1

Higher establishment success in more diverse groups of pygmy grasshoppers 1

under semi-natural conditions 2

LENA WENNERSTEN, JENNY JOHANSSON, EINAT KARPESTAM AND ANDERS FORSMAN1 3

Ecology and Evolution in Microbial Model Systems, EEMiS, School of Natural Sciences, 4

Linnaeus University, SE-391 82 Kalmar, Sweden 5

1Corresponding author: E-mail Anders.Forsman@lnu.se 6

Running title: DIVERSITY ENHANCES ESTABLISHMENT SUCCESS 7

Ecology (Report) accepted 2012-09-06 8

Abstract. Large founder groups and habitat match have been shown to increase establishment 9

success of reintroduced populations. Theory posits that the diversity of founder groups should 10

also be important, but this has rarely been investigated. Here, experimental introductions of 11

colour polymorphic Tetrix subulata pygmy grasshoppers into outdoor enclosures were used to 12

test whether higher phenotypic diversity promotes establishment success. We show that the 13

number of individuals present one year after introduction increases with colour morph 14

diversity in founder groups. Variance in establishment success did not decrease with 15

increasing founder diversity, arguing against an important contribution of sampling effects or 16

evolutionary rescue. Colour morphs in T. subulata co-vary with a suite of other functionally 17

important traits and utilize different resources. The higher establishment success in more 18

diverse founder groups may therefore result in part from niche complementarity. Variation in 19

establishment among groups was not associated with differences among source populations in 20

reproductive capacities. 21

22

Keywords: colonization; conservation biology; establishment; phenotypic diversity; 23

polymorphism; Tetrix subulata; translocation; variable founder groups.24

2

INTRODUCTION 25

Biological diversity is threatened by fragmentation of natural habitats, pollution, climate 26

change, and anthropogenic spread of species. In conservation projects, reintroductions of 27

locally extinct species or restocking of declining populations is used to protect biodiversity. 28

Theoretical and empirical studies concur that repeated introductions (Lockwood et al. 2005), 29

and larger founder groups (Drake et al. 2005) increase establishment success. Larger 30

populations reduce the risk of extinction due to demographic stochasticity (Lande 1988, 31

Simberloff 2009), and may also harbour more genetic variation allowing for faster adaptations 32

to new or changing conditions (Bell and Gonzales 2009). 33

Establishment success may depend on source population, due to differences in 34

reproductive life-history characteristics, genetic structure, local adaptations and environmental 35

similarities between the source habitat and the introduction site (Fagan et al. 2001, Roe et al. 36

2010), but also on degree of diversity in founder groups. It has been predicted that more 37

diverse groups should have a higher establishment success compared with monomorphic 38

groups (Forsman et al. 2008, Hughes et al. 2008, Wennersten and Forsman 2012). In 39

polymorphic groups, morphs may represent integrated phenotypes with alternative 40

combinations of ecologically important traits that utilize different niches (Forsman et al. 2008, 41

McKinnon and Pierotti 2010, Bolnick et al. 2011). The effects of such niche complementarity 42

(Bolnick et al. 2011) may be increased even further if different phenotypes facilitate the 43

success of other phenotypes, as suggested in the case of a higher biomass production in 44

mixtures compared to monocultures in the eelgrass Zostera marina (Reusch et al. 2005) and 45

the diatom Cylindrotheca closterium (Vanelslander et al. 2009). Establishment success may 46

increase also because diverse founder groups are more likely to include already pre-adapted 47

individuals that can survive the environmental conditions at the new site (i.e., a sampling 48

3

effect)(Gamfeldt and Kallstrom 2007), and because standing genetic variance increases the 49

likelihood of evolutionary rescue (Bell and Gonzales 2009). Under limited resources, groups 50

that consist of differently specialized morphs may also benefit from decreased intraspecific 51

competition (Forsman et al. 2008, Hughes et al. 2008, Caesar et al. 2010). 52

The prediction of higher establishment success in more variable founder groups has 53

very rarely been experimentally evaluated. Crawford and Whitney (2010) showed that 54

establishment success was higher in genetically more diverse groups of Arabidopsis thaliana 55

when seeds were planted in trays with soil in a greenhouse. Similarly, Gamfeldt et al. (2005) 56

showed higher establishment success in genetically more diverse founder groups of Balanus 57

improvisus barnacle larvae in the laboratory. Neither of these studies considered or recorded 58

phenotypic differences among individuals. 59

Here we test whether establishment success is promoted by variation in functionally 60

important traits (Forsman et al. 2008, Bolnick et al. 2011, Wennersten and Forsman 2012), 61

and whether it is influenced by source population. Experimental founder groups of colour 62

polymorphic Tetrix subulata pygmy grasshoppers were introduced to outdoor enclosures 63

under semi-natural conditions. We manipulated founder diversity by combining individuals 64

that belonged to similar or different colour morphs and tested for effects on establishment 65

success, quantified as population size one year after the introductions. T. subulata exhibits a 66

high degree of colour polymorphism. Morphs range from light grey via different shades of 67

brown to black, with some morphs being uniform and others mottled or patterned with stripes 68

or speckles (see Appendix A and illustrations availible elsewhere: Caesar et al. 2010, Forsman 69

et al. 2012). Results from dual-choice experiments indicate that there is no mate choice based 70

on colour morph and that females are polyandrous (Caesar et al. 2007). The genetic 71

mechanisms underpinning the variation in colour and pattern – and associated traits (see 72

4

below) – in pygmy grasshoppers are not known in any detail. However, results from captive 73

breeding, parent offspring resemblance studies, common garden and split-brood design rearing 74

experiments indicate that colour morphs are genetically determined and not influenced by 75

developmental plasticity (e.g., Karlsson et al. 2009, Forsman et al. 2011). Pygmy grasshopper 76

colour morphs represent integrated phenotypes that co-vary with other ecologically important 77

traits such as preferred body temperatures, thermal physiology, reproductive life-history (egg 78

and clutch size, inter-clutch interval), body size, predator avoidance behaviour, microhabitat 79

utilization and diet (e.g., Caesar et al. 2010, Forsman et al. 2011, Karpestam and Forsman 80

2011, Forsman et al. 2012 and references therein). For instance, darker morphs not only warm 81

up faster, they also prefer higher body temperatures than paler morphs (Forsman 2000). Both 82

evolutionary rescue and niche complementary may therefore promote the establishment 83

success of more diverse founder groups. Colour morph diversity has recently been shown to 84

increase the establishment success of T. subulata founder groups released into the wild 85

(Forsman et al. 2012), but that study did not control for a possible influence of immigration 86

into or emigration from the experimental sites after founder groups were released. Our use of 87

enclosures precludes such effects in the present study. 88

METHODS 89

Tetrix subulata (Orthoptera: Tetrigidae) is a small (up to 15 mm), ground-dwelling, diurnal 90

pygmy grasshopper species. It has a circumpolar distribution and inhabits biomes ranging 91

from tropical rainforests to arctic regions of Europe, Asia and North to South America 92

(Mexico) (Rehn and Grant 1955, Holst 1986). T. subulata occupies open areas with access to 93

open water or moist soil. They live on the soil surface and feed on algae, moss and dead partly 94

decayed animal and plant matter in the litter. Nymphs develop through five (males) or six 95

(females) instars before final moult (Holst 1986). Late instars and imagos hibernate and 96

5

emerge in spring when mating season begins in April-May in our study area in the southeast of 97

Sweden. Females produce multiple egg pods, with up to 35 eggs/pod, but do not survive for 98

more than one reproductive season (Holst 1986). 99

Outdoor enclosures and founder groups 100

In December 2008, we constructed 80 outdoor enclosures (120 cm in diameter, 60 cm high) 101

placed within a fenced area on a sandy boulder-ridge with pine and birch in Vassmolösa, in 102

the southeast of Sweden (see Appendix B for details). In May 2009, 480 adult T. subulata 103

collected from five populations in different biotopes were used to construct 80 founder groups, 104

each consisting of 6 individuals (4 females and 2 males), that were released into the enclosures 105

(Appendix C). To be able to evaluate effects of both within group diversity and source 106

population, we never mixed individuals from different source populations in the same group. 107

Founder groups differed in degree of colour pattern variation (Forsman et al. 2012). In the 108

most homogenous groups, all individuals belonged to the same colour morph, in the most 109

variable groups all six individuals instead belonged to different colour morphs. The 110

distribution among morphs of individuals used to stock the enclosures matched approximately 111

the relative frequencies in natural source populations. To avoid that any differences in viability 112

or reproductive capacity among morphs should mistakenly be interpreted as an effect of 113

variation, and to enable evaluation of the variance reducing effect of founder diversity, we 114

used 11 different colour morphs for the different homogenous propagules (n = 22). All morphs 115

used in homogenous groups were represented also in variable groups. Between the extremes of 116

one and six colour morphs, we used groups consisting of two, three, four, and five morphs. 117

We prioritized replicates with either low or high variation, at the expense of propagules with 118

an intermediate number of morphs. The exact combination of colour morphs included within 119

6

each group was chosen at random but subject to the constraint imposed by their relative 120

availability in the source populations. 121

One femur from each individual was preserved in 95% EtOH to enable forthcoming 122

genetic analysis of individuals and propagules. We often find grasshoppers in the wild with 123

only one hind leg, and since all individuals and founder groups were treated in the same way, 124

any increased mortality associated with removal of the femur should not bias our results. Prior 125

to release, each propagule was kept for 2-7 days in separate 10-L plastic buckets filled to one 126

third with a mixture of soil and peat and a tuft of hair cap moss Polytrichum commune for 127

housing during preparation and transport (Forsman et al. 2011). Each founder group was 128

randomly assigned to one of the 80 enclosures and released together with the soil and moss. 129

Enclosures were adjacent to each other and therefore exposed to the same weather 130

conditions and other abiotic factors. All enclosures also were prepared in the same way, such 131

that variability was low at the onset of the experiment. However, after one year, vegetation 132

cover, and the number and species composition of plants and invertebrates differed somewhat 133

(not quantified) among enclosures. These differences may have increased the variation in 134

establishment success, but should not have systematically influenced the results or biased the 135

conclusions, since experimental founder groups were randomly assigned to enclosures. 136

To quantify reproductive capacity, adult females (that were not included in the founder 137

groups) from each population (n = 17 – 54) were maintained in the laboratory for egg-laying. 138

Information on housing is provided elsewhere (Forsman et al. 2011). Egg pods were placed on 139

a moist cotton pad in a Petri-dish for incubation, and hatched nymphs per clutch were counted 140

and mean value per population calculated (Appendix C). 141

7

Establishment success 142

All enclosures were surveyed after three months and then again one year after introduction. At 143

the first preliminary inventory performed 19-20 and 24 August, 2009, the number of 144

individuals was estimated, primarily to identify empty enclosures and groups that had not 145

succeeded to survive or reproduce. At the second inventory, performed 26-29 April and 3-4 146

May, 2010 (i.e., one year after propagules were released into the enclosures), two persons 147

searched all enclosures under suitable weather conditions (at least 15oC, no rain, and not too 148

windy). Each enclosure was inventoried for at least five minutes, but searching continued for 149

another five minutes after the last individual was found, to ensure that no individuals remained 150

hiding in the vegetation. All individuals were caught, counted and classified by sex, age 151

(nymph or imago), and colour morph. All morph classifications were made by the same person 152

(LW) to avoid biases due to inter-observer differences in perception and classification. To 153

avoid false zeros and assess our ability to accurately estimate low numbers of individuals, 154

seven enclosures with no or few individuals at the first search in the morning were searched 155

again in the afternoon. The numbers of individuals found in each enclosure during the first and 156

second search were highly correlated (measurement repeatability was estimated from one-way 157

ANOVA with enclosure identity as fixed factor (Sokal and Rohlf 1981): F 6,13 = 3.87, η2= 158

95%, n = 7, P < 0.001). 159

Since T. subulata individuals live for one year only, grasshoppers found in spring 2010 160

were descendants of the original propagules. The number of individuals recorded in each 161

enclosure in spring 2010 was used as a measure of establishment success in the analyses 162

(Forsman et al. 2012). The number of colour morphs was recorded to quantify diversity in the 163

established population. 164

8

Statistical analyses 165

We used Generalized Linear Mixed Models (GLMMs) implemented using procedure 166

GLIMMIX in SAS (Littell et al. 2006) to test for effects of diversity (1-6 colour morphs), 167

source population (n = 5), and reproductive capacity (average number of nymphs per clutch 168

for each source population) on establishment success (number of individuals one year after 169

introduction). We modelled establishment success using Poisson distribution, suitable for 170

analyzing count data. Diversity and reproductive capacity were treated as fixed effects, and 171

source population as a random effect. We used the Kenward-Roger method to approximate 172

degrees of freedom. To evaluate the prediction that variation in establishment success should 173

decrease with increasing diversity in the founder groups, we computed an index of dispersion 174

(ratio of the variance to the mean number of individuals) and tested for an association with 175

number of morphs present in the founder groups. 176

To test whether the number of colour morphs in founder groups influenced diversity in 177

established experimental populations we estimated colour morph diversity in each sample, 178

expressed as residuals from least-squares linear regression of log number of colour morphs on 179

log number of individuals (Forsman et al. 2012). We evaluated the association between colour 180

morph diversity in the established population and number of colour morphs in the founder 181

group using Pearson correlation analysis. Finally, the null-hypothesis that the relative 182

frequency of different colour morphs in experimentally established populations was 183

independent of the relative frequency of colour morphs among individuals in the founder 184

groups was evaluated using contingency table analysis (Forsman et al. 2012). 185

RESULTS 186

At the first inventory, three months after release, 10 enclosures (12.5%) were empty. In the 187

majority (7/10) of these empty enclosures, founder groups with only one or two colour morphs 188

9

had been released. At the second inventory, one year after release, we caught 601 individuals 189

(average = 7.5 individuals/enclosure, range 0-56 individuals). At this time, 19 enclosures were 190

empty, including those 10 that were empty already in August 2009 and an additional seven of 191

which contained only one or two individuals in August 2009. Average morph diversity in 192

established populations was 3.8 (range 1-14) morphs per enclosure. 193

The average number of hatched nymphs per clutch ranged from 11.8 (SD = 6.2) in 194

Jordtorpsåsen to 23.3 (SD = 8.5) in Bredsättra, and differed significantly among the five 195

source populations (one-way Anova: F4, 128 = 3.54, P = 0.009) (Appendix C). 196

Establishment success increased with colour morph diversity in founder groups 197

The number of individuals present in the enclosures after one year increased significantly with 198

increasing colour morph diversity in the founder group (GLMM: F 1,30.53 = 12.99, P = 0.0011) 199

(Fig. 1), but was not influenced by the interaction between source population and colour 200

morph diversity (GLMM: F 4,1 = 3.34, P = 0.39). The effect of founder diversity was strong; 201

number of individuals present after one year ranged from 4.1 on average in enclosures where 202

monomorphic groups had been introduced to 11.9 (i.e., almost a three-fold increase) in 203

populations established by the most diverse founder groups that contained 6 different morphs 204

(Figure 1). Variation in reproductive capacity among source populations did not influence 205

number of individuals one year after the introduction (GLMM: F 1,1.449 = 0.14, P = 0.76) 206

(Appendix D). The results also indicated no significant random effect of source population 207

(estimate 0.0235, se 0.076). 208

Variation in establishment success among replicates did not decrease with increasing 209

morph diversity in the founder groups, the index of dispersion (ratio of the variance to the 210

mean number of individuals) instead tended to increase with increasing founder morph 211

diversity (rs = 0.77, N = 6, P = 0.07)(see also Fig. 1). 212

10

Colour morph diversity in established populations 213

Colour morph diversity in experimental populations (expressed as residuals from the least-214

squares linear regression of log number of colour morphs on log number of captured 215

individuals) was not associated with the number of morphs included in the introduced founder 216

groups (correlation on individual data points: r = 0.16, n = 61, P = 0.76; Least-squares linear 217

regression on mean values: F 1,4 = 0.10, P = 0.76, R2 = 0.03)(Appendix E). 218

The distribution of individuals among different colour morph categories in the samples 219

collected from established populations one year after the introductions was different from the 220

distribution among individuals included in the founding groups (χ2 = 78.45, df = 17, P < 221

0.0001), indicating that some colour morphs increased and others decreased in relative 222

frequency between generations (Appendix F). 223

DISCUSSION 224

In this study, we introduced founder groups of 6 T. subulata pygmy grasshoppers to 80 225

outdoor enclosures under semi-natural conditions, and demonstrated that the number of 226

individuals present after one year increased with increasing colour morph diversity in the 227

founder group (Fig. 1). This result is in accordance with expectations from theory (reviewed 228

by Forsman et al. 2008, Hughes et al. 2008, Bolnick et al. 2011, Wennersten and Forsman 229

2012), and in agreement with previous studies that report on higher establishment success and 230

increased population persistence in genetically more variable founder groups (Gamfeldt et al. 231

2005, Mattila and Seeley 2007, Agashe 2009, Crawford and Whitney 2010). However, in 232

comparison to these previous studies, our experiment was performed under more natural 233

conditions and investigated the influence of phenotypic variation in functionally important 234

traits (see also Forsman et al. 2012). 235

11

There are many ways and pathways by which polymorphism in colour pattern and 236

associated phenotypic traits may influence the ecological and evolutionary success of 237

populations. The higher establishment in more diverse founder groups may have been 238

mediated by reduced predation in polymorphic groups (Bond 2007), or by other factors 239

operating on colour pattern per se. However, pygmy grasshopper colour morphs represent 240

complex phenotypes that differ in a suite of behavioural, physiological and reproductive life-241

history characteristics (e.g., Caesar et al. 2010, Forsman et al. 2011, Karpestam and Forsman 242

2011, Forsman et al. 2012 and references therein). It is therefore possible that the greater 243

success of more diverse groups was driven by selection on traits that are genetically, 244

developmentally or functionally associated with colour pattern, such as development time or 245

disease resistance (for examples see reviews in True 2003, McKinnon and Pierotti 2010). 246

The shifts in relative frequencies of the different morphs between individuals in 247

founding groups and in established populations demonstrated in the present study (Appendix 248

F) are indicative of selection. We consider it unlikely that any of the introduced T. subulata 249

morphs was optimally suited for the enclosures. When different morphs are present in the 250

founder group, the probability increases that at least one of them has a combination of 251

reproductive, physiological and behavioural traits well suited for life in new or changing 252

habitats (Forsman et al. 2008, Hughes et al. 2008, Bolnick et al. 2011). However, that we did 253

not find an increased predictability and decreased variance in establishment success with 254

increasing founder diversity indicates that such a sampling effect did not play an important 255

role in our experiment. The increased establishment success with increasing founder diversity 256

may instead be attributed to niche complementarity (Reusch et al. 2005), mediated via 257

differences among morphs in thermal requirements, physiology, microhabitat use, and life-258

history characteristics. Vertebrate predators were excluded, but more diverse groups may have 259

12

been less susceptible to invertebrate predators, parasites and disease (Baer and Schmid-260

Hempel 1999). Since T. subulata colour morphs differ in diets (Karpestam and Forsman 261

2011), individuals in diverse groups may also have benefitted from decreased competition 262

(Caesar et al. 2010). However, pinpointing the exact mechanisms and selective agents that 263

mediate the effects of variation goes beyond the scope of the present study and will require a 264

different approach. 265

Since the experimental animals were confined to enclosures that represented a single 266

type of environment our present study did not mimic a natural situation but nevertheless 267

resembled, in several respects, conditions in the wild. The densities of individuals at the onset 268

(6 individuals/enclosure) and at the end of the experiment (up to 56 individuals/enclosure) are 269

within the range observed in the wild (Forsman et al. 2012), and considerably lower than those 270

that have been successfully used for breeding and common garden experiments (Forsman et 271

al. 2011 and references therein). The enclosures undoubtedly constrained the grasshoppers’ 272

mobility, but mark-recapture data suggest that free-ranging adult T. subulata are sedentary and 273

move only a few meters per day in the wild (Forsman and Appelqvist 1999). Moreover, 274

pygmy grasshoppers produce egg pods that may result in more than 30 hatchlings, and 275

nymphs probably remain in close vicinity to where they hatch. As for the levels of colour 276

morph diversity, the most extreme founder groups consisted of 6 individuals that represented 6 277

morphs. By comparison, representatives of up to 5 different morphs can be found among 278

offspring from within a single clutch (e.g., Forsman et al. 2007). Finally, the compositions of 279

food types, competitors and predators differed from the wild. Even though the enclosures used 280

in our present study did not completely mimic natural conditions, Forsman et al. (2012) 281

arrived at a similar result when more than 60 T. subulata founder groups were introduced 282

during a three-year period to different meadows, clear-cuts, pastures and coastal meadows in 283

13

the wild, indicating that the positive effect of founder diversity on establishment success is 284

manifest under natural conditions, and repeatable across habitat types. 285

The variation in reproductive capacity among source populations did not influence 286

establishment success. This negative outcome probably is not due to low statistical power and 287

insufficient variation in reproductive capacity among populations, since average number of 288

nymphs per clutch ranged from 11.8 in the population at Jordtorpsåsen to 23.3 nymphs in 289

Bredsättra (Appendix C). The highest number of individuals found in the enclosures after one 290

year was 56. Each propagule included four females, and each female may lay several egg pods 291

of up to 35 eggs per pod. It therefore seems likely that variation in establishment success 292

among populations was influenced more by mortality of founder females prior to egg-laying, 293

or by hatchability of eggs or mortality of nymphs due to pathogens, parasites or predation 294

from other invertebrates, than by differences in reproductive capacity. That we found no effect 295

of reproductive potential may also be due in part to the fact that the population–level estimates 296

of reproductive potential were based on data for leftover individuals from each population, 297

since they belonged to colour morphs that were not representative of those used for stocking 298

the enclosures. A low impact of reproductive capacity on establishment was reported in a 299

meta-analysis of studies of translocation of different animal species published between 1973-300

1986, where early breeding species with large clutches were found to be only slightly more 301

successfully established than late breeding species with small clutches (75 % and 62 % 302

respectively) (Griffith et al. 1989). However, reproductive potential of the species was not 303

significantly associated with establishment success when the same set was re-analyzed using a 304

comparative approach that accounted for phylogenetic relationships (Wolf et al. 1998). 305

Higher establishment success in more variable founder groups could be one 306

explanation as to why some of the most well known invasive species (e.g., the harlequin 307

14

ladybird, Harmonia axyridis, the zebra mussel, Dreissena polymorpha, the Spanish slug Arion 308

vulgaris, the lizard Anolis sagrei, the brown tree snake Boiga irregularis, and the cane toad 309

Bufo marinus) all show high levels of intra-specific colour pattern variation and have 310

undergone extreme range expansions (Savidge 1987, Johnson and Carlton 1996, Krafsur et al. 311

1997, Kolbe et al. 2004, Phillips and Shine 2006). Although these examples do not represent 312

tests of causality, the pattern is suggestive and deserves further investigation. It also remains to 313

be investigated whether it is variation in colour pattern per se, or polymorphism in other 314

functionally important traits associated with coloration that is important. That diversity 315

promotes establishment success may also explain why, despite the eroding effects of founder 316

events and drift in small peripheral populations, some studies have reported surprisingly high 317

levels of genetic variation in marginal populations (Simberloff 2009). 318

In the context of conservation biology, taking within-group diversity into consideration 319

may enhance the success of re-location projects, especially when small founder groups must 320

be used. Local adaptations that match the challenges imposed by the conditions in the new 321

habitat are probably crucial for establishment, while standing genetic variation is most likely 322

to determine long-term persistence (Agashe 2009). When Forsman et al., (2012) introduced T. 323

subulata into the wild, they found that variation present in founder groups positively affected 324

colour morph diversity in the established populations, such that it may contribute also to long-325

term persistence via evolutionary rescue (Bell and Gonzales 2009). In the present study, 326

however, relative colour morph diversity in the established populations was independent of 327

number of colour morphs in founding groups (Appendix E). 328

In conclusion, our present findings demonstrate, in accordance with expectations from 329

theory (Forsman et al. 2008, Hughes et al. 2008, Wennersten and Forsman 2012), that an 330

increased diversity among individuals in functionally important traits offers a means to 331

15

increase establishment success, and suggest that the role of individual variation deserves 332

increased consideration in applied conservation and re-introduction programs. Associations of 333

colour pattern with other ecologically important traits have been reported in several species in 334

addition to pygmy grasshoppers, including birds, fish, reptiles and mammals (McKinnon and 335

Pierotti 2010). The positive effect of diversity on establishment demonstrated here, and by 336

Forsman et al. (2012) under natural and different conditions in the wild, may therefore be 337

applicable also to other model systems and types of organisms. Future investigations should 338

attempt to disentangle the relative contribution of colour pattern itself and of other 339

functionally important traits associated with coloration. 340

ACKNOWLEDGMENTS 341

We are grateful to M. Karlsson, P. Tibblin and A. Månsson for helping with construction of 342

enclosures and setting the irrigation system. J. Eriksson, S. Forsman and H. Berggren helped 343

in the field. P. Ekdahl, P. Hallström and their colleagues at Kalmar Vatten AB, Kalmar, 344

Sweden, let up the place for the experiment and assisted with installation of electricity. O. 345

Hellgren and two anonymous reviewers commented on the manuscript. The study was 346

supported financially by The Swedish Research Council, The Swedish Research Council 347

Formas (grants to AF) and Linnaeus University, Kalmar. 348

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450

SUPPLEMENTAL MATERIAL 451

Appendix A 452

A photo showing examples of Tetrix subulata pygmy grasshopper colour morphs included in 453

the experiment is available in ESA’s Electronic Data Archive: Ecological Archives. 454

Appendix B 455

A description of the outdoor enclosures is available in ESA’s Electronic Data Archive: 456

Ecological Archives. 457

Appendix C 458

A table showing information of location, biotopes and reproductive capacity of the five Tetrix 459

subulata source populations used to construct founder groups is available in ESA’s Electronic 460

Data Archive: Ecological Archives. 461

Appendix D 462

A figure showing establishment success of Tetrix subulata founder groups as a function of 463

reproductive capacity in the source population is available in ESA’s Electronic Data Archive: 464

Ecological Archives. 465

Appendix E 466

A figure showing colour morph diversity in established populations as a function of the 467

number of morphs included in the founder group is available in ESA’s Electronic Data 468

Archive: Ecological Archives. 469

Appendix F 470

21

A figure showing the relationship between the relative frequencies of different colour morphs 471

among individuals in established populations and in experimental founder groups is available 472

in ESA’s Electronic Data Archive: Ecological Archives. 473

474

22

FIGURE LEGEND 475

476

FIG. 1. Number of Tetrix subulata individuals present in outdoor enclosures after one year as 477

a function of number of colour morphs included in founding propagules released the previous 478

year. Data for 80 founding groups each consisting of 6 (4 females and 2 males) individuals. 479

Numbers above horizontal axis indicate sample sizes (i.e., number of replicates). Diamonds 480

indicate means. 481

23

Colour morphs in founder group

1 2 3 4 5 6

Indi

vidu

als

year

N +

1

0

10

20

30

40

50

60

22 13 3 4 17 21

482