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Article

Assembly, Annotation, and Comparative Analysis of Mitochondrial Genomes in Trichoderma

by
Xiaoting Wang
,
Zhiyin Wang
,
Fanxing Yang
,
Runmao Lin
* and
Tong Liu
*
Key Laboratory of Green Prevention and Control of Tropical Plant Diseases and Pests, Ministry of Education, School of Tropical Agriculture and Forestry, School of Breeding and Multiplication (Sanya Institute of Breeding and Multiplication), Hainan University, Sanya 572025, China
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2024, 25(22), 12140; https://doi.org/10.3390/ijms252212140
Submission received: 13 October 2024 / Revised: 9 November 2024 / Accepted: 10 November 2024 / Published: 12 November 2024
(This article belongs to the Section Molecular Genetics and Genomics)
Browse Figures
Versions Notes

Abstract

:
Trichoderma is a widely studied ascomycete fungal genus, including more than 400 species. However, genetic information on Trichoderma is limited, with most species reporting only DNA barcodes. Mitochondria possess their own distinct DNA that plays a pivotal role in molecular function and evolution. Here, we report 42 novel mitochondrial genomes (mitogenomes) combined with 18 published mitogenomes of Trichoderma. These circular mitogenomes exhibit sizes of 26,276–94,608 bp, typically comprising 15 core protein-coding genes (PCGs), 2 rRNAs, and 16–30 tRNAs; however, the number of endonucleases and hypothetical proteins encoded in the introns of PCGs increases with genome size enlargement. According to the result of phylogenetic analysis of the whole mitogenome, these strains diverged into six distinct evolutionary branches, supported by the phylogeny based on 2830 single-copy nuclear genes. Comparative analysis revealed that dynamic Trichoderma mitogenomes exhibited variations in genome size, gene number, GC content, tRNA copy, and intron across different branches. We identified three mutation hotspots near the regions encoding nad3, cox2, and nad5 that caused major changes in the mitogenomes. Evolutionary analysis revealed that atp9, cob, nad4L, nad5, and rps3 have been influenced by positive selection during evolution. This study provides a valuable resource for exploring the important roles of the genetic and evolutionary dynamics of Trichoderma mitogenome in the adaptive evolution of biocontrol fungi.

1. Introduction

Trichoderma (Hypocreales) is a genus of biocontrol fungi that is ubiquitous in ecological environments. It has the capacity to control pathogenic microorganisms, nematodes, and insects, and has been used extensively in the fields of biological control and the development of biocontrol agents [1,2,3]. Trichoderma is rich in species diversity and is widely distributed, consisting of more than 400 species (of which 375 species have been effectively named by 2020) [4], and can be used as an ideal material for evolutionary research. However, genetic information on Trichoderma is limited, with most species reporting only DNA barcodes. The DNA barcodes ITS, rpb2, and tef1 are frequently used for the molecular identification of Trichoderma spp. [4]. A total of 357 species with unambiguous identification have been reported with DNA barcodes available in the National Center for Biotechnology Information (NCBI) nucleotide database (August 2022). Of these, 243 species (64.8%) have been reported with all three DNA barcode genes (ITS, tef1, and rpb2). DNA barcodes and a lack of genomic resources have hindered the understanding and species identification of Trichoderma.
Genetic and comparative genomic studies have shown that Trichoderma spp. continually reconstruct their genomes to enhance their ability to rapidly colonize and compete for nutrients and space in new habitats [5,6,7]. Intensive studies on the evolution of Trichoderma have revealed the existence of large-scale horizontal gene transfers in its genome [8] and that it gained a large number of genes during its evolution, as well as the existence of gene loss events, which contributed to the genomic diversity observed in Trichoderma spp. [7].
Previous studies on the evolution of Trichoderma have primarily been conducted at the nuclear genome level. Compared to nuclear genome studies, mitogenome data are easier to obtain and are more conducive to analyzing the genetics and evolution of Trichoderma, promoting our understanding of the diversity of Trichoderma. In 2020, Kwak et al. conducted a comparative analysis of the Trichoderma mitogenome from the perspective of mitochondrial evolution for the first time [9]. In response to intense selective pressures, organisms may have evolved adaptations that enable their survival under specific ecological conditions. This process involves site-specific amino acid substitutions that affect the protein structure and function [10]. In 2021, Kwak et al. proposed that the isolation of Trichoderma from a range of different environments (including soil, wood, living plants, and fungi), particularly those containing oxygen, was associated with the adaptive diversity of Trichoderma mitochondria [11]. Consequently, an analysis of the Trichoderma mitogenome and its associated diversity characteristics will facilitate an understanding of the evolutionary history of Trichoderma species.
Mitochondria are vital organelles that are pervasively distributed within eukaryotic cells and exert a pivotal influence on energy production, cellular respiration, cellular aging, and apoptosis [12]. Mitochondrial DNA (mtDNA) is a form of genetic information independent of the nuclear genome. It is characterized by its uniqueness, stability, and rapid evolution. Consequently, it is an ideal tool for examining the genetic diversity within fungal populations and identifying fungal species or isolates at the genetic level [13,14,15]. The fundamental structure of the fungal mitogenome includes protein-coding genes, transfer RNA (tRNA), ribosomal RNA (rRNA), introns, homing endonucleases, and other genes [16]. The mitogenome is characterized by its simple structure, small molecular weight, unique and stable sequences, and rapid mutation rate [17]. It has a high AT content, high copy number, low methylation levels, and low recombination rate [18,19]. The gene-coding region is relatively conserved, the intergenic region exhibits a high degree of variability, and the introns of the gene lead to high genomic polymorphisms [20]. Mitogenome sequences contain important genetic inforfmation on species differentiation and are widely used to study phylogenetic relationships among species [21,22]. By studying the structure of mitogenomes, we can determine the characteristics of ancestral mitogenomes and explore their evolution and adaptive evolution [23,24].
Studying the variations in the Trichoderma mitogenome in gene composition, structure, order, and other aspects can provide valuable insights into the evolution of species [9,11]. However, the available Trichoderma mitogenome data is limited, with only 18 complete Trichoderma mitogenomes reported in the NCBI up to October 2023. This limits our understanding of these fungal species. Therefore, more Trichoderma mitogenomes are required to facilitate an in-depth exploration of the genetic variation and evolution of Trichoderma species.
In this study, we report 42 Trichoderma mitogenomes combined with 18 published Trichoderma mitogenomes, yielding a total of 60 mitogenomes, and adding new molecular data for Trichoderma. The composition and structure of the Trichoderma mitogenome were analyzed and a phylogenetic tree was constructed using the complete mitogenome sequence. Subsequently, a comparative analysis was conducted on the genome size and gene number, GC content, arrangement and copy number of tRNA, and intron number of Trichoderma mitogenomes to explore the genetic variation in Trichoderma mitogenomes. In addition, a selection pressure analysis was performed on 15 conserved PCGs within the Trichoderma mitogenome. This study facilitates the understanding of the evolution of Trichoderma and contributes to revealing the significant role of vital genes in the adaptive evolution of Trichoderma.

2. Results

2.1. Identification of Trichoderma spp. Using Phylogenetic Analysis of ITS, tef1, and rpb2 Genes

We collected the barcode sequences of ITS, tef1, and rpb2 from 194 Trichoderma spp. (273 strains) for phylogenetic analysis, including sequences available in the NCBI Nucleotide database or inferred from genome assemblies (Supplementary Table S5, 01.seq.EF1.fa; 01.seq.ITS.fa; 01.seq.RPB2.fa [25]). Based on the phylogenetic tree (Figure 1), we identified new strains and reported strains that formed independent branches, such as T069, B02G, and T. breve voucher HMAS248844 formed one branch; FJ004, T. virens IMI 304061 Tvii3, T. virens Gv29-8 TviG2, T. virens GLi 39, and T. virens DAOM 167652 formed another branch, suggesting that T069 and B02G are T. breve strains and FJ004 is T. virens.
Sequence alignment between T069 and T. breve voucher HMAS248844 showed sequence identities of 0.99, 1.00, and 0.99 for ITS, tef1, and rpb2, respectively (Figures S1–S3). Based on the sequence alignment and phylogenetic analyses, we identified T069 as a T. breve strain. Similarly, for strains that were most closely related to lineages of known species, such as YN006, parallel to one branch of three T. pyramidale strains (ITEM 908 Tati9, Tpyle24, and T20), we analyzed the identity of gene sequences between YN006 and ITEM 908 Tati9, with 0.99 for ITS, 1 for tef1 and 0.98 for rpb2, suggesting that YN006 was a T. pyramidale strain. However, the current evidence cannot identify the species names of the five strains (YN065, FJ059, SRR12137155, HN143, and NM158) because of the high level of sequence identity between each of the five strains and several reported species (Supplementary Table S5). Finally, based on the phylogenetic relationships and sequence alignments, we identified or confirmed the species names of 37 fungal strains, excluding five strains (YN065, FJ059, SRR12137155, HN143, and NM158).

2.2. Trichoderma Mitogenome Organization and Features

In this study, we reported 42 Trichoderma mitogenomes, which were combined with 18 published Trichoderma mitogenomes from GenBank (accession numbers presented in Table 1), and 60 mitogenomes were annotated (Figure 2, Figure 3, Figure 4 and Figures S4–S6). Among the 42 newly reported Trichoderma mitogenomes, the complete mitogenomes of 13 Trichoderma species (T. breve, T. longibrachiatum, T. velutinum, T. zelobreve, T. brevicompactum, T. gracile, T. ghanense, T. zeloharzianum, T. pyramidale, T. subviride, T. asperelloides, T. citrinoviride, and T. cyanodichotomus) have been reported for the first time. The 60 Trichoderma mitogenomes were circular and exhibited considerable variations in genome size, ranging from 26,276 bp (T. breve AI337-ZX01-01-R02 and T. zelobreve FJ014) to 94,608 bp (T. cornu-damae KA19-0412C). The GC content of these Trichoderma mitogenomes ranged from 26.86% (T. brevicompactum HA032) to 28.29% (T. koningii SRR9599881). The annotation results revealed that the 60 Trichoderma mitogenomes encoded a set of 38–96 genes, including two rRNA genes (rnl and rns), 16–30 tRNA genes, and 15–68 protein-coding genes (PCGs) (Table 1). Each of these Trichoderma mitogenomes encodes 15 core PCGs, including three cytochrome c oxidase subunits (cox1-3), apocytochrome b (cob), three ATP synthase subunits (atp6, atp8-9), seven subunits of NADH dehydrogenase (nad1-6, nad4L), and ribosomal protein S3 (rps3). In addition, these Trichoderma mitogenomes encode GIY-YIG endonuclease, LAGLIDADG endonuclease, and hypothetical protein genes of unknown function. The number of GIY-YIG endonucleases ranged from 0 to 13, the number of LAGLIDADG endonucleases ranged from 0 to 30, and the number of hypothetical protein genes ranged from 0 to 11 (Figure 2, Figure 3, Figure 4 and Figure 5). Furthermore, these Trichoderma mitogenomes contained 0–53 introns, encoding rps3, GIY-YIG endonuclease, LAGLIDADG endonuclease, and hypothetical protein genes. Among them, the mitogenomes of some Trichoderma strains contained one or more introns in atp9, cox2, cob, cox1 genes, the intergenic region between nad5 and cob, and the intergenic region between rnl and nad2. Moreover, among the core protein-coding genes, the mitogenomes of some strains rarely contained introns compared to other genomes, including the nad2, nad4, and rps3 genes of T. cornu-damae Tcok, the nad3 genes of T. longibrachiatum PR001 and T. longibrachiatum XJ011, and the atp6 gene of Trichoderma sp. YN065 and T. gracile HK011 (Figure 6). In a previous study, only one rRNA gene (rns) was reported in T. asperellum B05 (TasB), and the atp9 gene was absent in T. gamsii KUC1747 (TgaK) [9]. However, our annotation identified the presence of the rnl gene of TasB and the atp9 gene of TgaK (Figure S4). Furthermore, the rps3 gene of T. cornu-damae KA19-0412C (TcoK) has not previously been reported to be located within rnl gene [26]. In contrast, our annotations indicated that the rnl gene of TcoK was significantly longer, and more coding genes were present in the intron besides the rps3 gene (Figure 1, Supplementary Table S3). We found that among the 60 Trichoderma mitogenomes in this study, 56 exhibited a single nucleotide overlap between the stop codon of the nad4L gene and the start codon of the nad5 gene, which was different from those in the four T. asperellum mitogenomes of TasB, TasF, DQ-1, and HL007. The length of the interval between nad4L and nad5 in their mitogenomes was 456 bp, except for TasF, which was 454 bp long (Supplementary Table S3).
For whole Trichoderma mitogenomes, the order of the tRNA genes in the genome is highly conserved; however, there are differences in the number and copy number of the tRNA genes. Trichoderma sp. FJ059 and Trichoderma sp. YN065 contained fewer tRNA genes, which lacked the trnT, trnE, trnL, trnA, and trnK genes. In addition, both strains contained only one trnM gene, whereas the other strains contained three trnM gene copies. Furthermore, T. cornu-damae Tock, T. koningiopsis AH009, and several other Trichoderma mitogenomes have been observed to possess four copies of the trnM gene. The mitogenome of T. ghanense SC106 contained two copies of the trnG gene, and the number of trnW copies was two in both T. velutinum FJ002 and T. velutinum ZJ051, whereas only one was predicted in the other strains. The mitogenomes of T. hamatum SRR24154105 and T. gamsii TgaK both had three copies of the trnR gene, whereas two trnR copies were present in all strains, with the exception of T. afroharzianum SRR10848483, which displayed a single copy of this gene. The mitogenome of T. koningiopsis SRR17548019 exhibited three copies of the trnS gene, with two copies of this gene being identified in all strains except T. afroharzianum TafA, which displayed only one copy of the trnS gene. Furthermore, most Trichoderma strains displayed multiple copies of trnF and trnQ in their mitogenomes (Figure 7).

2.3. Phylogenetic Analysis at Whole-Genome Level

Phylogenetic tree inferred from the whole mitogenome sequences of 59 Trichoderma strains based on ML methods. The 59 strains of Trichoderma were classified into six major evolutionary branches (A–F) (Figure 8). Branch A contained T. cornu-damae and T. brevicompactum; branch B contained T. virens and T. cyanodichotomus; branch C contained T. harzianum, T. afroharzianum, T. simmonsii, T. breve, T. zelobreve, T. pyramidale, and T. velutinum; branch D contained T. pseudokoningii, T. ghanense, T. reesei, T. longibrachiatum, T. citrinoviride, and T. gracile; branch E contained T. asperellum, T. hamatum, and T. asperelloides; branch F contained T. atroviride, T. subviride, T. gamsii, T. koningii, and T. koningiopsis.
In the inferred phylogenetic tree, node support between the five branches (B–F) was high (91% or 100%), except for evolutionary branch A, which had low node support with the other branches (74%) (Figure S7). Most nodes within each branch exhibited high levels of support, with values ranging from 85% to 100%. However, some of the branch nodes within branch C exhibited 70–85% support, whereas individual branches demonstrated less than 50% support. Notably, this branch encompassed 21 Trichoderma strains, representing the highest species diversity (Figure 8). In addition, the phylogenetic tree constructed based on 2830 single-copy genes of 48 Trichoderma nuclear genomes had a topology similar to that of the tree based on the mitogenome (Figure 8, Figures S7 and S8), supporting the capability to explore the divergence of Trichoderma species based on mitogenomes.

2.4. Comparative Analysis of Trichoderma Mitogenomes from Different Evolutionary Branches

Among the 58 Trichoderma mitogenomes (refer to 4.6, Materials and methods section), we identified 16,231 bp conserved sequences at the whole-mitogenome level, which represented highly conserved sequence identities, distinctly changed patterns, and mutational hot spots among mitogenomes from six distinct branches (Figure 9a). The results indicated that these mitogenomes exhibited a high level of conservation, with most identity values exceeding 90%. However, hotspots of variation were observed in nad3, cox2, and nad5. The principal regions of variation in branch A were observed in the nad3, nad5, and nad4 gene regions, branch B in the cox2, nad5, and nad4 gene regions, branch C in the nad5 gene region, branch D in the cox2 and nad5 gene regions, branch E in the cox2 and nad5 gene regions, and branch F in the nad3, nad5, and atp6 gene regions. These results revealed that the three PCGs among the Trichoderma mitogenomes could be considered potential molecular markers for phylogenetic analyses.
The principal component analysis supported the divergence of the six major branches of these mitogenomes (Figure 9b). Clustered mitogenomes from the six branches were clearly identified. The two strains from the early divergent branch A were located between strains from branches D and E. The strains from branches B, C, and D were separated. The strains from branches E and F were close to each other, which is consistent with their recent divergence in phylogeny.
To further explore the evolutionary features of the Trichoderma mitogenome, a comparative analysis was conducted of the GC content, genome size, number of coding genes, introns, and tRNAs of different evolutionary branches of the Trichoderma mitogenome. The GC content of the 60 Trichoderma mitogenomes ranged from 26.86% (T. brevicompactum HA032) to 28.29% (T. koningii SRR9599881), and that of the coding genes ranged from 15 (T. zelobreve FJ014, T. breve T069 and AI337-ZX01-01-R02) to 68 (T. cornu-damae Tcok) (Table 1). Correlation analysis revealed a negative correlation between GC content and Trichoderma mitogenome size and a positive correlation between coding gene number and Trichoderma mitogenome size (Figure 9c). The E (T. asperellum, T. hamatum, and T. asperelloides) and F (T. atroviride, T. subviride, T. gamsii, T. koningii, and T. koningiopsis) branches exhibited a higher GC content in the mitogenome. In contrast, the D (T. pseudokoningii, T. ghanense, T. reesei, T. longibrachiatum, T. citrinoviride, and T. gracile) branch displayed a larger genome size and a higher number of coding genes and introns (Figure 6 and Figure 9d).
Furthermore, T. cornu-damae Tcok from branch A had two introns in the intergenic region between the nad4 and atp8 genes, and the Trichoderma mitogenomes of branch D contained a single intron in the intergenic region between the nad4 and atp8 genes, which were different from the mitogenomes in other branches (Figure 6). Trichoderma mitogenomes in branch C were devoid of predicted introns in the nad5 and cob intergenic regions. In contrast, the mitogenomes of the other five branches contained introns in the nad5 and cob intergenic regions. The mitogenomes of certain strains belonging to branches E and F exhibited an increased number of copies of tRNA genes trnF, trnM, trnQ, trnL, and trnH (Figure 7).

2.5. Evolutionary Selection on PCGs of the Trichoderma Mitogenome

To determine whether the 15 core PCGs from Trichoderma mitogenomes are evolving under positive selection pressure or not, we calculated nonsynonymous/synonymous substitution rate ratios (dN/dS) for these genes using the CODEML program in PAML with site models M1 (neutral), M2 (selection), M7 (beta), and M8 (beta & ω). For the nad5 gene, the LRT statistic for comparing M7 [lnL(log likelihood value) = −7686.569999] and M8 (lnL = −7678.380104) is 16.37979 [2Δ = 2 × (7686.569999 − 7678.380104) = 16.37979], with a p-value = 2.774 × 10−4 using the Chi-square test (with df = 2). For the rps3 gene, we calculated the value of the LRT statistic between M7 and M8 (2Δ = 45.262836, p-value = 1.484 × 10−10, the Chi-square test with df = 2), as well as M1 and M2 (2Δ = 32.447006, p-value = 9 × 10−8) (Table 2, Supplementary Table S4). Moreover, we explored 18 sites in rps3 with values of dN/dS > 1, 11 sites were considered statistically significant (p ≥ 0.95), and 23 sites in nad5 with values of dN/dS > 1, and seven sites were considered statistically significant (p ≥ 0.95) (Figure 10). The results of the log-likelihood test indicated that atp9, cob, and nad4L genes were positively selected in Trichoderma mitogenome with a chi-squared test p-value < 0.05 (Supplementary Table S4).

3. Discussion

In the present study, we annotated and analyzed 60 Trichoderma mitogenomes. The mitogenomes of T. breve AI337-ZX01-01-R02 and T. zelobreve FJ014 (26,276 bp) were the smallest Trichoderma mitogenomes, followed by T. koningiopsis POS7 (TkoP) (27,560 bp) [27]. These Trichoderma mitogenomes generally included 15 core protein-encoding genes (cox1-3, cob, atp6, atp8-9, nad1-6, nad4L, and rps3) and two conserved ribosomal RNA genes (rnl and rns), which is consistent with previous reports on Trichoderma mitogenomes [11,27,28]. Furthermore, the order of the 15 core PCGs, two rRNA genes, and some tRNA genes in Trichoderma mitogenomes was highly conserved. A single nucleotide overlap between the stop codon of the nad4L gene and the start codon of the nad5 gene in 56 Trichoderma mitogenomes, consistent with the characteristics of Trichoderma mitogenomes reported previously [28]. Interestingly, all four strains that did not exhibit this common phenomenon in their mitogenomes were T. asperellum. The reason for this particularity of the species T. asperellum is worth further investigation.
This study newly reported 42 Trichoderma mitogenomes, and the mitogenome phylogeny revealed the phylogenetic placement of at least 30 species, supported by nuclear phylogeny at the whole-genome level (Figure 8). Ongoing advances in genomics and biotechnology have facilitated the sequencing and assembly of mitogenomes. As shown in this study, assemblers [29,30,31] can obtain complete mitogenomes based on the assembly of short or long sequence reads. Mitogenomic phylogeny offers the opportunity to identify Trichoderma spp. The molecular identification of Trichoderma spp. based on DNA barcodes with little genetic information remains challenging. A survey on the identification of two Trichoderma isolates based on the ITS, tef1, and rpb2 genes showed that 21% (10/47) of the experts correctly identified both strains [4]. Compared with the minimal size of 31 Mb for nuclear genomes [7,32,33,34,35,36], the sizes of almost all mitogenomes are less than 50 kb, with a maximal size of 94 kb [9,11,16,26,27,28,34,37,38,39,40], suggesting the easy and efficient use of mitogenomes for correct species identification.
Although fungal mitogenomes exhibit considerable variability in terms of size, structure, and gene order [20], the gene order of Hypocreales mitogenomes is conserved [41], suggesting that it is possible to infer phylogenetic relationships among Trichoderma species using one Fusarium strain as an outgroup. Researchers have frequently used the amino acid sequences of mitogenome core protein-encoding genes for phylogenetic analysis, resolving numerous ambiguous phylogenetic issues [42]. This approach has become the standard methodology for phylogenetic analyses [43]. In addition, studies have used mitochondrial intergenic region sequences to analyze phylogeny and identify molecular phylogenies within and between fungal species [44,45]. The core protein-coding genes, rRNA genes, and the majority of the tRNA gene sequences of the 60 Trichoderma mitogenomes analyzed in this study exhibited high levels of conservation (Supplementary Table S3). Considering the aforementioned findings, we used the mitochondrial whole-genome sequence for phylogenetic analysis, resulting in a phylogenetic tree that was largely concordant with the classification information for these Trichoderma species (Figure 8). Furthermore, the evolutionary branches of Trichoderma obtained by our research exhibited notable similarity to those reported in previous studies [4,7]. The detailed comparison of tree topologies between mitogenome and nuclear genome lineages showed little difference, such as the phylogenetic placement of branch A, which may be due to random sorting of ancestral lineages during the short internode, homoplasy in the mtDNA data, or both [46]. However, distinct branches were observed in the mitogenome and nuclear genome phylogenies (Figure 8). The phylogenetic analysis methods and results presented in this study serve as valuable references for the classification and identification of Trichoderma. In addition, this study identified three mutation hotspot regions in the nad3, cox2, and nad5 genes of the Trichoderma mitogenome. These three PCGs (nad3, cox2, and nad5) have the potential to serve as molecular markers for phylogenetic analyses, thereby clarifying taxonomic ambiguities associated with Trichoderma spp. The phylogenetic tree constructed from the concatenated sequences of nad3, cox2, and nad5 genes of 59 Trichoderma strains exhibits a topology similar to that of the mitogenome phylogeny (Figure 8 and Figure S9), which represents the ability to recognize strains from different branches.
The GC content of the Trichoderma mitogenomes analyzed in this study ranges from 26.86% to 28.29%, with 0–53 introns and 16–30 tRNA genes. Variations in the size of the Trichoderma mitogenome were primarily associated with the number and length of introns and accessory genes (Table 1, Figure 9, Supplementary Table S3). Trichoderma cornu-damae TcoK (94,608 bp) had the largest mitogenome and contained 53 introns, whereas the remaining mitogenomes ranged in size from 26,276 bp to 49,170 bp and contained introns ranging from 0 to 15, supporting the previous discovery of intronic regions as major size contributors in fungal mitogenomes [41]. The smallest mitogenomes of T. breve AI337-ZX01-01-R02, T. zelobreve FJ014, and another smaller Trichoderma mitogenome (T. breve T069) had no introns (Figure 2, Figure 3 and Figure S5). Furthermore, comparative analysis revealed that the genome size, number of genes, GC content, tRNA copy number, and number of introns in the mitogenomes of different Trichoderma branches exhibited notable variation. The mitogenomes of the E and F branches (T. asperellum, T. hamatum, and T. atroviride, etc.) of various biocontrol Trichoderma species exhibited high GC content, and some strains displayed multiple copies of tRNA within their mitogenomes. The mitogenome size of the D branch of the industrial fungus, T. reesei is larger, with a greater number of coding genes and introns. Except for T. cornu-damae Tcok, which had two introns in the nad4 and atp8 intergenic regions, only Trichoderma mitogenomes in the D branch contained one intron in the nad4 and atp8 intergenic regions (Figure 6, Figure 7 and Figure 9d). The genomic GC content of an organism is influenced by mutation bias, selection, and biased recombination associated with DNA repair. This can be used as an indicator reflecting evolutionary processes [47,48]. The high rate of evolution of the mitogenome allows the number and positioning of its introns to explain the different variabilities within different strains within the same genus or even within the same species [49]. Furthermore, according to previous reports [50], both conserved mitochondrial genes and highly variable regions in fungal mitogenomes are involved in host phenotypic plasticity. Therefore, the significant differences in the mitogenomes of biocontrol Trichoderma and industrial Trichoderma from different evolutionary branches revealed in this study provide important information for explaining the correlation between their differences in parasitic and biocontrol abilities, as well as for studying the adaptive evolution of Trichoderma.
Gene selection pressure analysis is an important method for understanding species adaptability, revealing the evolutionary paths of genes, and studying their inheritance and evolution [51]. It provides a perspective and method for gaining an in-depth understanding of biological adaptability. An analysis of coding gene sequences in human mitogenomes revealed that natural selection shaped regional mtDNA variations [52]. Variability has been found in the evolutionary rates of genes encoded in animal mitogenomes, which are influenced by parasitic lifestyle and locomotory capacity [53]. Our selection pressure analysis revealed that atp9, cob, nad4L, nad5, and rps3 were positively selected during evolution. There were multiple positive selection sites in the amino acid sequences of nad5 and rps3, with the observed changes at these sites exhibiting a high degree of correlation with the evolutionary branches of the phylogeny (Figure 10). Mutations in sequences from these positively selected sites in the mitogenome may be affected by lifestyle changes in Trichoderma spp. Ecophysiological and lifestyle changes may also have resulted in species diversification [1]. For species in branch C of the phylogenetic tree (Figure 8), divergent events may have occurred recently, and they possessed special mutations (Figure 10). Similar patterns were observed in species from branches D, E, and F (Figure 8 and Figure 10). Mutations in nad5 render it an ideal molecular marker for systematic evolution and taxonomic identification. It has been successfully used to study phylogeny and genetic variation in numerous organisms [54,55,56,57]. Sequence changes in genes such as the ancient gene rps3 may reflect the evolution of the fungal mitogenome [58]. Future studies on the evolutionary changes in genes and phenotypic traits may provide a comprehensive understanding of the evolution of positively selected genes in Trichoderma.

4. Materials and Methods

4.1. Molecular Identification of 60 Trichoderma Strains

Sixty Trichoderma strains were selected for mitogenome analysis. To correctly identify the species names of these strains, we performed molecular identification analysis based on three genes: ITS, tef1, and rpb2. An alignment of the ITS, tef1, and rpb2 sequences was performed using MUSCLE (version 3.8.31) [59]. The aligned genes were concatenated using the Perl script (global_alignment_single_copy_genes.pl; https://github.com/linrm2010/global_alignment_single_copy_genes/; accessed on 7 November 2022). Poorly aligned regions were removed using Gblocks (version 0.91b) [60]. Raxml-ng (version 1.1.0) [61] with a bootstrap value of 500 and MEGA (version X) [62] with a bootstrap value of 1000 were used to construct the phylogenetic tree [63] under the best model of GTR + I + G, which was identified using jModelTest (version 2.1.4) [64].

4.2. Assembly of the Trichoderma Mitogenomes

The total genomic DNA of each of the 29 strains was isolated from freeze-dried mycelia. DNA libraries were constructed, and the sequenced data were produced using the Illumina Xten platform at Biomarker Company (Beijing, China). PacBio HiFi sequencing data were produced for the T069 strain and nanopore sequencing data were produced for the HL201 strain. In addition, the reported Illumina short reads of 11 strains and BGISEQ-500 short reads of two strains were downloaded from NCBI. Based on these sequenced data of 42 strains, we used SPAdes (version 3.15.4) [29] for genome assembly, which yielded 40 mitogenomes, and used Hifiasm (version 0.16.1) [30] for genome assembly of the T069 strain, as well as used Canu (version 1.8) [31] for genome assembly of HL201 strain. During the assembly process, the mitochondrial DNA contigs were identified by aligning assembly sequences against previously reported Trichoderma mitogenomes, which showed high sequence similarity. For each strain, we may always obtain one mitochondrial contig containing overlapping sequences at the beginning and end of the contig. We manually improved the contig by removing overlapping sequences at the end. Finally, we obtained 42 mitogenomes, including 38 complete mitogenomes and four mitogenomes with gaps (Supplementary Table S1).

4.3. Trichoderma Mitogenome Annotation and Visualization

We annotated 42 Trichoderma mitogenomes reported in this study and 18 reported mitogenomes of Trichoderma from NCBI (Table 1). We uploaded the mitogenome sequence to MITOS (https://usegalaxy.org/root?tool_id=toolshed.g2.bx.psu.edu%2Frepos%2Fiuc%2Fmitos2%2Fmitos2%2F2.1.3%20galaxy0 (accessed on 2 January 2024)) [65], with the parameter “Reference: RefSeq 63 fungi; Genetic Code: 4 Mold” and MFannot webserver (https://megasun.bch.umontreal.ca/apps/mfannot/ (accessed on 2 January 2024)) [66] with the parameter “Genetic Code: 4 Mold” for basic gene prediction. For protein-coding gene structure prediction, we used IGV [67] to uncover the varied prediction results between MITOS and MFannot and aligned the predicted amino acids against the downloaded mitochondrial sequences from the NCBI refseq database (https://ftp.ncbi.nlm.nih.gov/refseq/release/mitochondrion/ (accessed on 2 January 2024)) using Basic Local Alignment Search Tool for Proteins (BLASTP) [68]. In addition, we submitted the amino acid sequence of the predicted protein-coding gene to NCBI CDD (https://www.ncbi.nlm.nih.gov/cdd (accessed on 2 January 2024)) [69] for domain annotation. For the annotation of non-coding RNAs, we used tRNAscan-SE (version 2.0, http://trna.ucsc.edu/tRNAscan-SE/ (accessed on 2 January 2024)) [70] with the parameters of “sequence source: other mitochondrial; Search mode: default; Genetic Code for tRNA Isotype Prediction: Mold & Protozoan Mito” to predict transfer RNAs (tRNAs), combined with the tRNA prediction results of MITOS and MFannot, the set of prediction results of the three methods was obtained as the annotation results of tRNA. Ribosomal RNAs (rRNAs) were identified based on the prediction results of MITOS, MFannot, Rfam (https://rfam.org/ (accessed on 2 January 2024)) [71], RNAweasel (https://megasun.bch.umontreal.ca/apps/rnaweasel/ (accessed on 2 January 2024)) [72], as well as the rRNA reference sequences collected by the authors.
For repeat sequence annotation, we used the Tandem Repeats Finder (https://tandem.bu.edu/trf/trf.html (accessed on 2 January 2024)) [73] to predict the tandem repeat sequences. We used SFMA (https://github.com/linrm2010/SFMA; accessed on 30 Juanary 2024) to mask the predicted gene sequence and submitted the masked sequence to DNA Analyzer Palindrome (http://palindromes.ibp.cz/#/en/palindrome (accessed on 2 January 2024)) [74] with the parameters of “Size: 6–30; Spacer: 0–10; Mismatches: 0” to identify the reverse repeat sequence.
We combined the above analysis results using SFMA to obtain the tbl file, then table2asn (https://ftp.ncbi.nlm.nih.gov/asn1-converters/by_program/table2asn/ (accessed on 2 January 2024)) was used to generate the GenBank file, which was uploaded to OGDRAW (https://chlorobox.mpimp-golm.mpg.de/OGDraw.html (accessed on 2 January 2024)) [75] to generate the mitogenome map.
The detailed operational procedures and scripts used can be viewed in the SFMA. Detailed information on the annotations of the 60 Trichoderma mitogenomes is provided in Supplementary Table S3, and nucleotide sequence data reported for 13 mitogenomes are available in the Third Party Annotation Section of the DDBJ/ENA/GenBank databases under the accession numbers TPA: BK068259-BK068263, BK068321-BK068328 (Table 1).

4.4. Identification of Orthologous Gene Clusters

Based on the annotated amino acid sequences of genes from 60 Trichoderma mitogenomes and Fusarium oxysporum Mh2-2 mitogenome [76] that was selected as an outgroup for phylogenetic analysis, we used OrthoFinder (version 2.5.4) [77] to identify the orthologous groups of these genes. The originally analyzed results showed that the nad2 and nad5 genes from some Trichoderma mitogenomes were classified into one group, which was manually improved. A total of 39 groups were identified, including 15 groups of single-copy genes (atp6, atp8-9, cob, cox1-3, nad1-6, nad4L, and rps3), three groups of GIY-YIG endonucleases, and seven groups of LAGLIDADG endonucleases (Supplementary Table S2).

4.5. Phylogenetic Analysis

The whole-genome sequences of 59 Trichoderma mitogenomes were used to reconstruct phylogenetic relationships. The previously reported T. harzianum TharA was not included in the sliding window analysis of sequence identities among the 60 mitogenomes, suggesting a significant difference between TharA and other mitogenomes. Trichoderma mitogenomes are circular, and the order of the 15 core protein-coding genes (PCGs; atp6, atp8-9, cob, cox1-3, nad1-6, nad4L, and rps3), 2 rRNA genes (rns and rnl), and some tRNA genes in the genome is highly conserved (Supplementary Table S3). For each genome, the linear genome sequence was obtained with the nad2 gene as the starting position of the whole genome sequence, which was submitted to the MAFFT webserver [78] for whole-genome sequence alignment. We used Gblocks (version 0.91b) [60] to remove low-quality aligned regions. The best model of GTR + I + G was identified using jModelTest (version 2.1.10) [64] to construct the phylogenetic tree with the parameter “-t ML -f -i -g 4 -AIC -BIC -a”. We used IQ-TREE (version 1.6.12) [79] to construct the maximum likelihood (ML) phylogenetic tree for the 59 mitogenomes with the parameter “-m GTR+I+G -b 1000”, setting Fusarium oxysporum mh2-2 as the outgroup. The ML phylogenetic tree was visualized using Archaeopteryx (version 0.9928) [80].
Of the 60 Trichoderma strains analyzed in this study, we obtained whole-genome sequences of nuclear genes from 48 strains (unpublished data). Based on the annotated amino acid sequences of nuclear genes from 48 Trichoderma genomes and F. oxysporum FO47 [81] that was selected as outgroup for phylogenetic analysis, we used OrthoFinder (version 2.5.4) [77] to identify 2830 single-copy genes and concatenated their alignments using the Perl script global_alignment_single_copy_genes.pl (https://github.com/linrm2010/global_alignment_single_copy_genes/; accessed on 7 November 2022). Poorly aligned regions were removed using Gblocks (version 0.91b) [60]. Raxml-ng (version 1.1.0) [61] with a bootstrap value of 500 was used to construct a phylogenetic tree under the best model of JTT+I+G+F, which was identified using ProtTest (version 3.4) [82].

4.6. Comparative Mitogenomic Analysis Across Trichoderma Species

The in-house Python script [83] was used to calculate the identity value of the conserved region sequences of Trichoderma mitogenomes using the sliding window approach and to visualize the results, excluding T. harzianum TharA (the reason discussed above) and T. afroharzianum SRR10848483. SRR10848483 showed extremely high variation at some sites owing to incompletely assembled mitogenome sequences, which may have led to incorrect results. Whole-genome alignment sequences were obtained using Gblocks and the T. breve T069 sequence was used as a reference. The window length was set to 100 bp with a step size of 10 bp. Identity values from the conserved sequence alignment results were used for principal component analysis (PCA) implemented in R (version 4.3.2).

4.7. Identification of Positively Selected Genes

For each core PCG, we used ClustalW (version 2.1) [84] to perform sequence alignment (excluding T. harzianum TharA, the reason as shown above; excluding T. afroharzianum SRR10848483, as an incomplete mitogenome with loss of the region encoding rps3; and excluding T. atroviride TatP, T. gamsii TgaK, and T. virens TviG, as the early appearance of the stop codon in rps3 resulted in the prediction of two short Open Reading Frames lacking the complete sequence of rps3; Figure S6, Supplementary Table S3). The positive selection pressure on 15 core PCGs was detected using the CODEML program in PAML (version 4.10.7) [85] with site models Model1 (neutral), Model2 (selection), Model7 (beta), and Model8 (beta & ω). The likelihood ratio test (LRT) was performed to compare the likelihood differences between Model1 and Model2, and Model7 and Model8. Genes with a p-value < 0.05 using the Chi-square test (with df = 2; Model1 vs. Model2, and Model7 vs. Model8) were considered to be evolving under positive selection in Trichoderma (Supplementary Table S4).

5. Conclusions

In the present study, 42 Trichoderma mitogenomes were newly reported. In total, 60 Trichoderma mitogenomes were annotated by combining 18 previously published genomes with the new dataset. This study elucidated the structural characteristics of Trichoderma mitogenomes and their diversity among different Trichoderma species. The phylogenetic tree constructed based on the entire mitogenome provided insights into the phylogenetic relationships among Trichoderma species. Comparative analysis revealed significant differences in the mitogenome size, gene number, GC content, tRNA copy number, and intron number of Trichoderma in different branches. Furthermore, we identified three PCGs (nad3, cox2, and nad5) as mutation hotspots in the Trichoderma mitogenome. These genes can be used as potential molecular markers for further phylogenetic analyses. The results of the selection pressure analysis demonstrated that atp9, cob, nad4L, nad5, and rps3 were subjected to positive selection during the evolutionary process. Our findings are beneficial for elucidating the phylogeny and evolutionary relationships of Trichoderma species, as well as for the development of potential molecular markers for phylogenetic analysis.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms252212140/s1.

Author Contributions

Conceptualization, X.W., R.L. and T.L.; methodology, R.L.; software, X.W., Z.W., F.Y. and R.L.; validation, X.W., Z.W. and F.Y.; formal analysis, X.W., Z.W., F.Y. and R.L.; investigation, X.W. and R.L.; resources, T.L.; data curation, X.W. and R.L.; writing—original draft preparation, X.W.; writing—review and editing, R.L. and T.L.; visualization, X.W. and R.L.; supervision, T.L.; project administration, R.L. and T.L.; funding acquisition, T.L. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Collaborative Innovation Center Project of Hainan University (XTCX2022NYB12).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article and Supplementary Table S5.

Acknowledgments

We thank Raja Asad Ali Khan, who worked in our lab, for his helpful comments on the article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. The phylogenetic tree of 194 Trichoderma species (273 strains). Gene sequences of ITS, tef1, and rpb2 were used to construct the phylogenetic tree based on the GTR + I + G model using Raxml-ng with a bootstrap value of 500. The best model for phylogenetic analysis was detected using jModelTest (version 2.1.10). The strains whose mitogenomes were newly reported in this study are shown in red.
Figure 1. The phylogenetic tree of 194 Trichoderma species (273 strains). Gene sequences of ITS, tef1, and rpb2 were used to construct the phylogenetic tree based on the GTR + I + G model using Raxml-ng with a bootstrap value of 500. The best model for phylogenetic analysis was detected using jModelTest (version 2.1.10). The strains whose mitogenomes were newly reported in this study are shown in red.
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Figure 2. Mitogenome map of T. cornu-damae TcoK (a), T. brevicompactum HA032 (b), T. virens FJ004 (c), T. cyanodichotomus SRR10917712 (d), Trichoderma sp. HN143 (e), Trichoderma sp. FJ059 (f), T. breve T069 (g), T. harzianum XJ023 (h), T. simmonsii AH003 (i), Trichoderma sp. NM158 (j), T. afroharzianum LTR-2 (k), and Trichoderma sp. YN065 (l). The outermost layer lists the gene composition of 15 core PCGs, 2 rRNA genes, and tRNA genes (represented by filled boxes in different colors).
Figure 2. Mitogenome map of T. cornu-damae TcoK (a), T. brevicompactum HA032 (b), T. virens FJ004 (c), T. cyanodichotomus SRR10917712 (d), Trichoderma sp. HN143 (e), Trichoderma sp. FJ059 (f), T. breve T069 (g), T. harzianum XJ023 (h), T. simmonsii AH003 (i), Trichoderma sp. NM158 (j), T. afroharzianum LTR-2 (k), and Trichoderma sp. YN065 (l). The outermost layer lists the gene composition of 15 core PCGs, 2 rRNA genes, and tRNA genes (represented by filled boxes in different colors).
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Figure 3. Mitogenome map of T. zelobreve FJ014 (a), T. pyramidale YN006 (b), Trichoderma sp. SRR12137155 (c), T. velutinum FJ002 (d), T. pseudokoningii TpsA (e), T. citrinoviride SRR18739368 (f), T. ghanense SC106 (g), T. gracile HK011 (h), T. reesei TreA (i), T. longibrachiatum XJ011 (j), T. asperelloides ZJ116 (k), and T. asperellum DQ-1 (l).
Figure 3. Mitogenome map of T. zelobreve FJ014 (a), T. pyramidale YN006 (b), Trichoderma sp. SRR12137155 (c), T. velutinum FJ002 (d), T. pseudokoningii TpsA (e), T. citrinoviride SRR18739368 (f), T. ghanense SC106 (g), T. gracile HK011 (h), T. reesei TreA (i), T. longibrachiatum XJ011 (j), T. asperelloides ZJ116 (k), and T. asperellum DQ-1 (l).
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Figure 4. Mitogenome map of T. hamatum YN047 (a), T. gamsii SRR5171276 (b), T. subviride YN021 (c), T. atroviride HL088 (d), T. koningiopsis HL201 (e), and T. koningii SRR9599881 (f).
Figure 4. Mitogenome map of T. hamatum YN047 (a), T. gamsii SRR5171276 (b), T. subviride YN021 (c), T. atroviride HL088 (d), T. koningiopsis HL201 (e), and T. koningii SRR9599881 (f).
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Figure 5. Comparative analysis of homologous gene families in the Trichoderma mitogenomes. The color-filled circle represents the presence of homologous genes, and the number represents the number of homologous genes. Blocks A–F were identified by phylogenetic relationships of Trichoderma species.
Figure 5. Comparative analysis of homologous gene families in the Trichoderma mitogenomes. The color-filled circle represents the presence of homologous genes, and the number represents the number of homologous genes. Blocks A–F were identified by phylogenetic relationships of Trichoderma species.
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Figure 6. Comparative analysis of intron in the Trichoderma mitogenomes. The genes are presented in order of their position in the genome below the squares. The color-filled square represents the presence of intron in the gene or intergenic region, and the number represents the number of intron. Blocks A–F were identified by phylogenetic relationships of Trichoderma species.
Figure 6. Comparative analysis of intron in the Trichoderma mitogenomes. The genes are presented in order of their position in the genome below the squares. The color-filled square represents the presence of intron in the gene or intergenic region, and the number represents the number of intron. Blocks A–F were identified by phylogenetic relationships of Trichoderma species.
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Figure 7. Comparative analysis of tRNA in the Trichoderma mitogenomes. The tRNAs are presented in order of their position in the genome below the circles. The color-filled circle represents the presence of the tRNA. Blocks A–F were identified by phylogenetic relationships of Trichoderma species.
Figure 7. Comparative analysis of tRNA in the Trichoderma mitogenomes. The tRNAs are presented in order of their position in the genome below the circles. The color-filled circle represents the presence of the tRNA. Blocks A–F were identified by phylogenetic relationships of Trichoderma species.
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Figure 8. Phylogenetic tree based on the mitogenome (left) and nuclear genome (right) of Trichoderma. For the mitogenome phylogeny, it was inferred from the whole mitogenome sequences of 59 Trichoderma strains, based on Maximum likelihood (ML) methods. The best model of GTR + I + G with a bootstrap value of 1000 replicates was used to construct the phylogeny, and the Fusarium oxysporum mh2-2 (FoxM) was used as an outgroup. The 59 Trichoderma strains were clustered into six main evolutionary branches (A–F), which were represented by different color blocks. Regarding the nuclear genome phylogeny, it was constructed based on single-copy genes from 48 Trichoderma nuclear genomes, with F. oxysporum FO47 as the outgroup. The bootstrap values of the tree nodes were coded with different colors.
Figure 8. Phylogenetic tree based on the mitogenome (left) and nuclear genome (right) of Trichoderma. For the mitogenome phylogeny, it was inferred from the whole mitogenome sequences of 59 Trichoderma strains, based on Maximum likelihood (ML) methods. The best model of GTR + I + G with a bootstrap value of 1000 replicates was used to construct the phylogeny, and the Fusarium oxysporum mh2-2 (FoxM) was used as an outgroup. The 59 Trichoderma strains were clustered into six main evolutionary branches (A–F), which were represented by different color blocks. Regarding the nuclear genome phylogeny, it was constructed based on single-copy genes from 48 Trichoderma nuclear genomes, with F. oxysporum FO47 as the outgroup. The bootstrap values of the tree nodes were coded with different colors.
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Figure 9. Comparison of characteristics between Trichoderma mitogenomes: (a) Consistent characterization of the Trichoderma mitogenomes. The identity value of the conserved region sequences of Trichoderma mitogenomes was calculated by sliding window analysis with a window length of 100 bp and a step size of 10 bp. The T. breve T069 sequence was used as the reference to identify the gene location information below the x-axis. (b) Principal component analysis (PCA) according to the conserved sequences of Trichoderma mitogenomes. (c) Relationship between GC content, coding gene number, and genome size for Trichoderma mitogenomes (excluding T. cornu-damae KA19-0412C in the relationship between GC content and genome size). (d) Comparative analysis of the GC content, gene size, and coding gene number of the Trichoderma mitogenomes of six evolutionary branches. Branches A–F and the representative colors in (a,b,d) are consistent with Figure 8.
Figure 9. Comparison of characteristics between Trichoderma mitogenomes: (a) Consistent characterization of the Trichoderma mitogenomes. The identity value of the conserved region sequences of Trichoderma mitogenomes was calculated by sliding window analysis with a window length of 100 bp and a step size of 10 bp. The T. breve T069 sequence was used as the reference to identify the gene location information below the x-axis. (b) Principal component analysis (PCA) according to the conserved sequences of Trichoderma mitogenomes. (c) Relationship between GC content, coding gene number, and genome size for Trichoderma mitogenomes (excluding T. cornu-damae KA19-0412C in the relationship between GC content and genome size). (d) Comparative analysis of the GC content, gene size, and coding gene number of the Trichoderma mitogenomes of six evolutionary branches. Branches A–F and the representative colors in (a,b,d) are consistent with Figure 8.
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Figure 10. Positive selection sites across nad5 (a) and rps3 (b) in Trichoderma mitogenome. Positive selection sites were identified using Bayes Empirical Bayes dN/dS values and labeled with symbols of “.”, “*” (p ≥ 0.95) and “#” (p ≥ 0.99). The different colors represent the A–F branches as shown in Figure 8. TcoK: T. cornu-damae TcoK, HA032: T. brevicompactum HA032, TviF: T. virens TviF, G-41: T. virens G-41, TviG: T. virens TviG, FJ004: T. virens FJ004, S7712: T. cyanodichotomus SRR10917712, HN143: Trichoderma sp. HN143, FJ059: Trichoderma sp. FJ059, TharC: T. harzianum TharC, XJ023: T. harzianum XJ023, TharM: T. harzianum TharM, TharP: T. harzianum TharP, AH003: T. simmonsii AH003, NM158: Trichoderma sp. NM158, TafA: T. afroharzianum TafA, LTR-2: T. afroharzianum LTR-2, S8483: T. afroharzianum SRR10848483, YN065: Trichoderma sp. YN065, WC045: T. breve WC045, T069: T. breve T069, R02: T. breve AI337-ZX01-01-R02, FJ014: T. zelobreve FJ014, TsiG: T. simmonsii TsiG, S7155: Trichoderma sp. SRR12137155, YN006: T. pyramidale YN006, FJ002: T. velutinum FJ002, ZJ051: T. velutinum ZJ051, TpsA: T. pseudokoningii TpsA, S9368: T. citrinoviride SRR18739368, SC106: T. ghanense SC106, TreA: T. reesei TreA, reesei: T. reesei reesei, R04: T. longibrachiatum AI337-ZX01-01-R04, PR001: T. longibrachiatum PR001, XJ011: T. longibrachiatum XJ011, HK011: T. gracile HK011, TasB: T. asperellum TasB, DQ-1: T. asperellum DQ-1, HL007: T. asperellum HL007, TasF: T. asperellum TasF, ZJ116: T. asperelloides ZJ116, S2116: T. asperelloides SRR19762116, T203: T. asperelloides T203, S7028: T. asperelloides SRR9837028, ThamA: T. hamatum ThamA, YN047: T. hamatum YN047, S4105: T. hamatum SRR24154105, TatA: T. atroviride TatA, TgaK: T. gamsii TgaK, S1276: T. gamsii SRR5171276, YN021: T. subviride YN021, TatP: T. atroviride TatP, HL088: T. atroviride HL088, TkoP: T. koningiopsis TkoP, S8019: T. koningiopsis SRR17548019, AH009: T. koningiopsis AH009, HL201: T. koningiopsis HL201, S9881: T. koningii SRR9599881.
Figure 10. Positive selection sites across nad5 (a) and rps3 (b) in Trichoderma mitogenome. Positive selection sites were identified using Bayes Empirical Bayes dN/dS values and labeled with symbols of “.”, “*” (p ≥ 0.95) and “#” (p ≥ 0.99). The different colors represent the A–F branches as shown in Figure 8. TcoK: T. cornu-damae TcoK, HA032: T. brevicompactum HA032, TviF: T. virens TviF, G-41: T. virens G-41, TviG: T. virens TviG, FJ004: T. virens FJ004, S7712: T. cyanodichotomus SRR10917712, HN143: Trichoderma sp. HN143, FJ059: Trichoderma sp. FJ059, TharC: T. harzianum TharC, XJ023: T. harzianum XJ023, TharM: T. harzianum TharM, TharP: T. harzianum TharP, AH003: T. simmonsii AH003, NM158: Trichoderma sp. NM158, TafA: T. afroharzianum TafA, LTR-2: T. afroharzianum LTR-2, S8483: T. afroharzianum SRR10848483, YN065: Trichoderma sp. YN065, WC045: T. breve WC045, T069: T. breve T069, R02: T. breve AI337-ZX01-01-R02, FJ014: T. zelobreve FJ014, TsiG: T. simmonsii TsiG, S7155: Trichoderma sp. SRR12137155, YN006: T. pyramidale YN006, FJ002: T. velutinum FJ002, ZJ051: T. velutinum ZJ051, TpsA: T. pseudokoningii TpsA, S9368: T. citrinoviride SRR18739368, SC106: T. ghanense SC106, TreA: T. reesei TreA, reesei: T. reesei reesei, R04: T. longibrachiatum AI337-ZX01-01-R04, PR001: T. longibrachiatum PR001, XJ011: T. longibrachiatum XJ011, HK011: T. gracile HK011, TasB: T. asperellum TasB, DQ-1: T. asperellum DQ-1, HL007: T. asperellum HL007, TasF: T. asperellum TasF, ZJ116: T. asperelloides ZJ116, S2116: T. asperelloides SRR19762116, T203: T. asperelloides T203, S7028: T. asperelloides SRR9837028, ThamA: T. hamatum ThamA, YN047: T. hamatum YN047, S4105: T. hamatum SRR24154105, TatA: T. atroviride TatA, TgaK: T. gamsii TgaK, S1276: T. gamsii SRR5171276, YN021: T. subviride YN021, TatP: T. atroviride TatP, HL088: T. atroviride HL088, TkoP: T. koningiopsis TkoP, S8019: T. koningiopsis SRR17548019, AH009: T. koningiopsis AH009, HL201: T. koningiopsis HL201, S9881: T. koningii SRR9599881.
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Table 1. General features of the Trichoderma mitogenomes.
Table 1. General features of the Trichoderma mitogenomes.
SpeciesAbbreviationAccessionSize (bp)GC (%)PCGsrRNAstRNAs
T. afroharzianumTafAON764439.1 *29,51127.7119224
T. afroharzianum LTR-2LTR-2BK06832329,06027.5117226
T. afroharzianum SRR10848483SRR10848483BK06825925,94328.1216222
T. asperelloides ICC 012SRR9837028BK06832428,62127.8917227
T. asperelloides PK1J2SRR19762116BK06832629,81028.0118227
T. asperelloides T203T203BK06826129,58327.9317225
T. asperelloides ZJ116ZJ116PP952381 29,70027.918227
T. asperellum B05TasBNC_037075.1 *29,99927.7817227
T. asperellum DQ-1DQ-1PP93370930,27627.8417227
T. asperellum FT101TasFCP084950.1 *30,28527.8417227
T. asperellum HL007HL007PP952395 30,27627.8417227
T. atroviride ATCC 26799TatAMN125601.1 *32,75828.1818229
T. atroviride HL088HL088PP952396 31,26527.9919229
T. atroviride P1TatPCP084942.1 *29,98128.119229
T. breve AI337-ZX01-01-R02AI337-ZX01-01-R02PP952387 26,27627.4715226
T. breve T069T069PP93371026,28527.4415226
T. breve WC045WC045PP952403 31,26627.4819226
T. brevicompactum HA032HA032PP95239329,48626.8618225
T. citrinoviride SRR18739368SRR18739368BK06832149,17027.1530225
T. cornu-damae KA19-0412CTcoKMW525445.1 *94,60827.9468226
T. cyanodichotomus SRR10917712SRR10917712BK06826041,05927.4426226
T. gamsii A5MHSRR5171276BK06832829,32628.2416229
T. gamsii KUC1747TgaKNC_030218.1 *29,30328.2517228
T. ghanense SC106SC106PP952402 38,00727.1823225
T. gracile HK011HK011PP952394 45,49527.1430224
T. hamatumThamAMF287973.1 *32,76327.6720229
T. hamatum SRR24154105SRR24154105BK06832730,16427.818228
T. hamatum YN047YN047PP952383 35,30727.7622228
T. harzianum CBS 226.95TharCMN564945.1 *27,63227.5516226
T. harzianum MUT3171TharMNC_052832.1 *29,79127.4218226
T. harzianum PAR3TharPMZ713368.1 *27,63127.5516226
T. harzianumTharAMT263519.1 *32,27727.7421228
T. harzianum XJ023XJ023PP952405 27,75727.5616226
T. koningii SRR9599881SRR9599881BK06826328,38928.2916228
T. koningiopsis SRR17548019SRR17548019BK06832231,02328.0518228
T. koningiopsis AH009AH009PP95238628,02628.1316226
T. koningiopsis HL201HL201PP952397 29,41228.1717227
T. koningiopsis POS7TkoPMT816499.1 *27,56027.6716226
T. longibrachiatum AI337-ZX01-01-R04AI337-ZX01-01-R04PP952388 36,93527.1723224
T. longibrachiatum PR001PR001PP952400 36,17227.524224
T. longibrachiatum XJ011XJ011PP952404 35,69427.5523225
T. pseudokoningiiTpsAOW971927.1 *45,11227.3728226
T. pyramidale YN006YN006PP952406 33,43428.0319227
T. reeseiTreANC_003388.1 *42,13027.2427225
T. reeseireeseiPP952401 42,13027.2427225
T. simmonsii AH003AH003PP95238527,81327.4416225
T. simmonsii GH-Sj1TsiGMZ292901.1 *28,66827.5817225
T. subviride YN021YN021PP952382 45,21627.7829228
T. velutinum FJ002FJ002PP95238939,67327.2127228
T. velutinum ZJ051ZJ051PP952380 39,75027.2226230
T. virens FJ004FJ004PP952390 26,58027.4716225
T. virens FT-333TviFCP071122.1 *31,08127.619224
T. virens G-41SRR9836993BK06826234,60127.2923225
T. virens Gv29-8TviGCP071114.1 *27,94327.7518225
T. zelobreve FJ014FJ014PP952391 26,27627.4715226
Trichoderma sp. FJ059FJ059PP952392 38,69326.9225216
Trichoderma sp. HN143HN143PP952398 30,23427.3418225
Trichoderma sp. M10SRR12137155BK06832530,36127.5817225
Trichoderma sp. NM158NM158PP952399 27,55027.5616225
Trichoderma sp. YN065YN065PP952384 30,40727.2418218
* The previously published Trichoderma mitogenomes are highlighted with asterisks.
Table 2. Log-likelihood values and parameter estimates for nad5 and rps3 genes in Trichoderma mitogenomes.
Table 2. Log-likelihood values and parameter estimates for nad5 and rps3 genes in Trichoderma mitogenomes.
GeneModelℓdN/dSEstimates of Parameters
nad5M1 (Nearly Neutral)−7693.4456390.0857p0 = 0.92632 (p1 = 0.07368)
ω0 = 0.01295
M2 (Positive Selection)−7693.4456390.0857p0 = 0.92632, p1 = 0.03912 (p2 = 0.03456) ω0 = 0.01295
M7 (beta)−7686.5699990.1102p = 0.02698, q = 0.21684
M8 (beta & ω > 1)−7678.3801040.0853p0 = 0.94591 (p1 = 0.05409)
p = 0.07077, q = 2.62016, ωs = 1.21899
rps3M1(Nearly Neutral)−4251.444640.1166p0 = 0.90017 (p1 = 0.09983)
ω0 = 0.01868
M2(Positive Selection)−4235.2211370.2012p0 = 0.89972, p1 = 0.07594 (p2 = 0.02434) ω0 = 0.02137
M7(beta)−4256.3833120.1153p = 0.03581, q = 0.27625
M8(beta & ω > 1)−4233.7518940.1757p0 = 0.96277 (p1 = 0.03723)
p = 0.09916, q = 1.40487, ωs = 3.16366
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Wang, X.; Wang, Z.; Yang, F.; Lin, R.; Liu, T. Assembly, Annotation, and Comparative Analysis of Mitochondrial Genomes in Trichoderma. Int. J. Mol. Sci. 2024, 25, 12140. https://doi.org/10.3390/ijms252212140

AMA Style

Wang X, Wang Z, Yang F, Lin R, Liu T. Assembly, Annotation, and Comparative Analysis of Mitochondrial Genomes in Trichoderma. International Journal of Molecular Sciences. 2024; 25(22):12140. https://doi.org/10.3390/ijms252212140

Chicago/Turabian Style

Wang, Xiaoting, Zhiyin Wang, Fanxing Yang, Runmao Lin, and Tong Liu. 2024. "Assembly, Annotation, and Comparative Analysis of Mitochondrial Genomes in Trichoderma" International Journal of Molecular Sciences 25, no. 22: 12140. https://doi.org/10.3390/ijms252212140

APA Style

Wang, X., Wang, Z., Yang, F., Lin, R., & Liu, T. (2024). Assembly, Annotation, and Comparative Analysis of Mitochondrial Genomes in Trichoderma. International Journal of Molecular Sciences, 25(22), 12140. https://doi.org/10.3390/ijms252212140

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