Research Article |
Corresponding author: Nan Song ( songnan@henau.edu.cn ) Academic editor: Martin Haase
© 2023 Te Zhao, Nan Song, Xingyu Lin, Yang Zhang.
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:
Zhao T, Song N, Lin X, Zhang Y (2023) Complete mitochondrial genomes of the slugs Deroceras laeve (Agriolimacidae) and Ambigolimax valentianus (Limacidae) provide insights into the phylogeny of Stylommatophora (Mollusca, Gastropoda). ZooKeys 1173: 43-59. https://doi.org/10.3897/zookeys.1173.102786
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In this study, we sequenced two complete mitogenomes from Deroceras laeve and Ambigolimax valentianus. The mitogenome of Ambigolimax valentianus represented the first such data from the family Limacidae. The lengths of the mitogenomes of Deroceras laeve and Ambigolimax valentianus were 14,773 bp and 15,195 bp, respectively. The entire set of 37 mitochondrial genes were identified for both mitogenomes. Compared with the mitogenome of Achatina fulica, the trnP_trnA tRNA cluster was rearranged in both Deroceras laeve and Ambigolimax valentianus. The secondary structures of tRNA and rRNA genes for the two species were predicted. Phylogenetic analyses based on amino acid sequences supported (1) monophyly of Stylommatophora, (2) division of Stylommatophora into the ‘achatinoid’ clade (i.e., the suborder Achatinina) and the ‘non-achatinoid’ clade (i.e., the suborder Helicina), (3) placement of the Orthurethra in the ‘non-achatinoid’ clade, and (4) monophyly of each of the superfamilies Helicoidea, Urocoptoidea, Succineoidea, Arionoidea, Pupilloidea and Limacoidea. The exemplars of Helicidae, Philomycidae and Achatinellidae displayed many more mitochondrial gene rearrangements than other species of Stylommatophora.
Gene rearrangement, Limacoidea, mitogenome, next generation sequencing, phylogeny
Typically, the metazoan mitochondrial genome (mitogenome) is a closed-circular and small (15–20 kb) genome encoding 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs) and two ribosomal RNAs (rRNAs) (
The Stylommatophora is the largest group within the pulmonate gastropods, containing 20,000 ± 1500 species (
Limacoidea is a superfamily of Stylommatophora that is subdivided into four families: Agriolimacidae, Limacidae, Boettgerillidae and Vitrinidae (
Specimens of Deroceras laeve and Ambigolimax valentianus were collected from Zunyi, Guizhou Province, China, in July, 2020. They were identified by checking their adult morphological characters and blasting the mitochondrial cox1 gene sequences in the BOLD system. The voucher specimens were deposited at the Henan Agricultural University, Zhengzhou, China, under the accession numbers MT-Zy20200701 and MT-Zy20200702. The specimens were preserved in absolute ethanol, and stored at -80 °C until DNA extraction. Total genomic DNA of the individual specimen was extracted with the TIANamp Genomic DNA Kit (TIANGEN BIOTECH CO., LTD), following the manufacturer’s protocol.
Genome sequencing was performed on an Illumina HiSeq2500 platform, with a strategy of 150 paired-end sequencing. Library generation for the Illumina Hiseq sequencing was carried out using the Illumina TruSeqTM DNA Sample Prep Kit (Illumina, San Diego, CA, USA), with 350 bp insert size. NGS QC Toolkit v.2.3.3 (
GetOrganelle v.1.7.5.2 (
Pairwise comparisons of gene order with the gene order of Achatina fulica (
Protein-coding genes were aligned individually using MUSCLE as implemented in MEGA 11 (with default settings) (
A total of 68 mollusk mitogenome sequences were used in the phylogenetic analyses, of which 59 species were included as ingroup to represent Stylommatophora. Four species from Systellommatophora, two species from Ellobiida and three species from Hygrophila were selected as outgroups (Suppl. material
The entire mitochondrial genomes of Deroceras laeve and Ambigolimax valentianus were 14,773 base pairs (bp) and 15,195 bp long, respectively. They contained the entire set of 37 genes usually present in the animal mitogenomes and had identical gene orders (Fig.
For the protein-coding genes, ATG (five for Deroceras laeve and four for Ambigolimax valentianus), ATT (six for Deroceras laeve and three for Ambigolimax valentianus) and ATA (one for Ambigolimax valentianus) were used as the start codons. For the cox2 gene and atp8 gene of Ambigolimax valentianus, GTG was the start codon. For the cox1 gene and cob gene of both Ambigolimax valentianus and Deroceras laeve, TTG was the start codon. All protein-coding genes terminated with the stop codon TAA or TAG, except for nad3 and nad4L of Ambigolimax valentianus, which had the incomplete stop codon T. For both species, Leu, Ile, Phe and Val were the most frequently used amino acids. Relative synonymous codon usage (RSCU) for 13 protein-coding genes of Deroceras laeve is shown in Fig.
All 22 tRNA genes were identified by both MITOS and ARWEN, and their lengths ranged from 61 to 68 bp. Most tRNA genes of both species can be folded into the classic clover-leaf structure (Fig.
ML and BI produced similar tree topologies. The monophyly of Stylommatophora was supported under both analyses (Figs
ML phylogenetic tree inferred with IQ-TREE using amino acid sequences of 13 PCGs. Numbers at the nodes are ultrafast bootstrap values (BS > 70). Blue indicates the newly sequenced species. The branch of Cepaea nemoralis is depicted as half of its original branch length. Scale bar represents substitutions/site.
Bayesian phylogenetic tree inferred in MrBayes using amino acid sequences of 13 PCGs (left), and gene order comparisons for the stylommatophoran mitogenomes (right). Numbers at the nodes in the tree are Bayesian posterior probabilities (PP > 0.9). Blue indicates the newly sequenced species. The branch of Cepaea nemoralis is depicted as half of its original branch length. Scale bar represents substitutions/site. In the mitogenome structure maps, gene rearrangements are highlighted by red.
The major differences between the ML and BI analyses were the positions of Xanthonychidae and the clade Orthalicoidea + Punctoidea. The ML analysis placed Xanthonychidae as sister to all other Helicoidea. Whereas, the BI analysis recovered Xanthonychidae as the sister group of a clade Geomitridae + Helicidae. The ML analysis placed Orthalicoidea + Punctoidea between a clade comprising (Clausilioidea + Pupilloidea) and Arionoidea comprising (Philomycidae + Arionidae). But this arrangement received no statistical support. The BI analysis recovered Orthalicoidea + Punctoidea between the clade (Clausilioidea + Pupilloidea) and a clade including Limacoidea, Helicarionoidea, Trochomorphoidea and Punctidae.
Large-scale changes in genomes are considered to be rare events (
Within Stylommatophora, the division of the order into Achatinina and Helicina is well accepted (
The present mitogenome data recovered Clausilioidea as a sister group of the orthurethran clade. This result contrasted with
The superfamily Punctoidea included the families Punctidae, Charopidae, Cystopeltidae, Discidae (“Endodontidae”), Helicodiscidae, Oopeltidae and Oreohelicidae (
Previous analyses based on the multiple gene fragments have demonstrated the monophyly of Helicoidea, which comprised the families Helicidae, Bradybaenidae, Xanthonychidae, Hygromiidae, Camaenidae, Polygyridae and Sagdidae (
For the taxon sampling of outgroups, we included four exemplars from Systellommatophora and two from Ellobiida. The monophyly of Systellommatophora and Ellobiida were consistently supported in both ML and BI analyses. Moreover, Systellommatophora and Ellobiida formed a sister-group relationship (BS = 100, PP = 1). Amphipulmonata Schrödl, 2014 was established as a clade containing Systellommatophora and Ellobiida (
The authors have declared that no competing interests exist.
No ethical statement was reported.
This study was funded by the National Natural Science Foundation of China (U1904104) and Zunyi Branch of Guizhou Tobacco Company Key Research Project (2020XM01). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
N.S. and T.Z. designed the research; N.S., X.L., Y.Z. and T.Z. performed the research and analyzed the data; N.S., T.Z. and X.L. wrote the paper. All authors have read and agreed to the published version of the manuscript.
Taxa included in this study, including GenBank Accession numbers and literature references
Data type: table (docx. file)
Relative synonymous codon usage (RSCU) for 13 protein-coding genes of Ambigolimax valentianus
Data type: figure (PDF file)
The secondary structures of tRNA genes inferred for the mitogenome of Ambigolimax valentianus
Data type: figure (PDF file)
The secondary structure of rrnL inferred for Ambigolimax valentianus
Data type: figure (PDF file)
The secondary structure of rrnS inferred for Ambigolimax valentianus
Data type: figure (PDF file)