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Accepted by M. Vences: 29 Apr. 2009; published: 1 Jun 2009 51
ZOOTAXA
ISSN 1175-5326 (print edition)
ISSN 1175-5334 (online edition)
Copyright © 2009 · Magnolia Press
Zootaxa 2122: 51–68 (2009)
www.mapress.com/zootaxa/Article
Two new species of shrub frogs (Rhacophoridae: Philautus) from the lowlands of
Sri Lanka
MADHAVA MEEGASKUMBURA1,2,3., KELUM MANAMENDRA-ARACHCHI2 &
ROHAN PETHIYAGODA2
1 Museum of Comparative Zoology, Harvard University, 26 Oxford Street, Cambridge MA 02138, USA
2 Wildlife Heritage Trust (WHT), P.O. Box 66, Mt Lavinia, Sri Lanka
3Corresponding author. E-mail: mmeegask@oeb.harvard.edu
Abstract
Two new species of Sri Lankan frogs of the genus Philautus are described. Species diagnoses are based on morphology,
morphometrics and mitochondrial DNA sequence data. Philautus tanu sp. nov. inhabits shrubs in open areas of the
lowland wet zone, while P. singu sp. nov. is found on shrubs in the understory of lowland and mid-elevation rainforests.
These descriptions bring the total number of valid Sri Lankan Philautus to 65 species, of which 46 are extant.
Key words: Rhacophorinae, taxonomy, molecular systematics, new species, conservation
Introduction
Following the discovery in Sri Lanka of a large radiation of Oriental tree-frogs of the genus Philautus
(Meegaskumbura et al. 2002), 37 new species have up to now been described as part of an on-going effort to
document this fauna (Manamendra-Arachchi & Pethiyagoda 2005; Meegaskumbura & Manamedra-Arachchi
2005; Meegaskumbura et al. 2007). The review and description of 27 new species by Manamendra-Arachchi
and Pethiyagoda (2005) though informed by a phylogeny, was based purely on morphology (given the
unavailability of molecular data for historical type material). However, Meegaskumbura & Manamendra-
Arachchi (2005) described eight additional new species using the General Lineage concept (de Quieroz,
1998), according to which species are regarded as independent evolutionary lineages based on multiple
criteria, such as genetic divergence, morphology, ecology and vocalization. Meegaskumbura et al. (2007)
added two new but extinct species discovered in historical museum collections, again adopting a purely
morphological approach. The island’s inventory of Philautus now stands at 63 species, of which 44 are extant.
Surveys in Sri Lanka since the early 1990s have shown that 19 of these, known today only from museum
specimens collected in the 19th and early 20th centuries, have since disappeared (Manamendra-Arachchi &
Pethiyagoda 2005; Meegaskumbura et al. 2007).
Here we continue to document the new species discovered in Sri Lanka as a result of exploratory work,
based on morphological, morphometric and molecular data, in the context of the General Lineage concept of
species.
Materials and methods
Field sampling and anatomical measurements were made as described in Manamendra-Arachchi &
Pethiyagoda (2005), except as mentioned below.
MEEGASKUMBURA ET AL.52 · Zootaxa 2122 © 2009 Magnolia Press
Morphological analysis. The suite of characters and character states used by Manamendra-Arachchi &
Pethiyagoda (2005) was analysed for all individuals. Measurements were made to the nearest 0.1 mm using
dial vernier calipers. These were distance between back of eyes (DBE); distance between front of eyes (DFE);
length of disk (DL) width of disk (DW); eye diameter (ED); eye-to-nostril distance (EN); eye-to-snout length
(ES); femur length (FEL); length of finger 1 (FLI); length of finger 2 (FLII); length of finger 3 (FLIII); length
of finger 4 (FLIV); pes length (FOL); head length (HL); head width (HW); length of inner metatarsal tubercle
(IML); internarial distance (IN); interorbital distance (IO); lower-arm length (LAL); posterior mandible-to-
eye distance (MBE); least distance from mandible to anterior eye (MFE); least distance from mandible to
nostril (MN); nostril-to-snout length (NS); palm length (PAL); snout–vent length (SVL); tibia length (TBL);
length of toe 1 (TLI); length of toe 2 (TLII); length of toe 3 (TLIII); length of toe 4 (TLIV); and length of toe
5 (TLV); diameter of tympanum (TYD); distance from tympanum to front of eye (TYE); length of upper arm
(UAW); and width of upper eyelid (UEW). Illustration of the webbing pattern follows Manamendra-Arachchi
& Pethiyagoda (2005). Measurements with high coefficients of variation or low repeatability were omitted
from the PCA analysis, for which the following were used: DBE, DFE, DL, DW, ED, EN, ES, FEL, FLI,
FLII, FLIII, FLIV, FOL, HL, HW, IN, IO, LAL, MBE, MFE, MN, NS, PAL, SVL and TBL.
Principal components analysis of the character correlation matrix was used to reduce dimensionality of
the continuous morphological variables and to identify those variables that best discriminate among
morphologically similar species (P. singu and P. tanu were compared to P. decoris and P. mittermeieri).
Various axis rotations were tested and one selected for optimal interpretability of variation among the
characters. For consistency, only mature males were used in this analysis. SYSTAT (Version 11.00.01 for
Windows XP) was used for statistical analysis.
Molecular analysis. One of the species described here is included in the phylogenetic analysis of
Meegaskumbura et al. (2002) and Meegakumbura & Manamendra-Arachchi (2005; reference number WHT
2658). A further species (WHT 6343) was added to the latter phylogeny. Only 12s rRNA and 16s rRNA
partial sequences were used to construct the phylogenetic tree, as was done in the above-mentioned works
(see Table 1 for details on species and their genbank accession numbers). Cytochrome-b data were used, in
addition, to determine the percentage divergences among sister taxa, and PCR amplification and alignment of
sequences were done as explained in Meegaskumbura & Manamendra-Arachchi (2005).
Data were analyzed using Bayesian, Maximum Likelihood (ML) and Maximum Parsimony (MP) criteria.
Here, we present only the Maximum Likelihood tree, which is identical to the Bayesian tree, together with one
of the two equally parsimonious trees. We used Bayesian inference as implemented in MrBayes (Huelsenbeck
& Ronquist 2001) to generate a phylogenetic hypothesis of relationships among the taxa and to estimate a
general time-reversible model of sequence evolution with gamma-distributed rate variation among sites and a
proportion of invariant sites (GTR+I+G). We ran four Metropolis-Coupled Markov Chain Monte Carlo
(MCMCMC) chains for 500,000 generations and the summed likelihood of the four chains converged on a
stationary value by 100,000 generations (the burn-in time). We used the frequency of clades in trees that were
sampled every ten generations from the last 250,000 generations as estimates of the posterior probabilities of
those clades (Huelsenbeck et al. 2001). Uniform priors were used throughout and branch lengths, topology,
and nucleotide substitution parameters were unconstrained. Maximum likelihood analysis used a GTR+I+G
model of nucleotide substitution with the parameters estimated from the Bayesian analysis. A single heuristic
search with Tree Bisection and Reconnection (TBR) branch swapping was conducted using PAUP*4.0b10
(Swofford, 2002). For tree searches under a Maximum Parsimony criterion we used 100 heuristic searches
with TBR branch-swapping and random taxon addition as implemented in PAUP*4.0b10. Two equally
parsimonious trees with tree scores of 1075 were recorded. A bootstrap analysis (1000 replicates, random
stepwise addition with 100 repetitions) to determine node support was also carried out within a maximum-
parsimony framework.
Once we identified the divergent mtDNA lineages and their sister taxa using the 12S and 16S rRNA gene
tree, to facilitate comparisons with published summaries of mitochondrial divergence among vertebrate sister
species (Johns & Avise 1998), we sequenced a fragment of the mitochondrial cytochrome-b gene from the
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TWO NEW SRI LANKAN SHRUB FROGS FROM LOWLANDS
species described herein and their sister species (Meegaskumbura & Manamendara-Arachchi 2005). A ~ 590
base-pair fragment was amplified using primers CB-J-10933, (5’- TATGTTCTACCATGAGGACAAATATC-
3’) and BSF4 (5’- CTTCTACTGGTTGTCCTCCGATTCA-3’) (Bossuyt & Milinkovitch 2000) under
standard PCR conditions: denaturation at 95° C for 40 s, annealing at 45° C for 40 s and extension at 72° C for
40 s, 35 cycles, with a final extension of 72° C for 5 min. Products were gel purified and sequenced on an ABI
377 or ABI 3100 automated sequencer following manufacturer’s protocols. Sequences were aligned using
translated amino acid sequences using Se-Al (ver. 2.0a11; Rambaut 1996).
TABLE 1. Reference numbers, and the Genbank accession numbers for the species used in the phylogenetic analysis.
Species Reference number Genbank Accession numbers
12s 16s
P. alto WHT2723 AY141781 AY141827
P. asankai WHT5107 FJ788141 FJ788160
P. a ur at us WHT2792 AY141789 AY141835
P. caeruleus WHT2511 AY141764 AY141810
P. cavirostris WHT3299 FJ788137 FJ788156
P. cf. sarasinorum WHT2484 AY141762 AY141808
P. cf. sarasinorum WHT2489 AY141763 AY141809
P. cf. sordidus WHT_H12 AY141791 AY141837
P. cf. sordidus WHT_H15 AY141792 AY141838
P. c ha rius FB AY141840 AY141794
P. decoris WHT3271 FJ788144 FJ788163
P. femoralis WHT2566 AY141771 AY141817
P. femoralis WHT2772 AY141785 AY141831
P. frankenbergi WHT2552 AY141768 AY141814
P. frankenbergi WHT2555 AY141769 AY141815
P. hallidayi WHT_H11 AY141793 AY141839
P. hoffmanni WHT3223 FJ788142 FJ788161
P. hoipolloi WHT2675 AY141776 AY141822
P. limbus WHT2690 AY141777 AY141823
P. limbus WHT2700 AY141779 AY141825
P. lunatus WHT3283 FJ788150 FJ788169
P. microtympanum WHT2558 AY141770 AY141816
P. mittermeieri KAN2 FJ788143 FJ788162
P. m oo reorum WHT3209 FJ788134 FJ788153
P. o cu ll ari s WHT2887 FJ788145 FJ788164
P. pappilosus WHT3284 FJ788151 FJ788170
P. pleurotaenia WHT3176 FJ788146 FJ788165
P. poppiae WHT5026 FJ788135 FJ788154
P. poppiae WHT2779 FJ788136 FJ788155
P. popularis WHT3191 FJ788149 FJ788168
P. procax WHT2786 AY141788 AY141834
P. sarasinorum WHT2481 AY141761 AY141807
to be continued.
MEEGASKUMBURA ET AL.54 · Zootaxa 2122 © 2009 Magnolia Press
Results
Morphometric analysis. Philautus tanu, P. singu, P. mittermeieri and P. decoris separate distinctly from each
other in morphological space (Fig. 1, Table 2). Principal components analysis shows that the four species are
distinguished by a combination of body size, finger lengths, and head dimensions. The PC(1) axis, which
explains 82 % of the variance, is a size axis (SVL loads most heavily and FLII least heavily, but all variables
have high, positive loadings on this axis; component loadings range from 0.879–0.765, suggesting that the
variation relates mostly to size). The PC(2) axis represents 8 % of the variance, with FLII (-0.570), FLIII (-
0.505), IO (0.359), TLII (-0.377), ES (0.359), FLI (-0.353), MBE (0.321), and ED (0.311) loading most
heavily. Three of the four species separate well on the PC(1) axis, P. decoris being the largest and P. tanu
being the smallest (P. singu and P. mittermieri overlap on this axis). Philautus singu separates from all other
species on the PC2 axis, while P. tanu separates from P. mittermieri, but not P. d eco ris , on this axis.
Molecular phylogenetics. The final dataset contains 12S and 16S rRNA mitochondrial gene sequences
from 54 putative species, 53 from the dataset analyzed by Meegaskumbura and Manamendra-Arachchi
(2005); plus one additional species. Fifty-one of these represent Sri Lankan Philautus, while three represent
Indian species (one, P. wynaadensis, is nested within the Sri Lankan clade, whereas the other two represent the
sister group to the Sri Lankan Philautus: see Fig. 2 in Meegaskumbura et al. 2002; Bossuyt et al. 2004). Out
of the 939 nucleotide positions sequenced, 867 were clearly allignable and were included in this analysis.
TABLE 1. (continued)
Species Reference number Genbank Accession numbers
12s 16s
P. schmarda WHT2715 AY141780 AY141826
P. signatus FB AY141795 AY141841
P. s im ba WHT3221 FJ788148 FJ788167
P. s in gu WHT2658 AY141773 AY141819
P. s ordidus WHT2699 AY141778 AY141824
P. sp. WHT2515 AY141765 AY141811
P. sp. WHT2540 AY141767 AY141813
P. sp. WHT2797 AY141790 AY141836
P. sp. WHT2667 AY141774 AY141820
P. sp. WHT2669 AY141775 AY141821
P. sp. WHT2525 AY141766 AY141812
P. sp. WHT2774 AY141786 AY141832
P. sp. WHT2729 AY141782 AY141828
P. sp. WHT2731 AY141783 AY141829
P. s te in eri WHT3210 FJ788138 FJ788157
P. stuarti WHT3207 FJ788139 FJ788158
P. stuarti WHT3208 FJ788140 FJ788159
P. t an u WHT6343 FJ788152 FJ788171
P. v ir id is WHT2627 AY141772 AY141818
P. v ir id is WHT2766 AY141784 AY141830
P. wynaadensis FB AY141796 AY141842
P. zorro WHT3175 FJ788147 FJ788166
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TABLE 2. Component loadings for axes 1 and 2 of the principal component analysis, variance explained and percentage
of total variance explained for Philautus tanu, P. singu, P. mittermeieri and P. decoris.
The Maximum Likelihood tree (from the Maximum Likelihood analysis) is rooted with two Indian taxa
(Philautus charius and P. signatus) that represent the sister group to the Sri Lankan Philautus radiation
(Meegaskumbura et al. 2002; Fig. 2). For the Bayesian analysis we ran 500,000 generations of the MCMCMC
algorithm and the summed likelihood of the four chains reached stationarity by 85,000 generations. Posterior
probabilities of clades shown at nodes in Fig. 2 represent the frequency of those clades in the 25,000 trees
sampled from the last 250,000 generations; clades with posterior probability of 50% or less were collapsed.
Parameters of the nucleotide substitution model for the most likely tree are as follows. Rate matrix: R(G-T),
0.0078; R(C-T), 0.6649; R(C-G), 0.0152; R(A-T), 0.0368; R(A-G), 0.2356; R(A-C), 0.0395. Nucleotide
frequency: A, 0.3493; C, 0. 0.2204; G, 0.1906; T, 0.2394. Rate variation: shape parameter for gamma
distributed rate variation among sites (alpha) = 0.745; proportion of invariant sites = 0.382. The maximum
likelihood tree found via a Tree Bisection and Reconnection branch-swapping heuristic search using the
above nucleotide substitution parameters in PAUP*v.4.0b10 has the same topology as the Bayesian tree, but
Axis 1 Axis 2
SVL 0.979 0.060
FEL 0.976 -0.056
HW 0.975 0.142
FOL 0.969 -0.118
EN 0.964 0.136
DBE 0.963 0.173
LAL 0.960 0.028
HL 0.957 0.212
MN 0.955 0.257
TBL 0.949 -0.041
PAL 0.942 -0.223
DFE 0.941 0.261
TLIII 0.938 -0.275
MFE 0.933 0.278
TLIV 0.918 -0.151
FLIV 0.908 -0.275
MBE 0.890 0.321
DW 0.885 -0.106
FLI 0.872 -0.353
TLII 0.870 -0.377
TLV 0.867 -0.216
ES 0.849 0.359
IO 0.824 0.490
IN 0.810 0.111
ED 0.796 0.311
FLIII 0.770 -0.505
FLII 0.765 -0.570
Variance explained by components 22.209 2.058
Percent of total variance explained 82.254 7.622
MEEGASKUMBURA ET AL.56 · Zootaxa 2122 © 2009 Magnolia Press
has slightly different branch-lengths. A heuristic search using the Parsimony criterion, TBR branch swapping
with 100 replicates with random taxon addition, and all characters unordered and weighted equally gave two
equally parsimonious trees. One of these is shown (Fig. 3) with the maximum parsimony bootstrap values at
nodes (with nodes having bootstrap values less than fifty percent collapsed). Bootstrap values towards the
base of the Sri Lankan radiation are low, which results in a basal polytomy. However, as expected, values
closer to the OTUs show higher bootstrap values, and relationships of taxa within these better-supported
clades are identical to those of the maximum likelihood analysis. The relationships of taxa of the clade from
which the two new species are described are also identical to the relationships from the maximum likelihood
analysis.
FIGURE 1. PC1 vs. PC2 factor scores of the principal components analysis of Philautus tanu, n. sp., P. singu n. sp., P.
mittermeieri and P. decoris, show these four species to separate well from each other in PC space. Most of the variation
is explained by the PC1 axis, which relates mainly to body size (P. decoris is the largest and P. tanu the smallest;
however, P. mittermeieri and P. singu overlap on this axis). The PC2 axis is mostly explained by finger, toe and head
dimensions; here, P. ta nu overlaps completely with P. decoris, but P. singu and P. mittermeieri do not.
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FIGURE 2. Maximum likelihood tree of 12s and 16s rRNA gene fragments, with posterior probabilities from the
Bayesian analysis shown at nodes. The two new species, Philautus tanu and P. singu (indicated by asterisks), form a
clade with P. mittermeieri and P. decoris.
MEEGASKUMBURA ET AL.58 · Zootaxa 2122 © 2009 Magnolia Press
FIGURE 3. Unweighted Maximum Parsimony tree of 12s and 16s rRNA gene fragments, with maximum parsimony
bootstrap values shown at the nodes. The two new species, Philautus tanu and P. singu (indicated by asterisks), form a
clade with P. mittermeieri and P. decoris.
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Philautus singu, new species
(Figs. 4–7)
Material examined. Holotype: mature male, 16.2 mm SVL, WHT6034, Kitulgala, alt. 101 m (6°59'N,
80°20'E), coll. 22 February 1999.
Paratypes: mature males, 16.6 mm SVL, WHT6340, Kottawa Forest Reserve (Galle), alt. 60 m (06°06’N,
80°20’E), coll. 3 June 1999; 16.1 mm SVL, WHT5977, Sinharaja World Heritage Site (near Kudawa), alt. 513
m (06°25’N, 80°25E), coll. 25 January 1999; 16.1 mm SVL, WHT6341, Kottawa Forest reserve (Galle), alt.
60 m (6° 5'N, 80°18’E), coll. 3 June 1999.
Diagnosis. Philautus singu is distinguished from all its Sri Lankan congeners by the following
combination of characters: size small, mature males 16.1-16.6 mm SVL; a prominent tubercle present on
upper eyelid; tympanum distinct; supratympanic fold distinct; canthal edges rounded; vomerine ridge absent;
throat, chest and belly granular.
FIGURE 4. Philautus singu (WHT 6340), in life, Kottawa Forest Reserve, Galle.
Description. Body slender. Head laterally convex above. Snout obtusely pointed in dorsal view, rounded
in lateral view. Canthal edges rounded. Loreal region concave. Interorbital space convex. Internasal space
concave. Nostrils oval. Pupil oval, horizontal. Tympanum distinct, oval, oblique, its outer rim distinct. Pineal
ocellus absent. Vomerine ridge absent. Tongue moderate, emarginate, sometimes (n = 1) bearing a rounded
lingual papilla. Supratympanic fold distinct. Cephalic ridges absent. Co-ossified skin on head absent. Upper
and lower arms short, thin. Fingers slender. Relative length of fingers, 1 < 2 < 4 < 3. Tips of fingers with discs
bearing circum-marginal grooves. Webbing and lateral dermal fringe absent on fingers. Subarticular tubercles
on fingers prominent, oval, single, sometimes absent on fingers III (n = 2) and IV (n = 2). Prepollex oval,
distinct. Two palmar tubercles, oval, distinct; outer tubercle bifid. Supernumerary tubercles present. Thigh,
shank slender. Toes thin, relative length 1 < 2 < 3 = 5 < 4 (holotype), (1 < 2 < 5 < 3 < 4, WHT6340), (1 < 2 <
3 < 5 < 4, WHT5977, WHT6341). Tips of toes with discs, with circum-marginal groves. Webbing present on
MEEGASKUMBURA ET AL.60 · Zootaxa 2122 © 2009 Magnolia Press
toes. Subarticular tubercles on toes prominent, oval, single, sometimes absent on toe V (n = 2). Inner
metatarsal tubercle distinct, oval. Outer metatarsal tubercle absent. Tarsal fold absent; some dermal tubercles
on outer edge of foot. Supernumerary tubercles on toes absent. Tarsal tubercle absent. Dorsal and lateral parts
of snout, between eyes, side of head, anterior part of back, posterior part of back, both upper and lower flanks
with scattered, glandular tubercles. Dorsolateral fold absent. Dorsal and lateral parts of upper arm, lower arm,
thigh, shank and foot with scattered glandular tubercles. Throat, chest, belly and ventral side of thigh granular;
underside of thigh smooth. Nuptial pad absent. Internal vocal slits present.
FIGURE 5. Philautus singu: a, lateral; b, dorsal; and c, ventral aspects, respectively, of head of holotype, male, WHT
6034, 16.2 mm SVL. Scale bar: 1 mm.
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FIGURE 6. Philautus singu: a, ventral aspect of left manus; b, ventral aspect of left pes; and c, semi-diagrammatic
representation of left-pes webbing pattern of the holotype, male,WHT 6034, 16.2 mm SVL. Scale bar: 1 mm.
Coloration in life. Dorsal and lateral parts of head and body brown (Fig. 4). Interorbital area dark brown.
A dark-brown “W”-shaped marking on mid-back. Upper flank brown, lower flank yellow with brown
pigments. Inguinal zone pale yellowish brown. Loreal region dark brown. Tympanic region blackish brown.
Upper half of tympanum dark brown, lower half pale yellowish light brown. Upper lip brown with pale-
yellow patches. Dorsal and lateral parts of forelimb pale yellow with brown pigments, lacking distinct
MEEGASKUMBURA ET AL.62 · Zootaxa 2122 © 2009 Magnolia Press
crossbars. Thigh and shank pale brown with three dark-brown crossbars. Dorsal side of foot pale brown with
darker patches. Posterior thigh pale brown. Throat and margin of throat with dark-brown pigments on a pale-
yellow background. Chest, belly, thigh and webbing on toes with dark-brown pigments and pale-yellow
patches.
Coloration in alcohol (based on holotype, WHT6034). Dorsal and lateral parts of head and body brown.
Interorbital area dark brown. A dark-brown “W”-shaped marking on mid-back. Upper flank brown, lower
flank yellow with brown pigments. Inguinal zone pale yellowish brown. Loreal region brown. Tympanic
region dark brown. Upper half of tympanum dark brown, lower half pale yellowish light brown. Upper lip
brown with pale-yellow patches. Dorsal and lateral parts of forelimb pale yellow with brown pigments; no
distinct crossbars. Thigh and shank pale brown with three dark-brown crossbars. Dorsal side of foot pale
brown with dark-brown patches. Posterior side of thigh pale brown. Throat and margin of throat with dark-
brown pigments on a pale-yellow background. Chest, belly, thigh and webbing on toes with dark-brown
pigments and pale-yellow patches.
Measurements of holotype (WHT6034, in mm): DBE, 6.3; DFE, 3.6; DL, 0.7; DW, 1.0; ED, 2.5; EN, 2.0;
ES, 2.3; FEL, 7.7; FL I, 1.2; FL II, 1.7; FL III, 3.0; FL IV, 2.2; FOL, 11.1; HL, 6.4; HW, 6.2; IML, 0.9; IN,
1.8; IO, 1.6; LAL, 3.2; MBE, 2.2; MFE, 4.0; MN, 5.7; NS, 1.2; PAL, 5.1; SVL, 16.2; TBL, 8.2; TL I, 1.3; TL
II, 1.7; TL III, 2.8; TL IV, 3.9; TL V, 2.8; TYD, 0.7; TYE, 0.7; UAW, 3.6; UEW, 2.2.
Etymology. The species name is Sinhala for horn, an allusion to the horn-like tubercles on the upper
eyelids of this frog; applied as a noun in apposition.
Remarks. Morphologically, Philautus singu is reminiscent of P. deco ris, P. mitte rmeie ri and P. ta nu , new
species. It is distinguished from P. decoris and P. mittermeieri, however, by the presence of a prominent
tubercle on the upper eyelid (absent in P. mittermeieri, P. decoris and P. tan u); by having the snout rounded in
lateral aspect (pointed in P. mittermeieri, and obtusely pointed in both P. decoris and P. tanu); by the absence
of a tarsal tubercle (present in P. mittermeieri and P. dec or is); absence of a lateral dermal fringe and webbing
on fingers (present in P. mitte rmeieri and P. decoris); and by the absence of a tarsal fold (presenct in both P.
mittermeieri and P. decor is).
Distribution. We observed males of P. singu perched on leaves of shrubs, 0.5-1.5 m above ground level,
in the rainforest understory. Although we recorded the species only from the Kottawa and Kitulgala Forest
Reserves, it probably occurs also in other rainforest patches in the wet-zone lowlands of Sri Lanka.
Philautus tanu, new species
(Figs. 7–10)
Material examined. Holotype: mature male, 13.5 mm SVL, WHT6348, Kanneliya Forest Reserve (near
Galle), alt. 45 m (6°15'N, 80°20'E), coll. 5 May 1999.
Paratypes: mature males, 13.6 mm SVL, WHT6343; 13.9 mm SVL, WHT6342, Pituwala (Galle), alt. 24
m (6°16'N, 80°12'E), coll. 4 March 1999.
Diagnosis. Philatus tanu is distinguished from all other Sri Lankan congeners by the following
combination of characters: small size, mature individuals 13.5–13.9 mm SVL; canthal edges rounded;
tympanum distinct; vomerine ridge absent; supratympanic fold absent; a very narrow dermal fold along mid-
dorsum, from tip of the snout to vent; venter granular; nuptial pads absent; vocal sac indistinct.
Description. Body stout. Head laterally convex. Snout obtusely pointed in both dorsal and lateral aspect.
Canthal edges rounded. Loreal region flat. Interorbital space flat. Internasal space flat. Nostrils oval. Pupil
rounded or (horizontally) oval. Tympanum distinct, oval, vertical. Pineal ocellus absent. Vomerine ridge
absent. Tongue moderate, emarginate, not bearing a lingual papilla. Supratympanic fold absent. Cephalic
ridges absent. Co-ossified skin on head absent. Both upper and lower arms slender. Fingers slender. Relative
length of fingers, 1 < 2 < 4 < 3. Tips of fingers with discs bearing circum-marginal grooves. Fingers without
lateral dermal fringe. Webbing on fingers absent. Subarticular tubercles on fingers prominent, oval, single
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(absent on finger IV; n = 2). Prepollex oval. Two palmar tubercles, oval, flat. Supernumerary tubercles present
on fingers I–III and on palm. Thigh and shank slender. Toes thin. Relative length of toes 1 < 2 < 5 < 3 < 4 or 1
< 2 < 3 < 5 < 4 (WHT6343, WHT6342). Tips of toes with discs bearing circum-marginal grooves. Webbing
on toes present. Subarticular tubercles on toes prominent, oval, single. Inner metatarsal tubercle distinct, oval.
Outer metatarsal tubercle absent. Tarsal fold absent. Supernumerary tubercles present on toes and on foot.
Tarsal tubercle absent. Dorsal and lateral parts of head and body shagreened. Both upper and lower parts of
flank granular. Dorsolateral dermal fold absent. Dorsal and lateral parts of upper arm, lower arm, thigh, shank
and foot smooth. A very narrow dermal fold present on mid dorsum, extending from tip of snout to vent.
Ventral parts of throat, chest, abdomen, both upper and lower arms, anterior thigh, shank, and foot granular.
Nuptial pad absent. Vocal sacs indistinct. Internal vocal slits present.
FIGURE 7. South-western Sri Lanka, showing the distribution of Philautus singu (squares) and P. t an u (circles), and
place-names cited in the text.
Coloration in life. Dorsal parts of head and body pale brown (Fig. 8). A dark-brown stripe about as wide
as pupil extends backwards from snout, fading away on mid-flank. About eight dark-brown stripes of varying
width on dorsum. Ground color of body creamy-light brown. A narrow creamy-brown stripe extends from
snout, over eye to flank. Lower flank pale yellow or white. Inguinal zone white with light brown pigments.
Loreal and tympanic regions dark brown. Both upper and lower lips light brown with dark-brown pigments.
Dorsal and lateral parts of both upper and lower arms, fingers, thigh, shank, foot and toes pale brown. Ventral
parts of head, body, upper and lower arms, fingers, thigh, shank, foot and toes white with scattered brown
pigments. Posterior edge of orbit light blue.
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FIGURE 8. Philautus tanu (WHT 6342), in life, Beraliya Forest Reserve, Pituwela.
Coloration in alchohol (description based on holotype, WHT6348). Dorsal and lateral parts of head and
body pale brown. Anterior half of upper flank dark brown, posterior half yellow or white. Lower flank pale
yellow or white. Inguinal zone white with light-brown pigments. Loreal and tympanic regions dark brown.
Both upper and lower lips white with dark-brown pigments. Dorsal and lateral parts of both upper and lower
arms, fingers, thigh, shank, foot and toes pale brown. Ventral parts of head, body, upper and lower arms,
fingers, thigh, shank, foot and toes white with scattered brown pigments.
Measurements of holotype (WHT6348, in mm): DBE, 4.6; DFE, 3.1; DL, 0.6; DW, 0.7; ED, 2.3; EN, 1.7;
ES, 2.6; FEL, 6.0; FL I, 0.8; FL II, 1.3; FL III, 2.1; FL IV, 1.6; FOL, 8.7; HL, 5.2; HW, 5.2; IML, 0.6; IN, 1.4;
IO, 1.6; LAL, 2.2; MBE, 1.6; MFE, 3.1; MN, 4.6; NS, 1.0; PAL, 3.7; SVL, 13.5; TBL, 6.8; TL I, 0.9; TL II,
1.1; TL III, 1.8; TL IV, 2.9; TL V, 1.7; TYD, 0.4; TYE, 0.7; UAW, 1.8; UEW, 1.4.
Etymology. The species name is Sinhala for ‘slender,’ a reference to the habitus of P. tanu; applied as a
noun in apposition.
Remarks. Philautus tanu morphologically resembles P. decoris, P. mittermeieri and P. singu new species,
but it can be distinguished from them as follows: no prominent tubercle on the upper eyelid (present in P.
singu); snout obtuse in lateral aspect (pointed in P. mittermeieri); tarsal tubercle absent (present in both P.
mittermeieri and P. de coris); no lateral dermal fringe or webbing on fingers (present in both P. mittermeieri
and P. decoris); no tarsal fold (present in both P. mittermeieri and P. deco ri s).
Distribution. We observed males of P. ta nu sitting on leaves of shrubs, 0.5-1.0 m above ground level, in
shrubs adjacent to (10-25 m from) the canopy covered forests. Philautus tanu was recorded from Kanneliya
(6°15'N, 80°20'E) and Pituwela (6°16'N, 80°12'E) Forest Reserves, and probably occurs also in other open
habitats close to patches of rainforest elsewhere in the wet-zone lowlands of Sri Lanka.
Discussion
Philautus tanu, a widespread but strictly lowland species, is basal to two other lowland species, P. singu, and
P. mittermeieri, and to a high elevation species, P. decoris, suggesting that the diversification in this clade
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occurred in the lowlands. Furthermore, P. t anu inhabits open shrub habitats and may be able to disperse freely
through secondary-forest corridors and suitable anthropogenic habitats. Thus, given the availability of
conducive habitat, P. tanu is likely to have a wider distribution than currently recorded. Philautus tanu
appears to be a highly cryptic species, usually calling from deep within shrubs, which makes these frogs
difficult to localize. This trait may represent an adaptation high predation pressure in its relatively open
habitat.
FIGURE 9. Philautus tanu: a, lateral; b, dorsal; and c, ventral aspects, respectively, of head of holotype, male, WHT
6348, 13.5 mm SVL. Scale bar: 1 mm.
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Philautus singu is distributed widely both in the lowlands and the mid-hill region. Its range overlaps
partly with that of P. mittermeieri, one of its sister species. Neither of these frogs is a strictly leaf litter dweller;
they do climb on to shrubs at night to vocalize. Philautus singu, like P. mittermeieri, is restricted to primary
and secondary forests with extensive canopy cover; we did not record either species from open habitats.
Interestingly, where they are syntopic, P. singu occupies a separate microhabitat from P. mittermeieri: 0.5–1.5
m above ground, and less than 0.5 m above ground, respectively. Thus, the two species may partition
resources to some extent.
FIGURE 10. Philautus tanu: a, ventral aspect of left manus; b, ventral aspect of left pes; and c, semi-diagrammatic
representation of the left-pes webbing pattern of the holotype, male, WHT 6348, 13.5 mm SVL. Scale bar: 1 mm.
Philautus tanu, P. singu, P. decoris and P. mittermeieri form a single clade (Figs. 2, 3), but the new species
are well differentiated genetically from the others (Tables 2, 3). Philautus singu is distinct from the three other
species by uncorrected percent genetic distances of 7.7–8.4 (combined 12s and 16s gene fragments) and
17.1–18.5 (cytochrome-b gene fragment). Similarly, Philautus tanu is distinct by uncorrected percent genetic
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distances of 7.9–8.7 (combined 12s and 16s gene fragments) and 18.2–22.6 (cytochrome-b gene fragment)
(Tables 3 & 4). These values are well over the 2% cytochrome-b genetic distance that indicates species-level
divergence in several groups of mammals (Bradley & Baker 2001); and that is exceeded by 90% of putative
sister species across a wide range of vertebrate taxa (Johns and Avise 1998), which adds confidence to our
recognition of these taxa at the species level. The 16s rRNA gene has been recognized as a suitable barcoding
gene for amphibians (Vences et al. 2005). For Mantellidae, a wide range of divergence among species,
ranging from 1-16.5% has been highlighted (Vences et al. 2005), and a 3% divergence has been proposed as a
species level threshold (Fouquet et al. 2007)
TABLE 3. Matrix of pairwise uncorrected percent divergences for 12s and 16s rRNA gene fragments among P.
mittermeieri, P. decoris, P. singu and P. tanu.
TABLE 4. Matrix of uncorrected percent divergences for cytochrome-b gene fragments among P. mittermeieri, P.
decoris, P. singu and P. tanu.
Acknowledgements
We thank James Hanken (Harvard University), Christopher J. Schneider (Boston University), Suyama
Meegaskumbura (University of Peradeniya, Sri Lanka) and two reviewers - Franky Bossuyt (Brussels Free
University) and an anonymous one - for comments that helped significantly to improve the manuscript;
Mohomed Bahir and Sudath Nanayakkara for fieldwork and support at WHT’s Agrapatana field station;
Sudesh Batuwita (WHT) for assistance in the field and hospitality while working in the lowland areas; Mark
Wilkinson, David Gower and Barry Clarke (The Natural History Museum, London) for facilities to work in
their institution, hospitality and valuable assistance; Prof. Punchi Meegaskumbura (University of Peradeniya)
for suggesting suitable Sinhala names for the species. This study was supported financially by National
Science Foundation (Grant No: 0345885 to CJS and JH) and National Geographic Society (Grant No: 7612-
04 to CJS). We are grateful to the Forest Department of Sri Lanka for permits to work in their reserves, and
accommodation during visits; and the Department of Wildlife Conservation Sri Lanka for collection and
export permits. MM is also grateful for the Ziff Environmental Postdoctoral Fellowship through the Harvard
University Center for the Environment (HUCE), which facilitated part of this work.
References
Bradley, R. D. & Baker, R. J. (2001) A test of the genetic species concept: Cytochrome-b sequences and mammals.
Journal of Mammalogy, 82, 960–973.
Bossuyt, F. & Milinkovitch, M. C. (2000) Convergent adaptive radiation in Madagascan and Asian Ranid frogs reveal
covariation between larval and adult traits. Proceedings of the National Academy of Sciences of the United States of
America, 97, 6585–6590.
Bossuyt F., Meegaskumbura, M., Beenaerts, N., Gower, D. J., Pethiyagoda, R., Roelants, K., Mannaert, A., Wilkinson,
P. mittermeieri P. decoris P. singu
P. decoris 2.0
P. s in gu 8.4 7.7
P. t an u 8.2 8.7 7.9
P. mittermeieri P. decoris P. singu
P. decoris 5.5
P. s in gu 17.1 18.5
P. t an u 21.3 22.6 18.2
MEEGASKUMBURA ET AL.68 · Zootaxa 2122 © 2009 Magnolia Press
M., Bahir, M. M., Manamendra-Arachchi, K., Ng, P.K.L., Schneider, C.J., Oommen, O.V. & Milinkovitch, M.C.
(2004) Local endemism within the western Ghats-Sri Lanka Biodiversity hotspot. Science, 306, 479–481.
de Queiroz, K. (1998) The General Lineage Concept of species. In: Howard, D. J. & Berlocher. S. H. (Eds.), Endless
forms: species and speciation. Oxford University Press, Oxford, pp. 57–75.
Huelsenbeck, J. P. & Ronquist, F. (2001) MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics, 17,
754–755.
Huelsenbeck, J. P., F. Ronquist, R. Neilsen & Bollback, J. P. (2001) Bayesian inference of phylogeny and its impact on
evolutionary biology. Science, 294: 2310–2314.
Johns, G.C. & Avise, J.C. (1998) A comparative summary of genetic distances in the vertebrates from the mitochondrial
cytochrome b gene. Molecular Biology and Evolution, 15, 1481–1490.
Manamendra-Arachchi, K. & Pethiyagoda, R. (2005) The Sri Lankan shrub frogs of the genus Philautus Gistel, 1848
(Ranidae: Rhacophorinae), with description of 27 new species. In: Yeo, C. J., Ng, P. K. L. & Pethiyagoda, R. (Ed.),
Contributions to biodiversity exploration and research in Sri Lanka. The Raffles Bulletin of Zoology, Supplement 12,
163–303.
Manamendra-Arachchi, K. & Pethiyagoda, R. (2006) Sri Lankan amphibians [in Sinhala]. WHT Publications, Colombo,
440 pp., 88 pl.
Meegaskumbura, M., F. Bossuyt, R. Pethiyagoda, K. Manamendra-Ararchchi, M. Bahir, M. C. Milinkovitch &
Schneider, C. J. (2002) Sri Lanka: an amphibian hotspot. Science, 298, 379.
Meegaskumbura M. & Manamendra-Arachchi K. (2005) Description of eight new species of shrub frogs (Ranidae:
Rhacophorinae: Philautus) from Sri Lanka. In: Yeo, C. J., Ng, P. K. L. & Pethiyagoda, R. (Ed.), Contributions to
biodiversity exploration and research in Sri Lanka. The Raffles Bulletin of Zoology, Supplement 12, 305–338.
Meegaskumbura, M., Manamendra-Arachchi, K., Schneider, C. J. & Pethiyagoda, R. (2007) New species amongst Sri
Lanka’s extinct shrub frogs. Zootaxa, 1397, 1–15.
Fouquet, A., Gilles, A., Vences, M., Marty, C., M. Blanc & Gemmell, N. J. (2007) Underestimation of species richness in
Neotropical frogs revealed by mtDNA analysis. PLoS ONE 2(10): e1109. doi:10.1371/journal.pone.0001109.
Rambaut, A. (1996) Se-Al: Sequence Alignment Editor. Available at http://tree.bio.ed.ac.uk/software/seal/
Swofford, D. L. (2002) PAUP, phylogenetic analysis using parsimony (and other methods), v. 4b10. Sinauer Associates,
Sunderland, MA.
Vences, M., Thomas, M., Bonett, R. M. & Vieites, D. R. (2005) Deciphering amphibian diversity through DNA
barcoding: chances and challenges. Philosophical Transactions of the Royal Society, B, 360, 1462 1859-1868.
Vences, M., Thomas, M., Van der Meijden, A., Y. Chiari & Vieites, D. R. (2005) Comparative performance of the 16s
rRNA gene in DNA barcoding of amphibians. Frontiers in Zoology, 360, 1859-1868.
