The second complete mitochondrial genome of Capillidium rhysosporum within the family Capillidiaceae, Entomophthorales

Abstract

The complete mitochondrial genome of the entomophthoroid fungus Capillidium rhysosporum (strain no.: ATCC 12588) was sequenced using next-generation sequencing technology. The assembled circular genome has a length of 46,756 base pairs with a GC content of 27.06%. Gene prediction identified 15 core protein-coding genes (PCGs), two rRNA genes, and 27 tRNA genes. Phylogenetic analysis confirmed that C. rhysosporum belongs to the Zoopagomycota clade and is closely related to C. heterosporum. This study presents the second complete mitochondrial genome within the family Capillidiaceae, contributing to the mitochondrial DNA database of entomophthoroid fungi.

Introduction

The genus Capillidium B. Huang & Y. Nie 2020 was recently distinguished from Conidiobolus (Brefeld 1884) based on its unique characteristic of capilliconidia arising from conidia (Nie et al. 2020). Subsequently, it was reclassified into the family Capillidiaceae Y. Nie, Stajich & K.T. Hodge 2022 based on molecular data, ancestral lifestyles, and morphological features (Gryganskyi et al. 2022). With the addition of two more species in 2022, there are now 10 known species in this genus (Nie et al. 2022).

In a previous study, we reported the mitochondrial genome of Capillidium heterosporum (Drechsler) B. Huang & Y. Nie 2020 as the first complete mitochondrial genome in the family of Capillidiaceae. Comparative analysis revealed it to have the second-highest number of introns among 22 basal fungi (Nie et al. 2019). Here, we present the sequencing and analysis of the second complete mitochondrial genome of Capillidiaceae, providing insights into phylogenetic relationships and evolutionary histories among entomophthoroid fungi.

Materials and methods

The ex-type strain ATCC 12588 of C. rhysosporum was obtained from the American Type Culture Collection (Manassas, VA), and this species was known for its rough zygospores, and was isolated from decaying plant debris near Laplace (38°32′ N, 76°59′ W), Louisiana, United States in 1952 (the original descriptive image were shown in Figure 1). Fungal cultures were incubated on PDA for seven days at 21 °C. Total genomic DNA was extracted from fresh fungal mycelia using a modified CTAB method (Watanabe et al. 2010). Duplicate specimens and genomic DNA were deposited at the Research Center for Entomogenous Fungi (RCEF), Anhui Agricultural University (www.ahau.edu.cn, Professor Bo Huang, Email: bhuang@ahau.edu.cn), Hefei, Anhui, China under the voucher number RCEF 20081012. The constructed paired-end libraries with 300 bp inserts was used for Illumina HiSeq X-ten sequencing (NextOmics Biosciences, Co., Ltd., Wuhan, China) following the manufacturer’s instructions (Bioo Scientific, Austin, TX, AIR™ Paired-End DNA Sequencing Kit). Quality assessment was performed to obtain clean reads from the raw sequencing data by FastQC 0.11.8 (Andrews 2010). The sequencing depth and coverage map were provided in the supplementary materials (Supplementary Figure S1). The mitogenome was assembled using Norgal 1.0 (Al-Nakeeb et al. 2017), and gene annotation was conducted as previously described (Zhang et al. 2017; Li et al. 2021), with tRNA genes identified using tRNAscan-SE v1.3.1 (Lowe and Eddy 1997). Circular mitogenome maps were drawn with PMGmap (Zhang et al. 2023, http://www. 1kmpg.cn/pmgmap). CPGView (http://www.1kmpg.cn/cpgview/) was utilized to illustrate the structures of intron-containing genes as shown in Supplementary Figure S2.

Figure 1.

Capillidium rhysosporum as found in maize-meal agar cultures; ×1000. (1–3) Stout conidiophores bearing globose conidia. (4–17) Detached globose conidia. (18–20) Conidia in two stages of repetitional development. (21, 22) Conidia germinating on agar already permeated with mycelium of fungus. (23) Hypha with two non-adjacent segments, a and b, that are ready to conjugate by union of their terminally swollen branches, c and d. (24) Same, 45 min. Later, the two segments, a and b, now nearly empty from migration of contents into the enlargement c. (25) Same, after another interval of 45 min; young zygospore now full-grown (Drechsler 1954).

Mitochondrial genome sequences from fungi in four phyla of Blastocladiomycota, Chytridiomycota, Mucoromycota, and Zoopagomycota were downloaded from GenBank, with two Ascomycetes (Yarrowia lipolytica and Colletotrichum acutatum), and one Basidiomycete (Lactifluus hygrophoroides) were chosen as outgroups. A combined mitochondrial gene dataset of 14 proteins (atp6, atp8, atp9, nad1, nad2, nad3, nad4, nad4L, nad5, nad6, cob, cox1, cox2, and cox3) was used for phylogenetic analysis. Individual amino acid sequences were aligned using MEGA v6.06 (Caspermeyer 2016) and then concatenated with SequenceMatrix v1.7.8 (Vaidya et al. 2011). The best model, GTR + G + I, for the maximum-likelihood (ML) analysis was selected based on the Akaike information criterion (AIC) using Modeltest 3.7 (Posada and Crandall 1998). A phylogenetic tree using the ML method was constructed with RAxML 8.1.17 with 1000 bootstrap replicates (Stamatakis 2014). The BI analysis was performed with MrBayes 3.2.2 (Ronquist and Huelsenbeck 2003). Phylogenetic trees were modified with FigTree 1.4 (Rambaut 2012).

Results

The complete mitogenome sequence of C. rhysosporum (GenBank accession number MZ286627, Nie et al. 2019) is 46,756 base pairs in length, comprising two ribosomal RNA genes (rnl and rns), 27 tRNA genes, 15 conserved protein-coding genes (PCGs), and one free-standing ORF. The PCGs include ATP synthase (atp6, atp8, and atp9), NADH dehydrogenase (nad1, nad2, nad3, nad4, nad4L, nad5, and nad6), ubiquinol cytochrome c reductase (cob), cytochrome c oxidase (cox1, cox2, and cox3), and ribosomal protein (rps3). Twenty-seven introns were detected in seven genes, with one in atp6 and nad4, two in nad1, three in cox2, five in nad5, seven in cox1, and eight in cob (Figure S2). The complete circular mitogenomes of C. rhysosporum are shown in Figure 2. In the phylogenetic tree (Figure 3), C. rhysosporum was located in the clade of Zoopagomycota, C. rhysosporum exhibited a close phylogenetic relationship with C. heterosporum, consistent with previous studies (Nie et al. 2019; Sun et al. 2020).

Figure 2.

Circular complete mitogenome map of Capillidium rhysosporum genes is represented by different colored blocks. Genes outside and inside the outermost black ring line are transcribed in a clockwise and counter-clockwise direction, respectively. The inner circles represent the genome scale, GC content and distributions of short tandem repeats, long tandem repeats and the dispersed repeats, respectively. The colored parabola in the center circle represents the dispersed repeats.

Figure 3.

The phylogenetic tree based on 14 mitochondrion-encoded proteins. The 25 fungal mitogenomes were used in this phylogenetic analysis: Allomyces macrogynus (U41288, Paquin and Lang 1996), Blastocladiella emersonii (DQ287690, Tambor et al. 2008), Capillidium rhysosporum (MZ286627, this article), Capillidium heterosporum (MK049352, Nie et al. 2019), Chytriomyces confervae (MK292704, van de Vossenberg et al. 2018), Conidiobolus chlamydosporus (MZ436168, Nie et al. 2021), Conidiobolus lichenicolus (MZ436169, Nie et al. 2021), Conidiobolus mycophagus (MZ436170, Nie et al. 2021), Conidiobolus polyspermus (MZ436171, Nie et al. 2021), Conidiobolus polytocus (MZ436172, Nie et al. 2021), Conidiobolus sp. (MN640580, Sun et al. 2020), Conidiobolus taihushanensis (MZ436173, Nie et al. 2021), Gigaspora rosea (JQ693395, Nadimi et al. 2012), Gigaspora margarita (JQ041882, Pelin et al. 2012), Harpochytrium sp. JEL94 (AY182005, Bullerwell et al. 2003), Harpochytrium sp. JEL105 (AY182006, Bullerwell et al. 2003), Microconidiobolus nodosus (MW795365, Cai et al. 2021), Mortierella verticillata (AY863211, Seif et al. 2005), Neoconidiobolus thromboides (MW795364, Nie et al. 2021), Rhizophagus fasciculatus (KM586389, Nadimi et al. 2016), Synchytrium endobioticum (MK292696, van de Vossenberg et al. 2018), Zancudomyces culisetae (AY863213, Seif et al. 2005). Colletotrichum acutatum (KR349346, Kim et al. 2016), Lactifluus hygrophoroides (MH319477, Li et al. 2019), and Yarrowia lipolytica (CP017559, Magnan et al. 2016) were served as outgroups. The newly sequenced mitogenome is denoted in bold. Maximum-likelihood bootstrap values (1000 replicates) for each clade are indicated along the branches. The scale bar refers to 0.1 nucleotide substitutions per character.

Discussion and conclusions

In this article, we reported another complete mitogenome of the genus Capillidium with 46,756 bp in total length. The number and length of fungal mitogenomic introns have an implication for inferring the fungal mitogenome size (Friedrich et al. 2012; Torriani et al. 2014), and we noted that the number of introns (27) was fewer than that of C. heterosporum (30). The rps3 gene encodes protein component of the ribosome for protein translation (Bullerwell et al. 2000), which was annotated in the mitogenome of C. rhysosporum. As a note, the mitogenome of C. rhysosporum is the second complete mitogenome in the family Capillidiaceae, which enhances our understanding of the evolution of this fungal group.

In summary, this study presents another complete mitochondrial genome within the family Capillidiaceae. We also conducted the phylogenetic analysis with other related entomophthoroid fungi based on the sequences of 14 protein coding genes, which reveal a close relationship with C. heterosporum. The comprehensive analysis of its mitochondrial genome contributes valuable insights into the phylogenetic relationships and evolutionary history of entomophthoroid fungi.

Funding Statement

This project was supported by the National Natural Science Foundation of China [No. 32370007].

Author contributions

Conceived and designed the experiments: BH. Performed the experiments: HL. Analyzed the data: HL and YN. Wrote the paper: HL, YN, and BH. All authors read and approved the final manuscript.

Ethical approval

This specimen is not an endangered or protected species. Therefore, permission is not required to collect this species and no ethical issues were involved in this study. The collection of this fungus was legal and reasonable. Information of this specimen and who identified it were included in the manuscript.

Disclosure statement

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

Data availability statement

The data that support the findings of this study are openly available in GenBank of NCBI (https://www.ncbi.nlm.nih.gov). The accession number of mitogenome is MZ286627. The associated BioProject, SRA, and BioSample numbers are PRJNA1030606, SRR27393659, and SAMN37911642, respectively.