Two New Species of Freshwater Planarian (Platyhelminthes, Tricladida, Dugesiidae, Dugesia ) from Southern China Exhibit Unusual Karyotypes, with a Discussion on Reproduction in Aneuploid Species

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Introduction
Although about 110 species of the freshwater planarian genus Dugesia Girard, 1850, have been reported from the major portion of the Old World and Australia, it was only recently that taxonomic studies started to unveil its rich biodiversity in China, which forms a potential distribution hotspot for this genus [1].From southern China, eight new species were described [2][3][4][5][6], while from northern China, thus far one new species was reported [7].In the present paper, we add two new species of Dugesia to the alreadyknown diversity in southern China by applying an integrative approach, including morphological, karyological, histological, and molecular information.
The uncommon karyotypes of the two new species, combined with the asexual reproduction of aneuploid animals, are evaluated in the context of the relationship between ploidy levels and reproductive modalities in the genus Dugesia.

Materials and Methods
2.1.Specimen Collection and Culturing.Specimens were collected from under stones in a stream with the help of a paintbrush.At collection, only 10 worms of D. pendula Chen & Dong, sp.nov.were collected in the field, none of which was sexually mature.With respect to D. musculosa Chen & Dong, sp.nov., over 50 asexual worms and seven sexual worms were collected.After collection, the worms were transferred to plastic bottles filled with stream water that during transportation to the laboratory were placed in a cooler filled with an ice bag.
In an automatic incubator, 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 seven days before being used for karyotype and histological studies and DNA extraction.Images of their external morphology were obtained by using a digital camera attached to a stereo-dissecting microscope.

Phylogenetic Analysis and Genetic Distances.
Procedures for DNA extraction, amplification, and sequencing followed Wang et al. [5,7].Fragments of the cytochrome c oxidase subunit I (COI) and internal transcribed spacer-1 (ITS-1) were amplified using the primer pairs COIF and COIR and 9F and ITSR, respectively [5,7,8].For both of the two new species, four specimens were used to extract DNA, from which COI and ITS-1 were amplified.
In total, 83 sequences of 40 Dugesia species from major portions of the geographic range of the genus were used to perform phylogenetic analyses and to calculate genetic distances, including the two new species, while Schmidtea mediterranea (Benazzi et al., 1975), S. polychroa (Schmidt, 1861), and Recurva postrema Sluys & Solà, 2013, were chosen as the outgroup taxa (Table 1).The sequences of Dugesia were selected for one or more of the following reasons: (1) they were obtained from representative species from the major geographic range of the genus Dugesia; (2) they were obtained from species with a well-established taxonomic status; (3) they were either of high quality and/or had a long length.These sequences-indicated by the species names-are represented as terminals in the phylogenetic trees that we generated.
Sequence analyses were done as described previously by Wang et al. [4][5][6].In brief, ITS-1 sequences were aligned online with MAFFT (Online Version 7.247, [9]) using the G-INS-i algorithm.Protein-coding sequences were translated into amino acid sequences in order to check for the presence of stop codons (with the help of the ninth NCBI's genetic code -Flatworm Mitochondrial).With respect to COI sequences, Translator X (http://translatorx.co.uk, [10]) was used, followed by MAFFT with FFT-NS-2 method, was checked by BioEdit 7.2.6.1 [11], and, thereafter, backtranslated to nucleotide sequences.Since automated removal of gap columns and variable regions has been reported to negatively affect the accuracy of the inferred phylogeny [12,13], the Gblocks option [14] was disabled.
Bayesian information criterion (BIC) was implemented in PartitionFinder 2 [15,16] to estimate the best-fit partition The genetic distances of COI and ITS-1 were calculated by MEGA 6.06 [19] with the Kimura 2-parameter substitution model [20,21].In order to obtain more accurate values for the genetic distances, COI sequences that had less than 600 bp were removed, since the study of Marques et al. [22] indicated that at least 600 bp of COI needs to be used in the determination of interspecific divergences and for species delineation, at least in the land planarian genus Obama Carbayo et al., 2013.

Histology and Karyology.
Histological sections were prepared, as described previously by Dong et al. [23].In brief, worms were fixed in Bouin's fluid for 24 h, and, subsequently, rinsed and stored in 70% ethanol.For the histological study, specimens were dehydrated in an ascending series of ethanol solutions, after which they were cleared in clove oil and embedded in synthetic wax.Serial sections were made at intervals of 6 μm and were stained with hematoxylin-eosin, or in hematoxylin and Cason's Mallory-Heidenhain stain [24].Histological preparations of specimens have been deposited in the Zoological Museum of the College of Life Science of Henan Normal University, Xinxiang, China (ZMHNU), and Naturalis Biodiversity Center, Leiden, the Netherlands (RMNH).
Air-drying method was used to obtain karyological preparations, according to the protocols described by Dong et al. [23] and Wang et al. [5].In brief, 5-10 individuals were randomly selected, with each of the worms being treated separately.Regenerating blastemas were obtained by transversely cutting each worm into 3 fragments (head, middle, and tail), which were cultured for 3 days.After 3 days, 4 blastemas (head, prepharyngeal, post-pharyngeal, and tail) were cut off transversely and placed on 4 separate slides and, thereafter, treated with a 0.02% colchicine solution at 4 °C for 3 h.Hereafter, the blastemas were placed in 0.1% KCl hypotonic solution for 3 h.Subsequently, the surplus hypotonic solution was removed from the slides, and then fixative fluid I (glacial acetic acid : absolute alcohol : deio-nized water in the ratios 3 : 3 : 4) was added and was removed after only a few seconds.Fixative fluid I was then added again for about 30 s, after which the blastemas were evenly hammered with an oversized needle in order to create a cell suspension, which spread over the glass slide.Before the slides were completely dry, fixative fluid II (glacial acetic acid : absolute alcohol as in 1 : 1 proportion) was drop-wise added to the preparations.When the slides had dried again, fixative fluid III (glacial acetic acid) was added for only a few seconds, after which it was removed, and the preparations were dried at room temperature.After 24 h, they were stained for 15 min with a 0.5% Giemsa solution, whereafter the slides were washed with deionized water and dried at room temperature.Hereafter, coverslips were attached with DPX mountant (MFD1266, MesGen Biotechnology Co., Ltd., Shanghai, China).
Mitotic metaphase chromosomes were observed and photographed under a compound microscope (ZEISS, Axio Scope A1) equipped with a CoolCube digital camera (Meta-Systems, Altlussheim, Germany).Karyograms were prepared using the IKAROS Karyotyping system (MetaSystems, Altlussheim, Germany, https://metasystems-international .com/en/products/ikaros/). Well-spread sets of metaphase plates from randomly selected individuals (13 from the D. pendula population and 25 from the D. musculosa population) were used to determine the ploidy level, centromeric indices, and relative lengths of the chromosomes.Based on IKAROS artificial intelligence (AI) for karyotyping, the curved or overlapping chromosomes were accurately measured and their centromeric position was calculated.Hereafter, the relative lengths of the chromosomes and centromeric positions were rechecked by manual calibration.In the calculations of the averages, the very few heteromorphic chromosomes and karyotypes were not included, in order to avoid misleading results and incorrect conclusions.Karyotype parameter measurements were executed as published previously by Chen et al. [25].Chromosomal nomenclature follows Levan et al. [26].

Results
3.1.Molecular Phylogeny and Genetic Distances.Phylogenetic trees were constructed using the alignment of 1418 base pairs (bp), including 774 bp for COI and 644 bp for ITS-1.Four specimens were examined for each of the two new species, Dugesia pendula and D. musculosa, and these showed no variation in either COI or ITS-1 sequences.
The BI and ML analyses generated trees that showed identical topologies, differing only in support values (Figure 1; Supplementary Figure S1).The Chinese species included in our analysis fall into two major groups, viz., an Eastern Palearctic/Oriental group and an Oriental/ Australasian group (pp = 0 99, bs = 77).The highest COI distance values between D. pendula and D. musculosa and their congeners were 22.76% and 23.00% (both with D. naiadis Sluys, 2013), respectively, while the lowest COI distance values were 6.07%(D. pendula with D. tumida) and 12.22% (D. musculosa with D. benazzii Lepori, 1951), respectively.Furthermore, there is a 16.13% COI difference between the two new species (Supplementary Table S1).With respect to ITS-  17.73% and 19.88%, respectively, and exhibited the lowest distance values with D. tumida (1.02%) and D. circumcisa (4.83%).For ITS-1, the molecular distance between the two new species was 7.02% (Supplementary Table S2).Thus, the separate species status of D. pendula and D. musculosa is well-supported by both molecular phylogenetics and genetic distances.Collection site and habitat.On 30 th December 2018, the specimens were collected from a stream in the Shiwan Dashan Mountain, close to the China-Vietnam border in Guangxi Zhuang Autonomous Region (Figures 2 and 3(a)), at an altitude of 103 m above sea level (a.s.l.), an air temperature of 14 °C, and water temperature of 18.5 °C.At collection, none of the worms was sexually mature in the field, yet, in total 13 ex-fissiparous worms sexualized in the laboratory.At this site, only specimens of D. pendula appeared to be present, and, thus, no other species was collected at this location.In the population of D. pendula, sexual individuals showed no variation in COI and ITS-1, while asexual individuals showed also no variation in COI and ITS-1.
Diagnosis.Dugesia pendula is characterized by the presence of the following features: symmetrical openings of the oviducts into the bursal canal; a long connecting duct between the seminal vesicle and diaphragm; small diaphragm; dorsally located seminal vesicle; penis papilla suspended from the dorsal wall of the male atrium; mixoploid karyotype, with diploid complements of 2n = 2x = 14 + 0 − 1 B-chromosome and triploid complements of 2n = 3x = 21 + 0 − 1 B-chromosome, with all chromosomes being metacentric.
Etymology.The specific epithet is derived from the Latin adjective pendulus, suspended, pendulous, and alludes to the penis papilla that suspends from the dorsal wall of the male atrium.
Karyology.A total of 140 metaphase plates were examined from six randomly selected specimens, in which 17 .In total, 66 metaphase plates exhibited B-chromosomes, while these were absent in 74 complements.Further, there were 25 metaphase plates with diploid complements and 115 triploid complements.Thus, the six specimens examined all exhibited aneuploid plus mixoploid chromosome complements with diploid (2n = 2x = 14 + 0 − 1 B-chromosome) and triploid (2n = 3x = 21 + 0 − 1 B-chromosome) sets.The haploid sets consisted of seven metacentric chromosomes and no or only one metacentric B-chromosome.A chromosomal plate and idiogram of the karyotype are shown in Figure 4. Karyotype parameters, including relative length, arm ratio, and centromeric index, are given in Table 2.
Description.Sexualized living specimens measured 14-21 mm in length and 1.4-1.6 mm in width.The triangular head is provided with two blunt auricles and two eyes, which are placed in pigment-free spots.Each pigmented eyecup houses numerous photoreceptor cells.The dorsal surface is yellowish-brown, yet the body margin is pale, while accumulations of pigment follow the outline of the pharyngeal pocket (Figure 3(b)); the ventral surface is paler than the dorsal one.
The pharynx is positioned at ca. 4/7 of the body length (as determined from the anterior margin), measuring about 1/6 th of the body length (Figure 3(b)); the mouth opening is located at the posterior end of the pharyngeal pocket.Outer pharyngeal musculature is composed of a subepithelial layer of longitudinal muscles, followed by a thin layer of circular muscles; an extra inner layer of longitudinal muscles was not observed.The inner pharyngeal musculature consists of a subepithelial and thick layer of circular muscle, followed by a thin layer of longitudinal muscle.
The hyperplasic, ventrally located ovaries occupy ca.1/3-1/2 of the dorsoventral space, with several scattered masses situated at a short distance behind the brain.From the ovaries, the infranucleated oviducts run ventrally in a caudal direction to the level of the genital pore, after which they curve dorsomedially to open separately and symmetrically into the ventral portion of the bursal canal (Figure 5).Cyanophil shell glands discharge their secretion into the vaginal region of the bursal canal, at the level of the oviducal openings (Figures 5, 6 The large, sac-shaped copulatory bursa is situated immediately posterior to the pharynx and occupies ca.1/2-2/3 of the dorsoventral space (in specimens LHCA 1, 2, 7, and 8), or almost the entire dorsoventral space (in specimens LHCA 3, 4, and 9).The bursa is lined by a stratified, columnar, vacuolated epithelium provided with basal nuclei and is devoid of any surrounding musculature (Figures 5(a) and 6(c)).The bursal canal arises from the posterodorsal wall of the bursa, after which it runs in a caudal direction to the left side of the male copulatory apparatus, whereafter the canal curves ventrad to communicate with the common atrium (Figure 5(a)).The narrow bursal canal is lined with cylindrical, infranucleated, ciliated cells and surrounded by a subepithelial layer 5 Journal of Zoological Systematics and Evolutionary Research of longitudinal muscles, followed by a layer of circular muscle.There is an extra outer layer of longitudinal musculature extending from the atrium to halfway along the bursal canal, forming the ectal reinforcement (Figure 5(a)).
The testes are situated dorsally and extend from the level of the hyperplasic ovaries to the posterior end.However, these testicular follicles are poorly developed and contain only small numbers of immature spermatozoa.Therefore, sperm is absent from the vasa deferentia.The sperm ducts are lined with nucleated cells and surrounded by a layer of circular muscles.At the level of the penis bulb, the sperm ducts curve towards the dorsal body surface, then penetrate the ventral wall of the penis bulb to open separately and symmetrically into the midlateral section of the seminal vesicle (Figures 5(b), 6(a), and 6(f)).The penis bulb is located directly anterior to the male atrium and positioned in the dorsal portion of the body; it is rather weakly muscularized.In contrast, the large, sac-shaped seminal vesicle is sur- A well-developed and long duct arises from the posterior wall of the seminal vesicle, after which it runs in a caudal direction, more or less parallel to the body surface.The interconnecting duct is lined with a nucleated epithelium and is surrounded by a thin layer of circular muscles.At the level of the penis papilla this interconnecting duct bends sharply towards the ventral body surface, after which it opens into the ejaculatory duct via a very small diaphragm.The diaphragm and the proximal, dorsal section of the ejaculatory duct receive the abundant secretion of erythrophil penis glands (Figures 5(b) and 6(a)-6(e)).The ejaculatory duct, which is lined with a cuboidal, infranucleated epithelium and is devoid of any discernible musculature, follows a central course through the penis papilla, opening at the The penis papilla is a cylindrical or barrel-shaped structure that suspends from the dorsal wall of the male atrium and 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.
The genital atrium is divided into a common atrium and a male atrium.The common atrium communicates with a gonoduct, which leads to the ventral gonopore; the gonoduct is lined by a tall, columnar epithelium and receives the openings of abundant cement glands (Figures 5, 6(b), 6(c), and 6(e)).
Reproduction.Ten worms were immature at collection, and under laboratory conditions reproduced asexually through the process of fission.But after about 8 months of rearing under laboratory conditions, the immature specimens continuously sexualized.After 12 months of culturing, 13 sexualized worms produced only two cocoons.The spherical cocoons (1.0 mm in diameter) were dark brownish and provided with a stalk, yet no juveniles hatched from these cocoons.
Remarks.Dugesia pendula is unique among its congeners in that it has its penis papilla suspending from the dorsal wall of the male atrium and, thus, the papilla has a fully vertical orientation.This contrasts strongly with the penis papillae in all other species of Dugesia, which are generally horizontally or more or less obliquely orientated, pointing in a ventrocaudal direction.A species that somewhat resembles D. pendula in the vertical orientation of the penis papilla is D. krishnaswamyi Kawakatsu, 1975, from India.However, in D. krishnaswamyi, the short penis papilla still has a slightly oblique, ventrocaudal orientation, while the penis bulb is very large and extends onto two large atrial folds [27], which are absent in D. pendula.Furthermore, D. krishnaswamyi is a blackish-brown species, in contrast to the yellowish-brown D. pendula.
In some specimens of D. nannophallus Ball, 1970, the penis papilla seemingly exhibits a vertical orientation [28].However, this is only due to the fact that the penis papilla of this species is highly asymmetrical, with a large dorsal lip and a small ventral lip, which contrasts with the symmetrical papilla of D. pendula.
Notably, D. pendula, D. tumida, and D. majuscula belong to a small clade in the phylogenetic tree (Figure 2), although each species occupies its own branch, while they are anatomically very different.In both of the two lastmentioned species, the ejaculatory duct has a subterminal, dorsal opening at the tip of the penis papilla.Furthermore, D. tumida possesses a symmetrical penial valve at the base of the penis papilla, while in D. majuscula, the posterior section of the bursal canal is expanded.All of these features are absent in D. Collection site and habitat.On 7 th April 2021, specimens were collected from a stream on the Southern Tianpin Mountain, Guangxi Zhuang Autonomous Region (Figures 2  and 7(a)), at an altitude of 237 m a.s.l.; the air temperature was 18 °C and the water temperature was 14 °C.At collection, approximately seven worms were sexual, while most of the worms were asexual in the field.Under laboratory conditions, the asexual worms were fissiparous, of which 5 exfissiparous specimens sexualized.Two other species were found at the same location from where D. musculosa was collected.However, these three species could be distinguished  Diagnosis.Dugesia musculosa is characterized by the presence of the following features: oviducts following highly asymmetrical trajectories before separately opening into the bursal canal; two diaphragms; a bursal canal provided with a highly powerful and thick layer of circular muscles, which extends from the copulatory bursa to its opening into the common atrium but also surrounds the common atrium and the gonoduct; left vas deferens opening into the midlateral section of the seminal vesicle, and the right sperm duct opening into the dorsolateral portion of the vesicle; karyotype exhibiting complicated aneuploid and diploid mosaicism (2n = 2x = 16 and 2n = 2x = 16 − 1 7th -1 8th ), with the haploid chromosome number being 8, and all chromosomes being metacentric.
Etymology.The specific epithet is derived from the Latin adjective musculosus, muscular, and alludes to the highly Karyology.From eight randomly selected specimens, a total of 141 metaphase plates were examined, in which 86 metaphase plates showed an aneuploid karyotype with 14 chromosomes (each of the chromosomes pairs 7 and 8 lost one chromosome, Figure 8(a)), and 46 plates had a diploid complement of 2n = 2x = 16 (Figure 8(b)), while the remaining 5 metaphase plates could not be determined and 4 heteromorphic metaphase plates exhibited irregular aneuploid karyotypes.All eight specimens exhibited complicated karyotypes, with diploid complements of 16 chromosomes and aneuploid complements of 14 chromosomes (7 th and 8 th chromosomes lost), while all chromosomes were metacentric.Thus, all of the eight specimens examined showed complicated aneuploid and diploid mosaicism, with 8 being the haploid chromosome number.A chromosomal plate and the idiogram of this karyotype are shown in Figure 8. Karyotype parameters, including relative length, arm ratio, and centromeric index, are given in Table 3.
Description.Sexualized living specimens measured 14-18 mm in length and 1.3-1.8mm in width.The triangular head is provided with two blunt auricles and two eyes, which are placed in pigment-free spots.Each pigmented eyecup houses numerous photoreceptor cells.The dorsal surface is darkbrown, with accumulations of pigment following the outline of the pharyngeal pocket, while the body margin is pale; the ventral surface is paler than the dorsal surface (Figure 7(b)).
The pharynx is positioned approximately at the midregion of the body, measuring about 1/6 th of the body length The hyperplasic ovaries occupy more than half of the dorsoventral space, with several scattered masses situated at a short distance behind the brain.From the ovaries, the oviducts run ventrally in a caudal direction.From about the level of the gonopore, the two ducts follow rather different trajectories.From thereon, the right oviduct curves medio-dorsally, and upon reaching the dorsal section of the body, it recurves in anterior direction, whereafter it   curves downwards to open into the posterior section of the bursal canal, near its point of communication with the common atrium.Thus, the oviduct approaches the bursal canal from a dorsal direction.In contrast, at the level of the gonopore, the left oviduct immediately starts to recurve in an anteromedial direction and approaches the ventral side of the bursal canal, into which it opens at a point that lies even closer to the opening of the bursal canal into the common atrium (Figures 9 and 10(a)).Cyanophil shell glands discharge their secretion into the vaginal region of the bursal canal around the oviducal openings (Figure 9).
A large, sac-shaped copulatory bursa is situated immediately behind the pharynx, extending backwards over a distance of about 600-800 μm, while occupying most of the dorsoventral space (Figures 9 and 10(c)).The bursa is lined by a stratified, columnar, vacuolated epithelium that is provided with basal nuclei and is devoid of any surrounding musculature.The bursal canal arises from the midposterior wall of the bursa, and, thereafter, immediately expands in diameter to become a wide or even very wide duct, which runs in a caudal direction to the left side of the male copulatory apparatus.The posterior section of the bursal canal curves ventrad to open into the broad common atrium (Figure 9).The bursal canal is lined with cylindrical, infranucleated, ciliated cells and surrounded by a subepithelial, thin layer of longitudinal muscles, followed by a very thick layer of circular muscle (Figures 9, 10(b), 11(a), and 11(b)), which measures about 70-100 μm in thickness on the ventral side of the bursal canal, and 45-63 μm on its dorsal side.This highly powerful and thick layer of circular muscle extends along the bursal canal from the copulatory bursa to the common atrium and even encompasses the gonoduct (Figures 9, 10 The dorsal testes are well-developed and provided with numerous mature spermatozoa and extend from the level of the hyperplasic ovaries to the posterior end of the body.At the level of the copulatory bursa, the vasa deferentia expand to form spermiducal vesicles, which are packed with sperm.At the level of the penis bulb, the sperm ducts curve dorsomedially, penetrate the penis The large, sac-shaped seminal vesicle is lined by a flat, nucleated epithelium and is surrounded by a layer of irregularly crosswise arranged muscle fibres, forming a kind of loose net that extends throughout the major portion of the penis bulb, the latter occupying most of the dorsoventral space (Figures 9(b), 11(e), and 11(f)).The seminal vesicle opens into the ejaculatory duct via two diaphragms, the first one being pointed and the second diaphragm being a blunt structure located more distally in the ejaculatory duct.Both diaphragms receive the openings of erythrophil penis glands (Figures 9(b) and 11(f)), while cyanophil glands discharge their secretion into a section of the ejaculatory duct immediately posterior to the second diaphragm.The wide ejaculatory duct opens at the tip of the penis papilla.The broad, conical penis papilla is covered with an infranucleated epithelium, which is underlain by a subepithelial layer of circular muscle, followed by a layer of longitudinal muscle fibres.
The penis papilla lies in a spacious male atrium that via a constriction opens into a large common atrium, the latter   brought back to the laboratory.After about 1 month of rearing under laboratory conditions, 5 immature specimens sexualized.During laboratory culture, none of the mature animals (sexual at collection or sexualized during culturing) produced any cocoons.
Remarks.Dugesia musculosa exhibits a combination of a number of features that sets it immediately apart from all of its congeners, in particular, the presence of (a) a bursal canal provided with a highly powerful and thick layer of circular muscles, which extends from the copulatory bursa to its opening into the common atrium but also surrounds the common atrium and the gonoduct, (b) two diaphragms, and (c) oviducts following highly asymmetrical trajectories before separately opening into the bursal canal.
There are several other species of Dugesia that exhibit a highly muscularized bursal canal, such as D. afromontana However, in none of these species, the circular muscle layer reaches the thickness of that of D. musculosa, while in these species the strong musculature does not extend onto the common atrium and the gonoduct.In some species of Dugesia, only a portion of the bursal canal is strongly muscularized.For example, in D. japonica, the vaginal region is surrounded by a hump of mesenchymal tissue traversed by several rows of longitudinal and circular muscle fibres [29][30][31], while in D. mertoni (Steinmann, 1914), only the proximal section of bursal canal, near the vaginal region, has a thick coat of circular muscles [32].In contrast, in D. musculosa, the entire bursal canal is enveloped by an extremely thick musculature.
The presence of two diaphragms is a rare condition among species of Dugesia and is found in only seven congeners, viz.It is interesting to note that two of these species are closely related to D. musculosa, viz., D. circumcisa and D. semiglobosa, as these belong to the same clade in our phylogenetic tree (Figure 1).However, each species occupies its own branch, while they are anatomically very different.For example, D. circumcisa shows a nozzle at the tip of the penis papilla, while D. semiglobosa exhibits a curious copulatory bursa lined with a complex stratified epithelium, features that are absent in D. musculosa.
Dugesia bijuga is only very distantly related to D. musculosa (Figure 1) and is also very different from the latter, in that, it possesses a highly glandular, barrel-shaped penis papilla and lacks the highly powerful and thick layer of circular muscles on the bursal canal, common atrium, and gonoduct.Similar to the situation in D. musculosa, the seminal vesicle of D. didiaphragma is surrounded also by a thick layer of intermingled musculature, but the gross morphology of its copulatory apparatus is rather different.Lastly, D. maghrebiana differs from D. musculosa in that its penis papilla is asymmetrical and is also provided with a knoblike extension.
In species of Dugesia, the left and right sperm ducts usually open symmetrically into the seminal vesicle.An exception is formed by D. circumcisa, in which the left sperm duct opens dorsally to the right one, which differs from the situation in D. musculosa.It is only in D. bifida Stocchino & Sluys, 2014, where the right sperm duct opens into the seminal vesicle dorsally to the left one, similar to the situation in D. musculosa.However, D. bifida is easily distinguished from D. musculosa by the peculiar course of its oviducts, which give rise to a common posterior extension [33].

Discussion
Molecular phylogenetic trees of previous studies showed that the Afrotropical/Mediterranean and Malagasy groups generally occupy basal positions in the phylogenetic tree of the genus Dugesia and that the Western Palearctic group shares a sister-group relationship with the Eastern Palearctic/Oriental/Australasian group [3-5, 7, 34], which basically agrees with the topology of our present tree (Figure 1).In the analyses of Stocchino et al. [34] and Song et al. [3], the Malagasy species did not form the branch that was sister to all other Dugesia included in the study, albeit with low support.In their tree, it was an Afrotropical/Mediterranean group that formed the sister group to all other Dugesia species included in the analysis.
Unfortunately, the amount of molecular data available for species of Dugesia is rather limited, while the number  1; Supplementary Figure S1), resulting in less stable relationships among Malagasy, Afrotropical (including Cameroon), and Mediterranean species.However, in the most recent historical-biogeographic analysis of the genus Dugesia, it was shown that the situation is even more complex, in that there are two separate Malagasy clades [1].In other words, Malagasy lineages do not form a single monophyletic group, with one of these Malagasy clades being sister to all other Dugesia species.The other Malagasy clade was sister to an Afrotropical-Eastern Mediterranean clade, which together shared a sister-group relationship with four major clades from the Afrotropics, Asia, Eastern Europe + Middle East, and Western Europe [1].For Oriental, Australasian, and Eastern Palearctic species of Dugesia, our BI and ML analyses generated trees with identical topologies, with only very few nodes being weakly supported.Furthermore, the taxonomic status of the two new species described herein is well-supported by molecular phylogenetics, thus complementing the anatomical evaluation.
The separate specific status of the two new species is supported also by their genetic distances.Dugesia pendula showed the lowest COI and ITS-1 distances with D. tumida, being 6.07% and 1.02%, respectively.Dugesia musculosa showed the lowest COI and ITS-1 distances with D. benazzii and D. circumcisa, being 12.22% and 4.83%, respectively.The COI and ITS-1 distances between the two new species were 16.13% and 7.02%, respectively.According to previous studies of the genus Dugesia, the lowest COI interspecific distance among species is commonly greater than 6%, while the lowest ITS-1 distance is usually more than 1% [6, 7 and references therein].Thus, based on these markers, the two new species are well-separated not only from their congeners but also from each other.
In species of Dugesia, the presence of B-chromosomes and monosomic chromosomes are generally considered to represent a case of aneuploidy.Apart from the situation in D. pendula, the presence of B-chromosomes and, thus, the condition of aneuploidy have been reported also for several other species of Dugesia, viz., D. aethiopica  [35,[41][42][43][44][45][46][47][48].
However, the karyotypes of these species are rather different, so the condition of aneuploidy and the presence of B-chromosomes are actually expressed in greatly different chromosome portraits.For example, in D. arabica, D. sicula, and D. ryukyuensis, B-chromosomes are part of a triploid karyotype, while in D. maghrebiana, D. aethiopica, and D. pendula, they form a component of a mixoploid karyotype.Further, it is noteworthy that the condition of aneuploidy is found in all three of the haploid karyotypes with either a basic chromosome number of 7, 8, or 9.
The karyotype of D. musculosa exhibits a complex diploid set (2n = 2x = 16 and 16-7 th -8 th ), with a basic chromosome number of 8. We surmise that this represents a case of aneuploidy with a monosomic condition.A similar case of diploid aneuploidy has been reported for one population of D. ryukyuensis, in which only one chromosome was absent [43].Furthermore, monosomy is also found in several populations of D. japonica and D. ryukyuensis, which exhibit triploid aneuploidy (loss of one or more chromosomes) [43].Furthermore, B-chromosomes were also observed in D. japonica and D. ryukyuensis populations [43].
In the genus Dugesia, populations of several species show other cases in which chromosomes are absent.For instance, one population belonging to D. gonocephala s. str.(Dugès, 1830) exhibited aneuploidy, with the majority of cells possessing 24 chromosomes, but some cells containing 23 or 25 chromosomes [49].Individual fissiparous specimens of D. aethiopica showed two complements, viz., 27 + 1 − 2 B-chromosome and 18 ± 1 + 0 − 1 B-chromosomes [35].Presently, it is difficult to formulate a good explanation for the abovementioned complex and aneuploid karyotypes (with certain chromosomes being absent), albeit that frequent occurrence of both mixoploidy and aneuploidy in Dugesia populations has been postulated to form an adaptive response to environmental pressures [35].

Figure 1 :
Figure 1: Molecular phylogenetic tree obtained from Bayesian analysis of the ITS-1 and COI concatenated dataset.Numbers at nodes indicate support values (pp/bs).New species indicated in red.Scale bar: substitutions per site.
rounded by a well-developed layer of intermingled muscle fibres.The seminal vesicle is located in the dorsal body region and is lined by a flat, nucleated epithelium (Figures 5(b) and 6(b)-6(e)).
bulb, and open separately into the posterior, distal portion of the seminal vesicle.The left sperm duct penetrates the posterolateral wall of the penis bulb to open into the midlateral portion of the seminal vesicle.The right sperm duct bends towards the dorsal body surface, and after recurving ventrad, it penetrates the antero-dorsal wall of the penis bulb to open into the laterodorsal portion of the seminal vesicle (Figures 9(b), 11(a), and 11(b)).Thus, the sperm ducts open somewhat asymmetrically into the seminal vesicle.

Table 1 :
GenBank accession numbers of COI and ITS-1 sequences used in molecular analyses.Journal of Zoological Systematics and Evolutionary Research schemes and models of the concatenated sequences.The best models for each gene and codon position were GTR + G for ITS-1, GTR + I + G for the first and second codon positions of COI, and HKY + I + G for the third codon position of COI.In the phylogenetic analyses, the concatenated dataset consisted of defined gene partitions.Bayesian inference anal- 1, D. pendula and D. musculosa showed the highest distance values with D. pustulata Harrath & Sluys, 2019 and D. naiadis, being

Table 2 :
Karyotype parameters (mean values and standard deviations) of Dugesia pendula.

Table 3 :
Karyotype parameters (mean values and standard deviations) of Dugesia musculosa.species for China is This leads to the situation that many known species of Dugesia cannot be represented in molecular phylogenetic trees.In turn, this several nodes to have low support values in BI and ML trees (Figure