Zoosyst. Evol. 98 (2) 2022, 233-243 | DO! 10.3897/zse.98.83184 ete Ee BERLIN 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! 1 Shenzhen Key Laboratory of Marine Bioresource and Eco-environmental Science, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen, Guangdong, China 2 School of Urban Planning and Design, Shenzhen Graduate School, Peking University, Shenzhen, China 3 State Key Laboratory of Protein and Plant Gene Research, Center for Bioinformatics, School of Life Sciences, Peking University, Beijing, China 4 Naturalis Biodiversity Center, P.O. Box 9517, Leiden, 2300 RA, Netherlands https://zoobank. org/E8 B38 FE7-A 904-43 D2-8024-EE0745E415C7 Corresponding author: Shuang-Fei Li (sfli@szu.edu.cn) Academic editor: Pavel Stoev # Received 7 March 2022 Accepted 10 June 2022 Published 29 June 2022 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). Copyright Liu, Y et a/. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distri- bution, and reproduction in any medium, provided the original author and source are credited. 234 In the present study we provide an integrative descrip- tion of another new species of Dugesia, which was col- lected from Guizhou Province located at a plateau in the southwest of China, on the basis of morphological, histo- logical and molecular data. Materials and methods Sample collection and culturing The specimens were collected in a rural streamlet at an al- titude of about 1,400 m in Qingzhen City, Guizhou Prov- ince, China (26°33.83'N, 106°13.65'E; Fig. 1). The ani- mals were collected from the underside of small pebbles in the riverbed and, thereafter, were transported to Shen- zhen 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 con- ditions, the animals multiplied by means of fission and, eventually, in total, about 30 specimens sexualised. 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.™ Mollusc DNA Isolation Kit (Ome- ga, Norcross, GA, USA). Cytochrome C oxidase subunit I (COJ) gene was amplified by polymerase chain reaction (PCR), using forward primer COJF: GCT CAT GGT TTA RTW ATG ATG ATT TTY TT and reverse primer COIJR: 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, 45s at 5 °C, 50 s at 7 °C and finally 7 min extension at 72 °C (Chen et al. 2015). DNA sequences were de- termined by Sanger sequencing in Beijing Genomics In- stitution (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). 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 & Sola, 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). CO/ 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). zse.pensoft.net Liu, Y et al.: New Dugesia from China 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 informa- tion 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 GIR+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 MR- BAYES 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 simul- taneous 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 devi- ation 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 val- ues. 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. Table 1. GenBank accession numbers of CO/ sequences used in the phylogenetic analyses. Species Col Species Col 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 KF 308774 D. ariadnae JN376142 D. japonica AB618487 D. aurea MK712632 _D. liguriensis MK712645 D. batuensis KF907819 D. majuscula MW533425 D. benazzii FJ646977, D. malickyi KF 308750 FJ646933 D. bifida KY498851 D. naiadis KF 308757 D. bijuga MH119630 D. notogaea FJ646993, FJ646945 D. circumcisa MZ147041 D. parasagitta KF 308739 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 KF 308797 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 MT1 76641 D. gemmulata sequence 2 OL632202* D. verrucula MZ147040 D. gemmulata sequence 3. OL632203* D. vilafarrei MK712648 *Sequences from this study. Zoosyst. Evol. 98 (2) 2022, 233-243 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 um. Specimens were stained in slightly modified Ca- son’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), Beying, China. Abbreviations used in the figures ag: auricular groove; au: auricle; be: 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 ep- ithelium; Im: longitudinal muscle; lod: left oviduct; Ivd: 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: pe- nis papilla; rod: right oviduct; rvd: right vas deferens; sg: shell glands; sv: seminal vesicle. Results Molecular phylogeny The amplified sequences of the CO/ sequence had a length of approximately 900 base pairs (bp). After GBLOCKS processing, the COJ dataset comprised 46 Operation- al Taxonomic Units (OTUs) and 762 bp in length. Nu- cleotide 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 230 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). Systematic Account Order Tricladida Lang, 1884 Suborder Continenticola Carranza, Littlewood, Clough, Ruiz-Trillo, Baguiia & Riutort, 1998 Family Dugesiidae Ball, 1974 Genus Dugesia Girard, 1850 Dugesia gemmulata Sun & Wang, sp. nov. https://zoobank. org/4C439299-E70E-442 1-98E9-6068C91A 1423 Material examined. Holotype: IZCAS PLA-0101, arural streamlet in Qingzhen City, Guizhou, China, 26°33.83'N, 106°13.65'E, 3 February 2018, coll. Zhong-Yin Sun, sag- ittal 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 sur- face 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 poste- ro-dorsally directed loop before connecting with a small diaphragm; ejaculatory duct opening terminally or sub- terminally; 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. zse.pensoft.net 236 Guizhou Province sampling locality. 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 (7 = 3; Fig. 3A, B), while asexual spec- imens ranged from 5.12 to 8.31 mm in length and 0.91 to 1.32 mm in width (n = 3). Sexualised and asexual in- dividuals 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). Dorsal body surface with a brown ground colour, over- lain with scattered black pigmentation and provided with zse.pensoft.net Figure 1. Locality and habitat of Dugesia gemmulata. A. Sampling locality in Qingzhen City, Guizhou, China; B, C. Habitat at Liu, Y et al.: New Dugesia from China 106°13.65'E e Wangjiadapo Mountain 26°33.83'N Dawenglin Reservoir Pratt S ey 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 to- tal body length, 1.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 epitheli- um (Fig. 4B). The outer epithelium of the pharynx is underlain by 1-3 subepithelial layers of circular mus- cles, followed by a thin layer of longitudinal muscle fibres (Fig. 4B). The inner pharynx epithelium, which is densely ciliated, 1s underlain by a thin subepithe- lial 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). Zoosyst. Evol. 98 (2) 2022, 233-243 -140 0.60 0.86/86 /31 0.81/71 0.66/62 0.87/85 0.77/86 0.63/64 0.80/77] [00/97 237 D. malickyi D. arcadia D. sagitta D. parasagitta D. elegans D. improvisa D. ariadnae D. effusa D. damoae —— D. vilafarrei D. cretica Western Palearctic 1.00/100-—— D. aurea 0.54/69) D. subtentaculata D. corbata 0.78/86 D. etrusca 1.00/98) | “8 D. ilvana aaa D. liguriensis D. gonocephala D. benazzii 1.00/100 D. hepta D. gemmulata 1 1.00/100 D. gemmulata 2 0.83/87 0.80/90 D. gemmulata 3 a D. umbonata ~ D. japonica 486 D. circumcisa | 1.00/97 D. sinensis Australasian D. semiglobosa n riental 0.95/97 0.85/89 161 D. notogaea : qomouenta — 0.60/70 D. deharvengi D. batuensis D. ryukyuensis 0.86/82 0.56/91 D. verrucula 0.61/66 LL D. constrictiva D. mae. 0.80/89; . bijuga D. pustulata si | Cameroon 1.00/100 — D. bifida er oes 1.00/100 D. gibberosa | Madagascar D. sigmoides D. naiadis D. afromontana Afrotropical and rege one South-West Palearctic 1.00/100 Recurva postrema Pa Schmidtea figuilefancs | Outgroup eS 0.2 Figure 2. Maximum likelihood phylogenetic tree topology based on CO/ sequence. Numbers at nodes indicate support values (pos- terior probability/bootstrap). Scale bar: substitutions per site. Testes could not be discerned. The large, elongated pe- nis bulb consists of intermingled longitudinal and circular muscle fibres. The vasa deferentia open asymmetrically into the seminal vesicle, with the right vas deferens open- ing 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 spec- imen PLA-0101 and into the postero-ventral part in spec- imen 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 nucle- ated epithelium and surrounded by 1-2 layers of longitu- dinal muscles (Figs 4C, E, 5A, C). From the postero-dor- sal 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 al- most vertically but then shows a postero-dorsally directed loop before connecting with a small diaphragm (Figs 4C, E, 5A, C). The very small diaphragm is lined by a nucleated epithelium, which is underlain by 1-2 layers of circu- lar muscle and is pierced by the openings of erythro- phil 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 lay- ers 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 ejacu- latory 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 ven- tral 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, fol- lowed 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 an- tero-lateral section of the ovary (Fig. 4A). From the ova- ries, 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). Imme- diately posterior to the gonopore, the oviducts turn me- dially to open asymmetrically into the female copulatory zse.pensoft.net 238 Liu, Y et al.: New Dugesia from China & 600 um 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. 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 1s situated immediately anterior to the penis bulb and is sur- rounded 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, zse.pensoft.net subsequently communicating with the dorsal portion of the common atrium (Figs 4E, F, 5C, D). The bursal ca- nal 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 Zoosyst. Evol. 98 (2) 2022, 233-243 259 anteriorly to its point of communication with the common atrium, carries a voluminous, ellipsoidal muscular swell- ing that measures about 348-481 um in anterior-posteri- or 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 200um Figure 4. Dugesia gemmulata, holotype PLA-0101, sagittal sections and reconstructions of the copulatory apparatus. A. Photomi- crograph showing ovaries and the opening of the oviduct; B. Photomicrograph showing musculature of the pharynx; C. Photomi- crograph 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. in a more loosely arranged, irregular and reticulated way (Figs 4D, F, 5B, D, 6). The male atrium is lined by an epithelium consisting of nucleated, cylindrical cells and is surrounded by a subep- ithelial layer of circular muscles, followed by 1—2 layers of longitudinal muscles. The male atrium communicates with the common atrium via a pronounced constriction zse.pensoft.net 240 Liu, Y et al.: New Dugesia from China Figure 5. Dugesia gemmulata, paratype PLA-0102, sagittal sections and reconstructions of the copulatory apparatus. A. Photomi- crograph 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. (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 ventral- ly through the gonopore, which is located at 1/3—-1/4 of the body length ( = 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 zse.pensoft.net of the shell glands (Figs 4E, 5A, C). Erythrophilic cement glands open into the ventral portion of the common atri- um 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 Zoosyst. Evol. 98 (2) 2022, 233-243 241 Figure 6. Dugesia gemmulata, paratype PLA-0103, horizontal sections. A, B. Photomicrographs showing the muscular swelling. 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. (202la, 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), 1.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 spe- cies 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 Sola et al. (2022, figs 2, 3), in which the basal position is not taken by the Afro- tropical and South-West Palearctic clade but by a Mada- gascan 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 rath- er low supporting values (0.61 pp; 66% bs), which may zse.pensoft.net 242 contribute to the fact that the Madagascan group does not form the basal clade in our tree. Interestingly, in the analysis of Sola 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 1t 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 sim- ilar 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 ejac- ulatory 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 ha- lo-like structure in the vaginal area of the bursal canal in D. japonica, which is also composed of mesenchymal tis- sue traversed by irregular muscle fibres. The same argu- ment holds true for the non-homologous relationship be- tween 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 ova- ries 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” Spe- cial 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 zse.pensoft.net Liu, Y et al.: New Dugesia from China aquatic feed” granted to S.F.L.; a Shenzhen Universi- ty 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 sup- port in the laboratory. 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