Ten specimens of the new species were collected from the intertidal zones of Rushan and Qingdao in Shandong Province, China. The live animals were photographed and preserved in 99% ethanol for molecular study and 4% formalin for morphological dissections. Type specimens were deposited at the Marine Biological Museum, Chinese Academy of Sciences (MBMCAS).

External morphology and internal anatomy were examined, dissected, and photographed using a dissecting microscope (ZEISS Discovery V20). For scanning electron microscopy (SEM) analysis, jaws and radulae were dissected from the buccal bulb. They were then cleaned with 10% NaOH, rinsed in distilled water, air-dried, coated with gold, and examined under SEM at an accelerating voltage of 5 kV.

Six individuals of the new species and two specimens of Pseudobornella orientalis were subjected to molecular analysis. Genomic DNA from each individual was extracted using the Column Genomic DNA Isolation Kit (Beijing TIANGEN, China) following to the manufacturer’s instructions. Polymerase chain reactions (PCRs) were conducted in a total volume of 25 μl, including 2 μl DNA template, 0.5 μl of each 10 mM primers, 0.5 μl of 10 mM dNTPs, 2.5 μl of 10× buffer, and 0.5 U Taq DNA polymerase. Thermal cycling was performed under the following conditions: 94 °C for 2 min (initial denaturation); followed by 30 cycles of 94 °C for 30 s (denaturation), 42 °C for 45 s (annealing), and 72 °C for 60 s (extension); and a final extension at 72 °C for 5 min. The cytochrome c oxidase subunit I gene (COI) was amplified by polymerase chain reaction (PCR) using the primers LCO1490 (forward: 5′-GGTCAACAAATCATAAAGA TATTGG-3′) and HCO2198 (reverse: 5′-TTAACTTCAGGGTGACCAAAAAATCA-3′) (Folmer et al. 1994), the 16S ribosomal RNA (16S rRNA) was amplified by the primers 16Sar (forward: 5’-CGCCTGTTTATCAAA AACAT-3’) and 16Sbr (reverse: 5’-CTCCGGTTTGAACTCAGATCA-3’) (Palumbi 1996), and the histone 3 gene (H3) was amplified by the primers HexAF (forward: 5’-ATGGCTCGTACCAAGCAGACGGC-3’) and HexAR (reverse: 5’-ATATCCTTGGGCATGATGGTGAC-3’) (Colgan et al. 1998). PCR products were verified on a GelGreen-stained 1.5% agarose gel and sequenced using the BigDye Terminator Cycle Sequencing Kit (v. 3.1 Applied Biosystems, USA) and an AB PRISM 3730 (Applied Biosystems, USA) automatic sequencer.

Three partial gene sequences (COI, 16S, and H3) were obtained from each specimen and deposited in the GenBank. Additionally, sequences of other dendronotid species were retrieved from GenBank and used for phylogenetic analyses (Table 1). Two tritoniid species, Marionia blainvillea (Risso, 1818) and Marioniopsis arborescens (Bergh, 1890), were used to root the phylogenetic tree.

Sequences were aligned with MAFFT (Katoh and Standley 2013) using ‘G-INS-i (accurate)’ strategy and normal alignment mode. Separate analyses were conducted for COI (634 bp), 16S (401 bp), H3 (302 bp), and concatenated data (1328 bp). Ambiguously aligned fragments of 16S alignment were removed using Gblocks (Talavera and Castresana 2007). Maximum-likelihood phylogenies (ML) were inferred using IQ-TREE (Nguyen et al. 2015) under Edge-unlinked partition model for 10,000 ultrafast bootstraps (Minh et al. 2013), as well as the Shimodaira–Hasegawa-like approximate likelihood ratio test (Guindon et al. 2010). Bayesian-inference (BI) phylogenies were inferred using MrBayes v. 3.2.6 (Ronquist et al. 2012) under partition model (2 parallel runs, 5 million generations), in which the initial 25% of sampled data were discarded as burn-in. The best-fit models of evolution (GTR+F+I+G4 for COI and 16S, GTR+F+I for H3) were determined by ModelFinder (Kalyaanamoorthy et al. 2017) using AIC criterion. Results were visualized using FigTree v. 1.4.3. The p-distances within and among each species grouping were estimated with MEGA 6 (Tamura et al. 2013) based on the Kimura 2-parameter (K2P) model (Kimura 1980).

The Automatic Barcode gap Discovery (ABGD) (Puillandre et al. 2012) and the Assemble Species by Automatic Partitioning (ASAP) (Puillandre et al. 2021) methods were used to assess the number of Pseudobornella species. The alignment from the fast-evolving COI gene was uploaded to the online servers of ABGD (https://bioinfo.mnhn.fr/abi/public/abgd/abgdweb.html) and ASAP (https://bioinfo.mnhn.fr/abi/public/asap), respectively. The analyses were performed with the model of Jukes-Cantor (JC69) with default settings.

Superfamily Dendronotoidea Allman, 1845

Family Dendronotidae Allman, 1845

 Pseudobornella qingdaoensis sp. nov.

https://zoobank.org/3213D127-1786-4FA9-9BE7-9FEE128E1538

Figs 1, 2, 3

Type material.

Holotype (Fig. 1A, B): China • 1 specimen, length alive 40 mm; dissected; Shandong Province, Qingdao, Golden Beach Park, intertidal zone; 35°57'03"N, 120°14'26"W; 18 Jan. 2025; Shu-Qian Zhang leg.; MBM288155. Paratypes 1–4: China • 4 specimens, lengths 35–40 mm; complete; complete; Shandong Province, Qingdao, Golden Beach Park, intertidal zone; 35°57'03"N, 120°14'26"W; 20 Jan. 2025; Shu-Qian Zhang leg.; MBM288156–MBM288159. Paratype 5 (Fig. 1C): China • 1 specimen, preserved length 10 mm; dissected; Shandong Province, Qingdao, Taiping Bay, intertidal zone; 11 Apr. 2024; Ju-Hao Wang leg.; MBM288160. Paratype 6 (Fig. 1D): China • 1 specimen, preserved lengths 20 mm; dissected; Shandong Province, Rushan, intertidal zone; 19 Apr. 2019; Ju-Hao Wang leg.; MBM288161. Paratypes 7–10: China • 4 specimens, preserved lengths 20 mm; complete; same collection data as paratypes 6; MBM288162–MBM288165.

Figure 1. 

Pseudobornella qingdaoensis sp. nov. A, B holotype, MBM288155, length 40 mm C paratype 5, MBM288160, preserved length 1.0 cm D paratype 6, MBM288161, preserved length 20 mm.

Diagnosis.

A large Pseudobornella reaching 40 mm in body length. Background color generally translucent yellow to dark brown, with numerous scattered orange to brown spots and white blotches. Radula formula 11 × 3.0.3.

Description.

External morphology (Fig. 1). Living animals up to 40 mm in length. Foot wide and tapering relatively abruptly towards the tail. Background color generally translucent yellow to dark brown, with numerous scattered orange to brown spots and white blotches. In some individuals, the white blotches forming two longitudinal streaks running along both sides of the dorsum. Anterior margin of head rounded. Each side of the mouth equipped with four or five smooth, tapering oral tentacles of varying size. Rhinophore sheath very elongate. Upper edge of each rhinophore sheath bearing four lateral digitiform branched papillae and one posterior papilla that is remarkably longer. Rhinophores with ~12 lamellae. Dorsal margin with four pairs of dorsolateral papillae, decreasing in size towards the posterior end of the foot. Each papilla with numerous unbranched gills attached to its inner side. Anus small, located on the right side of the dorsum between the first and second pair of dorsolateral appendages. Genital opening located on the right side, midway between the rhinophore sheath and the first dorsolateral appendage. Foot pale pink, scattered with numerous very small, indistinct darker spots, separated from the head by a long, transversal groove.

Internal anatomy. Jaws (Fig. 2A, B) elongate, inner margin (masticatory margin) terminating distally in a small, pointed process. Masticatory margin equipped with rows of short, cone-shaped rodlets. Radular (Fig. 2C–F) formula 11 × 3.0.3. Rachidian teeth stout, with a strong and bluntly pointed cusp and ~9–12 denticles on both sides of the cusp. Innermost lateral teeth with a very strong elongate and sharp cusp and serrated sides. Outer lateral teeth simple and hamate, and outermost one smallest.

Figure 2. 

Jaws, radulae of Pseudobornella qingdaoensis sp. nov. A jaw, MBM288155 B jaw masticatory border, MBM288155 C, D radula, MBM288155 E, F radula, MBM288161. Scale bars: 1 mm (A); 50 μm (B, C); 20 μm (D, F); 100 μm (E).

Reproductive system (Fig. 3) located in the front right corner of the body. Ampulla very large, sausage-shaped, one end connecting with ovotestis via a thin hermaphroditic duct, the other end divided into two different, thin ducts: the short oviduct entering the female gland mass and the second duct branching into a wide, short prostate which extends into a long, folded vas deferens, narrowly opening to a wide and stout penial sac. Two ducts departing from the genital aperture: vagina relatively long and simply folded, connecting with a small, rounded receptaculum seminis; second duct wider and shorter, connecting with a very large pyriform bursa copulatrix.

Figure 3. 

Reproductive system of Pseudobornella qingdaoensis sp. nov., with ovotestis and female gland mass removed, MBM288155. Abbreviations: am–ampulla; bc–bursa copulatrix; ga–genital aperture; hd–hermaphrodite duct; ov–oviduct, pr–prostate, ps–penial sac; rs–receptaculum seminalis; va–vagina; vd–vas deferens. Scale bar: 1 mm.

Etymology.

The new species is named after its type locality, Qingdao.

Distribution and habitat.

Known from Yellow Sea, China, and possibly also distributed in Japan (Kinoshita 2002; Nobuhiko 2017). It was found in the rocky intertidal zone, feeding on a hydroid (Kinoshita 2002).

The phylogenetic trees inferred using BI and ML criteria were generally congruent (Fig. 4). The genus Pseudobornella was recovered as monophyletic with high support (PP = 1, BS = 99). Within the genus, P. qingdaoensis sp. nov. formed an independent clade (PP = 1, BS = 100) from P. orientalis. This result supports the systematic placement of the new species in the genus Pseudobornella and its separation from P. orientalis.

Figure 4. 

Phylogenetic tree inferred by Bayesian analysis (BI) and maximum likelihood (ML) based on concatenated dataset of COI, 16S and H3 genes. Numbers adjacent to nodes refer to BI posterior probability (PP > 0.5) and ML bootstrap scores. Numbers in the vertical black bars indicate the results of ABGD and ASAP species delimitations.

The ABGD analysis resulted in the delimitation of two species. The prior maximal distance (p-distance) ranged between 0.0017 and 0.1. However, the ASAP analysis recovered three partitions with the lowest score (generally considered to be the most supported partition scheme) (Fig. 4; Suppl. material 1). The partition with the second highest score was chosen here as it avoided the splitting of two populations of P. orientalis with a pairwise genetic distance of only 0.6%. In both cases, P. qingdaoensis sp. nov. was separated from P. orientalis.

Based on the available molecular data, the analysis of a 634-bp fragment of the COI gene yielded a pairwise distance of 11.7–12.5% between P. qingdaoensis sp. nov. and P. orientalis, a divergence much higher than the known intraspecific variation (0–0.6%) (Suppl. material 2), further supporting the separation of P. qingdaoensis sp. nov. from P. orientalis.

The authors have declared that no competing interests exist.

No ethical statement was reported.

This work was financially supported by the National Natural Science Foundation of China (42090044), the Natural Science Foundation of Shandong Province, China (ZR2021MD072), and the Biological Resources Programme of Chinese Academy of Sciences (CAS-TAX-24-031).

SZ conceived and designed the project. SZ and JW collected and performed morphological examinations of the specimens. SZ conducted DNA sequencing, analyzed the molecular data, and drafted the manuscript.

Shuqian Zhang https://orcid.org/0000-0002-1521-5317

All of the data that support the findings of this study are available in the main text or Supplementary Information.