The complete mitochondrial genome of Neoporphyra hollenbergii (E. Y. Dawson) L.-E. Yang & J. Brodie, an endemic species from the Gulf of California, Mexico

Abstract

Neoporphyra hollenbergii is an endemic red alga from the Gulf of California, a region with notable cryptic diversity. We report its first complete mitochondrial genome and the first Bangiales mitogenome from the region. The circular mitogenome measures 31,565 bp (30.6% GC) and contains 50 genes, including 22 protein-coding genes, two rRNA genes, 24 tRNA genes, tatC, and orf813. The cox1 and rnl genes contain introns, reflecting red algal mitogenome complexity, while gene organization is conserved across Neoporphyra. Phylogenetic analyses place N. hollenbergii sister to N. perforata, clarifying regional Bangiales mitochondrial evolution.

Introduction

Neoporphyra hollenbergii (E.Y. Dawson) L.-E. Yang & J. Brodie (Rhodophyta, Bangiales) is an endemic species in the Gulf of California, Mexico, distributed along the central zone of the Baja California peninsula, from Punta Chivato to Bahía Agua Verde (López-Vivas et al. 2015). This species inhabits rocky coastal habitats in the mid- to high-intertidal zone, where thalli grow attached to hard substrata (López-Vivas et al. 2015). Phylogenetic relationships within the Bangiales are currently under revision because the boundaries among foliose Bangiales genera are not clear; more comprehensive analyses are required (Zuccarello et al. 2022; Li et al. 2025). The characterization and analysis of phylogenomic data have emerged as key tools contributing to taxonomic stability in the order (Li et al. 2025). In the Gulf of California, genetic diversity in the Bangiales is greater than expected, with molecular data revealing many cryptic species, some of which exhibit very restricted distributions (López-Vivas et al. 2015), possibly reflecting recent speciation processes. The study of mitogenomes from geographically under-studied regions such as the Gulf of California contributes to our understanding of relationships in the Bangiales and to our knowledge of the structure of red algal mitochondrial genomes.

Material and methods

Specimens of N. hollenbergii (Figure 1) were collected at its type locality, Bahía Agua Verde, B.C.S., Mexico (25°30′56.78″ N, 111°56′42.2″ W), and transported to the laboratory under cold conditions. A portion of the collected material was processed as herbarium voucher material, including pressed specimens and silica-gel-preserved tissue. Voucher specimens (FBCS 20349) were deposited at the Phycological Herbarium of the Universidad Autónoma de Baja California Sur. The remaining material was used to establish conchocelis cultures. In the laboratory, zygotospore release from zygotosporangium was induced by fragmenting fertile thallus portions with a sterile scalpel, and fragments from each thallus were isolated in Petri dishes. Cultures were maintained under controlled conditions at 20 °C, salinity 35 PSU, irradiance of 60 µmol photons m⁻² s⁻¹, 12 h light:12 h dark photoperiod, and nutrient-enriched seawater. Genomic DNA was extracted from cultured conchocelis material following the protocol of Healey et al. (2014) with minor modifications. Whole-genome sequencing was performed on the Illumina NovaSeq X Plus (PE150) at Novogene GmbH, Munich, Germany. Genomes were assembled de novo in Geneious Prime (v2025.2.2; https://www.geneious.com) using the Geneious Assembler. Assembled genomes were annotated in Geneious Prime using a local reference database built from Bangialean mitochondrial genomes available in NCBI. Reference mitogenomes were imported into Geneious Prime, and annotations of protein-coding genes, rRNA genes, tRNA genes, and open reading frames were transferred to the assembled mitochondrial genome based on sequence similarity. Protein-coding genes and open reading frames were further checked using BLASTx searches against Bangialean mitochondrial references.

Figure 1.

Specimens of Neoporphyra hollenbergii (FBCS 20349) collected at Bahía Agua Verde B.C.S., Mexico..

Codon usage analysis was conducted using the mitochondrial protein-coding genes of Neoporphyra hollenbergii and selected closely related Neoporphyra mitogenomes, including its sister taxon N. perforata and two additional Neoporphyra species, N. dentata and N. haitanensis. Coding sequences were analyzed in MEGA using the protozoan mitochondrial genetic code. Codon frequencies and relative synonymous codon usage (RSCU) values were calculated for each species. Codons exported by MEGA in RNA notation were converted to DNA notation for reporting, and stop codons were excluded from the interpretation of synonymous codon usage patterns. The genomic map was generated in OGDRAW v1.3.1 (Greiner et al. 2019). Phylogenetic analyses were performed using a concatenated dataset comprising 22 mitochondrial protein-coding genes and the mitochondrial rns gene from 19 Bangiales species, with three Gracilariales species used as outgroups (Supplementary Table 1). Individual genes were aligned using Clustal Omega (Sievers et al. 2011) in Geneious Prime v2025.2.2 and concatenated into a single matrix. Model selection was conducted in jModelTest v2.1.10 (Darriba et al. 2012) independently for each gene fragment (22 protein-coding genes and the mitochondrial rns region), and the best-fit substitution model for each partition is provided in Supplementary Table 2. Bayesian inference was performed in BEAST v2.7.5 with 10,000,000 generations using a partitioned scheme, where each gene fragment was assigned its corresponding substitution model as determined by jModelTest. Maximum likelihood analysis was conducted using RAxML v8 (Stamatakis 2014) with 1,000 bootstrap replicates. For the ML analysis, the GTR + I + G model was applied to the concatenated dataset as a single model across all partitions.

Results

The complete mitochondrial genome of Neoporphyra hollenbergii with GenBank Accession No. PX910200 (Figure 2) was assembled in a single contig with an average coverage of 46.15X (Figure S1). The complete mitochondrial DNA sequence (mtDNA) of N. hollenbergii is a single circular molecule of 31,565 bp, with a guanine-cytosine (GC) content of 30.6%. The genome contains 50 genes: 18 respiratory protein-coding genes (nad1, nad2, nad3, nad4, nad4L, nad5, nad6, sdh2, sdh3, sdh4, cob, cox1, cox2, cox3, atp4, atp6, atp8, and atp9), four ribosomal protein genes (rpl16, rps3, rps11, and rps12), two ribosomal RNA genes (rnl and rns), 24 tRNA genes, tatC gene, and 1 open reading frame, orf813, in the intron within rnl (Supplementary Table 3). All annotated genes exhibit canonical start codons (ATG), except for cox1 and cox2, which contain non-canonical initiation codons (GTG and TTG, respectively), a feature commonly reported in red algal mitochondrial genomes, including species in the Bangiales (Yang et al. 2015; Park and Lee 2020). The cox1 and rnl genes are interrupted by introns. Two genes, tatC and trnF(GAA), overlap by 10 bp. Codon usage analysis showed a conserved pattern among N. hollenbergii and the three compared Neoporphyra species. In N. hollenbergii, 5,306 sense codons were analyzed after excluding stop codons. Among these, 4,368 codons (82.32%) ended in A or T, whereas 938 codons (17.68%) ended in G or C. A similar preference for A/T-ending codons was observed in N. perforata, N. dentata, and N. haitanensis. The most strongly preferred codons in N. hollenbergii included TTA for leucine, AGA for arginine, TCT for serine, CCT for proline, GCT for alanine, and GGT for glycine. The complete codon usage and RSCU values are provided in Supplementary Table 4.

Figure 2.

The mitochondrial genome map of Neoporphyra hollenbergii (PX910200). External ring shows gene positions, with forward-strand genes annotated on the exterior and reverse-strand genes on the interior. Internal ring shows the %GC content. Gene categories are color-coded; see legend at bottom.

The phylogenetic reconstruction yielded a highly supported topology for both maximum likelihood bootstrap values and Bayesian posterior probabilities (Figure 3). This analysis confirms the monophyly of the genus. In the Neoporphyra clade, Neoporphyra hollenbergii was a sister species to Neoporphyra perforata from Baja California, Mexico, and California, USA, with strong bootstrap support (1/86). The Asian Neoporphyra species N. dentata, N. haitanensis, and N. seriata were the most closely related group to the American clade (1/100). Pyropia kanakaensis, Py. endiviifolia, and Py. pulchra formed a distinct clade related to Neoporphyra (0.98/100). The Neopyropia clade is related to Phycocalidia sp. from Myanmar (1/100). These two clades were distantly related to the basal branch of the Bangiales (.98/55) composed of Wildemania schizophylla, Bangia fuscopurpurea, B. atropurpurea, Porphyra purpurea, and P. umbilicalis (Figure 3).

Figure 3.

Maximum likelihood and Bayesian phylogeny of concatenated 22 protein-coding genes and the mitochondrial rns region with nineteen species in the Bangiales and Gracilariopsis and Gracilaria species as an outgroup. Node values indicate Bayesian posterior probabilities (left) and ML bootstrap values (right). GenBank accession numbers are shown after each taxon name in the tree. Accession numbers and source references for comparative mitochondrial genomes follow Li et al. (2025), Supplementary Table S1. The following mitochondrial genome sequences were used: Neoporphyra hollenbergii PX910200 (this study); Bangia fuscopurpurea OUPT01 NC_026905 (Bi et al. 2023, unpublished); Bangia atropurpurea SAG 1351-1 NC_062398 (Beveren et al. 2023, unpublished); Neoporphyra dentata NC_081968 (Lee et al. 2024, unpublished); Neoporphyra haitanensis NC_017751 (Mao et al. 2012); Neoporphyra perforata KJ708768 and KJ708769 (Hughey et al. 2014); Neoporphyra seriata OR123500 (Lee et al. 2024); Neopyropia fucicola NC_024288 (Hughey et al. 2014); Neopyropia tenera NC_021475 (Hwang et al. 2013); Neopyropia yezoensis MK695879 (Xu et al. 2019); Porphyra purpurea NC_002007 (Burger et al. 1999); Porphyra umbilicalis NC_018544 (Smith et al. 2012); Pyropia endiviifolia KU356193 (Xu et al. 2018); Pyropia kanakaensis NC_024289 (Hughey et al. 2014); Pyropia nitida NC_027616 (Harden et al. 2016); Pyropia pulchra MT588076 (Park and Lee 2020); Wildemania schizophylla NC_024579 (Silva and Hughey 2016); Phycocalidia Myanmar B LC761957 (San et al. 2023); Gracilariopsis lemaneiformis JQ071938 (Zhang et al. 2012); Gracilaria tenuistipitata KY054924 (Xu et al. 2019, unpublished); and Gracilaria salicornia KT373903 (Song et al. 2016).

Discussion and conclusion

This study reports the complete mitochondrial genome of Neoporphyra hollenbergii, representing the first mitogenome from a species in the Bangiales from the Gulf of California. The mitogenome composition and organization are highly conserved relative to other Neoporphyra species, containing the typical set of respiratory, ribosomal, and tRNA genes (Park and Lee 2020; Choi et al. 2022). The codon usage profile of N. hollenbergii was strongly biased toward codons ending in A or T, consistent with the low GC content of its mitochondrial genome. This pattern was shared with N. perforata, N. dentata, and N. haitanensis, suggesting that synonymous codon preference is conserved among closely related Neoporphyra mitogenomes. The presence of introns in the cox1 and rnl regions is consistent with the structural complexity commonly observed in red algal mitogenomes (Yang et al. 2015). Phylogenetic analyses reliably placed N. hollenbergii in the Neoporphyra clade, closely related to N. perforata, the geographically closest species with an available mitogenome (Hughey et al. 2014). These findings add to documented mitogenomic diversity in the Bangiales and support future research on red algal mitochondrial genome structure in the Gulf of California.

Funding Statement

This work was supported by the Secretary of Science, Humanities, Technology and Innovation (SECIHTI) through project CONACYT CB 2016 and the national scholarship (No. 833840) awarded to Gustavo Ceballos.

Disclosure statement

No potential conflict of interest was reported by the author(s). Specimen collection was carried out under the authorization of the relevant local authorities in Mexico (PAF/DGOPA-004). All sampling procedures complied with national and regional regulations. The study did not involve endangered or protected species.

Data availability statement

The data generated in this study are available at NCBI. The mitochondrial genome sequence was deposited in GenBank under accession PX910200. Raw Illumina sequencing reads were deposited in the NCBI Sequence Read Archive (SRA) under BioProject accession PRJNA1369157, BioSample accession SAMN53383584, and run accession SRR36181203.