PROJECT COORDINATION AND PARTNERSHIPS
The genetics work will be carried out by Michael Blouin at Oregon State University.  This project has been coordinated with the Hood River steelhead hatchery and research program, funded by Bonneville Power Administration and administered and implemented by the Oregon Department of Fish and Wildlife and the Warm Springs Tribes (project numbers 198805307, 198805308, 198805304 and 198805303).  These projects included operation and maintenance of the Oak Springs and Parkdale hatchery facilities, and operation and maintenance of the fish collection and handling facility at Powerdale Dam, as well as database management and data analysis on the part of ODFW.

LOCATION OF PROJECT
Steelhead samples were collected at Powerdale Dam, Hood River, under supervision of Rod French, ODFW, who also coordinated aging of scale samples.  All laboratory work and genetics data analysis  continues to be conducted in the laboratory of Michael Blouin at Oregon State University.

BACKGROUND AND RESULTS TO DATE FROM THE HOOD RIVER
The Hood River supports two populations of steelhead, a summer run and a winter run.  They spawned only above the Powerdale Dam, which is a complete barrier to all salmonids.  From 1991 to 2010 every adult passed above the dam was measured, cataloged and sampled for scales or fin snip.  Therefore, we have a DNA sample from every adult steelhead that went over the dam to potentially spawn in the Hood River from 1991 to 2010, when the dam was removed.  Similar numbers of hatchery and wild fish were passed above the dam during the last decade.  During the 1990's "old" domesticated hatchery stocks of each run (multiple generations in the hatchery, out-of-basin origin) were phased out, and conservation hatchery programs were started for the purpose of supplementing the two wild populations (using wild broodstock; hereafter F1 hatchery fish).  The winter-run F1s were started in 1991, and the summer-run F1s were started in 1997.  In a supplementation program such as this, wild-born broodstock are used as parents in the hatchery in an attempt to circumvent the low fitness induced by multiple generations of selection in the hatchery.  This 19 years of samples gave us the ability to estimate, via microsatellite-based pedigree analysis, the relative total reproductive success (adult to adult production) of hatchery (H) and wild (W) fish for two populations (summer and winter), over multiple brood years.  We now have an almost 4-generation pedigree that is complete for all anadromous fish. Note, however, that we are missing samples from resident fish that apparently are the parents of many steelhead.  We compared the relative success of two "old" hatchery stocks vs. wild fish (the winter run “Big Creek” stock and the summer run “Skamania” stock), and showed they have much lower total fitness than wild fish when both breed in the wild (Araki et al., 2007a).  In that paper we also concluded that the winter-run F1 were not significantly different from wild fish, based on 3 run years of data.  But in a subsequent analysis based on six run years of data the difference was significant, with the F1 winter run fish averaging about 85% the fitness of wild fish (Araki et al., 2007d).  

One problem with interpreting an observed difference in fitness between fish raised in a hatchery and fish raised in the wild is that the difference can have a genetic and/or environmental basis (because the H fish experienced a very different environment during the juvenile phase).  However, in Araki et al. (2007d) we were able to compare the first generation fish with second-generation hatchery fish raised in the same hatchery.  These data suggest that the second generation fish have ~55% the reproductive fitness of the first generation fish (Araki et al., 2007d).  Because both types of fish experienced identical environments, the difference between them must be genetically based.  This result also suggests that the decline in fitness that results from additional generations of selection in  the hatchery can occur very quickly.  

Thus, we have demonstrated a genetically-based effect of hatchery culture that reduces fitness in the wild and that accumulates with each generation of hatchery culture.  Nevertheless, even if captive-bred individuals are genetically different and produce fewer offspring than wild individuals, adding them to a wild population can still give a demographic boost without substantial harm to a wild population that is below carrying capacity if (1) the genetic effects do not persist into the next generation (i.e., natural selection purges the offspring generation of their deleterious alleles before they reproduce), and (2) enough captive-bred individuals are added each generation to make up for their lower productivity.   If the first condition is not true, however, genetic effects will accumulate over time, potentially leading to a downward spiral in the absolute fitness of the supplemented wild population.  Thus, one key question is whether the wild-born descendents of captive-bred fish are less reproductively successful than the descendents of wild fish. In Araki et al. (2009) we  analyzed the fitness of wild-born fish as a function of their parentage.  We found that wild-born offspring of two first-generation hatchery fish averaged 37% the fitness of the offspring of wild fish, while offspring of hatchery-by-wild crosses averaged 87% (Araki et al., 2009).  These results suggest that the hatchery genetic load is not purged from the wild-born population after a full generation of natural selection in the wild.  

We subsequently showed that F1 winter-run  hatchery fish make better broodstock than do wild fish (in terms of number of returning adult hatchery offspring produced)(Christie et al., 2012).  We also showed an interesting tradeoff in which wild broodstock that successfully produced many returning adult hatchery offspring, produced offspring that performed poorly in the wild and vice versa (Christie et al., 2012).  These two pieces of information strongly suggest that strong domestication selection was acting in the hatchery to make fish rapidly adapt to hatchery conditions. Interestingly, a similar pattern has now been observed in Chinook, so the result does not appear to be limited to steelhead (Ford et al., 2012).  One intriguing result from Christie et al. (2012)  is that we noticed a correlation between the size of the fitness tradeoff and the density at which the hatchery fish were raised.  This result suggests the hypothesis that the intensity of domestication selection increases with crowding in the hatchery.

To summarize the work to date on the Hood River, we have shown: (1) the older, multi-generation, summer and winter hatchery stocks from the Hood River had very low fitness relative to wild fish (10-30%).  This result is consistent with results of many other studies on old stocks (Berejikian and Ford, 2004; Araki et al., 2008). (2) first generation winter run fish have significantly lower fitness than wild fish (about 85%), second generation fish do even worse, and the effect is genetically based. (3) The genetic effects of hatchery culture identified for the winter-run stock persist into the first wild-born generation, with the fitness of wild-born fish depending on whether their parents were both wild, both hatchery or one of each.  Again, the common environment experienced by these three types of wild fish suggests a genetic effect.  Finally, the data in Christie et al. (2012) strongly suggest that the above effects result from rapid adaptation to the hatchery.

In addition to completing our main mission of analyzing the fitness of hatchery and wild fish and their descendents, we have also addressed several other applied and basic questions.  These topics include the effects of hatchery stock and resident fish on inbreeding and the effective size of the Hood River population (Araki et al., 2007b; Christie et al. 2012b, Christie et al. 2013a) and methodological work on methods for fitness estimation (Araki and Blouin, 2005; Christie et al., 2011), parentage assignment (Christie et al. 2013b) and for estimation of effective size (Araki et al., 2007c; Christie ).  We also tested what fraction of missing winter-run parents were residualized hatchery fish, and found that only a very few could have been.  Therefore, residualized hatchery fish are not a significant route of gene flow from the hatchery into the wild steelhead population (Christie et al., 2011).   Furthermore, that work illustrated how important are resident, wild fish to the genetic integrity of the anadromous,  steelhead populations.  We also recently reviewed the data from other studies on the relative reproductive success of early-generation hatchery fish and show that reduced fitness of even F1 stocks is a general phenomenon, and not just an unusual feature of steelhead, or the Hood River population (Christie et al., 2014).

CONTINUING AND FUTURE WORK
What are the selection pressures that operated to cause such rapid domestication in steelhead?
This is probably the most important question that needs to be answered.  It appears that strong selection in some part of the life cycle caused winter-run  fish to quickly evolve to be different than wild fish.  But we do not know which traits are involved.  The answer to that question would help identify ways to modify the hatchery experience in order to lessen those selection pressures.  We are taking the following approaches.
(1)  We have been testing the hypothesis that increased crowding in the hatchery increases the opportunity for domestication selection.  Results of the first three year's experiments did not support the hypothesis, in that raising fish at lower densities does not decrease the among-family component of body size at release (which is correlated with survival after release; unpub. data).  
(2) We are conducting similar experiments (as with density) to see if manipulating other environmental variables, such as the feeding method or flow regimes in the tanks, will result in making families more similar in size at release (which would reduce the opportunity for selection on body size after release).  
(3) We are attempting to find genes whose expression levels may be under selection in the hatchery.  Identifying such genes could then point us to the traits under selection, and thus to features of the hatchery that might be changed.  We are approaching this in two ways.  
  First, we have compared genome-wide expression levels in the offspring of hatchery and wild fish from the Hood River, and are repeating that experiment using H and W  fish from the Siletz River in order to see if the patterns are repeatable.  We found hundreds of genes are differentially expressed between offspring of H and W fish from the Hood River (Christie et al., 2015).  We found a similar but much less dramatic effect in a similar study using H and W fish from the Siletz River (unpub. data).  We are currently analyzing whether similar metabolic pathways appear to be under selection in both populations.
  Second, from our density experiments in past years we see substantial variation among families in growth rates.  We will examine gene expression in the siblings of families that are being assayed for growth rate.  Here we hope to identify any genes whose expression levels correlate at the family level with growth rate.  If any such genes also appear in the list of those differentially expressed between H and W fish, that would be compelling circumstantial evidence that those genes are under selection in the hatchery, and thus warrant further study.

REFERENCES CITED
Araki, H. and M.S. Blouin. 2005. Unbiased estimation of relative reproductive success of different groups: evaluation and correction of bias caused by parentage assignment errors. Molecular Ecology, 13:4907-4110.

Araki, H., W.R. Ardren, E. Olsen, B. Cooper and M.S. Blouin. 2007a. Reproductive success of captive-bred steelhead trout in the wild: evaluation of three hatchery programs in the Hood River. Conservation Biology 21:181-190.

Araki, H., R.S. Waples, W.R. Ardren, B. Cooper and M.S. Blouin. 2007b. Effective population size of steelhead trout: influence of variance in reproductive success, hatchery programs, and genetic compensation between life-history forms. Molecular Ecology 16:953-966

Araki, H., R.S. Waples and M.S. Blouin. 2007c. A potential bias in the temporal method for estimating Ne in admixed populations under natural selection. Molecular Ecology 16: 2261–2271

Araki, H., B. Cooper and M.S. Blouin. 2007d. Genetic effects of captive breeding cause a rapid, cumulative fitness decline in the wild. Science 318: 100-103.

Araki, H., B. Berejikian, M. Ford, and M.S. Blouin. 2008 Fitness of hatchery-reared salmonids in the wild. Evolutionary Applications 1:342-355.

Araki, H., B. Cooper and M.S. Blouin. 2009. Carry-over effect of captive breeding reduces reproductive fitness of wild-born descendents n the wild. Biology Letters doi: 10.1098/rsbl.2009.0315

Berejikian, B. A., and M. J. Ford. 2004. Review of the Relative Fitness of Hatchery and Natural Salmon. U.S. Dept. Commer., NOAA Tech. Memo. NMFS-NWFSC-61. 28 p. Northwest Fisheries Science Center, Seattle, WA.

Blouin, M.S. V. Thuillier, B. Cooper, V. Amarasinghe, L. Cluzel, H. Araki and C. Grunau. 2010. No evidence for large differences in genomic methylation between wild and hatchery steelhead trout (Oncorhynchus mykiss). Canadian Journal of Fisheries and Aquatic Sciences. 67: 217-224.



Christie, M., M. Ford and M.S. Blouin. 2014.  On the reproductive success of early-generation hatchery fish in the wild.  Evolutionary Applications, 7:883-896.

Christie MR, RA French, ML Marine and MS. Blouin. 2012b Effective size of a wild salmonid population is greatly reduced by hatchery supplementation.  Heredity, 109, 254–260

Christie MR, RA French, ML Marine and MS. Blouin. 2013a. Does inbreeding cause the reduced fitness of captive-born individuals in the wild? J. Heredity, doi: 10.1093/jhered/est076

Christie, M. R., M. L. Marine, R. A. French, and M. S. Blouin. 2012a. Genetic adaptation to captivity can occur in a single generation. Proc Natl Acad Sci U S A 109:238-242.

Christie, M.R., M.L. Marine and M.S. Blouin. 2011. Who are the missing parents? Grandparentage analysis identifies multiple sources of gene flow into a wild population. Molecular Ecology, 20, 1263–1276

Christie, MR, ML Marine, SE Fox, RA French and MS Blouin. 2015. A single generation of domestication heritably alters expression at hundreds of genes.  Nature Communications doi:10.1038/ncomms10676

Christie MR, Tennessen JA, Blouin MS (2013b) Bayesian parentage analysis with systematic accountability of genotyping error, missing data, and false matching. Bioinformatics 10.1093/bioinformatics/btt039

Ford,  M., A. Murdoch, and S. Howard. 2012. Early male maturity explains a negative correlation in reproductive success between hatchery spawned
salmon and their naturally spawning progeny. Conservation Letters. DOI: 10.1111/j.1755-263X.2012.00261.x