Marine biology
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The reef-building coral Acropora conditionally hybridize under sperm limitation
Research
Seiya Kitanobo1, Naoko Isomura2, Hironobu Fukami3, Kenji Iwao4 and Masaya Morita1
Cite this article: Kitanobo S, Isomura N, Fukami H, Iwao K, Morita M. 2016 The reefbuilding coral Acropora conditionally hybridize under sperm limitation. Biol. Lett. 12: 20160511. http://dx.doi.org/10.1098/rsbl.2016.0511
1
Sesoko Station, Tropical Biosphere Research Center, University of the Ryukyus, Sesoko, Motobu, Okinawa 905-0227, Japan 2 Department of Bioresources Engineering, Okinawa National College of Technology, 905 Henoko, Nago-City, Okinawa 905-2192, Japan 3 Faculty of Agriculture, Department of Marine Biology and Environmental Science, University of Miyazaki, Miyazaki, Japan 4 Akajima Marine Science Laboratory, Zamami, Okinawa 901-3311, Japan MM, 0000-0002-0170-1226
Received: 15 June 2016 Accepted: 28 July 2016
Subject Areas: evolution, ecology, developmental biology Keywords: hybridization, fertilization, coral, broadcast spawning, sperm limitation
Author for correspondence: Masaya Morita e-mail: [email protected]
Multi-specific synchronous spawning risks both sperm limitation, which reduces fertilization success, and hybridization with other species. If available sperm of conspecifics are limited, hybridization with heterospecific sperm could be an alternative. Some species of the reef-building coral Acropora produce hybrid offspring in vitro, and therefore hybridization between such species does sometimes occur in nature. Here, we report that the interbreeding species Acropora florida and A. intermedia preferentially bred with conspecifics at optimal gamete concentrations (106 cells ml21), but when sperm concentration was low (104 cells ml21), A. florida eggs displayed an increased incidence of fertilization by sperm of A. intermedia. However, A. intermedia eggs never crossed with heterospecific sperm, regardless of gamete concentrations. It appears that A. florida eggs conditionally hybridize with heterospecific sperm; in nature, this would allow A. florida to cross with later-spawning species such as A. intermedia. These results indicate that hybridization between some Acropora species could occur in nature according to the number of available sperm, and the choice of heterospecific sperm for fertilization could be one of the fertilization strategies in the sperm-limited condition.
1. Introduction
Electronic supplementary material is available at http://dx.doi.org/10.1098/rsbl.2016.0511 or via http://rsbl.royalsocietypublishing.org.
Low sperm densities during fertilization events, named ‘sperm limitation’, is a factor that can reduce the reproductive success of broadcast-spawning invertebrates [1,2], and thus many invertebrates spawn synchronously to increase fertilization success [3,4]. However, synchronous spawning of multiple species could increase the risk of interspecific hybridization [3]. Hybridization is considered an evolutionary dead end because it often results in progeny that are less fit or sterile, but alternatively it can result in hybrid vigour and increased adaptive potential [5]. To ensure that gametes are only fertilized by conspecifics even in the presence of heterospecifics, broadcast-spawning organisms have evolved gamete recognition. Many gamete-recognition proteins evolved rapidly (ratio of substitution rates at non-synonymous and synonymous sites: dN/dS . 1), ensuring the similarly rapid development of strict gamete recognition [6]. Genes coding for gamete-recognition proteins with distinctive features related to fertilization conditions (such as sperm concentration) also change over a relatively short time period, reflecting changes in fertilization
& 2016 The Author(s) Published by the Royal Society. All rights reserved.
species
A. florida
A. intermedia
spawning date and spawning time
3 June 2015 21.30 (two colonies)
3 June 2015 21.51 (one
a
f1, 21.38 (one colony)
colony) 22.01 (one
a
colony) ai35
f13
21.39 (two colonies) a f26, f11
22.09 (two colonies) a
i17, ai18 22.12 (two colonies) ai24 a
Colonies used in this study for fertilization experiments.
by counting the number of cells showing cell division. Larvae (2 days post fertilization) were fixed with 99.5% EtOH and stored at 2208C until DNA extraction could be performed.
(c) Sperm-choice test Paternity was determined using Acropora microsatellite marker 11745m3 ([18], electronic supplementary material, figure S1b). DNA was extracted from larvae as follows: fixed larvae were treated with 20 ml lysis buffer (1 mg ml21 proteinase K, 100 mM NaCl, 0.5% SDS, 10 mM EDTA and Tris-HCl pH 8.0) at 558C for 3 h, and then incubated at 988C for 5 min to terminate the reaction. The resultant suspensions were used as templates for PCR. PCR was performed using GoTaq green mix (5.0 ml GoTaq, 3.6 ml water, 0.6 ml 10 mM microsatellite marker 11745m3 [18] and 0.2 ml template). The PCR protocol of Shinzato et al. [18] was used, with some modifications. The protocol was performed for 2 min at 958C, followed by 35 cycles of (948C for 30 s, 588C for 90 s and 728C for 1 min), and a final elongation at 608C for 30 min. Amplicons were run on a 12% TAE (40 mM Tris-acetate ( pH 8.0) 1 mM EDTA) acrylamide gel (106 100 mm) at 150 V and 20 mA for 5 h. The gel was stained with GelRed. The expected ratio of fertilization if there were no preference between conspecifics and heterospecifics was calculated as follows: number of hybrids/(number of fertilized eggs/2).
(a) Coral species We used the coral species A. florida and A. intermedia, whose gametes are capable of bidirectional intercrossing [14]. Four colonies of each species were collected from Akajima Island (268120 N, 1278170 E) and maintained in aquaria until they spawned. Gamete bundles were collected and used in fertilization experiments: (i) sperm no-choice experiments and (ii) sperm-choice experiments.
(b) Fertilization experiments Fertilization experiments were conducted with sperm choice and no-choice conditions. In no-choice experiments, crossings between conspecifics or heterospecifics were performed with optimal sperm concentrations (106 cells ml21) [17]. In spermchoice experiments (electronic supplementary material, figure S1a), eggs were exposed to a mixture of sperm from conspecifics and heterospecifics at optimal (106 cell ml21) [17] or low sperm concentrations (104 cells ml21). The concentrations of conspecific and heterospecific sperm were equal (0.5 106 cells ml21 and 0.5 104 cells ml21 in high and low conditions, respectively). Fertilization rates in each condition were determined after 2 h
3. Results Spawning of the coral species A. florida and A. intermedia occurred on the same date but at different times; A. florida spawned 20 to 30 min before A. intermedia (table 1). The rates of crossings between heterospecifics varied (A. florida eggs A. intermedia sperm: 36.5 + 25.9%, A. intermedia eggs A. florida sperm: 34.7 + 30.1%, mean + s.d.; electronic supplementary material, table S1) but were uniformly high between conspecifics (A. florida eggs: 96.0 + 4.3%, A. intermedia: 95.9 + 4.3%, mean + s.d.; electronic supplementary material, table S1). In sperm-choice experiments, eggs of both species were primarily fertilized by conspecific sperm under optimal sperm concentration (figure 1; electronic supplementary material, figures S1b and S2, total fertilization rate of A. florida eggs: 94.6 + 4.7%, A. intermedia eggs: 95.0 + 5.8%, mean + s.d.). When sperm concentration was low, A. florida eggs showed an increased rate of heterospecific fertilization (figure 1, paired t-test, p , 0.05, t ¼ 22.93;
2
Biol. Lett. 12: 20160511
2. Material and methods
Table 1. Spawning date and time of the coral Acropora florida and Acropora intermedia in 2015.
rsbl.royalsocietypublishing.org
conditions [7]. In this manner, the risk of hybridization between synchronously spawning species decreases over time, but the gamete-recognition mechanisms of several species still allow some degree of hybridization with other species. The incidence of intercrossing between different species of broadcast-spawning coral remains a profound mystery. The gametes of several species of the reef-building coral genus Acropora are compatible (e.g. [8– 10]) and therefore synchronous spawning among such species might lead to hybridization in nature [8,11]. The presence of hybrids of Indo-pacific Acropora species is suspected [5]; experimentally derived hybrids of compatible species are fecund [12]. In light of this fact, it would seem likely that hybrids would reproduce and become abundant in nature, but there is no evidence that this is the case. Hybridization does not occur in the presence of conspecifics or occur conditionally. Sperm choice during fertilization events is likely to be related to prezygotic reproductive isolation. In a heterospecific mixture of gametes, sperm of both Caribbean and Indo-Pacific Acropora spp. preferentially fertilize the eggs of conspecifics [8,13]. However, the presence of hybrids, the Caribbean A. plorifera, indicates that fertilization by heterospecifics does sometimes occur. Moreover, hybrid larvae survive at the same rate as do non-hybrids [14]. These findings suggest that the conditions under which hybridization might occur in nature are conditional. In this study, we investigated the effects of sperm concentration and sperm preference on interspecies fertilization leading to hybridization in the reef-building coral Acropora. Acropora is vulnerable to environmental changes and experiences population fluctuations due to bleaching events, disease and so on [15]. Mass bleaching events have increased in frequency, and Acropora populations are becoming more isolated and are also likely to have been decreasing. When stressed, or under low population size the reproductive success of Acropora can be impacted, particularly where there is a limited amount of sperm available [10,16]. The relationship between sperm concentration and sperm preference was examined.
0.8
0.6
0.4
0 low optimal low optimal (106 cells ml–1) (104 cells ml–1) (106 cells ml–1) (104 cells ml–1)
A. florida eggs
A. intermedia eggs
Figure 1. Crossing rates in sperm-choice experiments. Sperm-choice fertilization trials were carried out in five combinations of A. florida or A. intermedia eggs (n ¼ 5 in each trial). In the optimal sperm concentration conditions, 0.5 106 cells ml21 of each of the heterospecific and conspecific sperm were added to egg suspensions to make a final concentration of 106 cells ml21. In low sperm concentrations, 0.5 104 cells ml21 of each sperm type were added to the suspension, making a final concentration of 104 cells ml21. A paired t-test was carried out to determine the significance of differences between optimal and low sperm concentrations. electronic supplementary material, figure S2, total fertilization rates of A. florida eggs: 71.8 + 19.8%, n ¼ 5), but A. intermedia eggs did not (figure 1; electronic supplementary material, figure S2; total fertilization rates of A. intermedia eggs: 93.4 + 6.8%, n ¼ 5).
Ethics. All procedures for coral treatment were approved by the University of the Ryukyus Animal Care Board, project number 28-21.
4. Discussion
Data accessibility. The data for this project are available in Dryad http://
This study indicates that hybridization between broadcastspawning species of Acropora might occur when low population numbers result in diminished sperm concentrations during fertilization events. When sperm concentration was low, heterospecific fertilization occurred (about 15–50%; electronic supplementary material, figure S1) between sperm of A. intermedia and eggs of A. florida, which releases gametes 20– 30 min earlier than A. intermedia. When Acropora
Authors’ contributions. S.K. and M.M. designed the study. S.K., H.F., N.I.,
dx.doi.org/10.5061/dryad.v8s04 [25]. K.I. and M.M carried out the experiment, and the statistical analyses. All authors contributed to writing the manuscript and have given final approval for publication. All the authors agree to be held accountable for the content.
Competing interests. We have no competing interests. Funding. This study was partly supported by Japan Society for the Promotion of Science (#26440225).
Acknowledgement. We thank two reviewers for helpful comments.
References 1.
2.
3.
Levitan DR, Petersen C. 1995 Sperm limitation in the sea. Trends Ecol. Evol. 10, 228 –231. (doi:10. 1016/S0169-5347(00)89071-0) Yund PO. 2000 How severe is sperm limitation in natural populations of marine free-spawners? Trends Ecol. Evol. 15, 10 –13. (doi:10.1016/S01695347(99)01744-9) Babcock R. 1995 Synchronous multispecific spawning on coral reefs: potential for hybridization and roles of gamete recognition. Reprod. Fertil. Dev. 7, 943–950. (doi:10.1071/RD9950943)
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Babcock RC, Bull GD, Harrison PL, Heyward AJ, Oliver JK, Wallace CC, Willis BL. 1986 Synchronous spawnings of 105 scleractinian coral species on the Great Barrier Reef. Mar. Biol. 90, 379–394. (doi:10. 1007/BF00428562) Richards ZT, Hobbs JPA. 2015 Hybridisation on coral reefs and the conservation of evolutionary novelty. Curr. Zool. 61, 132 –145. (doi:10.1093/czoolo/61. 1.132) Vacquier VD, Swanson WJ. 2011 Selection in the rapid evolution of gamete recognition proteins in
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marine invertebrates. Cold Spring Harb. Perspect. Biol. 3, a002931. (doi:10.1101/cshperspect. a002931) Levitan DR. 2012 Contemporary evolution of sea urchin gamete-recognition proteins: experimental evidence of density-dependent gamete performance predicts shifts in allele frequencies over time. Evolution 66, 1722– 1736. (doi:10.1111/j.15585646.2012.01608.x) Fogarty ND, Vollmer SV, Levitan DR. 2012 Weak prezygotic isolating mechanisms in threatened
3
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0.2
populations decreased in size, the number of gametes released decreased correspondingly, causing sperm concentration during spawning events to be insufficient to achieve high rates of fertilization [10]. Moreover, sperm are rapidly diluted after spawning [19], causing sperm limitation even when population numbers are high. Unfertilized eggs represent a wasted resource; being fertilized by the later-spawning species A. intermedia may thus be a preferable alternative for A. florida. Breeding with heterospecific sperm is likely to be a strategy through which A. florida avoids wasting resources on producing eggs that remain unfertilized. Hybridization often results in sterile progeny, but hybrids from A. florida eggs and A. intermedia sperm are in fact fertile and produce offspring [12]. In general, hybrid species exhibit decreased rates of sexual reproduction because of low fecundity and the difficulty of mating with other hybrids (e.g. [20]). Indeed, hybrids of Caribbean Acropora spp. seem to have low fecundity, but genetic data suggest that they may backcross with parent species [8,21,22]. However, F1 hybrids of Indo-Pacific A. florida eggs and A. intermedia sperm display almost normal fecundity of mother species [12]. Crosses produced by A. intermedia eggs and A. florida sperm exhibit low fecundity [12], and we showed that this type of hybridization did not occur with either optimal or low sperm concentrations. It seems A. florida may have retained the ability to be fertilized by heterospecific sperm because hybrids produced by such crossings are highly fertile. Hybridization is a mode of speciation in Acropora and other corals (e.g. [5,23,24]). The extent of natural hybridization is not currently understood in diverse Indo-Pacific coral communities; further fine-scale phylogenetic studies that integrate studies of reproductive biology should shed more light on this issue.
rsbl.royalsocietypublishing.org
preference rate of heterospecific sperm
p < 0.05
10.
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15.
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18.
19. Omori M, Fukami H, Kobinata H, Hatta M. 2001 Significant drop of fertilization of Acropora corals in 1999: an after-effect of heavy coral bleaching? Limnol. Oceanogr. 46, 704 –706. (doi:10.4319/ lo.2001.46.3.0704) 20. Mallet J. 2007 Hybrid speciation. Nature 446, 279–283. (doi:10.1038/nature05706) 21. Vollmer SV, Palumbi SR. 2002 Hybridization and the evolution of reef coral diversity. Science 296, 2023– 2025. (doi:10.1126/science.1069524) 22. Palumbi SR, Vollmer S, Romano S, Oliver T, Ladner J. 2012 The role of genes in understanding the evolutionary ecology of reef building corals. Evol. Ecol. 26, 317– 335. (doi:10.1007/S10682-0119517-3) 23. Veron JEN. 1995 Corals in space and time. London, UK: Comstock/Cornell. 24. Kenyon JC. 1997 Models of reticulate evolution in the coral genus Acropora based on chromosome numbers: parallels with plants. Evolution 51, 756–767. (doi:10.2307/2411152) 25. Kitanobo S, Isomura N, Fukami H, Iwao K, Morita M. 2016 Data from: The reef-building coral Acropora conditionally hybridize under sperm limitation. Dryad Data Repository. (doi:10.5061/ dryad.v8s04)
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37, 489– 517. (doi:10.1146/Annurev.Ecolsys.37. 091305.110136) Isomura N, Iwao K, Fukami H. 2013 Possible natural hybridization of two morphologically distinct species of Acropora (Cnidaria, Scleractinia) in the Pacific: fertilization and larval survival rates. PLoS ONE 8, e56701. (doi:10.1371/journal.pone.0056701) Haapkyla J, Melbourne-Thomas J, Flavell M, Willis BL. 2013 Disease outbreaks, bleaching and a cyclone drive changes in coral assemblages on an inshore reef of the Great Barrier Reef. Coral Reefs 32, 815 –824. (doi:10.1007/s00338-013-1029-x) Nozawa Y, Isomura N, Fukami H. 2015 Influence of sperm dilution and gamete contact time on the fertilization rate of scleractinian corals. Coral Reefs 34, 1199–1206. (doi:10.1007/s00338-015-1338-3) Oliver J, Babcock R. 1992 Aspects of the fertilization ecology of broadcast spawning corals: sperm dilution effects and in situ measurements of fertilization. Biol. Bull. 183, 409–417. (doi:10.2307/1542017) Shinzato C, Yasuoka Y, Mungpakdee S, Arakaki N, Fujie M, Nakajima Y, Satoh N. 2014 Development of novel, cross-species microsatellite markers for Acropora corals using next-generation sequencing technology. Front. Mar. Sci. 1, 11. (doi:10.3389/ fmars.2014.00011)
rsbl.royalsocietypublishing.org
9.
Caribbean Acropora corals. PLoS ONE 7, e30486. (doi:10.1371/journal.pone.0030486) Hatta M, Fukami H, Wang W, Omori M, Shimoike K, Hayashibara T, Ina Y, Sugiyama T. 1999 Reproductive and genetic evidence for a reticulate evolutionary history of mass-spawning corals. Mol. Biol. Evol 16, 1607–1613. (doi:10.1093/oxfordjournals.molbev. a026073) Willis BL, Babcock RC, Harrison PL, Wallace CC. 1997 Experimental hybridization and breeding incompatibilities within the mating systems of mass spawning reef corals. Coral Reefs 16, S53–S65. (doi:10.1007/s003380050242) Van Oppen MJ, Willis BL, Van Rheede T, Miller DJ. 2002 Spawning times, reproductive compatibilities and genetic structuring in the Acropora aspera group: evidence for natural hybridization and semipermeable species boundaries in corals. Mol. Ecol. 11, 1363–1376. (doi:10.1046/j.1365-294X.2002.01527.x) Isomura N, Iwao K, Morita M, Fukami H. In press. Spawning and fertility of F1 hybrids of the coral genus Acropora in the Indo-Pacific. Coral Reefs. (doi:10.1007/s00338-016-1461-9) Willis BL, van Oppen MJH, Miller DJ, Vollmer SV, Ayre DJ. 2006 The role of hybridization in the evolution of reef corals. Annu. Rev. Ecol. Evol. System
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The reef-building coral Acropora conditionally hybridize under sperm limitation
Research
Seiya Kitanobo1, Naoko Isomura2, Hironobu Fukami3, Kenji Iwao4 and Masaya Morita1
Cite this article: Kitanobo S, Isomura N, Fukami H, Iwao K, Morita M. 2016 The reefbuilding coral Acropora conditionally hybridize under sperm limitation. Biol. Lett. 12: 20160511. http://dx.doi.org/10.1098/rsbl.2016.0511
1
Sesoko Station, Tropical Biosphere Research Center, University of the Ryukyus, Sesoko, Motobu, Okinawa 905-0227, Japan 2 Department of Bioresources Engineering, Okinawa National College of Technology, 905 Henoko, Nago-City, Okinawa 905-2192, Japan 3 Faculty of Agriculture, Department of Marine Biology and Environmental Science, University of Miyazaki, Miyazaki, Japan 4 Akajima Marine Science Laboratory, Zamami, Okinawa 901-3311, Japan MM, 0000-0002-0170-1226
Received: 15 June 2016 Accepted: 28 July 2016
Subject Areas: evolution, ecology, developmental biology Keywords: hybridization, fertilization, coral, broadcast spawning, sperm limitation
Author for correspondence: Masaya Morita e-mail: [email protected]
Multi-specific synchronous spawning risks both sperm limitation, which reduces fertilization success, and hybridization with other species. If available sperm of conspecifics are limited, hybridization with heterospecific sperm could be an alternative. Some species of the reef-building coral Acropora produce hybrid offspring in vitro, and therefore hybridization between such species does sometimes occur in nature. Here, we report that the interbreeding species Acropora florida and A. intermedia preferentially bred with conspecifics at optimal gamete concentrations (106 cells ml21), but when sperm concentration was low (104 cells ml21), A. florida eggs displayed an increased incidence of fertilization by sperm of A. intermedia. However, A. intermedia eggs never crossed with heterospecific sperm, regardless of gamete concentrations. It appears that A. florida eggs conditionally hybridize with heterospecific sperm; in nature, this would allow A. florida to cross with later-spawning species such as A. intermedia. These results indicate that hybridization between some Acropora species could occur in nature according to the number of available sperm, and the choice of heterospecific sperm for fertilization could be one of the fertilization strategies in the sperm-limited condition.
1. Introduction
Electronic supplementary material is available at http://dx.doi.org/10.1098/rsbl.2016.0511 or via http://rsbl.royalsocietypublishing.org.
Low sperm densities during fertilization events, named ‘sperm limitation’, is a factor that can reduce the reproductive success of broadcast-spawning invertebrates [1,2], and thus many invertebrates spawn synchronously to increase fertilization success [3,4]. However, synchronous spawning of multiple species could increase the risk of interspecific hybridization [3]. Hybridization is considered an evolutionary dead end because it often results in progeny that are less fit or sterile, but alternatively it can result in hybrid vigour and increased adaptive potential [5]. To ensure that gametes are only fertilized by conspecifics even in the presence of heterospecifics, broadcast-spawning organisms have evolved gamete recognition. Many gamete-recognition proteins evolved rapidly (ratio of substitution rates at non-synonymous and synonymous sites: dN/dS . 1), ensuring the similarly rapid development of strict gamete recognition [6]. Genes coding for gamete-recognition proteins with distinctive features related to fertilization conditions (such as sperm concentration) also change over a relatively short time period, reflecting changes in fertilization
& 2016 The Author(s) Published by the Royal Society. All rights reserved.
species
A. florida
A. intermedia
spawning date and spawning time
3 June 2015 21.30 (two colonies)
3 June 2015 21.51 (one
a
f1, 21.38 (one colony)
colony) 22.01 (one
a
colony) ai35
f13
21.39 (two colonies) a f26, f11
22.09 (two colonies) a
i17, ai18 22.12 (two colonies) ai24 a
Colonies used in this study for fertilization experiments.
by counting the number of cells showing cell division. Larvae (2 days post fertilization) were fixed with 99.5% EtOH and stored at 2208C until DNA extraction could be performed.
(c) Sperm-choice test Paternity was determined using Acropora microsatellite marker 11745m3 ([18], electronic supplementary material, figure S1b). DNA was extracted from larvae as follows: fixed larvae were treated with 20 ml lysis buffer (1 mg ml21 proteinase K, 100 mM NaCl, 0.5% SDS, 10 mM EDTA and Tris-HCl pH 8.0) at 558C for 3 h, and then incubated at 988C for 5 min to terminate the reaction. The resultant suspensions were used as templates for PCR. PCR was performed using GoTaq green mix (5.0 ml GoTaq, 3.6 ml water, 0.6 ml 10 mM microsatellite marker 11745m3 [18] and 0.2 ml template). The PCR protocol of Shinzato et al. [18] was used, with some modifications. The protocol was performed for 2 min at 958C, followed by 35 cycles of (948C for 30 s, 588C for 90 s and 728C for 1 min), and a final elongation at 608C for 30 min. Amplicons were run on a 12% TAE (40 mM Tris-acetate ( pH 8.0) 1 mM EDTA) acrylamide gel (106 100 mm) at 150 V and 20 mA for 5 h. The gel was stained with GelRed. The expected ratio of fertilization if there were no preference between conspecifics and heterospecifics was calculated as follows: number of hybrids/(number of fertilized eggs/2).
(a) Coral species We used the coral species A. florida and A. intermedia, whose gametes are capable of bidirectional intercrossing [14]. Four colonies of each species were collected from Akajima Island (268120 N, 1278170 E) and maintained in aquaria until they spawned. Gamete bundles were collected and used in fertilization experiments: (i) sperm no-choice experiments and (ii) sperm-choice experiments.
(b) Fertilization experiments Fertilization experiments were conducted with sperm choice and no-choice conditions. In no-choice experiments, crossings between conspecifics or heterospecifics were performed with optimal sperm concentrations (106 cells ml21) [17]. In spermchoice experiments (electronic supplementary material, figure S1a), eggs were exposed to a mixture of sperm from conspecifics and heterospecifics at optimal (106 cell ml21) [17] or low sperm concentrations (104 cells ml21). The concentrations of conspecific and heterospecific sperm were equal (0.5 106 cells ml21 and 0.5 104 cells ml21 in high and low conditions, respectively). Fertilization rates in each condition were determined after 2 h
3. Results Spawning of the coral species A. florida and A. intermedia occurred on the same date but at different times; A. florida spawned 20 to 30 min before A. intermedia (table 1). The rates of crossings between heterospecifics varied (A. florida eggs A. intermedia sperm: 36.5 + 25.9%, A. intermedia eggs A. florida sperm: 34.7 + 30.1%, mean + s.d.; electronic supplementary material, table S1) but were uniformly high between conspecifics (A. florida eggs: 96.0 + 4.3%, A. intermedia: 95.9 + 4.3%, mean + s.d.; electronic supplementary material, table S1). In sperm-choice experiments, eggs of both species were primarily fertilized by conspecific sperm under optimal sperm concentration (figure 1; electronic supplementary material, figures S1b and S2, total fertilization rate of A. florida eggs: 94.6 + 4.7%, A. intermedia eggs: 95.0 + 5.8%, mean + s.d.). When sperm concentration was low, A. florida eggs showed an increased rate of heterospecific fertilization (figure 1, paired t-test, p , 0.05, t ¼ 22.93;
2
Biol. Lett. 12: 20160511
2. Material and methods
Table 1. Spawning date and time of the coral Acropora florida and Acropora intermedia in 2015.
rsbl.royalsocietypublishing.org
conditions [7]. In this manner, the risk of hybridization between synchronously spawning species decreases over time, but the gamete-recognition mechanisms of several species still allow some degree of hybridization with other species. The incidence of intercrossing between different species of broadcast-spawning coral remains a profound mystery. The gametes of several species of the reef-building coral genus Acropora are compatible (e.g. [8– 10]) and therefore synchronous spawning among such species might lead to hybridization in nature [8,11]. The presence of hybrids of Indo-pacific Acropora species is suspected [5]; experimentally derived hybrids of compatible species are fecund [12]. In light of this fact, it would seem likely that hybrids would reproduce and become abundant in nature, but there is no evidence that this is the case. Hybridization does not occur in the presence of conspecifics or occur conditionally. Sperm choice during fertilization events is likely to be related to prezygotic reproductive isolation. In a heterospecific mixture of gametes, sperm of both Caribbean and Indo-Pacific Acropora spp. preferentially fertilize the eggs of conspecifics [8,13]. However, the presence of hybrids, the Caribbean A. plorifera, indicates that fertilization by heterospecifics does sometimes occur. Moreover, hybrid larvae survive at the same rate as do non-hybrids [14]. These findings suggest that the conditions under which hybridization might occur in nature are conditional. In this study, we investigated the effects of sperm concentration and sperm preference on interspecies fertilization leading to hybridization in the reef-building coral Acropora. Acropora is vulnerable to environmental changes and experiences population fluctuations due to bleaching events, disease and so on [15]. Mass bleaching events have increased in frequency, and Acropora populations are becoming more isolated and are also likely to have been decreasing. When stressed, or under low population size the reproductive success of Acropora can be impacted, particularly where there is a limited amount of sperm available [10,16]. The relationship between sperm concentration and sperm preference was examined.
0.8
0.6
0.4
0 low optimal low optimal (106 cells ml–1) (104 cells ml–1) (106 cells ml–1) (104 cells ml–1)
A. florida eggs
A. intermedia eggs
Figure 1. Crossing rates in sperm-choice experiments. Sperm-choice fertilization trials were carried out in five combinations of A. florida or A. intermedia eggs (n ¼ 5 in each trial). In the optimal sperm concentration conditions, 0.5 106 cells ml21 of each of the heterospecific and conspecific sperm were added to egg suspensions to make a final concentration of 106 cells ml21. In low sperm concentrations, 0.5 104 cells ml21 of each sperm type were added to the suspension, making a final concentration of 104 cells ml21. A paired t-test was carried out to determine the significance of differences between optimal and low sperm concentrations. electronic supplementary material, figure S2, total fertilization rates of A. florida eggs: 71.8 + 19.8%, n ¼ 5), but A. intermedia eggs did not (figure 1; electronic supplementary material, figure S2; total fertilization rates of A. intermedia eggs: 93.4 + 6.8%, n ¼ 5).
Ethics. All procedures for coral treatment were approved by the University of the Ryukyus Animal Care Board, project number 28-21.
4. Discussion
Data accessibility. The data for this project are available in Dryad http://
This study indicates that hybridization between broadcastspawning species of Acropora might occur when low population numbers result in diminished sperm concentrations during fertilization events. When sperm concentration was low, heterospecific fertilization occurred (about 15–50%; electronic supplementary material, figure S1) between sperm of A. intermedia and eggs of A. florida, which releases gametes 20– 30 min earlier than A. intermedia. When Acropora
Authors’ contributions. S.K. and M.M. designed the study. S.K., H.F., N.I.,
dx.doi.org/10.5061/dryad.v8s04 [25]. K.I. and M.M carried out the experiment, and the statistical analyses. All authors contributed to writing the manuscript and have given final approval for publication. All the authors agree to be held accountable for the content.
Competing interests. We have no competing interests. Funding. This study was partly supported by Japan Society for the Promotion of Science (#26440225).
Acknowledgement. We thank two reviewers for helpful comments.
References 1.
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populations decreased in size, the number of gametes released decreased correspondingly, causing sperm concentration during spawning events to be insufficient to achieve high rates of fertilization [10]. Moreover, sperm are rapidly diluted after spawning [19], causing sperm limitation even when population numbers are high. Unfertilized eggs represent a wasted resource; being fertilized by the later-spawning species A. intermedia may thus be a preferable alternative for A. florida. Breeding with heterospecific sperm is likely to be a strategy through which A. florida avoids wasting resources on producing eggs that remain unfertilized. Hybridization often results in sterile progeny, but hybrids from A. florida eggs and A. intermedia sperm are in fact fertile and produce offspring [12]. In general, hybrid species exhibit decreased rates of sexual reproduction because of low fecundity and the difficulty of mating with other hybrids (e.g. [20]). Indeed, hybrids of Caribbean Acropora spp. seem to have low fecundity, but genetic data suggest that they may backcross with parent species [8,21,22]. However, F1 hybrids of Indo-Pacific A. florida eggs and A. intermedia sperm display almost normal fecundity of mother species [12]. Crosses produced by A. intermedia eggs and A. florida sperm exhibit low fecundity [12], and we showed that this type of hybridization did not occur with either optimal or low sperm concentrations. It seems A. florida may have retained the ability to be fertilized by heterospecific sperm because hybrids produced by such crossings are highly fertile. Hybridization is a mode of speciation in Acropora and other corals (e.g. [5,23,24]). The extent of natural hybridization is not currently understood in diverse Indo-Pacific coral communities; further fine-scale phylogenetic studies that integrate studies of reproductive biology should shed more light on this issue.
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