Origin, evolution, phylogeny and taxonomy of Pulex irritans

Fo Re iew Only 1O igin, e olu ion, phylogeny and axonomy o Pulex i i ans 2(Siphonap e a:Pulicidae). 3 4ANTONIO ZURITA1, ROCÍO CALLEJÓN1, ÁNGELA M. GARCÍA-SÁNCHEZ1, 5MARA URDAPILLETA2, MARCELA LARESCHI3 & CRISTINA CUTILLAS1 61Depa men o Mic obiology and Pa asi ology. Facul y o Pha macy. Uni e si y o 7Se ille. P o eso Ga cía González 2, 41012 Se ille, Spain. 8 2Na ional Ins i u e o T opical Medicine (INMET). Neuquén y Jujuy s/n, 3370 Pue o 9Iguazú - Misiones, A gen ina. 10 3Cen e o Pa asi ology and Vec o s S udies (CEPAVE) (CONICET CCT La Pla a- 11 UNPL). B 120 s/n e/ 60 y 64, 1900 La pla a, A gen ina. 12 13 14 15 * Co esponding au ho : 16 D . C is ina Cu illas 17 Depa men o Mic obiology and Pa asi ology. Facul y o Pha macy. Uni e si y o 18 Se ille. P o . Ga cía González 2, 41012 Se ille, Spain. 19 Phone: +34954556773 20 e-mail: [email p o ec ed] Page 1 o 49 Medical and Ve e ina y En omology Fo Re iew Only 21 Abs ac 22 Wi hin Pulicidae amily, he so-called human lea, Pulex i i ans Linnaeus, 1758 has 23 been he mos s udied species oge he wi h C. elis Bouché, 1835, since hey ha e a 24 cosmopoli an dis ibu ion oge he wi h he ac ha hese species a e closely ela ed 25 wi h humans. The main aim o his wo k was o ca y ou a compa a i e mo phome ic 26 and molecula s udy o wo di e en popula ions o P. i i ans (Spain and A gen ina). 27 Fo his pu pose, he ITS1, ITS2 o he DNA and pa ial cy och ome-c oxidase (cox1) 28 and cy och ome b (cy b) m DNA gene o hese axa we e sequenced. Fu he mo e, we 29 assessed he axonomy, o igin, e olu ion and phylogeny o P. i i ans. In ou s udy, 30 mo phome ic da a did no show signi ican di e ences be ween P. i i ans specimens 31 om Spain and A gen ina, e en when hese wo popula ions we e collec ed om 32 di e en hos s; howe e , we ound a conside able deg ee o molecula di e gence 33 be ween bo h popula ions based on nuclea and mi ochond ial ma ke s. Thus, we could 34 sugges ha P. i i ans, in con as o o he gene alis leas, main ain a ce ain deg ee o 35 mo phological simila i y, a leas be ween Wes e n Palea c ic and Neo opical a eas. 36 Fu he mo e, we p o ided he exis ence o wo well de ined geog aphical gene ic 37 lineages wi hin P. i i ans species sugges ing he exis ence o wo c yp ic species which 38 could be disc imina ed by PCR-linked RFLP. Page 2 o 49Medical and Ve e ina y En omology Fo Re iew Only 39 In oduc ion 40 Pulicidae (O de Siphonap e a) has emained as he mos s udied amily o leas o e 41 he wo ld since mos leas o medical o e e ina y impo ance, such as C enocephalides 42 elis, C enocephalides canis, Pulex i i ans o Xenopsylla cheopis, a e membe s o his 43 amily. Cu en ly, Pulicidae consis s o ou ibes, 21 gene a, and 167 species (Whi ing 44 e al., 2008). Some au ho s (Lewis, 1998) conside ed Pulicidae as including Tungidae; 45 howe e , Whi ing e al. (2008) placed his amily as a monophyle ic g oup and 46 phylogene ically dis an om Tungidae. Recen ly, K asno e al. (2015) placed 47 Pulicidae amily on he basis o he lea phylogeny oge he wi h Lep opsyllidae. These 48 au ho s sugges ed ha he mainly Palea c ic dis ibu ion and o igin o Pulicidae and 49 Lep opsyllidae and he mainly Nea c ic dis ibu ion o he mos de i ed amily 50 Ce a ophyllidae, indica es an eas wa d ea lie (p e-glacia ion) mig a ions om 51 Palea c ic o Nea c ic zones h ough he Be ing Land B idge. On he o he hand, 52 Pulicidae exhibi s an in e es ing di e si y o hos speci ici y pa e ns and ecological 53 habi s (Whi ing e al., 2008). Wi hin his amily, he so-called human lea, P. i i ans 54 Linnaeus, 1758 ( he ea lies lea species desc ibed) has been he mos s udied species 55 oge he wi h C. elis Bouché, 1835, since hey ha e a cosmopoli an dis ibu ion 56 oge he wi h he ac ha hese species a e closely ela ed wi h humans. P. i i ans 57 pa asi izes a wide a ie y o hos s, including oden s o bi ds (G aham e al., 2016); 58 howe e , i gene ally pa asi izes la ge wild and domes ic mammals, pa icula ly 59 ca ni o es, li es ock and humans (G a z, 1999). Fu he mo e, speci ic hos associa ions 60 a y be ween geog aphic egions (Hopla, 1980; Lewis, 1998). Thus, in he las yea s 61 se e al au ho s ha e assessed he ole o his species as a ec o o se e al diseases 62 ca ying ou epidemiological s udies based on he de ec ion and p e alence o ce ain 63 pa hogens such as Ye sinia pes is, Ricke sia elis o Ba onella sp. in P. i i ans Page 3 o 49 Medical and Ve e ina y En omology Fo Re iew Only 64 collec ed om di e en hos s and di e en geog aphical a eas (Bel ho e al., 2015; 65 Fon al o e al., 2017; Paloma e al., 2017). Acco ding o Yssou e al. (2014) he apid 66 and eliable iden i ica ion o leas a species le el is an essen ial componen o he igh 67 agains lea bo ne diseases in o de o es ablish epidemiological ela ionship be ween 68 lea species and hei bo ne zoono ic agen s. Thus, be o e o assess he p e alence o 69 ce ain pa hogens in leas i is needed o know accu a ely which species we a e s udying 70 in o de o es ablish app op ia e p e en ion and con ol s a egies. 71 Du ing he las i een yea s, molecula app oaches ha e con ibu ed o signi ican da a 72 abou he diagnos ic de e mina ion o genus and species o leas (Di ma & Whi ing, 73 2003; Vobis e al., 2004; Game schlag e al., 2008; Whi ing e al., 2008; Ma ugal e 74 al., 2013; Zu i a e al., 2016). Ne e heless, he speci ic di e en ia ion o leas is 75 gene ally based on a a ie y o mo phological c i e ia such as he shape and s uc u e o 76 hei complex geni alia o he dis ibu ion o se ae, spines and c enidia (Lina di & 77 San os, 2012; López Be izbei ia e al., 2016; Has i e e al., 2017). The combina ion o 78 molecula and mo phome ic da a has allowed o ind synonymies o c yp ic species on 79 leas (Zu i a e al., 2015; Zu i a e al., 2018). These s udies e ealed he exis ence o 80 ce ain gene ic plas ici y in he O de Siphonap e a which should be aken in o accoun 81 in o de o ca y ou u he axonomic s udies. 82 The main aim o his wo k was o ca y ou a compa a i e mo phome ic and molecula 83 s udy o wo di e en popula ions o P. i i ans (Spain and A gen ina). Fo his 84 pu pose, he ITS1, ITS2 o he DNA and pa ial cy och ome-c oxidase (cox1) and 85 cy och ome b (cy b) m DNA gene o hese axa we e sequenced. The axonomy, o igin, 86 e olu ion and phylogeny o P. i i ans a e discussed. 87 Page 4 o 49Medical and Ve e ina y En omology Fo Re iew Only 88 Ma e ials and me hods 89 Collec ion o samples 90 Fi y- i e leas om Se ille (sou hwes e n o Spain) we e collec ed o -hos om a 91 neglec ed ho se s able and nea o a child en school. On he o he hand, hi y- h ee leas 92 isola ed om one Andean ox (Lycalopex culpaeus) and h ee Sou h Ame ican g ay 93 oxes (Lycalopex g iseus) om San a C uz (sou h o A gen ina) we e ob ained h ough 94 he assis ance o colleagues (see Acknowledgemen s). In addi ion, wo indi iduals o C. 95 elis collec ed om dogs (Canis lupus amilia is) om A gen ina we e molecula ly 96 s udied only o compa a i e pu poses. 97 Fleas ob ained we e kep in Eppendo ubes wi h 70 % e hanol un il equi ed o 98 subsequen iden i ica ion and sequencing (Table 1). 99 Mo phological iden i ica ion 100 Flea specimens collec ed by us (Spain) we e classi ied by ou sel es whe eas hose leas 101 p o ided by ou colleagues (A gen ina) we e classi ied i s ly by hem (see 102 Acknowledgemen s) and hen mo phologically compa ed wi h ou specimens in ou 103 labo a o y. Fi s o all o mo phological analysis, whole specimens we e examined 104 unde op ical mic oscope, whe eas, lea legs we e cu o in o de o ca y ou he 105 pos e io DNA ex ac ion. Secondly, es o body p ese ed in ials wi h 70 % e hanol 106 we e clea ed wi h KOH, dehyd a ed in a g owing se ies o alcohol, diaphanized in 107 eugenol and moun ed on pe manen slides in Canada balsam o EUKITT o hei 108 de ailed examina ion unde op ic mic oscope. Pho og aphs we e aken by using a 109 Mic oscope Olympus BX51 equipped wi h Pho og aphic Came a Olympus DP71. 110 Diagnos ic mo phological cha ac e s o P. i i ans we e s udied by compa ing wi h 111 igu es, keys and desc ip ions gi en in Hopkins & Ro hschild (1953), Ba e a (1955), Page 5 o 49 Medical and Ve e ina y En omology Fo Re iew Only 112 Smi (1958) and Beaucou nu & Launay (1990). A e mo phological iden i ica ion, en 113 specimens om A gen ina (se en emales and h ee males) and ou een specimens 114 om Spain (six males and eig h emales) we e measu ed acco ding o i een di e en 115 pa ame e s (Table 2). Desc ip i e uni a ia e s a is ics (a i hme ic means, s anda d 116 de ia ions, and coe icien o a ia ion) o all pa ame e s we e de e mined o wo 117 popula ions (Spain and A gen ina) using IBM® SPSS® S a is ics p og am e sion 118 24.0.0.0 (Pa do & Ruiz, 2002). Fu he mo e, mo phome ic da a was explo ed using 119 mul i a ia e analysis in i e measu emen s (HL, HW, PROL, MESL, and METL) 120 (Table 2) by he p incipal componen analysis (PCA), a echnique o summa izing mos 121 o he a ia ion in a mul i a ia e da ase in ew dimensions (Rohl & Ma cus, 1993; 122 Klingenbe g, 1996; Duja din & Le Pon , 2004). The analyses we e ca ied ou using he 123 BAC .2 so wa e (Duja din, 2002; Vale o e al., 2009). 124 Molecula s udy 125 To al DNA was ex ac ed om leas using lea legs by he DNeasy Blood and Tissue 126 Ki (Qiagen) acco ding o he manu ac u e ’s p o ocol. Then, genomic DNA was 127 checked using an elec opho esis in 0.8 % aga ose gel elec opho esis in used wi h 128 SYBR Sa e. 129 All molecula ma ke s sequenced in his s udy (ITS1 and ITS2 DNA, cox1 and cy b 130 m DNA) we e ampli ied by polyme ase chain eac ion (PCR) using a he mal cycle 131 (Eppendo AG). PCR mix, PCR condi ions and PCR p ime s a e summa ized in Table 132 S1. The ITS1, ITS2, cox1 and cy b pa ial gene sequences ob ained om P. i i ans 133 om he wo geog aphical a eas we e deposi ed in GenBank da abase (Table 1). In 134 o de o compa e wi h o he Pulicidae species, i we e sequenced and analysed ITS1 and Page 6 o 49Medical and Ve e ina y En omology Fo Re iew Only 135 ITS2 DNA and cox1 and cy b m DNA pa il genes o C. elis isola ed om dogs (C. l. 136 amilia is) om La Pla a (A gen ina). 137 The PCR p oduc s we e checked on SYBR Sa e s ained 2 % T is–Bo a e–EDTA (TBE) 138 aga ose gels. Bands we e elu ed and pu i ied om he aga ose gel by using he QWiza d 139 SV Gel and PCR Clean-Up Sys em Ki (P omega). Once pu i ied, he p oduc s we e 140 sequenced by S ab Vida (Po ugal). To ob ain a nucleo ide sequence alignmen ile, we 141 used MUSCLE alignmen me hod (Edga , 2004) by he MEGA p og am e sion 5.2 142 (Tamu a e al., 2011). The ITS1 DNA in aindi idual a ia ion was de e mined by 143 sequencing ou o se en clones o wo specimens om Spain and one specimen om 144 A gen ina. The PCR p oduc s we e elu ed om he aga ose gel using he WIZARD® 145 SV Gel and PCR Clean-Up Sys em (P omega) and ans o ma ion was ca ied ou as 146 ci ed by Cu illas e al. (2009). Plasmids we e pu i ied using a Wiza d Plus SV 147 (P omega) and sequenced by S ab Vida (Po ugal) wi h an uni e sal p ime (M13). 148 A es ic ion map o he cox1 sequences o P. i i ans om Spain and A gen ina was 149 cons uc ed using The Sequence Manipula ion Sui e (S o ha d, 2000; a ailable a 150 h p://www.bioin o ma ics.o g/sms2/ es _map.h ml) in o de o iden i iy ce ain 151 endonucleases which could disc imina e be ween bo h geog aphical o igins by PCR- 152 linked andom- agmen -leng h polymo phism (RFLP). 153 In o de o assess he simila i y among all ma ke sequences o P. i i ans ob ained in 154 his s udy and o he Pulicidae species, we analyzed he numbe o base di e ences pe 155 sequence om be ween sequences s udied using no. o di e ences me hod o MEGA 5 156 p og am e sion 5.2 (Tamu a e al., 2011). The p og am DOTMATCHER om he 157 Eu opean Molecula Biology Open So wa e Sui e package (Rice e al., 2000) was used 158 o ind epea s wi hin he ITS1 sequences. Page 7 o 49 Medical and Ve e ina y En omology Fo Re iew Only 159 Phylogene ic ees we e in e ed using nucleo ide da a and pe o med using wo 160 me hods: Maximum Likelihood (ML) and Bayesian in e ences (B). Maximum 161 Likelihood ees we e gene a ed using he PHYML package om Guindon & Gascuel 162 (2003), whe eas Bayesian in e ences we e gene a ed using M Bayes-3.2.6 (Ronquis & 163 Huelsenbeck 2003). JMODELTEST (Posada 2008) p og am was used o de e mina e 164 he bes - i subs i u ion model o he pa asi e da a (ITS2, cox1 and cy b). Models o 165 e olu ion we e chosen o subsequen analyses acco ding o he Akaike In o ma ion 166 C i e ion (Huelsenbeck & Rannala 1997; Posada & Buckley, 2004). Fo he s udy o he 167 da ase con aining he conca ena ion o h ee ma ke s (ITS2, cox1 and cy b), analyses 168 based on BI we e pa i ioned by gene and models o indi idual genes wi hin pa i ions 169 we e hose selec ed by jModel es . Fo ML in e ence, bes - i nucleo ide subs i u ion 170 models included gene al ime- e e sible model wi h gamma-dis ibu ed a e a ia ion 171 GTR+G (ITS2) and gene al ime- e e sible model wi h gamma-dis ibu ed a e 172 a ia ion and a p opo ion o in a iable si es, GTR+I+G (cox1 and cy b). Suppo o he 173 opology was examined using boo s apping (heu is ic op ion) (Felsens ein 1985) o e 174 1000 eplica ions o assess he ela i e eliabili y o clades. The commands used in 175 M Bayes-3.2.6 o BI we e ns =6 wi h gamma a es (ITS2) and ns =6 wi h in gamma 176 a es (cox1 and cy b). Fo BI, he s anda d de ia ion o spli equencies was used o 177 assess i he numbe o gene a ions comple ed was su icien ; he chain was sampled 178 e e y 500 gene a ions and each da ase was un o 10 million gene a ions. Adequacy o 179 sampling and un con e gence we e assessed using he e ec i e sample size diagnos ic 180 in TRACER p og am e sion 1.6 (Rambau & D ummond, 2007). T ees om he i s 181 million gene a ions we e disca ded based on an assessmen o con e gence. Bu n-in 182 was de e mined empi ically by examina ion o he log likelihood alues o he chains. 183 The Bayesian Pos e io P obabili ies (BPP) a e pe cen age con e ed. Page 8 o 49Medical and Ve e ina y En omology Fo Re iew Only 184 The phylogene ic analyses, based on ITS2, cox1 and cy b m DNA sequences we e 185 ca ied ou using ou sequences and hose ob ained om GenBank da abase (appendix 186 1). Phylogene ic ees based on ITS2, cox1, cy b m DNA and conca ena ed (ITS2, cox1 187 and cy b) sequences we e oo ed including ou g oup species ep esen ing membe s o 188 he O de Mecop e a: Pano pa me idionalis. This choice was based on he he 189 combina ion o mo phological and molecula da a ob ained in o me s udies which 190 p o ided compelling e idences o a sis e g oup ela ionship be ween Mecop e a and 191 Siphonap e a (Whi ing, 2002; Whi ing e al., 2008). ITS1 sequence o P. me idionalis 192 o o he species o Mecop e a was no a ailable nei he by ampli ica ion o di e en 193 indi iduals no in any public da abase. Thus, no phylogene ic ee wi h o he 194 Siphonap e a species based on ITS1 sequences was cons uc ed, as well as his 195 molecula ma ke was disca ded o he conca ena ed da ase . The selec ion o lea axa 196 o he conca ena ed phylogene ic ee was limi ed o lea species whose ITS2, cox1 and 197 cy b sequences we e a ailable on GenBank da abase. 198 NETWORK ( 5.0.0.1) was used o c ea e in e -popula ion median-joining ne wo ks 199 (Bandel e al., 1999; a ailable a h p://www. luxus-enginee ing.com), o isualize he 200 e olu iona y ela ionships be ween cox1 and cy b haplo ypes. This app oach has been 201 shown o yield he bes - esol ed genealogies ela i e o o he oo ing and ne wo k 202 p ocedu es (Cassens e al., 2003). Page 9 o 49 Medical and Ve e ina y En omology Fo Re iew Only 347 Because o mo phological specializa ions, highly p omiscuous leas species such as P. 348 i i ans o C. elis which occu s on a wide a ie y o Ca ni o a, could show high le els 349 o gene ic a iabili y especially when we assess popula ions which pa asi ize di e en 350 hos s o hey a e se led in di e en geog aphical a eas. Van de Mesch e al. (2015) 351 sugges ed ha he hos speci ici y migh in luence he le el o in aspeci ic gene ic 352 di e gence since mo e gene alis pa asi e species will show a highe le el o 353 in aspeci ic gene ic a ia ion enabling hem o in es a b oade hos ange. This ac has 354 been ecen ly demons a ed by Ho nok e al. (2018) who ound high mi ochond ial 355 sequence di e gence in some synan h opic lea species such as C. elis o P. i i ans. 356 In he p esen s udy, mo phome ic da a showed sligh di e ences be ween P. i i ans 357 specimens om Spain and A gen ina. This esul was co obo a ed by PCA appea ing 358 he Spanish adul s o P. i i ans sligh ly bigge . This ac could be explained acco ding 359 o he di e en geog aphical o igins and/o di e en hos s. This is in ag eemen wi h 360 Med ede (1998) who cha ac e ized he human lea as a mono ypic axon, being 361 ine icien adi ional and classic mo phological me hods in sepa a ing i s popula ion 362 g oups. In ou s udy, mo phome ic esul s did no co espond wi h molecula and 363 phylogene ic ones since hese showed a high deg ee o nucleo ide di e gence be ween 364 indi iduals om bo h geog aphical o igins. Ou esul s a e in ag eemen wi h Ho nok e 365 al. (2018) who did no obse e mo phological di e ences among P. i i ans specimens 366 isola ed om humans and wild ca ni o es (badge , jackal and ox) om Hunga y and 367 C oa ia; howe e , hese au ho s ound a conside able deg ee o molecula di e gence 368 be ween bo h popula ions based on mi ochond ial ma ke s. These esul s disag ee wi h 369 K asno e al. (2015) who suppo ed he idea ha he p ocess o hos selec ion by leas 370 is de e mined by ecip ocal ela ionships be ween hos ai s and lea ai s. Thus, lea 371 species wi h simila ai s, independen ly o hei phylogene ic a ini ies, we e ound on Page 16 o 49Medical and Ve e ina y En omology Fo Re iew Only 372 he same hos species mo e o en han expec ed by chance om he en i e pool o lea 373 species. In his sense, u u e mo phome ic s udies o P. i i ans om di e en hos s 374 and con inen s would be necessa y. 375 The In e nal T ansc ibed Space 1 and 2 ibosomal DNA (ITS1 and ITS2) ha e been 376 shown o be wo o he bes molecula ma ke s o analyze gene ic ela ionships a he 377 species le el in a h opods (Monje e al., 2013; Zagoskin e al., 2014). 378 A he p esen wo k, we obse ed ha ITS2 sequences o P. i i ans we e ma kedly 379 sho e han ITS1 sequences, which has al eady been no iced in o he lea species such 380 as C. elis, S enoponia ipec ina a ipec ina a, C. canis and N. ascia us (Vobis e al., 381 2004, Zu i a e al., 2015; 2016 and 2018). Fu he mo e, he ITS1 DNA egions 382 e ealed a conside able leng h a ia ion be ween bo h geog aphical popula ion caused 383 by a long epe i i e egion o 86 bp leng h ha appea ed wice and once in specimens 384 collec ed om Spain. In e nal epea s in he ITS space s a e usual and ha e been 385 equen ly desc ibed. This ac ha e al eady no iced in leas by Game schlag e al. 386 (2008) who epo ed he exis ence o leng h di e ences be ween he ITS1 DNA o he 387 A ican and he Sou h Ame ican T. pene ans popula ions caused by he numbe o 388 epea s o a epe i i e egion o 99 bp. Fu he mo e, hese au ho s de ec ed epe i i e 389 sequences wi hin he ITS1 DNA egion o o he lea species such as C. elis, 390 Echidnophaga gallinacea, P. i i ans, Spilopsyllus cuniculi, and X. cheopis, 391 highligh ing ha hese epe i i e elemen s could se e as a aluable ool o 392 phylogeog aphic s udies. Ou s udy also ag ees wi h Gha ami e al. (2018) who ound 393 h ee epea ed uni s wi h a leng h o 98-99 bp and a andemly epea ed sequence wi hin 394 he ITS1 o P. i i ans popula ions isola ed om Khodabandeh and Mahneshan (I an). 395 Thus, hese au ho s sugges ed ha he di e en numbe and size o epe i i e uni s in 396 ITS1 may be he sign o de eloped ai s es ablishing plesiomo phic cha ac e s among Page 17 o 49 Medical and Ve e ina y En omology Fo Re iew Only 397 di e en popula ions. In P. i i ans, he numbe o hese uni s depends on ecological 398 condi ions (McKe n e al., 2008; Game schlag e al. 2008); u he mo e, in con as o 399 o he au ho s such as Vobis e al. (2004), Game schlag e al. (2008), Ma ugal e al. 400 (2013) and Zu i a e al. (2015) who no obse ed g ea ITS sequences di e ences 401 among se e al popula ions belonging o he same lea species, we ound high alues o 402 in aspeci ic a ia ion be ween bo h geog aphical popula ions in P. i i ans, especially 403 in ITS1 sequences (In aspeci ic simila i y anged om 95.9 % o 96.3 %). The 404 exis ence o wo gene ic lineages (Spain and A gen ina) was co obo a ed by ITS2 405 phylogene ic ee, hus bo h popula ions clus e ed sepa a ely based on hei 406 geog aphical o igin. Addi ionally, when ITS1 sequences o di e en specimens o P. 407 i i ans isola ed om di e en geog aphical a eas we e compa ed he highes alues o 408 nucleo ide di e gence we e obse ed in specimens om Nea c ic and Neo opical a eas 409 (Uni ed S a es and A gen ina), whe eas almos no di e ences we e obse ed among 410 indi iduals om Palea c ic and A o opical egions (Spain and Came oon) (Table 3). 411 These da a migh sugges a possible Ame ican o igin o his lea species since ances al 412 popula ions usually exhibi highe gene ic di e si y alues compa ed o ecen 413 popula ions ha ha e expanded in o no el e i o ies (Sa olainen e al., 2002). 414 Cox1 and cy b ma ke s ha e been used in lea s udies in he las i een yea s wi h 415 se e al pu poses. In o de o assess he phylogeog aphic s uc u e o ce ain popula ions 416 (Di ma & Whi ing, 2003), o s udy he phylogene ic di e si y o some species 417 (Law ence e al., 2014), o ca y ou a molecula cha ac e iza ion o ce ain species 418 (Zu i a e al., 2015; Zu i a e al., 2016) o e en o he econs uc ion o ances al hos 419 a ilia ion and biogeog aphic his o y o leas (Zhu e al., 2015). A he p esen s udy, we 420 ampli ied cox1 pa ial gene. The ob ained sequences showed a low alue o in aspeci ic 421 simila i y wi hin P. i i ans om Spain and A gen ina (91.5 % - 92 %), in con as o Page 18 o 49Medical and Ve e ina y En omology Fo Re iew Only 422 he high alues o simila i y obse ed o specimens om he same popula ion (> 99 423 %). Zu i a e al. (2016) obse ed alues o simila i y a ound 97 % be ween wo 424 congene ic species o leas (C. elis and C. canis) collec ed om di e en geog aphical 425 a eas. Recen ly, Ho nok e al. (2018) based on cox1 sequences o P. i i ans om 426 di e en hos s, obse ed wo di e ged mi ochond ial lineages be ween C oa ia and 427 Hunga y. The e o e, hese au ho s claimed abou he necessi y o ca y ou 428 supplemen a y s udies using a la ge scale sampling o P. i i ans om di e en hos s 429 and geog aphical a eas o conclude in his con ex . In ou s udy, he compa a i e s udy 430 o cox1 sequences o P. i i ans isola ed om di e en geog aphical egion showed he 431 lowes alues o nucleo ide di e gence among Palea c ic and Aus alian specimens, 432 whe eas, likewise ITS1 analysis, he highes alues o nucleo ide di e gence we e 433 obse ed when hese specimens we e compa ed wi h A gen inean popula ion 434 (Neo opical) (Table 4). These esul s would suppo he idea ha his species had a 435 Sou h Ame ican o igin. His o ically, DNA ba coding s udies on insec s and 436 in e eb a es ha e shown maximum in aspeci ic a ia ion anging om 3 o 3.9 % 437 (Ca ew e al. 2007). This high deg ee o polymo phism o in aspeci ic analysis could 438 be explained a ending o wide geog aphical locali ies whe e he samples we e 439 collec ed. Indeed, in ce ain g oups, such as amphibians, when se e al indi iduals o he 440 same species come om dis an geog aphical egions, in aspeci ic a ia ion can exceed 441 he in e speci ic a ia ion obse ed be ween species o he same genus, making i 442 di icul o he delimi a ion o species wi h only he sequence o cox1 (Vences e al. 443 2005). Howe e , in ou s udy, cy b sequence analysis con i med he exis ence o wo 444 highly di e gen mi ochond ial lineages wi hin P. i i ans (Spain and A gen ina) 445 e o ced by ibosomal esul s. Al hough we obse ed a high pe cen age o 446 in apopula ion simila i y in bo h geog aphical o igins (bi lowe in A gen ina) based on Page 19 o 49 Medical and Ve e ina y En omology Fo Re iew Only 447 cy b sequences, we no iced g ea e nucleo ide a iabili y in A gen ina han in Spain 448 wi h he exis ence o a highe numbe o haplo ypes in he Sou h Ame ican a ea. In his 449 case in aspeci ic simila i y obse ed (90 % o 92.2 %) we e simila o e en lowe han 450 hose obse ed be ween wo di e en congene ic species such as C. elis and C. canis 451 (90.6 %) o Xenopsylla sk jabini and Xenopsylla con o mis (92.5 %) (Table 5). These 452 high deg ees o m DNA in aspeci ic a iabili y could be explained by he ac ha 453 gene alis lea shows conside ably mo e in aspeci ic gene ic a ia ion han hos - 454 speci ic lea species. Fo a gene alis pa asi e, g ea e le els o gene ic a iabili y can 455 p o ide e olu iona y po en ial o local hos ace o ma ion (Gómez-Díaz e al., 2007). 456 P e ious examples ha e been epo ed o icks and lice pa asi izing sympa ic hos s 457 (McCoy e al. 2001; Johnson e al. 2002). Fo his eason, we analyzed he cox1 and 458 cy b in aspeci ic simila i y be ween Spania d and A gen inean popula ions o ano he 459 gene alis lea like C. elis, bu , su p isingly, we did no obse e di e ences be ween 460 hem. Cox1, cy b and conca ena ed phylogene ic ees ein o ced he idea o he 461 exis ence o wo geog aphical gene ic lineages wi hin P. i i ans. Thus, cox1 462 phylogene ic ee showed specimens collec ed om Palea c ic and Aus alian egion 463 (Spain, C oa ia, China, Hunga y and New Zealand) clus e ed oge he in he same clade 464 and sepa a ed om indi iduals collec ed om Neo opical egion (A gen ina). 465 Fu he mo e, hese esul s show no signi ican hos dependency since specimens 466 collec ed om Palea c ic and Aus alian a eas we e isola ed om di e en hos s (see 467 appendix 1). Likewise, cox1, cy b and conca ena ed phylogene ic ees showed wo well 468 suppo ed subclades wi hin P. i i ans based on geog aphical o igins (Spain and 469 A gen ina). 470 The phylogene ic analysis ca ied ou on he basis on ibosomal and mi ochond ial 471 DNA molecula ma ke s sugges s he exis ence o wo gene ic lineages (A gen ina, Page 20 o 49Medical and Ve e ina y En omology Fo Re iew Only 472 Sou h Ame ica and Spain, Eu ope) o P. i i ans popula ions and he minimum 473 spanning ne wo k showed all he cy b haplo ypes om A gen ina clus e ed oge he and 474 wi h s a -like pa e n a ound H2 haplo ype. Based on coalescen heo y (Sla kin & 475 Hudson, 1991) his s a opology showed ha P. i i ans popula ions had expe ienced a 476 signi ican popula ion expansion. A he cen e o he ne wo k is haplo ype 2, which 477 akes o e he highes p opo ion in he popula ion. This sugges s ha he haplo ype 2 478 should be he ances al haplo ype. This highe gene ic di e si y in A gen ina would 479 ein o ce he idea sugges ed by Buckland & Sadle (1989) ha P. i i ans, in con as 480 wi h o he human ec opa asi es, could ha e a Sou h Ame ican o igin eaching Wes e n 481 Palea c ic a ea h ough Be ingian and Asia ic ou es, a any ime du ing he Pos glacial. 482 This ac oge he wi h a ep oduc i e isola ion, could ha e o igina ed he exis ence o 483 wo c yp ic species wi hin P. i i ans. In spi e o ha , o con i m a possible Sou h 484 Ame ican o igin o his species, mo e axonomic, phylogene ic and phylogeog aphic 485 s udies o P. i i ans pa asi izing di e en hos s om di e en geog aphical a eas a e 486 needed. 487 In conclusion, he p esen s udy p o ides o he i s ime, compa a i e mo phome ic 488 and molecula da a o P. i i ans collec ed om Spain and A gen ina (Palea c ic and 489 Neo opical a eas). On he basis on mo phome ic esul s, we ound sligh di e ences 490 be ween bo h popula ions. Al hough we only assessed wo popula ions o P. i i ans in 491 his s udy, ou esul s could sugges he hypo hesis ha his lea species, in con as o 492 o he gene alis leas, main ain a ce ain deg ee o mo phological simila i y, a leas 493 be ween Wes e n Palea c ic and Neo opical a eas. Fu he mo e, based on molecula 494 and phylogene ic da a ob ained in his wo k we p o ided he exis ence o wo well 495 de ined geog aphical gene ic lineages wi hin P. i i ans species sugges ing he exis ence 496 o wo c yp ic species which could be disc imina ed by PCR-linked RFLP. Page 21 o 49 Medical and Ve e ina y En omology Fo Re iew Only 497 Acknowledgemen 498 The p esen wo k was suppo ed by a g an o he V Plan P opio de In es igación o he 499 Uni e si y o Se ille, Spain. 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(2017) New 572 eco d o a pho e ic lea associa ed wi h ea wigs (De map e a, A ixeniidae) and a 573 edesc ip ion o he ba lea Laga opsylla signa a (Siphonap e a, Ischnopsyllidae). 574 ZooKeys, 657, 67–69. 575 Hopkins, G.H.E. & Ro hschild, M. (1953) An Illus a ed Ca alogue o he Ro hschild 576 Collec ion o Fleas in he B i ish Museum (Na . His .). Vol. I. Tungidae and Pulicidae. 577 Camb idge Uni e si y P ess, Camb idge, UK. 578 Hopla, C. E. (1980) A s udy o he hos associa ions and zoogeog aphy o Pulex, pp. 579 185–207. In R. T aub and H. S a cke (eds.), In P oceedings o he In e na ional 580 Con e ence on Fleas Lewis, R. E., J. H. Lewis, & C. Mase . (1988) The leas o he 581 Paci ic No hwes . O egon S a e Uni e si y P ess, Co allis, OR.n Fleas, 1977, 582 Pe e bo ough, UK. A.A. Balkema, Ro e dam. 583 Ho nok, S., Beck, R., Fa kas, R., G ima, A., O an o, D., Kon schán, J., Takács, N., 584 Ho á h, G., Szőke, K., Szeke es, S., Majo os, G., Juhász, A., Salan , H., Ho mann- Page 25 o 49 Medical and Ve e ina y En omology Fo Re iew Only 716 Fig. 2. Fac o map co esponding o Pulex i i ans adul s om Spain and Sou h 717 Ame ica. Samples a e p ojec ed on o he i s (PC1, 71 %) and second (PC2, 14 %) 718 p incipal componen s. Each g oup is ep esen ed by i s pe ime e . 719 Fig. 3. Alignmen o he pa cial ITS1 DNA sequences o Pulex i i ans om Spain and 720 A gen ina. In ed and g een bold ype he 86 bp epea uni . Ve ical box in blue bold 721 ype indica es he polymo phic si es. Gaps gene a ed by alignmen (ma ked by a dash). 722 Fig. 4. Phylogene ic ee o Pulex i i ans om di e en geog aphical o igins (see Table 723 1) based on pa ial cy och ome c-oxidase 1 (cox1) gene o mi ochond ial DNA 724 sequences using he Bayesian (B) and Maximum Likelihood (ML) me hods and 725 Bayesian opology. The pe cen age o eplica e ees in which he associa ed axa 726 clus e ed oge he in he boo s ap es (1,000 eplica es) is shown on he b anches 727 (B/ML). Boo s ap alues lowe han 60 % a e no shown. The Bayesian Pos e io 728 P obabili ies (BPP) is pe cen age con e ed. 729 Fig. 5. Phylogene ic ee o Pulex i i ans om di e en geog aphical o igins (see Table 730 1) based on pa ial cy och ome b (cy b) gene o mi ochond ial DNA using he Bayesian 731 (B) and Maximum Likelihood (ML) me hods and Bayesian opology. The pe cen age o 732 eplica e ees in which he associa ed axa clus e ed oge he in he boo s ap es (1,000 733 eplica es) is shown on he b anches (B/ML). Boo s ap alues lowe han 60 % a e no 734 shown. The Bayesian Pos e io P obabili ies (BPP) is pe cen age con e ed. 735 Fig. 6. A minimum spanning ne wo k cons uc ed using 20 haplo ypes o mi ochond ial 736 cy b pa ial gene sequences o Pulex i i ans. The sizes o he ci cles a e p opo ional o 737 he numbe o haplo ypes ep esen ed and he numbe s co espond o he mu a ional 738 s eps obse ed be ween haplo ypes. H1 (2): P. i i ans om A gen ina; H2 (3): P. 739 i i ans om A gen ina; H3 (1): P. i i ans om A gen ina; H4 (1) P. i i ans om Page 32 o 49Medical and Ve e ina y En omology Fo Re iew Only 740 A gen ina; H5 (1): P. i i ans om A gen ina; H6 (1): P. i i ans om A gen ina; H7 741 (1): P. i i ans om A gen ina; H8 (10): P. i i ans om Spain; H9 (1): P. i i ans om 742 Spain. 743 Fig. 7. Phylogene ic ee o Pulex i i ans om di e en geog aphical o igins (see Table 744 1) based on conca ena ed In e nal T ansc ibed Space 2 (ITS2), pa ial cy och ome c- 745 oxidase 1 (cox1) and cy och ome b (cy b) gene o mi ochond ial DNA in e ed using he 746 Bayesian (B) and Maximum Likelihood (ML) me hods and Bayesian opology. The 747 pe cen age o eplica e ees in which he associa ed axa clus e ed oge he in he 748 boo s ap es (1,000 eplica es) is shown on he b anches. The Bayesian Pos e io 749 P obabili ies (BPP) a e pe cen age con e ed. 750 Fig. S1. Phylogene ic ee o Pulex i i ans om di e en geog aphical o igins (see 751 Table 1) based on he In e nal T ansc ibed Space 2 (ITS2) sequences using he 752 Bayesian (B) and Maximum Likelihood (ML) me hods and Bayesian opology. The 753 pe cen age o eplica e ees in which he associa ed axa clus e ed oge he in he 754 boo s ap es (1,000 eplica es) is shown on he b anches (B/ML). Boo s ap alues 755 lowe han 60 % a e no shown. The Bayesian Pos e io P obabili ies (BPP) is 756 pe cen age con e ed. Page 33 o 49 Medical and Ve e ina y En omology Fo Re iew Only ITS1 Loca ion/Coun y/Sample ID Species/Gende Hos Numbe o leas Base pai s (bp) Accession numbe Se ille/Spain/PI1 P. i i ans /1♂ - 1 962 LT797452 Se ille/Spain/PI1 (Clone 3) P. i i ans - - 968 LT853871 Se ille/Spain/PI1 (Clone 1-2, 4) P. i i ans - - 968 LT853872 Se ille/Spain/PI2 P. i i ans /1♀ - 1 876 LT797453 Se ille/Spain/PI4 P. i i ans /1♀ - 1 876 LT797454 Se ille/Spain/PI4 (Clone 2) P. i i ans - - 882 LT853866 Se ille/Spain/PI4 (Clone 3) P. i i ans - - 882 LT853867 Se ille/Spain/PI4 (Clone 5) P. i i ans - - 882 LT853869 Se ille/Spain/PI4 (Clone 6-7) P. i i ans - - 881 LT853868 Se ille/Spain/PI4 (Clone 4, 8) P. i i ans - - 882 LT853870 Se ille/Spain/PI5 P. i i ans /1♀ - 1 876 LT797455 Se ille/Spain/PI6 P. i i ans /1♂ - 1 962 LT797456 Se ille/Spain/PI7 P. i i ans /1♀ - 1 876 LT797457 Se ille/Spain/PI8 P. i i ans /1♀ - 1 876 LT797458 Se ille/Spain/PI10 P. i i ans /1♂ - 1 876 LT797459 Se ille/Spain/PI11 P. i i ans /1♀ - 1 876 LT797460 Se ille/Spain/PI12 P. i i ans /1♂ - 1 962 LT797461 Se ille/Spain/PI13 P. i i ans /1♀ - 1 876 LT797462 Se ille/Spain/PI14 P. i i ans /1♂ - 1 876 LT797463 San a C uz/A gen ina/ PI26-35 P. i i ans/6♂ 5♀ L. culpaeus and L. g iseus 10 796 LT797464 San a C uz/A gen ina/PI32 (Clone 1) P. i i ans L. culpaeus and L. g iseus - 796 LT853873 San a C uz/A gen ina/PI32 (Clone 3) P. i i ans L. culpaeus and L. g iseus - 796 LT853874 San a C uz/A gen ina/PI32 (Clone 2, 6, 8) P. i i ans L. culpaeus and L. g iseus - 796 LT853875 La Pla a/A gen ina/668, 670 C. elis/2♀ Canis lupus amilia is 2 668 LT853877 ITS2 Loca ion/Coun y/Sample ID Species/Gende Hos Numbe o leas Base pai s (bp) Accession numbe Se ille/Spain/PI1 P. i i ans /1♂ - 1 322 LT797448 Se ille/Spain/PI6 P. i i ans /1♂ - 1 322 LT797449 Se ille/Spain/ PI2,4,5,7,8,10-14 P. i i ans/3♂ 7♀ - 10 322 LT797450 San a C uz/A gen ina/ PI26-35 P. i i ans/6♂ 5♀ L. culpaeus and L. g iseus 10 324 LT797451 La Pla a/A gen ina/668, 670 C. elis/2♀ Canis lupus amilia is 2 327 LT853876 Cox1 Loca ion/Coun y/ID Species/Gende Hos Numbe o leas Base pai s (bp) Accession numbe /Haplo ype Se ille/Spain/PI2 P. i i ans /1♀ - 1 658 LT797468/H4 Se ille/Spain/PI6 P. i i ans /1♂ - 1 658 LT797469/H5 Se ille/Spain/PI1, 4-5, 7-8, 10-14 P. i i ans/4♂ 6♀ - 10 658 LT797470/H6 San a C uz/A gen ina/ PI30 P. i i ans /1♀ L. culpaeus 1 658 LT797465/H1 San a C uz/A gen ina/ PI33 P. i i ans /1♂ L. g iseus 1 658 LT797466/H2 San a C uz/A gen ina/ PI26, 27, 35 P. i i ans /3♀ L. culpaeus 3 658 LT797467/H3 La Pla a/A gen ina/668 C. elis/2♀ Canis lupus amilia is 2 601 LT853879 Cy b Loca ion/Coun y/ID Species/Gende Hos Numbe o leas Base pai s (bp) Accession numbe /Haplo ype Se ille/Spain/PI2 P. i i ans /1♀ - 1 374 LT797473/H9 Se ille/Spain/PI1, PI4-8, PI10-11, PI13-14 P. i i ans/4♂ 6♀ - 10 374 LT797474/H8 San a C uz/A gen ina/PI26, PI32 P. i i ans /2♀ L. culpaeus 2 374 LT797475/H1 San a C uz/A gen ina/PI27, PI29, PI35 P. i i ans /3♀ L. culpaeus 3 374 LT797476/H2 San a C uz/A gen ina/ PI28 P. i i ans /1♀ L. culpaeus 1 374 LT797477/H3 San a C uz/A gen ina/ PI30 P. i i ans /1♀ L. culpaeus 1 374 LT797478/H4 San a C uz/A gen ina/ PI31 P. i i ans /1♀ L. culpaeus 1 374 LT797479/H5 San a C uz/A gen ina/ PI33 P. i i ans /1♂ L. g iseus 1 374 LT797480/H6 San a C uz/A gen ina/ PI34 P. i i ans /1♀ L. culpaeus 1 374 LT797481/H7 La Pla a/A gen ina/668, 670 C. elis/2♀ Canis lupus amilia is 2 374 LT853878 Table 1. GenBank accession numbe s o ITS1, ITS2 and pa ial cy b, cox1 gene sequences o indi iduals o P. i i ans and C. elis ob ained in his s udy. Page 34 o 49Medical and Ve e ina y En omology Fo Re iew Only Pulex i i ans (Spain) Pulex i i ans (A gen ina) MIN MAX X SD CV MIN MAX X SD CV TLF(mm) 1.8 2.1 1.9 0.1 5 1.7 2.5 2.1 0.2 9 TLM(mm) 1.3 2.1 1.8 0.3 17 1.4 1.8 1.6 0.2 12 TWF(mm) 1.1 1.2 1.1 0.1 9 1.0 1.3 1.1 0.1 9 TWM(mm) 0.8 1.2 1.0 0.2 20 0.7 0.9 0.8 0.1 12 HLF(µm) 398 597 458 40 8 352 469 416 38 9 HLM(µm) 380 498 418 47 11 322 387 354 46 13 HWF(µm) 234 293 259 25 9 205 264 237 22 9 HWM(µm) 205 293 250 32 12 176 234 205 41 20 EL(µm) 82 147 119 21 17 70 147 110 26 23 EW(µm) 53 59 57 3 5 47 64 57 6 10 CRL(µm) 26 40 34 5 14 28 33 30 4 13 DASL(µm) 82 110 97 10 10 80 94 87 10 11 PROL(µm) 87 152 111 16 14 70 137 100 24 24 MESL(µm) 106 167 135 19 14 88 152 124 25 20 METL(µm) 134 182 162 15 9 100 182 145 24 16 Table 2. Mo phome ic analysis o en specimens o P. i i ans om A gen ina (se en emales and h ee males) and ou een specimens o P. i i ans om Spain (six males and eigh emales) assessed in his s udy. Indi iduals om bo h sexes ha e been pooled in bo h popula ions. TLF = o al emale leng h, TLM = o al male leng h, TWF = o al emale wid h, TWM = o al male wid h, HLF = o al leng h o he emale head, HLM = o al leng h o he male head, HWF = o al wid h o he emale head, HWM = o al wid h o he male head, EL = o al leng h o he spe ma heca, EW = o al wid h o he spe ma heca, CRL = o al leng h o he ex ended egion o he c oche , DASL = o al leng h o he do sal aedeagal scle i e, PROL= o al leng h o he p o ho ax, MESL = o al leng h o he meso ho ax, METL = o al leng h o he me a ho ax, MAX = maximum, MIN = minimum, SD = s anda d de ia ion. X = a i hme ic mean, CV = Coe icien o Va ia ion (pe cen age con e ed). In black bold ype pa ame e s which showed ce ain di e ences be ween bo h geog aphical o igins. Page 35 o 49 Medical and Ve e ina y En omology Fo Re iew Only ITS1 P. i i ans om Spain LT797452- LT797463 P. i i ans om A gen ina LT797464 P. i i ans om Uni ed S a es GQ387496. Hos unknown P. i i ans om Came oon EU169198. Human hos P. i i ans om I an KX822017. Hos unknown P. i i ans om Spain LT797452- LT797463 99.5-100* P. i i ans om A gen ina LT797464 95.9-96.3 100* P. i i ans om Uni ed S a es GQ387496. Hos unknown 94.0-95.4 94.4 - P. i i ans om Came oon EU169198. Human hos 99.5-99.8 96.2 95.1 - P. i i ans om I an KX822017. Hos unknown 99.6-99.9 96.5 93.9 99.6 - Table 3. In apopula ion* and in aspeci ic simila i y obse ed amog all he ITS1 sequences o Pulex i i ans om di e en geog aphical a eas ob ained in his wo k and om Genbank da abase. Values a e gi en in pe cen ages. Page 36 o 49Medical and Ve e ina y En omology Fo Re iew Only COX1 PI/Se ille (Spain)/ LT797468-70 (H4-H6) PI/San a C uz (A gen ina)/ LT797465-67 (H1-H3) P. i i ans/Spain/ Badge / KF479246 P. i i ans/Spain/ human/ KF479247 P. i i ans/New Zealand/dog/ KY048351 P. i i ans/Hunga y/ ox/ MG668624 P. i i ans/Hunga y/ badge / MG668626 P. i i ans/Hunga y/ jackal/ MG668627 P. i i ans/China/ poleca / MF000666 P. i i ans/C oa ia/ human/ MG668622 C. elis/ A gen ina/ LT853879 C. elis/Spain/ LN827896 C. canis/ I an/LN827901 E.. gallinacea/ Aus alia/ JN008921 E.. ibe ica/ Spain/ KF479239 PI/Se ille (Spain)/LT797468- 70 (H4-H6) *99.6- 99.8 PI/San a C uz (A gen ina)/ LT797465-67 (H1-H3) 91.4-92.0 *99.2- 99.8 P. i i ans/Spain/badge / KF479246 96.0-96.2 92.8-93.0 - P. i i ans/Spain/human/ KF479247 99.5-99.8 91.8-92.2 96.4 - P. i i ans/New Zealand/dog KY048351 99.8-100 91.5-91.8 96.0 99.7 - P. i i ans/Hunga y/ ox/ MG668624 96.0-96.2 93.2-93.4 98.6 96.4 96.0 - P. i i ans/Hunga y/badge / MG668626 96.0-96.2 93.2-93.4 98.6 96.4 96.0 100 - P. i i ans/Hunga y/jackal/ MG668627 96.0-96.2 93.2-93.4 98.6 96.4 96.0 100 100 - P. i i ans/China/poleca / MF000666 99.6-99.8 91.4-91.8 96.2 99.4 99.8 96.2 96.2 96.2 - P. i i ans/C oa ia/human/ MG668622 99.4-99.6 91.2-91.6 95.6 99.2 99.6 95.6 95.6 95.6 99.4 - C. elis/A gen ina/ LT853879 85.7-86.0 84.9-85.2 85.9 85.9 85.9 87.3 87.3 87.3 86.5 86.3 - C. elis/Spain/LN827896 85.7-86.0 84.9-85.2 85.9 85.9 85.9 87.3 87.3 87.3 86.5 86.3 100 - C. canis/I an/LN827901 86.4-86.5 85.2 86.4 86.7 86.4 87.3 87.3 87.3 86.9 86.7 97.7 97.7 - E. gallinacea/ Aus alia/JN008921 87.9-88.0 87.5 88.9 88.0 87.9 88.5 88.5 88.5 88.3 88.1 85.7 85.7 86.0 - E.. ibe ica/Spain/KF479239 88.7-88.9 89.2-89.5 89.9 88.9 88.7 89.6 89.6 89.6 88.7 88.9 86.7 86.7 87.2 93.5 - Table 4. In apopula ion (*), in aspeci ic and in e speci ic simila i y obse ed among all he pa ial cox1 m DNA gene sequences o Pulex i i ans om di e en geog aphical a eas ob ained in his wo k and o he Pulicidae species om GenBank da abase. Values a e gi en in pe cen ages. (PI = Pulex i i ans). Page 37 o 49 Medical and Ve e ina y En omology Fo Re iew Only CYTB PI/Se ille (Spain)/ LT797473-74 (H8-H9) PI/San a C uz (A gen ina)/ LT797475-76 (H1-H7) A. e inacei/ Spain/ LT604120 A. e inacei/ Co se (F ance)/ LT627350 C. elis/Spain/ LN897470 C. elis/ A gen ina/ LT853878 C. canis/I an/ LN897471 X. con o mis/ KM890723 X. sk jabini/ KM890718 X. cheopis/ Cana y Islands/ LT604122 S. cuniculi/ KM890622 S. gi a di/ KM890686 E. oschanini/ KM890719 PI/Se ille (Spain)/ LT797473-74 (H8-H9) *99.7- 100 PI/San a C uz (A gen ina)/ LT797475-81 (H1-H7) 90.9-92.2 *97.8- 99.7 A. e inacei/Spain/ LT604120 84.9-85.2 82.5-83.6 - A. e inacei/Co se (F ance)/ LT627350 85.5-85.8 82.5-83.6 98.9 - C. elis/Spain/ LN897470 82.0-82.3 80.6-81.2 84.7 85.2 - C. elis/A gen ina/ LT853878 82.0-82.3 80.6-81.2 84.7 85.2 100 - C. canis/I an/ LN897471 82.3 79.8-80.4 85.5 86.0 90.6 90.6 - X. con o mis/ KM890723 80.1-80.4 78.2-78.8 82.0 82.5 83.9 83.9 83.6 - X. sk jabini/ KM890718 79.8-80.1 77.7-78.2 81.7 82.5 84.4 84.4 82.8 92.5 - X. cheopis/Cana y Islands/ LT604122 75.8 72.6-73.4 79.8 80.1 82.0 82.0 81.2 79.6 80.9 - S. cuniculi/ KM890622 81.5 81.7-82.3 82.3 82.8 83.1 83.1 83.9 82.3 81.7 80.1 - S. gi a di/ KM890686 80.6-80.9 78.5-79.8 83.6 84.7 84.9 84.9 84.1 84.9 82.8 80.1 78.5 - E. oschanini/KM890719 84.9-85.2 81.7-82.3 84.1 84.7 83.6 83.6 83.9 83.9 83.1 78.5 82.0 79.3 - Table 5. In apopula ion (*), in aspeci ic and in e speci ic simila i y obse ed among all he pa ial cy b m DNA gene sequences o Pulex i i ans om di e en geog aphical a eas ob ained in his wo k and o he Pulicidae species om GenBank da abase. Values a e gi en in pe cen ages. (PI = Pulex i i ans). Page 38 o 49Medical and Ve e ina y En omology Fo Re iew Only ITS1 ITS2 Cy b cox1 PCR Mix PCR Bu e (5X) 10 µl 10 µl 10 µl 10 µl dNTPs (10mM) 2 µl 1 µl 1 µl 1 µl MgCl2 (25 mM) 6 µl 6 µl 4 µl 4 µl Fo wa d P ime (10 M) 5 µl 5 µl 5 µl 5 µl Re e se P ime (10 M) 5 µl 5 µl 5 µl 5 µl Templa e DNA 5 µl 5 µl 5 µl 5 µl goTaq DNA polyme ase 0,5 µl 0,5 µl 0,5 µl 0,5 µl Au ocla ed dis illed wa e o 100 µl 50 µl 50 µl 50 µl PCR P ime s Fo wa d P ime NC5 (Gasse e al., 1996) senITS2 (Vobis e al., 2004) Cy bF (Di ma &Whi ing, 2003) LCO1490 (Folme e al., 1994) Re e se P ime ITS1 e (Ma ugal e al.., 2013) ITS2R (Luche i e al., 2007) A5F (Di ma &Whi ing, 2003) HCO2198 (Folme e al., 1994) PCR Condi ions Ini ial Dena u ing 94 ºC o 5´ 94 ºC o 5´ 95 ºC o 12´ 96 ºC o 2´ Numbe o cycles 35 35 30 40 Dena u ing 94 ºC o 30´´ 94 ºC o 60´´ 95 ºC o 30´´ 94 ºC o 30´´ Annealing 58 ºC o 30´´ 55 ºC o 60´´ 40 ºC o 30´´ 50 ºC o 30´´ P ime ex ension 72 ºC o 90´´ 72 ºC o 60´´ 68 ºC o 2´ 72 ºC o 60´´ Final ex ension 72 ºC o 5´ 72 ºC o 10´ 68 ºC o 7´ 72 ºC o 7´ Table S1. PCR mix, p ime s and condi ions used o each molecula ma ke sequenced in his s udy. Page 39 o 49 Medical and Ve e ina y En omology Fo Re iew Only Figu e 1 254x190mm (96 x 96 DPI) Page 40 o 49Medical and Ve e ina y En omology Fo Re iew Only Figu e 2 138x90mm (96 x 96 DPI) Page 41 o 49 Medical and Ve e ina y En omology Fo Re iew Only Appendix 1 Lis o axa used in he analysis, including GenBank accession numbe s and hos in o ma ion. Species Family Hos Accession numbe Gen Region Sequence leng h Pulex i i ans Pulicidae Homo sapiens EU169198 ITS1 929 Pulex i i ans Pulicidae Homo sapiens GQ387496 ITS1 948 Pulex i i ans Pulicidae Unknown KX822017 ITS1 1,208 Oph halmopsylla ki i schenkoi Lep opsyllidae Unknown GQ161960 ITS2 474 Oph halmopsylla ex ema Lep opsyllidae Unknown GQ161956 ITS2 466 Amphipsylla quad a oides quad a oides Lep opsyllidae Unknown AY072642 ITS2 497 Lep opsylla sp. Lep opsyllidae Unknown EF504221 ITS2 459 Lep opsylla sp. Lep opsyllidae Unknown EF504223 ITS2 449 Neopsylla siboi C enoph halmidae Unknown AF353113 ITS2 479 Neopsylla e a u a C enoph halmidae Unknown AF353122 ITS2 479 Neopsylla s e ensi C enoph halmidae Unknown AY337033 ITS2 479 Neopsylla specialis C enoph halmidae Unknown AF353120 ITS2 479 Xenopsylla cheopis Pulicidae Ra us sp. DQ295061 ITS2 356 Xenopsylla cheopis Pulicidae Ra us sp. DQ295059 ITS2 356 Xenopsylla cheopis Pulicidae Ra us sp. LT604121 ITS2 358 C enocephalides elis Pulicidae Canis lupus amilia is LN827903 ITS2 327 C enocephalides canis Pulicidae Canis lupus amilia is LN827905 ITS2 327 C enocephalides canis Pulicidae Canis lupus amilia is LN864485 ITS2 327 A chaeopsylla e inacei Pulicidae E inaceus eu opaeus LT703438 ITS2 360 A chaeopsylla e inacei Pulicidae E inaceus eu opaeus LT604114 ITS2 361 Tunga pene ans Tungidae Homo sapiens DQ844716 ITS2 471 Tunga pene ans Tungidae Homo sapiens DQ844724 ITS2 473 Tunga imamilla a Tungidae Unknown AY425820 ITS2 470 S enoponia ipec ina a ipec ina a S enoponiidae Mus musculus LK937042 ITS2 332 S enoponia ipec ina a ipec ina a S enoponiidae Mus musculus LK937039 ITS2 332 S enoponia ipec ina a ipec ina a S enoponiidae Mus musculus LK937038 ITS2 332 Ci ellophilus esquo um dze ysuensis Ce a ophyllidae Unknown EU770316 ITS2 332 Ci ellophilus esquo um al aicus Ce a ophyllidae Unknown EU770312 ITS2 332 Nospsyllus ascia us Ce a ophyllidae Apodemus syl a icus LT158059 ITS2 318 Nosopsyllus ascia us Ce a ophyllidae Mu idae LT158060 ITS2 318 Nosopsyllus ba ba us Ce a ophyllidae Ra us sp. LN881537 ITS2 318 Pano pa me idionalis Pano pidae - LT604124 ITS2 1,121 Echidnophaga gallinacea Pulicidae O yc olagus cuniculus JN008921 Cox1 650 Echidnophaga my mecobii Pulicidae O yc olagus cuniculus JN008919 Cox1 649 Echidnophaga ibe ica Pulicidae O yc olagus cuniculus KF479239 Cox1 658 Echidnophaga sp. Pulicidae Mammal JN008922 Cox1 654 Echidnophaga ambulans ambulans Pulicidae Tachyglossus aculea us KR363632 Cox1 601 Xenopsylla cunicula is Pulicidae O yc olagus cuniculus KF479238 Cox1 658 Pulex i i ans Pulicidae Meles meles KF479246 Cox1 658 Pulex i i ans Pulicidae Homo sapiens KF479247 Cox1 658 Pulex i i ans Pulicidae Canis lupus amilia is KY048351 Cox1 658 Pulex i i ans Pulicidae Jackal MG668627 Cox1 489 Pulex i i ans Pulicidae Badge MG668626 Cox1 489 Pulex i i ans Pulicidae Fox MG668624 Cox1 489 Pulex i i ans Pulicidae Homo sapiens MG668622 Cox1 489 Pulex i i ans Pulicidae Vo mela pe egusna MF000666 Cox1 672 Spilopsyllus cuniculi Pulicidae O yc olagus cuniculus KF479236 Cox1 658 Spilopsyllus cuniculi Pulicidae O yc olagus cuniculus KF479237 Cox1 658 A chaeopsylla e inacei Pulicidae E inaceus eu opaeus LT604116 Cox1 658 A chaeopsylla e inacei Pulicidae E inaceus eu opaeus LT604115 Cox1 658 A chaeopsylla e inacei Pulicidae E inaceus eu opaeus LT627349 Cox1 658 A chaeopsylla e inacei Pulicidae E inaceus eu opaeus LT703440 Cox1 658 C enocephalides elis Pulicidae Canis lupus amilia is LN827896 Cox1 600 Page 48 o 49Medical and Ve e ina y En omology Fo Re iew Only C enocephalides elis Pulicidae Canis lupus amilia is LT853879 Cox1 600 C enocephalides elis elis Pulicidae Felis ca us KF684891 Cox1 601 C enocephalides elis s ongylus Pulicidae Canis lupus amilia is KF684876 Cox1 601 C enocephalides o ien is Pulicidae Canis lupus amilia is KF684871 Cox1 601 C enocephalides canis Pulicidae Canis lupus amilia is KP684210 Cox1 658 C enocephalides canis Pulicidae Canis lupus amilia is LN827901 Cox1 600 S enoponia ipec ina a ipec ina a S enoponiidae Mus musculus LK937072 Cox1 677 S enoponia ipec ina a ipec ina a S enoponiidae Mus musculus LK937071 Cox1 677 S enoponia ipec ina a ipec ina a S enoponiidae Mus musculus LK937073 Cox1 677 Nosopsyllus ascia us Ce a ophyllidae C ocidu a ussula LT158040 Cox1 658 Nosopsyllus ascia us Ce a ophyllidae Apodemus syl a icus LT158041 Cox1 658 Nosopsyllus ba ba us Ce a ophyllidae Ra us sp LN881549 Cox1 658 Nosopsyllus ba ba us Ce a ophyllidae Ra us sp LN881550 Cox1 658 Pano pa me idionalis Pano pidae - LT604125 Cox1 658 Pano pa me idionalis Pano pidae - LT604126 Cox1 658 S enoponia ipec ina a ipec ina a S enoponiidae Mus musculus LN897473 Cy b 374 Oph halmopsylla p ae ec a p ae ec a Lep opsyllidae Unknown KM890714 Cy b 369 C enocephalides elis Pulicidae Canis lupus amilia is LN897470 Cy b 374 C enocephalides elis elis Pulicidae Unknown KM890759 Cy b 369 C enocephalides canis Pulicidae Canis lupus amilia is LN897471 Cy b 374 C enocephalides elis dama ensis Pulicidae Unknown KM890641 Cy b 369 Xenopsylla cheopis Pulicidae Ra us sp. LT604122 Cy b 374 A chaeopsylla e inacei e inacei Pulicidae Unknown KM890725 Cy b 369 A chaeopsylla e inacei Pulicidae E inaceus eu opaeus LT604120 Cy b 374 A chaeopsylla e inacei Pulicidae E inaceus eu opaeus LT604117 Cy b 374 A chaeopsylla e inacei Pulicidae E inaceus eu opaeus LT627350 Cy b 374 Synopsyllus gi a di Pulicidae Unknown KM890686 Cy b 369 Xenopsylla con o mis con o mis Pulicidae Unknown KM890723 Cy b 369 Xenopsylla skj abini Pulicidae Unknown KM890718 Cy b 369 Xenopsylla amesis Pulicidae Unknown KM890637 Cy b 342 Echidnophaga oschanini Pulicidae Unknown KM890719 Cy b 369 Spilopsyllus cuniculi Pulicidae Unknown KM890622 Cy b 369 Cediopsylla inaequalis inaequalis Pulicidae Unknown KM890600 Cy b 369 Nosopsyllus ba ba us Ce a ophyllidae Ra us sp LN897460 Cy b 374 Nosopsyllus ba ba us Ce a ophyllidae Ra us sp LN897462 Cy b 374 Nosopsyllus ascia us Ce a ophyllidae Mu idae LT158049 Cy b 374 Nosopsyllus ascia us Ce a ophyllidae Apodemus syl a icus LT158043 Cy b 374 Nosopsyllus i anis heodo i Ce a ophyllidae Ge billus dasyu us KM890603 Cy b 369 Nosopsyllus lae iceps ellobii Ce a ophyllidae Unknown KM890720 Cy b 369 Pano pa me idionalis Pano pidae - LT604127 Cy b 374 Pano pa me idionalis Pano pidae - LT604128 Cy b 374 Page 49 o 49 Medical and Ve e ina y En omology