Aplacophoran traits in the late Ordovician septemchitonid polyplacophorans.

A sample of phosphatized, originally calcareous, mollusk shells from the Katian age uppermost Mójcza Limestone at its type locality yielded a few hundred polyplacophoran plates. The chelodids are very rare among them. Three septemchitonid species dominate. They represent a gradation from underived steep roof-like plates to almost cylindrical ones, leaving only a narrow ventral slit for the foot. Apparently, this represents the first step toward the extremely derived 'segmented clam' Bauplan of the Silurian Carnicoleus, with plates completely closed at the venter except for the mouth and anal openings. To enable growth, the plates became thinner and more flexible (or perhaps resorbed) along the dorsum. The tendency toward reduction of the ventral gap of the plates in the early Paleozoic septemchitonid polyplacophorans implies their lack of ability to cling to the substrate with a muscular foot. In compensation, their plates changed toward a more efficient protective function, covering the animal body sides more and more completely. This may explain the origin of the ventral furrow of extant solenogasters hiding the rudimentary foot. An opposite route was chosen by the coeval Acaenoplax lineage, in which the plates did not contact each other, exposing much of the soft body on the dorsum. In both cases the animals appeared to be worm-like, perhaps representing different ways of evolution from the Paleozoic chitons to the extant aplacophorans.

Preserved appendages in a Silurian binodicope: implications for the evolutionary history of ostracod crustaceans.

Ostracod crustaceans originated at least 500 Ma ago. Their tiny bivalved shells represent the most species-abundant fossil arthropods, and ostracods are omnipresent in a wide array of freshwater and marine environments today and in the past. Derima paparme gen. et sp. nov. from the Herefordshire Silurian Lagerstätte (~430 Ma) in the Welsh Borderland, UK, is one of only a handful of exceptionally preserved ostracods (with soft parts as well as the shell) known from the Palaeozoic. A male specimen provides the first evidence of the appendages of Binodicopina, a major group of Palaeozoic ostracods comprising some 135 Ordovician to Permian genera. The appendage morphology of D. paparme, but not its shell, indicates that binodicopes belong to Podocopa. The discovery that the soft-part morphology of binodicopes allies them with podocopes affirms that using the shell alone is an unreliable basis for classifying certain fossil ostracods, and knowledge of soft-part morphology is critical for the task. Current assignment of many fossil ostracods to higher taxa, and therefore the evolutionary history of the group, may require reconsideration.

Assessing phenotypic variation and plasticity of endemic gastropods from thermal water refugia using complex morphometric techniques: A case study of Lake Pețea melanopsids.

Understanding the underlying reasons for phenotypic plasticity and resulting morphological disparity is one of the key topics of evolutionary research. The phenotypic plasticity of extant and fossil melanopsids has been widely documented. Yet millennial-resolution, well-dated records from small aquatic habitats harboring endemics are scarce. The thermal spring-fed Lake Pețea is an ice age refugia harboring a unique endemic warm-water fauna. Subfossil melanopsids display incredible morphological variability from smooth to keeled, elongated to ribbed, shouldered forms. Numerous morphotypes have been considered as individual taxa with a fluent succession from the smooth elongated to the ribbed, shouldered types. This study presents an extensive morphometric analysis of subfossil melanopsids (ca. 3500 specimens) derived from stratified samples with an independent chronology. The aim was to separate morphotypes for investigations of temporal morphological disparity. Our results challenge the widely accepted hypothesis that proposes the evolution of shouldered, compressed, ribbed shells through a two-step process from smooth elongated spindle-shaped shells. Instead, it suggests that the subfossil shells belong to two distinct taxa present throughout the available stratigraphic data. The main components of shape variation, shape globularity, and shell coiling seem allometry-related. Ribs, striation, and keels appear randomly. High-spired spindle-shaped forms were considered to represent specimens of Microcolpia daudebartii hazayi. Bulkier low-spired and shouldered specimens represent phenotypes of Mi. parreyssii parreyssii. The collective and random distribution of morphotypes from the early stages of the lake's history also refutes the idea of a continuous transformation of the elongated forms into compressed, shouldered ones. Rather points to multiple events and environmental stimuli triggering development. Melanopsids appear in Late Glacial horizons, with Theodoxus prevostianus preferring temperatures above 23°C which may indicate the subordinate presence of hot water microhabitats in cooler waters.

Hydrodynamic performance of Ordovician archaeostracan carapaces.

The diversification of macroscopic pelagic arthropods such as caryocaridid archaeostracans was a crucial aspect of the Great Ordovician Biodiversification Event, and the plankton revolution. A pelagic mode of life has been inferred for caryocaridids from their common presence in black graptolitic shales alongside carapace morphologies that appear streamlined. However, the hydrodynamic performance within the group and comparisons with other archaeostracans were lacking. Here we use a computational fluid dynamics approach to quantify the hydrodynamic performance of caryocaridids, and other early Palaeozoic archaeostracans including Arenosicaris inflata and Ordovician ceratiocaridids. We show that streamlining of the carapace was an important factor facilitating a pelagic mode of life in caryocaridids, in reducing the drag coefficient and facilitating a broader range of lift coefficients at different angles of attack. However, comparable hydrodynamic performance is also recovered for some ceratiocaridids. This suggests that alongside carapace streamlining, adaptations to appendages and thinning of the carapace were also important for a pelagic mode of life in Ordovician caryocaridids.

Middle through late Holocene long-distance transport of exotic shell personal adornments in Central West Patagonia (southern South America). The archaeomalacological assemblage of Baño Nuevo 1.

The exchange of information and social interactions on broad spatial scales between human groups in the past can be studied through the provenance of key indicators of distant origin recorded at archaeological sites. The remains of shells of mollusk species, especially when crafted as elements of personal ornaments, express aspects of the behaviors and valuations for the populations that selected, transformed, and exchanged such items. In the southern cone of South America, past hunter-gatherer groups traveled long distances and interacted with communities distributed throughout the territory to acquire goods for technological use, visual display or considered highly valued materials. When recorded at distant locations, these goods of extra local origin are very informative regarding the differences between commonly used home ranges and the occasional access to remote spaces. We present the results of the analysis of the archaeomalacological assemblage of the Baño Nuevo 1 site, a cave with exceptional preservation conditions in Central West Patagonia. This site has yielded a diverse group of artifacts made of shells with origins from multiple distances, as well as evidence of the use of marine, freshwater, and terrestrial species. Its deposits, which extend over the last 11,000 years, reveal an antiquity of at least the middle Holocene for the acquisition, manufacture, use and transport of goods as personal ornaments from shells in the macroregion.

Differentiation of Bulinus senegalensis and Bulinus forskalii Snails in West Africa Using Morphometric Analysis.

PURPOSE: Accurate identification of medically important intermediate host and vector species is crucial for understanding disease transmission and control. Identifying Bulinus snails which act as intermediate host species for the transmission of schistosomiasis is typically undertaken using conchological and genital morphology as well as molecular methods. METHODS: Here, a landmark-based morphometric analysis of shell morphology was undertaken to determine its utility to distinguish the closely related and morphologically similar sister species Bulinus senegalensis and Bulinus forskalii. The method was developed to increase the accuracy of conchological morphology methods to identify Bulinus species in the field. Both species are found in West Africa, but only B. senegalensis is implicated in the transmission of urogenital schistosomiasis. RESULTS: We found when scaled down to the same length, 3-whorl and 4-whorl (juvenile) B. senegalensis shells had a longer spire, narrower body whorl and shorter aperture than B. forskalii. In contrast, 5-whorl (adult) B. senegalensis had a shorter spire, but still had a shorter aperture and narrower body whorl than B. forskalii. Canonical Variate Analysis (CVA) showed minimal overlap between B. senegalensis and B. forskalii for 3-whorl and 4-whorl shells, with a clear separation for 5-whorl shells. Overall, B. senegalensis had a consistently shorter aperture size and narrower body whorl than B. forskalii for all development stages. Spire length was variable depending on the stage of development, with 3-whorl and 4-whorl shells having the opposite trends of adult shells. CONCLUSIONS: Our study demonstrates the applicability of landmark-based morphometrics in distinguishing the medically important, Bulinus senegalensis from its morphologically similar sister species, Bulinus forskalii. We recommend using measurements based on spire length, penultimate whorl length, body whorl width and aperture size to differentiate B. senegalensis and B. forskalii, when used with the appropriate information for each shell's development stage.

Shell shape does not accurately predict self-righting ability in hatchling freshwater turtles.

Flat hydrodynamic shells likely represent an evolutionary trade-off between adaptation to an aquatic lifestyle and the instability of more rounded shells, thought beneficial for self-righting. Trade-offs often result in compromises, this is particularly true when freshwater turtles, with flatter shells, must self-right to avoid the negative effects of inverting. These turtles, theoretically, invest more biomechanical effort to achieve successful and timely self-righting when compared to turtles with rounded carapaces. This increase in effort places these hatchlings in a precarious position; prone to inversion and predation and with shells seemingly maladapted to the act of self-righting. Here, we examine hatchling self-righting performance in three morphologically distinct freshwater turtle species (Apalone spinifera, Chelydra serpentina and Trachemys scripta scripta) that inhabit similar environmental niches. We demonstrate that these hatchlings were capable of rapid self-righting and used considerably less biomechanical effort relative to adult turtles. Despite differences in shell morphology the energetic efficiency of self-righting remained remarkably low and uniform between the three species. Our results confound theoretical predictions of self-righting ability based on shell shape metrics and indicate that other morphological characteristics like neck or tail morphology and shell material properties must be considered to better understand the biomechanical nuances of Testudine self-righting.

Fractal-like geometry as an evolutionary response to predation?

Fractal-like, intricate morphologies are known to exhibit beneficial mechanical behavior in various engineering and technological domains. The evolution of fractal-like, internal walls of ammonoid cephalopod shells represent one of the most clear evolutionary trends toward complexity in biology, but the driver behind their iterative evolution has remained unanswered since the first hypotheses introduced in the early 1800s. We show a clear correlation between the fractal-like morphology and structural stability. Using linear and nonlinear computational mechanical simulations, we demonstrate that the increase in the complexity of septal geometry leads to a substantial increase in the mechanical stability of the entire shell. We hypothesize that the observed tendency is a driving force toward the evolution of the higher complexity of ammonoid septa, providing the animals with superior structural support and protection against predation. Resolving the adaptational value of this unique trait is vital to fully comprehend the intricate evolutionary trends between morphology, ecological shifts, and mass extinctions through Earth's history.

Imbricated shell sculpture in benthic bivalves.

Molluscan shells display a high diversity of external sculpture. Sculptural elements may be symmetrical, where both edges of an element are morphologically similar, or asymmetrical, where one edge is steeper than the other. Asymmetrical sculpture can be ratcheted, with the leading edges (those in the direction of locomotion or growth) less steep than the trailing edges, or imbricated (leading edges steeper than trailing edges). While the ratcheted sculpture is better known, the diversity of imbricated sculpture has remained largely unexplored. In a survey of extant benthic shell-bearing molluscs, we document imbricated sculpture primarily in epifaunal bivalves or on the exposed sectors of shells of semi-infaunal bivalves. Imbricated sculpture is particularly widespread in pteriomorphian bivalves, but it is absent in the subclade Mytiloidea as well as in highly mobile Pectinidae. It also occurs in many carditid bivalves (Archiheterodonta) and in phylogenetically scattered euheterodonts. In several infaunal bivalves including species of Cardites (Carditidae), Hecuba (Donacidae), and Chione (Veneridae), comarginal elements on the posterior sector are imbricated whereas anterior comarginal ridges are ratcheted. Imbricated sculpture in bivalves tends to be concentrated on the upper (left) valves of pectinids or on the posterior sector of both valves in archiheterodonts and euheterodonts. Imbricated sculpture is uncommon in gastropods, even in epifaunal species, but does occur in the collabral ridges in some Vasidae and a few other groups. Expression of imbricated sculpture does not depend on shell mineral composition or microstructure. The ecological distribution and within-shell pattern of expression of imbricated sculpture point to the likelihood that this type of asymmetrical sculpture is both widespread and potentially functional. Additionally, we present a potential methodology whereby shell sculpture categories (symmetrical, ratcheted, and imbricated) may be quantified by comparing the lengths of corresponding leading and trailing edges across the shell surface.

Skeletal repatterning enhances the protective capacity of the shell in African hinge-back tortoises (Kinixys).

Changes in the structural association of skeletal traits are crucial to the evolution of novel forms and functions. In vertebrates, such rearrangements often occur gradually and may precede or coincide with the functional activation of skeletal traits. To illustrate this process, we examined the ontogeny of African hinge-back tortoises (Kinixys spp.). Kinixys species feature a moveable "hinge" on the dorsal shell (carapace) that enables shell closure (kinesis) when the hind limbs are withdrawn. This hinge, however, is absent in juveniles. Herein, we describe how this unusual phenotype arises via alterations in the tissue configuration and shape of the carapace. The ontogenetic repatterning of osseous and keratinous tissue coincided with shifts in morphological integration and the establishment of anterior (static) and posterior (kinetic) carapacial modules. Based on ex vivo skeletal movement and raw anatomy, we propose that Kinixys employs a "sliding hinge" shell-closing system that overcomes thoracic rigidity and enhances the protective capacity of the carapace. Universal properties of the vertebrate skeleton, such as plasticity, modularity, and secondary maturation processes, contributed to adaptive evolutionary change in Kinixys. We discuss a hypothetical model to explain the delayed emergence of skeletal traits and its relevance to the origins of novel form-to-function relationships.

Temporal variation in climatic factors influences phenotypic diversity of Trochulus land snails.

Organisms with limited dispersal capabilities should show phenotypic plasticity in situ to keep pace with environmental changes. Therefore, to study the influence of environmental variation on the phenotypic diversity, we chose land snails, Trochulus hispidus and T. sericeus, characterized by high population variability. We performed long-term field studies as well as laboratory and common garden experiments, which revealed that temporal environmental changes generate visible variation in shell size and shape of these snails. Many shell measurements of T. hispidus varied significantly with temperature and humidity in individual years. According to this, the first generation of T. hispidus, bred in controlled laboratory conditions, became significantly different in higher spire and narrower umbilicus from its wild parents. Interestingly, offspring produced by this generation and transplanted to wild conditions returned to the 'wild' flat and wide-umbilicated shell shape. Moreover, initially different species T. hispidus and T. sericeus transferred into common environment conditions revealed rapid and convergent shell modifications within one generation. Such morphological flexibility and high genetic variation can be evolutionarily favored, when the environment is heterogeneous in time. The impact of climate change on the shell morphometry can lead to incorrect taxonomic classification or delimitation of artificial taxa in land snails. These findings have also important implications in the context of changing climate and environment.

Bivalves maintain repair when faced with chronically repeated mechanical stress.

Even though mollusks' capacity to repair shell damage is usually studied in response to a single event, their shells have to defend them against predatory and environmental threats throughout their potentially multi-decadal life. We measured whether and how mollusks respond to chronic mechanical stress. Once a week for 7 months, we compressed whole live California mussels (Mytilus californianus) for 15 cycles at ∼55% of their predicted one-time breaking force, a treatment known to cause fatigue damage in shells. We found mussels repaired their shells. Shells of experimentally stressed mussels were just as strong at the end of the experiment as those of control mussels that had not been experimentally loaded, and they were more heavily patched internally. Additionally, stressed shells differed in morphology; they were heavier and thicker at the end of the experiment than control shells but they had increased less in width, resulting in a flatter, less domed shape. Finally, the chronic mechanical stress and repair came at a cost, with stressed mussels having higher mortality and less soft tissue than the control group. Although associated with significant cost, mussels' ability to maintain repair in response to ongoing mechanical stress may be vital to their survival in harsh and predator-filled environments.

Transcriptome analysis of the mantle tissue of Pinctada fucata with red and black shells under salinity stress.

To understand the molecular responses of Pinctada fucata with different shell colors to salinity stress, we used transcriptome sequencing on the mantle of P. fucata with a black shell and red shell color under the salinity of 20, 35, and 50. The 414 and 2371 differentially expressed genes (DEGs) in P. fucata with a black shell under low- or high-salt stress, while there were 588 and 3009 DEGs in P. fucata with a red shell. KEGG pathway enrichment analysis showed that, under low salt stress, the DEGs of P. fucata with the black shell were significantly enriched in pathways MAPK signaling pathway, protein processing in endoplasmic reticulum, vitamin B6 metabolism, longevity regulating pathway-multiple species, estrogen signaling pathway and antigen processing and presentation, the DEGs of P. fucata with a red shell were significantly enriched in pathways vitamin B6 metabolism. Under high salt stress, the DEGs of P. fucata with a red shell were significantly enriched in pathways arginine biosynthesis. 11 DEGs were randomly selected for quantitative real-time PCR, and the results were consistent with the RNA-seq. In addition, under high salt stress, DEGs were enriched into some pathways related to osmotic regulation and immune defense of P. fucata with black shell and red shell, such as Glycolysis / Gluconeogenesis, AMPK signaling pathway, Beta-Alanine metabolism, Glycine, serine and threonine metabolism, MAPK signaling pathway and Phagosome. The study showed that high salt stress had a greater influence on P. fucata with two shell colors, and P. fucata with a black shell made a positive immune defense response. Our results will improve to further understand the salt tolerance mechanism of P. fucata with different shell colors.

Evaluation of remodeling and geometry on the biomechanical properties of nacreous bivalve shells.

Mollusks have developed a broad diversity of shelled structures to protect against challenges imposed by biological interactions(e.g., predation) and constraints (e.g., [Formula: see text]-induced ocean acidification and wave-forces). Although the study of shell biomechanical properties with nacreous microstructure has provided understanding about the role of shell integrity and functionality on mollusk performance and survival, there are no studies, to our knowledge, that delve into the variability of these properties during the mollusk ontogeny, between both shells of bivalves or across the shell length. In this study, using as a model the intertidal mussel Perumytilus purpuratus to obtain, for the first time, the mechanical properties of its shells with nacreous microstructure; we perform uniaxial compression tests oriented in three orthogonal axes corresponding to the orthotropic directions of the shell material behavior (thickness, longitudinal, and transversal). Thus, we evaluated whether the shell material's stress and strain strength and elastic modulus showed differences in mechanical behavior in mussels of different sizes, between valves, and across the shell length. Our results showed that the biomechanical properties of the material building the P. purpuratus shells are symmetrical in both valves and homogeneous across the shell length. However, uniaxial compression tests performed across the shell thickness showed that biomechanical performance depends on the shell size (aging); and that mechanical properties such as the elastic modulus, maximum stress, and strain become degraded during ontogeny. SEM observations evidenced that compression induced a tortuous fracture with a delamination effect on the aragonite mineralogical structure of the shell. Findings suggest that P. purpuratus may become vulnerable to durophagous predators and wave forces in older stages, with implications in mussel beds ecology and biodiversity of intertidal habitats.

Mechanical adaptation of brachiopod shells via hydration-induced structural changes.

The function-optimized properties of biominerals arise from the hierarchical organization of primary building blocks. Alteration of properties in response to environmental stresses generally involves time-intensive processes of resorption and reprecipitation of mineral in the underlying organic scaffold. Here, we report that the load-bearing shells of the brachiopod Discinisca tenuis are an exception to this process. These shells can dynamically modulate their mechanical properties in response to a change in environment, switching from hard and stiff when dry to malleable when hydrated within minutes. Using ptychographic X-ray tomography, electron microscopy and spectroscopy, we describe their hierarchical structure and composition as a function of hydration to understand the structural motifs that generate this adaptability. Key is a complementary set of structural modifications, starting with the swelling of an organic matrix on the micron level via nanocrystal reorganization and ending in an intercalation process on the molecular level in response to hydration.

Significance of the suture line in cephalopod taxonomy revealed by 3D morphometrics in the modern nautilids Nautilus and Allonautilus.

Assessing the taxonomic importance of the suture line in shelled cephalopods is a key to better understanding the diversity of this group in Earth history. Because fossils are subject to taphonomic artifacts, an in-depth knowledge of well-preserved modern organisms is needed as an important reference. Here, we examine the suture line morphology of all known species of the modern cephalopods Nautilus and Allonautilus. We applied computed tomography and geometric morphometrics to quantify the suture line morphology as well as the conch geometry and septal spacing. Results reveal that the suture line and conch geometry are useful in distinguishing species, while septal spacing is less useful. We also constructed cluster trees to illustrate the similarity among species. The tree based on conch geometry in middle ontogeny is nearly congruent with those previously reconstructed based on molecular data. In addition, different geographical populations of the same species of Nautilus separate out in this tree. This suggests that genetically distinct (i.e., geographically isolated) populations of Nautilus can also be distinguished using conch geometry. Our results are applicable to closely related fossil cephalopods (nautilids), but may not apply to more distantly related forms (ammonoids).

New species of latest Eocene/earliest Oligocene microgastropods (Heterobranchia: Orbitestellidae and Omalogyridae) from the Gries Ranch Formation, Lewis County, Washington State, USA.

Continued sampling of the latest Eocene to earliest Oligocene Gries Ranch Formation in Lewis County, Washington State, has yielded new heterobranch microgastropod species. Orbitestella kieli sp. nov., is the third fossil species of this genus and family Orbitestellidae from western North America. Two new species of Ammonicera, A. rolani sp. nov. and A. danieli sp. nov., are together only the second fossil record of this genus and the family Omalogyridae from the northeastern Pacific Ocean. New specimens of two previously recorded species, O. palaiopacifica Squires Goedert and A. benhami Squires Goedert, from early Eocene rocks of the Crescent Formation provide new data regarding shell morphology. The fossil record of both Ammonicera and Orbitestella in western North America is restricted to early Eocene to earliest Oligocene age rocks in Washington State.

Microstructural design for mechanical-optical multifunctionality in the exoskeleton of the flower beetle Torynorrhina flammea.

Biological systems have a remarkable capability of synthesizing multifunctional materials that are adapted for specific physiological and ecological needs. When exploring structure-function relationships related to multifunctionality in nature, it can be a challenging task to address performance synergies, trade-offs, and the relative importance of different functions in biological materials, which, in turn, can hinder our ability to successfully develop their synthetic bioinspired counterparts. Here, we investigate such relationships between the mechanical and optical properties in a multifunctional biological material found in the highly protective yet conspicuously colored exoskeleton of the flower beetle, Torynorrhina flammea Combining experimental, computational, and theoretical approaches, we demonstrate that a micropillar-reinforced photonic multilayer in the beetle's exoskeleton simultaneously enhances mechanical robustness and optical appearance, giving rise to optical damage tolerance. Compared with plain multilayer structures, stiffer vertical micropillars increase stiffness and elastic recovery, restrain the formation of shear bands, and enhance delamination resistance. The micropillars also scatter the reflected light at larger polar angles, enhancing the first optical diffraction order, which makes the reflected color visible from a wider range of viewing angles. The synergistic effect of the improved angular reflectivity and damage localization capability contributes to the optical damage tolerance. Our systematic structural analysis of T. flammea's different color polymorphs and parametric optical and mechanical modeling further suggest that the beetle's microarchitecture is optimized toward maximizing the first-order optical diffraction rather than its mechanical stiffness. These findings shed light on material-level design strategies utilized in biological systems for achieving multifunctionality and could thus inform bioinspired material innovations.

Combining genotypic and phenotypic variation in a geospatial framework to identify sources of mussels in northern New Zealand.

The New Zealand green-lipped mussel aquaculture industry is largely dependent on the supply of young mussels that wash up on Ninety Mile Beach (so-called Kaitaia spat), which are collected and trucked to aquaculture farms. The locations of source populations of Kaitaia spat are unknown and this lack of knowledge represents a major problem because spat supply may be irregular. We combined genotypic (microsatellite) and phenotypic (shell geochemistry) data in a geospatial framework to determine if this new approach can help identify source populations of mussels collected from two spat-collecting and four non-spat-collecting sites further south. Genetic analyses resolved differentiated clusters (mostly three clusters), but no obvious source populations. Shell geochemistry analyses resolved six differentiated clusters, as did the combined genotypic and phenotypic data. Analyses revealed high levels of spatial and temporal variability in the geochemistry signal. Whilst we have not been able to identify the source site(s) of Kaitaia spat our analyses indicate that geospatial testing using combined genotypic and phenotypic data is a powerful approach. Next steps should employ analyses of single nucleotide polymorphism markers with shell geochemistry and in conjunction with high resolution physical oceanographic modelling to resolve the longstanding question of the origin of Kaitaia spat.

Premating barriers in young sympatric snail species.

Sympatric coexistence of recently diverged species raises the question of barriers restricting the gene flow between them. Reproductive isolation may be implemented at several levels, and the weakening of some, e.g. premating, barriers may require the strengthening of the others, e.g. postcopulatory ones. We analysed mating patterns and shell size of mates in recently diverged closely related species of the subgenus Littorina Neritrema (Littorinidae, Caenogastropoda) in order to assess the role of premating reproductive barriers between them. We compared mating frequencies observed in the wild with those expected based on relative densities using partial canonical correspondence analysis. We introduced the fidelity index (FI) to estimate the relative accuracy of mating with conspecific females and precopulatory isolation index (IPC) to characterize the strength of premating barriers. The species under study, with the exception of L. arcana, clearly demonstrated preferential mating with conspecifics. According to FI and IPC, L. fabalis and L. compressa appeared reliably isolated from their closest relatives within Neritrema. Individuals of these two species tend to be smaller than those of the others, highlighting the importance of shell size changes in gastropod species divergence. L. arcana males were often found in pairs with L. saxatilis females, and no interspecific size differences were revealed in this sibling species pair. We discuss the lack of discriminative mate choice in the sympatric populations of L. arcana and L. saxatilis, and possible additional mechanisms restricting gene flow between them.