Absence of postzygotic isolating mechanisms: evidence from experimental hybridization between two species of tropical sea urchins

Absence of postzygotic isolating mechanisms: evidence from experimental hybridization between two species of tropical sea urchins 1 first-author †E-mail:aminur1963@gmail.com †E-mail:aminur1963@gmail.com aminur1963@gmail.com 2 1 3 1 3 1 Laboratory of Marine Biotechnology, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia Laboratory of Marine Biotechnology, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia Department of Chemistry, Biology and Marine Science, Faculty of Science, University of the Ryukyus, 1 Senbaru, Nishihara-cho, Okinawa 903-0213, Japan Department of Chemistry, Biology and Marine Science, Faculty of Science, University of the Ryukyus, 1 Senbaru, Nishihara-cho, Okinawa 903-0213, Japan Department of Aquaculture, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia Department of Aquaculture, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia Two reef margin species of tropical sea urchins, Echinometra sp. C (Ec) and Echinometra oblonga (Eo), occur sympatrically on Okinawa intertidal reefs in southern Japan. Hybridization between these species was examined through a series of cross-fertilization experiments. At limited sperm concentrations, where conspecific crosses reached near 100% fertilization, both heterospecific crosses showed high fertilization rates (81%–85%). The compatibility of the gametes demonstrated that if gamete recognition molecules are involved in fertilization of these species, they are not strongly species-specific. We found that conspecific crosses reached peak fertilization levels much faster than did heterospecific crosses, indicating the presence of a prezygotic barrier to hybridization in the gametes. Larval survival, metamorphosis, and juvenile and adult survival of hybrid groups were nearly identical to those of their parent species. Hybrids from crosses in both directions developed normally through larval stages to sexually mature adults, indicating that neither gametic incompatibility nor hybrid inviability appeared to maintain reproductive isolation between these species. In adults, Ec×Ec crosses gave the highest live weight, followed by Eo (ova)×Ec (sperm), Ec (ova)×Eo (sperm), and Eo×Eo. Other growth performance measures (viz., test size, Aristotle’s lantern length, and gonad index) of hybrid groups and their parental siblings showed the same trends. The phenotypic color patterns of the hybrids were closer to the maternal coloration, whereas spine length, tube-foot and gonad spicule characteristics, pedicellaria valve length, and gamete sizes showed intermediate features. Adult F 1 hybrids were completely fertile and displayed high fertilization success in F 1 backcrosses, eliminating the likelihood that hybrid sterility is a postzygotic mechanism of reproductive isolation. Conversely, intensive surveys failed to find hybrid individuals in the field, suggesting the lack or rarity of natural hybridization. This strongly suggests that reproductive isolation is achieved by prezygotic isolating mechanism(s). Of these mechanisms, habitat segregation, gamete competition, differences in spawning times, gametic incompatibility or other genetic and non-genetic factors appear to be important in maintaining the integrity of these species. Sea urchins Echinometra Hybridization Gamete compatibility Reproductive isolation Speciation Introduction 1 Significant differences in morphology, ecology, molecular phylogeny, mitochondrial DNA (mtDNA), and gamete compatibility have been combined to plot the borders of four closely related Echinometra species common all over the tropical Pacific. These have been designated as Echinometra A, B, C, and D (Uehara et al., 1990 1990 ; Matsuoka and Hatanaka, 1991 1991 ; Nishihira et al., 1991 1991 ; Metz and Palumbi, 1996 1996 ; Palumbi, 1996 1996 ; Rahman et al., 2001 2001 ; 2004a 2004a ; 2004b 2004b ; 2005 2005 ). mtDNA sequence data show that divergence among the Echinometra spp. in the central and west Pacific occurred over the past 1–3 million years (Palumbi, 1996 1996 ). Although the four Echinometra species are known as four distinct species, valid names for these species have been debated. Echinometra sp. B is now given the name Echinometra mathaei (Em) (Arakaki et al., 1998 1998 ), while Echinometra sp. D (Ed) belongs in the Echinometra oblonga (Eo) species complex, which may be composed of three cryptic species (Arakaki and Uehara, 1999 1999 ). The other two species, Echinometra sp. A (Ea) and Echinometra sp. C (Ec), have yet to be formally named (Rahman et al., 2000 2000 ; 2001 2001 ). For sympatric, sessile marine invertebrates that broadcast their gametes into the water column, an effective mechanism for reproductive isolation would be to shift spawning times or to have species-specific gamete interactions during fertilization. Among these factors, gametic incompatibility may be particularly important for maintaining reproductive isolation and species integrity in many free-swimming animals including echinoderms (Palumbi and Metz, 1991 1991 ; Byrne and Anderson, 1994 1994 ; Rahman et al., 2001 2001 ; 2004a 2004a ; 2004b 2004b ; Zigler and Lessios, 2003 2003 ; 2004 2004 ; Lessios, 2007 2007 ; Zigler, 2008 2008 ; Palumbi, 2009 2009 ). Conversely, postzygotic mechanisms such as the creation of nonviable larvae and infertile adults can also contribute to reproductive isolation (Coyne, 1992 1992 ; Knowlton, 1993 1993 ; Behrmann-Godel and Gerlach, 2008 2008 ). Among the Okinawan Echinometra , experimental hybridizations have been conducted between Eo and Ea, Ec and Ea, Eo and Em, and Ec and Em (Aslan and Uehara, 1997 1997 ; Rahman et al., 2000 2000 ; 2001 2001 ; 2004a 2004a ; Rahman and Uehara, 2004 2004 ). In each of these combinations, ova from the former species are easily fertilized by the sperm from the latter species, but the reciprocal crosses show very low percentages of fertilization even at a very high sperm concentration. Despite these asymmetries in fertilization, hybrids originating from crosses in either direction develop normally into adults that are fertile both among themselves and in backcrosses. Conversely, gametes of the two co-occurring species on Okinawan reef flats (Ea and Em) show a strong block to fertilization in both directions, but hybrids produced at higher sperm densities grow well and exhibit parental heterosis (Rahman et al., 2005 2005 ). These findings, as well as a lack of hybrids in the field, indicate that gene flow among these combinations is most likely to be minimized by prezygotic mechanisms, particularly segregation of their particular microhabitats and gametic incompatibility at realistic sperm concentrations. Among the four species of Echinometra , two reef margin species, Ec and Eo show distinct differences in adult morphologies and habitat preference (Table 1 1 ). Recent studies of the mitochondrial CO1 gene, and the entire molecule of the nuclear binding gene sequences revealed that these species are of recent origin and are probably less than three-million years old (Palumbi, 1996 1996 ). The gametes of these two species are reciprocally compatible (Uehara et al., 1990 1990 ), their breeding seasons overlap (Arakaki and Uehara, 1991 1991 ), and their microhabitats intermingle. In this paper, through detailed hybridization trials and phenotypic determination of their distinct traits, we determine how reproductive isolation occurs and in particular, how these two species maintain their genetic identity in the field. Data are expressed as mean±SD Ref.: 1. Tsuchiya and Nishihira ( 1984 1984 ); 2. Tsuchiya and Nishihira ( 1985 1985 ); 3. Uehara and Shingaki ( 1985 1985 ); 4. Arakaki and Uehara ( 1991 1991 ); 5. Nishihira et al. ( 1991 1991 ); 6. This study ( n =25 adults of each species) Parameter Ec Eo Ref. * * Habitat Burrows on the reef margin, slightly above Em and with a narrower range of distribution Deep burrows along narrow reef margin in surf break, slightly below Ec 1, 2, 5 Bathymetric range Intertidal, above mean low water level Intertidal, above mean low water level 1, 2, 5 Salinity and thermo-tolerance Higher tolerance to sudden temperature and salinity changes Lower tolerance to sudden temperature and salinity changes 4 Body size Moderate among Okinawan species of Echinometra Smallest among Okinawan species of Echinometra 3 Wet weight (g) 39.17±8.71 11.06±4.81 6 Test length (mm) 43.09±3.14 35.97±3.64 6 Test width (mm) 34.82±2.65 27.96±2.20 6 Spine length (mm) 16.56±0.87 18.43±1.04 6 Color Highly variable, spines mostly green, brownish-black, greenish-brown or whitish-gray; basal translucent, white ring Entirely black test and spines, basal ring of spine unclear 3, 6 Spicule shape   Tube-foot Triradiate Triradiate, bihamate, and triradiate-bihamate 3, 6   Gonad Triradiate, spindle, and bihamate Triradiate, spindle, bihamate, and spindle-triradiate 3, 6 Breeding season Apr.–Dec. (max. around late Sept.) May–Sept. (max. around mid Sept.) 4 Egg size (μm) 72.68±1.25 75.85±1.62 6 Sperm head size (μm) 6.01±0.45 8.14±0.61 6 Jelly layer thickness (μm) 18.13±3.49 15.67±3.76 6 Materials and methods 1 Sample collection and spawning 2 In total, 60 mature adults each of the sea urchin species Ec and Eo were collected from their respective habitats (Table 1 1 ) on the Sunabe coast of Okinawa Island, Japan (26°07′ N; 127°46′ E) at low tide by snorkeling and walking along the sea shore between July and September 2003, a period of reproductive activity for both species (Arakaki and Uehara, 1991 1991 ). They were maintained in closed aquaria in the laboratory of the Department of Chemistry, Biology, and Marine Science, University of the Ryukyus, Okinawa, Japan. Gametes were obtained from each sea urchin following the injection of 0.5 mol/L KCl solution into the coelomic cavity. Eggs were collected in sterilized filtered seawater (SFSW). “Dry” sperm were pipetted off the genital pores and kept in concentrated form in a refrigerator at 4–5 °C for not more than 2 h. Sperm concentration experiments 2 To determine the fertilization rates at different sperm concentrations for conspecific and heterospecific crosses, a 0.1-ml aliquot of diluted egg suspension (350–400 eggs) was kept in small glass vials with 0.8 ml of SFSW. Fresh “dry” sperm were quickly diluted in a series of eight 10-fold dilutions: 10 −1 –10 −8 . A 0.1-ml aliquot from each of these sperm solutions was then placed into the vials containing 0.9-ml egg suspensions, to bring their final volumes to 1 ml. This procedure was followed through a series ranging from 10 −2 –10 −9 diluted concentrations of sperm. Sperm concentrations of the 10 −4 dilution from each species were first measured by hemacytometer counts and then adjusted with the dilution series. Percent fertilization was estimated by counting the number of embryos, reaching 2–4 cells among the first 100 eggs examined. Gamete exposure time experiments 2 To assess the effects of gamete exposure time on fertilization in Ec, Eo, and their reciprocal hybrids, eggs were exposed to a limited sperm concentration at 10 −5 dilution of “dry” sperm (1.0×10 6 sperm/ml). A 2-ml aliquot of diluted egg suspension (3 500–4 000 eggs/ml) was kept in a small glass beaker with 16 ml of SFSW. Fresh dry sperm were diluted in a series of four 10-fold dilutions, following the protocols described in the above sperm dilution experiment. A 2-ml aliquot from a 10 −4 diluted sperm suspension was then poured into the beaker containing 18 ml of egg suspension, which ultimately constituted a 10 −5 diluted concentration of sperm. In each time interval (30, 60, 90, 120, 150, 180, 210, 240, 270, and 300 s), a 1-ml aliquot of the inseminated egg suspension was then gently pipetted into plastic cylinders, the bottoms of which had been fitted with 30 μ Nitex mesh. The cylinders were then rinsed 3–4 times with SFSW to remove excess sperm, and the eggs were resuspended in fresh SFSW. The method for estimating the percent fertilization was as described above. Cross-fertilization and embryonic development 2 Cross-fertilizations between pairs of Echinometra spp. were conducted using all possible combinations of egg and sperm at ambient temperature (27–28 °C) following the protocols described by Rahman et al. ( 2000 2000 ; 2005 2005 ). Following convention, the maternal species is named first. In each heterospecific cross, a conspecific fertilization was conducted using the same gametes as a control. Six replicate crosses were conducted between each pair of species and percent fertilization was calculated as above. Fertilized eggs were shifted to glass beakers and incubated in SFSW at ambient temperature (27–28 °C) until they attained the free-swimming blastula stage. They were then moved to glass bottles on 10 r/min rotating paddles. Both the conspecific and heterospecific crosses attained four-armed pluteus (feeding) larvae within 48 h after fertilization. Larval rearing and metamorphosis induction 2 The four-armed pluteus larvae from both conspecific and heterospecific crosses were reared in 400 or 800 ml glass bottles at a larval density of 1 individual per ml SFSW. Larvae were fed with a cultured phytoplankton, Chaetoceros gracillis, at densities of 1×10 4 –2×10 4 cells/ml, adjusting the food level every 2 d until metamorphic competence was attained (Rahman et al., 2000 2000 ). Six replicate trials were conducted for each cross and the survivals of hybrid and non-hybrid (conspecific) larvae were quantified and compared among the treatments. After 20–24 d of rearing, the mature larvae deemed competent were used for settlement tests. Induction of metamorphosis of competent larvae from all crosses was performed on coralline red algal stones, which were immersed in SFSW in petri dishes each containing 40 ml SFSW. Larval density was maintained at 1 individual in 2 ml SFSW using the method of Rahman and Uehara ( 2001 2001 ). In each trial, six replicate petri dishes (each with 20 competent larvae) were used per treatment and percent metamorphosis was estimated within 24–30 h using the same environmental conditions and procedures as for larval rearing. Culture of juveniles and adults 2 The newly produced juveniles of hybrids and their conspecific controls were reared in small aquaria (25 cm×20 cm×10 cm) with aerated seawater, and pieces of dead coral with coralline algae were provided as food (Rahman et al., 2000 2000 ; 2005 2005 ). This was continued for up to three months. The juveniles were then shifted to plastic aquaria (46 cm×55 cm×25 cm) supplied with aerated flow-through seawater. Dead coral covered with encrusting coralline algae was supplied as food. The stocking density was maintained at 30 individuals in each replicate aquarium. The cultures were continued for one year by which time the urchins attained sexual maturity. The survival rate (%) and growth performances of juveniles and adults were examined and compared among the hybrid groups and their parental controls. Morphological characteristics 2 Detailed morphological characteristics were recorded from one-year-old Ec×Ec, Eo×Eo, and their hybrids, including color patterns of oral and aboral spines and test, sizes of test, spine length, and Aristotle’s lantern, morphology of spicules in the tube-foot and gonad, pedicellariae valve length, and gamete size following the detailed procedures described by Rahman et al. ( 2004a 2004a ; 2004b 2004b ). F 1 backcrosses 2 After one year of rearing, the majority of reciprocal hybrids and their conspecific parents attained sexual maturity and contained mature gametes. To determine gametic compatibility among hybrids and conspecific controls, all gametes were reciprocally backcrossed at a limited sperm concentration (1.0×10 6 sperm/ml) following the methods in the above sperm concentration experiments and protocols described by Rahman and Uehara ( 2001 2001 ) and Rahman et al. ( 2004a 2004a ; 2004b 2004b ). Following convention, when referring to the backcrosses, the maternal species is named first. Six replicate crosses among the F 1 conspecifics and their F 1 hybrids were performed and the protocols including incubation and counting of fertilized eggs were the same as those described in the sperm concentration experiments. Hybrids in nature 2 To investigate the incidence of any natural hybridization between the two Echinometra species, field surveys were conducted along the Sunabe coast of Okinawa and the west coast of Sesoko Island, where both species occur sympatrically in adjacent microhabitats. About 400 individuals, suspected to be hybrids on the basis of color patterns, were collected and compared to the lab-cultured hybrids with respect to the above morphological characteristics. Data analysis 2 Percentage data were arcsine transformed, and replicates in which no eggs or all eggs were fertilized were given a value of 0.25 n and 1−0.25 n ( n is the number of observations) to improve the arcsine transformation (Zar, 1996 1996 ). A “Bartlett’s test” was used to analyze the homogeneity of variances (Bartlett, 1937 1937 ). When variances were not significantly heterogeneous and there were no major departures from normality, a one-/two-way analysis of variance (ANOVA) was carried out followed by the Tukey’s multiple comparison test. All statistical analyses were performed with the computerized statistical package SPSS 15.0. Untransformed data are presented in tables and figures. Results 1 Effects of sperm concentration and exposure time on fertilization success 2 Fertilization success of conspecific and heterospecific crosses was highly dependent on sperm concentrations (Fig. 1 1 ). Fertilization rates in conspecific (Eo×Eo and Ec×Ec) and heterospecific (Ec×Eo and Eo×Ec) crosses at higher sperm concentrations (1.0×10 7 –1.0×10 9 sperm/ml) were 100% or very near to 100%. At a limited (intermediate) sperm concentration (1.0×10 6 sperm/ml), where conspecific crosses reached nearly 100% fertilization, mean fertilization rates in Ec×Eo (85.33%) and Eo×Ec (81.83%) crosses were significantly (Tukey’s test, P < 0.05) lower than in either conspecific cross. Under lower sperm concentrations (≤1.0×10 5 sperm/ml), heterospecific crosses showed similar trends but progressively lower fertilization rates compared to conspecific crosses. Maternal species is named first in each cross. Data are expressed as mean±SD ( n =6). Mean values for each concentration data point with the same letters are not statistically significantly different (Tukey’s test, P > 0.05) Fertilization success of both conspecific and heterospecific crosses was significantly influenced by sperm-egg exposure time (i.e., the greater the exposure time, the higher the fertilization rate) (Fig. 2 2 ). At a limited sperm concentration (1.0×10 6 sperm/ml), the conspecific crosses reached high percentages of fertilization (98.0% in Ec×Ec and 99.2% in Eo×Eo) within a contact period of 90 s, whereas heterospecific Ec×Eo and Eo×Ec crosses took much longer (180 s for Ec×Eo and 210 s for Eo×Ec) to reach asymptotic levels of fertilization. Maternal species is named first in each cross. Fresh gametes from new individuals were used for each replicate. Data are expressed as mean±SD ( n =6). Mean values for each exposure time data point with the same letters are not statistically significantly different (Tukey’s test, P > 0.05) Larval, juvenile, and adult performances 2 Survival percentages of competent larvae of Ec×Eo and Eo×Ec hybrids were not significantly different from those of larvae of conspecific crosses (Tukey’s test, P > 0.05). Both reciprocal hybrids showed lower metamorphosis rates than either of the parental crosses but the values did not differ significantly (Table 2 2 ). The recovery rate of three-month-old juveniles of conspecific parents and their hybrids followed the same trends as metamorphosis (Table 2 2 ). Six replicate experiments were conducted in each cross for each performance measure. Data are expressed as mean±SD (range). Mean values in the same column having the same superscripts are not significantly different (Tukey’s test, P > 0.05) Three-month-old juvenile urchins that were transferred to a flow-through sea water system for advanced culture 2 Cross 3 Larva and juvenile urchins 4 Adult urchin (one-year-old) Survival (%) Metamorphosis (%) Recovery * * (%) Wet weight (g) Wet gonad weight (g) Gonad index (%) Survival (%) 2 Ec×Ec 78.50±2.08 a 88.33±5.16 a 71.46±2.23 a 9.21±0.83 a 1.33±0.06 a 14.52±0.70 a 86.67±3.34 a (75.25–81.00) (80.00–95.00) (68.75–74.25) (8.25–10.90) (1.26–1.50) (13.51–15.33) (83.33–90.00) 2 Ec×Eo 76.63±1.98 a 85.83±4.92 a 69.88±2.13 a 6.59±0.42 b 0.75±0.07 b 11.41±0.40 b 83.33±3.34 a (73.50–78.75) (80.00–90.00) (66.50–72.75) (6.01–7.23) (0.66–0.85) (10.79–11.98) (80.00–86.67) 2 Eo×Ec 77.08±2.22 a 86.67±4.08 a 70.33±2.04 a 6.77±0.45 b 0.79±0.08 b 11.68±0.35 b 84.45±3.35 a (74.25–79.50) (80.00–90.00) (67.75–73.50) (6.20–7.43) (0.69–0.90) (10.99–12.11) (80.00–86.67) 2 Eo×Eo 79.30±2.02 a 89.16±5.85 a 72.67±2.26 a 5.02±0.50 c 0.51±0.05 c 10.19±0.45 c 87.78±1.92 a (76.50–81.75) (80.00–95.00) (69.50–75.50) (4.40–5.90) (0.42–0.60) (9.38–11.13) (86.67–90.00) The mean live weight attained by one-year-old Ec×Ec urchins was significantly higher than the weight gained by Eo×Eo urchins (Tukey’s test, P < 0.05), while both reciprocal hybrids attained intermediate sizes. These values did not differ significantly from each other (Tukey’s test, P > 0.05). Wet gonad weight was significantly lower in the slow-growing Eo×Eo urchins than in the faster-growing Ec×Ec urchins. Both reciprocal hybrids had gonads of intermediate weight but did not differ significantly from each other (Tukey’s test, P > 0.05). The gonad index followed the same trends seen for wet gonad weight. Survival was the highest in Eo×Eo crosses followed by Ec×Ec, Eo×Ec, and Ec×Eo. Neither reciprocal hybrid showed significant differences in survival (Tukey’s test, P > 0.05) from their parental siblings and among themselves. However, survival and growth performance indicated that hybrids in both directions were viable but intermediate to their parents, under lab-reared conditions. Comparisons of phenotypic characteristics 2 Tests, spines, and Aristotle’s lanterns of one-year-old urchins were the largest in Ec×Ec crosses followed by Eo×Ec, Ec×Eo, and Eo×Eo progenies. Statistically significant differences (Tukey’s test, P < 0.05) were found in all parameters between Ec×Ec and Eo×Eo, but the hybrids showed intermediate sizes and did not differ significantly (Tukey’s test, P > 0.05) from each other (Table 3 3 ). Twenty adult specimens were measured for each treatment. Data are expressed as mean±SD (range). Mean values in the same column having the same superscripts are not significantly different (Tukey’s test, P > 0.05) Cross Test length (mm) Test width (mm) Test height (mm) Test volume (cm 3 ) Spine length (mm) Aristotle’s lantern length (mm) Aristotle’s lantern diameter (mm) 2 Ec×Ec 24.24±0.98 a 22.25±0.93 a 12.01±0.48 a 6.50±0.79 a 20.13±0.75 a 8.71±0.59 a 8.35±0.70 a (23.05–25.95) (21.20–23.82) (11.35–12.90) (5.55–7.97) (18.68–21.56) (7.60–9.80) (7.00–9.40) 2 Ec×Eo 22.17±0.58 b 20.56±0.78 b 10.91±0.40 b 5.01±0.44 b 17.81±0.64 b 8.04±0.57 b 7.71±0.58 b (20.90–22.92) (18.88–21.71) (10.03–11.39) (4.01–5.66) (16.25–19.22) (6.80–9.10) (6.60–8.90) 2 Eo×Ec 22.30±0.62 b 20.69±0.73 b 11.05±0.41 b 5.11±0.47 b 18.02±0.68 b 8.17±0.56 b 7.93±0.57 b (21.00–23.15) (19.00–21.95) (10.10–11.65) (4.12–5.92) (16.50–19.45) (7.00–9.20) (6.80–9.00) 2 Eo×Eo 20.80±0.68 c 19.35±0.42 c 10.20±0.60 c 4.07±0.43 c 15.10±0.50 c 7.33±0.57 c 7.20±0.64 c (19.80–21.92) (18.62–19.90) (9.32–11.20) (3.50–4.83) (13.25–17.02) (6.40–8.40) (6.20–8.20) The body coloration differed between adult conspecifics and hybrids. In Ec×Ec specimens, test and spine color was uniformly green, and each spine had a clear white ring at its base. Eo×Eo specimens had entirely dark tests and spines, and each spine had a faded basal white ring. The Ec×Eo hybrids were more similar to Ec×Ec conspecifics in having a greenish-dark test with greenish spines and each spine having a clear basal white ring. On the other hand, Eo×Ec specimens were closer to Eo×Eo specimens in having a quite dark test and spines with faded basal white rings. In terms of oral body coloration, Ec×Ec urchins had yellowish-green spines around the mouth and a greenish test color, whereas Eo×Eo urchins had an entirely dark test and spines around the mouth. Ec×Eo hybrids were more similar to Ec×Ec conspecifics, and Eo×Ec hybrids were similar to Eo×Eo conspecifics in these characters; i.e., the coloration of hybrids was maternally inherited. Tube-foot spicules in Ec×Ec urchins were always triradiate (100%), whereas those in Eo×Eo urchins were triradiate (90.29%), bihamate (3.67%), and triradiate-bihamate (6.04%) (Table 4 4 ). Tube-foot spicules of Ec×Eo hybrids were triradiate (83.38%), bihamate (2.19%), bihamate-like (3.86%), and triradiate-bihamate (10.57%), whereas those in Eo×Ec hybrids were triradiate (74.12%), bihamate (4.30%), bihamate-like (6.60%), and triradiate-bihamate (14.98%) (Table 4 4 ). The proportions of various types of tube-foot spicules in Ec×Eo and Eo×Ec hybrids differed significantly (Tukey’s test, P < 0.05) from their conpecific controls and showed higher proportions of intermediate features. Twenty individuals were randomly examined for each treatment with 10 tube-feet and 10 gonadal tissues per treatment. Data are expressed as mean±SD (range). Mean values in the same column having the same superscripts are not significantly different (Tukey’s test, P > 0.05) 2 Cross 4 Tube-foot spicule (%) 6 Gonad spicule (%) Triradiate Bihamate Bihamate-like Triradiate-bihamate Triradiate Spindle Spindle-like Spindle-bihamate Bihamate Bihamate-like 2 Ec×Ec 100 a 0 d 0 c 0 d 93.75±0.88 a 5.13±0.69 c 0 c 0 d 1.12±0.22 d 0 c (92.50–95.02) (3.85–6.18) (0.86–1.64) 2 Ec×Eo 83.38±1.76 c 2.19±0.40 c 3.86±0.72 b 10.57±1.19 b 80.06±1.41 c 4.22±0.59 d 3.12±0.66 b 7.67±0.66 b 2.07±0.23 c 2.88±0.45 b (80.90–86.21) (1.53–2.86) (2.72–5.06) (8.59–12.96) (78.02–82.69) (2.94–5.43) (2.17–4.18) (6.67–8.96) (1.68–2.56) (2.07–3.59) 2 Eo×Ec 74.12±1.71 d 4.30±0.66 a 6.60±0.65 a 14.98±1.08 a 72.05±1.74 d 5.98±0.43 b 4.31±0.50 a 10.45±0.74 a 3.36±0.50 a 3.85±0.57 a (70.25–76.71) (3.02–5.79) (5.16–7.79) (13.58–17.26) (69.89–76.14) (5.03–6.74) (3.08–4.94) (8.90–12.25) (2.33–4.10) (2.46–4.60) 2 Eo×Eo 90.29±1.14 b 3.67±0.46 b 0 c 6.04±0.78 c 84.01±1.80 b 8.12±0.71 a 0 c 4.91±0.76 c 2.96±0.55 b 0 c (88.21–92.38) (2.86–4.37) (4.76–7.69) (81.07–86.67) (6.93–9.85) (3.87–5.97) (2.10–3.69) Gonad spicules in Ec×Ec urchins were nearly all triradiate-shaped (93.75%; other spicules seen: spindle, 5.13%; bihamate, 1.12%), whereas those in Eo×Eo urchins were triradiate (84.01%), spindle (8.12%), spindle-triradiate (4.91%) and bihamate (2.96%) (Table 4 4 ). Gonad spicules in Ec×Eo hybrids were triradiate (80.06%), spindle (4.22%), spindle-like (3.12%), spindle-triradiate (7.67%) with a few bihamate (2.07%), and bihamate-like (2.88%) types, whereas spicules in Eo×Ec hybrids were triradiate (72.05%), spindle (5.98%), spindle-like (4.31%), spindle-triradiate (10.45%), bihamate (3.36%), and bihamate-like (3.85%). Although significant differences (Tukey’s test, P < 0.05) were recognized among the hybrids and their parental species, the proportions in the hybrids were intermediate. Four types of pedicellariae, tridentate, globiferous, ophiocephalous and triphyllous, were observed in both conspecifics and their reciprocal hybrids. Valve lengths of all four types of pedicellariae of Ec×Ec urchins were significantly (Tukey’s test, P < 0.05) larger than those of their corresponding types from Eo×Eo urchins (Table 5 5 ). Both the hybrids had intermediate sizes, but they differed significantly from each other. Twenty individuals were examined from each cross with 10 pedicellariae of each type from each individual. Data are expressed as mean±SD (range). Mean values in the same column having the same superscripts are not significantly different (Tukey’s test, P > 0.05) 2 Cross 4 Valve length (μm) Tridentate Globiferous Ophiocephalous Triphyllous 2 Ec×Ec 880.9±34.2 a 649.5±29.9 a 546.4±27.7 a 145.3±20.1 a (840.0–950.0) (590.0–680.0) (490.0–590.0) (110.0–180.0) 2 Ec×Eo 833.7±32.7 b 628.9±28.6 b 517.2±27.5 b 133.6±18.1 b (780.0–900.0) (580.0–670.0) (460.0–570.0) (100.0–170.0) 2 Eo×Ec 783.3±30.7 c 609.0±28.5 c 484.9±27.7 c 122.5±18.1 c (750.0–840.0) (570.0–660.0) (440.0–550.0) (90.0–160.0) 2 Eo×Eo 732.4±29.7 d 590.7±27.7 d 452.4±26.9 d 110.5±16.3 d (680.0–800.0) (530.0–630.0) (410.0–500.0) (80.0–140.0) Egg diameters of Ec×Ec urchins were the smallest among the four crosses while the Eo×Eo eggs were the largest. Hybrids had intermediate-sized eggs that differed significantly (Tukey’s test, P < 0.05) in size from the eggs of the conspecifics. But the hybrids did not differ significantly in size (Tukey’s test, P > 0.05) from each other (Table 6 6 ). The sizes of sperm heads were also the smallest in Ec×Ec urchins, and were significantly different (Tukey’s test, P < 0.05) among crosses, including between the hybrid groups. Therefore the gamete sizes of hybrids were intermediate between their parental controls. Six individuals were examined from each cross for each sex with 25 eggs and 25 sperm from each individual. Data are expressed as mean±SD (range). Mean values in the same column having different superscripts are significantly different (Tukey’s test, P < 0.05) 2 Cross 2 Gamete size (μm) Egg diameter Sperm-head length Ec×Ec 72.36±1.27 c (70.68–75.64) 6.04±0.54 d (5.00–7.00) Ec×Eo 74.36±1.28 b (72.54–76.88) 7.16±0.57 c (6.50–8.50) Eo×Ec 73.98±1.23 b (71.92–76.26) 6.83±0.59 b (6.00–8.00) Eo×Eo 75.74±1.54 a (73.16–78.12) 8.24±0.53 a (7.50–9.50) Existence of natural hybrids 2 Four hundred individuals with coloration patterns more or less intermediate between those of the two species were collected from the reef flats of the Sunabe and Sesoko coasts, where Ec and Eo were abundant and found close together. However, detailed comparisons of the above morphological characters revealed that none of these individuals were hybrids; that is, all could be assigned to either Ec or Eo. Molecular analyses (e.g., nuclear DNA) are needed to find out whether hybrids and their backcross individuals are occurring in the field, which have not been identified by morphological characteristics. Fertilization rates in F 1 backcrosses 2 Fertilization rates in F 1 backcrosses using the gametes of F 1 hybrids and their conspecific parents at a limited sperm concentration are shown in Table 7 7 . Eggs from both hybrids, Ec×Eo and Eo×Ec, yielded higher fertilization rates with Ec×Ec sperm (93.33% and 91.83%) than with Eo×Eo sperm (92.83% and 89.00%), indicating that Eo sperm were more discriminating than Ec sperm. However, backcrosses by sperm from Ec×Eo and Eo×Ec hybrids produced higher percentages of fertilization with Ec×Ec ova (85.83% and 81.17%) than with Eo×Eo ova (83.0% and 78.83%), again indicating that Eo ova appeared to be slightly less attractive than Ec ova. Thus, differences in fertilization rates among the F 1 conspecifics and their F 1 hybrids indicate discrimination differences in their gamete recognition genes. Each value represents six replicate crosses with gametes from new individuals in each replicate. Data are expressed as mean±SD (range). Mean values in the same column having different superscripts are statistically significant (Tukey’s test, P < 0.05) 2 Egg 4 Fertilization rate (%) Sperm (Ec×Ec) Sperm (Ec×Eo) Sperm (Eo×Ec) Sperm (Eo×Eo) Ec×Ec 98.67±1.63 a (96.0–100.0) 85.83±2.48 c (83.0–90.0) 81.17±2.99 b (77.0–85.0) 85.67±3.86 c (79.0–89.0) Ec×Eo 93.33±1.51 b (92.0–96.0) 97.67±1.21 a (96.0–99.0) 93.17±2.31 a (90.0–96.0) 92.83±1.72 b (90.0–95.0) Eo×Ec 91.83±1.60 b (90.0–94.0) 91.33±2.17 b (88.0–94.0) 95.17±2.32 a (92.0–98.0) 89.00±2.37 c (86.0–92.0) Eo×Eo 81.0±3.79 c (75.0–86.0) 83.0±2.37 c (80.0–86.0) 78.83±3.43 b (75.0–83.0) 99.67±0.52 a (99.0–100.0) Discussion 1 The gametes of two reef margin species, Ec and Eo, are reciprocally compatible ηUehara et al., 1990 1990 ). However, at a sperm concentration where conspecific crosses achieved 100% or near 100% fertilization, both heterospecific crosses showed 81%–85% fertilization success, and these values declined significantly with decreasing sperm concentrations. These impediments to fertilization in both heterospecific crosses indicate the presence of a protein-binding system for gamete recognition. Incompatibility of bindin and bindin-receptors might eventually lead to reproductive isolation, as proposed by Metz et al. ( 1994 1994 ), Metz and Palumbi ( 1996 1996 ), Biermann et al. ( 2004 2004 ), Lessios ( 2007 2007 ), Zigler ( 2008 2008 ), and Palumbi ( 2009 2009 ). These two species typically do not co-occur throughout their respective ranges, and Geyer and Palumbi ( 2003 2003 ) found more genetic separation of bindin genes in their sympatric than in their allopatric populations. In Okinawa, where they are sympatric, fertilization percentages over a broad range of sperm densities are almost identical between heterospecific crosses, while other combinations of gametes from the four Echinometra species show high asymmetries in fertilization: the ova of reef margin species are readily fertilized by sperm of reef flat species, but not vice versa (Uehara et al., 1990 1990 ; Rahman et al., 2001 2001 ; 2004a 2004a ; Rahman and Uehara, 2004 2004 ). In contrast, gametes of the two reef flat species, Ea and Em, are nearly incompatible (Rahman et al., 2004b 2004b ) which in this case suggests positive selection for gamete incompatibility to prevent hybridization in sympatry. Moreover, in F 1 backcrosses the Ec-Eo hybrids exhibited higher fertilization rates than Ea-Eo (Aslan and Uehara, 1997 1997 ), Ea-Ec (Rahman et al., 2001 2001 ), Em-Eo (Rahman et al., 2004a 2004a ), Em-Ec (Rahman and Uehara, 2004 2004 ), and Ea-Em (Rahman et al., 2004b 2004b ) hybrids. In other words, the two species show a high divergence in their bindins (Landry et al., 2003 2003 ; Geyer and Palumbi, 2005 2005 ). Similarly, although the two sympatric species of Caribbean sea urchins, Lytechinus willamsi and Lytechinus variegatus show a high divergence in bindins (Zigler and Lessios, 2004 2004 ), their gametes are reciprocally as compatible as those in F 1 backcrosses within a wide range of sperm concentrations. However, the lack of evidence for gamete incompatibility between Ec and Eo suggests that the observed differences in bindin do not significantly affect gamete interactions. There may be subtle fertilization effects that are not detected, but it is also possible that the monophyly of bindin is simply a result of the stochasticity of coalescence processes (Hudson and Turelli, 2003 2003 ; Zigler and Lessios, 2004 2004 ). Two sympatric species of asteroids belonging to the genus Patiriella , P . calcar and P . gunni , are reciprocally compatible and do not show any gametic incompatibility in either cross, despite the fact that they are morphologically and genetically distinct (Byrne and Anderson, 1994 1994 ). Therefore, reproductive isolation between Ec and Eo may have occurred before the evolution of gametic incompatibility because gamete recognition molecules accumulate over time as species diverge (McCartney and Lessios, 2002 2002 ; Lessios, 2007 2007 ; Zigler, 2008 2008 ). If this is true, it provides evidence for the eventual evolution of gamete incompatibility and speciation in these two species (Rahman et al., 2001 2001 ). The percentages of larval survival, metamorphosis, and juvenile and adult survival of the hybrid groups were similar to those of their parents, eliminating the probability that developmental incompatibility or hybrid inviability is a postzygotic mechanism of reproductive isolation. The surviving hybrids grew at the same rate as the conspecifics. Moreover, hybrids were as viable and fertile as the conspecifics, demonstrating that there are neither gametic nor postzygotic obstructions to introgression between them. These similarities are correlated with their higher genetic affinities compared to other closely related pairs of Okinawan Echinometra spp., where hybrids in one direction were as viable as conspecifics while hybrids in the other direction were less viable (Rahman et al., 2000 2000 ; 2001 2001 ; 2004a 2004a ; Rahman and Uehara, 2004 2004 ). The hybrids of two reef flat species, Ea and Em, showed inferior performances in larval and juvenile traits, but better performances in advanced stages (Rahman et al., 2004b 2004b ; 2005 2005 ). The progeny of reciprocal crosses between Strongylocentrotus droebachiensis and Strongylocentrotus pallidus produced viable gametes, and backcrosses to the parent species indicated that gene flow would run predominantly back to S . pallidus (Strathmann, 1981 1981 ). However, Addison and Hart ( 2005 2005 ) reported only small amounts of introgression from the reciprocal cross ( S . pallidus × S . droebachiensis ) and none at all from the more fertile cross of S . droebachiensis × S . pallidus . The higher compatibility of the gametes of Ec and Eo demonstrated that if gamete recognition molecules are involved in fertilization in these species, they are not strongly species-specific. Genetic divergence and speciation of Ec and Eo have occurred without gametic incompatibility and perhaps arose through ecological factors. The two species in the present study live comparatively close to each other but inhabit different microhabitats: Ec inhabits burrows in the reef margins whereas Eo inhabits deep burrows in surf breaking reef margins, positioned below those of Ec. During our field studies, we observed that Ec was more aggressive than Eo and always showed notable antagonistic behavior towards intruders by driving them away if the Eo was placed into a burrow inhabited by Ec. Tsuchiya and Nishihira ( 1985 1985 ) observed a similar phenomenon in two reef flat species, Ea and Em. There is little evidence of highly selective microhabitat assortment in sea urchin larvae. Cameron and Schroeter ( 1980 1980 ) observed that competent larvae of S. purpuratus settled to metamorphose indiscriminately on bacterial-covered substrates, and later microhabitat segregation of the juveniles occurred to avoid high selective mortality or to avoid predation. It was also observed that the majority of the larvae of Echinometra spp. settled indiscriminately on stones covered with coralline algae in the field and their juveniles were found in their respective microhabitats. However, this discrimination probably decreases most chances of cross-fertilization because, as shown in other broadcast spawning invertebrates, fertilization success decreases with the distance between spawning individuals (Levitan, 1998 1998 ). If gametes of one species are swept over spawning individuals of another species in adjacent areas, sperm concentration has a major effect on fertilization (Rahman et al., 2004a 2004a ). Moreover, individuals of Ec are occasionally found to intermingle with those of Eo, and in this case sperm concentration will not be a factor in avoiding hybridization. Consequently, it is still unclear whether microhabitat segregation, by itself, is enough to prevent introgression in these two congeners. The annual breeding seasons of Ec and Eo overlap extensively (Arakaki and Uehara, 1991 1991 ) (Table 1 1 ) and they can readily spawn during this time. There are no data on the precise spawning times of any echinoid (Pearse and Cameron, 1991 1991 ), nor is there any evidence or indication that different species of Echinometra spawn in different lunar cycles, as do some closely related sympatric species of Diadema (Muthiga, 2003 2003 ; Coppard and Campbell, 2005 2005 ). Therefore, temporal separation of spawning seasons is an unlikely mechanism for maintaining reproductive isolation (Byrne and Anderson, 1994 1994 ; Rahman and Uehara, 2004 2004 ). Nevertheless, peak spawning periods, as well as salinity and temperature tolerances differ between the two species (Arakaki and Uehara, 1991 1991 ) (Table 1 1 ). Different gametogenetic cycles observed through histological examinations (Aslan, 2000 2000 ), revealed that different individuals of Echinometra spp. spawned at different times as found for Em in Okinawa (Nishihira, 1975 1975 ), the Gulf of Suez (Pearse, 1969 1969 ), and at Rottnest Island (Pearse and Philips, 1968 1968 ). These factors, in addition to possible separation in daily spawning times or specific pheromonal spawning cues, could prevent hybridization between the two species (Rahman and Uehara, 2004 2004 ). Free-spawning invertebrates (e.g., coelenterates, polychaetes, mollusks, and echinoderms), including sea urchins, are recognized as having no obvious courtship or little premating behavior between sexes before reproduction (Metz et al., 1994 1994 ; Lamare and Stewart, 1998 1998 ; Landry et al., 2003 2003 ), even when habitats and spawning seasons may overlap as in Echinometra spp. (Arakaki and Uehara, 1991 1991 ; Rahman and Uehara, 2004 2004 ). Instead, gametes are released into the water column, and the most essential interaction is between eggs and sperm at fertilization. In these instances, reproductive isolation may arise by changes in the timing of gamete release (Lessios, 1984 1984 ) or clumping of conspecific adults (Billett and Hausen, 1982 1982 ). However, the behavioral components in reproduction that are considered to force rapid speciation in other animals are largely absent in sea urchins including those within the Echinometra spp. complex (Palumbi and Metz, 1991 1991 ; Rahman and Uehara, 2004 2004 ). If gametes of both species are in the water together and species specificity operates in gamete recognition and sperm-binding genes, there could be interspecific competition for fertilization success (Rahman et al., 2004a 2004a ). Our findings from the gamete exposure (contact) time experiment revealed that conspecific Ec×Ec and Eo×Eo crosses reached asymptotic levels of fertilization much faster than the heterospecific Ec×Eo and Eo×Ec crosses under a limited sperm concentration (Fig. 2 2 ). How such sperm competition could be achieved in broadcast spawning, externally fertilizing species such as Echinometra spp. is unclear. However, if it does occur in the field, and if conspecific sperm outcompetes heterospecific sperm for fertilization (Howard et al., 1998 1998 ), a mechanism for maintaining species integrity in sympatric Ec and Eo may be present. Evidence for such a system of gamete competition was found recently when gametes of these two species were mixed at low concentrations and nearly all parents of the resulting embryos were identified as conspecifics by the use of DNA markers (Geyer and Palumbi, 2005 2005 ). Although the expression of an intermediate phenotype by the lab-reared hybrids might be used to discover hybrids in the field, it would be hard to identify hybrids if the phenotype of only one parent is expressed or if the two parental species are morphologically similar. Coloration patterns of the hybrids tended to be maternally inherited. Conversely, other characters, such as test sizes, spine lengths, Aristotle’s lantern length, spicule morphology of the tube-foot and gonad, pedicellariae valve length, and gamete sizes, tended to be intermediate and could be used to distinguish easily hybrids from either parent. We searched for the distinctive intermediate phenotypes in the field, and although suggestive color morphs were observed, none were identified as hybrids and all of them were assigned to either Ec or Eo. However, without doing any genetic analysis (e.g., mtDNA analysis), we cannot rule out that some of these intermediate phenotypes were backcross individuals. As in other Echinometra , Ec and Eo are sympatric but hybrids are rare or absent (Geyer and Palumbi, 2005 2005 ). The only evidence of hybridization in the field is a single specimen out of 97 examined that had similar color and spicules characteristics of Em, but with the mtDNA of Eo (Palumbi et al., 1997 1997 ). This specimen may have represented a backcross, perhaps of several generations (Rahman et al., 2004a 2004a ). Moreover, although two species of the tropical long-spined sea urchins, Diadama savignyi and Diadama setosum , readily hybridize in the laboratory (Uehara et al., 1990 1990 ), and often occur in mixed populations in the field (Pearse, 1998 1998 ), allozyme analyses revealed that there is restricted introgression and hybrids occur only rarely in the field (Lessios and Pearse, 1996 1996 ). Based on mtDNA analyses, these two species separated between 6 and 10 million years ago (Lessios et al., 2001 2001 ) and are at least twice as old as the Pacific species of Echinometra (Rahman et al., 2004a 2004a ). Since the lab-reared hybrids are fully fertile in all these species, there is almost certainly some kind of effective isolating mechanism(s) separating them that does not involve hybrid viability and fertility. 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