3.3. Divergence times analysis
The divergence time analysis based on mitochondrial protein-coding genes suggested that T. saginata and T. asiatica should diverge at 1.10 Mya (0.80-1.41, 95% highest probability density) in the early Pleistocene period, which is consistent with the previous reports based on genomic genes (Michelet and Dauga, 2012; Wang et al., 2016); T. caixuepengi should diverge from T. pisiformis 5.49 Mya (3.87-7.19, 95% highest probability density) in the initial Pliocene period, which is close to the divergence time between E. shiquicus and E. multilocularis (4.12 Mya, 2.81-5.32, 95% highest probability density); T. tianguangfui on the other hand, originated 13.11 Mya (9.36-17.18, 95% highest probability density) in the middle Miocene period, which was earlier than the differentiation of its intermediate host, N. fuscus (4.98 Mya, 4.08-5.90, 95% highest probability density) (Fig. 3, Supplementary Fig. S2).
Discussion
The discovery of these two new parasites, T. caixuepengi andT. tianguangfui , highlights the species diversity of the family Taeniide, and further proved the true biodiversity characteristic of the QTP. Given the lack of human intervention and the rich diversity of wild host species, the present understanding of the species diversity within this family in QTP is apparently just a tip of the iceberg. This is not surprising, given the appreciable cryptic diversity so far uncovered within the taeniid family in Africa and northern latitudes (Lavikainen et al., 2011; Lavikainen et al., 2013; Terefe et al., 2014).
Here the whole mt genomes of both new species were sequenced, and were clearly different from all available Taenia mt genome sequences, verifying the validity of their species status. Their mt genomes were similar as those of other sequenced tapeworms with respect to length, nucleotide bias, and their tRNA, rRNA and protein-encoding genes composition (Fig. 1) (Jeon et al., 2005; Le et al., 2000; Nakao et al., 2003). Furthermore, the codon ATT was inferred as a more unusual start codon for the atp 6 gene of T. caixuepengi (Table 1), which is a common start codon used by Caenorhabditis elegans andAscaris suum (Okimoto et al., 1990).
T. caixuepengi larva is so far undetected in other animals, except plateau pika (O. curzoniae ), meanwhile, no other cysticerci have been found in plateau pika hitherto. Lagomorpha is the intermediate host of T. pisiformis and T. laticollis(Hallal-Calleros et al., 2016; Valdmann et al., 2004). Although similar in appearance and size to the vole, the plateau pika belongs to the Lagomorpha (Smith et al., 2019). The close phylogenetic relatedness of these three Taenia species (Fig. 2) is further highlighted by their high preference for lagomorphs as an intermediate host. Based on the divergence time and phylogeographic analyses, the extent pikas (genus Ochotona ) originated on the QTP in the middle Miocene, ~14 Mya (Wang et al., 2020). However, the rapid speciation of many Ochotona species, including O. curzoniae , occurred during the late Miocene and early Pliocene period (Wang et al., 2020), which almost coincided with the rapid uplift of the QTP (An et al., 2006; Li et al., 2007; Shi et al., 2015). Coincidentally, the evolutionary divergence time analysis in this study also suggests that both T. caixuepengi and E. shiquicus had evolved in the early Pliocene epoch, about 5.49 Mya and 4.12 Mya, respectively (Fig. 3). These almost synchronous events may not have happened by chance. Large-scale diversification of species is often provoked by abiotic factors, such as changes in the living environment and food supply (Benton, 2009). The uplift of the QTP from south to north provided climatic opportunities and food supply for the diversification of cold temperature-preferring pikas but led to the extinction of other warm temperature-preferring rodents (Wang et al., 2020).
For most free-living organisms, speciation is usually the result of genetic drift or adaptive differentiation between geographically separate populations (Turelli et al., 2001). For parasites, however, it has long been thought that sympatric speciation of parasites is common, mediated by ecological isolation caused by host switching within the same geographic region (de Meeƻs et al., 1998; Huyse et al., 2005; Paul, 2002). Therefore, we speculate that T. pisiformi s in the QTP (Li et al., 2013) may share a common ancestor with T. caixuepengi ; the split of the pika population caused the ecological isolation between their ancestral populations, which further resulted in the lack of gene flow between them due to intermediate host switching, and the eventual formation of two different species. It can also be speculated that the differentiation pattern between E. shiquicus and E. multilocularis is similar as that of T. caixuepengi and T. pisiformis .
Our evolutionary divergence time analysis suggests that the speciation of T. tianguangfui occurred in the middle Miocene period (~13.11 Mya) (Fig. 3) when the QTP was undergoing a slow uplift period (An et al., 2006). The timing of the divergence ofN. fuscus evolved from ~4.98 Mya (Supplementary Fig. S2), which also coincided with the rapid uplift of the QTP (An et al., 2006; Li et al., 2007; Shi et al., 2015). As the species spread in the QTP and Himalaya (Pradhan et al., 2019), the evolutionary origin of theNeodon spp., like the plateau pika, may well be due to changes in climate and food supply caused by the uplift of the QTP and Himalaya. The speciation of T. tianguangfui was earlier than that of its intermediate host, indicating that T. tianguangfui did not differentiate into a newTaenia species in order to adapt to the intermediate host, rather, it suggests that T. tianguangfui larva might not be limited to N. fuscus . Taenia crassiceps and T. martis have similar intermediate hosts range, infecting a variety of rodents, even humans and other primates (Deplazes et al., 2019). Given the close relationship between T. tianguangfui , T. crassiceps and T. martis , it also cannot be excluded thatT. tianguangfui may be infective to a variety of rodents other than N. fuscus , as well as humans and other primates. So far, a clear understanding of their evolutionary origin from these clues is elusive, thus, more data and investigation are needed to provide further insight.
Adult worms of the T. tianguangfui and T. caixuepengi have not yet been collected due to the difficulty in sampling endangered or protected carnivores. Plateau pikas and voles are the primary food source for wild canids across the QTP. Tibetan foxes are the obligate predator of plateau pikas, as their remains (plateau pikas) are often encountered in 99% of their feces (Smith et al., 2019). Wild canids, especially the red fox and the Tibetan fox, may well be important definitive hosts for T. tianguangfui and T. caixuepengi .
Adult or larval samples of tapeworm are easily damaged in the process of collection, freeze-thaw and processing, and the morphological features are mostly unidentifiable (Lavikainen et al., 2013). While mt genome data alone may not fully answer the scientific questions surrounding their evolutionary origins, it is the most cost-effective and accurate method. Recently, although laborious and costly, there have been an increasing whole genome sequencing and analyses for many tapeworm species. This kind of investigation, not only is it important to provide insights into their host adaptation and switching, evolution mechanisms through gene groups amplification, hosts-parasites interaction, immune regulation and nutrition, it also provides urgently needed resources for the identification of drug target and diagnostic molecular markers (International Helminth Genomes Consortium, et al., 2019; Wang et al., 2016). In the future, a lot of genomic data will be needed to study this fascinating group.
Conclusion
Finally, the mitochondrial genome sequence data adequately confirm the validity of the two new Taenia species named T. caixuepengi and T. tianguangfui , we have previously reported. The phylogenetic trees and divergence times analysis suggest thatT. caixuepengi evolve from its closest relative, T. pisiformis , in the initial Pliocene period (~5.49 Mya), due to the intermediate host switching caused by the rapid uplift of the QTP; T. tianguangfui be probably parasitic in a wide variety of rodents, and share a common ancestor with T. crassiceps , T. twitchelli and T. martis , splitting in middle Miocene period (~13.11 Mya).