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.

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 et al. (2000b) and Geng et al. (2007) for A. marina. 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 H₂O 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).