DOI: 10.5061/dryad.3r2280gss
title: Microsatellite data, chloroplast and nuclear rRNA sequences of Avicennia marina from Vietnam, Malaysia, and The Philippines

description: Mangrove forests maintain connectivity and stay genetically linked through
 ocean-dispersed propagules. Most propagules are dispersed over relatively
 short distances, but those reaching open waters can be transported over
 relatively long distances. Avicennia species exhibit a pronounced genetic
 structure across varying distances following a stepping stone migration
 model, with connectivity patterns linked to strength and direction of
 ocean-surface currents Here, we use present-day spatial genetic structure
 of A. marina populations as an imprint of connectivity. This should allow
 us to estimate their migration history in relation to coastal
 configuration and Holocene sea-level rise on the Sunda Shelf. We examined
 the genetic diversity, structure, as well as the demographic, and
 evolutionary history of establishment for ten A. marina populations across
 coastal stretches of Vietnam, using nuclear microsatellite markers in 558
 individual trees. Additionally, genome skimming of 24 samples allowed
 detailed analysis of complete chloroplast genome and nuclear ribosomal
 cistron sequences.
description: Genomic DNA was extracted from approximately 20 mg of each dried
 leaf tissue using the E.Z.N.A. SP plant DNA Mini kit (Omega bio-tek,
 Norcross, GA, USA). The concentration of individual samples ranged from 10
 – 200 ng/µl. The multiplexed PCR reactions consisted of 15 microsatellite
 markers: Avma1, Avma02, Avma03, Avma05, Avma6, Avma8, Avma10, Avma14,
 Avma17 (Geng et al., 2007); Am3, Am81, (Maguire et al., 2000a); Aa22,
 Aa23, Aa67 (Teixeira et al., 2003); and AMK6 (Triest et al.,
 2020)<em>.</em> Primers were fluorescence-labelled with four
 different dye-labels (6FAM/VIC/NED/PET), and a mixture of 0.2 µM of each
 primer. About 6.25 µl master mix (Qiagen Multiplex PCR kit), 1.25 µl
 primer mix, 2.5µl H<sub>2</sub>O, and 2.5µl of genomic DNA
 were used for multiplex PCR reactions. PCR was performed in a thermal
 cycler (Bio-Rad MyCycler) under the conditions of initial denaturation at
 95 °C for 15 min followed by 35 cycles of 30 sec denaturation at 95 °C, 90
 sec annealing at 57 °C, 80 sec elongation at 72 °C and a final extension
 of 30 min at 60 °C. All PCR products were separated on an ABI3730XL
 sequencer (Macrogen, Seoul, Korea) and allele sizes were determined with
 GeneMarker <em>v</em>.2.60 (SoftGenetics LLC, State College,
 USA). A “comparative analysis” of Southern Vietnam
 populations with <em>A. marina</em> from The Philippines
 (Leyte) and Peninsula Malaysia (Southwest and West) was done for an
 overall available set of nine microsatellite loci (out of abovementioned
 15 loci): Avma1, Avma02, Avma6, Avma8, Avma10, Avma14, Avma17, Am3 and
 Am81 (and used only for positioning in a combined PCoA and for
 evolutionary model testing with ABC-RF, see further under microsatellite
 analysis). For a complete chloroplast genome analysis
 and rRNA cistron, through genome skimming, we considered sixteen
 individuals covering all Vietnam sites (one in each transect from N1 to C8
 and double in S9 and S10). For comparative reasons, we newly included and
 analyzed seven individuals from the western coast of Leyte Island in The
 Philippines (one from each population AM5, AM6, AM8, AM9, AM14, AM15, AM16
 as mentioned in Triest et al., 2021a) and three individuals from the
 western coast of the Peninsular Malaysia (one from each population W1, W2,
 W3 as mentioned in Triest et al., 2021b). Genomic DNA extracts of 26
 samples were made at the Plant Biology and Nature Management (APNA) lab of
 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. If necessary, multiple DNA extractions
 of one sample were pooled and concentrated by ethanol precipitation. 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). 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) product was used
 for genome skimming of <em>de novo</em> chloroplast assembles.
 The <em>de novo</em> chloroplast assemblies were done at first
 for a good quality fresh <em>A. marina</em> sample from Kenya
 (Gazi Bay) using NOVOPlasty assembly at Kmer = 33 (Dierckxsens et al.,
 2017). All assemblies were executed by taking a single read from the
 dataset that originates from the targeted plastid as seed
 (<em>rbcL</em>) and taking 30% as subsample from the FASTA
 file with default parameters. Thereafter, the Illumina pair-end next
 generation sequencing (NGS) products are used for chloroplast assembles
 that were referenced to the existing annotated <em>A.
 marina</em> chloroplast genome (GenBank accession number MT108381
 from Fujian, China by Li et al., 2020) and appeared similar in genome
 structure and perfectly aligned with our reference sample from Kenya
 though did not align well with GenBank accession number MT012822
 (<em>A. marina</em> from Oman by Khan et al. in 2020,
 unpublished). All 26 individual samples (16 from Vietnam, 7 from The
 Philippines, and 3 from Malaysia) were assembled using ‘assemble to
 reference’ function in Geneious software. Illumina 2 x 300 bp paired-end
 were processed in Geneious Prime® 2024.0.5 (Biomatters Ltd.) to obtain
 complete chloroplast genome sequences. The assemblages averaged 13,316 –
 119,539 reads with a mean depth of reads ranging from 27 – 239 coverage.
 This approach facilitated the analysis without need to describe<em>
 de novo </em>chloroplast genome features and gene annotations. The
 26 consensus complete chloroplast sequences were aligned with mafft v7.388
 (Katoh and Standley, 2013). We further adjusted manually at sites with
 mononucleotide repeats and insertions/deletions (indels).
 Genome skimming allowed to assembler the nuclear ribosomal
 cistron (18S, ITS1, 5.8S, ITS2, and 26S)  from a 671 bp template sequence
 of <em>Avicennia marina </em>(Genbank AF365978, Schwarzbach
 and McDade, 2002) 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 four times
 as a seed in Geneious Prime® 2024.0.5 to progressively enlarge the
 flanking regions. A 5,772 bp nuclear ribosomal cistron was obtained for
 one sample (from C7 population) and used as a reference to map every
 sample, averaging 3,932 – 23,717 reads with a mean depth of reads ranging
 from 123 – 713 coverage. The generated consensus sequences of 5,772 bp
 length for a total of 26 samples (16 from Vietnam, 7 from the Philippines,
 and 3 from Malaysia) were aligned for comparative description of mutated
 positions using mafft v7.388 (Katoh and Standley, 2013).
description: # Microsatellite data, chloroplast and nuclear rRNA sequences of Avicennia
 marina from Vietnam, Malaysia, and The Philippines Dataset DOI:
 [10.5061/dryad.3r2280gss](10.5061/dryad.3r2280gss) ## Description of the
 data and file structure The microsatellite data comprise 15 loci for
 individual genotypes in 10 Avicennia marina populations of Vietnam. The
 chloroplast and nuclear rRNA cistron sequences  were obtained from NGS
 data of Avicennia marina individuals of Vietnam, Malaysia, and The
 Philippines  ### Files and variables #### File:
 Microsatellite\_data\_15\_loci\_10\_pops\_Vietnam.xlsx
 **Description:** nuclear microsatellites of Avicennia marina #####
 Variables * 15 loci * 10 populations  of Vietnam #### File:
 Suppl\_table\_S3\_cpDNA\_mutations.xlsx **Description:** Chloroplast DNA
 with 128 mutational changes, namely 35 transitions (ti), 43 transversions
 (tv), 9 insertion-deletions (indel) and 41 mononucleotide repeats (µsat)
 in Avicennia marina individuals of ten populations in Vietnam, three in
 West Malaysian Peninsula and seven in The Philippines. #### File:
 Suppl\_Table\_S4\_rRNA\_cistron.xlsx **Description:**  Incomplete
 homogenization at 21 nucleotide positions of the nuclear ribosomal RNA
 cistron (SSrRNA, ITS1, 5.8srRNA, ITS2, LSrRNA ) in Avicennia marina
 individuals of ten populations in Vietnam, three in West Malaysian
 Peninsula and seven in The Philippines. Colors indicate the positions with
 incomplete homogenizations for each region. ## Code/software Any tabular
 data software