DOI: 10.5061/dryad.vmcvdnctd
title: Rhizophora complete chloroplast genome sequences

description: Historical processes of long-distance migration and ocean-wide expansion
 feature the global biogeographic pattern of Rhizophora species. Throughout
 the Indian Ocean, R. stylosa and R. mucronata appear as a young
 phylogenetic group with expansion of R. mucronata towards the Western
 Indian Ocean (WIO) driven by the South Equatorial Current. Nuclear
 microsatellites revealed genetic patterns and breaks, however, estimating
 propagule dispersal routes requires maternally inherited cytoplasmic
 markers. Here, we examine the phylogeography of 21 R. mucronata
 provenances across a &gt;4,200 km coastal stretch in the WIO using R.
 stylosa as outgroup. Full length chloroplast genome (164,474 bp) and
 nuclear ribosomal RNA cistron (8,033 bp) sequences were assembled.
 Boundaries, junction point, sequence orientation and stretch between
 LSC/IRb/SSC/IRa/LSC showed no differences with the R. stylosa chloroplast
 genome. A total of 58 mutations in R. mucronata encompassing
 transitions/transversions, insertion-deletions and mononucleotide repeats
 revealed three major haplogroups. Haplonetwork, Bayesian ML and
 Approximate Bayesian Computation (ABC) analyses supported discrete
 historical migration events. An ancient haplogroup A in the Seychelles and
 eastern Madagascar was as divergent from other R. mucronata haplogroups as
 it was from R. stylosa. A star-like haplonetwork referred to recent range
 expansion of haplogroup B from northern Madagascar towards the African
 mainland coastline, including a single variant spanning &gt;1,800 km
 across the Mozambique Channel Area. Populations south of Delagoa Bight
 contained haplogroup C and originate from a unique bottleneck dispersal
 event. Divergence estimates of pre- and post-Last Glacial Maximum
 illustrated a recent emergence of WIO Rhizophora mangroves compared to
 other oceans. Connectivity patterns could be aligned with directionality
 of major ocean currents. Madagascar and the Seychelles each harbored
 haplogroups A and B, albeit among spatially separated populations,
 explained from a different migration era. Likewise, the Aldabra Atoll
 harbored spatially distinct haplotypes. Nuclear ribosomal cistron
 (8,033bp) variants corresponded to haplogroups and confirmed admixtures in
 the Seychelles and Aldabra. These findings shed new light on the origins
 and dispersal routes of R. mucronata lineages that have shaped their
 contemporary populations in large regions of the WIO, which may be
 important information for defining marine conservation units, both at
 ocean scale and at level of small islands.
description: We considered <i>Rhizophora</i> samples from 21
 mangrove populations distributed across the WIO (Table 1). Ten populations
 were located along a &gt;4200 km stretch of the eastern African
 continent and eleven populations on islands in the WIO. Samples were from
 remote islands of the Seychelles (both sides of Mahé and Isle Curieuse)
 and Aldabra Atoll (three sites), from Madagascar (two sites on eastern
 coastline, two at northern tip and one along western coastline) and on the
 East African mainland coastline from Kenya, Mozambique and Republic of
 South Africa (ten sites). These were collected or provided and
 morphologically identified as <i>R. mucronata</i> following
 Tomlinson (2016) For each sample site, fresh leaves were collected from
 individual adult trees and stored in individual bags with silica gel for
 transport. Genomic DNA extracts of 23 samples were made
 at the Plant Biology and Nature Management 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). 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 are used as the
 input file for <i>de novo</i> chloroplast assembles. The
 <i>de novo</i> chloroplast assemblies were done at first for
 three chloroplast genomes using NOVOPlasty assembly at Kmer = 33. All
 assemblies were executed by taking a single read from the dataset that
 originates from the targeted plastid as seed (<i>rbcL</i>) and
 taking 30% as subsample from the FASTA file with default parameters. These
 were compared to the existing annotated <i>R. stylosa</i>
 chloroplast genome (GenBank accession number MK070169 and appeared similar
 in genome structure and perfectly aligned with <i>R.
 stylosa</i> though did not align well with GenBank accession number
 MN307165 (<i>Rhizophora mucronata</i>, Wu, H. unpublished) or
 MW387538 (<i>Rhizophora apiculata</i>, Jiang, G.-F.
 unpublished). Therefore, the remaining samples were assembled using
 ‘assemble to reference’ function in Geneious software. Illumina 2 x 300 bp
 paired-end were processed in Geneious Prime v 2019.2.1 (© 2005-2019
 Biomatters Ltd.) to obtain complete chloroplast genome sequences. All
 assemblies used MK070169 (<i>R. stylosa</i>) as a reference
 genome and the mapping of reads of 23 samples were performed. This
 approach yielded identical results as obtained with Novoplasty and the
 assemblages using <i>R. stylosa</i> as a reference to map
 every sample, averaged 66,892 – 454,099 reads with a mean depth of reads
 ranging from 118 – 829 coverage.