Independent Evolutionary Origin of <i>fem</i> Paralogous Genes and Complementary Sex Determination in Hymenopteran Insects

<div><p>The primary signal of sex determination in the honeybee, the <i>complementary sex determiner</i> (<i>csd</i>) gene, evolved from a gene duplication event from an ancestral copy of the <i>fem</i> gene. Recently, other paralogs of the <i>fem</i> gene have been identified in several ant and bumblebee genomes. This discovery and the close phylogenetic relationship of the paralogous gene sequences led to the hypothesis of a single ancestry of the <i>csd</i> genetic system of complementary sex determination in the Hymenopteran insects, in which the <i>fem</i> and <i>csd</i> gene copies evolved as a unit in concert with the mutual transfers of sequences (concerted evolution). Here, we show that the paralogous gene copies evolved repeatedly through independent gene duplication events in the honeybee, bumblebee, and ant lineage. We detected no sequence tracts that would indicate a DNA transfer between the <i>fem</i> and the <i>fem1/csd</i> genes between different ant and bee species. Instead, we found tracts of duplication events in other genomic locations, suggesting that gene duplication was a frequent event in the evolution of these genes. These and other evidences suggest that the <i>fem1/csd</i> gene originated repeatedly through gene duplications in the bumblebee, honeybee, and ant lineages in the last 100 million years. Signatures of concerted evolution were not detectable, implicating that the gene tree based on neutral synonymous sites represents the phylogenetic relationships and origins of the <i>fem</i> and <i>fem1/csd</i> genes. Our results further imply that the <i>fem1</i> and <i>csd</i> gene in bumblebees, honeybees, and ants are not orthologs, because they originated independently from the <i>fem</i> gene. Hence, the widely shared and conserved complementary sex determination mechanism in Hymenopteran insects is controlled by different genes and molecular processes. These findings highlight the limits of comparative genomics and emphasize the requirement to study gene functions in different species and major hymenopteran lineages.</p></div

The evolutionary fate of <i>fem</i> gene substitutions in an evolutionary window predating the putative repeated gene duplications.

<p>(<b>a</b>) The expected evolutionary fate of <i>fem</i> substitutions in the paralogous genes <i>fem</i> and <i>csd</i>/<i>fem1</i> under the model of concerted evolution and repeated gene duplication. (<b>b, c</b>) The letters above the yellow boxes show the inferred amino acid changes in the Fem protein tree that evolved during the evolutionary window of the MRCA of ants and bees and the MRCAs of ants (<b>b</b>) and of bees (<b>c</b>). Letters above the blue boxes indicate the amino acid residues that are found at the homologous sites in the ancestral Csd/Fem1 protein sequence of the MRCA of ants (<b>b</b>) and of bees (<b>c</b>). Numbers above the letters designate the homologous sites in the Fem amino acid sequence alignment. Numbers before the boxes indicate the nodes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091883#pone.0091883.s002" target="_blank">Fig. S2</a>) used to infer the ancestral sequence information. aa denotes amino acid.</p

Events of gene conversion and recombination in the <i>fem</i> and <i>csd</i> sequences of <i>A. mellifera</i>, <i>A. dorsata</i> and <i>A. cerana</i>.

<p>Tracts of putatively recombined sequences were detected by seven different methods as indicated on the <i>x</i>-axis using the RDP 3.44 software program. The analysis was run on a single alignment of 71 <i>csd</i> and 4 <i>fem</i> sequences. (a) The number of concerted evolution events that refer to the DNA transfer between paralogous genes <i>fem</i> and <i>csd</i>. (b) The number of recombination events that identify events between sequences of the same gene. Falsely detected events (FDE) in (a) and (b) refer to biologically implausible events. The outgroup reference sequence, <i>B. terrestris fem</i>, was never involved in one of the detected events.</p

Two models for the evolutionary history of <i>fem</i> paralogous genes in ants and bees: (a) repeated gene duplication and (b) concerted evolution.

<p>Points in (<b>a</b>) denote gene duplication events giving rise to two gene copies. Connecting lines in (<b>b</b>) between branches indicate concerted evolution events resulting from unequal crossing over and/or gene conversion.</p

The <i>d_N</i> and <i>d_S</i> values and ratios for the interspecies comparisons of the <i>fem</i> and <i>fem1/csd</i> genes.

<p>Species names: <i>Amel</i>, <i>A. mellifera</i>; <i>Ador</i>, <i>A. dorsata</i>; <i>Acer</i>, <i>A. cerana</i>; <i>Bimp</i>, <i>B. impatiens</i>; <i>Bter</i>, <i>B. terrestris</i>; <i>Hsal</i>, <i>H. saltator</i>; <i>Pbar</i>, <i>P. barbatus</i>; <i>Acep</i>, <i>Atta cephalotes</i>; <i>Cflo</i>, <i>Camponotus floridanus</i>. SE: standard error.</p><p>*A one-tailed χ² - test was conducted using the absolute number of synonymous and nonsynonymous differences.</p

The evolutionary trajectory of <i>fem</i> gene substitutions in the evolutionary window that follows the putative gene duplication event in the <i>Bombus</i> lineage.

<p>(<b>a</b>) The expected evolutionary fate of <i>fem</i> substitutions in the paralogous genes <i>fem</i> and <i>fem1</i> under the models of concerted evolution and repeated gene duplication. (<b>b</b>) Deduced amino acid changes. The yellow box indicates the Fem protein, and the blue box indicates the Fem1 protein. Black letters above the boxes indicate the ancestral state of the amino acid residues found in the MRCA of <i>B. terrestris</i> and <i>B. impatiens</i>. Red letters in the red frame indicate the amino acid residues that evolved since the MRCA of <i>B. terrestris</i> and <i>B. impatiens</i> in the Fem protein. Blue letters in the blue frame indicate the amino acid residues that evolved since the MRCA of <i>B. terrestris</i> and <i>B. impatiens</i> in the Fem1 protein. Numbers before the boxes indicate the nodes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091883#pone.0091883.s002" target="_blank">Fig. S2</a>) used to infer the ancestral sequence information. aa denotes amino acid.</p

Number of gene conversion and recombination events in <i>B. terrestris</i> and <i>B. impatiens</i> sequences.

<p>Tracts of putatively recombined sequence were detected by the 7 methods as shown on the <i>x</i>-axis and using RDP 3.44 software program. The analysis was run on a single alignment of the <i>fem</i> and <i>fem1</i> sequences of the <i>B. terrestris</i>, <i>B. impatiens</i> and <i>A. mellifera fem</i> sequence. Gene conversion events refer to DNA transfers between the paralogous genes <i>fem</i> and <i>fem</i>1. Recombination events indicate transfer events between sequences of the same gene. Falsely detected events (FDE) refer to biologically implausible events (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091883#s4" target="_blank">Materials and Methods</a>).</p

Pseudogenes (<i>ps</i>) of the <i>fem</i> and <i>csd</i> genes in the ant, bumblebee and western honeybee genomes.

<p>(<b>a</b>) The orientation and location of the pseudogenes (<i>psfem</i>, <i>pscsd</i>). Boxes denote the genes or pseudogenes. The box length of pseudogenes indicates the relative degree of homology to the coding nucleotide sequences of the <i>fem</i> or <i>csd</i> genes. The phylogenetic relationship assignments are based on the lowest <i>d_S</i> estimates or the ancestral state. Numbers behind the bars indicate the genomic scaffold, linkage group or the GenBank accession number. (<b>b</b>) Evolutionary distance between duplicated <i>fem</i> and <i>fem1/csd</i> gene copies are presented in terms of pairwise synonymous divergence per synonymous site (<i>d_S</i>). Abbreviations<i>: Amel</i>, <i>A. mellifera</i>; <i>Bimp</i>, <i>B. impatiens</i>; <i>Bter</i>, <i>B. terrestris</i>; <i>Hsal</i>, <i>H. saltator</i>; <i>Pbar</i>, <i>P. barbatus.</i></p

RAD tag (SgrAI) derived SNPs from Bombus impatiens

RAD tag (SgrAI) derived SNPs from Bombus impatiens from Sadd et al. (2015) "The genomes of two key bumblebee species with primitive eusocial organisation