Tropical Biomedicine 37(1): 237–250 (2020)

Global genetic diversity of tapeworms

Hong, X., Liu, S.N., Xu, F.F., Han, L.L., Jiang, P., Wang, Z.Q., Cui, J.* and Zhang, X.* Department of Parasitology, School of Basic Medical Sciences, Zhengzhou University, 40 Daxue Road, Zhengzhou 450052, People’s Republic of China *Corresponding author e-mails: [email protected] (Zhang, X.), [email protected] (Cui, J.) Received 17 April 2019; received in revised form 16 November 2019; accepted 20 November 2019

Abstract. Spirometra larvae are etiological agents of human . However, the systematics of spirometrid cestodes has long been controversial. In order to determine the current knowledge on the evolution and genetic structure of Spirometra, an exhaustive population diversity analysis of spirometrid cestodes using the mitochondrial gene: cytochrome c oxidase subunit 1 (cox1) was performed. All publicly available cox1 sequences available in the GenBank and 127 new sequencing genes from China were used as the dataset. The haplotype identify, network, genetic differentiation and phylogenetic analysis were conducted successively. A total of 488 sequences from 20 host species, representing four spirometrid tapeworms (S. decipiens, S. ranarum, S. erinaceieuropaei and Sparganum proliferum) and several unclassified American and African isolates from 113 geographical locations in 17 countries, identified 45 haplotypes. The genetic analysis revealed that there are four clades of spirometrid cestodes: Clade 1 (Brazil + USA) and Clade 2 (Argentina + Venezuela) included isolates from America, Clade 3 contained African isolates and one Korean sample, and the remainders from Asia and Australia belonged to Clade 4; unclassified Spirometra from America and Africa should be considered the separate species within the ; and the of two Korea isolates (S. erinaceieuropaei KJ599680 and S. decipiens KJ599679) was still ambiguous and needs to be further identified. In addition, the demographical analyses supported population expansion for the total spirometrid population. In summary, four lineages were found in the spirometrid tapeworm, and further investigation with deeper sampling is needed to elucidate the population structure.

INTRODUCTION 2017). The most recent review concluded only 4 valid species of the genus (Kuchta and The Spirometra tapeworm belongs to the Scholz, 2017), however, many more nominal family and order Spirometra species have been described. . Adults live in the In addition, the sparganum isolates from intestine of felids and canids, however larvae America (Argentina, Brazil and USA) and (plerocercoids or spargana) parasitize in Africa (Ethiopia and South Sudan) were still body cavity and tissues of all tetrapode unclassified (Eberhard et al., 2015; Almeida groups (Okamoto et al., 2007). In humans, et al., 2016; Waeschenbach et al., 2017). the plerocercoid (sparganum) invades Therefore, there is a pressing requirement mainly subcutaneous tissues, but also eyes, to investigate the genetic diversity and brain, and central nervous system, causing phylogenetic relationship among the a serious parasitic zoonosis known as Spirometra species in order to establish sparganosis (Cui et al., 2011, 2017; Liu et al., the taxonomy of spirometrid tapeworm. 2015). Although with medical importance, the Generally, species identification of identification and taxonomy of Spirometra Spirometra tapeworm is based on the species has long been controversial (Faust morphology of adult worms, however the et al., 1929; Mueller, 1937; Okamoto et al., typical specimens obtained in practice 2007; Jeon et al., 2016a, 2018a; Zhang et al., were Spirometra larval forms. It is hard to

237 distinguish the morphological differences instructions. The cox1 gene was amplified in the plerocercoids among spirometrid by PCR using the primers designed by tapeworms. Therefore, more species Yanagida et al. (2010). The PCR products identification has preferred DNA borcoding were purified using the EasyPure PCR methods. Among all genetic markers that Purification Kit (Transgen, China) and have been applied in the molecular studies sequenced in both directions at the Genwiz of Spirometra, the mitochondrial cytochrome Company (Suzhou, China). All sequences c oxidase subunit 1 gene (cox1) has been the were deposited in the GenBank database most commonly used. This gene has been under accession numbers MK085765- widely used for the species identification K085891. (Lee et al., 1997; Miyadera et al., 2001; Liu et al., 2010; Simpson et al., 2012; Boonyasiri et Genetic diversity analysis al., 2013, 2014; Jongthawin et al., 2014; Jeon To perform a worldwide genetic variable et al., 2015, 2016a, 2018a; Holldorf et al., 2015; comparative analysis of Spirometra Petrigh et al., 2015; Thanchomnang et al., tapeworms, all available sequences of cox1 2016; Tang et al., 2017), genetic variation in the GenBank database were included (Okamoto et al., 2007; Zhang et al., 2014, in this study (Table 1). More specifically, 2015a; Jeon et al., 2016b, 2018b) and 366 sequenced cox1 genes of spirometrid phylogeny (Lee et al., 2007; Dai et al., 2012; tapeworms have been published, representing Wei et al., 2015; Zhang et al., 2015b, 2016). five species and several unclassified species: To date, 366 sequences of the cox1 gene S. decipiens (n=1), S. ranarum (n=1), S. of spirometrid tapeworms have been erinaceieuropaei (n=349), Sparganum deposited in the GenBank database, which proliferum (n=1) and Spirometra sp. (n=14). represent the isolates collected from different For S. erinaceieuropaei, 11 sequences were hosts and originated from more than 84 from Australia, three from Indonesia, one from geographical locations in 17 countries. These India, 12 from Thailand, 16 from Laos, one publicly available datasets offer good from Myanmar, three from Korea, seven opportunities to compare the intra- and from Japan, one from Vietnam, one from interspecific diversity of Spirometra Iran and 293 from mainland China. Two tapeworms. In this study, we intend to diphyllobothriid tapeworms, Diphyllo- perform an exhaustive genetic diversity bothrium scoticum (KY552883) and analysis of spirometrid cestodes using these Adenocephalus pacificus (KY552867) were available datasets, as well as with 127 newly used as outgroups according to the report added sequences of spargana isolated from by Waeschenbach et al. (2017). wild frogs in 29 geographical locations in The alignment of cox1 sequences was mainland China. performed in MEGA v.6.06 (Tamura et al., 2013) with the default settings. The variable sites, nucleotide compositions and pairwise MATERIALS AND METHODS distances were also estimated in MEGA v.6.06. The number of haplotypes, haplotype Sequencing the cox1 gene of Chinese diversity (Hd), and nucleotide diversity (Pi) isolates were analysed with DnaSP v.6 (Rozas et al., Wild frogs were collected from different 2017). The software Network v.5.0 (Bandelt locations in China from May 2014 until et al., 1999) was employed to draw a median- September 2018. The presence of spargana joining network to explore the relationships was examined as the methods described by among the detected haplotypes. To explore Wei et al. (2015). In total, 127 spargana were the levels of genetic differentiation among collected from 29 geographical locations the geographical populations, the pairwise (Table 1). Genomic DNA was extracted FST values between populations were from individual plerocercoids using the calculated by using Arlequin v.3.5 (Excoffier EasyPure Genomic DNA Kit (Transgen, et al., 2010). China) according to the manufacturer’s

238 Phylogenetic analysis isolates (KF740506 and KF740507) and six The phylogenetic pattern of all cox1 isolates from South Sudan were identified 2 haplotypes was estimated through two Haps; and the remaining S. erinaceieuropaei methods: maximum likelihood (ML) and isolates and a S. ranarum sample Bayesian inference (BI). The sequence constituted another 36 Haps. All samples evolution model was selected by jModelTest had high haplotype diversity (0.652±0.023) v0.2 (Darriba et al., 2012) under the Akaike accompanied by low nucleotide diversity information criterion (AIC). The ML analysis (0.01619±0.00156). The median-joining was performed in MEGA v.6.06. Confidence network analysis showed that Hap12 was the in each node was assessed by boot-strapping most prominent haplotype represent 55.3% (1000 pseudo-replicates). BI was performed of all sequences and originated from China, in MrBayes v.3.2 (Ronquist et al., 2012). The Thailand, Japan, Australia, Korea and Iran analysis consisted of two runs, each with (Fig. 1). Hap2 was shared by isolates from four MCMC chains running for 5 000 000 China, Thailand and Myanmar. Hap13 was generations, and sampling every 100th shared by isolates from China, Australia, generation. The neutrality tests using Japan and Indonesia. Hap14 was shared Tajima’s D (Tajima, 1989) and Fu’s FS (Fu, by isolates from China and Indonesia. The 1997) were also applied through Arlequin Chinese isolates (82.9% of the sequences v.3.5 as an assessment of possible population represented China samples) showed the most expansion. In addition, to estimate changes diversity of haplotypes (24 Haps). Samples in population size over time, and the time to from Japan and Korea shared 5 (Haps12-13, the most recent common ancestor (tMRCA), Haps19-21) and 4 (Hap3, Hap12, Haps17-18) a Bayesian Skyline Plot analysis (BSP) Haps, respectively. Isolates from Thailand implemented in BEAST v1.8.2 (Drummond (Hap2, Hap12 and Hap25), Indonesia et al., 2012) was performed. Using the (Haps13-15) and Laos (Haps22-24) revealed piecewise-constant skyline model, the 3 Haps. Worms from Australia (Haps12-13), molecular evolutionary rate of cox1 was Brazil (Haps6-7) and South Sudan (Happs9- fixed at 0.0225 substitutions per site per 10) possessed 2 Haps. The species from million years ago (Mya) as described Venezuela (Hap1), Argentina (Hap5), previously (Michelet et al., 2010). Ethiopia (Hap8), the USA (Hap11), India (Hap16) and Vietnam (Hap45) were identified as separate single haplotype. The pairwise RESULTS Fst values between isolates from different countries were calculated to estimate levels Haplotypic variability of genetic differentiation (Table 2). Most of Six sequences (AF096239, KT375455, the Fst values between American countries KT375456, KF656743, KF656744 and (Brazil, Argentina, Venezuela and USA) and KP738256) were excluded due to ambiguous Asian countries + Australia, American and alignment. The final alignment contained African countries, African countries and 487 sequences representing one Sparganum Asian countries + Australia were very high proliferum, one S. decipiens, one S. (above 0.5), although with little statistically ranarum, 12 unclassified Spirometra and significance (p < 0.05). 473 S. erinaceieuropaei. A total of 45 haplotypes (Haps) were identified from Phylogeny these 488 sequences (Table 1). Among the The model test suggested that the HKY+G haplotypes, Sparganum proliferum model was most suitable for the cox1 originated from Venezuela (AB015753), S. sequences. Both maximum likelihood and decipiens from Korea (KJ599679), three Bayesian inference analyses revealed four unclassified isolates from Argentina clades: Clades 1–4 (Fig. 2). The earliest (KF572950), Ethiopia (KM248530), and the divergence gave rise to isolates from Brazil USA (KY552892) were identified as and the USA (Clade 1) and then to a sample separate Haps; two unclassified Brazil from Argentina and a sample from Venezuela

239 2017 2017 2017 2014 2014 2013 2001 2015 2015 2007 2007 2007 2007 2007 2007 2007 2007 et al. et al. et al. 2012 2016 2016 2015 et al. 2018a et al. et al. 2015 et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. aeschenbach aeschenbach aeschenbach Miyadera Jeon Okamoto Almeida Eberhard Eberhard Okamoto Okamoto Boonyasiri Almeida Okamoto Okamoto Simpson Reference Petrigh Direct submission Boonyasiri Okamoto Okamoto Okamoto Jongthawin GenBank No. HQ699076 AJ308264-AJ308265 AJ308259-AJ308260 AJ308257-AJ308258 KM099139-KM099140 seqs No. of 1 haplotype Cox Hap1 1Hap3 AB015753 Hap2Hap13 1 1 KJ599679 1 MH298843 AJ308261 Eom Hap6Hap8Haps9-10Hap11Hap4 1 KF740506 6 1 KM248531-KM248536 KM248530 1 1 KY552892Hap45 W Haps13-15Hap16Hap12 3 1 AB278575-AB278577 KY552887 1 9 AB278574 KF539833-KF539841 W Hap7 1Hap12 KF740507 Hap13Hap13Hap13Hap13Hap13Hap12 2 1 1 AJ308263 2 AJ308262 2 1Hap2 1 JN367266 KY552886 W 2 Hap5 1 KF572950 Hap25 1 KC551943 1 sequences used in this study. Asterisks indicate sequences newly reported in this study 1 sequences used in this study. cox Host Homo sapiens Rhabdophis tigrinus Hoplobatrachus rugulosus Felis silvestris Leopardus pardalis Homo sapiens Pantherophis obsoletus Xenochrophis flavipunctatus C. familiaris C. familiaris H. sapiens L. pardalis Litoria caerulea Python bivittatus snake Tiger Vulpes velox Canis familiaris C. familiaris C. familiaris Ptyas korros Lycalopex gymnocercus Country of Venezuela Korea Myanmar Australia origin Brazil Ethiopia South Sudan USA China N/a Vietnam Indonesia India Thailand tapeworms and the corresponding sp. Argentina Spirometra Table 1. Table Taxa Sparganum proliferum Spirometra S. decipiens S. ranarum S. erinaceieuropaei

240 2014 2014 2007 2007 2007 2001 2007 2015a 2014 2015b 2016 2017 et al. et al. 2017 2009 2015 2007 2007 2012 et al. et al. et al. et al. . 2015 2010 et al. et al. et al. et al. et al. et al. et al. et al et al. et al. et al. et al. et al. et al. ino amasaki amasaki amasaki Jongthawin Jongthawin Eom Miyadera Badri Okamoto Direct submission Direct submission Lee Abe Lee Direct submission Dai Liu Zhang This study Zhang Zhang * -MK085891 * KC561784, KF988137 GQ866863-GQ866884 FJ886763-FJ886773 GQ999946-GQ999957 KF745143-KF745158 MK085765 KM605255-KM605342KP738228-KP738288 Zhang KT376494-KT376545 127 Haps22-24Hap2Hap17Hap19 16 KM099122-KM099137 1 1 KM099138 KJ599680 1Hap12Hap12 AB015754 1 2 KY009916 Hap12Hap20Hap12Hap12 1 1Hap14 AB369249Haps2, 4 AB369251Haps28-31 1Haps4, 12, 14, 26-27 1Hap12 AB522603 Y Haps2, 12 AB278573 Y 12 17 22 11 GU576892-GU576908 Ok 12 16 KF656735-KF656746 Wei Hap12Hap21Hap13 1 AF096238 1 1 AB369250 AB480297 Haps2, 12, 13, 32-38 Y Haps12, 39,Haps2, 4, 12, 14, 33, 40-42 88 52 32, 39, 43, 44 61 Haps2, 4, 12-14, Ptyas korros Hoplobatrachus rugulosus H. sapiens Elaphe climacophora catus F. catus F. N/aElaphe quadrivirgata H. sapiens E. quadrivirgata Felis catus Hap18C. familiaris Pelophylax nigromaculatus C. familiaris nigromaculatus P. 1Rana rugulosa Rana temporaria AF096237 Laos Myanmar Korea Japan Iran China Table 1 continued... Table

241 0.579 0.000 0.000 0.059 0.000 0.080 0.013 0.000 < 0.05) are in bold P 0.107 0.311 0.377 0.151 0.126 0.082 0.113 0.077 0.000 0.241 0.020 0.000 0.124 0.256 0.333 0.369 0.545 0.568 0.587 0.625 0.420 0.413 0.325 isolates. Significant comparisons ( 1.00 0.252 0.406 0.000 0.847 0.001 0.177 0.261 0.000 0.819 0.907 0.896 0.940 0.676 0.992 0.885 0.890 0.930 0.960 0.949 Spirometra st) values among F 0.000 1.000 1.0000.993 0.000 0.9941.000 0.992 1.000 0.000 1.000 0.895 0.000 1.000 1.000 1.000 0.990 1.000 0.544 0.960 1.000 0.832 0.281 0.400 0.520 1.000 0.765 1.000 0.000 0.353 0.510 0.248 1.000 1.000 1.000 0.874 0.903 0.841 0.849 0.883 0.814 0.924 0.940 0.903 0.915 0.931 0.891 1.000 1.000 1.000 0.990 1.000 0.121 0.558 1.000 0.084 0.159 0.086 0.091 1.000 0.597 0.000 1.000 1.0000.950 1.000 0.959 0.991 0.937 1.000 0.217 0.487 1.000 0.179 0.132 0.147 0.091 0.000 0.935 0.916 0.898 0.921 0.869 0.871 0.874 0.877 0.846 0.911 Table 2. Genetic differentiation ( Table Population1 Brazil2 Argentina 1 0.672 0.000 2 3 4 5 6 717 Iran 8 0.636 9 10 11 12 13 14 15 16 17 3 USA4 Venezuela5 South Sudan 6 0.669 Ethiopia 0.5117 China 1.000 0.000 0.658 8 Korea 0.562 9 Myanmar 0.824 10 Thailand 11 Japan 12 Australia 13 Indonesia 0.837 16 Vietnam 0.651 14 India15 Laos 0.654 0.940

242 Table 3. Neutrality tests results of spirometrid tapeworms. Significant level = 0.05. Number in parentheses is P value

Neutrality Tests Phylogroups Fu’s FS Tajima’s D Total population -5.40180 (0.17) -1.89942 (0.00)

Figure 1. Median-joining network of haplotypes of spirometrid tapeworms. Each haplotype is represented by a circle, with the area of the circle proportional to its frequency. The median vector is indicated by a solid black circle.

(Clade 2), next to isolates from Africa (South expansion was identified between 0.25–0.75 Sudan and Ethiopia) and a sample from Korea Mya by BSP (Fig. 3). (KJ599680) (Clade 3). The last divergence event separated isolates from Asia and Australia (Clade 4). However, within the DISCUSSION cluster of Clade 4, a sample from Korea (KJ599679) and a sample from Myanmar The systematics of Spirometra and the (MH298843) were classified as S. decipiens taxonomic position of species within it and S. ranarum in the GenBank database remain controversial (Faust et al., 1929; respectively. Neutrality tests based on Mueller, 1937; Okamoto et al., 2007; Kuchta Tajima’s D and Fu’s FS for the spirometrid et al., 2008; Zhang et al., 2019). Molecular tapeworms showed negative values, markers, especially the cox1, have played supporting a possible population expansion an important role in the studies on modern (Table 3). The result of Bayesian Skyline Plot parasite identification, genetic variation and analysis also supports a sudden population evolution over the past decade (Miyadera expansion for the total population: a sudden et al., 2001; Okamoto et al., 2007; Dai et al.,

243 Figure 2. Bayesian phylogenetic tree of spirometrid tapeworms based on the data set of cox1. The numbers along branches indicate posterior probabilities and only posterior probabilities above 0.6 are shown. The number in the parenthesis indicates the sampling size of each haplotype. The abbreviations of countries are designated as follows: Bra, Brazil; Arg, Argentina; Ven, Venezuela; S Sud, South Sudan; Eth, Ethiopia; Kor. Korea; Jap, Japan; Ira, Iran; Tha, Thailand; Indi, India; Aus, Australia; Vie, Vietnam; Indo, Indonesia; Mya, Myanmar; Lao, Laos; Chi, China.

2012; Boonyasiri et al., 2014; Eom et al., 2015; A total of 488 sequences representing Almeida et al., 2016; Zhang et al., 2018). Here, four species of spirometrid cestodes we performed the first comparative analysis (Sparganum proliferum, S. decipiens, S. of spirometrid cestodes from worldwide ranarum, and S. erinaceieuropaei) and using all available cox1 sequences deposited several unclassified Spirometra from in the GenBank as well as with 127 newly American and African identified 45 added sequences of spargana isolated from haplotypes. Sparganum proliferum, whose 29 geographical locations in mainland China. adult stage is still unknown, has been

244 Figure 3. Estimated demographic expansion of the spirometrid tapeworm population. A Bayesian skyline plot derived from the data set of cox1. The X-axis is in units of million years in the past, and the Y-axis is Ne × µ (effective population size × mutation rate per site per generation). The median estimates are shown as thick solid lines, and the purple areas show the 95% HPD limits. identified as a diphyllobothriidean cestode observations of an adult tapeworm. For the using cox1 and nuclear sdhB genes (Miyadera unclassified Spirometra, four isoaltes from et al., 2001). In this study, S. proliferum was America, one from Argentina, two from Brazil identified as a separate Hap, indicating that and one from the USA, were revealed as four this organism should be a valid species. separate Haps, and seven samples from Africa However, the phylogenetic analysis showed (South Sudan 6, Ethiopia 1) showed 3 Haps, that S. proliferum is a sister lineage to the indicating their specific taxonomy within Argentina isolate. Plerocercoid larvae the genus. The remaining sequences of S. collected from snake (Rhabdophis tigrinus) erinaceieuropaei from 11 countries (China, were identified as S. decipiens (KJ599679) Japan, Korea, Myanmar, Thailand, Indonesia, in Eom et al. (2015). Although the S. Laos, Vietnam, Iran, India and Australia) decipiens (KJ599679) was revealed as a constituted 37 additional Haps. The haplotype separate Hap, this species was inserted diversity was less than that with smaller into a group containing mainly Asia and sampling of S. erinaceieuropaei in China Australia S. erinaceieuropaei isolates in the (Zhang et al., 2015a, 2016). A possible reason phylogenetic tree, suggesting the ambiguous might be that the molecular marker used taxonomy of S. decipiens. The S. ranarum here is only a small portion of the cox1 (369 from Myanmar and isolates of S. bp), and the markers for Chinese samples in erinaceieuropaei from Thailand and different previous studies were the concatenated locations in China were identified as a complete genes of cox1 (1566 bp) and cytb single haplotype (Hap2), confirming that (1110 bp) (Zhang et al., 2015a, 2015b, 2016). S. ranarum is a synonym of S. The phylogenetic analyses supported the erinaceieuropaei. However, Jeon et al. existence of four lineages among spirometrid (2018a) confirmed a Spirometra species of tapeworms. America isolates belong to two Myanmar origin as S. ranarum based on cox1 clades: Clade 1 and Clade 2. Interestingly, in and nad1 genes as well as morphological comparison with the Argentina isolate, the

245 USA isolate has a closer relationship with of the genetic structure of spirometrid the Brazil isolates. In addition, sibling tapeworms needs further investigation relationships were revealed between the with deeper sampling, especially with Argentina isolate and S. proliferum. The third samples from America, Africa and other lineage only included African isolates, with scarce species, in the future. the exception of a Korea isolate (KJ599680), indicating the specific phylogenetic position of the African Spiromtra species. In CONCLUSION consideration of the above genetic analyses, the unclassified Spirometra from America In this study, all publicly available cox1 and Africa should be separate species within sequences in GenBank and 127 newly added the genus which could be due to high Fst sequences from China were used to explore values between the different geographical the genetic diversity analysis of spirometrid isolates, identified as individual haplotypes cestodes worldwide. A total of 488 sequences and distinct phylogenetic patterns. Never- of spirometrid tapeworms from 113 theless, the precise taxonomy of the geographical locations with different hosts unclassified isolates requires further in 17 countries identified 45 haplotypes. identification with more robust evidence in The network, genetic differentiation and the future. phylogenetic analyses revealed that (1) there A Korean isolate (KJ599680), which are four clades of spirometrid cestodes: Clade has been classified as S. erinaceieuropaei 1 included isolates from Brazil and USA, (Eom et al., 2015), was identified as member Clade 2 included isolates from Argentina of Clade 3 in the genetic analysis. The and Venezuela, Clade 3 contained African phylogenetic tree topology also indicated isolates and one Korean sample, Clade 4 that this species had a close relationship contain the remaining samples from Asia and with the African isolates. In addition, our Australia; (2) unclassified Spirometra from previous mitogenomic comparative analysis America and Africa should be considered of Spirometra also showed that the KJ599680 individual valid species within the genus; was distinct from other S. erinaceieuropaei (3) The taxonomy of two Korean isolates mitogenomic sequences from China and (S. erinaceieuropaei KJ599680 and S. Japan (Zhang et al., 2017). Therefore, the decipiens KJ599679) was still ambiguous taxonomy of the Korea isolate (KJ599680) and need to be further identified. In addition, was probably imprecisely identified in the demographical analyses supported GenBank and should be considered as an population expansion for all spirometrid independent species within the genus. tapeworms. Although S. decipiens (KJ599679) was identified as a separate haplotype, its Acknowledgements. We thank Mr. QW Wu, close relationship with most of the S. DJ Zeng, CH Du, JW Rao, Y Chen, YH Lin and erinaceieuropaei isolates (with exception of HH Huang, Ms. M Gao, HQ Li, M Peng, SZ KJ599680) has been firmly supported in the Wang, F Wu, SS Wang, M Tao and SQ Yi for phylogenetic analysis, indicating that it is collecting valuable specimens used in this hard to distinguish S. decipiens from S. study. This work was supported by National erinaceieuropaei using only the cox1 gene. Natural Science Foundation of China For S. erinaceieuropaei, although high (U1704189), China Postdoctoral Science haplotype diversity has been found, no Foundation (2018T110740), Henan Province obvious phylogenetic patterns were revealed Science and Technology Key Project among isolates from different hosts and (182102310075) and Backbone Teachers different geographical localities. Con- Development Program in Henan Province sidering that the distribution of Spirometra (2017GGJS011). species is cosmopolitan, the true pattern

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