Morphology of the shell of Happiella cf . insularis ( Gastropoda : Heterobranchia : Systrophiidae ) from three forest areas on Ilha Grande , Southeast Brazil

We conducted a study on shell morphology variation among three populations of Happiella cf. insularis (Boëttger, 1889) inhabiting different areas (Jararaca, Caxadaço, and Parnaioca trails) at Vila Dois Rios, Ilha Grande, Angra dos Reis, state of Rio de Janeiro, Brazil. Linear and angular measurements, shell indices representing shell shape, and whorl counts were obtained from images drawn using a stereomicroscope coupled with a camera lucida. The statistical analysis based on ANOVA (followed by Bonferroni’s test), Pearson’s correlation matrix, and discriminant analysis enabled discrimination among the populations studied. The variable that most contributed to discriminate among groups was shell height. Mean shell height was greatest for specimens collected from Jararaca, probably reflecting the better conservation status of that area. Good conservation is associated with enhanced shell growth. Mean measurements were smallest for specimens from Parnaioca, the most disturbed area surveyed. Mean aperture height was smallest for specimens from Parnaioca, which may represent a strategy to prevent excessive water loss. Discriminant analysis revealed that the snails from Jararaca differ the most from snails collected in the two other areas, reflecting the different conservation status of these areas: shells reach larger sizes in the localities where the humidity is higher. The similarities in shell morphology were greater between areas that are more similar environmentally (Caxadaço and Parnaioca), suggesting that conchological differences may correspond to adaptations to the environment.

Land snails are exceptionally diverse in morphology, for instance they display great polymorphism in shell color and variations in shell dimensions.For this reason, they are a good subject for evolutionary biology studies (CLARKE et al. 1978).Differences in size, morphology and growth rates are associated with ecological conditions, natural selection, and phylogenetic history (VERMEIJ 1971, CLARKE et al. 1978, EMBERTON 1994, 1995b, COOK 1997, PARMAKELIS et al. 2003, TESHIMA et al. 2003).According to GOULD (1984), the low mobility of land snails influences character variability.The literature shows that habitat alterations, which result in fragmentation, are an important factor affecting shell morphological differentiation (COOK 1997, GOODFRIEND 1986, EMBERTON 1982, 1994), which can be accelerated in degraded environments (CHIBA 2004, CHIBA & DAVISON 2007).
Ilha Grande, a continental island in the southern portion of the state of Rio de Janeiro, harbors large, continuous and conserved fragments of Atlantic Forest (ROCHA et al. 2006), which is among the most threatened biomes in the world (MYERS et al. 2000).Over 50% of Ilha Grande is covered by ombrophilous dense forest, now at different levels of regeneration (ALHO et al. 2002, OLIVEIRA 2002, ALVES et al. 2005, CALLADO et al. 2009) from disturbances caused by a range of human activities over the past five decades, being now a natural laboratory to study shell morphological differentiation induced by in environment conditions.
The focus of this study was to investigate variations in the morphology of the shell of Happiella cf.insularis in three different environments (Table I).This species was described by BOËTTGER (1889) based on a single shell collected from the type locality, Ilha das Flores, São Gonçalo city, Rio de Janeiro, where additional specimens have not been found (SANTOS et al. 2010).BOËTTGER's (1889) description, which was not accompanied by illustrations, highlighted the following diagnostic features: maximum diameter with 5.25 mm, shell height 2 mm, large umbilicus, one-fourth the size of the shell base; shell pebbleshaped, thin, white, polished, spire apex slightly prominent, with ½ whorls, slightly convex; borders distinct, mildly striated, last border over the third, approximately as wide as shell, less arched at top than bottom, angled below central region; In the present study, we analyzed the shell morphology of three populations of H. cf.insularis subjected to different environmental conditions, with the goal to assess variability in shell morphology, as detailed morphology and range of variation can prove useful for refining species diagnoses.

MATERIAL AND METHODS
The specimens used in this study were collected from three areas, known as the Jararaca, Caxadaço, and Parnaioca trails, located in Vila Dois Rios, on the ocean side of Ilha Grande, Municipality of Angra dos Reis, southern region of the state of Rio de Janeiro (23°04'25" to 23°13'10"S, 44°05'35" to 44°22'50"W).In each collecting site (Fig. 1), a distinct level of forest regeneration (VERA-Y-CONDE & ROCHA 2006) can be found, making them suitable for investigations on the influence of environmental factors on shell morphology.
Table I contains a summary of the environmental parameters measured at the three areas studied.
The analysis of shell morphological variation followed VALORVITA & VÄISÄNEN (1986), with some modifications.Descriptive statistics were performed for each variable in each group, and normality was tested using the skewness test.In cases when a given variable had asymmetrical distribution, the following transformation procedures were applied to normalize it as appropriate: e-base logarithm of X (Neperian logarithm) [1wd/ D, NW], square root of X [D/ud], sin X [MA, ah], cos X [SA, 1ww/2ww], tan X [ah/aw, ah/d], and reciprocal of X (i.e., 1/X) [1ww, 2ww, h/D, D/NW] (KREBS 1998, ZAR 1999), where X is the variable considered.
After normalization, each variable was standardized by reduction (SPIEGEL 1993) and compared using analysis of variance (ANOVA) followed by the Bonferroni's test.Differences  at p < 0.05 were considered statistically significant.The third decimal place was dropped and differences at p р 0.05 were considered statistically significant.
After the exclusion of the highly correlated variables (KLECK 1982, ENGELMAN 1997), discriminant analysis was performed on 15 variables: MA, CA, SA, SS, SPA, 1wd,h, ah,aw,sh, 1ww, 2ww, pW, NW, and TW.Preliminary Pearson's correlation matrix revealed a high correlation (r у 0.90) between the variables D, d, ud, and ah.These were removed from the analysis, and the variable h, representing all correlated variables excluded, was kept.Upon analysis, the variables MA, SA, sh, and 1ww were also removed, owing to their low contribution to group discrimination, as shown by the discriminant function coefficients.Statistical analyses were performed with the aid of the SYSTAT 7.0 statistical package (ENGELMAN 1997).

Shell morphometry
Table II shows the morphometric and meristic data of the 102 shells examined.The mean values of these features were lowest in specimens from the Parnaioca Trail.
The results of the ANOVA, distinguished among the three samples collected from Jararaca, Caxadaço, and Parnaioca, revealed significant differences in all linear and angular measure-ments, except for the mean maximum angle.Specimens from the Jararaca and Parnaioca trails differed significantly in mean columellar and mean spire angles, but the differences in these measurements between samples from Jararaca and Caxadaço and from Parnaioca and Caxadaço were not statistically significant.The shells from Jararaca differed from those from the Caxadaço and Parnaioca trails in the mean sutural and lower sutural angles; shells from Caxadaço and Parnaioca, however, were statistically similar with regards to these two variables.The Bonferroni's test revealed differences between samples from Parnaioca and Jararaca in nine morphological features, between Jararaca and Caxadaço samples in eight features, and between samples from Caxadaço and Parnaioca in seven features -i.e., samples from Caxadaço and Parnaioca were less dissimilar to each other than to the sample from Jararaca (Table III).
ANOVA revealed significant differences in the D/ud, ud/ h, ah/aw, 1wd/D, and D/NW ratios across samples.Bonferroni's test showed Parnaioca and Jararaca samples to differ in four of these ratios (D/ud, ud/h, 1wd/D, and D/NW), Caxadaço and Parnaioca samples to differ in three (D/ud, 1wd/D, and D/NW), and Jararaca and Caxadaço samples to differ on two of these ratios (ah/aw and D/NW) -i.e., differences in measurement ratios were most pronounced between samples collected from the Jararaca and Parnaioca trails, as were differences in linear measurements (Table III).
The mean total number of whorls (NW) differed significantly between the samples from Caxadaço (greater mean) and Parnaioca (Table III).The mean total number of teleoconch whorls (TW) differed significantly between the Jararaca and Parnaioca samples.The mean number of protoconch whorls (pW) differed significantly not only between samples from Jararaca and Caxadaço, but also between samples from Jararaca and Parnaioca (Table III).

Discriminant analysis
The discriminant analysis (Fig. 11) allowed the distinction of all three samples (Wilks's Lambda = 0.300, F = 6.689, df = 22, p = 0.000), particularly with respect to the sample from Jararaca, which differed the most from the others.The samples from Caxadaço and Parnaioca were more similar to each other than each was to the sample from Jararaca (Fig. 11).This analysis correctly classified 67% of the specimens (Fig. 11), with 34 out of 102 being incorrectly classified.
Table III.Results of ANOVA followed by Bonferroni's multiple comparison test, applied to linear and angular measurements, ratios, and number of whorls of Happiella cf.insularis specimens collected from the Jararaca (Jar), Caxadaço (Cax), and Parnaioca (Par) trails, Ilha Grande.Differences were considered statistically significant* at p р 0.05.For abreviations see Table II  The proportions of explanation were of 84.6% and 15.4% for the first and second discriminant functions, respectively.The coefficients also revealed the following variables to be major contributors to the degree of differentiation achieved with the first function: shell height (h), lower sutural angle (SS'), spire angle (SPA), aperture height (ah), number of protoconch whorls (pW) e number of teleoconch whorls (TW).

DISCUSSION
The morphology of the shell of H. cf.insularis fits the description for Systrophiidae perfectly: shell thin, translucent, polished, generally smooth, spire apex slightly prominent, discoid, simple peristomatic edge.According to BAKER (1925), Happiella shells are characterized by a very low spire and an umbilicus normally reduced to a small perforation.In his original description of H. cf.insularis, BOËTTGER (1889) reported a maximum diameter of 5.25 mm, very close to the mean values found for the Caxadaço and Parnaioca samples, and shell height of 2 mm, very close to the mean obtained for the sample from Parnaioca.Aperture height and aperture width were originally described as measuring 2 mm and 2.25 mm, respectively, with the latter measurement falling within the confidence interval of the sample from Jararaca.The original description of the number of whorls (3.5) is also within the confidence intervals of both Jararaca and Parnaioca samples.According to the original description, the umbilicus size is one-fourth the maximum diameter of the shell, a similar ratio to that found in our specimens.
Our results revealed significant differences among the three populations of H. cf.insularis examined, which may be explained by differences in the degree of forest conservation in each area surveyed.The original vegetation in the Caxadaço and Parnaioca areas has been disturbed, more dramatically so in the latter, where the vegetation was entirely slashed down in some areas to make way for the now disabled Vila Dois Rios-Jararaca Dam road and for a number of plantations that served the now defunct Cândido Mendes Penal Colony.Along the Caxadaço Trail, inhabited until the construction of the penal colony by local native fishermen, the environmental changes were less pronounced.The Jararaca Trail region, by contrast, is better preserved.It has a relatively undisturbed secondary forest in lower-altitude areas and primary forest in higher areas.This translates into a deeper leaf litter layer, a more closed canopy, and lower temperature (Table I).As in other instances (SHIMEK 1930, BOYCOTT 1934, CAIN 1977, CLARKE et al. 1978, TILLIER 1981, EMBERTON 1995b, COOK 1997, WELTER-SCHULTES 2000, TESHIMA et al. 2003), better conservation certainly influences the environmental conditions overall, including leaf litter quality, structure, humidity, and depth, which in turn influence mollusk morphology (GOULD 1968, PEAKE 1978, CIPRIANI 2007).Investi-gating Ainohelix editha (Adams, 1868) in Hokkaido, Japan, TESHIMA et al. (2003) demonstrated that shell size and growth rates are adaptations to the environmental conditions; CHIBA (2004), investigating the genus Mandarina Pilsbry, 1894 on the Bonin Islands, found that differentiation in shell shape and dimensions are accelerated in degraded environments.
In the present study, the smallest mean values were obtained for specimens collected from the Parnaioca Trail area (Table II), the most disturbed of the three areas (shallower leaf litter, more open canopy, higher temperature).The lower capacity of the local leaf litter to retain water is likely responsible for the smaller size of the snails.The smaller mean aperture height of the shell may represent a strategy to prevent excessive water loss (GOODFRIEND 1986).MACHIN (1967), PEAKE (1978), EMBERTON (1982), andGOODFRIEND (1986), along with other investigators, have reported that smaller specimens are found in terrestrial gastropod populations living in dry areas with a strong incidence of sunlight.
The mean shell diameter and number of whorls were greatest in snails collected from Jararaca (Table II), consistent with the hypothesis that higher humidity and lower temperatures promote increased rates of shell growth in terrestrial gastropods (GOULD 1984, GOODFRIEND 1986, EMBERTON 1994).BAUR (1988) concluded that the size of the shell of Chondrina clienta (Westerlund, 1883) increases in higher temperatures and lower population densities, as verified by ANDERSON et al. (2007) for Oreohelix cooperi (Binney, 1838).BAUR (1988) commented that the phenotypic plasticity found in C. clienta may be adaptive, as the genetic makeup of snails allows for different shell growth patterns under different environmental conditions.
The populations of the most disturbed areas -the Caxadaço and Parnaioca trails -are more similar to each other than to the population of the Jararaca Trail, which is the bestpreserved area (Fig. 11).The greater similarity observed between the Parnaioca and Caxadaço samples can be explained by the intermediate degree of conservation of the Caxadaço region, which is closer to the degree of conservation of the Parnaioca area than to that of the Jararaca region.
The findings of this study corroborate investigations conducted in other countries showing that morphological differentiation is a result of the isolation of populations in areas that are distinct in vegetation cover, dominant plant species, maximum altitude, and soil type -i.e., areas that offer different microhabitats.As CHIBA (2004) pointed out, degraded environments accelerate differentiation by eliciting new ecological interactions and new habitat conditions, thus subjecting species to a number of selective pressures.
We believe that a similar process has occurred in the areas investigated in the present study, where the different degrees of forest degradation, added to different degrees of moisture, contributed to the morphological differentiation of the three studied populations of H. cf.insularis.However, as Brazilian species of Systrophiidae are not yet well defined, we Morphology of the shell of Happiella cf.insularis from three forest areas ZOOLOGIA 31 (3): 230-238, June, 2014 have decided not to treat the three populations as separate species, and we recommend that they continue to be identified as H. cf.insularis until ongoing anatomical studies are concluded, and a decision on their taxonimical status is reached.

Morphology of the shell of Happiella cf. insularis (Gastropoda: Heterobranchia: Systrophiidae) from three forest areas on Ilha Grande, Southeast Brazil Amilcar Brum Barbosa 1 & Sonia Barbosa dos Santos 1,2
THIELE (1927)sed.Aperture elliptical-lunular, with small slit, aperture height with 2 mm, aperture width with 2.25 mm, simple peristomatic edge, with curved, spherical, sub-angular syphunculus[sic]protruding to right side of base.THIELE (1927), in addition to the type locality of H. cf.insularis, also listed it in Piracicaba (state of São Paulo), Blumenau (state of Santa Catarina) and Porto Alegre (state of Rio Grande do Sul); MORRETES (1949) also listed it only in Ilha dasFlores and SIMONE (2007)to Xanxerê and São Carlos (state of Santa Catarina).

Table I .
Summary of local environmental parameters from three areas (Jararaca, Caxadaço and Parnaioca trails) at Ilha Grande.