## 4 Evolution of Cadmium Transporters and Their Functions in Cadmium Accumulation
### Evolution of Cd Transporters
It is interesting that these two transporters underwent significantly different evolution courses. In the phylogenetic analysis, the orthologs of NRAMP5 proteins were grouped into two clusters (Figure 2A), with cluster 1 including green algae, Glaucophyta, Rhodophyta, and mosses, and cluster 2 containing green algae, hornnports, liverworts, lycophytes, ferns, gymnosperms, and angiosperms. Furthermore, cluster 2 was deeply branching. In the three sub-clusters of cluster 2, green algae formed a distinct sub-sub-cluster. The other two sub-clusters included lycophytes, ferns, and gymnosperms, while only the last sub-cluster included hornnports, liverworts, and angiosperms. In addition, the plants were clearly divided into two groups, monocots and eudicots, containing their specific ancestral gymnosperms and basal angiosperms (Figure 2A). It could be suggested that the orthologs of NRAMP5 in plants descended from a polyphyletic evolutionary lineage that originated in different ancestors. Furthermore, the divergent presence of green algae, lycophytes, ferns, and gymnosperms in the phylogenetic tree indicates a rampant occurrence of horizontal gene transfer during the evolution of the NRAMP5 orthologs, which has been previously evidenced in studies on the evolution of NRAMPs in bacteria [99] and OPTs in plants [97]. However, further studies are necessary to provide insight into the molecular mechanisms and adaptive roles of horizontal gene transfer events in the evolution of Cd transporters in plants. The evolution course of HMA2 is much simpler. The orthologs of HMA2 from algae, mosses, ferns, lycophytes, gymnosperms, and angiosperms formed a distinct cluster, but they were all basal to Rhodophyta species _Rhodocheate parvula_ (Figure 2B), thereby indicating that this transporter experienced an early evolution in plants. The angiosperm cluster was grouped into two sub-clusters consisting of monocots and eudicots, all of which were basal to _Amborella trichopoda_, which is the only living species in the sister lineage to all other flowering plants [100]. In addition, eudicots had a closer orthologous relationship with _Myristica fragrans_ (Figure 2B). These results indicate that the origin of the HMA2 transporter in both monocots and eudicots can be traced back to  
Figure 1: Similarity heat map of key membrane cadmium (Cd) transporters in different species. The Genesis software was used to estimate the similarities among protein sequences based on Table 1 and Table S1. Candidate protein sequences were selected by BLASTP searches that satisfied the following criteria: E value \(<10^{-10}\) and query coverage \(>50\%\). Colored squares indicate protein sequence similarity from 0 (blue) to 100% (red). White squares indicate that no homologous genes were found. _Amborella trichropoda_, which dates back to approximately 130 Mya. However, these species evolved separately thereafter and formed a monophyletic evolutionary lineage. Figure 2: Phylogenetic analysis of NRAMP5 (**A**) and HMA2 (**B**). The mRNA sequences of HMA2 and NRAMP5 were queried from the One Thousand Plant Transcriptome (oneKP) database [90]. The amino acid sequences of _Oryza sativa_ OsHMA2 and OsNRAMP5 were employed as the query sequences to access the transcriptome data with the criteria E-value \(<10^{-10}\) and coverage \(>50\%\), using BLASTP. The sequences were aligned with MAFFT, and the phylogenies were constructed with the online toolkit RAxML [101] of CIPRES [102]. Genes sampled from Chromia algae were used as the outgroup (in the shade of light grayish magenta) and the root of the tree, and the Interactive Tree of Life resource ([[http://www.itol.embl.de](http://www.itol.embl.de)]([http://www.itol.embl.de](http://www.itol.embl.de)), accessed on 24 February 2020) was used to annotate gene trees. Bootstraps (1–100) are displayed as the width of branches (1–10 px).