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.