FERNS RICHNESS ALONG ENVIRONMENTAL GRADIENTS IN AYER HITAM FOREST, PENINSULAR MALAYSIA

Fern species richness was assessed to ascertain the distributional pattern of the ferns as influenced by the environmental gradients in Ayer Hitam forest reserve, Peninsular Malaysia. The ferns were collected using a preferential non-random sampling technique. Temperature, humidity, light intensity and elevation data were obtained at each point of collection of the ferns. The influence of these environmental gradients on the distribution of the species was determined using Detrended Correspondence Analysis (DCA) and general linear model (GLM). A total of 67 ferns were identified at all the study sites. Two ferns namely Asplenium antiquum and Pteris longipinnula were first recorded in this study. In all the sites, light intensity ranges from 337.9 – 8098 lux, temperature ranges from 28.5 - 33 o C, humidity ranges from 66.24 - 88.80%, and elevation ranges from 14–33m. Tectaria singaporeana and Nephrolepis biserrata are the only species that are widely distributed across all the study sites. The DCA revealed that the light intensity influences the distribution of the ferns species in this forest. However, the GLM showed that temperature had a positive influence on the distribution of the ferns while light intensity had a negative influence on them.


INTRODUCTION
Ferns and lycophytes are known to be the second largest among the different groups of vascular plants due to having about 11000 species (PPG & Shmakov 2016). They are important ecological components of tropical forests by constituting more than 20% of the total plant cover (Linares-Palomino et al., 2008;Salazar et al., 2015). The diversity of ferns and lycophytes in biotic communities have been described as closely related with the environmental gradients of such communities and can be used as bioindicators of the environmental stability (Zuquim 2015). The availability of water (high humidity), light intensity and topography are mostly strongly correlated with the richness and diversity of ferns in many tropical and sub-tropical regions of the world (Krömer et al., 2013;Salazar et al., 2015). Although these ferns-environmental gradients relationships are still very complex and lots need to be done to fully understand the mechanisms involved (Kessler et al., 2014). Studies on the distribution of plants along elevation and climatic gradients have been more focused on angiosperms, only few have addressed the influence of these environmental gradients on ferns species distribution (Kessler, 2000(Kessler, , 2001Hemp, 2002). The elevational gradient has been used to assess the relationship between species ecology and their responses to climatic changes in the environments (Korner, 2000). In other words, the microclimatic factors such as temperature, light intensity and humidity explain the distribution of ferns species along elevation gradient (Othman et al., 2015). The distribution of ferns along elevation gradients has revealed that ferns species are richer at mid-elevation gradient comprising more epiphytes than terrestrials (Kessler et al., 2011;Krömer et al., 2013). All these environmental factors are products of the global climate change which has been reported to be one of the major factors responsible for the habitat change of many plants across the world (Sharpe, 2019). Most importantly, increase in global earth temperature has been predicted to lead to greater loss of plants' distributional ranges (Warren et al. 2018). Therefore, studies on the ferns species distribution along environmental gradients can be used to indicate the quality of forests (Othman et al., 2015). This study aimed to assess the diversity and richness of ferns along environmental gradients in Ayer Hitam forest, Johor, Malaysia. Their distribution patterns as influenced by the environmental conditions and their conservation statuses were elucidated in this study.

Study area
This scientific expedition was carried out in Ayer Hitam forest, which is a lowland reserved forest located in Muar, Johor, Malaysia (Fig. 1). Muar (Bandar Maharani) is known to be one of the most tourist visited cities in Johor, Malaysia. This town has an approximate land mass of 234,612 ha out of which 83% of it was reported to be covered by natural forests in the year 2010. In year 2019, almost 138ha of its natural forests were lost due to several anthropogenic factors (https://www.globalforestwatch.org). Muar can be described as a city with a year-round tropical climate. The mean monthly temperature ranges between 21.4 -32.7 o C. The city's mean monthly precipitation ranges from 112 to 238 mm. The people of Muar engage in the cultivation of some major cash and food crops including rubber, oil palm, coconut, cocoa, fruits and vegetables. Beside these, they are also involved in fish, poultry and livestock farming.

Plants Collection and Identification Techniques
The collection of the ferns was done using the preferential non-random sampling technique as described by Akomolafe and Rahmad (2019). Four different sampling sites were chosen for study within the forest based on ease of accessibility and based on the presence of any fern species there. The complete and healthy ferns fronds were collected and identified. At each point of plant collection, the light intensity and temperature were measured with an Urceri MT-912 light meter. Relative humidity was measured with a HT-86 humidity meter. Elevation and geographical coordinates of the sampling sites were measured using a hand-held Global Positioning System (GPS) device. The taxonomic identities, naturalization and conservation status of the ferns species were established using the flora of Peninsular Malaysia (Parris et al., 2010(Parris et al., , 2013(Parris et al., , 2020, fern flora of Malaysia (Piggott, 1988), International Plant Names Index (IPNI, 2020), Malaysia Biodiversity Information System (MyBIS), and International Union for Conservation of Nature and Natural resources (IUCN). The voucher specimens of the collected and identified ferns were deposited at the herbarium of the Universiti Sains Malaysia.

Data analyses
The ferns species richness of the forests was estimated using an incidence-based rarefaction and extrapolation analysis. This was accomplished with 500 bootstrap replicates using iNEXT software (Chao et al. 2016). The 95% confidence intervals of the species accumulation curves were used to determine the significance difference in the species richness. An overlap in the confidence intervals shows there is no significant difference in the species richness between the sites. This analysis is important because it quantifies the actual number of species in the sampled sites which might not have been adequately observed on the field. The similarity of the sites in terms of the fern species found there was analyzed using a Bray-Curtis index with single linkage algorithm in BioDiversity Pro software. Principal component analysis (PCA) was done to see the pattern of variations in the distribution of the ferns at the different sites. Detrended correspondence analysis (DCA) which is a multivariate analysis used by ecologists for arranging multiple species along environmental gradient was employed in this study. This analysis was able to isolate the important environmental factors closely related to the distribution of the fern species. In order to have further robust explanations on the factors that drive the fern species distribution along the environmental gradients, we did a simple general linear model (GLM) with the species richness as response variable and the environmental factors as explanatory variable. The PCA, DCA and GLM were achieved using PAST 3 software.

RESULTS AND DISCUSSION Influence of the Environmental Gradients on the Ferns richness and Diversity
Thirty-nine (39) fern species were identified at site 1 with the following ranges of environmental factors; light (337.9 -8098 lux), temperature (30.1 -32.5 o C), humidity (76.40 -84.27 %) and elevation (14 -28m). At site 2, fifteen (15) fern species were identified along the following ranges of environmental factors; light (414.9 lux), temperature (33 o C), humidity (66.24%) and elevation (15m). Also, at site 3, fourteen (14) ferns species were observed along the following environmental factors; light (588.8 -607.2 lux), temperature (28.5 -29.4 o C), humidity (84.16 -88.80%) and elevation (18 -27m). At site 4, ten (10) ferns species were observed having the following environmental ranges; light (1423 lux), temperature (29.0 o C), humidity (81.08%) and elevation (33m). In all the sites, a total of 67 fern species belonging to 14 families were enumerated (Table 1). Aspleniaceae and Lindsaeaceae had the highest number of species (12 and 11 respectively) while Hypodematiaceae and Marattiaceae are represented by only one species each (Fig. 2). Tectaria singaporeana is the only species found common to all the study sites. Nephrolepis biserrata is also the next species which was found common to only three out of the four study sites. The distribution of Tectaria singaporeana and Nephrolepis biserrata at most of the study sites shows that they are species that could adapt to the varying degrees of microclimates. The incidence-based rarefaction and extrapolation species curve shows that site 1 had the highest estimated species richness (90) which is significantly different from all the other sites (Fig. 3). Sites 2, 3 and 4 had the species richness of 30, 35 and 20 respectively. The wide difference between the observed and estimated species richness of the ferns species in these sites revealed the true state of what is available in the field and what could have been observed if the sampling was adequate (Zuquim 2015). As for the variations in the distribution of the species, the principal components (PCs) 1 and 2 accounted for a total variation of 47.15% and 29.47% respectively (Fig. 4). Site 1 and site 3 significantly contributed to the PC 1 while site 2 significantly contributed to PC 2. All the species encountered are within the 95% eclipse. The similarity among the sites in terms of the fern species is shown in the dendrogram (Fig. 5). This revealed a close relationship between site 2 and site 4 with the highest similarity index of 30%. The DCA analysis indicated that all the fern species in this forest were influenced more along the light intensity gradient than along other environmental factors measured (Fig. 6). The GLM revealed that the light intensity ( Figure 7A, r 2 = 0.009, P = 0.428, slope = -249.3, intercept = 1598), humidity ( Figure 7B, r 2 = 0.003, P = 0.645, slope = -0.557, intercept = 80.61) and elevation ( Figure 7C, r 2 = 0.000, P = 0.835, slope = -0.17, intercept = 17.62) had inverse effects on the distribution of the ferns. However, the temperature exhibited a direct relationship with the distribution of the ferns ( Figure 7D, r 2 = 0.007, P = 0.491, slope = 0.161, intercept = 30.86). The slight influence of humidity on the ferns observed in this study clearly supported the hypothesis that ferns species richness and diversity in tropical forests are promoted by water availability (Linares-Palomino et al. 2008;Krömer et al. 2013). In the same vein, the influence of temperature on the ferns species richness is described to be such that lower temperature decreases evapotranspiration rates and thereby increasing the humidity of the forests, hence increases the diversity and richness of the ferns (Krömer et al. 2013). The influence of elevation on the richness of ferns in Peninsular Malaysia has been reported earlier (Kessler et al. 2011). It was revealed that ferns richness increased with increasing elevation and this had a corresponding positive correlation with the environmental humidity. Although in our study, elevation is not the main environmental gradients influencing the ferns in the study sites. All these ferns were found at lower elevations. The importance of light intensity and temperature on the growth and distribution of ferns has also been reported by a study on ferns diversity along environmental gradients in Thailand (Sathapattayanon & Boonkerd 2006). They reported that low light intensity influenced the richness of ferns but negatively affected the diversity. This was also observed in this study whereby light intensity had an inverse relationship with the ferns richness. It is very significant to note that the elevation range of all the forests studied is between 14-33m. A similar study of ferns distribution in some urban areas of Pahang and Kedah, Malaysia has reported ferns to be mostly  200-500m (Othman et al. 2015). However, some of the ferns they reported as found at high elevations such as Dicranopteris linearis, Angiopteris evecta and Pityrogramma calomelanos were also found at lower elevations in this study. This revealed that those ferns are not restricted only to high elevations.

Conservation Status of the Ferns
The IUCN redlist status of all the observed ferns in this study revealed only four ferns namely Adiantum capillus-veneris, Nephrolepis undulata, Pteris vittata, and Dicranopteris linearis as least concern (Table 2). Others were categorized as either not recorded or not evaluated and this amount to 85% and 9% respectively (Fig. 8A). On the contrary, the Malaysia biodiversity redlist assessment status showed that 66% of the ferns are not evaluated, 4% have no records, 27% are least concern, 2% is data deficient and 1% is near threatened (Fig.  8B). Out of all these, two ferns namely Asplenium antiquum and Pteris longipinnula were first mentioned in this study. The assessment of the IUCN redlist status of ferns in this study further indicated that less attention has been given to the conservation status of ferns in Malaysia, and perhaps globally. This is because over 90% of the ferns were categorized under no record (NR) and not evaluated (NE) in the IUCN redlist. A similar observation was also reported concerning the ferns of Kedah, Malaysia whereby the IUCN status of the ferns were also lacking (Akomolafe & Rahmad 2019). The Malaysian biodiversity assessment report was also lacking in ferns species data due to over 60% of the ferns categorized as NE. This calls for an immediate comprehensive conservation assessment of ferns in Malaysia by both the local biodiversity agencies and IUCN. Also, Davallia solida which is classified as near threatened needs to be properly conserved in order to avoid it being vulnerable or endangered. Angiopteris helferiana which is popular in some Southern Asian countries (Lamichhane et al. 2019), was first reported in this study as a fern in Malaysia. All the newly mentioned ferns were not formally reported to be found in Malaysia by Parris et al. (2010) andPiggott (1988). They are also not mentioned in the Peninsular Malaysia biodiversity assessment portal. However, further taxonomic descriptions particularly using molecular techniques are needed to substantiate these newly mentioned species.

CONCLUSION
This study revealed that the ferns species identified at these studied forests were positively influenced mostly by low light intensity and increasing temperature. The influence of these environmental gradients on the distribution of the fern species at the various study sites of this forest revealed that any corresponding change in the ferns species richness and distribution could detect the change in the microclimatic conditions of the forests. Considering the paucity of information on the conservation statuses of the ferns in Malaysia, it is imperative for relevant government agencies and ecologists to pay close attention to the ecology and conservation of this group of plants.    Table 1.