Research Article |
Corresponding author: Daniel Jablonski ( daniel.jablonski@uniba.sk ) Academic editor: Umilaela Arifin
© 2022 Daniel Jablonski, Rafaqat Masroor, Sylvia Hofmann.
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:
Jablonski D, Masroor R, Hofmann S (2022) On the edge of the Shivaliks: An insight into the origin and taxonomic position of Pakistani toads from the Duttaphrynus melanostictus complex (Amphibia, Bufonidae). Zoosystematics and Evolution 98(2): 275-284. https://doi.org/10.3897/zse.98.79213
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The common Asian toad Duttaphrynus melanostictus (Schneider, 1799) complex has a wide distribution ranging from western foothills of the Himalaya to the easternmost range of the Wallacea, with the evidence of human-mediated introductions to some other areas. In the entire distribution range, the complex is formed by several evolutionary clades, distributed mostly in South-East Asia with unresolved taxonomy. In the northwestern edge of its distribution (Pakistan), the name D. melanostictus hazarensis (Khan, 2001) has been assigned to local populations but its biological basis remained, so far, understudied and unvalidated. Therefore, we re-evaluated the available genetic data (mitochondrial and nuclear) to show the relationships between Pakistani populations (including the type locality of D. m. hazarensis) and others from across the range. Our results showed that Pakistani populations are associated with one, deeply diverged, well-supported and widely distributed clade (so-called Duttaphrynus sp. 1 according to 16S, or clade B based on tRNAGly-ND3), that has already been detected in previous studies. This clade is further distributed in India, Nepal, Bangladesh, Malaysia, Singapore, and Indonesia and is characterized by a low level of genetic variability. This further suggests that both natural, as well as potential human-mediated dispersal, might have played an important role in setting up the current phylogeographic and distribution pattern of this clade. The clade is deeply divergent from other clades of the complex and represents a taxonomically unresolved entity. We here argue that the clade Duttaphrynus sp. 1/B represents a distinct species for which the name Duttaphrynus bengalensis (Daudin, 1802) comb. nov. is applicable, while the description of D. m. hazarensis does not satisfy the rules of the International Code of Zoological Nomenclature.
Bufo, Bufonidae, Indian subcontinent, invasions, phylogeography, taxonomy
The distribution of the Duttaphrynus melanostictus (Schneider, 1799) complex (
The Indian subcontinent and northern Pakistan form the western border of the D. melanostictus complex natural range where its distribution is limited to the edge of Shivalik Hills, i.e. to several districts of Khyber Pakhtunkhwa, Punjab, and Azad Jammu and Kashmir Provinces (
Due to the topographic heterogeneity and habitat diversity, it might be plausible that Pakistani amphibian populations of different genera with the Oriental affiliation may hold a different evolutionary history compared to their populations from other parts of South-East Asia. The significance of the far-western part of the Himalaya and the Indo-Gangetic plain for understanding the biogeography of taxa distributed across these geomorphological domains has been recently shown in several species groups (see Allopaa, Chrysopaa, Microhyla, Nanorana, Scutiger, Sphaerotheca;
Tissue samples of six individuals of the Duttaphrynus melanostictus complex from northern Pakistan were collected from five different localities of the Khyber Pakhtunkhwa Province during the field studies between 2018 and 2019. Individuals were documented photographically by DJ (Nikon D810, and 105 mm macro lens) and sampled for toe or blood before release or being deposited in scientific collections (Suppl. material
We amplified a fragment of the nuclear (nDNA) rhodopsin (Rho) gene (~316 bp), of the 16S rRNA (~560 bp), and approximately 469 bp of mitochondrial DNA (mtDNA) that encodes part of the cytochrome oxidase c subunit III (COIII), tRNA glycine (tRNAGly), and part of the NADH dehydrogenase subunit 3 (ND3) using the primers and PCR conditions following previous studies (
We complemented our new mitochondrial and nuclear DNA sequences dataset with published and unpublished sequence data from GenBank (for details see Suppl. material
New sequences were aligned in MEGA X v.11.0 (
Bayesian phylogenetic analyses were carried out with MrBayes v.3.2.6 (
For reasons of comparison, we also inferred the BI tree for 16S based on the secondary structure of the ribosomal RNA using RNAsalsa 0.8.1 (
To assess the evolutionary distance between our samples and the Duttaphrynus sister clades recovered from the phylogenetic analysis, we calculated the uncorrected p-distances for the 16S rRNA and tRNAGly-ND3 data set separately using Mega-X based on 1,000 bootstrap replications, with the pairwise deletion option, and by considering both transitions and transversions. For clades that correspond with the Pakistani population of D. melanostictus, and for the Rho sequence data we created a median-joining haplotype network using PopART (
The newly generated sequences of 16S, tRNAGly-ND3, and Rho from Pakistan belong to one, well-supported, monophyletic group in all investigated markers (Figs
The molecular phylogenetic reconstruction of the Duttaphrynus melanostictus complex on 16S rRNA (A) sensu terminology of
For 16S, populations from Pakistan correspond with the clade called Duttaphrynus sp. 1 (sensu
Uncorrected genetic distances (%) between investigated 16S clades of the Duttaphrynus melanostictus complex as shown in the Fig.
Clade p-distances (%) | cf. microtympanum | brevirostris | parientalis | melanostictus ? | melanostictus | Duttaphrynus sp. 2 | D. bengalensis comb. nov. (Duttaphrynus sp. 1) |
---|---|---|---|---|---|---|---|
cf. microtympanum | – | 0.7 | 0.7 | 0.8 | 0.6 | 0.9 | 0.9 |
brevirostris | 2.3 | – | 0.8 | 0.9 | 0.7 | 1.0 | 0.9 |
parientalis | 2.7 | 3.0 | – | 0.6 | 0.7 | 0.9 | 0.9 |
melanostictus ? | 2.9 | 3.4 | 2.3 | – | 0.6 | 0.8 | 0.8 |
melanostictus | 2.8 | 3.0 | 2.9 | 2.5 | – | 1.0 | 0.8 |
Duttaphrynus sp. 2 | 4.1 | 4.3 | 4.7 | 4.3 | 4.9 | – | 0.6 |
D. bengalensis comb. nov. (Duttaphrynus sp. 1) | 4.2 | 3.7 | 4.2 | 3.2 | 3.9 | 2.7 | – |
Phylogeographic reconstruction and distribution of major clades of the Duttaphrynus melanostictus complex across the species range based on 16S rRNA (A) and tRNAGly–ND3 (B) sequences available in GenBank and from own data (see Suppl. material
For tRNAGly-ND3, Pakistani sequences correspond to the clade B (sensu
Uncorrected genetic distances (%) between investigated tRNAGly-ND3 clades of the Duttaphrynus melanostictus complex as shown in the Fig.
Clade p-distances (%) | A | B | C | D | E | F | G |
---|---|---|---|---|---|---|---|
A | – | 3.4 | 3.4 | 2.8 | 2.7 | 3.2 | 2.9 |
B | 9.3 | – | 2.4 | 2.9 | 2.1 | 3.0 | 2.7 |
C | 9.3 | 5.6 | – | 2.6 | 2.3 | 2.6 | 2.4 |
D | 7.9 | 7.2 | 6.8 | – | 1.8 | 1.9 | 1.9 |
E | 7.4 | 4.9 | 5.4 | 4.2 | – | 1.7 | 1.6 |
F | 8.4 | 7.2 | 6.1 | 4.3 | 3.6 | – | 1.2 |
G | 8.0 | 6.7 | 5.9 | 4.7 | 3.6 | 2.7 | – |
The results on rhodopsin (Rho) confirm mitochondrial data. It shows that D. melanostictus from Pakistan represents an independent group (Fig.
Median-joining alleles network of sequences from the Duttaphrynus melanostictus complex and closely related members of western Palearctic Bufonidae based on rhodopsin (Rho) gene available in GenBank and from own data. The colors correspond to those used in Fig.
The most prominent results of our study are the following: (i) the “hazarensis” population from Pakistan belongs to a widely distributed and taxonomically so far unresolved clade (see below), (ii) Pakistani mitochondrial sequences represent unique haplotypes with a close relationship to other populations from northern parts of the Indian subcontinent, (iii) comparing the overall phylogeographic pattern, the clade Duttaphrynus sp. 1/clade B has been most likely artificially translocated to South-East Asia, probably from the Indian subcontinent, (iv) the name for the clade Duttaphrynus sp. 1/clade B is available and, thus, should be applied (see Taxonomy). These results are thus important for both, evolution and taxonomy of the D. melanostictus complex. However, they still underline previous conclusions that the complex needs comprehensive biogeographical research with taxonomic revision based on dense DNA sampling, especially from the Indian subcontinent (see
By re-analysing 16S sequences of the D. melanostictus complex we showed a pattern of three main, geographically widely distributed clades (called as Duttaphrynus melanostictus, Duttaphrynus sp. 1, and sp. 2 sensu
For the clade Duttaphrynus sp. 1/clade B, we revealed a phylogeographic structure resembling patterns detected in other widely distributed amphibians of the Indo-Gangetic plain. These species colonized Pakistan from the Oriental region, namely Microhyla nilphamariensis Howlader, Nair, Gopalan, Merilä, 2015 and Sphaerotheca maskeyi (Schleich & Anders, 1998) (
Recently, two basic biogeographic scenarios have been hypothesized for the origin of different amphibian taxa distributed in northern Pakistan: i) dispersal of ancestral lineages during the Miocene from East or Southeast China into the far northwestern part of the Himalaya-Tibet orogen along a warm temperate corridor that existed in significant parts of Paleo-Tibet’s interior (
It is well known that the D. melanostictus complex is considered an invasive organism, with several evolutionary clades that were introduced out of its native range (
All the hereby presented markers and overall phylogenetic pattern of the clade to which sequences of D. melanostictus from Pakistan belong to (see Figs
Following the recommendation of
We would like to thank our colleagues, friends and local people for their help during the field work in Pakistan. For technical and laboratory support we thank Martina Lawson and Jana Poláková. For recommendations regarding the taxonomy we thank Frank Tillack. We are also grateful to S. R. Chandramouli and one anonymous reviewer for their useful comments on the manuscript. This work was supported to DJ by the Slovak Research and Development Agency under the contract APVV-19-0076, and by the German Research Foundation (DFG, grant no. HO 3792/8-1) to SH.
Table S1
Data type: excel file
Explanation note: Dataset of sequences used for the study.