Multiple sequence alignments: Detection and isolation of a new member of Burkholderiaceae‑related endofungal bacteria from Saksenaea boninensis sp. nov., a new thermotolerant fungus in Mucorales

Methods:
Nucleotide sequences were aligned independently for each region using MAFFT v7.212 (Katoh and Standley, 2013). The obtained alignment blocks were subject to Gblocks 0.91b (Castresana, 2000) to remove poorly aligned positions with the relaxed selection setting described in Talavera & Castresana (2007) using the following parameters (-t = d -b2 = 9 -b3 = 10 -b4 = 5 -b5 = h). After automatically removing gaps, the alignment blocks were viewed using MEGA 6.06 software (Tamura et al., 2013) and poorly aligned positions at either end of the alignments were removed manually. Pairwise distances of the nucleotide sequences (ITS2, ITS1-5.8S-ITS2, LSU, and tef1) of the ex-type strains of seven Saksenaea spp. and the representative isolate S. boninensis Sak4 were calculated by MEGA 6.06 software (Tamura et al. 2013). Multiple sequence alignment of 16S rRNA gene of the family Burkholderiaceae was prepared for the phylogeny of a bacterial endosymbiont. Multiple sequence alignments of  ITS, LSU, and tef1 genes of Saksenaea spp. (Mucorales) were separately prepared for the phylogeny of a fungal host. Concatenated dataset of these genes were also prepared. All nucleotide sequences were retrieved from GenBank (See "Sequence_ID.csv" and taxon names of each alignment).  

Description of files:
A. Phylogeny of the family Burkholderiaceae (Bacterial endosymbiont):
1. Burkholderiaceae_16S_RAW.fasta
Non-aligned dataset of 16S rRNA gene of the family Burkholderiaceae.

2. Burkholderiaceae_16S_aligned.fasta
Aligned dataset of 16S rRNA gene of the family Burkholderiaceae.

B. Phylogenies of Saksenaea spp. (Fungal host):
1. Sequence_ID_v2.csv
Taxon names, accession numbers, and sequence ID for the concatenated multiple sequence alignment are listed.

2. Saksenaea_ITS_RAW_v2.fasta
Non-aligned dataset of ITS1-5.8S-ITS2 region of Saksenaea spp.  

3. Saksenaea_ITS_aligned_v2.fasta
Aligned dataset of ITS1-5.8S-ITS2 region of Saksenaea spp. Only used for ITS1-5.8S-ITS2 phylogeny.

4. Saksenaea_LSU_RAW_v2.fasta
Non-aligned dataset of LSU gene region of Saksenaea spp.  

5. Saksenaea_LSU_aligned_v2.fasta
Aligned dataset of LSU gene region of Saksenaea spp. Only used for LSU phylogeny.

6. Saksenaea_tef1_RAW_v2.fasta
Non-aligned dataset of tef1 gene region of Saksenaea spp.  

7. Saksenaea_tef1_aligned_v2.fasta
Aligned dataset of tef1 gene region of Saksenaea spp. Only used for tef1 phylogeny.

8. Saksenaea_ITS2_for_concatenated_RAW_v2.fasta
Non-aligned dataset of ITS2 region of Saksenaea spp. used for preparation of a concatenated dataset 1.

9. Saksenaea_ITS2_for_concatenated_aligned_v2.fasta
Aligned dataset of ITS2 region of Saksenaea spp. used for preparation of a concatenated dataset 1.

10. Saksenaea_LSU_for_concatenated_RAW_v2.fasta
Non-aligned dataset of LSU gene region of Saksenaea spp. used for preparation of concatenated datasets 1 and 2.

11. Saksenaea_LSU_for_concatenated_aligned_v2.fasta
Aligned dataset of LSU gene region of Saksenaea spp. used for preparation of concatenated datasets 1 and 2.

12. Saksenaea_tef1_for_concatenated_RAW_v2.fasta
Non-aligned dataset of tef1 gene region of Saksenaea spp. used for preparation of concatenated datasets 1 and 2.

13. Saksenaea_tef1_for_concatenated_aligned_v2.fasta
Aligned dataset of tef1 gene region of Saksenaea spp. used for preparation of concatenated datasets 1 and 2.

14. Saksenaea_ITS2_LSU_tef1_concatenated_dataset1.fasta
Concatenated dataset of three multiple sequence alignments (9, 11, and 13). This concatenated dataset was used for the main phylogeny of Saksenaea spp.

15. Saksenaea_ITS_for_concatenated_RAW_v2.fasta
Non-aligned dataset of ITS1-5.8S-ITS2 region of Saksenaea spp. used for preparation of a concatenated dataset 2.

16. Saksenaea_ITS_for_concatenated_aligned_v2.fasta
Aligned dataset of ITS1-5.8S-ITS2 region of Saksenaea spp. used for preparation of a concatenated dataset 2.
Blank sequences were inserted for five isolates of Saksenaea longicolla after the alignment.

17.Saksenaea_ITS_LSU_tef1_concatenated_dataset2.fasta
Concatenated dataset of three multiple sequence alignments (15, 11, and 13). This concatenated dataset was used for the main phylogeny of Saksenaea spp.

C. Pairwise distances of the ex-type strains of Saksenaea spp.
1. Saksenaea_ITS_type_RAW.fasta
Non-aligned dataset of ITS1-5.8S-ITS2 region of the ex-type strains of Saksenaea spp.

2. Saksenaea_ITS2_type_aligned.fasta
Aligned dataset of ITS2 region of the ex-type strains of Saksenaea spp.

3.Saksenaea_ITS_type_aligned.fasta
Aligned dataset of ITS1-5.8S-ITS2 region of the ex-type strains of Saksenaea spp. without Saksenaea longicolla.

4. Saksenaea_LSU_type_RAW.fasta
Non-aligned dataset of LSU gene region of the ex-type strains of Saksenaea spp.

5. Saksenaea_LSU_type_aligned.fasta
Aligned dataset of LSU gene region of the ex-type strains of Saksenaea spp.

6. Saksenaea_tef1_type_RAW.fasta
Non-aligned dataset of tef1 gene region of the ex-type strains of Saksenaea spp.

7. Saksenaea_tef1_type_aligned.fasta
Aligned dataset of tef1 gene region of the ex-type strains of Saksenaea spp.