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A Comparison of Genetic Variation Between an Anadromous Steelhead, Oncorhynchus mykiss, Population and Seven Derived Populations Sequestered in Freshwater for 70 Years

A Comparison of Genetic Variation Between an Anadromous Steelhead, Oncorhynchus mykiss, Population and Seven Derived Populations Sequestered in Freshwater for 70 Years
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   Environmental Biology of Fishes  69:  111–125, 2004.© 2004  Kluwer Academic Publishers. Printed in the Netherlands . A comparison of genetic variation between an anadromous steelhead, Oncorhynchus mykiss , population and seven derived populationssequestered in freshwater for 70 years Frank Thrower a , Charles Guthrie III a , Jennifer Nielsen b & John Joyce aa  National Marine Fisheries Service, Auke Bay Laboratory, 11305 Glacier Hwy, Juneau, AK 99801,U.S.A. (e-mail: frank.thrower@noaa.gov) b USGS, Alaska Science Center, Office of Biological Science, Anchorage, AK, U.S.A. Received 17 April 2003 Accepted 16 June 2003 Key words:  rainbow trout, DNA, microsatellites, allozymes Synopsis In1926canneryworkersfromtheWakefieldFisheriesPlantatLittlePortWalterinSoutheastAlaskacapturedsmalltrout, Oncorhynchusmykiss ,fromaportionofSashinCreekpopulatedwithawildsteelhead(anadromous O.mykiss )run. They planted them into Sashin Lake which had been fishless to that time and separated from the lower streamby two large waterfalls that prevented upstream migration of any fish. In 1996 we sampled adult steelhead fromthe lower creek and juvenile  O. mykiss  from an intermediate portion of the creek, Sashin Lake, and five lakes thathad been stocked with fish from Sashin Lake in 1938. Tissue samples from these eight populations were comparedfor variation in: microsatellite DNA at 10 loci; D-loop sequences in mitochondrial DNA; and allozymes at 73 lociknown to be variable in steelhead. Genetic variability was consistently less in the Sashin Lake population and allderived populations than in the source anadromous population. The cause of this reduction is unknown but it islikely that very few fish survived to reproduce from the initial transplant in 1926. Stockings of 50–85 fish into fiveother fishless lakes in 1938 from Sashin Lake did not result in a similar dramatic reduction in variability. We discusspotential explanations for the observed patterns of genetic diversity in relation to the maintenance of endangeredanadromous  O. mykiss  populations in freshwater refugia. Introduction Inrecentyearsmanystocksofsteelhead, Oncorhynchusmykiss , in the western United States have been listedas threatened or endangered under the EndangeredSpecies Act by the National Marine Fisheries Service(Busby et al. 1 ). In many cases, freshwater habitatdestruction has been cited as a principal factor of pop-ulationdeclineand,withoutsubstantialhabitatrestora-tion,thesedeclineswillprobablycontinue.Restorationof freshwater habitats can frequently take years or 1 Busby, P.J., T.C. Wainwright, G.J. Bryant, L.J. Lierheimer,R.S. Waples, F.W. Waknitz & I.V. Lagomarsino. 1996. Statusreview of west coast steelhead from Washington, Idaho, OregonandCalifornia.NOAATech.Memo.NMFS-NWFSC-27.261pp. decades and, in some cases, the continued risk to theremaining population requires some more drastic formof intervention to prevent extinction. In some cases(Flagg et al. 1995, Baugh & Deacon 1988) portions of the wild populations are brought into captivity whilehabitat restoration efforts are underway. However, themaintenance of wild populations in captivity is fraughtwith genetic pitfalls. The effective breeding size of these populations is frequently constrained by eco-nomicssincemaintainingcaptivepopulationsisexpen-sive,andthisexpenseisdirectlyrelatedtothenumbersmaintained. However, small populations are more sub- ject to genetic change through genetic drift (Falconer1981), inbreeding depression (Kincaid 1983), domes-tication selection (Reisenbichler & Brown 1995), andfounder effects (Luczynski et al. 1996).  112An alternative is to maintain endangered popu-lations in different natural environments that allowfor large breeding populations and natural reproduc-tion (Baugh & Decon 1988). This is rarely possi-ble especially for larger animals. For an anadromousspecies such as  O. mykiss , normal mortality rates inthe marine phase routinely exceed 90%. This highmortality can exceed the reproductive potential of an already endangered stock. To reduce this mortal-ity, pumped seawater systems and marine net-pensare currently used to maintain captive populations(Shaklee et al. 1995), however, this usually involvesartificial feeding and captive breeding and, concur-rently, the associated genetic risks. If the life cycle of a normally anadromous fish can be completed with-out the marine phase – and the ability to adapt toseawater is not lost after decades of freshwater seques-tration – then large, naturally reproducing populationsof endangered, normally anadromous fish might bemaintained in protected freshwater habitats until theirnative habitats are restored. This could reduce someof the genetic concerns (e.g. domestication selection,inbreeding depression) for captive populations.Long-term genetic change within specific popu-lations has not been studied extensively on a bio-chemical level because many of the tools we usetoday (starch gel electrophoresis and DNA sequenc-ing) have only been developed and used extensivelyin the last two or three decades. Thus, while manypopulations of animals have been maintained in acaptive state for many decades, no genetic recordexists of the populations srcinally brought into cap-tivity. Since most of these captive populations containrelatively small numbers of individuals, gene frequen-cies would most likely have changed due to foundereffects, genetic drift and domestication selection overthe decades. If the population has been maintainedas a large naturally breeding population in a nat-ural (although perhaps, not native) habitat that hasnot seen substantial disruption (either anthropogenicor natural), then it is more likely that gene frequen-cies of ‘neutral’ alleles might not have changed sub-stantially due to genetic drift and the loss of rarealleles would be minimal. Selection would presum-ably alter frequencies of alleles with high selectioncoefficients that were favored in the new environ-ment. While any substantial change in gene frequen-cies could be seen as undesirable, for some criticallyendangered populations, the only alternative may beextinction.  P o r t  W a l t e r   S a s  h  i  n   C  r e e  k N S W  E  1 km    L   i   t   t   l  e    P  o  r   t    W  a   l   t  e  r  AlaskaCanadaStudy area 200 km Weir  Sashin LakeRound Lake Barrier falls to upstream migrants1926 transplant1938 transplantsBetty LkFawn LkDavidof LkRezanof Lk  5 0  f i s h  e a c h                                                                                  8                                                                                 5                                                                                    f                                                                                 i                                                            s                                                                                  h    L  a   k  e    B  o  r  o  d   i  n  o Figure 1 . Map of Port Walter showing Sashin Creek study areaand indicating the initial transplant (1926) from the anadromousportion of the creek to Sashin Lake and the secondary transplants(1938) to five other barren lakes. The purpose of this study was to determine if long-term sequestration in fresh water of a normallyanadromous stock of fish would result in significantchanges in genetic variation that could preclude it as auseful methodology in the preservation of endangeredsteelhead populations. We compared genetic varia-tion within a wild, anadromous steelhead population(Sashin Creek) in Southeast Alaska with genetic varia-tion in a rainbow trout population from a semi-isolatedlake (Sashin Lake) in the same drainage that had beenestablished with a single transplant from the anadro-mousportionofSashinCreekin1926(70yearsearlier)(Anonymous 1939) (Figure 1). We also extended thiscomparison to include five other lake populations thathad been stocked with fish from Sashin Lake in 1938(approximately 60 years earlier), and a stream popula-tion in the intermediate section of Sashin Creek that isseparated by barrier falls from the anadromous portionof the creek and Sashin Lake. All of the study lakesand the intermediate stream section were barren of anyspeciesoffishatthetimeofstocking,areabovebarrierfallsthatprevententryofanyfishfrombelow,andhavenorecordsofsubsequenttransplants.Fishfromalleightpopulations (hereafter referred to as the ‘study’ popu-lations) were examined for variation at allozyme andmicrosatellitelociandmitochondrialDNAhaplotypes.The number of fish srcinally transplanted to SashinLake is unknown. A survey conducted in 1934 indi-cated the  O. mykiss  population in Sashin Lake to be  113large (thousands) so we assumed the initial stockingsizewaslargeorsurvivalwasquitehighinthefirstgen-eration. Stocking records report numbers stocked foreach of the five secondary transplants (Chipperfield 2 )and indicate the maximum breeding size of the sec-ondary transplants was small (50–85 fish). Given thatthe fish were stocked in July, and thus subject to natu-ral mortality for 10 months prior to first spawning andthat the sex ratios at stocking were probably unequal,we hypothesized that founder effects could have sub-stantially altered gene frequencies through loss of rarealleles and increased genetic homozygosity. All of thewatersheds in the study area remain pristine and cur-rently support population sizes of at least several hun-dred to several thousand fish (Thrower, pers. observ.). Materials and methods A weir on Sashin Creek was used to capture all adultanadromous steelhead in 1996 and 1997. Hoop nets,minnow traps and sport fishing gear were used tocapture resident fish in the study lakes and the inter-mediate section of Sashin Creek. Tissue samples forDNA extraction consisted of ventral fin clips of livefish. Samples from Sashin Creek and Sashin Lake in1996werecollectedfromadultfishandstoredin100%ethanol, whereas those from other populations consist-ing of mixtures of adults and juveniles were preservedby air drying. Tissue samples for allozyme analysiswere collected from a portion of the adult steelheadreturn in both 1996 and 1997, and from resident fishin 1997 with a separate collection from Sashin Lakemadein1996.Thesamplesfromanadromousfishwereplaced in  − 20 ◦ C freezers for 2 months and transferredto  − 70 ◦ C freezers until processed, whereas residentfish were kept alive during transit to Little Port Walterwheretissueswereremovedandplacedoniceforupto2h, transferred into liquid nitrogen for up to 3 months,and moved to  − 70 ◦ C freezers until processed.A seventh lake (Deer Lake), also initially stockedwith fish from Sashin Lake, was included for contrastbecause it is known to have had multiple introduc-tions of fish from outside the study area. At Deer Lake,allozymesamplesinthespringof1997,andDNAsam-ples in the spring of 1998, were collected from fishmigrating out of the lake (mostly smolts). 2 Chipperfield,W.A.1938.Memoforfiles,DistrictRangerU.S.Forest Service, July 30, Juneau, Alaska.  Laboratory analysis Mitochondrial DNA A total of 256 fish were examined for mtDNAhaplotype variability. DNA was extracted from asmall portion of dried fin tissue using Chelex 100resin (BioRad) following methods given in Nielsenet al. (1994a). We used conserved primers (S-pheand P2) to amplify a highly variable segment of trout mtDNA, including 188 base pairs (bp) of thecontrol region and 5bp of the adjacent phenylalaninetRNA gene. Double- and single-stranded amplifica-tions were performed using polymerase chain reaction(PCR). PCR products were sequenced directly andthe DNA visualized on X-ray film. DNA protocols,sequence for specific primers, and the complete con-trol region segment amplified in  O. mykiss  are givenin Nielsen et al. (1994b). Sequences were alignedusing MacDNASIS (Hatachi Software EngineeringCompany, Ltd.).  Microsatellites Ten nuclear microsatellite loci developed in other lab-oratorieswerechosenforthisstudybasedontheirhighlevel of polymorphism in previous studies of rainbowtrout and steelhead in our laboratory. The Omy-seriesof microsatellite loci were developed specifically for O. mykiss ; the One µ -series was developed for sockeyesalmon,  Oncorhynchus nerka ; Ots-series for chinook salmon,  Oncorhynchus tshawytascha ; Sfo-series forbrook trout  Salvelinus fontinalis ; and the Ssa-serieswas developed for Atlantic salmon,  Salmo salar  . Foreach locus, primer B was labeled according to pro-tocols given in Nielsen et al. (1994b). Amplificationof microsatellites followed the methods given inNielsen et al. (1997) using three fluorescent dyesand running all microsatellite gels on an ABI 373(Applied Biosystems) adapted for microsatellite anal-ysis.AllmicrosatellitegelswerereadusingABIPrismGenotyper Software (Applied Biosystems). All lociwere initially run individually as separate PCR reac-tionstodetermineallelicsizedistributionsintheAlaskarainbow trout. PCR products were then multiplexed onthe gels according to the protocol given in Table 1.The size reported here for each microsatellite allelewas equal to the size of the total product amplified(including amplified primer sequence). Allelic sizewas determined by two methods: (1) reference to theABI Genescan-500 size marker ladder and (2) known O. mykiss  DNA samples that were rerun on each gel.  114 Table1 . Multiplexconditionsusedforamplificationsof10microsatellitelociinsouthwestAlaska rainbow trout and steelhead.Anneal ( ◦ C) Locus (primer conc.)6Fam-blue Tet-green Hex-yellowMykiss A 56 One14 (0.14) Ots1 (0.17) One11 (0.06)Ssa85 (0.06) Sfo8 (0.10)Mykiss B 52 Omy77 (0.30) Ssa4 (0.55) Omy325 (0.11)One2 (0.055) One8 (0.13)Primer concentrations are given in parentheses. Binning of alleles was performed after an analysis of variance for size distributions of each allele at eachlocus identified by Genotyper. To ensure consistencyin both PCR reactions and scoring of microsatellites,7.8% of all samples were run again on different gelsand scored independently. Repeated runs were notincludedintheanalysisofvarianceperformedtoestab-lish allelic binning protocols. Alleles found in < 5% of thetotalstudypopulation(allsamplescombined)wereconsidered rare.  Allozymes Seventy-three allozyme loci known to be variable in O. mykiss  were screened in 612 fish (Appendix 2).Protein electrophoresis was conducted as describedby Aebersold et al. 3 Specific enzyme activities werestained according to Harris & Hopkinson (1976), orAebersold et al. 3 We followed Reisenbichler & Phelps(1989) and B. Baker (Washington Department of Fishand Wildlife, pers. commun.) for presumed loci forwhich data were obtained, the tissues in which theywere expressed, and the buffer systems with whichthey were resolved.  Data analysis To test for a recent genetic bottleneck, an analysis of allozyme and microsatellite data based on Cornuet &Luikart (1996) which examines differences betweenthe observed heterozygosity and expected heterozy-gosity based on the observed number of alleles usingboth an infinite alleles model and a stepwise muta-tion model was conducted on all study populations 3 Aebersold, P.B., G.A. Winans, D.J. Teel, G.B. Milner &F.M.Utter.1987.Manualforstarchgelelectrophoresis:Amethodfor the detection of genetic variation. U.S Dept. CommerceNOAA Tech. Rept. NMFS 61. 19 pp. using BOTTLENECK (version 1.2.02 (16.II.99) Piryet al. 4 ). Results Analysis of scale samples of anadromous steelheadindicates that smolting takes place at age three or fourin Sashin Creek steelhead. The smolts spend 2–3 yearsat sea and repeat spawners comprise 10–30% of theanadromous adults. Age validation of scale reading onresident fish in Sashin Lake by marking or tagging hasnot been accomplished, and reliable aging of older fishis difficult; however, resident males mature as earlyas age two and commonly at age three and femalescan mature at age three and age four. Maximum age isthought to be at least 8 and possibly substantially older(F. Thrower, unpubl. data).  Mitochondrial DNA Sashin Lake and all lake populations derived solelyfrom Sashin Lake, and the fish from the intermedi-ate section of Sashin Creek (Sashin Creek residents)were monomorphic for haplotype MYS1. Only theanadromous population collected from Sashin Creek and the Deer Lake population (that had multiple trans-plants of different srcins) showed any variation in theregion of the d-loop examined (Table 2). Anadromoussteelhead from Sashin Creek had four additional hap-lotypes and resident fish from Deer Lake had twoadditional haplotypes. One of the Deer Lake haplo-types (MYS10) was not found in the other study sites. 4 Piry, S., G. Luikart & J.M. Cornuet. BOTTLENECK:A program for detecting recent effective population size reduc-tions from allele data frequencies. Version 1.2.02 (16.II.1999).Available online. URL: http://www.ensam.inra.fr/URLB/ bottlenect/bottleneck.html.  115 Table 2 . Distribution of mtDNA haplotypes in Sashin Creek anadromous steelhead and sevenderived landlocked populations.Population mtDNA haplotype TotalMYS1 MYS3 MYS10 MYS12 MYS21 CLA1Sashin Cr. Anadromous 33 3 0 14 5 1 56Sashin Lake 26 0 0 0 0 0 26Sashin Cr. Residents 20 0 0 0 0 0 20Round Lake 20 0 0 0 0 0 20Betty Lake 20 0 0 0 0 0 20Davidof Lake 19 0 0 0 0 0 19Fawn Lake 20 0 0 0 0 0 20Rezanof Lake 49 0 0 0 0 0 49Deer Lake 22 3 1 0 0 0 26Total 229 6 1 14 5 1 256 Oneanadromoussteelhead(designatedSCS57)carrieda mtDNA sequence highly divergent from the other O. mykiss  haplotypes found in this study (Table 3).Alignment of this haplotype with other  Oncorhynchus sequences for the same segment of the mtDNA d-loop,showed close identity between this fish and a coastalcutthroat trout,  O. clarki clarki , from British Columbia(J. Nielsen, unpubl. data). Only a single variable sitedifferedbetweenthesequencederivedfromSCB57andour coastal cutthroat trout. Five additional sites differ-entiated both the British Columbia coastal cutthroatand SCS57 from a sequence derived from an interiorcutthroat ( O. clarki henshawi ) from Nevada.  Microsatellite DNA All 10 microsatellite loci were variable in at least oneof the study populations. Allelic variants ranged froma low of three per locus (One8) to a high of 15 (Ssa85).A total of 111 allelic variants for the 10 loci weredetected in the eight study populations (Appendix 1).Of these, 84 were unique (present in only one pop-ulation) or rare alleles (whose frequencies were lessthan or equal to 5% of all samples combined). TheDeer Lake samples, which were used for contrast, had17 additional unique alleles. The anadromous SashinCreeksamplescontained24rareand35uniquealleles,whereas the Sashin Lake resident population samplescontained only 15 rare alleles and one unique allele(Figure2).Whentheuniqueandrareallelesarepooledand adjusted for sample size, the anadromous fish hadon average one unique or rare allele per fish, whereasthe resident fish had only one unique or rare allele perfour fish.Differences between the Sashin Lake residents andthe secondary transplant populations were far less dra-matic. The Sashin Creek residents in the intermediateportion of the creek and the Round Lake populationhad a similar ratio of unique and rare alleles per fish asthe Sashin Lake population. The four other lake pop-ulations had ratios varying from eight fish per uniqueor rare allele in the case of Betty Lake to five fish perallele in Rezanof and Davidof lakes to about four fishper allele in the Fawn Lake population.Distributionofthe27commonalleleswasmoreuni-form and did not show a reduction as a result of the ini-tial transplant to Sashin Lake. They ranged from a lowof23inBettyLaketohighsof27inSashinLake,SashinCreekresidentsandRoundLake,comparedto26intheanadromousSashinCreekfish.Allthesecondarytrans-plant lake populations initiated with 50 fish had lostcommon alleles (from 1 to 4 per population) comparedto the secondary source population (Sashin Lake).  Allozymes Seventy-three loci were examined of which 18 werefound to be variable in at least one of the studypopulations and the remainder, 52, were invariant(Appendix 3). Within these 18 loci, 40 allelic vari-ants were found in the eight study populations. Onlysix of these 18 loci were polymorphic (all study pop-ulations combined). Fourteen common alleles weredetected among this range of loci and populations.There were 13 unique and two rare alleles among the18 variable loci. The anadromous steelhead samplehad eight unique and two rare alleles which, whencombined and adjusted for sample size, implies one
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