2urn:lsid:arphahub.com:pub:45048D35-BB1D-5CE8-9668-537E44BD4C7Eurn:lsid:zoobank.org:pub:91BD42D4-90F1-4B45-9350-EEF175B1727AZooKeysZK1313-29891313-2970Pensoft Publishers10.3897/zookeys.1097.8021680216Research ArticleAnnelidaClitellataLumbricidaeOpisthoporaMolecular systematicsPhylogenySystematicsTaxonomyCenozoicNeogeneEuropeThe 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 positioningCsuzdiCsabahttps://orcid.org/0000-0002-0319-78361InvestigationWriting - original draftWriting - review and editingKooJachoon2Data curationFormal analysisWriting - review and editingHongYonggeoworm@hanmail.nethttps://orcid.org/0000-0002-8093-97172ConceptualizationFunding acquisitionSupervisionWriting - original draftWriting - review and editingDepartment of Zoology, Eszterházy Károly Catholic University, Eger, HungaryDivision of Science Education and Institute of Fusion Science, College of Education, Jeonbuk National University, Jeonju 54896, Republic of KoreaDepartment of Agricultural Biology, College of Agriculture & Life Science, Jeonbuk National University, Jeonju 54896, Republic of Korea
Corresponding author: Yong Hong (yonghong@jbnu.ac.kr)
Academic editor: Sergei Subbotin
2022290420221097167181D1E40BF9-CFC9-5D3D-86B4-A4CDDA35DFE1C43A88B7-0CF9-46A1-8F5E-51B35E43C48865089070701202203042022Csaba Csuzdi, Jachoon Koo, Yong HongThis 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.http://zoobank.org/C43A88B7-0CF9-46A1-8F5E-51B35E43C488
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.
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
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 methodsSample 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.
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
*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).
Comparative analysis of gene organization of Eiseniaandrei and E.fetida mitogenomes (bp = base pairs).
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.
https://binary.pensoft.net/fig/678335
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.
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
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).
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).
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).
https://binary.pensoft.net/fig/678337Discussion
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%).
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
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).
ReferencesAkaikeH (1973) Information theory and an extension of the maximum likelihood principle. In: PetrowBNCsákiF (Eds) Proceedings of the 2nd International Symposium on Information., 267–281.AndréF (1963) Contribution à l’analyse expérimentale de la reproduction des lombriciens.97: 3–101.AvelM (1937) Delmas, Bordeaux, 76 pp.BankevichANurkSAntipovDGurevichAADvorkinMKulikovASLesinVMNikolenkoSIPhamSPrjibelskiADPyshkinAVSirotkinAVVyahhiNTeslerGAlekseyevMAPevznerPA (2012) Spades: A new genome assembly algorithm and its applications to single-cell sequencing.19(5): 455–477. https://doi.org/10.1089/cmb.2012.0021BlakemoreRJ (2008) VermEcology, Yokohama, 757 pp.BlakemoreRJ (2013) Earthworms newly from Mongolia (Oligochaeta, Lumbricidae, Eisenia).285: 1–21. https://doi.org/10.3897/zookeys.285.4502BolgerAMLohseMUsadelB (2014) Trimmomatic: A flexible trimmer for Illumina sequence data.30(15): 2114–2120. https://doi.org/10.1093/bioinformatics/btu170BooreJLBrownWM (1995) Complete sequence of the mitochondrial DNA of the annelid worm Lumbricusterrestris. Genetics 138: 423–433. https://doi.org/10.1093/genetics/138.2.423BouchéMB (1972) Numéro hors-série, Paris, 671 pp.ChangCHJamesSW (2011) A critique of earthworm molecular phylogenetics. Pedobiologia 54S: 3–9. https://doi.org/10.1016/j.pedobi.2011.07.015CsuzdiCsZicsiA (2003) Hungarian Natural History Museum, Budapest, 273 pp.DomínguezJVelandoAFerreiroA (2005) Are Eiseniafetida and Eiseniaandrei (Oligochaeta, Lumbricidae) different biological species? Pedobiologia 49(1): 81–87. https://doi.org/10.1016/j.pedobi.2004.08.005DomínguezJAiraMBreinholtJWStojanovićMJamesSWPérez-LosadaM (2015) Underground evolution: New roots for the old tree of lumbricid earthworms.83: 7–19. https://doi.org/10.1016/j.ympev.2014.10.024DonathAJühlingFAl-ArabMBernhartSHReinhardtFStadlerPFMiddendorfMBerntM (2019) Improved annotation of protein-coding genes boundaries in metazoan mitochondrial genomes.47(20): 10543–10552. https://doi.org/10.1093/nar/gkz833EastonEG (1983) A guide to the valid names of Lumbricidae (Oligochaeta). In: SatchellJE (Ed.) Earthworm Ecology - From Darwin to Vermiculture., 475–487. https://doi.org/10.1007/978-94-009-5965-1_41EisenG (1873) On Skandinaviens Lumbricider.30: 43–56. https://archive.org/details/biostor-135527EisenG (1879) On the Oligochaeta collected during the Swedish Expeditions to the Arctic regions in the years 1870, 1875 and 1876.15(7): 1–49.JaenikeJ (1982) “Eiseniafoetida” is two biological species.4: 6–8.KalyaanamoorthySMinhBQWongTKFvon HaeselerAJermiinLS (2017) ModelFinder: Fast model selection for accurate phylogenetic estimates.14(6): 587–589. https://doi.org/10.1038/nmeth.4285KatohKStandleyDM (2013) MAFFT multiple sequence alignment software version 7: Improvements in performance and usability.30(4): 772–780. https://doi.org/10.1093/molbev/mst010KumarSStecherGLiMKnyazCTamuraK (2018) MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms.35(6): 1547–1549. https://doi.org/10.1093/molbev/msy096LatifRMalekMCsuzdiCs (2017) When morphology and DNA are discordant: Integrated taxonomic studies on the Eiseniafetida/andrei complex from different parts of Iran (Annelida, Clitellata: Megadrili).81: 55–63. https://doi.org/10.1016/j.ejsobi.2017.06.007LehmitzRRömbkeJJänschSKrückSBeylichAGraefeU (2014) Checklist of earthworms (Oligochaeta: Lumbricidae) from Germany.3866(2): 221–245. https://doi.org/10.11646/zootaxa.3866.2.3LiuHXuNZhangQWangGXuHRuanH (2020) Characterization of the complete mitochondrial genome of Drawidagisti (Metagynophora, Moniligastridae) and comparison with other Metagynophora species.112(5): 3056–3064. https://doi.org/10.1016/j.ygeno.2020.05.020MalmAW (1877) Om daggmaskar, Lumbricina.1: 34–47.MartinPMartinez-AnsemilEPinderATimmTWetzelMJ (2016) World checklist of freshwater Oligochaeta species. [Available online at] http://fada.biodiversity.be/group/show/12 [accessed 09 October 2021]MillerMAPfeifferWSchwartzT (2010) Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In: 2010 Gateway Computing Environments Workshop (GCE), 1–8. https://doi: 10.1109/GCE.2010.5676129NguyenLTSchmidtHAvon HaeselerAMinhBQ (2015) IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies.32(1): 268–274. https://doi/org/10.1093/molbev/msu300PerelTS (1985) Specific Features of the Earthworm Fauna (Oligochaeta, Lumbricidae) in Altai Refugia of Nemoral Vegetation.283(3): 752–756. [In Russian]PerelTS (1998) Nauka, Moscow, 97 pp. [in Russian]PopV (1943) Hazai és külföldi lumbricidák a Magyar Nemzeti Múzeumban.34: 12–24.QiuJPBouchéMB (1998) Revision des taxons supraspecifiques de Lumbricoidea.3: 179–216.ReineckeAJViljoenSA (1991) A comparison of the biology of Eiseniafetida and Eiseniaandrei (Oligochaeta).11(4): 295–300. https://doi.org/10.1007/BF00335851ReynoldsJW (1977) Royal Ontario Museum, Toronto, 141 pp.RonquistFTeslenkoMvan der MarkPAyresDLDarlingAHöhnaSLargetBLiuLSuchardMAHuelsenbeckJP (2012) MrBayes 3.2: efficient bayesian phylogentic inference and model choice across a large model space.61(3): 539–542. https://doi.org/10.1093/sysbio/sys029RömbkeJAiraMBackeljauTBreugelmansKDominguezJFunkeEGrafNHajibabaeiMPerez-LosadaMPortoPGSchmelzRMViernaJVizcainoAPfenningerM (2016) DNA barcoding of earthworms (Eiseniafetida/andrei complex) from 28 ecotoxicological test laboratories.104: 3–11. https://doi.org/10.1016/j.apsoil.2015.02.010RosaD (1901) Un lombrico cavernicolo.4: 36–39. http://www.morebooks.unimore.it/site/home/la-produzione-scientifica/documento610066458.htmlSavignyJC (1826) Analyse d’un memoire sur les Lumbricus par Cuvier.5: 176–184.SchwarzG (1978) Estimating the dimension of a model.6(2): 461–464. https://doi.org/10.1214/aos/1176344136ShekhovtsovSPeltekSE (2019) The complete mitochondrial genome of Aporrectodearosea (Annelida: Lumbricidae). Mitochondrial DNA.4(1): 1752–1753. https://doi.org/10.1080/23802359.2019.1610091ShekhovtsovSVGolovanovaEVErshovNIPoluboyarovaTVBermanDIBulakhovaNASzederjesiTPeltekSE (2020) Phylogeny of the Eisenianordenskioldi complex based on mitochondrial genomes. European Journal of Soil Biology 96: e103137. https://doi.org/10.1016/j.ejsobi.2019.103137SimsRW (1983) The scientific names of earthworms. In: SatchellJE (Ed.) Earthwom Ecology: from Darwin to Vermiculture., 365–373. https://doi.org/10.1007/978-94-009-5965-1_40SimsRWGerardBM (1985) Brill, Leiden, 171 pp.ZhangLSechiPYuanMJiangJDongYQiuJ (2016) Fifteen new earthworm mitogenomes shed new light on phylogeny within the Pheretima complex. Scientific Reports 6(1): e20096. https://doi.org/10.1038/srep20096ZhangQLiuHZhangYRuanH (2019) The complete mitochondrial genome of Lumbricusrubellus (Oligochaeta, Lumbricidae) and its phylogenetic analysis. Mitochondrial DNA.4(2): 2677–2678. https://doi.org/10.1080/23802359.2019.1644242