A New Species of the Intertidal Limpet Eoacmaea (Patellogastropoda: Eoacmaeidae) from Yonaguni Island, Japan and Taiwan

© 2021 Academia Sinica, Taiwan Open Access A New Species of the Intertidal Limpet Eoacmaea (Patellogastropoda: Eoacmaeidae) from Yonaguni Island, Japan and Taiwan Tomoyuki Nakano1,*, Takenori Sasaki2, Takeharu Kosuge3, and Benny K.K. Chan4 1Seto Marine Biological Laboratory, Field Science Education and Research Centre, Kyoto University, 459 Shirahama, Nishimuro, Wakayama, 649-2211, Japan. *Correspondence: E-mail: [email protected] (Nakano) 2The University Museum, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail: [email protected] (Sasaki) 3Vietnam Japan University, Luu Huu Phuoc, My Dinh 1, Nam Tu Liem, Hanoi, Vietnam. E-mail: [email protected] (Kosuge) 4Biodiversity Research Center, Academia Sinica, Nankang, Taipei 11529, Taiwan. E-mail: [email protected] (Chan) Received 28 February 2021 / Accepted 18 August 2021 / Published 19 November 2021 Communicated by Nozawa Yoko A new species of intertidal limpet was identified from Yonaguni Island, Japan and southern Taiwan in the present study and described as Eoacmaea nivea n. sp. It was previously reported by different authors as Cellana mauritiana, or as Acmaeid sp. in Taiwan, and as Patelloida sp. in Yonaguni Island, Japan. Based on detailed morphological observations and molecular analyses (COI), E. nivea n. sp. is distinguished from other Eoacmaea species. Eoacmaea nivea n. sp. is presently known from southern Taiwan, and a population on Yonaguni Island, Japan that is very limited. The present study reports a total 11 species of patellogastropod limpet species in Taiwan. The seven species—Cellana grata, Cellana toreuma, Nipponacmea nigrans, Nipponacmea fuscoviridis, Lottia dorsuosa, Lottia luchuana and Lottia tenuisculpta—were found in the intertidal on rocky shores along the north to northeastern shores of Taiwan. The five species—Cellana radiata, Lottia luchuana, Scutellastra flexuosa, Patelloida saccharina and E. nivea n. sp.—can be found mostly within depressions in limestone substrate in southeastern Taiwan. Of these, only Lottia luchuana is found throughout Taiwan, and overall has a tropical, not warmtemperate, distribution. Key words: Mollusca, Gastropoda, Limestone, Biogeography, Patellogastropoda. BACKGROUND The limpets belonging to the order Patellogastropoda are common inhabitants of intertidal rocky shores throughout the world oceans, typically as important grazers in intertidal systems (Branch 1985a b). The genus Eoacmaea was earlier referred to as the Patelloida profunda group (Christiaens 1975; Lindberg and Vermeij 1985). Nakano and Ozawa (2007) revealed that this group is different from other species of the genus Patelloida both morphologically and genetically and designated it as a new genus Eoacmaea (Nakano and Ozawa 2007). Furthermore, Eoacmaea was found to be a basal branch among the recent Patellogastropoda. The new family Eoacmaeidae was established for this genus (Nakano and Ozawa 2007). However, there are long branch attraction problems due to the long insertions and deletions in Lottioidea of Patellogastropoda to construct the phylogenetic tree (Uribe et al. 2019; Ponder et al. 2020). Although the distinct mt genome organization of Eoacmaea is reported (Uribe et al. 2019), the placement of this family is still debated (Uribe et al. 2019; Ponder et al. 2020). Eoacmaeid limpets formerly assigned as Patelloida profunda group are limited to limestone in high to supratidal zone and widely distributed in South Africa, Indo-Pacific and Caribbean. The highest diversity of eoacmaeid limpets can be found in the Indo-West Pacific Citation: Nakano T, Sasaki T, Kosuge T, Chan BKK. 2021. A new species of the intertidal limpet Eoacmaea (Patellogastropoda: Eoacmaeidae) from Yonaguni Island, Japan and Taiwan. Zool Stud 60:68. doi:10.6620/ZS.2021.60-68. Zoological Studies 60:68 (2021) doi:10.6620/ZS.2021.60-68 1 © 2021 Academia Sinica, Taiwan (Christiaens 1975; Ponder and Creese 1980; Lindberg and Vermeij 1985; Kirkendale and Meyer 2004). A molecular phylogenetic analysis of the genus Eoacmaea based on the two mitochondrial genes, 16S and COI has been conducted and results revealed that Eoacmaea is genetically highly differentiated in each island and likely harbors undescribed species especially in IndoWest Pacific (Kirkendale and Meyer 2004). Among this undescribed diversity, so far only Eoacmaea javanica has been described from Java, Indonesia (Nakano et al. 2005). The present study describes a new species of Eoacmaea from Yonaguni Island, Japan and Taiwan (Kosuge and Sasaki 2002) based on molecular data and detailed morphological and anatomical characters. This species was previously reported as Cellana mauritiana, or as Acmaeid sp. in Taiwan (Oyama and Takemura 1959; Takenouchi 1993), and as Patelloida sp. in Yonaguni Island, Japan (Kosuge and Sasaki 2002). MATERIALS AND METHODS Sample collections Limpets were collected at intertidal shores from Yonaguni Island, Japan. In Taiwan, limpets were collected from Yeliu in northern coastline to Kenting in southern coastline (Fig. 1). The new Eoacmaea species described here was collected from the high intertidal to supratidal zones at two localities, the outlying Pacific Island, Green Island and at Jialershui, southern Taiwan. Fig. 1. Collection localities of Eoacmaea nivea n. sp. in southeastern Asia. page 2 of 12Zoological Studies 60:68 (2021) © 2021 Academia Sinica, Taiwan Living specimens were preserved in 95% ethanol. Details of the specimens are listed in table 1. DNA extraction, PCR amplification and DNA sequencing The protocol of DNA extraction, PCR amplification for COI and DNA sequencing followed Nakano and Ozawa (2007). Phylogenetic analyses Sequences were assembled and edited using ATGC. Sequences of COI, which lacked any indels across the data set, were aligned manually using MacClade 4.03 (Maddison and Maddison 2002), referring to translated amino acid sequences. Phylogenetic trees were constructed with neighbour-joining (NJ) (Saitou and Nei 1987), maximum parsimony (MP) (Felsenstein 1988) and Bayesian methods (Ronquist and Huelsenbeck 2003). Bayesian analysis was performed by MrBayes v. 3.1.2 (Ronquist and Huelsenbeck 2003). The choice of substitution model used in in Bayesian phylogenetic analyses, GTR + I + G, was selected using MrModelTest v.2.1 (Nylander 2004). The NJ bootstrap analysis consisted of 10,000 replicates. The MP bootstrap analysis comprised 1,000 replicates of a heuristic search with 10 random addition sequence replicates and TBR branch-swapping. MrBayes was run with the following settings (nst = 6, rate = invgamma, mcmc ngen = 10,000,000). Trees were sampled every 100 generations. The first 10% of trees were discarded as burn-in. Anatomy and radular morphology The radula from [paratype specimen RM28705] was dissected and placed in 10% KOH at approximately 60°C for 5 min and rinsed in distilled water. The radula was examined by scanning electron microscope (SEM). Terminology used in the morphological description is adopted from Lindberg and Vermeij (1985) and Nakano et al. (2005). RESULTS Molecular data In total, seven specimens were newly sequenced in this study. The aligned dataset of 658 bp characters including the outgroup species, Patella caerulea, P. ferruginea and P. vulgata, had 297 variable and 247 parsimony-informative characters. There were no indels across the data sets. All sequences obtained in this study have been deposited in GenBank under Accession numbers (LC598472–LC598478) (Table 1). Table 1. Species identification, localities, voucher numbers and GenBank accession numbers for specimens genetically analyzed in this study Species Locality Specimen voucher Accession number DNA-ID Eoacmaea nivea n. sp. Umahana-saki, Yonaguni Island, Okinawa, Japan UMUT-RM28704 LC598472 L708 Sanninu-dai, Yonaguni Island, Okinawa, Japan UMUT-RM28705 LC598473 L709 Umahana-saki, Yonaguni Island, Okinawa, Japan UMUT-RM28706 LC598474 L710 Green Island, Taiwan SMBL-V0595 LC598475 L826 Green Island, Taiwan SMBL-V0596 LC598476 L827 Green Island, Taiwan SMBL-V0597 LC598477 L828 Jialershui, Taiwan ASIZM0001712 LC598478 L1104 Eoacmaea javanica (Nakano, Aswan & Ozawa, 2005) Sedekan Beach, Java, Indonesia NUGB-L558 AB238506 L526 Eoacmaea conoidalis (Pease, 1868) Cook Islands UF296033 AB238505 L603 Eoacmaea ESU 1 Zanzibar UF296016 AB238511 L601 Eoacmaea mauritiana (Pilsbry, 1891) Mauritius UF296014 AB238510 L626 Eoacmaea albonotata (Smith, 1901) South Africa UF295658 AB238508 L605 Eoacmaea profunda (Deshayes, 1863) Reunion UF295652 AB238507 L607 Eoacmaea omanensis (Christiaens, 1975) Gulf of Oman UF295654 AB238509 L609 Patella caerulea Linnaeus, 1758 Ceuta, Spain NUGB-L653 AB238577 L653 Patella ferruginea Gmelin, 1791 Ceuta, Spain NUGB-L655 AB238578 L655 Patella vulgata Linnaeus, 1758 Millport, UK NUGB-L340 AB238580 L340 Abbreviations: UMUT; The University Museum, The University of Tokyo. SMBL; Seto Marine Biological Laboratory, Kyoto University. ASIZ; Biodiversity Research Museum, Academia Sinica. page 3 of 12Zoological Studies 60:68 (2021) © 2021 Academia Sinica, Taiwan Molecular phylogeny All phylogenetic trees—whether constructed using NJ, MP or Bayesian analyses—confirmed the monophyly of E. nivea n. sp. Two clades were confirmed; one consists of Eoacmaea javanica, E. nivea n. sp. and E. conoidalis, and the other comprises E. sp. from Zanzibar, E. mauritiana, E. albonotata and E. omanensis (Fig. 2). Eoacmaea profunda is in the basal branch among Eoacmaea species. Pairwise genetic distances of COI between the species ranged from 6.4–22.2 % in the genus Eoacmaea (Table 2). Eoacmaea nivea had 0.3–0.6% intraspecific variation. TAXONOMY Family Eoacmaeide Nakano and Ozawa, 2007 Genus Eoacmaea Nakano and Ozawa, 2007 Type species Patella profunda Deshayes, 1863 Diagnosis: Light-colored shell medium and high in profile. Lateral teeth of radula typically equal size and shape. Habitats limited to limestome in high intertidal to splash zone. Fig. 2. Phylogenetic tree of Bayesian analysis based on the mitochondrial COI gene (658 bp). The numbers above each branch are Bayesian posterior probabilities/ NJ bootstrap/ MP bootstrap. L526 E. javanica L708 L828 L827 L710 L709 L1104 L826 L603 E. conoidalis L601 E. sp. Zanzibar L626 E. mauritiana L605 E. albonotata L609 E. omanensis L607 E. profunda L653 P. caerulea L653 P. ferruginea L340 P. vulgata 0.05 substitutions/site E. nivea n. sp. 1.00/100/100 0.90/78/80 1.00/100/100 1.00/100/98 0.60/68/72 0.88/81/88 0.72/62/58 1.00/100/100 0.88/81/ - page 4 of 12Zoological Studies 60:68 (2021) © 2021 Academia Sinica, Taiwan Eoacmaea nivea n. sp. (Figs. 3–5) urn:lsid:zoobank.org:act:4F15A48A-538A-43D5-935BF4DF30407758 Cellana mauritiana in part, not Pilsbry, 1892: Oyama and Takemura, 1959, Fig. Cellana (2). 1, 2. Acmaeidae sp.: Takenouchi, 1993, Fig. 2C. Patelloida sp.: Kosuge and Sasaki, 2002, Figs. 3A–I. Eoacmaea sp.: Nakano, 2017, Fig. 9, Pl. 16 Material examined: Holotype, UMUT-RM28074 (Length 20.4 mm, width 16.5 mm, height 8.2 mm), Umahana-saki, Yonaguni Island, Japan. Paratype 1, UMUT-RM28705, Sanninu-dai, Yonaguni Island, Japan. Paratype 2, UMUT-RM28706, Umahana-saki, Yonaguni Island, Japan. Paratype 3, ASIZM0001712, Jialershui, Taiwan. Paratype 4, SMBL-V0595, Green Island, Taiwan. Paratype 5, SMBL-V0596, Green Island, Taiwan. Paratype 6, SMBL-V0597, Green Island, Taiwan. Shell morphology Shell (Fig. 3) conical. thickened and oval in outline (width/length = 0.78–0.81). Apex located at the anterior 47–53% of shell length, and dorsally pointed as the highest point of shell. Exterior is sculptured by thin radial ribs, which are variable in thickness from evenly to differentially ribbed conditions. Ribs granulate at intersections with growth lines and fine commarginal sculpture. Shell exterior color is almost white except corroded apex in part of examined specimens (UMUTRM28074). Margin of shell is roughened with minutely projected ends of radial ribs. From lateral view, anterior and posterior slopes almost straight. Ventral margin apparently concaved, not planar. From ventral view, shell interior smooth and lustrous. Shell muscle scar deeply impressed. Interior color white in the marginal area, and yellow to orange in inner area; depth and size of colored area variable among specimens; brown markings may be formed in apical area. Anatomy Mantle margin (mm: Fig. 4A–C) thick, circumpallial tentacles completely invisible in fixed specimens (may be retracted or tiny length). Foot muscular, well developed and stiffened. Pallial cavity (pc: Fig. 4A–C) possesses a large ctenidium (ctn: Fig. 4D) extending from posterior left to anterior right. A pair of small elongate osphradia attached on floor of pallial cavity. Attachment area of shell muscle (sm) U-shaped, and divided into bundles, more finely in posterior than in anterior. Mouth (m: Fig. 5A) surrounded by thick outer lip (oli) and laterally extended oral lappets (ola). Eyes (e: Fig. 5B) exist at bases of cephalic tentacles (ct). Head contains muscular buccal mass (bm). Radular sac (rds: Fig. 5B) forms two loops; first loop (L1) on left posterior, second loop (L2) on right anterior. End of radular sac (ers) enlarged and bulbous behind the buccal mass. Part of stomach (st: Fig. 4A, C) and intestine (i) visible as pale lines on darker background color of digestive glands (dg) which on dorsal surface of the animal. Pericardium (pd: Fig. 4A, C) located on the left anterior side of limpet and with a single auricle (au: Fig. 4D) and ventricle (vn). Right kidney (rk) discernible on the right side of the animal. Radular morphology The radular sac contains 75 rows of radular Table 2. Uncorrected p-distances within and among species 1234567891011 1. E. javanica 0.00 2. E. sp. Zanzibar 13.4 0.00 3. E. conoidalis 8.5 14.1 0.00 4. E. albonota 15.2 6.4 15.7 0.00 5. E. profunda 21.6 20.5 22.1 20.7 0.00 6. E. omanensis 16.0 8.5 15 7.8 21.1 0.00 7. E. mauritiana 14.3 13.8 13.8 5.4 19.8 6.8 0.00 8. E. nivea n. sp. 7.6–8.1 14.6–15.0 8.8–9.1 14.7–15.2 21.9–22.2 15.5–16.0 14.3–14.7 0.3–0.6 9. P. caerulea 27.5 28.4 26.9 27.5 32.5 27.5 27.4 27.6-28.1 0.00 10. P. ferruginea 29.1 30.1 29.0 30.2 32.6 29.6 28.6 29.3-29.6 12.3 0.00 11. P. vulgata 29.6 29.9 29.9 29.0 32.8 29.0 29.4 29.1-29.6 13.8 12.8 0.00 page 5 of 12Zoological Studies 60:68 (2021) © 2021 Academia Sinica, Taiwan segments in paratype UMUT-RM28075 (Fig. 6A–C). Rows 1–66 have thickly mineralized orange-brown teeth, rows 67–70 are weakly mineralized, and rows 71–75 are organic and translucent. Each radula row consists of three pairs of lateral teeth and two pairs of marginal teeth. The first teeth are thickest among three pairs; cusps (L1c: Fig. 6D) are almost in contact between the right and left teeth; posterior margins of Fig. 3. Shell morphology of Eoacmaea nivea n. sp. A–C, Holotype, UMUT-RM28704, Umahana-saki, Yonaguni Island, Japan; 20.4 × 16.5 × 8.2 mm. D–F, Paratype 1, UMUT-RM28705, Sanninu-dai, Yonaguni Island, Japan; 17.9 × 13.9 × 7.6 mm. G–I, Paratype 3, Jialershui, Taiwan; 11.8 mm × 10.1 mm × 4.9 mm. page 6 of 12Zoological Studies 60:68 (2021) © 2021 Academia Sinica, Taiwan Fig. 4. External view of animal. UMUT-RM28074. A, Dorsal view. B, Ventral view. C, Left lateral view. D, Enlarged view of ctenidium and adjacent organs, with mantle removed. Abbreviations: a, anus; acs, afferent ctenidial sinus; au, auricle; cpt, cephalic tentacle; ctn, ctenidium; dg, digestive gland; ecs, efferent ctenidial sinus; epc, end of pallial cavity; ft, foot; h, head; i, intestine; mm, mantle margin; pc, pallial cavity; pd, pericardium; ps, pedal sole; rk, right kidney; sm, shell muscle; st, stomach; vn, ventricle. Animal size: length = 15 mm, width = 12 mm. page 7 of 12Zoological Studies 60:68 (2021) © 2021 Academia Sinica, Taiwan their cusps blunt and roundly curved; shafts (L1s: Fig. 6D) stout and short; bases concaved on inner side and broadly fused with second and third teeth on outer posterior side. Second lateral teeth (L2: Fig. 6D, E) almost 50% of first teeth in width and about 55% in length; inner margins of bases are curved towards the midline of the radula. Third lateral teeth (L3) slightly wider than second teeth; bases are mostly hidden in the dorsal view, anterior sides are thinly extended along the first teeth. Posterior margins of cusps of second and third teeth blunt, rounded, and more strongly curved than those of first teeth; boundaries of cusps of second and third teeth discernible as distinct lines. Marginal teeth (M1, M2: Fig. 6F) tightly overlapped; cusps curved backwards; shafts elongate; bases concealed by anterolateral corners of basal plates of the next row of the radula; inner marginal teeth slightly wider than outer teeth. Basal plates (bp: Fig. 6D) tightly fused between right and left, considerably thickened, and wider than long (height/width = ca. 0.67); anterior margins weakly convex; anterolateral corners obliquely truncated and broadly extended laterally; posterolateral corners straight; posterior margins overlaid by next basal plates. Distribution: The species is restricted to calcareous Fig. 5. Ventral and internal view of animal. UMUT-RM28075. A, Ventral view of mouth region. B, Dorsal view of buccal mass and radular sac, with pallial and visceral organs removed. Abbreviations: bm, buccal mass; cpt, cephalic tentacle; e, eye; ers, end of radular sac; I, intestine; L1, first loop; L2, second loop; m, mouth; ola, oral lappet; oli, outer lip of mouth; rds, radular sac; sm, shell muscle. Size of buccal mass: length = 3.6 mm, width = 2.9 mm. page 8 of 12Zoological Studies 60:68 (2021) © 2021 Academia Sinica, Taiwan substrates in Yonaguni Island, Japan and eastern and southern Taiwan (note rock substrate in northern Taiwan is not calcareous). Habitat: The species lives in crevices and hollows on vertical limestone faces on exposed shores in the high intertidal to supratidal zone. Although it does not occur in dense communities in Yonaguni Island, Japan and Jialershui, Taiwan, the species is abundant on Green Island and Orchid Island, southeastern Taiwan. Etymology: The species name nivea is given for the white color of the shell. DISCUSSION Previous identification of Eoacmaea nivea n. sp. Oyama and Takemura (1959) firstly reported Eoacmaea nivea n. sp. from Taiwan as Cellana mauritiana in the family Nacellidae. However, judging from the shell morphology and anatomical characters, the identification of C. mauritiana in Oyama and Takemura (1959) is not correct. The genus Cellana has secondary gills, whereas E. nivea. n. sp. has a true Fig. 6. Radula. A–C, Radular sac cut into three portions. The numbers 1 to 75 represent the first to 75th rows from the anterior. D–F, Scanning electron micrographs of radular teeth. D, Enlarged view of single radular segment. E, Second and third cusps of lateral teeth. F, Marginal teeth. Abbreviations: bm, basal membrane; bp, basal plate; L1c, cusp of first lateral tooth; L1s, shaft of first lateral tooth; L2, second lateral tooth; L3, third lateral tooth; M1, first marginal tooth; M2, second marginal tooth. page 9 of 12Zoological Studies 60:68 (2021)