Loss of RXFP2 and INSL3 genes in Afrotheria shows that testicular descent is the ancestral condition in placental mammals - PubMed (original) (raw)

Loss of RXFP2 and INSL3 genes in Afrotheria shows that testicular descent is the ancestral condition in placental mammals

Virag Sharma et al. PLoS Biol. 2018.

Abstract

Descent of testes from a position near the kidneys into the lower abdomen or into the scrotum is an important developmental process that occurs in all placental mammals, with the exception of five afrotherian lineages. Since soft-tissue structures like testes are not preserved in the fossil record and since key parts of the placental mammal phylogeny remain controversial, it has been debated whether testicular descent is the ancestral or derived condition in placental mammals. To resolve this debate, we used genomic data of 71 mammalian species and analyzed the evolution of two key genes (relaxin/insulin-like family peptide receptor 2 [RXFP2] and insulin-like 3 [INSL3]) that induce the development of the gubernaculum, the ligament that is crucial for testicular descent. We show that both RXFP2 and INSL3 are lost or nonfunctional exclusively in four afrotherians (tenrec, cape elephant shrew, cape golden mole, and manatee) that completely lack testicular descent. The presence of remnants of once functional orthologs of both genes in these afrotherian species shows that these gene losses happened after the split from the placental mammal ancestor. These "molecular vestiges" provide strong evidence that testicular descent is the ancestral condition, irrespective of persisting phylogenetic discrepancies. Furthermore, the absence of shared gene-inactivating mutations and our estimates that the loss of RXFP2 happened at different time points strongly suggest that testicular descent was lost independently in Afrotheria. Our results provide a molecular mechanism that explains the loss of testicular descent in afrotherians and, more generally, highlight how molecular vestiges can provide insights into the evolution of soft-tissue characters.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1

Fig 1. Gene-inactivating mutations in RXFP2 in four afrotherian species.

(A) The exon-intron structure of the coding region of the RXFP2 gene is shown as boxes (exons, drawn to scale) and lines (introns, not drawn to scale). A vertical red line/arrowhead indicates a frameshifting deletion/insertion, with the number of deleted/inserted bases given above. Stop codon mutations are shown as a black vertical line. Splice site mutations are indicated by the mutated dinucleotide. A blue vertical line indicates a frame-preserving deletion. Red boxes are exons that are either deleted or accumulated numerous mutations that destroy any sequence similarity. All inactivating mutations were validated by unassembled genome sequencing reads stored in the SRA. Elephant, rock hyrax, and aardvark have an intact RXFP2 gene and are not shown. A filled star indicates mutations that we confirmed by PCR and Sanger sequencing in the lesser hedgehog tenrec; the exon 17 frameshift was also found in the greater hedgehog tenrec (S5A and S5B Fig). An open star indicates mutations that we confirmed by PCR and sequencing in the dugong, the sister species of the manatee (S5C and S5D Fig). (B-I) Examples of inactivating mutations and their validation by unassembled SRA reads. RXFP2, relaxin/insulin-like family peptide receptor 2; SRA, Sequence Read Archive.

Fig 2

Fig 2. Gene-inactivating mutations and critical amino acid mutations in INSL3.

(A) Functional domains of the INSL3 protein and the exon-intron structure of the INSL3 gene. Inactivating mutations are as in Fig 1, frame-preserving insertions/deletions are shown as blue lines/arrowheads. Exons but not introns are drawn to scale. Elephant, rock hyrax, and aardvark have an intact INSL3 and are not shown. (B) While the cape golden mole does not exhibit any gene-inactivating mutations (A), an INSL3 protein alignment of the A- and B-chain shows mutations (red background) at amino acids that are critical for structure and function of the mature hormone (gray background) [–48]. Disulfide bonds between Cys residues are indicated by blue lines. Residues that are affected by frameshifting deletions in the underlying tenrec or cape elephant shrew nucleotide sequence are indicated by asterisks. For these two species, we ignored these frameshifts and used the ancestral reading frame. Species in red font are testicond. Note that elephant and rock hyrax have no mutations at any of the critical sites. INSL3, insulin-like 3.

Fig 3

Fig 3. RXFP2 and INSL3 are only lost in several testicond afrotherian lineages.

The exon-intron structures of RXFP2 (18 coding exons) and INSL3 (2 coding exons) are shown. A yellow rectangle indicates an intact exon. Exons with inactivating mutations (stop codon or splice site mutations, frameshifts) are indicated in orange; completely deleted or highly diverged exons are indicated in red. Gray exons could not be examined because the respective genome assembly is incomplete at this locus (assembly gap). We could not examine the RXFP2 in the fragmented pangolin genome, because different parts of RXFP2 locus align to at least 5 different short scaffolds. Neither exons nor introns are drawn to scale. INSL3, insulin-like 3; RXFP2, relaxin/insulin-like family peptide receptor 2.

Fig 4

Fig 4. Evolution of RXFP2 and estimated time intervals during which RXFP2 came under neutral evolution.

Red boxes represent a lower and upper bound for an estimated time interval during which RXFP2 started to evolve neutrally (Materials and methods, S2 Table), which shows that the gene was lost independently at different time points. Gray boxes represent an estimated interval for species divergence times taken from TimeTree [57] (see also S4 Table). Species in blue font are testicond. The phylogenetic position of tenrecs, golden moles, elephant shrews is well supported by morphological and molecular characters [23, 24, 26]. Uncertain phylogenetic relationships are shown as a polytomy. Note that while the loss of RXFP2 in the elephant shrew is estimated to have happened at the base of the lineage, this loss is clearly independent from the much later loss in the tenrec and in the golden mole lineage. Evidence for the loss of RXFP2 in the dugong and the greater hedgehog tenrec was obtained by PCR and sequencing experiments (S5 Fig). RXFP2, relaxin/insulin-like family peptide receptor 2.

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Max Planck Society. Received by MH. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. German Research Foundation (grant number HI 1423/3-1). Received by MH. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Leibniz Association (grant number SAW-2016-SGN-2). Received by TL, HS, MH. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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