TEXT S9: YELLOW/MAJOR ROYAL JELLY PROTEIN FAMILY
Rick Overson, Martin Helmkampf, and Jürgen Gadau
School of Life Sciences, Arizona State University, Tempe, AZ 85287, United States of America
The yellow/major royal jelly protein family is a quickly evolving gene family which
curiously has been discovered in all insects investigated to date, as well as in some
bacterial and fungal species but in no other non-insect metazoan [1]. Yellow genes
function in diverse roles in development, locomotion, melanization, immune response,
and mating and courtship behavior [2-4]. An expansion of an ancestral gene similar to
the extant yellow-e3 has led to the formation of the major royal jelly protein (MRJP)
subfamily, which was first detected in Apis mellifera. In this species, members of the
MRJP subfamily have taken on a nutritional role (the production of royal jelly) which in
turn regulates reproductive division of labor in the colony [1,5]. A similar but apparently
independent expansion of major royal jelly protein-like genes has been detected in the
genome of the parasitoid wasp Nasonia vitripennis [6].
In the Atta cephalotes genome we detected a total of 21 yellow/MRJP genes, 13
of which are yellow genes and eight of which are similar to Apis mellifera MRJP genes,
using an approach described elsewhere (see section on cytoplasmic ribosomal proteins;
the MRJP genes of Apis mellifera served as additional reference genes). Of the 13
yellow genes, nine were identified as single-copy orthologs of the yellow genes of either
Drosophila melanogaster or Apis mellifera (Acep_Y-b,-c,-e,-e3,-g,-g2,-h,-y and -x2). In
contrast, the Apis mellifera and Nasonia vitripennis gene Y-x1 is partially represented by
three gene models: Acep_Y-x1_frag1 contains the first 2/3 of a complete gene (using
the Apis mellifera Y-x1 gene as a reference) while Acep_Y-x1_frag2 contains the exact
final 1/3 of the gene but it is present on a different scaffold. Acep_Y-x1_frag3 which is
located on the same scaffold as AcepY-x1_frag2 also contains the 3’ end of a Y-x1
gene. We included only Acep_Y-x1_frag1 in the phylogenetic analysis below as it is the
largest and most complete of the three. Whether this fragmentation is due to repeated
duplications and truncations or genome misassembly remains unknown. Finally, a gene
termed Acep_Y-1 without clear homology relations to other insect yellow genes was
also found. Of the eight MRJP-like genes we detected, three models (Acep_MRJPL1, 2, -3) posses the six exons of a typical complete MRJP or MRJPL gene (Acep_MRJPL3
is missing part of the sixth exon, but it seems to be artifactually cut short by the end of
the scaffold). The remaining four MRJP-like genes (Acep_MRJPL4, -5, -6, -7, -8) are
presumably pseudogenes as they contain missing exons, large indels, or lack open
reading frames.
To understand the evolution of the gene family, we performed a phylogenetic
analysis of yellow/MRJP genes across insect taxa. We started with the initial reference
set of Drosophila melanogaster yellow genes and retrieved homologous genes from the
genomes of Apis mellifera, Nasonia vitripennis and Tribolium castaneum from public
archives. A yellow gene from the bacterium Dienococus radiodurans was included to
serve as the outgroup for the analysis. Amino acid sequences of these genes and those
from Atta cephalotes (88 genes in total) were aligned with MAFFT v6 and the L-INS-i
algorithm [7]. Positions which were aligned ambiguously were removed using Aliscore
v1 [8] with default settings. This resulted in a final dataset containing 254 amino acid
positions. The evolutionary model with the best fit to this dataset, LG+G, was
determined by ProtTest [9] according to the Akaike Information Criterion corrected for
small sample size. Based on this model, a maximum likelihood tree was reconstructed
using RAxML v7.2.6 [10]. Nodal support values were obtained by the rapid bootstrap
algorithm as implemented in RAxML (500 replicates).
The tree (Fig. 1) reveals twelve gene subfamilies within insect yellow/MRJP
genes, most of which are characterized by a one-to-one orthologous relationship among
the five focal taxa (Y-b, -c, e, -g, -h, -y). Y-x1, -g2 and -e3 display expansions in
individual taxa, mainly Drosophila melanogaster and Nasonia vitripennis. Gene losses
are encountered rarely, with Y-x1 and Y-x2 being restricted to hymenopterans (although
the Y-1 to Y-5 genes in Tribolium castaneum might be co-orthologous to the
hymenopteran Y-x1 genes), and hymenopterans in turn lack Y-f (according to the tree,
the genes named Y-f in Apis mellifera and Nasonia vitripennis are probably misnamed
as they are part of the Y-c clade). Finally, the MRJP subfamiliy is restricted to
Hymenoptera, and characterized by independent expansions in all three represented
taxa, as all are more closely related to their intraspecific paralogues than to genes in
other taxa. Although only three complete MRJP genes could be identified in Atta
cephalotes, the existence of five putative pseudogenes indicates that the gene number
was originally in the same range as in the other two taxa. It may be that an ancestral
gene with the propensity to expand in this manner has done so independently to fulfill
varying roles. In the case of Apis mellifera MRJP genes, one of these roles is the
production of royal jelly. Their function in ants and parasitoid wasps, however, is
unknown.
The phylogenetic position of two orphaned genes, Atta cephalotes Y-1 and
Drosophila melanogaster Y-k could not be determined reliably. While nodal support
values for many yellow/mrjpl subfamilies is strong, only a few inter-clade relationships
could be resolved. These include the sister-group relationship between the Y-g and Yg2 genes, and within the well supported monophyly of the Y-b, -c, -f, -y and possibly Y-h
genes. Because this clade contains the originally described Y-y gene of Drosophila
melanogaster (reviewed in [11]), we refer to it as the yellow core group.
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Figure 1. Maximum likelihood tree of the yellow/mrjp genes found in the genomes of Atta cephalotes (Ac,
highlighted), Apis mellifera (Am), Nasonia vitripennis (Nv), Drosophila melanogaster (Dm) and Tribolium
castaneum (Tc). Support values > 50 based on 500 rapid bootstrap replicates are shown at the nodes of
the tree.