

Figure 9. Spicule types from Echinometra. A: Needle spicules in gonads of E. sp. EE. B:
Triradiate & shaped spicules in the gonads of E. sp. C. C: Triradiate spicules in tubefeet of E. oblonga. D: Bihamate spicules in tubefeet of E. sp. EE. Scale bars indicate 100 m.

4.3.2 Tubefeet spicules

The tubefeet spicules of E. spp. A, ZE, and EE were composed entirely of the bihamate type (100%). In E. mathaei, 76.92% of the population carries bihamate spicules, and the remaining 23.08% carry a mix of bihamate & triradiate spicules. E. sp. C presents the greatest variety of tubefeet spicules with four different spicules categories; triradiate, bihamate, a mix of bihamate & triradiate, and a final category with no spicules whatsoever (78.57%, 5.36%, 1.78%, and 14.29%, respectively). In E. oblonga 75% of the population exhibits triradiate spicules while the rest (25%), have no spicules in their tubefeet (Fig. 10).

36

100%

80%

60%

40%

20%

0% A B C D ZE EE

Figure 10. Shape of the spicules in the tubefeet of Echinometra from Okinawa, Zanzibar, and
Eilat. X axis represents species; Y axis represents percentage of occurrence. Sample size for E. sp. A, E. mathaei, E. sp. C, E. oblonga from Okinawa, E. sp. ZE from Zanzibar and E. sp. EE from Eilat is, n=4, n=13, n=56, n=8, n=51, and n=40 respectively. Color legend: Bihamate, Bihamate & Triradiate, Triradiate, None.

4.3.3 Gonad spicules

Gonad spicules were the most variable and diverse among all characters studied. This diversity is evident in the ten different character-states described from the studied populations. E. sp. A and E. mathaei presented the most homogenous spicule array with all spicules (100%) belonging to the needle type. E. sp. C and E. oblonga presented the most diverse array of gonad spicules. E. sp. C presented seven different spicule types including triradiate, triradiate & needle, none, multiple, needle, triradiate & , and bihamate (58.93%, 16.07%, 8.93%, 5.36%, 5.36%, 3.57%, and 1.79%, respectively). E. oblonga presented five different spicule types that included, triradiate & needle, bihamate, none, triradiate & , and needle & (37.5%, 25%, 12.5%, 12.5%, 12.5%, respectively). The gonad spicules in E. spp. ZE and EE are very similar in composition.

37

The three spicule types presented in each of these species are composed of the pure needle type or needle combinations. Spicule types in E. sp. ZE were needle, needle & , and needle & bihamate (60.78%, 27.46%, 11.76%, respectively). E. sp. EE spicule types were needle, needle & , and needle, bihamate & complex (70%, 20%, 10%, respectively) (Fig. 11).
100%

80%

60%

40%

20%

0% A B C D ZE EE

Figure 11. Shape of the spicules in the gonads of Echinometra from Okinawa, Zanzibar, and
Eilat. X axis represents species; Y axis represents percentage of occurrence. Sample size for E. sp. A, E. mathaei, E. sp. C, E. oblonga from Okinawa, E. sp. ZE from Zanzibar and E. sp. EE from Eilat is, n=4, n=13, n=56, n=8, n=51, and n=40 respectively. Color legend: Needle, Bihamate, Needle & Bihamate, Triradiate, None, Triradiate & Needle, Multiple, Triradiate & , Needle & , Needle, Bihamate & .

4.3.4 Sperm morphology

Two different sperm morphologies were observed in Echinometra from the Pacific and Indian-ocean populations. E. spp. oblonga, BE, and BO have an elongated sperm head, with a ratio of length over width of about six, whereas the rest of the Echinometra species have a more compact shape, with a ratio of length over width of

38

about three (Table 1). Populations of E. sp. EE display a typical compact sperm morphology (Fig. 12), similar to those of the Okinawa E. sp. C and E. mathaei (Fig. 12), and Hawaii E. sp. HA (Table 1). Differences between the sperm shape from Eilat and those from Mauritius, Bonin, and Guam, as well as the Okinawa E. sp. A, and E. oblonga, are significant (student t-test, p<0.05).

EB

BE

VE

EE

Figure 12 . Sperm of Okinawa Echinometra (A, B, C, D), Mauritius Echinometra (VE, EB, BE),
and Eilat Echinometra. A, B, C, D, VE, EB, BE, and EE denote Echinometra spp. A, mathaei, C, oblonga, Violet Echinometra, Echinometra sp. B-like, Black Echinometra, and Eilat Echinometra, respectively. The bar indicates 2 m. Okinawa and Mauritius pictures obtained from Arakaki et al. (1998).

39

Table 1 . Sperm head size (length and width) and shape (length/width) of Echinometra from Okinawa, Mauritius, Eilat, Bonin, Guam, and
Hawaii. The values indicate the mean S.D. A, B, C, D, EE, VE, EB, BE, BO, GU, and HA denote Echinometra spp. A, mathaei, C, oblonga, Eilat Echinometra, Violet Echinometra, Echinometra sp. B-like, Black Echinometra, Bonin Echinometra, Guam Echinometra, and Hawaii Echinometra, respectively. The number of sperm heads analyzed is indicated in parentheses. Sperm were obtained from one individual of each species for Okinawa and Mauritius, 5 individuals (30 sperm heads from each) for Eilat, and 4 individuals of each species for Guam and Hawaii. The data for Okinawa Echinometra were obtained from Arakaki 1989, Mauritius Echinometra from Arakaki et al. (1998), and Bonin, Guam and Hawaii Echinometra from Arakaki et al. (1999).

Okinawa A
(n = 50)

Eilat C D
(n = 50)

Mauritius VE
(n = 20)

Bonin BE
(n = 20)

Guam GU
(n = 80)

Hawaii HA
(n = 42)

B
(n = 50)

EE
(n = 150)

EB
(n = 20)

BO
(n = 20)

(n = 50)

Length (m) Width (m) Shape (L/W)

2.910.19 1.240.05 2.340.18

3.220.16 1.20.05 2.670.18

3.450.16 1.120.05 3.070.18

5.890.31 0.970.05 6.110.48

3.8960.340 1.2690.084 3.0770.287

3.250.13 1.340.04 2.430.12

3.160.16 1.270.05 2.490.15

6.280.40 1.060.06 5.970.55

6.890.45 1.010.06 6.340.56

2.700.17 1.190.06 2.270.18

3.160.21 1.190.08 2.670.26

40

4.4 Sex, length and morphology

The effect of sex on the different aspects of morphology was examined for each of the characters measured. However, no correlation between sex and character-state appearance or proportions was detected, as males and females seem to be evenly distributed among species. Length measurements and calculated proportions (L/W, L/H, and W/H) did not correlate with species or sex, as no significant differences were found throughout the comparisons of these parameters.

4.5 Phylogenetic relationship among sequences

CO fragments amplified were 544 bp long and had 92 variable sites. Out of the 192 individual CO sequences, 104 different haplotypes were detected. These haplotypes fell into five phylogenetic clades with strong geographic associations (Fig. 13). Among these five clades, clade A is a very well supported monophyletic group (100% bootstrap support using ML, NJ, and MP). Clades D and EZ are also well-supported monophyletic groups (93.7%, 91%, and 91% bootstrap support for clade D, and 92.6%, 97%, and 90% for clade EZ using ML, NJ, and MP, respectively). Support for the monophyly of clade C is slightly weaker (86.1% bootstrap support using ML, 92% using NJ, and 89% using MP). The monophyly of clade B was supported in values lower than other clades (75.5% bootstrap support using ML, 86% using NJ, and 72% using MP). Urchins belonging to clades A, B and D together form a cluster, although its monophyly is only weakly supported (51.6% bootstrap support using ML, 64% using MP, and no support using NJ). The monophyly of the cluster formed by clades B and D receives no support. However clades EZ, A, B, and D together form a cluster whose monophyly receives very strong

41

support (87% bootstrap support using ML, 93% using NJ, and 94% using MP). Population percentage sequence divergences are summarized in table 2. Within-species divergence ranged from ~0.12% in E. sp. A to ~0.69 in E. sp. EZ. E. sp. C had the largest between-species average divergences in regards to all other species. The divergence of E. sp. EZ from E. mathaei, E. oblonga and E. sp. A was always greater than the divergence among these three species.

Table 2. Percentage sequence divergence within and between populations. Distances were
calculated as P = nd/nt (where nd is the average number of different nucleotides between two sequences and nt is the total number of nucleotides examined). Diagonal: Differences within populations. Below diagonal: Differences between populations. Distance method: Tamura. P values are presented in the lower table. A, B, C, D, and EZ represent E. spp. A, mathaei, C, oblonga, and the clade of E. spp. ZE and EE, respectively.

A B C D EZ

A 0.122779 2.339844 4.981149 2.999006 3.008675

B 0.657618 4.061496 2.172522 2.579274

EZ

0.544963 3.717866 3.833730

0.587450 3.150892

0.688259

Using E. insularis as an outgroup, the four clades, corresponding to the four currently recognized IWP species (Palumbi 1996), are divided into two multi-species clades. One contains E. sp. C, a species found among several Pacific island archipelagoes (Palumbi 1996), and the other clades contain the widely distributed E. mathaei, E. oblonga and E. sp. A. The tree topology shows that the most recent cladogenic event was the split between the three IWP species, E. mathaei, E. oblonga, and E. sp. A. The immediately previous split was between the ancestors of the latter three species and the WIO and Red Sea species. The most ancient split was between the cluster of E. mathaei, E. oblonga, E.

42

sp. A and the WIO and Red Sea species, to the IWP E. sp. C. Zanzibarian and Eilatian urchins (E. sp. ZE and E. sp. EE, respectively) were found only on the strongly supported clade EZ. No apparent sub-divisions between the two were detected. Sex had no effect on clustering, and both males and females were present in all of the clades from the Okinawa, Eilat, and Zanzibar populations.

43

Figure 13. Neighbor Joining tree of the 104 CO haplotypes. Bootstrap support values for Maximum Likelihood, Neighbor Joining, and Maximum Parsimony are shown in the parentheses next to branches (dash indicates little or no bootstrap support). Clade names A, B, C, and D correspond to Echinometra species A, B (mathaei), C, and D (oblonga), respectively. Clade EZ represents Echinometra from Eilat and Zanzibar.

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4.6 Comparing morphological keys and molecular taxonomy

Out of the four Echinometra species, only E. sp. A can be identified based on a single morphological feature, due to its unique coloration of the spines (Fig. 5). This is not possible with any of the other species as some of their character-states are shared by other species. Comparing external morphological characters (spine color, milled rings, and skin of peristome), to molecular data, reveals substantial disagreements (lower than 50% correct morphological identification), with the exception of E. sp. A. Comparing molecularly assigned species to the entire set of morphological data (both external and internal features) reveals that 18.5% of the total (n=81) were described as unidentifiable (i.e. similar scores were reached for at least two species) and a mere 3.7% were described as misidentifications. Mismatches in identification only happened between E. sp. C and E. oblonga, with E. oblonga always overestimated. Based on their molecular attribution, the morphologically unidentified individuals were either E. mathaei (26.7% of the unidentified individuals) or E. sp. C (73.3% of the unidentified individuals). The best hierarchical design among the morphological characters was reached with color of spines predominance. The percentage of the unidentified individuals was reduced this way from 18.5% to only 4.9% of the population. No other hierarchical combination was able to reduce this value further. When the keys used to identify Okinawa urchins were applied on the Eilat and Zanzibar urchins using this method, all individuals were returned as unidentifiable.

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5. Discussion
The current study combined both morphological and molecular tools for the first time to test and infer relations among sea urchins from the genus Echinometra. Two Echinometra populations, never previously studied using molecular tools, from the WIO (Zanzibar) and the Red Sea (Eilat), were examined and compared with a previously studied population in Okinawa, resulting in an up-to-date revision of this genus in those regions. As detailed morphological data from the literature are available for the Okinawa species only, I address my discussion to these species (E. spp. A, C, mathaei, and oblonga) first. Having examined the existing definition conventions for Okinawa Echinometra, I then address my discussion to Echinometra from the two remaining localities, Eilat and Zanzibar. As previously mentioned, both these latter species are currently considered to be E. mathaei. The morphological identification features were divided into external and internal, as this is of major importance from an ecological point of view. The external morphological characters, which include color of spines, state of milled rings, and skin around the peristome, are the only available keys for an ecologist when conducting research in the field (e.g. density surveys, size distributions, habitat preferences). If an ecological study aims to be species-specific, the ability of these external features to provide definition of a species should be tested, as they alone are available for examination during field surveys. In many organisms, including sea urchins from the genus Echinometra, color patterns are among the most prominent external features (Mortensen 1943; Doan &

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Castone 2003). However, it is clear that spine color alone is insufficient to assert species delamination in most Okinawa Echinometra, since all but one share spine colors. The exception is E. sp. A, where a single color morph was observed. This color, referred to as white tips, never appeared in any of the other species, making it a viable marker for that species. In contrast to previously published data (Arakaki et al. 1998; Arakaki & Uehara 1999), two color morphs were detected in E. oblonga, black and dark brown. This finding strengthens the claim that spine color alone is insufficient in defining Echinometra species, as the newly-discovered dark brown morph of E. oblonga is also displayed by E. sp. C. The high plasticity of spine color and its subjective terminology limit the use of this feature as a defining character of these urchins. Subjective terminology is a common shortcoming of many morphological character descriptions found in the literature. Many of these terminologies are a byproduct of the attempt to subject a large variety to a general role. This problem is frequent in Mortensens (1943) Monograph of the Echinoidea. In his study, Mortensen often uses ambiguous terminology such as: usually not very bare, usually very bare, usually very small and not very small to describe skeletal features in the genus Tripneustes. Ambiguous terminology is also apparent in the literature regarding Echinometra from Okinawa (Arakaki et al. 1998; Arakaki & Uehara 1999). The skin around the peristome is one example of a character rendered uninformative due to such ambiguous and subjective terminology. This skin is as variable in color as the spines themselves (Fig. 5). However, as opposed to the spines in which eight distinct character-states are used to define their variety, in the latter, only two are used. Using either dark or bright to describe such great diversity in appearance, will inevitably lead to subjective terminology. When applied to a plastic

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feature such the color of the skin around the peristome, this subjective terminology may result in conflicting decisions even when evaluating twice the exact same individual by the same person. Differences in the appearance and proportions of character-states in comparisons between the current study and data obtained from the literature are common. In many cases, the new findings in the current study eliminate the role of a distinctive feature from a character that was previously regarded as such. Milled rings for example presented two character-states that proved to be only partially informative in distinguishing between the four Okinawan species. In E. sp. A and E. oblonga all individuals presented a single character-state (Fig. 7). While findings from these two species correspond well with the literature (Arakaki et al. 1998; Arakaki & Uehara 1999), it is in E. sp. C and E. mathaei that great differences were found. Arakaki et al. (1998) describes E. sp. C and E. mathaei as having completely dark and completely bright milled rings respectively, making this character useful in delineating Okinawan species. However, in this study I found that both species feature bright and dark rings simultaneously (Fig. 7). The reason for these differences may be attributed to the low number of samples obtained by Arakaki et al. (a total of n=21 for both species, in comparison to n=69 samples collected in the current study). Small sample sizes are often mentioned by many authors as a significant constraint on their findings (e.g. McCartney et al. 2000; Lessios et al. 2001). The current study demonstrates that the relatively small number of samples previously obtained for Okinawan urchins was insufficient to cover all variations in the population. It is however possible to use this character to make decisions, to some extent, in E. sp. A and E. oblonga. For example, if one selects an urchin in the field and its milled rings are dark it

48

will be possible to say that this urchin is not E. sp. A, since dark milled rings are present in all but E. sp. A. The same is true for a brightly-ringed urchin, which can be any but E. oblonga. Zanzibar and Eilat Echinometra present great similarities in all external morphological features. Populations from both these locations share some unique features such as the faded character-state of the milled rings that is not apparent in any of the Okinawa urchins. In other characters, Zanzibar and Eilat Echinometra show highly similar proportions of character-states, further implying similarities between the two. One example of such similarity is seen in the character skin of peristome, where both populations present predominantly dark-skinned individuals and a mere few brightskinned ones (Fig. 8) in highly similar proportions. The spine coloration of urchins from these two localities may provide some explanation as to why these populations were mistaken for E. mathaei. Although Zanzibar Echinometra are generally darker than the ones found in Eilat, in both localities the brown morph comprises a substantial part of the population. Out of the four Okinawa species, E. mathaei, and to some extent E. sp. C, are the only species to share this coloration pattern. This similarity, although very limited, may be one of the reasons why these urchins have been regarded as the same species. However, as other color morphs are not shared among these species, the value of spine color as an attributing factor in this case is diminished. From examining external morphological features it is possible to conclude that only Okinawa E. sp. A can be identified with certainty based on its prominent whitetipped spines. As for the other species, it is also clear that external features alone are insufficient and other, internal features must be utilized as well. In Zanzibar and Eilat

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Echinometra external morphological features imply similarities between the two on the one hand and segregation from the Okinawa species on the other. However, additional delineating features are required in order to conclude that the latter are indeed separate species from their Okinawa counterparts. The introduction of internal morphological features increases the resolving power among the species of Echinometra. These characters, which include spicules and sperm morphology, present clearly definable character-states that use relatively objective descriptions and allow the adoption of a quantitative approach. However, in order to examine these characters laboratory work is needed, often involving dissection of the urchins in order to extract internal components such as gonads. In addition the individuals need to be sexually mature. The fact that these analyses can not be done in the field, as well as the consequences of such observations (the urchins examined are ultimately killed), renders these methods inappropriate for most ecological research. Applying such techniques in areas where Echinometra are scarce may present further complications, in light of nature conservation efforts, as the effect of such sampling on the sea urchin community structure may be considerable. However, the use of such an approach is inevitable in any attempt to define the borders such between closely-related species. The effect of larger sample sizes is perhaps best illustrated in the internal morphological features. The four character-states of tubefeet spicules found in Okinawa Echinometra are composed of combinations of two spicule-types (Fig. 10). Except for E. sp. A, comparisons of spicule assemblages in the tubefeet of the other three Okinawa species were in poor agreement with previously published data (Arakaki et al. 1998). Nevertheless, an intriguing pattern emerges from these disagreements. In both E. mathaei

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and E. sp. C the same character-states were present in the current study as in previously published data; however proportions of these character-states within species differed. Obviously, a larger number of samples is more likely to better represent the proportion of natural states; where a larger number of samples was used, a greater variety of characterstates was found. In E. oblonga for instance, two character-states are described in this work whereas four character-states are described elsewhere (Arakaki et al. 1998; Arakaki & Uehara 1999) based on a sample size twice as large. Gonad spicules present a significantly larger number of character-states in comparison to those found in the tubefeet (ten and four character-states, respectively). This increased variety allows for greater resolution; however, it also calls for attention as to the stability of this character (Fig. 11). Two species are in complete agreement with the literature, E. sp. A and E. mathaei. In the case of the latter, an important conclusion to solve the question of this characters stability could be reached based on this observation. In this species, the small sample size used by Arakaki et al. (1998) proved to be insufficient in determining the real proportions of tubefeet spicules (based on the current study findings). Yet, in the case of gonad spicules, both studies gave the same results despite the large differences in sample size. This may indicate that gonad spicules are relatively more stable than tubefeet spicules, as suggested by Arakaki et al. (1998). As with the external characters, ambiguous and generalized character-state descriptions were encountered here as well. Both Arakaki et al. (1998) and Arakaki & Uehara (1999) use the character-state multiple to describe any spicule combination in E. oblonga, pooling together possibly separated character-states. The outcome of that generalization was that although larger sample sizes were obtained in the previous studies (Arakaki et al.

51

1998, n=16; Arakaki & Uehara 1999, n=16, and n=8 in the current study), a greater variability was observed in the current one. Therefore it is not just the sample size criterion that must be met, but also the meticulousness in examining the samples and the level of delineation imposed on each character. Finally, Zanzibar and Eilat Echinometra presented a high resemblance in both tubefeet and gonad spicules. Populations from the two localities are composed entirely of the bihamate spicule type in the tubefeet. The relatively large sample sizes, n=51 from Zanzibar and n=40 from Eilat, validates the presence of this sole character-state, providing further evidence of dissimilarities between urchins from these populations and E. mathaei. The gonad spicule combinations in Eilat and Zanzibar Echinometra are almost identical. They share two out of three character-states in similar proportions and, in both, the pure needle character-state predominates. The only difference is in the appearance of the small shaped spicule in about 10% of the Eilat population. On the other hand, the evident bihamate spicule in both Zanzibar and Eilat Echinometra is in striking contrast to the Okinawa E. mathaei that lacks this spicule altogether. These findings further support the view that E. mathaei differs from the Zanzibar and Eilat urchins and should not be viewed as the same. Sex and speciation have been intimately bound in the definition of the biological species concept (Landry et al. 2003; Palumbi 1992). It has been shown that in many species, sperm size is related to male fitness (Radwan 1996) and is subject to selection (Gomendio & Roldan 1991; LaMunyon & Ward 2002). Panhuis et al. (2001) suggested that sexual selection might be acting when species are diverging and that within-species sexual selection may drive the development of reproductive isolation. As in other mass-

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spawning invertebrates, sea urchins are known to have little pre-mating behavior or communication (Lamare & Stewart 1998). Therefore, gametic incompatibility is likely to be an important pre-zygotic mechanism of isolation (Palumbi 1992), especially when habitats and spawning seasons may overlap such as in Echinometra (Arakaki & Uehara 1991). Such reproductive isolation may eventually lead to the formation of a new species. Although more work is needed to clarify the role of sperm morphology in speciation, it is clear that variations in sperm morphology can be used, along with other markers, as a robust morphological character for species delimitation and identification (Arakaki & Uehara 1991; Landry et al. 2003). Sperm size in sea urchins can be polymorphic among populations within species (Arakaki et al. 1998); however, a large variation in length (i.e. more than two-fold) usually represents differences in species (Chia et al. 1975; Amy 1983; Raff et al. 1990; Landry et al. 2003). The primary difference lies in the tip of the sperm head and the shape of the nucleus (Arakaki et al. 1998). In the present study, the sperm morphology of the Eilat Echinometra resembled those of three other Echinometra species described in the literature. The fact that there was no significant difference in sperm morphology between E. spp. C, HA, and mathaei (Table 2) and the Eilat Echinometra, suggests that they might be similar or closely-related species. On the other hand, the significant differences in sperm morphology between the Eilat Echinometra and the other species (E. spp. A, VE, EB, BE, BO, GU, and oblonga) strongly suggest that they are separate species. Among these, a striking difference in sperm morphology appears between the Eilat Echinometra and E. sp. BE and E. oblonga (Fig. 12). This character distinguishes Eilat Echinometra

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from Okinawa E. sp. C and E. oblonga, and highlights the need for further examination of the validity of this morph as a new Echinometra species

5.1 The effect of sex on the morphological characters

Morphological dimorphism in respect to sex is a well-documented phenomenon in the literature (Morton 1965; Bult et al. 2008; Fonteneau et al. 2009). However, sexual delimitating features based on external morphology remain elusive in sea urchins. In the current study, none of the morphological characters examined correlated with sex. Therefore, finding easy-to-measure characters to determine sex in sea urchins remains an important task, due to the ecological (Muthiga 2005) and economic (Sloan 1985) implication of such findings.

A
97 93 100 86 91 92 0.005 E. sp. EE & E. sp. ZE

B
1% seq. difference 63 E. sp. A E. sp. B (mathaei) E. sp. D (oblonga) 78

E. mathaei

E. sp. nov. A
E. mathaei

E. sp. nov. A

E. oblonga

E. sp. C

98 E. sp. nov. C

Figure 14 . Neighbor-Joining trees showing the phylogenetic relationships among clades of CO

sequences of Indo-Pacific Echinometra species. A. Tree obtained from the current study based on sequences originating from 192 individuals. Intraspecific variation is represented by the size of the triangle drawn for each clade. B. Tree obtained from Palumbi et al. (1997) based on sequences originating from 200 individuals. Intraspecific variation is represented by the depth of the box drawn for each clade. For both A and B, bootstrap values are shown above branches.

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5.2 Phylogenetic relationships and comparison to morphological data

Phylogenetic reconstruction of the CO sequences revealed five distinct clades (Fig. 13). Combining sequences obtained from GeneBank into the reconstruction, allowed the assigning of species identity to the monophyletic clades. Based on these results, several conclusions could be drawn: First, the Eilat Echinometra and Zanzibar Echinometra form a strongly supported monophyletic clade with no internal clustering. The obvious separation and relatively long distance between this clade (EZ) and the rest of the Echinometra species suggests that they are a different and new species. Additionally, the lack of internal clustering in this clade suggests a single species exists in these locations. This new species will be referred to, from now on, as E. sp. nov. EZ. Claims that E. sp. nov. EZ is the same as E. mathaei are clearly refuted. Second, the robustness of the current findings is strongly supported by the cladding pattern among the four Okinawa Echinometra (E. spp. A, C, mathaei, and oblonga). Relations between these four clades, or species, display a very high resemblance to data published by Palumbi et al. (1996), and provide strong confirmation for the current findings, as demonstrated in figure 14. Third, cladding revealed a clear geographic pattern, with Pacific populations (Okinawa) being separated from Indian-Ocean populations (Eilat and Zanzibar) (Fig. 13). However, E. sp. nov. EZ is closer to the Okinawa species A, mathaei and oblonga than to E. sp. C. The reason for this clustering is unclear. It can be hypothesized that these relations among clades are rooted in the early days of speciation of these species; however, insights into this enigma may only be achived following the application of population genetics tools. Fourth, urchins did not cluster to form clades according to sex, as males and females were evenly distributed among and within the

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clades. Clustering by sex would have cast a doubt on the current findings as a tool to delimit species. Finally, as previously reported, and confirmed in the current study, the most ancestral clade among the IWP Echinometra is that of E. sp. nov. C (the same as E. sp. C) (Palumbi 1996). In Palumbis data this is followed by E. oblonga, and then a cluster of three clades from E. mathaei and E. sp. nov. A (Fig. 14). However, data obtained in the current study suggest that the new clade of E. sp. nov. EZ, is ancestral to the Okinawa E. spp. A, mathaei and oblonga.

5.3 Biogeography and cladogenic events

Rooted phylogenies allow the identification of derived species. The geographic locality of such species in relation to phylogenetically basal taxa can be used to test biogeographic theories, and to further our understanding of the series of evolutionary events leading to biogeographic patterns. However, implementing molecular tools in studies of speciation of marine organisms is particularly challenging, due to the high dispersal potential of some planktonic larvae and the difficulty in determining barriers to gene flow in the ocean (Palumbi 1992, 1994; McCartney et al. 2000). One of the early papers dealing with speciation of marine organisms was by Mayr (1954). In his study Mayr extracted taxonomic and distributional information from Mortensens (192851) monograph on the Echinoidea. Mayr concluded that, in addition to the emergence of the Central American land bridge, oceanic barriers consisting of deep or cold water were likely causes of speciation in several tropical shallow water genera of sea urchins. Based on morphological differentiation between geographical populations, he interpreted these differences as a sign of the early stages of allopatric speciation. A long allopatric stage,

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he believed, was necessary to achieve reproductive isolation between two populations since, at the time, such isolation was only thought to be the product of divergence at many loci scattered throughout the genome. Mayr concluded that only once reproductive isolation had been achieved, could species be found in sympatry. Mayr also noted that both the early phase of geographical speciation (divergence between allopatric populations), and the late phase (highly differentiated species occurring sympatrically), were evident in the genus Echinometra. He followed Mortensen in the belief that only one (or at most two) species of this genus occurred in the IWP. However, as previously discussed here, there are currently four species of Echinometra recognized from the IWP (Uehara et al. 1986; Matsuoka & Hatanaka 1991; Palumbi & Metz 1991). Furthermore, a relatively recent discovery showed that reproductive isolation between species can be achieved through sperm and egg incompatibility caused by mutations in a few loci, as seen in the gene coding to the binding protein (Metz et al. 1994; Palumbi & Metz 1991). These new findings suggest that in spite of Mayrs belief that speciation can occur only in an allopatric way, sympatric speciation is also possible. The current study demonstrates even further speciation in the genus Echinometra, placing the WIO and Red Sea populations as separate but closely-related species to those of the IWP. If the timing of the vicariant events leading to the foundation of E. sp. nov. EZ could be evaluated, then the processes driving this speciation could be hypothesized. Such new hypotheses could then be used to accept or refute Mayrs and later workers such as Palumbi and Lessios theories on speciation processes. Genetic data can help to establish the approximate timing of species divergence. Although this requires a taxonomically-appropriate calibration (Martin et al. 1992),

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information on the genetic distance can sometimes be useful to distinguish between alternative biogeographic mechanisms. For many marine species, including sea urchins from the genus Echinometra, using genetic divergence between species separated by closure of the Isthmus of Panama serves as a calibration to estimate dates of cladogenic events. The time of the final isthmus closure is placed by geological evidence at about 3 mya, with the more exact value of 3.1 mya considered as probable (Duque-Caro 1990). An isthmus-related vicariant event is a reasonable assumption in cases such as those of the sea urchin genus Echinometra (McCartney et al. 2000), in which clades that appeared more recently than the separation of the tropical eastern Pacific and the Atlantic are confined to either ocean. McCartney et al. (2000) calculated a rate of 3.49% Kimuracorrected sequence divergence per million years, based on average pairwise divergence between E. vanbrunti from the eastern Pacific and both E. lucunter and E. viridis from the Atlantic. Based on such calculations the entire set of cladogenic events in the genus Echinometra were summarized by Palumbi and Lessios (2005) as follows: E. mathaei, E. oblonga and E. sp. A diverged 12 million years ago. This cluster split from E. sp. C and the Easter Island endemic E. insularis at about the same time. In the Caribbean, the sympatric species E. lucunter and E. viridis diverged ~1.5 million years ago. The eastern Pacific E. vanbrunti differs from other Pacific species by ~13% nucleotide differences in COI, corresponding to separation of ~3.5 million years. Findings in the current study place the split of E. sp. nov. EZ between the separation of the cluster of E. mathaei, E. oblonga and E. sp. A from E. sp. C, therefore dating this split to the Pleistocene or late Pliocene, around 1-2 mya.

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The separation of the four IWP species as well as the split of E. sp. nov. EZ 1-2 mya, indicates that the WIO and not just the IWP Echinometra populations (Palumbi 1994, 1996) were influenced by the active speciation that took place during this period of glaciations. Isolation between local populations due to decreased sea levels and the alteration of currents was possible during these times. It is difficult to point to a particular sea-level fluctuation as the cause of each cladogenic event since there were approximately 12 major and many smaller-scale glaciations in the Pleistocene (Crowley & North 1991). However, little is known on the geographical barriers that existed between the WIO and the IWP during the Pleistocene or late Pliocene, although such barriers must be hypothesized due to the currently reported allopatric populations. Lessios et al. (2001) reported two relatively recent genetic breaks in Diadema paucispinum and Diadema setosum, two pantropical sea urchin species, between the West Pacific and the Indian Oceans. D. paucispinum-a is limited to the central and western Pacific, whereas D. paucispinum-b is predominantly found in the Indian Ocean. D. setosum present two populations separated by large and significant Fst values and by the presence of one DNA site diagnostic between populations from the two oceans. Although these genetic breaks suggest the occasional existence of a barrier between the two oceans, they can not be assumed to be similar to the barriers that split Echinometra as the former breaks are dated to only 0.17-0.9 mya. Nearly all previous genetic comparisons between the western Pacific and Indian Ocean populations of marine organisms reported some degree of genetic differentiation (Benzie & Stoddart 1992; Benzie et al. 1993; McMillan & Palumbi 1995; Lavery et al. 1996; Miya & Nishida 1997; Williams & Benzie 1997, 1998; Chenoweth et al. 1998; Duke et al. 1998; Duda & Palumbi 1999; Lessios et al.

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1999; Barber et al. 2000). The generally accepted explanation for divergence between conspecific populations from the two oceans is that during low sea water levels in the Pleistocene (Audley-Charles 1981; Galloway & Kemp 1981), there were repeated restrictions to the passage of Pacific water between Australia and Southeast Asia that lasted long enough for differentiation to arise. However, it is clear that further sampling from locations along the Indian Ocean, as well as the implementation of population genetic tools, are needed to answer the questions regarding the modes of speciation between the Pacific and Indian Oceans. The Red Sea first opened to the Indian Ocean in the late Miocene (Girdler & Styles 1974) or early Pliocene (Braithwaite 1987; Girdler & Southern 1987). Along with more widely distributed species, it contains an endemic fauna (Ekman 1953; Gohar 1954; Briggs 1974, 1995), including at least 19 endemic species of echinoderms (Campbell 1987). Klausewitz (1968), Por (1978), and Briggs (1995) have attributed this endemicity to possible isolation of the Red Sea during major glaciations. The strait of Bab-elMandab, at the entrance to the Red Sea, is only 125 m deep. This shallow entrance is likely to have become emergent during low sea-level stands and to have separated the Red Sea from the Indian Ocean. McMillan and Palumbi (1995) estimated from mtDNA sequence comparisons that a Red Sea endemic species of butterfly fish was separated from its Indian Ocean sister species no earlier than 850,000 years ago. Lessios et al. (2001) dated the separation of two D. setosum lineages to pre-Pleistocene times. In contrast, in Echinometra no separated species or lineages have been observed, suggesting a strong connectivity between the Red Sea and the WIO. Timing of colonization, connectivity through larval transport, and the geographical boundaries of E. sp. nov. EZ,

60

are currently left unanswered. Further locations along the Red Sea and the WIO must be sampled and population genetic tools utilized in order to try and solve some of these questions.

5.4 Morphological keys and molecular attribution

A thorough examination of the morphological keys for identification revealed that, of the four Okinawa species currently recognized, only one species can be clearly identified based on external features. This species, E. sp. A, also bears stable internal characteristics such as tubefeet and gonad spicules. The other three species carry no such distinct external features and tend to share character-states, making their delineation, based on a single feature, practically impossible. This lack of clearly definable external features is an obstacle to many ecological studies. For example, conducting population density surveys require quick and reliable identification tags, as hundreds of individuals may be surveyed daily. However, in respect to concordance with the molecular data, including internal morphological features, the analysis yielded high levels of congruence. Such high levels of congruence do not always exist; Flot et al. (2008) for example, found little agreement between putative species boundaries inferred from molecular data and morphology delimitation in corals from the genus Seriatopora. Moreover, even in sea urchins from the genus Echinometra, morphology and genetics are often inconsistent (Lessios 1980). However, even in the current study ~20% of the population was described at first as unidentifiable. This happens when a set of character-states ascribes an individual to two different species, an equal number of times. This similar scoring is actually the artifact of character-states being shared by several individuals. Multiplying

61

the score given to the color of spines by a factor of two, was effective in solving this similar scores problem. The reason why spine color improves resolution (the number of unidentified individuals was reduced from ~20% to ~5%) is due to the fact that in this feature, character-states are only shared by a maximum of two individuals. Incorporating internal morphological features to improve species identification is of little use in field work. However, using such features to reach identifications with high levels of confidence is absolutely crucial in molecular studies. At the base of most molecular studies, at the species or population level, lies morphologically-based species identification (Lessios 1980; Palumbi & Metz 1991). Without stable markers to correctly identify specimens, it would be meaningless to conduct studies at either species or community levels for example. Yet it seems that many authors of molecular studies tend to ignore many of the problems associated with species identification. Biased sampling due to small sample sizes, insufficient keys for identification, and narrow geographical scales to describe species range, appear to be common practice in many molecular studies. Chen et al. (2008) analyzed only twenty-nine samples when attributing morphology to molecular data in the genus Seriatopora, all collected around Taiwan. These authors found good correlations among some of the features and ascribed their findings to the entire South-East Asia region. However, their findings were soon contradicted as new data from Okinawa, New Caledonia, and the Philippines, suggested that no correlation exists between genetics and morphology in the same set of characters (Flot et al. 2008). The latter authors suggested that the small sample size and its collection on a limited geographical scale, as the probable reasons for their conflicting results with Chen et al. (2008). Table 1 illustrates the problem of having insufficient keys

62

for identification. This problem is perhaps greater that the previously discussed biased sampling. As insufficient sampling could easily be corrected; ascribing molecular barcodes under the wrong species definition are much harder to track. Moreover, only on rare occasions are published genomic barcodes scrutinized in later studies. Therefore, it is of great importance to use the largest possible set of identifying features when ascribing barcodes to newly-established species. Urchins from Eilat and Zanzibar share many common features. Moreover, their indisputable cladding as a strongly supported monophyletic group further emphasizes the similarities between the two. However, an attempt to tie these morphological similarities to their obvious molecular attribution resulted in strong disagreements. The reason for this discordance probably lies in the morphological identification keys used. Since specific keys for the Eilat and the Zanzibar Echinometra are currently not available, the set of Okinawa keys was used. The set of Okinawa morphological keys failed to place E. sp. nov. EZ with any of the four Okinawa species. This further suggests that E. sp. nov. EZ should be viewed as a different species from its Okinawa counterparts.

6. Summary
In this study, a combined approach applying morphological and molecular tools was used to test and infer relations among sea urchins from the genus Echinometra. Four closely-related sympatric species of this genus, found in Okinawa, were used to test the general concordance between species inferred by DNA barcoding and known morphological keys. Two Echinometra populations, never previously studied at the

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molecular level, from the WIO (Zanzibar) and the Red Sea (Eilat), were examined for species identification and phylogenetic reconstruction. The results of this study ratify current morphological keys for identifying the four closely-related species of Echinometra found in Okinawa. However, an in-depth examination of the characters used for identification of these species revealed a much higher diversity compared to previous reports. No potent external feature was found capable of delineating theses sibling species, with the exception of E. sp. A. The latter can easily be identified based on its distinct spine coloration. Molecular phylogenetics of the Okinawa Echinometra strongly reconfirmed their current attribution into four distinct species. The phylogeny and systematics of Echinometra from Eilat and Zanzibar, based on CO sequences, are presented here for the first time. Based on this taxonomy and supported by morphological comparisons, I found that Echinometra from both Eilat and Zanzibar had been mistakenly identified as E. mathaei. Molecular and morphological evidence suggests that Echinometra from Eilat and Zanzibar are very closely-related, and may be the same species. Urchins from the different sites around Zanzibar did not cluster, suggesting that a single species exists in this location. Based on the reconstructed phylogeny and comparisons to existing phylogenies of the genus Echinometra, it is suggested that the splitting of E. sp. nov. EZ occurred 1-2 mya in the Pleistocene or late Pliocene. These populations from the WIO and the Red Sea in regard to Echinometra from the rest of the worlds oceans reassert Ernst Mayrs ideas on allopatric speciation. Further examination of the vicariant events that led to the creation of this new species might shed new light on the geological events that drive speciation in this region.

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7. Reference
Amy RL (1983) Gamete sizes and developmental time tables of five tropical sea urchins. Bulletin of Marine Science 33:173-176 Audley-Charles MG (1981) Geological history of the region of Wallaces Line. Pp. 24-35 in T. C. Whitmore, ed. Wallaces line and plate tectonics. Clarendon Press, Oxford, U.K. Arakaki Y, T Uehara (1991) Physiological adaptations and reproduction of the four types of Echinometra mathaei (Blainville). In Biology of Echinodermata (ed. T. Yanagisawa, I. Yasumasu, C. Oguro, N. Suzuki & T. Motokawa), pp. 105-112. Rotterdam: Balkema Arakaki Y, Uehara T, I Fagoonee (1998) Comparative studies of the genus Echinometra from Okinawa and Mauritius. Zoological Science 15:159-168 Avise JC, K Wollenberg (1997) Phylogenetics and the origin of species. Proceedings of the National Academy of Sciences of the United States of America 94:7748-7755 Bak RPM, G van Eys (1975) Predation of the sea urchin Diadema antillarum Philippi on living coral. Oecologia 20:111-115 Barber PH, Palumbi SR, Erdmann MV, MK Moosa (2000) Biogeography: a marine Wallaces line? Nature 406:692-693 Benzie JAH, JA Stoddart (1992) Genetic structure of crown-of-thorns starfish (Acanthaster planci) in Australia. Marine Biology 112:631-639 Benzie JAH, Ballment E, S Frusher (1993) Genetic structure of Penaeus monodon in Australia: concordant results from mtDNA and allozymes. Aquaculture 111:89-93 Besansky NJ, Severson DW, MT Ferdig (2003) DNA barcoding of parasites and invertebrate disease vectors: what you dont know can hurt you. Trends in Parasitology 19:545-6 Birkeland C, RH Randall (1981) Facilitation of coral recruitment by echinoid excavations. Proceedings of the 4th International Coral Reef Symposium 1:695-698 Blainville HM (1825) Dictionnaire des sciences naturelles, dans lequel un trait mthodiquement des diffrences tetres de la nature 37:93-120 Blaxter ML (2003) Counting angels with DNA. Nature 421:122-4

65

Blaxter ML (2004) The promise of a DNA taxonomy. Philosophical Transactions of the Royal Society of London, B 359:669-79 Braithwaite CJR (1987) Geology and palaeogeography of the Red Sea region. Pp. 22-44 in A. J. Edwards, and S. M. Head, eds. Red Sea. Pergamon Press, Oxford, U.K. Briggs JC (1974) Marine zoogeography. McGraw-Hill, New York. Briggs JC (1995) Global biogeography. Elsevier, Amsterdam. Brumfield RT, MJ Braun (2001) Phylogenetic relationships in bearded manakins (Pipridae: Manacus) indicate that male plumage color is a misleading taxonomic marker. The Condor 103(2):248-258 Bult G, Irschick DJ, G Blouin-Demers (2008) The reproductive role hypothesis explains trophic morphology dimorphism in the northern map turtle. Functional Ecology 22(5):824-830 Buyck B (1999) Taxonomists are an endangered species in Europe. Nature 401:321 Chen C, Dai CF, Plathong S, Chiou CY, CA Chen (2008) The complete mitochondrial genomes of needle corals, Seriatopora spp. (Scleractinia: Pocilloporidae): an idiosyncratic atp8, duplicated trnW gene, and hypervariable regions used to determine species phylogenies and recently diverged populations. Molecular Phylogenetics and Evolution 46:19-33 Chenoweth SE, Hughes JM, Keenan CP, S Lavery (1998) When oceans meet: a teleost shows secondary intergradation at an Indian-Pacific interface. Proceedings of the Royal Society of London series B-Biological Sciences. 265:415-420 Chia FS, Atwood D, B Crawford (1975) Comparative morphology of echinoderm sperm and possible phylogenetic implications. American Zoologist 15:533-565 Claridge MF, Dawah HA, MR Wilson, (1997) Species: The Units of Biodiversity. Chapman & Hall, London Clark AM, FW Rowe (1971) Monograph of shallow-water Indo-West Pacific echinoderns. Brithis Museum (Natural History) Publications 690:1-239 Crowley TJ, GR North (1991) Paleoclimatology. Oxford Univ. Press, New York. Daly HV (1995) Endangered species: doctoral students in systematic entomology. American Entomologist 1995:55-9 Campbell AC (1987) Echinoderms of the Red Sea. Pp. 215-232 in A. J. Edwards, and S. M. Head, eds. Red Sea. Pergamon Press, Oxford, U.K.

66

Dart JKG (1972) Echinoids, algal lawn and coral recolonization. Nature (Lond.) 39(2):50-51 Darwin C (1859) On the origin of species. John Murray publications, United Kingdom Doan TM, TA Castone (2003) Using morphological and molecular evidence to infer species boundaries within Proctoporus bolivianus Werner (Squamata: Gymnophthalmidae). Herpetologica 59(3):432-449 Dobzhansky T (1937) Genetics and the Origin of Species. Columbia University Press, New York Doyle JJ, BS Gaut (2000) Evolution of genes and taxa: a primer. Plant Molecular Biology 42:1-6 Duda TF, SR Palumbi (1999) Population structure of the black tiger prawn, Penaeus monodon, among western Indian Ocean and western Pacific populations. Marine Biology 134:705-710 Duke NC, Benzie JAH, Goodall JA, ER Ballment (1998) Genetic structure and evolution of species in the mangrove genus Avicennia (Avicenniaceae) in the Indo-West Pacific. Evolution 52:1612-1626 Duque-Caro H (1990) Neogene stratigraphy, paleoceanography and paleobiogeography in northwest South America and the evolution of the Panama seaway. Palaeogeography, Palaeoclimatology, Palaeoecology 77:203-234 Edmondson CH (1935) Hawaii reef and shore fauna. Bishop Museum Press, Honolulu, HI Ereshefsky M (2001) The Poverty of the Linnaean Hierarchy: A Philosophical Study of Biological Taxonomy. Cambridge University Press. Cambridge, England Eakin CM (1996) Where have all the carbonates gone? A model comparison of calcium carbonate budgets before and after the 1982-1983 EI Nino at Uva Island in the eastern Pacifc. Coral Reef 15:109-119 Ekman S (1953) Zoogeography of the sea. Sidgwick and Jackson, Ltd, London. Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783-791 Fishelson L (1971) Ecology and distribution of the benthic fauna in the shallow waters of the Red Sea. Marine Biology 10:113-133

67

Flot JF, Licuanan WY, Nakano Y, Payri C, Cruaud C, S Tillier (2008) Mitochondrial sequences of Seriatopora corals show little agreement with morphology and reveal the duplication of a tRNA gene near the control region. Coral Reefs 27:789-794 Floyd R, E Abebe (2002) Nematode molecular barcodes. Soil Biodiversity 8:3 Floyd RM, Abebe E, Papert A, ML Blaxter (2002) Molecular barcodes for soil nematode identification. Molecular Ecology 11:839-50 Folmer O, Black M, Hoeh W, Lutz R, R Vrijenhoek (1994) DNA primers for amplification of mitochondrial Cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3:294-299 Fonteneau F, Paillisson JM, L Marion (2009) Relationships between bird morphology and prey selection in two sympatric Great Cormorant Phalacrocorax carbo subspecies during winter. UMR CNRS Ecobio, Universit de Rennes 1, Campus Beaulieu, 35042 Rennes cedex, France Galloway RW, EM Kemp (1981) Late Cainozoic environments in Australia. Pp. 5180 in A. Keast, ed. Ecological biogeography of Australia. Junk, The Hague Geyer LB, SR Palumbi (2003) Reproductive character displacement and the genetics of gamete recognition in tropical sea urchins. Evolution 57:1049-1060 Girdler RW, P Styles (1974) Two stage Red Sea floor spreading. Nature 247:7-11 Girdler RW, TC Southern (1987) Structure and evolution of the northern Red Sea. Nature 330:716-721 Gohar HAF (1954) The place of the Red Sea between the Indian Ocean and the Mediterranean. Publication Hidrobioloji Arastirma Enstitutusu Istanbul Universite Series B 2:1-40 Glynn PW, Wellington GM, C Birkeland (1979) Coral reef growth in the Galapagos: limitation by sea urchins. Science 203:47-49 Gomendio M, ERS Roldan (1991) Sperm competition influence sperm size in mammals. Proceedings of the Royal Society of London series B-Biological Sciences 243:181185 Harris JD (2003) Can you bank in GenBank? Tends in Ecology and Evolution 18:317-9 Hebert PDN, Cywinska A, Ball SL, JR deWaard (2003a) Biological identifications through DNA barcodes. Proceedings of the Royal Society of London series BBiological Sciences 270:313321

68

Hebert PDN, Ratnasingham S, JR deWaard (2003b) Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings of the Royal Society of London series B-Biological Sciences (Suppl.) 270:S96-9 Hendler G, Miller JE, Pawson DL, Kier PM (1995) Sea stars, Sea Urchins, and Allies. Echinoderms of Florida and the Caribbean. Smithsonian Institution Press, Washington and London. P. 199 Hendrixon BE, JE Bond (2005) Testing species boundaries in the Antrodiaetus unicolor complex )Araneae: Mygalomorphae: Antrodiaetidae): Paraphyly and cryptic diversity. Molecular Phylogenetics and Evolution 36:405-416 Hopkins GW, RP Freckleton (2002) Declines in the numbers of amateur and professional taxonomists: implications for conservation. Animal Conservation 5:245-9
Hull DL (1977) The ontological status of species as evolutionary units. In: Foundational Problems in the Special Sciences (Ed. R. Butts and J. Hintikka), pp. 91-102, D. Reidel Publishing Company, Dordrecht, Holland.

Jarman SN, NG Elliott (2000) DNA evidence for morphological and cryptic Cenozoic speciations in the Anaspididae, living fossils from the Triassic. Journal of Evolutionary Biology 13:624-633 Jennings S, NVC Polunin (1996) Impacts of fishing on tropical reef ecosystems. Ambio 25:44-49 Kelso D (1970) A comparative morphological and ecological study of two species of sea urchin genus Echinometra in Hawaii. PhD dissertation, Dept. of Biology. University of Hawaii, Honolulu. 112 pp. Klausewitz W (1968) Remarks on the zoogeographical situation of the Mediterranean and the Red Sea. Annali di Museo Civico di Storia Naturale di Giacomo Doria 77:323328 Knowlton N (1993) Sibling species in the sea. Annual Review of Ecology and Systematics 24:189-216 Kurtzman CP (1994) Molecular taxonomy of the yeasts. Yeast 10:1727-1740 Lamare MD, BG Stewart (1998) Mass spawning by the sea urchin Evechinus chloroticus (Echinodermata: Echinoidea) in a New Zealand fiord. Marine Biologgy 132:135140 Lammers TG (1999) Plant systematics today: all our eggs in one basket? Systematic Botany 24:4946

69

LaMunyon CW, S Ward (2002) Evolution of larger sperm in response to experimentally increased sperm competition in Caenorhabditis elegans. Proceedings of the Royal Society of London series B-Biological Sciences 269:11251128 Landry C, Geyer LB, Arakaki Y, Uehara T, SR Palumbi (2003) Proceedings of the Royal Society of London series B-Biological Sciences 270:18391847 Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, DG Higgins (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947-2948 Laval LG, S Schneider (2005) Arlequin ver. 3.0: An integrated software package for population genetics data analysis. Evolutionary Bioinformatics Online 1:47 50 Lavery S, Moritz C, DR Fielder (1996) Indo-Pacific population structure and evolutionary history of the coconut crab Birgus latro. Molecular Ecology 5:557-570 Lawrence JM (1975) On the relationship between marine plants and sea urchins. Oceanography and Marine Biology Annual Review 13:213-286 Lawrence JM (1983) Alternate states of populations of Echinometra mathaei (De Blainville) (Echinodermata: echinoidea) in the Gulf of Suez and the Gulf of Aqaba. Bulletin of the Institute of Oceanography and Fisheries 9:141-147 Lessios HA (1980) Divergence in allopatry: molecular and morphological differentiation between sea urchins separated by the Isthmus of Panama. Evolution 35(4):618-634 Lessios HA, Kessing BD, Robertson DR, G Paulay (1999) Phylogeography of the pantropical sea urchin Eucidaris in relation to land barriers and ocean currents. Evolution 53:806-817 Lefbure T, Douady CJ, Gouy M, J Gibert (2006) Relationship between morphological taxonomy and molecular divergence within Crustacea: Proposal of a molecular threshold to help species delimitation. Molecular Phylogenetics and Evolution 40:435-447 Lipscomb D, Platnik N, Q Wheeler (2003) The intellectual content of taxonomy: a comment on DNA taxonomy. Trends in Ecology and Evolution 18:65-6 Mallet J (1995) A species definition for the modern synthesis. Trends in Ecology and Evolution 10:294-299 Mallet J, K Willmott (2003) Taxonomy: renaissance or tower of babel? Trends in Ecology and Evolution 18:57-59

70

Martin AP, Naylor GJP and SR Palumbi (1992) Rates of mitochondrial DNA evolution in sharks are slow compared to mammals. Nature 357: 153-155 Mathews LM, Schubart CD, Neigel JE, DL Felder (2002) Genetic, ecological, and behavioural divergence between two sibling snapping shrimp species (Crustacea: Decapoda: Alpheus). Molecular Ecology 11:14271437 Matsuka N, T Hatakana (1991) Molecular evidence for the existence of four sibling species within the sea-urchin, Echinometra mathaei in Japanese waters and their evolutionary relationships. Zoological Science 8:121-133 Mayden RL (1997) A hierarchy of species concepts: the denouement in the saga of the species problem. In: Claridge, M.F., Dawah, H.A., Wilson, M.R. (Eds.), Species: The Units of Biodiversity. Chapman & Hall, London, pp. 381-424 Mayr E (1942) Systematics and the Origin of Species from the Viewpoint of a Zoologist.Columbia University Press: New York McAllister D (2000) Biodiversity awareness: people, museums and the web. Biodiversity 1:38-9 McCartney MA, Keller G, HA Lessios (2000) Dispersal barriers in tropical oceans and speciation in Atlantic and eastern Pacific sea urchins of the genus Echinometra. Molecular Ecology 9(9):1391-1400 McClanahan TR (1994) Kenyan coral reef lagoon fish: effects of fishing, substrate complexity, and sea urchins. Coral Reefs 13:231-241 McClanahan TR, D Obura (1995) Status of Kenyan coral reefs. Coastal Management 23:57-76 McClanahan TR, SH Shafir (1990) Causes and consequences of sea urchin abundance and diversity in Kenyan coral reef lagoons. Oecologia 83:362-370 McClanahan TR, B Kaunda-Arara (1996) Creation of a coral-reef marine park: Recovery of fishes and its effect on the adjacent fishery. Conservation Biology 10:1187-1199 McClanahan TR, NA Muthiga (2001) The Ecology of Echinometra. In Jangoux, M. & M. Lawrence (eds), Edible Sea Urchins. Development in Aquaculture and Fisheries-32. Amsterdam, Elsevier: 225243 McMillan WO, SR Palumbi (1995) Concordant evolutionary patterns among Indo-west Pacific butterflyfishes. Proceedings of the Royal Society of London series BBiological Sciences 260:229-236

71

Metz EC, Kane RE, Yanagimachi H, Palumbi SR (1994) Fertilization between closely related sea urchins is blocked by incompatibilities during sperm-egg attachment and early stages of fusion. Biological Bulletin 187:23-34 Mishler BD, EC Theriot (2000) A critique from the Mishler and Theriot Phylogenetic Species Concept: Monophyly, Apomorphy, and Phylogenetic Species Concepts. In: Species Concepts and Phylogenetic Theory (Ed. Q. D. Wheeler and R. Meier), pp. 119-132, Columbia University Press, New York Miya M, M Nishida (1997) Speciation in the open ocean. Nature 389:803-804 Miya M, M Nishida (2000) Use of mitogenomic information in teleostean molecular phylogenetics: a tree-based exploration under the maximum-parsimony optimality criterion. Molecular Phylogenetics and Evolution 17:437-55 Mortensen TH (1943) Monograph of the Echinoidea: Camarondonta. C. A. Reitzel, Copenhagen, Denmark. Morton WM. (1965) The taxonomic significance of the kype in American salmonids. Copeia 4:1419 Motokawa T (1991) Introduction to the symposium Echinometra a complex under speciation. Biology of Echinodermata, edited by T. Yanagisawa, I. Yasumasu, C. Oguro, N. Suzuki and T. Motokawa. Balkema Press, Rotterdam, p. 89 Muthiga NA, TR McClanahan (1987) Population changes of a sea urchin (Echinometra mathaei) on an exploited fringing reef. African Journal of Ecology 25:18 Muthiga NA (2005) Testing for the effects of seasonal and lunar periodicity on the reproduction of the edible sea urchin Tripneustes gratilla (L) in Kenyan coral reef lagoons. Hydrobiologia 549:5764 Neill JB (1988) Experimental analysis of burrow defense in Echinometra mathaei (de Blainville) on an Indo-West Pacific reef flat. Journal of Experimental Marine Biology and Ecology 115:127136 Niemi GJ (1985) Patterns of morphological evolution in bird genera of New World and Old World peatlands. Ecology 66(4):1215-1228 Nishihira M, Sato Y, Arakaki Y, M Tsuchiya (1991) Ecological distribution and habitat preference of four types of the sea urchin Echinometra mathaei on the Okinawan coral reefs. Pp 91-104 in T. Yanagisawa, I. Yasumasu, C. Oguro, N. Suzuki, and T. Motokawa, eds. Biology of Echinodermata. Balkema Press, Rotterdam, The Netherlands Palumbi SR, E Metz (1991) Strong reproductive isolation between closely related tropical sea urchins (genus Echinometra). Molecular Biology and Evolution 8:227-239 72

Palumbi SR (1992) Marine speciation on a small planet. Trends in Ecology and Evolution 7:114117 Palumbi SR (1994) Genetic divergence, reproductive isolation, and marine speciation. Annual Review of Ecology and Systematics 25:547572 Palumbi SR (1996) What can molecular genetics contribute to marine biogeography? An urchins tale. Journal of Experimental Marine Biology and Ecology 203:75-92 Palumbi SR, Grabowsky G, Duda T, Geyer L, N Tachino (1997) Speciation and the evolution of population structure in tropical Pacific sea urchins. Evolution 51:1506 1517 Palumbi SR, HA Lessios (2005) Evolutionary animation: How do molecular phylogenies compare to Mayrs reconstruction of speciation patterns in the sea? Proceedings of the National Academy of Sciences 102:6566-6572 Panhuis TM, Butlin R, Zuk M, T Tregenza (2001) Sexual selection and speciation. Trends in Ecology Evolution 10:228231 Pochon X, Pawlowski J, Zaninetti L, R Rowan (2001) High genetic diversity and relative specificity among Symbiodinium- like endosymbiotic dinoflagellates of sortid foraminiferans. Marine Biology 139:10691078 Por FD (1978) Lessepsian migration. Springer-Verlag, Berlin. Posada D, KA Crandall (1998) MODELTEST: testing the model of DNA substitution. Bioinformatics 14:817-818 Radwan J (1996) Intraspecific variation in sperm competition success in the bulb mite: a role for sperm size. Proceedings of the Royal Society of London series B-Biological Sciences 263:855859 Raff RA, Herlands L, Morris VB, J Healy (1990) Evolutionary modification of echinoid sperm correlates with developmental mode. Development Growth & Differentiation 32:283291 Rasband WS, ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://rsb.info.nih.gov/ij/, 1997-2008 Rozas J, Snchez-DelBarrio JC, Messeguer X, R Rozas (2003) DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 19:2496-2497 Russo AR, (1977) Water flow and the distribution and abundance of Echinoids (Genus Echinometra) on a Hawaiian reef. Australian Journal of Marine and Freshwater Research 28:693-702

73

Sammarco PW, Levinton JS, JC Ogden (1974) Grazing and control of coral reef community structure by Diadema antillarum Phillipi (Echinodermata: Echinoidea): a preliminary study. Journal of Marine Research 32:47-53 Schander C, E Willassen (2005) What can biological barcoding do for marine biology? Marine Biology Research 1:79-83 Seberg O, Humphries CJ, Knapp S, Stevenson DW, Petersen G, Scharff N, NM Andersen (2003) Shortcuts in systematics? A commentary on DNA-based taxonomy. Trends in Ecology and Evolution 18:63-5 Sloan NA, (1985) Echinoderm fisheries of the world: a review. In: Keegan, B.F & B.D. S. OConnor (eds), AA Balkema, Rotterdam, 109-124 Sneath PHA, RR Sokal (1973) Numerical Taxonomy. W. H. Freeman and Company, San Francisco Sket B (1999) The nature of biodiversity in hypogean waters and how it is endangered. Biodiversity and Conservation 10:1319-1338 Stoeckle M (2003) Taxonomy, DNA, and the bar code of life. BioScience 53:796-7 Tamura K, M Nei (1993) Estimation of the Number of Nucleotide Substitutions in the Control Region of Mitochondrial-DNA in Humans and Chimpanzees. Molecular Biology and Evolution 10:512-526 Tamura K, Dudley J, Nei M, S Kumar (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24:15961599 Tautz D, Arctander P, Minelli A, Thomas RH, AP Vogler (2002) DNA points the way ahead in taxonomy. Nature 418:479 Tautz, D., Arctander, P., Minelli, A., Thomas, R.H., A.P. Vogler (2003) A plea for DNA taxonomy. Trends in Ecology and Evolution 18:70-4 Tsuchiya M, M Nishihira (1984) Ecological distribution of two types of sea urchin, Echinometra mathaei (Blainville), on Okinawan reef flat. Galaxea 3:131-143 Tsuchiya M, M Nishihira (1985) Agonistic behavior and its effect on the dispersion pattern in the two types of the sea urchin, Echinometra mathaei (Blainville). Galaxea 4:37-48 Tsuchiya M, M Nishihira (1986) Re-colonization process of two types of the sea urchin, Echinometra mathaei (Blainville), on the Okinawan reef flat. Galaxea 5:283-294

74

Uehara T, M Shingaki (1984) Studies on the fertilization and development in the two types of Echinomerru marhaei from Okinawa. Zoological Science Vol. 1, pp. 1008 Uehara T, M Shingaki (1985) Taxonomic studies in the four types of sea urchin, Echinometra mathaei from Okinawa, Japan. Zoological Science 2:1009 Uehara T, Shingaki M, K Taira (1986) Taxonomic studies in the sea urchin, genus Echinometra, from Okinawa and Hawaii. Zoological Science 3:1114 Uehara T, Asakura H, Y Arakaki (1990) Fertilization blockage and hybridization among species of sea urchins. In Advances in Invertebrate Reproduction 5 Ed by M Hoshi and O Yamashita, Elsevier Sci Publisher BV, pp 305-310 Wiens JJ, TA Penkrot (2002) Delimiting species using DNA and morphological variation and discordant species limits in spiny lizards (Sceloporus). Systematic Biology 51:69-91 Will KW, D Rubinoff (2004) Myth of the molecule: DNA barcodes for species cannot replace morphology for identification and classification Cladistics 20:47-55 Williams ST, JAH Benzie (1997) Indo-West Pacific patternsnof genetic differentiation in the high-dispersal starfish Linckia laevigata. Molecular Ecology 6:559-573 Williams ST, JAH Benzie (1998) Evidence of a biogeographic break between populations of a high dispersal starfish: congruent regions within the Indo-west Pacific defined by color morphs, mtDNA, and allozyme data. Evolution 52:87-99 Wilson KH (1995) Molecular biology as a tool for taxonomy. Clinical Infectious Diseases 20:192 208 Zar JH (1999) Biostatistical analysis, 4th edn. Prentice Hall, Englewood Cliffs, NJ

75

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67

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