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. 2022 Apr 29;1097:167–181. doi: 10.3897/zookeys.1097.80216

The complete mitochondrial DNA sequences of two sibling species of lumbricid earthworms, Eiseniafetida (Savigny, 1826) and Eiseniaandrei (Bouché, 1972) (Annelida, Crassiclitellata): comparison of mitogenomes and phylogenetic positioning

Csaba Csuzdi 1, Jachoon Koo 2, Yong Hong 2,✉
PMCID: PMC9848864  PMID: 36761685

Abstract

Composting earthworms of the genus Eisenia play an important role in soil ecosystems. However, taxonomic classification of this genus, especially the sibling species Eiseniafetida and Eiseniaandrei, is complicated because of their morphological similarity. In this study, we assessed the utility of the complete mitochondrial genome (mitogenome) for identification and differentiation of the two species. The complete mitogenomes of E.andrei and E.fetida were 15,714 and 16,560 bp, respectively. They contained 37 genes, comprising 13 protein-coding genes (PCGs), two rRNA genes, 22 tRNA genes, and a putative non-coding region, as observed in other earthworms. Sequence comparisons based on the complete nucleotide sequences excluding the non-coding region showed 85.8% similarity, whereas the predicted amino acid sequences of the 13 PCGs were 92.7% similar between the two species. In particular, distinct features were found in the non-coding regions of the mitogenomes. They include a control region associated with putative mitogenome replication and an extended sequence. The extended sequence showed significant differences between the two species and other known earthworm species, suggesting its potential as a feasible molecular marker for species identification. Phylogenetic analysis of the 36 mitogenomes of earthworm species corroborated the monophyly of the genus Eisenia and the taxonomic distinctness of the sibling species pair, E.fetida and E.andrei.

Keywords: Compost worms, mitogenome, Oligochaeta, phylogeny, sibling species

Introduction

The earthworm species Eiseniafetida was described as Enterionfetidum by Savigny (1826). Eisen (1873) relegated this species to his newly described genus Allolobophora Eisen, 1873 and remarked that it is easily recognized by its peculiar color pattern consisting of reddish-brown bands separated by yellowish intersegments. Later, Malm (1877) selected Enterionfetidum as the type species of the genus Eisenia Malm, 1877. For a long time, the characteristic striated pattern was a primary identifiable characteristic of the species until Avel (1937) recognized that the classical Eiseniafetida existed in two morphological variants: a typical striped form and an evenly pigmented form that might represent a separate species (Avel 1937).

André (1963) carried out breeding experiments with earthworms and recognized that reproductive isolation exists between the striped and evenly colored forms of E.fetida, and that the crossbred offspring are sterile. Consequently, he described the uniformly pigmented form as Eiseniafetidavar.unicolor. Variety names proposed after 1961 were considered intrasubspecific and invalid; therefore, Bouché (1972) proposed a new name for var. unicolor, Eiseniafetidaandrei Bouché, 1972. Since then, various authors have treated the subspecies E.f.andrei differently. Reynolds (1977), Sims (1983), Easton (1983), Csuzdi and Zicsi (2003), and Blakemore (2008, 2013) regarded it as a color morph and synonym of Eiseniafetida (Savigny, 1826). Others, such as Sims and Gerard (1985), Qiu and Bouché (1998), Lehmitz et al. (2014), and Martin et al. (2016), considered E.fetida and E.andrei to be two distinct valid species.

Eiseniafetida is an important composting worm and ecotoxicological test organism (Domínguez et al. 2005; Römbke et al. 2016). Therefore, intensive studies have been carried out since the early 1980s to determine whether the two types of E.fetida (striped and unicolor) represent two morphological variants or two separate species. The first clear indication that E.fetida and E.andrei might represent two separate species was presented by Jaenike (1982) who used an electrophoretic survey to demonstrate complete reproductive isolation between the two species. Later, Reinecke and Viljoen (1991) and Domínguez et al. (2005), using crossbreed experiments, reported complete reproductive isolation between the two species (no viable cocoons were observed in interspecific crosses) and noted that the two species differed in their life histories. Furthermore, E.andrei exhibited higher reproduction rates.

Recently, Römbke et al. (2016) carried out a detailed barcoding study of the Eiseniafetida / E.andrei complex using samples from 28 laboratories in 15 countries. The two species formed two distinct clades on the neighbor-joining tree, and the E.fetida clade consisted of two subclades, fetida 1 and fetida 2. The mean uncorrected p-distances were 14.2% between fetida1 and andrei, 14.3% between fetida2 and andrei, and 11.2% between the two fetida subclades; these values exceed the species-level threshold suggested by Chang and James (2011). Therefore, Römbke et al. (2016) concluded that the complex consists of three taxa: E.andrei and two cryptic taxa, E.fetida 1 and E.fetida 2. Moreover, they found that E.andrei was always correctly identified from its morphology, whereas E.fetida was often misidentified as E.andrei (Römbke et al. 2016).

It is worth mentioning that the native range of E.fetida and E.andrei is unknown. All of the above-mentioned studies were based on laboratory stocks or specimens collected from compost or manure heaps. Perel (1998) hypothesized that the native range of E.fetida is somewhere in the forest-steppe zone of Central Asia; therefore, Latif et al. (2017) barcoded 62 new specimens of this complex collected from different anthropogenic and natural habitats in Iran. Surprisingly, all Iranian material appeared in the E.andrei clade, irrespective of striped or uniform pigmentation. Moreover, the E.andrei clade showed high genetic structuring in contrast to the almost uniform genetic composition found by Römbke et al. (2016). Automatic barcode gap discovery (ABGD) analysis identified two species corresponding to Eiseniaandrei and Eiseniafetida with high genetic structuring inside both species, but neither of the subclades reached the unambiguous species threshold [15% K2P distance according to Chang and James (2011)].

Comparison of mitogenomes may reveal important genome-level characteristics, helping us understand genome structure, gene order, phylogenetic relationships, and evolutionary lineages. The earthworm mitogenome is a circular, double-stranded, covalently closed DNA molecule containing 13 protein-coding genes (PCGs), two ribosomal RNA genes (rRNAs), 22 transfer RNA genes (tRNAs), and one non-coding region (Zhang et al. 2016). Although Lumbricidae is the most important earthworm family in the Northern Hemisphere temperate zone and contains many widespread and invasive cosmopolitan species, only a few complete or nearly complete mitogenomes are available for this family (Boore and Brown 1995; Shekhovtsov and Peltek 2019; Zhang et al. 2019; Shekhovtsov et al. 2020). In the present study, we sequenced the complete mitochondrial genome of the sibling species E.fetida and E.andrei to clarify its taxonomic position and to gain a better understanding of the mitogenomes of Lumbricidae.

Material and methods

Sample preparation and DNA extraction

Adult E.andrei were collected from a farm in Sangseo-myeon, Buan-gun, Jeollabuk-do, Korea (33°41'23.80"N, 126°38'33.67"E; 40 m a.s.l.) on March 26, 2021. Eiseniafetida adults were collected near a house at Seolcheon-myeon, Muju-gun, Jeollabuk-do, Korea (33°58'00.61"N, 127°47'47.88"E; 408 m a.s.l.) on April 2, 2021, and preserved in 99% ethanol until DNA extraction. A voucher specimen of each species was deposited at Jeonbuk National University, Jeonju City, Korea, under accession numbers JBNU0011 and JBNU0012. Total genomic DNA was prepared from a small portion of body segments of a single adult earthworm using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). The remaining tissue was stored at -20 °C in 90% ethanol to preserve the specimens.

TruSeq DNA Library construction

The sequencing library was prepared by random fragmentation of genomic DNA, followed by 5’ and 3’ adapter ligations. Briefly, 100 ng genomic DNA was fragmented using adaptive focused acoustic (AFA) technology (Covaris Inc., Woburn, MA, USA). The fragmented DNA was end-repaired and ligated to TruSeq indexing adapters using the Illumina TruSeq DNA Nano Library Prep Kit according to the manufacturer’s instructions (Illumina Inc., San Diego, CA, USA). The resulting libraries were quantified through a qPCR-based assay using the KAPA Library Quantification Kit for Illumina Sequencing platforms according to the manufacturer’s instructions (Kapa Biosystems, Woburn, MA, USA). The libraries were qualified using an Agilent Technologies 2200 TapeStation (Agilent Technologies, Santa Clara, CA, USA).

DNA sequencing and assembly

Paired-end (2 × 150 bp) sequencing was performed using an Illumina HiSeq-X platform (Illumina Inc., USA) at Macrogen Inc. (Seoul, Korea). For each species, > 39 million reads (5.1–5.9 Gb) were generated. To reduce bias in the analysis, adapter trimming and quality filtering were performed using Trimmomatic version 0.36 (Bolger et al. 2014). After filtering, the number of total reads of E.andrei and E.fetida was > 29 million (4.4 Gb) and > 34 million (5.2 Gb), respectively. De novo assembly of raw sequencing reads was performed using various k-mer lengths in SPAdes version 3.13.0 (Bankevich et al. 2012). Mitochondrial contigs were assembled into a single contig using BlastN alignment (https://blast.ncbi.nlm.nih.gov/Blast.cgi) against the Lumbricusterrestris Linnaeus, 1758 mitogenome (GenBank accession number, NC_001673) as the reference sequence. The assembled mitochondrial sequences for E.andrei were connected to a single circular molecule, whereas the conformation of the contig for E.fetida was unclear because the 12 bp TA-repeat sequence overlapped at both ends. This region corresponds to heteroplasmic tandem repeats in the mitochondrial control region (Liu et al. 2020). To close the circular genome, pairs of PCR primers (5’-ACCACCAGAGTTCTCGTTCG-3’ and 5’-GCCAATATCGGCCCAAAACC-3’) were designed to amplify the control region. The reaction was performed in an nTaq-tenuto (Enzynomics Inc., Seoul, Korea) with the following program: 95 °C for 3 min; 35 cycles of 95 °C for 20 s, 55 °C for 30 s, and 72 °C for 1 min; and a final extension of 5 min at 72 °C. The amplicons were directly sequenced using Sanger sequencing (Macrogen Inc., Seoul, Korea) to determine the complete mitogenome of E.fetida.

Mitogenome annotation

The annotation and visualization of mitochondrial genomes were performed using the online MITOS software (Donath et al. 2019), and manual curation was performed using BLAST searches in the NCBI database for various earthworm mitochondrial genomes deposited in NCBI (Table 1). A comparative map of mitochondrial genomes was created using Geneious Prime 2021 software (https://www.geneious.com). The cox1 sequence was used as an anchor for linearized maps of the mitochondrial genomes. The annotated complete genome sequences were registered in GenBank under accession numbers OK513069 for E.andrei and OK513070 for E.fetida. The associated biosample numbers were SAMN26185682 for E.andrei and SAMN26185683 for E.fetida. All sequencing datasets, including SRA, are available in the NCBI BioProject database under the accession number PRJNA769829.

Table 1.

List of Megadrili mitogenomes used in this study.

Species Genbank No. Total length (bp) *Non-coding region (bp) Topology
Amynthasaspergillus KJ830749 15,115 565 Circular
Amynthascarnosus KT429008 15,160 601 Circular
Amynthascorticis KM199290 15,126 573 Circular
Amynthascucullatus KT429012 15,122 569 Circular
Amynthasgracilis KP688582 15,161 582 Circular
Amynthashupeiensis KT429009 15,069 477 Circular
Amynthasinstabilis KT429007 15,159 577 Circular
Amynthasjiriensis KT783537 15,151 618 Circular
Amynthaslongisiphonus KM199289 15,176 491 Circular
Amynthasmoniliatus KT429020 15,133 562 Circular
Amynthaspectiniferus KT429018 15,188 618 Circular
Amynthasredactus KT429010 15,131 572 Circular
Amynthasrobustus KT429019 15,013 432 Circular
Amynthasrongshuiensis KT429014 15,086 546 Circular
Amynthasspatiosus KT429013 15,152 595 Circular
Amynthastriastriatus KT429016 15,160 582 Circular
Amynthasyunoshimensis LC573969 15,109 581 Circular
Metaphirecalifornica KP688581 15,147 567 Circular
Metaphireguillelmi KT429017 15,174 594 Circular
Metaphirehilgendorfi LC573968 15,186 649 Circular
Metaphirevulgaris KJ137279 15,061 484 Circular
Duplodicodrilusschmardae KT429015 15,156 595 Circular
Perionyxexcavatus EF494507 15,083 504 Circular
Tonoscolexbirmanicus KF425518 15,170 595 Circular
Aporrectodearosea MK573632 15,086 512 Circular
Lumbricusrubellus MN102127 15,464 433 Circular
Lumbricusterrestris U24570 14,998 384 Circular
**Eiseniabalatonica MK642872 14,589 - Linear
**Eisenianana MK618511 14,599 - Linear
**Eisenianordenskioldi MK618509 14,572 - Linear
**Eisenianordenskioldi MK618510 14,592 - Linear
**Eisenianordenskioldi MK618513 14,567 - Linear
**Eisenianordenskioldi MK642867 14,576 - Linear
**Eisenianordenskioldi MK642868 14,556 - Linear
**Eisenianordenskioldipallida MK618512 14,567 - Linear
**Eisenianordenskioldipallida MK642869 14,553 - Linear
**Eiseniaspelaea MK642870 14,738 - Linear
**Eiseniatracta MK642871 14,589 - Linear
Eiseniaandrei OK513069 15,714 1151 Circular
Eiseniafetida OK513070 16,560 1988 Circular
Drawidajaponica KM199288 14,648 3 Circular
Pontoscolexcorethrurus KT988053 14,835 318 Circular
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*Putative non-coding region between trnR and trnH.

** Incomplete mitochondrial genome sequence lacking the entire non-coding region and trnR.

Phylogenetic analyses

To clarify the phylogenetic position of the two species, the available complete or near-complete mitogenomes were obtained from GenBank, comprising 24 species of Megascolecidae, 14 species of Lumbricidae, and one species of Rhinodrilidae. Drawidajaponica (Michaelsen, 1892) from the exquisiclitellate family Moniligastridae was used as the outgroup.

Two sets of sequence matrices were composed: one containing the PCGs, 12S, and 16S RNA genes, and the other consisting only of PCGs. Sequences were aligned with MAFFT ver. 7 (Katoh and Standley 2013) using the G-INS-i option and concatenated in MegaX (Kumar et al. 2018); the resulting matrices were 13,505 and 11,241 bp, respectively. The protein-coding alignment was translated into amino acid sequences and aligned in MAFFT ver. 7 using the G-INS-i option; the resulting matrix was with 3714 amino acid positions.

The best-fitting evolutionary model for each partition (PCG, 16S, 12S) was selected using ModelFinder (Kalyaanamoorthy et al. 2017) implemented in the IQTree web server (http://iqtree.cibiv.univie.ac.at/) by applying the Akaike information criterion (AIC; Akaike 1973) and Bayesian information criterion (BIC; Schwarz 1978). GTR + I + Γ was selected as the best-fitting evolutionary model for PCGs and 12S RNA, TIM2 I + Γ was selected for 16S RNA, and MtMAM I + Γ for the amino acid sequences.

Bayesian inference of the phylogeny was estimated with MrBayes v.3.2.6 (Ronquist et al. 2012) as implemented in CIPRES Science Gateway V. 3.3. (Miller et al. 2010). The analysis was performed with default parameters, and each of the two independent runs was set to 10 million generations and sampling every 1000th generation (10,000 trees). Twenty percent of the trees were discarded as burn-in, and the remaining trees were combined and summarized in a 50% majority-rule consensus tree. As the TIM2 model was not implemented in MrBayes, the closest complex model GTR + I + Γ was used instead. Maximum likelihood phylogenetic inference was performed using the IQTree web server with default options (Nguyen et al. 2015http://iqtree.cibiv.univie.ac.at/).

Results

The complete mitochondrial genomes of Eiseniafetida and Eiseniaandrei consisted of 16,560 and 15,714 base pairs, respectively. The setup of the mitogenomes of both species followed the typical Bauplan of the earthworm mitogenome assembly, consisting of 13 PCGs, 22 transfer RNAs, two ribosomal RNA genes, and a control region (Fig. 1; Table 2).

Figure 1.

Figure 1.

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Comparison of mitogenomes of Eiseniaandrei and E.fetida. The map is based on sequence similarity and was constructed using Geneious Prime 2021 software. Sequence similarity is represented by green (100%), brown (30–99%), and red (<30%). cox1 was used as an anchor to linearized genomes. Organization of mitochondrial genes is shown in Table 2. Non-coding region is defined as the region between trnR and trnH.

Table 2.

Comparative analysis of gene organization of Eiseniaandrei and E.fetida mitogenomes (bp = base pairs).

Gene Strand E.fetida E.andrei Similarity
Size (bp) start/stop codon Size (bp) start/stop codon
cox1 + 1540 ATG/T 1540 ATG/T 86%
trnN + 61 61 98%
cox2 + 687 ATG/TAG 687 ATG/TAA 86%
trnD + 61 61 89%
atp8 + 163 ATG/T 160 ATG/T 78%
trnY + 63 63 95%
trnG + 63 64 92%
cox3 + 778 ATG/T 778 ATG/T 86%
trnQ + 69 69 91%
nad6 + 469 ATG/T 469 ATG/T 85%
cytb + 1140 ATG/TAA 1140 ATG/TAA 85%
trnW + 62 63 90%
atp6 + 696 ATG/TAA 696 ATG/TAA 82%
trnR + 61 63 93%
*NC + 1988 1151 60%
trnH + 62 62 90%
nad5 + 1722 ATG/TAA 1722 ATG/TAA 83%
trnF + 62 63 92%
trnE + 63 63 95%
trnP + 64 64 94%
trnT + 65 63 - 97%
nad4L + 297 ATG/TAA 297 ATG/TAA 88%
nad4 + 1359 ATG/TAG 1359 ATG/TAG 83%
trnC + 65 65 97%
trnM + 63 63 100%
rrnS + 794 794 94%
trnV + 64 63 94%
rrnL + 1282 1278 89%
trnL + 62 63 94%
trnA + 62 62 94%
trnS + 67 67 94%
trnL + 64 62 95%
nad1 + 919 ATG/T 919 ATG/T 85%
trnI + 64 64 97%
trnK + 65 65 97%
nad3 + 354 ATG/TAG 354 ATG/TAG 82%
trnS + 64 64 97%
nad2 + 1003 ATG/T 1003 ATG/T 81%
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* Non-coding regions between trnR and trnH.

All genes were encoded on the heavy DNA strand, and both genomes showed biased base composition, with 63.5% AT and 36.4% GC content in E.fetida and 62.8% and 37.2% in E.andrei.

The overall mitogenome sequence similarity between the two species was 80.8%, and it increased to 85.8% when the control region was excluded. The 13 PCGs were 78%–86% similar (Table 2). Among the PCGs, nad4l showed the highest similarity (88%) and atp8 the lowest (78%). The average similarity of the 13 PCGs between the two species was 84%.

However, the deduced amino acid sequences of the 13 PCGs showed, on average, 92.7% similarity between the species; COX1 was the most similar (99.4%) and ATP8 the most dissimilar (79.6%) (Table 3). Sequence variation between the two species was lower at the amino acid level than at the DNA level. In particular, cox1 showed 86% similarity at the DNA level but more than 99% similarity at the amino acid level.

Table 3.

Comparison of deduced amino acid sequences of 13 protein-coding genes between Eiseniaandrei and E.fetida.

Protein Eiseniafetida Eiseniaandrei Similarity (%)
cox1 513 aa 513 aa 99.4
cox2 228 aa 228 aa 95.2
atp8 54 aa 53 aa 79.6
cox3 259 aa 259 aa 97.7
nad6 156 aa 156 aa 93.6
cytb 379 aa 379 aa 96.0
atp6 231 aa 231 aa 93.1
nad5 567 aa 573 aa 90.8
nad4l 98 aa 98 aa 92.9
nad4 452 aa 452 aa 92.0
nad1 306 aa 306 aa 92.6
nad3 117 aa 117 aa 92.3
nad2 334 aa 334 aa 89.1
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Phylogenetic reconstruction of the available Lumbricidae complete or nearly complete mitogenomes using the 13 PCGs and the 12S and 16S RNA genes highly supported the Lumbricidae family (1 posterior probability and 100% bootstrap support). In addition, the genus Eisenia was resolved monophyletic, and the close relationship of the E.fetida/andrei species pairs was confirmed (Fig. 2). Interestingly, the included Eisenia sequences formed two well-supported subclades: one consisting of the European E.spelaea (Rosa, 1901) and the E.fetida/andrei species pair, and the other comprising the Asian taxa of the E.nordenskioldi (Eisen, 1879) species complex (including E.tracta Perel, 1985 and E.nana Perel, 1985), and the Asian specimens of the European E.balatonica (Pop, 1943). A nearly identical tree topology was obtained using the translated amino acid sequences. The only notable difference was in the swapped position of Perionyxexcavatus Perrier, 1872 and Tonoscolexbirmanicus within the Megascolecidaeae clade (Fig. 3).

Figure 2.

Figure 2.

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Phylogenetic analysis of 42 Megadrili species, including E.andrei and E.fetida, based on nucleotide sequences of 13 protein-coding genes and the 12S and 16S RNA genes. The numbers above branches present Bayesian posterior probabilities/maximum likelihood bootstrap values (values under 0.75 and 75% are not shown).

Figure 3.

Figure 3.

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Phylogenetic analysis of 42 Megadrili species, including E.andrei and E.fetida, based on translated amino acid of 13 protein-coding genes. The numbers above branches are Bayesian posterior probabilities/maximum likelihood bootstrap values (values under 0.75 and 75% are not shown).

Discussion

The mitogenomes of E.fetida and E.andrei show the same setup as other lumbricid mitogenomes (Boore and Brown 1995; Shekhovtsov and Peltek 2019; Zhang et al. 2019; Shekhovtsov et al. 2020). The nucleotide composition of the mitogenomes was also similar to that of other Lumbricidae species: the AT content of E.fetida and E.andrei (63.5% and 62.8%, respectively) was comparable to that in Lumbricidae species (59.88–65.69%), including Eisenianordenskioldi, E.balatonica, E.tracta, E.spelaea, Lumbricusterrestris Linneaus, 1758, and Aporrectodearosea (Savigy, 1826) (Shekhovtsov et al. 2020). Zhang et al. (2016) reported higher AT contents in other earthworm families; for example, Megascolecidae has an AT content of 62.6–67.6%, and the Moniligastridae (Drawidajaponica) genome has an AT content as high as 69.7%. However, the mitogenomes of E.fetida (16,560 bp) and E.andrei (15,714 bp) were larger than those of other lumbricid species, such as L.terrestris (14,998 bp), L.rubellus Hoffmeister, 1845 (15,464 bp), and Ap.rosea (15,089 bp). These size differences are primarily due to the extreme length variation of the non-coding region (Shekhovtsov et al. 2020). The length of the non-coding region was 1988 bp in E.fetida and 1152 bp in E.andrei and significantly longer than those of known mitogenomes of other earthworm species [from 318 bp in Pontoscolexcorethrurus (Müller, 1857) to 649 bp in Metaphirehilgendorfi (Michaelsen, 1892); Table 1]. In addition, slight differences were observed in the coding regions. The atp8 gene consists of 54 amino acids in fetida and 53 amino acids in andrei, whereas nad5 comprises 567 amino acids in fetida and 573 amino acids in andrei.

The family Lumbricidae is well-known for its notoriously polyphyletic genera (Domínguez et al. 2015). Unfortunately, only 16 complete or nearly complete Lumbricidae mitogenomes are available in GenBank (including our two new sequences), which prevents us from reaching a comprehensive conclusion on Lumbricidae phylogeny. However, our phylogenetic reconstructions using the available complete or nearly complete mitogenomes corroborated the monophyly of the family Lumbricidae and Eisenia (Domínguez et al. 2015; Shekhovtsov et al. 2020), the genus with the most mitogenome sequences (13 sequences) reported, including the type species Eiseniafetida. It is interesting to note that the E.fetida and E.andrei clade along with the Central European E.spelaea is distant from the Asian E.nordenskioldi species complex, E.tracta, and E.nana.

Perel (1998) hypothesized that the native range of E.fetida is somewhere in the forest-steppe zone of Central Asia, and that the species originally occurred under the bark of fallen logs. In addition, Latif et al. (2017) found surprisingly high morphological and genetic variability of E.andrei in northwestern Iran, which demonstrates that the native range of both species is somewhere in western Central Asia. This could explain their closer affinity to the Central European E.spelaea than to the Siberian–Far Eastern E.nordenskioldi species group.

Eiseniafetida and E.andrei are sister taxa in both tree topologies (Figs 2, 3), and the branch length between E.fetida and E.andrei is similar to those of other species on the trees. This supports their distinct species status. However, considering the genetic p-distances of the studied mitogenomes (Table 4), the E.fetida/E.andrei species pair showed the second smallest genetic distance (14.1%), whereas the p-distance between L.rubellus and L.terrestris was 18.9% or even larger between the two closely related species E.nana and E.tracta (19.2%).

Table 4.

Genetic p-distances of the Lumbricidae mitogenomes.

Lumbricusterrestris (U24570)
Lumbricusrubellus (MN102127) 0.189
Aporrectodearosea (NC046733) 0.238 0.231
Eiseniafetida (OK513070) 0.245 0.244 0.223
Eiseniaandrei (OK513069) 0.245 0.243 0.217 0.141
Eisenianana (MK618511) 0.251 0.246 0.231 0.222 0.224
Eiseniatracta (MK642871) 0.245 0.238 0.221 0.209 0.212 0.192
Eisenianordenskioldi (MK618509) 0.260 0.255 0.238 0.233 0.236 0.205 0.204
Eisenianordenskioldi (K618513) 0.246 0.241 0.221 0.213 0.216 0.202 0.179 0.212
Eisenianordenskioldi (MK618510) 0.246 0.24 0.221 0.216 0.217 0.202 0.178 0.213 0.138
Eisenianordenskioldi (MK642867) 0.252 0.249 0.229 0.224 0.225 0.194 0.187 0.206 0.198 0.199
Eisenianordenskioldi (MK642868) 0.257 0.251 0.234 0.226 0.227 0.199 0.196 0.211 0.204 0.199 0.194
Eisenianordenskioldi (MK642869) 0.25 0.245 0.232 0.221 0.22 0.195 0.187 0.204 0.197 0.193 0.191 0.170
Eisenianordenskioldi (MK618512) 0.258 0.25 0.232 0.228 0.232 0.205 0.196 0.218 0.201 0.202 0.196 0.200 0.199
Eiseniabalatonica (MK642872) 0.252 0.248 0.228 0.225 0.224 0.217 0.2 0.225 0.206 0.206 0.209 0.219 0.214 0.220
Eiseniaspelaea (MK642870) 0.264 0.261 0.252 0.221 0.218 0.25 0.245 0.257 0.243 0.242 0.249 0.252 0.251 0.255 0.250
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Conclusion

On the basis of the mitogenomic analysis of E.fetida and E.andrei, we can conclude that, although the reproductive isolation between the two taxa is not complete, they should be considered as two independently evolving phylogenetic lineages and, consequently, two separate species.

It is clear that mitogenomes, owing to their highly conserved and highly variable regions, are useful in understanding earthworm systematics at the species and genus/family levels. Addition of other species in future analyses will help to further elucidate the phylogenetic relationships within earthworm families.

Acknowledgements

This study was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2019R1I1A3A01060987).

Citation

Csuzdi C, Koo J, Hong Y (2022) The complete mitochondrial DNA sequences of two sibling species of lumbricid earthworms, Eisenia fetida (Savigny, 1826) and Eisenia andrei (Bouché, 1972) (Annelida, Crassiclitellata): comparison of mitogenomes and phylogenetic positioning. ZooKeys 1097: 167–181. https://doi.org/10.3897/zookeys.1097.80216

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