Sequence Evolution and Expression of the Androgen Receptor and Other Pathway-Related Genes in a Unisexual Fish, the Amazon Molly, Poecilia formosa, and Its Bisexual Ancestors

Abstract

The all-female Amazon molly (Poecilia formosa) originated from a single hybridization of two bisexual ancestors, Atlantic molly (Poecilia mexicana) and sailfin molly (Poecilia latipinna). As a gynogenetic species, the Amazon molly needs to copulate with a heterospecific male, but the genetic information of the sperm-donor does not contribute to the next generation, as the sperm only acts as the trigger for the diploid eggs' embryogenesis. Here, we study the sequence evolution and gene expression of the duplicated genes coding for androgen receptors (ars) and other pathway-related genes, i.e., the estrogen receptors (ers) and cytochrome P450, family19, subfamily A, aromatase genes (cyp19as), in the Amazon molly, in comparison to its bisexual ancestors. Mollies possess-as most other teleost fish-two copies of the ar, er, and cyp19a genes, i.e., arα/arβ, erα/erβ1, and cyp19a1 (also referred as cyp19a1a)/cyp19a2 (also referred to as cyp19a1b), respectively. Non-synonymous single nucleotide polymorphisms (SNPs) among the ancestral bisexual species were generally predicted not to alter protein function. Some derived substitutions in the P. mexicana and one in P. formosa are predicted to impact protein function. We also describe the gene expression pattern of the ars and pathway-related genes in various tissues (i.e., brain, gill, and ovary) and provide SNP markers for allele specific expression research. As a general tendency, the levels of gene expression were lowest in gill and highest in ovarian tissues, while expression levels in the brain were intermediate in most cases. Expression levels in P. formosa were conserved where expression did not differ between the two bisexual ancestors. In those cases where gene expression levels significantly differed between the bisexual species, P. formosa expression was always comparable to the higher expression level among the two ancestors. Interestingly, erβ1 was expressed neither in brain nor in gill in the analyzed three molly species, which implies a more important role of erα in the estradiol synthesis pathway in these tissues. Furthermore, our data suggest that interactions of steroid-signaling pathway genes differ across tissues, in particular the interactions of ars and cyp19as.

Fig 1. Structural comparison of P. formosa Ars with those of other species.

The numbers within each box indicate the percent amino acid identity of each putative domain (NTD: N-terminal domain, DBD: DNA binding domain (blue color), LBD: ligand binding domain (green color)), relative to P. mexicana Arα. The numbers above each box indicate the amino acids position of each domain. P. formosa had two alleles of both Arα and Arβ, pointing to its hybrid origin.

Fig 2

Polymorphic nucleotide positions in the coding region of arα (A) and arβ (B) among the 3 analyzed Poecilia species. Red triangles indicate non-synonymous substitutions among the ancestral alleles of P. mexicana and P. latipinna origin. The blue round point represents non-synonymous substitutions caused by mutation within a single lineage. The green capital letters below the alignment in NTD of arβ represent additional amino acids at a position with an insertion/deletion event. Green stars indicate positions, at which P. mexicana was heterozygous (C/T). Dots in the alignment indicate identity with P. mexicana. Colored boxes indicate different functional domains within the protein (Green: NTD, Blue: LBD, Orange: DBD). Abbreviation in figure, Me: P. mexicana, Fo: P. formosa, La: P. latipinna.

Fig 3

Phylogenetic tree of ars (A), ers (B) and cyp19as(C) in Poeciliidae and other vertebrates. Amino acid sequences were used for phylogenetic analysis with MrBayes. Labels provide Bayesian posterior probabilities. Teleost fish typically possess two copies of all genes. Note that Danio rerio has lost arα. This phylogenetic tree clearly demonstrates that the two gene copies emerged in the course of the fish-specific whole-genome duplication (FSGD, Meyer and Schartl 1999). It also confirms the hybrid origin of P. formosa, as they are heterozygous and possess one allele per locus from each of its ancestors. See S1 Table for accession numbers.

Fig 4. Gene expression profiling.

This profiling presents each single gene expression distribution through all 3 tissues (ovary, gill, brain) among P. formosa and its ancestors. Expression levels are normalized relative to P. mexicana ovary expression data of 6 different pathway genes, arα (A), arβ (B), cyp19a1 (C), cyp19a2 (D), erα (E), erβ1 (F). They are shown on a logarithmic scale. Red stars indicate that expression has not been detected in our study. * 0.01≤P≤0.05; ** P< 0.01.