Temporal fluctuation in north east Baltic Sea region cattle population revealed by mitochondrial and Y-chromosomal DNA analyses

RESEARCH ARTICLE Tempo al Fluc ua ion in No h Eas Bal ic Sea Region Ca le Popula ion Re ealed by Mi ochond ial and Y-Ch omosomal DNA Analyses Ma ianna Niemi 1,2 *, Auli Bläue 1,3 , Te hi Iso-Tou u 1 , Janne Ha jula 1,3 , Ve onica Nys öm Edma k 4¤ , E e Rannamäe 5 , Lembi Lõugas 6 , An i Sajan ila 2 , Ke s in Lidén 7 , Jussi-Pekka Taa i sainen 3 1Bio echnology and Food Resea ch, MTT Ag i ood Resea ch Finland, Jokioinen, Finland, 2Uni e si y o Helsinki, Depa men o Fo ensic Medicine, Helsinki, Finland, 3Depa men o A chaeology, Uni e si y o Tu ku, Tu ku, Finland, 4Depa men o Zoology, S ockholm Uni e si y, S ockholm, Sweden, 5Ins i u e o His o y and A chaeology, Uni e si y o Ta u, Ta u, Es onia, 6Ins i u e o his o y, Tallinn Uni e si y, Tallinn, Es onia, 7A chaeological Resea ch Labo a o y, S ockholm Uni e si y, S ockholm, Sweden ¤Cu en add ess: Depa men o Bioin o ma ics and Gene ics,Swedish Museum o Na u al His o y, S ockholm,Sweden *ma ianna.niemi@helsinki. i Abs ac Backg ound Ancien DNA analysis o e s a way o de ec changes in popula ions o e ime. To da e, mos s udies o ancien ca le ha e ocused on hei domes ica ion in p ehis o y, while only a limi ed numbe o s udies ha e analysed la e pe iods. Con e sely, he gene ic s uc u e o mode n ca le popula ions is well known gi en he unde aking o se e al molecula and popula ion gene ic s udies. Resul s Bones and ee h om ancien ca le popula ions om he No h-Eas Bal ic Sea egion da ed o he P ehis o ic (La e B onze and I on Age, 5 samples), Medie al (14), and Pos - Medie al (26) pe iods we e in es iga ed by sequencing 667 base pai s (bp) om he mi o- chond ial DNA (m DNA) and 155 bp o in on 19 in he Y-ch omosomal UTY gene. Compa i- son o ma e nal (m DNA haplo ypes) gene ic di e si y in ancien ca le (45 samples) wi h mode n ca le popula ions in Eu ope and Asia (2094 samples) e ealed 30 ancien m DNA haplo ypes, 24 o which we e sha ed wi h mode n b eeds, while 6 we e unique o he an- cien samples. O se en Y-ch omosomal sequences de e mined om ancien samples, six we e Y2 and one Y1 haplo ype. Combined da a including Swedish samples om he same pe iods (64 samples) was compa ed wi h he occu ence o Y-ch omosomal haplo ypes in mode n ca le (1614 samples). PLOS ONE | DOI:10.1371/jou nal.pone.0123821 May 20, 2015 1/16 OPEN ACCESS Ci a ion: Niemi M, Bläue A, Iso-Tou u T, Ha jula J, Nys öm Edma k V, Rannamäe E, e al. (2015) Tempo al Fluc ua ion in No h Eas Bal ic Sea Region Ca le Popula ion Re ealed by Mi ochond ial and Y- Ch omosomal DNA Analyses. PLoS ONE 10(5): e0123821. doi:10.1371/jou nal.pone.0123821 Academic Edi o : Yong-Gang Yao, Kunming Ins i u e o Zoology, Chinese Academy o Sciences, CHINA Recei ed: Augus 29, 2014 Accep ed: Ma ch 7, 2015 Published: May 20, 2015 Copy igh : © 2015 Niemi e al. This is an open access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed. Da a A ailabili y S a emen : The analysed sequences a e a ailable in he GenBank, accession numbe s KF233429-KF233528. All ele an da a a e wi hin he pape and i s Suppo ing In o ma ion iles. Funding: This esea ch was unded by Academy o Finland ( he p ojec decision numbe 128451), h p:// www.aka. i/en-GB/A/. Wo k o MN was also unded by Na ional Popula ion Gene ics Doc o al P og amme, unded by he Minis y o Educa ion and Cul u e and he Academy o Finland, h p://www.oulu. i/biology/PopGenSchool/. Ca le bone samples om Conclusions The di e si y o haplog oups was highes in he P ehis o ic samples, whe e many haplo- ypes we e unique. The Medie al and Pos -Medie al samples also show a high di e si y wi h new haplo ypes. Some o hese haplo ypes ha e become equen in mode n b eeds in he No dic Coun ies and No h-Wes e n Russia while o he haplo ypes ha e emained in only a ew local b eeds o seem o ha e been los . A empo al shi in Y-ch omosomal haplo- ypes om Y2 o Y1 was de ec ed ha co esponds wi h he appea ance o new m DNA haplo ypes in he Medie al and Pos -Medie al pe iod. This sugges s a eplacemen o he P ehis o ic m DNA and Y ch omosomal haplo ypes by new ypes o ca le. In oduc ion A chaeological and mi ochond ial DNA e idence indica e ha ca le we e domes ica ed om he au och (Bos p imigenius)[1–5], abou 10,000 yea s ago in he Fe ile C escen [6]. F om he Fe ile C escen , domes ic ca le sp ead o Sou h Eas e n Eu ope a ound 8,800 Be o e P es- en (BP), o Cen al Eu ope a ound 7,000 BP, and o No h Cen al Eu ope a e 6,700 BP [7]. Domes ic ca le eached sou he n Scandina ia by 6,000 BP [8], Es onia by 4,100 BP [7] and i- nally Finland in he no he n Bal ic Sea egion by 3,000 BP [9]. The oldes adioca bon da ed emains o ca le in Finland da e back o 3086 ± 30 BP [9]. Molecula analyses o mi ochond ial DNA and he Y-ch omosome can be used o ace bo ine ma e nal and pa e nal lineages, espec i ely [10,11]. Va ia ion in he hype a iable e- gion o he mi hochond ial D-loop de ines he majo i y o au ine ca le, as well as some mi- ochond ial lineages o Nea Eas e n au ochs and many I alian au ochs [12], o belong o he T mega-haplog oup, including he haplog oups T, T1, T2, T3, and T4 [1,5,13–15]. A s udy o he whole mi ochond ial DNA has sugges ed an addi ional haplog oup, T5, de ined by si es ou side o he D-loop egion [15]. Th ee o he haplog oups ha e been iden i ied in au ine ca le, whe e he closes in phylogeny o haplog oup T is haplog oup Q [10], di e ing by one diagnos ic SNP si e in he hype a iable egion (posi ion 15953 in V00654) [15]. Haplog oup Q has been ound a low equency in mode n Sou h Eu opean ca le b eeds [10,15]. The dis- ibu ion o haplog oup Q has been hypo hesised o indica e a pa allel Nea eas e n o igin o haplog oups T and Q, whe e Q ep esen s a mino domes ica ed lineage [16]. Haplog oup P ha has only been iden i ied in no he n and cen al Eu opean au ochs, and in a couple o sca e ed au ine samples, di e ged om T and Q p io o hei spli [15,17]. The oldes di- e ging b anch in he m DNA phylogeny is he e y a e haplog oup R ha has only been iden i ied in local I alian ca le b eeds [16]. The gene ic di e si y o he T haplog oup is highes in he Nea and Middle Eas ca le pop- ula ions, whe e ou haplog oups T, T1, T2, and T3 exis [1,14], indica ing a Nea Eas e n o i- gin o au ine ca le, which is also suppo ed by nuclea ma ke analyses ha show highe a iabili y in he Nea Eas han in o he egions [1,18]. Eu opean domes ic ca le ca y he same ou haplog oups as Nea Eas ca le, bu wi h T3 p edomina ing in Eu ope a leas om he Neoli hic pe iod onwa ds [11,14,17,19,20]. Haplog oup T1 is qui e equen ac oss he Medi e anean coun ies [3,21], and p edominan and almos ixed in A ica [14]. Haplog oup T4 de i es om T3 and has hus a only been de ec ed in Asian and Yaku ian ca le om Rus- sian Sibe ia [11]. The s a -like pa e ns o he T3-cen e ed haplo ypes de ec ed in mode n and Mi ochond ial and Y-Ch omosomal DNA Analyses o N-E Bal ic Sea Ca le PLOS ONE | DOI:10.1371/jou nal.pone.0123821 May 20, 2015 2/16 Es onia we e pa o he g an p ojec no 8156 o Es onian Science Founda ion, h p://www.e ag.ee/ ahas amine/e g andid/. The unde s had no ole in s udy design, da a collec ion and analysis, decision o publish, o p epa a ion o he manusc ip . Compe ing In e es s: The au ho s ha e decla ed ha no compe ing in e es s exis . Neoli hic Eu opean ca le popula ions ha e been sugges ed o esul om pos -domes ic accu- mula ion o mu a ions [14,19]. A no h—sou h g adien o gene ic di e si y has been de ec ed in mode n Eu opean ca le (Bos au us), [11,18], including he Y-ch omosome [11]. A single nucleo ide polymo phism in in on 19 o he UTY gene (UTY19) can be used o dis inguish be ween he wo Y-ch omosom- al haplo ypes, Y1 and Y2 [22]. Whe eas Y1 is he domina ing haplo ype in mode n Wes e n and No he n Eu opean b eeds, haplo ype Y2 domina es in Sou h Eu opean b eeds [22], wi h a clea di iding zone in cen al Eu ope [23]. Apa om he geog aphical a ia ion, a empo al luc ua ion in Y1 and Y2 haplo ype equencies has been de ec ed, mainly om Swedish an- cien bulls and au ochs, sugges ing ha a ia ion in p esen -day equencies o Y1 and Y2 hap- lo ypes is likely due o ecen demog aphic e en s [24]. The aim o his s udy was o explo e empo al popula ion a ia ion by ma e nally and pa e - nally inhe i ed ma ke s in ca le om he No h Eas Bal ic Sea egion (N-EBSR), and o com- pa e ancien popula ions wi h mode n b eeds. Haplo ype da a om 45 ancien m DNA and 7 Y-ch omosome samples was used oge he wi h con empo a y da a om 2094 m DNA [10,11,15,16,20] and 1614 mode n [22–24] and 71 ancien Y-ch omosomes [24–26] samples. The da a indica es clea changes in he N-EBSR ca le popula ions om la e B onze/I on Age o mode n imes. Ma e ials and Me hods Ancien ca le bones A o al o 77 ca le bones we e selec ed o aDNA analysis om di e en si es ac oss Finland and Es onia and in he own o Vybo g in he Lening ad Region in no h-wes e n Russia (Fig A in S1 File). The samples o his s udy we e om museum collec ions held a 1.) The Na ional Boa d o An iqui ies, 2.) Museum o Raisio (Ha kko), 3.) The Museum Cen e o Tu ku, 4.) Ålands Museum, 5.) Museum o Viljandi, 6.) Pä nu Museum, 7.) Saa emaa Muse- um, 8.) Uni e si y o Tu ku, 9.) S . Pe e sbu g, Ins i u e o he Ma e ial Cul u e His o y, Rus- sian Academy o Sciences, 10.) Tallinn Uni e si y, and 11.) Uni e si y o Ta u (Table A in S1 File). All necessa y pe mi s we e ob ained o he desc ibed s udy, which complied wi h all ele an egula ions. The samples om Vybo g de i e om he Medie al and Pos -Medie al pe iods, du ing which Vybo g was pa o Finland. The ea lies bones (2 samples) a ailable o his s udy de i e om he La e B onze Age (700–500 BC) om he island o Saa emaa, Es onia. The es o he P ehis- o ic samples da es o he La e I on Age (800–1200 AD). To e i y ha each indi idual wi hin one si e and pe iod was sampled only once, samples de i ing om he same side o he animal we e selec ed, o he size and age o he indi idual was used o sepa a e indi iduals. Whene e possible, me aca pals we e p e e ed as me aca pals a e used o os eologically de e mine he sex o he animal [27,28]. F om 77 samples ini ially selec ed, a o al o 18 bones o ee h om he P e- his o ic pe iod (700 BC-1200 AD), 24 om he Medie al pe iod (1200–1550 AD), and 34 om he Pos -Medie al pe iod (1550–1800 AD) we e used o aDNA analyses. One sample ha was adioca bon da ed as mode n was omi ed om u he aDNA analysis. A o al o 21 skele al samples we e adioca bon-da ed a he Labo a o y o Ch onology o he Finnish Museum o Na - u al His o y (LUOMUS), Uni e si y o Helsinki (Table A in S1 File). Radioca bon da ed samples co e ed all bones and ee h om non-dis inc cul u al laye s ha we e used o aDNA analyses. DNA ma ke s and labo a o y me hods To de e mine he m DNA haplog oups T, T1, T2, T3, T4, and T5 [15], a combina ion o h ee agmen s yielding 486 bp o sequence co e ing he m DNA D-loop om posi ion 16031 o Mi ochond ial and Y-Ch omosomal DNA Analyses o N-E Bal ic Sea Ca le PLOS ONE | DOI:10.1371/jou nal.pone.0123821 May 20, 2015 3/16 178 [GenBank: V00654] and a 181 bp sequence om he ND5 gene (posi ion 12 911 o 13 091 [GenBank: V00654]) we e analysed. An addi ional 77 bp D-loop agmen (posi ions 15936– 16012 in V00654), de e mining haplo ype Q, was analysed om one sample (H01, B To 4). As a Y-ch omosomal haplo ype ma ke , a 155 bp sequence om in on 19 in he UTY gene was analysed ( he ans e sion G>T a posi ion 423 in [GenBank: AY936543], de ining haplo ypes Y1 o Y2) [22]. DNA ex ac ion [29], PCR me hods and sequencing o PCR p oduc s we e as desc ibed in [30]. B ie ly, 0.2–0.5 ml o bone powde was suspended in 900 μl 0.5M EDTA, 100 μl 10M u ea and 5 μl p o einase K (20 mg/ml), and incuba ed wi h cons an shaking a 55°C o e nigh . DNA om he concen a ed supe na an (Amicon-4 30K cen i ugal il e uni s, Me ck Millipo e) was ex ac ed wi h a QIAquick PCR Pu i ica ion Ki (Qiagen, Sweden) acco ding o manu ac u e ’s ins uc ions. App oxima ely 5–10 μL o DNA ex ac was used in he PCR pe o med wi h he Ho S a Taq DNA polyme ase Ki (Qiagen, Sweden) wi h an inclu- sion o 0.4 mM dNTP, 0.2 μM o each p ime and 0.25 uni s (U) o U acil DNA Glycosylase (UNG, Sigma-Ald ich). The PCR p og am included ini ial s eps o 37°C o 10 min and 95°C o 15 min ollowed by 55 h ee-s ep cycles o 94°C o 30s, AT°C o 40s and 72°C o 1 min and 10 min a 72°, whe e AT s ands o a speci ic annealing empe a u e o each p ime pai (Table B in S1 File). P ime s and success a es o aDNA analyses (Tex A in S1 File, Table B in S1 File) a e p o ided in he Suppo ing In o ma ion. Au hen ici y o ancien ca le DNA The au hen ici y o aDNA analyses was con olled in a ious s eps o he labo a o y wo k- low and he analyses we e epea ed in independen ancien DNA labo a o ies. All 45 ancien sam- ples included in he s a is ical analyses we e ex ac ed a leas wice (MTT Ag i ood Resea ch Finland, Jokioinen, Finland, S ockholm Uni e si y, S ockholm, Sweden and Depa men o Fo- ensic Medicine, Uni e si y o Helsinki, Helsinki, Finland). Each pa icipa ing ancien DNA labo a o y ollowed gene al guidelines o ancien DNA wo k such as sepa a e space o sample p epa a ion and ancien DNA wo k, sepa a e p e- and pos -PCR a eas, ai -con olled s e ile aDNA wo k space, wea ing o p o ec i e clo hing, using disposable ools, pipe es wi h ae osol esis an il e ips and ea ing equipmen and wo king su aces wi h bleach and ul a- iole i adia ion equen ly. To ensu e he au hen ici y o he m DNA and Y-ch omosomal sequences, and o de ec pos- sible PCR e o s, each DNA agmen o each sample was sequenced om a leas wo di e en PCR eac ions wi h DNA de i ed om di e en ex ac ions. The sample was conside ed o be ep oducible when consis en sequences o each DNA agmen we e ob ained om a leas h ee ampli ica ions. The consis en sequences we e e i ied om wo ex ac ions in analyses done a leas in wo independen aDNA labo a o ies. O e lapping p ime s speci ic o ca le DNA we e designed o p e en c oss eac i i y wi h human DNA (Tex A in S1 File, Table B in S1 File). Nega i e con ols we e applied o all s eps in he aDNA ex ac ion and ampli ica ion. A p e iously analysed mammo h sample [31] was used as a posi i e con ol when he i s i e samples we e ex ac ed. The mammo h sample was sui able as a posi i e con ol as i is ancien and i s sequence clea ly di e s om ca le. Fo u he analyses, sequences om aDNA samples ob ained om di e en ex ac ions and ampli ica ions, p o en iden ical by a leas wo independen aDNA labo a o ies we e used. One sample was no epea able and was hus excluded om analyses (Table A in S1 File). Fo six samples only pa ial m DNA was success ully ampli ied. Consequen ly, hey we e omi ed om he s a is ical analyses. As ampli ica ion om 25 samples (including one mode n sample, Table A in S1 File) yielded no DNA, a o al o 45 samples emained o s a is ical analyses (Table A in S1 File). Mi ochond ial and Y-Ch omosomal DNA Analyses o N-E Bal ic Sea Ca le PLOS ONE | DOI:10.1371/jou nal.pone.0123821 May 20, 2015 4/16 S a is ical analysis The m DNA sequences om he 45 success ully sequenced ancien ca le we e aligned sepa- a ely o he 486 bp D-loop and he 181 bp ND5 gene sequences using CLUSTALW [32] whe e penal ies used we e 10 o gap opening, 0.20 o gap ex ension, and 5 o gap dis ances. The combina ion o he sequenced egions is e e ed o below as he 667 bp haplo ype e- gion. A CLUSTALW alignmen was also pe o med o he se en success ully ampli ied Y- ch omosomal 155 bp sequences. The analysed sequences a e a ailable in GenBank, accession numbe s KF233429-KF233528. The Reduced Median-joining Ne wo k (RMN o be mos conse a i e ε= 0) was cons uc ed acco ding o he algo i hm desc ibed by Bandel , Fo s e and Rohl [33] wi h NETWORK 4.6.0.0 [33]. The opology ob ained in RMN was con i med wi h he Maximum likelihood (ML) and Bayesian Ma ko Chain Mon e Ca lo (MCMC) analyses using jModel es 2.1 [34], PhyML 3.0 [35] and M Bayes 3.2 [36]. Bo h he ML and he MCMC ee along wi h he de ailed s a is ical me hods a e p esen ed in Suppo ing In o ma ion (Tex A in S1 File, Fig B in S1 File). DnaSP ( e sion 5) [37] was used o calcula e he gene ic di e si y es ima es based on he 486 bp D-loop sequences. Numbe o haplo ypes (h), haplo ypic di e si y (Hd), numbe o seg- ega ing si es (S), nucleo ide di e si y (π), Tajima’s D (D), and a e age numbe o nucleo ide di e ences (K) we e calcula ed o each popula ion. To app oxima e he le el o bias in he di- e si y es ima es caused by he e och oni y in he da ase when pooling samples o di e en ages, co ec ed π hμ [38] was calcula ed wi h mu a ion a es o 34 and 53% pe million yea s and gene a ion leng hs o 5 and 7 yea s (uppe and lowe anges as calcula ed om Nea -Eas e n ca le in [39]). In o de o p o ide da es o he samples when calcula ing π hμ , adioca bon da es we e used and he used da es we e andomly assigned o co e he ange o con ex o samples da ed by con ex . In o de o compa e ancien ca le di e si y o mode n ca le popula ions, a numbe o addi- ional sequences om Eu ope, Nea Eas and No h Asia we e included in he popula ion di e - si y analysis. These sequences ha e p e iously been desc ibed and analysed [10,11,15,16,20]. The size o he common aligned m DNA sequence in his compa ison was 245 bp om a o al o 2139 indi iduals. This da ase was hen used in wo app oaches. Fi s , o explo e he empo al luc ua ion in haplo ypes wi hin he N-EBSR, 49 mode n ca - le samples om i e na i e N-EBSR b eeds (No he n, Wes e n, and Eas e n Finnca le, Es o- nian Red and Es onian Na i e [11,16]), along wi h he 45 ancien ca le analysed he e we e ex ac ed om he aligned 245 bp da ase . These 94 N-EBSR samples we e g ouped in o h ee empo al coho s; P ehis o ic and Medie al (n = 19), Pos -Medie al (n = 26), and Mode n (n = 49) and in o wo g oups: 1) he mos equen 245 bp haplo ype ound among he en i e 2139 da ase (563 samples) and 2) he es o he haplo ypes. The second app oach was used o explo e he appea ance and equency o ancien haplo ypes among 2094 mode n ca le di ided in o en geog aphical egions (N-EBSR, Scandina ia, Wes - e n Eu ope, Sou he n Eu ope, Sou h-Eas e n Eu ope, Eas e n Eu ope, Wes e n Russia, Cen al Russia, Sibe ia, and Nea Eas /Cen al Asia). Fo his app oach, he 2094 mode n samples we e g ouped in o h ee haplo ype g oups: 1) he mos common 245 bp haplo ype in he en i e da ase (563 ou o 2139 samples), 2) he es o he haplo ypes ound among 45 ancien N-EBSR ca le, and 3) o he haplo ypes no ound in ancien da a. The p ocedu e was used o s udy he dis ibu- ion o ancien haplo ypes among con empo a y ca le. No e ha he P ehis o ic haplo ypes we e excluded he e as mos o he P ehis o ic haplo ypes we e no p esen in con empo a y da a. Pea son’s chi-squa e es , as implemen ed in SPSS .11.5.0, was conduc ed o es o di e - ences in equencies o m DNA haplo ypes in bo h app oaches, be ween he empo al coho s and he geog aphical egions. Mi ochond ial and Y-Ch omosomal DNA Analyses o N-E Bal ic Sea Ca le PLOS ONE | DOI:10.1371/jou nal.pone.0123821 May 20, 2015 5/16 Six y-nine samples we e u he analysed o he Y ch omosomal SNP in UTY19, which di - e en ia es ca le Y ch omosomes in o haplo ypes Y1 and Y2 [22]. The se en samples success- ully analysed o he Y1/Y2 ma ke we e analysed o empo al luc ua ion wi h he Swedish ancien (n = 64) and Fennoscandian mode n (No he n, Wes e n, and Eas e n Finnca le, Swedish Red, Red polled, Fjallna a and Moun ain ca le, n = 41) da a gi en in [22–25]. The combined da a om Fennoscandian bulls we e di ided in o ou empo al g oups: I on Age (n = 8 [25]), Medie al (n = 37 his s udy and [24,25]), Pos -Medie al (n = 19 his s udy and [24]), and mode n (n = 28 [22] and [11] as epo ed in [23]). To compa e he empo al analy- ses in Fennoscandia o Cen al Eu ope, da a om Medie al bulls (n = 14, [26]) om Swi ze - land was analysed oge he wi h da a om mode n Swiss b eeds (B aun ieh, Eh inge , and Simmen al, n = 39, [22] and [11,40] as epo ed in [23]). In o de o make wide geog aphical compa isons, Y1/Y2 in o ma ion om 127 mode n Eu asian b eeds (n = 1614 [22,24] and [23] combining he da a o [11,40–42]) we e included. Da a om a o al o 1692 bulls was di ided in o nine geog aphical egions ( he No dic coun- ies, Wes e n Eu ope, Sou he n and Cen al Eu ope, Sou h Eas e n Eu ope, Eas e n Eu ope, Nea -Eas and Cen al Asia, Wes e n Russia, Cen al Russia and Sibe ia). A Pea son’s chi-squa e es , as implemen ed in SPSS .11.5.0, was conduc ed o es o di - e ences in equencies o Y1 and Y2 be ween he empo al coho s (Fennoscandia and Swi ze - land) and geog aphical egions. In cases whe e 20% o mo e o he g oups had expec ed coun s less han 5, Fishe ’s exac p obabili y wo- ailed es was used ins ead. Resul s Radioca bon da ing A o al o 21 samples we e adioca bon da ed. Th ee samples appea ed o be om a la e pe iod han expec ed based on he con ex da ing while one sample om an I on Age con ex u ned ou o be mode n (Table A in S1 File). Os eological analysis The me ical analysis o me aca pals e ealed h ee males and 12 emales while wo me aca - pals we e inde e minable and i e me aca pals we e oo agmen ed o be analysed by os eo- logical me hods (Table A in S1 File). The esul s om he Y-ch omosomal UTY19 we e in acco dance wi h he os eological analyses as none o he samples aken om emale me aca - pals ampli ied wi h Y-ch omosomal p ime s. Two male me aca pals we e con i med and one inde e minable me aca pal was de e mined as male by Y-ch omosomal ampli ica ion (Table A in S1 File). M DNA haplo ypes Using DnaSP, 30 haplo ypes we e ound among he ancien ca le, including one sample p o- iding only pa ial in o ma ion. Twen y-nine haplo ypes, including he ull 667 bp sequence, we e used o u he analysis. When analysing he phylogeny o hese 29 haplo ypes, Bayesian MCMC, ML and RMN analyses ga e simila opologies (Fig 1, Median-joining ne wo k o he 29 ancien mi ochond ial haplo ypes (g ey-black) wi h 43 mode n e e ence haplo ypes (whi e), and Fig B in S1 File). All o he ancien haplo ypes we e assigned o he au ine hap- log oups acco ding o he known diagnos ic posi ions o ca le m DNA [10,11,14,15,20] (Tex AinS1 File). One sample was assigned o au ine mac o-haplog oup Q while he es o he samples we e assigned o he au ine mac o-haplog oup T (Fig 1, Tex A in S1 File). The 28 an- cien haplo ypes in mac o-haplog oup T we e u he di ided in o haplog oups T2 (one Mi ochond ial and Y-Ch omosomal DNA Analyses o N-E Bal ic Sea Ca le PLOS ONE | DOI:10.1371/jou nal.pone.0123821 May 20, 2015 6/16 haplo ype) and T3 (17 haplo ypes) and sub-haplog oups T3b (9 haplo ypes) and T1 (one hap- lo ype) (Fig 1, B and C Figs in S1 File, Tex A in S1 File). The sample p o iding pa ial in o ma- ion was assigned o T2 (Tex A in S1 File, Fig C in S1 File). Analysis o popula ion di e si y The m DNA di e si y in he Finnish, Es onian, and Vybo g ancien ca le da a a e summa ized in Table 1. The nucleo ide di e si y o he en i e da a se was 0.969. Wi hin each ancien em- po al ca le coho he mi ochond ial haplo ype di e si y es ima es (s, h, Hd, K, and π) indica e a high di e si y (Table 1). The haplo ype di e si y was highes in P ehis o ic ca le (Hd = 1.000) and sligh ly lowe in Medie al and Pos -Medie al ca le (Hd = 0.956 and 0.972, espec i ely). Nucleo ide di e si y a ied among pe iods wi h he highes obse ed di e si y (π=7.41 10 –3 ) in he P ehis o ic popula ion (Table 1). The bias in nucleo ide-di e si y es ima e caused by he - e och oni y was low, less han 1.5% in all empo al coho s (Table 1). Tajima’s D alue was neg- a i e o all empo al coho s wi h a signi ican ly nega i e p alue o he Pos -Medie al pe iod and he whole ancien ca le da ase sugges ing a popula ion expansion in Finland including Vy- bo g and he Bal ic egion (Table 1). Haplog oup T3 and sub-haplog oup T3b o med a s a -like phylogeny o haplo ypes, wi h majo haplo ypes H17 and H05 o T3 and T3b, espec i ely. The highes haplo ype di e si y was de ec ed in he oldes and smalles sample, om he P ehis o ic pe iod. A di e en se o haplo ypes was ound om he Medie al and Pos -Medie al samples (Fig 1). Tempo al m DNA analyses Signi ican empo al luc ua ions in he equency o m DNA haplo ypes in he N-EBSR ca le we e de ec ed (Pea son Chi-Squa e es , n = 94, χ 2 = 13.1, d = 4, p = 0.011). He e he mos Fig 1. Median-joining ne wo k o he 29 ancien mi ochond ial haplo ypes (g ey-black) wi h 43 mode n e e ence haplo ypes (whi e). Median-joining ne wo k (ε= 0) shows molecula ela ionships be ween 30 ancien haplo ypes (H01-H03 and H05-H30). Majo haplog oups (T1, T2, T3, T5 and Q) and sub-haplog oups (T1 , T3b) a e de ined by inclusion o 43 mode n e e ence haplo ypes om [10,15]. Each ci cle ep esen s one m DNA haplo ype whe e he size is p opo ional o he numbe o indi iduals in ha haplo ype. Black diamonds ep esen hypo he ical haplo ypes. The leng h o he b anches is p opo ional o he numbe o mu a ions be ween he haplo ypes excep he b anch be ween Bos au us and Bos indicus (32 mu a ions), which is sho ened o i in he pic u e. Haplo ypes om he P ehis o ic, Medie al, and Pos -Medie al pe iods a e indica ed in black, da k g ey, and ligh g ey, espec i ely. doi:10.1371/jou nal.pone.0123821.g001 Mi ochond ial and Y-Ch omosomal DNA Analyses o N-E Bal ic Sea Ca le PLOS ONE | DOI:10.1371/jou nal.pone.0123821 May 20, 2015 7/16 common 245 bp haplo ype inc eased in equency mo e han wice om Medie al o Pos -Me- die al and mo e han h ice om Pos -Medie al o mode n ime (g ey in Fig 2b in Fig 2, Dis i- bu ion o ancien N-EBSR ca le m DNA haplo ypes in mode n Eu asian ca le popula ions). Consequen ly, he p opo ion o o he haplo ypes dec eased h ough ime (colou ed and whi e pa e ns in Fig 2b). Nea ly hal o hese o he haplo ypes in con empo a y ca le we e no ound in ancien coho s (whi e in Fig 2b); and hus he p opo ion o he ancien haplo ypes (o he han he mos common) in mode n N-EBSR is app oxima ely 20% (colo ed pa e ns in Fig 2b). The p opo ion o unique ancien haplo ypes (unique among 2139 samples) was high- es in he P ehis o ic sample (black in Fig 2b). Geog aphical m DNA analyses The mos common 245 bp haplo ype (including he ancien 667 bp haplo ypes H05, H06, H11, H17, H24, and H26, Table C in S1 File) was ound in mos mode n Eu opean and Russian b eeds wi h a equency anging om 16 o 63% wi hin geog aphical egions (Table 2). The o he ancien haplo ypes had mo e es ic ed occu ences and equencies, less han 1.6% among he 2094 mode n ca le da ase (Table C in S1 File). The e we e signi ican di e ences in appea ance and equency o ancien haplo ypes among en geog aphical egions o con empo a y ca le (Pea son Chi-Squa e es , n = 2094, χ 2 = 355, d = 18, p<0.001). The p opo ion o ancien haplo ypes was highes in con empo a y N-EBSR ca le and Wes e n Russian ca le (Table 2), while he p opo ion o haplo ypes no ound in ou ancien sample inc eased wi h geog aphical dis ance showing highes p opo ions in Sou h and Sou h-Eas Eu ope, and Nea Eas /Cen al Asia (Table 2, indica ed in whi e in Fig 2a). Table 1. Summa y s a is ics o m DNA a ia ion in ancien No h Eas Bal ic Sea egion ca le om P ehis o ic, Medie al, and Pos -Medie al pe iods. Ancien No h Eas Bal ic Sea egion ca le P ehis o y, 700 BC-1200 AD Medie al, 1200–1550 AD Pos -Medie al, 1200–1800 AD To al N51426 45 S91522 33 h51120 29 Hd 1.000 0.956 0.972 0.969 K3.600 3.055 2.788 2.951 θs4.320 4.717 5.765 7.736 D-1.184 -1.437 -1.869*-2.067* π7.41 6.29 5.74 6.07 π hμa 7.35 6.28 5.73 6.04 Bias a 0.84% 0.13% 0.10% 0.43% π hμb 7.30 6.28 5.73 6.03 Bias b 1.45% 0.22% 0.17% 0.74% N is numbe o indi iduals sampled; S is he numbe o seg ega ing si es (excluding indels); h is he numbe o haplo ypes; Hd is he haplo ype di e si y; K is he a e age numbe o di e ences; θsis‘The a’de i ed om he obse ed numbe o seg ega ing si es (S); D is Tajima0s D s a is ic alue whe e s a is ical significances P<0.05 is ma ked wi h *.πis he nucleo ide di e si y*10 –3 ; The P ehis o ic coho includes wo samples om La e B onze Age and h ee samples om La e I on Age. a Based on gene a ion leng h o 7 yea s and mu a ion a e o 43% pe million yea s b Based on gene a ion leng h o 5 yea s and mu a ion a e o 53% pe million yea s doi:10.1371/jou nal.pone.0123821. 001 Mi ochond ial and Y-Ch omosomal DNA Analyses o N-E Bal ic Sea Ca le PLOS ONE | DOI:10.1371/jou nal.pone.0123821 May 20, 2015 8/16 Y-ch omosomal analysis UTY19 allele equencies in Fennoscandian ca le (Table D in S1 File) di e ed signi ican ly be- ween empo al coho s (Chi-Squa e es , p<0.001). Type Y2 was domina ing in bo h he I on Age (7/8) and he Medie al pe iod (36/37), wi h no s a is ical di e ence in allele equencies be ween he wo pe iods (Fishe ’s Exac es , p = 0.327). The p opo ion o Y1 inc eased signi i- can ly om he Medie al (1/37) o he Pos -Medie al pe iod (9/19, Fishe ’s Exac Tes , p<0.001) and hen again om he Pos -Medie al pe iod (9/19) o Mode n imes (33/41, Fish- e ’s Exac Tes p = 0.015). The Y1 ype was ixed in mos con empo a y Fennoscandian na i e b eeds wi h only one excep ion whe e Y2 was domina ing (8/9), iz. in one Finnish b eed, he Eas e n Finnca le. Fig 2. Dis ibu ion o ancien N-EBSR ca le m DNA haplo ypes in mode n Eu asian ca le popula ions. Haplo ype dis ibu ion in ancien Finnish, Es onian and Wes e n Russian (Vybo g a he sho e o Bal ic Sea) ca le popula ions om he La e B onze Age, I on Age, Medie al, and Pos -Medie al pe iods is indica ed wi h pie cha s a he igh side o he map (2B, see Table C in S1 File). Se en een ancien haplo ypes ound in mode n Eu asian popula ions (Table C in S1 File) a e indica ed by pie cha s wi h co esponding pa e ns (see key) on he map (2A). The mode n haplo ypes no ound in ancien ca le a e coun ed oge he and indica ed in whi e. Coun s o unique ancien haplo ypes no ound in mode n popula ions a e indica ed in black. doi:10.1371/jou nal.pone.0123821.g002 Table 2. Dis ibu ion o N-EBSR ancien haplo ypes in mode n Eu opean and Asian ca le b eeds. N-EBSR Scandina ia Wes e n Eu ope Sou he n Eu ope Sou h- Eas e n Eu ope Eas e n Eu ope Wes e n Russia Nea Eas and Cen al Asia Cen al Russia Sibe ia To al Common H 31 23 93 334 8 13 16 4 12 14 548 63.3% 28.4% 38.1% 21.7% 16.0% 50.0% 61.5% 16.0% 37.5% 58.3% 26.2% O he Ancien H 10 27 17 51 7 2 9 1 10 2 136 20.4% 33.3% 7.0% 3.3% 14.0% 7.7% 34.6% 4.0% 31.3% 8.3% 6.5% H no ound in Ancien da a 8 31 134 1152 35 11 1 20 10 8 1410 16.3% 38.3% 54.9% 75.0% 70.0% 42.3% 3.8% 80.0% 31.3% 33.3% 67.3% To al 49 81 244 1537 50 26 26 25 32 24 2094 Figu es ep esen he coun and pe cen age o mode n ca le da a om en geog aphical egions g ouped in h ee haplo ype (H) g oups acco ding o he appea ance o he haplo ypes in ancien N-EBSR da a: The mos common 245 bp haplo ype (Common H), o he ancien haplo ypes ound in Pos - Medie al o Medie al pe iods and haplo ypes no ound (H no ound) in ancien No h-Eas Bal ic Sea egion ca le. doi:10.1371/jou nal.pone.0123821. 002 Mi ochond ial and Y-Ch omosomal DNA Analyses o N-E Bal ic Sea Ca le PLOS ONE | DOI:10.1371/jou nal.pone.0123821 May 20, 2015 9/16 44. Schlumbaum A, Tu gay M, Schible J. Nea Eas m DNA haplo ype a ian s in Roman ca le om Au- gus a Rau ica, Swi ze land, and in he Swiss E olène b eed. Anim Gene . 2006; 37: 373–375. PMID: 16879349 45. Soininen AM. Vanha maa alou emme: Maa alous ja maa alous äes ö Suomessa pe innäisen maa a- louden loppukaudella 1720-lu ul a 1870-lu ulle. His o iallisia u kimuksia 96. Helsinki: Suomen His o - iallinen Seu a; 1974. 46. Li R, Xie WM, Chang ZH, Wang SQ, Dang RH, Lan XY, e al. Y ch omosome di e si y and pa e nal o i- gin o Chinese ca le. Mol Biol Rep. 2013; 40: 6633–6636. doi: 10.1007/s11033-013-2777-y PMID: 24065546 47. Li R, Zhang XM, Campana MG, Huang JP, Chang ZH, Qi XB, e al. Pa e nal o igins o Chinese ca le. Anim Gene . 2013; 44: 446–449. doi: 10.1111/age.12022 PMID: 23347145 48. Edg en T, Den Fö his o iska iden. Finlands his o ia I. Esbo: Schild s; 1998. 49. Soininen AM. Pohjois-Sa on asu aminen keski- ja uuden ajan aih eessa. His o iallisia u kimuksia 58. Helsinki: Suomen His o iallinen Seu a; 1981. 50. O man E. Ru al condi ions. In: Helle K, edi o . The Camb idge His o y o Scandina ia. Volume 1. P e- his o y o 1520. Camb idge: Camb idge Uni e si y P ess; 2003. pp. 250–311. 51. Tö nblom L. Medel iden. Finlands his o ia 1. Ekenäs: Schild s; 1998. 52. Kala T, Kaljundi L, K eem J, Leimus I, Ma kus K, Mänd A, e al. Ees i ajalugu II: Ees i keskaeg. Ta u: Ta u Ülikooli ajaloo ja a heoloogia ins i uu ; 2012. 53. Mu oma S. Suu e kuolon uode 1696–1697. In: Ka onen P, edi o . “Pane leipään puole pe äjäis ä”— nälkä ja pula uode Suomen his o iassa. Uni e si y o Jy äskylä: Suomen his o ian julkaisuja 19; 1994. pp. 25–42. 54. Decke JE, McKay SD, Rol MM, Kim J, Molina Alcalá A, Sons ega d TS, e al. Wo ldwide pa e ns o ances y, di e gence, and admix u e in domes ica ed ca le. PLoS Gene 2014 Ma 27. doi: 10.1371/ jou nal.pone.0005541 Mi ochond ial and Y-Ch omosomal DNA Analyses o N-E Bal ic Sea Ca le PLOS ONE | DOI:10.1371/jou nal.pone.0123821 May 20, 2015 16 / 16