Journal of The Malacological Society of London Molluscan Studies

Journal of Molluscan Studies (2019) 85: 262–270. doi:10.1093/mollus/eyz008

Advance Access publication date: 14 March 2019 Downloaded from https://academic.oup.com/mollus/article-abstract/85/2/262/5380598 by University of Patras, Library & Information Service user on 16 May 2019

Viviparous reproduction in the land snail Idyla (: ) from Greece: a disadvantageous inheritance?

Anna Sulikowska-Drozd1, Konstantina Apostolopoulou2, Sinos Giokas2 and Menno Schilthuizen3 1Department of Invertebrate Zoology and Hydrobiology, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland; 2Department of Biology, University of Patras, Patras, Greece; and 3Naturalis Biodiversity Center, Leiden, the Netherlands

Correspondence: A. Sulikowska-Drozd; e-mail: [email protected] (Received 21 August 2018; editorial decision 17 December 2018)

ABSTRACT Viviparity in land snails occurs mainly in species living in tropical or harsh mountain habitats, both of which are characterized by high humidity. The clausiliid Idyla bicristata (Rossmässler, 1839) is an exception. This viviparous snail inhabits the Mediterranean region, which is characterized by long, dry summers. Here, based on field and laboratory breeding observations and experiments, we describe the phenology and fecundity of I. bicristata in detail. The species follows a seasonal pattern of activity: snails are active from October until April (but not in January), and aestivate during the summer months. Reproduction occurs in autumn and spring, with a peak in October, when about 50% of adults were found to be gravid, with an average of 4.9 embryos/adult. In laboratory cultures, adults usually produced neonates twice a year, and the annual fecundity of a pair averaged 15.7 neonates. Reproductive maturity was attained 6.5–7 months after birth. We also investigated the effect of a two-month long experimental drought on gravid snails. We found that the reproductive period was long and flexible, with a low number of indivi- duals being gravid at a given time (in comparison to other viviparous snails). This strategy may reduce the risk of failure and may be a response to demanding climatic conditions. The widespread occurrence of viv- iparous reproduction in related taxa, which inhabit mainly mountainous areas (the Mentissoideinae group), is also discussed.

INTRODUCTION habitats, particularly in species with a very low reproductive rate Giving birth to live young (viviparity) has evolved in many aquatic (Heller, 2001; Bengtsson & Baur, 1993; Maltz & Sulikowska- and terrestrial groups with internal fertilization (Blackburn, Drozd, 2014). However, when inferring the selective pressures 1999; McGhee, 2011; Royle, Smiseth & Kölliker, 2012). The under which current patterns have originated it is not enough to selective pressures that led to viviparity are uncertain; possible consider the present ecological conditions in which living vivipar- explanations include the greater likelihood of offspring surviving ous species occur (see Blackburn, 1999). inside the parent when predation is acute and/or behavioural Among land snails, door-snails (Clausiliidae) have become a thermoregulatory benefits. In reptiles, for example, viviparity model group for evolutionary research due to their high species might have evolved in cold and unpredictable climates where richness, complex shell morphology and diversity of life-history maternal manipulation of the thermal conditions of embryogenesis traits (van Moorsel, Dijkstra & Gittenberger, 2000; Uit de is most advantageous (Shine & Berry, 1978; Blackburn, 2006). Weerd et al.,2006; Maltz & Sulikowska-Drozd, 2008; Giokas, In contrast to the amount of scientific attention paid to the Páll-Gergely & Mettouris, 2014; Kornilios, Stamataki & adaptive significance of parental care in vertebrates, only a limited Giokas, 2015). The mode of reproduction has been well docu- number of studies have investigated the evolution of viviparity in mented for several clausiliid species from Europe (e.g. Baur & terrestrial invertebrates. In land gastropods, for example, vivipar- Baur, 1992; Mamatkulov, 2007; Sulikowska-Drozd, 2009; ity is widespread and occurs in at least 30 families (Heller, 2001). Szybiak, 2010; Sulikowska-Drozd, Maltz & Kappes, 2013; In land snails viviparity is often considered to decrease offspring Szybiak, Gabała&Leśniewska, 2015). Among these, viviparity mortality caused by drought and predation. According to Tompa has been recorded in Balea perversa (L., 1758), Alinda biplicata (1979), viviparity may enhance successful reproduction in geo- (Montagu, 1803), Vestia turgida (Rossmässler, 1836), and graphical regions where the onset of the rainy season is unpredict- Ruthenica filograna (Rossmässler, 1836). Several other species able. The incidence of viviparity is greater in extreme habitats, exhibit short-term retention of eggs (e.g. Sulikowska-Drozd, such as exposed rock walls, and in the tropics (Baur, 1994; Heller, 2009; Sulikowska-Drozd, Maltz & Stachyra, 2012; Sulikowska- 2001). Viviparity may also facilitate the colonization of new Drozd & Maltz, 2016).

© The Author(s) 2019. Published by Oxford University Press on behalf of The Malacological Society of London, all rights reserved. For Permissions, please email: [email protected] REPRODUCTION OF THE CLAUSILIID IDYLA

In the temperate climate of Central Europe viviparous reproduc- seemstobedoubtful(Table1). Depending on the time of collection, the tion in clausiliids follows a clear phenological pattern (Mamatkulov, uterus of a viviparous snail might be either temporarily empty or filled 2007; Sulikowska-Drozd, 2009; Szybiak, 2010; Sulikowska-Drozd with eggs (not shelled embryos). et al.,2013): embryos are present in the reproductive tract for only a Among viviparous clausiliids, several species occur in the mountain- short period, when temperatures are mild and humidity is high (usu- ous ( = more humid) regions of southern and south-eastern Europe, ally from May to July). Consequently, gravid individuals have never with one of these species, Idyla (Idyla) bicristata (Rossmässler, 1839), Downloaded from https://academic.oup.com/mollus/article-abstract/85/2/262/5380598 by University of Patras, Library & Information Service user on 16 May 2019 been recorded in winter months. To date, no attempts have been being also widespread in lower-elevational regions near the made to study the reproductive activity of viviparous clausiliids native Aegean Sea, mostly in Greece and Turkey (Welter-Schultes, to the hotter and drier climates of the Mediterranean and Pontic 2012). It may be that the dry and hot summers characteristic regions of Europe. Even in species that have been thoroughly studied of this snails’s natural habitat has an adverse effect on its’ anatomically (e.g. Likharev, 1962; Nordsieck, 1975, 1994; Neubert, reproduction; land snails avoid the effects of desiccation by 1993; Neubert & Menkhorst, 1994), the focus has been exclusively on retracting their bodies deep into the shell and this may be hin- anatomical structures of possible taxonomic value, such as the male dered if embryos are present in thereproductivetract(Fig.1). genitalia and the bursa copulatrix. Consequently, observations on the Here, we analyse the reproductive phenology of I. bicristata, dynamics of embryo development in the female tract of the reproductive under both natural and laboratory conditions, and investigate system are fragmentary. Moreover, in several cases the classification of the effect of drought on gravid snails and on the embryos pre- clausiliids as oviparous or viviparous (formerly known as ovoviviparous) sent in their reproductive tract.

Table 1. Reproductive strategies of selected Clausiliidae (formerly subfamily Mentissoideinae) as recorded in the literature.

Taxon Region References Embryos in genital tract

Armenica laevicollis banki Neubert & Turkey Neubert & Menkhorst (1994): ovoviviparous, 6 embryos in the uterus + Menkhorst, 1994 Armenica laevicollis laevicollis (De Charpentier, Turkey Neubert & Menkhorst (1994: fig. 4): embryos in the genital system + 1852) Armenica laevicollis paphlagonica Nordsieck, Turkey Neubert & Menkhorst (1994: fig. 5): embryos in the genital system + 1975 Boettgeria crispa (Lowe, 1831) Madeira Groh & Hemmen (1984): ovoviviparity detected in collected in July + Boettgeria deltostoma (Lowe, 1831) Madeira Groh & Hemmen (1984): ovoviviparity detected in animals collected in + December Boettgeria lowei (Albers, 1852) Madeira Mandahl-Barth (1951): ovoviviparity (no details) + Elia (Megaleuxina) derasa (Mousson, 1863) Transcaucasia Likharev (1962): viviparous; one specimen contained 20 embryos + Elia (Caucasica) ossetica (Mousson, 1863) Caucasus Likharev (1962): viviparous (no details) + Elia (Caucasica) somchetica (L. Pfeiffer, 1846) Caucasus Likharev (1962), as genus Euxina, Schileyko (2000: fig. 910): uterus usually + contains a number of eggs Euxina lessonae (Issel, 1865) Iran Likharev (1962: fig. 101): viviparous, 5 embryos in uterus + Euxina pontica (Retowski, 1887) [=Euxina North Turkey Likharev (1962): oviparous, but fig. 103: genital tract with several egg-like ? promta A. Schmidt, 1868] structures Euxinastra (Odonteuxina) harchbelica, Pall- Turkey Páll-Gergely (2010): 7 embryos were found (whorls up to 4) in the ovaries + Gergely, 2010) (sic!) of the 2 dissected specimens Filosa filose (Mousson, 1863) Georgia Likharev (1962): viviparous (no details) + Galeata schwerzenbachi (L. Pfeiffer, 1848) Asia Minor Schileyko (2000: fig. 914): a well-developed embryo with brown shell + consisting of 3 whorls in uterus Idyla (Idyla) bicristata (Rossmässler, 1839) Rhodope Mtns, Schileyko (2000): ovoviviparous (no details) + Greece Idyla (Strigilidyla) liebegottae Nordsieck, 1994 Turkey Schileyko (2000): oviparous (no details) ? Macroptychia africana (Melvill & Ponsonby, Africa Neubert (2002): 3 embryos in the uterus + 1899) Likharevia gustavi (Boettger, 1880) Talysh Mtns, Nordsieck (1975): ovoviviparous (no details) + Azerbaijan Quadriplicata aggesta f. alpestris Nordsieck, Caucasus Nordsieck (1975): ovoviviparous (3 embryos in free oviduct) + 1975 Strigileuxina reuleauxi (Boettger, 1887) Georgia Likharev (1962): viviparous (no details) + Sumelia Nordsieck, 1994 Turkey, the Nordsieck (1994): ovoviviparous + Pontic Mtns Sumelia boniferae Neubert, 1993 Turkey, the Páll-Gergely (2010), pers. comm. (2018): eggs found in genital tract ? Pontic Mtns Sumelia rolli Nordsieck, 1975 Turkey, the Neubert (1993: fig. 3): egg-like structures in genital system; no comments ? Pontic Mtns provided

Symbols: +, presence confirmed; ?, reproductive strategy uncertain.

263 A. SULIKOWSKA-DROZD ET AL. Downloaded from https://academic.oup.com/mollus/article-abstract/85/2/262/5380598 by University of Patras, Library & Information Service user on 16 May 2019

Figure 1. X-ray microcomputed tomography images of Idyla bicristata, coll. Naturalis Biodiversity Center. A. Gravid individual with developing embryos in situ. B. Cervix with basal and dorsal keels. C. Interior of the body whorl with clausilium (visualization by M.T. Walczak).

MATERIAL AND METHODS 50 × 50 cm (see Giokas & Mylonas, 2002). While counting adult and juvenile snails, their activity (such as feeding and crawling) Fieldwork was also recorded. A field study was conducted at the Archaeological Site of Nemea, Peloponnisos, Greece (37°48.551′N; 22°42.754′E), where the local population of Idyla bicristata is confined to a small (c. 1.5 m × Estimation of population density and dispersion 30 m), northwest-facing area at the ruins of the Temple of Zeus. We estimated population density, and using two dispersion indi- Snails were found on the surface of boulders and inside crevices. ces, assessed population spatial patterns. The dispersion indices Another clausiliid species (Albinaria grisea) was also present at the used, Green’s index (GI) (Green, 1966) and the standardized Temple, but the two species were not found to be strictly syntopic Morisita’s index (MIS) (Morisita, 1959; Smith-Gill, 1975), are (i.e. they occurred in different parts of the site). Fieldwork began among the best available for describing spatial patterns because in October 2015 and ended in October 2016, with a 3-month they are nearly independent of population density and sample size summer break (June–August). Each month the population of (Krebs, 1999).These indices primarily examine the deviation from I. bicristata was sampled once by visual searching of randomly a random (Poisson) distribution (Krebs, 1999). Negative values of selected quadrats. Sampling was done at the same time (c. 13:00 h) GI indicate a uniform pattern, zero values indicate a random dis- on the last week of each month, under similar weather conditions tribution and positive values indicate a clumped pattern. GI was (i.e. on non-rainy days). The number of quadrats in each of the 10 calculated using PASSaGE software (Rosenberg & Anderson, sampling occasions was set to 20 and the size of each quadrat was 2011). Smith-Gill (1975) proposed MIS as an improvement to

264 REPRODUCTION OF THE CLAUSILIID IDYLA

Morisita’s index (Morisita, 1959) and adopted an absolute scale RESULTS from −1to+1. MIS = 0 when patterns are random, MIS > 0 when patterns are clumped and <0 when patterns are uniform. Field data MIS was calculated using PAST software (Hammer, Harper & Snails were active for seven months of the year (i.e. from October Ryan, 2001). We used ANOVA tests with SPSS (IBM, 2017)to to April, but not in January). Feeding and crawling was observed

fi Downloaded from https://academic.oup.com/mollus/article-abstract/85/2/262/5380598 by University of Patras, Library & Information Service user on 16 May 2019 investigate if the following measures varied signi cantly between in the field, but no copulation was detected on sampling days. immature and mature snails: (1) mean population density; (2) Aestivation occurred from May to September, when Idyla bicristata mean values of GI and (3) mean values of MIS. usually retreated into crevices. Mean population density was found to be 4.728 individuals/ 0.25 m2. Mean density of immature individuals was significantly = = Dissections lower than that of mature individuals (F1,18 8.604, P 0.010) (Table 2). As expected, since observed density can be also related A study on the dynamics of reproduction was conducted on the to weather conditions (see Giokas & Mylonas, 2002), density fluc- material collected from Nemea on a monthly basis. The number of tuations across months were observed (Fig. 2); except for the individuals sampled each time varied between 14 (May 2016) and month of May, mature individuals were always found to be more 38 (October 2015) individuals. An additional sample was collected abundant than immature individuals. Immature clausiliid snails in late August 2017. Snails were killed in boiling water and then may hide more easily in crevices (Giokas & Mylonas, 2002) and preserved in 70% ethanol. Each individual was photographed for this might in part explain why immature snails were always further morphometric study, and then part of the shell was observed at lower densities than mature ones. removed and the reproductive system was dissected and examined The study population always showed a clumped spatial distribu- for embryos. When detected, embryos were removed, counted and tion. The vast majority of individuals (90%) were present in clus- photographed. Two developmental stages were distinguished: ters, each of about 10–15 specimens; these clusters were usually embryos covered by an egg envelope with CaCO3 crystals in the inside or around small rock crevices. The remaining individuals outer layer (E1) and embryos without such an envelope (E2). (10%) were found individually on rock surfaces (i.e. were not clus- tered). Both GI and MIS showed a high level aggregation. Aggregation was higher in April and May (i.e. at the onset of aes- Laboratory cultures tivation). We found no significant difference between immature = = and mature individuals for mean values of GI (F1,18 0.535, P A laboratory culture was established on 22 October 2015 with 40 0.475) and MIS (F1,18 = 0.001, P = 0.996) (Table 2). adult Idyla snails from Nemea. Pairs of snails were kept in separate boxes containing paper tissue and a piece of limestone rock as a source of calcium. The boxes were sprayed with water to ensure – ° constant humidity. The temperature was kept at 20 25 C. Snails 2 ’ were provided initially with a vegetable diet of lettuce and cucum- Table 2. Mean density (inds/0.25 m ), mean values of Green s (GI) and standardized Morisita’s (MIS) indices of dispersion and associated SD for ber, but this remained untouched. The number of adults declined immature (IMA), mature (MAT) and all (TOT) individuals of Idyla bicristata between October 2015 and February 2016 from 40 to 29 indivi- studied. duals. From February 2016 snails were offered lichens scraped from well shaded places on a concrete wall and this food was Maturity Density SD GI GI (SD) MIS MIS (SD) accepted. After the change in diet the mortality of individuals was IMA 1.617 0.600 0.289 0.297 0.559 0.081 very low. Altogether, 26 individuals sampled in the field (F0) remained alive until the end of our observations (17 May 2017). MAT 3.111 1.406 0.375 0.189 0.559 0.036 The reproduction of the subsequent generation (F1) was assessed TOT 4.728 1.196 0.615 0.410 0.563 0.050 between March 2016 and May 2017 (8 pairs). The boxes were scrutinized at least weekly. In periods of reproductive activity the recorded neonates were transferred every 3–4 days to separate containers. The data on fecundity were aggregated for monthly periods and expressed as means/replicate (pair of snails). 7.0 IMA The height of the shells of 14 juveniles (kept in two groups of 7 MAT inds), was measured twice each month until shells were fully devel- 6.0 oped. The first births of neonates were recorded for these two groups. The remaining juveniles were kept in separate boxes at 5.0 high density (30–40 inds/box).

4.0

Experimental drought 3.0

In May 2017 42 snails from the laboratory culture were exposed Mean Density to an experimental drought: the damp tissue paper was removed 2.0 from the laboratory boxes and water spraying and feeding were stopped. The duration of the experiment was varied and there 1.0 were five treatments: 18 days (5 inds), 64 days (9 inds), 111 days (4 inds), 178 days (12 inds) and 190 days (12 inds). After the drought period, all snails except the group subjected to drought for 190 0.0 Nov-15 Dec-15 Jan-16 Feb-16 Mar-16 Apr-16 May-16 Sep-16 Oct-16 days were preserved in ethanol and dissected. In the dissected snails, the number and developmental stage of embryos (E1, E2) Month observed were recorded. Twelve individuals exposed to 190 days Figure 2. Mean monthly density (individuals/0.25 m2) of mature (grey fill) of drought were sprayed with water to check their survival and and immature (black fill) specimens of Idyla bicristata, as observed during their ability to give birth to neonates. the field study.

265 A. SULIKOWSKA-DROZD ET AL.

60 6 The annual fecundity of a pair averaged 15.7 neonates (range = (38) % brooding adults 1–38). average no. of embryos 50 5 Snails born in the laboratory in March 2016 (F1 generation) began to produce neonates in September of the same year. (29) 40 4 During nine months of almost constant reproductive activity, 16

(22) Downloaded from https://academic.oup.com/mollus/article-abstract/85/2/262/5380598 by University of Patras, Library & Information Service user on 16 May 2019 (31) adults produced 107 juveniles. On average, a pair of F1 snails pro- = – 30 3 duced 13.4 juveniles (range 2 21). (17) Under laboratory conditions the time required for the complete development of the shell ranged from 2.5 to 3.5 months. In the

20 2 Brooding adults (%) adults Brooding (26) embryos of Number fastest growing individuals, the formation of the apertural barriers began 1.5 months from birth, at a shell height of c. 10 mm. In the 10 1 slowest growing individuals, the formation of apertural barriers (25) (21) (23) (14) – – (17) began 3 months from birth. Idyla bicristata produced neonates after 0 0 6.5–7 months from birth. The juveniles kept at high densities had Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct very variable growth rates and often exhibited shell deformation Month (i.e. with a scalar form of the last whorl). Figure 3. Seasonality of reproduction in Idyla bicristata, showing the per- centage of adults retaining embryos and mean number of embryos/snail in consecutive months; the number of dissected snails per sample is shown Drought exposure within parentheses. All adult I. bicristata exposed to drought survived. However, intra- uterine embryos found in these snails suffered high mortality when the drought lasted more than two months (Table 3). When the 10 drought exposure lasted 64 days, three out of five brooding indivi- duals contained dead and partially dissolved E2 embryos. When 9 drought exposure lasted 178 days, six out eight brooding snails 8 had dead E2 embryos. In snails subjected to 111 days of drought, followed by watering, E1 embryos were found in the reproductive 7 tract (Fig. 7). In one case, seven E1 embryos were encountered 6 together with four dead E2 embryos (Fig. 7D). After 190 days, only three of 16 snails produced offspring, of which only two were 5 alive. 4

3 DISCUSSION Number of individuals of Number 2 The timing of reproduction is one of the most important life his- 1 tory traits in terrestrial gastropods living in seasonal climate regimes. Land snails inhabiting the Mediterranean region respond 0 12345678910 to the strong climatic seasonality of the region and exhibit quite predictable oscillation in their activity patterns (Lazaridou- Number of embryos Dimitriadou & Scardelis, 1997; Heller, 2009). The several heli- Figure 4. Number of embryos found in the reproductive tract of adult coids studied to date have two main patterns of reproduction: Idyla bicristata collected in the field. short-lived species breed in autumn and long-lived species breed in autumn or in spring–summer (Lazaridou-Dimitriadou & Scardelis, 1997). Autumn breeding commences with the first rains Reproduction was recorded mostly in autumn (Fig. 3). Embryos and ceases when temperatures fall. On the other hand, spring were found only in 34 of the 263 adults dissected (12.9%). The breeding commences when mean monthly temperature rises over number of embryos retained by an adult ranged from 1 to 10 10 °C and ceases with the onset of the arid periods (Staikou, = = (mean 4.0; SD 2.4) (Fig. 4). The proportion of brooding snails Lazaridou-Dimitriadou & Farmakis, 1988). Our field observations and the average number of embryos/gravid snail was highest in indicate that Idyla bicristata is principally an autumn breeding spe- > October 2015 at 50% and 4.9 embryos, respectively. Of the two cies, although a small number of individuals may also reproduce types of embryos found in the dissected snails, those with already in spring. In Nemea, I. bicristata faces two climatically challenging developed shells (E2) were the most frequently observed. periods, a dry summer, and a cold winter, when the minimum dai- However, broods containing only E1 embryos or both stages of ly temperatures drop below 0 °C (minimum −5.2 °C). According embryos were also recorded (Fig. 5). Embryonic shell height to the climatic data available for the area (source: Nemea’s local = reached on average 2.2 mm (maximum 2.7 mm) shell width a meteorological station, part of the National Observatory of = mean of 1.2 mm (maximum 1.4 mm). Snails collected in Athens), frost was recorded for a total of 36 days in the winter of January, February, April and May did not contain embryos. 2015/2016. Idyla bicristata was found to be inactive during the January sampling. The highest proportion of gravid individuals Laboratory cultures was recorded in October, suggesting that autumn is the main reproductive season for this species. For clausiliids belonging to Individuals collected in the field (26 adults kept in pairs; F0 gener- the genus Albinaria from Greece, Schilthuizen & Lombaerts (1994) ation) reproduced repeatedly during the observation period; no and Giokas & Mylonas (2002) have recorded a single, long season copulation was observed. A pair of snails produced between 11 of reproductive and feeding activity, extending from mid-October and 54 neonates. Neonates were produced twice a year, in two to late April. In contrast, the activity of Cristataria genezarethana,a reproductive seasons in autumn and spring (Fig. 6). The number clausiliid occurring in Israel, lasts only a few days per year; this of neonates encountered during a single observation (every 3–4 species takes an exceptionally long time (11 years) to reach adult days during the reproductive period) averaged 3.4 (median = 3). size (Heller & Dolev, 1994). Albinaria and Cristataria produce eggs

266 REPRODUCTION OF THE CLAUSILIID IDYLA Downloaded from https://academic.oup.com/mollus/article-abstract/85/2/262/5380598 by University of Patras, Library & Information Service user on 16 May 2019

Figure 5. Dissected examples of adult Idyla bicristata collected in the field, showing the two types of embryos (E1 and E2) found in the reproductive tract. A. Adult with E1 embryos. B. Adult with E2 embryos. C, E. E2 embryos. D. E1 embryo. Scale bar = 1 mm.

60 F0 generation which require a long period of incubation (c. 3 weeks). As clausiliid F1 generation eggs are not resistant to water loss, it is essential that the timing of 50 egg-laying is synchronized with the rainy period, which extends 40 from October to December (Heller & Dolev, 1994; Giokas & Mylonas, 2002). The timing of aestivation does not differ between 30 oviparous Albinaria and viviparous I. bicristata and lasts from May until September (Giokas & Mylonas, 2002; and this paper). 20 While embryonic development in I. bicristata seems to coincide

Number of neonates of Number with the autumn activity period, retention of eggs was not 10 recorded even during late August. This suggests that in this spe- 0 cies, viviparity does not provide a means by which offspring are produced right at the beginning of the rainy season, as postulated for some other subtropical gastropods (see Heller, 2001). The spring months, with only few individuals retaining eggs, have only a Figure 6. Reproduction in the laboratory culture of Idyla bicristata. minor importance for reproduction. In contrast, in viviparous and Observations were made on the F0 generation (13 pairs) from October 2015 to egg-retaining clausiliids living in temperate Europe, embryonic May 2017 and on the F1 generation (8 pairs) from March 2016 to May 2017.

267 A. SULIKOWSKA-DROZD ET AL.

Table 3. Effects of drought exposure on the reproduction of Idyla bicristata.

No. of dry days Drought period No. of No. of inds No. of embryos/ No. of E1 No. of E2 embryos No of inds with No. of neonates inds with embryos adult (range) embryos (alive/dead) neonates (alive/dead)

18 19 May–6 June 2017 5 2 1–40 5/0 –– Downloaded from https://academic.oup.com/mollus/article-abstract/85/2/262/5380598 by University of Patras, Library & Information Service user on 16 May 2019 64 5 Apr.–8 June 2017 9 5 1–60 15/6 –– 178 19 May–13 Nov. 2017 12 8 1–50 6/16 –– 111 19 May–7 Sept. 2017 4 3 1–11 8 0 / 6 0* 0* 190 19 May–25 Nov. 2017 12 No data No data No data No data 3* 2 / 5*

*Snails watered after drought period.

Figure 7. Adult Idyla bicristata from the laboratory culture dissected shortly after exposure to the experimental drought. A–C. Reproductive tract. D, E. Eggs and embryos removed from the reproductive tract. Scale bar = 1 mm.

268 REPRODUCTION OF THE CLAUSILIID IDYLA development starts in spring and juveniles are produced until late (longevity, age at maturity, adult size) (Lazaridou & Chatziioannou, summer (Sulikowska-Drozd, 2009; Sulikowska-Drozd et al.,2013; 2005). Similarly, significant differences were observed in the popula- Sulikowska-Drozd & Maltz, 2016). In the well-studied case of Alinda tion dynamics and grow pattern of the clausiliid Vestia gulo in the biplicata, the onset of brooding coincides with an increase of humidity Carpathians (Sulikowska-Drozd, 2011). The time required for and temperature in May (Sulikowska-Drozd et al.,2013). The differ- growth in I. bicristata was not studied in the field. However, our ence in the reproductive activity of I. bicristata in Nemea between observations of the laboratory cultures showed that shell develop- Downloaded from https://academic.oup.com/mollus/article-abstract/85/2/262/5380598 by University of Patras, Library & Information Service user on 16 May 2019 October 2015 and October 2016 is consistent with Lazaridou- ment lasts only 2.5–3.5 months, and after a subsequent 4-month Dimitriadou & Scardelis’s (1997) that long living, iteroparous species phase, snails begin to produce neonates. If growth rates in Nemea are quite plastic in their response to the climatic differences from one are comparable, snails born in October should be fully grown adults year to the next. Moreover, mating and egg laying are shorter and at the onset of aestivation in May. However, in the field juveniles more synchronous in the population living along the seashore than in are present throughout the year, and this suggests that there is far those inhabiting inland areas, where these activities may take place more variation in the developmental times in wild populations. almost in all seasons, except during winter (Lazaridou-Dimitriadou & Our results indicate that the seasonal dynamics of reproduction Scardelis, 1997). It should be noted that the proportion of gravid indi- can obscure the identification of reproductive mode. It is tempting viduals in the populations of A. biplicata or Vestia turgida during the to classify gastropods into different reproductive modes, but this height of the reproductive season is much higher (80–90%) should not be attempted if the animals have not been collected in (Sulikowska-Drozd, 2009; Sulikowska-Drozd et al.,2013), than that multiple seasons. For adult I. bicristata from Nemea the likelihood recorded for I. bicristata in Nemea during its reproductive peak in of collecting gravid individual is rather low (12.9%) and fluctuates October (50%). This suggests that possibly in the more unpredictable during the year. The accurate classification of species as being and stressful Mediterranean climatic regimes the species safeguards its oviparous or viviparous requires adequate sampling during recog- success by not ‘putting all its eggs in one basket’ (diversified bet- nized periods of reproductive activity or laborious captive breed- hedging strategy, see Olofsson,Ripa&Jonzén,2009). ing efforts. The humidity of the laboratory boxes had a beneficial effect on the reproduction of Idyla. During the first year in captivity, the reproductive output of the F0 generation was confined to two peri- ods, while in the F1 generation offspring were produced almost ACKNOWLEDGEMENTS continuously. Less strict seasonality has also been observed in The study of Idyla bicristata reproduction was initiated during a laboratory colonies of Alinda biplicata (Maltz & Sulikowska-Drozd, Synthesis Scholarship (NL-TAF-5042) held by ASD at Naturalis, 2014). We found that the annual fecundity of Idyla in laboratory Leiden, with MS as the supervisor. We thank the Corinth’s cultures reached c. 15.7 offspring per pair. Vestia turgida (mean = Ephorate of Antiquities and the personnel of the Archaeological 11.7 neonates/ind.) and A. biplicata show a similar level of fecund- Site of Nemea for allowing and facilitating sampling. We are ity, with between 17 and 33 neonates/pair having been recorded grateful to Dirk van der Marel (Naturalis) and Michał Walczak (Sulikowska-Drozd, 2009; Maltz & Sulikowska-Drozd, 2014). (Silesia University) for producing the CT scans and visualization These findings are in sharp contrast to the high fecundity of the of the shell of I. bicristata. We are indebted to Ewa Janowska and East Asian clausiliids, Tauphaedusa sheridani and T. tau, which have Karolina Chaniecka (University of Lodz) for their help in main- been found to produced more than 160 neonates/pair annually taining the laboratory culture and processing photographs for the and can retain up to 11 embryos at a time in the reproductive tract manuscript. Additional financial support for the project was pro- (Sulikowska-Drozd et al., 2018). The mean number of embryos vided by the Endless Forms group of Naturalis Biodiversity Center found in gravid I. bicristata was 4 (maximum 10), while in V. turgida and by internal funds from the University of Lodz, Poland. it averages 6.5 (maximum 12), and in A. biplicata 9.4 (maximum 20) (Sulikowska-Drozd, 2009; Sulikowska-Drozd et al., 2013). Long-term aestivation in the natural population of snails may require other physiological mechanisms than our experimentally- induced desiccation. 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