DOI: 10.5061/dryad.v9s4mw6t2
title: Microsatellite data of Avicennia marina from Gazi Bay, Kenya

description: Mangrove ecosystems along the East African coast are often characterized
 by a disjunct zonation of seaward and landward Avicennia marina. This
 disjunct zonation may be maintained through different positions in the
 tidal frame, yielding different dispersal settings. The spatial
 configuration of the landscape and coastal processes such as tides and
 waves are expected to largely influence the extent of propagule transport
 and subsequent regeneration. We hypothesized that landward sites would
 keep a stronger genetic structure over short distance in comparison to
 enhanced gene flow among regularly flooded seaward fringes. We tested this
 hypothesis from densely vegetated A. marina transects of a well-documented
 mangrove system (Gazi Bay, Kenya) and estimated local gene flow and
 kinship-based fine scale genetic structure. Ten polymorphic microsatellite
 markers in 457 A. marina trees revealed no overall significant difference
 in levels of allele or gene diversities between sites that differ in
 hydrological proximity. Genetic structure and connectivity of A. marina
 populations however indicated an overall effect of geographic distance and
 revealed a pronounced distinction between channels and topographic
 setting. Migration models allowed to infer gene flow directionality among
 channels, and indicated a bidirectional steppingstone between seaward and
 nearest located landward stands. Admixed gene pools without any fine-scale
 structure were found within the wider and more exposed Kidogoweni river
 estuary, suggesting open systems. Elevated kinship values and structure
 over 5 to 20 m distance were only detected in two distant landward and
 seaward transects near the mouth of the Mkurumuji River, indicating local
 retention and establishment. Overall, our findings show that patterns of
 A. marina connectivity are explained by hydrological proximity, channel
 network structure, and hydrokinetic energy, rather than just their
 positioning as disjunct landward or seaward zones.
description: Genomic DNA was
 extracted from approximately 20 mg of dried leaf tissue using the E.Z.N.A.
 SP plant DNA Mini kit (Omega bio-tek, Norcross, GA, USA). A multiplex
 polymerase chain reaction (PCR) consisted of in total 10 microsatellite
 markers (Appendix 1). Six of the markers were previously developed by
 Maguire<i> </i>et al. (2000b) and Geng et al. (2007) for
 <i>A. marina. </i>To ensure high resolution of genotyped
 individuals, we developed four new primers for polymorphic microsatellite
 markers using source material from Gazi Bay. For the development of these
 new markers an Illumina paired-end library was constructed and sequenced
 using the Illumina HiSeq platform at Macrogen (Seoul, Republic of Korea).
 SSR_pipeline (Miller et al., 2013) was used to find microsatellites. Out
 of 19,3 million 100 bp paired end reads, 1,4 million pairs were
 successfully joined by the module joinseqs. The module SSR_search found
 5178 dinucleotide SSRs with at least 10 repeats, 362 trinucleotide SSRs
 with at least 8 repeats and 227 tetranucleotide SSRs with at least 6
 repeats. We used Batchprimer 3 (You et al, 2013) to design primers and 56
 primer-pairs were selected for synthesis on the basis of number of repeats
 and expected fragment length. Using Multiplex Manager (Holleley and
 Geerts, 2009) we added 4 new polymorphic loci to the previously existing
 multiplex to form one single multiplex reaction of 10 amplifiable primer
 pairs. Primers were fluorescence-labelled with 4 different dye-labels
 (6FAM/VIC/NED/PET) and a primer mix was made by mixing 0.2 µM of each
 primer together. Multiplex PCR reactions consisted of 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. PCR was performed in
 a thermal cycler (Bio-Rad MyCycler) with the following conditions: an
 initial denaturation of 95°C for 15 minutes followed by 35 cycles of: 30
 seconds denaturation at 95°C, 90 seconds annealing at 57°C and 80 seconds
 elongation at 72°C followed by a final extension of 30 minutes at 60°C.
 PCR products were separated on an ABI3730XL sequencer (Macrogen, Seoul,
 Korea) and allele sizes were determined with GeneMarker V2.60
 (SoftGenetics LLC, State College, USA).