Biodiversity Data Journal :
Research Article
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Corresponding author: Huei-Ping Shen (shenhp@tbri.gov.tw)
Academic editor: Samuel James
Received: 26 Jan 2024 | Accepted: 13 Apr 2024 | Published: 09 May 2024
© 2024 Chih-Han Chang, Huei-Ping Shen, Emma Sherlock, Csaba Csuzdi
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:
Chang C-H, Shen H-P, Sherlock E, Csuzdi C (2024) A review of the earthworm Amynthas masatakae (Beddard, 1892) (Clitellata, Megascolecidae), with designation of two new synonyms. Biodiversity Data Journal 12: e119599. https://doi.org/10.3897/BDJ.12.e119599
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Correct and timely identification of an invasive species during quarantine or at an early stage of invasion before establishment or spread is critical for preventing biological invasions. However, taxonomic confusion of potential invasive earthworm species caused by incorrect taxonomic treatment or reckless taxonomic work has made it difficult to properly recognize potential invasion threats. Through analyzing publicly available DNA sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene, we confirmed the validity of the specific status of Amynthas masatakae (Beddard, 1892), a peregrine earthworm species in East Asia with the potential to spread to other regions of the world, and designated two new synonyms of A. masatakae: Amynthas tralfamadore Blakemore, 2012 syn. nov. and Amynthas scaberulus Sun and Jiang, 2021 syn. nov. Additionally, the name A. triastriatus usualis Dong, Jiang, Yuan, Zhao and Qiu, 2020 is nomenclaturally unavailable since it was published in an electronic journal without ZooBank registration and an explicit statement establishing a new nominal taxon. Specimens described under this unavailable name actually belong to A. masatakae. Inadequate literature review and erroneous species identities associated with sequences in GenBank have caused even more problems in the already confusing earthworm taxonomy.
earthworm, Amynthas masatakae, Amynthas robustus, Amynthas triastriatus, taxonomy
The invasion of non-native earthworms is one of the main threats to forest ecosystems around the world (
While there have been ample studies focusing on European invasive earthworms, from their taxonomic identity to ecological impacts, studies focusing on invasive pheretimoid earthworms, especially their taxonomy, are scarce (e.g.
Similar to A. carnosus in its distribution and invasion status, Amynthas masatakae (Beddard, 1892) is a cosmopolitan species found in Japan, Korea, China and Taiwan, and has the potential to spread to other regions of the world. However, our current knowledge on A. masatakae, both morphological and genetic, does not allow correct identification of this species by most researchers, making confirmation of potential invasion a challenging task. Amynthas masatakae had long been regarded as a junior synonym of Amynthas robustus (Perrier, 1872) (
Historically, A. masatakae was only reported once in Taiwan (
Earthworms were collected throughout Taiwan during 2005–2019. The specimens were anesthetized in 10% ethyl alcohol and then preserved in 95% ethyl alcohol. They are deposited in the earthworm collection at the Taiwan Biodiversity Research Institute, Jiji, Nantou, Taiwan. The following specimens were selected for phylogenetic analysis (Table
GenBank accession numbers of COI sequences of specimens used in this study.
Species | Locality | Voucher no. | GenBank accession no. |
Amynthas gracilis (Kinberg, 1867) | Douliou, Yunlin, western Taiwan | Gra1 | OR801254 |
Lake Shuangli, Guningtou, Kinmen | Gra2 | OR801255 | |
Central Boulevard, Nangan, Matsu | MTS5 | OR801251 | |
Nanao, Ilan, northeastern Taiwan | East617 | OR801252 | |
Fuhsing, Taoyuan, northern Taiwan | LLS43 | OR801253 | |
Amynthas masatakae (Beddard, 1892) | Chilai mountain house, Hualien, eastern Taiwan | East567 | OR801241 |
Amynthas robustus (Perrier, 1872) | Meilan Forest Road, Kaohsiung, southern Taiwan | laut1 | OR801247 |
Meilan Forest Road, Kaohsiung, southern Taiwan | laut2 | OR801248 | |
Meilan Forest Road, Kaohsiung, southern Taiwan | laut3 | OR801249 | |
Baibaohsi Agricultural Road, Hualien, eastern Taiwan | East348 | OR801242 | |
Lijia Forest Road, Taitung, eastern Taiwan | East382 | OR801243 | |
Fanpaoshan Forest Road, Ilan, northeastern Taiwan | East422 | OR801244 | |
Hubaotan, New Taipei City, northern Taiwan | East686 | OR801245 | |
Wutai, Pingtung, Southern Taiwan | PT001 | OR801246 | |
Perionyx excavatus (Perrier, 1872) | Guangfu Forest Road, Hualien, eastern Taiwan | East341 | OR801250 |
In addition to the newly-collected specimens, we examined the morphology of the type specimens of A. masatakae archived in the Natural History Museum in London, UK (BMNH 1904.10.5 912-3) on 14 May 2011 (by CHC) and 23 Jul. 2014 (by HPS).
Muscle tissues were taken from the posterior 10–20 segments of the specimens and then preserved in 95% ethyl alcohol at -20oC. DNA extraction was conducted using the Tissue Genomic DNA Extraction Mini Kit (Favorgen Biotec, Pingtung, Taiwan). Polymerase chain reaction for COI was carried out using the primers LCO1490 and HCO2198 (
COI sequences of A. masatakae, A. robustus, A. scaberulus, A. triastriatus, Amynthas aspergillum (Perrier, 1872), Amynthas corticis (Kinberg, 1867), A. gracilis, Metaphire californica (Kinberg, 1867) and Metaphire schmardae (Horst, 1883) were retrieved from GenBank including those of A. masatakae from Kyushu, Japan and A. robustus from the Ryukyus, Japan recently reported by
COI sequences published by
Taxon name in Blakemore’s publication | Source | Locality | Voucher no. |
Amynthas tralfamadore sp. nov. | Korea | WO2 | |
Amynthas masatakae (Beddard, 1892) | Japan | WO35 | |
Amynthas masatakae (Beddard, 1892) | Korea | w28b | |
Amynthas tralfamadore Blakemore, 2012 |
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Korea | w29 |
Amynthas tralfamadore Blakemore, 2012 |
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Korea | w30 |
Amynthas masatakae (Beddard, 1892) |
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Korea | H3 |
Sequences were aligned using the default settings of ClustalX 2.0 (
BMNH = Natural History Museum, London, UK
CN = China
IND = India
JP = Japan
KR = Korea
TW = Taiwan
As mentioned earlier, A. masatakae was regarded as a junior synonym of A. robustus for a long time (
A comparison of characters among A. masatakae (Beddard, 1892) from Japan, Korea and Taiwan, A. robustus (Perrier, 1872) from Taiwan, A. tralfamadore Blakemore, 2012 from Korea and A. triastriatus (Chen, 1946), A. triastriatus “usualis” Dong et al. (2020) and A. scaberulus Sun and Jiang, 2021 from China.
Species | A. masatakae | A. masatakae | A. masatakae | A. masatakae | A. robustus as Pheretima lauta | A. tralfamadore | A. scaberulus | A. triastriatus “usualis” | A. triastriatus |
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Locality | Japan | Japan | Korea | Taiwan | Taiwan | Korea | China | China | China |
Length (mm) | 127 | 135–189 | 102–130 | 105–138 | 125–203 | 125 | 129–166 | 120–150 | 110 |
Segments | 90 | 110–125 | 114–130 | 96–138 | 88–129 | 125 | 129–131 | 108–111 | 88 |
Diameter (mm) | 6 | 4.5–7 | 5.5–7 | 4–7.5 | 6.5–7.5 | – | 4.5–6 | 4.9–6.8 | 7 |
First dorsal pore | – | 12/13 | 11/12 | 11/12 | 11/12 | 10/11 | 10/11 or 11/12 | 11/12 | 10/11 |
Setal number | |||||||||
III | – | – | 20–30 | – | – | – | 21–38 | 20–26 | 34 |
VI | – | 34–36 | 39–45 (VII) | 34–41 (VII) | – | – | 36–42 (V) | 24–30 (V) | – |
VIII | – | 40–43 | 40–50 | – | 47–55 | – | 46–52 | 30–34 | 36 (IX) |
XII | – | – | 47–57 | – | – | 46–50 | – | – | – |
XX | – | 44–51 | 54–64 | 41–49 | – | ca. 70 | 56–62 | 40–48 | – |
XXV | – | – | – | – | 55 (XXVI) | ca. 70 | 52–74 | 55–60 | 38 |
Between male pores | – | 8–9 | 13–15 | 13–15 | 18–21 | ca. 15 | 14–20 | 12–16 | 12 |
Genital papillae | |||||||||
Preclitellar | VIII and IX (= 2 medial to each spermathecal pore) | 1 medial to each spermathecal pore | 2 medial to each spermathecal pore | 2 medial to each spermathecal pore | 1 medial to each spermathecal pore and paired presetal on ventral VIII–IX | 2 medial to each spermathecal pore | 1–2 medial to each spermathecal pore | 14–18 from postsetal VII to presetal IX | 2 medial to each spermathecal pore |
Postclitellar | – | 2 medial to each male pore | 2 medial to each male pore | 2 medial to each male pore | 1–2 medial to each male pore and 1–2 presetal on ventral XVIII | 2 medial to each male pore | 1–2 medial to each male pore | 2 medial to each male pore and 2 presetal on ventral XVIII | 2 medial to each male pore |
Spermathecal pores | 2 pairs (7/8/9) | 2 pairs (7/8/9) | 2 pairs (7/8/9) | 2 pairs (7/8/9) | 2 pairs (7/8/9) | 2 pairs (7/8/9) | 2 pairs (7/8/9) | 2 pairs (7/8/9) | 2 pairs (7/8/9) |
Spermathecae | small, diverticulum longer than ampulla | ampulla 1.5 mm long, diverticulum much longer than ampulla, no seminal chamber | ampulla small, diverticulum longer than ampulla with distended ental end forming a large seminal chamber | ampulla oval-shaped, diverticulum with a slender stalk and a long seminal chamber | ampulla large, diverticulum long with a slender duct and a rod-like seminal chamber | ampulla round on short duct with rounded clavate diverticulum | ampulla ball-shaped, stout duct as long as ampulla; diverticulum shorter than the main axis, distal ⅓ dilated into a peach-shaped seminal chamber | ampulla oval‐shaped, diverticulum short with terminal 1/2 dilated into an oval‐shaped glossy seminal chamber | ampulla large, heart-shaped, diverticulum long, with ental half enlarged as seminal chamber |
Hearts | – | – | 4 pairs in X–XIII | – | – | 4 pairs in X–XIII | 4 pairs in X–XIII | 4 pairs in X–XIII | 4 pairs in X–XIII |
Intestinal caeca | XXVI | XXVIII, serriformed ventral margin | XXVI, simple without indentations | XXVII, simple with saw-shaped border | XXVI, simple with serrated ventral border | XXVII, simple in having a paler rugose and capillaried interior face | XXVII, simple, or with tiny incisions on ventral margins | XXVII, simple with short pointed saccules on ventral margin | simple, smooth |
Seminal vesicles | small in XI–XII | weak and thin in XI–XII | small in XI–XII | large in XI–XII | XI–XII | larger in XI and smaller in XII | underdeveloped in XI–XII | XI–XII, second pair more developed | small in XI–XII |
Prostate glands | absent | absent | absent or rudimentary | absent or rudimentary | XVI–XIX | aborted | degenerated or developed | small, rudimentary or absent | absent |
Prostatic ducts | short and bent muscular | large, muscular, bent | small, bow-shaped | slender, bent | long, slender, bent | short, muscular | bent | S-shaped | U-shaped, stout |
Accessory glands | pear-shaped | large, long-stalked | large, long-stalked | – | stalked | small, stalked | stalked | – | stalked |
Amynthas triastriatus (Chen, 1946) is an earthworm endemic to China. Its original description is based on a single specimen found in Mt. Omei, Sichuan, central China (
Five apparent clades among our target species/sequences, clades A–E, can be superficially recognized in the COI tree (Fig.
Phylogenetic tree inferred from the Maximum Likelihood analysis of the DNA sequences of the cytochrome c oxidase subunit I (COI) gene. Sequences acquired in this study and those available from
Clade A includes A. masatakae specimens from Korea (w28b and H3) and Japan (WO35) reported by
Uncorrected p-distances (lower-left) and Kimura’s two-parameter distances (upper-right) among clades A–E, based on the COI gene.
Clade A | Clade B | Clade C | Clade D | Clade E | |
Clade A | – | 0.066 | 0.103 | 0.105 | 0.167 |
Clade B | 0.062 | – | 0.100 | 0.110 | 0.169 |
Clade C | 0.094 | 0.092 | – | 0.125 | 0.180 |
Clade D | 0.097 | 0.100 | 0.113 | – | 0.161 |
Clade E | 0.148 | 0.150 | 0.159 | 0.144 | – |
Clade C consists of sequences from type specimens of A. scaberulus from China (Fig.
In addition to the sequences/specimens mentioned above, 36 sequences in clades A and B clearly came from misidentification, including 16 sequences, one from China and 15 from Japan, identified as A. robustus, 18 sequences from China identified as A. triastriatus and two sequences, one from China and the other from India, identified as A. gracilis (Fig.
When applying integrative taxonomic criteria widely used in pheretimoid earthworms (
Clade C consists of four sequences of A. scaberulus from China, including the holotype of the nominal species. However, A. scaberulus described by
To sum up, clades A, B and C in our phylogenetic analysis are conspecific and their identity should be A. masatakae. This species has frequently been incorrectly identified as A. robustus, A. triastriatus or A. gracilis, as evidenced in many GenBank sequences we analyzed. Molecular data support that A. masatakae and A. robustus are separate species (Fig.
Our synthesis provided strong evidence that both A. masatakae and A. robustus are peregrine species mainly found in East Asia. Amynthas masatakae is primarily reported in Japan, Korea and China, with only a few cases in Taiwan and India, whereas A. robustus is common in China and Taiwan, rare in Japan (except the Ryukyu Archipelago (
Both A. robustus and A. masatakae in these areas have long been confounded with A. triastriatus by Chinese authors (e.g.,
Together with the newly-collected specimens in Taiwan, our finding that A. traistriatus in
Structures of spermatheca (A) and prostate gland (B). A1 Fig. 4A in
As the first study in which “DNA barcoding” and “earthworm” appear in the same article,
We urge researchers to be more careful when using sequences in GenBank, especially when relying on DNA barcodes for species identification, and taxonomists who generate sequences to take full responsibility for the sequences they submit to GenBank and to make necessary corrections throughout their academic career. For GenBank users, stop simply relying on the scientific name shown on the sequence page, even when it appears to be identified by a well-known taxonomist. Read the literature of those sequences and other sequences with the same species label and conduct preliminary analyses before deciding which sequences to use. It will always be the responsibility of users to check the identity of specimens and the integrity of their sequence data (
For species identification of A. masatakae in the future, a relatively easy molecular approach is to use three sequences from each of clades A, B and C as reference sequences and conduct a quick phylogenetic analysis (Neighbor-joining, Maximum Likelihood, etc.). Any of the GenBank sequences in clades A, B and C in our phylogenetic tree (Fig.
We are grateful to Takafumi Nakano and Parin Jirapatrasilp for their constructive comments and suggestions, and to Wen-Jay Chih and Po-Wei Yo for helping with the laboratory work. This study was supported financially by the National Science and Technology Council, Taiwan (MOST 111-2621-B-002 -005 -MY3) and the Ministry of Education, Taiwan (Yushan Fellow Program) to CHC, and by the Taiwan Biodiversity Research Institute, Ministry of Agriculture, Taiwan to HPS.
No ethical principles were violated when providing this study.
HPS and CHC conceived and designed the study. HPS collected specimens. CHC performed experiments and analyzed data. HPS and CHC wrote the manuscript. All authors reviewed and edited the manuscript. All authors have read and agreed to the version of the manuscript.
The issues of sympatric subspecies, distribution of lineages A and B and parthenogenetic polymorphisms reported by Dong et al. (2020).