The changing epitome of species identification - DNA barcoding

Abstract

The discipline taxonomy (the science of naming and classifying organisms, the original bioinformatics and a basis for all biology) is fundamentally important in ensuring the quality of life of future human generation on the earth; yet over the past few decades, the teaching and research funding in taxonomy have declined because of its classical way of practice which lead the discipline many a times to a subject of opinion, and this ultimately gave birth to several problems and challenges, and therefore the taxonomist became an endangered race in the era of genomics. Now taxonomy suddenly became fashionable again due to revolutionary approaches in taxonomy called DNA barcoding (a novel technology to provide rapid, accurate, and automated species identifications using short orthologous DNA sequences). In DNA barcoding, complete data set can be obtained from a single specimen irrespective to morphological or life stage characters. The core idea of DNA barcoding is based on the fact that the highly conserved stretches of DNA, either coding or non coding regions, vary at very minor degree during the evolution within the species. Sequences suggested to be useful in DNA barcoding include cytoplasmic mitochondrial DNA (e.g. cox1) and chloroplast DNA (e.g. rbcL, trnL-F, matK, ndhF, and atpB rbcL), and nuclear DNA (ITS, and house keeping genes e.g. gapdh). The plant DNA barcoding is now transitioning the epitome of species identification; and thus, ultimately helping in the molecularization of taxonomy, a need of the hour. The 'DNA barcodes' show promise in providing a practical, standardized, species-level identification tool that can be used for biodiversity assessment, life history and ecological studies, forensic analysis, and many more.

Keywords: Biodiversity; Conservation genetics; DNA barcoding; Molecular markers; Plant taxonomy; Species identification.

Figure 1

Sizes of plant genomes. Sizes (bp) vs. chromosome numbers (2n) of plant genomes from different taxa and compared to human genome size (3.2 × 10⁹ bp).

Figure 2

rDNA sequence domains of tandem gene clusters (about 10 kb each) of nuclear and organellar rDNAs of different organisms and organelles (cp and mt). (a) Pre-rDNA in the genome and (b) the translated ribosomal subunits. Abbreviations: ETS – external transcribed spacer, ITS – internal transcribed spacer, NTS – non transcribed spacer, mt – mitochondrion; cp – chloroplast, nu – nucleus, 5.8S to 28S – ribosomal subunits of nucleoproteins of rRNAs.

Figure 3

Sample of ITS phylogeny. ITS cladogaram of Legume trees. ITS sequences of twenty-two species were analyzed by BioEdit (Hall, 1999), and ML (Maximum Likelihood; Hillis et al., 1994) cladogaram was edited by MEGA4 (Tamura et al., 2007) (×1000 bootstrap). The three subfamilies of Fabaceae and the substitution rate (0.1) are indicated.

Figure 4

Samples of ITS sequence polymorphism. Sequence alignment of ITS sequences of rDNAs of twenty-two Legume trees studied with low (a) (180–270 nt) and high (b) (270–360 nt) sequence similarities.

Figure 5

cpDNA. Comparative sizes (bp) of total plant cpDNAs of different taxa (with NCBI accession numbers; Altschul et al., 1997). (1) Gymnosperm Cycas taitungensisNC_009618. (2) Transition species between gymnosperrm and angiosperms Amborella trichopodaNC_005086. (3) Platanus occidentalisNC_008335. (4) Vitis viniferaNC_007957. (5) Nuphar advenaNC_008788. (6) Nymphaea albaNC_006050. (7) Liriodendron tulipiferaNC_008326. (8) Morus indicaNC_008359. (9) Prunus persicaNC_014697. (10) Populus trichocarpaNC_009143. (11) Populus albaNC_008235. (12) Arabidopsis thalianaNC_000932. (13) Unicellular green alga Euglena gracilisNC_001603. (14) Zea maysNC_001666. (15) Hordeum vulgareNC_008590. (16) Triticum aestivumNC_002762. (17) Oryza sativa JaponicaNC_001320. (18) Oryza sativa indicaNC_008155. (19) Equisetum arvenseNC_014699. (20) Marchantia polymorphaNC_001319. (21) Lathyrus sativusNC_014063. (22) Welwitschia mirabilisNC_010654. (23) Cedrus deodaraNC_014575. (24) The longest-living plant Pinus longaevaNC_011157. (25) Durinskia balticaNC_014287. (26) Pinus monophyllaNC_011158. (27) Gnetum parvifoliumNC_011942. (28) Ephedra equisetinaNC_011954. (29) Cathaya argyrophyllaNC_014589 (described in 1955). (30) Cuscuta obtusifloraNC_009949. (31) Euglena longaNC_002652. (32) Epifagus virginianaNC_001568.

Figure 6

Cladogram of total cpDNAs. ML (Maximum Likelihood; Hillis et al., 1994) cladogaram of total cpDNA genomes with 1000× bootstrap values (MEGA4; Tamura et al., 2007). Polyphyletic Dicots of Angiosperm are not labeled; monocots (○) show four cpDNA lineages including the individual Acorus. Gymnosperm clades (green) show three lineages: (1) Gnetum (▾) – Podocarpus (●) and the Pinaceae species of Pinus, Picea, Abies, Cathaya and Cedrus. Cathaya (▴) (NC_014589) has the smallest cpDNA genome (107.122 bp) of Coniferales. This clade shows close genetic distance to the bryophyte Nothoceros; (2) Taxodiaceae including Taiwania, Taxus, and Cunninghamia, which clade is close to eudicot Monsomia and Geranium; and (3) the evolutionarily youngest gymnosperm Cepahlotaxaceae (Cephalotaxus) of Coniferales. Water submerged plants of monocot Elodea and dicot Ceratophyllum (◊); and parasite plants with reduced cpDNA genomes of Cuscuta and Epifagus (●) are also labeled. Scale (0.05) shows relative genetic distances based on substitution rate. Accession numbers are available at NCBI (http://www.ncbi.nlm.nih.gov) (Altschul et al., 1997) and CGP (http://chloroplast.ocean.washington.edu). For computing about 21 million nucleotides an eight core computer with 24 GB RAM was used running for 2–3 weeks.

Figure 7

The mtDNA (mitome) sizes (bp) of organisms in increasing order including the only wood available Cycas (13). Human mtDNS (1), and lower plants (2–7) are indicated (NCBI accession #, Altschul et al. 1997). (1) Homo s.NC_012920. (2) Mesostigma v.NC_008240. (3) Chara v.NC_005255. (4) Physcomitrella p.NC_007945. (5) Megaceros ae.NC_012651. (6) Marchantia p.NC_001660. (7) Phaeoceros l.NC_013765. (8) Brassica n.NC_008285. (9) Silene l.NC_014487. (10) Arabidopsis th.NC_001284. (11) Beta v.NC_002511. (12) Citrullus l.NC_014043. (13) Cycas taitungensisNC_010303. (14) Nicotiana t.NC_006581. (15) Triticum ae.NC_007579. (16) Sorghum b.NC_008360. (17) Carica p.NC_012116. (18) Oryza s. J.NC_011033. (19) Oryza s. I.NC_007886. (20) Zea lux.NC_008333. (21) Oryza r.NC_013816. (22) Zea m.NC_007982. (23) Zea pren.NC_008331. (24) Zea parv.NC_008332. (25) Tripsacum d.NC_008362. (26) Vitis v.NC_012119. (27) Cucurbita p.NC_014050. (28) Cucumis sativus. (29) Cucumis melo with the largest mtDNA. The mtDNAs of Human (1), Cucurbits (12, 27, 28, 29), and gymnosperm Cycas (13) are indicated.

Figure 8

Samples’ (60 nt) sequence polymorphism of pPongy2 LTR retrotransposons. Spread and sequence diversity of RT (reverse transcriptase) gene of pPongy2, a Ty3-gypsy-like LTR-retrotransposon though the evolution of gymnosperms from Gingo → Podocarpus (248 My) → Araucaria (230 My) → Taxus (206 My) → Pinaceae (180 My). Sequence # AJ290647.1 was downloaded from NCBI and aligned by BioEdit program (Hall, 1999).

Figure 9

The pPongy2 cladogram. Fast Minimum Dendrogram edited by NCBI server (Altschul et al., 1997) shows the spread among-and-within species of RT (reverse transcriptase) gene of pPongy2, a Ty3-gypsy-like LTR-retrotrasnposon though the evolutionary lineage of gymnosperms from Gingo → Podocarpus (248 My) → Araucaria (230 My) → Taxus (206 My) → Pinaceae (180 My). Genetic distance (scale 0.005) and branch length are indicated, and gymnosperm species are labeled with different color symbols. The accession numbers of taxon included in analyses were Araucaria araucana (AJ290651, AJ290652, AJ290653, AJ290654, AJ290655), Ginkgo biloba (AJ290656), Picea abies (AJ290585, AJ290586, AJ290591, AJ290592, AJ290593, AJ290594), Pinus pinaster (AJ290605, AJ290606), Pinus pumila (AJ290616), Pinus sibirica (AJ290623, AJ290626, AJ290629, AJ290630, AJ290631), Podocarpus totara (AJ290647, AJ290648, AJ290649, AJ290650), Taxus baccata (AJ290640, AJ290641, AJ290642, AJ290643, AJ290644, AJ290645).

Figure 10

Size correlations between cpDNA and mtDNA genomes show a shift from green algae (Chara vulgaris) with high cpDNA/mtDNA ratio (2.73) through mosses of Physcomitrella patens (1.16) and Marchantia polymorpha (0.65) toward flowering plants of dicots to monocots with exception of Vitis. The decreasing ratio of cpDNA/mtDNA indicates an enlarging mtDNA during the evolution: Spirodela polyrhiza (0.74); Brassica napus (0.69); Daucus carota (0.55); Helianthus annuus (0.50); Arabidopsis thaliana (0.42); Lotus japonicus (0.40); Vaccinium macrocarpon (0.38); Glycine max (0.38); Vigna radiata (0.38); Huperzia lucidula (0.37); Ricinus communis (0.32); Sorghum bicolor (0.30); Triticum aestivum (0.29); Liriodendron tulipifera (0.29); Oryza sativa Japonica (0.274); Oryza sativa Indica (0.273); Zea mays (0.25); Oryza rufipogon (0.24); Phoenix dactylifera (0.22); Vitis vinifera (0.21). NCBI (Altschul et al., 1997) data were plotted by XY plot of Microsoft Windows Xcel program.

Figure 11

Changes of organelle genome sizes during the evolution (mtDNA – mitochondrial DNA; cpDNA chloroplast DNA) (see Fig. 10). NCBI (Altschul et al., 1997) data were plotted by Microsoft Windows Xcel program. Monocots are labeled with open symbols.