Comparative Study of the Disjunct Zonation Pattern of the Grey Mangrove Avicennia Marina (forsk.) Vierh. in Gazi Bay (kenya)

The disjunct zonation pattern of Avicennia marina (Forsk.) Vierh., frequently observed along the Kenyan coast as a landward and a seaward zone, has been studied on a morphological level and complemented with preliminary genetic data. The objective was to describe the two zones in-depth in order to provide a background ecological setting that may be used in explanations on the emergence of such a bimodal zonation. The vegetation structure in the two zones is described by means of the Braun-Blanquet relevé and PCQ-methods, rootand leaf-morphological characteristics, and environmental variables to provide a background ecological setting. The two zones show considerable differences in physiognomy of A. marina trees, and indicate that the differences may constitute an environmental response. Results show that in the landward A. marina zone, tree heights, leaf sizes, density, and total length of pneumatophores tend to be smaller as compared to the same attributes of the trees in the seaward zone. Differences in leaf stomatal density and in aboveand below-ground pneumatophore length, the latter closely related to the effect of inundation, were observed. This suggests a considerable adaptability and plasticity with respect to environmental conditions. The hypothesis for the genetic analysis was that mangrove fringes as close as 105 m from one another constitute a same genetic pool. Preliminary genetic analysis using 48 amplified DNA fragments (RAPD Randomly Amplified Polymorphic DNA) from 37 landward and seaward trees revealed significantly different frequencies for four DNA fragments, indicating that there might be less contact and genetic interchange between the two zones than within each zone. Whether this phenomenon, coupled to the different environmental conditions, can lead to further divergence cannot be answered yet. Of all mangrove zonation patterns described worldwide by various authors, that of Avicennia marina (Forsk.) Vierh. provides an incentive for particular analysis because of its disjunct nature (Watson, 1928; Macnae, 1968, 1969; Johnstone, 1983; Smith, 1987a, 1992). In Kenya this species displays a disjunct zonation pattern across the intertidal zone (Dahdouh-Guebas et al., 2002). Avicennia marina is present in the most landward as well as seaward fringe of the mangrove forest at certain locations along the Kenyan coast. The same pattern has been reported for other specific regions of the world; however, it has been described visually or superficially without intensive sampling or in-depth comparisons. Walter and Steiner (1936) described the higher tidal A. marina trees as “exceptions” of seaward specimens. Other authors (e.g., Macnae, 1968; Johnstone, 1983; Smith, 1992) reported a “double zonation” of A. marina, but did not go beyond stating this observation or mentioning as personal observations that tree height or leaf size varies between the upper and lower intertidal zones. This paper provides a more rigorous comparison of the landward and seaward A. marina fringe on a morphological level (such as tree height, leaf size, root density, etc.), as an ecological setting framing the emergence of the two disjunct zones. We also provide preliminary findings on genetic differentiation between the observed zones. Rather than aiming at a generalization from BULLETIN OF MARINE SCIENCE, VOL. 74, NO. 2, 2004 238 this type of research, we try to unveil the structure of a local mangrove and comparable forests. The genus Avicennia L. (Avicenniaceae) comprises eight species, occupying diverse mangrove habitats (Tomlinson, 1986; Duke, 1991). Avicennia marina is the species with the widest distribution, with phenological trends according to latitude (Duke, 1990), and it is the only representative of the genus in Kenya (Kokwaro, 1985; Tomlinson, 1986). It is often considered to play an important pioneering role in plant succession (e.g., Osborne and Berjak, 1997). Some taxonomic variation within A. marina has been proposed in reviews by Bakhuizen van den Brink (1921) and Moldenke (1960, 1967). These authors recognized seven varieties, which are mainly based on geographic segregation (Tomlinson, 1986). Duke (1991) found that there were only three varieties within A. marina. Of all varieties, A. marina (Forsk.) Vierh. var. marina Moldenke is the only one known to have a limited range in East Africa (Tomlinson, 1986; Duke, 1991). However, the identification of Avicennia species in East Africa has always been restricted to A. marina. For a clear description of the species we refer to Duke (1991). In Gazi Bay (Kenya), as well as other sites in Kenya such as Lamu (pers. obs.), A. marina is observed both in the landward and seaward mangrove fringe (e.g., Dahdouh-Guebas et al., 1998, 2002; Ochieng and Erftemeijer, 2002) and physiognomic differences of trees between these two zones are apparent. The aim of this study was to investigate the disjunct zonation pattern of A. marina across the mangrove belt in our study site and to compare the plant attributes from the two zones. Our hypothesis was that phenotypic plasticity within the species may allow diverse physiognomy due to different environmental conditions in seaward vs. landward zones. In the first approach, we compare the rooting system, leaf morphology and physiognomy in the two Avicennia zones. Then, we investigate whether the observed differences between the zones correlate with selected environmental factors that may lead to physiological or morphological plasticity. Since the Avicennia zones considered in this study are located as close as 105 m from each other, we hypothesized that they are part of only one population, with no significant genetic differences. We do not address the vegetation causes of the emergence of the two zones, but describe the ecological setting as reflected in physiognomy, morphology, and preliminary genetic differentiation. MATERIALS AND METHODS STUDY LOCATION.—Gazi Bay (4°25 ́S, 39°30 ́E), Kenya, an open estuary located about 50 km south of Mombasa, is fed by two seasonal rivers, the Kidogoweni and the Mkurumuji (Fig. 1). All ten East-African mangrove species can be found in Gazi Bay, including A. marina, Bruguiera gymnorrhiza (L.) Lam., Ceriops tagal (Perr.) C.B. Robinson, Heritiera littoralis Dryand., Lumnitzera racemosa Willd, Pemphis acidula Forst., Rhizophora mucronata Lam., Sonneratia alba Sm., Xylocarpus granatum Koen, and X. moluccensis (Lamk.) Roem. However, the species diversity and abundance across the intertidal complex varies with location within and among mangrove forests (Dahdouh-Guebas et al., 2002). STRUCTURE OF THE VEGETATION.—In an undisturbed part of the mangrove forest along the major creek that connects the Kidigoweni with the bay, the width of the mangrove belt was surveyed using a transect between the landward terrestrial vegetation and the creek, hereafter called “Transect 1” (Fig. 1). Both on aerial photography and on the field, this transect proved to be representative for the disjunct pattern of A. marina (Fig. 1C). The Braun-Blanquet relevé method (Westhoff and Van der Maarel, 1978; Van der Maarel, 1979) was used in 5 × 5 m quadrats at 10 m intervals between quadrats to generate a qualitative description of the vegetation. Along the same transect the Point-Centered Quarter Method (PCQM) of Cottam and Curtis (1956) was used to describe DAHDOUH-GUEBAS ET AL.: DISJUNCT ZONATION OF GREY MANGROVE 239 the vegetation quantitatively (cf. Cintrón and Schaeffer Novelli, 1984). The diameter D130 [term according to Brokaw and Thompson (2000), but formerly referred to as DBH, the diameter at breast height] of trees with a D130 greater than 2.5 cm was measured. Common mangrove tree Figure 1. A) Map of the Kenyan coast showing B) a close-up of our study site Gazi Bay; and C) an aerial photograph from 1992 showing the location of Transects 1, 2, and 3. Basic coastal features were redrawn from Dahdouh-Guebas et al. (2000) and Slim (1993). BULLETIN OF MARINE SCIENCE, VOL. 74, NO. 2, 2004 240 anomalies (prop roots, forking stems) that inhibited a normal D130 measurement were dealt with as described by Cintrón and Schaeffer Novelli (1984). The tree height was measured using a clinometer. The topographic gradient was determined at 10 m intervals along the transect by use of a theodolite, with the known maximum water height above datum, as indicated in tide tables available from the Kenya Port Authority, as a benchmark. Elevations were expressed as “meters above datum” and the error of the theodolite was experimentally recorded as 3 cm. Each zone was additionally sampled by a transect running approximately parallel with the water line, entirely within each of the zones: one, 100 m transect, hereafter called “Transect 2,” was placed in the most landward Avicennia zone at an average of 3.16 m above datum, the other of 80 m length, hereafter called “Transect 3,” in the most seaward Avicennia zone at an average of 1.32 m above datum. Along these, vegetation analysis was carried out as before. COMPARISON OF HEIGHT AND SALINITY.—Soil salinity was measured with a precision of 1 at 10 m intervals along Transect 1 using an ATAGO refractometer. The mean height of A. marina trees per PCQM sample point, which consists of four quadrants in which the closest tree was surveyed (see Cintrón and Schaeffer Novelli, 1984), was then compared to the salinity. COMPARISON OF ROOT SYSTEM.—In each of the A. marina zones along Transect 1, quadrats of 1 m2 were established with an interspace of 25 cm (n = 12 in the landward zone, and n = 10 in the seaward zone). Within each quadrat the number of pneumatophores was counted and their aboveground length was recorded. In addition, four randomly chosen pneumatophores per quadrat were excavated down to the cable root to measure their belowground length. Since little variation in belowground length was observed within these areas of 1 m2, the average of the four measurements was added to each of the aboveground length measurements to obtain the total pneumatophore length. For each of the above variables and parameters, the correlation with the height above datum was calculated using Spearman s̓ rank correlation coefficient (rs) as described by Sokal and Rohlf (1981) and Kent and Coker (1992). Because of the non-normality within the data set, the significance of the observed differences in root characteristics between the landward and seaward A. marina zones for each of these factors was tested with the Mann-Whitney U-test (Sokal and Rohlf 1981; Kent and Coker 1992). COMPARISON OF LEAF MORPHOLOGY.—One thousand leaves were collected haphazardly from all the trees in each A. marina zone to investigate leaf morphology (n = 1000 in the landward zone, and n = 1000 in the seaward zone). For each leaf the following characteristics were measured: petiole length, lamina length, and maximal lamina width. Similar to the comparative analysis of the root system, between-zone differences pertaining to leaf characteristics were calculated using the Mann-Whitney U-test (loc. cit.). Stomatal density was investigated in the two A. marina zones in view of the different inundation regimes and salinity levels. For this purpose, leaves from 16 trees were taken in each zone. On each tree the fifth leaf pair of a branch was sampled and one of the two leaves on this position was collected, thus avoiding young, not fully developed leaves. Following collection in the field, leaves were frozen and further treated in the laboratory within 2 mo of collection. Leaf epidermis was removed by first boiling the leaves for 1 min in a 10% potassium hydroxide solution (Sidhu, 1975), then transferring them into cold water in a Petri dish. The epidermis strips were then washed in sodium hypochlorite to bleach impurities. Stomatal densities could thus be investigated using the epidermis strips from the abaxial leaf sides. The stomatal density was calculated by counting the stomata within the cells of a grid with known dimensions and visualized within the ocular of the microscope. Since it is known that within one species stomatal density can vary due to environmental conditions (Al-Farrajii, 1983), and it may even vary on the surface of one leaf (e.g., distance to central vein), the epidermis strips were always taken from the same location on a leaf. GENETIC DIFFERENTIATION.—The genetic identity of a total of 37 randomly sampled A. marina trees from the landward (n = 17) and seaward zone (n = 20) of the forest was studied to test for genetic differences between zones. Leaf samples were transported to the laboratory in Brussels for DNA analysis within 48 hrs of collection. DNA extraction was carried out according to Abeysinghe et al. (1999) and amplification conditions according to Williams et al. (1993). Genomic DNA DAHDOUH-GUEBAS ET AL.: DISJUNCT ZONATION OF GREY MANGROVE 241 of four individuals from A. marina were amplified using 45 arbitrary decanucleotide primers obtained from Operon Technologies, Inc. (U.S.) to find out which primers show polymorphism. A final selection of 11 primers was made on the basis of their reproducibility. The reproducibility of all amplified products was tested by repeating the same reaction at least twice. Since RAPD markers (Randomly Amplified Polymorphic DNA) dominate, a particular DNA band (locus), which is generated from the genome of one individual but absent from a second individual of the same species, represents a polymorphism. The amplification products for all samples were compared to each other and screened for the presence or absence of specific markers. To assess the genetic structure within and among Avicennia populations, all 48 amplified DNA fragments were compared at relative frequencies. Genetic parameters (allele frequencies, Fst and genetic distance) were calculated with the software program RAPD-SURV (now named AFLP-SURV; Vekemans, 2002; Vekemans et al., 2002). This program computes, for each population and locus, the fraction of individuals in the population with the marker (1-x) and without the marker (x), then the estimated frequency of the marker allele (1-q) and of the null allele (q). All estimators are from Lynch and Milligan (1994).