Evolutionary origin and genomic organisation of runt-domain containing genes in arthropods
© Duncan et al; licensee BioMed Central Ltd. 2008
Received: 22 September 2008
Accepted: 25 November 2008
Published: 25 November 2008
Gene clusters, such as the Hox gene cluster, are known to have critical roles in development. In eukaryotes gene clusters arise primarily by tandem gene duplication and divergence. Genes within a cluster are often co-regulated, providing selective pressure to maintain the genome organisation, and this co-regulation can result in temporal or spatial co-linearity of gene expression. It has been previously noted that in Drosophila melanogaster, three of the four runt-domain (RD) containing genes are found in a relatively tight cluster on chromosome 1, raising the possibility of a putative functional RD gene cluster in D. melanogaster.
To investigate the possibility of such a gene cluster, orthologues of the Drosophila melanogaste r RD genes were identified in several endopterygotan insects, two exopterygotan insects and two non-insect arthropods. In all insect species four RD genes were identified and orthology was assigned to the Drosophila sequences by phylogenetic analyses. Although four RD genes were found in the crustacean D. pulex, orthology could not be assigned to the insect sequences, indicating independent gene duplications from a single ancestor following the split of the hexapod lineage from the crustacean lineage.
In insects, two chromosomal arrangements of these genes was observed; the first a semi-dispersed cluster, such as in Drosophila, where lozenge is separated from the core cluster of three RD genes often by megabases of DNA. The second arrangement was a tight cluster of the four RD genes, such as in Apis mellifera.
This genomic organisation, particularly of the three core RD genes, raises the possibility of shared regulatory elements. In situ hybridisation of embryonic expression of the four RD genes in Drosophila melanogaster and the honeybee A. mellifera shows no evidence for either spatial or temporal co-linearity of expression during embryogenesis.
All fully sequenced insect genomes contain four RD genes and orthology can be assigned to these genes based on similarity to the D. melanogaster protein sequences. Examination of the genomic organisation of these genes provides evidence for a functional RD gene cluster. RD genes from non-insect arthropods are also clustered, however the lack of orthology between these and insect RD genes suggests this cluster is likely to have resulted from a duplication event independent from that which created the insect RD gene cluster. Analysis of embryonic RD gene expression in two endopterygotan insects, A. mellifera and D. melanogaster, did not show evidence for coordinated gene expression, therefore while the functional significance of this gene cluster remains unknown its maintenance during insect evolution implies some functional significance to the cluster.
Runt-domain (RD) containing proteins are transcriptional regulators that are able to activate or repress transcription dependent on the biological context of the cell  and are involved in cell fate specification and cell differentiation during development in metazoans . The RD, a DNA binding domain, consists of a 128 amino acid motif that mediates DNA binding, heterodimerisation with a β-subunit (similar to the mammalian CBFβ proteins)  and also nucleotide binding  Outside of the RD itself, the only conserved motif in this protein family is a C-terminal pentapeptide (VWRPY, or I/LWRPF) which is thought to mediate the interaction with the transcriptional co-repressor Groucho .
The number of RD genes reported in metazoan genomes varies, and it is generally acknowledged that the RD genes have been independently duplicated in the chordate and insect lineages [6, 7]. Studies of sea urchin (Strongylocentrotus purpuratus) runt and the mammalian runt orthologues Runx1, Runx2 and Runx3 have shown that RD proteins play an essential role in actively cycling cells and regulate cell proliferation prior to terminal differentiation . All three RD proteins in humans are associated with disease, particularly cancer. Runx1, for example, is required for haematopoiesis and mutations in this gene cause leukaemia. Runx2 is required for bone development, and Runx3 for growth and differentiation of the gastric epithelium . The Caenorhabditis elegans and S. purpuratus runt genes are also expressed in the gut [9, 10], implying that runx3 may be the ancestral runx gene in chordates , although this is not supported by molecular data .
The number of RD genes found in ecdysozoan genomes also varies, with a single RD gene in C. elegans, and three in the mosquito Anopheles gambiae . In Drosophila, four RD genes are present in the genome: runt and lozenge, which have been very well characterised and two uncharacterised RD containing genes, CG42267 (previously CG15455) and CG34145 .
In Drosophila, runt is involved in many developmental processes, including embryonic segmentation [11, 12], sex determination [13, 14] and neurogenesis [15, 16]. Runt is initially expressed in a broad domain in the centre of the syncitial blastoderm embryo regulated by bicoid [17, 18] and tailless , and is required for activation of sex-lethal, the major switch in Drosophila sex-determination . Runt is a pair-rule gene but also has gap gene like properties . During segmentation runt is expressed in the blastoderm with a dual segment periodicity, becoming expressed in seven stripes of cells. The expression of each of the seven stripes appears to be regulated independently by the gap genes, but possibly also by the maternal coordinate gene bicoid . Other pair-rule genes, particularly hairy, act to maintain the runt expression pattern [17, 18]. Later in the segmentation cascade runt is expressed with segmental periodicity, resulting in the formation of the secondary stripe pattern. This pattern is dependent on all of the pair-rule genes except sloppy-paired . Following segmentation runt is expressed in a subset of cells in the developing nervous system and determines cell fate in specific neural lineages .
The other well-characterised Drosophila RD protein, lozenge, also has roles in several developmental processes. It has been shown to function in the development of antennae and tarsal claws, as well as haematopoiesis and female fertility . Lozenge was first identified on the basis of its role in eye development and is critical for the differentiation of both photoreceptor neurons and non-neuronal cone cells [21, 22]
Gene clusters, such as the Hox gene complex, are often ancient and are found in animals separated by hundreds of millions of years of evolution. While gene clustering as a phenomenon appears more common in genomes of higher complexity, for instance in mammalian as compared to insect genomes, very ancient clusters such as those involving the wnt family of genes  and the Fox gene transcription factors  have been reported across evolution. In the well-studied cases of the Hox, Wnt and Fox clusters, genes within the cluster show coordinated expression during embryogenesis, implying the genes may be regulated by shared cis-regulatory elements, which invoke stabilising selection to maintain the gene cluster. For the majority of gene clusters, however, it is not clear whether the genomic organisation is maintained as a result of functional constraints, or whether it is merely by chance that no chromosomal rearrangements disrupting the complex have become fixed.
It has been previously noted that of the Drosophila RD genes, runt, CG34145 and CG42267 are closely linked on chromosome 1 , and are likely to have evolved as a result of tandem gene duplication. The close linkage of these genes may be due to the recent evolution of multiple RD genes in Drosophila or alternatively may indicate the presence of functional constraints that retain these genes in close proximity. It is possible to distinguish between these possibilities by comparison of the genomic organisation amongst species that are sufficiently diverged to have had numerous chromosomal rearrangements become fixed, resulting in a shuffling of both the order and spacing of genes.
A comprehensive survey of runt domain containing genes in a wide range of arthropods is now possible due to the availability of genome sequence for several key species. Here we identify the RD genes in the genomes of several endopterygotan insects, including the twelve Drosophila species , Apis mellifera (the honeybee) , Nasonia vitripennis (the jewel wasp) , Tribolium castaneum (the red flour beetle) Bombyx mori (the silkmoth)  and Aedes aegypti (the yellow fever mosquito) . The genomes of two fully sequenced exopterygotan insects, Acyrthosiphon pisum (the pea aphid)  and the Pediculus humanus (the human body louse)  were also included in this analysis. In addition two non-insect arthropod genome sequences are now available: the crustacean Daphnia pulex  and the chelicerate Ixodes scapularis . We examine the phylogenetic relationships and genomic organisations of the RD genes in these organisms, and present evidence for a putative conserved runt complex in insects. To determine if the close genomic proximity of these RD genes in insects relates to functional conservation of regulatory elements, the embryonic expression profiles of the four RD genes were examined in D. melanogaster and the hymenopteran insect A. mellifera.
Identification of a runt gene complex in Drosophila species
Three of the four RD genes in D. melanogaster are clustered within 163 kb on chromosome 1 and are likely to have arisen by tandem gene duplication . There is very tight linkage between CG42267 and CG34145, but there are three annotated functionally unrelated genes that lie between CG34145 and runt. Lozenge is separated from the other three genes by a distance of over 11 Mb.
To determine whether the genomic organisation of this 'runt complex' is the result of functional constraint or has been maintained purely by chance, we utilised the recent release of the twelve Drosophila species genome sequences . These species are approximately 40 – 60 million years diverged , and extensive gene shuffling within the chromosome arms has occurred even between moderately diverged genomes such as D. melanogaster and D. yakuba [25, 36].
The orthologues of CG34145, CG42267 and Runt are all found tightly linked (within 140 – 225 kb) in all 12 Drosophila species. In contrast, the orthologue of lozenge is usually present on the same chromosome but not closely linked to the other three RD genes. In species where scaffolds have been assigned to chromosome groups, this complex falls on the X chromosome (chromosome 1 in D. melanogaster).
Together, the phylogenetic and genomic organisation supports the duplication of the RD genes prior to the radiation of the Drosophila lineage, and the resultant cluster of RD genes shows remarkable stability since the divergence of Drosophila species 60 million years ago .
Identification of a runt gene complex in other insect species
A survey of other fully sequenced insect genomes [26–28, 31, 32, 38], shows that aspects of this runt gene complex are conserved, not only in the endopterygotan insects A. mellifera, A. aegypti, B. mori and T. castaneum, but also in the exopterygotan insects P. humanus (a Phthiapteran) and A. pisum (a Hemipteran). In all of these insects four RD genes were identified, and phylogenetic analyses (Fig. 3) were used to assign orthology to these genes. Where RD genes were incompletely or incorrectly annotated, particularly in the genomes of T. castaneum, A. aegypti, A. pisum and B. mori, annotation was performed manually with the assistance of Augustus  and GenomeScan , in all cases the presence of both the RD and C-terminal VWRPY motif was confirmed (the C-terminal pentapeptide in A. pisum XP001950158 is modified to IWRPF, and followed by an additional 5 amino acids prior to the stop codon). Proteins that were manually annotated in this way are indicated with an asterisk on the phylogeny presented in Fig. 3 [see Additional file 2 for multiple sequence alignment and phylogeny for endopterygotan insects, and Additional file 3 for multiple sequence alignment for both endopterygotan and exopterygotan insects].
Data for A. aegypti and A. pisum were inconclusive due to gaps in the genome assemblies, but the orthologues of both runt and CG34145 were found on the same contig in close proximity, consistent with that seen in the other insects. In all cases the transcriptional orientation of these three core RD genes is completely conserved.
Interestingly, in the species where the four RD genes are tightly linked (A. mellifera, B. mori and P. humanus), the orthologue of lozenge lies proximal to the orthologue of runt. This contrasts to the remainder of species where lozenge often lies distal to the RD cluster, separated by a megabase or more.
Expression of the RD genes: comparison of Drosophila and A. mellifera
The persistence of an intact RD gene cluster in all insects examined, despite more than 400 million years of evolution, and significant changes in life history and developmental modes, implies some functional relevance to the organisation of these genes. Analysis of g ene expression patterns has previously provided important insight into the nature of selective forces acting on gene clusters . The expression patterns of runt and lozenge are well-understood in D. melanogaster, but the expression patterns of the other two RD genes remains uncharacterised. As the Drosophila RD complex shows evidence of dispersal, of lozenge in particular, we also chose to examine the expression patterns of all four RD genes in the basally branching endopterygotan insect, A. mellifera, which possesses a tight cluster of all four RD genes (Fig. 4).
The expression of Drosophila lozenge (Fig. 5g) is not consistent with that previously reported; a previous study demonstrated that by embryonic stage 7 lozenge expression is restricted to the anterior of the embryo in a sub-population of prohaemocytes, which terminally differentiate to form crystal cells (which are involved in innate immunity and wound healing) by embryonic stage 10 . In this study lozenge mRNA is initially maternally provided and is localised to nuclei at early stages, expression later in development is ubiquitous. This difference in expression patterns is probably because the previous study used an enhancer fragment linked to a reporter gene to determine the expression pattern. A. mellifera lozenge (Fig. 5h) expression is detected very early (stage 1) in the anterior region of the embryo and is then detected in a distinct cell type, that develop from head mesoderm (Fig. 5h). These may be differentiating crystal cells, which develop from the head mesoderm in Drosophila . By stage 9 (just prior to hatching), Amlz is expression is detected throughout the embryo (data not shown).
The embryonic expression of the other two RD genes in Drosophila, CG34145 (Fig. 5c) and CG42267 (Fig. 5e), have not been previously described. At all embryonic stages expression of both of these genes is ubiquitous, with in situ hybridisation of imaginal discs also showing no evidence of localised expression (data not shown). Both Am GB11654 and Am GB19482, the A. mellifera orthologues of these genes, are also expressed ubiquitously throughout late stages of embryonic development (Fig. 5d and 5f).
While clustered genes often show coordinated expression, neither the Drosophila nor A. mellifera RD genes (Fig. 5) demonstrate strong coordination of either temporal or spatial expression during embryonic development.
The evolution of RD proteins in metazoa and arthropods
The presence of a runt domain cluster (Fig. 4) in all insect species examined, together with the phylogenetic data presented in Fig. 3, suggests that the duplication of the RD genes occurred prior to the divergence of the insect lineage into these two branches. In order to examine the evolution of these RD genes, and this cluster in a wider context, the genome sequence of two non-insect arthropods were utilised. Examination of the genome sequences of the crustacean D. pulex and the chelicerate I. scapularis clearly demonstrates multiple RD genes in both of these species, contradicting previous assertions that non-insect arthropods have only a single RD gene . The water flea D. pulex has four RD genes, and two of these fall on the same scaffold (Fig. 4). In contrast I. scapularis only has two RD genes, and at present these fall on two independent scaffolds. Extensive searches of the I. scapularis genome have been unable to identify any further orthologues, however this could be due to the preliminary nature of the genome assembly. Two RD genes have also been identified in the spider Cupiennius salei , and one in the two-spotted spider mite, Tetranychus urticae .
Phylogenetic analysis indicates that the duplication of the RD genes has occurred independently in the deuterostome and arthropod lineages, and that the duplication events that gave rise to the multiple vertebrate runx genes, have occurred independently of the duplications giving rise to the multiple RD genes in the sea urchin, S. purpuratus. Somewhat surprisingly however, the phylogeny also indicates independent duplication events have given rise to the chelicerate RD genes and the crustacean/insect RD genes. The phylogeny indicates a single origin for chelicerate RD genes independent of the duplications that gave rise to the RD genes in Daphnia and in insects. Supporting this is the expression pattern of the RD gene cloned from Tetranychus urticae (the two-spotted spider mite), which clearly suggests a role for Tu-run in segmentation, but also in limb specification , a role not seen for either the A. mellifera or Drosophila orthologues of runt, but consistent with expression of Cs-runt in C. salei.
Interestingly, a single D. pulex gene (Dp290554) groups with the clade of insect RD proteins, while the other three D. pulex genes form an independent clade. This seems to indicate that runt is the ancestral protein in crustaceans and chelicerates. However, the crustacean/insect clade on the whole is not well resolved, as by necessity, this phylogeny only included 125 amino acids of the RD as two of the D. pulex sequences are incorrectly annotated and are missing 10 amino acids of the RD, thus reducing the informational content of the alignment.
In this study we identified four RD genes in the twelve sequenced Drosophila genomes  and in several non-dipteran endopterygotan insects including A. mellifera, A. aegypti, B. mori, and T. castaneum. Four RD genes were also identified in the exopterygotan insects, P. humanus and A. pisum, and the crustacean D. pulex. Two different arrangements of RD genes were observed in these species: a semi-dispersed cluster was seen in Drosophila species and in T. castaneum; and an intact, tight cluster with few or no intervening genes was seen in A. mellifera, B. mori and P. humanus. In the other species examined, the genome assembly was fragmented such that one or more RD genes was localised to a different contig and thus no assertions about cluster organisation could be made. In species with an intact RD gene cluster, lozenge was found to be located proximal to runt, while in the species with a semi-dispersed cluster lozenge was often localised to the same chromosome, but separated from the core cluster by megabases of DNA. The genomic organisation of the remaining three RD genes, including gene spacing and transcriptional orientation, was remarkably conserved.
The number and identity of intervening genes in this cluster varies; between two and seven genes lie between the CG34145 and runt orthologues in Drosophila species, including Cyp6v1. Cyp6v1 is a P450 enzyme, and is part of a large gene family that is prone to duplication . Hydra and CG1835 are localised between CG34145 and runt in D. melanogaster, D simulans, D. yakuba and D. erecta, these two genes are localised to an orphan scaffold in D. sechellia (scaffold 600), but this is likely a problem with the genome assembly. Hydra is found exclusively in the melanogaster sub-group, with no orthologues found in any other dipterans or insects (data not shown), suggesting recent evolution of this gene. Conversely CG1835 is also found in D. willistoni suggesting at least two independent losses of this gene (in the obscura group and D. ananassae) A fourth gene is found in this region in the melanogaster species group, however this gene is unannotated in D. melanogaster and therefore may represent a conserved regulatory sequence rather than a protein coding gene. CG42267 and CG34145 are in much tighter linkage, and the majority of Drosophila species have either one or no intervening genes in this region, other than D. persimilis which has three. Based on established taxonomic relationships between Drosophila species it is likely that there has been multiple gains or losses of genes in these genomic regions, although this effect may be exaggerated due to inaccuracies in the genome assemblies or gene prediction models. In all but one non-dipteran insect species, no intervening genes were identified in the RD gene cluster, the only exception being in B. mori where five annotated genes fall between BmCG42267 and BmCG34145. The presence of intervening genes in the Dipteran RD cluster, seemingly unrelated in either expression or function, is consistent with the RD complex being considered a genomic regulatory block  and is reminiscent of the Hox complex in Drosophila, where invasion of cuticle genes into the ANT-C complex between labial and proboscipedia has been attributed to the accelerated rate of evolution in this lineage, and may have contributed to splitting of the Hox complex in Drosophila .
The presence of an intact cluster in the endopterygotan insects A. mellifera and B. mori, and the exopterygotan insect P. humanus, strongly supports the phylogenetic data that duplication of the RD genes occurred prior to the radiation of insect species, and implies that the ancestral insect genome had a very tight cluster of four RD genes. The semi-dispersed gene cluster seen in Drosophila and T. castaneum may indicate that the functional constraint holding lozenge in the gene cluster has been lost or modified in the Coleopteran and Dipteran lineages. The pattern of RD gene dispersal in these species is similar to that seen for the Fox family of transcription factors, where relaxation of selection on the cluster has resulted in cluster fragmentation . The dissolution of such clusters also may be the result of accelerated genome evolution associated with Drosophila lineages . However, the T. castaneum genome is not noted to have undergone particularly rapid evolution  and consistent with this, the T. castaneum genome has just a single Hox complex, with no evidence of dispersal . A recent study has shown that while up to 91% of orthologues remain in synteny between D. melanogaster and the most diverged Drosophila species, D. virilis synteny is only 3% between D. melanogaster and B. mori, and 10% between D. melanogaster and A. mellifera . This, along with the high observed rate of turnover of intervening genes within the RD cluster, would indicate that the conservation of genomic organisation seen for RD genes across insect genomes would be very unlikely to have occurred simply by chance, and is likely to have been maintained by selection, presumably in favour of retaining function.
Functionally related genes are known to have, in some cases, conserved genomic organisation . In general, genes that have evolved as a result of tandem gene duplications tend to maintain a similar function to their parental copy due to sharing of the same regulatory elements , and this sharing of regulatory elements can facilitate the evolution of co-regulation . To examine the possibility of co-regulation acting as a selective force to maintain the RD gene cluster, the expression of the four RD genes was examined in D. melanogaster (which houses a semi-dispersed RD cluster) and in A. mellifera (which houses an intact 'ancestral' RD cluster) as a means of investigating functional conservation of RD gene orthologues between these species. In situ hybridisation did not reveal any strong evidence for spatial or temporal co-regulation of RD gene orthologues during Drosophila or A. mellifera embryogenesis. Although it is possible that localization or expression of the protein (which would not be detected by in situ hybridisation) is altered independently of that of its mRNA. Three of the four RD genes are expressed ubiquitously in the blastoderm stage of embryogenesis in Drosophila, while the fourth, runt is expressed in a pair-rule pattern in seven stripes. Therefore the cluster of RD genes in insects could be driving co-regulation of expression at the blastoderm stage, while runt expression is negatively regulated by fushi tarazu , however this is not the case for A. mellifera. It is possible that overlapping rather than shared regulatory elements are responsible for the retention of the gene cluster. However, this scenario would also appear unlikely; while the runt enhancer in Drosophila covers a distance of approximately 14 kb , the intergenic distance between CG34145 and runt averages around 100 kb across Drosophila species, and the number of intervening genes in this region varies markedly, highlighting that this genomic region is free to evolve.
There has been much speculation about the origin of the RD proteins, and it is largely accepted that the RD genes have been independently duplicated in the deuterostome and protostome lineages [6, 7]. However, the question of RD gene evolution in arthropods has not been addressed specifically. Phylogenetic analyses presented here support the notion of gene duplication prior to the radiation of insect species, as all insect RD proteins could be placed into four orthologue groups. However, two non-insect arthropods whose genomes have been sequenced completely, the chelicerate I. scapularis and the crustacean D. pulex, also have multiple RD genes, and phylogenetic analyses support the notion that these genes have been independently duplicated in insect and non-insect arthropod lineages. This phylogeny implies that the ancestor of arthropods had a single RD gene. However, gene conversion events are known to result in phylogenetic analyses that overestimate the incidence of gene duplication. This is an issue for linked genes, such as those generated by tandem gene duplication , and has resulted in spurious phylogenies for insect gene families .
The phylogeny presented here indicates that there have been multiple duplications of RD genes across bilaterian evolution, and as there is no evidence for the duplicated copies decaying into pseudogenes, these duplicated copies are likely to have undergone rapid divergence resulting in subfunctionalisation or neofunctionalisation of the protein functions. This kind of rapid divergence could be due to the chromosomal location of the ancestral gene, as it is known that areas of the genome are more prone to this phenomenon, such as the X chromosome in Drosophila, where the RD gene complex is localised . Consistent with the hypothesis of rapid divergence following duplication, there is clear evidence for neofunctionalisation of the duplicated genes, in both ecdysozoan and deuterostome species. For instance, the C. elegans RD gene and one of the sea urchin RD genes, which are most closely related to mammalian runx3, are both expressed in the developing gut, a role not seen in the cnidarian Nematostella. This has led to the assertion that runx3 may be the ancestral RD gene in chordates , although this hypothesis is yet to be supported by molecular data .
To date there has been no discussion about the evolution of RD genes in arthropods, and the phylogenetic analyses presented here indicates that the last common ancestor of all arthropods had a single RD gene, which has been independently duplicated in the chelicerate and crustacean/insect lineages. All arthropod RD genes possess a runt DNA binding domain and a Groucho binding domain, suggesting that these proteins are able to act as transcriptional regulators, and both runt and lozenge have been shown to modulate developmental gene expression in Drosophila . The functions of CG34145 and CG42267 are unknown, although CG42267 was identified as having a role in the control of cell survival in an RNAi screen  and it has been suggested that cross-talk between RD proteins and the cell cycle may modulate cell cycle progression ; this would also be consistent with the ubiquitous expression of CG42267 and CG34145 during embryogenesis in Drosophila and A. mellifera . Interestingly, one of the two sea urchin RD genes is also highly expressed in proliferating cells during embryogenesis , perhaps supporting an ancestral role for the RD genes in cell-cycle modulation.
The phylogenetic data indicates that runt is the ancestral RD gene in insects and crustaceans. In insects runt has a key role in segmentation , and orthologs of runt have been shown to be involved in the segmentation of two chelicerates [46, 47], raising the possibility that one of the functions of the single RD gene in the last common ancestor of all arthropods was a role in segmentation, and that the duplication and diversification of the RD genes has served to recruit the transcriptional co-repressor Groucho into new developmental niches, such as eye development .
RD genes are present in the genomes of organisms throughout metazoa, often found in multiple copies. Phylogenetic evidence supports the notion that these duplication events leading to multiple genomic copies of RD genes have occurred independently in the deuterostome and ecdysozoan lineages. Within arthropods, RD genes are also clustered, however phylogenetic data supports the independent duplication of RD genes in the chelicerate and crustacean/insect lineages. The RD genes in insects bear all the hallmarks of a functional gene cluster, in particular the very tight association of three of the four RD genes; CG42267, CG34145 and runt. However, in situ hybridisation of RNA expression did not suggest any temporal or spatial co-regulation of these RD genes in either Drosophila, which has a semi-dispersed cluster formation or in A. mellifera, which has an intact complex. It is possible that the retention of such a complex is not associated with functional constraints but is merely an artefact of tandem gene duplication; however, given the accelerated genome evolution observed in both Drosophila and mosquito species , but not T. castaneum , this would seem unlikely. While the significance of this RD gene cluster in insect species remains unknown the persistence of this cluster across all insect species implies functional importance.
Apis mellifera were cultured using standard techniques in Dunedin, New Zealand. A. mellifera embryos were collected from frames removed from nucleus boxes containing small A. mellifera colonies.
Phylogenetic analyses and cluster identification
Full-length RD metazoan protein sequences were obtained from relevant genome databases [25–28, 30–34], or from NCBI. Unannotated RD proteins were predicted with the assistance of Augustus , and GeneScan . Where required, runt domains were extracted from full length protein sequences using the hmmer suite of programs , against the Pfam runt hmm model (PF00853, 138 amino acids) .
Multiple alignments were carried out using ClustalX . The multiple alignments were analysed using MrBAYES v3.1.2  under the Jones model with default priors. The Jones model was chosen as the most appropriate model of amino acid substitution after preliminary analyses using MrBAYES with mixed models. The Monte Carlo Markov Chain search was run with four chains over 1000000 generations with trees sampled every 1000 generations. The first 250000 trees were discarded as 'burn-in'.
Identification and cloning of RD genes
RNA was extracted from A. mellifera embryos using the RNeasy Mini Kit (Qiagen). cDNA was generated using Superscript II reverse transcriptase (Invitrogen) and an oligo-dT primer. The cDNA was used as a template for amplification of putative RD genes using the following oligonucleotide primers:
GB 16431r GTGTCCCGACGGAAGAACAGT
GB 15836f AAGCGGTAGAGGAAAGAGTT
GB 15836r GGTGAAGACCTTGAAAGTGA
GB 11654 ATGCACTTACCGGAGGGCCCACTA
GB 11654 CTCGTGCTCGAGTCGCCCTAGTAG
Amplified DNA fragments were purified using the High Pure PCR purification kit (Roche) and cloned into pCRII-TOPO (Invitrogen). The sequence and orientation of each cloned gene fragment was confirmed by DNA sequencing.
In situ hybridisation
A. mellifera embryos were collected and fixed as described . Antisense or sense digoxigenin (DIG) labelled RNA probes were produced by in vitro transcription from linearised DNA templates containing cDNA fragments. In situ hybridisation on A. mellifera embryos were performed as previously described .
The authors acknowledge the contribution of JR Alcock for discussions about portions of this data. The authors wish to acknowledge support from, and useful discussions with, the staff and students of the Laboratory for Evolution and Development. This work was funded by a RSNZ Marsden Grant (UOO401) to PKD.
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