Mitochondrial DNA variation of Drosophila obscura (Diptera: Drosophilidae) across Europe

This is a sel -a chi ed e sion o an o iginal a icle. This e sion may di e om he o iginal in pagina ion and ypog aphic de ails. Au ho (s): Ti le: Yea : Ve sion: Copy igh : Righ s: Righ s u l: Please ci e he o iginal e sion: CC BY 4.0 h ps://c ea i ecommons.o g/licenses/by/4.0/ Mi ochond ial DNA a ia ion o D osophila obscu a (Dip e a: D osophilidae) ac oss Eu ope © 2022 Ins i u e o En omology, Biology Cen e, Czech Academy o Sciences Published e sion E ić, Pa le; S amenko ić-Rasak, Ma ina; D agiće ić, Milan; Kanka e, Maa ia; Wallace, Megan A.; Sa ić Veselino ić, Ma ija; Jelić, Mihailo E ić, P., S amenko ić-Rasak, M., D agiće ić, M., Kanka e, M., Wallace, M. A., Sa ić Veselino ić, M., & Jelić, M. (2022). Mi ochond ial DNA a ia ion o D osophila obscu a (Dip e a: D osophilidae) ac oss Eu ope. Eu opean Jou nal o En omology, 119, 99-110. h ps://doi.o g/10.14411/eje.2022.011 2022 99 Final o ma ed a icle © Ins i u e o En omology, Biology Cen e, Czech Academy o Sciences, České Budějo ice. An Open Access a icle dis ibu ed unde he C ea i e Commons (CC-BY) license (h p://c ea i ecommons.o g/licenses/by/4.0/). EUROPEAN JOURNAL OF ENTOMOLOG Y EUROPEAN JOURNAL OF ENTOMOLOGY ISSN (online): 1802-8829 h p://www.eje.cz ion o he au ho s). Among obscu a g oup species i o ms a monophyle ic iad wi h he wo mos ela ed species: D. ambigua and D. isi s (Bachmann & Spe lich, 1993; Gao e al., 2007). Al hough hese h ee species li e in sympa y, D. obscu a is mo e common (S amenko ić-Radak e al., 2003) especially a highe al i udes compa ed o he o he wo, which a e a ely collec ed he e (long- e m obse a- ion o he au ho s). Species om he obscu a g oup, especially Ame ican D. pseudoobscu a and Eu opean D. subobscu a, ha e long se ed as models in e olu iona y biology (Ande son e al., 1975; K imbas, 1993; Powell, 1997; Schae e e al., 2003; Balanyá e al., 2006; Sa ić Veselino ić e al., 2019). The gene ic a ia ion o na u al popula ions o D. sub- obscu a has been well desc ibed using pheno ypic, ch o- mosomal, and molecula ma ke s (K imbas, 1993; Pascual Mi ochond ial DNA a ia ion o D osophila obscu a (Dip e a: D osophilidae) ac oss Eu ope PAVLE ERIĆ 1, 5 , MARINA STAMENKOVIĆ-RADAK 2, 5 , MILAN DRAGIĆEVIĆ 1 , MAARIA KANKARE 3, 5 , MEGAN A. WALLACE 4, 5, * , MARIJA SAVIĆ VESELINOVIĆ 2, 5  and MIHAILO JELIĆ 2, 5 1 Ins i u e o Biological Resea ch “Siniša S anko ić”, Na ional Ins i u e o Republic o Se bia, Uni e si y o Belg ade, Bule a despo a S e ana 142, 11000 Belg ade, Se bia; e-mails: [email p o ec ed], [email p o ec ed] 2 Uni e si y o Belg ade, Facul y o Biology, S uden ski g 16, 11000 Belg ade, Se bia; e-mails: [email p o ec ed], [email p o ec ed], [email p o ec ed] 3 Depa men o Biological and En i onmen al Science, Uni e si y o Jy äskylä, Su on ie 9, 40014 Jy äskylä, Finland; e-mail: maa ia.kanka e@jyu. i 4 Ins i u e o E olu iona y Biology, Uni e si y o Edinbu gh, Cha lo e Aue bach Road, Kings Buildings, Edinbu gh EH9 3FL, UK 5 The Eu opean D osophila Popula ion Genomics Conso ium (D osEU) Key wo ds. Cy b, gene ic a ia ion, popula ion expansion, phylogeog aphy Abs ac . D osophila obscu a is a common ui l y ha inhabi s he empe a e o es s o Eu ope. While i is abundan in he no h compa ed o o he D osophila, i s densi y dec eases sou hwa ds, whe e i is g adually eplaced by o he D osophila species. This s udy desc ibes a ia ion in he mi ochond ial Cy b gene o D. obscu a om se e al Eu opean popula ions. We obse ed a la ge numbe o haplo ypes, oge he wi h he s uc u ing o gene ic a ia ion. Gene ic a ia ion is highe in he wes whe e O1 and e- la ed di e gen haplo ypes domina e. In he eas , he O2 haplo ype is mos equen , oge he wi h haplo ypes ha ecen ly a ose om i . In he cen al pa o he species ange, bo h O1 and O2 a e equally p esen , along wi h many o he s. These da a e eal signs o popula ion expansions ha p obably happened ea lie in he wes , and mo e ecen ly in he eas . Though ou conclusions a e based on only one gene ic ma ke , limi ing he powe o he analysis, he esul s imply ei he pos glacial expansion om wo unique sou ces o , mo e likely, eas wa ds s epping-s one expansion. This s udy adds impo an in o ma ion on gene ic a ia ion and phylogeog aphy o he obscu e biology o D. obscu a, a species ha has he po en ial o become an in e es ing model in e olu iona y biology and conse a ion gene ics. * Cu en add ess: College o Li e and En i onmen al Sciences, Uni e si y o Exe e , Pen yn Campus, Co nwall TR10 9FE, UK; e- mail: M.A.Wallace@exe e .ac.uk INTRODUCTION D osophila obscu a, he nomina e species o he obscu a g oup, is a Palea c ic ui l y whose dis ibu ion ex ends om Sou he n Eu ope o he cen al Fennoscandia, and om Wes e n Eu ope o cen al Asia (Lako aa a & Sau a, 1971; B ehm & K imbas, 1991). In Eu ope, i s ela i e abundance dec eases om no h o sou h (B ehm & K im- bas, 1991). In sou he n Finland, i is he mos abundan D osophila species (Lako aa a & Sau a, 1971). Along wi h D. subobscu a, D. obscu a is he one o he wo mos common D osophila species in G ea B i ain, al hough D. subobscu a ou numbe s i du ing mos mon hs (Sho ocks, 1975; Begon, 1978). In he Cen al Balkans, among obscu- a g oup species, i is much less abundan han D. subob- scu a (S anić e al., 2002; Pa ko ić-Lučić e al., 2012), bu is eadily collec ed a highe al i udes (long- e m obse a- Eu . J. En omol. 119: 99–110, 2022 doi: 10.14411/eje.2022.011 ORIGINAL ARTICLE 100 E ić e al., Eu . J. En omol. 119: 99–110, 2022 doi: 10.14411/eje.2022.011 de aul pa ame e s (Thompson e al., 1994). The i nal analysis included 185 sequences. A Median Joining ne wo k (Bandel e al., 1999) was calcu- la ed and plo ed in R 4.1.0 (R De elopmen Co e Team, 2018), using he pegas package (Pa adis, 2010). Epsilon was se o 0, o pu he ewes possible median haplo ypes in he ne wo k. A Cy b sequence o D. ambigua (IFS R42, collec ed a M . R anj in 2014) was used as an ou g oup. The ne wo k was plo ed o show only single al e na i e mu a ional s eps be ween median haplo ypes. Bayesian in e ence o phylogeny implemen ed in Beas 2.6.2 (Bouckae e al., 2019) was used o in e ee opology and hen o cons uc a phylogene ic ee. Sequences o closely e- la ed species D. ambigua and D. is is (NCBI accession numbe : EF216284.1) we e used as ou g oups o oo he ee. We selec ed he bes - i subs i u ion model o hese da a using likelihood a io es s and Akaike in o ma ion c i e ion (Akaike, 1973), imple- men ed in jModelTes 2.1.10 (Posada, 2008). The bes - i model was a Tamu a-Nei model o nucleo ide subs i u ion wi h a signi i - can p opo ion o in a iable si es (I) and gamma (G) dis ibu ed among-si e a e he e ogenei y (TnR + I + G). The Ma ko chain e al., 2001; F aga a e al., 2010; Sa ić Veselino ić e al., 2019). Pa icula ly in e es ing is he pa e n o mi ochon- d ial (m DNA) a ia ion ound in na u al popula ions o D. subobscu a, which has p o en o be an excellen model o s udying he selec i e o ces ha main ain sympa ic m DNA a ia ion (Jelić e al., 2015; Sa ić Veselino ić e al., 2019; Ku balija No ičić e al., 2020). In con as o D. subobscu a, limi ed da a is a ailable on gene ic a ia ion in na u al popula ions o i s sympa - ic coun e pa D. obscu a. Ch omosomal in e sion poly- mo phism has been s udied in 13 iso emale s ains (IFSs) collec ed ac oss he Eu opean con inen (B ehm & K im- bas, 1991). The sample size was limi ed, bu he numbe o de ec ed in e sions would sugges ha ch omosomal a ia ion in D. obscu a is simila o ha in D. subobscu a. Gene ic a ia ion has also been assessed by enzyme loci in mul iple popula ions om Fennoscandia (Lako aa a & Sau a, 1971) whe e a la ge numbe s o polymo phic loci we e obse ed. Howe e , so a , m DNA a ia ion has no been assessed in his species. The e is also a lack o D. ob- scu a popula ion s udies ha co e wide geog aphic a eas. This species is easily collec ed in he wild, easily b ed in he labo a o y, and is a p omising model o s udying his- o ical and adap i e p ocesses ha ha e shaped he gene ic a ia ion o na u al popula ions o he Palea c ic. In his pape , we desc ibe a ia ion in an 893 bp long sequence o he mi ochond ial Cy och ome b (Cy b) gene om se e al Eu opean popula ions o D. obscu a. We e- co d signi i can gene ic di e en ia ion among di e en egions o Eu ope, as well as di e en le els o wi hin- popula ion a ia ion. We discuss ou i ndings in ligh o colonisa ion om glacial e ugia, admix u e, and mo e ecen p ocesses ha could ha e in l uenced he obse ed pa e n o a ia ion. MATERIALS AND METHODS Samples o D. obscu a we e collec ed om ou Eu opean coun ies: Se bia, Finland, Ge many, and Sco land, UK ( e e ed o as ou popula ions in he u he ex ), co e ing a wide geo- g aphic ange (Fig. 1). Samples om Se bia co e se e al dis inc locali ies. Table 1 con ains in o ma ion on he collec ion si es, da e o collec ion, and he numbe o specimens. DNA was ex ac ed using a me hod ha en iches genomic DNA wi h m DNA (Ma inez e al., 1992). Fo Se bian samples, ex ac- ion was done om he F1 p ogeny o he emales collec ed in he wild. Ex ac ion was conduc ed sepa a ely o he p ogeny o di e en emales. Fo he es o he samples, DNA was ex ac ed om wild-caugh indi iduals ha had been kep in e hanol, and he i nal s ep o alkaline lysis was excluded o ob ain enough DNA. An 893 bp agmen co esponding o he mi ochond ial Cy b gene was PCR ampli i ed and sequenced wi h he ollowing p ime s: Cy b-F 5’-TTAT GGTT GATT ATTA CGAA-3’ and Cy b-R 5’- CAAA ACAT ATGC TTAT TCAA-3’ (Gao e al., 2007). The PCR cycling condi ions consis ed o an ini ial dena u a ion s ep a 94°C o 3 min, 35 cycles: a 94°C o 50 s, 51.5°C o 1 min, and 72°C o 1 min; wi h a i nal ex ension a 72°C o 3 min. Ampli i ed p oduc s we e pu i i ed using he QIAquick PCR Pu i- i ca ion ki (QIAGEN, Hilden, Ge many), and sequencing eac- ions we e pe o med using bo h p ime s (Mac ogen inc. Ams e - dam, The Ne he lands). The ob ained sequences we e aligned in BioEdi .7.2.5 (Hall, 2011), using he Clus alW algo i hm wi h Fig. 1. Map o sampling loca ions o D osophila obscu a h ough- ou Eu ope. A magni i ed map o Se bia is gi en bo om igh since mo e sampling loca ions a e p esen in close p oximi y. Base map was aken om Google Maps, 2021 [h ps://www.google.com/maps /@51.0375553,24.7087047,2977433m/da a=!3m1!1e3!5m1!1e4], June 8, 2021. Table 1. Sampling de ails. No. males/ emales – numbe o sam- pled males/ emales. Coun y / Popula ion Locali y Code Coo dina es o sampling si es Sampling yea No. males / emales Se bia M . GočSG 43°33´29.10˝N 20°45´17.40˝E2015 24 emales 43°32´54.00˝N 20°47´11.30˝E Se bia M . Ta a ST 43°56´58.09˝N 19°21´27.18˝E2016 34 emales 43°55´04.37˝N 19°25´13.19˝E Se bia M . S a a SS 43°22´27.34˝N 22°37´02.28˝E2016 20 emales 43°20´12.76˝N 22°41´42.99˝E Se bia M . Maljen MS 44°06´18.31˝N 19°58´56.55˝E2017 2 emales Finland Akaa FA 61°08´58.69˝N 23°31´26.33˝E2018 30 males/6 emales Ge many Foh be g GF 48°13´04.43˝N 07°49´09.10˝E2015 17 males/4 emales Sco land, UK Edinbu gh SC 55°55´23.13˝N 03°10´24.02˝W 2017– 2019 35 males/13 emales 101 E ić e al., Eu . J. En omol. 119: 99–110, 2022 doi: 10.14411/eje.2022.011 Mon e Ca lo (MCMC) sea ch was s a ed om andom ees and was un o 5 × 108 gene a ions, and he cu en ee was sa ed o i le e e y 1000 gene a ions. This gene a ed an ou pu o 5 × 105 ees. A e disca ding 108 s a es (20% o samples) as ‘‘bu n-in’’, he emaining samples we e summa ized in o a 50% majo i y- ule consensus ee, wi h clade pos e io p obabili ies o app oxi- ma e he pos e io dis ibu ion o he phylogeny, calcula ed using T eeAnno a o 2.1.2 (BEAST package). The i nal ee was isu- alized and edi ed wi h FigT ee 1.3.1 (h p:// ee.bio.ed.ac.uk/ so wa e/ i g ee). Mixing and con e gence among chains, as well as s abiliza ion o likelihood and pa ame e alues, we e assessed using he e ec i e sampling size c i e ion (ESS alues > 200 we e conside ed accep able) in T ace 1.7 (Rambau e al., 2018). We hen calcula ed nucleo ide and haplo ype di e si y. Taji- ma’s D (Tajima, 1989), Fu’s Fs (Fu, 1997), Fu and Li’s D-F (Fu & Li, 1993), and Fay and Wu’s H (Fay & Wu, 2000) es s we e used o es depa u e om mu a ion-d i equilib ium. We used se - e al es s due o hei di e ing s a is ical app oaches, and powe o in e demog aphic his o y (Ramos-Onsins & Rozas, 2002). This was impo an since popula ions di e ed in he numbe o analysed sequences. Fo Fu and Li’s and Fay and Wu’s es s, a Cy b sequence om he mos closely ela ed species D. ambigua (IFS R42, collec ed a M . R anj in 2014) se ed as an ou g oup. A McDonald-K ei man es (McDonald & K ei man, 1991) was implemen ed o compa e he a io o non-synonymous o synony- mous change wi hin and be ween species o D. obscu a and D. ambigua. Changes in popula ion size we e examined by calcu- la ing he obse ed and expec ed pai wise di e ences (misma ch dis ibu ion) (Roge s & Ha pending, 1992). The abo e pa am- e e s and es s we e conduc ed using DNASP .6.0 (Lib ado & Rozas, 2009). An ex ended Bayesian skyline plo (EBSP) (Heled & D ummond, 2008) was applied o addi ionally in e demo- g aphic his o y using BEAST2 (Bouckae e al., 2019), wi h an app op ia e subs i u ion a e o D. melanogas e m DNA (Haag- Liau a d e al., 2008), and an assump ion o 4 gene a ions pe yea (Begon, 1978) which co esponds o he clock a e o 0.248. The leng h o he Ma ko chains was se o 109 o he EBSP, logging he pa ame e s e e y 3000 i e a ions. Bu n-in was se o disca d 25% o he samples. T ace (Rambau e al., 2018) was used o assess he con e gence o he chains. An analysis o molecula a iance (AMOVA) was implemen - ed in o de o pa i ion a ia ion be ween and wi hin popula ions ha ep esen he ou geog aphical egions. An AMOVA was also conduc ed sepa a ely o he Se bian popula ion ha is com- p ised o ou locali ies (wi h he exclusion o MS locali y whe e only wo indi iduals we e a ailable). Pai wise FST indices we e calcula ed using A lequin .3.5.1.2 (Exco i e & Lische , 2010). The Man el es (Man el, 1967) in A lequin .3.5.1.2 was used o es o a signi i can isola ion-by-dis ance co ela ion. The Man el es compa es FST gene ic dis ances wi h log e- ans o med Eu- clidean spa ial dis ances in kilome e s. Ano he o m o man el es was employed, using wo ma ices o log e coo dina es, one o longi ude and one o la i ude, o be compa ed o he FST ma- ix. All samples we e es ed o he p esence o Wolbachia. A PCR assay using 16S DNA Wolbachia-speci i c p ime s (O’Neill e al., 1992) was used wi h a sligh modi i ca ion o he PCR condi ions (Ga cía-Ma ínez e al., 1998). D osophila is is wi h known in ec ion s a us se ed as a posi i e con ol (E ić e al., 2019). To exclude he possible p esence o o he ma e nally- ansmi ed bac e ia (Hu s & Jiggins, 2005) we conduc ed mic obiome se- quencing. Two samples we e made, each using 10 emales o igi- na ing om 10 di e en andomly chosen IFSs om Se bia. One sample included he O1 haplo ype and 9 o he s ha sha e i s spe- ci i c subs i u ion on posi ion 828. The o he included he O2 hap- lo ype and 9 o he s wi h i s speci i c a ian on posi ion 828. A he ime o he analysis, hese lines had been kep in he labo a o- y o 5 gene a ions. DNA was ex ac ed om pooled indi iduals using a p e iously published p o ocol o mic obiome sequencing in D osophila (Kapun e al., 2020). The mic obiome sequencing was pe o med by Fisabio (Valencia, Spain) and included Illu- mina 16S V3–V4 amplicon lib a y p epa a ion and MiSeq 300 bp pai ed-end sequencing. P ime sequences [ o wa d: CCT ACG GGN GGC WGC AG, e e se: GAC TAC HVG GGT ATC TAA TCC (Klindwo h e al., 2013)] we e emo ed wi h BBDuk (h ps://jgi.doe.go /da a-and- ools/bb ools/) using kme leng h 15 and allowing o 2 misma ches. The gene a ed da a was analysed in R 4.1.0 (R De elopmen Co e Team, 2018) using dada2 o e o es ima ion, sequence denoising, me ging, and chime a e- mo al (Callahan e al., 2016) as desc ibed p e iously (Be ibaka e al., 2021). In sho , sequences we e i s immed o 240 bp o o wa d and 210 bp o e e se eads, and all sequences con ain- ing mo e han 2 o 4 expec ed e o s ( o o wa d and e e se eads espec i ely) we e disca ded. A e denoising, sequence pai s we e me ged using a minimum o e lap o 20 bases wi hou misma ches, and all sequences sho e han 400 o longe han 428 we e disca ded. Taxonomy assignmen up o genus le el was pe o med using he Sil a 132 (h ps://www.a b-sil a.de/docu- men a ion/ elease-132/) da abase wi h he RDP Nai e Bayesian Classi i e algo i hm (Wang e al., 2007) as implemen ed in dada2 wi h de aul pa ame e s. RESULTS Among he 185 ob ained sequences, we de ec ed 72 di e en haplo ypes o he Cy b gene (NCBI accession numbe s: MZ337620.1–MZ337804.1). The e we e 73 seg ega ing si es, among which wo had h ee al e na ing nucleo ides. Among all seg ega ing si es, 59 we e synony- mous, and 16 non-synonymous subs i u ions (Table S1). Mo eo e , 43 changes we e obse ed only once, while 32 we e obse ed in mul iple indi iduals. One speci i c sub- s i u ion (G o A on posi ion 828 which changes aline o isoleucine) is o pa icula in e es due o i s p esence in 73 indi iduals. The a ian wi h aline is pa icula ly e- quen in Se bia (0.875), ollowed by Finland (0.750), and Ge many (0.524), whils i is a e in Sco land, UK (0.083). The lowes di e si y was eco ded in Se bia (π = 0.0146, hd = 0.546), while he highes was in Sco land (π = 0.0387, hd = 0.964, Table 1). The majo i y o haplo ypes we e sin- gle ons. A Median Joining ne wo k o D. obscu a Cy b haplo- ypes is p esen ed in Fig. 2. The O1 haplo ype is he mos common in Sco land, oge he wi h a la ge numbe o e y di e gen haplo ypes ha sha e i s speci i c subs i u ion on posi ion 828. Also, Sco ish haplo ypes a e mo e closely ela ed o he ou g oup species D. ambigua sequence (Fig. 2). The O2 haplo ype is pa icula ly equen in Se bia, whe e mos o he haplo ypes sha e i s speci i c subs i u ion on posi ion 828 and di e in only one mu a ional s ep om O2. In addi ion o he O1 and O2 haplo ypes, O3 is also ob- se ed in mo e han wo sampled indi iduals. O3 sha es a speci i c subs i u ion a posi ion 828 wi h he O1 haplo ype. Excep o O1, which was no obse ed in Finland, hese h ee mos equen haplo ypes a e p esen in all analysed egions. Haplo ype O2 is he mos equen o e all, maybe due o he o e ep esen a ion o samples om Se bia. 102 E ić e al., Eu . J. En omol. 119: 99–110, 2022 doi: 10.14411/eje.2022.011 Apa om spli ing ou g oup species as di e en clades, he Bayesian ee (Fig. S1) shows one sequence om Sco - land (O65) as a monophyle ic clade (PP = 1), and a ew shallow nodes spli ing sis e haplo ypes (PP > 0.7). A e ha , he phylogene ic ela ionships could no be esol ed con i den ly, as he pos e io p obabili y is e y low o he es o he nodes (PP < 50%). The esul s o Tajima’s D, Fu’s Fs, Fu and Li’s D-F, and Fay and Wu’s H a e p esen ed in Table 2. Nega i e alues we e obse ed in all cases, he majo i y o which we e s a- is ically signi i can . The McDonald-K ei man es (Table 3) showed ha he a io o non-silen o silen a ia ion was g ea e wi hin species han be ween species. A s a is ically signi i can depa u e om neu ali y was obse ed o he Se bian popula ion (SR) and he Ge man (GF) popula ion. G aphs o misma ch dis ibu ion a e p esen ed in Fig. 3. All g aphs show some aggedness o he obse ed unc- ion. A unimodal dis ibu ion wi h a peak a 0 obse ed pai wise di e ences is ound o sequences om Se bia. A unimodal dis ibu ion wi h a peak a 2–3 obse ed pai - wise nucleo ide di e ences is ound in Sco land. A bimod- al dis ibu ion was obse ed o sequences om Finland, Ge many, and o al Eu ope. Visual inspec ion shows ha only samples om Sco land show a good i o a popula ion expansion scena io in con as o cons an popula ion size. Fig. 2. Median Joining Ne wo k based on an 893bp agmen o he Cy b gene in D osophila obscu a. The size o ci cles co esponds o he numbe o indi iduals wi h he obse ed sequence. Dashes on lines ep esen nucleo ide subs i u ions. Dashed lines ep esen al e na i e one mu a ional s ep e icula ions. Di e en geog aphical egions a e p esen ed wi h di e en colou s indica ed in he legend wi hin he i gu e. Table 2. Pa ame e s o gene ic di e si y and Tajima’s D, Fu’s Fs, Fu and Li’s D-F, and Fay and Wu’s H es s ha measu e depa u e om mu a ional-d i equilib ium. Sample loca ion Di e si y Tajima Fu Fu & Li Fay & Wu πhd D PFs PFPDP H P Se bia, SR 0.00146 0.546 –2.451 ** –23.208 *** –3.846 *–3.673 *–13.839 ** Finland, FA 0.00178 0.560 –2.110 *–6.241 *–2.670 * –2.277 ns –9.305 ** Ge many, GF 0.00279 0.816 –1.661 ns –2.429 ns –1.860 ns –1.514 ns –7.316 * Sco land UK, SC 0.00387 0.964 –2.273 ** –42.140 *** –2.322 ns –1.684 ns –15.638 * π – nucleo ide di e si y; hd – haplo ype di e si y; ns – nonsigni i can ; *P < 0.05; **P < 0.02; ***P < 0.001, h esholds o P alues a e gi en based on he DNASP so wa e; signi i can alues a e gi en in bold le e s. 103 E ić e al., Eu . J. En omol. 119: 99–110, 2022 doi: 10.14411/eje.2022.011 EBSP es ima es o he ime o demog aphic expansion a e gi en in Fig. 4. The analysis wi h all sequences includ- ed shows ha popula ion expansion s a ed a ound 3000 yea s ago (Fig. 4a). When analysing he Se bian popula- ion he g aph gi es signs o ecen expansion ha s a ed a ound 1200 yea s ago bu wi h no able unce ain y (Fig. 4b). The EBSP plo o samples om Sco land es ima es popula ion expansion o ha e s a ed a a ound 8000 yea s ago, bu he con i dence in e al ge s wide as we go back in ime (Fig. 4c). The Ge man sample was e y small and he EBSP unc ion did no de ec pas popula ion expan- sion, as he Ma ko chain did no con e ge e en when we inc eased he numbe o i e a ions (Fig. 4d). The popula- ion om Finland, despi e he small sample size, showed signs o a mild popula ion expansion anging om 3000 o 2000 yea s in he pas , hough wi h a wide con i dence in e al (Fig. 4e). The esul s o he AMOVA show ha 13.24% o he o al a ia ion is p esen among popula ions (FST = 0.1324, P < 0.001). Pai wise FST alues and he signi i cance o hei di e ence om ze o a e p esen ed in Table 4. The mos di e en ia ed popula ions a e Se bia and Sco land, while Table 3. Resul s o he McDonald-K ei man es . Sample loca ion NF, P G P G (W) P G(Y) P SR, Se bia 8.826 ns 5.756 *5.411 *3.851 * FA, Finland 1.824 ns 0.680 ns 0.133 ns 0.116 ns GF, Ge many 10.714 *5.956 *5.457 *3.879 * SC, Sco land UK 4.550 ns 2.581 ns 2.412 ns 1.335 ns N – neu ali y index; F, P – Fishe ’s exac es P- alue ( wo ailed); G – es ; G (W), G(Y) – G es wi h Williams’ and Ya es’ co ec ion espec i ely; ns – nonsigni i can ; *P < 0.05; **P < 0.02; ***P < 0.001, h esholds o P alues a e gi en based on he DNASP so wa e; signi i can alues a e gi en in bold le e s. Fig. 3. Misma ch dis ibu ions o Cy b gene in D osophila obscu a popula ions. The solid line is he obse ed pai wise di e ences, he do ed line ep esen s he expec ed dis ibu ion unde he cons an popula ion size model, and he dashed line ep esen s he expec - ed alues unde he popula ion g ow h-decline model. 104 E ić e al., Eu . J. En omol. 119: 99–110, 2022 doi: 10.14411/eje.2022.011 he leas di e en ia ed a e hose om Se bia and Finland. Ge many shows a simila le el o di e en ia ion o all h ee o he popula ions. Fo he Se bian popula ion, whe e ou locali ies we e a ailable, he AMOVA shows a g ea simila i y be ween he samples. Only 2.27% o he a ia- ion is obse ed among di e en geog aphic locali ies om Se bia (FST = 0.02269, P < 0.05). Signi i can isola ion by dis ance is no obse ed in ou da a se . When geog aphical dis ances a e used in he Man- el es , a posi i e co ela ion was obse ed ( = 0.304757; P = 0.330740), which was no s a is ically signi i can . I la i ude is conside ed, he co ela ion is nega i e, and also non-signi i can ( = –0.234862; P = 0.667790). The high- es co ela ion was obse ed o longi ude ( = 0.735025; P = 0.082810), which, hough no s a is ically signi i can , shows signs o possible popula ion di e en ia ion along he Eas -Wes cline. All samples we e nega i e o he p esence o Wol- bachia. Mic obiome sequencing o a ailable IFSs shows a comple e absence o ma e nally ansmi ed bac e ia (no a single ead was eco ded o Wolbachia, Spi oplasma, Mic ospo idia, o Ricke sia) ha could in l uence m DNA a ia ion (Fig. 5). Lac obacillus oge he wi h Ace obac e comp ise mo e han 99% o he mic obio a in bo h samples while Rals onia comp ises less han 0.5% and all he o he gene a ep esen less han 0.1%. Fig. 4. Popula ion expansion o D osophila obscu a es ima ed wi h EBSP, o he o al sample (a), Se bian popula ion (b), Sco ish popula ion (c), Ge man popula ion (d), and Finnish popula ion (e). Con i dence in e als a e gi en as shaded EBSP. Time is gi en in million yea s. Fig. 5. P esence o bac e ial gene a in D osophila obscu a sam- ples, ob ained by mic obiome sequencing o 16S V3–V4 egion. O1–828 a con ains indi iduals wi h he O1 a ian a he 828 h nucleo ide and O2-828 a con ains indi iduals wi h he O2 a ian a he 828 h nucleo ide. Table 4. Pai wise popula ion di e en ia ion calcula ed by FST al- ues. Se bia Finland Ge many Finland 0.00174 ns Ge many 0.08485 *** 0.02939 * Sco land 0.2371 *** 0.14119 *** 0.05841 *** ns – non signi i can ; *P < 0.05; **P < 0.02; ***P < 0.001; signi i can alues a e gi en in bold le e s. 105 E ić e al., Eu . J. En omol. 119: 99–110, 2022 doi: 10.14411/eje.2022.011 DISCUSSION In his s udy, we analyse nucleo ide a ia ion o he mi- ochond ial Cy b gene in a widesp ead Eu opean D oso- phila species, h oughou i s ange. Ou esul s show ha D. obscu a possesses a high le el o m DNA a ia ion bo h wi hin and be ween popula ions. The nucleo ide di e si y wi hin popula ions obse ed o he D. obscu a Cy b gene (0.00146–0.00387) is gene ally highe compa ed o o he D osophila species: D. melanogas e (0.0009), D. simu- lans (0.0003), D. yakuba (0.0014) (Balla d & K ei man, 1994) and D. subobscu a (0.00087) (E ić e al., 2019). Se- quences o m a complex haplo ype ne wo k wi h se e al s a -shaped subne wo ks, wi h a geog aphical s uc u ing o gene ic a ia ion ac oss Eu ope, mos ly ac oss he Eas - Wes axis. In e es ingly, as O1 and O2 equencies di e in he Eas and Wes , so do he equencies o he o he haplo- ypes ha sha e hei speci i c a ian s on he 828 h nucleo- ide o he Cy b gene. Va ia ion is especially high in he wes e n pa o his species ange. The haplo ypes sha ing he O1 a ian on posi ion 828 a e mo e di e gen om one ano he han he haplo ypes sha ing he O2 a ian . Haplo- ypes sha ing he O2 828 a ian , in all bu one case, di e om O2 by only one mu a ional s ep. Haplo ypes O1 and O2 show simila equencies in Cen al Eu ope. Al hough he numbe o indi iduals sampled om his a ea was e- s ic ed, and only one si e om Ge many was sampled, i is in ui i e o conclude ha he haplo ype dis ibu ion in Cen al Eu ope is somewhe e be ween eas e n and wes - e n popula ions. The obse ed geog aphical s uc u ing o m DNA a ia ion in D. obscu a is especially in e es ing when compa ed o he sympa ic D. subobscu a, which shows geog aphic homogenei y in m DNA a ia ion ac oss i s en i e ange. Du ing he pas 40 yea s, s udies o mo e han 30 popula ions o D. subobscu a ha e shown ha wo main haplo ypes a e almos equally p esen in all popula- ions and ha he e a e less equen popula ion-speci i c haplo ypes (Ku balija No ičić e al., 2020). Cy och ome b is highly conse ed and he sequences a e e y simila , so pos e io p obabili ies we e low o all bu a ew nodes. Bayesian in e ence did no econs uc he Cy b phylogeny wi h high con i dence, bu we did d aw a ew conclusions om he analysis. The Bayesian ee shows ha haplo ypes ha sha e a polymo phism a posi ion 828 wi h he O1 haplo ype, and ha a e equen in he wes , a e indeed olde and close o D osophila ambigua. The ee also shows ha sequences ha sha e he 828 O2 poly- mo phism a e g ouped oge he , bu wi h a low pos e io p obabili y. The obse ed excess o single ons and a la ge numbe o seg ega ing si es a e esponsible o he nega i e al- ues o he a ay o pa ame e s ha measu e he depa u e o haplo ype dis ibu ion om mu a ion-d i equilib ium. This esul implies ei he popula ion expansion o pu i y- ing selec ion. Posi i e alues o neu ali y indices om he McDonald-K ei man es indica e an excess o non-silen polymo phism compa ed o di e gence. This excess o amino acid polymo phism in he m DNA, ela i e o di e - gence, is gene ally p esen in mice and humans (Nachman e al., 1994, 1996; Temple on, 1996), and also in D oso- phila (Kaneko e al., 1993; Balla d & K ei man, 1994; Rand e al., 1994), pa icula ly o he Cy b gene (Balla d & K ei man, 1994; E ić e al., 2019). The obse ed pa e n o non-silen polymo phism can be in e p e ed (Rand & Kann, 1996, 1998) in he ligh o he nea ly neu al model (Oh a, 1992a, b) ha p edic s he accumula ion o mildly dele e ious alleles ha pe sis o sho ime wi hin a popu- la ion and do no con ibu e o di e gence (Nachman, 1998; Wein eich & Rand, 2000; Meiklejohn e al., 2007). Olde , sligh ly dele e ious haplo ypes canno become p ogeni- o s o new lineages due o na u al selec ion (G an , 2015). This could be he case o he haplo ype pa e n obse ed in Sco land. Recen inc eases in e ec i e popula ion size can also gene a e a e ac ual e idence o posi i e selec ion i subs i u ions a e sligh ly dele e ious and i he e is no se- lec ion upon synonymous codon use (Ey e-Walke , 2002). Fig. 6. Maps o p oposed pos glacial expansion scena ios o D osophila obscu a. The non-con inuous s a ing poin o he a ows indi- ca es unce ain y in he e ugial sou ce. (a) Colonisa ion om a leas wo di e en sou ces. Di e en colou s indica e di e en e ugia; (b) Expansion om wes e n peninsulas wi h g adual coloniza ion by eas wa ds s epping-s one expansion. G adual colou change indica es a dec ease in a ia ion. 106 E ić e al., Eu . J. En omol. 119: 99–110, 2022 doi: 10.14411/eje.2022.011 This may be he case o he O2 haplo ype, and o he hap- lo ypes cha ac e is ic o Eas e n Eu ope, whose ne wo k e l ec s only ecen expansion. Misma ch dis ibu ion analysis shows unimodal dis i- bu ions o he Se bian popula ion, wi h low misma ch alues. Unimodal dis ibu ions wi h high misma ch alues a e de ec ed o Sco land. A unimodal dis ibu ion o mis- ma ches, which e l ec s demog aphic expansion, mo es o highe alues as mu a ions accumula e o e ime in a popula ion (G an , 2015), which implies ea lie popula ion expansion in he wes e n ange o he species. The analysis o in ec ion s a us wi h ma e nally ans- mi ed mic oo ganisms is impo an since hey sha e he same mode o inhe i ance wi h m DNA. They can o en con ound he in e ence o e olu iona y his o y ob ained by m DNA ma ke s, as a ia ion is al e ed by selec ion ac ing on hese mic oo ganisms (Hu s & Jiggins, 2005). Impo an ly, all ou samples we e nega i e o Wolbachia. Addi ionally, we conduc ed mic obiome sequencing on he Se bian samples, and ma e nally ansmi ed bac e- ia we e also excluded (Spi oplasma, Mic ospo idia, and Ricke sia). Al hough we canno exclude hei p esence in non- es ed samples, he main haplo ypes ha a e p esen Eu ope-wide we e co e ed wi hin ou sample. While Lac- obacillus and Ace obac e , which we e de ec ed in ou samples, a e common bac e ia also ound in IFSs o D. su- bobscu a and D. melanogas e kep in he labo a o y, D. obscu a shows less di e si y o bac e ial gene a compa ed o hese wo species (Be ibaka e al., 2021). Wha his o ical p ocesses could ha e shaped he ob- se ed pa e n o m DNA a ia ion in D. obscu a? The di e en composi ions o haplo ypes in he eas and he wes could imply pos glacial colonisa ion om a leas wo di e en sou ces wi h an admix u e in cen al Eu ope (Fig. 6a). The Balkan Peninsula would be he hypo he i- cal sou ce o he O2 haplo ype, while he sou ce o he O1 haplo ype could be he Ibe ian o Apennine peninsula. The Cen al Eu opean popula ion pe ec ly ma ches his scena io, as i possesses eas e n and wes e n lineages in almos equal equency. In addi ion, g aphs o misma ch dis ibu ion show wo peaks ha co espond o he peaks o wo dis inc lineages. In ha espec , pos glacial colonisa- ion o his species would ollow he hedgehog’s scena io (Hewi , 2004). Al hough ou EBSP in e als o popula- ion expansion a e e y wide, he esul s imply ha he expansion p obably happened ea lie in he Wes han he Eas . Then he ques ion a ises as o why expansion a e he las glacial maximum happened a di e en imes om di e en glacial e ugia, and why he e is such a di e ence in he le el o a ia ion be ween di e en e ugial sou ces? One migh imagine ano he scena io, o pos glacial ex- pansion om wes e n peninsulas, o wes e n c yp ic e u- gia, and hen g adual colonisa ion o he Eu opean con- inen by eas wa ds s epping-s one ange expansion (Fig. 6b). In he Eas , he O2 haplo ype migh ha e inc eased i s equency o he de imen o o he haplo ypes due o he bo leneck e ec , and subsequen ly gene a ed an a ay o young single on haplo ypes in i s ecen expansion. In his scena io, pos glacial colonisa ion o his species would be mo e simila o he b own bea ’s scena io (Hewi , 2004). This hypo hesis is suppo ed by he ea lie expansion o D. obscu a in he wes , app oxima ely 8000 yea s ago, while he O2 haplo ype ne wo k migh e l ec only ecen expan- sion a e i s colonisa ion o he highlands o he Balkan Peninsula. These expansions may also include hose ha gene ally happen yea ly a e win e o summe con ac- ions, which a e also obse ed in D. subobscu a (Cas o e al., 2010; Ch is ie e al., 2010; E ić e al., 2019). Signs o ancien his o ical expansions may also ha e been masked o e by annual con ac ions and expansions. I is also im- po an o unde s and ha he ime o he expansion gene - a ed by EBSP used in his s udy is jus a ough es ima- ion based on a cons an numbe o gene a ions pe yea (Begon, 1978) which could ha e a ied h ough ime and ac oss di e en egions. We should also s ess a speci i c aspec o his species’ bi- ology, which could be esponsible o he obse ed pa e n o s uc u e, and le el, o gene ic a ia ion. In he Balkan Peninsula, D. obscu a is ound in g ea numbe s only in highe al i udes, while mo ing o he no h i is equen ly ound in he lowlands oo. The ac ha i is he mos com- mon D osophila species in sou he n Fennoscandia (La- ko aa a & Sau a, 1971) implies ha i is adap ed o colde clima es. So, i is expec ed o sou he n D. obscu a o show di e en popula ion size dynamics compa ed o no he n Eu ope. On he o he hand, ecological condi ions could be simila since ege a ion and clima e in highe al i udes in he sou h a e simila o hose om no he n Eu ope. Popu- la ions om he sou h a e mo e isola ed and p one o ge- ne ic d i . Al hough popula ions can each high numbe s o indi iduals in he summe , hey a e es ic ed o small geog aphical a eas and less p one o gene l ow in he op i- mal pa o he season ha could es o e a ia ion. To mo e accu a ely deciphe he popula ion his o y ha has caused he obse ed pa e n o a ia ion in D. obscu- a m DNA mo e sampling is needed, especially om he Apennine and he Ibe ian Peninsula, whe e D. obscu a is mo e likely o be ound in highe al i udes. Fu he analysis should also be conduc ed wi h addi ional gene ic ma ke s, bo h mi ochond ial and nuclea (B i o & Edwa ds, 2009). This species’ dis ibu ion in highe al i udes and la i udes also makes i a po en ial model o s udying e olu ion- a y change due o global wa ming. Al hough D osophila l ies can mig a e easily, woodland habi a s sui able o D. obscu a canno be o med a he pace dic a ed by global wa ming. Addi ionally, in some egions, he e is no much space le o he l ies o mo e, ei he o highe al i udes o no hwa ds. Being e y abundan , and easy o collec (pa - icula ly in no he n Eu ope), designa e and main ain in he labo a o y, oge he wi h i s ich m DNA polymo phism, makes his species a p omising model o hese so s o e olu iona y s udies. ACKNOWLEDGEMENTS. This esea ch is i nanced by he Minis- y o Educa ion, Science and Technological De elopmen o he Republic o Se bia (451-03-9/2021-14/200178 o MSR, MSV, and MJ; 451-03-9/2021-14/ 200007 o PE and MD). The esea ch