Abstract
From 1996 to 2002, we studied the body size, measures of reproductive strategy (relative clutch mass and delayed reproduction at sexual maturity), and reproductive output (clutch frequency and annual egg production) of female European Pond turtles, Emys orbicularis, at two sites separated by 12 km in central Mediterranean Tuscany (San Rossore and Camp Darby, central northern Italy). Females did not reproduce at the first appearance of external sexual characters, but reproduced at larger sizes, probably as older turtles. Among years, reproductive females were more common than were non-reproductive females, yet both groups had similar body sizes. Body size (carapace length and width, plastron length and width, shell height and body mass) varied between localities and among years. Body size differed between reproductive and non reproductive females in Camp Darby, but not in San Rossore females. Shell volume did not vary among years, nor between localities, nor between reproductive status. Reproductive females had higher body condition indices (BCI) than did non-reproductive females, while BCI did not differ between females laying one clutch and females laying multiple clutches. Clutch size did not vary among years. One clutch per year was much more frequent than multiple clutches, and multiple clutches were more frequent in Camp Darby than in San Rossore females, likely due to differences in population structures between sites.
Introduction
Organismal fitness may be implied through their phenotypes, including survivorship and reproductive output or success. In many oviparous reptiles, reproductive output (e.g., clutch size, clutch frequency, hatchling survivorship) is affected by female body size, where large females produce larger clutches than smaller females produce (Seigel and Collins, 1993; Ford and Seigel, 1994; Rowe, 1994), by latitudinal and altitudinal gradients that affect clutch frequency (and probably reproductive success) (Christiansen and Moll, 1973; Iverson et al., 1993; Zuffi et al., 2007), and by lipid stores rather than body size per se (Aubret et al., 2002; Lourdais, Bonnet and Doughty, 2002; Bulté, Irschick and Blouin-Demers, 2008; Warner et al., 2008). Reproductive output may also depend on the availability of nesting habitat (Rovero and Chelazzi, 1996; Zuffi and Odetti, 1998), nest predation (Zuffi and Rovina, 2006), hatching rate, and hatchling survivorship (Marco, Dìaz-Paniagua and Hidalgo-Vila, 2004). Furthermore, reproductive success may require adaptation of parental body size and reproductive mode to geographic, ecological, and habitat variables (e.g., wetland availability and predator distribution; Bonnet et al., 2001; Lagarde et al., 2003; Lanszki, Molnár and Molnár, 2006; Zuffi et al., 2007; Herrel et al., 2008).
The chelonian shell can impose often, but not always (Lovich et al., 2012), physical, anatomical constraints on their reproductive abilities (Congdon and Gibbons, 1987; Lee, 1996; Bonnet et al., 2001) when compared to other reptiles. Recent studies demonstrated that habitat (Claude et al., 2003; Zuffi et al., 2007; Rivera, 2008), genetic isolation (Chiari et al., 2009), and feeding (Herrel, O’Reilly and Richmond, 2002; Bulté, Irschick and Blouin-Demers, 2008) significantly influence the overall size and shape of terrestrial tortoises and freshwater turtles. The size and shape of the carapace and plastron appear to influence the volume available for egg formation, imposing a trade-off between egg size and clutch size (Berry and Shine, 1980; Congdon and van Loben Sels, 1991; Iverson et al., 1993; Zuffi, Odetti and Meozzi, 1999; Zuffi et al., 2007). Consequently, studying close populations enables tests for a) measuring phenotypic plasticity (Rowe, 1994), b) assessing the extent of adaptation to a fluctuating resource (e.g., abiotic factors and food availability), and c) characterizing the biological advantage of phenotypic plasticity (Finkler, Steyermark and Jenks, 2004). In fact, comparing life-history traits within and among populations offers insights into the degree of variation and adaptive patterns relative to ecological constraints (Gibbons, Greene and Patterson, 1982; Rowe, 1994; Lomolino, 2005). Inter- and intra-population comparison and relationships of turtle growth rates have been studied for a long time, and can account for patterns of variability of life history traits (Berry and Shine, 1980; Iverson and Smith, 1993; Rowe, 1994).
Within fragmented and distant habitats, varying environmental conditions and evolutionary separation times may have led to variations in a) body size, shape and colour (Zuffi et al., 2007), b) sexual size dimorphism (Forsman and Shine, 1995; Lovich et al., 2010), and c) reproductive frequency and reproductive success (Brockelman, 1975; Elgar and Heaphy, 1989; Rowe, 1994; Zuffi, Di Benedetto and Foschi, 2004). Comprehending such biological patterns is particularly valuable for determining the relative importance of natural selection and phenotypic plasticity (Berry and Shine, 1980; Bonnet et al., 2001; Finkler, Steyermark and Jenks, 2004; Goodman, 2006; Zuffi et al., 2007). In addition, environment and cladogenesis affect the evolution of turtle shell (Claude et al., 2003; Chiari et al., 2009), which markedly influences life-history patterns (Gibbons, Greene and Patterson, 1982; Congdon and Gibbons, 1983; Claude et al., 2003).
We studied the European pond turtle (Emys orbicularis) because it is a long-living reptile (Rollinat, 1934; Fritz, 2003), abundant in many Italian areas (Zuffi, 2000), can lay multiple clutches per year (Zuffi and Odetti, 1998; Kotenko, 2000), and whose reproductive variation strongly correlates to localities and habitat features (Mitrus and Zemanek, 1998; Zuffi, Odetti and Meozzi, 1999; Kotenko, 2000; Zuffi et al., 2007). Despite the species being listed in the EU Habitat Directive and in several European Red Lists, its biology still deserves further research and information.
Our research was aimed at documenting the variation of life history traits of two close populations of E. orbicularis experiencing the same climate. We wish to answer the following questions: i) do turtles of proximate populations differ in body size? ii) do size differences convey information on female reproductive status (i.e., reproductive females are bigger than non-reproductive females)?, iii) does clutch frequency remain constant among years within and between populations? and iv) does reproductive output vary between close populations?
Materials and methods
Study areas
The study populations were at Tenuta di San Rossore (SR) (average data: 9 m asl, 43°42′51″N, 10°18′30″E; females) and at the US Army Camp Darby (CD) areas (average data: 10 m asl, 43°38′03″N, 10°20′11″E; females), both in the Natural Regional park of “Migliarino San Rossore Massaciuccoli” (Pisa, Tuscany, central Italy). Actual geographic limits of sampling area are 43°43′52.75″N, 10°18′51.20″E (northernmost limit); 43°42′1.98″N, 10°17′36.08″E (northwestern limit), 43°37′45.51″N, 10°20′47.22″E (southeastern limit) and 43°36′38.66″N, 10°19′27.00″E (southernmost limit) (fig. 1). We considered freshwater turtles under study as belonging to two different populations due to the following reasons. Sampling areas are very close (from a maximum of 13 km to a minimum of 7 km), are separated by River Arno (circa 200 m wide) and about 300 to 1500 m of houses and agricultural fields on each side of River Arno. Despite the closeness of the sites, the sites differed in vegetation and human structures (present research). The northern area, San Rossore, has human access by only park rangers, and is characterized by several habitats, many of them favoured by Emys: i) retrodunal marshy areas, often bordered by mixed woods (Quercus and Pinus), ii) artificial drainage canals, crossing West to East and North to South, bordering and/or passing through wooded areas; iii) isolated ponds, mainly located in the forested areas; some also in clearings of woods or external to them.
Geographic position of Emys orbicularis study areas near Pisa, Italy. Pins represent the extreme geographic position of northern site (San Rossore, SR) and southern site (Camp Darby, CD) (43°43′52.75″N, 10°18′51.20″E, northernmost limit; 43°42′1.98″N, 10°17′36.08″E, northwestern limit; 43°37′45.51″N, 10°20′47.22″E, southeastern limit; 43°36′38.66″N, 10°19′27.00″E, southernmost limit). The River Arno separates the two sites.
Citation: Amphibia-Reptilia 36, 4 (2015) ; 10.1163/15685381-00003009
The southern area, Camp Darby, is totally protected by external electric fences and military personnel, is part of a much larger area, the Tenuta di Tombolo. Since the establishment of U.S. military community in the 1970s, the introduction of a dozen fallow deer (Dama dama) led to a population of about 900 deer, which exceeded 1400 in the 1990s (Parco internal reports; A. Perfetti, pers. comm.). These herbivores overgrazed and substantially changed the herbaceous, bushy and arboreal physiognomy of the area, virtually eliminating all small and medium plants. The water system is predominantly based on rectilinear, North-South and West-East oriented canals. Ponds are limited in number (circa ), of small size (circa 0.1-0.2 ha each) and distributed almost exclusively in the wooded part of Camp Darby.
Historical scenario and genetic assignment
The Tuscan coastline reached its current shape about 3000 years ago (Pasquinucci et al., 2001; Carboni et al., 2010; Pasquinucci and Menchielli, 2012), but the superficial waters (ponds and other wetlands) have been drained by humans for more than 2000 years (Pasquinucci et al., 2001; Pasquinucci and Menchielli, 2012). Habitat has been affected by humans for centuries, strongly reducing available habitat for Emys orbicularis, by increasing human settlements and forest cutting. Thus, distribution of suitable habitats for amphibians and for freshwater turtles has been changed quite rapidly since their original formation (3500-3000 yrs BP) and present distribution and status of wet natural areas may be considered, without doubt, a very recent phenomenon. Although the genetic status of these local populations has not been investigated, pond turtle populations of the Italian west coasts belong to haplotype V (Lenk et al., 1999).
Reproductive assessment
Each captured female was marked with notches on shell marginal scales following the method of Stubbs et al. (1984) and numbered white labels on both sides of carapace for recognition at a distance (Zuffi, Odetti and Meozzi, 1999). We examined 510 E. orbicularis adult females distinguished from males using secondary sexual characteristics, including their plain (single colour) and large plastron, a tail shorter than half of plastron length, a carapace length > 9 cm, a more domed carapace than in males, and pale head colouration (Zuffi, Odetti and Meozzi, 1999). The minimum straight carapace length at which we could distinguish sexes from these characters in these populations of pond turtles E. orbicularis was 100 mm (Zuffi and Odetti, 1998). However, the smallest female to experience her first ovulation in this study had a carapace length of 118 mm (with follicular ova larger than 12 mm diameter, or shelled eggs; Zuffi et al., 2015). During the ovulation and egg-laying periods (early May to late July; see Zuffi et al., 2007 and references therein), we assessed the reproductive condition (reproductive females, RF, and non-reproductive females, NRF) of a smaller sample of females (), using inguinal palpation to detect gravid or non-gravid status (which is not 100% accurate), ultrasonography (for detecting follicles, vitellogenesis and oviductal eggs), and X-ray radiography to detect shelled eggs and quantify clutch size (Gibbons and Greene, 1979; Hinton et al., 1997; Keller, 1998; Zuffi, Odetti and Meozzi, 1999). Consequently, the sample size of females that bore follicles () differed from sample size of gravid females (). We weighed laid eggs, calculated clutch mass as the sum of egg masses, and calculated relative clutch mass [(clutch mass/pre-ovulating female body mass) × 100]. These data were arcsin transformed to correct for non-normal distributions. The number of females varied from 1996 to 2002, as 53, 87, 86, 34, 27, 12 and 29, respectively.
Body size and body condition index
We measured turtles with standard methods, following Zuffi, Odetti and Meozzi (1999): including carapace length (straight line) and width, plastron length and width, shell height, tail length (from cloacal opening to tail tip), and body mass (BM). The measurement resolution of the digital calliper and electronic pesola were ±1 mm and ±1 g, respectively. To calculate body condition index (BCI), we used the ellipsoid formula (Loehr, Hofmeyr and Henen, 2007) as π × carapace length × shell height × carapace width/6000 to estimate shell volume (SV), then we calculated BCI (BM/SV, as g/cm3). BM was controlled to SV with an ANCOVA to determine if groups (sites, years, sites and years) differ. BCI was used to express biometrically the physical characteristics of females as a single variable.
Age estimation and statistical analyses
We estimated individual age by averaging the carapace growth rings counted on the first and second costal scutes of both sides. This method differed from the method used by Keller, Andreu and Ramo (1998), who counted left pectoral or abdominal scutes of the plastron. The maximum number of detectable growth rings was 20, but older females may have lost or worn scute lamellae, and we likely underestimated their age. Thus the estimated age ranged from younger classes up to 18-20 years (but see Keller, Andreu and Ramo, 1998; Fritz, 2003) was correlated to all morphological variables using Spearman’s bivariate correlation (correlation coefficient, r): body mass had the higher r (r = 0.855, , ). The best fit line was logarithmic model: (adj. R2 = 0.736, ANOVA F1,34 = 89.144, ). We used this equation to estimate age for heavier (larger and older) individuals.
We first tested all variables for normality and log-transformed non-normal variables to try to meet normal distributions. Overall sample differences were tested with parametric or non-parametric tests according to the subsequent normal or non-normal distributions. First, dependent variables were compared between the two localities via Students’ t-tests or one way-ANOVA (yearly differences in size). MANOVA was used to evaluate variation in reproductive status (i.e., RF vs NRF) with body size, body condition, year and locality as independent factors. Following ANOVA and MANOVA, we used the Tukey post-hoc test to distinguish which samples differed.
We used standardized residuals, from linear regressions against body mass (independent variable) of clutch size, clutch mass and egg mass to analyse variation in reproductive output. Analysis of variation of normal and non-normal data between populations or among clutch frequencies (e.g., two clutches, one clutch, no clutch) was performed with Student’s t test or Kruskal-Wallis test, respectively. Kurtosis and skewness were calculated using significance of critical values (http://mvpprograms.com/help/mvpstats/distributions/SkewnessCriticalValues). Values are presented as mean ± 1 SD. Significance level was set at . Statistical analyses were performed with SPSS 21.0 (SPSS Inc., Chicago IL, USA).
Results
Female size and mass
We sampled 124 NRF and 260 RF: 45 NRF and 90 RF in San Rossore (SR), and 79 NRF and 170 RF in Camp Darby (CD), respectively. Reproductive and non-reproductive females of SR were similar in size for all shell measures (Student’s t test, table 1). At CD, reproductive females were larger and heavier than NRF in all the selected parameters (table 1). For reproductive females, SR females were larger than CD females in carapace length and height, plastron width, and body mass [with the exception of carapace width, , all with P ranging from 0.043 (plastron width) to 0.0001 (all the other variables)]. Similarly for NRF, SR females were larger than CD females in all variables (with ) with the exception of carapace width ().
Mean (± 1 standard deviation) adult female body size (mm), mass (g), shell volume (cm3), and body condition index (BCI = body mass/shell volume) from two close sites (SR = San Rossore, CD = Camp Darby) near Pisa, Italy. Statistical differences between sites (top versus bottom) and between reproductive and non-reproductive females within sites (final column), based on Students’ t-tests (n = sample size, P = probability), with ** & * emphasising highly significant & significant results, respectively).
Shell volume had a normal distribution (average 396.04 ± 86.13 cm3; , Kolmogorov-Smirnov , ), and was similar between NRF and RF of San Rossore, while it differed in Camp Darby females (table 1). Shell volume did not vary interannually, even when looking at all possible interactions. Post-hoc tests, however, revealed that shell volume in 1999 and 2000 was larger than in 1996, 1997 and 1998 (Tukey’s honestly significant difference test; , < 0.0001, = 0.001 and = 0.002, = 0.003, and = 0.015 respectively).
Body condition index (BCI)
The correlation between body mass and shell volume was very marked (CD site, 0.939, , ; SR site, 0.875, , ). ANCOVA analysis (BM as dependent variable, SV as covariate, site and year as factors and in interaction) gave significant results (model, 10 df, ), with a strong effect of locality (1 df, ), year (6 df, ) and shell volume (1 df, ), but no interaction between locality and year (2 df, ). The BCI of sexually mature females was ⩾1.147 and normal (, Kolmogorov-Smirnov , ). BCI was higher in RF than in NRF (table 1), and varied significantly only between females producing one clutch and those non reproductive (RFtwoclutches 1.158 ± 0.095, RFoneclutch 1.155 ± 0.098, NRF 1.102 ± 0.089; ANOVA, F2,191 = 2.463, ; LSD post-hoc test RFoneclutch vs NRF, ). However, the CD RF had BCI significantly lower than that of SR RF (Mann-Whitney , ; table 1).
Clutch-size variability between areas and among years
Median clutch size did not differ between sites, and distributions were not platykurtotic or leptokurtotic (SR and CD kurtosis ). However, both SR and CD distributions were skewed or asymmetric. Furthermore, clutch size did not vary among years at both sites (table 2). Females bearing one clutch (via palpation, ultrasonography and x-rays, ) were more frequent than those females producing two clutches (), even if not significantly different (72 vs 13 in SR and 103 vs 20 in CD; Chi Square test = 0.035, 1 df, ). Both sites had similar frequencies of multiple clutches (CD = 19.41% and SR = 18.05%).
Mean (± 1 standard deviation) clutch size among years at two close sites (SR = San Rossore, CD = Camp Darby) near Pisa, Italy. n = sample size; H, Kruskal-Wallis test; P = probability.
Age and reproductive output
The smallest female that became reproductive, was at a 118 mm carapace length, a body mass of 280 g (fig. 2), and at an age of about 14 or 15 years. The estimated age of adult females was greater for SR females than that for CD females (41.9 ± 8.9 yrs vs 35.1 ± 7.8 yrs, respectively; Students’ [not homogeneous variances] test = 8.647, ).
Relationship of body mass (g) to carapace length (mm) for gravid (dark grey 568 circles) and non-gravid (light grey circles) female Emys orbicularis. The dotted lines represent the lower limits for reproductive females.
Citation: Amphibia-Reptilia 36, 4 (2015) ; 10.1163/15685381-00003009
Standardized residuals of clutch size, clutch mass and egg mass did not differ between SR and CD (table 3). However, relative clutch mass (RCM) differed between areas, with CD females having a higher RCM than SR females (table 3). The distribution of RCM was significantly kurtotic in CD than in SR (CD kurtosis = −0.132, ; SR kurtosis = −0.695, ).
Mean (± 1 standard deviation) of clutch size, clutch mass, egg mass (analyses on standardized residuals) and relative clutch mass (arcsin transformed) between the two close sites (SR = San Rossore, CD = Camp Darby; t = Student’s t-test; P = probability value).
Discussion
Female body size variability
Differences in body size among populations are a widespread pattern in most chelonian species, and may be related to latitude (Iverson et al., 1993; Sacchi et al., 2007) and climate or site-dependent reproductive strategies and habitat features (Rowe, 1994; Forsman and Shine, 1995; Zuffi, Di Benedetto and Foschi, 2004; Dodd and Dreslik, 2008; Rivera, 2008). More specifically, population differences in body size have been found between very close areas, less than 15 km (Zuffi, Di Benedetto and Foschi, 2004; Zuffi et al., 2007), but also among localities at a larger scale (from 37 to 115 km: Rowe 1994; more than 700 km: Zuffi et al., 2007; 150-250 km: Lovich et al., 2010). Our results show clearly that variability of freshwater turtle body size may be tracked also at a small scale, given a long term research effort. Such long-term studies of size variation may help explain ecological, evolutionary, and perhaps human influences on the success of chelonians and similar oviparous ectotherms.
Habitat effects and proximal causes
Why did we find small but statistically different body sizes (and by correlation age) between two close populations of E. orbicularis? Different treatments of areas and habitats, as result of the deep modifications started from the Romans till present times (Pasquinucci et al., 2001), likely shaped current habitats and might be the first proximal cause of the differences between San Rossore and Camp Darby females. Specifically, CD was deeply changed when the US military base was established (1951), with severe tree-cutting along canals, the building of several structures and drainage canals, and the introduction of fallow deer. This may have induced a different amount of water and food availability, even if these factors have been not monitored before and after the base construction. In parallel, the tree cutting and canal construction, with many bulldozers and tractors, should have altered the original habitats (prior to the US military base), especially those of nesting areas (in open areas, close to wet areas) (G. Tognetti, pers. com.); it may have destroyed nests and caused the death of many large individuals hibernating in the canals. Furthermore, factors other than size, e.g., as food income (Ottonello, Salvidio and Rosecchi, 2005), or parasite load (Satorhelyi and Sreter, 1993) could affect offspring quality, clutch frequency and offspring viability of reproduction (Gibbons, Greene and Patterson, 1982; Pearse and Avise, 2001; Galeotti et al., 2005). We must experiment to evaluate whether these size and rate differences explain why CD females are small, especially the non-reproductive, CD females. Earlier studies (Zuffi et al., 2006) suggest the same pattern exists among male E. orbicularis of the two populations, and that likely either food intake and growth rates are slower at CD, or that mortality rates differ between the two sites.
Female body size and reproductive output relationships
The body size (e.g., body mass)-clutch size relationship was evident as in most species (Congdon et al., 1987; Zuffi et al., 2007; Rollinson and Brooks, 2008). However, differences of body size in reproductive females at the two sites did not correspond to differences in reproductive output (e.g., both populations have a similar clutch size, and clutch size did not differ among years), supporting the general findings (e.g. clutch size correlated to body size) of many turtle populations (Kotenko, 2000; Lovich et al., 2012). Yet, an inverse relationship is evident when considering resource allocation strategy (e.g. relative clutch-mass investment) (Zuffi et al., 2007), a known pattern also in other turtle populations (Congdon and Gibbons, 1983). The larger clutch mass for SR females is consistent with a body size effect (Gibbons et al., 1981; Rowe, 1994). The higher relative clutch mass of CD females emphasizes a different matter: smaller females tend to invest more, relative to their body size, in reproduction than do larger females (Rowe, 1994; Zuffi et al., 2007). A higher relative clutch mass can contribute, along with egg laying success, hatching rate (Zuffi and Odetti, 1998) and hatchling survivorship, to a higher lifetime reproductive success, that it could represent a bit higher fitness in CD females than in SR females, or a compensation for higher disturbance, and possibly higher mortality in early life stages, at CD. Nevertheless, we do not actually hypothesise if such a situation might lead to a differential, higher, density of CD population vs the SR population. To address these questions we need more data and analyses.
It is also reasonable to consider why CD females produced eggs at a smaller body size and at younger age than SR females. Fallow deer at CD, disturbance pond and canal margins in crucial places for pond turtle nesting (Zuffi and Rovina, 2006), and nest digging (Zuffi et al., 1999), destroying nests and reducing hatching success This may have caused the anticipation of female sexual maturation at a relatively smaller body size and the higher frequency of double clutching. Possible adaptive benefits of laying multiple clutches are larger the number of clutches and lesser the probability of mechanical destruction of all the nests (Zuffi et al., 1999; Zuffi and Rovina, 2006). It is however also possible that we missed the reproduction of a few SR NRF because they had already laid their eggs when measured (e.g., we suppose that large females may even reproduce earlier due to larger nutrient reserves, and to the higher BCI of SR females). Despite this, our relatively large sample size and captures quite early in the reproductive season, should have limited a bias towards the larger, NRF of SR; we should have detected a similar result in CD females.
Long term studies contribution
Long term studies on freshwater turtle reproduction have been relatively uncommon but there have been several (Congdon et al., 1987; Iverson and Smith, 1993; Rowe, 1994; Keller et al., 1998; Finkler, Steyermark and Jenks, 2004; Rollinson and Brooks, 2008; Hensley et al., 2010). However, data on the temporal variations of morphological and reproductive patterns in European pond turtles are limited to a few studies (Keller, 1998; Keller et al., 1998; Mitrus, 2006; Zuffi et al., 2007). Despite this low number of long-term studies, it clearly emerged that large, prolonged, data sets are powerful in determining population dynamics (Keller et al., 1998; Mitrus, 2006), individual characteristics (Zuffi, Odetti and Meozzi, 1999; Zuffi, Di Benedetto and Foschi, 2004) and ecological variability. In chelonians that produce multiple clutches each year, clutch size and clutch frequency depend on many factors, such as maternal age (Congdon et al., 2003), body size (Gibbons et al., 1981), the duration of active season, latitude (Iverson et al., 1993; Iverson and Smith, 1993; Zuffi, Di Benedetto and Foschi, 2004; Zuffi et al., 2007), and local ecological factors (i.e., predator abundance; Lanszki et al., 2006; Zuffi and Rovina, 2006). Without the long-term studies of E. orbicularis at SR and CD (Zuffi, Odetti and Meozzi, 1999; Zuffi, Di Benedetto and Foschi, 2004; Zuffi and Rovina, 2006; Zuffi et al., 2007, 2015), we would know very little about its spatial and temporal variation in size, rates, reproduction and demography in response to habitat variation.
Concluding remarks
The unknown age of most of the large, heavy and old turtles or tortoises limits our predictive models of turtle demography and life-history traits (Cordero Rivera and Fernandez, 2004). The estimated age of females in our study area was compatible with the size of the studied European pond turtles (Keller et al., 1998; Fritz, 2003). The significant size and age differences between CD and SR were related to different population structures of the two sites. We do not completely understand why this difference occurs, and additional research may help in explaining the patterns we observed. The size, age and reproductive differences could be induced by local differences in food availability and consumption, with habitat disturbances (e.g., impacts of deer on vegetation, and human production of canals and ponds) increasing the small fish, invertebrates and vegetation available to small E. orbicularis, but not to larger E. orbicularis (e.g., of SR). Similarly, the deer-disturbed habitat and increased pond and canal structure of CD may reduce competition for basking sites, facilitating the survival of younger, smaller individuals at CD. By addressing these aspects of E. orbicularis biology at these two sites, we will have a better understanding of their biology, and a better understanding how to manage their populations in human-transformed landscapes.
Acknowledgements
We wish particularly to thank the U.S. Air Force Personnel and the Italian Military Officer P. Bellinvia at Camp Darby, the Direction and staff of Parco di Migliarino, S. Rossore, Massaciuccoli for study permissions and logistic support. M.E. Fabbri, M. Giusti, F. Odetti, L. Rovina, A. Teti provided valuable help in the field. S. Citi (Veterinary Clinic Department, University of Pisa) kindly provided ultrasonographic and X-ray analyses. Miss G. Candolino and Mr. D.N. Vinsanto provided useful discussions on a previous draft of the manuscript. A special thank to the three anonymous referees, to Sylvain Ursenbacher and Uwe Fritz who highly improved previous versions of this paper with comments, criticisms and many useful suggestions and literature records.
References
Aubret F., Bonnet X., Shine R., Lourdais O. (2002): Fat is sexy for females but not males: the influence of body reserves on reproduction in snakes (Vipera aspis). Horm. Behav. 42: 135-147.
Berry J.F., Shine R. (1980): Sexual size dimorphism and sexual selection in turtles (order Testudines). Oecologia 44: 185-191.
Bonnet X., Lagarde F., Henen B.T., Corbin J., Nagy K.A., Naulleau G., Balhoul K., Chastel O., Legrand A., Cambag R. (2001): Sexual dimorphism in steppe tortoises: influence of the environment and sexual selection on body shape and mobility. Biol. J. Linn. Soc. 72: 357-372.
Brockelman W.Y. (1975): Competition, the fitness of offspring, and optimal clutch size. Am. Nat. 109: 677-699.
Bulté G., Irschick D.J., Blouin-Demers G. (2008): The reproductive role hypothesis explains trophic morphology dimorphism in the northern map turtle. Funct. Ecol. 22: 824-830.
Carboni M.G., Bergamin L., Di Bella L., Esu D., Pisegna Cerone E., Antonioli F., Verrubbi V. (2010): Palaeoenvironmental reconstruction of late Quaternary foraminifera and molluscs from the ENEA borehole (Versilian plain, Tuscany, Italy). Quaternary Research 74: 265-276.
Chiari Y., Hyseni C., Fritts T.H., Glaberman S., Marquez C., Gibbs J.P., Claude J., Caccone A. (2009): Morphometrics parallel genetics in a newly discovered and endangered taxon of Galápagos Tortoise. PlosOne 4: e6272.
Christiansen J.L., Moll E.O. (1973): Latitudinal reproductive variation within a single subspecies of painted turtle, Chrysemys picta bellii. Herpetologica 29: 152-163.
Claude J., Paradis E., Tong H., Auffray J.-C. (2003): A geometric morphometric assessment of the effects of the environment and cladogenesis on the evolution of the turtle shell. Biol. J. Linn. Soc. 79: 485-501.
Congdon J.D., Breitenbach G.L., Van Loben Sels R.C., Tinkle D.W. (1987): Reproduction and nesting ecology of snapping turtles (Chelydra serpentina) in Southeastern Michigan. Herpetologica 43: 39-54.
Congdon J.D., Gibbons J.W. (1983): Relationships of reproductive characteristics to body size in Pseudemys scripta. Herpetologica 39: 147-151.
Congdon J.D., Gibbons J.W. (1987): Morphological constraint on egg size: a challenge to optimal egg size theory? Proc. Natl. Acad. Sci. USA 84: 4145-4147.
Congdon J.D., van Loben Sels R.C. (1991): Growth and body size in Blanding’s turtles (Emydoidea blandingi): relationships to reproduction. Can. J. Zool. 69: 239-245.
Congdon J.D., Nagle R.D., Kinney O.M., van Loben Sels R.C., Quinter T., Tinkle D.W. (2003): Testing hypotheses of aging in long-lived painted turtles (Chrysemys picta). Experim. Gerontol. 38: 765-772.
Cordero Rivera A.C., Fernandez C.A. (2004): A management plan for the European pond turtle (Emys orbicularis) population of the Louro river basin (Northwestern Spain). Biologia 59: 161-171.
Dodd C.K., Dreslik M.J. (2008): Habitat disturbances differentially affect individual growth rates in a long-lived turtle. J. Zool., Lond. 275: 18-25.
Elgar M.A., Heaphy L.J. (1989): Covariation between clutch size, egg weight and egg shape: comparative evidence for chelonians. J. Zool., Lond. 219: 137-152.
Finkler M.S., Steyermark A.C., Jenks K.E. (2004): Geographic variation in snapping turtle (Chelydra serpentina serpentina) egg components across a longitudinal transect. Can. J. Zool. 82: 102-109.
Ford N.B., Seigel R.A. (1994): An experimental study of the trade-offs between age and size at maturity: effects of energy availability. Funct. Ecol. 8: 91-96.
Forsman A., Shine R. (1995): Sexual size dimorphism in relation to frequency of reproduction in turtles (Testudines: Emydidae). Copeia 1995: 727-729.
Fritz U. (2003): Die Europäische Sumpfschildkröte (Emys orbicularis). Laurenti Verlag, Bielefeld.
Galeotti P., Sacchi R., Pellitteri Rosa D., Fasola M. (2005): Female preference for fast-rate, high-pitched calls in Hermann’s tortoises Testudo hermanni. Behav. Ecol. 16: 301-308.
Gibbons J.W., Greene J.L. (1979): X-ray photography: a technique to determine reproductive patterns of freshwater turtles. Herpetologica 35: 86-89.
Gibbons J.W., Greene J.L., Patterson K.K. (1982): Variation in reproductive characteristics of aquatic turtles. Copeia 1982: 776-784.
Gibbons J.W., Semlitsch R.D., Greene J.L., Schubauer J.P. (1981): Variation in age and size at maturity of the slider turtle (Pseudemys scripta). Am. Nat. 117: 841-845.
Goodman B.A. (2006): Costs of reproduction in a tropical invariant-clutch producing lizard (Carlia rubrigularis). J. Zool., Lond. 270: 236-243.
Hensley F.R., Jones T.R., Maxwell M.S., Adams L.J., Stevenson Nedella N. (2010): Demography, terrestrial behavior, and growth of sonora mud turtles (Kinosternon sonoriense) in an extreme habitat. Herpetol. Monogr. 24: 174-193.
Herrel A., Huyghe K., Vanhooydonck B., Backeljau T., Breugelmans K., Grbac I., Van Damme R., Irschick D.J. (2008): Rapid large-scale evolutionary divergence in morphology and performance associated with exploitation of a different dietary resource. Proc. Natl. Acad. Sci. USA 105: 4792-4795.
Herrel A., O’Reilly J.C., Richmond A.M. (2002): Evolution of bite performance in turtles. J. Evol. Biol. 15: 1083-1094.
Hinton T.G., Fledderman P.D., Lovich J.E., Congdon J.D., Gibbons J.W. (1997): Radiographic determination of fecundity: is the technique safe for developing turtle embryos? Chel. Cons. Biol. 2: 409-414.
Iverson J.B., Balgooyen C.P., Byrd K.K., Lyddan K.K. (1993): Latitudinal variation in egg and clutch size in turtles. Can. J. Zool. 71: 2448-2461.
Iverson J.B., Smith G.R. (1993): Reproductive ecology of the painted turtle (Chrysemys picta) in the Nebraska sandhills and across its range. Copeia 1993: 1-21.
Keller C. (1998): Assessment of reproductive state in the turtle Mauremys leprosa: a comparison between inguinal palpation and radiography. Wildlife Res. 25: 527-531.
Keller C., Andreu A.C., Ramo C. (1998): Aspects of the population structure of Emys orbicularis hispanica from southwestern Spain. Mertensiella 10: 147-158.
Kotenko T.I. (2000): The European pond turtle (Emys orbicularis) in the Steppe zone of the Ukraine. In: Die Europäische Sumpschildkröte, p. 87-106. Hödl, W., Rössler, M., Eds, Stapfia 69, 149. Landesmuseums, Linz.
Lagarde F., Bonnet X., Henen B., Nagy K., Corbin J., Lacroix A., Trouvé C. (2003): Plasma steroid and nutrient levels during the active season in wild Testudo horsfieldi. Gen. Comp. Endocrin. 134: 139-146.
Lanszki J., Molnár M., Molnár T. (2006): Factors affecting the predation of otter (Lutra lutra) on European pond turtle (Emys orbicularis). J. Zool., Lond. 270: 219-226.
Lee M.S.Y. (1996): Correlated progression and the origin of turtles. Nature 379: 812-815.
Lenk P., Fritz U., Joger U., Wink M. (1999): Mitochondrial phylogeography of the European pond turtle, Emys orbicularis (Linnaeus 1758). Mol. Ecol. 8: 1911-1922.
Loehr V.J.T., Hofmeyr M.D., Henen B.T. (2007): Annual variation in the body condition of a small, arid-zone tortoise, Homopus signatus signatus. J. Arid Environ. 71: 337-349.
Lomolino M.V. (2005): Body size evolution in insular vertebrates: generality of the island rule. J. Biogeogr. 32: 1683-1699.
Lourdais O., Bonnet X., Doughty P. (2002): Costs of anorexia during pregnancy in a viviparous snake (Vipera aspis). J. Exp. Zool. 292: 487-493.
Lovich J.E., Madrak S.V., Drost C.A., Monatesti A.J., Casper D., Znari M. (2012): Optimal egg size in a subotpimal environment: reproductive ecology of female Sonora mud turtle (Kinosternon sonoriense) in cental Arizona, USA. Amphibia-Reptilia 33: 161-170.
Lovich J.E., Znari M., Baamrane M.A.A., Naimi M., Mostalih A. (2010): Biphasic geographic variation in sexual size dimorphism of turtle (Mauremys leprosa) populations along an environmental gradient in Morocco. Chelon. Conserv. Biol. 9: 45-53.
Marco A., Díaz-Paniagua C., Hidalgo-Vila J. (2004): Influence of egg aggregation and soil moisture on incubation of flexible-shelled lacertid lizard eggs. Can. J. Zool. 82: 60-65.
Mitrus S. (2006): Spatial distribution of nests of the European pond turtle Emys orbicularis (Reptilia: Testudines: Emydidae), from long-term studies in central Poland. Zoologische Abhandlungen (Dresden) 55: 95-102.
Mitrus S., Zemanek M. (1998): Reproduction of Emys orbicularis (L.) in central Poland. Mertensiella 10: 187-191.
Ottonello D., Salvidio S., Rosecchi E. (2005): Feeding habits of the European pond terrapin Emys orbicularis in Camargue (Rhône delta, Southern France). Amphibia-Reptilia 26: 562-565.
Pasquinucci M., Menchielli S. (2012): Landscape transformation in North coastal Etruria. In: Landscape Archaeology Between Art and Science. From a Multi- to an Interdisciplinary Approach, p. 179-196. Kluiving S.J., Guttmann-Bond E., Eds, Amsterdam University Press.
Pasquinucci M., Menchielli S., Mazzanti R., Marchisio M., D’Onofrio L. (2001): Coastal archaeology in North Etruria. North coastal Etruria. Geomorphologic, archaeological, archive, magnetometric and geoelectrical researches. Revue Archéom. 25: 187-201.
Pearse D.E., Avise J.C. (2001): Turtle mating system: behavior, sperm storage and genetic paternity. J. Hered. 92: 206-211.
Rivera G. (2008): Ecomorphological variation in shell shape of the freshwater turtle Pseudemys concinna inhabiting different aquatic flow regimes. Integr. Comp. Biol. 48: 769-787.
Rollinat R. (1934): La vie des reptiles de la France centrale. Libraire Delagrave, Paris.
Rollinson N., Brooks R.J. (2008): Optimal offspring provisioning when egg size is “constrained”: a case study with the painted turtle Chrysemys picta. Oikos 117: 144-151.
Rovero F., Chelazzi G. (1996): Nesting migrations in a population of the European pond turtle Emys orbicularis (L.) (Chelonia, Emydidae) from central Italy. Ethol. Ecol. Evol. 8: 297-304.
Rowe J.W. (1994): Egg size and shape variation within and among Nebraskan painted turtle (Chrysemys picta bellii) populations: relationships to clutch and maternal body size. Copeia 1994: 1034-1040.
Sacchi R., Pupin F., Pellitteri Rosa D., Fasola M. (2007): Bergmann’s rule and the Italian Hermann’s tortoises (Testudo hermanni): latitudinal variations of size and shape. Amphibia-Reptilia 28: 43-50.
Satorhelyi T., Sreter T. (1993): Studies on internal parasites of tortoises. Parasitol. Hung. 26: 51-55.
Seigel R., Collins J. (1993): Snakes: Ecology and Behavior. McGraw-Hill, New York.
Stubbs D., Hailey A., Pulford E., Tylor W. (1984): Population ecology of European tortoises: review of field techniques. Amphibia-Reptilia 5: 57-68.
Warner D.A., Bonnet X., Hobson K.A., Shine R. (2008): Lizards combine stored energy and recently acquired nutrients flexibly to fuel reproduction. J. Anim. Ecol. 77: 1242-1249.
Zuffi M.A.L. (2000): Conservation biology of the European pond turtle, Emys orbicularis, of Italy. In: Die Europäische Sumpfschildkröte, p. 219-228. Hödl, W., Rössler, M., Eds., Stapfia 69 149, Linz.
Zuffi M.A.L., Celani A., Foschi E., Tripepi S. (2007): Reproductive strategies and body shape in the European pond turtle (Emys orbicularis) from contrasting habitats in Italy. J. Zool., Lond. 271: 218-224.
Zuffi M.A.L., Citi S., Foschi E., Marsiglia F., Martelli E. (2015): Into a box interiors: clutch size variation and resource allocation in the European pond turtle. Acta Herpetol. 10: 39-45.
Zuffi M.A.L., Di Benedetto M.F., Foschi M.E. (2004): The reproductive strategies in neighbouring populations of the European pond turtle, Emys orbicularis, in central Italy. Ital. J. Zool. 71 (Suppl. 2): 101-104.
Zuffi M.A.L., Odetti F. (1998): Double egg deposition in the European pond turtle, Emys orbicularis, from central Italy. Ital. J. Zool. 65: 187-189.
Zuffi M.A.L., Odetti F., Batistoni R., Mancino G. (2006): Morphometrics and genetics in the study of variability pattern of the European pond turtle, Emys orbicularis. Ital. J. Zool. 73: 363-372.
Zuffi M.A.L., Odetti F., Meozzi P. (1999): Body size and clutch-size in the European pond turtle, Emys orbicularis, from central Italy. J. Zool., Lond. 247: 139-143.
Zuffi M.A.L., Rovina L. (2006): Habitat characteristics of nesting areas and of predated nests in a Mediterranean population of the European pond turtle, Emys orbicularis. Acta Herpetol. 1: 37-50.
Footnotes
Associate Editor: Uwe Fritz.
