Research Article |
Corresponding author: Zi-mei Dong ( dzmhjx@163.com ) Corresponding author: Guang-wen Chen ( chengw0183@sina.com ) Academic editor: Tom Artois
© 2021 Lei Wang, Jin-zi Chen, Zi-mei Dong, Guang-wen Chen, Ronald Sluys, De-zeng Liu.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Wang L, Chen J-z, Dong Z-m, Chen G-w, Sluys R, Liu D-z (2021) Two new species of Dugesia (Platyhelminthes, Tricladida, Dugesiidae) from the tropical monsoon forest in southern China. ZooKeys 1059: 89-116. https://doi.org/10.3897/zookeys.1059.65633
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Two new species of the genus Dugesia (Platyhelminthes, Tricladida, Dugesiidae) from the tropical monsoon forest in southern China are described on the basis of an integrative taxonomic study involving morphology, karyology, histology, and molecular analyses. The new species Dugesia circumcisa Chen & Dong, sp. nov. is characterised by asymmetrical openings of the oviducts; right vas deferens opening at anterior portion of the seminal vesicle and the left one opening at mid-lateral portion of the seminal vesicle; two diaphragms in ejaculatory duct, the latter being ventrally displaced and opening at the tip of the penis papilla, which is provided with a nozzle; wide duct connecting male atrium and common atrium; chromosome complement triploid with 24 metacentric chromosomes. The other new species, Dugesia verrucula Chen & Dong, sp. nov., is characterised by the large size of the living worm, usually exceeding 3.5 cm in length; asymmetrical openings of the oviducts; subterminal opening of ventrally displaced ejaculatory duct; vasa deferentia symmetrically opening into the postero-lateral portion of the seminal vesicle; well-developed duct between the seminal vesicle and diaphragm; single dorsal bump near the root of the penis papilla; bursal canal with pleated wall and spacious posterior section; unstalked cocoons; chromosome complement diploid with 16 metacentric chromosomes. Inter-specific molecular distances and their positions in the phylogenetic tree reveal that D. circumcisa and D. verrucula are clearly separated from their congeners.
Genetic distance, karyology, molecular phylogeny, monsoon forest, new species, taxonomy
Approximately 96 species of the freshwater planarian genus Dugesia Girard, 1850 are distributed in a major portion of the Old World and Australia (
On 1 January 2019, the specimens were collected in the Shiwan Dashan Mountain National Natural Reserve in Guangxi Province, where some animals were collected from under stones in a freshwater stream, while others were collected from under stones in a pond under a waterfall (for sampling localities, see Fig.
Total genomic DNA was extracted from specimens by using the QIAamp DNA Mini Tissue Kit (Qiagen, Germany), according to the manufacturer’s protocols. The primers BarS and COIR were used for amplification of fragments of the Cytochrome c oxidase subunit I (COI) (
In order to determine whether the presumed new species are molecularly different from other species of Dugesia, we performed a phylogenetic analysis and calculated genetic distances. The ingroup included the two new species, as well as 28 other Dugesia species from major portions of the geographic range of the genus. Schmidtea mediterranea (Benazzi et al., 1975) was chosen as the outgroup taxon (for GenBank accession numbers, see Table
GenBank accession numbers of COI and ITS-1 sequences used for molecular analyses.
Species | GenBank | |
---|---|---|
CO I | ITS-1 | |
D. aethiopica | KY498845 | KY498785 |
D. afromontana | KY498846 | KY498786 |
D. ariadnae | KC006972 | KC007048 |
D. arcadia | KC006971 | KC007044 |
D. batuensis | KF907818 | KF907815 |
D. benazzii | FJ646977+FJ646933 | FJ646890 |
D. bengalensis | FJ646897 | |
D. bifida | KY498851 | KY498791 |
D. bijuga | MH119630 | |
D. circumcisa | MZ147041 | MZ146782 |
D. cretica | KC006976 | KC007050 |
D. deharvengi | KF907820 | KF907817 |
D. elegans | KC006984 | KC007063 |
D. gibberosa | KY498857 | KY498803 |
D. hepta | FJ646988+FJ646943 | FJ646902 |
D. japonica | FJ646990 | FJ646904 |
D. majuscula | MW533425 | MW533591 |
D. naiadis | KF308756 | |
D. notogaea | FJ646993+FJ646945 | FJ646908 |
D. pustulata | MH119631 | |
D. ryukyuensis | AF178311 | FJ646910 |
D. semiglobosa | MW525210 | MW526992 |
D. sicula | FJ646994 + FJ646947 | DSU84356 |
D. sigmoides | KY498849 | KY498789 |
D. sinensis | KP401592 | |
D. subtentaculata | FJ646995 +FJ646949 | DSU84369 |
D. umbonata | MT176641 | MT177211 |
D. verrucula | MZ147040 | MZ146760 |
S. mediterranea | JF837062 | AF047854 |
Nuclear ribosomal markers were aligned online with MAFFT (Online Version 7.247) using the G-INS-i algorithm (
Mr Bayes v 3.2 (
The genetic distances of COI and ITS-1 were calculated by MEGA 6.06 (
Histological sections were prepared as described previously by
Karyological preparations were obtained by air-drying, following methods described by
The concatenated sequences included 846 base pairs (bp) for COI and 732 bp for ITS-1. The populations of both D. circumcisa and D. verrucula showed no variation in either COI or ITS-1.
The phylogenetic trees obtained by BI and ML from the concatenated dataset showed similar topologies and supported nodes (Fig.
The separate species status of D. circumcisa and D. verrucula is supported also by the genetic distances between the species included in our analysis, albeit that COI distances vary greatly among species (Suppl. material
The highest distance value between D. circumcisa and its congeners is 24.65% (with D. batuensis Ball, 1970 and D. sicula Lepori, 1948), while the lowest distance value is 11.20% (with D. umbonata). With respect to D. verrucula, the highest distance value between this species and its congeners is 26.09% (with D. aethiopica Stocchino et al., 2002), while the lowest distance value is 15.47% (with D. umbonata). Furthermore, there is a 17.15% difference between the two new species.
With respect to ITS-1, D. circumcisa and D. verrucula show highest distance values with D. sicula, which are 18.65% and 17.57%, respectively. Furthermore, their lowest distance values are with D. majuscula, which are 4.8% and 2.77%, respectively. For this marker, the molecular distance between the two new species is 4.98% (Suppl. material
Suborder Continenticola Carranza, Littlewood, Clough, Ruiz-Trillo, Baguñà & Riutort, 1998
Family Dugesiidae Ball, 1974
Holotype.
Dugesia circumcisa is characterised by the presence of the following features: right vas deferens opening at anterior portion of the seminal vesicle, and the left one opening at lateral portion of the vesicle, with the left sperm duct opening dorsally to the right one; two diaphragms in ejaculatory duct, the distal one receiving secretion of penial glands; ejaculatory duct with ventral course through penis papilla and with terminal opening; small nozzle at tip of penis papilla; wide duct connecting male and common atrium; asymmetrical openings of the oviducts into the bursal canal; chromosome complement triploid, with 24 metacentric chromosomes.
The specific epithet is derived from the Latin adjective circumcisus, ‘pruned of excess, sheared on all sides’, and alludes to the appearance of the tip of the penis papilla.
Approximately 20 animals were collected from a freshwater stream on the Shiwan Dashan Mountain (Fig.
Each of the five, randomly selected specimens exhibited triploid chromosome complements. In a total of 100 metaphase plates examined, 86 chromosome complements were triploid with 2n = 3× = 24 chromosomes, with all chromosomes being metacentric (Fig.
Karyotype parameters (mean values and standard deviations) of Dugesia circumcisa; m: metacentric.
Chromosome | Relative length | Arm ratio | Centromeric index | Chromosome type |
---|---|---|---|---|
1 | 18.15±0.86 | 1.18±0.12 | 46.13±2.38 | m |
2 | 15.53±0.95 | 1.23±0.13 | 45.08±2.44 | m |
3 | 13.83±0.15 | 1.34±0.12 | 43.01±2.26 | m |
4 | 12.53±0.44 | 1.25±0.11 | 44.64±2.09 | m |
5 | 11.59±0.52 | 1.29±0.09 | 44.10±1.67 | m |
6 | 10.48±0.14 | 1.60±0.28 | 39.22±4.07 | m |
7 | 9.55±0.20 | 1.28±0.16 | 44.19±2.99 | m |
8 | 8.35±0.43 | 1.15±0.12 | 46.73±2.39 | m |
In asexual living specimens, body 8–15 mm in length and 0.8–1.5 mm in width, while in sexualised specimens the body measures 15–22 mm in length and 1.5–2.3 mm in width. Head of low triangular shape and provided with two auricles, as well as two eyes located in pigment-free patches (Fig.
Pharynx situated in the mid-region of the body, measuring ca. 1/5th of the body length (Fig.
Dugesia circumcisa A transverse section of pharynx of paratype YWSZ11, showing musculature B sagittal section of paratype YWSZ8, showing poorly developed ovary C sagittal section of paratype YWSZ9, showing hyperplasic ovaries and poorly developed testes D sagittal section of paratype
The ventral ovaries are located at a short distance behind the brain and dorso-medially to the ventral nerve cords. The development of the ovaries differs greatly between specimens. In some animals the ovaries are rather small or even poorly developed (Fig.
The large sac-shaped copulatory bursa, which occupies the entire dorso-ventral space, is lined by a vacuolated epithelium with basal nuclei and is almost devoid of any surrounding musculature (Figs
Dugesia circumcisa A sagittal section of holotype YWSZ2, showing bursal canal and copulatory bursa B sagittal section of holotype YWSZ2, showing opening of the right vas deferens into seminal vesicle, and two diaphragms C transverse section of paratype YWSZ11, showing left vas deferens, seminal vesicle, and ejaculatory duct D transverse section of paratype YWSZ11, showing right vas deferens E sagittal section of holotype YWSZ2, showing ejaculatory duct F sagittal section of holotype YWSZ2, showing small nozzle at tip of penis papilla G sagittal section of paratype YWSZ9, showing small nozzle at tip of penis papilla and the duct connecting male and common atrium H horizontal section of paratype YWSZ12, showing small nozzle at tip of penis papilla. Abbreviations: bc: bursal canal; ca: common atrium; cb: copulatory bursa; d: diaphragm; ed: ejaculatory duct; go: gonopore; lvd: left vas deferens; ma: male atrium; no: nozzle; ph: pharynx; pp: penis papilla; rvd: right vas deferens; sv: seminal vesicle. Scale bars: 100 μm.
The small, dorsally located testes are poorly developed and provided with immature spermatozoa (Fig.
Dugesia circumcisa Sagittal reconstruction of the copulatory apparatus of holotype YWSZ2. Abbreviations: bc: bursal canal; cb: copulatory bursa; cg: cement glands; d1: first diaphragm; d2: second diaphragm; ed: ejaculatory duct; go: gonopore; lod: left oviduct; lvd: left vas deferens; mo: mouth; no: nozzle; ph: pharynx; pp: penis papilla; rod: right oviduct; rvd: right vas deferens; sg: shell glands; sv: seminal vesicle. Scale bar: 100 μm.
The large and well-developed penis bulb occupies the major part of the dorso-ventral space and is composed of intermingled muscle fibres (Figs
Dugesia circumcisa Sagittal reconstruction of the copulatory apparatus of paratype YWSZ9. Abbreviations: bc: bursal canal; cb: copulatory bursa; cg: cement glands; d1: first diaphragm; d2: second diaphragm; ed: ejaculatory duct; go: gonopore; lod: left oviduct; lvd: left vas deferens; no: nozzle; pg: penial glands; pp: penis papilla; rod: right oviduct; rvd: right vas deferens; sg: shell glands; sv: seminal vesicle. Scale bar: 100 μm.
From the point of the large expansion, the ejaculatory duct changes its vertical orientation and starts to run more or less parallel to the body surface or attains an oblique, ventro-caudal orientation, thus basically conforming to the particular orientation of the penis papilla. The narrow section of the ejaculatory duct that runs between the large expansion and the tip of the penis papilla is lined by an infranucleated epithelium and follows a ventral course through the papilla, opening terminally at its tip (Figs
The asymmetrical penis papilla is covered by an infranucleated epithelium, which is underlain by a subepithelial layer of circular muscle, followed by a layer of longitudinal muscle fibres. The penis papilla almost completely occupies the small male atrium, the latter communicating with the common atrium via a wide duct (Figs
Generally, there is only one diaphragm present in the ejaculatory duct of species of Dugesia, and only a few species exhibit two diaphragms, such as D. bijuga Harrath & Sluys, 2019, D. machadoi de Beauchamp, 1952, D. mirabilis de Vries, 1988, D. maghrebiana
In species with two diaphragms, the proximal diaphragm is usually small and basically formed by a non-glandular constriction of the seminal vesicle, while the true diaphragm is a larger structure that receives the secretion of penial glands, as is usual for the diaphragm in species of Dugesia. The situation in D. circumcisa is slightly different in that the proximal diaphragm is not small but consists of a well-developed valve.
Generally, in species of Dugesia the openings of the left and right sperm ducts into the intrabulbar seminal vesicle are located at ca. the same level. However, in D. circumcisa the vasa deferentia open asymmetrically into the seminal vesicle. Such asymmetrical openings have been reported explicitly for D. bifida Stocchino & Sluys, 2014, in which the sperm ducts open halfway into the vesicle, with the right duct opening dorsally to the left one. This contrasts with the situation in D. circumcisa, in which the right sperm duct opens into the anterior portion of the seminal vesicle and the left duct opens through the mid-lateral wall of the seminal vesicle, with the left sperm duct opening dorsally to the right one.
The characteristic nozzle at the tip of the penis papilla in D. circumcisa is paralleled in D. bakurianica Porfirjeva, 1958, D. bijuga, and perhaps also in D. sinensis. From that perspective, it is interesting that D. bijuga also possesses two diaphragms (see above). However, other parts of the male copulatory apparatus of D. circumcisa are different from that of D. bijuga (e.g., glands opening at a major portion of the blunt penis papilla, as well as atrial folds in the latter species). Furthermore, in the phylogenetric tree, D. circumcisa is far removed from D. bijuga (Fig.
Another characteristic feature of D. circumcisa is the rather wide and long duct connecting the male atrium with the common atrium. Generally, in species of Dugesia the male atrium opens more or less directly into the common atrium, without interpolation of a well-defined duct. Apart from D. circumcisa, other exceptions to this ground-plan condition can be found in D. bactriana de Beauchamp, 1959, D. bengalensis Kawakatsu, 1983, D. bifida, D. capensis Sluys, 2007, and D. colapha Dahm, 1967. However, other characters prevent synonymisation of D. circumcisa with any of these species, which in the case of D. bifida is supported also by completely different positions in the phylogenetic tree (Fig.
Holotype.
Dugesia verrucula is characterised by the presence of the following features: large size of the live worm, usually exceeding 3.5 cm in length; asymmetrical openings of the oviducts into the bursal canal; subterminal opening of the ventrally displaced ejaculatory duct; vasa deferentia symmetrically opening into the postero-lateral portion of the seminal vesicle; well-developed duct between seminal vesicle and diaphragm; single dorsal bump near root of penis papilla; bursal canal with pleated wall and spacious posterior section; unstalked cocoons; chromosome complement diploid with 16 metacentric chromosomes.
The specific epithet is derived from the Latin verrucula, small wart, and alludes to the dorsal bump on the penis papilla; the specific epithet is treated as an adjective.
The average annual temperature of the tropical monsoon rain forest in the Shiwan Dashan Mountain National Natural Reserve ranges between 20 and 25 °C, while in the coldest month (January), the average temperature is ca. 10–13 °C. The worms were collected from a pond under a waterfall on the Shiwan Dashan Mountain, with a water temperature of 8.4 °C, while air temperature was 5.5 °C (Fig.
Each of the six, randomly selected specimens exhibited diploid chromosome complements. In a total of 100 metaphase plates examined, 85 chromosome complements were diploid with 2n = 2× = 16 chromosomes, with all chromosomes being metacentric (Fig.
Karyotype parameters (mean values and standard deviations) of Dugesia verrucula; m: metacentric.
Chromosome | Relative length | Arm ratio | Centromeric index | Chromosome type |
---|---|---|---|---|
1 | 18.72±0.66 | 1.31±0.16 | 43.54±3.00 | m |
2 | 15.81±0.56 | 1.25±0.12 | 44.75±2.62 | m |
3 | 13.88±0.42 | 1.47±0.27 | 41.35±3.36 | m |
4 | 12.31±0.29 | 1.34±0.15 | 43.03±2.63 | m |
5 | 11.1±0.33 | 1.59±0.30 | 39.89±4.45 | m |
6 | 10.22±0.46 | 1.19±0.09 | 45.81±1.87 | m |
7 | 9.49±0.32 | 1.48±0.25 | 41.49±3.28 | m |
8 | 8.47±0.53 | 1.32±0.18 | 43.36±3.22 | m |
The body of live, sexual specimens measures 2.8–3.9 mm in length and 1.8–2.8 mm in width. Low-triangular head provided with two blunt auricles and two eyes, which are located in the centre of the head and placed in pigment-free spots and house numerous retinal cells (Fig.
Pharynx situated in the mid-region of the body and measuring ca. 1/5th of the body length (Fig.
Dugesia verrucula A sagittal section of paratype
Ventral ovaries located at a short distance behind the brain, occupying ca. 1/6th of the dorso-ventral space (Fig.
The large, sac-shaped copulatory bursa occupies the entire dorso-ventral space, and is lined by a vacuolated epithelium with basal nuclei (Fig.
Dugesia verrucula A sagittal section of holotype ZJYA5, showing the testes B transverse section of paratype ZJYA7, showing vasa deferentia and seminal vesicle C sagittal section of holotype ZJYA5, showing subterminal opening of ejaculatory duct at tip of penis papilla D sagittal section of holotype ZJYA5, showing dorsal bump and copulatory bursa E sagittal section of holotype ZJYA5, showing seminal vesicle, diaphragm in the ejaculatory duct, dorsal bump, and copulatory bursa F sagittal section of holotype ZJYA5, showing seminal vesicle, dorsal bump, and copulatory bursa G sagittal section of paratype ZJYA11, showing subterminal opening of ejaculatory duct and dorsal bump H sagittal section of paratype ZJYA11, showing gonopore, ejaculatory duct, and diaphragm. Abbreviations: bc: bursal canal; ca: common atrium; cb: copulatory bursa; d: diaphragm; db: dorsal bump; ed: ejaculatory duct; go: gonopore; lvd: left vas deferens; mo: mouth; pp: penis papilla; rvd: right vas deferens; sv: seminal vesicle; te: testis; vd: vas deferens. Scale bars: 100 μm.
The bursal canal is lined with columnar, nucleated, ciliated cells and is surrounded by a thin subepithelial layer of longitudinal muscles, followed by a thicker layer of circular muscle. An ectal reinforcement layer of longitudinal muscles runs from the vaginal region to ca. halfway along the bursal canal (Figs
Dugesia verrucula Sagittal reconstruction of the copulatory apparatus of holotype ZJYA5. Abbreviations: bc: bursal canal; cb: copulatory bursa; cg: cement glands; d: diaphragm; db: dorsal bump; ed: ejaculatory duct; go: gonopore; lod: left oviduct; mo: mouth; pg: penial glands; ph: pharynx; pp: penis papilla; rod: right oviduct; sg: shell glands; sv: seminal vesicle; vd: vas deferens. Scale bar: 100 μm.
The well-developed testes are situated dorsally and provided with mature spermatozoa (Fig.
The vasa deferentia expand to form large spermiducal vesicles, filled with sperm (Figs
Dugesia verrucula Sagittal reconstruction of the copulatory apparatus of paratype ZJYA11 A sagittal reconstruction of male copulatory apparatus B sagittal reconstruction of female copulatory apparatus. Abbreviations: bc: bursal canal; cb: copulatory bursa; cg: cement glands; d: diaphragm; db: dorsal bump; ed: ejaculatory duct; epg: extrabulbar penial glands; go: gonopore; lod: left oviduct; mo: mouth; pg: penial glands; ph: pharynx; pp: penis papilla; rod: right oviduct; sg: shell glands; sv: seminal vesicle; vd: vas deferens. Scale bar: 100 μm.
The ejaculatory duct arises from the antero-dorsal wall of the seminal vesicle and, subsequently, curves downwards towards the ventral root of the penis papilla. Near the ventral root of the papilla the ejaculatory duct is provided with a small diaphragm and changes its more or less vertical orientation by turning towards the tip of the penis papilla, having a subterminal opening at its tip (Figs
Because of the ventrally displaced course of the ejaculatory duct, the penis papilla is asymmetrical, with its dorsal lip being considerably larger than the ventral lip. The conical or sub-cylindrical penis papilla has an oblique, ventro-caudad orientation and is covered by an infranucleated epithelium, which is underlain by a subepithelial layer of circular muscle, followed by a layer of longitudinal muscle fibres. Near its dorsal root, the penis papilla is provided with a pronounced bump (Figs
The most characteristic feature of D. verrucula is the permanent dorsal bump on its penis papilla, a similar character being known only from Dugesia gibberosa Stocchino & Sluys, 2017. However, the latter is provided with two dorsal bumps on its penis papilla instead of one, while its ejaculatory duct opens terminally at the tip of the papilla (
Apart from the penial bump and the subterminal opening of the ejaculatory duct, other characteristic features of D. verrucula are the asymmetrical penis papilla with ventrally displaced ejaculatory duct (character 1, state 1 in
Inter-specific genetic distances of both COI and ITS-1 reveal that D. circumcisa and D. verrucula are well-separated from their congeners. The lowest COI distance values between D. circumcisa and D. verrucula and other congeners are 11.20% and 15.47%, respectively, while the distance between the two new species is 17.15% (Suppl. material
The topology of our phylogenetic tree (Fig.
The two new species show a haploid number of n = 8 metacentric chromosomes, thus conforming to the situation that in the genus Dugesia the basic chromosome number is 7, 8 or 9 (
The number of 8–11 juveniles hatching from a single cocoon of D. verrucula falls at the higher end of the range as reported for other sexual species of Dugesia, such as D. benazzii (8–10 hatchlings), D. etrusca (8–10), D. hepta Pala et al., 1981 (8–10), D. cretica (Meixner, 1928) (4–15) (
In contrast, D. circumcisa never produced cocoons and only showed asexual reproduction by means of fission, which corresponds with its poorly developed or hyperplasic ovaries and the triploid chromosome complement. It has been established that in such abnormal ovaries the oocytes are anomalous (
This work was supported by the National Natural Science Foundation of China (grant numbers: 32070427, 31570376, 31471965, u1604173), the Major Public Welfare project of Henan Province (grant: 201300311700) and by the Puyang Field Scientific Observation and Research Station for Yellow River Wetland Ecosystem, Henan Province.
Figure S1
Data type: Phylogenetic tree
Explanation note: Phylogenetic tree obtained from ML analysis of the concatenated dataset. Numbers at nodes indicate support values (bootstrap). Scale bar: substitutions per site.
Table S1
Data type: Genetic distances
Explanation note: Genetic distances for COI. Highest and lowest distance values between the two new Chinese species and Oriental-Australasian congeners indicated in red and blue, respectively. Purple: distance value between the two new species.
Table S2
Data type: Genetic distances
Explanation note: Genetic distances for ITS-1. Highest and lowest distance values between the two new Chinese species and Oriental-Australasian congeners indicated in red and blue, respectively. Purple: distance value between the two new species.