Zoosyst. Evol. 100 (4) 2024, 1191-1200 | DOI 10.3897/zse.100.131019 Ate BERLIN Description of a new freshwater mussel species of Pletholophus, Simpson, 1900 (Bivalvia, Unionidae) from Guangdong, China Yu-Ting Dai’*, Zhong-Guang Chen", Cheng-Lin Hu’, Shan Ouyang!, Xiao-Chen Huang’, Xiao-Ping Wu! 1 School of Life Sciences, Nanchang University, Nanchang 330031, China https://zoobank. org/C9F1030C-0564-4960-9378-38D9IS4SACE05 Corresponding authors: Xiao-Chen Huang (xiaochenhuang@hotmail.com, xchuang@ncu.edu.cn); Xiao-Ping Wu (xpwu@ncu.edu.cn) Academic editor: Matthias Glaubrecht # Received 2 July 2024 # Accepted 5 August 2024 Published 28 August 2024 Abstract The Pearl River Basin, China’s second-largest freshwater basin, hosts a significant diversity of species and a highly endemic fresh- water mussel fauna. In this study, a new species from the Liuxi River in Guangzhou, Guangdong, China, Pletholophus guang- zhouensis sp. nov., is described based on morphological diagnostic features and molecular phylogenetics. The glochidia shells of the new species are subtriangular, medium-sized, and have a styliform hook on the ventral angle of each valve. Phylogenetic analyses based on the CO/ and 28S rRNA gene fragments indicated that Pletholophus guangzhouensis sp. nov. 1s the sister to Pletholophus tenuis + Pletholophus reinianus. The pairwise uncorrected COI p-distance analysis demonstrated genetic distances ranging from 5.27% (between P. guangzhouensis sp. nov. and P. tenuis) to 11.06% (between P. guangzhouensis sp. nov. and P. honglinhensis). Our findings suggest a significant underestimation of the diversity of freshwater mussel species in Guangdong. Further field col- lections and systematic studies are necessary to fully explore the biodiversity of this region. Furthermore, integrative classification methods and genetic research are essential for informing the development of effective conservation strategies. Key Words conservation, glochidia, molecular systematics, morphological characters, taxonomy Introduction Freshwater bivalves (Bivalvia, Unionidae) are well-known for providing important ecosystem functions and services, including nutrient cycling, habitat structure, substrate and food web modification, and serving as environmental monitors (Vaughn 2018). Furthermore, their stable bioge- ography, characterized by low dispersal and restriction to freshwater habitats, makes them invaluable for elucidat- ing past geological and hydrological events (Zieritz et al. 2021). The life cycle of Unionidae is unique, involving parasitic larvae (glochidia) that must attach to vertebrate hosts, primarily freshwater fish, before becoming sessile * These authors contributed equally to this paper. adults. This distinctive life cycle has likely contributed sig- nificantly to the rapid diversification of this group (Barn- hart et al. 2008). However, freshwater mussels represent one of the most threatened faunal groups on a global scale (Bohm et al. 2021), as they are highly impacted by human activities, climate change, and water loss (Aldridge et al. 2022). In recent decades, freshwater mussels have expe- rienced a significant decline, with both species loss and reductions in abundance (Karatayev et al. 2012; Vaughn 2018). This highlights the importance of further research into their diversity, distribution, and evolution. China exhibits both high species diversity and a high- ly endemic mussel fauna (Zieritz et al. 2018; Liu et al. Copyright Dai, Y.-T. 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, distribution, and reproduction in any medium, provided the original author and source are credited. 1192 2022). Nevertheless, field investigations and studies of freshwater bivalves in China exhibit a geographic bias, with the majority of research concentrated in the middle and lower reaches of the Yangtze River (Liu et al. 2022; Wu et al. 2022). In recent years, an expanding body of research has revealed that the Pearl River Basin, China’s second-largest freshwater basin, hosts numerous previ- ously unidentified and distinct species (Dai et al. 2023). For example, several new species have recently been discovered in the Guangxi Zhuang Autonomous Region, situated along the Pearl River Basin, including Postolata guangxiensis Dai, Huang, Guo & Wu, 2023; Pseudocu- neopsis yangshuoensis Wu & Liu, 2023; P. wuana Liu & Wu, 2023; and P. Jongjiangensis Liu & Wu, 2024 (Dai et al. 2023; Dai et al. 2024; Liu et al. 2024). This observa- tion prompted the hypothesis that Guangdong Province, another significant region through which the Pearl River Basin flows, may also be rich in unique species. Howev- er, there is a paucity of mussel diversity surveys and stud- ies in Guangdong, particularly over the past decade (Liu and Duan 1991; Hu 2005; He and Zhuang 2013; Zhang et al. 2013; Dong et al. 2017). Pletholophus Simpson, 1900, belongs to the Union- inae Rafinesque, 1820, in the family Unionidae Rafin- esque, 1820. This genus was established by Simpson (1900) as a subgenus of Cristaria Schumacher, 1817, with Cristaria (Pletholophus) discoidea (Lea, 1834) (by original designation) designated as the type species. Dang et al. (1980) elevated Pletholophus to the generic level and included three species: Pletholophus swinhoei (Adams, 1866), Pletholophus inangulatus (Haas, 1910a), and Pletholophus discoideus (Lea, 1834). All of these are considered synonyms of Cristaria tenuis (He & Zhuang, 2013). Based on the COJ + 28S rRNA phylogenies, the species Cristaria tenuis (Griffith & Pidgeon, 1833) has been recently reassigned to Pletholophus and separated from Cristaria (Lopes-Lima et al. 2017). Lopes-Lima et al. (2020) considered Pletholophus reinianus (Martens, 1875) to be a valid species based on the analysis of CO/ and 28S rRNA gene fragments. Recently, Bogan et al. (2023) summarized the taxonomy and diversity of Ano- dontini in Vietnam, identifying a new species, Plethol- ophus honglinhensis Bogan, Do, Froufe & Lopes-Lima, 2023, based on molecular and morphological evidence. Currently, the genus is recognized to comprise three valid species: Pletholophus tenuis (Griffith & Pidgeon, 1833), Pletholophus reinianus (Martens, 1875), and Plethol- ophus honglinhensis Bogan, Do, Froufe & Lopes-Lima, 2023. Pletholophus tenuis has been recorded as wide- spread in southeastern Asia, ranging from the Yangtze River south to Cambodia (Bogan et al. 2023). In contrast, the distribution of P. reinianus and P. honglinhensis 1s more restricted, with P. reinianus endemic to southern Japan and P. honglinhensis found exclusively in a coastal basin south of Hanoi, Vietnam (Lopes-Lima et al. 2020; Bogan et al. 2023). In this study, we discovered a distinct species of fresh- water mussel in the Liuxi River, Guangzhou, China. After zse.pensoft.net Dai, Y.-T. et al.: A new species of Pletho/ophus examining the shell morphology of this unique species, as well as referring to the literature (e.g., Heude 1875, 1877a, 1877b, 1878, 1879, 1880a, 1880b, 1881, 1883, 1885; Simpson 1900, 1914; Haas 1969; Brandt 1974; Liu et al. 1979; He and Zhuang 2013) and MUSSELp online database (see http://mussel-project.uwsp.edu), we were unable to match it to any of the recorded species. Sub- sequently, a new species of Pletholophus was described based on a combination of morphological characters and the COI + 28S rRNA gene phylogenies. We provide mor- phological descriptions, glochidia descriptions, localities, and photographs for this new species. Materials and methods Specimen sampling, identification, and deposition In January 2021, a total of 10 specimens were collect- ed from the Liuxi River (23°32'02"N, 113°35'03"E) in Guangzhou City, Guangdong, China (Figs 1, 2). A dig- ital vernier caliper with an accuracy of + 0.01 mm was used to measure the length, height, and width of the type series of the new taxa. Live specimens were euthanized with 100% ethanol and then separated into soft tissue and shells. The adductor muscle was used for subse- quent DNA extraction. The remaining soft parts were preserved at —80 °C. All voucher specimens were de- posited in the Museum of Biology, Nanchang University (NCUMB), China. Scanning electron microscopy of glochidia The glochidial mass was stored in 96% ethanol and sub- sequently washed with deionized water. It was then trans- ferred into a 5% NaOH solution and allowed to rest for approximately two hours to remove any residual tissue. Following the deionized water wash, the glochidia were observed under an optical microscope to ascertain their cleanliness and the integrity of their shells. The sample preparation process was completed using anhydrous eth- anol for storage purposes. Prior to scanning electron mi- croscopy, the samples were dried in a clean environment for a minimum of eight hours, after which their surfaces were sprayed with gold. Subsequently, the samples were subjected to examination via scanning electron micros- copy (SEM) (Quanta 200FEG03040702, USA) (Shu and Wu 2005b; Sayenko et al. 2023). Molecular phylogenetic analyses The Qiagen Genomic DNA kit (Qiagen, Hilden, Germa- ny) was employed to extract total genomic DNA from the excised tissue following the instructions provided by the manufacturer. The quality and concentration of the DNA L193 Zoosyst. Evol. 100 (4) 2024, 1191-1200 Figure 1. Distribution map of Pletholophus guangzhouensis sp. nov. were checked using 1% agarose gel electrophoresis and NanoDrop 2000 (Thermo Scientific, USA). We amplified and sequenced fragments from the mitochondrial cyto- chrome c oxidase subunit-I gene (COI) (LCO22me2 + HCO700dy2) (Walker et al. 2007) and the nuclear 28S ribosomal RNA gene (28S) (D23F + D4RB) (Park and Foighil 2000). The polymerase chain reaction (PCR) was conducted using a 25 uL mixture of 2 x Taq Plus Mas- ter MixII (Vazyme, China) (12.5 wL), ddH,O (9.5 pL), 10 uM primers (1 uL each), and genomic DNA (1 uL, about 100 ng/uL). Thermal cycling was started at 98 °C for 10s, followed by 35 cycles of 94 °C for 1 min, anneal- ing at 50 °C for 1 min, extension at 72 °C for 1 min, and then a final extension at 72 °C for 7 min. The PCR prod- ucts were sequenced commercially by Sangon Biotech (Shanghai, China). The newly obtained sequences have been deposited in GenBank, and their accession numbers are provided in Table 1. Two datasets were constructed in this study: (1) the COI dataset (11 sequences; 600 bp); and (ii) the COJ + 28S rRNA dataset (67 sequences; 1,009 bp) (Table 1). All PCGs were codon-aligned by MUSCLE ver. 3.6 (https://www.drive5.com/muscle/; Edgar 2004) imple- mented in MEGA ver. 10.1.6 (http://www.megasoft- ware.net; Kumar et al. 2018), whereas 28S rRNA were aligned in MAFFT ver. 7 (https://mafft.cbrc.jp/align- ment/software/; Katoh et al. 2019) using the Q-INS-i algorithm. We used Gblocks ver. 0.91b (http://gensoft. pasteur. fr/docs/gblocks/0.91b/; Castresana 2000) to ex- clude ambiguous areas of the alignment for each gene. DnaSP ver. 6 (http://www.ub.edu/dnasp/; Rozas et al. 2017) was used to calculate the number of haplotypes. The best-fit model for each gene and gene partition was calculated by PartitionFinder2 ver. 2.3.4 (Lanfear et al. 2017), based on the corrected Akaike Information Cri- terion (AICc) and using a heuristic search algorithm. The program proposed the division of the concatenated dataset into three partitions, comprising partitions for the 28S gene and each of the three codon positions of the COI gene. The best-fit model was determined to be GTR + 1+ G for the first and third codon positions of COI, GTR for the second position of COJ, and GTR + G for 28S. Maximum-likelihood (ML) analyses were performed in IQ-TREE (Nguyen et al. 2015) with the ML + rap- id bootstrapping method and 10,000 replicates. Bayes- ian inference (BI) analyses were conducted in MrBayes (Ronquist et al. 2012). Four simultaneous runs with four independent Markov Chain Monte Carlo (MCMC) algo- rithms were implemented for 10 million generations, and trees were sampled every 1000 generations with a burn-in of 25%. The convergence was checked with the average standard deviation of split frequencies < 0.01 and the po- tential scale reduction factor (PSRF) ~ 1. Inter- and intra-specific distances based on the CO/ dataset were calculated in MEGA X using the uncorrect- ed p-distance. Standard error estimates were obtained by 1000 bootstrapping replicates. zse.pensoft.net 1194 Table 1. List of sequences used in phylogenetic analyses. (*) Sequenced from this study. Taxon UNIONIDAE Rafinesque, 1820 Unioninae Rafinesque, 1820 Cristariini Lopes-Lima, Bogan, & Froufe, 2017 Amuranodonta kijaensis Moskvicheva, 1973 Anemina arcaeformis (Heude, 1877) Beringiana beringiana (Middendorff, 1851) Beringiana japonica (Clessin, 1874) Beringiana fukuharai San, Hattori & Kondo, 2020 Beringiana gosannensis San, Hattori & Kondo, 2020 Buldowskia flavotincta (Martens, 1905) Buldowskia suifunica (Lindholm, 1925) Buldowskia iwakawai (Suzuki, 1939) Buldowskia kamiyai San, Hattori & Kondo, 2020 Buldowskia shadini (Moskvicheva, 1973) Cristaria bellua (Morelet, 1866) Cristaria clessini (Kobelt, 1879) Cristaria plicata (Leach, 1814) Cristaria truncata Dang, Thai & Pham, 1980 Pletholophus honglinhensis Bogan, Do, Froufe & Lopes-Lima, 2023 Pletholophus reinianus (Martens, 1875) Pletholophus tenuis (Griffith & Pidgeon, 1833 Pletholophus tenuis (Griffith & Pidgeon, 1833 Pletholophus tenuis (Griffith & Pidgeon, 1833 Pletholophus tenuis (Griffith & Pidgeon, 1833 Pletholophus tenuis (Griffith & Pidgeon, 1833) Pletholophus guangzhouensis Dai, Chen, Huang & Wu, sp. nov.* Pletholophus guangzhouensis Dai, Chen, Huang & Wu, sp. nov.* Pletholophus guangzhouensis Dai, Chen, Huang & Wu, sp. nov.* Pletholophus guangzhouensis Dai, Chen, Huang & Wu, sp. nov.* Sinanodonta angula (Tchang, Li & Liu, 1965) Sinanodonta calipygos (Kobelt, 1879) Sinanodonta lauta (Martens, 1877) Sinanodonta lucida (Heude, 1877) Sinanodonta schrenkii (Lea, 1870) Sinanodonta tumens (Haas, 1910) Sinanodonta pacifica (Heude, 1878) Sinanodonta woodiana (Lea, 1834) Parreysiinae Henderson, 1935 Scabies crispata (Gould, 1843) Trapezidens exolescens (Gould, 1843) MARGARITIFERIDAE Henderson, 1929 Gibbosula laosensis (Lea, 1863) Margaritifera margaritifera (Linnaeus, 1758) Ss re wes ws Results Molecular analyses Four COI haplotypes and one 28S haplotype were iden- tified in the 10 sequenced specimens from Guangzhou, Guangdong. The CO/ dataset had an aligned length of 600 characters, with 95 variable sites and 42 parsimony informative sites. The CO/J + 28S dataset, which had un- dergone trimming and concatenation, consisted of 1,009 characters, comprising 600 bp of CO/ and 409 bp of 28S. There were 383 variable sites and 307 parsimony infor- mative sites. The ML and BI trees based on the CO/ + 28S data- set exhibited largely congruent topologies, except for two nodes containing polytomies in the BI tree (Fig. 3). In both trees, Pletholophus Simpson, 1900, occupied a zse.pensoft.net Dai, Y.-T. et al.: A new species of Pletho/ophus COI 28S rRNA Country MK574204 MK574473 Russia MG462936 MG595463 China MT020557 MT020799 Japan MT020576 MT020803 Japan MT020567 MTO20801 Japan MT020584 MT020802 Japan MT020537 MT020804 South Korea MK574190 MK574460 Russia MT020523 MTO020806 Japan MT020525 MT020808 Japan MK574197 MK5 74467 Russia ON704642 ON695893 Laos MT020592 MT020810 Japan MG462956 MG595484 China OP491287 OP499826 Vietnam OR912962 OR913009 Vietnam MT020603 n/a Japan KX822658 KX822614 Vietnam MT020599 LC519084 Japan MTO20600 LC519085 Japan MT020601 KX822614 Japan MT020602 KX822614 Japan PP945818 PP956591 China PP945819 PP956591 China PP945820 PP956591 China PP945821 PP956591 China MG463053 MG595580 China MT020623 MT020833 Japan MT020616 MT020834 Japan MG463066 MG595589 China MT020618 MT020837 South Korea MT020622 MT020838 Japan MG463052 MG595599 China MG463080 MG595608 China MG288632 MG552824 Thailand KX230532 KX230559 Thailand JX497731 KT343741 Laos KX550089 KX550093 Russia distinct position in the subfamily Unioninae and was the sister group with Sinanodonta + Beringiana (BS/ BPP = 98/1.0) (Fig. 3). Within Pletholophus, specimens from Guangzhou, Guangdong, represent a distinct taxon and were recovered as sisters to Pletholophus tenuis + Pletholophus reinianus, with high nodal support (BS/BPP = 98/1). The pairwise uncorrected CO/ p-distance analy- sis demonstrated genetic distances ranging from 5.27% (between this species and P. tenuis) to 11.06% (between this species and P. honglinhensis) (Table 2). This species shared a closer relationship with P. tenuis. It occupies a unique phylogenetic position and displays distinctive morphological characteristics (Fig. 3; Table 3), which are described herein as Pletholophus guangzhouensis sp. nov. Moreover, our results resolved the phylogenetic relationship within Pletholophus as (P. honglinhensis + (P. guangzhouensis sp. nov. + (P. tenuis + P. reinianus))). Zoosyst. Evol. 100 (4) 2024, 1191-1200 1195 Table 2. Average intraspecific (bold) and interspecific uncorrected p-distance (% + S.E.) for CO/ sequences of species in Plethol- ophus Simpson, 1900. Taxa 1 2 3 4 1. P. guangzhouensis sp. 0.42 + 0.18 nov. 2. P. tenuis 5.27 + 0.89 0.67 + 0.20 3. P. reinianus 5.30 + 0.93 5.68 + 0.93 n/c 4. P. honglinhensis 11.06-+ 1.32 1075-44125 1152-23132 n/c Table 3. Analyzed conchological characters of Pletholophus species. Characteristic descriptions of P. tenuis, P. reinianus, and P. honglinhensis are referenced from published works (Simpson 1900; Haas 1969; He and Zhuang 2013; Bogan et al. 2023) and the MUSSELp online database. Conchological P. guangzhouensis features Shell shape Oval P. honglinhensis Slightly rectangular to elongate oval Shell thickness Thin Thin P. reinianus P. tenuis Elliptical or slightly Evenly elliptical rhomboid Rather thin Thin but strong Shell color Greenish-yellow in young Brown to black Greenish or brownish Yellowish-green individuals, darkish-brown in old individuals Umbo 1/4 of shell length, 1/3 of shell length, inflated, 1/3 of shell length, 1/3 of shell length, compressed, as high as not elevated above the compressed, as heigh as — compressed, as heigh as hinge line dorsal margin hinge line hinge line Umbo cavity Rather shallow, open Shallow, open Rather shallow, open Shallow, open Posterior ridge Developed Prominent but not sharp Developed Almost wanting Surface sculpture Fine and dense growth Growth lines Three faint darker ridges; — Feebly rayed throughout; lines; two faint ridge on the posterior dorsal; a few elegant, feebly rays Reduced to mere raised threads One tooth on both valves, long and narrow Pseudocardinal teeth Lateral teeth One long, thin lamellar tooth Right valve with a long, narrow lateral tooth; left valve with a straight and on the posterior slope with finer growth lines a few slight plications; finer growth lines Linear pseudocardinal § Wanting or reduced to mere incach valve raised threads One tooth, high and triangular Anterior tooth well developed, posterior tooth reduced well developed tooth Nacre colour Bluish-white, iridescent Taxonomy Family Unionidae Rafinesque, 1820 Subfamily Unioninae Rafinesque, 1820 Tribe Cristariini Lopes-Lima, Bogan & Froufe, 2017 Genus Pletholophus Simpson, 1900 Type species. Pletholophus tenuis (Griffith & Pidgeon, 1833) Pletholophus guangzhouensis Dai, Chen, Huang & Wu, sp. nov. https://zoobank. org/E435D35E-CE14-4F27-A726-6346A AOECF3A Fig. 2 Material examined. Holotype CHINA * 9; Guang- dong, Guangzhou City, Conghua District, Liuxi River; 23°32'02"N, 113°35'03"E; 9 January 2021; leg. local peo- ple; ex. Y. T. Dai & L. Guo; 24 NCU_XPWU_PGU01. White, becoming bluish- iridescent toward the posterior margin One tooth on both valves scarcely developed Bluish-white, iridescent behind One tooth on both valves, slender Bluish-white Paratypes CHINA * 9 shells; same collection data as for the holotype; specimen vouchers are shown in Table 4. Diagnosis. Periostracum greenish-yellow in young 1n- dividuals, darkish-brown in old individuals; with fine and dense growth lines and two faint ridges on the posterior dorsal; periostracum often painted with a few elegant, feebly rays. Hinge undeveloped. Beak cavities shallow, open. In both valves, only one peudocardinal and lateral tooth. Peudocardinal teeth reduced to mere raised threads, lateral teeth long and narrow. Nacre bluish-white, irides- cent. Glochidia hooked, subtriangular in shape, medium size, shell length less than shell height. The surface of glochidia have deep and dense small holes. Shell description. Shell medium-sized, not inflated, thin but strong. Length 34.47-51.19 mm, width 6.55— 16.21 mm, height 22.86-33.76 mm (Table 4). Shell ovoid, anterior rounded, short, posterior long and wide, slightly obtuse angle, posterior ridge developed. Umbo not prominent, compressed, as high as dorsal margin, located at 1/4 of the dorsal margin, and often eroded. Dorsal margin straight, rear end curved down-wards, zse.pensoft.net 1196 Dai, Y.-T. et al.: A new species of Pletho/ophus Figure 2. The shell morphology of Pletholophus guangzhouensis sp. nov. A. holotype; B—J. paratypes. with a low wing behind; ventral margin weakly curved. Periostracum greenish-yellow in young individuals, dark- ish-brown in old individuals; with fine and dense growth lines and two faint ridges on the posterior dorsal; peri- ostracum often painted with a few elegant, feebly rays. Lines arranged in irregular concentric circles. Hinge un- developed. Beak cavities shallow, open. In both valves, only one peudocardinal and lateral tooth. Peudocardinal teeth reduced to mere raised threads, and lateral teeth long and narrow. Mantle attachment scars on the edge of shells obvious. Both anterior adductor muscle scars and posterior adductor muscle scars shallow, irregularly cres- cent-shaped. Nacre bluish-white, iridescent. Glochidia morphology description. Glochidial shells typically anodontin hooked shells and subtriangular in shape, with the ventral angle slightly protruding dorsally. Medium size, length 0.226 + 0.003 mm, height 0.247 + 0.015, shell length less than shell height. The ventral an- gle of each glochidia valve with an anchor-shaped styli- form hook. The hook covered by lanceolate macrospines arranged in 2—3 diagonal rows near the ventral terminus and reduced to a single row distally. Microspines and mi- cropoints cover the entire ventral terminus and less than zse.pensoft.net one-third of the hook lateral lobes. The fossae on the shell surface deep and dense, with distinct small holes. Etymology. The name of this species 1s derived from Guangzhou City, in which its type locality is located. For the common name of Pletholophus guangzhouen- sis, We recommend “Guangzhou micro tooth mussel” (English) and “Guang Zhou Wei Chi Bang” (J (Ar iE) (Chinese). Table 4. Shell measurements of Pletholophus guangzhouensis sp. nov. Measurements are in millimeters (mm). Status of Specimen voucher Shell Shell Shell specimen length width height Holotype 24 NCU_XPWU_PGU0O1 50.86 15.51 26.72 Paratype 24 NCU_XPWU_PGU02 50.56 15.42 33.76 Paratype 24 NCU_XPWU_PGU03 45.71 11.96 29.87 Paratype 24_NCU_XPWU_PGU04 49.32 15.41 33.11 Paratype 24 NCU_XPWU_PGU05 48.85 15.70 32.54 Paratype 24 NCU_XPWU_PGU06 51.19 16.21 33.22 Paratype 24 NCU_XPWU_PGUO7 48.95 15.19 32.38 Paratype 24 NCU_XPWU_PGU08 47.46 14.98 31.02 Paratype 24 NCU_XPWU_PGU09 40.87 11.58 23.98 Paratype 24 NCU_XPWU_PGU10 34.47 6.55 22.86 Zoosyst. Evol. 100 (4) 2024, 1191-1200 88 fo FYetholophus guangzhouensis : Sp. nov. 98 Pe 100;) 98 9 ML 98 99 100) 100 62 99 0.09 .. Pletholophus tenuis -ovwneeee Pletholophus reinianus ‘Pletholophus honglinhensis Sinanodonta lauta oe Sinanodonta pacifica -- Sinanodonta woodiana -- Sinanodonta calipygos a Sinanodonta tumens -- Sinanodonta schrenkii 100 ~~ — Sinanodonta lucida 77 mentee Sinanodonta angula 93 umn Beringiana fukuharai 100 vooemnme Boringiana gosannensis te Be Beringiana beringiana 69 -——- Beringiana japonica 96 pro = Cristaria plicata Cristaria truncata Cristaria clessini Cristaria bellua Buldowskia iwakawai - Buldowskia kamiyai ~~ Buldowskia flavotincta Buldowskia shadini Buldowskia suifunica ~- Anemina arcaeformis --- Amuranodonta kijaensis 400 ~rrmmemnmnmnemanmamennns TADEZIAENS EXOIESCENS Scabies crispata 100 Honnnnnanmnnnnnnmnnnmnrnns bbOSuUIa laosensis ~-« Margaritifera margaritifera [enn 1197 ee Bl Parreysiinae Figure 3. Maximum likelihood (ML) and Bayesian inference (BI) trees of Unionidae based on the CO/ + 28S dataset. Gibbosula laosensis and Margaritifera margaritifera from the family Margaritiferidae were used as outgroups. Support values above the branches are the posterior probability and bootstrap support, respectively. Distribution. The species is endemic to the Liuxi Riv- er, located in Conghua District, Guangzhou City, Guang- dong Province. Discussion Our morphological and molecular analyses provide compelling evidence that the freshwater mussels from Guangzhou, Guangdong, represent a new species of Pletholophus within the tribe Cristariini of the subfamily Unioninae. Species belonging to the Cristariini exhibit high levels of cryptic diversity, rendering it challenging to distinguish them based solely on morphological char- acteristics (He and Zhuang 2013; Lopes-Lima et al. 2020; Bogan et al. 2023). Our study has once again highlight- ed the importance of utilizing an integrative approach in generic classification. In our phylogenetic trees, Plethol- ophus guangzhou sp. nov. formed a well-supported clade in Pletholophus and has large genetic distances from its congeneric species, supporting it as a distinct species (un- corrected COI p-distance = 5.27% ~ 11.06%; Table 2). The phylogenetic relationships of genera in the Cristariini align with previous studies in most topologies (Lopes-L- ma et al. 2020; Bogan et al. 2023). Our CO/ + 28S phy- logenies showed the position of Buldowskia, Anemina, and Amuranodonta at the base of Cristariini (Fig. 3). Nevertheless, previous studies have inferred from the COI + 28S dataset that Cristaria was placed at the base of the clade in Cristariini (Lopes-Lima et al. 2020; Bogan et al. 2023). The incongruencies between topologies are likely due to incomplete lineage sorting, insufficient tax- on sampling, and varying rates of genome evolution and mutation (Perkins et al. 2017). To resolve the intergener- ic relationships within this tribe, it is recommended that more comprehensive taxon sampling and an increased number of informative loci be utilized. The morphologic analysis is in alignment with the mo- lecular data. Pletholophus is distinguished from other gen- era in Cristariini by its slender pseudocardinal teeth. For example, Sinanodonta lacks any evidence of hinge teeth, while Cristaria typically possesses only well-developed lateral teeth (Simpson 1914; Bogan et al. 2023). The new species, Pletholophus guangzhou sp. nov., can be distin- guished from its congeneric species by its oval shell shape, weakly curved ventral margin, faint rays, and two faint ridges on the posterior dorsal (Fig. 2; Table 3). Plethol- ophus tenuis is taller and has a more rounded ventral mar- gin compared to other species within Pletholophus. In con- trast, P. honglinhensis possesses a more elongated shell. Therefore, P. tenuis and P. honglinhensis can be readily distinguished from their congeneric species based on shell morphology. Pletholophus guangzhou sp. nov. is morpho- logically similar to P. reinianus but can be distinguished zse.pensoft.net 5/19/2021 t HV |mag| WD - spot 100 pr 5/19/2021 del 12:18:39 PM ETD 20.00 kV|800 x} 10.3 mm 3.0 \ “AS (d) Sy 5/19/2021 det HV mag WD {spot 12:21:09 PM ETD 20.00 kV|2 400 x 10.3 mm} 3.0 t HV | ma WD _ spot M ETD 20.00 kV/500 x} 10.3 mm _3.0 6/19/2021 det HV mag WD 12:21:50 PM ETD 20.00 kV/20 000 x 10.3 mm_3.0 Dai, Y.-T. et al.: A new species of Pletho/ophus 7 oe | 200 pm ] 5/19/2021 det HV mag WD spot 100 um: label 12:23:08 PM ETD 20.00 kV/1 200 x 10.2 mm| 3.0 label 0) spot ————— 5m 5/19/2021 det HV mag WD |spot 20 um: 12:19:18 PM ETD/20.00 kV|5 000 x 10.2 mm| 3.0 label Figure 4. SEM microphotographs of Pletholophus guangzhouensis sp. nov. glochidia. A. Closed valves of glochidia; B. Open valves of glochidia; C. Hinge of glochidia; D. Hook of glochidia; E. Microspines on the ventral margin of glochidia; F. Pores on exterior glochidial valve surfaces. by its more developed pseudocardinal teeth and the pres- ence of two faint ridges (versus reduced pseudocardinal teeth and three faint darker ridges in P. reinianus). In this study, we provide morphological descriptions of the glochidia of Pletholophus guangzhou sp. nov., which have proven useful for interpreting the phyloge- netic relationships among freshwater mussels (Hoggarth 2000; Sayenko 2006; Sayenko et al. 2020). The glochidia shells of P. guangzhou are subtriangular, medium-sized, and have a styliform hook on the ventral angle of each valve (Fig. 4). These characteristics are consistent with those observed in the majority of species within the sub- family Unioninae (Wu et al. 1999a, 1999b; Cmiel et al. 2021; Sayenko et al. 2023). The majority of Margaritife- ridae species, as well as the Ambleminae and Gonideinae within the Unionidae, lack hooks (Shu and Wu 2005a; Xu et al. 2013; Wu et al. 2018; Vikhrev et al. 2019;Cmiel et al. 2021). Furthermore, the glochidia of Margaritiferidae are notably small and semicircular, as observed in Mar- garitifera dahurica (Cmiel et al. 2021) and Gibbosula rochechouartii (unpublished data from our laboratory). The size of glochidia can aid in taxonomic classification (Cmiel et al. 2021), while their shape (including aspects such as symmetry and vertical/horizontal elongation) provides valuable taxonomic characteristics that can be utilized in the reconstruction of paleoenvironments (Pfeiffer and Graf 2015; Chernyshev et al. 2020). Given the plasticity of freshwater mussel shells, it 1s increas- ingly necessary to incorporate glochidia morphology and anatomical characters into mussel taxonomic studies. zse.pensoft.net In light of the ongoing global biodiversity loss, the as- sessment and monitoring of species, along with the detec- tion of new species, are of paramount significance (Dal et al. 2024). The discovery of the new freshwater mussel taxon serves to confirm the high diversity and endemic nature of the mussel fauna in Guangdong. Nevertheless, the high levels of urbanization in the area may result in significant habitat loss for the mussels, thereby threaten- ing their survival. Integrative classification methods and genetic research will inform the development of effective conservation strategies, enabling management based on a more accurate understanding of the unique evolutionary relationships of imperiled freshwater organisms. Acknowledgments We are grateful to Prof. Matthias Glaubrecht as well as two reviewers, Dr. Arthur Bogan and Dr. Ivan N. Bolotov, for their helpful comments. This study was supported by the National Natural Science Foundation of China (No. 32100354 and No. 31772412) and the Jiangxi Provincial Natural Science Foundation (No. 20232BAB205067). References Dang N, Thai T, Pham V (1980) Identification of freshwater inverte- brates of North Vietnam. Hanoi. Sciences and Technology Publish- ing Co, Vietnam. Zoosyst. Evol. 100 (4) 2024, 1191-1200 Aldridge DC, Ollard IS, Bespalaya YV, Bolotov IN, Douda K, Geist J, Haag WR, Klunzinger MW, Lopes-Lima M, Mlambo MC, Riccardi N, Sousa R, Strayer DL, Torres SH, Vaughn CC, Zajac T, Zieritz A (2022) Freshwater mussel conservation: A global horizon scan of emerging threats and opportunities. 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