Humidity and seasonality drives body size patterns in males of the bush cricket Isophya rizeensis Sevgili, 2003 (Orthoptera: Tettigoniidae: Phaneropterinae)

Insect Science (2013) 00, 1–14, DOI 10.1111/1744-7917.12027

ORIGINAL ARTICLE

Humidity and seasonality drives body size patterns in malesof the bush cricket Isophya rizeensis Sevgili, 2003(Orthoptera: Tettigoniidae: Phaneropterinae)

Selim S. Caglar, Cagasan Karacaoglu, A. Cem Kuyucu and Ismail K. SaglamDepartment of Biology, Ecological Sciences Research Laboratories, Faculty of Science, Hacettepe University, Beytepe 06800, Ankara,

Turkey

Abstract Two primary patterns of body size variation have been recorded in ectothermsin relation to latitudinal/altitudinal shifts. In some, body size increases with increasinglatitude/altitude whereas, in others, body size decreases with increasing latitude/altitude.This clinal variation is generally assumed to be caused by local adaptation to environmentalconditions however the selective variable(s) (temperature, humidity, diet quality, etc.) isstill heavily debated. Here we investigate geographic variation in body size of dark and palecolor morphs of males of the bush-cricket Isophya rizeensis collected from 15 locationsalong an elevation gradient ranging from 350 to 2 500 m. Using an information theoreticalapproach we evaluate the relative support of four different hypotheses (the temperature sizerule, the moisture gradient hypothesis, the seasonal constraint hypothesis, and the primaryproductivity hypothesis) explaining body size variation along the altitudinal gradient. Bodysize variation in pale color morphs showed a curvilinear relationship with altitude whiledark color morphs showed no variation in body size. Body size variation in pale colormorphs was highly correlated with precipitation and temperature seasonality values thusgiving strong support for the moisture gradient and seasonal constraint hypothesis. Ourresults reinforce the importance of gradients in humidity and seasonality over temperaturein the creation of altitudinal body size clines and the role of selection for resistance tostress factors in the establishment of these clines. Whether a body size cline is observedor not might also depend on the phenotypic properties of the individuals, like coloration.

Key words altitude, Bergmann’s rule, converse Bergmann’s rule, Fırtına Valley,information–theoretic, temperature size rule

Introduction

As with many other ectotherms, insects show a mixed pat-tern of body size variation with environmental gradientsalong latitude/altitude (Blanckenhorn & Demont, 2004;

Correspondence: Ismail K. Saglam, Department of Biol-ogy, Ecological Sciences Research Laboratories Faculty of Sci-ence, Hacettepe University, Beytepe 06800, Ankara, Turkey.Tel: +90 312 297 6435; fax: +90 312 299 2028; email:[email protected]

Whitman, 2008; Chown & Gaston, 2010). Chown andGaston (2010) reviewed 74 insect species from 9 orders,in which 30 species showed an increase in body size withlatitude/altitude (Bergmann’s rule), whereas 32 speciesshowed a decrease in body size with latitude/altitude (con-verse Bergmann’s rule). In addition, 10 species showed noclear pattern of variation, while, in 2 species, body sizeshowed a curvilinear relationship.

Observed patterns of body size increase with lati-tude/altitude (i.e., Bergmannian size clines) are gener-ally accepted to be driven directly by temperature differ-ences along spatial gradients (Atkinson, 1994; Atkinson

C© 2013 Institute of Zoology, Chinese Academy of Sciences1

2 S.S. Caglar et al.

& Sibly, 1997). However, whether the observed patternsare an adaptive response (Partridge & Coyne, 1997; Reeveet al., 2000) or a form of phenotypic plasticity known asthe temperature size rule (Kingsolver & Huey, 2008) isstill debated. The temperature size rule states that the slopeof the thermal reaction norm for body size in ectothermsis negative (Van der Have & De Jong, 1996; Kingsolver &Huey, 2008) therefore ectotherms reared at high temper-atures will be smaller relative to those reared at low tem-peratures. Historically, the notion that Bergmannian sizeclines predominates the ectothermic world comes fromlaboratory studies showing the validity of the tempera-ture size rule for over 80% of the studied species (Ray,1960; Atkinson, 1994). However, more detailed studiesof insect thermal reaction norms show that body sizeis actually maximized at medium temperatures resultingin curvilinear reaction norms when insects are reared atbroad temperature ranges (Moreteau et al., 1997; Karanet al., 1999) and that different environments can modifythe temperature of maximum value (i.e., the maxima ofthe thermal reaction curve) (Morin et al., 1999). There-fore, at least for insects both plasticity of thermal reactionnorms (the temperature size rule) and adaptive responses(temperature of maximum value) seem to play a role inthe observed Bergmannian size clines.

Although most of the attention has been given to tem-perature as the proximate cause of Bergmannian sizeclines in insects, recent studies have shown that this pat-tern can also be caused by gradients in moisture (rain-fall and humidity) (Kennington et al., 2003; Stillwellet al., 2007). Studies show that large body sizes evolverapidly in response to low versus high humidity conditions(Kennington et al., 2003) and in the seed feeding beetleStator limbatus, Bergmannian size clines were driven bygradients in precipitation (humidity) rather than by tem-perature mediated selection on body size (Stillwell et al.,2007, 2008).

On the other hand, the predominant factor shapingconverse Bergmannian size clines (i.e., decreasing bodysize patterns with latitude/altitude) seems to be sea-sonal constraints, as opposed to temperature or humidity(Blanckenhorn & Demont, 2004; Whitman, 2008). Sea-sonal environments, constrain the amount of time or re-source necessary for growth and development (Johansson& Rowe, 1999; Johansson et al., 2001). As a result bodysize decreases at higher latitudes/altitudes as seasonalityincreases (Masaki, 1967; Fischer & Fiedler, 2002; Chown& Klok, 2003; Lehmann & Lehmann, 2008; Bidau et al.,2012). There is broad agreement that converse Bergman-nian size clines are often adaptive and genetic as smallersize and shorter development times of high latitude pop-ulations are also expressed in common garden labora-

tory settings (Berner & Blanckenhorn, 2006; Schutze& Clarke, 2008). Since seasonality is a more powerfullimiting factor for univoltine species than multivoltineones (Roff, 1980, 1992) converse Bergmannian size clinesare expected to be seen more often in univoltine insects(Walters & Hassall, 2006).

However, many factors besides temperature, humidity,and seasonal constraints can affect body size patterns ininsects. For example, diet can have an important impacton the outcome of clinal body size patterns with bodysize increasing or decreasing with latitude depending onthe quality and diversity of food resources (Diamond &Kingsolver, 2010; Ho et al., 2010). In addition, any char-acteristic of an individual’s capacity to attain and main-tain high body temperatures will affect optimal body size(Ahnesjo & Forsman, 2003; Pereboom & Biesmeijer,2003). For example, dark coloration, by increasing ther-mal capacity (Forsman, 1997; Forsman et al., 2002) canallow an organism to speed up its development withouta reduction in body size or may even result in increasedbody size (Ahnesjo & Forsman, 2003). Moreover, it hasbeen shown that along elevation gradients darker individ-uals are more prevalent at higher altitudes (cooler envi-ronments) while paler individuals are more readily foundat lower altitudes (known as Bogert’s rule, Gaston et al.,2009 or sometimes referred to as converse of Gloger’srule, Rapoport, 1969). This in turn can complicate bodysize patterns along an altitudinal gradient if color is nottaken into consideration.

In addition all of these factors are not mutually exclu-sive as interactions between temperature, seasonality, andvoltinism can produce nonlinear body size clines such asthe classic saw-tooth pattern (Roff, 1980, 1983; Mousseau& Roff, 1989) or the U-shaped latitudinal body size pat-terns observed in Odonata (Johansson, 2003; De Blocket al., 2008). Blanckenhorn and Demont (2004) suggestthat these two patterns might be part of a continuumof adaptive responses to temperature (Bergmannian sizeclines) or seasonal constraints (converse Bergmanniansize clines) acting jointly with countergradient variation(the ability of an organism to adjust to seasonal constraintsby evolving faster growth rates, Conover & Schultz, 1995)to produce the variety of clines observed.

Here, we report a body size trend with altitude in malesof the color polymorphic bush-cricket Isophya rizeensisdiffering from common expectations. Like most univol-tine species whose life cycle is limited by seasonal timeconstraints, we would expect I. rizeensis to show a de-crease in body size with increased elevation (i.e., theconverse Bergmann’s rule). However, while dark colormorphs of the species showed no body size pattern withaltitude, pale color morphs showed a concave pattern,

C© 2013 Institute of Zoology, Chinese Academy of Sciences, 00, 1–14

Humidity and seasonality drives body size 3

with body size reaching maximum values at mid alti-tudes. Therefore, pale color morphs would seem to obeythe temperature size rule observed in laboratory studiespredicting a curvilinear thermal reaction norm for bodysize.

To determine which environmental factor(s) might bedriving the observed pattern of body size variation inpale color morphs, we used an information–theoretical ap-proach together with high-resolution geographic climaticdatasets to evaluate the relative support of four differenthypotheses: (H1) the temperature size rule, which predictsa curvilinear relationship between body size and tem-perature; (H2) the moisture gradient hypothesis, whichpredicts a decrease in body size with increasing hu-midity/rainfall; (H3) the seasonal constraint hypothesis,which predicts a negative relationship between body sizeand seasonality; and (H4) the primary productivity hy-pothesis, which predicts a positive relationship betweenbody size and productivity. In addition, to evaluate whya body size pattern with altitude was lacking in darkcolor morphs, we tested for differences in several climaticvariables (temperature, temperature seasonality, precipi-tation) and plant productivity values between distribu-tional areas of the two-color morphs.

Material and methods

The species

The bush cricket I. rizeensis is a univoltine, rela-tively large (17.7–25.0 mm) flightless ground-living bushcricket endemic to Anatolia (Sevgili, 2003). The speciesis found only within and around the Fırtına Valley in theNortheast Mountain Ranges of Turkey occurring from350 m above sea level to 2 500 m (Fig. 1) (Saglam &Caglar, 2005). I. rizeensis overwinters in the egg stagewith nymph emergence occurring from mid to late Mayat lower altitudes (below 1500 m) and early to mid June athigher altitudes (over 1500 m). Development time is con-stant at all altitudes as maturation is completed in approx-imately 3 weeks and adults survive until mid July/earlyAugust (below 1 500 m) or late August/early September(over 1 500 m) (Saglam, 2004). Distribution of I. rizeensiswithin the Fırtına Valley is not continuous, but shows afragmented spatial pattern with the species appearing onlyin certain habitat patches made up of forb/herb and shrubvegetation (Saglam & Caglar, 2005). The most strikingfeature of this species is the high variability it shows inboth dorsal and ventral coloration, a feature, which is mostapparent in males. Moreover, this variation in colorationwas shown to have a distinct clinal pattern opposite to that

expected from converse Gloger’s/Bogert’s rule (Rapoport,1969; Gaston et al., 2009) with dark color morphs appear-ing in the warmer lowlands (350–1 000 m) and pale colormorphs appearing in the cooler subalpine zones of theKackar Mountains (1 000–2 500 m) (Saglam & Caglar,2005).

Sampling and field work

All bush crickets were collected during the months ofJune, July, and August of 2006. A total of 399 adult maleswere hand collected from 15 localities (i.e., subpopula-tions) at various altitudes within the Fırtına Valley (Fig. 1).All specimens were brought to the laboratory in individ-ual cages, killed using cyanide vials, photographed andlater preserved in 70% alcohol at 4◦C. Males were clas-sified into two distinct color morphs (dark and pale) ac-cording to dorsal coloration. In the dark color morph,dorsal coloration of the abdomen was usually black withyellow or red markings. On occasion, it was possible tofind dark green individuals with black markings. How-ever, these were infrequent (a total of 17 of 153). Thepale color morph showed relatively little intravariation asdorsal coloration of the abdomen was usually light greenwith two distinct yellow bands. Occasionally, it was pos-sible to find yellowish brown individuals. However, aswith the dark green color morph, frequencies were low(15 of 246). Therefore, we chose a two-category classifi-cation instead of a more detailed one since pale and darkcoloration seemed to be the most significant distinctionbetween color morphs. Coordinates of sampling localities(i.e., subpopulations) together with information on alti-tude, number of individuals collected from each localityand their respective color classes are given in Table 1.

Climatic and environmental data

Climatic data for each locality were extracted from theWorldClim dataset of interpolated global climate layers(approximately 1-km spatial resolution) developed frommonthly climate measurements of long-term observa-tions (1950–2000) of temperature and precipitation fromweather stations around the globe (Hijmans et al., 2005).Using a geographical information system (GIS), three bio-climatic variables; mean temperature of the active season(MTAS, derived from mean monthly temperatures fromMay to September), mean precipitation of the active sea-son (MPAS, derived from mean monthly precipitationfrom May to September), and TS (coefficient of varia-tion of mean monthly temperatures), were extracted foreach locality. Primary productivity of the active season



Fig. 1 Locations of the 15 sampling sites of dark (D1–D5) and pale (P1–P10) color morphs of male Isophya rizeensis along an elevationgradient within the Fırtına Valley, Camlıhemsin-Rize, Turkey. Detailed information of sampling sites is given in Table 1.

(PPAS) for each locality was measured by using thenormalized difference vegetation index (NDVI) of themonths of May to September derived from 1-min reso-lution time series maps (16-day periodicity) of MODISNDVI recorded from 2001 to 2004.

For each of the four hypotheses aiming to explain theobserved pattern of body size with altitude in pale colormorphs, we selected an environmental variable that wepredicted was most likely to underlie the given hypothesis.The hypotheses and their associated variables are as fol-lows: (H1) the temperature size rule, MTAS was chosen as

the predictive variable since body size patterns caused bythe temperature size rule or selection for differential tem-peratures are assumed to be a direct response to develop-mental temperatures; (H2) moisture gradient hypothesis,MPAS was chosen as the predictive variable representinggradients in moisture; (H3) seasonal constraint hypothe-sis, TS was chosen as the predictive variable representingseasonality of temperature; (H4) primary productivity hy-pothesis, mean May to September NDVI (PPAS) a mea-sure of plant productivity during the active season, wasused as the predictive variable.



Table 1 Summary data detailing altitude and coordinates of collection sites, color class of subpopulations, number of samplescollected from each subpopulation, means, and standard errors of the three morphometric characters measured, mean PC1 scores ofeach subpopulation and means of environmental variables at each altitude.

ALT PL PH TW MTAS MPASLOC LAT LON CL N PC1 TS PPAS

(m) (mm) (mm) (mm) (◦C) (mm)

D1 450 40.9856 40.9646 DARK 22 3.51 ± 0.03 1.35 ± 0.02 3.89 ± 0.04 − 0.94 65.90 18.62 88.40 441.66D2 620 40.9819 40.9662 DARK 11 3.51 ± 0.05 1.40 ± 0.02 4.00 ± 0.05 − 0.65 65.54 18.56 85.20 441.66D3 850 40.9406 40.9850 DARK 56 3.49 ± 0.05 1.58 ± 0.02 4.04 ± 0.04 − 0.77 73.70 17.78 63.60 332.84D4 880 40.9166 40.9456 DARK 15 3.41 ± 0.03 1.46 ± 0.02 3.94 ± 0.03 − 0.71 74.33 17.82 62.00 444.96D5 980 40.9075 40.9479 DARK 41 3.43 ± 0.04 1.52 ± 0.03 4.02 ± 0.04 − 0.86 75.66 17.58 59.00 573.18P1 830 40.9235 40.9505 LIGHT 24 3.61 ± 0.03 1.52 ± 0.02 3.92 ± 0.04 − 0.16 73.34 17.98 64.80 432.10P2 1030 40.8880 40.9297 LIGHT 28 3.78 ± 0.04 1.59 ± 0.02 4.13 ± 0.04 0.38 76.58 17.54 57.80 429.62P3 1260 40.8630 40.9342 LIGHT 39 3.77 ± 0.03 1.68 ± 0.02 4.21 ± 0.04 0.66 77.80 17.02 54.00 540.38P4 1300 40.8638 40.9501 LIGHT 14 3.74 ± 0.05 1.69 ± 0.03 4.11 ± 0.07 0.38 78.00 16.10 53.00 570.52P5 1500 40.8705 40.9385 LIGHT 26 3.86 ± 0.04 1.70 ± 0.02 4.29 ± 0.04 1.02 77.67 15.66 53.20 540.38P6 1610 40.8420 40.9453 LIGHT 20 3.88 ± 0.04 1.77 ± 0.02 4.33 ± 0.04 1.23 78.25 15.60 52.00 431.26P7 1900 40.8548 41.0125 LIGHT 12 3.74 ± 0.06 1.75 ± 0.02 4.39 ± 0.06 1.04 76.46 13.44 54.80 538.90P8 1930 40.7998 40.9217 LIGHT 17 3.77 ± 0.05 1.65 ± 0.02 4.31 ± 0.04 0.81 76.92 12.98 53.60 448.72P9 2070 40.7995 40.9588 LIGHT 31 3.56 ± 0.03 1.66 ± 0.02 4.14 ± 0.03 0.05 76.74 12.56 54.60 512.00P10 2300 40.7915 40.9574 LIGHT 30 3.58 ± 0.03 1.64 ± 0.02 4.12 ± 0.03 0.00 75.81 10.86 57.80 512.00

LOC, locality, codes refer to positions on Fig. 1; ALT, altitude; LAT, latitude; LON, longitude; CL, color; PL, pronotum length;PH, pronotum height; TW, tegmina width; TS, temperature seasonality; MTAS, mean temperature of the active season; MPAS, meanprecipitation of the active season; PPAS, primary productivity of the active season.

Morphometric measurements

Three external morphological characters (PH, prono-tum height; PL, pronotum length; TW, tegmina width)were chosen as body size measures. All characters werephotographed using a Leica MZ-7.5 stereoscopic zoomdissection microscope with a DC-300 digital camera sys-tem, digitalized, archived and measured using TPSdigsoftware (Rohlf, 2003). Of the 399 males, 386 individ-uals gave clear imagery and, therefore, only these maleswere used in the subsequent analyses.

Before analyses, all characters were screened for uni-variate normality (Kolmogorov–Smirnov test) and equal-ity of error variances (Levene’s test). In order to reduce thenumber of variables and to extract an allometric size vec-tor, a principal components analysis (PCA) was performedusing the variance–covariance matrix. PC1 explained 68%of the variance in pronotum height (PH), pronotum length(PL), and tegmina width (TW) and factor loadings forall characters were high (>0.80) and positive. Therefore,PC1 scores can be taken as a reliable estimate of overallbody size and were used as the dependent variable in allanalyses.

Means and standard errors of each measured morpho-metric character and mean PC1 scores representing bodysize at each locality for male I. rizeensis are given in

Table 1 along with means of climatic variables at eachlocality.

Analyses

The relationship between body size, color pattern andaltitude was analyzed using the full data set under thefollowing model: PC1 = Color + Altitude + Altitude2.Color pattern was entered as a categorical variable and al-titude was included as a continuous variable. A quadraticterm for altitude was added to the model after examina-tion of subpopulation means, which showed a curvilineardistribution with altitude.

Correlations between environmental variables and alti-tude were examined by calculating Pearson’s product–moment correlation coefficients (Sokal & Rohlf,1995).

Differences in climatic properties and primary pro-ductivity of distributional areas of dark and pale colormorphs were analyzed by conducting separate nonpara-metric Mann–Whitney–U tests for each environmentalvariable. Subpopulation means of environmental vari-ables were used as data points in the analyses. In additionfor each subpopulation, we also calculated the minimum,maximum, and coefficient of variation of each climaticvariable and for the primary productivity index (NDVI).



Following Gilchrist et al. (2004) and Stillwell et al.(2007), the relationship between body size pattern of palecolor morphs and environmental variables was analyzedusing population means rather than individuals in order toavoid the nonindependence of individuals between sites.As a first step, we performed explanatory analyses byconducting separate regression analysis between PC1 andthe four environmental variables (MTAS, MPAS, TS, andPPAS). Bivariate plots of PC1 with environmental vari-ables were screened for possible nonlinear relationshipsand a quadratic term was added to the regression equationwhen nonlinearity was found.

To evaluate the relative support of competing hypothe-ses explaining variation in body size, we used an infor-mation theoretic approach (Burnham & Anderson, 2002;Diniz-Filho et al., 2008; Gur & Kart Gur, 2012). UsingPC1 (body size) as the dependent variable, we built gener-alized linear models using all possible combination of en-vironmental variables. Models including quadratic termsfor environmental variables, which showed a nonlinearrelationship with body size, were also included in theanalysis. In total, we built 22 different models predictingthe relationship of body size in response to environmentalvariables (Table 2). Relative support of different modelswas calculated using Akaike’s Information Criterion cor-rected for small sample size (AICc). This model selectionprocedure chooses models based on the trade-off betweenthe amount of variance explained and the number of pa-rameters included in the model (Burnham & Anderson,2002) and is calculated as

AICc = n × ln (σ 2) + [2K × (K + 1)/(n − K − 1)].

Here, n is the number of observations, ln is the naturallogarithm, σ 2 is the error variance, and K is the numberof parameters in the model (number of predictor variablesplus the intercept and error term). Models explaining themost variation with the fewest predictive variables havethe lowest AICc scores. Therefore, the model with the low-est AICc score is accepted to be the most parsimonious(best) model. For each model, we calculated �AICc (dif-ference between AICc of each model and the best model)and Akaike weights (AICc wi) describing the probabil-ity of the different models given the data (Burnham &Anderson, 2002). Models were considered to have strongstatistical support if �AICc was <2.

We examined for the presence of spatial autocorrelationby drawing spatial correlograms based on Moran’s I spa-tial autocorrelation coefficient (Diniz-Filho et al., 2008)using SAM (Spatial Analysis in Macroecology, Version4.0, Rangel et al., 2010). We computed correlograms us-

ing the residuals of the two best models based on theoutcome of the AICc procedure (see Results). We couldnot detect any significant spatial autocorrelation in theresiduals of our best models (i.e., none of the Moran’s I co-efficients were significant at the 5% level after Bonferronicorrection, results not shown). Therefore, using nonspatialregression models in the calculation of AICc values werejustified.

Results

Altitudinal variation in male body size of I. rizeensis

Body size of I. rizeensis males varied significantly withaltitude (β = 0.003 ± 0.0004, t = 5.44, P < 0.001)and this variation also had a strong quadratic component(β = −8.993 × 10−7 ± 1.604 × 10−7, t = −5.606,P < 0.001). Coloration also had a significant effect onbody size (β = 1.034 ± 0.119, t = 8.703, P < 0.001)and comparison between adjusted least square means be-tween color morphs indicated that pale color morphs wereon average larger than dark color morphs (F1,382 = 75.733,P < 0.001; LSMdark = − 0.392, LSMpale = 0.642). Over-all, the full model (PC1 = Color + Altitude + Altitude2)was able to explain 43.3% of the total variation in bodysize (F3,382 = 99.326, P < 0.001). Likelihood ratio testsshowed that the quadratic model (PC1∼ Color + Altitude+ Altitude2, lnL = −435.918) was a statistically moresignificant fit than the model containing only the linearcomponent (PC1∼ Color + Altitude, lnL = − 451.175)(χ2 = 30.514, df = 1, P < 0.001).

When mean body size of I. rizeensis from each local-ity is plotted against altitude, it can clearly be seen thatthe response of body size to altitude is driven by palecolor morphs as black color morphs showed no signifi-cant variation in mean body size along its distributionalrange (Fig. 2). In addition since very little overlap wasobserved between the distribution of dark and pale colormorphs we conducted separate regression analyses foreach morph. Results were similar to the ANCOVA analy-sis as dark color morphs showed no significant correlationwith altitude in either the linear (β < 0.001, t = 0.364,P = 0.716, r2 ≤ 0.001) or the quadratic model (Alt: β =0.004 ± 0.003, t = 1.196, P = 0.234; Alt2: β < 0.001,t = −1.165, P = 0.246, r2 = 0.011). Pale color morphsshowed no significant correlation with altitude in the lin-ear model (β < −0.001, t = −0.322, P = 0.747, r2 ≤0.001) however the quadratic model was highly signifi-cant (Alt: β = 0.006 ± 0.0007, t = 8.519, P < 0.001;Alt2: β = 8.993 × 10−7 ± 1.604 × 10−7, t = −8.637,P < 0.001, r2 = 0.232).



Table 2 Candidate environmental models explaining variation in body size in pale color morphs of males of Isophya rizeensis. Sign ofassociation between environmental variables and body size for the two best models are given in parenthesis.

No Model K r2 AICc �AICc L(d|x) AICc wi

1 PC1∼MPAS (−) 3 0.519 15.598 0.000 1.000 0.4712 PC1∼TS (+) 3 0.459 16.780 1.182 0.554 0.2613 PC1∼MTAS + MTASˆ2 4 0.629 19.003 3.405 0.182 0.0864 PC1∼MPAS + PPAS 4 0.586 20.098 4.500 0.105 0.0505 PC1∼MTAS + MPAS 4 0.576 20.357 4.759 0.093 0.0446 PC1∼TS + MPAS 4 0.521 21.563 5.965 0.051 0.0247 PC1∼TS + PPAS 4 0.503 21.935 6.337 0.042 0.0208 PC1∼TS + MTAS 4 0.464 22.693 7.095 0.029 0.0149 PC1∼PPAS 3 0.006 22.870 7.272 0.026 0.01210 PC1∼MTAS 3 0.001 22.915 7.317 0.026 0.01211 PC1∼TS + MTAS + MPAS 5 0.678 26.591 10.993 0.004 0.00212 PC1∼MTAS + MPAS + MTASˆ2 5 0.661 27.108 11.510 0.003 0.00113 PC1∼MTAS + PPAS + MTASˆ2 5 0.654 27.308 11.710 0.003 0.00114 PC1∼MTAS + MPAS + PPAS 5 0.628 28.044 12.446 0.002 0.00115 PC1∼MTAS + PPAS 4 0.008 28.842 13.244 0.001 0.00116 PC1∼TS + MPAS + PPAS 5 0.590 29.021 13.423 0.001 0.00117 PC1∼TS + MTAS + PPAS 5 0.503 30.927 15.329 <0.001 <0.00118 PC1∼TS + MTAS + MPAS + PPAS 6 0.722 40.141 24.543 <0.001 <0.00119 PC1∼MTAS + MPAS + PPAS + MTASˆ2 6 0.707 40.663 25.065 <0.001 <0.00120 PC1∼TS + MTAS + PPAS + MTASˆ2 6 0.701 40.868 25.270 <0.001 <0.00121 PC1∼TS + MTAS + MPAS + MTASˆ2 6 0.679 41.575 25.977 <0.001 <0.00122 PC1∼TS + MTAS + MPAS + PPAS + MTASˆ2 7 0.722 70.129 54.531 <0.001 <0.001

TS, temperature seasonality; MTAS, mean temperature of the active season; MPAS, mean precipitation of the active season; PPAS,primary productivity of the active season; K, number of parameters in the model (including intercept and error term); r2, Coefficient ofdetermination; AICc, Akaike’s information criterion corrected for small sample size; �AICc, the difference between the AICc of eachmodel and the model with the lowest AICc; L(d|x), Likelihood of the model given the data; AICc wi, Akaike weights.

Correlations between altitude and environmentalvariables

All climatic variables were strongly correlated with al-titude and with each other (Table 3). However, there wasno correlation between primary productivity (PPAS) andaltitude or any other climatic variable. Both mean tem-perature (MTAS) and precipitation (MAPS) of the activeseason showed a strong negative relationship with alti-tude, whereas temperature seasonality (TS) showed theopposite trend.

When mean environmental conditions of the distribu-tional ranges of dark versus pale color morphs are com-pared, we can deduce that dark color morphs inhabit muchmore humid, hot, and less seasonal environments whereaspale color morphs are subjected to colder, drier, and moreseasonal conditions (Table 4). Mean primary productivityin the active season was higher in the distributional rangeof pale color morphs than that of dark color morphs, butdifferences were not significant (Table 4).

Comparing environmental hypotheses explaining bodysize variation in pale color morphs

Univariate regression analysis revealed significant as-sociations between body size and all environmental vari-ables, except primary productivity of the active season(PPAS: β = 0.001 ± 0.003, t = 0.212, P = 0.836, r2 =−0.119, Fig. 3B). Body size was positively associatedwith temperature seasonality (TS: β = 0.231 ± 0.088,t = 2.606, P = 0.031, r2 = 0.392, Fig. 3C), while theassociation was negative for mean precipitation of the ac-tive season (MPAS: β = –0.093 ± 0.032, t = −2.941,P < 0.019, r2 = 0.459, Fig. 3D). Body size was alsosignificantly associated with mean temperature of the ac-tive season (MTAS). However, this association also had astrong quadratic component (MTAS: β = 2.431 ± 0.705,t = 3.446, P < 0.011; MTAS2: β = −0.083 ± 0.024,t = −3.444, P < 0.011, r2 = 0.523). Body size initiallyincreased with increasing temperatures, reached a max-imum at intermediate temperatures (14–16◦C) and then



Fig. 2 Mean ± SE of PC1 scores (body size index) of dark and pale color morphs of male Isophya rizeensis along an altitudinalgradient within the Fırtına Valley, Camlıhemsin-Rize, Turkey.

Table 3 Correlation coefficients among altitude and environ-mental variables.

ALT TS MTAS MPAS

TS 0.661*MTAS − 0.975** − 0.512*MPAS − 0.701* − 0.994** 0.557*PPAS 0.340 0.407 − 0.351 −0.398

ALT, altitude; TS, temperature seasonality; MTAS, mean tem-perature of the active season; MPAS, mean precipitation of theactive season. *P < 0.05, **P < 0.001.

declined sharply at temperatures over 16◦C (Fig. 3A).Model comparisons using likelihood ratio tests showedthat the quadratic model (PC1∼ MTAS + MTAS2, lnL =−1.502) was a statistically more significant fit thanthe model containing only the linear component (PC1∼MTAS, lnL = −6.548) (χ2 = 9.912, df = 1, P = 0.0016).

To evaluate the relative support of different environ-mental variables (hypotheses) in explaining body sizevariation in pale color morphs of I. rizeensis, we com-pared 22 environmental models using the AICc criteria(Table 2). The best environmental model had only onevariable, MPAS and explained 52% of the variation inbody size. The AICc criteria also gave strong support forthe second model (�AICc = 1.182), which was com-

posed of TS explaining 46% of the variation in body size.AICc wi suggested that there was a 47% chance that themodel featuring only MPAS was the best model, whereasthe model featuring only TS had a 26% chance of beingthe best model. �AICc for all other models were sub-stantially higher than 2 (third best model had a �AICcscore of 3.405) and, according to AICc wi, none of thesemodels had higher than 0.08% chance of being the bestmodel.

Discussion

The results of our study revealed the presence of two dif-fering responses of body size to altitudinal gradients inmales of I. rizeensis depending on color pattern. Darkcolor morphs showed no significant variation in bodysize along its distributional range, whereas pale colormorphs showed a curvilinear pattern of body size withaltitude (Fig. 2). Although this pattern matches the em-pirical patterns of thermal reaction norms in insects (thetemperature size rule), our results suggest that the bestcandidate environmental factors explaining body sizevariation in pale color morphs were gradients in moisture(as measured by MPAS) and seasonality (as measuredby TS) and not temperature per se. In addition, primaryproductivity showed no pattern with either altitude orbody size. Hence, the availability and quality of food



Table 4 Comparison between environmental properties of the distributional areas of dark versus pale color morphs. Z scores andP values correspond to results of separate nonparametric Mann–Whitney–U tests.

Variables Mean Min. Max. CV Z P

TS Dark 71.026 65.540 75.660 6.894 − 2.694 0.007Pale 76.757 73.340 78.250 1.864

MTAS (◦C) Dark 18.072 17.580 18.620 2.667 2.694 0.007Pale 14.974 10.860 17.980 15.900

MPAS (mm) Dark 71.640 59.000 88.400 19.519 2.697 0.007Pale 55.560 52.000 64.800 6.787

PPAS Dark 446.860 332.840 573.180 19.059 − 0.737 0.461Pale 495.588 429.620 570.520 11.003

does not seem to be a limiting factor within the elevationgradient.

Limitations of the study

The information–theoretic approach we have undertaken in this paper enables us to compare the rela-tive support of different hypothesis (i.e., climatic vari-ables) without the need for setting up formal experiments(Burham & Anderson, 2002). Nevertheless the presentdata underhand does not allow us to distinguish whetherbody size variation results directly from the influenceof these factors or from another correlated yet uncon-sidered variable or whether these factors act through se-lection or plasticity in producing the observed patternsof body size variation. Doing so would require conduct-ing common garden experiments in controlled conditions.Therefore, our results and all discussion of the data willbe evaluated in light of this apparent weakness of thestudy.

The effect of moisture gradients (humidity/rainfall) onbody size

According to our results based on the AICc model selec-tion procedure, the best environmental variable explain-ing body size variation in pale color morphs of I. rizeensiswas MPAS thus giving support to the moisture gradienthypothesis. MPAS showed a highly significant negativecorrelation both with body size and with altitude. This isin accordance with recent publications, which also shownegative correlations between body size and moisture (hu-midity/rainfall), and emphasize the relative importance ofmoisture gradients on body size clines along altitudinalor latitudinal gradients (Bidau & Martı, 2007b; Stillwellet al., 2007).

One way in which gradients in moisture could resultin body size clines is trough resistance to desiccation(Stillwell, 2010; Gunderson et al., 2011). Larger individ-uals are more resistant to desiccation due to having asmaller surface to volume ratio (Gaston & Chown, 1999;Chown & Gaston, 2010). Throughout the distributionalrange of I. rizeensis, both precipitation and temperaturedecrease with increasing altitude resulting in cooler albeitdrier conditions. Dry and cool conditions are found to behighly correlated with desiccation stress in Drosophilaspecies (Parkash et al., 2005, 2008, 2009) and numer-ous studies have found positive correlations between in-creased body size and desiccation resistance via reducedwater loss in arthropods (e.g., Lighton et al., 1994; Le La-gadec et al., 1998; Addo-Bediako et al., 2001; Kærsgaardet al., 2004).

However body size variation in I. rizeensis need notresult from desiccation resistance as gradients in pre-cipitation can also elucidate a response through its ef-fect on the available amount of sunlight (Harris et al.,2012). Helioterms like I. rizeensis can quickly becomeinactive during cloudy conditions and rainfall (Chap-pell & Whitman, 1990; Lactin & Johnson, 1998), re-ducing the available time and energy allocated to growthand development, which in turn might lead to smallerbody sizes in areas subjected to higher precipitationlevels.

The effect of seasonality gradients on body size

Apart from MPAS, AICc model selection procedurealso suggested that the best environmental model explain-ing body size variation in pale color morphs of I. rizeen-sis could be TS. Contrary to MPAS, TS showed a highlysignificant positive correlation both with body size andaltitude. However, the effect of seasonality was directlyopposite from that predicted by the seasonal constraint



Fig. 3 Relationship between body size (PC1) of pale color morphs and the four environmental variables along an altitudinal gradientwithin the Fırtına Valley, Camlıhemsin-Rize, Turkey. (A) mean temperature of the active season (MTAS), (B) primary productivity ofthe active season (PPAS), (C) temperature seasonality (TS), and (D) mean precipitation of the active season (MPAS).

hypothesis as formulated for univoltine ectotherms(Walters & Hassall, 2006).

In insects with generation times on par with thegrowing season, such as I. rizeensis, an increase inseasonality usually leads to a reduction in body size,since development time is heavily constrained. This pat-tern is well supported for many univoltine Orthopteraspecies (Cıplak et al., 2008; Lehmann & Lehmann, 2008;Whitman, 2008; Bidau et al., 2012). However, the generalmodel linking development with available time for growthassumes constant growth rates within species. Some in-sects have the ability to adjust to seasonal time constraintsby evolving faster growth rates (i.e., countergradient vari-ation, Ayres & Scriber, 1994; Nylin, 1994; Nygren et al.,2008; Scharf et al., 2009). Therefore, one possible way forI. rizeensis individuals to reach larger body sizes in theface of increasing seasonality (hence, reduced growing

time) would be an overcompensation in growth rates.However, this assertion needs to be empirically tested.

Increasing seasonality can also result in larger bodysizes possibly due to better survival under more heteroge-neous and less predictable environments, where resourceavailability is heavily restricted and stress factors suchas starvation and desiccation increase (Cushman et al.,1993; Arnett & Gotelli, 1999; Ashton, 2001; Stillwellet al., 2007). Therefore, an increase in body size of palecolor morphs of I. rizeensis could be a response to stresscaused by unpredictability in temperature acting in con-juncture with stress caused by a reduction in humiditylevels (desiccation resistance). Similar positive body sizeclines as a result of seasonality in temperature have beenshown in seed feeding beetles (Stillwell et al., 2007) andin melanopline grasshoppers (Bidau & Martı, 2007a, b,2008). However, as in this study whether these patterns



are the direct result of selection to elevated stress levelsor a plastic response is not clear (Stillwell & Fox, 2009;Bidau et al., 2012).

No body size trend in dark color morphs

Dark color morphs of male I. rizeensis did not showany substantial variability in body size with altitude, eventhough MPAS and TS actually showed higher amounts ofvariability within the dark color morphs range (Table 4).Evidence gained from pale color morphs suggests thatbody size variation in I. rizeensis results from desiccationstress and temperature variability. Therefore, the advan-tages gained by dark coloration might nullify the need forincreased body size in dark color morphs. The level ofmelanization is known to be highly correlated with des-iccation resistance (Parkash et al., 2008, 2009) as darkerindividuals are able to store higher levels of body watercontent and have a higher tolerance to dehydration thanpale individuals (Parkash et al., 2012). In addition, ther-mal capacity and heating rates of dark individuals are usu-ally higher than pale individuals (Forsman, 1997; Forsmanet al., 2002) giving them an advantage under conditionsof low ambient temperature and limited/unpredictable ra-diative regimes (Watt, 1968; Huey & Kingsolver, 1989;Jong et al., 1996).

Conclusion

Our results give support to other studies showing the rela-tive importance of humidity and/or seasonality gradientsin shaping body size clines along altitudinal/latitudinalgradients as body size variability in pale color morphs ofI. rizeensis was driven by MPAS and TS. Our results alsogive support to the assumption that selection for desicca-tion resistance or selection for resistance to stress factorsmight be an important factor in the production of bodysize clines along altitudinal/latitudinal gradients. On aside note, we also show that whether body size patternsshow a strong or weak correlation with altitude might alsodepend on phenotypic properties of individuals like col-oration. However, further research conducting commongarden or transplant experiments are necessary to eluci-date if body size patterns are driven by selection or resultfrom plasticity.

Acknowledgments

This research was funded by the Hacettepe UniversityScientific Research Center Project No. 03 02 601 012.We would like to thank Dr. Hakan Gur for his valuable

input in the analysis and for critically evaluating the paper.We would also like to thank Dr. Cagatay Tavsanoglu forhis suggestions concerning the final manuscript.

Authors’ contributions

SSC took part in planning of the study and contributed tothe discussion. CK is responsible for extracting and orga-nizing the climatic and environmental data. Contributedto the discussion. ACK is responsible for measurementof morphological characters and preparation of the data.Contributed to the introduction and discussion. IKS de-signed the study and determined the main hypotheses tobe tested. IKS also conducted all statistical analyses andwrote the manuscript.

Disclosure

None of the authors have any conflict of interest to de-clare and none of the authors hold any financial interestregarding the study outlined in the paper.

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Accepted March 11, 2013


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Humidity and seasonality drives body size patterns in males of the bush cricket Isophya rizeensis Sevgili, 2003 (Orthoptera: Tettigoniidae: Phaneropterinae)