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Research Article
Phylogeographic pattern and taxonomic revision of the Kaloula baleata species complex (Amphibia, Anura, Microhylidae) with description of two new species from Indochina
expand article infoVladislav A. Gorin, Nikolai L. Orlov§, Andrey M. Bragin|, Parinya Pawangkhanant#, Konstantin D. Milto§, Dac Xuan Le|, Tan Van Nguyen¤, Christophe Dufresnes«, Chatmongkon Suwannapoom, Nikolay A. Poyarkov|
‡ Lomonosov Moscow State University, Moscow, Russia
§ Russian Academy of Sciences, St. Petersburg, Russia
| Joint Vietnam - Russia Tropical Science & Technology Research Center, Hanoi, Vietnam
¶ University of Phayao, Phayao, Thailand
# Rabbit in the Moon Foundation, Ratchaburi, Thailand
¤ Duy Tan University, Da Nang, Vietnam
« Université des Antilles, Paris, France
Open Access

Abstract

Despite increased attention by molecular taxonomists, the herpetofauna of Southeast Asia still hides many undescribed species among far-ranging taxa. In this study, we re-examine the mitochondrial diversity of painted frogs of the microhylid genus Kaloula, based on ~2,455 bp of published and new 12S and 16S rRNA sequences, and describe two new species from the tropical forests of southern Vietnam and central Laos based on integrative evidence. These species, which belong to the K. baleata complex, feature species-level mitochondrial divergence (> 4.4% at 16S rRNA) and are both morphologically well-differentiated from each other and from the recently described K. indochinensis, to which they were previously confounded. Comparative examinations also indicate distinct male advertisement calls and unique coloration features. Based on genetic barcoding, we preliminarily revise the species distribution ranges in the K. baleata complex, which support a general pattern of biogeographic partitioning that has been widely retrieved among the Indochinese amphibians studied so far. Molecular diversity within K. baleata further suggests genetic structure across Sundaland, namely three shallow mitochondrial haplogroups worthy of fine-scale phylogeographic and taxonomic investigations. Furthermore, our study highlights the propensity of amphibian species “hidden in plain sight,” even among recently studied taxa, thus calling for caution when specifying type specimens—the type series of K. indochinensis, described in 2013, includes specimens of the one of the new species. Our study emphasizes the continued need for thorough herpetological surveys even in supposedly well-known parts of Indochina and sets the ground for future research in Kaloula painted frogs, notably to test evolutionary and taxonomic hypotheses with genomic loci.

Key Words

16S rRNA, bioacoustics, DNA barcoding, Laos, morphology, Southeast Asia, systematics, taxonomy, Vietnam

Introduction

Many widely distributed amphibian genera in Asia have undergone a considerable increase in their numbers of recognized species since the turn of the 21st century. For example, among microhylids of the genus Microhyla Tschudi, 1838, the number of recognized species jumped from 22 to 51 within two decades (Poyarkov et al. 2020a; Zhang et al. 2022; Trofimets et al. 2024). The number of species within the genus Micryletta Dubois, 1987, rose from three in the 20th century to 13 species nowadays (Suwannapoom et al. 2020; Poyarkov et al. 2021a; Sankar et al. 2022). The genus Kalophrynus Tschudi, 1838, included from 13 to currently 27 recognized species (Fukuyama et al. 2021). Two new microhylid genera were discovered in Southeast Asia in the past several years, namely, Siamophryne Suwannapoom, Sumontha, Tunprasert, Ruangsuwan, Pawangkhanant, Korost & Poyarkov, 2018 (one single known species) and Vietnamophryne Poyarkov, Suwannapoom, Pawangkhanant, Aksornneam, Duong, Korost & Che, 2018 (six species described so far; see Suwannapoom et al. 2018; Poyarkov et al. 2018a, 2021b; Gorin et al. 2021; Frost 2024).

Quite a few genera of Asian microhylids, however, have so far evaded the focus of taxonomic studies, and their species numbers thus remained relatively stable. One notable example is the genus Kaloula Gray, 1831, also known as Asian painted frogs (Frost 2024). Kaloula is a widely distributed genus of microhylids, with 18 species recognized up to date. Painted frogs inhabit East Asia, from Korea and northeastern China to Southeast Asia, including Indochina to the Sunda Islands and the Philippines to the south, and Bangladesh and northeastern India to the west (Othman et al. 2022; Frost 2024). The genus Kaloula is characterized by the following morphological attributes: pupil round; tongue oval, entire, and free behind; two transverse ridges across the palate in front of the pharynx; a strong bony ridge behind each choanae; the first finger shorter than the second and the outer toe shorter than the third; medium body size (SVL 35–60 mm); smooth or slightly rough dorsum with irregular dark markings; tips of the fingers dilated into disks and truncated; fingers free of the web; nearly full webbing on toes in males and reduced webbing in females (e.g., Gray 1831; Sengupta et al. 2009; Gorin et al. 2021). Recent contributions to the diversity of this genus include the review by Blackburn et al. (2013), which focused on Kaloula species inhabiting Southeast Asian islands, mainly the Philippine Archipelago. In this study, the authors conducted phylogenetic analysis on 140 Kaloula individuals from all over Southeast Asia and indicated the presence of at least six undescribed lineages of putative species status. Following the study of Blackburn et al. (2013), two new species of the K. baleata complex were described from Indochina, namely K. indochinensis Chan, Blackburn, Murphy, Stuart, Emmett, Ho & Brown, 2013 (Chan et al. 2013), and from Peninsular Malaysia, namely K. latidisca Chan, Grismer & Brown, 2014 (Chan et al. 2014). In both cases, authors provided only limited morphological data to substantiate species divergence, did not provide any bioacoustic data, and based their phylogenetic estimations entirely on the previously published mtDNA data of Blackburn et al. (2013).

In parallel, a new species, K. nonggangensis Mo, Zhang, Zhou, Chen, Tang, Meng & Chen, 2013, was described from the Guangxi Province of China (Mo et al. 2013) on the basis of morphological and genetic data. More recently, the subspecies K. baleata ghoshi Cherchi, 1954, endemic to the Andaman Islands, was elevated to full species status based on morphological differentiation, accompanied by new data on natural history, bioacoustics, and distribution (Chandramouli and Prasad 2018). Lastly, the phylogeography of the genus Kaloula in East Asia was re-summarized by Othman et al. (2022), based mostly on previously published data, and without emphasizing undescribed diversity.

In this work, we present an updated mitochondrial DNA (hereafter mtDNA)-based genealogy for the genus Kaloula, combining sequences available in GenBank with new sequences obtained from an extensive sampling in Indochina, as well as integrative taxonomic revisions in the K. baleata species complex, specifically for two populations from central Laos and southern Vietnam previously assigned to K. indochinensis (Chan et al. 2013). These populations are deeply divergent in the mitochondrial phylogeny and feature morphological and bioacoustic differentiation from related taxa, hence substantiating their description as new species.

Materials and methods

Sample collection

Fieldwork in Vietnam was conducted by NAP, AMB, and VAG in 2009–2023; fieldwork in Laos was conducted by NLO and KDM in 2009. We obtained geographic coordinates and elevation data using a Garmin GPSMAP 60CSx (USA) and recorded them in the WGS84 datum. The specimens collected were initially fixed in 4% formalin for 24 h and later transferred to 75% ethanol for storage; muscle or liver tissues were taken prior to the fixation in formalin and preserved in 95% ethanol for genetic analysis. Specimens were subsequently deposited in the herpetological collections of the Zoological Museum of Lomonosov Moscow State University (ZMMU, Moscow, Russia) and of the Zoological Institute, Russian Academy of Sciences (ZISP, St. Petersburg, Russia).

Laboratory methods

For the molecular phylogenetic analyses, total genomic DNA was isolated using the standard phenol-chloroform-proteinase K extraction procedures with consequent isopropanol precipitation for a final concentration of about 1 mg/ml (protocols followed the works of Hillis et al. 1996; Sambrook and Russell 2001). We visualized the isolated total genomic DNA using agarose electrophoresis in the presence of ethidium bromide. We measured the concentration of total DNA in 1 μL using NanoDrop 2000 (Thermo Scientific) and consequently adjusted it to ca. 100 ng DNA/μL.

We amplified mtDNA fragments covering the 16S rRNA gene and the adjacent tRNA genes to obtain a 1,003 bp-long continuous fragment. The 16S rRNA gene is widely used for biodiversity surveys in amphibians (Vences et al. 2005; Vieites et al. 2009) and has been analyzed in the most recent phylogenetic studies on Microhylinae (Matsui et al. 2011; Garg et al. 2019; Gorin et al. 2020, 2021). We performed DNA amplification in 20 μL reactions using ca. 50 ng genomic DNA, 10 nmol of each primer, 15 nmol of each dNTP, 50 nmol additional MgCl2, Taq PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.1 mM MgCl2, and 0.01% gelatine), and 1 unit of Taq DNA polymerase. Primers used in PCR and sequencing include 16sL-2188 (CTGACCGTGCAAAGGTAGCGTAATCACT) and 16H-1 (CTCCGGTCTGAACTCAGATCACGTAGG) (Hedges et al. 1994; Matsui et al. 2005). The PCR conditions involved an initial denaturation step of 5 min at 94 °C, followed by 43 cycles of denaturation for 1 min at 94 °C, primer annealing for 1 min using the TouchDown program, reducing 1 °C every cycle, extension for 1 min at 72 °C, and a final extension step for 5 min at 72 °C (Gorin et al. 2020, 2021).

PCR products were loaded onto 1.0% agarose gels in the presence of ethidium bromide and visualized in agarose electrophoresis. When distinct bands were obtained, we purified PCR products using 2 μL of a 1:4 dilution of ExoSapIt (Amersham) per 5 μL of PCR product prior to cycle sequencing. A 10 μL sequencing reaction included 2 μL of template, 2.5 μL of sequencing buffer, 0.8 μL of 10 pmol primer, 0.4 μL of BigDye Terminator version 3.1 Sequencing Standard (Applied Biosystems), and 4.2 μL of water. The cycle sequencing used 35 cycles of 10 sec at 96 °C, 10 s at 50 °C, and 4 min at 60 °C. We purified the cycle sequencing products by ethanol precipitation. We carried out sequence data collection and visualization on an ABI 3730xl Automated Sequencer (Applied Biosystems). We deposited the obtained sequences in GenBank under the accession numbers PQ268497PQ268520.

Phylogenetic analyses

To reconstruct the matrilineal genealogy, we used newly obtained 16S rRNA sequences of Kaloula spp. from Laos and Vietnam and the sequences of 12S rRNA and 16S rRNA mtDNA fragments of the K. baleata species complex members from Thailand, Malaysia, and Indonesia, as well as other Kaloula species obtained from GenBank. Table 1 summarizes the information on GenBank Accession Numbers, museum vouchers, and the locality of origin for the sequences used in this study. We also added sequences of Uperodon taprobanicus (Parker, 1934) as a sister group; a sequence of Kalophrynus interlineatus (Blyth, 1855) was used to root the tree; the final alignment included 2,455 bp. In total, we obtained data for 16S rRNA for 233 specimens, which included 15 out of 18 currently recognized Kaloula species, including the topotype specimens of K. baleata (Müller, 1836) (type locality: Java, Indonesia) (see Table 1); geographic distribution of the sampled populations of the K. baleata species complex is shown in Fig. 1.

Table 1.

Museum voucher information, geographic localities, and GenBank accession numbers of specimens and sequences used in this study. Exact locality information is unknown for specimens obtained via the pet trade or those published in some earlier works. Locality numbers given in brackets in bold after the locality name correspond to the numbers shown in Fig. 1.

No. Species Locality Museum / Sample ID Accession numbers Reference
Ingroup 12S rRNA 16S rRNA
Kaloula baleata species complex
1 Kaloula discordia sp. nov. Vietnam, Dong Nai Prov., Cat Tien NP (1) ZMMU-A-4642 PQ268510 this work
2 Kaloula discordia sp. nov. Vietnam, Dong Nai Prov., Cat Tien NP (1) ZMMU-A-4739 PQ268511 this work
3 Kaloula discordia sp. nov. Vietnam, Dong Nai Prov., Cat Tien NP (1) ZISP 15285 PQ268512 this work
4 Kaloula discordia sp. nov. Vietnam, Dong Nai Prov., Cat Tien NP (1) ZMMU-A-8134 PQ268513 this work
5 Kaloula discordia sp. nov. Vietnam, Dong Nai Prov., Cat Tien NP (1) ZMMU-A-8135 PQ268514 this work
6 Kaloula discordia sp. nov. Vietnam, Lam Dong Prov., Loc Bac (2) ZMMU NAP-02826 PQ268515 this work
7 Kaloula discordia sp. nov. Vietnam, Lam Dong Prov., Loc Bac (2) ZMMU NAP-03384 PQ268516 this work
8 Kaloula laosensis sp. nov. Laos, Khammouane Prov., Nakai-Nam Theun NP (3) ZISP 15284 PQ268517 this work
9 Kaloula laosensis sp. nov. Laos, Khammouane Prov., Nakai-Nam Theun NP (3) ZMMU-A-8144 PQ268518 this work
10 Kaloula indochinensis Vietnam, Dak Lak Prov., Yok Don NP (4) ZMMU-A-8147 PQ268497 this work
11 Kaloula indochinensis Vietnam, Dak Lak Prov., Yok Don NP (4) ZMMU-A-8148 PQ268498 this work
12 Kaloula indochinensis Vietnam, Gia Lai Prov., Krong Pa (5) ROM32925 KC180032 de Sa et al. 2012
13 Kaloula indochinensis Vietnam, Gia Lai Prov., Krong Pa (5) ZISP-Gia Lai 1 PQ268499 this work
14 Kaloula indochinensis Vietnam, Gia Lai Prov., Krong Pa (5) ZISP-Gia Lai 2 PQ268500 this work
15 Kaloula indochinensis Vietnam, Gia Lai Prov., Krong Pa (5) ROM32925 KC822572 Blackburn et al. 2013
16 Kaloula indochinensis Vietnam, Gia Lai Prov., Krong Pa (5) ROM32932 KC822573 Blackburn et al. 2013
17 Kaloula indochinensis Vietnam, Gia Lai Prov., Krong Pa (5) ROM32943 KC822574 Blackburn et al. 2013
18 Kaloula indochinensis Vietnam, Gia Lai Prov., Kon Ka Kinh NP (6) ZMMU-A-6315 PQ268501 this work
19 Kaloula indochinensis Vietnam, Gia Lai Prov., Kon Ka Kinh NP (6) ZMMU-A-6316 PQ268502 this work
55 Kaloula latidisca Malaysia, Kedah, Gubir (7) LSUHC5074 KC822576 Blackburn et al. 2013
56 Kaloula latidisca Thailand, Satun Prov., Baan Suan Tondin (8) ZMMU-A-8145 PQ268503 this work
57 Kaloula latidisca Thailand, Suratthani Prov., Khao Sok (9) ZMMU-A-8146 PQ268504 this work
20 Kaloula baleata clade 1 Indonesia, Java isl., Jawa Barat (10) TNHC67086 KC822569 Blackburn et al. 2013
21 Kaloula baleata clade 1 Indonesia, Bali Prov., Bali isl. (11) JAM3232 KC822570 Blackburn et al. 2013
22 Kaloula baleata clade 1 Indonesia, Sumatra, Way Jepara (12) ZMMU A-04412 PQ268507 this work
23 Kaloula baleata clade 1 Indonesia, Sumba (13) KUHE32313 AB634629 AB634687 Matsui et al. 2011
24 Kaloula baleata clade 1 Philippines, Palawan isl. (14) ACD1303 KC822582 Blackburn et al. 2013
25 Kaloula baleata clade 1 Philippines, Palawan isl. (14) ACD1307 KC822583 Blackburn et al. 2013
26 Kaloula baleata clade 1 Indonesia, Sulawesi (–) JAM3573 KY132185 Alexander et al. 2017
27 Kaloula baleata clade 1 Indonesia, Sulawesi Tengah Prov., Pulau Batudaka (15) JAM-3853 KC822568 Blackburn et al. 2013
28 Kaloula baleata clade 1 Indonesia, Sulawesi Tengah Prov., Donggala (16) LSUMZ-83998 KC822566 Blackburn et al. 2013
29 Kaloula baleata clade 1 Indonesia, Sulawesi Utara Prov., Bogani Nani Wartabone NP (17) LSUMZ-83999 KC822567 Blackburn et al. 2013
30 Kaloula baleata clade 2 Malaysia (–) FRIM1066 KC822579 Blackburn et al. 2013
31 Kaloula baleata clade 2 Malaysia (–) DWNP975 KC822580 Blackburn et al. 2013
32 Kaloula baleata clade 2 Malaysia, Johor, Pulau Aceh (18) LSUHC5712 KC822577 Blackburn et al. 2013
33 Kaloula baleata clade 2 Malaysia, Sabah, Sepilok (19) LSUHC6156 KC822578 Blackburn et al. 2013
35 Kaloula baleata clade 2 Malaysia, Sabah, Kinabalu (20) ZMHA10028 GU154880 Das and Haas 2010
34 Kaloula baleata clade 2 Malaysia, Sarawak, Kuching (21) ID8317 KC822581 Blackburn et al. 2013
36 Kaloula baleata clade 2 Malaysia, Pahang, Taman Negara (22) ZMMU-A-6152 PQ268508 this work
37 Kaloula baleata clade 2 Malaysia, Pahang, Taman Negara (22) ZMMU-A-6153 PQ268509 this work
38 Kaloula baleata clade 2 Indonesia, Java, Bogor (23) released (Java) PQ268505 this work
39 Kaloula baleata clade 2 Indonesia, Java, Bogor (23) released (Java) PQ268506 this work
40 Kaloula baleata clade 3 Myanmar, Tanintharyi Div., Yeybu (24) USNM-587865 MT608909 Mulcahy et al. unpublished
41 Kaloula baleata clade 3 Myanmar, Tanintharyi Div., Yeybu (24) USNM-587869 MT608910 Mulcahy et al. unpublished
42 Kaloula baleata clade 3 Myanmar, Tanintharyi Div., Yeybu (24) USNM-587870 MT608911 Mulcahy et al. unpublished
43 Kaloula baleata clade 3 Myanmar, Tanintharyi Div., Yeybu (24) USNM-587871 MT608912 Mulcahy et al. unpublished
44 Kaloula baleata clade 3 Myanmar, Tanintharyi Div., Yeybu (24) USNM-587602 MT608913 Mulcahy et al. unpublished
45 Kaloula baleata clade 3 Myanmar, Tanintharyi Div., Yeybu (24) USNM-587603 MT608914 Mulcahy et al. unpublished
46 Kaloula baleata clade 3 Myanmar, Tanintharyi Div., Yeybu (24) USNM-587863 MT608915 Mulcahy et al. unpublished
47 Kaloula baleata clade 3 Myanmar, Tanintharyi Div., Yeybu (24) USNM-587864 MT608916 Mulcahy et al. unpublished
48 Kaloula baleata clade 3 Myanmar, Tanintharyi Div., Yeybu (24) USNM-587866 MT608917 Mulcahy et al. unpublished
49 Kaloula baleata clade 3 Myanmar, Tanintharyi Div., Yeybu (24) USNM-587867 MT608918 Mulcahy et al. unpublished
50 Kaloula baleata clade 3 Myanmar, Tanintharyi Div., Yeybu (24) USNM-587868 MT608919 Mulcahy et al. unpublished
51 Kaloula baleata clade 3 Myanmar, Tanintharyi Div., Yeybu (24) USNM-587604 MT608920 Mulcahy et al. unpublished
52 Kaloula baleata clade 3 Myanmar, Tanintharyi Div., Yeybu (24) USNM-587861 MT608921 Mulcahy et al. unpublished
53 Kaloula baleata clade 3 Myanmar, Tanintharyi Div., Yeybu (24) USNM-587862 MT608922 Mulcahy et al. unpublished
54 Kaloula baleata clade 3 Myanmar, Tanintharyi Div., Yeybu (24) USNM-586944 MG935845 Mulcahy et al. 2018
Other Kaloula species
58 Kaloula borealis South Korea, Jeju isl. KUHE-33139 AB634688 Matsui et al. 2011
59 Kaloula borealis South Korea, Jeju isl. NIBRAM-000123 JQ815297 Jeong et al. 2013
60 Kaloula borealis South Korea, Jeju isl. NIBRAM-100348 JQ815298 Jeong et al. 2013
61 Kaloula borealis - JQ692869 Hwang and Lee, 2012
62 Kaloula borealis China, Beijing, Xiangshan KIZHERP-0173 JX678908 Li et al. 2012
63 Kaloula borealis China, Beijing, Xiangshan KIZHERP-0174 JX678909 Li et al. 2012
64 Kaloula conjuncta Philippines, Negros isl. RMB 2252, PNM / CMNH AY326064 Darst and Canatella, 2003
65 Kaloula conjuncta Philippines, Luzon isl., Palola ACD 769 KC822537 Blackburn et al. 2013
66 Kaloula conjuncta Philippines, Mindanao isl., Bukidnon Prov. ACD 996 KC822536 Blackburn et al. 2013
67 Kaloula conjuncta Philippines, Semirara isl., Antique Prov. KU301849 KC822532 Blackburn et al. 2013
68 Kaloula conjuncta Philippines, Semirara isl., Antique Prov. KU301854 KC822533 Blackburn et al. 2013
69 Kaloula conjuncta Philippines, Polillo isl., Quezon Prov. KU303279 KC822527 Blackburn et al. 2013
70 Kaloula conjuncta Philippines, Luzon isl., Laguna Prov. KU320031 KC822538 Blackburn et al. 2013
71 Kaloula conjuncta Philippines, Mindoro isl., Mindoro Occidental Prov. KU323280 KC822535 Blackburn et al. 2013
72 Kaloula conjuncta Philippines, Mindoro isl., Mindoro Oriental Prov. RMB509 KC822534 Blackburn et al. 2013
73 Kaloula conjuncta Philippines, Luzon isl., Quezon Prov. TNHC59628 KC822539 Blackburn et al. 2013
74 Kaloula conjuncta Philippines, Luzon isl., Albay Prov. TNHC62972 KC822528 Blackburn et al. 2013
75 Kaloula conjuncta Philippines, Luzon isl., Albay Prov. TNHC62973 KC822526 Blackburn et al. 2013
76 Kaloula conjuncta Philippines, Luzon isl., Albay Prov. TNHC62975 KC822530 Blackburn et al. 2013
77 Kaloula conjuncta Philippines, Luzon isl., Albay Prov. TNHC62976 KC822529 Blackburn et al. 2013
78 Kaloula conjuncta Philippines, Luzon isl., Albay Prov. TNHC62986 KC822531 Blackburn et al. 2013
79 Kaloula conjuncta Philippines, Camigun Sur isl., Camiguin Sur Prov. KU309658 KC822588 Blackburn et al. 2013
80 Kaloula conjuncta Philippines, Mindanao isl., Davao del Sur Prov. TNHC59632 KC822521 Blackburn et al. 2013
81 Kaloula conjuncta Philippines, Mindanao isl., Davao del Sur Prov. TNHC59635 KC822520 Blackburn et al. 2013
82 Kaloula conjuncta Philippines, Mindanao isl., Davao del Sur Prov. TNHC59636 KC822522 Blackburn et al. 2013
83 Kaloula conjuncta Philippines, Mindanao isl., Davao del Sur Prov. TNHC59669 KC822524 Blackburn et al. 2013
84 Kaloula conjuncta Philippines, Mindanao isl., Davao del Sur Prov. TNHC59670 KC822523 Blackburn et al. 2013
85 Kaloula conjuncta Philippines, Mindanao isl., Davao City Prov. TNHC59870 KC822525 Blackburn et al. 2013
86 Kaloula conjuncta Philippines, Negros isl., Negros Oriental Prov. KU328639 KC822518 Blackburn et al. 2013
87 Kaloula conjuncta Philippines, Negros isl., Negros Oriental Prov. KU328640 KC822519 Blackburn et al. 2013
88 Kaloula kalingensis Philippines, Palaui isl., Cagayan Prov. KU328643 KC822603 Blackburn et al. 2013
89 Kaloula kalingensis Philippines, Palaui isl., Cagayan Prov. KU328644 KC822604 Blackburn et al. 2013
90 Kaloula kalingensis Philippines, Luzon isl., Kalinga Prov. TNHC60118 KC822605 Blackburn et al. 2013
91 Kaloula kalingensis Philippines, Luzon isl., Kalinga Prov. RMB2210 KC822608 Blackburn et al. 2013
92 Kaloula kalingensis Philippines, Luzon isl., Kalinga Prov. RMB3137 KC822609 Blackburn et al. 2013
93 Kaloula kalingensis Philippines, Luzon isl., Kalinga Prov. TNHC59647 KC822610 Blackburn et al. 2013
94 Kaloula kokacii Philippines, Luzon isl., Camarines del Norte Prov. KU313824 KC822596 Blackburn et al. 2013
95 Kaloula kokacii Philippines, Luzon isl., Camarines Sur Prov. KU328629 KC822595 Blackburn et al. 2013
96 Kaloula kokacii Philippines, Luzon isl., Albay Prov. KU328630 KC822590 Blackburn et al. 2013
97 Kaloula kokacii Philippines, Luzon isl., Quezon Prov. KU328634 KC822593 Blackburn et al. 2013
98 Kaloula kokacii Philippines, Luzon isl., Camarines Sur Prov. TNHC62684 KC822594 Blackburn et al. 2013
99 Kaloula kokacii Philippines, Luzon isl., Albay Prov. TNHC62685 KC822589 Blackburn et al. 2013
100 Kaloula kokacii Philippines, Luzon isl., Albay Prov. TNHC62687 KC822591 Blackburn et al. 2013
101 Kaloula kokacii Philippines, Luzon isl., Sorsogon Prov. TNHC62688 KC822592 Blackburn et al. 2013
102 Kaloula mediolineata Thailand, Tak Prov. KUHE35178 AB634631 AB634689 Matsui et al. 2011
103 Kaloula mediolineata Vietnam, Gia Lai Prov. ROM32838 KC822571 Blackburn et al. 2013
104 Kaloula mediolineata Thailand, Nakhorn Ratchasima Prov. KU328285 KC822508 Blackburn et al. 2013
105 Kaloula mediolineata Thailand, Nakhorn Ratchasima Prov. KU328280 KC822509 Blackburn et al. 2013
106 Kaloula mediolineata Thailand, Ubon Ratchatani FMNH265819 KC822510 Blackburn et al. 2013
107 Kaloula nonggangensis China, Guanxi Prov., Nonggang NHMG-1106040 KC567231 Chen et al. 2013
108 Kaloula nonggangensis China, Guanxi Prov., Nonggang NHMG-1108035 KC567232 Chen et al. 2013
109 Kaloula nonggangensis China, Guanxi Prov., Nonggang NHMG-1108036 KC567233 Chen et al. 2013
110 Kaloula nonggangensis China, Guanxi Prov., Nonggang NHMG-CHN-T20120901 KC567234 Chen et al. 2013
111 Kaloula picta KUHE UN AB634686 Matsui et al. 2011
112 Kaloula picta USFS56931 KC180019 de Sa et al. 2012
113 Kaloula picta Philippines, Luzon isl., Cavite Prov. DLSUD021 KP298039 Brown et al. 2015
114 Kaloula picta Philippines, Palawan isl., Palawan Prov. ACD1206 KC822541 Blackburn et al. 2013
115 Kaloula picta Philippines, Leyte isl., Leyte Prov. ACD1242 KC822563 Blackburn et al. 2013
116 Kaloula picta Philippines, Palawan isl., Palawan Prov. ACD1261 KC822542 Blackburn et al. 2013
117 Kaloula picta Philippines, Palawan isl., Palawan Prov. ACD1304 KC822540 Blackburn et al. 2013
118 Kaloula picta Philippines, Luzon isl., Pampanga Prov. ACD1390 KC822546 Blackburn et al. 2013
119 Kaloula picta Philippines, Leyte isl., Leyte Prov. ACD1501 KC822562 Blackburn et al. 2013
120 Kaloula picta Philippines, Luzon isl., Cavite Prov. DLSUD020 KC822552 Blackburn et al. 2013
121 Kaloula picta Philippines, Camiguin Sur Isl., Camiguin Sur Prov. KU301873 KC822565 Blackburn et al. 2013
122 Kaloula picta Philippines, Luzon isl., Laguna Prov. KU326261 KC822554 Blackburn et al. 2013
123 Kaloula picta Philippines, Luzon isl., Laguna Prov. KU326262 KC822555 Blackburn et al. 2013
124 Kaloula picta Philippines, Luzon isl., Quezon Prov. KU326265 KC822548 Blackburn et al. 2013
125 Kaloula picta Philippines, Luzon isl., Cagayan Prov. RMB4223 KC822551 Blackburn et al. 2013
126 Kaloula picta Philippines, Luzon isl., Cagayan Prov. RMB4224 KC822550 Blackburn et al. 2013
127 Kaloula picta Philippines, Leyte isl., Leyte Prov. RMB4294 KC822564 Blackburn et al. 2013
128 Kaloula picta Philippines, Leyte isl., Leyte Prov. RMB4344 KC822561 Blackburn et al. 2013
129 Kaloula picta Philippines, Bohol isl., Bohol Prov. TNHC56430 KC822559 Blackburn et al. 2013
130 Kaloula picta Philippines, Bohol isl., Bohol Prov. TNHC56431 KC822560 Blackburn et al. 2013
131 Kaloula picta Philippines, Cebu isl., Cebu Prov. TNHC56468 KC822543 Blackburn et al. 2013
132 Kaloula picta Philippines, Mindanao isl., Davao del Sur Prov. TNHC59653 KC822558 Blackburn et al. 2013
133 Kaloula picta Philippines, Mindanao isl., Davao del Sur Prov. TNHC59654 KC822557 Blackburn et al. 2013
134 Kaloula picta Philippines, Mindanao isl., Davao del Sur Prov. TNHC59871 KC822556 Blackburn et al. 2013
135 Kaloula picta Philippines, Luzon isl., Albay Prov. TNHC62470 KC822549 Blackburn et al. 2013
136 Kaloula picta Philippines, Luzon isl., Zambales Prov. TNHC62471 KC822547 Blackburn et al. 2013
137 Kaloula picta Philippines, Polillo isl., Quezon Prov. USNM-FS56932 KC822545 Blackburn et al. 2013
138 Kaloula picta Philippines, Polillo isl., Quezon Prov. USNM-FS-56936 KC822544 Blackburn et al. 2013
139 Kaloula pulchra Thailand, Nong Khai KUHE 22206 AB634632 AB634690 Matsui et al. 2011
140 Kaloula pulchra Thailand, Kanchanaburi, Thong Pha Phum KUHE 35171 AB201183 AB201194 Matsui et al. 2005
141 Kaloula pulchra Myanmar, Sagaing USFS34083 KC180025 de Sa et al. 2012
142 Kaloula pulchra Vietnam, Cat Ba ZMMU-A-4761-1 PQ268519 this work
143 Kaloula pulchra Vietnam, Cat Ba ZMMU-A-4761-2 PQ268520 this work
144 Kaloula pulchra Bangladesh, Sylhet IABHU-3781 / Kpul-Bd1 AB530543 Hasan et al. 2012
145 Kaloula pulchra Bangladesh, Bandarban IABHU 3783 / Kpul-Bd2 AB530544 Hasan et al. 2012
146 Kaloula pulchra Myanmar KIZHERP0439 JX678910 Li et al. 2012
147 Kaloula pulchra Thailand, Ranong - AB530633 Hasan et al. 2014
148 Kaloula pulchra Indonesia, Sulawesi, Makassar - AB530639 Hasan et al. 2014
149 Kaloula pulchra Malaysia VUB0677 EF017955 Van Bocxlaer et al. 2006
150 Kaloula pulchra - NC006405 Zhang et al. 2005
151 Kaloula pulchra Myanmar KIZHERP3003 JX678902 Li et al. 2012
152 Kaloula pulchra China, Yunnan, Jinghong KIZHERP0121 JX678903 Li et al. 2012
153 Kaloula pulchra China, Yunnan, Mengla KIZHERP0129 JX678904 Li et al. 2012
154 Kaloula pulchra China, Guangxi, Qinzhou KIZHERP0171 JX678907 Li et al. 2012
155 Kaloula pulchra Thailand, Phang Nga P994 KR827832 Grosjean et al. 2015
156 Kaloula pulchra China, Hainan GRE119/G027 KR827833 Grosjean et al. 2015
157 Kaloula pulchra Myanmar, Tanintharyi USNM:Herp:586945 MG935846 Mulcahy et al. 2018
158 Kaloula pulchra Myanmar, Tanintharyi USNM:Herp:586946 MG935847 Mulcahy et al. 2018
159 Kaloula pulchra Myanmar, Sagaing USNM:Herp:520322 MG935848 Mulcahy et al. 2018
160 Kaloula pulchra Myanmar, Sagaing USNM:Herp:520326 MG935849 Mulcahy et al. 2018
161 Kaloula pulchra Myanmar, Bago MBM-USNMFS35512 MG935850 Mulcahy et al. 2018
162 Kaloula pulchra Myanmar, Yangon MBM-JBS19849 MG935851 Mulcahy et al. 2018
163 Kaloula pulchra Myanmar, Sagaing USNM:Herp:523967 MG935852 Mulcahy et al. 2018
164 Kaloula pulchra Myanmar, Mandalay MBM-USNMFS36482 MG935853 Mulcahy et al. 2018
165 Kaloula pulchra China, Guangxi, Nonggang NHMG<CHN>:1106008 KC567235 Mo et al. 2013
166 Kaloula pulchra China, Guangxi, Nonggang NHMG<CHN>:1106009 KC567236 Mo et al. 2013
167 Kaloula pulchra China - AF315162 Jiang and Zhou., 2001
168 Kaloula pulchra Southeast Asia ZCYK-Kpul1 LC640532 Kambayashi et al. 2022
169 Kaloula pulchra Bangladesh - MN477194 Rony et al. unpublished
170 Kaloula pulchra Vietnam, Ha Tinh Prov., Huong Son AMCC 106697 DQ283397 Frost et al. 2005
171 Kaloula pulchra pet trade RdS 02 DQ283398 Frost et al. 2005
172 Kaloula pulchra SIH-09 AY330893 Hoegg et al. 2004
173 Kaloula pulchra Thailand ACD1538 KC822621 Blackburn et al. 2013
174 Kaloula pulchra Laos, Khammouane Prov., Boudalapha FMNH225128 KC822620 Blackburn et al. 2013
175 Kaloula pulchra Laos, Bolikhamxay Prov., Thaphabat FMNH225129 KC822619 Blackburn et al. 2013
176 Kaloula pulchra Malaysia, Selangor Prov. JAM1753 KC822616 Blackburn et al. 2013
177 Kaloula pulchra Malaysia, Pahang Prov., Tioman isl. JAM1857 KC822615 Blackburn et al. 2013
178 Kaloula pulchra Malaysia, Pahang, Pulau Tioman LSUHC3869 KC822575 Blackburn et al. 2013
179 Kaloula pulchra Malaysia (continental) LSUHC3870 KC852906 Blackburn et al. 2013
180 Kaloula pulchra Indonesia, Sumatra isl. MF0766 KC822623 Blackburn et al. 2013
181 Kaloula pulchra Indonesia, Sumatra isl. MF0812 KC822624 Blackburn et al. 2013
182 Kaloula pulchra China NHMS3208 KC822614 Blackburn et al. 2013
183 Kaloula pulchra Indonesia, Sulawesi isl., Propinsi Sulawesi Selatan TNHC59422 KC822618 Blackburn et al. 2013
184 Kaloula pulchra Indonesia, Sulawesi isl., Propinsi Sulawesi Selatan TNHC59423 KC822617 Blackburn et al. 2013
185 Kaloula pulchra Vietnam TZ629 KC822622 Blackburn et al. 2013
186 Kaloula pulchra China - AF315130 Jiang and Zhou., 2001
187 Kaloula rigida Philippines, Luzon isl., Benguet Prov. ACD1570 KC822636 Blackburn et al. 2013
188 Kaloula rigida Philippines, Luzon isl., Isabela Prov., San Mariano ACD1954 KC822631 Blackburn et al. 2013
189 Kaloula rigida Philippines, Luzon isl., Isabela Prov., San Mariano ACD2043 KC822632 Blackburn et al. 2013
190 Kaloula rigida Philippines, Luzon isl., Isabela Prov., San Mariano ACD2044 KC822634 Blackburn et al. 2013
191 Kaloula rigida Philippines, Luzon isl., Kalinga Prov. ACD2032 KC822633 Blackburn et al. 2013
192 Kaloula rigida Philippines, Luzon isl., Sierra Madres ACD660 KC822626 Blackburn et al. 2013
193 Kaloula rigida Philippines, Luzon isl., Kalinga Prov., Balbalan ACD756 KC822628 Blackburn et al. 2013
194 Kaloula rigida Philippines, Luzon isl., Isabela Prov., San Mariano KU326470 KC822635 Blackburn et al. 2013
195 Kaloula rigida Philippines, Luzon isl., Cagayan Prov., Pagudpud KU328628 KC822627 Blackburn et al. 2013
196 Kaloula rigida Philippines, Luzon isl., Cagayan Prov., Gattaran RMB4226 KC822625 Blackburn et al. 2013
197 Kaloula rigida Philippines, Luzon isl., Kalinga Prov., Balbalan TNHC59644 KC822629 Blackburn et al. 2013
198 Kaloula rigida Philippines, Luzon isl., Kalinga Prov., Balbalan TNHC60119 KC822630 Blackburn et al. 2013
199 Kaloula rugifera China, Sichuan, Mianyang KIZHERP071601 JX678911 Li et al. 2012
200 Kaloula rugifera China, Sichuan, Mianyang KIZHERP071602 JX678912 Li et al. 2012
201 Kaloula rugifera China, Sichuan, Mianyang CIB20110802028 KC567237 Mo et al. 2013
202 Kaloula verrucosa China, Sichuan, Huidong CIB20090349 KC567238 Mo et al. 2013
203 Kaloula verrucosa China, Sichuan, Huidong CIB20090351 KC567239 Mo et al. 2013
204 Kaloula verrucosa China, Yunnan, Chuxiong KIZHERP2015 JX678900 Li et al. 2012
205 Kaloula verrucosa China, Yunnan, Wuliangshan KIZHERP2020 JX678901 Li et al. 2012
206 Kaloula verrucosa China NMNS 3246 KC822507 Blackburn et al. 2013
207 Kaloula walteri Philippines, Mindanao isl., Bukidnon Prov., Malagaylay ACD994 KC822640 Blackburn et al. 2013
208 Kaloula walteri Philippines, Polillo isl., Quezon Prov., Polillo KU303285 KC822642 Blackburn et al. 2013
209 Kaloula walteri Philippines, Luzon isl., Quezon Prov., Lucban KU327338 KC822639 Blackburn et al. 2013
210 Kaloula walteri Philippines, Luzon isl., Quezon Prov., Tayabas RMB3701 KC822641 Blackburn et al. 2013
211 Kaloula walteri Philippines, Luzon isl., Quezon Prov., Lucban TNHC59667 KC822637 Blackburn et al. 2013
212 Kaloula walteri Philippines, Luzon isl., Quezon Prov., Lucban TNHC60116 KC822638 Blackburn et al. 2013
213 Kaloula sp. Philippines, Luzon isl., Laguna Prov. ACD1692 KC822602 Blackburn et al. 2013
214 Kaloula sp. Philippines, Luzon isl., Sierra Madres ACD2479 KC822606 Blackburn et al. 2013
215 Kaloula sp. Philippines, Luzon isl., Isabela Prov., Palanan ACD650 KC822607 Blackburn et al. 2013
216 Kaloula sp. Philippines, Luzon isl., Laguna Prov., Los Banos ACD943 KC822597 Blackburn et al. 2013
217 Kaloula sp. Philippines, Luzon isl., Laguna Prov., Los Banos ACD945 KC822598 Blackburn et al. 2013
218 Kaloula sp. Philippines, Luzon isl., Laguna Prov., Los Banos FMNH267555 KC822599 Blackburn et al. 2013
219 Kaloula sp. Philippines, Luzon isl., Aurora Prov., Aurora Memorial NP RMB750 KC822601 Blackburn et al. 2013
220 Kaloula sp. Philippines, Luzon isl., Aurora Prov., Aurora Memorial NP RMB783 KC822600 Blackburn et al. 2013
221 Kaloula sp. Philippines, Panay isl., Antique Prov., Sibalom GVAG253 KC822515 Blackburn et al. 2013
222 Kaloula sp. Philippines, Panay isl., Antique Prov., Sibalom GVAG255 KC822514 Blackburn et al. 2013
223 Kaloula sp. Philippines, Sibuyan isl., Rombolon Prov. KU328607 KC822511 Blackburn et al. 2013
224 Kaloula sp. Philippines, Sibuyan isl., Rombolon Prov. KU328608 KC822512 Blackburn et al. 2013
225 Kaloula sp. Philippines, Panay isl., Antique Prov. MG012 KC822513 Blackburn et al. 2013
226 Kaloula sp. Philippines, Panay isl., Antique Prov. TNHC56341 KC822516 Blackburn et al. 2013
227 Kaloula sp. Philippines, Panay isl., Antique Prov. TNHC56343 KC822517 Blackburn et al. 2013
228 Kaloula sp. Philippines, Samar isl., Eastern Samar Prov., Taft KU310699 KC822587 Blackburn et al. 2013
229 Kaloula sp. Philippines, Leyte isl., Leyte Prov., Danao KU328632 KC822584 Blackburn et al. 2013
230 Kaloula sp. Philippines, Leyte isl., Leyte Prov., Danao KU328633 KC822585 Blackburn et al. 2013
231 Kaloula sp. Philippines, Leyte isl., Leyte Prov., Baybay KU328645 KC822586 Blackburn et al. 2013
232 Kaloula sp. Philippines, Panay isl., Antique Prov., Sibalom MG0000 KC822611 Blackburn et al. 2013
233 Kaloula sp. Philippines, Panay isl., Antique Prov., Sibalom MG0001 KC822612 Blackburn et al. 2013
234 Kaloula sp. Philippines, Panay isl., Antique Prov., Sibalom MG0002 KC822613 Blackburn et al. 2013
Outgroups
235 Uperodon taprobanicus Bandgladesh, Mymensingh Ktap-Bd AB530545 Hasan et al. 2012
236 Uperodon taprobanicus Sri Lanka KUHE-37252 AB634633 AB634691 Matsui et al. 2011
237 Uperodon taprobanicus - AY948729 Roelants et al. 2007
238 Kalophrynus interlineatus KIZ-HERP-0169 JX678906 Li et al. 2012
Figure 1. 

Geographic origins of the specimens of the Kaloula baleata species complex included in phylogenetic analyses. Dots in the symbols denote type localities. Symbol colors correspond to those used in Fig. 2. Numbers near symbols correspond to the locality numbers given in Table 1. The white rectangle represents the area of inset map detailing the distribution of the K. baleata species complex in Indochina in Fig. 5.

We initially aligned nucleotide sequences using ClustalX 1.81 (Thompson et al. 1994) with default parameters and then optimized them manually in BioEdit 7.0.5.2 (Hall 1999) and MEGA 11.0 (Tamura et al. 2013). We utilized ModelFinder (Kalyaanamoorthy et al. 2017) to determine the most suitable evolutionary models for our data set analysis. According to the Akaike Information Criterion (AIC), the best-fitting models of DNA evolution for 12S rRNA, tRNA-Val, and 16S rRNA genes of mtDNA are GTR+I+G, K2P+G, and GTR+I+G, respectively. We determined mean uncorrected genetic distances (p-distances) between sequences with MEGA 11.0.

We inferred the matrilineal genealogy using Bayesian Inference (BI) and Maximum Likelihood (ML) approaches. We conducted BI using MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003). Metropolis-coupled Markov chain Monte Carlo (MCMCMC) analyses were run with one cold chain and three heated chains for one million generations and sampled every 1,000 generations. We performed two independent MCMCMC runs, and the initial 100 trees were discarded as burn-in. We assessed confidence in tree topology based on the frequency of nodal resolution (posterior probability; BI PP) (Huelsenbeck and Ronquist 2001). We used IQ-TREE (Nguyen et al. 2015) to reconstruct ML-trees. A total of 10,000 ultrafast bootstrap replications for ML analysis (UFB) (Minh et al. 2013) assessed the confidence in tree topology for ML analysis. In both datasets, we regarded tree nodes with BI PP and UFB values over 0.95 to be sufficiently resolved a priori. We considered BI PP and UFB values between 0.95 and 0.90 as tendencies. Lower values were considered to indicate unresolved nodes (Huelsenbeck and Hillis 1993; Minh et al. 2013).

Morphological analysis

Measurements were taken using a digital caliper under a light dissecting microscope to the nearest 0.01 mm, subsequently rounded to 0.1 mm. The adult morphometrics and character terminology follow Poyarkov et al. (2018b): (1) snout-vent length (SVL; measured from the tip of the snout to cloaca); (2) head length (HL; measured from the tip of snout to hind border of jaw angle); (3) snout length (SL; measured from the anterior corner of eye to the tip of snout); (4) eye length (EL; measured as the distance between anterior and posterior corners of the eye); (5) nostril-eye length (N-EL; measured as the distance between the anterior corner of the eye and the nostril center); (6) head width (HW; measured as the maximum width of head on the level of mouth angles in ventral view); (7) internarial distance (IND; measured as the distance between the central points of nostrils); (8) interorbital distance (IOD; measured as the shortest distance between the medial edges of eyeballs in dorsal view); (9) upper eyelid width (UEW; measured as the maximum distance between the medial edge of eyeball and the lateral edge of upper eyelid); (10) tympanum length, measured as the horizontal tympanum diameter (TMP); (11) forelimb length (FLL; measured as the length of straightened forelimb to the tip of third finger); (12) lower arm and hand length (LAL; measured as the distance between elbow and the tip of third finger); (13) hand length (HAL; measured as the distance between the proximal end of outer palmar (metacarpal) tubercle and the tip of third finger); (14) first finger length (1FL; measured as the distance between the tip and the distal end of inner palmar tubercle); (15) inner palmar tubercle length (IPTL; measured as the maximum distance between proximal and distal ends of inner palmar tubercle); (16) outer palmar tubercle length (OPTL; measured as the maximum diameter of outer palmar tubercle); (17) third finger disk diameter (3FDD); (18) hindlimb length (HLL; measured as the length of straightened hindlimb from groin to the tip of fourth toe); (19) tibia length (TL; measured as the distance between the knee and tibiotarsal articulation); (20) foot length (FL; measured as the distance between the distal end of tibia and the tip of fourth toe); (21) first toe length (1TOEL), measured as the distance between the distal end of inner metatarsal tubercle and the tip of first toe; (22) fourth toe disk diameter (4TDD); (23) outer metatarsal tubercle length (OMTL). Webbing and subarticular tubercle formulas follow those of Savage (1975). We took all measurements on the right side of the examined specimen. We determined the sex of specimens by examining the presence of eggs in the abdomen of a dissected specimen.

These characters were measured in specimens from Khammouane Province, Laos, and Dong Nai and Lam Dong provinces, Vietnam, as well as specimens of K. indochinensis from the type locality (Gia Lai Province, central Vietnam). We performed a Principal Component Analysis (PCA) in Statistica v10.0 (StatSoft, Inc. 2011) to examine overall morphological variation among the populations of K. indochinensis sensu stricto and the populations of Kaloula spp. from central Laos and southern Vietnam. We conducted PCA using SVL and size-corrected values for characters 2–23 (their ratios to SVL), following the methodology of Nishikawa et al. (2007). The first two principal components (i.e., those that explained the highest proportion of variance) were extracted for display with an ordination plot.

Furthermore, we compare external morphological characters based on information taken from the literature (e.g., Chan et al. 2013, 2014; Mo et al. 2013).

Bioacoustic analysis

Male advertisement calls of Kaloula sp. were recorded in Cat Tien National Park, near Nam Cat Tien Village, Tan Phu District, Dong Nai Province, Vietnam, on August 4, 2023 (11.410618°N, 107.419334°E; altitude 119 m a.s.l.); the record was taken at an air temperature of 24.0 °C. Male advertisement calls (described as calls hereafter) were recorded with an Apple iPhone X (iPhone 10); the calls were recorded at a distance of approximately 0.1–0.2 m from the calling males; an HTC-2 Digital LCD Thermometer Hygrometer with an outdoor sensor attached was used to take ambient temperatures and humidity at the calling site after the recording. The software Raven Pro 1.6 (http://www.birds.cornell.edu/raven) was used to analyze the recorded calls. Audio spectrograms were calculated with a fast Fourier transform (FFT) of 512 points, 90% overlap, and 135 Hz grid spacing using the Hanning window. The terminology of call analysis and description using a call-centered approach (defining uninterrupted units as calls whenever they are separated by long silent intervals) follows Koehler et al. (2017). We examined oscillograms (waveforms) and audio spectrograms of vocalizations and measured the call duration (in milliseconds—ms), intercall interval duration (ms), call repetition rate (calls/s and calls/min), number of notes per call (notes/call), harmonic frequency (kHz), and dominant frequency (kHz). We qualitatively compared the call of Kaloula sp. from Cat Tien National Park with the call parameters of K. indochinensis sensu stricto from Gia Lai Province, Vietnam, given by Nguyen et al. (2022).

Results

Genealogical mitochondrial relationships

The trees recovered by the BI and ML analyses featured essentially similar topologies, with the only differences being the relationships between the higher K. baleata clades. Our mtDNA-genealogy confirms the monophyly of the genus Kaloula (0.98/96; hereafter node support values are given for BIPP/UFB, respectively) with respect to its sister genus Uperodon Duméril & Bibron, 1841 (Fig. 2). Relationships between different groups within Kaloula, however, were not statistically supported. The East Asian diversification of Kaloula, including K. borealis (Barbour, 1908), K. nonggangensis, K. verrucosa Boulenger, 1904, and K. rugifera Stejneger, 1924 (1.0/100), is suggested as a sister group with respect to all other Kaloula species, but this topology got strong support only in the ML analysis (0.62/98). Kaloula pulchra Gray, 1831, is recovered as a sister species to all remaining species of Kaloula, again without significant nodal support by BI PP (0.62/98). All the remaining Kaloula species are grouped in three major clades, with their respective branching being unresolved (0.73/50): (1) the K. baleata species complex; (2) K. mediolineata Smith, 1917; (3) the Kaloula species found across the Southeast Asian Islands. In the latter, populations from the Philippine Archipelago and adjacent Southeast Asian islands, including K. picta (Duméril & Bibron, 1841), K. kalingensis Taylor, 1922, K. kokacii Ross & Gonzales, 1992, K. conjuncta (Peters, 1863), K. rigida Taylor, 1922, K. walteri Diesmos, Brown & Alcala, 2002, together with unnamed candidate species (noted sp.), are grouped in a clade, again with little support by BI PP (0.63/100). Kaloula rigida is paraphyletic with respect to K. walteri in the 12S-16S tree. The sister species of the K. baleata species complex is given as K. mediolineata, again with little support (0.73/50). The K. baleata species complex is monophyletic (1.0/100) and features two major clades (Fig. 2): (1) One clade is composed of K. latidisca from Peninsular Malaysia and southern Thailand (1.0/100), together with three divergent lineages of K. baleata sensu lato (1.0/100) from the Sunda Islands, Sulawesi, Palawan, and southern Myanmar; (2) the second clade (0.99/99) comprises populations from eastern Indochina, including two unnamed candidate species (labelled Kaloula sp.), one from southern Vietnam (1.0/100) and one from central Laos (1.0/100), the latter being most closely related to K. indochinensis from central Vietnam (1.0/100).

Figure 2. 

Genealogical relationships of the genus Kaloula based on the analysis of mtDNA fragments, including 12S rRNA, tRNAVal, and 16S rRNA gene sequences. Numbers at tree nodes correspond to PP/UFB support values, respectively. Black circles correspond to well-supported (PP ≥ 0.95 or UFB ≥ 90) nodes. Numbers in bold following sample names correspond to localities in Fig. 1 and Table 1. The information on GenBank Accession Numbers, museum vouchers, and localities of origin for sequences used in this study is summarized in Table 1. The holotypes of Kaloula discordia sp. nov. and Kaloula laosensis sp. nov. are marked with asterisks (*H). Photographs by N.A. Poyarkov and N.L. Orlov.

Kaloula baleata sensu lato, which monophyly is robustly supported only in the ML analysis (0.83/98), features three geographically circumscribed mitochondrial lineages (see Fig. 2): (1) K. baleata 1 is distributed in Palawan Island of the Philippines and Sulawesi, Sumba, Bali, central Java, and southern Sumatra Islands of Indonesia; (2) K. baleata 2 is distributed in Sarawak and Sabah in Borneo and eastern Peninsular Malaysia, including the island of Tioman, as well as western Java, in sympatry with K. baleata 1; (3) K. baleata 3 is confirmed from a single locality in Tanintharyi Region, southern Myanmar (see Fig. 1).

Mitochondrial distances

The uncorrected genetic p-distances for the 16S rRNA gene fragment among members of the genus Kaloula are available in Table 2. Interspecific distances among Kaloula species varied from 1.43% (between K. verrucosa and K. nonggangensis) to 14.62% (between K. rugifera and an undescribed species, Kaloula sp. from Panay, Philippines). Intraspecific distances were high in several currently recognized species, including K. kokacii (up to 3.39%), but especially in K. indochinensis from the K. baleata complex, where populations from Khammouane Province, Laos, as well as from Dong Nai and Lam Dong provinces, Vietnam, showed p-distances from the closest taxon above 4.45% and 4.33%, respectively. These 16S distances are much higher than the proposed threshold of 3% for species-level divergence in anurans (Vieites et al. 2009), which was also recently supported to delimit species based on instances of reproductive isolation (Dufresnes et al. 2021). Accordingly, these distances are higher than the p-distances observed between some sister species pairs in the K. baleata complex, namely 3.0% between K. baleata and K. latidisca. We also report a high interspecific variation in K. baleata, namely 2.72% between K. baleata 1 and K. baleata 2.

Table 2.

Uncorrected average interspecific (below and above diagonal) and intraspecific (on the diagonal) genetic p-distances for the 16S rRNA mtDNA gene fragment (in percentage) are given for species of the genus Kaloula.

Species 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
1 Kaloula discordia sp. nov. 0.39 4.45 6.17 5.41 5.40 5.47 6.29 4.33 7.00 6.74 9.57 11.17 8.14 6.25 6.98 7.17 6.08 7.45 7.00 6.65 12.77 9.41 10.61 6.54
2 Kaloula laosensis sp. nov. 4.45 0.22 4.77 6.81 6.59 7.03 7.09 6.28 7.24 8.14 10.74 12.08 7.93 7.65 8.95 8.47 7.47 6.78 7.01 7.89 14.01 10.69 10.64 8.39
3 K. indochinensis 6.17 4.77 0.37 7.24 7.25 6.85 8.20 6.26 7.90 7.45 11.87 12.98 8.37 7.92 8.82 8.70 6.86 6.96 7.29 7.84 15.09 11.44 11.07 7.81
4 K. baleata 1 5.41 6.81 7.24 1.37 2.72 2.08 3.22 4.79 6.64 7.07 9.72 10.85 8.05 6.90 6.71 7.37 6.24 8.19 7.21 6.72 12.46 9.26 11.25 7.57
5 K. baleata 2 5.40 6.59 7.25 2.72 2.56 2.65 3.96 5.36 6.74 7.29 10.26 11.31 7.92 7.35 7.14 7.74 6.76 8.19 7.04 6.82 12.59 9.67 10.59 7.84
6 K. baleata 3 5.47 7.03 6.85 2.08 2.65 0.00 3.00 4.98 6.65 6.44 9.10 10.74 7.57 6.86 6.80 7.06 5.99 7.80 6.51 6.26 13.66 9.00 9.95 7.34
7 K. latidisca 6.29 7.09 8.20 3.22 3.96 3.00 1.45 6.04 7.28 7.47 9.71 11.01 8.89 7.41 7.26 7.74 7.13 8.43 7.82 7.24 14.05 9.41 11.83 7.66
8 K. mediolineata 4.33 6.28 6.26 4.79 5.36 4.98 6.04 0.55 5.89 5.43 8.74 10.49 6.12 5.70 6.95 6.05 5.61 6.81 5.46 5.39 12.30 8.49 10.02 4.97
9 K. borealis 7.00 7.24 7.90 6.64 6.74 6.65 7.28 5.89 0.00 7.43 10.12 11.18 2.07 7.07 7.92 7.46 6.44 2.76 1.93 6.78 13.40 9.91 10.11 7.75
10 K. conjuncta 6.74 8.14 7.45 7.07 7.29 6.44 7.47 5.43 7.43 1.39 8.37 9.78 8.18 5.08 8.59 4.56 3.53 7.70 8.00 3.56 11.65 8.05 7.06 2.57
11 K. kalingensis 9.57 10.74 11.87 9.72 10.26 9.10 9.71 8.74 10.12 8.37 2.07 6.18 10.05 9.39 11.22 8.23 7.55 9.74 9.43 7.52 10.16 4.43 11.20 7.78
12 K. kokacii 11.17 12.08 12.98 10.85 11.31 10.74 11.01 10.49 11.18 9.78 6.18 3.39 11.41 10.64 11.73 9.29 9.26 10.64 10.51 8.25 9.62 4.48 12.17 9.25
13 K. nonggangensis 8.14 7.93 8.37 8.05 7.92 7.57 8.89 6.12 2.07 8.18 10.05 11.41 0.00 7.73 8.60 8.77 8.05 2.76 1.43 8.35 14.14 10.02 11.05 8.14
14 K. picta 6.25 7.65 7.92 6.90 7.35 6.86 7.41 5.70 7.07 5.08 9.39 10.64 7.73 0.31 9.40 3.83 3.15 7.54 8.18 3.51 11.78 8.75 9.18 4.56
15 K. pulchra 6.98 8.95 8.82 6.71 7.14 6.80 7.26 6.95 7.92 8.59 11.22 11.73 8.60 9.40 0.68 8.88 8.98 8.56 7.93 9.27 14.15 10.63 12.93 8.41
16 K. rigida 7.17 8.47 8.70 7.37 7.74 7.06 7.74 6.05 7.46 4.56 8.23 9.29 8.77 3.83 8.88 1.90 2.73 8.56 7.99 2.45 9.90 7.82 7.74 3.58
17 K. cf. rigida 6.08 7.47 6.86 6.24 6.76 5.99 7.13 5.61 6.44 3.53 7.55 9.26 8.05 3.15 8.98 2.73 n/c 7.59 7.45 1.92 10.72 7.78 6.82 3.88
18 K. rugifera 7.45 6.78 6.96 8.19 8.19 7.80 8.43 6.81 2.76 7.70 9.74 10.64 2.76 7.54 8.56 8.56 7.59 0.00 1.98 8.12 14.62 9.33 10.70 7.98
19 K. verrucosa 7.00 7.01 7.29 7.21 7.04 6.51 7.82 5.46 1.93 8.00 9.43 10.51 1.43 8.18 7.93 7.99 7.45 1.98 0.27 7.48 13.70 9.19 10.44 7.68
20 K. walteri 6.65 7.89 7.84 6.72 6.82 6.26 7.24 5.39 6.78 3.56 7.52 8.25 8.35 3.51 9.27 2.45 1.92 8.12 7.48 0.30 9.38 6.72 6.61 3.19
21 Kaloula sp. Panay 12.77 14.01 15.09 12.46 12.59 13.66 14.05 12.30 13.40 11.65 10.16 9.62 14.14 11.78 14.15 9.90 10.72 14.62 13.70 9.38 0.00 8.51 12.80 10.47
22 Kaloula sp. East Luzon 9.41 10.69 11.44 9.26 9.67 9.00 9.41 8.49 9.91 8.05 4.43 4.48 10.02 8.75 10.63 7.82 7.78 9.33 9.19 6.72 8.51 1.85 10.44 7.51
23 Kaloula sp. Samar & Leyte 10.61 10.64 11.07 11.25 10.59 9.95 11.83 10.02 10.11 7.06 11.20 12.17 11.05 9.18 12.93 7.74 6.82 10.70 10.44 6.61 12.80 10.44 1.56 7.78
24 Kaloula sp. Sibuyan 6.54 8.39 7.81 7.57 7.84 7.34 7.66 4.97 7.75 2.57 7.78 9.25 8.14 4.56 8.41 3.58 3.88 7.98 7.68 3.19 10.47 7.51 7.78 1.66

Morphology

Given the limited number of morphometric characters provided in the original description of K. indochinensis in Chan et al. (2013), we did not incorporate their data in our analysis, which further avoids measurer bias. Consequently, the morphological data on K. indochinensis relies only on our specimens from near the type locality of this species in Kon Ka Kinh National Park in Gia Lai Province, central Vietnam, as well as in Yok Don National Park in Dak Lak Province, central Vietnam. These specimens were confirmed as K. indochinensis by the genetic analysis (Fig. 2). The first two principal components (PCs) of the PCA explain 48.72% (PC1: 28.68%, PC2: 20.04%) of the variation among specimens of K. indochinensis, Kaloula sp. from central Laos, and Kaloula sp. from southern Vietnam (Fig. 3). The specimens of each population/candidate species form distinct groups in the morphospace, without overlapping, and also with respect to sex (Fig. 3).

Figure 3. 

Scatterplot of first two PCs in the principal component analysis (PCA) on morphological data from Kaloula indochinensis, Kaloula discordia sp. nov. from southern Vietnam, and Kaloula laosensis sp. nov. from Khammouane, central Laos, overlayed by convex hull polygons; filled circles indicate male specimens, and empty circles indicate female specimens. The morphological data used in the analysis is presented in Table 3.

Bioacoustics

The advertisement call of Kaloula sp. from southern Vietnam is documented based on the recordings of one calling male individual. Males of Kalophrynus interlineatus (Blyth, 1855), Occidozyga martensii (Peters, 1867), and Microhyla mukhlesuri Hasan, Islam, Kuramoto, Kurabayashi & Sumida, 2014, were also calling at the same habitat. The calls of Kaloula sp. are slow and low-pitched, resonant, booming sounds that resemble the sound of a bicycle horn to the human ear. Call parameters are shown in Table 3, and the sonograms and waveforms of the call are presented in Fig. 4. The call consists of a single note with 11–13 pulses with an average duration of 190.8 ms (144.8–260.6 ms, N = 23). Calls were repeated at a rate of one call per second, 56.5 (42–71) calls per minute, and had an average intercall interval of 1,222.9 ms (327.1–8,954.4 ms, N = 23). The fundamental frequency was not evident, and the average dominant frequency was 0.30–0.45 kHz. Three harmonics were detected at 1.0, 1.2, and 1.5 kHz (Table 3, Fig. 4). The call of the Laos population of Kaloula sp. was not recorded.

Table 3.

Comparison of male advertisement calls for Kaloula discordia sp. nov. and other members of the K. baleata species complex. NR: not recorded.

Species Kaloula discordia sp. nov. Kaloula indochinensis Kaloula baleata Kaloula ghoshi
Locality Cat Tien NP, Tan Phu District, Dong Nai Province, Vietnam Kon Von II village, Dak Roong Commune, K’Bang District, Gia Lai Province, Vietnam Kinabalu, Sabah, Malaysia Hut Bay, Little Andaman Island
Coordinates 11.411°N, 107.419°E 14.543°N, 108.414°E NR NR
Number of calls measured 23 40 NR NR
Call duration (ms) 186.1 (144–214) 215.6 (194–250) 280–300 400 (320–640)
Intercall interval (ms) 1539.4 (851–8954) 789.3 (481–1627) NR 300 (240–530)
Call repetition rate (call/s) 1.0 (0.75–1.21) 1.0 (0.76–1.25) NR NR
Call repetition rate (call/minute) 56.5 (42–71) 60.1 (45.9–75.2) NR NR
Notes/call 1 1 NR NR
Pulse/call 11–13 1* Described as pulsed 7–25
Dominant frequency (kHz) 0.38 (0.30–0.45) 0.38 (0.34–0.43) 1–3 1
2nd harmonic (kHz) 1.0 0.8 NR NR
3nd harmonic (kHz) 1.2 1.2 NR NR
4nd harmonic (kHz) 1.5 1.37 NR NR
Temperature (°C) 24.0 20.5 23.0 29.1
Source this study Nguyen et al. (2022) Malkmus et al. (2002) Chandramouli and Prasad (2018)
Figure 4. 

The call of Kaloula discordia sp. nov. from Cat Tien NP, Dong Nai Province, Vietnam. A. 33 s waveform of relative amplitude (Rel. amp.) and corresponding spectrogram over time; B. 1.5 s waveform and corresponding spectrogram of two calls, obtained from the last two calls in (A). Data on acoustic characters is summarized in Table 4.

Systematics

The two populations of Kaloula spp. from Laos and southern Vietnam belong to the K. baleata species complex and are characterized by divergent mitochondrial lineages and distinctive morphologies. Their amount of 16S divergence (Table 2), together with the substantial morphological differentiation between them and the closely related species K. indochinensis (Fig. 3), suggests a species-level split. Based on our mitochondrial genealogy, the species from Khammouane Province of Laos is the sister species of K. indochinensis, while the species from southern Vietnam is the sister species of a clade including K. indochinensis and the Laos species (Fig. 2). In addition to diagnostic morphological differences (see below), the three Indochinese species of the K. baleata species complex also feature stable differences in coloration, as well as differences in bioacoustic characteristics for the studied Vietnamese species. The distribution of all members of the K. baleata species complex in Indochina, following our results, is detailed in Fig. 5. Given the congruence of morphological, bioacoustic, and molecular data in suggesting a deep divergence of the Laos and southern Vietnam Kaloula sp. populations from all currently recognized species in the genus, we herein describe them as two new species of Kaloula.

Figure 5. 

Known distribution of the Kaloula baleata species complex members in Indochina. Dots within symbols denote type localities. Symbol colors correspond to mitochondrial DNA lineages (Figs 1, 2. Note that the tentative assignment of the populations that were not sequenced is based on geographic proximity and/or examination of morphological or photographic data from these populations). Symbol numbers correspond to locality numbers given in Appendix 1.

Kaloula discordia Poyarkov, Gorin, Bragin & Nguyen, sp. nov.

Figs 2, 4, 6, 7, 8, 9C, Tables 3, 4

Chresonymy

Kaloula baleata [partim]—Orlov et al. (2002: 99); Nguyen et al. (2005: 43); Orlov and Ananjeva (2007: 148); Nguyen et al. (2009: 94).

Kaloula indochinensis [partim]—Chan et al. (2013: 334, 2014: 577); Chandramouli and Prasad (2018: 52); Poyarkov et al. (2021b: 39); Vassilieva (2021: 72–73); Holden (2023: 149).

Holotype

ZMMU A-8134 (field number NAP-14688), adult male, from a pond in Cat Tien National Park, along the road from park headquarters to the Bau Sau Lake, Dong Nai River valley, Dac Lua Commune, Tan Phu District, Dong Nai Province, Vietnam, collected by N.A. Poyarkov, A.M. Bragin, and V.A. Gorin on 15 June, 2024 (11.44121°N, 107.41312°E; elevation 137 m a.s.l.) (Figs 6, 7C).

Figure 6. 

Holotype of Kaloula discordia sp. nov. (ZMMU A-8134), adult male. A. Dorsal view; B. Ventral view; C. Head in a lateral view; D. Plantar view of left foot; E. Volar view of left hand. Photographs by N.A. Poyarkov.

Figure 7. 

Breeding habitats of Kaloula discordia sp. nov. (A, B) and lateral view of holotype of Kaloula discordia sp. nov. in situ (ZMMU A-8134) (C). Photographs by A.M. Bragin.

Paratypes

(n = 14). ZMMU A-8135–A-8140 (field numbers NAP-14689–14694), six adult males with collection data same as the holotype; ZMMU A-8141–A-8143 (field numbers NAP-14688–14694), three adult males with collection data same as the holotype; ZISP 15285–15287 (field numbers NAP-14687, NAP-14695–14696), three adult males with collection data same as the holotype; ZMMU A-4739 (field number NAP-01674), adult male from Cat Tien National Park, Dong Nai River valley, Dac Lua Commune, Tan Phu District, Dong Nai Province, Vietnam, collected by N.A. Poyarkov on June 1, 2011 (11.44576°N, 107.38673°E; elevation 128 m a.s.l.); ZMMU A-4642 (field number NAP-00630), adult female from Cat Tien National Park, Dong Nai River valley, Dac Lua Commune, Tan Phu District, Dong Nai Province, Vietnam, collected by N.A. Poyarkov and A.B. Vassilieva on June 2, 2009 (11.44576°N, 107.38673°E; elevation 128 m a.s.l.) (Fig. 8).

Figure 8. 

Variation in dorsal coloration in paratypes of Kaloula discordia sp. nov. Scale bar equals 5 cm. Photographs by N.A. Poyarkov.

Referred materials

(n = 3). ZMMU-A-4602-1–3 (field numbers NAP-02010, NAP-02086, and NAP-02122), adult female and two subadult females from Cat Tien National Park, Dong Nai River valley, Dac Lua Commune, Tan Phu District, Dong Nai Province, Vietnam, collected by E.A. Galoyan on August 22, 2011, September 15, 2011, and November 11, 2011 (11.419503°N, 107.426442°E; elevation 117 m a.s.l.).

Diagnosis

Kaloula discordia sp. nov. is distinguished from its congeners by the following combination of morphological characters: (1) medium body size (SVL 42.9–56.2 mm); (2) eyes comparatively small (eye length comprising 60%–75% of snout length); (3) dorsally uniform dark olive-brown; (4) beige-gray ventrally with irregular white mottling on belly and limbs; (5) pale yellow or orange-brown 8-shaped patch on either side of the neck posterior to eyes; (6) yellowish to orange axillary patch present; (7) grayish to beige-yellow inguinal patch present edged with black markings; (8) from gray to beige spot on tibiotarsal articulation present; (9) dark interorbital bar absent; (10) enlarged, widened finger disks (3FDD 6.5%–7.5% of SVL), ca. 1.27 times wider than toe disks; (11) finger subarticular tubercle formula: 1:1:2:2; (12) toe subarticular tubercle formula: 1:1:2:2:2; (13) two metacarpal tubercles not in touch with each other; (14) two metatarsal tubercles, outer metatarsal tubercle rounded, smaller than elongated inner metatarsal tubercle.

Description of the holotype

(Fig. 6). Adult male in a good state of preservation, habitus robust, head wider than long (HW/HL 1.27), snout projecting beyond lower jaw, gently rounded in lateral view (Fig. 6C); truncated in dorsal view; top of head flat; upper eyelid lacking supraciliary tubercles; eye length less than snout length (EL/SL 0.74) and less than interorbital distance (EL/IOD 0.76); pupils round; nostrils rounded, placed more towards the lateral sides of the snout, located closer to tip of snout than to eye, relatively close to each other (IND/IOD 0.60); supratympanic fold flat, glandular, rather thin; tympanum not visible (Fig. 6C); dorsal surfaces of body and limbs with sparse tubercules, getting denser backwards; ventral surfaces of body and limbs with flat tubercules (Fig. 6A, B). Cloacal opening unmodified, directed posteriorly. Forelimbs relatively long, more than a half of hind limb length (FLL/HLL 0.53); hand long, comprising more than a half of lower arm length (HAL/LAL 0.60) and almost half of forelimb length (HAL/FLL 0.49); fingers rather robust, notably flattened in cross section; relative finger lengths: I<II<IV<III; fingers free of webbing; terminal digits flattened forming wide transversely expanded disks (Fig. 6E); finger subarticular tubercles distinct, large and round, finger subarticular tubercle formula 1:1:2:2; inner metacarpal tubercle elongate; outer metacarpal tubercule triangular-shaped, dilated, bigger than inner metacarpal tubercle (OPTL/IPTL1.18), two metacarpal tubercles not in touch with each other (Figs 6E, 9C); hindlimbs robust, relatively short, not much longer than body length (HLL/SVL 1.16); relative toe lengths: I<II<V<III<IV; tarsal fold on inner surface of tarsus absent; tips of all toes widened, forming rounded terminal disks; all toe disks having dorso-terminal groves; the disk on toe IV the largest (Figs 6D, 9C); toe webbing well developed between all toes, reaching disks at all toes except toe IV; webbing formula i1-2ii1.5-2.5iii1.5-3iv3-1.5v; toe subarticular tubercules distinct, rounded; toe subarticular formula 1:1:2:2:2; two metatarsal tubercles, inner metatarsal tubercle elongated, oval; outer metatarsal tubercle smaller, rounded (Figs 6D, 9C).

Figure 9. 

Morphological details of feet and hands in the three species of the Kaloula baleata species complex of eastern Indochina. A. Volar view of the left hand and plantar view of the right foot of Kaloula laosensis sp. nov.; B. Volar view of the left hand and plantar view of the right foot of K. indochinensis; C. Volar view of the left hand and plantar view of the right foot of Kaloula discordia sp. nov. Scale bar equals 5 mm. Drawings by N.A. Poyarkov.

Coloration

In life, the dorsal surfaces of the head and body olive-brown, dorsal surfaces of the limbs grayish-olive with grayish-white spots (Fig. 6A); flanks of the body and lateral sides of the head grayish olive; orange-brown 8-shaped patches on the neck posterior to eyes; bright-orange axillary patch continuing on elbows (Fig. 7C); grayish-white inguinal patch; small grayish-beige spots near tibiotarsal articulation; ventral surfaces of head and body gray, darker on the throat near the jaw; belly and ventral surfaces of limbs pinkish with weak white mottling (Fig. 6B). Iris golden with brown reticulations ventrally and dorsally from the pupil (Figs 6C, 7C). In preservation after one year of storage in ethanol, dorsal coloration faded to dark gray, light patches became less pronounced, and they faded to light gray or grayish-beige, though the pattern generally remained unchanged.

Measurements of the holotype

(in mm): SVL 51.5; HL 11.7; HW 14.8; SL 5.5; EL 4.1; N-EL 3.9; IND 3.2; IOD 5.3; UEW 3.0; FLL 31.9; LAL 26.4; HAL 15.7; HLL 60.0; TL 45.0; FL 30.2; IPTL 3.0; OPTL 3.5; 1FL 8.1; 1TOEL 7.9; OMTL 2.1; 3FDD 3.6; 4TDD 2.3.

Variation

Table 4 presents the morphometric variation of the type series. Fig. 8 displays the variation in dorsal coloration of the paratypes. In general, all paratypes agree well with the description of the holotype, differing only in the brightness of light inguinal and axillary markings. In male paratypes ZISP 15285 and ZMMU A-8138, the right foot is poorly developed; in male paratype ZMMU A-8139, the right hand is poorly developed.

Table 4.

Measurements (in mm) of specimens of Kaloula discordia sp. nov., Kaloula laosensis sp. nov., and K. indochinensis. For character abbreviations, see the Materials and methods section. Abbreviations: F – adult female; M – adult male; SF – subadult female; SM – subadult male.

Species Sample ID Sex Status SVL HL SL EL N-EL HW IND IOD UEW FLL LAL HAL 1FL IPTL OPTL 3FDD HLL TL FL OMTL 1TOEL 4TDD
Kaloula discordia sp. nov. ZMMU-A-8134 M holotype 51.5 11.7 5.5 4.1 3.9 14.8 3.2 5.3 3.0 31.9 26.4 15.7 8.1 3.0 3.5 3.6 60.0 14.7 30.2 2.1 7.9 2.3
K. discordia sp. nov. ZMMU-A-4739 M paratype 52.0 11.9 5.2 4.0 3.9 15.2 3.2 5.3 2.9 33.4 26.8 15.8 8.2 3.0 3.4 3.8 62.7 15.3 31.4 2.5 7.7 2.9
K. discordia sp. nov. ZISP 15285 M paratype 48.0 10.8 5.1 3.1 3.7 13.7 3.1 5.0 2.8 31.2 24.1 15.0 7.8 2.9 3.3 3.2 58.0 13.4 28.9 2.5 7.9 2.6
K. discordia sp. nov. ZMMU-A-8135 M paratype 49.6 11.3 5.3 3.4 3.8 14.0 3.2 4.9 2.7 31.5 25.3 15.3 7.9 2.6 3.3 3.5 59.0 14.1 29.7 2.4 7.6 2.8
K. discordia sp. nov. ZMMU-A-8136 M paratype 45.4 10.6 4.9 3.3 3.4 13.8 3.0 4.8 2.7 31.4 24.2 14.6 7.8 2.9 2.8 3.3 57.4 14.1 27.9 2.2 7.2 2.4
K. discordia sp. nov. ZMMU-A-8137 M paratype 42.9 10.3 4.9 3.0 3.0 13.7 3.0 4.5 2.6 30.8 24.0 14.9 7.8 2.3 3.1 3.0 56.3 13.1 28.5 2.0 7.1 2.4
K. discordia sp. nov. ZMMU-A-8138 M paratype 48.7 11.1 5.3 3.7 3.5 14.7 3.2 4.9 2.9 31.1 25.0 15.4 7.9 2.4 3.0 3.5 60.6 14.4 30.5 2.2 7.4 2.3
K. discordia sp. nov. ZMMU-A-8139 M paratype 49.5 11.2 5.1 3.1 3.5 15.0 3.1 4.7 3.0 31.7 24.9 14.2 7.4 2.6 3.1 3.3 60.5 13.8 31.0 1.9 7.5 2.7
K. discordia sp. nov. ZMMU-A-8140 M paratype 47.2 10.5 5.0 3.4 3.3 15.1 3.0 4.9 2.7 27.9 23.0 14.1 6.8 2.7 3.3 3.5 57.6 14.7 27.4 1.9 6.9 2.8
K. discordia sp. nov. ZISP 15286 M paratype 52.6 12.2 5.8 3.8 3.8 15.7 3.2 5.6 2.8 32.9 26.3 15.6 7.9 3.2 3.6 4.0 61.9 15.1 31.1 2.3 8.2 3.4
K. discordia sp. nov. ZISP 15287 M paratype 49.1 10.6 5.3 3.6 3.5 14.5 3.1 5.0 2.7 31.2 24.6 15.4 8.1 2.8 3.1 3.4 60.6 14.3 29.7 2.6 8.2 2.6
K. discordia sp. nov. ZMMU-A-8141 M paratype 50.0 11.3 5.5 3.5 3.7 15.3 3.2 5.0 3.0 31.8 25.2 15.2 7.5 2.6 3.0 3.3 60.5 14.3 31.0 1.9 7.4 2.6
K. discordia sp. nov. ZMMU-A-8142 M paratype 52.2 11.7 5.1 3.6 3.2 15.3 3.2 5.0 2.8 31.9 25.1 15.6 7.9 2.5 3.0 3.6 61.0 14.1 30.8 1.9 7.2 2.7
K. discordia sp. nov. ZMMU-A-8143 M paratype 51.5 11.7 5.5 3.4 3.6 15.0 3.2 5.1 2.7 32.1 25.4 15.2 7.9 2.9 3.1 3.7 58.5 13.4 29.9 2.4 7.5 2.6
K. discordia sp. nov. ZMMU-A-4642 F paratype 56.2 12.8 6.2 4.1 4.2 17.0 3.8 5.9 3.0 35.9 28.2 16.3 9.3 3.2 3.7 4.0 67.4 16.7 33.3 3.0 9.0 3.0
K. discordia sp. nov. ZMMU-A-4602-1 F 56.4 13.0 5.4 4.2 4.0 16.9 3.7 6.0 3.0 35.8 28.6 16.6 10.0 3.4 3.4 3.8 67.6 17.2 33.7 3.0 9.1 2.9
K. discordia sp. nov. ZMMU-A-4602-2 SF 25.3 7.0 3.1 2.5 2.8 7.5 1.9 3.2 1.5 15.5 11.9 7.0 3.8 2.0 1.8 1.7 30.2 7.4 13.9 - 3.5 1.5
K. discordia sp. nov. ZMMU-A-4602-3 SF 35.0 8.3 3.8 3.0 2.8 10.2 2.6 3.6 1.9 23.2 14.5 10.3 5.9 2.2 2.2 2.6 40.2 9.6 20.4 1.4 4.5 2.0
Kaloula laosensis sp. nov. ZISP 15284 M holotype 44.0 10.6 4.6 3.3 3.2 12.6 2.7 4.3 2.4 27.6 21.7 13.0 7.5 2.7 2.8 3.4 49.3 11.6 25.4 2.0 7.3 2.2
K. laosensis sp. nov. ZMMU-A-8144 F paratype 54.8 12.4 5.3 5.0 5.2 16.5 3.6 5.7 3.0 33.8 28.3 17.8 10.4 3.2 3.7 4.3 66.9 16.4 32.5 2.8 8.4 2.8
Kaloula indochinensis ZMMU-A-8147 M 45.3 11.0 4.9 3.9 3.3 14.7 3.1 5.0 2.8 31.5 23.7 13.9 8.1 3.1 3.4 3.8 55.2 14.0 27.2 2.19 7.2 2.3
K. indochinensis ZMMU-A-8148 M 50.9 11.6 5.3 3.4 3.8 15.1 3.0 4.5 2.5 32.5 24.8 14.6 7.9 2.9 3.1 3.3 57.3 14.5 27.5 2.72 8.1 2.4
K. indochinensis ZMMU-A-8149 M 42.3 9.8 4.8 3.4 3.3 12.0 2.9 4.5 2.4 28.1 20.4 12.6 7.8 2.4 2.6 2.9 47.9 10.9 24.9 2.15 6.2 2.2
K. indochinensis ZMMU-A-8150 M 46.3 10.7 4.9 3.5 3.5 13.0 3.1 4.5 2.6 29.9 22.4 13.9 7.9 2.6 3.0 3.4 50.7 12.2 26.2 2.59 7.0 2.4
K. indochinensis ZMMU-A-8151 M 48.5 11.2 5.2 3.9 3.5 15.7 3.1 4.7 2.7 31.1 24.0 14.6 8.2 3.2 3.3 3.7 55.1 12.9 27.6 2.44 7.4 2.7
K. indochinensis ZMMU-A-8152 M 49.1 11.9 5.3 4.0 3.8 15.0 3.3 5.1 2.8 30.6 24.4 14.9 8.7 3.1 3.2 3.6 55.9 12.7 27.5 2.35 7.4 2.3
K. indochinensis ZMMU-A-8153 M 48.8 11.8 4.9 4.0 3.6 15.3 3.0 5.0 2.7 31.3 24.3 14.5 7.8 2.9 3.3 3.6 53.0 12.7 26.2 2.80 6.6 2.5
K. indochinensis ZMMU-A-8154 M 49.8 11.9 5.5 3.9 3.5 16.3 3.4 5.0 2.7 32.0 24.3 15.1 8.4 2.7 3.1 3.8 54.5 12.9 27.5 2.62 7.4 2.5
K. indochinensis ZMMU-A-6316 M topotype 50.4 11.7 5.2 4.0 3.7 14.9 3.2 4.9 2.9 28.1 22.8 14.5 8.6 3.2 3.3 3.1 54.7 12.4 27.0 2.75 6.8 2.3
K. indochinensis ZMMU-A-6317 M topotype 47.3 10.8 5.1 3.8 3.3 14.1 3.0 4.9 2.9 26.7 22.0 13.9 8.0 2.7 2.9 2.8 52.2 12.2 25.7 2.89 5.9 1.8
K. indochinensis ZMMU-A-6318 M topotype 51.1 11.6 5.4 3.9 3.5 16.2 3.3 5.2 2.7 31.7 25.2 15.5 8.9 3.4 3.4 3.5 55.8 13.0 27.5 2.84 7.1 2.2
K. indochinensis ZMMU-A-6319 M topotype 59.6 12.7 5.8 4.1 4.0 17.7 3.4 5.8 3.1 34.3 28.6 18.1 10.4 3.9 4.4 4.2 63.9 15.0 31.6 2.79 8.5 2.8
K. indochinensis ZMMU-A-6320 M topotype 51.3 12.3 5.3 4.0 3.8 15.9 3.4 5.3 2.9 30.5 24.9 15.7 9.2 3.3 3.2 3.4 55.4 13.1 27.3 2.79 7.7 2.2
K. indochinensis ZMMU-A-6321 M topotype 50.0 12.3 5.3 4.0 3.7 16.1 3.4 5.4 3.2 28.4 24.0 14.7 8.8 2.9 3.4 3.4 55.7 13.4 27.5 3.22 7.0 2.1
K. indochinensis ZMMU-A-6322 M topotype 50.5 12.4 6.0 4.2 4.2 16.5 3.6 5.7 3.0 30.4 24.9 15.7 9.4 3.5 3.5 3.9 57.8 13.9 28.8 2.88 7.8 2.4
K. indochinensis ZMMU-A-6323 M topotype 51.7 11.4 5.4 4.0 3.7 15.0 3.6 5.9 3.3 31.3 25.3 15.8 9.2 3.6 3.9 3.2 58.0 14.1 28.0 2.85 7.4 2.4
K. indochinensis ZMMU-A-6324 M topotype 50.2 12.4 5.5 4.0 3.5 15.7 2.9 5.0 3.1 29.1 23.6 14.6 8.2 3.3 3.4 3.2 55.7 13.6 26.5 2.97 7.0 2.2
K. indochinensis ZMMU-A-6325 M topotype 48.2 10.7 5.1 3.9 3.7 14.3 3.3 5.1 3.0 28.8 23.1 14.4 7.8 2.7 2.9 3.4 53.9 12.4 26.3 2.65 6.7 2.1
K. indochinensis ZMMU-A-6315 F topotype 55.2 13.1 6.2 4.3 4.4 17.3 3.4 5.8 3.1 32.9 27.9 18.0 9.8 3.2 3.8 4.0 62.7 14.8 31.9 3.08 8.4 2.5
K. indochinensis ZMMU-A-8155 SF 36.1 8.7 4.4 3.4 2.9 11.6 2.6 3.9 2.1 24.8 18.8 11.7 5.9 2.2 2.4 3.0 41.6 10.4 21.3 1.69 5.7 2.1

Tadpole morphology

Vassilieva (2021) provided morphometric data and a detailed description of tadpoles of Kaloula discordia sp. nov. (as K. indochinensis). Vassilieva (2021) noted that tadpoles of ‘K. indochinensis’ from Cat Tien NP, Dong Nai Province, southern Vietnam (corresponding to Kaloula discordia sp. nov.) differ from K. indochinensis sensu stricto from Kon Ka Kinh NP, Gia Lai Province, and Chu Mon Ray NP, Kon Tum Province, central Vietnam, by having comparatively longer spiracle tubes (markedly longer than the vent tubes), a narrower mouth relative to body width, more developed tail musculature (tail base width more than a third of the body width), and a rather contrasting tail coloration.

Distribution and natural history

Kaloula discordia sp. nov. is currently reliably known from Dong Nai, Lam Dong, Tay Ninh, and Binh Phuoc provinces of Southern Vietnam and was also reported from the adjacent Mondulkiri Province of eastern Cambodia; the known distribution of the new species is shown in Fig. 5, and the locality information is detailed in Appendix 1. The single Cambodian record of the new species from Phnom Prich Wildlife Sanctuary in Mondulkiri Province, originally reported as K. indochinensis by Chan et al. (2013) and Holden (2023), can be confidently assigned to Kaloula discordia sp. nov. based on the external morphology and coloration of the recorded specimen and the geographic proximity of the locality to the population of Kaloula discordia sp. nov. in Binh Phuoc Province of southern Vietnam. The new species was recorded mostly from relatively low elevations of ca. 70–300 m a.s.l.; in Bao Loc forestry of Lam Dong Province, the new species was recorded at elevations up to 550 m a.s.l. Kaloula discordia sp. nov. is restricted to lowland and hilly, seasonally dry, semi-deciduous, and evergreen monsoon forests of southern Vietnam (see Vassilieva et al. 2016). Male frogs were recorded calling from small temporary pools and flooded areas from May to November (Fig. 7A, B); the peak of the breeding season coincides with heavy rains in June–August. The new species is semi-fossorial and quite elusive and is usually active only after periods of heavy rains; frogs are aestivating during dry periods in leaf litter, underground borrows, or tree hollows (Vassilieva et al. 2016; Holden 2023). Diet consists of ants and beetles (Vassilieva et al. 2016); the new species is a good climber; males can call when perching a few meters above ground level (Holden 2023). The actual distribution of K. discordia sp. nov. is still insufficiently known, but we suppose that this species is likely to occur in those provinces of southern Vietnam where forests similar in composition are still preserved, including Dak Nong, Binh Duong, Binh Thuan, and Ba Ria-Vung Tau provinces. Syntopic species of amphibians included Microhyla butleri Boulenger, 1900; M. heymonsi Vogt, 1911; M. mukhlesuri; Kaloula pulchra; Fejervarya limnocharis (Gravenhorst, 1829); Occidozyga martensii; Polypedates megacephalus Hallowell, 1861; and Rhacophorus annamensis Smith, 1924.

Comparisons

Kaloula discordia sp. nov. most closely resembles other frogs of the K. baleata species complex in overall morphology; most specifically, it is similar to K. indochinensis, with which it was previously confused. From K. indochinensis, the new species can be distinguished by having generally smaller eye size in males (EL mean 3.5 ± 0.3 mm [n = 14] vs. mean 3.9 ± 0.2 mm [n = 18]; EL/SVL 0.071 ± 0.004 vs. 0.079 ± 0.004); smaller finger I in males (1FL mean 7.8 ± 0.4 mm [n = 14] vs. mean 8.5 ± 0.7 mm [n = 18]; 1FL/SVL 0.16 ± 0.01 vs. 0.17 ± 0.007); generally smaller inner palmar tubercules in males (IPTL mean 2.8 ± 0.3 mm [n = 14] vs. mean 3.1 ± 0.4 mm [n = 18]; IPTL/SVL 0.056 ± 0.004 vs. 0.062 ± 0.005); and generally smaller outer palmar tubercules in males (OPTL mean 3.2 ± 0.2 mm [n = 14] mm vs. mean 3.3 ± 0.4 mm [n = 18]; OPTL/SVL 0.064 ± 0.004 vs. 0.067 ± 0.005); generally longer hindlimbs in both sexes (HLL mean 59.6 ± 1.9 mm [n = 14] in males, mean 67.5 ± 0.2 mm [n = 2] in females vs. mean 55.2 ± 3.3 mm [n = 18] in males; 62.7 mm [n = 1] in female; HLL/SVL 1.21 ± 0.04 vs. 1.12 ± 0.03 in both sexes); smaller metatarsal tubercule in males (OMTL mean 2.2 ± 0.3 mm [n = 14] vs. mean 2.7 ± 0.3 mm [n = 18]); by having two metacarpal tubercles, see Fig. 9C (vs. three metacarpal tubercles, see Fig. 9B); by having two subarticular tubercles on toe IV, see Fig. 9C (vs. three well-developed subarticular tubercles, see Fig. 9B); and by having olive dorsal coloration (vs. chocolate-brown to dark grayish-brown).

From K. baleata (morphological data taken from Chan et al. 2013), Kaloula discordia sp. nov. can be distinguished by having generally smaller eyes in both sexes (EL mean 3.5 ± 0.3 mm [n = 14] in males, mean 4.2 ± 0.01 mm [n = 2] in females vs. mean 4.1 ± 0.4 mm [n = 10] in males, mean 4.5 ± 1.7 mm [n = 2] in females); larger distance between nares in both sexes (IND mean 3.1 ± 0.1 mm [n = 14] in males, mean 3.7 ± 0.1 mm [n = 2] in females vs. mean 2.7 ± 0.4 mm [n = 10] in males, mean 3.1 ± 0.30 mm [n = 2] in females); wider finger III disks in both sexes (3FDD mean 3.5 ± 0.3 mm [n = 14] in males, mean 3.9 ± 0.1 mm [n = 2] in females vs. mean 1.7 ± 0.2 mm [n = 10] in males, mean 2.1 ± 0.6 mm [n = 2] in females); smaller metatarsal tubercule in males (OMTL mean 2.2 ± 0.3 mm [n = 14] vs. mean 3.2 ± 0.2 mm [n = 10]); and by having two subarticular tubercles on toe IV, see Fig. 9C (vs. three well-developed subarticular tubercles).

From K. latidisca (morphological data taken from Chan et al. 2014), the new species can be distinguished by having shorter head in males (HL mean 11.2 ± 0.6 mm [n = 14] vs. mean 14.4 ± 0.8 mm [n = 4]); narrower head in males (HW mean 14.7 ± 0.7 mm (n = 14) vs. mean 18.7 ± 1.1 mm [n = 4]); smaller distance between nares in males (IND mean 3.1 ± 0.1 mm [n = 14] vs. mean 4.0 ± 0.1 mm [n = 4]); smaller distance between eyes in males (IOD mean 5.0 ± 0.2 mm [n = 14] vs. mean 5.7 ± 0.4 mm [n = 4]); smaller eye in males (EL mean 3.5 ± 0.3 mm [n = 14] vs. mean 4.6 ± 0.3 mm [n = 4]); smaller metatarsal tubercle in males (OMTL mean 2.2 ± 0.3 mm [=14] vs. mean 3.4 ± 0.1 mm [n = 4]); and by having two subarticular tubercles on toe IV, see Fig. 9C (vs. three subarticular tubercles).

Furthermore, Kaloula discordia sp. nov. differs from K. aureata Nutphand, 1989, by having an olive dorsum with no dark reticulations (vs. golden dorsum with dark brown reticulations); from K. borealis by having wider finger disks (vs. finger tips slightly dilated but not forming wide disks); by yellowish blotches on flanks absent (vs. present); and by olive dorsal coloration (vs. gray-brown). The new species is further diagnosed from K. conjuncta by the stratified coloration on flanks absent (vs. present) and by having distinct outer metatarsal tubercle (vs. weak or indistinct). Kaloula discordia sp. nov. differs from K. ghoshi by having axillary and inguinal light spots (vs. absent); tubercles on dorsum and venter (vs. smooth skin or with small granules dorsally); and olive dorsal coloration (vs. orange-brown). The new species further differs from K. kalingensis by having tubercles on the dorsum (vs. dorsum smooth); by having a distinct outer metatarsal tubercle (vs. indistinct); yellow or orange axillary and inguinal spots (vs. usually absent or small and red if present); lacking light pericloacal ring (vs. present); from K. kokacii by tuberculated dorsum (vs. smooth); axillary and inguinal spots (vs. absent); from K. mediolineata by lacking dorsolateral stripes (vs. present); sacral medial stripe absent (vs. present); and finger disks widened (vs. finger disks slightly dilated but not forming wide disks). Kaloula discordia sp. nov. is distinguished from K. nonggangensis by lacking protuberant tubercles on the upper surface of finger tips (vs. present); from K. picta by lacking dorsolateral stripes (vs. present); by the stratified coloration on flanks absent (vs. present); and by having wide finger disks (vs. finger disks slightly dilated but not forming wide disks). The new species further differs from K. pulchra by the absence of dorsolateral stripes (vs. always present); by having axillary and inguinal spots (vs. absent); from K. rigida by having wide finger disks (vs. finger disks slightly dilated but not forming wide disks); by the stratified coloration on flanks absent (vs. present); by the absence of dorsolateral stripes (vs. present); and by having axillary and inguinal spots (vs. absent). Kaloula discordia sp. nov. differs from K. rugifera by having wide finger disks (vs. slightly expanded small finger disks); by having axillary and inguinal spots (vs. absent); from K. verrucosa by having wide finger disks (vs. slightly expanded small finger disks); by having axillary and inguinal spots (vs. absent); from K. walteri by having wide finger disks (vs. slightly expanded small finger disks); by the stratified coloration on flanks absent (vs. present); having axillary and inguinal spots (vs. absent); and by having a distinct outer metatarsal tubercle (vs. indistinct or absent). Kaloula discordia sp. nov. is geographically isolated from other members of the K. baleata species complex and most of the other congeners, except K. pulchra, with which it occurs in sympatry everywhere throughout its range, and, possibly, K. mediolineata, with which the new species might co-occur in the Tay Ninh, Binh Phuoc, and Ba Ria-Vung Tau provinces of southern Vietnam.

Acoustic data

For a comparison of the male advertisement calls of Kaloula discordia sp. nov. with K. indochinensis sensu stricto, see Table 3; data for K. indochinensis are taken from Nguyen et al. (2022). The power call parameters of Kaloula discordia sp. nov. and K. indochinensis are quite similar, with the dominant frequency of the call being the same in the two species (0.38 kHz, range 0.34–0.43 kHz vs. 0.38 kHz, range 0.30–0.45 kHz, respectively) (Table 3). At the same time, Kaloula discordia sp. nov. had a slightly shorter call duration (186.1 ms, range 144–214 ms) than K. indochinensis (215.6 ms, range 194–250 ms) and a significantly longer intercall interval: 1539.4 ms (range 851–8954 ms) in Kaloula discordia sp. nov. vs. 789.3 ms (range 481–1627 ms) in K. indochinensis. These differences have to be taken cautiously as the records of calling of the two species were taken with different ambient temperatures (20.5 °C for K. indochinensis and 24.0 °C for the new species), which also could contribute to the observed differences in call parameters between the two species. Nguyen et al. (2022) did not calculate the number of pulses per call in K. indochinensis and noted the presence of one pulse per call in this species (Nguyen et al. 2022: Table 2), while we recorded 11–13 pulses per call in Kaloula discordia sp. nov. These differences actually result from the different terminology used by Nguyen et al. (2022) and our study, as the waveform oscillograms in Nguyen et al. (2022) clearly show the presence of several pulses in each call.

Etymology

The specific epithet “discordia” is a noun in apposition, in the nominative case, given in reference to the Roman mythological goddess Discordia. According to the poet Hesiod, this goddess personified not only strife and discord but also competition and labor (Hesiod, Theogony: 20–24, 226–230; see Most 2006). The duality of this name echoes the two aspects of the discovery of the new species. The first aspect is the authors’ hard work and laborious approach in collecting data for the description of the new species. The second challenge pertains to the authors’ internal struggle to choose a politically correct and neutral name for the new species. In modern taxonomy, international teams often face the common challenge of strife and competition; however, this can also lead to overall scientific progress. We recommend “South Vietnamese Painted Frog” as the common name in English, “Yuzhnovietnamskiy Bychiy Uzkorot” as the common name in Russian, and “Ễnh ương Nam bộ” as the common name in the Vietnamese language.

Conservation status

At present, the new species is reliably known only from four localities in southern Vietnam and a single locality in eastern Cambodia (Fig. 5; Appendix 1). The main threats to this species in Vietnam are habitat loss and degradation. The new species is restricted to the lowland monsoon tropical forests of southern Vietnam; it should be noted that these forests during the last 40 years have been subjected to greater anthropomorphic conversion (including logging, agriculture, road construction, and other human activities) than other areas in Vietnam (e.g., De Koninck 1999; Kuznetsov and Kuznetsova 2011; Laurance 2007; Meijer 1973; Meyfroidt and Lambin 2008). The range of the new species covers several nature conservation areas of southern Vietnam and Cambodia, including Cat Tien NP (Dong Nai Biosphere Reserve), Bu Gia Map NP, Lo Go-Xa Mat NP (Vietnam), and Phnom Prich Wildlife Sanctuary (Cambodia). Given the lack of comprehensive studies on the adjacent territories, we suggest Kaloula discordia sp. nov. be classified as Data Deficient (DD) according to the IUCN’s Red List categories (IUCN 2019).

Kaloula laosensis Poyarkov, Orlov, Gorin & Milto, sp. nov.

Figs 2, 9A, 10, 11, 12, Table 4

Chresonymy

Kaloula baleata [partim]—Orlov and Ananjeva (2007: 148); Nguyen et al. (2009: 94).

Kaloula indochinensis [partim]—Chan et al. (2013: 334, 2014: 577); Teynié et al. (2014: 29); Chandramouli and Prasad (2018: 52); Poyarkov et al. (2021b: 39); Holden (2023: 148).

Holotype

ZISP 15284 (field label ZISP 199), adult male, Na Home Village, Bouphala District, near the border of Nakai-Nam Theun National Park, Khammouane Province, Laos, collected by N.L. Orlov, S.N. Nguyen, and K.D. Milto on June 18, 2009 (17.544528°N, 105.695278°E; elevation 174 m a.s.l.).

Paratype

(n = 1). ZMMU A-8144 (field label ZISP 200), adult female from the same location and with the same collection data as the holotype.

Diagnosis

Kaloula laosensis sp. nov. is distinguished from its congeners by the following combination of morphological attributes: (1) medium size (SVL ranging 44.0–54.8 mm); (2) eyes comparatively large (eye length comprising 75%–100% of snout length); (3) dark-brown dorsally, with numerous irregular dark blotches forming pericloacal ring; (4) gray or beige ventrally with dense white mottling on belly and limbs; (5) orange triangular patch on either side of the neck posterior to eyes; (6) bright-orange axillary patch present; (7) orange inguinal patch present; (8) bright-orange butterfly-shaped blotch above cloaca present; (9) gray spot on tibiotarsal articulation present; (10) dark interorbital bar present; (11) enlarged, widened finger disks (7.8%–7.9% of SVL), ca. 1.53 times wider than toe disks; (12) finger subarticular tubercle formula: 1:1:2:2; (13) toe subarticular tubercle formula: 1:1:2:2:2; (14) three metacarpal tubercles, median metacarpal tubercle in contact with inner metacarpal tubercle; (15) two metatarsal tubercles, inner metatarsal tubercle ca. three times larger than outer metatarsal tubercle; (16) three small supernumerary tubercles at the basis of toes I, III, IV.

Description of the holotype

(Fig. 10). Adult male in a good state of preservation, habitus robust; head wider than long (HW/HL 1.19); snout projecting beyond lower jaw, truncated in dorsal and lateral views (Fig. 10C); top of head flat; upper eyelid lacking supraciliary tubercles; eye length less than snout length (EL/SL 0.75) and less than interorbital distance (EL/IOD 0.77); pupils round; nostrils rounded, placed more towards the lateral sides of snout, located closer to tip of snout than to eye, relatively close to each other (IND/IOD 0.63); supratympanic fold flat, tubercular; tympanum not visible (Fig. 10C); dorsal surfaces of body and limbs with sparse tubercules, getting denser backwards; ventral surfaces of body and limbs almost smooth (Fig. 10A, B). Cloacal opening unmodified, directed posteriorly. Forelimbs relatively long, more than a half of hind limb length (FLL/HLL 0.63); hand long, comprising more than a half of lower arm length (HAL/LAL 0.6) and almost half of forelimb length (HAL/FLL 0.47); fingers rather robust, flattened in cross section; relative finger lengths: I<II<IV<III; no webbing between fingers; terminal digits flattened into very wide transversely expanded T-shaped disks (Fig. 10E); finger subarticular tubercles distinct, protuberant, large and round, finger subarticular formula: 1:1:2:2; metacarpal tubercles three (Figs 9A, 10E), inner metacarpal tubercle elongate, flattened; outer metacarpal tubercule oval, dilated, slightly bigger than inner (OPTL/IPTL 1.05), median metacarpal tubercle the smallest, oval-shaped, contacting both the inner and outer metacarpal tubercles (Figs 9A, 10E); hindlimbs robust, relatively short, not much longer than body length (HLL/SVL 1.12); relative toe lengths: I<II<V<III<IV; tarsal fold on inner surface of tarsus absent; tips of all toes widened, forming terminal oval-shaped disks (Figs 9A, 10D); all toe disks having dorso-terminal groves; the disk on toe IV the largest; toe webbing well developed between all toes, reaching disks at all toes except toe IV; webbing formula i1.5-2ii1.5-2.5iii1.5-3iv3-2v; toe subarticular tubercules distinct, rounded, protruding; toe subarticular formula: 1:1:2:2:2; two metatarsal tubercles, inner metatarsal tubercle elongated, oval, shovel-shaped; outer metatarsal tubercle smaller, rounded; three small supernumerary tubercles at the basis of toes I, III, IV (Figs 9A, 10D).

Figure 10. 

Holotype of Kaloula laosensis sp. nov. (ZISP 15284), adult male. A. Dorsal view; B. Ventral view; C. Head in a lateral view; D. Plantar view of left foot; E. Volar view of left hand. Photographs by V.A. Gorin.

Coloration

In life, dorsal surfaces of head and body dark brown with contrasting black spots and blotches; black interorbital bar between the upper eyelids; irregular black blotch on the snout; a series of black blotches forming a L-shaped chevron pattern in the scapular region; elongated black blotches on mid-dorsum, body flanks, and sacral area, forming a black pericloacal ring with irregular borders (Figs 2, 11); dorsal surfaces of limbs grayish brown with dark-brown to black blotches; flanks of body and lateral sides of head grayish brown; on lateral sides of belly getting dark-brown with white spots and blotches; pale orange patches on the neck posterior to eyes; bright reddish-orange axillary patch edged with dark brown; bright reddish-orange inguinal patch edged with black; bright reddish-orange patch above cloaca in the center of dark pericloacal ring (Fig. 2); small grayish-beige spots near tibiotarsal articulation; ventral surfaces of head and body gray, darker near the jaw; belly and ventral sides of limbs densely mottled with white spots and dots (Fig. 10); iris chocolate brown with copper sparkles dorsally and ventrally (Fig. 11). In preservation after 15 years of storage in ethanol, dorsal coloration faded to dark grayish-brown; dark markings on dorsum are well descernible; light patches became less pronounced and faded to yellowish or beige in color, though the coloration pattern generally remained unchanged (Fig. 10).

Figure 11. 

Dorsolateral (A) and frontal (B) views of the holotype of Kaloula laosensis sp. nov. in situ (ZISP 15284, male). Photographs by N.L. Orlov.

Measurements of the holotype

(in mm): SVL 44.0; HL 10.6; HW 12.6; SL 4.6; EL 3.3; N-EL 3.2; IND 2.7; IOD 4.3; UEW 2.4; FLL 27.6; LAL 21.7; HAL 13.0; HLL 49.3; TL 37.0; FL 25.4; IPTL 2.7; OPTL 2.8; 1FL 7.5; 1TOEL 7.3; OMTL 2.0; 3FDD 3.4; 4TDD 2.2.

Variation

Morphometric variation of the type series is presented in Table 4. A dorsal view of a female paratype specimen is presented in Fig. 12. In general, the female paratype specimen agrees well with the description of the holotype, diverging only in body size, which is larger than in the holotype (SVL 54.8 mm). Female paratype ZMMU-A-8144 has comparatively fewer dark markings on the dorsum (Fig. 12), but generally, the coloration pattern is similar to that of the holotype. Holden (2023: 148, fig. 152) published a photo of the new species from Laos as ‘K. indochinensis,’ which has bright-red axillary, inguinal, and pericloacal patches and lighter grayish-brown dorsal coloration.

Figure 12. 

Dorsal coloration in a single paratype of Kaloula laosensis sp. nov. (ZMMU-A-8144, female) Scale bar equals 5 cm. Photograph by V.A. Gorin.

Tadpole morphology

Currently, data on the larval morphology of Kaloula laosensis sp. nov. are lacking.

Distribution and natural history

Kaloula laosensis sp. nov. is currently reliably known only from two localities in the Khammouane Province of Laos; the distribution of the new species is shown in Fig. 5, and the locality information is detailed in Appendix 1. In Nakai-Nam Theun NP and in Hin Nam No NP, Khammouane Province, Laos, the new species was recorded from limestone evergreen tropical forests at relatively low elevations of ca. 100–300 m asl. The data on the natural history of Kaloula laosensis sp. nov. is scarce; it inhabits primarily lowland forests and is quite secretive, emerging only after heavy rains from June to September, when depressions in the forest floor are flooded and form temporary pools. The new species is likely associated with limestone karst landscapes of central Laos; the actual distribution of Kaloula laosensis sp. nov. is unknown, but we suppose that this species is likely to occur in adjacent provinces of Vietnam (Ha Tinh and Quang Binh), which harbor limestone forests similar in composition. Details about the new species’ reproductive biology and diet are unknown.

Comparisons

Kaloula laosensis sp. nov. most closely resembles other members of the K. baleata species complex in overall morphology, and comparison with these species appears to be the most pertinent. Most specifically, the new species superficially resembles K. indochinensis, with which it was previously confused. From K. indochinensis, Kaloula laosensis sp. nov. can be distinguished by having relatively longer head in males (HL/SVL 0.24 [n = 1] by our data or mean 0.25 ± 0.1 [n = 4] reported by Chan et al. 2013) vs. mean 0.23 ± 0.1 [n = 18] by our data or [n = 25] reported by Chan et al. 2013); relatively wider disk of finger III in males (3FDD/HAL 0.26 ± 0.1 [n = 1] vs. mean 0.23 ± 0.1 [n = 18]); relatively longer first toe in males (1TOEL/FL 0.29 ± 0.1 [n = 1] vs. mean 0.26 ± 0.1 [n = 18]); by having three metacarpal tubercles with median metacarpal tubercle touching both the inner and outer metacarpal tubercles, see Fig. 9A (vs. median metacarpal tubercle small, not touching the inner metacarpal tubercle, see Fig. 9B); by having two subarticular tubercles on toe IV, see Fig. 9A (vs. three well-developed subarticular tubercles, see Fig. 9B); by having supranumerary tubercles at the basis of toes I, III, and IV, see Fig. 9A (vs. supranumerary tubercles absent, see Fig. 9B); by the presence of numerous contrasting black markings on dorsum (vs. absent or indistinct); and brownish dorsal coloration (vs. chocolate to dark grayish-brown).

From K. baleata, Kaloula laosensis sp. nov. can be distinguished by having a relatively larger distance between nares in males (IND/HW 0.21 [n = 1] by our data or [n = 6] reported by Chan et al. (2013) vs. 0.18 ± 0.1 [n = 10] reported by Chan et al. (2013)); wider finger III disks in males (3FDD 3.4 ± 0.1 mm [n = 1] by our data) or mean 3.1 ± 0.3 mm [n = 6] reported by Chan et al. (2013) vs. 1.7 ± 0.2 mm [n = 10] reported by Chan et al. (2013)); by having two subarticular tubercles on toe IV, see Fig. 9A (vs. three well-developed tubercles); and by having supranumerary tubercles at the basis of toes I, III, and IV, see Fig. 9A (vs. supranumerary tubercles absent).

From K. latidisca, Kaloula laosensis sp. nov. can be distinguished by having relatively shorter heads in males (HL/SVL 0.24 [n = 1] by our data or mean 0.25 ± 0.1 [n = 4] reported by Chan et al. (2013) vs. mean 0.27 ± 0.1 [n = 4] reported by Chan et al. (2014)); relatively smaller distance between eyes in males (IOD/HW 0.34 [n = 1] by our data or mean 0.33 ± 0.1 [n = 6] reported by Chan et al. (2013) vs. mean 0.30 ± 0.1 [n = 4] reported by Chan et al. ((2014); by the presence of numerous black markings on the dorsum (vs. black markings on dorsum absent or few); and by having two subarticular tubercles on toe IV (vs. three well-developed tubercles).

From Kaloula discordia sp. nov. (described above), the new species can be distinguished by having a relatively longer head in males (HL/SVL 0.24 [n = 1] by our data or mean 0.25 ± 0.2 [n = 6] reported by Chan et al. (2013) vs. mean 0.22 ± 0.2 [n = 14]); relatively shorter snout in males (SL/HL 0.43 [n = 1] by our data or mean 0.40 ± 0.1 [n = 6] reported by Chan et al. (2013) vs. mean 0.47 ± 0.1 [n = 14]); relatively narrower head in males (HW/HL 1.19 [n = 1] vs. mean 1.31 ± 0.2 [n = 14]); wider finger III disks (3FDD/HAL 0.26 [n = 1] vs. mean 0.23 ± 0.10 [n = 14]); shorter toe I (1TOEL/FL 0.29 [n = 1] vs. mean 0.25 ± 0.1 [n = 14]); by having three metacarpal tubercles, see Fig. 9A (vs. two metacarpal tubercles, see Fig. 9C); by having supranumerary tubercles at the basis of toes I, III, and IV, see Fig. 9A (vs. supranumerary tubercles absent, see Fig. 9C); by the brown coloration of the dorsum (vs. olive); and by the presence of numerous black markings on the dorsum (vs. black markings on dorsum absent or few).

Furthermore, Kaloula laosensis sp. nov. differs from K. aureata by having a brownish dorsum with no reticulations (vs. golden dorsum with dark brown reticulations); from K. borealis by having wide finger disks (vs. finger tips dilated but not forming wide disks); by the absence of yellowish blotches on flanks (vs. present); an by brownish dorsal coloration (vs. gray-brown). Kaloula laosensis sp. nov. further differs from K. conjuncta by the stratified coloration on flanks absent (vs. present); by having distinct outer metatarsal tubercle (vs. weak or indistinct); from K. ghoshi by the presence of axillary and inguinal spots (vs. axillary and inguinal spots absent); by tuberculated dorsum and venter (vs. smooth or with small flat granules dorsally); by brownish dorsal coloration (vs. orange-brown). The new species differs from K. kalingensis in that it has tubercles on the dorsum (vs. dorsum smooth), a distinct outer metatarsal tubercle (vs. indistinct), and yellow or orange axillary and inguinal spots (vs. usually absent or small and red if present). Kaloula laosensis sp. nov. further differs from K. kokacii by the presence of dorsal tubercles, axillary and inguinal spots, and a light pericloacal ring (vs. absent). The new species differs from K. mediolineata by dorsolateral stripes and sacral stripe absent (vs. present); by having enlarged, wide finger disks (vs. finger tips slightly dilated but not forming wide disks); from K. nonggangensis by protuberant tubercles on the upper surface of finger tips absent (vs. present); from K. picta by the absence of dorsolateral stripes and stratified coloration on flanks (vs. present); by having enlarged, wide finger disks (vs. finger tips slightly dilated but not forming wide disks). The new species can be readily diagnosed from K. pulchra by the absence of dorsolateral stripes (vs. present) and by having axillary and inguinal spots (vs. absent). Kaloula laosensis sp. nov. further differs from K. rigida by having enlarged, wide finger disks (vs. finger tips slightly dilated but not forming wide disks), by the absence of dorsolateral stripes and stratified coloration on flanks (vs. present), and by having axillary and inguinal light spots (vs. absent). The new species differs from K. rugifera by having enlarged, wide finger disks (vs. slightly dilated small finger disks) and by having axillary and inguinal spots (vs. absent) and is further different from K. verrucosa by having very wide finger disks (vs. small finger disks) and by having axillary and inguinal spots (vs. absent). The new species differs from K. walteri by having very wide finger disks (vs. slightly dilated small finger disks), by the absence of stratified coloration on flanks (vs. presence), by having axillary and inguinal light spots (vs. absent), and by having a distinct outer metatarsal tubercle (vs. indistinct or absent). Except for K. pulchra, which can be found in central Laos in syntopy with the new species, Kaloula laosensis sp. nov. is geographically separated from most of its relatives.

Acoustic data

The male advertisement call of Kaloula laosensis sp. nov. has not been recorded, and the bioacoustic data on this species is absent.

Etymology

The specific epithet “laosensis” is an adjective in the nominative case, given in reference to the new species’ distribution in central Laos. The name also mirrors the specific epithet of K. indochinensis, with which the new species was previously confused. We recommend “Laotian Painted Frog” as the common English name, “Laosskiy bychiy uzkorot” as the common name in Russian, “Ễnh ương Lào” as the common name in Vietnamese, and “ອື່ງຢາງລາວ” (“Ung Yang Lao”) as the common name in Lao languages.

Conservation status

At present, the new species is known only from two localities in Khammouane Province of Laos (Fig. 5). The main threats to this species in Laos are habitat loss and degradation due to intensified logging and deforestation, namely Nakai-Nam Theun NP and Hin Nam No NP. We propose that the IUCN’s Red List categories (IUCN 2019) classify Kaloula laosensis sp. nov. as Data Deficient (DD) due to the absence of comprehensive studies in the adjacent territories.

Comments

We assume, based on the distribution of the new species, that several specimens from the type series of K. indochinensis in the original description by Chan et al. (2013), collected in Khammouance Province of Laos and labeled as ‘K. indochinensis Laos’ or ‘Laos OTU’ throughout the text, actually represent Kaloula laosensis sp. nov. These specimens include six adult males stored in the Field Museum of Natural History (FMNH), Chicago, USA, under voucher numbers FMNH 270360–65. The lack of genetic information on these specimens in the original description of K. indochinensis prevented Chan et al. (2013) from recognizing their taxonomic distinctiveness.

Discussion

Until recently, K. baleata sensu lato was reported to inhabit a vast area in Southeast Asia, including territories of Indonesia, Malaysia, India, Thailand, Laos, and Vietnam (Blackburn et al. 2013; Chandramouli and Prasad 2018). This was until Blackburn et al. (2013), who published the first comprehensive phylogeny of the genus, revealed that K. baleata is a complex of (presumably) cryptic species, which motivated morphological investigations and taxonomic revisions (Chan et al. 2013, 2014). Our discovery of Kaloula discordia sp. nov. from Southern Vietnam and Kaloula laosensis sp. nov. from Central Laos follows the recent descriptions of two new species from Indochina: K. indochinensis (see Chan et al. 2013) and K. latidisca (see Chan et al. 2014), as well as the elevation of K. ghoshi to full species status (see Chandramouli and Prasad 2018). It is quite remarkable that the populations from southern Vietnam and central Laos, which we herein describe as two new species, were well-known to the authors of previous taxonomic studies of this group (Chan et al. 2013, 2014). In fact, Chan et al. (2013) included six specimens from central Laos (FMNH 270360–65) in their morphological study, assigning them to K. indochinensis and including them in the type series of this species, but without assessing their genetic divergence from topotypic specimens of K. indochinensis from central Vietnam. Similarly, Chan et al. (2013) did not include specimens of the K. baleata complex members from southern Vietnam or adjacent Cambodia in their analysis. However, they included these localities in the proposed distribution range of ‘K. indochinensis,’ likely based on photo records, likewise assuming their conspecificity. In essense, our study thus unravelled diversity in the K. baleata complex that was “hidden in plain sight” and thereby emphasized the need for careful genetic and phenotypic surveys in presumably widespread species of Southeast Asian amphibians. For example, the common practice of including specimens from different localities in a type series may be misleading. Here, the type locality of K. indochinensis described by Chan et al. (2013) from Gia Lai Province of Vietnam and the type locality of Kaloula laosensis sp. nov. in Central Laos are located in biogeographically and climatically different parts of Indochina, separated from each other by a straight distance of over 500 km (see Poyarkov et al. 2021b, 2023). Hence, including specimens from both areas in a type series without DNA-barcoding at least some representative specimens from each locality appears an unnecessary gamble.

Different research groups have developed a tradition of providing extensive datasets of morphological characters for Asian anurans (e.g., Poyarkov et al. 2014, 2020b, 2021a; Meegaskumbura et al. 2015; Garg et al. 2019; Biju et al. 2020; Suwannapoom et al. 2018; Brakels et al. 2023). However, some researchers continue to present limited datasets of seemingly handpicked morphological characters. In the description of K. walteri by Diesmos et al. (2002), authors provided data on a total of 21 morphometric characters, including complete measurements of fore- and hindlimbs. Mo et al. (2013) reduced the number of studied characters to 14 and lacked data on fingers, toes, and respective disks in their description of K. nonggangensis. In the description of K. indochinensis by Chan et al. (2013), the number of studied characters further decreased to 11, with no morphometric data on forelimbs and only partial data on hindlimbs. In the following paper, Chan et al. (2014) described K. latidisca and provided the same set of 11 characters. Finally, in the most recent taxonomic paper on the genus Kaloula (Chandramouli and Prasad 2018), the authors provided measurements on a set of 17 characters of K. ghoshi. None of the respective papers, however, explains or at least mentions the observed reduction in the number of studied morphometric characters. The lack of data on the morphology of these relatively newly described species makes it difficult to compare datasets from different studies and hampers further research into species diversity in Kaloula frogs (see the Morphology and Comparisons subsections in the Results section). Since amphibian taxonomy relies heavily on morphological descriptions, we emphasize the utmost importance of providing comprehensive and complete datasets for newly discovered and documented species that can then be re-used in future integrative studies.

Our discovery of two new species of the K. baleata complex from Indochina corroborates the idea that many species of Southeast Asian amphibians currently considered to be widely distributed may instead still encompass unrecognized diversity, hidden by a lack of comprehensive field surveys and/or the aforementioned deficiency of comprehensive morphological and genetic data (e.g., Chen et al. 2018; Hasan et al. 2019; Gorin et al. 2020; Suwannapoom et al. 2020; Poyarkov et al. 2020a; Lyu et al. 2023; Liu et al. 2024; Trofimets et al. 2024). Here, the new species of Kaloula from the Cat Tien National Park in southern Vietnam appears particularly remarkable. Cat Tien National Park covers one of the few remaining massifs of lowland monsoon forests in Vietnam and likely represents one of the best herpetologically studied areas in Vietnam (Yushchenko et al. 2023b). Herpetological exploration of this territory has continued for over 40 years with numerous expeditions and long-term monitoring programs by Vietnamese, Russian, German, USA, British, and Canadian herpetologists, the results of which were summarized in a monograph (Vassilieva et al. 2016). With 45 species of amphibians and 107 species of reptiles recorded in the park, Cat Tien NP is unanimously recognized as an important local center of herpetofaunal diversity (Geissler et al. 2011, 2015; Vassilieva et al. 2013, 2016; Holden and Poyarkov 2021; Yushchenko et al. 2023a,b). The new Kaloula species from Cat Tien NP thus illustrates the need for herpetological surveys even in presumably well-known Southeast Asian regions and species.

Moreover, our study provides new information on the evolution, diversity, and distribution of other members of the K. baleata species complex, specifically K. latidisca and K. baleata. Chan et al. (2014) described K. latidisca from Kedah, northern Peninsular Malaysia, based on phylogenetic data from Blackburn et al. (2013). In Chan et al.’s (2014) phylogenetic reconstruction, K. latidisca is suggested as a sister lineage to K. baleata sensu lato, which included samples from Java, Palawan, Borneo, Sulawesi, and southern Peninsular Malaysia. Chan et al. (2014) noted that the populations of southern Peninsular Malaysia and Borneo may not be conspecific with either K. baleata or K. latidisca, but they refrained from further taxonomic revisions and did not discuss the diversity and extent of distribution of members of the K. baleata complex in the Thai-Malay Peninsula. First, our data showed relatively low, although species-level, sequence differentiation at the 16S rRNA gene between K. latidisca and the different lineages of K. baleata (p = 3.00%–3.96%; Table 2). Second, the monophyly of K. baleata with respect to K. latidisca is only moderately supported in our analyses (0.83/98, see Fig. 2). Finally, the mitochondrial distribution of K. latidisca and the three lineages of K. baleata partially overlap with each other, which requires investigating and potentially updating their respective ranges (see Fig. 1). Specifically, we hypothesize the following.

  1. Our mitochondrial data suggests that K. latidisca inhabits southern peninsular Thailand, including Satun and Suratthani provinces (see Fig. 1); numerous localities of the K. baleata complex from the Isthmus of Kra in the north to the Thai-Malay national border in the south likely belong to this species, which is penetrating to Malaysia only in the state of Kedah (see Fig. 5).
  2. The populations of the K. baleata complex from Tioman Island in southern Peninsular Malaysia, as well as from the Terengganu and Pahang states in northern Peninsular Malaysia, all belong to the K. baleata lineage 1 and thus appear conspecific with the populations from western Java based on their lack of mitochondrial differentiation. It appears that the Titiwangsa Mountain Range separates the K. baleata clade 1 and K. latidisca ranges in Peninsular Malaysia and southern Thailand.
  3. The K. baleata population from the Tanintharyi Region of southern Myanmar features distinct mtDNA (lineage 3) that is genealogically closer to the mtDNA lineage(s) found in K. baleata sensu stricto (lineage 2) than to K. latidisca (Fig. 2). At the same time, this lineage likely extends to the Tenasserim Mountains/Tanintharyi Region north of the Isthmus of Kra (Figs 1, 5). This would imply that K. latidisca effectively separates the ranges of the K. baleata mtDNA lineages restricted to Sundaland and the Tenasserim Mountains north of the Isthmus of Kra.

These distribution patterns, combined with low genetic divergence in the 16S rRNA gene and unclear morphological differentiation between K. latidisca and K. baleata, stress the need for further integrative studies to clarify the distribution of these species and the taxonomic relevance of their mitochondrial diversity.

Our new data on the diversity and distribution in the K. baleata species complex may have important biogeographic implications. In eastern Indochina, the three Kaloula species (K. indochinensis, Kaloula laosensis sp. nov., and Kaloula discordia sp. nov.) group into a clade that is sister to the clade including all other populations of the complex, from the Thai-Malay Peninsula and Southeast Asian islands. The distribution areas of the three Indochinese species correspond well to the main biogeographic regions elaborated by Poyarkov et al. (2021b, 2023). Specifically, the range of K. indochinensis is associated with the Central Annamites region, while the newly described Kaloula laosensis sp. nov. and Kaloula discordia sp. nov. are restricted to the Northern Annamites and the Central-South Vietnam Lowlands, respectively. In the K. baleata-K. latidisca clade, the distribution of K. baleata lineage 1 encompasses Wallacea (Sulawesi, Sumba, and Palawan islands), Java, and Sumatra, while K. baleata lineage 2 extends over Borneo and eastern Peninsular Malaysia but also on Java (although it may be a result of a human-associated dispersal). At the same time, the range of K. baleata lineage 3 is restricted to the North Tenasserim Region, namely north of the Isthmus of Kra, while K. latidisca likely inhabits the South Tenasserim south of Kra and the South Thai-Malayan Lowlands Region. The diversification of these lineages and its association with paleographic events shall greatly benefit from phylogeographic analyses that implement nuclear markers, first to be able to assess their genomic divergence and to test for potential hybridization and introgression in areas of mitochondrial sharing.

Acknowledgements

The fieldwork in Vietnam was completed within the frameworks and with partial financial support from the research project E-1.2 of the Joint Vietnam-Russia Tropical Science and Technology Research Center for 2024 (Task No. 3). Permission to conduct fieldwork in Vietnam was granted by the Bureau of Forestry, the Ministry of Agriculture and Rural Development of Vietnam, and local administrations. Permission to conduct fieldwork and collect specimens in Cat Tien National Park was granted by Agreement #37/HD on the scientific cooperation between Cat Tien National Park and the Joint Vietnam-Russia Tropical Science and Technology Research Centre. Permissions to conduct fieldwork and collect specimens were granted by the Department of Forestry, Ministry of Agriculture and Rural Development of Vietnam (permit numbers #547/TCLN-BTTN; #432/TCLN-BTTN; #822/TCLN-BTTN; #142/SNgV-VP; #1539/TCLN-DDPH; #1700/UBND.VX); the Forest Protection Departments of the People’s Committees of Gia Lai Province (permit numbers #530/UBND-NC; #1951/UBND-NV), Phu Yen Province (permit number #05/UBND-KT); by the Department of Forestry, Ministry of Agriculture and Forestry of Lao PDR (permit number #271/08, form #511); and by the Institute of Animals for Scientific Purpose Development (IAD), Bangkok, Thailand (permit number U1-01205-2558). Specimen collection protocols and animal operations followed the Institutional Ethical Committee of Animal Experimentation of the University of Phayao, Phayao, Thailand (certificate number UP-AE61-01-04-0022). We thank Valentina F. Orlova and Roman A. Nazarov (ZMMU) for permission to examine specimens under their care. We thank the members of MSU HerpLab, including T. Matsukoji, S. S. Idiiatullina, N. S. Kliukin, D. V. Arkhipov, and E. N. Solovyeva, for their support and assistance. NAP and AMB are grateful to Andrei N. Kuznetsov, Svetlana P. Kuznetsova, Hoi Dang Nguyen, and Leonid P. Korzoun for their support and organization of fieldwork. We express our sincere gratitude to Peter Mikulíček and two anonymous reviewers for numerous helpful comments and suggestions, which allowed us to improve the previous version of the manuscript.

This work was supported by the Russian Science Foundation to N.A. Poyarkov (Grant No. 22-14-00037, specimen collection and preservation, molecular and morphological analyses, data analyses) and the Thailand Science Research and Innovation Fund and the University of Phayao (Unit of Excellence 2025 on Aquatic animals biodiversity assessment (Phase I)) to C. Suwannapoom.

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Appendix 1

Table A1.

List of localities of the Kaloula baleata complex members in Indochina appearing on Fig. 5. Symbols: (1) = Locality number on Fig. 5; (2) = Verified by morphology data (yes/no); (3) = Verified by molecular data (yes/no).

Species (1) (2) (3) Location Sources
K. laosensis sp. nov. 1 yes yes Nakai-Nam Theun NP, Khammouan, Laos (type locality) Chan et al. (2013); our data
K. laosensis sp. nov. 2 yes no Hin Nam No NP, Khammouane, Laos Luu et al. (2016)
K. indochinensis 3 yes no Kiat Ngong, Xepian NBCA, Champasak, Laos Chan et al. (2013)
K. indochinensis 4 yes no Chu Mom Ray NP, Kon Tum, Vietnam Jestrzemski et al. (2013)
K. indochinensis 5 yes yes Kon Chu Rang NR, Gia Lai, Vietnam (type locality) Chan et al. (2013); our data
K. indochinensis 6 yes yes Kon Ka Kinh NP, Gia Lai, Vietnam Vassilieva et al. (2016); our data
K. indochinensis 7 yes no An Khe, Gia Lai, Vietnam Chan et al. (2013); our data
K. indochinensis 8 yes no Song Hinh, Phu Yen, Vietnam Do et al. (2017)
K. indochinensis 9 yes no Tuy Hoa, Phu Yen, Vietnam Do et al. (2017)
K. indochinensis 10 yes yes Yok Don NP, Dak Lak, Vietnam our data
K. discordia sp. nov. 11 yes yes Phnum Prech WS, Mondulkiri, Cambodia Chan et al. (2013)
K. discordia sp. nov. 12 yes no Bu Gia Map NP, Binh Phuoc, Vietnam Vassilieva et al. (2016); our data
K. discordia sp. nov. 13 yes yes Bao Lam, Lam Dong, Vietnam Vassilieva et al. (2016); https://www.inaturalist.org/observations/212830145
K. discordia sp. nov. 14 yes yes Cat Tien NP, Dong Nai, Vietnam (type locality) our data; https://www.inaturalist.org/observations/168961087
K. discordia sp. nov. 15 yes no Lo Go-Xa Mat NP, Tay Ninh, Vetnam Vassilieva et al. (2016); our data
K. baleata Clade 3 16 yes no Thungyai Naresuan East, Umphang, Tak, Thailand our data
K. baleata Clade 3 17 yes no Thong Pha Phum, Kanchanaburi, Thailand https://www.inaturalist.org/observations/180645435
K. baleata Clade 3 18 yes no Suan Phueng, Ratchaburi, Thailand our data
K. baleata Clade 3 19 yes no Kaeng Krachan NP, Phetchaburi, Thailand https://www.inaturalist.org/observations/153399059; https://www.inaturalist.org/observations/176926007
K. baleata Clade 3 20 no yes Yeybu, Tanintharyi, Myanmar Zug et al. (2018)
K. baleata Clade 3 21 yes no Saphan, Prachuap Khiri Khan, Thailand https://www.inaturalist.org/observations//218052579
K. latidisca 22 yes no Saeng, Surathani, Thailand Chan-ard et al. (2011); our data
K. latidisca 23 yes no Khao Sok NP, Surat Thani, Thailand https://www.inaturalist.org/observations/70379448; https://www.inaturalist.org/observations/191660698
K. latidisca 24 yes yes Kapong, Phangnga, Thailand https://www.inaturalist.org/observations/161810790
K. latidisca 25 yes no Khao Lak-Lam Ru NP, Phang Nga, Thailand Pauwels et al. (2000); https://www.inaturalist.org/observations/194115334
K. latidisca 26 yes no Thalang, Phuket, Thailand https://www.inaturalist.org/observations/202954715
K. latidisca 27 yes no Khao Phanom Bencha NP, Krabi, Thailand our data
K. latidisca 28 yes no Ban Na San, Surat Thabi, Thailand https://www.inaturalist.org/observations/83178477
K. latidisca 29 yes no Tha Sala, Nakhon Si Thammarat, Thailand https://www.inaturalist.org/observations/190990980
K. latidisca 30 yes no Khao Luang NP, Nakhon Si Thammarat, Thailand Chan-ard et al. (2011); our data
K. latidisca 31 yes no Khao Pu-Khao Ya NP, Phatthalung, Thailand https://www.inaturalist.org/observations/92061585
K. latidisca 32 yes no Namtok Khao Chong, Trang, Thailand Chan-ard et al. (2011); our data
K. latidisca 33 yes no Yan Ta Khao, Trang, Thailand https://www.inaturalist.org/observations/55886801
K. latidisca 34 yes yes Ton Nga Chang WS, Songkhla, Thailand https://www.inaturalistchancc.org/observations/114582513
K. latidisca 35 yes yes Tha Le Ban NP, Satun, Thialnd our data
K. latidisca 36 yes no Tarutao Island, Satun, Thialnd Nidup et al. (2013); our data
K. latidisca 37 yes no Bang Lang NP, Yala, Thailand https://www.inaturalist.org/observations/165320389
K. latidisca 38 yes yes Gubir, Kedah, Malaysia (type locality) Chan et al. (2014)
K. baleata Clade 1 39 yes yes Kampung Merapunh Lama, Pahang, Malaysia Chan et al. (2014)
K. baleata Clade 1 40 yes no Hulu Terengganu, Terengganu, Malaysia Badli-Sham et al. (2023)
K. baleata Clade 1 41 yes no Kuala Tahan, Pahang, Malaysia https://www.inaturalist.org/observations/116005614
K. baleata Clade 1 42 yes yes Tioman Island, Pahang, Malaysia Grismer et al. (2006)
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