Morphological and molecular characterisation of Paranoplocephala ...

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Morphological and molecular characterisation of Paranoplocephala ...

Syst Parasitol (2007) 66:55–71DOI 10.1007/s11230-006-9059-1ORGINIAL PAPERMorphological and molecular characterisationof Paranoplocephala buryatiensis n. sp. andP. longivaginata Chechulin & Gulyaev, 1998 (Cestoda:Anoplocephalidae) in voles of the genus ClethrionomysVoitto Haukisalmi Æ Lotta M. Hardman ÆMichael Hardman Æ Juha Laakkonen ÆJukka Niemimaa Æ Heikki HenttonenReceived: 26 January 2006 / Accepted: 12 March 2006 / Published online: 15 September 2006Ó Springer Science+Business Media B.V. 2006Abstract A new species, Paranoplocephalaburyatiensis n. sp. (Cestoda:Anoplocephalidae), isdescribed from the grey-sided vole Clethrionomysrufocanus (Sundevall) in the Republic of Buryatia(Russian Federation) and compared with P. longivaginataChechulin & Gulyaev, 1998, a parasiteof the red vole C. rutilus (Pallas) in the same region.P. buryatiensis n. sp. and P. longivaginataboth have an exceptionally long vagina and cirrus,unique features among known species of ParanoplocephalaLühe, 1910. The new species differsfrom P. longivaginata primarily by its wider andmore robust body, lower length/width ratio ofmature proglottides, tendency of testes to occur intwo separate groups, seminal receptacle of a differentshape and the position of the cirrus-sac withrespect to the ventral longitudinal osmoregulatorycanal. The cytochrome oxidase subunit I (COI)sequence data support the independent status ofthese species, and show that they form a monophyleticassemblage within Paranoplocephala(sensu lato). Assuming cospeciation, an indirectV. Haukisalmi (&) Æ L. M. Hardman ÆM. Hardman Æ J. Laakkonen Æ J. Niemimaa ÆH. HenttonenFinnish Forest Research Institute, Vantaa ResearchUnit, PO Box 18, FIN-01301Vantaa, Finlande-mail: voitto.haukisalmi@metla.ficalibration using host speciation dates estimated arate of mtDNA substitution of 1.0–1.7% pairwise(0.5–0.85% per lineage) sequence divergence permillion years. A faunistic review of Paranoplocephalaspecies in C. rufocanus and C. rutilus inthe Holarctic region is presented.IntroductionParanoplocephala Lühe, 1910 (sensu lato) (Cestoda:Anoplocephalidae) includes at least 35species, most of which parasitize arvicoline rodents(voles and lemmings) in the Holarctic region(Haukisalmi, Henttonen, & Hardman, 2006;Haukisalmi, Henttonen, Niemimaa, & Rausch,2002; Tenora, Murai, & Vaucher, 1986). Paranoplocephalaspp. usually exhibit a relatively highhost-specificity, with most species being confinedto a single or a few congeneric host species (seeDiscussion). Some of the apparently host-generalistspecies of Paranoplocephala have turned outto include multiple cryptic and host-specific specieswhen subjected to molecular phylogeneticanalyses (Haukisalmi, Wickström, Henttonen,Hantula, & Gubányi, 2004; Wickström, Haukisalmi,Varis, Hantula, Fedorov, & Henttonen,2005). Cryptic species seem to be common inanoplocephalid cestodes due to the scarcity ofdiagnostic characters, including the absence of123


56 Syst Parasitol (2007) 66:55–71rostellum and hooks (Ba et al., 1993; Baverstock,Adams, & Beveridge, 1985; Hu, Gasser, Chilton,& Beveridge, 2005).P. longivaginata Chechulin & Gulyaev, 1998 isa morphologically distinctive species describedfrom the red vole Clethrionomys rutilus (Pallas)in South-Central Siberia, but is also known fromthe red squirrel Sciurus vulgaris (Linnaeus) in thesame region (Chechulin & Gulyaev, 1998) andfrom the grey-sided vole C. rufocanus (Sundevall)in eastern Siberia (Haukisalmi & Wickström,2005; Wickström, Haukisalmi, Varis, Hantula, &Henttonen, 2005).We have recently surveyed the helminths ofC. rufocanus and C. rutilus in the Republic ofBuryatia (Russian Federation) and found bothvoles to be parasitised by P. longivaginata-likecestodes. The cestodes from C. rufocanus differedslightly but consistently from those in C. rutilus,suggesting the presence of two host-specific species.The species from C. rufocanus is describedhere as P. buryatiensis n. sp., and compared morphometricallywith P. longivaginata from C. rutilus.Mitochondrial cytochrome oxidase subunit I(COI) sequence data are provided for P. buryatiensisn. sp. and P. longivaginata to define theirtaxonomic status and phylogenetic position withrespect to other Paranoplocephala species inarvicoline rodents and to other anoplocephalinesmore broadly. Additionally, a faunistic review ofParanoplocephala species in C. rufocanus and C.rutilus throughout the Holarctic region is compiledbased on studies by the present research group withthe help of several collaborators (Table 3).Materials and methodsCestodesH. Henttonen, J. Laakkonen and J. Niemimaacollected voles and cestodes from the Republic ofBuryatia, Russian Federation, during 11–20 August2004 as part of a joint research programmeon rodent-borne zoonotic viruses with the AgriculturalAcademy of Buryatia. The seven studysites (Ganzurinov, Kamensk, Maloje Kolesova,Nizhnaya Ivolga, Pasolskaya, Utochkina Pad andVerhnaya Berezovka) are located near thesouthern coast of Lake Baikal (see Table 1 forcoordinates). The host material examined forhelminths included 63 specimens of Clethrionomysrufocanus, 17C. rutilus, 36Microtus cf. fortisBüchner and 19 M. oeconomus (Pallas). Rodentswere snap-trapped, placed in a cooled styrofoambox and dissected the same day.Thirty-seven specimens of P. buryatiensis n. sp.were obtained, of which 15 gravid specimens wereTable 1 The GenBank and museum accession numbers for Paranoplocephala buryatiensis n. sp. and P. longivaginataCestode species(Host species)Region, locality Co-ordinates GenBankacc. no.Type- and voucherspecimen acc. no.(UAM = host acc. no.)P. buryatiensis n. sp.(C. rufocanus)P. buryatiensis n. sp.(Microtus sp.)P. longivaginata(C. rutilus)Buryatia, Pasolskaya 51°58¢N, 106°20¢E DQ445259 USNPC 97137 (holotype),USNPC 97138 (paratype)Buryatia, PasolskayaDQ445262Buryatia, PasolskayaDQ445264Buryatia, PasolskayaDQ445265Buryatia, Kamensk 52°00¢N, 106°35¢E DQ445266N-E Siberia (Kolyma River),Kontakt Station61°50¢N, 147°39¢E AY568202,AY568203USNPC 91095(UAM 80007)Buryatia, Kamensk USNPC 97139Buryatia, Utochkina Pad 51°57¢N, 107°28¢E DQ445267 USNPC 97140Buryatia, Verhnaya Berezovka 51°54¢N, 107°41¢E DQ445261Buryatia, PasolskayaDQ445260Buryatia, PasolskayaDQ445263N-E Siberia (Omolon River),40 km W of Magadan59°41¢N, 150°20¢E USNPC 91094(UAM 80797)123


Syst Parasitol (2007) 66:55–71 57examined morphometrically (13 from C. rufocanus,2 from M. cf. fortis). Eight specimens of P. longivaginatawere collected from C. rutilus, of whichfive were sampled for morphometric data. No otherParanoplocephala species were found from volesof the genus Clethrionomys Tilesius, but cestodessimilar to P. omphalodes (Hermann, 1783)occurred in Microtus spp.The molecular analysis of P. longivaginata-likecestodes was performed on five specimens fromC. rufocanus and four specimens from C. rutilusfrom Buryatia (Table 1). In addition, the analysisincluded one specimen from C. rufocanus fromthe Kolyma River region in eastern Siberia,Russia (Kontakt Station; Table 1), designated asP. longivaginata in the phylogenetic analysis ofWickström et al. (2005). The latter specimen,collected by the Beringian Coevolution Project(Hoberg et al., 2003), was stained and mountedfor a specific identification but not included in thedescription of the new species. Samples of hostsfrom which the eastern Siberian specimens wererecovered have been deposited in the Universityof Alaska Museum, Fairbanks (UAM; Table 1).Molecular analysisWe performed a molecular phylogenetic analysisin order to understand the relationships ofP. longivaginata and P. buryatiensis n. sp. withother members of Paranoplocephala (sensu lato)and selected anoplocephalines. Genomic DNAwas isolated from tissues fixed and stored in70–96% ethanol, and a 641-bp region of COI wasamplified and sequenced following the protocoland using primers listed in Wickström et al.(2003, 2005) and Haukisalmi et al. (2004).Sequences of other exemplar anoplocephalinesand cyclophyllidean outgroups (Taeniidae andHymenolepididae) were downloaded from Gen-Bank (see Appendix 1) to provide an extendedcomparative framework and root.Sequences were assembled and edited usingAlign IR TM Sequence Assembly and AlignmentSoftware (LI-COR Inc., Nebraska, USA).Consensus sequences were aligned in Clustal W(Thompson et al., 1994) with default gap penalties.Alignments were translated to their aminoacid residues using the platyhelminth geneticcode (Telford et al., 2000) and adjusted by eye tominimise stop codons in MacClade v4.0 (Maddison& Maddison, 2000). Gapped positions weretreated as missing data.Maximum parsimony methods were employedto identify optimal topologies within PAUP* v.4.0b10 (Swofford, 2002). All characters weretreated as unordered, received equal weighting,and the heuristic search algorithm ‘‘tree-bisection-reconnection’’(TBR) was used to identifyoptimal topology(ies). Taxonomic congruenceamong equally parsimonious trees was summarisedthrough strict consensus. Nodal support wasestimated through majority-rule consensus of1000 nonparametric bootstrap pseudoreplicates(Felsenstein, 1985). Nodes present in >90% ofpseudoreplicates were considered strongly supported.Rate estimationGiven that P. longivaginata and P. buryatiensis n.sp. are each known exclusively from differentspecies of Clethrionomys, they may be the productsof a cospeciation event (Hafner & Page,1995). Calibrated rates are lacking for platyhelminthsbecause they do not have a fossil record.We seized an opportunity to estimate a rateof mtDNA substitution in these worms by anindirect method assuming cophyly with theirhosts. We calculated the age of the speciation oftheir hosts (C. rufocanus and C. rutilus) usingGTR + G corrected distances of cytochrome bsequences (GenBank: AF272632, AF272635,AY309416, AY305263 and AF429810) and usedthe Microtus oeconomus rate of 6–10% sequencedivergence per million years (Brunhoff et al.,2003) to estimate the age of their cladogenesis.Assuming cospeciation, the age estimate of thehosts also applies to the speciation of the lineagesrepresented today by P. longivaginata andP. buryatiensis n. sp. We calibrated the platyhelminthrate based on estimated ages of host speciationat 6% and 10% sequence divergence permillion years and using HKY85 correcteddistances.Prior to calibration and node age estimation, wetested for rate heterogeneity with the likelihoodratio test (LRT) (Goldman, 1993; Sanderson, 1998)123


Syst Parasitol (2007) 66:55–71 59665052601009410 changesParanoplocephala blanchardiParanoplocephala primordialisDiandrya composita100 Paranoplocephala macrocephalaParanoplocephala omphalodesParanoplocephala kalelaiParanoplocephala etholeni100 Paranoplocephala buryatiensis n.sp.100 Paranoplocephala buryatiensis n.sp.100 Paranoplocephala longivaginataParanoplocephala longivaginata100 Paranoplocephala alternataParanoplocephala arcticaParanoplocephala fellmani99 Anoplocephaloides variabilisParanoplocephala krebsiParanoplocephala oeconomiAnoplocephaloides lemmi52Paranoplocephala nordenskioeldiParanoplocephala gracilisParanoplocephala serrata5850Anoplocephaloides dentata74Anoplocephaloides kontrimavichusiAndrya rhopalocephalaNeandrya cuniculi98 Anoplocephala magnaAnoplocephala perfoliataMonoecocestus americanusMoniezia sp.89Moniezia expansaMoniezia benedeniProgamotaenia ewersiProgamotaenia macropodisMosgovoyia pectinataRodentolepis nanaHymenolepididaeHymenolepis diminutaEchinococcus multilocularisEchinococcus granulosusTaeniidaeTaenia asiaticaTaenia soliumParanoplocephalasensu strictoFig. 1 Strict consensus phylogram and approximate hostranges of three equally parsimonious topologies resultingfrom the phylogenetic analysis of the COI sequencealignment. Numbers above or below nodes are percentagesof 1,000 nonparametric bootstrap pseudoreplicates. Nodesrecovered in < 50% of pseudoreplicates are not shown123


Syst Parasitol (2007) 66:55–71 61Figs. 2–6 Paranoplocephala buryatiensis n. sp. fromClethrionomys rufocanus from Buryatia. 2, 3. Scolex. 4.Transverse section of mature proglottis. 5. Terminal genitalducts and early uterus in mature proglottis. 6. Fullydevelopeduterus in pregravid proglottis. Scale-bars: 2–4, 6,0.50 mm; 5, 0.30 mm123


62 Syst Parasitol (2007) 66:55–71Figs. 7–9 Mature proglottides of Paranoplocephalaburyatiensis n. sp. 7. Ex Clethrionomys rufocanus fromBuryatia (holotype). 8. Ex Microtus sp. from Buryatia. 9.Ex C. rufocanus from north-eastern Russia (KolymaRiver). Scale-bars: 7–9, 0.50 mmusually overlaps proximal vagina dorsally; coveredby layer of poorly stained cells.Vagina very long (0.47–0.65, 0.56, n = 24), 1.5–2.0 times length of cirrus-sac, tube-like, of uniformwidth (0.04–0.05), clearly distinct fromseminal receptacle; positioned posterior or postero-ventralto cirrus-sac. Vagina covered externallyby dense layer of small, intensely stainedcells; cells around proximal vagina larger thanthose around other parts of vagina; internally,vagina covered by delicate, fairly long microtrichesexcept in its distal part, which usuallyforms short dilation. Vagina usually curved ortwisted, especially in proximal part. Seminalreceptacle transversely elongate, pyriform orasymmetrical, 0.20–0.45 (0.30, n = 25) long and0.12–0.26 (0.18, n = 25) wide in mature proglottides;maximum length 0.35–0.55 (0.46, n = 10) inpostmature proglottides. Vitellarium asymmetricallybilobed, long (0.15–0.30, 0.21, n = 27)123


Syst Parasitol (2007) 66:55–71 63Table 2 The main morphometric data forParanoplocephala buryatiensis n. sp. and P.longivaginata. Values of P. longivaginata, which are nearlyor completely non-overlapping with those of P.buryatiensis n. sp., have been indicated in boldCestode species P. buryatiensis n. sp. P. longivaginata P. longivaginataHost species C. rufocanus C. rutilus C. rutilusGeographical origin Buryatia South-Central Russia Buryatia(including Buryatia)Source Present study Chechulin & Gulyaev,1998Present studyBody, length 148–277 200–210 198–225 (n = 3)Body, maximum width 3.0–5.0 1.7 2.1–2.7 (n = 3)Mature proglottides, width 1.7–2.9 0.9–1.0 1.1–1.8 (n = 14)Mature proglottides, l/w ratio 0.13–0.28 0.47 0.25–0.51 (n = 10)Scolex, diameter 0.66–0.77 0.50–0.55 0.65–0.77 (n = 4)Suckers, diameter 0.24–0.35 0.24–0.26 0.25–0.33 (n = 16)Neck, length 0.4–0.8 – 0.5–0.7 (n = 4)Neck, minimum width 0.36–0.65 – 0.33–0.41 (n = 4)Testes, total number 46–77 45–55 34–56 (n = 7)Longitudinal ventral canals, width 0.038–0.090 0.05 0.045–0.085 (n = 14)Cirrus-sac, length* 0.26–0.42 0.23–0.30 0.30–0.43 (n = 4)Ovary, width 0.6–1.1 0.4–0.5 0.4–0.7 (n = 7)Vitellarium, width 0.23–0.41 0.12–0.20 0.21–0.36 (n = 7)Vitellarium, position (index of asymmetry) 0.37–0.46 – 0.41–0.51 (n = 7)Vagina, length 0.47–0.65 0.45–0.53 0.46–0.54 (n = 4)Vagina/cirrus-sac ratio 1.5–2.0 1.5–2.0 ca. 1.5Seminal receptacle, length* 0.20-0.45 0.11–0.13 0.15–0.35 (n = 12)Egg, length 0.041–0.055 0.048–0.050 0.037–0.053 (n = 20)n, Number of measurements* Combining mature and postmature proglottidesrelative to its width (0.23–0.41, 0.31, n = 27),positioned poral with respect to mid-line of proglottisand ovary, overlapping posterior part ofovary. Mehlis’ gland large (0.09–0.12), ovoid orspherical, situated between lobes of vitellarium.Ovary 0.64–1.10 (0.81, n = 27) wide, coarselylobulate, slightly poral, covering ca. 2/3 of spacebetween longitudinal ventral canals or 29–46%(35%, n = 25) of mature proglottis width.Uterus appears in premature proglottides asfine, complex reticulum, positioned ventral toother organs, completely overlaps ovary (but notvitellarium) and extends bilaterally beyond longitudinalosmoregulatory canals. In postmatureproglottides, uterus develops complex internaltrabeculae and irregular marginal sacculationsand diverticula; at this stage uterus covers practicallywhole medulla. Testes remain in postmatureand early pregravid proglottides overlappingdeveloping uterus; cirrus-sac and vagina persist inpregravid proglottides. Various internal structuresdisintegrate fully or partly in gravid proglottides.Eggs 41–55 (48, n = 64) · 32–44(37, n = 7) lm wide, spherical in surface view,ovoid in side view. Pyriform apparatus with2 slender horns crossing each other.RemarksParanoplocephala buryatiensis n. sp. and P. longivaginataare both characterised by an exceptionallylong vagina and cirrus (the length of theformer being at least 1.5 times the cirrus-sac;Figs. 7–9), a unique feature among all knownParanoplocephala species. Therefore, P. buryatiensisn. sp. is compared here only with P. longivaginata.P. buryatiensis n. sp. is a wider-bodied, morerobust cestode than P. longivaginata (Table 2)with a low length/width ratio of mature proglottidescompared with the latter species. In addition,there is a smaller proportion of testes123


64 Syst Parasitol (2007) 66:55–71Figs. 10–11 Paranoplocephala longivaginata from Clethrionomys rutilus from Buryatia. 10. Scolex. 11. Mature proglottis.Scale-bars: 10, 0.50 mm; 11, 0.30 mmanterior to the ovary in P. buryatiensis, and thereis a tendency of testes to occur in two separategroups in the new species, but not in P. longivaginata.There are also consistent differences inthe shape of the seminal receptacle (transversallyelongate in P. buryatiensis, versus longitudinallyelongate in P. longivaginata), and the position ofthe cirrus-sac (overlapping the ventral longitudinalcanal in P. longivaginata, versus not so in P.buryatiensis). The cirrus was usually fully evertedin P. longivaginata; this condition was never seenin P. buryatiensis (cf. Figs. 7–9 and 11).The specimens of P. buryatiensis from C. rufocanusin eastern Siberia (Kolyma River region)corresponded morphologically with those fromthe type-locality. In addition, the specimens fromMicrotus sp. (one case) did not differ noticeablyfrom those from the type-host (cf. Figs. 7–9).The prevalence of P. buryatiensis in Buryatia(host C. rufocanus) was 27%, being higher inmature voles (males 47%, females 31%) than inimmature voles (19%). The prevalence was evidentlyvery low in eastern Siberia (Kolyma Riverregion), since only a single infection of P. buryatiensiswas encountered among the grey-sidedvoles and other rodents examined in that region(Wickström et al., 2005; unpublished data of theBeringian Coevolution Project).DiscussionParanoplocephala species of Clethrionomysrufocanus and C. rutilusBased on the material examined by us (with thehelp of numerous collaborators; Table 3), thereare six species of Paranoplocephala (sensu lato)inClethrionomys rufocanus and C. rutilus in theHolarctic region, two in the former and four in thelatter host species. In any given locality the speciesnumber is low, ranging between zero and two.The following Paranoplocephala species havealso been reported as parasites of C. rufocanusand/or C. rutilus in Siberia and north-westernRussia: P. caucasica (Kirshenblat, 1938), P. macrocephala(Douthitt, 1915), P. microti (Hansen,1947), P. montana (Kirshenblat, 1941) andP. omphalodes (e.g. Egorova & Nadtochii, 1975;Gubanov & Fedorov, 1970; Ryzhikov et al., 1978;Yushkov, 1995). However, we believe that noneof these reports refers to the correct/valid Paranoplocephalaspecies. Firstly, P. macrocephalaand P. microti are currently known to be parasitesof voles of the genus Microtus Schrank (bothcestode species) and geomyids (P. macrocephala)in North America (Haukisalmi & Henttonen,2003; Haukisalmi et al., 2004), although both123


Syst Parasitol (2007) 66:55–71 65Table 3 Records of Paranoplocephala spp. in Clethrionomys rufocanus (Crufo) and C. rutilus (Cruti) based on published and unpublished studies by us and ourcollaborators. Notice that C. rufocanus does not occur in the Nearctic, and that no C. rutilus specimens were examined from North-East China (Fenglin) or South-East Russia (Siziman Bay). The study localities have been ordered by the mean latitude (from west to east). N, North; E, East; S, South; W, West; C, CentralRegion PALAEARCTIC NEARCTICLocality(Country)Lapland(N Finland,N Norway)Lower TunguskaRiver(N-C Russia)Buryatia(S-C Russia)Fenglin(N-E China)SizimanBay*(S-E Russia)Omolon/KolymaRivers(N-E Russia)Chukotka(N-E Russia)Alaska(N-W USA)Source 1 2 3 4 5 6 6 6Host species Crufo Cruti Crufo Cruti Crufo Cruti Crufo Crufo Crufo Cruti Crufo Cruti CrutiSample size >1000 >1000 36 73 63 17 28 16 c.130 c.450 18 67 >1000Paranoplocephala batzliiHaukisalmi, Henttonen, &Hardman, 2006P. buryatiensis n. sp. + +P. gracilis Tenora & Murai, 1980 + +P. kalelai (Tenora, Haukisalmi& Henttonen, 1985)P. longivaginata Chechulin &Gulyaev, 1998++ +P. primordialis (Douthitt, 1915) +(+) #Sources: 1 Tenora et al., 1985; Haukisalmi, 1986; V. Haukisalmi &H. Henttonen, unpubl2 A. Lavikainen & V. Haukisalmi, unpubl3 Present study4 E. Kallio & V. Haukisalmi, unpubl5 P. Niemelä, H. Henttonen & V. Haukisalmi, unpubl6 Unpublished material of the Beringian Coevolution Project (Hoberg et al., 2003)* Khabarovsk Region# Exceptional (one case), primarily a parasite of Microtus miurus Osgood (see Haukisalmi et al., 2006)123


66 Syst Parasitol (2007) 66:55–71have frequently (and probably erroneously) beenreported as parasites of various rodents in Eurasia(e.g. Ryzhikov et al., 1978). Based on molecularmethods, P. omphalodes has been shown to beprimarily a parasite of Microtus voles in Europe(Haukisalmi et al., 2004). In northern Siberia andAlaska there is another, morphologically similarspecies (P. jarrelli Haukisalmi, Henttonen &Hardman, 2006) parasitising the root/tundra voleMicrotus oeconomus (see Haukisalmi et al.,2006), but the status of the P. omphalodes-likecestodes from C. rufocanus and C. rutilus remainsunknown. Finally, P. caucasica and P. montanaare poorly known taxa, regarded here as speciesinquirendae and which cannot be separated morphologicallyfrom any of the related species(Haukisalmi & Henttonen, 2005; Haukisalmiet al., 2006). It is possible that some Paranoplocephalaspecies from C. rufocanus and C. rutilusin Siberia and north-western Russia actuallyrepresent P. buryatiensis n. sp., P. longivaginataor P. kalelai (Tenora, Haukisalmi & Henttonen,1985), but this cannot be verified since the earlierfindings were without morphological descriptionsor vouchered specimens and were not comparedto related species.Of the six Paranoplocephala species recognisedhere (Table 3), P. buryatiensis n. sp. and P. kalelaiare more or less host-specific parasites of C. rufocanus,and P. longivaginata is largely restricted toC. rutilus, whereas P. gracilis Tenora & Murai,1980 and P. primordialis (Douthitt, 1915) are truehost-generalists of Microtus and Clethrionomysvoles (and occasionally other rodents) in Europeand North America, respectively (Haukisalmi &Henttonen, 2001, and unpublished data; Tenora &Murai, 1980). The helminths of other genera ofClethrionomyini (Eothenomys Miller, PhaulomysThomas and Alticola Blanford) are poorly knownand no host-specific anoplocephalid cestodes haveso far been reported from them, despite theirrelatively high species diversity in South-East Asiaand Japan (Asakawa & Harada, 1989). Therecords of P. macrocephala and P. omphalodesfrom the eastern Eurasian Alticola spp. (Ryzhikovet al., 1978) are probably erroneous (see above).C. rufocanus and C. rutilus have widely overlappingdistributions throughout northern Eurasia(the latter occurs also in North America). Despiteconsiderable geographical and habitat overlap,the helminth faunas of these species are largelydisjunct (Table 3; Asakawa, 1989; Haukisalmi,1986), probably reflecting their divergent phylogeneticposition within the Clethrionomyini (below).For example, despite considerable samplingeffort, the host-specialist P. kalelai Tenora et al.,1985 has never been found in the sympatric C.rutilus in Fennoscandia although it is common inits primary host, C. rufocanus (see Haukisalmi,1986; Tenora et al., 1985; and our large subsequentdataset). In addition, Anoplocephaloidesdentatoides Sato, Kamiya, Tenora and Kamiya,1993 seems to be specific to C. rufocanus despitethe presence of C. rutilus in Hokkaido, Japan(Sato et al., 1993). The non-overlapping hostdistributions of P. buryatiensis n. sp. and P. longivaginatagive further support for the helminthologicaldistinction of C. rufocanus, C. rutilusand allied species.The combined geographical distribution of P.buryatiensis n. sp. and P. longivaginata is knownto span from South-Central to North-East Siberia(Table 3) and is evidently non-overlapping withother host-specific Paranoplocephala species ofClethrionomys voles. The western distributionlimits of P. buryatiensis and P. longivaginataremain undefined, although we know that neitheroccurs in northern Europe despite the presence oftheir primary host species (Haukisalmi, 1986;Haukisalmi, Henttonen, & Tenora, 1987; andunpublished data). Their eastern distributionlimit could be located at the Kolyma/Omolonrivers, forming roughly the western border of theformer Beringia, since neither species has beenfound from easternmost Russia (Chukotka) orAlaska (Table 3; Haukisalmi et al., 2006). Sinceboth C. rufocanus and C. rutilus show majorphylogeographical splits in eastern Asia (Iwasaet al., 2000; Iwasa, Kartavtseva, Dobrotvorsky,Panov, & Suzuki, 2002), it is possible that theabsence of P. buryatiensis and P. longivaginata ineasternmost Siberia is due to the divergence oftheir hosts at this biogeographical border (seealso Brunhoff et al., 2003; Galbreath & Cook,2004), which has possibly led to lineage sorting ofcestodes in small, isolated host populations (cf.123


Syst Parasitol (2007) 66:55–71 67Haukisalmi & Henttonen, 2001). No Paranoplocephalaspecies are known from C. rufocanus orC. rutilus in Hokkaido, Japan (Asakawa et al.,1983; Asakawa, 1989).However, the apparent absence of P. buryatiensisand P. longivaginata in some of the hostpopulations studied here (Table 3) may be due tothe combined effect of small sample size andspatially and/or temporarily varying prevalence ofthese cestodes. A subsequent collection of volehelminths in northern Buryatia in 2005 (Henttonenand Haukisalmi et al., unpublished data)revealed a very low prevalence of P. buryatiensisin C. rufocanus (1.5%, n = 69), compared withthe prevalence (27%) seen in the previous year ina more southern region in Buryatia (presentmaterial). Thus, the geographical distribution ofP. buryatiensis is evidently very patchy, a featureshared by some other anoplocephalid cestodes ofarvicoline rodents (Haukisalmi & Henttonen,1999, 2001). The patchiness of P. buryatiensis wasevident also on a smaller geographical scale(among study localities) in the present material(results not shown).Evolutionary history of Paranoplocephalaburyatiensis n. sp. and P. longivaginataThe genetic and morphologic data strongly suggestthat P. buryatiensis n. sp. and P. longivaginata forman exclusive clade among the species studied here.However, cytochrome b sequences suggest thattheir primary hosts (Clethrionomys rufocanus andC. rutilus, respectively) belong to different cladeswithin Clethrionomyini (Cook, Runck, & Conroy,2004; Luo et al., 2004). C. rutilus belongs to a cladecomposed of C. californicus (Merriam), C. gapperi(Vigors), C. glareolus (Schreber) and Alticolamacrotis (Radde), whereas C. rufocanus groupswith the Japanese endemics Phaulomys andersoni(Thomas) and P. smithii (Thomas) (both oftenassigned to Eothenomys). Thus, Clethrionomys,aspresently defined, is paraphyletic with respect toPhaulomys and Alticola (and possibly Eothenomys),and, according to Luo et al. (2004), C. rufocanusshould actually be assigned to Phaulomyswith P. smithii and P. andersoni. Based on the fossilevidence (Chaline & Graf, 1988), the divergence ofC. rufocanus probably took place ca. 0.7 mya in theFar East, where its closest relatives (P. smithii andP. andersoni) still exist.Since Paranoplocephala longivaginata-likespecies have not been found in Clethrionomysspecies other than C. rufocanus and C. rutilus (orfrom any Phaulomys or Alticola species), thescenario of host-parasite cospeciation requiresthat several extinctions and/or ‘missing the boat’events (Paterson & Gray, 1997) have taken place.Alternatively, P. buryatiensis n. sp. and P. longivaginatamay have diverged more recently due toa colonisation event between C. rufocanus and C.rutilus (from either direction) and the widelyoverlapping geographical distributions and feedingstrategies of these voles support such a scenario.If we consider these two scenarios withinthe context of the genetic differences observedbetween P. buryatiensis and P. longivaginata(7.05±0.47%), cospeciation provides a rate (1.0–1.7% pairwise divergence per million years) thatis comparable to other heterothermic metazoans,whereas the host-switch scenario (using the minimumage of 0.7 mya for C. rufocanus) requires arate of evolution that is faster than the fastest ofhomeothermic vertebrates, at approximately 10%pairwise divergence per million years. These datafavour the explanation that P. buryatiensis and P.longivaginata have codiverged with their hosts,rather than originating from a more recent hostcolonisation episode. Since this explanation impliesmultiple apparent ‘sorting events’, we predictthat the Clethrionomyini will be found tohost yet unknown species of P. longivaginata-likecestodes when subjected to comprehensive faunisticand taxonomic scrutiny.The COI sequence data alone do not provideconvincing resolution of the phylogenetic relationshipsof P. buryatiensis + P. longivaginata withrespect to the other Paranoplocephala species(Fig. 1). However, several moderately supportedrelationships were suggested by the combined sequencedata sets (COI + 28S + ITS1) of Wickströmet al. (2005) and, based on their results, theprobable sister species for the P. longivaginatacladeis P. primordialis, a host-generalist ofClethrionomys and Microtus voles (and occasionallysome other rodents) in North America. Thesethree species were generally associated with123


Syst Parasitol (2007) 66:55–71 69Table 4 continuedLength GenBank. SourceP. arctica 638 AY181508 Wickström et al., 2003P. blanchardi 641 AY604729 Wickström et al,. unpubl.P. etholeni 588 AY568214 Wickström et al., 2005P. fellmani 641 AY568200 Wickström et al., unpubl.P. gracilis 641 AY395680 Wickström et al., unpubl.P. kalelai 641 AY181513 Haukisalmi et al., (2004)P. krebsi 590 AY568216 Wickström et al., 2005P. macrocephala 641 AY181515 Haukisalmi et al., (2004)P. nordenskioeldi 641 AY568204 Wickström et al., 2005P. oeconomi 641 AY568205 Wickström et al., 2005P. omphalodes 608 AY181549 Haukisalmi et al. (2004)P. primordialis 427 AY568218 Wickström et al., 2005P. serrata 561 AY568220 Wickström et al., 2005Progamotaenia ewersi 375 AJ716022 Hu et al., 2005P. macropodis 375 AJ716035 Hu et al., 2005HymenolepididaeHymenolepis diminuta 641 AF314223 von Nickisch-Rosenegk et al., 2001Rodentolepis nana 641 AB033412 Nakao et al., 2000TaeniidaeEchinococcus granulosus 641 AF297617 Le et al., 2002E. multilocularis 641 AB018440 Fukunaga, unpubl.Taenia asiatica 641 AF445798 Jeon et al., 2005T. solium 641 AB086256 Nakao et al., 2003ReferencesAsakawa, M. (1989). Helminth fauna of Japanese Microtidaeand Muridae. Honyurui Kagaku (MammalianScience), 29, 17–35 (in Japanese)Asakawa, M., Harada, M. (1989). Faunal and zoogeographicalstudy on the internal parasites of the Japanesered-backed volve [sic], Eothenomys spp. Bulletinof the Biogeographical Society of Japan, 44, 199–210(in Japanese)Asakawa, M., Yokoyama, Y., Fukumoto, S., & Ueda, A.(1983). A study of the internal parasites of Clethrionomysrufocanus bedfordiae (Thomas). JapaneseJournal of Parasitology, 32, 399–411Ba, C. T., Wang, X. Q., Renaud, F., Euzet, L., Marchand,B., & De-Meeus, T. (1993). Diversity and specificity incestodes of the genus Moniezia: Genetic evidence.International Journal for Parasitology, 23, 853–857Baverstock, P. R., Adams, M., & Beveridge, I. (1985).Biochemical differentiation in bile duct cestodes andtheir marsupial hosts. Molecular Biology and Evolution,2, 321–337Brunhoff, C., Galbreath, K. E., Fedorov, V. B., Cook, J.A., & Jaarola, M. (2003). Holarctic phylogeography ofthe root vole (Microtus oeconomus): Implications forlate Quaternary biogeography of high latitudes.Molecular Ecology, 12, 957–968Chaline, J., & Graf, J. D. (1988). Phylogeny of the Arvicolidae(Rodentia): Biochemical and paleontologicalevidence. Journal of Mammalogy, 69, 22–33Chechulin, A. I., & Gulyaev, V. D. (1998). Paranoplocephalalongivaginata sp. n. (Cyclophyllidea: Anoplocephalidae)- a new cestode from rodents of the WesternSiberia. Parazitologiya, 32, 352–356 (in Russian)Conroy, C. J., & Cook, J. A. (1999). MtDNA evidence forrepeated pulses of speciation within arvicoline andmurid rodents. Journal of Mammalian Evolution, 6,221–245Cook, J. A., Runck, A. M., & Conroy, C. J. (2004). Historicalbiogeography at the crossroads of the northerncontinents: Molecular phylogenetics of red-backedvoles (Rodentia: Arvicolinae). Molecular Phylogeneticsand Evolution, 30, 767–777Egorova, T. P. & Nadtochii, E. V. (1975). [Helminths ofsome rodents of the Kolyma Mountains.] In:Gel’mintologicheskie Issledovaniya Zivotnyh i Rastenii.(Trudy Biologo – Pochvennogo Insitituta ANS-SSR), Vol. 26, pp. 33–45 (in Russian)Felsenstein, J. (1985). Confidence limits on phylogenies: Anapproach using the bootstrap. Evolution 39, 783–791.Galbreath, K. E., & Cook, J. A. (2004). Genetic consequencesof Pleistocene glaciations for the tundra vole(Microtus oeconomus) in Beringia. Molecular Ecology,13, 135–148Goldman, N. (1993). Statistical test of models of DNA substitution.Journal of Molecular Evolution, 36, 182–198Gubanov, N. M., & Fedorov, K. P. (1970). Helminth faunaof mouse-like rodents in Yakutia. In: Cherepanov,A.I. (Eds.), Fauna Sibirii (pp. 18–47). Nauka: Novosibirsk,(in Russian)123


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