Microstructural Details in Shells of the Gastropod Genera Carychiella and Carychium of the Middle Miocene

Microstructural details in shells of the gastropod genera Carychiella and Carychium of the Middle Miocene

ADRIENNE JOCHUM, THOMAS A. NEUBAUER AND MATHIAS HARZHAUSER

Jochum, A., Neubauer, T. A. & Harzhauser, M. 2015: Microstructural details in shells of the gastropod genera Carychiella and Carychium of the Middle Miocene. Lethaia, DOI: 10.1111/let.12134.

Microstructural details are revealed via scanning electron microscopy (SEM) in two carychiid species from the early Middle Miocene of Styria, SE Austria. The proto- conchs of the shells of Carychiella eumicrum (Bourguignat 1857) and Carychium gibbum (Sandberger 1875) show different types of microstructure on the embryonic shell during ontogeny. Total, superficial punctate structure on the shell of Carychi- ella eumicrum contrasts with the protoconch–teleoconch demarcation (p/t boundary) observed on the protoconch of Carychium gibbum. Both species exhibit aragonitic microstructure. Diagenetic effects, prismatic, homogeneous and crossed lamellar microstructures are detectible in both species. Rheomorphic folding and dense pitting within the columella of Carychiella eumicrum suggest a structure– function relationship for tensile strength and bulk weight reduction in carychiid snails. We hypothesize that total superficial pitting on the shell of C. eumicum, seen here for the first time in the Carychiidae, suggests paedomorphosis as a life-history strategy to palaeoecological conditions of the Rein Basin during the early Middle Miocene. □ Carychiella, Carychium, middle Miocene, mollusc assemblage, Rein Basin (Styria Austria), shell structure.

Adrienne Jochum [[email protected]], Naturhistorisches Museum der Burgergemeinde Bern, Bernastr. 15, Bern CH-3005, Switzerland Institute of Ecology and Evolution, University of Bern, Baltzerstrasse 6, Bern CH-3012, Switzerland; Thomas A. Neubauer [[email protected]], and Mathias Harzhauser [[email protected]], Department of Geology and Paleontology, Natu- ral History Museum Vienna, Burgring 7, Vienna A-1010, Austria; manuscript received on 29/04/2014; manuscript accepted on 11/03/2015.

The Carychiidae represent a fascinating taxon of die- potential new characters for phylogenetic analysis in hard, terrestrial gastropods. Although it is assumed congruence with recent molecular analyses of extant they have a sketchy 330- to 300-million-year-old fos- species (Weigand & Jochum 2010; Weigand et al. sil record (Tracy et al. 1993; Bandel 1994), they are 2011, 2012a,b, 2013) and fossil Carychiidae. well represented in the rich mollusc assemblages of Carychium Muller€ 1773, frequently dominates the Neogene of Europe (Boettger 1870; Wenz 1923; mollusc assemblages and often characterizes com- Strauch 1977; Meijer 1986; Prisyazhnyuk & Stworze- munities, such as the ‘Carychium beds’ of the Vienna wicz 1995; Stworzewicz 1999; Harzhauser & Kowalke Basin (Jirıcek & Senes 1974; Bernor et al. 2004). 2002; Bernor et al. 2004; Binder 2004; Harzhauser & Carychiidae are considered facies indicators for Binder 2004; Harzhauser & Piller 2004; Frank 2006; moist lakeshore habitats during the warm temperate Salvador 2015; Harzhauser et al. 2014a). As the to marginally tropical early Middle Miocene Bade- Carychiidae are amongst the earliest gastropod nian stage (Lueger 1981; Harzhauser & Kowalke groups to have completed the transition from a mar- 2002). Extant Carychium inhabit shady, moist, inter- ine/estuarine habitat to a terrestrial mode of life, stitial layers of leaf, grass and wood litter of mesic they provide a model for studying the adaptations environments in riparian zones, damp meadows, and ecology characterizing the radiation of terrestrial mountain forests and tropical woodlands. The Cary- pulmonates (Barker 2001). The purpose of this study chiidae show a Holarctic distribution (Pilsbry 1948; was to examine and report discernible microstruc- Morton 1955) and today comprise two genera: Cary- tures found in carychiid fossils of this well-docu- chium (epigean) and Zospeum (troglobitic). mented mollusc assemblage of the early Middle Understanding the Carychiidae has as much to do Miocene. In this study using SEM, we describe shell with carychiid evolutionary history, ontogeny, bio- microstructures preserved in the two known cary- chemistry and palaeoecology as with their reciprocal chiid species of the Rein Basin, Styria, SE Austria inﬂuences in the constructional morphology of the (Harzhauser et al. 2014a). This investigation is part remarkably thin and translucent carychiid shell. of an ongoing search for ecological information and Luchtel et al. (1997) emphasized that gastropod shell

DOI 10.1111/let.12134 © 2015 Lethaia Foundation. Published by John Wiley & Sons Ltd 2 A. Jochum et al. LETHAIA 10.1111/let.12134 structure varies from species to species, and within comprise a microstructure identifiable and charac- each species, distinctive details are specifically con- teristic for specific taxa at the genus to family levels figured for general shape, pigment, mineral organi- (Hedegaard & Wenk 1998; Fuchigami & Sasaki zation and other features. Kano et al. (2008) 2005; Medakovic & Popovic 2012). Although knowl- observed that the smaller the snail’s body size, the edge of the structure and composition of the organic more simplified anatomical structures become and components constituting the biominerals within the that a more specialized ecology is required to sustain crystalline composites has grown considerably, that it. Within the Ellobioidea, the degree of inner shell of their fossilization process is still sparse (Guzman resorption varies within the different sub-families, et al. 2009). Moreover, the specificity of structural whereby shell sculpture is considered most informa- organization reflects structure–function relation- tive at lower taxonomic levels and varies between ships, which have recently become increasingly use- genera (Martins 2007). To comprehend the Cary- ful in designing models for material sciences chiidae and their role as baseline taxa in molluscan (Ehrlich et al. 2011). assemblages, it is essential to recognize and compare Our knowledge of mineralogy in the Carychiidae the peculiarities of shell structure in both extant and is limited to three studies (Medakovic et al. 1999; fossil taxa. Although advanced assessment tech- Slapnik & Medakovic 2007; Medakovic & Popovic niques such as X-ray diffraction (XRD), scanning 2012), whereby X-ray powder diffraction (XRD) was electron microscopy (SEM), nanocomputer tomog- used on Zospeum shells to investigate mineral com- raphy (NanoCT), transmission electron microscopy position and phase fractions. A high concentration (TEM) and atomic force microscopy (AFM) have of aragonite and a small fraction of calcite in the given many authors insight into the complex struc- shells were detected. As calcite is a better insulator ture of calcareous mollusc shells (Berman et al. than aragonite, these authors attributed the calcite 1993; Hedegaard & Wenk 1998; Chateigner et al. fraction to adaptation to the fluctuating tempera- 2000; Hess et al. 2008; De Paula & Silveira 2009; tures in certain cave habitats. As very little is known Fryda et al. 2009; Furuhashi et al. 2009; Guzman about ellobioid, let alone extant or fossil carychiid et al. 2009; Vendrasco et al. 2010; Rodrıguez-Navar- microstructure, our investigation presents new ro et al. 2012; Hickman 2013), very few studies (Jo- information about the microstructure of the Cary- chum 2011; Medakovic & Popovic 2012; Jochum chiidae. et al. 2013) have been conducted at the microstructural level on terrestrial ellobioid taxa. It is well Carychiid taxonomy and systematics known that environmental influences play a pivotal role in shell mineralization and thus, influence shell As fossil and extant carychiid gastropods are known form, development and repair (McMahon & to be highly morphologically variable (Watson & Whitehead 1987; De Paula & Silveira 2009; Medak- Verdcourt 1953; Strauch 1977; Burch & Van Dev- ovic & Popovic 2012; Hickman 2013). As the Cary- ender 1980; Stworzewicz 1999; Weigand & Jochum chiidae inhabit semi-subterranean, subterranean and 2010; Weigand et al. 2012a,b; Weigand et al. 2013), extreme subterranean habitats, some existing 980 m they have been subject to frequent taxonomic re- below the surface (Weigand et al. 2013), their shells evaluation. Consequently, the plethora of forms has harbour a potential reservoir of valuable informa- systematically been shuffled and reshuffled into gen- tion. era and sub-genera, based on the degree of character Mollusc shells are polycrystalline complexes of expression in shell size, shape, form, dentition, whorl calcium carbonate, diverse proteins rich in acidic number, whorl height and, most specifically, the amino acids and glycoproteins (Lowenstam & Wei- degree of sinuosity of the columellar lamella ner 1989; Berman et al. 1993; Hedegaard & Wenk (Strauch 1977; Lueger 1981; Prisyazhnyuk & Stwor- 1998). Gastropod and bivalve shells demonstrate a zewicz 1995; Salvador 2015). For the herein treated comparable range of shell mineralogy and micro- carychiids, we follow the latest taxonomic and sys- structure (Carter et al. 1998), whereby gastropod tematic revisions of Miocene freshwater deposits by shells comprise an outer organic periostracal sheet Harzhauser et al. (2014a,b). Although recent molec- and an inner carbonate layer (Saleuddin & Petit ular analyses focusing on phylogenetic and phylo- 1983; Slapnik & Medakovic 2007). Their rigidity and geographical studies of extant Carychiidae have durability (texture) are governed by hierarchically answered some profound questions regarding cary- organized layered carbonates (Hickman 2013) in the chiid phylogeny (Weigand & Jochum 2010; Weigand form of calcite, aragonite and vaterite (rarest form) et al. 2011, 2012a,b, 2013), shell microstructural such that the fractions of minerals, grain morphol- investigations of extant and fossil material are still ogy and aggregation and crystal orientation requisite to support these studies in an integrative, LETHAIA 10.1111/let.12134 Middle Miocene shell microstructures 3 contemporary taxonomic approach (Jochum 2011; Jochum et al. 2013). Material and methods

Faunal composition and palaeoecological The shell dimensions of Carychiella eumicrum range setting from a height of 0.95 to 1.1 mm and a width of 0.45 to 0.5 mm. The larger Carychium gibbum Recently, Harzhauser et al. (2014a) performed a shows a height ranging from 1.40 to 1.45 mm and comprehensive survey on all mollusc fauna of the a width of 0.75 to 0.85 mm (Harzhauser et al. Lake Rein (Fig. 1), including an up-to-date system- 2014a). atic concept, critical synonymy lists, revisions of Two microscopically well-preserved specimens of many taxa and descriptions of several new species. Carychiella eumicrum (Bourguignat 1857) and six Along with the two species of Carychiidae comprising specimens with the exposed columella of Carychium this present study, this work revealed a moderately gibbum (Sandberger 1875) were selected and diverse assemblage including 47 gastropod species mounted on 13-mm aluminium pin stubs and (12 freshwater, 35 terrestrial) and one bivalve. coated with gold–palladium in a sputter coater. Shell During the early Middle Miocene, the basin har- microstructures were examined on the shell surfaces, boured a geologically short-lived wetland with interfaces and fractured sections of the shells using ephemeral ponds, altogether referred to as Lake the JEOL JSM-6610LV (Germany) scanning electron Rein. More precise information on the palaeoenvi- microscope (SEM) at the Natural History Museum ronment was recently inferred from the mollusc Vienna (NHMW, Austria). In this study, we describe fauna (Harzhauser et al. 2014a). These authors the preserved microstructures according to the emphasize that the freshwater molluscs comprise a nomenclature established by Gregoire (1972), Carter large number of pulmonate gastropods, indicating a & Clark (1985) and Carter (1990). These descrip- shallow and stagnant water body with densely vege- tions were recently modiﬁed by Genio et al. (2012) tated banks and limited riverine inﬂux. Moist wood- (Table 1). land habitats fringing Lake Rein are indicated by We interpret carychiid shell microstructures and most of the terrestrial taxa. The herein discussed discernible textures using SEM images collated from carychiids, Carychiella eumicrum (Bourguignat the vast pool of literature spanning geological time- 1857) and Carychium gibbum (Sandberger 1875), scales. We use the following abbreviations to desig- most likely settled the permanently moist to inun- nate structures: Hom, homogeneous structure; Lam, dated habitats near the lakeshore. laminar (tablet) structure; Le, lamellar structure; Li,

B D

Fig. 1. A, political units of Austria. B, province of Styria with indication of studied region (grey area corresponds to the extension of the Alps). C, distribution of Miocene freshwater deposits in the marginal Styrian Basin and nearby intramontane basins. D, geological units of the Rein Basin with indication of localities mentioned in the text (main coal-mining area at the Tallak hill shaded). Maps modiﬁed after Ebner & Gr€af (1979), Ebner (1983), Weber & Weiss (1983), Flugel€ & Neubauer (1984), Sachsenhofer et al. (2003) and Harzhauser et al. (2012, 2014a,b). 4 A. Jochum et al. LETHAIA 10.1111/let.12134

Table 1. Modiﬁed deﬁnitions by Genio et al. (2012) of shell microstructure from Carter (1990).

Microstructure Description

Simple prismatic (SP) First-order prisms that show low-to-moderate length–width ratios: individual first-order prisms lack fan-like arrangements of their second-order structural subunits; the boundaries between adjacent simple prisms are generally well defined Nacreous Polygonal to round aragonitic tablets arranged in broadly continuous, regular, mutually parallel laminae, by definition, this structure is always aragonitic Homogeneous (Hom) More or less equidimensional, irregularly shaped crystallites or crystal morphotypes lacking clear first-order structural arrangement except for possible accretion banding Crossed lamellar (CL) First-order lamellae of thin mutually parallel laths or rods, with two non-horizontal dip directions of their elongate sub-units in adjacent lamellae. The structure is co-marginal, if the first-order lamellae are parallel to the shell margin, radial if perpendicular Complex crossed lamellar (CCL) Crossed structure with three or more non-vertical dip directions of other elongate structural units. It differs from crossed lamellar in that the largest sub-units are not concentrically elongate first-order lamellae but are much less regular and more variable in form Intersected cross-platy A crossed structure with only two predominant dip directions in which the first-order lamellae consist of short, rhomboidal platy crystallites laminated structure; Gr, granular structure; and SP, densely punctate. A pattern of regular pits (Fig. 3C) simple prismatic structure. is located on the planar surface emanating from the The studied material is housed at the Natural His- interface of the outer, columellar shell layer. tory Museum Vienna, Austria, under the collection By close inspection of the pitted rheomorphic number NHMW 2012/0154. folds, still smaller equidistantly arranged, distinct pinprick-like pores symmetrically pock a series of non-joining segments of a lightly discernible track- Results like pattern of horizontal meshwork. These non- fibrillar (at least with SEM) ‘caterpillar feet-like’ Descriptions of shell microstructures tracks are co-marginally concentrated on the mid- outermost section of the shell fracture. They demon- Carychiella eumicrum (Bourguignat 1857) (Figs 2A– strate an irregular pattern. Although no reference to F, 3A–C, 5A, B): the outer layer of the shell shows these tracks in other molluscs is found in the litera- homogeneous structure (Fig. 2F) consisting of gran- ture, they do not resemble growth lines or micro- ular and shoal-like formations. Shallow semi-ellipti- structural details described by previous authors. We cal pits are regularly distributed over the entire shell speculate this pattern is due to a Middle Miocene surface. These pits show a generally granular, homo- bacterium, fungus, parasite or commensal inter- geneous structure embedded by larger, aragonitic, loper. From a constructional perspective, the irregu- rhomboidal laminate (RLi) tablets described by Car- lar positioning of these peculiar microstructures ter (1990, p. 611) as ‘rods, laths, blades or tablets’ suggests no function–structure relationship. (Fig. 2E, F). Faint transverse rheomorphic striations The strongly diagenetic deteriorated outer layer of intersperse the cavities and follow the curvature of the ventral side of the columella is comprised of the shell. homogeneous structure, whereby a nacreous ‘arago- The region of dense calcium carbonate (Fig. 3A, nitic laminar structure consisting of polygonal to B), constituting the columella at the second abapical rounded tablets arranged in a regularly formed, par- columellar fold, demonstrates an inner and outer allel sheet’ (Carter & Clark 1985, p. 55) can be dis- layer. The outer layer (Fig. 3B) is composed of sim- cerned. Laminate laths and tablets loosely flank the ple prismatic laminar shell microstructure. At the perimeters of this structure. homogeneous, horizontal interface between the two Carychium gibbum (Sandberger 1875) (Fig. 4A): layers (Fig. 3B), individual crystals show prismatic The surface structure of C. gibbum reveals a different rhomboidal and tabular form. The curvature of the morphology than Carychiella eumicrum. The proto- inner layer shows strongly diagenetically altered conch and, in a few cases, the early teleoconch crossed lamellar structure abutting a region of diage- (Fig. 4B) are marked by the regular pattern of char- netically altered simple prismatic microstructure. acteristic carychiid punctation (Jochum 2011; Jo- The surrounding structure (Fig. 3B, right side) is chum et al. 2013). In contrast to Carychiella prismatic, but the exact underlying morphology is eumicrum, the second protoconch whorl bears the difficult to ascertain. clear line indicating the demarcation zone of larval The ventral surface (Fig. 3A) of the columella torsion and the reorganization of shell secretion encompassing the second abapical columellar fold is between the protoconch and teleoconch (the p/t LETHAIA 10.1111/let.12134 Middle Miocene shell microstructures 5

EF Hom Hom

RLi Hom Str

RLi

Fig. 2. SEM images of Carychiella eumicrum (Bourguignat 1857). A–F, shell with total superﬁcial punctate structure. A, ventral view of shell. B, punctate ultimate whorl. C, bulbous protoconch with punctate structure. D, regular pattern of semi-elliptical pits and diagenetic rubble. E, contextual view of shell and pit morphology showing RLi, rhomboidal- and blade-shaped nacreous laminate tablets; Hom, shoal of granular homogeneous structure initiating depth of pit mixed with diagenetic rubble; and Str, rheomorphic transverse striations. F, close-up view of RLi, rhomboidal- and blade-shaped nacreous laminate tablet, concave to the shoal of Hom, homogeneous shell structure; granular and diagenetic homogenous structure surrounds the pit. boundary) (Hickman 2013). This signiﬁcant feature gibbum. Revealed is a plane of tabular aragonite was observed only in one specimen of Carychiella e- prism comprised of vertical crystalline sub-units umicrum from this assemblage. constituting the simple prismatic laminar shell The edge of the fractured teleoconch (Fig. 4A, C– microstructure on the inner marginal side of the F) shows discernible microstructure of Carychium shell (Fig. 4C, E, F). Below this layer is a stratum of 6 A. Jochum et al. LETHAIA 10.1111/let.12134

A homogeneous structure marked by a bifurcated ribbon of potential accretion banding (Fig. 4C). As this banding oddly bifurcates and is oblique to the shell growth surfaces, it could possibly be organic material attached to the fossil shell surface. Slightly SP discernible, co-marginal crossed lamellar microstructure of thin, mutually aligned laths or lamellae are arranged parallel to the point of bifurcation of Hom the aforementioned banding. At the endpoint of the CL accretion banding ribbon is an isolated, superim- PStr posed ropey structure of mineral sheets built up of laths (Fig. 4C, D). These laths show a regularity of crystal thickness in an arrangement of successive sheets of obliquely aligned crossed lamellar structure surrounded by homogeneous structure and diage- B netic rubble (Fig. 4D). SP A second fractured shell of C. gibbum provided a clearer and different perspective of microstructures at approximately the same region along the edge of the shell (Fig. 4E–F). This specimen shows sheets of simple prismatic structure merging into microstructure described by Hedegaard (1997, fig. 6) as ‘first order lamellae perceived as irregular Lam aggregates of intergrown pieces of simple crossed CL lamellar structure’. Cliffs of simple prismatic structure border a zone of either crossed lamellar structure or an interface of continuation of the simple prismatic layer with variable diagenetic alteration below (Fig. 4E). The crossed lamellar structure C shows different orientation of the cleavage planes (Fig. 4E). Simple prismatic structure is indicated by a plane of tabular crystal aragonite (Fig. 4F). C. gibbum here presents Hedegaard’s (1997) aforementioned complex of crossed lamellar structure at the inner shell PpTr Lam margin. Additionally, transverse jagged sheets of cross-lamellar structure (Fig. 4F) superficially resemble those described by Chateigner et al. (ICP, fig. 7b, 2000) for intersected cross-platy structure. A Lam Hom star-like complex of acicular bodies is embedded in a mass of diagenetic rubble (Fig. 4F). It is not clear whether this formation is due to intrinsic prismatic arrangement, whether the bodies are themselves ar- Fig. 3. SEM images of columellar structure of Carychiella eumicrum (Bourguignat 1857). A, contextual view of interior of colu- tefacts or whether these structures comprise a con- mella showing SP, simple prismatic structure at the inner shell stellation of randomly assembled prisms amassed in margin; CL, laths of crossed lamellar structure eroded by diage- diagenetic rubble. netic alteration; Hom, homogeneous structure; PStr, punctate structure on ventral interface. B, close-up view of SP, diageneti- A regular pattern of wedge-shaped planes of ara- cally altered simple prismatic structure; Lam, nacreous aragonitic gonitic structure (Fig. 5A–D) is present in different laminar structure; CL, underside of fracture showing laths of densities on the columellar lamellae of both species. crossed lamellar structure eroded by diagenetic alteration; C, ventral interface of columellar fracture showing PpTr, fine pin- This structure has been consistently observed on the prick tracks on rheomorphic folds of planar surface of PStr, columellar lamellae in extant Carychiidae and likely punctate structure (tensile function?) on ventral interface; Lam, aids in traction during mobility (Jochum 2011; Jo- nacreous laminate laths and tablets intersected by homogeneous structure. chum et al. 2013). LETHAIA 10.1111/let.12134 Middle Miocene shell microstructures 7

pits P/t

edge

P/t

CL CL SP ACCB

DR DR Hom CL

SP ACC

CL SP CL

CL SP

Fig. 4. SEM images of Carychium gibbum (Sandberger 1870–1875). A, shell showing inspection zones. B, characteristic carychiid protoconch pitting and protoconch–teleoconch (p/t) boundary of larval reorganization C, shell fracture showing ACCB, accretion banding of homogeneous structure; SP, simple prismatic structure at the inner shell margin and CL, superimposed mineral sheets of crossed lamellar structure. D, shell fracture (rightmost section of C) in context showing regularity of thickness of CL laths; Hom, homogeneous structure and DR, diagenetic rubble. E, SP, simple prismatic structure at the inner shell margin; CL, crossed lamellar layer. F, tabular forms of Sp, simple prismatic structure; CL, transverse sheets of intergrown, crossed lamellar structure; ACC, acicular crystal arrangement in diagenetic rubble. European fossil biotopes inhabited concurrently by Discussion one to three Carychium or Saraphia species. Our investigation here agrees with this ﬁnding in that Carychiella eumicrum was found inhabiting the Protoconch morphology and structure same biotope with the larger Carychium gibbum. Strauch (1977) reported that one of ﬁve known spe- Moreover, although no direct phylogenetic relation- cies of the genus Carychiella is usually found in ship was determined within the few fossil forms 8 A. Jochum et al. LETHAIA 10.1111/let.12134

Wss Wss

Wss

Fig 5. SEM images of Wss, wedge-shaped aragonitic structure. A, B, columellar lamella of Carychiella eumicrum (Bourguignat 1857). C, D, columellar lamella of Carychium gibbum (Sandberger 1870–1875). representing Carychiella in his investigations, gibbum here (Harzhauser et al. 2014a,b) (Fig. 3A). Strauch (1977) hypothesized that Carychiella most Stworzewicz (1999) observed pitting on the proto- plausibly originated from a common ancestor stem- conch of Carychium rhenanum (Strauch 1977) ming from the Oligocene (33.9–23 Mya). although Strauch (1977) did not describe this feature Although comparable morphological analyses of in this species nor in any other in his extensive Carychiella eumicrum (Bourguignat 1857) (Fig. 2A) analysis of Miocene carychiids. Moreover, Stworze- are documented by Strauch (1977, pl. 14, fig. 13) wicz (1999) equally did not report similar finds on and Prisyazhnyuk & Stworzewicz (1995) from Opole the shells of Carychiella eumicrum material she (Poland), Stworzewicz (1999, fig. 5–6) from examined from Bełchatow, Opole or Tuchorice. On Bełchatow (Poland) and Tuchorice (Czech Repub- the other hand, and contrary to these observations, lic), and Salvador (2015, fig. 1) from Sandelzhausen, it is notable that Harzhauser et al. (2014b) recently Mainburg (Germany) (2013, fig. 1), the specimens found punctate microstructure on the protoconch examined here and presented by Harzhauser et al. and early teleoconch of shells of C. eumicrum from (2014a, 2014b), pl. 6, fig. 5) differ significantly in Tuchorice. that the entire shell is densely punctate beyond the The pronounced, globular protoconch (Fig. 2C) protoconch (Fig. 2B, C). This total pitting of the of Carychiella eumicrum (Harzhauser et al. 2014a,b; shell is a structural feature new to both fossil and pl. 6, figs 5, 6), although not uncommon to lesser recent Carychiidae. degrees for extant Carychiidae, is indicative of onto- Superficial semi-ellipsoidal pitting (Fig. 2D) has genetic development derived from a large egg con- been consistently observed in the protoconch of taining a large yolk supply (Lima & Lutz 1990). Doll extant Carychiidae (Jochum 2011; Jochum et al. (1982) and Bulman (1990) confirm that the egg cap- 2013) as well as in the protoconch of Carychium sule of extant Carychium tridentatum (Risso 1826) is LETHAIA 10.1111/let.12134 Middle Miocene shell microstructures 9

‘gigantic’ compared to the adult, encompassing of Mellopegma georginensis (Runnegar & Jell 1976) about one-fifth the maximal length of the adult shell from the Middle Cambrian of Australia (Vendrasco (Doll 1982). This observation holds equally well for et al. 2010; pl. 1, fig. 10). These tablets within the the relationship of protoconch to adult shell length pits of C. eumicrum are rhomboidal- and blade in Carychiella eumicrum here (Fig. 2A). Together, shaped (Fig. 2E, F), suggesting similarity with the these two structural features are striking in that they nacre tablet forms known in extant bivalves (Taylor fail to consistently demonstrate the usual proto- et al. 1969; fig. 10, Pinctada margaritifera; Wada conch–teleoconch boundary (the p/t boundary), 1972; fig. 8, Pinna attenuata) and from fossils indicating when hatching occurred during the het- described by Vendrasco et al. (2010, text-fig. 4E–J) erotectonic constructional processes of shell secre- dating from the Middle Cambrian. Additionally, the tion (Hickman 2013). This line is associated with moderately advancing homogeneous wave of struc- larval reorientation and the accompanying ontoge- ture initiating the depth of each pit (Fig. 2E) sug- netic shift in shell morphology during metamorpho- gests a merging of tabular structure as described by sis. The p/t boundary, however, is well marked on Vendrasco et al. (2010, fig. 4H–J). the shells of Carychium gibbum (Fig. 3A–B). A likely The microstructure comprising the outer surface similar constructional transition was reported on the of the columella (Fig. 3A, B) of Carychiella eumi- shells for Carychiella puisseguri (Truc 1972) of the crum consists of simple prismatic structure. The Early Pliocene from Celleneuve (Herault), France interior, crossed lamellar structures show the ‘plate- (Strauch 1977). Our observations suggest a partial like elongated aspect’ of lamellae described for Conus developmental and/or transitory stage for the genus omaria by Rodrıguez-Navarro et al. (2012, fig. 1E, Carychiella of the Lake Rein. F). The regular punctate microstructure observed at Shell microstructure the lower ventral face of the Carychiella eumicrum columella (3A, C), just below the second abapical Microstructure in molluscs is characterized by the columellar fold, is reminiscent of similar superficial morphology of crystal units and their mode of ori- pock-like structures found on shells of brachiopods entation and layering arrangement. The most com- and diverse molluscs. Waren & Gofas (1996, fig. 5a) mon extant shell microstructures (spherulitic reported external pitting in the monoplacophoran prismatic, crossed lamellar, nacre and foliated cal- Veleropilina reticulata. Vendrasco et al. (2010) cite) are known since the Middle Cambrian (Runne- described a pattern of regular pitting structure on gar 1985; Vendrasco et al. 2010). Carter (1979) the interior surface of the shell in Figurina specimens detected microstructural similarities in brachiopods (pl. 4, fig. 8) and in specimens of Corystos thorntoni- and mollusc shells, suggesting a similarity in biomin- ensis (Pl. 5, figs. 7–8) from the Middle Cambrian. eralization processes which may have derived from These authors attributed this punctate morphology an early common ancestor (Vendrasco et al. 2010). to either an accelerated rate of organic matrix degra- Five terms describe the different levels of crystal dation in relation to the dissolution of shell structure aggregation in molluscan shell microstructures: or to shell diagenesis. For Carychiella here, we homogeneous, prismatic, spherulitic (grained), hypothesize with Jochum (2011) that a function– crossed lamellar and complex crossed lamellar structure relationship, similar to that carried by tra- (Gregoire 1972; Carter 1990; Chateigner et al. 2000). becular bone in vertebrate skeletons, is an intrinsic Carter (1990) referred to microstructural modes of element of design. The vast network of pores most crystal aggregations as ‘rods, tablets, laths and plausibly serves to underscore shell strength while blades’. Simple crossed lamellar structures are com- reducing shell weight during the unwieldy, semi- monly found in gastropods and are always aragonitic rotary manoeuvering of the carychiid snail in action. (Bøggild 1930; Taylor et al. 1969; Carter 1990; In addition, the densely pitted microstructure most Hedegaard 1990; Genio et al. 2012). Crossed lamel- likely enhanced tensile strength in the columella of lar calcite is known in some patellogastropods Carychiella eumicrum during the tractional exertion (Fuchigami & Sasaki 2005). of the columellar muscle on the columella. More- The pits and the surrounding superficial surface over, it can be assumed that Carychiidae from the of Carychiella eumicrum (Fig. 2E, F) present a rubbly Middle Miocene manoeuvered in the same charac- homogeneous structure (Fig. 2E, F). The tabular ele- teristic, semi-rotational jerking movements observed ments of C. eumicrum here are morphologically sim- in extant species when they circumvent obstacles or ilar to the laminate, rhombic type, aragonitic enter tight crevices in wood or rocks. nacreous tablets (Taylor & Weeden 2000; Vendrasco Our SEM analyses suggest that homogeneous et al. 2010) observed on imprints of internal moulds microstructure is prevalent in both carychiid species. 10 A. Jochum et al. LETHAIA 10.1111/let.12134

Carter & Clark (1985, p. 63) defined this seemingly 2012). For example, if tininess of C. eumicrum amorphous structure as ‘aggregations of more or less (and all Carychiella species) promoted increased equidimensional, irregularly shaped crystallites lack- survival by wedging into tight crevices to avoid ing clear first-order structural arrangement except predation or desiccation in extreme environmental for possible accretion banding’. Irregularly shaped situations, then an apparent trade-off may have crystallites are present on the protoconch of Carychi- encompassed their avoidance in reaching the mini- ella eumicrum, while a large ribbon of accretion mum possible size/age for sexual maturation. Thus, banding is clearly visible on the shell fracture of in this context, a selective pressure favouring a shift Carychium gibbum. Although accretion banding has towards paedomorphosis via progenesis is plausible often been observed in extant Carychiidae (Jochum (Diz et al. 2012). In a recent model, Edeline et al. personal observation), it is not necessarily an ele- (2013) observed that warming-induced body down- ment of repair but rather an ‘extra’ manifestation of sizing (Bergmann’s rule and the temperature-size conchiolin fabric. The mechanisms responsible for rule) was a primary determinant of life-history why and where it appears are not yet clear. trends in river fish spanning decades and encom- The ‘intersected cross-platy’ variety of crossed passing 52 species. These authors determined that lamellar microstructure may be demonstrated by interspecific competition favoured small body size C. gibbum here (Fig. 4F). This form, however, is and that this played an advantageous role in high perplexing in that a microstructural consensus is dif- diversity communities. In the light of Harzhauser ficult to reach with SEM here. For example, trans- et al. (2014a,b) survey depicting the diverse assem- verse jagged sheets of crossed lamellar structure blage of 47 gastropod species of the Lake Rein resemble those described for ‘intersected cross-platy’ environment, Carychiella eumicrum factors specifi- reported by Chateigner et al. (2000, ICP, fig. 7b). cally, with the larger member of the genus Carychi- Hedegaard (1990), on the other hand, observed this um, Carychium gibbum as a potential competitor in microstructure only in vetigastropods. Chateigner this context. et al. (2000) found it to be identical to the ‘Type II Paedomorphosis, on the other hand, has been crossed lamellar structure’ reported by Batten found to be reversible over relatively long time inter- (1975). Methods such as TEM or the implementa- vals (i.e. millions of years) in a recent phylogenetic tion of a wider sample basis from the same facies of study of spelerpine salamanders, suggesting that Lake Rein would shed more light on this type of CL metamorphosis and adult traits can re-evolve after microstructure. being evolutionarily lost (Bonett et al. 2013). Hereby, genes were found to counteract time by Small shell size and paedomorphosis in assuming alternate functions. Carychiella Paedomorphosis also occurs in small-sized, vent- seep bathymodiolin mussels found at organic falls The minute (max. 1.1 mm shell height), completely (Genio et al. 2012). The bathymodiolin genus Idas is punctate shell of Carychiella eumicrum, suggests a known from wood and whale-falls dating from the paedomorphic life strategy. To understand the sig- Eocene to Miocene (Amano & Little 2005). In the nificance and singular context of paedomorphism light of their extreme habitat status, Idas favours a and small body size in C. eumicrum, a cursory dis- transitory, ephemeral lifestyle much like the extant cussion of the literature is necessary. terrestrial Carychiidae (i.e. Zospeum in caves, Cary- Paedomorphosis involves the heterochronic pro- chium in semi-subterranean habitats). Genio et al. cess by which descendants resemble the juveniles of (2012) hypothesize that the small size of Idas may their ancestors (Bhullar et al. 2012). It is a frequent well reflect an adaptation to ephemeral habitats that mechanism of phenotypic change (Gould 1977; disappear in a few months or a few years. These McNamara 1997), whereby larvae do not complete authors attribute the evolution of paedomorphic metamorphosis and individuals reach sexual matu- forms to the distinct ecological selective pressures at rity while retaining a larval morphology. Paedo- organic falls such as space limitation, community morphosis is largely driven by ecological factors productivity and ephemeralism. Truncated lifespans, (Diz et al. 2012; Bonett et al. 2013). Moreover, accommodated by a trade-off of resource allocation paedomorphosis via progenesis promotes early during early development, would likely favour somatic maturation and an abbreviated ontogeny reproduction rather than growth. In agreement with (Bhullar et al. 2012). Although small size alone Ockelmann & Dinesen (2011) and Genio et al. does not result in paedomorphic morphology, it (2012), it is plausible that ‘a short generation time has factored in discussions about the evolution of and small population size are preconditions for the paedomorphosis (Bhullar et al. 2012; Diz et al. possibility of an accelerated evolution’. Specifically, LETHAIA 10.1111/let.12134 Middle Miocene shell microstructures 11

Carychiella eumicrum likely represents this scenario decomposer communities that most effectively min- for Lake Rein. eralize their own litter (Wardle 2002; Ayres et al. As this study revealed the only known carychiid to 2006, 2009; Vivanco & Austin 2008; Strickland et al. morphologically suggest paedomorphosis, further 2009), it is fundamental to consider this relationship investigation of other fossil assemblages containing in the light of direct biochemical influences exerted Carychiella species may reveal more occurrences in the individual mineralization of shells as well as in from the Neogene. The question remains open the subsequent taphonomic processes of both Cary- whether the observed microstructural aspects and chiella eumicrum and Carychium gibbum facies. protoconch form of C. eumicrum are indicatory of a potential transitory stage of C. eumicrum forms known from Bełchatow, Opole, Tuchorice and San- Conclusions delzhausen. In this context, it is interesting that Strauch (1977) observed C. eumicrum finds fre- The presented data provide additional evidence that quently associated with other, larger Carychiella spe- shell microstructures are similar (i.e. within similar cies at different geological periods: Miocene morphotypes) throughout diverse molluscan groups (eumicrum, crossei) and Pliocene (marinae, puisseg- through geological time (Runnegar 1985; Vendrasco uri). Strauch (1977) also observed apparent construc- et al. 2010). This study reveals instances of consis- tional transitions on the shells of C. puisseguri (Truc tency with extant Carychiidae such as pitting on the 1972) of the Early Pliocene from Celleneuve. A con- protoconch and wedge-shaped planes of aragonitic temporary investigation emphasizing morphological structure on the columella in fossils of two carychiid and anatomical studies of Carychiella’s proposed genera from the Middle Miocene. The Carychiidae closest extant relative, Carychium sibiricum (Westerl- of the Lake Rein show similar microstructures and und 1897) (Strauch 1977; Stworzewicz 1999), could textures in the same parts of the shell of different well reveal valuable, ecologically contextual informa- genera. Our SEM images show the most common tion in this smallest of extant epigean Carychiidae. crystallographical aragonitic microstructural units according to Carter (1990). These structures include Shell morphology reflects ecology homogeneous, simple prismatic and crossed lamellar structure. However, we conclude that although SEM Constructional shifts in shell morphology, such as is effective in detecting these structure types, it is not the p/t boundary observed in Carychium gibbum, sufficient enough to reveal finer structural relation- often correlate with ecological transitions during ships such as crystallographical texture in fossil cary- ontogeny (Jackson et al. 2007; Hickman 2013). In chiid shells. Also, as mineralogical diagenetic addition, as similar shell ornamentation in the Cary- alteration is highly variable within a single layer of chiidae may reflect similar shifts in gene expression fossil shell structure, mineralogy and chemical com- profiles (Gregoire 1972), shell structure may show position (Barskov et al. 1997; Guzman et al. 2009), similar gene regulatory transitions in response to more carychiid shells would need to be investigated ecological and functional requirements (Jackson for palaeoecological reconstructions based on these et al. 2007; Hickman 2013). structures for the Lake Rein. As the organic matrix Mechanisms influencing fossil and extant terres- of the periostracum provides a key source of ontoge- trial gastropod shell structure are manifold. Less netic and ecological information setting Carychiella apparent but none the less influential for shell struc- eumicrum of Lake Rein apart, future research using ture are those involving cross-ecosystem interactions X-ray diffraction of shell tissue and TEM investiga- such as species-specific coupling of plants with soil tions of the periostracal layer would greatly augment in extant and paleobiotic communities. Plant species these studies. Carychiella eumicrum specifically rep- influence the soil biota, including carychiid snails, resents a highly context-dependent dynamic for via changes in the quality and quantity of organic which ontological, biochemical, genetic and ecologi- material entering the substrate (Bardgett & Wardle cal interactions at many different levels contributed 2010). Ions used for biomineralization are derived to producing either: (1) an ecophenotype capable of from the diet or directly from the environment exploiting a potentially novel and extreme habitat; (Weiner 2008), whereby the onset of shell minerali- or (2) it represents an autapomorphic condition (i.e. zation may be correlated to feeding requirements character state derived via natural selection) in (Eyster & Morse 1984). As extant carychiid species ancient carychiid phylogeny. demonstrate a marked affinity towards specific plant As protoconch pitting has been consistently communities (Morton 1955; Lozek 1957), and plant observed in microstructural investigations of recent communities and species preferentially select for Carychiidae, including the troglobitic genus Zospe- 12 A. Jochum et al. LETHAIA 10.1111/let.12134 um (Jochum 2011; Jochum et al. 2013), it can be received support from the SYNTHESYS Project http://www.synthesys.info/, which is financed by the European Community assumed that this frequent ellobioid microstructural Research Infrastructure Action under the FP7 ‘Capacities’ Pro- characteristic (Martins 2007) appears more fre- gram. It contributes to the FWF-project P 25365–B25 ‘Freshwa- quently in the fossil record than reported thus far. ter systems in the Neogene and Quaternary of Europe: Changes in gastropod biodiversity, provinciality, and faunal gradients’. More importantly, this consistency within the Cary- We are grateful to David Reid and Luciana Genio for their con- chiidae suggests that pitting is a microstructural structive input, the anonymous reviewers and the editor, Peter character demonstrating low phenotypic plasticity Doyle, who provided valuable suggestions for improving the and thus reflects evidence of strong genetic control quality of the article. in both extant and fossil species. In addition, as previous investigations (Strauch 1977; Prisyazhnyuk & Stworzewicz 1995; Stworzewicz 1999) predate con- References temporary technological refinements, the degrees of Amano, K. & Little, C.T.S. 2005: Miocene whale-fall community from Hokkaido, northern Japan. Paleogeography, Paleoclima- technological advancement (SEM in this case) as tology, Paleoecology 215, 345–356. well as the preservation potential of the shell have Ayres, E., Dromph, K.M. & Bardgett, R.D. 2006: Do plant spe- much, if not all, to do with this discrepancy. Up to cies encourage soil biota that specialize in the rapid decomposition of their litter? Soil Biology and Biochemistry 38, now, five records show pitting on the protoconch 183–186. and early teleoconch of the shells of fossil Carychii- Ayres, E.H., Steltzer, S., Berg, S. & Wall, D.H. 2009: Soil biota dae dating from the Miocene. These include Carych- accelerate decomposition in high-elevation forests by specializ- ing in the breakdown of litter produced by the plant species ium rhenanum (Strauch 1977) (from Bełchatow) above them. Journal of Ecology 97, 901–912. and Harzhauser et al.’s (2014b) observations in Bandel, K. 1994: Triassic Euthyneura (Gastropoda) from St. Cas- specimens of Carychiopsis schwageri (Reuss 1868), sian Formation (Italian Alps) with a discussion on the evolution of the Heterostropha. Freiberger Forschungshefte C452, Carychiopsis prisyazhnyuki (Stworzewicz 1999), 79–100. 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Palaeontographia Italica 89, & Butlin 2010; Weigand et al. 2013), it can be 3–36. assumed that fossil Carychiidae, comprising these Bhullar, B.A.S., Lobon, J.M., Racimo, F., Bever, G.S., Rowe, T.B., Norell, M.A. & Abzhanov, A. 2012: Birds have paedomorphic Neogene assemblages, responded phenotypically in dinosaur skulls. Nature 487, 223–226. the same manner to paleoenvironmental circum- Binder, H. 2004: Terrestrial, freshwater and brachyhalin Gastro- stances. Hence, the multitude of carychiid fossil sub- poda from the Lower Miocene deposits of Oberdorf (Steier- mark, Osterreich).€ Annalen des Naturhistorischen Museums in genera and the diversity of fossil species assigned to Wien 105A, 189–229. them most likely well reflect the same skewed Boettger, O. 1870: Revision der terti€aren Land- und Susswasser-€ dynamic. Subsequently, a falsely sufficient and mis- Versteinerungen des nordlichen€ Bohmens.€ Jahrbuch der k.k. Geologischen Reichsanstalt 20, 283–302. leading understanding of species comprising these Bøggild, O.B. 1930: The shell structure of the molluscs. Det Kon- Neogene assemblages is likely. Work discussing the gelige Danske Videnskabernes Selskabs Skrifter, Naturvidenska- broad spectrum of morphological and intraspecific belige og Mathematiske Afdeling 9, 231–325. Bonett, R.M., Steffen, M.A., Lambert, S.M., Wiens, J.J. & Chip- variability in extant Carychiidae is currently in pro- pindale, P.T. 2013: Evolution of paedomorphosis in pleth- gress. odontid salamanders: ecological correlates and re-evolution of metamorphosis. Evolution 68, 466–482. Acknowledgements. – We thank Dan Topa for his expertise with Bourguignat, M.J.R. 1857: Amenites Malacologiques, LXIV. Du the SEM at the NHMW. We also thank Angel Diz, Robert Hersh- genre Carychium. Revue et Magasin de Zoologie 9, 209–232. ler, Carole Hickman and Silvia de Paula for their expedient help Bulman, K. 1990: Shell variability in Carychium tridentatum in literature acquisition. Special gratitude goes to Timo Noack (Risso, 1826) and its importance for infraspecific taxon- for technical help with the image processing. This research omy (Gastropoda, Pulmonata: Ellobiidae). Malakologische LETHAIA 10.1111/let.12134 Middle Miocene shell microstructures 13

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