Baja California peninsula (Moffatt and Thomson 

 1975; Moffatt 1977). 



Hybridization and hybrid survival experiments 

 have been widely used as indices of divergence and 

 have made valuable contributions as a tool in the 

 definition of phylogenetic relationships (Hubbs 

 1967, 1970). In an attempt to further illuminate 

 the relationship between the grunions, we made 

 artificial and reciprocal crosses and we report on 

 the first successful reciprocal hybridization of 

 Leuresthes tenuis and L. sardina. 



Materials and Methods 



Adult grunions, although easily obtained in 

 large numbers, are difficult to maintain and 

 transport alive. On 18 March 1976 (2330 PST), 

 milt from six California grunion males was col- 

 lected at Scripps Beach, La Jolla Calif., mixed in 

 the beaten yolks of two hen eggs (Bratanov and 

 Dikov 1961), and transported to El Golfo de Santa 

 Clara, Sonora, Mexico. The milt-yolk mixture, 

 maintained between 16° and 20 °C, was used to 

 fertilize the eggs from 8 to 10 Gulf grunion females 

 obtained at El Golfo on the following day ( 19 

 March) during a spawning run which began about 

 1700 MST. During this same run Gulf grunion 

 milt was collected from 6 to 7 males, transported in 

 the same manner and used to fertilize California 

 grunion eggs from about 10 females obtained dur- 

 ing a run that night at La Jolla at 0115 PST (20 

 March). One prior and four subsequent attempts 

 to hybridize the grunions were made during the 

 1975, 1976, and 1977 spawning seasons, but these 

 were unsuccessful because one or both grunions 

 failed to spawn. 



The female grunions were rinsed thoroughly in 

 clean seawater before their eggs were stripped 

 directly into the milt-yolk mixture. The mixture 

 was diluted slightly with fresh seawater to in- 

 crease sperm motility, gently agitated, and kept 

 cool until the end of the spawning run. The eggs 

 were then strained, rinsed with seawater, and 

 placed in plastic refrigerator containers between 

 moist paper towels for transport and incubation. 



Conspecific control embryos of each species were 

 obtained by mixing eggs and milt in a bucket of 

 seawater, one-third full, and did not involve the 

 transportation or preservation of milt in hen yolk. 

 When spawning individuals were plentiful, as at 

 the Gulf grunion run on 19 March, six to nine 

 males were stripped per one female in order to 



achieve maximum fertilization levels (Moffatt 

 1977). 



Both sets of hybrid fertilized eggs and the con- 

 specific controls of L. tenuis (18 March) and L. 

 sardina (19 March) were transported from San 

 Diego, Calif., to the University of Arizona at Tuc- 

 son aboard commercial airlines. Upon arrival (22 

 h postfertilization in L. sardina x L. tenuis and L. 

 sardina controls; 13 h in L. tenuis x L. sardina; 

 and 32 h inL. tenuis controls) each set of eggs was 

 inspected. Their development was monitored daily 

 thereafter. 



Both California grunion spawning runs were 

 sparse at La Jolla. Therefore, the greatest portion 

 of eggs and sperm available were devoted to the 

 hybridization experiments and a low conspecific L. 

 tenuis sample size resulted. Consequently, the de- 

 velopmental and hatching data reported herein 

 for these embryos are a compilation of these few 

 controls and egg sets obtained on other occasions, 

 incubated at 20°C from 12 h postfertilization (Mof- 

 fatt 1977). 



Yolk-sac larvae of the two hybrids and the con- 

 specific controls were placed in separate tanks 

 containing artificial seawater and raised on newly 

 hatched Artemia, freeze-dried marine zooplank- 

 ton, commercial staple food, and frozen Artemia 

 nauplii. Larvae of the hybrids and controls were 

 maintained for nearly 5 mo although initial mor- 

 tality rates (first 2 mo) in all groups were high 

 ( >90% ). On 19 August, 141 days posthatching, the 

 aquaria air lines were fouled by compressor oil and 

 the few remaining hybrids and controls died. Only 

 two L. tenuis x L. sardina and nine L. sardina x 

 L. tenuis individuals survived to a size ( >12 mm) 

 at which the scale rows could be counted. This is 

 not to imply that scales might not have been pre- 

 sent prior to this time, merely that no attempt was 

 made to count them. 



Results 



At 22 h postfertilization, cleavage had pro- 

 gressed to the gastrula stage in L. sardina x L. 

 tenuis embryos as it had in theL. sardina controls. 

 The L. tenuis x L. sardina hybrids had reached a 

 32-cell blastodic stage at 13 h postfertilization as 

 do L. tenuis embryos. 



Artificial fertilization levels in the conspecific 

 controls fell between 85 and 99'^ during the peaks 

 of their spawning seasons when male to female 

 ratios of 6 or 9:1 were available. The fertilization 



477 



