Research Article |
Corresponding author: Chatmongkon Suwannapoom ( chatmongkonup@gmail.com ) Corresponding author: Nikolay A. Poyarkov ( n.poyarkov@gmail.com ) Academic editor: Peter Mikulíček
© 2024 Vladislav 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.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Gorin VA, Orlov NL, Bragin AM, Pawangkhanant P, Milto KD, Le DX, Nguyen TV, Dufresnes C, Suwannapoom C, Poyarkov NA (2024) Phylogeographic pattern and taxonomic revision of the Kaloula baleata species complex (Amphibia, Anura, Microhylidae) with description of two new species from Indochina. Herpetozoa 37: 391-420. https://doi.org/10.3897/herpetozoa.37.e137394
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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.
16S rRNA, bioacoustics, DNA barcoding, Laos, morphology, Southeast Asia, systematics, taxonomy, Vietnam
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 (
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 (
In parallel, a new species, K. nonggangensis Mo, Zhang, Zhou, Chen, Tang, Meng & Chen, 2013, was described from the Guangxi Province of China (
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 (
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 (
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
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 (
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 PQ268497–PQ268520.
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
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.
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) |
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PQ268510 | this work | |
2 | Kaloula discordia sp. nov. | Vietnam, Dong Nai Prov., Cat Tien NP (1) |
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PQ268511 | this work | |
3 | Kaloula discordia sp. nov. | Vietnam, Dong Nai Prov., Cat Tien NP (1) |
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PQ268512 | this work | |
4 | Kaloula discordia sp. nov. | Vietnam, Dong Nai Prov., Cat Tien NP (1) |
|
PQ268513 | this work | |
5 | Kaloula discordia sp. nov. | Vietnam, Dong Nai Prov., Cat Tien NP (1) |
|
PQ268514 | this work | |
6 | Kaloula discordia sp. nov. | Vietnam, Lam Dong Prov., Loc Bac (2) |
|
PQ268515 | this work | |
7 | Kaloula discordia sp. nov. | Vietnam, Lam Dong Prov., Loc Bac (2) |
|
PQ268516 | this work | |
8 | Kaloula laosensis sp. nov. | Laos, Khammouane Prov., Nakai-Nam Theun NP (3) |
|
PQ268517 | this work | |
9 | Kaloula laosensis sp. nov. | Laos, Khammouane Prov., Nakai-Nam Theun NP (3) |
|
PQ268518 | this work | |
10 | Kaloula indochinensis | Vietnam, Dak Lak Prov., Yok Don NP (4) |
|
PQ268497 | this work | |
11 | Kaloula indochinensis | Vietnam, Dak Lak Prov., Yok Don NP (4) |
|
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) |
|
PQ268499 | this work | |
14 | Kaloula indochinensis | Vietnam, Gia Lai Prov., Krong Pa (5) |
|
PQ268500 | this work | |
15 | Kaloula indochinensis | Vietnam, Gia Lai Prov., Krong Pa (5) | ROM32925 | KC822572 |
|
|
16 | Kaloula indochinensis | Vietnam, Gia Lai Prov., Krong Pa (5) | ROM32932 | KC822573 |
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|
17 | Kaloula indochinensis | Vietnam, Gia Lai Prov., Krong Pa (5) | ROM32943 | KC822574 |
|
|
18 | Kaloula indochinensis | Vietnam, Gia Lai Prov., Kon Ka Kinh NP (6) |
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PQ268501 | this work | |
19 | Kaloula indochinensis | Vietnam, Gia Lai Prov., Kon Ka Kinh NP (6) |
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PQ268502 | this work | |
55 | Kaloula latidisca | Malaysia, Kedah, Gubir (7) | LSUHC5074 | KC822576 |
|
|
56 | Kaloula latidisca | Thailand, Satun Prov., Baan Suan Tondin (8) |
|
PQ268503 | this work | |
57 | Kaloula latidisca | Thailand, Suratthani Prov., Khao Sok (9) |
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PQ268504 | this work | |
20 | Kaloula baleata clade 1 | Indonesia, Java isl., Jawa Barat (10) | TNHC67086 | KC822569 |
|
|
21 | Kaloula baleata clade 1 | Indonesia, Bali Prov., Bali isl. (11) | JAM3232 | KC822570 |
|
|
22 | Kaloula baleata clade 1 | Indonesia, Sumatra, Way Jepara (12) |
|
PQ268507 | this work | |
23 | Kaloula baleata clade 1 | Indonesia, Sumba (13) | KUHE32313 | AB634629 | AB634687 |
|
24 | Kaloula baleata clade 1 | Philippines, Palawan isl. (14) | ACD1303 | KC822582 |
|
|
25 | Kaloula baleata clade 1 | Philippines, Palawan isl. (14) | ACD1307 | KC822583 |
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|
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 |
|
|
28 | Kaloula baleata clade 1 | Indonesia, Sulawesi Tengah Prov., Donggala (16) | LSUMZ-83998 | KC822566 |
|
|
29 | Kaloula baleata clade 1 | Indonesia, Sulawesi Utara Prov., Bogani Nani Wartabone NP (17) | LSUMZ-83999 | KC822567 |
|
|
30 | Kaloula baleata clade 2 | Malaysia (–) | FRIM1066 | KC822579 |
|
|
31 | Kaloula baleata clade 2 | Malaysia (–) | DWNP975 | KC822580 |
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|
32 | Kaloula baleata clade 2 | Malaysia, Johor, Pulau Aceh (18) | LSUHC5712 | KC822577 |
|
|
33 | Kaloula baleata clade 2 | Malaysia, Sabah, Sepilok (19) | LSUHC6156 | KC822578 |
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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 |
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|
36 | Kaloula baleata clade 2 | Malaysia, Pahang, Taman Negara (22) |
|
PQ268508 | this work | |
37 | Kaloula baleata clade 2 | Malaysia, Pahang, Taman Negara (22) |
|
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 |
|
|
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 |
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63 | Kaloula borealis | China, Beijing, Xiangshan | KIZHERP-0174 | JX678909 |
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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 |
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66 | Kaloula conjuncta | Philippines, Mindanao isl., Bukidnon Prov. | ACD 996 | KC822536 |
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67 | Kaloula conjuncta | Philippines, Semirara isl., Antique Prov. | KU301849 | KC822532 |
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68 | Kaloula conjuncta | Philippines, Semirara isl., Antique Prov. | KU301854 | KC822533 |
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69 | Kaloula conjuncta | Philippines, Polillo isl., Quezon Prov. | KU303279 | KC822527 |
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70 | Kaloula conjuncta | Philippines, Luzon isl., Laguna Prov. | KU320031 | KC822538 |
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71 | Kaloula conjuncta | Philippines, Mindoro isl., Mindoro Occidental Prov. | KU323280 | KC822535 |
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72 | Kaloula conjuncta | Philippines, Mindoro isl., Mindoro Oriental Prov. | RMB509 | KC822534 |
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73 | Kaloula conjuncta | Philippines, Luzon isl., Quezon Prov. | TNHC59628 | KC822539 |
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74 | Kaloula conjuncta | Philippines, Luzon isl., Albay Prov. | TNHC62972 | KC822528 |
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75 | Kaloula conjuncta | Philippines, Luzon isl., Albay Prov. | TNHC62973 | KC822526 |
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76 | Kaloula conjuncta | Philippines, Luzon isl., Albay Prov. | TNHC62975 | KC822530 |
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77 | Kaloula conjuncta | Philippines, Luzon isl., Albay Prov. | TNHC62976 | KC822529 |
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78 | Kaloula conjuncta | Philippines, Luzon isl., Albay Prov. | TNHC62986 | KC822531 |
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79 | Kaloula conjuncta | Philippines, Camigun Sur isl., Camiguin Sur Prov. | KU309658 | KC822588 |
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80 | Kaloula conjuncta | Philippines, Mindanao isl., Davao del Sur Prov. | TNHC59632 | KC822521 |
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81 | Kaloula conjuncta | Philippines, Mindanao isl., Davao del Sur Prov. | TNHC59635 | KC822520 |
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82 | Kaloula conjuncta | Philippines, Mindanao isl., Davao del Sur Prov. | TNHC59636 | KC822522 |
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83 | Kaloula conjuncta | Philippines, Mindanao isl., Davao del Sur Prov. | TNHC59669 | KC822524 |
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84 | Kaloula conjuncta | Philippines, Mindanao isl., Davao del Sur Prov. | TNHC59670 | KC822523 |
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85 | Kaloula conjuncta | Philippines, Mindanao isl., Davao City Prov. | TNHC59870 | KC822525 |
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86 | Kaloula conjuncta | Philippines, Negros isl., Negros Oriental Prov. | KU328639 | KC822518 |
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87 | Kaloula conjuncta | Philippines, Negros isl., Negros Oriental Prov. | KU328640 | KC822519 |
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88 | Kaloula kalingensis | Philippines, Palaui isl., Cagayan Prov. | KU328643 | KC822603 |
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89 | Kaloula kalingensis | Philippines, Palaui isl., Cagayan Prov. | KU328644 | KC822604 |
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90 | Kaloula kalingensis | Philippines, Luzon isl., Kalinga Prov. | TNHC60118 | KC822605 |
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91 | Kaloula kalingensis | Philippines, Luzon isl., Kalinga Prov. | RMB2210 | KC822608 |
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92 | Kaloula kalingensis | Philippines, Luzon isl., Kalinga Prov. | RMB3137 | KC822609 |
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93 | Kaloula kalingensis | Philippines, Luzon isl., Kalinga Prov. | TNHC59647 | KC822610 |
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94 | Kaloula kokacii | Philippines, Luzon isl., Camarines del Norte Prov. | KU313824 | KC822596 |
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95 | Kaloula kokacii | Philippines, Luzon isl., Camarines Sur Prov. | KU328629 | KC822595 |
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96 | Kaloula kokacii | Philippines, Luzon isl., Albay Prov. | KU328630 | KC822590 |
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97 | Kaloula kokacii | Philippines, Luzon isl., Quezon Prov. | KU328634 | KC822593 |
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98 | Kaloula kokacii | Philippines, Luzon isl., Camarines Sur Prov. | TNHC62684 | KC822594 |
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99 | Kaloula kokacii | Philippines, Luzon isl., Albay Prov. | TNHC62685 | KC822589 |
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100 | Kaloula kokacii | Philippines, Luzon isl., Albay Prov. | TNHC62687 | KC822591 |
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101 | Kaloula kokacii | Philippines, Luzon isl., Sorsogon Prov. | TNHC62688 | KC822592 |
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102 | Kaloula mediolineata | Thailand, Tak Prov. | KUHE35178 | AB634631 | AB634689 |
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103 | Kaloula mediolineata | Vietnam, Gia Lai Prov. | ROM32838 | KC822571 |
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104 | Kaloula mediolineata | Thailand, Nakhorn Ratchasima Prov. | KU328285 | KC822508 |
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105 | Kaloula mediolineata | Thailand, Nakhorn Ratchasima Prov. | KU328280 | KC822509 |
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106 | Kaloula mediolineata | Thailand, Ubon Ratchatani | FMNH265819 | KC822510 |
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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 |
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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 |
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115 | Kaloula picta | Philippines, Leyte isl., Leyte Prov. | ACD1242 | KC822563 |
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116 | Kaloula picta | Philippines, Palawan isl., Palawan Prov. | ACD1261 | KC822542 |
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117 | Kaloula picta | Philippines, Palawan isl., Palawan Prov. | ACD1304 | KC822540 |
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118 | Kaloula picta | Philippines, Luzon isl., Pampanga Prov. | ACD1390 | KC822546 |
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119 | Kaloula picta | Philippines, Leyte isl., Leyte Prov. | ACD1501 | KC822562 |
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120 | Kaloula picta | Philippines, Luzon isl., Cavite Prov. | DLSUD020 | KC822552 |
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121 | Kaloula picta | Philippines, Camiguin Sur Isl., Camiguin Sur Prov. | KU301873 | KC822565 |
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122 | Kaloula picta | Philippines, Luzon isl., Laguna Prov. | KU326261 | KC822554 |
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123 | Kaloula picta | Philippines, Luzon isl., Laguna Prov. | KU326262 | KC822555 |
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124 | Kaloula picta | Philippines, Luzon isl., Quezon Prov. | KU326265 | KC822548 |
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125 | Kaloula picta | Philippines, Luzon isl., Cagayan Prov. | RMB4223 | KC822551 |
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126 | Kaloula picta | Philippines, Luzon isl., Cagayan Prov. | RMB4224 | KC822550 |
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127 | Kaloula picta | Philippines, Leyte isl., Leyte Prov. | RMB4294 | KC822564 |
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128 | Kaloula picta | Philippines, Leyte isl., Leyte Prov. | RMB4344 | KC822561 |
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129 | Kaloula picta | Philippines, Bohol isl., Bohol Prov. | TNHC56430 | KC822559 |
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130 | Kaloula picta | Philippines, Bohol isl., Bohol Prov. | TNHC56431 | KC822560 |
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131 | Kaloula picta | Philippines, Cebu isl., Cebu Prov. | TNHC56468 | KC822543 |
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132 | Kaloula picta | Philippines, Mindanao isl., Davao del Sur Prov. | TNHC59653 | KC822558 |
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133 | Kaloula picta | Philippines, Mindanao isl., Davao del Sur Prov. | TNHC59654 | KC822557 |
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134 | Kaloula picta | Philippines, Mindanao isl., Davao del Sur Prov. | TNHC59871 | KC822556 |
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135 | Kaloula picta | Philippines, Luzon isl., Albay Prov. | TNHC62470 | KC822549 |
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136 | Kaloula picta | Philippines, Luzon isl., Zambales Prov. | TNHC62471 | KC822547 |
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137 | Kaloula picta | Philippines, Polillo isl., Quezon Prov. | USNM-FS56932 | KC822545 |
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138 | Kaloula picta | Philippines, Polillo isl., Quezon Prov. | USNM-FS-56936 | KC822544 |
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139 | Kaloula pulchra | Thailand, Nong Khai | KUHE 22206 | AB634632 | AB634690 |
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140 | Kaloula pulchra | Thailand, Kanchanaburi, Thong Pha Phum | KUHE 35171 | AB201183 | AB201194 |
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141 | Kaloula pulchra | Myanmar, Sagaing | USFS34083 | KC180025 | de Sa et al. 2012 | |
142 | Kaloula pulchra | Vietnam, Cat Ba |
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PQ268519 | this work | |
143 | Kaloula pulchra | Vietnam, Cat Ba |
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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 |
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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 |
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152 | Kaloula pulchra | China, Yunnan, Jinghong | KIZHERP0121 | JX678903 |
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153 | Kaloula pulchra | China, Yunnan, Mengla | KIZHERP0129 | JX678904 |
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154 | Kaloula pulchra | China, Guangxi, Qinzhou | KIZHERP0171 | JX678907 |
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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 |
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166 | Kaloula pulchra | China, Guangxi, Nonggang | NHMG<CHN>:1106009 | KC567236 |
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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 |
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174 | Kaloula pulchra | Laos, Khammouane Prov., Boudalapha | FMNH225128 | KC822620 |
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175 | Kaloula pulchra | Laos, Bolikhamxay Prov., Thaphabat | FMNH225129 | KC822619 |
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176 | Kaloula pulchra | Malaysia, Selangor Prov. | JAM1753 | KC822616 |
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177 | Kaloula pulchra | Malaysia, Pahang Prov., Tioman isl. | JAM1857 | KC822615 |
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178 | Kaloula pulchra | Malaysia, Pahang, Pulau Tioman | LSUHC3869 | KC822575 |
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179 | Kaloula pulchra | Malaysia (continental) | LSUHC3870 | KC852906 |
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180 | Kaloula pulchra | Indonesia, Sumatra isl. | MF0766 | KC822623 |
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181 | Kaloula pulchra | Indonesia, Sumatra isl. | MF0812 | KC822624 |
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182 | Kaloula pulchra | China | NHMS3208 | KC822614 |
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183 | Kaloula pulchra | Indonesia, Sulawesi isl., Propinsi Sulawesi Selatan | TNHC59422 | KC822618 |
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184 | Kaloula pulchra | Indonesia, Sulawesi isl., Propinsi Sulawesi Selatan | TNHC59423 | KC822617 |
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185 | Kaloula pulchra | Vietnam | TZ629 | KC822622 |
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186 | Kaloula pulchra | China | - | AF315130 | Jiang and Zhou., 2001 | |
187 | Kaloula rigida | Philippines, Luzon isl., Benguet Prov. | ACD1570 | KC822636 |
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188 | Kaloula rigida | Philippines, Luzon isl., Isabela Prov., San Mariano | ACD1954 | KC822631 |
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189 | Kaloula rigida | Philippines, Luzon isl., Isabela Prov., San Mariano | ACD2043 | KC822632 |
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190 | Kaloula rigida | Philippines, Luzon isl., Isabela Prov., San Mariano | ACD2044 | KC822634 |
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191 | Kaloula rigida | Philippines, Luzon isl., Kalinga Prov. | ACD2032 | KC822633 |
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192 | Kaloula rigida | Philippines, Luzon isl., Sierra Madres | ACD660 | KC822626 |
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193 | Kaloula rigida | Philippines, Luzon isl., Kalinga Prov., Balbalan | ACD756 | KC822628 |
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194 | Kaloula rigida | Philippines, Luzon isl., Isabela Prov., San Mariano | KU326470 | KC822635 |
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195 | Kaloula rigida | Philippines, Luzon isl., Cagayan Prov., Pagudpud | KU328628 | KC822627 |
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196 | Kaloula rigida | Philippines, Luzon isl., Cagayan Prov., Gattaran | RMB4226 | KC822625 |
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197 | Kaloula rigida | Philippines, Luzon isl., Kalinga Prov., Balbalan | TNHC59644 | KC822629 |
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198 | Kaloula rigida | Philippines, Luzon isl., Kalinga Prov., Balbalan | TNHC60119 | KC822630 |
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199 | Kaloula rugifera | China, Sichuan, Mianyang | KIZHERP071601 | JX678911 |
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200 | Kaloula rugifera | China, Sichuan, Mianyang | KIZHERP071602 | JX678912 |
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201 | Kaloula rugifera | China, Sichuan, Mianyang | CIB20110802028 | KC567237 |
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202 | Kaloula verrucosa | China, Sichuan, Huidong | CIB20090349 | KC567238 |
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203 | Kaloula verrucosa | China, Sichuan, Huidong | CIB20090351 | KC567239 |
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204 | Kaloula verrucosa | China, Yunnan, Chuxiong | KIZHERP2015 | JX678900 |
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205 | Kaloula verrucosa | China, Yunnan, Wuliangshan | KIZHERP2020 | JX678901 |
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206 | Kaloula verrucosa | China | NMNS 3246 | KC822507 |
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207 | Kaloula walteri | Philippines, Mindanao isl., Bukidnon Prov., Malagaylay | ACD994 | KC822640 |
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208 | Kaloula walteri | Philippines, Polillo isl., Quezon Prov., Polillo | KU303285 | KC822642 |
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209 | Kaloula walteri | Philippines, Luzon isl., Quezon Prov., Lucban | KU327338 | KC822639 |
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210 | Kaloula walteri | Philippines, Luzon isl., Quezon Prov., Tayabas | RMB3701 | KC822641 |
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211 | Kaloula walteri | Philippines, Luzon isl., Quezon Prov., Lucban | TNHC59667 | KC822637 |
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212 | Kaloula walteri | Philippines, Luzon isl., Quezon Prov., Lucban | TNHC60116 | KC822638 |
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213 | Kaloula sp. | Philippines, Luzon isl., Laguna Prov. | ACD1692 | KC822602 |
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214 | Kaloula sp. | Philippines, Luzon isl., Sierra Madres | ACD2479 | KC822606 |
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215 | Kaloula sp. | Philippines, Luzon isl., Isabela Prov., Palanan | ACD650 | KC822607 |
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216 | Kaloula sp. | Philippines, Luzon isl., Laguna Prov., Los Banos | ACD943 | KC822597 |
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217 | Kaloula sp. | Philippines, Luzon isl., Laguna Prov., Los Banos | ACD945 | KC822598 |
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218 | Kaloula sp. | Philippines, Luzon isl., Laguna Prov., Los Banos | FMNH267555 | KC822599 |
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219 | Kaloula sp. | Philippines, Luzon isl., Aurora Prov., Aurora Memorial NP | RMB750 | KC822601 |
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220 | Kaloula sp. | Philippines, Luzon isl., Aurora Prov., Aurora Memorial NP | RMB783 | KC822600 |
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221 | Kaloula sp. | Philippines, Panay isl., Antique Prov., Sibalom | GVAG253 | KC822515 |
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222 | Kaloula sp. | Philippines, Panay isl., Antique Prov., Sibalom | GVAG255 | KC822514 |
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223 | Kaloula sp. | Philippines, Sibuyan isl., Rombolon Prov. | KU328607 | KC822511 |
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224 | Kaloula sp. | Philippines, Sibuyan isl., Rombolon Prov. | KU328608 | KC822512 |
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225 | Kaloula sp. | Philippines, Panay isl., Antique Prov. | MG012 | KC822513 |
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226 | Kaloula sp. | Philippines, Panay isl., Antique Prov. | TNHC56341 | KC822516 |
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227 | Kaloula sp. | Philippines, Panay isl., Antique Prov. | TNHC56343 | KC822517 |
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228 | Kaloula sp. | Philippines, Samar isl., Eastern Samar Prov., Taft | KU310699 | KC822587 |
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229 | Kaloula sp. | Philippines, Leyte isl., Leyte Prov., Danao | KU328632 | KC822584 |
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230 | Kaloula sp. | Philippines, Leyte isl., Leyte Prov., Danao | KU328633 | KC822585 |
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231 | Kaloula sp. | Philippines, Leyte isl., Leyte Prov., Baybay | KU328645 | KC822586 |
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232 | Kaloula sp. | Philippines, Panay isl., Antique Prov., Sibalom | MG0000 | KC822611 |
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233 | Kaloula sp. | Philippines, Panay isl., Antique Prov., Sibalom | MG0001 | KC822612 |
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234 | Kaloula sp. | Philippines, Panay isl., Antique Prov., Sibalom | MG0002 | KC822613 |
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Outgroups | ||||||
235 | Uperodon taprobanicus | Bandgladesh, Mymensingh | Ktap-Bd | AB530545 | Hasan et al. 2012 | |
236 | Uperodon taprobanicus | Sri Lanka | KUHE-37252 | AB634633 | AB634691 |
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237 | Uperodon taprobanicus | – | - | AY948729 | Roelants et al. 2007 | |
238 | Kalophrynus interlineatus | – | KIZ-HERP-0169 | JX678906 |
|
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.
We initially aligned nucleotide sequences using ClustalX 1.81 (
We inferred the matrilineal genealogy using Bayesian Inference (BI) and Maximum Likelihood (ML) approaches. We conducted BI using MrBayes 3.1.2 (
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
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 (
Furthermore, we compare external morphological characters based on information taken from the literature (e.g.,
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
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.
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.
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.
The uncorrected genetic p-distances for the 16S rRNA gene fragment among members of the genus Kaloula are available in Table
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 |
Given the limited number of morphometric characters provided in the original description of K. indochinensis in
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
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
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 |
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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
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
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
Kaloula baleata [partim]—
Kaloula indochinensis [partim]—
(n = 14).
(n = 3).
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.
(Fig.
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.
In life, the dorsal surfaces of the head and body olive-brown, dorsal surfaces of the limbs grayish-olive with grayish-white spots (Fig.
(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.
Table
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. |
|
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. |
|
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. |
|
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. |
|
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. |
|
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. |
|
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. |
|
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. |
|
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. |
|
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. |
|
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. |
|
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. |
|
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. |
|
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. |
|
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. |
|
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. |
|
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. |
|
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. |
|
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. |
|
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. |
|
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 |
|
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 |
|
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 |
|
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 |
|
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 |
|
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 |
|
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 |
|
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 |
|
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 |
|
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 |
|
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 |
|
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 |
|
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 |
|
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 |
|
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 |
|
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 |
|
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 |
|
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 |
|
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 |
|
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 |
|
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 |
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.
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.
From K. baleata (morphological data taken from
From K. latidisca (morphological data taken from
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.
For a comparison of the male advertisement calls of Kaloula discordia sp. nov. with K. indochinensis sensu stricto, see Table
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
At present, the new species is reliably known only from four localities in southern Vietnam and a single locality in eastern Cambodia (Fig.
Kaloula baleata [partim]—
Kaloula indochinensis [partim]—
(n = 1).
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.
(Fig.
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
(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.
Morphometric variation of the type series is presented in Table
Currently, data on the larval morphology of Kaloula laosensis sp. nov. are lacking.
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.
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
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
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
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
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.
The male advertisement call of Kaloula laosensis sp. nov. has not been recorded, and the bioacoustic data on this species is absent.
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.
At present, the new species is known only from two localities in Khammouane Province of Laos (Fig.
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
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 (
Different research groups have developed a tradition of providing extensive datasets of morphological characters for Asian anurans (e.g.,
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.,
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.
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
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 (
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.
List of localities of the Kaloula baleata complex members in Indochina appearing on Fig.
Species | (1) | (2) | (3) | Location | Sources |
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K. laosensis sp. nov. | 1 | yes | yes | Nakai-Nam Theun NP, Khammouan, Laos (type locality) |
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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 |
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K. indochinensis | 4 | yes | no | Chu Mom Ray NP, Kon Tum, Vietnam |
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K. indochinensis | 5 | yes | yes | Kon Chu Rang NR, Gia Lai, Vietnam (type locality) |
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K. indochinensis | 6 | yes | yes | Kon Ka Kinh NP, Gia Lai, Vietnam |
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K. indochinensis | 7 | yes | no | An Khe, Gia Lai, Vietnam |
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K. indochinensis | 8 | yes | no | Song Hinh, Phu Yen, Vietnam |
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K. indochinensis | 9 | yes | no | Tuy Hoa, Phu Yen, Vietnam |
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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 |
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K. discordia sp. nov. | 12 | yes | no | Bu Gia Map NP, Binh Phuoc, Vietnam |
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K. discordia sp. nov. | 13 | yes | yes | Bao Lam, Lam Dong, Vietnam |
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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 |
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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 |
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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 |
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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 |
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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 |
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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 |
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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 |
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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) |
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K. baleata Clade 1 | 39 | yes | yes | Kampung Merapunh Lama, Pahang, Malaysia |
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K. baleata Clade 1 | 40 | yes | no | Hulu Terengganu, Terengganu, Malaysia |
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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 |
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