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Mitochondrial DNA B Resour
Mitochondrial DNA B Resour
Mitochondrial DNA. Part B, Resources
2380-2359
Taylor & Francis

10.1080/23802359.2024.2399925
2399925
Version of Record
Data Note
Mitogenome Report
Characterization and phylogenetic analysis of the complete mitochondrial genome of the red alga, Gracilaria eucheumatoides Harvey 1860 (Gracilariales: Gracilariaceae) from Vietnam
T. V. Nguyen et al.
https://orcid.org/0000-0003-4778-1927
Nguyen Tu Van a
https://orcid.org/0000-0002-7736-8845
Duc Luong Thien a
https://orcid.org/0009-0001-9121-4260
Thi Le Trang a
Lee Dae-Sung b
https://orcid.org/0000-0002-7125-5460
Yoon Moongeun b
https://orcid.org/0000-0002-7647-3766
Kim Keun-Yong c
https://orcid.org/0000-0002-1849-6089
Tran Biet Thanh c
https://orcid.org/0009-0005-4205-6218
Woo Jiyoung b
a Department of Ecology, Institute of Tropical Biology, Vietnam Academy of Science and Technology, Ho Chi Minh, Vietnam
b National Marine Biodiversity Institute of Korea, Seocheon, Republic of Korea
c Department of Genetic Analysis, AquaGenTech Co., Ltd., Busan, Republic of Korea
Supplemental data for this article can be accessed online at https://doi.org/10.1080/23802359.2024.2399925.

CONTACT Jiyoung Woo jywoo1022@mabik.re.kr National Marine Biodiversity Institute of Korea, Seocheon 33662, Republic of Korea
9 9 2024
2024
9 9 2024
9 9 11961200
27 5 2024
28 8 2024
KnowledgeWorks Global Ltd.9 9 2024
published online in a building issue9 9 2024
© 2024 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
2024
The Author(s)
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.

Abstract

The marine red alga, Gracilaria eucheumatoides, is economically significant for its agar production and pharmacologically active compounds. This study reveals its complete mitochondrial genome (mitogenome), sequenced using Illumina’s next-generation technology. The mitogenome is a 25,909 bp circular molecule with a G + C content of 27.21%, comprising 24 protein-coding genes, two ribosomal RNA genes, 24 transfer RNA genes, and one open reading frame (ORF) with an unidentified function. Both gene structure and composition are highly conserved within Gracilaria. The phylogenetic analyses fully support a close relationship of G. eucheumatoides with other Gracilaria species, as well as its sister relationship with G. urvillei. This mitogenome sequencing effort of G. eucheumatoides provides crucial data for future phylogenetic research on marine red algae.

Keywords

Mitogenome
phylogeny
red algae
Gracilariaceae
Gracilaria eucheumatoides
National Marine Biodiversity Institute of Korea (MABIK) 10.13039/501100009338 2024M00500 Vietnam National Foundation for Science and Technology Development (NAFOSTED) 10.13039/100007224 106.06-2019.338 This work was supported by the National Marine Biodiversity Institute of Korea (MABIK) from in-house Research Programs under Grant [2024M00500]; Vietnam National Foundation for Science and Technology Development (NAFOSTED) under Grant [106.06-2019.338].
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pmc1. Introduction

The red alga, Gracilaria eucheumatoides Harvey 1860, regarded as a synonym of Hydropuntia eucheumatoides (Harvey) Gurgel & Fredericq 2004, is a marine species in the family Gracilariaceae, found in the western Pacific and the southeastern Asia (Guiry and Guiry 2024). This species holds economic significance due to its production of agarans for the agar industry, and its bioactive compounds with potential pharmaceutical application (Smit 2004). High morphological variability among species of the Gracilariaceae presents challenges for species identification using traditional methods (Núñez-Resendiz et al. 2015).

The application of molecular-based taxonomy through complete mitochondrial genome (mitogenome) has become an invaluable approach to resolve taxonomic challenges (Yuan et al. 2017). This study presents the sequencing, annotation, and characterization of the complete mitogenome of G. eucheumatoides, which can be useful in exploring the phylogeny of marine red algae.

2. Materials and methods

A fresh specimen of G. eucheumatoides was collected in May 2023 from Son Hai village, Ninh Thuan province, Vietnam (11°25′13.0″N, 109°00′54.0″E) and preserved in absolute ethanol before being deposited in the Institute of Tropical Biology, Vietnam Academy of Science and Technology (https://vast.gov.vn/web/vietnam-academy-of-science-and-technology, Tu Van Nguyen, nvtu.itb@gmail.com) under a voucher no. VNM00029419 (Figure 1). The morphology of the collected specimen was characterized by its thalli forming dense clusters that were compressed and thick, and ranged from dichotomous to pinnate with dentate margins (Tsutsui et al. 2005; Kim et al. 2022).

Figure 1. Specimen image of the red alga, Gracilaria eucheumatoides collected from Son Hai village, Ninh Thuan province, Vietnam. Arrows point to the G. eucheumatoides clumps with compressed and thick branches, ranging from dichotomous to pinnate with dentate margins. This photograph was taken by Tu Van Nguyen on 20 April 2023.

Genomic DNA (gDNA) of G. eucheumatoides was extracted from thallus, using the E.Z.N.A.® Plant DNA Kit (Omega Bio-tek Inc., Norcross, GA), following the manufacturer’s protocol. The quality of the extracted gDNA was evaluated using NanoDrop One Microvolume UV-Vis Spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE). Four overlapped long-range PCR amplifications covering the entire mitogenome of G. eucheumatoides, with amplicon sizes ranging from approximately 7300–8900 bp, were carried out using the Invitrogen™ Platinum™ SuperFi™ PCR kit (Thermo Fisher Scientific Inc., Wilmington, DE) and primer sets specifically designed for this study (Supplementary Table S1). These primers were designed by targeting highly conserved regions identified through alignment of all mitogenomic sequences from Gracilariaceae species in the NCBI (National Center for Biotechnology Information) GenBank database (Supplementary Table S2), with Hydropuntia urvillei (GenBank accession number MZ336095) used as the reference to anchor primer positions. PCR amplifications were conducted using the ProFlex™ PCR System (Thermo Fisher Scientific Inc., Wilmington, DE). The successful amplified products were pooled together and underwent library preparation with the Illumina DNA Prep kit (Illumina Inc., San Diego, CA) for next-generation sequencing. The prepared library was sequenced on an Illumina iSeq100 sequencing system (Illumina Inc., San Diego, CA) with 150-bp paired-end runs (2 × 150).

A total of 1,256,840 raw reads obtained were processed by trimming adapters, filtering out low quality reads (Q < 20) and short reads (<50 bp), and removing duplicates, using Geneious Prime v.2023.2.1 (Biomatters, Auckland, New Zealand). The clean reads were assembled de novo using Geneious Prime with default parameters. Contigs longer than 10 kbp were validated by aligning them with the annotated mitogenome of G. rangiferina (GenBank accession number MH396020) using the MAFFT alignment tool (Katoh and Standley 2013) in Geneious Prime with default parameters. The contigs showing the highest similarity to the G. rangiferina mitogenome were selected as the draft mitogenome sequence. This draft sequence was then used as a reference to map the clean reads using the ‘Map to Reference’ function in Geneious Prime with default parameters. A total of 956,323 reads were mapped to the draft sequence with the average coverage depth of ×4772 (Supplementary Figure S1). The consensus sequence, determined by the highest quality base call at each nucleotide position, was annotated using the Open Reading Frame Finder web server (https://www.ncbi.nlm.nih.gov/orffinder/) and the MITOS web server (Bernt et al. 2013). Transfer RNA (tRNA) genes were identified by tRNAscan-SE 1.21 (Lowe and Eddy 1997). The RNAweasel tool was used to validate the RNA annotations and detect introns (Lang et al. 2007). The arrangement and direction of the annotated genes were illustrated using GenomeVx (Conant and Wolfe 2008). The annotated sequence was submitted to the GenBank database and accessible under the accession number, OR778082.

Eight mitogenomic sequences of Gracilaria species, including G. eucheumatoides, were selected for phylogenetic analyses based on their high sequence similarity to G. eucheumatoides, as determined by a percent identity matrix following MAFFT alignment using Geneious Prime v.2023.2.1 (Table 1). Gracilariopsis chorda (KC875851) and Gracilariopsis tenuifrons (MZ336099) were designated as the outgroups. The nucleotide sequences of all protein-coding genes (PCGs) shared among these 10 mitogenomic sequences were first extracted, then aligned using MAFFT within Geneious Prime, and finally concatenated for constructing the maximum-likelihood (ML) tree and performed the Bayesian inference (BI) analysis. Both analyses employed the GTRGAMMAI model, selected by jModelTest v.2 (Darriba et al. 2012). The ML tree was generated using RAxML v.7.0.4 (Stamatakis 2006; Stamatakis et al. 2008) with 1000 nonparametric bootstrap (BS) inferences. The BI analysis was carried out with MrBayes v.3.1.2 (Ronquist et al. 2012), running for 1,000,000 generations until the average standard deviation of split frequencies fell below 0.01, discarding the first 25% of samples as burn-in. TreeView X v. 0.5.0 (Page 2002) was used for tree visualization.

Table 1. List of accession number and publication used in the phylogenetic tree.

Genus	Species	GenBank accession number	Reference	
Gracilaria	Gracilaria cervicornis	MZ336080	Lyra et al. (2021)	
Gracilaria domingensis	MZ336085	Lyra et al. (2021)	
Gracilaria gracilis	MH396019	Iha et al. (2018)	
Gracilaria ferox	MH396018	Iha et al. (2018)	
Gracilaria edulis	MG592725	Sedanza et al. (2020)	
Gracilaria eucheumatoides	OR778082	This study	
Gracilaria rangiferina	MH396020	Iha et al. (2018)	
Gracilaria urvillei	MZ336095	Lyra et al. (2021)	
Gracilariopsis (outgroups)	Gracilariopsis chorda	KC875851	Yang et al. (2014)	
Gracilariopsis tenuifrons	MZ336099	Lyra et al. (2021)	

3. Results

The complete mitogenome of G. eucheumatoides is a circular DNA molecule of 25,909 bp, containing 51 genes: 24 PCGs, two ribosomal RNA (rRNA) genes, 24 tRNA genes, and one open reading frame (ORF) with an unidentified function (Figure 2). The mitogenome has a G + C content of 27.21%. All PCGs start with an ATG codon and terminate with TAA or TAG codon. The tRNA gene for trnI, situated between nad4 and nad5, is interrupted by a 456 bp long intron (Supplementary Figure S2).

Figure 2. The circular mitogenome map of Gracilaria eucheumatoides analyzed in this study. Genes encoded on the forward strand and reverse strands are illustrated outside the circle and inside the circle, respectively.

The ML tree shows that G. eucheumatoides clusters with other three Gracilaria species: G. rangiferina, G. edulis, and G. urvillei with full support (BS = 100% and posterior probability, PP = 1) (Figure 3). In addition, it fully supports the sister relationship between G. eucheumatoides and G. urvillei.

Figure 3. Maximum-likelihood (ML) tree based on concatenated nucleotide sequences from all protein-coding genes of complete mitogenomes of species within the family Gracilariaceae. The newly assembled mitogenome of Gracilaria eucheumatoides was highlighted in bold. Two species belonging to the genus Gracilariopsis were selected as the outgroups. Numbers above each node correspond to ML bootstrap probabilities followed by the Bayesian posterior probabilities. Scale bar represents nucleotide substitutions per site.

4. Discussion and conclusions

The mitogenomic features of G. eucheumatoides are consistent with the typical compositions observed in red algal species. Compared to its congeners, G. eucheumatoides exhibits a highly conserved mitogenomic structure and composition with only minor variations. The mitogenomic composition of G. eucheumatoides are similar to those of G. rangiferina, G. edulis, and G. urvillei, except for the absence of an ORF and trnI genes in G. edulis and the triplication of trnY, trnR, and trnS in G. rangiferina (Iha et al. 2018; Sedanza et al. 2020; Lyra et al. 2021). The gene order between trnA and trnN in G. eucheumatoides is similar to that in G. rangiferina, G. edulis, and G. urvillei but inverted in other species, with G. cervicornis notably lacking trnY (Iha et al. 2018; Lyra et al. 2021). However, these observed differences might be due to annotation artifacts. Automated tools, despite their robustness, can lead to discrepancies, particularly across species with varying genomic complexity. Manual curation, experimental verification, or further comparative analysis is necessary to confirm whether these differences are genuine or result from annotation errors. The phylogenetic analyses demonstrate a close relationship of G. eucheumatoides with other Gracilaria species, and support for its sister relationship with G. urvillei.

Supplementary Material

Supplementary Materials R2.docx

Acknowledgements

Special thanks to AquaGenTech Co. Ltd., Republic of Korea, for providing expert consultation and support during the experimental design stage, troubleshooting issues, refining the project, participating in investigations and data analysis, as well as drafting and refining the manuscript.

Author contributions

Conception and design of the work: TVN, KYK, MGY, BTT, and JYW; investigation and data analysis: TDL, TTL, DSL, BTT; data interpretation: TVN, KYK, and BTT; writing – original draft preparation: TVN and BTT; writing – review and editing: TVN, TDL, TTL, DSL, MGY, KYK, BTT, and JYW; resources: TVN, TDL, and TTL; supervision and funding acquisition: JYW and TVN. All authors agreed to be accountable for all aspects of the work and approved the final version of the manuscript.

Ethics statement

This study was approved by the Ethics Committee of Institute of Tropical Biology, Vietnam Academy of Science and Technology, Vietnam. This species is not endangered or collected in nature reserves, so it did not need any specific permissions for sample collection or access to lands during field work.

Disclosure statement

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

Data availability statement

The mitogenomic sequence data that support the findings of this study are openly available in GenBank database of NCBI under the accession number, OR778082. The associated BioProject, SRA, and BioSample numbers are PRJNA1037608, SRR26779580, and SAMN38194450, respectively.
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