Bostrom et a\ Hybridization between two serranids, Ccphalopholis fulvo and Paranlhias lurcifcr 653 



assess hybridization (Campton, 1987). Through the analy- 

 sis of multiple loci it is possible to identify F, and post-F, 

 hybrids, as well as to detect the introgression of alleles 

 between species by hybrid backcrossing. It became evident 

 however, that analysis of nuclear DNA loci is necessary 

 in studies of hybridization as a means to overcome some 

 of the sampling restrictions of allozyme analysis and to 

 provide a sur\'ey of biparentally inherited genes ( Verspoor 

 and Hammar, 1991). Mitochondrial DNA (mtDNA) has 

 also been used extensively in studies of hybridization. Be- 

 cause mtDNA is maternally inherited in fishes, analysis of 

 the molecule allows one to identify the maternal parent of 

 F, hybrids, as well as the sexual preferences of Fj hybrids 

 and their offspring (Dowling et al., 1996). 



The key to using molecular markers to identify hybrid- 

 ization is to find multiple independent nuclear loci and 

 a mitochondrial gene region that have unique alleles in 

 each putative parent species (Dowling et al., 1996). An Fj 

 hybrid would be heterozygous at all nuclear loci and have 

 a mitochondrial haplotype identical to one parent species 

 I Campton, 1987; Dowling et al., 1989). A backcrossed indi- 

 vidual would be heterozygous at some diagnostic nuclear 

 loci and homozygous at others. Therefore the power of 

 demonstrating an F, hybrid, as opposed to a backcross 

 or pure parent individual, increases with the number of 

 nuclear loci examined. 



In this study, genetic information from four diagnos- 

 tic allozyme loci, two diagnostic nuclear DNA loci, and 

 three diagnostic mtDNA gene regions, was used to assess 

 hybridization and introgression between Cephalopholis 

 fulva and Paranthias furcifer in Bermuda waters. 



Materials and methods 



A total of 51 Cephalopholis fulva (Linnaeus, 1758), three 

 C. cruentata (Lacepede, 1802), and 37 Paranthias furci- 

 fer (Valenciennes, 1828) were collected from Bermuda 

 with baited handlines or rotenone solution. In addition, 

 six C. fulva and two C. cruentata were sampled from 

 Navassa Island. Fifteen putative hybrids were captured 

 by Bermudian fishermen using handlines or lobster traps. 

 Cephalopholis cruentata was in-cluded in the study as a 

 possible parent species of the putative hybrid and three 

 Epinephelus guttatus (Linnaeus, 1758) specimens from Lee 

 Stocking Island. Bahamas, were used in the preliminary 

 mitochondrial DNA study. 



Specimens were frozen upon capture and stored at - 

 20°C or -80°C and transported to the laboratory for analy- 

 sis. For mitochondrial and nuclear DNA analysis, muscle 

 tissues were removed and placed in storage buffer (0.25M 

 EDTA, 20':^ DMSO and saturated with NaCli. For allo- 

 zyme analysis, 1.5-cm'^ pieces of liver and muscle tissue 

 were separately homogenized in 250 pL of chilled (4°C) 

 grinding buffer (0.1 M Tris, 0.9 mM EDTA, and 0.05 mM 

 NADP*, pH 7.2). Samples were centrifuged for 3 min at 

 16,000 xg and stored at -80°C or analyzed immediately. 



Genomic DNA was isolated from a l.O-cm'' piece of mus- 

 cle tissue by using the phenol/chloroform protocol of Win- 

 nepenninckx et al. (1993) with the following modifications. 



CTAB (hexadecyltrimethylammonium bromide) was not 

 added to the extraction and phenol was added immediately 

 following incubation of the tissue at 37°C. DNA was pre- 

 cipitated by the addition of 0.04 volume of 5M NaCl and 1.0 

 volume of isopropanol. DNA was resuspended in 150 pL 

 of sterile O.IX TE (Tris-EDTA) and stored at -20°C. 



Morphological analyses 



Specimens of C. fulva and P. furcifer used in the mor- 

 phological analysis were obtained from and measured 

 at the National Museum of Natural History (Table 1). 

 Ten putative hybrids were suitable for morphological 

 analysis. The remaining five samples were not properly 

 preserved and morphological analysis was not possible. 

 Hybrid specimens as well as a small number of the C. 

 fulva and P. furcifer were frozen. Specimens obtained from 

 the National Museum of Natural Historv were fixed in for- 



