SciELO - Scientific Electronic Library Online

 
vol.40 issue4Comparative transcriptome profile of the leaf elongation zone of wild barley (Hordeum spontaneum) eibi1 mutant and its isogenic wild typeExpression analysis on 14-3-3 proteins in regenerative liver following partial hepatectomy author indexsubject indexarticles search
Home Pagealphabetic serial listing  

Services on Demand

Journal

Article

Indicators

Related links

Share


Genetics and Molecular Biology

Print version ISSN 1415-4757On-line version ISSN 1678-4685

Genet. Mol. Biol. vol.40 no.4 Ribeirão Preto Oct./Dec. 2017  Epub Oct 23, 2017

http://dx.doi.org/10.1590/1678-4685-gmb-2016-0308 

Genetics of Microorganisms

The complete mitochondrial genome of Engyodontium album and comparative analyses with Ascomycota mitogenomes

Xiao-Long Yuan#1 

Xin-Xin Mao#1 

Xin-Min Liu1 

Sen Cheng2 

Peng Zhang1 

Zhong-Feng Zhang1 

1Tobacco Research Institute of Chinese Academy of Agricultural Sciences, Qingdao, China

2Shanghai Tobacco Group Company Limited, Shanghai, China


Abstract

Engyodontium album is a widespread pathogen that causes different kinds of dermatoses and respiratory tract diseases in humans and animals. In spite of its perniciousness, the basic genetic and molecular background of this species remains poorly understood. In this study, the mitochondrial genome sequence of E. album was determined using a high-throughput sequencing platform. The circular mitogenome was found to be 28,081 nucleotides in length and comprised of 17 protein-coding genes, 24 tRNA genes, and 2 rRNA genes. The nucleotide composition of the genome was A+T-biased (74.13%). Group-II introns were found in the nad1, nad5, and cob genes. The most frequently used codon of protein-coding genes was UAU. Isoleucine was identified as the most common amino acid, while proline was the least common amino acid in protein-coding genes. The gene-arrangement order is nearly the same when compared with other Ascomycota mitogenomes. Phylogenetic relationships based on the shared protein-coding genes revealed that E. album is closely related to the Cordycipitaceae family, with a high-confidence support value (100%). The availability of the mitogenome of E. album will shed light on the molecular systematic and genetic differentiation of this species.

Keywords: Engyodontium album; mitochondrial genome; comparative analysis; phylogenetic analyses

Introduction

The Engyodontium album fungus is a member of the Cordycipitaceae family and it characterized by cottony, white colonies that produce numerous dry, tiny conidia. Evidence suggests that E. album can infect a wide range of invertebrates and vertebrates with a cosmopolitan distribution, including arthropods, reptiles, birds, mammals, and humans (Zimmermann, 2007). Infections caused by E. album can induce mild to severe disease, including eczema vesiculosum (Hoog, 1972), granulomatous skin lesions, brain abscesses (Seeliger, 1983), and keratitis (McDonnell et al., 1984). In addition, some patients are even infected without being directly exposed to this fungus, e.g., by using an E. album product bassianin (Tucker et al., 2004). With the incidence of E. album infection increasing throughout the world, it is necessary to explore the molecular characteristics and phylogenetics of E. album for effective therapeutic strategies. Unfortunately, the taxonomy of E. album genus remains unsettled.

Mitochondria are responsible for cellular respiration and energy production in eukaryotic organisms (Henze and Martin, 2003). Mitochondrial DNA (mtDNA) is typically circular and has its own replication machinery that is usually regulated by the nuclear genome (Hu et al., 2004). Owing to their high mutation rates, small sizes, and lack of recombination, mtDNAs have been widely used as informative molecular markers for phylogenetic analyses and species identification (Botero-Castro et al., 2013). Recently, mtDNA was also used for DNA barcoding to facilitate identification in the fields of population genetics, comparative genomics, and evolutionary genomics (Kurbalija Novicic et al., 2015; Qiu et al., 2013). The mitochondrial genomes of fungi have been used as genetic markers for identification and classification purposes (Beaudet et al., 2013). In 1997, Canadian researchers defined the goals of the fungal mitochondrial genome project as being to analyze the genome structure, gene content, and evolution of gene expression in fungal mitochondria (Paquin et al., 1997). Fungal mitochondrial genomes are closed, circular-DNA molecules with lengths ranging from 10 to 80 kb and encode a respiratory chain subunit gene, an ATP synthase complex subunit gene, and ribosomal RNA and tRNA genes (Paquin et al., 1997). As of November, 2016, 339 fungal mitochondrial genomes had been deposited in the National Center for Biotechnology Information (NCBI) database. The mitochondrial genomes of Heterakis gallinae and Heterakis beramporia were amplified by Wang et al. (2016) to develop useful markers for their systematic- and population-genetics study. Liu et al. (2014) sequenced the complete mitochondrial genome of Micrura ignea and made comparisons with other nemertean mitogenomes. However, the complete mitochondrial genome sequence remains unavailable for the genus Engyodontium.

In this study, we completely sequenced the E. album mitogenome to characterize and classify it. We also analyzed the gene content and structure, as well as codon utilization associated with protein-coding genes (PCGs). Other fungal mitogenomes were comparatively analyzed to gain additional insights into their gene content, structure, organization, and phylogenetic relationships.

Materials and Methods

Sample collection and DNA extraction

E. album (strain: ATCC-56482), isolated from a human brain abscess causing death in a female patient (Seeliger, 1983), was purchased from BeiNa Biological Technology Co., Ltd. (Suzhou, China). The strain was cultured at 24 °C in ATCC 200 Yeast Mold Agar medium (BD 271120). Fungus samples were collected after washing twice with sterile water and then stored at −80 °C. Total genomic DNA was isolated from the spores and mycelium using the E.Z.N.A. Fungal DNA Kit (Omega), according to the manufacturer’s instructions. The integrity of the genomic DNA was checked on a 1% agarose gel, and the concentration was detected using a NanoDrop 2000 UV-Vis spectrophotometer (NanoDrop).

Sequence assembly, annotation, and analysis

E. album mtDNA was sequenced using an Illumina HiSeq2000 instrument and assembled using SPAdes software, version 3.6.1 (Bankevich et al., 2012). The Bandage 0.7.1 program was used to check the assembly path and confirm the E. album mtDNA formed a circular molecule (Wick et al., 2015). Moreover, iterative mitochondrial baiting was used to further verify the accuracy of the sequence from head to tail. PCGs were annotated using NCBI’s ORF-finder program (https://www.ncbi.nlm.nih.gov/orffinder/). Analysis of tRNA genes was conducted with the tRNAscan-SE 1.21 Search Server (http://lowelab.ucsc.edu/tRNAscan-SE/) (Lowe and Eddy, 1997). Complete ribosomal RNA genes were identified by alignment with the Lecanicillium saksenae mitogenome (GenBank accession no. KT585676) through BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The circular genome map was constructed using OGDRAW (http://ogdraw.mpimp-golm.mpg.de/cgi-bin/ogdraw.pl) (Lohse et al., 2007). The codon-usage frequency for each amino acid was determined with CodonW (Peden, 2000). The complete sequence of E. album mtDNA was deposited in GenBank under accession no. KX061492. Comparative analyses of the nucleotide sequence of each PCG and ribosomal DNA genes were conducted for Acremonium chrysogenum, Fusarium oxysporum, Hypocrea jecorina, L. saksenae, and Metacordyceps chlamydosporia. Strand bias was characterized by determining AT skewing and GC skewing, calculated using the relationships (A%–T%)/(A%+T%) and (G%–C%)/(G%+C%), respectively. Mitochondrial genome sequences were compared using the Blast Ring Image Generator (BRIG; Tablizo and Lluisma, 2014), with E. album mtDNA serving as the reference sequence. To estimate the evolutionary-selection constraints on genes in the Hypocreales and Ascomycota taxa, common PCGs were chosen to calculate the ratio of nonsynonymous and synonymous changes (Ka/Ks). Codon alignments were performed before pairwise Ka, Ks, and Ka/Ks ratios were calculated using DnaSP software, version 5 (Librado and Rozas, 2009).

Phylogenetic analysis

To determine the phylogenetic location of E. album, currently available complete or near-complete mitochondrial genomes of fungi were used for phylogenetic analysis. The clade including Phaeosphaeria nodorum and Sporothrix schenckii was set as the outgroup. A global analysis was performed using 13 shared PCGs (nad1–nad6, nad4L, cox1–cox3, atp6, atp8, and atp9) among E. album and other related mitochondrial genomes. These genes were individually aligned using the default settings of MAFFT (Katoh et al., 2005), and then these 13 alignments were concatenated using CLUSTAL X software, version 1.81 (Thompson et al., 2002). Finally, a phylogenetic tree was constructed using RAxML version 8.1.12 and MrBayes 3.2, using the general time-reversible model (Stamatakis, 2014; Huelsenbeck and Ronquist, 2001). For each node of the ML tree, bootstrap support was calculated using 1000 replicates. For the Bayesian tree, the initial 10% of values were discarded as burn-in and 4 simultaneous chains were run for 10,000,000 generations.

Results

Genome organization, structure, and composition

The complete mt genome of E. album is a circular molecule of 28,081 bp containing 17 PCGs, 24 transfer RNA genes, and 2 ribosomal RNA genes. All mt genes of E. album are transcribed in the same direction. The average base composition of the complete E. album mitogenome is 37.39% A, 14.65% C, 11.21% G, and 36.74% T. Therefore, the nucleotide composition of the E. album mt genome is biased toward A+T (74.14%). The composition of the E. album mt genome sequence was found to be strongly skewed away from A, in favor of T (AT skew = –0.01), and the GC skew was 0.14, as observed with those of other Cordycipitaceae family members. Moreover, Figure 1 shows that the mitogenome includes 24 tRNA genes and 2 rRNAs genes (large and small subunits).

Figure 1 Mitochondrial genome map of Engyodontium. album. Genes are transcribed in a clockwise direction. 

Protein-coding genes

The E. album mitochondrial genome encodes 17 proteins. Among these, seven protein-coding genes (PCGs) are involved in oxidative phosphorylation (nad1nad6, nad4L), three genes encode different subunits of the cytochrome c oxidase complex (cox1–cox3), three genes encode different subunits of ATP synthase (atp6, atp8, and atp9), one gene encodes the cytochrome b subunit (cob), one gene encodes a ribosomal protein (rps3), and two genes encode open reading frames (ORFs), namely ORF77 and ORF148. Group-II introns were found in the nad1, nad5, and cob genes. Moreover, all PCGs in the mt genome start with ATG, 13 genes (nad2, nad3, nad4L, nad5, nad6, atp6, atp8, atp9, rps3, cox3, cob, ORF77, and ORF148) use TAA as the termination codon, and four genes (cox1, cox2, nad1, and nad4) end with TAG (Table 1).

Table 1 List of annotated mitochondrial genes in E. album. 

Gene Position Length (bp) Start/stop codons Anticodons
rrnL 154–2397, 4026–4559 2244
rps3 2656–3930 1275 ATG/TAA
tRNA-Thr [T] 4602–4672 71 TGT
tRNA-Glu [E] 4678–4750 73 TTC
tRNA-Met [M1] 4934–5006 73 CAT
tRNA-LeuUUN [L1] 5009–5090 82 TAA
tRNA-Ala [A] 5097–5168 72 TGC
tRNA-Phe [F] 5172–5244 73 GAA
tRNA-Lys [K] 5245–5317 73 TTT
tRNA-LeuCUN [L2] 5336–5418 83 TAG
tRNA-Gln [Q] 5426–5498 73 TTG
tRNA-His [H] 5520–5592 73 GTG
tRNA-Met [M2] 5713–5785 73 CAT
nad2 5778–7472 1695 ATG/TAA
nad3 7473–7892 420 ATG/TAA
atp9 7996–8220 225 ATG/TAA
cox2 8371–9120 750 ATG/TAG
tRNA-ArgCGN [R1] 10007–10077 71 ACG
nad4L 10481–10750 270 ATG/TAA
nad5 10750–13813 3064 ATG/TAA
cob 13968–16179 2212 ATG/TAA
tRNA-Cys [C] 16219–16288 70 GCA
cox1 16548–18149 1602 ATG/TAG
orf77 18234–18464 231 ATG/TAA
tRNA-ArgAGN [R2] 18627–18697 71 TCT
orf148 19104–19550 447 ATG/TAA
nad1 19659–21102 1444 ATG/TAG
nad4 21187–22644 1458 ATG/TAG
atp8 22725–22871 147 ATG/TAA
atp6 22929–23708 780 ATG/TAA
rrnS 24092–25559 1468
tRNA-Tyr [Y] 25655–25739 85 GTA
tRNA-Asp [D] 25744–25816 73 GTC
tRNA-SerAGN [S1] 25818–25901 81 GCT
tRNA-Asn [N] 25916–25986 71 GTT
cox3 26020–26829 810 ATG/TAA
tRNA-Gly [G] 26859–26930 72 TCC
nad6 27018–27683 666 ATG/TAA
tRNA-Val [V] 27701–27772 72 TAC
tRNA-Ile [I] 27774–27845 72 GAT
tRNA-SerUCN [S2] 27847–27931 85 TGA
tRNA-Trp [W] 27936–28007 72 TCA
tRNA-Pro [P] 28009–28081 73 TGG

The relative synonymous codon usage (RSCU) value is a measure of the synonymous codons present in a coding sequence. If there is no codon-usage bias, the RSCU values equal 1.00. A codon that is used less frequently than expected will have an RSCU value of < 1.00, whereas a codon used more frequently than expected will have an RSCU value of > 1.00 (Sharp et al., 1986). The results from the E. album mitogenome indicated that almost all amino acids (except for Met) showed codon-usage bias. The most frequently used codon in PCGs was UAU, followed by AUU and UAA, which is consistent with the (A+T)-rich content of the E. album mitogenome. CGC was the least used codon. Ile is the most commonly encoded amino acid in the E. album mitogenome, while Pro is the least common (Table 2).

Table 2 Number of codons and codon usages in mt protein-coding genes of E. album. 

Amino acid Codon N RSCU Amino acid Codon N RSCU
Phe [F] UUU 462 1.55 Tyr [Y] UAU 624 1.61
UUC 135 0.45 UAC 150 0.39
Leu-UUN [L] UUA 475 2.61 Ter [end] UAA 482 1.61
UUG 136 0.75 UAG 262 0.88
Leu-CUN [L] CUU 182 1.00 UGA 154 0.51
CUC 45 0.25 His [H] CAU 137 1.57
CUA 174 0.96 CAC 37 0.43
CUG 81 0.44 Gln [Q] CAA 104 1.13
Ile [I] AUU 570 1.43 CAG 80 0.87
AUC 150 0.38 Asn [N] AAU 442 1.53
AUA 474 1.19 AAC 137 0.47
Met [M] AUG 160 1.00 Lys [K] AAA 469 1.38
Val [V] GUU 160 1.42 AAG 213 0.62
GUC 42 0.37 Asp [D] GAU 146 1.62
GUA 183 1.63 GAC 34 0.38
GUG 65 0.58 Glu [E] GAA 159 1.33
Ser-UCN [S] UCU 124 1.22 GAG 81 0.68
UCC 73 0.72 Cys [C] UGU 139 1.17
UCA 110 1.08 UGC 99 0.83
UCG 37 0.36 Trp [W] UGG 107 1.00
Pro [P] CCU 43 1.51 Arg-CGN [R] CGU 33 0.47
CCC 18 0.63 CGC 12 0.17
CCA 33 1.16 CGA 40 0.57
CCG 20 0.70 CGG 26 0.37
Thr [T] ACU 85 1.10 Arg-AGN [R] AGA 169 2.39
ACC 65 0.84 AGG 144 2.04
ACA 121 1.56 Ser-AGN [S] AGU 151 1.48
ACG 39 0.50 AGC 116 1.14
Ala [A] GCU 67 1.72 Gly [G] GGU 71 1.46
GCC 22 0.56 GGC 25 0.51
GCA 47 1.21 GGA 62 1.27
GCG 20 0.51 GGG 37 0.76

N: number of codons. RSCU: relative synonymous codon usage

Transfer and ribosomal RNA genes

Twenty-four tRNAs were recognized in the mt genome of E. album, were interspersed between the rRNA- and PCGs, and ranged from 70 to 85 bp in length. Of these tRNAs, two forms each were identified for tRNA-Arg (AGN and CGN), tRNA-Ser (UCN and AGN), and tRNA-Leu (UUN and CUN). Taking into account their relative proximities, the tRNA genes could be considered to cluster into three groups: TEMLAFKLQHM (trnT-TGT, trnE-TTC trnM1-CAT, trnL1-TAA, trnA-TGC, trnF-GAA, trnK-TTT, trnL2-TAG, trnQ-TTG, trnH-GTG, and trnM2-CAT), YDSN (trnY- GTA, trnD-GTC, trnS1-GCT, and trnN-GTT), and VISWP (trnV-TAC,trnI-GAT, trnS2-TGA, trnW-TCA, and trnP-TGG), with the exception of four trn genes (trnR, trnL, trnR2, and trnC) that were scattered as single genes throughout the mt genome. All 24 tRNA genes were predicted to have the typical cloverleaf structure, except for tRNA-Tyr (UAU), tRNA-Ser (UCN and AGN), and tRNA-Leu (UUN and CUN) (Figure 2). These five tRNAs adopt a special structure that is widely found in the Sordariomycetes class and is a common feature for Hypocreales species. The E. album rrnL (16S rRNA) gene is located between tRNA-Pro and rps, while rrnS (12S rRNA) is located between atp6 and tRNA-Tyr. The lengths of the rrnS and rrnL genes are 1,468 bp and 2,244 bp, respectively, and their A+T contents are 65.46% and 67.98%, respectively.

Figure 2 Predicted tRNA structures of E. album

Comparative analysis with other mt genomes

To better understand the gene contents and structure of this species in the Hypocreales order, which consists of six families, the mt genomes from L. saksenae (Cordycipitaceae), Fusarium oxysporum (Nectriaceae), Hypocrea jecorina (Hypocreaceae), Metacordyceps chlamydosporia (Clavicipitaceae), and Acremonium chrysogenum (Hypocreales incertae sedis) were chosen for comparative analysis. The genomes were similar in size, with the exception of F. oxysporum (Table 3). The results showed that genome size ranged from 25 kb to 42 kb. The AT-skew values for these species were all negative, while the GC-skew values were positive. As shown in Table 3, the AT-skew value of E. album is fairly close to that of M. chlamydosporia.

Table 3 Composition and skewing in the mitochondrial genomes of Hypocreales. 

Species Size (bp) A% C% G% T% AT skewing GC skewing
Ach 27,266 35.9 11.0 15.5 37.6 –0.02 0.17
Eal 28,081 36.7 11.2 14.7 37.4 –0.01 0.14
Fox 33,396 34.3 14.2 16.8 34.7 –0.01 0.08
Hje 42,130 37.0 12.2 15.1 35.8 0.02 0.11
Lsa 25,919 36.5 11.6 14.9 37.0 –0.01 0.12
Mch 25,615 35.6 12.7 15.6 36.2 –0.01 0.10

Ach: Acremonium chrysogenum; Fox: Fusarium oxysporum; Hje: Hypocrea jecorina; Lsa: Lecanicillium saksenae; Mch: Metacordyceps chlamydosporia.

Our results showed clear differences in the gene contents of the mitogenomes studied (Table 4). They all contain genes encoding components of the oxidative-phosphorylation machinery, subunits of the cytochrome c-oxidase complex of ATP synthase, and the cytochrome b subunit. However, the rps genes were absent from the A. chrysogenum mitogenome. ORFs were present in both F. oxysporum and E. album. Therefore, the gene contents in Hypocreales are highly conserved.

Table 4 Comparison of G + C content (%) of the protein-coding and rRNA genes of mitochondrial genomes of Hypocreales species. 

Gene or region Ach Fox Hje Lsa Mch Eal
cox1 27.14 32.27 26.02 30.48 32.39 31.61
cox2 27.25 28 26.77 28 28.93 34.47
cox3 29.38 32.35 30.74 30 30.99 28.76
cob 28.41 29.58 28.25 28.6 31.2 26.74
nad1 27.69 27.57 25.63 24.64 27.39 25.13
nad2 22.8 24.42 24.2 22.28 25 21.7
nad3 21.98 23.91 23.43 21.67 27.54 24.52
nad4 23.14 25.59 25.71 23.88 25.72 23.18
nad4L 22.96 24.07 23.33 24.07 25.93 24.44
nad5 25.56 27.51 26.94 25.42 29.32 25.8
nad6 23.09 23.07 22.7 19.91 23.16 18.69
atp6 27.25 26.72 27.56 25.03 27.35 25.13
atp8 20.41 21.09 20.92 23.13 20.41 20.41
atp9 31.14 34.22 34.31 32.89 36 32
orf77 26.56 23.38
orf148 23.71
rrnS 35.43 37.67 35.18 35.32 35.34 34.54
rrnL 26.99 33.94 31.39 27.43 27.98 31.82
rps 21.5 19.02 16.67 19.29 16.22
EmtG 26.54 31.06 27.24 26.53 28.28 25.87

Ach: Acremonium chrysogenum; EmtG: entire mitochondrial genome; Fox: Fusarium oxysporum; Hje: Hypocrea jecorina; Lsa: Lecanicillium saksenae; Mch: Metacordyceps chlamydosporia

Comparison of the Hypocreales mtDNA sequences revealed that they were fairly well conserved, with almost 80% sequence identity in the genomic regions shared with that of E. album and only major differences existing in the regions containing the tRNA-Arg (8.8k–12k), nad5 (11.5k–12.5k), cob (14.4k–15.6k), orf148 and orf77 (18.1k–19.9k), and nad1 (20.3k–20.5k) genes. In addition, no gene-module rearrangement occurred in these species, as can be seen in the BRIG map (Figure 3).

Figure 3 Genome-similarity comparison ring constructed using BRIG software. 

Phylogeny analysis

To investigate the phylogenetic position of E. album and the inner relationships of the order Hypocreales, phylogenetic trees were constructed using the nucleotide sequences of 13 PCGs from 20 complete mitochondrial genomes that belong to the Ascomycota division. The phylogenetic trees reconstructed using the ML and Bayes algorithms revealed different clades, which represented five orders, including Hypocreales, Pleosporales, Eurotiales, Glomerellales, and Ophiostomatales (Figure 4). The species in three different families, namely Nectriaceae (F. oxysporum and Gibberella moniliformis), Hypocreaceae (H. jecorina and Trichoderma harzianum), and Clavicipitaceae (M. chlamydosporia and Metarhizium anisopliae), branched in the same clade and then clustered with the Acremonium implicatum and A. chrysogenum species. The species in the Hypocreales order all clustered within the same clade. E. album was located with species in the Cordycipitaceae family with a strong node-supporting value (100% for ML and 1 for Bayes). Examination of the pairwise Ka/Ks ratio for the 13 common PCGs in the Hypocreales and Ascomycota taxa demonstrated that all these genes have undergone purifying selection (Ka/Ks < 1) (Figure 5). Among the species in the Hypocreales order, the Ka/Ks ratio was higher in the cox1 (0.409), cox2 (0.329), and nad6 (0.263) genes than in other genes, while among the species in the Ascomycota division, the most variable genes were nad6 (0.597), cox1 (0.579), and nad5 (0.504).

Figure 4 Phylogenetic tree of Ascomycota species. The numbers shown beside the branches indicate ML bootstrap probabilities from 1000 replicates. 

Figure 5 Ka/Ks ratio of pair-wise comparison among the species in the Hypocreales and Ascomycota according to 13 common PCGs. 

Discussion

Many fungi have a significant adverse impact on global human and animal health (Campbell and Johnson, 2013). A particularly important example is the Cordycipitaceae family of fungi (Menzies and Turkington, 2015). E. album is a widespread species that poses allergic, pathogenic, or toxic risks to humans and mammals (Siegel and Shadduck, 1990; Goettel et al., 2001; Tucker et al., 2004; Balasingham et al., 2011). Despite advances in sequencing and bioinformatics technologies, only limited characterization of their mitogenomes has been conducted. Here, we sequenced the whole mitochondrial genome of E. album, and then compared its genome structure, content, and phylogenetic relationships with other fungal mitogenomes. The mitochondrial genome of E. album is a circular DNA molecule of 28,081 bp in length. This size is comparable to that of previously sequenced mitogenomes of members of the Hypocreales order, such as A. chrysogenum (27,266 bp) (Eldarov et al., 2015), L. saksenae (25,919 bp) (Xin et al., 2017), and M. chlamydosporia (25,615 bp) (Ghikas et al., 2006). The average AT content of the E. album complete mitogenome is 74.13%, just like the A+T contents reported for A. Chrysogenum (74.13%) and L. saksenae (74.13%) (Xin et al., 2017). The E. album mitogenome gene arrangement is identical to that of other Cordycipitaceae family members, such as Ophiocordyceps sinensis (Li et al., 2015), Beauveria pseudobassiana (Oh et al., 2015), Cordyceps militaris (Sung, 2015), and Hirsutella minnesotensis (Zhang et al., 2016). In addition, the PCGs of the E. album mt genome were inferred to start with ATG, which is consistent with the arrangement in the mt genomes of other Cordycipitaceae family members (Oh et al., 2015; Sung, 2015). The gene-structure comparison showed that E. album has the same gene order and shares homology with the highly conserved mt genomes found within other members of the Hypocreales order. Like other mitogenomes, the rrnS and rrnL genes are located between atp6 and tRNA-Lys, and between tRNA-Pro and rps, respectively. The GC contents of the E. album rrnS and rrnL genes are 34.54% and 31.82%, respectively, which is within the range of other Cordycipitaceae mitogenomes (Table 4).

For decades, there has been considerable debate concerning the validity of the taxonomical classification of the Engyodontium species. Regarding E. album, it was previously included in the Beauveria genus. In 1940, this genus was renamed Tritirachium and reclassified as a member of the Moniliaceae family. However, E. album was later re-assigned to the Engyodontium genus (Hoog, 1972). Due to insufficient morphological features, the phylogenetic framework of Engyodontium has been little explored, even though the sequences of the 18S and 28S ribosomal RNA genes, the nuclear ribosomal internal transcribed spacer, and the cox1 gene sequences are available (Seifert, 2009; Schoch et al., 2012). Alternatively, mt genome sequences may provide reliable genetic markers in examining the taxonomic status of E. album. Phylogenetic analysis indicated that species in Nectriaceae, Hypocreaceae, Clavicipitaceae, and Cordycipitaceae are well resolved. As a member of the Cordycipitaceae family, E. album showed, as expected, a close genetic relationship with the Cordycipitaceae family. This finding was also supported by AT/GC-skew values and sequence differences in PCGs at both the nucleotide and amino acid levels among five representative Hypocreales species. However, no exact data exist yet regarding other lineages of Hypocreales. Therefore, it would be meaningful if a comprehensive phylogeny of Hypocreales is performed in the future, after more mt genome data become available, especially the mitogenome sequences of genera with currently incomplete sequences, such as Engyodontium and Elaphocordyceps.

In conclusion, the complete nucleotide sequence of the E. album mt genome was determined in this study. Comparative analysis showed that the structure, organization, and gene content of E. album mtDNA are highly similar to that of species in the Cordycipitaceae family. The availability of the complete mt genome sequence of E. album provides novel genetic markers for exploring cryptic/sibling species relating to the Hypocreales order; for preventing infection; and for further studies of the epidemiology, biology, population genetics, and phylogenetic systematics of E. album.

Acknowledgments

This work was supported by funding from the Agricultural Science and Technology Innovation Program of CAAS (grant no. 20603020001002).

References

Balasingham S, Chalkias S, Balasingham A, Saul Z, Wickes BL and Sutton DA (2011) A case of bovine valve endocarditis caused by Engyodontium album. Med Mycol 49:430-434. [ Links ]

Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, et al. (2012) SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455-477. [ Links ]

Beaudet D, Nadimi M, Iffis B and Hijri M (2013) Rapid mitochondrial genome evolution through invasion of mobile elements in two closely related species of arbuscular mycorrhizal fungi. PloS One 8:e60768. [ Links ]

Botero-Castro F, Tilak M, Justy F, Catzeflis F, Delsuc F and Douzery EJ (2013) Next-generation sequencing and phylogenetic signal of complete mitochondrial genomes for resolving the evolutionary history of leaf-nosed bats (Phyllostomidae). Mol Phylogenet Evol 69:728-739. [ Links ]

Campbell CK and Johnson EM (2013) Identification of Pathogenic Fungi. John Wiley & Sons Press, New York, 337 p. [ Links ]

Eldarov MA, Mardanov AV, Beletsky AV, Dumina MV, Ravin NV and Skryabin KG (2015) Complete mitochondrial genome of the cephalosporin-producing fungus Acremonium chrysogenum. Mitochondrial DNA 26:943-944. [ Links ]

Ghikas DV, Kouvelis VN and Typas MA (2006) The complete mitochondrial genome of the entomopathogenic fungus Metarhizium anisopliae var. anisopliae: Gene order and trn gene clusters reveal a common evolutionary course for all Sordariomycetes, while intergenic regions show variation. Arch Microbiol 185:393-401. [ Links ]

Goettel MS, Hajek AE, Siegel JP and Evans HC (2001) Fungi as biocontrol agents: Progress, problems and potential. In: Butt TM, Jackson C and Magan N (eds) Fungi as Biocontrol Agents: Progress Problems and Potential. CABI Press, Wallingford, pp 347. [ Links ]

Henze K and Martin W (2003) Evolutionary biology: Essence of mitochondria. Nature 426:127-128. [ Links ]

Hoog GD (1972) The genera Beauveria, Isaria, Tritirachium and Acrodontium gen. nov. Stud Mycol 1:1-41. [ Links ]

Huelsenbeck JP and Ronquist F (2001) MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17:754-755. [ Links ]

Hu M, Chilton NB and Gasser RB (2004) The mitochondrial genomics of parasitic nematodes of socio-economic importance: Recent progress, and implications for population genetics and systematics. Adv Parasitol 56:133-212. [ Links ]

Katoh K, Kuma K, Toh H and Miyata T (2005) MAFFT version 5: Improvement in accuracy of multiple sequence alignment. Nucleic Acids Res 33:511-518. [ Links ]

Kurbalija Novicic Z, Immonen E, Jeli M, AnDelkovic M, Stamenkovic-Radak M and Arnqvist G (2015) Within-population genetic effects of mtDNA on metabolic rate in Drosophila subobscura. J Evol Biol 28:338-346. [ Links ]

Li Y, Hu XD, Yang RH, Hsiang T, Wang K, Liang DQ, Liang F, Cao DM, Zhou F, Wen G, et al. (2015) Complete mitochondrial genome of the medicinal fungus Ophiocordyceps sinensis. Sci Rep 5:13892. [ Links ]

Librado P and Rozas J (2009) DnaSP v5: A software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25:1451-1452. [ Links ]

Liu GH, Zhou DH, Zhao L, Xiong RC, Liang JY and Zhu XQ (2014) The complete mitochondrial genome of Toxascaris leonina: Comparison with other closely related species and phylogenetic implications. Infect Genet Evol 21:329-333. [ Links ]

Lohse M, Drechsel O and Bock R (2007) Organellar Genome DRAW (OGDRAW): A tool for the easy generation of high-quality custom graphical maps of plastid and mitochondrial genomes. Curr Genet 52:267-274. [ Links ]

Lowe TM and Eddy SR (1997) tRNAscan-SE: A program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 25:955-964. [ Links ]

McDonnell PJ, Werblin TP, Sigler L and Green WR (1984) Mycotic keratitis due to Beauveria alba. Cornea 3:213-216. [ Links ]

Menzies J and Turkington T (2015) An overview of the ergot (Claviceps purpurea) issue in western Canada: Challenges and solutions. Can J Plant Pathol 37:40-51. [ Links ]

Oh J, Kong WS and Sung GH (2015) Complete mitochondrial genome of the entomopathogenic fungus Beauveria pseudobassiana (Ascomycota, Cordycipitaceae). Mitochondrial DNA 26:777-778. [ Links ]

Paquin B, Laforest MJ, Forget L, Roewer I, Wang Z, Longcore J and Lang BF (1997) The fungal mitochondrial genome project: Evolution of fungal mitochondrial genomes and their gene expression. Curr Genet 31:380-395. [ Links ]

Peden, JF (2000) Analysis of Codon Usage. University of Nottingham Press, Nottingham, 215 p. [ Links ]

Qiu F, Kitchen A, Beerli P and Miyamoto MM (2013). A possible explanation for the population size discrepancy in tuna (genus Thunnus) estimated from mitochondrial DNA and microsatellite data. Mol Phylogenet Evol 66:463-468. [ Links ]

Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL, Levesque CA, Chen W, Bolchacova E, Voigt K and Crous PW (2012) Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for fungi. Proc Natl Acad Sci U S A 109:6241-6246. [ Links ]

Seeliger HPR (1983) Infections of man by opportunistic molds - Their identification and nomenclature of their diseases. Mycoses 26:587-598. [ Links ]

Seifert KA (2009) Progress towards DNA barcoding of fungi. Mol Ecol Resour 9:83-89. [ Links ]

Sharp PM, Tuohy TM and Mosurski KR (1986) Codon usage in yeast: Cluster analysis clearly differentiates highly and lowly expressed genes. Nucleic Acids Res 14:5125-5143. [ Links ]

Siegel J and Shadduck J (1990) Safety of microbial insecticides to vertebrates: Humans. In: Laird M, Lacey LA and Davidson EW (eds) Safety of Microbial Insecticides. CRC Press, Boca Raton, pp 102-112. [ Links ]

Stamatakis A (2014) RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30:1312-1313. [ Links ]

Sung GH (2015) Complete mitochondrial DNA genome of the medicinal mushroom Cordyceps militaris (Ascomycota, Cordycipitaceae). Mitochondrial DNA 26:789-790. [ Links ]

Tablizo FA and Lluisma AO (2014) The mitochondrial genome of the red alga Kappaphycus striatus (“Green Sacol” variety): Complete nucleotide sequence, genome structure and organization, and comparative analysis. Mar Genomics 18:155-161. [ Links ]

Thompson JD, Gibson TJ and Higgins DG (2002) Multiple Sequence Alignment Using ClustalW and ClustalX. Current Protocols in Bioinformatics 2.3:2.3.1-2.3.22. [ Links ]

Tucker DL, Beresford CH, Sigler L and Rogers K (2004) Disseminated Beauveria bassiana infection in a patient with acute lymphoblastic leukemia. J Clin Microbiol 42:5412-5414. [ Links ]

Wang BJ, Gu XB, Yang GY, Wang T, Lai WM, Zhong ZJ and Liu GH (2016) Mitochondrial genomes of Heterakis gallinae and Heterakis beramporia support that they belong to the infraorder Ascaridomorpha. Infect Genet Evol 40:228-235. [ Links ]

Wick RR, Schultz MB, Zobel J and Holt KE (2015) Bandage: Interactive visualisation of de novo genome assemblies. Bioinformatics 31:3350-3352. [ Links ]

Xin B, Lin R, Shen B, Mao Z, Cheng X and Xie B (2017) The complete mitochondrial genome of the nematophagous fungus Lecanicillium saksenae. Mitochondrial DNA A DNA Mapp Seq Anal 28:52-53. [ Links ]

Zhang YJ, Zhang S and Liu XZ (2016) The complete mitochondrial genome of the nematode endoparasitic fungus Hirsutella minnesotensis. Mitochondrial DNA A DNA Mapp Seq Anal 27:2693-2694. [ Links ]

Zimmermann G (2007) Review on safety of the entomopathogenic fungi Beauveria bassiana and Beauveria brongniartii. Biocontrol Sci Technol 17:553-596. [ Links ]

Associate Editor: Zhong-Feng Zhang

Received: November 29, 2016; Accepted: May 07, 2017

Send correspondence to Zhong-Feng Zhang. Tobacco Research Institute of the Chinese Academy of Agricultural Sciences, 11 Keyuan Fourth Road, Qingdao 266101, P.R.China. E-mail: zhangzhongfeng@caas.cn.

#

These authors contributed equally to this study and share first authorship.

Creative Commons License License information: This is an open-access article distributed under the terms of the Creative Commons Attribution License (type CC-BY), which permits unrestricted use, distribution and reproduction in any medium, provided the original article is properly cited.