# A phylogenomic and morphometric reassessment of Ovulidae highlights host-related shell shape evolution

E Nocella, G Fassio, T Lemarcis, P Zaharias, D Tamagnini, N de Leo, N

Puillandre, M V Modica, M Oliverio

► To cite this version:

E Nocella, G Fassio, T Lemarcis, P Zaharias, D Tamagnini, et al.. A phylogenomic and morphometric reassessment of Ovulidae highlights host-related shell shape evolution. Molecular Phylogenetics and Evolution, 2026, 217, pp.108545. 10.1016/j.ympev.2026.108545. hal-05504572

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# A phylogenomic and morphometric reassessment of Ovulidae highlights host-related shell shape evolution

E. Nocella<sup>a,b,*</sup>, G. Fassio<sup>a</sup>, T. Lemarcis<sup>c</sup>, P. Zaharias<sup>c</sup>, D. Tamagnini<sup>a</sup>, N. de Leo<sup>a</sup>, N. Puillandre<sup>c</sup>, M.V. Modica<sup>b,1</sup>, M. Oliverio<sup>a,1</sup>

<figure><img src="image_3.png" /></figure>

## ARTICLE INFO

**Keywords:**

Exon capture

Corallivory

Morphometric analyses

Eco-evolution

Gastropods

## ABSTRACT

The egg-cowries comprise a family of specialised carnivorous caenogastropods (Ovulidae Fleming, 1828) that feed by browsing on octocorals, hexacorals and stylasterid hydrozoans. A recent phylogenetic study, employing a Sanger-based multilocus nucleotide dataset, revealed numerous instances of morphological-molecular incongruence and raised the need for a revision of the current classification. To test alternative hypotheses of ovulid classifications, we applied a phylogenomic approach relying on an Exon-capture methodology to 41 samples from 26 genera and 41 species, representative of the subfamily- and genus-level diversity. Tree reconstructions were performed using both Maximum Likelihood and Bayesian inference methods, resulting in a total of five well-supported subfamily-level clades. The genera *Jenneria* and *Pseudocypraea* formed a monophyletic clade, sister to *Pedicularia*, altogether here considered as forming the subfamily Pediculiinae. The monophyly of Ovulinae was upheld, incorporating *Quasisimnia*. Prionovolvinae as traditionally recognised proved to be a melting pot of diverse genera and species, including *Ovula isibasi*, challenging conventional morphology-based classifications. To further investigate morphological variation within Ovulidae, we conducted 2D geometric morphometric analyses to assess shape differences. Groups emerging from Principal Component Analysis (PCA) aligned with the traditional, morphology-based classification. The weak phylogenetic signal associated with the morphometric patterns suggested that phylogeny had only a marginal influence on the morphological evolution of this clade, in contrast to the much stronger role played by host adaptation. This phylogenetic reassessment based on robust genomic data, not only helped to refine our taxonomic framework but also underscores the power of molecular insights in investigating complex evolutionary relationships within families such as the Ovulidae. Furthermore, the morphometric multivariate analysis proved efficient in revealing patterns and addressing eco-evolutionary links.

## 1. Introduction

Ovulidae, Fleming, 1828 or egg-cowries, comprise a widespread family of specialised corallivorous gastropods that feed by browsing on polyps and tissues of anthozoans, mostly octocorals (Anthozoa: Octocorallia: Malacalcyonacea and Scleralcyonacea) or, less frequently, hexacorals (Anthozoa: Hexacorallia: Antipatharia and Scleractinia); a small group is instead associated with stylasterid hydrozoans

(Hydrozoa: Hydroidolina: Anthoathecata: Stylasteridae) (Lorenz and Fehse, 2009, Schiaparelli et al., 2005; Tazioli et al., 2007). Ovulids are predominantly tropical, with few species living in temperate waters and the highest species richness recorded in the Indo-West Pacific (Wilson 1998). The family, as currently conceived, includes 321 species (MoluscaBase, 2025). However, Schiaparelli et al. (2005) already suggested that the actual number of living species might be drastically reduced (more reasonably estimated as closer to 100 than to 300), a suggestion

<footnote><sup>a</sup> Department of Biology and Biotechnologies "Charles Darwin", Sapienza University of Rome, Rome, Italy</footnote>

<footnote><sup>b</sup> Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, Napoli, Italy</footnote>

<footnote><sup>c</sup> Institut Systématique Evolution Biodiversité (ISYEB), Muséum national d'Histoire naturelle, CNRS, Sorbonne Université, EPHE, Université des Antilles, 57 rue Cuvier, CP 26, 75005 Paris, France</footnote>

<footnote>* Corresponding author at: Department of Biology and Biotechnologies "Charles Darwin", Sapienza University of Rome, Rome, Italy.

E-mail addresses: elisa.nocella@uniromal1.it (E. Nocella), giulia.fassio@uniromal1.it (G. Fassio), thomas.lemarcis@mnhn.fr (T. Lemarcis), paul.zaharias@mnhn.fr (P. Zaharias), davide.tamagnini@uniromal1.it (D. Tamagnini), naomi.deleo@uniromal1.it (N. de Leo), nicolas.puillandre@mnhn.fr (N. Puillandre), mariavittoria.modica@szn.it (M.V. Modica), marco.oliverio@uniromal1.it (M. Oliverio).</footnote>

<footnote n="1">¹ These authors contributed equally to this work and share senior authorship.</footnote>
supported by the molecular data of Nocella et al. (2024b). Rosenberg (1992) related the proliferation of nominal taxa of ovulids to the remarkable variation in mantle colour and shell shape among these organisms. In living specimens of most species, the shell is almost entirely covered by the mantle that camouflages the gastropod on its cnidarian host (Fig. 1), and a few species have aposematic colorations (Rosenberg, 1992). The species of the genus *Pedicularia* differ from the remaining egg-cowries by not covering their shell with the mantle (Lorenz and Fehse, 2009) and in their radular morphology (Simone, 2004). On this basis, *Pedicularia*, together with a few morphologically similar taxa that nonetheless cover the shell with the mantle (e.g., the genera *Pseudocypraea* and *Jenneria*) have been classified by Fehse (2007) and Lorenz and Fehse (2009) in a separate family *Pediculariidae* Gray, 1853. Recent analyses based on molecular data have prompted a deep revision of the taxonomy of the Ovulidae (Nocella et al., 2024b; Schiaparelli et al., 2005; Zvonareva et al., 2020). In particular, a recent study on this family (Nocella et al., 2024b), based on a multilocus Sanger-based molecular phylogeny, proposed a drastic reorganization of the classification at the subfamily level, with five subfamilies (Pediculariinae, Prionovolvinae, Simniinae, Aclyvolvinae, and Ovulinae), and two lineages (*Jenneria* and *Pseudocypraea*) seemingly deserving subfamily recognition on their own. Additionally, Nocella et al. (2024b) suggested that host association played a major role in shaping ovulid evolution. It is therefore possible that the incongruences between traditional morphology-based classifications and phylogenetic patterns inferred from molecular analyses reflect a stronger association of morphological variation with ecological contexts than with phylogenetic relatedness.

To test this hypothesis, we have first assembled the largest genome scale dataset for the egg-cowries, including representatives of all known subfamily-level lineages, by applying an exon-capture strategy to target thousands of loci with Next-Generation Sequencing (NGS) techniques. NGS has transformed phylogenetics by enabling rapid, broad-scale marker acquisition; in particular, transcriptome-informed exon capture retrieves hundreds of loci from genomic DNA to support robust

phylogenetic inference (Lemmon and Lemmon, 2013; McCormack et al., 2013; Bi et al., 2012; Hugall et al., 2016). Additionally, target-capture enrichment represents a cost-effective approach for sequencing genomic data across diverse taxa in phylogenomic studies and has been applied using high-throughput sequencing in various organisms, including plants, vertebrates, insects, and spiders (Branstetter et al., 2017; Hamilton et al., 2016; Hughes et al., 2021; McLay et al., 2021; Smith et al., 2014). In the malacological field, sequence capture methods are now emerging for phylogenomic investigations spanning genera, families, and superfamilies (e.g., Abdelkrim et al., 2018; Goulding et al., 2023).

Then, to investigate morphological variation within Ovulidae, we performed 2D geometric morphometric analyses to assess interspecific shape differences. Morphometrics, the quantitative study of biological shape variation and its covariation with other variables (Adams et al., 2004), enables the direct quantification and statistical analysis of morphological differences. Despite shells being a rich source of morphological traits, this approach has been relatively underutilized in gastropods (e.g., Cruz et al., 2012; Doyle et al., 2018). Geometric morphometrics based on landmark data provides a powerful tool to distinguish taxa, particularly in groups such as Ovulidae, where morphology-based taxonomy often conflicts with molecular phylogenies (Nocella et al., 2024b). This study aimed to analyse intra- and inter-subfamilial morphological variability in Ovulidae and to identify key variables influencing shell morphology. Under the hypothesis that ovulid morphological variation is more closely associated with host use than with phylogenetic relatedness, we would expect a weak or absent phylogenetic signal and a stronger correlation between shell shape variation and host traits in the quantitative analyses.

## 2. Material and methods

### 2.1. Material examined

Most of the samples were collected during multiple expeditions led by the Muséum National d'Histoire Naturelle, Paris (MNHN, expedition s.mnhn.fr), while the rest of the samples (acronyms BAU) belong to the malacological collections at the Dept. of Biology and Biotechnologies 'Charles Darwin' of Sapienza University of Rome.

Samples were selected based on integrative taxonomy and phylogenetic results obtained by Nocella et al. (2024b) in order to cover as much as possible the subfamily-level and the genus-level diversity of the egg-cowries, among available samples. Specimens from tropical, subtropical and temperate areas were included (Fig. 2), with a specific focus on the Indo-Pacific region. Samples were collected from the lower intertidal to approximately 500 m depth and specifically fixed in the field for molecular analyses. Whole gastropod genomic DNA was extracted from tissue samples with the Macherey-Nagel NucleoSpin 96 Tissue Kit following the manufacturer's protocol (at the Service de Systématique Moléculaire of the MNHN), or with a 'salting out' protocol (modified from Aljanabi and Martinez, 1997 after Fassio et al., 2023) (at Sapienza University of Rome). DNA samples were quantified using a Qubit 2.0 Fluorometer, Invitrogen. All the samples showing a DNA concentration < 2 µg/ml were discarded.

In total, 63 ovulid samples were selected for exon-capture analysis, covering 27 genera and 47 species. Six outgroups were also included: *Trivia arctica* (Pulteney, 1799), *Triviidae*; *Alvania lineata* Risso, 1826, *Rissoidae*; an undetermined species of *Lamellaria* Montagu, 1816, *Velutinidae*; *Tonna galea* (Linnaeus, 1758), *Tonnidae*; *Littoriaria nebulosa* (Lamarck, 1822), *Littorinidae*; an undetermined species of *Epitonium* Röding, 1798, *Epitoniidae*.

### 2.2. Identification of the targeted exon and post-sequencing assembly

As part of a larger project, the exon probes used for the Ovulidae were selected to cover the whole diversity of the Neogastropoda and

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Living specimens Ovulidae on host. (A) *Naviculavolva kurziana* on Isididae, New Caledonia. (B) *Phenacovolva rosea* on Neptheidae, Papua New Guinea. (C) *Crenavolva traillii* on Euplexauridae, New Caledonia. (D) *Pseudosimnia carnea* on Alcyoniidae, Corse. (E) *Pedicularia vanderlandi* on Stylasteridae. (F) *Prosimnia semperi* on Melitheidae, New Caledonia. Photo credits: Laurent Charles (MNHN).</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Global map featuring sampling locations of the Ovulidae dataset (created using QGIS Geographic Information System v 3.28.11). See Table S1 for locality details.</figcaption></figure>

close allies. The protocol followed for the exon designation and sequencing is detailed in Lemarcis et al. (2025). Briefly, the chromosome-level and annotated available genome from *Conus ventricosus* (Conidae) was used (Pardos-Blas et al., 2021) in combination with 15 transcriptomes representative of the taxon diversity. Comparison of the genome and transcriptome allowed the identification of 11,125 loci corresponding to exons, from which probes were synthesized. The probes were then used to capture the corresponding loci in the DNA extract of the 69 individuals, then sequencing them using an Illumina approach.

Similarly, the bioinformatic pipeline follows Lemarcis et al. (2025). To assemble the reads and maximize the number of contigs reconstructed, both SPAdes v3.8.1 (Bankevich et al., 2012) and Trinity (Grabherr et al., 2011) were used independently. The two datasets of contigs were merged before a clustering step using cap3 (Huang and Madan, 1999) and cd-hit v4.6.2.5. Reads were mapped on the transcripts using Bowtie2 (Langmead and Salzberg, 2012).

A blastn search (Altschul et al., 1990) was then performed querying the assembled contigs of each sample against the 1125 exon sequences from *Conus ventricosus*. Blast hits were filtered using an in-house python script to remove sequences with less than 4X of coverage on more than 70 % of the exon sequence length and/or with an estimated heterozygosity > 2 SDs. At this step, when several contigs were available for one sample and one exon, only the contig with the best coverage (or the longest, when the coverage was identical) was retained.

## 2.3. Phylogenetic analyses and temporal calibration

An initial alignment using MAFFT v7.520 (Katoh and Standley 2013) with the option «addfragments» was performed for each of the 1,125 exons. To translate the nucleotide alignments into proteins, a blastx search (Altschul et al., 1997) querying each *Conus* exon against the proteins annotated from the genome (Pardos-Blas) was performed to identify the starting position of the corresponding ORF. An in-house python script was then used to translate all the alignment in the correct ORF, remove the sequences shorter than 60 % of the length of the *Conus* exon and trim the positions that contained more than 40 % of missing data. Alignments with stop codons were then checked manually. We produced two datasets, containing respectively nucleotide (NT) and amino acid (AA) sequences. Exon alignments with fewer than 21 ingroup sequences were excluded, resulting in a dataset of 785 exons. Initial concatenated analyses of both the NT and AA datasets were performed using IQ-TREE 2 (Minh et al., 2020), with ModelFinder (Kalyaanamoorthy et al., 2017) selecting the best-fit substitution model

and 100 non-parametric bootstrap replicates. The same procedure was applied to each exon individually in the NT dataset. Species tree inference was conducted using ASTRAL (Zhang and Mirarab, 2022) on the set of exon trees.

The NT, AA, and ASTRAL trees were largely congruent, except for the placement of Aclyvolviinae, which remained unsupported across all analyses. Due to this instability, all exon trees were manually inspected for artifacts potentially caused by long branches (likely contaminants) or paralogy. We identified 93 exons with suspicious patterns, most consistent with paralogy (e.g., repeated subfamily relationships and atypical placements of outgroups). In cases of duplicated clades, the smaller clade was removed, resulting in the elimination of sequences from 80 exon alignments (Table S2) and the exclusion of 13 exons (fewer than 21 ingroup sequences). The final dataset consisted of 772 high-quality exons, totalling 26,184 sequences.

We then reanalysed this cleaned dataset using the same pipeline (concatenated NT, AA, and ASTRAL). Additionally, we ran PhyloBayes (Lartillot et al., 2009) to check whether the -cat -gtr model on the NT dataset would better deal with the position of Aclyvolviinae. The position of Aclyvolviinae remained unresolved across all four species trees (Supplementary Figs. S1-S4). However, the AA concatenated analysis provided the strongest bootstrap support, placing Aclyvolviinae as sister to Prionovolvinae. In contrast, the NT tree showed stronger bootstrap support for recent (intra-subfamily) relationships. Based on these results, we conducted a final maximum likelihood analysis on the NT dataset, but we constrained Aclyvolviinae as sister to Prionovolvinae.

The sequences analysed in this study are available on the InDores platform at the following link: Doi: 10.48579/PRO/XEP3EX.

We identified 4 reliable fossil occurrences of Ovulidae in the literature, suitable for calibration of relevant nodes in our time-calibrated

<caption>**Table 1**</caption>

The four time-ranges used as calibrations, with their references, and the corresponding 95% HPD obtained in the time inference.

<table><thead><tr><td>Node</td><td>Fossil records</td><td>Calibration, in mya</td><td>References</td><td>Results 95 % HPD</td></tr></thead><tbody><tr><td>Ovulidae</td><td>Early Eocene</td><td>47.8–56</td><td>Fehse, 2001</td><td>53–56.7</td></tr><tr><td>Pedicularia</td><td>Late Eocene</td><td>33.9–37.8</td><td>Ladd, 1972</td><td>33.8–37.6</td></tr><tr><td>Volva</td><td>Late Pliocene</td><td>2.58–3.6</td><td>Schilder, 1932</td><td>2.7–3.7</td></tr><tr><td>Pellasimnia</td><td>Late Eocene</td><td>33.9–37.8</td><td>Beu and Marshall, 2011</td><td>33.6–36.8</td></tr></tbody></table>
phylogenetic reconstruction (Table 1). We used MCMCTree (Rannala and Yang, 2007; Yang and Rannala, 2006) to perform Bayesian inference of divergence times (and their highest posterior density, HPD) on the NT dataset using the final ML (constrained) topology and following the protocol defined by the authors (dos Reis and Yang, 2019). The approximate likelihood step was run under a HKY85 model of substitution (the most complex one implemented in MCMCTree) and both the autocorrelated and independent log-normally distributed rates models were used to date the tree, with an MCMC chain ran for 2,020,000 iterations.

## 2.4. Geometric morphometrics and morphological analyses

Geometric morphometrics was used to extract morphological data for investigating shell shape variability among species since this technique of morphological quantification is particularly suitable for evolutionary analyses (Tamagnini et al., 2021, Chatar et al., 2024). The species included in this study encompass almost the entire morphological variation in shell shape of the ovulids; representativeness of the dataset is particularly robust, considering the broad variation across the family, but the relative stability of shape within species (Lorenz and Fehse, 2009). Each ingroup sample was photographed in a ventral view, ensuring a consistent orientation across all images, at the MNHN using the BK imaging system with a Canon 5DSr. Shell pictures are available on the MNHN collection website (https://science.mnhn.fr/institution/mnhn/collection/im/item/search). Landmarks were extracted using tpsDig v.1.40 (Rohlf, 2015), a freeware program available at http://life.bio.sunysb.edu/morph and digitized to capture shell morphology, with a particular focus on the aperture, following a landmark configuration similar to those used in previous morphological research on gastropod shells (e.g., Cruz et al., 2012; Avaca et al., 2013).

Digitized landmark coordinates were analysed using PAST v.4.03 (Hammer et al., 2001), available at http://folk.uio.no/ohammer/past. A Procrustes transformation was applied to normalize for isometric size variation, translation, and rotation, superimposing all specimens within a common coordinate system (Adams et al., 2004). This approach facilitated the analysis of shape variations, by removing the impact of size and orientation differences.

Subsequently, a Principal Component Analysis (PCA) was performed on the resulting Procrustes coordinates to summarize the main axes of shape variation (see Table S3 for details and Fig. S5 for landmarks configuration). The PCA results were visualized in two ways: the first highlighting the relationship to the phylogenomic subfamilial classification, and the second focusing on host morphological traits. This approach allows for a comprehensive exploration of shell shape variability within the studied taxa. As traits for coral species, we have selected two that were more likely to bear effects on ovulid shell shape and could more easily be defined for the involved taxa: slenderness and rigidity. Slenderness (states: slim or broad) has been defined visually based on the shape of the colony of the actual host identified for that ovulid (using the trait of the cnidarian host species, genus or family where appropriate). Rigidity (states: rigid, semi-rigid or soft) has been derived from the Coral trait Database (available at www.coraltraits.org), an open source research initiative that makes observations and measurements of corals publicly accessible. See Table 2 for the correspondence between number IDs and the Ovulidae species analysed.

To further assess the evolutionary patterns of shell shape, a phylogenetic signal analysis was conducted. The phylogenetic signal measures the degree to which morphological traits are influenced by phylogeny, reflecting the tendency of closely related species to resemble each other (Münkemüller et al., 2012). This analysis was performed in R (version 4.0.1) using the geomorph package (Adams and Otárola-Castillo, 2013; Baken et al., 2021), incorporating both the time calibrated phylogenetic tree (see below) and the shape data. This step aimed to explore the relationship between shell shape evolution and phylogeny among the studied taxa. Finally, a Procrustes ANOVA in a phylogenetic context (i.

<caption>**Table 2**</caption>

Numerical IDs used in the morphometry dataset and the corresponding Ovulidae species and voucher catalogue numbers.

<table><thead><tr><td>ID</td><td>Morphometry</td><td>Species</td><td>Voucher</td></tr></thead><tbody><tr><td>1</td><td></td><td>Aclyvolva coarctata</td><td>MNHN-IM-2013-54314</td></tr><tr><td>2</td><td></td><td>Aclyvolva sp.</td><td>MNHN-IM-2013-69669</td></tr><tr><td>3</td><td></td><td>Amonovula piriei</td><td>MNHN-IM-2013-53904</td></tr><tr><td>4</td><td></td><td>Calpurnus verrucosus</td><td>MNHN-IM-2007-32154</td></tr><tr><td>5</td><td></td><td>Contrasimnia formosana</td><td>MNHN-IM-2007-36343</td></tr><tr><td>6</td><td></td><td>Contrasimnia xanthochila</td><td>MNHN-IM-2013-61951</td></tr><tr><td>7</td><td></td><td>Crenavolva aureola</td><td>MNHN-IM-2013-54048</td></tr><tr><td>8</td><td></td><td>Crenavolva traillii</td><td>MNHN-IM-2013-85207</td></tr><tr><td>9</td><td></td><td>Cyphoma gibbosum</td><td>MNHN-IM-2013-8520</td></tr><tr><td>10</td><td></td><td>Dentiovula dorsuosa</td><td>MNHN-IM-2013-4461</td></tr><tr><td>11</td><td></td><td>Dentiovula eizoi</td><td>MNHN-IM-2013-54265</td></tr><tr><td>12</td><td></td><td>Dentiovula oryza</td><td>MNHN-IM-2019-18294</td></tr><tr><td>13</td><td></td><td>Diminovula culmen</td><td>MNHN-IM-2013-47924</td></tr><tr><td>14</td><td></td><td>Habuprionovolva sp.1</td><td>MNHN-IM-2013-17977</td></tr><tr><td>15</td><td></td><td>Habuprionovolva sp.2</td><td>MNHN-IM-2013-63255</td></tr><tr><td>16</td><td></td><td>Hiatavolva depressa</td><td>MNHN-IM-2013-18146</td></tr><tr><td>17</td><td></td><td>Jenneria pustulata</td><td>BAU-4297</td></tr><tr><td>18</td><td></td><td>Margovula anulata</td><td>MNHN-IM-2013-55743</td></tr><tr><td>19</td><td></td><td>Naviculavolva kurziana</td><td>MNHN-IM-2019-8310</td></tr><tr><td>20</td><td></td><td>Ovula isibashii</td><td>MNHN-IM-2013-68826</td></tr><tr><td>21</td><td></td><td>Ovula ovum</td><td>MNHN-IM-2007-32155</td></tr><tr><td>22</td><td></td><td>Ovulidae Gen. sp.</td><td>MNHN-IM-2013-68846</td></tr><tr><td>23</td><td></td><td>Pedicularia elegantissima</td><td>MNHN-IM-2019-18318</td></tr><tr><td>24</td><td></td><td>Pedicularia granulata</td><td>MNHN-IM-2013-9609</td></tr><tr><td>25</td><td></td><td>Pedicularia vanderlandi</td><td>MNHN-IM-2013-53610</td></tr><tr><td>26</td><td></td><td>Pellasimnia angasi</td><td>MNHN-IM-2019-18300</td></tr><tr><td>27</td><td></td><td>Pellasimnia brunneiterma</td><td>MNHN-IM-2013-53962</td></tr><tr><td>28</td><td></td><td>Phenacovolva lahainaensis</td><td>MNHN-IM-2013-1738</td></tr><tr><td>29</td><td></td><td>Phenacovolva rosea</td><td>MNHN-IM-2013-18076</td></tr><tr><td>30</td><td></td><td>Procalpurnus semistriatus</td><td>MNHN-IM-2013-4426</td></tr><tr><td>31</td><td></td><td>Prosimnia semperi</td><td>MNHN-IM-2013-51443</td></tr><tr><td>32</td><td></td><td>Pseudocypraea adamsonii</td><td>MNHN-IM-2013-3598</td></tr><tr><td>33</td><td></td><td>Pseudosimnia carnea</td><td>BAU-4169</td></tr><tr><td>34</td><td></td><td>Quasisimnia hirasei</td><td>MNHN-IM-2007-38930</td></tr><tr><td>35</td><td></td><td>Quasisimnia robertsoni</td><td>MNHN-IM-2013-44511</td></tr><tr><td>36</td><td></td><td>Sinnialena uniplicata</td><td>MNHN-IM-2013-57299</td></tr><tr><td>37</td><td></td><td>Sinnia spelta</td><td>MNHN-IM-2019-18138</td></tr><tr><td>38</td><td></td><td>Sinnia sp.</td><td>MNHN-IM-2013-60781</td></tr><tr><td>39</td><td></td><td>Takasagovolva honkakujiana</td><td>MNHN-IM-2007-36346</td></tr><tr><td>40</td><td></td><td>Volva kilburni</td><td>MNHN-IM-2019-18296</td></tr><tr><td>41</td><td></td><td>Volva volva</td><td>MNHN-IM-2007-33691</td></tr></tbody></table>

e., PGLS – Phylogenetic Generalized Least Squares) was performed to evaluate the effect of the ecological factor “host traits” on shell shape variation. This analysis assumed a Brownian Motion evolutionary model (i.e., conditions of morphological neutral evolution), with the dependent variable represented by all the principal components of the PCA performed on the Procrustes coordinates.

# 3. Results

## 3.1. Phylogenetic reconstruction

Out of the 69 selected samples, seven were discarded due to unsuccessful amplification. In particular, this was due to either having zero exons after filtering (five samples) or being unmistakably identified as contaminated during a preliminary analysis (two samples). To assemble the final dataset, we retained the sample with the highest number of exons for each species, discarding 15 replicates. Therefore, our final dataset included 41 ovulid samples belonging to 41 species and 26 genera, plus six outgroups, for a total of 47. For sample details see Table S1.

The PhyloBayes analysis ran for 30 days on four chains, corresponding to approximately 10,000 points. Chain 2 did not converge even after 10,520 points, suggesting that it could be stuck in a local maximum, so we discarded it. A burn-in of 2,000 points was applied to the three remaining chains, and convergence was checked using PhyloBayes' tracecomp function. All parameters had good ESS values, and
the discrepancy values ("rel_diff") were mostly below 0.3, indicating overall good convergence. The bpcomp function was used to test for discrepancies observed across all bipartitions (< 0.1) and to compute a consensus tree (Supplementary Figs. S1-S4).

Comparison of the major nodes among all trees (ML, BI and Astral) revealed no significant inconsistencies (Fig. 3), except for the position of Aclyvolviinae (see MandM). The monophyly of a group including all ovulids was strongly supported with Bootstrap (BS) = 100, and Posterior Probability (PP) = 1 values. Most of the subfamilies' arrangement was found to be in agreement with the phylogeny of Nocella et al. (2024b).

A large clade of core Prionovolvinae was defined (BS = 100, PP = 1), also encompassing a species, *Amphiperas isibasii* Kuroda, traditionally ascribed as *Ovula isibasii* to a different subfamily (Ovulinae, see e.g., Okutani, 2000). The monophyly was also strongly supported for the subfamilies Ovulinae (BS = 100, PP = 1), Simniinae (BS = 100, PP = 1), Aclyvolvinae (BS = 100, PP = 1) and Pediculariinae (BS = 100, PP = 1). Within pediculariines, the genera *Jenneria* and *Pseudocypraea* were identified as sister lineages (BS = 100, PP = 1), contrary to the results presented in Nocella et al. (2024b), and both are sister to *Pedicularia* (BS

= 100, PP = 1).

Within Simniinae, the genus *Simnia* (the type genus of the subfamily) did not prove monophyletic, being split into two lineages: one comprising *Simnia* sp. and another represented by the type species of the genus, *Simnia spelta*.

The genus *Quasisimnia* was recovered as the sister to the subfamily Ovulinae (BS = 100, PP = 1).

Among the Prionovolvinae, two genera as traditionally conceived, did not prove monophyletic. The genus *Dentiovula* was retrieved as polyphyletic and split into three lineages: one represented by *Dentiovula eizoi*, sister to MNHN-IM-2013-54265 Ovulidae Gen. sp. (BS = 100, PP = 1), another represented by *Dentiovula dorsuosa*, and a third one including the species *Dentiovula oryza*. Similarly, the genus *Habuprio-novolva* was split into two lineages: one including *Habuprionovolva* sp. 1, and one represented by *Habuprionovolva* sp. 2.

When represented by multiple species, all the other genera (i.e. not discussed above) were found monophyletic.

<figure><img src="image_6.png" /></figure>

**Fig. 3.** Phylogenetic relationships of the family Ovulidae: Maximum Likelihood tree based on the nucleotide dataset, with Aclyvolvinae constrained as sister to Prionovolvinae (see MandM). Numbers at nodes indicate ingroup branch support values (bootstrap values and posterior probability, respectively; support values are shown only when at least one of them is ≥ 95; black circles at nodes indicate maximum support, BS = 100, PP = 1). Colours at clades and taxa indicate the proposed subfamilial classification of the included taxa. Shells of representative taxa, not to scale (from top-left): BAU-4297 *Jenneria pustulata*; MNHN-IM-2013-3598 *Pseudocypraea adamsonii*; MNHN-IM-2019-18318 *Pedicularia elegantissima*; MNHN-IM-2019-22810 *Simnia spelta*; MNHN-IM-2013-8520 *Cyphoma gibbosum*; MNHN-IM-2019-18296 *Volva kilburni*; MNHN-IM-2007-32155 *Ovula ovum*; MNHN-IM-2013-54314 *Aclyvolva coarctata*; MNHN-IM-2013-69669 *Aclyvolva* sp.; MNHN-IM-2013-68826 *Ovula isibasii*; MNHN-IM-2013-85207 *Crenavolva traillii*; MNHN-IM-2019-8310 *Naviculavolva kurziana*; MNHN-IM-2013-61951 *Contrasimnia xanthochila*. Photograph credits: Elisa Nocella.
## 3.2. Temporal calibration

All our calibration nodes were dated in the MCMCTree output congruently with the corresponding fossil data (Fig. 4; see Table 1 for 95 % HPD). The analysis estimated the origin of the family Ovulidae at 54 mya (95 % HPD 53–57) during the Ypresian (Early Eocene). The origin of the Pediculariinae was dated at 45 mya (95 % HPD 40–50) during the Upper Eocene. The subfamily Ovulinae (including the genus *Quasisimnia*) was estimated as having originated 47 mya (95 % HPD 43.3–51.5) during the lower Eocene. The origin of the Prionovolvinae was dated at 39 mya (95 % HPD 32.5–46.7) between Eocene and Oligocene. The Simniinae were estimated as having originated during the Upper Eocene, with the node dated at 42.6 mya (95 % HPD 31.6–52.3). The Aclyvolvinae was recovered to be the most recently diverged subfamily, with the origin estimated around 23 mya (95 % HPD 9.6–36.5) between Miocene and Oligocene. The results are consistent with those obtained by Nocella et al. (2024b), although most nodes appear older. This is likely due to the smaller number of species included in the present phylogeny (Venditti et al., 2006); however, this difference does not affect the subsequent analyses.

## 3.3. Morphometric differentiation across clades and hosts

PC1 explained 45 % of the shape variance, describing a morphological pattern characterized by a wider aperture and irregular outline (e.g., genus *Pedicularia*) for extreme positive scores, and a narrow aperture for negative scores (e.g., *Amonovula piriei* and *Contrasimnia formosana*). Concerning genomic-based classification, PC1 mainly discriminated Pediculariinae from Prionovolvinae, while host traits differentiated between slim rigid hosts and broad soft hosts.

PC2 explained 34 % of the shape variance, capturing variation in slenderness, with elongate forms associated with positive scores and broader shapes with negative scores. In terms of genomic-based classification, PC2 discriminated most Ovulinae from Pediculariinae, while host traits differentiated between semi-rigid and strictly rigid hosts.

The PCA plot illustrating differences among subfamilies is shown in Fig. 5, while the PCA plot portraying associations with hosts and their traits is presented in Fig. 6.

Phylogenetic signal analyses showed a significant, yet weak impact of phylogenetic relationships on shell shape evolution of Ovulidae (K = 1.2986, p-value = 0.001). By contrast, PGLS analysis returned a strong

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Time calibrated phylogenetic reconstruction, with Bayesian inference of divergence times and their HPD, obtained using MCMCTree on the molecular dataset. Numbers are the estimated ages of that node (in mya); bars at nodes indicate 95% HPD of ages. Coloured vertical bars indicate the proposed subfamilial classification (see legend) of the embraced clades.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** PCA of shell morphometrics, showing shell shape variation and phylogenomic-based subfamilial classification. Subfamilies are described by five different colors: Ovulinae = red, Prionovolvinae = green, Simniinae = yellow, Aclyvolvinae = blue, Pediculariinae = purple). The extremes of shape variations are shown by means of wireframe deformations (in black). Each number corresponds to an Ovulidae species (see Table 2). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

and significant effect of host traits on shell shape evolution, explaining almost half of total shape variance in shape evolution in ovulids ($R^2 = 39.6\%$, Z-score = 3.40, p-value = 0.001).

# 4. Discussion

## 4.1. Taxonomic implications of the phylogenetic analysis

The phylogenetic trees obtained with the exon-capture dataset (Fig. 3) are significantly more resolved and supported than those previously published on the Ovulidae (e.g. Schiaparelli et al., 2005; Zvonareva et al., 2020; Nocella et al., 2024b).

Out of 26 genera represented in our dataset (65 % of total existent), a total of four (Ovula, Simnia, Dentiovula, and Habuprionovolva) were not monophyletic as traditionally conceived, as suggested by Nocella et al. (2024b). Notably, among these, the genera Ovula and Simnia are the type genera of the subfamilies Ovulinae, and Simniinae, respectively.

Our analysis provided strong support for the recent subfamily classification proposed by Nocella et al. (2024b): (i) the monophyly of a group encompassing all the egg cowries (ovulids and pediculariids) was strongly supported. In the currently accepted classification of extant Pediculariinae (MolluscaBase 2025, largely based on Lorenz and Fehse, 2009) they include only the genera Pedicularia, Lunovula, and Pedicypraedia, while Jenneria and Pseudocypraea are assigned to the subfamily Cypraeidiinae F. A. Schilder, 1927. However, in contrast to the most recent Sanger-based phylogeny by Nocella et al. (2024b), and to

earlier studies that did not resolve the position of Pediculariinae among other egg-cowry lineages (Schiaparelli et al., 2005; Zvonareva et al., 2020), our phylogenomic analysis revealed that Jenneria and Pseudocypraea form a well-supported clade with the three Pedicularia species, which together are sister to all remaining egg-cowries. Therefore, we propose their classification within the subfamily Pediculariinae Gray, 1853. Jenneria and Pseudocypraea likely represent lineages with plesiomorphic ovulid features in ecology and morphology (Nocella et al., 2024b). In this framework, while we did not find substantial evidence supporting or rejecting the proposal to classify Pedicularia and its allies in a separate family, Pediculariidae (e.g., Lorenz and Fehse 2009), our results rather suggest the adoption of a conservative classification within the family Ovulidae, ranking this clade as a subfamily Pediculariinae. (ii) In agreement with Nocella et al. (2024b), the subfamily Ovulinae J. Fleming, 1822 represents a monophyletic group only if excluding Ovula isibasi. We suggest the inclusion of O. isibashi within Prionovolvinae (with actual genus allocation to be further assessed). (iii) The genus Quasisimnia was recovered as the sister to the Ovulinae; we suggest incorporating Quasisimnia within the subfamily Ovulinae.

## 4.2. Shell shape evolution

The PCA plot in Fig. 5 clearly highlights the challenges in classifying ovulids based solely on morphology, both at the subfamily and genus levels. As expected, Pedicularia spp. (Fig. 5 N° 23–25) are positioned far from all remaining ovulids, including Jenneria pustulata (17) and
<figure><img src="image_9.png" /><figcaption>**Fig. 6.** PCA of shell morphometrics, showing shell shape variation and host trait categorization. Host shape (slenderness) information is described by two different colors (Broad = green, Slim = orange), while host rigidity is described by four different symbols (soft = triangle, rigid = circle, semi-rigid = diamond, both soft and semi-rigid = star). See Supplementary Table S1 for coral traits details (numbers corresponding to Ovulidae species as in Fig. 5). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

*Pseudocypraea adamsonii* (32), which morphologically appear closer to some Prionovolvinae. The latter, in turn, exhibit considerable morphological variability, as illustrated by the green polygon that overlaps those of both the Ovulinae and the Simniinae. Although molecular analyses unequivocally place *Ovula isibasi* (20) within Prionovolvinae (see above), in terms of shell shape this species, apart from obvious difference in size, is nearly indistinguishable from *Ovula ovum* (21), reinforcing the idea that integrative taxonomy and molecular phylogenetics are essential for an accurate classification of these gastropods. Similarly, *Hiatavolva depressa* (Prionovolvinae, Fig. 5 N° 16) and *Simnia* sp. (Simniinae, Fig. 5 N° 38) appear to overlap in shell shape. The only specimen of *Simnia* sp. available for this study was a juvenile, which may have influenced the morphometric results; however, this may highlight ontogenetic changes in shell shapes, which may be worthy of further study (Bandel, 1991).

The PCA plot in Fig. 6 (where for each ovulid species, the traits of its host are portrayed, when available) reveals that ovulid shell shapes are more clearly associated with host traits in the morphospace. With the exception of *Procalpurnus semistriatus* and *Diminovula culmen* (Fig. 6 N° 13 and 30), host types classified as “broad” and “slim” appear to be associated with distinct shell shape morphologies. Notably, Pedicularinae, which are clearly morphologically separated from the other ovulids, are the only taxa feeding on slim, rigid corals (the hydrozoan family Stylasteridae).

*Volva kilburni* (Fig. 6 N° 40) has been reported as the only ovulid species feeding on two different coral orders (Malacalcyonacea and Scleralcyonacea; Lorenz and Fehse, 2009). However, its overlap in morphospace with *Volva volva* suggests that the association with

malacalcyonaceans might be a case of misidentification and that *V. kilburni*, like *V. volva*, exclusively feeds on soft corals of the order Scleralcyonacea.

Our results also provide hints on host associations for taxa with unknown diets. Based on both morphological similarity and phylogenetic proximity, it is likely that *Pseudocypraea adamsonii* (Fig. 6 N° 32) feeds on scleractinians (broad, rigid corals), similar to its close relative *Jenneria pustulata*, as also suggested by Nocella et al. (2024b). Likewise, we suggest that *Contrasimnia formosana*, *Habuprionovolva* sp. 2, *Ovula isibasi* and *Ovulidae* Gen. sp. (Fig. 6 N° 5, 15, 20, 22) feed on broad soft corals. In contrast, *Aclyvolva* sp., *Quasisimnia hirasei*, and *Simnia* sp. (Fig. 6 N° 2, 34, 38) might be associated with slim, semi-rigid corals.

Our phylogenetic signal and PGLS analyses reveal that shell shape evolution in Ovulidae is more influenced by ecological interactions than by their phylogenetic history. Although the phylogenetic signal was statistically significant (K = 1.2986, p = 0.001), it was relatively weak, suggesting that phylogenetic inertia alone does not account for much of shell shape variation across taxa. In contrast, the effect of host morphology, used here as a proxy for ecological interaction (see Table S1), was both highly significant and substantially more explanatory (PGLS: R² = 39.6 %, Z = 3.40, p = 0.001). This indicates that most of the observed variation in shell shape can be attributed to differences in host traits, supporting the hypothesis that shell morphology of egg-cowries is strongly shaped by ecological adaptation to specific host morphologies. This explains how some aspects of the traditional (morphology-based) systematics of egg-cowries might have been severely biased by misleading adaptive homoplasies.

Our results align with growing evidence from both terrestrial and
marine systems indicating that ecological factors can be stronger drivers of morphological evolution than phylogenetic history. In a classic terrestrial example, Mahler et al. (2013) demonstrated that lizard species of the genus *Anolis* repeatedly evolved similar morphologies in response to equivalent ecological conditions on different Caribbean islands, despite independent evolutionary origins. Similarly, in an aquatic context, Muschick et al. (2012) demonstrated that cichlid fishes from separate lineages developed strikingly similar body shapes when occupying equivalent ecological niches within adaptive radiations. Echoing these patterns, our analysis of Ovulidae shows that shell shape variation is more closely linked to host morphology (probably a good proxy for ecological interaction) than to phylogenetic relatedness.

Furthermore, our findings align with the sequential evolution scenario proposed by Nocella et al. (2024a, 2024b) suggesting that host association played a key role in shaping corallivorous gastropod evolution and shell morphology, potentially overriding phylogenetic constraints. Such ecological specialization may reflect ovulid functional demands related to camouflage, attachment, or mobility on particular host surfaces, reinforcing the adaptive nature of shell shape evolution in this group.

## 5. Conclusions

Compared to a Sanger-based sequencing of a few genes, applying an exon capture strategy represents a notable improvement in refining the reconstruction of the phylogenetic relationships of the Ovulidae. Almost all the deepest nodes in the trees are strongly supported. This outcome conclusively underscores that ovulids' shell and mantle cannot be taken as a reliable universal proxy for systematic classification. In particular, the geometric morphometric analyses revealed a limited phylogenetic signal in shell shape, indicating that morphological evolution in this group is only marginally influenced by shared ancestry. Instead, shape variation appears to be more strongly associated with ecological factors such as host association. Consequently, any hypotheses regarding the ovulid systematics and evolution should be formulated and validated within a robust molecular phylogenetic framework. Now that a substantial amount of data on this family is becoming available, an integrative taxonomic approach should be adopted by combining multiple datasets such as shell morphology, host associations, and genetic data in order to refine the classification at the subfamily, genus, and species levels.

### CRediT authorship contribution statement

**E. Nocella:** Writing – original draft, Visualization, Methodology, Investigation, Formal analysis. **G. Fassio:** Writing – review & editing, Conceptualization. **T. Lemarcis:** Writing – review & editing, Investigation. **P. Zaharias:** Writing – original draft, Formal analysis. **D. Tamagnini:** Writing – original draft, Data curation. **N. de Leo:** Writing – original draft, Data curation. **N. Puillandre:** Writing – review & editing, Funding acquisition. **M.V. Modica:** Writing – review & editing, Funding acquisition, Conceptualization. **M. Oliverio:** Writing – original draft, Funding acquisition, Conceptualization.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgments

Many of the specimens sequenced herein were collected under the auspices of the Our Planet Reviewed Tropical Deep Sea Benthos programs at the MNHN (expeditions AURORA 2007; CORSICABENTHOS 3; EXBODI, DOI: 10.17600/11100080; ILES DU SALUT; KANACONO, DOI:

10.17600/16003900; KARUBENTHOS 2, DOI: 10.17600/15005400; KAVIENG 2014, DOI: 10.17600/14004400; KOUMAC 2.3; NanHai 2014; PANGLAO 2004; PAPUA NIUGINI, DOI:10.17600/18000841; SALOMONBOA 3, DOI: 10.17600/7100070; SPANBIOS, DOI: 10.17600/18000701; WESTERN AUSTRALIA 2011; ZhongSha 2015). These expeditions operated under the regulations then in force in the countries in question and satisfy the conditions set by the Nagoya Protocol for access to genetic resources. We are grateful to Philippe Maestrati (MNHN) and Laurent Charles (MNHN), for providing precious photographic material. We thank Dario Zuccon (MNHN) for his help with molecular sequences, and Barbara Buge (MNHN) for her help in curating the MNHN samples. Raimondo Villa and Daniela Pica (SZN) are acknowledged for their help with the taxonomic identification.

### Benefit-sharing

A research collaboration was developed with scientists from the countries providing samples; all collaborators are included as co-authors. The results of research have been shared with the provider communities.

### Ethics approval statement

The manuscript presents research on animals that do not require ethical approval for their study.

### Funding sources

This research was funded by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement no. 865101) to N.P., and by Sapienza University of Rome (2021 SEED-3840974 grant) to M.O. Partial funding was also provided by the Doctoral School in Evolutionary and Environmental Biology of Sapienza University of Rome to E.N., and by institutional funding from the Stazione Zoologica Anton Dohrn to M.V. M. and E.N.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ympev.2026.108545.

### Data availability

https://www.indores.fr/

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