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Research Article
Distribution and systematics of the cosmopolitan Amynthas carnosus complex (Crassiclitellata, Megascolecidae) from eastern Asia
expand article infoAnne Charis N. Han‡§, Yufeng Zhang§, Pu Miao|, Shaolong Wu, Nengwen Xiao#, Mingyan Qin‡§, Huifeng Zhao§¤, Donghui Wu‡¤, Nonillon M. Aspe«
‡ Northeast Normal University, Changchun, China
§ Langfang Normal University, Langfang, China
| Henan Province Tobacco Company, Luoyang, China
¶ Hunan Province Tobacco Company, Changsha, China
# Chinese Research Academy of Environmental Sciences, Beijing, China
¤ Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun, China
« Mindanao State University at Naawan, Naawan, Philippines

Corresponding author: Huifeng Zhao ( zhaohf@lfnu.edu.cn )

Corresponding author: Donghui Wu ( wudonghui@iga.ac.cn )

Academic editor: Pavel Stoev
© 2024 Anne Charis N. Han, Yufeng Zhang, Pu Miao, Shaolong Wu, Nengwen Xiao, Mingyan Qin, Huifeng Zhao, Donghui Wu, Nonillon M. Aspe.
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.
Citation: Han ACN, Zhang Y, Miao P, Wu S, Xiao N, Qin M, Wu D, Zhao H, Aspe NM (2024) Distribution and systematics of the cosmopolitan Amynthas carnosus complex (Crassiclitellata, Megascolecidae) from eastern Asia. Zoosystematics and Evolution 100(3): 1061-1073. https://doi.org/10.3897/zse.100.119292
ZooBank: urn:lsid:zoobank.org:pub:541660A7-7B6A-4432-AEF5-C689737C0A3C
Open Access

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 between 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 VIII, a pair of racemose prostates opening through male pores in XVIII, and testes contained within testis sacs (Aspe 2016). They are known to be widely distributed and predominantly 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 China, 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 occasionally three pairs in segments 6/7–8/9, with genital markings typically closely paired mid-ventral and presetal in VIII–IX and often also in XVIII–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, including Jeju Island and Dagelet Island (Ulleung-do) (Kobayashi 1938; Blakemore 2013b, 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 is 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 (Carrera-Martínez and Snyder 2016). Therefore, the known distribution 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 original 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 possibility 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 species synonymy was caused by the erroneous assignment of names, which has added to the complexity of this species’ identity. Further information about A. carnosus’ provisional synonyms is listed and discussed by Blakemore (2012).

Amynthas pingi was previously considered a questionable synonym of A. carnosus, “as it is, on average, a larger worm with several other differences that presently exclude it from A. carnosus” (Blakemore 2012). However, 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 is only 132 mm long, and a later onset of intestine origin and septal glands, now also proven false). There, he also pointed out errors in Gates (1939) redefinition 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’ tubercles 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 separate species from A. carnosus. In which case, A. carnosus would likely suggest prevalence in China, from which A. pingi (= A. carnosus) was reported to have been abundantly 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 indication of the prevalent range of A. carnosus in the country, as was suggested by Chen (1936) and concluded by Kobayashi (1936).

Preliminary attempts at using the DNA barcodes of A. carnosus specimens from Japan and South Korea were carried out (Blakemore 2013b). Two new subspecies have been established, namely, A. carnosus naribunji Blakemore 2013 from Naribunji, Ulleung-do (Dagelet Island, South Korea), and A. carnosus roki Blakemore and Lee 2013 from Incheon (South Korea). Preliminary DNA data for taxon identification and phylogenetic relationships were also explored, yet a rather deficient description 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 morphological 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 habitat 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. Collections near the sites with surface castings were also taken into account. The earthworms collected were preserved and stored in 100% ethanol.

Table 1.
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Collection information for sampling areas and specimens.

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

DNA extraction, amplification, and sequencing

Total genomic DNA was extracted from the muscle tissue of the posterior part using the TIANGEN Genomic DNA Kit (China) following the manufacturer’s instructions. Regions of the cytochrome c oxidase subunit I (COI) were amplified using the polymerase chain reaction (PCR). The mixture (total volume 25 μl) contained 1 μl DNA and 17.25 μl sterile ddH2O, 2.0 μl of dNTP, 2.5 μl of buffer, 0.25 μl TransGen EasyTaq-polymerase and 1.0 μl of Primer HCO1490 (5-GGTCAACAAATCATAAAGATATTGG-3) (Folmer et al. 1994), and 1.0 μl of Primer COIE (5-TATACTTCTGGGTGTCCGAAGAATCA-3) (Bely and Wray 2004). The cycling 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 visualized 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. (Beijing) for Sanger sequencing using an ABI 3730 automated 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 (Stamatakis 2014), using the default rapid hill-climbing algorithm 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 Pontodrilus litoralis as an outgroup. Pairwise distance analysis among A. carnosus subspecies and between COI sequences of the other 10 Amynthas species downloaded from GenBank was conducted using MEGA5 (Tamura et al. 2011) with the Kimura-2 parameter model (Kimura 1980).

Morphological examination and identification

Fixed specimens were brought to the laboratory for external and internal examination using a stereomicroscope (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 diagnoses 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 inter-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 subspecies 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 between 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.

Table 2.
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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
1 A. 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 18–19 21 0
8 A. corticis 16–20 17–19 19–20 19 18–19 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

Also, a specimen identified as “A. carnosus carnosus” 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.

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 subspecies 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 is 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).

On the one hand, A. c. naribunji’s current distribution pattern has been expanded because of its new record in northern China (Beijing and Liaoning). Still, future investigations 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 (NIBR-IV0000261264 providing DNA w56) from Incheon Great Park (Blakemore and Lee 2013) has inter-subspecific distance values of 8.1–10% from A. c. carnosus and 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-Martínez and Snyder 2016: 297. Chang et al. 2016: 505.

Amynthas pingi (Stephenson, 1925) – Sims and Easton 1972: 235. Blakemore 2013c: 112.

Material examined

Specimen IDs: 362R1_01, 02, 04, 05, 06, 07, 09, seven matures from Nanshan Park, Jinzhou, Liaoning; 533R70_08, 09, 10, three matures from Jinjiang Mt. Park, Dandong, Liaoning; LFXHR7_02, 04, 05, three matures 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 Municipality; 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 in Shangqiu Normal University, Liangyuan District, Shangqiu, Henan.

Diagnosis

Length 105–210 mm. Spermathecal pores having four pairs in 5/6/7/8/9, rarely 3 pairs in 6/7/8/9, with pre-intersegmental hemispherical arc (spermathecal papillae). Dorsal pores typically from 12/13. Pre-clitellar genital 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 XXVII.

Figure 2. 

Amynthas carnosus carnosus variations on the number of spermathecal pores and spermathecae: four pairs (A, D) (specimen ID 362R1_06), three pairs (B, E) (specimen ID LFXHR7_05), five pairs (C, F) (specimen ID HNLNR2_05). Scale bars: 1 mm.

Figure 3. 

Pre-clitellar genital marking (arrows) variations of Amynthas carnosus. A. Modified fig. 1 of the variations of pre-clitellar 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).

Figure 4. 

Post-clitellar genital marking (arrows) variations of Amynthas carnosus. A. Modified fig. 2 of the variations of post-clitellar genital markings from Kobayashi (1936); B. This study. B1, B2. (specimen IDs 362R1_07 and HNLNR2_04); B3 (specimen ID 362R1_04); B4, B5. (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).

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-Martínez and Snyder 2016). However, prior to this study, no other specimen with five pairs was recorded elsewhere.

Distribution

China (Liaoning, Beijing, Tianjin, Hebei, 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 (Blakemore 2012), widely spaced ones are mostly observed with the A. carnosus specimen from China, which resembles 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 markings of the Liaoning specimens (Dandong, DNA 533R) match those of the South Korean specimen from Geoman (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: table S2. External characters such as the number of spermathecal pores and segment locations of pre-clitellar genital markings match among specimens from different countries. However, internal character variations are observed in the position of the intestinal caeca, which was reported to begin at XXVII and extend to XXIII or XXIV (Blakemore, 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 anterior 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 between 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 species identification.

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.

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 genital making variations in the A. c. carnosus from China comply with Kobayashi’s Types IIII, VI, and VIII (see Fig. 3), while the post-clitellar genital marking variations 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).

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 presented (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-clitellar genital markings absent (complying with Kobayashi Type I in Blakemore 2013a); if present, one to three pairs; pre-setal in VIII and IX when one or two pairs and having a post-setal pair in VIII when three pairs (complying with Kobayashi’s Types III, IV, and VIII); asymmetrical patterns also present (Fig. 7). Male pores superficial in XVIII having “disc-like” genital markings paired posterio-median to male pores (Fig. 8).

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. 

A. 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.

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).

Description

Length 185–228 mm. Color of preserved specimens may have varying shades of brown but dorsum 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. Segments 115–137. Prostomium epilobous. First dorsal pore on 12/13. Clitellum annular at XIV–XVI without setae or dorsal pores. Setal arrangement perichaetine, setae between 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 (spermathecal papillae). Pre-clitellar genital markings circular in shape, latero-ventral (0.25–0.29 mm), randomly located in pairs (three pairs/two pairs/one pair, a total 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 lateral margin (with ventral distance 0.26–0.29 mm) on large circular porophores. Post-clitellar genital markings distinguishably 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 absent. Esophageal gizzard within VIII−X. Intestinal origin at XV (or XIV). Intestinal caeca simple, paired in XXVII, extending anteriorly to XXII. 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 botuliform. Accessory glands sessile and round.

Seminal vesicles paired in XI and XII, large, smooth, yellowish, posterior pair larger but not as obvious compared to A. c. carnosus, each with a dorsal lobe. Ovaries present. Prostate glands paired in XVIII, large, lobulated, covering XVI−XX; ducts thick and large, U-shaped. Accessory glands round, sessile, or slightly lobed, corresponding 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 morphological description of A. carnosus naribunji in the original 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 IX. In contrast, A. c. carnosus is typically medium to smaller size, with mostly only four genital markings (two pairs) or less, either wide or closely-spaced pre-clitellar (Table 3).

Table 3.
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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 A. carnosus naribunji A. carnosus roki
Length 105–340 mm 185–228 mm 175–300 mm
No. of segments 96–179 121–137 136
No. of setae between mp 16–20 17–20 14
Male pore round or elliptical porophores on large circular porophores, superficial on small mounds within concentric rings
Distance between Male pores (circumference apart) 0.25–0.29 0.26–0.29 0.30
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 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
Distance between spermathecal pores (circumference apart) 0.28–0.30 0.29–0.30 NA
Pre-clitellar gm (circumference apart) closely paired or widely spaced pre-setal in VIII and IX, mid-ventral (mostly two pairs, total of 1–4 GMs) widely spaced, randomly located in pairs (three pairs or two pairs or one pair, a total of 2–6 GMs) absent
Post-clitellar gm (circumference apart) up to three pairs of genital markings near male pores, pre-/post-setal in VXIII and pre-setal in XIX. Second pair, post-setal more medial than the first distinguishably paired, post-setal in XVIII, mid-ventral to male pore absent
Spermathecae (circumference apart) typically four pairs in VI–IX, first pair often slightly smaller; or three pairs in VII–IX or five in IV−IX four pairs in VI−IX four pairs in VI−IX
Ampulla and Duct (circumference apart) Wrinkled; Ovate to narrowly ovate Wrinkled; Ovate to narrowly ovate Narrowly ovate*
Prostate gland (circumference apart) racemose at XVIII, covering XV (or XVI)–XX racemose at XVIII, covering XVI−XX racemose in XVIII
Intestinal caeca (circumference apart) paired in XXVII, simple, extending to XX, or XI, or XIII paired in XXVII, simple, extending to XXII Simple from XXVII
Gizzard (circumference apart) VIII−X VIII−X NA
Intestine XV (or XIV) XV NA

The distinctive character of having “a pair of genital markings posterio-median to male pores” in A. c. naribunji may distinguish it from those of A. c. carnosus (Blakemore, 2013a). However, it is now undeniable that this seemingly “distinctive character” was likewise observed 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 Kobayashi’s Types I and II markings” as a distinctive character for the A. c. carnosus subspecies, A. c. naribunji. Although this might be the case, one cannot ignore the certain degree of dissimilarity between the two subspecies’ morphological characters (e.g., the shape of male pore porophore and the distinct shape of the genital markings in A. c. naribunji, described as “disc-like” by Blakemore). As such, applications of state-of-the-art methodologies such as high-throughput sequencing or geometric morphometric (Marchán et al. 2020) may be adopted that go beyond traditional methods of taxonomic diagnosis (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 distinctive 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 subspecies appears to differ from the nominal taxon’s neotype and from other synonyms in Blakemore.” Aside from the external morphological differences, slight internal character differences were also observed (Table 3).

According to Blakemore (2012), “the genital marking 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 asymmetrical 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 possess 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 of morphological as well as genetic variations in different geographical occurrences of this species can be further elucidated. Still, a more thorough investigation should be carried 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 compared 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 National Science and Technology Fundamental Resources Investigation Program of China (2018FY100301).

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Supplementary material

Supplementary material 1 

Taxa list with the accession numbers and comparison of morphological characters

Anne Charis N. Han, Yufeng Zhang, Pu Miao, Shaolong Wu, Nengwen Xiao, Mingyan Qin, Donghui Wu, Huifeng Zhao, Nonillon M. Aspe

Data type: docx

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 users 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.
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