Two new species of Rhabdocoela (Polycystididae and Trigonostomidae) from China Author Hu, Xiao-Zhou Author Chen, Yu-Si Author Zhong, Lin-Hong Author Xie, Yao-Hang Author Feng, Wei-Tao Author Zhang, Yu Author Wang, An-Tai text Zootaxa 2019 2019-11-11 4695 4 351 366 journal article 24922 10.11646/zootaxa.4695.4.3 f9db7b4c-11e2-4abb-86a0-8217d52cb61c 1175-5326 3534784 1CF2E78F-8B1A-4E53-9BB7-FAD3D5F06FDD Trigonostomum sinensis Wang & Hu n. sp. ( Figs. 4–6 ) Locality. Same as A. sinensis n. sp. Material examined. Holotype : PLA-Tr001, whole-mounted specimen. Paratypes : PLA-Tr002–003, wholemounted slides of stylet; PLA-Tr004–006, serially-sectioned (sagittal) specimens. Description. Matured individuals are 0.9–1.3 mm in length and 110–170 μm in width (n=3), rod-shaped, and without pigment ( Fig. 4A ). The triangular invagination (“proboscis”) is situated at the anterior end. Tuft of sensory bristles lay at the front ( Fig. 6A ). The paired kidney-shaped eyes are located posterior to the pharynx. The distance between eyes is 31–50 μm (n=3, Fig. 4 A–B, 5A, 6A). The spheroid shape pharynx (125–162 μm in length and 80–97 μm in width, n=3) is located posterior to the eyes ( Fig. 4 A–B, 5A, 6A). Two bundles of adenal rhabdites are present in the posterior region on both sides of the body ( Fig. 4B & F , 5A & C–D, 6A). The paired spheroid testes (65–84 μm in length and 33–60 μm in width, n=3), are situated laterally posterior to the pharynx ( Fig. 4 B–C, 5G–H, 6A). A pair of long seminal vesicles (35–51 μm in length, n=3), is located at the posterior end of the body ( Fig. 4 B–C & E, 5A & D–H, 6A–B). The spheroid prostate vesicle (31–41 μm in length and 27–34 μm in width, n=3) contain two types of secretions. It connects to a seminal duct (fusion of two ends of seminal vesicles) at its anterior end and to the copulatory organ at its posterior end ( Fig. 4 B–C & E, 5A & C–H, 6A–B). The copulatory organ consists of a stylet and two "T"-shaped plates. The long-tubular stylet (52–54 μm in length; 5–8 μm in midpoint diameter; 12–14 μm in basal diameter, n=3, Fig. 6D ), bends 120° at 25% of the stylet from the base ( Fig. 4 B–C & E & I–J, 5C–E, 6A–C) and extends straight at its distal end. There is an aperture at one side of the distal end and a hook bridging other two structures of the stylet. The two "T"-shaped plates are connected to each other and surround the stylet. The "T" plate I is 29–31 μm in length and 10–13 μm in width (n=3, Fig. 6E ), and is hook-shaped at its distal end, while the "T" plate II has a similar length but only half the width of "T" plate I ( Fig. 6F ). The genital pore is located ventrally at 86% of the body. The paired long-tubular vitellaria (336–480 μm in length, n=3) are situated dorsally at both sides of the body, extending from the pharynx to the posterior end ( Fig. 4 A–C, 5E–H, 6A–B). The female bursa is relatively large (90–142 μm in length and 56–74 μm in width, n=3), and is situated at the middle of the body. It connects to the ovaries via a bursal appendage. The bursal appendage (99–102 μm in length, n=3) is coiled tube. Its funnel-shaped anterior end (22–24 μm in diameter, n=3, Fig. 4 C–D & G–H, 5B & G, 6A–B & G) has a sheath structure. It tapers towards posterior end, and twists about 360° into a helix in its middle part (7–9 μm in width), and bifurcates into six microtubes. The microtubes of the bursal appendage enter the ovaries, which are located laterally behind the testes. The paired ovaries, spheroid in shape, is 60–78 μm in length and 25–32 μm in width (n=3) ( Fig. 4 B–D, 5B & E–H, 6A–B). Remarks. So far, 20 species within the genus Trigonostomum have been recorded globally. The characteristics of copulatory organ (CO) and bursal appendage (BA) are main criteria for identification and classification of the species within this genus. The CO and BA of the new species Trigonostomum sinensis n. sp. are highly similar to those of T. tillicum from the Northeast Pacific and T. vanmecheleni from Italy . Their stylets are all in long-tubular shape, with a spur at the anterior end and a sharp distal end surrounded by a mantle that forms two plates. Their bursal appendages are in coiled tubular shape, with several micro-tubes at posterior end. However, there are still some obvious differences among them. For instance, the stylet of T. tillicum and T. vanmecheleni curve around 90°, while T. sinensis n. sp. in this study curves around 120° at anterior end. The distal end part of BA in T. tillicum splits into six tubes with swollen vesicle-like structure. However, the swollen part is absent in the new Trigonostomum species from China . Besides, in T. tillicum , the length of the stylet and bursal appendage are 80–92 µm and 230–300 μm, respectively ( Van Steenkiste & Leander 2017 ), which are much larger than those of T. sinensis n. sp. ( Table 3 ) in this study. As such, it is evident that T. sinensis n. sp. is a new species within the genus Trigonostomum . TABLE 3. Comparison on T. sinensis n. sp. and similar species (μm)

Species	Body length	Length of ST	Length of BA	STA	BAD	SST	Reference
T. sinensis n. sp.	(μm) 0.9–1.3	(μm) 52–54	(μm) 99–102	120°	6 tubes	No	This study
T. tillicum	1.1–1.9	80–92	230–300	90°	6 tubes	Yes	Van Steenkiste &
T. vanmecheleni	0.9	48–55	105–126	90°	6 tubes	No	Leander, 2017 Artois et al ., 2013

STA: angle of the curve of proximal stylet; BAD: distal structure of bursa appendage; SST: swollen vesicle-like structure of tubes FIGURE 4 . Trigonostomum sinensis n. sp : (A) live specimen; (B) squeezed live specimen; (C–F) reproductive organs (partim); (G–H) bursal appendage; (I–J) copulatory organ. Molecular and phylogenetic analyses. The NCBI nucleotide BLAST results indicated that 18S and 28S rDNA sequences of Alcha sinensis n. sp. are most similar to those of Alcha evelinae (18S: ~ 98% identity; 28S: ~94% identity). The 18S and 28S rDNA phylogenetic analyses ( Figs 7–8 ) showed that two specimens of A. sinensis n. sp. clustered together and form a clade with Alcha evelinae , which agrees well with the morphological comparison between these two closely related species. As such, the molecular data support the establishment of Alcha sinensis n. sp. as a new species. As for Trigonostomum sinensis n. sp. , its 18S and 28S rDNA sequences are most similar to T. tillicum (18S: ~ 99%; 28S: ~98%). The 18S rDNA phylogenetic analyses showed that the two specimens of T. sinensis n. sp. clustered together with T. tillicum , at a short evolutionary distance from T. tillicum . The 28S rDNA phylogenetic tree revealed a similar result, but generated a somewhat longer distance between T. sinensis n. sp. and T. tillicum , supporting the establishment of T. sinensis n. sp. as a new species within the genus Trigonostomum . FIGURE 5 . Trigonostomum sinensis n. sp : (A) whole mount; (B–C) whole mount, showing the reproductive organs (partim); (D–H) serially-sectioned (sagittal) specimens. Discussion. Before this study, only one species has been recorded within the genus Alcha since its description seventy years ago ( Marcus, 1949 ). The species has been documented from Kenya ( Jouk & De Vocht 1989 ), Mexico ( Artois et al . 2013 ), and California ( Karling & Schockaert 1977 ) as well as Brazil . The identification of the new species, namely A. sinensis n. sp . , is the first time this genus has been found in Asia. As both the morphology and the phylogenetic analyses indicated that this species is closely related to A. evelinae , it would be important to know whether this new species also has a cosmopolitan distribution. The species of Trigonostomum have a global distribution, having been discovered in Iceland , Norway , Germany , England , the United Kingdom , Australia , France , Brazil , USA , Africa, Greece , Pacific Ocean and so on ( Willems et al . 2004 ). The identification of Trigonostomum sinensis n. sp. represents the first description within the genus Trigonostomum in China . FIGURE 6 . Trigonostomum sinensis n. sp : (A) habitus from a live animal; (B) sagittal reconstruction of atrial organs, showing reproductive organs (partim); (C) copulatory organ; (D) stylet; (E) plate I; (F) plate II; (G) bursal appendage. In 1924, based on the morphology of the bursal appendage, Meixner divided the species of Trigonostomum into the penicillatum type (TPT) and the setigerum type (TST). According to the design of copulatory organ, which is the main classification method in Trigonostomum , Willems (2004) categorized 18 species of this genus into the following groups: (1) group 1A, including T. venenosum and T. messoplanoides , is characterized by long and narrow stylet that makes a proximal turn of 270°, and the mantle forms a narrow ring and bears one long, slender spine; (2) group 1B, including T. coronatum , T. watsoni , T. penicillatum , T. lilliei , T. breitfussi , T. mirabile , T. nataschae . , and T. prytherchiv , is characterized by the wider and shorter stylet, with a turn of 90° or 180°, while the mantle is divided into several pointed plates. Thereafter Artois (2013) and Van Steenkiste (2017) also respectively described T. vanmecheleni and T. tillicum into this group; and (3) group 2, including T. franki , T. armatum , T. setigerum , T. australis , T. galapagoensis and T. tori is characterized by a spirally coiled and narrow stylet, which is followed by a mantle. Moreover, two species, namely T. denhartogi & T. spinigerum , have not been categorized into any group. FIGURE 7. The phylogenetic tree of 18S rDNA sequences of generated with Maximum Likelihood method (bootstrap=1000, GTR+F+I+G4). In this study, a phylogenetic analysis of ten species ( Trigonostomum sinensis n. sp. included) within the genus Trigonostomum indicated that five species from group 2: T. franki , T. tori , T. setigerum T. armatum and T. sinensis n. sp. , clustered together and form a well-supported clade, which agrees well with traditional categorization based on morphology. However, the ungrouped species ( T. denhartogi ) and four species from group 1B ( T. watsoni , T. penicillatum , T. vanmecheleni , T. tillicum ) show a discrepancy between morphology and molecular-sequence analysis. In the molecular cladogram, T. penicillatum forms an independent group, and T. denhartogi clusters with T. watsoni and T. vanmecheleni to form a clade, which is quite different from traditional taxonomy. We take the morphologies of the copulatory organ and bursal appendage into account, to classify the species of Trigonostomum in more detail. As shown in table 4, 21 species were divided into four types . The species in type 4 are actually from the previous group 2. As mentioned above, this group is characterized by a coiled stylet enveloped by the mantle over its entire length. In more detail, the mantle distally splits into two spiny plates with terminal hook. Its bursal appendage (BA) contains 2 tubules that curved proximally over 270°or 360° and bears a straight distal part. These characteristics are clearly different from any species of the other three types . The original group 1 species and the two undetermined species together can be divided into types 1, 2, and 3. Most of these species have a long-tube stylet that bends proximally 90°, 180° or 270°, and the mantle surrounds only the distal part of the stylet. Here we mainly categorized them by notable characters, namely the number of plates/tubes in the mantle and bursal appendage. Both the mantles of type 1 and type 3 split into a variable number of plates, but the BA structure of type 1 is crown-like and its distal part splits into nine or ten tubes. As for type 3, its BA consists of 2 irregular coiled tubes. In type 2, the mantle consists of 2 "T" plates, while the BA is a coiled tube, with its distal part splits into 6 microtubes. When the copulatory organ and bursal appendage were jointly used as the basis of classification, the result agrees with the 18S and 28S rDNA molecular results. In the future, we still need more morphological and molecular information about the species within the genus, to verify and supplement the new classification proposed in this study.