The evolutionary history and ecological adaptation of Capulus danieli (Littorinimorpha, Capulidae)

Capulus danieli, a distinct member of Capulidae, with a limpet-shaped shell, exhibits a unique ecological behaviour by attaching and drilling onto the shells of scallops, distinguishing itself from other members of the gastropod class, offering a compelling case for evolutionary and ecological study. This study initially obtained the complete mitochondrial genome of C. danieli through second-generation sequencing. In addition, 25 species closely related to C. danieli were selected to establish phylogenetic analysis using Maximum Likelihood (ML) and Bayesian Inference (BI) methods. Furthermore, a divergence time tree of Capulidae was constructed based on the analysis of the 16S rRNA gene sequence of 11 Capulidae species. The results showed that the mitochondrial genome of C. danieli is similar to most known neogastropods, confirming the first record of this species in China. The phylogenetic analysis also revealed a close evolutionary relationship between C. danieli (Family Capulidae) and Ficus subintermedia, Ficus variegata (Family Ficidae) within the Order Littorinimorpha. The divergence time estimation suggested that C. danieli diverged approximately 52.29 million years ago. The genus Capulus of mollusks exhibits morphological plasticity, adapting their form to better suit their parasitic lifestyle. This adaptability may aid in their survival and reproduction on various hosts. The adaptive changes in the shell morphology of Capulus species in response to the morphology of their host shells can be considered an example of co-evolution.

mitochondrial genome; phylogeny; divergence time estimation

Capulidae belongs to the Phylum Mollusca, Class Gastropoda, Subclass Caenogastropoda, Order Littorinimorpha, is a widely distributed marine gastropod. It comprises eighteen acknowledged genera, the majority of which exhibit a coiled form Author-formatted, not peer-reviewed document posted on 06/11/2024. DOI:  https://doi.org/10.3897/arphapreprints.e141013 morphology. However, a considerable subset of Capulidae species also exhibit a limpet-like form, demonstrating the family's morphological diversity (Fassio et al. 2020a). Within the uncoiled faction, the genus Capulus (established by Montfort in 1810) stands out as the most extensively distributed and comprises a minimum of twenty extant species according to contemporary taxonomical consensus (Worms 2024). Capulidae represents an exemplary case of extreme limpetization among gastropods (Simone 2018). Capulid shell plasticity is associated with a broad range of feeding ecology. Many coiled species within the Capulidae are obligate suspension feeders, capitalizing on water currents generated by their hosts to facilitate feeding mechanisms (Iyengar 2007). Capulus danieli is a relatively large species within the genus, with diameters ranging from 20 to 30 mm (Orr 1962). Characterized by its low, limpet-shaped form, this species possesses a unique shell structure with a protoconch composed of smooth, planispiral whorls, followed by a cap-shaped teleoconch featuring a large, horseshoe-shaped muscle scar (Ponder and Lindberg 1997). The species of the genus Capulus show significant variations in morphology, and this variation is related to the morphology of the host shells they parasitize (Beu et al. 2004). Ecologically, C. danieli shows intriguing behaviours, notably its attachment and drilling onto the shells of scallops, reflecting a xenomorphic sculpture mirroring the form of its host (Orr 1962). Despite its ecological significance, research on C. danieli remains limited. Due to the rarity of most capulid species and the infrequent collection of live specimens, only a few studies have attempted to explore the phylogenetic relationship of Capulidae. The first phylogeny of capulids was produced by Fassio et al. (2015), who also explored the larval ecology of some Antarctic species. More recently, Fassio et al. (2020b) proposed a new phylogenetic hypothesis using a taxonomic framework based on six genera to address the Indo-West Pacific diversity of Hyalorisia. Additionally, Fassio et al. (2020a) conducted an ancestral state reconstruction analysis on a time-calibrated phylogenetic tree within the family Capulidae, suggesting that capulids evolved from a coiled suspension feeder lineage, with a significant evolutionary shift to kleptoparasitism occurring in the family ancestor. Here, we present the first characterisation of the mitochondrial genome of C. danieli to elucidate its gene function, phylogenetic relationships with limpet-like gastropods, and divergence time estimation. This study advances our understanding of C. danieli's evolutionary history and ecological adaptations.

Sample collection and identification Four specimens of C. danieli were collected in December 2022 from Yangjiang, Guangdong Province, China (21°85′N; 111°95′E). The individual capture was not feasible, the specimens were all found on the surface of shells of the economically significant scallop Amusium pleuronectes (Figure 1). Capulus specimens were relatively rare, with approximately one found in every 30 scallops. Fresh tissue was stripped from the shell, digestive glands were removed, and foot muscle tissue was Author-formatted, not peer-reviewed document posted on 06/11/2024. DOI:  https://doi.org/10.3897/arphapreprints.e141013 preserved in anhydrous ethanol for subsequent experiments. The species identification was based on the classification and comparison of the morphological characteristics of the specimens by Fassio et al. (2020a) and Orr (1962), according to its ovate, reddish-brown shell, irregular concentric growth lines on the surface and degradation, curls backward apex. By blasting in GenBank through the 16S in the mitochondrial gene of the species, and the percent identity with C. danieli being 99.43%, it was determined that the species was C. danieli. Currently, there are no records of this species in China; previous records mainly documented Capulus dilatatus A. Adams, 1860, Capulus kawamurai Habe, 1992, and Capulus otohimeae Habe, 1946 (Liu 2008;Zhang 2008). These records appear morphologically similar to the species described in this paper, but due to the lack of actual specimens and molecular information, it is impossible to determine whether they are synonyms. Figure 1. C. danieli and its host A. pleuronectes. A, The back view of C. danieli. B, The ventral view of C. danieli. C, The lateral view of C. danieli. D, C. danieli attached to A. pleuronectes. E, The shell of A. pleuronectes with a drill hole from C. danieli. (Scales 10mm). DNA extraction, library preparation and next generation sequence One specimen had its foot muscle extracted (2g) for DNA extraction. The genomic DNA (gDNA) was extracted using the Qiagen DNeasy® Blood & Tissue kit (Qiagen, Hilden, Alemanha) and pre-grinding in liquid nitrogen. The concentration of the extracted DNA was tested using a Qubit dsDNA HS assay kit from Sangon (Shanghai, China), and its integrity was confirmed using 1% agarose gel electrophoresis. Subsequently, library preparation and next-generation sequencing were performed by Sangon Biotech (Shanghai) Co., Ltd. For the library preparation, 500 ng of the quantified DNA was randomly fragmented using Covaris (Woburn, USA). The Hieff NGS® MaxUp II DNA Library Prep Kit for Illumina from YEASEN (Shanghai, China) was utilized for the following steps. The process included repairing the ends and adding a 3ʹ end A tail, followed by the ligation of adaptors using an enhancer and Fast T4 DNA ligase. Index primers were added through PCR, and the resulting amplified product (approximately 400 bp) was selected using DNA selection beads. Author-formatted, not peer-reviewed document posted on 06/11/2024. DOI:  https://doi.org/10.3897/arphapreprints.e141013 The concentration and size of the library were confirmed using the Qubit 4.0 (Thermo, Waltham, USA) and 2% agarose gel electrophoresis, respectively, and the libraries were pooled and loaded onto a Novaseq 6000 from Illumina (San Diego, USA) or DNBseq-T7 from BGI (Shenzhen, China) sequencer using a 2 × 150 bp paired-end sequence kit, following the manufacturer's instructions (Dierckxsens et al. 2017). Sequence assembly and annotation of the mitochondrial genome Raw sequencing data of at least 6 GB was used for subsequent analyses. All of the raw reads were trimmed by Fastp (0.36) (Chen et al. 2018). SPAdes software (version 3.15) (Bankevich et al. 2012) was used to assemble the raw sequence reads into contigs. The candidate mitochondrial contig with lapped bases between the start and end of the contig was selected as a circular genome from raw contigs. Finally, the lapped bases were dropped from the candidate contig to generate a complete mitochondrial genome. NCBI-blast tblastn and Hmmer software search for protein-coding genes from scaffolds against the protein database and MiTFi (1.1) was used to annotate tRNA and rRNA genes. Systematic analysis A total of 25 species were curated for the construction of a phylogenetic tree based on the complete mitochondrial genome sequences. The genetic data for 23 species encompassing families Haliotidae, Neritidae, Patellidae, Nacellidae, Calyptraeidae, Muricidae, Ficidae, Naticidae, Strombidae, Struthiolariids, and Xenophoridae were obtained from the NCBI database (refer to Table 1). The selection of certain species was predicated on their morphological resemblance to C. danieli (Vermeij 2017), it contains all the limpet-like families of gastropods mentioned in Vermeij's 2017 article with mitochondrial genomes. Further selections were made based on their inferred phylogenetic proximity to C. danieli as delineated by the comparative alignment of their mitochondrial genomes accessible via NCBI. Additionally, two bivalve species, Chlamys farreri and Mizuhopecten yessoensis, were incorporated as outgroup, with GenBank accession numbers EF473269.1 and FJ595959.1 respectively. Prior to tree construction, PhyloSuite v1.2.3 (Zhang et al. 2020; Xiang et al. 2023) was utilised to extract the protein-coding genes (PCGs) from each sequence, followed by multiple sequence alignment using MAFFT (Abascal et al. 2010; Katoh and Standley 2013). The alignment results of the protein-coding gene sequences were optimised using MACSE (Ranwez et al. 2018), and Gblocks (Talavera and Castresana 2007) were employed for sequence pruning. The isolated PCGs were then concatenated to form a unified dataset. ModelFinder (Shapiro et al. 2006; Kalyaanamoorthy et al. 2017) was employed to segregate the data into appropriate partitions and to identify the most suitable evolutionary model. The phylogenetic tree was constructed using both the Maximum Likelihood (ML) method in IQ-TREE v2.2.0 (Nguyen et al. 2015) and Bayesian Inference (BI) in MrBayes v3.2.7a (Ronquist et al. 2012). To evaluate the robustness of the branches, 5000 bootstrap replicates were performed for the highest scoring ML tree. In the Bayesian analysis, Markov Chain Monte Carlo (MCMC) simulations were initiated for Author-formatted, not peer-reviewed document posted on 06/11/2024. DOI:  https://doi.org/10.3897/arphapreprints.e141013 1,000,000 generations, with data collection occurring every 1000 generations. The initial 25% of the MCMC sampled data was excluded as burn-in to ensure the accuracy of the posterior estimates. Table 1. List of the mitochondrial genome of 25 species analyzed in this study and their GenBank accession numbers. Subclass Family Species Length (bp) GenBank accession Vetigastropoda Haliotidae Haliotis iris 17131 KU310895.1 Haliotis rubra 16907 AY588938.1 Neritimorpha Neritidae Clithon sowerbianum 15919 MT230542.1 Theodoxus fluviatilis 15667 MT628587.1 Patellogastropoda Patellidae Patella ferruginea 14400 MH916654.1 Patella vulgata 14808 MH916653.1 Nacellidae Cellana radiata 16194 MH916651.1 Cellana toreuma 16268 ON018805.1 Nacella clypeater 16742 KT990124.1 Nacella magellanica 16663 KT990125.1 Caenogastropoda Calyptraeidae Desmaulus extinctorium 16608 OQ511529.1 Muricidae Concholepas concholepas 15495 JQ446041.1 Indothais sacellum 15237 NC063938.1 Capulidae Capulus danieli 15640 NC084349.1 Ficidae Ficus subintermedia 16255 OR522697.1 Ficus variegata 15736 NC056153.1 Naticidae Neverita didyma 15629 NC046594.1 Notocochlis qualtieriana 15176 NC046705.1 Strombidae Canarium labiatum 15843 NC084213.1 Laevistrombus canarium 15626 NC053786.1 Struthiolariidae Struthiolaria papulosa 15475 NC059921.1 Xenophoridae Onustus exutus 16043 MK327366.1 Xenophora japonica 15684 MW244823.1 Autobranchia (Outgroup) Pectinidae Mizuhopecten yessoensis 20964 FJ595959.1 Chlamys farreri 20889 EF473269.1 Estimation of differentiation time of Capulidae We employed the 16S rRNA gene sequences of 11 Capulidae species (Table 2) to estimate the divergence time within the Capulidae family, utilizing the topological structure of the Bayesian phylogenetic tree as a reference framework. The analysis was conducted using BEAST v2.7.6 software (Drummond et al. 2012), employing a relaxed clock model. The Yule process was selected to model the prior branch evolution rate of the tree. To incorporate fossil evidence, we referenced the oldest species confidently assignable to the genus Capulus, specifically Capulus onyxoides (Cossmann, 1879†), which dates back to the Ypresian period (Lower Eocene, 56–47.8 Ma). Similarly, we referenced Capulus (Hyalorisia) nettlesi (Robinson, 1983†) from the Upper Eocene period (41.2–33.9 Ma) for the genus Hyalorisia (Fassio et al. 2020a). Markov chain Monte Carlo (MCMC) analysis was executed with 100 million generations with samples taken every 1000 generations. The TreeAnnotator v1.8.4 component of the BEAST software package was used to discard the first 25% of aging samples as burn-in. Convergence of the chain was confirmed using Tracer v.1.7 (Rambaut et al. Author-formatted, not peer-reviewed document posted on 06/11/2024. DOI:  https://doi.org/10.3897/arphapreprints.e141013 2018), ensuring effective sample size (ESS) values greater than 200. The resulting divergence time estimates were validated against Timetree fossil records, and the

were reported to verify the accuracy. We used TVBOT as a graphic beautification tool (https://www.chiplot.online/tvbot.html) (Peng et al. 2022). Table 2. List of the 16S rRNA genes of Capulidae species analyzed in this study and their GenBank accession numbers.

Characteristics of C. danieli's mitochondrial genome The complete mitochondrial genome of C. danieli spanned 15,640 bp (GenBank accession number: NC084349.1) and comprised a typical circular, closed, double-stranded molecule with a control region. The coding region contains a total of 37 coding genes, including 13 protein-coding genes, 22 tRNA, and 2 rRNA. The non-coding region contains a total of 23 gene intervals, with a combined length of 829 bp. The contents of four bases were A: 31.57%, T: 39.55%, G: 14.92%, C: 13.96%. The A + T content was 71.12% and the G + C content was 28.88% (Figure 2). Species name Length (bp) GenBank accession Capulus danieli 524 MT525840.1 Capulus ungaricus 525 MT525803.1 Hyalorisia tosaensis 405 MT525849.1 Hyalorisia galea 526 MT525835.1 Cryocapulus subcompressus 760 KR364850.1 Torellia exilis 529 MT525847.1 Torellia smithi 714 KR364868.1 Torellia insignis 769 KR364865.1 Torellia mirabilis 745 KR364856.1 Trichamathina violaceus 529 MT525806.1 Trichamathina bicarinata 529 MT525846.1 Author-formatted, not peer-reviewed document posted on 06/11/2024. DOI:  https://doi.org/10.3897/arphapreprints.e141013 Figure 2. Gene map of the complete mitogenomes for C. danieli. The bar plots (turquoise) in the inner circle represent the depth of base sequencing. The outermost gene element contains forward transcription genes within its inner circle, while the outer circle contains reverse transcription genes. Phylogenetic relationship The phylogenetic analysis of the gastropod species was conducted utilizing two prominent methods: The Bayesian Inference (BI) method and Maximum Likelihood (ML). The approaches were used to construct a phylogenetic tree of 13 PCGs sequences across 25 species, with two species of C. farreri and M. yessoensis as outgroup (Figure 3). The phylogenetic tree contained 13 families, each forming a monophyletic group. This pattern is consistent with prior research, which has established the monophyletic nature of these groups (Zhong et al. 2020; Qi et al. 2024; Ma et al. 2024). A comprehensive examination of the tree revealed a specific evolutionary hierarchy: ((Neritimorpha + Patellogastropoda) + Vetigastropoda + Caenogastropoda). This structure underscores the basal position of the Haliotidae family within the Vetigastropoda as the outermost branch of the tree. The remaining gastropods were primarily divided into two major clades: (Neritimorpha + Patellogastropoda) and Caenogastropoda. C. danieli exhibited the closest genetic affinity with Ficus, a member of the Littorinimorpha. C. danieli had a distant phylogenetic relationship with the limpet-like shell species Neritimorpha, Patellogastropoda, and Caenogastropoda, indicating that it does not belong to the same primary branch. The recurrent appearance of limpet-like shells in multiple branches of the phylogenetic tree suggests multiple independent evolutionary events within the Gastropoda. Statistically, limpet-like shells have been documented in at least 54 families of gastropods (Vermeij 2017). Author-formatted, not peer-reviewed document posted on 06/11/2024. DOI:  https://doi.org/10.3897/arphapreprints.e141013 Figure 3. The phylogenetic tree was constructed based on the Maximum Likelihood (ML) and Bayesian Inference (BI) of 13 protein-coding genes. The numbers displayed above branches were Bayesian posterior probabilities, and the numbers below branches represent bootstrap support. The red triangle represents the species C. danieli of this study. Divergence time estimation We reconstructed a divergence time tree of Capulidae based on the analysis of the 16S to explore its evolutionary process (Figure 4). Through its evolution history, the family Capulidae had undergone numerous transitions towards the development of limpet-shaped shells. The divergence time tree indicated that the Capulidae family originated in the Callovian stage (Middle Jurassic) period approximately 155.25 million years ago. This finding establishes a significant temporal benchmark for the family's emergence. The family's evolution to its first limpet-like shell was exemplified by the branch representing Cryocapulus subcompressus, which occurred around 123.95 Mya (Early Cretaceous). Subsequently, the second evolution transition to the limpet-like shell took place approximately 82.65 million years ago (Late Cretaceous), leading to a divergence between the limpet-shaped genus Trichamathina and the spiral genus Torellia. A further divergence event occurred about 66.44 million years ago (Late Cretaceous), distinguishing the genus Hyalorisia from the genus Capulus. The genus Trichamathina, characterized by its limpet-like shell, is primarily defined by symmetrical and enlarged body whorls, yet it retains certain features reminiscent of the coiled tower shape, indicating a distinct evolutionary pathway. About 66.44 million years ago (Late Cretaceous), there was a divergence between Hyalorisia and Capulus. Although there are differences in the taxonomic status of the two species, Author-formatted, not peer-reviewed document posted on 06/11/2024. DOI:  https://doi.org/10.3897/arphapreprints.e141013 they show similar and highly capped shell-like characteristics, which further highlights the diversity and complexity of the evolution of the family. Finally, during the Eocene (Paleogene), about 52.29 to 36.03 million years ago, the differentiation of Capulidae reached its peak, and Capulidae gradually evolved a shell type similar to that of limpets. This period marked an important stage in the evolution of the family and provided valuable information about its evolution. Figure 4. Estimating the divergence time of the family Capulidae based on 16S. Bars indicate 95% highest posterior density intervals for node ages (Ma), and number at node the median (Ma).

The distribution of C. danieli C. danieli was originally identified in New Caledonia in the South Pacific (Beu et al. 2004; Fassio et al. 2020a), and its distribution records also exist in Japan (Okutani et al. 2017). This study presents the first report of C. danieli in China, specifically in the vicinity of the South China Sea in Yangjiang City, Guangdong Province. C. danieli (Crosse, 1858) is a species widely distributed in the Western Pacific, ranging from central Japan (Okutani et al. 2017), the Philippines to southern Australia (Garrard 1961), including northern New Zealand (Beu et al. 2004). In Australia, there are fossil records of C. danieli, discovered by Tate (1893) in the Miocene and Pliocene strata of Victoria and South Australia. The fossil records in New Zealand mainly come from the Landguard Sand and Te Piki Member in the Wanganui region, and it expanded from Australia to New Zealand during the Pleistocene period (Beu et al. 2004). Its dispersal methods may be two fold: one is through the dispersal of its larvae, and the Author-formatted, not peer-reviewed document posted on 06/11/2024. DOI:  https://doi.org/10.3897/arphapreprints.e141013 other is due to its host scallops, which are also found in the same distribution where this species is detected. C. danieli might have a pelagic larval stage, and these larvae can drift in the ocean and settle in suitable environments (Beu et al. 2004).The findings from this study have confirmed Beu's hypothesis..this diffusion mechanism enables the species to cross the ocean in the absence of physical barriers. The presence of C. danieli found in the Philippines, the South China Sea of China, and Japan may be influenced by the Japan Current. The distribution of scallops to which it attaches is also found in the Philippines, the South China Sea of China, and Japan. The possibility of its spread through the migration of scallops cannot be excluded. More evidence also requires an increase in fossil records. Characteristics of the mitogenomes In this study, the whole genome sequence of C. danieli was obtained by high-throughput sequencing technology, with an assembled length of 15600 bp. The length of the whole genome sequence of other gastropods in this study was 14400–17131 bp (Table 1). In most cases, the mitochondrial genome of gastropods is usually between 14,000 and 18,000 bp (Chen et al. 2023; Kim et al. 2023; Qu et al. 2024). The length of the mitochondrial genome of C. danieli was within the normal range of gastropods. The mitochondrial genome AT content of C. danieli was 71.12%. Among the gastropods in this study, the lowest AT content was Haliotis rubra (59.1%), and the highest was Ficus subintermedia (74.2%). The AT content of C. danieli was higher than that of Vetigastropoda (59.1%–59.8%) and similar to that of most Caenogastropoda (67.8%–74.2%). Generally, the AT content of gastropods is about 60%–75% (Chen et al. 2023; Kim et al. 2023). The high content of AT makes them more susceptible to base mutations. Due to its susceptibility to replication errors, which can lead to an increased rate of polymorphism under environmental stress (Broughton and Reneau 2006). Phylogenetic implications Mitochondrial DNA sequences are increasingly being employed in phylogenetic studies, owing to their significance in elucidating evolutionary relationships among organisms (Dhorne-Pollet et al. 2020). In this study, a phylogenetic tree was constructed based on PCGs of 25 species, specifically focusing on gastropod mollusks that exhibit the closest genetic and morphological affinities (limpet-like shell) with C. danieli. The purpose is to understand the taxonomic status of C. danieli and its evolutionary relationship with limpet-like gastropods. Results showed : C. danieli was not clustered with other limpet-like species but was grouped with F. subintermedia and F. variegata (Ficidae) to form one subclade. According to the available records, Ficidae does not include any limpet-like species. Additionally, among the sister branches of (C. danieli + F. subintermedia and F. variegata), the families Naticidae, Strombidae, Struthiolariidae, and Xenophoridae also do not have limpet-like species. In contrast to other aforementioned families, Capulidae is the only family that has evolved shell-shaped mollusks. Throughout the Phanerozoic eon, which spans from the Cambrian to the Neogene periods, gastropods have experienced a multitude of morphological transformations, with the evolution of limpet-like shells being particularly prevalent Author-formatted, not peer-reviewed document posted on 06/11/2024. DOI:  https://doi.org/10.3897/arphapreprints.e141013 in marine environments (Vermeij 2017). The Capulidae family, known for its highly limpet-shaped shells, has seen numerous transitions to this form (Fassio et al. 2020a). Although C. danieli shares morphological similarities with these limpet-like gastropods, it was found to be only distantly related. This instance of convergent evolution is primarily attributed to the influence of specific environmental pressures (Vermeij 2001). Becoming a limpet-like shell simplified shell structure of C. danieli, which is less suited to intense interspecific competition and predation. According to the ecological niche and lifestyle of C. danieli, we can roughly summarize the benefits of its transformation into a limpet-like shell: The limpet-like shell form minimizes water flow resistance, maximizes the attachment surface area, and reduces the potential for dislodgement (Vermeij 2017), which is particularly adapted to the obligate sedentary parasitic lifestyle of C. danieli on the surface of scallops. It is known from the phylogenetic tree of the Conidae family constructed based on 16S that the recurrent emergence of the limpet-like shell within the family Capulidae, as exemplified by the Cryocapulus, Capulus + Hyalorisia, and Trichamathina lineages. The process of becoming a limpet-like shell appears intermittently and repeatedly in the family Capulidae, due to the complex evolutionary pressures that have shaped the diversity of shell morphologies within this family. Molecular clock analysis estimated the Capulidae family originated in the Middle Jurassic period, specifically during the Callovian stage, approximately 155.25 million years ago. However, Fassio et al. proposed a more recent origin, dating Capulidae to 112.87 million years ago (Fassio et al. 2020a). This divergence predates the oldest known Capulidae fossil record by several million years (Saul and Squires 2008), indicating a gap between molecular and paleontological evidence that necessitates further investigation to elucidate the early evolutionary narrative of Capulidae. The divergence time for C. danieli is dated to around 52.29 million years ago during the Palaeocene-Eocene transition, a period marked by global warming and elevated sea surface temperatures (Tripati et al. 2001). The paleoenvironmental conditions of this era, characterized by transient eutrophication and ocean acidification (Scheibner et al. 2005; Alegret and Ortiz 2007; Scheibner and Speijer 2008), likely exerted selective pressures favouring a parasitic lifestyle in C. danieli. Such rapid climatic shifts can precipitate significant alterations in faunal composition across various habitats, presenting novel evolutionary prospects. Eutrophication can lead to alterations in the composition of primary producers in marine ecosystems, such as algal blooms. As the availability of suspended particulate matter diminishes and humus concentrations increase, gastropods may shift from filter feeding to alternative feeding strategies (Vermeij 2001). Poulin and Randhawa have discussed the evolution of parasitic behavior as a response to environmental pressures. When the main food sources become scarce or the energetic cost of feeding escalates, organisms may adopt parasitic lifestyles to persist (Poulin and Randhawa 2015). This ecological transition may have facilitated the adoption of a limpet-like shell form in C. danieli, enhancing nutrient access through kleptoparasitism of bivalves. Author-formatted, not peer-reviewed document posted on 06/11/2024. DOI:  https://doi.org/10.3897/arphapreprints.e141013 The morphological changes The species of the genus Capulus show significant variations in morphology, and this variation is related to the morphology of the host shells they parasitize. For instance, their shell morphology might change according to the shape of the host, forming the so-called xenomorphic sculpture (Orr 1962). Some samples of C. danieli attached to host shells with distinct radiating ribs develop corresponding shell edge depressions (Beu et al. 2004). In this study, the samples were attached to the smooth surface of scallop shells and did not form xenomorphic sculpture. However, the scallops to which they were attached showed concentric growth line patterns, and similar patterns were also formed on the C. danieli that parasitized on their surface. This proves that the species of the Capulus genus typically have morphological characteristics adapted to parasitic life.

In this study, we have documented the mitochondrial genome of C. danieli, representing the inaugural comprehensive mitochondrial genome sequence recorded within the family Capulidae. We analysed the characteristics of the mitochondrial sequence of C. danieli. Additionally, we conducted a phylogenetic analysis to infer the evolutionary nexus between C. danieli and limpet-like gastropods. Molecular clock estimates were utilised to approximate the divergence timeframe of the Capulidae lineage, concurrently delineating the evolutionary trajectory leading to the adoption of a limpet-like shell morphology.Additionally, we have summarized the relationship between its morphological plasticity and its host, suggesting that although the species exhibits a variety of external morphologies, the primary reason is co-evolution with the host's form. This species was discovered for the first time in China , and our study has summarized its distribution range, speculating that the influence of the Japan Current has shaped its current distribution area. The collective findings of this study substantially augment our comprehension of the evolutionary narrative and genetic underpinnings of C. danieli, casting new light on its distinctive ecological adaptations within the marine ecosystem.

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