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study, F1 and F2 hybrid families of coho salmon (Oncorhynchus kisutch) were ... notre étude des familles hybrides F1 et F2 de saumons coho (Oncorhynchus ...
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Two generations of outbreeding in coho salmon (Oncorhynchus kisutch): effects on size and growth Erin K. McClelland, James M. Myers, Jeffrey J. Hard, Linda K. Park, and Kerry A. Naish

Abstract: Outbreeding is a potential genetic risk in Pacific salmon (Oncorhynchus spp.) when aquaculture practices introduce nonnative domesticated fish to wild environments, making interbreeding with wild populations possible. In this study, F1 and F2 hybrid families of coho salmon (Oncorhynchus kisutch) were created using a captive freshwater aquaculture strain and a locally derived hatchery population that is integrated with naturally spawning fish. Intermediate growth was detected in F1 and F2 hybrids from crosses reared in captivity; both generations had mean weight and length values between those of the parent populations after their first year (p < 0.05). In the early life history stages, maternal effects increased alevin growth in progeny of hatchery dams relative to those of captive dams (p < 0.001). Aquaculture control families showed greater growth rates than hybrids in late summer of their 1st year and in the following spring (p < 0.05), while the hatchery controls had lower growth rates during the first summer (p < 0.05). Line cross analysis indicated that changes in additive and dominance interactions, but not unfavorable epistatic interactions, likely explain the differences in weight, length, and growth rate observed in hybrids of these stocks of coho salmon. Résumé : La reproduction des saumons du Pacifique (Oncorhynchus spp.) hors des conditions de consanguinité pose un risque génétique potentiel lorsque les pratiques d’aquaculture introduisent des poissons domestiqués non indigènes dans les milieux naturels, permettant ainsi la reproduction croisée avec les populations sauvages. Nous avons créé dans notre étude des familles hybrides F1 et F2 de saumons coho (Oncorhynchus kisutch) à partir d’une race d’eau douce d’aquaculture gardée en captivité et une population de pisciculture d’origine locale qui s’est intégrée aux poissons qui fraient en nature. Dans les élevages des croisements faits en captivité, les hybrides F1 et F2 ont une croissance intermédiaire; les deux générations ont des valeurs de masse et de longueur moyennes comprises entre celles des populations parentales après leur première année (p < 0,05). Durant les premiers stades du cycle biologique, les effets maternels augmentent la croissance des alevins dans la progéniture des pères provenant de pisciculture par rapport à celle des pères en captivité (p < 0,001). Les familles témoins en aquaculture ont des taux de croissance plus élevés que ceux des hybrides à la fin de l’été de leur première année et au printemps suivant (p < 0,05), alors que les témoins provenant de pisciculture ont des taux de croissance plus faibles durant leur premier été (p < 0,05). Une analyse des croisements entre les lignées indique que les changements dans les interactions additives et les interactions de dominance, mais non les interactions épistatiques défavorables, expliquent les différences de masse, de longueur et de taux de croissance observées chez les hybrides de ces stocks de saumons coho. [Traduit par la Rédaction]

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Introduction The maintenance of the genetic processes leading to the differentiation of populations is an important consideration in conservation, especially if remedial actions such as supplementation or introduction are implemented. The genetic composition of two populations may diverge significantly from each other over relatively short time frames

(e.g., Quinn et al. 2001). Such divergence is of particular concern in salmon where hatchery stocks may have genetically diverged from their founder wild stocks (Reisenbichler and Rubin 1999; Waples 1999) and where fish have been transferred extensively between basins and across biogeographical boundaries (Utter 2001), making interbreeding between previously genetically isolated populations possible. One potential outcome of such interbreeding is outbreeding

Received 31 August 2004. Accepted 5 May 2005. Published on the NRC Research Press Web site at http://cjfas.nrc.ca on 14 October 2005. J18290 E.K. McClelland1 and K.A. Naish. School of Aquatic and Fishery Sciences, University of Washington, Box 355020, Seattle, WA 98195, USA. J.M. Myers, J.J. Hard, and L.K. Park. National Marine Fisheries Service, Northwest Fisheries Science Center, Conservation Biology Division, 2725 Montlake Boulevard E., Seattle, WA 98112, USA. 1

Corresponding author (e-mail: [email protected]).

Can. J. Fish. Aquat. Sci. 62: 2538–2547 (2005)

doi: 10.1139/F05-159

© 2005 NRC Canada

McClelland et al.

depression, the reduction in fitness following hybridization between unrelated populations. The genetic basis of outbreeding depression resulting from phenotypic divergence can reflect simple additive genetic differences, dominance interactions among alleles within loci, or epistatic interactions among alleles at different loci (Lynch and Walsh 1998). In general, for traits where all variation is additive, hybrids will display phenotypes midway between parental values (Falconer and Mackay 1996). Dominance interactions are thought to play only a small role in fitness changes of outbred individuals (Lynch and Walsh 1998). Disruption of additive effects and dominance interactions may reduce fitness and growth characteristics as early as the first (F1) hybrid generation. Changes in epistatic interactions resulting from the breakup of coadapted loci are generally not seen until the second (F2) hybrid generation or later when parental genomes undergo recombination. Therefore, findings of heterosis, or hybrid vigor, in the F1 generation may be an unreliable predictor of the outcome of hybridization over successive generations (Lynch and Walsh 1998). This point has important implications for aquaculture and supplementation of natural reproduction because outbreeding is commonly employed in broodstock development to counter the negative effects of inbreeding, and outbreeding frequently results in heterosis in the first hybrid generation (e.g., Heschel and Paige 1995). However, examples of heterosis followed by outbreeding depression have been seen across species (e.g., Edmands 1999; Marr et al. 2002). To better understand the consequences of outbreeding, multigenerational studies are required. Increased genetic distance between parental populations is thought to result in greater severity of outbreeding depression (Emlen 1991; Lynch 1991). This hypothesis is supported by empirical work on copepods where survivorship and numbers of individuals reaching metamorphosis decreased with increased genetic distance (Edmands 1999). However, analysis across a wide range of species indicates that while reproductive compatibility may be correlated with parental divergence to some degree, genetic divergence may not be the best predictor of hybrid fitness (Edmands 2002; E.K. McClelland and K.A. Naish, unpublished). In fishes, most work on outbreeding has focused on firstgeneration hybrids (e.g., Bams 1976; Einum and Fleming 1997; Cooke et al. 2001). After one generation of hybridization, decreases were seen in homing ability in pink salmon (Oncorhynchus gorbuscha) (Bams 1976), in physiological performance in largemouth bass (Micropterus salmoides) (Cooke et al. 2001), and in salinity tolerance in kokanee (Oncorhynchus nerka) hybrids (Foote et al. 1992). Only a few fish studies have evaluated outbred populations through the F2 or subsequent generations (e.g., Gharrett et al. 1999; Sheffer et al. 1999; Gilk et al. 2004). In a study on the endangered Gila topminnow (Poeciliopsis o. occidentalis), reductions in size were seen in the F2 hybrids of certain parental populations (Sheffer et al. 1999). In pink salmon, F1 hybrids within an odd-year line showed a decrease in adult return rate, while no reduction was seen within the F1 hybrids of an even-year line (Gilk et al. 2004). These differences in survival may be due to interannual variation of ocean and river conditions. Alternatively, different genetic interactions may have evolved in the odd- and even-year

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lines resulting in a different response to outbreeding (Gilk et al. 2004). Smoker et al. (2004) found genetically based differences in development rate but no evidence for reduced survival to adulthood in first- or second-generation hybrids among three populations of Alaskan coho salmon (Oncorhynchus kisutch). The studies on pink salmon have been very revealing. However, few data exist to demonstrate the relationship between changes in phenotypic traits and genetic distance, and it is unknown if observations on pink salmon are applicable across salmonid species. Here, we attempt to add to this knowledge by examining the consequences of outbreeding between genetically distinct coho salmon populations. Coho salmon are an important commercial species along the west coast of the United States and Canada. Many coho salmon runs are maintained through supplementation and some populations have received such an influx of hatchery fish that native fish can no longer be distinguished (Johnson et al. 1991). If domestication selection in the hatchery leads to genetically distinct fish, subsequent interbreeding with wild fish can have negative consequences on that wild population (Reisenbichler and Rubin 1999). We examined the consequences of outbreeding between two genetically distinct populations of coho salmon identified as originating from different evolutionarily significant units (ESUs) (Weitkamp et al. 1995). One population was derived from a fast-growing aquaculture strain. The second population was derived from hatchery fish that are released to the wild. The hatchery population produces particularly large adult fish, and we used the two populations to examine the potential for reduced growth rate and size in crossbred offspring. A number of growth traits were monitored over both F1 and F2 hybrid generations with the pure parental lines as controls. Outbreeding was considered to have a negative effect on hybrids if the hybrids showed reduced mean growth rate and size or increased variance for these traits compared with the parental lines. We also sought to determine, as far as possible, whether any reduced growth or size detected between these two populations could be attributed to changes in additive effects or dominance interactions or to breakup of coadapted gene complexes.

Methods Parental strains Two lines of coho salmon were used to create two generations of hybrid families. One parental line was derived from the Satsop River population. Satsop River coho are in the Lower Columbia / Southwest Washington Coast ESU (Weitkamp et al. 1995). Broodstock for this experiment were collected from a hatchery on Bingham Creek, a tributary to the Satsop River. The hatchery incorporates indigenous wild broodstock into the hatchery each spawning season (Ashbrook and Fuss 1996). The second parental line was derived from an aquaculture strain, Domsea broodstock (Myers et al. 1999). Domsea broodstock originated in 1973 and 1974 from the Skykomish River (Wallace River Hatchery), which is part of the Puget Sound ESU (Weitkamp et al. 1995). This ESU is separated from the Lower Columbia / Southwest Washington Coast ESU with a genetic distance of GST = 0.043 (Teel et al. 2003). © 2005 NRC Canada

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The Domsea line is used primarily for aquaculture and has been selectively bred for over 12 generations for fast growth, high fecundity, and early maturation at two years of age (Myers et al. 1999). Despite breeding protocols designed to minimize inbreeding, the inbreeding coefficient after 10 generations was estimated to be 22.8% and fish showed a loss of genetic variability at neutral loci (Myers et al. 2001). However, inbreeding depression does not appear to have had a detectable influence on the growth performance of the stock (Myers et al. 2001). Line crosses Crosses were established in each of two brood years: 1999 and 2001. Single parent matings with reciprocal crosses between Satsop and Domsea fish were used to create 30 F1 hybrid families in the 1999 brood. The F2 crosses established in 2001 were created with progeny from the 1999 F1 cross. A two by two design was used to create 10 F2 families from the F1. Four families were F1 (D × S) × F1 (S × D) crosses, four families were from the reciprocal cross, and two families were from F1 (D × S) × F1 (D × S) crosses (throughout the paper, D stands for Domsea and S stands for Satsop with the sire stock given first). Control crosses of pure Satsop and pure Domsea fish were recreated in 2001 from the parent populations for a total of 20 families per line. An additional 20 families of each F1 reciprocal cross were established in 2001 to provide a comparison among parental, F1, and F2 generations raised in a common environment. Rearing Fish were maintained in a captive freshwater environment throughout the course of this experiment. Alevins were incubated in Heath trays until their yolk sacs were completely absorbed. Families were randomly distributed throughout the incubator stacks to avoid bias owing to stack position. F0 and F1 families, brood year 1999 Following yolk absorption, 600 fish from individual families were transferred into separate 100 L tanks. After 60 days of rearing, tank densities were reduced to 400 fish per tank. In August 2000, the 1999 brood fish were marked to distinguish families and transferred to new tanks (1500 L) with two families per tank (one family in each tank was identified by the excision of the adipose fin). In February 2001, families from the reciprocal F1 crosses and the Satsop pure cross were marked by cold-branding (Mighell 1969) and reared communally in a large broodstock tank (10 000 L). F0, F1, and F2 families, brood year 2001 Following yolk absorption, 500 individuals from each family were transferred to 1000 L tanks with one family per tank. In August 2002, 200 juveniles from each of the 2001 brood families were cold-branded and pooled into two large broodstock tanks (10 000 L). Distribution of individuals was random among tanks with approximately half of each family in each tank. Fish were maintained at a constant temperature of 10 °C throughout. Feeding regimens were based on percent body weight according to the manufacturer’s recommendations (Skretting, Vancouver, British Columbia). This

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feeding regime is based on feed conversion efficiency rather than maximizing growth performance. Trait measurements Three performance metrics were measured: weight, length, and growth rate. These traits were selected because of their association with other important fitness traits in salmon. Growth at certain critical periods in the spring and autumn prior to smoltification plays an important role in determining the age at which some salmon species smolt (e.g., Beckman and Dickhoff 1998) and reach maturity (e.g., Shearer and Swanson 2000). Spring growth rates have a significant relationship to smolt to adult return (Beckman et al. 1998). Female size is highly correlated with fecundity (Fleming and Gross 1990). Traits were evaluated in a single, captive freshwater environment. At the alevin stage, 20 fish per family were selected at random, removed from each incubation tray, and preserved in 10% buffered formalin. The yolk was separated from the embryo. Yolks and embryos were dried in an incubator for 20–24 h at 70 °C. Wet and dry weights were recorded for both yolks and embryos. The first set of samples was taken approximately 50 days after fertilization. Samples were collected every 14 days thereafter, for a total of three sets of data, until the alevins had completely absorbed their yolks. For alevin samples, conversion efficiency was calculated as the change in body weight over a specified time period divided by the change in yolk weight over the same time period (Kinnison et al. 1998). Growth rates were also calculated for alevins based on the daily change in dry embryo weight. Once yolks were absorbed, weight and length data was collected from 25–100 fish per family beginning in April 2002 and every 6 weeks thereafter until fish reached 1 year of age. Sample sizes for each sampling period were determined by power analysis to limit type II error to 10% with an α level of 0.05. Two final measurements on all remaining fish (approximately 100 fish per family) were taken in June and October 2003. Growth rates were calculated based on the average daily change in body weight. Statistical techniques Data from the 1999 brood year were not included as part of these analyses, since the data collected from the F1 hybrids showed a similar trend to that seen in the F1 in the 2001 brood year. For all statistical analyses, the F1 hybrids with Domsea dams (S × D) were treated as a separate cross type from the F1 hybrids with Satsop dams (D × S) to identify maternal genetic effects or effects owing to common maternal environment, since several factors such as age at spawning, diet, and time spent in salt water differed between the two parental populations. The F2 hybrids were not separated by origin of maternal parent, as there was no statistical difference between F2 families whose dams came from different F1 crosses. Equality of means between parental, F1, and F2 cross types for weights, lengths, and growth rate was tested using a nested analysis of variance (ANOVA) (SPSS version 10.0; SPSS Inc., Chicago, Illinois). Families were nested within cross types to partition variance owing to family and tank effects before testing for differences between cross types. © 2005 NRC Canada

McClelland et al. Fig. 1. Mean alevin wet embryo weight. Crosses shown are the Satsop control, the Domsea control, F1 hybrids with Domsea dams (F1 (S × D)), F1 hybrids with Satsop dams (F1 (D × S)), and F2 hybrids. Error bars are ±1 SD. Points are offset for visibility. The same pattern is evident for wet embryo weights and wet and dry yolk weights.

Family and tank effects could not be evaluated separately, since families were not split between tanks at early stages (prior to August 2002). Large differences in means were observed between cross types, and thus, a coefficient of variation (CV) was calculated for each family. CVs were then averaged across families within a cross to determine a CV for each cross type. Equality of the CVs between cross types was tested using ANOVA (SPSS version 10.0). For all ANOVAs, significance levels were adjusted for multiple comparisons using a simple Bonferroni correction to keep test-wise α at 0.05. Additionally, the percent reduction in the total sum of squares (%TSS = factor sum of squares/total sum of squares) was calculated for each ANOVA. For each trait, a joint-scaling test was used to evaluate the fit of the line cross data to an additive and an additivedominance model following the procedure outlined in Hard et al. (1992) and Lynch and Walsh (1998). Data from four line crosses were used in the model: the two parental populations and the F1 hybrids and F2 hybrids. Owing to the limited number of lines used in this experiment, models that incorporated epistatic interactions could not be tested. The model was evaluated using data from the 2001 brood year crosses for weight, length, and growth rate at all juvenile and subadult sampling periods. Alevin data were not used in this analysis owing to the strong maternal influence seen at this life stage.

Results Alevin comparisons Maternal effects were evident in the hybrid crosses at the alevin stage. F1 (D × S) hybrids were not significantly different in embryo or yolk weight from the Satsop control fish (ANOVA, p = 1.0 for each sample date). Alevin of Satsop dams had yolk and embryo weights that were significantly greater than those of alevins of Domsea dams or of F1 hybrid dams (ANOVA, p < 0.001, with the exception of dry embryo weight on day 52 after fertilization, where p > 0.05). The F2 alevins were not significantly different from the Domsea control alevins in embryo and yolk weight

2541 Fig. 2. Alevin specific growth rate for the following cross types: the Satsop control, the Domsea control, F1 hybrids with Domsea dams (F1 (S × D)), F1 hybrids with Satsop dams (F1 (D × S)), and F2 hybrids. Growth rate over a specified time period is (∆ average body weight) × (number of days)–1 × 100. Error bars are ±1 SD. Within the same sample date, cross types with different letters are significantly different from each other (ANOVA, p < 0.05). Where letters are the same or absent, groups are not significantly different.

(ANOVA, p > 0.05 for each sample date) (Fig. 1). Satsop dams had significantly greater lengths from other lines (ANOVA, F[2,0.05] = 314.49, p < 0.001), which likely explains some of the differences in the early size of offspring. Alevin weights seem to have been influenced more by initial weight, attributed to maternal effects (i.e., egg size), rather than by differential growth prior to complete yolk absorption. Growth rates differed between all groups over the entire alevin life stage (days 50–72 after fertilization: ANOVA, F[4,0.05] = 15,66, %TSS = 0.60, p < 0.001) with progeny of Satsop dams having a higher growth rate than those of Domsea or F1 dams. However, the higher growth rates in the Satsop control and F1 (D × S) fish were not consistent over the entire alevin stage (Fig. 2). For the period just prior to yolk absorption (days 64–72), there were no significant differences in growth rates between families (ANOVA, F[4,0.05] = 2.11, %TSS = 0.17, p = 0.097) (Fig. 2). Further, there were no significant differences in conversion efficiency between cross types (days 50–64: ANOVA, F[4,0.05] = 1.30, %TSS = 0.12, p = 0.286; days 64–72: ANOVA, F[4,0.05] = 0.43, %TSS = 0.04, p = 0.789), indicating that yolk was not converted into body mass at a faster rate in any particular cross type. Alevin data were not used to determine if outbreeding depression was present in hybrids or used as part of the jointscaling test because maternal effects were seen at this life history stage. Juvenile and subadult comparison The Satsop and F1 (D × S) crosses comprised the largest fish on day 132 after fertilization (weight: ANOVA, F[4,0.05] = 10.25, %TSS = 0.69, p < 0.001; length: ANOVA, F[4,0.05] = 11.38, %TSS = 0.72, p < 0.001), while the Domsea control fish were the smallest (Fig. 3; Table 1). Al© 2005 NRC Canada

2542 Fig. 3. Average body weight for the 2001 brood. Crosses shown are the Satsop control, the Domsea control, F1 hybrids with Domsea dams (F1 (S × D)), F1 hybrids with Satsop dams (F1 (D × S)), and F2 hybrids. (a) Data from the 1st year (days 132– 329); (b) data from the 2nd year (days 377–686). Error bars are ±1 SD. Points are offset for better visibility of data. The same pattern is evident in the length data.

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ings. By the end of the first year (day 377 after fertilization), both F1 and F2 crosses had weights and lengths between those of the control lines (weight: ANOVA, F[4,0.05] = 3.53, %TSS = 0.44, p < 0.001 (Fig. 3); length: ANOVA, F[4,0.05] = 4.40, %TSS = 0.49, p < 0.001 (Table 1)). The Satsop control tended to have the lowest CV for both weight and length over most of this study period (Table 2). However, the CV for either trait was only significantly different between cross types on days 377 and 686 postfertilization (ANOVA, p < 0.05 for both sample dates). On day 377, the Satsop fish had a lower CV for both length and weight than all other crosses; this relationship was significant in comparison with the F1 (S × D) crosses and also in comparison with the F2 cross for length. On day 686, the Satsop fish had the highest CV for both traits, and in this case, the relationship was significant in comparison with all other cross types with the exception of the F1 (S × D) cross. Line cross analysis A simple additive genetic model proved to be inadequate to explain divergence between the populations in weight, length, and growth rate. An additive-dominance model adequately explained the variance between lines for weight 2 2 (χ1,0.05 , p > 0.05), length (χ1,0.05 , p > 0.05), and growth rate 2 (χ1,0.05, p > 0.05) at all sampling dates. Inclusion of the dominance parameter significantly improved the fit of the model 2 in each case (χ1,0.05 , p < 0.05). This analysis indicates that even where trait values for the F1 and F2 hybrids fell between those of the parent lines, the hybrid values departed significantly from the midparent value. In the F1 hybrids, deviations from the expected line mean for both weight and length were in the direction of the maternal parent during the first year (prior to day 329) and in the direction of the male parent during the second year. F2 hybrids were lower than the expected line mean for weight and length prior to day 377. No pattern was seen in direction of deviation for growth rates of hybrid lines.

Discussion

though the Domsea broodstock juveniles were initially smaller than all other crosses, by the end of the first year (day 329), the Domsea control had the greatest average weight (ANOVA, F[4,0.05] = 4.55, %TSS = 0.50, p < 0.001) (Fig. 3) and length (ANOVA, F[4,0.05] = 5.05, %TSS = 0.53, p < 0.001) (Table 1). Between days 230 and 329, late summer to early fall of the first year of growth, the Domsea control families experienced high growth rates (Fig. 4), which resulted in a change in order of both weight and length rank-

Average weights and lengths of both F1 and F2 hybrids from the 2001 brood year fell between the average values for each of the two parental strains during the juvenile and subadult phases. Data from the F1 hybrids in the 1999 brood followed a trend similar to that observed in 2001 (data not shown). Joint-scaling tests to assess the contributions of additive and dominance effects to the variation between lines indicate that a model incorporating additive and dominance effects adequately explained the variability observed among the strains and their first- and second-generation hybrids. Thus, composite epistatic interactions do not appear to be a significant factor contributing to the phenotypic variation seen in this study, and we conclude that even in the F2 generation, variation in trait means can be attributed to changes in additive effects or dominance interactions within loci rather than to epistatic interactions or linkage between loci. Differences in phenotypes between the Domsea and Satsop strains resulting from different selection regimes for growth, size, and maturation may explain the population differentiation and resulting reduction in trait values of hybrids observed in this study. However, selection and other factors © 2005 NRC Canada

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Table 1. Mean weights (g) and lengths (mm) (±1 SD in parentheses) and sample sizes for all 2001 broodyear crosses. Cross type Sample date (days after fertilization) 26 February 2001 (84) 15 April 2002 (132)

5 June 2002 (183)

24 July 2002 (230)

29 October 2002 (329)

16 January 2003 (377)

1 February 2003 (424)

28 June 2003 (571)

21 October 2003 (686)

Weight n Weight Length n Weight Length n Weight Length n Weight Length n Weight Length n Weight Length n Weight Length n Weight Length n

Satsop

Domsea

F1 (S × D)

F1 (D × S)

F2

11.1 (1.70)a 24 1.62 (0.47)a 49.50 (4.55)a 75 5.26 (2.15)ac 69.10 (12.20)ac 75 12.04 (4.59)a 97.7 (16.76)a 150 24.23 (7.94)a 125.00 (13.18)a 137 27.49 (8.84)a 129.60 (13.40)a 121 38.81 (15.01)a 144.4 (18.91)a 254 98.72 (46.03)a 195.30 (31.15)a 216 158.50 (80.12)a 230.2 (40.15)a 168

8.2 (1.0)bc 27 1.11 (0.39)b 44.50 (4.98)b 75 4.50 (2.02)ab 68.40 (10.64)ac 75 12.30 (6.10)a 93.8 (18.45)ab 150 48.92 (20.62)b 161.50 (27.46)ab 150 58.75 (30.23)b 168.70 (32.10)b 151 99.16 (46.75)b 192.6 (35.08)b 305 243.40 (100.96)b 260.30 (33.01)b 289 338.43 (110.98)b 293.3 (31.83)b 233

11.2 (1.9)cd 21 1.69 (0.46)a 50.8 (3.76)a 75 5.63 (2.09)c 71.90 (10.13)a 75 13.16 (5.57)a 97.5 (18.23)a 150 36.67 (16.67)c 145.50 (25.04)ab 149 44.87 (22.65)cd 153.80 (26.59)c 148 71.21 (39.16)c 174.01 (31.01)c 307 185.00 (81.63)c 241.5 (33.42)c 275 268.52 (96.41)c 275.90 (33.152)c 224

7.0 (1.0)a 27 1.13 (0.35)b 45.2 (4.42)b 75 4.92 (2.16)bc 66.1 (11.45)bc 75 10.38 (4.52)b 93.10 (15.68)ab 150 28.82 (15.83)a 131.90 (22.42)b 144 39.03 (25.23)c 142.80 (29.96)d 150 53.24 (33.58)d 155.6 (31.63)d 309 140.59 (72.20)d 218.60 (34.74)d 303 212.02 (92.08)d 255.00 (36.05)d 261

8.6 (1.1)bd 33 1.13 (0.31)b 45.30 (3.56)b 275 4.33 (1.86)b 64.00 (10.88)b 275 9.70 (4.70)b 90.10 (15.57)b 550 33.68 (18.66)c 138.7 (27.18)ab 547 45.85 (27.54)d 153.4 (31.48)c 551 67.52 (39.40)c 169.40 (32.66)c 551 183.04 (84.53)c 241.70 (33.12)c 1005 282.03 (104.90)c 282.6 (33.73)c 835

Note: Within the same sample date and trait, cross types with different letters are significantly different from each other (ANOVA, p < 0.05). A Bonferroni correction was used to adjust for multiple comparisons.

contributing to phenotypic differences between populations have not been sufficient to produce markedly different genetic backgrounds in these lines. Where variation is due to only additive effects, CV in hybrids should fall between values seen in parental lines. However, where phenotypic variation includes epistatic interactions, more allele combinations are possible in the hybrid offspring than in the parents, which is expected to result in higher CVs in the hybrids (Lynch and Walsh 1998). Domsea fish, which have experienced selective pressure for increased growth and size, were expected to be less variable at these traits. However, the Satsop fish displayed the lowest amount of variation at the end of the first year, although they were the most variable at the end of the second year. The CVs of the two hybrid generations fell between the values of the parental lines at the end of the second year. At some sampling periods, hybrids did displayed greater variation than either parental line; however, results were not always statistically significant. One explanation for these results is that epistatic interactions have not played a significant role in the divergence of these populations as suggested above. Maternal effects appear to have dominated early size in both generations. Alevins of Satsop dams were significantly larger than were alevins of Domsea or hybrid dams. Previous studies on salmon have found a correlation between female size, egg size, and offspring size at early life history

stages (Fleming and Gross 1990; Kinnison et al. 1998). Thus, the fact that Satsop females were larger than both Domsea and F1 females likely accounts for the size differences among alevins of the two strains. This pattern of early maternal effects on pre-absorption weights is consistent with other studies (e.g., Kinnison et al. 1998). Maternal influences, primarily egg size, have generally been found to be greatly reduced during the first 120 days after ponding (Iwamoto 1982). In this study, maternal effects on length and weight were not seen beyond the first summer of growth (day 230). The F2 hybrids had weights and lengths that were significantly different from parental values over most of the juvenile and subadult sampling period, but there were generally no significant differences in growth rate. A similar trend was observed in the F1 generation, although results were not always statistically significant. Based on the results of the joint-scaling test, it does not appear that epistatic interactions between loci associated with growth rate, weight, and length have detectable effects on size or growth in these populations of coho salmon. However, depending on the rates of recombination between loci affecting the trait under study, it may take several generations to fully evaluate loss of favorable epistatic interactions (Lynch and Walsh 1998), and it is possible that such interactions would be detected if this experiment were continued. Models of outbreeding sug© 2005 NRC Canada

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Fig. 4. Specific growth rate for the 2001 brood. Crosses shown are the Satsop control, the Domsea control, F1 hybrids with Domsea dams (F1 (S × D)), F1 hybrids with Satsop dams (F1 (D × S)), and F2 hybrids. Growth rate over a specified time period is (∆ average body weight) × (number of days)–1 × 100. Error bars are ±1 SD. Within the same sample date, cross types with different letters are significantly different from each other (ANOVA, p < 0.05). Where letters are the same or absent, groups are not significantly different.

Table 2. CVs for length (mm) and weight (g) (±1 SD in parentheses) for all broodyear 2001 crosses. Cross type Sample date (days after fertilization) 15 May 2002 (132) 5 June 2002 (183) 24 July 2002 (230) 29 October 2002 (329) 16 January 2003 (377) 1 February 2003 (424) 28 June 2003 (571) 21 October 2003 (686)

CV

Satsop

Domsea

F1 (S × D)

F1 (D × S)

F2

Weight Length Weight Length Weight Length Weight Length Weight Length Weight Length Weight Length Weight Length

26.64 (1.69) 8.96 (1.59) 39.86 (5.68) 17.44 (2.03) 37.24 (6.80) 16.09 (5.90) 32.69 (7.61) 10.49 (1.70) 31.79 (1.58)a 10.14 (0.97)a 37.87 (6.21) 12.93 (1.47) 42.961 (1.88) 15.05 (2.53) 49.88 (4.80)a 17.12 (2.18)a

32.09 10.01 41.94 15.16 47.25 19.29 40.70 16.71 49.96 18.77 46.99 17.99 41.29 12.59 31.99 10.69

31.29 (4.23) 9.78 (1.68) 43.68 (11.87) 16.78 (5.71) 43.49 (4.58) 16.74 (3.13) 54.38 (11.60) 16.87 (1.75)b 64.60 (6.58)b 21.04 (1.75)b 63.08 (6.21) 20.10 (1.94) 51.08 (1.56) 15.74 (1.43) 43.70 (3.33) 14.20 (2.60)

21.92 (6.08) 7.19 (1.69) 36.96 (3.40) 14.00 (2.47) 41.53 (8.81) 17.89 (4.84) 45.83 (4.39) 17.26 (0.59) 50.56 (5.27) 17.32 (0.98) 55.14 (5.06) 17.82 (1.64) 43.85 (3.18) 13.81 (1.18) 36.03 (3.93)b 11.99 (0.85)

24.28 (4.99) 7.16 (1.47) 39.29 (9.39) 16.25 (2.63) 46.31 (8.11) 16.17 (3.48) 45.99 (12.87) 16.63 (4.78) 53.51 (13.43) 18.04 (4.46)b 53.11 (12.93) 17.7 (4.06) 44.24 (6.74) 13.04 (1.94) 35.4 (4.99)b 11.57 (1.05)b

(6.79) (0.76) (2.95) (0.84) (4.85) (1.55) (1.26) (1.38) (1.94) (1.64) (6.53) (2.34) (4.02) (1.11) (5.74)b (1.03)b

Note: The coefficients were calculated as CV = (SD/mean) × 100 averaged across families within a cross type. Within the same sample date and trait, cross types with different letters are significantly different from each other (ANOVA, p < 0.05). A Bonferroni correction was used to adjust for multiple comparisons.

gest that increasing recombination rates increases the magnitude of outbreeding depression (Edmands and Timmerman 2003). Unfortunately, no empirical studies have examined the effects of recombination over multiple generations, and ideally, hybrids and backcrosses would be raised in both parental environments to accurately discern between simple genetic differences and epistatic interactions among alleles at different loci (Lynch and Walsh 1998). The effects of outbreeding are likely to depend on the environment in which the hybrids are raised (Burton 1987;

Waser et al. 2000). In this study, all variation was evaluated in a freshwater aquaculture environment. The increased growth rates, and subsequently greater weights and lengths, of the Domsea control fish are most likely due to past selection for growth and maturity in the Domsea broodstock in a freshwater captive environment. The Domsea broodstock has been reared as a captive broodstock for over 12 generations and subjected to deliberate selection for high growth rates, increased size, and early maturation (Myers et al. 1999). Thus, these fish are likely adapted to a closed culture © 2005 NRC Canada

McClelland et al.

environment, while the Satsop stock incorporates naturally spawning fish every year and is adapted to a more variable natural environment that involves 18 months of free-ranging migration at sea. Absence of the saltwater stage of the life cycle during this experiment may have had an impact on the growth of the Satsop fish, but a prior study on growth in coho raised in fresh and salt water demonstrated higher growth rates in individuals raised entirely in fresh water than in those transferred to salt water following smoltification (Silverstein and Hershberger 1994). However, a caveat of this finding was that the stress associated with the transfer process may have negatively affected growth in the saltwater group (Silverstein and Hershberger 1994). Although outbreeding depression has certainly been detected in captivity (Burton 1987; Edmands 1999; Gharrett et al. 1999), some authors have suggested that the full extent of outbreeding depression will not be observed unless individuals are raised in the wild, a presumably more stressful environment (Montalvo and Ellstrand 2001). We expect the Satsop fish to perform better in a wild environment than do the Domsea fish, but we have not explicitly tested this prediction in our study. Further, more stressful natural conditions might affect the fitness of the hybrid crosses differently than the relatively benign captive environment. A number of studies have examined outbreeding depression in wild environments. Loss of local adaptation resulting in poor homing ability was observed in hybrids of wild pink salmon from two different river systems (Bams 1976). Similarly, other studies on the same species have shown dramatic decreases in survival in second-generation hybrids in fish released to the wild (Gharrett et al. 1999; Gilk et al. 2004). On the other hand, no differences were seen in smolt to adult survival of recently derived hatchery coho salmon released to the sea (Smoker et al. 2004) or of farmed, wild, or hybrid Atlantic salmon (Salmo salar) (Einum and Fleming 1997). Therefore, experimentation under wild environmental conditions may be necessary to observe the fitness outcomes of releasing domesticated fish into the wild and would help to better quantify the impact that environmental variation has in the expression and magnitude of outbreeding depression. It should also be noted that the genetic mechanism of outbreeding depression might change as growth is assessed in different environments. For example, it has been suggested that genetic control of growth in salmon is different in fresh water than in salt water (Silverstein et al. 1998). The results of the joint-scaling test indicated that additive and dominance interactions are the major contributors to the variation between populations in growth in the captive environment. However, this result does not exclude the possibility that epistatic interactions play a role in the differentiation of growth traits among populations in the wild. Heterosis was a potential outcome following outbreeding for the F1 generation, since the Domsea broodstock has experienced elevated rates of inbreeding (Myers et al. 2001). Any decreases in growth performance owing to inbreeding may be the result of increased homozygosity and the unmasking of deleterious recessive alleles while favorable dominance interactions are lost (Falconer and Mackay 1996; Wang et al. 2002). Outbreeding has the potential to increase variation in inbred populations, masking the effects of deleterious alleles and, to some extent, counteracting inbreeding

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depression (Falconer and Mackay 1996). In a previous experiment, inbred full-sib matings within the Domsea broodstock showed a significant decrease in weight when compared with outbred half-sib matings (Myers et al. 2001). However, it appears that the increase in the level of inbreeding reported in the Domsea stock has not led to marked inbreeding depression in captivity, and hybridization with the genetically divergent Satsop population led to phenotypic change reflecting additive and dominance effects that did not involve heterosis in the F1 hybrids. Little is known about the relationship between outbreeding depression and genetic distance in fishes. The studies of pink salmon appear to fit the predictions that the severity of outbreeding depression will increase with genetic distance. Odd- and even-year pink salmon are completely reproductively isolated over their natural geographic range and the experiments carried out using these populations were designed to maximize the chance of detecting effects of outbreeding depression (Beacham et al. 1988; Gharrett et al. 1999). The genetic distance between these pink salmon populations, measured as GST = 0.020 (Beacham et al. 1988), was lower than that seen between the ESUs from which the populations used in our study were derived, GST = 0.043 (Teel et al. 2003). However, not only was outbreeding depression detected in pink salmon, but Gharrett et al. (1999) provided a rare example of a decrease in fitness that probably reflects time since divergence. At least two studies involving different species of salmonids found no evidence of outbreeding depression among populations with closer genetic relationships but with divergent life histories. Smoker et al. (2004) observed no differences in smolt to adult survival of recently derived hatchery coho salmon released to sea, and Einum and Fleming (1997) detected no evidence for outbreeding depression in farmed, wild, or hybrid Atlantic salmon. Utter (2001) has proposed that life history differentiation, rather than genetic distance, is a more likely predictor of the likelihood of outbreeding depression in salmonids. Indeed, McKay and Latta (2002) have shown that divergence at neutral markers is not a good predictor of divergence at quantitative traits and that FST tends to be lower than QST. Merila and Crnokrak (2001) also showed that QST tends to be greater than FST, although they found a positive correlation between the two values. Both of these findings on the increase of QST compared with FST support the conclusion that genetic differentiation is often higher in life history traits than at neutral loci owing to action of divergent natural selection. The findings of this study add to a growing body of literature that demonstrates a mainly negative effect of hybridizing different salmon populations. Collectively, these studies support the need for caution in releases and transfers of domesticated stocks and for a greater monitoring of hatchery– wild interactions. To ensure that outbreeding depression will be minimized in hybrids of donor or recipient populations, steps should be taken to minimize genetic divergence between hatchery and wild fish by taking local broodstock into the hatchery each year or else by minimizing opportunities for hatchery and wild individuals to interbreed. Multigenerational monitoring studies are also necessary because the fitness outcome of outbreeding cannot reliably be predicted © 2005 NRC Canada

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based on the first generation of hybrids only (Edmands 1999; Gharrett et al. 1999). In cases where multigenerational breeding studies are not feasible, efforts should focus on gathering relevant demographic and life history information through monitoring hatchery and natural populations following supplementation.

Acknowledgements We thank the members of the Aquaseed Corporation, suppliers of the Domsea broodstock, particularly P. Heggelund, G. Hudson, and P. Munsell, for providing fish and rearing them for the 1st years of this experiment. Thanks also to G. George at the Big Beef Creek Field Station for help with rearing fish. Thanks to R. Iwamoto and M. Purcell for help in the setup of the 1999 crosses and to J. Miller for field support throughout this project. We also extend our thanks to all of the volunteers who helped in the field. K.A.N. was partially supported by an National Research Council fellowship and E.K.M. was supported by the H. Mason Keller Endowment.

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