Viviparous gastropods keep embryos within their genital tract, hidden as the whole soft body, within a parental shell. Consequently, during birth, shelled offspring pass through the aperture of the parental shell, which results in a trade-off between the dimensions of the aperture and the embryo size. The mechanical obstacles involved in this process mirror the selection of obstetrics experienced by humans, as well as the pelvic constraints recorded in other tetrapods. The repeated evolution of viviparity in Clausiliidae, the family of land snails, which produce complex apertural barriers, gave us the opportunity to explore different ways of overcoming this evolutionary conflict. Most viviparous species are adapted by broadening of the shell canal and/or reduction of embryonic width. Some species reduce apertural barriers to the extent that they are no longer functional, whilst others produce unusual flexible, “soft shelled” embryos. The latter adaptation, recorded here for the first time, allows to overcome the “obstetrical dilemma” with minimal change in the outline of the adult shell and apertural barriers. The thickness and organomineral structure of flexible embryonic shells were investigated, compared to the typical “hard” shell of clausiliid embryos, which are rigid and unpliable already in the genital tract of the parent.
Article
“Obstetrical dilemma” in viviparous snails
https://doi.org/10.21203/rs.3.rs-1707751/v1
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Clausiliidae
Gastropoda
land snails
reproduction
shell microstructure
In evolutionary biology, the term obstetrical dilemma refers to a human challenging birth. It is the trade-off between antagonistic selection pressures acting in females: the first, for a larger birth canal, permitting successful passage of a large-brained neonate, and the second, for a smaller pelvic dimension, required for bipedal locomotion [1, 2]. The evolutionary compromise between these opposing selective mechanisms has led to some adaptations that reduce maternal and perinatal mortality from obstructed labour. Among them, the curved, multirotational trajectory of the fetus through the maternal pelvis, flexion of fetal head, and open fontanelles, are the most important adaptations resulting from obstetric selection. A tight fit between the dimensions of the birth canal and those of the brain and body of the offspring is maintained even in a changing environment by the offspring matching its growth trajectory to metabolic signals of the maternal phenotype [3].
Obstetric selection, i.e. antagonistic pressures on offspring size and maternal morphology, may function in any animal with a skeleton that limits the space available for embryo development or hinder their delivery through a narrow, relatively inflexible passage. A pelvic constraint model was presented for tetrapods [4], which convincingly explains a functional upper size limit for progeny both in live-bearing and egg-laying species [5, 6]. Another group of animals most likely experiencing some kind of obstetrical dilemma are shelled gastropods. However, they have seldom been studied regarding size and shape selection related to reproduction mode [7].
The trade-off between antagonistic selection pressures imposed by reproductive traits may affect a large number of land snail taxa, because viviparity (previously called ovoviviparity) or embryo-retention occur in at least 30 families of stylommatophorans, hermaphroditic land snails [8, 9]. On the other hand, the great diversity of shell geometry limits the distribution of the acute examples of obstetric selection to snails with a relatively small shell mouth or strong aperture barriers that can obstruct the passage of embryo. This kind of morphology is typical for Clausiliidae, the stylommatophoran family known for their elaborate apertural barriers [10] and containing many viviparous or embryo-retaining species [11].
Clausiliid shells consist of many whorls (often 10–12) and are usually high and slender. Their unique character is a complex closing apparatus (clausiliar) within the body whorl that includes several lamellae and plicae, and a single calcareous plate (clausilium), connected to the shell columella via a flexible stalk [10]. The shape of the clausilium plate corresponds well to the cross-cut of the shell canal at its narrowest point. Although the main functions of apertural barriers are debatable [11, 12], it is obvious that the clausilium blocks the passage to a shell canal when the snail body is retracted, and thus may confer an antipredator benefit. When a snail is active, its head with a genital opening emerges from the shell passing between lamellae and plicae of apertural barriers, and pushing the clausilium aside. During parturition, the shelled neonate has to pass through the constriction of apertural barriers, which implicates opposing selective pressures operating on embryo size and size of the shell canal [7].
Viviparous species are known in several subfamilies of Clausiliidae, yet the strategy seems to be most frequent in the species-rich subfamily Phaedusinae. Recently, the phylogeny of the subfamily and ancestral reproductive modes in the selected lineages were reconstructed [13]. It was clearly showed that the basal taxa of the group were oviparous, while viviparity evolved several times independently. That study included 21 viviparous or embryo-retaining taxa in the analysis [13] and more than 70 viviparous species in the subfamily have been reported in other papers [14, 15, 16]. The high taxonomic richness of viviparous Phaedusinae gave us a good opportunity to study the relationship between shell morphology and obstetric selection.
As the molecular data suggest a repeated and independent evolution of viviparity among the Phaedusinae, constraints associated with the live-bearing strategy may have been overcome in different ways. In this paper, we investigate a handful of adaptations that allow for the passage of the embryo through the occlusion of apertural barriers in the parental shell. Some of these were mentioned briefly in taxonomic papers [17], and we review these and illustrate them for comparative purposes in several clausiliid species: Reinia variegata (A. Adams, 1868), Reinia ashizuriensis Azuma, 1968, Stereophaedusa addisoni (Pilsbry, 1901), Tauphaedusa sheridani (L. Pfeiffer, 1865), Euphaedusa steetzneri (Pilsbry, 1919), E. cylindrella (Heude 1886). The main goal, however, is to present a novel adaptation concerning the structure of the embryonic shell found in Oospira miranda (Loosjes & Loosjes-van Bemmel, 1973) and Zaptyx ventriosa (Schmacker & Boettger, 1891), that was previously entirely overlooked.
The studied viviparous clausiliids developed four types of morphological adaptations that facilitate the delivery of embryos through the shell aperture. Compared to hypothetical ancestral Phaedusinae, the most extreme changes in shell shape and structure are exemplified by members of the Reinia genus, arboreal species from Japan (Fig. 1). The shell outline changed in these species from spindle-shaped to more conical and stouter, the number of whorls decreased to about six, and the aperture widened, giving an overall subulinid-like appearance. Adaptations in R. variegata also included almost full reduction of the clausiliar apparatus that consisted of only vestigial lamellae (Fig. 1F). The clausilium is lacking in this species.
Another adaptation concerns the shape of the embryonic shell, which becomes very narrow in some viviparous species. This feature is conspicuous because embryonic whorls (“protoconch”) are retained in clausiliids as apical whorls of the adult shell. The apical whorls being significantly narrower than the first whorls of the teleoconch are clear evidence that the growth trajectory in S. addisoni has changed abruptly after birth (Fig. 2A–D). Other examples include E. cylindrella and E. steetzneri, in which both the protoconch and the teleoconch are very narrow. However, at the borderline between these, the shell axis is slightly bent, possibly as a result of obstruction during birth (Fig. 2E–L).
The third type of adaptation is the widening of the shell canal in the body whorl, allowing for easier passage of the embryo between the lamellae and plicae of the apertural barriers. In this case, the outline of the shell changes only slightly, giving the body whorl a more convex appearance, though the apertural barriers are reshaped substantially: the clausiliar includes a broad clausilium plate and a spirally ascending inferior lamella (Fig. 3A–D). These modifications result in a much more spacious shell canal in the body whorl that can accommodate the transfer of a large embryo (compare neonatal size and clausilium width in S. addisoni and E. sheridani; Table 2). In contrast to these characteristics, the narrow clausilium plate and straight ascending inferior lamella were traditionally recognized as indicators of oviparous reproduction.
Many species of viviparid clausiliids adopted these three types of modification, which may sometimes co-occur within one species. A wide clausilium accompanies a narrow apex in some taxa. Transitions between these adaptations are also recognized. For example, R. ashizurensis has a stout shell with a low number of whorls that resemble its congener, R. variegata, but also has fully developed apertural barriers with a broad clausilium plate and a spirally ascending inferior lamella (Fig. 1A–C).
Development of a flexible embryonic shell
The fourth type of adaptation found in Phaedusinae concerns the structure of embryonic shells. It has been recorded in O. miranda and Z. ventriosa.
Oospira miranda is a dextral, often decollated, ground-dwelling species from Vietnam (Fig. 4A). The species is viviparous: embryos were found within a parental shell during microCT scanning of museum specimens (Fig. 4B); in laboratory culture, neonates were observed immediately after live birth (Fig. 4C–D). Morphological characters recognized in the adult shell, i.e., a wide apex (= wide embryonic shell), straightly ascending inferior lamella, and a narrow clausilium plate (Fig. 3G-H), seemed to exclude the possibility of live-bearing reproduction, as embryos are too large to pass through the shell canal at the narrowest point. The height and width of the neonatal shell significantly exceeds the width of the clausilium plate in this species (Table 2). Under closer examination, the shell was found to be thin and delicate, which we referred to as a “soft shell”. In direct examination, the neonatal shell of O. miranda resembles cellophane, which may keep a given shape for a long time but under slight pressure becomes distorted.
A similar adaptation was identified in Z. ventriosa, a Taiwanese species with a very wide apex, never decollated (Fig. 4E–F), a straight ascending inferior lamella, and a narrow clausilium plate (Fig. 3E–F). This species produces neonates in laboratory culture (Fig. 4G–H). The shell of such freshly delivered juveniles, when gently touched with laboratory tweezers, became dented, but not fractured. More intense and stronger pressing can break this dentation.
These initial observations carried out during the maintenance of the laboratory culture suggested that shortly after birth, the neonatal shells of O. miranda and Z. ventriosa have flexible walls. These “soft-shells” seem to be highly malleable during the entire embryonic development and delivery through apertural barriers. Shortly after birth, they harden. The physical properties of the embryonic shell were further investigated by means of microcomputed tomography and scanning electron microscopy.
Microcomputed tomography
We scanned ’soft-shelled‘ neonates of O. miranda and Z. ventriosa together with ’hard-shelled‘ embryos and neonates of S. addisoni and T. sheridani, in order to compare the density and thickness of the shells (Fig. 5).
Preliminary observations using the two-dimensional X-ray photographs showed a difference in thickness and density between S. addisoni and Z. ventriosa (Fig. 5M, enlarged in N and O, respectively). MicroCT scans of O. miranda and S. addisoni were performed using the same scanning and reconstruction parameters. The 3D visualization confirmed the difference between density and shell thickness of these two species (Fig. 5P).
Due to variations in wall thickness within the neonatal shell (e.g., between the first and the last whorls), it is not possible to precisely determine the thickness of the shell wall. The accuracy of the measurement is also limited by the resolution of the microCT scans, especially in the case of the relatively large neonates of O. miranda and Z. ventriosa. When scanning the whole embryonic shell of Z. ventriosa (approximately 4 mm in height), the size of the voxel was approximately 1 µm. Thus, the shell thickness cannot be determined down to the micrometer, but it can be estimated from a few to a dozen microns. A direct comparison between virtual microCT sections of samples scanned under the same conditions shows a significant difference between the ’soft–shelled‘ and ’hard-shelled‘ taxa (Fig. 5A–L). The ’hard-shelled‘ samples (both embryos dissected from the reproductive tract of a parent and neonates) have a shell wall of 30–40 µm thick. We examined ’soft-shelled‘ O. miranda samples that differed in size (the exact time of birth of each of the cultured neonates is unknown). It appeared that the larger (older) the neonate, the thicker the shell. The shell of the largest of the studied O. miranda was up to 20µm thick. However, the shell wall of this relatively large juvenile (several millimeters in height) still did not reach the thickness of the small ’hard-shelled‘ T. sheridani embryo, which was already about 30–40 µm thick during the retention in the genital tract and was stiff and rigid. The neonates of O. miranda and Z. ventriosa were much larger than the embryos and neonates of S. addisoni and R. variegata (Table 2), however, the former taxa has much thinner shells.
Scanning electron microscopy
After the non-invasive microCT scan, embryos and neonates were scanned using SEM (Fig. 6). The different properties of the shells of Z. ventriosa and O. miranda vs. S. addisoni and R. variegata were already visible during the preparation of the analysis. Under vacuum conditions, the soft shells of Z. ventriosa and O. miranda shrank and crumpled, creating a cellophane-like surface (Fig. 6A). Embryos and neonates of S. addisoni and R. variegata did not require any special preparation and their shell shape remained unchanged under the vacuum conditions applied during the SEM examination (Fig. 6D–E). To reduce the shell deformations, the next group of thin-shelled neonates was freeze-dried prior to SEM analyses (Fig. 6B–C).
The SEM studies allowed for complementary measurements of the shells. In the broken fragments of Z. ventriosa and O. miranda, the thickness of the shell wall ranged from 2–3 µm (Fig. 6F, G, I, J, L, M) to 18 µm in the largest neonate of O. miranda (Fig. 6O). The shells of S. addisoni (Fig. 6H, K) and R. variegata (Fig. 6N) are several times thicker.
All analyzed samples have a thin (< 1 µm) layer of periostracum. Beneath that, the aragonite shells are composed of lamellae with alternate orientations that were recognized as crossed lamellar microstructure, typically reported in adult gastropods [18, 19]. The crossed lamellar part of the gastropod shell is usually constructed from a few macrolayers of different orientation of lamellae [18]. The number of macrolayers in the ’soft‘ and ’hard‘ embryonic shells varies and is correlated with the thickness of the shell (compare the thin wall of O. miranda (Fig. 6L) and the number of alternating macrolayers in R. variegata (Fig. 6Q)).
The gastropod shell is an example of an external skeleton that also functions as a defence against various external threats, such as predation or desiccation in terrestrial environments. However, such a skeleton always has its most vulnerable part, the aperture, which allows the anterior soft parts of the animal to pass out of the shell for crawling or feeding. The smaller the area of the aperture, the better the protective function of the shell, thus additional structures constricting the area of the aperture, such as apertural barriers (e.g., teeth or folds), have evolved in many land snail families. They probably deter small-sized predators that try to enter the shell [20]. There are also other hypotheses that explain their importance. For example, stabilization of the shell during locomotion, protection of pallial organs against pressure from neighbouring organs, or division of the mantle cavity into the respiratory and excretory parts have been discussed [21]. In any case, development of apertural barriers is costly in terms of energy and calcium reserves; moreover, it was claimed that these structures may interfere with the reproductive strategy of snails [11]. Land snails produce three types of eggs with different shape flexibility: non-calcified, partly calcified, and heavily calcified [22]. The non-calcified or partly calcified eggs are typical of gastropods with strong apertural barriers; such eggs are pliable and can be compressed during passage through the aperture and regain their original shape afterwards. In contrast, the shape and size of aperture provide a mechanical constraint for passing heavily calcified eggs and for the shelled neonates produced by viviparous taxa [11]. In such a situation, strong selection towards a wide aperture and a smooth (without teeth or lamellae) internal surface of the shell canal is expected.
Considering these relationships, it is intriguing that clausiliid snails with well-developed apertural barriers, have acquired a viviparous reproductive mode and can produce offspring that are relatively large in comparison to the aperture of the adult. In this study, we showed that shell morphology of viviparous clausiliids resulted from the evolutionary compromise between protection (possibly against predators) and giving birth to shelled juveniles. This idea was initially explored for closely related species of the Clausiliinae subfamily, which have a significant increase of shell canal patency in the body whorl of viviparous taxa, in contrast to the narrow passage through apertural barriers in oviparous species [7]. Viviparity evolved repeatedly in clausiliids of the Phaedusinae subfamily [13], each time overcoming the mechanical constraints of apertural barriers, which makes this family an interesting model group for studying obstetrical selection in gastropods. The extreme stage of adaptation to viviparity with the complete reduction of the clausilium, was found among Phaedusinae in Reinia variegata. According to available phylogenies, R. variegata belongs to the lineage which includes only viviparous taxa [13, 15], however most of them (e.g., R. ashizurensis), did not lose the clausilium [23], and represent less advanced adaptations to viviparity. Reduction of apertural barriers occurred also in members of other clausiliid subfamilies, for example in Balea perversa L., Macroptychia africana (Melvill & Ponsonby, 1899) (subfamily Clausilinae), and Temesa clausilioides (Reeve, 1849) (Peruiniinae). This suggests that similar ecological drivers and developmental constraints affect the whole family.
In the Phaedusinae, a modification of apertural barriers including a broad clausilium plate and spirally ascending inferior lamella, was recognized as being associated with viviparous reproduction [17]. Contrastingly, a narrow clausilium plate and a straight ascending lamella, were regarded as indicative of oviparity. The occurrence of the broad clausilium plate in viviparous species was not explicitly referred there to as an adaptation allowing for overcoming the mechanical constraints for passage of the neonate, yet it was clearly assumed. Broad clausilium plates occur, among others, in species classified within the genera Phaedusa, Reinia, Euphaedusa, and Tauphaedusa. Despite a similar morphology of apertural barriers in these taxa, they are not closely related [13, 15]. Apertural barriers were modified in response to selective pressure imposed by the reproductive mode.
Due to recent advances in our understanding of the diversity of reproductive modes in Phaedusinae, it is known that some taxa with a narrow clausilium plate are also viviparous and produce shelled neonates [16]. This phenomenon, which is contrary to the opinion mentioned above [17], can be explained by the unusual properties of the embryonic shell, i.e., its high flexibility. In general, the gastropod shell is an organo-mineral composite with crossed lamellar microstructure of at least four orders of hierarchy (1st order lamellae are composed of layers of 2nd order lamellae. Each 2nd order lamella is formed of tens of thousands of 3rd order lamellae. The 3rd order lamellae are formed from numerous 4th order particles, surrounded by a thin organic sheath [19]). Nanoindentation tests of the gastropod shells revealed that the combination of aragonite and organic matrix in the crossed lamellar microstructure has several times greater fracture resistance than abiotic aragonite [19, 24, 25]. Most likely, the thin organically enriched layer of biomineral composite (Fig. 6L-Q) that forms the embryonic shells of O. miranda and Z. ventriosa may be flexible enough to withstand squeezing through the apertural barriers during birth and not to crack. After parturition, the ‘soft shells’ regain their shape. So, the tight fit between the size of the shell canal and the width of the embryonic shell is mitigated by the unusual properties of the embryonic shell. It appeared that species capable of producing flexible-shelled embryos, successfully overcome the necessity of adapting apertural barriers to parturition and kept the small patency of the shell canal. This adaptation remained unnoticed for so long possibly because these unique shell properties disappear within a few hours after birth. According to the phylogeny reconstructed in our study and other available data [13, 15], O. miranda and Z. ventriosa represent separate lineages within the Phaedusinae, so flexible shelled embryos in these snails must have evolved convergently. In light of this finding, the identification of a viviparous reproductive mode in a particular species based solely on adult shell morphology, for example the width of clausilium, should finally be dismissed. Figure 7 visualizes the distribution of species with ’soft shelled‘ embryos within the subfamily (Fig. 7).
While our study on obstetrical selection gives a new perspective for malacology, the link between life history evolution and skeleton morphology has already been explored in mammals and other vertebrates [1, 6, 21, 28]. Studies have mainly focused on the sexually dimorphic pelvis in tetrapods, both viviparous and oviparous. According to the pelvic constraint model [4], the maximal egg size in reptile taxa is related to the size of their pelvic girdle aperture. Additionally, the small size of the anal gap in the turtle shell leads to a constraint when laying eggs [27]. In reptiles, in addition to female morphological characteristics, also differences in egg-shell properties (brittle-shelled vs. pliable-shelled eggs) should also be considered in relation to evolution of reproductive mode. Most squamates and some turtles produce soft-shelled eggs with a leathery outer covering [29, 30]. The recent discovery of soft-shelled eggs in Mesozoic dinosaurs and the reconstruction of the ancestral state of the eggshell composition that included all known fossil eggshell types, has revealed that the first dinosaur egg was soft-shelled, while the calcified eggshell evolved later [31]. Soft-shelled reptile eggs are sensitive to desiccation and physical deformation, but are not prone to getting stuck in a narrow pelvic aperture. In contrast, brittle-shelled eggs cannot undergo deformation to pass through the pelvic aperture. However, the possible constraints resulting from the production of rigid eggs, are mitigated because females might use pelvic kinesis to pass them at oviposition [6, 27]. This adaptation closely resembles the increased flexibility of pelvic joints before labour in women and the accompanying malleable fontanelles of newly born human babies [2]. Flexible, ’soft-shelled‘ embryos in viviparous snails, which we observed in clausiliids, are a similar response to obstetric selection. The spectrum of shell calcification in embryos has yet to be revealed by a more widespread investigation in other viviparous gastropod taxa.
The repeated evolution of viviparity in clausiliids, which clearly required overcoming the mechanical constraints, implies that this reproductive mode was advantageous under different ecological conditions, even if adaptation to live bearing weakens the protective function of apertural barriers. Retention of developing embryos in the genital tract protects them from many environmental stressors and after birth, juveniles can actively avoid dry or submerged places and seek food immediately. In contrast, egg clutches, if not hidden in protective microhabitats (in soil, under bark), are vulnerable to drowning or desiccation. The proximate factors driving the switch in reproductive mode is still unknown, but it can be speculated that for small and middle-sized, iteroparous gastropods such as clausiliids, the increase in reproductive success through higher fecundity has not evolved; in contrast, they should invest more resources in survival of a single progeny. This may be achieved by producing larger eggs, better provided with nutrients in oviparous species or by retaining developing embryos in the genital tract.
The viviparity of the studied taxa was positively verified by X-ray screening of a large material reported in the previous study [16] or by direct observation in laboratory. The species (except E. cylindrella) were cultured and bred for several years at the Department of Invertebrate Zoology and Hydrobiology of the University of Łódź. For their original localities and shell sizes, see Tables 1–2; adult shells are presented in Figs. 1A, D, 2 A, E, I, 4 A, E. The studied snails never laid viable eggs with early stages of embryo development; instead they delivered neonates which began moving and feeding immediately after birth (life-bearing reproduction). The newborn snails were preserved in ethanol for shell size and shell thickness measurements.
| Species name | Collection data: country, locality name, coordinates, collector(s) | In laboratory culture since: |
|---|---|---|
| Tauphaedusa sheridani (L. Pfeiffer, 1865) | Taiwan Keelung City, Dawulun 25.159, 121.709 Shu Ping Wu | November 2015 |
| Reinia variegata (A. Adams, 1868) | Japan Ogago, Hachijo, Tokyo 33.1019, 139.776 Takumi Saito | June 2016 |
| Reinia ashizuriensis Azuma, 1968 | Japan Kochi, Tosashimizu City, Cape Ashizuri 32.724, 133.013 Yuki Miki, Osamu Miura, Daishi Yamazaki | June 2016 |
| Euphaedusa steetzneri (Pilsbry, 1919) | China Sichuan, Wenchuan County, 31.4085, 103.532 T. Hirano, B. Ye | September 2018 |
| Oospira miranda (Loosjes & Loosjes-van Bemmel, 1973) | Vietnam Ninh Binh, Cuc Phuong National Park 20.2949, 105.67 Anna Drozd, Thuy Dieu Dinh | May 2019 |
| Stereophaedusa addisoni (Pilsbry, 1901) | Japan Kagoshima, Kagoshima Shrine, Hayato, Kirishima 31.7534, 130.738 Rei Ueshima, Takahiro Asami, Anna Drozd | September 2019 |
| Zaptyx ventriosa (Schmacker & Boettger, 1891) | Taiwan Pingtung County, Manchou Townshio, Gankou Village 21.9926, 120.836 Chung-Chi Hwang | November 2019 |
The randomly selected neonates / hatchlings were photographed using a Leica M205C stereomicroscope, with a Leica DFC 295 camera. The height and width of the shell (SH, SW) were measured using the Leica Application Suite V4.5 microscope software.
Several individuals of each species were examined anatomically and shelled embryos found in the genital tract were carefully extracted for measurements. For dissection, the niku-nuki method was used [32], allowing for the extraction of soft body parts of the specimen poured beforehand with boiling water, without breaking the shell.
To examine shell structure and thickness, we used microcomputed Tomography (microCT) and scanning electron microscopy (SEM).
To preserve embryos and neonates for scanning, we fixed specimens overnight in 2.5% glutaraldehyde solution and were then transferred to 30%, 50% and 70% ethanol [33]. Finally, they were embedded in eppendorf tubes filled with 0.5% low melting temperature agarose. The agarose gel immobilizes the samples for microCT scans and prevents them from drying out.
MicroCT data were collected using the Zeiss XRadia MicroXCT-200 system, equipped with a 90 kV/8W tungsten X-ray source, in the Laboratory of Microtomography, Institute of Paleobiology, Polish Academy of Sciences, Warsaw, Poland. Radial projections were reconstructed using XMReconstructor software. The virtual sections and 3D visualizations were prepared with the XM3DViewer and Avizo Fire software. The shell thickness measurements of microCT sections were carried out using XM3DViewer. To compare the shell density of the studied species, we used the same scanning and reconstruction parameters for all specimens. For our purpose, we did not need absolute density values, but only comparability between the scans, so we use the term ‘relative density’ [34]. The detailed parameters of the scans are given in the supplementary online materials (SOM).
Scanning electron microscope (SEM) studies were conducted at the Institute of Paleobiology, PAS, Warsaw, Poland, using Quattro S (Thermo Fisher Scientific) equipment. The samples were investigated in high vacuum or in low vacuum (50 Pa) mode, in the 2–5 keV accelerating voltage range. Samples investigated in high vacuum mode were carbon coated prior investigations. The 10 nm carbon layer was deposited using a CCU-010 high vacuum sputter (Safematic). The samples were fixed directly onto the stubs using silver cement. Fixing was followed by freeze-drying (c.a. -20°C) using silica gel as a desiccant.
For the evolutionary framework of this study, the single gene phylogeny was generated covering Phaedusinae species kept in our laboratory. We used a subset of the 28S marker dataset available in BOLD (dx.doi.org/https://doi.org//10.5883/DS-PHAEDUSA), supplemented with sequences of five new individuals representing three species (S. addisoni, E. steetzneri, and Z. ventriosa), which were isolated and amplificated following the same procedure [13]. Newly obtained sequences are deposited in GenBank under accession numbers: XXXXXX-XXXXXX/
The phylogeny of the 28S marker was reconstructed using the maximum likelihood (ML) approach in RAxML 8.2.12 [35]. The substitution model was set to GTRGAMMAX, following the specification of Bayesian information criterion obtained from ModelTest-NG v0.1.7 [36]. Supports were obtained using a thorough bootstrap with autoMR setting.
Acknowledgments: The research was founded by Polish National Science Centre (NCN project no. 2016/21/B/NZ8/03086). We are grateful to colleagues who provided living snails for this project and gave access to laboratories. The authors wish to thank Dr Paweł Bącal for help with FESEM analysis and preparation of samples and to Dr Tomasz Mamos for molecular analysis. We also thank Dr Stephen Venn for the linguistic correction of the manuscript.
Author contributions: ASD conceived the study and provided data on reproductive strategies of snails; TKM preserved material for scanning; KJ conducted the scanning and measured shell thickness. AS and KJ wrote the paper and prepared figures.
Data availability statement: The data is available upon a reasoned request to the corresponding author.
Consent for publication: Not applicable.
Additional Information: The authors declare that they have no competing interests.
- Rosenberg, K.R. The Evolution of Modern Human Childbirth. Yearb. Phys. Anthropol. 35, 89–124 (1992).
- Haeusler, M. et al. The obstetrical dilemma hypothesis: there’s life in the old dog yet. Biol. Rev. 96, 2031–2057 (2021).
- Wells, J.C.K. Between Scylla and Charybdis: renegotiating resolution of the ‘obstetric dilemma’ in response to ecological change. Philos. Trans. R. Soc. B 370, 20140067. 10.1098/rstb.2014.0067 (2015).
- Congdon, J.D. & Gibbons, J.W. Morphological constraint on egg size: A challenge to optimal egg size theory? Proc. Natl. Acad. Sci. U.S.A. 84, 4145–4147 (1987).
- Sinervo, B. & Licht, P. Proximate Constraints on the Evolution of Egg Size, Number, and Total Clutch Mass in Lizards, Science 252, 1300–1302 (1991).
- Cordero, G.A. Is the Pelvis Sexually Dimorphic in Turtles? Anat. Rec. 301, 1382–1389 (2018).
- Sulikowska-Drozd, A., Walczak, M. & Binkowski, M. Evolution of shell apertural barriers in viviparous land snails (Gastropoda: Pulmonata: Clausiliidae). Can. J. Zool. 92, 205–213 (2014).
- Tompa, A.S. Studies on the reproductive biology of gastropods: Part 1. The systematic distribution of egg retention in the subclass Pulmonata (Gastropoda). J. Malacol. Soc. Aust. 4, 113–120 (1979).
- Baur, B. Parental care in terrestrial gastropods. Experientia 50, 5–14 (1994).11 Maltz, T.K. & Sulikowska-Drozd, A. Life cycles of clausiliids of Poland – knowns and unknowns. Ann. Zool. 58, 857–880 (2008).
- Nordsieck, H. Worldwide door snails (ConchBooks, 2007).
- Pokryszko, B.M. Land snail apertural barriers – adaptation or hindrance? (Gastropoda: Pulmonata). Malakol. Abh. Staatl. Mus. Tierk. Dresd. 18, 239–248 (1997).
- Páll-Gergely, B., Hettyey, A., Turóci, Á. & Fehér, Z. Evidence that reduction of the apertural closing apparatus has no decisive effect on water loss in the land snail family Clausiliidae (Gastropoda: Stylommatophora). Biol. J. Linn. Soc. 136, 120–126 (2022).
- Mamos, T., Uit de Weerd, D., von Oheimb, P.V. & Sulikowska-Drozd, A. Evolution of reproductive strategies in the species-rich land snail subfamily Phaedusinae (Stylommatophora: Clausiliidae). Mol. Phylogenet. Evol. 158, 107060;10.1016/j.ympev.2020.107060 (2021).
- Loosjes, F.E. Monograph of the Indo-Australian Clausiliidae (Gastropoda, Pulmonata, Clausiliidae, Phaedusinae). Beaufortia 31, 1-224 (1953).
- Motochin, R., Wang, M. & Ueshima, R. Molecular phylogeny, frequent parallel evolution and new system of Japanese clausiliids land snails (Gastropoda: Stylommatophora). Zool. J. Linn. Soc. 181, 795–845 (2017).
- Sulikowska-Drozd, A., Duda, P. & Janiszewska, K. Micro-CT screening of old shell collections helps to understand the distribution of viviparity in the highly diversified clausiliid clade of land snails. Sci. Rep. 10, 60; 10.1038/s41598-019-56674-7 (2020
- Nordsieck, H. Revision of the system of the Phaedusinae from Mainland China with the description of new taxa (Gastropoda: Stylommatophora: Clausiliidae). Arch. Molluskenkd. 129, 25–63 (2001).
- Dauphin, Y. & Dennis, A. Structure and composition of the aragonitic crossed lamellar layers in six species of Bivalvia and Gastropoda. Comp. Biochem. Physiol., Part A Mol. Integr. Physiol. 126: 367–377 (2000).
- Salinas, Ch. et al. Enhanced toughening of the crossed lamellar structure revealed by nanoindentation. J. Mech. Behav. Biomed. Mater. 76, 58–68 (2017).
- Gittenberger, E. Adaptations of the aperture in terrestrial gastropod-pulmonate shells. Neth. J. Zool. 46, 191–205 (1996).
- Suvorov, A.N. Morpho-functional analysis of the closing apparatus in two clausiliid species (Gastropoda, Pulmonata). Zool. Zhurnal 70, 21–31 (1991).
- Tompa, A.S. A comparative study of the ultrastructure and mineralogy of calcified land snail eggs (Pulmonata: Stylommatophora). J. Morphol. 150, 861–888 (1976).
- Minato, H. Genital Studies of the Japanese Land Snails – XXI. Clausiliidae (7): The Genus Pictophaedusa. Venus 42, 331–343 (1983).
- Ji, H., Li, X. & Chen, D. Cymbiola nobilis shell: Toughening mechanisms in a crossed-lamellar structure. Sci. Rep. 7, 40043; 10.1038/srep40043 (2017).
- Deng, Z., Jia, Z. & Li, L. Biomineralized Materials as Model Systems for Structural Composites: Intracrystalline Structural Features and Their Strengthening and Toughening Mechanisms. Adv. Sci. 9, 2103524; 10.1002/advs.202103524 (2022).
- Oufiero, C.E. & Gartner, G.E.A. The effect of parity on morphological evolution among phrynosomatid lizards. J. Evol. Biol. 27, 2559–2567 (2014).
- Hofmeyr, M.D., Henen, B.T. & Loehr, V.J.T. Overcoming environmental and morphological constraints: egg size and pelvic kinesis in the smallest tortoise, Homopus signatus. Can. J. Zool. 83, 1343–1352 (2005).
- Trevathan, W. Primate pelvic anatomy and implications for birth. Philos. Trans. R. Soc. B 370, 20140065; 10.1098/rstb.2014.0065 (2015).
- Shine, R. Reptilian Reproductive Modes: The Oviparity-Viviparity Continuum. Herpetologica 39, 1–8 (1983).
- Deeming, D.C. Nesting environment may drive variation in eggshell structure and egg characteristics in the Testudinata. J. Exp. Zool. 329, 331–342 (2018).
- Norell, M.A. et al. The first dinosaur egg was soft. Nature 583, 406–410 (2020).
- Fukuda, H., Haga, T. & Tatara, Y. Niku-nuki: a useful method for anatomical and DNA studies on shell-bearing molluscs. Zoosymposia 1, 15–38 (2008).
- Metscher, B.D. X-Ray Microtomographic Imaging of Intact Vertebrate Embryos. Cold Spring Harb. Protoc.10.1101/pdb.prot067033 (2011).
- Chatzinikolaou, E., Keklikoglou, K. & Grigoriou, P.. Morphological Properties of Gastropod Shells in a Warmer and More Acidic Future Ocean Using 3D Micro-Computed Tomography. Front. Mar. Sci. 8, 645660;10.3389/fmars.2021.645660 (2021).
- Stamatakis, A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30, 1312–1313 (2014).
- Darriba, D. et al., ModelTest-NG: A New and Scalable Tool for the Selection of DNA and Protein Evolutionary Models. Mol. Biol. Evol. 37, 291–294 (2019).
No competing interests reported.