Flexible embryonic shell allies large offspring size and anti-predatory protection in viviparous snails

The studied viviparous clausiliids developed four types of morphological adaptations that facilitate the delivery of embryos through the shell aperture: (1) reduction of the clausiliar apparatus, (2) decrease of embryonic shell width, (3) widening of the shell canal, and (4) development of a flexible embryonic shell.

Reduction of the clausiliar apparatus

Members of the Reinia genus, arboreal species from Japan (Fig. 1), show the most advanced adaptations to live-bearing compared to hypothetical ancestral Phaedusinae. The shell shape in these species is more conical than fusiform, the number of whorls decreases, and the aperture widens. One of the species, R. variegata, features almost full reduction of the clausiliar apparatus that consists of only vestigial folds (Fig. 1F). This species also lacks the clausilium, so the entrance through the aperture is unprotected.

Decrease of embryonic shell width

Another adaptation concerns the shape of the embryonic shell (“protoconch”), which becomes very narrow in some viviparous species. This feature is conspicuous because embryonic whorls remain in the adult shell as apical whorls. For instance in S. addisoni (Fig. 2A–D), the apical part being much narrower than the first whorls of the teleoconch is a clear evidence that the growth trajectory has changed abruptly after birth. Other examples include E. cylindrella and E. steetzneri, in which both the protoconch and the teleoconch are very narrow, yet at the borderline between these parts, the shell axis is slightly bent (Fig. 2E–L). We suppose that this feature develops as a result of obstruction during birth.

Widening of the shell canal

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. A substantial difference to egg-laying species concerns the apertural barriers: the clausiliar includes a broad clausilium plate and a spirally ascending inferior lamella (Fig. 3A–D). These modifications result in a spacious shell canal in the body whorl, for example in S. addisoni and E. sheridani, that can accommodate the transfer of a large embryo. Table 1 presents neonatal size in these species (shell width ca. 1.2 mm), which is very similar to their clausilium width (ca. 1.1–1.2 mm).

Most viviparid clausiliids develop one of these three types of modification; some adaptations co-occur within a single species, for example a wide clausilium accompanies a narrow apex. Interestingly, the Reinia genus includes taxa with a gradual escalation of viviparity-related adaptations: R. ashizurensis, with a stout shell shape and a low number of whorls, has fully developed apertural barriers with a broad clausilium plate (Fig. 1A–C), while its congener, R. variegata, has reduced apertural barriers (Fig. 1D–F).

Development of a flexible embryonic shell

The fourth type of adaptation found in Phaedusinae concerns the structure of the embryonic shells. We report this adaptation in O. miranda and Z. ventriosa.

Oospira miranda is a dextral, often decollated, ground-dwelling species from Vietnam (Fig. 4A). The species is viviparous: during microCT scanning of museum specimens, we found embryos within a parental shell (Fig. 4B); in laboratory culture, we observed neonates 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 (mean values: 5.19 mm, 3.59 mm) evidently exceeds the width of the clausilium plate in this species (1.97 mm) (Table 1). However, under closer examination, we found the shell to be thin and delicate, which we refer 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 becomes distorted already under slight pressure.

A similar adaptation exists in Z. ventriosa, a Taiwanese species with a very wide apex, never decollated, a straight ascending inferior lamella, and a narrow clausilium plate (Figs. 3E,F, 4E,F). This species produces neonates in laboratory culture (Fig. 4G–H). The dimensions of the neonates (mean values: height 3.37 mm, width 2.51 mm) exceed at last twofold the width of the clausilium plate (1.08 mm). The shells 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, that we made during the maintenance of the laboratory culture, suggested that 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 period and delivery through apertural barriers, hardening shortly after birth. We further investigated the physical properties of the embryonic shell 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). The 3D visualization of O. miranda and S. addisoni (the same microCT scanning and reconstruction parameters) 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 second 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 3.5 mm in height), the size of the voxel was approximately 1 µm. Thus, we cannot determine the shell thickness down to the nearest micron, but we can estimate it from a few to a dozen microns. A direct comparison between virtual microCT sections of specimens scanned under the same conditions shows a clear difference between the ‘soft-shelled’ and ‘hard-shelled’ taxa (Fig. 5G–L). The ’hard-shelled’ neonates have a shell wall of 30–40 µm thick. We examined the sequence of three ’soft-shelled’ O. miranda specimens that differed in size (the exact time of birth of each of the cultured neonates is unknown, ca. 1–2 days). The larger (older) the neonate was, the thicker the shell. The shell of the largest of the studied O. miranda was up to 20 µm 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, stiff and rigid during the retention in the genital tract. The neonates of O. miranda and Z. ventriosa were much larger than the embryos and neonates of S. addisoni and R. variegata (Table 1), however, the former taxa has much thinner shells.

Scanning electron microscopy

After the non-invasive microCT scan, we scanned embryos and neonates 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, we freeze-dried the next group of thin-shelled neonates 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, we recognized the aragonite shells composed of lamellae with alternate orientations (i.e. crossed lamellar microstructure), which is typical for adult gastropods. The crossed lamellar part of the gastropod shell usually includes a few macrolayers of different orientation of lamellae²². The number of macrolayers in the ‘soft’ and ‘hard’ embryonic shells varies and possibly influences the thickness of the shell [compare to the thin wall of O. miranda (Fig. 6L) and the number of alternating macrolayers in R. variegata (Fig. 6Q)].

Phylogeny reconstruction

IQ-TREE phylogeny reconstruction clearly shows that the viviparous strategy occurs independently many times within Phaedusinae (Fig. 7). The development of ‘soft-shelled’ neonates in O. miranda and Z. ventriosa cannot be explained by descent of these species from a common ancestor. Similarly, the widening of the shell canal and the decrease in embryonic shell width evolved repeatedly within the studied group. Almost complete reduction of the clausiliar occurred only in R. variegata.

Source: Ecology - nature.com