Research Article |
Corresponding author: Guang-wen Chen ( chengw0183@sina.com ) Academic editor: Yasen Mutafchiev
© 2021 Lei Wang, 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, Dong Z-m, Chen G-w, Sluys R, Liu D-z (2021) Integrative descriptions of two new species of Dugesia from Hainan Island, China (Platyhelminthes, Tricladida, Dugesiidae). ZooKeys 1028: 1-28. https://doi.org/10.3897/zookeys.1028.60838
|
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.
Histology, karyology, molecular distance, molecular phylogeny, taxonomy
Approximately 94 species of the freshwater planarian genus Dugesia Girard, 1850 have been reported from a major portion of the Old World and Australia (
Specimens were collected during 2016–2018 from under stones in springs with the help of a paint brush (for sampling localities, see Fig.
Histological sections were prepared as described by
Karyological preparations were made by means of the air-drying technique, following
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
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 (
GenBank accession numbers of COI and ITS-1 sequences used for molecular analysis.
Species | GenBank | Species | GenBank | ||
---|---|---|---|---|---|
COI | ITS-1 | COI | ITS-1 | ||
D. aethiopica | KY498845 | KY498785 | D. improvisa | KC006987 | KC007065 |
D. afromontana | KY498846 | KY498786 | D. japonica | FJ646990 | FJ646904 |
D. arcadia | KC006971 | KC007044 | D. liguriensis | FJ646992 | FJ646907 |
D. ariadnae | KC006972 | KC007048 | D. majuscula | MW533425 | MW533591 |
D. batuensis | KF907818 | KF907815 | D. malickyi | KC006990 | KC007069 |
D. benazzii | FJ646977 + FJ646933 | FJ646890 | D. naiadis | KF308756 | |
D. bengalensis | FJ646897 | D. notogaea | FJ646993 + FJ646945 | FJ646908 | |
D. bifida | KY498851 | KY498791 | D. ryukyuensis | AF178311 | FJ646910 |
D. cretica | KC006976 | KC007050 | D. semiglobosa | MW525210 | MW526992 |
D. damoae | KC006979 | KC007057 | D. sicula | FJ646994+FJ646947 | DSU84356 |
D. deharvengi | KF907820 | KF907817 | D. sigmoides | KY498849 | KY498789 |
D. effusa | KC006983 | KC007058 | D. sinensis | KP401592 | |
D. elegans | KC006984 | KC007063 | D. subtentaculata | FJ646995 + FJ646949 | DSU84369 |
D. gibberosa | KY498857 | KY498803 | D. umbonata | MT176641 | MT177211 |
D. gonocephala | FJ646986 + FJ646941 | FJ646901 | S. mediterranea | JF837062 | AF047854 |
D. hepta | FJ646988 + FJ646943 | FJ646902 |
Nuclear ribosomal markers were aligned online with MAFFT (Online Version 7.247) using the G-INS-i algorithm (
MrBayes v3.2 (
Genetic distances were calculated for CO1 and ITS-1 with the help of MEGA 6.06 (
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.
Phylogenetic tree obtained from Bayesian analysis of the concatenated dataset. Numbers at nodes indicate support values (posterior probability/bootstrap). ∗: Bootstrap value not applicable to the node, because of different topologies of trees obtained by BI and ML methods. Scale bar: substitutions per nucleotide position.
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
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
Order Tricladida Lang, 1884
Suborder Continenticola Carranza, Littlewood, Clough, Ruiz-Trillo, Baguñà & Riutort, 1998
Family Dugesiidae Ball, 1974
Genus Dugesia Girard, 1850
Holotype
:
Paratypes
:
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.
The specific epithet is derived from the Latin semis, half, and globosus, spherical, and alludes to the hemispherical penis papilla.
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.
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.
Karyotype parameters (mean values and standard deviations) of Dugesia semiglobosa; m: metacentric.
Chromosome | Relative length | Arm ratio | Centromeric index | Chromosome type |
---|---|---|---|---|
1 | 17.43 ± 0.93 | 1.12 ± 0.09 | 47.31 ± 1.83 | m |
2 | 14.71 ± 0.46 | 1.19 ± 0.09 | 45.88 ± 1.69 | m |
3 | 13.69 ± 0.45 | 1.30 ± 0.18 | 43.86 ± 3.53 | m |
4 | 12.93 ± 0.46 | 1.34 ± 0.14 | 43.21 ± 2.54 | m |
5 | 11.78 ± 0.26 | 1.29 ± 0.11 | 43.82 ± 2.12 | m |
6 | 10.80 ± 0.17 | 1.27 ± 0.06 | 44.33 ± 1.35 | m |
7 | 9.92 ± 0.53 | 1.28 ± 0.10 | 44.24 ± 1.97 | m |
8 | 8.72 ± 0.64 | 1.21 ± 0.05 | 45.40 ± 1.02 | m |
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.
Pharynx situated in the mid-region of the body, measuring ~ 1/5th of the body length (Fig.
Dugesia semiglobosa A transverse section of paratype JWT 6, showing longitudinal and circular muscles in outer and inner pharyngeal musculature B transverse section of paratype JWT 7, showing hyperplasic ovaries. C sagittal section of holotype JWT 5, showing testes and small ovary D transverse section of paratype JWT 7, showing symmetrical openings of the oviducts E transverse section of paratype JWT 6, showing spermiducal vesicles F transverse section of paratype JWT 7, showing openings of the vasa deferentia into the seminal vesicle. The anterior is to the front in A, B, D, E, F, and anterior is to the right in C. Abbreviations: bc: bursal canal; ca: common atrium; cm: circular muscles; in: intestine; lm: longitudinal muscles; lod: left oviduct; ov: ovary; rod: right oviduct; spv: spermiducal vesicle; sv: seminal vesicle; te: testis; vd: vas deferens. Scale bars: 100 μm.
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.
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
The small, dorsally located testes are well developed and provided with mature spermatozoa (Fig.
Dugesia semiglobosa A sagittal section of holotype JWT 5, showing reniform seminal vesicle, two diaphragms in the ejaculatory duct, ventral course of the ejaculatory duct, and hemispherical penis papilla B sagittal section of holotype JWT 5, showing copulatory bursa. C transverse section of paratype JWT 7, showing copulatory bursa D sagittal section of holotype JWT 5, showing bursal canal and its surrounding musculature E sagittal section of holotype JWT 9, showing small copulatory bursa, and mouth F horizontal section of paratype JWT 4, showing hemispherical penis papilla. The anterior is to the right in A, B, D, E, F and anterior is to the front in C. Abbreviations: bc: bursal canal; ca: common atrium; cb: copulatory bursa; cm: circular muscles; d: diaphragm; ed: ejaculatory duct; in: intestine; lm: longitudinal muscles; m: mouth; ma: male atrium; pp: penis papilla; sv: seminal vesicle; vd: vas deferens. Scale bars: 100 μm.
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.
Dugesia semiglobosa. 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; in: intestine; m: mouth; od: oviduct; pp: penis papilla; sg: shell glangs; sv: seminal vesicle; te: testis; vd: vas deferens. Scale bar: 100 μm.
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
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
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.
Dugesia semiglobosa exhibits a combination of three characteristic features (ventrally displaced ejaculatory duct, absence of duct intercalated between seminal vesicle and diaphragm, 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
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 (
Holotype
:
Paratypes
:
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.
The specific epithet is derived from the Latin adjective majusculus, somewhat larger, and alludes to expanded portion of the bursal canal.
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.
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.
Karyotype parameters (mean values and standard deviations) of Dugesia majuscula; m: metacentric.
Chromosome | Relative length | Arm ratio | Centromeric index | Chromosome type |
---|---|---|---|---|
1 | 17.76 ± 0.82 | 1.20 ± 0.08 | 45.66 ± 1.69 | m |
2 | 14.95 ± 0.72 | 1.15 ± 0.08 | 46.61 ± 1.67 | m |
3 | 13.30 ± 0.33 | 1.19 ± 0.04 | 45.70 ± 0.74 | m |
4 | 12.27 ± 0.31 | 1.25 ± 0.09 | 44.68 ± 1.81 | m |
5 | 11.47 ± 0.46 | 1.31 ± 0.17 | 43.58 ± 3.37 | m |
6 | 10.77 ± 0.44 | 1.19 ± 0.10 | 46.06 ± 2.07 | m |
7 | 10.21 ± 0.57 | 1.20 ± 0.08 | 45.77 ± 1.50 | m |
8 | 9.28 ± 0.34 | 1.25 ± 0.17 | 44.73 ± 3.13 | m |
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.
Pharynx positioned in the mid-region of the body and measuring ~ 1/6th of the body length (Fig.
Dugesia majuscula A transverse section of paratype YHYQ8, showing longitudinal and circular muscles in outer and inner pharyngeal musculature B horizontal section of paratype YHYQ 7, showing hyperplasic ovaries C transverse section of paratype YHYQ8, showing opening of the left oviduct D sagittal section of paratype YHYQ10, showing dorsal testes E transverse section of paratype YHYQ8, showing course of a vas deferens F sagittal section of paratype YHYQ3, showing opening of a vas deferens into 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.
Ovaries hyperplasic, with several scattered masses at a short distance behind the brain, extending backwards over a distance of at least 800 μm (Fig.
The well-developed testes are situated dorsally and extend from the level of the ovaries to the posterior end of the body (Fig.
Dugesia majuscula A sagittal section of holotype YHYQ4, showing seminal vesicle, ejaculatory duct, small diaphragm, penis papilla, and copulatory bursa B Sagittal section of holotype YHYQ4, showing seminal vesicle, ejaculatory duct, penis papilla, and copulatory bursa C sagittal section of paratype YHYQ5, showing terminal and dorsal opening of ejaculatory duct D sagittal section of paratype YHYQ9, showing bursal canal and its musculature. The anterior is to the right in A, B, C, D. Abbreviations: bc: bursal canal; cb: copulatory bursa; cm: circular muscles; d: diaphragm; ed: ejaculatory duct; in: intestine; lm: longitudinal muscles; ma: male atrium; pp: penis papilla; sv: seminal vesicle; vd: vas deferens. Scale bars: 100 μm.
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
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.
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
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 (
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 differences. 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 (
The topology of our phylogenetic tree (Fig.
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.
It is well-known that sexualized specimens from originally fissiparous populations generally develop hyperplasic ovaries (cf.
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 (
Within the genus Dugesia, the basic chromosome number is seven, eight, or nine (
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, 1964 and D. siamana Kawakatsu, 1980 (
It has been suggested that polyploidization is adaptive in harsh climatic conditions and extreme environments (
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.
This work was supported by grants from the National Natural Science Foundation of China (nos. 32070427, 31570376, 31471965, and u1604173) and the Program for Innovative Research Team in University of Henan Province (18IRTSTHN022). We are grateful to Dr. G. A. Stocchino (University of Sassari, Italy) for information and discussion on karyology in the genus Dugesia. We thank are grateful to Drs F. Carbayo, P. Boll, and two anonymous reviewers for their constructive criticisms, which improved the manuscript.
Figure S1. Phylogenetic tree obtained from ML analysis of the concatenated dataset
Data type: phylogenetic tree
Explanation note: Numbers at nodes indicate support values (bootstrap). Scale bar: substitutions per site.
Table S1. Primers used for amplification, with their annealing temperatures
Data type: molecular data
Table S2. Genetic distances for COI
Data type: molecular data
Explanation note: Highest and lowest distance values between the two new Chinese species and Oriental-Australasian congeners indicated in blue and red, respectively. Purple: distance value between the two new species.
Table S3. Genetic distances for ITS-1
Data type: molecular data
Explanation note: Highest and lowest distance values between the two new Chinese species and Oriental-Australasian congeners indicated in blue and red, respectively. Purple: distance value between the two new species.