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).