Biodiversity Data Journal : Taxonomic Paper
PDF
Taxonomic Paper
First records of the fanged frogs Limnonectes bannaensis Ye, Fei & Jiang, 2007 and L. utara Matsui, Belabut & Ahmad, 2014 (Amphibia: Anura: Dicroglossidae) in Thailand
expand article infoChatmongkon Suwannapoom, Ke Jiang§, Yun-He Wu|, Parinya Pawangkhanant, Sengvilay Lorphengsy, Tan Van Nguyen#, Nikolay A. Poyarkov¤,«, Jing Che|
‡ Division of Fishery, School of Agriculture and Natural Resources, University of Phayao, Phayao, Thailand
§ CAS Key Laboratory of Mountain Ecological Restoration and Bioresource Utilization & Ecological Restoration and Biodiversity Conservation Key Laboratory of Sichuan Province, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, Sichuan, China
| State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, China
¶ Division of Biotechnology, School of Agriculture and Natural Resources, University of Phayao, Phayao, Thailand
# Department of Species Conservation, Save Vietnam’s Wildlife,, Ninh Binh, Vietnam
¤ Faculty of Biology, Department of Vertebrate Zoology, Moscow State University, Moscow, Moscow, Russia
« Laboratory of Tropical Ecology, Joint Russian-Vietnamese Tropical Research and Technological Center, Hanoi, Vietnam
Open Access

Abstract

Background

The taxonomic status of the Thai populations belonging to the Limnonectes kuhlii species complex is controversial, due to phenotypic similarity in the cryptic species complex. Recently, some studies on this group in Thailand have discovered four new species: L. taylori, L. megastomias, L. jarujini and L. isanensis. Even so, the diversity of this group is still incomplete.

New information

Based on an integrative approach encompassing genetic and morphological analyses, we conclude that the Limnonectes populations from Nan Province (northern) and Yala Province (southern) of Thailand are conspecific with L. bannaensis Ye, Fei & Jiang, 2007 and L. utara Matsui, Belabut & Ahmad, 2014, respectively. These are the first records of these species in Thailand. Our study highlights the importance of using DNA sequence data in combination with morphological data to accurately document species identity and diversity. This is especially important for morphologically cryptic species complexes and sympatrically occurring congeners.

Keywords

Nan Province, Yala Province, 16S rRNA, Cryptic species, Species complex

Introduction

Limnonectes Fitzinger, 1843 is the most species rich genus of Asian frogs of the family Dicroglossidae presently comprising 78 species distributed throughout East and Southeast Asia (Frost 2021). This genus is generally characterised by a morphological crypsis and contains several potentially undescribed cryptic species, especially within widespread species complexes, such as the L. kuhlii (Tschudi 1838) complex (McLeod 2010, Suwannapoom et al. 2016). In terms of a systematic framework, the L. kuhlii species complex in Thailand had been previously explored and four species are currently recorded within the country (McLeod 2008Matsui et al. 2010b, McLeod et al. 2012). According to established records, L. megastomias McLeod, 2008 is only found in Nakhon Ratchasima and Sa Kaeo Provinces of eastern Thailand. Two other species (L. taylori Matsui, Panha, Khonsue & Kuraishi, 2010 and L. jarujini Matsui, Panha, Khonsue & Kuraishi, 2010), occur in the mountain ranges of western Thailand, with L. taylori documented from northern and L. jarujini from southern parts of the country (Matsui et al. 2010a). Furthermore, Limnonectes isanensis McLeod, Kelly & Barley, 2012 is only known to occur in Phu Luang National Park, Loei Province, Thailand (McLeod et al. 2012).

During the course of our recent herpetological surveys conducted in the northern and southern parts of Thailand from 2017-2018, we collected several Limnonectes specimens from previously not examined populations in Nan and Yala Provinces. Based on detailed morphological comparisons and a phylogenetic analyses, we confirm here the presence of two Limnonectes species previously unreported for Thailand: L. bannaensis Ye, Fei, Xie & Jiang, 2007 and L. utara Matsui, Belabut & Ahmad, 2014, respectively. Therefore, we report L. bannaensis and L. utara as two new records of amphibian species for Thailand, provide morphological and morphometric descriptions of the collected specimens and remark on the natural history of these species, based on our field observations.

Materials and methods

Sampling

Field surveys were conducted in Bo Kluea District, Nan Province in December 2017, in Bannang Sata District, Yala Province in August 2018 (Fig. 1). A total of 11 specimens of Limnonectes bannaensis and four specimens of L. utara were collected. Liver tissue samples of all specimens were taken and preserved in 95% ethanol for molecular analysis. The specimens were fixed with 10% formalin for 24 hours and subsequently transferred to 70% ethanol. Specimens and tissues were subsequently deposited at the herpetological collections of the School of Agriculture and Natural Resources, University of Phayao (AUP), Phayao, Thailand.

Figure 1.  

Map showing the type-localities of Limnonectes kuhlii species complex in Thailand. Circles = Type localities of L. taylori (red), L. jarujini (purple), L. megestomias (orange) and L. isanensis (brown). Blue square (L. bannaensis) and yellow square (L. utara) represent the two new distribution records in Thailand reported here.

Molecular analysis

Genomic DNA was extracted from liver tissues preserved in 95% ethanol using the standard phenol-chloroform extraction protocol (Sambrook et al. 1989). Partial fragments of the mitochondrial 16S rRNA were amplified for all samples via the polymerase chain reaction (PCR) using the following primers: 16SAR (5'-CGCCTGTTTAYCAAAAACAT-3') and 16SBR (5'-CCGGTYTGAACTCAGATCAYGT-3'). PCR amplifications were performed in a 25 µl reaction volume with the following cycling conditions: initial denaturing step at 95°C for 4 min, 35 cycles of denaturing at 94°C for 40 s, annealing at 55°C for 30 s, extending at 72°C for 1 min and a final extension step at 72°C for 10 min. PCR products were directly sequenced using an ABI 3730xl DNA automated sequencer with both forward and reverse primers.

Matrilineal genealogies were reconstructed to examine genealogical relationships amongst Limnonectes, based on the 16S rRNA gene fragment. Homologous sequences of the related species of Limnonectes and those of the outgroups (Fejervarya limnocharis (Gravenhorst) and Fejervarya iskandari Veith, Kosuch, Ohler & Dubois were downloaded from GenBank (see Table 1).

Table 1.

Sequences and voucher specimens of Limnonectes and outgroup taxa used in molecular analyses for this study with sampling localities.

#

Species

Voucher

Locality

GenBank

Reference

1

L. bannaensis

CIB 200901116

China, Yunnan, Jinghong

AB526312

Matsui et al. 2010

2

L. bannaensis

FMNH 255140

Laos, Huaphahn, Vieng Tong

HM067133

McLeod 2010

3

L. bannaensis

FMNH 258519

Laos, Phongsaly, Phongsaly

HM067158

McLeod 2010

4

L. bannaensis

VNMN A.2015.41

Vietnam, Ha Giang, Vi Xuyen

HM067246

McLeod 2010

5

L. bannaensis

AMNH 106430

Vietnam, Vinh Phuc, Tam Dao

HM067272

McLeod 2010

6

L. bannaensis

KIZ 024971

China, Yunnan, Xishuangbanna, Mengla, Yiwu

KU599847

Suwannapoom et al. 2016

7

L. bannaensis

KIZ 024970

China, Yunnan, Xishuangbanna, Mengla, Yiwu

KU599848

Suwannapoom et al. 2016

8

L. bannaensis

KIZ 011793

China, Yunnan, Xishuangbanna, Mengla, Bubang

KU599849

Suwannapoom et al. 2016

9

L. bannaensis

KIZ 011726

China, Yunnan, Xishuangbanna, Mengyang

KU599850

Suwannapoom et al. 2016

10

L. bannaensis

KIZ 011727

China, Yunnan, Xishuangbanna, Mengyang

KU599851

Suwannapoom et al. 2016

11

L. bannaensis

KIZ 022207

China, Guangxi, Hulong, Pinglongshan

KU599856

Suwannapoom et al. 2016

12

L. bannaensis

KIZ 011608

Vietnam, Thanh Hoa, Quan Hoa

KU599857

Suwannapoom et al. 2016

13

L. bannaensis

KIZ YPX18365

Vietnam, Quang Tri, Bac Huong Hoa

KU599861

Suwannapoom et al. 2016

14

L. bannaensis

AUP-00481

Thailand, Nan, Bo Kluea

MZ493348

This study

15

L. bannaensis

AUP-00484

Thailand, Nan, Bo Kluea

MZ493349

This study

16

L. bannaensis

AUP-00485

Thailand, Nan, Bo Kluea

MZ493350

This study

17

L. bannaensis

AUP-00488

Thailand, Nan, Bo Kluea

MZ493351

This study

18

L. cintalubang

KUHE 47859

Malaysia, Borneo, Sarawak, Serian

AB981409

Matsui et al. 2010a

19

L. fragilis

CIB 20081089

China, Hainan, Wuzhi Shan

AB526315

Matsui et al. 2010

20

L. fujianensis

CIB ZJ 200806223

China, Jiangxi, Zixi

AB526311

Matsui et al. 2010

21

L. isanensis

KUHE 19284

Thailand, Loei, Phu Luang

AB526314

Matsui et al. 2010

22

L. isanensis

KUHE 19320

Thailand, Loei, Phu Luang

AB558955

Matsui et al. 2010a

23

L. jarujini

KUHE 19514

Thailand, Kanchanaburi, Sangkhla Buri

AB558940

Matsui et al. 2010a

24

L. jarujini

KUHE 19690

Thailand, Surat Thani, Khlong Saeng

AB558950

Matsui et al. 2010a

25

L. longchuanensis

KIZ048424

China, Yunnan, Dehong, Longchuan

KU599867

Suwannapoom et al. 2016

26

L. longchuanensis

KIZ048527

China, Yunnan, Yingjiang, Tongbiguan

KU599869

Suwannapoom et al. 2016

27

L. kuhlii

GMU unnumbered

Indonesia, Java, Purwerojo

AB526316

Matsui et al. 2010

28

L. megastomias

FMNH 266221

Thailand, Sa Kaew, Pang Si Da

HM067184

McLeod 2010

29

L. megastomias

KU 307760

Thailand, Nakon Ratchasima

HM067201

McLeod 2010

30

L. namiyei

KUHE L0809191

Japan, Okinawa, Okinawajima

AB526309

Matsui et al. 2010

31

L. quangninhensis

IEBR 3969

Vietnam, Quang Ninh, Hai Ha

KY595927

Pham et al. 2017

32

L. quangninhensis

IEBR 3970

Vietnam, Quang Ninh, Hai Ha

KY595928

Pham et al. 2017

33

L. selatan

KUHE54079

Malaysia, Genting, Pahang

AB981384

Matsui et al. 2010

34

L. selatan

KUHE54080

Malaysia, Genting, Pahang

AB981385

Matsui et al. 2010

35

L. taylori

KUHE 19101

Thailand, Chiang Mai, Doi Inthanon

AB558929

Matsui et al. 2010a

36

L. taylori

KUHE 19868

Thailand, Chiang Mai, Tha Ton

AB981390

Matsui et al. 2010a

37

L. utara

KUHE54064

Malaysia, Larut, Perak

AB981377

Matsui et al. 2010

38

L. utara

KUHE54065

Malaysia, Larut, Perak

AB981378

Matsui et al. 2010

39

L. utara

AUP 01705

Thailand, Yala, Bannang Sata

MZ493344

This study

40

L. utara

AUP 01706

Thailand, Yala, Bannang Sata

MZ493345

This study

41

L. utara

AUP 01707

Thailand, Yala, Bannang Sata

MZ493346

This study

42

L. utara

AUP 01708

Thailand, Yala, Bannang Sata

MZ493347

This study

Outgroup

43

F. limnocharis

AMNH A-161230

Vietnam, Nghe An, Con Cuong, Pu Mat

AY843588

Faivovich et al. 2005

44

F. iskandari

UI unnumbered

Indonesia, Java, Banyuwangi

AB526324

Matsui et al. 2010

Trees were reconstructed using Bayesian Inference (BI) and Maximum Likelihood (ML). JMODELTEST v.2.1.7 (Darriba et al. 2012) was used to select an appropriate nucleotide substitution model for BI. The GTR+G model was chosen as the best-fit model following the Bayesian Information Criterion (BIC, Posada 2008). The CIPRES web server (Miller et al. 2010) was selected to implement BI. The Monte Carlo Markov chain length was run for 10,000,000 generations and sampled every 1,000 generations. A burn-in value of 25% was used. Convergence was assessed by the average standard deviation of split frequencies (below 0.01) and the ESS values (over 200) in TRACER v.1.5 (Rambaut A and Drummond A 2007). ML was performed using RAxML with 1,000 bootstrap replicates (Stamatakis et al. 2008).

Morphometric analysis and morphological comparisons

Morphometric measurements were taken using digital callipers to the nearest 0.1 mm, following Matsui (1984) and McLeod 2008) abbreviations of the morphometric traits are as follows: snout-vent length (SVL), horizontal eye diameter (ED), eye nostril distance (END), rostrum length distance (RLD), thigh (femur) length (FEL), foot length (FOL), head length (HL), head width (HW), internarial distance (IN), interorbital width (IO), lower arm length (LAL), mandible-nostril distance (MN), palm length (PAL), relative finger length (RFL), relative toe length (RTL), shank (tibia) length (TBL), tympanum diameter (TD) and upper eyelid width (UEW). The digital-webbing formulae followed Savage (1975). Morphological comparisons were made with specimens of morphologically related congeners, deposited at the University of Phayao (AUP).

Data resources

Molecular phylogeny

Sequencing generated a total of 492 base pairs (bp) of 16S rRNA for Limonectes bannaensis and L. utara. All newly-generated sequences were submitted to GenBank (Accession numbers MZ493344-MZ493351, see Table 1). Interspecific uncorrected p-distances between the newly-discovered population of L. bannaensis collected from Nan Province in Thailand and the other known species of Limnonectes varied from 6.3% (in relation to L. quangninhensis) to 12.1% (in relation to L. cintalubang) (Suppl. material 1). The uncorrected p-distance between the newly-found populations of L. bannaensis from Nan Province and the topotypic L. bannaensis (Mengyang, Yunnan, China) is 2.1%. Both ML and BI analyses recovered the Nan population nested within a strongly supported clade, together with topotypic L. bannaensis (see Fig. 2). The newly-discovered population of L. utara from Yala Province, Thailand and the congeners varied from 5.8% (in relation to L. selatan) to 13.5% (in relation to L. cintalubang) (see Suppl. material 1). The uncorrected p-distance between the newly-discovered populations of L. utara from Yala Province and the topotypic L. utara (Larut, Perak, Malaysia) is 0.2%. Both ML and BI analyses recovered the Yala population within a strongly supported clade, together with topotypic L. utara (Fig. 2).

Figure 2.  

BI tree resulting from 492 bp length fragment of mitochondrial 16S rRNA gene for Limnonectes species and outgroups. Bayesian posterior probabilities (BPP) > 95%/ML inferences (ML-BS) > 80% are shown for each node; “-” denotes low support of Bayesian posterior probabilities and bootstrap support < 80% in one analysis, no values on branches represent low support in both analyses. The scale bar represents 0.02 nucleotide substitutions per site.

Taxon treatments

Limnonectes bannaensis Ye, Fei, Xie & Jiang, 2007

Materials    Download as CSV 
  1. scientificName:
    Limnonectes bannaensis
    ; class:
    Amphibia
    ; order:
    Anura
    ; family:
    Dicroglossidae
    ; genus:
    Limnonectes
    ; specificEpithet:
    bannaensis
    ; scientificNameAuthorship:
    Ye, Fei, Xie & Jiang, 2007
    ; country:
    Thailand
    ; countryCode:
    TL
    ; stateProvince:
    Nan
    ; locality:
    Doi Phu Kha
    ; verbatimElevation:
    750
    ; verbatimLatitude:
    19°03'21.3"N
    ; verbatimLongitude:
    101°10'47.8"E
    ; eventDate:
    17 December, 2017
    ; fieldNotes:
    collected by C. Suwannapoom, P. Pawangkhanant
    ; individualCount:
    1
    ; sex:
    male
    ; lifeStage:
    adult
    ; catalogNumber:
    AUP-00481
    ; language:
    en
    ; collectionCode:
    Amphibians
    ; basisOfRecord:
    Preserved Specimen
  2. scientificName:
    Limnonectes bannaensis
    ; individualCount:
    1
    ; sex:
    adult male
    ; catalogNumber:
    AUP-00482
    ; basisOfRecord:
    Preserved Specimen
    ; dynamicProperties:
    collection date, collector and Location as the AUP-00481
  3. scientificName:
    Limnonectes bannaensis
    ; individualCount:
    1
    ; sex:
    adult male
    ; catalogNumber:
    AUP-00483
    ; basisOfRecord:
    Preserved Specimen
    ; dynamicProperties:
    collection date, collector and Location as the AUP-00481
  4. scientificName:
    Limnonectes bannaensis
    ; individualCount:
    1
    ; sex:
    adult male
    ; catalogNumber:
    AUP-00484
    ; basisOfRecord:
    Preserved Specimen
    ; dynamicProperties:
    collection date, collector and Location as the AUP-00481
  5. scientificName:
    Limnonectes bannaensis
    ; individualCount:
    1
    ; sex:
    adult male
    ; catalogNumber:
    AUP-00485
    ; basisOfRecord:
    Preserved Specimen
    ; dynamicProperties:
    collection date, collector and Location as the AUP-00481
  6. scientificName:
    Limnonectes bannaensis
    ; individualCount:
    1
    ; sex:
    adult male
    ; catalogNumber:
    AUP-00486
    ; basisOfRecord:
    Preserved Specimen
    ; dynamicProperties:
    collection date, collector and Location as the AUP-00481
  7. scientificName:
    Limnonectes bannaensis
    ; individualCount:
    1
    ; sex:
    adult male
    ; catalogNumber:
    AUP-00487
    ; basisOfRecord:
    Preserved Specimen
    ; dynamicProperties:
    collection date, collector and Location as the AUP-00481
  8. scientificName:
    Limnonectes bannaensis
    ; individualCount:
    1
    ; sex:
    adult male
    ; catalogNumber:
    AUP-00488
    ; basisOfRecord:
    Preserved Specimen
    ; dynamicProperties:
    collection date, collector and Location as the AUP-00481
  9. scientificName:
    Limnonectes bannaensis
    ; individualCount:
    1
    ; sex:
    adult female
    ; catalogNumber:
    AUP-00489
    ; basisOfRecord:
    Preserved Specimen
    ; dynamicProperties:
    collection date, collector and Location as the AUP-00481
  10. scientificName:
    Limnonectes bannaensis
    ; individualCount:
    1
    ; sex:
    adult male
    ; catalogNumber:
    AUP-00490
    ; basisOfRecord:
    Preserved Specimen
    ; dynamicProperties:
    collection date, collector and Location as the AUP-00481
  11. scientificName:
    Limnonectes bannaensis
    ; individualCount:
    1
    ; sex:
    adult female
    ; catalogNumber:
    AUP-00491
    ; basisOfRecord:
    Preserved Specimen
    ; dynamicProperties:
    collection date, collector and Location as the AUP-00481

Description

Morphological characters of specimens from Nan Province agreed with the descriptions by Ye et al. (2007). Large body size, with males SVL of 80.7 mm (n = 9) and females SVL of 75.4 mm (n = 2). The complete morphometric description of each specimen is presented in Suppl. material 2. They are morphologically distinct in comparison between sexes. Males can be distinguished from females by the dorsal skin texture of the male appearing to be smoother, with less tubercles, supratympanic fold dark brown, indistinct, throat heavily pigmented. Head longer than wide (males HL of 36.8 mm, HW 34.9 mm, n = 9 and females HL of 34.9 mm, HW 32.9 mm, n = 2). Fore limbs robust, relatively short, fingers moderately slender, finger length formula: II< I < IV < III (Fig. 3D), toe length formula: I < II < V < III < IV (Fig. 3E), tips of toes expanded into round elevated pads lacking grooves, toe webbing well-developed, complete, webbing formula: I 0 – 0 II 0 – 0 III 0 – 0 IV 0 – 0 V. Skin on dorsum weakly granulated with few fine folds on the back and a few small rounded tubercles scattered on the rear of the dorsum, ventrally smooth. Colouration in life: black stripes present on areas around the folds (Fig. 3A and C), dorsum light red brown with confluent dark brown markings (Fig. 3A and B and Fig. 4), dark transverse bars on upper surface of hind limbs, side of head and lateral surfaces of body lighter brown, lower lip white marbled with brown, belly white with brown vermiform markings, dark brown bar between eyes edged with thin yellowish-brown bars, lower half of iris golden, upper half brown, separated by a dark brown horizontal band, nuptial pad white. Colouration in preservative: after three years in preservative, the colouration pattern did not change, dorsal and lateral body colouration faded to brown, dark brown bars on upper lip turned less distinct, lower lip turned dark with light mottling, ventre immaculate, ventral portions of limbs mottled around margins, palmar and plantar surfaces turned dark brown.

Figure 3.  

Male of Limnonectes bannaensis (AUP-00485) in life. A. Dorsal view; B. Ventral views; C. Dorsal view of leg (notice the tubercles); D. Palmar view of hand; E. Ventral view of foot.

Figure 4.  

Colour variations of Limnonectes bannaensis A. Dorsal view of male (AUP-00481); B. Dorsal view of female (AUP-00491).

Distribution

This species is known from southern China, northern and central Vietnam and northern Laos (Frost 2020). This is the first record for Thailand, ca. 266 km southwest from the type locality in Jinghong City, Mengla County, Yunnan Province, China (Ye et al. 2007).

Ecology

Specimens were found between 19:00 to 21:00 h in small rocky streams (Fig. 5). Most specimens were found in the water. The surrounding habitat was secondary evergreen forest of medium growth. Other anuran species found in sympatry include: Limnonectes taylori, Kurixalus bisacculus (Taylor), Leptobrachium cf. huashen Fei & Ye, Leptobrachella cf. minima (Taylor) and Amolops cremnobatus Inger & Kottelat.

Figure 5.  

Habitat of Limnonectes bannaensis in Bo Kluea District, Nan Province, northern Thailand.

Limnonectes utara Matsui, Belabut & Ahmad, 2014

Materials    Download as CSV 
  1. scientificName:
    Limnonectes utara
    ; class:
    Amphibia
    ; order:
    Anura
    ; family:
    Dicroglossidae
    ; genus:
    Limnonectes
    ; specificEpithet:
    utara
    ; scientificNameAuthorship:
    Matsui, Belabut & Ahmad, 2014
    ; country:
    Thailand
    ; countryCode:
    TL
    ; stateProvince:
    Yala
    ; locality:
    Bannang Sata
    ; verbatimElevation:
    680
    ; verbatimLatitude:
    6°11'39.5"N
    ; verbatimLongitude:
    101°18'28.2"E
    ; eventDate:
    21 August, 2018
    ; fieldNotes:
    P. Pawangkhanant, C. Suwannapoom
    ; individualCount:
    1
    ; sex:
    female
    ; lifeStage:
    adult
    ; catalogNumber:
    AUP-01706
    ; language:
    en
    ; collectionCode:
    Amphibians
    ; basisOfRecord:
    Preserved Specimen
  2. scientificName:
    Limnonectes utara
    ; individualCount:
    1
    ; sex:
    female
    ; lifeStage:
    adult
    ; catalogNumber:
    AUP-01706
    ; basisOfRecord:
    Preserved Specimen
    ; dynamicProperties:
    collection date, collector and Location as the AUP-01705
  3. scientificName:
    Limnonectes utara
    ; individualCount:
    1
    ; sex:
    male
    ; lifeStage:
    adult
    ; catalogNumber:
    AUP-01707
    ; basisOfRecord:
    Preserved Specimen
    ; dynamicProperties:
    collection date, collector and Location as the AUP-01705
  4. scientificName:
    Limnonectes utara
    ; individualCount:
    1
    ; sex:
    male
    ; lifeStage:
    adult
    ; catalogNumber:
    AUP-01708
    ; basisOfRecord:
    Preserved Specimen
    ; dynamicProperties:
    collection date, collector and Location as the AUP-01705

Description

Morphological characters of specimens from Yala Province agreed with the description by Matsui et al. (2014): Body size moderate, with males SVL of 70.7 mm (n = 2) and females SVL of 46.1 mm (n = 2). The complete morphometric description of each specimen is presented in Suppl. material 2. Head slightly longer than wide (males HL of 32.8 mm, HW 29.8 mm, n = 2 and females HL of 20.0 mm, HW 19.6 mm, n = 2). Snout obtusely pointed in dorsal view, obtuse in profile, projecting beyond the lower jaw. Eye diameter shorter than snout length, canthus rostralis rounded, loreal region sloping and concave, nostril dorsolaterally orientated, placed closer to tip of snout than to eye, internarial distance equal to upper eyelid width. Fore limb robust, relatively short and moderately slender fingers, finger length formula: II< I < IV < III (Fig. 6F), toe length formula, I < II < V < III < IV (Fig. 6E), tips of toes expanded into round, elevated pads lacking grooves, toe webbing complete, webbing formula, I 0 – 0 II 0 – 0 III 0 – 0 IV 0 – 0 V.

Figure 6.  

Male of Limnonectes utara (AUP-01708) in life. A. Dorsal view; B. Lateral view of head; C. Dorsolateral view of head; D. Dorsal view of leg (notice the tubercles); E. Ventral view of foot; F. Ventral view of hand.

Skin on dorsal surfaces of head, fore limbs and body feebly crenulate, skin of body flanks rough with moderately, roundish and non-pearl tipped tubercles, skin around vent, knees and shanks distinctly tuberculate, covered with moderately, small, low tubercles with translucent spinules, ventral surfaces smooth, pair of faint, but broken dorsolateral folds extending from posterior of eye to vent.

Colouration in life: dorsum light brown with confluent dark brown markings (Fig. 6A), head with narrow light bands placed anteriorly to the dark interorbital bar, blackish-brown stripe on canthus rostralis, sides of head pale brown with dark markings. Ventral surfaces of hand and foot dark brown (Fig. 6E and F). Colouration in preservative: after two years in preservative, dorsal colouration slightly faded, but other than that, no obvious change in colour pattern has occurred.

Distribution

Prior to these records, this species was considered endemic to Peninsular Malaysia. This is the first country record for Thailand, ca. 158 km northeast from the type locality [Bukit Larut (= Larut Hill), Perak State, Peninsular Malaysia] (Matsui et al. 2014).

Ecology

Specimens were found after 20:00 h in small rocky streams. Most specimens were found in the water. All specimens were collected in evergreen forests along hillside streams and small tributaries varying in width from 1 m to 2 m (Fig. 7). Other syntopic anuran species include: Limnonectes plicatellus (Stoliczka), Nyctixalus pictus (Peters) and Rhacophorus rhodopus Liu & Hu.

Figure 7.  

Habitat of Limnonectes utara in Bannang Sata District, Yala Province, southern Thailand.

Discussion

In this study, we examined newly-collected samples of Limnonectes species related to the L. kuhlii species complex, from previously not surveyed areas in northern and southern Thailand. From a biogeographic perspective, according to Matsui et al. (2010a), L. taylori was thought to be the unique representative of the L. kuhlii species complex in northern Thailand, whereas L. jarujini was believed to occur in the southern part of the country, the biogeographic distribution between these species being located between Thong Pha Phum and Khao Laem National Parks in Kanchanaburi Province. With the exception of L. bannaensis and L. utara, it was already known which other species of the L. kuhlii complex occur in northern and southern Thailand; therefore, it is not a result that can be obtained from phylogenetic analysis. Actually, phylogeny corroborates the identification of the collected specimens and, thus, demonstrates that L. bannaensis and L. utara occur in northern and southern Thailand, respectively. Our new records of L. bannaensis and L. utara from Thailand increase to 20 the number of Limnonectes species occurring in the country.

Our study and others like this (e.g. Suwannapoom et al. 2016) further highlight the importance of using molecular data in combination with traditional morphological characteristics. This is especially important for species complexes whose members have sympatric distribution, which is the case with the L. kuhlii complex. We recorded sympatric occurrence of L. taylori and L. bannaensis, which were observed sharing the same habitats at Bo Kluea, Nan Province, northern Thailand. Consistent with the findings of previous studies involving the Limnonectes species complex (e.g. Suwannapoom et al. 2016), our results demonstrate that species living in sympatry are not necessarily close relatives (i.e. sister taxa).

These two sympatric members of the Limnonectes species complex in Bo Kluea, Nan Province, are difficult to distinguish from each other, based only on morphological evidence. The application of molecular methods is crucial for reliable identification and can guide morphological re-examinations, further elucidating fine-scale differences in morphological characteristics that represent species-specific variations. Identification of tadpoles, juveniles and adult females still remains challenging in the field. Our study underscores that the herpetofaunal diversity of Thailand still remains underestimated and also illustrates the special role of evergreen forests with regard to biodiversity conservation in the country.

Acknowledgements

This work was supported by the Unit of Excellence 2021 on Genetic diversity assessment of widely distributed aquatic animals and herpetology from Thailand (UoE64003), University of Phayao and the Thailand Research Fund (DBG6180001) to C.S., the International Partnership Program of Chinese Academy of Sciences (CAS) (152453KYSB20170033), Southeast Asia Biodiversity Research Institute, CAS (Y4ZK111B01: 2017CASSEABRIQG002) and the Animal Branch of the Germplasm Bank of Wild Species, CAS (Large Research Infrastructure Funding) to J.C. and partially supported by the Russian Science Foundation (RSF grant № 19-14-00050, data analysis) to N.A.P. Specimens were collected under approval from the Institute of Animals for Scientific Purposes Development (IAD), which issued the fieldwork permission (No. 610104022). This research study was also granted permission by the Department of National Parks, Wildlife and Plant Conservation (DNP).

References

Supplementary materials

Suppl. material 1: Genetic distance between species of Limnonectes. 
Authors:  Chatmongkon Suwannapoom
Data type:  Table
Brief description: 

The pairwise uncorrected p-distance (%) of 16S rRNA gene between species of Limnonectes.

Suppl. material 2: Measurement (in mm) and proportions of the series of Limnonectes bannaensis and L. utara 
Authors:  Chatmongkon Suwannapoom
Data type:  Table
Brief description: 

Measurements (in mm) and proportions of the series of Limnonectes bannaensis from Nan Province and L. utara from Yala Province. (M = Male, F = Female; N/a = Not applicable; for other abbreviations, see Materials and Methods).