Microsatellites and chloroplast genome sequences of the mangrove (<em>Avicennia marina</em>) from Western Indian Ocean

Avicennia marina (Forssk.) Vierh is considered the most widely distributed mangrove species and a major constituent of Indo-West Pacific mangroves. To understand spatial patterns of genetic diversity in this species, and the role of ocean currents and historical events in shaping these patterns, we examined population genetic structure, maternal phylogeography, and colonization history across the species’ Western Indian Ocean range. The data sets include samples from 34 populations of Kenya, Tanzania, Mozambique, South Africa, Madagascar, Mayotte Island, Europa Island, Aldabra atoll, and the Granitic Seychelles + outgroups of the Red Sea and Southeast Asia. The data set includes 1,150 genotyped trees using 18 nuclear microsatellites for conducting population genetic analyses, including structure, migrate, barrier, and Bayesian origin models. The data set also includes 50 chloroplast sequences (+ 1 reference) of a nearly complete genome (at least one individual from each population) to investigate maternal phylogeography and infer lineage origins from a haplotype network. Methods Study area and sampling A total of 1079 individuals of Avicennia marina from natural mangrove forests were collected in 34 locations of the West Indian Ocean (WIO), of which 22 were located along the continental eastern African coastline from a northernmost site in Lamu, Kenya, to the species' southern range limit site of Wavecrest, South Africa. Twelve populations were located on Madagascar, Europa Island, Comores (Mayotte Island), the Aldabra atoll, and the granitic Seychelles (Mahé, St. Pierre, and Curieuse island). For comparative reasons only and as an outgroup in diverse analyses, we considered an additional 71 individual trees outside the WIO from a Red Sea population (Saudi Arabia), the East Indian Ocean (western Malaysian Peninsula), and the East Sea (northern Vietnam). For NGS-based chloroplast genome assemblage, a subset of 50 individuals, at least one from each population, was used and included 2 individuals from extra WIO sites (Ile St Marie in East Madagascar and Nahoon in South Africa) and 9 individuals outside the WIO as outgroup region (Red Sea, Bangladesh, southern Malay Peninsula, southern and central Vietnam, and Philippines). At each site, fresh leaves per tree were collected, dried, and stored in bags with silica gel for transport. The distance between sampled individual trees ranged between 10 and 100 m, and were taken along transects either parallel or perpendicular to the shore or estuarine river. Samples of A. marina were made in cooperation with and by local researchers that obtained permits from their authorities. Microsatellite analysis and data quality Total genomic DNA extractions were performed on 20-30 mg leaf material following the method as in Triest et al. (2025). We selected 18 microsatellite loci out of 38 tested for A. marina (Maguire et al., 2000; Geng et al., 2007; Triest et al., 2020) and Avicennia alba (Teixeira et al., 2003) based on amplification, polymorphism, and data quality. These were Am3a, Am40b, Am49b, Am81a, Aa23b, Aa67b, Avma1a, Avma2a, Avma6a, Avma8a, Avma10a, Avma14b, Avma17a, AMK3b, AMK6b, AMK10b, AMK18b, and AMK34b assembled in two sets (a, b) of a multiplex polymerase chain reaction (PCR) following conditions as detailed in Triest et al. (2025). Geng, Q. F., Lian, C. L., Tao, M., Li, Q., and Hogetsus, T. (2007). Isolation and characterization of 10 new compound microsatellite markers for a mangrove tree species, Avicennia marina (Forsk.) Vierh. (Avicenniaceae). Mol. Ecol. Notes 7, 1208–1210. https://doi.org/10.1111/j.1471-8286.2007.01834.x Maguire, T.L., Edwards, K.J., Saenger, P., and Henry, R. (2000). Characterisation and analysis of microsatellite loci in a mangrove species, Avicennia marina (Forsk.) Vierh. (Avicenniaceae). Theor. Appl. Genet. 101, 279–285. https://doi.org/10.1007/s001220051480 Triest, L., Van der Stocken, T., Akinyi, A.A., Sierens, T., Kairo, J., and Koedam, N. (2020) Channel network structure determines genetic connectivity of landward–seaward Avicennia marina populations in a tropical bay. Ecol. Evol. https://doi.org/10.1002/ece3.6829 Triest, L., Phan Thi Thuy Hang, Luong Quang Doc, Bousquet-Mélou, A., Bich T. N. Do, Sierens, T., Dahdouh-Guebas, F., Koedam, N., and Van der Stocken, T. (2025). Migration history of Avicennia marina populations: a legacy of mangrove expansion on the Sunda Shelf. Frontiers Marine Sc.: Sec. Marine Evolutionary Biology, Biogeography and Species Diversity 12. https://doi.org/10.3389/fmars.2025.1565908 Chloroplast genome assembly, data quality, and alignment The analysis of complete chloroplast sequences through genome skimming followed the detailed protocol as mentioned in Triest et al. (2025). Briefly, genomic DNA extracts were processed for next-generation sequencing (NGS) analysis. Paired-end reads were assembled to a reference (an annotated A. marina chloroplast genome with GenBank accession number MT108381 from Fujian, China, by Li et al., 2020) in Geneious Prime® 2024.0.5 (Biomatters Ltd., Auckland, New Zealand) that perfectly aligned with our reference sample from Kenya (used by Dierckxsens et al., 2017). All mutational steps were recorded, however we encountered ‘heterozygosity’ in many coding genes, due to either two different copies of cpDNA (a biparental plastid inheritance) or caused by nuclear copies of plastid DNA (NUPTs). Therefore, we omitted those ambiguous coding regions and kept only mutational differences of the non-ambiguous cpDNA sequences for further analysis. Ambiguous sequences were detected in 27 genes of the Large Single Copy (LSC) region, including matK, rpoC1, rpoC2, rpoB, psbD, psbC, trnS, atpE, atpB, rbcL, accD, psaI, ycf4, cemA, petA, psbJ, psbL, psbF, psbE, rps11, rpl36, infA, rps8, rpl14, rpl16, rpl2, and rpl23. After removing likely nuclear plastid DNA insertions (NUPTs), 87 mutations were retained in 41 WIO individuals - 45 substitutions (transitions or transversions) and 42 mononucleotide repeats or indels. When including outgroups from the Red Sea and Southeast Asia, a total of 342 mutations were identified: 223 substitutions and 119 mononucleotide repeats or indels. Dierckxsens, N., Mardulyn, P., Smits, G. (2017). NOVOPlasty: de novo assembly of organelle genomes from whole genome data. Nucleic Acids Res 45:e18. https://doi.org/10.1093/nar/gkw955. Li, H., Ma, D., Li, J., Wei, M., Zheng, H., and Zhu, X. (2020). Illumina sequencing of complete chloroplast genome of Avicennia marina, a pioneer mangrove species. Mitochondrial DNA B Resour. 5(3), 2131-2132. https://doi.org/10.1080/23802359.2020.1768927 Triest, L., Phan Thi Thuy Hang, Luong Quang Doc, Bousquet-Mélou, A., Bich T. N. Do, Sierens, T., Dahdouh-Guebas, F., Koedam, N., and Van der Stocken, T. (2025). Migration history of Avicennia marina populations: a legacy of mangrove expansion on the Sunda Shelf. Frontiers Marine Sc.: Sec. Marine Evolutionary Biology, Biogeography and Species Diversity 12. https://doi.org/10.3389/fmars.2025.1565908