The complete mitochondrial genome of fungal endosymbiont, Ophiocordycipitaceae sp., isolated from Ricania speculum (Hemiptera: Ricaniidae)

Abstract

Ricania speculum is called as black planthopper, belonging to the family Ricaniidae. We have determined a mitochondrial genome of fungal endosymbiont of R. speculum, belonging to the family Ophicocordycipitaceae. The circular mitogenome of this fungus is 66,785 bp including 32 protein-coding genes, 2 ribosomal RNA genes, and 19 transfer RNAs. Its GC ratio is 30.6%. A phylogenetic tree presented that this fungus is clustered with fungi of two genera, Hirsutella and Ophiocordyceps.

Keywords:

• Endosymbiont
• fungi
• mitochondrial genome
• Ophiocordycipitaceae
• Ricania speculum

Ricania speculum (Walker, 1851) is called as black planthopper belonging to the family Ricaniidae of the order Hemiptera. It has been known that insect species in this family contains fungal symbionts or pathogens (Mueller 2011). For example, Scolypopa australis has entomopathogen, Lecanicillium muscarium (Marshall et al. 2003) and Ricania japonica contains yeast-like symbionts (Hongoh and Ishikawa 2000). When we conducted de novo assembly using NGS raw reads obtained from a R. speculum sample collected in Jungtosa Template in Jeollabuk-do Province in Korea (35.6585365 N, 126.887853E; the specimen is stored in Gyeongsang National University, Korea, Accession number: 2018-HJ-56), a circular fungal mitogenome sequence was obtained together with a R. speculum mitogenome. There is no sequence variation on this fungal mitochondrial genome based on the alignment of raw reads, reflecting that the amount of this fungus inside R. speculum occupies relatively large and homogeneous group; it can be a fungal symbiont of R. speculum.

DNA was extracted using DNeasy Blood &Tissue Kit (QIAGEN, Hilden, Germany). Raw sequences obtained from Illumina HiSeq2000 (Macrogen Inc., Korea) were filtered by Trimmomatic 0.33 (Bolger et al. 2014) and de novo assembled by Velvet 1.2.10 (Zerbino and Birney 2008) and gaps were closed with SOAPGapCloser 1.12 (Zhao et al. 2011), BWA 0.7.17, and SAMtools 1.9 (Li et al. 2009; Li 2013). Geneious R11 11.1.5 (Biomatters Ltd, Auckland, New Zealand) was used to annotate mitochondrial genome with referring mitogenomes of Tolypocladium inflatum (NC_036382; Zhang et al. 2017) and Fusarium oxysporum (MN259514; Kwon et al. 2019).

The mitogenome of fungal symbiont of R. speculum (MT019333) is 66,785 bp long and its GC ratio is 30.3%. It contains 32 protein-coding genes (PCGs), 2 ribosomal RNA genes (rRNAs), and 19 transfer RNA genes (tRNAs). Number of tRNAs in this mitochondrial genome is smaller than the other fungal mitochondrial genomes missing three types of tRNAs: tRNA-Ala, tRNA-Gln, and tRNA-Tyr. Thirteen out of 32 PCGs are identified as endonuclease, which is one of the contributors to expand Aspergillus and Penicillium mitochondrial genomes (Joardar et al. 2012; Park et al. 2019).

We inferred the phylogenetic relationship of six representative mitogenomes belonging to the family Ophiocordycipitaceae with Fusarium graminearum (Nectriaceae) and Heterodermia speciosa (Physciaceae) as outgroup species. Multiple sequence alignment was conducted by MAFFT 7.450 (Katoh and Standley 2013) based on concatenated the 12 conserved genes selected based on annotation of the eight mitochondrial genomes. A maximum likelihood phylogenetic tree was constructed using MEGA X (Kumar et al. 2018). Phylogenetic trees present that our species is clustered with Hirsutella and Ophiocordyceps spp. (Figure 1; Simmons et al. 2015). While rRNA of this mitogenome displays that 85.94% identity against Tolypocladium inflatum as a best hit, which is incongruent with phylogenetic tree. This mitogenome is one of the examples which shows power of NGS technologies to understand unknown fungal symbionts in insect (e.g. aphid mitogenome from Nymphaea colodata genome (Park et al. accepted) and Stegobium paniceum mitogenome from the truffle (Tuber melanosporum) whole genomes (Park et al. in preparation) as well as contributing fungal mitogenome sequence resources for further diverse researches.

Figure 1. Maximum likelihood (1,000 bootstrap repeats) phylogenetic tree of 12 conserved genes, ATP6, ATP8, COB, COX1, COX2, COX3, NAD1, NAD3, NAD4, NAD4L, NAD5, and NAD6, originated from the six mitochondrial genomes of Ophiocordycipitaceae and two outgroup species: Ophiocordycipitaceae sp., Ophiocordyceps sinensis, Hirsutella rhossiliensis, Hirsutella thompsonii, Hirsutella minnesotensis, and Tolypocladium inflatum and Fusarium graminearum PH-1and Heterodermia speciose as two outgroup species. Gray line indicates family of six fungal species used in the tree. The numbers above branches indicate bootstrap support values of a maximum likelihood phylogenetic tree.

Disclosure statement

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

Additional information

Funding

This research was supported by “Cooperative Research Program for Agriculture Science & Technology Development [Project No. PJ013389012020]” Rural Development Administration, Republic of Korea.