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The low copy nuclear region, RPB2 as a novel DNA barcode region for species identification in the rattan genus Calamus (Arecaceae)

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Abstract

Taxonomic complexities, like environmental plasticity and homoplasy, make precise identification challenging in Calamus, the genus of spiny climbing palms of the subfamily Calamoideae (Arecaceae). In the present study, the species discriminatory power of twelve potential DNA barcode regions (rbcL, matK, psbA-trnH, rpoC, rpoB, psbK-psbI, atpF-atpH, psbZ-trnfM, ITS1, ITS2, PRK, and RPB2) were evaluated in 21 species of Calamus from the Western Ghats region of India, using distance, tree, and similarity based statistical methods. Except for the low copy nuclear region, RPB2, none of the tested plastid loci or nuclear loci ITS, either singly or in combinations, could discriminate all the species of Calamus due to low substitution rate of plastid regions and multiple copies of ITS respectively. The RPB2 locus showed highest species resolution with 96% accuracy in similarity based analysis, indicating its potential and efficiency as a barcode locus for the genus. The putative “Calamus gamblei complex” based on overlapping morphology was successfully resolved as six distinct, though closely related, species. The analysis also indicates that C. delessertianus is a morphological variant of C. dransfieldii. In spite of being a low copy nuclear gene region, RPB2 provided an efficient barcode to delineate Calamus species and has the potential to further extend its use as a prospective barcode to other Palm genera.

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Data Availability Statement

All the nucleotide sequence data generated in the study  was submitted to NCBI and the list of accession numbers are provided as supplementary table S1.

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Acknowledgements

We thank Kerala Forest Department, Govt. of Kerala and Karnataka Forest Department, Govt. of Karnataka for their permission to collect samples. The financial support received from Kerala State Council for Science Technology and Environment (KSCSTE) is also acknowledged.

Funding

The financial support for this study was received from Kerala State Council for Science, Technology and Environment (KSCSTE) (Grant No. KFRI RP 617/2011).

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Authors and Affiliations

  1. Forest Genetics and Biotechnology Division, Kerala Forest Research Institute, Peechi, Thrissur, Kerala, 680653, India

    Anoja Kurian, Suma Arun Dev & E. M. Muralidharan

  2. Forest Ecology and Biodiversity Conservation Division, Kerala Forest Research Institute, Peechi, Thrissur, Kerala, 680653, India

    Vadakkethil Balakrishnan Sreekumar

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  1. Anoja Kurian
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  2. Suma Arun Dev
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Contributions

AK: contributed towards conducting wet lab experiments, sample collections from the field, writing of the paper and analysing the data, SAD: involved in writing of the paper and designing the project, SVB: sample collections from the field site, identification and writing of the paper, MEM: involved in coordination of project and writing of the paper

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Correspondence to E. M. Muralidharan.

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Supplementary Fig.1.

PCR amplified products of the analysed DNA barcode loci (TIFF 202 kb)

Supplementary Fig.2.

Multiple Sequence Alignment showing species specific differences in the Calamus species using RPB2 (TIFF 2057 kb)

Supplementary Fig.3.

Multiple Sequence Alignment showing 300bp deletion in the C. hookerianus and C. pseudotenuis using psbA-trnH. (TIFF 580 kb)

Supplementary Fig.4.

Multiple Sequence Alignment showing transversions in the Calamus gamblei species complex (TIFF 1553 kb)

Supplementary Fig.5.

Neighbor-joining tree (NJ) of the genus Calamus using RPB2 based on p-distance using MEGA v.6.0 with bootstrap percentage value shown below (TIFF 53 kb)

Supplementary Fig.6.

Phylogenetic tree with posterior probability using RPB2 of the genus Calamus (TIFF 553 kb)

Supplementary Fig.7.

Phylogenetic tree with posterior probability using RPB2 of the family Arecaceae (TIFF 114 kb)

Supplementary Table S1.

List of samples collected for DNA Barcoding, their location and GenBank Accession numbers (DOC 118 kb)

Supplementary Table S2.

List of sequences downloaded from GenBank and their Accession numbers. (DOC 29 kb)

Supplementary File 1.

Multiple Sequence Alignment of RPB2 (MAS 50 kb)

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Kurian, A., Dev, S.A., Sreekumar, V.B. et al. The low copy nuclear region, RPB2 as a novel DNA barcode region for species identification in the rattan genus Calamus (Arecaceae). Physiol Mol Biol Plants 26, 1875–1887 (2020). https://doi.org/10.1007/s12298-020-00864-5

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