Cover Single.cdr BIOTROPIA Vol. 27 No. 2, 2020: 153 - 161 DOI: 10.11598/btb.2020.27.2.1198 LIMITED SEED DISPERSAL MAY SHAPE GENETIC STRUCTURE OF Hydnophytum formicarumJACK. POPULATIONS IN MANGROVE ECOSYSTEM** ABDUL SHUKOR YUSOFF, WAN BAYANI WAN OMAR AND SHAHRUDIN ROHANF Faculty of Science and Marine Environment, Universiti Malaysia Terengganu, 21030 Kuala Nerus, Terengganu, Malaysia Received 03January 2019 / Accepted 22 January 2019 ABSTRACT Hydnophytum formicarum is an epiphytic plant, commonly distributed throughout Southeast Asia. However, its distribution is currently declining due to forest fragmentation and the subsequent habitat changes which may have also consequently affected the genetic structure of H. formicarum populations. Hence, this study aimed to understand the genetic variation and differentiation within and among populations of this species. Using Random Amplified Polymorphic DNA (RAPD) markers, the genetic variation and genetic differentiation among three populations were assessed in order to identify potential conservation management strategies for this species in the mangrove ecosystem in Malaysia. Ten highly reproducible primers were used in the population analysis, resulting in a total of 221 discernible fragments. Genetic variation among populations was high at 98% identified polymorphic fragments. AMOVA showed significant genetic differentiation among the populations (OPT = 0.554, p 0.001), with pairwise genetic distances between the populations ranging from 0.495 - 0.589. PCoA clustering analysis separated the populations according to their geographical locations. The high genetic variation within a population, high genetic differentiation between populations and clear separation in the cluster analysis indicated a restricted seed dispersal of the species. Keywords: AMOVA, epiphyte, myrmecophyte, RAPD, Setiu Wetlands INTRODUCTION Epiphytes are plants growing on other plants without directly harming their hosts (Zotz 2016). It is one major plant groups contributing largely to worldwide plant diversity. Vascular epiphytes alone constitute about 10% of overall global vascular floristic composition (Brown et al. 2015) and are distributed extensively across the world. Some epiphytic species have been recorded as myrmecophytes due to their association with ants (Chomicki & Renner 2015). Epiphyte studies have also revealed that these plants are particularly sensitive to environmental and habitat changes. Hydnophytum formicarum Jack is one epiphyte of the Rubiaceae family that is identified as a **This paper was presented at the 3rd International Conference on Tropical Biology 2018, 20-21 September 2018, Bogor, West Java, Indonesia myrmecophyte due to its close association with ants as nesters and dispersers (Hosoishi et al. 2018). Hydnophytum formicarum is a species among 93 others in the genus Hydnophytum, under subtribe Hydnophytinae, which also considered as the world’s most species-rich ant plant group (Chomicki & Renner 2015). This species is distributed natively in Southeast Asia (Huxley 1978). Notably, the species is widely distributed in the tropical region, and no record can be found on this species in the temperate region. This species favours coastal trees, mangroves and lowland swamp savannahs and hill savannahs (Huxley 1978). Giesen et al. (2006) have described this species as a mangrove- associated species as its abundance is prominent in this ecosystem. In Singapore, H. formicarum has been listed as critically endangered, with few in numbers currently present and no other surviving myrmecophytic epiphytes of the Rubiaceae family (Lok & Tan 2009). 153 BIOTROPIA Vol. 27 No. 2, 2020 The association of Rubiaceous epiphytes including Hydnophytum with their ant occupants is called symbiosis or mutualism (Huxley 1980). Notably, ants of the Iridomyrmex spp. and Crematogaster spp. were found dwelling in the epiphytes domatia (Huxley 1980). Ant’s occupation on the epiphytes were highly observed in parts of the plants including the stem, leaf or root of domatia (Chomicki & Renner 2015). Ants also act as dispersers for their host epiphytes. For instance, the ant species Philidris nagasau (Dolichoderinae) was doing an obligate farming of some epiphytes from genus Squamellaria in Fiji (Chomicki & Renner 2016). However, the epiphytes and ants mutualism can also be affected when other changes occur such as habitat shift and also the morphological changes of the host (Chomicki & Renner 2017). Habitat deterioration and fragmentation has affected the distribution and genetic structure of the epiphyte species Crepis triasii (Asteraceae) in the Mediterranean Islands (Mayol et al. 2012) and Hedyotis chrysotricha (Rubiaceae) in China (Yuan et al. 2012). Along the east coast of Peninsular Malaysia, aggressive coastal development has affected the coastline (Muslim et al. 2011; Ahmad et al. 2014). In that same region, in the state of Terengganu, an abundance of H. formicarum has been observed in the coastal islands with high proclivity to be affected by the aforementioned changes. Hence, this fragmentation threat has directed the assessment of the genetic structure of H. formicarum on island populations along the coasts. Studies on the genetic structure of plant’s island population have discovered that their spread is in accordance with the species’ geographical distribution (Oiki et al. 2001; Zhang et al. 2018). However, when there is intermixing of genetic groups, dispersers were found to normally play their roles (Godoy & Jordano 2001; Schidegger et al. 2012). Geographical barriers and isolation by distance (IBD) of populations separated by bodies of water or oceanic barriers have been thoroughly studied (Dias et al. 2016; Levy et al. 2016) as these factors will eventually contribute to species’ gene flow (Lee & Thomas 2011). One study on the genetic structure of plant populations in an island ecosystem (Hufford et al. 2013) (discovered a strong genetic differentiation within and among island populations and apparent IBD between islands. A high variation within island populations and significant genetic differentiation among populations were also observed in the epiphytic orchids (Mallet et al. 2014). Similarly, the geographical isolation has limited the seed and pollen dispersal of Banksia arborea in terrestrial islands (Nistelberger et al. 2015). As island populations are relatively smaller in comparison to mainland populations, the genetic variation in island populations should be less (Frankham 1996), a theory that was substantiated by Hufford et al. (2013) and Laukkanen et al. (2014). Since no study using molecular markers has yet been conducted for H. formicarum, this study aimed to investigate the genetic variation and differentiation within and among populations for this species as these will provide basic information for management plans focusing in the conservation of this species. Specifically, this study will determine and compare the genetic variation of this species in Pulau Telaga Tujuh, Pulau Layat and Pulau Redang populations. MATERIALS AND METHODS Plant Sampling Leaves were sampled from 21 individual plants (one leaves per individual), of the 7 individual plants from each of the three different island mangrove forests in Terengganu on the east coast of Peninsular Malaysia. Pulau Telaga Tujuh (PT) and Pulau Layat (PL) are nearby small mangrove islands, while Pulau Redang (PR) is a tourism island with a fragmented mangrove ecosystem located in the district of Kuala Terengganu, approximately 30 km away from Setiu Wetlands (Fig. 1). Each H. formicarum leaf was collected from different phorophytes. Leaves were preserved in zip-lock bags with silica gel and then frozen in -20 °C conditions prior to genetic material extraction. Molecular Analysis DNA was extracted from the frozen leaf samples using modified CTAB method (Doyle & Doyle 1990) after leaf grinding into lyophilized form using liquid nitrogen. The DNA concentration was estimated using 154 Genetic structure of H. formicarum populations — Yusoff et al. BioDrop (Denville Scientific) spectrophoto¬ meter. Ten out of 20 screened universal RAPD (Random Amplified Polymorphic DNA) primers by Operon Technologies were chosen in this study (Table 1). The reproducibility of the selected primers was ensured by replicating the amplification process during optimization. Genomic DNA was amplified in a reaction volume of 20 pL consisting of template DNA, IX reaction buffer (Promega), 2 mM MgCh (Promega), 0.1 mg/mL BSA (Promega), 0.25 mMdNTPs (Promega), 0.25 pM of each primer (OPA, 1st Base) and 0.5 units of Taq polymerase (Promega). The DNA was amplified using thermocycler (Applied Biosystems) at 30 and 45 cycles for 180 sec of initial denaturation and then 30 sec of denaturation at 95 °C, 30 sec annealing temperature at 32.6 °C to 37.7 °C, 60 sec of extension and then 420 sec of final extension at 72 °C. The PCR products were electrophoresed in 1.5% Agarose gel and TBE buffer and ran for 90 min at 60 V. The gel was then stained using Diamond® Nucleic Acid dye (Promega) and was finally visualized and photo¬ graphed using the Bio Rad gel documentation system. Data Analyses The amplified fragments were scored as 1 if present and 0 if absent and then assembled into a data matrix. The Principal Coordinate Analysis (PCoA) cluster analysis based on genetic distance was performed using PAST 3 software (Hammer et al. 2001). Analysis of the molecular variance (AMOVA) (Excoffier et al. 1992) was used to partition the total genetic diversity between and among populations. The popula¬ tion genetic differentiation indicator, pairwise OPT of AMOVA, was also calculated. AMOVA and pairwise OPT were both performed using GENALEX V6.1 (Peakall & Smouse 2006). Pulau Redang South C hina Sea Pulau Layat I Pulau Tclaga TujuhrV - . i Figure 1 Map of the Hydnophytum formicarum populations in Terengganu Table 1 Primer names and sequences chosen for PCR No. Primer name Primer sequence 5’ to 3’ Nucleotide length C+G content (%) 1 OPA-01 CAGGCCCTTC 10-mer 70.0 2 OPA-03 AGTCAGCCAC 10-mer 60.0 3 OPA-05 AGGGGTCTTG 10-mer 60.0 4 OPA-07 GAAACGGGTG 10-mer 60.0 5 OPA-09 GGGTAACGCC 10-mer 70.0 6 OPA-11 CAATCGCCGT 10-mer 60.0 5 OPA-13 CAGCACCCAC 10-mer 70.0 8 OPA-15 TTCCGAACCC 10-mer 60.0 9 OPA-17 GACCGCTTGT 10-mer 60.0 10 OPA-19 CAAACGTCGG 10-mer 60.0 155 BIOTROPIA Vol. 27 No. 2, 2020 RESULTS AND DISCUSSION Genetic Variation A total of 221 discernible fragments (Fig. 2) from the 21 individual plants of H. formicarum were produced at an average of 22.1 fragments per primer. The amplified fragment sizes ranged from 150 to 2500 base pairs (bp). Using the primer OPA17, the fragments showed 98% polymorphism, with Pulau Telaga Tujuh scoring the highest percentage (89.5%), followed by Pulau Redang (78.11%) and Pulau Layat (60.11%) (Table 2) indicating that Pulau Telaga Tujuh has the highest genetic variation since a high percentage of polymorphism in a population means a high genetic variation (Oiki et al. 2001; Boneh et al. 2003). Moreover, genetic variation is positively correlated with population size (Frankham 1996). This high genetic variation was also found within the island population of epiphytic orchids (Mallet et al. 2014). The Principal Coordinate Analysis (PCoA) further showed that the individuals from the three different populations were genetically different (Fig. 3). Furthermore, the RAPD marker confirmed the genetic variation among the plants’ population (Monaghan & Halloran 1996). The percentage of polymorphism in Pulau Redang, an island located at sea, was 18% higher than Pulau Layat even though Pulau Layat was located just meters away from Pulau Telaga Tujuh, which had the highest polymorphism percentage. The relatively low genetic variation in Pulau Redang compared to Pulau Telaga Tujuh is possibly due to its oceanic location, which may have limited the gene flow from the mainland resources (Maki 2001). Islands that are closer to mainland, like Pulau Telaga Tujuh are more likely to act as allelic sink (Curto et al. 2017). Presumably, Pulau Layat is a newly established population, considering its low genetic variation and possibly genetic diversity (Hagen & Hamrick 1998). Since the lowest polymorphism percentage was in this population, it is considered as newly founded (Ouborg et al. 1999). However, in this study, no further investigation was done to validate the reasons for the low genetic variation in Pulau Layat. The PCoA cluster analysis exhibited that the individuals were grouped in accordance with the populations where the samples were collected. The results from this study suggest that gene flow between populations was highly restricted. Geographical barriers that have formed may have impeded the dispersal of seeds, ultimately preventing the gene flow from occurring in a wider region (Monaghan & Halloran 1996). However, the epiphytic ferns of Hawaiian Islands were generally not genetically distinct between island populations, possibly due to high interisland gene flow (Ranker 1992). In contrast, the epiphytic orchids sampled from island populations in Puerto Rico, Dominican Republic and Cuba, had patterns of genetic variation more similar within the islands than with populations of other islands (Ackerman & Ward 1999). A study on the Jumellea epiphytic orchid also showed that the genetic variation between two islands populations differed significantly (Blambert et al. 2016). >500bp JOOObp - ISOObp - lOOObp - — I kbp PT1 PT2 PT3 PT4 PT5 PT6 PT7 PL1 PL2 PL3 PL4 PL5 PL6 PL7 PR I PR2 PR3 PR4 PR5 PR6 PR7 C lOObp Figure 2 Photograph of Diamond® stained agarose gel of RAPD fragments using primer OPA 17 DNA samples from Pulau Telaga Tujuh (PT), Pulau Layat (PL) and Pulau Redang (PR) Note: Lanes 1 kbp and 100 bp are the markers, while lane C is the control. 156 Genetic structure of H. formicarum populations — Yusoff et al. Table 2 Genetic variability estimates for populations of H. formicarum from RAPD analysis No. Code Locality N n No. of loci No. of polymorphic loci % of polymorphism 1. PT Pulau Telaga Tujuh > 3000 7 137 123 89.05 2. PL Pulau Layat > 100 7 101 65 60.11 3. PR Pulau Redang 7 7 121 99 78.11 Notes: N = estimated population size; n = number of sampled individual plants of H. formicarum. o