Review of tapeworms of rodents in the Republic of Buryatia , with emphasis on anoplocephalid cestodes

Examination of ca. 500 rodents [Microtus spp., Myodes spp., Cricetulus barabensis (Pallas), Apodemus peninsulae Th omas] from 14 localities in the Republic of Buryatia (Russian Federation) revealed a minimum of 11 cestode species representing Anoplocephaloides Baer, 1923 s. str. (1 species), Paranoplocephala Lühe, 1910 s.l. (5 species), Catenotaenia Janicki, 1904 (2 species), Arostrilepis Mas-Coma & Tenora, 1997 (at least 2 species) and Rodentolepis Spasskii, 1954 (1 species). At least 5 of these species are previously unknown from the region. Th e taxonomic and phylogenetic position of Buryatian Paranoplocephala species was defi ned by cytochrome oxidase I (COI) sequences (mtDNA). Th e phylogenetic analysis also confi rmed the status of Parandrya Gulyaev & Chechulin, 1996 as a junior synonym of Paranoplocephala s.l. Th e species diversity of anoplocephalid cestodes was signifi cantly lower in Buryatia and North-East Siberia (6-7 species) than in Europe (17 species). Th e connections of the anoplocephalid fauna of Buryatia seem to be closer with Beringia (North-East Siberia and Alaska) than with Europe. Th e present study demonstrated high spatial variation (patchiness) among study sites in cestodes of Buryatian rodents, with the exception of the ubiquitous Arostrilepis horrida (von Linstow, 1901)-complex.


Introduction
Th e rodent fauna of the Republic of Buryatia (Russian Federation) represents a mixture of Asian, northern Palaearctic and Holarctic species.Of the Buryatian rodents, the grey-sided vole Myodes rufocanus (Sundevall) (syn.Clethrionomys rufocanus), the red vole Myodes rutilus (Pallas) (syn.Clethrionomys rutilus) and the root vole Microtus oeconomus (Pallas) are distributed throughout northern Eurasia, the latter two extending into north-western North America.More southern Palaearctic elements include the reed vole Microtus fortis Büchner, Maximowicz's vole Microtus maximowiczii (Schrenk), the striped dwarf hamster Cricetulus barabensis (Pallas) and the Korean fi eld mouse Apodemus peninsulae Th omas.Biogeographically the location of Buryatia is interesting because the east-west taiga corridor between the Lake Baikal and Mongolian steppe has probably been a major migration pathway for the Palaearctic fauna and fl ora (de Lattin 1967;Mikkola 1988).
Th e helminths of rodents in the Lake Baikal basin, including Buryatia, have been the subject of two major faunistical studies, i.e. those of Machul'skii (1958) and Zhaltsanova (1992).In addition, four recent descriptions of new species of anoplocephalid cestodes have included material from Buryatia (Chechulin and Gulyaev 1998;Gulyaev and Chechulin 1996;Gulyaev and Krivopalov 2003;Haukisalmi et al. 2007a).In terms of species diversity, anoplocephalid cestodes are the dominant helminth group in arvicoline rodents in Buryatia and elsewhere.However, some of the anoplocephalid cestodes previously reported from Buryatian rodents [i.e.Paranoplocephala macrocephala (Douthitt, 1915) and P. microti (Hansen, 1947)] are now known to be restricted to the Nearctic region (Haukisalmi and Henttonen 2003;Haukisalmi et al. 2004).Moreover, the apparently widespread Paranoplocephala omphalodes (Hermann, 1783) has been shown to include multiple species (Haukisalmi et al. 2004), and it is not known which of these species occur in Buryatia.Similar taxonomical problems are present in catenotaeniid cestodes.For example, Catenotaenia pusilla (Goeze, 1782) probably does not occur in Microtus Schrank and Apodemus Kaup in Buryatia, because it is presently regarded as a specifi c parasite of Mus musculus (L.) (Tenora and Murai 1975).
Th e present study reviews the cestode fauna of Buryatian rodents based on new extensive collections and includes recent advances in the taxonomy of anoplocephalid cestodes.Th e Buryatian cestode fauna of rodents is compared with those of Europe and north-eastern Siberia.Mitochondrial DNA sequences are used for defi ning the taxonomic and phylogenetic position of the Paranoplocephala species of Buryatian voles.

Material and methods
Rodents were trapped at 14 study sites of Buryatia in August in 2004 and2005.Th e location of Buryatia, study sites therein, and the rodent material sampled from each locality are given in Fig. 1 and Table 1.

Host species Locality
Aden We used snap-traps (Finnish metal mouse trap), which were set in the late afternoon and evening, and checked early next morning.In the fi eld, rodents were placed in a Styrofoam box with coolers, kept in a refrigerator in a laboratory, and dissected the same day.Th e cestodes found were fi rst allowed to relax in tap water, then fi xed fl at (without pressure) in 70% ethanol, and those used for morphology stained later with Mayer's haemalum, Semichon's acetocarmine or ironacetocarmine, cleared in eugenol and mounted in Canada balsam.Voucher specimens (whole-mounts) of all identifi ed cestode species from Buryatia have been deposited in the Museum of Southwestern Biology, University of New Mexico, USA (MSB) and the United States National Parasite Collection, Maryland, USA (USNPC).
Most of the comparative cestode materials from Chukotka, North-East Siberia, were collected in connection with the Beringian Coevolution Project (BCP; see Cook et al. 2005;Hoberg et al. 2003).
Seven specimens of Paranoplocephala Lühe, 1910 from Buryatia were sequenced for the partial cytochrome oxidase I (COI) gene and embedded in a larger COI sequence data set of Paranoplocephala spp., originating primarily from Wickström et al. (2005).GenBank numbers for the COI sequences are given in Fig. 2. For the extraction, amplifi cation and sequencing of DNA, see Wickström et al. (2003Wickström et al. ( , 2005) ) and Haukisalmi et al. (2004).
A recent molecular phylogenetic analysis (Haukisalmi et al. 2009) suggested that A. dentata-like cestodes include at least fi ve species (plus A. dentatoides) in the Holarctic region: four species in western Eurasia and one in the rest of Eurasia and Beringia (north-eastern Siberia and Alaska).Based on the cytochrome oxidase I (mtDNA) sequences, the A. dentata-like cestodes from Buryatia (hosts Myodes rufocanus and Microtus fortis) group strongly, and are therefore conspecifi c, with the Holarctic species  (Haukisalmi et al. 2009).However, it is not yet known if this Holarctic species is conspecifi c with either of the two related species described in North America, i.e.Anoplocephaloides infrequens (Douthitt, 1915) and A. troeschi (Rausch, 1946) from Geomys bursarius (Shaw) (Geomyidae) and Microtus pennsylvanicus (Ord), respectively.
In the Palaearctic, A. dentata-like cestodes are characteristically parasites of voles of the genera Microtus and Chionomys.However, in addition to Microtus gregalis (Pallas), M. maximowiczii and M. oeconomus, Machul'skii (1958) and Zhaltsanova (1992) reported P. brevis or P. dentata from Myodes rufocanus, M. rutilus, Apodemus peninsulae and Cricetulus barabensis in Buryatia.In the present study, A. cf.dentata was found from Microtus fortis and Myodes rufocanus.It is probable that all A. dentata-like cestodes in Buryatia represent a single species, with the occurrences in Myodes, Apodemus and Cricetulus being accidental.
Because there are few morphological features useful for the diagnosis of A. dentatalike cestodes, sequence data (COI, 28S rRNA) have proved to be necessary tools in the taxonomy of this species complex (Haukisalmi et al. 2009).
Th e Buryatian specimens form a monophyletic group that is sister to the divergent North Alaskan subclade of P. jarrelli.Th e topology and high statistical support of the tree suggests that these two clades are both independent species, forming a sister group for the true, Holarctic P. jarrelli.
Paranoplocephala cf.omphalodes from Buryatia may be primarily associated with the Asian Microtus species, i.e.M. fortis and M. maximowiczii, because it is not known from M. oeconomus outside Buryatia.Paranoplocephala cf.omphalodes will be later described as a new species.

Paranoplocephala blanchardi (Moniez, 1891)
Paranoplocephala blanchardi (Moniez, 1891) sensu Tenora et al. (1985b) is primarily a parasite of Microtus spp. in Europe (Haukisalmi et al. 1994;Tenora et al. 1985b) and western Siberia, extending at least to the Novosibirsk region in the east (Chechulin 1998).Th e conspecifi city of two Buryatian specimens of P. blanchardi (from Microtus fortis) with the European specimens is confi rmed by the present molecular phylogenetic analysis (Fig. 2).Th e Buryatian records thus represent the easternmost fi nding of this species.Despite considerable sampling eff ort, P. blanchardi has not been found in north-eastern Siberia (Magadan region, Chukotka; unpublished BCP records).P. blanchardi was found at a single study site (prevalence 17%) (Table 1).

Paranoplocephala feodorovi (Gulyaev & Chechulin, 1996)
Paranoplocephala feodorovi was originally described as Parandrya feodorovi Gulyaev & Chechulin, 1996 from Arvicola terrestris (L.) (primary host), Microtus agrestis and M. maximowiczii from the Novosibirsk region in the south of West Siberia (type locality) and Zabaikalsk in Buryatia (Gulyaev and Chechulin 1996; see Chechulin 1998 for further records in the latter region).Only a single specimen from Microtus fortis conforming to the description of P. feodorovi was encountered during the present study, which supports the secondary role of Microtus spp. in the epidemiology of this cestode.Morphologically, P. feodorovi is closely related to P. blanchardi, the main diff erence being the antiporal extent of the testes (extending across the antiporal ventral longitudinal osmoregulatory canal in P. blanchardi, but not in P. feodorovi).However, P. feodorovi is not genetically related to P. blanchardi.In the COI phylogram, P. feodorovi was associated with P. oeconomi, appearing as one of the basal lineages within Paranoplocephala (Fig. 2).Th e phylogenetic position of P. feodorovi supports the hypothesis that Parandrya Gulyaev & Chechulin, 1996 is a synonym of Paranoplocephala s.l., as suggested by Haukisalmi and Henttonen (2000) and Haukisalmi and Wickström (2005).
Paranoplocephala buryatiensis Haukisalmi, Hardman, Hardman, Laakkonen, Niemimaa & Henttonen, 2006 P. buryatiensis, described from Buryatian material, is primarily a parasite of Myodes rufocanus (Haukisalmi et al. 2007a), although it was found occasionally in Microtus fortis in the present material.Its known range spans from Buryatia to north-eastern Siberia.However, it does not occur in the westernmost populations of Myodes rufocanus in northern Fennoscandia (Haukisalmi et al. 2007a).P. buryatiensis is morphologically and genetically closely related to P. longivaginata Chechulin & Gulyaev, 1998 from Myodes rutilus (below); they form a strongly supported clade in the mtDNA phylogenies (Fig. 2 and Haukisalmi et al. 2007a).Haukisalmi et al. (2007a) provided evidence for phylogenetic codivergence of P. buryatiensis and P. longivaginata with their primary hosts.
P. buryatiensis occurred in Myodes rufocanus at four of the eight study sites, the prevalence in Pasolskaya (43%) being higher than in the other sites (0-10%).It also occurred in Microtus fortis at two study sites, where it also parasitized the primary host Myodes rufocanus.
P. longivaginata occurred at fi ve of the seven study sites, but always at a low prevalence.
In the present material, Arostrilepis horrida-like cestodes occurred commonly in all Microtus and Myodes species and accidentally in C. barabensis but were absent in A. peninsulae.Th e data of Zhaltsanova (1992) show an equally wide host spectrum for Arostrilepis horrida-like cestodes in Buryatia.Th ree species have been described within the A. horrida-complex, i.e. A. horrida from an unknown rodent host, A. beringiensis Kontrimavichus & Smirnova, 1991 from Lemmus sibiricus and A. microtis Gulyaev & Chechulin, 1997 from Microtus spp.(see Kontrimavichus andSmirnova 1991 andGulyaev andChechulin 1997).However, applying molecular methods, the BCP has demonstrated a complex of at least 10 species of A. horrida-like cestodes that are poorly diff erentiated morphologically (Cook et al. 2005, and K. Galbreath and E. P. Hoberg, unpublished).For example, there may be fi ve Arostrilepis-species in Myodes spp.(species previously assigned to Clethrionomys) and two species in Microtus spp. in the Holarctic region.Th e situation is complicated by the fact that it is not known which one (if any) of them is the true A. horrida.Th erefore, it is not possible to assign the present specimens to a particular species of Arostrilepis.However, it is probable that the Buryatian material includes at least one undescribed species, i.e. that/those occurring in Myodes spp.

Rodentolepis sp.
Rodentolepis sp. from Cricetulus barabensis was characterized by 22-24 fraternoid hooks.In this respect it resembles Rodentolepis fraterna (Stiles, 1906) (see Genov 1984) and R. sinensis (Oldham, 1929) (see Ryzhikov et al. 1978), although the hooks of the present species are somewhat longer (0.020-0.032 mm) than those of R. fraterna.In addition, the scolex of the present cestode is signifi cantly larger (0.24-0.26 mm) than that of R. sinensis, and its eggs are signifi cantly longer (0.060-0.075 mm) than those of R. fraterna.It is probable that Rodentolepis sp. from Buryatia represents an undescribed, host-specifi c species of C. barabensis.Th e high variation in hook length suggests that the material may include more than one species.Zhaltsanova (1992) reported R. straminea (Goeze, 1782) from C. barabensis and Myodes rufocanus from Buryatia, but that identifi cation is dubious, because many forms, including R. fraterna and R. nana (Siebold, 1852), have been synonymized with R. straminea in the Soviet/Russian literature (see Ryzhikov et al. 1978).
(earlier known as Anoplocephaloides variabilis-like cestodes), appears to be absent in Buryatia, although it occurs widely in Europe and North America, and sporadically in North-East Siberia (Haukisalmi et al. 2008).Th e high species diversity and endemism in Europe is partly due to the high taxonomic diversity of voles in that region; of the 14 vole and lemming species considered in the present comparison, seven occur exclusively in Europe or western Eurasia.If we restrict the comparison to the vole species with a wide Palaearctic or Holarctic distribution (Microtus oeconomus, Myodes rufocanus and M. rutilus), Europe (eight spp.) still shows somewhat higher species diversity than Buryatia (four species) and North-East Siberia (six spp.).Th e diff erences in species diversity may also refl ect the more extensive research and use of molecular methods for the European fauna.However, high European species diversity has also been indicated for anoplocephalid cestodes of lagomorphs (Spasskii 1951), suggesting that this phenomenon may have a more general biogeographical and/or historical explanation.Th e existing phylogenetic data indicate a possible European origin for Microcephaloides Haukisalmi, Hardman, Hardman, Rausch & Henttonen, 2008(see Haukisalmi et al. 2008) and Paranoplocephala s. str.(see Haukisalmi et al. 2004), suggesting that Europe may have acted as a centre of origin for several independent anoplocephalid lineages.However, the phylogenetic relationships among various anoplocephalid lineages are still unresolved, which impedes more comprehensive analyses on the historical biogeography of anoplocephalid cestodes of arvicoline rodents.
Th e faunistic connections of Buryatia seem to be slightly closer to North-East Siberia than to Europe; Buryatian anoplocephalids shared three and two species with these regions, respectively.Moreover, an unexpected connection is seen between Buryatia and eastern Beringia; namely, the sister clade/species of the Buryatian P. cf. omphalodes occurs in the northernmost Alaska, but probably not elsewhere in North America.Neither clade has been found in extensive studies in the North-East Siberia (BCP), suggesting that the present distribution represents a relict of an earlier continuous distribution.On the continental level, the Buryatian taiga corridor has probably not played a signifi cant role as a migration pathway among anoplocephalid cestodes of rodents; only one species with a continent-wide distribution (A.cf.dentata) occurred in Buryatia.Moreover, A. cf.dentata also occurs also in North-Central Siberia (Tunguska river region; Haukisalmi et al. 2009) and may not necessarily have utilized the Buryatian corridor.
Th e present and earlier studies (Haukisalmi et al. 2008, Haukisalmi et al. 2004, Hu et al. 2005) show that molecular markers, such COI sequences, are necessary tools in the taxonomy of anoplocephalid cestodes, which lack a rostellum and hooks and which can be devoid of reliable distinguishing features.It can be estimated that 50-60% of species diversity of anoplocephalid cestodes of voles and lemmings in the Holarctic region would have remained undetected if the taxonomy had been based on morphological features only (Table 2 and unpublished records of the BCP and present authors).No molecular systematic studies exist for catenotaeniids but because they also lack anchoring devices, additional, cryptic species diversity is expected to occur also within this family.
Th e present results demonstrate high variation among study sites in cestodes of Buryatian rodents, with the exception of the ubiquitous A. horrida-complex (Table 1).Haukisalmi and Henttonen (1999) showed that patchiness is a predictable feature of certain helminths of rodents and that such patches may be remarkably persistent.Th e BCP studies also reveal fi ne-scaled spatial variation in many anoplocephalid cestodes of voles and lemmings in Alaska and adjacent regions (Haukisalmi et al. 2009, Haukisalmi et al. 2004, Wickström et al. 2003).Patchiness has an obvious implication for assessment of the biodiversity cestodes: species and genetic diversity may be much underestimated in samples that are not spatially comprehensive.In addition, helminths may be very specialized in a certain cohort(s) and/or sex of the host, related to seasonally changing heterogeneity of rodent populations (Haukisalmi et al. 1995), which should also be considered in the estimation of parasite diversity.

Fig. 2 .
Fig. 2. A neighbour-joining reconstruction of partial cytochrome oxidase I (mtDNA) sequences of Paranoplocephala spp., Andrya rhopalocephala and Neandrya cuniculi from lagomorphs were used as an outgroup.Th e labels show the GenBank number for each sequence.Values at nodes show the percentage from 10000 bootstrap replicates.
Ganzorig et al. (1999)demus peninsulae had 29-34 uterine branches, which corresponds with Catenotaenia afghanaTenora, 1977, C. californica Dowell, 1953, C.  neotomae Babero & Cattan, 1983 and C. peromysci Smith, 1954.However, the cestodes found by us have a clearly divided testicular fi eld, a feature which occurs in C. californica but not in the three other species mentioned above.Compared with the present material, C. californica has markedly wider body (0.75-0.83 mm in the present specimens) and smaller scolex (ca.0.38 mm in the present specimens).Since C. californica, C. neotomae and C. peromysci are Nearctic parasites of heteromyid and sigmodontid (Neotominae) rodents, it is unlikely that they would occur in a murid rodent in Eurasia.Th e brief original description of C. afghana was based on material from two unrelated host species, Cricetulus migratorius (Pallas) and Alticola sp. from Afghanistan, and it was not stated which was the type host.Moreover, no type specimen was designated(Tenora 1977).Ganzorig et al. (1999)later redescribed C. afghana from Lasiopodomys brandtii (Radde) from Mongolia but it is probable that the specimens assigned to C. afghana include more than one species.Th e specimens from Buryatian A. peninsulae may therefore represent another undescribed species of Catenotaenia.Voucher specimen: MSB Endo 158 from A. peninsulae (Kamensk).

Table 2 .
Host and geographic distribution of anoplocephalid cestodes of voles and lemmings in Europe, and My.rutilus(ruti).Th e Arctic lemmings of the genera Lemmus and Dicrostonyx have been excluded, because they are absent in Buryatia.Th e primary host species in each region (when evident) have been in indicated in bold.