Zoosyst. Evol. 100 (3) 2024, 1061-1073 | DOI 10.3897/zse.100.119292 Gee Te BERLIN Distribution and systematics of the cosmopolitan Amynthas carnosus complex (Crassiclitellata, Megascolecidae) from eastern Asia Anne Charis N. Han’, Yufeng Zhang*, Pu Miao°, Shaolong Wu*, Nengwen Xiao°, Mingyan Qin? , Huifeng Zhao*°®, Donghui Wul®, Nonillon M. Aspe’ 1 State Environmental Protection Key Laboratory of Wetland Ecology and Vegetation Restoration, School of Environment, Northeast Normal University, Changchun 130117, China Hebei Key Laboratory of Animal Diversity, College of Life Science, Langfang Normal University, Langfang 065000, China Henan Province Tobacco Company, Luoyang 471000, China Hunan Province Tobacco Company, Changsha 410004, China State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing 100012, China Dn oO FP W PY Key Laboratory of Wetland Ecology and Environment, State Key Laboratory of Black Soils Conservation and Utilization, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun 130102, China 7 College of Marine and Allied Sciences, Mindanao State University at Naawan, Naawan 9023, Misamis Oriental, Philippines https://zoobank. org/541660A 7-7B6A-4432-AEF5-C689737C0A3C Corresponding authors: Huifeng Zhao (zhaohf@lfnu.edu.cn); Donghui Wu (wudonghui@iga.ac.cn) Academic editor: Pavel Stoev # Received 23 January 2024 # Accepted 17 June 2024 @ Published 1 August 2024 Abstract Pheretimoid earthworms, Amynthas carnosus, were collected from Northeast and North China. An update on the distribution and systematics of the A. carnosus complex in eastern Asia using both morphological and molecular data is provided. Three subspecies, A. carnosus carnosus, A. carnosus naribunji, and A. carnosus roki, are confirmed. Comparisons of morphological characters be- tween the subspecies of A. carnosus are provided. Our results support the subspecies assignment with an intraspecific K2P genetic distance of not greater than 10% using the mitochondrial cytochrome c oxidase subunit I (COI). In addition, a re-description of the morphology of A. carnosus naribunji is presented here. Key Words DNA barcoding, earthworm, morphological characters, K2P, Megascolecidae Introduction Pheretimoids are a group of earthworms belonging to the family Megascolecidae (Oligochaeta) characterized by having a perichaetine setal arrangement, a meronephridial excretory system, a single gizzard around segment VUI, a pair of racemose prostates opening through male pores in XVUI, and testes contained within testis sacs (Aspe 2016). They are known to be widely distributed and pre- dominantly occur in East and Southeast Asia. Amynthas and Metaphire, two of the most speciose pheretimoid genera, have species with a wider range of distribution and have become well established outside their native ranges (McCay et al. 2020; Chang et al. 2021). In Chi- na, Amynthas, with around 450 species, and Metaphire, with around 130 species, account for 88.9% of the total number of earthworm species in the country (Aspe 2016; Jiang and Qiu 2018). Amynthas carnosus Goto & Hatai, 1899 is known to be one of the cosmopolitan pheretimoid species (Blakemore 2009). They are characterized by four pairs of obvious spermathecal pores in segments 5/6—8/9, or occasional- ly three pairs in segments 6/7—8/9, with genital markings typically closely paired mid-ventral and presetal in VITI— Copyright Han, A.C.N. et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 1062 IX and often also in XVII—XIX (Chen 1933; Blakemore 2012). Its distribution has been reported in Japan, from Kyushu to Tohoku and Hokkaido (Goto and Hatai 1899; Kobayashi 1936; Ohfuchi 1937; Easton 1981), in Nara and Hikone (Blakemore 2013a), and in South Korea, in- cluding Jeju Island and Dagelet Island (Ulleung-do) (Ko- bayashi 1938; Blakemore 201 3b, c). In China, the species has been reported in several provinces (Xiao 2019), but such claims are deemed questionable because of the lack of information about where the specimens were collected. So far, the published record of A. carnosus in China 1s in Hainan (Sun 2013) and Shanghai (Zhang et al. 2016), which only provided molecular data. In the USA, the Species was reported near Manhattan in Kansas (Carre- ra-Martinez and Snyder 2016). Therefore, the known dis- tribution of this species is in South Korea, Japan, China, and North America. There has been an underlying confusion regarding A. carnosus morphology in the past due to its poor origi- nal account and successive misdescriptions. The problem with the original description by Goto and Hatai (1899) was that there were three pairs of spermathecal pores in 5/6/7/8, but the spermathecae were stated to be in 7, 8, and 9, suggesting they exited in 6/7/8/9 with the possi- bility of missing a pair. Nevertheless, Ohfuchi (1937), in a more detailed account, showed the species to have four pairs of spermathecal pores in 5/6/7/8/9. This then caused other character traits (e.g., dorsal pores, genital markings, segment count, and so on) to be misnumbered as well (Blakemore 2012). Not to mention that the spe- cles synonymy was caused by the erroneous assignment of names, which has added to the complexity of this spe- cies’ identity. Further information about 4. carnosus’ provisional synonyms is listed and discussed by Blake- more (2012). Amynthas pingi was previously considered a ques- tionable synonym of A. carnosus, “as it 1s, on average, a larger worm with several other differences that presently exclude it from A. carnosus” (Blakemore 2012). How- ever, Blakemore (2013a) re-examined the London type mature A. pingi specimen labeled as “Pheretima pingi 1924.11.29.5 HOLOTYPE (sic) Nanking, China Don. Prof. C. Ping” and refuted those differences (e.g., the supposed larger size in A. pingi, now known to be false as the type 1s only 132 mm long, and a later onset of in- testine origin and septal glands, now also proven false). There, he also pointed out errors in Gates (1939) redef- inition of Pheretima pingi, such as having “lower setal counts, mistaken septa, hearts, and spermathecal pores that he insisted were posterior in segments 5—8 (but that are now shown to be in the intersegmental furrows of 5/6/7/8/9 in the types of both A. pingi and A. carnosus). Moreover, the assumption that the ‘characteristic’ tuber- cles were nephridial (Goto and Hatai 1899) was more likely due to Monocystis infestation, as indicated by both Gates (1939) and Blakemore (2013a), hence dismissing the possible justification of retaining A. pingi as a sepa- rate species from A. carnosus. In which case, A. carnosus would likely suggest prevalence in China, from which zse.pensoft.net Han, A.C.N. et al.: Distribution and systematics of eastern Asian Amynthas carnosus A. pingi (= A. carnosus) was reported to have been abun- dantly distributed in Nanking (Stephenson 1931). Given this and with the incorporation of the new investigation of A. carnosus specimens in Northeast and North China (reported here), it may further support a possible indica- tion of the prevalent range of A. carnosus in the country, as was suggested by Chen (1936) and concluded by Ko- bayashi (1936). Preliminary attempts at using the DNA barcodes of A. carnosus specimens from Japan and South Korea were carried out (Blakemore 2013b). Two new subspe- cies have been established, namely, A. carnosus naribunji Blakemore 2013 from Naribunji, Ulleung-do (Dagelet Is- land, South Korea), and A. carnosus roki Blakemore and Lee 2013 from Incheon (South Korea). Preliminary DNA data for taxon identification and phylogenetic relation- ships were also explored, yet a rather deficient descrip- tion of A. carnosus naribunji’s morphological characters by Blakemore (2013a) was presented, which then makes it more of a molecular taxon. This paper provides an update on the taxonomic status of the A. carnosus complex in East Asia using both mor- phological and molecular data, as well as a report on the present distribution of this species in China. In addition, an update on the morphological diagnosis of A. carnosus carnosus and a re-description of A. carnosus naribunji are presented. Materials and methods Sampling Earthworm specimens were collected during the summer of 2022 and 2023, around the months of May and July, in Northeast China and the neighboring provinces. The collection sites chosen were mainly based on three hab- itat types, including forests, farmlands, and urban parks (Table 1). Earthworm samples were also collected in a nature reserve area on Changbai Mountain. Earthworms were collected through digging and hand sorting. Collec- tions near the sites with surface castings were also taken into account. The earthworms collected were preserved and stored in 100% ethanol. DNA extraction, amplification, and sequencing Total genomic DNA was extracted from the muscle tis- sue of the posterior part using the TIANGEN Genomic DNA Kit (China) following the manufacturer’s instruc- tions. Regions of the cytochrome c oxidase subunit I (COI) were amplified using the polymerase chain reac- tion (PCR). The mixture (total volume 25 wl) contained 1 wl DNA and 17.25 ul sterile ddH,O, 2.0 wl of dNTP, 2.5 ul of buffer, 0.25 wl TransGen EasyTaq-polymerase and 1.0 ul of Primer HCO1490 (5-GGTCAACAAAT- CATAAAGATATTGG-3) (Folmer et al. 1994), and 1.0 wl of Primer COIE (5-TATACTTCTGGGTGTC- Zoosyst. Evol. 100 (3) 2024, 1061-1073 Table 1. Collection information for sampling areas and specimens. 1063 Sampling ID Location Latitude, Longitude Specimen number 362R Liaoning Prov., Jinzhou Pref., Nanshan Park 41.0718°N, 121.1479°E 8 533R Liaoning Prov., Dandong Pref., Jinjiang Mt. Park 40.1312°N, 124.3746°E 12 534R Liaoning Prov., Dandong Pref., Kundian County, Beishan Park 40.7319°N, 124.7780°E 10 551R Liaoning Prov., Huludao City, Longwan Park 40.7143°N, 120.8415°E 10 LFXH Hebei Prov., Xianghe County, Zhuti Park 39.7774°N, 116.9816°E 7 LFSF Hebei Prov., Langfang Pref., Anci Dist., Langfang Normal University 39.5222°N, 116.6654°E 1 E28, E29 Tianjin Municipality, Dongli Dist., Anonymous Park 39.0836°N, 117.3125°E 2 BJCY Beijing Municipality, Chaoyang Dist., Lvfeng Park 39.8760°N, 116.5800°E 1 BJTZ Beijing Municipality, Tongzhou Dist., 39.8760°N, 116.7250°E 1 HNLNR2, HNLNGR, HNLNNG Henan Prov., Luoyang Pref., Luoning County 34.4363°N, 111.6398°E 4 HNSQ Henan Prov., Shangqiu Pref., Liangyuan Dist. 34.4291°N, 115.6183°E 3 CGAAGAATCA-3) (Bely and Wray 2004). The cy- cling profile was as follows: firstly, initial denaturation for 5 min at 95 °C; secondly, denaturation for 30 sec at 95 °C, annealing for 30 sec at 51 °C, and extension for 45 sec at 72 °C for 35 cycles; thirdly, final extension for 5 min at 72 °C. PCR amplifications were confirmed by electrophoresis in 1% agarose gel, which were visu- alized by SAGECREATION Gel Documentation and Image Analysis System Equipment, and Sage software was used for capturing the image. DNA samples were sent to Tianyi Huiyuan Biotechnology Co., Ltd. (Bei- jing) for Sanger sequencing using an ABI 3730 auto- mated sequencer. Data analysis The raw data were corrected manually in BioEdit (Hall 1999), and the exported fasta files were aligned using Clustal W (Thompson et al. 1994). COI sequences from Genbank labeled as A. carnosus have also been included in the analysis (Suppl. material 1: table S1). A phylogenetic tree was constructed using the maximum likelihood method (ML) performed in RAXML 8.0 (Sta- matakis 2014), using the default rapid hill-climbing al- gorithm and the GTRGAMMA model to search for the best tree. Clade support was assessed using 1,000 rapid bootstrap replicates. The tree was rooted using Pon- todrilus litoralis as an outgroup. Pairwise distance anal- ysis among A. carnosus subspecies and between COI sequences of the other 10 Amynthas species downloaded from GenBank was conducted using MEGAS (Tamura et al. 2011) with the Kimura-2 parameter model (Kimu- ra 1980). Morphological examination and identification Fixed specimens were brought to the laboratory for external and internal examination using a stereomicro- scope (ZEISS) and ZEN 3.3. Pro software was used for image capture and to aid in identifying and measuring small organs and other characters. The generic diagno- ses and taxonomic assignments to the subspecies level follow Blakemore (2012, 2013b) and Blakemore and Lee (2013). References to figures from the cited papers are listed in lowercase (fig. or figs), and figures in this paper are noted with an initial capital (Fig. or Figs). The following abbreviations are used: Ag accessory gland; mp male pore; re receptacle; Amp ampula; P prostomium; sp spermathecal pore; Gm genital marking; prg prostate gland; SV seminal vesicles. Results and discussion Molecular characterization A total of 66 COI sequences had been sequenced and submitted to Genbank (Accession numbers: PP067669— PP067734). Results of the K2P analysis using COI show that the three intraspecific taxa of A. carnosus have in- ter-subspecific genetic distances that are between 7% and 10% (Table 2). Meanwhile, the genetic distance between A. carnosus and other species in the same genus Is greater than 16% (16-22%). A study by Dong et al. (2019) revealed a genetic distance of 10.7—11.4% between two subspecies of Amynthas shengtangmontis. A. s. shengtangmontis and A. s. minusculus, which showed to be more than 1% and less than 15%. The intra-specific pairwise distances of subspe- cies A. c. naribunji and A. c. roki from A. c. carnosus are 7-8% and 9-10%, respectively (Table 2). In other studies, the interspecific distances in the same genus ranged be- tween 17-23% (Sun 2013), 16-23% (Huang et al. 2007), 15-16% (Admassu et al. 2006), 16-22% (Novo et al. 2009), 15-28% (Chang et al. 2008), and 14.7—-25% (Dong et al. 2019), which are all in agreement with our results. Also, a specimen identified as “A. carnosus carno- sus” in Hainan (China) by Sun (2013) (cf. Dong et al. 2019; KF205962) is seen to have diverged greatly from the A. carnosus taxa (Fig. 1), having a pairwise distance to the remaining A. carnosus taxa of 22-24% (Table 1), which could possibly suggest a misidentification of this Species or subspecies. zse.pensoft.net 1064 Han, A.C.N. et al.: Distribution and systematics of eastern Asian Amynthas carnosus Table 2. Percentage of K2P distance of the three subspecies of A. carnosus with inclusion of other pheretimoid species (values in %). Species 1 2 3 4 5 6 7 8 9 10 11 12 13 1A. carnosus carnosus 0-1 2 ‘A. carnosus naribunji 7-8 0-1 3 A. carnosus roki 9-10 8 0 4 A. carnosus carnosus (KF205962) 23-24 22-23 24 0 5 A. daeari 20 19 20 22 0 6 A. gageodo 17-18 19-20 18-19 21 21 0 7 A. gracilis 19 19-20 20-21 19 18-1921 0 8 A. corticis 16-20 17-19 19-20 19 1819 18-20 18-20 0-7 9 A. fuscatus 17-20 17-22 18-22 19-20 18-20 20-23 18-21 17-20 0-15 10 A. tokioensis 20-21 19-21 22-23 23 16 20-21 20-21 18-21 18-22 0-1 11 A. maximus 19 19 20 20 18 18 22 17-18 16-19 20-21 0 12 A. shengtangmontis 20-21 19 20 21 20 20 20-21 18-19 19-21 22-23 23 0 13 A. robustus 20-22 21-22 21-23 17-22 21-22 22-23 20 17-21 17-21 23-24 24 19-22 0-21 subspecies Country 362R1_01_Liaoning 362R1_02_Liaoning 362R1_04 Liaoning 362R1_05_Liaoning 362R1_06_Liaoning 362R1_07_Liaoning 362R1_08_Liaoning 362R1_09_Liaoning 533R55_02_Liaoning 533R55_03_Liaoning 533R55_04_Liaoning 533R55_05_Liaoning 533R70_08_Liaoning 533R70_09_Liaoning 533R70_10_Liaoning 533R71_04_Liaoning 533R71_06_Liaoning 533R71_07_Liaoning 533R71_08_ Liaoning 533R71_10_Liaoning 534R1_02_Liaoning 534R1_03_Liaoning 534R1_09_Liaoning 534R3_02_Liaoning 534R3_03_Liaoning 534R3_04_Liaoning 534R3_05_Liaoning 534R3_06_Liaoning 534R3_10_Liaoning §51R11_06_Liaoning 951R11_09 Liaoning 551R3_01_Liaoning 551R3_02_Liaoning 551R3_03_Liaoning 551R3_04_Liaoning 551R3_05_Liaoning 551R3_06_Liaoning 551R3_07_Liaoning 551R3_08_Liaoning 551R3_09 Liaoning 551R3_10_Liaoning BJCY_42_Beijing BJTZ02_00_Beijing E28_01_Tianjin E29_05_Tianjin HNLNGR_I2_04_Henan HNLNNG_1I1_06_Henan HNLNR2_04_Henan HNLNR2_05_Henan HNLNR2_06_Henan HNSQ_07_Henan HNSQ_13_Henan HNSQ_15_Henan KF205962_carnosus KP030699_ Shanghai KP030702_Shanghai KP030703_Shanghai KP030728_Shanghai KP030732_Shanghai KT252956_unknown KT429008_ Shanghai SP SOSOOOOOOOSOOOOOOOOR REE EEREEEE @ A1yNIhas Cainosus Ccainosus A A1ynihas Canosus (Okt Be A7y0has Carnosus Nariouryf subspecies Country subspecies Country 362R1_01_Liaoning = 362R1_02_Liaoning LFSF_013_Hebei LFXH_R10_01_Hebei LFXH_R2_01_Hebei LFXH_R2_03_Hebei LFXH_R2_04_Hebei LFXH_R2_05_Hebei LFXH_R6_02_Hebei LFXH_R6_03_Hebei LFXH_R7_02_Hebei LFXH_R7_04_Hebei LFXH_R7_05_Hebei LFXH_R8_03_Hebei LFXH_R9_05_Hebei w56_roki_Incheon H4_roki_Busan WO24_Incheon W0O32_Sammock W0O34_Sido WO67_Jeju w37_Geoman w54_naribunji_Ullungdo w55_Incheon w57_Incheon JET112-115_Hikone LC721143_Kyoto LC726524_Kyoto AB542452_ Aichi AB542453_Nara AB542454_ Osaka AB543184_Nara KP030728_Shanghai KP030732_Shanghai KT429008_Shanghai PHERRERPRERERRRERRRERERERERERERERERERERER REG eccccooouuununuexe es * PRP RPE EREEEEE 5 ee ee TT AB542453_Nara AB542454_Osaka w56_roki_Incheon A KT252956_A_carnosus 551R11_06_Liaoning 551R11_09_Liaoning 551R3_01_Liaoning 551R3_02_Liaoning 551R3_03_Liaoning 551R3_04_Liaoning 551R3_05_Liaoning 551A3_06_Liaoning | 551R3_07_Liaoning 551R3_08_Liaoning 551R3_09_Liaoning 551R3_10_Liaoning w54_naribunji_Ullungdo BJCY_42_Beijing 0.03 KF205962_carnosus AB608782_Pontodrilus_litoralis Figure 1. The geographical distribution of the A. carnosus complex in eastern Asia and its corresponding phylogenetic tree based on COI using the maximum likelihood method. Color coding: red for China, purple for South Korea, and green for Japan. Molecular data show a divergence among the subspe- cies of A. carnosus (Fig. 1), which comprise the DNA samples provided from China, Japan, and South Korea. It also shows little genetic variation of A. c. carnosus, as shown by the absence or having of very short branches within the clade that 1s composed of the three countries. The same is observed in the other two subspecies that occur in China and South Korea. Here, the geographic representation shows a wide distribution of the species in eastern China (Fig. 1). zse.pensoft.net On the one hand, 4. c. naribunji’s current distribution pattern has been expanded because of its new record in northern China (Beijing and Liaoning). Still, future inves- tigations and additional sampling sites must be explored to be able to have a thorough understanding of the origin of this species and its migration pattern across countries. Moreover, the specimen of A. c. roki (NI- BR-IV0000261264 providing DNA w56) from Incheon Great Park (Blakemore and Lee 2013) has inter-subspe- cific distance values of 8.1-10% from A. c. carnosus and Zoosyst. Evol. 100 (3) 2024, 1061-1073 7.2-7.5% from A. c. naribunji, respectively. Alternatively, a specimen labeled “A. carnosus” from China (KT252956; the detailed location is unknown) was grouped with the A. c. roki specimens from South Korea, with a 100% bootstrap value. Intersubspecific distances among A. c. carnosus and A. c. naribunji are 9-10% and 8%, respectively. As no data on the exact location and morphological descriptions of A. c. roki in China have been reported, further sampling of A. c. roki needs to be done in the future. Morphological characterization Family Megascolecidae Rosa, 1891 Genus Amynthas Kinberg, 1867 Amynthas carnosus carnosus Goto & Hatai, 1899 Perichaeta carnosa Goto & Haitai, 1899: 15. Pheretima carnosa — Kobayashi 1936: 115. Amynthas carnosus — Sims and Easton 1972: 235. Blakemore 2012: 36; 2013a: 58; 2013c: 101. Carrera-Martinez and Snyder 2016: 297. Chang et al. 2016: 505. Amynthas pingi (Stephenson, 1925) — Sims and Easton 1972: 235. Blakemore 2013c: 112. 1065 Material examined. Specimen IDs: 362R1_01, 02, 04, 05, 06, 07, 09, seven matures from Nanshan Park, Jinzhou, Lia- oning; 533R70_ 08, 09, 10, three matures from Jinjiang Mt. Park, Dandong, Liaoning; LFXHR7_02, 04, 05, three ma- tures from Zhuti Park, Xianghe County, Langfang, Hebei; LFSF_013, one mature from Langfang Normal University, Anci District Langfang, Hebei; E29 05, one mature from an anonymous park in Dongli District, Tianjin Municipal- ity; HNLNR2_ 04, 05, 06, three matures from the tobacco field in Xiaojie Town, Luoning County, Luoyang, Henan; HNSQ_07, 13, 15, three matures under the bushes tn Shangq- iu Normal University, Liangyuan District, Shangqiu, Henan. Diagnosis. Length 105-210 mm. Spermathecal pores having four pairs tn 5/6/7/8/9, rarely 3 pairs in 6/7/8/9, with pre-intersegmental hemispherical arc (spermathecal papil- lae). Dorsal pores typically from 12/13. Pre-clitellar geni- tal markings typically with two pairs, pre-setal in VIII and IX; these genital markings paired either widely or closely apart (B1 and B2, Fig. 3); Post-clitellar genital markings prominent, up to three pairs median to male pores; first pair pre-setal on XVIII, slightly median to male pores; second pair post-setal and more medial than the first; third pair pre-setal in XIX (Fig. 4). Male pores superficially paired in XVIII close to the lateral margin on round or elliptical porophores (Fig. 4). Ampulla ovate to narrowly ovate (Fig. 2D-F). Intestinal caeca simple at XX VII. men ID 362R1_ 06), three pairs (B, E) (specimen ID LFXHR7_05), five pairs (C, F) (specimen ID HNLNR2_05). Scale bars: 1 mm. zse.pensoft.net 1066 Han, A.C.N. et al.: Distribution and systematics of eastern Asian Amynthas carnosus Figure 3. Pre-clitellar genital marking (arrows) variations of Amynthas carnosus. A. Modified fig. 1 of the variations of pre-clitel- lar genital markings from Kobayashi (1936); B. This study. B1. (specimen ID 362R1_01); B2. (specimen ID 533R70_09), and B3. (specimen ID HNLNR2_05) comply with the “permissible variations” [termed by Blakemore (2012)] of Kobayashi’s (1936) Type III (red), VI (blue), and VIII (green). Variations. For the A. c. carnosus from China, the number of spermathecal pores and spermathecae are variable: 14 out of 16 specimens typically have four pairs in 5/6/7/8/9, one specimen has three pairs in 6/7/8/9 (LFXHR7_05), and another one has five pairs in 4/5/6/7/8/9 (HNLNR2_05) (Fig. 2). However, despite these variations, molecular analyses have shown them to belong in the same clade with little genetic divergence within the clade (Table 2, Fig. 1). Two specimens from South Korea and one specimen from the USA have three pairs of spermathecal pores in 6/7/8/9 (Kobayashi 1936; Carrera-Martinez and Snyder 2016). However, prior to this study, no other specimen with five pairs was record- ed elsewhere. zse.pensoft.net Distribution. China (Liaoning, Beijing, Tianjin, He- bei, Henan, and Shanghai), Japan (Kyushu, Honshu, and Hokkaido), and South Korea (Incheon, Jeju Island). Remarks. Detailed descriptions of A. carnosus were reported by Kobayashi (1936), Ohfuchi (1937), and Blakemore (2012, 2013a, c). Rather than typical closely spaced mid-ventral pre-clitellar genital markings (Blake- more 2012), widely spaced ones are mostly observed with the A. carnosus specimen from China, which re- sembles those Hikone specimens from Japan (Tokyo An- 460-DNA JET-112) [cf. fig. 3 by Blakemore (2013a)]. In contrast, the closely spaced pre-clitellar genital mark- ings of the Liaoning specimens (Dandong, DNA 533R) match those of the South Korean specimen from Geoman Zoosyst. Evol. 100 (3) 2024, 1061-1073 I II ef 288826 © £6 & Pete es em ees «+ * wt eee ee ee -=—=2=< «= © «= = = « «| VI = SS 6 £26 © «=| 66 &e =Ssereres Sees ee Ill “= 62 © © = «= &@ & Vill 1067 IV V ==] = «eee « = eee] * © © ee ee ee =e ee Se se 68 S&S @ +t t+ = © © = ©F © © lar genital markings from Kobayashi (1936); B. This study. B1, B2. (specimen IDs 362R1_07 and HNLNR2_ 04); B3 (specimen ID 362R1_04); B4, BS. (specimen IDs 362R1_01 and 533R70_10) comply with the “permissible variations” [termed by Blakemore (2012)] of Kobayashi’s (1936) Type II (red), IV (blue), and V (green). (NIBR IV261234-DNA w37) and the Japanese neotype of A. carnosus (Tokyo An435) [cf. fig. 2 by Blakemore (2013a)]. A comparison of characters from the specimens of China, Japan (a neotype NSMT An435 from the Tokyo Museum) (Blakemore 2012), and the USA (Kansas) (Chang et al. 2016) is presented in Suppl. material 1: ta- ble S2. External characters such as the number of sperma- thecal pores and segment locations of pre-clitellar genital markings match among specimens from different coun- tries. However, internal character variations are observed in the position of the intestinal caeca, which was reported to begin at XX VII and extend to XXIII or XXIV (Blake- more, 2012; 2013b; 2013c; Chang et al. 2016), while the intestinal caeca in the Chinese specimens extends up to XX, XXI (or XXIII) (Fig. 5), 2-3 segments more anteri- or than those from the two previous accounts. Moreover, some character measurements that were not presented in the other two accounts, such as the ventral distances be- tween male pores (0.25—0.29 mm), spermathecal pores (0.28-0.30 mm), and genital markings (latero-ventrally with 0.21—0.29 mm distance apart or mid-ventrally with 0.08 mm distance apart), were added to further aid spe- cies identification. Kobayashi (1936), in his thorough investigation of A. carnosus, presented “permissible” variations on the pre-clitellar and post-clitellar genital markings [text- figs. 1-2 in Kobayashi (1936)]. The pre-clitellar geni- tal making variations in the A. c. carnosus from China comply with Kobayashi’s Types III, VI, and VIII (see Fig. 3), while the post-clitellar genital marking varia- tions comply with Kobayashi’s Types II, IV, and V (see Fig. 4). It is important to take note that genital marking patterns can also be considered a distinctive character for species identification (e.g., Nguyen et al. 2020; Aspe et al. 2021). zse.pensoft.net Han, A.C.N. et al.: Distribution and systematics of eastern Asian Amynthas carnosus Figure 5. Amynthas carnosus carnosus intestinal caeca showing the segment length variations: A. (specimen ID 533R70_10); B. (specimen ID 362R1_05), and C. (specimen ID HNLNR2_05). Scale bars: 1 mm. Amynthas carnosus naribunji Blakemore, 2013 Amynthas pingi: Blakemore 2013a: 60, figs 4, 5. Material examined. Specimen IDs: 551R3 (01-10), 10 adults from Longwan Park, Huludao Pref., Liaoning Prov. One specimen from Beijing is a juvenile and was poorly preserved. Thus, its morphological examination was not performed. However, the molecular data is pre- sented (DNA ID: BJCY_42, Fig. 1). Diagnosis. Spermathecal pores four pairs in 5/6/7/8/9, located latero-ventrally (0.29-0.30 mm), each with pre-intersegmental hemispherical arc (spermathecal papillae) anterior to intersegments (Fig. 6). Pre-clitel- lar genital markings absent (complying with Kobayashi Type I in Blakemore 201 3a); if present, one to three pairs; pre-setal in VHI and IX when one or two pairs and hav- ing a post-setal pair in VIII when three pairs (complying with Kobayashi’s Types HI, IV, and VIII); asymmetrical patterns also present (Fig. 7). Male pores superficial in XVIUI having “disc-like” genital markings paired poste- rio-median to male pores (Fig. 8). Description. Length 185—228 mm. Color of preserved Specimens may have varying shades of brown but dor- sum generally dark brown in pre-clitellar region to brown in post-clitellar region, fading to lighter brown towards posterior end with darker clitellum, while ventrum part is paler/fleshy color. Clitellum width 5.8-8.3 mm. Seg- ments 115—137. Prostomium epilobous. First dorsal pore on 12/13. Clitellum annular at XIV—XVI without setae or dorsal pores. Setal arrangement perichaetine, setae be- tween male pore 18-19. Female pore single and circular, midventral at XIV. Spermathecal pores large, having four pairs in 5/6/7/8/9 and widely-spaced, latero-ventral (0.29- 0.30 mm) in pre-intersegmental hemispherical arc (sper- mathecal papillae). Pre-clitellar genital markings circu- lar in shape, latero-ventral (0.25—0.29 mm), randomly located in pairs (three pairs/two pairs/one pair, a total zse.pensoft.net of 2-6 genital markings), or asymmetrically located on one side (1-2 genital markings), about 0.38-0.59 mm in diameter. Male pores superficially paired in XVIII close to later- al margin (with ventral distance 0.26—0.29 mm) on large circular porophores. Post-clitellar genital markings dis- tinguishably paired, post-setally in XVIII, mid-ventral to male pore, 0.42—0.69 mm in diameter. Septa 4/5—7/8 and 10/11—14/15 thickened, 8/9/10 ab- sent. Esophageal gizzard within VII—X. Intestinal origin at XV (or XIV). Intestinal caeca simple, paired in XX VII, extending anteriorly to X XII. Last hearts in XIII. Four pairs of spermathecae in VI-IX. Ampulla ovate, wrinkled; ducts short and stout. Diverticula reaching one- third to half of ampulla with a slender stalk and a wider seminal chamber; seminal chamber elongated or botuli- form. Accessory glands sessile and round. Seminal vesicles paired in XI and XII, large, smooth, yellowish, posterior pair larger but not as obvious com- pared to A. c. carnosus, each with a dorsal lobe. Ovaries present. Prostate glands paired in XVIII, large, lobulat- ed, covering XVI—XX; ducts thick and large, U-shaped. Accessory glands round, sessile, or slightly lobed, corre- sponding to each genital marking around male pore area. Distribution. Northern China (Liaoning, Beijing) and South Korea (Ulleung Island). Remarks. There is not much of a thorough morpho- logical description of A. carnosus naribunji in the orig- inal account of Blakemore (2013a) (see Suppl. material 1: table S3) aside from its single illustration of paired post-clitellar genital markings in the male pore area and a spermathecal pore with corresponding spermathecae shown in fig. 4 by Blakemore (2013c). Notable features of A. c. naribunji in comparison with A. c. carnosus were its slightly larger size with lengths of 185-228 mm, typically wide-spaced pre-clitellar genital markings with a maximum number of six (three pairs) to at least three genital markings; pre-setal/post-setal in VIII and pre-setal in [X. In contrast, A. c. carnosus 1s Zoosyst. Evol. 100 (3) 2024, 1061-1073 1069 Figure 6. A. carnosus naribunji (specimen ID 551R3_01): Prostomium (A); Male pore with posterio-median paired genital marking (B); Seminal vesicles (C); Intestinal caeca (D); spermathecae (E); Prostate gland with U-shaped think duct (F); and Spermathecal pores with three pairs of pre-clitellar genital markings (G). Scale bars: 1 mm. Figure 7. 4. carnosus naribunji pre-clitellar genital marking variations, A-C. Complying with Kobayashi’s (1936) Types III, IV, and VIII, respectively; D-F. Displays asymmetrical patterns. Scale bars: 1 mm. typically medium to smaller size, with mostly only four The distinctive character of having “a pair of genital genital markings (two pairs) or less, either wide or close- markings posterio-median to male pores” in A. c. na- ly-spaced pre-clitellar (Table 3). ribunji may distinguish it from those of A. c. carnosus zse.pensoft.net 1070 Han, A.C.N. et al.: Distribution and systematics of eastern Asian Amynthas carnosus Figure 8. Image comparison of male pore porophores and paired posterio-median genital markings between A. carnosus carnosus. A, B. (specimen IDs 362R1_07 and HNLNR2_04) and A. carnosus naribunji from Liaoning; C—E. (specimen IDs 551R3_01, 551R3_09, and 551R3_06) and Ulleungdo, F. (specimen ID w54). Table 3. Character comparison among A. c. carnosus, A. c. naribunji, and A. c. roki. The asterisk stands for the figure in Blakemore and Lee 2013a, p. 131, yet there is no definite description. Characters A. carnosus carnosus Length 105-340 mm No. of segments 96-179 No. of setae between mp 16-20 Male pore round or elliptical porophores Distance between Male pores 0.25-0.29 (circumference apart) Spermathecal pore 6/7/8/9 (three pairs, rarely); 5/6/7/8/9 (four pairs); 4/5/6/7/8/9 (five pairs, rarely); with pre-intersegmental spemathecal papillae Distance between 0.28-0.30 spermathecal pores (circumference apart) Pre-clitellar gm (circumference closely paired or widely spaced pre-setal in VIII apart) and IX, mid-ventral (mostly two pairs, total of 1-4 GMs) up to three pairs of genital markings near male pores, pre-/post-setal in VXIIl and pre-setal in XIX. Second pair, post-setal more medial than the first typically four pairs in VI-IX, first pair often slightly smaller; or three pairs in VIX or five in IV-IX Wrinkled; Ovate to narrowly ovate Post-clitellar gm (circumference apart) Spermathecae (circumference apart) Ampulla and Duct (circumference apart) Prostate gland (circumference apart) Intestinal caeca racemose at XVIII, covering XV (or XVI)-XX paired in XXVII, simple, extending to XX, or (circumference apart) Xl, or XiIll Gizzard (circumference apart) VIII-X Intestine XV (or XIV) (Blakemore, 2013a). However, it is now undeniable that this seemingly “distinctive character” was likewise ob- served in the A. c. carnosus specimens from Liaoning and Henan (Fig. 8). Here, we dismiss the former notion of Blakemore (2013a) “to consider for exclusion Ko- bayashi’s Types I and II markings” as a distinctive char- acter for the A. c. carnosus subspecies, A. c. naribunji. Although this might be the case, one cannot ignore the zse.pensoft.net A. carnosus naribunji A. carnosus roki 185-228 mm 175-300 mm 121-137 136 17-20 14 superficial on small mounds within concentric rings 0.30 on large circular porophores, 0.26-0.29 5/6/7/8/9 (four pairs) with pre- intersegmental spermathecal papillae post-intersegmental pores 5/6/7/8/9 (four pairs) with post-intersegmental spermathecal papillae 0.29-0.30 NA widely spaced, randomly located in absent pairs (three pairs or two pairs or one pair, a total of 2-6 GMs) distinguishably paired, post-setal in absent XVIII, mid-ventral to male pore four pairs in VI-IX four pairs in VI-IX Wrinkled; Ovate to narrowly ovate Narrowly ovate* racemose at XVIII, covering XVI-XX racemose in XVIII paired in XXVII, simple, extending to Simple from XXVII XXxll VIII-X NA XV NA certain degree of dissimilarity between the two subspe- cies’ morphological characters (e.g., the shape of male pore porophore and the distinct shape of the genital mark- ings in A. c. naribunji, described as “disc-like” by Blake- more). As such, applications of state-of-the-art method- ologies such as high-throughput sequencing or geometric morphometric (Marchan et al. 2020) may be adopted that go beyond traditional methods of taxonomic diagnosis Zoosyst. Evol. 100 (3) 2024, 1061-1073 (which in this case is rather insufficient to “quantify” the degree of variations). Nevertheless, the incorporation of genetic data such as DNA barcoding and calculating K2P intraspecific distances (as conducted in this study) may aid in suggesting subspecies delineation. Amynthas carnosus roki Blakemore & Lee, 2013 Material examined. In China, only molecular data is available in the Genbank (Accession No. KT252956). Description. See Blakemore and Lee 2013: 129-132. Distribution. South Korea, China. Morphological comparison among the subspecies of A. carnosus A list of character comparisons between A. c. carnosus, A. c. naribunji, and A. c. roki is summarized in Table 3. Most of the distinctive characters for A. c. carnosus and A. c. naribunji are discussed above. As for A. c. roki, “distinctive characters are the tendency to have large size (175-300 mm) post-intersegmental spermathecal pores with U-shaped spermathecal papillae. Obvious distinc- tive character accounts for the lack of genital markings, thereby complying with Kobayashi’s Types II and I” (Blakemore and Lee 2013). Blakemore and Lee (2013) added further: “On these characters, the present subspe- cies appears to differ from the nominal taxon’s neotype and from other synonyms in Blakemore.” Aside from the external morphological differences, slight internal char- acter differences were also observed (Table 3). According to Blakemore (2012), “the genital mark- ing variation in A. carnosus allowed for by Kobayashi in his detailed and most thorough account is excessive, rather representing a congery of morphs, if not separate species’’, this can be said out from the preliminary DNA results forming new taxa as A. c. naribunji and A. c. roki (Blakemore 2013a; Blakemore and Lee 2013). Genital marking variations as observed in our specimens here did appear to have variations exceeding that of Kobayashi’s permissible” genital marking variations (with asymmetri- cal GMs also observed). Conclusion Our results agree with Blakemore’s subspecies assignment of A. c. naribunji and A. c. roki (additional molecular data only). Furthermore, the attempt to justify the presumed “distinctive character” of having a pair of genital markings posterio-median to male pores in A. c. naribunji, which is distinct from the A. c. carnosus as noted by Blakemore (2013a, c), has now been invalidated, given that both A. c. naribunji and A. c. carnosus specimens in China pos- sess this same character. Here, a pairwise distance for the A. carnosus subspecies was shown to be 10% and below. With new occurrences of A. carnosus in China, patterns 1071 of morphological as well as genetic variations in different geographical occurrences of this species can be further elu- cidated. Still, a more thorough investigation should be car- ried out by conducting a broader sampling collection in the country, which may contribute to new distribution records. Moreover, with the updated and detailed morphological descriptions of A. c. naribunji provided here, elaborate data can now be used to clarify morphological distinctions. Thus, similar specimens from any region may now be com- pared genetically to an increasingly refined formulation of A. carnosus, its subspecies, and even synonymous species. Acknowledgments We thank the reviewer, Parin Jirapatrasilp, for his valuable suggestions. This study was supported by the National Natural Sciences Foundation of China (42071059); the Northeast Asia Biodiversity Research Center (2572022DS09); by the China National Tobacco Corporation of Science and Technology Major Projects (110202201018 [LS-02]); by the “One Belt, One Road” international scholarship from the Ministry of Science and Technology of China (DL2023130001L); and by the President’s International Fellowship Initiative from the Chinese Academy of Sciences (2024VCA0009); the Na- tional Science and Technology Fundamental Resources Investigation Program of China (201 8FY 100301). References Admassu B, Juen A, Traugott M (2006) Earthworm primers for DNA- based gut content analysis and their cross-reactivity in a multi-spe- cies system. Soil Biology & Biochemistry 38(6): 1308-1315. https:// doi.org/10.1016/j.soilbio.2005.08.019 Aspe NM (2016) The geographic distribution of the genera in the Pheretima complex (Megascolecidae) in eastern Asia and the Pacific region. Katyo Monthly 48: 39-45. Aspe NM, Manasan RE, Manlavi AB, Ma. Patiluna LE, Sebido MA, Obusan MCM, Simbahan JF, James SW (2021) The earthworm fauna of Palawan, Philippines with description of nineteen new pheretimoid species (Clitellata, Megascolecidae). Journal of Natural History 55(11—12): 11-12, 733-779. https://doi.org/10.1080/00222 933.2021.1923849 Bely AE, Wray GA (2004) Molecular phylogeny of naidid worms (Annelida, Clitellata) based on cytochrome oxidase I. Molecular Phylogenetics and Evolution 30(1): 50-63. https://doi.org/10.1016/ S$1055-7903(03)00180-5 Blakemore R (2009) Cosmopolitan Earthworms—A Global and Historical Perspective. 257—283. https://doi.org/10.1002/9780470455203.ch14 Blakemore R (2012) Amynthas carnosus (Goto & Hatai, 1899) re- described on its neotype (Oligochaeta, Megadrilacea, Megasco- lecidae). Journal of Species Research 1(1): 35-46. https://doi. org/10.12651/JSR.2012.1.1.035 Blakemore R (2013a) Megascolex (Perichaeta) diffringens Baird, 1869 and Pheretima pingi Stephenson, 1925 types compared to the Amyn- thas corticis (Kinberg, 1867) and A. carnosus (Goto & Hatai, 1899) (Oligochaeta, Megadrilacea, species-groups Megascolecidae). zse.pensoft.net 1072 Journal of Species Research 2(2): 99-126. https://doi.org/10.12651/ JSR2013.2.2.099 Blakemore R (2013b) Jeju-do earthworms (Oligochaeta, Megadri- lacea)-Quelpart Island revisited. Journal of Species Research 2(1): 15-54. https://doi.org/10.12651/JSR.2013.2.1.015 Blakemore R (2013c) Ulleung-do earthworms - Dagelet Island revisited. Journal of Species Research 2(1): 55-68. https://doi.org/10.12651/ JSR.2013.2.1.055 Blakemore R, Lee W (2013) Survey of Busan Oligochaeta earthworms supported by DNA barcodes. Journal of Species Research 2(2): 127— 144. https://dot.org/10.12651/JSR.2013.2.2.127 Carrera-Martinez R, Snyder B (2016) First report of Amynthas carnosus (Goto & Hatai, 1899) (Oligochaeta, Megascolecidae) in the Western Hemisphere. Zootaxa 4111(3): 297-300. https://doi.org/10.11646/ zootaxa.4111.3.7 Chang C-H, Lin S-M, Chen J-H (2008) Molecular systematics and phylogeography of the gigantic earthworms of the Metaphire for- mosae species group (Clitellata, Megascolecidae). Molecular Phy- logenetics and Evolution 49(3): 958-968. https://doi.org/10.1016/j. ympev.2008.08.025 Chang C-H, Snyder B, Szlavecz K (2016) Asian pheretimoid earth- worms in North America north of Mexico: An illustrated key to the genera Amynthas, Metaphire, Pithemera, and Polypheretima (Clitellata, Megascolecidae). Zootaxa 4179(3): 495-529. https://do1. org/10.11646/zootaxa.4179.3.7 Chang C-H, Bartz M, Brown G, Callaham M, Cameron E, Davalos A, Dobson A, Gorres J, Herrick B, Ikeda H, James S, Johnston M, McCay T, McHugh D, Minamiya Y, Nouri Aiin M, Novo M, Ortiz-Pachar J, Pinder R, Szlavecz K (2021) The second wave of earthworm invasions in North America: Biology, environmental impacts, management and control of invasive jumping worms. Bi- ological Invasions 23(11): 1-32. https://doi.org/10.1007/s10530- 021-02598-1 Chen Y (1933) A preliminary survey of the earthworms of the Lower Yangtze Valley. Contributions from the Biological Laboratory of the Science Society of China 9: 177-296. Chen Y (1936) On the terrestrial Oligochaeta from Szechuan II with the notes on Gates’ types. Contributions of Biological Laboratory of Science Society of China. Zoology 11: 269-306. Dong Y, Law MMS, Jiang J, Qiu J (2019) Three new species and one subspecies of the Amynthas corticis-group from Guangxi Zhuang Autonomous Region, China (Oligochaeta, Megasco- lecidae). ZooKeys 884: 23-42. https://doi.org/10.3897/zook- eys.884.30988 Easton EG (1981) Japanese earthworms: A synopsis of the Megadrile species (Oligochaeta). Bulletin of the British Museum (Natural His- tory). Zoology : Analysis of Complex Systems, ZACS 40: 33-65. Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA prim- ers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3: 294-299. Gates GE (1939) On some species of Chinese earthworms with special reference to specimens collected in Szechuan by Dr. D.C. Graham. Proceedings of the United States National Museum 85: 405-507. https://doi.org/10.5479/si.00963801.3040.405 Goto S, Hatai S (1899) New or imperfectly known species of earth- worms. No. 2. Annotationes Zoologicae Japonenses 3: 13-24. zse.pensoft.net Han, A.C.N. et al.: Distribution and systematics of eastern Asian Amynthas carnosus Hall TA (1999) BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95-98. https://doi.org/10.14601/phyto- pathol_mediterr-14998u1.29 Huang J, Xu Q, Sun Z, Tang G, Su Z (2007) Identifying earthworms through DNA barcodes. Pedobiologia 51(4): 301-309. https://doi. org/10.1016/j.pedobi.2007.05.003 Jiang J, Qiu J (2018) Origin and evolution of earthworms belonging to the family Megascolecidae in China. Shengwu Duoyangxing 26(10): 1074-1082. https://doi.org/10.17520/biods.2018105 Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide se- quences. Journal of Molecular Evolution 16(2): 111-120. https:// doi.org/10.1007/BF01731581 Kobayashi S (1936) Distribution and some external characteristics of Pheretima (Ph.) carnosa (Goto et Hatai) from Korea. Science Re- port of the Tohoku Imperial University 11: 115-138. Kobayashi S (1938) Earthworms of Korea I. Science Report of the To- hoku Imperial University 13: 89-170. Marchan DF, Novo M, Sanchez N, Dominguez J, Diaz Cosin DJ, Fernandez R (2020) Local adaptation fuels cryptic speciation in terrestrial annelids. Molecular Phylogenetics and Evolution 146: 106767. https://do1.org/10.1016/j.ympev.2020. 106767 McCay TS, Brown G, Callaham Jr MA, Chang C-H, Davalos A, Dob- son A, Gorres JH, Herrick BM, James SW, Johnston MR, McHugh D, Minteer T, Moore J-D, Nouri-Aiin M, Novo M, Ortiz-Pachar J, Pinder RA, Richardson JB, Snyder BA, Szlavecz K (2020) Tools for monitoring and study of peregrine pheretimoid earth- worms (Megascolecidae). Pedobiologia 83: 150669. https://doi. org/10.1016/j.pedob1.2020. 150669 Nguyen TT, Nguyen NQ, Lam DH, Nguyen AD (2020) Six new spe- cies of the genus Metaphire Sims & Easton, 1972 (Annelida, Oli- gochaeta, Megascolecidae) from southeastern Vietnam. The Raffles Bulletin of Zoology 68: 220-236. https://doi.org/10.26107/RBZ- 2020-0019 Novo M, Almodovar A, Diaz Cosin DJ (2009) High genetic divergence of hormogastrid earthworms (Annelida, Oligochaeta) in the central Iberian Peninsula: Evolutionary and demographic implications. Zoologica Scripta 38(5): 537-552. https://doi.org/10.1111/j.1463- 6409.2009.00389.x Ohfuchi S (1937) On the species possessing four pairs of spermathecae in the genus Pheretima, together with the variability of some exter- nal and internal characteristics. Saito Ho-On Kai Museum Research Bulletin: 31-136. Sims RW, Easton EG (1972) A numerical revision of the earthworm genus Pheretima auct. (Megascolecidae, Oligochaeta) with the rec- ognition of new genera and an appendix on the earthworms collected by the Royal Society North Borneo Expedition. Biological Journal of the Linnean Society, Linnean Society of London 4(3): 169-268. https://doi.org/10.1111/j.1095-8312.1972 tb00694.x Stamatakis A (2014) RAxML version 8: A tool for phylogenetic anal- ysis and post-analysis of large phylogenies. Bioinformatics 30(9): 1312-1313. https://doi.org/10.1093/bioinformatics/btu033 Stephenson J (1925) Oligochaeta from various regions, including those collected by the Mount Everest Expedition 1924. Proceed- ings of the Zoological Society of London 95: 879-907. https://doi. org/10.1111/j.1469-7998 1925 .tb07109.x Zoosyst. Evol. 100 (3) 2024, 1061-1073 Stephenson J (1931) Oligochaeta from Burma, Kenya and other parts of the world. Proceedings of the Zoological Society of London 101: 33-92. https://doi.org/10.1111/j.1469-7998.1931.tb06185.x Sun J (2013) Taxonomy and Molecular Phylogeny of Amyn- thas Earthworms from China. Shanghai: Shanghai Jiao Tong University. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Sudhir K (2011) MEGAS: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony meth- ods. Molecular Biology and Evolution 28(10): 2731-2739. https:// doi.org/10.1093/molbev/msr121 Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight ma- trix choice. Nucleic Acids Research 22(22): 4673-4680. https://doi. org/10.1093/nar/22.22.4673 Xiao N (2019) Terrestrial Earthworms (Oligochaeta, Opisthopora) of China. Elsevier Inc, China, 395 pp. https://doi.org/10.1016/B978-0- 12-815587-5.09986-8 Zhang L, Sechi P, Yuan M-L, Jiang J, Dong Y, Qiu J (2016) Fifteen new earthworm mitogenomes shed new light on phylogeny within the Pheretima complex. Scientific Reports 6(1): 20096. https://doi. org/10.1038/srep20096 1073 Supplementary material | Taxa list with the accession numbers and comparison of morphological characters Authors: Anne Charis N. Han, Yufeng Zhang, Pu Miao, Shaolong Wu, Nengwen Xiao, Mingyan Qin, Donghui Wu, Huifeng Zhao, Nonillon M. Aspe Data type: docx Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://do1.org/10.3897/zse.100.119292.suppl1 zse.pensoft.net