Sequence alignment and phylogenetic analysis

The sequences used for alignments and phylogenetic trees were obtained in Phytozome database (http://phytozome.jgi.doe.gov) except for P. taeda that were obtained from Congenie database (http://congenie.org/). P. pinaster AS1 [GenBank:{"type":"entrez-protein","attrs":{"text":"ADU02856","term_id":"315272542","term_text":"ADU02856"}}ADU02856]; and AS2 [GenBank:{"type":"entrez-protein","attrs":{"text":"ADK13052","term_id":"300432594","term_text":"ADK13052"}}ADK13052] protein sequences were obtained from GenBank at the NCBI. For P. pinaster AS3 [PGC:geneCapture_all_rep_c7631] and AS5 [PGC:geneCapture_all_rep_c8956/geneCapture_all_rep_c10521 we used sequences obtained in the course of this work and deposited in the Pine Gene Capture database (PGC, http://www.scbi.uma.es/pgc/). Finally for P. pinaster AS4 [SPDB: sp_v3.0_unigene97582/sp_v3.0_unigene8248] we used the sequence obtained from our transcriptomic database SustainPineDB [20] (http://www.scbi.uma.es/sustainpinedb/).

The CLUSTALW program was used for sequence alignments [50]. The phylogenetic tree was constructed with full-length AS amino acid sequences using the neighbor-joining method [51] with 1000 bootstrap replications. The evolutionary distances were computed using the JTT matrix-based method [52] and are in the units of the number of amino acid substitutions per site. The rate variation among sites was modeled with a gamma distribution (shape parameter = 1). All ambiguous positions were removed for each sequence pair. The positions not presented in all the sequences were eliminated. Finally there were a total of 509 positions in the final dataset. All of these analyses were conducted in MEGA6 [53].