Macquarie University PURE Research Management System

This is the peer reviewed version of the following article:

Yadav, S., Stow, A. and Dudaniec, R.Y. (2020), Elevational partitioning in species distribution, abundance and body size of Australian alpine (Kosciuscola). Austral Ecology, vol. 45, no. 5, pp. 609-620.

which has been published in final form at: doi.org/10.1111/aec.12876

This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions.

1 Elevational partitioning in species distribution, abundance and body size of Australian

2 alpine grasshoppers (Kosciuscola)

3

4 Sonu Yadav1*, Adam Stow1, Rachael Y. Dudaniec1

5

6 1 Department of Biological Sciences, Macquarie University, North Ryde, 2109, NSW,

7 Australia

8

9 *Corresponding author:

10

11 Sonu Yadav, Department of Biological Sciences, Macquarie University, North Ryde, 2109,

12 NSW, Australia, Email- [email protected]

13

14 Acknowledgments

15 We thank Kate D. L. Umbers for help with field work protocols and useful discussions. We

16 thank Justin McNab for help with fieldwork, Rachel Slatyer for providing useful information

17 on the species occurrence and Giselle Muschett for helping with species identification. We

18 thank Youning Su from the Australian National Collection, CSIRO museum, Canberra

19 for providing access to type specimens. This project was funded by Macquarie University

20 with start-up funding to R.Y.D., a Macquarie graduate student research grant to S.Y, a

21 Holsworth Wildlife Research Endowment grant from the Ecological Society of Australia (to

22 SY), the Theodore J. Cohn Research Fund from the Orthopterist’s Society (to SY) and the

23 Joyce W. Vickery Scientific Research Fund from The Linnean Society of NSW (to SY). All

24 procedures were performed in accordance with the ethical guidelines of Macquarie 25 University, Australia, and sampling permissions were obtained from local government

26 authorities and national parks (License number: SL101832). 27 Abstract

28 Changes in the physical environment with elevation can influence species distributions and

29 their morphological traits. In mountainous regions, steep temperature gradients can result in

30 patterns of ecological partitioning among species that potentially increases their vulnerability

31 to climate change. We collected data on species distributions, relative abundance and body

32 size for three species of the genus Kosciuscola (K. usitatus, K. tristis, and K.

33 cognatus) at three locations within the mountainous Kosciuszko National Park in Australia

34 (Thredbo, Guthega and Jagungal). All three species showed differences in their distributions

35 according to elevation, with K. usitatus ranging from 1400 m asl to 2000 m asl, K. tristis

36 from 1600 m asl to 2000 m asl, and K. cognatus from 1550 m asl to 1900 m asl. Decreasing

37 relative abundance with increasing elevation was found for K. usitatus, but the opposite

38 pattern was found for K. tristis. The relative abundance of K. cognatus did not change with

39 elevation but was negatively correlated with foliage cover. Body size decreased with

40 elevation in both male and female K. usitatus, which was similarly observed in female K.

41 tristis and male K. cognatus. Our results demonstrate spatial partitioning of species

42 distributions and clines in body size in relation to elevational gradients. Species-specific

43 sensitivities to climatic gradients may help to predict the persistence of this grasshopper

44 assemblage under climate change.

45

46 Keywords

47 Australian Alps, Kosciuscola, , relative abundance, body size 48 Introduction

49 Alpine species are often adapted to narrow thermal ranges, making them sensitive to

50 increasing temperatures (Laurance et al, 2011; Williams et al, 2008) and more likely to be

51 adversely affected by climate change (Dirnböck et al, 2011; La Sorte & Jetz, 2010). Alpine

52 ecosystems show sharp clines in climatic characteristics leading to changes in the

53 composition and diversity of biota at fine spatial scales (Beccacece et al, 2016; Hilt &

54 Fiedler, 2005; Sundqvist et al, 2013; Zenker et al, 2015). Changes in species composition and

55 diversity along elevation gradients have been reported for several taxa (moths: Brehm &

56 Fiedler, 2003; dung beetles: Herzog et al, 2013; plants: Mota et al, 2018; birds: Rahbek,

57 1997; Zenker et al, 2015), and climate, topography and productivity are among the major

58 drivers of such changes (Currie & Kerr, 2008; Lomolino, 2001; Ramirez-Bautista &

59 Williams, 2019). Species’ physiological tolerances and microhabitat usage are further

60 contributors (Birkett et al, 2018; Röder et al, 2017; Wachter et al, 1998; Werenkraut &

61 Ruggiero, 2014), whereby specialist species with narrow distributions and narrow thermal

62 niches are expected to show increased sensitivity to shifts in climatic conditions (Hoffmann,

63 2010). Therefore, knowledge of the fine-scale distributions of species within alpine

64 environments is necessary for assessing their vulnerability to climate change.

65

66 Species distributed along environmental gradients are often observed with morphological

67 changes such as intraspecific colour polymorphism and variation in body size, particularly in

68 (Sheridan & Bickford, 2011; Whitman, 2008; Yadav et al, 2018). Body size is an

69 important trait related to fitness, survival, growth, and physiology (Chown & Gaston, 2010;

70 Kleiber, 1947) and may therefore influence the relative success of certain species within

71 species assemblages in variable environments. Body size in ectotherms, including insects, is

72 generally positively correlated with temperature with smaller individuals in cooler climates 73 (Berner & Blanckenhorn, 2006; Buckley et al, 2014; Mousseau, 1997; Telfer & Hassall,

74 1999), however no single consistent pattern has been found across the insect taxa (Brehm et

75 al, 2018; Maveety & Browne, 2014). This inconsistency indicates that the underlying

76 mechanisms controlling body size may vary across species and locations.

77 Insects frequently show sexual size dimorphism, with females typically being the

78 larger sex (Chown & Gaston, 2010) including in Orthoptera (Hochkirch & Gröning, 2008).

79 Males and females may show distinct variations in body size along environmental gradients,

80 potentially due to sex-specific selection pressures (Fairbairn, 1997; Stamps, 1993; Teder &

81 Tammaru, 2005) or altered developmental periods along temperature gradients (Fairbairn et

82 al, 2007; García-Navas et al, 2017; Laiolo et al, 2013). Due to a strong association between

83 environmental variables (for e.g. temperature) and variation in dimorphism in insects

84 (Davidowitz et al, 2004; Stillwell et al, 2007b), the pattern of dimorphism may vary or shift

85 with changing environmental conditions. Understanding sex-specific variation in body size

86 along elevation gradients may therefore provide insight into the environmental mechanisms

87 generating clinal distributions within species assemblages.

88

89 Alpine ecosystems are particularly susceptible to climate change (Gobiet et al, 2014; IPCC,

90 2014) and the Australian Alps are no exception. Since 1979 the Australian Alps have

91 experienced a decline in precipitation by 6%, an increase in temperature at an average of

92 0.4°C since 1954 along with a substantial decline in snow depth (Hennessy et al, 2003;

93 Nicholls, 2005; Sánchez-Bayo & Green, 2018; Wahren et al, 2013). The Australian alpine

94 bioregion is further threatened by fire, sub-alpine vegetation encroachment and impacts of

95 invasive species (Hoffmann et al, 2019). Such threatening processes have been linked to

96 alterations in the survival rates, distributions and population dynamics of endemic alpine

97 species (Green, 2010; Nicholls, 2005). For invertebrates, overwintering survival rates under 98 reduced snow cover may be decreased because snow acts as a buffer from fluctuating

99 temperatures and insulates eggs during cold winter months (Bale & Hayward, 2010). Despite

100 the potential consequences of climate change to alpine species, studies of these assemblages

101 are few in the Australian Alps (Hoffmann et al, 2019), especially for invertebrates, in spite of

102 their value in assessing ecological responses to shifting thermal regimes (Hassall, 2015;

103 Khamis et al, 2014; Yadav et al, 2019).

104

105 Here we examine the effect of temperature-elevation gradients on Kosciuscola grasshoppers

106 in terms of their occurrence, relative abundance and body size. We focus on three of five

107 species of the genus: K. usitatus, K. tristis, and K. cognatus (Orthoptera: ), which

108 all co-occur, are flightless, and endemic to the Australian Alps. Our three focal species are

109 common throughout the Kosciuszko alpine region ranging from montane woodlands to alpine

110 areas (Slatyer et al, 2016). Field observations suggests that the species may differ in

111 elevational range, with K. cognatus occupying the broadest range (Tatarnic et al, 2013) and

112 K. tristis having the narrowest range of the five species of Kosciuscola. (Slatyer et al, 2014).

113 Further observations similarly indicate that K. cognatus occupies low to middle elevation

114 areas (900-1700m), K. tristis middle to high elevations (1500-2000m) and K. usitatus (1400-

115 2200m) from middle to the highest of elevations (Campbell & Dearn, 1980). Moreover,

116 previous work has found that these grasshopper species show variation in cold tolerance

117 limits (Slatyer et al, 2016), with K. usitatus having the highest cold tolerance (CTmin= −3

118 °C), followed by K. tristis (CTmin= −2 °C) and K. cognatus (CTmin= −1 °C). Despite these

119 patterns there has been no systematic survey to date on the fine-scale elevational distributions

120 and abundances of Kosciuscola species, which are required to better characterise the current

121 structure of this species assemblage.

122 123 Here, we examine how the distribution, relative abundance and body size of three

124 Kosciuscola grasshopper species vary along elevation gradients in the Kosciuszko alpine

125 region in the Australian Alps. We collected data during a single season from replicate

126 transects along elevational gradients across three mountain regions (Thredbo, Guthega and

127 Jagungal). We test if (1) Kosciuscola species differ in occurrence and relative abundance

128 along temperature, elevation and vegetation gradients, and (2), whether intra-specific

129 variation in body size among males and female Kosciuscola is related to temperature-

130 elevation gradients.

131

132 Materials and methods

133 Study species

134 All three focal grasshopper species have non-overlapping generations (Campbell & Dearn,

135 1980; Umbers et al, 2013). Adults lay eggs during autumn and summer and hatching takes

136 place in spring (Campbell & Dearn, 1980; Umbers et al, 2013). Of the other two species, K.

137 tasmanicus only occurs in Tasmania, and K. cuneatus is observed to be absent from the

138 higher elevational zones of Kosciuszko National Park (Tatarnic et al, 2013) and thus were

139 excluded from our study. Sexual dimorphism in body size is present in all three species with

140 females having a larger body size than males (Slatyer et al, 2016; Umbers et al, 2013).

141

142 Study sites and sample collection

143 Grasshoppers were collected over a two-week period in late February to early March 2017 in

144 the Kosciuszko alpine region within the south-east of New South Wales of Australia (Fig 1).

145 Transects were constructed along an elevational range of 1,400 to 2,000m, with time-based

146 searches conducted at 50-metre increments in altitude. Samples were collected from a total of

147 48 sites across three locations: 1) Thredbo (-36.5°S, 148.3 °E), 2) Guthega (-36.4°S, 148.38 148 °E) and 3) Jagungal (-36.14°S, 148.38 °E; Table S1). We searched for the three focal species

149 within a ~30m2 plot for 20 mins by two people (40 minutes in total; except in 11 sites where

150 the number of people varied from 3-5; see Tables S2, S3, S4 for species specific details).

151 Transect length ranged from 500-600 meters and depended on the steepness of the elevation

152 gradient for each transect.

153

154 In Thredbo, we collected grasshoppers from three transects with a total of 23

155 sampling points (T1= 13 points, ~400m length, T2= 4 points, ~500m length, and T3= 6

156 points, ~250m length) with an elevational gradient ranging from ~1,400-2,000 m a.s.l (Fig.

157 1). A total of three transects were laid in Guthega with 18 sampling points (T1= 7 points, ~

158 170m, T2= 5 points, ~180m, and T3= 6 points, ~250m) with an elevational range of ~1,600-

159 1,850 m a.s.l (Fig. 1) and in Jagungal one transect was covered (T1 = 7 points, ~350m length)

160 and an elevation range of ~1,550-1,900 m a.s.l. (Fig. 1). Only one transect was sampled in

161 Jagungal due to poor accessibility. Spatial coordinates (latitude, longitude) were recorded at

162 each transect point using a handheld GPS device (Garmin Etrex10).

163

164 Grasshoppers were captured using small plastic jars and the time of the day, species name

165 and sex were recorded from live specimens upon processing. To further confirm species

166 identification, features such as eye colour, cheek pattern, pronotum shape, body shape and

167 size were compared against type specimens stored in the Australian National Insect

168 Collection (CSIRO, Canberra). Hind leg femur length was measured on the left side for each

169 live specimen using Vernier calipers (precision to 0.02 mm), which is a commonly-used

170 proxy for body size in grasshoppers (Masaki, 1967; Mousseau & Roff, 1989). All samples

171 were stored in 95% ethanol for DNA preservation.

172 173 Environmental data

174 Environmental variables were chosen based on field observations, expert opinion and

175 knowledge of the species ecology and thermal tolerance (Slatyer et al, 2016; Slatyer et al,

176 2014; Umbers et al, 2013). Temperature, precipitation (including rainfall or snow cover) and

177 topography are known to affect reproduction, growth, activity, and distribution of insects,

178 including grasshoppers (Buckley et al, 2013; Buckley & Nufio, 2014; Hodkinson, 2005;

179 Joern & Gaines, 1990; Roland & Matter, 2016; Yadav et al, 2018). Foliage cover has also

180 been linked with movement, egg laying (Clark, 1967), the distribution of morphs (Yadav et

181 al, 2018) as well as adaptive genetic variation in grasshoppers (Yadav et al, 2019).

182 Maximum summer temperature (‘Temperature’ from here on) and annual rainfall

183 (‘Precipitation’ from here on) were obtained from ANUCLIM 6.1 (Lyons et al, 2017; Xu &

184 Hutchinson, 2013) and resampled to a 30m resolution. Elevation data were derived from a

185 Digital Elevation Model (DEM) obtained from Geoscience Australia at a 30m resolution

186 (Table S1 for per site information). Foliage Projective Cover (FPC) refers to the percentage

187 of land obscured by woody vegetation and trees, thus FPC provides a fine-scale measurement

188 of vegetation cover. FPC was obtained from the Terrestrial Ecosystems Research Network

189 AusCover database (TERNAusCover, 2011) at a resolution of five meters. To extract FPC

190 values, a buffer of 30m was used around each sampling site from which cell values were

191 averaged using the raster (Hijmans & van Etten, 2014) package in R (Team, 2017).

192 Correlations between environmental variables were examined using Pearson correlation

193 method and highly correlated variables (r >0.80) were removed from further analysis.

194

195 Temperature, precipitation and FPC association with elevation

196 We examined whether temperature, precipitation and FPC changed significantly with

197 elevation within our three study regions by fitting a linear mixed effect model (LMM) with 198 elevation as a fixed effect and the region (Thredbo, Guthega and Jagungal) as a random

199 intercept. The LMMs were fitted using the lmer function of the lme4 package (Bates et al,

200 2015) and goodness of fit was examined by calculating conditional R2 using the

201 r.squaredGLMM function in MUMIn (Barton, 2018). The statistical significance of the fixed

202 effect in LMM was examined using ANOVA with a Chi-square test in the R package car

203 (Fox & Weisberg, 2011). Diagnostic plots for each LMM were examined to ensure model

204 assumptions were met. We conducted all analyses in R.

205

206 Relative abundance along elevation and FPC

207 The relative abundance of each species at each site was recorded as capture rate per minute,

208 calculated as the number of grasshoppers caught divided by the number of searchers

209 multiplied by the total number of minutes spent collecting. This approach of obtaining

210 relative abundance estimates is commonly applied, for example in insects (Lancaster et al,

211 2015; Yadav et al, 2018) and salamanders (Dudaniec & Richardson, 2012). Due to the

212 correlation between elevation and precipitation, and elevation and temperature, we used

213 elevation as a proxy for temperature-precipitation in all further models (see Results section).

214 The influence of elevation and FPC on relative abundance in the three sampled mountain

215 regions pooled together was evaluated by fitting a LMM. Models were fitted individually for

216 each species with elevation and FPC as a fixed effect and region and time of the day as

217 random effects. Time of the day was used as a random effect to account for possible bias in

218 the number of samples collected due to variations in temperature and weather differences

219 during the day. Model significance were assessed using Chi-square test.

220

221 Body size correlation with elevation 222 To test if body size of each species changed significantly with elevation and to account for

223 sex-specific differences, we first fitted the model with body size (measured from femur

224 length) as a response variable, elevation as a predictor, sex as an interaction term with

225 elevation and region and site as a random effect. We then examined the change in body size

226 along elevation gradients for male and females separately for each of the three species,

227 pooling data from all transects sampled according to each mountain region. A LMM was

228 fitted using femur length (body size proxy) as a response variable and elevation as a fixed

229 effect, with region and site as random effects. In addition to region, site was also used as a

230 random effect to account for non-independence of individuals from the same site. The

231 correlation between body size and FPC was not examined due to less variation in FPC at

232 some of the sites (Table S1).

233

234 Results

235 Temperature, precipitation and FPC association with elevation

236 Precipitation was positively correlated with elevation (χ2 = 1154.5, DF=1, P = <0.0001, R2 =

237 0.96). Temperature (χ2 = 1749.5, DF=1, P = <0.0001, R2 = 0.97) and FPC (χ2 = 4.51, DF=1, P

238 = 0.03, R2 = 0.20) both showed a significant negative association with elevation (Fig. S1).

239

240 Relative abundance with elevation and FPC

241 The relationship between relative abundance and elevation was not consistent across species.

242 For K. usitatus, relative abundance decreased with elevation (χ2 = 6.72, DF=1, P= 0.009, R2 =

243 0.26, Fig. 2A) with lower relative abundance at higher elevations. The relative abundance of

244 K. tristis increased with elevation (χ2 =17.61, DF=1, P = <0.0001, R2 = 0.44; Fig. 2B). No

245 significant correlation was evident between the relative abundance of K. cognatus and

246 elevation (χ2= 0.006, DF=1, P= 0.93; Fig. 2C). Similarly, the relationship between relative 247 abundance and FPC varied across species. K. cognatus showed a significant negative

248 correlation with FPC (χ2= 4.7, DF=1, P=0.03, R2 = 0.36; Fig. 2D), with higher abundances at

249 sites with low vegetation cover, however relative abundance was not significantly correlated

250 with FPC for K. usitatus (P= 0.10) or K. tristis (P= 0.80).

251

252 Body size change with elevation and sex

253 The relation between body size and elevation varied between species and sexes. The

254 interaction term was non-significant for K. usitatus (P = 0.14) showing that the change in

255 body size with elevation was not influenced by sex differences, and sexes similarly showed a

256 decline in body size with elevation (male N = 174, P= 0.0001, R2 = 0.34 and female N =237,

257 P=0.026, R2 = 0.04, Table 1, Fig. 3A,3B). However, the interaction effect of sex and

258 elevation was significant for K. tristis (P=0.009, Table 1) showing that the observed change

259 in body size was affected by sex, with only females significantly declining in body size with

260 elevation (females: N=91, P= 0.03, R2 = 0.21; males: N=188, P= 0.99, R2 = < 0.01; Table 1,

261 Fig. 3C, Fig. S2A). For K. cognatus the interaction effect of sex and elevation was also

262 significant (P=0.045) but in contrast, male body size significantly declined with increasing

263 elevation (females: N=128, P= 0.97, R2 <0.01; males: N=143, P= 0.019, R2 =0.70, Table 1,

264 Fig. 3D, Fig. S2B).

265

266 Discussion

267 Here we characterise changes in the occurrence and relative abundance of Kosciuscola

268 grasshoppers along elevational gradients in the Australian Alps. Elevation was strongly

269 associated with temperature and precipitation in the sampled regions, and was the dominant

270 driver of change in the relative abundance of K. usitatus and K. tristis, which occur at higher

271 elevations. In contrast, the relative abundance of the lower elevation species, K. cognatus, 272 was not significantly influenced by elevation, but decreased in relative abundance with

273 increasing foliage cover. Furthermore, we found sex-specific associations between body size

274 and elevation, with both male and female K. usitatus, female K. tristis and male K. cognatus

275 showing declines in body size. Our study highlights that elevational gradients strongly

276 associated with climatic variables and vegetation gradients can influence the relative

277 abundance and body size of grasshoppers at fine spatial scales, which may be indicative of

278 species and sex-specific sensitivities to climate change and habitat degradation.

279 280 Relative abundance and elevation

281 The relative abundance of K. tristis was greater at higher elevations, but the opposite effect

282 was found for K. usitatus, and K. cognatus showed no relationship. In ectotherms, including

283 insects, thermal preferences and habitat variation along elevation gradients may affect

284 community structure and composition (Birkett et al, 2018; Röder et al, 2017). In Kosciuscola,

285 the differential patterns of relative abundance we observe in the three focal species are likely

286 due to differences in their thermal tolerances along elevational gradients, as shown in K.

287 usitatus, which maintains higher cold tolerance compared to K. tristis (Slatyer et al, 2016). In

288 addition, interspecific differences in heat tolerance have been observed, whereby populations

289 above the tree line are more tolerant to heat compared to lower-elevation populations (Slatyer

290 et al, 2016). Furthermore, a previous study on K. cognatus, K. usitatus and Praxibulus sp. by

291 Dearn (1977), showed a decline in egg production per female at higher elevations, which may

292 act to partially explain the decline in relative abundance with elevation in K. usitatus.

293

294 For insects to cope with variable thermal regimes in alpine ecosystems it is beneficial to

295 regulate body temperature via colour polymorphism in order to exploit particular elevation

296 zones (Trullas et al, 2007). The increase in relative abundance of K. tristis at higher 297 elevations may be assisted by its documented colour changing behaviour that occurs

298 primarily in response to ambient temperature, where males change colour from turquoise blue

299 to black at lower temperatures (Umbers, 2011; Umbers et al, 2013). The maintenance of

300 different colour morphs in species along elevational gradients has been shown in several

301 insect species (e.g. Dearn, 1990; Hodkinson, 2005; Valverde & Schielzeth, 2015), and may

302 be assist thermoregulation (Harris et al. 2013), particularly at higher elevations (Umbers et al.

303 2013 .

304

305 Vegetation cover along the sampled transects was not a significant predictor of abundance in

306 K. usitatus and K. tristis but was significant for K. cognatus, whereby a large number of

307 individuals were observed at sites with lower vegetation cover (Fig. 2D). The higher relative

308 abundance of K. cognatus at lower vegetation site is unlikely to be a consequence of

309 increased detectability in areas of low vegetation cover as other species collected from the

310 same sites did not show significant change in abundance with FPC. In grasshoppers,

311 vegetation cover is highly important for egg laying, early instar survival, food resources

312 (Clark, 1967) and basking which in turn is very important for thermoregulation (Chappell &

313 Whitman, 1990; Kearney et al, 2009). Grasshoppers maintain preferred body temperature

314 through basking (Chappell & Whitman, 1990; Samietz et al, 2005), therefore vegetation

315 cover can affect thermoregulatory behaviour (principal means of controlling body

316 temperature with respect to ambient temperature) by altering the availability of basking sites.

317 The degree to which ectotherms can withstand changes in their ambient thermal environment

318 is crucial in regulating their abundance (Willott & Hassall, 1998). Vegetation cover has been

319 found to be an important predictor of the relative abundance and population dynamics of

320 other grasshopper species (Anderson, 1964; Kemp et al, 1990; Yadav et al, 2018). Our

321 previous study on a widespread grasshopper, Phaulacridium vittatum detected associations of 322 candidate loci putatively under selection with foliage cover, highlighting its potential role in

323 shaping adaptive genetic variations (Yadav et al, 2019). The absence of a significant

324 relationship in K. usitatus and K. tristis with FPC is likely due to lower variability in the

325 foliage cover in the species preferred elevation zones. A further examination of the

326 correlation in other areas might shed light on the interspecific differences in the relative

327 abundance change with FPC.

328

329 Body size variation along the elevation gradient

330 Temperature and precipitation gradients associated with elevation are important for

331 thermoregulation and resource availability, which in turn may exert selective constraints on

332 body size in alpine grasshopper communities (Laiolo et al, 2013; Whitman, 2008). At higher

333 elevations with lower temperatures, heat gain in insects may dominate over heat conservation

334 and insects with smaller body size may rapidly gain and lose heat (Kubota et al, 2007). The

335 capacity for rapid heat gain and loss in small ectothermic species leads to rapid changes in

336 body temperature under variable conditions. This is likely to confer wider thermal tolerances

337 (Peters & Peters, 1986), allowing small ectothermic insects to occupy higher elevations. Our

338 observation of declining body size with elevation may indicate selection for more efficient

339 thermoregulation at lower temperatures, however further investigation in to how body size

340 variation affects basal metabolism is required.

341

342 As univoltine species, the three studied Kosciuscola grasshopper species may experience

343 growth limitation at higher elevations due to a shorter season (i.e. summer period).

344 Constraints on resources and season length at higher elevations may lead to variation in the

345 timing of egg hatching, with implications for developmental periods in grasshoppers (Berner

346 & Blanckenhorn, 2006; Berner et al, 2004) and in other insects (Hodkinson, 2005). Short 347 season length may reduce a species growth period, and since this is positively correlated with

348 body size (Roff, 1980), short seasons may lead to smaller body size (Blanckenhorn &

349 Demont, 2004). Adult body size and fecundity may be particularly limited in univoltine

350 species that are sensitive to the length of a single season, as opposed to multivoltine species

351 that have a longer time period to grow and reproduce (Chown & Gaston, 1999; Honěk, 1993;

352 Horne et al, 2018; Kozłowski et al, 2004). In addition, grasshoppers at higher elevations may

353 experience later hatching compared to those at lower elevations (Mousseau, 1997), leading to

354 further shortening of the season length for higher elevation populations. Such patterns have

355 not been studied in Kosciuscola but may contribute to the overall differences in body size we

356 found according to elevation.

357

358 While most insects show sexual size dimorphism, geographic trends in body size change

359 between males and females may also differ (Stillwell et al, 2010; Stillwell et al, 2007a). We

360 found sex-specific patterns of body size change with elevation for K. tristis and K. cognatus

361 suggesting that sex-specific selection and fitness pressures may act on the sexes. Our results

362 are consistent with those of a previous study that examined femur length of only female K.

363 cognatus and K. usitatus in the same study area (Dearn, 1977), which failed to observe a

364 change in body size with elevation for K. cognatus, but found a negative correlation for K.

365 usitatus. Sex-specific clines in body size across geographical scales have been documented,

366 yet their underlying causes are not clear (reviewed in Blanckenhorn et al, 2006b).

367 One explanation is that environmental gradients can influence males and females

368 differently, creating sex specific clines (for e.g. Laiolo et al, 2013; Teder & Tammaru, 2005).

369 Elevational gradients in our study area are strongly correlated with precipitation and

370 temperature, and our results suggest that these variables affect body size in one sex more than

371 the other, as indicated for K. tristis and K. cognatus. The absence of a significant correlation 372 between female K. cognatus body size with elevation may indicate that females benefit from

373 a more consistent body size than males due to female-specific reproductive trade-offs with

374 environmental pressures. Such patterns have been found in several other insects for example

375 in grasshopper Chorthippus parallelus (Laiolo et al, 2013) and in seed beetle Callosobruchus

376 maculatus (Stillwell & Fox, 2007), whereby environment pressures such as temperature and

377 development time constrain variation in female body size than males.

378

379 The extent of sexual selection on males and natural selection on females for increased body

380 size, and therefore increased fecundity, may further lead to sex-specific body size clines

381 (Blanckenhorn et al, 2006a; Teder, 2014). The predominance of female-biased sexual size

382 dimorphism in Orthoptera (Hochkirch & Gröning, 2008) and in our study system suggests

383 that fecundity selection is an important selection pressure in this Order, which often results in

384 a change in body size in females compared to males (Laiolo et al, 2013) as observed in K.

385 tristis. Taken together, the evolution of male and female body size may follow a divergent

386 evolutionary trajectory as a consequence of processes such as sexual selection on males to

387 attain maturity at smaller sizes, fecundity selection on females, or sex-specific plasticity due

388 to ecological pressures (Fairbairn, 2013; Fairbairn et al, 2007; García-Navas et al, 2017),

389 leading to a decoupling of body size evolution between the sexes.

390

391 Conclusion

392 Our study suggests that fine-scale elevation gradients and habitat variables can significantly

393 influence relative abundances and body size clines in alpine insect species. Our results imply

394 that a further change in climatic and vegetation conditions may lead to changes in species

395 distributions and fitness related traits such as body size. While these patterns were observed

396 only in one season, statistical support was robust, though a greater sampling effort across 397 years with a longer sampling period throughout the season would further strengthen our

398 conclusions across spatial and temporal scales. An understanding of species distributions and

399 abundances in alpine regions is necessary to monitor responses to future change, and our

400 study exemplifies the use of invertebrate species for measuring these processes. In the

401 Australian Alps this is particularly important as the region is characterised by relatively flat

402 and rounded mountains with shallow elevational gradients, (1,600-2,228m on the mainland;

403 Tatarnic et al, 2013), so the opportunity for alpine species to migrate towards cooler, higher

404 elevations under climate-change induced temperature increases is restricted (Tatarnic et al,

405 2013). Furthermore, the results of our study provide a basis for investigating molecular

406 signatures of local adaptation across species in this system, which can inform the broader

407 conservation management of alpine species under environmental change. 408 References

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685

686

687

688 689 Table and Figure

690 Table 1: Correlations between body size and elevation in three Kosciuscola species, shown

691 for males and females individually and for the interaction between sex and elevation

692 (indicated by * mark). ‘Region’ indicates the number of regions analysed in the model.

693

Sex Observations Region Estimate ± SE Chisq DF P K. usitatus Male 174 3 -0.002 ± 0.00 14.295 1 0.0001

Female 237 3 -0.001 ± 0.00 4.93 1 0.02 Elevation*Sex 411 3 - 2.17 1 0.14 K. tristis Male 188 2 - 0 1 0.99

Female 91 2 -0.003 ± 0.001 4.27 1 0.03 Elevation*Sex 279 2 - 6.7162 1 0.009 K. cognatus Male 143 2 -0.012± 0.00 5.5 1 0.01

Female 128 2 - 0.00006 1 0.97 Elevation*Sex 271 2 - 4.016 1 0.045

Collection sites −36.00

Collection sites Collection sites −36.00 − 36.0 −36.05 Collection sites −36.05

− 36.0 −36.1 Jagungal−36.10

Longitude −36.10 Longitude

Collection sites Jagungal −36.35 T1 −36.15 −36.15 −36.2 −36.2 −36.36 0 2 4km −36.20 −36.20

148.30T1 148.35 148.40 Latitude 148.30 148.35 148.40 Latitude −36.3 −36.37 Guthega Jagungal Thredbo Guthega Jagungal Thredbo Longitude atitude L T2 Longitude GuthGuthegaega −36.38 −atitude 36.4 L Longitude −36.4 T3

Collection− 36.39sites 0 1 2km −36.5 Thredbo −36.49 −36.40 Thredbo T2

148.35 148.36 148.37 148.38 148.39 148.40 −36.6 Latitude −36.6 0 10 20km T3 −36.51 Guthega T1Jagungal Thredbo Longitude 148.1 148.2 148.3 148.4 148.5 Latitude Longitude−36.53 Guthega Jagungal Thredbo 148.1 148.2 148.3 148.4 148.5 Latitude 0 2 4km

694 Longitude Guthega Jagungal Thredbo 148.20 148.25 148.30 695 Figure 1: Collection sites for all three species across the three sampledLatitude mountain regions Guthega Jagungal Thredbo

696 (Thredbo, Guthega and Jagungal) shown on a relief map of Kosciuszko National Park.

697

698

699

700

701 (A) 1.2 (C) 1.00

0.80.9 0.75

area

GuthegaGuthega area 0.6 0.4 JagungalJagungal GuthegaGuthega ThredboThredbo 0.50 JagungalJagungal ThredboThredbo

Relative abundance abundance Relative Relative 0.3 Relative abundance Relative 0.25 0.0

0.0 1400 1600 1800 2000 0.00 Elevation (m asl) 1400 1600 1800 2000 Elevation (m asl)

(B) (D) 1.00

0.8 0.75

area area GuthegaGuthega GuthegaGuthega 0.50 JagungalJagungal JagungalJagungal 0.4 Thredbo Thredbo ThredboThredbo Relative abundance Relative

Relative abundance Relative 0.25

0.0

0.00 0 20 40 60 1400 1600 1800 2000 FPC (%) 702 Elevation (m asl)

703 Figure 2: The relative abundance of the three Kosciuscola grasshopper species varied in

704 response to elevation and Foliage Projective Cover (FPC %). (A) K. usitatus; (B) K. tristis;

705 (C) K. cognatus; (D) K. cognatus vs FPC. Regions were pooled for analysis. (A) (B)

13 K. usitatus male K. usitatus female

16 12

15 11

14 10 Femur length (mm) Femur Femur length (mm) Femur

13 9

1400 1600 1800 1400 1600 1800 Elevation (m asl) Elevation (m asl)

12.0 (D) (C) K. cognatus male K. tristis female 16 11.5

11.0 14

10.5 Femur length (mm) Femur Femur length (mm) Femur 12

10.0

1600 1700 1800 19000 1600 1700 1800 1900 2000 Elevation (m asl) 706 Elevation (m asl)

707 Figure 3: Significant changes (P<0.05) in sex-specific body size with elevation in three

708 species of Kosciuscola: K. usitatus A) male and B) female; C) K. tristis female; D) K.

709 cognatus male.

710