Polypedates leucomystax - Four-lined Tree Frog

Seen a brown frog clinging onto a surface in an urban area?

**Heard a loud, nasal 'quack' in the evening?**

You have likely encountered a Four-lined Tree Frog!

FOUR-LINED TREE FROG
Polypedates leucomystax (Gravenhorst, 1829)

Figure 1. A Four-lined Tree Frog, with a plain back, clinging onto the wall of a building at the Singapore Botanic Garden. (Source: Everlyn Julya Koh) Figure 2. A Four-lined Tree Frog, with four distinct brown stripes on its back, holding onto the stalk of a plant. (Source: Thomas Brown, Wikimedia Commons, used in accordance to terms of Fair Use)

1. INTRODUCTION

An ubiquitous and widespread frog species across Southeast Asia, including Singapore, the Four-lined Tree Frog (Polypedates leucomystax) is one of the most commonly-encountered frog species in Singapore. This nocturnal native species can be found in both natural and urban landscapes, typically heard or seen near pools of water in the evening where they congregate and produce loud, characteristic calls.¹ While there are at least seven other frog species that may be encountered in the urban areas of Singapore, the Four-lined Tree Frog is the only frog species amongst those that is arboreal and often observed off the ground, clinging onto surfaces of artificial structures (e.g. wall, glass) or holding onto the twigs, stems or leaves of shrubs and small trees.² Interestingly, the Four-lined Tree Frog may have obtained its common name from the four dark stripes extending down the back of some individuals. However, there are also individuals which do not possess such stripes and instead have a plain or spotted back, though they are not as common in Singapore.³ Like many members of the Family Rhacophoridae, this frog adopts a rather peculiar reproductive strategy where eggs are deposited in a foam nest built just above the water surface.⁴ This species has also been said to form a species complex with other cryptic species.⁵

TABLE OF CONTENTS

2. BIOLOGY

2.1. Diet

The Four-lined Tree Frog is generally considered an opportunistic predator and has a diet differing between the tadpoles and adult life stages. Within water bodies where food is scarce, exotrophic tadpoles are known to feed on almost any food items that are available, and larger tadpoles may feed on almost any organism that they can overpower. This include both live and dead tadpoles belonging to the same or different species, as well as decomposing plant material such as fallen leaves and bark.⁶ The adults are carnivorous and forage singly within low vegetation for small invertebrates which can include insects from orders Coleoptera (beetles) and Orthoptera (crickets, locusts and grasshoppers).⁷ Adults also sometimes prey on small vertebrates such as geckos. During a hunt, the frog is observed to be stealthy and ambushes prey at close proximity.

2.2. Predation & Parasite

The Four-lined Tree Frog faces changing predation pressures as it metamorphoses from a tadpole to an adult, transitioning from largely facing aquatic predators to terrestrial ones. The main predator of tadpoles are nymphs of Order Odonata (dragonflies and damselflies) that dwell in the same water body as tadpoles.⁸ Tadpoles of the Four-lined Tree Frog are not known to exhibit notable anti-predatory defensive behaviours apart from hiding in decomposing plant materials and fleeing from potential predators.⁸ The adults may be preyed upon by a variety of terrestrial predators which include birds, snakes and lizards. In Singapore, there has been two reports of the Four-lined Tree Frog being attacked by the Painted Bronzeback (Dendrelaphis pictus), a snake that is often encountered in urban areas and is known to consume frogs and lizards.⁹ ¹⁰ ¹¹ When held by the snake, the Four-lined Tree Frog was reported to emit a series of loud, squeaky distress calls, an anti-predatory defensive behaviour typically exhibited by many species of frogs when held in order to startle the predator.¹² ¹³ In both reports of attack, it was however not known if the frogs have been successfully subdued and consumed by the snake.¹⁴ ¹⁵

The Four-lined Tree Frog may also carry various protozoan parasites (e.g. Cepedea dimidiata, Entamoeba ranarum, Trypanosoma sp.) in its guts, rectum and blood.¹⁶ While the parasitic amoeba Entamoeba ranarum has been said to cause amoebiasis in these frogs ¹⁷ , Cepedea dimidiata may simply be a commensal and likely brings no negative effect on the frog.¹⁶ In Singapore, tapeworms of genus Spirometra have been found in the gut of Four-lined Tree Frog. In Thailand, the Four-lined Tree Frog has also been found to carry a parasitic nematode (Rhabdias kongmongthaensis).¹⁸

Figure 3. A Four-lined Tree Frog being held at the nape by a Painted Bronzeback in Singapore. (Source: Tan & Low (2018), used in accordance to terms of Fair Use)

3. LIFE CYCLE & REPRODUCTION

3.1. Mating

In Singapore which has a tropical rain forest climate (based on Köppen climate classification), the Four-lined Tree Frog breeds year-round as there is no distinct dry season which may halt breeding. Locally, female Four-lined Tree Frogs have been found with an enlarged oviduct throughout the year. Eggs have also been sighted in almost all months across the year.¹⁹

Mating begins with the male frogs congregating on the vegetation near a stagnant water body to form a calling group.¹ ¹⁹ Such a water body may be a natural or man-made pond, ephemeral puddle or even water-containing flower pot and an urn.¹⁹ Only the male of this species emits advertisement calls and such calls are often produced while the male frog is arboreal (Video 1). Typically heard in the evening when this nocturnal frog is most active, these calls have been described as a "loud, widely-spaced nasal quack, and sometimes a low throaty chuckle".¹ ²⁰ The call of the Four-lined Tree Frog is very distinct and often unmistakable among those produced by urban-adapted frog species in Singapore.

Mating in the Four-lined Tree Frog has also been said to be largely driven by female mate selection. While females do not produce advertisement calls, females may produce a weak reciprocal call in response to advertisement calls emitted by a male. ²¹ On many occasions, the advertisement call responded to have been found to belong to the largest and heaviest male among all calling males.²¹ In response to the female's reciprocal call, this male may then continue to persistently call throughout the evening while making its calls more apparent by increasing the intensity and duration of each single note. Such calls that contain a higher acoustic energy may signal that the caller is in good health.²¹ The female may then choose to approach this calling male.

When a male successfully chases an approaching female, amplexus ensues which involves the male frog hitching securely onto the back of the female by clasping tightly at the female's arms or armpit regions (Figure 4).²² Interestingly, it has also been observed that the presence of a female in the vicinity typically causes adjacent males to terminate calling.²² The female frog will then carry the clasped male to the oviposition (egg-deposition) site.

3.2. Oviposition

Much like many frog species from the Family Rhacophoridae, the Four-lined Tree Frog constructs foam nest at the oviposition site where ivory-white eggs will be deposited into during a breeding event. The foam nest can be created on overhanging vegetation above a stagnant water body (Figure 5) or attached to a surface at the edge of a water body (Figure 6).⁶ ²³ Typically measuring 10 cm in length, the foam nest is created from the skin secretion of both female and male frogs, which will be wiped downwards to the cloaca region using their hind legs.²⁴ This foam nest is said to serve many important functions such as protecting the egg clutch against pathogens, predation and dehydration.²⁵ ²⁶ In 2017, an antimicrobial peptide (brevinin-2 type) has indeed been isolated from the foam nest of the Four-lined Tree Frog which is believed to guard the egg clutch from pathogens.²⁴ Eggs of the Four-lined Tree Frog are externally-fertilized. Once the foam nest is large enough to cover both the cloaca of female and male frogs, the female frog begins depositing as many as 150–900 eggs per clutch into the foam nest while the male frog will simultaneously release sperms into the foam nest to fertilize the eggs.²⁷

During a breeding event, other males may continue to contest for a chance to breed by attempting to dislodge the clasped male of the amplecting pair. In some cases, one female may simultaneously engage in amplexus with multiple males (Figure 5), a polyandry phenomenon said to be rare among frogs.²⁸ ²⁹ ¹⁹ This contestation between males can result in physical wrestling where the first male may attempt to physically drive away the second male while the second male attempts to remove the first. Should attempts by the second male to dislodge the first male fail, he may simply grasp onto the first male. More males may be added to a mating group so long as the foam nest is large enough to cover the cloaca of all males for successful sperm deposition.²⁹ Throughout the contestation, the female frog is usually unaffected by the brawl and will continue to deposit eggs into the foam nest.²⁹ Hence, it may be interesting to note that while the female frog may have actively selected her first male partner during courtship (which may have the best physical conditions among all calling males), there was no apparent effort made by her to ensure that her eggs are solely fertilized by this male. Once a breeding event terminates, female and male frogs will go on separate ways. It is not known if the Four-lined Tree Frog exhibits parental care to the egg clutch.

Eggs may hatch after four days into tadpoles with external gills. These tadpoles will then maneuver out of the foam nest and fall into the water beneath where they live until metamorphosis.³⁰ ²³ Tadpoles have been observed to require up to seven weeks from point of hatching to metamorphose into an adult frog.³⁰ Some tadpoles may leave the aquatic medium as a metamorph, with tail not reabsorbed. The vacant foam nest may then be disintegrated during heavy rain.

Video 1. Documentation of Four-lined Tree Frog calling in Singapore, 3rd February 2012 evening. (Source: Leong Tzi Ming, used in accordance to terms of Fair Use)

Figure 4. Smaller male (top) and larger female (bottom) adult Four-lined Tree Frogs in amplexus. (Source: Nick Baker, Permission pending; Annotation: Everlyn Julya Koh)

Figure 5. Multiple male Four-lined Tree Frogs clasping onto one female during oviposition. (Source: Feng & Narins (1991), used in accordance to terms of Fair Use)

Figure 6. A foam nest constructed on the inner walls of an artificial structure. (Source: W.A. Djatmiko, Wikimedia Commons, used in accordance to terms of Fair Use)

4. HABITAT & RANGE

4.1. Habitat

The Four-lined Tree Frog is notable for being an adaptable opportunist and a successful human commensal, with its range intimately following human activities which have been said to provide suitable living and breeding habitat for the frog.³¹ The frog has been found in vegetation in almost all forms of built-up habitats, including agricultural land, ditches, man-made ponds, parks and gardens, and even within buildings. This species has also been found to inhabit forest edge habitats and occasionally deep within the forest.³¹ ³ Within a patch of vegetation, the frog is typically observed on a tree, shrub or long grass. Tadpoles are aquatic and may be found in still water bodies with a silty base that has some exposure to the sun.

4.2. Range

Figure 7. Global distribution map of Four-lined Tree Frog showing its extant native range (highlighted in orange) and areas where it has been introduced (circled in purple). (Source: Diesmos et al. (2004), used in accordance to terms of Fair Use)

The Four-lined Tree Frog is widely distributed and abundant throughout Southeast Asia and has a native extant range stretching from Bangladesh, Thailand to the many islands of Indonesia and the Philippines (Figure 7).³² It has also been introduced into the Okinawa island of the Ryukyu archipelago, Japan, with the first sighting reported in 1964. Since 2009, wild populations have been established on more than 20 islands in Japan.³³

In Singapore, the Four-lined Tree Frog is also widespread and abundant across the island in both natural and built-up environments. In green spaces, the frog can be commonly seen in urban parks and gardens such as in Singapore Botanic Garden and Lower Peirce Reservoir Park, and in forest reserves and green buffer areas such as Bukit Timah Nature Reserve and various nature parks (e.g. Hindhede, Dairy Farm). They may even inhabit wetland and swamp habitats such as the Sungei Buloh Wetland Reserve and Nee Soon Swamp Forest respectively. Populations have also been reported on offshore islands, such as Pulau Ubin, Pulau Tekong and Semakau Island.³⁴ In largely urbanized spaces, individuals have also been reported in several areas within the National University of Singapore.

5. CONSERVATION, THREATS AND USES

According to the IUCN Red List of Threatened Species, the Four-lined Tree Frog has been listed as 'least concerned' with a stable population that is not critically fragmented.³² This species has been listed as such since it has a large range, is tolerant of a wide variety of habitat and is believed to have a big population that is not likely to be decreasing rapidly enough to be justify it being placed in a more threatened category.³²

Locally, this species has been considered 'common' by the Lee Kong Chian Natural History Museum and is hence not listed within the Singapore Red Data Book. While this frog species is not exposed to disturbances at an extent that could lead to its decline, the supposedly heavy use of pesticides around residential areas might endanger local sub-populations. This is especially problematic should individuals consume prey coated with pesticides or when pesticide is absorbed through the skin of the frog. This frog species is also involved in the international pet trade and is a popular captive frog species in many European and American regions.³⁵ ³⁶ However, the collection of wild individuals for pet trade has not reach a critical level that warrants critical attention. This species have also been used in traditional medicine and consumed as a form of bush meat in certain areas where it occur. For example, in Assam, India, the meat of this frog have traditionally been boiled and consumed with a belief that it cures asthma.³⁷

The primary conservation concern for the Four-lined Tree Frog is the cryptic diversity within the species complex since the phylogenetic relationship within the species complex still remains largely unresolved. This can pose serious conservation implications as some taxa within the complex might be in a predicament which requires conservation efforts. However, there needs to be trustworthy identification of each species within the complex before any at-risk species may be identified and conservation methods can be crafted. Hence, further research into the taxonomy, population size and distribution trends of this species is definitely needed.³¹

6. MORPHOLOGY

6.1. Description

TADPOLE & METAMORPH

The tadpoles of the Four-lined Tree Frog can grow to 40–50 mm in total length (Figure 8). The body of the tadpole is slightly flattened from the top, and is elongated in shape with eyes on each side of the body (Figure 9).²⁰ The mouth of the tadpole is ventral and downward-facing. The tail of the tadpole is narrowly pointed, especially towards the end. The dorsal skin of the tadpole is greyish-green or brownish in colouration and has a patchy appearance.²⁰ The ventral skin of the tadpole is white or cream coloured and the gut may be prominently seen. Tadpole of the Four-lined Tree Frog may be loosely distinguished from tadpoles of other species by the distinct white dot at the tip of its snout. In the late metamorphic stages, the tadpole may leave the aquatic medium as a metamorph and come on land while still staying close to the water body. A metamorph presents intermediate features between a tadpole and adults, typically with the front and back limbs fully-emerged but has yet to fully reabsorb its tail (Figure 10).


Figure 8. Lateral view of the Four-lined Tree Frog tadpole with emerged hind limb. (Source: Meegaskumbura et al. (2011), used in accordance to terms of Fair Use; Annotations: Everlyn Julya Koh)	Figure 9. Dorsal view of the Four-lined Tree Frog tadpole with emerged hind limbs. (Source: Nick Baker. Permission pending; Annotations: Everlyn Julya Koh)

Figure 10. A Four-lined Tree Frog metamorph that has fully-emerged and developed front and hind limbs but has not fully reabsorbed tail. (Source: Jonathan Hakim, permission pending; Annotation: Everlyn Julya Koh)

ADULT

The adult Four-lined Tree Frog is of a small to moderately large size, measuring 30–75 mm in snout-vent length (SVL) (Figure 11).³⁸ The body of adults may be slender to slightly stout, with limbs that are long and thin. The head of the frog is longer than wide and has a round-tip snout that has sharp edges. The tympanum (eardrum) of the frog is apparent and is slightly smaller than the eye diameter.²⁰ The skin of this frog is largely smooth on the back but may have rough granules on the belly. There exists a thin fold of skin that stretches horizontally from each eye to above the tympanum to behind the arm. There is also a thin, white ridge on outer edge of forearm. Tips of the fingers and toes are dilated into large, round flattened disks which act as suction pads.³⁸ The fingers are also not webbed while toes are half-webbed.


Figure 11. Dorso view of the Four-lined Tree Frog (striped morph). (Source: Pavel Kirillov, Wikimedia Commons, used in accordance to terms of Fair Use; Annotation: Everlyn julya Koh)	Figure 12. Lateral view of the Four-lined Tree Frog (striped morph). (Source: Thomas Brown, Wikimedia Commons, used in accordance to terms of Fair Use; Annotation: Everlyn Julya Koh)

There are generally three colour morphs in the Four-lined Tree Frog: striped (Figure 12), mottled (Figure 13), and plain (Figure 14) morphs.³⁹ ¹ In Peninsular Malaysia and Singapore, most individuals are of the striped morph. In other regions such as Java, the mottled morph seems to be most prevalent while in other areas such as Bali, the plain morphs are most prevalent.³ The striped morph, which are most commonly seen in Singapore, has light or dark yellowish-brown skin on the back with four distinct longitudinal dorsal stripes.¹ The mottled morph has isolated brown specks over its entire back while the plain form has neither stripes nor specks. Across all three morphs, the belly skin is whitish or cream coloured that is clear or with dark spots on the throat. The limbs of all three morphs also have dark horizontal bars, though these bars may not be distinct in the plain morph.³⁸


Figure 12. Striped morph of the Four-lined Tree Frog. (Source: Wildlife Singapore, permission pending)	Figure 13. Mottled morph of the Four-lined Tree Frog. (Source: W.A. Djatmiko, Wikimedia Commons, used in accordance to terms of Fair Use)	Figure 14. Plain morph of the Four-lined Tree Frog, encountered at the National University of Singapore (Source: Everlyn julya Koh)

The sex of the Four-lined Tree Frog may often be determined by body size (Figure 15). Females are much larger in size than males, measuring 57–75 mm and 37–50 mm respectively.²⁰ Males also have bodies that are more slender than those of females. Males also possess a yellowish, oval nuptial pad covering the dorsal surface of the first finger which serves to provide a firmer grip onto a female during amplexus.³⁸

Figure 15. Male (striped morph) and female (mottled morph) Four-lined Tree Frog in amplexus. Abdomen of female frog is swollen with eggs. (Source: W.A. Djatmiko, Wikimedia Commons, used in accordance to terms of Fair Use; Annotation: Everlyn Julya Koh)

6.2. Diagnosis

The genus Polypedates consist of 24 different species, but only the Four-lined Tree Frog may be found locally.⁴⁰ In Singapore, this species is also the only Tree Frog species in an urban environment. The Four-lined Tree Frog may be distinguished from other urban-adapted frog species in Singapore by having brown skin on its back, a relatively slender body, an obtusely-pointed round snout, and large rounded flattened disks which act as suction pads at the tip of all digits.

Outside of Singapore, such as in Borneo, the Four-lined Tree Frog may occur in the same regions as other members of the same genus.²⁰ The Four-lined Tree Frog may then be distinguished from some other members from the same genus using the following diagnostic characters:

Species	Photograph labelled with diagnostic features	Distribution
Four-lined Tree Frog (Polypedates leucomystax)	Figure 16. Four-lined Tree Frog (Source: Pavel Kirillov, Wikimedia Commons, used in accordance to terms of Fair Use; Annotation: Everlyn julya Koh)	Throughout Southeast Asia, including Singapore
Collette's Tree Frog (Polypedates colletti)	Figure 17. Collette's Tree Frog has a pointed snout while the Four-lined Tree Frog has a blunt snout. (Source: Inger et al. (2017), used in accordance to terms of Fair Use; Annotations: Everlyn Julya Koh)	Endemic to Borneo
Green-eyed Tree Frog (Polepedates chloropthalmus)	Figure 18. Green-eyed Tree Frog has green eyes while the Four-lined Tree Frog do not has green eyes. (Source: Das (2005), used in accordance to terms of Fair Use; Annotations: Everlyn Julya Koh)	Endemic to Borneo
File-eared Tree Frog (Polypedates otilophus)	Figure 19. File-eared Tree Frog has serrated projections above its tympanum and a sharply-pointed projection at the edge of jaw while the Four-lined Tree frog do not have these projections. (Source: Inger et al. (2017), used in accordance to terms of Fair Use; Annotations: Everlyn Julya Koh)	Throughout most parts of Southeast Asia (Absent in Singapore)
Dark-eared Tree Frog (Polypedates macrotis)	Figure 20. Dark-eared Tree Frog has a thick dark stripe extending from behind its eyes to over its tympanum and shoulder while the Four-lined Tree Frog has light brown flanks. (Source: Inger et al. (2017), used in accordance to terms of Fair Use; Annotations: Everlyn Julya Koh)	Throughout most parts of Southeast Asia (absent in Singapore)

7. TAXONOMY

7.1. Taxonavigation

Kingdom	Animalia
Phylum	Chordata
Class	Amphibia
Order	Anura
Family	Rhacophoridae
Genus	Polypedates
Species	Polypedates leucomystax

7.2. Etymology

The species name 'Polypedates leucomystax' has a Greek origin. The word 'Polypedates' is derived from the Greek noun 'pēdētēs' which means a leaper or a dancer, or from the Greek verb 'pēdanto' which means to leap or jump. Added with the English prefix 'poly-' which means 'many' in Greek, 'Polypedates' may mean multiple leapers or dancers which aptly described the arboreal lifestyle of members in the genus where members leap from branches to branches. The word 'leucomystax' is derived from the Greek adjective 'leucos' which means white, and the Greek noun 'mystax' which means an upper lip or a moustache. The species epithet 'leucomystax' is hence attributed to the extended white stripe present above the upper lips of this frog species.

Term	Origin and meaning
Poly	Greek noun for 'many'.
Pedates	Greek noun 'pēdētēs' meaning 'a leaper or dancer', or Greek verb ' pēdanto' meaning 'to leap or dance'.
Leuco	Greek adjective 'leucos' meaning 'white'.
Mystax	Greek noun 'mystax' meaning 'an upper lip' or 'a moustache'.

7.3. Original description & Type information

Despite being presently known as Polypedates leucomystax, this species was first described as Hyla leucomystax by Johann Ludwig Christian Gravenhorst in 1829 from a specimen obtained from Java, Indonesia. His original description of the species in Latin may be loosely translated into the species having a 'rust coloured dorsal skin', 'granular skin at the underside of the abdomen and hip' and 'the presence of an extended white line at the lateral edge of the upper jaw' (Figure 21).⁴¹ Based on the original description, one cannot be sure what system or species concept was used in delimiting this species. This is often the case in species descriptions as authors rarely provide information on the system or concept which have been used in delimiting the particular species.

The holotype of P. leucomystax, obtained from Java, Indonesia, is currently stored at the Breslau Museum, presumably the Museum of Natural History University of Wroclaw.⁴²

Figure 21. An excerpt from the original description of Polypedates leucomystax as Hyla leucomystax by Johann Ludwig Christian Gravenhorst in 1829. (Source: Gravenhorst (1829), used in accordance to terms of fair use)

7.4. Synonyms

A synonym is a scientific name given to a species that currently has a different scientific name. Synonyms may come about when taxonomists describe a species that has already been described. Through the years, the phylogenetic position of P. leucomystax has been reconsidered many times, leading to the species having faced multiple genus and epithet changes. Just nine years after being originally described in the genus Hyla by Gravenhorst, this species was moved to genus Polypedates by Tsudi in 1838. For the next 30 years, this species has been moved back and forth through Hyla and Polypedates. However, for most of its history, P. leucomystax had been places into the genus Rhacophorus. It is only until 1987 that the species has been placed back into genus Polypedates. The synonyms of the species are as follow:⁴⁰

Hyla leucomystax Gravenhorst, 1829
Hyla sexvirgata Gravenhorst, 1829
Hyla quadrilineata Wiegmann, 1834
Polypedates leucomystax Tschudi, 1838
Hyla leucopogon Tschudi, 1838
Hyla quadrivirgata Tschudi, 1838
Polypedates rugosus Duméril and Bibron, 1841
Polypedates quadrilineatus Günther, 1859 "1858"
Limnodytes celebensis Fitzinger, 1861 "1860"
Rhacophorus maculatus var. quadrilineata Boulenger, 1882
Hylorana longipes Fischer, 1885
Polypedates maculatus quadrilineatus Fischer, 1885
Rhacophorus leucomystax Boulenger, 1889
Rhacophorus leucomystax leucomystax Mocquard, 1890
Rhacophorus leucomystax quadrilineatus Mocquard, 1890
Rhacophorus leucomystax var. sexvirgata Boettger, 1894
Rhacophorus leucomystax quadrilineata Werner, 1903
Rhacophorus maculatus leucomystax Annandale, 1912
Rhacophorus maculatus himalayensis Annandale, 1912
Hyla wirzi Roux, 1927
Rhacophorus (Polypedates) leucomystax Bourret, 1927
Rhacophorus (Polypedates) quadrilineatus Bourret, 1927
Rhacophorus kampeni Ahl, 1927
Rhacophorus (Rhacophorus) kampeni Ahl, 1931
Rhacophorus (Rhacophorus) leucomystax Ahl, 1931
Rhacophorus (Rhacophorus) himalayanus Ahl, 1931
Rhacophorus (Rhacophorus) leucomystax leucomystax Wolf, 1936
Rhacophorus (Rhacophorus) wirzi Forcart, 1946
Rhacophorus leucomystax quadrilineatus Inger, 1954
Polypedates teraiensis Dubois, 1987
Polypedates leucomystax Dutta, 1997
Rhacophorus teraiensis Ao, Bordoloi & Ohler, 2003

7.5. Common names

As the striped morph of P. leucomystax is most commonly seen in Singapore, most Singaporeans primarily adopt the common name 'Four-lined Tree Frog' when referring to this species. However, other common names such as 'Common Tree Frog', 'Java Whipping Frog', and 'White-lipped Tree Frog' are also popularly used in neighbouring countries such as Malaysia and Indonesia where this species also commonly occur. It may also be interesting to note that while most common names shown are English names, there are also non-English common names attributed to this species. The common names of the species are as follow:⁴⁰

Bamboo Tree Frog
Brown Tree Frog
Common Tree Frog
Four-lined Tree Frog
House Tree Frog
Jar Tree Frog
Java Whipping Frog
Malayan House Frog
Malayan Tree Frog
Stripe Tree Frog
White-lipped Tree Frog
Shiroagogaeru (Japanese)
Palakang Saging (Tagalog)

7.5. Species complex

Many lineages of animals that occupy a wide geographical range may sometimes encompass cryptic species, and Polypedates leucomystax is one of such species.⁵ In the case of the Polypedates leucomystax species complex, this group consist of frogs species which are particularly tough to be distinguished morphologically and genetically from the 'true' P. leucomystax with type locality Java, Indonesia. Some members from the genus Polypedates such as P. macrotis and P. maculatus were also once considered part of the P. leucomystax species complex, though both are treated as distinct species today.⁵

To examine the ambiguity in phylogenetic relationship between cryptic species within the P. leucomystax species complex, a phylogenetic study was conducted by Kuraishi et al. in 2013 which analysed the phylogenetic relationship of groups within the species complex using Polypedates specimen collected from diverse areas of Southeast Asian and South Asian countries. In this study, several mitochondrial genes (12S rRNA, tRNA^val and 16S rRNA) and concatenated nuclear DNA genes (BDNF, NCX, POMC, Rag1, Rhod, SCF and Tyr) of Polypedates specimen were used for Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian analyses. Bayesian trees were then generated to show relationship between Polypedates species. ⁵

Based on the Bayesian tree generated using mtDNA genetic sequences (Figure 22), the P. leucomystax species complex can be said to consist of six major clades with each clade represented by one species: South China Clade (P. cf. mufus 1), North China Clade (P. brauen), Indochina Clade (P. megacephalus), Sunda Clade (P. leucomystax), Laos Clade (P. cf. mufus 2) and Malay Clade (P. sp.). However, the Bayesian tree (Figure 22) still suggest substantial ambiguity amongst species within the complex, with many nodes where these six major clades originate from having relatively low bootstrap support values, hence suggesting that these clades are not particularly stable. This ambiguity may also be highlighted by the Malay Clade being embedded within a polytomy, suggesting that phylogenetic position of this clade within the tree has not yet been resolved. The Bayesian tree combining analysis of both mitochondria DNA and nuclear DNA (Figure 23) provided a largely similar relationship and conclusion. However, instead of a polytomy as highlighted by analysis using mitochondra DNA, analysis using concatenated nuclear DNA shows that P. sp. from the Malay clade had diverged prior to P. macrotis. Although this relationship may be substantially supported by a BPP of 1.0, this support value may drop with additional data and specimen used since the Malay clade had a small number of samples used. Thus, there needs to be further work carried out on the taxonomy on P. leucomystax species complex to better resolve such cryptic relationship between the species.

Figure 22. A Bayesian tree constructed using 2005 bp mitochondrial 12S rRNA, tRNA^val and
16S rRNA genes for specimen used in the study. The numbers at the end of each branch represent sample numbers which the scientists have assigned to each of the specimen. Numbers above the branches are bootstrap support (BS) values for the maximum parsimony (MP), maximum likelihood (ML) and Bayesian posterior probability (BPP) (MPBS/MLBS/BPP). Asterisks mark nodes with MPBS and MLBS ‡ 70% and BPP ‡ 0.95. (Source: Kuraishi et al. (2013), used in accordance to terms of fair use)

Figure 23. Bayesian trees constructed using 2005-bp sequence of the mitochondrial 12S rRNA, tRNAval and 16S rRNA genes (left) and a 4696-bp concatenated sequence of the nuclear BDNF, NCX, POMC, Rag1, Rhod, SCF and Tyr genes (right). Numbers above the branches are bootstrap support (BS) values for the maximum parsimony (MP), maximum likelihood (ML) and Bayesian posterior probability (BPP) (MPBS/MLBS/BPP) in the mtDNA tree (left) and BPP in the nuDNA tree (right). (Source: Kuraishi et al. (2013), used in accordance to terms of fair use)

7.5. Genetic sequence/barcode

To create a phylogeny between samples or specimens, universal marker genes are typically used. For amphibians, commonly used genes in constructing a phylogeny include mitochondria genes such as the Cytochrome Oxidase Subunit 1 (COI) gene, 16S rRNA and tRNA^val. For Polypedates leucomystax, the DNA sequence of COI gene, and 16S rRNA and tRNA^val may be obtained from Genbank database here and here respectively.

8. SYSTEMATICS

The family Rhacophoridae, or "Old-World Tree Frog", is an exceptionally diverse amphibian family in Asia. This family is a well-supported monophyletic clade, with 18 genera and 413 species spreading all across Asia with a disconnected presence in Africa.⁴³ ⁴⁰ The most recent phylogeny of family Rhacophoridae has been elucidated and published by Chan et al. in 2018.⁴⁴

In this study, Chan et al. (2018) had carried out a comprehensive multi-loci phylogeny of Rhacophoridae using molecular data from 327 specimen representing an estimated 247 species from all 18 genera of the family. For all specimen, two mitochondria genes (16S rRNA and Cytochrome-b) and two nuclear DNA genes (RAG-1 and Tyrosinase) were analysed. In particular, the 16S gene was used because it is said to an universal DNA barcoding marker gene in amphibians, and it is also popularly used in species delimitation.⁴⁵ ⁴⁶ These sequences were mostly compiled from Genbank with a small proportion of sequences isolated from specimens collected in Malaysia. The sequences from the four genes were concatenated and phylogenies were estimated using maximum likelihood and Bayesian analysis. The Bayesian phylogeny (Figure 24) was created using BEAST v2.4.8. The maximum likelihood phylogeny (Figure 25) was created using W-IQ-TREE phylogenomic software and model-testing function was used to conclude the best-fit model for each gene partitioned under Bayesian information criterion. To test other possible topological hypotheses, the Shimodaira-Hasegawa-like approximate likelihood ratio test (SH-aLRT) was used and branch support was evaluated using Ultrafast Bootstrap Approximation. For the Bayesian phylogeny (Figure 24), maximum boostrap support value for IQ-TREE SH-aLRT and Ultrafast are both 100% while maximum Bayesian posterior probability is 1.0. For maximum likelihood phylogeny (Figure 25), blue circle at a node indicate a bootstrap support (BS) of > 90 while nodes indicated with red circle indicate BS < 90. To evaluate species boundaries, the Automatic Barcode Gap Discovery (ABGD) method was also used and the 16S gene sequences were used in the analysis.⁴⁷

Both the Bayesian and maximal likelihood phylogenies constructed by this study show largely the same topologies with most nodes having high support values. From the Bayesian phylogeny (Figure 24), the genus Taruga is shown to be the sister genus to Polypedates with maximum bootstrap support (100/100/1.0). This topology may also be observed with high bootstrap support in the maximum likelihood phylogeny (Figure 25). This topology has also been shown to be well-supported by Meegaskumbura et al. (2010) with Bayesian posterior probability of 1.0 and maximum parsimony bootstrap support of 100% when the authors newly described the genus Taruga.⁴⁸ Despite having differences in some morphological characters such as the shape of the cranium, both genus are still relatively similar genetically and in terms of lifestyle where frogs from both genus are closely associated with being arboreal. With these evidences, one may confidently consider Polypedates and Taruga to be sister genus.

Within genus Polypedates, the topologies within the P. leucomystax species complex is relatively well-supported with all BS > 90. However, this study has shown P. leucomystax to be a well-supported sister species to P. cf. mutus (Figure 25) instead of P. megacephalus (Figure 22 & 23). This discrepancy in topology further highlights the controversy mired within the taxonomy of P. leucomystax complex and that more data should be analysed in order to clarify the topology between these cryptic species. Furthermore, unlike Kuraishi et al. (2013), the authors for this study have also considered P. impresus and P. teraiensis to be part of the species complex with the topologies of these two species having high BS > 90. Since P. impresus has been included in phylogenetic studies of the species complex as early as that of Kuraishi et al. (2013), this discrepancy may be due to different sampling efforts and not due to species being newly added into the complex.⁵ ⁴⁹

Figure 24. Bayesian phylogenetic tree at the generic level. Node support is denoted as: IQ-TREE SH-aLRT bootstrap support/Ultrafast bootstrap support/Bayesian posterior probability. The genus Polypedates is boxed up using an orange rectangle while the node bootstrap support value for monophyly has been underlined in orange. (Source: Chan et al. (2018), used in accordance to terms of fair use)

Figure 25. A section of maximum likelihood phylogeny of the family Rhacophoridae estimated using 3483 bp of sequence data from four genes. Circles at nodes denote ultrafast bootstrap support values from the IQ-TREE analysis (blue = BS > 90; red = BS < 90). Polypedates leucomystax has been boxed up using an orange rectangle.(Source: Chan et al. (2018), used in accordance to terms of fair use)

9. GLOSSARY

Word	Meaning
Nocturnal	Active beyond dusk and before dawn.
Cryptic species	Individuals that are morphologically very similar to one another but belong to different species.
Species complex	A group consisting of closely-related species that are so similar in morphology and genetics to the extend that the boundaries between them are ambiguous.
Opportunistic predator	A predator that feeds whenever a prey is available.
Exotrophic tadpole	A tadpole that needs to feeds on other organism to grow and metamorphose.
Commensalism	An interaction between two organisms by which one benefits and the other acquire neither benefit nor harm.
Advertisement calls	The vocalisation created for the purpose of attracting a mate.
Amplexus	The mating posture of a frogs and toads, where the male clasp onto the back of a female.
Oviposition	The laying of eggs.
Cloaca	A short tube at the vent of the frog which connects internally to the genital, bladder and rectum.
Polyandry	A form of mating structure whereby a female organism has more than one male mate.
Ventral	The underside or abdominal side of an organism.
Dorsal	The upperside or back of an organism.
Species delimitation	The process by which species boundaries are decided.
Holotype	A single type specimen which the new species is being described and named upon.
Monophyletic clade	A group of organism that encompasses a common ancestor and all of its descendant.

10. REFERENCES

Footnotes
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