Odd-Paired is Involved in Morphological Divergence of Snail-Feeding Beetles.

Body shape and size diversity and their evolutionary rates correlate with species richness at the macroevolutionary scale. However, the molecular genetic mechanisms underlying the morphological diversification across related species are poorly understood. In beetles, which account for one-fourth of the known species, adaptation to different trophic niches through morphological diversification appears to have contributed to species radiation. Here, we explored the key genes for the morphological divergence of the slender to stout body shape related to divergent feeding methods on large to small snails within the genus Carabus. We show that the zinc-finger transcription factor encoded by odd-paired (opa) controls morphological variation in the snail-feeding ground beetle Carabus blaptoides. Specifically, opa was identified as the gene underlying the slender to stout morphological difference between subspecies through genetic mapping and functional analysis via gene knockdown. Further analyses revealed that changes in opa cis-regulatory sequences likely contributed to the differences in body shape and size between C. blaptoides subspecies. Among opa cis-regulatory sequences, single nucleotide polymorphisms on the transcription factor binding sites may be associated with the morphological differences between C. blaptoides subspecies. opa was highly conserved in a wide range of taxa, especially in beetles. Therefore, opa may play an important role in adaptive morphological divergence in beetles.

Out of the ancient lake: Multiple riverine colonizations and diversification of the freshwater snails in the genus Semisulcospira around Lake Biwa.

Ancient lakes are a hotspot of biodiversity. Freshwater species often experience spectacular species radiation after colonizing lakes from riverine habitats. Therefore, the relationship between the fauna of the ancient lakes and the surrounding riverine system has a special significance in understanding their origin and evolutionary history. The study of ancient lake species often focused on the lake colonization of riverine species. In contrast, far less attention has been placed on the reverse direction: the riverine colonization of the lake species, despite its importance in disentangling their complex evolutionary history. The freshwater snails in the genus Semisulcospira involve endemic groups that radiated in the ancient Lake Biwa. Using genetics and fossil records, we inferred that the ancestors of these lake-endemic Semisulcospira snails historically colonized the riverine habitats at least three times during the Middle Pleistocene. Each colonization resulted in the formation of a new lineage that was genetically and morphologically distinct from other lineages. Further, one of these colonizations was followed by hybridization with a cosmopolitan riverine species, which potentially facilitated the population persistence of the colonizers in the new environment. Despite their complex histories, all these colonizers were currently grouped within a single species, Semisulcospira kurodai, suggesting cryptic diversity in this species. This study highlights the significance of the riverine colonizations of the lake species to fully understand the diversification history of freshwater fauna in and around the ancient lakes.

Using pose estimation to identify regions and points on natural history specimens.

A key challenge in mobilising growing numbers of digitised biological specimens for scientific research is finding high-throughput methods to extract phenotypic measurements on these datasets. In this paper, we test a pose estimation approach based on Deep Learning capable of accurately placing point labels to identify key locations on specimen images. We then apply the approach to two distinct challenges that each requires identification of key features in a 2D image: (i) identifying body region-specific plumage colouration on avian specimens and (ii) measuring morphometric shape variation in Littorina snail shells. For the avian dataset, 95% of images are correctly labelled and colour measurements derived from these predicted points are highly correlated with human-based measurements. For the Littorina dataset, more than 95% of landmarks were accurately placed relative to expert-labelled landmarks and predicted landmarks reliably captured shape variation between two distinct shell ecotypes ('crab' vs 'wave'). Overall, our study shows that pose estimation based on Deep Learning can generate high-quality and high-throughput point-based measurements for digitised image-based biodiversity datasets and could mark a step change in the mobilisation of such data. We also provide general guidelines for using pose estimation methods on large-scale biological datasets.

Retinoids promote penis development in sequentially hermaphroditic snails.

Sexual systems are surprisingly diverse, considering the ubiquity of sexual reproduction. Sequential hermaphroditism, the ability of an individual to change sex, has emerged multiple times independently across the animal kingdom. In molluscs, repeated shifts between ancestrally separate sexes and hermaphroditism are generally found at the level of family and above, suggesting recruitment of deeply conserved mechanisms. Despite this, molecular mechanisms of sexual development are poorly known. In molluscs with separate sexes, endocrine disrupting toxins bind the retinoid X receptor (RXR), activating ectopic male development in females, suggesting the retinoid pathway as a candidate controlling sexual transitions in sequential hermaphrodites. We therefore tested the role of retinoic acid signaling in sequentially hermaphroditic Crepidula snails, which develop first into males, then change sex, maturing into females. We show that retinoid agonists induce precocious penis growth in juveniles and superimposition of male development in females. Combining RXR antagonists with retinoid agonists significantly reduces penis length in induced juveniles, while similar treatments using retinoic acid receptor (RAR) antagonists increase penis length. Transcripts of both receptors are expressed in the induced penis. Our findings therefore show that retinoid signaling can initiate molluscan male genital development, and regulate penis length. Further, we show that retinoids induce ectopic male development in multiple Crepidula species. Species-specific influence of conspecific induction of sexual transitions correlates with responsiveness to retinoids. We propose that retinoid signaling plays a conserved role in molluscan male development, and that shifts in the timing of retinoid signaling may have been important for the origins of sequential hermaphroditism within molluscs.

One, two or three? Integrative species delimitation of short-range endemic Hemicycla species (Gastropoda: Helicidae) from the Canary Islands based on morphology, barcoding, AFLP and ddRADseq data.

Hemicycla mascaensis and H. diegoi are short-range endemics that occur allopatrically in small areas in the Teno Mountains in the western part of Tenerife (Canary Islands). Both taxa have been recognised as distinct species based on differences in shell morphology and genital anatomy. Preliminary molecular analyses using mitochondrial markers suggested a potential paraphyly of H. diegoi with regard to H. mascaensis. We here use multilocus AFLP data and ddRADseq data as well as distribution data, data on shell morphology and genital anatomy to assess the status of these taxa using phylogenetic analyses, species tree reconstruction and molecular species delimitation based on the multispecies coalescent as implemented in BFD* and BPP in an integrative approach. Our analyses show that, based on the analysis of multilocus data, the two taxa are reciprocally monophyletic. Species delimitation methods, however, tend to recognise all investigated populations as distinct species, albeit neither lending unambiguous support to any of the species hypotheses. The comparison of the anatomy of distal genital organs further suggests differentiation within H. mascaensis. This highlights the need for a balanced weighting of arguments from different lines of evidence to determine species status and calls for cautious interpretations of the results of molecular species delimitation analyses, especially in organisms with low active dispersal capacities and expected distinct population structuring such as land snails. Taking all available evidence into account, we favour to recognise H. mascaensis and H. diegoi as distinct species, acknowledging, though, that the recognition of both taxa as subspecies (with possibly a third yet undescribed) would also be an option as morphological differentiation is within the limits of other land snail species that are traditionally subdivided into subspecies.

Genetic and morphological divergence between Littorina fabalis ecotypes in Northern Europe.

Low dispersal marine intertidal species facing strong divergent selective pressures associated with steep environmental gradients have a great potential to inform us about local adaptation and reproductive isolation. Among these, gastropods of the genus Littorina offer a unique system to study parallel phenotypic divergence resulting from adaptation to different habitats related with wave exposure. In this study, we focused on two Littorina fabalis ecotypes from Northern European shores and compared patterns of habitat-related phenotypic and genetic divergence across three different geographic levels (local, regional and global). Geometric morphometric analyses revealed that individuals from habitats moderately exposed to waves usually present a larger shell size with a wider aperture than those from sheltered habitats. The phenotypic clustering of L. fabalis by habitat across most locations (mainly in terms of shell size) support an important role of ecology in morphological divergence. A genome scan based on amplified fragment length polymorphisms (AFLPs) revealed a heterogeneous pattern of differentiation across the genome between populations from the two different habitats, suggesting ecotype divergence in the presence of gene flow. The contrasting patterns of genetic structure between nonoutlier and outlier loci, and the decreased sharing of outlier loci with geographic distance among locations are compatible with parallel evolution of phenotypic divergence, with an important contribution of gene flow and/or ancestral variation. In the future, model-based inference studies based on sequence data across the entire genome will help unravelling these evolutionary hypotheses, improving our knowledge about adaptation and its influence on diversification within the marine realm.

Putative chromosomal rearrangements are associated primarily with ecotype divergence rather than geographic separation in an intertidal, poorly dispersing snail.

Littorina saxatilis is becoming a model system for understanding the genomic basis of ecological speciation. The parallel formation of crab-adapted ecotypes that exhibit partial reproductive isolation from wave-adapted ecotypes has enabled genomic investigation of conspicuous shell traits. Recent genomic studies suggest that chromosomal rearrangements may enable ecotype divergence by reducing gene flow. However, the genomic architecture of traits that are divergent between ecotypes remains poorly understood. Here, we use 11,504 single nucleotide polymorphism (SNP) markers called using the recently released L. saxatilis genome to genotype 462 crab ecotype, wave ecotype and phenotypically intermediate Spanish L. saxatilis individuals with scored phenotypes. We used redundancy analysis to study the genetic architecture of loci associated with shell shape, shape corrected for size, shell size and shell ornamentation, and to compare levels of co-association among different traits. We discovered 341 SNPs associated with shell traits. Loci associated with trait divergence between ecotypes were often located inside putative chromosomal rearrangements recently characterized in Swedish L. saxatilis. In contrast, we found that shell shape corrected for size varied primarily by geographic site rather than by ecotype and showed little association with these putative rearrangements. We conclude that genomic regions of elevated divergence inside putative rearrangements were associated with divergence of L. saxatilis ecotypes along steep environmental axes-consistent with models of adaptation with gene flow-but were not associated with divergence among the three geographical sites. Our findings support predictions from models indicating the importance of genomic regions of reduced recombination allowing co-association of loci during ecological speciation with ongoing gene flow.

Lineage Identification Affects Estimates of Evolutionary Mode in Marine Snails.

In order to study evolutionary pattern and process, we need to be able to accurately identify species and the evolutionary lineages from which they are derived. Determining the concordance between genetic and morphological variation of living populations, and then directly comparing extant and fossil morphological data, provides a robust approach for improving our identification of lineages through time. We investigate genetic and shell morphological variation in extant species of Penion marine snails from New Zealand, and extend this analysis into deep time using fossils. We find that genetic and morphological variation identify similar patterns and support most currently recognized extant species. However, some taxonomic over-splitting is detected due to shell size being a poor trait for species delimitation, and we identify incorrect assignment of some fossil specimens. We infer that a single evolutionary lineage (Penion sulcatus) has existed for 22 myr, with most aspects of shell shape and shell size evolving under a random walk. However, by removing samples previously classified as the extinct species P. marwicki, we instead detect morphological stasis for one axis of shell shape variation. This result demonstrates how lineage identification can change our perception of evolutionary pattern and process. [Genotyping by sequencing; geometric morphometrics; morphological evolution; Neogastropoda; phenotype; speciation; stasis.].

Global Diversification Dynamics Since the Jurassic: Low Dispersal and Habitat-Dependent Evolution Explain Hotspots of Diversity and Shell Disparity in River Snails (Viviparidae).

The Viviparidae, commonly known as River Snails, is a dominant group of freshwater snails with a nearly worldwide distribution that reaches its highest taxonomic and morphological diversity in Southeast Asia. The rich fossil record is indicative of a probable Middle Jurassic origin on the Laurasian supercontinent where the group started to diversify during the Cretaceous. However, it remains uncertain when and how the biodiversity hotspot in Southeast Asia was formed. Here, we used a comprehensive genetic data set containing both mitochondrial and nuclear markers and comprising species representing 24 out of 28 genera from throughout the range of the family. To reconstruct the spatiotemporal evolution of viviparids on a global scale, we reconstructed a fossil-calibrated phylogeny. We further assessed the roles of cladogenetic and anagenetic events in range evolution. Finally, we reconstructed the evolution of shell features by estimating ancestral character states to assess whether the appearance of sculptured shell morphologies was driven by major habitat shifts. The molecular phylogeny supports the monophyly of the three subfamilies, the Bellamyinae, Lioplacinae, and Viviparinae, but challenges the currently accepted genus-level classification in several cases. The almost global distribution of River Snails has been influenced both by comparatively ancient vicariance and more recent founder events. In Southeast Asia, Miocene dispersal was a main factor in shaping the modern species distributions. A recurrent theme across different viviparid taxa is that many species living in lentic waters exhibit sculptured shells, whereas only one strongly sculptured species is known from lotic environments. We show that such shell sculpture is habitat-dependent and indeed evolved several times independently in lentic River Snails. Considerably high transition rates between shell types in lentic habitats probably caused the co-occurrence of morphologically distinct shell types in several lakes. In contrast, directional evolution toward smooth shells in lotic habitats, as identified in the present analyses, explains why sculptured shells are rarely found in these habitats. However, the specific factors that promoted changes in shell morphology require further work. [biogeographical analyses; fossil-calibrated phylogeny; fossil-constrained analyses; Southeast Asia; stochastic character mapping.].

Physiological alterations in Pseudosuccinea columella (Mollusca: Gastropoda) after infection by Heterorhabditis baujardi LPP7 (Rhabditida: Heterorhabditidae).

The snail Pseudosuccinea columella participates in the distribution of Fasciola hepatica in the environment by acting as its intermediate host. Therefore, the control of this lymnaeid is one of the ways to prevent hepatic fascioliasis. The objective of this study was to evaluate the susceptibility of P. columella to infective juveniles (IJs) of the entomopathogenic nematode (EPN) Heterorhabditis baujardi in laboratory conditions, as well as to investigate aspects related to the biochemistry and histopathology of snails exposed or not to the EPNs during three weeks. The EPN exposure induced significant reductions in the concentrations of glucose, total proteins and glycogen (gonad-digestive gland complex) in the snails during the onset of the infection, with the levels being restored as the infection progresses. These alterations were accompanied by increased hemolymph activities of aminotransferases and lactate dehydrogenase, as well as the concentrations of uric acid after the first and second weeks of the experiment. The histopathological analyses of the exposed snails revealed cell necrosis at the end of the first week, tissue inflammatory reactions one and two weeks after exposure, and degeneration three weeks afterward in comparison with the unexposed snails. Finally, scanning electronic microscopy revealed proliferation of fibrous connective tissue three weeks after exposure. The results indicate that P. columella is susceptible to H. baujardi. The exposure favored the establishment of a negative energy balance, increased the activity of enzymes related to tissue damages and promoted accumulation of nitrogen compounds in the host snails. Additionally, was observed in P. columella exposed to the EPNs, significant tissue lesions, and demonstrated the strong pathogenic potential of H. baujardi, indicating its possible application for biological control of this snail.

Adaptive divergence in shell morphology in an ongoing gastropod radiation from Lake Malawi.

BACKGROUND: Ecological speciation is a prominent mechanism of diversification but in many evolutionary radiations, particularly in invertebrates, it remains unclear whether supposedly critical ecological traits drove or facilitated diversification. As a result, we lack accurate knowledge on the drivers of diversification for most evolutionary radiations along the tree of life. Freshwater mollusks present an enigmatic example: Putatively adaptive radiations are being described in various families, typically from long-lived lakes, whereas other taxa represent celebrated model systems in the study of ecophenotypic plasticity. Here we examine determinants of shell-shape variation in three nominal species of an ongoing ampullariid radiation in the Malawi Basin (Lanistes nyassanus, L. solidus and Lanistes sp. (ovum-like)) with a common garden experiment and semi-landmark morphometrics. RESULTS: We found significant differences in survival and fecundity among these species in contrasting habitats. Morphological differences observed in the wild persisted in our experiments for L. nyassanus versus L. solidus and L. sp. (ovum-like), but differences between L. solidus and L. sp. (ovum-like) disappeared and re-emerged in the F1 and F2 generations, respectively. These results indicate that plasticity occurred, but that it is not solely responsible for the observed differences. Our experiments provide the first unambiguous evidence for genetic divergence in shell morphology in an ongoing freshwater gastropod radiation in association with marked fitness differences among species under controlled habitat conditions. CONCLUSIONS: Our results indicate that differences in shell morphology among Lanistes species occupying different habitats have an adaptive value. These results also facilitate an accurate reinterpretation of morphological variation in fossil Lanistes radiations, and thus macroevolutionary dynamics. Finally, our work testifies that the shells of freshwater gastropods may retain signatures of adaptation at low taxonomic levels, beyond representing an evolutionary novelty responsible for much of the diversity and disparity in mollusks altogether.

Responses of Leptinaria unilamellata (Gastropoda: Subulinidae) to Novel Micro-Environmental Conditions: Shift in Shell Morphology and the Balance between Protection against Desiccation and Reproductive Success.

In the present study, we observed that Leptinaria unilamellata responds to changes in microclimatic conditions through shifts in shell morphology. Over three laboratory generations, shell differences between two populations, representing distinct morphotypes, became less evident. Only F1 generations from both populations showed shell morphometry very similar to the field parental snails, suggesting maternal effect. Snails from the locality with higher values of rainfall, relative humidity and evaporation index and smaller values of temperature and insolation produced more and larger hatchlings. Snails from the locality with less favorable climatic conditions presented shells traits that offer protection against desiccation, but reduce reproductive success. These snails showed smaller offspring production and faster response to a desiccation regime, through changes in conchiometrics. In addition, the results of the present study suggest that the spire index plays a less important role in determining protective properties of the shell of L. unilamellata, in response to desiccation risk, compared to aperture dimension. As shell aperture dimension is an important trait related to resistance to desiccation, and at the same time to reproduction, plastic responses to environmental conditions promoting the balance between survival and reproductive success are critical for the species adaptive success.

Phylogeography of Bellamya (Mollusca: Gastropoda: Viviparidae) snails on different continents: contrasting patterns of diversification in China and East Africa.

BACKGROUND: Species diversity is determined by both local environmental conditions that control differentiation and extinction and the outcome of large-scale processes that affect migration. The latter primarily comprises climatic change and dynamic landscape alteration. In the past few million years, both Southeast Asia and Eastern Africa experienced drastic climatic and geological oscillations: in Southeast Asia, especially in China, the Tibetan Plateau significantly rose up, and the flow of the Yangtze River was reversed. In East Africa, lakes and rivers experienced frequent range expansions and regressions due to the African mega-droughts. To test how such climatic and geological histories of both regions relate to their respective regional species and genetic diversity, a large scale comparative phylogeographic study is essential. Bellamya, a species rich freshwater snail genus that is widely distributed across China and East Africa, represents a suitable model system to address this question. We sequenced mitochondrial and nuclear DNA for members of the genus from China and used published sequences from Africa and some other locations in Asia to investigate their phylogeny and distribution of genetic diversity. RESULTS: Our phylogenetic analysis revealed two monophyletic groups, one in China and one in East Africa. Within the Chinese group, Bellamya species show little genetic differentiation. In contrast, we observe fairly deep divergence among the East African lakes with almost every lake possessing its unique clade. Our results show that strong divergence does not necessarily depend on intrinsic characteristics of a species, but rather is related to the landscape dynamics of a region. CONCLUSION: Our phylogenetic results suggest that the Bellamya in China and East Africa are independent phylogenetic clades with different evolutionary trajectories. The different climate and geological histories likely contributed to the diverging evolutionary patterns. Repeated range expansions and regressions of lakes likely contributed to the great divergence of Bellamya in East Africa, while reversal of the river courses and intermingling of different lineages had an opposite effect on Bellamya diversification in China.

Riverscape genetic variation, migration patterns, and morphological variation of the threatened Round Rocksnail, Leptoxis ampla.

Within riverine systems, headwater populations are hypothesized to harbour higher amounts of genetic distinctiveness than populations in the main stem of a river and display increased genetic diversity in large, downstream habitats. However, these hypotheses were mostly developed with insects and fish, and they have not been tested on many invertebrate lineages. Pleuroceridae gastropods are of particular ecological importance to rivers of eastern North America, sometimes comprising over 90% of macroinvertebrate biomass. Yet, virtually nothing is known of pleurocerid landscape genetics, including whether genetic diversity follows predictions made by hypotheses developed on more mobile species. Moreover, the commonly repeated hypothesis that intraspecific morphological variation in gastropods results from ecophenotypic plasticity has not been well tested on pleurocerids. Using 2bRAD-seq to discover single nucleotide polymorphisms, we show that the threatened, Cahaba River endemic pleurocerid, Leptoxis ampla, has limited gene flow among populations and that migration is downstream-biased, conflicting with previous hypotheses. Both tributary and main stem populations harbour unique genomic profiles, and genetic diversity was highest in downstream populations. Furthermore, L. ampla shell morphology was more correlated with genetic differences among individuals and populations than habitat characteristics. We anticipate similar genetic and demographic patterns to be seen in other pleurocerids, and hypotheses about gene flow and population demographics that were based on more mobile taxa often, but not always, apply to freshwater gastropods. From a conservation standpoint, genetic structure of L. ampla populations suggests distinctive genetic diversity is lost with localized extirpation, a phenomenon common across the range of Pleuroceridae.

Role of ancient lakes in genetic and phenotypic diversification of freshwater snails.

Endemic organisms of ancient lakes have been studied as models to understand processes of speciation and adaptive radiation. However, it remains unclear how ancient lakes play roles in genetic and phenotypic diversity of freshwater mollusks. In the present study, we focus on viviparid freshwater snails in the ancient lakes of East and Southeast Asia (Japan and China) to address this question. Using molecular phylogenetic analyses based on mitochondrial (COI, 16S) and nuclear genes (18S, 28S, H3), we show that patterns of species diversification in viviparid lineages. Colonization to ancient lakes occurred independently in China and Japan at least four times, with subsequent diversification into more than two species within each lake group. Morphological analyses of fossil related viviparids suggest parallel phenotypic evolution occurred in the different lakes and ages. Each lake contained a single lineage which was phenotypically diversified relative to those from other sites. Using genome-wide SNPs obtained by MIG-seq, we also examined the genetic structure of three Japanese viviparids, including two endemic species of ancient Lake Biwa. The results suggest that these two species diversified from the population of the third species living in wetlands surrounding the lake. These findings suggest that rapid diversification of lineages and phenotypic divergence can occur in ancient lakes compared to other habitats. Formation of large lakes probably promotes speciation and phenotypic divergence as a result of adaptation into different microhabitats. High numbers of ancient lakes could be a driver of species diversity in Asian viviparid snails.

Molecular phylogeny of Chondrocyclus (Gastropoda: Cyclophoridae), a widespread genus of sedentary, restricted-range snails.

The genus Chondrocyclus Ancey, 1898 contains the majority of southern African members of the Cyclophoridae, a large family of operculate land snails. We present the first molecular phylogeny of the genus based on two mitochondrial genes (16S and CO1) and complement this with an appraisal of morphological characters relating to the shell and soft parts. Worn shells on which some descriptions and records of different species were based appear to be indistinguishable morphologically, creating taxonomic confusion. We show that Chondrocyclus s.l. underwent two major radiations, one Afromontane and the other largely coastal. Accordingly, we recommend a revision recognising two genera. Chondrocyclus s.s. contains four monophyletic lineages, each characterized by a combination of morphological features. The Afromontane group is shown to be a species complex; relationships within this complex could not be resolved due to insufficient DNA sequence data. The molecular data confirms the monophyly of seven currently recognised species and provides evidence for at least twelve undescribed species; the morphological data are broadly consistent with this finding. The morphological data suggest that the two species from countries to the north of South Africa should be removed from the genus, and that Chondrocyclus sensu lato is endemic to South Africa. The historical biogeography of this group of microhabitat specialists with poor dispersal abilities contributes an additional, phylogenetically independent taxon to our understanding of the processes generating biodiversity in southern Africa, a natural laboratory for palaeobiogeography. All taxa are narrow-range endemics, underlining the importance of conserving South Africa's threatened forest habitats.

Integrating multilocus DNA data and 3D geometric morphometrics to elucidate species boundaries in the case of Pyrenaearia (Pulmonata: Hygromiidae).

To accurately delimit species the use of multiple character types is essential as all speciation processes are not equally reflected in different data (e.g. morphological, molecular or ecological characters). With the introduction of geometric morphometrics methods and advances in 3D technology, a comprehensive combination of molecular and morphological data has been enabled in groups where exhaustively quantifying and measuring morphological shape change was not possible before such as gastropod shells. In this study, we combined multilocus coalescent species delimitation methods with 3D geometric morphometrics of shell shape to delimit species within the land snail genus Pyrenaearia. A new taxonomic scheme was constructed for the genus identifying ten species. Two nominal species were synonymized and a hitherto unrecognized cryptic species was identified. Our findings support the importance of combining multiple lines of evidence as molecular and morphological data on their own do not yield the same information. Further, the integration of morphological and molecular data shows the importance of allometry in shell shape and suggests a combined effect of population history and selection in different environments on shells morphological variation. Our new taxonomy and phylogenetic reconstruction suggest that, besides the glacial cycles of the Pleistocene, passive dispersal and rock substrate complexity could also have been involved in the speciation of the genus.

Locking of the operculum in a water snail: Theoretical modeling and applications for mechanical sealing.

How can a water snail lock its door by an operculum? In this theoretical and experimental combined research, we revealed this by dissection, modeling and validation with a 3D printed technique. The operculum is a corneous or calcareous trapdoor-like sheet which attaches to the upper surface of the water snail's foot. It can plug the shell aperture by retracting the soft body when a predator or environmental threat is encountered. For a water snail (Pomacea canaliculata), the operculum can be locked in its shell rapidly. By optical microscope images, we found the operculum of P. canaliculata is a multilayered disk with a thicker center and thinner edge, which may be functionally influential for successful closing and opening the trapdoor. We filmed the locking in opercula of living snails, and designed an experiment to measure the deformation of opercula on the dead samples. We propose one mathematical model to describe the connections among geometry, sectionalized stiffness and the force for locking. By using 3D printing technique, we designed an operculum inspired locking mechanism to validate the theories we proposed. Under the same normal force, the water leakage rate of the bio-inspired structure can be reduced to 99% compared to the disk with uniform thickness. Our results reveal that the snail's operculum not only develops a light-weight trapdoor, but a locking mechanism which could serve as a valuable model for designing compliant locking mechanisms.

Connecting pattern to process: Growth of spiral shell sculpture in the gastropod Nucella ostrina (Muricidae: Ocenebrinae).

Shell morphology is a well-suited and underused system to examine the development of novel forms. The three-dimensional structure produced (the shell) is separate from the largely two-dimensional tissue that secretes it (the mantle), allowing us to disentangle the pattern from the process. Despite knowing a great deal about the mechanics of shell secretion (process), and the variety of shell shapes that exist (pattern), no effort has been made to understand how the mantle changes to produce different shell shapes. We investigated this question in the dimorphic snail Nucella ostrina, which exhibits both smooth and ribbed shells to determine how ribs are formed by the mantle. Rib thickenings are produced only in the outer calcitic shell layer and secreted by the distal Outer Mantle Epithelium (OME) with increased acid phosphatase activity. The evenly thick inner aragonitic layers are secreted by the proximal OME which expresses acid phosphatase. Here we show that locally thicker ribs in N. ostrina are produced by changing the dimensions of the distal OME: elongation in the direction of growth and increased cell height. This should increase the amount of shell material secreted, producing locally thicker shell (ribs). Preliminary evidence suggests this mechanism may be widespread in gastropods.

Evolutionary lineages of marine snails identified using molecular phylogenetics and geometric morphometric analysis of shells.

The relationship between morphology and inheritance is of perennial interest in evolutionary biology and palaeontology. Using three marine snail genera Penion, Antarctoneptunea and Kelletia, we investigate whether systematics based on shell morphology accurately reflect evolutionary lineages indicated by molecular phylogenetics. Members of these gastropod genera have been a taxonomic challenge due to substantial variation in shell morphology, conservative radular and soft tissue morphology, few known ecological differences, and geographical overlap between numerous species. Sampling all sixteen putative taxa identified across the three genera, we infer mitochondrial and nuclear ribosomal DNA phylogenetic relationships within the group, and compare this to variation in adult shell shape and size. Results of phylogenetic analysis indicate that each genus is monophyletic, although the status of some phylogenetically derived and likely more recently evolved taxa within Penion is uncertain. The recently described species P. lineatus is supported by genetic evidence. Morphology, captured using geometric morphometric analysis, distinguishes the genera and matches the molecular phylogeny, although using the same dataset, species and phylogenetic subclades are not identified with high accuracy. Overall, despite abundant variation, we find that shell morphology accurately reflects genus-level classification and the corresponding deep phylogenetic splits identified in this group of marine snails.