Micro-evolution of toxicant tolerance: from single genes to the genome's tangled bank

Abstract

Two case-studies published 55 years ago became textbook examples of evolution in action: DDT resistance in houseflies (Busvine) and the rise of melanic forms of the peppered moth (Kettlewell). Now, many years later, molecular studies have elucidated in detail the mechanisms conferring resistance. In this paper we focus on the case of metal tolerance in a soil-living arthropod, Orchesella cincta, and provide new evidence on the transcriptional regulation of a gene involved in stress tolerance, metallothionein. Evolution of resistance is often ascribed to cis-regulatory change of such stress-combatting genes. For example, DDT resistance in the housefly is due to insertion of a mobile element into the promoter of Cyp6g1, and overexpression of this gene allows rapid metabolism of DDT. The discovery of these mechanisms has promoted the idea that resistance to environmental toxicants can be brought about by relatively simple genetic changes, involving up-regulation, duplication or structural alteration of a single-gene. Similarly, the work on O. cincta shows that populations from metal-polluted mining sites have a higher constitutive expression of the cadmium-induced metallothionein (Mt) gene. Moreover, its promoter appears to include a large degree of polymorphism; Mt promoter alleles conferring high expression in cell-based bioreporter assays were shown to occur at higher frequency in populations living at polluted sites. The case is consistent with classical examples of micro-evolution through altered cis-regulation of a key gene. However, new data on qPCR analysis of gene expression in homozygous genotypes with both reference and metal-tolerant genetic backgrounds, show that Mt expression of the same pMt homozygotes depends on the origin of the population. This suggests that trans-acting factors are also important in the regulation of Mt expression and its evolution. So the idea that metal tolerance in Orchesella can be viewed as a single-gene adaptation must be abandoned. These data, added to a genome-wide gene expression profiling study reported earlier shows that evolution of tolerance takes place in a complicated molecular network, not unlike an internal tangled bank.

Fig. 1

Cartoon of single-gene-based tolerance evolution. Tolerance may be achieved by mutations in the architecture of the promoter of a gene encoding a protein contributing to stress tolerance. Such cis-regulatory change may be due to alterations in the number or relative position of transcription factor-binding sites caused by single nucleotide substitutions, indels, recombination or transposition. If these mutations result in constitutive or induced over-expression of stress-combatting genes they may cause a fitness advantage to the phenotype and be favored by selection

Fig. 2

Box plots representing the basal (constitutive) and induced (exposed to cadmium in green algae at a nominal concentration of 1 μmol per g dry weight) expression of metalothionein in Orchesella cincta homozygous for the A1, A2 and C alleles of the metallothionein promoter, in populations derived from a reference site (Roggebotzand) and a metal-polluted site (Plombières). Metallothionein gene expression was measured by qPCR, expressed relative to beta-actin and logarithmically transformed. Averages were taken of three (pMtA1 and pMtA2 homozygotes and six (pMtC homozygotes) replicates. Means within a genotype bearing the same letter (a–c) do not differ significantly from each other, based on the Tukey HSD post-hoc test

Fig. 3

Cartoon illustrating the network of molecular interactions involved with gene expression. Black dots indicate the multiple sites in the DNA where mutations can lead to enhanced stress tolerance. These changes can involve non-synonomous mutations in the coding region of a gene (causing an altered protein) and mutations in the 5′ cis-regulatory region of the gene (causing more or less protein). In addition, a multitude of trans-acting factors can affect the expression of the gene, and mutations may affect the expression or the structure of such proteins. In addition, the phenotypic effects of these mutations are shaped by genetic interactions with other genes, e.g. due to linkage disequilibrium, pleiotropy and epistasis. So the analysis of toxicant tolerance must take a genome-wide, network perspective