Evolution of Reproductive Strategies in the Species-Rich Land Snail Subfamily Phaedusinae (Stylommatophora: Clausiliidae)

Title Evolution of reproductive strategies in the species-rich land snail subfamily Phaedusinae (Stylommatophora: Clausiliidae)

Authors Mamos, Tomasz; Uit de Weerd, Dennis; von Oheimb, Parm Viktor; Sulikowska-Drozd, Anna

Date Submitted 2021-03-08 Molecular Phylogenetics and Evolution 158 (2021) 107060

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Molecular Phylogenetics and Evolution

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Evolution of reproductive strategies in the species-rich land snail subfamily Phaedusinae (Stylommatophora: Clausiliidae)

Tomasz Mamos a,b, Dennis Uit de Weerd c,d, Parm Viktor von Oheimb e,f, Anna Sulikowska-Drozd a,* a University of Lodz, Faculty of Biology and Environmental Protection, Department of Invertebrate Zoology and Hydrobiology, Banacha 12/16, 90-237 Lodz, Poland b University of Basel, Zoological Institute, Vesalgasse 1, 4051 Basel, Switzerland c Faculty of Science, Department of Environmental Sciences, Open Universiteit, P.O. Box 2960, NL-6401 DL Heerlen, the Netherlands d Naturalis Biodiversity Center, P.O. Box 9517, NL-2300 RA Leiden, the Netherlands e Life Sciences Department, The Natural History Museum, Cromwell Road, London SW7 5BD, United Kingdom f Museum für Naturkunde – Leibniz Institute for Evolution and Biodiversity Science, Invalidenstraße 43, 10115 Berlin, Germany

ARTICLE INFO ABSTRACT

Keywords: Most of the present knowledge on animal reproductive mode evolution, and possible factors driving transitions Viviparity between oviparity and viviparity is based on studies on vertebrates. The species rich door snail (Clausiliidae) Oviparity subfamily Phaedusinae represents a suitable and unique model for further examining parity evolution, as three Embryo-retention different strategies, oviparity, viviparity, and the intermediate mode of embryo-retention, occur in this group. Repeated evolution The present study reconstructs the evolution of reproductive strategies in Phaedusinae based on time-calibrated Molecular phylogenetics Gastropoda molecular phylogenetics, reproductive mode examinations and ancestral state reconstruction. Our phylogenetic analysis employing multiple mitochondrial and nuclear markers identifieda well-supported clade (including the tribes Phaedusini and Serrulinini) that contains species exhibiting various reproductive strategies. This clade evolved from an oviparous most recent common ancestor according to our reconstruction. All non-oviparous taxa are confined to a highly supported subclade, coinciding with the tribe Phaedusini. Both oviparity and viviparity occur frequently in different lineages of this subclade that are not closely related. During Phaedusini diversifi cation, multiple transitions in reproductive strategy must have taken place, which could have been promoted by a high fitness of embryo-retaining species. The evolutionary success of this group might result from the main tenance of various strategies.

1. Introduction 2019). Yet, the selective factors that led to the evolution of viviparity are not well understood. In squamates, viviparity might have evolved as an The evolution of novel phenotypes that provide access to new adaptation to cold climate where viviparous females can use thermo ecological niches can increase the diversificationrate of lineages (Yoder regulatory behaviour to shorten embryonic developmental time and to et al., 2010). One key innovation, which may stimulate reproductive reduce the exposure of embryos to stressful environmental conditions isolation and rapid speciation, is viviparity, a reproduction strategy with (Shine, 1983). This so-called cold-climate hypothesis, linking viviparity extended investment per single progeny and release of live young (Zeh with lower temperatures characteristic for high latitudes and altitudes, and Zeh, 2000, Helmstetter et al., 2016). Transitions from oviparity to acquired much but not unequivocal support (Hodges, 2004, Watson viviparity attracted much attention as important evolutionary changes et al., 2014, Ma et al., 2018). This explanation is likely not applicable to in the phylogenies of several animal taxa, mainly vertebrates (e.g. most non-squamate animals, yet the herpetological perspective pre Reynolds et al., 2002, Mank and Avise 2006, Warren et al., 2008, Pyron dominates in studies on the evolution of parity. It is thus necessary to and Burbrink, 2014, Furness and Capellini, 2019, Horreo et al., 2019). employ research on further, phylogenetically distant animal groups, Viviparity has originated around 150 times among vertebrates, while where transitions to viviparity are less known but common (e.g. Meier species with bimodal reproduction are extremely rare (Horreo et al., et al., 1999, Wong et al., 2013, Ostrovsky et al., 2016), to provide further

* Corresponding author. E-mail address: [email protected] (A. Sulikowska-Drozd). https://doi.org/10.1016/j.ympev.2020.107060 Received 24 July 2020; Received in revised form 22 December 2020; Accepted 24 December 2020 Available online 28 December 2020 1055-7903/© 2021 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). T. Mamos et al. Molecular Phylogenetics and Evolution 158 (2021) 107060 insights into selection pressures driving switches in reproductive mode. the oviparous Megalophaedusini and the ‘ovoviviparous’ Phaedusini In aquatic gastropods, for example, the evolution of viviparity may have tribes; the latter group was characterised by a broad clausilium plate. In had an effect on ecological and evolutionary success. The relatively his subsequent publications, this taxonomical division was not main common occurrence of this reproductive mode in freshwater snails as tained (Nordsieck, 2007, 2011). Meanwhile, evidence was provided that opposed to marine snails (Glaubrecht, 2006), can be seen as an adap a wide patency of apertural barriers is a common adaptation to the de tation to a harsher and less predictable habitat (Kohler¨ et al., 2004). livery of live young in clausiliids of different genera (Sulikowska-Drozd While it is widely accepted that oviparity is the ancestral condition in et al., 2014). This is congruent with growing awareness of common Gastropoda and the most common mode of reproduction in land snails, homoplasy in these complex structures (van Moorsel et al., 2000, Uit de the fertilized eggs of some taxa undergo a partial or complete embryonic Weerd et al., 2004). Recent findingsindicated a link between phylogeny development in the reproductive tract (Tompa, 1979ab, Heller, 2001, and reproduction mode for Phaedusinae from Japan (Motochin et al., Ostrovsky et al., 2016). This strategy is known as embryo-retention but 2017). Among six main clades, two included only oviparous and two it corresponds to viviparous reproduction sensu Blackburn (1999) if live only ‘ovoviviparous’ taxa; in the remaining two clades both strategies young are released; the latter has also been termed ‘ovoviviparity’ occurred (Motochin et al., 2017; note that embryo-retaining species (Tompa, 1979ab, Nordsieck, 2002, Maltz and Sulikowska-Drozd, 2008). were not distinguished and possibly regarded as oviparous). The lack of suitable oviposition sites in certain habitats, such as exposed Given this background, the present study aims to reconstruct the rock-walls, was hypothesised as a factor stimulating the evolution of evolution of reproductive strategies in the door snail subfamily Phae viviparity in land snails (Baur, 1994). The frequent occurrence of this dusinae based on a combination of time-calibrated molecular phyloge reproductive mode in arboreal gastropods from tropical regions, e.g. netics, reproduction mode examinations, and ancestral state Achatinellidae and Partulidae, may have the same cause (Cowie, 1992, reconstruction. Baur, 1994, Price et al., 2015). Data on reproductive traits in land snails are fragmentary but suggest 2. Material and methods independent origins of embryo-retention and viviparity in approxi mately 30 families of Stylommatophora (Tompa, 1979a). On a finer 2.1. Material studied scale, however, the evolution of parity has never been investigated in a phylogenetic framework. The species-rich stylommatophoran land snail Material was collected from various parts of Eurasia (Fig. 1a). Our family Clausiliidae (door snails) provides a suitable model for such dataset includes representatives of all three Phaedusinae tribes: Serru research, as three modes of reproduction occur in this group: oviparity, linini, Phaedusini and Synprosphymini. Locality and specimen details embryo-retention (i.e. oviparity with extended embryonic development including specimen images are available in the Barcode of Life Data in the reproductive tract) and viviparity (e.g. Loosjes, 1953, Likharev, Systems (BOLD) dataset DS-PHAEDUSA [dx.doi.org/https://doi.org// 1962, Maltz and Sulikowska-Drozd, 2008, Szybiak et al., 2015). Clau 10.5883/DS-PHAEDUSA]. siliids have already been the focus of evolutionary studies on shell Individual clausiliids used for DNA sequencing were either collected chirality and complex apertural barriers, including the unique structure in the wild (F0 generation) or from the F1 generation of laboratory that is the clausilium (e.g. van Moorsel et al., 2000; Uit de Weerd et al., colonies (see DS-PHAEDUSA). For 12 Phaedusa angustocostata and 2004; Uit de Weerd et al., 2006; Kornilios et al., 2015). Phaedusa ramelauensis specimens, DNA isolates were provided by F. Clausiliids, which are distributed in Eurasia, Africa, South America Kohler¨ (see Kohler¨ and Mayer, 2016 for extraction methodology) and for and two of the Greater Antilles (Hispaniola and Puerto Rico), have 11 of them, COI sequences from Kohler¨ and Mayer (2016) were used (see become subject of several molecular phylogenetic studies (e.g. Uit de DS-PHAEDUSA). Weerd and Gittenberger, 2013; Feher´ et al., 2013; Motochin et al., The present study follows the suprageneric classificationof Bouchet 2017). Among these, the work of Uit de Weerd and Gittenberger (2013) et al. (2017). At genus-level and below, we follow Motochin et al. (2017) included all major lineages and largely verified the traditional, mostly for all taxa revised by them, and Nordsieck (2007) for the remaining shell-based classification into subfamilies (see Zilch, 1954, Nordsieck, taxa, with the single exception of the genus Renschiphaedusa, which is 1978, 2007). As a result, seven recent subfamilies of Clausiliidae regarded as a synonym of Phaedusa following Kohler¨ and Mayer (2016). (Clausiliinae, Alopiinae, Garnieriinae, Laminiferinae, Neniinae, Perui Subgenus and subspecies names are only used for selected taxa. niinae, Phaedusinae) are currently recognized (Bouchet et al., 2017). The Phaedusinae, which comprise the three tribes Serrulinini, 2.2. DNA processing and analysis Phaedusini and Synprosphymini (Bouchet et al., 2017), and which include around 600 known species (Nordsieck, 2007; Hunyadi and DNA was isolated from a total of 141 ethanol-preserved individuals. Szekeres, 2016, Pall-Gergely´ and Szekeres, 2017), are the most speciose We amplifiedseven markers: two mitochondrial markers, the barcoding subfamily of clausiliids (Nordsieck, 2007). The range of this subfamily fragment of the cytochrome c oxidase subunit I gene (COI, ca. 650 bp) consists of two isolated parts: (1) western Eurasia (Pontic-Caucasian- and a fragment of the 16S ribosomal RNA gene (ca. 390 bp), and five Hyrcanian region) harbouring Serrulinini, and (2) East and Southeast nuclear markers, fragments of the 28S ribosomal RNA (ca. 950 bp), Asia harbouring Phaedusini and Synprosphymini. Molecular studies histone H3 (326 bp) and histone H4 (266 bp) genes, and the complete have so far yielded only limited insight into the phylogenetic relation internal transcribed spacers 1 (ITS1, ca. 650 bp) and 2 (ITS2, ca. 300 ships within Phaedusinae and respective diversification ages. Uit de bp); see Appendix, Tables S1 and S2 for details. Weerd and Gittenberger (2013) showed that the East and Southeast Obtained sequence data were tested for contamination via BLASTN Asian tribe Phaedusini constitutes a well-supported clade nested within (Altschul et al., 1990) and verified as Clausiliidae. Heterozygous sites the western Eurasian Serrulinini. According to their study, the lineage were only observed for ITS1 and ITS2; in such cases, only one version leading to the Phaedusini arose 22.7 Ma in western Eurasia and spread was used. GENEIOUS 6.1.4 (https://www.geneious.com; Biomatters eastwards prior to the aridification of the interlaying region. Pre- 2013) was used for sequence assembly, editing and quality trimming Pleistocene fossils of Phaedusinae are known from western Eurasia (the latter included the removal of the partial H3-H4 spacer region from (Europe north of the Tethys) only (Nordsieck, 2015). the H3 and the H4 sequences). All generated and processed sequences The diversity of reproduction modes in Phaedusinae was reported by were deposited in GenBank and are available in DS-PHAEDUSA Loosjes (1953), Nishi (1980), Minato (1983, 1994), Nordsieck (2001, including metadata. Altogether, 682 new DNA sequences were gener 2002), and Sulikowska-Drozd et al. (2020). Nordsieck (1998, 2001, ated in this study (125 sequences of COI, 126 of 16S, 98 of 28S, 71 of 2002) regarded certain shell characters to be correlated with repro ITS1, 52 of ITS2, 103 of H3 and 107 of H4). ductive modes and distinguished in his taxonomical system at that time Alignments of sequence data, including those for subsequent

2 T. Mamos et al. Molecular Phylogenetics and Evolution 158 (2021) 107060

Fig. 1. Sampling localities and phylogeny of studied taxa. (a) Sampling localities of studied Phaedusinae specimens with information on reproductive strategies (legend in figure);coloured ellipses indicate geographical areas. (b) Examples of Phaedusinae species with different reproduction strategies including respective eggs or neonates; from the left: Oospira vanbuensis, O. formosensis, and Tauphaedusa sheridani. (c) Time-calibrated Bayesian maximum clade credibility tree of Phaedusinae, Garnieriinae and outgroup taxa (not shown) based on sequence data from 7 markers (main analysis). Node ages were inferred from common ancestral heights. Bayesian posterior probabilities (only values ≥ 0.50 shown) and bootstrap values (only values ≥ 50 shown) from Maximum Likelihood framework (see Fig. S5b) are provided at the respective nodes. For outgroup taxa, highest posterior density, and sample IDs see Fig. S4. Circles at branch tips indicate present reproduction modes (no circle: unknown), pie charts at nodes indicate ancestral reproduction strategies based on the maximum parsimony reconstruction (black: oviparous, grey: embryo-retaining, white: viviparous). Taxon names are highlighted in colour according to the geographical area of sampling localities, as shown in (a). analyses, were performed using MAFFT with automatic settings select 2018) for the full datasets of each single-marker (Fig. S1). In order to ing the best algorithm for each dataset (Katoh et al., 2005; all alignments assess potential discrepancies in phylogenetic signal between different are deposited in the Dryad repository). The full datasets of all single genes, individual phylogenies were reconstructed using the full datasets markers (see DS-PHAEDUSA) were tested for substitution saturation of each single-marker in a Maximum Likelihood (ML) approach through with DAMBE 5.3 (Xia, 2013) using the index proposed by Xia et al. RAxML 8.2.8 (Stamatakis, 2014). The best-scoring ML trees were pro (2003). The test revealed little saturation under the assumption of a duced using the GTR + I + G substitution model. Bipartition information symmetrical tree for all markers and for most markers also under the was drawn from the phylogenies obtained with the rapid hillclimbing assumption of an asymmetrical tree (Table S3). Molecular distances tree search algorithm. Statistical supports were estimated with thorough were presented as trees using the Neighbor-Joining method (Saitou and bootstrap tests set to 1000 repetitions. These analyses did not reveal Nei, 1987) with the Kimura 2-parameter model (Kimura, 1980) and substantially conflicting patterns between individual markers, while 1000 bootstrap replicates (Felsenstein, 1985) in MEGA X (Kumar et al., they provided a generally poor resolution for deeper divergences

3 T. Mamos et al. Molecular Phylogenetics and Evolution 158 (2021) 107060

(Fig. S2). culture; egg incubation lasts shorter than 2 weeks; eggs with shelled embryos found in dissected animals, see Fig. 1b for examples). 2.3. Reconstruction of time-calibrated phylogeny We furthermore attempted to classify taxa according to their repro ductive mode by means of X-rays of individuals (see taxa with data For the reconstruction of a time-calibrated phylogeny, the full source ‘X-ray studies’ in Table S6). The X-ray studies were carried out in dataset of sequences (153 individuals; see DS-PHAEDUSA) was reduced. the μ-CT laboratories of the Institute of Palaeontology, Polish Academy This reduced dataset (58 individuals; see individuals labelled in black in of Science, Warsaw, Poland, and of the University of Silesia, Chorzow,´ Fig. S3) comprised a single individual per taxon except for Mega Poland. This method allowed to rule out oviparous reproduction lophaedusa pinguis platyauchen, Phaedusa cf. lypra, Phaedusa stenothyra, whenever X-rayed individuals were gravid with shelled embryos (see Leptacme sykesi and Megalophaedusa (cf.) martensi, which showed rela Sulikowska-Drozd et al., 2020). For distinguishing embryo-retaining tively high levels of intraspecific variation, and Phaedusa ramelauensis, and viviparous taxa, embryo size and size distribution were taken into where euphallic and aphallic specimens were studied (see DS- account; embryonic shells of more than 2 whorls were regarded as ev PHAEDUSA). For these taxa, 2–3 individuals were included. idence for viviparity. All specimens that were used for X-ray study were In order to apply the molecular clock calibration of Uit de Weerd and also utilized for DNA sequencing. For Phaedusa angustocostata and Gittenberger (2013) to our data, we combined the reduced dataset of P. ramelauensis, viviparity was assumed based on Kohler¨ and Mayer 28S, H3 and H4 markers, with the dataset from Uit de Weerd and Git (2016). tenberger (2013) and carried out an initial phylogenetic analysis following Uit de Weerd and Gittenberger (2013) (see Appendix, Tables 2.5. Reconstruction of ancestral reproduction strategies S4, S5 and Fig. S3). In addition, a ML analysis was performed in RAxML 8.2.8 using the same settings as for the single-markers phylogeny re While changes in reproductive strategy took place in various animal constructions (Fig. S5a). Based on the results from this initial analysis, a groups over evolutionary times, such transitions are presumably rela set of three secondary calibration points was selected for following main tively rare events. Based on this assumption, a maximum parsimony analysis (Table S4, Fig. S3). approach was chosen to reconstruct the evolution of reproductive stra For the main phylogenetic analysis, we combined the reduced tegies among Serrulinini and Phaedusini. Synprosphymini were not dataset with fiveindividuals from the subfamily Clausiliinae from Uit de included in this analysis given that no data on their reproduction were Weerd and Gittenberger (2013) (see Appendix, Fig. S3). The phylogeny available. The analysis was run in the program Mesquite 3.6 (Maddison was reconstructed using Bayesian Inference in BEAST 2.5 (Bouckaert and Maddison, 2018). ‘Oviparous’, ‘embryo-retaining’ and ‘viviparous’ et al., 2019), using all 7 markers produced in the study, for details see were coded as ordered states in a multistate analysis, with embryo- Appendix. All single markers were treated as separate partitions retaining treated as an intermediate state between oviparous and following the partitioning scheme selected by PartitionFinder 2.1.1. viviparous. The character states were mapped on a subset of 1000 trees (Lanfear et al., 2012), evolutionary models were selected using bMo from the main analysis using multiple maximum parsimony re deltest (Bouckaert and Drummond, 2017; results in Table S5). The constructions (MPR option) per tree. The results were summarized on Effective Sampling Size of each parameter was verifiedto be above 200 the consensus tree from the main analysis. In addition, we calculated the in Tracer 1.7 (Rambaut et al., 2018). In order to assess bootstrap support number of transitions between the alternative reproductive strategies values (BSVs), we used the ML approach in RAxML 8.2.8 with the same for each of the 1000 trees, using the Pscores command in PAUP* 4.0 settings as for the single-markers phylogeny reconstructions. (Swofford, 2001).

2.4. Identification of reproductive strategies 3. Results

Living individuals of 33 Phaedusinae taxa (see taxa with data source 3.1. Time calibrated reconstruction of phylogeny ‘Laboratory culture’ in Table S6) were held in laboratory culture at the Dept. Invertebrate Zoology and Hydrobiology, University of Lodz, Our phylogenetic analysis (Fig. 1c; see Fig. S4 for HPD and Fig. S5b Poland, in order to identify their reproductive strategy. Observations for ML results) supports the monophyly of a group formed by the Ser were conducted on individuals kept in climatic chambers (POL-EKO rulinini and the Phaedusini (clade SE + PH; BPP = 1.0, BSV = 96). The ◦ KK350 TOP) under controlled temperature (15–21 C) and natural light phylogenetic relationships of the Synprosphymini remain less clear; our conditions. Individuals from different populations were bred separately, analysis positions them as sister group of the Garnieriinae, however, summing up to 42 laboratory colonies. Snails were kept in pairs or without good support (BPP = 0.92, BSV = 57). groups of three in plastic containers (300 cm3) with cellulose tissue and Within clade SE + PH, the studied representatives of the Serrulinini fragments of limestone as source of calcium, and were fed with lettuce, form two successively basal lineages. The more basal of them consists of cucumber and zucchini. Humidity in the containers was almost constant Pontophaedusa funiculum and diverged from its sister group in the Late and close to 100% due to regular spraying. During the firstmonths of the Eocene, 37.3 Ma (95% HPD: 48.9–25.8). The less basal lineage, which is study, the containers were inspected every 2–3 days for the presence of composed of Caspiophaedusa perlucens and Pravispira semilamellata, eggs and neonates, which were immediately transferred to separate diverged from its sister group in the Oligocene, 33.2 Ma (95% HPD: containers with damp cellulose tissue. Freshly deposited eggs were 43.6–23.3). The remaining lineages form the well-supported clade PH checked every three days for hatching. Later, the reproduction was (BPP = 1.0, BSV = 93, 95% HPD: 36.8–20.0), which corresponds to the monitored weekly and progeny was preserved in ethanol. Additionally, tribe Phaedusini. This group diversified into eight major lineages mature individuals (3–10 per taxon) were preserved in ethanol and (PH1–PH8) in the Oligocene, with largely unresolved interrelationships. dissected to determine whether they retained developing embryos in the Five of those lineages (PH1, PH3, PH4, PH7 and PH8) lead to supra reproductive tract. Each taxon was observed for at least one year. Based specific clades, each well-supported in our analysis (BPP ≥ 0.96, BSV on these long term observations and dissections, taxa were classifiedinto 66–100). Clade PH1, which is composed of species from Vietnam, China three categories according to their reproductive mode (see Sulikowska- and East Timor, diversified mainly through the Miocene. In this clade, Drozd, 2009): viviparous taxa (litters of neonates observed regularly in Euphaedusa porphyrea is the sister group of all other taxa, which form culture; large shelled embryos found in dissected animals), oviparous two subclades. Clade PH2 only consists of Acanthophaedusa ookuboi from taxa (clutches of eggs observed regularly in culture; egg incubation lasts China. Lineages PH3–PH6 are composed of species living in Vietnam. 2 weeks or longer; shelled embryos never found in dissected animals) The most species-rich group of these in our analysis is PH4 with a and embryo-retaining taxa (clutches of eggs observed regularly in diversification spanning through the Miocene. Lineage PH3 contains

4 T. Mamos et al. Molecular Phylogenetics and Evolution 158 (2021) 107060

Hemiphaedusa polydona and Hemiphaedusa billeti, which split in the viviparous reproduction and an arboreal lifestyle among Phaedusini Miocene. Lineage PH5 only consists of P. stenothyra and lineage PH6 is from Japan. As none of the oviparous species studied by Motochin et al. represented by the single species Oospira antibouddah. Lineage PH7, (2017) was regarded as a tree-dweller, it may be assumed that the snails which diversified during the Miocene, includes only species found in require soil for egg-laying. This would be in line with the hypothesis of Taiwan. Clade PH8, which began to diversify 25.1 Ma (95% HPD: Baur (1994), that viviparity can allow reproducing successfully in hab 17.41–32.46), comprises numerous species from Japan, Vietnam, and itats unavailable for oviparous land snail species. Outside Japan, how Taiwan and consists of four lineages (J1–J4) with poorly resolved in ever, this pattern is not universal given that the oviparous species terrelationships. Clade J1, which includes several species from all three Oospira vanbuensis is arboreal (personal observation). Besides, we as areas, diversified in the Miocene according to our analysis. Within the sume that so-called arboreal clausiliids retreat to the soil during the dry clade, Tauphaedusa sheridani from Taiwan and Tauphaedusa tau from or winter season, both in Japan and in Indochina. Japan form the sister group of all other taxa. Lineage J2 consists of While viviparous animal species may produce less progeny than their species of the genus Reinia from Japan and Taiwan, which diverged in oviparous relatives, the survival rate of their offspring is presumably the Pleistocene, and lineage J3 consists only of Changphaedusa horikawai higher (see Wourms, 1981 for fishes). For clausiliids, however, such an from Taiwan. Clade J4, which includes only species from Japan, diver evolutionary trade-off has not yet been found (Sulikowska-Drozd, 2009, sified in the Miocene. This clade consists of two subclades that corre Szybiak et al., 2015, Sulikowska-Drozd et al., 2018a,b); for species spond to the genera Stereophaedusa and Megalophaedusa. differing in reproduction mode, the available evidence does not suggest considerable fecundity differences (neither observed in laboratory 3.2. Reproduction strategies in studied Phaedusinae and reconstruction of conditions nor in population dynamics in the field). ancestral reproduction strategies Oviparity is likely the ancestral mode of reproduction in stylomma tophoran land snails (Tompa, 1979ab, Heller, 2001) and it predominates Among the studied Phaedusinae taxa, we identified16 as oviparous, in clausiliids (Maltz and Sulikowska-Drozd, 2008). All studied Serruli 18 as viviparous and three as embryo-retaining; we left 12 taxa without nini species are exclusively oviparous and, to our knowledge, no vivip conclusively determined reproductive strategy (Fig. 1a, c, Table S6). arous species have been identifiedin this tribe. Two oviparous lineages Focussing on the well-supported clade of Phaedusini and Serrulinini are basal to the Phaedusini in our phylogenetic analysis (Pontophaedusa (clade SE + PH), none of the reproduction modes was uniquely syna and Caspiophaedusa/Pravispira) and, based on our reconstruction, this pomorphic for a lineage within this clade. According to our ancestral reproductive strategy is ancestral for the whole clade SE + PH. It should state reconstruction (Fig. 1c, Table S7), clade SE + PH evolved from an be noted, however, that the Serrulinini exhibit a significantvariation in oviparous most recent common ancestor. This state was found as most the structure of egg envelopes, which is unique among oviparous clau parsimonious for the respective node in each of the 1000 trees. Our data siliids. In contrast to the typical, partly calcified clausiliid egg, where are less clear about the ancestral reproductive strategy of the Phaedusini discrete crystals of CaCO3 are dispersed randomly in the jelly layer (clade PH). The most often found ancestral state for this group across the (Tompa, 1976, Maltz and Sulikowska-Drozd, 2008), Pontophaedusa 1000 trees examined was oviparous (50%), followed by viviparous funiculum produces a hard egg envelope (Pall-Gergely´ and Nemeth,´ (39%) and embryo-retaining (11%). Of the major subclades distin 2008). The spiral deposition of calcium crystals in the egg membranes of guished within this clade, clade PH1 evolved from a viviparous ancestor, Caspiophaedusa and Pravispira also stands out from the usual pattern according to our analyses, a condition that was reversed in the oviparous (Stolarski et al., 2019). Further insights in the evolution of reproduction Oospira vanbuensis. Clade PH3 consists entirely of taxa without conclu strategies among the Phaedusinae might be gained from the Synpros sively determined reproductive strategy. The ancestral states of clades phymini, whose reproduction biology remains still unknown. Future PH4, PH7, and PH8 differ among trees. For clade PH8, the result of our studies should thus focus particularly on this group. analysis was 40% oviparous, 14% embryo-retaining and 43% viviparous The reproductive mode of the most recent common Phaedusini (3% node absent). The total number of transitions in reproductive ancestor remains ambiguous in our analysis (only 50% of the trees strategies within clade SE + PH was between 13 and 16. support oviparity) due to the insufficientresolution of the relationships between lineages PH1–PH8. The proportion between species of different 4. Discussion reproductive modes in the whole clade is yet to be surveyed. For the Phaedusini subclade PH8, which contains species from the 4.1. Reproductive modes of recent Phaedusinae and inference of ancestral Japanese Archipelago and Taiwan, our dataset partly overlaps with the strategies molecular phylogeny of Motochin et al. (2017). In the subset of Japanese clausiliids included in our analysis, oviparous species predominate, yet, We showed that similar reproduction modes evolved independently all sublineages (J1–J4) include viviparous taxa. Our results confirmthe among the studied Phaedusinae. Both oviparity and viviparity occur relatively close relationship of the exclusively viviparous genus Tau frequently in not closely related lineages of the East and Southeast Asian phaedusa and the reproductively diverse genus Zaptyx. According to Phaedusini. According to our analysis multiple switches must have taken Motochin et al. (2017), the genus Zaptyx includes oviparous and place during the group’s diversification, which indicates a high evolu ‘ovoviviparous’ taxa. The former reproductive mode predominates, tionary plasticity. which could suggest its ancestral origin. The distribution of viviparity If viviparity would have evolved as an adaptation to cold climates, as among different subgenera of Zaptyx (Motochin et al., 2017) may indi it was suggested for squamates in the cold-climate hypothesis (Shine, cate a high evolutionary plasticity of parity-modes in this group. During 1983), one may expect viviparous snail species to be more common in laboratory observations, we found the intermediate strategy of embryo- colder regions. The majority of viviparous stylommatophoran species, retention for two Zaptyx species (Z. hachijoensis and Z. kikaiensis), which however, occurs in the tropics and subtropics (Tompa, 1979b) and were classified as ‘ovoviviparous’ and oviparous, respectively in Moto thermoregulatory behaviour has never been recorded in land snails. chin et al. (2017). This is in line with our hypothesis on the significant Acknowledging that a test for potential correlations between tempera role of embryo-retention in the repeated evolution of viviparity in ture and reproduction mode is beyond the scope of the present paper, Phaedusini (see below). Our ancestral state reconstruction showed that our study does not reveal an obvious latitudinal pattern in the distri viviparity or embryo retention could be ancestral for the entire J1 bution of different strategies among Phaedusini. This may suggest that lineage (59% and 39%, respectively). In contrast, a predominance of no strategy is clearly superior in the macroclimatic context. However, oviparity was found for the ancestor of lineage J4 (71%). From the two oviparous and viviparous species may differ in their distribution at groups within this lineage, Megalophaedusa and Stereophaedusa, the microhabitat level. Motochin et al. (2017) noticed a correlation between former includes only oviparous species, while the latter includes a

5 T. Mamos et al. Molecular Phylogenetics and Evolution 158 (2021) 107060 similar number of ‘ovoviviparous’ and oviparous species according to could have provided the Phaedusini access to new ecological niches and Motochin et al. (2017). Based on our analyses, which include only four thus boosted their diversification into all main lineages in a relatively Stereophaedusa species, oviparity is the most probable ancestral mode in short time window. The exact factors, which promote shifts between this group and at least two switches between reproductive strategies strategies, however, are yet to be discovered. have to be assumed. A remarkable findingof our study concerns clade PH1, which occurs 4.2. Phaedusinae systematics and biogeography in Southeast Asia. All except one of the species in this clade are vivip arous, and our analysis assumes a viviparous most recent common The present study challenges the division of the Phaedusinae into the ancestor of the clade in 100% of the analysed trees. The oviparous tribes Serrulinini, Phaedusini, and Synprosphymini (Bouchet et al., Oospira vanbuensis is deeply nested in clade PH1 with high support 2017). In our molecular phylogeny, the Serrulinini species Pontophae values, which indicates a reversal from viviparity to oviparity during the dusa funiculum forms the sister group to the Phaedusini and all other evolution of this species. To our knowledge, this is the firstevidence for Serrulinini. Our analysis thus confirms the paraphyly of Serrulinini, such a reversal of parity in land snails. suggested already by Nordsieck (2007) and shown by Uit de Weerd and A reversal to a previous reproductive strategy seems to be very rare Gittenberger (2013), and endorses the classificationof Pontophaedusa in in animals. In vertebrates, viviparity has evolved repeatedly but it is a separate tribe as suggested by Bouchet et al. (2017). Our phylogenetic highly disputed if oviparity can re-evolve (Blackburn, 2006, 2015, Pyron reconstruction also showed that the Synprosphymini represent a and Burbrink, 2014, King and Lee, 2015, Recknagel et al., 2018, Furness phylogenetically very distinct lineage, which may be more closely and Capellini, 2019). According to King and Lee (2015), a reversal may related to the Garnieriinae than to other Phaedusinae. Given that the be possible only for a finite time window after the evolution of vivi respective node was not well-supported and that the knowledge of parity, as an accumulation of mutations irreversibly degrades the ge deeper relationships in the family Clausiliidae remains limited (see Uit netic pathways related to oviparity. In squamates, for example, the de Weerd and Gittenberger, 2013), however, future studies expanding evolution of viviparity requires the loss of the eggshell, which protects the set of molecular markers are needed to confirm this result. Many the embryo from desiccation but restricts gas exchange if the embryo is Phaedusini genera that inhabit Southeast Asia (e.g. Oospira, Phaedusa) kept in the reproductive tract (Griffith et al., 2015). In consequence, do not represent monophyletic groups in our analysis and require a during reversal towards oviparity, the developing embryo may not be taxonomic revision. The changes introduced to the Phaedusinae system drought resistant enough to survive in ambient environments (Griffith by Motochin et al. (2017) are supported by our study. et al., 2015, Shine, 2015). Almost 200 Phaedusinae species and subspecies occur on the Japa In contrast, the egg envelope of land snails, which provides me nese Islands (Minato, 1994, Motochin et al., 2017), of which 16 were chanical support and calcium necessary for the embryonic shell forma included in our analysis. Our findings indicate that the most recent tion, does not hinder gas exchange and because of this, does not protect common ancestor of the clade that includes all Japanese taxa (clade from desiccation (Tompa, 1979ab). As a consequence, the egg envelope PH8) occurred at the end of the Oligocene, ca. 25 Ma, while intense may keep its basic structure during the shift towards viviparity. The diversification of Japanese lineages started about 15 Ma, which corre embryos of some viviparous clausiliids (e.g. Alinda biplicata, Idyla lates to the full isolation of the archipelago due to the opening of the bicristata and Stereophaedusa jacobiana) are initially enveloped in an egg Okhotsk Sea (ca. 20 Ma) and the Sea of Japan (ca. 15 Ma) (see Tojo et al., membrane with calcium carbonate crystals, which resembles the struc 2017 and references therein). The Taiwanese species Tauphaedusa ture of the partly calcifiedegg of oviparous taxa (Maltz and Sulikowska- sheridani clusters among the Japanese taxa, which suggests dispersal Drozd, 2012, Sulikowska-Drozd et al., 2018a,b, 2019). The presence of from Japan to Taiwan. Potential migration routes between Japan and these crystals suggests that the ability of egg shell production could be continental Asia via Taiwan developed about 5 Ma, and again during maintained in these species. In individuals of Reinia variegata, Tau Pliocene and Pleistocene glaciation peaks (Shih et al., 2006). The rela phaedusa tau and Changphaedusa horikawai that were dissected in the tively recent split between the Japanese and Taiwanese members of the present study, however, no calcium carbonate crystals were present in genus Reinia (ca. 2 Ma) suggests that dispersal along an ice-age land the egg membrane. While these data are still preliminary, such histo bridge may have been involved. However, migration might have also logical differences between viviparous taxa from different clades may be occurred over sea probably via ocean currents, as it was suggested by related to the multiple independent origins of their reproduction strat Motochin et al. (2017) for clausiliid species from isolated islands. The egy. Possible differences in egg membrane structure and in the sources same mechanism was also proposed for other terrestrial animal groups of embryo nutrition among viviparous taxa from different phylogenetic (Kobayashi et al., 2014, He et al., 2015). Notably in our phylogeny, lineages, should be investigated in future studies. Furthermore, future Zaptyx hachijoensis, which is endemic to the oceanic Izu Islands south research should focus on potential differences between the reproduction east of Japan’s main island Honshu, is relatively closely related to of Oospira vanbuensis and other oviparous Phaedusini in order to Messageriella gargomyi from southern Vietnam. This relationship sug examine whether the oviparity of this species really results from a gests dispersal in the opposite direction to the major Kuroshio ocean reversal of parity. current, probably via migrating birds, a mechanism that has already The frequent reproduction mode switches during Phaedusini evolu been suggested for other clausiliids (Gittenberger et al., 2006). tion could have been promoted by a high fitness of species with an in termediate strategy. Embryo-retention, was recorded in two major 5. Conclusion Phaedusini lineages (clades PH7 and PH8). Selection towards it was likely involved in the repeated shifts of reproductive modes. Embryo- The present study represents the first reconstruction of the evolu retention could represent a stepping stone, from which selection can tionary history of parity in land snails with advanced analytical tools. act in either direction, maintaining evolutionary plasticity. In embryo- Oviparity is likely the ancestral reproduction strategy of the clade con retaining species, the genetic heritage of their oviparous ancestors sisting of Phaedusini and Serrulinini (clade SE + PH). The diversification may still be preserved, while adaptations to a delayed delivery of em of Phaedusini is associated with many switches in reproduction mode. bryos have evolved. These adaptations may include serous subepithelial The evolutionary success of this group might thus not only depend on cells in the free oviduct, which secrete products that provide oncotic the evolution of viviparity but also on the maintenance of various pressure and create a suitable environment for embryos (Maltz et al., strategies. 2017), and a widening of the shell channel, which allows passing of Data accessibility shelled embryos (Sulikowska-Drozd et al., 2014). Newly produced sequences are released in GenBank under accession The evolutionary plasticity regarding their reproductive strategy numbers: COI MW378723 - MW378858; 16S, MW378859 - MW378984;

6 T. Mamos et al. Molecular Phylogenetics and Evolution 158 (2021) 107060

28S, MW379089 - MW379186; H3, MW378986 - MW379088; H4, Blackburn, D.G., 2015. Evolution of viviparity in squamate reptiles: Reversibility – MW379187 - MW379254 (sequences shorter then 200bp given in sup reconsidered. J. Exp. Zool. 324B, 473 486. https://doi.org/10.1002/jez.b.22625. Bouchet, P., Rocroi, J.-P., Hausdorf, B., Kaim, A., Kano, Y., Nützel, A., Parkhaev, P., plementary data 2) ; ITS1, MW379307 - MW379377; ITS2, MW379255 - Schrodl,¨ M., Strong, E.E., 2017. Revised classification, nomenclator and typification MW379306. All sequences, metadata, locality and specimen details of gastropod and monoplacophoran families. Malacologia 61, 1–526. https://doi. including specimen images are available in the BOLD dataset DS- org/10.4002/040.061.0201. Bouckaert, R., Vaughan, T.G., Barido-Sottani, J., Duchêne, S., Fourment, M., PHAEDUSA (dx.doi.org/10.5883/DS-PHAEDUSA). Alignments pro Gavryushkina, A., et al., 2019. BEAST 2.5: An advanced software platform for vided in supplementary data 2. 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