Mitochondrial genome of Salix cardiophylla and its implications for infrageneric division of the genus of Salix

Abstract Salix cardiophylla was a member of the genus of Salix in family Salicaceae with unique morphological traits, and once recognized as a separate genus, Toisusu Kimura. Here, we sequenced and assembled the complete mitochondrial genome of S. cardiophylla, which was 735,173 bp in length, including 56 genes, 28 protein-coding genes, 3 rRNA genes, 25 tRNA genes, and one large inverted repeat regions with length of 13,603 bp. Phylogenetic analysis based on 26 mitochondrial CDS confirmed that S. cardiophylla is a member of Salix, and support its merge into Salix in aspect of our new insights on mitogenome phylogenomics.

Salix cardiophylla Trautv. & C.A.Mey. is deciduous tree up to 20 m high found in north to central Honshu and southern Kurils of Japan, growing along the gravel river. Salix cardiophylla belongs to the genus Salix (family Salicaceae), because of its unique reproductive organs, such as the pendulous female catkins and the deciduous styles, it was once treated as a separate genus, i.e., Toisusu Kimura (Barkalov and Kozyrenko 2014). The genus Toisusu was then merged into Salix afterwards based on plastid and nuclear sequence evidence (Azuma et al. 2000;Ohashi 2001;Chen et al. 2010). However, merge of Toisusu into Salix based on phylogentic inference of mitochondrial sequence is lack.
Here, we used a modified CTAB method (Sahu et al. 2012), the complete mitochondrial genomic DNA was extracted from young leaves of S. cardiophylla which grown in Honshu, Japan (36 N, 138 E). Voucher specimens were stored in the Herbarium of Kunming Institute of Botany (Accession no: T. Tanaka s.n., cult.). The genomic library for Illumina paired-end (PE) sequencing was constructed using the Illumina Hiseq X Ten sequencer. A total of ca. 31.2 million generated and this sequence has been deposited in the NCBI Sequence Read Archive (SRA) with accession number SRR12534676. MiSeq raw reads were assembled via NOVOPlasty v2.7.2 with a kmer of 39 (Dierckxsens et al. 2016). Taking the S. paraflabellaris mitogenome as the reference sequence Huang et al. 2019), the annotation of genome assembled was carried out with Geneious v2020.1.1 software (Kearse et al. 2012).
To explore the controversial systematic position of S. cardiophylla, four Salix species and two outgroup Populus species with mitogenome sequence were downloaded from GenBank (Kersten et al. 2016;Wei et al. 2016;Ye et al. 2017;Choi et al. 2017;Chen et al. 2019;Huang et al. 2019). Ultimately, 26 mitochondrial CDS shared by all these seven species were aligned by software MAFFT v7.47 (Katoh and Standley 2013). After manual adjustment, the maximum likelihood (ML) tree was constructed using the software IQ_TREE 1.6.2 (Nguyen et al. 2015) based on an array with the aligned seven sequences, branch support was estimated by bootstrap value and SH-like approximate likelihood ratio (SHAlrt) (Guindon et al. 2010) with 10,000 replicates under HKY þ F model according to Bayesian information criterion by the software Model Finder (Kalyaanamoorthy et al. 2017). Our ML revealed that five Salix species formed a robust monophyletic clade, confirmed that S. cardiophylla is a member of Salix and support the treatment of merging it back into Salix based on our new insights from mitogenome phylogenomic inference (Figure 1). This work provides a valuable source of data for the study of the controversial systematic position of S. cardiophylla.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
The research was supported by NSFC, grant No. [31960050, 31560062] and the 2019 Major Project on Biodiversity Conservation of Ministry of Ecology and Environment.

Data availability statement
The data that support the findings of this study are openly available in NCBI at https://www.ncbi.nlm.nih.gov, reference number MT806745, SRR12534676.