The determinants of queen size in a socially polymorphic ant

In social animals, body size can be shaped by multiple factors, such as direct genetic effects, maternal effects, or the social environment. In ants, the body size of queens correlates with the social structure of the colony: colonies headed by a single queen (monogyne) generally produce larger queens that are able to found colonies independently, whereas colonies headed by multiple queens (polygyne) tend to produce smaller queens that stay in their natal colony or disperse with workers. We performed a cross‐fostering experiment to investigate the proximate causes of queen size variation in the socially polymorphic ant Formica selysi. As expected if genetic or maternal effects influence queen size, eggs originating from monogyne colonies developed into larger queens than eggs collected from polygyne colonies, be they raised by monogyne or polygyne workers. In contrast, eggs sampled in monogyne colonies were smaller than eggs sampled in polygyne colonies. Hence, eggs from monogyne colonies are smaller but develop into larger queens than eggs from polygyne colonies, independently of the social structure of the workers caring for the brood. These results demonstrate that a genetic polymorphism or maternal effect transmitted to the eggs influences queen size, which probably affects the social structure of new colonies.     

Chemical communication of queen supergene status in an ant

Traits of interest to evolutionary biologists often have complex genetic architectures, the nature of which can confound traditional experimental study at single levels of analysis. In the fire ant Solenopsis invicta, the presence of a Mendelian ‘supergene’ is both necessary and sufficient to induce a shift in a fundamental property of social organization, from single‐queen (monogyne) to multiple‐queen (polygyne) colonies. This selfish genetic element, termed the Social b (Sb) supergene, contains > 600 genes that collectively promote its fitness by inducing the characteristic polygyne syndrome, in part by causing polygyne workers to accept only queens bearing the Sb element (a behaviour termed ‘worker Sb discrimination’). Here, we employ a newly developed behavioural assay to reveal that polygyne workers, many of which bear the Sb element, employ chemical cues on the cuticle of queens to achieve worker Sb discrimination, but we found no evidence for such pheromonally mediated worker Sb discrimination in monogyne workers, which universally lack the Sb element. This polygyne worker Sb discrimination was then verified through a ‘green beard’ effect previously described in this system. We thus have demonstrated that the Sb element is required both for production of relevant chemical cues of queens and for expression of the behaviours of workers that collectively result in worker Sb discrimination. This information fills a critical gap in the map between genotype and complex phenotype in S. invicta by restricting the search for candidate genes and molecules involved in producing this complex social trait to factors associated with the Sb element itself.     

Covariation between colony social structure and immune defences of workers in the ant Formica selysi

Several ant species vary in the number of queens per colony, yet the causes and consequences of this variation remain poorly understood. In previous experiments, we found that Formica selysi workers originating from multiple-queen (=polygyne) colonies had a lower resistance to a fungal pathogen than workers originating from single-queen (=monogyne) colonies. In contrast, group diversity improved disease resistance in experimental colonies. This discrepancy between field and experimental colonies suggested that variation in social structure in the field had antagonistic effects on worker resistance, possibly through a down-regulation of the immune system balancing the positive effect of genetic diversity. Here, we examined if workers originating from field colonies with alternative social structure differed in three major components of their immune system. We found that workers from polygyne colonies had a lower bacterial growth inhibitory activity than workers from monogyne colonies. In contrast, workers from the two types of colonies did not differ significantly in bacterial cell wall lytic activity and prophenoloxidase activity. Overall, the presence of multiple queens in a colony correlated with a slight reduction in one inducible component of the immune system of individual workers. This reduced level of immune defence might explain the lower resistance of workers originating from polygyne colonies despite the positive effect of genetic diversity. More generally, these results indicate that social changes at the group level can modulate individual immune defences.     

Kin structure and queen execution in the Argentine ant Linepithema humile

Every spring, workers of the Argentine Ant Linepithema humile kill a large proportion of queens within their nests. Although this behaviour inflicts a high energetic cost on the colonies, its biological significance has remained elusive so far. An earlier study showed that the probability of a queen being executed is not related to her weight, fecundity, or age. Here we test the hypothesis that workers collectively eliminate queens to which they are less related, thereby increasing their inclusive fitness. We found no evidence for this hypothesis. Workers of a nest were on average not significantly less related to executed queens than to surviving ones. Moreover, a population genetic analysis revealed that workers were not genetically differentiated between nests. This means that workers of a given nest are equally related to any queen in the population and that there can be no increase in average worker–queen relatedness by selective elimination of queens. Finally, our genetic analyses also showed that, in contrast to workers, queens were significantly genetically differentiated between nests and that there was significant isolation by distance for queens.     

Sex investment ratios in monogyne and polygyne populations of the fire ant Solenopsis invicta

Both monogyne (single queen per colony) and polygyne (multiple queens per colony) populations of the fire ant Solenopsis invicta are good subjects for tests of kin selection theory because their genetic and reproductive attributes are well-characterized, permitting quantitative predictions about the degree to which sex investment ratios should be female-biased if workers and not queens control reproductive allocation. In the study populations, an investment ratio of 3 females: 1 male is predicted (a proportional investment in females of 0.75) in the monogyne form, whereas a proportional investment in females between 0.637 and 0.740 is expected in the polygyne form. To test these predictions, colonies from a single population of each social form were collected and censused during three different seasons. Consistent with their alternative modes of colony founding, monogyne colonies invested more in reproduction (sexual production) and less in growth/maintenance (worker production) than did the polygyne colonies. Overall, the sex investment ratios were female-biased in both forms, although there was considerable seasonal variation. After adjusting for sex-specific energetic costs, the proportional investment in females was 0.607 in the monogyne population, a value in between those expected under complete control by either the queen or the workers. However, when combined with data from four other previously studied monogyne populations in the U.S.A., the mean investment ratio did not differ significantly from the value predicted if workers have exclusive control. In the polygyne population, the proportional investment in females of 0.616 was consistent with the level of female bias expected under partial to complete worker control, although the potential influence of two confounding factors — possible contact with monogyne colonies and the preponderance of sterile diploid males — weakens this conclusion somewhat. Taken as a whole, the sex investment ratios of monogyne and polygyne populations of S. invicta are consistent with at least partial worker control. Of several ultimate and proximate explanations that have been proposed to explain inter-colonial variation in the sex investment ratio, only the effect of the primary sex ratio (female-determined eggs: male-determined eggs) laid by the queen appears to account for the observed variation among monogyne colonies. In the polygyne population, there is limited support for the hypothesis that greater resource abundance favors investment in females.     

Queen reproduction, chemical signalling and worker behaviour in polygyne colonies of the ant Formica fusca

Potential reproductive conflicts are common in social insects and may occur between and within castes. In multiqueen (polygyne) colonies of ants, reproductive conflicts among coexisting queens may be resolved by aggressive interactions and/or by pheromonal signalling. Pheromonal signals may be directed at workers, which may adjust the reproductive shares of queens behaviourally. Workers are expected to favour a queen that is more likely to be their mother and to produce offspring of close kin to them. We used microsatellite markers to study reproductive partitioning between cobreeding Formica fusca queens in a laboratory experiment where workers received a choice between two queens. We expected queens of this species to communicate their reproductive status by chemical communication, because no aggressive interactions between queens have been observed. We found that queens of different reproductive status (with majority, minority or no production) differed in their cuticular hydrocarbon (CHC) profiles. The fecundity of a queen was associated with worker behaviour; the higher a queen's fecundity, the more attention she received from workers. Our results suggest that a queen fecundity signal is encoded in her CHC profile and acts as a pheromone for workers, which respond to the signal by discriminating between queens. Copyright 2002 Published by Elsevier Science Ltd on behalf of The Association for the Study of Animal Behaviour     

Social context predicts recognition systems in ant queens

Recognition of group‐members is a key feature of sociality. Ants use chemical communication to discriminate nestmates from intruders, enhancing kin cooperation and preventing parasitism. The recognition code is embedded in their cuticular chemical profile, which typically varies between colonies. We predicted that ants might be capable of accurate recognition in unusual situations when few individuals interact repeatedly, as new colonies started by two to three queens. Individual recognition would be favoured by selection when queens establish dominance hierarchies, because repeated fights for dominance are costly; but it would not evolve in absence of hierarchies. We previously showed that Pachycondyla co‐founding queens, which form dominance hierarchies, have accurate individual recognition based on chemical cues. Here, we used the ant Lasius niger to test the null hypothesis that individual recognition does not occur when co‐founding queens do not establish dominance hierarchies. Indeed, L. niger queens show a similar level of aggression towards both co‐foundresses and intruders, indicating that they are unable of individual recognition, contrary to Pachycondyla. Additionally, the variation in chemical profiles of Lasius and Pachycondyla queens is comparable, thus informational constraints are unlikely to apply. We conclude that selection pressure from the social context is of crucial significance for the sophistication of recognition systems.     

Has gene expression neofunctionalization in the fire ant antennae contributed to queen discrimination behavior?

Queen discrimination behavior in the fire ant Solenopsis invicta maintains its two types of societies: colonies with one (monogyne) or many (polygyne) queens, yet the underlying genetic mechanism is poorly understood. This behavior is controlled by two supergene alleles, SB and Sb, with ~600 genes. Polygyne workers, having either the SB/SB or SB/Sb genotype, accept additional SB/Sb queens into their colonies but kill SB/SB queens. In contrast, monogyne workers, all SB/SB, reject all additional queens regardless of genotype. Because the SB and Sb alleles have suppressed recombination, determining which genes within the supergene mediate this differential worker behavior is difficult. We hypothesized that the alternate worker genotypes sense queens differently because of the evolution of differential expression of key genes in their main sensory organ, the antennae. To identify such genes, we sequenced RNA from four replicates of pooled antennae from three classes of workers: monogyne SB/SB, polygyne SB/SB, and polygyne SB/Sb. We identified 81 differentially expressed protein‐coding genes with 13 encoding potential chemical metabolism or perception proteins. We focused on the two odorant perception genes: an odorant receptor SiOR463 and an odorant‐binding protein SiOBP12. We found that SiOR463 has been lost in the Sb genome. In contrast, SiOBP12 has an Sb‐specific duplication, SiOBP12b′, which is expressed in the SB/Sb worker antennae, while both paralogs are expressed in the body. Comparisons with another fire ant species revealed that SiOBP12b′ antennal expression is specific to S. invicta and suggests that queen discrimination may have evolved, in part, through expression neofunctionalization.     

Nutritional versus genetic correlates of caste differentiation in a desert ant

1. In many ant species, caste differentiation stems from trophic differences at the larval stage. Adult workers that feed larvae have great control over the allocation of colony resources to growth (production of workers) versus reproduction (production of queens). However, larval caste fate may also be constrained very early on through direct genetic effects or non‐genetic maternal effects. 2. Here, we combined isotopic and genetic analyses to study the developmental origin of queens and workers in a desert‐dwelling ant, Cataglyphis tartessica (Amor & Ortega, 2014). Queens do not found new colonies alone but rather disperse with workers. As the latter are always wingless, selection pressures on specific queen traits such as flight ability have become relaxed. Though the phylogenetically related species, C. emmae (Forel, 1909) only produces winged queens much larger than workers, C. tartessica produces two types of small queens relative to workers: brachypterous (short‐winged) queens and permanently apterous ergatoid (wingless and worker‐like) queens. 3. Upon emergence, workers and ergatoids have similar δ15N isotopic values, which were lower than those of brachypters, suggesting the latter are fed more protein as larvae. Microsatellite analyses indicated that: (i) colonies are mostly monogynous and monandrous; (ii) both ergatoids and brachypters are equally related to workers; and (iii) in the few polyandrous colonies, patrilines were evenly represented across workers, brachypters and ergatoids. 4. Overall, there was no evidence of genetic caste determination. We suggest that, in contrast to brachypters, ergatoids are selfish individuals that escape the nutritional castration carried out by workers and develop into queens in spite of the colony's collective interests.     

Selective male mortality in the red imported fire ant, Solenopsis invicta

Males in polygyne populations of Solenopsis invicta are primarily sterile diploids and thought to not express the Gp-9 gene coding for a pheromone-binding protein affecting complex social behavior. We examined an aspect of the breeding system hitherto not considered--male Gp-9 genotypes in relation to sperm stored in queens. Four sites with varying frequencies of sympatric monogyne and polygyne colonies were sampled, including sexuals, workers, and broods from four colonies. Most queens were heterozygotes storing B sperm. Although predicted to be common, only 14 of 504 males were B or BB genotypes, suggesting strong selection. Increased frequency of polygyne colonies at each site paralleled increases in queens with b sperm (1.9-32.8%) and of noninseminated queens. The presence of both B and b sperm in 1.9-18.9% of queens, genotype profiles of colonies, and genotypes of offspring from individual queens suggest some frequency of multiple mating. The bb genotype, rather than an obligate, developmental lethal, was present in some queens and common in alates, workers, and brood. Selective mortality of sexuals may affect multiple aspects of the breeding system, including female-mediated dispersal, mating success, and gene flow.     

A comparison of queen oviposition rates from monogyne and polygyne fire ant, Solenopsis invicta, colonies

Abstract. The oviposition rate of individual queens of Solenopsis invicta Buren (Hymenoptera: Formicidae) in relation to their weight and number of queens present in the colony was investigated by direct 2 h observations. There is a strong positive correlation between the weight of a queen and its oviposition rate in both monogyne and polygyne colonies. However, the number of eggs laid per mg queen is higher for moonogyne queens than for polygyne queens. This difference is more evident when the total weight of queens present in a colony is considered. The individual queen oviposition rate is negatively correlated with the number of queens in the colony. In addition, the weight loss per egg laid is significantly greater for polygyne than for monogyne queens, probably due to differences in egg size. These data suggest that oviposition is more efficient in monogyne than in polygyne queens at the individual level; however, at the colony level, polygyne colonies produce significantly more eggs. Comparison of colony level efficiency predicts that polygyne colonies must have at least nine queens to compete reproductively with a mature monogyne queen. Therefore, oligogyny does not appear to be a viable strategy for S.invicata.     

Successful establishment of the invasive fire ant Solenopsis invicta in Taiwan: insights into interactions of alternate social forms

Aim Understanding the factors underlying the successful establishment of invasive ant species is critical for developing quarantine strategies to prevent additional invasions as well as for determining how such species overcome the selective pressures in invaded areas. Although several studies have revealed differences in the social organization and population genetics of invasive ants in their native and introduced ranges, few studies have considered the potential interactions between alternate social forms within newly‐invaded areas simply because many invasive ants are characterized as polygyne or unicolonial. Location Taoyuan and Chiayi County, Taiwan. Methods Both social forms (polygyne and monogyne) of the red imported fire ant, Solenopsis invicta, occur in two separately invaded areas in Taiwan (Taoyuan and Chiayi). We employed intensive sampling methods and diagnostic polymerase chain reaction assays to determine the distribution of these two social forms in both infested areas in Taiwan. Results The distribution of social forms differs dramatically between the two infested areas, consistent with different invasion histories regarding the original make‐up of individuals comprising the initial founder group. The Taoyuan population likely was colonized initially by ants of both social forms, with the subsequent spread characterized by continuous outward movement of the two forms, particularly the monogyne form. In contrast, the initial founders of the Chiayi population likely were of the polygyne social form only, suggesting the monogyne social form in this population appeared only recently and likely arose directly from polygyne queens converting to the alternate social form. Main conclusions Our results provide detailed insights into the invasion history of S. invicta in Taiwan and suggest that the distinct reproductive biology of the two social forms may have shaped the current distribution of S. invicta in these infested areas and that the dynamics of two forms may affect the long‐term persistence and potential for spread of this pest ant species.     

SIMPLE GENETIC BASIS FOR IMPORTANT SOCIAL TRAITS IN THE FIRE ANT SOLENOPSIS INVICTA

Variation in queen phenotype and reproductive role in the fire ant Solenopsis invicta has been shown to have a simple genetic basis in a single introduced population in the United States. The evidence consists of an association between this variation and queen genotype at Pgm-3, a phosphoglucomutase-encoding gene. In the present study, we surveyed Pgm-3 allele and genotype frequencies in diverse populations from the native and introduced ranges of this ant to learn whether this simple genetic basis for reproductive traits is a general feature of the species or a genetic anomaly in introduced ants stemming from a recent bottleneck or the invasion of novel habitats. No egg-laying queens living in polygyne (multiple-queen) nests possessed the homozygous genotype Pgm-3^a/a in any of the study populations, yet nonreproductive females from such nests (workers as well as queens that had not yet initiated oogenesis) possessed this genotype at moderate frequencies. Remarkably, Pgm-3^a/a was the most common genotype among all classes of females, including egg-laying queens, in monogyne (single-queen) nests from all populations studied. Genotype proportions at Pgm-3 in polygyne populations typically departed strongly from the proportions expected under Hardy-Weinberg equilibrium, whereas those in monogyne populations did not. These patterns establish that a single mendelian gene influences queen reproductive role in S. invicta and that this gene uniformly is under strong directional selection in the polygyne social form only. Moreover, the perfect association of Pgm-3 genotype and reproductive role in all populations, combined with the known function of phosphoglucomutase in insect metabolism, suggest that this gene may directly influence queen phenotypes rather than merely serving as a marker for a linked gene that causes the effects.     

Immune priming and pathogen resistance in ant queens

Growing empirical evidence indicates that invertebrates become more resistant to a pathogen following initial exposure to a nonlethal dose; yet the generality, mechanisms, and adaptive value of such immune priming are still under debate. Because life‐history theory predicts that immune priming and large investment in immunity should be more frequent in long‐lived species, we here tested for immune priming and pathogen resistance in ant queens, which have extraordinarily long life span. We exposed virgin and mated queens of Lasius niger and Formica selysi to a low dose of the entomopathogenic fungus Beauveria bassiana, before challenging them with a high dose of the same pathogen. We found evidence for immune priming in naturally mated queens of L. niger. In contrast, we found no sign of priming in virgin queens of L. niger, nor in virgin or experimentally mated queens of F. selysi, which indicates that immune priming in ant queens varies according to mating status and mating conditions or species. In both ant species, mated queens showed higher pathogen resistance than virgin queens, which suggests that mating triggers an up‐regulation of the immune system. Overall, mated ant queens combine high reproductive output, very long life span, and elevated investment in immune defense. Hence, ant queens are able to invest heavily in both reproduction and maintenance, which can be explained by the fact that mature queens will be protected and nourished by their worker offspring.     

A formal assessment of gene flow and selection in the fire ant Solenopsis invicta

Abstract.— Recent studies of the introduced fire ant Solenopsis invicta suggest that introduced polygyne (with multiple queens per nest) populations are strongly influenced by male-mediated gene flow from neighboring monogyne (single queen per nest) populations and selection acting on a single locus, general protein-9 (Gp-9) . This investigation formally tests this hypothesis and determines if these processes can account for the genotypic structure of polygyne S. invicta . To increase the statistical power of this test, we considered the genotypes of polygyne queens and workers at both Gp-9 and the closely linked, selectively neutral locus Pgm-3 . We then constructed and analyzed a novel mathematical model to delimit the effects of monogyne male gene flow and selection on the joint genotypes at the Pgm-3/Gp-9 superlocus. Using this framework, a hierarchical maximum-likelihood method was developed to estimate the best-fitting gene flow and selection parameters based on the fit of our model to data from both the current study and an earlier one of the same population. In each case, selection on polygyne queens and workers alone, with no monogyne male gene flow, provides the most parsimonious explanation for the observed genotype frequencies. The apparent discrepancy between this result and the empirical evidence for monogyne male gene flow indicates that undocumented factors, such as other forms of selection in polygyne males or workers, are operating in introduced polygyne S. invicta .     

Unusual Behavior of Polygyne Fire Ant Queens on Nuptial Flights

This study reports previously undescribed behavior of fire ant queens (Solenopsis invicta) on their nuptial flights. We captured large numbers of alate (winged) queens flying at low altitudes in dense swarms that were virtually devoid of males. We assayed the genotypes of these alate queens at the locus Gp-9, which exhibits strong genotype frequency differences between monogyne (single-queen) and polygyne (multiple-queen) populations, and found that almost all of these low-flying queens originated from polygyne colonies. Comparisons of mtDNA haplotype distributions of these queens to those of alates leaving polygyne nests suggest that the flying queens had not dispersed more than a few hundred meters. Moreover, the proportion of flying queens that were mated did not differ significantly from the proportion of reproductive queens that were mated within the same sites. Thus the flight behavior appears to occur subsequent to mating. We suggest that the flying queens are sampling the local environment in order to select a suitable landing site. Such a site would contain established polygyne nests into which the queens may be adopted as new reproductives.     

The interaction of food distribution and the caste composition of an ant colony (Myrmica rubra L.)

The distribution of food between members of a Myrmica rubra L. Society was investigated by varying the ratios of queens, workers and medium-sized larvae. Observations revealed patterns in colony behaviour which could be of importance in a polygyne system.
Queens had little effect upon the rate of food transmission, but the worker/larva ratio was of significance. Many workers effectively fed all larvae present in a colony, but a small number of workers fed only a few. If larvae and/or queens were in abundance, the workers were partly deprived of access to them. Competition between the queens and larvae for food and worker attention occurred when their numbers were high. In this situation, queens fed themselves while the workers cared for the larvae. The significance of overcrowding, not only upon the administration of food, but upon the queen effect acting on the workers to stimulate or inhibit worker egg-laying and brood-rearing, is discussed.     

Habitat age, breeding system and kinship in the ant Formica fusca

In polygyne ants (multiple queens per colony) factors that affect the distribution and survival of queens may play a key role in shaping the population-wide mating system and colony kin structure. The aim of this paper was to study the breeding system in two populations of different age in the facultatively polygyne ant Formica fusca. Both the observed numbers of queens, and the relatedness patterns among queens, workers and colony fathers were compared in two adjacent populations (ages 17 years and > 100 years) in Southern Finland. The results showed that both the mating system and colony kin structure differed between the study populations. In the old population the relatedness among workers, queens and colony fathers was high. The queens were also related to their mates, resulting in significant inbreeding in workers, but not in queens. Finally, the number of queens per colony fluctuated between years, suggesting queen turnover, and nest-mate queens shared their reproduction unequally (reproductive skew). In the younger population relatedness among queens and workers was lower than in the old population, and the colony fathers were unrelated. Furthermore, inbreeding was absent, and no conclusive evidence was found for reproductive skew among nest-mate queens. Finally, the number of queens per colony appeared more stable between years, although queen turnover occurred also in this population. The observed differences in dispersal and mating behaviour are discussed in the light of a potential connection between population age and habitat saturation.     

Multiple mating and supercoloniality in Cataglyphis desert ants

In social organisms, the breeding system corresponds to the number of breeders in a group, their genetic relationships, and the distribution of reproduction among them. Recent, genetically based studies suggest an amazing array of breeding system and reproductive strategies in desert ants of the genus Cataglyphis. Using highly polymorphic DNA microsatellites, we performed a detailed analysis of the breeding system and population genetic structure of two Cataglyphis species belonging to the same phylogenetic group: C. niger and C. savignyi. Our results show that both species present very different breeding systems. C. savignyi colonies are headed by a single queen and populations are multicolonial. Remarkably, queens show one of the highest mating frequency reported in ants (M_p = 9.25). Workers can reproduce by both arrhenotokous and thelytokous parthenogenesis. By contrast, colonies of C. niger are headed by several, multiply mated queens (M_p = 5.17), and they are organized in supercolonial populations made of numerous interconnected nests. Workers lay arrhenotokous eggs only. These results illustrate the high variability in the socio‐genetic organization that evolved in desert ants of the genus Cataglyphis. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 104 , 866–876.     

Experimental Conversion of Colony Social Organization in Fire Ants (<Emphasis Type="Italic">Solenopsis invicta</Emphasis>): Worker Genotype Manipulation in the Absence of Queen Effects

Colony social organization in the fire ant Solenopsis invicta appears to be under strong genetic control. In the invasive USA range, polygyny (multiple queens per colony) is marked by the presence of the Gp-9 ^b allele in most of a colony’s workers, whereas monogyny (single queen per colony) is associated with the exclusive occurrence of the Gp-9 ^B allele. Ross and Keller, Behav Ecol Sociobiol 51:287–295 (2002) experimentally manipulated social organization by cross-fostering queens into colonies of the alternate form, thereby changing adult worker Gp-9 genotype frequencies over time. Although these authors showed that social behavior switched predictably when the frequency of b-bearing adult workers crossed a threshold of 5–10%, the possibility that queen effects caused the conversions could not be excluded entirely. We addressed this problem by fostering polygyne brood into queenright monogyne colonies. All such treatment colonies switched social organization to become polygyne, coincident with their proportions of b-bearing workers exceeding 12%. Our results support the conclusion that polygyny in S. invicta is induced by a minimum frequency of colony workers carrying the b allele, and further confirm that its expression is independent of queen genotype or history, worker genotypes at genes not linked to Gp-9, and colony genetic diversity.