Niviventer undetermined

New Data to the Earthworm Fauna of the Korean Peninsula with Redescription of Eisenia koreana (Zicsi) and Remarks on the Eisenia nordenskioldi Species Group (Oligochaeta, Lumbricidae) Yong Hong1 and Csaba Csuzdi2,* 1Department of Agricultural Biology, College of Agriculture & Life Sciences, Chonbuk National University, Jeonju, Republic of Korea 2Department of Zoology, Eszterházi Károly College, Eger, Hungary (Received November 19, 2014; Accepted September 29, 2015) Yong Hong and Csaba Csuzdi (2016) The earthworm fauna of the Korean peninsula, especially the Northern part (Democratic People’s Republic of Korea) is still poorly known. Altogether 148 earthworm taxa are reported from Korea of which 22, including some uncertain records, belong to the Holarctic family Lumbricidae. From these 22 lumbricid taxa only eight are thought to be autochthonous, including the widely distributed Eisenia nordenskioldi species group which consists of six highly diverged DNA lineages. Due to a lack of material, the phylogenetic affinities of the Korean E. nordenskioldi s.l. specimens have not been determined so far. Here we report on the first lumbricid records from the Northern part of the peninsula (DPRK) after the description of the endemic earthworm species Eisenia koreana (Zicsi, 1972) together with a redescription of its type specimens. The supposedly autochthonous Eisenia nordenskioldi species group is briefly reviewed and the specimens collected recently in South Korea (Republic of Korea) were DNA barcoded and compared to the published lineages from Siberia (Russia). The redescription of the type specimen of Eiseniella koreana confirmed its inclusion in the genus Eisenia. Barcoding of the unpigmented Korean E. nordenskioldi specimens revealed that they form an independent clade placed quite far from the previously published unpigmented E. nordenskioldi forms and might represent a distinct species. Key words: North Korea, South Korea, Earthworm fauna, DNA barcode, Eisenia nordenskioldi species group. *Correspondence: E-mail: [email protected] BACKGROUND The systematic earthworm research in the Korean Peninsula dates back to the work of Shinjiro Kobayashi (1934, 1936a, b) who later summarized the earthworm fauna of Korea, reporting 38 species including 10 lumbricids (Kobayashi 1938, 1941). After Kobayashi’s pioneering work until the last decade, there was only one lumbricid species described from the peninsula; Eiseniella koreana Zicsi, 1972 from Taesong Mt., Pyongyang, North Korea. In the family Megascolecidae, Song and Paik (1969, 1970a, 1970b, 1971, 1973) presented data on the earthworm fauna of the Korean Peninsula including descriptions of four new species. At the beginning of the new millennia Hong (2000) reviewed the lumbricid fauna of Korea reporting seven species including some doubtful records (i.e. Aporrectodea tuberculata (Eisen, 1874) or Bimastos beddardi (Michaelsen, 1894)). Unfortunately, at that time the sole endemic lumbricid species E. koreana was not listed. From the early 2000’s, due to the work of Yong Hong and Sam James and later Robert Blakemore, the number of the earthworm taxa reported from the Korean Peninsula has rapidly grown to 94 (Blakemore 2006), later to 106 (Hong and James 2009) and recently reached 148 (Blakemore et al. 2012, Blakemore 2014, Blakemore et al. 2014, Blakemore et al. 2015). However, most of the recent works deal with species belonging to the family Megascolecidae and are almost exclusively Zoological Studies 55: 12 (2016) doi:10.6620/ZS.2016.55-12 1 from South Korea. From the family Lumbricidae after Zicsi’s work (1972) only four taxa were described from Korea: Eisenia gaga Blakemore, 2012, Eisenia sindo Blakemore, 2012, Eisenia japonica vaga Blakemore, 2013 and most recently Eisenia muuido Blakemore, 2015, all from South Korea. Here we report on earthworms collected on a trip by the second author to North Korea in 1988 and several other lumbricid specimens collected in different parts of South Korea by the first author. Furthermore, we have redescribed E. koreana (Zicsi, 1972) from an examination of the type material housed in the Hungarian Natural History Museum, Budapest. This work was undertaken as Blakemore and Park (2012) reported two new Eisenia species from South Korea and mentioned that E. koreana had not been recorded since the original description and also that the original account of Zicsi (1972) is incomplete in several ways. The widely distributed Eisenia nordenskioldi species complex has recently been analysed using DNA barcodes and ITS2 sequences (Shekhovtsov et al. 2013). In this work six highly diverged E. nordenskioldi lineages were identified apart from the unpigmented E. n. pallida Malevich, 1956 which represented also an independent lineage. The inter-lineage genetic distances (K2P) for COI gene were between 13.6% and 19.1% suggesting that some of the lineages are beyond the intraspecific distance limit determined by Chang and James (2011). Since the analysis by Shekhovtsov et al. (2013) did not include the recently reported Mongolian specimens (Blakemore & Park 2012, Blakemore 2013) nor representatives from the Korean Peninsula, to determine the position of the Korean E. nordenskioldi s.l. specimens we reanalysed Shekhovtsov et al. (2013) data complemented with the newly sequenced Korean specimens and the published DNA sequences of Blakemore and Park (2012) and Blakemore (2013b) (Table 1). Table 1. Species and COI references/GenBank accession numbers used in this study Species Accession/Identification Number Reference Eisenia andrei AY874509 Pérez-Losada et al. 2005 Eisenia andrei AY874510 « Eisenia andrei JN870085 Voua Omoto et al. 2013 Eisenia andrei JN870087 « Eisenia andrei JN870088 « Eisenia andrei JN870086 « Eisenia cf andrei w11 Jeju Blakemore and Lee 2013 Eisenia cf japonica WO19 large NIBRIV0000250888 Korea « Eisenia fetida FJ214228 Lund et al. 2009 Eisenia fetida H6 Busan Blakemore and Lee 2013 Eisenia fetida JX531566 Shekhovtsov et al. 2013 Eisenia fetida WO12 S1 from Jeju Island Korea Blakemore and Lee 2013 Eisenia fetida FJ214216 Lund et al. 2009 Eisenia fetida AY874516 Pérez-Losada et al. 2005 Eisenia fetida AY874517 Pérez-Losada et al. 2005 Eisenia fetida cf xanthurus W018 from mainland Korea Blakemore and Lee 2013 Eisenia gaga Holotype WM1 INV 0000245509 Blakemore and Park 2012 Eisenia gaga Paratype WM3 INV 0000245513 « Eisenia gaga Paratype1 WM2 INV 0000245511 « Eisenia japonica AB542698 Minamiya et al. unpublished Eisenia japonica japonica JET170 An-417 Enoshima topotype Blakemore and Lee 2013a Eisenia japonica JET124 Tokyo An-466.1 Nogeyama « Eisenia japonica vaga H5 holotype from Busan « Eisenia japonica vaga WO20 paratype IV0000250889 « Eisenia japonica w18b Jeju Blakemore 2013a Eisenia nordenskioldi clade 1 JX531495 Shekhovtsov et al. 2013 Eisenia nordenskioldi clade 1 JX531498 « Eisenia nordenskioldi clade 1 JX531503 « Eisenia nordenskioldi clade 2 JX531465 « page 2 of 15Zoological Studies 55: 12 (2016) MATERIALS AND METHODS Earthworms were collected by the diluted formaldehyde method (Raw 1959), with digging and searching under stones, mosses, and the bark of fallen logs. The collecting sites in North Korea were scattered around three centres; the Baekdu Mts close to the Chinese border, the Myohynag Mts in the central mountainous region and Haeju, close to the South Korean border. In South Korea the collection sites are scattered all over the country and covered mainly agricultural fields. The specimens collected were killed in 75% ethanol and fixed in 4% formaldehyde solution, then transferred into 75% ethanol and deposited in the earthworm collection of the Hungarian Natural History Museum (HNHM) and National Institute of Biological Resources, Korea (NIBR). For molecular studies, some specimens from the newer samples were placed into 96% ethanol. Partial sequences of COI gene were obtained from three specimens representing the Eisenia nordenskioldi species complex following the protocol described in Szederjesi and Csuzdi (2015). Sequences were compared with the GenBank nucleotide database using blastn 2.2.14 algorithm (Altschul et al. 1997). Additional DNA sequences representing the different E. nordenskioldi lineages (Shekhovtsov et al. 2013), Eisenia japonica, Eisenia fetida and Eisenia andrei were downloaded from the GenBank (Table 1). The DNA sequences were aligned with ClustalW (Thompson et al. 1994) implemented in MEGA 6.06 (Tamura et al. 2013) using the default settings. Maximum Likelihood analysis was carried out with MEGA 6.06 (Kumar et al. 2008) using GTR + G + I model selected by the Akaike Information Criteria (Akaike 1974) implemented in MEGA 6.06. Bootstrap support was estimated from 1000 replicates. Bayesian analysis was carried out with BEAST 1.8.0 (Drummond and Rambaut 2007) using Metropolis coupled Markov chain Monte Carlo simulations for 10 million generations, sampling a tree in every 1000 generations. After Species Accession/Identification Number Reference Eisenia nordenskioldi clade 2 JX531475 Shekhovtsov et al. 2013 Eisenia nordenskioldi clade 2 JX531483 « Eisenia nordenskioldi clade 2 JX531485 « Eisenia nordenskioldi clade 3 JX531542 « Eisenia nordenskioldi clade 3 JX531550 « Eisenia nordenskioldi clade 3 JX531553 « Eisenia nordenskioldi clade 3 JX531564 « Eisenia nordenskioldi clade 3 JX531565 « Eisenia nordenskioldi clade 4 JX531487 « Eisenia nordenskioldi clade 4 JX531488 « Eisenia nordenskioldi clade 4 JX531489 « Eisenia nordenskioldi clade 5 JX531515 « Eisenia nordenskioldi clade 5 JX531518 « Eisenia nordenskioldi clade 5 JX531519 « Eisenia nordenskioldi clade 5 JX531521 « Eisenia nordenskioldi clade 6 JX531526 « Eisenia nordenskioldi clade 6 JX531535 « Eisenia nordenskioldi clade 6 JX531536 « Eisenia nordenskioldi Korea 1 KM593696 New Eisenia nordenskioldi Korea 2 KM593697 New Eisenia nordenskioldi Korea 3 KM593698 New Eisenia nordenskioldi mongol Holotype wo63 Blakemore 2013b Eisenia nordenskioldi mongol Paratype wo64 « Eisenia nordenskioldi onon Holotype wo65 « Eisenia nordenskioldi pallida JX531522 Shekhovtsov et al. 2013 Eisenia nordenskioldi pallida JX531523 « Eisenia sindo WO25 Holotype IV0000246435 Blakemore 2013b Eisenia sindo WO26 Paratype P1 IV0000246436 « Enchytraeus albidus GU902047 Erseus et al. 2010 Table 1. (continued) page 3 of 15Zoological Studies 55: 12 (2016) removing the first 2,000 trees as burn-in, the remaining 8,000 sampled trees were analyzed with TreeAnnotator v1.8.0 and visualized by FigTree 1.4.0 (Rambaut 2012). Pairwise genetic distances were calculated in MEGA 6.0.6 using the Kimura 2-Parameter model. RESULTS Class Oligochaeta Family Lumbricidae Rafinesque-Schmaltz, 1815 Aporrectodea cf. trapezoides (Dugès, 1928) Lumbricus trapezoides Dugès, 1828: 289. Allolobophora caliginosa f. trapezoides: Kobayashi 1941: 150. Aporrectodea trapezoides: Blakemore 2008: 534 (for complete synonymy). Aporrectodea trapezoides: Blakemore 2013a: 39. Aporrectodea trapezoides: Blakemore and Lee 2013: 134. Aporrectodea trapezoides: Blakemore 2014: 11. Material examined: South Korea. Jeollanamdo, Sinan-gun, Huksando Island, 10.08.2009, 1 ex. Leg. Y. Hong., Jeollabuk-do, Gunsan-si, Sunyudo Island, 01.10.2009, 9 ex. Leg. Y. Hong., Chungcheongnam-do, Boryeong-si, Ungcheon, 11.06.2008, 5 ex. Leg. Y. Hong., Jeju-do, Jejusi, 30.05.2008, 1 ex. Leg. Y. Hong., Jeollabukdo, Gunsan-si, Muyedo Island, 06.10.2009, 5 juv. ex. Leg. Y. Hong., Gangwon-do, Jeongsungun, Jeongsun, 04.07.2009, 1 ex. Leg. Y. Hong., Jeollanam-do, Jangheung-gun, Jangheung, 23.09.2009. 2 ex. Leg. Y. Hong., Incheon-si, Ongjin-gun, Baekryeong-do Island, 28.08.2008, 2 ex. Leg. Y. Hong., Chungcheongbuk-do, Okcheongun, Okcheon, 23.08.2007, 3 ex. Leg. Y. Hong., Jeollabuk-do, Gunsan-si, Sunyudo Island, 26.04.2013, 34 ex. Leg. Y. Hong., Jeollabukdo, Buan-gun, Dongjin, 23.05.2013, 1 ex. Leg. Y. Hong (NIBRIV0000323392)., Jeollabuk-do, Buan-gun, Dongjin, 12.10.2013, 16 ex. Leg. Y. Hong., Incheon-si, Gangwha-gun, Gangwhado, 03.05.2013, 8 ex. Leg. Y. Hong. Remarks: All of the specimens examined have dark-brown colour on dorsum that is typical for Ap. trapezoides but neither of them has clearly band-shaped tubercles (Fig. 1). Bimastos parvus (Eisen, 1874) Allolobophora parva Eisen, 1874: 46. Bimastos parvus: Kobayashi 1941: 155. Bimastos parvus: Blakemore 2008: 537 (for complete synonymy). Bimastos parvus: Blakemore 2014: 11. Material examined: North Korea. Before Haeju, 17.07.1988, 10 ex. Leg. Cs. Csuzdi., Haeju, Su-Yong waterfall, 16.07.1988, 12 ex. Leg. Cs. Csuzdi., South Korea. Incheon-si, Ongjin-gun, Baekryeong-do Island, 28.08.2008, 3 ex. Leg. Y. Hong., Jeollabuk-do, Gunsan-si, Sunyudo Island, 17.10.2013, 3 ex. Leg. Y. Hong (NIBRIV0000323393)., Jeollabuk-do, Buangun, Dongjin, 23.05.2013, 1 ex. Leg. Y. Hong., Jeollabuk-do, Buan-gun, Dongjin, 12.10.2013, 3 ex. Leg. Leg. Y. Hong., Jeollabuk-do, Muju-gun, Muju, 26.10.2013, 7 ex. Leg. Leg. Y. Hong. Remarks: B. parvus is perhaps of North American origin (Gates 1972) but it has been widely introduced in Europe and also in Asia (Gates 1972, Blakemore 2008, Szederjesi et al. 2013a, 2013b). Eisenia japonica japonica (Michaelsen, 1892) Allolobophora japonica Michaelsen, 1892: 230. Allolobophora japonica: Kobayasi 1936b: 183. Allolobophora japonica f. typica: Kobayashi 1941: 151. Eisenia japonica: Blakemore and Grygier 2011: 270 (for complete synonymy). Eisenia japonica: Blakemore 2013a: 42. Material examined: South Korea. Jeju-do, 01.06.2008, 5 ex. Leg. Y. Hong. Remarks: Our specimens are unpigmented, greyish. The biometry varies between 46 × 3 mm, segment No. 98 and 72 × 4 mm segment No. 120. Clitellum on 1/n 23-31, tubercles two distinct maize kernel-shaped protuberances on 27, 29. Genital papillae completely missing. Fig. 1. Aporrectodea trapezoides (Dugés, 1828); ventrolateral view of the clitellar region. page 4 of 15Zoological Studies 55: 12 (2016) Eisenia vaga Blakemore, 2013 stat. nov. Eisenia japonica vaga Blakemore, 2013: 132. Eisenia japonica vaga: Blakemore 2014: 11. Material examined: South Korea. Incheonsi, Ongjin-gun, Baekryeong-do Island, 28.08.2008, 2 ex. Leg. Y. Hong., Chungcheongnam-do, Cheongyang-gun, Cheongyang, 17.06.2008, 1 ex. Leg. Y. Hong., Jeju-do, Jeju-si, 30.05.2008, 3 ex. Leg. Y. Hong. Remarks: This is the first record of E. j. vaga after its original description. Our specimens agree well with the original description except the somewhat shorter clitellum, presence of slight red pigmentation on dorsum and the somewhat smaller biometry. Length 32-35 × 2 mm, segment No. 110122. Clitellum ½ 24-31, tubercles two dorsally connected maize kernel-shaped protuberances on 27, 29. Two pairs of prominent genital papillae around ab on 25, 26 (Fig. 2). Setal arrangement after clitellum aa:ab:bc:cd:dd = 7:1.25:5:1:23. Vesicles four pairs in 9-12, but those in 9 and 10 are much smaller. Spermathecae 9/10-10/11 roundish clearly stalked and filled with sperms, open laterally in cd. Calciferous glands small in 11-12, typhlosole moderate T-shaped, nephridial bladders elongated sausage-shaped with slightly curved ectal part. The morphological difference between E. japonica and E. vaga are quite scanty (biometry and the distribution of genital papillae) however the genetic distance between japonica and vaga (Fig. 13, 14, Table 2) clearly indicates that they represent independent species. Eisenia koreana (Zicsi, 1972) Eiseniella koreana Zicsi, 1972: 129. Eisenia koreana: Perel 1979: 82. Eisenia koreana: Easton 1983: 480. Eisenia koreana: Blakemore and Park: 2012: 299. Material examined: Holotype. HNHM/12715 with label data: Eiseniella koreana sp. n. typus, Phenjan, 22.V.1970. Leg. S. Mahunka u. H. Fig. 2. Eisenia vaga Blakemore, 2013b; ventrolateral view of the clitellar region. Table 2. Pairwise nucleotide K2P differences (in %) between the investigated earthworm species/lineages species 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1. E. sindo 0.01 2. E. n. cf. pallida 0.225 0 3. E. n. onon 0.238 0.165 - 4. E. n. mongol 0.249 0.179 0.082 0 5. E. nordenskioldi_Korea 0.24 0.231 0.19 0.217 0.008 6. E. nordenskioldi_6 0.235 0.215 0.207 0.207 0.219 0.005 7. E. nordenskioldi_5 0.23 0.213 0.176 0.203 0.229 0.219 0.068 8 .E. nordenskioldi_4 0.228 0.199 0.216 0.219 0.2 0.215 0.214 0.027 9. E. nordenskioldi_3 0.226 0.224 0.196 0.203 0.178 0.212 0.204 0.212 0.046 10. E. nordenskioldi_2 0.248 0.222 0.21 0.222 0.223 0.209 0.207 0.145 0.201 0.052 11. E. nordenskioldi_1 0.236 0.187 0.129 0.151 0.222 0.205 0.163 0.204 0.206 0.195 0.065 12. E. j. vaga 0.244 0.21 0.204 0.206 0.215 0.236 0.226 0.237 0.222 0.248 0.219 0.057 13. E. j. japonica 0.23 0.228 0.205 0.204 0.235 0.224 0.234 0.239 0.224 0.247 0.223 0.208 0.123 14. E. gaga 0.206 0.25 0.241 0.253 0.226 0.228 0.229 0.237 0.209 0.247 0.238 0.235 0.239 0.005 15. E. andrei 0.24 0.234 0.23 0.225 0.262 0.195 0.235 0.253 0.249 0.237 0.219 0.213 0.217 0.258 0.007 16. E. fetida 0.241 0.22 0.237 0.229 0.266 0.224 0.218 0.239 0.249 0.238 0.217 0.202 0.222 0.249 0.16 0 In the diagonal (bold) the within lineage differences are shown. K2P values shaded in light grey refer to subspecific differences, in darkgrey to infra-subspecific differences according to Szederjesi et al. (2011). page 5 of 15Zoological Studies 55: 12 (2016) Steinmann., Paratypes. HNHM/7000 3 adult + 2 preadult complete specimen, 2 adult specimen with missing tail, 1 juvenile and 1 tail part with label: Eiseniella koreana paratypus, Phenjan 22.V.70. Leg. S. Mahunka u. H. Steinmann. New material: North Korea. Haeju, SuYong waterfall, 16.07.1988. Leg. Cs. Csuzdi (HNHM/16919 1 juv. ex). Redescription: External characters. Holotype 44 mm long and 2.8 mm wide. Number of segments 100. Paratypes 41-52 mm long and 2.83 mm wide. The tail is prominently quadrangular. Number of segments 125-130. Colour pale with slight hints of previous pigmentation. Prostomium epilobous ½ closed. First dorsal pore at intersegmental furrow 4/5. Setae closely paired. Setal arrangement behind clitellum: aa:ab:bc:cd:dd = 5.7:1.2:5.2:1:8.6 (Fig. 3). Male pores on segment 15, surrounded by glandular crescents, slightly protruding into segments 14 and 16. Female pores as small slits on 14, just above the setae b. Nephridial pores irregularly alternate between setal line b and above d. Clitellum on segments 25-31 but segment 24 also modified slightly (Fig. 4). Tubercula pubertatis as glandular trench on segments 27-⅔29, 29 (Fig. 5). Genital papillae on Holotype; 10 cd, 10 ab Rhs, 11 cd, 16, 26-28, 30 ab. Paratype 1; 11 cd, 9, 10 ab Rhs, 11 cd Lhs, 16, 25, 30. Paratype 2; 23 ab Rhs, 17 ab Lhs, 11 cd, 16, 26-28, 30 ab. Spermatophore at 22/23 on Holotype. Internal characters: Septa 7/8-8/9 thickened, 6/7, 9/10-10/11 slightly strengthened, and also 12/13-14/15 somewhat stout. Testes and funnels paired in segments 10-11, free. The entalmost part of the sperm ducts somewhat coiled and slightly thickened. Seminal vesicles in 9-10 small, 1112 larger. Spermathecae drop-shaped in 9/1010/11 with external openings close to the middorsal line. Ovarium in 13, elongated and slightly flattened. Ovarian funnel large, with undulated rim, ovisac small, pending from 13/14 just above Fig. 3. Eisenia koreana (Zicsi, 1972) setal arrangement. aa = distance between the ventral setal lines, ab = distance between the ventrolateral setal lines, cd = distance between the dorsolateral setal lines, dd = distance between the dorsal setal lines. Fig. 4. Eisenia koreana (Zicsi, 1972) ventrolateral view of the fore-body. Fig. 5. Eisenia koreana (Zicsi, 1972) ventrolateral view of the clitellar region. page 6 of 15Zoological Studies 55: 12 (2016) the ovarian duct. Calciferous glands in 11-12 with slightly more developed diverticula in segment 12. Paired hearts appear in segments 7-11, with a pair of small extraoesophageal vessel in 12. Nephridial bladders simple, slightly elongated sac-shaped (Fig. 6). Crop in segments 15-16, and gizzard in segments 17-18. Typhlosole bilobed, moderate (Fig. 7). Longitudinal muscle layer is of pinnate type. Remarks: On the basis of the gizzard (in 1718) and the position of the female pores (above setae b) koreana is surely fits into the genus Eisenia (e.g. Perel 1979, Easton 1983) and seems to be very close to E. gaga Blakemore, 2012 (in Blakemore & Park 2012) described from Gageodo Island, S. Korea. The only substantial differences found from this species are the size and segment number (41-52 × 2.8-3 mm vs. 80-105 (diameter not noted in gaga) and 100-130 vs. 124-162). E. koreana is close also to E. sindo Blakemore, 2012 from Sindo Island, Incheon, S. Korea but differs from it in the smaller biometry and presence of a typhlosole (lacking in sindo). As the two recently described species were mainly defined by their DNA barcodes, adequately preserved North Korean specimens would needed to clear the relationship between E. koreana, E. sindo and E. gaga. Eisenia nordenskioldi cf. pallida Malevic, 1956 Eisenia nordenskiöldi f. typica: Kobayashi 1941: 148 (misidentification). Eisenia nordenskioldi species complex: Blakemore 2008: 580. Eisenia nordenskioldi pallida: Blakemore 2014: 12. Notes: E. nordenskioldi represents a quite heterogeneous species complex with different polyploid races (Perel 1997) and several, genetically quite diverged lineages (Shekhovtsov 2013). According to Perel (1997) in the Korean Peninsula the slightly pigmented form E. nordenskioldi pallida Malevic, 1956 occurs which, as stated by Blakemore (2013b), might be a synonym of Helodrilus (Allolobophora) acystis Michaelsen, 1903 from Turkestan. Material examined: North Korea. Myohyang Mts. Kuchung, 1000 m, 06.1985, 1 ex. Leg. Z. Tóth (HNHM/16902)., Baekdu Mts. near Pochon, 13.07.1988, 1 ex. Leg. Cs. Csuzdi (HNHM/16904)., Baekdu Mts. near Samjiyon, 12.07.1988, 3 ex. Leg. Cs. Csuzdi (HNHM/16905)., between NamChon and Haeju, 17.07.1988, 3 ex. Leg. Cs. Csuzdi (HNHM/16920)., Haeju, Su-Yong waterfall, 16.07.1988, 1 ex. Leg. Cs. Csuzdi (HNHM/16921)., Myohyang Mts., near Hyangsan, 07.07.1988, 3 ex. Leg. Cs. Csuzdi (HNHM/16922). South Korea. Jeju-do, Jeju-si, 30.05.2008. 2 ex., 31.05.2008, 1 ex. Leg. Y. Hong., Incheon-si, Ongjin-gun, Baekryeong-do Island, 28.08.2008, 5 ex. Leg. Y. Hong., Incheon-si, Gangwha-gun, Gangwhado, 03.05.2013, 5 ex. Leg. Y. Hong., Jeollabukdo, Gunsan-si, Sunyudo Island, 26.04.2013, 2 ex. Leg. Y. Hong., Jeollabuk-do, Gunsan-si, Muyeodo Island, 18.10.2013, 1 ex. Leg. Y. Hong., Daegu-si, 03.08.1965, 1 ex. Leg. M.J. Song, Gyeongsangbuk-do, Gimcheon, 16.07.1966, 1 ex. Leg. M.J. Song. Remarks: None of our specimens possessed red pigmentation even alive. Their colour is greyish, more dark on the dorsum at the head. Setae closely paired (Figs. 8, 9), the biometry varies between 44-74 × 3-4.5 mm, segment No. 80-141. Clitellum 25, 26-33, 1/n 34, tubercles ½ 29, 29-½ 31, ½ 32. Number of papillae before clitellum varied, but they are constant on the clitellum around ab 27-33, (34). (Table 3). Four pairs of vesicles in 9-12 but the first two pairs in 9-10 are rather small. Spermathecae in 9/10-10/11 drop-shaped open just near to the dorsal pores. Fig. 6. Eisenia koreana (Zicsi, 1972) nephridial bladder. Fig. 7. Eisenia koreana (Zicsi, 1972) typhlosole. dv = dorsal vessel. page 7 of 15Zoological Studies 55: 12 (2016) Fig. 8. Eisenia nordenskioldi cf. pallida Malevic, 1956; setal arrangement of the Korean specimens. aa = distance between the ventral setal lines, ab = distance between the ventrolateral setal lines, cd = distance between the dorsolateral setal lines, dd = distance between the dorsal setal lines. Fig. 9. Eisenia nordenskioldi cf. pallida Malevic, 1956 setal arrangement of the type specimen. aa = distance between the ventral setal lines, ab = distance between the ventrolateral setal lines, cd = distance between the dorsolateral setal lines, dd = distance between the dorsal setal lines. Table 3. Main morphological characters of the Eisenia nordenskioldi subspecies Species Reg. No. Biometry mm, segment No. Clitellum Tubercles Papillae E. n. pallida Korea 16905 74 × 4 132 (25)26-1/n34 29-1/n32 ab 10-12,14,16,22-24,27-34 16902 55 × 3 113 (26)27-33 29-31 ab 11,12,16,23-24,27-33 16904 58 × 4 141 27-33 ½29-½32 ab 9-12,16-17,24-34 16922 42 × 3 123 (26)27-33 29-31 ab 25-33 16903 57 × 4.5 80 (24)25-1/n34 29-31 ab 9-13,16,25-34 16921 47 × 3 136 27-33 29-31 ab 27-32 (not fully adult) Gangwon-do 50 × 3.5 115 26-33 29-31 ab 16,23-24,27-32,34 E. n. pallida Malevic, 1956 ? (25)26,27-33 29-31 ab 24-25,27-34 E. n. acystis (Michaelsen, 1903) 90 × 4 126 26-33 29-30, 1/n31 abcd 9-12 E. n. polypalpillata Perel, 1969 55-80 × 4-5 102-137 26,27-32 ½ 28,29-½31,31 ab 16-18,21-23,26,32-34 E. tracta Perel, 1985 47-138 × 5-12 111-168 26-33 29-½32 ab 8-10,16,17,23-36,37 cd 11,12 E. ventripapillata Perel, 1985 40-90 × 3-4 111-139 26-33 ½28,28-30,½31 ab 10,11,26-32 cd 10,(11) E. n. mongol Blakemore, 2013 80-110 × ? 131-142 (25)26-33 29-31 ab 7, 11, 26, 27-32,33 cd 8-11 E. n. onon Blakemore, 2013 100 × ? 170 (24)25-33 28-31 ab 11-12 page 8 of 15Zoological Studies 55: 12 (2016) Typhlosole moderate, bilobed or T-shaped. The above description agrees well with the original description of E. n. papillda. Especially the shared papillar segments are characteristic; therefore we regard our Korean specimens to be identical to E. n. pallida described from environs of Vladivostok (Russia), just ca. 150 km NE from the North Korean border. Helodrilus (Allolobophora) acystis Michaelsen, 1903 is also a similar species however, E. n. pallida clearly differs from it in the setal arrangement (in pallida dd is always less than ½ circumference, in acystis dd = 7/13 circumference) and in the distribution of the genital papillae (in acystis they are confined to segments 9-12, in pallida they are more widespread). Therefore we agree with the opinion of Perel (1997: 70) that H. (A) acysis Michaelsen, 1903 represents a different (sub) species. To clear the positions of the different species/subspecies in the Eisenia nordenskioldi species complex a thorough morphological/ molecular analysis of the species involved are needed (cf. Blakemore 2013b). Eisenia nordenskioldi nordenskioldi (Eisen, 1879) Allolobophora nordenskioldi Eisen, 1879: 6. Eisenia nordenskioldi nordenskioldi: Perel 1997: 69. Eisenia nordenskioldi nordenskioldi: Blakemore 2013b: 2. Notes: To compare with the Korean E. nordenskioldi material we have obtained several specimens from Siberia (Russia) belonging to the nominal subspecies. Material examined: Russia, South Siberia, Tyumen, 18.07.2013, 4 ex. Leg. V. Chernov. Remarks: All the four specimens are dark red especially on dorsum, with paler spots on both side around 9-11 cd. Setae closely paired, size of the single complete specimen 100 × 7 mm, segment No. 143. Clitellum 27-½33, tubercles 29-31. Genital papillae around setae ab on segments 8, 27-31. (Table 2). Four pairs of vesicles in 9-12, the first two pairs in 9-10 are somewhat smaller than the last two. Spermathecae in 9/10-10/11 elongated, irregular sac-shaped open near to the dorsal pores. Typhlosole small, tubular protuberance of the intestine. E. n. nordenskioldi clearly differs from E. n. pallida apart from the pigmentation also in the distribution of genital the papillae, the shape of the typhlosole and setal arrangement (Fig. 10). However the real differences among the closely related dark-red pigmented species in the E. nordenskioldi species group need still to be worked out (Table 4). Eisenia lagodechiensis (Michaelsen, 1910) Helodrilus nordenskiöldi lagodechiensis Michaelsen, 1910: 18. Eisenia lagodechiensis: Kvavadze 1985: 169. Eisenia lagodechiensis: Perel 1997: 72. E. nordenskioldi lagodechiensis: species incertae sedis Blakemore 2013b: 21. Material examined: Russia, Dagestan, 05.07.1989, 45 ex. Leg. A. Zicsi, Cs. Csuzdi (HNHM/11047), Dagestan, 09.07.1989, 42 ex. Leg. A. Zicsi, Cs. Csuzdi (HNHM/11057), Dagestan, 10.07.1989, 11 ex. Leg. A. Zicsi, Cs. Csuzdi (HNHM/11063). Remarks: Our material completely agrees with that of Kvavadze (1985) from around Lagodechi. All the specimens were red violet alive however, in the preserved material most of the pigmentation bleached out. Setae moderately paired, biometry of the worms varies between 66115 × 3.5-5 mm, segment No. 117-135. Clitellum 25, 26-34, tubercles ½28-½32, 32. Genital papillae around setae cd on 9-11, ab on segments 25-37, 38 (Table 2). Four pairs of vesicles in 9-12, the first two pairs in 9-10 are smaller than the last two pairs. Spermathecae in 9/10-10/11 elongated sacFig. 10. Eisenia nordenskioldi nordenskioldi (Eisen, 1879) setal arrangement. aa = distance between the ventral setal lines, ab = distance between the ventrolateral setal lines, cd = distance between the dorsolateral setal lines, dd = distance between the dorsal setal lines. page 9 of 15Zoological Studies 55: 12 (2016)