1Illuminating the genetic relationships within soldier-producing aphid colonies is an 2 essential element of any attempt to explain the evolution of the altruistic soldier caste. 3Pemphigus spyrothecae is a soldier-producing aphid that induces galls on the leaf 4 petioles of its host (trees of the genus Populus). At least a quarter of the aphids within 5 the clonally-produced gall population are morphologically and behaviourally distinct 6 first-instar soldiers that defend the gall population from predation. Using field 7 trapping and microsatellites, we investigated the degree of clonal mixing within 8 natural gall populations. Field trapping in the UK showed that all the migrants of P. 9 spyrothecae and of two other Pemphigus species were wingless first-instar soldiers. 10The average degree of mixing estimated from trapping P. spyrothecae migrants was 11 0.68% (range = 0-15%). Microsatellite genotyping of 277 aphids from thirteen galls 12 collected in Italy revealed an average mixing level of 10.4% (range = 0-59%). Six 13 galls contained more than one clone (range = 2-5). Non-kin aphids were not restricted 14 to the soldier caste but were evenly distributed across instars. An additional gall, from 15 which 527 occupants were genotyped, contained 12 non-kin aphids distributed among 16 9 clones, showing that clonal diversity can be high even when mixing is very low. 17These observations suggest that although soldiers migrate regularly and can moult and 18 reproduce within foreign galls, clonal mixing in this species is generally low and is 19 unlikely to provide a barrier to the evolution of investment by the aphid clones in an 20 altruistic soldier caste.

Background: Because the systems of social organisation in the various species of Pemphigus aphids span the continuum from asociality through to advanced sociality (typified by the possession of morphologically specialised soldiers), the genus is an ideal model clade in which to study the influence of ecology on the origins of eusociality. We made detailed study of the ecology of three gall-dwelling species that show clear differences in their levels of social behaviour. To elucidate evolutionary relationships and to attempt to estimate the number of origins of sociality, we also created a phylogeny based on sequences spanning the mitochondrial genes Cytochrome Oxidase I and II for nine species of Pemphigus.

Soldier-producing aphids have evolved at least nine separate times. The larvae of soldier-producing species can be organized into three general categories: monomorphic larvae, dimorphic larvae with a reproductive soldier caste, and dimorphic larvae with a sterile soldier caste. Here we report the discovery of a novel soldier type in an undescribed species of Pseudoregma that is morphologically similar to P. bambucicola. A colony of this species produced morphologically monomorphic first-instar larvae with a defensive behavioral dimorphism. These larvae attacked natural predators, and larval response to a simple assay, placing the tips of forceps in front of larvae, was correlated with this attacking behavior. Approximately one third of the first-instar larvae in the colony attacked and this proportion was uncorrelated with the time of day, the ambient temperature, or the diel migratory behavior of the aphids. Migrating larvae rarely attacked. Attacking behavior was correlated with another defensive behavior, hind-leg waving. Attackers were more likely to possess the next-instar skin, suggesting that they were older than non-attackers. This is the first example of a possible within-instar age polyethism in soldier-producing aphids. Canonical variates analysis of seven morphological measurements failed to discriminate between attacking and non-attacking larvae. The monomorphic larvae share some morphometric characteristics in common with the soldiers of P. bambucicola and other characteristics in common with normal larvae. We discuss these results with respect to the evolution and loss of soldier castes in the tribe Cerataphidini.