Philippine Journal of Science 148 (S1): 167-180, Special Issue on Genomics ISSN 0031 - 7683 Date Received: 02 Apr 2019

Complete Mitochondrial Genome and Novel Gene Organization of Ryssota otaheitana (: Chronidae), and its Implications on the Phylogeny

Amor M. Damatac II1* and Ian Kendrich C. Fontanilla1,2

1Institute of Biology, University of the Philippines, Diliman, Quezon City 1101 Philippines 2Philippine Genome Center, University of the Philippines, Diliman, Quezon City 1101 Philippines,

Whole mitochondrial genomes (mitogenomes) have been increasingly used as markers for phylogenetic inferences. In the Philippines, a marine snail from the Conidae family has been the only species to date whose mitogenome has been fully sequenced, despite the country’s rich malacofaunal diversity particularly in the terrestrial environment. In this study, we sequenced the complete mitogenome of the Ryssota otaheitana (Chronidae), an endemic Philippine pulmonate species. The mitogenome is 13,888 bp in length and encodes the typical 37 genes – including 13 protein-coding, 22 tRNA, and 2 rRNA genes. Short intergenic spacers were found – including a reduced, 42-bp putative control region. Although the gene content is conserved, R. otaheitana showed a novel mitogenome organization involving the translocation of tRNASerine and NADH dehydrogenase subunit 4 gene segment. Combined with available stylommatophoran mitogenomes, different datasets from concatenated sequences of protein-coding and rRNA genes were used for phylogenetic reconstructions. Bayesian Inference (BI) and Maximum Likelihood (ML) trees congruently supported several monophyletic clades within the Stylommatophora such as the , Urocoptoidea, , and Limacoidea sensu lato (syn. ‘limacoid clade’). R. otaheitana is grouped with Deroceras reticulatum in the lower Stylommatophora and forms the monophyletic Limacoidea sensu lato. Both sequence-based phylogeny and gene order comparisons have led us to a hypothesis that Limacoidea sensu lato is at the base of the ‘non-achatinoid’ clade while supporting the division of ‘achatinoid and ‘non-achatinoid’ groups in the Stylommatophora. This study reports the first complete mitogenome from the Chronidae family, which can be used further for the molecular phylogeny of Philippine snails and gastropods at large.

Keywords: mitogenome, molecular phylogenetics, pulmonate snail, sequencing

INTRODUCTION (Pilsbry 1900); however, groupings at the lower level were unclear due to inconsistent findings from morphological The suborder Stylommatophora comprises the majority datasets (Shileyko 1979, Nordsieck 1986, Tillier, 1989). of pulmonate gastropods and terrestrial mollusks in Given the challenges of traditional techniques, their the world (Barker 2001). Its higher classification was lineage reconstructions have later shifted towards the traditionally based on the excretory system anatomy use of molecular characters due to their higher resolving *Corresponding Author: [email protected] power. Wade et al. (2001) provided the first extensive molecular phylogeny of the Stylommatophora, which

167 Special Issue on Genomics Damatac and Fontanilla: Complete Mitogenome and Novel Gene Organization

showed an unexpected separation into ‘achatinoid’ and sequenced to date. Phylogenetic studies involving whole ‘non-achatinoid’ groups. Such analyses have increased mitogenomes have already been done on various in the past years primarily due to the extent of molecular groups (Cameron 2014, Weigert et al. 2016). A concern data to produce large sets of discrete, heritable, and in those studies, however, is the impact of incomplete unambiguous characters that can be effectively used for taxon sampling on the reconstruction of evolutionary systematic inferences (Hillis 1987, Grauer and Li 2000). relationships. Tree topologies could vary depending on the taxa included, and therefore hypotheses derived from Mitochondrial DNA has been steadily used as a standard phylogenies can be affected by sampling of taxa (Nabhan molecular marker to infer historical relationships among and Sarkar 2011). Obtaining the mitogenome sequences taxa. Its advantages such as being small, haploid with no of underrepresented taxa such as Philippine snails and recombination, being maternally inherited, and having comparing them with other mitogenomes would not fast mutation rates make it suitable for the phylogenetic only provide baseline information on their phylogenetic reconstruction of divergent groups (Curole and Kocher position with respect to other species worldwide but also 1999). Today, the development of cost-efficient high add valuable knowledge to the current phylogeny of snails. throughput sequencing technologies has made it easier to obtain the whole mitochondrial genome (or mitogenome) Here, we present the complete mitogenome of the endemic and use it as a marker for phylogenetic analysis (Boore snail – Ryssota otaheitana Ferrusac, 1821 – characterize et al. 2005). The mitogenome practically offers a larger the features of its mitochondrial DNA and construct a set of characters; therefore, it has a higher chance to phylogenetic tree of the species. R. otaheitana (syn. R. approximate relationships than just using an individual to ovum Valenciennes, 1854) is the largest of its and few genes from the mitochondrial DNA. This approach is distributed in Luzon and the Visayas islands (Faustino of using mitogenomes may support existing phylogenies 1930, Springsteen and Leobrera 1986, Solatre 2004). and provide new answers to some unresolved gaps in Locally known as bayuku, it is considered as bush meat evolutionary history (Boore and Brown 1998, Boore 1999). and a delicacy in some parts of the country (Solatre 2004, Scheffers et al. 2012). This study provides the first In the Philippines, the phylogeny of gastropods mitogenome of a chronid land snail from the Philippines particularly of those found in the terrestrial habitats and contributes to the taxon sampling of the currently is poorly understood. The distinct geological history available gastropod mitogenomes. of the archipelago has resulted to a high number of endemic species, including many pulmonate land snails that have restricted biogeographic distribution in the country (Faustino 1930, Heaney 1986, Springsteen MATERIALS AND METHODS and Leobrera 1986). The stylommatophoran Ryssota (Chronidae family) includes endemic representatives in the Philippines (Faustino 1930, Springsteen and Leobrera Sample Collection and DNA Extraction 1986). In some studies, the genus is considered as part of A voucher specimen of R. otaheitana was collected the Helicarionidae family due to the former inclusion of from Mt. Makiling, Laguna, Philippines. The snail was the Chronidae in the Helicarionidae (Solem 1978, Tillier identified following Springsteen and Leobrera (1986) and 1989, Sosa et al. 2014). Most recent classifications, Abbott (1989), and the whole sample was kept in –80 °C however, have separated the two families and placed at the DNA Barcoding Laboratory, Institute of Biology, them in separate superfamilies and thus consequently University of the Philippines Diliman. Approximately 25 dissociates the genus from Helicarionidae (Hausdorf 1998, mg of foot tissue was excised for DNA extraction using Bouchet et al. 2005). There is limited molecular data on DNeasy Blood and Tissue Kit (Qiagen) following the the Ryssota since most studies made on the taxon have manufacturer’s protocol. relied on traditional taxonomic methods (De Chavez and De Lara 2011, Sosa et al. 2014). It is therefore important Polymerase Chain Reaction (PCR) and Sequencing to provide such data to understand the phylogeny of the Two PCR approaches were employed in this study. genus, its family, and Philippine land snails in general. The first approach involved the amplification of three overlapping long fragments using pulmonate-specific As of March 2019, the National Center for Biotechnology Information (NCBI) database contains 169 gastropod primers designed by White et al. (2011) (Table 1). The mitogenomes, of which 25 are stylommatophorans. This PCR was carried out in a total volume of 50 μl containing is far insufficient given that gastropods have an estimated 10 μl of 5x Ranger Reaction Buffer (Bioline), 1 μL of number of 40,000 to 100,000 species (Boss 1971, Gotting each primer (20 mM), 1 μl of Ranger DNA Polymerase (Bioline), 27 μl of ultrapure water, and 10 μl of template 1974). In the Philippines, only the marine snail Conus tribblei (Conidae family) has its entire mitogenome DNA (10ng/ μl). The amplification conditions were: 3

168 Special Issue on Genomics Damatac and Fontanilla: Complete Mitogenome and Novel Gene Organization

Table 1. Primers used in the PCR approach. For long PCR, a forward F437 and reverse primers were used when the genes are coded in CRNCTGTTTANCAAAAACATAGYY the same direction while either both forward or both reverse R972 primers were used when the genes are coded in the opposite CCGGTCTGAACTCAGATCATGT directions. All primers were designed by White et al. (2011) F515 except for LEP primers by Smith et al. (2007). GGCCGCAGTACMYTGACTGTGCDAAGGTAGC Region Primer sequence (5’–3’) R972 CCGGTCTGAACTCAGATCATGT Long PCR cytb F384 cox1- cytb F844 GGNTAYGTNYTDCCDTGRGGNCARAT GGWTTTATTGTNTGRGCDCAYATRTTTAC R809 R809 GCRTANGCRAANARRAARTAYCAYTCNGG GCRTANGCRAANARRAARTAYCAYTCNGG cox2 F301 cytb- rrnS F384 AYHGGNCAYCARTGRTAYTG GGNTAYGTNYTDCCDTGRGGNCARAT R580 F302 ATYTCNGARCAYTGNCCRTA AAACTRGGATTAGAKACCCYAYTAT rrnS F302 rrnS- cox1 R735 AAACTRGGATTAGAKACCCYAYTAT TACYYYTACTDTGTTACGACTT R735 R839 TACYYYTACTDTGTTACGACTT GTAAAYATRTGHGCYCANACAATAAAWCC F143 Short PCR GGTGCCAGCADYCGCGGYCAWACC cox1 LEP F1 R695 ATTCAACCAATCATAAAGATATTGG RGRRTGACGGGCGATTTGTRCAC LEP R1 cox1- rrnL F1281 TAAACTTCTGGATGTCCAAAAAATCA TAGGDYTDKCWGGHATRCCNCGNCG F14 R437 WYTCNACDAAYCAYAAAGAYATTGG RRCTATGTTTTTGNTAAACAG R839 GTAAAYATRTGHGCYCANACAATAAAWCC min at 95 ⁰C, 34 cycles of 10 s at 98 ⁰C, 45 s at 45 ⁰C F253 and 6–8 minutes at 68 ⁰C, and 10 min at 72 ⁰C. The PCR GGNGCNCCWGAYATRAGHTTYCC R698 products were sent to the DNA Sequencing Core Facility ATRTADACYTCNGGRTGHCCRAARAAYCA of the Philippine Genome Center, University of the F466 Philippines Diliman for purification, library preparation, TTRGGDGCNATTAATTTTATYAC and sequencing. Genomic libraries generated by Nextera R1052 XT protocol (Illumina) were sequenced on a MiSeq TCYAAHGAWGARTTWGAHARNACAATWCC (Illumina) platform with 2x300 bp run. F698 TGRTTYTTYGGDCAYCCNGARGTHTAYAT The second approach involved the amplification of R1052 overlapping short segments that span the tips of the long TCYAAHGAWGARTTWGAHARNACAATWCC PCR fragments. The 50 uL PCR mix contained 10 μL of F844 5x My Taq PCR buffer (Bioline), 1.5 μl of each primer GGWTTTATTGTNTGRGCDCAYATRTTTAC (10 mM), 2.5 μl of DMSO, 0.5 μl of MgCl (50mM), R1052 2 TCYAAHGAWGARTTWGAHARNACAATWCC 0.25 μl of MyTaq DNA Polymerase (Bioline), 30.75 μl of ultrapure water, and 3 μl of DNA template (10 ng/ F698 TGRTTYTTYGGDCAYCCNGARGTHTAYAT μl). The amplification conditions were: 3 min at 94 ⁰C, R1281 42 cycles of 40 s at 95 ⁰C, 45 s at 45 ⁰C and 1 min at 65 CGNCGNGGYATDCCWGMHARHCCTA ⁰C, and 5 min at 72 ⁰C. The products were purified using F844 the QIAquick® Gel Extraction Kit (Qiagen) and sent to GGWTTTATTGTNTGRGCDCAYATRTTTAC First BASE Laboratories (Malaysia) for bidirectional R1281 sequencing. CGNCGNGGYATDCCWGMHARHCCTA rrnL STY arm CTTCTCGACTGTTTATCAAAAACA Mitogenome Assembly and Annotation STY brm The Illumina sequencing of three libraries resulted in GCCGGTCTGAACTCAGATCAT 570,826 to 1,243,608 raw reads. Quality control was carried out in CLC Genomics Workbench 11.0.1 (https:// www.qiagenbioinformatics.com/) to trim and filter reads

169 Special Issue on Genomics Damatac and Fontanilla: Complete Mitogenome and Novel Gene Organization

with adapters and bases of low quality. High-quality The start and end of each gene were manually adjusted by paired-end reads with a quality score of ≥ Q30 were aligning the preliminary annotations with genes from other merged and normalized in Geneious 11.1.4 (https://www. pulmonate mitogenomes. The Geneious ORF finder set with geneious.com) using the bbmerge and bbnorm algorithms, invertebrate mitochondrial DNA as the genetic code was respectively. De novo assembly of the three libraries was also used to call the limits of protein-coding genes (PCGs). also performed in Geneious using its own assembler with default settings. The consensus mitogenome assembly was Mitogenome Comparison and Alignment validated using the short capillary sequences, particularly The mitogenome features such as mitogenome content and along overlaps of the three fragments where low coverage size, nucleotide composition, location of intergenic non- and few disagreements were observed. coding regions, and gene organization were determined. The initial annotation of the mitogenome was determined These characteristics were compared with other existing using MITOS web server (Bernt et al. 2013). The tRNA genomes from NCBI. All available mitogenomes from the genes were further located using ARWEN (Laslett and order Stylommatophora (n = 25) and one ellobiid outgroup Canback 2008) and tRNAscan-Se (Lowe and Eddy 1997). based on the classification of Bouchet et al. (2005) were

Table 2. List of gastropod mitochondrial genomes from NCBI used in this study. Order Family Organism Name Genome Size(bp) Accession Stylommatophora Achatinidae Achatina fulica 15057 NC_024601 Achatinellidae Achatinella mustelina 16323 NC_030190 Bradybaenidae Aegista aubryana 14238 NC_029419 Aegista diversifamilia 14039 NC_027584 Dolicheulota formosensis 14237 NC_027493 Mastigeulota kiangsinensis 14029 NC_024935 Clausiliidae Albinaria caerulea 14130 NC_001761 Camaenidae Camaena cicatricosa 13843 NC_025511 Cerionidae incanum 15177 NC_025645 Cerion uva 15043 NC_034226 Geomitridae Cernuella virgata 14147 NC_030723 Helicidae Cepaea nemoralis 14100 NC_001816 Cylindrus obtusus 14610 NC_017872 Helix aspersa 14050 NC_021747 Agriolimacidae Deroceras reticulatum 14048 NC_035495 Pupillidae Gastrocopta cristata 14060 NC_026043 Pupilla muscorum 14149 NC_026044 Philomycidae Meghimatium bilineatum 13972 NC_035429 Microceramus pontificus 14275 NC_036381 Orthalicidae Naesiotus nux 15197 NC_028553 Orcula dolium 14063 NC_034782 Polygyra cereolus 14008 NC_032036 Praticolella mexicana 14153 NC_032079 Succineidae Succinea putris 14092 NC_016190 Vertiginidae Vertigo pusilla 14078 NC_026045 Outgroup 14246 NC_012434 Ovatella vulcani 14,247 NC_016175 bidentata 14,135 NC_016168 Systellomatophora Peronia peronii 13,968 NC_016181 Basommatophora Biomphalaria glabrata 13,722 NC_005439

170 Special Issue on Genomics Damatac and Fontanilla: Complete Mitogenome and Novel Gene Organization used in the analyses (Table 2). The nucleotide sequences These parameters were similar for BI except that it used of protein-coding and rRNA genes were extracted from the Bayesian Information Criterion metric and only models genomes, and each gene was aligned separately in Geneious. available in MrBayes. Data blocks were set according to the The PCGs were aligned based on their translation, while gene name and its codon position (for PCGs). rRNA gene alignments were based on their nucleotide The trees were estimated using the optimal partition scheme sequences. Ambiguous regions in the alignments were and models applied to each concatenated dataset (Chernomor removed using the default setting of Gblocks online server et al. 2015; Table I). ML analyses were employed in IQ- (Castresana 2000). Finally, the gene alignments were put Tree v.1.6.10 (Nguyen et al. 2014), and the support values together using FasConCAT-G v1.04 (Kück and Meusemann were determined using the ultrafast bootstrap with gene 2010) to generate two concatenated datasets: one that has and site resampling strategy of 1000 replicates (Hoang et protein coding and rRNA genes (PCG+rRNA) and another al. 2017). On the other hand, BI inferences were conducted with PCGs only. The datasets were subjected to saturation in MrBayes v3.2.6 (Ronquist and Huelsenbeck 2003). test using the Xia method as implemented in DAMBE ver. The trees were estimated using two independent runs of a 5.0.25 (Xia and Xie 2001). Markov Chain Monte Carlo with 10,000,000 generations. Each simultaneous run has four chains with 0.1 burning Phylogenetic Analyses temperature and samples a tree every 500 generations. A Phylogenetic reconstructions were conducted using the ML 25% burn-in was set and the posterior probabilities were and BI methods. Prior to each analysis, the best partition calculated as support. An average standard deviation of schemes and their corresponding optimal models were split frequencies below 0.01 served as the basis to accept the determined using PartitionFinder 2.1.1 (Lanfear et al. 2016). convergence of the runs. FigTree v1.4.4 was used to visualize For ML, the search was defined by all models, corrected all trees (Rambaut, 2018). Bootstrap support and posterior Akaike Information Criterion metric, linked branch lengths, probability values of ≥ 95 were considered as strong support. and a greedy partition search algorithm (Lanfear et al. 2012).

Figure 1. The mitochondrial genome of Ryssota otaheitana. Arrows indicate the direction of transcription. Blue, red, and pink arrows represent the protein-coding genes, ribosomal RNAs, and transfer RNAs, respectively.

171 Special Issue on Genomics Damatac and Fontanilla: Complete Mitogenome and Novel Gene Organization

RESULTS AND DISCUSSION 12.7% C, 14.8% G, and 40.1% T – with a total of 72.5% AT content. Coinciding with this composition is the detection of strand asymmetry in the mitogenome. The forward strand Mitogenome Size and Content has a negative AT skew (–0.106) and positive GC skew The whole mitogenome of Ryssota otaheitana (Genbank (0.076), similar to almost all pulmonates reported (White accession number MK716255) is 13,888 bp in length and et al. 2011). Francino and Ochman (1997) hypothesized contains 13 PCGs, 22 transfer RNA (tRNA), and two ribosomal that differences in the gene expression between two strands RNA (rRNA) genes (Figure 1). It has the typical mitogenome may lead to changes in the composition and distribution composition found among metazoans, and its length is within of nucleotides, as more frequent expressions are likely the size range of previously sequenced pulmonates (Boore to increase the deamination of nucleotides. In particular, 1999, White et al. 2011). The genes are mostly encoded in the mitogenomes may undergo spontaneous A and C the forward strand except for cox3, tRNAThr, tRNASer, nad3, deaminations during DNA replication leading to G and tRNAMet, rrnS, tRNAGlu, tRNAArg, atp6, tRNAAsn, atp8, tRNALeu, U mutations, respectively (Xia 2012). These changes do and tRNAGln (Table 3). Genes encoded on the same strand not only affect the overall nucleotide composition but also are grouped exclusively, thus forming the two gene clusters: result in an asymmetrical distribution of nucleotides in the tRNAIle- tRNAHis and cox3-tRNAGln. strands. In mollusks, different patterns of strand asymmetry The nucleotide composition and AT content of the are observed, which postulates the changing of leading and mitogenome sequence is similar to other pulmonates lagging strand roles in the mitochondrial DNA (Boore 2006, reported (White et al. 2011, Liu et al. 2012). It has 32.4% A, Grande et al. 2008, Rawlings et al. 2010).

Table 3. The mitochondrial genome features of Ryssota otaheitana. Gene Strand Length Start codon Stop codon Anticodon Intergenic nucleotides cox1 F 1,527 TTG TAA –2 tRNAVal F 63 TAC –21 rrnL F 1,009 0 tRNALeu F 68 TAG +5 tRNAPro F 65 TGG –3 tRNAAla F 61 TGC +4 nad6 F 450 ATG TAA –8 nad5 F 1,674 ATG TAA –22 nad1 F 903 ATG TAG +2 nad4L F 309 ATG TAA –28 cytb F 1,122 ATG TAA –11 tRNAAsp F 63 GTC –8 tRNACys F 66 GCA –2 tRNAPhe F 67 GAA +2 cox2 F 666 ATG TAA +2 tRNATyr F 61 GTA –5 tRNATrp F 65 TCA 0 tRNAGly F 64 TCC –7 tRNASer F 58 GCT +4 nad4 F 1,313 ATG TA –7 tRNAHis F 73 GTG –1 tRNAGln R 60 TTG +19 tRNALeu R 70 TAA –32 atp8 R 183 ATG TAA 0 tRNAAsn R 62 GTT –29 atp6 R 672 ATG TAA –1 tRNAArg R 71 TCG –3 tRNAGlu R 71 TTC 0 rrnS R 709 –2 tRNAMet R 64 CAT –21 nad3 R 369 ATA TAA –3 tRNASer R 56 TGA 0 tRNAThr R 62 TGT –10 cox3 R 789 ATG TAG 0 tRNAIle F 65 GAT +2 nad2 F 954 ATG TAA –35 tRNALys F 70 TTT –7

172 Special Issue on Genomics Damatac and Fontanilla: Complete Mitogenome and Novel Gene Organization

Overlapping and Noncoding Regions PCGs, Transfer RNAs, and Ribosomal RNAs The dynamics between overlapping and noncoding All PCGs were identified by MITOS except the atp8 gene. regions significantly affect the mitogenome size of a The gene was located based on prior information that atp8 species (Grande et al. 2008, Doublet et al. 2015). So far, R. is usually flanked by tRNALeu and tRNAAsn in euthyneurans otaheitana has one of the smallest mitogenomes compared (White et al. 2011). Using ORF Finder, a putative atp8 with other pulmonates in NCBI (13,650–16,708), and gene (183 bp) was found between these two tRNAs in the this can be attributed to the presence of frequently large reverse strand of the mitogenome; however, nucleotide overlapping regions and less common intergenic spacers. BLAST search showed no similarity to other atp8 genes Overlapping regions are observed in 23 junctions across in the NCBI database. To resolve this, we examined the the mitogenome of R. otaheitana, which has a size range coding sequence of the putative gene and discovered the of 1–35 bp and a total length of 268 bp. Both PCGs Methionine-Proline-Glutamine peptide at the N-terminus and tRNAs are involved in the gene overlaps, contrary that is a conserved feature of a functional atp8 gene in to previous reports that gene overlaps mostly involved several metazoans (Gissi et al. 2008). Furthermore, protein tRNAs (Grande et al. 2008, Cunha et al. 2009, Rawlings BLAST search against UNIPROT database revealed that et al. 2010). Intergenic spacers, albeit less frequent than the translated nucleotides of the putative gene have a match, the overlapping regions, make up 0.59% (82bp) of the albeit weak, with the atp8 protein from Albinaria caerulea mitogenome. These noncoding regions are generally with 36% identity and 3e–2 e-value. This poor match is short, having most intergenic spacers ≤ 5 bp in length with consistent with other studies that had difficulties in atp8 exceptions. These exceptions include a 42 bp intergenic annotation due to high sequence divergence as a result of region between cox3 and tRNAIle, which is determined more relaxed selection (Śmietanka et al. 2010, Uliano-Silva as the potential origin of replication (POR) or control et al. 2016; Egger et al. 2017). region. The region is assumed as the POR based on several reasons: first, it is the largest noncoding region Most PCGs start with the standard ATG codon except cox1 in the mitogenome; second, it is located at the boundary (TTG) and nad3 (ATA). The codon TTG has been commonly between the two gene clusters, which may signify its role used as a start codon by the cox1 gene in pulmonates as the site of replication; third, its composition has higher while ATA, albeit uncommon, is also used by other PCGs AT content (85.7%) than the rest of the mitogenome; and (White et al. 2011). The most frequent stop codon is TAA lastly, most studies have located the POR flanked between with exceptions from cox3 (TAG), nad1 (TAG), and nad4 cox3 and a tRNA gene (White et al. 2011, Gaitan-Espitia (TA). Incomplete termination codon is completed by post- et al. 2013). transcriptional polyadenylation and is commonly observed in small gastropod mitogenomes (Yamazaki et al. 1997, Grande

Figure 2. Putative secondary structures of tRNA genes in R. otaheitana mitogenome. Dash indicates correct base pairing while plus and dot indicate mismatches.

173 Special Issue on Genomics Damatac and Fontanilla: Complete Mitogenome and Novel Gene Organization

Figure 3. Maximum likelihood tree inferred from the PCG dataset under the optimal partition schemes and best-fit models strategy. The topology of ML and BI trees are identical. The values near the nodes indicate the ultrafast bootstrap and posterior probability support values of ML and BI trees, respectively. The tree is rooted on pulmonate non-stylommatophorans. The scale bar represents the number of nucleotide substitutions per site.

Figure 4. Maximum likelihood tree inferred from the PCG+rRNA dataset under the optimal partition schemes and best-fit models strategy. The topology of ML and BI trees have minimal differences (see Figure I for BI tree). The values near the nodes indicate the ultrafast bootstrap and posterior probability support values of ML and BI trees, respectively. The tree is rooted on pulmonate non-stylommatophorans. The scale bar represents the number of nucleotide substitutions per site. et al. 2008). Codon usage bias is observed among the PCGs The rRNAs are flanked by tRNA genes. The large rRNA having most amino acids prefer codons with high AT content. subunit (rrnL) is 1,009bp in length and located in the Particularly in the third codon position, the strong preference forward strand between tRNAVal and tRNALeu, while the for these codons coincides with the nucleotide compositional small ribosomal subunit (rrnS) is 708bp long and is situated bias in the mitogenome. in the reverse strand between tRNAMet and tRNAGlu. Their

174 Special Issue on Genomics Damatac and Fontanilla: Complete Mitogenome and Novel Gene Organization locations corroborate other reported mitogenomes of fulica is the sole representative of the ‘achatinoid’ group, euthyneurans (White et al. 2011, Wang et al. 2014). The a strong support value (PCG+rRNA: BS = 79, PP = 0.996; tRNAs are spread in both strands of the mitogenome and PCG: BS = 80, PP = 1.0) for the ‘non-achatinoid’ clade vary in size from 56 to 73 bp. Almost all tRNAs follow was generated in BI trees, which indicates a stable split the classic cloverleaf structure except both tRNASer genes of the derived ‘non-achatinoid’ Stylommatophora from that lack DHU arms (Figure 2). Moreover, Watson-Crick the basal A. fulica. Groenenberg et al. (2017) generated a base pair mismatches are also observed at the acceptor arm similar phylogeny using the 13 mitochondrial PCGs and of several tRNAs. Yokobori et al. (1995) mentioned that showed A. fulica as sister to the ‘non-achatinoid’ taxa at most nucleotides involved in the mismatches are portions the base of the Stylommatophora. of overlapping genes, which is in congruence with the Several groups within the Stylommatophora appeared to mitogenome in this study. The high occurrence of gene be fully supported based on both datasets (BS = 100, PP overlaps is a result of a selective pressure to favor smaller = 1.0). These clades were the Helicoidea, Urocoptoidea, mitogenome size (Doublet et al. 2015). Fortunately, repair Orthurethra, and Limacoidea sensu lato. The monophyly of mechanisms for the tRNA occur since correct base pairing these groups was not surprising since several studies based is essential to the functionality of the tRNA itself (Yokobori on different molecular datasets have already established and Pääbo 2005, Doublet et al. 2015). these major groups within the Stylommatophora (Wade et al. 2001, 2006; Razkin et al. 2015). Most recent pieces of Phylogenetic Analyses molecular evidence based on the mitogenome data have A total of 26 stylommatophoran species and five outgroup also supported these monophyletic groups. Minton et al. taxa were used for the construction of phylogenetic trees (2016) produced a monophyletic tree of the Helicoidea in the study. Two datasets comprising 10,018 and 8,979 based on a concatenated dataset of 13 PCGs. Using the bp were generated from the concatenation of 13 PCGs same dataset, Groenenberg et al. (2017) displayed a with (PCG+rRNA) or without (PCG) ribosomal RNA monophyletic Helicoidea and Orthurethra while Yang genes, respectively. Based on the Xia test, little saturation and colleagues (2019) recovered the additional groupings under a symmetrical tree was detected in both datasets. such as Urocoptoidea and Arionoidea. In the study, three The substitution saturation index (Iss) values were distinct splits within the ‘non-achatinoid’ clade have significantly smaller than the critical Iss (Iss.c) values been noted: the most derived clade of Helicoidea and (0.0.5670 < 0.9057 for PCG+rRNA and 0.6617 < 0.8581 Urocoptoidea (PCG+rRNA: BS =100, PP = 1.0; PCG: BS for PCG only); thus, both datasets were considered useful = 99, PP = 1.0); the clade of Orthurethra and Albinaria for the construction of phylogenetic trees. caerulea of Clausillioidea (PCG+rRNA: BS = 99, PP = 1.0; PCG: BS = 94, PP = 1.0), and the most basal group The trees based on PCG and PCG+rRNA are shown in of Limacoidea sensu lato (BS = 100, PP = 1.0). Such Figures 3 and 4, respectively. Both ML and BI trees had affinities are different from the topological relationships nearly identical topologies when the same dataset was used. generated from rRNA gene cluster data (Wade et al. 2001, All trees were rooted to non-stylommatophoran snails – 2006). On the other hand, except for the third group that Biomphalaria glabrata (Basommatophora), Peronia peronii is first reported in this study, the first two groupings (Systellomatophora), Myosotella myosotis (Ellobiidae), are consistent with recent phylogenies based on the Ovatella vulcani (Ellobiidae), and mitogenome (Groenenberg et al. 2017, Yang et al. 2019). (Ellobiidae) – according to the proposed classification of Bouchet et al. (2005). These species belong to groups that are So far, only the mitogenome of D. reticulatum has been members of the Pulmonata and sister to the Stylommatophora. reported as a member of the Limacoidea sensu lato and A monophyletic clade of the Stylommatophora was generated included in recent phylogenetic analyses. Ahn et al. in all trees, similar to previous molecular phylogenies (Wade (2017) first reported the mitogenome of D. reticulatum et al. 2001; 2006; Klussmann-Kolb et al. 2008; Groenenberg and showed its close relationship with members of the et al. 2017). In all datasets, the suborder was recovered with Orthurethra. Yang et al. (2019), however, recovered the 100% support from bootstraps (BS) in ML and posterior species as the most basal Stylommatophora and even probabilities (PP) in BI. ancestral to A. fulica. This study provides R. otaheitana as an additional representative for the informal clade, which Within the Stylommatophora, the trees from different increases the taxon sampling in the group and improves the datasets shared similar topologies and few differences. lineage position of the Limacoidea sensu lato within the All trees consistently recovered Achatina fulica as the tree. In this study, all trees resulted in the clustering of the most basal taxon giving rise to a separate clade of more R. otaheitana and D. reituculatum with full support values. derived stylommatophorans. This separation coincides The clade was consistently recovered as the most basal with the division of ‘achatinoid’ and ‘non-achatinoid’ offshoot after the ‘achatinoid’ A. fulica, which indicates groups coined by Wade et al. (2001, 2006). Although A.

175 Special Issue on Genomics Damatac and Fontanilla: Complete Mitogenome and Novel Gene Organization

Figure 5. Linearized mitochondrial genome organization of some species included in the study. Similar genes are represented by a particular color. Genes encoded on the reverse strand are underlined.

Limacoidea sensu lato as the ancestral lineage within the analysis provides another basis for lineage construction ‘non-achatinoid’ Stylommatophora. This study is first to (Rokas and Holland 2000). show such topology in which the Limacoidea sensu lato The mitogenome of R. otaheitana presented a novel is basal to the ‘non-achatinoid’ Stylommatophora. gene organization (Figure 5). Comparing its gene order Other problematic nodes include the position of with close relatives based on the generated phylogeny, R. Meghimatium bilineatum of the Arionoidea, Naesiotus otaheitana exhibited two rearrangements: a transposition of Ala Pro Ser nux of the , and Succinea putris of the tRNA and tRNA , and the translocation of tRNA and Gly His Elasmognatha due to low bootstrap support in all trees. nad4 genes between tRNA and tRNA . On the one hand, Albeit always basal to the Helicoidea and Urocoptoidea the first translocation was also observed in A. fulica and Ala Pro clade, their affinities vary depending on the dataset. The all outgroups, suggesting that a tRNA -tRNA order is minor discrepancy can either be attributed to the insufficient an ancestral gene arrangement for the stylommatophorans, number of taxa for higher level phylogeny or the differences which further supports the basality of the ‘achatinoid’ and in the evolutionary rates of the genes (Hedtke et al. 2006). Limacoidea sensu lato within the Stylommatophora. On The second scenario is now prevented in concatenated data the other hand, the second arrangement was not found in matrices through partition models, which allow each gene any other species to date, indicating that this arrangement or codon position to evolve under its best-fit evolutionary might be an autapomorphic character state to the genus model (Chernomor et al. 2015). Support values such as or possibly a shared characteristic with other closely BS and PP are also used as a standard measure to the related taxa. The latter cannot be determined since its accuracy of the phylogenetic tree (Hedtke et al. 2006). All closest relative in the tree, D. reticulatum, while a fellow trees in this study were generated under partition schemes member of the Limacoidea sensu lato, still belongs to a and optimal models determined by PartitionFinder, while different superfamily. The two rearrangements were not BS and PP were calculated in both ML and BI analyses, observed in D. reticulatum, but this was expected since respectively. In general, some high-level clades with one the Limacoidea sensu lato is a relatively diverse group. representative each had low nodal supports while consistent Interestingly, the gene organization of D. reticulatum was groupings were recovered with high supports in both identical to Albinaria caerulea, Microceramus pontificus, datasets. Consequently, this stresses the need to sequence and Cernuella virgata – which suggests the affinity of the more mitogenomes in order to improve tree reconstructions, limacoid species to other derived taxa in the ‘non-achatinoid’ particularly in the underrepresented groups. Stylommatophora. Moreover, A. fulica had an almost similar gene order to these three species except that their gene order Gene Order had a tRNAPro-tRNAAla transposition. Instead, the gene While nucleotide sequence-based phylogeny supported organization of A. fulica is identical to the ellobiids Ovatella some of the existing phylogenies and provided interesting vulcani and Auriculinella bidentata, the systellomatophoran insights into the diversification of the Stylommatophora, Peronia peronii, and the basommatophoran Biomphalaria few subdivisions within the order are still problematic glabrata. All these information suggest that the ancestral possibly due to incomplete taxon sampling. Alternatively, stylommatophoran gene organization is observed in A. the mitogenome offers other genomic features such as fulica and further supports that the species being basal to gene order that in conjunction with the conventional the rest of the ‘non-achatinoid’ clade is congruent with

176 Special Issue on Genomics Damatac and Fontanilla: Complete Mitogenome and Novel Gene Organization previous studies that split the Stylommatophora into the new question as to whether its gene order should be two clades. Groenenberg et al. (2017) came up with the considered an apomorphic or autapomorphic character same conclusion from observing a tRNAAla-tRNAPro gene state for the genus Ryssota and its close relatives. order in A. fulica similar to distantly related outgroups. In the study, it can be hypothesized that the tRNAPro-tRNAAla gene order is an apomorphic character to recent members of the Limacoidea and derived major groups such as the ACKNOWLEDGMENTS Clausilioidea, Orthurethra, Urocoptoidea, and Helicoidea This study was supported by the Department of Science – which provides further evidence to the affinity of the and Technology (DOST) and the Science Education Limacoidea sensu lato lineage within the derived ‘non- Institute through its Accelerated Science and Technology achatinoids’. Based on this study, the Limacoidea sensu Human Resource Development Program thesis grant. The lato lineage might have served as the basal group for the authors would also like to thank Dr. Ernelea P. Cao and diversification of ‘non-achatinoids’ given that its members her laboratory for additional financial assistance. possessed both ancestral (tRNAAla-tRNAPro) and derived (tRNAPro-tRNAAla) character states through R. otaheitana and D. reticulatum, respectively. Furthermore, the gene order of D. reticulatum can be hypothesized as the ancestral APPENDIX gene arrangement for ‘non-achatinoids’ given that it is widely observed among other derived groups such as the The complete appendices section of the study is accessible Clausillioidea and Urocoptoidea (Groenenberg et al. 2017). at http://philjournsci.dost.gov.ph

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Table I. The partition schemes and best-fit models generated by Table II. The partition schemes and best-fit models generated by PartitionFinder for the maximum likelihood analyses. PartitionFinder for the Bayesian Inference analyses. Subset Best model Partition names Subset Best Partition names model 1 GTR+I+G atp6_1cp, atp8_1cp, nad3_1cp 1 GTR+I+G nad4L_1cp, nad6_1cp, nad2_1cp, 2 GTR+I+G atp6_2cp nad4_1cp, nad5_1cp, 3 TRN+G atp6_3cp, nad3_3cp nad1_1cp, atp8_1cp, atp6_1cp, nad3_1cp 4 TVM+G atp8_2cp, nad3_2cp 2 GTR+I+G atp6_2cp, nad3_2cp, nad4_2cp, nad5_2cp, 5 TRN+G atp8_3cp, cox3_3cp nad1_2cp 6 GTR+I+G cox1_1cp 3 HKY+G atp6_3cp, nad3_3cp, cox3_3cp, atp8_3cp 7 GTR+I+G cox1_2cp 4 GTR+I+G atp8_2cp, nad4L_2cp, nad2_2cp, 8 TRN+I+G cox1_3cp nad6_2cp 9 GTR+I+G cytb_1cp, cox2_1cp 5 GTR+I+G cox1_1cp 10 GTR+G cytb_2cp, cox2_2cp 6 GTR+I+G cox1_2cp 11 TIM+I+G cox2_3cp 7 HKY+G cox1_3cp, cytb_3cp, cox2_3cp 12 GTR+I+G cox3_1cp 8 GTR+I+G cox3_1cp, cytb_1cp, cox2_1cp 13 TVM+I+G cox3_2cp 9 GTR+I+G cox3_2cp, cytb_2cp, cox2_2cp 14 TIM+G cytb_3cp 10 HKY+G nad4L_3cp, nad6_3cp, nad2_3cp, nad1_3cp, nad5_3cp, 15 GTR+I+G nad1_1cp, nad5_1cp nad4_3cp 16 GTR+I+G nad4_2cp, nad1_2cp, nad5_2cp 11 GTR+I+G rrnL, rrnS 17 K81UF+G nad1_3cp 18 GTR+I+G nad6_1cp, nad2_1cp 19 GTR+I+G nad2_2cp 20 TRN+G nad2_3cp 21 GTR+I+G nad4_1cp 22 TRN+G nad4_3cp, nad5_3cp 23 GTR+G nad4L_1cp 24 GTR+G nad4L_2cp 25 HKY+G nad4L_3cp, nad6_3cp 26 TVM+G nad6_2cp 27 GTR+I+G rrnL 28 GTR+I+G rrnS

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Figure I. Bayesian inference tree inferred from the PCG+rRNA dataset under the optimal partition schemes and best-fit models strategy.

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