Morphometric study of Kalophrynus palmatissimus at two forest reserves in Malaysia

Abstract A research study on morphometrics of Kalophrynus palmatissimus (commonly known as Lowland Grainy Frog) at Ayer Hitam Forest Reserve (AHFR), Selangor and Pasoh Forest Reserve (PFR), Negeri Sembilan was carried out from 12 November 2016 to 13 September 2017. The study was to examine data on the morphometric traits of K. palmatissimus at the two forest reserves. 15 morphometric traits of K. palmatissimus that were taken by using vernier calipers. Frog surveys were done by using 15 and 18 nocturnal 400 m transect lines with an interval distance of 20 m at AHFR and PFR, respectively. The GPS coordinates for all frog samples were recorded to ensure the precise geographic location. In addition, five climatic data were recorded. The results showed that most morphometric traits in AHFR (n = 34) and PFR (n = 31) were positively correlated with each other. On the other hand, climatic factor, which was soil pH, had a significant positive influence on most of the morphometric traits (p < .01), except for tympanum diameter and upper eyelid width (p ≥ .05). Meanwhile, the temperature had a significantly negative influence on all morphometric traits (p < .01). General linear model (GLM) analysis showed that snout‐vent length (SVL) influenced most morphometric traits (F ≤ 80.86, p < .01), except for hand length (HAL: F = 0.299, p > .05). Later, it was found that the snout‐vent length of K. palmatissimus at AHFR was slightly larger than at PFR (AHFR: μ = 37.00 mm, SE = 1.16 c.f. PFR: μ = 30.29 mm, SE = 1.07). It showed that there were variations in morphometric traits of K. palmatissimus at AHFR and PFR. From PCA analysis, morphometric traits are grouped into two components for AHFR and PFR, respectively. In AHFR, head length, eye diameter, head width, internarial distance, interorbital distance, forearm length, tibia length, foot length, and thigh length were strongly correlated, while snout length and eye‐nostril distance were strongly correlated. In PFR, eye diameter, head width, internarial distance, interorbital distance, foot length, and thigh length were strongly correlated, though snout length and eye‐nostril distance were strongly correlated, hence, suggested that all morphometric traits grow simultaneously in K. palmatissimus with eye‐nostril distance (EN), and snout length (SL) growing almost simultaneously at AHFR (r = .91) and PFR (r = .97). There is still a lack of available information regarding the distribution and morphometric studies of K. palmatissimus in Malaysia, especially at AHFR and PFR. This study showed 15 different morphometric traits of K. palmatisssimus between AHFR and PFR, with K. palmatissimus at AHFR were found to be slightly larger than at PFR.


| BACKG ROU N D
The genus Kalophrynus is reported to consist of 25 nominal species with the greatest diversity in Borneo (Zug, 2015 (Zug, 2015).
These species are usually found at relatively low elevations, and the highest known record was 1,006 m above sea level (a.s.l) for K. robinsoni (Dring, 1979). Manthey and Grossmann (1997) reported that it usually inhabit or habituate undisturbed lowland rainforests and bamboo stands. Meanwhile, their reproduction sites include waterfilled bamboo stumps and other cavities (IUCN, 2016). However, its phylogenetic relations with other Microhylidae family are unclear (Frost et al., 2006), and now it is considered to represent a distinct subfamily, Kalophryninae (Frost, 2009). Phylogenetic relationships are assessed either by using a classical approach or molecular approach, or a combination of both approaches. The classical approach refers to morphological characteristics and morphometric traits. Morphometric traits of herpetological organisms are necessary for species delineation, phylogenetic analyses, and understanding of evolutionary changes in an organism's physical characteristics, but yet there are few consistencies in physical measurements and descriptions across, or even within taxonomy (Dubois, 2010). Arbour and Brown (2014) assigned that the accurate measurement of amphibian morphometrics is essential for taxonomy, studies of growth and development, and studies of fluctuating asymmetry. According to Singleton et al. (2011), morphometric or morphometry is a quantitative analysis form, which is a concept that divided into size and shape. In frogs, 12-16 morphometric traits are used to distinguish species (Chan et al., 2011;Kiew, 1984;Watters et al., 2016).
In this study, K. palmatissimus from two populations: Ayer Hitam Forest Reserve (AHFR), Selangor and Pasoh Forest Reserve (PFR), Negeri Sembilan, studied for 15 morphometric traits. According to Matsui (1984), there were 15 body measurements of K. palmatissimus were taken in millimeters (mm), since there is a lack of study on the detailed information concerning the morphometric traits differentiation of these species in Peninsular Malaysia especially at AHFR and PFR. Though several studies conducted, the studies only measured 13 morphometric traits (Kiew, 1984) and two morphometric traits  for species from Ayer Hitam Forest Reserve (AHFR), Selangor with fewer individuals (n = two). Furthermore, previous studies only focused on morphometric traits of K. palmatissimus with different populations separately (Kiew, 1984). In the aspect of environmental parameters, air temperature (°C), light intensity (lx), wind (m/s), soil pH, and relative humidity (%RH) of two forest reserves were determined and recorded. Therefore, the objectives of this study are to determine the morphometric traits differences of K. palmatissimus at the two previously studied forest reserves (AHFR and PFR) and the potential correlations among morphometric traits and climatic factors.

| Study sites
The project conducted at two study sites, which were Ayer Hitam Forest Reserve (AHFR), Selangor and Pasoh Forest Reserve (PFR), Negeri Sembilan from 12 November 2016 to 13 September 2017.
The Ayer Hitam Forest Reserve (AHFR) is 15 to 233 m above sea level with its highest peak at Bukit Permatang Kumbang. The Rasau River at the south and Bohol River at the north are the primary sources of irrigation in this forest (Paiman & Amat, 2007;Shamsudin et al., 2015). The three studied compartments of AHFR were Compartment 12, Compartment 13, and Compartment 15 ( Figure 1). Meanwhile, the core area of Pasoh Forest Reserve (PFR), which is approximately 600 ha, is still covered with oldgrowth forest. However, most of the surrounding areas of this forest logged in the past. Therefore, this forest is an example of a regenerating lowland forest. The PFR has an area of approximately 140 km 2 , covered with lowland dipterocarp forest and hill dipterocarp forest at its north-eastern boundary. The three studied PFR compartments were Compartment 21, Compartment 22, and Compartment 32 ( Figure 2). information regarding the distribution and morphometric studies of K. palmatissimus in Malaysia, especially at AHFR and PFR. This study showed 15 different morphometric traits of K. palmatisssimus between AHFR and PFR, with K. palmatissimus at AHFR were found to be slightly larger than at PFR.

K E Y W O R D S
climatic factors, forest reserve, Kalophrynus palmatissimus, morphometrics, transect lines

| Sampling of Kalophrynus palmatissimus
During this study, a visual encounter survey (VES) was utilized as the sampling method. VES helps to maximize the observation of frogs in each survey so that the optimum number of frog species per survey can be achieved (Crump & Scott, 1994). Surveys on K. palmatissimus were conducted at two different study areas which were located at walking trails and river banks based on the accessibility of these areas. Fifteen and eighteen 400 m nocturnal transect lines with an interval distance of 20 m were established for frog surveys in AHFR and PFR, respectively. Gillespie (1997) recommended that 400 m to 500 m transect lines along riparian habitat were the preferred distance for a VES to study threatened frog species.
The visual encounter survey procedure consisted of an active search for frogs in a randomized walk at a steady pace from November 2016 to September 2017. At AHFR, the frog surveys were conducted for eight nights per month within a five months of the sampling period. In PFR, the surveys were conducted for 12 nights per month within a six-month sampling period. The duration for each survey was between three and four hours, which started at 2030 and finished at 0000, involving two to three personnel to search for the presence of K. palmatissimus.
The coordinates of captured K. palmatissimus were also taken by using a GPS device (Garmin GPSMAP 64S Handheld) to ensure precise geographic location records. Prior to the survey, climatic factors, such as macroclimates and microclimates data (i.e., temperature, wind, light intensity, humidity, and soil pH) were recorded by using meteorological apparatus (Extech Meter for Humidity, Temperature, Airflow and Light Level 45179) and pH soil device (pH soil and moisture tester Takemura DM-15 for soil pH).
Captured frogs were stored in plastic containers (12 cm × 7 cm) and brought back to the on-site laboratories. The captured frogs were weighed by using a weighing scale (PESOLA Weighing Scale), and photographs of the frogs at every viewpoint were taken for species identification. Species identification was made by referring to the online database, namely "Amphibia.my: Amphibians & Reptiles of Peninsular Malaysia," and reference books entitled "Amphibians and Reptiles of the Seribuat Archipelago of Peninsular Malaysia" by Grismer (2011) and "The Amphibian Fauna of Peninsular Malaysia" by Berry (1975).

| Morphometric traits measurement
Fifteen morphometric traits of K. palmatissimus were taken in millimeter (mm) units of the length, according to Matsui (1984)  After taken all of the measurements, blue nail polish was used as a dye to mark the tibia of each K. palmatissimus before released it back at the captured area. It was done to avoid any recapture of K. palmatissimus individual. The frogs were ethically handled during the study (UPM/IACUC/AUP-R007/2018).

| Data analysis
To evaluate sampling effort and relate the morphometric traits of K. palmatissimus between two forest reserves, individuals of this species were assessed based on the presence or absence at the study areas (walking trails and river banks). All statistical analyses were done separately for individuals from AHFR and PFR. The independent samples t test was performed to evaluate sexual dimorphism since it is known that K. palmatissimus is a sexually dimorphic animal (Kiew, 1984). According to Tolosa et al. (2015), sexual dimorphism in anuran species are body size (female is larger than male), tympanum size (male has tympanum size larger than eye while the female has tympanum size equal or smaller than the eye), and the throat coloration, which is a dark-spotted coloration in males, while light color in females. Pearson's correlation coefficient test was used to address the association between 15 morphometric traits to evaluate the significant correlation of morphometric traits within them. Meanwhile, the association between climatic factors, which are microclimates data and morphometric traits, were calculated by using Spearman's    Table 5). The soil pH A (a macroclimate factor) had significantly influenced the head length and foot length (Table 5); however, the soil pH B (a microclimate factor) had significantly influenced snout length, eye-nostril distance, and upper eyelid width (Table 5) (AHFR and PFR) had significantly influenced the eye diameter, internarial distance, and thigh length (Table 5). Meanwhile, the head width and tibia length of K. palmatissimus were significantly influenced by the interaction between sex and SVL (  Figure 5). Four morphometric traits, snout-vent length, tympanum diameter, upper eyelid width, and hand length, were not included in any components due to the low value of loading factors (≥0.40 on more than one component).

| RE SULTS
In    and vegetation. It is known that morphometric differentiation of amphibians is correlated by climatic and ecological conditions (Amor et al., 2009) and a combination of genetic and environmental factors (Savage et al., 2015;Stock et al., 2008).  (Hessen et al., 2013).

| D ISCUSS I ON
In turn, body size affects survival, locomotion, and reproductive success (Wikelski & Romero, 2003). Therefore, the temperature can be an important selective factor, and testing on how physiological traits, morphology, and locomotion reaction to different thermal environments, can provide a better understanding of local adaptations of anurans (Angilletta et al., 2004). Anderson and Johnson (2018) argued that soil pH was found to be important as an abiotic factor that affects the growth and survival of Marbled Salamanders. As soil acidity increased, the salamander body development and survival will decline. At AHFR, male and female K. palmatissimus demonstrated statistically significant differences for only head length (HL), eye diameter (ED), and head width (HW). The statistical results were the same for PFR, except for snout-vent length (SVL), internarial distance (IND) and thigh length (THL) also showed a significant difference. The study proposed that there was sexual dimorphism between males and females K. palmatissimus at AHFR and PFR. Sexual dimorphism in anuran is where the male and female differ in body size and this difference is attributed to sexual selection (Bell & Zamudio, 2012).
However, a limitation in this study is that the species of K. palmatissimus is restricted to only undisturbed areas. For example, the 50-hectare plot, which is one of the study areas in Compartment 32 at PFR, has relatively high stem density, where large numbers of trees have been logged over the recent years. Manokaran and LaFrankie (1990)

| CON CLUS IONS
The present study showed the comparison of 15 morphometric traits of K. palmatisssimus at two forest reserves (AHFR and PFR), with K. palmatissimus at AHFR were slightly larger than at PFR. Furthermore, the females and males of K. palmatissimus were found to be sexually dimorphic. Still, all morphometric traits had simultaneous growth, and the temperature had a negative influence on all morphometric traits, whereas soil pH had a positive effect on a few morphometric traits. It suggested that the information from this study could contribute to a better understanding of K. palmatissimus morphologies characteristics and the influence of climatic factors (soil pH and temperature) on morphological characteristics of K. palmatissimus at the two forest reserves. This could help future conservation programs and management to protect this endemic species from extinction.