Diversity of Parasitic Peltogastrid Barnacles (Crustacea: Cirripedia: Rhizocephala) on Hermit Crabs in Korea

© 2019 Academia Sinica, Taiwan Open Access Diversity of Parasitic Peltogastrid Barnacles (Crustacea: Cirripedia: Rhizocephala) on Hermit Crabs in Korea Jibom Jung1,§, Ryuta Yoshida2,§, and Won Kim1,* 1School of Biological Sciences, Seoul National University, 1, Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea. *Correspondence: Tel: +82028806695. E-mail: [email protected] (Kim). E-mail: [email protected] (Jung) 2Tateyama Marine Laboratory, Marine and Coastal Research Center, Ochanomizu University, Kou-yatsu, Tateyama, Chiba 294-0301, Japan. E-mail: [email protected] §JJ and RY contributed equally to this work. Received 3 April 2019 / Accepted 18 September 2019 / Published 11 November 2019 Communicated by Benny K.K. Chan We performed a diversity study on parasitic barnacles (Crustacea: Cirripedia: Rhizocephala: Peltogastridae) that parasitize hermit crabs in Korea. Their morphological, ecological, molecular (cytochrome c oxidase subunit I, 16S rRNA), and biogeographical characteristics were examined. Three species were identified based on GenBank sequences and the external morphology of the externa. In addition, this study proposes four new candidate species. This is the first report on the family Peltogastridae from Korea. Six hermit crab species were found to be new hosts to peltogastrids. Korean peltogastrids are less prevalent on their host hermit crabs than those from Japan are, especially in the west coast of Korea. Peltogasterella gracilis is widely distributed throughout Korea, Peltogaster lineata is located on the east coast, and Peltogaster postica is only located on Jeju Island. Key words: Parasitic barnacle, Paguroidea, Morphology, DNA barcode, Ecology, Biogeography. Citation: Jung J, Yoshida R, Kim W. 2019. Diversity of parasitic peltogastrid barnacles (Crustacea: Cirripedia: Rhizocephala) on hermit crabs in Korea. Zool Stud 58:33. doi:10.6620/ZS.2019.58-33. BACKGROUND Cirripedia barnacles are categorized into three superorders: Acrothoracica (Nielsen et al. 2016), Thoracica with various life styles (Yusa et al. 2013 2018; Buckeridge et al. 2018), and parasitic Rhizocephala (Yoshida et al. 2012). Rhizocephalan barnacles in the family Peltogastridae are highly adapted to decapod parasites, mostly infecting hermit crabs. Of the 46 species and 17 genera in this family (Boyko and Boxshall 2019), 28 species from 8 genera are parasitic on the abdomen of hermit crabs (McDermott et al. 2010; Yoshida et al. 2011 2013 2015; Yoshida and Naruse 2016). The peltogastrid taxa exhibit extremely degenerated internal and external organ systems without sensory, respiratory, digestive, or excretory systems in the adult stage (Høeg 1992). In addition, the adults lack the segmentation and appendages observed in other crustaceans (Høeg and Lützen 1995). Owing to the externa, which gives the appearance of a membranous sac without any distinct morphological characteristics, morphological identification of Rhizocephala is mainly based on the internal morphological characteristics based on paraffin sectioning of the externa and larval morphology (Yoshida et al. 2011). Recently, DNA sequencing has also provided additional support for taxonomic study of this group of animals (Yoshida et al. 2012 2014). Several new peltogastrid species have recently been reported based on morphological and molecular differences (Yoshida et al. 2011 2013 2015; Noever et al. 2016; Yoshida and Naruse 2016). Peltogastrids have been reported from Europe (Rathke 1842; Reinhard 1942; Høeg and Lützen 1985) and East Asia, including southeast Russia (Kashenko Zoological Studies 58: 33 (2019) doi:10.6620/ZS.2019.58-33 1 © 2019 Academia Sinica, Taiwan and Korn 2003), Taiwan (Yoshida et al. 2012), and Japan (Shiino 1943; Utinomi and Kikuchi 1966; Nagasawa et al.1996; Yoshida et al. 2014). However, despite previous studies conducted in and near Korea, including plenty of systematics studies on hermit crabs (the peltogastrid’s host) (Kim 1973; Oh 2000; Kim and Son 2006; Jung and Kim 2014 2015 2016), no peltogastrids have been reported in this region. We conducted a taxonomic study of Korean hermit crab fauna and found some peltogastrid individuals attached to the abdomen of hermit crabs. To identify these samples, we assessed the external morphology of the externa and performed DNA barcoding. The results identified some peltogastrids that were not previously reported as parasitizing specific hermit crab species (see McDermott et al. 2010; Yoshida et al. 2014). We follow the methods in Yoshida et al. (2014), who described the ecological and biogeographical characteristics of Japanese peltogastrids with morphological and molecular identification. Herein we present results on morphological, ecological, biogeographical, and molecular characteristics of Korean peltogastrids. MATERIALS AND METHODS We examined the abdomens of 4,716 individual Korean paguroid specimens belonging to 48 species (Table 1) collected from 40 sites (administrative district, Si(city) or Gun(country)) deposited into the Laboratory of Systematic and Molecular Evolution (EVOSYS) and Marine Arthropods Depository Bank of Korea (MADBK) of Seoul National University, and the Ewha Womans University Natural History Museum. All specimens examined in the present study were preserved in 70%–99% ethyl alcohol. The morphological examination methods for Korean peltogastrids generally followed those described by Yoshida et al. (2014) and Jung and Kim (2017). The shape of the externa, position and prominent rate of the mantle, shape of mantle opening, and presence of a chitinous shield in Korean peltogastrid specimens were examined using a macroscope and MZ8 dissection microscope (Leica, Wetzlar, Germany). Photographs were taken with a D200 digital camera (Nikon, Tokyo, Japan) and processed using a Helicon Focus software (Helicon Soft Ltd., Kharkov, Ukraine). Shield length (SL) was recorded to indicate the size of the host hermit crab specimen, measured from the tip of the rostrum to the midpoint of the posterior margin of the shield using a CD6CSX digital caliper (Mitutoyo, Kawasaki, Japan) to the nearest 0.1 mm. Between one quarter and one-third of the externa tissue was excised from the posterior end of each peltogastrid specimen to extract the total DNA using a QIAamp DNA Micro Kit (QIAGEN, Hilden, Germany). Universal primers LCO1490 and HCO2198 were used to amplify the mitochondrial COI gene (cytochrome c oxidase subunit I, Folmer et al. 1994). 16SH2 and 16SL2 primers were used (Schubart et al. 2000) to amplify the mitochondrial 16S rRNA gene. The polymerase chain reaction (PCR) solution included 1 µL DNA template, 1 µL of each primer (10 µM), 0.3 µL Go Taq DNA polymerase (Promega, Madison City, WI, USA), 5 µL 5x color Go Taq reaction buffer, 1 µL dNTP mixture (10 mM) and 15.7 µL distilled H2O (total 25 µL). The amplification protocol followed that previously described by Yoshida et al. (2014). The size of the PCR products was observed in 1% agarose gels. PCR products were analyzed in an ABI 3730 automated sequencer (Applied Biosystems, Foster City, CA, USA). The molecular analysis methods followed those described by Jung et al. (2018a). The mitochondrial sequences of the collected specimens obtained by DNA extraction were checked and edited using SeqMan 5.0 (DNASTAR, Madison City, WI, USA) and DNA Sequence Polymorphism (DnaSP) version 6 software (Rozas et al. 2017); they were aligned using a ClustalW interface ClustalW in MEGA7 (MEGA, PA, USA) (Kumar et al. 2016). The mitochondrial sequences of the Korean peltogastrids plus 11 peltogastrid species from GenBank (Table 2) were used in a molecular evolutionary genetics analysis using MEGA7. Maximum likelihood analyses of COI and 16S rRNA sequences of the selected peltogastrids were performed based on the Tamura-Nei (TN93) (Tamura and Nei 1993) and Hasegawa-Kishino-Yano (Hasegawa et al. 1985) models, respectively, with 5 rate categories of gamma distribution (+G) and invariable sites (+I), which had the lowest Bayesian Information Criterion (BIC) scores in the Model Selection option of MEGA7. The consistency of topologies was assessed using bootstrap values with 1,000 replications. Interspecific and intraspecific sequence variations were obtained based on the K2P distance in MEGA7. RESULTS Based on the shape of the externa and mitochondrial sequences of the peltogastrids, we found 42 peltogastrid individuals belonging to 10 species from 15 collection sites (Fig. 1) on 17 host Korean hermit crab species (Table 2). Among the Korean peltogastrids, only seven and five species could be distinctly identified by morphological characteristics and molecular sequences, respectively; their morphological page 2 of 18Zoological Studies 58: 33 (2019) © 2019 Academia Sinica, Taiwan Table 1. Individual number and infestation rate (%) of peltogastrids on the Korean hermit crab hosts examined in this study Host hermit crab Species Total individuals Infested individuals Infestation rate Areopaguristes japonicas 35 0 0.0% Areopaguristes nigroapiculus 42 1 2.3% Ciliopagurus krempfi 2 0 0.0% Clibanarius virescens 5 0 0.0% Dardanus arrosor 66 0 0.0% Dardanus crassimanus 4 0 0.0% Dardanus impressus 16 0 0.0% Dardanus lagopodes 2 0 0.0% Dardanus pedunculatus 8 0 0.0% Diogenes deflectomanus 4 0 0.0% Diogenes edwardsii 49 0 0.0% Diogenes nitidimanus 15 0 0.0% Diogenes penicillatus 4 0 0.0% Elassochirus cavimanus 75 0 0.0% Lophopagurus (Australeremus) triserratus 1 0 0.0% Paguristes acanthomerus 3 0 0.0% Paguristes digitalis 30 0 0.0% Paguristes ortmanni 382 1 0.2% Paguristes seminudus 1 0 0.0% Paguristes versus 2 0 0.0% Pagurus brachiomastus 100 1 1.0% Pagurus constans 34 0 0.0% Pagurus filholi 670 9 (Peltogaster lineata: 1, P. postica: 6, Peltogasterella gracilis: 2) 1.3% Pagurus gracilipes 36 0 0.0% Pagurus japonicus 93 3 3.2% Pagurus lanuginosus 239 5 2.1% Pagurus maculosus 133 4 1.5% Pagurus middendorffii 47 1 2.1% Pagurus minutus 1244 2 0.2% Pagurus nigrivittatus 10 0 0.0% Pagurus nigrofascia 46 0 0.0% Pagurus ochotensis 68 1 1.5% Pagurus parvispina 8 1 12.5% Pagurus pectinatus 313 3 1.0% Pagurus proximus 175 1 0.4% Pagurus quinquelineatus 3 0 0.0% Pagurus rathbuni 13 1 7.7% Pagurus rectidactylus 2 0 0.0% Pagurus rubrior 260 5 1.9% Pagurus similis 18 0 0.0% Pagurus simulans 274 0 0.0% Pagurus spina 28 1 3.6% Pagurus trigonocheirus 109 0 0.0% Pagurus undosus 12 0 0.0% Pomatocheles jeffreysi 4 0 0.0% Porcellanopagurus nihonkaiensis 13 2 15.4% Discorsopagurus maclaughlinae 13 0 0.0% Discorsopagurus tubicola 5 0 0.0% Total 4,716 42 0.9% page 3 of 18Zoological Studies 58: 33 (2019) © 2019 Academia Sinica, Taiwan Table 2. Geographical locations of and sample information on the Korean peltogastrids and others in this study in the order of species name, host species, location, and specimen number Species Host Species Location Specimen Number Briarosaccus auratum* Lithodes aequispinus Alaska, US YPM 74383 Briarosaccus regalis* Paralithodes camtschaticus Alaska, US YPM 74281 Dipterosaccus indicus* Calcinus morgani Okinawa, Japan NSMT-Cr 22224 Dipterosaccus shiinoi* Calcinus vachoni Okinawa, Japan NSMT-Cr 22236 Ommatogaster nana* Diogenes leptocerus Okinawa, Japan Peltogaster lineata Pagurus brachiomastus Yangyang, Korea MADBK 430101_001 Pagurus filholi Busan, Korea MADBK 160707_043 Pagurus nigrivittatus* Shirahama, Japan NSMT-Cr 22303 Peltogaster paguri* Pagurus bernhardus Gullmar Fjord, Sweden ZMUC CRU-9909 Peltogaster postica Pagurus filholi Jeju, Korea MADBK 160707_009 MADBK 160707_052 MADBK 430102_001 Pagurus minutus* Okinawa, Japan Peltogaster aff. ovalis Pagurus japonicus* Wakayama, Japan NSMT-Cr 22308 Pagurus ochotensis Busan, Korea MADBK 160714_013 Pagurus parvispina Goseong, Korea EWUNHM DP 20151217063 Pagurus rathbuni Yangyang, Korea EVOSYS 260720#013 Pagurus rubrior Busan, Korea EVOSYS 260717#012 EWUNHM DP 20151125023 Seogwipo, Korea EVOSYS 260717#038 MADBK 160717_007 Yeosu, Korea EVOSYS 260717#033 Peltogaster aff. reticulatus Pagurus proximus Shinan, Korea MADBK 160718_040 Peltogaster sp. 1 Pagurus japonicus Jeju, Korea MADBK 160710_012 Seogwipo, Korea EVOSYS 260710#021 MADBK 160710_002 Peltogaster sp. 2 Areopaguristes nigroapiculus Pohang, Korea EVOSYS 260510#008 Peltogaster sp. 3 Paguristes ortmanni Pohang, Korea MADBK 160513_050 Peltogaster sp. 4 Pagurus minutus Namhae, Korea MADBK 160706_065 MADBK 160706_125 Peltogaster sp. Areopaguristes japonicus* Areopaguristes japonicus Chiba, Japan Peltogaster sp. Paguristes ortmanni* Paguristes ortmanni Chiba, Japan Peltogasterella gracilis Pagurus filholi Gyeongju, Korea MADBK 160707_039 Yeosu, Korea MADBK 430103_001 Pagurus lanuginosus Busan, Korea MADBK 160712_040 Taean, Korea EVOSYS 260712#023 Uljin, Korea MADBK 160712_020 Ulsan, Korea MADBK 160712_006 Pagurus maculosus Gangneung, Korea MADBK 160722_004 Pohang, Korea MADBK 160722_013 Ulleoung, Korea MADBK 160722_023 MADBK 160722_024 Pagurus middendorffii Goseong, Korea MADBK 160713_007 Pagurus nigrivittatus* Shizuoka, Japan NSMT-Cr 22310 Pagurus pectinatus Busan, Korea MADBK 160715_016 Uljin, Korea MADBK 160715_005 Yeongdeok, Korea EWUNHM DP 20151202019 Pagurus spina Ulleoung, Korea MADBK 160726_003 Peltogasterella aff. gracilis Porcellanopagurus nihonkaiensis Jeju, Korea MADBK 160730_002 Peltogasterella sensuru* Pagurixus pseliophorus Okinawa, Japan RUMF-ZC 03035 Peltogasterella sulcata* Pagurus cuanensis Gullmar Fjord, Sweden ZMUC CRU-9910 Septosaccus sp.* Diogenes tumidus Penghu, Taiwan NMNS-6795-004 page 4 of 18Zoological Studies 58: 33 (2019) © 2019 Academia Sinica, Taiwan Species Individual Number COI Accession Number 16S rRNA Accession Number Briarosaccus auratum* 1 KR812208 KR812156 Briarosaccus regalis* 1 KR812198 KR812177 Dipterosaccus indicus* 1 AB618607 AB778078 Dipterosaccus shiinoi* 1 AB742438 AB778083 Ommatogaster nana* 1 AB602398 Peltogaster lineata 1 MK604142 MK604159 1 MK604143 MK604160 1 AB778060 AB778092 Peltogaster paguri* 1 FJ481958 Peltogaster postica 4 MK604144-MK604146 MK604161, MK604162 1 MK604147, MK604148 (two externae) MK604163, MK604164 (two externae) 1 MK604149 MK604165 1 AB602392 AB778105 Peltogaster aff. ovalis 1 AB778063 AB778095 1 1 1 1 1 1 1 1 Peltogaster aff. reticulatus 1 Peltogaster sp. 1 1 1 1 Peltogaster sp. 2 1 Peltogaster sp. 3 1 MK604150 MK604166 Peltogaster sp. 4 1 MK604151 MK604167 1 Peltogaster sp. Areopaguristes japonicus* 1 AB778074 AB778111 Peltogaster sp. Paguristes ortmanni* 1 AB778075 AB778112 Peltogasterella gracilis 1 1 MK604152 MK604168 1 MK604153 MK604169 2 1 MK604154 MK604170 1 MK604155 1 MK604156 MK604171 1 1 1 1 1 AB778077 AB778114 1 MK604157 MK604172 1 1 1 MK604158 MK604173 Peltogasterella aff. gracilis 2 Peltogasterella sensuru* 1 LC013692 LC013700 Peltogasterella sulcata* 1 FJ481955 Septosaccus sp.* 1 AB688772 *: NCBI sequence Table 2. (Continued) page 5 of 18Zoological Studies 58: 33 (2019) © 2019 Academia Sinica, Taiwan characteristics and status used for this identification are shown in figure 2 and table 3. All the peltogastrid sequence data in this study are available in GenBank (Table 2); their phylogenies are shown in figures 3 and 4. We report the discovery of three peltogastrid species and four candidates for new species from Korea. Fig. 1. Distribution map of Korean peltogastrids. Peltogaster postica Peltogaster lineata Peltogaster aff. ovalis Peltogaster sp. 2 Peltogaster sp. 4 Peltogaster aff. reticulatus Peltogasterella gracilis Peltogasterella aff. gracilis Peltogaster sp. 3 Taean Peltogaster sp. 1 Goseong Gangneung Uljin Yeongdeok Pohang Gyeongju Ulsan Shinan Busan Yeosu Namhae Seogwipo Jeju Ulleung page 6 of 18Zoological Studies 58: 33 (2019) © 2019 Academia Sinica, Taiwan TAXONOMY Peltogaster lineata Shiino, 1943 (Fig. 5A) Peltogastridae Lilljeborg, 1859 Peltogaster Rathke, 1842 Material examined: On Pagurus brachiomastus: 1 Ind., Yangyang, 37°55'49.00"N 128°47'25.00"E, Scuba, 10 Apr. 2014, Coll. Park JH, MADBK 430101_001, host: male, SL 4.7 mm. On P. filholi: 1 Ind., Busan, 35°8'16.83"N 129°9'37.01"E, 8 Oct. 2015, Coll. Jung J, MADBK 160707_043, host: male, SL 3.4 mm. Hosts: P. brachiomastus, P. filholi, P. nigrivittatus, P. maculosus (Paguridae). Distribution: Japan, Korea. Remarks: Morphologically, the externa on the examined materials showed a single oval shape of whole externa, terminal located and slightly projected mantle, circular mantle opening, and presence of a chitinous shield, in close agreement with the original description (Shiino 1943) and re-description of Peltogaster lineata by Yoshida et al. (2014). In Korean P. lineata, the projection of the mantle aperture is slightly elevated. In addition, the mitochondrial sequence of the examined material (MK604142, MK604143, MK604159, MK604160) is similar to the DNA sequences (AB778060, AB778092) of the species reported by Yoshida et al. (2014). The present study reports Pagurus brachiomastus as the fifth host of Peltogaster lineata after Pagurus japonicus, P. filholi, P. nigrivittatus, and Fig. 2. Semi-diagrammatic figures of mantle aperture of Korean peltogastrids. A, Peltogaster lineata; B, Peltogaster postica; C, Peltogaster aff. ovalis; D, Peltogaster aff. reticulatus; E, Peltogaster sp. 2; F, Peltogasterella gracilis; a, mantle opening; b, mantle. Comparative degree of mantle projection and shape of mantle opening are the most important characters on morphological identification of this study. Table 3. Morphological characters of the externa of the Korean peltogastrids (modified from Yoshida et al. 2014) Species Whole externa Mantle Chitinous shield Projection Opening Position Peltogaster lineata Oval Slightly elevated Circular or U-shaped Termina Yes Peltogaster postica Irregularly elongate Elevated tube U-shaped Terminal or subterminal Yes Peltogaster aff. ovalis Oval Elevated U-shaped Terminal Yes Peltogaster aff. reticulatus Oval Elevated tube X-shaped Terminal Yes Peltogaster sp. 2 Oval Slightly elevated Slit-shaped Terminal Yes Peltogasterella gracilis Elongate, gregarious Elevated Circular Termina No page 7 of 18Zoological Studies 58: 33 (2019) © 2019 Academia Sinica, Taiwan P. maculosus (Shiino 1943; Yoshida et al. 2014). In addition, the specimen of Peltogaster lineata (MADBK 430101_001) was found in Yangyang, Korea, much farther north (37°55'N) than the previous record in southern Japan (35°09'N) (Shiino 1943; Yoshida et al. 2014). Furthermore, this is the first report of this species collected from outside of Japanese waters. The mantle of the Pagurus filholi specimen (MADBK 160707_043) has a U-shaped opening, similar to that of Peltogaster postica. However, the former’s mitochondrial sequence (MK604143, MK604160) differed from that of P. postica, but it constituted a single clade of sequences with those of P. lineata in this study (MK604142, MK604159) and previous studies (AB778060, AB778092) (Figs. 3, 4). In addition, Yoshida et al. (2014) reported that P. lineata can parasitize Pagurus filholi. Therefore, the present study identified this specimen as Peltogaster lineata based on molecular and ecological characteristics. Peltogaster postica/host: Pagurus filholi MK604147 Peltogaster postica/host: Pagurus filholi MK604148 Peltogaster postica/host: Pagurus filholi MK604149 Peltogaster postica/host: Pagurus filholi MK604144 Peltogaster postica/host: Pagurus filholi MK604145 Peltogaster postica/host: Pagurus filholi MK604146 Peltogaster postica/host: Pagurus minutus Japan AB602392* Peltogaster sp. 4/host: Pagurus minutus MK604151 Peltogaster paguri KT208574* Briarosaccus auratum KR812208* Briarosaccus regalis KR812198* Peltogaster lineata/host: Pagurus nigrivittatus Japan AB778060* Peltogaster lineata/host: Pagurus brachiomastus MK604142 Peltogaster lineata/host: Pagurus filholi MK604143 Peltogaster aff. ovalis AB778063* Peltogaster sp./host: Areopaguristes japonicus AB778074* Peltogaster sp. 3/host: Paguristes ortmanni MK604150 Peltogaster sp./host: Paguristes ortmanni AB778075* Dipterosaccus indicus AB618607* Dipterosaccus shiinoi AB742438* Ommatogaster nana AB602398* Septosaccus sp./host: Diogenes tumidus AB688772* Peltogasterella sensuru LC013692* Peltogasterella gracilis/host: Pagurus nigrivittatus Japan AB778077* Peltogasterella gracilis/host: Pagurus lanuginosus MK604153 Peltogasterella gracilis/host: Pagurus filholi MK604152 Peltogasterella gracilis/host: Pagurus maculosus MK604156 Peltogasterella gracilis/host: Pagurus pectinatus MK604157 Peltogasterella gracilis/host: Pagurus lanuginosus MK604155 Peltogasterella gracilis/host: Pagurus lanuginosus MK604154 Peltogasterella gracilis/host: Pagurus spina MK604158 100 52 100 100 96 99 100 100 100 100 63 99 54 73 77 46 34 23 100 67 84 0.10 Fig. 3. Maximum likelihood rooted trees of COI sequences of Peltogastridae from Korea and NCBI. The number next to the branches is the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test. *means NCBI sequence. page 8 of 18Zoological Studies 58: 33 (2019) © 2019 Academia Sinica, Taiwan Peltogaster postica Yoshida and Osawa in Yoshida, Osawa, Hirose, and Hirose, 2011 (Fig. 5B, 5C) Material examined: On Pagurus filholi: 4 Inds., Jeju, 33°31'38.25"N 126°35'4.60"E, 31 Oct. 2010, Coll. Kang SH, MADBK 160707_009, host: 3 males, SL 3.14.0 mm, 1 female, SL 3.3 mm; 1 Ind. (2 externae), Jeju, 33°31'23.00"N 126°33'31.60"E, 4 May 2016, Coll. Jung J, MADBK 160707_052, host: male, SL 3.7 mm; 1 Ind., same as MADBK 160707_052, MADBK 430102_001, host: male, SL 3.2 mm. Host: P. filholi, P. minutus, P. nigrivittatus, P. angustus (Paguridae). Distribution: Southern Honshu and Okinawa (Japan), Taiwan, Korea. Remarks: The externa of examined materials had morphological characteristics, such as irregularly elongated body, terminal-posited elevated mantle, U-shaped mantle opening, and chitinous shield similar to the original description (Yoshida et al. 2011) and redescription of Peltogaster postica by Yoshida et al. Fig. 4. Maximum likelihood rooted trees of 16S rRNA sequences of Peltogastridae from Korea and NCBI. The number next to the branches is the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test. *means NCBI sequence. Peltogaster postica/host: Pagurus filholi MK604165 Peltogaster postica/host: Pagurus filholi MK604163 Peltogaster postica/host: Pagurus filholi MK604162 Peltogaster postica/host: Pagurus filholi MK604161 Peltogaster postica/host: Pagurus filholi MK604164 Peltogaster postica/host: Pagurus minutus Japan AB778105* Peltogaster sp. 4/host: Pagurus minutus MK604167 Briarosaccus auratum KR812156* Briarosaccus regalis KR812177* Peltogaster lineata/host: Pagurus nigrivittatus Japan AB778092* Peltogaster lineata/host: Pagurus brachiomastus MK604159 Peltogaster lineata/host: Pagurus filholi MK604160 Peltogaster paguri FJ481958* Peltogaster aff. ovalis AB778095* Peltogaster sp./host: Areopaguristes japonicus AB778111* Peltogaster sp. 3/host: Paguristes ortmanni MK604166 Peltogaster sp./host: Paguristes ortmanni AB778112* Peltogasterella sensuru LC013700* Peltogasterella sulcata FJ481955* Peltogasterella gracilis/host: Pagurus nigrivittatus Japan AB778114* Peltogasterella gracilis/host: Pagurus filholi MK604168 Peltogasterella gracilis/host: Pagurus lanuginosus MK604170 Peltogasterella gracilis/host: Pagurus lanuginosus MK604169 Peltogasterella gracilis/host: Pagurus maculosus MK604171 Peltogasterella gracilis/host: Pagurus pectinatus MK604172 Peltogasterella gracilis/host: Pagurus spina MK604173 Dipterosaccus indicus AB778078* Dipterosaccus shiinoi AB778083* 100 100 100 100 97 68 99 91 65 69 59 98 99 99 99 94 0.20 page 9 of 18Zoological Studies 58: 33 (2019) © 2019 Academia Sinica, Taiwan The sites at which Korean peltogastrids were collected show that the peltogastrids exhibit some habitat preferences. A total of 15 and 21 Korean peltogastrid specimens were collected from the eastern and southern coasts of Korea, respectively (Fig. 1). However, only two individuals were collected from the western coast of Korea, in spite of the high abundance of hosts (Jung et al. 2018b). The lack of peltogastrids on the western coast of Korea might be related to the development rate of larvae in the environment. Kashenko and Korn (2003) demonstrated that low temperature and low salinity negatively affect the development of larvae of the peltogastrid Peltogaster reticulata. The water on the western coast of Korea is Pagurus filholi 100% Korea Pagurus filholi 20% Pagurus nigrivittatus 70% Pagurus maculosus 10% Japan Pagurus filholi 50% Pagurus brachiomastus 50% Korea Pagurus filholi 58% Pagurus nigrivittatus 6% Pagurus maculosus 30% Pagurus middendorffii 6% Japan Pagurus filholi 30% Pagurus nigrivittatus 15% Pagurus minutus 55% Japan Pagurus angustus 100% Taiwan Peltogaster lineata Peltogaster postica Peltogasterella gracilis Pagurus filholi 13% Pagurus lanuginosus 33% Pagurus maculosus 20% Pagurus middendorffii 7% Pagurus pectinatus 20% Pagurus spina 7% Korea Fig. 7. Pie chart of the prevalence of hermit crab species for each peltogastrids in Korea, Japan, and Taiwan. The pie chart of Japan was derived from Nagasawa et al. (1996) and Yoshida et al. (2011 2014). The pie chart of Taiwan was derived from Yoshida et al. (2012). page 16 of 18Zoological Studies 58: 33 (2019) © 2019 Academia Sinica, Taiwan cold in the winter due to its shallow depth and has low salinity due to a great inflow of fresh water (Park et al. 2017). These factors could decrease the development rate of larvae and the abundance of peltogastrids on the western coast of Korea. Korean peltogastrids infest their host hermit crabs at a lower rate than those from Japan. In Japan, Yoshida et al. (2014) found 87 individual peltogastrids (6 species) on 2,282 individual hermit crabs (23 species), an infestation rate of 3.8%. However, the infestation rate by Korean peltogastrids is lower, at 0.9% (Table 1). According to Kashenko and Korn (2003), Korean peltogastrids may have a lower infestation rate because the water temperature in Korea is lower than in Japan. The high infestation rates (> 7%) for Pagurus parvispina, P. rathbuni, and Porcellanopagurus nihonkaiensis might be biased by the small sample size. CONCLUSIONS In this study, we demonstrated the presence of peltogastrid parasitic barnacles in Korea using morphological and molecular analysis and found six new species of hermit crabs that are peltogastrid hosts. In addition, we noted that the host prevalence of Korean peltogastrids differs slightly from what has been previously reported. Furthermore, this study slightly expanded the distribution range of some peltogastrids species. This is the first systematic study on peltogastrid species in Korea, and it emphasizes the need for further studies on Korean peltogastrids to identify their morphological, molecular, ecological, and biogeographical characteristics. List of abbreviations SL, shield length. COI, cytochrome c oxidase subunit I. EVOSYS, Laboratory of Systematics and Molecular Evolution. MADBK, Marine Arthropod Depository Bank of Korea. Acknowledgments: This work was supported by a grant from the Marine Biotechnology Program (20170431) funded by the Ministry of Oceans and Fisheries, Korea. We thank Dongu Lee, Beong-ju Che and Ji-hyeon Shin (School of Biological Sciences, Seoul National University, Seoul, Republic of Korea) for their help with specimen finding and molecular analysis. We also appreciate Dr. Su-Yuan Seo (Ewha Womans University Natural History Museum, Ewha Womans University, Seoul, Republic of Korea) for assistance with hermit crab samples from the Ewha Womans University Natural History Museum. The authors would like to thank Enago (www.enago.co.kr), Mrs. Hyunsoon Kim, and Noah Last (Third Draft Editing) for their English language reviews. Authors’ contributions: Jibom Jung and Ryuta Yoshida contributed equally to this work. Ryuta Yoshida conceived of the study. Jibom Jung and Ryuta Yoshida designed the study. Jibom Jung and Won Kim performed the field work and analyzed the specimens. Jibom Jung, Ryuta Yoshida, and Won Kim contributed to the acquisition of the sequence data. Jibom Jung and Ryuta Yoshida contributed to the analysis and interpretation of the sequence data. Jibom Jung and Won Kim drafted the manuscript. Ryuta Yoshida participated in critical revisions to the manuscript. Competing interests: The authors declare that they have no conflict of interest. Availability of data and materials: All sequence data in this study are available in GenBank. All examined and sampled specimens are available from the Laboratory of Systematic and Molecular Evolution (EVOSYS), Marine Arthropods Deposited Bank of Korea (MADBK) of Seoul National University, and Ewha Womans University Natural History Museum, Seoul, Republic of Korea. Consent for publication: Not applicable. Ethics approval consent to participate: Not applicable. REFERENCES Boschma H. 1927. On the larval forms of Rhizocephala. Proc Sect Sci Kon Akad Wetensch Amsterdam 30:293–297. Boyko CB, Boxshall G. 2019. 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