Tapeworms of the genus Taenia (Linnaeus, 1758) include parasites of mammals that use carnivores as definitive hosts, and herbivores (or omnivores) as intermediate hosts. Humans may be infected by both adult and larval forms (of various species), causing taeniosis or cysticercosis respectively. As with all species of tapeworm, transmission from host to host follows a trophic pathway via ingestion. However, species of Taenia are unique amongst the Cestoda in requiring two obligate mammalian hosts for transmission and life cycle completion; egg to herbivore, herbivore to carnivore, the cestode matures in the carnivore and releases fertilized eggs . Taenia also enters humans trophically, through the inadvertent consumption of eggs or larval stages present in undercooked meat. Human Taenia species include T. saginata Goeze, 1782, T. asiatica Eom & Rim, 1993, and T. solium Linnaeus, 1758, with the following zoonotic species also found in humans: T. taeniaeformis (Batsch, 1786), T. crassiceps (Zeder, 1800), T. multiceps Leske, 1780, and T. serialis (Gervais, 1847). Species of Taenia cause significant health problems and considerable socio-economic losses when infecting humans and livestock ; human neurocysticercosis is the most common helminth infection of the central nervous system and has considerable societal impact in endemic areas [3–5]. The genus is widespread globally, with hotspots of prevalence in humans related to diet, social conditions, cultural practices and poverty. In the wild prevalence is dictated by specific predator-prey interactions, and there is little doubt their influence on host ecology is significant [6, 7].
Historically, as an old genus, Taenia (Cestoda, Taeniidae) has become somewhat of a 'catch-all' taxon for tapeworm systematics, with upwards of 70 nominal species having been attributed to Taenia. Species circumscription based on morphology has been, and remains, problematic. Approximately 42 valid species and 3 subspecies are recognized currently , circumscribed predominantly on the basis of adult morphology; other names persist and largely arise from descriptions of larval stages or the use of some of the many synonyms [6, 8–10]. There are conflicting estimates of phylogeny from morphology and various molecular markers, thus preventing a full understanding of the evolutionary history with their hosts through space and time. Taenia is one of only two genera in the Taeniidae. The other genus in the family, Echinococcus, is also of importance as it causes morbidity in humans and livestock [11–13]. As a result of their importance, species of these two genera have been studied extensively, but relatively little is understood about their biology in the context of molecular ecology, epidemiology or control. Much of this comes from an inability to quickly, or accurately, diagnose species or to track populations. In many endemic areas diseases caused by human Taenia are often categorized as 'neglected tropical diseases' .
As is widely recognized for many parasites, knowledge of life cycles, mechanisms and dynamics of transmission and infection, all form the basis for effective control strategies [12, 14–16]. Molecular tools are increasingly used to develop these areas of knowledge, and here we take a comparative mitogenomic approach to evaluate mitochondrial (mt) DNAs as a source of new molecular markers.
Mitochondrial genes are amongst the most popular markers for molecular-based approaches to ecology, population genetics and evolutionary biology and have been popular targets for molecular-based methods of species identification [17–19]. Multiplex PCR approaches to diagnose mixed infections of Taenia are also being developed and are targeted for PCR-RFLP analysis using mt cytochrome c oxidase subunit I (cox 1) and cyt b [20, 21]. Certain genes and gene regions have become popular choices as representative mitochondrial markers, because they are bordered by regions of sequence conservation (e.g. cox 1) and 'universal' (or at least broadly conserved) PCR primer sets can be readily designed. This is also the case for helminths, where mt gene fragments have been used routinely for population genetics, ecology and diagnostics [22, 23]. More recently, the ability to readily characterize complete helminth mtDNAs, by means of long PCR and a variety of sequencing techniques [24, 25], has prompted an assessment of entire genomes as a source for phylogenetic analysis [26–29], and their comparison to reveal variation within and between genes in order to develop novel (or optimise existing) molecular markers [30, 31]. Mitochondrial genomes of bilaterian animals are short, circular DNA molecules typically 14-16 kb in length, without introns and with short intergenic regions. Gene content is highly conserved with typically 12 protein coding genes in Platyhelminthes (they lack the gene for ATP synthase subunit 8, atp 8), two ribosomal subunits and 22 tRNAs. Mitochondrial gene rearrangement is not uncommon in flatworms, occurring within the genus Schistosoma [32, 33], in some monogeneans  and appears to be different in at least some turbellarians . Evidence suggests that gene order is otherwise generally conserved in tapeworms (Cestoda) and flukes (Trematoda) .
Complete mtDNAs offer variation over multiple levels of organization, from gene content and gene order to variation in amino acids and nucleotides, offering opportunities to resolve both recent and ancient divergence events. Here we characterize the complete mt genomes of T. multiceps Leske, 1780, T. hydatigena Pallas, 1766 and T. pisiformis (Bloch, 1780), each of intrinsic interest. Coenurosis is a debilitating disease caused by the metacestode (larval forms) of T. multiceps and is common in sheep and other herbivores. Humans may also be infected with the metacestode occasionally ; infections occur when eggs are ingested via the fecal-oral route of transmission. Infective eggs hatch, and the liberated oncospheres cross the membrane of the small intestine and migrate within the body, typically ending up in the central nervous system, mesentery and visceral organs [37, 38]. Within the host's tissues, the oncospheres mature into coenuri or cysticerci causing the metacestodiasis. Amongst carnivores, each of these 3 species is cosmopolitan in canines and T. hydatigena and T. pisiformis are also found in felines. In China, sheep are the most common hosts for metacestodes of T. multiceps and T. hydatigena, and lagomorphs are the most common hosts of T. pisiformis tapeworms; their distribution across China and surrounding countries is extensive [39–41]. Each of the species characterized here is found globally, and mixed infections with other taeniids are common. For accurate diagnosis, there is a need to identify individual species from mixed infections of tapeworms whether in intermediate or definitive hosts. Differentiating adult worms using morphology alone requires taxonomic expertise, but to differentiate amongst mixed populations of eggs and larvae requires molecular techniques . Once developed, molecular techniques can be readily applied to portions of adult worms, larvae, eggs and environmental samples and should aid in accurate, rapid identification.
Among the genus Taenia, complete mt genome sequences are already available for T. asiatica, T. saginata, T. solium and T. crassiceps [32, 43, 44]. We use these sequences with the new data to achieve three goals. Firstly, we evaluate the potential for complete mt genomes in estimating the phylogeny of the genus and revealing its evolutionary history. Taenia is a species-rich genus with a widespread distribution but there are conflicting estimates of phylogeny from morphology and various molecular markers. Secondly, we use comparative mitogenomics to highlight regions of nucleotide variation amongst Taenia species to investigate whether mitochondrial gene fragments currently used as molecular markers offer the best regions for characterization, whether for species recognition or other molecular-based applications. Third, we take an in silico approach to developing PCR primer pairs designed to amplify short fragments of mtDNAs for all species (for which entire mt genomes have been characterized), with a view to providing primers that will work for all Taenia species capturing high levels of variation in mtDNAs, and we test some of these primers to demonstrate their efficacy.