International Journal of Genomics
Volume 2021 (2021), Article ID 6652508, 20 pages
https://doi.org/10.1155/2021/6652508

Research Article

A Comparative Analyses of the Complete Mitochondrial Genomes of Fungal Endosymbionts in Sogatella furcifera, White-Backed Planthoppers

Nak Jung Choi,¹ Hong Xi,2,3 and Jongsun Park2,3

¹Crop Foundation Division, National Institute of Crop Science, RDA, Wanju, Republic of Korea
²InfoBoss Inc., 301 room, 670, Seolleung-ro, Gangnam-gu, Seoul, Republic of Korea
³InfoBoss Research Center, 301 room, 670, Seolleung-ro, Gangnam-gu, Seoul, Republic of Korea

Correspondence should be addressed to Nak Jung Choi; njchoi@korea.kr and Jongsun Park; starflr@infoboss.co.kr

Received 29 December 2020; Revised 3 April 2021; Accepted 8 May 2021; Published 9 June 2021

Academic Editor: Ertugrul Filiz

Copyright © 2021 Nak Jung Choi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Sogatella furcifera Horvath, commonly known as the white-backed planthoppers (WBPH), is an important pest in East Asian rice fields. Fungal endosymbiosis is widespread among planthoppers in the infraorder Fulgoromorpha and suborder Auchenorrhyncha. We successfully obtained complete mitogenome of five WBPH fungal endosymbionts, belonging to the Ophiocordycipitaceae family, from next-generation sequencing (NGS) reads obtained from S. furcifera samples. These five mitogenomes range in length from 55,390 bp to 55,406 bp, which is shorter than the mitogenome of the fungal endosymbiont found in Ricania speculum, black planthoppers. Twenty-eight protein-coding genes (PCGs), 12 tRNAs, and 2 rRNAs were found in the mitogenomes. Two single-nucleotide polymorphisms, two insertions, and three deletions were identified among the five mitogenomes, which were fewer in number than those of four species of Ophiocordycipitaceae, Ophiocordyceps sinensis, Hirsutella thompsonii, Hirsutella rhossiliensis, and Tolypocladium inflatum. Noticeably short lengths (up to 18 bp) of simple sequence repeats were identified in the five WBPH fungal endosymbiont mitogenomes. Phylogenetic analysis based on conserved PCGs across 25 Ophiocordycipitaceae mitogenomes revealed that the five mitogenomes were clustered with that of R. speculum, forming an independent clade. In addition to providing the full mitogenome sequences, obtaining complete mitogenomes of WBPH endosymbionts can provide insights into their phylogenetic positions without needing to isolate the mtDNA from the host. This advantage is of value to future studies involving fungal endosymbiont mitogenomes.

1. Introduction

Sogatella furcifera Horvath commonly known as the white-backed planthopper (WBPH) is a planthopper belonging to the infraorder Fulgoromorpha [1] and suborder Auchenorrhyncha [2]. It has migrated to temperate climates from subtropical regions and become a major pest in rice fields across East Asia [3–6]. In particular, migration from China to Japan via Korean peninsula has highlighted the extent of its spread across the region [7]. Sogatella furcifera has already been registered in the National Species List of Korea [8] indicating that this species has been frequently found within the country. It damages rice plants by feeding directly on them, producing a characteristic symptom, hopper burn [9]. Because of the importance of WBPH as a threat to agriculture, the mitochondrial genome (mitogenome) as well as whole genome sequences of S. furcifera has been sequenced successfully [10, 11]. The fundamental background of WBPH genomic research is, therefore, well established. For example, the complete genome sequence of the Cardinium bacterial endosymbiont of S. furcifera was also completed from the same raw reads generated by the whole genome project [12]. Another bacterial endosymbiont of WBPH, Wolbachia, which alters host reproductions by parthenogenesis, feminization, male-killing, and induction of cytoplasmic incompatibility in arthropods [13], also causes the cytoplasmic incompatibility in WBPH together with Cardinium endosymbiont [14].

Besides these bacterial endosymbionts, fungal endosymbiont has been identified using PCR method in planthopper, Ricania japonica [15]. This yeast-like endosymbiont uses the enzyme uricase to recycle uric acid secreted by the host species, assisting in metabolic processes [15]. In addition, yeast-like symbionts have been identified in Nilaparvata lugens, a brown planthopper [16, 17] which also support the host’s uric acid metabolism [18]. However, there was no sequence information of this endosymbiont until the complete fungal mitogenome was obtained from the raw reads of Ricania speculum, a black planthopper [19]. This mitogenome was identified as an Ophiocordycipitaceae species by comparing already known several complete mitogenomes in this family [19]. This result suggests that next-generation sequencing technology that provides a large number of short reads can be used to provide evidence for the existence of endosymbiont species using DNA extracted from insect species. These results draw comparison to previous studies that have successfully identified a multiple number of complete organelle or bacterial genomes from one NGS library [12, 19–37].

Here, we reported the first complete mitogenomes of fungal WBPH endosymbiont from five WBPH samples isolated in Korea and China. The five mitogenomes display 55,390 to 55,406 bp in length, shorter than that of R. speculum [19]. The numbers of intraspecific variations among the five mitogenomes are fewer in number than those of the four Ophiocordycipitaceae species. Phylogenetic analysis based on conserved PCGs across Ophiocordycipitaceae mitogenomes displays that the five mitogenomes were clustered with that of R. speculum, forming an independent clade. Once additional planthopper fungal endosymbiont mitogenomes become available, their phylogenetic relationships as well as evolutionary histories based on their complete mitogenomes will become clearer.

2. Materials and Methods

2.1. DNA Preparation and Genome Sequencing of Four WBPH Samples

All four WBPH samples were captured at two places in Korea (Table 1). One individual of WBPH was frozen with liquid nitrogen using 1.5 ml microtube and then was ground using a plastic pestle. The Quick-DNA Miniprep Plus Kit (Zymo Research, USA) was used for extracting DNA. Genome sequencing was performed using NovaSeq6000 at Macrogen Inc., Korea, from the extracted DNA of four WBPH samples with constructing a 350 bp pair-end library.

Table 1: List of five WBPH fungal endosymbiont mitogenomes.

No.
Name
Sample location
NCBI accession
Length (bp)
GC ratio (%)
No. of PCGs
No. of tRNAs
No. of rRNAs
Reference

1
KR
321-2, Daesong-ri, Geumnam-myeon, Hadong-gun, Gyeongsangnam-do, Republic of Korea
MW115131
55,393
30.7
28
16
2
This study

2
KR.1D
1411-6, Wolga-ri, Gunnae-myeon, Jindo-gun, Jeollanam-do, Republic of Korea
MW373710
55,406
30.7
28
16
2
This study

3
KR.5D
MW373711
55,390
30.7
28
16
2
This study

4
KR.11D
MW376862
55,393
30.7
28
16
2
This study

5
WGS
University of Science and Technology of China, Anhui province, China
BK059186
55,393
30.7
28
16
2
SRR3954848 [11]

2.2. Assembly and Annotation of the Five Fungal WBPH Endosymbiont Mitogenomes

De novo assembly, with confirmation, was accomplished with Velvet v1.2.10 [38] after filtering raw reads using Trimmomatic v0.33 [39]. After obtaining mitogenome contig sequences with the condition that sequence coverage is more than 60x, gaps were filled with GapCloser v1.12 [40], and all bases from the assembled sequences were confirmed by checking each base in the alignment (tview mode in SAMtools v1.9 [41]) against the assembled mitogenome generated with BWA v0.7.17 [42]. The circular form of mitogenomes was confirmed by the pair-end reads connecting both sides of mitogenomes. All these bioinformatic analyses were conducted under the environment of the Genome Information System (GeIS; http://geis.infoboss.co.kr/) like the previous studies of mitogenomes [19, 21–24, 26, 28, 30, 32, 33, 36, 43–91].

Geneious Prime® 2020.2.4 (Biomatters Ltd, Auckland, New Zealand) was used for mitogenome annotation with referring to the mitogenome of R. speculum fungal endosymbiont (NC_049089) [19] by transferring annotations while correcting exceptional cases, including missing start or stop codons. Also, the “FindORF” function in Geneious Prime® 2020.2.4 together with BLAST v2.2.24 [92] was also utilized to find novel PCGs including LAGLIDADG endonucleases. tRNAs were predicted and confirmed using tRNAScan-SE v2.0.6 [93].

2.3. Identification of Sequence Variations on the Complete Mitogenomes of WBPH Fungal Endosymbionts

Single-nucleotide polymorphisms (SNPs) and insertions and deletions (INDELs) were identified using the “Find variations/SNP” function implemented in Geneious Prime® 2020.2.4 (Biomatters Ltd, Auckland, New Zealand) based on multiple sequence alignment of the five mitogenomes of WBPH fungal endosymbionts conducted by MAFFT v7.450 [94]. Each identified variation was manually checked to understand which mitogenome has them.

2.4. Identification of Simple Sequence Repeats (SSRs)

Simple sequence repeats (SSRs) were identified on the mitogenome sequence using the pipeline of the SSR database (SSRDB; http://ssr.pe.kr/; Park et al., in preparation). Based on the conventional definition of an SSR on an organelle genome, monoSSR (1 bp) to hexaSSR (6 bp), the total length of SSRs on the mitogenome exceeds 10 bp. Owing to the different criteria of SSRs on organelle genomes [95–101], we adopted the criteria used in various organelle genome analyses [21, 44, 102–104], as follows: the monoSSR (unit sequence length of 1 bp) to hexaSSR (6 bp) are used as normal SSRs, and heptaSSR (7 bp) to decaSSR (10 bp) are defined as extended SSRs. Among the normal SSRs, pentaSSRs and hexaSSRs for which the number of unit sequences is 2 are classified as potential SSRs.

2.5. Construction of Phylogenetic Trees

Five conserved PCGs, including ATP8, CO2, NAD3, NAD4, and NAD4L, from 26 fungal mitogenomes including the five mitogenomes assembled in this study and one outgroup species, Fusarium graminearum, were aligned independently using MAFFT v7.450 [94] and concatenated using the Perl script, one of the component of GenomeArchive® (http://www.genomearchive.info) [105]. The model test was conducted with jModelTest v2.1.5 [106]. The neighbor-joining (NJ) and maximum-likelihood (ML) trees were reconstructed in MEGA X [107]. In the ML analysis, a heuristic search was used with nearest-neighbor interchange (NNI) branch swapping, general time reversible (GTR) model, and uniform rates among sites. All other options used the default settings. Bootstrap analyses with 1000 pseudoreplicates were conducted with the same options. The posterior probability of each node was estimated by Bayesian inference (BI) using the MrBayes v3.2.7 [108] plug-in implemented in Geneious Prime® 2020.2.4. The HKY85 model with gamma rates was used as a molecular model. A Markov chain Monte Carlo (MCMC) algorithm was employed for 1,100,000 generations, sampling trees every 200 generations, with four chains running simultaneously. Trees from the first 100,000 generations were discarded as burn-in.

3. Results and Discussions

3.1. Complete Mitogenome of Fungal WBPH Endosymbionts

We successfully assembled fungal endosymbiont mitogenomes from four WBPH samples isolated in Korea and China and one public dataset of NGS raw reads (Table 1). This is the first WBPH fungal endosymbiont mitogenome identified. Their lengths ranged from 55,390 bp to 55,406 bp (Table 1), which is shorter than that of R. speculum (66,785 bp) [19]. In these mitogenomes, there were 28 protein-coding genes (PCGs), 12 tRNAs, and 2 rRNAs (Table 2). Some of the PCGs found were LAGLIDADG endonucleases, which are usually found in intronic regions of various fungal mitogenomes, contributing to the expansion of their length [19, 55, 108–112]. In comparison to the previously sequenced mitogenome of the fungal endosymbiont of R. speculum, there were slightly fewer PCGs and tRNAs found in the WBPH endosymbiont mitogenomes. There were three fewer PCGs for three reasons: the smaller number of LAGLIDADG endonucleases, the absence of one endonuclease and a GIY-YIG endonuclease, and the presence of two additional PCGs—a hypothetical protein and a LAGLIDADG/HNH endonuclease. This particular configuration of PCGs is usually identified in other fungal mitogenomes; for example, two mitogenomes of Fusarium oxysporum (GenBank accessions are MN259514 and MN259515) display two completely different PCGs in each mitogenome [54, 56]. There are also five fewer tRNAs because of the different configurations: tRNA-Asp, tRNA-Cys, tRNA-Ile, and two tRNA-Ser (also found in the mitogenome of the fungal symbiont of R. speculum [19]). This difference in configuration of tRNAs between two different fungal symbionts suggests that tRNA configuration may not be critical because essential tRNAs absent in the fungal mitogenome can be supported from the nuclear genome [113].

Table 2: List of genes in WBPH fungal endosymbiont mitogenomes.

No.
Name
Type
Start position
End position
Length (bp)
Strand
No. of exons

1
Cytochrome b
CDS
160
8182
975
Reverse
5

2
LAGLIDADG/HNH endonuclease
CDS
7429
7776
348
Reverse
1

3
NADH dehydrogenase subunit 5
CDS
8432
10,405
1974
Reverse
1

4
NADH dehydrogenase subunit 4 L
CDS
10,405
10,680
276
Reverse
1

5
Cytochrome c oxidase subunit II
CDS
10,896
13,952
894
Reverse
6

6
ATP synthase F0 subunit c
CDS
14,052
14,237
186
Reverse
1

7
NADH dehydrogenase subunit 3
CDS
14,315
14,968
447
Reverse
2

8
LAGLIDADG endonuclease
CDS
14,683
14,880
198
Reverse
1

9
NADH dehydrogenase subunit 2
CDS
14,969
18,193
1431
Reverse
2

10
Hypothetical protein
CDS
15,626
16,390
765
Reverse
1

11
tRNA-Met
tRNA
18,210
18,283
74
Reverse
1

12
tRNA-His
tRNA
18,325
18,398
74
Reverse
1

13
tRNA-Leu
tRNA
18,485
18,569
85
Reverse
1

14
tRNA-Lys
tRNA
18,570
18,642
73
Reverse
1

15
tRNA-Phe
tRNA
18,643
18,715
73
Reverse
1

16
tRNA-Leu
tRNA
19,673
19,755
83
Reverse
1

17
tRNA-Met
tRNA
19,758
19,830
73
Reverse
1

18
tRNA-Glu
tRNA
19,904
19,976
73
Reverse
1

19
Large subunit rRNA
rRNA
20,067
27,337
4793
Reverse
3

20
Ribosomal protein S3
CDS
20,672
21,916
1245
Reverse
1

21
tRNA-Pro
tRNA
27,400
27,471
72
Reverse
1

22
NADH dehydrogenase subunit 6
CDS
27,677
28,441
765
Reverse
1

23
tRNA-Gly
tRNA
28,544
28,614
71
Reverse
1

24
Cytochrome c oxidase subunit III
CDS
29,224
32,387
795
Reverse
3

25
Intron-encoded endonuclease aI1
CDS
29,932
30,138
207
Reverse
1

26
Hypothetical protein
CDS
30,395
30,664
270
Reverse
1

27
Alpha-beta-hydrolase
CDS
30,423
30,686
264
Forward
1

28
tRNA-Asn
tRNA
32,437
32,508
72
Reverse
1

29
Small subunit rRNA
rRNA
34,254
35,615
1362
Reverse
1

30
ATP synthase F0 subunit a
CDS
35,918
38,001
762
Reverse
2

31
ATP synthase F0 subunit 8
CDS
38,061
38,237
177
Reverse
1

32
NADH dehydrogenase subunit 4
CDS
38,620
40,035
1416
Reverse
1

33
NADH dehydrogenase subunit 1
CDS
40,119
42,582
1107
Reverse
4

34
tRNA-Arg
tRNA
42,726
42,799
74
Reverse
1

35
Cytochrome c oxidase subunit I
CDS
42,809
55,293
1314
Reverse
7

36
LAGLIDADG endonuclease
CDS
43,660
44,880
1221
Reverse
1

37
LAGLIDADG endonuclease
CDS
45,125
46,054
930
Reverse
1

38
LAGLIDADG endonuclease
CDS
46,857
48,248
1392
Reverse
1

39
LAGLIDADG endonuclease
CDS
48,620
49,483
864
Reverse
1

40
LAGLIDADG endonuclease
CDS
49,525
50,814
1290
Reverse
1

41
LAGLIDADG endonuclease
CDS
50,865
51,734
870
Reverse
1

42
Hypothetical protein
CDS
54,290
54,850
561
Forward
1

Several PCGs in the fungal mitogenomes have been invaded by introns multiple times. For example, COX1 contains three introns, and COB has five introns in the Hirsutella thompsonii mitogenome [114]. This phenomenon contributes to increased fungal mitogenome: Aspergillus pseudoglaucus and Aspergillus egyptiacus are longer than the other Aspergillus mitogenomes because of the presence of many introns on major PCGs [55, 115]. The fungal mitogenomes examined in this study also present many introns on PCGs including COB, COX1, NAD1, ATP8, COX3, COX2, and NAD2 (Figure 1), which is a major reason for the expansion of fungal mitogenomes together with endonucleases.

Figure 1: Complete mitogenome of WBPH fungal endosymbionts. Blue circle indicates fungal endosymbiont mitogenome, yellow arrows are protein-coding genes, purple arrows are tRNAs, and red arrows mean rRNAs. Direction of arrows indicates direction of transcription. Each gene name was displayed with lines directing to the corresponded arrows. Numbers displayed outside of blue circle mean base pair.

The gene order of WBPH and R. speculum fungal symbiont mitogenomes was the same when PCGs except endonucleases and rRNAs are considered. However, intron structures of COX1, COX2, NAD2, NAD3, NAD5, and ATP synthase F0 subunit present different configurations between the two mitogenomes (Figure 2). The intron structures of NAD5 and NAD2 present reduce of a reduction in the number of exons via removal of intron regions in the WBPH fungal endosymbiont mitogenome (Figures 2(a) and 2(d)), whereas those of COX2, NAD3, and the ATP synthase F0 subunit display insertions of one intron into the WBPH fungal endosymbiont mitogenome (Figures 2(b), 2(c), and 2(e)). This indicates that the reduction in the total length of the WBPH fungal symbiont mitogenome is not primarily caused by reducing the number of exons, unlike in Aspergillus mitogenomes [55, 116]. In addition, COX1, which contains the largest number of exons in these mitogenomes, lost the sixth and seventh exons of the R. speculum fungal endosymbiont mitogenome in the mitogenome of WBPH endosymbiont (Figure 2(f)). However, the total length of COX1, including the introns of WBPH fungal endosymbionts, is longer than that of R. speculum fungal endosymbionts by 1 kb (Figure 2(f)), reflecting complex events that occurred during the evolution of both mitogenomes. Additional studies are required to identify the correct exons of the COX1 gene of this fungal endosymbiont. For example, alignment of RNA-Seq raw reads against this mitogenome could provide expressed regions in this mitogenome.

(a)

(b)

(c)

(d)

(e)

(f)

Figure 2: Schematic diagram of exon/intron structure of protein-coding genes displaying different configurations between fungal endosymbiont mitogenomes of WBPH and R. speculum. Orange-colored diagrams indicate components of WBPH fungal endosymbionts, and blue-colored diagrams are those of R. speculum fungal endosymbionts. Exon/intron structures of (a) NAD5, (b) COX2, (c) NAD3, (d) NAD2, (e) ATP synthase F0 subunit, and (f) COX1 are displayed.

Once more fungal symbiont mitogenomes are available, patterns of presence and absence of tRNAs, additional endonucleases, and intron structures of PCGs in endosymbiont mitogenomes will elucidate a detailed evolutionary history of these genes.

3.2. Identification of Intraspecific Variations on Fungal WBPH Endosymbiont Mitogenomes

We identified two SNPs, three insertions, and two deletions via multiple sequence alignments of the five fungal mitogenomes (Table 3). One of two SNPs was identified in KR.5D WBPH and changed leucine (L) to glutamine (Q) in the ATP synthase F0 subunit (Table 3). One 10 bp insertion in the intergenic space was found in KR.1D WBPH, while the remaining two insertions and all three deletions were 1 to 3 bp in length (Table 3).

Table 3: List of intraspecific variations identified on the five WBPH fungal endosymbiont mitogenomes.

No.
Type
Coordination of multiple sequence alignments
Strains
Base changes
Position

1
Insertion
4209-4210
KR.1D, KR.5D
- to CC
Intergenic

2
Insertion
27,476-27,486
KR.1D
- to TGGGCCCCCC
Intergenic

3
SNP
27,487
KR.1D
A to C
Intergenic

4
Deletion
32,727-32,728
KR.5D
CC to -
Intergenic

5
SNP
37,574
KR.5D
A to T
L to Q in ATP synthase F0 subunit

6
Insertion
38,727
KR.1D
- to G
Intergenic

7
Deletion
38,728-38,730
KR.5D
GGG to -
Intergenic

The proportions of these intraspecific SNPs, insertions, and deletions in these fungal mitogenomes were 0.0036%, 0.020%, and 0.012%, respectively. The proportion of insertions and deletions was higher than that of SNPs. Interestingly, there is geographical variation in the fungal symbiont mitogenomes. The mitogenome of WBPH endosymbionts used in the whole genome sequencing (WGS) and the KR.11D isolate were identical to that of KR, while the other three WBPHs captured in other locations in Korea displayed intraspecific variations. The sample used in the WGS originated from the University of Science and Technology of China (Anhui province, China), indicating that KR 11D and KR WBPH samples obtained in Korea have migrated from the similar region to the WGS sample. However, further analyses of their complete mitogenomes or whole genomes will be needed to provide more supportive data for identifying their origins.

There is a relatively small number of intraspecific SNPs and INDELs identified from these fungal mitogenomes in comparison to those of other fungal mitogenomes, for example, 16 to 17 SNPs (0.055% to 0.0582%) and 22 to 27 INDELs (0.075% to 0.092%) on Aspergillus flavus [52, 53] and 62 SNPs (0.15%) and 181 INDELs (0.43%) on Fusarium oxysporum f.sp. lactucae [56]. They are also fewer than those identified in insect mitogenomes [10, 22, 23, 43, 45–51].

Based on 25 available complete fungal mitogenomes in Ophiocordycipitaceae, four species, Ophiocordyceps sinensis, Hirsutella thompsonii, Hirsutella rhossiliensis, and Tolypocladium inflatum, contain more than one complete fungal mitogenome (Table 4). We investigated intraspecific variations in the mitogenomes of these four species (Table 5). There are significantly more INDELs than SNPs identified in the four fungal species, a trend identical to that observed in the four mitogenomes of fungal endosymbiont WBPH with the exception of their absolute amounts. Moreover, there were at least three times more SNPs and INDELs in these fungal mitogenomes than that in the fungal symbiont of WBPHs. This phenomenon can be explained by two major factors: first, the geographical distribution or genetic background of WBPH samples is relatively limited in comparison to those of the four fungal species, and second, the surroundings of fungal endosymbionts are less dynamic than those of normal fungal species, causing low selection pressure from the environment. This second factor is supported by two studies: first, the bacterial genome of aphid endosymbiont Buchnera aphidicola (Aphis gossypii) displays a low level of intraspecific variation in comparison to those of host mitogenome (Bae et al., under revision), and second, the whole genome of endosymbiont of Pediculus humanus capitis also shows low-level intraspecific variations in comparison to those of their whole genomes [117].

Table 4: List of available complete fungal mitogenomes in Ophiocordycipitaceae.

No.
Species
NCBI accession
Length (bp)
GC ratio (%)
No. of PCGs
No. of tRNAs
No. of rRNAs
Reference

1
Ophiocordyceps sinensis
NC_034659
157,539
30.2
88
27
2
Unpub.

2
Ophiocordyceps sinensis
MH400233
157,584
30.2
76
27
2
Unpub.

3
Ophiocordyceps sinensis
KP835313
157,510
30.2
6
23
2
[123]

4
Hirsutella thompsonii
NC_040165
62,509
29.8
30
27
2
[114]

5
Hirsutella thompsonii
MH367296
65,332
30.3
32
27
2
[114]

6
Hirsutella thompsonii
MH367295
60,362
30.0
29
27
2
[114]

7
Hirsutella rhossiliensis
MG979071
62,949
28.3
33
26
2
[129]

8
Hirsutella rhossiliensis
NC_030164
62,483
28.2
24
26
2
Unpub.

9
Hirsutella vermicola
NC_036610
53,793
25.3
27
25
2
[130]

10
Hirsutella minnesotensis
NC_027660
52,245
28.4
30
25
2
[131]

11
Tolypocladium sp.
MN583265
46,466
26.1
15
26
2
[132]

12
Tolypocladium guangdongense
MT471267
46,102
26.1
30
27
2
[133]

13
Tolypocladium ophioglossoides
NC_031384
35,159
27.5
19
25
2
[134]

14
Tolypocladium cylindrosporum
NC_046839
34,698
27,0
24
26
2
[135]

15
Tolypocladium inflatum
NC_036382
25,328
27.8
15
25
2
[136]

16
Tolypocladium inflatum
KY924880
25,238
27.8
15
25
2
[136]

17
Tolypocladium inflatum
KY924881
25,328
27.8
15
25
2
[136]

18
Tolypocladium inflatum
KY924882
25,328
27.8
15
25
2
[136]

19
Tolypocladium inflatum
KY924883
24,793
27.8
15
25
2
[136]

20
Ophiocordycipitaceae sp.
NC_049089
66,785
30.6
31
17
2
[19]

Mitogenome annotation of this genome (KP835313) seems not to be complete because several major genes, such as COX1, NAD1, NAD5, and COB, which have many introns on fungal mitogenomes that were not annotated as CDS.

Table 5: Number of intraspecific variations identified from four fungal species belonging to Ophiocordycipitaceae.

No.
Species
No. of mitogenomes
Aligned length (bp)
No. of SNPs
SNP coverage (%)
No. of INDELs
INDEL coverage (%)

1
Ophiocordyceps sinensis
3
157,606
16
0.010
144
0.091

2
Hirsutella thompsonii
3
66,635
281
0.42
6489
9.74

3
Hirsutella rhossiliensis
2
64,858
7
0.01
2008
3.10

4
Tolypocladium inflatum
5
25,338
30
0.12
375
1.48

3.3. Identification and Comparative Analysis of Simple Sequence Repeats on the Five WBPH Fungal Endosymbiont Mitogenomes

Simple sequence repeats (SSRs) identified from organellar genomes have been utilized as molecular markers in various species such as plant species [99, 118–122], suggesting that SSRs on fungal endosymbiont mitogenomes can be used as molecular markers to identify the geographical origins of WBPH. In total, 23 normal and 6 extended SSRs were identified from fungal endosymbiont mitogenomes (Figure 3(b)), with the exception of the fungal endosymbiont mitogenome of WBPH KR.1D which displays 24 normal and 6 extended SSRs (Table 6). The fungal endosymbiont mitogenome of WBPH KR.1D has one more monoSSR (Table 6) with a unit sequence of C and length of 15 bp caused by one insertion (Table 3). In addition, 140 potential SSRs were also identified in the five mitogenomes (Table 6). SSRs identified in the mitogenome were distributed evenly (Figure 3(a)), suggesting that there was no hot spot of SSRs in these fungal mitogenomes.

Figure 3: Numbers and distribution of SSRs on the mitogenome of fungal endosymbiont of WBPH KR. (a) Black circle indicates fungal endosymbiont mitogenome, yellow arrows are protein-coding genes, purple arrows are tRNAs, and red arrows mean rRNAs. SSRs, extended SSRs, and potential SSRs are displayed with yellow, green, and light green colors, respectively. (b) The number of SSRs along with SSR types is displayed. Blue color indicates SSRs and extended SSRs, and orange color means potential SSRs.

Table 6: List of SSRs identified from the five fungal mitogenomes of WBPH endosymbionts.

SSR type
KR
KR.1D
KR.5D
KR.11D
WGS

MonoSSR
8
9
8
8
8

DiSSR
8
8
8
8
8

TriSSR
2
2
2
2
2

TetraSSR
4
4
4
4
4

PentaSSR
0
0
0
0
0

HexaSSR
1
1
1
1
1

HeptaSSR
5
5
5
5
5

OctaSSR
1
1
1
1
1

NonaSSR
0
0
0
0
0

DecaSSR
0
0
0
0
0

Subtotal
29
30
29
29
29

PentaPotentialSSR
101
101
101
101
101

HexaPotentialSSR
39
39
39
39
39

Subtotal
140
140
140
140
140

The length of the identified SSRs is relatively short (a maximum length of 18 bp; Figure 4(a)) in comparison to those of other fungal species in the same family: Ophiocordyceps sinensis (up to 24 bp) [123], as well as fungal species in the other families, such as Pestalotiopsis fici (up to 45 bp) [124]. Moreover, the maximum length of SSRs identified from the mitogenome of R. speculum (NC_049089) [19] was 18 bp, suggesting that this short SSR length can be linked to the evolution of endosymbiont mitogenomes.

(a)

(b)

(c)

Figure 4: Comparative analysis of simple sequence repeats on mitogenome of WBPH fungal endosymbiont. (a) Number of SSRs, extended SSRs, and potential SSRs along with its length. -axis indicates SSR length (bp), and -axis means the number of SSRs. Blue-colored bars indicate SSRs and extended SSRs, and orange bars mean potential SSRs. (b) Number of SSRs, extended SSRs, and potential SSRs based on positions, genic, intronic ORF, intergenic, and intronic. (c) Number of genic SSRs along with genes (blue bars) as well as length of genes (orange line). -axis is genes containing SSRs, and -axis indicates the number of SSRs (left) and gene length (bp; right).

Out of 191 normal SSRs, extended SSRs, and potential SSRs, 84 (43.98%) are located in the genic region (genic and intronic ORF categories in Figure 4(b); Table 7). The intronic ORF position indicates the location of the PCGs placed at the introns of other PCGs, most of which are LAGLIDADG endonucleases (Table 2). Nearly half of the SSRs are in PCGs, which are conserved in comparison to intron and intergenic regions, indicating that these SSRs can be utilized for distinguishing species level or even higher rank. In the intergenic region, there were 61 SSRs (31.94%), and in comparison, only 24 SSRs (12.57%) in the intergenic region (Figure 4(b); Table 7). These SSRs are located in relatively nonconserved regions in comparison to PCG regions, suggesting that these SSRs can be used to distinguish intraspecific levels, such as population or geographical origins. Once more endosymbiont mitogenomes are available in the near future, these SSRs can be evaluated for their use in identification of species and their geographical origin as well as evolutionary history of their mitogenomes.

Table 7: List of SSRs identified on fungal endosymbiont mitogenome of WBPH KR.

No.
Name
SSR type
Type
Start
End
Unit sequence
Repeat number
Genes

1
M0000001
Normal SSR
MonoSSR
4209
4219
C
11
(Intron)Cob

2
M0000002
Normal SSR
MonoSSR
5548
5558
A
11
Cob

3
M0000003
Normal SSR
MonoSSR
22314
22323
T
10
Large subunit ribosomal RNA

4
M0000004
Normal SSR
MonoSSR
22482
22492
T
11
Large subunit ribosomal RNA

5
M0000005
Normal SSR
MonoSSR
28442
28451
T
10

6
M0000006
Normal SSR
MonoSSR
32715
32728
C
14

7
M0000007
Normal SSR
MonoSSR
38315
38327
G
13

8
M0000008
Normal SSR
MonoSSR
40035
40044
T
10
NAD4

9
D0000001
Normal SSR
DiSSR
5788
5797
TA
5
(Intron)Cob

10
D0000002
Normal SSR
DiSSR
13708
13721
AT
7
(Intron)COX2

11
D0000003
Normal SSR
DiSSR
20068
20077
AT
5
Large subunit ribosomal RNA

12
D0000004
Normal SSR
DiSSR
20646
20657
AT
6
(Intron)large subunit ribosomal RNA

13
D0000005
Normal SSR
DiSSR
27266
27275
TA
5
Large subunit ribosomal RNA

14
D0000006
Normal SSR
DiSSR
37995
38004
TA
5
ATP synthase F0 subunit a

15
D0000007
Normal SSR
DiSSR
40430
40441
AT
6
NAD1

16
D0000008
Normal SSR
DiSSR
55352
55361
TA
5

17
T0000001
Normal SSR
TriSSR
35829
35840
ATT
4

18
T0000002
Normal SSR
TriSSR
52780
52791
ATA
4
(Intron)COX1

19
Te0000001
Normal SSR
TetraSSR
24721
24732
ATTT
3
Large subunit ribosomal RNA

20
Te0000002
Normal SSR
TetraSSR
42598
42609
TTTA
3

21
Te0000003
Normal SSR
TetraSSR
48477
48488
ATAA
3
(Intron)COX1

22
Te0000004
Normal SSR
TetraSSR
52709
52720
AATA
3
(Intron)COX1

23
P0000001
Potential SSR
PentaSSR
586
595
TTGT
2
(Intron)Cob

24
P0000002
Potential SSR
PentaSSR
1923
1932
TAATA
2
(Intron)Cob

25
P0000003
Potential SSR
PentaSSR
3523
3532
TAAAA
2
(Intron)Cob

26
P0000004
Potential SSR
PentaSSR
4116
4125
TTGTC
2
(Intron)Cob

27
P0000005
Potential SSR
PentaSSR
5068
5077
ATAAT
2
(Intron)Cob

28
P0000006
Potential SSR
PentaSSR
6540
6549
TAATG
2
(Intron)Cob

29
P0000007
Potential SSR
PentaSSR
6646
6655
ATTTT
2
(Intron)Cob

30
P0000008
Potential SSR
PentaSSR
6714
6723
TTTT
2
(Intron)Cob

31
P0000009
Potential SSR
PentaSSR
7636
7645
AGCAA
2
LAGLIDADG/HNH endonuclease, (Intron)Cob

32
P0000010
Potential SSR
PentaSSR
9109
9118
AAGTT
2
NAD5

33
P0000011
Potential SSR
PentaSSR
9264
9273
ATAA
2
NAD5

34
P0000012
Potential SSR
PentaSSR
10305
10314
AGACA
2
NAD5

35
P0000013
Potential SSR
PentaSSR
10857
10866
ATTCA
2

36
P0000014
Potential SSR
PentaSSR
11369
11378
AGATA
2
COX2

37
P0000015
Potential SSR
PentaSSR
12511
12520
TTATA
2
(Intron)COX2

38
P0000016
Potential SSR
PentaSSR
12600
12609
TAAGA
2
(Intron)COX2

39
P0000017
Potential SSR
PentaSSR
12710
12719
AAGCG
2
(Intron)COX2

40
P0000018
Potential SSR
PentaSSR
12796
12805
TTAAC
2
(Intron)COX2

41
P0000019
Potential SSR
PentaSSR
13303
13312
TAATA
2
COX2

42
P0000020
Potential SSR
PentaSSR
14946
14955
AAAAG
2
NAD3

43
P0000021
Potential SSR
PentaSSR
16747
16756
TCGAG
2
(Intron)NAD2

44
P0000022
Potential SSR
PentaSSR
17383
17392
TCATT
2
NAD2

45
P0000023
Potential SSR
PentaSSR
17427
17436
AATAA
2
NAD2

46
P0000024
Potential SSR
PentaSSR
17514
17523
AAATG
2
NAD2

47
P0000025
Potential SSR
PentaSSR
17685
17694
AATAA
2
NAD2

48
P0000026
Potential SSR
PentaSSR
18083
18092
AATAC
2
NAD2

49
P0000027
Potential SSR
PentaSSR
18109
18118
TATT
2
NAD2

50
P0000028
Potential SSR
PentaSSR
18121
18130
ATAGA
2
NAD2

51
P0000029
Potential SSR
PentaSSR
18400
18409
TTATG
2

52
P0000030
Potential SSR
PentaSSR
18819
18828
GATA
2

53
P0000031
Potential SSR
PentaSSR
19078
19087
ATTTT
2

54
P0000032
Potential SSR
PentaSSR
19190
19199
TTGTA
2

55
P0000033
Potential SSR
PentaSSR
19302
19311
ATAAT
2

56
P0000034
Potential SSR
PentaSSR
19613
19622
AACT
2

57
P0000035
Potential SSR
PentaSSR
19629
19638
TATT
2

58
P0000036
Potential SSR
PentaSSR
19904
19913
TAGAC
2
tRNA-Glu

59
P0000037
Potential SSR
PentaSSR
20384
20393
TTATT
2
Large subunit ribosomal RNA

60
P0000038
Potential SSR
PentaSSR
20968
20977
TTATT
2
RPS3, (Intron)large subunit ribosomal RNA

61
P0000039
Potential SSR
PentaSSR
21146
21155
TGTAT
2
RPS3, (Intron)large subunit ribosomal RNA

62
P0000040
Potential SSR
PentaSSR
21173
21182
TATTA
2
RPS3, (Intron)large subunit ribosomal RNA

63
P0000041
Potential SSR
PentaSSR
21730
21739
TTATT
2
RPS3, (Intron)large subunit ribosomal RNA

64
P0000042
Potential SSR
PentaSSR
22038
22047
TTTTA
2
(Intron)large subunit ribosomal RNA

65
P0000043
Potential SSR
PentaSSR
22121
22130
TTATT
2
(Intron)large subunit ribosomal RNA

66
P0000044
Potential SSR
PentaSSR
22606
22615
TAATA
2
Large subunit ribosomal RNA

67
P0000045
Potential SSR
PentaSSR
23512
23521
AAGAC
2
Large subunit ribosomal RNA

68
P0000046
Potential SSR
PentaSSR
23962
23971
TTTTC
2
Large subunit ribosomal RNA

69
P0000047
Potential SSR
PentaSSR
24453
24462
AATTA
2
Large subunit ribosomal RNA

70
P0000048
Potential SSR
PentaSSR
24501
24510
ATTTA
2
Large subunit ribosomal RNA

71
P0000049
Potential SSR
PentaSSR
25211
25220
TTTAC
2
Large subunit ribosomal RNA

72
P0000050
Potential SSR
PentaSSR
25357
25366
TTTT
2
Large subunit ribosomal RNA

73
P0000051
Potential SSR
PentaSSR
26181
26190
CATTT
2
(Intron)large subunit ribosomal RNA

74
P0000052
Potential SSR
PentaSSR
27224
27233
ATTTC
2
Large subunit ribosomal RNA

75
P0000053
Potential SSR
PentaSSR
27700
27709
TTAAG
2
NAD6

76
P0000054
Potential SSR
PentaSSR
27844
27853
ATAAT
2
NAD6

77
P0000055
Potential SSR
PentaSSR
28013
28022
TAAAA
2
NAD6

78
P0000056
Potential SSR
PentaSSR
29119
29128
TCCCC
2

79
P0000057
Potential SSR
PentaSSR
29273
29282
CAGTA
2
COX3

80
P0000058
Potential SSR
PentaSSR
29973
29982
TGAT
2
Intron-encoded endonuclease aI1, (Intron)COX3

81
P0000059
Potential SSR
PentaSSR
30860
30869
AGTG
2
(Intron)COX3

82
P0000060
Potential SSR
PentaSSR
32618
32627
TCCCC
2

83
P0000061
Potential SSR
PentaSSR
33397
33406
TAAAT
2

84
P0000062
Potential SSR
PentaSSR
33416
33425
ATGGT
2

85
P0000063
Potential SSR
PentaSSR
33916
33925
AGAGA
2

86
P0000064
Potential SSR
PentaSSR
34842
34851
AATT
2
Small subunit ribosomal RNA

87
P0000065
Potential SSR
PentaSSR
36680
36689
TTAAA
2
(Intron)ATP synthase F0 subunit a

88
P0000066
Potential SSR
PentaSSR
36993
37002
TTAAA
2
(Intron)ATP synthase F0 subunit a

89
P0000067
Potential SSR
PentaSSR
37021
37030
ATTTT
2
(Intron)ATP synthase F0 subunit a

90
P0000068
Potential SSR
PentaSSR
37070
37079
AAGGA
2
(Intron)ATP synthase F0 subunit a

91
P0000069
Potential SSR
PentaSSR
37736
37745
ATTTG
2
ATP synthase F0 subunit a

92
P0000070
Potential SSR
PentaSSR
38246
38255
TATTT
2

93
P0000071
Potential SSR
PentaSSR
38820
38829
ACAAT
2
NAD4

94
P0000072
Potential SSR
PentaSSR
38940
38949
ATAAA
2
NAD4

95
P0000073
Potential SSR
PentaSSR
40209
40218
TTCAG
2
NAD1

96
P0000074
Potential SSR
PentaSSR
40498
40507
AATAC
2
NAD1

97
P0000075
Potential SSR
PentaSSR
40724
40733
GTTA
2
(Intron)NAD1

98
P0000076
Potential SSR
PentaSSR
41115
41124
AATGG
2
(Intron)NAD1

99
P0000077
Potential SSR
PentaSSR
41463
41472
AATAT
2
NAD1

100
P0000078
Potential SSR
PentaSSR
41867
41876
TACAA
2
(Intron)NAD1

101
P0000079
Potential SSR
PentaSSR
41973
41982
ATATT
2
NAD1

102
P0000080
Potential SSR
PentaSSR
42415
42424
TAGTT
2
(Intron)NAD1

103
P0000081
Potential SSR
PentaSSR
43163
43172
TACAC
2
(Intron)COX1

104
P0000082
Potential SSR
PentaSSR
43808
43817
TATTT
2
LAGLIDADG endonuclease (QPC56057.1), (Intron)COX1

105
P0000083
Potential SSR
PentaSSR
44007
44016
AATTT
2
LAGLIDADG endonuclease (QPC56057.1), (Intron)COX1

106
P0000084
Potential SSR
PentaSSR
44079
44088
ATAT
2
LAGLIDADG endonuclease (QPC56057.1), (Intron)COX1

107
P0000085
Potential SSR
PentaSSR
44160
44169
TTATA
2
LAGLIDADG endonuclease (QPC56057.1), (Intron)COX1

108
P0000086
Potential SSR
PentaSSR
44359
44368
TAATT
2
LAGLIDADG endonuclease (QPC56057.1), (Intron)COX1

109
P0000087
Potential SSR
PentaSSR
47717
47726
TGTTT
2
LAGLIDADG endonuclease (QPC56054.1), (Intron)COX1

110
P0000088
Potential SSR
PentaSSR
48411
48420
ATATA
2
(Intron)COX1

111
P0000089
Potential SSR
PentaSSR
48965
48974
TATAT
2
LAGLIDADG endonuclease (QPC56060.1), (Intron)COX1

112
P0000090
Potential SSR
PentaSSR
49852
49861
TATTT
2
LAGLIDADG endonuclease (QPC56055.1), (Intron)COX1

113
P0000091
Potential SSR
PentaSSR
50038
50047
ATAAA
2
LAGLIDADG endonuclease (QPC56055.1), (Intron)COX1

114
P0000092
Potential SSR
PentaSSR
50572
50581
AAATA
2
LAGLIDADG endonuclease (QPC56055.1), (Intron)COX1

115
P0000093
Potential SSR
PentaSSR
50686
50695
CATAG
2
LAGLIDADG endonuclease (QPC56055.1), (Intron)COX1

116
P0000094
Potential SSR
PentaSSR
50901
50910
TATTT
2
LAGLIDADG endonuclease (QPC56059.1), (Intron)COX1

117
P0000095
Potential SSR
PentaSSR
52105
52114
ATAG
2
(Intron)COX1

118
P0000096
Potential SSR
PentaSSR
52165
52174
TATTT
2
(Intron)COX1

119
P0000097
Potential SSR
PentaSSR
53150
53159
TTTAC
2
(Intron)COX1

120
P0000098
Potential SSR
PentaSSR
53214
53223
ATAT
2
(Intron)COX1

121
P0000099
Potential SSR
PentaSSR
53261
53270
TTATA
2
(Intron)COX1

122
P0000100
Potential SSR
PentaSSR
53525
53534
ATATT
2
(Intron)COX1

123
P0000101
Potential SSR
PentaSSR
55085
55094
ATAT
2
COX1

124
H0000001
Potential SSR
HexaSSR
1409
1420
ATTTAG
2
(Intron)Cob

125
H0000002
Potential SSR
HexaSSR
1544
1555
GAATTA
2
(Intron)Cob

126
H0000003
Potential SSR
HexaSSR
1819
1830
TTAATC
2
(Intron)Cob

127
H0000004
Potential SSR
HexaSSR
2353
2364
ATTTT
2
(Intron)Cob

128
H0000005
Normal SSR
HexaSSR
2548
2565
AAATAT
3
Cob

129
H0000006
Potential SSR
HexaSSR
2996
3007
TTTTTA
2
(Intron)Cob

130
H0000008
Potential SSR
HexaSSR
5935
5946
TTTATT
2
(Intron)Cob

131
H0000009
Potential SSR
HexaSSR
6512
6523
TAAATC
2
(Intron)Cob

132
H0000011
Potential SSR
HexaSSR
7506
7517
GATTA
2
LAGLIDADG/HNH endonuclease, (Intron)Cob

133
H0000012
Potential SSR
HexaSSR
8965
8976
AACTA
2
NAD5

134
H0000013
Potential SSR
HexaSSR
9972
9983
ATCCC
2
NAD5

135
H0000014
Potential SSR
HexaSSR
12348
12359
TAAAT
2
(Intron)COX2

136
H0000015
Potential SSR
HexaSSR
12475
12486
AAAGT
2
(Intron)COX2

137
H0000016
Potential SSR
HexaSSR
13479
13490
ATTTA
2
(Intron)COX2

138
H0000017
Potential SSR
HexaSSR
17949
17960
GTTAAT
2
NAD2

139
H0000018
Potential SSR
HexaSSR
17975
17986
TAAAAA
2
NAD2

140
H0000019
Potential SSR
HexaSSR
19353
19364
TAATAC
2

141
H0000020
Potential SSR
HexaSSR
21110
21121
TTTTAA
2
RPS3, (Intron)large subunit ribosomal RNA

142
H0000023
Potential SSR
HexaSSR
22403
22414
TATGCC
2
Large subunit ribosomal RNA

143
H0000024
Potential SSR
HexaSSR
23824
23835
TCCGCA
2
Large subunit ribosomal RNA

144
H0000025
Potential SSR
HexaSSR
24585
24596
GAACT
2
Large subunit ribosomal RNA

145
H0000026
Potential SSR
HexaSSR
26510
26521
AAATA
2
(Intron)large subunit ribosomal RNA

146
H0000027
Potential SSR
HexaSSR
27040
27051
TATTTT
2
Large subunit ribosomal RNA

147
H0000028
Potential SSR
HexaSSR
27669
27680
TTTAT
2
NAD6

148
H0000029
Potential SSR
HexaSSR
28253
28264
TATTAA
2
NAD6

149
H0000030
Potential SSR
HexaSSR
31008
31019
TCTGA
2
(Intron)COX3

150
H0000031
Potential SSR
HexaSSR
34196
34207
TAGTT
2

151
H0000032
Potential SSR
HexaSSR
36802
36813
GTGTA
2
(Intron)ATP synthase F0 subunit a

152
H0000034
Potential SSR
HexaSSR
37885
37896
AGATAA
2
ATP synthase F0 subunit a

153
H0000035
Potential SSR
HexaSSR
41287
41298
ATTTAA
2
NAD1

154
H0000036
Potential SSR
HexaSSR
44425
44436
TCCATC
2
LAGLIDADG endonuclease (QPC56057.1), (Intron)COX1

155
H0000037
Potential SSR
HexaSSR
45779
45790
TCCATC
2
LAGLIDADG endonuclease (QPC56058.1), (Intron)COX1

156
H0000038
Potential SSR
HexaSSR
46175
46186
TATTTA
2
(Intron)COX1

157
H0000039
Potential SSR
HexaSSR
46345
46356
TTATT
2
(Intron)COX1

158
H0000040
Potential SSR
HexaSSR
46609
46620
TTAATA
2
(Intron)COX1

159
H0000041
Potential SSR
HexaSSR
47358
47369
ATAAAC
2
LAGLIDADG endonuclease (QPC56054.1), (Intron)COX1

160
H0000042
Potential SSR
HexaSSR
50889
50900
TTTTAA
2
LAGLIDADG endonuclease (QPC56059.1), (Intron)COX1

161
H0000043
Potential SSR
HexaSSR
53483
53494
CTTAT
2
(Intron)COX1

162
H0000044
Potential SSR
HexaSSR
54105
54116
TTACCC
2
(Intron)COX1

163
H0000045
Potential SSR
HexaSSR
55364
55375
TTCT
2

164
cHp0000001
Extended SSR
HeptaSSR
898
911
AATTATA
2
(Intron)Cob

165
cHp0000002
Extended SSR
HeptaSSR
13979
13992
AATAATA
2

166
cHp0000003
Extended SSR
HeptaSSR
15909
15922
GGTATTT
2
Hypothetical protein, (Intron)NAD2

167
cHp0000005
Extended SSR
HeptaSSR
34242
34255
TTATAA
2
Small subunit ribosomal RNA

168
cHp0000006
Extended SSR
HeptaSSR
44930
44943
ATTATT
2
(Intron)COX1

169
O0000001
Extended SSR
OctaSSR
40662
40677
TTCATAT
2
(Intron)NAD1

In the genic region, 84 SSRs were distributed in 24 different genes consisting of 21 PCGs, 2 rRNAs, and 1 tRNA (Figure 4(c); Table 7). The large subunit RNA contained the most SSRs and the genes COX1, COX3, NAD3, two LAGLIDADG endonucleases, intron-encoded nuclease aI1, hypothetical protein, and tRNA-Glu contained the fewest (Figure 4(c); Table 7). Considering the length of these genes, some, including large submit RNA, NAD2, LAGLIDADG endonuclease (QPC56057.1), NAD1, NAD6, ATP synthase F0 subunit a, and LAGLIDADG/HNH endonuclease, displayed a relatively large number of SSRs (Figure 4(c); Table 7). Meanwhile, the remaining genes have a relatively low number of SSRs. This inequality of SSR distribution in PCGs can be another useful characteristic for developing efficient molecular markers. In addition, SSRs in PCGs are known to affect the functions of those PCGs especially for adaptation to environmental factors in fungi [125–127], suggesting that these SSRs can also affect the functions of mitochondrial PCGs.

3.4. Phylogenetic Analysis of 25 Fungal Mitogenomes of Ophiocordycipitaceae

We constructed bootstrapped maximum-likelihood (ML) and Bayesian inference (BI) phylogenetic trees using 26 fungal mitogenomes consisting of 5 mitogenomes used in this study, 25 mitogenomes in the Ophiocordycipitaceae family, and 1 outgroup species (Fusarium graminearum) [128]. Due to the incomplete annotation of the Ophiocordyceps sinensis fungal mitogenome (KP835313), five PCGs, NAD5, COB, COX1, NAD1, and NAD4, containing introns are not correctly annotated. Only five conserved PCGs, ATP8, COX2, NAD2, NAD3, and NAD4L, were selected and aligned individually. Subsequently, this alignment was concatenated to construct three phylogenetic trees.

Five fungal endosymbiont mitogenomes of WBPH were well clustered with another fungal symbiont mitogenome of R. speculum (NC_049089) [19] with high supportive values (Figure 5). This indicates taxonomic similarity between the R. speculum endosymbiont and the five WBPH endosymbionts, suggesting that other fungal endosymbionts may also be independently clustered with other fungal species in the sample family, Ophiocordycipitaceae. In terms of evolution, it can be explained by the two hypotheses: (i) independent evolution once this endosymbiont entered the host insect species or (ii) independent taxonomic groups of Ophiocordycipitaceae entering into the host insect species multiple times during evolution. To determine which hypothesis is more likely, we would need more endosymbiont mitogenomes from various host insect species of infraorder Fulgoromorpha and suborder Auchenorrhyncha as well as mitogenomes from neighboring noninsect endosymbiont fungal species.

Figure 5: Phylogenetic trees of 26 fungal complete mitogenomes in Ophiocordycipitaceae. Neighbor-joining (bootstrap repeat is 10,000) and maximum-Likelihood (bootstrap repeat is 1000) phylogenetic trees as well as Bayesian inference tree (1,100,000 generations) of 26 fungal mitogenomes of Ophiocordycipitaceae. Phylogenetic tree was displayed based on the maximum-likelihood tree. The numbers above branches indicate bootstrap support values of maximum-likelihood and neighbor-joining phylogenetic trees and posterior possibility value of Bayesian inference tree, respectively. Scientific names inside the parenthesis indicate those of host species.

Four fungal species used to investigate intraspecific variations in mitogenomes, Hirsutella thompsonii, Hirsutella rhossiliensis, Ophiocordyceps sinensis, and Tolypocladium inflatum, also display rigid clades covering all mitogenomes of each species with high supportive values (Figure 5). Three mitogenomes of Ophiocordyceps sinensis were clustered with the longest branch length among the four species, of which Hirsutella thompsonii had the second longest (Figure 5). These branch lengths were not proportional to the ratio of SNPs and INDELs (Table 4). The topology of the Tolypocladium genus in the trees was not congruent between the ML and BI trees with low bootstrap values (Figure 5), indicating that additional conserved gene sequences are required to resolve this clade properly.

4. Conclusions

We successfully elucidated the five complete mitogenomes of the fungal endosymbiont of WBPH from various sources of NGS raw reads obtained from WBPH samples. These five complete mitogenomes show common and their own characteristics in comparison to the previously elucidated complete mitogenome of the R. japonica fungal endosymbiont [19]. There were fewer intraspecific variations in the five WBPH endosymbiont mitogenomes in comparison to those identified from the four Ophiocordycipitaceae fungal species, Ophiocordyceps sinensis, Hirsutella thompsonii, Hirsutella rhossiliensis, and Tolypocladium inflatum. This can be explained by the narrow geographical distribution and/or genetic background and the low selection pressures of endosymbionts. We identified 191 SSRs were from each WBPH fungal symbiont complete mitogenomes, except for the WBPH_KR.1D mitogenome, which presented an additional SSR. These SSRs are relatively short in length (a maximum length of 18 bp) compared to those of other fungal mitogenomes. Nearly half of the SSRs are in the genic region, suggesting that these SSRs may be more conserved and they may affect the functionality of PCGs. Based on the phylogenetic trees of 5 conserved PCGs of 26 fungal mitogenomes, including one outgroup species, WBPH fungal endosymbiont mitogenomes were clustered with that of R. speculum with high supportive values. This suggests that these insect-hosted fungal endosymbionts have been evolved independently from the other fungal species in the Ophiocordycipitaceae family. Owing to the advantages of NGS raw reads, which can detect sequences from unknown or unexpected organisms [12, 19–37], we successfully identified the complete mitogenomes of WBPH fungal endosymbionts within the NGS raw reads, suggesting that we can understand their phylogenetic positions of fungal symbiont with high resolution without the need to isolate the symbiont from the host. Furthermore, our study shows that NGS raw reads of insects generated in the future can be used to pinpoint further fungal endosymbionts that have previously been difficult to identify. This method could provide novel insights into their phylogenetic positions as well as interactions with their host species.

Data Availability

Mitochondrial genome sequence used in this study can be accessed via accession numbers MW115131, MW373710, MW373711, MW376862, and BK059186 in the NCBI GenBank.

Conflicts of Interest

The authors declare that they have no competing interests.

Acknowledgments

This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (project title: Population Analysis and Development Technology to Predict Change of White Back Planthopper Population, Project No. PJ01386402),” Rural Development Administration, Republic of Korea. We also would like to thank Editage (http://www.editage.co.kr) for English language editing.

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