W. A paradigmatic example of a diachronic explanation is that of Wong and Wolfe in

W. A paradigmatic example of a diachronic explanation is that of Wong and Wolfe in “Birth of gene cluster by adaptive gene relocation” [108].Clustering of six genes involved in allantoin utilisation is demonstrated to be a relatively recent novelty appearing at one specific stage of the evolution of the genus Saccharomyces. This novelty coincides with the ability to grow in anaerobic conditions and with the inability to utilise urate as a nitrogen source (a process that generates reactive oxygen species), due to the concomitant loss of the genes encoding urate oxidase and the urate/ xanthine transporter [108,109]. Not surprisingly three other genes (orthologues of xanA, uaX and uaW of A. nidulans, [100]), also necessary for xanthine and urate utilisation, are lost together with the appearance of the allantoin utilisation cluster (my own unpublished observations). An allantoin specific transporter gene, DAL4 , integrated in the cluster, originated from a duplication of the uracil transporter gene FUR4, concomitantly with the birth of the cluster [108]. The clustering of three genes involved in nitrate get Caspase-3 Inhibitor assimilation (encoding the transporter, nitrate reductase, and nitrite reductase) in a number of fungi (including Eurotiales among the Ascomycetes and at least some Basidiomycetes) but not in others, has been interpreted as a result on horizontal transmission of the whole cluster from an Oomycete to the ancestor of Dikarya or perhaps even earlier, as the genes (but not as a cluster) are present in Mucoromycotina [110]. Episodes of de-clustering would have repeatedly occurred among the Dikarya. The assimilation of nitrate has been studied in detail in three ascomycetes, A. nidulans [2,95,96], N. crassa [111] and a member of the Saccharomycotina, Pichia angusta (Hansenula polymorpha). In the latter organism the genes of the nitrate utilisation pathway are completely clustered. This cluster comprises not only the three genes mentioned above, but also two Cys6Zn2 transcription factors (Yna1 and Yna2 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27689333 [112]), which are different from the orthologous pathway-specific A. nidulans NirA and N. crassa NIT4 transcription factors. The regulatory patterns of A. nidulans and N. crassa are very similar, not withstanding the fact that no clustering is extant in N. crassa. Figure 4 compares the clusters of A. nidulans and P. angusta. I can see no obvious explanation for the assimilation of two novel transcription factor genes into the cluster of Pichia angusta. While horizontal transmission is a suitable explanation for the ancestral presence of the cluster, there is no clear rationale either for de-clustering or for assimilation of new genes into the cluster. The cluster has been functionally characterised in another member of the Saccharomycotina, Arxula (Blastobotrys) adeninivorans, where it includes two transporter genes but not the transcription factor genes [113]. Arxula is a basal clade of the Saccharomycotina [114], which supports a secondary clustering of the regulatory genes occurring after the divergence of Arxula and Pichia. Complete clustering of the nitrate assimilation pathway is found in yet another nitrate utilisingScazzocchio Fungal Biology and Biotechnology 2014, 1:7 http://www.fungalbiolbiotech.com/content/1/1/Page 13 ofFigure 4 Comparison of the nitrate assimilation gene cluster in A. nidulans and P. angusta. White, nitrate transporter, yellow nitrate reductase, blue nitrite reductase, green transcription factors. In A. nidulans a second.