By means of an integrated approach, including molecular, morphological, anatomical and histological data, we describe a new species of freshwater flatworm of the genus Dugesia from southwest China, representing the third species recorded for the country. Morphologically, the new species, Dugesia umbonata Song & Wang, sp. nov., is particularly characterised by the presence of a muscularised hump immediately antero-dorsally to a knee-shaped bend in its bursal canal and by an ejaculatory duct that opens subterminally through the dorsal side of the penis papilla. Four molecular datasets (18S rDNA; ITS-1; 28S rDNA; COI) facilitated determination of the phylogenetic position of the new species, which belongs to a clade comprising other species from the Australasian and Oriental regions. We also analysed the structure of its major nervous system by means of the acetylcholinesterase (AChE) histochemical method and compared these results with data available for three other species of Dugesia.

Molecular phylogeny, taxonomy, histochemistry, acetylcholinesterase (AChE)

The genus Dugesia Girard, 1850 currently comprises about 93 valid species and exhibits a distribution that comprises a major part of the Old World and Australia (see Sluys and Riutort 2018: fig. 13B). China covers a vast territory, covering six temperature zones, thus resulting in various types of climate. Therefore, it is to be expected that variations in climate and other physico-geographical conditions have resulted in a rich biodiversity of freshwater planarians, including species of Dugesia. However, the Dugesia fauna of China is very poorly known, thus far only being represented by two species. For a long time, Dugesia japonica Ichikawa & Kawakatsu, 1964 was the only species recorded for this country (Chen et al. 2001). It was only recently that a second species for the genus was described from southern China, viz. Dugesia sinensis Chen & Wang, 2015 (Chen et al. 2015).

In the present study, we describe a third, new species of Dugesia from southwest China on the basis of both molecular and morphological data. In addition, we document the structure of the major nervous system of the new species on the basis of a histochemical study using acetylcholinesterase (AChE) and compare its nervous system with those documented for three other species of Dugesia. Information on the structure of the nervous system may be useful for future phylogenetic and taxonomic studies.

The substitution saturation tests of the COI datasets (nos. 7 to 12 in Suppl. material 3: Table S3) revealed that gaps and ambiguous codes significantly influenced the accuracy of the tests. The results reveal that the COI dataset is actually substitutionally saturated (Iss > Iss.c; no. 7 in Suppl. material 3: Table S3) and, therefore, not suitable for phylogenetic reconstruction. The main reason for this resides in the presence of two rather short sequences, which will cause a great number of gaps during alignment. Inclusion of only the fully-resolved sites of the COI dataset (nos. 8, 10 and 12 in Suppl. material 3: Table S3) reduces the analysis to only a few sites and, thus, cannot represent the true level of substitution saturation. Hence, the real saturation level in the COI dataset is most likely closer to the results obtained with the aCOI dataset (nos. 13 and 14 in Suppl. material 3: Table S3) than that it reflects its current results (no. 7 in Suppl. material 3: Table S3), because numerous gaps have a greater influence during determination of the saturation than two missing sequences (D. batuensis and D. deharvengi). This implies that the COI dataset is more likely to experience little or no substitution saturation and, thus, would be reliable and valid and, in principle, suitable for phylogenetic analysis. Although the third codon position showed substitution saturation to a greater or lesser extent (nos. 17 and 18 in Suppl. material 3: Table S3), its inclusion, nevertheless, is to be preferred as, without it, the phylogenetic trees exhibit rather low resolution.

In the phylogenetic trees generated during the present study, D. umbonata occupies a single branch that shares a sister-group relationship either with D. japonica (in trees based on the 18S rDNA, 28S rDNA, COI and concatenated datasets; Fig. 1 and Suppl. material 4: Figs S1–S5) or with a clade formed by a group of species from the Australasian and Oriental regions (in the case of ITS-1; Suppl. material 4: Fig. S6). The long branch of D. umbonata in all phylogenetic trees signals its high divergence from other species and supports its separate specific status.

In the phylogenetic trees obtained from the separate 18S rDNA, 28S rDNA and COI datasets or from the concatenated dataset, the sister-group relationship between D. umbonata and D. japonica is robust, strongly suggesting that these two species belong to the same monophyletic group and are each other’s closest relative (Fig. 1 and Suppl. material 4: Figs S1–S5).

It is unusual that five phylogenetic trees (based on 18S rDNA, 28S rDNA, COI, ITS-1 and the concatenated dataset; Fig. 1 and Suppl. material 4: Figs S1–S6) show rather different topologies. However, the trees resulting from the concatenated dataset (Fig. 1 and Suppl. material 4: Fig. S1) should be the most reliable ones, as these are based on all four genes, while a partition analysis was also applied. One could argue that the low support values are due to incongruence amongst the various molecular markers and that a concatenated dataset could be analysed with exclusion of COI. When this would result in higher support values, it would underscore removal of COI from the concatenated dataset. In point of fact, we did perform a phylogenetic analysis on such a restricted concatenated dataset which, surprisingly, had precisely the opposite effect, i.e. lowering the support values. Therefore, we kept COI in our concatenated dataset.

With respect to the trees independently generated from each of the four sequences, it should be noted that the reliabilities of 18S rDNA and 28SrDNA are lower than those of ITS-1 and COI, due to the limited number of 18S and 28S rDNA sequences involved in the analysis and the fact that the last-mentioned sequences are more conservative during evolution than ITS-1 and COI, implying that the latter provide more molecular signals for our level of analysis. That is also the reason why the phylogenetic trees based on COI and ITS-1 have topologies similar to those based on the concatenated dataset (Fig. 1 and Suppl. material 4: Figs S1–S3, S6). In the 18S rDNA tree, the clade comprising D. umbonata and D. japonica clusters with Afrotropical and Cameroon species (Suppl. material 4: Fig. S4), while it clusters with species from the western Palearctic region in the 28S rDNA tree (Suppl. material 4: Fig. S5). However, due to the low reliability of these trees (see above), we believe that D. umbonata and D. japonica actually belong to the Australasian and Oriental species group, as shown in the phylogenetic trees based on COI, ITS-1 and the concatenated dataset.

Although the major purpose of our phylogenetic analysis was to determine the phylogenetic position and taxonomic status of the new species D. umbonata, inclusion of four gene sequences of no less than 39 species of Dugesia in our BI and ML analyses makes it worthwhile to compare the topology of our tree with those generated in several other, recent studies. Evidently, the different molecular markers used in the different studies, including ours, complicate proper evaluation and, therefore, we refrain from drawing any conclusions on the evolutionary relationships between the species.

In the tree of Harrath et al. (2019: fig. 2), the species D. bijuga Harrath & Sluys, 2019 and D. pustulata Harrath & Sluys, 2019 from Cameroon cluster together and are sister to a group constituted by Asian, Australian and European species. It is noteworthy that, in our phylogenetic trees based on COI, D. bijuga clusters with an Oriental species, viz. D. batuensis, albeit with rather low support (0.60 pp; 33% bs; Suppl. material 4: Figs S2, S3). However, in our other trees, based on ribosomal genes and the concatenated dataset, these two Cameroon species share the same affinities as revealed by Harrath et al. (2019), in that they cluster with Australasian, Oriental and Western Palearctic species.

Although we included in our analyses these two species of Dugesia from Cameroon that were recently described by Harrath et al. (2019), the topology of our tree, based on the concatenated dataset, is more similar to the one of Stocchino et al. (2017), which was based on ITS-1 and COI, than to the tree published by Harrath et al. (2019), based on 18S rDNA and COI. With respect to the biogeographic groupings identified in these studies, the five biogeographic groups indicated in our trees, based on the concatenated dataset, are basically similar to those in Harrath et al. (2019) and Stocchino et al. (2017). However, it should be noted that, although in the present study we included more genetic markers and a larger number of species, as compared with the studies of Stocchino et al. (2017) and Harrath et al. (2019), the number of Dugesia species for which molecular data are available (~40) is still rather small. Therefore, robust hypotheses on the historical biogeography of the genus Dugesia and the testing of previously-formulated ones (for an overview, see Harrath et al. 2019) may only be well possible when more molecular data have become available, notably from more and also better resolved markers.

Frequently, species of Dugesia are characterised only by a unique, diagnostic combination of features, while each of these characters separately occurs in many congeners. Much more rarely, a species exhibits an unambiguous apomorphic character or character state through which it can easily and quickly be diagnosed. Fortunately, Dugesia umbonata is an example of the last-mentioned situation, as it features a peculiar muscularised hump on the postero-dorsal portion of its bursal canal, a structure thus far unknown from any species of Dugesia. In addition, D. umbonata exhibits the unique condition in which its ejaculatory duct opens subterminally through the dorsal side of the penis papilla. Although other species of Dugesia may also show subterminal openings (see Stocchino et al. 2017 and references therein), these are located on the ventral side of the penis papilla, with the only exception of D. hepta Pala et al., 1981, in which the opening is located laterally (Stocchino et al. 2005). Therefore, the condition in D. umbonata (subterminal and dorsal opening of ejaculatory duct) represents an apomorphy.

Other noteworthy features of D. umbonata are the asymmetrical openings of the oviducts and the presence of a duct between the seminal vesicle and the diaphragm, representing character states 11-1 and 5-1, respectively, in the phylogenetic analysis of Sluys et al. (1998). Amongst the already-described species of Dugesia, the combination of these two character-states occurs only in D. notogaea Sluys & Kawakatsu, 1998 and D. sudanica Dahm, 1971 (Dahm 1971; Sluys et al. 1998). However, both species are different from D. umbonata as, for example, neither of them exhibits the muscular hump on the bursal canal.

In view of the fact that, in most of our molecular analyses and particularly in the tree based on the concatenated dataset (Fig. 1), D. umbonata was the sister-species of D. japonica and that the latter occurs also in China (Kawakatsu et al. 1995), it is opportune to discuss the similarities and differences between these two species in some more detail. In particular, it is noteworthy that specimens of D. japonica have been described in which the vaginal region of the bursal canal is surrounded by a “…very wide halo-like structure…[consisting of] …mesenchymal tissue traversed by several coarse rows of longitudinal muscles and less-developed radial ones” (Kawakatsu et al. 1976: p. 85). This is reminiscent of the muscularised hump in D. umbonata (cf. Kawakatsu et al. 1976: fig. 3), but frequently, this thick coat of muscles in D. japonica gradually expands and thereafter also gradually decreases in diameter towards the copulatory bursa (cf. Kawakatsu et al. 1979: fig. 3), while it is developed also ventrally to the bursal canal (cf. Kawakatsu et al. 1976: figs 5, 8; Kawakatsu et al. 1980: fig. 2A). This contrasts with the situation in D. umbonata, in which the muscular hump is confined to the dorsal side of the bursal canal and also has a much more clearly-defined shape and proportions. Evidently, other differences also preclude assignment of our newly-collected specimens to D. japonica. For example, in D. japonica, the oviducts open symmetrically into the bursal canal, whereas in D. umbonata, the oviducal openings are highly asymmetrical. Another clear difference between both species concerns the location of the subterminal opening of the ejaculatory duct at the tip of the penis papilla. Dugesia japonica expresses the condition that is rather common amongst a group of Dugesia species distributed in the Palearctic, Afrotropical, Oriental, Sino-Japanese and Australian regions, i.e. a subterminal ventral opening (cf. Sluys et al. 1998), whereas D. umbonata exhibits the unique character state of a subterminal dorsal opening.

Hyperplasic ovaries are a common feature of sexualised planarians from originally fissiparous populations (cf. Stocchino et al. 2012 and references therein) and, thus, were expected to occur in the sexualised specimens of D. umbonata. Regularly, hyperplasic ovaries in such ex-fissiparous specimens go together with under-developed testes or even with complete absence of testicular follicles (cf. Stocchino et al. 2012 and references therein). The latter condition is present also in D. umbonata as we were unable to discern testis follicles in any of the specimens examined, coinciding with the fact that, in our cultures, we never found cocoons.

The major nerve system of D. umbonata, as documented above, may be compared with three of its congeners for which the structure of the nerve system was described, viz. Dugesia gonocephala (Dugès, 1830), D. japonica and D. sinensis Chen & Wang, 2015. As studies on these three species and D. umbonata were done with different techniques (e.g. various immunocytochemical methods, acetylcholinesterase (AChE) histochemistry, traditional histology), some caution should be taken in comparing their results. However, as our focus is on the gross morphology of the nervous system, it is well possible to compare the neuroanatomical results of these studies with our results on D. umbonata.

The shape of the planarian brain probably is correlated with the shape of the head, with triangular heads tending to have brains composed of thickened anterior portions of the ventral nerve cords that form two lobes or cerebral ganglia and truncate heads having more rounded, bilobed cerebral ganglia (Hyman 1951; Reuter et al. 1995), while in land planarians, the ventral nerve plate extends to the anterior tip and may expand into the lunate head of species of Bipalium Stimpson, 1858 (Hyman 1951; Rieger et al. 1991). In that respect, the shape of the brain is similar in all four species of Dugesia in that it consists of the thickened anterior portions of the ventral nerve cords, which are connected by a major commissure at their anterior ends, while the eyes are located at a short distance posterior to this commissure and at the lateral margins of the thickened lobes (cf. Ude 1908; Umesono et al. 2011; Chen et al. 2015). In all four species, the lateral nerves communicate with the marginal nerves, which are much less condensed than the ventral nerve cords or the transverse and lateral branches and basically consist of a peripheral submuscular nerve plexus (cf. Chen et al. 2015: fig. 16). In D. gonocephala, D. japonica and D. sinensis, the major ventral nerve cords are not confluent in the very tail end of the body (Ude 1908; Nishimura et al. 2010; Chen et al. 2015), whereas in D. umbonata the cords pass into each other (Fig. 8A, C).

In D. gonocephala, Ude (1908) counted 47 transverse commissures and 51–64 lateral branches, while in D. japonica (46–54 lateral branches; 37–40 transverse commissures; Nishimura et al. 2010) and D. umbonata (43–47 lateral branches; 38–42 transverse commissures; this study), the numbers are somewhat lower. For D. sinensis, the numbers are considerably lower, viz. 21–24 transverse commissures and 28 lateral branches (from Chen et al. 2015: fig. 16).

For the number of transverse commissures between the two ganglia and the number of lateral branches arising from the brain, the following data have been reported, respectively: D. gonocephala: 8, 8 (Ude 1908); D. sinensis: 7–8, 9–10 (from Chen et al. 2015: fig. 16; Chen et al. 2015, respectively); D. japonica: 6, ± 9 (Nishimura et al. 2010; Umesono et al. 2011). These figures are in the same order of magnitude as the branches in D. umbonata, viz. 6–7 transverse commissures between the ganglia and 9–11 lateral branches arising from each cerebral ganglion.

This study was supported by Special Funds for the Cultivation of Guangdong College Students’ Scientific and Technological Innovation (“Climbing Program” Special Funds; grant no. pdjh2020b0509); a Shenzhen Science and Technology Application Demonstration project (grant no. KJYY20180201180253571); a China Undergraduate Training Program for Innovation and Entrepreneurship (grant no. 201910590033) and the Shenzhen University Innovation Development Fund (grant no. 2019272). We are grateful to Xiao-Zhou Hu for collecting the samples, Zhong-Yin Sun for culturing the animals in lab and Yi-Tao Lin for assistance with the PCR. We thank Dr. Fang-Luan Gao (Fujian Agriculture and Forestry University, China), Dr. Yu Zhang (Shenzhen University, China) and Si-Yu Zhang for their kind help and useful suggestions on molecular phylogeny. Yuki Oya from Hokkaido University (Japan) is also thanked for making available literature on Japanese Dugesia species. We also thank Jun-Yu Li, Jia-Jia Chen, Ying Yang and Ming-Qi Wu for their kind support in the laboratory. Wei-Xuan Li would like to express his gratitude to all members, as well as supervisors, in Wang’s lab for their companionship and support during this study and, more importantly, during his four-year academic journey at Shenzhen University. We thank reviewer Dr. M. Álvarez-Presas and also an anonymous reviewer for their constructive comments and encouragement.