{"paper_id":"222657d7-e4f2-432e-8da6-b10a8e6de52b","body_text":"The Evolutionary History and Ecological Adaptation of\nCapulus danieli (Littorinimorpha, Capulidae)\nYaoyu Xie 1, Yida Han 1, Menghao Jia1, Linxuan Cai 1, Bin Zhao2, Zhenlin Hao 1*,\nYing Tian 13*\n1 Key Laboratory of Mariculture & Stock Enhancement in North China Sea, Ministry\nof Agriculture and Rural Affairs, Dalian Ocean University, Dalian, Liaoning 116023,\nChina\n2 College of Environment and Resources, Dalian Minzu University, 18 Liaohe West\nRoad, Dalian 116600, PR China\n3 Dalian shell museum ,Dalian, Liaoning 116023, China\nCorresponding author: Zhenlin Hao (haozhenlin@dlou.edu.cn); Ying Tian\n(tianying@dlou.edu.cn)\nAbstract\nCapulus danieli, a distinct member of Capulidae, with a limpet-shaped shell, exhibits\na unique ecological behaviour by attaching and drilling onto the shells of scallops,\ndistinguishing itself from other members of the gastropod class, offering a compelling\ncase for evolutionary and ecological study. This study initially obtained the complete\nmitochondrial genome of C. danieli through second-generation sequencing. In\naddition, 25 species closely related to C. danieli were selected to establish\nphylogenetic analysis using Maximum Likelihood (ML) and Bayesian Inference (BI)\nmethods. Furthermore, a divergence time tree of Capulidae was constructed based on\nthe analysis of the 16S rRNA gene sequence of 11 Capulidae species.\nThe results showed that the mitochondrial genome of C. danieli is similar to most\nknown neogastropods, confirming the first record of this species in China. The\nphylogenetic analysis also revealed a close evolutionary relationship between C.\ndanieli (Family Capulidae) and Ficus subintermedia, Ficus variegata (Family Ficidae)\nwithin the Order Littorinimorpha. The divergence time estimation suggested that C.\ndanieli diverged approximately 52.29 million years ago. The genus Capulus of\nmollusks exhibits morphological plasticity, adapting their form to better suit their\nparasitic lifestyle. This adaptability may aid in their survival and reproduction on\nvarious hosts. The adaptive changes in the shell morphology of Capulus species in\nresponse to the morphology of their host shells can be considered an example of\nco-evolution.\nKeywords\nmitochondrial genome; phylogeny; divergence time estimation\nIntroduction\nCapulidae belongs to the Phylum Mollusca, Class Gastropoda, Subclass\nCaenogastropoda, Order Littorinimorpha, is a widely distributed marine gastropod. It\ncomprises eighteen acknowledged genera, the majority of which exhibit a coiled form\nAuthor-formatted, not peer-reviewed document posted on 06/11/2024. DOI:  https://doi.org/10.3897/arphapreprints.e141013\n\nmorphology. However, a considerable subset of Capulidae species also exhibit a\nlimpet-like form, demonstrating the family's morphological diversity (Fassio et al.\n2020a). Within the uncoiled faction, the genus Capulus (established by Montfort in\n1810) stands out as the most extensively distributed and comprises a minimum of\ntwenty extant species according to contemporary taxonomical consensus (Worms\n2024). Capulidae represents an exemplary case of extreme limpetization among\ngastropods (Simone 2018). Capulid shell plasticity is associated with a broad range of\nfeeding ecology. Many coiled species within the Capulidae are obligate suspension\nfeeders, capitalizing on water currents generated by their hosts to facilitate feeding\nmechanisms (Iyengar 2007). Capulus danieli is a relatively large species within the\ngenus, with diameters ranging from 20 to 30 mm (Orr 1962). Characterized by its low,\nlimpet-shaped form, this species possesses a unique shell structure with a protoconch\ncomposed of smooth, planispiral whorls, followed by a cap-shaped teleoconch\nfeaturing a large, horseshoe-shaped muscle scar (Ponder and Lindberg 1997). The\nspecies of the genus Capulus show significant variations in morphology, and this\nvariation is related to the morphology of the host shells they parasitize (Beu et al.\n2004).\nEcologically, C. danieli shows intriguing behaviours, notably its attachment and\ndrilling onto the shells of scallops, reflecting a xenomorphic sculpture mirroring the\nform of its host (Orr 1962). Despite its ecological significance, research on C. danieli\nremains limited.\nDue to the rarity of most capulid species and the infrequent collection of live\nspecimens, only a few studies have attempted to explore the phylogenetic relationship\nof Capulidae. The first phylogeny of capulids was produced by Fassio et al. (2015),\nwho also explored the larval ecology of some Antarctic species. More recently, Fassio\net al. (2020b) proposed a new phylogenetic hypothesis using a taxonomic framework\nbased on six genera to address the Indo-West Pacific diversity of Hyalorisia.\nAdditionally, Fassio et al. (2020a) conducted an ancestral state reconstruction analysis\non a time-calibrated phylogenetic tree within the family Capulidae, suggesting that\ncapulids evolved from a coiled suspension feeder lineage, with a significant\nevolutionary shift to kleptoparasitism occurring in the family ancestor.\nHere, we present the first characterisation of the mitochondrial genome of C.\ndanieli to elucidate its gene function, phylogenetic relationships with limpet-like\ngastropods, and divergence time estimation. This study advances our understanding of\nC. danieli's evolutionary history and ecological adaptations.\nMaterials and Methods\nSample collection and identification\nFour specimens of C. danieli were collected in December 2022 from Yangjiang,\nGuangdong Province, China (21°85′N; 111°95′E). The individual capture was not\nfeasible, the specimens were all found on the surface of shells of the economically\nsignificant scallop Amusium pleuronectes (Figure 1). Capulus specimens were\nrelatively rare, with approximately one found in every 30 scallops. Fresh tissue was\nstripped from the shell, digestive glands were removed, and foot muscle tissue was\nAuthor-formatted, not peer-reviewed document posted on 06/11/2024. DOI:  https://doi.org/10.3897/arphapreprints.e141013\n\npreserved in anhydrous ethanol for subsequent experiments.\nThe species identification was based on the classification and comparison of the\nmorphological characteristics of the specimens by Fassio et al. (2020a) and Orr\n(1962), according to its ovate, reddish-brown shell, irregular concentric growth lines\non the surface and degradation, curls backward apex. By blasting in GenBank through\nthe 16S in the mitochondrial gene of the species, and the percent identity with C.\ndanieli being 99.43%, it was determined that the species was C. danieli. Currently,\nthere are no records of this species in China; previous records mainly documented\nCapulus dilatatus A. Adams, 1860, Capulus kawamurai Habe, 1992, and Capulus\notohimeae Habe, 1946 (Liu 2008;Zhang 2008). These records appear morphologically\nsimilar to the species described in this paper, but due to the lack of actual specimens\nand molecular information, it is impossible to determine whether they are synonyms.\nFigure 1. C. danieli and its host A. pleuronectes.\nA, The back view of C. danieli. B, The ventral view of C. danieli. C, The lateral view of C. danieli.\nD, C. danieli attached to A. pleuronectes. E, The shell of A. pleuronectes with a drill hole from C.\ndanieli. (Scales 10mm).\nDNA extraction, library preparation and next generation sequence\nOne specimen had its foot muscle extracted (2g) for DNA extraction. The genomic\nDNA (gDNA) was extracted using the Qiagen DNeasy® Blood & Tissue kit (Qiagen,\nHilden, Alemanha) and pre-grinding in liquid nitrogen. The concentration of the\nextracted DNA was tested using a Qubit dsDNA HS assay kit from Sangon (Shanghai,\nChina), and its integrity was confirmed using 1% agarose gel electrophoresis.\nSubsequently, library preparation and next-generation sequencing were performed by\nSangon Biotech (Shanghai) Co., Ltd. For the library preparation, 500 ng of the\nquantified DNA was randomly fragmented using Covaris (Woburn, USA). The Hieff\nNGS® MaxUp II DNA Library Prep Kit for Illumina from YEASEN (Shanghai,\nChina) was utilized for the following steps. The process included repairing the ends\nand adding a 3ʹ end A tail, followed by the ligation of adaptors using an enhancer and\nFast T4 DNA ligase. Index primers were added through PCR, and the resulting\namplified product (approximately 400 bp) was selected using DNA selection beads.\nAuthor-formatted, not peer-reviewed document posted on 06/11/2024. DOI:  https://doi.org/10.3897/arphapreprints.e141013\n\nThe concentration and size of the library were confirmed using the Qubit 4.0 (Thermo,\nWaltham, USA) and 2% agarose gel electrophoresis, respectively, and the libraries\nwere pooled and loaded onto a Novaseq 6000 from Illumina (San Diego, USA) or\nDNBseq-T7 from BGI (Shenzhen, China) sequencer using a 2 × 150 bp paired-end\nsequence kit, following the manufacturer's instructions (Dierckxsens et al. 2017).\nSequence assembly and annotation of the mitochondrial genome\nRaw sequencing data of at least 6 GB was used for subsequent analyses. All of the\nraw reads were trimmed by Fastp (0.36) (Chen et al. 2018). SPAdes software (version\n3.15) (Bankevich et al. 2012) was used to assemble the raw sequence reads into\ncontigs. The candidate mitochondrial contig with lapped bases between the start and\nend of the contig was selected as a circular genome from raw contigs. Finally, the\nlapped bases were dropped from the candidate contig to generate a complete\nmitochondrial genome. NCBI-blast tblastn and Hmmer software search for\nprotein-coding genes from scaffolds against the protein database and MiTFi (1.1) was\nused to annotate tRNA and rRNA genes.\nSystematic analysis\nA total of 25 species were curated for the construction of a phylogenetic tree based on\nthe complete mitochondrial genome sequences. The genetic data for 23 species\nencompassing families Haliotidae, Neritidae, Patellidae, Nacellidae, Calyptraeidae,\nMuricidae, Ficidae, Naticidae, Strombidae, Struthiolariids, and Xenophoridae were\nobtained from the NCBI database (refer to Table 1). The selection of certain species\nwas predicated on their morphological resemblance to C. danieli (Vermeij 2017), it\ncontains all the limpet-like families of gastropods mentioned in Vermeij's 2017 article\nwith mitochondrial genomes. Further selections were made based on their inferred\nphylogenetic proximity to C. danieli as delineated by the comparative alignment of\ntheir mitochondrial genomes accessible via NCBI.\nAdditionally, two bivalve species, Chlamys farreri and Mizuhopecten yessoensis,\nwere incorporated as outgroup, with GenBank accession numbers EF473269.1 and\nFJ595959.1 respectively. Prior to tree construction, PhyloSuite v1.2.3 (Zhang et al.\n2020; Xiang et al. 2023) was utilised to extract the protein-coding genes (PCGs) from\neach sequence, followed by multiple sequence alignment using MAFFT (Abascal et al.\n2010; Katoh and Standley 2013). The alignment results of the protein-coding gene\nsequences were optimised using MACSE (Ranwez et al. 2018), and Gblocks\n(Talavera and Castresana 2007) were employed for sequence pruning. The isolated\nPCGs were then concatenated to form a unified dataset. ModelFinder (Shapiro et al.\n2006; Kalyaanamoorthy et al. 2017) was employed to segregate the data into\nappropriate partitions and to identify the most suitable evolutionary model. The\nphylogenetic tree was constructed using both the Maximum Likelihood (ML) method\nin IQ-TREE v2.2.0 (Nguyen et al. 2015) and Bayesian Inference (BI) in MrBayes\nv3.2.7a (Ronquist et al. 2012). To evaluate the robustness of the branches, 5000\nbootstrap replicates were performed for the highest scoring ML tree. In the Bayesian\nanalysis, Markov Chain Monte Carlo (MCMC) simulations were initiated for\nAuthor-formatted, not peer-reviewed document posted on 06/11/2024. DOI:  https://doi.org/10.3897/arphapreprints.e141013\n\n1,000,000 generations, with data collection occurring every 1000 generations. The\ninitial 25% of the MCMC sampled data was excluded as burn-in to ensure the\naccuracy of the posterior estimates.\nTable 1. List of the mitochondrial genome of 25 species analyzed in this study and their\nGenBank accession numbers.\nSubclass Family Species Length (bp) GenBank\naccession\nVetigastropoda Haliotidae Haliotis iris 17131 KU310895.1\nHaliotis rubra 16907 AY588938.1\nNeritimorpha Neritidae Clithon sowerbianum 15919 MT230542.1\nTheodoxus fluviatilis 15667 MT628587.1\nPatellogastropoda Patellidae Patella ferruginea 14400 MH916654.1\nPatella vulgata 14808 MH916653.1\nNacellidae Cellana radiata 16194 MH916651.1\nCellana toreuma 16268 ON018805.1\nNacella clypeater 16742 KT990124.1\nNacella magellanica 16663 KT990125.1\nCaenogastropoda Calyptraeidae Desmaulus extinctorium 16608 OQ511529.1\nMuricidae Concholepas concholepas 15495 JQ446041.1\nIndothais sacellum 15237 NC063938.1\nCapulidae Capulus danieli 15640 NC084349.1\nFicidae Ficus subintermedia 16255 OR522697.1\nFicus variegata 15736 NC056153.1\nNaticidae Neverita didyma 15629 NC046594.1\nNotocochlis qualtieriana 15176 NC046705.1\nStrombidae Canarium labiatum 15843 NC084213.1\nLaevistrombus canarium 15626 NC053786.1\nStruthiolariidae Struthiolaria papulosa 15475 NC059921.1\nXenophoridae Onustus exutus 16043 MK327366.1\nXenophora japonica 15684 MW244823.1\nAutobranchia\n(Outgroup)\nPectinidae Mizuhopecten yessoensis 20964 FJ595959.1\nChlamys farreri 20889 EF473269.1\nEstimation of differentiation time of Capulidae\nWe employed the 16S rRNA gene sequences of 11 Capulidae species (Table 2) to\nestimate the divergence time within the Capulidae family, utilizing the topological\nstructure of the Bayesian phylogenetic tree as a reference framework. The analysis\nwas conducted using BEAST v2.7.6 software (Drummond et al. 2012), employing a\nrelaxed clock model. The Yule process was selected to model the prior branch\nevolution rate of the tree.\nTo incorporate fossil evidence, we referenced the oldest species confidently\nassignable to the genus Capulus, specifically Capulus onyxoides (Cossmann, 1879†),\nwhich dates back to the Ypresian period (Lower Eocene, 56–47.8 Ma). Similarly, we\nreferenced Capulus (Hyalorisia) nettlesi (Robinson, 1983†) from the Upper Eocene\nperiod (41.2–33.9 Ma) for the genus Hyalorisia (Fassio et al. 2020a). Markov chain\nMonte Carlo (MCMC) analysis was executed with 100 million generations with\nsamples taken every 1000 generations. The TreeAnnotator v1.8.4 component of the\nBEAST software package was used to discard the first 25% of aging samples as\nburn-in. Convergence of the chain was confirmed using Tracer v.1.7 (Rambaut et al.\nAuthor-formatted, not peer-reviewed document posted on 06/11/2024. DOI:  https://doi.org/10.3897/arphapreprints.e141013\n\n2018), ensuring effective sample size (ESS) values greater than 200. The resulting\ndivergence time estimates were validated against Timetree fossil records, and the\nresults were reported to verify the accuracy. We used TVBOT as a graphic\nbeautification tool (https://www.chiplot.online/tvbot.html) (Peng et al. 2022).\nTable 2. List of the 16S rRNA genes of Capulidae species analyzed in this study and\ntheir GenBank accession numbers.\nResults\nCharacteristics of C. danieli's mitochondrial genome\nThe complete mitochondrial genome of C. danieli spanned 15,640 bp (GenBank\naccession number: NC084349.1) and comprised a typical circular, closed,\ndouble-stranded molecule with a control region. The coding region contains a total of\n37 coding genes, including 13 protein-coding genes, 22 tRNA, and 2 rRNA. The\nnon-coding region contains a total of 23 gene intervals, with a combined length of 829\nbp. The contents of four bases were A: 31.57%, T: 39.55%, G: 14.92%, C: 13.96%.\nThe A + T content was 71.12% and the G + C content was 28.88% (Figure 2).\nSpecies name Length (bp) GenBank\naccession\nCapulus danieli 524 MT525840.1\nCapulus ungaricus 525 MT525803.1\nHyalorisia tosaensis 405 MT525849.1\nHyalorisia galea 526 MT525835.1\nCryocapulus subcompressus 760 KR364850.1\nTorellia exilis 529 MT525847.1\nTorellia smithi 714 KR364868.1\nTorellia insignis 769 KR364865.1\nTorellia mirabilis 745 KR364856.1\nTrichamathina violaceus 529 MT525806.1\nTrichamathina bicarinata 529 MT525846.1\nAuthor-formatted, not peer-reviewed document posted on 06/11/2024. DOI:  https://doi.org/10.3897/arphapreprints.e141013\n\nFigure 2. Gene map of the complete mitogenomes for C. danieli. The bar plots\n(turquoise) in the inner circle represent the depth of base sequencing. The outermost\ngene element contains forward transcription genes within its inner circle, while the\nouter circle contains reverse transcription genes.\nPhylogenetic relationship\nThe phylogenetic analysis of the gastropod species was conducted utilizing two\nprominent methods: The Bayesian Inference (BI) method and Maximum Likelihood\n(ML). The approaches were used to construct a phylogenetic tree of 13 PCGs\nsequences across 25 species, with two species of C. farreri and M. yessoensis as\noutgroup (Figure 3). The phylogenetic tree contained 13 families, each forming a\nmonophyletic group. This pattern is consistent with prior research, which has\nestablished the monophyletic nature of these groups (Zhong et al. 2020; Qi et al. 2024;\nMa et al. 2024). A comprehensive examination of the tree revealed a specific\nevolutionary hierarchy: ((Neritimorpha + Patellogastropoda) + Vetigastropoda +\nCaenogastropoda). This structure underscores the basal position of the Haliotidae\nfamily within the Vetigastropoda as the outermost branch of the tree. The remaining\ngastropods were primarily divided into two major clades: (Neritimorpha +\nPatellogastropoda) and Caenogastropoda. C. danieli exhibited the closest genetic\naffinity with Ficus, a member of the Littorinimorpha. C. danieli had a distant\nphylogenetic relationship with the limpet-like shell species Neritimorpha,\nPatellogastropoda, and Caenogastropoda, indicating that it does not belong to the\nsame primary branch. The recurrent appearance of limpet-like shells in multiple\nbranches of the phylogenetic tree suggests multiple independent evolutionary events\nwithin the Gastropoda. Statistically, limpet-like shells have been documented in at\nleast 54 families of gastropods (Vermeij 2017).\nAuthor-formatted, not peer-reviewed document posted on 06/11/2024. DOI:  https://doi.org/10.3897/arphapreprints.e141013\n\nFigure 3. The phylogenetic tree was constructed based on the Maximum Likelihood\n(ML) and Bayesian Inference (BI) of 13 protein-coding genes. The numbers displayed\nabove branches were Bayesian posterior probabilities, and the numbers below\nbranches represent bootstrap support. The red triangle represents the species C.\ndanieli of this study.\nDivergence time estimation\nWe reconstructed a divergence time tree of Capulidae based on the analysis of the 16S\nto explore its evolutionary process (Figure 4). Through its evolution history, the\nfamily Capulidae had undergone numerous transitions towards the development of\nlimpet-shaped shells. The divergence time tree indicated that the Capulidae family\noriginated in the Callovian stage (Middle Jurassic) period approximately 155.25\nmillion years ago. This finding establishes a significant temporal benchmark for the\nfamily's emergence.\nThe family's evolution to its first limpet-like shell was exemplified by the branch\nrepresenting Cryocapulus subcompressus, which occurred around 123.95 Mya (Early\nCretaceous). Subsequently, the second evolution transition to the limpet-like shell\ntook place approximately 82.65 million years ago (Late Cretaceous), leading to a\ndivergence between the limpet-shaped genus Trichamathina and the spiral genus\nTorellia. A further divergence event occurred about 66.44 million years ago (Late\nCretaceous), distinguishing the genus Hyalorisia from the genus Capulus. The genus\nTrichamathina, characterized by its limpet-like shell, is primarily defined by\nsymmetrical and enlarged body whorls, yet it retains certain features reminiscent of\nthe coiled tower shape, indicating a distinct evolutionary pathway. About 66.44\nmillion years ago (Late Cretaceous), there was a divergence between Hyalorisia and\nCapulus. Although there are differences in the taxonomic status of the two species,\nAuthor-formatted, not peer-reviewed document posted on 06/11/2024. DOI:  https://doi.org/10.3897/arphapreprints.e141013\n\nthey show similar and highly capped shell-like characteristics, which further\nhighlights the diversity and complexity of the evolution of the family. Finally, during\nthe Eocene (Paleogene), about 52.29 to 36.03 million years ago, the differentiation of\nCapulidae reached its peak, and Capulidae gradually evolved a shell type similar to\nthat of limpets. This period marked an important stage in the evolution of the family\nand provided valuable information about its evolution.\nFigure 4. Estimating the divergence time of the family Capulidae based on 16S. Bars\nindicate 95% highest posterior density intervals for node ages (Ma), and number at\nnode the median (Ma).\nDiscussion\nThe distribution of C. danieli\nC. danieli was originally identified in New Caledonia in the South Pacific (Beu et al.\n2004; Fassio et al. 2020a), and its distribution records also exist in Japan (Okutani et\nal. 2017). This study presents the first report of C. danieli in China, specifically in the\nvicinity of the South China Sea in Yangjiang City, Guangdong Province. C. danieli\n(Crosse, 1858) is a species widely distributed in the Western Pacific, ranging from\ncentral Japan (Okutani et al. 2017), the Philippines to southern Australia (Garrard\n1961), including northern New Zealand (Beu et al. 2004). In Australia, there are fossil\nrecords of C. danieli, discovered by Tate (1893) in the Miocene and Pliocene strata of\nVictoria and South Australia. The fossil records in New Zealand mainly come from\nthe Landguard Sand and Te Piki Member in the Wanganui region, and it expanded\nfrom Australia to New Zealand during the Pleistocene period (Beu et al. 2004). Its\ndispersal methods may be two fold: one is through the dispersal of its larvae, and the\nAuthor-formatted, not peer-reviewed document posted on 06/11/2024. DOI:  https://doi.org/10.3897/arphapreprints.e141013\n\nother is due to its host scallops, which are also found in the same distribution where\nthis species is detected. C. danieli might have a pelagic larval stage, and these larvae\ncan drift in the ocean and settle in suitable environments (Beu et al. 2004).The\nfindings from this study have confirmed Beu's hypothesis..this diffusion mechanism\nenables the species to cross the ocean in the absence of physical barriers. The\npresence of C. danieli found in the Philippines, the South China Sea of China, and\nJapan may be influenced by the Japan Current. The distribution of scallops to which it\nattaches is also found in the Philippines, the South China Sea of China, and Japan.\nThe possibility of its spread through the migration of scallops cannot be excluded.\nMore evidence also requires an increase in fossil records.\nCharacteristics of the mitogenomes\nIn this study, the whole genome sequence of C. danieli was obtained by\nhigh-throughput sequencing technology, with an assembled length of 15600 bp. The\nlength of the whole genome sequence of other gastropods in this study was\n14400–17131 bp (Table 1). In most cases, the mitochondrial genome of gastropods is\nusually between 14,000 and 18,000 bp (Chen et al. 2023; Kim et al. 2023; Qu et al.\n2024). The length of the mitochondrial genome of C. danieli was within the normal\nrange of gastropods. The mitochondrial genome AT content of C. danieli was 71.12%.\nAmong the gastropods in this study, the lowest AT content was Haliotis rubra\n(59.1%), and the highest was Ficus subintermedia (74.2%). The AT content of C.\ndanieli was higher than that of Vetigastropoda (59.1%–59.8%) and similar to that of\nmost Caenogastropoda (67.8%–74.2%). Generally, the AT content of gastropods is\nabout 60%–75% (Chen et al. 2023; Kim et al. 2023). The high content of AT makes\nthem more susceptible to base mutations. Due to its susceptibility to replication errors,\nwhich can lead to an increased rate of polymorphism under environmental stress\n(Broughton and Reneau 2006).\nPhylogenetic implications\nMitochondrial DNA sequences are increasingly being employed in phylogenetic\nstudies, owing to their significance in elucidating evolutionary relationships among\norganisms (Dhorne-Pollet et al. 2020). In this study, a phylogenetic tree was\nconstructed based on PCGs of 25 species, specifically focusing on gastropod mollusks\nthat exhibit the closest genetic and morphological affinities (limpet-like shell) with C.\ndanieli. The purpose is to understand the taxonomic status of C. danieli and its\nevolutionary relationship with limpet-like gastropods. Results showed : C. danieli was\nnot clustered with other limpet-like species but was grouped with F. subintermedia\nand F. variegata (Ficidae) to form one subclade. According to the available records,\nFicidae does not include any limpet-like species. Additionally, among the sister\nbranches of (C. danieli + F. subintermedia and F. variegata), the families Naticidae,\nStrombidae, Struthiolariidae, and Xenophoridae also do not have limpet-like species.\nIn contrast to other aforementioned families, Capulidae is the only family that has\nevolved shell-shaped mollusks.\nThroughout the Phanerozoic eon, which spans from the Cambrian to the\nNeogene periods, gastropods have experienced a multitude of morphological\ntransformations, with the evolution of limpet-like shells being particularly prevalent\nAuthor-formatted, not peer-reviewed document posted on 06/11/2024. DOI:  https://doi.org/10.3897/arphapreprints.e141013\n\nin marine environments (Vermeij 2017). The Capulidae family, known for its highly\nlimpet-shaped shells, has seen numerous transitions to this form (Fassio et al. 2020a).\nAlthough C. danieli shares morphological similarities with these limpet-like\ngastropods, it was found to be only distantly related. This instance of convergent\nevolution is primarily attributed to the influence of specific environmental pressures\n(Vermeij 2001). Becoming a limpet-like shell simplified shell structure of C. danieli,\nwhich is less suited to intense interspecific competition and predation. According to\nthe ecological niche and lifestyle of C. danieli, we can roughly summarize the\nbenefits of its transformation into a limpet-like shell: The limpet-like shell form\nminimizes water flow resistance, maximizes the attachment surface area, and reduces\nthe potential for dislodgement (Vermeij 2017), which is particularly adapted to the\nobligate sedentary parasitic lifestyle of C. danieli on the surface of scallops.\nIt is known from the phylogenetic tree of the Conidae family constructed based\non 16S that the recurrent emergence of the limpet-like shell within the family\nCapulidae, as exemplified by the Cryocapulus, Capulus + Hyalorisia, and\nTrichamathina lineages. The process of becoming a limpet-like shell appears\nintermittently and repeatedly in the family Capulidae, due to the complex\nevolutionary pressures that have shaped the diversity of shell morphologies within\nthis family.\nMolecular clock analysis estimated the Capulidae family originated in the\nMiddle Jurassic period, specifically during the Callovian stage, approximately 155.25\nmillion years ago. However, Fassio et al. proposed a more recent origin, dating\nCapulidae to 112.87 million years ago (Fassio et al. 2020a). This divergence predates\nthe oldest known Capulidae fossil record by several million years (Saul and Squires\n2008), indicating a gap between molecular and paleontological evidence that\nnecessitates further investigation to elucidate the early evolutionary narrative of\nCapulidae.\nThe divergence time for C. danieli is dated to around 52.29 million years ago\nduring the Palaeocene-Eocene transition, a period marked by global warming and\nelevated sea surface temperatures (Tripati et al. 2001). The paleoenvironmental\nconditions of this era, characterized by transient eutrophication and ocean\nacidification (Scheibner et al. 2005; Alegret and Ortiz 2007; Scheibner and Speijer\n2008), likely exerted selective pressures favouring a parasitic lifestyle in C. danieli.\nSuch rapid climatic shifts can precipitate significant alterations in faunal composition\nacross various habitats, presenting novel evolutionary prospects. Eutrophication can\nlead to alterations in the composition of primary producers in marine ecosystems,\nsuch as algal blooms. As the availability of suspended particulate matter diminishes\nand humus concentrations increase, gastropods may shift from filter feeding to\nalternative feeding strategies (Vermeij 2001). Poulin and Randhawa have discussed\nthe evolution of parasitic behavior as a response to environmental pressures. When\nthe main food sources become scarce or the energetic cost of feeding escalates,\norganisms may adopt parasitic lifestyles to persist (Poulin and Randhawa 2015). This\necological transition may have facilitated the adoption of a limpet-like shell form in C.\ndanieli, enhancing nutrient access through kleptoparasitism of bivalves.\nAuthor-formatted, not peer-reviewed document posted on 06/11/2024. DOI:  https://doi.org/10.3897/arphapreprints.e141013\n\nThe morphological changes\nThe species of the genus Capulus show significant variations in morphology, and this\nvariation is related to the morphology of the host shells they parasitize. For instance,\ntheir shell morphology might change according to the shape of the host, forming the\nso-called xenomorphic sculpture (Orr 1962). Some samples of C. danieli attached to\nhost shells with distinct radiating ribs develop corresponding shell edge depressions\n(Beu et al. 2004). In this study, the samples were attached to the smooth surface of\nscallop shells and did not form xenomorphic sculpture. However, the scallops to\nwhich they were attached showed concentric growth line patterns, and similar patterns\nwere also formed on the C. danieli that parasitized on their surface. This proves that\nthe species of the Capulus genus typically have morphological characteristics adapted\nto parasitic life.\nConclusions\nIn this study, we have documented the mitochondrial genome of C. danieli,\nrepresenting the inaugural comprehensive mitochondrial genome sequence recorded\nwithin the family Capulidae. We analysed the characteristics of the mitochondrial\nsequence of C. danieli. Additionally, we conducted a phylogenetic analysis to infer\nthe evolutionary nexus between C. danieli and limpet-like gastropods. Molecular\nclock estimates were utilised to approximate the divergence timeframe of the\nCapulidae lineage, concurrently delineating the evolutionary trajectory leading to the\nadoption of a limpet-like shell morphology.Additionally, we have summarized the\nrelationship between its morphological plasticity and its host, suggesting that although\nthe species exhibits a variety of external morphologies, the primary reason is\nco-evolution with the host's form. 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