Composting earthworms of the genus Eisenia play an important role in soil ecosystems. However, taxonomic classification of this genus, especially the sibling species Eisenia fetida and Eisenia andrei, 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.

Compost worms, mitogenome, Oligochaeta, phylogeny, sibling species

The earthworm species Eisenia fetida was described as Enterion fetidum 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 Enterion fetidum 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 Eisenia fetida 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 Eisenia fetida var. unicolor. Variety names proposed after 1961 were considered intrasubspecific and invalid; therefore, Bouché (1972) proposed a new name for var. unicolor, Eisenia fetida andrei 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 Eisenia fetida (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.

Eisenia fetida 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 Eisenia fetida / 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 Eisenia andrei and Eisenia fetida 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.

The complete mitochondrial genomes of Eisenia fetida and Eisenia andrei 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. 

Comparison of mitogenomes of Eisenia andrei 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.

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.

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 Perionyx excavatus Perrier, 1872 and Tonoscolex birmanicus within the Megascolecidaeae clade (Fig. 3).

Figure 2. 

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. 

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).

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 Eisenia nordenskioldi, E. balatonica, E. tracta, E. spelaea, Lumbricus terrestris Linneaus, 1758, and Aporrectodea rosea (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 (Drawida japonica) 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 Pontoscolex corethrurus (Müller, 1857) to 649 bp in Metaphire hilgendorfi (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 Eisenia fetida. 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.

Eisenia fetida 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%).

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.

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