Morphology, genetic characterization and phylogeny of Moniliformis tupaia n. sp. (Acanthocephala: Moniliformidae) from the northern tree shrew Tupaia belangeri chinensis Anderson (Mammalia: Scandentia)

A new species of Moniliformis, M. tupaia n. sp. is described using integrated morphological methods (light and scanning electron microscopy) and molecular techniques (sequencing and analysing the nuclear 18S, ITS, 28S regions and mitochondrial cox1 and cox2 genes), based on specimens collected from the intestine of the northern tree shrew Tupaia belangeri chinensis Anderson (Scandentia: Tupaiidae) in China. Phylogenetic analyses show that M. tupaia n. sp. is a sister to M. moniliformis in the genus Moniliformis, and also challenge the systematic status of Nephridiacanthus major. Moniliformis tupaia n. sp. represents the third Moniliformis species reported from China.


Introduction
The northern tree shrew Tupaia belangeri chinensis Anderson (Mammalia: Scandentia: Tupaiidae) is a novel ideal animal model for human disease, due to its small size, easy breeding, rapid reproduction and close genetic relationship to primates (Xu et al., 2012(Xu et al., , 2013;;Xiao et al., 2017;Tang et al., 2018;Wang et al., 2021).Tupaia belangeri chinensis is omnivorous, eating fruits, seeds, insects and small vertebrates, which is mainly distributed in southwest China (including Yunnan and Sichuan Provinces) (Xiang and Yang, 2014) and can act as the intermediate and definitive host for some helminth parasites and protozoa (Brack et al., 1987;Tian et al., 1989;Xiang et al., 2010;Xiang and Yang, 2014).However, our present knowledge of the species composition of the acanthocephalans of the northern tree shrew is very limited.To date, only Prosthenorchis sp.(Archiacanthocephala: Oligacanthorhynchidae) has been reported from T. belangeri chinensis (Tian et al., 1989).
In the present study, some acanthocephalan specimens were collected from T. belangeri chinensis in China.In order to accurately identify these acanthocephalan specimens to species level, the detailed morphology of these specimens was studied using light and scanning electron microscopy.Moreover, the nuclear small subunit ribosomal DNA (18S), internal transcribed spacer (ITS) and large subunit ribosomal DNA (28S), and mitochondrial cytochrome c oxidase subunit 1 (cox1) and subunit 2 (cox2) genes were sequenced and analysed.Phylogenetic analyses were also performed based on the 18S + cox1 sequence data using maximum likelihood (ML) and Bayesian inference (BI) methods, to clarify the phylogenetic relationships between this species and its congeners.

Morphological observation
Acanthocephalans were isolated from the intestine of the northern tree shrew T. belangeri chinensis in Kunming, Yunnan Province, China.Specimens were washed and kept in cold water for several hours until the proboscis everted, and then stored in 80% ethanol until studied.For light microscopical studies, specimens were made in impermanent mount slide and cleared in lactophenol.Photomicrographs were recorded using a Nikon® digital camera coupled to a Nikon® optical microscopy.For scanning electron microscopy (S.E.M.), specimens were post-fixed in 1% OsO4, dehydrated via an ethanol series and acetone, and then critical point dried.The specimens were coated with gold and examined using a Hitachi S-4800 scanning electron microscope at an accelerating voltage of 20 Kv.Measurements (range, followed by the mean in parentheses) are given in micrometres unless otherwise stated.

Phylogenetic analyses
Phylogenetic analyses were performed based on the 18S + cox1 sequence data using maximum likelihood (ML) inference with IQ-TREE and Bayesian inference (BI) with Mrbayes 3.2 (Ronquist et al., 2012;Nguyen et al., 2015), respectively.Polyacanthorhynchus caballeroi Diaz-Ungria & Rodrigo, 1960 (Polyacanthocephala: Polyacanthorhynchida) was treated as the out-group.The in-group included 15 species of the class Archiacanthocephala representing 6 different genera belonging to 3 orders Gigantorhynchida, Moniliformida and Oligacanthorhynchida.The detailed information of acanthocephalan species included in the present phylogenetic analyses is provided in Table 1.
We used a built-in function in IQTREE to select a best-fitting substitution model for the sequences according to the Bayesian information criterion (Posada and Crandall, 2001).The GTR + F + I + G4 model was identified as optimal nucleotide substitution model.Reliabilities for ML tree were tested using 1000 bootstrap replications and BI tree was tested using 10 million generations.In the ML tree, bootstrap support (BS) values ⩾90 were considered as fully supported; whereas BS values ⩾70 and <90 were considered as generally supported.In the BI tree, Bayesian posterior probabilities (BPP) ⩾0.90 were considered as fully supported, whereas BPP values ⩾0.70 and <0.90 were considered as generally supported.

Phylogenetic analyses
Phylogenetic trees of the class Archiacanthocephala constructed from the 18S + cox1 sequence data using ML and BI methods have almost identical topology (Fig. 4).The representatives of Archiacanthocephala were divided into three major clades.Clade I included species of Macracanthorhynchus, Nephridiacanthus, Oligacanthorhynchus and Oncicola, representing the order Oligacanthorhynchida.Among them, the phylogenetic results showed N. major (Bremser, 1811) clustered together with M. ingens (Von Linstow, 1879).Clade I contained species of Moniliformis, representing the order Moniliformida.Clade III included species of Mediorhynchus, representing the order Gigantorhynchida.In the genus Moniliformis, M. tupaia n. sp.showed sister relationship with M. moniliformis.

Discussion
The present specimens collected from the northern tree shrew T. belangeri chinensis belong to the genus Moniliformis (Moniliformida: Moniliformidae), due to the pseudosegmented trunk, the very small cylindrical proboscis, the double-walled proboscis receptacle, the very long lemnisci and the presence of 8 spherical cement glands (Travassos, 1917;Van Cleave, 1923, 1953;Southwell and Macfie, 1925;Yamaguti, 1963;Schmidt, 1972;Amin, 1987).The genus Moniliformis currently comprises 19 species mainly parasitic in mammals (Amin, 2013;Amin et al., 2016Amin et al., , 2019;;Martins et al., 2017;Gomes et al., 2020;Lynggaard et al., 2021).Among them, only M. moniliformis and Moniliformis sp.XH-2020 have been reported in China (Chen, 1933;Chandler, 1941;Dai et al., 2022).The proboscis of the new species has 14 spiral longitudinal rows of 7-8 simple rooted hooks each, which is similar to the proboscis of following species M. acomysi Ward andNelson, 1967, M. cryptosaudi, M. moniliformis, M. saudi andM. siciliensis Meyer, 1932.Moniliformis tupaia n. sp.can be easily distinguished from M. acomysi by its much longer proboscis and lemnisci (proboscis 0.37-0.44mm and lemnisci 5.00-9.32mm long in the male of new species vs proboscis 0.19-0.36mm and lemnisci 2.73-4.42mm long in the male of M. acomysi).Moniliformis tupaia n. sp.differs from M. cryptosaudi and M. saudi by having larger cement glands (854-1829 long in the new species vs 312-811 long in the latter two species).Moreover, M. cryptosaudi and M. saudi are both parasitic in hedgehogs (Erinaceomorpha: Erinaceidae) in Saudi Arabia and Iraq, but the new species parasitizes the northern tree shrew T. belangeri chinensis in China.Furthermore, molecular analysis revealed strong genetic divergence (25.9-26.9%difference in nucleotides in the cox1 region) between the new species and M. cryptosaudi and M. saudi.Moniliformis siciliensis is a poorly known acanthocephalan species only reported from the garden dormouse Eliomys quercinus Linnaeus (Mammalia: Rodentia) in the Italian island of Sicily (Meyer, 1932;Petrochenko, 1958).The new species differs from M. siciliensis in having shorter lemnisci (5.00-9.32mm long in the former vs about 10.0 mm in the latter) and different localities and hosts.

Parasitology
The class Archiacanthocephala currently includes 4 orders, namely Gigantorhynchida, Moniliformida, Oligacanthorhynchida and Apororhynchida (Amin, 2013).However, the phylogenetic relationships of the 4 orders remain unclear, due to a lack of genetic data of some taxa, especially the order Apororhynchida.The previous phylogenetic study using 18S or 18S + cox1 genetic data suggested a close affinity between Moniliformida and Gigantorhynchida (Amin et al., 2013(Amin et al., , 2020)).However, our phylogenetic results based on the 18S + cox1 data suggested Moniliformida is a sister to Oligacanthorhynchida, rather than Gigantorhynchida, which are consistent with some previous studies based on cox1 or 18S data (Gomes et al., 2020;Rodríguez et al., 2021;Amin et al., 2021Amin et al., , 2022)).In the order Oligacanthorhynchida, the present phylogeny displayed Nephridiacanthus major nested in representatives of Macracanthorhynchus (clustered together with M. ingens), which challenged the current systematic position of this species.The present results agreed well with the previous phylogenetic study based on cox1 data (Rodríguez et al., 2021).In the genus Moniliformis, our molecular phylogenetic analyses indicate that M. tupaia n. sp. is a sister to M. moniliformis.

Table 1 .
Species of Archiacanthocephala with their detailed information of genetic data included in the phylogenetic analyses