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Research Article
Description of a new freshwater mussel species of Pletholophus, Simpson, 1900 (Bivalvia, Unionidae) from Guangdong, China
expand article infoYu-Ting Dai‡, Zhong-Guang Chen‡, Cheng-Lin Hu‡, Shan Ouyang‡, Xiao-Chen Huang‡, Xiao-Ping Wu‡
‡ Nanchang University, Nanchang, China

Corresponding author: Xiao-Chen Huang ( xchuang@ncu.edu.cn )

Corresponding author: Xiao-Ping Wu ( xpwu@ncu.edu.cn )

Academic editor: Matthias Glaubrecht
© 2024 Yu-Ting Dai, Zhong-Guang Chen, Cheng-Lin Hu, Shan Ouyang, Xiao-Chen Huang, Xiao-Ping Wu.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Dai Y-T, Chen Z-G, Hu C-L, Ouyang S, Huang X-C, Wu X-P (2024) Description of a new freshwater mussel species of Pletholophus, Simpson, 1900 (Bivalvia, Unionidae) from Guangdong, China. Zoosystematics and Evolution 100(4): 1191-1200. https://doi.org/10.3897/zse.100.131019
ZooBank: urn:lsid:zoobank.org:pub:C9F1030C-0564-4960-9378-38D9848ACE05
Open Access

Abstract

The Pearl River Basin, China’s second-largest freshwater basin, hosts a significant diversity of species and a highly endemic freshwater mussel fauna. In this study, a new species from the Liuxi River in Guangzhou, Guangdong, China, Pletholophus guangzhouensis 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 COI and 28S rRNA gene fragments indicated that Pletholophus guangzhouensis sp. nov. is 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 collections 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 biogeography, characterized by low dispersal and restriction to freshwater habitats, makes them invaluable for elucidating 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 adults. This distinctive life cycle has likely contributed significantly to the rapid diversification of this group (Barnhart et al. 2008). However, freshwater mussels represent one of the most threatened faunal groups on a global scale (Böhm 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 experienced 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 highly endemic mussel fauna (Zieritz et al. 2018; Liu et al. 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 previously 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; Pseudocuneopsis yangshuoensis Wu & Liu, 2023; P. wuana Liu & Wu, 2023; and P. longjiangensis Liu & Wu, 2024 (Dai et al. 2023; Dai et al. 2024; Liu et al. 2024). This observation prompted the hypothesis that Guangdong Province, another significant region through which the Pearl River Basin flows, may also be rich in unique species. However, there is a paucity of mussel diversity surveys and studies 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 Unioninae Rafinesque, 1820, in the family Unionidae Rafinesque, 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. Ðặng 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 COI + 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 COI and 28S rRNA gene fragments. Recently, Bogan et al. (2023) summarized the taxonomy and diversity of Anodontini in Vietnam, identifying a new species, Pletholophus 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 Pletholophus honglinhensis Bogan, Do, Froufe & Lopes-Lima, 2023. Pletholophus tenuis has been recorded as widespread 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 is 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 freshwater mussel in the Liuxi River, Guangzhou, China. After 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. Subsequently, a new species of Pletholophus was described based on a combination of morphological characters and the COI + 28S rRNA gene phylogenies. We provide morphological 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 collected from the Liuxi River (23°32'02"N, 113°35'03"E) in Guangzhou City, Guangdong, China (Figs 1, 2). A digital 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 subsequent DNA extraction. The remaining soft parts were preserved at −80 °C. All voucher specimens were deposited in the Museum of Biology, Nanchang University (NCUMB), China.

Figure 1. 

Distribution map of Pletholophus guangzhouensis sp. nov.

Figure 2. 

The shell morphology of Pletholophus guangzhouensis sp. nov. A. holotype; B–J. paratypes.

Scanning electron microscopy of glochidia

The glochidial mass was stored in 96% ethanol and subsequently washed with deionized water. It was then transferred 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 ethanol for storage purposes. Prior to scanning electron microscopy, 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 microscopy (SEM) (Quanta 200FEG03040702, USA) (Shu and Wu 2005b; Sayenko et al. 2023).

Molecular phylogenetic analyses

The Qiagen Genomic DNA kit (Qiagen, Hilden, Germany) 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 were checked using 1% agarose gel electrophoresis and NanoDrop 2000 (Thermo Scientific, USA). We amplified and sequenced fragments from the mitochondrial cytochrome 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 µL mixture of 2 × Taq Plus Master MixII (Vazyme, China) (12.5 µL), ddH2O (9.5 µL), 10 µM primers (1 µL each), and genomic DNA (1 µL, about 100 ng/μL). Thermal cycling was started at 98 °C for 10 s, followed by 35 cycles of 94 °C for 1 min, annealing 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 products 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.

Table 1.
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List of sequences used in phylogenetic analyses. (*) Sequenced from this study.

Taxon COI 28S rRNA Country
UNIONIDAE Rafinesque, 1820
Unioninae Rafinesque, 1820
Cristariini Lopes-Lima, Bogan, & Froufe, 2017
Amuranodonta kijaensis Moskvicheva, 1973 MK574204 MK574473 Russia
Anemina arcaeformis (Heude, 1877) MG462936 MG595463 China
Beringiana beringiana (Middendorff, 1851) MT020557 MT020799 Japan
Beringiana japonica (Clessin, 1874) MT020576 MT020803 Japan
Beringiana fukuharai San, Hattori & Kondo, 2020 MT020567 MT020801 Japan
Beringiana gosannensis San, Hattori & Kondo, 2020 MT020584 MT020802 Japan
Buldowskia flavotincta (Martens, 1905) MT020537 MT020804 South Korea
Buldowskia suifunica (Lindholm, 1925) MK574190 MK574460 Russia
Buldowskia iwakawai (Suzuki, 1939) MT020523 MT020806 Japan
Buldowskia kamiyai San, Hattori & Kondo, 2020 MT020525 MT020808 Japan
Buldowskia shadini (Moskvicheva, 1973) MK574197 MK574467 Russia
Cristaria bellua (Morelet, 1866) ON704642 ON695893 Laos
Cristaria clessini (Kobelt, 1879) MT020592 MT020810 Japan
Cristaria plicata (Leach, 1814) MG462956 MG595484 China
Cristaria truncata Dang, Thai & Pham, 1980 OP491287 OP499826 Vietnam
Pletholophus honglinhensis Bogan, Do, Froufe & Lopes-Lima, 2023 OR912962 OR913009 Vietnam
Pletholophus reinianus (Martens, 1875) MT020603 n/a Japan
Pletholophus tenuis (Griffith & Pidgeon, 1833) KX822658 KX822614 Vietnam
Pletholophus tenuis (Griffith & Pidgeon, 1833) MT020599 LC519084 Japan
Pletholophus tenuis (Griffith & Pidgeon, 1833) MT020600 LC519085 Japan
Pletholophus tenuis (Griffith & Pidgeon, 1833) MT020601 KX822614 Japan
Pletholophus tenuis (Griffith & Pidgeon, 1833) MT020602 KX822614 Japan
Pletholophus guangzhouensis Dai, Chen, Huang & Wu, sp. nov.* PP945818 PP956591 China
Pletholophus guangzhouensis Dai, Chen, Huang & Wu, sp. nov.* PP945819 PP956591 China
Pletholophus guangzhouensis Dai, Chen, Huang & Wu, sp. nov.* PP945820 PP956591 China
Pletholophus guangzhouensis Dai, Chen, Huang & Wu, sp. nov.* PP945821 PP956591 China
Sinanodonta angula (Tchang, Li & Liu, 1965) MG463053 MG595580 China
Sinanodonta calipygos (Kobelt, 1879) MT020623 MT020833 Japan
Sinanodonta lauta (Martens, 1877) MT020616 MT020834 Japan
Sinanodonta lucida (Heude, 1877) MG463066 MG595589 China
Sinanodonta schrenkii (Lea, 1870) MT020618 MT020837 South Korea
Sinanodonta tumens (Haas, 1910) MT020622 MT020838 Japan
Sinanodonta pacifica (Heude, 1878) MG463052 MG595599 China
Sinanodonta woodiana (Lea, 1834) MG463080 MG595608 China
Parreysiinae Henderson, 1935
Scabies crispata (Gould, 1843) MG288632 MG552824 Thailand
Trapezidens exolescens (Gould, 1843) KX230532 KX230559 Thailand
MARGARITIFERIDAE Henderson, 1929
Gibbosula laosensis (Lea, 1863) JX497731 KT343741 Laos
Margaritifera margaritifera (Linnaeus, 1758) KX550089 KX550093 Russia

Two datasets were constructed in this study: (i) the COI dataset (11 sequences; 600 bp); and (ii) the COI + 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) implemented in MEGA ver. 10.1.6 (http://www.megasoftware.net; Kumar et al. 2018), whereas 28S rRNA were aligned in MAFFT ver. 7 (https://mafft.cbrc.jp/alignment/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 exclude 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 Criterion (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 + I + G for the first and third codon positions of COI, GTR for the second position of COI, and GTR + G for 28S.

Maximum‐likelihood (ML) analyses were performed in IQ-TREE (Nguyen et al. 2015) with the ML + rapid bootstrapping method and 10,000 replicates. Bayesian inference (BI) analyses were conducted in MrBayes (Ronquist et al. 2012). Four simultaneous runs with four independent Markov Chain Monte Carlo (MCMC) algorithms 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 potential scale reduction factor (PSRF) ~ 1.

Inter- and intra-specific distances based on the COI dataset were calculated in MEGA X using the uncorrected p-distance. Standard error estimates were obtained by 1000 bootstrapping replicates.

Results

Molecular analyses

Four COI haplotypes and one 28S haplotype were identified in the 10 sequenced specimens from Guangzhou, Guangdong. The COI dataset had an aligned length of 600 characters, with 95 variable sites and 42 parsimony informative sites. The COI + 28S dataset, which had undergone trimming and concatenation, consisted of 1,009 characters, comprising 600 bp of COI and 409 bp of 28S. There were 383 variable sites and 307 parsimony informative sites.

The ML and BI trees based on the COI + 28S dataset exhibited largely congruent topologies, except for two nodes containing polytomies in the BI tree (Fig. 3). In both trees, Pletholophus Simpson, 1900, occupied a 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 COI p-distance analysis 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))).

Figure 3. 

Maximum likelihood (ML) and Bayesian inference (BI) trees of Unionidae based on the COI + 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.

Table 2.
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Average intraspecific (bold) and interspecific uncorrected p-distance (% ± S.E.) for COI sequences of species in Pletholophus Simpson, 1900.

Taxa 1 2 3 4
1. P. guangzhouensis sp. nov. 0.42 ± 0.18
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 10.75 ± 1.25 11.52 ± 1.32 n/c
Table 3.
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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 features P. guangzhouensis P. honglinhensis P. reinianus P. tenuis
Shell shape Oval Slightly rectangular to elongate oval Elliptical or slightly rhomboid Evenly elliptical
Shell thickness Thin Thin Rather thin Thin but strong
Shell color Greenish-yellow in young individuals, darkish-brown in old individuals Brown to black Greenish or brownish Yellowish-green
Umbo 1/4 of shell length, compressed, as high as hinge line 1/3 of shell length, inflated, not elevated above the dorsal margin 1/3 of shell length, compressed, as heigh as hinge line 1/3 of shell length, compressed, as heigh as 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 lines; two faint ridge on the posterior dorsal; a few elegant, feebly rays Growth lines Three faint darker ridges; on the posterior slope with a few slight plications; finer growth lines Feebly rayed throughout; finer growth lines
Pseudocardinal teeth Reduced to mere raised threads One long, thin lamellar tooth Linear pseudocardinal incach valve Wanting or reduced to mere raised threads
Lateral teeth One tooth on both valves, long and narrow Right valve with a long, narrow lateral tooth; left valve with a straight and well developed tooth Anterior tooth well developed, posterior tooth reduced One tooth, high and triangular
Nacre colour Bluish-white, iridescent White, becoming bluish-iridescent toward the posterior margin One tooth on both valves, slender One tooth on both valves scarcely developed
Bluish-white Bluish-white, iridescent behind

Taxonomy

Family Unionidae Rafinesque, 1820

Subfamily Unioninae Rafinesque, 1820

Tribe Cristariini Lopes-Lima, Bogan & Froufe, 2017

Pletholophus Simpson, 1900

Type species

Pletholophus tenuis (Griffith & Pidgeon, 1833)

Pletholophus guangzhouensis Dai, Chen, Huang & Wu, sp. nov.

https://zoobank.org/E435D35E-CE14-4F27-A726-6346AA0ECF3A
Fig. 2

Material examined

Holotype China • ♀; Guangdong, Guangzhou City, Conghua District, Liuxi River; 23°32'02"N, 113°35'03"E; 9 January 2021; leg. local people; ex. Y. T. Dai & L. Guo; 24_NCU_XPWU_PGU01. Paratypes China • 9 shells; same collection data as for the holotype; specimen vouchers are shown in Table 4.

Table 4.
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Shell measurements of Pletholophus guangzhouensis sp. nov. Measurements are in millimeters (mm).

Status of specimen Specimen voucher Shell length Shell width Shell height
Holotype 24_NCU_XPWU_PGU01 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_PGU07 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

Diagnosis

Periostracum greenish-yellow in young individuals, 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, iridescent. 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, with a low wing behind; ventral margin weakly curved. Periostracum greenish-yellow in young individuals, 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. Lines arranged in irregular concentric circles. Hinge undeveloped. 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 crescent-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 angle of each glochidia valve with an anchor-shaped styliform 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 micropoints cover the entire ventral terminus and less than 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 is derived from Guangzhou City, in which its type locality is located. For the common name of Pletholophus guangzhouensis, we recommend “Guangzhou micro tooth mussel” (English) and “Guang Zhou Wei Chi Bang” (广州微齿蚌) (Chinese).

Distribution

The species is endemic to the Liuxi River, located in Conghua District, Guangzhou City, Guangdong 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 characteristics (He and Zhuang 2013; Lopes-Lima et al. 2020; Bogan et al. 2023). Our study has once again highlighted the importance of utilizing an integrative approach in generic classification. In our phylogenetic trees, Pletholophus 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 (uncorrected 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-Lima et al. 2020; Bogan et al. 2023). Our COI + 28S phylogenies 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 taxon sampling, and varying rates of genome evolution and mutation (Perkins et al. 2017). To resolve the intergeneric 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 molecular data. Pletholophus is distinguished from other genera 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 distinguished 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). Pletholophus tenuis is taller and has a more rounded ventral margin compared to other species within Pletholophus. In contrast, 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 morphologically similar to P. reinianus but can be distinguished by its more developed pseudocardinal teeth and the presence 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 phylogenetic 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 subfamily Unioninae (Wu et al. 1999a, 1999b; Ćmiel et al. 2021; Sayenko et al. 2023). The majority of Margaritiferidae 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;Ćmiel et al. 2021). Furthermore, the glochidia of Margaritiferidae are notably small and semicircular, as observed in Margaritifera dahurica (Ćmiel et al. 2021) and Gibbosula rochechouartii (unpublished data from our laboratory). The size of glochidia can aid in taxonomic classification (Ćmiel 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 is increasingly necessary to incorporate glochidia morphology and anatomical characters into mussel taxonomic studies.

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.

In light of the ongoing global biodiversity loss, the assessment and monitoring of species, along with the detection of new species, are of paramount significance (Dai 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 threatening 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).

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1 Yu-Ting Dai and Zhong-Guang Chen contributed equally to this paper.
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