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Journal of Genetics (2020)99:17 Ó Indian Academy of Sciences

https://doi.org/10.1007/s12041-020-1177-z (0123456789().,-volV)(0123456789().,-volV)

RESEARCH ARTICLE

Molecular phylogeny and evolution of (: ) on the basis of mitochondrial (16S, COI) and nuclear markers (18S, 28S): an overview

VIJAYA SAI AYYAGARI* and KRUPANIDHI SREERAMA

Department of Biotechnology, Vignan’s Foundation for Science, Technology and Research (Deemed to be University), Guntur 522 213, *For correspondence. E-mail: [email protected].

Received 13 December 2018; revised 19 November 2019; accepted 3 December 2019

Abstract. The phylogenetic relationships among the major groups of Pulmonata were studied by the information derived from a concatenated dataset consisting of mitochondrial (16S and COI) and nuclear (18S and 28S) markers. are recovered as monophyletic. as paraphyletic due to the emergence of taxa from and lower Heterobranchia. The major groups of Pulmonata, namely , , , Otinoidea, and are recovered as monophyletic. Monophyly of was not confirmed due to the variable position of Siphonarioidea and . Evolutionary divergence times for different taxa were also estimated using a relaxed molecular clock method in Bayesian evolutionary analysis by sampling trees (BEAST). The common ancestor of Heterobranchia and was originated in the period and the common ancestors of Euthyneura and Pulmonata were originated in the and lower periods, respectively.

Keywords. pulmonata; heterobranchia; phylogeny; evolution; 16S marker; 18S marker; 28S marker; cytochrome oxidase I.

Introduction dorsal bodies are the synapomorphic characters of the Pulmonata. Whereas the presence of Rhinophoral nerve is a According to Bouchet and Rocroi (2005), Gastropoda is diagnostic character found only in and divided into six main . Of these, Heterobranchia is Opisthobranchia (Haszprunar and Huber 1990; Ponder and divided into lower Heterobranchia, Opisthobranchia and Lindberg 1997). Pulmonata. Opisthobranchia and Pulmonata are grouped Although there are several synapomorphic characters that together as ‘Euthyneura’. The diagnostic character that support the monophyly of different groups in Hetero- unites Pulmonata and Opisthobranchia is the ‘Pentagan- branchia, there are still incongruencies in their relationships glionate’ condition marked by the acquisition of an addi- when reconstructed using various molecular markers. For tional ‘Parietal ganglia’ (Haszprunar and Huber 1990). Loss example, in phylogenetic studies, Euthyneura is nonmono- of , hyperstrophy of (Haszprunar phyletic and recovered paraphyletic due to Pyramidellidae 1985; Dayrat and Tillier 2002), anatomy of the genital (Grande et al. 2004a, 2008; Klussmann-Kolb et al. 2008; system (Haszprunar 1988) are a few of the synapomorphic Dinapoli and Klussmann-Kolb 2010; Dayrat et al. 2011; characters of the Euthyneura. In contrast, lower Hetero- Dinapoli et al. 2011) and recovered monophyletic in the branchia lacks parietal ganglion and are ‘Triganglionate’ study of Wade and Mordan (2000). In a few studies, Pul- (Haszprunar 1985). Further, Opisthobranchia and Pul- monata recovered paraphyletic (Klussmann-Kolb et al. monata are distinctly divided from each other on the basis 2008; Dinapoli and Klussmann-Kolb 2010; Dayrat et al. of synapomorphic characters of the central nervous system. 2011) and polyphyletic (Grande et al. 2004a, b, 2008; Of these, presence of procerebrum, cerebral gland and Vonnemann et al. 2005;Sunet al. 2016). Similar is the case

Electronic supplementary material: The online version of this article (https://doi.org/10.1007/s12041-020-1177-z) contains supplementary material, which is available to authorized users. 17 Page 2 of 14 Vijaya Sai Ayyagari and Krupanidhi Sreerama with Opisthobranchia (Grande et al. 2004a, b, 2008; Von- Materials and methods nemann et al. 2005; Klussmann-Kolb et al. 2008; Dinapoli and Klussmann-Kolb 2010; Dinapoli et al. 2011; Sun et al. Taxon sampling and identification 2016). The lack of congruency in the results among different phylogenetic studies is mainly due to the differences in the Nine different of gastropods belonging to Pulmonata number of taxa incorporated, number of loci used, com- and Caenogastropoda were collected from different geo- pleteness of the locus and substitution saturation related graphical locations of India, from August 2014 to April 2016 issues etc. (table 1 in electronic supplementary material at http://www. So far, a comprehensive phylogeny of Pulmonata avail- ias.ac.in/jgenet/). The sequences of most of the species are able is that of Dayrat et al.(2011), on the basis of infor- new to science. In the present study, a total of 70 taxa were mation derived from 16S, 18S (complete) and cytochrome included, of which 10 (nine genera) were sequenced and the oxidase I (COI). However, they did not include 28S rDNA in sequences of the remaining 60 were retrieved from GenBank their phylogenetic analyses. Nuclear markers need to be (table 1). All the collected specimens were identified by the included for studying the deeper divergences as they evolve taxonomic experts at the Zoological Survey of India, slowly compared to mitochondrial markers (Brown et al. Kolkata. 1979; Dinapoli et al. 2006; Lajmi et al. 2019; Sil et al. 2019). Further, in their study, the taxa from Stylom- matophora were poorly represented. Apart from Dayrat et al. Genomic DNA extraction, amplification and sequencing (2011) there were few phylogenetic studies in which (i) major pulmonate group(s) were not included (Grande Genomic DNA was extracted using NucleoSpin Tissue kit et al. 2004a, b; Vonnemann et al. 2005; Klussmann-Kolb (Macherey Nagel) from the foot muscle tissues of the et al. 2008; Dinapoli and Klussmann-Kolb 2010); (ii) taxa following the manufacturer’s instructions. For a few isolates, from a few pulmonate groups were scantily represented genomic DNA was isolated by employing a slightly modi- (Grande et al. 2004a, b; Vonnemann et al. 2005;Jo¨rger et al. fied procedure of Sokolov (2000) and Ayyagari et al.(2017). 2010); and (iii) nucleotide sequences for one or more A total of six loci were amplified: 16S, COI, 18S, 5.8S, ITS- markers were missing for few pulmonate taxa (Klussmann- II and 28S rDNA. List of primer sequences and cycling Kolb et al. 2008;Jo¨rger et al. 2010). conditions are provided in table 2 in electronic supplemen- Hence, in the present study phylogenetic relationships tary material. among Pulmonata (in particular) and Heterobranchia (at Polymerase chain reaction (PCR) amplicons were cleaned large) were evaluated by including more number of available up using ExoSAP-IT (GE Healthcare) following the manu- pulmonate sequences from GenBank representing different facturer’s instructions and were sequenced using the primers pulmonate groups along with the sequences obtained in the that were used for PCR at regional facility for DNA present study by means of information derived from mito- Fingerprinting, India. The obtained forward and reverse chondrial (16S and COI) and nuclear (18S and 28S) markers. strand chromatograms were aligned using DNA Baser Further, in most of the studies, Euthyneura is rendered v4.20.0 (Heracle BioSoft S.R.L. Romania). The sense strand paraphyletic due to the emergence of taxa from lower sequences were submitted to NCBI GenBank and were Heterobranchia in particular to Pyramidellidae and assigned accession numbers (table 1). Glacidorboidea (Klussmann-Kolb et al. 2008; Dinapoli and Klussmann-Kolb 2010; Dayrat et al. 2011; Dinapoli et al. 2011). Hence, a few taxa from these two groups were Data analyses included in the present study to ascertain their phylogenetic positions. The nucleotide sequences obtained from GenBank were Gastropods have a rich record traced back to refined before performing multiple sequence alignment (Dinapoli and Klussmann-Kolb 2010). This (table 3 in electronic supplementary material). Multiple enables the study of the evolution of different groups in sequence alignment was carried out using the inbuilt Muscle Pulmonata to those for which fossil records are not (Edgar 2004) programme with default parameters in MEGA available. Dinapoli and Klussmann-Kolb (2010) have v6.06 (Tamura et al. 2013). The following datasets were estimated the divergence times of different groups in created to determine the nucleotide substitution models and Heterobranchia. However, in their study taxa from major extent of substitution saturation: dataset 1, original groups in Pulmonata were poorly represented. Further, unmasked multiple sequence alignment; dataset 2, sequence there are no comprehensive evolutionary analyses on alignment masked under ‘less stringent’ option in Gblocks; Pulmonata with increased number of taxa to date. As a dataset 3, sequence alignment masked under ‘more strin- result, in the present study divergence times for major gent’ option in Gblocks. groups in Pulmonata and Heterobranchia were estimated Multiple sequence alignment file was checked for regions with increased number of pulmonate taxa in addition to of ambiguous alignment by online Gblocks server (Castre- phylogenetic analyses. sana 2000) under ‘less stringent’ and ‘more stringent’ Phylogeny and evolution of Pulmonata Page 3 of 14 17

Table 1. List of taxa used in the present study with their taxonomic classification and GenBank accession numbers.

Taxon 16S 18S COI 28S

Caenogastropoda 1 globosa* KT716350* KU365379* KX514446* KU748527* 2 bengalensis* KT716349* KU365378* FJ405877 KU748526* Lower heterobranchia 3 piscinalis FJ917248 FJ917222 FJ917267 FJ917224 4 Cornirostra pellucida FJ917249 FJ917215 FJ917282 FJ917225 Orbitestellidae 5 Orbitestella sp. EF489333 EF489352 EF489397 EF489377 6 punctocaelatus EF489318 GQ845186 EF489393 EF489370 7 solidula EF489319 AY427516 DQ238006 AY427481 Pyramidellidae 8 sp. EF489332 EF489351 EF489396 EF489376 9 sp. GU331950 GU331940 GU331959 GU331930 10 ventricosa FJ917255 FJ917213 FJ917274 FJ917235 11 rusticus FJ917264 FJ917211 FJ917284 FJ917227 Opisthobranchia 12 Toledonia globosa EF489327 EF489350 EF489395 EF489375 13 hydatis EF489323 AY427504 DQ238004 AY427468 Aplysiomorpha 14 californica AF192295 AY039804 AF077759 AY026366 15 auricularia AF156132 AY427503 AF156148 AY427467 16 bullata AF156127 AY427502 AF156143 AY427466 17 dolabrifera AF156133 DQ237960 AF156149 DQ237973 18 antillarum FJ917425 FJ917441 FJ917483 FJ917466 19 viridis AJ223398 AY427499 DQ237994 AY427462 20 Plakobranchus ocellatus JX272649 AY427497 DQ471270 AY427459 21 nigricans EU140843 AY427500 DQ237995 AY427463 - 22 perversa KJ022803 AY427496 AF249809 AY427458 S.F Pleurobranchoidea 23 Tomthompsonia antarctica EF489330 AY427492 DQ237992 AY427452 24 peroni EF489331 AY427494 DQ237993 AY427455 S.F Bathydoridoidea 25 Bathydoris clavigera AF249222 AY165754 AF249808 AY427444 Pulmonata Amphiboloidea 26 sp. GU331952 GU331942 GU331961 GU331932 27 solida JQ228494 DQ093440 HQ660002 DQ256741 (Salinator solida) 28 concinna EF489300 EF489334 EF489378 EF489353 29 Siphonaria capensis EF489301 EF489335 EF489379 EF489354 Hygrophila Chilinoidea 30 sp. EF489305 EF489338 EF489382 EF489357 31 neritoides EF489307 EF489339 EF489384 EF489359 32 lacustris EF489311 AY282592 AY282581 KU341306 33 acuminata* KX060742* KU726620* KX514444* KX060749* 34 EF489314 EF489345 EF489390 EF489367 35 truncatula HQ659899 HQ659965 HQ660031 AY465066 36 auricularia AF485646 Z73980 EU818827 AY465067 Ancylidae 37 fluviatilis EF489312 AY282593 AY282582 EF489365 17 Page 4 of 14 Vijaya Sai Ayyagari and Krupanidhi Sreerama

Table 1 (contd) Taxon 16S 18S COI 28S

38 fuscus EU038346 AY282599 AY282588 DQ256734 39 tropicus EF489313 AY282594 AY282583 EF489366 40 planorbis EF489315 EF012192 EF012175 EF489369 41 convexiusculus* MF004204* KU365382* KX514439* KX060747* 42 alexandrina DQ084847 U65225 DQ084825 AF435693 43 AY198102 BGU65224 DQ084824 AF435694 44 anceps HQ659902 HQ659968 HQ660034 AF435689 ( anceps) 45 exustus AY577471 AY282598 AY282587 AF435662 46 acuta AY651241 AY282600 AY282589 EF489368 Otinoidea 47 EF489310 EF489344 EF489389 EF489363 48 phillipensis FJ917263 FJ917210 FJ917283 FJ917229 Ellobioidea 49 minimum EF489308 EF489341 EF489386 EF489361 50 ornatus HQ659888 HQ659954 HQ660020 DQ279994 51 myosotis HQ659887 HQ659953 EF489385 EF489360 Onchidiidae 52 Onchidium verrucosum HQ659903 AY427522 EF489391 AY427487 53 floridana HQ659903 HQ659969 EF489392 AY427486 54 AY345048 X70211 AY345048 KM281087 Veronicellidae 55 alte* KT716351* KU365377* KX514440* KU992691* 56 Laevicaulis (Filicaulis) alte* KX060745* KU365380* KX514443* KX060748* Stylommatophora ; 57 putris HQ659927 HQ659993 HQ660059 AY014057 Cochlicopoidea; 58 lubrica GU331954 GU331944 GU331963 KU341313 ; 59 fulica* KX060743* KU365375* KX514441* KU992690* Achatina fulica* KP317640 – – – Achatina fulica* KP317641 – – – Achatina fulica* KP119753 – – – ; 60 rotundatus FJ917265 FJ917212 FJ917285 FJ917240 Helicarionoidea; 61 indica* KX060744* KU365381* KX514445* KX060746* 62 Unidentified Ariophantidae* KY231965* 63 Cryptozona bistrialis* KT716352* KU365376* KX514442* KX378390* Cryptozona bistrialis* KP327718* – – – Cryptozona bistrialis* KY231964* – – – ; 64 reticulatum FJ917266 AY145373 FJ917286 FJ917241 ; 65 silvaticus AY947380 AY145365 AY987918 AY145392 66 Arion ater HQ659926 HQ659992 HQ660058 AY014144 ; 67 nemoralis KC967199 AJ224921 AY546270 KU341312 68 arbustorum JF717810 AY546383 AY546263 AY014136 69 aspersa EU912832 X91976 HQ203052 AY014128 ( aspersa) Helicoidea; 70 obvia GU331953 GU331943 GU331962 GU331933

* Taxa sequenced in the present study. Phylogeny and evolution of Pulmonata Page 5 of 14 17 options. Nucleotide substitution models for the datasets were electronic supplementary material) in the concatenated determined using jModelTest v2.1.10 (Darriba et al. 2012) dataset. The results thus obtained were compared (table 2). on the basis of Bayesian information criterion (BIC) (ta- The phylograms were annotated using iTOL v3, http://itol. bles 4–6 in electronic supplementary material). embl.de/ (Letunic and Bork 2016). Substitution saturation was determined for all the three datasets using the entropy based test of Xia et al.(2003) implemented in DAMBE v6.4.48 (Xia 2013) (tables 7–9 in Molecular evolution electronic supplementary material). Dataset I showed satu- ration for 16S, 18S and 28S. While datasets II and III did not Dataset II was used for estimating the divergence times of show saturation for these three markers. Dataset II was the taxa in BEAST v1.10.4 (Suchard et al. 2018). In BEAUti chosen for carrying out phylogenetic analyses with a view of (from BEAST package), nucleotide substitution models were not losing much of the sequence information. For COI, unlinked for each of the four partitions. Four calibration codon 3 was excluded due to saturation. Codon positions 1 points were used in the present study: (i) Heterobranchia, and 2 of COI gene from dataset II were combined and used divergence time was set to 399 ± 11.5 million years (Di- for phylogenetic and evolutionary analyses. napoli 2009). This is on the basis of the fossil record of The following substitution models were determined: 16S Palaeocarboninia jankei which is considered to be the oldest (reduced dataset and complete dataset), HKY?1?G; 18S, Heterobranchia from the Middle period (Bandel SYM?I?G; 28S, GTR?I?G; COI (codon positions 1 and 2 and Heidelberger 2002; Dinapoli 2009). (ii) Acteonoidea, combined), GTR?I?G. The final alignment of the com- the node age was set to 210 ± 6 million years (Tracey et al. bined dataset consisted of 1692 nucleotides (16S (reduced 1993; Dinapoli 2009); (iii) Lymnaeidae, the fossil age was dataset), 282 nucleotides; 18S, 423 nucleotides; 28S, 595 set to 145 million years (Remigio and Blair 1997; Dayrat nucleotides; COI, 392 nucleotides). Incongruence length et al. 2011); and (iv) , 140 million years (Jo¨rger difference (ILD) test was carried out in PAUP* 4.0a159 et al. 2010). (Swofford 2002). The combined dataset of 16S, 18S, 28S For 16S, nucleotide substitution model of HKY?I?G and and COI gave a P value of 0.01. Hence, phylogenetic for the rest of the three markers, GTR?I?G model was analyses were carried out on concatenated datasets. implemented. The clock model was set to ‘uncorrelated relaxed molecular clock’ (Drummond et al. 2006) with ‘log normal’ distribution and ‘Yule Process’ of Phylogenetic analyses (Gernhard 2008) was set as Tree prior. MCMC was run independently for five times with each run for 20 million Bayesian inference (BI) of phylogeny (figure 1) was carried generations with sampling for every 1000 generations. out in MrBayes v3.2.5 (Ronquist et al. 2012). Phylogenetic LogCombiner v1.10.4 was used for removing a burnin of analyses were carried out on concatenated datasets (16S ? 2000 from each of the five tree files and for generating a 18S ? 28S ? COI) (figures 1&2), as well as on individual combined tree file. A best tree was generated with all the datasets consisting of 16S complete dataset (figure 1 in annotations incorporated using TreeAnnotator v1.10.4. The electronic supplementary material), 18S (figure 2 in elec- combined log file was imported to Tracer v1.6 for assessing tronic supplementary material), 28S (figure 3 in electronic the ESS. It was found to be[200 for all the parameters. The supplementary material) and COI (codons 1 and 2 com- consensus tree generated was visualized using FigTree bined) (figure 4 in electronic supplementary material) for 15 v1.4.2 (Rambaut 2012). Divergence times estimated from million and 10 million generations, respectively with two the present study (with and without COI) were compared independent runs. Sampling was done at the end of every with the divergence times obtained from the studies of 1000th generation with 25% of initial samples were dis- Dinapoli (2009) and Medina et al. (2011) (table 10 in carded as burnin. At the end of 15 million generations, the electronic supplementary material). average standard deviation of the split frequencies for con- catenated dataset as well as for individual markers was found to be \0.005. TRACER v1.6 (Rambaut et al. 2014) was Results used to determine the effective sample size (ESS) and was found to be [500. Phylogenetic analyses Maximum likelihood analysis (figure 2) was carried out in raxmlGUI1.5b2 (Silvestro and Michalak 2012) on the con- Heterobranchia was found to be monophyletic with strong catenated dataset divided into four partitions set under node support (figures 1–2). Lower Heterobranchia and GTR?I?G model for 1000 bootstrap replications under Opisthobranchia did not form a distinct monophyletic . ‘ML ? rapid bootstrap’ mode. The branch lengths were Opisthobranchia formed a series of basal lineages intermitted allowed to vary for each of the partitions. Phylogenetic by taxa from lower Heterobranchia. Euthyneura was para- analyses were also carried out in two scenarios, namely with phyletic due to the emergence of a few taxa from lower (figures 1&2) and without COI gene (figures 5&6 in Heterobranchia. Pulmonata recovered paraphyletic due to 17 Page 6 of 14 Vijaya Sai Ayyagari and Krupanidhi Sreerama the emergence of Sacoglossa as a sister clade to Siphonari- However, in the study of Dinapoli (2009), Acteonoidea oidea in BI phylogram (figure 1) whereas it recovered emerged in the upper period (83.68 mya). monophyletic without bootstrap support in ML analysis Euthyneura and Pulmonata originated in the Carboniferous (figure 2). and lower Triassic periods, respectively. There are differ- Monophyly of Basommatophora was rejected in the pre- ences in the divergence times obtained in the present study sent study due to the positions of ‘Amphiboloidea’ and when compared with the divergence times from the studies ‘Siphonarioidea’. Hygrophila formed a monophyletic clade of Dinapoli (2009) and Medina et al. (2011) (table 10 in with strong node support. Chilinoidea emerged basal to a electronic supplementary material). monophyletic clade consisting of superfamilies Lymnaeidae, Acroloxidae and Planorboidea. Acroloxoidea and Physidae were in an unresolved relationship due to tree topology. Discussion Planorboidea is nonmonophyletic due to the position of (Acroloxoidea) in Bayesian phylogram Molecular phylogeny (figure 1). In ML analysis (figure 2), it is monophyletic. Ancylidae (Ancylus fluviatilus and )is Eupulmonata: The concept of the monophyly of Eupul- nonmonophyletic rendering Planorbidae paraphyletic. monata (Bouchet and Rocroi 2005) consisting of Otinoidea, Eupulmonata was paraphyletic due to the association of Ellobioidea, Systellommatophora and Stylommatophora is Amphiboloidea with Ellobiidae (figure 2). In BI analysis, it supported in BI analysis (figure 1). Eupulmonata recovered was monophyletic. Otinoidea was monophyletic with strong monophyletic in several studies (Klussmann-Kolb et al. node support. It emerged as a sister clade to Systellom- 2008; Dinapoli and Klussmann-Kolb 2010; Holznagel et al. matophora. Ellobiidae was monophyletic in BI analysis 2010;Jo¨rger et al. 2010; Dinapoli et al. 2011; Golding 2012; whereas in ML analysis, the taxa were in unresolved rela- Gaita´n-Espitia et al. 2013). It is nonmonophyletic in the tionship with each other due to the tree topology. Systel- study of Dayrat et al.(2011) due to the placement of Sty- lommatophora formed a monophyletic clade. lommatophora and Systellommatophora in different clades. Stylommatophora emerged as a distinct monophyletic clade with strong node support in Bayesian and maximum Stylommatophora: Stylommatophora harbours around 80% of likelihood analyses. Achatinidae was the basal taxon in 30,000–35,000 living terrestrial molluscs with 103 families Stylommatophora. Endodontidae was the next basal lineage (Bouchet and Rocroi 2005; Razkin et al. 2015). As per in Stylommatophora. The remaining Stylommatophoran taxa Bouchet and Rocroi (2005), Stylommatophora is divided were arranged in three distinct clades in BI and ML phylo- into two subclades: Elasmognatha and Orthurethra, and an grams; though the order of arrangement of clades was informal group Sigmurethra. The group includes organisms slightly different. From table 2, it was evident that there that are terrestrial and possess two pairs of retractile were slight differences in the topologies of the trees recon- and the eyes are present at the tip of posterior tentacles. This structed with and without COI gene in the concatenated character is also seen in Systellommatophora (Dayrat and dataset. Tillier 2002). The monophyly of Stylommatophora is sup- ported by the presence of a long pedal gland placed beneath a membrane, occurrence of retractile tentacles and the Molecular evolution acquisition of a secondary ureter (Dayrat and Tillier 2002). In comparison with the present study, Stylommatophora is The topology of the tree obtained in the present study (fig- under-represented in previous studies (Klussmann-Kolb ure 3) is slightly different from the Bayesian tree (figure 1). et al. 2008; Dinapoli and Klussmann-Kolb 2010;Jo¨rger The results of the present study suggest the common et al. 2010; Dayrat et al. 2011). It recovered monophyletic ancestor for Heterobranchia and Caenogastropoda originated with strong node support and similar topologies in BI (fig- in the Silurian period (440.71 mya). This is almost similar to ure 1) and ML (figure 2) phylograms. It was also recovered the results obtained from Dinapoli (2009), where the com- monophyletic in the previous studies (Wade and Mordan mon ancestor for the Caenogastropoda and Heterobranchia 2000; Dayrat and Tillier 2002; Grande et al. 2004a; Wade originated *425.13 mya in the Silurian period. et al. 2006; Grande et al. 2008; Klussmann-Kolb et al. 2008; Heterobranchia and lower Heterobranchia (Orbitestellidae Holznagel et al. 2010,Jo¨rger et al. 2010; Dayrat et al. 2011). ? Valvatoidea) were originated in the lower Devonian and Sigmurethra is nonmonophyletic due to the inclusion of upper Devonian periods, respectively. The common ancestor (Orthurethra) and for Euthyneura originated in the Carboniferous period. (Succineidae). Achatina fulica (Achatinoid clade) emerged Nudipleura was the basal-most lineages in Opisthobranchia, basal to the rest of the stylommatophoran taxa (nonachati- which originated in the upper Triassic period. Acteonoidea noid taxa, Orthurethra and Succineidae). In the present originated in the upper Triassic around 208.38 mya. This is study, only one taxon Achatina fulica belonging to the more or less similar to the fossil age (210 ± 6 mya) for family Achatinidae under the Achatinoid clade is available. Acteonoidea, used as a calibration point in the present study. This has emerged as a basal taxon to the rest of the Phylogeny and evolution of Pulmonata Page 7 of 14 17

Figure 1. Bayesian phylogram constructed using the sequence information obtained from the concatenated dataset II consisting of 16S, 18S, 28S and COI (codons 1 and 2 combined) markers. Indicated on the nodes is the posterior probability (PP) value (only PP value greater than 0.5 are shown). 17 Page 8 of 14 Vijaya Sai Ayyagari and Krupanidhi Sreerama

Figure 2. ML tree constructed using the sequence information obtained from the concatenated dataset II consisting of 16S, 18S, 28S and COI (codons 1 and 2) markers. Indicated on the nodes is the bootstrap (BS) value (only BS values obtained [50 are shown). Phylogeny and evolution of Pulmonata Page 9 of 14 17

‘nonachatinoid’ taxa. In the future, more number of taxa Lymnaeidae and Planorbidae act as an intermediate for from different families needs to be incorporated to obtain trematodes that causes infection to and humans clear picture on the relationships within Stylommatophora. (Remigio 2002; Jørgensen et al. 2004; Correa et al. 2010). Basommatophora is nonmonophyletic in the present study Systellommatophora: As per Bouchet and Rocroi (2005), (figures 1&2). This is due to the placement of Siphonari- Systellommatophora (Gymnomorpha) consists of two oidea and Amphiboloidea in different clades separately from superfamilies: Onchidioidea and . Onchid- Hygrophila. This is in line with the system of classification ioidea includes Onchidiidae. Veronicelloidea includes proposed by Tillier (1984). In this classification, subclass Veronicellidae (Vaginulidae) and . In the pre- Pulmonata was divided into three orders, namely sent study, Systellommatophora (Onchidiidae and Veroni- Archeopulmonata, Basommatophora and Stylommatophora. cellidae) formed a monophyletic clade. It was also recovered and Siphonariidae were placed in the super- monophyletic in the studies of Holznagel et al.(2010); family Amphiboloidea in the order ‘Archeopulmonata’. The Jo¨rger et al. (2010); Dayrat et al.(2011). However, there are remaining families from the currently known Hygrophila no synapomorphic characters that define Systellom- were placed separately from Amphiboloidea and matophora (Dayrat and Tillier 2002). In most of the phylo- Siphonariidae in the order ‘Basommatophora’. Basom- genetic studies, Veronicellids are not represented. As a matophora also recovered nonmonophyletic in previous result, the monophyly of Systellommatophora was unknown studies (Klussmann-Kolb et al. 2008; Dinapoli and Kluss- (Thollesson 1999; Grande et al. 2004a, b; Vonnemann et al. mann-Kolb 2010; Holznagel et al. 2010;Jo¨rger et al. 2010; 2005; Grande et al. 2008; Klussmann-Kolb et al. 2008; Dayrat et al. 2011; Dinapoli et al. 2011). Dinapoli and Klussmann-Kolb 2010; Dinapoli et al. 2011). Fe´russac (1819) introduced the concept of ‘Geophila’ for Hygrophila: As per Bouchet and Rocroi (2005), informal uniting Onchidiidae, Veronicellidae and Stylommatophora. group ‘Basommatophora’ consists of a clade Hygrophila. The synapomorphies that support the monophyly of the This is divided into four superfamilies, namely Chilinoidea, Geophila are the location of the eyes at the tip of posterior Acroloxoidea, and Planorboidea. Chilinoidea tentacles, acquisition of a long pedal gland located upon the includes two families, Chilinidae and Latiidae. Planorboidea floor of the visceral cavity and the presence of an additional includes two families, Planorbidae and Physidae (Bouchet unpaired jaw (Dayrat and Tillier 2002). Veronicellids are and Rocroi 2005). In the present study, Hygrophila is characterized by the presence of several unique anatomical monophyletic. It was also recovered monophyletic in the features related to the reproductive system (distinctive penial studies of Klussmann-Kolb et al.(2008); Dinapoli and apparatus and a female pore on the right hyponotum) Klussmann-Kolb (2010); Holznagel et al.(2010); Jo¨rger (Dayrat et al. 2011). Onchidiids are without an internal et al.(2010); Dinapoli et al.(2011); Golding (2012). Inter- shell. They are and are predominantly estingly, Hygrophila is nonmonophyletic in the study of inhabit marine , although few of them inhabit terres- Dayrat et al.(2011). trial and brackish (Dayrat 2009). Further, Onchids exhibit the secondary loss of pulmonary vessels and an Lymnaeidae: Lymnaeidae formed a monophyletic clade (fig- unpaired jaw (unique to Geophila) (Dayrat and Tillier 2002). ures 1&2). Lymnaeidae and Lymnaeoidea were also recov- Occurrence of albumen gland spatially separated is found ered monophyletic in the study of Dayrat et al. (2011). In the only in onchidiids and ellobiids, and is not seen in studies dealing with the phylogeny of Heterobranchia at the Opisthobranchia. The process of spermatogenesis and sper- broad level, Lymnaeoidea is poorly represented (Klussmann- matozoids in onchidiids is similar to Pulmonata than with Kolb et al. 2008; Dinapoli et al. 2011) and in some, they are Opisthobranchia (Tillier 1984). not represented (Thollesson 1999; Dinapoli and Klussmann- The concept of Geophila is rejected in the present study Kolb 2010). A comprehensive evaluation of the phylogeny due to the emergence of Stylommatophora and Systellom- of Lymnaeidae was carried out by Correa et al.(2010)by matophora in different clades. Veronicellidae recovered means of 16S, ITS-1 and ITS-II. Like other major groups of monophyletic (figures 1&2). It was also recovered mono- Heterobranchia, intrafamilial relations of Lymnaeidae are phyletic in the study of Dayrat et al.(2011). Onchidiidae obscure due to homoplasy of anatomical characters (Correa recovered monophyletic in the present study (figures 1&2). et al. 2010). Onchidioidea recovered monophyletic in the studies of Vonnemann et al.(2005); Grande et al.(2008) and Kluss- Ancylidae and Planorbidae (Ancyloplanorbidae): Ancylidae and mann-Kolb et al. (2008). Planorbidae are morphologically distinct on the basis of the shape of the shell and also on the basis of the anatomy Basommatophora: This is the largest assemblage of pul- of reproductive organs. However, Hubendick (1978) sug- monates that live in freshwater habitats. It contains *300 gested the union of Ancylidae and Planorbidae on the species; of these, Lymnaeidae, Physidae and Planorbidae basis of the characters of the reproductive organs and comprise *90% of the total species in Basommatophora created a new family ‘Ancyloplanorbidae’ (Jørgensen (Correa et al. 2010). Members of this group, e.g. et al. 2004). 17 Page 10 of 14 Vijaya Sai Ayyagari and Krupanidhi Sreerama

Ancyloplanorbidae is monophyletic both in BI (figure 1) and ML (figure 2) analyses. In the study of Jørgensen et al. (2004), Ancyloplanorbidae emerged paraphyletic due to the association of Acroloxidae with Ancylidae. Further, Lae- phora in vapex fuscus (Ancylidae) emerged within Planorbidae sep- arately from Ancylus fluviatilus (Ancylidae) (figure 1). This is almost similar to the results obtained by Morgan et al.

bility (PP) and bootstrap (2002), where monophyletic Ancylidae emerged within Planorbidae. 28S

? Molecular evolution 18S Systellommatophora (no bootstrap support) ? ? Euthyneura: The common ancestor of Euthyneura was originated in the Carboniferous period of the Paleozoic era around 329 mya (figure 1). However, this is in contrast to the results from the study of Dinapoli (2009), where the Sacoglossa

? point of origin of Euthyneura was traced back to the upper Triassic period. The present result defies the proposal of the origin of Euthyneura ‘not before Mesozoic’ (Fryda et al. 2008; Dinapoli 2009). BI analysis (0.7). InSiphonarioidea ML analysis, it emerged as a sister clade to analysis (no bootstrap support) and ‘Siphonarioidea’ Pulmonata: The common ancestor for Pulmonata was traced Paraphyletic Monophyly rejected due to variable positions of ‘Amphiboloidea’ back to the Lower Triassic period (figure 3). This is in contrary to the views of Kiel and Bandel (2001); Dinapoli (2009), where it was believed that Pulmonata was nonexis- tent in Triassic. In the study of Dinapoli and Klussmann- Kolb (2010), the common ancestor for Pulmonata was traced back to lower . Pulmonata further diverged to Hygrophila and to rest of the pulmonate taxa in the upper and middle Triassic periods, respectively (figure 3).

Hygrophila: The common ancestor for Hygrophila originated in the upper Triassic period (*218 mya) (figure 3). How- ever, in the study of Dinapoli (2009), Hygrophila repre- sented by Acroloxus lacustris (Acroloxoidea) and (Chilinoidea) is nonmonophyletic and originated in upper Cretaceous. Lymnaeidae originated in the upper

COI (codons 1 and 2 combined) 16S Jurassic period around 144.9 mya (figure 3). This was sim- ? ilar to the fossil age set as a calibration point for Lymnaei-

28S dae. Remigio and Blair (1997) estimated the evolutionary ? origin of Lymnaeidae in a phylogenetic frame work on the 18S

? basis of 16S rDNA. They proposed the origin for Lymnaeids in the upper Jurassic; similar to the findings of the present 16S study. Planorbidae (paraphyletic) originated around 111 mya in the lower Cretaceous period (figure 3). This is in deviance (no bootstrap support) ‘Amphiboloidea’ and ‘Siphonarioidea’ with the fossil records of Planorbidae, which suggests its origin around 200–300 mya (Jørgensen et al. 2004).

Siphonariidae: Siphonariidae originated in the upper Creta- ceous period (figure 3). The result is in line with the fossil record of Siphonaria, which dated back to upper Cretaceous

Comparison of the results obtained in the present study for phylogenetic analyses on concatenated dataset with and without COI gene. Posterior proba (Dayrat et al. 2011). In the study of Dinapoli (2009), Siphonarioidea originated around 16.38 mya; whereas in the case of Medina et al.(2011), Siphonariidae originated in the 1 Heterobranchia2 Pulmonata Monophyletic Paraphyletic in BI analysis; Monophyletic in ML analysis Monophyletic 3 Eupulmonata4 Stylommatophora5 Systellommatophora Monophyletic Monophyletic6 Monophyletic in Ellobioidea7 BI and Paraphyletic Otinoidea in ML analysis Monophyletic in BI analysis. Unresolved in Monophyletic ML (0.8/70); analysis emerged as sister clade to Systellommatophora Monophyletic Paraphyletic (0.9/63); emerged as sister clade to Systellommato Paraphyletic in BI analysis. Unresolved in ML analysis Monophyletic Monophyletic in BI analysis (0.9); paraphyletic in ML 8 Hygrophila9 Basommatophora Monophyly rejected due to variable Monophyletic positions of Monophyletic 11 Planorbidae Paraphyletic Paraphyletic 10 Lymnaeoidea Monophyletic Monophyletic Table 2. (BS) values were indicated in parenthesis as (PP/BS). period (282 mya). Phylogeny and evolution of Pulmonata Page 11 of 14 17

Figure 3. Maximum clade credibility tree displaying the ages (in million years) of the nodes estimated in BEAST obtained from the concatenated dataset II consisting of 16S, 18S, 28S and COI (codons 1 and 2 combined). Node ages are given above the nodes, posterior probability values are given below the nodes.

Stylommatophora: Stylommatophora originated in the middle stylommatophorans appeared with certainty in the upper Cre- Jurassic period (166 mya) (figure 3). Subsequent divergences taceous period. However, Bandel (1997) later proposed the of common ancestors as well as the appearance of few species appearance of Stylommatophora during per- were seen from the early Cretaceous onwards until of iod. Results from the present study do not support Bandel and Cenozoic era. As per Bandel and Riedel (1994), Riedel (1994), and Bandel (1997) due to the origin of 17 Page 12 of 14 Vijaya Sai Ayyagari and Krupanidhi Sreerama

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