Two new enchytraeid species from Jeju Island, Korea (Annelida, Clitellata)

Abstract The enchytraeid fauna of three areas in Jeju Island (Korea) was studied, and comparative morphological and molecular taxonomic examinations (based on CO1, ITS and H3 sequences) were performed on nine samples collected in 2016. Twenty-two enchytraeid species were recorded and identified. The descriptions of two new species (Achaetamultisacculatasp. n. and Fridericiafloriformissp. n.) are presented in this paper. The main diagnostic features of A.multisacculatasp. n. are: three pairs of pyriform glands per segment, clitellum with two “baguette-like” packages of glands, dorsal blood vessel from VII, secondary pharyngeal glands absent, oesophageal appendages well developed, two pairs of preclitellar nephridia, the reproductive organs (except the spermathecae in V) shifted one segment forward. The main features of F.floriformissp. n. are that they are large worms, have up to 2–4 chaetae in bundles, strong body wall, thick cuticle, five pairs of preclitellar nephridia, c-type coelomo-mucocytes sometimes with some refractile vesicles, chylus cells in XII–XV, sperm funnels approximately twice as long than wide, spermathecae with long ectal duct without glands, ampullae surrounded distally by about 9–12 sessile diverticula of varying size. Molecular phylogenetic analyses supported the morphological results and confirmed the status of the two new species.


Introduction
The investigation of the previously unknown enchytraeid fauna of Korea has been continuing since 2007. Results have been published in four previous papers that yielded a total of 19 species new to science (Dózsa-Farkas and Hong 2010;Christensen and Dózsa-Farkas 2012;Dózsa-Farkas et al. 2015;. For Jeju Island, the fauna of Hallasan National Park (Mount Hallasan) was studied and published separately . In this paper, further faunistic results from the lowland areas of Jeju Island, outside the Hallasan National Park, are presented, including two new species. The morphological studies are supplemented with molecular taxonomic analyses targeting the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene, the nuclear ribosomal ITS region and the nuclear histone 3 (H3) gene, as in earlier studies (Dózsa-Farkas et al. 2015. For this, morphologically similar species and species described previously from Korea have been selected.

Study sites
Jeju Island (Jeju Province) encompasses 1,848 km 2 and is the largest island in South Korea. It was formed by volcanic eruptions approximately 2 million years ago. The center of its area is occupied by Mt. Hallasan. The island has a humid subtropical climate, making it warmer than the rest of South Korea. Winters are cool and dry while summers are hot, humid, and sometimes rainy. One of our study areas, Jocheon-eup, is a wetland and currently a candidate for designation as a Ramsar Wetland City, while the two other areas have relatively stronger human impact, being both popular sites for tourists.
The three study areas and nine sites within these areas are listed below. All samples were collected in 2016 by Yong Hong, similarly as in our parallel study regarding Mt. Hallasan

Methods of morphological examination
Soil samples were refrigerated until processing. Worms were extracted from the soil by the wet funnel method (O'Connor 1962). Enchytraeids were first observed and measured alive, and subsequently fixed in 70% ethanol. Some of the fixed specimens were stained with borax-carmine, and then passed through an ethanol dehydration series (from 70% to absolute), mounted temporarily in clove oil, then permanently in Euparal between two coverslips. Hence the worms were observable from both sides (Schmelz 2003). All the important morphological characters were recorded in vivo, drawn and photographed [Axio Imager.A2 microscope with DIC (differential interference contrast) illumination, AxioCam MRc 5 (Zeiss) digital camera, Axiovision software]. The whole-mounted specimens were reexamined and photographed as well. In all micrographs presented in this study, the orientation of specimens is the same: the head is either on the left side or at the top of the picture.
The holotypes and two paratypes are deposited in the National Institute of Biological Resources, Korea (NIBRIV). The remaining paratypes ("P", together with slide numbers) and further studied materials are deposited at the Department of Systematic Zoology and Ecology, ELTE Eötvös Loránd University, Hungary.

Morphological results
In total, 22 enchytraeid species belonging to seven genera were found in the samples (Table 1), among which two are new to science: Achaeta multisacculata sp. n. and Fridericia floriformis sp. n. With the two new species described here, the Korean fauna consists of 36 recorded terrestrial enchytraeid species to date. Additionally, one terrestrial polychaete, Hrabeiella periglandulata Pižl & Chalupský, 1984, was recorded at site 8. Diagnosis. The new species can be recognized by the following combination of characters: (1) small, slender worms (2.5-4.2 mm long and 160-220 μm wide at clitellum in vivo), segments 25-31; (2) six pyriform glands per segment in general; (3) clitellum weakly developed, interrupted middorsally and midventrally, with two elongate, "baguette-like" packages of gland cells on each dorso-lateral side; (4) dorsal blood vessel from VII; (5) pharyngeal glands at 4/5-6/7 connected dorsally, with ventral lobes and no secondary glands; (6) two pairs of preclitellar nephridia; (7) pars tumida of midgut from XII-XVI, extending over 2-3 segments, circumferal; (8) sperm funnels small, barrel-shaped, collar well developed about as wide as funnel body; (9) male pores in XI, ventro-lateral, each pore surrounded by small inconspicuous glands; (10) spermathecae free, confined to V with an asymmetrical dilation of ampulla and the ental tube ending in an oval reservoir.

Description of the new species
Description. Small, slender worm (Fig. 2E). Holotype (fixed) 3.2 mm long, 190 μm wide at VIII and 200 μm wide at clitellum (fixed), 31 segments. Paratypes 2.5-4.2 mm long, 155-200 μm wide at VIII and 160-220 μm wide at clitellum in vivo; 2.4-3.6 mm long, 150-210 μm wide at VIII and 160-210 μm wide at clitel-   Micrograph of Achaeta multisacculata sp. n. A Head lateral view (b = brain, knob on brain marked with black arrow, first dorsal pyriform glands marked with white arrow) B Brain dorsal view (knob on brain marked with arrow) C Cuticle thicker dorsally than ventrally, lateral view D Transverse body wall striation by strong ring muscles E Forepart of body to VII, lateral view (b = brain, ph = pharynx, oe = oesophageal appendages, dorsal pyriform glands marked with black arrows, lateral pyriform glands marked with wider white arrows, ventral pyriform glands marked with narrower white arrows) F Pyriform glands in IV-IX lateral view G Clitellar glands of holotype, lateral view (dorso-laterally 2 elongate, "baguette-like" packages of hyalocytes marked with black arrows, granulocytes ventro-laterally marked with white arrow) H Granular clitellar glands in transverse rows ventrally, lateral view (male openings marked with arrow) I Two baguette-like packages of clitellar glands (marked with arrows, in the middle hyalocytes, on the margins granulocytes) J Segments III-VIII, lateral view (oe = oesophageal appendages with meandering canal, marked with black arrow, dv = origin of dorsal vessel, n = first nephridium, ganglia of ventral nerve cords marked with white arrows) K Segments IV-VIII of paratypes NIBRIV0000813659, No. 2459 lateral view (p = pharyngeal glands, oe = oesophageal glands, spermatheca marked with arrow) A, D-F, H-J in vivo, B-C, G, K fixed, stained. Scale bars: 50 μm, in H, I: 20 μm.
Morphologically similar species. There are only three species (F. paraunisetosa Xie et al., 2000, F. ventrochaetosa Nagy, Dózsa-Farkas & Felföldi, 2018and F. callosa Eisen, 1878 among all Fridericia species, which possess more diverticula of spermathecae and the lateral chaetal bundles absent or incomplete, varying with 0, 1 or maximum 2 chaetae. Fridericia paraunisetosa can easily be distinguished from F. floriformis sp. n. based on the following characters: smaller size (5.0-7.8 mm long vs. 8-17.3 mm, fixed), lateral chaetal bundles absent, ventrally only one chaeta per bundle (vs. 2-4 chaetae ventrally and 0-2 laterally), dorsal pores only from XVIII (vs. from VII), brain incised anteriorly (vs. convex), oesophageal appendages stout and unbranched (vs. with branches at the end) (Xie et al. 2000). Fridericia ventrochaetosa could be distinguished from the new species by the total absence of the lateral chaetae and having spermathecal diverticula with stalk (vs. sessile) . The new species is similar to F. callosa in most traits (e.g., body size, segment number, strong body wall, thick cuticle, chaetal arrangement, number of preclitellar nephridia, position of chylus cells, the length of sperm), but the main differences between the two species are: in F. callosa the collar of sperm funnel not narrower than funnel body (Fig. 6A) (vs. narrower, Fig. 5A, B), seminal vesicle 2-3 segment large (vs. only in XI and not conspicuous). The spermathecae very variable in F. callosa (probably species complex) with or without diverticula, and the maximum number  of diverticula is 6 (Eisen 1878(Eisen , 1879Christensen and Dózsa-Farkas 1999;Schmelz 2003). From the material collected in Siberia in 1994, some stained slides were prepared now. On these slides, it was visible that the few diverticula are oriented towards the proximal ampullar part (Fig. 6B-C) in contrast to the spermathecae of the new species, which always have many diverticula or diverticula-like protrusions surrounding the ampullae (Fig. 5F-J).

Results of molecular analysis
In total, 9, 13 and 9 new sequences were determined from various Achaeta and Fridericia species in the case of ITS, CO1 and H3, respectively. Additional sequences determined in previous studies (Erséus et al. 2010;Dózsa-Farkas et al. 2015Dózsa-Farkas andFelföldi 2017, 2018;Nagy et al. 2018) were also used for comparison (Table 2). However, unfortunately, we failed to amplify the H3 gene from specimens of Achaeta multisacculata sp. n., which was probably due to the improper hybridization of PCR primer sequences to the extracted genomic DNA. Results of the molecular analyses confirmed that the two new species are genetically separate from morphologically similar species and species described previously from Korea and their sequences form distinct lineages on the phylogenetic trees (Figs 7, 8). This was also supported by interspecific sequence distances, since in the case of the

Discussion
Earlier we studied and described the enchytraeid fauna of Hallasan National Park (Mt. Hallasan) from Jeju Island ). In the present study, we investigated nine new samples collected from other areas of Jeju Island. This time 22 enchytraeid species were found, two of which are new to science. According to the studied samples, sites 8 and 9 (Youngnuni-orum) were the most species-rich (with ten and seven detected species), harboring the two new species, Achaeta multisacculata sp. n. and Fridericia floriformis sp. n. (both new species were found only in this area) ( Table 1). This could be explained probably with the meadow habitat, since the other samples were collected from forest habitats. Results of molecular analyses confirmed the status of the two new species.
Four species (Achaeta macroampullacea, Xetadrilus jejuensis, X. aphanoides, Fridericia cf. paroniana) which were described from Mt. Hallasan previously, were found also in the lowland areas of Jeju Island. Xetadrilus aphanus did not occur in the Hallasan National Park, so the present record from Dongbaekdongsan, Jocheon-eup (site 2) is new for the Korean fauna. The comparison of the three Xetadrilus species (a genus established by Schmelz et al. 2011) is given in    Christensen and Dózsa-Farkas 2012;Dózsa-Farkas et al. 2015) were also found in the present study. It seems that these species are characteristic members of the Korean enchytraeid fauna, and that the genus Hemienchytraeus has a wide geographic distribution within the country. Fridericia cf. paroniana was found in this study and also in Mt. Hallasan, and the differences from F. paroniana were discussed in . At site 6, a species very similar to F. sphaerica Dózsa-Farkas et al., 2015 occurred, but according to the results of molecular analysis, it is different from F. sphaerica. Unfortunately, we found only two specimens from this putatively new species, and we will try to solve its taxonomic status later (therefore we referred to it now as F. cf. sphaerica).
As mentioned above, the specimens of Achaeta multisacculata sp. n. did not possess any mature eggs, although the extraction of worms from soil samples was carried out several times from autumn to January. In contrast, Achaeta macroampullacea specimens mostly had mature eggs, so it can be assumed that A. multisacculata sp. n. belongs to that enchytraeid group where the worms reproduce only in certain seasons, as e.g. most Mesenchytraeus species (Dózsa-Farkas 1996).
Before 2007, the Korean enchytraeids were completely unknown. Results of subsequent studies (Dózsa-Farkas and Hong 2010; Christensen and Dózsa-Farkas 2012;Dózsa-Farkas et al. 2015; indicated that the fauna is species rich. Including the findings in this paper, we described 23 species new for science and 13 new records for the Korean fauna. Thus, the Korean fauna now consists of 36 recorded terrestrial enchytraeid species. The high species number could be explained by the diverse geographic relief of the area which would result in many different microhabitats with differing microclimates, providing both for subtropical and temperate species (e.g., the typical tropical and subtropical Hemienchytraeus species or the widely distributed European Fridericia bulboides and Hemifridericia parva) suitable conditions to flourish. We think that some worms are introduced species, e.g., the two terrestrial polychaetes, Parergodrilus heideri Reisinger, 1925 detected in a previous survey (Dózsa-Farkas and Hong 2010) and Hrabeiella periglandulata (a typical European taxon) which was detected in this study for the second time in Korea (only at site 8). Probably Xetadrilus aphanus, an enchytraeid species described from Brazil (Schmelz et al. 2011), is also an introduced species. Unfortunately, detailed biogeographical conclusions cannot be drawn yet regarding the Korean enchytraeid fauna, since the fauna of several areas has not been studied yet or is under study; furthermore some morphologically identical material (e.g., Fridericia seoraksani, F. sphaerica), possibly representing cryptic species, requires further analysis.