Description of a new freshwater mussel species of Pletholophus , Simpson, 1900 (Bivalvia, Unionidae) from Guangdong, China

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 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 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.


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).
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 1875Heude , 1877aHeude , 1877bHeude , 1878Heude , 1879Heude , 1880aHeude , 1880bHeude , 1881Heude , 1883Heude , 1885;;Simpson 1900Simpson , 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.

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.

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), ddH 2 O (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.
All PCGs were codon-aligned by MUSCLE ver.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.

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))).Paratypes China • 9 shells; same collection data as for the holotype; specimen vouchers are shown in Table 4.
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 34.47-51.19mm, width 6.55-16.21mm, height 22.86-33.76mm (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,  (Simpson 1900;Haas 1969;He and Zhuang 2013;Bogan et al. 2023)  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(Wu et al. , 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.
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.
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.
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 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 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.
x i R i v e r

Figure 3 .
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 1 .
List of sequences used in phylogenetic analyses.(*) Sequenced from this study.

Table 3 .
Analyzed conchological characters of Pletholophus species.Characteristic descriptions of P. tenuis, P. reinianus, and P. honglinhensis are referenced from published works