Adaptive molecular evolution of the two-pore channel 1 gene TPC1 in the karst-adapted genus Primulina (Gesneriaceae)

Abstract

Background and aims: Limestone karst areas possess high floral diversity and endemism. The genus Primulina, which contributes to the unique calcicole flora, has high species richness and exhibit specific soil-based habitat associations that are mainly distributed on calcareous karst soils. The adaptive molecular evolutionary mechanism of the genus to karst calcium-rich environments is still not well understood. The Ca²⁺-permeable channel TPC1 was used in this study to test whether its gene is involved in the local adaptation of Primulina to karst high-calcium soil environments.

Methods: Specific amplification and sequencing primers were designed and used to amplify the full-length coding sequences of TPC1 from cDNA of 76 Primulina species. The sequence alignment without recombination and the corresponding reconstructed phylogeny tree were used in molecular evolutionary analyses at the nucleic acid level and amino acid level, respectively. Finally, the identified sites under positive selection were labelled on the predicted secondary structure of TPC1.

Key results: Seventy-six full-length coding sequences of Primulina TPC1 were obtained. The length of the sequences varied between 2220 and 2286 bp and the insertion/deletion was located at the 5' end of the sequences. No signal of substitution saturation was detected in the sequences, while significant recombination breakpoints were detected. The molecular evolutionary analyses showed that TPC1 was dominated by purifying selection and the selective pressures were not significantly different among species lineages. However, significant signals of positive selection were detected at both TPC1 codon level and amino acid level, and five sites under positive selective pressure were identified by at least three different methods.

Conclusions: The Ca²⁺-permeable channel TPC1 may be involved in the local adaptation of Primulina to karst Ca²⁺-rich environments. Different species lineages suffered similar selective pressure associated with calcium in karst environments, and episodic diversifying selection at a few sites may play a major role in the molecular evolution of Primulina TPC1.

Keywords: Ca2+-permeable channel; Primulina; TPC1; calcium; karst; molecular evolution; positive selection.

Fig. 1.

Bayesian phylogenetic tree of TPC1 from Primulina species. Bayesian posterior probabilities above 0·5 are labelled at the nodes.

Fig. 2.

Sliding window plots of the Z-scores of radically changed amino acid properties showing regions under positive destabilizing selection (Z-score values >3·09) in Primulina TPC1.

Fig. 3.

Kyte–Doolittle hydropathy plots for AtTPC1 and PhTPC1. aa, amino acid.

Fig. 4.

Topology model of P. heterotricha TPC1 (PhTPC1) secondary structure and the location of the potential positive selection sites. Red and blue sites represent amino acids that evolved under positive selection, and the sites identified by at least three methods are shown in red. EF hand domains are shown as squares. Insertion or deletion regions in the N-terminal region are shown as diamonds. The codon number of each positively selected site is marked near the dotted line. Numbers without brackets correspond to the positions of positively selected sites in the sequence alignment used in selection analyses, and numbers within brackets correspond to the positions of positively selected sites in full-length sequences of PhTPC1.

Fig. 5.

Amino acids in the same evolutionary positions showing strong selection signatures at the nucleic acid and amino acid levels. Results obtained with PAML (M8), SLAC, FEL, MEME, FUBAR and TreeSAAP (at least one property under selection) are marked with a black box at sites showing positive selection. Sites with at least three properties under selection in TreeSAAP are marked with an asterisk. Background colours represent amino acid properties: polar positive (yellow), polar negative (red), polar neutral (green), non-polar neutral (purple), non-polar aliphatic (blue) and non-polar aromatic (pink).The three typical soil types – karst, Danxia and acid soils – where the species grow are marked with circles, triangles and squares, respectively.