The complete mitogenome of Orcula dolium (Draparnaud, 1801); ultra-deep sequencing from a single long-range PCR using the Ion-Torrent PGM
© The Author(s) 2017
Received: 10 January 2017
Accepted: 23 March 2017
Published: 4 April 2017
With the increasing capacity of present-day next-generation sequencers the field of mitogenomics is rapidly changing. Enrichment of the mitochondrial fraction, is no longer necessary for obtaining mitogenomic data. Despite the benefits, shotgun sequencing approaches also have disadvantages. They do not guarantee obtaining the complete mitogenome, generally require larger amounts of input DNA and coverage is low compared to sequencing with enrichment strategies. If the mitogenome could be amplified in a single amplification, additional time and costs for sample preparation might outweigh these disadvantages.
A sequence of the complete mitochondrial genome of the pupilloid landsnail Orcula dolium is presented. The mitogenome was amplified in a single long-range (LR) PCR and sequenced on an Ion Torrent PGM (Life Technologies). The length is 14,063 nt and the average depth of coverage is 1112 X. This is the first published mitogenome for a member of the family Orculidae. It has the typical metazoan makeup of 13 protein coding genes (PCGs), 2 ribosomal RNAs (12S and 16S) and 22 transfer RNAs (tRNAs). Orcula is positioned between Pupilla and the Vertiginidae as the sister-group of Gastrocopta and Vertigo, together. An ancestral gene order reconstruction shows that Orthurethra in contrast to other Stylommatophora, have tRNA-H before tRNA-G and that the gene order in the ‘non-achatinoid’ clade is identical to that of closely related non-stylommatophoran taxa.
We show it is feasible to ultra-deep sequence a mitogenome from a single LR-PCR. This approach is particularly relevant to studies that have low concentrations of input DNA. It results in a more efficient use of NGS capacity (only the targeted fraction is sequenced) and is an effective selection against nuclear mitochondrial inserts (NUMTS). In contrast to previous studies based in particular on 28S, our results indicate that phylogeny reconstructions based on complete mitogenomes might be more suitable to resolve deep relationships within Stylommatophora. Ancestral gene order reconstructions reveal rearrangements that characterize systematic groups.
List of included mitogenomes (accessed 2016-10-17)
Albinaria caerulea (Deshayes, 1835)
Cepaea nemoralis Linnaeus, 1758
Cornu aspersum (Müller, 1774)
Cylindrus obtusus (Draparnaud, 1805)
Succinea putris Linnaeus, 1758
This study was carried out on a specimen of Orcula dolium dolium (RMNH 114169) collected in 2009 in SW Berchtesgaden (Bayern, Germany). DNA was extracted with a Qiagen DNA tissue kit. Total yield of DNA extracted was ~13 ug (DNA conc. 66.7 ng/ul with an elution volume of 200 ul). Using universal barcoding primers  a partial sequence (655 nt) of Cytochrome Oxidase subunit I (COI) was obtained using the procedure described in . This sequence was used to design specimen specific primers (Orcula_529_COI_F 5′-CTAAGACTATTTGTGTGGTCGATCTTA-3′ and Orcula_336_COI_R 5′-TCTAGACCTAATCAAAAGAACAAATGAAG-3′) to amlify the complete mitogenome of O. dolium. An amplicon with a length of 13,871 nt was obtained with GoTaq long PCR Master Mix (Promega) using the manufacturers protocol. Thermocycling profile was 2 m. at 94 °C, followed by 40 cycles of 30 s. at 94 °C, 15 m. at 65 °C, 10 m. at 72 °C. The PCR product was gel purified with the Wizard SV gel and PCR cleanup-system (Promega) and subsequently checked on a BioAnalyzer 2100 using a DNA 12000 chip (Agilent). The Orcula library was part of a combined run in which different samples were pooled. The purified amplicon was enzymatically digested and individual samples were ligated with a unique Ion Express Barcode Adapter (Life Technologies) using the NEBNext Fast DNA & Library Prep Set for Ion Torrent (New England Biolabs), following the manufacturers instructions. After ligation samples were quantified on the Bioanalyser 2100 using a DNA High sensitivity chip (Agilent). An equimolair pool was prepared of the highest possible concentration. This equimolair pool was diluted according to the calculated template dilution factor to target 10–30% of all positive Ion Sphere Particles. Template preparation and enrichment was carried out with the Ion Touch 2 system, using the OT2 400 kit (Life Technologies), according to the manufactures protocol (7218RevA0). The enriched Ion Sphere Particles were prepared for sequencing on a Personal Genome Machine (PGM) with the Ion PGM 400 Sequencing kit as described in the protocol using a 316v2 chip.
Quality check and assembly
Reads from the Orcula library were separated from the pool based on their unique barcode tag by the Ion Torrent Server. Quality was checked using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Reads shorter than 35 nt or with a phred score below 28 were removed with Fastx_trimmer (http://hannonlab.cshl.edu/fastx_toolkit/). A ‘de novo’ assembly was carried out with Spades v.2.5.1.  and reads were mapped against available orthurethran sequences (KC185403-05, KU525108) using Geneious v.7.1.7 . Finally ‘de novo’ contigs larger than 1 kb (for gene order assessment) and mapping assemblies were merged (see Additional file 1: Figure S1) and used as reference for iterative mapping (medium sensitivity, no fine tuning, not trimmed before mapping; other parameters left at default) of the quality checked reads (again using Geneious v.7.1.7). The reason for this two-step approach was that the initial ‘de novo’ assembly did not result in a contig of expected length. To confirm the final assembly (and enclosed gene order) a selection of matching reads (~ten fold downsampling) was analysed with another assembler, MITObim v. 1.8  (using the 655 nt COI sequence as seed bait) and with Spades 2.5.1.  again also.
The position of the protein coding genes (PCGs) was determined by alignment with available stylommatophoran mitogenomes (Table 1) and by locating start and stop codons. The identification of the ribosomal RNAs was done with BLAST searches. Arwen and Mitos  were used to locate the tRNAs and the secondary structures were all generated with Arwen . The contig sequence was annotated in Geneious .
A prerequisite for understanding (mitochondrial) gene rearrangements is a robust, well rooted phylogeny. Representatives of the clades Hygrophila and Eupulmonata were selected as outgroup because these taxa are supposed to be closely related to, but not part of the ingroup [3, 4, 21]. For each of the 13 PCGs alignments were made with TranslatorX ; a program that aligns nucleotide sequences based on their corresponding amino acid translations. Translator X was run with the MAFFT alignment module  and the invertebrate mitochondrial genetic code; other settings were left at default. A supposed ‘copy’ of ND4L  for Naesiotus nux (GenBank accession number NC_028553) was excluded because there are no stylommatophoran homologs known to align it with. Ribosomal genes were individually aligned in Geneious, again using MAFFT  and conserved datablocks were selected with Gblocks  using default settings. The PCG and ribosomal alignments were concatenated in Geneious and exported as a phylip formatted matrix (27 taxa, 13,419 nucleotides). With 41 datablocks specified (each codon position and the ribosomal RNAs), PartitionFinder v.1.1.1  estimated the best partitioning scheme (33 partitions) and nucleotide substitution models. A Bayesian analysis (two simultaneous MCMC runs 10 M generations each) was conducted with MrBayes 3.2.3 hosted on the CIPRES Science Gateway . Inspection of the parameter files with Tracer v.1.5  showed proper mixing of the MCMC (effective sampling size values > 200). The first 2500 trees (25% of each tree-file) were discarded as burnin. Majority rule consensus trees were visualised and edited in FigTree v.1.4.0 . The procedure was repeated with the amino acid alignments (13 datablocks, 4190 amino acids) from TranslatorX . The estimated partitioning scheme now consisted of six partitions and the analysis was carried out with 2.5 M generations to keep computation time tenable. Nucleotide and amino acid datamatrices were analysed separately to see if both would yield phylogenies with similar topologies. Abbreviations: BPP = Bayesian Posterior Probability.
Mitochondrial gene arrangements
An ancestral gene order reconstruction  was done using the Maximum Likelihood Gene Order analysis (MLGO) web server (http://www.geneorder.org/server.php) using the phylogeny obtained in the previous step (outgroup reduced to Biomphalaria) as a fixed tree (ie. Small Parsimony Problem, using SPP option).
For the classification and the nomenclature of the various taxa, the review by Bouchet and Rocroi  served as the primary starting point. Historic classifications such as the Pilsbry-Baker system [31, 32] are not exhaustively discussed. The more recent literature is dealt with only when it contains additional data. Ever since the rise of phylogenetic systematics, the increase of more detailed anatomical analyses and in particular the quickly growing quantity of molecular data, the classification of the gastropods has been in a confusing transitional state. In the monograph of Zilch  for example, published before the rise of phylogenetic systematics had started, the classis Gastropoda is subdivided into the subclasses Prosobranchia and Euthyneura. The latter subclassis contains the ordines Basommatophora and Stylommatophora, whereas the ordo Stylommatophora is further split into the subordines Orthurethra, Heterurethra and Sigmurethra. Many of these names are still in use, but often not for exactly the same group of species. Next to the classical taxonomical categories, new nominal taxa names were more recently introduced, like ‘clade’ and ‘informal group’. Bouchet et al.  for example, in a recent nomenclatorial monograph on gastropod classification and nomenclature, use ‘Informal Group Pulmonata’ as a partial synonym of Zilch’s Euthyneura. The Orthurethra are accepted by  as a ‘Subclade’, which is identical with Zilch’s subordo of that name. There are many more discrepancies, however. For a more recent contribution to this subject, see . Here we do not aim at a summary of the changing and sometimes conflicting views regarding gastropod phylogeny.
Selection of outgroup
Recent studies have shown that the traditionally accepted nominal taxa Pulmonata and Opisthobranchia are not monophyletic, what necessitated a new classification of the Euthyneura . Within that taxon these authors accept Panpulmonata and Eupulmonata, with the Stylommatophora belonging to the Eupulmonata. The Informal Group Basommatophora is assigned to a paraphyletic group Panpulmonata. Next to the selection of markers and taxa, outgroup selection strongly affects our concept of Euthyneuran phylogeny, as is evident from the literature [1, 3, 4, 21, 34–36]. Despite the differences, all these studies show that the clades Hygrophila (Lymnaeoidea & Planorboidea), Systellommatophora (Veronicelloidea) and Eupulmonata (Ellobioidea) are closely related to, but separate from the Stylommatophora (i.e. these are eligible outgroup taxa).
Comparison with existing classifications
The number of Stylommatophora with known mitogenomes is still limited, but representatives of most of the major groups are available now. This allows for a preliminary comparison.
Initially, Orthurethra was considered a ‘primitive’ group of Stylommatophora [31, 40]. Molecular analyses showed however, that the Orthurethra are a more derived taxon [7, 37]. Bouchet and Rocroi  accept five orthurethran superfamilies, one of which is the Pupilloidea. Complete mitogenomic sequences are available only for Achatinelloidea  and for Pupilloidea, i.e. for Pupillidae and Vertiginidae  and now additionally for Orculidae.
The Orthurethra and the Pupilloidea are both monophyletic. The Vertiginidae, represented by Gastrocopta and Vertigo are the sister-group of the Orculidae (Fig. 2). Recently, Gastrocopta (Gastrocoptinae) was excluded from the ‘vertiginid’-clade (Vertiginidae), mainly based on 28S sequence data; that phylogeny  showed two clades: one consisting of Pupilla and Vertigo and another with Orcula and Gastrocopta, albeit poorly supported. These sister-group relationships, as well as the exclusion of Gastrocoptinae from Vertiginidae are rejected by our data (Fig. 2).
In the Orthurethra (and Stylommatophora in general) most mitochondrial gene rearrangements occurred in the region between CytB and ATP8. Except for that region, the gene order of Pupilloidea is identical to that in Albinaria (Fig. 2). In the Achatinelloidea a number of rearrangements was found that are not observed in Pupilloidea (most prominent is the transposition of the region tRNA-F_COII_tRNA-YHG; Fig. 2). Both orthurethran superfamilies have the gene order tRNA-HG (or at least tRNA-H before tRNA-G) in which they deviate from other Stylommatophora (which have tRNA-GH). We hypothesize that the arrangement tRNA-HG is an apomorphic character state for the Orthurethra.
The MLGO result predicted tRNA-YHWG as the ancestral gene arrangement for Orthurethra (requiring additional steps for all orthurethrans except Vertigo). Alternatively tRNA-YHGW would have been equally parsimonious (also nine steps) and is more likely, given that tRNA-YHG is observed in Achatinella, Pupilla and Gastrocopta. Therefore the latter scenario was adopted in Fig. 4. In either reconstruction the shift from tRNA-GH to tRNA-H_before_G must have taken place early in orthurethran history. Additionaly, for the Pupilloidea, tRNA-W was transposed independently in Orcula and Vertigo, tRNA-DC in Pupilla, and tRNA-Q from L-strand to H-strand in Gastrocopta (Figs. 2 and 4).
The mitogenome of Achatinella mustelina is said to be “similar to those of other pulmonates” . We assume that statement refers to gene composition, not gene order, because one of the largest transpositions recorded for Stylommatophora is seen in Achatinella. The latter taxon has transposed tRNA-F_COII_tRNA-YHG (assuming it was not the larger fragement tRNA-WQL2_ATP8-ATP6_tRNA-RE_12S), tRNA-W and tRNA-N (Figs. 2 and 4).
The Heterurethra sensu Pilsbry  or the subclade Elasmognatha according to  when Athoracophoroidea are included next to Succineoidea, were classified in a clade with Acavoidea (Leucotaenius) and Orthalicoidea (Placostylus) [7, 37], albeit poorly supported. Our analyses support (BPP = 0.92/0.97; Fig. 3) a sister-group relation between Elasmognatha (Succinea: Succineoidea) and Orthalicoidea (Naesiotus). In agreement with previous studies [7, 37] our data indicate that Succineoidea and Orthalicoidea together are the sister-group of the combined Urocoptoidea and Helicoidea (Fig. 3). Of the three gene rearrangements within Succinea (Figs. 2 and 4) none is shared with Naesiotus.
A peculiar feature of Naesiotus nux (genbank access.nr. KT821554) is a ‘duplication’ of ND4L (positioned between tRNA-L and tRNA-P; ). Since this ‘copy’ (sequence divergence > 60%) was not present in other Stylommatophora it could not be included in our analyses. It might partly explain the increased size of the mitogenome of N. nux compared to that of other Stylommatophora (Table 1).
Of this nominal taxon the Cerionidae and Clausiliidae, which were included by , are represented. Earlier studies showed the polyphyly of this group [7, 37]. Our phylogeny reconstructions (Fig. 3) also reject the hypothesis of a close relation between Cerionidae and Clausiliidae.
Recently, Urocoptidae and Cerionidae were shown to constitute a monophyletic group (based on 28S sequence data) for which the superfamily Urocoptoidea was introduced . Urocoptoidea and Helicoidea (= Sigmurethra) are sister-groups (BPP = 1.0; Fig. 3). Vaught  classified only the Clausiloidea as Mesurethra.
The Sigmurethra sensu Pilsbry  was already shown to be paraphyletic [7, 37]. Our data reconfirm that Sigmurethra is not monophyletic (Fig. 3). Bouchet and Rocroi  refer to the this nominal taxon as an ‘Informal Group’, which may remain in use as long as no preferential alternative has been advocated.
In our analyses, Achatina fulica is the sister-group of the ‘non-Achatinoid’ clade. Stylommatophora (‘Achatinoid’ and ‘non-Achatinoid’ clades) is a monophyletic group (BPP = 1.0; Fig. 3). The mitogenomic gene arrangement of Achatina differs from that of the other Stylommatophora (Fig. 2), but it is identical to that of the outgroup genera Biomphalaria and Planorbarius, which are classified with the Planorboidea of the clade Hygrophila. Unlike the representatives of the ‘non-Achatinoid clade’, Achatina has the gene order tRNA-LAP (between 16S and ND6), which also occurs in more distantly related outgroups (Pedipes, Myosotella and Rhopalocaulis; Fig. 2). Therefore, using the principle of outgroup comparison, we hypothesize that the ancestral mitochondrial gene order of the Stylommatophora has been identical to the arrangement currently found in Achatina and in some taxa of the clade Hygrophila.
Our data show a strongly supported (BPP = 1.0; Fig. 3) basal split in the ‘non-Achatinoid clade’ with on the one hand Orthurethra (Achatinelloidea, Pupilloidea) + Clausilioidea and on the other hand Elasmognatha (= Heterurethra) + Orthalicoidea + Urocoptoidea + Helicoidea. In the latter clade Elasmognatha + Orthalicoidea is the sister-group of Urocoptoidea + Helicoidea.
Rearrangements of PCGs are thus far only observed in the Helicoidea and Achatinelloidea. Separation of ND6 and ND5 by a tRNA from the L-P-A region and transposition of tRNA-T + COIII might indeed be an apomorphy for the Helicidae, as suggested by ; the recently added mitogenome of Cornu aspersum  confirms this. We hypothesize that the gene arrangement tRNA-GH before tRNA-YW is an apomorphic character state characterizing Bradybaenidae. The transposition of ND3 is not observed in the other bradybaenids and at the moment being, is unique to Aegista. The mitogenomic gene arrangement of Camaenidae might be in between that of an helicoid ancestor and that of Bradybaenidae; tRNA-Y is still at the ancestral position, whereas tRNA-W is already transposed to the ‘bradybaenid’ location (albeit still on the heavy strand). Wang et al. concluded that the mitogenomic gene order of Camaena cicatricosa differs from that in other stylommatophores in the position of COII and the tRNA’s C, F, D, G, H and W . The single transposition of tRNA-D is a more parsimonious explanation for the first four ‘differences’. The latter three could subsequently be explained by a single transposition of tRNA-W after tRNA-GH or the transposition of tRNA-GH (as a single unit) before tRNA-W. Movement of two consecutive tRNA’s as a single unit is not uncommon, as is exemplified by tRNA-YW in Succinea and tRNA-AP in Cerion (Fig. 2).
Camaenidae and Bradybaenidae have been considered confamilial by Scott , whereas both nominal taxa are paraphyletic according to others [7, 50]. According to our analyses, Camaenidae and Bradybaenidae form a clade within the Helicoidea (Fig. 3), leaving no place for a separate superfamily Camaenoidea, which was accepted by [51, 52].
The mitochondrial gene arrangements of Albinaria (Clausilioidea) and Naesiotus (Orthalicoidea) are identical, despite the fact that both taxa belong to different basal groups within the ‘non-Achatinoid’ clade. We hypothesize that this is the ancestral mitogenomic gene order of the ‘non-Achatinoid clade’ and that a rearrangement from tRNA-LAP to tRNA-LPA characterized the most recent common ancestor of this clade.
On the chosen sequence strategy
Amplification of the complete mitochondrial genome in a single LR-PCR with target specific primers for the snail species tested here was straightforward. Nevertheless, other studies show that full mitogenomic amplifications can be stochastic  and the method will not work with degraded material. Alternatively the mitochondrial fraction can be enriched by physical isolation (e.g. centrifugation in CsCl-gradient; requires significant amounts of starting material) or by target capture approaches, in which mitochondrial sequences are isolated by hybridisation to biotinylated probes . Some studies abandon mitochondrial enrichment steps entirely and shotgun sequence (pools of) DNA extracts instead [41, 47, 55–58]. Each of these methods have their own strengths and weaknesses. In studies where multiple extracts are pooled and that do not make use of indexing tags, additional controls are necessary to check against ‘chimeric’ assemblies (especially when closely related taxa are involved). To this end (and for linking the obtained mitogenomes to species or vouchers) multiple ‘bait’ sequences have to be determined a priori [55, 56, 58]. A mitogenome assembled with pooled DNA from a large number of individuals of the same species , is inevitably artificial. Enrichment of the mitochondrial fraction by LR-PCR (and labeling using indexing tag sequences) is expensive and potentially time-consuming. With the capacity of current generation sequencers mitochondrial enrichment strategies might seem outdated. Advantages of the here demonstrated method are that it ensures obtainment of the complete mitochondrial locus from even minute amounts of starting material, there are no negative costs associated with increasing genome sizes (no mitochondrial to nuclear ratio effect) and it diminishes the risk of erroneoulsy sequencing NUMTs.
This study shows that a complete mitogenome can be amplified and sequenced with high coverage from a single LR-PCR. The approach might be especially relevant in situations where only small amounts of starting material are available. Phylogeny reconstructions based on entire mitogenomes are promising to resolve deep level relationships within Stylommatophora, that could not be resolved using only 28S sequence data. Well supported groups from previous studies based on ten-fold less sequence data [7, 37] were reconfirmed. The region between COII and ATP8 is apparently a hot spot for rearrangements in stylommatophoran mitogenomes. The ‘Achatinoid’ clade is a basal branch in Stylommatophora and has the same mitochondrial gene arrangement as closely related non-Stylommatophoran taxa (especially from the clade Hygrophila). Rearrangements in mitochondrial gene order can characterize different stylommatophoran ranks, e.g. (non-) ‘Achatinoid’ clade, Orthurethra, Helicidae and Bradybaenidae.
We are grateful to M. Kolet for assisting with the fieldwork for this project.
The molecular work was funded in part by Fonds Economische Structuurversterking (FES).
Availability of data and materials
The annotated mitogenome of Orcula dolium is available from GenBank under accesssion number KJ867421.
Experimental concept and design: DSJG. Labwork: ED, DSJG. Data analyses: DSJG. Manuscript writing: DSJG, EG, JH, ED. Figures: DSJG. All authors contributed to the analysis of data and writing of the manuscript and approved the final manuscript.
The authors declare that they have no competing interests.
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