1. Hybridization and Gametic Incompatibilities between Two Diverged Species of Indo-Pacific Sea Urchins, Echinometra sp. A and Echinometra mathaei M. Aminur Rahman, Tsuyoshi Uehara, Yuji Hiratsuka and Md. Sirajul Islam Faculty of Science, University of the Ryukyus, Okinawa, Japan

2. Background and Objectives Tropical sea urchins genus Echinometra, the most abundant echinoids in the warm Indo-Pacific, occur sympatrically on Okinawan intertidal reefs. They are widely distributed from Japan to Australia, and from Mexico to the Gulf of Suez. Extensive morphological, ecological, and molecular studies show four independent gene pools of Echinometra in the Indo-Pacific. Molecular phylogenies indicate Echinometra split in the last 1-3 million years. E. sp. B is now recognized as E. mathaei (Em) while E. sp. D as E. oblonga (Eo), which include at least 3 cryptic species. Taxonomic description and designation of Echinometra sp. A (Ea) and E. sp. C (Ec) are yet to be made. In this study, two most distinct species, Ea and Em were examined for potential hybridization through a series of cross-fertilization and hybrid rearing experiments in order to: determine comparative fertility between the conspecific and heterospecific crosses. examine the gametic compatibility, developmental compatibility and reproductive capacity of the hybrids with the aim of elucidating the speciation process between them. understand how these two sympatric species prevent or at least reduce the incidence of hybridization in the field. determine the compatibility of gametes and gene flows among the hybrids and conspecific controls through F1 backcrossing. characterize hybrid F1 phenotypes and determine the presence/absence of these phenotypes in likely hybrid zones. investigate which mechanisms (pre- or postzygotic) maintain the genetic integrities of Ea and Em despite their closer proximity and sympatry.

3. Table 1. Parental Echinometra sp. A (Ea) and E. mathaei (Em). Summary of characters relevant to identification and species integrity; Mean  SD, n = 25. Echinometra sp. AEchinometra mathaei HabitatMoat and tide poolsShallow excavated burrows on reef flat. Bathymetric rangeIntertidal, below mean low water level (M.L.W.S.) Intertidal, both above and below M.L.W.S. Salinity and Thermo-tolerance Low tolerance to sudden temperature and salinity changes High tolerance to sudden temperature and salinity changes Breeding seasonApril-December (max. around late September) April-September (max. around late September) ColorEntirely white to greenish or brownish black, spines with white tip and distinct basal white ring Color very variable, spine mostly brown and greenish brown without white tip, and indistinct basal ring Body sizeBiggest among Okinawan EchinometraBigger among Okinawan Echinometra Wet weight (g)50.79±12.7544.55±7.38 Test diameter (mm)47.43±4.59 45.49±5.66 Spine length (mm)22.54±1.1118.43±1.04 Spicules: a. TubefeetC-like or bihamateBihamate, bihamate-like, and triradiate b. GonadSpindleSpindle, and spindle-like Egg size (µm)67.46 ± 0.8769.61 ± 1.12 Sperm head size (µm)4.08 ± 0.435.15 ± 0.47 Thickness of jelly layer (µm)23.93 ± 3.1720.98 ± 3.55

4. Aboral (upper) and oral (lower) color patterns of adult Echinometra spp.: A) Echinometra sp. A; B) E. mathaei.

5. A) Collection site, B) Habitat, and C) Schematic distribution pattern of Echinometra sp. A (Ea) and E. mathaei (Em) at Sesoko coast of Okinawa Island.

6. Cross-fertilization Gametes were obtained from Ea and Em after induced spawning by inter-coelomic injection of 0.5 M KCl. Eggs were collected from the gravid females and washed in SFSW three times. Dry sperm were collected directly from the gonopores. Cross-fertilization was conducted using all possible combinations of ova and sperm at 27-28 o C. To determine fertilization rates at various sperm concentrations, 0.1-ml diluted egg suspensions (350-400 eggs) were placed in small vials with 0.8- ml SFSW. Fresh "dry" sperm was diluted in a series of 10-fold dilutions: 10 -1 -10 -7. From each of these sperm solutions, 0.1-ml aliquots were placed into the vials containing 0.9-ml egg suspensions to bring their final volumes to 1 ml. Sperm concentration from 10 -4 dilution was counted by hemacytometer and adjusted with the dilution series. Fertilization rate was estimated by counting the number of embryos reaching 2-4 cells among the first 100 eggs.

7. Fertilization levels as a function of sperm concentration for conspecific (Ea x Ea, Em x Em) and heterospecific (Ea x Em, Em x Ea) crosses; maternal species first. Mean  SD, n = 6.

8. Larval development of Echinometra spp.

9. Rearing of larvae of Ea, Em and their reciprocal hybrids till attaining competent stage 22-24 days post-fertilization in glass bottles containing SFSW on 10 rpm rotating rollers: A) 400 ml glass bottles; B) 800 ml glass bottles.

10. Induction of metamorphosis of competent larvae of Ea, Em and their reciprocal hybrids on coralline algal rocks immersed into petri dishes containing SFSW.

11. Rearing of the juveniles of Ea x Ea, Em x Em and hybrids up to three months in aerated aquaria and coralline red algae on limestone served as food: A) Plastic aquaria; B) Glass aquaria.

12. Growth and development of juveniles of Echinometra spp. up to three months on coralline red algal stones.

13. Comparison of larval and juvenile performances among Ea x Ea, Em x Em and their reciprocal hybrids; Mean  SD, n = 6. Bars with the same letter are not significantly different (Tukey’s test, P > 0.05).

14. Culturing of the adults of Ea x Ea, Em x Em and hybrids up to 1 year in plastic aquaria with aerated flow through seawater and coral skeletons covered with encrusting coralline algae served as food.

15. Aboral (left) and oral (right) color patterns of Ea, Em and their reciprocal hybrids, 1 year after metamorphosis; maternal species first: A) Ea x Ea; B) Em x Em; C) Ea x Em; D) Em x Ea.

16. Comparison of adult performances among Ea x Ea, Em x Em and their reciprocal hybrids; Mean  SD, n = 6. Bars with the same letter are not significantly different (Tukey’s test, P > 0.05).

17. Fig. 4. Comparison of test sizes and spine lengths of Ea x Ea, Em x Em and their reciprocal hybrids, 1 year after metamorphosis. Mean  SD, n = 20. Bars with the same letter are not significantly different (Tukey’s test, P > 0.05).

18. Types and percentages of spicules in tubefeet of 1-year-old Ea x Ea, Em x Em and their reciprocal hybrids; Mean  SD, n = 20 x 10. Bars with the same letter are not significantly different (Tukey’s test, P > 0.05).

19. Types and percentages of spicules in gonads of 1-year-old Ea x Ea, Em x Em and their reciprocal hybrids; Mean  SD, n = 20 x 10. Bars with the same letter are not significantly different (Tukey’s test, P > 0.05).

20. Morphology and valve length (VL) of 4 types of pedicellaria in Ea x Ea, E x Em and their reciprocal hybrids; Mean  SD, n = 20 x 10. Bars with the same letter are not significantly different (Tukey’s test, P > 0.05).

21. Gamete sizes of mature Ea x Ea, Em x Em and their reciprocal hybrids. Twenty individuals were examined from each cross with 25 eggs and 25 sperm from each individual; Mean  SD in µm. Bars with the same letter are not significantly different (Tukey’s test, P > 0.05).

22. Fertilization levels in backcrosses among 1-year-old lab-reared F 1 generation of Ea, Em and their reciprocal hybrids at limited sperm concentration (1.1 x 10 6 sperm/ml); Mean ± SD, n = 6. Egg from Sperm fromEa x EaEa x EmEm x EaEm x Em Ea x Ea 92.5 ± 3.162.2 ± 2.365.3 ± 3.12.5 ± 1.1 Ea x Em 51.7 ± 2.769.2 ± 3.167.7 ± 4.657.2 ± 2.6 Em x Ea 54.2 ± 3.365.2 ± 2.871.2 ± 3.359.7 ± 2.7 Em x Em1.7 ± 0.869.0 ± 4.672.7 ± 3.396.7 ± 2.0

23. Phenotypic characteristics of lab-produced hybrids used as morphological markers to identify hybrids from the field. Distinctive featuresEa x EmEm x Ea Aboral body colorEa-likeEm-like Oral body colorEa-likeEm-like Spine colorEa-like with distinct white tipEm-like with faint white tip Spine base ring colorEa-like Tubefoot spiculeIntermediate Gonad spiculeIntermediate Pedicellaria valve lengthIntermediate Sperm size/formIntermediate Egg sizeIntermediate 300 individuals with more or less intermediate coloration were collected from the reef flats of Sesoko coasts where both Ea and Em are sympatrically intermingled. Detailed comparisons of the above characters revealed that none of these individuals were hybrids, that is, all could be assigned to either Ea or Em.

24. Conclusions The strong block to fertilization in either heterospecific cross between Ea and Em indicate the presence of protein-binding system for gamete recognition. Such a system might eventually lead to complete gametic incompatibility and reproductive isolation. Successful culturing of adult hybrids in lab-experiments suggests that developmental incompatibility or hybrid inviability are not large enough to maintain species integrity. Gametes of the F1 hybrids are fertile, minimizing the possibility that hybrid infertility is a postzygotic mechanism of reproductive isolation. Strong agonistic behavior along with differences in microhabitat preference between Ea and Em probably decreases the most chances of cross-fertilization and introgression in the field. Sperm conc. used to produce hybrids in our study may be higher than would be encountered in natural conditions, which may contribute to reproductive isolation between Ea and Em. Possible separation in diel spawning times or specific pheromonal spawning cues, provide exogenous factors to prevent near simultaneous spawning that could lead to hybridization between the two species. As the breeding season of Ea and Em overlap, there have the higher possibility of their gametes to meet in the water column. If it does occur in the field and if conspecific sperm outcompetes heterospecific sperm for fertilization, a mechanism for maintaining species integrity in sympatric Ea and Em may be present. Intensive surveys fail to find hybrids, suggesting the lack of natural hybridization in the field. Prezygotic barrier such as strong gamete incompatibility and probably differences in microhabitat and spawning times, gamete competition appear to be important for maintaining genetic integrity of these two species.

25. Grateful Thanks to: Director and Staff of the Tropical Biosphere Research Center, Sesoko Marine Station, Univ. of the Ryukyus for providing space and facilities in culturing urchins. Japan Society for Promotion of Science (JSPS) for financial support. Organizing Committee of the 8 th International Small Islands Studies Association (ISISA 2004) Conference for invitation and nice hospitality. Thank You All