Addition to the known diversity of Chinese freshwater planarians: integrative description of a new species of Dugesia Girard, 1850 (Platyhelminthes, Tricladida, Dugesiidae)

Yi Liu; Xiao-Yu Song; Zhong-Yin Sun; Wei-Xuan Li; Ronald Sluys; Shuang-Fei Li; An-Tai Wang

doi:10.3897/zse.98.83184

Abstract

The present paper describes a new species of freshwater flatworm of the genus Dugesia from Guizhou province, China, based on an integrative approach, combining morphological, histological and molecular information. This new species, Dugesia gemmulata Sun & Wang, sp. nov., is characterized by the ventral part of the most posterior section of the bursal canal being provided with a voluminous, ellipsoidal muscular swelling; sac- or egg-shaped seminal vesicle situated near the ventral body surface in anterior portion of the penis bulb; postero-dorsal wall of seminal vesicle communicating with a narrow duct that first runs almost vertically but then shows a postero-dorsally directed loop before connecting with a small diaphragm; an ejaculatory duct opening terminally or subterminally; an asymmetrical penis papilla, with its dorsal lip being provided with a bump; oviducts opening asymmetrically into female copulatory apparatus, with the left oviduct opening into the common atrium and the right oviduct opening into the vaginal section of the bursal canal. Molecular phylogenetic analyses revealed that the new species belongs to a clade comprising species from the Australasian and Oriental regions, while it shares a sister-group relationship with D. umbonata Song & Wang, 2020, a species characterized by a muscular swelling on the dorsal side of its bursal canal.

Key Words

anatomy, freshwater planarian, histology, molecular phylogeny, taxonomy

Introduction

Freshwater planarians of the genus Dugesia Girard, 1850 (Platyhelminthes, Tricladida, Dugesiidae) currently comprise about 100 nominal species distributed in a large part of the Old World and Australia (Sluys and Riutort 2018: fig. 13B). For a long period of time, Dugesia japonica Ichikawa & Kawakatsu, 1964 was the only known Dugesia species from mainland China (cf. Kawakatsu et al. 1995). However, over the past few years renewed attention paid to this group of animals has rapidly increased the number of Chinese Dugesia by seven more species, viz., D. sinensis Chen & Wang, 2015, D. umbonata Song & Wang, 2020, D. semiglobosa Chen & Dong, 2021, D. majuscula Chen & Dong, 2021, D. circumcisa Chen & Dong, 2021, D. verrucula Chen & Dong, 2021, and D. constrictiva Chen & Dong, 2022 (Chen et al. 2015; Song et al. 2020; Wang et al. 2021a, b, 2022).

In the present study we provide an integrative description of another new species of Dugesia, which was collected from Guizhou Province located at a plateau in the southwest of China, on the basis of morphological, histological and molecular data.

Materials and methods

Sample collection and culturing

The specimens were collected in a rural streamlet at an altitude of about 1,400 m in Qingzhen City, Guizhou Province, China (26°33.83'N, 106°13.65'E; Fig. 1). The animals were collected from the underside of small pebbles in the riverbed and, thereafter, were transported to Shenzhen University. The animals were reared as described in Song et al. (2020). Only 15 specimens were collected at the beginning, none of which was sexually mature. During about two years of rearing under laboratory conditions, the animals multiplied by means of fission and, eventually, in total, about 30 specimens sexualised.

Figure 1.

Locality and habitat of Dugesia gemmulata. A. Sampling locality in Qingzhen City, Guizhou, China; B, C. Habitat at sampling locality.

DNA extraction, amplification, sequencing and phylogenetic analysis

After starvation for three days, total DNA was extracted independently from three asexual fissiparous individuals, using the E.Z.N.A.^TM Mollusc DNA Isolation Kit (Omega, Norcross, GA, USA). Cytochrome C oxidase subunit I (COI) gene was amplified by polymerase chain reaction (PCR), using forward primer COIF: GCT CAT GGT TTA RTW ATG ATG ATT TTY TT and reverse primer COIR: GWG CAA CAA CAT ART AAG TAT CAT (Chen et al. 2015). PCR was performed as follows: denaturation of 5 min at 94 °C, followed by 35 cycles of 50 s at 94 °C, 45 s at 5 °C, 50 s at 7 °C and finally 7 min extension at 72 °C (Chen et al. 2015). DNA sequences were determined by Sanger sequencing in Beijing Genomics Institution (BGI, Shenzhen, China) and by using the same primers as used for the amplification. All new sequences have been uploaded in GenBank, NCBI (Table 1).

Table 1.

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GenBank accession numbers of COI sequences used in the phylogenetic analyses.

Species	COI	Species	COI
Recurva postrema	KF308763	D. gibberosa	KY498857
Schmidtea mediterranea	JF837062	D. gonocephala	FJ646941, FJ646986
Dugesia aethiopica	KY498845	D. hepta	MK712639
D. afromontana	KY498846	D. ilvana	FJ646989, FJ646944
D. arcadia	KC006969	D. improvisa	KF308774
D. ariadnae	JN376142	D. japonica	AB618487
D. aurea	MK712632	D. liguriensis	MK712645
D. batuensis	KF907819	D. majuscula	MW533425
D. benazzii	FJ646977, FJ646933	D. malickyi	KF308750
D. bifida	KY498851	D. naiadis	KF308757
D. bijuga	MH119630	D. notogaea	FJ646993, FJ646945
D. circumcisa	MZ147041	D. parasagitta	KF308739
D. corbata	MK712637	D. pustulata	MH119631
D. constrictiva	MZ871766	D. ryukyuensis	AB618488
D. cretica	KC006974	D. sagitta	KC007006
D. damoae	KF308768	D. semiglobosa	MW525210
D. deharvengi	KF907820	D. sicula	KF308797
D. effusa	KF308780	D. sigmoides	KY498849
D. elegans	KC006985	D. sinensis	KP401592
D. etrusca	MK712651	D. subtentaculata	MK712561
D. gemmulata sequence 1	OL632201*	D. umbonata	MT176641
D. gemmulata sequence 2	OL632202*	D. verrucula	MZ147040
D. gemmulata sequence 3	OL632203*	D. vilafarrei	MK712648

Phylogenetic trees were generated on the basis of COI data of the following ingroup taxa: the new species Dugesia gemmulata Sun & Wang, sp. nov. (described below) and 41 other species of the genus Dugesia (Table 1). For outgroups, we used two other dugesiid taxa, viz., Recurva postrema Sluys & Solà, 2013 and Schmidtea mediterranea Benazzi et al., 1975. All sequences were downloaded from GenBank, NCBI (Table 1).

Methods and parameters for identification of open reading frames and substitution saturation assessment were the same as in Song et al. (2020). COI sequences were translated into amino acids, using genetic code 9 (echinoderm and flatworm mitochondrial genetic code), after which they were aligned by MUSCLE (Edgar 2004) in the TRANSLATOR X server (Abascal et al. 2010). Regions of ambiguous alignments were excluded by GBLOCKS (Talavera and Castresana 2007), using the less stringent selection option (smaller final blocks, gap positions within the final blocks, and less strict flanking positions allowed). JMODELTEST 2.1.7 (Darriba et al. 2012) was used to estimate the best-fit evolution model for the Bayesian Inference method (BI); the GTR+I+G model was selected by applying the Akaike information criterion (AIC). MODELFINDER (online version: http://iqtree.cibiv.univie.ac.at/; accessed 18 Jan. 2022; Kalyaanamoorthy et al. 2017) was used to find the best-fit evolution model for the Maximum Likelihood method (ML); the GTR+F+I+G4 model was selected by applying the Bayesian Inference criterion (BIC).

Phylogenetic trees were constructed by ML and BI methods, using IQ-TREE 2.1.3 (Minh et al. 2020) and MRBAYES 3.2.6 (Ronquist et al. 2012), respectively. For ML, ultrafast bootstrap analysis with 10,000 replications was used to assess the confidence level of the nodes (Hoang et al. 2018). For BI, the Markov Chain Monte Carlo (MCMC) algorithm was run for 7,000,000 generations in four simultaneous chains, sampling every 7,000 generations. In order to ensure that the chains had reached the stationary region, we terminated the analysis when the average standard deviation of split frequencies was consistently lower than 0.01; the first 25% of the generated trees were discarded as burn-in to obtain the consensus tree and posterior probability values. We checked the .p file of each run in TRACER v1.7.1 (Rambaut et al. 2018) to ensure that the effective sample size (ESS) values of each parameter were above 200.

Histology

Histological sections were made using three-day starved animals in the same procedure as described in Song et al. (2020). Histological sections were made at intervals of 6 μm. Specimens were stained in slightly modified Cason’s Mallory-Heidenhain stain solution (see Yang et al. 2020). Hereafter, slides were mounted with neutral balsam (Shanghai Yuanye Biotechnology Co., Ltd.). Histological glass slides were deposited in the Institute of Zoology, Chinese Academy of Science (IZCAS), Beijing, China.

Abbreviations used in the figures

ag: auricular groove; au: auricle; bc: bursal canal; ca: common atrium; cb: copulatory bursa; ceg: cement glands; cm: circular muscle; d: diaphragm; du: duct; e: eye; ed: ejaculatory duct; go: gonopore; ie: inner epithelium; lm: longitudinal muscle; lod: left oviduct; lvd: left vas deferens; ma: male atrium; ms: muscular swelling; od: oviduct; oe: outer epithelium; ov: ovary; pb: penis bulb; pg: penis glands; ph: pharynx; pp: penis papilla; rod: right oviduct; rvd: right vas deferens; sg: shell glands; sv: seminal vesicle.

Results

Molecular phylogeny

The amplified sequences of the COI sequence had a length of approximately 900 base pairs (bp). After GBLOCKS processing, the COI dataset comprised 46 Operational Taxonomic Units (OTUs) and 762 bp in length. Nucleotide saturation analysis was conducted on the fully resolved sites only, while all three codon positions were included. The critical Iss.c values (Iss.cSym=0.717; Iss.cAsym=0.428) were both greater than the observed Iss values (0.141), suggesting little substitution saturation in our dataset (Xia et al. 2003).

The phylogenetic trees constructed by BI and ML methods have identical topologies (Fig. 2). Three individuals of Dugesia gemmulata cluster together with very high support values (1.00 posterior probability – pp; 100% bootstrap – bs; Fig. 2), while they share a sister-group relationship with D. umbonata Song & Wang, 2020 that is well supported (0.80 pp; 90% bs; Fig. 2). The clade comprising D. gemmulata and D. umbonata is sister to D. japonica Ichikawa & Kawakatsu, 1964 with high support (0.87 pp; 91% bs; Fig. 2), and together they cluster with D. circumcisa Chen & Dong, 2021 with 81% bs (Fig. 2). These four species together form a clade that shares a sister-group relationship (88% bs) with a clade composed of the two species D. sinensis Chen & Wang, 2015 and D. semiglobosa Chen & Dong, 2021 (Fig. 2). This clade of six species clusters with a clade of seven other species from Australian and Oriental regions (from D. notogaea Sluys & Kawakatsu, 1998 to D. majuscula in Fig. 2) with 0.85 pp and 89% bs. This entire Australasian-Oriental clade is sister to a Western Palearctic clade (from D. malickyi De Vries, 1984 to D. hepta Pala et al., 1981 in Fig. 2). The major clade formed by the two sister-clades of the Australasian-Oriental regions and the Western Palearctic shares a sister-group relationship with two species from Cameroon (D. bijuga Harrath & Sluys, 2019 and D. pustulata Harrath & Sluys, 2019), while these two clades together are sister to a small clade of three species from Madagascar. The most basal clade in our tree is formed by a clade comprising four species from the Afrotropical and South-West Palearctic regions (from D. naiadis Sluys, 2013 to D. sicula Lepori, 1948 in Fig. 2).

Figure 2.

Maximum likelihood phylogenetic tree topology based on COI sequence. Numbers at nodes indicate support values (posterior probability/bootstrap). Scale bar: substitutions per site.

Systematic Account

Order Tricladida Lang, 1884

Suborder Continenticola Carranza, Littlewood, Clough, Ruiz-Trillo, Baguñà & Riutort, 1998

Family Dugesiidae Ball, 1974

Genus Dugesia Girard, 1850

Dugesia gemmulata Sun & Wang, sp. nov.

https://zoobank.org/4C439299-E70E-4421-98E9-6068C91A1423

Material examined

Holotype : IZCAS PLA-0101, a rural streamlet in Qingzhen City, Guizhou, China, 26°33.83'N, 106°13.65'E, 3 February 2018, coll. Zhong-Yin Sun, sagittal sections on 20 slides.

Paratypes : IZCAS PLA-0102, ibid., sagittal sections on 30 slides; IZCAS PLA-0103, ibid., horizontal sections on 18 slides.

Habitat

Specimens were collected from a rural streamlet (26°33.83'N, 106°13.65'E) at an altitude of about 1,400 m above sea level (a.s.l.) in Qingzhen City, Guizhou, China (Fig. 1A). The animals were collected from the underside of small pebbles in the riverbed (Fig. 1B, C), which had a water depth of 20–50 cm. Water temperature was about 19.9 °C, while air temperature was about 21.8 °C.

Diagnosis

Dugesia gemmulata is characterised by the following characters: ventral part of the most posterior section of the bursal canal, just anteriorly to its point of communication with the common atrium, provided with a voluminous, ellipsoidal muscular swelling; sac- or egg-shaped seminal vesicle situated near the ventral body surface in anterior portion of the penis bulb; postero-dorsal wall of seminal vesicle communicates with a narrow duct that first runs almost vertically but then shows a postero-dorsally directed loop before connecting with a small diaphragm; ejaculatory duct opening terminally or subterminally; penis papilla asymmetrical, with dorsal lip provided with a bump; oviducts opening asymmetrically into female copulatory apparatus, with the left oviduct opening into the common atrium and the right oviduct opening into the vaginal section of the bursal canal.

Etymology

The specific epithet is derived from the Latin gemmula, small bud, and alludes to the muscular swelling on the ventral wall of the bursal canal.

Description

Body size of live, sexualised specimens ranged from 16.12 to 22.75 mm in length and 1.93 to 2.87 mm in width (n = 3; Fig. 3A, B), while asexual specimens ranged from 5.12 to 8.31 mm in length and 0.91 to 1.32 mm in width (n = 3). Sexualised and asexual individuals exhibited no differences in appearance, except for the body size. The following measurements are based on sexualised individuals. At a distance varying between 0.46 mm and 0.56 mm from the anterior body margin, two eyes are present in the middle of the low-triangular head, situated in pigment-free patches (n = 3; Fig. 3C). Each kidney-shaped eyecup contains numerous retinal cells. Unpigmented auricular grooves are marginally placed just posteriorly to the blunt auricles (Fig. 3C).

Figure 3.

Dugesia gemmulata. A. Habitus of live, sexualised animal in dorsal view; B. Habitus of live, sexualised animal in ventral view; C. Anterior end, dorsal view; D. Anterior end, ventral view, showing ovaries.

Dorsal body surface with a brown ground colour, overlain with scattered black pigmentation and provided with a thin, pale median line that runs from anterior to the eyes to the tail end (Fig. 3A–C); ventral surface much paler than dorsal surface (Fig. 3D).

The cylindrical pharynx lies more or less in the middle of the body and measures about 1/7 of the total body length, i.e., about 2.07–2.77 mm in length and 0.16–0.32 mm in width (n = 3; Fig. 3A). The entire pharynx is covered with a nucleated epithelium (Fig. 4B). The outer epithelium of the pharynx is underlain by 1–3 subepithelial layers of circular muscles, followed by a thin layer of longitudinal muscle fibres (Fig. 4B). The inner pharynx epithelium, which is densely ciliated, is underlain by a thin subepithelial layer of longitudinal muscle fibres, followed by 3–5 layers of circular muscles (Fig. 4B). The mouth opening is located at a distance of about 1/3–1/2 of the body length, as measured from the posterior body margin; the mouth is situated at the posterior end of the pharyngeal cavity (n = 3; Fig. 3B).

Figure 4.

Dugesia gemmulata, holotype PLA-0101, sagittal sections and reconstructions of the copulatory apparatus. A. Photomicrograph showing ovaries and the opening of the oviduct; B. Photomicrograph showing musculature of the pharynx; C. Photomicrograph showing copulatory bursa, seminal vesicle, interconnecting duct, penis glands, ejaculatory duct, penis papilla, male atrium and cement glands; D. Photomicrograph showing bursal canal and muscular swelling; E. Reconstruction male copulatory apparatus; F. Reconstruction female copulatory apparatus.

Testes could not be discerned. The large, elongated penis bulb consists of intermingled longitudinal and circular muscle fibres. The vasa deferentia open asymmetrically into the seminal vesicle, with the right vas deferens opening into the mid-anterior portion of the seminal vesicle in specimen PLA-0101 and into the mid-dorsal portion in specimen PLA-0102, while the left vas deferens opens into the postero-dorsal part of the seminal vesicle in specimen PLA-0101 and into the postero-ventral part in specimen PLA-0102 (Figs 4E, 5C). The sac- or egg-shaped seminal vesicle is situated near the ventral surface in the anterior portion of the penis bulb; it is lined with a nucleated epithelium and surrounded by 1–2 layers of longitudinal muscles (Figs 4C, E, 5A, C). From the postero-dorsal wall of the seminal vesicle arises a narrow duct, lined with a glandular, nucleated epithelium and surrounded by 1–3 layers of circular muscle fibres, which first runs almost vertically but then shows a postero-dorsally directed loop before connecting with a small diaphragm (Figs 4C, E, 5A, C).

Figure 5.

Dugesia gemmulata, paratype PLA-0102, sagittal sections and reconstructions of the copulatory apparatus. A. Photomicrograph showing copulatory bursa, seminal vesicle, diaphragm, penis glands, penis papilla, male atrium, common atrium, shell glands, cement glands and gonopore; B. Photomicrograph showing bursal canal and muscular swelling; C. Reconstruction male copulatory apparatus; D. Reconstruction female copulatory apparatus.

The very small diaphragm is lined by a nucleated epithelium, which is underlain by 1–2 layers of circular muscle and is pierced by the openings of erythrophil penial glands (Figs 4C, E, 5A, C). The diaphragm leads to a broad ejaculatory duct, which is lined by an infranucleated epithelium that is underlain with 1–3 layers of circular muscle fibres (Figs 4C, E, 5A, C).

The ejaculatory duct runs more or less centrally through the penis papilla and opens subterminally through the ventral wall of the penis papilla in specimens PLA-0101 and PLA-0103, while in specimen PLA-0102 the ejaculatory duct runs a ventrally displaced course and opens at the tip of the papilla (Figs 4C, E, 5A, C). The penis papilla is asymmetrical, in that its dorsal lip is larger than the ventral one, while the former carries a bump (Figs 4C, E, 5A, C). The papilla is covered by a thin, nucleated epithelium, which is underlain by a layer of longitudinal muscles, followed by 2–3 layers of circular muscles, the latter being thicker at the base of penis papilla (Figs 4C, E, 5A, C).

The ovaries are hyperplasic, with several scattered masses distributed in the body region directly posterior to the brain, filling up the entire dorso-ventral space (Fig. 4A). The left oviduct arises from the mid-lateral section of the ovary, while the right duct arises from a more antero-lateral section of the ovary (Fig. 4A). From the ovaries, the oviducts, which are lined with an infranucleated epithelium, run backwards and continue their course on either side of the pharyngeal pocket (Figs 4F, 5D). Immediately posterior to the gonopore, the oviducts turn medially to open asymmetrically into the female copulatory apparatus, with the left oviduct opening into the common atrium and the right oviduct opening more dorsally into the vaginal section of the bursal canal (Figs 4E, 5C).

The large, irregularly egg-shaped copulatory bursa is situated immediately anterior to the penis bulb and is surrounded by a thin layer of intermingled longitudinal and circular muscle fibres (Figs 4C–F, 5). The bursal canal arises from the posterior surface of the copulatory bursa and runs caudally to the left of the copulatory apparatus, subsequently communicating with the dorsal portion of the common atrium (Figs 4E, F, 5C, D). The bursal canal is lined by a nucleated, glandular epithelium, which is underlain by a subepithelial layer of longitudinal muscle fibres, followed by 2–5 layers of circular fibres. The coat of circular fibres on the ventral side of the bursal canal varies from 3–5 layers, while that on the dorsal side is thinner, being 2–3 layers thick. Furthermore, the ventral part of the most posterior section of the bursal canal, just anteriorly to its point of communication with the common atrium, carries a voluminous, ellipsoidal muscular swelling that measures about 348–481 μm in anterior-posterior direction (n = 3; Figs 4D, F, 5B, D, 6). The swelling consists of irregular, nucleated mesenchymal cells and is surrounded by a coat of intermingled muscles; there are also muscle fibres traversing the swelling in all directions in a more loosely arranged, irregular and reticulated way (Figs 4D, F, 5B, D, 6).

Figure 6.

Dugesia gemmulata, paratype PLA-0103, horizontal sections. A, B. Photomicrographs showing the muscular swelling.

The male atrium is lined by an epithelium consisting of nucleated, cylindrical cells and is surrounded by a subepithelial layer of circular muscles, followed by 1–2 layers of longitudinal muscles. The male atrium communicates with the common atrium via a pronounced constriction (Figs 4C–E, 5A, C, D). The common atrium is lined with an infranucleated epithelium, which is underlain by 1–2 layers of circular muscle, followed by a layer of longitudinal muscle. The common atrium opens ventrally through the gonopore, which is located at 1/3–1/4 of the body length (n = 3), as determined from the posterior body margin (Figs 4E, F, 5A, C, D).

Massive shell glands are distributed posteriorly to the common atrium and near the vaginal section of the bursal canal (Figs 4E, 5A, C). Near its opening into the common atrium, the bursal canal receives the cyanophilic secretion of the shell glands (Figs 4E, 5A, C). Erythrophilic cement glands open into the ventral portion of the common atrium and into the gonoduct (Figs 4C, E, 5A, C).

Discussion

In our phylogenetic tree, the three individuals of Dugesia gemmulata occupy a single branch that shares a sister-group relationship with D. umbonata (Fig. 2). The long branch of D. gemmulata unequivocally indicates its high divergence from other species, while its phylogenetic position reveals that it belongs to a clade comprising other Dugesia species from the Australasian and Oriental regions. The topology of our phylogenetic tree (Fig. 2) basically corresponds with those generated in previous studies, except for some nodes.

The Australasian and Oriental clade is formed by three major branches that basically coincide with the topology of the trees of Song et al. (2020: figs 1, S2, S3) and Wang et al. (2021a, 2021b, 2022: figs 2). However, in the trees of Wang et al. (2021a, 2021b, 2022: figs 2), D. japonica clusters with the clade comprising D. majuscula, D. verrucula and D. constrictiva, whereas in our tree (Fig. 2) it forms a clade with D. gemmulata and D. umbonata. Similarly, in the tree of Song et al. (2020) D. japonica shares a sister-group relationship with D. umbonata. This discrepancy may be due to the different gene segments analysed by both ourselves and Song et al. (2020), i.e., complete mitochondrial genome (AB618487), while only a partial mitochondrial sequence (FJ646990) was used in Wang et al. (2021a, b, 2022).

Furthermore, the clade formed by the four species D. deharvengi Kawakatsu & Mitchell, 1989, D. ryukyuensis Kawakatsu, 1976, D. batuensis Ball, 1970, and D. notogaea Sluys & Kawakatsu, 1998 receives only low support in our phylogenetic tree (0.60 pp; 70% bs), whereas this clade is robust in the trees of Song et al. (2020: fig. 1; 0.99 pp; 92% bs) and Wang et al. (2021a, 2021b, 2022: figs 2; 1.00 pp; 81% bs; 1.00 pp; 95% bs; 1.00 pp; 74% bs, respectively).

In our phylogenetic tree, a large clade comprising species from Cameroon, Western Palearctic, Australasian and Oriental regions is sister to a Madagascan clade, and together cluster with an Afrotropical and South-West Palearctic clade. This topology is the same as in Song et al. (2020: fig. 1), but differs from other trees in Song et al. (2020: figs S2, S3) and from those in Wang et al. (2021a, 2021b, 2022: figs 2) and Solà et al. (2022, figs 2, 3), in which the basal position is not taken by the Afrotropical and South-West Palearctic clade but by a Madagascan clade. However, in our tree the Madagascan clade comprises only three taxa, while the Afrotropical and South-West Palearctic clade contains only four OTUs. Thus, these two clades have only low representation, as compared to the other three major clades. Additionally, the node connecting the Madagascan group with the large clade comprising 35 species of Dugesia shows only rather low supporting values (0.61 pp; 66% bs), which may contribute to the fact that the Madagascan group does not form the basal clade in our tree. Interestingly, in the analysis of Solà et al. (2022) it was shown that there are actually two Malagasy clades of which only one occupied a basal position in the phylogenetic tree.

Dugesia gemmulata exhibits an apomorphic feature that sets it immediately apart from all of its congeners, viz., the presence of a muscular swelling on the ventral side of its bursal canal. In contrast to D. umbonata, this muscular swelling is confined to the ventral side of the bursal canal, whereas in the former there is on the same posterior section of the bursal canal a structurally similar swelling or hump on the dorsal side of the duct. It is noteworthy that both species are characterised by the presence of a seminal vesicle that is located in the very antero-ventral section of the penis bulb and that from the dorsal wall of the vesicle arises a duct that shows a postero-dorsal loop before communicating with a small diaphragm. However, in D. gemmulata this loop is much more pronounced. In both species the oviducts open asymmetrically into the bursal canal. A clear difference between both species concerns the opening of the ejaculatory duct. Dugesia gemmulata exhibits a terminal or a subterminal ventral opening, whereas in D. umbonata the ejaculatory duct has a subterminal dorsal opening, the latter representing an apomorphic condition within the genus Dugesia. It is interesting that in the phylogenetic tree the sister-group relationship between D. gemmulata and D. umbonata is robust, thus strongly supporting their close affinity, which may explain the correlation between their anatomical characters, notably the presence of a muscular hump or swelling on the bursal canal.

Song et al. (2020) already argued that the muscular hump in D. umbonata is not homologous with the halo-like structure in the vaginal area of the bursal canal in D. japonica, which is also composed of mesenchymal tissue traversed by irregular muscle fibres. The same argument holds true for the non-homologous relationship between the ventral muscular swelling on the bursal canal of D. gemmulata and the halo-like structure in D. japonica.

Similar to the situation in D. umbonata, the sexualised specimens of D. gemmulata possessed hyperplasic ovaries and were devoid of testicular follicles, a condition that is not uncommon among such animals (cf. Song et al. 2020 and references therein).

Acknowledgements

This study was supported by the Special Funds for the Cultivation of Guangdong College Students’ Scientific and Technological Innovation (“Climbing Program” Special Funds; grant no. pdjh2021b0429) granted to X.Y.S.; a Shenzhen Special Project for Sustainable Development (grant no. KCXFZ20201221173404012) and the 2021 Shenzhen Special Fund for Agricultural Development (fishery) Agricultural high-tech project “Development and demonstration application of algae protein replacing aquatic feed” granted to S.F.L.; a Shenzhen University Innovation Development Fund (grant no. 2021248) granted to Y.L.. We are grateful to Long-Jie Tian for assistance with the DNA extraction and for drawing the map in Fig. 1. We also thank Jia-Jia Chen for kind support in the laboratory.

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