Zoosyst. Evol. 100 (1) 2024, 167-182 | DOI 10.3897/zse.100.114016 yee BERLIN Integrative description of a new species of Dugesia (Platyhelminthes, Tricladida, Dugesiidae) from southern China, with its complete mitogenome and a biogeographic evaluation Ying Zhu!, JiaJie Huang', Ronald Sluys*, Yi Liu’, Ting Sun!, An-Tai Wang!, Yu Zhang! 1 Shenzhen Key Laboratory of Marine Bioresource and Eco-environmental Science, College of Life Science and Oceanography, Shenzhen University, Shenzhen, Guangdong, China 2 Naturalis Biodiversity Center, P.O. Box 9517, 2300 RA Leiden, Netherlands 3 Guangdong Engineering Research Center for Marine Algal Biotechnology, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen, Guangdong, China https://zoobank. org/808B9FAB-975 D-4A 59-8 D9F-18E7F 4A 176D3 Corresponding author: Yu Zhang (biozy@szu.edu.cn) Academic editor: Pavel Stoev # Received 13 October 2023 Accepted 10 January 2024 Published 16 February 2024 Abstract A new species of freshwater flatworm of the genus Dugesia from Guangdong Province in China is described through an integra- tive approach, including molecular and morphological data, as well as mitochondrial genome analysis. The new species, Dugesia ancoraria Zhu & Wang, sp. nov., is characterised by: (a) a highly asymmetrical penis papilla, provided with a hunchback-like dorsal bump; (b) a short duct between seminal vesicle and ejaculatory duct; and (c) a postero-ventral course of the ejaculatory duct, which opens to the exterior at the subterminal, ventral part of the penis papilla. The molecular phylogenetic tree obtained from the concat- enated dataset of four DNA markers (18S rDNA, ITS-1, 28S rDNA, COI) facilitated determination of the phylogenetic position of the new species, which shares a sister-group relationship with a small clade, comprising D. notogaea Sluys & Kawakatsu, 1998 from Australia and D. bengalensis Kawakatsu, 1983 from India. The circular mitogenome of the new species is 17,705 bp in length, in- cluding 12 protein coding genes, two ribosomal genes, and 22 transfer RNAs. Via analysis of gene order of mitochondrial genomes, the presently available pattern of mitochondrial gene rearrangement in the suborder Continenticola is discussed. Key Words biogeography, Dugesia, mitogenome, molecular phylogeny, taxonomy Introduction From the approximately 110 known species of Dugesia, thus far only 12 species have been recorded The distributional range of freshwater planarians of the genus Dugesia Girard, 1850 covers a large part of the Old World and Australia (cf. Sluys and Riutort 2018, fig. 13B). The historical biogeography of the genus has at- tracted the attention of planarian specialists for already a good number of years (cf. Sluys et al. 1998 and refer- ences therein), culminating in the most recent analysis, which could make use of a time-calibrated phylogenetic tree (Sola et al. 2022). from China, namely, D. japonica Ichikawa & Kawakat- su, 1964; D. ryukyuensis Kawakatsu, 1976; D. sinensis Chen & Wang, 2015; D. umbonata Song & Wang, 2020; D. semiglobosa Chen & Dong, 2021; D. majuscula Chen & Dong, 2021; D. circumcisa Chen & Dong, 2021; D. verrucula Chen & Dong, 2021; D. constrictiva Chen & Dong, 2021; D. gemmulata Sun & Wang, 2022; D. adun- ca Chen & Sluys, 2022; and D. tumida Chen & Sluys, 2022. The present study adds a new species of Dugesia to Copyright Zhu, Y. et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distri- bution, and reproduction in any medium, provided the original author and source are credited. 168 the Chinese fauna by describing it through an integrative approach, involving morphological, molecular phyloge- netic and mitogenomic analyses. Among these methods, morphological characters, especially the anatomy of the copulatory apparatus, form the main source for the de- scription and identification of the new species. Since the mitogenome is characterized by strict gene homology and uniparental inheritance without recombi- nation, and contains genes that evolve at different rates, mitochondrial gene order is considered as a strong genet- ic marker for resolving the phylogenetic position of new species (Rosa et al. 2017). Unfortunately, mitochondri- al genomic information on freshwater planarians 1s still highly limited. Therefore, we expanded our taxonomic study by including also the sequencing and annotation of the complete mitogenome of the new species Dugesia an- coraria Zhu & Wang, sp. nov. and compare its gene order with that of other species in the suborder Continenticola Carranza et al., 1998 for which such information is cur- rently available from GenBank. Materials and methods Sample collection and culturing Specimens were collected from a narrow artificial canal running from Wenshan lake in Shenzhen city, Guangdong Province, China (22°31'55"N, 113°56'21"E) on 10 May 2021 (Fig. 1). A 200-um-mesh sieve was used to collect Cladophora algae, to which the worms were attached. The contents of the mesh sieve were washed into a bucket us- ing habitat water, and then transported to the laboratory of Shenzhen University for further analysis and culturing. The flatworms were reared in a glass aquarium (21 cm x 15 cm; depth 18 cm) at room temperature (23—26 °C). The culture was aerated, and the flatworms were fed daily with Daphnia. DNA extraction, amplification, sequencing and phylogenetic analysis After starvation for three days, total DNA was extracted from three sexual individuals using the E.Z.N.A.™ Mol- lusc DNA Isolation Kit (Omega, Norcross, GA, USA). Four gene fragments, namely 18S ribosomal gene (18S rDNA), 28S ribosomal gene (28S rDNA), ribosomal internal transcribed spacer-1 (ITS-1), and cytochrome Table 1. Primer sequences used for PCR amplification. Zhu, Y. et al.: A new species of Dugesia from Southern China C oxidase subunit I (COI), were amplified by polymerase chain reaction (PCR). We used 2xTaq Plus Master Mix IT (Vazyme, China) to amplify 18S rDNA, 28S rDNA, ITS- 1, and COI. Primers used for amplification and the PCR protocol are listed in Table 1. Forward and reverse DNA strands were determined by Sanger sequencing either at BGI (Guangzhou, China) or TsingKe Biotech (Beijing, China). All new sequences have been uploaded to Gen- Bank, NCBI (Table 2). To determine the phylogenetic position of the new spe- cies within the genus Dugesia, we generated datasets con- sisting of marker gene sequences (18S rDNA, 28S rDNA, ITS-1, and COI; see Table 2) of the new species Dugesia ancoraria and available sequences of other Dugesia spe- cies from GenBank, NCBI, as well as two outgroup spe- cles, viz., Recurva postrema Sluys & Sola, 2013 (ITS-1 sequence not available in GenBank, NCBI), and Schmid- tea mediterranea (Benazzi, Bagufia, Ballester, Puccinelli & del Papa, 1975). Nuclear ribosomal markers were aligned with MAFFT (online version 7: MAFFT alignment and NJ / UPGMA phylogeny (cbrc.jp), Katoh et al. 2017) using the E-INS-1 algorithm, while mitochondrial coding gene COI was aligned by MASCE v2.03 (Ranwez et al. 2018). In order to check for the absence of stop codons, COI sequenc- es were translated into amino acids by ORFFINDER in NCBI, applying genetic code 9 before alignment, after which regions of ambiguous alignments were removed by Gblocks v0.91b (Talavera and Castresana 2007), using the same parameters as specified in Li et al. (2019). For ribosomal DNA, sequences were excluded by ClipKIT (Steenwyk et al. 2020) with kpic-gappy mode to keep parsimony-informative and constant sites and to remove highly gappy sites. Final length of the alignments was 693 base pairs (bp) for COI, 1,388 bp for 18S rDNA, 1,383 bp for 28S rDNA, and 591 bp for ITS-1. To ensure sequences’ validity, the substitution saturation test (Xia et al. 2003; Xia and Lemey 2009) in DAMBE6 software (Xia 2017) was used to evaluate the nucleotide substitu- tion saturation of four datasets, followed by the assembly of a multi-gene concatenated dataset (with the order 18S trDNA-28S rDNA-ITS-1—COlI), using SequenceMatrix v1.8 (Vaidya et al. 2011). Sequences that were shorter or not available in GenBank were completed with “-”. We used PartitionFinder2 (Lanfear et al. 2017) to evaluate the best-fit evolution models by estimating independent mod- els of molecular evolution for subsets of sites that were deemed to have evolved in similar ways. Gene Primer Sequence (5'-3') Reference PCR protocol COl COIEFMF Forward: GGW GGK TTT GGW AAW TG 94 °C 5 min, 35x (94 °C 50 s, COIRSong Reverse: GWG CAA CAA CAT ART AAG TAT CAT 50 °C 45 s, 72 °C 45 s); 72 °C 7 min ITS-1 ITSOF Forward: GTA GGT GAA CCT GCG GAA GG Baguna et al. 1999 O8:°C-S mie 3098 C30*s: ITSR Reverse: TGC GTT CAA ATT GTC AAT GAT C 46 °C 45 s, 72 °C 30s); 72 °C 7 min 18S rDNA 18S 1F — Forward: TAC CTG GTT GAT CCT GCC AGT AG Carranza et al. 1996 94 °C 5 min, 40x (95 °C 50 s, 18S 9R_ Reverse: GAT CCT TCC GCA GGT TCA CCT AC 50° C,45's,.72°°C 50's)7 72°C. 7 min 28S rDNA 28S 1F Forward: TAT CAG TAA GCG GAG GAA AAG _ Alvarez-Presas et al. 2008 94 °C 5 min, 40x (94 °C 50 s, 28S 6R Reverse: GGA ACC CCT TCT CCA CTT CAG T 52 CA5's: J2°C bO-shef 26-7 min zse.pensoft.net Zoosyst. Evol. 100 (1) 2024, 167-182 Table 2. GenBank accession numbers of sequences for species taxa used in the phylogenetic analyses. Species Col Recurva postrema KF308763 Schmidtea mediterranea JF837062 Dugesia adunca O0L505739 aenigma KC006968 aethiopica KY498845 afromontana KY498846 ancorarial* OR326966 ancoraria2* OR326967 ancoraria3* OR326968 arabica OL410620 arcadia KC006969 ariadnae JN376142 aurea MK712632 batuensis KF907819 D. D. D. D. £3 D. ‘Bf D. D. D. D. D. benazzii FJ646977, FJ646933 D. bengalensis - D. bifida KY498851 D. bijuga MH119630 D. circumcisa MZ147041 D. constrictiva MZ871766 D. corbata MK712637 D. cretica KC006974 D. damoae KF308768 D. deharvengi KF907820 D. effusa KF308780 D. elegans KC006985 D. etrusca MK712651 D. gemmulata OL632201 D. gibberosa KY498857 D. gonocephala FJ646941, FJ646986 D. granosa OL410634 D. hepta MK712639 D. ilvana FJ646989, FJ646944 D. improvisa KF308774 D. japonica AB618487 Dz liguriensis MK712645 D. majuscula MW533425 D. malickyi KF308750 D. naiadis KF308757 D. notogaea FJ646993, FJ646945 D. parasagitta KF308739 D. pustulata MH119631 D. ryukyuensis AB618488 D. sagitta KCO007006 D. semiglobosa MW525210 D. sicula KF308797 D. sigmoides KY498849 D. sinensis KP41592 D. subtentaculata MK712561 D. tubgalis OM281843 D. tumida OL505740 D. umbonata MT176641 D. vilafarrei MK712648 D. verrucula MZ147040 ITS-1 18S rDNA 28S rDNA - KF 308691 MG45274 AF047854 U31085 MG457267 OL527659 - KC007043 KF 308698 = KY498785 KY498822 KY498806 KY498786 KY498823 KY498807 OR296750 OR198141 OR225689 OR296751 OR198142 OR225690 OR296752 OR198143 OR225691 0OK587374 0K646637 0K491342 KC007047 KF 308694 0K491318 KC007049 0K646636 OK491317 MK713027 - MK712523 KF907816 0K646630 KF907823 MK713037 0K646628 MK712509 FJ646897 - - KY498791 KY498843 KY498813 - MH113806 - MZ146782 - - MZ869023 = = MK713029 - MK712525 KC007055 KF 308697 - KC007057 0K646619 0K491310 KF907817 - KF907817 KC007058 0K646618 0K491311 KC007063 KF 308695 0K491313 FJ646898 OK646617 0K491312 KY498803 KY498842 KY498819 FJ646901 DQ666002 DQ665965 KY498795 KY498833 KY498816 MK713035 OK646612 MK712512 FJ646903 OK646608 0K491334 KC007065 KF 308696 0K491304 FJ646906 D83382 DQ665966 FJ646907 OK646615 0K491353 MW533591 - 7 KC007069 OK646585 OK491294 OK587343 0K646581 OK491293 FJ646908 KJ599713 KJ599720 KC007073 OK646577 = OK587366 MH113807 OK491355 FJ646910 AF050433 DQ665968 KC007085 OK646567 0K491320 MW526992 : 7 FJ646915 KF 308693 DQ665969 KY498789 KY498827 KY498811 MK712995 AFO13155 MK712493 0OK587337 OK646555 OK491285 OL527709 7 = MT177211 MT177214 MT177210 MK712997 OM281820 MK712511 MZ146760 - = *this study. Phylogenetic trees were constructed by Maximum Likelihood (ML) and Bayesian Inference (BI) methods. For ML, standard bootstrap analysis with 1,000 replica- tions was performed by IQ-TREE v1.6.2 (Nguyen et al. 2015). BI was performed in MrBayes v3.2.6 (Ronquist et al. 2012) with two simultaneous runs of one cold and three hot chains. Each run for the concatenated dataset was performed for 1,000,000 generations, sampling ev- ery 1,000 generations. We checked the resulting param- eter file of each run in TRACER v1.7.1 (Rambaut et al. 2018) to ensure that the effective sample size (ESS) val- ues of each parameter were above 200. zse.pensoft.net 170 Mitochondrial DNA extraction, amplification, sequencing and phylogenetic analysis After having been starved for three days, the mitochondrial DNA of an asexual specimen of D. ancoraria was extract- ed (due to absence of sexual specimens at that time) using Animal mitochondrial DNA column extraction kit (PCR Grade; BioLebo Technology, Beijing China), followed by amplification of mitochondrial DNA using a REPLI-g Midi Kit (QIAGEN, Hilden, Germany). We compared the COI gene of the three sexual individuals with that of the asexual individual for mitochondrial extraction via mega- blast and, thus, found that they were perfectly identical. Paired-end sequencing was conducted on the Illumina His- eq 2500 platform (BGI, Guangzhou, China). The mitoge- nome sequences were assembled using MitoFinder (Allio et al. 2020). The functional regions of these genes were annotated and verified according to Huang et al. (2022). However, both MITOS (online version: MITOS Web Serv- er (uni-leipzig.de), Bernt et al. 2013) and tRNAscan-SE (Lowe and Chan 2016; ) failed to annotate trnT in the mi- togenome of D. ancoraria. Therefore, trnT was identified manually on the basis of homology comparisons with other species in the family Dugesiidae Ball, 1974. Seven species belonging to Dugesiidae, five to Geoplanidae Stimpson, 1857, and two species belonging to Planariidae Stimpson, 1857 were chosen for the construction of the mitochondrial tree (Table 3). As outgroup taxon we used the maricolan species Obrimoposthia wandeli (Hallez, 1906), which is a member of Uteriporidae Diesing, 1862 (Table 3). Multi- ple sequences alignments (MSA) for protein coding genes (PCGs) and ribosomal genes were carried out using MA- SCE v2.03, which translates the nucleotides to amino ac- ids before alignment. Hereafter, MSAs were trimmed by Gblocks v0.91b. Substitution saturation tests for each PCG were performed using DAMBEG®. Subsequently, Sequence- Matrix v1.8 was used to combine the alignments. The best- fit model for each PCG was selected by PartitionFinder2. ML analysis was conducted by IQ-TREE v1.6.2. For BI, MrBayes v3.2.6 was applied with 2,000,000 generations, sampling every 2,000 generations. Gene rearrangement scenarios including reversals, transpositions, reverse trans- positions, reversal and tandem-duplication-random-losses (TDRL) among all species were analysed using the soft- ware CREx (Bernt et al. 2007) on the CREx web server (http://www.pacosy. informatik.uni-leipzig.de/crex) based on common intervals. Zhu, Y. et al.: A new species of Dugesia from Southern China Histology For the morphological analysis, the flatworms were starved for three days prior to the preparation of histolog- ical sections according to procedures described by Song et al. (2020). Briefly, histological sections were made at intervals of 6 um and were stained with modified Cason’s Mallory-Heidenhain stain solution (see Yang et al. 2020). Hereafter, slides were mounted onto glass slides with neu- tral balsam (Yuanye Biotechnology, Shanghai, China) and sealed with a coverslip. Preparations registered with PLA codes were deposited in the Institute of Zoology, Chinese Academy of Sciences (IZCAS), while histological slides registered with RMNH.VER. codes will be deposited at Naturalis Biodiversity Center, Leiden, The Netherlands. Abbreviations used in the figures au: auricle; be: bursal canal; ca: common atrium; cb: copulatory bursa; cm: circular muscle; d: diaphragm; db: distal bulge; du: duct; e: eye; ed: ejaculatory duct; esv: extension seminal vesicle; go: gonopore; hb: hunch- back bump; ie: inner epitheltum; Im: longitudinal mus- cle; lod: left oviduct; Ivd: left vas deferens; ma: male atrium; od: oviduct; oe: outer epithelium; ov: ova- ry; pg: penis glands; ph: pharynx; pp: penis papilla; rod: right oviduct; rvd: right vas deferens; sg: shell glands; sv: seminal vesicle. Results Molecular phylogeny The phylogenetic trees obtained by BI and ML from the concatenated dataset (with the order 18S rDNA-28S trDNA-ITS-1—COI) showed similar topologies and sup- ported nodes (Fig. 2). In this tree, the terminals for D. ancoraria grouped together and did not group with any other species of Dugesia included in our molecular anal- ysis. It is noteworthy that the new species D. ancoraria formed a well-supported clade with D. notogaea Sluys & Kawakatsu, 1998 and D. bengalensis Kawakatsu, 1972 (Fig. 2, 100% bootstrap [bs] in ML; Suppl. material 1, 1.00 posterior probability [pp] in BI). Dugesia notogaea is an Australian species, while D. bengalensis inhabits a Table 3. Species and corresponding GenBank accession numbers of mitochondrial genomes used for mitochondrial analysis. Species GenBank Amaga expatria MT527191 Bipalium kewense NC045216 Crenobia alpina KP208776 Dugesia ancoraria* OR400685 Dugesia japonica NC016439 Dugesia constrictiva OK078614 Dugesia ryukyuensis AB618488 *this study. zse.pensoft.net Species GenBank Obama sp. NC026978 Obrimoposthia wandeli NCO50050 Parakontikia ventrolineata MT081960 Phagocata gracilis KP090060 Platydemus manokwari MT081580 Schmidtea mediterranea JX398125 Zoosyst. Evol. 100 (1) 2024, 167-182 Guangdong Province 171 113°56'21" | 22°31'55" pling locality. portion of India. As suggested by the high support value (99% bs; 1.00 pp), these three species are closely relat- ed and share a sister-group relationship with D. adunca Chen & Sluys, 2022 from Guangxi province. Then, these four species are supported as sister to a small clade com- posed of D. ryukyuensis Kawakatsu, 1976 and D. batuen- sis Ball, 1970 (96% bs; 1.00 pp). Mitochondrial genome The complete, circular mitochondrial genome of Dugesia ancoraria 1s 17,705 bp in length, and includes 12 of the 13 protein-coding genes of mitochondrial genomes (atp8 was not found), two ribosomal RNA (rRNA) genes, and 22 transfer RNA (tRNA) genes, which are arranged as follows: coxl-E-nad6-nad5-S2-D-R-cox3-I-Q-K-atp6-V- nad 1-W-cox2-P-nad3-A-nad2-M-H-F-rrnS-L 1-Y-G-S1- rrnL-L2-T-C-N-cob-nad4l-nad4. GC content is 23.77%, while a positive GC skew ([G-C]/[G+C] = 0.323) indicat- ed the occurrence of more Gs than Cs (Fig. 3). Both the ML and BI trees obtained from 12 protein coding genes (PCGs) have highly supported clades, ex- cepting one node with a bootstrap support lower than 70%. Since the topologies of the ML and BI trees are basically identical, we integrated them into one phy- logenetic tree. In the integrated tree, Crenobia alpina (Dana, 1766) and Phagocata gracilis (Haldeman, 1840) together form a clade that shares a sister-group relation- ship with a clade that is composed of two smaller clades, one comprising land planarians (Geoplanidae) and the other constituted by dugesiid freshwater planarians (Dugesiidae). The latter family forms a well-support- ed monophyletic group, in which D. ancoraria is sister to D. ryukyuensis with high support values (100% bs; 1.00 pp) (Fig. 4). zse.pensoft.net 172 95/1.00 85/1.00 53/0.77 66/1.00 *«/0.72 52/0.96 100/1.00 83/1.00 100/1.00, 96/1.00 100/1.00 53/0.97 100/1.00 61/0.96 */1.00 97/1.00 b8/7.00 99/1.00 97/1.00 99/1.00 51/0.66 98/0.96 100/1.00, 0.05 100/1.00 100/1.00 100/1.00 100/1.00 D. bifida D. afromontana 66/0.94 100/1.00 100/1.00) p. 100/1.00) D D. naiadis 0.05 100/1.00 54/0.94 92/1.00 D. gibberosa D. sigmoides Zhu, Y. et al.: A new species of Dugesia from Southern China D. ancoraria1 @ 1004.00} 1D. ancoraria2? @ 100/1.00 D. ancoraria3 @ D. notogaea D. bengalensis D. adunca 99/1.00 96/1.00 100/1.00 D. ryukyuensis D. batuensis Australasian and D. deharvengi D. gemmulata D. umbonata D. japonica Oriental D. sinensis D. semiglobosa D. circumcisa D. constrictiva D. verrucula D. tumida D. majuscula D. benazzii D. hepta D. gonocephala 94/1.00 D. etrusca D. ilvana D. liguriensis D. subtentaculata D. aurea coo D. corbata D. vilafarrei D. tubqalis 94/1.00 88/0.95 */* | O5/* Western ; Palearctic D. parasagitta D. sagitta D. aenigma D. arcadia D. malickyi aaian D. elegans 6o71.00. D. effusa D. damoae D. improvisa D. ariadnae D. cretica 81/1.00 100/1.00' D. bijuga | Cameroon D. pustulata Madagascan D. granosa OK Afrotropical and aethiopica arabica ¢ South-west Palearctic D. sicula Schmidtea mediterranea Recurva postrema | Outgroup Figure 2. Maximum likelihood phylogenetic tree topology inferred from the concatenated dataset (18S rDNA, ITS-1, 28S rDNA and COI). Numbers at nodes indicate support values (bootstrap/ posterior probability). Asterisks (*) indicate support values lower than 50% bs/0.50 pp, or posterior probability not applicable to this node, because of different topologies of trees generated by BI and ML methods. Scale bar: substitutions per site. The gene order of rRNAs, PCGs and tRNAs of D. an- coraria and other species used in our phylogenetic anal- ysis are shown in Fig. 4. Some tRNAs absent in previous publications, such as those from Platydemus manokwari de Beauchamp, 1963 and Parakontikia ventrolineata (Dendy, 1892), had been successfully annotated using MITOS (Bernt et al. 2013). Our results show that the or- ders of PCGs and ribosomal genes are conserved among species belonging to the triclad suborder Continenticola and are arranged as follows: cox] -nad6-nad5-cox3-atp6- nad 1-cox2-nad3-nad2-rrmS-rmL-cob-nad4l-nad4. In contrast, the order of tRNAs is highly variable. Within the cluster of Dugesiidae species, D. ancoraria shares an identical gene order with D. constrictiva. An analysis of gene order rearrangements with CREx suggests that only one transposition (trnN) occurred from D. ancoraria to D. ryukyuensis and also one transposition (trnE) from D. ryukyuensis to D. japonica. Except for a transposi- tion of trnE, a tandem-duplication-random-loss (TDRL) event is required for the transformation from D. japonica to Schmidtea mediterranea (Benazzi et al., 1975). With respect to the Geoplanidae, Amaga expatria Jones & Sterrer, 2005 and Obama sp. share the same gene order. Besides a transposition of trnF, a transposition of traM and trnH linkage is needed to go from C. alpina to Bipa- lium kewense Moseley, 1878. Two inverse transpositions, namely trnL2 and trnT, occurred from Bipalium kewense zse.pensoft.net to Obama sp. and Amaga expatria, and from Obama sp. to Platydemus manokwari, resulting in almost the same gene order shared by B. kewense and P. manokwari, with the only exception being the position of trnC (Fig. 4). Systematic account Order Tricladida Lang, 1884 Suborder Continenticola Carranza, Littlewood, Clough, Ruiz-Trillo, Baguiia & Riutort, 1998 Family Dugesiidae Ball, 1974 Genus Dugesia Girard, 1850 Dugesia ancoraria Zhu & Wang, sp. nov. https://zoobank.org/137337E1-0D52-4288-A 3A 3-7E8C80241A74 Material examined. Holotype: PLA-0251, a narrow artificial canal of Wenshan lake, Shenzhen city, Guang- dong Province, China, 22°31'55"N, 113°56'21"E, 10 May 2021, coll. MY Xia and co-workers, sagittal sections on 14 slides. Paratypes: PLA-0252, ibid., sagittal sections on 12 Slides; PLA-0253, ibid., transverse sections on 35 slides; RMNH. VER.21525.1, ibid., sagittal sections on 12 slides. Habitat. Specimens were collected from a narrow ar- tificial canal running from Wenshan lake (22°31'55"N, Zoosyst. Evol. 100 (1) 2024, 167-182 vON ©. c S 173 Figure 3. Arrangement of the mitochondrial genome of Dugesia ancoraria. Outer circle: annotation of genes, with protein-coding genes, ribosomal RNAs and transfer RNAs represented by cyan, orange, and red, respectively. Intermediate circle: sequencing cov- erage, with green colour indicating coverage greater than 95% average coverage. Inner circle, with the blue colour indicating GC content and the thin orange circle indicating 50% of GC content. The picture in the middle represents an individual of D. ancoraria. 113°56'21"E), which is located in Shenzhen city, Guang- dong Province, China (Fig. 1A). The animals were col- lected from Cladophora algae, as well as the stone wall of the canal, which had a water depth of 20-30 cm; wa- ter temperature was about 23 °C. Thirty specimens were collected, none of which was sexually mature. However, after six months of rearing under laboratory conditions, about 20 specimens eventually attained sexual maturity. Diagnosis. Dugesia ancoraria is characterised by the following characters: highly asymmetrical penis papilla, provided with a hunchback-like dorsal bump; vasa defer- entia opening symmetrically into the mid-lateral section of the more or less ellipsoidal seminal vesicle, which may give rise to a narrow dorsal extension; long and narrow duct connecting seminal vesicle with small diaphragm; ejaculatory duct with a subterminal opening through the ventral surface of the penis papilla; asymmetrical ovidu- cal openings, with the right oviduct opening into a section of the bursal canal that bends ventrally to communicate with the common atrium; the left oviduct opens into the bursal canal at the point where the latter meets the com- mon atrium. Etymology. The specific epithet is derived from Latin adjective ancorarius, of the anchor, and alludes to the pe- nis papilla, which has a hunchback-shape, reminiscent of an anchor, more or less. Description. Sexualized specimens measured 8.43— 9.11 mm in length and 1.13—1.18 mm in width (n = 4; zse.pensoft.net 174 Zhu, Y. et al.: A new species of Dugesia from Southern China Schmidtea mediterranea Dugesia japonica Ougesia constrictiva Dugesia ryukyuensis Dugesia ancoraria Parakontikia ventrolineata Platydemus manokwari Obama sp. H Amaga expatria ! Bipalium kewense Crenobia alpina Phagocata gracilis Obrimoposthia wandeli 1 SERED BE M/ HIF P A FIGE M| H/F au Dugesiidae oO fe) = = o) = Geoplanidae 8 a ‘al i Planarioidea rl A 5 w/ 3] Pp | 18! w | r ~) lela] ali x/ 4] 3/3 a) F Figure 4. Possible mechanisms of mitochondrial gene rearrangement in Continenticola estimated using CREx, with reference to phylogenetic relationships. On the left-hand side, phylogenetic tree obtained from Maximum likelihood and Bayesian analysis of the concatenated dataset for the protein-coding and rRNA genes within mitochondrial genomes; numbers at nodes indicate support values (pp/bs); Asterisks (*) indicate that bootstrap is not applicable to these nodes because of different topologies of trees generated by BI and ML methods. Pink lines connect species that share the same gene order. On the right-hand side, changes of gene order in mitochondrial genomes in several species of triclad; protein-coding genes in blue, tRNA genes in white, rRNA genes in grey; lines with different colours indicate transpositions of different genes; red lines indicate tandem duplication random loss (TDRL) events between species, while the orange colour indicates where TDRL events occurred. Fig. 5A, B). Head of low triangular shape with blunt aur- icles. At the level of the auricles there is a pair of black, bean-shaped eyes, located in pigment-free areas. The distance between the eyes and the lateral body margin is about 0.36—0.46 mm, while the size of the eyecups varies between 210-230 um. Each eyecup contains numerous retinal cells. The ground colour of the dorsal surface is brown, dot- ted with dark brown and white specks; ventral surface much paler than dorsal surface; the body margin is pale (Fig. 5A, B). The cylindrical pharynx is positioned at about 1/2 of the body and measures about 1/5 of the total body length; the mouth opening 1s situated at the posterior end of the pharyngeal pocket. The musculature of the pharynx con- sists of an outer, subepithelial layer of circular muscle, followed by a layer of longitudinal muscle, while the inner musculature 1s composed of a thick, subepithelial layer of circular muscle, followed by 2-3 layers of lon- gitudinal muscle. The gonopore is situated at about 1/5 of the length of the body, as measured from the posterior body margin (Fig. 5D). The globular ovaries are located at 1/6 — 1/7 of the dis- tance between the brain and the root of pharynx. From the ovaries, the nucleated oviducts run ventrally in a caudal direction and open separately and asymmetrically into the female reproductive apparatus. Posterior to the gonopore, the right oviduct turns antero-medially and then opens into a section of the bursal canal that bends ventrally to communicate with the common atrium. The left oviduct opens into the bursal canal at the point where the latter meets the common atrium. (Figs 6A, 9B). A large sac-shaped copulatory bursa is situated imme- diately behind the pharyngeal pocket and occupies the entire dorso-ventral space; it is lined with a layer of vac- uolated, nucleated cells (Figs 6B, 9B). From the bursa, the bursal canal runs in a caudal direction dorso-lateral- zse.pensoft.net ly to the male copulatory apparatus. At the level of the gonopore, the bursal canal curves rather sharply down- wards, thus giving rise to a more or less vertically ori- ented section that opens through the dorsal wall of the common atrium (Figs 6C, 9B). The bursal canal is lined by a nucleated, columnar glandular epithelium, which is underlain with a layer of longitudinal muscles, followed by 1—4 layers of circular muscles. Along the ventral coat of muscle, ectal rein- forcement is present in the form of a single layer of lon- gitudinal muscle running from about the opening of the canal into the common atrium to about 1/3 of the length of the bursal canal (Fig. 9B). Shell glands discharge their cyanophil secretion into the most ventral section of the vertically running portion of the bursal canal, with some glands even discharging into the common atrium (Figs 6B, C, 9B). The large, near-globular testicular follicles are sit- uated dorsally and extend posteriorly from a short distance behind the brain to well beyond the copula- tory apparatus. The male atrium comprises most of the dorso-ventral space of the body (Figs 6A, 9A, B). The large and oval-shaped penis bulb is composed of inter- mingled longitudinal and circular muscle fibres. The penis papilla has a more or less oblique, postero-ven- tral orientation or even a vertical orientation, and has a striking shape (Figs 6A, 9A). The papilla is markedly asymmetrical as a result of the course of ejaculatory duct, which opens to the exterior through the poste- ro-ventral wall of the penis papilla. Furthermore, near its root, the papilla has a dorsal bump, which gives it a hunchback appearance (Figs 6A, 9A). The degree of development of this dorsal bump differs between spec- imens. In the holotype it is highly developed (Fig. 6A), while in paratype PLA-0104 it is somewhat smaller, albeit still well-developed (Fig. 8A), but in paratype PLA-0102 the bump is practically absent (Fig. 7A). Zoosyst. Evol. 100 (1) 2024, 167-182 175 j | ' | ' | { 1 ' 7 \ ‘ { i i Figure 5. External morphology of Dugesia ancoraria. A. Living sexual animal in dorsal view; B. Living sexual animal in ventral view; C. Anterior end, dorsal view; D. Ventral view of rear end, showing pharynx, mouth and gonopore. Scale bars: 500 um. In addition, the asymmetrical appearance of the penis papilla is enhanced by the fact that the distal portion of the dorsal lip of the papilla gives rise to another bulge, which may be swollen or drawn-out to a greater or less- er extent. In paratype RMNH.VER.21525.1, it is a rath- er long-drawn bulge (Fig. 8A), whereas in the holotype and paratype PLA-0102 it is more rounded (Fig. 7A). The papilla is covered by a thin, nucleated epithelium, which is underlain with a well-developed, subepithelial layer of circular muscle, followed by a layer of longitu- dinal muscle at the ventral root. The vasa deferentia have expanded to form spermiducal vesicles that are packed with sperm. At the level of the pe- nis bulb, the ducts recurve, while decreasing in diameter, run postero-medially for some distance and, thereafter, re- curve anteriad before opening separately into the mid-lat- eral portion of the seminal vesicle (Figs 6A, 9A, B). The vesicle has a more or less ellipsoidal shape, while its dor- sal wall may form a narrow extension, which was present in all specimens examined, excepting paratype RMNH. VER.21525.1. The seminal vesicle is lined by a ciliated, nucleated epithelium. A long and narrow duct connects zse.pensoft.net 176 Zhu, Y. et al.: A new species of Dugesia from Southern China Figure 6. Dugesia ancoraria, holotype PLA-0101, sagittal sections, anterior to the right. A. Photomicrograph showing copulatory bursa, ejaculatory duct, penis papilla, and seminal vesicle; B. Photomicrograph showing copulatory bursa, bursal canal, and go- nopore; C. Photomicrograph showing copulatory bursa, and bursal canal. Scale bars: 100 um. the seminal vesicle with the small diaphragm, which com- municates with the ejaculatory duct. The diaphragm re- ceives the abundant secretion of erythrophil penial glands. From the diaphragm, the ejaculatory duct curves strongly postero-ventrally to open subterminally through the ven- tral epithelium of the penis papilla, thus giving rise to a highly asymmetrical papilla with a large dorsal lip and a small ventral lip (Figs 6A, 9A). Particularly the blunt tip of the dorsal lip of the penis papilla is penetrated by the numerous openings of orange-staining glands. zse.pensoft.net The male atrium is lined by a nucleated epithelium. The dorsal part of the male atrium is surrounded by a layer of circular muscle, followed by 1-2 layers of lon- gitudinal muscle, while a subepithelial layer of circular muscle, followed by a layer of longitudinal muscle con- stitutes the musculature on the ventral part of the atrium. The male atrium communicates with the common atrium via a broad opening. The common atrium is lined with a nucleated epithelium, which is underlain by 2-3 layers of circular muscle (Figs 6A, 9). Zoosyst. Evol. 100 (1) 2024, 167-182 ie) Figure 7. Dugesia ancoraria, paratype PLA-0102, sagittal sections. A. Photomicrograph showing ejaculatory duct, penis papilla, and seminal vesicle; B. Photomicrograph showing copulatory bursa, bursal canal, and gonopore. Scale bars: 100 um. Discussion Molecular phylogeny and biogeography In our phylogenetic tree (Fig. 2), the terminals for D. anco- raria grouped together, while they did not group with any other species of Dugesia included in our molecular analy- sis. Thus, the molecular analysis already suggested that D. ancoraria concerns a new species of Dugesia, which was supported by the morphological study (see below). In the phylogenetic trees obtained from the concatenat- ed dataset (Fig. 2), the sister-group relationship between D. ancoraria on the one hand and D. notogaea and D. bengalensis on the other hand is consistent and is support- ed by high bootstrap values, strongly suggesting that these three species form a monophyletic group. It 1s notewor- thy that D. ancoraria from southern China, shares only a distant relationship to other Dugesia species from China, including D. constrictiva, D. verrucula, D. majuscula, D. circumcisa, D. semiglobosa, D. umbonata, D. gemmu- lata and D. tumida, but is most closely related to D. noto- gaea from Australia and D. bengalensis from India. The clade comprising D. ancoraria, D. notogaea and D. ben- galensis shares a sister-group relationship with D. adunca, then is sister to a small clade comprising D. ryukyuensis from Japan and D. batuensis from peninsular Malaysia, and then further clusters with D. deharvengi, which is ba- sically in agreement with the results of Chen et al. (2022). However, according to Liu et al. (2022), D. deharvengi shares a sister-group relationship with D. notogaea first, and then clusters with a group comprising D. ryukyuensis and D. batuensis, which could be due to the absence in the species phylogeny of the COI sequence of D. bengalensis. Actually, in the phylogenetic tree generated solely on COI sequences, D. adunca shared a sister-group relationship with D. deharvengi, albeit with low support, while D. ryukyuensis and D. batuensis clustered with a clade con- sisting of D. majuscula, D. verrucula, D. constrictiva and D. tumida with very low support (data not shown here), instead of clustering with D. notogaea and D. bengalensis, zse.pensoft.net Figure 8. Dugesia ancoraria, paratype RMNH.VER.21525.1, sagittal sections. A. Photomicrograph showing ejaculatory duct, penis papilla, and seminal vesicle; B. Photomicrograph showing copulatory bursa, common atrium, and gonopore; C. Photomicro- graph showing copulatory bursa, and bursal canal. Scale bars: 100 um. zse.pensoft.net Zoosyst. Evol. 100 (1) 2024, 167-182 d Pg A sg sg hb 179 aa s Pee — lod ed d a — a. . _— SV = Figure 9. Dugesia ancoraria, Sagittal reconstruction of the copulatory apparatus of the holotype. A. Male copulatory apparatus; B. Female copulatory apparatus. Scale bars: 100 um. as in concatenation-based analyses. Furthermore, since different genes may exhibit highly variable rates of evo- lution, phylogenies inferred from single genes, with only limited evolutionary information, are often inconsistent. Altogether, our results suggested that concatenation-based analyses resulted in more resolved phylogenetic trees. With respect to the geographical distribution within China of other species of Dugesia, in relation to the dis- tribution of D. ancoraria, the following should be noted. Dugesia japonica has a wide distribution, as it has been reported from the eastern, southern and northern regions of China, while the remaining 11 species are found only in southern China. Among these species, D. semiglobosa and D. majuscula were documented in Hainan province, D. circumcisa and D. adunca in Guangxi province, with these two provinces being relatively close to the locali- ty of D. ancoraria in Guangdong. Dugesia tumida and D. sinensis occur also in Guangdong Province, while they share only a very distant relationship with the new spe- cies D. ancoraria. Other species, like D. umbonata, were found in Jiangsu province and D. gemmulata in Guizhou. Although these two species are geographically far distant from each other, they share a close relationship. The close relationship between Chinese D. ancoraria and Australian D. notogaea, with the latter being the sis- ter-species of Malaysian D. bengalensis, is interesting from a historical biogeographic perspective. This pattern of relationships basically agrees with that uncovered by Sola et al. (2022), in which D. notogaea also fell into an Asian clade, including specimens from China, Malaysia, Thailand, Japan, and Indonesia. These authors surmised that this pointed to anthropochore dispersal of Dugesia from Asia to Australia, as they considered Wallace’s Line to indicate an unsurmountable biological barrier for natural arrival of the genus in Australia (see also Ali and Heaney 2022). Excluding unlikely jump dispersal, earlier hypoth- eses that Dugesia naturally spread from Southeast Asia to Australia (Sluys et al. 1998) foundered on the paleogeo- graphical evolution of the Indo-Australian archipelago. According to paleogeographical reconstructions, the river systems of Asia on the one hand and those of Australia/New Guinea on the other hand have never been in contact, not even during the Pleistocene when the sea level was much lower (Sluys et al. 2007). Nevertheless, the distribution of Dugesia 1s repeated by the equally remarkable distribution in Asia and Australasia of portions of the camaenid land snails (Scott 1997; Cuezzo 2003), while the earliest nauti- loids from Australia share major characteristics with Asiat- ic species (Stait and Burrett 1987). Evidently, similarity in these distributional patterns does not imply that they orig- inated during the same period in geological history. Future studies on Dugesia from China and the Indo-Australian archipelago would be very interesting, as these may shed light on the biogeographic history of the region. zse.pensoft.net 180 Annotation of trnT In our mitogenome analysis, trnT was the only tRNA that could not be automatically annotated by MITOS. However, through translating nucleotides of 12 PCGs to amino acid with Expacy (http://web.expasy.org/ translate/), 63 sites of threonine, which is coded by ACN, were found. These results thus support the existence of trnT, which is required for the reading of the triplet of the genetic code (ACN). Therefore, we annotated trnT manually, based on homology comparisons with other species in the family Dugesiidae. Specifically, we aligned the complete mitogenome of D. ancoraria with three species belonging to family Dugesiidae, namely D. japonica, D. ryukyuensis and D. constrictiva and found a homologous sequence (60 bp) among these five species, which 1s particularly conserved at 5’ end (AGAA) and 3’ end (TTCTT). In addition, the position of trnT of all reported species belonging to Dugesiidae 1s very conserved and 1s situated between trnL2 and trnC. Interestingly, the putative trnT in D. ancoraria is also located between trnL2 and trnC, providing another line of evidence in support of the annotation. Furthermore, we predicted the secondary structure of trnT manually and presented this through RNAalifold WebServer (http:// rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNA fold. cgi) and were surprised to find that the predicted result is not a typical cloverleaf structure with an absence of the DHU stem. By comparing the free energy between the two predicted structures, we found that the free energy with DHU stem (-1.20 kcal/mol) is higher than the one without DHU stem (-4.50 kcal/mol). The absence of the DHU stem in trnT also occurred in several other species of Tricladida, such as Dugesia japonica, D. ryukyuensis, Crenobia alpina, Obama sp. and Schmidtea mediterranea. (Sakai and Sakaizumi 2012; Sola et al. 2015; Ross et al. 2016). To the best of our knowledge, P. gracilis 1s the only Tricladida species reported to possess trnT with a DHU loop. Therefore, absence of the DHU stem in trnT could be a common phenomenon among species of the Tricladida. Mitochondrial gene order of suborder Continenticola In addition, some common features can be discovered in the mitochondrial gene order of the investigated species. Among the five species of Geoplanidae, locations of trnT are variable, in that transpositions of trnT occur in each of two adjacent species in the mitogenome tree, from B. kewense to P. ventrolineata. Besides, trnF is located at 3’ downstream of nad4, with the only exception being Crenobia alpina. The gene rearrangement of the maricolan Obrimoposthia wandeli differs considerably from species belonging to the suborder Continenticola. Therefore, transformation of gene order in O. wandeli to species of the Continenticola may require multiple rearrangements, including reversals, trans- positions, and TDRL. Similarly, several TDRL events are zse.pensoft.net Zhu, Y. et al.: A new species of Dugesia from Southern China required to go from the Geoplanoidea gene order to those of the Dugesiidae species. It is also noteworthy that two species (D. ancoraria and D. constrictiva) with identical mitochondrial gene order occur in two separate clades. Since gene rearrangements appear to be rare events that may not arise independently in separate lineages (Boore 1999), it is likely that the ancestor of Dugesia (indicated by a triangle in Fig. 4) may have had a pattern identical to that of D. ancoraria and D. constrictiva, implying that in the course of evolutionary history only a transposition of trnE occurred in D. japonica as well as transposition of trnN in D. ryukyuensis. However, the small number of species for which mitogenomic datasets are currently available, make it presently impossible to test this hypothesis. Morphological comparisons A highly asymmetrical penis papilla with both a proxi- mal as well as distal dorsal bumps is the most charac- teristic feature of Dugesia ancoraria. Similar bumps are known only from D. gibberosa Stocchino & Sluys, 2017. However, in D. gibberosa the penis papilla has a differ- ent, ventro-caudal orientation, while its ejaculatory duct Opens terminally at the tip of the papilla, in contrast to the subterminal opening in D. ancoraria. Moreover, in D. gibberosa the bursal canal is surrounded by a very thick layer of circular muscle, while its ectal reinforce- ment extends more than halfway along the bursal canal. In contrast, the bursal canal musculature in D. ancoraria is thinner and the ectal reinforcement weakly developed. Furthermore, D. ancoraria and D. gibberosa are far re- moved from each other in the phylogenetic tree (Fig. 2), thus corroborating their separate taxonomic status. Our molecular analyses, particularly the concatenated dataset, showed that D. ancoraria shares a sister-group relationship with Australian D. notogaea and Malaysian D. bengalensis. Morphologically, all three species have an asymmetrical penis papilla, with the dorsal lip being thicker than ventral lip, and a duct between seminal ves- icle and ejaculatory duct. In addition, D. ancoraria and D. notogaea share the condition in which the oviducts Open asymmetrically into the bursal canal. However, there are also clear differences between these three spe- cies. For example, in D. bengalensis and D. ancoraria, the ejaculatory duct has a subterminal opening at the tip of the penis papilla, whereas D. notogaea exhibits a ter- minal opening. In D. bengalensis and D. notogaea, the vasa deferentia open through the postero-lateral roof of the seminal vesicle, whereas in D. ancoraria the ducts open into the mid-lateral portion of the vesicle. Although D. gibberosa is the only other species with two clear dorsal bumps on the penis papilla, there are a number of Dugesia species that deserve some compari- son with D. ancoraria, viz., D. astrocheta Marcus, 1958, D. austroasiatica Kawakatsu, 1985, and D. tamilensis Kawakatsu, 1980. Dugesia astrocheta has a clear, prox- imal hunchback bump on its very asymmetrical penis Zoosyst. Evol. 100 (1) 2024, 167-182 papilla, while there is also some indication of a distal bump or bulge (cf. Sluys 2007, fig. 4A). But even when there is indeed such a distal bulge, the species differs in other details from D. ancoraria. For example, in D. an- coraria there is a relatively long duct interposed between the seminal vesicle and the diaphragm, whereas this duct is virtually absent in D. astrocheta. In D. austroasiati- ca there seems to be a rather flexible distal bulge on the dorsal lip of the penis papilla that receives the secretion of glands (cf. Kawakatsu et al. 1986, fig. 3). Apart from the absence of the hunchback, proximal penial bump in D. austroasiatica, there are also other differences which signal that it differs from D. ancoraria. For example, in the latter the oviducts open asymmetrically into the bursal canal, whereas D. austroasiatica has symmetrical ovidu- cal openings. The penis papilla of D. tamilensis resem- bles that of D. ancoraria in that it is highly asymmetrical, with the ejaculatory duct opening also at the postero-ven- tral wall of the papilla, while its dorsal lip is provided also with a distal bulge. However, in this species the oviducts also open symmetrically into the bursal canal, in contrast to the asymmetrical oviducal openings in D. ancoraria. Acknowledgements This study was supported by grants from Cultivation of Guangdong College Students’ Scientific and Tech- nological Innovation (“Climbing Program” Special Funds; grant no. pdjh2023b0449), China Undergraduate Training Program for Innovation and Entrepreneurship (grant no. S202210590072) and the Shenzhen Univer- sity Innovation Development Fund (grant no. 2021258), as well as grants from the Scientific and Technical In- novation Council of Shenzhen Government (grant nos. jcyj20210324093412035 and kcxfz20201221173404012) and Special Program of Key Sectors in Guangdong Uni- versities (grant no. 2022ZDZX4040). We are grateful to Meng-yu Xia for assistance with sample collection. References Ali JR, Heaney LR (2022) Alfred R. Wallace’s enduring influence on biogeographical studies of the Indo-Australian archipelago. Journal of Biogeography 50(1): 32-40. https://doi.org/10.1111/jbi.14470 Allio R, Schomaker-Bastos A, Romiguier J, Prosdocimi F, Nabholz B, Delsuc F (2020) MitoFinder: Efficient automated large-scale extraction of mitogenomic data in target enrichment phylogenom- ics. Molecular Ecology Resources 20(4): 892-905. https://doi. org/10.1111/1755-0998.13160 Bernt M, Merkle D, Ramsch K, Fritzsch G, Perseke M, Bernhard D, Schlegel M, Stadler PF, Middendorf M (2007) CREx. inferring ge- nomic rearrangements based on common intervals. Bioinformatics (Oxford, England) 23(21): 2957-2958. https://doi.org/10.1093/bio- informatics/btm468 Bernt M, Donath A, Juhling F, Externbrink F, Florentz C, Fritzsch G, Putz J, Middendorf M, Stadler PF (2013) MITOS: Improved de 181 novo metazoan mitochondrial genome annotation. Molecular Phy- logenetics and Evolution 69(2): 313-319. https://doi.org/10.1016/j. ympev.2012.08.023 Boore JL (1999) Animal mitochondrial genomes. Nucleic Acids Re- search 27(8): 1767-1780. https://doi.org/10.1093/nar/27.8.1767 Chen GW, Wang L, Wu F, Sun XJ, Dong ZM, Sluys R, Yu F, Yu-wen YQ, Liu DZ (2022) Two new species of Dugesia (Platyhelminthes, Tricladida, Dugesiidae) from the subtropical monsoon region in Southern China, with a discussion on reproductive modalities. BMC Zoology 7(25): 1-20. https://do1.org/10.1186/s40850-022-00127-8 Cuezzo MG (2003) Phylogenetic analysis of the Camaenidae (Mol- lusca: Stylommatophora) with special emphasis on the American taxa. Zoological Journal of the Linnean Society 138(4): 449-476. https://doi.org/10.1046/j.1096-3642.2003.00061.x Huang JJ, Liao YY, Li WX, Li JY, Wang AT, Zhang Y (2022) The com- plete mitochondrial genome of a marine triclad Miroplana shen- zhensis (Platyhelminthes, Tricladida, Maricola). Mitochondrial DNA. Part B, Resources 7(6): 927-929. https://doi.org/10.1080/23 802359 .2022.2079102 Katoh K, Rozewicki J, Yamada KD (2017) Mafft online service: Mul- tiple sequence alignment, interactive sequence choice and visual- ization. Briefings in Bioinformatics 20(4): 1160-1166. https://doi. org/10.1093/bib/bbx108 Kawakatsu M, Takai M, Oki I, Tamura S, Aoyagi M (1986) A note on an introduced species of freshwater planarian, Dugesia austroasiatica Kawakatsu, 1985, collected from culture ponds of Tirapia mossam- bica in Saga City, Kyasht, Japan (Turbellaria, Tricladida, Paludico- la). Bulletin of Fuji Women’s College no. 24, ser. II: 87-94. Lanfear R, Frandsen PB, Wright AM, Senfeld T, Calcott B (2017) Par- titionFinder 2: New methods for selecting partitioned models of evolution for molecular and morphological phylogenetic analyses. Molecular Biology and Evolution 34(3): 772-773. https://doi. org/10.1093/molbev/msw260 Li WX, Sluys R, Vila-Farré M, Chen JJ, Yang Y, Li SF, Wang AT (2019) A new continent in the geographic distribution of the genus Oregoniplana (Platyhelminthes: Tricladida: Maricola), its rediscovery in South Africa and its molecular phylogenetic position. Zoological Journal of the Linnean Society 187(1): 82-99. https://do1. org/10.1093/zoolinnean/zlz013 Liu Y, Song XY, Sun ZY, Li WX, Sluys R, Li SF, Wang AT (2022) Addition to the known diversity of Chinese freshwater planarians: Integrative description of a new species of Dugesia Girard, 1850 (Platyhelminthes, Tricladida, Dugesiidae). Zoosystematics and Evo- lution 98(2): 233-243. https://doi.org/10.3897/zse.98.83184 Lowe TM, Chan PP (2016) tRNAscan-SE On-line: Search and Con- textual Analysis of Transfer RNA Genes. Nucleic Acids Research 44(W1): W54—W57. https://doi.org/10.1093/nar/gkw413 Nguyen LT, Schmidt HA, Haeseler A, Minh BQ (2015) IQ-TREE: A fast and effective stochastic algorithm for estimating maxi- mum-likelihood phylogenies. Molecular Biology and Evolution 32(1): 268-274. https://doi.org/10.1093/molbev/msu300 Rambaut A, Drummond AJ, Xie D, Baele G, Suchard MA (2018) Posterior summarisation in Bayesian phylogenetics using Tracer 1.7. Systematic Biology 67(5): 901-904. https://doi.org/10.1093/sysbio/syy032 Ranwez V, Douzery EJP, Cambon C, Chantret N, Delsuc F (2018) MACSE v2: Toolkit for the alignment of coding sequences account- ing for frameshifts and stop codons. Molecular Biology and Evo- lution 35(10): 2582-2584. https://doi.org/10.1093/molbev/msy 159 zse.pensoft.net 182 Ronquist F, Teslenko M, Van der Mark P, Ayres DL, Darling A, Hohna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61(3): 539-542. https://doi.org/10.1093/sysbio/sys029 Rosa MT, Oliveira DS, Loreto ELS (2017) Characterization of the first mitochondrial genome of a catenulid flatworm: Stenostomum leu- cops (Platyhelminthes). Journal of Zoological Systematics and Evo- lutionary Research 55(2): 98-105. https://doi.org/10.1111/jzs.12164 Ross E, Blair D, Guerrero-Hernandez C, Alvarado AS (2016) Com- parative and transcriptome analyses uncover key aspects of coding and long noncoding RNAs in flatworm mitochondrial genomes. G3 Genes|Genomes|Genetics 6(5): 1191-1200. https://doi.org/10.1534/ 23.116.028175 Sakai M, Sakaizumi M (2012) The complete mitochondrial genome of Dugesia japonica (Platyhelminthes; Order Tricladida). Zoological Science 29(10): 672-680. https://doi.org/10.2108/zsj.29.672 Scott B (1997) Biogeography of the Helicoidea (Mollusca: Gastro- poda: Pulmonata): land snails with a Pangean distribution. Journal of Biogeography 24(4): 399-407. https://doi.org/10.1111/j.1365- 2699.1997.00106.x Sluys R (2007) Annotations on freshwater planarians (Platyhelmin- thes Tricladida Dugesiidae) from the Afrotropical Region. Tropical Zoology 20(2): 229-257. Sluys R, Riutort M (2018) Planarian diversity and phylogeny. In: Rink JC (Ed.) Planarian Regeneration: Methods and Proto- cols. Methods in Molecular Biology, vol 1774, Humana Press, Springer Science+Business Media, New York, 1-56. https://doi. org/10.1007/978-1-4939-7802-1_ 1 Sluys R, Kawakatsu M, Winsor L (1998) The genus Dugesia in Aus- tralia, with its phylogenetic analysis and historical biogeography (Platyhelminthes, Tricladida, Dugestidae). Zoologica Scripta 27(4): 273-289. https://doi.org/10.1111/j. 1463-6409. 1998 .tb00461.x Sluys R, Grant LJ, Blair D (2007) Freshwater planarians from arte- sian springs in Queensland, Australia (Platyhelminthes, Tricladi- da, Paludicola). Contributions to Zoology 76(1): 9-19. https://doi. org/10.1163/18759866-0760 1002 Sola E, Alvarez-Presas M, Frias-Lopez C, Littlewood DTJ, Rozas J, Riutort M (2015) Evolutionary analysis of mitogenomes from para- sitic and free-living flatworms. PLoS ONE 10(3): 1-20. https://doi. org/10.1371/journal.pone.0120081 Sola E, Leria L, Stocchino GA, Bagherzadeh R, Balke M, Daniels SR, Harrath AH, Khang TF, Krailas D, Kumar B, Li MH, Maghsoudlou A, Matsumoto M, Naser N, Oben B, Segev O, Thielicke M, Tong X, Zivanovic G, Riutort M (2022) Three dispersal routes out of Africa: The puzzling biogeographical history in freshwater planarians. Journal of Biogeography 49(7): 1219-1233. https://doi.org/10.1111/jb1.14371 Song XY, Li WX, Sluys R, Huang SX, Li SF, Wang AT (2020) A new species of Dugesia (Platyhelminthes, Tricladida, Dugesiidae) from China, with an account on the histochemical structure of its major nervous system. Zoosystematics and Evolution 96(2): 431-447. https://do1.org/10.3897/zse.96.52484 Stait B, Burrett C (1987) Biogeography of Australian and Southeast Asian Ordovician nautiloids. In: McKenzie GD (Ed.) Gondwana zse.pensoft.net Zhu, Y. et al.: A new species of Dugesia from Southern China Six: Stratigraphy, Sedimentology and Paleontology. Washington DC: American Geophysical Union, 21-28. https://doi.org/10.1029/ GM041p0021 Steenwyk JL, Buida TJ III, Li Y, Shen XX, Rokas A (2020) ClipKIT: A multiple sequence alignment trimming software for accurate phylogenomic inference. PLoS Biology 18(12): 1-17. https://doi. org/10.1371/journal.pbio.3001007 Talavera G, Castresana J (2007) Improvement of phylogenies after re- moving divergent and ambiguously aligned blocks from protein se- quence alignments. Systematic Biology 56(4): 564-577. https://dol1. org/10.1080/10635150701472164 Vaidya G, Lohman DJ, Meier R (2011) SequenceMatrix: Concatenation software for the fast assembly of multi-gene datasets with character set and codon information. Cladistics 27(2): 171-180. https://doi. org/10.1111/j.1096-0031.2010.00329.x Xia X (2017) DAMBE¢®: New tools for microbial genomics, phyloge- netics, and molecular evolution. The Journal of Heredity 108(4): 431-437. https://doi.org/10.1093/jhered/esx033 Xia X, Lemey P (2009) Assessing substitution saturation with DAM- BE. In: Lemey P, Salemi M, Vandamme A (Eds) The phylogenetic handbook: a practical approach to phylogenetic analysis and hy- pothesis testing. Cambridge University Press, Cambridge, 615-630. https://do1.org/10.1017/CBO9780511819049.022 Xia X, Zheng X, Salemi M, Chen L, Wang Y (2003) An index of substitution saturation and its application. Molecular Phyloge- netics and Evolution 26(1): 1-7. https://doi.org/10.1016/S1055- 7903(02)00326-3 Yang Y, Li JY, Sluys R, Li WX, Li SE Wang AT (2020) Unique mating behavior, and reproductive biology of a simultaneous hermaphro- ditic marine flatworm (Platyhelminthes, Tricladida, Maricola). In- vertebrate Biology 139(1): 1-10. https://doi.org/10.1111/ivb.12282 Supplementary material | Bayesian inference phylogenetic tree topology Authors: Ying Zhu, JiaJie Huang, Ronald Sluys, Yi Liu, Ting Sun, An-Tai Wang, Yu Zhang Data type: docx Explanation note: Bayesian inference phylogenetic tree topology inferred from the concatenated dataset (18S rDNA, IT-1, 28S DNA and COI). Numbers at nodes indicate support values (posterior probability). Scare bar: substitutions per site. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zse.100.114016.suppl1