Univerzita Karlova v Praze

Prodovdeck fakulta
Katedra zoologie
Molekulrn fylogeografie lna obecnho
Tinca tinca (Linnaeus, 1758)
Zdenk Lajbner
Disertan prce
kolitel: RNDr. Petr Kotlk, Ph.D.
FG AV R, v.v.i., Libchov
Praha 2010
Prohlasuii, ze isem predlozenou disertacni praci vypracoval samostatn s pouzitim citovan
literatury. Dilci publikace byly zkompletovany spolecn se imenovanmi spoluautory. Prohlasuii,
ze isem nepredlozil tuto praci ani zadnou ieii cast k ziskani iinho nebo steinho akademickho
titulu.
V Praze, 21. prosince 2010 ...........................
Zdenk Laibner

3RGNRYiQt
Na prvnim mist dkuii RNDr. Petru Kotlikovi, Ph.D., z Ustavu zivocisn Iyziologie a genetiky
(UZFG) AV CR, v.v.i., za trplivost, kterou se mnou ml iiz od dob mho magisterskho studia
asniz se zhostil i vedeni m prace disertacni. Rovnz mu dkuii za vsestrannou podporu, iiz se mi
ziehostranydostavalo.
Dkuii ProI. Ing. Petru Rabovi, DrSc. a vsem kolegm z Laboratoe genetiky ryb UZFG
AV CR, v.v.i., v Libchov, za vytvoeni piiemnho pracovniho prostedi, trval pisun inspirace
acennrady.
V neposledni ad dkuii spoluautorm publikaci i vsem dalsim lidem, ktei mi bhem
tvorby disertacni prace iakkoliv pomohli a to pedevsim pi shromazovani vzork lin z celho
svta.
Velmi rad za trplivost, s niz tolerovala m zaneprazdnni, dkuii i m rodin.
Tato prace vznikla za Iinancni podpory Ministerstva skolstvi mladeze a tlovchovy Cesk
republiky-proiekt cislo LC06073 a vzkumnch zamr pidlench Ustavu zivocisn Iyziologie
a genetiky Akademie vd Cesk republiky -proiekt cislo AV0Z50450515, Pirodovdeck Iakult
Univerzity Karlovy v Praze - proiekt cislo 21620828, Fakult rybastvi a ochrany vod Jihocesk
Univerzity v Ceskch Budiovicich -proiekt cislo 6007665809 a Interni grantov agentury UZFG
AV CR v.v.i. -proiekty cislo 05/22 a 08/13.
Summary
ThetenchTincatinca (Linnaeus,1758)isavaluedtableIishnativetoEuropeandAsia,butwhich
is now widely distributed in many temperate Ireshwater regions oI the world as the result
oIhuman-mediated translocations. Spatial genetic analysis applied to sequence data Irom Iour
unlinked loci (three nuclear introns and mitochondrial DNA) deIined two groups oI populations
that were little structured geographically but were signiIicantly diIIerentiated Irom each other,
anditidentiIiedlocationsoImaiorgeneticbreaks,whichwereconcordantacrossgenesandwere
driven by distributions oI two maior phylogroups.This pattern most reasonably reIlects isolation
in two principal glacial reIugia and subsequent range expansions, with the Eastern and Western
phylogroups remaining largely allopatric throughout the tench range. However, this
phylogeographic variation was also present in European cultured breeds studied and some
populations at the western edge oI the native range contained the Eastern phylogroup. Thus,
naturalprocesseshaveplayedanimportantroleinstructuringtenchpopulations,buthuman-aided
dispersal have also contributed signiIicantly, with the admixed genetic composition oI cultured
breedsmostlikelycontributingtotheintrogression.
We have then designednovel PCR-RFLP assays oI twonuclear-encoded markers and one
mitochondrial DNA (mtDNA) marker, which allow a rapid identiIication oI the morphologically
undistinguishableWestern and Eastern phylogroup and also oI three geographical mtDNA clades
within the Eastern phylogroup.The method will enable researchers and also Iishery practitioners
to rapidly distinguish genetically divergent geographical populations oI the tench and will be
useIul Ior monitoring the introduction and human-mediated spread oI the phylogroups in wild
populations,IorcharacterizationoIculturedstrainsandinbreedingexperiments.
The population genetic test based on analyses oI variation at introns oI nuclear genes,
microsatellites, allozymes and mitochondrial DNA in populations Irom two postglacial lakes
within the contact zone oI both phylogroups did not detect robust linkage and Hardy-Weinberg
disequilibria that would not be limited to individual loci and would be concordant between
populations. This test, based on the expectation that in the presence oI strong barriers
toreproduction the hybrid populations would show genome-wide associations among alleles
andgenotypes, supports the hypothesis oI Iree interbreeding between the two phylogroups
oItench.ThereIore,althoughthephylogroupsmaybeconsideredasseparatephylogeneticspecies,
thepresentdatasuggestthattheyIormasinglespeciesunderthebiologicalspeciesconcepts.
Obsah
1. Uvod...............................................................................................................................................6
1.1. Vvoi sladkovodni ichtyoIauny..............................................................................................6
1.2. Hybridizace a ieii evolucni vznam........................................................................................9
1.3. Vliv clovka na genetickou strukturu populaci.....................................................................10
1.4. Lin obecn Tinca tinca (Linnaeus, 1758) iako modelov druh............................................13
1.5. Cile prace..............................................................................................................................15
1.6. Seznam pouzit literatury:....................................................................................................16
2. Publikace......................................................................................................................................26
2.1. Human-aided dispersal has altered but not erased the phylogeography oI the tench...........26
2.2. PCR-RFLP assays to distinguish the Western and Eastern phylogroups in wild and cultured
tench Tinca tinca. ........................................................................................................................60
2.3. Lack oI reproductive isolation between the Western and Eastern phylogroups oI the tench.
......................................................................................................................................................65
3. Shrnuti vsledku a ieiich vznam.................................................................................................78
3.1 Seznam pouzit literatury:.....................................................................................................80
1. vod
1.1. Vvoj sladkovodn ichtyofauny
Sladkovodni ichtyoIauna, stein iako veskera biota, byla v prubhu ctvrtohor siln ovlivnna
periodickmi zmnami klimatu zpusobenmi zeimna Milankovicovmi cykly (Dynesius
a Jansson 2000). Takto isou oznacovany zmny obzn drahy Zem a sklonu zemsk osy,
kter ovlivnuii mnozstvi slunecniho zareni dopadaiiciho na zemsk povrch. S prichodem
chladnch obdobi, kdy se zvtsovala plocha pokryta kontinentalnim ledovcem, populace
teplomilnch organismu ustupovaly ze severnii polozench oblasti, vikariantn se stpily
a nakonec prezivaly pouze v oblastech, kde nalezly vhodn podminky pro zivot, tzv. glacialnich
reIugiich (Hewitt 2004). V tepleisich obdobich se tyto druhy opt rozsirily, pri cemz dochazelo
ke kontaktu populaci do t doby izolovanch v iednotlivch reIugiich, kter se tak mohly opt
krizit (hybridizovat) (HoIreiter a kol. 2004). Analogicky reagovaly chladnomiln organismy
na otepleni (Makhrov a Bolotov 2006: Stewart a kol. 2010). Dlouhodob oddleni populaci muze
vyvolat tzv. alopatrickou speciaci (Mayr 1963). V pripad alopatrick speciace dochazi ke vzniku
reprodukcn izolacnich mechanismu obvykle pomaleii, nez v pripad speciace sympatrick (napr.
McCune a Loveioy 1998: Noor 1999: Turelli a kol. 2001: Matute 2010). Jsou-li proto odstranny
migracni bariry, puvodn oddlen populace casto hybridizuii, coz v pripad absence silnch
reprodukcnich barir muze vst ke genetick homogenizaci (Olden a kol. 2004). Hybridizace ie
u organismu s vnisim oplozenim, iako isou ryby, bzna a to i u druhu, kter byly izolovany
po stovky tisic i nkolik milionu let (Hubbs 1955: Schwartz 1981: Laibner a kol. 2009).
Hybridizace a z ni plynouci introgredovan populace vsak mohou zustat geograIicky omezen
na nov osidlen areal a s prichodem dalsi klimatick zmny vyhynout, aniz by geneticky
ovlivnily populace reIugialni a narusily tak ieiich evolucni divergenci (HoIreiter a kol. 2004).
Neiruznisi studie dokladaii, ze klimatick zmny provazeiici konec posledniho glacialu
byly (v geologickm mritku) pomrn rychl (Kasse a kol. 2005: SteIIensen a kol. 2008). Tani
ledovcu zpusobilo neien zvednuti hladin oceanu a oddleni napr. Britskch ostrovu od pevniny,
ale treba i naplnni Baltskho more, ci zvtseni a propoieni Cernho more s Kaspickm
a Stredozemnim (Chepalyga 2007). Pocatek Holocnu tak mozna zaznamenali i starovci
kronikari iako onu .biblickou potopu' (Ryan a kol. 1997). Slabnouci tlak ledovcu na zemsk
podlozi rovnz spustil geologick procesy, v ieiichz dusledku se nkter oblasti nesteinomrn
zdvihaly, coz v nkterch oblastech probiha dodnes (Peltier 2004). Dochazelo k vraznm
zmnam v ricnich sitich, spoiovani a rozdlovani povodi a ke vzniku iezer. Na rozdil od dob
ledovch, kter se mi. proievuii aridizaci klimatu, byla postglacialni Holarkticka kraiina nahle plna
6
kapaln vody a ryby i dalsi sladkovodni organismy mohly do ruzn miry rozsirovat sv arealy,
pricemz svoii roli hraly vchozi .startovni pozice' iednotlivch druhu (napr. poloha glacialnich
reIugii), ieiich ekologick naroky i disperzni schopnosti (Birks a Ammann 2000).
Bnrescu (1991), predevsim na zaklad chorologickch a paleontologickch dat,
identiIikoval hlavni glacialni reIugia a kolonizacni cesty, iimiz po otepleni sladkovodni ryby
osidlily rozsahl oblasti Evropy. Neivznamnisi glacialni reIugia pro vtsinu druhu stredni
a zapadni Evropy se zreim nachazela v povodi Dunaie a pripadn dalsich pritoku Cernho
a Kaspickho more (Durand a kol. 1999: Kotlik a Berrebi 2001: Kotlik a kol. 2004). Diky rutinni
aplikaci molekularnich genetickch markeru a v souvislosti s rozvoiem populacni genetiky,
Iylogenetiky a pozdii pak predevsim IylogeograIie (Avise a kol. 1987, 2000) doslo
k vznamnmu narustu detailnich znalosti o tom, iak se postglacialni evropska ichtyoIauna
Iormovala (Hewitt 2004). FylogeograIie se iako disciplina na pomezi Iylogenetiky a biogeograIie
zabva principy a procesy, kter ridi geograIickou distribuci genealogickch linii mezi populacemi
steinho nebo blizce pribuznch druhu (Avise 2000). Prestoze IylogeograIie metodicky tak
preklenula propast mezi molekularni Iylogenetikou a populacni genetikou a vyuziva proto
metodickch pristupu, kter vznikly pro potreby tchto dvou oboru, cela rada metod byla navrzena
specieln pro potreby IylogeograIie (viz. dale).
Ackoliv bylo v populacni genetice v minulosti vyuzivano predevsim variability
v elektroIoretick mobilit proteinu (napr. alozymu), ktera na tomto poli zauiima vznamnou
ulohu dodnes (Hamilton 2009), k bourlivmu rozvoii IylogeograIie doslo az diky masovmu
rozsireni studia DNA a zeimna vynalezu polymerazov retzov reakce (PCR). FylogeograIie,
podobn iako Iylogenetika, zpocatku vyuzivala predevsim haploidni, mimoiadernou (organelovou)
DNA, v pripad zivocichu mitochondrialni DNA (mtDNA) (Avise 1998). Jelikoz se mtDNA ddi
tmr vhradn po matersk linii, ziistuieme timto zpusobem predevsim maternalni historii (Avise
1998). V mtDNA obratlovcu zpravidla nedochazi k rekombinaci, ie zde ctvrtinova eIektivni
velikost populace oproti iadern casti genomu (Birky a kol. 1989) a mnozstvi generaci potrebnch
ke koalescenci ie tedy relativn nizsi (relativn blizsi posledni spolecn predek) (Moore 1995).
Dlouhodobou strukturaci populaci lze tedy lpe odhalit pomoci iednopohlavn ddn mtDNA
(ale napr. i Y chromozomu) nez studiem srovnateln variabilniho useku rekombinuiici iadern
DNA (nDNA). Studie omezen na mtDNA vsak selhavaii v pripad hybridizace. Problmy
pri interpretaci dat ziskanch studiem iednopohlavn ddn DNA muze sehrat i vyssi mira
Iilopatrie (ti. vrnosti mistu narozeni) iednoho z pohlavi a pod. (napr. BonIil a kol. 2005). Proto ie
dulezit kombinovat vsledky ziskan analzou mtDNA s vhodn zvolenmi iadernmi markery
(Avise 2000).
7
Mira divergence mezi mitochondrialnimi liniemi byla zpocatku ziistovana pomoci
restrikcnich enzymu a tvorby restrikcnich map (Brown a Vinograd 1974). Dalsi, kvalitativni
i kvantitativni skok prinesla moznost analzy genetick variability na urovni sekvenci DNA a ieii
zpristupnni sirok vdeck vereinosti, predevsim diky poklesu ceny a dostupnosti Iormou servisni
sluzby. Rostouci obliba sekvencnich markeru se odrazila i ve vvoii novch metod zpracovani dat.
Ziskan IylogeograIick vzory byly zpocatku popisovany hlavn graIicky a interpretovany
intuitivn (Avise 2000). Jednim z prvnich pokusu o standardizaci IylogeograIick analzy byla
takzvana .Nested clade analysis' (Templeton a kol. 1995). Tato metoda si rychle ziskala znacnou
oblibu, ale v soucasnosti ie iiz vytlacena pokrocileisimi statistickmi pristupy (Knowles 2009)
zalozenmi predevsim na hodnoceni koalescencni (Kingman 1982) pravdpodobnosti
alternativnich IylogeograIickch modelu, casto s vyuzitim principu bayesiansk statistiky (napr.
Kuhner a kol. 1995: Wakeley a Hey 1997, Beerli a Felsenstein 1999: souhrn viz. Nielsen
a Beaumont 2009). Alternativni pristup nabizi uplatnni teorie koalescence (Kingman 1982)
pri simulaci genovch stromu uvnitr stromu populacnich (Knowles a Maddison 2002), kter
umoznuie napriklad program Mesquite (Maddison 2008). Zvlastni misto zauiimaii metody
prostorov analzy genetickch dat (Guillot a kol. 2009), iako ie napriklad SAMOVA (Dupanloup
a kol. 2002), ieiichz hlavni vhodou ie, ze odstranuii problm se zarazovanim iedincu
do diskrtnich populaci, cimz ie zatizena rada iinch pristupu.
Krom sekvenci DNA se ve IylogeograIii vyuziva opakovani motivu tandemovch repetic
(mikrosatelity) a iednonukleotidovch polymorIismu (SNPs) rozmistnch v ruznch oblastech
iadernho genomu. Vznam SNPs zreim nadale poroste v souvislosti s rostouci dostupnosti
genomickch dat, produkovanch pomoci sekvenacnich metod nov generace (Avise 2010,
Emerson a kol. 2010).
8
1.2. Hybridizace a jej evolucn vznam
U ryb ie hybridizace pomrn castm ievem, kter ma nezridka za nasledek genovou introgresi
mezi druhy nebo mezi geneticky odlisnmi populacemi (Turner 1999). Vlivem introgrese cizich
genu mohou populace ztratit dulezit adaptace umoznuiici iim Iungovat v prostredi, kter obvaii
(AllendorI a kol. 2001). Hybridizace muze tak vst k outbredni depresi (napr. narusenim
kompatibility mezi geny v ruznch populacich), snizeni plodnosti (Iertility) a zivotaschopnosti
(viability) ci k situaci, kdy pocetnisi z hybridizuiicich taxonu (populaci, druhu) geneticky
.vstreba' ten vzacnisi (prehled viz. Laibner 2004: Laibner a kol. 2009). V dusledku hybridizace
muze v urcitch pripadech dochazet tak k polyploidizaci, vzacn i k ruznm odchylkam
od sexualniho rozmnozovani a vytvareni slozitch hybridnich komplexu nebo dokonce ke vzniku
asexualn se mnozicich klonalnich linii.
S narustem vyuziti molekularn genetickch metod ziistuieme, ze navzdory drivisim
predpokladum ie hybridizace u ryb velmi rozsirenm Ienomnem s vznamnm dopadem na ieiich
evoluci (Avise 1994: Haig 1998). V nkterch pripadech vsak muze bt metodologicky obtizn
odlisit genovou introgresi od nekompletniho oddleni genealogickch linii (tzv. .incomplete
lineage sorting') a to predevsim tehdy, ie-li pouzito mal mnozstvi genetickch markeru, navic
s neznamou speciIicitou vuci hybridizuiicicim druhum (Avise a Robinson 2008). Nekompletni
oddleni genealogickch linii ie situace, kdy v urcitm genu maii oba druhy spolecn dv nebo
vice alel aniz mezi nimi dochazi k hybridizaci (alely tedy zddily od spolecnho predka: Degnan
a Rosenberg 2009).
I studium hybridizace proslo v poslednich desetiletich bourlivm rozvoiem. MorIologick
znaky pri ieii detekci casto selhavaii, coz bylo ziistno iiz diky prvnim analzam genetickch,
napr. alozymovch dat (Valenta a kol. 1979: Verspoor a Hammar 1991). Studium mtDNA potom
prineslo moznost urcit prevladaiici smr hybridizace (napr. kombinace samec iednoho druhu
a samice druhho druhu: Laibner a kol. 2009), zatimco studium sekvenci DNA umoznilo lpe
detekovat napriklad historickou hybridizaci, ktera muze stat za vznikem nkterch recentnich
taxonu (Dowling a Secor 1997). Detailni studium hybridizace spada predevsim do pole pusobnosti
populacni genetiky a vyuziva tedy predevsim statistickch metod vyviiench pro tuto vdeckou
disciplinu. V soucasnosti ie vsak iiz k dispozici velk mnozstvi pokrocilch statistickch metod
vyvinutch specieln k testovani konkrtnich hypotz spoiench s hybridizaci, mezi nimiz zauiima
vznamn postaveni bayesianska metoda v programu NewHybrid (Anderson a Thompson 2002).
K odhaleni novch pripadu hybridizace vsak casto dochazi v pracich IylogeograIickch
a Iylogenetickch (napr. Tsigenopoulos a kol. 2002: Markova a kol. 2010).
9
1.3. Vliv clovka na genetickou strukturu populac
V obdobi od konce 18. stoleti nkdy oznacovanm terminem antropocn (Crutzen 2002), kdy se
lidsk aktivity stavaii celosvtov iednim z hlavnich Iaktoru ovlivnuiicich zivotni prostredi, se vliv
clovka na rozsireni organismu neustale stupnuie, coz podnitilo vlnu zaimu ohledn evolucnich
a ekonomickch souvislosti (napr. Guisan a Thuiller 2005: Halpern a kol. 2008). Lidska cinnost
ovlivnuie biologickou evoluci celou radou zpusobu (Palumbi 2001). V nkterch pripadech muze
vst az k lokalnimu ci dokonce globalnimu vymirani druhu (Martin 1966: Brooks a kol. 2002:
Courchamp a kol. 2006: Gillespie a kol. 2008), avsak v iinch pripadech muze naopak umoznit
rozsireni druhu do novch oblasti, coz ovsem tak casto zpusobuie nechtn zmny biodiverzity
(Elton 1958: Carlton a Geller 1993: Mooney a Clealand 2001: Rahel 2002: Clavero a Garcia-
Berthou 2005). Vodni ekosystmy isou lidskou cinnosti ovlivnny zvlast vznamn a sladkovodni
biotopy isou tmi neiohrozenisimi vubec (Jenkins 2003: Xenopoulos a kol. 2005). Presto ochrana
vodni biologick diverzity za tou suchozemskou znacn zaostava (Brooks a kol. 2006: Nei a kol.
2009).
Vlivu clovka nezustava usetrena ani geneticka variabilita a strukturace populaci ryb,
kter clovk ovlivnuie iak primo (manipulaci s rybami) tak i neprimo (ovlivnovanim ieiich
zivotniho prostredi). Za prim vlivy lze povazovat praktiky rybarskho hospodareni ve volnch
vodach. Rybari se pri lovu specializuii na urcit druhy ryb a ieiich velikostni skupiny. Bylo
prokazano, ze takovato zamrna selekce u neicastii lovench populaci morskch ryb muze
zpusobit zmenseni prumrn velikosti ieiich tla pri dosazeni pohlavni zralosti (napr. Hutchings
a Reynolds 2004: Swain a kol. 2007: Miethe a kol. 2010), coz muze nasledn zvysovat i ieiich
prirozenou mortalitu (Swain 2010). Ve sladkch volnch vodach dochazi vlivem rybolovu,
nechtnch uniku ryb z akvakultury iakozto i zamrnho vysazovani ryb do volnch vod
k vraznm zmnam populaci i v druhovm slozeni rybich spolecenstev (Kottelat a FreyhoI 2007).
Podpurnm vysazovanim ie suplovan ubytek pocetnosti populaci nkterch druhu ryb. Vysazovan
nasady isou obvykle produkty umlho vtru a odchovu, kdy dochazi k vznamnmu zasahu
do genoIondu populace, nebot doide k velmi uspsnmu rozmnozeni nkolika malo vybranch
iedincu (Fraser 2008). Ryby casto mivaii velmi vysokou plodnost, kterou predstavuii az miliony
iiker (Penaz 1995). Preziti ranch vvoiovch stadii ryb, ktera se vyznacuii neivyssi mortalitou,
ie mnohonasobn vyssi v lihnich, nez volnch vodach (Ferguson 2007). Vliv umlho vtru
na genetickou variabilitu zdroiov populace ie tak znacn a v nkterch ohledech dokonce
srovnateln s tzv. .pruchodem hrdlem lahve' (Hansen a kol. 2000).
10
Presto ie podpora prirozen reprodukce rybami, ieiichz rodice vsak museii pochazet z tze
oblasti, zreim tim neivhodnisim zpusobem, kterm lze suplovat snizovani pocetnosti populaci
ryb, kter isou pod silnm rybarskm tlakem. Volbou vhodn metodiky tak lze riziko dalsiho
poklesu genetick variability divokch populaci snizit (Ferguson 2007: McClure a kol. 2008:
Ballou a kol. 2010). Skutecnost, ze isou nasadov ryby casto transportovany a vysazovany
v oblastech znacn vzdalench od mist ieiich puvodu, vsak vede k hybridizaci a genetick
homogenizaci voln ziiicich populaci a ke smvani prirozenho IylogeograIickho vzoru (Olden
2004: Taylor 2004: Sanz a kol. 2006: Ferguson a kol. 2007: Mabuchi a kol. 2008: Randi 2008:
MuhlIeld a kol. 2009). Jest vraznisi dopad na puvodni populace muze mit vypoustni
kulturnich a domestikovanch linii thoz druhu z akvakultury. Chovn linie isou obvykle
predmtem plemenitby a casto isou slechtny na konkrtni uzitkov vlastnosti. Diky neuvazen
podpore prirozen reprodukce tak divok populace mohou ztratit sv lokalni adaptace,
prizpusobivost a plasticitu, coz vede ke snizovani ieiich reprodukcni zdatnosti Iitness
(napr. AllendorI a kol. 2001: Araki a kol. 2008: Hutchings a Fraser 2008: Fraser a kol. 2010:
Marie a kol. 2010).
Zvlastnim pripadem ie vysazovani druhu mimo oblast ieiich prirozenho vskytu. Prestoze
isou dnes iiz dobre znama mnoha rizika, ktera s sebou introdukce exotickch druhu prinasi
(napr. Pimentel a kol. 2000, 2005), v pripad ryb k ni casto stale dochazi zamrn a to predevsim
u hospodarsky vznamnch druhu (Lintermans 2004). Detailni znalost introdukovanch populaci
ie nutnm predpokladem pro schopnost vrohodn odhadnout mozn dopad na mistni ekosystmy
i miru nebezpeci pro autochtonni organismy plynouci z pravdpodobnosti naturalizace exotickch
druhu.
Neprimch antropogennich vlivu na genetickou strukturu populaci ryb ie mnoho a mnoho
iich zreim ani dosud nebylo rozpoznano. Ve sv disertacni praci tyto vliv tchto ievu nestuduii
a tak se k nim vyiadrim ien velmi strucn. Casto se iedna o dalsi vlivy, kter smvaii habitatovou
segregaci druhu a indukuii hybridizaci. Vznamn vliv na genetickou strukturu populaci ryb proto
maii vodni dila (PoII a kol. 2007). Patri mezi n iak propoiovani puvodn oddlench povodi
kanaly, tak Iragmentace prehradami nebo iezy. Diky zmnam charakteru vod iak
na makrohabitatov tak mikrohabitatov urovni muze dochazet k prostorovmu i casovmu
prekryvu vtru ruznch druhu ryb (napr. Balon 1992). Vrazna zmna charakteru substratu,
hloubky, proudni a teploty muze vst neien k vraznm zmnam ve druhovm slozeni obsadky
(De Leeuw a kol. 2007: Schmutz a kol. 2007), ale i k tvrd selekci urcitch Ienotypu, ci kolapsu
reprodukcn izolacnich mechanizmu a masov mezidruhov hybridizaci (napr. Balon 1992).
Dalsim prikladem muze bt hybridizace ceina obecnho s plotici obecnou (Abramis brama x
Rutilus rutilus) na mnoha lokalitach po cel Evrop (Hayden a kol. 2010). Krom vodnich staveb
11
clovk svou cinnosti indukuie zmny obsahu rozptlench castic a rozpustnch latek ve vod.
Tyto Iaktory mohou mit podstatn vliv napr. na inkubaci iiker, parazitaci ryb ai. (LaIIerty 2008:
Roni a kol. 2010). Znecistni vod neurohumoraln aktivnimi latkami muze u nkterch druhu
indukovat hermaIroditismus nebo zvrat pohlavi (Jobling a Tyler 2003). To vse muze vst neien
k nabouravani reprodukcn izolacnich mechanismu, ale i k vznamnm zmnam v genetick
strukture populaci a slozeni ichtyoIauny volnch vod vubec. V clovkem narusench
ekosystmech potom mohou mit tendenci dominovat nepuvodni (invazni) druhy (Litchman 2010).
12
1.4. Ln obecn 1inca tinca (Linnaeus, 1758) jako modelov druh
Lin obecn ie domestikovanm (Bilio 2007) cyprinoidem, iehoz IylogeograIicka historie dosud
byla navzdory sirokmu arealu rozsireni a vznamu v evropsk akvakulture (Susta 1884: SteIIens
1995) prakticky neznama. Neistarsi pisemn zaznam o ryb, zvan .tinca' vytvoril
neipravdpodobnii Decimius Magnus Ausonius roku 370 n. l. v poemu o rece Mosella protkaiici
soucasnou severni Francii (bvalou Galii) a slovo .tinca' ie pravdpodobn Galskho puvodu
(Giovio 1524). V Cechach ie lin chovan v rybnicich iako doplnkova ryba spolu s kaprem
minimaln od pocatku 18. stoleti (Susta 1884). Tento systm ieho produkce existuie v mnoha
oblastech dodnes (SteIIens 1995). Prestoze ie lin obecn i nyni hospodarsky vznamnou
a komercn zaiimavou rybou, ktera zaziva intenzivni domestikaci (Hulata 1995: Gela a kol. 1998),
ve velk casti puvodniho arealu, kter pravdpodobn saha od Velk Britanie pres Kaspick umori
po iezero Baikal (Brylinska a kol. 1999a), se stale iest rozmnozuie prirozen.
Hlavnim cilem m disertacni prace bylo ziistit, do iak miry ie soucasna IylogeograIicka
struktura prirodnich populaci ovlivnna transIerem ryb v dusledku rybarskho hospodareni
a do iak miry odrazi prirozen demograIick a biogeograIick zmny, ke kterm dochazelo
v souvislosti s vvoiem klimatu v prubhu ctvrtohor (Hewitt 2004).
Z pohledu zoologick systematiky ie lin obecn iedinm ziiicim druhem rodu Tinca
Cuvier, 1816. Jiz Kryzanovskii (1947) iei oddlil do zvlastni podceledi Tincinae, coz vsak mnozi
systematici pozdii zpochybnovali (viz. Howes 1991). V ramci nadceledi Cyprinoidea, coz ie
velmi druhov bohata skupina recentnich sladkovodnich ryb (Chen a Mayden 2009), ie ieho
postaveni stale neiasn (Briolay a kol. 1998) a prilis svtla do rekonstrukce ieho genealogie
nevnesly ani recentni molekularn-Iylogenetick studie. Na zaklad vsledku studie zalozen
na kombinaci dat ziskanch sekvenaci mitochondrialni kontrolni oblasti, cytochromu b
a 16S rDNA byl Gillesem a kol. (2001) zarazen do blizkosti neistarobyleisich podceledi
Rasborinae a Cyprininae, iako sesterska skupina vsech ostatnich druhu celedi Cyprinidae.
V soucasnosti isou tyto skupiny povazovany za samostatn celedi a lin byl oddlen
do monotypick celedi Tincidae, kter se zdaii bt Iylogeneticky neiblizsi celedi Acheilognathidae
a Tanichthyidae (Chen a Mayden 2009).
Paleontologicky ie rod Tinca dolozen od stredniho miocnu z Nmecka (Gaudant 1980,
Bhme 2002). Obrhelova a Obrhel (1987) uvadii vskyt tohoto rodu na uzemi bval CSSR
(Filakovo, SK) az z pliocnu. Znamo ie celkem asi devt Iosilnich druhu rodu Tinca (Cern 1995).
Ve Iosilnim zaznamu na uzemi CR uvadi Obrhelova (1977) zastupce blizce pribuznch rodu
(Protothvmallus oligocn: Palaeotinca spodni az stredni miocn). Neistarsi zminovana Iosilie
druhu Tinca tinca pochazi ze svrchniho pliocnu Italie (Pieragnoli 1931 citovan Brylinskou a kol.
13
1999b), avsak tento nalez dle nkterch autoru vyzaduie veriIikaci (Brylinska a kol. 1999b). Bzn
se vsak lin obecn vyskytuie od pleistocnu v Holandsku, Nmecku, Polsku, Rakousku
a Madarsku (Gaudant 1979: Bhme a Ilg 2010) a Britanii (Lister a kol. 1990), v povodi
Kaspickho a Cernho more (Lebedv 1960) a na zapadni Sibiri (Shtylko 1934 citovan Brylinskou
a kol. 1999a). Glacialy pravdpodobn prezival v nkolika iizn polozench reIugiich a bhem
interglacialu, stein tak iako v postglacialu, opakovan rekolonizoval vodnatmi toky
za ustupuiicim ledovcem preglacialni areal (Thienemann 1950). Skandinavsk poloostrov
pravdpodobn osidlil bhem sladkovodni Iaze vvoie Baltskho more (Thienemann 1925, 1950),
zvan Ancylov iezero
1
(De Geer 1890) pred ~10,700-8,500 lety (Birck 2008).
Lina obecnho lze v soucasnosti nalzt na vsech kontinentech krom Antarktidy, za coz
vdci rozsahlm introdukcim (Welcomme 1988: Brylinska a kol.1999b). Problm v rekonstrukci
prirozenho arealu rozsireni predstavuii predevsim nezaznamenan introdukce provadn na ieho
hranicich, kter isou velmi tzko odlisiteln od spontanniho sireni podminnho napr. eIektem
.river capture'
2
. Prvni zaznamy o introdukcich linu v ramci Evropy pochazeii z pocatku 18.stoleti
(Kennedy & Fitzmaurice 1970). Je vsak velmi pravdpodobn, ze k nim dochazelo iiz drive.
Molekularnimi markery, iimiz byl lin dosud studovan na populacni urovni, isou alozymy
(Slechtova a kol. 1995: Kohlmann a Kersten 1998), mikrosatelity (Kohlmann a Kersten 2006:
Kohlmann a kol. 2010) a restrikcnimi enzymy stpen useky mtDNA (Lo Presti a kol. 2009). Tyto
studie isou vsak pouze lokalniho charakteru a neprinaseii komplexni vhled do IylogeograIick
historie druhu. Nktera ziistni byla navic shledana obtizn interpretovatelnmi, s neivtsi
pravdpodobnosti z duvodu absence sirsiho IylogeograIickho kontextu (viz. napr. Kohlmann
a kol. 2010).
1
Jako Ancylov iezero byla tato etapa vvoie Baltskho more nazvana diky sladkovodnimu mlzi kamomilu ricnimu
(Ancvlus fluviatilis), kter se tam v tto etap hoin vyskytoval.
2
Proces, kdy v dusledku eroze, ci zablokovani puvodniho koryta, reka nebo ieii cast zmni smr svho toku tak,
ze nasledn tece do iinho povodi. Vsechny organismy obvaiici tuto cast reky zmni svou prislusnost k novmu
povodi spolecn s ni. K tomuto geologickmu ievu zcela iist dochazelo ve zvsen mire v postglacialnich
obdobich, kdy masy vody z taiicich horskch ledovcu zpusobovaly zvsenou erozi, vytvareni priledovcovch
iezer, docasnch toku a novch ricnich spoieni, cimz umoznily migraci chladnomiln sladkovodni Iauny
do novch oblasti a genetick tok mezi docasn izolovanmi populacemi (Arkhipov a kol. 1995).
14
1.5. Cle prce
Cilem moii disertacni prace bylo provst IylogeograIickou studii lina v ramci celho ieho arealu
rozsireni s pouzitim ruznch molekularnich markeru, identiIikovat zodpovdn evolucni procesy
a rekonstruovat historick udalosti, kter mly na IylogeograIii lina rozhoduiici vliv. Dale bylo
mm cilem z tchto dat urcit vliv rybarskho hospodareni na IylogeograIii lina a ziskat podklady
pro inIormovan management lina ve volnch vodach i v akvakulture. Z praktickho hlediska bylo
uzitecn navrhnout metodiku pro identiIikaci geneticky a IylogeograIicky odlisnch populaci,
ktera umozni dalsi zpresnovani nasich znalosti o evolucni historii lina vcetn vlivu clovka. Moie
disertacni prace ie proto tvorena tremi ucelenmi studiemi, kter vsak na sebe vzaiemn uzce
navazuii. Vsechny tri studie isem zvereinil v impaktovanch vdeckch casopisech.
Clnek 1. Laibner Z., Linhart O., Kotlik P. (in press) Human-aided dispersal has altered but not
erased the phylogeography oI the tench. Evol. Appl.
(doi:10.1111/i.1752-4571.2010.00174.x).
Touto publikaci, ktera ie publikovana v casopise Evolutionarv Applications, predstavuii vsledky
IylogeograIick studie lina v celm arealu ieho rozsireni zalozen na sekvencich DNA iednoho
mitochondrialniho a trech iadernch genu. Krom prirozenho arealu hodnotim i IylogeograIii
znamch introdukci a tak rady chovnch linii.
Clnek 2. Laibner Z., Kotlik P. (Early View: 20 SEP 2010) PCR-RFLP assays to distinguish the
Western and Eastern phylogroups in wild and cultured tench Tinca tinca. Mol. Ecol. Resour.
(doi: 10.1111/i.1755-0998.2010.02914.x).
Clanek zvereinn v casopise Molecular Ecologv Resources popisuie nov RFLP markery,
kter isem vyvinul pro rychlou identiIikaci IylogeograIicky odlisnch populaci lina a shrnuie
moznosti ieiich vyuziti pri charakterizaci populaci lina v chovech i ve volnch vodach.
Clnek 3. Laibner Z., Kohlmann K., Linhart O., Kotlik P. (2010) Lack oI reproductive isolation
between the Western and Eastern phylogroups oI the tench. Rev. Fish Biol. Fisher. 20, 289-300.
(doi: 10.1007/s11160-009-9137-y).
S vyuzitim alozymovch, mikrosatelitovch a nov vyvinutch RFLP markeru v tto praci
zvereinn v casopise Reviews in Fish Biologv ana Fisheries testuii existenci reprodukcn-
izolacnich mechanizmu v oblasti postglacialniho kontaktu a hybridizace dvou geneticky odlisnch
populaci lina.
15
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25
2. Publikace
2.1. Human-aided dispersal has altered but not erased the phylogeography of
the tench.
Laibner Zdenk, Linhart Otomar, Kotlik Petr (v tisku)
Evolutionarv Applications.
(doi:10.1111/i.1752-4571.2010.00174.x)
26
Human-aided dispersal has altered but not erased
the phylogeography of the tench
Zdenk Laibner
1,2
,OtomarLinhart
3
andPetrKotlik
1
1LaboratoryoIFishGenetics,DepartmentoIVertebrateEvolutionaryBiologyandGenetics,InstituteoIAnimal
Physiology and Genetics, Academy oI Sciences oI the Czech Republic, Libchov, Czech Republic
2 DepartmentoIZoology,FacultyoIScience,CharlesUniversity,Prague,CzechRepublic
3 University oI South Bohemia, Cesk Budiovice, Faculty oI Fisheries and Protection oI Waters, Research Institute oI
Fish Culture and Hydrobiology at Vodany, Czech Republic.

Correspondence: Zaenk Laibner. LaboratorvofFishGenetics.DepartmentofJertebrateEvolutionarvBiologvana
Genetics.InstituteofAnimalPhvsiologvanaGenetics.AcaaemvofSciencesoftheCzechRepublic.Rumburska89.
277 21 Libchov. Czech Republic. e-mail.z.laibner(seznam.cz.phone.420315639516.fax.420315639510.

Abstract
Human-aided dispersal can result in phylogeographical patterns that do not reIlect natural
historical processes, particularly in species prone to intentional translocations by humans. Here
we use a multiple-gene sequencing approach to assess the eIIects oI human-aided dispersal
on phylogeography oI the tench Tinca tinca, a widespread Eurasian Ireshwater Iish with a long
history in aquaculture. Spatial genetic analysis applied to sequence data Irom Iour unlinked loci
and 67 geographic localities (38382 gene copies per locus) deIined two groups oI populations
that were little structured geographically but were signiIicantly diIIerentiated Irom each other,
anditidentiIiedlocationsoImaior geneticbreaks,whichwereconcordantacrossgenesandwere
drivenbydistributionsoItwophylogroups.ThispatternmostreasonablyreIlectsisolationintwo
maiorglacialreIugiaandsubsequentrangeexpansions,withtheEasternandWesternphylogroups
remaining largely allopatric throughout the tenchrange. However, thisphylogeographic variation
was also present in all 17 cultured breeds studied and some populations at the western edge
oIthenative range contained the Eastern phylogroup. Thus, natural processes have played
an important role in structuring tench populations, but human-aided dispersal have also
contributed signiIicantly, with the admixed genetic composition oI cultured breeds most likely
contributingtotheintrogression.
Keywords
Intron:mtDNA:secondarycontact:speciesrange:stocking:Tincatinca
27
Introduction
Determining the eIIects oI human-aided dispersal and how it overlays with natural distributional
changes is essential Ior the eIIective protection oI species throughout their native ranges.
TranslocationsthatoccurwithinthelimitsoIthenaturaldistributionoIaspeciesdonotextendits
range but instead superimpose new genetic signatures on the natural diversity patterns iI they
involve genetically divergent populations or domestic breeds(Taylor 2004: Ferguson et al. 2007:
Stone et al. 2007: Mabuchi et al. 2008: Randi 2008: MuhlIeld et al. 2009). The impacts oI such
translocations are thereIore more diIIicult to detect. Molecular phylogeography oIIers here
apowerIultool,whichcanalsobeusedtoresolvethecryptogenicnatureoIspecieswhosestatus
in a given area may be either native or introduced but where clear evidence Ior either origin is
absent(Carlton1996).
Theinternationaltradeandhuman-aidedtransportprovidesaneIIectivedispersalmechanism
in many aquatic organisms, and Ireshwater Iishes in particular. Up until now, phylogeographic
studies oI European Ireshwater Iishes were largely Iocused on species that were not targets
oIaquaculture (e.g. Durand et al. 1999: Kotlik and Berrebi 2001: lechtova et al. 2004:
Bohlen et al. 2007: ediva et al. 2008). Few economically important species have been studied
phylogeographically across their ranges, but even in those cases the Iocus has been primarily
onputative native populations, assuming (or hoping Ior) negligible phylogeographic contribution
oI human-aided dispersal (see Nesbo et al. 1999: TriantaIyllidis et al. 2002: Van Houdt et al.
2005).Astheresult,phylogeographicinIormationisstilllackingIormanycommonIishes,despite
theirroleinIreshwatercommunitiesandeconomicimportance.
One such domesticated Iish (Bilio 2007) with poorly known genetic structure (Kohlmann et
al. 2010:LoPrestietal.2010)despitetheancienthistoryintheEuropeanaquacultureandcuisine
(Giovio 1524: Lebedev1960: SteIIens 1995: Garcia-Berthouetal. 2007)isthetench Tinca tinca
(Linnaeus,1758).ThetenchiswidelydistributedbetweentheBritishIslesandIberianPeninsula
inthewesttocentralSiberiaintheeast(Fig.1),butbecauseithasbeenincultivationinEurope
Ior a long time (usta 1884: SteIIens 1995), itsexactnativerangeisdiIIiculttodiscern:insome
areas (e.g. Spain: Garcia-Berthou et al. 2007: Italy: Gherardi et al. 2008: Turchini and De Silva
2008),itmaybeeithernativeorintroducedbutclearevidenceIoreitheroriginisabsent(i.e.itis
cryptogenic there).There are records oI tench introduction outside its native range Irom as early
as the 18th century (e.g. to Ireland: Kennedy and Fitzmaurice 1970), and since then, introduced
populations have been established on all continents except Antarctica (Welcomme 1988: Bryliska
etal. 1999).Insomecountriesitisevenconsideredasaninvasive,potentiallyharmIulspeciesdue
concerns over competition with native Iish (e.g. Rowe 2004: Stokes et al. 2004: Hesthagen
andSandlund2007:Roweetal. 2008:DeVaneyetal.2009).
28
Distribution oI genetic diversity oI Ireshwater Iishes is largely controlled by the island-like
nature oI their habitats (Bernatchez and Wilson 1998), and the present-day phylogeographic
patterns oI temperate species have been shaped primarily by isolation in multiple glacial reIugia
during the last glacial maximum (18 000-23 000 years ago), Iollowed by range expansion and
drainageisolation.ManywidelydistributedtemperateIreshwaterIishspeciesthereIoreshowdeep
phylogeographicsubdivisions(e.g.Durandetal. 1999:Bernatchez2001:KotlikandBerrebi2001:
Van Houdt et al. 2005: Kotlik et al. 2008: HnIling et al. 2009). However, some species display
only a limited or shallow phylogeographic structure, which is usually interpreted as the result oI
arecentdispersionIromonlyoneglacialreIugium(TriantaIyllidis
Figure 1 Putativenative(olive)andpartoInon-native(violet)distributionrangeoIthetench.Largeareas
wheretheoriginisconsideredambiguousarehighlightedbyorange.LocationsoImaiorIreshwaterglacial
reIugiainEurope,Western/Atlantic(R1),Danubian(R2)andPonto-Caspian(R3),areindicated.Sampling
countriesare labelled(codes:B,Belgium:BG, Bulgaria:BIH,BosniaandHerzegovina:CH,Switzerland:
CZ,CzechRepublic:D,Germany:EST,Estonia:GB,GreatBritain:H,Hungary:I,Italy:P,Portugal:RO,
Romania:S,Sweden:SK,Slovakia).
ReIerences to the map: Urchinov 1995: Bryliska et al. 1999: MitroIanov and Petr 1999: Savvaitova
andPetr1999:Economidisetal.2000:Wangetal.2004:InnalandErkakan2006:HestagenandSandlund
2007:Popov2009:Mamilovetal.2010.
etal. 2002:Bohlenetal.2007).
Alternatively,itcanpointtostrongeIIectsoIhuman-aidedtranslocations(HnIlingetal.2009).
The present study uses a multiple-gene sequencing approach (Brito and Edwards 2008)
andbarrier-detection statistics to test iI the range-wide genetic variation oI the tench shows
asigniIicant phylogeographic structure that can be explained by natural processes during the last
glacial-interglacialcycle.TenchoccupyallmaiorIreshwaterregionsinEurope,sothatitshouldbe
possible to identiIy the contribution oI diIIerent reIugia (Fig. 1) to its present-day distribution.
However, iI human-aided dispersal signiIicantly altered recent evolutionary history oI the tench,
the haplotypes could have been re-distributed among populations, wiping out any natural
phylogeographic structure (Sanz et al. 2006). Captive breeding can produce admixed gene pools,
increasing the homogenizing eIIect oI human-aided dispersal. To assess this eIIect oI hatchery
practices, in addition to putative native populations we also sampled various cultured strains
andknownintroducedpopulationsoutsidethenativerange.
29
Materials and methods
Sampling
Sampled populations were chosen to cover the maiority oI the natural range oI the tench in Europe
and Asia. Fin tissue samples were stored in 95 ethanol. A total oI 225 individuals were collected
Irom 76 populations and included 25 hatchery stocks and several known introductions (Fig. 2:
Appendix A). A single specimen (MNHN 00001357) Irom the collection oI the Museum National
d'Histoire Naturelle in Paris, France, was sampled. We also analysed 16 Czech and Ioreign
cultured tench breeds maintained in the live gene bank oI the Research Institute oI Fish Culture
and Hydrobiology in Vodnany, Czech Republic (Gela et al. 1998: Flaishans et al. 1999: Gela et al.
2006), and an Italian regional breed, the Golden hump tench oI Poirino highland (Gasco et al.
2010).
Data collection
Introns oI three nuclear genes and a complete sequence oI one mitochondrial gene (Table 1) were
analysed by polymerase chain reaction (PCR) ampliIication Irom genomic DNA and direct
sequencing. Total genomic DNA was extracted with QIAGEN DNeasy

Tissue Kit. The PCR

conditions Iollowed standard methods (Tsigenopoulos and Berrebi 2000: Machordom and Doadrio
2001). The resulting PCR products were puriIied using the Millipore Montage PCR centriIugal
Iilter devices and were directly sequenced with the ABI PRISM BigDye Terminator Cycle
Sequencing Kit (Applied Biosystems) and puriIied using DyeEx Spin Kit (Qiagen). The extension
products were run on ABI 3730 or 3730xl automated sequencers. Sequences were assembled using
SEQMAN II (DnaStar Inc.) with the deIault options. All sequence traces were inspected visually
to check the accuracy oI the heterozygous base calls (Hare and Palumbi 1999). Nucleotide
sequences oI each unique haplotypes were deposited in the GenBank database under the accession
nos HM167935-HM167965.
A part oI nuclear DNA containing the second intron oI the actin gene (Act) was ampliIied
and sequenced using primers Act-2-R and Act-2-F described by Atarhouch et al. (2003). The intron
oI the gene coding Ior the ATP synthase beta subunit (ATPase) was ampliIied and sequenced using
the primers described by Jarman et al. (2002). The Iirst intron oI the gene coding Ior the S7
ribosomal protein (RpS7) was ampliIied and sequenced using the primers S7RPEX1F
and S7RPEX2R (Chow and Hazama 1998). Haplotypes were inIerred Irom diploid sequence traces
(Clark 1990: Won and Hey 2005) and veriIied by the use oI IastPHASE (Scheet and Stephens
2006). The entire mitochondrial cytochrome b gene (Cvtb) was ampliIied with the primers GluF
and ThrR described by Machordom and Doadrio (2001) and sequenced with newly designed
Iorward (5' - AAACAACCCAACAGGACT - 3') and reverse sequencing primers
(5' - CAAATAGGAAATATCATTCTG - 3').
30

Figure 2 Geographical distribution oI maior clades and SAMOVA groups. CladeW is shown in red and
cladeEinblueIorATPase(a),Act (b)andRpS7 (c).ForCvtb (d),cladeWisinred,cladeEAinblue,clade
EC in green and clade EI in yellow.The same colours are used Ior the SAMOVA groups (e). Boxed data
pointstotherightandleItoIthemapsin(b)through(e)representidentitiesIortwositesinNorthAmerica
andinChinaandNewZealand,respectively|see(a)|.ForexacthaplotypedistributionandIrequenciessee
AppendixA.
31
Data analyses
Sequence analvsis
For each locus, we estimated the haplotype and nucleotide diversities and their variances (Nei
1987). To explore whether intragenic recombination may have aIIected the patterns oI variation
at Act, ATPase and RpS7. we used the Iour-gamete test (Hudson and Kaplan 1985). McDonald-
Kreitman (1991) test was perIormed Ior Cvtb to test Ior deviation Irom neutrality using
an outgroup species and comparing diIIerent tench clades against each other. Tinca tinca is
the only species in the Iamily Tincidae, so that a sharpbelly species, Hemiculter leucisculus, Irom
a related Iamily Cultridae (Chen and Mayden 2009) has been used as the outgroup (GenBank
Accession no. AF095608). All the calculations were perIormed using DNASP, version 4.50.3
(Rozas et al. 2003).
Phvlogenetic ana network analvses
Rooted phylogenies were reconstructed by the maximum-likelihood criterion (ML) using PhyML
version 3.0.1 (Guindon and Gascuel 2003). We used Akaike inIormation criterion and iModelTest
version 0.1 (Posada 2008) to identiIy the HKYG model as the most suitable model oI DNA
substitution Ior the Cvtb data and the TrN model Ior the RpS7 data. Sharpbelly RpS7 sequence
was not available, so that a sequence (AY325789) oI the rosy bitterling, Rhoaeus ocellatus,
Irom another related Iamily Acheilognathidae was used to root the RpS7 tree. The robustness
oI the trees was assessed by the approximate likelihood ratio test (Anisimova and Gascuel 2006)
and by bootstrap resampling (1000 replicates: Felsenstein 1985) using PhyML. A haplotype
network was constructed Ior each gene by the statistical parsimony (Templeton et al. 1992)
as implemented in TCS version 1.21 (Clement et al. 2000).
Inference of aemographic historv
To examine past population dynamics we calculated two commonly used summary statistics D
(Taiima 1989) and Fs (Fu 1997) with DnaSP and ARLEQUIN version 3.11 (ExcoIIier et al. 2005).
Their signiIicance was tested by generating random samples under constant population size using
a coalescent simulation conditioned on the number oI polymorphic sites (Ramirez-Soriano et al.
2008). For neutral markers, signiIicant negative values can be expected in cases oI population
expansion (Taiima 1989: Fu 1997).
As another way oI assessing signatures oI reIugial expansion, we considered the distribution
oI the number oI pairwise nucleotide diIIerences (mismatch distribution) by contrasting observed
distributions with those expected Irom models oI population size change. We tested whether
the data Iitted the sudden demographic expansion model (Rogers and Harpending 1992)
32
or the instantaneous range expansion model (ExcoIIier 2004), using ARLEQUIN. The models
were Iitted to the data by a generalized non-linear least-square approach, which allowed
the estimation oI the parameter t t/2, the expansion time scaled by the mutation rate (Schneider
and ExcoIIier 1999). A parametric bootstrapping approach (Schneider and ExcoIIier 1999) was
used to obtain the probability that the observed data conIorm to the model using the sum oI square
deviations (SSD) between the observed and expected mismatch distribution as a test statistic.
We considered a wide range oI estimated Cvtb mutation rates Ior Iishes oI about 0.0050.125
substitutions per site per Myr, published by Dowling et al. (2002) and Burridge et al. (2008),
respectively.
Spatial genetic analvsis
Two complementary barrier-detection methods were applied to identiIy any discontinuities
in the geographic distribution oI genetic variation (Guillot et al. 2009). The geographical
component oI the phylogeographic pattern was Iirst assessed by the spatial analysis oI molecular
variance using SAMOVA version 1.0 (Dupanloup et al. 2002). The advantage oI SAMOVA is that
it removes bias in population designation because it does not make a priori group distinction
Ior genetic analyses. It employs a simulated annealing procedure using geographical locations
oI the sampling sites to cluster the sites into a user-deIined number oI groups (K), so that
the proportion oI total genetic variance between groups (F
CT
) is maximized and the proportion
oI variation among sites within groups (F
SC
) minimized.
Maior barriers to the distribution oI genetic variation were then estimated by the Monmonier`s
(1973) maximum diIIerence algorithm implemented in BARRIER version 2.2 (Manni et al. 2004),
based on a matrix oI the pairwise net genetic distances among sampling sites generated Irom DNA
sequences using ARLEQUIN. The algorithm was applied to a network connecting
the geographical coordinates oI the sampling locations computed using Delaunay triangulation
(Manni et al. 2004). Analyses were perIormed separately Ior each locus but on the same
geographical network and the results were then combined to identiIy barriers supported
by multiple loci: the locus ATPase was excluded due to its limited geographical coverage.
33
Coalescent simulation
We conducted a series oI simulation experiments to evaluate iI a natural population that was
Iounded by unrelated clades at the end oI the Younger Dryas, and has been isolated Irom other
populations since then, may still carry haplotypes Irom diIIerent clades. This situation would
correspond, Ior example, to tench populations inhabiting lakes in deglaciated areas oI northern
Europe (see Laibner et al. 2010). In each experiment, we simulated 10 000 coalescent trees using
Mesquite version 2.5 (Maddison 2008: Maddison and Maddison 2008) to estimate the distribution
oI the time to the most recent common ancestor (TMRCA) in such a population, and we counted
the trees deeper than 3000 generations, approximately corresponding to the end oI the Younger
Dryas c. 11,500 years ago (Muscheler et al. 2008) and the generation time oI Iour years (Monich
1953: Pekar 1965). We parameterized the simulations by Iemale eIIective population size (N
ef
)
values corresponding to known population densities oI tench (c. 100500 individuals per hectare:
Lusk et al. 1998) and a lake area between 10 and 400 hectares, and assuming an equal sex ratio
(Monich 1953) and the ratio oI the eIIective population size to the adult census size, N
e
/N, oI 0.3
(Turner et al. 2006). We Iocused on the Iemale component oI population, which is represented
in our data by mtDNA variation, because oI its relatively shallower coalescence-time depth
and thereIore shorter expected TMRCA compared with autosomal loci. For values oI N
ef
yielding
the number oI deep trees that was less than 5 oI all the trees simulated assuming that N
ef
,
we considered it unlikely that a population with that eIIective number oI Iemales would still
contain haplotypes Irom diIIerent clades unless the haplotypes were recently redistributed among
populations through human-mediated movement. On the other hand, a high number oI deep trees
(i.e. more than 95) would indicate that there is no need to invoke recent gene Ilow as the likely
explanation Ior the coexistence oI divergent clades in such population, which could be the result
oI natural post-glacial contact. Although these simulation experiments make simpliIying
assumptions that may not be realistic, they generate ideal benchmarks Ior interpreting the observed
data.
34
Results
Sequence variation
The levels oI polymorphism among sequences obtained Ior each oI the Iour genes (38430 gene
copies per gene) are summarized in Table 1. There were Iive short (5 bp) insertion/deletion
(indel) polymorphisms segregating at the RpS7 locus (Table 1) that were not associated
with simple sequence repeats and could be unambiguously aligned. OI these, a two-base deletion
was inIerred to have occurred along the branch leading to clade W (see below) and a single-base
deletion along the branch leading to clade E. Data sets Irom neither Act, ATPase nor RpS7 showed
evidence oI homoplasy and they all passed the Iour-gamete test, indicating that recombination
has not aIIected the patterns oI variation at the nuclear genes in our study. The McDonald-
Kreitman test provided no evidence oI selection on the coding sequence oI the Cvtb gene (P~0.05).
Genealogical and geographical relationships
The phylogenetic and network analyses split the range-wide data set Ior the mitochondrial Cvtb
into two distinct phylogroups (clades W and E) separated with 1.6 oI genetic distance
(Fig. 3e,I), translating to a divergence time oI about 64x10
3
1600x10
3
years ago. The Western
phylogroup was Iound in Europe between the British Isles and Poland whereas the Eastern
phylogroup was present Irom Europe throughout Asia to China, with a broad zone oI overlap
with the Western phylogroup in Europe (Fig. 2d). While clade W showed very little internal
structure, clade E was partitioned into three subclades (Fig. 3e,I). The maiority oI haplotypes were
in the clade EA, while the other two clades had very restricted distributions: the EC haplotypes
in the Anzalee lagoon oI the Caspian Sea in Iran, and the EI haplotype in the Iskar River
oI the Danube River drainage in Bulgaria (Fig. 2d).
We constructed a phylogenetic network Ior each nuclear DNA locus and a phylogenetic tree
oI the RpS7 haplotypes (Fig. 3ad). The most salient Ieature oI the inIerred genealogies
is the complete lineage sorting oI nuclear genes between the two phylogroups in that all genes
are distinguished into two clades W and E and the divergence between the phylogroups based
on sequences oI the nuclear Act, ATPase and RpS7 genes is geographically concordant
with mitochondrial Cvtb sequences (Fig. 2ad). Nuclear DNA loci and mtDNA thus display
striking similarities, showing a strong genealogical concordance across the distribution range
oI the tench. Changes in mtDNA and the three nuclear loci are concordant also across the contact
zone between the two phylogroups, with only Iiner-scale diIIerences being evident in phylogroup
Irequencies among sites (Fig. 2ad).
35
The introduced populations in Turkey and China carried at all loci only clade E haplotypes,
as did the overseas introduction to the state oIWashington. However, the non-native populations
in BosniaandHerzegovina,inNewZealandandinQuebeccarriedatoneormorelocihaplotypes
IrombothcladeWandcladeE(Fig.2ad).
The phylogeographical variation observed among the tench populations was present also
in the cultured breeds, with the exception oI Cvtb clades EC and EI that had very restricted
geographical distributions. Each one oI the 16 cultured breeds in the Vodany live gene bank
as wellastheItalianregionalbreedcarriedhaplotypesIrombothcladesWandEatoneormore
loci, including the seven regional Czech breeds, three European breeds (German, Romanian
andHungarian),threeexperimentalbreedsandthreeornamentalbreeds(AppendixA).
Figure 3 Haplotype relationships. Clade E is shown in blue and clade W in red Ior ATPase (a), Act (b)
and RpS7 (c, d). For Cvtb (e, I), clade W is in red, clade EA in blue, clade EC in green and clade EI
in yellow. The networks were constructed under the 95 maximum parsimony criterion and the size
oI the circles is proportional to the haplotype Irequency: small empty circles represent unobserved
haplotypes.The maximum-likelihood phylograms are shown with bootstrap (Irom 1000 replicates)/ aLRT
support Ior maior partitions in the RpS7 (d) and Cvtb (I) phylogenies, with branch lengths proportional
to thescalebarwiththeunitbeingameannumberoInucleotidechangespersite.
36
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37
Population demographic history
The D and Fs statistics were negative Ior the maior Cvtb clades W and E as well as Ior clades EA
and EC, reIlecting the excess oI rare mutations compared to the expectation under constant
population size, and Ior clades W, E and EA this diIIerence was signiIicant (Table 1). A similar
pattern was observed at the Act and RpS7 genes, with a number oI D and Fs values being large
and negative, and with signiIicant results Ior both Act clades and the RpS7 clade E (Table 1).
For all Iour genes and clades W and E as well as Ior Cvtb clades EA and EC there was also
a good Iit (P (simulated SSD observed SSD) ~ 0.1) between the observed and the expected
mismatch distribution Irom at least one expansion model (Table 1). The t values obtained Ior Cvtb
clades W (0.373) and EA (3.000) translate into an expansion time oI about 130831 134 years ago
and 10 517262 927 years ago, respectively.
Spatial genetic structure
The SAMOVA analyses identiIied a signiIicant two-group spatial structure Ior each locus (Fig. 2e),
with approximately 65 to 100 oI the genetic variation proportioned between the two groups
(Cvtb: F
CT
, 0.687, P0.05: F
SC
, 0.606, P0.001: nuclear DNA loci: F
CT
, 0.6671.000, P0.001: F
SC
,
0.0000.080, P0.001). Assuming a Iour-group scenario Ior Cvtb placed the Anzalee population
(clade EC) and the Iskar population (clade EI) in their own separate groups (Fig. 2e), yielding
higher F
CT
(0.791, P0.001) and lower F
SC
values (-0.095, P0.001) than those observed Ior this
gene in the two-group scenario. Interestingly, one SAMOVA group was deIined in the way that its
distribution was clearly partitioned into distinct sets oI sites which belonged to that same group but
which were not geographically adiacent (i.e. the British, one Swedish and the Spanish
and Portuguese sites were placed in the same group with sites Irom eastern Europe and Asia:
Fig. 2e).
The BARRIER analysis overlaying Iive maior barriers Ior each locus identiIied several
discontinuities with a support Irom multiple loci (Fig. 4). The longest break divided the tench
distribution into a western part and an eastern part and was Iully supported by two loci
and partially by all three loci (Fig. 4), depending on the local patterning oI clades in the contact
zone between the Western and Eastern phylogroups (Fig. 2bd). Another barrier separated
the Spanish and Portuguese sites Irom the rest oI the sites with a complete support oI all loci.
The third barrier separated the British sites Irom the other sites with a support oI two loci,
and the Iourth barrier separated the Swedish site Lake re si Irom the other sites in Sweden
and around the Baltic Sea, with a complete support Irom two loci and a partial support oI all loci
(Fig. 4). Additional three short breaks supported by two loci were identiIied in central Europe
(Fig. 4), Iollowing the transitions between phylogroups in that region (see Fig. 2e).
38
TMRCA distribution
The simulations oI the TMRCA assuming N
ef
oI 730 produced Iewer than 5 oI coalescent trees
that were deeper than 3000 generations. We thereIore consider it unlikely that an isolated
population with this eIIective number oI Iemales or smaller that was Iounded by unrelated mtDNA
clades at the end oI the Younger Dryas (assuming the generation time oI Iour years) would still
contain haplotypes Irom diIIerent clades, unless the haplotypes were recently redistributed among
populations by human-mediated movement. However, Ior any N
ef
larger than that, there was
greater than 5 chance that the TMRCA predated the origin oI the population, and Ior N
ef
larger
than 4000, more than 95 oI all coalescent trees were deeper than 3000 generations. The eIIective
number oI Iemales oI 4000 would translate to an adult census size oI c. 25 000 individuals
assuming an equal sex ratio and the ratio N
e
/N oI 0.3, which would correspond to a lake area
oI c. 250 hectares, assuming the population density oI 100 individuals per hectare.
Figure 4 European phylogeographic breaks identiIied in tench data by BARRIER using the Monmonier`s
algorithm. Thin lines, Delaunay triangulations: thick lines, barriers supported by at least two loci.
The thickness oI the diIIerent barriers and their segments is proportional to the number oI loci that
supported them (two or three).
39
Discussion
Pleistocene phylogeographical subdivision
The statistical method in SAMOVA detected a signiIicant phylogeographical pattern driven by
the spatial orientation oI the Western and Eastern phylogroups, with high congruence between
mtDNA and nuclear DNA loci (Fig. 2e). The barrier detection method in BARRIER revealed
a well-supported genetic break crossing central Europe in a north-south direction (Fig. 4),
paralleling the transition between the phylogroups (Fig. 2ad). These results together provide
evidence oI a strong geographical component to the present phylogeographical pattern in the tench
that is highly concordant among unlinked loci.
The distribution oI highly divergent, reciprocally monophyletic phylogroups is strongly
reminiscent oI phylogeographic discontinuities modulated by reIugial isolation (Taberlet et al.
1998: Hewitt 2000). It seems thus likely that, aIter the last glacial maximum, the Western
phylogroup originated Irom the western European reIugium, whereas the Eastern phylogroup
originated Irom an eastern European or western Asian reIugium. This conclusion is in accordance
with previous phylogeographic studies indicating putative Ireshwater reIugia in drainages oI
the Atlantic tributaries and oI Rhone River (Durand et al. 1999: Nesbo et al. 1999: Kotlik
and Berrebi 2001) and in the Black and Caspian Sea basins (Bnrescu 1991: Kotlik and Berrebi
2001: Kotlik et al. 2004, 2008). The importance oI the Ponto-Caspian reIugium is supported
by the Iindings oI tench Iossils Irom glacial deposits in the Black Sea basin (Lebedev 1960). It is
interesting that a distinct Cvtb clade EC occurred in the southern Caspian Sea and only there,
although the widespread clade EA occurred in the northern Caspian Sea, and all tench Irom both
sites carried the same nuclear DNA haplotypes (Fig. 2ad). Furthermore, clade EI occurred only
at one site in the Iskar River basin in the lower Danube River drainage, where again only
widespread nuclear DNA haplotypes were present (Fig. 2ad). This shows hitherto undescribed
complexities in the distribution oI reIugia within the Ponto-Caspian region and the Danube River,
and lineage sorting and/or gene Ilow between them.
The signatures oI population expansion in both phylogroups are consistent with a history
oI post-glacial dispersion Irom Iormerly isolated reIugia. The estimates oI times Irom population
expansion are approximately consistent with an expansion Iollowing the last glacial maximum. II,
on the other hand, the signiIicant tests reIlected recent introductions, the time estimates should
indicate much more recent expansion. The higher age oI the expansion oI the Eastern phylogroup
than oI the Western phylogroup is congruent with phylogeographical evidence Irom other Iishes
that the geographic range occupied by the Eastern phylogroup was much less directly aIIected
by recent glacial advances than the western European drainages (Bernatchez 2001).
40
Fourteen sites in central and northern Europe were assigned to one SAMOVA group by some
loci and to the other SAMOVA group by the other loci (Fig. 2e), and admixed sites carrying
haplotypes oI both phylogroups were observed over a large area between, roughly, Belgium
and Estonia (Fig. 2ad). Changes in mtDNA and the three nuclear loci are concordant across
the contact zone, supporting that this is not a matter oI primary contact and selection on some
oI the markers but rather oI a secondary contact oI populations Irom diIIerent reIugia. But can this
introgression be caused entirely by human-aided dispersal? Our TMRCA simulations indicated
that there is no need to invoke recent gene Ilow as a likely explanation Ior the presence oI both
phylogroups even in relatively small populations. Furthermore, the location oI the tench contact
zone matches phylogeographical subdivisions in other species where expanding populations
Irom diIIerent reIugia meet in the same area (e.g. Taberlet et al. 1998: Hewitt 2000). We thereIore
consider it unlikely that the overlap between the phylogroups at the sites in central Europe has
been entirely caused by human transport and release. Rather, it most likely represents a region
oI natural post-glacial contact between lineages Irom the eastern and western reIugia.
Evidence for human-aided dispersal
On the other hand, the contact zone is very broad and spans across several watershed divides,
and there is Iairly high amount oI introgression in western Europe (Fig. 2bd). The SAMOVA
analysis even placed sites Irom three western European regions that contained particularly high
proportions oI the Eastern phylogroup into the same group with the sites Irom eastern Europe
and Asia (Fig. 2e). These sites were located on Iberian Peninsula, in Britain and in Sweden,
and they were separated Irom the other western sites with a BARRIER support oI several loci
(Fig. 4). All tench Irom the three sites in Spain and Portugal contained exclusively the Eastern
phylogroup, which strongly speaks in Iavour oI the hypothesis that tench are not a native species
on the Iberian Peninsula (Garcia-Berthou et al. 2007: Ribeiro et al. 2009), and points to the eastern
Europe or Asia as their likely source. This demonstrates the ability oI detailed phylogeographic
studies such as ours to resolve the status oI cryptogenic species where other evidence Ior either
native or introduced origin is absent (Carlton 1996). The lack oI phylogeographic resolution
means, however, that we cannot conIirm or reiect the native status oI the populations in Italy
(Gherardi et al. 2007: Turchini and De Silva 2008). The absence oI strong genetic separation Irom
more northern sites (Fig. 2e and 4) suggests that tench colonization oI Italy is most likely oI post-
glacial origin.
41
Another site in western Europe that only contained Eastern alleles is Lake re si in southern
Sweden. It may suggest that this population escaped admixture, but it may also be that the sample
oI only one Iish (Iour loci) was not enough to detect the Western phylogroup iI it was present
in low Irequency.
The British sites were separated Irom the other western sites by BARRIER, but they carried
a mixture oI the Eastern and Western phylogroups, which was reIlected by their SAMOVA
assignment to both groups, depending on the locus (Fig. 2e). This is probably a result oI human
introduction oI the Eastern phylogroup to the British Isles as this phylogroup occurs in much lower
Irequency in western Europe. It could also be a natural colonization by both phylogroups but it
would require almost complete replacement oI the Eastern phylogroup in western Europe
(see Searle et al. 2009).
Cultured breeds and introgression
The above evidence strongly suggests that human-aided dispersal has altered the phylogeographic
structure oI the tench. This implies either that tench Irom geographically remote populations were
used Ior stocking, or that local source breeds carried the opposite phylogroup. Interestingly,
we Iound that although the cultured breeds originating Irom diIIerent parts oI Europe diIIered
in the Irequencies oI the Western and Eastern phylogroups (Appendix A), all oI them carried
haplotypes oI both phylogroups. ThereIore, supplemental stocking with these or genetically related
breeds would increase the probability oI introgression between the phylogroups. Our recent study
looked Ior evidence oI a reproductive isolation in a post-glacial lake inhabited by both
phylogroups but we Iound no results that would point towards barriers to their interbreeding
(Laibner et al. 2010). Furthermore, at many sites within the contact zone we observed individuals
oI apparently hybrid ancestry (see Fig. 2bd). The putative hybrids were heterozygous Ior alternate
phylogroups, or were homozygous but Ior diIIerent phylogroups at diIIerent loci and/or carried
mtDNA oI the opposite phylogroup (data not shown). Finally, that both phylogroups characterized
all oI the examined breeds supports that populations oI mixed origin can persist without strong
negative Iitness consequences at least under cultured conditions. ThereIore, the admixed genetic
composition oI the cultured breeds most likely contributed to the introgression between
the phylogroups in natural habitats.
42
Phylogeography of known introductions
There is no record as to the geographical origin oI tench in the Neretva River in Bosnia
and Herzegovina, which is in the eastern Adriatic Sea basin where tench do not naturally occur
(Glamuzina 2006). The presence oI both phylogroups in the Neretva population shows that it is
may have descended either Irom introductions Irom the adiacent Danube River drainage where
both phylogroups occur (Fig. 2), or Irom genetically admixed hatchery stocks.
In Turkey, tench are probably native to some river drainages within the Black Sea basin
(Brylinska et al. 1999) but it have been introduced to water systems oI central and western Turkey
(Korkmaz and Zencir 2005: Innal and Erk`akan 2006). The six putative non-native populations
in Turkey (Appendix A) contained exclusively haplotypes oI the Eastern phylogroup (Fig. 2bd),
which made them indistinguishable Irom the other sites in the eastern part oI the range (Fig. 2e
and 4). This points to a local source oI this introduction or to a distant source but within the range
oI the Eastern phylogroup.
The introduced population in China also carried only the Eastern phylogroup (Fig. 2ad).
Tench were introduced in large parts oI China during the 20
th
century (Walker and Yang 1999:
Huang et al. 2001), most probably Irom the Itrysh River drainage in northern China where tench
naturally occur (Fig. 1). Interestingly, European cultured breeds originating Irom the live gene
bank in Vodnany were recently imported to China to serve as a source Ior stocking into open
waters throughout China (Wang et al. 2004). II those breeds carry both phylogroups, as did all
breeds in that gene bank that we examined, this practice is likely to induce introgression
oI the European genes into the native populations oI the tench in Asia.
The Iirst introduction oI tench Irom Europe to the United States occurred in 1877 (Baird
1879). By 1896, their descendants had been distributed to at least 36 states, and subsequent
introductions to North America Iollowed, including to Canada in 1986 (Quebec: Dumont et al.
2002). Both these introductions used tench Irom Germany (Baughman 1947: Fuller et al. 1999:
Nico and Fuller 2010). Consistent with this, the population Irom Quebec contained both
phylogroups and was placed in the same SAMOVA group with German and other western
European sites (Fig. 2e). However, the Silver Lake population in the state oI Washington contained
only the Eastern phylogroup and it was grouped with the Eastern sites by SAMOVA (Fig. 2e). This
suggests that this population originated Irom yet another introduction to the United States
that occurred in the state oI Washington in 1909 (Wydoski and Whitney 2003), and which would
have involved an unknown but most likely an eastern European or Asian source.
43
New Zealand tench were introduced several times in 19
th
century Irom Tasmania (Allport
1866: Abbott 1868: Arthur 1881: Thomson 1922: Hicks 2003), to where they had been
successIully introduced Irom England in 1858 (Allport 1866, 1868). The North Island population
contained both phylogroups (Fig. 2ad) and it was placed in one SAMOVA group by one locus
and to the other SAMOVA group by other loci (Fig. 2e). We were unable to acquire samples
Irom Tasmania but these results suggest that England already had the Eastern phylogroup
in 19
th
century, placing an upper limit on the time oI its introduction to the British Isles.
Conclusions
The diIIiculty oI disentangling the conIounding eIIects oI secondary dispersal Irom the impact
oI natural historical processes presents a persistent challenge Ior studies on the historical
biogeography, particularly oI species prone to intentional translocation by humans. Our study
highlights that Ior such species it may be useIul to consider the eIIects oI anthropogenic Iactors
as iuxtaposed on the natural phylogeographic structure rather than viewing these as mutually
exclusive causes oI the observed genetic and distribution patterns. We showed that natural
historical processes have played an important role in genetically structuring the tench populations
and that their signatures can still be detected across multiple genes. On the other hand,
we demonstrated that human-aided dispersal signiIicantly contributed to the recent evolutionary
history oI the tench and that the admixed genetic composition oI cultured breeds most likely
enhances introgression between genetically diIIerentiated populations. It appears likely that iI the
current practices in open-water Iisheries management continue, the human-aided migration will
eventually erase the natural phylogeographic pattern Ior large parts oI the tench range. It is also
possible that, by increasing their adaptive variation, the hybridization would enhance the invasive
potential oI the admixed populations outside the native range, including into novel niches
not occupied in the native range (Lucek et al. 2010). Within the native range, phylogroups
descended Irom diIIerent reIugia would likely show physiological adaptations to diIIerent selective
environments. Stocking with individuals oI the opposite phylogroup or the mixed ancestry may
disrupt such adaptations, which can lead to reduction in Iitness oI wild populations (see Araki et al.
2008: Hutchings and Fraser 2008: Fraser et al. 2010: Marie et al. 2010: Ior numerous examples
Irom salmonids). Such impacts might substantially reduce the evolutionary potential oI wild
populations and aIIect their chance oI persistence (Stockwell et al. 2003: Frankham 2005).
44
Acknowledgements
We thank all the colleagues who assisted with sample collections, especially Adamek Z.,
Akbarzadeh A., Alavi M.S.H., Apostolou A., Bohlen J., Bolding B., Buras P., Cook I., Cern J.,
Dahlberg M., De Gelas K., Desloges C., Desloges S., Doadrio I., Dumont P., Dzuba B., Ekmekci
F.G., Flaishans M., GaIIaroglu M., Gallardo J.M., Gante H.F., Gasco L., Gomulka P., Gualtieri M.,
Henshaw A., Hertig A., Hicks B.J., Hubenova T., Kamler E., Kohlmann K., Korte E., Korwin-
Kossakowski M., Kosco J., Lopatin O., Mamilov N., Memis D., Navodaru I., Nico L.G., Paaver T.,
Pekarik L., Persat H., Petr T., Piackova V., Polli B., Rossi S., Sudakova N., Sychrova O., Svatora
M., Vandeputte M., Vassilev M., and Wang J. We thank Choleva L. Ior his assistance with nuclear
markers selection and Markova S., Pelikanova S., Sediva A., Bohlen-Slechtova V., Janko K., Rab
P., and many others Ior their advice. The work was supported by the Ministry oI Education, Youth
and Sports oI the Czech Republic (LC06073, MSM6007665809), by the Academy oI Sciences oI
the Czech Republic (IRP IAPG AV0Z50450515 and IGA UZFG/05/22), and by the Czech Science
Foundation (206/09/1154).
45
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55
AppendixA:Originoftenchspecimenswithhaplotypecodesandfrequencies.
Locality * Basin Water body Country
f
Coordinates Haplotype codes (counts) NYear
Latitude Longitude CvtB Act RpS7 ATPase
Open waters (non-Iarm sites)
Linkebeek Scheldt /
North Sea
ArtiIicial
pond
B 50.77 4.33 EA1(1),
W1(3)
E1(2),
W1(8)
E1(2),
W2(1),
W3(5)
- 5 2005
Osikovica Danube /
Black Sea
Iskar
tributary
BG 42.94 24.00 EI1(5) E1(10) E1(10) E1(2) 5 2005
Blagoevgrad Struma /
Aegean Sea
Struma BG 42.02 23.09 EA1(1) E1(2) E1(1),
E2(1)
- 1 2005
Karaotok
Park Prirode
Neretva /
Adriatic Sea
Canal Sunca
|
BIH 43.05 17.80 W1(1) E1(1),
W1(1)
W3(1),
W4(1)
- 1 2005
Stolac Neretva /
Adriatic Sea
Bregava | BIH 43.08 17.96 EA1(3) E1(2),
W1(4)
W2(3),
W3(1),
W4(2)
- 3 2005
Noyan Saint
Lawrence
River/Atlantic
Ocean
Richelieu
River |
CDN 45.12 -73.26 W1(3) E1(1),
W1(5)
E1(1),
W2(4),
W3(1)
- 3 2005
Zurich Rhine / North
Sea
Zurich CH 47.30 8.62 EA1(1),
W1(4)
E1(2),
W1(8)
E1(1),
W2(6),
W3(1)
- 5 2005
Lugano Po / Adriatic
Sea
Lugano CH 45.98 8.97 W1(2) E1(2),
W1(6)
W2(2),
W3(1),
W5(1)
- 4 2006
Olomouc Danube /
Black Sea
Morava CZ 49.61 17.25 - - - E1(2) 1 1863
Kokorin Elbe / North
Sea
Psovka CZ 50.44 14.58 EA1(1),
W1(1)
E1(1),
W1(3)
W1(1),
W2(1)
- 2 2003
Felchow Oder / Baltic
Sea
Grosser
Felchowsee
D 53.06 14.13 W1(1) E1(2),
W1(6)
W1(3),
W2(1)
W1(2) 5 1997
Haaven Wesser /
North Sea
Hunte D 53.09 8.21 W1(3),
W5(1)
W1(8) E1(1),
W1(3),
W2(4)
W1(2) 4 2004
Hessen Rhine / North
Sea
Rhine D 49.92 8.32 W3(4) E1(3),
W1(5)
E1(4),
W2(2),
W3(2)
- 4 2005
Pln Schwentine /
Baltic Sea
Vierer see D 54.13 10.47 EA1(1),
W1(1)
- - - 2 2007
Dllner Heide Oder / Baltic
Sea
Kleiner
Dllnsee
D 52.98 13.57 EA1(2),
W1(2)
E1(1),
W1(7)
W3(6) - 5 1996
Guadalupe Guadiana /
Atlantic
Ocean
Guadalupeio E 39.44 -5.31 EA1(1) E1(2) E1(2) - 1 2006
Vnnu Narva / Baltic
Sea
Emaigi EST 58.83 27.00 EA1(5) E1(6),
W1(4)
E1(10) - 5 2005
Priay Rhne /
Mediterranean
Sea
Ain F 46.00 5.27 W1(2) E1(1),
W1(3)
W1(1),
W3(1)
- 2 2005
Belley Rhne /
Mediterranean
Sea
Rhne F 45.78 5.81 W1(2) W1(4) W1(4) W1(2) 2 2005
Grardmer Rhine / North
Sea
Grardmer F 48.07 6.87 W1(2) W1(4) E1(1),
W3(3)
- 2 2005
Warbutts Ouse / North
Sea
ArtiIicial
pond
GB 54.05 -1.01 EA3(1),
W1(1)
E1(3),
W1(1)
E1(3),
W1(1)
- 2 2005
StillingIleet Ouse / North
Sea
ArtiIicial
pond
GB 53.86 -1.09 EA1(2) E1(3),
W1(1)
E1(2),
W2(1),
W4(1)
- 2 2005
56
Locality * Basin Water body Country
f
Coordinates Haplotype codes (counts) NYear
Latitude Longitude CvtB Act RpS7 ATPase
Cascina
Belgiardino
Po / Adriatic
Sea
Adda (Cavo
Roggione)
I 45.28 9.48 W1(3) E1(2),
W1(4)
W1(2),
W2(1),
W3(2),
W4(1)
- 3 2005
Ghazian Caspian Sea Anzalee
lagoon
IR 37.47 49.33 EC1(3),
EC2(1),
EC3(1)
E1(10) E1(3),
E3(1)
E1(2) 5 2005
Sadyrbay Tengiz -
Korgalzhyn
Korgalzhyn KZ 50.59 70.29 EA1(3) E1(6) E1(6) E1(2) 3 2005
Hamilton Waikato /
Tasman Sea
Hamilton
Lake |
NZ -37.80 175.28 EA1(1),
W1(2)
E1(8) E1(4),
W3(2)
E1(2) 4 2003
-2005
Lentiscais Teio / Atlantic
Ocean
Teio P 39.73 -7.49 EA1(1) - - - 1 2007
Satopy-
Samulewo
Pregel / Baltic
Sea
Saina PL 54.08 21.06 EA6(1),
EA7(1)
,W1(3)
E1(1),
E2(1),
W1(8)
E1(2),
W1(2),
W2(1),
W3(5)
W1(2) 5 2006
Kurowo Vistula /
Baltic Sea
Narew PL 53.12 22.80 EA1(2) E1(1),
W1(3)
E1(3),
W1(1)
- 2 2005
Tulcea Danube /
Black Sea
Danube delta RO 45.00 29.00 EA1(3),
EA4(1)
E1(8) E1(8) - 4 2004
Astrakhan Volga /
Caspian Sea
Volga RUS 46.41 48.00 EA1(4),
EA8(1)
E1(10) E1(10) E1(2) 5 2006
Vabacken Bve / North
Sea
re si S 58.31 12.13 EA1(1) E1(2) E1(2) E1(2) 1 2007
Stockholm Mlaren /
Baltic Sea
Mlaren S 59.33 18.07 EA1(1),
W1(2)
E1(3),
W1(3)
E1(4),
W1(1),
W3(1)
- 3 2007
Brringe Sege / Baltic
Sea
Havgrdssin S 55.49 13.36 EA1(3),
W2(1)
E1(4),
W1(4)
E1(2),
W1(2),
W2(1),
W3(1)
- 4 2007
Moravsk
Svt Jan
Danube /
Black Sea
Dlh luky SK 48.59 17.00 EA1(1),
W1(1)
E1(3),
W1(1)
E1(1),
W2(1),
W3(2)
- 2 2006
Buzica Danube /
Black Sea
Ida SK 48.55 21.08 EA1(2) W1(4) E1(2),
W3(2)
- 2 2006
Michalovce Danube /
Black Sea
Zemplinska
Sirava
SK 48.76 22.07 EA1(1) E1(1),
W1(1)
E1(2) - 1 2006
Gabcikovo Danube /
Black Sea
Star les SK 47.77 17.73 EA1(2),
W3(1)
E1(4) W1(1),
W3(3)
- 3 2004
-2005
Oborin Danube /
Black Sea
Laborec SK 48.54 21.90 EA1(2) E1(4) E1(4) - 2 2006
Sapanca Sakarya /
Black Sea
Sapanca gl
|
TR 40.71 30.28 EA1(4),
EA5(1)
E1(10) E1(10) - 5 2006
rencik Yenice Irmagi
/ Black Sea
Abant gl | TR 40.60 31.28 EA1(2) E1(4) E1(4) - 2 2006
Gedikli Gksu /
Mediterraean
Sea
Beysehir
gl |
TR 37.91 31.33 EA1(3) E1(6) E1(6) - 3 2006
Kprky Kizil Irmak /
Black Sea
Kprky
baraii |
TR 39.57 33.43 EA1(2) E1(4) E1(4) - 2 2006
Kirikkale Kizil Irmak /
Black Sea
Kapulukaya
baraii |
TR 39.69 33.46 EA1(2) E1(4) E1(4) - 2 2004
Toklumen Kizil Irmak /
Black Sea
HirIanli
baraii |
TR 39.13 33.71 EA1(2) E1(4) E1(4) - 2 2005
Kirinti Aksu Cayi /
Mediterraean
Sea
Kovada gl
|
TR 37.65 30.87 EA1(3) E1(6) E1(6) - 3 2006
57
Locality * Basin Water body Country
f
Coordinates Haplotype codes (counts) NYear
Latitude Longitude CvtB Act RpS7 ATPase
Savincy Donets /
Azov Sea
Siverskyi
Donets
UA 49.38 37.02 EA1(4) E1(8) E1(8) - 4 2006
Gola Pristan Dnipro /
Black Sea
Dnipro delta UA 46.31 32.31 EA1(4) E1(8) E1(8) E1(4) 4 2006
Senkove Donets / Azov
Sea
Krasnyi
Oskol
UA 49.51 37.69 EA1(2) E1(4) E1(4) E1(2) 2 2006
Medical Lake Columbia
River / PaciIic
Ocean
Silver lake | USA 47.54 -117.65 EA1(5) E1(10) E1(10) E1(2) 5 2005
Fish farms
Plovdiv Maritsa /
Aegean Sea
Fish pond BG 42.15 24.72 EA1(2) - - - 2 2007
Vegas del
Guadiana
Guadiana /
Atlantic
Ocean
Fish pond E 38.89 -6.88 EA1(5) E1(10) E1(10) E1(2) 5 2006
Mionnay Rhne /
Mediterranean
Sea
Fish pond F 45.90 4.92 W1(2) W1(4) W1(1),
W2(1),
W3(2)
- 2 2005
Bouligneux Rhne /
Mediterranean
Sea
Fish pond F 46.02 4.99 W1(1),
W2(1)
E1(2),
W1(2)
W3(2) - 2 2005
Perugia Tiber /
Tyrrhenian
Sea
Trasimeno
Lake
I 43.15 12.10 W1(2) W1(4) W3(4) W1(2) 2 2005
Mincio,
BonIerraro
di Sorga
Po / Adriatic
Sea
Garda Lake I 45.55 10.70 W1(3) E1(1),
W1(4),
W2(1)
W1(2),
W2(2),
W3(2)
- 3 2005
Zabieniec Vistula /
Baltic Sea
Fish pond PL 52.05 21.03 W1(1),
W5(1)
E1(3),
W1(1)
E1(1),
W3(3)
W1(2) 2 2005
Wuhan Yangtze River
/ East China
Sea
Fish pond PRC 30.56 114.37 EA1(3),
EA2(1)
E1(8) E1(2) E1(2) 4 2004
Italian regional breed
Ceresole
d Alba
Po / Adriatic
Sea
Fish pond I 44.80 7.82 W1(2) E1(1),
W1(3)
W1(3),
W3(1)
- 2 2005
Live gene bank in Vodany
lbrds
Hluboka, new
stock
Elbe / North
Sea
Fish pond CZ 49.05 14.43 EA1(2),
W1(1)
E1(1),
W1(5)
E1(1),
W1(2),
W3(3)
- 3 2004
Hluboka,
old stock

Elbe / North
Sea
Fish pond CZ 49.05 14.43 EA1(3) E1(2),
W1(4)
W2(2),
W3(4)
- 3 2004
Mariansk
Lazn
Elbe / North
Sea
Fish pond CZ 49.97 12.70 EA1(3) E1(3),
W1(3)
E1(4),
W3(2)
- 3 2005
Tabor
(Milevsko),
new stock
Elbe / North
Sea
Fish pond CZ 49.45 14.36 EA1(2),
W1(2)
E1(2),
W1(4)
E1(2),
W3(2)
- 4 2004
Tabor,
old stock
Elbe / North
Sea
Fish pond CZ 49.40 14.69 EA1(3) E1(2),
W1(4)
E1(2),
W1(1),
W3(3)
- 3 2004
Velk
Mezirici
Elbe / North
Sea
Fish pond CZ 49.35 16.02 EA1(1),
W1(1),
W4(1)
E1(2),
W1(4)
E1(2),
W1(4)
- 3 2004
Vodnany Elbe / North
Sea
Fish pond CZ 49.15 14.18 EA1(3) E1(4),
W1(2)
E1(5),
W2(1)
- 3 2004
Eurpbrds
Knigswartha
(Germany)
Elbe / North
Sea
Fish pond D 51.31 14.33 EA1(1),
W1(1)
E1(1),
W1(9)
E1(1),
W1(5),
W2(2)
- 5 2004
58
Locality * Basin Water body Country
f
Coordinates Haplotype codes (counts) NYear
Latitude Longitude CvtB Act RpS7 ATPase
Romania Danube /
Black Sea
Fish pond RO EA1(1)
, B1(3)
E1(5),
W1(3)
E1(2),
W1(1),
W3(3)
- 4 2004
Hungaria Danube /
Black Sea
Fish pond H EA1(5) E1(2),
W1(8)
E1(1),
W3(3)
- 5 2004
Exprmtlbrds
Leather 92 Fish pond CZ W2(3) E1(1),
W1(5)
E1(6) - 3 2004
Synthetic Fish pond CZ EA1(3) E1(3),
W1(3)
E1(2),
W2(2),
W3(2)
- 3 2004
Gynogenetic Fish pond CZ EA1(3) W1(6) E1(4),
W3(1),
W4(1)
- 3 2004
Ormtlbrds
Golden Fish pond CZ EA1(2) E1(2),
W1(2)
E1(1),
W1(3)
- 2 2004
Blue Fish pond CZ EA1(2) E1(1),
W1(3)
E1(1),
W1(1),
W3(2)
- 2 2004
Alampic Fish pond CZ EA1(2) E1(1),
W1(3)
E1(1),
W1(2),
W3(1)
- 2 2004
* For the geographical breeds in the live gene bank, locality identiIies the original source
oI the breed, while Ior the experimental and ornamental breeds only the breed name is given.
The countries are coded as Iollows: Belgium (B), Bulgaria (BG), Bosnia (BIH), Canada (CDN),
Switzerland (CH), Czech Republic (CZ), Germany (D), Spain (E), Estonia (EST), France (F),
Great Britain (GB), Hungary (H), Italy (I), Iran (IR), Kazakhstan (KZ), New Zealand (NZ),
Portugal (P), Poland (PL), China (PRC), Romania (RO), Russia (RUS), Sweden (S), Slovakia
(SK), Turkey (TR), Ukraine (UA), United States oI America (USA).
| Known introduced population.
InIormation about the source population.
59
2.2. PCR-RFLP assays to distinguish the Western and Eastern phylogroups in
wild and cultured tench 1inca tinca.
Laibner Zdenk, Kotlik Petr
Molecular Ecologv Resources. (Early View: published online 20 SEP 2010)
(doi: 10.1111/i.1755-0998.2010.02914.x)
60
MOLECULAR DI AGNOSTI CS AND DNA TAXONOMY
PCR-RFLP assays to distinguish the Western and Eastern
phylogroups in wild and cultured tench Tinca tinca
Z. LAJBNER* and P. KOTLI

K*
*Laboratory of Fish Genetics, Department of Vertebrate Evolutionary Biology and Genetics, Institute of Animal Physiology and
Genetics, Academy of Sciences of the Czech Republic, Rumburska 89, 277 21 Libechov, Czech Republic, Department of Zoology,
Faculty of Science, Charles University, 128 44 Prague, Czech Republic
Abstract
The tench Tinca tinca is a valued table sh native to Europe and Asia, but which is now widely distributed in many
temperate freshwater regions of the world as the result of human-mediated translocations. Fish are currently being trans-
planted between watersheds without concern for genetic similarity to wild populations or local adaptation, and efcient
phylogeographic markers are therefore urgently needed to rapidly distinguish genetically distinct geographical popula-
tions and to assess their contribution to the hatchery breeds and to the stocked wild populations. Here, we present a new
method to distinguish recently discovered and morphologically undistinguishable Western and Eastern phylogroups of
the tench. The method relies on PCR-RFLP assays of two independent nuclear-encoded exon-primed intron-crossing
(EPIC) markers and of one mitochondrial DNA (mDNA) marker and allows the rapid identication of the Western and
Eastern phylogroup and also of three geographical mtDNA clades within the Eastern phylogroup. Our method will enable
researchers and shery practitioners to rapidly distinguish genetically divergent geographical populations of the tench and
will be useful for monitoring the introduction and human-mediated spread of the phylogroups in wild populations, for
characterization of cultured strains and in breeding experiments.
Keywords: EPIC, exon-primed intron-crossing marker, mtDNA, stocking
Received 14 July 2010; revision received 6 August 2010; accepted 7 August 2010
The tench Tinca tinca is a valued table sh, native to
Europe and Asia between the British Isles and central
Siberia (Brylin ska et al. 1999), but it is now widely distrib-
uted in many temperate freshwater regions of the world
as the result of human-mediated translocations (Welcom-
me 1988). Up until recently, there has been only limited
information about population structure of the tench
throughout its vast native range (Kohlmann et al. 2010),
resulting in the absence of efcient phylogeographic
markers to monitor the genetic and geographical origin
of hatchery stocks, such that sh are being transplanted
from one watershed to another without concern for
genetic similarity to neighbouring wild populations or
adaptation to local environment. Therefore, for successful
shery management and sustainable exploitation of
tench, it is extremely important to develop easy markers
that would enable researchers and shery practitioners
to rapidly distinguish genetically distinct geographical
populations and to assess their contribution to the hatch-
ery breeds and to the stocked wild populations.
Recently, phylogeographic analysis of DNA
sequences of several exon-primed intron-crossing (EPIC)
markers and of a mitochondrial DNA (mtDNA) gene dis-
covered within the Eurasian range of the tench two geo-
graphical clades, a Western phylogroup (clade W) found
in Europe from the British Isles to Poland and an Eastern
phylogroup (clade E) distributed from Europe through-
out Asia to China (Z. Lajbner, O. Linhart and P. Kotl k,
unpublished manuscript). Within the Eastern phylo-
group, populations with mtDNA (but not nuclear mark-
ers) distinct from the major Eastern clade (EA) were
found in a southern tributary of the Danube River in
Bulgaria (clade EI) and in the southern part of the Caspian
Sea basin in Iran (clade EC). Separation into the phylo-
groups most reasonably reects long-term evolutionary
isolation of populations in different parts of the native
range, but we have shown that tench of both phylo-
groups can freely interbreed with one another when in
contact in wild populations (Lajbner et al. 2010). The
presence of both phylogroups in cultured breeds of dif-
ferent geographical origins and the occurrence of EA
haplotypes at the western edge of the native range (e.g.
in Spain and Britain) showed that extensive admixture
Correspondence: Zdenek Lajbner, Fax: (420) 315 639 510;
E-mail: z.lajbner@seznam.cz
2010 Blackwell Publishing Ltd
Molecular Ecology Resources (2010) doi: 10.1111/j.1755-0998.2010.02914.x
61
has occurred both in wild populations and in hatcheries,
largely as a consequence of shery practices that ignore
the genetic structure among and within populations.
To facilitate the assessment of admixture among wild
and cultured tench, we have developed a rapid and ef-
cient method that distinguishes between the nuclear
and mtDNA genomes of the phylogroups. The method
further enables distinguishing between the three
mtDNA clades (EA, EI and EC) in the Eastern phylo-
group. The method relies on polymerase chain reaction
(PCR) restriction fragment length polymorphism (RFLP)
assays of two independent nuclear-encoded EPIC mark-
ers, the second intron of the actin gene (Act) and the
rst intron of the gene coding for the S7 ribosomal pro-
tein (RpS7), and of one mtDNA marker, the cytochrome
b gene (Cytb). Examination of DNA sequences of all
known haplotypes for each of these three markers found
in a survey of 225 tench from a wide range of geograph-
ical regions (GenBank accession nos HM167935
HM167938, HM167941HM167965) indicated that the
Western and Eastern phylogroup could be reliably dis-
tinguished by cleavage of Act by Eco52I restriction endo-
nuclease, of RpS7 by NdeI endonuclease and of Cytb by
AluI and MboI endonucleases. In addition, cleavage of
Cytb using AluI distinguishes the EI clade and cleavage
with MboI the EC clade.
To validate the new assays, tench genomic DNA was
extracted from ethanol-preserved muscle tissue or n
clips using DNeasy Tissue Kit (Qiagen, Valencia, CA,
USA). A 335-bp amplicon containing the Act intron was
PCR-amplied with the EPIC primers described by
Atarhouch et al. (2003), and a 923927-bp amplicon (928
total bp of the alignment) containing the RpS7 intron with
the EPIC primers published by Chow & Hazama (1998).
A 1226-bp part of the mitochondrial DNA containing the
entire Cytb was amplied with the primers located in
anking tRNAs as described by Machordom & Doadrio
(2001). Primer names and sequences are listed in Table 1.
The 25 lL PCRs contained 0.5 concentration of PPP
Master Mix (2 stock solution: 150 mM Tris-HCl [pH 8.8],
40 mM (NH
4
)
2
SO
4
, 0.02% Tween 20, 5 mM MgCl
2
, 400 lM
of each dNTP, 100 U mL Taq-Purple DNA polymerase,
stabilizers, additives; Top-Bio, Prague, Czech Republic),
10 pmol of each primer, 0.2 lg of template DNA and
demineralized water to the nal volume. To facilitate
high-throughput analysis, the amplication in a PTC 200
Peltier thermal cycler (MJ Research, Watertown, MA,
USA) was performed using the same PCR program for
all markers, which contained 5 min of initial denatur-
ation at 95 C, six cycles of a touch-down prole of 1-min
denaturation at 94 C, 1 min 30 s at 6056 C with the
annealing temperature lowered by 2 C after every two
cycles and 2-min elongation at 72 C, followed by 30
cycles with the annealing temperature held at 54 C. For
the RFLP analyses, 4 lL of the PCR products were
digested for 10 h at 37 C in 10 lL reaction volumes con-
taining 4 lL of demineralized water and 1 lL of endonu-
clease buffer with 1 lL of the endonuclease Eco52I, AluI,
MboI (Fermentas, Vilnius, Lithuania) or NdeI (New
England Biolabs, Ipswich, MA, USA) and then
deactivated at 65 C for 20 min. Restriction fragments
were separated on 2% agarose gels containing 2 lL of
GoldView (SBS Genetech, Shanghai, China).
Digesting the amplicons of individuals carrying all
known haplotypes for each of the three genes, including
heterozygotes for each nuclear gene, yielded the pre-
dicted RFLP proles (Fig. 1). The restriction digestion of
the 1226-bp amplicon of Cytb by MboI at four cleavage
sites (345, 642, 876, 946 bp) resulted in ve fragments (70,
234, 280, 297, 345 bp) in the Eastern phylogroup, except
in clade EC, which does not have the 642-bp and 946-bp
sites but has a 132-bp site and had a four-band pattern
(132, 213, 350, 531 bp). The Western phylogroup also has
the 132-bp site but does not have the 946-bp site and had
a unique ve-band pattern (132, 213, 234, 297, 350 bp).
The digestion of Cytb by AluI at three cleavage sites (184,
891, 1168 bp) resulted in four fragments (58, 184, 277,
707 bp) in the Eastern phylogroup, except in the EI clade,
which does not have the 1168-bp site and had a three-
band pattern (184, 335, 707 bp). The Western phylogroup
does not have the 891-bp site and had a different three-
band pattern (58, 184, 984 bp), except in one of the haplo-
types (W2), which only has the 1168-bp site and had a
two-band pattern (58, 1168 bp).
Table 1 Primers and their sequences
Marker (amplicon size) Primer Sequence (5 to 3) Reference
Cytb (1226 bp) GluF AACCACCGTTGTATTCAACTACAA Machordom & Doadrio (2001)
ThrR ACCTCCGATCTTCGGATTACAAGACCG Machordom & Doadrio (2001)
RpS7 (923927 bp)* S7RPEX1F TGGCCTCTTCCTTGGCCGTC Chow & Hazama (1998)
S7RPEX2R AACTCGTCTGGCTTTTCGCC Chow & Hazama (1998)
Act (335 bp) Act-2-F GCATAACCCTCGTAGATGGGCAC Atarhouch et al. (2003)
Act-2-R ATCTGGCACCACACCTTCTACAA Atarhouch et al. (2003)
*Length variation because of gaps.
2010 Blackwell Publishing Ltd
2 MOLECULAR DI AGNOSTI CS AND DNA TAXONOMY
62
The digestion of the 923927-bp amplicon of RpS7
(size range is because of gaps) by NdeI yielded a two-
band pattern for the Eastern phylogroup and a three-
band pattern for the Western phylogroup (Fig. 1b). There
is a cleavage site at 110 bp common to both phylogroups
that produced a small fragment (110 bp) in both phylo-
groups and a second, large fragment (813817-bp) in the
Eastern phylogroup. An additional cleavage site at
478 bp that is absent in the Eastern phylogroup produced
two fragments of an intermediate size (368, 446448 bp)
in the Western phylogroup.
The digestion of the 335-bp amplicon of Act by Eco52I
at a single cleavage site (182 bp), which is absent in the
Eastern phylogroup, resulted in two fragments (153,
182 bp) in the Western phylogroup (Fig. 1c).
The amplication of Act under the above-mentioned
conditions produced a second major amplicon of a lar-
ger molecular size (approximately 580 bp) than our
marker, plus a minor amplicon (approximately 430 bp).
The genome of teleost shes contains multiple paralo-
gous actin genes (Venkatesh et al. 1996), and these addi-
tional amplicons thus were most probably derived from
paralogous annealing of the EPIC primers. The extra
amplicons were not cleaved with Eco52I, so that for reli-
able genotyping of our EPIC marker, they did not need
to be removed (see Fig. 1c). Occasionally, the amplica-
tion of RpS7 also yielded up to three minor amplicons
of different molecular sizes in addition to the expected
amplicon, some of which were present in multiple indi-
viduals (most notably amplicons of approximately 580
and 1600 bp), but these minor amplicons were not
cleaved by NdeI and did not interfere with the genotyp-
ing.
All individuals with previously sequenced amplicons
(Cytb: n = 17; RpS7: n = 11; Act: n = 11) were correctly
genotyped by the new method, which demonstrates its
accuracy. An additional 71 tench, originated from two
lakes in Germany where the Western and Eastern phylo-
group (EA clade) coexist (Lajbner et al. 2010), were
screened by these assays (only 35 sh for Cytb) to verify
the efciency of the protocol. All specimens were unam-
biguously genotyped as either the Western phylogroup
(Cytb: n = 32; RpS7: n = 55; Act: n = 42) or Eastern phylo-
group (Cytb: n = 3; RpS7: n = 4; Act: n = 1) or heterozyg-
otes (RpS7: n = 12; Act: n = 28). Thus, the new RFLP
assays are a robust and rapid method to distinguish the
Western and Eastern phylogroup and also the three
mtDNA clades in the Eastern phylogroup of the tench.
The assays will be useful for monitoring the human-med-
iated dispersal of the phylogroups among wild popula-
tions currently occurring via aquaculture, for
characterization and identication of cultured strains,
and in breeding experiments.
Acknowledgements
The authors thank Silvia Markova for laboratory assistance and
for advice. The work was supported by the Ministry of Educa-
tion, Youth and Sports of the Czech Republic (LC06073) and by
the Academy of Sciences of the Czech Republic (IRP IAPG
AV0Z50450515 and IGA UZFG 05 22).
References
Atarhouch T, Rami M, Cattaneo-Berrebi G et al. (2003) Primers for EPIC
amplication of intron sequences for sh and other vertebrate popula-
tion genetic studies. BioTechniques, 35(4), 676682.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Cytb (MboI) Cytb (AluI) RpS7 (NdeI) Act (Eco52I)
500
500
1000
100
bp
100
bp
(a) (b) (c)
Fig. 1 Agarose gels showing diagnostic restriction fragment patterns. (a) Cytb assays. Lanes 1, 5 and 10: 100-bp ladder molecular
weight standard (500- and 1000-bp bands highlighted); lane 2: Western phylogroup digested with MboI; lane 3: EC clade digested with
MboI; lane 4: other Eastern clades digested with MboI; lane 6: W2 haplotype digested with AluI; lane 7: other Western haplotypes
digested with AluI; lane 8: EI clade digested with AluI; lane 9: other Eastern clades digested with AluI. (b) RpS7 assay with NdeI. Lanes 11
and 15: 100-bp ladder; lane 12: Western phylogroup; lane 13: Western Eastern heterozygote; lane 14: Eastern phylogroup. (c) Act assay
with Eco52I. Lanes 16, 20 and 24: 100-bp ladder (500-bp band highlighted; note different version of ladder than in panels a and b); lanes
1719: native PCR product; lane 17: Western phylogroup; lane 18: Western Eastern heterozygote; lane 19: Eastern phylogroup; lanes
2123: target amplicon (335-bp) puried by gel-extraction prior to endonuclease treatment; lane 21: Western phylogroup; lane 22:
Western Eastern heterozygote; lane 23: Eastern phylogroup. Note that fragments with size under 100 bp are difcult to visualize but this
does not affect scoring.
2010 Blackwell Publishing Ltd
MOLECULAR DI AGNOSTI CS AND DNA TAXONOMY 3
63
Brylin ska M, Brylin ski E, Bnin ska M (1999) Tinca tinca (Linnaeus, 1758).
In: The Freshwater Fishes of Europe, 5 I: Cyprinidae 2 I (ed. Banarescu
PM), pp. 229302. AULA-Verlag, Wiesbaden.
Chow S, Hazama K (1998) Universal PCR primers for S7 ribosomal
protein gene introns in sh. Molecular Ecology, 7(9), 12551256.
Kohlmann K, Kersten P, Panicz P, Memis D, Flajshans M (2010) Genetic
variability and differentiation of wild and cultured tench populations
inferred from microsatellite loci. Reviews in Fish Biology and Fisheries, 20,
279288. doi:10.1007/s11160-009-9138-x
Lajbner Z, Kohlmann K, Linhart O, Kotl k P (2010) Lack of reproductive
isolation between the Western and Eastern phylogroups of the tench.
Reviews in Fish Biology and Fisheries, 20, 289300. doi:10.1007/s11160-
009-9137-y
Machordom A, Doadrio I (2001) Evidence of a Cenozoic Betic-Kabilian
connection based on freshwater sh phylogeography (Luciobarbus,
Cyprinidae). Molecular Phylogenetics and Evolution, 18(2), 252263.
Venkatesh B, Tay BH, Elgar G, Brenner S (1996) Isolation, characterization
and evolution of nine puffersh (Fugu rubripes) actin genes. Journal of
Molecular Biology, 259(4), 655665.
Welcomme RL (1988) International introductions of inland aquatic species.
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nization of the United Nations, Rome, Italy.
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4 MOLECULAR DI AGNOSTI CS AND DNA TAXONOMY
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2.3. Lack of reproductive isolation between the Western and Eastern
phylogroups of the tench.
Laibner Zdenk, Kohlmann Klaus, Linhart Otomar, Kotlik Petr (2010)
Reviews in Fish Biologv ana Fisheries 20, 289-300.
(doi: 10.1007/s11160-009-9137-y)
65
RESEARCH PAPER
Lack of reproductive isolation between the Western
and Eastern phylogroups of the tench
Zdenek Lajbner

Klaus Kohlmann

Otomar Linhart

Petr Kotl k
Received: 2 November 2008 / Accepted: 21 September 2009 / Published online: 14 November 2009
Springer Science+Business Media B.V. 2009
Abstract The Eurasian range of the tench distribu-
tion is subdivided into deeply divergent Western and
Eastern phylogroups evidenced by nuclear and mito-
chondrial DNA sequence markers. A broad zone of
overlap exists in central and western Europe, sug-
gesting post-glacial contact with limited hybridisa-
tion. We conducted a population genetic test of this
indication that the two phylogroups may represent
distinct species. We analysed variation at introns of
nuclear genes, microsatellites, allozymes and mito-
chondrial DNA in populations from two postglacial
lakes within the contact zone in Germany. The test is
based on the expectation that in the presence of
strong barriers to reproduction, a hybrid population
will show genome-wide associations among alleles
and genotypes from each phylogroup even after
hundreds of generations of interbreeding. In contrast
to this expectation, no consistent signicant devia-
tions from linkage and HardyWeinberg equilibria
were found. Samples from both lakes did show
signicant disequilibria but they were limited to
individual loci and were not concordant between
populations, and were not robust to the method used.
The single consistent association can be attributed to
physical linkage between two microsatellite loci.
Thus, results of our study support the hypothesis of
free interbreeding between the two phylogroups of
tench. Therefore, although the phylogroups may be
considered as separate phylogenetic species, the
present data suggest that they are a single species
under the biological species concept.
Keywords Allozymes Microsatellites
mtDNA Disequilibrium Hybridization
Tinca tinca
Introduction
Most freshwater sh species in Eurasia show phylog-
eographic subdivisions of their geographic ranges that
developed in response to recurrent isolation in glacial
Z. Lajbner (&) P. Kotl k
Department of Vertebrate Evolutionary Biology
and Genetics, Institute of Animal Physiology
and Genetics, Academy of Sciences of the Czech
Republic, Rumburska 89, 277 21 Libechov,
Czech Republic
e-mail: z.lajbner@seznam.cz
Z. Lajbner
Department of Zoology, Faculty of Science,
Charles University, 128 44, Prague, Czech Republic
K. Kohlmann
Department of Aquaculture and Ecophysiology,
Leibniz-Institute of Freshwater Ecology and Inland
Fisheries, Mueggelseedamm 310, P.O. Box 850119,
12561 Berlin, Germany
O. Linhart
University of South Bohemia, C

eske Budejovice,
Research Institute of Fish Culture and Hydrobiology
at Vodnany, Zatis 728/II, 389 25 Vodnany,
Czech Republic
123
Rev Fish Biol Fisheries (2010) 20:289300
DOI 10.1007/s11160-009-9137-y
66
refugia during the Pleistocene (Hewitt 2004). These
range shifts and the accompanying demographic
changes resulted in divergence between refugial
populations, which in some species may have pro-
ceeded towards speciation (see Bernatchez and Wil-
son 1998; Knowles 2001; Carstens and Knowles
2007). A recent phylogeographic study of the tench
Tinca tinca (L.) demonstrated that the Eurasian range
of this species is subdivided into deeply divergent
Western and Eastern phylogroups (i.e. clades with
geographically adjacent distributions), evidenced by
phylogenetic analysis of sequence variation at introns
of nuclear genes (actin, S7 ribosomal protein and ATP
synthase beta subunit) and at a mitochondrial (mt)
DNA gene (cytochrome b) (Lajbner et al. 2007).
Similar to other European freshwater sh species
(Durand et al. 1999; Nesb et al. 1999; Kotl k and
Berrebi 2001), these phylogroups probably diverged
after a colonization of western Europe from the Black
Sea basin during a Pleistocene interglacial and their
subsequent separation by cold periods, conforming to
the chub paradigm pattern (Hewitt 2004). A broad
zone of overlap was formed upon the geographic
contact between the tench phylogroups in central and
western Europe following their postglacial expansion
from two principal freshwater refugia, the Ponto-
Caspian refugium (Banarescu 1991; Kotl k et al.
2004) and the western European refugium (e.g.
Durand et al. 1999; Nesb et al. 1999; Kotl k and
Berrebi 2001). The sequence divergence between the
tench phylogroups at mtDNA (1.3% for cytochrome b
gene) approaches divergence among distinct sh
species (Hendry et al. 2000a) and it roughly corre-
sponds to a separation time for at least 750,000 years
(see Waters et al. 2007). Allopatric speciation takes
usually a prolonged time (McCune and Lovejoy 1998)
but there are exceptions (Near and Benard 2004), and
in sympatry mating barriers can evolve in just few
generations (Hendry et al. 2000b). The existence of
broad contact zone with the presence of individuals of
apparently hybrid ancestry (i.e. heterozygous for
alternate phylogroup-specic alleles at nuclear loci
or homozygous at a nuclear locus but with mtDNA of
the opposite phylogroup) suggests that there is
incomplete reproductive isolation between the tench
phylogroups. On the other hand, the fact they have
remained effectively allopatric outside the contact
zone suggests the existence of mechanisms restricting
introgression.
The present study examines the existence of
barriers to reproduction between the Western and
Eastern phylogroups of tench in two lakes within the
zone of their postglacial contact. The Grosser
Felchowsee and Kleiner Dollnsee lakes are situated
in north-eastern Germany in an area covered by the
Scandinavian ice sheet during the Weichselian gla-
ciation (Ehlers et al. 2004). The phylogeographic
study revealed the presence of markers characteristic
of the Western as well as the Eastern phylogroup in
both lakes (Lajbner et al. unpublished). The lakes
could only be colonised by tench after the deglaci-
ation, and the populations inhabiting the lakes today
most likely were not founded before the end of the
Younger Dryas (Jahns 2000), about 11,500 years ago
(Muscheler et al. 2008). Nevertheless, the period
since then corresponds to roughly 3,000 generations
of tench, and the populations founded by a mixture of
the Western and Eastern phylogroups should not
show genome-wide associations among alleles and
genotypes from each phylogroup if they have been
freely interbreeding (Nagylaki 1976, 1977; Barton
and Gale 1993). If, on the contrary, the phylogroups
show genetic incompatibilities in terms of differential
frequency or viability of homospecic versus heter-
ospecic crosses, the populations should display
genome-wide linkage and HardyWeinberg disequi-
libria, even after the many generations of interbreed-
ing (e.g. Lajbner et al. 2009). To rectify this issue,
tench sampled from these lakes were genotyped for
two introns of nuclear genes (actin and S7 ribosomal
protein) and for a mtDNA. These three markers are
fully diagnostic between the two phylogroups in that
they posses alternate, DNA sequence-based classes of
alleles (Lajbner et al. 2007), which were distin-
guished here by restriction fragment length polymor-
phism (RFLP) assay. To provide a broader genomic
coverage of markers, these data were analysed along
with genotypes for the same individuals at additional
fourteen genetic loci with unknown phylogroup
specicity (allozyme and microsatellite), which were
scored by starch electrophoresis (allozymes for the
Felchowsee individuals) or were available from
earlier studies (Kohlmann and Kersten 1998, 2006;
Kohlmann et al. 2007, 2009).
The principal question addressed by this study is
whether there is evidence of two genetically distinct
reproductive units in each lake. More specically, the
results are used to determine whether the lakes show
290 Rev Fish Biol Fisheries (2010) 20:289300
123
67
signs of a hybrid population structure (i.e. purebred
Western, purebred Eastern, F
1
hybrids etc.), and if
there are consistent (between lakes and across loci
and methods) deviations from linkage and Hardy
Weinberg equilibria in these lakes that could be
attributed to the existence of barriers to merging of
gene pools of the two founding phylogroups.
Materials and methods
Data acquisition
The study was conducted on 49 tench collected from a
wild population inhabiting Lake Grosser Felchowsee
(160 ha; 533
0
N, 148
0
E). This sampling was supple-
mented by an additional 19 individuals from Lake
Kleiner Dollnsee (25 ha; 5259
0
N, 1334
0
E). Both
lakes are situated in Germany in the north-eastern part
of the Oder River drainage of the Baltic Sea basin, in
the lowland area rich in lakes and wetlands. There
have probably never been introductions of foreign
tench into the lakes or supportive articial reproduc-
tion of the indigenous tench from the lakes (T. Lowe,
Lake Felchowsee owner, personal communication).
Recently conducted large scale phylogeographic
study (Lajbner et al. 2007) gives a possibility to
discriminate the two evolutionary lineages of tench
on the basis of two nuclear markers and one
mitochondrial marker, which can be easily scored
by RFLP. A restriction map was generated for each
marker using the CLC Free Workbench 4.5.1 (CLC
bio) from haplotype sequences of T. tinca (Lajbner
et al. unpublished) and endonucleases digesting the
markers at lineage specic positions were selected.
The phylogroup specic restriction endonuclease
Eco52I was selected for the 2nd intron of the actin
gene and MboII for the 1st intron of the S7 ribosomal
protein (RPS7) gene. Both endonucleases were
predicted to yield phylogroup-specic RFLP proles
following digestion of the respective polymerase
chain reaction (PCR) products due to restriction sites
present only in one of the phylogroups. The restric-
tion endonuclease AluI was predicted to yield
phylogroup-specic RFLP proles following diges-
tion of a PCR product of the mtDNA cytochrome b
gene due to a diagnostic restriction site, which
unambiguously identied each individual to either
Eastern or Western phylogroup maternal ancestry.
Total DNA of sh from Felchowsee and Dollnsee
was extracted from ethanol-preserved muscle tissue
or from n clips by using Dneasy Tissue Kit
(Qiagen). A part of nuclear DNA containing 2nd
intron of actin gene (336 bp) was amplied using
primers Act-2-R and Act-2-F designed by Touriya
et al. (2003) and another part containing 1st intron of
RPS7 gene (900 total bp) using primers S7RPEX2R
and S7RPEX1F (Chow and Hazama 1998). In
addition, an approximately 1,225 bp long portion of
mtDNA containing entire gene for cytochrome b was
amplied for sh from Felchowsee using primers
GluF and ThrR (Machordom and Doadrio 2001). For
the sake of simplicity, the PCR program was unied
for all markers and contained 5 min of initial
denaturation at 95C, touch-down prole of 1 min
at 94C, two cycles at 6056C (2C/cycle) for 1 min
30 s, and 2 min at 72C followed by 30 cycles with
annealing temperature held at 54C. The PCR
reaction mix consisted of 12.5 mm
3
of Top-Bio
PPP Master Mix (Top-Bio, Prague, Czech Republic),
10 pmol of each primer, 0.2 lg of DNA and demi-
neralised water up to 25 mm
3
. For RFLP analysis,
4 mm
3
of the PCR products were digested for 10 h at
37C in 10 mm
3
volumes containing 4.7 mm
3
of
demineralised water and 1 mm
3
of Y
?
/Tango buffer
with 0.3 mm
3
of the restriction endonucleases AluI,
Eco52I or MboII (Fermentas, Vilnius, Lithuania) for
fragments in the same order as listed above and than
deactivated at 65C for 20 min. Restriction fragments
were separated on 2% agarose gel containing 2 mm
3
of GoldView (SBS Genetech, Shanghai, China).
Samples from Felchowsee were analysed for their
genetic variability in 11 enzymatic systems repre-
senting 24 gene loci by horizontal starch gel electro-
phoresis (Aebersold et al. 1987) (Table 1). Staining
of all but two enzymes followed the standard
procedures of Shaw and Prasad (1970) and the
modied protocol of Vuorinen (1984). Creatine
kinase was visualised using general protein staining
(amido black). Superoxide dismutase appeared as
light, whitish spots on gels stained for alcohol
dehydrogenase, glycerol-3-phosphate dehydrogenase
or phosphoglucomutase, respectively. Enzyme band-
ing patterns were read by using the tench gene
nomenclature of S

lechtova et al. (1995), which

follows the rules suggested by Shaklee et al.
(1990). Alleles were named according to their
relative electrophoretic mobilities.
Rev Fish Biol Fisheries (2010) 20:289300 291
123
68
Additional raw data for the same allozyme loci for
sh from Dollnsee and 6 microsatellite loci for sh
from both lakes were taken from studies of Kohlmann
and Kersten (1998, 2006) and Kohlmann et al. (2007,
2009). Microsatellite locus MTT8 was inferred to
contain a null allele (Kohlmann et al. 2009) and was
therefore discarded from most analyses. Allelic
frequencies of MTT6 and MTT2 were compared
with raw data of Kohlmann et al. (2009) from Las
Vegas del Guadiana sh farm in Spain (Bada),
Wuhan in China (Chin) and Lake Sapanca in Turkey
(Turk) that appeared to contain only Eastern phylo-
group alleles (Lajbner et al. unpublished).
Data analyses
For each population, variation at polymorphic loci
was summarized as Neis unbiased expected hetero-
zygosity or gene diversity (Nei 1987).
Three types of exact test of HardyWeinberg
equilibrium were conducted for each population by
using Genepop 4.0 (Rousset 2008), which all assume
the same null hypothesis (random union of gametes)
but differ in the construction of the rejection zone. In
the exact probability test (e.g. Haldane 1954; Weir
1996), the probability of the observed sample is used
to dene the rejection zone, and the P-value of the
test corresponds to the sum of the probabilities of all
tables (with the same allelic counts) with the same or
lower probability. Two variants of the more powerful
score test (U test) were run, which assumed, respec-
tively, heterozygote excess or heterozygote de-
ciency as the alternative hypothesis to panmixia
(Rousset and Raymond 1995). The Markov chain
algorithm to estimate without bias the exact P-value
of this test (Guo and Thompson 1992) was conducted
by 1,000 batches of 20,000 iterations following
20,000 dememorization steps.
Wrights F-statistics was used as another means of
quantifying the conformity of genotype frequencies to
HardyWeinberg proportions and to test the existence
of geographical subdivision of populations. Two
parameters were estimated for the polymorphic loci
according to Weir and Cockerham (1984) with the
Genetix software package, v.4.05 (Belkhir et al.
2004). The inbreeding coefcient F
IS
was estimated
by the estimator f, and the xation index F
ST
by the
estimator h (Weir and Cockerham 1984). The signif-
icance of the multilocus estimates was assessed by a
permutation test using a 20,000 randomised data set
generated by permuting the alleles among individuals
for F
IS
and the individuals among the samples for F
ST
(Dallas et al. 1995; Balloux and Lugon-Moulin 2002).
Exact multilocus tests for association between
alleles were also computed using software MLD
(Zaykin et al. 1995) allowing haplo-diploid data
Table 1 Enzymes and tissues examined, number of loci screened and buffer systems used
Enzyme E.C. number Abbreviation Tissue Number of loci Buffer
a
Aspartate aminotransferase 2.6.1.1. mAAT Muscle 2 B
sAAT Muscle/liver 1 B
Alcohol dehydrogenase 1.1.1.1. ADH Liver 1 C
Creatine kinase 2.7.3.2. CK Muscle 1 A
Glycerol-3-phosphate dehydrogenase 1.1.1.8. G3PDH Muscle/liver 2 C
Glucose-6-phosphate isomerase 5.3.1.9. GPI Muscle/liver 2 A
Isocitrate dehydrogenase 1.1.1.42. mIDHP Muscle 2 C
sIDHP Liver 2 B
Lactate dehydrogenase 1.1.1.27. LDH Muscle/liver 3 B
Malate dehydrogenase 1.1.1.37. mMDH Muscle/liver 2 B
sMDH Muscle/liver 2 B
Phosphogluconate dehydrogenase 1.1.1.44. PGDH Liver 1 B ? C
Phosphoglucomutase 5.4.2.2. PGM Muscle/liver 2 C
Superoxide dismutase 1.15.1.1. SOD Liver 1 C
a
Buffers: A Triscitric acid, pH 8.5 (gel) and lithium hydroxideboric acid, pH 8.1 (tray; Ridgway et al. 1970), B Citric acid
morpholine, pH 6.5 (Clayton and Tretiak 1972, modied by Vuorinen 1984), C Triscitric acid, pH 7.1 (Shaw and Prasad 1970)
292 Rev Fish Biol Fisheries (2010) 20:289300
123
69
combination. In this test, the proportion of 20,000
permuted multilocus genotypic arrays as probable or
less probable than the sample forms an estimate of the
signicance level. Pairwise genotypic and allelic
linkage disequilibria were calculated by Black and
Krafsur (1985) method of Linkdos (Garnier-Gere and
Dillmann 1992) implemented in Genetix 4.05 (Belk-
hir et al. 2004), and using Cockerham and Weirs
(1977) coefcient of gametic disequilibrium for each
pair of loci (D
ij
) and Weirs (1979) correlation
coefcient between alleles at two loci (R
ij
). The
signicance of the genotypic linkage disequilibria was
calculated using 10,000 permutations of genotypes
among individuals within each population while
signicance of allelic associations were estimated by
the chi-square test (Weir 1979). Pairwise genotypic
associations among all loci (nuclear-encoded and
mitochondrial) were also calculated by Linkdos
(Garnier-Gere and Dillmann 1992) as implemented
in Genepop 4.0 (Rousset 2008). Contingency tables
were created for all pairs of loci for each lake and a G
test was computed for each table using the Markov
chain algorithm of Raymond and Rousset (1995).
Bayesian method and the program NewHybrids
v.1.1 (Anderson and Thompson 2002) were used for
quantifying the level of certainty that each individual
belongs to each of pre-specied genotype classes.
The method does not require that allele frequencies of
each of the species are known and the loci used do
not necessarily need to be diagnostic. Rather, it uses a
Markov chain Monte Carlo simulation to integrate
over possible values of the model parameters (i.e. the
proportion of individuals from the different genotype
classes and the allele frequencies of each species),
and estimates the posterior probability that an indi-
vidual belongs to each genotype class (Anderson and
Thompson 2002). The inheritance model imple-
mented assumes that a sample has been drawn from
a mixed population of two species with unknown
proportions of individuals from the different hybrid
classes. Although it is generally possible to consider
as many hybrid classes as needed, the nite number
of available loci is not sufcient to reliably distin-
guish between these numerous categories, and it is
thus more appropriate to concentrate on the early
generation hybrid classes (Boecklen and Howard
1997; Epifanio and Philipp 1997; Rieseberg and
Linder 1999). Therefore, only those genotype classes
that could occur after up to three generations of
crossing between the parental species were allowed
(pure Western phylogroup, pure Eastern phylogroup,
F
1
hybrid, F
2
hybrid, BC
1
to Western phylogroup,
BC
1
to Eastern phylogroup and their crosses). Three
separate NewHybrids analyses were run, the rst for
the Felchowsee and Dollnsee together, the second for
the Felchowsee alone, and the third for the Dollnsee
alone. For all analyses the Markov chain was run with
a burn-in period of 100,000 iterations and 1,000,000
iterations following the burn-in, and assuming unin-
formative Jeffreys-type priors on the parameters.
Each analysis was run several times to assess
convergence. The analysis was considered to have
converged upon a stationary distribution if the
independent runs generated similar results.
We determined the number of subpopulations of
tench within each lake using a statistical procedure
(Pritchard et al. 2000) that attempts to minimize
disequilibrium (HardyWeinberg and linkage) within
groupings. The number of subpopulations (K) with the
highest posterior probability was estimated by using
the program Structure 2.2 (Pritchard et al. 2000;
Falush et al. 2003, 2007). The admixture model and
the option of correlated allele frequencies between
populations (also called the F-model) were selected
because it is considered the superior model for
detecting structure even among closely related pop-
ulations (Falush et al. 2003). Markov Chain Monte
Carlo runs consisted of 100,000 burn-in iterations
followed by 1,000,000 iterations. We explored K in
the range from one to nine and performed 10 runs for
each K value in each lake separately.
Results
HardyWeinberg and linkage equilibrium
Tench populations fromFelchowsee and Dollnsee were
moderately but signicantly differentiated (F
ST
=
0.012, P\0.05). Samples from each lake were there-
fore treated separately in the analyses.
Heterozygote deciencies measured by the
inbreeding coefcient and by the exact tests of
HardyWeinberg equilibrium (probability and scores
test) revealed a signicant deviation of genotype
frequencies from HardyWeinberg proportions in
both lakes. However, the loci that showed deviations
in Felchowsee were not the same as the loci that
Rev Fish Biol Fisheries (2010) 20:289300 293
123
70
showed signicant patterns in Dollnsee (Table 2).
Multilocus heterozygote deciencies measured by the
inbreeding coefcient revealed a signicant deviation
from HardyWeinberg proportions in Felchowsee
(F
IS
= 0.079, P\0.01) but not in Dollnsee (F
IS
=
0.040, P[0.05). The exclusion of locus MTT8 from
this analysis slightly reduced the values of the
inbreeding coefcient but did not reduce the levels
of signicance of these ndings in Felchowsee
(F
IS
= 0.059, P\0.05) or in Dollnsee (F
IS
=
0.032, P[0.05). The signicant indication of mul-
tilocus heterozygote deciency is further suported in
Felchowsee (P\0.001) but not in Dollnsee (P[
0.05) by the multilocus score test under the hetero-
zygote deciency alternative hypothesis. The result
remained signicant after the exclusion of MTT8
locus in Felchowsee (P\0.05) and insignicant in
Dollnsee (P[0.05).
Multilocus test of linkage disequilibria detected
signicant associations in both lakes (MLD exact
test, P\0.05). However, few signicant pairwise
genotypic disequilibria were found (Table 3) and no
signicant cytonuclear disequilibria were detected.
Only the disequilibrium between microsatellite loci
MTT2 and MTT6 was consistently signicant in both
lakes, independent on the method used (Table 3). The
associated alleles of these loci were the same in both
lakes (Table 4), and they were indistinguishable in
size from alleles xed in three putatively pure Eastern
populations (Bada, Chin and Turk) analysed by
Kohlmann et al. (2009).
Population and hybrid structure
Partitioning of the tench populations in two distinct
reproductive units (i.e. subpopulations) was not
supported by the Structure analysis. Although the
prior parameters for the F model (gamma distribution
with mean 0.01 and SD 0.05) were chosen to allow
the existence of two populations even with very
similar allele frequencies, in both populations the
highest log likelihood value (Felchowsee: mean
-1,168.467, SD 0.327; Dollnsee: mean -406.750,
SD 0.565) was found for K = 1. Log likelihood
values for the population consisting of two (Fel-
chowsee: mean -1,175.483, SD 4.080; Dollnsee:
mean -407.420, SD 0.258) or three separate repro-
ductive units (Felchowsee: mean -1,190.433, SD
9.279; Dollnsee: mean -408.840, SD 0.559) were
lower than for K = 1, and higher values of K
received progressively decreasing probabilities.
The NewHybrids analysis did not detect any
individual that could be classied as either purebred
Western or purebred Eastern or any of the early-
generation hybrids. Instead, the analyses assigned all
individuals from both lakes as advanced backcrosses
to Western phylogroup. Genotypically, this category
corresponds to the product of mating of the rst-
generation backcrosses of a backcross-to-Western
type among themselves. The posterior probability of
assignment to this category was similar for individ-
uals from Felchowsee (mean 0.884, SD 0.066) and
Dollnsee (mean 0.735, SD 0.021) and for a pooled
sample (0.923, SD 0.039).
Discussion
Despite their high evolutionary divergence (1.3% for
cytochrome b gene) that compares with genetic
distance between species (Avise et al. 1998), two
phylogroups of tench emanating from different
Pleistocene refugia form a broad contact zone in
Europe composed of individuals of mixed ancestry
(Lajbner et al. unpublished). The present study found
evidence that these phylogroups are not separated by
strong barriers to reproduction and that they merged
back into a single population after colonization of the
same postglacial lake.
If the phylogroups evolved genetic incompatibil-
ities during their refugial isolation, the current hybrid
populations should consistently display signicant
associations among alleles and genotypes from each
phylogroup caused by lowered frequency and/or
viability of crosses between the phylogroups (e.g.
Caputi et al. 2007; Stadler et al. 2008; Lajbner et al.
2009). If, on the contrary, they freely interbreed,
current populations that were postglacially founded
by both phylogroups should not show genome-wide
associations anymore as the result of inter-locus
recombination over the many generations of inter-
breeding (see Templeton 2006).
Results of the various analyses in this study
strongly supported the hypothesis of free interbreed-
ing between the phylogroups as opposed to strong
barriers to reproduction. The Structure analysis
(Pritchard et al. 2000) suggested that tench in each
lake corresponded with the highest probability to a
294 Rev Fish Biol Fisheries (2010) 20:289300
123
71
T
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,
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-
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i
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296 Rev Fish Biol Fisheries (2010) 20:289300
123
73
single population at equilibrium (HardyWeinberg
and linkage). The NewHybrids analysis (Anderson
and Thompson 2002) did not detect any individual
that could be classied as either purebred Western or
purebred Eastern or any of the early-generation
hybrids, and it assigned all individuals from both
lakes as advanced backcrosses to the Western phylo-
group. This suggests that the data does not t well the
NewHybrids model assuming two hybridizing popu-
lations, which is consistent with the result of the
Structure analysis.
The conformity of genotype frequencies to Hardy
Weinberg proportions could not be rejected for the
majority of loci by any of the methods. Although the
samples from both lakes showed signicant disequi-
libria at some loci, the patterns were not concordant:
the loci that showed deviations from the Hardy
Weinberg or linkage equilibrium in Felchowsee were
not the same as the loci that showed a signicant
disequilibrium in Dollnsee. Furthermore, whether a
locus showed a signicant deviation was dependent on
the statistics used to quantify the conformity of the
samples to the HardyWeinberg proportions (F
IS
vs.
exact test) or on the method used to estimate the
signicance of linkage disequilibria (permutations vs.
Markov chain Monte Carlo approximation). Kohl-
mann et al. (2009) explained the heterozygote de-
ciency found in Felchowsee at the microsatellite locus
MTT8 by the presence of a null allele not amplied
with the current primers, resulting in a biased estimate
of population genotype frequencies at this locus.
Of all pairwise comparisons, only the microsatel-
lite loci MTT2 and MTT6 were in signicant linkage
disequilibrium in both lakes and this pattern was not
dependent on the method. Because no other pair of
loci showed consistent disequilibria, it seems most
reasonable to attribute the observed association
between MTT2 and MTT6 to strong physical linkage
of these loci (Page and Holmes 1998). The relative
location of the markers in the tench genome is,
however, currently unknown and this hypothesis
needs further testing. Furthermore, the comparison
with purebred Eastern populations suggest positive
association of alleles at these loci within phylogroups.
Although the tench phylogroups formed a broad
contact zone during their postglacial dispersions, they
have remained effectively allopatric outside the
contact zone. This is especially evident in the Eastern
phylogroup that shows no signs of introgression with
genes from the Western phylogroup throughout its
broad distribution between the Danube River in the
west to Lake Baikal in the east (Lajbner et al.
unpublished). This is remarkable given that the
present study demonstrated that the phylogroups are
not reproductively isolated and their gene pools have
effectively merged in mixed populations. Further-
more, it is reasonable to expect that similar patterns
to that observed today were created by dispersion
from the refugia also following earlier glacial max-
ima because the mtDNA divergence between the
phylogroups covers multiple glacial-interglacial
cycles (Hewitt 2004). What could have caused the
refugial populations to retain their genetic integrity in
face of recurrent contacts and interbreeding? The
answer may be found in the dynamics of species
response to changing climate. If, as appears to be the
case, most species responded to glacier advances by
extinction of populations in northerly areas with only
populations in the vicinity or refugia surviving
(Hewitt 2004), the admixed populations outside the
refugia would have been extirpated at the onset of
each glaciation, protecting the genetic purity of
refugia (Hofreiter et al. 2004).
Table 4 Allelic associations between alleles at the microsat-
ellite loci MTT2 and MTT6 expressed as correlation coef-
cients (R
ij
)
Alleles Felchowsee Dollnsee
R
ij
P R
ij
P
236-160 0.6672 0.0001 0.7379 0.0017
236-164 0.2161 0.3592
236-168 0.1483 0.5293
236-170 -0.1424 0.3188 -0.4323 0.0667
236-172 -0.4675 0.0011 -0.5994 0.0110
236-174 -0.1123 0.4319 -0.2965 0.2084
236-176 -0.0707 0.6208
240-160 -0.6672 0.0001 -0.7379 0.0017
240-164 -0.2161 0.3592
240-168 -0.1483 0.5293
240-170 0.1424 0.3188 0.4323 0.0667
240-172 0.4675 0.0011 0.5994 0.0110
240-174 0.1123 0.4319 0.2965 0.2084
240-176 0.0707 0.6208
Signicant probability values (at the 0.05 level) are in bold and
alleles present in the three presumably pure Eastern
populations (Bada, Chin and Turk) are in italics
Rev Fish Biol Fisheries (2010) 20:289300 297
123
74
The tench phylogroups can be considered separate
species according to the phylogenetic species concept
(Mishler and Theriot 2000; Wheeler and Platnick
2000). Some genetic disequilibria were detected in
natural hybrid populations but the pattern is not
consistent between populations and across methods
as would be expected in case of a strong reproductive
barrier. Thus, the present data suggest that the tench
can be considered a single species under the biolog-
ical species concept (Mayr 1942, 1963).
The fact that tench phylogroups can interbreed but
remained distinct in the refugia has important
practical implications. For example, populations in
the vicinity of the refugia may show physiological
adaptations to different selective environments that
could be lost if these populations were extirpated
(e.g. by anthropogenic habitat change) or admixed by
stocking with individuals of the opposite phylogroup
or the mixed ancestry (Allendorf et al. 2001). On the
other hand, since the two phylogroups readily form
mixed populations without detectable negative tness
consequences and tench are widely used for aqua-
culture, controlled genetic experiments can be envis-
aged to identify the genes underlying important
physiological or structural phenotypes (Nikinmaa
and Waser 2007). Furthermore, many tench popula-
tions in Europe are admixed, which offers a unique
opportunity to identify the genetic basis of pheno-
typic traits by the admixture mapping approach
(Buerkle and Lexer 2008). Tench phylogroups,
therefore, represent unique genetic resources and a
valuable new model for applied genetic research.
Acknowledgments The authors thank Petra Kersten for
laboratory assistance and Silvia Markova and Petr Rab for
advice. The work was supported by the Ministry of Education,
Youth and Sports of the Czech Republic (LC06073,
MSM6007665809), and by the Academy of Sciences of the
Czech Republic (IRP IAPG AV0Z50450515, IGA UZFG/05/
22 and IGA UZFG/08/13).
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3. Shrnut vsledku a jejich vznam
Prostorova geneticka analza variability sekvenci ctyr nezavislch genu odhalila dv hlavni
skupiny populaci lina obecnho. Kazda z nich ie ien malo geograIicky strukturovana, ale vzaiemn
se vznamn lisi (Laibner a kol. v tisku). Lokalizovala rovnz hlavni geograIick bariry v toku
genu (shoduiici se ve vice genech), kter isou zpusobeny pouze castecn se prekrvaiicim
rozsirenim dvou relativn vzdalench evolucnich linii-Zapadni a Vchodni. Tyto linie maii
pravdpodobn puvod v odlisnch glacialnich reIugiich, ze kterch kolonizovaly sv soucasn
arealy. Geny obou evolucnich linii isou pritomny v evropskch kulturnich liniich. Vchodni
evolucni linie se navic vyskytuie ve volnch vodach zapadni Evropy, kde v nkterch oblastech
dokonce pocetn prevlada nad Zapadni linii. Toto ie s neivtsi pravdpodobnosti dusledek migrace
zprostredkovan lidmi.
Ryby, lina nevyiimaie, isou casto vysazovany daleko od mista ieiich puvodu, nehled
na genetickou podobnost a lokalni adaptace. Nezridka dokonce dochazi k zarybnovani volnch
vod populacemi z chovu, coz vede k ieiich krizeni s puvodnimi populacemi a genov introgresi.
Diky rozsahlmu vysetreni svtov populace linu se mi podarilo navrhnout restrikcni enzymy,
kter speciIicky stpi tri diagnostick geny (1 mitochondrialni a 2 iadern), a iiz podle
elektroIoretickho vzoru tak lze snadno a rychle ziistit, k iak patri evolucni linii (Laibner a Kotlik
2010). Tato metoda umozni iak vdeckm pracovnikum tak i rybarskm praktikum rychle
identiIikovat geneticky odlisn populace lina a bude uzitecna tak pro monitoring lidmi
zprostredkovanho sireni evolucnich linii v divokch populacich, pro genetickou charakterizaci
kulturnich linii a pro krizici experimenty.
Populacn geneticka analza intronu iadernch genu, mikrosatelitu, alozymu
a mitochondrialni DNA v populacich dvou postglacialnich iezer v zon kontaktu mezi Zapadni
a Vchodni linii v Nmecku sice ukazala iednotliv, statisticky vznamn vazebn nerovnovahy,
ty vsak byly omezeny na iednotliv lokusy, v naprost vtsin nebyly shodn pro ob iezera a lisily
se tak v zavislosti na pouzit statistick metod (Laibner a kol. 2010). Test byl zalozen
na predpokladu, ze v pripad existence silnch reprodukcnich barir budou v hybridni populaci
mezi alelami a genotypy pozorovateln nenahodn vztahy odrazeiici ieiich Iylogenetick puvod
a to i navzdory moznosti krizeni v zon kontaktu. M vsledky naopak ukazuii, ze evolucni linie
lina neisou eIektivn reprodukcn izolovan a nepredstavuii tak samostatn biologick druhy.
Odliseni eIektu prirozench historickch procesu od vsledku sekundarniho sireni predstavuie
pro studium historick biogeograIie trvalou vzvu, coz plati dvoinasob v pripad hospodarsky
vznamnch druhu zamrn rozsirovanch clovkem. Moie prace ukazuie, ze u takovch druhu
muze bt uzitecn na antropogenni migraci pohlizet iako na proces komplementarni k prirozen
78
IylogeograIick strukturaci, spise nez oba ievy povazovat za vzaiemn se vylucuiici priciny
soucasn genetick a prostorov populacni struktury. Z mch vsledku ie zreim, ze prirozen
historick procesy hraii dulezitou roli v genetick strukturaci soucasnch populaci lina a ieiich
stopy isou stale ziistiteln na ruznch genech (Laibner a kol. v tisku). Oproti tomu isem ukazal,
ze clovkem zprostredkovan migrace vznamn ovlivnily nedavnou evolucni historii lina,
pricemz smsn genetick slozeni kulturnich linii ma pravdpodobn podil na zvsen introgresi
mezi geneticky odlisnmi populacemi (Laibner a kol. v tisku).
Vsledky tto studie by se mohly uplatnit pri modelovani sireni druhu, prispt ke zpresnni
odhadu adaptability konkrtnich populaci a ke zvseni eIektivity konzervacnich strategii
budoucnosti (Scoble a Lowe 2010). Pretrvavaiicim stereotypem v obhospodarovani sladkch
volnch vod ie ieiich zarybnovani nepuvodnimi druhy ryb, rybami z umlho vtru, kdy neni
bran ohled na geograIick puvod rodicu, ci primo rybami z akvakultury. Pokud budou stavaiici
praktiky obhospodarovani pokracovat, povazuii za pravdpodobn, ze clovkem zprostredkovana
migrace smaze prirozen IylogeograIick vzor v podstatn casti linem obvanho arealu. Tak ie
mozn, ze hybridizace (napr. zvsenim adaptivni variability) zesili invazni potencial smisench
populaci mimo prirozen areal (viz. Lucek a kol. 2010). Je tak pravdpodobn, ze populace
v blizkosti reIugialnich oblasti mohou disponovat Iyziologickmi adaptacemi na odlisna zivotni
prostredi, kter by vsak mohly bt ztraceny pokud by tyto populace vyhynuly napriklad v dusledku
destrukce habitatu, nebo pokud by byly kontaminovan iedinci z opacn evolucni linie nebo
smisenho puvodu (napr. AllendorI a kol. 2001). Tyto vlivy tak mohou postupn redukovat
evolucni potencial divokch populaci a snizovat ieiich sance na preziti tvari v tvar globalnim
antropogennim zmnam zivotniho prostredi (Stockwell a kol. 2003: Frankham 2005).
Jelikoz se vsak ob evolucni linie krizi (Laibner a kol. 2010), kontrolovan genetick
experimenty by mohly pomoci identiIikovat klicov geny zodpovdn za dulezit Iyziologick
ci strukturni Ienotypy (napr. Nikinmaa a Waser 2007). Mnoho populaci lina ve volnch vodach ie
smisench, coz spolu s moznosti identiIikovat speciIick markery (Laibner a Kotlik 2010) nabizi
unikatni prilezitost identiIikovat genetickou podstatu Ienotypovch znaku s vyuzitim koncepce
primsovho mapovani (Buerkle a Lexer 2008). Evolucni linie lina, kter isem obievil, tedy
predstavuii unikatni genetick zdroie a cenn nov model pro aplikovan genetick vzkum
s moznosti uplatnni vsledku v praxi. Rychlost rustu, konverze krmiva a dalsi geneticky
podminn vlastnosti ryb uzce souvisi s rentabilitou chovu, pricemz identiIikace geneticky
odlisnch evolucnich linii umozni inIormovan a cilen slechtitelsk zasahy.
79
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