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Ecological Entomology (1999) 24, 432±442

Social biology and sex ratios of the eusocial gall- inducing Kladothrips hamiltoni

BRENDA D. KRANZ, MICHAEL P. SCHWARZ, 1 2 LAURENCE A. MOUND andBERNARD J. CRESPI School of Biological Sciences, Flinders University, Australia, 1CSIRO Entomology, Canberra, Australia and 2Department of Biosciences, Simon Fraser University, Canada

Abstract. 1. Thrips comprise the only order besides where females are diploid and males are haploid. This makes them useful for studying the roles of and ecology in social evolution. 2. Kladothrips hamiltoni is one of six species of Australian gall-inducing thrips that have been identi®ed as eusocial. Galls are initiated by a single foundress, who rears her brood and remains within the enclosed gall for life. The adults of both sexes of her ®rst brood cohort are morphologically distinct from the second generation, comprising a nondispersing soldier caste. The foundress and sib-mated soldiers jointly produce a second, dispersing generation, approximately 60±80% of which are produced by the soldiers. Mean per capita egg production of female soldiers is less than 33% that of the foundress. 3. Adult eclosion of soldiers is protandrous but the overall sex ratio of the soldiers lacks bias (52% male). Protandry of soldiers increases the probability that female soldiers will be inseminated soon after their eclosion and therefore lay fertilised, female eggs. The lack of bias could be due to a balance between local resource competition and local mate competition. Gender-speci®c defensive behaviour of soldiers with their enemies may also be important in explaining this unexpected sex ratio. 4. The dispersing generation has an overall extreme female bias (5.6% male). Soldier incest increases relatedness between females more than between males, such that the foundress is more related to her granddaughters than her daughters, and female soldiers are more related to their daughters than their sons (assuming within-gall relatedness < 1). A female bias in the offspring of soldiers should be preferred by both the foundress and soldiers as they are more related to soldier- produced dispersing females than any other thrips in the gall. Female bias in the dispersing generation will also reduce local mate competition between males. Both soldier incest and local mate competition may therefore contribute to the extreme female bias in the dispersing generation. 5. Selection pressures for in gall-inducing thrips appear to be more similar to those in gall-inducing aphids and naked mole rats than to those in Hymenoptera. Key words. Galls, , incest, local mate competition, local resource competition, sex ratio, thrips.

Correspondence: Brenda Kranz, School of Biological Sciences, Flinders University of South Australia, GPO Box 2100, Adelaide S.A. 5001, Australia. E-mail: brenda.kranz@¯inders.edu.au

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Sociality and sex ratio in a galling thrips 433

Introduction For these reasons, and because of the role that soldiers play in gall defence, Crespi(1992a,b) described Oncothrips tepperi Thysanoptera is the only order other than Hymenoptera where and O. habrus as eusocial, following the de®nition of Wilson all females are diploid and males are haploid (Stannard, 1968; (1971). Crespiand Mound (1997) further described Lewis, 1973; Crespi, 1991; Chapman & Crespi, 1998). This K. hamiltoni and other species with soldiers as eusocial, using makes thrips useful insects for studying the interplay between the de®nition of Crespi and Yanega (1995). haplodiploidy and kin selection in social evolution, because The aim of the study reported here was to examine the life- haplodiploidy has been postulated to be a key factor shaping history and social biology of K. hamiltoni in detail. Brood the evolution of sociality in Hymenoptera (Hamilton, 1964; development of the soldier and dispersing generations, Trivers & Hare, 1976). Kin selection theory predicts that for reproductive contributions of the foundress and soldiers to haplodiploid females, the genetic payoffs for altruism towards the dispersing brood, and the sex ratio of each generation were sisters are greater than for rearing daughters or sons, and lower investigated. The results are discussed in terms of kin selection still for helping to rear brothers, whereas males should and sex ratio biasing factors such as local mate competition concentrate on direct reproduction (Trivers & Hare, 1976); (Hamilton, 1967) and local resource competition (Clark, however, the payoffs for rearing siblings depend on sex 1978). allocation and the reproductive value of males and females (see reviews by Bourke & Franks, 1995; Crozier & Pamilo, 1996). Materials and methods Six species of tubuliferan thrips that induce galls on Australian have been identi®ed as eusocial (Crespi, Study organism 1992a; Crespi& Mound, 1997). These thripshave a morphologically distinct soldier caste (reduced wings, anten- Kladothrips hamiltoni adults are about 2 mm long and nae, and melanisation; enlarged forelegs) that ®ghts invaders. induce tubular galls about 30 mm long by approximately 3 mm Phylogenetic analyses suggest that in these thrips in diameter. They occur on Acacia cambagei, a tree that grows may have had two independent origins (Mound et al., 1996; along episodic waterways in the Lake Eyre Basin region of Crespi& Mound, 1997; Crespi et al., 1997, 1998). Gall- central Australia (Mound & Crespi, 1995; Mound et al., 1996). inducing thrips are of particular interest in assessing the roles The basic life-cycle of K. hamiltoni is described by Mound and of ecology and genetics in the evolution of social behaviour in Crespi (1995) and is summarised here. Galls are initiated by a haplodiploids because their life-cycle and ecology differ single macropterous female. Following closure of the gall, the markedly from many other social insects. For example, thrips foundress lays eggs that develop into micropterous adults, with do not forage or nurse their young as do eusocial enlarged forelegs and reduced cuticular melanisation (sol- Hymenoptera. This is because galls act as nutrient sinks so diers), that defend against invaders, particularly Koptothrips that, as long as thrips feed on the cell contents of the gall, a and lepidopteran larvae. The foundress dies some time supply of nutrients is ensured until gall or plant senescence following soldier eclosion into adults. Most female soldiers (Raman, 1987; Ananthakrishnan & Gopichandran, 1993). have some ovarian development and lay eggs following death There may be selective pressure for some individuals to of the foundress. These eggs develop into macropterous specialise in gall defence, however. Gall-inducing thrips are dispersing adults that become the foundresses and their mates prone to attack by kleptoparasites and other enemies; galls of the next generation of galls. form highly attractive food and shelter resources in the arid habitats where eusocial thrips occur (Crespi, 1992a,b; Crespi & Mound, 1997; Crespi et al., 1997). Study site and ®eld collections Thrips are unusual among galling insects because the adult foundress lays her eggs after being enclosed within the gall. Kladothrips hamiltoni galls were collected from A. camba- There are currently 21 described species of tubuliferan gall- gei at two sites along Arckaringa Creek (27°56¢S, 134°44¢E inducing thrips in the genera Oncothrips, Kladothrips, and and 28°6¢S, 135°5¢E), approximately 1100 km north of Onychothrips (Mound, 1971; Mound & Crespi, 1995; Mound Adelaide, South Australia, in the southern Lake Eyre Basin. et al., 1996; Crespi & Mound, 1997). Species with a soldier One site was adjacent to Arckaringa Creek Homestead and the caste belong to the genera Kladothrips and Oncothrips (Mound other was 40 km SE of this site, where Arckaringa Creek et al., 1996). In these species, galls are usually initiated by a crosses the Coober Pedy to Oodnadatta road. Arckaringa Creek single female and the ®rst brood consists only of soldiers. This is usually a dry creek bed but ¯oods on average every 2± brood is followed by a second, dispersing generation in which 3 years, usually in late spring or early summer (November to the adults are morphologically indistinguishable from the gall February). Temperatures in Oodnadatta, the closest weather foundress. These dispersing adults are probably produced by station to the sites, range from ± 2.2 to 50.7 °C, the median both the foundress and soldiers, although the relative monthly rainfall ranges from 2.0 to 9.9 mm, and the median contributions of foundresses and soldiers have not been annual rainfall is 64.7 mm (Commonwealth of Australia, quanti®ed. Female soldiers are inseminated and lay eggs, 1998). although their fecundity is lower than that of their mother Collections were made at the Arckaringa Homestead site (Crespi, 1992a; Mound & Crespi, 1995; Kranz et al., 1998). between 7 December 1995 and 14 May 1996 (Table 1), and

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434 Brenda D. Kranz et al.

Table 1. Summary data for galls censused between December 1995 and May 1996. n is the number of galls censused. Live foundress is the proportion of galls whose foundress was alive out of all galls sampled. Brood size is the mean number of eggs, larvae, pupae, and adult soldiers. Other data are means with standard errors.

Live Proportion male Proportion Date n foundress Eggs 1st instars 1st instars Soldiers male soldiers Brood size

7 Dec 1995 7 0.92 1.6 6 0.8 0 ± 0 ± 2 6 0.8 28 Dec 1995 10 0.80 17.1 6 2.5 2.9 6 0.7 0.75 6 0.08 0 ± 23 6 3.0 6 Jan 1996 3 1.00 23.7 6 4.7 3.7 6 1.8 0.52 6 0.02 1.3 6 0.7 0.75 6 0.25 37 6 5.3 15 Jan 1996 6 0.80 23.0 6 5.1 7.2 6 1.2 0.48 6 0.15 3.8 6 0.4 0.96 6 0.04 39 6 5.3 30 Jan 1996 5 0.80 28.2 6 4.5 7.6 6 2.4 0.44 6 0.09 12.6 6 1.4 0.49 6 0.13 65 6 4.8 30 Mar 1996 7 0.14 51.4 6 7.9 29.9 6 5.2 0.07 6 0.03 27.9 6 3.4 0.56 6 0.05 164 6 16 30 Apr 1996 13 0.00 30.9 6 7.2 22.6 6 5.0 0.08 6 0.02 23.9 6 1.4 0.48 6 0.04 127 6 15 14 May 1996 5 0.00 13.4 6 5.7 14.4 6 3.7 0.11 6 0.08 23.4 6 3.2 0.54 6 0.10 74 6 11

from both sites on 20 January, 31 May 1997, and 25 February Ovarian development and insemination status 1998. For each collection, approximately 20 galls from each of two to six trees were detached from branches, placed in plastic Gall foundresses and soldiers from the 25 February 1998 bags, and kept on ice during transport to Flinders University. In collections were dissected to assess ovarian development. the laboratory, galls were either stored temporarily at 8 °C, Adults from fresh galls were dissected in 0.9% saline solution frozen at ± 70 °C, or preserved in acetic acid-glycerol-alcohol using watchmaker forceps. The abdomen was pulled apart (AGA; glacial acetic acid : glycerol : 60% ethanol, 1 : 1 : 10). carefully by holding the posterior end of the thorax and the terminal segments of the abdomen to display the ovaries and spermatheca or testes. The length of the three largest oocytes Demographic and sex ratio study was measured to an accuracy of 6 2.5 mm, and the number of chorionated oocytes [brown oocytes with the hexagonal Five to 30 intact galls were censused from each collection reticulation characteristic of many tubuliferan eggs (Heming, period. All eggs, ®rst and second instars, pupae (including pre- 1991)] was counted. Insemination status of females was pupae), micropterous adults (soldiers), and gall founders were determined by observing the spermatheca, using a Zeiss rinsed from their gall with 60% alcohol into a 4-cm Petri dish, Universal compound microscope (Carl Zeiss, Oberkochen, with 1 cm grids etched into the bottom, and counted under a Germany) with differential interference contrast microscopy Leica dissecting microscope (Leica AG, Heerbrugg, (Nomarski) optics at 4003 magni®cation. The density of Switzerland). sperm in the spermathecae of each of 10 female soldiers was Individuals from most frozen galls, all galls stored in acetic estimated and ranked on an ordinal scale from zero to ®ve, acid : glycerol : alcohol, and mature fresh galls were placed in a where zero was uninseminated and ®ve was very densely 100-mm plankton mesh sieve and sonicated (Soniclean 30 A + , packed with sperm. Sperm were not counted as they were on Transtek systems, Adelaide, Australia) for 10 min to remove several focal planes when observed under Nomarski illumina- old exuviae, excrement, and in some cases fungal hyphae, so tion. that individuals could be identi®ed to stage and ®rst instars could be sexed using chaetotaxy. First-instar larvae were sexed by mounting them dorsally on microscope slides in Hoyer's Spatial and feeding constraints of brood mounting medium, oven drying at 50 °C for at least 24 h, and examining the chaetotaxy of the ninth abdominal segment at In order to assess whether female soldiers were competing 2003 magni®cation with an Olympus BH2 compound light with each other for reproductive opportunities as a result of microscope (Olympus Optical, Tokyo, Japan). First-instar spatial or feeding constraints within the gall, the percentage of females of all thrips have three pairs of setae on the dorsal gall volume occupied by brood (all eggs, larvae, pupae, and aspect of their ninth abdominal segment whereas males have adults) was determined from a sample of 16 galls, collected on four (Heming, 1991). [Note that Crespi (1993) did not include 30 March 1996 when all soldiers appeared to have eclosed and the smallest pair of lateral setae found in both sexes and the brood size was at its largest (Table 1). The internal volume consequently referred to tubuliferan females as having two, of each gall was determined by removing all brood and debris and males three, pairs of setae.] While these setae could be from the gall interior, weighing the empty gall to an accuracy examined on specimens mounted in 0.9% saline and examined of 6 0.1 mg, packing the gall interior with a clay of known immediately, resolution of the setae improved after lipids had density, reweighing and converting to volume. The mass of the been cleared from the larvae. Adults, other than those dissected brood was measured to 6 0.001 mg using a Mettler UMT2 (see below), were cleared in Hoyer's medium, as for ®rst microbalance (Mettler-Toledo AG, Greifensee, Switzerland). instars, and sexed by viewing the fustis of females or Brood density was determined by displacing ®ve replicates of phallobase of males (Mound & Heming, 1991). approximately 200 individuals of total known mass, represent-

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Sociality and sex ratio in a galling thrips 435 ing all brood stages, in 0.9% saline (to minimise osmotic grasshoppers (observed on two occasions in the ®eld) or birds. stress). These holes probably accounted for the decline in mean brood In addition to gall volume, the internal surface areas of the size by May (Table 1). Dead adult soldiers and live, immature same galls were estimated using simple geometry, by assuming brood of the dispersing generation were still present in all galls the galls to be cylinders. The length and maximum width of 10 censused at this time. Many galls were observed to have individuals for each brood stage were measured to an accuracy dropped from trees and still contained live second-instar larvae of 6 2.5 mm, under a compound light microscope, after of the dispersing generation. mounting in 0.9% saline. For soldiers, ®ve individuals of each sex were measured to account for gender-speci®c size differences. The lengths and widths of each individual were Longevity of foundress multiplied and averaged with others of the same develop- mental stage to give an approximate footprint size for each The gall foundress was alive in 92% of all galls sampled on brood stage. From these footprints, the percentage of the total 7 December 1995, 14% of galls sampled on 30 March 1996, surface area of the gall that the brood occupied was estimated. and in no galls by and after 30 April 1996 (Table 1). The foundress was dead in 45% of all galls before the ®rst adult female soldier eclosed, 52% of all galls with up to two female Data analyses soldiers, and in all galls containing nine or more female soldiers (maximum of 17 females in a gall). All data were analysed using SPSSq version 8.0 (SPSS Inc., Chicago, Illinois). Homogeneity of variances was analysed using Levene's test. T-tests, assuming either homogeneity of Soldier emergence and brood sizes variances or not, were used where appropriate, and for the tests where homogeneity could not be assumed, Levene's and the In the following section, foundress and soldier clutch sizes statistics for unequal variances are speci®ed. Sex-ratio data are estimated by comparing the number of brood produced by were arcsin transformed prior to statistical analyses. the foundress just prior to the ®rst eclosion of soldiers with the total number of brood once all soldiers had eclosed. These data are used to determine when the foundress began laying eggs Results destined to become dispersers and what proportions of the dispersing brood were produced by the foundress and soldiers. Gall initiation and development Only data from the 1995 and 1996 collections are used in this analysis. Gall initiation by Kladothrips hamiltoni occurred in early The soldier generation was protandrous in both the ®rst summer (December) for all years, following rain and instar and adult stages (Tables 1 and 2). Data from the galls subsequent new phyllode growth on A. cambagei. Females collected on 28 December 1995 were pooled with a single gall usually initiated galls alone but 6% of unparasitised galls also without any eclosed soldiers from 6 January 1996, to contained a macropteran male (7 of 115 galls examined). As investigate brood size just prior to the ®rst eclosion of soldiers three of the galls that included a male founder were too young (Table 1). The mean brood size for these galls was 24.8 (6 3.5 to contain any brood other than eggs, no meaningful statistics SE, n = 11 galls), which was not signi®cantly different from the could be developed on possible differences in brood develop- mean number of adult soldiers (24.9 6 1.3, n = 25) once all had ment and sex ratio between those galls with and without male eclosed (t-test, assuming unequal variances Levene's F = 8.7, founder. P < 0.01, t12.9 = ± 0.03, P = NS). There was no obvious Only data from the 1995 and 1996 collections were used for mortality of larvae prior to soldier eclosion, as juvenile moults analysing brood (i.e. all eggs, larvae, pupae, and adult soldiers) in fresh, unparasitised galls could be distinguished easily from development and brood size, because the time intervals dead juveniles in galls that had been left for several weeks after between the other collections were too large for reliable collection. Consequently, it is likely that the foundress laid interpretations to be made (Table 1). Foundresses in galls most of her soldier-destined eggs prior to the eclosion of any of collected on 7 December 1995 had probably commenced egg these as adults, and most of the eggs laid following soldier laying within the previous 2 or 3 weeks, because the interval eclosion were destined to become the macropterous, dispersing between egg laying and emergence of ®rst-instar larvae in later generation. collections was approximately 2 weeks (Table 1, Fig. 1a). The The mean brood size (all eggs, larvae, pupae, and adult ®rst adult soldiers had eclosed in galls collected on 6 January soldiers) following male soldier eclosion but prior to any 1996. All soldiers appeared to have eclosed by the 30 March female soldier eclosion, from two galls collected on 6 January 1996 collection, because the mean number of soldiers per gall 1996 and ®ve galls collected on 15 January 1996 (Table 1), plateaus by this date (Table 1, Fig. 1b) and a one-way ANOVA was 48 (6 6.1, n = 7), which is signi®cantly more than the revealed that there was no signi®cant difference in the number mean number of all adult soldiers (t-test, assuming unequal of the soldier adults per gall for the 30 March, 30 April, and variances Levene's F = 19.9, P < 0.001, t6.6 = ± 3.72, P < 0.01). 14 May samples (F2,24 = 1.126, P = NS). By 14 May 1996, galls The difference in these brood sizes implies that, on average, often had holes, presumably from chewing by acridoid the foundress laid a minimum of about 23 eggs that were

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Fig. 1. Mean sex ratio (proportion of males) 6 SE of (a) ®rst-instar larvae and (b) adult soldiers for the 1995±96 season. In (a), soldiers, foundress-produced dispersers, and soldier-produced dispersers refer to the morph into which the larvae develop for that time period. In (a) and (b), the dashed line across 0.5 indicates sex-ratio parity.

destined to become macropterous dispersers (48 minus mean Therefore, on average the foundress and soldiers produced a number of all adult soldiers). Once all soldiers had eclosed, the maximum of approximately 42 and 77% of the dispersing mean brood size was 127 (6 11.0, n = 25), indicating that there brood, respectively. was an average of approximately 100 dispersing brood in the gall (mean total brood size minus mean number of adult soldiers). The mean number of eggs laid by the soldiers Reproductive output of foundress and female soldiers collectively at their peak egg-laying time, after the foundress had died, was 58.7 (6 0.26, n = 6). From all these data, it Ovarian development of foundresses was compared with follows that, on average, the foundress produced a minimum of that of female soldiers from 35 galls collected on 25 February approximately 23% of the dispersing brood (minimum number 1998. At this time, the mean brood size was 47 (6 3), the mean of dispersers produced by the foundress: total number of number of adult male soldiers was 5.1 (6 0.6), and the mean dispersers), and the soldiers collectively produced a minimum number of adult female soldiers was 3.3 (6 0.4). The oocyte of 58% of the dispersing brood (mean number of eggs at sizes of the foundress and soldiers were compared using an soldiers' peak laying time: total number of dispersers). unbalanced ANOVA nested design as there were unequal

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Sociality and sex ratio in a galling thrips 437 numbers of adult thrips in each gall. Gall was designated as a brood size, the distribution of the data was examined and a random factor for the analysis. Adult morph (foundress or linear regression of arcsin-transformed sex ratio with the soldier) was treated as a factor nested within gall so that the number of soldiers was performed. Adult soldier broods with a effect of gall was removed before analysing the effect of adult sex ratio of 1 (all male) occurred only in galls with less than morph. Only oocytes greater than 50 mm in length were ®ve soldiers, which is consistent with protandry, so these data included in the analysis, in order to exclude newly eclosed were removed from the analyses. Visual inspection of the sex- soldiers with immature ovaries. Foundresses had signi®cantly ratio distribution indicated that the data were unimodal, and longer oocytes (mean of three largest = 263 6 11 mm, n = 30) there was no signi®cant relationship between sex ratio (arcsin than did soldiers [141 6 23 mm, n = 26; ANOVA (adult within transformed) and number of soldiers (linear regression gall): gall F30,24 = 1.285, P = NS, adult F1,24 = 21.83, P < 0.001; F1,67 = 0.365, B = ± 0.002, P = NS). The mean sex ratio of the Fig. 2a]. dispersing brood was determined by including only those galls Analyses of the number of chorionated oocytes of the with adult female soldiers and a dead foundress, in order to be foundress and soldiers also utilised a nested design, but the certain of excluding all ®rst-instar larvae destined to be data were ordinal and not amenable to ANOVA. These data soldiers. In contrast to the soldier generation, ®rst instars of the could not be blocked by gall for Friedman's nonparametric dispersing brood were highly female biased (mean analysis because some galls had only one type of adult morph r = 0.056 6 0.013; also Tables 1 and 2). This sex ratio is not and most galls had multiple soldiers. Hence, a chi-squared signi®cantly different from the mean sex ratio of the gall analysis was performed, where data were grouped as 0, 1, and founders (mean founder r = 0.034 6 0.012, n = 115; t-test using

> 1 chorionated oocytes per individual. Foundresses had arcsin-transformed data, t135 = ± 0.974, P = NS). signi®cantly more chorionated oocytes than inseminated Male-destined eggs in the dispersing generation did not soldiers (mean per foundress = 1.83 6 0.29, n = 24; mean per appear to be laid in any particular order by the soldiers, as 2 soldier = 0.93 6 0.29, n = 14; c 2 = 5.96, P = 0.050; Fig. 2b). there was no correlation between sex ratio of the dispersing The foundress also laid a larger mean number of eggs per gall generation and the number of females (Pearson's r = 0.092, before female soldiers eclosed (18.6 6 2.8, n = 18 galls) than P = NS, n = 32 galls) or brood size (r = 0.081, P = NS, n = 32). the per capita number of soldier-produced eggs after the As the number of dispersers produced by the soldiers was foundress had died (6.0 6 0.94, n = 49 galls; t-test, assuming between about 59 and 79 individuals (58 and 77% of the total unequal variances Levene's F = 10.2, P < 0.01, t20.9 = 4.28, dispersing brood; see above), it follows that, collectively, P < 0.001; Fig. 2c). female soldiers produced about three to four males (both Inseminated female soldiers that had little or no ovarian minimum and maximum brood size 3 sex ratio). As the mean development (all oocytes < 50 mm) appeared to have a lower number of female soldiers in a gall was 9.4 (6 0.70), it appears density of sperm in their spermathecae than those with that, on average, less than half produced a son and the rest developing oocytes. There was a positive correlation between produced only daughters or did not reproduce at all. sperm density within the spermatheca of inseminated soldiers and the mean size of their three largest oocytes (Spearman's rank correlation coef®cient, r = 0.81, P < 0.01, n = 9). Spatial and feeding constraints of brood

The mean percentage of the gall volume occupied by brood Sex ratio in mature galls was 21 6 2%, n = 16. The mean percentage of the inner surface area of the gall occupied by all brood in the While the soldier generation was protandrous, there was same galls was 60 6 10%. By extrapolation, the mean overall parity in sex ratio once all soldiers had eclosed percentage of the surface area of these galls occupied by (Tables 1 and 2; Fig. 1). To investigate any patterns in the sex thrips once all the dispersing brood had developed into second ratio of soldiers, such as split sex ratios or a relationship with instars (as all dispersers appear to be just prior to leaving their

Table 2. One sample t-tests for assessing mean proportion of males (r) against 0.5, using arcsin-transformed data for statistical analyses.

Brood nr SE d.f. tP

Initial 1st instars 1 8 0.750 0.082 7 2.851 0.025 Initial soldiers 2 13 0.745 0.085 12 3.486 0.004 All adult soldiers 3 25 0.519 0.026 24 1.495 0.148 All adult soldiers 4 24 0.510 0.035 23 0.292 0.773 Dispersing 1st instars 5 22 0.056 0.013 21 33.32 < 0.001

1First instars destined to be soldiers from galls collected in December 1995. 2Adult soldiers from galls collected in January 1996. 3Adult soldiers from galls collected between March and May 1996. 4Adult soldiers from all galls where brood > 75. 5Dispersing ®rst instars when female soldiers were > 0 and foundress was dead. Soldiers are protandrous but lack sex ratio bias and the dispersing brood is highly female biased.

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gall) was 95 6 16%. As brood were observed to walk over and that local resource competition could be intense between the nudge each other to reach a feeding site, rather than be in thrips, particularly late in colony development. constant contact with the surface of the gall, these data suggest

Discussion

Kladothrips hamiltoni is one of the six species of Australian gall-inducing thrips that are known to be eusocial (Crespi & Mound, 1997). As in most gall-inducing thrips on Acacia, galls are usually induced by a single female that produces an initial, nondispersing generation of morphologically distinct soldiers. For K. hamiltoni, the data indicate protandry, but a lack of overall sex ratio bias, among soldiers. All other eusocial thrips studied so far have a female bias in the soldier generation (Crespi, 1992a, 1993; B. Kranz, unpublished). A second, highly female biased, generation produced predominantly by the soldiers, disperses and overwinters. While no data have been collected on how overwintering occurs in K. hamiltoni,it is likely that second-instar larvae or pupae overwinter in the soil litter until the following rains, because the dispersing generation does not become adult while in galls that are still attached to the host tree. In the closely related gall-inducing species K. rugosus and K. acaciae (Crespi et al., 1998; Morris et al., 1999), second-instar larvae can survive in soil for several months under laboratory conditions (D. Morris, unpublished). The sex ratio of the dispersing generation was highly female biased and not signi®cantly different from the sex ratio of gall founders. These sex ratios suggest that both sexes suffer similar mortality rates during overwintering and gall initiation. This is most likely to occur though predation or the inability to secure a viable gall-inducing site, such as if wind dispersal resulted in thrips being isolated from their host plants. No data have been collected for overwintering and gall-induction behaviour in K. hamiltoni, but in O. tepperi, complete sealing of the gall takes up to 10 days from commencement of feeding, and during this time spiders and have been observed to attack and kill the thrips (B. Kranz, unpublished). Incest between soldiers results in the foundress being more related to the offspring of the soldiers (the grandchildren) than the foundress's own offspring (assuming that there is at least some outbreeding occurring in the dispersing generation), and female soldiers being more related to their daughters than their sons (see Herre, 1985; Fig. 3). Consequently, both the foundress and soldiers should choose a female-biased sex ratio in the soldier-produced dispersing generation, because both gain greatest ®tness returns by investing in the production of these female dispersers. A female-biased sex ratio resulting from high levels of inbreeding between dispersers, which is evident from microsatellite analysis (T. Chapman and

Fig. 2. Box plots summarising distributions in reproductive output of the foundress and female soldiers: (a) length of oocytes (> 50 mm), (b) number of chorionated eggs, (c) per capita egg production. Bold line in box = median, box = 25th and 75th percentiles, bars = range. The foundress had signi®cantly larger oocytes (P < 0.001), more chorionated eggs (P = 0.05), and greater per capita egg production (P < 0.001) than female soldiers.

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Sociality and sex ratio in a galling thrips 439

Fig. 3. Pedigree showing minimum life-for-life relatedness between (a) the foundress, her soldier offspring, and disperser grandchildren and (b) the soldiers and their offspring. Relatedness values are those predicted for a singly mated, outbred foundress and singly sib-mated soldiers. As inbreeding is high in K. hamiltoni, actual relatedness values will be higher than those shown. As a result of incest between soldiers, the foundress is more related to her granddaughters than daughters, and female soldiers are more related to their daughters and nieces than sons.

B. Crespi, unpublished), will also reduce local mate competi- the greater reproductive output of the foundress than soldiers in tion although local mate competition could also contribute to K. hamiltoni and other eusocial gall-inducing thrips, although the female bias of the dispersers independently (Herre, 1985). optimal skew theories could be relevant to understanding the As foundresses are more related to granddaughters than origin of soldiers in thrips. daughters, and soldiers do not have the option of leaving the The foundress could be in¯uencing the sex ratio of the gall to become foundresses, optimal skew theories (Reeve & soldiers' brood by producing male soldiers ®rst. Protandry in Ratnieks, 1993; Vehrencamp, 1983; Keller & Reeve, 1994; soldiers maximises the probability that female soldiers are Reeve & Keller, 1995) may not be relevant to understanding mated as soon as they are reproductively mature and will lay

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fertilised, female eggs. It appears, in fact, that more than half daughters by r = 1, there should be high ®tness returns for the soldiers are almost exclusively laying fertilised eggs. Of defending offspring, particularly when the sex ratio of the course, as inbreeding increases, so does the relatedness dispersers is highly female biased. The inbreeding within between individuals, and the relative gain for the foundress K. hamiltoni also means that the relatedness asymmetry in investing in grandchildren, rather than producing more between males and daughters, and males and siblings, is dispersing brood, will be reduced. Empirical and modelling reduced such that there should also be increased bene®t in analyses of relatedness and sex ratio in this and other gall- male soldiers defending siblings compared to males in inducing thrips, particularly a comparison of those galls with a outbreeding species (Hamilton, 1972). Gender-speci®c beha- single foundress and those with a founder of both sexes, will vioural observations of K. hamiltoni soldiers and their enemies provide insight into these ®tness pay-offs and how sex ratio is may be critical to understanding the selection for the sex ratio manipulated. in the soldiers. The sex-ratio parity of soldiers could be The highest average percentage of the gall volume occupied explained at least partially if male soldiers were found to be by soldiers and dispersing brood was 21%. More importantly, superior to female soldiers in defending their gall's occupants. gall thrips need and appear to compete for access to feeding In terms of both intra-colony relatedness and social and egg-laying sites on the interior surface of the gall, such that behaviour, K. hamiltoni and other highly inbred eusocial thrips their packing must be able to accommodate free movement of (Chapman & Crespi, 1998) appear to be more similar to some individuals within the gall, and the functional space they gall-inducing, diploid clonal aphids (Stern & Foster, 1996, compete for is the surface area of the gall. The estimate that 1997) and diploid naked mole rats (Jarvis & Bennett, 1993; brood occupied 60% of a mature gall's surface area compares Jarvis et al., 1994) than to social Hymenoptera. Gall-inducing with 78% for a circle, inside and just touching the sides of a thrips and aphids, and naked mole rats, have nests that are rich square (determined by simple geometry). Therefore, competi- in food resources. The value of these nests as concentrated tion for feeding and egg-laying sites is potentially extreme, at food sources may reduce the threshold for the selection of least once female soldiers are reproductive. Moreover, the individuals that are specialised in defending the nest (Jarvis extrapolated estimate that an average of 95% of the gall's et al., 1994; Stern & Foster, 1997). Thrips, aphids, and naked surface area was occupied by thrips once all dispersers had mole rats all meet the ecological criteria for the evolution of become second instars, suggests that such intense competition eusociality suggested by Alexander et al. (1991), and the for feeding sites could be one explanation for brood leaving the speci®c predictions of Crespi (1994), which are independent of gall prior to maturity. Modelling the levels of resource haplodiploidy (but not of kin selection): a lifetime of food± competition to which thrips are subject at various stages of shelter coincidence, strong selection for defence, and the colony development would help to elucidate the impact of ability of some individuals to defend. spatial and feeding competition on sex ratio and colony Hamilton (1967) (see also Hartl, 1971; Wrensch, 1993) structure. proposed a biofacies for with regular full sib- If local resource competition operates on female soldiers, mating. The biofacies is: (1) a highly female-biased primary there will be selection for a male bias, but local mate sex ratio, (2) arrhenotokous reproduction, (3) at least one male competition between the male soldiers will be extreme, in every batch of offspring, (4) gregarious development of because male soldiers are able to mate with many females siblings, (5) protandry and multiple mating by males, (6) and the number of available mates is limited. Local mate females mate soon after eclosion, (7) males do not emigrate competition selects for a female bias, so it is possible that local from batch, (8) females store sperm in their spermathecae. resource competition and local mate competition are operating Soldiers of K. hamiltoni, which mate only with siblings, ®t this simultaneously to produce the 1 : 1 sex ratio in soldiers. It is biofacies, including multiple mating, which is suggested by the also possible that the high levels of inbreeding between correlation between ovarian development and the density of dispersers could in¯uence sex ratio through a preponderance of sperm in the spermatheca. The sex-ratio parity of the soldiers is diploid males, as in some hymenopterans (reviewed by Crozier an enigma, however. The high inbreeding coef®cient in & Pamilo, 1996), but there is no evidence of male diploidy soldiers (T. Chapman and B. Crespi, unpublished) and the from microsatellite analyses in soldiers of O. tepperi, small number of founding males, both of which imply that the O. habrus,orK. hamiltoni (Chapman & Crespi, 1998; dispersing/founding generation is also highly incestuous, T. Chapman, pers. comm.). suggest that the dispersing generation should also ®t these The protandry and overall sex ratio parity of the soldier biofacies, but the dispersers are not protandrous and their brood could also be due to the role that male soldiers play in offspring (the soldiers) lack sex-ratio bias. This is possibly the gall defence, however behavioural analyses on O. tepperi and only species thus far identi®ed, even among other gall- O. habrus found that male soldiers were less likely to attack inducing thrips, that ®ts the criteria of Hamilton's (1967) Koptothrips than were females (Crespi& Mound, 1997). The model, but where the sex ratio differs markedly from the relevance to K. hamiltoni of how soldiers in these two species prediction. defend is not clear because the data were collected opportu- While local resource competition and local mate competi- nistically, while observing the thrips for other purposes, so the tion could be acting simultaneously in opposite directions, the experimental design lacked rigour. More notably, both these overall lack of sex-ratio bias in the soldier generation is far species have a female bias in the soldier generation (Crespi, from understood. Manipulating local resource competition or 1992a, 1993). As haploid males are related to their diploid local mate competition within galls experimentally would be

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Sociality and sex ratio in a galling thrips 441 useful for testing this hypothesis. The novel in Crespi, B.J. & Yanega, D. (1995) The de®nition of eusociality. K. hamiltoni requires further behavioural study and compar- Behavioural Ecology, 6, 109±115. ison with closely related species in order that the relevance of Crozier, R.H. & Pamilo, P. (1996) Evolution of Social Colonies. this species to the evolution of social behaviour in haplodiploid Sex Allocation and Kin Selection. Oxford University Press, Oxford. insects might be understood better. Hamilton, W.D. (1964) The genetical evolution of social behaviour, II. Journal of Theoretical Biology, 7, 1±52. Hamilton, W.D. (1967) Extraordinary sex ratios. Science, 156, 477± 488. Acknowledgements Hamilton, W.D. (1972) Altruism and related phenomena, mainly in social insects. Annual Review of Ecology and Systematics, 3, 193± We thank Serena Williams, station owner of the Arckaringa 232. 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