The mitochondrial genome of Monacha cartusiana is the first complete mitochondrial sequence described for the pulmonate snail genus Monacha and for the family Hygromiidae. The identified mitogenome has a length of 13,894 bp and encodes 13 proteins, 22 tRNAs, and two rRNAs. A phylogenetic analysis of available mitogenomes from representatives of helicoid families shows a sister group relationship of Hygromiidae and Geomitridae, which have been recently recognised as separate families.

Carthusian snail, Helicoidea, mitogenome, phylogeny, Stylommatophora

Mollusca is the second largest animal phylum after Arthropoda in terms of the number of named species, with the class Gastropoda as the most speciose group with approximately 95,000 species (Ponder et al. 2020). Stylommatophoran pulmonates constitute the most species-rich gastropod order with an estimated number of about 30,000 species (Mordan and Wade 2008). Although the monophyly of Stylommatophora within panpulmonate heterobranchs is relatively well established (Jörger et al. 2010; Ponder et al. 2020), the phylogenetic relationships between stylommatophoran families are still debated (White et al. 2011; Gaitán-Espitia et al. 2013; Razkin et al. 2015; Doğan et al. 2020; Ponder et al. 2020).

Mitogenome sequences are of great importance in molecular phylogenetic studies (Moritz et al. 1987), especially to infer evolutionary relationships at species level (Avise et al. 1987); this is also the case within Mollusca (Boore 1999). The analysis of mito­genomes may thus provide additional evidence related to stylommatophoran phylogeny (White et al. 2011; Parmakelis et al. 2013; Minton et al. 2016a). The number of available stylommatophoran mitogenomes has increased in the last two decades, from three at the end of the 20^th century (Hatzoglou et al. 1995; Terrett et al. 1996; Yamazaki et al. 1997) to 35 in recent years (Yang et al. 2019; Doğan et al. 2020). However, considering the number of recognised extant families within the Stylommatophora (117 according to Bouchet et al. 2017), the number of stylommatophoran mitogenomes still is very small and new mitogenomes, especially from families for which no, or very few, mito­genomes are available, are worth publishing. Hitherto, two mitogenomes were available for the Hygromiidae, viz. Cernuella virgata (Da Costa, 1778) and Helicella itala (Linnaeus, 1758), published by Lin et al. (2016) and Romero et al. (2016), respectively. However, these two species have recently been transferred from the Hygromiidae to the Geomitridae (Razkin et al. 2015; Neiber et al. 2017; Bouchet et al. 2017), so that the Hygromiidae, very rich in species, is left without any available mitogenome.

The hygromiid genus Monacha Fitzinger, 1833 is widespread in the western Palae­arctic from western Europe to North Africa, Iran, and Arabia. It includes a large number of nominal species and shows its highest diversity in south-eastern Europe and Turkey (Hausdorf 2000a, 2000b; Welter-Schultes 2012). Although most of the Monacha species occur in rather narrow areas (Welter-Schultes 2012; Neiber and Hausdorf 2017), Monacha cartusiana (O.F. Müller, 1774), the type species of the genus, is widely distributed and can be found in almost the whole of Europe excluding its north-eastern fringes (Scandinavia, Russia, Baltic States, Belarus, northern Ukraine) (Welter-Schultes 2012; Pieńkowska et al. 2018). The mitogenome of this species will facilitate the future identification of species within the genus and the understanding of their phylogenetic relationships, as is the case with other families of terrestrial pulmonate snails (González et al. 2016; Groenenberg et al. 2017; Korábek et al. 2019; Doğan et al. 2020). Hence, in this paper, we present the complete mitogenome of M. cartusiana and analyse its phylogenetic position within the superfamily Helicoidea.

The specimen of Monacha cartusiana used for this research was collected in Ostrowiec Świętokrzyski (Poland) by Mariusz Gwardjan on 03.07.2015. It was identified by the sequence of the cytochrome c oxidase subunit I gene fragment (coI) of M. cartusiana in GenBank (KX258398) deposited by Pieńkowska et al. (2016). Total genomic DNA was extracted following Pieńkowska et al. (2015). The sequencing of the M. cartusiana mitogenome (for gene acronyms see Table 3) was started using four pairs of primers complementary to the conservative regions of coI (Folmer et al. 1994), 16S rRNA (Palumbi et al. 1991), coII (Hugall et al. 2002) and cytb (Merritt et al. 1998), the missing fragments between them were identified by primer walking (Lin et al. 2016). The primers used for the amplification of mtDNA are listed in Table 1.

The mitogenome was annotated using the MITOS Web Server (Bernt et al. 2013). For the phylogenetic analysis we used a concatenated sequence alignment of 12 protein coding genes (PCGs; excluding atp8), and 2 rRNAs (12S rRNA and 16S rRNA). Every set of 14 sequences was separately aligned using CLUSTAL W (Thompson et al. 1994) implemented in BIOEDIT v. 7.0.6 (Hall 1999; BioEdit 2017). The length of the alignment after combining the 14 gene sequences was for each species 14,287 bp. For the phylogenetic analysis we used all mitogenome sequences deposited in GenBank for species of the superfamily Helicoidea (Table 2). The mitogenome of Theba pisana (MH362760) was not annotated, so we designated the individual PCGs and rRNAs by aligning the whole T. pisana sequence with the extracted sequences of species belonging to the family Helicidae. Each of the T. pisana PCGs was tested for start and stop codons with ORF Finder (2004). Mitogenomes of two arionoid species (Arion vulgaris and Meghimatium bilineatum, Table 2) were used as the outgroup.

Phylogenetic analysis was performed using maximum likelihood (ML) as implemented in the online version of IQ-TREE (Trifinopoulos et al. 2016). ML analysis was done using 14 partitions. Best substitution models were inferred according to the Bayesian information criterion (BIC) for each of the partitions by ModelFinder (Kalyaanamoorthy et al. 2017) implemented in IQ-TREE. The TVM+F+I+G4 model was selected for nd1, nd2, nd4, nd5, atp6, and 16S rRNA; TPM3u+F+I+G4 for nd3; K3Pu+F+G4 for nd4l; TPM3+F+I+G4 for nd6; K3Pu+F+I+G4 for cytb, and coII; TIM+F+I+G4 for coI; GTR+F+I+G4 for coIII, and 12S rRNA. ML trees were constructed under 1,000 ultrafast bootstrap replicates (Minh et al. 2013) and with Shimodaira-Hasegawa-like approximate likelihood ratio test with 1,000 replicates (SH-aLRT; Guindon et al. 2010). A Bayesian inference (BI) analysis was performed with MrBayes v. 3.2.6 (Ronquist et al. 2012). Four Monte Carlo Markov chains were run for 1 million generations, sampling every 100 generations (the first 25% of trees were discarded as “burn-in”). Ultrafast bootstrap support, SH-aLRT support (both expressed in percentages) and posterior probability (PP) values obtained on 50% majority rule consensus Bayesian tree were mapped on the ML tree of concatenated sequences. The ML tree was visualized using FigTree v. 1.4.3 (Rambaut 2016).

The complete mitogenome of M. cartusiana was deposited in GenBank under accession number MW485067. With 13,894 bp in length, it was one of the shortest mito­genomes known in Helicoidea, which ranged from 13,798 bp (Camaena poyuensis) to 14,795 bp (Theba pisana) (Table 2). The mitogenome included: 13 PCGs, 22 tRNA genes and two rRNA genes (Fig. 1, Table 3), typical for most metazoan mitogenomes. The base composition of the M. cartusiana mitogenome was: 30.26% A, 37.95% T, 16.94% G and 14.85% C, i.e. with a bias towards A and T (68.21% content of A-T). These values differ from other helicoid species, but fit into the range previously reported for helicoids, especially when compared with the A-T values for C. virgata (65.96%) and H. itala (66.22%) (Doğan et al. 2020: table S3). The total length of all PCGs was 10,404 bp (74.88% of the entire mitogenome), and they had different start and stop codons, which also vary among helicoid mitogenomes (Table 4). Some of the stop codons TAA were generated by posttranscriptional polyadenylation (as in Groenenberg et al. 2012 and Yang et al. 2016). Nine PCGs were encoded in the “plus” direction (nd1, nd2, nd4, nd4l, nd5, nd6, cytb, coI, coII) and four in the “minus” direction (coIII, atp6, atp8, nd3). Furthermore, 14 tRNA and one rRNA were encoded in the “plus” direction and eight tRNA and one rRNA in the “minus” direction (Table 3). Additionally, seven intergenic regions (with noncoding sequences) were identified with a total length of 295 bp (the longest was 70 bp while the shortest 19 bp) (Fig. 1). The gene order in M. cartusiana mitogenome was exactly the same as in C. virgata and H. itala (geomitrid species). Yet, the polygyrid Practicolella mexicana differed in four places and helicid species in seven (Table 5). The species representing the Camaenidae formed three groups with the same order of genes, but each of these groups differed in gene order from species from Hygromiidae, Geomitridae, Helicidae, and Polygyridae (Table 5).

Figure 1. 

Circular diagram of the mitochondrial genome of Monacha cartusiana (GenBank acc. no. MW485067). Genes encoded in the “plus” and the “minus” directions are shown outside and inside the circle, respectively. Particular gene types are marked with different colours: red – PCGs coding I, II, and III subunits of cytochrome c oxidase; green – PCGs coding NADH dehydrogenase family; orange – PCGs coding ATPase family; yellow – sequence coding cytochrome b; purple – tRNAs coding sequences; blue – rRNA coding genes. Noncoding sequences are mapped on a small inner circle. The circular diagram was created with GenomeVx (Conant and Wolfe 2008).

Phylogenetic analyses of the stylommatophoran mitogenomes (González et al. 2016; Romero et al. 2016) showed them in a well-supported clade among Panpulmonata (with PP and bootstrap values 1 and 99, respectively). Previous mitogenome phylogenies of stylommatophoran superfamilies (Groenenberg et al. 2017; Harasewych et al. 2017; Yang et al. 2019; Doğan et al. 2020) showed a clade of Helicoidea separate from other superfamilies, although mitogenomes of only 11 stylommatophoran superfamilies (Yang et al. 2019) out of 26 listed by Bouchet et al. (2017) are represented in GenBank. According to Bouchet et al. (2017), Helicoidea includes 17 families but hitherto phylogenetic relationships could be analysed only for three or four of them, namely Helicidae, Camaenidae, Geomitridae, and Polygyridae (González et al. 2016; Lin et al. 2016; Minton et al. 2016a; Harasewych et al. 2017; Doğan et al. 2020).

For the phylogenetic analysis, a concatenated alignment of 12 PCGs (excluding atp8, because it was too short, too variable, and not annotated in the mitogenome of Cernuella virgata) and 2 rRNAs (12S and 16S) was used. The dataset included 19 helicoid species (Table 2) yielding the ML tree shown in Fig. 2. The Bayesian tree (not shown) had the same topology.

Figure 2. 

Maximum likelihood (ML) tree of mitochondrial genomes of species representing the superfamily Helicoidea (see Table 1). Mitogenome sequences included all PCGs (except atp8) and two rRNA genes were 14,287 positions in length. Ultrafast bootstrap support values (%), SH-aLRT support values (%) and Bayesian posterior probabilities are indicated next to the branches. The tree was rooted with sequences of Arion vulgaris (MN607980) and Meghimatium bilineatum (MG722906) mitogenomes deposited in GenBank by Doğan et al. (2020) and Yang et al. (2019), respectively.

The mitogenome of M. cartusiana allows to add Hygromiidae to the previous analyses of Helicoidea families. It shows up in a clade with mitogenomes of the geomitrid species, Cernuella virgata and Helicella itala, confirming the close relationships of two families, i.e., Hygromiidae and Geomitridae (Razkin et al. 2015). The mitogenome of the helicid Cylindrus obtusus of the subfamily Ariantinae forms a branch separated from the subfamily Helicinae (Fig. 2). This was also noted in previous phylogenetic analyses (Korábek et al. 2019; Doğan et al. 2020). Moreover, Camaenidae are separated into two clades i.e., Bradybaeninae and Camaeninae, treated frequently as two separate families (Lin et al. 2016; Minton et al. 2016a; Harasewych et al. 2017). Our results agree with the division of Helicidae and Camaenidae into subfamilies (Bouchet at al. 2017). However, the five helicid and seven camaenid species (Table 2, Fig. 2) represent only a tiny fraction of these speciose families. Therefore, more helicoid and stylommatophoran mitogenomes are urgently needed.

We are grateful to Ondřej Korábek (Charles University, Prague, Czech Republic) and two anonymous reviewers for their constructive remarks, Thierry Backeljau (Royal Belgian Institute of Natural Sciences, Brussels, Belgium) for his editorial help, Robert A.D. Cameron (University of Sheffield, UK) for revising the English text, and Krzysztof Duda (Adam Mickiewicz University, Poznań) for help in preparing figures for print.