Assortative fertilization in Chlamys farreri and Patinopecten yessoensis and its implication in scallop hybridization.

ABSTRACT Assortative mating and reduced hybrid fitness are typically invoked to explain the stability of hybrid zones and the specification of two closely related taxa. Such examples of mate preferences in nature, especially for broadcast free- spawning marine invertebrates, and the understanding of the underlying mechanisms responsible for this positive selection are still lacking. This study documents another example of assortative fertilization by genome in situ hybridization in reciprocal crosses of scallops Chlamys farreri and Patinopecten yessoensis. Females produced eggs selected sperm nonrandomly but showed significant gamete preference of conspecifics over heterospecifics, with Chlamys farrei females using average 89.1% of conspecific sperms and the Patinopecten yessoensis using average 94.3% in natural spawning conditions (temperature 17[degrees]C, salinity 31.5 [per thousand]). This conspecific gamete advantage may be related to the coordinated communication between interacting gametes, and the conspecific bindin genotype selection during the communicational process may also be involved. Two environmental factors, temperature and salinity were also tested to determinate to what extent they affect the patterns of assortative fertilization between these two species. The result showed that temperature seemed to have more influence on assortative fertilization than salinity did. At the temperatures of 11[degrees]C, 17[degrees]C, 22[degrees]C and 26[degrees]C, the average conspecific sperms preference of Chlamys farreri eggs was 80.0%, 89.1%, 95.1% and 95.5% respectively, whereas at the salinity of 24 [per thousand], 28 [per thousand], 31.5 [per thousand] and 35.5 [per thousand], the average mating preference was 97.2%, 91.2%, 89.1% and 99.5% respectively. These results may have some bearings on the condition dependent alteration in the inter gamete recognition system. Alternatively, it may be simply the byproduct of different sperm tolerance ability under different conditions. The implication it may provide to hybrid production was also discussed.

KEY WORDS: scallop, assortative fertilization, Chlamys farreri, Patinopecten yessoensis

INTRODUCTION

Many theories have been developed to interpret the formation of reproductive isolation and hence the speciation process between two sympatric closely related taxa in recently years. (Palumbi & Metz 1991, Metz et al. 1994, McCartney et al. 2000, Knowlton 1993, Coyne 1992) These theories generally fall into two categories: pre- and post-zygotic barriers, Pre-zygotic barriers include mechanisms such as mate choice, genital incompatibility, spatial and temporal habitat differences and gamete incompatibility. Postzygotic barriers, on the other hand, include mechanisms such as embryo inviability and hybrid sterility (Dobzhansky et al. 1977, Templeton 1989, Lessios 1998). Whereas, assortative fertilization or gamete preference, which is commonly regarded as a post-mating, pre-zygotic isolating mechanism, has only recently come to prominence and is increasingly accepted as another important possible barrier to hybridization and gene flow between closely related species (Bierne et al. 2002, Geyer & Palumbi 2005).

Assortative fertilization is a subtype of fertilization barrier that depends upon characteristics of the female and male such that gametes from like or unlike parents have a greater or lesser than random chance of uniting (Markow 1997). Evidence of such kind of gamete preference has been recorded in many terrestrial species such as Drosophila (Chang 2004, Markow 1997), crickets (Howard & Gregory 1993, Howard et al. 2002) and flour beetles (Robinson et al. 1994, Wade et al. 1995). These studies indicated without exception that conspecific sperm precedence, or biased use of sperm from males of one species might occur when a female was exposed to sperm from males of multiple species. Possible mechanism underlying was also highly varied. In some cases, it involves compatibility of genitalia or gamete morphology. For example, in Rhododendron, overgrowth or undergrowth of heterospecific pollen tubes yields few hybrid embryos (Williams & Rouse 1990). In other cases, it seems to be related to functional or physiological interactions between sperms and the female reproductive tract (Markow 1997). In ladybird beetles heterospecific sperm are inactivated in storage in female reproductive tract (Katakura 1986, 1997). In Drosophila conspecific sperm just incapacitates or displaces heterospecific sperm during the storage. But for the broadcast tree-spawning marine invertebrate, in which courtship and other complex mating behaviors that facilitate mate choice are generally non-existent, the opportunity for conspecific sperm preference and the possible mechanism has been largely ignored. To date, only few marine invertebrates have been studied for such gamete preference (Bierne et al. 2002, Palumbi 1999, Geyer & Palumbi 2005, Yung & McCartney 1994), and the possible mechanism seemed even more complicated than that of terrestrial species because of the smaller arena, the surface of gametes and the seawater between them, in which the assortative fertilization could take place. Here we present another example of assortative fertilization in external fertilization species of marine bivalves.

Hybridization between scallops Chlamys farreri and Patinopecten yessoensis, both of which are economically important shellfish species along the coast of China and Japan, has been intensively studied in recent years with the aim of improving Chlamys farreri germ quality from high rate of mortality and disease susceptibility (Yang et al. 2004, Zhou et al. 2005). No apparent gametic incompatibility was found in crosses of both directions between these two species either in small-scale laboratory experiment or in larger scale breeding program (Yang et al. 2004). The putative hybrid was believed to be able to live successfully through planktonic larval stages and grow into full adults with heterosis in growth and disease resistance (Yang et al. 2004), but in some cases of hybridization, contamination was detected by genetic examination of the progeny despite much care being taken in the hybridization process (Lu et al. in press). The same phenomenon was found in the hybridization of many oyster species of Crassostrea (Allen & Gaffney 1993, Allen et al. 1993). Sometimes the contamination rate is comparatively high given the fact that the concentration of contaminated sperm (if exists) was much lower than that of heterspecific sperm added in hybridization. We suspect assortative fertilization may be responsible for this problem. So The aim of this study was to investigate (1) if there also exists assortative mating phenomenon between species of scallops as found in other marine invertebrates; (2) to what degree the presence of conspecific sperms affect the cross-fertilization success of interspecific sperm and (3) to what extent this assortative fertilization can be affected by environmental factors, such as temperature and salinity, so that measures may be taken to improve the scallop hybridization program.

MATERIALS AND METHODS

Scallops and Gametes

Chlamys farreri were collected from the Jiaonan area of Qingdao and Patinopecten yessoensis were obtained from the Dalian coast of Bohai Sea in early April 2005. Both species were transported to the laboratory and temperature and feeding were adjusted to synchronize maturation among species. All gametes were obtained by induced spawning with drying and thermal shock. Oocytes from individual females were gently sieved and placed into separate beakers after the quality was checked by light microscope and no fertilization was confirmed, counted using a Coulter cell counter and adjusted to 1 x [10.sup.6] per female. Sperms from individual C. farreri and P. yessoensis were placed into separate beakers. Same treatment was conducted and adjusted to 1 x [l0.sup.8] per male.

Experimental Design

Four gamete mixtures were produced in the experiment: A mix of oocytes from 20 female C. farreri with a finally total number of 2 x [10.sup.7], a mix of oocytes from 20 female P. vessoensis with a finally total number of 2 x [10.sup.7], a mix of spermatozoa from 20 male C. farreri with a finally total number of 8 x [10.sup.8] and a mix of spermatozoa from 20 male P. yessoensis with a finally total number of 8 x [l0.sup.8]. All the mixtures were then further divided into equal amount of portions, each used for one cross. This guarantees almost equal amount of gametes used in each cross and obtain a 40:1 sperm: egg ratio for the fertilization. The six crosses were conducted at temperature 17[degrees] and salinity 31.5 [per thousand] (natural spawning condition): C. farreri ([female]) x C. farreri ([male]); C. farreri ([female]) x P. yessoensis ([male]); P. yessoensis ([female]) x P. yessoensis ([male]); P. vessoensis ([female]) x C. farreri([male]), C. farreri([female]) x [P. yessoensis ([male]) + C. farreri ([male]) 1; P. yessoensis ([female]) x [P. yessoensis ([male]) + C. farreri ([male])]. The first four crosses were used as positive controls and the later two allowed for interspecies sperm competition with equal amount of sperm from both species added. Another six sperm competition crosses were conducted the same way with the C. farreri ([female]) x [P. yessoensis ([male]) + C. farreri ([male])| under 3 levels of temperature (11[degrees]C, 22[degrees]C and 26[degrees]C) at the salinity 31.5 [per thousand], and 3 levels of salinity (20 [per thousand], 28 [per thousand] and 35.5 [per thousand] at the temperature 17[degrees]C. The temperature was maintained by using a bio-chemical incubator (LI5-2, SHELLAB) and the salinity was maintained by just adding fresh water or crude salt. All the crosses were performed in four replicates and conducted simultaneously.

Larval Development and Genetic Confirmation

Fertilization was allowed to proceed for a minimum of 50 min in each cross before all the eggs were transferred to a culture tank with a density of 30 eggs/mL. Normal culturing procedure was followed afterwards. Fertilization success was determined by examining at least 200 oocytes by light microscope at 60-90 min post-insemiantion. Two replicate subsamples were collected from each duplicate of the crosses for each sampling. The larvae were collected after they reached early trochophore stage and the hatching rate was also determined.

The metaphase chromosome was prepared following the procedure described by Wang et al. (1990). In brief, the embryos were exposed to 25% sea water for about 50 min after 40 min of culture in 0.04% colchicines at room temperature, then they were fixed with Carnoy fixatives (ethanol: glacial acetic acid = 3:1), and stored at -20[degrees]C until use. The fixed embryos were dissociated into fine pieces by pipetting in 50% acetic acid in a 1.5 mL microcentrifuge tube. The resultant cell suspension was dropped onto hot-dry glass slides and air-dried. Chromosome preparations were preserved in a moist chamber until use.

The GISH (genome in situ hybridization) procedure followed the protocol described by Leitch et al. (1994) and Takahashi et al. (1997) with some modifications. Briefly, chromosome slides were denatured in a mixture containing 75% formamide and 2 x SSC for 2~3 min at 72[degrees]C, dehydrated through an ice-cold ethanol gradient (70%, 90% and 100%), 5 min each, and air-dried. Genomic probe mixture was denatured for 5 min at 80[degrees]C, followed by immediately being put on ice for at least 10 min. The probe hybridization mix was applied to the slides and in situ hybridization was carried out at 37[degrees]C for 14~18 hr. The slides were washed twice in 2 x SSC, and 50% formamide at 42[degrees]C for 1 0 min, 1 x SSC at 42[degrees]C for 10 min and finally in 2 x SSC at room temperature for 10 min after the hybridization. Biotinylated probes were detected with fluorescein isothiocyanate conjugated avidin DCS (FITC) (VECTOR) for 1h at 37[degrees]C, counterstained with Vectashield mounting medium with propidium iodide (PI) (VECTOR) for 40 min at 37[degrees]C. Hybridization signals were detected by using a Nikon E-800 microscope equipped with the appropriate filter sets for FITC and PI.

Statistical Analysis

The percentage of hybrid larvae was determined by counting the hybrid rate in at least 200 metaphase chromosomes displayed by GISH. Two replicate subsamples were collected from each duplicate of the crosses for each sampling. These percentages were compared with random expectation by a [chi square] test of the observed number of hybrid offspring versus the number expected, if sperm use was random. The expectations were based on the measured concentrations of sperm used in each cross and the average larval hatching rate of each control. It was calculated as EP = S1 x HI/(S1 + S2) x H2, where SI and S2 are the respective sperm concentrations, and H1 and H2 are the respective average larvae hatching rates. Hybrid probabilities were calculated according to Palumbi (1999) as SR/(SR + 1), where SR (L1/L2) x (S2/S1). L1 and L2 are the number of larvae sired by heterospecific and conspecific males and S1 and S2 are the respective sperm concentrations. The percentage of hybrid larvae from each sperm competitive cross in changed conditions was also compared by a [chi square] test with that of in natural spawning conditions.

RESULTS

Fertilization and Genetic Confirmation in Control Crosses

In this part, four independent crosses with replicates were performed as positive control. Almost no difference in fertilization and the larval hatching rates was found among the crosses of C. farreri ([female]) x C. farreri ([male]); C. farreri ([female]) x P. yesonensis ([male]); P. yesonensis ([female]) x P. yesonensis ([male]) and P. yesonensis ([female]) x C. farreri ([male]). All crosses showed high levels of fertilization rates and larval hatching rates (Table 1). This is in agreement with what found by Yang in 2004. Genetic confirmation by GISH showed that all offspring from crosses of C. farreri ([female]) x P. yessoensis ([male]) and P. yessoensis ([female]) x C. ([male]) were hybrid (Fig. 1b). No evident gamete incompatibility was found in these two species.

[FIGURE 1 OMITTED]

Fertilization and Genetic Confirmation In Sperm Competitive Crosses

High fertilization and the larval hatching rates were also found in the two crosses of C. farreri ([female]) x [P. yesonensis ([male]) + C. farreri ([male])l and P. yesonensis ([female]) x [P. yesonensis ([male]) + C. farreri ([male])] at control condition (Table 1). But genetic confirmation by GISH showed that the hybrid rates of the offspring from these crosses departed significantly from equal species contribution (P < 0.005) when sperm choice was available. The average hybrid rates were found to be 10.9% in farreri ([female]) x [P. yesonensis ([male]) + C. farreri ([male])] and 5.7% in P. yesonensis ([female]) x [P. yesonensis ([male]) + C. farreri ([male])] (Table 2, Fig. 2). The expected hybrid rates based on the measured concentrations of sperm used in each cross and the mean larval hatching rate of each control were 49.3% and 48.6% respectively if the sperm use was random. The hybridization probability was 0.112 and 0.055 respectively. Females of both C. farreri and P. yessoensis showed strong biased sperm use when their oocytes were exposed to mixed sperms from the two species.

[FIGURE 2 OMITTED]

Environmental Effect of Assortative Fertilization

The fertilization and larval hatching rate in crosses of C. farreri ([female]) x [P. yesonensis ([male]) + C. farreri ([male])] decreased radically when the temperature or the salinity came to extreme with a less than 60% fertilization rate and hatching rate was found at salinity 31.5%c (the data not shown). The hybrid rate in crosses of C. farreri ([female]) x [P. yesonensis ([male]) + C. farreri ([male])] decreased significantly with increasing temperature (Table 3, Fig. 2). At the temperatures of 11[degrees]C, 22[degrees]C and 26[degrees]C, the mean hybrid rates were 20.0%, 4.9% and 4.5% respectively. These departed significantly from the mean hybrid rate obtained in natural spawning condition (P < 0.05). The salinity also showed significant impact on the assortative fertilization in the same manner with the mean hybrid rates of 3.8%, 8.8% and 0.5% at the salinity of 24 [per thousand], 28 [per thousand], and 35.5 [per thousand] (Table 3, Fig. 2). Temperature seemed to have more influence on assortative fertilization than salinity did.

DISCUSSION

Assortative fertilization is believed to be a very important mechanism in explaining barriers to gene flow and hybridization between many sympatric closely related species (Bierne et al. 2002). However, it is not our interest in this study to investigate whether it is also important in the speciation or the reproductive isolation between Chlamys farreri and Patinopecten yessoensis. Because the asynchronous spawning, which alone would suffice to prevent any form of hybridization in nature, has been intensively developed between these two species. The peak spawning time of C. farreri along north coast of China was from early May to mid June at water temperature of 16[degrees] to 22[degrees]C (Wang et al. 1993), whereas P. vessoensis spawned in a colder water temperature of 8[degrees]C to 8.5[degrees]C commonly from late March to mid April (Wang et al. 1993). Instead, this study was designed to test whether this mating preference also exists in C. farreri and P. yessoensis and how much of the heterospecific sperm fertilization success was influenced at the presence of conspecific sperm.

The heterozygote deficiency obtained in these sperm competition crosses in the natural spawning condition may be explained by either (1) assortative fertilization (gamete choice) : (2) differential fertilization (fertilization failure) or abortion. But the lack of heterozygote deficiency in the control crosses favors the assortative fertilization hypothesis because differential fertilization or abortion should have occurred both in control and sperm competition crosses. Moreover, abortion percentages seem insufficient to produce the observed heterozygote deficiency in the sperm competition crosses. Indeed, in the worst case, if we hypothesize that all the aborted embryos in our crosses were hybrids, then the hybrid rates won't reach the rate of expected (P < 0.05) when the sperm use was random. Assortative fertilization must therefore have occurred in C. farreri and P. vesonensis with C. farreri eggs using average 89.1% of conspecific sperms and P. yesonensis eggs using average 94.3% of conspecific sperms in the control condition (temperature 17[degrees]C and salinity 31.5 [per thousand]). These are consistent with those of other marine invertebrates, which have recently been investigated. In sea urchins of Echinometra oblonga and E. sp.C, an average of 87.8% E. sp.C eggs were fertilized by conspecific sperms when exposed to equal amount sperm of both species (Geyer & Palumbi 2005). In the mussels Mytilus edulis and M. galoprovincialis, the total conspecific sperm use of both females was 77% when all four gametes were mixed together (Bierne et al. 2002). Conspecific sperm precedence in free spawning invertebrates shows that the simple surfaces of eggs and sperm provide ample opportunity for egg choice and sperm competition even in the absence of intricate behavior or complex reproductive morphologies.

Mechanistically, to date, only few attempts were made to investigate the mechanism of sperm biased use in free-spawning invertebrates. In sea urchins, Metz et al. (1994) demonstrated that the specificity of fertilization between E. oblonga and E. mathaei is controlled by the interaction of the sperm protein bindin with a receptor on the egg surface. Palumbi (1998, 1999) further elucidated that different bindin alleles confer different fertilization properties in free spawning sea urchin Echinometra mathaei, and fertilization differences reflect male bindin genotype and are under the control of both female and male genotypes. Unfortunately, till now no detailed compositional or functional studies on sperm protein bindin were carried out in scallops, but a similar cell-cell interaction mechanism may, as well, be functioning in the assortative mating system in Chlamys farreri and Patinopecten yessoensi; if this were true, then the fact that the assortative fertilization can be influenced by environmental factors may provide another clue and possibility to further understanding of the mechanism behind the mating preference system. As shown in this study, the mean hybrid rate obtained in these crosses varied from 0.5% to 20.0% as temperature and salinity changed. It may suggest that the egg-sperm recognizing system may be slightly disturbed when fertilization condition changes. What event may take place in this recognizing system when the environmental factors change and what elements on the gamete surface may be involved in this process are now of great interest to us. But alternatively, it may just be the byproduct of different sperm tolerance under different conditions. Because the different sperm tolerance of the two species may influence the practical relative sperm concentration when the sperm competition took place. Whatever it is, assortative fertilization seemed to occur despite the temperature and salinity. Because even the highest mean hybrid rate of 20.0% at the temperature of 11[degrees]C was still likely to be the result of assortative fertilization in consideration of the same concentration of conspecific and heterospecific sperm added. Unfortunately, as no control data of hatching rate was available in each cross under these changed conditions, we can't speculate further.

Although much remains unknown about the underlying mechanism of assortative mating system until now, the phenomenon itself has much implication for our practical hybrid production. In the hybridization of Chlamys farreri and Patinopecten yessoensis, especially in a large scale breeding program, conspecific sperm contamination can cause problems that affect the efficiency of hybridization. The contamination can come from many sources such as; careless handling of fishery tools, sperm from natural seawater via water supply system and a small proportion of hermaphroditic individuals in scallops. In some cases, the heterspecific sperm was added prior to the female spawning with the intention to decrease the chance of self fertilization. But it commonly results in little amelioration. From the present study, we know that the preaddition of heterspecific sperm before spawning won't necessarily decrease the self fertilization rate because of the conspecific sperm precedence. So more care, such as parent scallops selection or more careful handling of fishery tools, should be taken prior to or during the hybridization to minimize the sperm contamination. Another implication we can get from the study is that, because environmental factors can affect the assortative fertilization in the competitive fertilization system, some measurements, such as fertilization at lower temperatures, fertilization in a optimum water salinity and pH value can be taken to increase the hybrid rate in hybridization, if contamination cannot be avoided.

ACKNOWLEDGMENTS

The authors thank Dr. Junda Lin, Florida Institute of Technology, for his critical review on this manuscript and Dr. Huang Xiaoting, Ocean University of China, for her technical assistance in the GISH technique. This work was completed in the Key Laboratory for Sustainable Utilization of Marine Fisheries Resources of Ministry of Agriculture and supported by the High Technology Research and Development Programme of China (2003AA603022) and National Basic Research Program of China (G1999012009)

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ZHENGMING LU, (1,2) AIGUO YANG, (2) QINGYIN WANG, (2) * ZHIHONG LIU (2) AND LIQING ZHOU (2)

* Corresponding author. E-mail: [e-mail]

(1) Life Sciences and Technology College, Ocean University of China, Qingdao, People's Republic of China, 266003; (2) Key Laboratory for Sustainable Utilization of Marine Fisheries Resources, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, People's Republic of China 266071

TABLE 1.
The mean fertilization rates and the
larvae hatching rates ([+ or -]SE) in
six crosses in natural spawning
conditions (temperature 17[degrees]C and
salinity 31.5[per thousand]). C: Chlamys
farreri and P: Patinopecten yessoensis
(n = 8)

Crosses                         Fertilization     Larvae Hatching
                                    Rate                Rate

C([female]) x C([male])       96.5 [+ or -] 2.6   91.2 [+ or -] 1.9
C([female]) x P([male])       95.3 [+ or -] 2.3   90.0 [+ or -] 1.9
P ([female]) x P ([male])     98.0 [+ or -] 1.7   92.0 [+ or -] 1.1
P([female]) x C([male])       96.2 [+ or -] 2.0   89.5 [+ or -] 2.2
C ([female]) x
  [P ([male]) + C ([male])]   97.4 [+ or -] 3.4   90.6 [+ or -] 2.5
P([female]) x                 94.0 [+ or -] 3.1   87.0 [+ or -] 1.6
  [P([male]) + C([male])]

TABLE 2.
The mean hybrid rate ([+ or -] SE) of sperm competition crosses in
natural spawning conditions (temperature 17[degrees]C
and salinity 31.5[per thousand]). The percentage was
compared with random expectation by a [chi square] test of the
observed number of hybrid offspring versus the
number expected if sperm use was random.
Hybridization probability was calculated as SR/(SR + 1),
SR is the average proportion of hybrids observed,
corrected for differences in
sperm concentration. (n = 8)

                                    Hybrid                     [chi
                     Hybrid          Rate     Hybridization  square]
Crosses             Rate (%)       Expected    Probability   Test (P)

C([female])x    10.9[ + or -] 1.6    48.6        0.112       P < 0.005
[P([male])+
C([male])]

P([female])x     5.7 [+ or -] 1.0    47.9        0.055       P < 0.005
[P([male])+
C([male])]

TABLE 3.
The mean hybrid rate ([+ or -]SE) of sperm competition crosses in
different conditions. The percentages were compared with random
expectation by a [chi square] test of the observed number of
hybrid offspring in each condition versus the number observed
in natural spawning condition (n = 8).

Environmental          Level                 Hybrid
Factors                 Set                 Rate (%)

Temperature        11[degrees]C       20.0 [+ or -] 3.2 (b)
                   17[degrees]C       10.9 [+ or -] 1.6 (a)
                   22[degrees]C        4.9 [+ or -] 1.1 (c)
                   26[degrees]C        4.5 [+ or -] 0.9 (c)
Salinity        24.0 [per thousand]    3.8 [+ or -] 1.3 (b)
                28.0 [per thousand]    8.8 [+ or -] 1.7 (a)
                31.5 [per thousand]   10.9 [+ or -] 1.6 (a)
                35.5 [per thousand]    0.5 [+ or -] 0.9 (b)

Means within the same factor, treatments sharing a common
superscript letter in the same column were not significantly
different (P > 0.05) as determined by [chi square] test.