Laboratory hybridization between Crassostrea ariakensis and C. sikamea

Journal of Shellfisheries Research, August, 2009 by Fei Xu, Guofan Zhang, Xiao Liu, Shoudu Zhang, Bin Shi, Ximing Guo

ABSTRACT To understand possible reproductive interaction between Crassostrea ariakensis (Fujita, 1913) and C. sikamea (Amemiya, 1928), which coexist in estuaries of China and Japan, we conducted 2 x 2 factorial crosses between the two species. Asymmetry in fertilization success was observed, where C. sikamea eggs can be fertilized by C. ariakensis sperm, and the reciprocal cross resulted in no fertilization. Fertilization success in C. sikamea female x C. ariakensis male (SA) crosses was lower than that in the two intraspecific crosses and produced larvae that had similar growth rate as their maternal species during the first nine days because of maternal effects. After that, genome incompatibility casted negative effects on the growth and survival of the hybrid larvae. Most hybrid larvae died during metamorphosis, but a small number of spat survived. Genetic analysis revealed that the survived SA spat contained DNA from both species and were true hybrids. This study demonstrates that hybridization between C. ariakensis and C. sikamea is possible in one direction.

KEY WORDS: Crassostrea ariakensis, C. sikamea, oyster, hybridization, reproductive isolation, internal transcribed spacer

INTRODUCTION

Species form because of prolonged reproductive isolation. Hybridization occurs when two species meet and the reproductive isolation is incomplete. Hybridization zones may exist where two closely related species overlap in distribution. Studying reproductive isolation and potential interaction among closely related species is important to our understanding and management of genetic diversity. It may also shed light on mechanisms of reproductive isolation and speciation.

There are at least five Crassostrea oyster species naturally occurring along the coast of China. They include Crassostrea gigas, C. ariakensis, C. sikamea, C. angulata, and C. hongkongensis (Wang et al. 2006). These species often occur in the same estuary. In North China, C. ariakensis coexist with C. gigas. In Central and South China, C. ariakensis, C. hongkongensis, C. angulata and C. sikamea may be found in the same estuary in various combinations. In Nantong, for example, two species, C. ariakensis and C. sikamea, live closely together on the same oyster reef (Guo et al. 2008). Genetic identification using multiple genetic markers failed to detect any hybrids among 578 oysters collected from Nantong (Wang et al. 2008). C. gigas, C. ariakensis, and C. sikamea also coexist in Ariake Sea, Japan, without any detectable hybrids (Hedgecock et al. 1999).

The absence of naturally occurring hybrids between C. ariakensis and C. sikarnea is interesting and raises the question whether hybridization between the two species is possible. Reports on hybridization among Crassostrea oysters appeared almost every decade since the 1920s. Some species can be easily hybridized, such as C. gigas and C. angulata (Imai & Sakai 1961, Menzel 1974, Numachi 1977, Soletchnik et al. 2002, Batista et al. 2007). The ease to hybridize C. gigas and C. angulata has led to questions about their status as two independent species (Huvet et al. 2002). Hybridization has been reported between some other species without genetic confirmation of the hybrids or the parental species (Gaffney & Allen 1993). Zhou et al. (1982) reported success in hybridizing three species, C. gigas, C. rivularis, and C. plicatula without genetic confirmation. The identity of these species is now in question because they have been frequently misidentified. The name C. rivularis was used for 2 species (C. hongkongensis and C. ariakensis), and C. plicatula has been rejected as a taxonomic species (Wang et al. 2004, Wang et al. 2007, Wang & Guo 2008a). As far as we can determine, no hybridization between C. ariakensis and C. sikamea has been reported. In this study, we conducted laboratory hybridization between C. ariakensis and C. sikamea, and used molecular markers to confirm the identity of parental species and hybrids. Here we report that the two species can hybridize in one direction.

MATERIALS AND METHODS

Oysters and Gametes

Sexually mature oysters were obtained in mid-June 2007 from Xiaomiaohong oyster reef, Nantong, China. After collection, oysters were transported to Qingdao and cultured in the laboratory of Institute of Oceanology, Chinese Academy of Sciences. Single oysters were separated from clusters and cleaned. Because C. ariakensis and C. sikamea differ considerably in size and shape, initial identification was done based on shell morphology. After gametes were collected, tissues from all parents used for hybridization were fixed for subsequent confirmation with genetic markers (Wang & Guo 2008a).

Parental oysters were opened, and gametes were obtained by dissection. Before working on an individual, all tools and containers 'were thoroughly washed with freshwater to avoid cross-contamination. Gonad of each opened oyster was sampled and examined under a light microscope to determine sex. Four females and three males from each species were chosen for gamete collection. Eggs from the four females were rinsed with sand-filtered seawater and pooled into one beaker. Egg suspension was passed through a 90-[micro]m nylon screen, rinsed on a 25-[micro]m nylon screen, and resuspended in seawater. Sperm from each male were washed into separate beakers and diluted until suspensions from the three males showed about the same density or cloudiness. Sperm from the three males were then pooled in equal volume.