Research Article |
Corresponding author: Gaurang G. Gowande ( gaurang.gowande@gmail.com ) Academic editor: Alexander Haas
© 2021 Gaurang G. Gowande, Harshal S. Bhosale, Pushkar U. Phansalkar, Mandar Sawant, Zeeshan A. Mirza.
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:
Gowande GG, Bhosale HS, Phansalkar PU, Sawant M, Mirza ZA (2021) On the systematics and the phylogenetic position of the poorly known, montane dragon-lizard species Pseudocalotes austeniana (Annandale, 1908) (Squamata, Agamidae, Draconinae). Evolutionary Systematics 5(1): 141-150. https://doi.org/10.3897/evolsyst.5.67137
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The montane agamid species Pseudocalotes austeniana has had a complicated taxonomic history, as the species was initially described as a member of the genus Salea Gray, 1845. Later, the species was placed in a monotypic genus Mictopholis Smith, 1935, which was erected only to include this species; however, the species was later on transferred to the genus Pseudocalotes Fitzinger, 1843, owing to the morphological similarities, and lack of strong characters to diagnose the genus Mictopholis. Nonetheless, its precise phylogenetic and systematic position has remained unresolved due to the lack of molecular sequence data. During a herpetological expedition to Arunachal Pradesh, specimens of P. austeniana were collected from the hills near the type locality. The mitochondrial 16S rRNA, ND2 and ND4, and the nuclear RAG1 regions were subjected to molecular phylogenetics. Maximum Likelihood and Bayesian Inference gene trees revealed that P. austeniana is a member of the subfamily Draconinae. The analyses showed that the genus Pseudocalotes is polyphyletic, and P. austeniana was embedded within the genus Japalura Gray, 1853 sensu stricto. We here, thus, propose to transfer the species P. austeniana to the genus Japlaura, as Japalura austeniana comb. nov. Biogeographic and evolutionary significance of the findings are discussed.
Arunachal Pradesh, generic re-allocation, Japalura, Himalayas, northeast India
The montane agamid species Pseudocalotes austeniana (Annandale, 1908) has had a complicated taxonomic history.
During a herpetological expedition to Arunachal Pradesh, we collected specimens of P. austeniana (Fig.
Two specimens, an adult male (CESL1212) and a juvenile (CESL1204), were collected by hand and euthanized using halothane, after which, they were fixed in 6% formaldehyde solution for 48 hours. The fixed specimens were washed with water to remove traces of formaldehyde, following which, they were transferred to 70% ethanol for long-term preservation. Thigh muscle tissue was taken from the specimens immediately after euthanasia, and the tissues were stored in ~99.9% molecular grade ethanol for DNA extraction. The specimens and the tissues were deposited at the collections facility at CES. The specimens were photographed in life prior to euthanasia, and in preservation using Canon 100 mm macro lens, mounted on Canon 70D, illuminated with two Canon 430EX-II external flashes (Canon Inc., Tokyo, Japan).
Total genomic DNA was isolated from the preserved muscle tissues by using Qiagen DNAeasy kits following protocols provided by the manufacturer. A fragment each of the three mitochondrial 16S rRNA gene (16S), Nicotinamide Adenine Dinucleotide Hydrogenase (NADH) subunit 2 (ND2), NADH subunit 4 (ND4) gene and one nuclear Recombination Activating Gene 1 (RAG1) was amplified using published primers (
We downloaded the relevant mitochondrial genomic sequences by
Maximum Likelihood (ML) and Bayesian Inference (BI) trees based on the concatenated mitochondrial dataset were inferred to assess the relationships within the genus Pseudocalotes and the subfamily Draconinae, in general, and to determine the phylogenetic position of P. austeniana within the subfamily. The coding ND2 and ND4 regions were partitioned per-codon position, whereas the non-coding 16S region was not partitioned per-codon position, ML trees were reconstructed using W-IQ-TREE (
The partitioning schemes and the model of sequence evolution used for each partition, for all genes and their partitions, across all analyses, is summarized in Table
Optimum partitioning scheme and model choice for each partition. *BI analyses had only 6 partitions instead of 7 for the mitochondrial dataset, as the codon position ND4 2nd was merged with 16S at partition 4; whereas in the nuclear dataset the codon position RAG1 3rd was merged with RAG1 2nd .
Partition | Codon position | Model | |
Mitochondrial | ML | BI | |
1 | ND2 1st | GTR+I+G | GTR+I+G |
2 | ND2 2nd | TN | GTR+I+G |
3 | ND2 3rd | TN+I+G | GTR+I+G |
4 | 16S | TIM2+I+G | GTR+I+G |
5 | ND4 1st | TIM3+I+G | GTR+G |
6 | ND4 2nd | TVM+G | * |
7 | ND4 3rd | K3Pu+I+G | HKY+I+G |
Nuclear | ML | BI | |
1 | RAG1 1st | TIM3+G | HKY+G |
2 | RAG1 2nd | TIM3+G | K80+G |
3 | RAG1 3rd | TIM3+G | * |
The ML and BI phylogenetic trees inferred from the mitochondrial ND4 region (Figure
ML+BI phylogenetic tree based on the 577 bp long mitochondrial ND4 dataset, built under the models TIM3+F+G4, GTR+F+I+G4, TPM3u+F+I+G4 (ML) and GTR+G, GTR+I+G and HKY+I+G (BI) for codon positions 1, 2 and 3 respectively. Numbers at the nodes indicate ML ultrafast ultrafast bootstrap values/BI posterior probabilities.
Pseudocalotes austeniana, nevertheless, did not cluster with any of the two clades, and was instead recovered as sister to Draco spp. (ML ultrafast bootstrap values 91, BI posterior probability 0.99). The ML and BI phylogenies inferred from the concatenated dataset were used to elucidate the position of P. austeniana within the subfamily Draconinae. The species was recovered as a member of the genus Japalura sensu stricto, sister to a clade containing the species J. kumaonensis Annandale, 1907, J. tricarinata Blyth, 1853 and the type species J. variegata Gray, 1853. The relationship was well-supported in the ML analysis (ultrafast bootstrap value 88), but less strongly in the BI analysis (posterior probability 0.81). The (Ptyctolaemus + (Japalura + Draco)) relationship was very well supported (ML ultrafast bootstrap values 100, BI posterior probabilities 1). In the most recent revision of Japalura sensu lato,
Maximum Likelihood phylogenetic tree based on the 2062 bp long mitochondrial (ND2+16S+ND4) concatenated dataset revealing the phylogenetic position of Japalura austeniana comb. nov. Numbers at the nodes indicate ML ultrafast bootstrap values.
The genus Pseudocalotes was found to be polyphyletic (Figs
It seems to be necessary to objectively define the genus Pseudocalotes sensu stricto. It appears that the genus Pseudocalotes is used by authors as per convenience, and the species which cannot be objectively allotted to specific genera are tentatively placed in this genus. Most species of the P. kakhienensis clade were earlier placed in a genus different than Pseudocalotes, but were later transferred to Pseudocalotes. For example, P. kaulbacki (Smith, 1937), a junior subjective synonym of P. kingdonwardi (Smith, 1935) was described as a member of Japalura. Pseudocalotes kakhienensis (Anderson, 1879) has been variously placed in the genera Oriocalotes, Acanthosaura, Calotes, and Salea, until it was transferred to Pseudocalotes (
With the increase in the availability of molecular sequence data for multiple species of the agamid subfamily Draconinae, our understanding of the systematic positions and the evolutionary relationships between the genera and their species is rapidly improving, as has been recently evidenced by the description of multiple new genera from south and southeast Asia (
Furthermore, northeast India lies at the interface of southeast Asia and mainland India, thus, enriching the molecular and the morphological databases, especially using the taxa from northeast India and adjoining Himalayan regions is of extreme importance, as these may be useful in testing hypotheses pertaining to the origin and dispersal, into or Out-of India, of the lizards of the subfamily Draconinae.
Molecular phylogenetic reconstructions reveal that the genus Pseudocalotes is polyphyletic in nature. The montane agamid species Pseudocalotes austeniana was not recovered as a member of the genus Pseudocalotes, but instead was found to cluster with Japalura spp. The position of P. austeniana within Pseudocalotes is untenable, and we propose to transfer the species to Japalura sensu stricto as Japalura austeniana comb. nov.
The authors are grateful to Arunachal Pradesh Forest Department, for kindly issuing the necessary permits to carry out the surveys: (CWL/Gen/173/2018-19/Pt.V11/2421-33 and CWL/Gen/173/2018-19/Pt.V11/2434-43). The authors thank Evolutionary Ecology and Biogeography lab at CES, IISc for providing the facilities for molecular analyses. GG is indebted to Sonali Joshi, Fergusson College, and Dhanashree Paranjpe and Ankur Patwardhan, Abasaheb Garware College, for being extremely supportive. GG also acknowledges the support of The Principal, Anasaheb Garware College and the Principal, Fergusson College. The study was funded by the Rufford Small Grants to GG. HB extends his heartfelt gratitude to Shripad Halbe and Brihat Bharatiya Samaj for the generous support in funding the expedition. Thanks are also due to Sandesh Kadur and Debabrata Phukon for their help with logisitics and to Deepak Apte and Rahul Khot (
A list of species, their corresponding sequences and accession numbers used in this study
Data type: phylogenetic, genomic, specimen
Explanation note: A table containing a list of species, the corresponding specimens and GenBank(R) accession numbers for each of the genes used in the phylogenetic analyses.
Figure S1
Data type: phylogenetic, images phylogenetic, images
Explanation note: ML+BI tree topology based on 1020 bp long nuclear RAG1 dataset.