The complete mitochondrial genome of the benthic diatom Pleurosigma inscriptura

Abstract

Pleurosigma inscriptura M. A. Harper 2009 is a marine diatom in Naviculales (Bacillariophyceae) order distributed in New Zealand, South America, Argentina, and Korea. We assembled the complete mitochondrial genome sequence of Pleurosigma inscriptura (38,013 bp), and annotated 34 protein-coding genes, 25 transfer RNAs, and 2 ribosomal RNAs. We analyzed a maximum-likelihood tree using conserved 34 mitochondrial genes from Bacillariophyta species. In the mitochondrial phylogeny, P. inscriptura showed a strong monophyletic relationship with Haslea nusantara and Navicula ramosissima.

Keywords:

• Pleurosigma inscriptura
• Bacillariophyceae
• mitochondrial genome
• phylogenetic analysis

Pleurosigma inscriptura M. A. Harper 2009, belongs to order Naviculales (Bacillariophyceae), is a kind of marine pennate diatom which is one of the important primary producers (Nelson et al. 1995; Harper et al. 2009) . Pleurosigma lives in a benthic environment, and includes epiphytic species with gliding movement over the surface area (Happey-Wood and Jones 1988; Sterrenburg 1991; Grossi et al. 2004; Al-Handal and Wulff 2008; Poulin et al. 2014). Pleurosigma was characterized by sigmoid raphe having a convex valve, and striated areola (Harper et al. 2009; Sar et al. 2012; Al-Handal et al. 2014; Poulin et al.2014). The valve outline of P. inscriptura is lanceolate with a gently sigmoid raphe, and their raphe angle is smaller than other Pleurosigma species (Hasle and Syvertsen 1996; Harper et al. 2009). Pleurosigma inscriptura is distributed in New Zealand, South America, Argentina, and Korea (Sar et al. 2013; Harper et al. 2012; Lee and Park 2015). Since P. inscriptura was reported as a novel species (Harper et al. 2009), there is no report about molecular data in public databases (e.g. NCBI). In this study, we analyzed the mitochondrial genome of P. inscriptura (MW566731) isolated from Gijang, Busan, Korea (35°13′05.9″N, 129°13′47.3″E). The culture strain was deposited at Marine Ecological Genomics Lab. in Kyungpook National University (isolate MEG005; [email protected]).

To establish monoclonal strain of the target species, single cell isolation was performed by customized Pasteur pipettes (glass) under light microscope, and cultured under L1 medium (L1 media kit, https://ncma.bigelow.org/MKL150L). The isolate was identified by light microscopic (LM; Nikon ECLIPSE Ts2, Nikon, Tokyo, Japan) and scanning electron microscopic (SEM; Hitachi SU8220; Hitachi Ltd., Tokyo, Japan) observation based on previously described morphological characteristics (Harper et al. 2009; Lee and Park 2015). The morphological characteristics of P. inscriptura MEG005 are as follows: length of apical axis 104–115 μm (avg. 111 μm, n = 100), width of transapical axis 15–25 μm (avg. 21 μm, n = 100; Supplementary Figure S1(A)), lanceolate valves, 3.6° raphe angle (Supplementary Figure S1(B)), a small oval central nodule with no hyaline areas (Supplementary Figure S1(C)), 20 striae in 10 μm and small triangle of internal polar raphe ends (Supplementary Figure S1(D)).

Figure 1. Maximum-likelihood (ML) tree using 34 concatenated mitochondrial proteins from eighteen Bacillariophyta and two Ochrophyta (outgroup) species.

We have done DNA extraction using the DNeasy Plant Mini-DNA kit (Qiagen, Hilden, Germany). Genome sequencing of P. inscriptura (NCBI BioProject PRJNA717901; SRR14087012, 12.5 Gbp) and their de novo genome assembly were conducted by Illumina Novaseq6000 (Illumina, San Diego, CA; 150 bp paired-end library), and SPAdes assembler (v3.14.2; Bankevich et al. 2012), respectively. Mitochondrial genome contigs of P. inscriptura were sorted by local BLASTn search (e-value cutoff = 1.e − 05) with the mitochondrial genome of Berkeleya fennica (KM886611) as reference, and re-assembled as a circular mitochondrial genome from the sorted contigs. The circular form of mitochondrial genome (MW566731) was validated using the read-mapping method (bowtie2 v2.4.2; Langmead and Salzberg 2012).

CLC Main Workbench v20.0 (CLC Bio, Aarhus, Denmark) was used to annotate mitochondrial genes that manually predicted by BLASTx search (NCBI nr database; e-value cutoff = 1.e − 05) with codon table 4 (The Mold, Protozoan, and Coelenterate Mitochondrial Code). Ribosomal RNAs (rRNAs) were predicted by the RNAmmer 1.2 Server (Lagesen et al. 2007). Transfer RNAs (tRNAs) were analyzed by tRNA scan-SE 2.0 (Lowe and Chan 2016; Chan and Lowe 2019) and ARAGORN programs (Laslett and Canback 2004), respectively. A total 34 protein-coding genes, 25 transfer RNAs, 2 rRNAs is annotated in the mitochondrial genome of P. inscriptura (MW566731). Intron sequences of protein coding genes are present in rps3 (two introns), nad2 and nad11 (one intron).

To construct the concatenated alignment of mitochondrial genes, conserved 34 protein-coding genes from 18 Bacillariophyta and two outgroup species were used, and each homologous gene set was aligned by MAFFT (v7.313; Katoh and Toh 2008) with default options. The maximum-likelihood (ML) tree using the concatenated alignment was constructed by IQ-tree program (v1.6.12; Nguyen et al. 2015) with the options as follows: the gene partition information (-q), the model test (-m TEST), and ultrafast bootstrapping with 1000 replications (-bb 1000). Pleurosigma inscriptura is monophyly with Haslea nusantara and Navicula ramosissima (bootstrap support 100%; Figure 1), and this clade is clustered with the clade of Berkeleya fennica, Fistulifera solaris, Didymosphenia geminata, Halamphora coffeaeformis, and Phaeodactylum tricornutum (bootstrap support 100%). In the clade, almost all species belong to Naviculales order but D. geminate and P. tricornutum are Cymbellales and Bacillariophyta classis incertae sedis, respectively (Guiry and Guiry 2021). In previous studies, D. geminate and P. tricornutum show monophyletic relationship with Naviculales order that the phylogenetic tree was constructed by 27 mitochondrial protein-coding genes (Aunins et al. 2018). Therefore, we postulate that D. geminate and P. tricornutum could be regarded as the Naviculales order. More mitochondrial genome samples in Naviculales order will provide a better understanding of phylogenetic relationship between genera.

Acknowledgments

The authors thank Hyun Shik Cho (Marine Ecological Genomics Lab., Department of Oceanography, Kyungpook National University, Daegu) for technical support.

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 at https://www.ncbi.nlm.nih.gov/genbank/, reference number ‘MW566731’. Raw reads of genome sequencing data in this study were uploaded to the NCBI Sequence Read Archive (SRA) database (BioProject PRJNA717901; SRR14087012).

Additional information

Funding

This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea Government (MSIT) [2020R1C1C1010193].