Zoosyst. Evol. 100 (2) 2024, 425-436 | DOI 10.3897/zse.100.113972 ee BERLIN Description of a new species of the genus Cultellus Schumacher, 1817 (Bivalvia, Pharidae) from the South China Sea, based on integrative taxonomy Yanan Yu', Yingyi Jiao’, Junlong Zhang!*>:4 1 Laboratory of Marine Organism Taxonomy and Phylogeny, Qingdao Key Laboratory of Marine Biodiversity and Conservation, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China 2 Laoshan Laboratory, Qingdao, China 3 Marine Biological Museum, Chinese Academy of Sciences, Qingdao 266071, China 4 University of Chinese Academy of Sciences, Beijing 100049, China https://zoobank. org/A 9F39F 7B-24A D-44CC-A61C-73D9BF4066CE Corresponding author: Junlong Zhang (zhangjl|@qdio.ac.cn) Academic editor: Thomas von Rintelen # Received 11 October 2023 Accepted 19 March 2024 Published 9 April 2024 Abstract The present study describes a new species within the genus Cu/tellus Schumacher, 1817 collected from the South China Sea. An in- tegrative taxonomic approach incorporating morphological comparisons, geometric morphometrics and genetic analyses was used to identify and differentiate the new species. Cultellus exilis sp. nov. 1s distinguished from its congeners by its slender, fragile and trans- lucent valves, curved posteroventral margins and relatively large protractor scars. The geometric morphometric analyses, based on out- lines data, indicated that samples of Cultellus exilis sp. nov. clustered together and were distinctly separated from other species. Multi- ple species delimitation results, based on the mitochondrial COI gene, support the separation of Cu/tellus exilis sp. nov. from its related congeners. Phylogenetic analyses of a nuclear (28S rRNA) and two mitochondrial (COI, 16S rRNA) genes using Maximum Likelihood and Bayesian Inference methods revealed that the species belongs to the genus Cu/tellus. The superfamily Solenoidea Lamarck, 1809, which includes the families Solenidae Lamarck, 1809 and Pharidae H. Adams & A. Adams, 1856, exhibits closer affinity to the family Hiatellidae Gray, 1824 than to Solecurtidae d’ Orbigny, 1846. Furthermore, we found that the genus Si/iqua was clustered alongside the genera Ensiculus and Phaxas as a sister clade, which contradicts the current systematics of the subfamily within the family Pharidae. This work highlights the utility of integrative taxonomy for species identification, recognition and phylogenetic investigation. Key Words geometric morphometrics, integrative taxonomy, Pharidae, phylogeny, Solenoidea, species delimitation Introduction Signorelli 2021). Solenidae was regarded as a family by H. & A. Adams, containing two subfamilies Soleninae (includ- The superfamily Solenoidea Lamarck, 1809 including two families, namely Solenidae Lamarck, 1809 and Pharidae H. Adams & A. Adams, 1856, constitutes a collective of ben- thic bivalves that belong to the order Adapedonta Cossmann & Peyrot, 1909 (Cosel 1993; Bieler et al. 2010; Carter et al. 2011). These molluscs are commonly referred to as “razor clams” or “jackknife clams” due to their narrow-elongated shells and sharp edges (Saeedi et al. 2013; Giacomino and ing Solen, Solena, Ensis) and Pharinae (including Pharus, Pharella, Cultellus, Siliqua, Macha = Solecurtus, Azor = Azorinus, Siliquaria = Tenagodus, Novaculina) (Adams and Adams 1858). Cosel (1993) elevated the superfamily Sole- noidea and allocated two families Solenidae and Pharidae within it, based on the number of central teeth. Solecurtidae d’Orbigny, 1846 and Psammobiidae J. Fleming, 1828 were currently taken as families within the superfamily Telli- Copyright Yu, 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, distribu- tion, and reproduction in any medium, provided the original author and source are credited. 426 noidea Blainville, 1814 (Bieler et al. 2010). Huber (2010) documented that the family Solenidae encompasses the genera Neosolen, Solen and Solena, while the family Pha- ridae comprises 14 genera including Afrophaxas, Cultel- lus, Ensiculus, Ensis, Phaxas, Sinucultellus, Orbicularia, Pharella, Sinonovacula, Novaculina, Nasopharus, Pha- rus, Sinupharus and Siliqua. Furthermore, Novaculininae Ghosh, 1920 was considered a junior synonym of Pharelli- nae within the family Pharidae by Bolotov et al. (2018). At present, this systematics has been widely accepted. The genus Cultellus was initially proposed by Schum- acher (1817) to accommodate the jackknife clam Cultel- lus magnus Schumacher, 1817 = C. maximus (Gmelin 1791). The shells of this genus are of medium to large size, exquisitely elongated and slender, with the umbos located closer to the anterior end. Coen (1933) established the subgenus Cu/te/lus (Cultrensis) Coen, 1933 to accom- modate Cultellus (Cultrensis) adriaticus Coen, 1933. This species is characterised by its straight dorsal margin and two lines extending from the umbo to the posterior end (Thiele 1992), whereas this species was regarded as a junior synonym of Phaxas pellucidus (Pennant, 1777) (McKay and Smith 1979). Adams (1861) observed that Cultellus cultellus (Linnaeus, 1758) exhibits significant differences in characters compared to other species with- in the genus Cul/tellus. It has more curved margins and a shorter internal rib. Hence, he proposed a new genus Ensiculus H. Adams, 1861 within the family Pharidae for this species. Huber (2010) suggested that the genus Cultellus currently comprises five valid species, C. atten- uatus Dunker, 1862, C. hanleyi Dunker, 1862, C. maxi- mus, C. subellipticus Dunker, 1862 and C. vitreus Dunker, 1862. Three of these species are found along the coast of China: C. maximus (Gmelin 1791) reported from Taiwan island, C. subellipticus Dunker, 1862 distributed in the South China Sea and C. attenuatus Dunker, 1862 wide- spread in the Yellow Sea, the East and South China Seas (Liu 2008; Xu and Zhang 2008). Nonetheless, the generic allocations have yet to be validated by molecular analysis. Razor clams are highly valued for their delectable taste and nutrition richness. In recent years, there has been an increasing interest amongst scholars in investigating the ar- tificial breeding of these species (Zeng et al. 2010; Xu et al. 2013; Jiang et al. 2017; Li et al. 2022). However, the phylogeny of the genus Cul/te//us and even the superfamily Solenoidea has received limited attention. Previous studies that utilised single gene fragments or Random Amplified Polymorphic DNA (RAPD) methods consistently indicated that Cultellus belongs to Pharidae, a family closely related to Solenidae (Chen et al. 2005; Wu et al. 2008; Bolotov et al. 2018). Phylogenetic analysis, based on the mitochondri- al genes, revealed a close relationship between the genera Cultellus and Sinonovacula, indicating their membership in the same family (Yuan et al. 2012; Yu et al. 2016). Anal- ysis of two nuclear genes (18S and 28S) unveiled that the superfamilies Solenoidea and Hiatelloidea formed sister clades (Taylor et al. 2007). Bieler et al. (2014) demonstrat- ed a well-supported clade between Hiatellidae and Sole- noidea by utilising more genes (COI, 16S, 28S, 18S, H3) for zse.pensoft.net Yu, Y. et al.: A new species of the genus Cultellus constructing the phylogenetic tree. A recent mitochondrial phylogenomic study further indicated a closer relationship between Solenoidea and Hiatelloidea compared to Solecur- tidae (Li et al. 2022). Given that the aforementioned studies were conducted on a limited number of taxonomic groups, the phylogenetic relationships of the taxa within the Sole- noidea remain inadequately investigated. In May 2021, a previously unknown jackknife clam was collected from the South China Sea using the Agassiz trawl and subsequently preserved at the Institute of Oceanology, Chinese Academy of Sciences IOCAS). In this study, an integrative taxonomic approach incorporating molecular and geometric morphometrics was employed to identify and differentiate this species. Additionally, historical spec- imens collected in the last century and currently housed at the Marine Biological Museum, Chinese Academy of Sci- ences (MBMCAS) were also identified as this species. This methodology enables a more comprehensive understand- ing of the evolutionary relationships between species and a more reliable delimitation of species. Morphological ex- amination and genetic analyses revealed that the specimen represented a hitherto undescribed species that belongs to the genus Culte/lus. Herein, we formally describe and il- lustrate the new species, which adds to the known species diversity of the genus Culte/lus from Chinese waters. Materials and methods Specimen collection and morphological analyses The specimen was collected from the shallow water in the South China Sea using the Agassiz trawl (Fig. 1). Ocean Data View software version 5.4.0 (http://odv.awi.de) was used to plot the collection locations. The newly-sampled specimen was preserved in 75% ethanol solution and de- posited in the Marine Biological Museum, Chinese Acad- emy of Sciences (MBMCAS), Qingdao, China, alongside other historical shell specimens. The shells were observed under a Zeiss SteREO Discovery.V12 stereomicroscope (Zeiss, Wetzlar, Germany) and photographs were cap- tured using a Canon EOS6D camera. Shell measurements were taken to the nearest 0.01 mm using a Vernier caliper. Abbreviations used in this study: L, shell length; H, shell height; W, width of right valve. Geometric image acquisition Each sample was assigned to a uniform numbering and the right inner surface of each shell was photographed using a Sony ILCE-7RM4. To minimise random errors caused by factors like angle and lighting, the camera was securely fixed on a camera stand during photography. The type specimen images of C. attenuatus, C. hanleyi, C. subellipticus and C. vitreus were provided by the Nat- ural History Museum, London. The outline data used for morphometric analysis were captured by ImageJ v.2 (Schneider et al. 2012) software to ensure consistency in Zoosyst. Evol. 100 (2) 2024, 425-436 60°N (ee 40°N 2s = 60°E 90°E point selection direction. Subsequently, the above data were imported into Mathematica v.13.2 and uniformly sampled using the Polly.Morphometrics.12.4 package to obtain 150 semi-landmarks for each sample. Geometric morphometric analyses Initially, Generalised Procrustes Analysis (GPA) was con- ducted on the obtained data, in order to eliminate the influ- ence of non-shape differences caused by sample size, photo size, position etc. Subsequently, Thin Plate Splines analysis (TPS) and Principal Component Analysis (PCA) were per- formed on the transformed data to convert the shape varia- tion of all data points into a smaller number of uncorrelated principal component variation indicators. Then, the first three principal components were selected as representatives of morphological variation and analysed using Linear Dis- criminant Analysis (LDA). All the above data analyses were executed using Past v.4.12 software (Hammer et al. 2001). DNA extraction and PCR amplification Genomic DNA of specimens used in the present study was extracted from the foot of the animals using the Marine Animal Genomic DNA Extraction Kit (Tiangen Biotech, Beijing, China) following the manufacturer’s instructions 120°E Figure 1. The location of Cultellus exilis sp. nov. in the South China Sea. The red dot represents holotype and the blue dots represent paratypes. Ocean Data View 150°E 180°E and frozen at -20 °C. The mitochondrial DNA cytochrome c oxidase subunit I (COI) region was amplified using uni- versal primers LCO1490 and HCO2198 (Folmer et al. 1994). The mitochondrial 16S rRNA gene was amplified using 16Sa (Xiong and Kocher 1991) and 16Sb (Edge- combe et al. 2002) and the nuclear 28S rRNA gene was amplified using 28Sa and 28Sb (Whiting et al. 1997). Poly- merase chain reaction (PCR) amplification was carried out in a total reaction volume of 25 ul, consisting of 12 ul of 2x Es TaqMasterMix (Dye) (CWBio Co., Ltd, Beijing, Chi- na), 1 pl of each primer, 2 ul of template DNA and 9 ul of DNase-free ddH,O. The amplification conditions consisted of an initial denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 46 °C for 30 s (52 °C for 28S rRNA), extension at 72 °C for 1 min and a final extension at 72 °C for 10 min. Sequencing and data analysis Amplification products were detected using agarose gel electrophoresis and subsequently sequenced by Sangon Biotech (Shanghai, China). The obtained sequences were uploaded and compared to the existing sequences in GenBank (www.ncbi.nlm.nih.gov/Genbank) using the Basic Local Alignment Search Tool (BLAST) provided by the National Center for Biotechnology Information. To conduct phylogenetic analyses, available sequence zse.pensoft.net 428 data for the superfamily Solenoidea were retrieved from GenBank (Suppl. material 1: table S1). Hiatella arctica (Linnaeus, 1767) and Solecurtus divaricatus (Lischke, 1869) were regarded as the outgroup taxa. The sequences of each gene fragment were independently aligned using MAFFT v.7 (Katoh and Standley 2013) with the G-INS-i and Q-INS-i algorithms for the protein-coding and ribo- somal regions, separately. The sequences of three genes from the same individual were concatenated into a single sequence in SequenceMatrix v.1.8 (Vaidya et al. 2011). The best-fitting evolutionary model of the concatenated dataset was selected using the Akaike Information Crite- rion, as implemented in jModelTest 2.1.10 (Darriba et al. 2012). Phylogenetic trees were constructed based on the Maximum Likelihood (ML) using IQ-TREE v. 2.1.3 with bootstrap values for 2000 replicates (Minh et al. 2020). Bayesian Inference (BI) analysis was performed using MrBayes v.3.2.7, with three parallel runs of five million generations each, sampling every 1000 generations and burn-in set to 25% (Ronquist et al. 2012). Multiple species delimitation methods were used to tn- vestigate the hypothesis that the specimen represents a dis- tinct species. The COI data of 30 homologous sequences were analysed using Automated Barcode Gap Discovery (ABGD) carried out by the web-based interface (available at https://bioinfo.mnhn. fr/abi/public/abgd/abgdweb.html/) (Puillandre et al. 2012) using the Kimura 2 parameter substi- tution model (TS/TV = 2.0), prior for the maximum value of intraspecific divergence ranging between 0.001 and 0.1, 10 recursive steps and a relative gap width (X) of 1.0. Bayesian implementation of the Poisson Tree Processes (bPTP) spe- cies delimitation model was analysed (Zhang et al. 2013) at the web server of the Heidelberg Institute for Theoretical Studies, Germany (http://species.h-its.org/) using ML phy- logenetic trees as input data. Markov Chain Monte Carlo (MCMC) runs were performed for 100,000 steps and sam- pled every 100 steps with burn-in set to 0.1. Removing the outgroup in initial runs did not affect delimitation results. The General Mixed Yule Coalescent (GMYC) model (Pons et al. 2006) was used in PyR8S of 1TaxoTools v.0.1 (Vences et al. 2021) with default parameters to determine the species of analysed individuals, based on ultrametric time trees de- rived from single-locus data. Pairwise comparisons of the p-genetic distances, based on COI genes, were also calcu- lated by MEGA X (Kumar et al. 2018). Results Systematics Superfamily Solenoidea Lamarck, 1809 Family Pharidae H. Adams & A. Adams, 1856 Genus Cultellus Schumacher, 1817 Type species. Solen maximus Gmelin, 1971 (by monotypy). zse.pensoft.net Yu, Y. et al.: A new species of the genus Cultellus Cultellus exilis sp. nov. https://zoobank.org/4F4A CFDD-3083-4EF7-900E-C259F51ECE95 Figs 2, 3, 4F Type specimens. Holotype: MBM229032, one complete in- dividual, collected on 28 May 2021 by Agassiz trawl on the research vessel “TAN KAH KEE” (muddy bottom, depth 55 m). Paratype: MBM264485, one complete specimen, collected from the Beibu Gulf, China, January 1962 (muddy bottom, depth 55 m); MBM264488, two complete speci- mens, collected from the Beibu Gulf, China, December 1959 (Habitat unknown); MBM264497, one complete specimen, collected from Haimen, Guangdong Province, China, March 1954 (Habitat unknown); MBM264500, one complete spec- imen, collected from Shanwei, Guangdong Province, China, January 1995 (Habitat unknown); MBM229040, one com- plete specimen, collected from Hainan Province, China, Jan- uary 1959 (muddy bottom, depth 91.5 m). Type locality. Neritic zone of the South China Sea (depth 55 m, 20°1'13.44"N, 117°10'45.84"E); Muddy bottom. Etymology. The specific epithet “exilis” is derived from the Latin, referring to its slender shell, which is a remarkable difference from other species in this genus. Description. Shell medium in size, flattened, elon- gate, fragile, glazed, translucent, equivalve, inequilateral. Some specimens are covered with various-sized bubbles on their surface (Figs 2A—D, 3A—D). Umbo depressed, weakly prosogyrous, situated at anterior 2/9 of shell. An- terior area short, elliptical, posterior area elongated, both ends gaping, anterior end slightly turned-up, posterior end slightly more pointed than anterior end; antero-dor- sal margin downward sloping, postero-dorsal margin almost straight, ventral margin rather arcuate, poste- ro-ventral margin more curved than antero-ventral, mid- dle-ventral margin almost straight. Periostracum of valve surface yellowish; shell surface with fine, dense, regular and non-isometric co-marginal growth lines, without ra- dial lines; lunule and escutcheon absent. Ligament short, but strong, dark brown and situated opisthodetic. Interior shell off-white, with yellowish periostracum in margin. Each valve with one white, strong, thin, straight, internal radial rib extending from umbonal area to anterior end, forming ca. 21° angle with antero-dorsal margin (Fig. 2H-—J). Anterior adductor muscle scar subtrigonal, obvious, near internal rib; posterior adductor scar falciform, almost straight near the dorsal margin; pedal retractor scars rhom- bic, situated at the corner between umbo and internal rib; pallial sinus shallow, pallial lines connected to adductor scars. Left valve with three strong and projecting cardinal teeth, central tooth bifid at the top and confluent at the base, without lateral tooth (Fig. 2H); right valve with two long and strong cardinal teeth, anterior protruding downwards, poste- rior pointing to the back end, without lateral tooth (Fig. 21). Siphons short and bifurcated, situated at posterior area; gills transversely folded; foot strong, depressed, truncat- ed, situated at anterior area. Measurement. Holotype: MBM229032: L= 34.66 mm, H = 10.35 mm, W = 2.27 mm. Paratype: MBM264485: L= Zoosyst. Evol. 100 (2) 2024, 425-436 Figure 2. Cultellus exilis sp. nov. (holotype) A, B. MBM229032, L = 34.66 mm; C, D. Exterior views of left and right valve; E, F. Interior views of left and right valve; H. Hinge of left valve; I. Hinge of right valve; J. Internal radial rib of left valve. 45.42 mm, H = 12.65 mm, W = 2.37 mm; MBM264488: L=61.51 mm, H=16.26mm, W=3.73 mm; MBM264497: L=77.05 mm, H=21.14mm, W =4.72 mm; MBM264500: L=73.30 mm, H=19.74mm, W =4.52 mm; MBM264601: L= 44.32 mm, H = 12.42 mm, W = 2.33 mm. Remarks. The shells of Cu/te/lus are relatively less elongated compared to those of the other genera of So- lenoidea. Typically, the shells of Cultellus have rounded anterior and posterior ends, an anteriorly located umbo, strong internal ribs and three cardinal teeth on the left zse.pensoft.net 430 Yu, Y. et al.: A new species of the genus Cultellus Figure 3. Cultellus exilis sp. nov. (paratypes) A-D. MBM264485; E-—H. MBM264488; I-L. MBM264497; M—P. MBM264500; Q-T. MBM229040. Scale bars: 10 mm. zse.pensoft.net Zoosyst. Evol. 100 (2) 2024, 425-436 valve and two on the right (Thiele 1992). The remarkable morphologic similarity amongst Cultell/us members makes species identification challenging. The distinguishing fea- tures that separate Cultellus exilis sp. nov. from other spe- cies in this genus are its particularly slender, fragile and translucent shells. Additionally, the new species differs from the C. maximus (Fig. 4E) in that its posterior end is notably narrower than its anterior end (Gmelin 1791). It closely resembles C. attenuatus (Fig. 4A) and C. vitreus (Fig. 4D). However, C. attenuatus (Fig. 4A) differs from the new species by the anterior area of the shell, which is obviously wider than the posterior part. Additionally, the posterior end of C. vitreus is slightly truncated (Fig. 4D), whereas Cultellus exilis is more curved. C. subellipticus (Fig. 4C) differs from the new species by the posterior area of the shell, which 1s much wider than the anterior part (Dunker 1862; Clessin 1888). Compared to this new spe- cies, the length-to-height ratio of C. hanleyi is 3.3—-3.4:1 (Fig. 4B), whereas the ratio of Cultellus exilis is 3.5—3.8:1. Geometric morphometrics of shell outlines. Prin- cipal Component Analysis (PCA) was conducted on the Progrustes alignment outline data of 32 samples repre- senting six species. The results revealed that the first three principal components accounted for a cumulative contri- bution rate of 96.88% (PCA1 80.79%, PCA2 13.97% and PCA3 2.12%), indicating that they can represent the major morphological differences amongst the samples. Accord- ing to the extreme distortion state of the thin plate spline plots, the main difference of all samples along the PCA1 axis occurs in the relative height of the shell (Fig. 5B, C). Differences in the PCA2 axis mainly involve the length- to-height ratio of the shells. Shells were increasingly elon- gated from the PCA2+ to PCA2- direction (Fig. 5A, D). Considering the distortion observed along both the PCA1 and PCA2 axes, the main variation in the outlines of the Six Species within the genus Cu/te/lus is the relative length and height of the shell. Discriminant analysis of all data indicated that the first two variance values explain 96.98% of the overall variation. A Discriminant analysis plot clear- ly demonstrated that Cultellus exilis sp. nov. is distinctly separated from the other species, forming its own cluster (Fig. 6). Therefore, the geometric morphometric analysis results, based on outlines, support distinguishing the new species from other species within the genus Cu/tellus. Species delimitation analyses. All species delimita- tion analyses, namely ABGD, bPTP and GMYC, con- ducted on the COI sequences, resulted in the delimita- tion of eleven species in the superfamily Solenoidea. The analysis confirmed that Cultellus exilis sp. nov. 1s a dis- tinct species from other Culte/llus species. Sequences of the genus Cul/te/lus were delimited into four species, 1.e. C. attenuatus, C. subellipticus, C. maximus and Cultellus exilis sp. nov. (Fig. 7). In addition, based on the available molecular data, the analysis of a 581-bp fragment of the COI gene yielded a 12.2%—13.9% pairwise distance be- tween Cultellus exilis sp. nov. and other congeners, a di- vergence higher than the intraspecific variation (0O—1.7%) of the genus Cultellus (Suppl. material 1: table S2). 431 Phylogenetic analyses. The best-fitting evolutionary model of the concatenated dataset (COI, 16S and 28S) was selected as GIR+G+I using the Akaike Information Crite- rion implemented in jModelTest 2.1.10. The evolutionary relationships amongst the razor clams were depicted on a phylogenetic tree constructed using the ML and BI meth- ods. These trees exhibited highly similar topologies (Fig. 8). Phylogenetic trees obtained from the datasets were completely resolved and highly supported, as indicated by posterior probability (PP) or bootstrap scores (BS). Cultellus exilis sp. nov. and the two other Culte/lus species formed a clade within the family Pharidae. The sequences of Cultellus exilis sp. nov. recovered a well-supported lin- eage distinct from the other congeners. Notably, Ensiculus cultellus and Phaxas pellucidus did not cluster together with the genus Cultellus. Instead, they occupied differ- ent branch positions in both ML and BI trees. On the ML tree, Ensiculus cultellus clustered with Phaxas pellucidus and formed a sister clade with the genus Siliqua. While on the BI tree, Ensiculus cultellus grouped with the genus Siliqua, constituting a sister lineage to Phaxas pellucidus. All phylogenetic results strongly supported the close re- lationship between pharids and solenids (PP = 0.99; BS = 92). Furthermore, the family Hiatellidae was found to be closely related as a sister to the superfamily Solenoidea (PP = 1; BS = 100). The best-fitting evolutionary mod- el of the COI sequence was GTR+G. The phylogenetic tree inferred using ML criteria, based on single gene COI (Fig. 8), showed a similar overall topology. Discussion In this paper, we present a new species of razor clam that was confirmed using an integrative taxonomic approach involving shell morphological comparisons, geometric morphometrics and genetic analysis. This species can be morphologically distinguished from other congeners by its slender valve, more curved posteroventral margin and relatively larger protractor scar (Fig. 4). The geometric morphometric analyses, based on outline data, revealed that samples of Cultellus exilis sp. nov. were clustered to- gether and clearly separated from other species of this ge- nus (Fig. 6). The molecular analysis demonstrated that this species belongs to the genus Culte/lus, but is genetically distinct from other known Culte/lus species (Figs 7, 8). The genus Culte/lus currently encompasses five valid extant species that inhabit the waters of the Indo-West Pacific, with most ranging from tropical to temperate seas (Okutani 2000; Swennen et al. 2001; Min et al. 2004; Thach 2005; Thach 2007; Xu and Zhang 2008; Huber 2010; Coan and Petit 2011; Poppe 2011). The new spe- cies, Cultellus exilis sp. nov., was also found from this region (Fig. 1). The paratype specimens were previously misidentified as C. attenuatus, but our study corrected the misidentifications using integrative taxonomy. Geometric morphometrics has been used to quantify morphologi- cal changes in the process of biological evolution since zse.pensoft.net 432 Yu, Y. et al.: A new species of the genus Cultellus : h Museum (Natural History) i98G103 /2 SYNTYPE Cultelius hanley! Dunkec 1862 C?Feb). PZ.S. (1861): 421 nm? 14, Loc. — 7 Coll. Cummings Ace. 1829 Figure 4. A. Cultellus attenuatus Dunker, 1862. Two syntypes, NHMUK 20240145: L = 54.7 mm, H = 13.9 mm; L = 45.2 mm, H = 12.2 mm; B. Cultellus hanleyi Dunker, 1862. One syntype, NHMUK 1986103: L = 54.1 mm, H = 16.1 mm; C. Cultellus su- bellipticus Dunker, 1862. One syntype, NHMUK 20240146: L = 46.3mm, H = 16.0 mm; D. Cultellus vitreus Dunker, 1862. One syntype, NHMUK 20240147: L = 39.5 mm, H = 10.9 mm; E. Cultellus maximus (Gmelin, 1791). MBM264615: L = 33.6 mm, H= 11.3 mm; FE, Cultellus exilis sp. nov. MBM229032: L = 34.66 mm, H = 10.35 mm. B Cultellus attenuatus @ Cultellus exilis sp. nov. @ Cultellus hanleyi @ Cultellus maximus @ Cultellus subellipticus Cultellus vitreus PCA 2 (13.97%) me UTI TTT " pace TUTTO TNMVIIONN HEU eit PCA 1 (80.79%) Figure 5. Principal component and thin plate spline analyses, based on outlines of the genus Cultellus. A. The extreme distortion of the outlines in the negative of PC2; B. The extreme distortion of the outlines in the positive of PC1; C. The extreme distortion of the outlines in the negative of PC1; D. The extreme distortion of the outlines in the positive of PC2. Each colour of the dot in the principal component analysis diagram represents a species. zse.pensoft.net Zoosyst. Evol. 100 (2) 2024, 425-436 3.0 -6.0 LDA 2 (24.51%) -9.0 433 6.0 Cultellus attenuatus Cultellus exilis sp. nov. Cultellus hanleyi Cultellus maximus Cultellus subellipticus Cultellus vitreus LDA 1 (72.47%) Figure 6. Scatter plot of discriminant analysis of the genus Cultellus. Each colour of the dot in the principal component analysis diagram represents a species. Z1IC01 Solecurtus divaricatus Z3IC02 Solecurtus divaricatus 0.2 BivAToL-195 Hiatella arctica 211012-1 Cultellus attenuatus 220309-2 Cultellus attenuatus 220309-1 Cultellus attenuatus 211012-2 Cultellus attenuatus 220726-5 Cultellus attenuatus 211119-10 Cultellus subellipticus 211119-9 Cultellus subellipticus 210805-3 Cultellus exilis sp. nov MN119650 Cultellus maximus jJC04 Sinonovacula rivularis JJC03 Sinonovacula rivularis JJC02 Sinonovacula rivularis YC05 Sinonovacula constricta | YCO8 Sinonovacula constricta | YC07 Sinonovacula constricta MW311108 Pharella acutidens MwW311109 Pharella acutidens FSJC03 Siliqua radiata FSJC04 Siliqua radiata DZC02 Solen grandis DZC03 Solen grandis 210707-1 Solen strictus 210707-2 Solen strictus 210914-5 Solen strictus ABGD Et EE 0, a nn ee eee bPT? i en nn ee eee GMYCHE EE 0 a nn nn 2 eee Figure 7. Phylogenetic tree obtained by the Maximum Likelihood (ML), based on COI gene sequences. Numbers adjacent to nodes refer to ML bootstrap scores (BS < 50 represented by “*’’). The results of three species delimitation methods are shown on the right of the figure (Each species is represented by a single colour). its proposal (Jacobson and Moyers 1993). Although it is not as widely used in molluscs as other methods, some studies have shown that geometric morphometrics anal- ysis 1s suitable for explaining the convergent evolution of bivalves (Serb et al. 2011; Sherratt et al. 2016), dis- covering taxonomic information for species identification (Marinho and Arruda 2021) etc. This study highlights the importance of using integrative taxonomy for species identification and phylogenetic studies. In recent years, integrative taxonomy has been proven to be more reliable for biological taxonomy and species identification, espe- cially for morphologically similar or indistinguishable species (Zhang et al. 2018a, 2018b; Sorensen et al. 2020; Zhang et al. 2020). Furthermore, integrative taxonomy facilitates phylogenetic analysis and exploration of evo- lutionary relationships amongst closely-related species (Sun et al. 2016; Liao et al. 2021). Integrative taxonomy will play a significant role in the taxonomy and phyloge- ny of marine organisms in the future. The findings of this study are largely consistent with the previous molecular research on the phylogenetic relationships of the superfamily Solenoidea. Our phy- logenetic trees, constructed using mitochondrial (COI, 16S) and nuclear (28S) genes, revealed that Cultellus, zse.pensoft.net 434 0.99/91 io0 , Z1C01 Solecurtus divaricatus L702 Selecurtus divaricatus 0.2 Yu, Y. et al.: A new species of the genus Cultellus 211012-1 Cultellus attenuatus 220309-1 Cultellus attenuatus 220309-2 Cultellus attenuatus 220726-5 Cultellus attenuatus 211012-2 Cultellus attenuatus 211119-10 Cultellus subellipticus 211119-9 Cultellus subellipticus 210805-3Cultellus exilis sp. nov. JiC02. Sinonovacula rivularis J1C03 Sinonovacula rivularis C04 Sinonovacula rivularis YC05 Sinonovacula constricta YC08 Sinonovacula constricta YC07 Sinonovacula constricta MW311108 Pharella acutidens MW311109 Pharella acutidens F8JC03 Siliqua radiata FsJco4 Siliqua radiata bzC02 Solen grandis DzC03 Solen grandis 210707-1 Solen strictus 210707-2 Solen strictus 210914-5 Solen strictus BivAToL-195 Hiatella arctica Pharidae Solenidae g Hiatellidae I Solecurtidae Figure 8. Phylogenetic tree inferred by Bayesian Inference analysis (BI) and Maximum Likelihood (ML), based on concatenated dataset of COI, 16S and 28S genes. Bayesian posterior probability and Maximum Likelihood bootstrap scores (left and right, re- spectively. “-” Sinonovacula, Siliqua, Pharella, Ensiculus and Phaxas belong to the family Pharidae. Interestingly, it appears that Cultellus cultellus (= Ensiculus cultellus) and Cultel- lus (Cultrensis) adriaticus (= Phaxas pellucidus) do not actually belong to the genus Cul/tellus, validating the mor- phological views proposed by Adams (1861) and McKay and Smith (1979). Both BI and ML results demonstrated that Pharidae and Solenidae formed a sister group. Fur- thermore, Pharidae, Solenidae and Hiatellidae clustered into a monophyletic group (Fig. 8), indicating that Sole- noidea and Hiatellidae belong to the order Adapedonta. Bieler et al. (2014) pointed out the common character- istics amongst the species of Adapedonta, including the absence of the oesophageal lip, a simple transverse ridge pattern of the right wall sorting area and the presence of a ridge posteriorly bordering the right wall sorting area. Compared to previous studies, our study incorporated more molecular information and taxonomic groups, fur- ther verifying the reliability of the current systematics (Taylor et al. 2007; Bieler et al. 2014; Bolotov et al. 2018; Li-ep al. 2022); Moreover, Bolotov et al. (2018) found that the genus Cultellus clustered together with Sinonovacula and Pharel- la in a phylogenetic tree of the family Pharidae, based on the concatenation of three genes (COI+16S+28S). They classified the genera Novaculina, Sinonovacula, Pharella and Cultellus as the subfamily Pharellinae. In our study, we observed that the genus Si/iqgua formed a sister clade with the genera Ensiculus and Phaxas. These findings col- lectively suggest that the current taxonomy of the fami- ly Pharidae is indeterminate and needs to be verified and reassessed by taking into account additional information. zse.pensoft.net represents different branch position on ML and BI trees) are shown above the branch. Acknowledgements We are grateful to Dr. Tom S. White and Dr. Andreia Salvador from the Natural History Museum, London for kindly providing photos of type materials of Cultellus at- tenuatus, C. hanleyi, C. subellipticus and C. vitreus. We also express our gratitude to Hao Wang, Huijie Liu and Yuyan Zhang from the Institute of Oceanology, Chinese Academy of Sciences, and the crews of RV TAN KAH KEE of Xiamen University, for their efforts and assistance in collecting samples for this study. 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Copyright notice: This dataset 1s 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.100.113972.suppl1