AsPECTSOF NAl11RAL AND ARTIFICIAL HYBRIDIZATIO N BETWEENBROWN TROUT AND ATLANTIC SALMON
IN NEWFOUNDLAND
by
Colin McGowan.B.SC.Agr.
A'IbeslftSubmi ttedinPartIalFulfillmentof the Requirementsfor the Degreeof
Muter of SCience
Department of Biochemistry Memorial Uc.lvenlty of Newfoundland.
St.John's,Newfoundland
April 1992
1+1
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Abstract
1\voproject s were und er takento obtain informat ion at themolecular.
organ is mal and ecologicallevels of organiz ationconcerningthecauses anddynamicsof natural hybridizationbetweenbrowntrout(Salntolrlllla L.)and Atlanticsalmon(Sa/mQ sa larL.)in Newfoundl and .
(1)Proteinelectrophor esis and mitochondrialDNAanalysi swere used to detect the frequencyand directionof naturalhybridizationbetween thesespecies in nine Newfoundlandrivers . Intotal.37 hyhrids were discovered Inasam ple of 792 juve nilefis h fora regionalfrequencyof 4.1 %.Localfreque ncie sran ged from0to 18.7%and were significa n tly het erogeneous.All of the hybrids sam pled were producedfrom maunge betweenfemale brown trou tandmaleAtlanUc salmon.
(2)The relativeviabilityof hybrids producedusing anadrornouebrown trout.Atlan ticsalmon grnse and sexuallymatureAtlantic salmon parr from a Newfoundland riverwas inves tiga ted. Thesperm ofsexually maturesalmon parr perfonnedequal ly wellcom pa red toanadrom ous salmon sperm when fertilizing both salmo n and trouteggs.Hatching successwas high in allcrosses and controls.Byfirst feeding. hyb rids producedusing brown trout eggs had suffered higher mortality and were smaller insize than the reciprocal hybridand both parentalcon trols.
Hybridsproduced usingsalmoneggsexhib ited highvtabnuyand by first feedingweregreaterin size than both parental controls.A comparisonof hatching time and length of hat chi ng period suggests a paternal influence Inem bryo development.
Possible reasons for the breakdown of prereproductive Isolating mechanisms betweenNewfoundlandbrowntrout and Atlanticsalmon are consideredIn lightof thecontrastingres ults obtainedfrom the two
tnvesugauons.Reproductive characteristicsof the populaUons involved appear to havea major Influenc eon thedyn amics of hybridization between these species in Newfound lan d. It is proposed that an abu n dan ce of sexuallymatureAtlantic salmon parr in Newfoundland str eam s is responsible for both the frequency and direction of hybridizationobserved in th isstudy.Possible reason s forthe durerences Inviability ofreciproc al hybrids and the significance of results wtt h respec t to theaquacul tureind us try are also discussed.
I"
Ackn owledge men ts
Iamdeeply indebted to the many people who have asetetedmein completing this work.I thank my supervisor. Dr.WilIlrun S.Dav'tdsou.
for his ideas. support. gutdnnce and friendship in helping me to reach this point.Mycolleaguesin the laboratory have been Sylvia Bartlett, VictoriaBtrt-Frtesen.MichaelCutler. John Goodier and HellenMcVeigh.
Theyhavesacrificed their time for me In both thelab nod the field and haveencouraged me with their enthusiasm.
My supervisory committeehas been Dr. GeneHerzberg.who provided usefu l comments on statisticalanalysisand experimentaldesign. and Dr.JohnGibson. whose helpIn the field has been of great value. Both have provided useful comments in the revision of lhls and ouicr manuscripts.
This project has benefitted from frequent discussions with Tim Birt, Steve Carr and Dave Innes. Rex. Porterand MichnelO'Connellntthe Departmentof Fisheries and Oceans,St.John's.Newfoundland have been very helpfuland supportive.The assi stanceof Jim Deverau xand Ross Wilsonat the Ocean Sciences CentreinLogyBaywasalso greatly appreciated.
I would liketo thank Lesley Howes,Wayne Donnelly and my parents.
KenandIsabelle McGowan. for their moral support.
"
Contents
Abstract Acknowledgements Conten ts List of Tables List ofFigures
Chapter 1 General Introduction 1.1.Organisma!Hybridization
1.2.The StudyofHybrtd tea tton 1.3.Theories of Hybridization 1.3 .1.Ingenera}
1.3.2.10fish
t.4. BiochemicalApproach to the Studyof Hy bridizati on 1.5.Hyb ridizationIn theSubfamily Salmonlnae 1.6.Atl an ticSalmonand BrownTrout 1.6.1. In general
1.6.2.InNewfou n d land 1.7.Objec tivesofthePrese n t Study
iii tv vii viii
4
10 10 12 16
Chapter 2
Natural HybridizationBetween Atlantic salmon and BroWllTroutinNewf oun dl an d 2.1 .Introduction
2.2.Materials and Methods 2.2.1.Sample collection 2.2.2.HybridIden tifica tion
2.2.3.ldenuflcattcnof hybrid maternal species 2.2.3.1 .DNApreparation
2.2.3.2.AmpUficnlion of cytochromebgene 2.2.3.3.Sequencing of the cytochrome b gene 2.2.4. Statistical analysis
2.3.Results
2.3.1.Hybridizationfrequency 2.3.2.Hybrid matemal species 2.4.Discussion
Chapter 3
ArtificialHybridizatJon Between Newfoundland BrownTrl)utand AtlanticSalmon
3.1.Int rod uc tion 3.2.Materialsand Methods 3.2.1.Notation
\9
20 20 23 2-1 3\ 3\
33 35 35 35 39 39
42 43 43
3.2.2.Breedi ng stock
3.2. 3.Spawni n g and exper ime n tal design 3.2 .4. Incubation andrearing 3.2.5.Length meas ureme n t 3.2.6. Stattsucalanalysts 3.3.Results 3.4.Discu ssion
Cbapter4 Summary and Overview
4.I.Natural Hybridization 4.2.ArtificialHybridization
Reference s Appendlz:
43 44 44 48 48 49 57
62 67
69 82
List of Tables
Table 2.1. Samplesites and thenumber of Allantic salmon.
brO\VI1'routandhybridscollectedat each. 38 Table3.1.Parent soffamilics.numberofeggs perfam il yand
relative pcetucn ofeach family In incubato r.
Incu batorconstsredofeight egg-trays stacked verucanvontop of one another with eachtra y
divided into four cells. - - 1 - 7
Table3.2.Parents of famllies[c). number of cggsreruuzcd ineachfamily(no). mortalityfromfertilization to hatchi ng(mh)and mortality from hatching to cgg-sacabsorption[mf]with percentagesIn brackets. 50 Table3.3.Mean percentage survival(m )from fertiUzalionto
hatch in ginclu din g thenumber offam ili es(1.11and
therange of percentages(r). 52
Table3.4.Chi-square andPvalues(in brackets) for the comparisonsof survivalfrom fertl1lzatlonto hatchingincludingthetotalfrequency of survivors (s) and mortalities(m)for each type of cross. 53 Table3.5.Mean percentagesurvival1m) from hatchingto
egg-sac absorptionincluding the number of families (n) and the range ofpercentages[rio 54 Tab le3.6.Chi-squareandPvalues(inbrackets) for the
comparisonsof su rvivalCrom hatchin g to egg-sac absorptionIncludingthetotalfrequency of survivors(5)andmort al iti es(m)for eachtype of
cross. 55
Table3.1.Degree-dayat wh ic h50%of eggs hatched (h)and period from begin n ingto 100% hatched (p) In=
numberoffamilies). 56
Table3.8.Mean length(I)in mm.of fish Immedtately after egg-sac absorptioninc ludingstandard deviation (sdl, range (r)and numberof Individualsmeasured (n). 58
List ofF.lgures
FIgure 1.1River systemsIn whichbrown trout are knownto havebeenintroduced.AlScottish brown trout":
l.Dildo pond.2.Topsail ponds.3.Rennie'sMill river.4.Waterford river,5.PettyHarbour ponds.
B)Germanbrown trout;6.Whlteway'spond.7.Robin's pond.8.Hodgewater pond.C)EnglIshbrown trout:
9.Maln river.(Frost 1938, TheEvenlTtgHerald..
St.John·s.Nfld.Peb.23, 1892)
·Other introductionsof Scottish brown trout may havebeen madeIntheSt. John'sarea.but are not listedhere due toambiguitiesinthe historical
reJXlrts 14
Figure1.2.Present Distribution ofbrowntrout in Newfound land.
1.Middle brook,2.Broad lake,3.Spread Eagle river 4.Dildo pond,5.Hearts Delight river.6.Hearts Conten t river.7. Hal fway brook,8.North river.
9.South river, 10.Maloney'sriver.II. North Ann river.12.Quarry brook.13.Seal Cove river.
14. Manuel's river.15.'ropsan river,16.Broad Cove river.17.Beachy Cove river. 18. Main river.
19.whuewaye pond.20.Robin'spond.21. Rennie's MI1l river.22. Waterford river. 23.Petty Harbour ponds,24. BayBulls river.25.Lowerpond.26.Mobile river.27.Tors Cove pond.28.Cape Broyleriver.
29.Renews river.30. Chance Cove river.31. Salmonier river.32.Collnetriver. 33. North Harbour river. 34.North East Placentia river. 35. RedHarbour river. 17 Figure 2.1.Location of sampled rivers:1.Moblle river.2.Cape
Broyle river,3. Renewsriver. 4.Salmonler river, 5.NorthEastPlace n tia river.6.Spread Eagle river.
7.Heart s Delight river.8. North river.9.NorthAnn
river. 21
Figure 2.2 .Zymogcams of loci used to Identifybrowntrout (SalmotJUttal. Atlantic salmon(Sa/mo solar) and theirFI hybrids.APhos p hoglucom u tase (EC 5.4.2.'2;Pgn-l,21.B.otuccse-e-pnospnare
Isomerasetsc5.3.1.9:Gplo l . 2 . 3 ). 25
Figure 2.3.Ph otographsof aymogramsused to identifybrowntrout fSaImotnJUa ).Atlanti c sal mon(Sa lm o salar), and theirFl hybrids .A}Phosph oglucomutase (EC 5.4.2 .2:Pgrn·1.21.BI Otccoee-e-phosphate
isomerasetee5.3.1. 9: Gpj-1.2.3). 27
Figure2.4.Nucleotide sequen cesof segmen tsof thecytoch romeb genes of browntro ut(Sa lntotruttaland Atlantic salmon(Salmosala rl.Numbersreferto the rela tive codon positionsfor thecyt ochrome b moleculeas det erm inedbyKocheretal.(1989) .
•Indica tes differenc esusedto Identify species. 29 Figure 2.5.Photograph of anau to-ra diogra ph used to determin e
the DNAsequenceforasegment of thecytochr omeb gene. sequencessh ownarethoseofabrowntrout (&limotruttal,two hybrids.and an Atlantic salmon(Sa lmo salar).
•in dica tedifferencesused to iden tify maternal
species . 36
Figure 3.1.SitesonNort h East PlacentiariverwhereAladu lt anadromousbrown troutandB)adult anadromous Atlantic salmon and sexuallymaturemaleAtlan tic
salmon juveniles werecollected. 45
Figure A.I. Location ofsample sites on Al theMobileriver(sit es a andbl,B) the CapeBroy leriverand Cl the Renews river.
(.'" sample site.scale",1:50000 )
FigureA.2.Location of sample sites onA)the Salmonlerriver.
B)the Spread Eagle riverand C}theHeartsDelight river.
(.'" sample site,scale=1:50000)
Figure A.3 .Locationof sample siteson Althe Northriverand B) the Nort h Arm river.
(. '" sample site.eealee 1:50000)
83
85
87
figureA.4.Locationofsamplesites en the Nort h EastPlacentia river(sites a.b andc).
(scale=I:50000) 89
Ch ap ter1
General Introduction
1.1.Organlsmal Hybridization
Hybridization. atthe organiza tionallevelof tileorganis m.has been definedbyMayr(1971 ) asthe crossingoftwoindiv idualsbelo ngiJlg totwo unWcencturctpopulationsthathave secon.dartlycome into contact. 1l1is includes both heteroepecrncandconspcctnc hybridization: the tatter involving individuals who belong to the same species. but whose resp ective popu lations have undergo nesomedegreeof genetic divergence and are consideredto be dilTerentsubsp ecies.racesorforms.Although thedeflnition refers topopulationsasnatural. artificial hybrids canbe produced when gametesof dilTerent popula tions . bethey na tural or dom esticated. are broughtinto contactthro ugh human interventio n. 111e termsecondarilyspecifiestha thybridizing individualshave come into contactas ares ult of conditio ns which contrad ictthosemaintai ningthe reproductive integrity ofa population. This isbrough t about bythelos s ordistur banceofsomephysical orbiologicalbarrier whichhas existed be tween twopopulations orspeciesandhas permitted them todiverge genetical lyover time.
The extentof hybridization mayvarydependingonthecase.In general.
naturalhybridization inthe animal kingdomIs rare. When produced.
hybrids are usuallylow in frequ ency. low in viability. sterile or sufficien tly dissimil ar to eitherparentalpopulationtha tback crossl ngIs even les slikelytooccur (Mayr19711.Whenhybridization is rare.the ecologicalor gene ticiso lation between closely rela ted pop ula tion s is con s idered ttl bestrong. In contrast. a lackofisolationbetween
hybridizing populationscan resultInintrogression.In which there is a transfer and incorporation of genetic material from one species intothe genome of another (Billingtonetal.1988;Campton 1987:Dowlingetal.
19891.Ahybridswarm Is produced whentwo species orpopulations hybridize on a continuous basis withconstantexchange of genetic material betweenthetwo parental extremes (GyllenstenetaI.1985a:
Mayr 1971). Hybrtdzonesare narrow regions in which genetically distinct populationsmeet and hybridize.forming a cline between thetwo parental extremes(Barton andHewitt19a5).
Mayr (l9711 considered hybridiza tion to be of littlesignificancewith respectto the evolutionof higheranimals.However.hybridization and introgression can ultimately lead to a decrease in overall diversity (Nelsonand Soule 1987).Therefore. an understandingof the processis essential for biologicalconeervarton.
1.2.The Study ofHybridization
Studiesofhybridlzatlon generallyfall Into three categoriesof research covering the genetics. performance andecology of hybrids . Although the focus of aparti cul ar studymayfall in toone ofthese. the resultsare oftenrelevant to aJI three.
Geneticresearch concentrates oneither the product ofhybridization or causes of hybrid stertltty.Emb ryos resultingfroma hybridmating can be parthenogenic.in whichthepronu cleu s ofeither thesperm (gynogenesis) or the ova (androgenesis)is elimina tedduringfert illzation. orhybrid. In which there Is a geneticcontrib ution from both paren ts (Chevassus 1963). Zygotes can also bedtplotd. tr:lploldor tetraploid depending onthe ploidyofthe parental gametes or as a result of polyspenny (Chevassus 1983). The fertilityof a hybr:ld canvary from completelysterile . with
abnormal gonadal(Buss and Wright19571. gametic (Lincoln 19EII) or zygot~e(Bus sandMiller 196 7) develop men t. tocompletelyfertilewith!';e ability to produceviableF2and F3gen era tion s .backcrosswtn-either parentalspecies.orevenhybridizewith athirdspecies (Buss an d Wright 19571. Cytologicalstudies of hybrid sterility haveInvcsng atcdproblem s that arisedurtogmeiosis.Abnormali tiesduringsynaps is. meioticspindle formalion or the first anaphase ofmetosts.are often the ca use ofinvia ble gametes (White197 3).
Asecond branc h of research focu ses on theorgarus mal level of organization and is conce rn ed with the relative appearance and performance of hybrids with respectto theirparent alspecies .These types of stud iesmeasure theviability ofhybridsfrom zygotetoad u lt and assess characteristicssuch as morphology. growthrat e. fertility and disease re sistance. Heterosis . a phenomenon in which highly heterozygous individ uals.suchas hybrid s.exhibitsuper ior perform anc e compared to their rela tively homozygous parents (Brown 1970: Mayr 1971:Kozlov1972:Mil'shteinand Popova1972 :Leary clat.1984).is often thesubject ofsuch hybridiza tionexperiments,Thedevelopmentof new breeds.sterilehybrid stra ins or thetransferof beneficial traitsfrom one species toanothe rcan be of significanteconomicimportanc e.
The study of hybridization at the ecological levelof organization examines population Iso lating mech an isms and their appare nt breakdown. 'Postmatlng' reproductive isolation isachieved through hybrid sterility. hybrid inviabilit y.physiological differences between hybridizingindividu alsor biochemical differences between hybridizing gamet eswhich prevent zygoteform a tion (Mayr 1971:Garbe rs 1989).
This type of isolation is for the most part theresult of stochastic processes whichhaveresultedin thegeneticdivergence.and subsequent incompatibility of hybridizing genotypes. It does not prevent hybridizationfrom occurring.but the facttha t this tncompaubtuty exists.
and that croeetng indi viduals waste gametesandlea ve fewer offspring.
drives the selection of'prema tlng' reproductiveisolatingmech anisms, Pre matingisolation involves themechan ical. behaviouralandspa tial barriers which prevent attempte d matlngsbetweenunlikeindividuals (Mayr 197 11. The result ofa breakdown in premating isola tionIs hybridization and the studyof naturalhybridizationis, for themost part.
the studyof the cause sorconditionswhich leadtothis breakdown and the subsequen t effects.
1.3.Theories of Hybridization
1.3,1.Tngeneral
In many cases. closely related speciesare similarin theirreproductive morphology and behaviours.Their respecti vebreeding timesand places may overlap as can their physical requirements for successful reprodu ction .Insuchcases. premating isolation is susceptibl e to disturbancesand maybreakd own. Mayr (1971) lists severalconditions or circumstances whichmay facilitate the breakdown ofpremating isolation andexplain some of the broader trends observed in theanimal kingdom.
Hybridization tends tobemorecommon amongexternally fertillzed organisms, such as fish.thanitis among Internally fertilIzed species.
such as terrestrialvert ebra tes. During external fertiliza tion.spermmay be carried by watercurrentsto the ova of another species spawning nearby.Also.the ova from onefemalecan be fertilized by several males at once as the ability of a femaleto restrict access to her ovaIs much more limited than it is for Internallyfertilized females.
The strength.or weaknessof the mating bond between individuals may
influ encethepotenti al for hybridization.Inmany spcctce. olTsprlng requ ireaconsiderableamount ofcareon behalfof oneor both parentsif reprodu cti onis tobesuccessful. Conseq uently.It is import an t that individ ualsbe discri minating whenselec Ung a matein order to avoid mistakes whichmay later prove costly. In otherspecies.mati ng bonds areweak wit h parentalinvolvem ent ending shortlyaftercopulation.
Individuals may not spendasmuch time selecti ng a su itablemate and accident al hybridization maybe more common.
If there isconsiderabledisparityin the abu n dance of differentspecies.
rarespecies may havetroublefinding suitabl emates.Asreproductiv e drives bu il d.rareindi vi d ualsmay become receptive toinapprop riate sti mu liand mate with thewrong speciesrather thannot mate atall.
Ifspecies arespatially.or geographically isolat ed. therewillbe no selective reenforcement of other prematlng isolatin g mechan isms. Circu ms tanceswh ich bringsuc h populationstogetherare often the ca use of hybridization.Degradat ionor limitation ofavai lable habitatmay force different species to use the same area for reproduction. Introduction ofaspecies intoanareawhereIt is not normallyfound.
often resultsin its hybrldlzation with relat ed .native species.
1.3.2.In.fi.~h
Much ofwhat has been summarized byMayr(l97l)concern ingthe causes of hybridization in theanlmal kingdom. was first ident ifi ed by Hubbs (1955).who.with his colleagues . spen t fifteen years documenting cases of natural hybridizationbetweenfishspecies. In general .Hubbs (19551foundhybridization to be much less frequentwithin the marin e environm en t than among freshwaterfishspecies. Thisbroadtrend was attributed to the relative Instability of lakes and riverswUh respect to the sea.Also.during ttmee ofchange. dispersal routes tosu ita ble habitat arc
not as limiting In themar ine environmentas theyareIn freshwater.
Hubbs(1955)also predictedthattheincidence of hybridization shou ld incr easewith latitude.suchthatthehighes tfreq uencieswilloccu r Inth e nctercucfreshwater regions ,which havebeenst rongly effectedbyglacial advancesduringthePleist ocen e.and decre ase toward theeq uatorial regions.
In addition LO the above. Hu bbs (1955)iden tified four ecological circumstan ces assoc iated with an increased tendencyof fish speciesto hybridize.(1)Fish speciesreproductivelyisolated byenvironmentalcues.
may breed together where a disturbance,eith er naturalor human ind uced.has rendered the environment In terme dia te. (2) Spawning habitat is llmited suchtha t distinct species of breedingfish are forced intoclose proximity.(3)There is considerabledisparityin theabundance of relatedspecies.(4) A specieshas been introducedinto an areawher e It is notnative.These cond itions are frequentlycited aspossiblereasons for the breakdownof reproductiveisolatio nwhen natural fish hybrids are discovered. However,it Is usuallydifficulttoSingle outapredominant cause In a particular case. Thisis partlydue tothe facttha tna t u ral ly occurring hybrids are rare and difficult to find.Inman ycases.the only infonnattonavaila ble Is the occurrence of hybrids and sometimes the freq uency.
1.4 . 81ochem.1calApproach totheStudyof Natural Hybridiza t ion SincetheIn trod u ction or biochemical techniquesto populationgenetics.
many suspected hybrtds have beenconfinned and many new cases of hybridization discovered.Contraryto the contentionof Hubbs (1955) that fish hybrtds are almostuniversallyintennedlate In morphologyto their parental species, Nyman (l970) found that in many ca ses.
morphological charactersare similar to eitherparent andtoa much
lowerdegree. Interm ediate.Leary et al.(1983)observed that thehybrid prod uct of brook trout(Sa lLlelinus !ontinal[s) and bull trout {SalLle/(nus conjl uen tusl had consisten tlyhigh meristiccountswit h respecttothe parental controls and attributedth is to theirrela tively slow embryonic developm ent. Also.many closelyrelatedspeciesmayoverlap in their morphometricand meristic chara cte rs making thepositive Identification of a hybrid difficult (Nyman 1970). In proteinsystems.enzymes arc expressed by sin glecodom inan t genes andhybrids usuallyexpress a complete summationof both parental genotypes {Nyman19701.Thus the application of protein electro phores is has provided a morescnstnvc mean sbywhich hybrids can beidentified.
Recen tly developed molecular tech n iques . which can be used to char acte rizethe mitochondrialDNA (mtONA) ofagivenspecies.have provided a means of obtainingadditional infonnatlonabout natural hybridization(Cam pton 1987).MtDNAisa circ ular. double -st randed.
extra -n u clear genome that does not recombine.It is generally considered tobematernallyinherited(Gyllenstenetat. I985b; Hutchisonctat.
1974). Gyllens te n et al. (1991) haveshown that paternal inheritanceof mtDNAcanoccu r in higher vertebrates. but that less than one In 1000 molecules are so derived. Also. repeated backcrosslng of female hybrids with the paternalspeciesisrequired (23 to 26 generationsIn mice) before enoughpaternal mtDNA is present In an individualto be detectedusing highlysensitive techniquessuchas the polymerasechain reac tion(PCRI (Gyllensten et al. 1991). Characterization of a given species'mtDNAcan beachieved byobserving the patterns genera tedon a gel whenthe molecule is cut with a particularrestriction en zyme (restriction fragment length polymorphisms or RFl.Ps) (Gyllenstenand Wlison 1987), or by direct nucleotideseq uencing of a mtDNA gene (Kocher ct al. 1989:
McVeigh et al. 19911.Therefore .by typing the mtDNA of a givenhybrid.
thematernal species canbedetermined.
several studies have already exploited thesepropertiesto iden tify ca ses of lntrogrcssion and understandmoreabout thecausesand dynamics of natural hybridization . Avise and Saunders (1984) used RFLPs to determine the maternal speciesof naturally occu rring hybr idsbetween speciesof sunfish (genus LepomLs). Theydiscovered a tend enc y for hybridization to occur between parental speciesdUTering greatly In abundance and a tendencyfor therare species to con tribu te the female parent. Theapparent associationbetweenrelative species abundance and direction of hybridization was attributed to the reproductive behaviourof these species.Within this genus. males construct and protectnestsand are subsequentl yselectedby females. In theabsence of conspeclfic males,rare femalesmaychoose tomatewithcongeneric males Instead(Avise and Saunders 1984). Herkel et al.(1990) found that naturallyoccurringhybridsproduced by northern pike(Esox lucius) andchai n pickerel(Esox niger)always hadch ainpickerelasamaternal parent.However. previous experiments in the lab had shown that hybridsproduced using northern pike femalesdo not progress beyond the zygotic stagewhereasth ereciprocal cross is'perfectly viable(Buss and Miller1967). Gyllenstenet al.(1985a) combined allozyme and mtDNA data to show that themalesandfemalesof Introducedand nativecu tt h roat trout subspecies (Oncorhynchusclarki lewtsf and O.
clarki bouvieri) contributed equallyto hybrid swarms. BlIIingtonetal. (1988)discoveredevidence oftn trogreeeton between twoStizostedlon speciesIn the great lakes. Inthiscase.two fishpossessing walleye (Stizostedlon vitreum) nuclear genotypes were found to have eauger (Stfzostedloncanadense)mtDNA genotypes.
1.6 .Hybridizati onintheSubfamilySalmonlnae
Fishrepreeenungthe su bfamil y Salmoninae.arecomm onty referred to
as salmo ni dsor the sarmons.troutsandcharrs. Severalcxam ptceof natu ral hybridiza tioncan befound within thissub family.Innort he rn Labrador. acetic chart- (SaIl1€'linus alpU1us) nave been reporte d to hybridizewith bot hbroo ktrou t(5.Jonttn allsJ(Hammaretat1991)and laketrout(Salvelinusnama!Ptush1(Hanunaretal. 1989).Brook troutIs alsoknown tohybridizenatu ral lywith brown trout(Salmotrutta)to producethe steri le'uger trout' (Brown19661.CamptonandUtter 119 8 51 identified natural hyb rids between eteethead trout (Oncorhynch us mykiss)andcut t h roat trout(0.cla rki )In two Puget Sou ndstreams. Natural hybridizationhasalsobeen disc overed betwee nchin ook salmon (Oncorhynchustshawyt sch a)and coho salmon(Oncorhynchuskisutch)In NorthernCaliforn ia(Bartley et at1990).Numerous examplesof natural hybridizationbetwee n brown trout(5.tndtal and Atlantic salmon(Salmo salar)havebeenreportedinboth Europe{Payneetal.1972:Solomon andChild 1978;Crozie r1984:Garciadeteanueandverspoor1989;
Hurrelland Price1991:Jan ssonet al.19911andNort h America(Beland etal.19 8 1;verspoor 19 881.Theabilityorsalm onlds toprodu ceviable tn terspecresandin tergenertc hybridsund erartificialconditionshas also beenwelldocumented (Chevassus 1979 ).
Salmon ids are orsu bs tan tial economic value, eupporttng both a conunercial andrecreation al fishery.Their impo rtancehas alsogrown recently with r-espect to the worldwide aqua c ult ure ind us try.
Consequently,the re hasbeenastro ng desi reon thepart of governmen t agencies andsportfulhe nnento enhance existin gpop u lations as wellas tryto increasespeci es diversit yin popular fishingrivers. Hatch ery reared fish have often beenIntroduced into riversor lakeswhere theydo not naturally occur.Such Introductionscan put native fish populations at risk through thepotential ofhybridization and tntrogrcseton.A dtsturbtngexamp leof thl.s wastheIntroductionof coastal rainbow trout (0. mykfss) intoseveral inlan d lakes throughoutthe western United
10
States.Hybr1dizaUonand subsequentint rogression ofintrod ucedfish withnativecutth roattroutto.darid)hasresulte din the perm anentloss ofseveralunique.locallyad a pted gene pools(Be nke197 21.
Examplessuchas the aboveha ve resu ltedIn a much morecautious approa ch to the managementofsalmonIds.with agreate rem p has ison the pro tecti onora population'sgeneticintegri ty(Billingsl ey1981:Stahl 19 87).However.a rapidly growing salmonidaq uacult u reind us try has raised concern over the poten tialeffect ofacciden talintrod uctions.
Damagetosea pen s canresult in thereleaseofseveralthousandfishat atime.Theimpactofthese'domesticated'strains on wild populationsis thesu bjec t of cons id erableectenttn c debate(Hanse net aI.199 1).
1.6.Atlantic Salmon and Brown Trout
1.6.l.ingenera l
Brown trout(Sa/mDtrutta)and Atlanticsalmon(Salmosalanareth e twopredominan tspecteeofthegenusSalmo.They arefoundnatu rall y
throughouttheNorth AUanUcdrainagewithAtlanti c salmonoccurring in
bothEuropeand NorthAmerica (Sco tt andCros sman1973)andbrown trou tbeinghistOricallyres tricted totheeasternAUanUc (Macertmmon and Marshall1968).Both specieshave been introducedtoregion s outsidetheir naturalrange whereself-sustain ingpopulationshavebeen es tablis hed (Crossman1984:McDowall 1984).Naturalized populationsof brown trout cannow befou ndth ro ughout NorthAmerica (MacCrinunon andMarsh all1968).
Brown troutand AtlanUc salmon have a sim1lar life-history and reprod uct ive biology.Both spawn Inthe (ail.withfemales burytngtheir fertilizedova Inthegravelly substrateofa freshwaterstre am. Embryos
II
develop during the winter. hatchingby earlysp rin g as 'atevtne'and emerging from thegravelas 'fry'once their egg· sacs have been absorbed.
Avari a ble number of yearsarespent in the fluvialenvi ronme nt 0\5 juvenilesor 'par r ' before migrating downstream Into either a marine environment (in the case of anadromous fish) or a lake lin the caseof lacuslrian or land-lockedpopulations}. Some brown trout and Aunuuc salmon complete their life-cycle in the Fluvtal environment. After spending oneor more wintersatsea or in a lake. mature adult nsu ascend a river (usuallytheir natal stream) to reproduce. Unlike Ihe Pacific salmons. which characteristicallydie after spawning. both brown trout and Atlantic salmon areueroparous.and may spawn againin Inter years. Both species also display an alternativelife-historypattern III which males can mature sexually within the river as parr and successfully fertilize larger.adult females (Jones and King 1952: Dalley et al. 1983: L'Abee-Lund etat.19891.
Atlantic salmon exhibit relatively little variability when compared to browntrout.Throughoutitsrange,Atlantic salmon is morphologically uniform,whereas brown trout Is polytypic and etten ctassmed Into distinct morphologicalandecological races(Benke 1972. Fergusonand Mason 1981). In a genetic analysis of Atlantic salmon across itsentire range.Slabl (1987)found nine out of 38 sampledloc i to be polymorphic and a genetic distance between European and North American population of 0.04. In comparison,Steven and McAndrew (lWOI surveyed brown trout populations In SCotland alone and found that 13 out of 34 lociwere polymorphic with a genetic distance between some populations of 0.05.
Gyllenstenand Wilson (1987) analysed restriction frogment length polymorphtsms (RFLPs)ofthe mitochondrial DNA(mtDNA) to detennlnc a genetic divergenceof approximately 6.0% between brown trout and Atlantic salmon. Similarly,McVeigh et al. (1991) observed a 5.2%
12
nucleotidesequence divergenceofthe mitochondrialcytochrome b gene between thesetwospeci es.The latterstudyalso discovereda biastoward tran sition base su bs ti t u tions over transve reto ns andno amino acid substitutions within the cyt ochro m e b gene. This is con si d ered characteristicof species that have divergedrecentlyfrom a comm on ancestor (McVeighetat.19911.Incon tra st toits uniform morphology.
thekaryotypeofAtl an t ic salmon is variable with a diploidchrom osom e numberrangingbetween54and 58and an arm number of 72 or 74 (Hartley 1987). Browntrout have a stablediploid chromosome numberof 80 with 100 arms (Chevassus1919) .The Atlanticsalmon karyotypehas probablybeenderivedfrom abrown trout likekaryotype through centric fusionsand pencentric inversio ns (Benke 197 2;Hartley198 7 ).
Theability of brown trout and Atlantic salmonto producevia ble hybrids under artificialcondit io nswasfirst established In the earlybreeding experiments of Ashworth and Ashworth (1853.fromDangel et aI. 1973l.
Sincethattime.several researchershaveInvestigatedthe potenUal of this cross for both stock en han ce ment andaq ua cultu re purpose s (Cbevassu e1979). The possibilityof natural hybridizationhas always been postulated.However.overl ap of morphological characte risticsanda tenden cyfor hybrids to resembleeitherone species or the other made positiveidentlficatJon in the field difficult(Nym an 19701.The existence of naturallyoccurring brown troutxAtlantic salmon hybridswasfirst confinned byPayne et aI.(1972). using biochemicalmakers.
1.6 .2.In NewJOWtdland
Atlantic salmon are native to and distributed throughout theisland of Newfoundland (Porter et aI.1974;Scott and Crossman1973).There are two morphologically distinctforms of salmon found in Newfoundland; a residen t.freshwaterIorm.comm only referred to asth e 'cuanarucbe' ,and
13
an anadromoueform.There is evidenceofgenetic isola tion between these types where they coexistIverspcorandCole 1989;Birtctnl.1991 ).
Atlanticsalmon supportbotha commercialand recreationalfisheryIn Newfoundland.ThereIsalsoasmaJl.but growing salmonidaquaculture in d u stryonth~is land.
Several different strainsofbrown trout havebeen introd uced to the Islan dof Newfoundland.Between1886 and 1888theLochLeven trout wasimportedfrom Scotlandbythe NewfoundlandGameFish Protection Societyandused to stock lakes in and arou ndthe St. John'sarea (Andrews 196 5).These fis hwer e also in troducedtothePett yHar bour ponds. ponds drainingIn to the Top sail river andSou t h DildoPon d , Trin ityBay(TheEveningHerald .St.John's.Njld.Feb.23.18921.Germ an bro wn troutwerebro ug h t to Newfo un dlan d In 1892and plante d In Whiteway 's pondandRob in'spond nearTor ba y andinto Hodge wa ter pond.near Whltbum(Fr ost1938). Itis also reported thatbrown trout CromEnglan d were brought to theislandsome tlmein 1905or1906 and pu tinto Clement'spon dand Miller's pond ICormerl y Lee'spond)near Port ugal CoveIFrost I 938).Figu re I.} Indicate sthe riversystems where browntroutarebelieved to havebeenIn trod uced.
BrowntroutinNewfllundlandarestill commonlyreferredtoasGerman. LochLeven . Scottishor English.eventhough thereIsno mcrpnologtcel mean soCteUlngonetypeCromanother(Frost 1938.1940).Popuiauonsof SCottishbrowntro utcanstilibefoundintheTopsail system:iso lat ed by impassiblewate rfalls.ThelakesIn Torbayand Portugal Cove.In which theGe rm an andEnglish browntro u t were Int rod uce d respe ctivel y.are also isola ted bywat erfalls an dstillcontai n repre s e ntativesoCthes e strain s{Figu re1.1l.
Since the Origi na lin t rod uc tion s.there hasbeen no managem ent
o r
brown trout on the Island oCNewfou n dlan d. Adap tat ion to an
- - - - - --- - -- - . _ --- - -_ ._---- --_ ...- --
14
Figure1.1.
River sys tems in which brown trout are knownto havebeen introduced.Al Scottishbro wn trout": 1. Dildo pond. 2. Topsail ponds. 3. Rennie'sMlll river.4. Waterford liver. 5. Petty Harbour ponds. BIGe nnan brown trout:6. whtteway's pond.
7. Robin's pond.8. Hodgewater pond. Cl EngUsh brown trout:
9.Main river. (Frost 1938.TheEvening Herald.St.John's, NJld.Peb.23.1892)
·Ollter lntroductiortSoj Scottish brown trout may have been madeinlheSt. John'sarea. but are notlistedhere dueto ambiguitiesinthe historical.reports
15
16
anedromous life-cycle has allowed this species to colonize several rivers both on and off the Avalon Peninsula.Populations of brown trout may be established as far north as Middle Brook. BonavtstaBay and as far west as the Red Harbour River on the BurinPeninsula (Porter ctal. 1974) (FIgure 1.2), Historically. brown trout have been of little economic importance and are often considered a pest in Newfoundland. Howe ver.
withrecentdeclines in Atlantic salmon stocks. anadromous brown trout are becoming a popular alternative for the angler.
Widespreadnatural hybridization between brown trout and Atlantic salmon In Newfoundland was first reported by Ver spoor (19881.No formal investigation ofartificially crossed brown trout and Atlantic salmon fromNewfoundland has ever been undertaken.
1.7.Obje c t ive s ofthe prese ntst udy
The objective of the present studywasto gain a deeper understanding of the causes and dynamics of natural hybridization between brown trout and Atlantic salmon on the island of Newfoundland. Twoprojects were undertaken to obtain infonnation at the molecular.organtsmat and ecologicallevels of organisation. (II In the field. several sympatric populations of brown trout and Atlantic salmon were sampled and analysed using protein electrophoresis in order to determine the frequency of hybridization at both a local and regional scale. The relative matemal contribution of each species to hybrid matlngs was then established through mtDNA analysis. (2) In the laboratory. artificial hybridization experiments compared the relative viability of the reciprocal hybrids with respect to their parental controls. This tnformatton was used to interpret resultsobtaIned from the field survey conceming the direction and frequency of hybrid matlngs In the wild. In addition.the performanceof hybrids was assessed with respect to their potential in the aquaculture industry.
17
Figure 1.2.
Present distribution of brown trout in Newfoundland.1.Middle brook.
2. Broad lake.3.Spread Eagleriver.4.Dildo pond. 5.Hearts Delight river,6.Heart s Contentriver, 7.Halfwaybrook.8.North river, 9.Southriver, 10.Maloney's river,11. North Annriver,12.QUiltry brook.13.Seal Cove river.14.Manuels river,15. Topsailriver.
16.Broadcove river.17.Beachy Cove river,18.Main liver.
19 . Whiteway's pond.20. Robi n's pond.21.Rennie'sMill river, 22.Waterfordriver,23. Petty Harbourponds,24.BayBullsriver.
25.Lowerpond.26.Mobileriver,21.TorsCovepond, 28,Cape Broyte river,29.Renews river.30.ChanceCove river.31. Salmonlerriver.
32. Colinet river.33. NorthHar bo ur river.34.Nort h East Placen tia river.35.RedHarbour river.
18
19 Chapter 2
Natural Hybridization Between Atlantic Salmon and BrownTroutinNewfoundland
2.1.Introduction
Nat ural lyoccurringhybri dizatio n betweenbrown trout ISalm otruLta) andAtlantic salmon (Salowsalar )was first reportedon the Avalo n peninsu laofNewfoundlandbyVerspoor(1988 ).At thetime. the ObSClVCd hybrid freque ncy of 0.9% was relative lyhighwhencornperedtovalue sof 0.3to0.4% recor ded IntheUnite dKin gdom (Paynectat.1972:Solom on andChil d 197 8: Crozier1984).Thi s differencewasattribut edtothe fact thatbrown troutarenot indigeno usto NorthAmerica(MacCrhnmon and Marshall1968, Ve rs poor 1988).
More recently,these species have beenfound tohybri d ize atfr equenci es of2.3%inSpanishrivers (Garci a de Leantaand Verspoor1989),13%in Swedis hrivers {JanssonetaI.19911 and 1.4% InEngli sh rivers (Hurrell and Pri ce 1991) whereboth browntrout andAtlanticsalmonarenati ve.
Suchhighfrequencies suggestthat specificecologicalconditio ns maybe responsib leforthebreakdown ofprereproductiveIsolating mech an is m s.
InNewfound lan d.factors otherthan the Intr oductionof brown trou tmay act to enco u rage natu ral hybri dizationbetweenthesespecies.
The studypresen tedIn thischap tercombinesproteinelectro phoresis and mtDNA analysis to determineboththe frequ ency and the directionof natural hybri diza tio n between brown tro utand Atlanti c salm on In Newfoundlan d.This Informationis usedIn conju nction withIn formation presen tedin chapterthreetoIdentify possiblereason s for the breakdown
20
of prereproductiveisolating mechanisms between these speclee in the wild.
2.2.Mat eri als and Meth ods
2.2.1.Sample coUection
Atlanticsalmon and brown troutparr weresampledfrom nineriver systems on theAvalon peninsula of Newfoundland during a period from Augus t 24.1990 toNovem be r 30.1990 (Figure 2.11.Some of the rivers were sampled at more thanonesite.The locations of each sample siteon allriversarepresentedin the appendix.Each oftheseliver systems has been reported tohaveanadromouspopulations of both brown trout and AtlanticSalmon (Porter et aI.1974;T.R.Porterand R. J. Gibson.
Department of Fisheries and Oceans. St. John's, Nfld.. personal communication).No introductions of eitherbrown trout orAtlan tic salmon have beenmade into any of the riversin this study.All salmon populationsarenaturaland all brown trout populationshave colonized therivers by way of theocean.
Parr werecollected usinga 12 volt.back-packelectrofisher. At most sample sites. shocked fish were pickedup from the waterusing a small netattached to the end ofthe anodethen transferredtoabucketof water.Th{s techniquewasmost effective when samplingtheriver In a downstream direction with the current pushing stunned fishinfro nt of theperson sampling.In some cases parr were driven downstream.using theetectronsher.intoa beach seine whichwas stretchedacross the river and securedon both banks.
Once ca ptured.fish were killed In the field using an overdose of anaesthe tic.Four tabl etsof Alka-seltzer(1300 mg acetylsalicacid.7664
21
Figure2.1,
Locationofsampledrivers;I.Mobileriver.2.Cape Broyle river.
3. Renewsriver, 4.Salmonier river, 5.Nort h EastPlacen tia river.
6. SpreadEagleriver,7.Heart sDelight river.8.No rthriver.
9.NorthArmriver.
22
23
mg heattreatedsodi umbicar bonate,-1-000mgcitric acid;Miles Cana da.
Etobicoke.OnL)weread dedto the water'liththe fish. Carbondioxide produced by thetablets firs t renderedthe fish unconsciousand kill ed afterappro ximatelytenminut es. Oncedead.fish were packedonleeand tran sport ed backtoSL John'swhere they weremeasu red. classifiedas either brown troutor Atlan ti c salmo n. then individuallynumbered . packaged andstoredat·7c:PC.
2.2.2. Hybrid identification
Hybrid swere identi fiedusing alloeymeelectrop horesis.Skeletal mu scl e (app roximately 0.2 grams) washomogenized in an equalvolumeof 0.01 M Tri s·HCllpH7.5) andsubjec ted toce nt ru ugunonat 10.000x:g forone minut e.The su pernatantoreachsamp lewas appliedtogels using0.5x 0.3cm wicksmade (rom Whatman(Maidst on e. U.K.)num ber 4 filter paper. Toprevent protein degradation.samp leswerekept onice atall Urnes duringtheirpreparation.Extra supernatant was storedat ·70oC.
Sampleswere subj ected to electr ophor esisthrough an 11% horlzon tnl electroetarch gel (Conn aught laborat ori es. Windsor. Ont. I using a discontinuous Trts-cttncaci d/borate buffer systemdescri bed byRidgwa y etal.(1970). The electr odebu fferwas 0.06M UOH and 0.3 M HaBOa andwasrecycled after eachrunbymi xin gtogeth erthe por tio n s (rom the anodeandcathodereservoirs. Thegel buffer was0.0 3 M Trts,0.005M citricacid.0.0006 M LiOHand0.003M HaB0 3.Gelswere 20 ern wide.
10 em long and 0.5cmIn thic k ness.Gelswererunata consta ntvoltage of300 V (or 2.5to 3.0 hours and werechJlled between twoglasscooling pl ates through whi ch cold water was cons tantly flowin g. After electro phore si s,gelswereslic edinhalfso th attwozymog rarn scouldbe obtaine dfrom onegel.
Gelswerestai ned forthe enzymes glucose-a -pho sph ate Isomerase(EC
24
5.3.1.9 : Gpi-1,2.3)andph oe phogt ucom utaseIEC5.4.2.2;Pgnt-1,2) using standard techniques describedby Harri sand Hop kinson(976 ) with some adjus tme nts.ForPgn.25 mgofglucose.t-phoep hate (containing 1%gtuccse-t -e-dsphosph at elwasdissolved In 7.5mlof0.1M Tti.s·HCl (pH 7.4)with0.5mlof2%MgC12•0.5m1of 1%nicotinami deadenine dln uc leo Ude phospha teINADP).0.5mlof1%3·(4.5·dime th ylt hlazo l.2.
yll·2. 5· dlphe nyt tetrazolium bromide:thiazolylblue1MTI1 and 0.5mlof 0.4% phen az lne methos u lfate (PMS). To this.5pi of glucos e-G.
phosphate dehydrogenase(250 units/milwasaddedbefo re mixing in 7.5 mlof2%agar (at about 55OC).Thiswaspoured overthe gel and placed In thedarkwhile developing.The stainforpgt wasthe same except that 10 mg ofIructose-a -pbosphatewas used Instead of the glucoee-t- phosp ha tewith 1%gtucoee-f -a -dtphcsphate.Thesestains detect atotal of Ave Indepe nde ntgeneticloci.four ofwhich are diagnostic fordetecting hybrid s of brown trou tand Atlantic salmon (Vuorinen and PUronen 1984).These enzymeshavebeen usedtoiden tifynatu ralhybridsInboth Europe(Crozier 1984:Garciade LeaniZandverspoor1989:Hurrelland PrIce1991: Jansson el al.1991j and North America {Belan det al,1981i vers poce19881. Eachspeciesisfixedatfourofthefiveloci for protein s withdUTerentelectro phore tic mobilities.Hybridsexpressthesumofboth parental types(Fig ures 2.2and 2.3).
2.2.3.Identtfi<a_ of hybrid matemalspectes
The maternal lineageof hybridfish was ide n tifiedby amplifying a segmen tofthe mUochondrialcytochrome b geneusing the polymerase chainreactio n (PCR) (Saiki et al. 1988).followed by directnucleotide sequen cing The cytochrome bgene was chosenas Ithas already been characterisedfor both Atlantic salmon and brown trout by McVeigh et aI.
(lWl). Dtagnceuc segmentsused to Identifythe mtDNA of each species are showninFigu re 2.4. The preparationofDNA from skeletalmuscle.
25
Figure 2.2.
Zymograms of loci usedto Identify browntrout(Salmatrotta).
Atlantic salmon(Salmo salar) ,and their Fl hybrids.
Al Phosphoglucomutas eIEC 5.4.2.2:Pgn-l.21.B}Olucoee-Scpbospbate isomerasetEe5.3 .1 .9 ; Gpf·l .2.3 1.
Locus
Locus
1,2
26
Phosphoglucomutase
Hybrid
Glucose-6-phosphate isomerase
Hybrid
Locus
1,2
Locus
Figure2.3.
Photographsof zymograms used to identify brown troutlSafmo trottal.Atlanticsalmon(Salmosetorl,andtheir FIhybrids.
AIPhosphoglucomutaselEe5.4.2.2:Pgrn-1.21. BIOlucose-S'phoep hateisomerase(Ee5.3.1.9:Gpl-l,2,3).
A
B
S.trutta
28
hybrid
hybrid
S.salar
s .
trutta29
Fig u re 2.4.
Nucleotidesequencesofsegmentsofthe cytochrome bgenes of brown trout(Sa lmo trulta) and Atlanticsalmon(Salma salar).
Numbers refertotherelativecodonpositionsforthe cytochromeb molecule as determinedbyKocheret al.(1989).
•indicate s differences usedtoIden tify species.
30
110 10 0
5almg t0 1t ta : 5'-TTATATAG:TAGGAACCATA;TA;AGTCCTCGGGCGAT~TG -3 ' Sa1moGal ar : 5'- TT ATATAGATAGGAACCAT AATAAAGT CCTCGGGCGATGT G-3' Sa l IDa trutta:5' -TATATAAATACAGATAAAGAAGAAAGATGCTCCGTTAGCGT- 3
'0
I Salmosala r : 5'-T ATATAAATACAGAT AAA GAA GAAA GAT GCT CCGTT AGCGT - ) '80 70
Salmo trutta:5'-GAATGTTTCGGATGAG'I'CAGCCGTAGCTAACATCTCGGCAA-J
. .
ISaba salar: S'-GAATCTTACGGATGAGTCAGCCATAGCTAACATCTCGGCAA-3I
31
amplificati on and direct sequence analysis of the mit ochondrial cytochrome bgene wereperformed USingtechniques described in detail byBartlett and Davidson(19 9 1).
2.2.3 .1.DNApreparation
CrudeDNA extract ions wer e carriedout in1.5ml mtcroccn trttuge lubes using 0.2to 0.5g of skeletalmuscle.Sampleswere Brst homogenizedin 400)-IIofguanldl nl umextraction buffer(4 Mguantdtruumthiocyanate.
25 roMsodiumcitra tepH7.0.0.5% Sarkosy l.0.1Mz-rncrcaptocma nou.
To this.15pIof 2 M sodiu macetate (pH 4.1),400}-IIof Trte-saturatcd phenol and200piof chloro fonn/isoamyl alcohol (21:1v/v)were added.
The homogena tes werevigorously mixed . incu bate d on icc for 15 minute s.then subjectedto centrifugationat 100 00 x g for20min u tes at 4OC.The clear.aqueous phase (about400pi )of each sample was transferr ed to freshrmcroceruntugetu bescontaini ngan equalvolumeof isopropan ol. mixed and incubated at·200C for atleastone hour, allowing the DNA to precipit ate. Sampleswere then subjected to centrifugati onat1000 0 x gfor 20minu tes at 4°C. Pelletswere washed withcold 70% ethanol, subjec ted to centrifugationat 10000xgfor 15 minutesand the ethanoldiscarde d.The finalDNA pelletwas thendried under a vacuumandresuspendedIn 20 to 50pIof'IEbuffer (10 mM Tris·HCl.1 mMdisodiumethylenediaminetetraacetatcIEOTAI,adj usted to pH8.0withNaOH )dependi ngonsizeofthe pellet.Once dissol ved.
samples were storedat 4°C.
2.2.3.2.Ampllflcati on ofcytochromebgene
Amplificati on of themitochondri alcytochromeb genewascarriedoutin twosteps usingthe CetusDNAThennal Cycler{Per ki nElmer. Norw alk ,
en.
First , asymmetri campIillcati on ofboththe light andheavyDNA strands(dou ble stranded amplifica tion )wascarriedout. Thi swas32
followed byanasymm etri camp li fica ti o n ofthe heavy DNA st ran d only (single strand edamplificati on) . In both amplific a ti on steps .reactions were run withou t DNAas a con trol for possiblecontamina tion.
Thedouble strandedamplification reac tion mixture consi sted of 2.5]11 of lOx amplificationbuffer (500 mM KCI,100 roM tris -HelpH 9.0. 15 mM MgC12' 0.1% gelatin[w/vl.1%Triton X-tOO:Promega. Madison,WII, 0.5piof40 mM deoxynucleosldetriphosphate{dN1P} stocksolution(10 mM clA1P.10 mM dGW.lOm Mde1P.10 mM dTIP; Pharmacla. Dorval.
Que. l. LO jJl of lOpM light strand primer It-primer. 5', CCATCCMCATClCACCATGATGAAA·3').1.0 fllof10
JIM
heavy stran d primer(H-primer:S'·CCCCTCAGMTGATATITGTCCTCA·3').20.0)11of sterilewater,0.2)11 of Tag-DNA polymerase (Promega .Madison.WI). and 1.0)11ofDNA The reactionmixturewas covered with a dropof mineral 011to preventeva pora tion thenrun through a thermal cycle in whichthe DNAwasdenatured at 920Cfor45 seconds,theprimerswereannealed to the single strandedtemplateDNAat50°C for 45 second s and the new DNAstran d extendedalong thetem pla te at 720(: for 1 minute an d30 seconds.Thiscyclewas repeat ed 30 times.The double stranded amplification product was isola ted by electrophores is . Fifteen microliters ofeach samplewas mixedwith 5,1 oftracking dye (50%sterile glycerol,5xgel buffer.0.05 %bromophenol blue)and run through a 2%low meltingpoint. NuSeive agarose gel (FMC BloProducts, Rockland.ME)us in g a continuousTrislsodlumace tat e(TA) buffer system (40 roMTria, 30 mM sodium acetat e.titratedto pH 7.4 wit h HCn.Gels were 60mlinvolu mewith5)'1 of ethidlum bromide (10 mg / ml l added to make the DNA fluorescentover ultraviolet {V.V.) light.
Theywererunataconstant voltage of65 V for 45 to 60 minutes.The amplUled DNA product.see n over U.V.light as atight band, wascu t out of the gel.melted at 700C in 50 to 100j11of sterilewater ldependingon theIn tens ity of the band) and used forth easymmetrtc amplifica tion of the cytochrome b heavy DNAstra nd .
33
The reaction mixture for the singlestranded arnpuncattouwas made with 10/'1oflOx amplificationbuffer, 2J11 of dN1P stock solution.4
pi
of 0.1fM lightprimer.4pi
of10 JIM heavy primer.75fliofsterile water.0.2
pi
of Taq·ONA polymeraseand 5)11 of double stranded amplification product.This reaction mixturewasnor coveredby a drop of all and was run through the same cycle as described above. After runpUficntion.the product was verifiedbyrunning 5JlI (wtm 2;11 of tracking dye)througha regular 2% nucleic acid grade agarose gel using the TA buffer system described above. This get was also 60 mt in volume with 5)-'1cmtdturn bromide so that the amplification product couldbeseen over U.V.light.Before sequencing. the single stranded amplification product was desalted to remove any residual nucteoudes.primers and bulTersalts.
Thiswasaccomplished by centrifugal dialysis usingCcntricon ·30 filters (Ameon ltd.. Oakville.Ont.l.The remaining 95 jllof samplewas transferredto thetop of the filter with 2 ml of distilledwater and subjected to centrifugation at 3500 x g for 20 minutes. This was repeated twice. the third time at 6000 x gfor 60 minutes.The final desalted product (about 60jIl) was stored at 4°C and used for sequencing.
2.2.3.3. Sequencing of the cytochrome b gene
Sequencing of the amplified cytochrome b gene heavy strand was carried out using the Sanger method (Sanger et aI.19711 and following the manufacturer's directions forthe Scquenase. version 2.0.DNA sequencing kit (UnitedStates BiochemicalCorp.. Cleveland, OHI. Ught primer was annealed to the heavy strand template In a mixture consisting of 7
fl
of desalted DNA.1pi
of10j1M light primer and 2)11 of 5x Sequenase buffer (200 mM liis·HCIpH 7.5 .100 mM MgC12' 250 mM NaCl). This mixturewasheated In the thermal cycler to 65°C for ten minutes then slowlycooledto 300c overa 30 minute period. To the10f '
of annealedDNA. I pIof0.1MdithJothreHoJ (DTI1. 2)llo(labelingmJx 10.15 I'M dG11'.0.15 I'M <!TIP and0.15 I'MdC11'l.0.51"of20
I'M
S"labeled dAlP (5fCIIna 10 mM Tricln e/l mM OTIbuffer:DuPont.
Bosto n.MAl. 2fldilu te Scquenase (DNApolyme rasediluted 1:8in enzymedilu tion bu fferwhichwas 10mMTris-HCI pH7.5 .5mM DTI and0.5mg/mlbovine serumalbumin) and I
fli
Mnbuffer(0.15 M Sodiu mrsccurare.0.1MMnC~1wasad ded(or afinalvolumeof16.5pI.Mer afive minuteinc ubationat roomtem perat u re.fouraliquots of 3.5 fleach were transferred tofour separatetenntnatlonreaction tubes containing2.5,P1ofeitherdideoxyC1P(80 JIMdOlP.80 JIM dC1P.80 )'M clA11'.SOI'MdTll'.8I'M ddGll'and 50 mM NaCII.ddAll' ISOI'Mof dGlP.dC'l'P.dAlPand
errr .
BflMddA1Pand50mM NaCI).ddCTP (80 pMof dOW.dCW.dAlPanddTIP.8fMddCTPand 50mMNaCOor ddTIP(SO)'Mof dGll'.dC11'.dAll'and dTIP.8 I'M ddTll'and 50 mM NaCI).Thisreaction was Incubatedat370C forOveminutesafterwhich 4flof stop solution(95%fonnamide.20mM EDTA0.05%Bromophenol blue.0.05%xylene cyanolFF')wasadded toeac h tube. Inuned Jat e ly prior toloadin g onsequenci ng gel sampleswerehea tedto 750Cfortwo mmutesthen quicklyplacedonIce.Sequencing products were separated on a 6% denaturing polyac rylami degel (7Murea.5.7% acrylamlde.0.3%bts-acrytemtde.0.1 M Trts.0.1M boneacfd.2mMEDTA. 0.08 %ammoniumpersulfate.3.3 mMtetra-meth y tethylenedtamtneITE MEDI).Gelswere60mlin volu me andwereleft to polymerizeovernight.Before loading the samples.gels werepre-run untiltheyhadwarmed to atem pe ra tur e of500c. Samples wereseparatedthro ugh thegelat a constantpowerof35 watts for1.5to 2hours.Afterarun.gelswereIlxedIna one litre solu ttonof10%
methanoland10% acetic acid(or fifteen minutes.drieddownon to 3 nun chro ma togrnphypaper (Whatman. Maidstone.U.K) using a geldrier nodexposed toauto-radiographyfilm(X-OmatRP diagnosticfilm:Kodak.
Rochester. NY) for two to three days before deveio ptng. The DNA sequence.determinedfromthe auto -radtogmph.identified the matern al speciesofahybrtdfish (Figure2.51.
2.2.4.StQtis dcalAnalysis
Sta tistical analysisinvolved a 2x9contingency tableanalysis totest for regional het erogeneity of local hybrid frequencies. This was determin ed by calculatingaGstatis ticadjus tedforsmall numberswith WilHam'scorrec tion (Sokal and Rohlf 1981).Gwasconsidered significant atalevel ofP<0.05.
2.3.Results
2.3.1.HybrldlzatiOllJ requency
Overall.792 Atlanticsalmon and brown trout were sampled.37 of whichwere detemtinedtobehybrids for a regionalfreque ncyof 4.7%
(Table2.1). Hybrid s were found inallriver systems exceptone;the MooneRiver.LocalIrequencieeofhybrid ization at the various sample sitesrangedbetween0.0 and 18.7%(Ta ble 2.1)andweresignificantly heterogeneou s (2 " 9contingen cytable:GadJ..38.03. P<0.001.d.Le81.
This suggests that the conditions responsi bleforhybrid ization are variable from onelocation toanother.However.IIthe samplefromthe HeartsDelightriver . wherethe hybrid frequency wasparticularlyhigh.is excluded from the analysts. frequency differences are no longer significan t (2x8contingency table:GadJ=13.49.P<0.10.d.f.=8),
All hybrids expresseda complete summation ofboth parental genotype s at each of the fo ur diagnosti c loci. There was no evidence of
36
Figure 2.5.
Photograph of anauto-radiogra ph usedto determinethe DNAsequen ce fora segment ofthecytochrome b gene.Sequencesshown arethose ofa brown trout (Sa lmotrutta),twohybrids.andanAtl antic salmonlSa Imosala r]•
•Indicatesdifferen cesused toIdentifymatern al species.
37
hybrid
s saiar
-
38
Table 2.1.
Sample sites and the number of Atlantic salmon,brown troutand hybrids collectedat eecn.
Sample sit. Salmon Trout Hybrids
No.
•
1- MobileR.
34 26 0.0
b. 77 46 31 0.0
total 111 72 3' 0.0
2.Cape Broyle R. 33 16
,.
6.13.Renews R. 78 53 21 5.1
4.Salmon!erR. 75 15 58 2.7
5.N.E. Placentia R.
34 25 2. '
b. 43 19 19 11.6
32 18 14 0.0
total 10' 45 58 5.5
6.spread Eagle R. 79 74 1.3
7.Hearts Delight R. 80 32 33 15 18.7
8.NorthR.
".
107 13 4.89.North Arm R. 101 4' 51 1.0
Overall 7.2 393 362 37 4.7
39
tntr ogreseton . Althoughverepoor and Hammer (1991) have suggested that introgression between these speciesis possible. backcrceetng Is considered unlikely due to disruptions of meiotic pairing In fertile F 1 hybrids (Johnson and Wright 1986).
2.3.2. Hybridmaternalspecies
AIl of the hybrids had mitochondrial cytochrome b genes characteristic of brown trout.Therefore. all hybrids werethe products of matings between female brown trout and male Atlantic salmon.
2.4.Discuss ion
The overallhybridfrequency of 4.7%observed In this study was much higher than the 0.9% reportedby Verspoor (1988) for the sameregion.
The lower estimate in the earlier survey Is probably the result of sampling biases. Verspoor (1988) analyzed only those fish IdenUfied morphologically as Atlantic salmon and included samples from rivers where no brown trout had ever been reported. Morphologically. hybrids can resemble either species (Jones 1948;Nyman 1970)so surveysmust Include a random sample of both brown trout and Atlantic salmon in order to obtain an accurate estimation of hybrid frequency.In addition.
low hybrid viability may prevent many fish from reachingadult size.The present survey only considered samples of juveniles.whereasverepoor (19881 included parr. smelts and adult fish.If only samples of parr are considered and those from rivers known not to contain brown trout are excluded. then the hybrid frequency in the survey by verspoor (1988) becomes 2.2%which is more similar to the results presented here.
Croeler (1984) considered only brown trout and sampled both adult and juvenile fish In detenninlng a hybrid frequency of 0.4% In the Lough
40
Neoghsystem ofNorthern Ireland.The combinedsurveysof Payne et al.
(1972) andSolomonand Child (1978 ) proposed a 0.3%hybridfrequency for the United Kingdom.However thesestudies onlyconsideredadult Atlanti csalmon caughtoffshore In commercial fishingnets.
Itis probab lymore appro priate to compare resultsof thepresentst udy withthose whichhave sampledonlyjuvenileAtlantic salmonor brown trout.In North America .Belandet al. (1981)sam pled juvenilesof both speciesfrom theStewtackeRiver.Nova Scotia and foundonehybri dout of56fish forafrequencyof 1.8%.HurrellandPrice(199 1)sampled both Atlantic salmon and brown trout parr when estimating a hybrid frequencyof1.4%for rivers insouthwes tEngland . Garcia de Leaniz andVerspoor(1989) foundthat 2.3%of Atlanticsalmonparr sampledin Spanish riverswere bybrtds. Jansson etal.(1991) discoveredhybrid frequencies of13% In samplesof brown tro utand Atlantic salmon parr from the RiverCronan.Sweden. withlocal freque nciesas highas 23%.
However. the last two cases may have ha d higher than norrnat hybridizationfrequenciesdueto former Introductionsor Atlantic salmon in therivers whichweresam pled.
It is difficultto say whetherfrequenciesof hybridiZation are actually higherInNorth America.wherebrowntrout are introduced.than they are In Europe.where bothspecies are naturallysympatrtc. Hybridization betweenthese speciesIs possiblein North America only becauseof the introductionof browntrou t.What Is not obviousis how prereproductive Isolatingmechanismsbetween these species have been compromised suchthatextensivehyb ridizat ion is occurringIn bath Eu ro pe andNorth America.MtDNAanalysis provtdee additio nal Infonnation and permitsa closerexaminationof possibleexplanations.
Hybridization between brown trout and Atlantic salmon in Newfo undland appears to be unidirectional.with Atlan ticsalm onmales
alwaysfertiliz ingbrown trout females. This biasin directi oncouldbethe res ultof differences inthe che motactic responsesof salmon and trout sperm to theeggs of a different species. The eggs of Echinodermata species (starfish.sea cucumbers and brittlestars)areknownto secrete chemicalatt ractan ts whichguidethe sperm to the eggan d arcthought to bespecies- specific [Miller1985:Garbers 1989).Trouteggs maybe able toetten astrongch emotac ticres ponseIn salmonsperm.whe reas th e factors secretedbysalmoneggs may not have as strong an effect on trout sperm. Theexis tence of spermchemotaxis In teleos tfishhas yet to be clearl ydemonstrated (Hart 1990). however indirect evidence of ch emotaxi sInthe Rosy barb (Barousconchonlus) has been discovered (Amanze and Iyengar 1990). Whether or not sperm cbemctaxrs representsa reproductivebarrier between closely relatedfishspecies is also unknown.Theobserva tionofna tu ral hybrids in Europewhichhave Atlantic salmon mothers (Youngson et ar. 1992 ; H. Jansson.
Laxforskning stnstttutet.Atvkarl eby.Sweden .personalconun unlca Uonj considerablyweakens thishypothesiswith respect toAtlantic salmon x brown trouthybridization .
Asimplerexplanation for this observa tion wouldbe that there isa dUTerence inviability between the reciprocal crosses such tha t only hybridswithbrown trout mothers are sUrviving long enough to be sam pled. Suchwasthe case fornatural hybridization betweennorthern pike(Esox lucius)and chainpickerel(Esox niger)In whichthe maternal parentof all hybrid fish was a chain pickerel(Herkeet al. 19901.
Previous experiments in thela bor atory had shownthat hybrids produced using chain pickerelfem ale s are viable.whereasthe reciprocal cross is completelyinviable(Buss and Miller19671.
To addressthis question with respect tothe results presentedabove.
breeding experimentswere undertaken to comparetherela tiveviability of reciprocal brown troutx Atlantic salmon hybrids .Thisstudy Is the subject of chapter 3.
42 Chapter 3
ArtlflclalHy bri diza tion BetweenNewfoundland Brown Trout and AUaDtlc SalmoQ
3.1, Introduction
Artificialhybrid iza tion between browntrout lSalmotrotta)andAtlantic sal mon (Sabno salar)has been ofbothscientificand comm erctat Intere st for over100years. Severalstudieshave Investig a ted the morp hological appearanceof hybrids(Day188 2:Jones1948), theirfertility(Alm19 55 : Piggins 19 70)and theirbioch emicalgenetics(Nyman1970: Nygrenetal.
1972:Vuortnen and Piironen19 84;Johnsonand Wrigh t1986).
Breeding espertme ntecomparing the relative vtabilttyofreciprocal hybridsandtheirparental con trolshave had variable results.Some Ilnd thathybrids producedusingbrowntrouteggs and Atlanticsalmon milt perform best (RefsUeandGjedrem 19751.whereas others have had greater successwithhybridsproduced using eggsfrom Atlan ticsalmon orAtlantic salmongrtlse(Hofer 1909(fromRefstJeandGjerern 19751:
Ahn1955:Piggins 1970). Itislikely thatagooddeal depends onth e strni nsoffishusedas wellasthequal ityofeggsand miltat the time of fertilization. Inareview ofthe litera ture.Chevassus(1979)concl uded that hybrids with brown trout mothersare grea terInviability.butthat bothcrossesperform aswellasorbetterthantheparentalcon trols.
The followi ng st udy In vestigated the relative viabilityof hybrids produced usingana dromou sbrowntrou t. Atlan ticsalmongrtleeand sexu ally ma ture Atlan tic salmonparr fromNewfound land.Hat ch a bility.
survivalandgrowthare consi de re d during thefres h waterperioduntil
first feeding.Also.some qualitativeobservationson theperformance of hybridsafterthi sstagearepresented.
2.2.MaterlalsllDdMethods
2.2 .1.Notation
The followingnotation is usedin orderto simplifyidentification of the various familiesand Individual fish. Eachtndivtdual is identifiedby its spec ies (T ::: browntrout. S=Atlantic salmon and SP =sexually mature salmon parr)and a number (except forthesexually maturesalmon parr whose milt waspooled). All crosseslistthefemale parentalspecies first.
Forexam pl e, Tl x T4 representsthe familyproducedby thefemale browntro u t 1an d themale brown trout 4,13xSPrepr esentsa hybrid crossproduced using browntr out eggs and the pooledmiltfr om six sex u al ly matu re salm on par r.When crossesare referred to in more generalterms.numbersare not used.
2.2 ,2 ,Breedingstock
Breeding stock was obtained from the North East PlacentiaRiver locatedonthe Avalon Peninsula of Newfoundland (47016'N,53°50'W).
Anadromous brown trout werecaptured at a counting fenceIn lateJu ly and transported liveto the OceanSCiencesCentre,MemorialUniver sity, St. John's.At the time, no secondarysexual characteristicswereevi den t on the fish makingIdentificationof males and females difficult. Fourteen fish were collected,threeofwhich turnedout to be male,Therewasone mortality durtng transport.
Atlanticsalmoncouldno t be collectedfromch e counting fenceat this
44
timewith out suffering highmortality.Itwas suspected thatwarmwater temperatures In July made the trip too difficult for the fish. Consequently,ripe anadromous Atlanticsalmonandsexuallymature male Atlanticsalmonparr were captu redin earlyNovember whenthe WOlterwas much colder.They were seinedfromanursery stream further up river from thecountingfenc e andtransportedback to St. John's witho utany problems. By th istime. thebrown trou twere also ripe and ready to spawn. Figure3.1Indicatesthe locationson the riverwhere the brown troutandAtlantic salmonwere collected.
3.2.3. Spawning and experimentaldesign
FJsh were artificiallyspawned onNovembe r 15. 1990. Eggs andmilt were notpooled except forthe miltof slx sexual lymaturemale Atlantic salmon parr.In s tead . theeggs ofeachfemale were dtvidedequallyinto five famili es cons isti ng of two con trols.twohybridcrosses and a single family us ingthe pooled miltfrom the salmonparr. No famHy was dupli cate d and ea chwasIncu ba tedseparatelyso that individualswhose gametes were abnonnaHy low in viability could be Identified and eliminatedfrom the results. The sizeof eachfamilywasestimatedby counti ngthenumber of eggs inonefamily for eachfemale andthe numbers are rounded to the near es t ten.Table3.1 lIIu s t ra tes the experime n tal des ign as wellas thesize andpositionof eachfamUy inthe incuba tor.Three anadromo us females and three anadromo us males of each spec ies were used along withthe pooledmiltof thesexu al lymature salmonparr.Overall.30 families wereprod uce d.
3.2.4 . Incubationandrooling
Fert ilized eggs wer e incu bated using recirc ula ted water with tem peraturesrangingfrom5.5 to8.7°C and amean temperatureof
Figure3.1 .
Site son the North East Placenti ariverwhereA)adu lt anad rom ous brown trout and B) adult anadrom ou eAtlanticsalmonandsexual ly mature male Atlantic salmon Juvenil es were collec ted .
'6
.7
Table3.1.
Pare nts of families, nWllber of eggs pe r fallily and relative position of each family in incubator.Inc ub at or con.1ated of eight eg g - t r ay s stacked vertically on top of one another with each tray dividedinto four cells.
Tray Cells
c
51x54 51x55 82x54 52 x56
360 360 600 600
53x56 53x55 52 xSP 51xSP
370 370 600 360
51xT4 51xTS 52 xT4 52xT6
360 360 600 600
53X T4 53 xT2 53xSP
370 370 370
Tlx54 T! x55 T2 x54 T2x56
37 0 370 500 500
TJ x56 T3 x55 T! xSP T2xSP
310 310 370 500
TI X T4 T!xT5 T2xTe T2xT6
370 370 500 500
T3xT'" T]xTS T]xSP
310 310 310
