International Journal for Parasitology 43 (2013) 427–437

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International Journal for Parasitology

journal homepage: www.elsevier.com/locate/ijpara

Molecular phylogeny of the genus Taenia (Cestoda: Taeniidae): Proposals

for the resurrection of Hydatigera Lamarck, 1816 and the creation of a new
genus Versteria q
Minoru Nakao a,⇑, Antti Lavikainen b, Takashi Iwaki c, Voitto Haukisalmi d,e, Sergey Konyaev f,
Yuzaburo Oku g, Munehiro Okamoto h, Akira Ito a
a
Department of Parasitology, Asahikawa Medical University, Asahikawa, Hokkaido 078-8510, Japan
b
Infection Biology Program/Department of Bacteriology and Immunology, Haartman Institute, P.O. Box 21, FI-00014 University of Helsinki, Finland
c
Meguro Parasitological Museum, Shimomeguro, Meguro-ku, Tokyo 153-0064, Japan
d
Finnish Forest Research Institute, Vantaa Research Unit, P.O. Box 18, FI-01301 Vantaa, Finland
e
Finnish Museum of Natural History, P.O. Box 17, FI-00014 University of Helsinki, Finland
f
Institute Systematics and Ecology of Animals, Siberian Branch Russian Academy of Sciences, Novosibirsk 630091, Russia
g
Department of Parasitology, School of Veterinary Medicine, Faculty of Agriculture, Tottori University, Koyama, Tottori 680-8553, Japan
h
Section of Wildlife Diversity, Center for Human Evolution Modeling Research, Primate Research Institute, Kyoto University, Inuyama, Aichi 484-8506, Japan

a r t i c l e i n f o a b s t r a c t

Article history: The cestode family Taeniidae generally consists of two valid genera, Taenia and Echinococcus. The genus
Received 29 September 2012 Echinococcus is monophyletic due to a remarkable similarity in morphology, features of development and
Received in revised form 23 November 2012 genetic makeup. By contrast, Taenia is a highly diverse group formerly made up of different genera.
Accepted 25 November 2012
Recent molecular phylogenetic analyses strongly suggest the paraphyly of Taenia. To clarify the genetic
Available online 18 February 2013
relationships among the representative members of Taenia, molecular phylogenies were constructed
using nuclear and mitochondrial genes. The nuclear phylogenetic trees of 18S ribosomal DNA and concat-
Keywords:
enated exon regions of protein-coding genes (phosphoenolpyruvate carboxykinase and DNA polymerase
Molecular phylogeny
Taenia
delta) demonstrated that both Taenia mustelae and a clade formed by Taenia parva, Taenia krepkogorski
Hydatigera and Taenia taeniaeformis are only distantly related to the other members of Taenia. Similar topologies
Versteria gen. nov were recovered in mitochondrial genomic analyses using 12 complete protein-coding genes. A sister rela-
tionship between T. mustelae and Echinococcus spp. was supported, especially in protein-coding gene
trees inferred from both nuclear and mitochondrial data sets. Based on these results, we propose the res-
urrection of Hydatigera Lamarck, 1816 for T. parva, T. krepkogorski and T. taeniaeformis and the creation of
a new genus, Versteria, for T. mustelae. Due to obvious morphological and ecological similarities, Taenia
brachyacantha is also included in Versteria gen. nov., although molecular evidence is not available. Taenia
taeniaeformis has been historically regarded as a single species but the present data clearly demonstrate
that it consists of two cryptic species.
Ó 2013 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction nearly 50 species (Loos-Frank, 2000; Hoberg, 2006; Rossin et al.,

The order Cyclophyllidea, which includes 15 families, is the
largest taxonomic group within the Cestoda (Jones et al., 1994).
The cyclophyllidean family, Taeniidae, is generally accepted to
be composed of two valid genera, Taenia Linnaeus, 1758 and Echi-
nococcus Rudolphi, 1801. Although Echinococcus is a small genus
containing nine species (Nakao et al., 2007), Taenia consists of
q
Nucleotide sequence data reported in this paper are available in DDBJ/EMBL/
GenBank databases under the accession numbers AB731615-53, AB731675,
AB731726-27, AB731758-62, AB732957-60 and AB745096-98.
⇑ Corresponding author. Tel.: +81 166682422; fax: +81 166682429.
E-mail address: nakao@asahikawa-med.ac.jp (M. Nakao).
2010; Haukisalmi et al., 2011). Species of Taenia are abundant
in Africa but only one endemic species has been found in South
America (Rossin et al., 2010) and there are no endemic species
in Australia (Loos-Frank, 2000). The cosmopolitan species of Tae-
nia are of medical and veterinary importance because the adult
tapeworms are responsible for intestinal infections (taeniasis)
and the metacestode larvae for systemic infections (cysticercosis
and coenurosis) in domestic animals and humans. Terrestrial
mammals are essential for the life cycles of Taenia spp. Members
of the order Carnivora mainly act as definitive hosts, and their
prey belonging to the orders Rodentia, Lagomorpha and Cetartio-
dactyla as intermediate hosts (Loos-Frank, 2000; Hoberg, 2006).
Humans serve as definitive hosts specifically for Taenia solium

0020-7519/$36.00 Ó 2013 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.ijpara.2012.11.014
428 M. Nakao et al. / International Journal for Parasitology 43 (2013) 427–437
Linnaeus, 1758, Taenia saginata Goeze, 1782 and Taenia asiatica
Eom & Rim, 1993, which are referred to as the ‘‘human-Taenia’’
spp. (Hoberg, 2006). The adult tapeworms of Taenia spp. living
in the small intestine of definitive hosts release their egg-filled
proglottids into the environment. The eggs are ingested by inter-
mediate hosts and develop into metacestodes in various internal
organs and body cavities.
The phylogenies of cyclophyllidean tapeworms have been
widely examined at familial, generic and specific levels, using cla-
distic analyses based on morphological characters (Hoberg et al.,
1999, 2000, 2001). The family Taeniidae was suggested to be the
sister group to the Paruterinidae and Metadilepididae, and to be
closely related to the puzzling genus, Dasyurotaenia Beddard,
1912, occurring in Australian marsupials (Hoberg et al., 1999).
The morphological phylogenies also demonstrated that the genus
Taenia is monophyletic and that Taenia mustelae Gmelin, 1790,
occurring in weasels, is sister to the remaining species (Hoberg
et al., 2000). The human-Taenia spp. have close relationships with
the African endemic species of Taenia from hyenas and lions, sug-
gesting a hypothesis that early hominids acquired carnivore-de-
rived tapeworms through dietary and behavioral changes from
herbivory to scavenging and carnivory (Hoberg et al., 2001).
There are few studies on the molecular phylogeny of cyclo-
phyllidean tapeworms at family level (von Nickisch-Rosenegk
et al., 1999; Foronda et al., 2004). The lack of comprehensive
molecular data makes it difficult to determine an appropriate out-
group for studies of taeniid phylogeny. Most molecular analyses
have been focused on the elucidation of relationships within the
genus Taenia or Echinococcus due to their importance as zoonotic
pathogens (Bowles and McManus, 1994; Bowles et al., 1995;
Okamoto et al., 1995a; Gasser et al., 1999; Nakao et al., 2007;
Zhang et al., 2007; Hüttner et al., 2008; Lavikainen et al., 2008;
Jia et al., 2010, 2012; Liu et al., 2011; Knapp et al., 2011). Recent
phylogenetic analyses using many taeniid taxa have clearly dem-
onstrated all the members of Echinococcus to be monophyletic
(Lavikainen et al., 2008; Knapp et al., 2011), supporting their phe-
notypic similarities in morphology and development. On the con-
trary, Taenia appears to be a highly diversified assemblage.
Phylogenies inferred from mitochondrial and nuclear genes indi-
cate that Taenia is a paraphyletic group, in which T. mustelae is a
sister taxon to the species of Echinococcus (Lavikainen et al.,
2008; Knapp et al., 2011). A clade consisting of Taenia parva Baer,
1926 and Taenia taeniaeformis (Batsch, 1786) was also found to
be distantly related to the other taeniids (Knapp et al., 2011).
Taxonomic concepts including subfamilies and tribes have been
proposed to subdivide the diversity of taeniid tapeworms (Hoberg
et al., 2000). Apart from Echinococcus, there are still disagreements
in the classification of taeniid genera (Abuladze, 1964; Verster,
1969; Schmidt, 1986; Rausch, 1994). The genera Cladotaenia Cohn,
1901, Paracladotaenia Yamaguti, 1935, Anoplotaenia Beddard, 1911,
Dasyurotaenia Beddard, 1912 and Insinuarotaenia Spasskii, 1948
have been placed until recently in the Taeniidae, but these are
now classified in other families (Rausch, 1994). Historically, the
nominal genera Multiceps Goeze, 1782, Taeniarhynchus Weinland,
1858, Hydatigera Lamarck, 1816, Fossor Honess, 1937, Tetratirotae-
nia Abuladze, 1964 and Monordotaenia Little, 1967 were created
within Taeniidae, but all of them were eventually synonymized
with Taenia by Verster (1969). Subsequently, Fimbriotaenia Kor-
nyushin & Sharpilo, 1986 was established, but was subsequently
synonymized with Taenia by Rausch (1994). Multiceps, Hydatigera,
Tetratirotaenia and Fimbriotaenia were characterized mainly by
their larval morphology, whereas Taeniarhynchus, Monordotaenia
and Fossor were differentiated by variations in the shape or num-
bers of rows of rostellar hooks. Among these synonymized genera,
Hydatigera sensu Abuladze (1964) is still presently used, mainly in
post-Soviet states (Bessonov et al., 1994; Shimalov, 2009). Molecu-
lar systematic analyses based on nuclear protein-coding genes cor-
roborate Hydatigera as a valid genus (Knapp et al., 2011).
The present study aimed to obtain additional molecular data to
reconfirm the paraphyly of Taenia, which was inferred from nucle-
ar protein-coding genes (Knapp et al., 2011). For this purpose, 18S
ribosomal DNA (rDNA) and whole mtDNA were further sequenced
in representative taxa of Taenia and Echinococcus. The resultant
phylogenetic trees enable us to propose the resurrection of Hyda-
tigera for T. parva, T. taeniaeformis and Taenia krepkogorski (Schultz
& Landa, 1934) and the creation of a new genus, Versteria, for T.
mustelae. The ubiquitous T. taeniaeformis has been historically re-
garded as a single species, but the present multi-locus approach
clearly demonstrates that it includes cryptic species.
2. Materials and methods
2.1. Taxa and DNA sequences used
A total of 38 taeniid samples representing 16 species of Taenia
and nine species of Echinococcus as well as an outgroup, Dipylidium
caninum (Linnaeus, 1758) (Cyclophyllidea: Dipylidiidae), were
used for the construction of a molecular phylogeny (Table 1). The
samples were generally the same individuals as those used in pre-
vious studies (Nakao et al., 2002, 2007; Lavikainen et al., 2008;
Knapp et al., 2011). In the case of T. taeniaeformis, the representa-
tive isolates BMM, SRN, TtaFi and ACR were added because previ-
ous studies suggested a possibility that at least two cryptic
species might exist within this taxon (Iwaki et al., 1994; Nonaka
et al., 1994; Okamoto et al., 1995a, 1995b; Azuma et al., 1995; Lavi-
kainen et al., 2008; Galimberti et al., 2012; Jia et al., 2012). The
developmental stage of each sample was recorded in the individual
entry of nucleotide databases (Table 1). The isolate name of each
sample was also added as an identifier for the database record.
For each sample, nuclear DNA sequences of 18S rDNA, phospho-
enolpyruvate carboxykinase (pepck) and DNA polymerase delta
(pold) were determined. Most sequences of the protein-coding
genes have already been published (Knapp et al., 2011). On the
other hand, whole mitochondrial genome sequencing was per-
formed in 12 species. The previously published sequences of tae-
niid mitochondrial genomes (Le et al., 2000, 2002; Nakao et al.,
2002, 2003, 2007; Jeon et al., 2005, 2007; Jia et al., 2010; Liu
et al., 2011) were also utilized in this study.
2.2. DNA amplification and sequencing
As reported previously (Lavikainen et al., 2008; Knapp et al.,
2011), genomic DNA (gDNA) was extracted from ethanol-pre-
served samples (larval tissues or adult proglottids) by using a
DNeasy Blood & Tissue kit (QIAGEN, Hilden, Germany), and then
stored at 20 °C until further use for PCR. In one exceptional case,
the gDNA of Echinococcus felidis Ortlepp, 1937 was extracted from
eggs, which were isolated from lion faeces (Hüttner et al., 2008).
The almost complete sequence of nuclear 18S rDNA of each
taeniid species was amplified by high-fidelity PCR using the pri-
mer pair WormA and WormB (Littlewood and Olson, 2001). The
PCR was performed in a final volume of 50 lL containing 1 lL of
template DNA (100 ng), 0.25 lM of each primer, 2.5 mM of
each dNTP, 1 unit of PrimeSTAR GXL DNA polymerase (TaKaRa
BIO INC., Otsu, Shiga, Japan) and the manufacturer-supplied reac-
tion buffer. A three-step thermal process consisting of 98 °C for
10 s, 55 °C for 15 s and 68 °C for 150 s was repeated 35 times
to amplify DNA fragments ranging in length from 2.4 to 2.8 kb.
Due to the presence of multiple copies of 18S rDNA, each of
the PCR amplicons was cloned into a pGEM-T plasmid vector
(Promega Corp., Madison, WI, USA) and then introduced into
 M. Nakao et al. / International Journal for Parasitology 43 (2013) 427–437 429

Table 1
Tapeworm taxa and their DNA sequences used in this study.

Species (isolates) Localities Database accession numbers of DNA sequences

18S rDNA pepck pold mtDNA (References)
Taenia
T. solium Ecuador AB731615 FN567996a FN568367a
T. solium China NC_004022 (Nakao et al., 2003)b
T. saginata Belgium AB731616 FN567997a FN568368a
T. saginata Belgium NC_009938 (Jeon et al., 2007)b
T. asiatica Taiwan AB731617 FN567998a FN568369a
T. asiatica Korea NC_004826 (Jeon et al., 2005)b
T. crassiceps Canada AB731618 FN567999a FN568370a NC_002547 (Le et al., 2000)b
T. hydatigena China AB731619 FN568000a FN568371a
T. hydatigena China NC_012896 (Jia et al., 2010)b
T. serialis Australia AB731620 FN568001a FN568372a AB731674 (This study)b
T. multiceps Unknown AB731621 FN568002a FN568373a
T. multiceps China NC_012894 (Jia et al., 2010)b
T. ovis New Zealand AB731622 FN568003a FR869707a AB731675 (This study)b
T. madoquae Kenya AB731623 FR869699a FR869705a AB731726 (This study)b
T. laticollis Finland AB731624 FR869697a FN869703a AB731727 (This study)b
T. martis Croatia AB731625 FR852569a FN869706a AB731758 (This study)b
T. twitchelli Russia AB731626 FR852568a FN869710a AB731759 (This study)b
T. parva Spain AB731627 FR869700a FN869708a AB731760 (This study)b
T. taeniaeformis
sp. A (BMM) Belgium AB731628 AB731644 AB731649 AB745096 (This study)c
sp. A (SRN) Japan AB731629 AB731645 AB731650 AB745097 (This study)c
sp. A China NC_014768 (Liu et al., 2011)b
sp. B (ACR) Japan AB731630 AB731646 AB731651 AB745098 (This study)c
sp. B (TtaFi) Finland AB731631 AB731647 AB731652 AB731761 (This study)b
T. krepkogorski China AB731632 AB731648 AB731653 AB731762 (This study)b
T. mustelae (TmuFi9) Finland AB731633 FR869698a FR869704a AB732957 (This study)b
T. mustelae (Hokkaido) Japan AB732960 (This study)c
Echinococcus
E. multilocularis Japan AB731634 FN567985a FN568356a NC_000928 (Nakao et al., 2002)b
E. shiquicus China AB731635 FN567986a FN568357a NC_009460 (Nakao et al., 2007)b
E. vogeli Colombia AB731636 FN567987a FN568358a NC_009462 (Nakao et al., 2007)b
E. oligarthrus Panama AB731637 FN567988a FN568359a NC_009461 (Nakao et al., 2007)b
E. felidis Uganda AB731638 FN567989a FN568360a AB732958 (This study)b
E. granulosus China AB731639 FN567990a FN568361a
E. granulosus England NC_008075 (Le et al., 2002)b
E. equinus England AB731640 FN567991a FN568362a
E. equinus England AF346403 (Le et al., 2002)b
E. ortleppi Argentina AB731641 FN567992a FN568363a NC_011122 (Nakao et al., 2007)b
E. canadensis Peru AB731642 FN567993a FN568364a
E. canadensis Kazakhstan NC_011121 (Nakao et al., 2007)b
Dipylidium
D. caninum Unknown AB731643 FR869702a FR869711a AB732959 (This study)b
a
Nucleotide sequences published in our previous report (Knapp et al., 2011).
b
Complete nucleotide sequences of mitochondrial genomes.
c
Nucleotide sequences of cox1 gene.

Escherichia coli strain JM109. At least three colonies were picked
from an agar plate and their insert DNAs were sequenced. After
confirming the low heterogeneity of the clonal sequences, one
randomly selected sequence was used for phylogenetic
reconstruction.
The nuclear protein-coding genes, pepck and pold, in T. taeniae-
formis and T. krepkogorski were amplified as reported previously
(Knapp et al., 2011). The whole mtDNA of each taeniid species
was determined using noncontiguous sequence islands (Nakao
et al., 2003). In brief, the initial amplifications of mtDNA were car-
ried out using primers designed from conserved regions and the
resultant sequences allowed us to design new primers whereby
the remaining unknown regions were amplified. All sequences ob-
tained, encompassing the complete mtDNA, were compiled into a
single sequence.
The DNA sequencing of cloned plasmid inserts and direct PCR
products was carried out using a BigDye terminator cycle sequenc-
ing kit and ABI genetic analyzers 310 and 3500 (Applied Biosys-
tems, Foster City, CA, USA). Plasmid universal primers and PCR
primers were used as sequencing primers. The long template DNAs
were read by primer walking.
2.3. Reconstruction of molecular phylogeny
A total of 29 operational taxonomic units (OTUs) consisting of
26 species were used for the multiple sequence alignment of 18S
rDNA (Table 1). The alignment program T-coffee (Notredame
et al., 2000) was employed, together with the optional program
R-coffee especially designed for RNA (Taly et al., 2011). The original
generated alignment was edited with the software package MEGA
5 (Tamura et al., 2011) to completely delete gaps. The trimmed
alignment was used as a working data set for nuclear 18S rDNA
(a total of 1,841 nucleotide sites).
The sequences of both pepck and pold genes were aligned with T-
coffee using 29 OTUs of 26 species (Table 1). As reported previously
(Knapp et al., 2011), putative introns were manually excluded from
each of the alignments. The resulting exon alignments of pepck
(1,452 sites) and pold (921 sites) were concatenated to make a data
set for nuclear protein-coding genes (2,373 sites). The appropriate-
ness of the concatenation was assessed by the incongruence length
difference (ILD) test (Farris et al., 1995) in the software PAUP 4.0b
(Sinauer Associates, Sunderland, MA, USA) using a heuristic tree
search with 1,000 replicates.
430 M. Nakao et al. / International Journal for Parasitology 43 (2013) 427–437
In the case of mitochondrial data sets, 27 OTUs of 26 species
(Table 1) were used for assembling the alignments of amino acid
and nucleotide sequences. The accurate alignment of mtDNA se-
quences among distantly related Taenia spp. is difficult due to
the strong bias of AT-richness. Nucleotide alignments, therefore,
were made under the guidance of amino acid alignments. Each of
12 protein-coding genes from complete mitochondrial genomes
was translated into amino acid sequences using the echinoderm
mitochondrial genetic code (Nakao et al., 2000; Telford et al.,
2000). The deduced amino acid sequences were aligned gene-by-
gene using T-coffee. The program CodonAlign 2.0 (Hall, 2004) sub-
sequently created nucleotide alignments according to the amino
acid alignments. All of the amino acid alignments were concate-
nated to make a data set for mitochondrial proteins (3,388 sites).
Using PAUP 4.0b, all of the sites of third codon positions were re-
moved from the nucleotide alignments to avoid synonymous sub-
stitution saturation. All of the corrected alignments were
concatenated to prepare a data set for mitochondrial protein-cod-
ing genes (6,776 sites).
The sequence characteristics of the nuclear and mitochondrial
genes were examined using MEGA 5. Pairwise divergence values
and their means within each genus were calculated under the Kim-
ura 2-parameter model (Kimura, 1980) with a gamma setting of
0.5. The pairwise divergence of deduced amino acid sequences
was computed using JTT model (Jones et al., 1992) with a gamma
setting of 0.5.
MEGA 5 was also employed to select nucleotide and amino acid
substitution models for each data set. The nucleotide model
HKY + G + I (Hasegawa et al., 1985) was applied to the 18 rDNA
data set, and GTR + G + I (Tavaré, 1986) to the sets of nuclear and
mitochondrial protein-coding genes. The amino acid model
JTT + G + I (Jones et al., 1992) was selected for the set of mitochon-
drial proteins. Phylogenies were reconstructed with the maxi-
mum-likelihood (ML) method as implemented in the program
PhyML 3.0 (Guindon et al., 2010) using each of the data sets with
its appropriate substitution model. The initial phylogenetic tree
was estimated using the BIONJ algorithm (Gascuel, 1997), and
the robustness of ML trees was tested by bootstrapping with 500
replicates. An approximate likelihood ratio test (aLRT) using SH-
like branch supports (Shimodaira and Hasegawa, 1999; Guindon
et al., 2010) was conducted to evaluate tree topologies. Branches
with aLRT values of more than 0.95 were considered to be
significant.
Alternative phylogenies were inferred by the Bayesian program,
MrBayes 3.2.1 (Ronquist et al., 2012) using the same data sets and
substitution models as those of ML analyses. The Markov chain
Monte Carlo (MCMC) analysis was run for 1 million generations
and sampled every 100 generations to estimate the posterior prob-
abilities of phylogenetic trees. The run produced 10,000 trees, of
which the initial 1,000 trees were treated as burn-in. Convergence
was assessed using the program Tracer 1.5 (http://
tree.bio.ed.ac.uk).
The ML and Bayesian analyses were conducted at least twice for
each data set to verify the consistency of results. The graphical
Table 2
Characteristics of alignment data sets used for phylogenetic reconstruction.
Data sets Na
Nuclear 18S rDNA (without gaps) 1,841
Nuclear pepck + pold (without introns) 2,373
Mitochondrial protein genes (without 3rd codon positions) 6,776
Mitochondrial proteins (amino acids) 3,388
a
N, number of sites examined; V, number of variable sites; P, number of parsimony-informative sites.
b
The means of pairwise divergence values within each genus.
viewer, FigTree 1.3 (http://tree.bio.ed.ac.uk), was used to draw
phylogenetic trees. All of the trees were rooted using the outgroup
taxon, D. caninum.
Since the outgroup used is distant from the Taeniidae (Hoberg
et al., 1999), unrooted trees were also constructed by ML analyses
to illustrate the relatedness of leaf nodes. After removing the se-
quence data of D. caninum from each of the data sets, unrooted
phylogenies were computed by PhyML 3.0 using appropriate sub-
stitution models. Their robustness was tested by bootstrapping
with 500 replicates.
2.4. Physical voucher specimens
Voucher specimens used for the taxonomic revision of Taenia
are listed in Supplementary Table S1. These were deposited in
the Meguro Parasitological Museum, Tokyo, Japan and the Zoolog-
ical Museum, Finnish Museum of Natural History, Helsinki, Fin-
land. The DNA of the other specimens of Taenia and Echinococcus
spp. were provided by various researchers over a long time period,
and no vouchers were retained.
3. Results
3.1. Sequence characteristics
The sequence characteristics of alignment data sets used for
phylogenetic analyses are summarized in Table 2. The data set of
nuclear protein-coding genes, pepck and pold, was more variable
than the set of nuclear 18S rDNA, suggesting that the former set
is more informative. The ILD test showed that the gene partitions
of the data set of pepck and pold were homogeneous (P = 0.480).
The data sets of mitochondrial protein-coding genes and their de-
duced proteins showed the highest variability. In all data sets, var-
iable and parsimony-informative sites were more abundant in
Taenia than in Echinococcus. The means of pairwise divergence val-
ues were also higher in Taenia.
3.2. Phylogenies inferred from nuclear genes
A phylogeny inferred from the data set of 18S rDNA is illus-
trated in Fig. 1A. A monophyletic grouping of Taenia spp. was
shown by ML analysis, but bootstrapping and aLRT values revealed
that the tree topology was not robust. Based on the branching pat-
tern, three major groups were defined as clades Ia, Ib and II. Clade II
consisting of T. taeniaeformis, T. krepkogorski and T. parva was sister
to the other members of Taenia except for T. mustelae. The BMM,
SRN, ACR and TtaFi isolates of T. taeniaeformis sensu lato (s.l.)
(Okamoto et al., 1995a; Lavikainen et al., 2008) were separated
into two divergent groups, which were temporarily designated as
sp. A and sp. B. Both of them were closely related to T. krepkogorski.
A sister relationship between T. mustelae and the other Taenia spp.
was not supported due to the low bootstrap value for the corre-
sponding node. A Bayesian analysis using the data set of 18S rDNA
Taenia Echinococcus Pairwise divergenceb
V P (%) V P (%) Taenia Echinococcus
260 146 (7.9) 154 38 (2.1) 0.044 0.025
720 489 (20.6) 109 23 (1.0) 0.131 0.013
2,605 1,879 (27.7) 1,365 614 (9.1) 0.206 0.088
1,749 1,375 (40.6) 1,059 512 (15.1) 0.424 0.175
 M. Nakao et al. / International Journal for Parasitology 43 (2013) 427–437 431

(A) 98
(100) T. madoquae (B) 99
(100) T. madoquae
56 78
( ) T. serialis (99) T. serialis
*
99 99
( ) T. multiceps (100) T. multiceps
*
88
T. asiatica Clade Ia 100
T. asiatica Clade Ia
(100) (100)

93 100 T. saginata 100 100 T. saginata

(100) (100) (100) (100)
T. ovis T. ovis
50
63 T. solium (70) T. solium
(94) 94 83
(100) T. martis (77)
T. martis
96
T. twitchelli Clade Ib (100) T. twitchelli Clade Ib
100 100
(100) T. crassiceps (100) T. crassiceps
66
(97) T. hydatigena T. hydatigena
61 61
(98) 70 T. laticollis (81) 86 T. laticollis
(95) (100)
98
(100) BMM T. mustelae
T. taeniaeformis
42
( ) SRN sp. A
* 88
53 100 ACR (100)
(58) ( ) T. taeniaeformis Clade II
67
* 100 TtaFi sp. B
(100) (100)
T. krepkogorski 100
(100) Echinococcus
T. parva
9 species
T. mustelae

100
(100) BMM
100
(100)
T. taeniaeformis
Echinococcus 92
sp. A
(100) SRN
9 species
100 ACR T. taeniaeformis
(100) Clade II
100 100 TtaFi sp. B
(100) (100)
T. krepkogorski
T. parva
0.05 0.05

Fig. 1. Phylogenetic trees of taeniid cestodes inferred from nuclear DNA sequences by using maximum likelihood and Bayesian analyses. Phylograms are depicted only by the
ML analysis, because the resultant topology was the same as that of the Bayesian analysis. Scale bars represent the estimated number of substitutions per site. An outgroup
(Dipylidium caninum) has been omitted from the trees. Values of each node are ML bootstrap percentages. Closed circles represent branches supported by SH-like aLRT values
of more than 0.95. Bayesian posterior probability percentages are shown in parentheses. Asterisks denote nodes collapsing to polytomies in the Bayesian analysis. (A)
Phylogram from 18S rDNA. (B) Phylogram from the protein-coding genes pepck and pold (concatenated exons).

also showed a similar topology to that of the ML analysis. However,
several internal nodes of clades Ia and II were collapsed in the
Bayesian analysis (Fig. 1A).
Another phylogenetic tree was reconstructed using the data set
of the protein-coding genes, pepck and pold (Fig. 1B). Clades Ia, Ib
and II were consistently supported by both ML and Bayesian anal-
yses, but clade II moved to a distinct position sister to all of the
other taeniid spp. including Echinococcus. In the ML analysis, the
sister relationship between clades Ia and Ib was not robust, as indi-
cated by the low bootstrap value. The internal topologies of the
three clades were identical to those inferred from 18S rDNA. The
phylogeny based on the protein-coding genes further demon-
strated that T. mustelae is sister to Echinococcus spp., as reported
previously (Knapp et al., 2011). The tentative separation of sp. A
and sp. B of T. taeniaeformis s.l. was highly supported by both the
ML and Bayesian inferences.
Two unrooted trees were also inferred by ML analyses using the
nuclear gene data sets of 18S rDNA and the protein-coding genes,
pepck and pold. The positions of leaf nodes and their clusters were
quite similar in both of the resultant trees; however, bootstrap
supports at most internal nodes were lower in the 18S rDNA tree
(Supplementary Fig. S1). The unrooted tree based on the protein-
coding genes clearly demonstrated that clade II, T. mustelae and
Echinococcus spp. are distantly related to the other members of
the Taeniidae.
3.3. Phylogenies inferred from mitochondrial genes
The gene arrangement of taeniid mitochondrial genomes could
not be used for phylogenetic inference because all of the genomes
examined in this study showed the same arrangement. All of the
protein-coding genes of taeniid mitochondrial genomes were,
therefore, utilized for phylogenetic analyses. As shown in Fig. 2A,
clades Ia, Ib and II were highly supported by both ML and Bayesian
analyses using the amino acid data set of mitochondrial proteins.
Clade II retained a sister position to the other members of Taenia
spp. except for T. mustelae. Both analyses also depicted a sister rela-
tionship between T. mustelae and Echinococcus spp., but in the case
of the ML analysis it was not highly supported by bootstrapping,
nor by aLRT.
The nucleotide data set of mitochondrial protein-coding genes
excluding the third codon positions was also subjected to ML and
Bayesian analyses. Both statistics yielded robust supports for
clades Ia, Ib and II, and the resultant phylogeny was identical to
that derived from the protein data set (Fig. 2B), apart from the
positions of Taenia hydatigena Pallas, 1766 and Taenia laticollis
Rudolphi, 1819.
Moreover, unrooted ML trees were inferred from the data sets
of mitochondrial protein-coding genes. The positions of leaf nodes
and their clusters were almost the same in both amino acid and
nucleotide trees (Supplementary Fig. S2). Significant bootstrap
432 M. Nakao et al. / International Journal for Parasitology 43 (2013) 427–437

(A) 98
(100) T. madoquae
(B) 99
(100) T. madoquae
99 100
(100) T. serialis (100) T. serialis
100 100
(100) T. multiceps (100) T. multiceps
96 T. asiatica Clade Ia 96 T. asiatica Clade Ia
(100) (100)
100 100
100 T. saginata 100 (100) T. saginata
(100) (100)
(100)
70 T. ovis T. ovis
(97) 51
(81)
54 T. solium T. solium
(95)
T. hydatigena T. hydatigena
96
96 (100) 29
(100) T. laticollis T. laticollis
(53)
100 100
(100) T. twitchelli (100) T. twitchelli
100 100
(100) Clade Ib
(100) T. martis Clade Ib 96
T. martis
92 (100)
(100) T. crassiceps T. crassiceps
100
100
(100) T. taeniaeformis sp. A (100) T. taeniaeformis sp. A
100 100
(100) T. taeniaeformis sp. B (100) T. taeniaeformis sp. B
100 Clade II 100 Clade II
(100) (100)
T. krepkogorski T. krepkogorski

T. parva T. parva

T. mustelae T. mustelae

58 58
(93) (61)

100 100
(100) (100) Echinococcus
Echinococcus
9 species 9 species

0.2 0.2

Fig. 2. Phylogenetic trees of taeniid cestodes inferred from mitochondrial genome sequences by using maximum likelihood and Bayesian analyses. The phylograms are
depicted only by the ML analysis, because the resultant topology was the same as that of the Bayesian analysis. Scale bars represent the estimated number of substitutions per
site. An outgroup (Dipylidium caninum) was omitted from the trees. Values of each node are ML bootstrap percentages. Closed circles represent branches supported by SH-like
aLRT values of more than 0.95. Bayesian posterior probability percentages are shown in parentheses. (A) Phylogram from deduced amino acids of 12 protein-coding genes. (B)
Phylogram from 12 protein-coding genes (excluding third codon positions).

values at most internal nodes supported the existence of different Table 3

clusters in Taenia. These mitochondrial unrooted trees also showed Intraspecific variation within Taenia taeniaeformis and Taenia mustelae in the
complete sequences of mitochondrial cox1 gene.
that clade II, T. mustelae and Echinococcus spp. were distantly re-
lated to the other members of taeniid cestodes. Comparisons Pairwise divergences
The branch lengths of the mitochondrial phylogenetic trees T. taeniaeformis sp. A
were far longer than those of the nuclear trees. The length from Between isolates BMM and SRN 0.017
an ancestral node can be used as a yardstick for exploring specific T. taeniaeformis sp. B
Between isolates TtaFi and ACR 0.009
status or validity of cryptic species. As shown in Fig. 2A and B, the
T. taeniaeformis sensu lato
branch lengths for T. taeniaeformis sp. A and sp. B were almost Between sp. A and sp. B 0.116–0.127
equal to or longer than those of the following sister species pairs: T. mustelae
Taenia madoquae (Pellegrini, 1950) and Taenia serialis (Gervais, Between isolates TmuFi9 and Hokkaido 0.028
1847), Taenia twitchelli Schwartz, 1924 and Taenia martis (Zeder,
1803) and T. asiatica and T. saginata.
sp. B (TtaFi and ACR), but the values between sp. A and sp. B ex-
3.4. Intraspecific variations in T. taeniaeformis and T. mustelae ceeded 0.1. In the case of T. mustelae, the value between the Finnish
and Japanese isolates (TmuFi9 and Hokkaido) was low. A ML tree
Pairwise divergence values of the mitochondrial cytochrome c made from the cox1 sequences showed that the isolates of T. muste-
oxidase subunit 1 (cox1) gene were utilized to numerically evalu- lae form a distinct clade (Supplementary Fig. S3), as previously
ate intraspecific variation in T. taeniaeformis s.l. and T. mustelae. shown by Lavikainen et al. (2008) using Finnish and Siberian
Four isolates of T. taeniaeformis s.l. (Okamoto et al., 1995a; Lavikai- isolates.
nen et al., 2008) and two isolates of T. mustelae (Iwaki et al., 1996;
Lavikainen et al., 2008) originating from different localities were
used for this evaluation. As shown in Table 3, the divergence values 4. Discussion
of the complete cox1 sequences between the Belgian and Japanese
isolates of T. taeniaeformis sp. A (BMM and SRN) were as low as The sampling and identification of Taenia spp. are difficult be-
those between the Finnish and Japanese isolates of T. taeniaeformis cause many of the carnivorous definitive hosts are endangered or
 M. Nakao et al. / International Journal for Parasitology 43 (2013) 427–437
Table 4
Selected morphological characters of the Taeniidae. Morphometric data were obtained from Abuladze (1964), Bray (1972), D’Alessandro and Rausch (2008), Freeman (1956),
Iwaki et al. (1994, 1995), Loos-Frank (2000), López-Neyra and Soler Planas (1943), Ortlepp (1934), Rausch and Bernstein (1972), Verster (1965, 1969), Xiao et al. (2005), and
original descriptions and redescriptions deposited in Global Cestode Database (University of Connecticut, http://tapewormdb.uconn.edu/).
Taxonomic units (newly Strobilar Large hook Small hook
proposed genera) lengths (mm)d lengths (lm) lengths (lm)
Echinococcus 1.7–12 20–60 16–47
Tentative Taeniaa 100–10,000 85–420 47–247
Clade II (Hydatigera)b 31–600 267–530 182–360
T. mustelae group 70–270 12–28
(Versteria)c
a
Excluding Taenia macrocystis, Taenia retracta, Taenia rileyi and Taenia selousi, all of which have strobilocercus-type metacestode stage. The measurements of rudimentary
hooks of Taenia asiatica were also excluded. The larval forms of coenurus and fimbriocercus were treated as cysticerci.
b
Consisting of Taenia krepkogorski, Taenia parva and Taenia taeniaeformis.
c
Consisting of Taenia mustelae and Taenia brachyacantha. The large and small hooks were indistinguishable.
d
Only the greatest length of each species taken into account.
protected. Nevertheless, previous molecular phylogenetic studies
using limited numbers of taeniid taxa have suggested that Taenia
is a highly diverse group (Lavikainen et al., 2008; Jia et al., 2012)
and that taxonomic revisions are necessary in the genus (Lavikai-
nen et al., 2008; Knapp et al., 2011). This study applied a multi-lo-
cus approach to clarify the molecular phylogeny of taeniid
cestodes. ML and Bayesian phylogenies inferred from data sets of
nuclear 18S rDNA, nuclear protein-coding genes and mitochondrial
genomes clearly demonstrate that both T. mustelae and a clade
formed by T. parva, T. krepkogorski and T. taeniaeformis are distantly
related to the other members of Taenia. However, statistical sup-
ports were relatively lower in the ML phylogenies than in the
Bayesian phylogenies. The lower reliability is probably due to the
small number of taxa and the use of a distantly related outgroup.
In particular, the low resolution of the 18S rDNA tree suggests that
the target sequences do not contain sufficient phylogenetic infor-
mation at the generic level.
In the present phylogeny, D. caninum was used as an outgroup.
It is still unknown which family of the Cyclophyllidea would be
optimal for taeniid cestodes. The selection of an outgroup is crucial
to deriving a robust phylogeny (Farris, 1982). The genus Dasyuro-
taenia is morphologically most similar to the Taeniidae (Hoberg
et al., 1999), but its DNA sequence data are presently unavailable.
Smith (1994) has warned of the risk of using a single outgroup tax-
on for rooting a molecular tree. In the present study, however, add-
ing another more distant outgroup (e.g., Hymenolepidae) would
probably not improve the phylogenetic trees. To overcome this de-
fect, unrooted ML trees were constructed. Although assumptions
about ancestry cannot be made from the unrooted trees, the relat-
edness of leaf nodes and their clusters separated by long branches
strongly support the necessity of taxonomic revision within Taenia.
Species of Taenia have been described using a traditional classi-
fication system based on a morphological or typological species
concept, while a phylogenetic or cladistic species concept high-
lights evolutionary relationships among organisms (Nixon and
Wheeler, 1990). A character-based phylogenetic reconstruction is
needed to show a hierarchy of common ancestry, especially for a
taxonomically problematic group. In this study, molecular phylog-
enies demonstrated Taenia to be paraphyletic, except for the result
of nuclear 18S rDNA. In cladistic taxonomy, synapomorphic traits
are important for making rational decisions in classifying organ-
isms. The monophyly of Taenia has hitherto been identified by
the larval character (the cysticercus, coenurus, strobilocercus and
fimbriocercus), but other synapomorphies are not evident (Hoberg
et al., 2000). The present molecular analyses clarified the following
monophyletic clades within members of Taenia.
Clade Ia, including the human-Taenia spp., is a robust group in
which members are closely related to each other. The utilization
of the order Cetartiodactyla (Bovidae and Suidae) as intermediate
hosts is characteristic for this clade. Another clade Ib, consisting
433
Scolex Rostellum Sucker No. of Larval forms
diameter (lm) diameter (lm) diameter (lm) testes
205–422 56–250 63–200 12–80 Hydatid
500–1540 154–790 173–530 70–1,200 Cysticercus
560–1,183 400–918 165–491 360–670 Strobilocercus
268–477 55–180 60–186 83–149 Cysticercus
of T. martis, T. twitchelli and Taenia crassiceps (Zeder, 1800), is also
highly robust, and members of the order Rodentia specifically
serve as intermediate hosts for them. Neither of the two clades cor-
responds to previously proposed genera within the Taeniidae, sug-
gesting that most of the former genera are invalid. The
phylogenetic position of these clades and the pattern of their host
selection are well supported by a phylogeny based on morphology
(Hoberg et al., 2000, 2001; Hoberg, 2006). However, a direct
comparison of topologies between the molecular and the morphol-
ogy-based trees is difficult due to the lower number of taxa in
molecular data sets. The present study also showed that T. hydati-
gena and T. laticollis are related to clades Ia and Ib (Figs. 1 and 2).
These cladistic relationships enable us to define the genus Taenia
sensu stricto (s.s.) as a monophyletic group presently comprising
T. hydatigena, T. laticollis and the members of clades Ia and Ib.
The position of other members of Taenia s.s. needs to be con-
firmed in further studies. Taenia hyaenae Baer, 1926, Taenia crocu-
tae Mettrick & Beverley-Burton, 1961, Taenia gonyamai Ortlepp,
1938, Taenia omissa Lühe, 1910, Taenia simbae Dinnik & Sachs,
1972, Taenia parenchymatosa Pushmenkov, 1945 and Taenia polya-
cantha Leuckart, 1856 have already been suggested as belonging to
this group in morphological phylogenies (Hoberg et al., 2000, 2001;
Hoberg, 2006). Previously published phylogenies based on short
mtDNA sequences also suggest that Taenia arctos Haukisalmi, Lavi-
kainen, Laaksonen & Meri, 2011, Taenia krabbei Moniez, 1879, Tae-
nia pisiformis (Bloch, 1780) and Taenia regis Baer, 1923 should be
included (Lavikainen et al., 2008, 2010). Definitive hosts for Taenia
s.s. commonly belong to the Carnivora (canids, felids, hyaenids,
mustelids and ursids), but host-switching to primates probably oc-
curred in the ancestral lineages of human-Taenia spp. in Africa due
to the shift of feeding to carnivory in early hominids, as suggested
by Hoberg et al. (2001). The molecular phylogenetic information on
the African species in hyaenids (T. hyaenae and T. crocutae) and fe-
lids (T. simbae) is needed to clarify this crucial event in human his-
tory. In a minority of the members of Taenia s.s. examined in this
study, the asexual proliferation of larvae occurs in a different man-
ner, including the coenurus of Taenia multiceps Leske, 1780 and T.
serialis, the fimbriated form of T. martis and T. twitchelli (Kornyu-
shin and Sharpilo, 1986) and the exogenous budding form of T.
crassiceps (Freeman, 1962). Taenia talicei Dollfus, 1960, a species re-
cently validated by Rossin et al. (2010), also has a multicephalic
fimbriocercus. Larval morphogenesis seems to be correlated with
the evolutionary history of Taenia s.s. but further confirmation is
required.
Phylogenetic analyses using nuclear 18S rDNA and mitochon-
drial protein-coding genes suggested that the members of Taenia
s.s. are sister to clade II consisting of T. parva, T. krepkogorski and
T. taeniaeformis. In a phylogeny inferred from nuclear protein-cod-
ing genes, clade II was placed as sister to all the other taeniid taxa
including Echinococcus. This topology clearly indicates the para-
434 M. Nakao et al. / International Journal for Parasitology 43 (2013) 427–437
phyly of Taenia. Species of clade II utilize carnivores (felids and viv-
errids) as definitive hosts and rodents as intermediate hosts. As
shown in Table 4, large rostellar hooks and cysticerci with a con-
spicuously segmented strobila (strobilocercus) are major charac-
teristics of the clade. The primary use of felids as definitive hosts
and the unique structure of the strobilocercus are in accord with
features of the genus Hydatigera Lamarck, 1816. Wardle and McLe-
od (1952) placed the following six species in Hydatigera: T. taeniae-
formis, T. parva, T. laticollis, Taenia balaniceps Hall, 1910 (a species
inquirenda in Verster, 1969), Taenia lyncis Skinker, 1935 (synony-
mized with Taenia rileyi Loewen, 1929 by Verster, 1969) and Taenia
macrocystis (Diesing, 1850). Yamaguti (1959) further added T.
krepkogorski to the genus. Finally, Abuladze (1964) redefined the
members of Hydatigera as follows: T. taeniaeformis, T. krepkogorski,
T. rileyi and Taenia hyperborea von Linstow, 1905. The last species,
however, was treated as a junior synonym of T. crassiceps by
Rausch (1959). Among these nominal taxa, our molecular phylog-
eny revealed that T. laticollis and T. crassiceps are entirely unrelated
to Hydatigera, as here conceived.
Another phylogenetically distinct species is T. mustelae. Phylog-
enies inferred from both nuclear and mitochondrial protein-coding
genes demonstrated that T. mustelae is sister to Echinococcus, while
this species was recognized as the most basal lineage among the
members of Taenia in the phylogeny based on nuclear 18S rDNA.
The basal position of T. mustelae is also shown in a morphological
phylogeny (Hoberg et al., 2000). Mustelids and rodents in the Hol-
arctic region serve as definitive and intermediate hosts for T. muste-
lae, respectively (Loos-Frank, 2000). Another morphologically very
similar species, Taenia brachyacantha Baer & Fain, 1951, occurs in
African striped weasels distributed in sub-Saharan Africa. Verster
(1969) suggested that T. mustelae and T. brachyacantha might be
even conspecific. The miniaturization of adult tapeworm is appar-
ent in both species. As shown in Table 4, the strobila, which consists
of a few dozen proglottids, is among the shortest (up to 27 cm in T.
mustelae) species of Taenia (Iwaki et al., 1994, 1995), and the rostel-
lum is armed with extremely small hooks (18–28 lm in length).
The lengths of the hooks, and especially the diameters of the scolex,
rostellum and suckers, fall into the same range as those of Echino-
coccus (Table 4). These structures are in general larger in the other
species of Taenia, including species with a similar or smaller strob-
ilar size (see Loos-Frank, 2000). An additional character, which
could differentiate T. mustelae and T. brachyacantha from the other
species of Taenia, is the small number of testes (Table 4), although
there is some overlap with T. martis and Taenia cf. intermedia (Tae-
nia martis americana in Loos-Frank, 2000). According to the original
description, the uterus of T. brachyacantha is saccular (Baer and
Fain, 1951), resembling in structure that of Echinococcus. Baer and
Fain, 1951, however, also found branched uteri in additional spec-
imens and suggested that saccular structure might indicate imma-
turity. Interestingly, the cysticercus of T. mustelae is monocephalic
in the Palearctic region (Murai, 1982; Iwaki et al., 1995, 1996) but
in the Nearctic region, polycephalic metacestodes occur as well
(Locker, 1955; Freeman, 1956; Mahrt and Chai, 1972; Rockett
et al., 1990), suggesting the existence of cryptic species or subspe-
cies. As shown in this study and in Lavikainen et al. (2008), low ge-
netic divergences among the Palearctic isolates of T. mustelae
suggest intraspecific differentiation. Historically, T. mustelae was
named based on specimens from European weasels (Freeman,
1956), but Taenia tenuicollis Rudolphi, 1819, now regarded as a ju-
nior synonym of T. mustelae, has been reported repeatedly in North
America (Locker, 1955). Further isolates from the Nearctic region
should be examined to clarify the species status of T. mustelae. As
regards T. brachyacantha in sub-Saharan Africa, the metacestode
stage is unknown (Loos-Frank, 2000).
Based on the resultant molecular phylogenies, we propose the
resurrection of Hydatigera for clade II. At present, the members of
Hydatigera are restricted to the three species of Hydatigera taeniae-
formis (Batsch, 1786) Lamarck, 1816, Hydatigera krepkogorski
Schultz & Landa, 1934 and Hydatigera parva (Baer, 1926) Wardle
& McLeod, 1952. The strobilocercus is a possible synapomorphy
in Hydatigera. The genus is also characterized by large rostellar
hooks and a narrow host specificity, restricted to felids, viverrids
(definitive hosts) and rodents (intermediate hosts). A polycephalic
metacestode called a polystrobilocercus or coenurostrobilocercus
(Murai et al., 1989) occurs in H. parva and H. krepkogorski. Accord-
ing to the list of Taenia spp. whose metacestodes are strobilocerci
(Loos-Frank, 2000), potential additional candidates for Hydatigera
are as follows: T. rileyi, T. macrocystis, Taenia retracta von Linstow,
1903 and Taenia selousi Mettrick, 1962. However, T. rileyi and T.
macrocystis differ from the species definitively assigned here to
Hydatigera in having terminal genital ducts that pass between lon-
gitudinal osmoregulatory canals (instead of passing them ven-
trally; see Verster, 1969). Rausch (1981) used the term
hemistrobilocysticercus for the metacestode of T. rileyi. A bicephal-
ic strobilocercus of Taenia sp. from rats in Malaysia (Kamiya et al.,
1987) is also included as a candidate for Hydatigera. The systematic
assignment of these candidates should be confirmed by further
molecular phylogenetic analyses.
Phylogenetic reconstructions have become fundamental tools
for species discovery (Brooks and McLennan, 2002). As shown in
the example of T. saginata and T. asiatica (Hoberg, 2006), sister
species relationships uncovered in phylogenies accelerate the
evaluation of their specific status from the multiple viewpoints
of natural history, biogeography, ecology, reproductive biology,
population genetics and morphology. Genetic yardsticks or DNA
barcoding approaches based on pairwise comparisons of sequence
divergence cannot serve as a universal proxy for species
delimitation, but such screenings can be utilized to identify cryp-
tic species, particularly in groups with limited morphological
characters (Hebert et al., 2004). Phylogenetic sequence compari-
sons in this study show that H. taeniaeformis includes two cryptic
species, named tentatively sp. A and sp. B. A similar conclusion
has been independently drawn by Jia et al. (2012), who compared
the sequences of taeniid mitochondrial genomes. Based on the
experimental data of Iwaki et al. (1994) and Nonaka et al.
(1994), we conclude that sp. A is H. taeniaeformis s. s. because
sp. A infects mice and rats from which Cysticercus fasciolaris
Rudolphi, 1808, a historical synonym for H. taeniaeformis, has
been found. The second species, sp. B, which uses voles as inter-
mediate hosts will be published as a new species in a separate pa-
per. Another study using mtDNA barcoding has revealed that sp. B
is sister to a minor clade limited geographically to the Apennine
Peninsula (Galimberti et al., 2012), but the exact composition of
the clade is still unknown.
Finally, we propose the erection of a new genus, Versteria, for T.
mustelae because this species is more closely related to Echinococ-
cus than to Taenia s.s. and Hydatigera. Versteria mustelae comb. nov.
differs distinctively from the members of Taenia s.s. in its morpho-
logical miniaturization, especially in its very small rostellar hooks.
Due to obvious morphological and ecological similarities, we pro-
pose the inclusion of T. brachyacantha in Versteria, although molec-
ular data are not available. Further molecular systematic studies
are required to confirm the validity of Versteria brachyacantha
comb. nov. Ecological, physiological and genetic relationships be-
tween Versteria and Echinococcus are important in elucidating the
evolutionary history of Echinococcus, a taeniid genus of a major
zoonotic significance.
It may be argued that the present molecular taxonomy of tae-
niid cestodes is premature due to the lack of critical taxa. However,
it will play an initiating role in creating a framework for phyloge-
netic and evolutionary studies to elucidate relationships between
the parasites and their mammalian hosts.
 M. Nakao et al. / International Journal for Parasitology 43 (2013) 427–437
5. Summary of proposed genera in the Taeniidae
5.1. Hydatigera Lamarck, 1816 (emended) (syn. Reditaenia Sambon,
1924)
Type species: Hydatigera taeniaeformis (Batsch, 1786) Lamarck,
1816. Synonyms: Taenia taeniaeformis (Batsch, 1786), Cysticercus
fasciolaris Rudolphi, 1808 and Hydatigera himalayotaenia Malhotra
& Capoor, 1982. For other possible synonyms, see Wardle and
McLeod (1952).
Other species: Hydatigera krepkogorski Schulz & Landa, 1934
(syn. Taenia krepkogorski (Schulz & Landa, 1934) Verster, 1969)
and Hydatigera parva (Baer, 1924) Wardle & McLeod, 1952 (syn.
Taenia parva Baer, 1924).
Generic diagnosis: Strobila from small to medium-sized, with
numerous proglottids. Immature and mature proglottids wider
than long, gravid proglottids elongate. Scolex with two rows of
large rostellar hooks. Genital pores irregularly alternating, opening
at middle of proglottid margin. Terminal genital ducts pass longi-
tudinal osmoregulatory canals ventrally. Female glands posterior,
median. Ovary bilobed. Vitellarium posterior to ovary, transversely
elongated. Testes numerous, mostly anterior and lateral to female
organs; may be confluent posterior to vitellarium. Uterus appears
as median longitudinal stem; gravid uterus with several lateral
branches, often with secondary branching. Metacestode monoce-
phalic or polycephalic strobilocercus.
Remarks: The most characteristic features of Hydatigera are the
strobilocercus-type metacestode that parasitizes rodents and large
rostellar hooks. There is, however, considerable overlap in the
length of rostellar hooks with Taenia endothoracicus (Kirshenblat,
1948), T. laticollis, T. macrocystis and Taenia pseudolaticollis Verster,
1969. Additionally, T. macrocystis, T. retracta, T. rileyi and T. selousi
resemble Hydatigera spp. by having a strobilocercus-type metaces-
tode. Of these, T. rileyi, T. selousi and T. endothoracicus resemble
Hydatigera spp. by using rodents as intermediate hosts. A possible
characteristic feature of Hydatigera may be the ventral position of
genital ducts with respect to the longitudinal osmoregulatory ca-
nals. In most species of Taenia s.s. the ducts are located between
the canals. Of the species mentioned above, only in T. selousi are
the genital ducts ventral and in T. retracta their position has not
been documented.
5.2. Versteria gen. nov.
Type species: Versteria mustelae (Gmelin, 1790) comb. nov. Syn-
onyms: Taenia mustelae Gmelin, 1790, Fimbriotaenia mustelae
(Gmelin, 1790) Kornyushin & Sharpilo, 1986 and Taenia tenuicollis
Rudolphi, 1819.
Other species: Versteria brachyacantha (Baer & Fain, 1951)
comb. nov. (syn. Taenia brachyacantha Baer & Fain, 1951).
Generic diagnosis: Strobila short. Immature and mature pro-
glottids wider than long, gravid proglottids elongate. Scolex, rostel-
lum and suckers small; rostellum with two rows of very small
hooks. Genital pores irregularly alternating, opening roughly at
middle of proglottid margin. Terminal genital ducts pass longitudi-
nal osmoregulatory canals ventrally. Female glands posterior, med-
ian. Ovary bilobed. Vitellarium posterior to ovary, transversely
elongated. Vaginal sphincter absent. Testes relatively few, mostly
anterior and lateral to female organs; may be confluent posterior
to vitellarium. Uterus appears as median longitudinal stem; gravid
uterus with several lateral branches. Metacestode monocephalic or
polycephalic cysticercus (metacestode unknown for V.
brachyacantha).
Remarks: Versteria spp. are distinctive, especially with their
miniature rostellar hooks, but also with small sizes of scolex,
435
rostellum and suckers, a short strobila and small number of testes.
There is, however, some overlap with Hydatigera and Taenia s.s. in
the length of strobila and the number of testes (Table 4).
Etymology: The new genus is named after Anna Verster (1931–
1994), a distinguished South African tapeworm taxonomist. Her
revision of the genus Taenia (1969) has remained as the most influ-
ential paper in the taxonomy of this genus.
Acknowledgements
The following persons and a project are acknowledged for pro-
viding taeniid specimens for this study: Philip S. Craig, Ping-Chin
Fan, Robin B. Gasser, David D. Heath, Heikki Henttonen, Marion
Hüttner, Donald P. McManus, Pedro L. Moro, Jiamin Qiu, Thomas
Romig, Peter M. Schantz and Beringian Coevolution Project. This
study was supported by Grants-in-Aid for Scientific Research
(Nos. 22590376, 21406009, 24406011, 21256003 and 24256002)
from Japan Society for the Promotion of Science (JSPS) to M.N.,
M.O. and A.I., and by JSPS-Asia/Africa Scientific Platform Fund
(2006–2011) and the Special Coordination Fund for Promoting Sci-
ence and Technology from the Ministry of Education, Culture,
Sports, Science & Technology in Japan (MEXT) (2010–2012) to
A.I. V.H. has been supported by NSF PBI award Nos. 0818696 and
0818823. S.K. worked under RFBR research projects (Nos. 11-04-
00870-a, 12-04-10171-k and 12-04-31203).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ijpara.2012.11.
014.
References
Abuladze, K.I., 1964. Principles of cestodology. Taeniata of Animals and Man and
Diseases Caused by Them, vol. IV. Izdatel’stvo Nauka, Moskow (English
translation, Israel Program for Scientific Translations, 1970).
Azuma, H., Okamoto, M., Oku, Y., Kamiya, M., 1995. Intraspecific variation of Taenia
taeniaeformis as determined by various criteria. Parasitol. Res. 81, 103–108.
Baer, J.G., Fain, A., 1951. Cestodes nouveaux du Congo Belge. Acta Trop. 8, 59–63.
Bessonov, A.S., Movsessian, S.O., Abuladze, K.I., 1994. On the classification and
validity of superspecies taxons of the cestodes of the suborder Taeniata Skrjabin
et Schulz, 1937. Helminthologia 31, 67–71.
Bowles, J., Blair, D., McManus, D.P., 1995. A molecular phylogeny of the genus
Echinococcus. Parasitology 110, 317–328.
Bowles, J., McManus, D.P., 1994. Genetic characterization of the Asian Taenia, a
newly described taeniid cestode of humans. Am. J. Trop. Med. Hyg. 50, 33–44.
Bray, R.A., 1972. The cestode Taenia krepkogorski (Schulz & Landa, 1934) in the
Arabian sand cat (Felis margarita Loche, 1758) in Bahrain. Bull. Br. Mus. Nat.
Hist. (Zool.) 24, 183–194.
Brooks, D.R., McLennan, D.A., 2002. The Nature of Diversity: An Evolutionary Voyage
of Discovery. University of Chicago Press, Chicago.
D’Alessandro, A., Rausch, R.L., 2008. New aspects of Neotropical polycystic
(Echinococcus vogeli) and unicystic (Echinococcus oligarthrus) echinococcosis.
Clin. Microbiol. Rev. 21, 380–401.
Farris, J.S., 1982. Outgroups and parsimony. Syst. Zool. 31, 328–334.
Farris, J.S., Källersjö, M., Kluge, A.G., Bult, C., 1995. Testing significance of
congruence. Cladistics 10, 315–319.
Foronda, P., Casanova, J.C., Vallanders, B., Martinez, E., Feliu, C., 2004. Molecular
systematics of several cyclophyllid families (Cestoda) based on the analysis of
18S ribosomal DNA gene sequences. Parasitol. Res. 93, 279–282.
Freeman, R.S., 1956. Life history studies on Taenia mustelae Gmelin, 1790 and the
taxonomy of certain taenioid cestodes from Mustelidae. Can. J. Zool. 34, 219–
242.
Freeman, R.S., 1962. Studies on the biology of Taenia crassiceps (Zeder, 1800)
Rudolphi, 1810 (Cestoda). Can. J. Zool. 40, 969–990.
Galimberti, A., Romano, D.F., Genchi, M., Paoloni, D., Vercillo, F., Bizzarri, L., Sassera,
D., Bandi, C., Genchi, C., Ragni, B., Casiraghi, M., 2012. Integrative taxonomy at
work: DNA barcoding of taeniids harboured by wild and domestic cats. Mol.
Ecol. Resour. 12, 403–413.
Gascuel, O., 1997. BIONJ: an improved version of the NJ algorithm based on a simple
model of sequence data. Mol. Biol. Evol. 14, 685–695.
Gasser, R.B., Zhu, X., McManus, D.P., 1999. NADH dehydrogenase subunit 1 and
cytochrome c oxidase subunit I sequences compared for members of the genus
Taenia (Cestoda). Int. J. Parasitol. 29, 1965–1970.
436 M. Nakao et al. / International Journal for Parasitology 43 (2013) 427–437
Guindon, S., Dufayard, J.F., Lefort, V., Anisimova, M., Hordijk, W., Gascuel, O., 2010.
New algorithms and methods to estimate maximum-likelihood phylogenies:
assessing the performance of PhyML 3.0. Syst. Biol. 59, 307–321.
Hall, B.G., 2004. Phylogenetic Trees Made Easy. A How-To Manual. Sinauer
Associates Inc., Massachusetts.
Hasegawa, M., Kishino, H., Yano, T., 1985. Dating of the human-ape splitting by a
molecular clock of mitochondrial DNA. J. Mol. Evol. 22, 160–174.
Haukisalmi, V., Lavikainen, A., Laaksonen, S., Meri, S., 2011. Taenia arctos n. sp.
(Cestoda: Cyclophyllidea: Taeniidae) from its definitive (brown bear Ursus
arctos Linnaeus) and intermediate (moose/elk Alces spp.) hosts. Syst. Parasitol.
80, 217–230.
Hebert, P.D., Penton, E.H., Burns, J.M., Janzen, D.H., Hallwachs, W., 2004. Ten
species in one: DNA barcoding reveals cryptic species in the neotropical
skipper butterfly Astraptes fulgerator. Proc. Natl. Acad. Sci. USA 101, 14812–
14817.
Hoberg, E.P., 2006. Phylogeny of Taenia: species definitions and origins of human
parasites. Parasitol. Int. 55 (Suppl.), S23–S30.
Hoberg, E.P., Alkire, N.L., de Queiroz, A., Jones, A., 2001. Out of Africa: origins of the
Taenia tapeworms in humans. Proc. Biol. Sci. 268, 781–787.
Hoberg, E.P., Jones, A., Bray, R.A., 1999. Phylogenetic analysis among the families of
the Cyclophyllidea (Eucestoda) based on comparative morphology, with new
hypotheses for co-evolution in vertebrates. Syst. Parasitol. 42, 51–73.
Hoberg, E.P., Jones, A., Rausch, R.L., Eom, K.S., Gardner, S.L., 2000. A phylogenetic
hypothesis for species of the genus Taenia (Eucestoda: Taeniidae). J. Parasitol.
86, 89–98.
Hüttner, M., Nakao, M., Wassermann, T., Siefert, L., Boomker, J.D., Dinkel, A., Sako, Y.,
Mackenstedt, U., Romig, T., Ito, A., 2008. Genetic characterization and
phylogenetic position of Echinococcus felidis (Cestoda: Taeniidae) from the
African lion. Int. J. Parasitol. 38, 861–868.
Iwaki, T., Nonaka, N., Okamoto, M., Oku, Y., Kamiya, M., 1994. Developmental and
morphological characteristics of Taenia taeniaeformis (Batsch, 1786) in
Clethrionomys rufocanus bedfordiae and Rattus norvegicus from different
geographical locations. J. Parasitol. 80, 461–467.
Iwaki, T., Abe, N., Shibahara, T., Oku, Y., Kamiya, M., 1995. New distribution record of
Taenia mustelae Gmelin, 1790 (Cestoda) from the least weasel Mustela nivalis in
Hokkaido. Jpn. J. Parasitol. 81, 796.
Iwaki, T., Abe, N., Shibahara, T., Oku, Y., Kamiya, M., 1996. Developmental study of
Taenia mustelae in the intermediate and definitive hosts, with a note on the life
cycle of T. mustelae in Hokkaido. Jpn. J. Parasitol. 82, 840–842.
Jeon, H.K., Kim, K.H., Eom, K.S., 2007. Complete sequence of the mitochondrial
genome of Taenia saginata: comparison with T. solium and T. asiatica. Parasitol.
Int. 56, 243–246.
Jeon, H.K., Lee, K.H., Kim, K.H., Hwang, U.W., Eom, K.S., 2005. Complete sequence
and structure of the mitochondrial genome of the human tapeworm, Taenia
asiatica (Platyhelminthes; Cestoda). Parasitology 130, 717–726.
Jia, W.Z., Yan, H.B., Guo, A.J., Zhu, X.Q., Wang, Y.C., Shi, W.G., Chen, H.T., Zhan, F.,
Zhang, S.H., Fu, B.Q., Littlewood, D.T., Cai, X.P., 2010. Complete mitochondrial
genomes of Taenia multiceps, T. hydatigena and T. pisiformis: additional
molecular markers for a tapeworm genus of human and animal health
significance. BMC Genomics 11, 447.
Jia, W., Yan, H., Lou, Z., Ni, X., Dyachenko, V., Li, H., Littlewood, D.T., 2012.
Mitochondrial genes and genomes support a cryptic species of tapeworm
within Taenia taeniaeformis. Acta Trop. 123, 154–163.
Jones, A., Bray, R.A., Khalil, L.F., 1994. Order Cyclophyllidea van Beneden in Braun,
1900. In: Khalil, L.F., Jones, A., Bray, R.A. (Eds.), Key to the Cestode Parasites of
Vertebrates. CAB International, Wallingford, pp. 305–307.
Jones, D.T., Taylor, W.R., Thornton, J.M., 1992. The rapid generation of mutation data
matrices from protein sequences. Comput. Appl. Biosci. 8, 275–282.
Kamiya, M., Ooi, H.K., Ohbayashi, M., Ow-Yang, C.K., 1987. Bicephalic larval cestode
of Taeniidae from rats in Malaysia. Jpn. J. Vet. Res. 35, 275–282.
Kimura, M., 1980. A simple method for estimating evolutionary rates of base
substitutions through comparative studies of nucleotide sequences. J. Mol. Evol.
16, 111–120.
Knapp, J., Nakao, M., Yanagida, T., Okamoto, M., Saarma, U., Lavikainen, A., Ito, A.,
2011. Phylogenetic relationships within Echinococcus and Taenia tapeworms
(Cestoda: Taeniidae): an inference from nuclear protein-coding genes. Mol.
Phylogenet. Evol. 61, 628–638.
Kornyushin, V.V., Sharpilo, L.D., 1986. A new taeniid genus (Cestoda) parasitic in
Mustelidae (in Russian). Vestn. Zool. 3, 10–16.
Lavikainen, A., Haukisalmi, V., Lehtinen, M.J., Henttonen, H., Oksanen, A., Meri, S.,
2008. A phylogeny of members of the family Taeniidae based on the
mitochondrial cox1 and nad1 gene data. Parasitology 135, 1457–1467.
Lavikainen, A., Haukisalmi, V., Lehtinen, M.J., Laaksonen, S., Holmström, S.,
Isomursu, M., Oksanen, A., Meri, S., 2010. Mitochondrial DNA data reveal
cryptic species within Taenia krabbei. Parasitol. Int. 59, 290–293.
Le, T.H., Blair, D., Agatsuma, T., Humair, P.F., Campbell, N.J., Iwagami, M., Littlewood,
D.T., Peacock, B., Johnston, D.A., Bartley, J., Rollinson, D., Herniou, E.A., Zarlenga,
D.S., McManus, D.P., 2000. Phylogenies inferred from mitochondrial gene orders
– a cautionary tale from the parasitic flatworms. Mol. Biol. Evol. 17, 1123–1125.
Le, T.H., Pearson, M.S., Blair, D., Dai, N., Zhang, L.H., McManus, D.P., 2002.
Complete mitochondrial genomes confirm the distinctiveness of the horse–
dog and sheep–dog strains of Echinococcus granulosus. Parasitology 124, 97–
112.
Littlewood, D.T., Olson, P.D., 2001. Small subunit rDNA and the Platyhelminthes:
signal, noise conflict and compromise. In: Littlewood, D.T., Bray, R.A. (Eds.),
Interrelationships of the Platyhelminthes. Taylor & Francis, London, pp. 262–278.
Liu, G.H., Lin, R.Q., Li, M.W., Liu, W., Liu, Y., Yuan, Z.G., Song, H.Q., Zhao, G.H., Zhang,
K.X., Zhu, X.Q., 2011. The complete mitochondrial genomes of three cestode
species of Taenia infecting animals and humans. Mol. Biol. Rep. 38, 2249–2256.
Locker, B., 1955. The identification of Taenia tenuicollis Rudolphi, 1819, in North
America. J. Parasitol. 41, 51–56.
Loos-Frank, B., 2000. An up-date of Verster’s (1969) ‘Taxonomic revision of the
genus Taenia Linnaeus’ (Cestoda) in table format. Syst. Parasitol. 45, 155–183.
López-Neyra, C.R., Soler Planas, M.A., 1943. Revision del genero Echinococcus Rud y
descripción de una especie nueva parárita intestinal del perro en Almería. Rev.
Ibér. Parasit. 3, 169–194.
Mahrt, J.L., Chai, S.J., 1972. Parasites of red squirrels in Alberta. Can. J. Parasitol. 58,
639–640.
Murai, É., 1982. Taeniid species in Hungary (Cestoda: Taeniidae). II. Larval stages of
taeniids parasitizing rodents and lagomorphs. Miscnea. Zool. Hung. 1, 27–44.
Murai, É., Tenora, F., Staněk, M., 1989. Atypical strobilocercus (Cestoda: Taeniidae) –
a parasite in experimental stocks of Ondatra zibethicus (Rodentia). Miscnea.
Zool. Hung. 5, 21–27.
Nakao, M., McManus, D.P., Schantz, P.M., Craig, P.S., Ito, A., 2007. A molecular
phylogeny of the genus Echinococcus inferred from complete mitochondrial
genomes. Parasitology 134, 713–722.
Nakao, M., Sako, Y., Ito, A., 2003. The mitochondrial genome of the tapeworm Taenia
solium: a finding of the abbreviated stop codon U. J. Parasitol. 89, 633–635.
Nakao, M., Sako, Y., Yokoyama, N., Fukunaga, M., Ito, A., 2000. Mitochondrial genetic
code in cestodes. Mol. Biochem. Parasitol. 111, 415–424.
Nakao, M., Yokoyama, N., Sako, Y., Fukunaga, M., Ito, A., 2002. The complete
mitochondrial DNA sequence of the cestode Echinococcus multilocularis
(Cyclophyllidea: Taeniidae). Mitochondrion 1, 497–509.
Nixon, K.C., Wheeler, Q.D., 1990. An amplification of the phylogenetic species
concept. Cladistics 6, 211–223.
Nonaka, N., Iwaki, T., Okamoto, M., Ooi, H.K., Oku, Y., Ohbayashi, M., Kamiya, M.,
1994. Infectivities of four isolates of Taenia taeniaeformis to various rodents. J.
Vet. Med. Sci. 56, 565–567.
Notredame, C., Higgins, D.G., Heringa, J., 2000. T-Coffee: a novel method for fast and
accurate multiple sequence alignment. J. Mol. Biol. 302, 205–217.
Okamoto, M., Bessho, Y., Kamiya, M., Kurosawa, T., Horii, T., 1995a. Phylogenetic
relationships within Taenia taeniaeformis variants and other taeniid cestodes
inferred from the nucleotide sequence of the cytochrome c oxidase subunit I
gene. Parasitol. Res. 81, 451–458.
Okamoto, M., Ito, A., Kurosawa, T., Oku, Y., Kamiya, M., Agatsuma, T., 1995b.
Intraspecific variation of isoenzymes in Taenia taeniaeformis. Int. J. Parasitol. 25,
221–228.
Ortlepp, R.J., 1934. Echinococcus in dogs from Pretoria and vicinity. Onderstepoort J.
Vet. Sci. Anim. Ind. 3, 97–108.
Rausch, R.L., 1959. Studies on the helminth fauna of Alaska. XXXV. On the identity of
certain cestodes (Taeniidae) from foxes. Proc. Helminthol. Soc. Wash. 26, 125–131.
Rausch, R.L., 1981. Morphological and biological characteristics of Taenia rileyi
Loewen, 1929 (Cestoda: Taeniidae). Can. J. Zool. 59, 653–666.
Rausch, R.L., 1994. Family Taeniidae Ludwig, 1886. In: Khalil, L.F., Jones, A., Bray,
R.A. (Eds.), Key to the Cestode Parasites of Vertebrates. CAB International,
Wallingford, pp. 665–672.
Rausch, R.L., Bernstein, J.J., 1972. Echinococcus vogeli sp. n. (Cestoda: Taeniidae) from
the bush dog, Speothos venaticus (Lund). Z. Tropenmed. Parasitol. 23, 25–34.
Rockett, J., Seville, R.S., Kingston, N., Williams, E.S., Thorne, E.T., 1990. A cestode,
Taenia mustelae, in the black-footed ferret (Mustela nigripes) and the white-
tailed prairie dog (Cynomys leucurus) in Wyoming. Proc. Helminthol. Soc. Wash.
57, 160–162.
Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L., Darling, A., Höhna, S., Larget,
B., Liu, L., Suchard, M.A., Huelsenbeck, J.P., 2012. MrBayes 3.2: efficient Bayesian
phylogenetic inference and model choice across a large model space. Syst. Biol.
61, 539–542.
Rossin, M.A., Timi, J.T., Hoberg, E.P., 2010. An endemic Taenia from South America:
validation of T. talicei Dollfus, 1960 (Cestoda: Taeniidae) with characterization
of metacestodes and adults. Zootaxa 2636, 49–58.
Schmidt, G.D., 1986. Handbook of Tapeworm Identification. CRC Press, Florida.
Shimalov, V.V., 2009. Hydatigerosis and strobilocercosis in the Republic of
Belarus and their medical significance. Med. Parazitol. (Mosk) 78, 59–62 (in
Russian).
Shimodaira, H., Hasegawa, M., 1999. Multiple comparisons of log-likelihoods with
applications to phylogenetic inference. Mol. Biol. Evol. 16, 1114–1116.
Smith, A.B., 1994. Rooting molecular trees: problems and strategies. Biol. J. Linn.
Soc. 51, 279–292.
Taly, J.F., Magis, C., Bussotti, G., Chang, J.M., Di Tommaso, P., Erb, I., Espinosa-
Carrasco, J., Kemena, C., Notredame, C., 2011. Using the T-Coffee package to
build multiple sequence alignments of protein, RNA, DNA sequences and 3D
structures. Nat. Protoc. 6, 1669–1682.
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5:
molecular evolutionary genetics analysis using maximum likelihood,
evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28,
2731–2739.
Tavaré, S., 1986. Some probabilistic and statistical problems in the analysis of DNA
sequences. Lect. Math. Life Sci. 17, 57–86.
Telford, M.J., Herniou, E.A., Russell, R.B., Littlewood, D.T., 2000. Changes in
mitochondrial genetic codes as phylogenetic characters: two examples from
the flatworms. Proc. Natl. Acad. Sci. USA 97, 11359–11364.
Verster, A., 1965. Review of Echinococcus species in South Africa. Onderstepoort J.
Vet. Res. 32, 7–118.
 M. Nakao et al. / International Journal for Parasitology 43 (2013) 427–437
Verster, A., 1969. A taxonomic revision of the genus Taenia Linnaeus, 1758 s. str..
Onderstepoort J. Vet. Res. 36, 3–58.
von Nickisch-Rosenegk, M., Lucius, R., Loos-Frank, B., 1999. Contributions to the
phylogeny of the Cyclophyllidea (Cestoda) inferred from mitochondrial 12S
rDNA. J. Mol. Evol. 48, 586–596.
Wardle, R.A., McLeod, J.A., 1952. The Zoology of Tapeworms. Univ. Minnesota Press,
Minneapolis.
Xiao, N., Qiu, J., Nakao, M., Li, T., Yang, W., Chen, X., Schantz, P.M., Craig, P.S., Ito, A.,
2005. Echinococcus shiquicus n. sp., a taeniid cestode from Tibetan fox and
plateau pika in China. Int. J. Parasitol. 35, 693–701.
437
Yamaguti, S., 1959. Systema helminthum. The Cestodes of Vertebrates, II.
Interscience Publishers Inc., New York.
Zhang, L., Hu, M., Jones, A., Allsopp, B.A., Beveridge, I., Schindler, A.R., Gasser,
R.B., 2007. Characterization of Taenia madoquae and Taenia regis from
carnivores in Kenya using genetic markers in nuclear and mitochondrial
DNA, and their relationships with other selected taeniids. Mol. Cell. Probes
21, 379–385.