Recovery of Gila Trout Descended from South Diamond Creek from Recently Hybridized Populations in the Mogollon Creek Drainage Robb F. Leary Fred W. Allendorf and Naohisa Kanda Wild Trout and Salmon Genetics Laboratory Report 99/1 Division of Biological Sciences The University of Montana Missoula, Montana 59812 Abstract: Gila trout, Oncorhynchus gilae, were extirpated from South Diamond Creek in 1995 by a fire and rain event. Prior to this, Gila trout from South Diamond Creek were used to establish populations in the Mogollon Creek drainage, but in 1995 it was discovered these populations were composed of Gila trout and descendants of first generation hybrids between Gila and rainbow trout, 0. mykiss. Since South Diamond Creek represents one of only four native Gila trout populations remaining, it was decided to attempt to recover Gila trout from the Mogollon Creek drainage populations. Fish from Mogollon Creek and its tributary Trail Canyon were transported to the Mescalaro National Fish Hatchery in 1996. In 1997, pair matings (one male and one female) were performed using the sexually mature fish from Mogollon Creek and Trail Canyon. After spawning the fish were sacrificed, and electrophoretic analysis of the products of seven protein coding loci known to distinguish Gila and rainbow trout was used to determine the genetic characteristics of the fish spawned and the likelihood that an individual was a Gila trout. This procedure was repeated using the sexually mature fish in 1998. In 1997, the electrophoretic data indicated that the fish spawned from both streams were mainly a mixture of Gila trout and first generation backcrosses to Gila trout. Thus, progeny produced this year from Gila trout by Gila trout matings represent a valuable resource for conservation and restoration efforts. Protein data from the fish spawned in 1998 indicated the extent of hybridization had increased. There was evidence of second generation backcrosses to Gila trout among the fish from both streams. Because of this, there was a good chance that some fish that electrophoretically appeared to be Gila trout actually were of hybrid origin. Fish produced this year, therefore, should be excluded from conservation and restoration efforts. Alternatively, each individual's genotype at 40 or more nuclear DNA regions that distinguish Gila and rainbow could be determined to ensure only Gila trout are used. 1 INTRODUCTION Gila trout, Oncorhynchus gilae, are native to the Gila River drainage in New Mexico and Arizona and the Verde River drainage, Arizona (Miller 1950; Minckley 1973; Behnke 1992). The species is currently listed as endangered under the Endangered Species Act (United States Fish and Wildlife Service 1989). It is believed to be extinct from the Verde River drainage (United States Fish and Wildlife Service 1993) and analysis of samples collected from 1995 through 1997 indicated only four native populations exist in the Gila River drainage: Main Diamond Creek and its replicate McKnight Creek in the Mimbres River drainage, a replicate of South Diamond Creek in the Mogollon Creek drainage, Whiskey Creek, and Spruce Creek and its replicate in Big Dry Creek (Leary and Allendorf 1998). Reasons for the large reduction in abundance of Gila trout, in numbers of individuals and populations, are due to human alteration of the environment and the introduction of non-native fishes (Miller 1961; United States Fish and Wildlife Service 1993). Human disturbances have resulted in a reduction of vegetation, increased sedimentation and water temperature in streams, and reduced stream flows. Non- native salmonids such as brown trout, SaImo trutta, are thought to result in the elimination of Gila trout populations through predation and competition (Dowling and Childs 1992; United States Fish and Wildlife Service 1993). Introduced rainbow trout, 0. mykiss, have eliminated many Gila trout populations through introgression and the formation of random mating hybrid swarms (Loudenslager et al. 1986; United States Fish and Wildlife Service 1993; Leary and Allendorf 1998; Riddle et al. 1998). That is, populations in which essentially all fish are of hybrid origin. The above factors have isolated the remaining native Gila trout populations from each other in small headwater streams. This fragmentation jeopardizes their continued existence. The populations can be severely reduced in size or eliminated by natural events such as drought, fire, or flood. Electrophoretic analysis of proteins indicated that substantial genetic divergence exists among the native Gila trout populations (Leary and Allendorf 1998). This divergence is mainly due to the presence of variant alleles in only one population but at high frequency and the presence of variant alleles in multiple populations but at highly variable frequencies. Preserving the genetic variation represented by the native Gila trout populations, therefore, will require ensuring the continued existence of all of them. In 1995, Gila trout were extirpated from South Diamond Creek by a fire and rain event. Furthermore, electrophoretic analysis of proteins indicated that the replicate populations of South Diamond Creek Gila trout in the Mogollon Creek drainage were now comprised of Gila trout and descendants of first generation hybrids between Gila and rainbow trout (Leary and Allendorf 1998). Without human intervention, therefore, it 2 was considered highly likely that within a few more generations the Mogollon Creek drainage populations would become hybrid swarms and the valuable genetic resource represented by South Diamond Creek Gila trout would be permanently lost. Thus, a plan was developed and implemented to recover Gila trout from the Mogollon Creek drainage. This paper presents the results of the recovery attempt. METHODS In 1996, a combined total of 600 trout were collected using electrofishing from the lower reaches of Mogollon Creek and its tributary Trail Canyon. The fish were transported by helicopter to hatchery trucks and then they were brought to the Mescalaro National Fish Hatchery where they were raised as separate Mogollon and Trail Canyon groups. After electrofishing, the streams were treated with antimycin A to remove all remaining fish for subsequent reintroduction of Gila trout. Pair matings (one male and one female) were petformed in 1997 at the Mescalaro National Fish Hatchery using the sexually mature fish from the Mogollon and Trail Canyon groups. Each family egg lot was incubated separately and all parents were sacrificed for electrophoretic analysis. Labelled eye, liver, and muscle tissue from each parent was sent frozen to the Museum of Southwestern Biology, University of New Mexico, and then to the Wild Trout and Salmon Genetics Laboratory, University of Montana, for electrophoretic analysis. After electrophoretic analysis, only those egg lots that appeared to be produced from Gila trout by Gila trout matings were retained. The remaining egg lots were sacrificed. This procedure was repeated in 1998. Since parents were sacrificed after spawning, in terms of individuals the fish spawned in 1997 and 1998 are completely different groups. Electrophoresis and data analysis Horizontal starch gel electrophoresis was used to determine each fish's genetic characteristics (genotypes) at seven loci (genes) coding for proteins present in muscle, liver, or eye tissue that previous results indicated distinguish Gila and rainbow trout (Leary and Allendorf 1998): alcohol dehydrogenase (ADH*, liver), fumarate hydratase (FH-1*, liver), glyceraldehyde-3-phosphate dehydrogenase (GAPDH-4*, eye), lactate dehydrogenase (LDH-C*, eye), phosphoglucomutase (PGM-1*, muscle), phosphoglycerate kinase (PGK-2*, muscle), and tripeptide aminopeptidase (PEPB*, eye). Electorphoretic procedures were identical to those used by Leary and Allendorf (1998). We used three methods to examine the extent of hybridization in the fish spawned. First, the proportion of Gila and rainbow trout genes in the Trail Canyon and Mogollon Creek fish, treated separately, was estimated by averaging the frequency of Gila and rainbow trout alleles over all seven loci. Contingency table chi-square analysis 3 was used to test for heterogeneity of the proportion of Gila and rainbow trout alleles between the fish spawned in 1997 and 1998 from the same stream. The procedure of Cockerham and Weir (1977) was used to test for a positive association between the presence of a Gila trout allele at one locus and another (gametic phase disequlibrium) for all possible pairs of loci in the fish spawned treated separately by stream and year. The amount of gametic phase disequilibrium would provide an insight into how recently in the past hybridization was initiated, and thus the likelihood that some fish spawned may be Gila trout. In Gila trout, rainbow trout, and first generation hybrids (Gila x rainbow trout, F1), gametic phase disequilibrium relative to its maximum value will have a value of one. With random mating, it will decrease to 0.5 in second generation hybrids (F1 x F1 = F2) and first generation backcrosses (Gila trout x F : rainbow trout x F Each additional generation of hybridization 1 = F1BG 1 = F1BR)• will decrease gametic phase disequilibrium by one-half its former value. Thus, in third generation hybrids (F2 x F2 = F3) and second generation. backcrosses (Gila trout x F18G = F2BG rainbow trout x FiBR = F28R) it will be 0.25, in fourth generation hybrids (F3 x F3 = F4) and third generation backcrosses (Gila trout x F28G = F_ 38G:, rainbow_ _ trouttrout_ _ x F