DOI: 10.5061/dryad.nvx0k6dp7
title: Potential causes and consequences of rapid mitochondrial genome evolution in thermoacidophilic Galdieria (Rhodophyta)

description: The Cyanidiophyceae is an early-diverged red algal class that thrives in
 extreme conditions around acidic hot springs. Although this lineage has
 been highlighted as a model for understanding the biology of extremophilic
 eukaryotes, little is known about the molecular evolution of their
 mitochondrial genomes (mitogenomes). To fill this knowledge gap, we
 sequenced five mitogenomes from representative clades of Cyanidiophyceae
 and identified two major groups, here referred to as Galdieria-type
 (G-type) and Cyanidium-type (C-type). G-type mitogenomes exhibit the
 following three features: (i) reduction in genome size and gene inventory,
 (ii) evolution of unique protein properties including charge, hydropathy,
 stability, amino acid composition, and protein size, and (iii) distinctive
 GC-content and skewness of nucleotides. Based on GC-skew-associated
 characteristics, we postulate that unidirectional DNA replication may have
 resulted in the rapid evolution of G-type mitogenomes. The high
 divergence of G-type mitogenomes was likely driven by natural
 selection in the multiple extreme environments that Galdieria species
 inhabit combined with their highly flexible heterotrophic metabolism. We
 speculate that the interplay between mitogenome divergence and adaptation
 may help explain the dominance of Galdieria species in diverse extreme
 habitats.
description: Gene
 alignments were constructed using MAFFT version 7 with the default
 options. Using IQ-TREE with 1,000 ultrafast bootstrap replications,
 maximum likelihood (ML)-based phylogenetic analysis and bootstrap methods
 (MLB) were conducted. The model test option integrated in IQ-TREE
 automatically selected the evolutionary
 models. <b><i>            </i></b>The ‘Independent-Samples Kruskal-Wallis Test’ was used to evaluate hypotheses. The null hypothesis was that the distribution of factor (e.g., amino acid composition) is identical among taxa from two ingroup clades (i.e., <i>Cyanidium</i>-type and <i>Galdieria</i>-type) and outgroup taxa. If the null hypothesis is rejected, pairwise comparisons among three groups were applied. Sixteen conserved mitochondrial<b><i> </i></b>genes (<i>sdh</i>C, <i>atp</i>4, <i>atp</i>6, <i>atp</i>8, <i>atp</i>9, <i>cob</i>, <i>cox</i>1, <i>cox</i>2, <i>cox</i>3, <i>nad</i>1, <i>nad</i>2, <i>nad</i>3, <i>nad</i>4, <i>nad</i>4L, <i>nad</i>5, <i>nad</i>6) that all encoded in mitogenomes of 13 species were chosen to test amino acid properties. <b><i>            </i></b>The amino acid compositions between species of each gene were calculated using the CODEML program in the PAML4 package. The alignments of each gene and their ML trees were used for pairwise comparisons and all results were summarized in electronic supplementary material. Only conserved sites excluding any gaps were selected from the protein<b> </b>alignment of conserved genes for analysis. Total 3,845 sites were chosen from 4,825 sites of the concatenated protein alignment.<b> </b>The<b> </b>average proportions of each group’s 20 amino acids were measured, and values were visualized in bar plots with standard error bars.             To compare protein properties, GRAVY (grand average of hydropathy), aliphatic index, and instability index were calculated by ProtParam implied in ExPASy, and the results were summarized in electronic supplementary material. The mean values of each feature were illustrated by a bar plot (‘ggplot2’ in R) with standard error bars.             For the EGT genes and transit peptide prediction,<b><i> </i></b>nuclear genomes of <i>G. sulphuraria</i> 074W and <i>Cz. merolae</i> 10D were used as the reference genomes to find mitochondrion-derived nuclear-encoded genes. EGT genes were further verified based on homologous search by MMSeqs2 and KEGG pathway. Transit peptide sequences in EGT candidates were predicted by TargetP for their potential localizations.
description: Supplementary files: description of all files, alignment files (*.fasta, *.phy) and result files of phylogenetic analyses (*.treefile) used in figures are provided.