Evolution of eusociality and the soldier caste in termites: a validation of the intrinsic benefit hypothesis

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1 doi:1.1111/j x Evolution of eusociality and the soldier caste in termites: a validation of the intrinsic benefit hypothesis E. A. ROUX & J. KORB Lehrstuhl Biologie I, Universität Regensburg, Regensburg, Germany Keywords: eusociality; evolution; indirect fitness benefit; Isoptera; soldier; sterile caste. Abstract In termites the evolution of reproductive altruism is not based on a particularly high relatedness between nestmates. For the evolution and maintenance of the ancestral sterile soldier caste, the benefits generated by the soldiers presence must compensate the loss of the soldiers reproductive potential. To study the impact of soldiers on colony s fitness, we manipulated the proportion of soldiers to nonsoldiers in colonies of the dry-wood termite Cryptotermes secundus. Soldier-less colonies were obtained by removing soldiers and inhibiting their development with an extract of soldier heads. The colonies were set up for 1 year in experimental nests in the field. Soldierless colonies produced fewer soldiers. The reduction of soldiers neither affected colony survival nor helper growth, but fewer dispersing sexuals were produced in soldier-less than in control colonies. This confirms what was only supposed so far, that in termites soldiers are maintained for their intrinsic benefit to cost ratio. Introduction Reproductive altruism (i.e. the decrease of one s own potential to reproduce in favour of another individual s reproduction), and complete sterility in particular, was long considered to be an evolutionary enigma as it appeared to contradict our understanding of classical Darwinian selection (Darwin, 1859). Now, kin selection is the central paradigm involved to explain the evolution and maintenance of altruism (Hamilton, 1964, 1972): altruistic behaviour will be evolutionary favoured when the relatedness between altruist and beneficiary is higher than the ratio of costs (reproductive loss of the altruist) to benefits (reproductive gain of the beneficiary). This means that reproductive altruism (as exhibited in cooperatives societies composed mainly of subfertile or sterile individuals) can evolve in groups of related individuals when (i) the relatedness is very high and/or (ii) helping is at low costs and/or yields high benefits. The importance of relatedness for the evolution of eusociality is well established and confirmed for eusocial Correspondence: Estelle Roux, Lehrstuhl Biologie I, Universität Regensburg, Universitätsstr. 31, 934 Regensburg, Germany. Tel.: ; fax: ; estelle.roux@biologie.uni-regensburg.de species with a high relatedness among siblings; this can arise either because of a haplodiploid genetic system (social Hymenoptera and thrips) or to clonal reproduction (aphids, polyembryonic wasps). The genetic system of diploid organisms do not predispose particularly the evolution of helping (naked mole-rats: Braude, 2; shrimps: Duffy, 1996; termites: Reilly, 1987; Thompson & Hebert, 1998; Husseneder et al., 1999) although within group relatedness can still be high (naked mole-rats: Reeve et al., 199; termites: Bulmer & Traniello, 22; Goodisman & Crozier, 22). In these systems, ecological factors affecting the benefits and costs of helping are claimed to account for the evolution of altruism (Lin & Michener, 1972; Alexander et al., 1991; Crespi, 1994). The diploid eusocial taxa share three traits thought to lower the costs of helping (Alexander et al., 1991): (i) gradual development, (ii) living in an environment in which independent breeding is difficult, and (iii) living in a long lasting, defensible nest. Gradual development offers the opportunity to start helping before the adult stage, and an individual can thus increase its indirect fitness early in life at a low cost. When independent breeding is difficult, because suitable nests are rare and/ or predation is high, individuals can be selected to help at the nest and maximize their indirect fitness before attempting to breed. A stable (i.e. long lasting), defensible 869

2 87 E. A. ROUX AND J. KORB nest offers protection to both brood and resources against conspecific usurpation and predators, securing the efforts of the nest holders. Regardless of the genetical predisposition, most eusocial taxa nest in cavities that are difficult to invade and easy to protect (thrips: Crespi, 1992; ants: Hölldobler & Wilson, 199; aphids: Itô, 1989). This may indicate the importance of defense for the evolution of eusocial systems. It is remarkable that a defensive morph did not evolve in all taxa (e.g. mole-rats, beetles and most ants), and when it occurs, not with the same frequency within each taxon. In termites, a caste specialized on defense evolved that has no equivalent in other eusocial taxa (Noirot & Pasteels, 1987; Noirot, 199). The soldier caste is unique both in its exclusive defensive function and its development. This caste is ancestral in termites and evolved prior to a true worker caste (i.e. a sterile helping caste). Unlike the soldiers found in the other eusocial insects (aphids, thrips and ants), this caste is monophyletic in termites (Noirot & Pasteels, 1987). The evolution of soldiers remains hypothetical, as there are only few clues about the life of termite ancestors. This uncertainty is caused by the absence of extant subsocial prototermites and by a controversy concerning the phylogeny of the basal families (Thompson et al., 2; Eggleton, 21; Grandcolas & D Haese, 22). Three hypotheses exist to account for the origin of termite soldiers. First, Yamamura (1993) developed a model of the evolution of soldiers based on increased relatedness systems (see also Higashi et al., 2). However, this model followed Bartz (1979), and erroneously assumed high inbreeding for termites (Husseneder et al., 1999). Secondly, based on observations of secondary reproductives with soldier traits ( soldier neotenics ) in Zootermopsis (Termopsidae), Myles (1986, 1988) suggested that soldier traits originated among neotenics as adaptations for fighting for the natal breeding position. This hypothesis, however, was criticized by Roisin (1999) who pointed out two problems. First, nonsoldier neotenics are common in Zootermopsis and there is no evidence for an actual role of weaponry in interindividual fights for the breeding position (but see Thorne et al., 23). Secondly, it is difficult to account for the loss of soldier traits in the neotenics of all nontermopsid termites (Roisin, 1999). The third hypothesis states that soldiers evolved for their intrinsic benefit (Noirot, 199; Roisin, 1999). This means, following Hamilton s rule, that soldiers get enough indirect fitness benefits to counter-balance the loss of their reproductive potential. Up to now soldier defense has only been reported in some species of derived termites (reviewed in Noirot & Darlington, 2) and was never quantified. However, in ancestral termites (Termopsids and Kalotermitids; Eggleton, 21), the main protection of colonies is the wood they nest in and feed upon (Abe, 1987; Lenz, 1994) and thus, soldiers do not seem to be their prime defensive force. As workers remain totipotent (thereafter helpers) in these families, soldiers raise an evolutionary puzzling question: why does one group of individuals completely give up reproduction (Jeon & Choe, 23)? The aim of this study was to test experimentally whether soldiers in an ancestral termite increase the fitness of their colony under field conditions. We focused on the Kalotermitid species Cryptotermes secundus (Hill) for which the costs and benefits of helpers are now well documented (Korb & Lenz, 24; J. Korb, unpubl. data). Colonies average 1 3 individuals, consisting mainly of nymphs and helpers (i.e. individuals beyond the third instars without wing buds or eyes which assume the role of workers, but are totipotent) (Korb & Lenz, 24). Soldiers of this genus are characterized by phragmotic heads, and in C. secundus, as in most species, they retain sharp and powerful mandibles (Gay & Watson, 1982). The production of soldiers is regulated in a seemingly adaptive way, so that dead soldiers are replaced within a few weeks (e.g. Springhetti, 1969). To investigate the impact of soldiers on the reproductive success of their colony, we compared various fitness parameters of colonies with and without soldiers. Therefore, we experimentally produced soldier-less colonies by chemically simulating the presence of soldiers (Korb et al., 23). Material and methods Cryptotermes secundus colonies were collected from mangroves near Darwin Harbor (Northern Territory, Australia) in 1999, 2 and 21. After their composition was determined, colonies were set up in wood blocks (Pinus radiata D. Don) adjusted to colony size (Lenz, 1994; Korb & Lenz, 24) and brought back to the field for 1 year. Colonies were then collected prior to the annual nuptial flights and their new composition was determined. First, 36 colonies that were not treated and used as controls were set up in 1999 and 2. Secondly, we set up 18 experimental soldier-less colonies by removing all soldiers and inhibiting soldier re-development with extracts of soldier heads (Korb et al., 23) in July and August 21. The extract consisted of four soldier heads squashed in 1 ml of methylenchloride. The solution was allowed to evaporate on a squared filter paper (length 1.2 cm). For each colony, we applied three extracts in total. One, when we set up the colonies, a second when we brought the colonies back to the field in September and the third about 6 months later. The advantage of soldier head extract as an inhibitor of soldier development, compared with a more classical juvenile hormone antagonist, is that the former does not induce the development of intercastes (Korb et al., 23). Thus, while soldier production is inhibited, helpers and nymphs develop normally. Three fitness parameters were measured after 1 year, namely colony survival, helper growth, and the production of dispersing sexuals.

3 Evolution of soldiers in termites 871 Helper growth was the production of helpers between the two census events. The production of sexuals was the number of late nymph instars and alates present at the end of the experiment. Nymphs can undergo regressive molts (Noirot, 1985); however, all late nymph instars present shortly before the nuptial flight will become alates (J. Korb, unpubl. data), so we counted them as dispersing sexuals. It was not possible to set up enough control colonies in 21 (N ¼ 15) because of the time constraint inherent to colony collection. However, the three parameters measured did not differ between the controls of 21 and those of the previous years (survival: N ¼ 39, Fisher exact test, n.s.; helper growth: ANCOVA, F 1,36 ¼.94, n.s.; production of sexuals: ANCOVA, F 1,36 ¼.85, n.s.) thus, possible change in environmental conditions experienced by controls and soldier-less colonies are unlikely to account for the variance observed. Statistical analysis Colony survival between treatments was compared with Fisher exact test. The production of soldiers in control colonies was the difference of soldiers between the second and first census. For soldier-less colonies, the production of soldiers was the difference of soldiers between the second and first census, prior to the removal of soldiers. Thus, the production of soldiers in control and soldier-less colonies could be compared. Colony size affects the development of colonies of C. secundus (Korb & Lenz, 24), as has also been shown for Cryptotermes brevis (Walker) (Lenz, 1994). However, there was no correlation between colony size and soldier production (Spearman rank correlation, N ¼ 46, R ¼ ).151, n.s.), therefore we used a Mann Whitney U-test to compare the soldier production of control and soldier-less colonies. In contrast to soldier production, helper growth and the production of sexuals were correlated with colony size (Spearman rank correlation, N ¼ 46, respectively R ¼ ).345, P <.5 and R ¼.648, P <.1). Thus, to test these variables with respect to colony treatment we performed an analysis of covariance (ANCOVA) with colony size as covariate. This ANCOVA followed Good (2) and a linear regression was used to express the variable, say helper growth, as a function of colony size. The usual F-ratios were calculated but they were tested by a Monte-Carlo procedure with 1 permutations (Manly, 1997). Results were expressed as the probability P that the permuted data sets would provide a F equal to or greater than the observed F. Results Only four soldier-less colonies, of 17 that survived, did not produce any soldiers. To check whether the inhibition with soldier head extract was nevertheless effective, we compared the production of soldiers between controls and soldier-less colonies (Mann Whitney, N ¼ 46, Z ¼ )2.568, P <.5). Soldier-less colonies produced significantly fewer soldiers than controls (Fig. 1). Thus the inhibition was effective, although it did not suppress the development of soldiers completely over the whole period of the experiment. Soldier presence and survival Low proportions or the absence of soldiers had no significant effect on the survival of soldier-less colonies compared with controls [18 (94%) soldier-less colonies and 29 (8.5%) controls survived; N ¼ 54, Fisher exact test, n.s.]. In the eight colonies that died, one soldierless and seven controls, no evidences of predators or competitors were found. The presence of dried dead bodies indicated that abiotic factors, such as low or high temperatures or low humidity, caused mortality. However, an alive ant colony or a cockroach family was found in the surviving nests of 12 (41%) controls and four (23%) soldier-less colonies. The proportion of intruders did not differ between the two treatments (N ¼ 46, v 2 I ¼.821, n.s.). There was no direct contact between the termites and these intruders, and the colonies did not seem to be affected in their survival or growth. Soldier production Control Soldier less Fig. 1 Comparison of soldier production between control colonies (open box, N ¼ 29) and soldier-less colonies (filled box, N ¼ 17). Less soldiers were produced in soldier-less colonies (Mann Whitney U-test, N ¼ 46, Z ¼ )2.568, P ¼.5).

4 872 E. A. ROUX AND J. KORB Soldier presence and helper growth Only 13 (45%) controls and five (29%) soldier-less colonies increased in size, and for both treatments helper growth was small (Fig. 2). In most colonies, we observed a decrease in colony size, and within each treatment the range of helper growth was large. The helper growth decreased on averaged of )14 ± 15 and )42 ± 114 helpers (mean ± SE) in control and soldier-less colonies, respectively. These helper growths did not differ significantly (ANCOVA, F 1,43 ¼ 5.35, n.s.). Soldier presence and production of sexuals (nymphs and alates) At the end of the experiment, dispersing sexuals were present in 17 (89%) control and seven (41%) soldierless colonies (Fig. 3). The production of nymphs and alates was almost three times higher in controls than in soldier-less colonies (mean ± SE ¼ 29 ± 53 in controls, 11 ± 2 in soldier-less ). This difference was highly significant (ANCOVA, F 1,43 ¼ 8.7, P <.5). Discussion In this study, we investigated the effect of soldiers on the reproductive success of termite colonies to test whether soldiers are maintained for their intrinsic benefit/cost ratio (Roisin, 1999). We found that, under natural conditions, the presence of soldiers was positively associated with the production of sexuals (nymphs and Helper growth Colony size at start Soldier less Control Fig. 2 The helper growth of control and soldier-less colonies correlated with the initial colony size (Spearman rank correlation, N ¼ 46, R ¼ ).345, P <.5). When this factor was accounted for, there was no difference between the growth of control and soldierless colonies (ANCOVA, F 1,43 ¼ 5.35, n.s.). 5 6 Production of sexuals Colony size at start Soldier less Control 5 6 Fig. 3 The production of sexuals (nymphs and alates) of control and soldier-less colonies correlated with the initial colony size (Spearman rank correlation, N ¼ 46, R ¼.648, P <.1). More sexuals were produced in control than in soldier-less colonies (ANCOVA, F 1,43 ¼ 8.7, P <.5). alates). In contrast, the presence of soldiers had no effect on both the survival and the helper growth of the colony. These results demonstrate that soldiers achieve indirect benefits by promoting the production of related sexuals. However, the dynamics and mechanisms of this positive effect are unclear, as the good survival and helper growth of soldier-less colonies indicate that defense is not of prime importance. The intrinsic benefit of soldiers A positive effect of soldiers on alate production has never been reported before. Comparisons with previous studies that manipulated the soldier to worker ratio are difficult to assess because all were done in unnatural conditions (laboratory), where soldiers are not or less important (Springhetti, 1969; Lüscher, 1973; Haverty, 1979; Haverty & Howard, 1981; Waller & La Fage, 1988; Hahn, 1993; Fei & Henderson, 22). Furthermore, only very few studies included predators (Wells & Henderson, 1993; Cornelius & Grace, 1996; Matsuura, 22) and only one study aimed at testing the effect of soldiers on colony s fitness (Thorne et al., 22). However, the development of soldiers was not inhibited in the soldier removal treatment, so no conclusions can be drawn about the effect of soldiers. A claim for a stimulating effect of soldiers on sexual production on a proximate level is, however, not new. Soldiers have often been cited to stimulate the production of neotenics (i.e. secondary reproductives developed

5 Evolution of soldiers in termites 873 within a colony from nonadult instars via a single molt) (Lüscher, 1973; Hahn, 1993; Henderson, 1998). However, when one traces back the literatures, this was never investigated (Springhetti, 1969, 197, 1971, 1985). Furthermore, subsequent studies failed to find any effect of soldiers on the development of reproductives (Lüscher, 1973; Hahn, 1993). Thus, the stimulation of neotenic development by soldiers remains a speculation and cannot explain our results at the proximate level. At the ultimate level, soldiers are expected to increase investment in alates by enhancing colony survival via colony defense (Noirot, 199; Roisin, 1999; Noirot & Darlington, 2). The lack of soldiers might affect alate production in more indirect ways, by altering the growth pattern of soldier-less colonies. According to caste optimization theory, the production of sexuals is maximized by the investment in defense that yields the optimum growth pattern (Oster & Wilson, 1978). Inferring from this, one would predict that the helper growth of soldierless colonies, with a poor defensive force, should have been suboptimal compared with control colonies. However, this was not the case in the present experiment. This result, together with the fact that only very few sexuals were produced, may indicate that a colony without soldiers is able to maintain its growth, but not with sufficient efficiency to produce sexuals. Basic information on soldier behaviour and their effectiveness at defense are reported in only few studies (McMahan, 1974; Kriston et al., 1977; Traniello, 1981; Wells & Henderson, 1993; Matsuura, 22), and is sometimes merely anecdotal (e.g. Reinhard & Clément, 22). Our results do not confirm the soldiers defensive role, as the survival rate of soldier-less colonies was not different from that of controls. Three, nonmutually exclusive, explanations can account for this good survival: (i) predators are absent or extremely rare (J. Korb & E. A. Roux, unpubl. data, as reported for Neotermes tectonae Damm.; Kalshoven, 1959); (ii) helpers took over defense of the nest against invasions (Thorne, 1982; Prestwich, 1984), however, no signs of intrusion, such as tunnels with a separate entering hole, were found; and (iii) intraspecific competition, which seems to be a major threat for damp-wood termite colonies (Shellman-Reeve, 1994, 1997), was possibly prevented in our set-up of the experiment, as there were no cracks in the wood block. Our results for C. secundus support the hypothesis that soldiers have an intrinsic benefit by enhancing the production of sexuals (Roisin, 1999). Their role in defense seems to be only occasional given the low predation and competition pressures. However, soldiers may affect alate production by playing a role in the development and growth of the colony. Soldiers: a new role? In all other eusocial systems, soldiers have a positive effect on colony survival (e.g. aphids: Foster & Rhoden, 1998; thrips: Crespi & Abbot, 1999; ants: Hasegawa, 1993). In ants this effect can be achieved by other means than defense (e.g. food storage, Hasegawa, 1993). Can termite soldiers also have more than a defensive function? To our knowledge no data support this hypothesis. Behavioural observations showed that soldiers do not differ very much from old helpers even in term of defensive behaviour (Roux & Korb, 22). Interestingly, soldiers seem to receive and give proctodeal trophallaxis in equal proportion (E. A. Roux, unpubl. data) although they cannot feed by themselves. One can speculate whether during these exchanges, soldiers provide a substance with a signalling function of the colony s status (e.g. colony size or nutritional condition). In the model system C. secundus, helpers choose to develop into sexuals when the chances of inheriting the breeding position are low (e.g. large colony or food shortage, Korb & Lenz, 24). A soldier signal could provide such decisive information on colony status. Considering this new hypothesis, two scenarios can account for the nondevelopment of alates after soldiers were replaced in soldier-less colonies. (i) The decision for helpers to disperse occurs in January/February (J. Korb, unpubl. data), when the inhibiting effect started to decline (Korb et al., 23) and none or very few soldiers were present. (ii) Young soldiers may not provide the same information as old soldiers. In both cases, the information conveyed by a lack of soldiers or young soldiers might indicate the incipient stage of the colony. In such a case, helpers would not disperse because the chances of inheriting the nest are non-negligible (J. Korb, unpubl. data). In other taxa, sterility of defenders is rare, polyphyletic in origin and apparently has always evolved secondarily (ants: Baroni Urbani & Passera, 1996; aphids: Stern & Foster, 1997; thrips: Chapman et al., 22). In diploid eusocial taxa, defense is typically performed by large individuals (mole rat: O Riain & Jarvis, 1997; snapping shrimp: Duffy et al., 22) and, in the mole rat, these large defenders are the most likely to inherit the breeding position (O Riain & Jarvis, 1997). Our results for the ancestral Kalotermitid C. secundus indicate that soldiers are maintained for their indirect fitness benefits by increasing the reproductive output of the colony. This advantage alone, when present in the prototermite, could have favoured the evolution of soldiers. Evidence for the role of soldiers, both in terms of behaviour and ecology, is still scarce in primitive termites. In the future, studies on ecological factors investigating colony composition and development would greatly improve our understanding of eusocial evolution in termites. Comparative studies of basal taxa would help to elucidate whether our results reflect a secondarily evolved mechanisms or a more general phenomenon.

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