Evolution of the Gene Network Underlying Wing Polyphenism in Ants Ehab Abouheif and Gregory A. Wray

Animal collections. We collected queened colonies of Pheidole morrisi, Myrmica americana, Crematogaster lineolata, and Neoformica nitidiventris from central Long Island, New York, USA. Colonies were maintained according to standard protocols (1)

Cloning and sequencing. We isolated fragments of wg, sd, and sal from Pheidole morrisi, as well as wg from Crematogaster lineolata, Myrmica americana, and Neoformica nitidiventris, using PCR after reverse transcription of larval mRNA. We cloned and sequenced these fragments to confirm their identity, and performed gene tree analyses to confirm their orthology with Drosophila wg, sd, and sal. We used the following PCR primers and conditions: forward and reverse primers for wg are: 5’-ATGTGGTGGGGSATYGCCAA-3’, 5’-ACYTCGCAGCACCARTGGAA-3’. Forward and reverse primers for sd are: 5’- GACGARGGYAARATGTACGG-3’, 5’-TTDAYCATRTACTCGCACAT-3’. Forward and reverse primers for sal are: 5’-CAYACBGGHGAGCGNCCCTT-3’, 5’-GTRTGNGTNCGGTAGTGCAT-3’. We used the following PCR conditions: 3 min at 94 °C (1X), 1 min 94 °C, 1min 58.5 °C, 1 min 72 °C (5X), 1 min 94 °C, 1min 63.5 °C, 1 min 72 °C (30X), and 10 min 72 °C for wg; 3 min at 94 °C (1X), 1 min 94 °C, 1min 47.5 °C, 1 min 72 °C (5X), 1 min 94 °C, 1min 52.5 °C, 1 min 72 °C (30X), and 10 min 72 °C for sd; and 3 min at 94 °C (1X), 1 min 94 °C, 1min 59.5 °C, 1 min 72 °C (5X), 1 min 94 °C, 1min 64.5 °C, 1 min 72 °C (30X), and 10 min 72 °C for sal.

In situ hybridization and Immunolocalization. Embryos and larvae were fixed and processed for: in situ hybridization using digoxigenin-labelled riboprobes (2) for Pmwg, Pmsd, and Pmsal; or for immunolocalization (3) using DLL (4), UBX (5), EXD (6), and EN (7).

Hormone applications. We observed the development of unfertilized eggs, and topically applied methoprene, a juvenile hormone analogue, to the abdomen of mated adult queens (at 2 mg/ml in acetone) and worker larvae (at 5 mg/ml in acetone) of Pheidole morrisi (8, 9).

Phylogenetic analyses. We aligned wg sequences using Clustal X (10), and used this alignment to estimate maximum parsimony (11), neighbor joining (12), and maximum likelihood (13) trees with 10 000 bootstrap replicates (14) using PAUP* version 4.0b10 (15). Bombyx mori (GenBank D14169) and Drosophila melanogaster (GenBank S67383) were used as outgroup taxa for all analyses.

Supplemental Figure 1. Polyphenic expression profiles of six wing-patterning genes in final instar larvae of Pheidole morrisi. A, Caste determination. We confirmed that castes in Pheidole morrisi are determined at three switch points during development (8, 19). The first switch point is genetically controlled: fertilized eggs develop into diploid females, unfertilized eggs develop into haploid males. The second switch point is environmentally controlled: if embryos experience appropriate shifts in photoperiod and temperature, they experience a pulse of JH during embryogenesis and develop into queens, if not, they develop into sterile workers. The third and final switch is also environmentally controlled: if worker larvae experience the appropriate diet, they experience a pulse of JH and develop into soldiers, if not, they become workers. B-C: scanning electron micrographs of adult queens, soldiers, and workers, respectively. E-F: diagrams of final instar wing discs in reproductives, soldiers, and workers, respectively, indicating the position and orientation of the forewing disc (fwd) and hindwing disc (hwd), as well as their position relative to the three leg discs (L1, L2, L3) in soldier and minor worker larvae; anterior to the top. H-Y: polyphenic expression of six gene products in three castes. Note that ant wing imaginal discs are more similar to those of butterflies than to those of Drosophila, in that they are folded along their dorsal-ventral margin, and do not specify part of the body wall (4, 19). First column shows expression in the fore- and hindwing discs of winged reproductives (H, K, N, Q, T, W). In all cases, expression is conserved relative to the forewing in Drosophila. Second column shows expression in the soldier forewing disc (I, L, O, R, U, X). Expression of the first five gene products (I, L, R, O, U) are also conserved relative to the those in the Drosophila forewing. In contrast, expression of sal, the sixth gene product, is absent from that region of the disc that will give rise to the soldier forewing (compare W and X, white arrows). sal is, however, expressed in the hinge region (compare W and X, black arrows), which does not give rise to the wing proper. No expression was detected in the expected position of the soldier hindwing disc (I, L, O, U, X), or in the expected positions of the minor worker fore- and hindwing discs (J, M, P, S, V, Y). To ensure that absence of gene expression in vestigial discs, or in expected positions of vestigial discs, was not an artifact of incomplete probe penetration, we used two criteria: first, where applicable, gene expression had to be evident in the leg discs or CNS within the same individual (e.g. J-S); and second, reproductive larvae (wing discs) placed in the same vial as soldier and minor worker larvae had to show the expected expression.

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Supplemental Figure 2. Embryonic and early larval gene expression in Pheidole morrisi. wg is expressed in the anterior half of each future segment along the anterior-posterior axis: A, ventro-lateral view; and B, ventral view of flattened specimen, anterior to the left. C, Embryonic expression of DLL is shown in appendage disc primordia (1, 2, 3) prior to separation into the three leg and two wing discs, as well as in neuroblasts (nb): ventro-lateral view, anterior to the right. D, Larval expression of DLL in the three leg primordia (L1, L2, L3) in first instar worker larva: lateral view, anterior to the top. The expression profiles of both gene products are conserved relative to those in Drosophila (14).

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Supplemental Figure 3. Polyphenic expression profiles of three wing-patterning genes in final instar larvae of Neoformica nitidiventris, Crematogaster lineolata, and Myrmica americana. A-F: scanning electron micrographs of adult queens and workers of Neoformica nitidiventris, Crematogaster lineolata, and Myrmica americana, respectively. G-L: diagrams of wing discs in final instar larvae, as for Figure 2, showing fore- and hindwing discs (fwd, hwd) or fore- and hindwing pads (fwp, hwp) and the three leg discs (L1, L2, L3). M-X: expression of UBX and EXD in reproductives and workers of Neoformica nitidiventris, Crematogaster lineolata, and Myrmica americana. In all cases, expression in reproductives and workers was conserved relative to those of the forewing in Drosophila. Y-BB: expression of EN in reproductives and workers of Neoformica nitidiventris and Crematogaster lineolata. En was conserved in reproductives of these two species relative to those in the forewing in Drosophila (Y, AA), but could not be detected in vestigial wing discs in workers (Z, BB). Both En antibodies (Mab4D9 and 4F11) failed to stain Myrmica americana larvae.

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