Integrative descriptions of two new species of Dugesia from Hainan Island, China (Platyhelminthes, Tricladida, Dugesiidae)

Abstract Two new species of the genus Dugesia (Platyhelminthes, Tricladida, Dugesiidae) from Hainan Island of China are described on the basis of morphological, karyological and molecular data. Dugesia semiglobosa Chen & Dong, sp. nov. is mainly characterized by a hemispherical, asymmetrical penis papilla with ventrally displaced ejaculatory duct opening terminally at tip of penis papilla; vasa deferentia separately opening into mid-dorsal portion of intrabulbar seminal vesicle; two diaphragms in the ejaculatory duct; copulatory bursa formed by expansion of bursal canal, lined with complex stratified epithelium, which projects through opening in bursa towards intestine, without having open communication with the gut; mixoploid chromosome complement diploid (2n = 16) and triploid (3n = 24), with metacentric chromosomes. Dugesia majuscula Chen & Dong, sp. nov. is mainly characterized by oviducts opening asymmetrically into female reproductive system; hyperplasic ovaries; expanded posterior section of bursal canal; vasa deferentia separately opening into mid-dorsal portion of seminal vesicle; asymmetrical penis papilla due to ventral course of ejaculatory duct, which has subterminal and dorsal opening at tip papilla; mixoploid chromosome complement diploid (2n = 16) and triploid (3n = 24); chromosomes metacentric. Apart from their anatomy, separate species status of the two new species is supported also by their genetic distances and by their positions in the phylogenetic tree. The sexualization process may have been induced by the lower temperatures, in comparison with their natural habitat, under which the worms were cultured in the laboratory.


Introduction
Approximately 94 species of the freshwater planarian genus Dugesia Girard, 1850 have been reported from a major portion of the Old World and Australia (Sluys and Riutort 2018;Song et al. 2020). Thus far, 22 species of Dugesia are known from the Oriental region (Sluys et al. 1998), while only three species have been recorded from China, viz., D. japonica Ichikawa & Kawakatsu, 1964, D. sinensis Chen & Wang, 2015, and D. umbonata Song & Wang, 2020(Kawakatsu et al. 1976Chen et al. 2015;Song et al. 2020). Hainan Island is the largest tropical island of China and forms part of the Indo-Burma biodiversity hotspot (Mittermeier et al. 2005(Mittermeier et al. , 2011 and represents an endemic bird area (Stattersfield et al. 1998). Although Liu (1993, map on p. 158) recorded the occurrence of Dugesia on Hainan Island from three localities, no information was provided on the taxonomic identity of the species. Therefore, in this paper we describe for the first time two new species of Dugesia from Hainan Island, which also form new species to the planarian fauna of China, on the basis of an integrative taxonomic approach, using morphological, karyological, and molecular data.

Specimen collection and culturing
Specimens were collected during 2016-2018 from under stones in springs with the help of a paint brush (for sampling localities, see Fig. 1). The worms were transferred to plastic bottles filled with spring water, which were placed in a cooler, containing an ice bag, during transportation to the laboratory. In the laboratory, the planarians were cultured in autoclaved tap water at 20 °C and fed with fresh beef liver once per week. The worms were starved for at least one week before being used in karyotype studies, histological studies, or DNA extraction. Images of the external appearance of the worms were obtained by using a digital camera attached to a stereo-microscope.

Histology and karyology
Histological sections were prepared as described by Dong et al. (2017). In brief, worms were killed with 1% nitric acid and thereafter fixed in Bouin's fluid for ~ 24 h, then rinsed in 70% ethanol, and, subsequently, dehydrated in an ascending series of ethanol solutions, after which the animals were cleared in xylene and embedded in synthetic paraffin. Serial sections were made at intervals of 6-8 μm and were stained with haematoxylin-eosin. Images were acquired by a digital camera attached to a compound microscope. Preparations of specimens have been deposited in the Zoological Museum of the College of Life Science of Henan Normal University (ZMHNU), Xinxiang, China.
Karyological preparations were made by means of the air-drying technique, following Dong et al. (2017). Well-spread sets of metaphase plates from five randomly selected individuals were used for karyotype analysis; karyotype parameter measurements were carried out as described by Chen et al. (2008). Chromosomal nomenclature follows Levan et al. (1964).

DNA extraction, amplification, sequencing, and phylogenetic analysis
Total genomic DNA was extracted by using the QIAamp DNA Mini Tissue Kit (Qiagen, Germany), according to the manufacturer's protocols. Fragments of the Cytochrome C oxidase subunit I (COI) and internal transcribed spacer-1 (ITS-1) were amplified using specific primers (see Suppl. material 2: Table S1 for sequences and annealing temperatures). Premix Ex TaqHot Start Version (TaKaRa, Otsu Japan) was used for the polymerase chain reaction (PCR). Amplifications were conducted in a final volume of 30 μL under the following conditions: 5 min at 94 °C, 35 cycles of 40 s at 94 °C, an annealing step for 30 s, and 1min at 72 °C, and 5 min at 72 °C as a final extension (for annealing temperatures see Suppl. material 2: Table S1). Purification of PCR products and sequencing were done by GENEWIZ (Tianjin, China). Sequencing reactions were performed with the same primers used to amplify the fragments. All specimens were sequenced for both forward and reverse DNA strands. Chromatograms were visually checked. In the case of Dugesia majuscula Chen & Dong, sp. nov., we were able to extract DNA from no less than 12 specimens. With respect to D. semiglobosa Chen & Dong, sp. nov., we could only extract DNA from four specimens. For both of the two new species COI and ITS-1 were amplified.
In order to determine whether the presumed new species are molecularly different from other Dugesia species, we performed a phylogenetic analysis. In the ingroup we included the two new species as well as 28 other Dugesia species from the Oriental, Australasian, Mediterranean, Eastern and Western Palearctic regions; the freshwater species Schmidtea mediterranea Benazzi et al., 1975 was chosen as outgroup taxon, since this genus forms the sister-group of the genus Dugesia (Álvarez-Presas et al. 2008; for GenBank accession numbers, see Table 1).
Nuclear ribosomal markers were aligned online with MAFFT (Online Version 7.247) using the G-INS-i algorithm (Katoh and Standley 2013), and checked by using BioEdit v7.2.6.1. For alignments of the protein-coding COI sequences we used the TranslatorX pipeline (http://translatorx.co.uk; Abascal et al. 2010). Nucleotide sequences were translated into amino acid sequences (Translation table 9), followed by MAFFT, using the FFT-NS-2 progressive alignment method, and checked by using BioEdit v7.2.6.1, and then back-translated to nucleotide sequences. Since automated removal of gap columns and variable regions had been reported to negatively affect the accuracy of the inferred phylogeny (Dessimoz and Gil 2010;Tan et al. 2015), the Gblocks option (Talavera and Castresana 2007) was disabled. The concatenated sequences for the phylogenetic analysis were in the order ITS-1+ COI and consisted of a total of 1473 bp, including 4.4% missing data. In the concatenated sequences, missing data had been coded as "?".
MrBayes v3.2 (Ronquist et al. 2012) and RaxML v8.2.10 (Stamatakis 2014) were used to infer phylogenies with the Bayesian Inference (BI) and Maximum-likelihood (ML) methods, respectively. For BI a run of 3 million generations and 25% burn-in was used under the GTR+I+G model. For the ML analysis, 10,000 replicates were performed under the GTR+I+G model. BI and ML trees were visualized and edited using Figtree v1.4.3.
Genetic distances were calculated for CO1 and ITS-1 with the help of MEGA 6.06 (Tamura et al. 2013) under the Kimura 2-parameter substitution model (Lázaro et al. 2009;Solà et al. 2013).

Molecular phylogeny
We obtained fragments of sequences of COI and ITS-1 from 12 specimens of D. majuscula and four specimens of D. semiglobosa. The final alignments of these fragments of the nuclear ribosomal internal spacer ITS-1 and the mitochondrial gene COI were 676 and 816 base pairs (bp), respectively. In the two populations of D. majuscula and D. semiglobosa, there was no variation in COI and ITS-1.
The phylogenetic trees and their supporting values resulting from the analysis of the concatenated dataset are very similar for both ML and BI, differing only in nodes weakly supported in at least one of the methodologies ( Fig. 2; Suppl. material 1: Figure S1).The new species D. majuscula and D. semiglobosa occupy separate branches that are clearly differentiated from their congeners. Interestingly, these two species from Hainan Island are not each other's closest relatives, as D. semiglobosa shares a sister-group relationship Table 1. GenBank accession numbers of COI and ITS-1 sequences used for molecular analysis.

Species
GenBank Species  GenBank  COI  ITS-1  COI  ITS- with D. sinensis, forming a clade that is part of a polytomy that comprises also three other branches, viz. D. majuscula, D. umbonata, and a clade comprising five species from the Oriental and Australasian regions (D. deharvengi Kawakatsu & Mitchell, 1989, D. batuensis Ball, 1970, D. ryukyuensis Kawakatsu, 1976, D. bengalensis Kawakatsu, 1983, D. notogaea Sluys & Kawakatsu, 1998. In addition to the fact that the new species occupy separate branches in the phylogenetic tree, separate species status of D. majuscula and D. semiglobosa is supported also by our analysis of the genetic distances among the species included in our study, albeit that both COI and ITS-1 distances vary greatly among species (Suppl. material S2, S3). With respect to COI, the highest distance value between D. majuscula and its Oriental-Australasian congeners is 22.02%, while the lowest distance value is 13.68%. The highest distance value between D. semiglobosa and the Oriental-Australasian congeners is 21.06%, while the lowest distance value is 10.61%. Furthermore, there is a 19.52% difference between the two new species.
With respect to ITS-1, the highest distance value between D. majuscula and its Oriental-Australasian congeners is 12.52%, while the lowest distance value is 5.82%. The highest distance value between D. semiglobosa and its Oriental-Australasian congeners is 15.74%, while the lowest distance value is 9.74%. For this marker the distance between the two new species is 5.83% (Suppl. material 4: Table S3). Diagnosis. Dugesia semiglobosa is characterized by the following features: hemispherical, asymmetrical penis papilla with ventrally displaced ejaculatory duct opening terminally at tip of penis papilla; absence of duct intercalated between seminal vesicle and diaphragm; vasa deferentia separately opening into mid-dorsal portion of intrabulbar seminal vesicle; two diaphragms in the ejaculatory duct; symmetrical openings of oviducts into bursal canal; copulatory bursa formed by expansion of bursal canal, lined with complex stratified epithelium, which projects through opening in bursa towards intestine, without having open communication with the gut; mixoploid chromosome complement diploid (2n = 16) and triploid (3n = 24); chromosomes metacentric.
Etymology. The specific epithet is derived from the Latin semis, half, and globosus, spherical, and alludes to the hemispherical penis papilla.
Habitat and reproduction. Specimens were collected from Jiuwentang volcano spring at an altitude of 80 m a.s.l. and with a water temperature of 23 °C. This spring is the third largest volcano spring in China, while it is also its largest selenium-rich spring (Fig. 3A, B). None of the animals was sexually mature at collection. However, after having been kept under laboratory conditions for ~ 150 days, the animals sexualized and laid cocoons. Newly laid cocoons are yellow, but turn dark brown after 2 to 3 days. Cocoons are spherical in shape (1 mm in diameter) and provided with a stalk. Thus far, none of the cocoons hatched, thus, most likely being infertile.
Karyology. Each of the five, randomly selected specimens exhibited mixoploid chromosome complements. In a total of 100 metaphase plates examined, 67 exhibited diploid chromosome portraits of 2n = 2x = 16, while in 20 plates chromosome complements were triploid with 2n = 3x = 24 chromosomes (Fig. 4); chromosome complements of the remaining 13 plates could not be determined, due to either lack of well-dispersed chromosomes or over-dispersed sets of chromosomes. All chromosomes were metacentric; karyotype parameters, including relative length, arm ratio, and centromeric index, are given in Table 2. The first pair of chromosomes is clearly larger than others, being 1.8 times larger than the shortest chromosome. Chromosomal plates and idiogram are shown in Fig. 4.
Description. Body of living asexual specimens is 4-6 mm in length and 0.72-0.85 mm in width, while in sexualized animals the body is 8-12 mm in length and 1.25-1.51 mm in width. Two eyes located in the center of the head, being situated in pigment-free patches. Each pigmented eye cup houses numerous photoreceptor cells. Head of low triangular shape and provided with two blunt auricles. Body light brown dorsally, excepting the pale body margin and accumulations of pigment following the outline of the pharynx. Ventral surface is paler than the dorsal one (Fig. 3C).
Pharynx situated in the mid-region of the body, measuring ~ 1/5 th of the body length (Fig. 3C). Mouth opening located at the posterior end of the pharyngeal pocket. Outer pharyngeal musculature composed of a subepidermal layer of longitudinal muscles, followed by a thick layer of circular muscles; extra inner layer of longitudinal muscles is absent (Fig. 5A). The inner pharyngeal musculature consists of a subepithelial layer of circular muscle, followed by a layer of longitudinal muscle, the former being thicker than the latter (Fig. 5A).
In specimen JWT-7 the ventrally placed ovaries are clearly hyperplasic and fused to form a single mass that extends into the lateral regions of the body (Fig. 5B). In other specimens examined (JWT-2, JWT-3, JWT-5, JWT-6, JWT-10), the gonads are generally atypical, in that they are very small (Fig. 5C). Only in specimen JWT-8 the ovaries are more or less of normal size. In general, ovaries are situated at a short distance behind the brain.
From the ovaries the oviducts run ventrally in a caudal direction to the level of the genital pore, after which they curve dorso-medially to open separately and symmetrically into the ventral portion of the bursal canal, close to its communication with the atrium (Figs 5D, 7).
The small, dorsally located testes are well developed and provided with mature spermatozoa (Fig. 5C). Testicular follicles are arranged on either side of the midline of the body in nine or ten longitudinal zones, extending from the posterior level of the ovaries to almost the posterior end of the body. The vasa deferentia, filled with spermatozoa, expand to form spermiducal vesicles at the level of the pharynx that occupy < 1/3 rd of the dorso-ventral space (Fig. 5E). At the level of the penis bulb the vasa deferentia decrease in diameter and bend sharply towards the dorsal body surface  rather large and blunt diaphragm is located in the proximal portion of the ejaculatory duct (Figs 6A, 7). The ejaculatory duct is lined by an infranucleated epithelium; we were unable to discern any musculature around the duct. The ejaculatory duct follows a noncentral, ventrally displaced course through the penis papilla, opening at its tip, thus resulting in an asymmetrical penis papilla in which the dorsal lip is much larger than the ventral one (Figs 6A, 7). The complete penis, comprising papilla and bulb, is nearly spherical, with the penis papilla being a hemispherical structure that is covered with an infranucleated epithelium, which is underlain by a subepithelial layer of circular muscle, followed by a layer of longitudinal muscle fibres (Fig. 6A). The penis papilla almost completely occupies the male atrium, the latter communicating with the common atrium via a slight constriction (Figs 6E, F, 7). The common atrium opens to the exterior via a gonoduct, which is lined by a columnar epithelium and receives the openings of abundant cement glands.
From its point of communication with the common atrium, the bursal canal gradually expands in diameter, meanwhile curving anteriad, while running on the left side of the male copulatory apparatus (Figs 6D, 7). The bursal canal is lined with columnar, nucleated, ciliated cells and is surrounded by a subepithelial layer of longitudinal muscles, followed by a layer of circular muscle (Figs 6D, 7). An ectal reinforcement layer of longitudinal muscles runs from the vaginal region to approximately halfway along the bursal canal. Shell glands discharge their erythrophil secretion into the vaginal region of the bursal canal, near the oviducal openings.
More or less dorsally to the penis bulb, the bursal canal first decreases somewhat in diameter but thereafter greatly expands to give rise to a more or less globular structure immediately in front of the male complex. This globular structure may be called a copulatory bursa since it occupies the same position as in other species of Dugesia, or freshwater planarians in general. The bursa is lined with a complex type of stratified epithelium. The basal portion of this epithelium consists of more or less cuboidal, nucleated cells and is basically a continuation of the lining epithelium of the rest of the bursal canal, albeit that there the cells are columnar. This basal layer is followed by a thick zone of stratified, non-nucleated squamous epithelium, leaving very little room for any lumen within the bursa. The cells of this squamous layer have an irregular shape, while those in the top zone, near the lumen, are vacuolated and provided with granular, cyanophil inclusions. The bursa is surrounded by a layer of longitudinal muscles (Figs  6C, 7). However, at one point this muscle layer is interrupted because of the presence of an opening in the bursa. This opening is located, more or less, at the antero-dorsal wall of the bursa. A portion of the squamous inner lining of the bursa projects through the opening and approaches and/or touches portions of the gut that are in its proximity. However, in none of the specimens examined we discerned an open connection between bursa and intestine.
Discussion. The curious copulatory bursa of D. semiglobosa is unparalleled among species of Dugesia, or freshwater planarians in general. Generally, copulatory bursae are lined with an epithelium consisting of tall columnar, vacuolated, and nucleated cells, while they are surrounded by only a very weak musculature. The structure of the bursa of D. semiglobosa differs considerably from this ground-plan condition, as it is basically an expanded continuation of the bursal canal, albeit with a simpler coat of muscles and a more complex lining epithelium.
phragm, terminal opening of ejaculatory duct) that is found in only nine congeners, viz., D. annandalei Kaburaki, 1918, D. damoae De Vries, 1984, D. didiaphragma De Vries, 1988, D. elegans De Vries, 1984, D. gibberosa Stocchino & Sluys, 2017, D. maghrebiana Stocchino et al. 2009, D. malickyi De Vries, 1984, D. naiadis Sluys, 2013 sinensis. Among these nine species, D. semiglobosa most closely resembles D. didiaphragma and D. maghrebiana in that these species also possess two diaphragms, in contrast to all other species mentioned. Presence of two diaphragms is a rare condition among species of Dugesia and is only known from three other species, viz., D. bijuga Harrath & Sluys, 2019, D. machadoi de Beauchamp, 1952, and D. mirabilis De Vries, 1988. However, in these three last-mentioned species the ejaculatory duct runs a central course through the penis papilla, in contrast to the ventral trajectory in D. semiglobosa. Further, there are ample other features that preclude assignment of our specimens to either of these Afrotropical species. Neither is it possible to assign our animals to D. didiaphragma or D. maghrebiana as they lack the large seminal vesicle enclosed by a highly muscularized, elongated penis bulb of the former and the knob-like extension on the penis papilla of the latter.
It is interesting to note that in all species in possession of two diaphragms, the small proximal diaphragm basically is formed by a non-glandular constriction of the seminal vesicle, while the true diaphragm is a larger structure and receives the secretion of penial glands, as usual for the diaphragm of species of Dugesia. The same situation applies to the two diaphragms in D. semiglobosa. It is noteworthy that in D. mirabilis both the proximal and distal diaphragm are glandular (De Vries 1988). Paratypes: ZMHNU-YHYQ1, ibid. sagittal sections on 45 slides; ZMHNU-YHYQ2, ibid., sagittal sections on 18 slides; ZMHNU-YHYQ3, ibid., sagittal sections on 24 slides; ZMHNU-YHYQ5, ibid., sagittal sections on 32 slides; ZMHNU-YHYQ 6, horizontal sections on 18 slides; ZMHNU-YHYQ 7, horizontal sections on12 slides; ZMHNU-YHYQ8, ibid., transverse sections on 24 slides; ZMHNU-YHYQ9, ibid., sagittal sections on 15 slides; ZMHNU-YHYQ10, ibid., sagittal sections on 19 slides.
Diagnosis. Dugesia majuscula is characterized by the following features: oviducts opening asymmetrically into female reproductive system; hyperplasic ovaries; expanded posterior section of bursal canal; vasa deferentia separately opening into mid-dorsal portion of seminal vesicle; asymmetrical penis papilla due to ventral course of ejaculatory duct, which has subterminal and dorsal opening at tip papilla; mixoploid chromosome complement diploid (2n=16) and triploid (3n = 24), with metacentric chromosomes.
Etymology. The specific epithet is derived from the Latin adjective majusculus, somewhat larger, and alludes to expanded portion of the bursal canal.
Habitat and reproduction. Animals were collected from a freshwater stream in the Yingge Mountains, at a water temperature of 19 °C and an altitude of 584 m a.s.l. (Fig. 8A, B). Approximately 40 individuals were collected in February 2018, none of which was sexually mature. During the first period of 80-90 days (March to May) in the laboratory culture, worms only showed asexual reproduction by means of fission. However, during the following period of 40-60 days, seven individuals sexualized, while after another 180 days, 1/3 of the animals sexualised, although thus far they have not produced any cocoons.
Karyology. Each of the five, randomly selected specimens exhibited mixoploid chromosome complements. In a total of 100 metaphase plates examined, 75 exhibited diploid chromosome complements of 2n = 2x = 16, while in 18 plates chromosome complements were triploid with 2n = 3x = 24 chromosomes (Fig. 9); chromosome complements of the remaining seven plates could not be determined, due to either lack of well-dispersed chromosomes or over-dispersed sets of chromosomes. All chromosomes were metacentric; karyotype parameters, including relative length, arm ratio, and centromeric index, are given in Table 3. The first pair of chromosomes is clearly larger than the others, being 1.86 times larger than the shortest chromosome. Chromosomal plates and idiogram are shown in Fig. 9.
Description. Body size of living asexual specimens is 5-8 mm in length and 0.65-0.78 mm in width, while in sexualized specimens the body measures 10-16 mm in length and 1.05-1.36 mm in width. Head of low triangular shape, provided with two blunt auricles and two eyes located in pigment-free patches (Fig. 8C). Each pigmented eye cup is provided with numerous retinal cells. Dorsal surface brown with numerous dark spots (Fig. 8C); ventral surface paler than dorsal one and provided with only scattered small, dark spots. Pharynx positioned in the mid-region of the body and measuring ~ 1/6 th of the body length (Fig. 8C); the mouth opening is situated at the hind end of the pharyngeal pocket. The pharyngeal outer musculature is composed of an outer, subepithelial layer of longitudinal muscles, followed by a layer of circular muscles. An extra longitudinal muscle layer internally to the circular muscles is absent (Fig. 10A). Inner pharyngeal musculature composed of an outer layer of longitudinal muscles and a subepithelial layer of circular muscles, the latter being thicker than the outer layer (Fig. 10A).
Ovaries hyperplasic, with several scattered masses at a short distance behind the brain, extending backwards over a distance of at least 800 μm (Fig. 10B) and occupying the entire dorso-ventral space. In live animals, the hyperplasic ovaries are visible through the dorsal body surface as two short, transparent stripes (Fig. 8C). From the ovaries, the oviducts run ventrally in a caudal direction to the level of the genital pore where they turn dorso-medially to open separately and asymmetrically into the female reproductive system. In point of fact, the right oviduct opens either into the most postero-ventral part of the expanded portion of the bursal canal or somewhat more ventrally (YHYQ-5), while the left oviduct opens into the common atrium, a distinct female atrium being absent (Figs 10C, 12).
The well-developed testes are situated dorsally and extend from the level of the ovaries to the posterior end of the body (Fig. 10D). Upon approaching the level of the penis bulb, composed of intermingled muscle fibers, the vasa deferentia curve dorso-mediad and near the postero-dorsal wall of the penis bulb the sperm ducts abruptly bend ventrad to penetrate the wall of the penis bulb. Within the bulb, the sperm ducts open separately and symmetrically into the mid-dorsal portion of a large reniform seminal vesicle (Figs 10E, F, 12). The sperm ducts are lined with nucleated cells and are surrounded by a layer of circular muscles. The seminal vesicle The anterior is to the front in A, C, E and anterior is to the right in B, D, F. Abbreviations: bc: bursal canal; ca: common atrium; cb: copulatory bursa; cm: circular muscles; go: gonopore; ho: hyperplasic ovaries; lm: longitudinal muscles; lod: left oviduct; ma: male atrium; pp: penis papilla; sv: seminal vesicle; te: testis; vd: vas deferens. Scale bars: 100 μm. opens into the ejaculatory duct via a very small diaphragm positioned at a level that corresponds with the root of the penis papilla (Figs 11A, 12). The ejaculatory duct runs ventrally through the penial papilla and opens to the exterior by means of subterminal dorsal opening at the tip of the papilla (Figs 11A-C, 12). The ejaculatory duct is lined by a cuboidal epithelium; we were unable to discern any musculature around the ejaculatory duct.
The cylindrical penis papilla is covered by an epithelium that is underlain with a subepithelial layer of circular muscle, followed by a layer of longitudinal muscle fibers. As a result of the ventral course of the ejaculatory duct, the penis papilla is asymmetrical, with its dorsal lip being much larger than the ventral one. The basal part of the penis papilla has an oblique, ventro-caudal orientation, after which the penis papilla makes a bend, so that the rest of the papilla has a more or less horizontal orientation (Figs 11A-C, 12). The papilla almost completely occupies the male atrium, which communicates with the common atrium via a more or less pronounced constriction (Fig. 12). In turn, the latter communicates with a gonoduct, lined with a columnar epithelium and receiving the openings of abundant cement glands, which leads to the ventral gonopore. The large sac-shaped copulatory bursa, which occupies the entire dorso-ventral space, is lined by a vacuolated epithelium provided with basal nuclei. There is hardly any musculature surrounding the bursa (Figs 11A-C, 12). From the postero-dorsal wall of the bursa, the bursal canal runs in a caudal direction to the left side of the male copulatory apparatus. Dorsally to the male atrium the bursal canal expands, particularly in dorso-ventral direction, to form a spacious posterior section that communicates with the common atrium. The bursal canal is lined with cylindrical, infranucleated, and ciliated cells and is surrounded by a subepithelial layer of longitudinal muscles, followed by a well-developed layer of circular muscle and a thin layer of longitudinal muscles. The outer longitudinal muscles constitute the ectal reinforcement layer, which extends from the vaginal region to approximately halfway along the bursal canal (Figs 11D, 12). Erythrophil shell glands open into the vaginal region of the bursal canal around the oviducal openings. Discussion. Our present task of comparing the features of D. majuscula with already known species of Dugesia is made considerably easier by the fact that it exhibits the unusual character of a subterminal dorsal opening of the ejaculatory duct at the tip of the penis papilla, which thus far has been reported for only one other species, viz., D. umbonata from southwest China (Song et al. 2020). In view of the sister-group relationship between these two species (Fig. 2), this subterminal, dorsal opening of the ejaculatory duct may be hypothesized to represent a synapomorphy for D. umbonata and D. majuscula.
Although D. umbonata exhibits also asymmetrical oviducal openings and a ventrally displaced ejaculatory duct, as in D. majuscula, it differs from the latter notably in the presence of a large, muscularized hump on its bursal canal, among other differ- Figure 12. Dugesia majuscula. Sagittal reconstruction of the copulatory apparatus of the holotype (anterior is to the right). Abbreviations: bc: bursal canal; cb: copulatory bursa; cg: cement glands; d: diaphragm; ed: ejaculatory duct; go: gonopore; lod: left oviduct; pp: penis papilla; rod: right oviduct; sg: shell glangs; sv: seminal vesicle; vd: vas deferens. Scale bar: 100 μm. ences. A subterminal opening of the ejaculatory duct is not uncommon among species of Dugesia, but in most cases the openings are ventral, in contrast to the dorsal opening in D. majuscula and D. umbonata. The only exception is D. hepta Pala et al., 1981 but in this species the ejaculatory duct exits at the dorso-lateral tip of the penis papilla, while in this species the ejaculatory duct is dorsally displaced, in contrast to the ventral course in D. majuscula and D. umbonata. Furthermore, in D. hepta the oviducts open symmetrically into the bursal canal (Stocchino et al. 2005), whereas in D. majuscula and D. umbonata the oviducal openings are asymmetrical.

Molecular phylogeny and distances
The topology of our phylogenetic tree (Fig. 2) basically accords with results from previous phylogenetic analyses (Lázaro et al. 2009;Solà et al. 2013;Stocchino et al. 2017;Song et al. 2020). Detailed comparison between these results and our tree are complicated since the various studies included different sets of species as well as different molecular markers, while it also serves little purpose as our sole aim was to assess the taxonomic status of the two new Chinese species. Song et al. (2020) already noted that because of the rather low number of Dugesia species for which molecular information is available, it is presently not well possible to analyse the historical biogeography of the genus.
For being able to interpret genetic distances between presumably new species in the delimitation of species boundaries, it is necessary to have well-supported information on distances between already well-known species. Presently, our information on genetic distances between species of Dugesia is rather limited. Harrath et al. (2019) found that for COI, the lowest distance value between species was 7%, while Stocchino et al. (2017) reported distances usually greater than 10% among species from Madagascar. Furthermore, Lázaro et al. (2009) and Solà et al. (2013) showed, respectively, that the lowest distance value between Mediterranean species generally is greater than 10% or 6%. With respect to the new species D. majuscula and D. semiglobosa, the lowest genetic distance value between these species and their Oriental-Australasian congeners was 13.68% and 14.26%, respectively, while there is a 19.52% difference between the two new species (Suppl. material 3: Table S2).The lowest distance values reported for ITS-1 were 6% between Malagasy species (Stocchino et al. 2017), and 7% and 1% for Mediterranean species (Lázaro et al. 2009;Solà et al. 2013). With respect to D. majuscula and D. semiglobosa, the lowest distance values between these species and other Oriental-Australasian congeners were 5.82% and 9.74%, respectively. For this marker, the distance between the two new species was 5.83% (Suppl. material 4: Table S3). Thus, these inter-species genetic distances for COI and ITS-1 support both the molecular phylogenetic and the anatomical results, which already indicated that D. majuscula and D. semiglobosa are well-separated from their congeners.

Morphology
It is well-known that sexualized specimens from originally fissiparous populations generally develop hyperplasic ovaries (cf. Song et al. 2020 and references therein), excepting D. benazzii and D. etrusca, in which ex-fissiparous individuals did not develop hyperplasic ovaries (Stocchino et al. 2012;. Furthermore, in such sexualized specimens with hyperplasic ovaries, testes may be under-developed or even be completely absent. However, such is not the case in D. semiglobosa and D. majuscula, as in both species the sexualized individuals possessed well-developed testes, while their vasa deferentia contained ample sperm, a condition that was previously observed also in Dugesia bifida Stocchino & Sluys, 2014(Stocchino et al. 2014. Nevertheless, in the two new species cocoons were either not produced or were infertile. It is noteworthy that in both new species from Hainan Island the vasa deferentia follow a characteristic course by first turning towards the dorsal body surface and then recurving ventrad and, subsequently, opening through the dorsal wall of the seminal vesicle. This specific course of the sperm ducts is uncommon among species of Dugesia and thus far had been reported only from D. bengalensis. However, in other features D. bengalensis is rather different from both D. semiglobosa and D. majuscula. For example, in D. bengalensis the sperm ducts open into the anterior portion of the seminal vesicle, while the ejaculatory duct has a subterminal ventral opening and the oviducal openings are symmetrical (Kawakatsu et al. 1983).

Karyology
Within the genus Dugesia, the basic chromosome number is seven, eight, or nine (Stocchino et al. 2004). With respect to karyotypes with a haploid number of n = 8, there are species in which all chromosomes are metacentric and species in which some chromosomes are not metacentric. Since in D. semiglobosa and D. majuscula all chromosomes are metacentric, we shall restrict our discussion to those species that also exhibit chromosome complements with a basic number of eight, metacentric chromosomes. Such a chromosome portrait is present in the following species: D. gonocephala (Dugès, 1830), many Sardinian populations of D. benazzii Lepori, 1951, D. etrusca labronica Lepori, 1950, D. sagitta Schmidt, 1861, D. elegans, D. indonesiana Kawakatsu, 1973, D. japonica, and presumably also in D. colapha Dahm, 1967(cf. Dahm 1967Benazzi and Gourbault 1975;Kawakatsu et al. 1976;Ball 1979;De Vries 1984;Deri et al. 1999;Pala et al. 1999;Stocchino 2018). Evidently, none of these species is anatomically similar to D. semiglobosa or D. majuscula, otherwise we would have included these in our comparative morphological discussions (see above).
In view of the fact that both D. semiglobosa and D. majuscula exhibited mixoploid chromosome complements, it is interesting to note that such a combination of diploid and triploid metacentric chromosomes in a basic set of eight chromosomes has been reported also for D. japonica Ichikawa & Kawakatsu, 1964and D. siamana Kawakatsu, 1980(Oki et al. 1981Stocchino et al. 2004 and references therein). Mixoploid complements have been reported also for other species of Dugesia with a different basic number of chromosomes, such as n=7 or n=9, which we shall here not further discuss.
It has been suggested that polyploidization is adaptive in harsh climatic conditions and extreme environments (Stebbins 1971). In a study on the freshwater planarians Phagocata vitta Dugès, 1830, Polycelis felina Dalyell, 1814, and Crenobia alpina Dana, 1776, Lorch et al. (2016 reported that mean chromosome numbers increased with latitude, with higher latitudes coinciding with harsher climatic conditions. In contrast, mean chromosome numbers decreased with increasing temperature, temperature ranges, precipitation, and net primary production. Furthermore, populations with high chromosome numbers, due to polyploidization, tended to reproduce asexually, whereas populations with relatively low ploidy levels reproduced sexually. Nevertheless, these results and conclusions may not be well applicable to the two new Chinese Dugesia's, since both species are mixoploid and possess hyperplasic ovaries. It is highly likely that the hyperplasic ovaries in the sexualized, ex-fissiparous animals prevented normal reproduction. In such abnormal ovaries the oocytes are also anomalous (Harrath et al. 2014), thus preventing regular oogenesis. That the cause of the infertility lies in the hyperplasic ovaries is supported by the fact that the testes are well-developed and that, thus, spermatogenesis presumably is regular. That gonadic anomalies effectuate infertility is known also from other species of Dugesia (cf. Stocchino et al. 2012;Harrath et al. 2017). Nevertheless, it is known that in some species of Dugesia ex-fissiparous individuals are able to produce fertile cocoons (Harrath et al. 2013;Stocchino and Manconi 2013 and references therein;Stocchino et al. 2014) but, apparently, D. semiglobosa and D. majuscula do not have that capacity.
The fact that animals from both species sexualized under laboratory conditions suggests that these conditions induced, or at least were favourable to sexualization. In our laboratory cultures we used a lower temperature than that of their habitat, in that in our cultures water temperature was 20 °C, whereas temperature in the springs was 23 °C and 19 °C. Furthermore, the specimens were collected in the coldest month of the year, viz., February. In Hainan Island, the average temperature during the coldest period usually is 18-22 °C, a situation that lasts for ~ 40 days. However, the temperatures in other months of the year are considerably higher than the temperature at which we collected our specimens. Especially, average temperatures from May to October reach 26-35 °C, with the highest temperature frequently being higher than 39 °C. This means that most of the time the two new species experience a relatively warm environment, in contrast to the relatively cold laboratory cultures. At collection, none of the specimens exhibited any signs of reproductive organs, while in the laboratory they sexualized at a lower temperature than they usually experience in their natural surroundings, with D. semiglobosa even producing cocoons, albeit inviable ones. This suggests that the sexualization process was induced by the lower temperatures that the worms experienced in the laboratory.