Morphological and molecular analyses of parasitic barnacles (Crustacea: Cirripedia: Rhizocephala) in Korea: preliminary data for the taxonomy and host ranges of Korean species

Morphological and molecular analyses of Korean rhizocephalan barnacle species were performed to examine their host ranges and taxonomy. Morphological examination and molecular analysis of mtDNA cox1, 16S, and nuclear 18S rRNA sequences revealed nine rhizocephalan species from three genera of the two families, Sacculinidae and Polyascidae. Phylogenetic analysis of molecular sequences revealed two new species candidates in the genus Parasacculina, and three Sacculina species (S. pilosella, S. pinnotherae, and S. imberbis) were transferred to the genus Parasacculina. Examination of host ranges revealed higher host specificity and lower infestation rates in Korean rhizocephalan species than rhizocephalans from other geographic regions. This is the first report of the taxonomy, species diversity, and host ranges of Korean parasitic rhizocephalan barnacles based on their morphological and molecular analyses. More information from extensive sampling of parasitic barnacles from a wide range of crustacean host species is necessary to fully understand their taxonomy, prevalence on decapod hosts, and phylogenetic relationships among major rhizocephalan taxa.

Phylogenetic relationships among rhizocephalan species were inferred for each of the three genes using maximum likelihood (ML) analysis and Bayesian inference (BI) implemented in RaxML version 8 (Stamatakis, 2014) andMrBayes v3.2.6 (Ronquist, Huelsenbeck &Teslenko, 2011), respectively. Phylogenetic trees were modified by MEGA 10. Maximum likelihood analyses of cox1, 16S, and 18S rDNA sequences were performed based on the Tamura-Nei (TN93) (Tamura & Nei, 1993), general time reversible (Tavaré, 1986), and Kimura 2-parameter (Kimura, 1980) models, respectively, with a gamma distribution (+G) and invariable sites (+I) rate categories based on Bayesian Information Criterion (BIC) scores model using the Model Selection option of MEGA10. The robustness of individual nodes in the ML trees was assessed by analysis of 1,000 bootstrap replications. Interspecific and intraspecific sequence divergences were estimated based on the K2P distance matrix in MEGA10.

RESULTS
Based on morphological examination (shape and number of externae and mantle aperture) and mitochondrial sequence information, we identified 38 rhizocephalan individuals belonging to nine species, three genera, and two families isolated from eight decapod hosts species collected from 16 sites ( Fig. 1; Table 2). All rhizocephalans identified by this study except Parasacculina pinnotherae comb. nov. were first reported from Korea. Detailed information regarding the Korean rhizocephalan species and their externa morphology is provided in Table 3.     Sacculinidae Lilljeborg, 1861Sacculina Thompson, 1836 Sacculina confragosa Boschma1933 ( Fig. 2A Diagnosis of the externa: whole externa mostly single and occasionally double, wrinkled cordiform with flat half-oval-shaped left and right lobes divided by an outer mid-groove and inner mid-ridge; outermost part of the robe wrinkled. Mantle well elevated, tube-shaped, and vertically slightly wrinkled with a circular opening at the extremity. Remarks: Morphological characteristics of the examined materials correspond with their original description (Boschma, 1933) except for the number of externa. Some of our specimens (Korea 6, Korea 12) had double externae (15% of total examined individuals), whereas others had a single externa. This type of variation in the number of externa has been reported in a previous study (Shiino, 1943). This species is found most abundantly parasitizing medium-sized individuals of host crab species. Further study is needed to determine if this species is a predominant parasitic form on medium-sized host individuals.   Boschma, 1931, S. pugettiae (Shiino, 1943, S. reinhardi (Boschma, 1955), and S. pilosella) were previously reported to parasitize Pugettia spp.. Morphological characteristics of the examined specimen correspond with the original description of S. pillosella (Van Kampen & Boschma, 1925  Diagnosis of the externa: whole externa smooth or slightly wrinkled, single or double, and flat oval or cordiform in shape; each outer-posterior margin elevated into a conical shape. Mantle slightly elevated and vertically wrinkled with a small round opening at the extremity. Remarks: Two Sacculina species (S. pertenuis (Boschma, 1933) and S. pinnotherae) have been reported to be parasitic on Pinnotheres spp.. Morphological characters of examined specimens correspond with the original description of S. pinnotherae (Shiino, 1943).

Taxonomic accounts and morphological features of Korean rhizocephalan species
However, phylogenetic analysis of mtDNA cox 1, 16S, and nuclear 18S rDNA sequences placed this species within the genus Parasacculina (Figs. 3A-3C), not in the genus Sacculina. Therefore, we treated this species as a member of the genus Parasacculina (see Discussion for more details). The host crab (Arcotheres sinensis) is known to parasitize bivalves, so P. pinnotherae comb. nov. is a secondary parasite that is rare in the ocean (McDermott, 2009).

Parasacculina shiinoi (Lützen et al., 2016)
Materials examined: 1 ind., Namhae (34.9 N 127.9 E), Korea 25, host: cl 8.8 mm; 1 ind., Namhae (34.9 N 127.8 E), Korea 26, host: cl 11.1 mm. Remarks: The examined specimens had a single externa, but detailed morphological characteristics could not be determined because of the immaturity of the specimens examined. Lützen et al. (2016) reported that Sacculina upogebiae parasitizes Upogebia species. Molecular analysis of mtDNA 16S rDNA sequences revealed that this species grouped with P. shiinoi (GenBank accession no: KF539761: Fig. 3B) with very high sequence identity (98.9%). Diagnosis of the externa: whole externa single, smooth or slightly wrinkled, and oval in shape. Mantle large, elevated, and vertically wrinkled with circular opening at the extremity. Remarks: Parasacculina leptodiae and P. sinensis have been reported to be parasites of Leptodius affinis, the most phylogenetically similar host species to M. distinguendus among the currently known hosts of Rhizocephala. However, the specimens examined in this study differ in morphology and molecular sequences from P. leptodiae and P. sinensis. This species has a single externa, whereas P. leptodiae has multiple externae. In addition, this species has a large, elevated mantle aperture, but P. leptodiae and P. sinensis have a flat mantle (Guérin-Ganivet, 1911;Boschma, 1933). Phylogenetic analysis clearly showed that the cox 1, 16S, and 18S rDNA sequences of this species are different from those of P. leptodiae and P. sinensis and all other Parasacculina species included in the analyses (Figs. 3A-3C). Therefore, we considered this species to be a new species candidate of the genus Parasacculina (see Discussion for more details). Diagnosis of the externa: whole externa smooth or slightly wrinkled, single, and flatcordiform shaped with flat half-oval-shaped left and right lobes divided by an outer mid-groove and inner mid-ridge; outermost part of the robe smooth or slightly wrinkled. Mantle tube-shaped, elevated, and vertically wrinkled with slit-shaped opening at the extremity.

Parasacculina sp. 2 (Fig. 2E)
Remarks: Morphological characteristics of the examined materials correspond with their original description (Okada & Miyashita, 1935) except for the number of externa and the host species. All specimens examined in this study had a single externa, whereas P. gregarius has multiple externae. In addition, the host species (H. sanguineus and H. takanoi) differ from the host species reported for P. gregarius, namely E. sinensis. Nevertheless, this species is likely P. gregarius because 18S rDNA sequences of these specimens were identical to the GenBank sequences of P. gregarius (Fig. 3C). In addition, individual variation in the number of externa of rhizocephalans has also been reported previously (Reinhard, 1942;Shiino, 1943;Høeg & Lützen, 1985).
In phylogenetic trees (Figs. 3A-3C), Polyascus cf. gregarious was clustered with P. planus that is commonly found in Japan and Taiwan. These two species are similar in having a flat-cordiform shaped externa, but different in some aspects of morphology and host species: the former has an elevated mantle and single externa, while the latter has an underdeveloped mantle and multiple externae (Boschma, 1933). In addition, the Varunidae crabs (H. sanguineus and H. takanoi) are used as P. cf. gregarious hosts, whereas the Grapsidae crabs (Grapsus albolineatus and Metopograpsus messor) are known as P. planus host (Tu, Chan & Jeng, 2009). Morphological and host range variation among rhizocephalan species has been reported by previous studies (Høeg & Lützen, 1985;Jung, Yoshida & Kim, 2019), and thus further studies with broader taxon sampling of P. gregarious and P. planus are needed to confirm an accurate species delimitation in their morphology and host range.

Phylogenetic relationships among rhizocephalan species
Since only Sacculinidae and Polyascidae species were found in this study, we focused on phylogenetic relationships among rhizocephalan species in these two families. Totals of 34 cox1 (555 bp), 33 16S rDNA (474 bp), and 35 18S rDNA (1002 bp) sequences were used for phylogenetic analysis, and the resulting ML and Bayesian trees were consistent with each other in that Sacculinidae and Polyascidae were monophyletic (Figs. 3A-3C). In all phylogenetic trees, the sequences of Korean rhizocephalans species nested and/or clustered with sequences of the same species retrieved from GenBank (Figs. 3A-3C).
Parasacculina sp. 1 and 2 were recognized as new species candidates because they did not show sister relationships with other Parasacculina species (Figs. 3A-3C). In the 16S and 18S DNA trees, they were placed at different positions and separated from P. leptodiae and P. sinensis, which share the host family and have similar morphological characteristics ( (Fig. 3A). Interspecific sequence differences of the two new species candidates from other Polyascidae species were 18.1-32.1% for cox1, 14.0-28.8% for 16S rDNA, and 1.9−4.7% for 18S rDNA. In contrast to the high interspecific sequence divergences discovered, there were no individual variations in cox1, 16S, and 18S rDNA sequences among Parasacculina sp. 1 specimens.

DISCUSSION
In this study, we identified nine species of Korean rhizocephalans from eight host decapod species using morphological and molecular analyses. Close examination of host ranges revealed that Korean rhizocephalan species have a different host prevalence than reported for rhizocephalan species from other geographic regions. In Korea, rhizocephalans were firstly found from three decapod hosts, i.e., Hemigrapsus takanoi, Macromedaeus distinguendus, and Pugettia intermedia. We also found that most Korean rhizocephalans showed high host specificity, parasitizing only one host, except Polyascus cf. gregarius that was found on two crab species (Fig. 4). The notable differences in host range between geographic isolates (i.e., rhizocephalans from Korea and other geographic regions) might be due to geographical variation in host species diversity and abundance or insufficient information about the geographic origins of host crab species as proposed by Jung, Yoshida & Kim (2019). In addition, unlike Korean S. confragosa individuals that were all found on only one grapsid crab species, Gaetice depressus, the Japanese form is known to parasitize three crab species, G. depressus, Pachygrapsus crassipes, and Cyclograpsus intermedius. Furthermore, Japanese P. yatsui parasitizes not only G. depressus, but also P. crassipes (Tsuchida, Lützen & Nishida, 2006;Kobayashi et al., 2018), whereas the Korean form of P. yatsui was found only on Hemigrapsus sanguineus. We could not determine if other crab species including P. crassipes and C. intermedius are potential hosts of Korean S. confragosa and P. yatsui because of the limited pool of crab host species examined in this study. Extensive taxon sampling of decapod hosts and their parasitic barnacles is needed to obtain a complete understanding of the host ranges of rhizocephalan barnacles and the distribution and prevalence of host-parasite associations.
The decapod host infestation rate of Korean rhizocephalan barnacles was much lower than that reported for Japanese species. In Japan, 35 individuals representing three rhizocephalan species were found in 354 individuals of three crab species, corresponding to an infestation rate of 9.9% (Tsuchida, Lützen & Nishida, 2006). By contrast, the infestation rate of Korean rhizocephalans was substantially lower at 1.2% on average (Table 1). Species richness and extent of host usage by parasitic barnacles are tightly correlated to the availability of host species (species diversity and abundance; Kamiya et al., 2014). Differences in the extent of host usage by rhizocephalan barnacles between the two geographic regions are likely due to differences in host species diversity and abundance, as well as the sample size of examined materials (e.g., total numbers of individuals and host species). Since we examined the prevalence of rhizocephalans on all decapod hosts (a total of 3,262 host individuals inspected), our result is likely an accurate estimate of the infestation rate. On the other hand, this prevalence difference between Korea and Japan may be originated from salinity, season, host sex and size (Mouritsen et al., 2018) or biogeographical differences (Kim et al., 2020). In a previous study, the infestation rate of Korean hermit crabs by rhizocephalans was reported to be 0.9% (Jung, Yoshida & Kim, 2019), which is similar to the infestation rate observed in this study. The unexpectedly high infestation rates (>50%) of Pachygrapsus crassipes and Arcotheres sinensis are due to strong bias from the very small sample size (one to four individuals) examined. The marine ecosystems in different geographic regions display different assemblages of barnacles (Kim et al., 2020) and thus extensive sampling of parasitic barnacles from a wide range of decapod host species is necessary to better understand their prevalence, infection intensity, and host range specificity (Mouritsen et al., 2018).
In addition to their host ranges, morphological and molecular analyses in this study provided insights into the taxonomy of Korean rhizocephalan barnacle species. Phylogenetic trees recognized four monophyletic rhizocephalan families, i.e., Polyascidae, Sacculinidae, Peltogastridae, and Peltogasterellidae, consistent with previous molecular analysis (Høeg et al., 2019) and morphology-based classification. Polyascidae is characterized by multiple externa and reproduces asexually (Glenner, Lützen & Takahashi, 2003), whereas Sacculinidae is characterized by single externa and sexual reproduction. Peltogastridae and Peltogasterellidae species mainly parasitize hermit crabs, and Peltogastridae is distinguished from Peltogasterellidae by the presence of the chitinous shield on its middle part of externae (Høeg et al., 2019). Two new species candidates in the genus Parasacculina (Parasacculina sp. 1 and Parasacculina sp. 2) were recognized based on molecular phylogenetic analyses. These species were distinct from their congeneric species, P. leptodiae and P. sinensis, based on phylogenetic analyses of mtDNA (16S rDNA) and nuclear (18S rDNA) sequences (Figs. 3B-3C) even though they are morphologically indistinguishable and were found in the same host species. These two species are genetically distinct cryptic species. Furthermore, we transferred three Korean Sacculina species (i.e., Sacculina imberbis, S. pilosella, and S. pinnotherae) to the genus Parasacculina because they grouped with Parasacculina species in mtDNA cox1, 16S, and 18S rDNA phylogenetic trees (Figs. 3A-3C). This new taxonomic replacement is consistent with previous studies that transferred several Japanese and Chinese Sacculina species to Parasacculina based on molecular evidence (Tsuchida, Lützen & Nishida, 2006;Glenner et al., 2010;Høeg et al., 2019).
Comparison of the external cuticles of Korean species with previously published morphological data provided new insight into the taxonomic status of the families Sacculinidae and Polyascidae. Although Høeg et al. (2019) showed that Sacculinidae and Polyascidae are phylogenetically distinct, the original descriptions of Polyascidae (Høeg et al., 2019) did not specify morphological characters differentiating this family from Sacculinidae. For example, Høeg et al. (2019) noted that polyascids have a smooth or almost smooth external cuticle, but some polyascid species (P. pinnotherae comb. nov. and P. yatsui) in the present study had wrinkled cuticles (Figs. 2C, 2F). In addition, Høeg et al. (2019) mentioned that Polyascus species have multiple externae, but Polyascus cf. gregarius in this study had only a single externa (Fig. 2G). These results indicate that the morphological characteristics of external cuticles, previously considered to be taxonomically valid features, are highly variable and cannot be used as diagnostic characters. Future comparative analyses of morphological characters along with molecular sequences are necessary to confirm the taxonomic status of Sacculinidae and Polyascidae and the taxonomic replacement of the three Korean Sacculina species in the genus Parasacculina.

CONCLUSIONS
In conclusion, this is the first report of the taxonomy, species diversity, and host ranges of Korean parasitic rhizocephalan barnacles based on morphological and molecular analyses. We identified nine parasitic barnacle species, including two new species candidates in the genus Parasacculina, in Korea. In addition, we found higher host specificity and lower infestation rates for Korean rhizocephalan species than reported for rhizocephalan species from other geographic regions. Nevertheless, the results of this study are based on preliminary data derived from limited taxon sampling in a narrow geographic range in Korea. Additional data from extensive samplings of parasitic barnacles from a wide range of crustacean host species are necessary to better understand the taxonomy, prevalence, host usage, and phylogenetic relationships of rhizocephalan species.