REPRODUCTIVE BIOLOGY AND GROWTH OF TWO VALLONIA SPECIES IN LABORATORY CONDITIONS ( GASTROPODA : EUPULMONATA : VALLONIIDAE )

Reproduction and growth of Vallonia pulchella (O. F. Müller) and V. costata (O. F. Müller) were studied in laboratory conditions. Their ellipsoid and singly laid eggs are among the smallest heavily calcified (mean size 0.68×0.67×0.52 and 0.69×0.67×0.54 mm in V. pulchella and V. costata, respectively). V. pulchella reached morphological maturity (lip completion) at 3.25–3.50 whorls (mean 3.35), 49 to 166 days after hatching (85). The first egg (sexual maturity) was laid at 3.25–3.50 whorls (3.35), 50–283 days after hatching (162). The life span ranged from 628 to 940 days (779), the time elapsing between the last egg and death was 12–184 days (111). In V. costata the lip was completed at 3.25–3.50 whorls (3.37), 42 to 183 days after hatching (120). The first egg was laid at 3.25–3.50 whorls (3.42), 131–290 days after hatching (193). The life span ranged from 360 to 841 days (709) and the time elapsing between the last egg and death was 11–692 days (210). The egg production rate and juvenile life span in individuals kept in isolation were the same as in those kept in pairs or larger groups. The growth pattern was similar in the two species: fast phase followed by slow phase. However, the formation of ribs on the shell surface of V. costata probably slowed down its growth rate. The life history traits shared by the two Vallonia species are determined growth, iteroparity, longevity and predominantly selfing reproductive mode. The greater lifetime fecundity, shorter incubation period, faster growth and longer life span predestine V. pulchella to be a better coloniser than V. costata. KEy words: land snails, reproduction, self-fertilisation, longevity, lifetime fecundity, growth rate

They are ground-dwelling snails, rarely climbing more than a few centimetres above the soil surface.They occur in a wide variety of habitats (gErbEr 1996) but become rare in the highlands (KErnEy 1999).They usually inhabit open places where they often co-occur, although V. pulchella prefers grassy, base-rich, humid places like meadows on floodplains, moist pastures, marshes, and dune slacks, while V. costata is typical of short-turfed swards, natural cliffs and screes, disused quarries and old walls (KErnEy 1999).V. costata can also be found in humid, close-canopy forests (HorsáK et al. 2010), and often selects calcium-richer sites compared to V. pulchella.Both species can occur abundantly in synanthropic habitats (KosińsKa 1979).
The only data on the biology of Vallonia are wHitnEy's (1938) observations on the reproductive cycle of V. pulchella, fragmentary data on the bionom-ics of V. pulchella and V. costata (gassiEs 1849, stErKi 1893, fröMMing 1954), and an unpublished thesis by isKrzyńsKa (1986).The aim of this paper was to compare the life histories of the two closely related species, based on laboratory observations.

MATERIAL AND METHODS
The initial material for the laboratory culture (10 adults of each species) was collected on the 2nd October 2010 in the ruins of the castle Grodztwo (50°47'26.9"N,16°02'09.6"E,440 m a.s.l.) in Kamienna Góra (Central Sudetes, SW.Poland).Thirty five of the hatched individuals of each species constituted the original laboratory population.The laboratory observations lasted till the 30th of March 2014.The individuals of each species were distributed as follows: 15 single, 4 pairs and 3 groups of 4 snails each.
The snails were kept in Petri dishes and in containers of a size depending on the number of inhabitants (dishes of 5-10 cm diameter, and plastic containers of 6×7.5×5 cm and 12×7.5×5cm), on humid tissue paper with litter brought from the habitat.The dishes were checked and cleaned at least once a week and during periods of intense observations (e.g.reproductive period) -every day or two.Eggs and newly hatched snails were removed to separate dishes.The snails were kept in a climatic chamber at constant conditions of temperature (day 22ºC, night 18ºC) and relative humidity (80%), with a 12 : 12 h light regime (fluorescent lighting).They were fed lettuce and sometimes carrots.Water and food were supplied as needed.Dolomite tablets constituted the source of calcium.
Laboratory observations included lifetime fecundity, egg-laying, egg morphometrics, incubation, hatching, growth rate, maturity and life span.Growth was assessed by counting whorls every 30 days using EHrMann's (1933) method.The maximum of reproduction (fecundity) was assessed by counting the total number of eggs produced by selected individuals of each species during their lifetime.Hatching success was estimated based on the hatching percentage.Two-way variance analysis (ANOVA) was applied to detect significant differences in the reproductive parameters between individuals kept singly and in pairs/groups as well as between the species.
To perform scanning electron microscope observations, the shells and egg shells of each species were gold-coated and SEM photos were taken using the microscope EVO LS 15, Zeiss.

RESULTS
In the laboratory both V. costata and V. pulchella laid eggs singly, under or between layers of damp tissue paper, on the bottom of the container, under litter and lettuce leaves.Newly laid eggs were milky-white, ellipsoid and rather uniform in size (Table 1).The egg surface consisted of a continuous heavily calcified layer, which developed as geometric forms (Figs 1-2).The size of calcite crystals on the external   egg surface in V. costata ranged from 3.621 to 5.096 µm (mean = 4.198, SD = 0.432, n = 18), whereas in V. pulchella it varied more widely between 1.118 and 5.070 µm (mean = 2.765, SD = 1.349, n = 18).The differences were not statistically significant (p = 0.823).The differences in thickness of the egg shells and cross-sections through fractured eggs in V. pulchella and V. costata are shown in Figs 3-5.
There were no statistically significant differences (p > 0.05) in any of the studied reproduction parameters between individuals kept in isolation and those living in pairs or groups.Therefore, the results were pooled; the comparison of all the life cycle parameters between V. costata and V. pulchella is shown in Table 1.
The growth pattern in V. costata and V. pulchella was similar (Fig. 6).Two phases could be distinguished: I -fast phase in snails from 1.0 to 3.0 whorls, II -slow phase in snails above 3.0 whorls.Considerable differences were observed in the growth rate in the first phase: the mean growth rate in V. pulchella was faster than in V. costata, which showed a greater individual variation during both phases.
In V. costata during the fast phase formation of one whorl took 28-113 days (mean = 56.9,SD = 28.7,n = 11), while in V. pulchella The shell of V. costata bears regular, widely spaced, coarse ribs on both the upper and lower surfaces.During the life span of ca. 2 years (maximum 841 days, Table 1) the ribs are easily weathered, making old shells difficult to identify; the ribs are usually better preserved inside the umbilicus (Figs 7-10).

DISCUSSION
All available data on the life history of V. costata and V. pulchella are summarised in Table 2.Where the same features have been studied, there is a broad measure of agreement among the studies; this study adds precision and more features.
Life strategy data are known in only 2% of all terrestrial gastropods (HEllEr 2001) and for European land-snail species the proportion is ca.6% (Maltz 2003).Our comparative study adds to the knowledge of life histories of two closely related species of Vallonia.Both represent iteroparous mode of reproduction, i.e. animals reproduce during one season and then live on to reproduce again (HEllEr 2001).Among land snails this kind of reproduction is common in clausiliids (Maltz & suliKowsKa-drozd 2008) and in some helicids (HEllEr 2001).In molluscs iteroparous species are long-lived (HEllEr 2001) and it has been also noted that short life spans are more frequent among very minute gastropods than among large ones (HEllEr 1990).Both Vallonia species, as well as Lauria cylindracea (HEllEr et al. 1997), are clearly exceptions to this pattern as these minute snails live longer than two years.
Uniparental reproduction is regular in Vallonia and was observed through several consecutive generations in this and previous studies (wHitnEy 1938, isKrzyńsKa 1986).Self-fertilisation was proposed as the mode of reproduction since in aphallic individuals gametes of both sexes were simultaneously found in individual acini of hermaphroditic gland in V. pulchella (wHitnEy 1941).In a closely related Acathinula aculeata self-fertilisation was also suggested (boycott 1917, stEEnbErg 1918).Our laboratory-bred Vallonia were aphallic and reproduced by self-fertilisation, since we did not observe copulating snails, and egg production and juvenile life span in individuals reared in isolation were the same as in those kept in pairs or larger groups.Aphallism is extremely common in Vallonia and was observed in all examined individuals of V. pulchella and V. peteri (watson 1920, wHitnEy 1941, aKraMowsKi 1976, scHilEyKo 1984, isKrzyńsKa 1986), while male cop-Figs 7-10.Shells of V. costata: 7 -apical view of fresh shell, 8 -apical view of old shell, 9 -umbilical view of fresh shell, 10 -umbilical view of old shell ulatory organs were recorded in the vast minority of V. excentrica and V. costata (stEEnbErg 1918, watson 1920, gittEnbErgEr 1977, scHilEyKo 1984, barKEr 1985, giusti & ManganElli 1986).The small number of euphallic individuals coupled with the ability to self-fertilise indicate that this mode of reproduction is predominant in both Vallonia species, also in the wild.Stylommatophoran eggs show different degrees of calcification: heavily calcified, partly calcified and uncalcified (toMPa 1976).The heavily calcified egg is sometimes viewed as an ancestral character in this group and is thought to be a terrestrial adaptation (toMPa 1976).However, the function of the calcified shell is still debateable.Some of the smallest eggs are calcified and others are not, while it appears that the larger ones are all heavily calcified which may have to do with providing the half-liquid egg contents with mechanical support once a certain size is exceeded (toMPa 1976).Various theories also suggest prevention of desiccation, protection from predators and physiological buffering (Mazzini et al. 1984).In land snails the smallest hard-shelled eggs known so far belong to Truncatellina cylindrica (Vertiginidae) (0.52-0.58 mm, MyzyK 2005) and to Vallonia (Vallonidae) (not exceeding 0.9 mm, Table 2).The diameter of most calcified eggs varies from 1 mm in Discus rotundatus (Endodontidae) to 51 mm in Megalobulimus popelairianus (Acavidae) (standEn 1917, KuźniK-KowalsKa 1999).Though the eggs of both Vallonia species do not differ in size, their surface structure is slightly different (Figs 1, 2), with minor differences in the size of calcite crystals (Fig. 3).toMPa (1976) did not notice differences between the two Vallonia species, but found egg shell ultrastructure to be species-specific in some other closely related species.
It is commonly believed that the fecundity of minute terrestrial gastropods is limited by the fact of producing only few eggs at any one time (HEllEr 2001).This, for example, is true of species which lay eggs singly: Carychium tridentatum, Punctum pygmaeum and Vertigo spp.(Morton 1954, baur 1989, bulMan 1990, PoKryszKo 1990, MyzyK 2011).In these species the mean number of eggs produced per individual lifetime is small and ranges from five or six (C.tridentatum and P. pygmaeum) to 25 (Vertigo moulinsiana).In V. pulchella it is higher, with the mean number of 38 eggs.On the other hand, D. ruderatus, having shell diameter twice as large as Vallonia, lays approximately 12 eggs in 2-5 batches per lifetime (KuźniK-KowalsKa 2006).Therefore, the relative egg size, the number of eggs per batch and during lifetime depend not only on the size (HEllEr 1990) but also on the longevity of adult snails.In Vallonia the life span is relatively long, of ca. 2 years (maximum 940 days in V. pulchella), while in other species it ranges from 170 days (P.pygmaeum) to 580 days (D. ruderatus).In contrast, L. cylindracea, a minute, litter-dwelling snail, whose life span is even longer (4-5 years in the wild), does not follow this pattern since its average number of hatchlings per adult is five to six (HEllEr et al. 1997).
It is, however, known that snails with long periods of egg retention tend to produce fewer eggs per lifetime than do oviparous snails of similar size (baur 1989).For example, brooding clausiliids usually produce fewer offspring per reproductive event than their oviparous cousins (suliKowsKa-drozd & Maltz 2013).The same may be true of minute species with different reproductive strategies: among all known ovoviviparous or egg-retaining species the number of offspring is small and does not exceed 8 (Pupilla muscorum, PoKryszKo 2001) but usually is 5-6 (Punctum pygmaeum or L. cylindracea, baur 1989cylindracea, baur , HEllEr et al. 1997, respectively) , respectively) or 3-7 (Pyramidula pusilla, gErMain 1930).In strictly oviparous species living in different habitats this number is higher, of up to ca. 38 eggs in V. pulchella, 20 in V. costata, 25 in Vertigo moulinsiana and 15 in V. pusilla (PoKryszKo 1990, MyzyK 2011).
In the laboratory the time to hatching varied but agreed with that observed in the other studies (wHitnEy 1938, isKrzyńsKa 1986).Its duration depends on temperature (e.g.MyzyK 2011) or may be a consequence of a different duration of the egg retention period (suliKowsKa-drozd & Maltz 2013).In oviparous small-sized species incubation periods also vary widely (Table 2, bulMan 1990, PoKryszKo 1990, KuźniK-KowalsKa 1999, 2006).This cannot be solely attributed to temperature differences, especially considering the constant laboratory conditions.The shells of newly hatched snails varied in the number of whorls, and their range was larger in V. costata than in V. pulchella (Table 1).Similarly as in the case of P. bidentata and Discus species (KuźniK-KowalsKa 2006, KuźniK-KowalsKa & roKsEla 2009), some differences may be explained by the variation of the non-ribbed, translucent, embryonic shell in V. pulchella versus hatchlings with a few ribs on definitive whorls in V. costata (isKrzyńsKa 1986).
The two species of Vallonia differ in their growth rate, but the growth pattern in both of them follows some general rules; the shape of growth curves is similar to other species with determined growth (Maltz 2003, KuźniK-KowalsKa & roKsEla 2009, ProćKów et al. 2013).However, the time to lip formation is longer in V. costata than in V. pulchella (Table 1).The development of regular and relatively large ribs on the shell surface of V. costata may explain the differences.
In several studies both V. pulchella and V. costata were regarded as pioneer species of anthropogenic sites which undergo secondary succession (barga-więcławsKa 1997, baucz-Malij 2001, Maltz 2009, 2011).Among terrestrial gastropods pioneer species may possess any of several life history strategies in various combinations (bEngtsson & baur 1993).As seen in this study both species of Vallonia show very similar life cycle parameters (Table 1).Nevertheless, the higher lifetime fecundity, shorter incubation period, faster growth and longer, significantly different life span, predestine V. pulchella to be a better coloniser.This is recognisable in the distribution of the species.Although both have wide-temperate distributions in Holarctic, V. pulchella was additionally successfully introduced to Africa, Asia and Australia (gErbEr 1996, stanisic 1998, Mitra et al. 2005, griffitHs & florEns 2006) and V. costata is known from South Africa and Japan (sasaKi 2008, HErbErt 2010).On the other hand, V. costata exhibits a wider ecological amplitude, i.e. it is usually more frequent at high altitudes in Switzerland, but in the north of the British Isles it is mainly found in coastal regions and only rarely in the highlands, but V. pulchella is slightly more tolerant against heat (wEltEr-scHultEs 2012).

Table 2 .
Life history traits of Vallonia species