Introduction

Tapeworms of the genus Taenia (Cyclophyllidea: Taeniidae) are parasites of mammals, with carnivores or humans as definitive hosts and mostly herbivores and humans as intermediate hosts (Carabin et al. 2005). Since the 1700s, nearly 100 nominal species have been proposed or described based on either adult or larval forms (Abuladze 1964). The actual number of valid species now appears to be close to 42 (and four subspecies) (Loos-Frank 2000; Hoberg 2006). Many of these have a global socio-economic impact by causing morbidity in humans and domestic livestock (Hoberg 2002; Murell 2005). Because of its medical and veterinary significance, the genus Taenia has become a focus of intensive epidemiological, ecological, and taxonomic studies.

Traditionally, identification of taeniids to species has been based on morphological criteria together with ecological and biological aspects such as host specificity (Abuladze 1964). The development of molecular genetic techniques has provided alternative tools for the characterization of taeniid species and for the investigation of their relationships.

The most comprehensive hypotheses regarding the phylogenetic relationships within Taenia were presented by Hoberg et al. (2000) and Hoberg (2006). These were necessarily based on morphological and biological characteristics: of the 42 valid species, most remain genetically uncharacterized. Several molecular phylogenies that include some Taenia species have been proposed (e.g., Bowles and McManus 1994; Okamoto et al. 1995; de Queiroz and Alkire 1998; von Nickisch-Rosenegk et al. 1999; Lavikainen et al. 2008; Jia et al. 2010; Liu et al. 2011; Knapp et al. 2011). Data derived from mitochondrial (mt) gene sequences were used for most of these molecular studies. However, Knapp et al. (2011) used nuclear markers based on protein-coding genes. Bowles and McManus (1994), De Quieroz and Alkire (1998), and Zhang et al. (2007) used a portion of the nuclear 28S ribosomal RNA gene as well as a mitochondrial fragment. Concern has been expressed that mitochondrial data alone might yield misleading results because of the mode of inheritance of the mt genome. For example, discrepancies were noted between nuclear and mitochondrial trees for the genus Echinococcus (Knapp et al. 2011). In general, sequencing of organisms is time consuming or economically costly. Indeed, next-generation sequencing technologies (e.g., Illumina) permit the rapid sequencing of a large amount of sequences. However, the analysis of a huge amount of data remains after this sequencing, and this kind of apparatus is still expensive. Nuclear ribosomal genes have been found satisfactory in the past for inferring phylogenies of trematodes (reviewed in Olson and Tkach 2005; Blair 2006) and have been used in other groups of cestodes (Campos et al. 1998; Olson et al. 2001; Foronda et al. 2004; Waeschenbach et al. 2007). Here, we investigate whether full-length nuclear 18S rRNA genes are suitable for inferring phylogenies of Taenia species and whether these are congruent with phylogenies based on mt sequence data. We sequenced the complete nuclear 18S rRNA gene from specimens of seven Taenia spp., Taenia multiceps, Taenia hydatigena, Taenia pisiformis, Taenia saginata, Taenia asiatica, Taenia solium, and Taenia taeniaeformis. Using these sequences, we inferred phylogenetic relationships within the genus Taenia and compared the results with those from previously published studies that have used molecular or morphological data.

Materials and methods

Parasite specimens and DNA extraction

One specimen of T. multiceps was collected from a dog infected experimentally with Coenurus cerebralis from naturally infected sheep (Huangcheng Sheep Breeding Company, Gansu Province) in our laboratory. One specimen of T. pisiformis cyst was collected from the liver of a white rabbit at a slaughterhouse in Linyi City, Shandong Province. Specimens of T. asiatica, T. solium, and T. saginata were collected from fecal samples of patients with taeniasis after anthelmintic treatment (Dali, Yunnan Province). These species were identified primarily based on morphological traits, including the number, size, and shape of the rostellar hooks, according to Verster (1969), Loos-Frank (2000), and Huang and Sheng (2006). A fragment from each tapeworm and the protoscolex from the cyst of T. pisiformis were washed with cold phosphate-buffered saline and frozen in liquid nitrogen. Genomic DNA was extracted using the Genomic DNA Purification Kit (Qiagen, Germany) according to the manufacturer′s instructions and stored at −20 °C. The genomic DNA of T. taeniaeformis was from Dr. Dyachenko′s lab (Institute for Infectious Diseases and Zoonoses, Ludwig-Maximilians-University of Munich, Germany).

Amplification of 18S rDNAs

A pair of primers “full-length Cestoda 18S” (FLC18SF) (5′-tcc tgc cag tag tca tat gc-3′) and FLC18SR (5′-ctt gtt acg act ttt act tcc tct-3′) was designed for the amplification of taeniid 18S rDNAs based on the complete 18S rRNA gene sequences of cyclophyllid tapeworms published in GenBank (Olson et al. 2003; Foronda et al. 2004; Waeschenbach et al. 2007). Polymerase chain reactions were performed in a 50-μl volume of the following reaction mixture: 10 mM Tris–HCl (pH 8.4), 50 mM KCl, 3 mM MgCl2, 250 mM of each dNTP, 25 pmol of each primer, and 2 U Taq polymerase (TaKaRa, China) under the conditions: 94 °C, 4 min, then 30 cycles of 94 °C, 40 s; 52 °C, 30 s; 72 °C, 2 min and 20 s, followed by a final step at 72 °C for 10 min. The amplicons were directly sequenced or cloned into the pGEM-T Easy vector (Promega, USA) for sequencing. Sequencing was performed in Sangon Biotech (Sangon, China) Co., Ltd. with primer walking.

Sequence alignment and phylogenetic analysis

The complete 18S rDNA sequences were aligned using ClustalW within MEGA 5.0 (Tamura et al. 2011). Settings used were: gap opening penalty = 15, gap extension penalty = 6.66, and delay divergent cutoff = 30 %. The resulting alignment was manually checked (Thompson et al. 1994). Percent pairwise divergences of nucleotides were calculated using MEGA 5.0. The 18S rDNA sequences of seven Taenia species were used for phylogenetic analysis. The 18S rDNA sequence of Echinococcus granulosus (GenBank accession number GQ260092) was utilized as the outgroup. Three methods, namely maximum likelihood (ML), neighbor joining (NJ), and maximum parsimony (MP), were used for phylogenetic reconstructions. The ML, NJ, and MP methods were carried out using the Tamura 3-parameter model (Tamura 1992), Maximum Composite Likelihood model (Tamura et al. 2004), and max–mini branch-and-bound algorithm (Purdom et al. 2000), respectively, and all analyses were implemented in MEGA 5.0 (Tamura et al. 2011). The consensus tree was obtained after bootstrap analysis, with 1,000 replications for NJ, MP, and ML trees, with values above 50 % reported.

Ethics statement

All experiments using rabbits, sheep, and dogs were undertaken under strict Chinese experimental animal permission clearances, and animals at all times were treated in accordance with animal ethical procedures and guidelines for animal husbandry of the Institutional Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

Results and discussion

The 18S ribosomal RNA gene sequences of T. multiceps, T. saginata, T. asiatica, T. solium, T. pisiformis, T. hydatigena, and T. taeniaeformis were 2,574, 2.608, 2,583, 2,599, 2,642, 2,531, and 2,891 bp in size, respectively. The alignment of these seven sequences consisted of 2,910 sites (Fig. 1). Five variable regions could be recognized in the 18S gene sequences of all seven taxa (Wuyts et al. 2001). They are V1 (alignment positions 180–248), V2 (280–365), V3 (822–1,220), V4 (2,172–2,482), and V5 (2,851–2,907), respectively. Among them, V1 is quite short, whereas V3 and V4 are the longest. V1 and V4 are the most variable (Fig. 1). Almost all differences between sequences (insertions/deletions and base substitutions) were found within the variable regions: the conserved regions are therefore not shown in Fig. 1. The G+C content of sequences ranged from 52.67 to 55.79 % (T. hydatigena at one extreme but T. saginata at the other). All nucleotide sequences in this study have been deposited in the GenBank database under the accession numbers GQ260088–GQ260091 for T. asiatica, T. multiceps, T. hydatigena, and T. solium and JQ609338–JQ609340 for T. saginata, T. pisiformis, and T. taeniaeformis, respectively.

Fig. 1
figure 1

Alignment of the most variable portions of the 18S RNA gene sequences of T. multiceps, T. saginata, T. asiatica, T. solium, T. pisiformis, T. hydatigena, and T. taeniaeformis. Dots indicate nucleotide similarity among the seven taxa and hyphens, alignment gaps. The numbers refer to alignment positions

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Pairwise distances in these 18S rDNA sequences among seven Taenia spp. were 0.78–14.96 %, using the Maximum Composite Likelihood model (Tamura et al. 2004). The 18S rDNAs of T. saginata and T. asiatica were highly similar to each other (with 99.2 % identity). These two species also share high similarity in all molecular studies using mitochondrial or nuclear DNA sequences (e.g., Bowles and McManus 1994; de Queiroz and Alkire 1998; Lavikainen et al. 2008; Jia et al. 2010; Knapp et al. 2011; Liu et al. 2011), reflecting their well-known sister relationship.

Elsewhere in the tree (Fig. 2), there is strong concordance with previous studies that used nuclear or mitochondrial genes. T. taeniaeformis lies more or less basal within Taenia in most studies that include it. Exceptions to this are the mt cox1 tree in De Queiroz and Alkire (1998) and studies that include Taenia mustelae, which is sometimes placed basal in Taenia or as sister to Echinococcus (de Queiroz and Alkire 1998; Lavikainen et al. 2008; Knapp et al. 2011). Where the following species have been included, the relationship (((T. asiatica, T. saginata), T. multiceps), T. solium) is always seen. The clade of T. pisiformis and T. hydatigena lies between the cluster comprising T. asiatica and other three related species and T. taeniaeformis. The placement of these two relative to each other varies between studies. Here, they are sisters (Fig. 2). Jia et al. (2010) also found this relationship based on complete mt genomes. Partial mt genome sequences (Lavikainen et al. 2008; Jia et al. 2010) placed T. pisiformis as sister to T. solium, a relationship not found in any other studies.

Fig. 2
figure 2

Phylogenetic tree of 18S rDNA sequences of Taenia species inferred by the methods of maximum likelihood [using the Tamura 3-parameter model (Tamura 1992)], neighbor joining [using Maximum Composite Likelihood models (Tamura et al. 2004)], and maximum parsimony [using max–mini branch-and-bound algorithm (Purdom et al. 2000)] with bootstrapping and 1,000 replications. Numbers at nodes represent the percentage of bootstrap replications (values >50 % reported)

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Phylogenies based on morphology (e.g., Fig. 2 in Hoberg et al. 2000) place T. saginata and T. asiatica as sisters. Their morphological tree contains 30 species, making comparisons difficult with molecular trees that include a much sparse sampling of taxa. However, the molecular grouping of T. multiceps and T. solium in a clade with T. saginata and T. asiatica is more or less congruent with the morphological tree. T. taeniaeformis lies near the base of the morphological tree, as it does in molecular trees. Morphology places T. pisiformis and T. hydatigena well apart, but within the larger clade that contains T. asiatica, T. saginata, T. multiceps, and T. solium. Molecular data from additional species of Taenia are required to be able to fully evaluate the relative merits of morphological and molecular data. Here, we have shown that sequences from the nuclear 18S ribosomal RNA gene have considerable promise as sources of phylogenetic information within the genus Taenia. Furthermore, given that almost all the variable sites lie within defined variable portions of that gene, it will be appropriate and economical to sequence only those regions for additional species of Taenia.