Analyses of the Complete Mitochondrial Genome of Paraconiothyrium sp. and Gene Rearrangement Diversity in the Pleosporales

Abstract

The Pleosporales is the most predominant order in the Dothideomycetes class, which contains over 4700 species that function in a variety of ways. The material used in this research was previously isolated from the Chinese white wax scale insect, and it was determined to be a Paraconiothyrium genus species that belonged to the Pleosporales order. For further molecular analysis, we assembled the complete mitochondrial genome of Paraconiothyrium sp. based on short reads of BGISEQ sequencing and subreads from Pacbio sequencing. The results showed that it was 42,734 bp in length and contained 8 open reading frames, 12 protein-coding genes and 31 non-coding genes. Phylogenetic analysis showed it was affiliated to the Pleosporales order and formed a sister relationship with Pithomyces chartarum. Compared to the seven other species in the Pleosporales order, Paraconiothyrium sp. has generally conserved gene content and structure, while the homologous blocks and gene order were shown to be significantly rearranged, in accordance with the species diversity in the Pleosporales order. In this study, we presented the first mitochondrial genome of Paraconiothyrium fungi to be reported, and we also showed gene order diversity in the Pleosporales order. These findings will lay the foundation for further species studies regarding molecular diversity and our understanding of species characteristics in the Paraconiothyrium genus.

1. Introduction

The Pleosporales order contains more than 4700 species, which account for nearly one-quarter of the Dothideomycetes class [1]. These species are present in various habitats, and they can exist as symbionts, parasites or endophytes of plant stems, plant leaves and insects [1,2]. As a member of the Pleosporales order, the Paraconiothyrium genus was found to be widely distributed, having been discovered in environments ranging from marine to soil. Additionally, instead of living an independent lifestyle, most species in this genus survive through reliance on a wide range of hosts, including plants, insects, etc. [3]. Commonly regarded as pathogens of plants, members of the Paraconiothyrium species can infect palm trees (Phoenix theophrasti), apple trees, pear trees and vines and cause spots or trunk damage [4,5]. These pathogens were found to affect humans with immunodeficiencies [6]. Moreover, they were found to exist inside insects, such as Acrida cinerea and Diaspidiotus sp. In insects, they were found to produce multiple chemical compounds, which were thought to be beneficial to the host insect without causing any disease [7,8]. Previously, we successfully isolated the fungi Paraconiothyrium sp. YMF1.07793 from the Chinese white wax scale insect (Ericerus pela) and revealed the existence of a symbiotic relationship between the fungus symbiont and the host based on amino acid and vitamin metabolic complementation [9].

The insect E. pela is well known as one of the most famous resource insects in China. It has great economic value due to the white wax it produces [10,11,12,13,14,15,16], which has been widely applied in Chinese medicine, the cosmetics industry and wax-printing products over the past thousand years. According to fossils of scale insects, the ancestors of scale insects were thought to dwell on the ground, and during this period, they came into contact with many microorganisms, especially when taking in nutrition from macrofungi or living in litter underground. Their transition from the ground to higher branches of plants with the prosperity of angiosperms thus led to a series of changes, including in their metabolism and physiology, and some microorganisms were subsequently retained as symbionts of insects. Paraconiothyrium sp. YMF1.07793 is considered to serve as a symbiont fungus in E. pela, although in most cases, Paraconiothyrium species are known to be pathogenic endophytes of plants [3].

Pleosporales species are exposed to diverse environments, and this environmental restriction consequently leads to diversification in the lifestyle and morphology of fungi in order for them to adapt. Therefore, an increasing number of unusual morphological characteristics of Pleosporales species are emerging [17]. For instance, species belonging to the Leptosphaeriaceae family are lightly pigmented and have multi-septate ascospores. Most Lophiostomataceae species usually contain ascomata that possess a compressed apex. Members of the Sporormiaceae family are heavily pigmented and are characterized by multi-septate ascospores which possess germ slits. Due to various inhabitation factors, diversified lifestyles and complicated morphological characteristics, some members of families such as the Venturiaceae family were miscategorized as belonging to the Pleosporales order [17,18]. At the same time, the formation of some morphological features of Pleosporales could be attributed to convergent evolution, such as fruiting body shapes [19]. Thus, it is difficult to identify the phylogenetic classification of these species using their morphological features. As has been established, the identification of Pleosporales species was originally based on their typical morphological characteristics, such as the presence of ascomata and hamathecium [20]. In this case, the diversity of the morphological characteristics in this order means they cannot be used for species identification and other corresponding studies. With the rapid development of sequencing technology that is currently taking place, genomic data could certainly provide information for species determination in a more precise way.

Compared with nuclear DNA, the mitochondrial genome has specific properties, such as smaller genome size, higher evolutionary rate and fewer gene recombination, which make it suitable for use in evolutionary biology research. With the advance in sequencing technology, fungal mitochondrial genomics has made great development in recent years and was applied to phylogenetic questions, including Pleosporales species [21]. Considering that Paraconiothyrium sp. is also a fungal species that belongs to the Pleosporales order, compared to its large and complicated nuclear genome, it is seemingly more suitable for use in analysis carried out with mitochondrial genomic data for the detection of its variety at the molecular level and phylogenetic relationships with other Pleosporales species. In this study, the whole mitochondrial genome of the fungi Paraconiothyrium sp. YMF1.07793 was assembled. Further analyses regarding species synteny and phylogeny were carried out for the determination of phylogenic positions and molecular diversity in Pleosporales species. This comparative analysis provided a more comprehensive perspective on the detection of species diversity within the Pleosporales order.

2. Methods

2.1. DNA Extraction and Sequencing

The fungus Paraconiothyrium sp. YMF1.07793 was isolated from E. pela in Yunnan Province (25°06′ N, 102°76′ E). It was deposited in the Microbial Library of the Germplasm Bank of Wild Species from Southwest China under the voucher number YMF1.07793. Genomic DNA of Paraconiothyrium sp. YMF1.07793 was extracted using CTAB lysis buffer [22].

The DNBSEQ library for the BGISEQ platform was constructed through high-density DNA nanochip technology. The SMRTbell library with 10–15 Kb fragments for the PacBio library was constructed using the SMRTbell Express Template Preparation Kit 2.0 (Pacific Biosciences, Menlo Park, CA, USA). After quality detection and assessment, the genomic DNA was sequenced based on both the BGISEQ and PacBio platforms.

2.2. Mitochondrial Genome Assembly

Here, 3907 Mb of raw data were obtained. The length of sequencing was 150 bp. The raw data from BGISEQ short reads were polished using the SOAPnuke software [23] to remove low-quality sequences and adaptors. Then, short reads were assembled into the mitochondrial genome using the SPAdes software (v3.11.0.) [24].

Then, all the subreads from PacBio sequencing were aligned with the mitochondrial genome assembled from BGISEQ short reads via local BLAST to obtain the mitochondrial subreads. These filtered subreads were assembled into the mitochondrial genome using Racon coupled with miniasm [25]. The mitochondrial genome was submitted to the GenBank database with the accession number OM617730.

2.3. Gene Annotation

Genome annotation was conducted using the MITOS web server and MFannot tool (v1.1). The annotation results were processed with additional artificial revision. The locations and lengths of essential mitochondrial genes were predicted using the MITOS WebServer (http://mitos2.bioinf.uni-leipzig.de/index.py, accessed on 1 March 2022). Open reading frames (ORFs) were detected using the ORF finder (https://www.ncbi.nlm.nih.gov/orffinder/, accessed on 1 March 2022). The complete mitochondrial Genome circular map was created on the webserver Organellar genome-draw (http://chlorobox.mpimp-golm.mpg.de/OGDraw.html, accessed on 1 March 2022).

2.4. Gene Component and Structure Analyses

The secondary structures of tRNA genes were depicted using the tRNAscan-SE search server (http://lowelab.ucsc.edu/tRNAscan-SE/index.html, accessed on 1 March 2022) with the default parameter settings. The tandem repeat units of the mitochondrial genome were analyzed using the Tandem Repeats Finder online server (http://tandem.bu.edu/trf/trf.html, accessed on 1 March 2022). The relative synonymous codon usage (RSCU) of Paraconiothyrium sp. was analyzed by importing all the sequences of mitochondrial protein-coding genes (PCGs) into the CodonW software (v1.4.2). The introns were identified from the annotation result of the MITOS web server and MFannot tool (v1.1). The analysis of the base composition skew was conducted manually according to the formula AT-skew = [A − T]/[A + T] and the formula GC-skew = [G − C]/[G + C] [26].

2.5. Phylogenetic Analysis

Phylogenetic analysis was carried out based on the 12 PCGs of the complete mitochondrial genome of Paraconiothyrium sp. and the 11 other species from the Pleosporales order (Table S1). The genome sequences were downloaded from the NCBI. Rhynchosporium orthosporum from the Ploettnerulaceae family was used as an outgroup. Multiple sequence alignment of the 12 homologous PCGs was performed using MUSCLE, and the redundant fragments were deleted manually [27]. A phylogenetic tree was constructed using Mega software (v.11) with the maximum likelihood method. The model applied was “cpREV + G + F”. The model with the lowest score for the Bayesian Information Criterion was selected.

2.6. Synteny Analysis

Based on phylogenetic analysis, we adopted the first eight Pleosporales species (which had a relatively close phylogenetic relationship with Paraconiothyrium sp.) in Table S1 to conduct synteny analysis. On the basis of the eight mitochondrial genomes downloaded from the NCBI, gene synteny analysis was carried out at the nucleic acid level using Mauve (v2.1.1).

2.7. Gene Rearrangement Analysis

Gene rearrangement analysis was conducted with the same eight Pleosporales species in synteny analysis. The essential mitochondrial genes (PCGs, tRNAs and rRNAs) were used for gene rearrangement analysis. Based on the eight mitochondrial genomes obtained from GenBank, the gene order of the selected species was indicated using colored fragments. The same types of genes were marked with the same color.

3. Results

3.1. General Genomic Features

The final complete mitochondrial genome assembled from PacBio subreads was 42,734 bp in length. There was one large sequence fragment with a length of 15,381 bp, which had the opposite direction to the mitochondrial genome assembled from short reads, indicating that there were differences between the mitochondrial genomes obtained from short reads and PacBio subreads.

The overall GC content was 27.86%, and the overall AT content was 72.14%. Additionally, the base composition was as follows: 35.77% A, 36.21% T, 14.14% C and 13.87% G (Table S2). The mitochondrial genome was composed of 8 open reading frames (ORFs) (orf100, orf112, orf113, orf125, orf133, orf144, orf215 and orf271), 12 protein-coding genes (PCGs) and 31 non-coding genes (Figure 1).

The 12 PCGs included 7 NADH dehydrogenase genes (nad1, nad2, nad3, nad4, nad4L, nad5 and nad6), 3 cytochrome c oxidase genes (cox1, cox2 and cox3), 1 cytochrome c reductase gene (cob) and 1 ATP synthase synthesis gene (atp6). In addition to the 12 PCGs, 31 non-coding genes were identified, which included 2 ribosomal RNA genes (rrnL and rrnS) and 29 transfer RNA genes that transferred 18 amino acids (

Table 1. Mitochondrial genome components of Paraconiothyrium sp.

Gene	Position (bp)	Length (bp)	Direction	Anti-/Start Codons	A + T%
trnR	140–210	71	-	TCT	73.24
trnC	3051–3120	70	+	GCA	61.43
cox1	5663–6543	881	+	ATG	65.72
cox2	6872–7537	666	+	ATG	67.26
trnR	9070–9141	72	+	CCT	70.83
trnL	9775–9857	83	+	TAG	57.83
trnQ	9990–10,061	72	+	TTG	66.67
trnH	10,338–10,410	73	+	GTG	57.54
trnR	11,460–11,530	71	+	TCG	74.65
nad4L	11,581–11,850	270	+	ATG	75.18
nad5	11,850–14,032	2183	+	ATG	69.54
trnV	15,072–15,145	74	+	TTA	62.16
trnR	15,208–15,267	60	+	CCT	73.33
nad4	15,474–16,909	1436	+	ATT	72.63
atp6	17,935–18,700	766	+	AAC	72.98
cob	19,966–21,031	1066	+	ATG	68.76
trnC	22,029–22,098	70	+	GCA	62.85
cox3	22,348–23,640	1293	+	ATG	71.77
nad2	23,766–25,535	1770	+	ATG	73.79
nad3	25,713–26,099	387	+	ATA	74.16
trnV	27,281–27,353	73	+	TAC	63.02
nad1	27,958–28,789	832	-	ACA	70.68
trnM	30,993–31,064	72	-	CAT	61.11
trnF	31,106–31,178	73	-	GAA	58.91
trnA	31,746–31,817	72	-	TGC	65.28
trnE	31,847–31,919	73	-	TTC	53.43
trnL	32,285–32,367	83	-	TAA	59.04
trnM	32,647–32,719	73	-	CAT	60.28
trnM	32,736–32,806	71	-	CAT	53.52
trnT	32,828–32,898	71	-	TGT	56.34
rrnL	33,303–36,429	3127	-		65.94
trnP	36,523–36,595	73	-	TGG	53.43
trnS	36,746–36,830	85	-	TGA	63.53
trnR	37,176–37,247	72	-	ACG	59.72
trnI	37,251–37,322	72	-	GAT	61.11
trnW	37,554–37,627	74	-	TCA	67.56
trnS	37,887–37,966	80	-	GCT	61.25
trnD	38,204–38,276	73	-	GTC	53.43
trnG	38,279–38,349	71	-	TCC	54.93
trnK	38,359–38,430	72	-	TTT	66.67
trnV	38,458–38,530	73	-	TAC	61.65
nad6	38,559–39,147	589	-	ATG	77.75
trnN	39,523–39,593	71	-	GTT	64.79
trnY	39,662–39,746	85	-	GTA	63.53
trnL	41,253–41,329	77	-	CAA	62.33
rrnS	41,351–42,716	1366	-		65.30

). The introns and intergenic regions were 23,747 bp in total, which accounted for 55.6% of the whole genome. There were four introns in the mitochondrial genome, and all the introns belonged to group I (GIY-YIG and LAGLIDADG).

3.2. Protein-Coding Genes and Codon Usage

The total length of the 12 PCGs was 12,139 bp, which accounted for 28.4% of the whole genome. The 12 PCGs had different sequence length sizes. The nad4L gene had the shortest sequence length of 270 bp, while the nad5 gene had the longest sequence length of 2183 bp. Except for introns within genes and the stop codons, the mitochondrial genome of Paraconiothyrium sp. encoded 3508 amino acids in total.

Within the 12 PCGs, there were three types of initiated codons. In total, 10 PCGs had the ORFs initiated with ATN codons (cox1, cox2, cox3, cob, nad2, nad4L, nad5 and nad6 with ATG, nad3 with ATA and nad4 with ATT), while the other 2 PCGs (nad1 and atp6) had the ORFs initiated with ACA and AAC, respectively.

According to the statistics of relative synonymous codon usage (RSCU), UUU of phenylalanine (Phe), UUA of Leucine (Leu) and UAU of tyrosine (Tyr) were the highest-frequency codons among all 12 PCGs. Leucine (Leu) accounted for the largest proportion of amino acid composition, 17.3%, which was followed by l-isoleucine (Ile) with 11.7% and tyrosine (Tyr) with 8.4%, while Tryptophan (Trp) accounted for the smallest proportion of 1.1% (Figure 2).

3.3. rRNAs and tRNAs

The mitochondrial genome contained two ribosomal RNA genes, which included the large subunit ribosomal RNA gene (rrnL), which was located between trnT and trnP, and the small subunit ribosomal RNA gene (rrnS), which was located between orf215 and trnR. The length of rrnL was 3127 bp, while the length of rrnS was 1366 bp. The A + T contents of rrnL and rrnS were 65.94% and 65.3%, respectively.

The mitochondrial genome of Paraconiothyrium sp. contained 29 tRNA genes which ranged from 60 bp (trnR) to 85 bp (trnS and trnY). According to the analysis result from tRNAscan-SE, 20 standard aa were decoded by the remaining 28 tRNAs (including 1 possible suppressor tRNA), except for 1 tRNA with an unknown isotype. The tRNAs that conveyed trnL, trnS and trnY possessed irregular secondary structures. Additionally, the other 21 tRNAs presented typical cloverleaf secondary structures, comprising one amino-acyl (AA) arm, one TΨC (T) arm, one anticodon (AC) arm, one dihydorouridine (DHU) arm and one variable (V) loop (Figure S1).

3.4. Phylogenetic Analysis

According to the phylogenetic analysis, the 12 species belonging to the Pleosporales order, including Paraconiothyrium sp. YMF1.07793, were clustered into the main clade of the phylogenetic tree, and Rhynchosporium orthosporum, which belongs to the Ploettnerulaceae family, was separated as the outgroup. Paraconiothyrium sp. YMF1.07793 and Pithomyces chartarum formed a sister group with a 100% bootstrap value. In contrast, species belonging to the Pleosporaceae family in the Pleosporales order (Bipolaris sorokiniana, Bipolaris oryzae, Bipolaris cookei, Stemphylium lycopersici and Alternaria alternata) were shown to have relatively distant phylogenetic relationships with Paraconiothyrium sp. YMF1.07793 (Figure 3).

3.5. Synteny Analysis

The synteny analysis of species from eight families in the Pleosporales order showed that the total number of homologous blocks in each mitochondrial genome varied from 13 (Coniothyrium glycines, Corynespora cassiicola, Phaeosphaeria nodorum, Shiraia bambusicola) to 16 (Pithomyces chartarum). Four homologous blocks existed in all eight species. The relative positions and sizes of these homologous blocks were dramatically different in each species. In particular, the last block in Stemphylium lycopersici, colored purple in the figure, was the largest homologous block (Figure 4).

3.6. Gene Rearrangement

According to gene arrangement analysis, the gene order of tRNAs from rrnL to the first PCG was generally conserved among the seven species, except for Paraconiothyrium sp., in which nad1 was inserted within 10 tRNAs. The gene order from the last PCG nad6 to trnP also exhibited similar conservation among all eight species (Figure 5). It was found that gene rearrangement frequently occurred among PCGs (mostly cox genes and nad genes) in the mitochondrial genomes. Additionally, this rearrangement took place in a variety of patterns. By comparing Paraconiothyrium sp. with its sister group, Pithomyces chartarum, it could be found that one of the most obvious differences was between nad4L and cox2, in which four tRNA genes were inserted in Paraconiothyrium sp., and this also led to a total increase in gene number. However, Pithomyces chartarum was mainly composed of a succession of PCGs. The other obvious gene order difference was shown in Paraconiothyrium sp., in which a series of genes (cox3-trnC-cob-atp6) was inserted between nad2 and nad4, while Pithomyces chartarum was composed of a series of nad genes.

4. Discussion

In this study, we assembled the complete mitochondrial genome of Paraconiothyrium sp. YMF1.07793 isolated from E. pela based on subreads from the PacBio sequence. This is the first completely annotated mitochondrial genome of fungi from the Didymosphaeriacea family in the Pleosporales order. It provides a valid fungi mitochondrial genome that will contribute to further species diversity studies regarding both parasitism and symbiosis. In terms of its basic mitochondrial genome characteristics, Paraconiothyrium sp. YMF1.07793 is similar to other species in the Pleosporales order, as it exhibits normal mitochondrial gene content and a moderately sized mitochondrial genome among the Pleosporales order.

In the Pleosporales order, the mitochondrial genome size varies greatly, from the smallest mitochondrial genome size of Pyrenophora tritici-repentis being 16,417 bp to the largest one of Pyrenophora teres f. teres being 221,815 bp. These species were shown to exhibit similar gene content but dramatically different mitochondrial genome sizes, which means there is no explicit correlation between mitochondrial genome size and gene content. According to the statistics of mitochondrial genome component of Pyrenophora teres f. teres (221,815 bp in length), Stemphylium lycopersici (75,911 bp in length) and Paraconiothyrium sp. (42,734 bp in length), the total length of essential genes (PCGs, tRNAs and rRNAs) were 40,747 bp and 23,772 bp, and 18,987 bp separately. Apparently, these essential genes did not respond to the variation of mitochondrial genome size. Additionally, from the statistics of some Pleosporales species, we found that species with larger mitochondrial genome sizes always had more introns (Table S3). This evidence showed that differences in mitochondrial genome length could be related to the variation in intergenic regions and introns [28,29,30]. Moreover, two important Pleosporales species, Leptosphaerulina chartarum and Curvularia trifolii, isolated from tobacco, could serve as symbiont endophytic fungi to assist in the growth of tobacco and help tobacco tissue to resist biotic or abiotic stress [31]. They were originally considered to cause global crop diseases. According to a previous study, the mitochondrial genomes of the two species were both of moderate size, being 68,926 bp and 59,100 bp, respectively. As symbionts, these two endophytic fungi contain complex metabolic regulation mechanisms and should contain a large number of regulatory mechanisms that reflect relatively large mitochondrial genome sizes. However, they have medium-sized mitochondrial genomes, meaning there is no obvious relation between the mitochondrial genome size and a fungus’s lifestyle, no matter whether it is an independent organism in an external environment or whether it lives as a parasite in vivo in hosts. This could also prove the view mentioned above: that intergenic regions, as well as introns, influence the size of a mitochondrial genome. Considering the fact that some species could be endophytic fungi that lack some genes and functions which could be supplemented by the host, this lack does have a possibility of resulting in variations in mitochondrial genomes (including variations in genome size).

In the mitochondrial genome of Paraconiothyrium sp., the codons of UUU, UUA and UAU were in the highest frequency (Figure 2), as these codons are completely comprised of A and U (T), it could reveal the high A + T content of PCGs. According to the statistics of base composition, not just PCGs but the complete mitochondrial genome had high A + T content. Additionally, the high A + T content of the mitochondrial genome could be a general phenomenon of most fungal species [32]. Additionally, other studies reported that this high A + T content could represent an adaptation to nitrogen deficiency, which was in accordance with the fact that the host insect, which contained endogenous fungi inside, was always in lack of nitrogen [33]. Moreover, mutations are more likely to take place in sequences with high A + T content, since the A − T base pair (which contains two hydrogen bonds) is less stable than the G − C base pair (which contains three hydrogen bonds) [34].

In terms of the detailed mitochondrial genome characteristics of Paraconiothyrium sp. YMF1.07793, it contained 12 functional protein-coding genes (3 cytochrome c oxidase genes, 7 NADH dehydrogenase genes, an ATPase 6 gene and a cytochrome b gene) and 31 non-coding genes (29 tRNAs and 2 rRNAs), which were essentially identical to the mitochondrial genomes in other Pleosporales-order species. According to the annotation results, the atp8 gene was missing, and there was only an atp6 gene in Paraconiothyrium sp.; we did not find an atp8 gene in any of the other Pleosporales-order species. Therefore, we believed that there was no atp8 gene present in Paraconiothyrium sp. However, some species in the Dothideomycetes class, such as Zymoseptoria tritici and Zasmidium cellare, could contain an ATPase 8 gene and an ATPase 9 gene, which could possibly indicate that these species require a more complex energy metabolism and that they face multiple types of environmental stress [35]. Considering that we isolated Paraconiothyrium sp. from the scale insect E. pela and it was found that the two species had a symbiotic relationship, we determined that the deficiency regarding energy and nutrition in the fungi could possibly be supplemented by its insect host, and this could explain the lack of an atp8 gene in Paraconiothyrium sp.

In addition to the general conservation in gene content among Pleosporales-order species, based on an analysis regarding synteny and gene rearrangement, Paraconiothyrium sp. did undergo significant rearrangement regarding homologous blocks and gene orders, which implied a significant amount of gene rearrangement occurred. However, the rearrangement of gene order and homologous blocks occurred to totally different extents: homologous blocks were greatly rearranged, which was much more obvious than the extent to which changes to gene orders occurred. Although synteny analysis had adopted the whole mitochondrial genome (including mitochondrial genes and intergenic regions) into the analysis, the analysis of gene order rearrangement only took essential genes (PCGs, tRNAs and rRNAs) into consideration. It seemed like intergenic regions could be the reason for such great variations among species, but we did not find obvious evidence supporting this. According to the analysis result, only four homologous blocks were exhibited in all eight species. However, it could be found that there did exist a series of homologous blocks in several species that showed conservative gene composition of Pleosporales order. Generally, the tRNAs in the mitochondrial genomes of Pleosporales species were classified into two groups: the large clusters of tRNAs located beside the PCGs, and a few tRNAs inserted into PCGs. The two groups were also found in the Eurotiomycetes class and Sordariomycetes class [22]. Significantly, except for Coniothyrium glucines [36], the gene nad1 in all the species we adopted was located at the border of the gene cluster, and the genes around were distributed in two complement strains. Additionally, in many cases, trnV was located similarly at the border as well (Figure 5), which made these genes (including the repeat sequences around) potential recombinational hotspots for Pleosporales species because the gene itself as well as repeat sequences could contribute to gene shuffling (in Paraconiothyrium sp. there were repeat sequences in the region of nad1-trnV and the repeat sequences were detected by the website https://www.novoprolabs.com/tools/repeats-sequences-finder, accessed on 22 April 2022) [37,38,39]. As more and more unprecedented Pleosporales species and new lineages are continuously emerging, analyses regarding gene rearrangement and homologous blocks are still worthy of detecting to find out recombinational hotspots and rearrangement patterns a step further.

However, it is worth noting that the mitochondrial genome of Paraconiothyrium sp. YMF1.07793 was assembled based on PacBio sequences, and it showed a sequence fragment in the opposite direction to that assembled from BGISEQ sequences. We found this fragment had no influence on gene orders.

Species belonging to the Pleosporales order are widely distributed and live in a variety of habitats, from living in natural environments, e.g., marine [40,41,42,43], to existing as endosymbionts inside host insects, and Paraconiothyrium sp. has been proved to serve as an essential nutrition provider of E. pela according to our previous study [10]. Regardless of whether the responses related to fungi’s metabolic pathways are caused by environments or by hosts, their responses would lead to a requirement of energy, which is generally controlled by mitochondrion. Perhaps this also implies the genetic variation, including mitochondrial gene variation to some extent, and could finally result in species diversity.

Interestingly, compared to the Pleosporales-order species whose rearrangement has always been related to PCGs, in some other species, gene rearrangement related to other regions, such as non-coding genes, takes place. For instance, Saissetia coffeae, another scale insect belonging to the Coccoidea family, has been confirmed to have fragments filled with tRNAs that are involved in gene rearrangement [33]. It implied that the mitochondrion of hosts and endogenous fungi were under different regulation mechanisms.

In terms of high-level classification, despite the fact that analysis based on mitochondrial DNA sequences has been proved to be an essential tool in solving the remaining taxonomy issues, considering mutual regulation and cooperation that occurs between nuclear genomes and mitochondrial genomes, nuclear genomic data still need to be taken into consideration in the future. Equipped with multiple genomic data and typical morphological knowledge, more comprehensive and precise detection concerning species evolution and diversity in the Pleosporales order could take place.