We investigated the phylogeography of B. gymnorhiza using complete chloroplast genomes and nuclear ribosomal DNA sequences from 14 provenances spanning the granitic Seychelles, Aldabra Atoll, Madagascar, and the African coast. Maximum-likelihood phylogenies and haplotype network analyses identified three Evolutionary Significant Units corresponding to the Seychelles, Aldabra, and Africa/Southwest Madagascar, highlighting a within-species conservation issue. Chloroplast and rRNA sequences are provided here in fasta format.

Study area

We sampled Bruguiera from 14 mangrove populations across the WIO. Nine populations were located along a 2800 km coastal stretch of the eastern African mainland, from northern Mozambique to the southern range limit in South Africa. Five populations were sampled from remote islands: two on opposite sides of Mahé Island (Seychelles), two from distinct lagoon parts of the Aldabra Atoll, and one from the southwestern coast of Madagascar. At each sample site, fresh leaves were collected from an individual adult tree, air-dried, and stored in bags with silica gel for transport. These 14 WIO sites were previously included in cpDNA and rRNA cistron sequences or nuclear microsatellites of Rhizophora mucronata (Triest et al., 2021; DOI: 10.5061/dryad.vmcvdnctd) and Avicennia marina (Triest et al., 2026; DOI: 10.5061/dryad.hmgqnk9wr).

DNA extraction and next generation sequencing

Genomic DNA extracts of 14 samples were made at the Vrije Universiteit Brussel (VUB) and processed for next generation sequencing analysis using the E.Z.N.A. SP plant DNA Mini Kit (Omega biotek, Norcross, GA, USA). Quantity and purity (260/280 and 260/230 ratios) of the DNA were determined using a Nanodrop one Spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Extractions were repeated for samples with a 260/280 ratio of less than 1.8 and/or a concentration lower than 5 ng/ul. An Illumina paired-end library was constructed using the TruSeq nano DNA Kit. After passing quality inspection (DNA concentration between 5 to 15ng/ul), the constructed library (TruSeq Nano DNA Kit) was sequenced by 300 bp x 2 paired-end sequencing in an Illumina MiSeq platform (Macrogen, Seoul, South Korea).

Chloroplast genome assembly, alignment and comparative analysis

Raw data was filtered out to remove the joint sequence and low-quality reads to obtain high-quality clean data. The Illumina pair-end next generation sequencing (NGS) products are used as the input file for de novo chloroplast assembles. A de novo chloroplast assembly was done at first for sample ALD2 (that showed the highest number of reads = 5 096,666 million) using NOVOPlasty assembly at Kmer = 33 (Dierckxsens et al., 2017). Assemblies were executed by taking a single read from the dataset that originates from the targeted plastid as seed (rbcL) and taking 30% as subsample from the FASTA file with default parameters. This assemble then was compared to the annotated B. gymnorhiza chloroplast genome (GenBank accession number MW402841 and appeared similar in genome structure and aligned over 163,411 bp. Thereafter, as an alternative and effective method, Illumina 2 x 300 bp paired-end of fourteen samples were processed in Geneious Prime v 2025.1.2 (© 2005-2025 GraphPad Software LLC d.b.a Geneious). Each sample was assembled using the ‘assemble to reference’ function in Geneious software with MW402841 as reference genome to obtain complete chloroplast genome sequences. This approach yielded assemblages that averaged 175,968 – 571,966 reads with a mean depth of reads ranging from 313 – 1039 coverage.

Because all B. gymnorhiza samples aligned well to the reference with a similar number and position of genes, CDS, and non-coding tRNA and rRNA, we refer to the annotated cp genome of MW402841 in Genbank and circular visualisation in Ruang-areerate et al. (2021).

Nuclear rRNA cistron assembly

The nuclear ribosomal cistron (18S, ITS1, 5.8S, ITS2 and 26S), including upstream and downstream flanking regions, was assembled from a Bruguiera gymnorhiza 625 bp sequence (Genbank HM366082, Sun and Lo, 2011) containing an internal transcribed spacer 1 (partial sequence), 5.8S ribosomal RNA gene (complete sequence) and internal transcribed spacer 2 (partial sequence) and subsequently used three times as a seed in Geneious Prime v 2025.1.2 to progressively enlarge the 18S, 26S and both (upstream and downstream) flanking regions. A >8,000 bp nuclear ribosomal cistron was obtained for one sample (ALD2) and further used as a reference to map every sample, averaging 5,584–46,644 reads with a mean depth of reads ranging from 203–1,787 coverage. The generated consensus sequences of 7,777 bp length for WIO samples were aligned for comparative description of mutated positions

Cited references

Dierckxsens, N., Mardulyn, P., and Smits, G. 2017. NOVOPlasty: de novo assembly of organelle genomes from whole genome data. Nucleic Acids Research 45: e18. https://doi.org/10.1093/nar/gkw955

Ruang-Areerate, P., Kongkachana, W., Naktang, C., Sonthirod, C., Narong, N., Jomchai, N., Maprasop, P., Maknual, C., Phormsin, N., Shearman, J.R., Pootakham, W., and Tangphatsornruang, S. 2021. Complete chloroplast genome sequences of five Bruguiera species (Rhizophoraceae): comparative analysis and phylogenetic relationships. PeerJ 9: e12268. https://doi.org/10.7717/peerj.12268

Sun, M. and Lo, E. Y. 2011. Genomic markers reveal introgressive hybridization in the Indo-West Pacific mangroves: a case study PLoS ONE 6(5): e19671. https://doi.org/10.1371/journal.pone.0019671