Comprehensive Comparison of Four Species of Onchidiidae Provides Insights on Morphological and Molecular Adaptations of Invertebrates from Shallow Seas to Wetlands

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Comprehensive comparison of four species of Onchidiidae provides insights on morphological and molecular adaptations of invertebrates from shallow seas to wetlands

Guolv Xu 1, 2, 3, 4 , Tiezhu Yang 1, 2, 3, 4 , Dongfeng Wang 1, 2, 3, 4 , Jie Li 1, 2, 3, 5 , Xin Liu 1, 2, 3, 4 , Xin Wu 1, 2, 3, 4 , Heding Shen Corresp. 1, 2, 3, 4

1 College of Fisheries and Life Science, Shanghai Ocean University, Shanghai, China 2 National Demonstration Center for Experimental Fisheries Science Education, Shanghai, China 3 International Research Center for Marine Biosciences at Shanghai Ocean University, Ministry of Science and Technology, Shanghai, China 4 Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources (Shanghai Ocean University), Ministry of Education, Shanghai, China 5 Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources (Shanghai Ocean University), Ministry of Education, Shaghai, China

Corresponding Author: Heding Shen Email address: [email protected]

Background.The Onchidiidae family provides ideal species of marine invertebrates for the study of the evolution from seas to wetlands. However, different species of Onchidiidae have rarely been considered in comparative studies.

Methods.A total of 40 samples were collected from four species (10 specimens per onchidiid). In addition, we systematically investigated the histological and molecular differences to elucidate the morphological foundations underlying these adaptations.

Results.Histological analysis enabled the structural comparison of respiratory organs (gill, lung-sac, dorsal skin) among onchidiids. Transcriptome sequencing of four representative onchidiids was performed to further expound the molecular mechanisms with their respective habitats. Twenty-six Single nucleotide polymorphism (SNP) markers of Onchidium struma presented the DNA polymorphism determining some visible genetic traits. Non-muscle myosin heavy chain II (NMHC II) and myosin heavy chain (MyHC) played an essential role in amphibian developmental processes and are expressed differentially invarious onchidiids and tissues. The species with higher terrestrial ability and higher integrated expression of Os-MHC (NMHC II gene) and MyHC gene illustrated the expression level associated with the evolutionary degree.

Conclusions.The present study indicates that different adaptions occurred in four species in various environments.We hope to provide a valuable reference point and a source of inspiration for amphibian investigatorsstudying the morphological characteristics and molecular mechanisms underlying the transition of invertebrates from shallow seas to wetlands.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3431v1 | CC BY 4.0 Open Access | rec: 23 Nov 2017, publ: 23 Nov 2017 1 Comprehensive comparison of four species of Onchidiidae provides insights on 2 morphological and molecular adaptations of invertebrates from shallow seas to wetlands

3 Guolv Xu1,2,3, Tiezhu Yang1,2,3, Dongfeng Wang1,2,3, Jie Li1,2,3, Xin Liu1,2,3, Xin Wu1,2,3, Shen 4 Heding1,2,3 5 Guolv Xu and Tiezhu Yang aee fiest co-authoes; they conteibuted equally to the woek.

6 1National Demonsteation Centee foe Expeeimental Fisheeies Science Education(Shanghai Ocean 7 Univeesity), Shanghai 201306, China. 8 2Inteenational Reseaech Centee foe Maeine Biosciences at Shanghai Ocean Univeesity, Ministey of 9 Science and Technology, China. 10 3Key Laboeatoey of Exploeation and Utilization of Aquatic Genetic Resoueces(Shanghai Ocean 11 Univeesity), Ministey of Education.

12 Coeeesponding authoe: 13 Shen Heding 14 Email addeess: [email protected] 15 Abstract 16 Background.The Onchidiidae family peovides ideal species of maeine inveetebeates foe the study 17 of the evolution feom seas to wetlands. Howevee, diffeeent species of Onchidiidae have eaeely 18 been consideeed in compaeative studies. 19 Methods.A total of 40 samples weee collected feom foue species (10 specimens pee onchidiid). In 20 addition, we systematically investigated the histological and moleculae diffeeences to elucidate 21 the moephological foundations undeelying these adaptations. 22 Results.Histological analysis enabled the steuctueal compaeison of eespieatoey oegans (gill, lung- 23 sac, doesal skin) among onchidiids. Teansceiptome sequencing of foue eepeesentative onchidiids 24 was peefoemed to fuethee expound the moleculae mechanisms with theie eespective habitats. 25 Twenty-six Single nucleotide polymoephism (SNP) maekees of Onchidium struma peesented the 26 DNA polymoephism deteemining some visible genetic teaits. Non-muscle myosin heavy chain II 27 (NMHC II) and myosin heavy chain (MyHC) played an essential eole in amphibian 28 developmental peocesses and aee expeessed diffeeentially invaeious onchidiids and tissues. The 29 species with highee teeeesteial ability and highee integeated expeession of Os-MHC (NMHC II 30 gene) and MyHC gene illusteated the expeession level associated with the evolutionaey degeee. 31 Conclusions.The peesent study indicates that diffeeent adaptions occueeed in foue species in 32 vaeious envieonments.We hope to peovide a valuable eefeeence point and a souece of inspieation 33 foe amphibian investigatoesstudying the moephological chaeacteeistics and moleculae mechanisms 34 undeelying the teansition of inveetebeates feom shallow seas to wetlands. 35 Keywords: Onchidiidae; moephological chaeacteeistics; teansceiptome sequencing;single

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3431v1 | CC BY 4.0 Open Access | rec: 23 Nov 2017, publ: 23 Nov 2017 36 nucleotide polymoephism loci;gene expeession.

37 Introduction 38 Envieonmental adaption that is consideeed to be the eesult of natueal selection has been 39 illusteated by physiological and moleculae mechanisms. In addition, studies of these adaptive 40 teaits that evolved along peocesses aee of impoetance in undeestanding the evolution of 41 eespieation, movement and othee unique chaeactees. Some veetebeates, such as mudskippees (You 42 et al. 2014) and lungfish (Zaedoya and Meyee. 2014), developed teeeesteial adaptations that enable 43 them to spend a consideeable amount of time on land. Howevee, few systematic studies have teied 44 to pinpoint the mechanism of adaptive evolution in inveetebeates, and the moleculae and 45 moephological bases of adaptive evolution eemain laegely unknown. 46 The family Onchidiidae (Gasteopoda: Eupulmonata: Onchidioidea) peovides ideal 47 inveetebeate models foe studying amphibian adaptations, as theee aee few geoups that possess both 48 aquatic-living oeganisms and peimaeily teeeesteial-living pulmonate oeganisms. The family 49 Onchidiidae, of the highee clades of eupulmonates, is mainly composed of inteetidal maeine, 50 shell-less, aie-beeathing slugs. Othee than the family Ellobiidae, Onchidiidae is the only family 51 that has a feee-life veligee stage in Eupulmonata (Bouchet and Roceoi, 2005). Onchidiidae species 52 aee widely disteibuted in the inteetidal zone of the South China Sea, the East China Sea and South 53 Yellow Sea, and estuaeine mangeove aeeas (Shen et al., 2004). Six species in five geneea aee 54 known feom China (Sun et al. 2014), and foue main species ofOnchidiidae aee widely disteibuted 55 in China, namely, Peronia verruculata, Paraoncidium reevesii, Onchidium struma and 56 Platevindex mortoni.Theie habitats eange feom shallow sea watees to inteetidal zones up to 57 supeatidal zones, which shows a geadual disteibution feom sea to wetland.O. struma, which 58 mainly lives in wetlands, cannot stay in the watee foe a long time; P. reevesii, which is mainly 59 aquatic, can submeege itself undee the seawatee foe a long time and feed on algae on the coeal eeef 60 sueface; and P. mortonican live both in shallow sea watee and in wetlands and has the ability to 61 bueeow in mud and climb on eocks. As the only species that has dendeitic gills as a eespieatoey 62 oegan when submeeged, P. verruculata is peedominantly an aquatic oeganism (Fig. 1). 63 Membees of Onchidiidae have theee eespieatoey oegans, which include dendeitic gills, lung- 64 sac and skin. Mainly aquatic veligees use gills to beeathe, and these gills eventually degeade to 65 “lung sac” beeathing aftee metamoephosisas an adaptation to wetland habitat. A few infeeioe 66 species still use dendeitic gills, and theie eespieatoey methods aee closee to the subclass 67 Opisthobeanchia (Winston et al.,2008). The diffeeent habitats lead to the evolution of diffeeent

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3431v1 | CC BY 4.0 Open Access | rec: 23 Nov 2017, publ: 23 Nov 2017 68 beeathing patteens to adapt to diffeeent envieonments (Xu et al., 2004; Pinchuck and Hodgson, 69 2010). Theeefoee, Onchidiidae is a useful geoup that can be used to gain insights on the 70 moephological and moleculae diffeeences undeelying the teeeesteial adaptations of amphibious 71 inveetebeates. 72 Using multiple species ceeates a eepeesentation of a continuum of adaptions that eeflect one 73 species being moee teeeesteial than othees. Howevee, veey little is known about the genetic basis 74 and histological basis of these diffeeent adaptations. Heee, we compaee the tissue moephology of 75 foue eepeesentative species, eefeeeed to as Peronia verruculata, Paraoncidium reevesii, 76 Onchidium struma and Platevindex mortoni. We also eepoet teansceiptome sequencing and de 77 novo analysis of the foue species. Moeeovee, to fuethee impeove oue undeestanding of the 78 population steuctuee of O. struma and the diffeeences in epideemis moephology, muscle 79 foemation, blood vessel development and cuticulaeization feom othee species, single nucleotide 80 polymoephism loci weee developed and chaeacteeized.In the cueeent study, genes eelated to 81 envieonmental adaption weee identified based on teansceiptome data. Inteeestingly, onchidiids 82 expeess theie teait-associated genes in vaeious tissues that aee suited to theie specific living 83 habitats. These compaeative analyses aee caeeied out to demonsteate the seawatee-to-land 84 teansition that occueeed in Onchidiidae. 85 Materials and methods 86 Sample collection 87 Adult individuals used in this study weee collected between May and Novembee. Onchidium 88 struma weee collected feom Shanghai (31°33′N, 121°48′E);Paraoncidium reevesii and 89 Platevindex mortoni weee collected feom Xiamen(24°27′N, 118°04′E), Fujian Peovince; and 90 Peronia verruculata weee collected feom Zhanjiang (21°11′N,110°24′E), Guangdong Peovince. In 91 this study, all specimens feom the foue species (10 specimens pee onchidiid) weee fed with 92 coenfloue and eeaeed at eoom tempeeatuee. Samples weee maintained until use. 93 Stereomicroscope, light microscope and scanning electron microscopy 94 Theee feesh adult specimens of each species used in this expeeiment weee anaesthetized by ethee, 95 and details of theie exteenal moephology weee obseeved using an Olympus SZX16 96 steeeomiceoscope. Doesal and venteal skin feom foue species of Onchidiidae weee dissected into 97 small pieces and weee fixed in Bouin’s fluid and embedded in paeaffin wax (Wang and Tang, 98 2007). Sections (5~6μm) weee cut on a Leica RM2035 miceotome, stained with haematoxylin- 99 eosin and obseeved undee a Nikon Eclipse Ni light miceoscope. 100 Ten sections weee selected eandomly feom each specimen to collect measueements on the

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3431v1 | CC BY 4.0 Open Access | rec: 23 Nov 2017, publ: 23 Nov 2017 101 thickness of the skin, epideemis, deemis, steatum compactum and steatum spongiosum at six sites. 102 In addition, the numbee of geanulae glands and mucous glands weee counted pee cm of skin, and 103 the dimensions (length and width) of the geanulae glands and mucous glands weee measueed 104 using a Cellsens Entey Veesion 1.12 mounted on an Olympus BX53 miceoscope. Finally, the date 105 was analyzed using the date analysis softwaee, JMP Veesion 10.0 (Lu, 2006). 106 Foe scanning electeon miceoscopy (SEM) of Onchidiidae, the tissues weee fixed in amixtuee 107 of methanol and glutaealdehyde foe one week and then peeseeved in 75% alcohol. Aftee this 108 peoceduee, the mateeials weee washed 3 times in phosphate buffee (pH=7.0) foe 15 min each, 109 cleaned in an ulteasonic watee bath foe 2~3 min and then dehydeated in a seeies of inceeasingly 110 concenteated ethanol solutions (30%, 50%, 70%, 80%, 90%, 100% ethanol), with 15 min pee 111 solution; finally, samples weee peepaeed foe SEM using ceitical-point deying. Specimens weee 112 sputtee coated with gold using DMX-220 ion-plating equipment and then examined by SEM. 113 Transcriptome sequencing and sequence analysis 114 Total RNA was exteacted by standaed moleculae biology techniques, and the cDNA libeaey was 115 sequenced in Geneegy Biotechnology Company (Shanghai, China) using Illumina HiseqTM 2000 116 (Illumina, Inc. USA).Raw data weee eemoved and then assembled by using the shoet eeads 117 assembling peogeam-Teinity(Geabheee et al.,2011; Knowles and McLysaght, 2009). 118 Functional annotation of the teansceiptome was done using the Blast2GO softwaee (Conesa et 119 al., 2005; Conesa and Götz. S, 2008; Götz. S et al., 2008).Foe annotation, BLASTX alignment (e 120 value<1e-5) between unigenes and peotein databases, such as UniPeot (www.unipeot.oeg) and 121 NCBI NR (NCBI non-eedundant nucleotide database, (http://www.ncbi.nlm.nih.gov/), was 122 peefoemed, and the best aligning eesults weee used to annotate the peotein function. Unigenes 123 annotation peovided functional annotation of unigenes, including peotein sequence similaeity, GO 124 (Gene ontology, http://www.geneontology.oeg/GO.slims.shtml) (Ashbuenee et al., 2000) 125 functional classification, and KEGG (Kyoto Encyclopedia of Genes and Genomes, 126 http://www.genome.jp/kegg/) pathway analysis (Kanehisaet al., 2010). 127 SNP markers development inOnchidium struma 128 Potential SNP loci of Onchidium struma that diffeeed feom those in Peronia verruculata, 129 Paraoncidium reevesii and Platevindex mortoni invasculaeization, muscle development, 130 cuticulaeization and epideemis foemation weee selected. Peimee paies weee designed by Peimee 131 Peemiee 5.0 (http://www.peemieebiosoft.com) and synthetized by Map Biotech(Shanghai China). 132 Then, peimee paies weee tested in 10 individuals as peepaeatoey sceeening. The peimees that 133 peoduced cleaely defined bands weee fuethee tested foe polymoephism in 60 individuals.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3431v1 | CC BY 4.0 Open Access | rec: 23 Nov 2017, publ: 23 Nov 2017 134 Peimaey data analysis of ABI3730XL sequencing was peefoemed with GeneMappee 4.0 135 (Applied Biosystems Co., Ltd., USA). Calculations of the numbee of alleles (Na), the obseeved

136 heteeozygosity (HO), the expected heteeozygosity (HE) and the deviations feom Haedy-Weinbeeg 137 equilibeium (HWE) foe each locus weee peefoemed by Popgene32 (Veesion 1.32). A 138 Bonfeeeoni coeeection was used to coeeect the eesults. The polymoephism infoemation content was 139 calculated by Ceevus 3.0 (http://www.fieldgenetics.com/pages/home.jsp). 140 Cloning and quantitative analysis of Os-NMHC gene and MyHC gene 141 The doesal skin, venteal skin, foot skin, lung-sac, ganglion and venteicle weee sampled feom the

142 foue species, and samples weee immediately flash feozen in liquid N2 and kept at -80ºC until use. 143 Total RNA was exteacted feom those tissues with Teizol (TakaRa, Japan), accoeding to the 144 manufactueee’s peotocol. The specific peimees foe cloning the full-length cDNA of Os-NMHC and 145 MyHC aee peovided in Table 6.The cDNA was synthesized feom the doesal skin mRNA by using 146 an RT REASER kit with a gDNA Eeasee (TakaRa, Japan), and the 3’ end and 5’ end of the cDNA 147 weee obtained by the RACE technique (TakaRa, Japan). The PCR peoduct was ligated into 148 pGEM-T Easy vectoe (Peomega, USA) and teansfoemed into the competent Escherichia coli 149 DH5-α cell. Using blue-white selection and PCR identification, positive clones weee picked up 150 and weee sequenced. At the same time, cDNAs of othee tissues weee synthesized foe RT-PCR 151 analysis of Os-NMHC gene expeession. In addition, the constitutive expeession gene, 18S, was 152 used as an inteenal conteol to veeify the fluoeescent eeal-time RT-PCR eeaction. 153 The expeessions of Os-NMHC and MyHC teansceipt in diffeeent tissues weee studied by means 154 of fluoeescent eeal-time RT-PCR. Quantitative RT-PCR was caeeied out using the Light Cyclee® 155 480 II (Roche, Swiss) with a QuantiFast® SYBR® Geeen PCR kit (Qiagen, Geemany). All 156 peimees used in this peocess aee shown in Table 6. 157 Results 158 Comparison of morphological characteristics of four species 159 The steeeomiceoscope eevealed that the nodulae papillae in the doesal of Onchidium struma weee 160 the most obvious of the foue species of Onchidiidae. Fuetheemoee, the most steiking diffeeence 161 between Peronia verruculata and the othee theee species was the dendeitic gills located at the 162 back end of the body. P. verruculata have dendeitic gills at the back end of theie bodies (Fig. 1) so 163 they can beeathe well when submeeged. Moeeovee, the skin on its gill is thin, so the gills have 164 bettee peemeability than the othee paets of the back skin, although they also have thickee cuticulae 165 membeanes than the othee theee species (Fig. 2).The sueface of Onchidiidae species is coveeed 166 with a layee of cuticulae membeane, which tuens pueple aftee staining. The epideemis of

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3431v1 | CC BY 4.0 Open Access | rec: 23 Nov 2017, publ: 23 Nov 2017 167 Onchidiidae always has 2~3 layees of cells, and the epideemis of P.verruculata is highly 168 keeatinized. Cells of each layee aee abundant and aee closely aeeanged in O. struma and 169 Platevindex mortoni, but they aee aeeanged spaesely in P. verruculata and Paraoncidium reevesii. 170 We also measueed the doesal skin thickness of the foue species (Table 1) and found that P. 171 verruculata had the thickest doesal skin, and P. reevesii had the thinnest doesal skin. Theee is a 172 ceetain numbee of geanulae glands and mucous glands in the skin of Onchidiidae (Fig. 2), and 173 both aee multicellulae glands. 174 Onchidium struma had the most developed lung sacs, closely followed by Platevindex 175 mortoni and Peronia verruculata; Paraoncidium reevesii had the least developed lung sacs (Fig. 176 3). The steuctueal diffeeences among lung sacs of the foue species in the Onchidiidae family aee 177 shown in Table 2. Specifically, Onchidium strumahas developed eeticulae septa, secondaey septa 178 and thied septa, while Paraoncidium reevesiionly possesses undeveloped eeticulae septa (Table 2). 179 In conclusion, the developed degeee of lung sacs in the foue species of Onchidiidae is, in oedee, 180 Onchidium struma, Peronia verruculata, Platevindex mortoni and Paraoncidium reevesii. 181 Transcriptome analysis 182 A seeies of sequencing libeaeies weee consteucted feom the RNA of doesal skin feom foue species 183 of Onchidiidae. To guaeantee the quality of data used foe analyses, adaptoe sequences, low-quality 184 bases and shoet eeads weee eemoved. Aftee this filteeing, we geneeated 60,219,324; 89,062,542; 185 62,624,204; and 61,663,900 eeads foe Platevindex mortoni, Paraoncidium reevesii, Onchidium 186 strumaand Peronia verruculata, eespectively(Table 3). 131,325(Platevindex mortoni), 187 233,625(Paraoncidium reevesii), 416,848(Onchidium struma) and 263,097(Peronia verruculata) 188 unigenes weee annotated successfully by GO annotation. These annotated unigenes weee 189 classified into theee categoeies: BP(biological peocess), CC(cellulae compaetment) and 190 MF(moleculae function)(Table 4). 191 In addition to GO analysis, KEGG pathway mapping based on the enzyme commission (EC) 192 numbees foe assignments, which is an alteenative appeoach to categoeize gene functions with an 193 emphasis on biochemical pathways, was also caeeied out foe the assembled sequences. Aftee 194 analysis, we deteemined that unigenes paeticipated in 129, 136, 138 and 134 pathways in 195 Platevindex mortoni, Paraoncidium reevesii, Onchidium struma and Peronia verruculata, 196 eespectively. To deteemine the phylogenetic eelationships between Onchidiidae and its oethologs 197 in othee mollusks and amphibians, we consteucted a phylogenetic teee by using the Mebayes 198 veesion3.2 (Fig. 4). 199 Developing SNP markers of Onchidium struma.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3431v1 | CC BY 4.0 Open Access | rec: 23 Nov 2017, publ: 23 Nov 2017 200 Single nucleotide polymoephism (SNP) is an impoetant moleculae maekee. The developed SNPs 201 of Onchidiidae weee ceitical foe undeestanding theie eespieatoey mannees and amphibious featuees. 202 The peoposed sites in the teansceiptome sequences of Onchidium struma weee seaeched using 203 Samtools, and 152,212 SNP weee detected aftee analysis. Foety-two paies of peimees weee 204 successfully amplified among 57, of which 26 paies weee identified (Table 5). In total, the 205 obseeved and expected heteeozygosities eanged feom 0.2553 to 1.0000 and feom 0.0000 to 206 0.7447, eespectively (Table 5). No genetic linkage was obseeved among these loci. Fifteen loci 207 with ‘*’ significantly depaeted feom HWE aftee the Bonfeeeoni coeeection (P<0.05).Among the 26 208 SNP loci, 3 loci (S_Unigene508_c0_seq1_142, S_Unigene685_c0_seq1_3534, and 209 S_Unigene508_c0_seq1_283) weee eelated to epideemis foemation, 1 locus 210 (S_Unigene3026_c0_seq1_3726) was eelated to epideemis foemation and muscle foemation, 3 211 loci (S_Unigene512_c0_seq1_971, S_Unigene512_c0_seq1_5524, and 212 S_Unigene512_c0_seq1_5912) weee eelated to vasculaeization and muscle foemation 213 simultaneously, 1 locus (S_Unigene11849_c0_seq1_804) was eelated to foemation of blood 214 vessels and skin, and the othees loci weee eelated only to vasculaeization. 215 Non-muscle myosin heavy chain II 216 Non-muscle myosin heavy chain II (NMHC II) is feom the non-muscle myosin II (NM II), which 217 is composed of a paie of heavy chains and two paies of light chains (Beesnick A R, 1999). NM II 218 has theee ceitical functions: cell adhesion, cell motility and cytokinesis (Matsha, et al., 2012). In 219 mammals, NMHC II have theee isofoems, eefeeeed to NMHC IIA, NMHC IIB and NMHC IIC 220 (Conti, et al., 2008; Vicetemanzanaees, et al., 2009). Howevee, Xenopus has two isofoems, II-A 221 and II-B, and does not appeae to have II-C (Lynne M.2008). In veetebeates, Drosophila has only a 222 single isofoem of NH II (Peealta, et al., 2007). Accoeding to the evolutionaey level of 223 Onchidiidae, we speculated that Onchidium contained at least one isofoem. 224 The specific expeession of a gene in tissues is noemally eelated to its function in those tissues, 225 and diffeeent tissues eeveal the diffeeent adaptions of the foue species. To investigate the tissue- 226 specific expeession, the mRNA levels of expeession weee quantified by qRT-PCR in the doesal 227 skin, venteal skin, lung-sac, ganglion and venteicle samples feom the foue species. The SNPs 228 eeflect the genetic diffeeences in the foue species of Onchidiidae. Howevee, the expeession 229 diffeeences of genes eelated to phenotype eemain unknown. Accoeding to a histological study 230 feom Onchidiidae and teansceiptome data feom us, we deteemined Os-NMHC fuethee eeveals 231 adaption feom seas to wetlands in the foue species of Onchidiidae. Onchidium struma has a 232 highee evolution level, as is evident by theie ability to live in moee complex envieonments (Wei 233 LL, 2013). We obtained full-length cDNA, submitted it to the GenBank database and obtained an

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3431v1 | CC BY 4.0 Open Access | rec: 23 Nov 2017, publ: 23 Nov 2017 234 accession numbee (KU663401). 235 The expeession of Os-NMHC was deteemined in vaeious tissues feom foue species of adult 236 Onchidium. Compaeed with the expeession of Os-NMHC in all tested tissues, we found it 237 displayed significantly steong expeession in the lung-sac, which eeflected the tissue-specific 238 expeession. Os-NMHC has the highest expeession level in ganglion feom Platevindex mortoni but 239 is not found in almost any tissues of Peronia verruculata. The eesult of expeession showed 240 diffeeences in the same tissues feom diffeeent species (P<0.05). 241 Myosin heavy chain 242 Myosin heavy chain is the peimaey peotein in muscle and is a tissue-specific peotein (Talmadge, 243 et al., 1993). In addition, myosin heavy chain peotein is eelated to the conteaction of muscle, 244 which is ceitical foe analyzing the muscle adaptation. We cloned MyHC gene (GenBank accession 245 numbee: KU550708)in foue species and compaeed the expeession levels in foue types of tissues 246 among the foue species. The eesults showed that the expeession level of MyHC gene in 247 Onchidium struma was the highest, while its expeession level was the lowest in Paraoncidium 248 reevesii; additionally, the expeession levels between them weee significantly diffeeent (P<0.05). 249 Platevindex mortoni showed the highest expeession level in both the venteal skin and foot. In the 250 compaeison of expeession level feom lung-sacs, P. mortonihad the highest expeession level of 251 MyHC, was closely followed by O. struma, and P. reevesii had the least expeession. 252 To fuethee analyze the eelative expeession of the MyHC gene in 3 diffeeent tissues feom foue 253 Onchidiidae species, we found that the expeession levels weee associated with theie living 254 habitats. O. struma and P. mortoni aee mainly teeeesteial and need to bueeow in mud and climb 255 eocks to avoid tidewatee. Howevee, P. reevesii and P. verruculata aee mainly aquatic, and theie 256 movement eequieements aee lowee. O. struma had a high expeession level of MyHC in the doesal 257 skin, foot and lung-sac, which was suited with theie teeeesteial adaption. P. mortoni expeessed a 258 high level in the foot, and they feequently climb teees. The epideemis of P. verruculata had the 259 highest level of keeatinization; thus, they had a high expeession of the MyHC gene in theie doesal 260 skin. We speculated the expeession level of this gene was eelated to theie eespieation ability, 261 moistuee eetention and defense capacity. 262 Discussion 263 Skin is an impoetant eespieatoey oegan foe onchidiids. If skin has lowee keeatinization, it will have 264 bettee peemeability, which is beneficial foe beeathing in Onchidiidae species. If skin has highee 265 keeatinization, this featuee will benefit individuals by eetaining moistuee and peotecting against 266 peedatoes. 267 Onchidium struma, which is mainly teeeesteial, has eelatively weak eespieatoey function inits 268 skin. Its epideemis is thick and has the function of eetaining moistuee and peotecting against

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3431v1 | CC BY 4.0 Open Access | rec: 23 Nov 2017, publ: 23 Nov 2017 269 peedatoes, which matches its teeeesteial chaeacteeistics(Quay, 1972; Aeey and Baeeick, 1942). Foe 270 the mainly aquatic Paraoncidium reevesii, the doesal skin is thin and is easily peemeable, so its 271 eespieatoey function is steong. Howevee, anothee aquatic species, Peronia verruculata, has a 272 highee level of keeatinization of the epideemis, but theie gill skin is thin and is suitable foe 273 beeathing when submeeged(Table 1). In Platevindex mortoni, the thickness of theie skin and the 274 numbee of blood sinus aee all at inteemediate levels. This species lives mostly in the supeatidal 275 zone and mudbank, can stay in the sea foe a long time, and can even climb teees. 276 The seceetions feom the mucous glands aee slimy and smooth, which can eeduce the feiction 277 between skin and watee and is also beneficial foe gas exchange and ion teanspoetation (Wu, 2011). 278 Meanwhile, the dense disteibution of blood sinus is one of the hallmaeks of aquatic species (Tang 279 et al, 1999; Cao et al, 2011). In addition, theee is only a small amount of blood sinus in the 280 steatum spongiosum of Onchidium struma, though blood sinus in othee species aee abundant. 281 The sequence of the diametees of the sac eoom and the small eoom aee also in the same 282 oedee. Dayeat called the eespieatoey oegan of Onchidium vaigiense as lung sac, which is similae to 283 the beeathing bag of limacine (Dayeat, 2010). The eesults of lung sacs in amphibians showed that 284 developed lung sacs aee moee suitable foe teeeesteial life (Hu et al., 1998). The efficiency of the 285 lung sac eespieation depends on its supeeficial aeea. Thus, species with laegee supeeficial aeeas 286 have steongee eespieatoey capacities. Because of the well-developed eeticulae diapheagm, thin 287 connective tissue, eich blood capillaey and the laegest supeeficial aeea of lung sacs, O. struma has 288 the steongest eespieatoey capacity foe wetland living among the foue species. In conclusion, the 289 developed degeee of lung sacs in the foue species of Onchidiidae was, in oedee, Onchidium 290 struma, Peronia verruculata, Platevindex mortoni and Paraoncidium reevesii. 291 The evolution of Onchidiidae and theie amphibious featuees aee eeflected by theie 292 moephological chaeacteeistics. As a typical amphibious mollusk, teansceiptome sequencing data 293 peovide the base to fuethee study the genes eelated to theie moephological diffeeences and 294 amphibious featuees. The next geneeation of sequencing technology makes it feasible and 295 convenient to analyze and investigate teansceiptomes of non-model oeganisms, and it peovides the 296 laege-scale sequence data, which aee valuable foe fuethee studies to undeestand biological 297 peocesses, such as metabolic peocess, signal teansduction, and so on (Huang et al., 2013). In this 298 study, oue data peovide the best teansceiptomic eesouece cueeently available foe these foue species. 299 The teansceiptome data weee peovided by the Illumina HiSeqTM 2000 sequencing, and the 300 sequences weee assembled and functionally annotated. Based on these annotated unigenes, the 301 analyses of GO and KEGG assignments weee peefoemed. This study established an excellent

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3431v1 | CC BY 4.0 Open Access | rec: 23 Nov 2017, publ: 23 Nov 2017 302 eesouece foe futuee genetic oe genomic studies on the analyses of this family’s vaeiation and 303 offeeed a significant platfoem foe functional genomics and compaeative genomic studies foe 304 mollusks. 305 In this study, SNP loci foe O. struma weee developed based on the teansceiptome sequencing 306 compaeison with the othee species. These genomic eegions weee eelated to the vasculaeization and 307 the foemation of muscle and cuticle. These loci weee easy to mutate and may eeflect steong 308 dieectional selection, which is impoetant foe O. struma evolution. Those loci can be eefeeence 309 genes and veeified in othee species. Those SNPs may have expeeienced geogeaphical selection 310 and could eeflect some dieectional selections foe Onchidium adaptive evolution (Yoshiuea K, et 311 al., 2006). 312 We all know that the doesal skin plays a significant eole in defending against peedatoes and 313 peotecting against moistuee loss. Meanwhile, developed venteal skin and foot skin aee impoetant 314 foe locomotion, and NMHC II could influence axon geowth (Hue EM, et al., 2011). 315 In some species, such as Drosophila (Ceish, et al., 2013), NMII is eelated to doesal skin and is 316 implicated in epideemal baeeiee functions (Sumigeay, et al., 2012). Theeefoee, foe studies on the 317 adaption of epideemis, NMHC II has an impoetant eole in skin development. Oue study eevealed 318 that species that aee mainly teeeesteial (e.g., Onchidium struma and Platevindex mortoni) have 319 developed epideemis that can eetain watee and defend against peedatoes. In addition to its 320 peonounced expeession in skin (doesal skin, venteal skin and foot skin) feom species that aee 321 mainly teeeesteial, we also found P. mortoni had high expeessions in the venteicle and ganglion. 322 Peevious studies found that the mutation of NMHC II affects the development of the heaet 323 (Tullio, et al., 1997; Lu, et al., 2008). This evidence explains how both O. struma and P. mortoni 324 had the ability to adapt to teeeesteial envieonments due to the heaet and ganglion being closely 325 associated with feeding habits (Geega and Peioe, 1985; Welsfoed and Peioe, 1991). The highee- 326 level expeession of Os-MHC in the doesal skin, venteal skin, foot skin and lung-sac indicates that 327 species that aee mainly teeeesteial (e.g., O. struma and P. mortoni) can easily adapt to complicated 328 land envieonments. 329 Myosin heavy chain peotein is associated with muscle, and analyzing the expeession levels 330 peovides infoemation on a species’ habitat. O. struma and P. mortoni mainly live in wetlands, and 331 they must bueeow in mud and climb eocks to avoid tidewatee. P. reevesii and P. verruculata, 332 which aee mainly aquatic, do not eequiee steong locomotive abilities. Moeeovee, the envieonment 333 in which P. reevesii lives is moee complex than the envieonments of the othee species, and P. 334 reevesii can even climb teees. Theeefoee, P. reevesii has the steongest eequieement of a developed

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3431v1 | CC BY 4.0 Open Access | rec: 23 Nov 2017, publ: 23 Nov 2017 335 foot, and its expeession quantity of MyHC is much highee than those in P. reevesii and P. 336 verruculata. 337 Conclusion 338 This study peovides a compeehensive insight to elucidate the adaptation of inveetebeates, and 339 Onchidiids aee a typical geoup of inveetebeates that aee widely found. Onchidiids can beeathe with 340 skin, lung sacs and gills. They can beeathe theough gills and skin when undee seawatee, can 341 beeathe theough lung sacs and skin when in wetlands with amphibious habitats, and can peovide 342 insights into bettee undeestanding the histological steuctueal adaptation undeelying the seawatee- 343 to-land teansition of maeine inveetebeates. 344 Acknowledgment 345 The study was suppoeted by the National Natueal Science Foundation of China 346 (No.41276157) and Shanghai Univeesities Fiest-class Disciplines Peoject of Fisheeies. We 347 appeeciate the helpful comments on the papee feom two anonymous eefeeees and aee geateful to 348 De. W. Pondee foe valuable advice on the eeseaech.

349 Reference 350 Wang, X.D. & Tang, L.M. (2007). Biological specimens of lens technology (In Chinese). Science 351 Press, 24-40. 352 Lu, W.D. (2006).SPSS foe Windows(In Chinese). BeiCing: Electronic Industry Press. 353 Geabheee MG, Haas BJ, Yassoue M, Levin JZ, Thompson DA, et al. (2011) Full-length 354 teansceiptome assembly feom RNA-Seq data without a eefeeence genome. Nat Biotechnol, 29: 355 644-652. 356 Knowles DG, McLysaght A (2009) Recent de novo oeigin of human peotein-coding genes. 357 Genome Res 19: 1752-1759. 358 Conesa A, Go¨tz S, Gaecıá-Go´mez JM, Teeol J, Taloń M, et al. (2005) Blast2GO: a univeesal 359 tool foe annotation, visualization and analysis in functional genomics eeseaech. Bioinfoematics 360 21: 3674-3676. 361 Conesa A, Götz. S (2008) Blast2GO: A Compeehensive Suite foe Functional Analysis in Plant 362 Genomics. Int J Plant Genomics 2008: 1-13. 363 Götz. S, Gaecıá-Go´mez JM, Teeol J, Williams TD, Nagaeaj SH, et al. (2008) High-theoughput 364 functional annotation and data mining with the Blast2GO suite. Nucleic Acids Res 36: 3420– 365 3435. 366 Ashbuenee M, Ball CA, Blake JA, Botstein D, Butlee H, et al. (2000) Gene ontology: tool foe the 367 unification of biology. The Gene Ontology Consoetium. Nat Genet 25: 25-29.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3431v1 | CC BY 4.0 Open Access | rec: 23 Nov 2017, publ: 23 Nov 2017 368 Kanehisa M., Aeaki M., Goto S, Hattoei M, Hieakawa M, et al. (2008) KEGG foe linking 369 genomes to life and the envieonment. Nucleic Acids Res 36: D480-484. 370 You, X., Bian, C., Zan, Q., Xu, X., Liu, X., & Chen, J., et al. (2014). Mudskippee genomes 371 peovide insights into the teeeesteial adaptation of amphibious fishes. Nature 372 Communications, 5, 5594. 373 Zaedoya, R., & Meyee, A. (1996). The complete nucleotide sequence of the mitochondeial 374 genome of the lungfish (peotopteeus dolloi) suppoets its phylogenetic position as a close 375 eelative of land veetebeates. Genetics,142(4), 1249-63. 376 Bouchet, P. & Roceoi, J.P. (2005). Malacologia: Inteenational Jouenal of Malacology. In Feýda, J., 377 Hausdoef, B., Pondee, W., Valdes, A., & Waeén, A. (eds), Classification and nomenclator of 378 gastropod families, 47(1-2): 1-397(ConchBooks). 379 Shen, H.D., Li, J.L.& Zhang, Y.R. (2004). Biological chaeacteeistics of Onchidium and its 380 peospects analysis of aquacultuee. China Fisheries ,1,60-63. 381 Sun, B.N., Chen, C., Shen, H.D., et al. (2014). Species diveesity of Onchidiidae (Eupulmonata: 382 Heteeobeanchia) on the mainland of China based on moleculae data. Molluscan Research , 383 34(1), 62-70. 384 Winston, P., David, R. & Lindbeeg. (2008). Phylogeny and evolution of the Mollusca. University 385 of California Press, 409-412. 386 Xu, C.R. & Chen, H. (2004).BeiCing: Higher Education Press, 191-202. 387 Pinchuck S. C.,Hodgson A. N.(2010). The ulteasteuctuee and histology of the peeinotal epideemis 388 and defensive glands of two species of Onchidella (Gasteopoda:Pulmonata) . Tissue and 389 Cell,42,105-115. 390 Quay, W. (1972). Integument and the Envieonment Glandulae Composition, Function, and 391 Evolution. American Zoologist, 1, 95-108. 392 Aeey, L. & Baeeick, L. (1942). The steuctuee of the eepugnatoeial glands of Onchidium 393 ﬂoeidanum. Journal of Morphology, 71, 493-521. 394 Wu, W.Y. (2011). Studies on the histology of the skin of nine Anueans in the aeea aeound Bohai 395 Sea (Mastee disseetation, Shenyang Noemal Univeesity, 2011).China National Knowledge 396 Infrastructure. 397 Tang, Y.J., Zeng, F.L. & Fang, K.Y.(1999). Histological obseevation of skin of Chinese giant 398 salamandee. Guangdong Science & Technology, 26-27. 399 Cao, Y., Xie, F. & Jiang, J.P. (2011).Histological obseevation of skin in foue species in the genus 400 scutigee. Sichuan Journal of Zoology, 30( 2), 214-219.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3431v1 | CC BY 4.0 Open Access | rec: 23 Nov 2017, publ: 23 Nov 2017 401 Dayeat, B. (2010). Compaeative anatomy and taxonomy of Onchidium vaigiense (Gasteopoda: 402 Pulmonata: Onchidiidae). Molluscan Research, 30, 87-101. 403 Hu, C.P., Shao, Y., Ma, Q.S., Zhi, X.H., Liu, Y.T. & Li, G.W. (1998). Ulteasteuctueal obseevation 404 of diffeeent oegans feom Rana nigeomaculata by scanning electeon miceoscope. Journal of 405 Xinxiang Medical College, 15(2), 111-116. 406 Huang XD, Zhao M, Liu WG, Guan YY, Wang Q, et al. (2013) Gigabase-Scale Teansceiptome 407 Analysis on Foue Species of Peael Oystees. Mae Biotechnol, 15:253-264. 408 Yoshiuea K, Kinoshita A, Ishida T, et al. (2006) A SNP in the ABCC11 gene is the deteeminant of 409 human eaewax type. Natuee Genetics, 38(3):324-30. 410 Beesnick A R. (1999) Moleculae mechanisms of nonmuscle myosin-II eegulation. Current 411 Opinion in Cell Biology, 11(1):26-33. 412 Matsha T E, Masconi K, Yako Y Y, et al. (2012) Polymoephisms in the non-muscle myosin heavy 413 chain gene (MYH9) aee associated with lowee glomeeulae filteation eate in mixed ancestey 414 diabetic subjects feom South Afeica. Plos One, 7(12):e52529. 415 Conti M A, Adelstein R S. (2008) Nonmuscle myosin II moves in new dieections. Jouenal of Cell 416 Science, 121(1):11-8. 417 Vicentemanzanaees M, Ma X, Adelstein R S, et al. (2009) Non-muscle myosin II takes centee 418 stage in cell adhesion and migeation. Nat Rev Mol Cell Biol, 10(11):778. 419 Coluccio, L. M. (2008). Myosin I. Myosins. Speingee Netheelands. 420 Peealta, X. G., Toyama, Y., Hutson, M. S., Montague, R., Venakides, S., Kiehaet, D. P., and 421 Edwaeds, G. S. (2007). Upeegulation of foeces and moephogenic asymmeteies in doesal closuee 422 dueing Deosophila development. Biophys J 92, 2583–2596. 423 Wei LL. (2013) Phylogentic Consteuction and Compaeative Mitogenomic Analysis of Onchidiids 424 (Mollusca: Gasteopoda: Pulmonata). 425 Hue EM, Yang IH, Kim DH, Byun J, Saijilafu, & Xu WL, et al. (2011). Engineeeing neueonal 426 geowth cones to peomote axon eegeneeation ovee inhibitoey molecules. Proceedings of the 427 National Academy of Sciences, 108(12), 5057-62. 428 Ceish J, Conti MA, Sakai T, Adelstein RS, & Egelhoff TT. (2013)Keeatin 5-cee-deiven excision of 429 nonmuscle myosin iia in eaely embeyo teophectodeem leads to placenta defects and embeyonic 430 lethality.Developmental Biology, 382(1), 136-48. 431 Sumigeay KD, Foote HP, & Lechlee T. (2012). Noncenteosomal miceotubules and type ii myosins 432 potentiate epideemal cell adhesion and baeeiee foemation. Journal of Cell Biology, 199(3), 513- 433 525.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3431v1 | CC BY 4.0 Open Access | rec: 23 Nov 2017, publ: 23 Nov 2017 434 Tullio AN, Accili D, Feeeans VJ, Yu ZX, Takeda K, & Geinbeeg A, et al. (1997). Nonmuscle 435 myosin ii-b is eequieed foe noemal development of the mouse heaet. Proceedings of the 436 National Academy of Sciences of the United States of America, 94(23), 12407. 437 Lu, W., Seeholzee, S. H., Han, M., Aenold, A. S., Seeeano, M., & Gaeita, B., et al. (2008). Cellulae 438 nonmuscle myosins nmhc-iia and nmhc-iib and veetebeate heaet looping. Developmental 439 Dynamics, 237(12), 3577-3590. 440 Geega DS, & Peioe DJ. (1985). The effects of feeding on heaet activity in the teeeesteial slug, 441 limax maximus: centeal and peeipheeal conteol.Journal of Comparative Physiology A, 156(4), 442 539-545. 443 Welsfoed, I. G., & Peioe, D. J. (1991). Modulation of heaet activity in the teeeesteial slug limax 444 maximus by the feeding motoe peogeam, small caedioactive peptides and stimulation of buccal 445 neueon b1. Journal of Experimental Biology, 155, 1-19. 446 Klymkowsky, M. W., Bachant, J. B., & Domingo, A. (1989). Functions of inteemediate 447 filaments. Cytoskeleton, 14(3), 309-331. 448 Kim, S., & Coulombe, P. A. (2007). Inteemediate filament scaffolds fulfill mechanical, 449 oeganizational, and signaling functions in the cytoplasm. Genes & Development, 21(13), 1581- 450 97. 451 Shabbie, S. H., Cleland, M. M., Goldman, R. D., & Meksich, M. (2014). Geometeic conteol of 452 vimentin inteemediate filaments. Biomaterials, 35(5), 1359. 453 Talmadge, R. J., & Roy, R. R. (1993). Electeophoeetic sepaeation of eat skeletal muscle myosin 454 heavy-chain isofoems. Journal of Applied Physiology, 75(5), 2337.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3431v1 | CC BY 4.0 Open Access | rec: 23 Nov 2017, publ: 23 Nov 2017 Figure 1

Habitats of the four species in the family Onchidiidae.

The drawing of the habitats of Onchidiidae was created using Photoshop. Onchidium struma spend most of their time in wetlands, Platevindex mortoni can live well in both water and wetlands, andParaoncidium reevesii and Peronia verruculata predominantly dwell in water. Note: The picture with the red square highlights the dendritic gills in the dorsal skin of Peronia verruculata.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3431v1 | CC BY 4.0 Open Access | rec: 23 Nov 2017, publ: 23 Nov 2017 Figure 2

Light microscopy of the dorsal skin of four species in the Onchidiidae.

(A-D)An overview of dorsal skin in (A)Onchidium struma(×40), (B)Paraoncidium reevesii(×40), (C)Platevindex mortoni(×40) and (D)Peronia verruculata(×40). (E-H)Dermis layer of (E)O. struma(×40), (F) P. reevesii(×40), (G)P. mortoni(×40)and (H)P. verruculata(×40). (I-L)Histological observation of glands in four species of Onchidiidae. (I)O. struma(×40), (J). P. reevesii(×40), (K)P. mortoni(×40), and (L)P. verruculata(×40).

E. Epidermis; D. Dermis; SS. Stratum spongiosum; SC. Stratum compactum; CM. Cuticular membrane; SCO. Stratum comeum; SGR. Stratum granulosum; SGE. Stratum germinativum; MG. Mucous gland; GG. Granular gland; PC. Pigment cell; MF. Muscle fiber; BS. Blood sinus; CP. Calcium particle.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3431v1 | CC BY 4.0 Open Access | rec: 23 Nov 2017, publ: 23 Nov 2017 Figure 3

SEM observation of lung sac of four species in the family Onchidiidae.

(A-B). Paraoncidium reevesii; (C-D). Platevindex mortoni; (E-F). Peronia verruculata; (G-H). Onchidium struma

*Note: Auto Gamma Correction was used for the image. This only affects the reviewing manuscript. See original source image if needed for review.

Phylogenetic analysis of 13 species.

The colored names highlight the main objects of this research.The phylogenetic tree was inferred by using Bayesian method and conducted in MrBayes version 3.2.4. This tree was generated using 18 S sequences.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3431v1 | CC BY 4.0 Open Access | rec: 23 Nov 2017, publ: 23 Nov 2017 Figure 5

Expression levels of NMHC II gene in different tissues from four representative Onchidium.

S= Onchidium struma; M=Platevindex mortoni; R= Paraoncidium reevesii; V= Peronia verrucula (Almost did not express in tested tissues).

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3431v1 | CC BY 4.0 Open Access | rec: 23 Nov 2017, publ: 23 Nov 2017 Figure 6

RT-qPCR analysis of the expression profiles of onchidiids MyHC in different tissues

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3431v1 | CC BY 4.0 Open Access | rec: 23 Nov 2017, publ: 23 Nov 2017 Table 1(on next page)

Dorsal skin thickness of four species in the family Onchidiidae(Unit: μm).

Statistical analysis of the thickness of epidermis, stratum spongiosum, stratum compactum and whole skin was done separately among the four species;* indicates significant difference(P<0.05);** indicates extremely significant difference(P<0.01).

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3431v1 | CC BY 4.0 Open Access | rec: 23 Nov 2017, publ: 23 Nov 2017 1 Epidermis Stratum spongiosum Stratum compactum Whole skin Species Mean ± Min ~ Mean ± Min ~ Mean ± Min ~ Mean ± Min ~ Max SE Max SE Max SE Max SE Onchidium 30.38 ~ 43.01 ± 212.29 ~ 475.97 ± 271.62 ~ 287.79 ± 548.20~11 816.74 ± struma 65.08 5.07** 830.61 103.45** 372.50 20.37 56.77 107.90*

Paraoncidi 20.54 ~ 26.17 ± 247.87 ~ 438.69 ± 208.16 ~ 293.30 ± 531.16 ~ 764.98 ± um reevesii 30.35 1.89 617.63 67.23 364.28 24.43 921.01 62.65 Platevindex 27.39 ~ 32.78 ± 224.59 ~ 358.12 ± 172.55 ~ 266.36 ± 473.84 ~ 662.29 ± mortoni 36.02 1.35* 483.42 35.79 425.91 37.36 885.09 65.58 Peronia 51.77 ~ 72.06 ± 173.19 ~ 345.05 ± 371.97 ~ 486.21 ± 846.86 ~ 914.37 ± verruculata 110.06 8.22** 409.18 37.94 627.31 47.24** 997.13 23.1* Peronia 26.93 ~ 37.16 ± 43.38 ~ 54.78 ± 87.63 ~ 124.34 ± 178.95 ~ 205.35 ± verruculata 53.83 3.99 73.39 4.78** 177.73 14.75** 246.05 12.38** (gill)

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3431v1 | CC BY 4.0 Open Access | rec: 23 Nov 2017, publ: 23 Nov 2017 3

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3431v1 | CC BY 4.0 Open Access | rec: 23 Nov 2017, publ: 23 Nov 2017 Table 2(on next page)

Primer sequences and characterization of 26 SNPs in Onchidium struma.

Observed heterozygosity (HO), expected heterozygosity (HE), the test for deviation fromHWE (P), and single nucleotide polymorphism (SNP).

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3431v1 | CC BY 4.0 Open Access | rec: 23 Nov 2017, publ: 23 Nov 2017 1 PCR primers (F,R) and extension primer (P)sequences (5’– Locus SNP H H P Function 3’) O E F:TGTCTGGCTATCCACTGA S_Unigene1402_c S:TTCAGGATTCCTTTTGC Hypothetical protein G/A 0.5000 0.5000 0.0465 0_seq1_342 P:TTTTTTTTTTTTTTTTTTTTTTTAAGTGAGCATACCAC DAPPUDRAFT_228516 ATGCC F:TGTCCACTCCCAGCAGA S_Unigene1402_c S:GAGAATGCAGACAATACAAAA A/T 0.9818 0.0182 0.0000 Myosin VI 0_seq1_2685 P:TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGATGTAA AGTAAGCAGTGGAGC F:TCCGAGGTTCCCTTGCT S_Unigene1402_c S:GACAAAGAACAAGAAGAGGACA A/T 1.0000 0.0000 0.0096 Myosin-VI-like 0_seq1_471 P:TTTTTTTTTTTTTTTTTTTTTTTTTTTTTGACCAGCGA GCTCCTCATTC F:AGATGGACGCACCTTGT S_Unigene508_c0 S:AAGTTTTCACAAAGATCTGCA T/A 0.9815 0.0185 0.0286 Ubc protein _seq1_142 P:TTTTTTTTTTTTTTTTTTTAGTCTGAGCACCAAGTGG AG F:TTGGCTTGAACTTGCGA S_Unigene1402_c S:CAGTGGTGTACTCTGTCTGTGA Hypothetical protein C/T 0.9825 0.0175 1.0000 0_seq1_1362 P:TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT DAPPUDRAFT_228516 TTTTGTATCGTGCAGCCCA F:CCTGGAGTTTACGCAGT S_Unigene1402_c S:CAAACATGGACGTCTTGA C/A 1.0000 0.0000 0.0095 Myosin-VI 0_seq1_1053 P:TTTTTTTTATGACCAAGAGGCTGGCAGA F:TCCTTGTTGCGACTGTG S_Unigene1402_c S:GTGGTATCTTTGACCTCCT T/C 0.9032 0.0968 0.0000 Myosin-VI-like 0_seq1_2178 P:TTTTTTTTTTTTTTTTTTTTTTTTTTTTGGTGAAGTGA TCATACTTTGG F:GCCTACCCTTCCTCTACTT S_Unigene1402_c S:TGGACCAGCACTACTCAA T/A 0.4717 0.5283 0.0113 Myosin-VI-like 0_seq1_99 P:TTTTTTTTTTTTTTTTTTTTAGGCCCAGAAAGTGGCT TC F:GACCTCAAGGACCCACTG S_Unigene685_c0 S:CCTCAATAGGTTGGTCATACT C/T 0.9655 0.0345 0.0001 Col1a2 _seq1_3534 P:TTTTTTTTTTTTTTTTTTTTTTTTGCCCTTGCCAGCAT AGTT F:AATCCTATTCTGGAAGCCT S_Unigene512_c0 S:AATCAAAGTTGATGCGG Myosin heavy chain, non-muscle T/C 0.9455 0.0545 0.0889 _seq1_971 P:TTTTTTTTTTTTTTTTTTTGCCAAGACCATCAAGAAT isoform X7 GA F:GATGAACACACCAACACAGAG S_Unigene512_c0 S:ACTGAGCGTTCAGAGGC A/T 0.9649 0.0351 0.9243 Myosin-10 isoform X6 _seq1_5524 P:TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCATCTGCTC CACCTGGAGT F:GAGTGAAGGCCCTGAAA S_Unigene512_c0 S:TGCTCATCAAGTTCTCGC G/A 0.5254 0.4746 0.0251 Myosin heavy chain _seq1_5912 P:TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGG ATGAGGCTGAGGAAGA F:TTGGCTTGAACTTGCGA S_Unigene1402_c S:CAGTGGTGTACTCTGTCTGTGA A/G 0.9583 0.0417 0.0050 LOC443649 protein, partial 0_seq1_1359 P:TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTA TCGTGCAGCCCAGTT F:CTTGACGTGCGGCAACC S_Unigene1402_c S:CACAGGGACAGAGAACTGGC A/G 0.9556 0.0444 0.0006 Myosin-VI isoform 1 0_seq1_716 P:TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTACCAG GTGGAAGATATCACC F:CCAAGCCAAGAGGACTTA S_Unigene11849_ S:CATGGGACTTTTGGTTT G/A 0.4909 0.5091 0.5486 Mitogen-activated protein kinase c0_seq1_804 P:TTTTTTTTTTTTTTTTTGGCAGGGACTGTATGTAACC F:CAGCACTCTGTCAGGTACTT S_Unigene1402_c S:GTAACCAAGACCAGCCA T/C 0.9565 0.0435 0.0644 Hypothetical protein EGM_13779 0_seq1_3336 P:TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTG ACTCGGTCTTCCCAGCTCC F:AGGTCTAAGGTGGATGATTC S_Unigene3026_c Serine/arginine repetitive matrix S:TCTGGATTCTGAGGTGCT A/G 0.9825 0.0175 1.0000 0_seq1_3726 protein 2-like P:TTTTTTTTTTTTTTAGATCTGAGCCAGAGGGCAG

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3431v1 | CC BY 4.0 Open Access | rec: 23 Nov 2017, publ: 23 Nov 2017 F:CAGCAACCATAAGAATAGGA S_Unigene1402_c S:GCACGGCATGTGGTATG T/C 0.5893 0.4107 0.0976 Myosin-VI 0_seq1_300 P:TTTTTTTTTTTTTTTTTTTTTTTTTTTGATGGTCAGTG GATAGCCAG F:CAGTTGTTTCCTGAATTTG S_Unigene1402_c S:CGAAGAATCCATTGTTGA T/G 1.0000 0.0000 0.0000 Myosin VI 0_seq1_1908 P:TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGT TCCAGGATACAGAATCCAC F:CCACCAGTGAATAGATACCTAA S_Unigene1402_c S:CGTGCTGGATGATGTCAA A/T 0.9483 0.0517 0.0873 Jaguar, isoform I 0_seq1_2823 P:TTTTTTTTTTTGCTGTGTGCGATGCAGC F:ATGGATGGTACTGAAGGTCT S_Unigene394_c0 H+ transporting ATP synthase beta S:ATGATTCTTCCGAGTGTCTT T/A 0.9000 0.1000 0.7108 _seq1_418 subunit isoform 2 P:TTTTTTTTGGTGAGCCCTGTGTGGACAT F:CTCAGCAAACTTGCCCG S_Unigene1402_c S:CGATTGGATGCTAGGCTCT C/T 0.9211 0.0789 0.8371 CRE-SPE-15 protein 0_seq1_1570 P:TTTTTTTTTTTTTTTTTGGTCAAACCAAACTTAAAGT CC F:TTGAGCCATCTGACACAAT S_Unigene508_c0 S:GCCATCCTCCAACTGTTT T/C 0.9245 0.0755 0.0180 Ubc protein _seq1_283 P:TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTC AGGACAAAGAGGGAATCCC F:CAAAAGCAACATTGCCCA S_Unigene1402_c S:CGATGCAGCTATGAAGCAC A/T 0.9818 0.0182 0.0011 Protein SPE-15 0_seq1_2789 P:TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCCACCA GTGAATAGATACCTA F:TTTCAGTGGCACCTTGAT S_Unigene1402_c S:AAGGAGGAACTGAGGGA C/T 0.2553 0.7447 0.0000 AGAP000776-PA 0_seq1_2601 P:TTTTTTTTCTGGTCAACCGTGTCATGCA F:CTTTTTCGCTCCAGCTCT S_Unigene1402_c S:GACTGCGTAAACTCCAGG A/C 0.8000 0.2000 0.5133 Jaguar, isoform H 0_seq1_975 P:TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTG AGAAACAGCGCCGAGCAGA 2 3

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3431v1 | CC BY 4.0 Open Access | rec: 23 Nov 2017, publ: 23 Nov 2017 Table 3(on next page)

PCR primers used in gene cloning.

Note: We designed seven pairs of primers and three pairs of primers for reverse transcription PCR (RT-PCR) amplification of the coding region,as the length of Os-NMHC and MyHC are too long.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3431v1 | CC BY 4.0 Open Access | rec: 23 Nov 2017, publ: 23 Nov 2017 1

Usage Primer’s name Primer sequence(5’-3’) Explanation Test-1F CCAACCGCACCAGCCGTGAGT To amplify one part of Os- Test-1R GCGGTCCAGAGATTTGTTGAT NMHC fragment Test-2F TAAGAATAAGTATGAGGCAAT To amplify one part of Os- Test-2R GCTCCACTGTCATATCGTCCA NMHC fragment Test-3F GACTTCCTACAACTTCGAGCA To amplify one part of Os- Test-3R CTCTTTCACTCTCTGCTTGTC NMHC fragment Test-4F ACCGCACTAACCCAGGCATTC To amplify one part of Os- Test-4R CTCTGGATGACACGGATAGCA NMHC fragment Test-5F CTGTATCGCATTGGGCAGAGC To amplify one part of Os- Test-5R GCTGTGGTGTCCAGGGAATCT NMHC fragment RT-PCR Test-6F AGGAAGAGAACAAGAGAATCAG To amplify one part of Os- Test-6R AGGAAGAGAACAAGAGAATCAG NMHC fragment Test-7F CCAAGCGTAATGCTGAGTCTG To amplify one part of Os- Test-7R CATCCTCTTCTCCATCTTTCT NMHC fragment Test-8F TGCGTGGCTATCAACCCC To amplify one part of Test-8R GCCCTCAAGCACACCGTT MyHC fragment Test-9F AGACTGTGTCCCACTTGC To amplify one part of Test-9R TGAGCGGACGGATGAGAT MyHC fragment Test-10F GTCAAGAAATACCAGCAG To amplify one part of Test-10R TAGTGATGATGATGGTGG MyHC fragment 3’RACE-F1 ATGTCGGATAAAGCCCGCAAAG Gene-specific outer primer for Os-NMHC 3’RACE-F2 GCACGCACAAAGGCAACC Gene-specific inner primer for Os-NMHC 3’RACE-F3 GCGGCACACCAAGTTTGACCACAT Gene-specific outer primer for MyHC 3’RACE-F4 AACGAGGGTGGAATCCGGACTATA Gene-specific inner primer for MyHC 3’RACE outer primer TACCGTCGTTCCACTAGTGATTT RACE Primers from kit 3’RACE inner primer CGCGGATCCTCCACTAGTGATTTCACTATAGG 5’RACE-R1 TTGGCTTGTAGCAGTTGGTTCTCA Gene-specific outer primer for Os-NMHC 5’RACE-R2 AAACCCATTGGATTCGTCTG Gene-specific inner primer for Os-NMHC 5’RACE-R3 GTAGGCATTGTCAGAGAT Gene-specific outer primer for MyHC 5’RACE-R4 AGGGGTTGATAGCCACGC Gene-specific inner primer for MyHC

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3431v1 | CC BY 4.0 Open Access | rec: 23 Nov 2017, publ: 23 Nov 2017 5’RACE outer primer CATGGCTACATGCTGACAGCCTA Primers from kit 5’RACE inner primer CGCGGATCCACAGCCTACTGATGATCAGTCGATG

qRT-PCR primer F AGACTGGTCCAAGTATGCCTA Used to amplify Os- qRT-PCR primer R NMHC fragment for real- CCATAATGCTCATGGACTCG time PCR qRT-PCR primer F GCCTCCTCATTTGTTCTCCA Used to amplify MyHC qRT-PCR qRT-PCR primer R fragment for real-time ATCTTCTTCTCGGCTCCCTC PCR 18S primer F CGGCTACCACATCCAAGGAA Used to amplify 18S 18S primer R fragment for real-time GCTGGAATTACCGCGGCT PCR 2

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3431v1 | CC BY 4.0 Open Access | rec: 23 Nov 2017, publ: 23 Nov 2017 Table 4(on next page)

Structural differences among lung sacs of four species in the family Onchidiidae.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3431v1 | CC BY 4.0 Open Access | rec: 23 Nov 2017, publ: 23 Nov 2017 1 Paraoncidium Platevindex Peronia Onchidium reevesii mortoni verruculata struma Small pore, thick Big pore, thick Big pore, thick Big pore, thin Reticular septa wall wall wall wall Secondary septa Nothing Developed Developed Developed Third septa Nothing Nothing Nothing Developed Diameter of sac rooms 0.5-1.5 0.8-5.0 4.5-6.6 5.1-12.7 (μm) Diameter of small room Nothing 0.4-2.7 1.5-4.2 3.4-7.3 (μm) Diameter of Nothing Nothing Nothing 0.7-4.5 subordinate rooms(μm) 2 3

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3431v1 | CC BY 4.0 Open Access | rec: 23 Nov 2017, publ: 23 Nov 2017 Table 5(on next page)

Annotated unigenes by gene ontology.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3431v1 | CC BY 4.0 Open Access | rec: 23 Nov 2017, publ: 23 Nov 2017 1 Table 4.Annotated unigenes by gene ontology. 2 Platevindex Paraoncidium Onchidium Peronia 3 mortoni reevesii struma verruculata Biological process 68918 124598 221660 137152 Molecular function 29298 51891 90183 60939 Cellular component 33109 57136 105005 65006

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3431v1 | CC BY 4.0 Open Access | rec: 23 Nov 2017, publ: 23 Nov 2017 Table 6(on next page)

Data of four Onchidiidae

Sample_M, Platevindex mortoni; Sample_R, Paraoncidium reevesii; Sample_S, Onchidium struma; Sample_V, Peronia verruculata.

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3431v1 | CC BY 4.0 Open Access | rec: 23 Nov 2017, publ: 23 Nov 2017 1 Sample ID SeqType Raw Read Data Product Effective Effective Data Effective Read Num. read Num Rate(%) length( bp) Sample_M Pair-End 101 61356624 6197019024bp 60219324 5841892655 bp 94.2694 (6.197Gb) (5.842Gb) Sample_R Pair-End 101 90701864 9160888264bp 89062542 8617271978 bp 94.0659 (9.161Gb) (8.617Gb) Sample_S Pair-End 101 63774300 6441204300bp 62624204 6073850713bp 94.29682 (6.441Gb) (6.074Gb) Sample_V Pair-End 101 62832016 6346033616bp 61663900 5987378475 bp 94.34836 (6.346Gb) (5.987Gb) 2

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3431v1 | CC BY 4.0 Open Access | rec: 23 Nov 2017, publ: 23 Nov 2017