Journal of Molecular Evolution

, Volume 86, Issue 3–4, pp 216–239 | Cite as

Application of Chloroplast Phylogenomics to Resolve Species Relationships Within the Plant Genus Amaranthus

  • Erika Viljoen
  • Damaris A. Odeny
  • Martin P. A. Coetzee
  • Dave K. Berger
  • David J. G. Rees
Original Article


Amaranthus species are an emerging and promising nutritious traditional vegetable food source. Morphological plasticity and poorly resolved dendrograms have led to the need for well resolved species phylogenies. We hypothesized that whole chloroplast phylogenomics would result in more reliable differentiation between closely related amaranth species. The aims of the study were therefore: to construct a fully assembled, annotated chloroplast genome sequence of Amaranthus tricolor; to characterize Amaranthus accessions phylogenetically by comparing barcoding genes (matK, rbcL, ITS) with whole chloroplast sequencing; and to use whole chloroplast phylogenomics to resolve deeper phylogenetic relationships. We generated a complete A. tricolor chloroplast sequence of 150,027 bp. The three barcoding genes revealed poor inter- and intra-species resolution with low bootstrap support. Whole chloroplast phylogenomics of 59 Amaranthus accessions increased the number of parsimoniously informative sites from 92 to 481 compared to the barcoding genes, allowing improved separation of amaranth species. Our results support previous findings that two geographically independent domestication events of Amaranthus hybridus likely gave rise to several species within the Hybridus complex, namely Amaranthus dubius, Amaranthus quitensis, Amaranthus caudatus, Amaranthus cruentus and Amaranthus hypochondriacus. Poor resolution of species within the Hybridus complex supports the recent and ongoing domestication within the complex, and highlights the limitation of chloroplast data for resolving recent evolution. The weedy Amaranthus retroflexus and Amaranthus powellii was found to share a common ancestor with the Hybridus complex. Leafy amaranth, Amaranthus tricolor, Amaranthus blitum, Amaranthus viridis and Amaranthus graecizans formed a stable sister lineage to the aforementioned species across the phylogenetic trees. This study demonstrates the power of next-generation sequencing data and reference-based assemblies to resolve phylogenies, and also facilitated the identification of unknown Amaranthus accessions from a local genebank. The informative phylogeny of the Amaranthus genus will aid in selecting accessions for breeding advanced genotypes to satisfy global food demand.


Phylogenomics Chloroplast Amaranthus Barcode 



The authors wish to thank the Department of Science and Technology of South Africa, the National Research Foundation and the Professional Development Program of the Agricultural Research Council (ARC) in South Africa for providing funding for the PhD study from where this work originated. The authors also thank Dr Charles Hefer at the ARC for bioinformatics support. The authors thank Mr Willem Jansen van Rensburg and his staff at the ARC Vegetable and Ornamental Plant Institute for providing the Amaranthus germplasm set (SAG) as well as plant maintenance. The authors thank the Core Facility team at the ARC Biotechnology Platform for DNA sequencing.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

239_2018_9837_MOESM1_ESM.docx (333 kb)
Supplementary material 1 (DOCX 332 KB)


  1. Achigan-Dako EG, Sogbohossou OE, Maundu P (2014) Current knowledge on Amaranthus spp.: research avenues for improved nutritional value and yield in leafy amaranths in sub-Saharan Africa. Euphytica 197:303–317CrossRefGoogle Scholar
  2. Akond M, Islam S, Wang X (2013) Genotypic variation in biomass traits and cell wall components among 35 diverse accessions of Amaranthaceae family. J Appl Phytotechnol Environ Sanit 2:37–45Google Scholar
  3. Alamgir M, Kibria M, Islam M (2011) Effects of farm yard manure on cadmium and lead accumulation in Amaranth (Amaranthus oleracea L.). J Soil Sci Environ Manag 2:237–240Google Scholar
  4. Alemayehu RF, Bendevis MA, Jacobsen SE (2015) The potential for utilizing the seed crop amaranth (Amaranthus spp.) in East Africa as an alternative crop to support food security and climate change mitigation. J Agron Crop Sci 201:321–329CrossRefGoogle Scholar
  5. Barrett CF, Davis JI, Leebens-Mack J, Conran JG, Stevenson DW (2013) Plastid genomes and deep relationships among the commelinid monocot angiosperms. Cladistics 29:65–87CrossRefGoogle Scholar
  6. Bell KL, de Vere N, Keller A, Richardson RT, Gous A, Burgess KS, Brosi BJ (2016) Pollen DNA barcoding: current applications and future prospects 1. Genome 59:629–640CrossRefPubMedGoogle Scholar
  7. Bezeng B, Davies TJ, Daru BH, Kabongo RM, Maurin O, Yessoufou K, van der Bank H, Van der Bank M (2017) Ten years of barcoding at the African Centre for DNA barcoding. Genome 60:629–638CrossRefPubMedGoogle Scholar
  8. Braukmann TW, Kuzmina ML, Sills J, Zakharov EV, Hebert PD (2017) Testing the efficacy of DNA barcodes for identifying the vascular plants of Canada. PLoS ONE 12:e0169515CrossRefPubMedPubMedCentralGoogle Scholar
  9. Brenner DM, Baltensperger DD, Kulakow PA, Lehmann JW, Myers RL, Slabbert MM, Sleugh BB (2000) Genetic resources and breeding of Amaranthus. In: Janick J (ed) Plant breeding reviews, vol 19. Wiley, New York, pp 227–285Google Scholar
  10. Burgess KS, Fazekas AJ, Kesanakurti PR, Graham SW, Husband BC, Newmaster SG, Percy DM, Hajibabaei M, Barrett SC (2011) Discriminating plant species in a local temperate flora using the rbcL + matK DNA barcode. Methods Ecol Evol 2:333–340CrossRefGoogle Scholar
  11. Chan K, Sun M (1997) Genetic diversity and relationships detected by isozyme and RAPD analysis of crop and wild species of Amaranthus. TAG 95:865–873CrossRefGoogle Scholar
  12. Chaney L, Mangelson R, Ramaraj T, Jellen EN, Maughan PJ (2016) The complete chloroplast genome sequences for four Amaranthus species (Amaranthaceae). Appl Plant Sci 4:1600063CrossRefGoogle Scholar
  13. Chung H-J, Jung JD, Park H-W, Kim J-H, Cha HW, Min SR, Jeong W-J, Liu JR (2006) The complete chloroplast genome sequences of Solanum tuberosum and comparative analysis with Solanaceae species identified the presence of a 241-bp deletion in cultivated potato chloroplast DNA sequence. Plant Cell Rep 25:1369–1379CrossRefPubMedGoogle Scholar
  14. Costea M, Brenner DM, Tardif FJ, Tan YF, Sun M (2006) Delimitation of Amaranthus cruentus L. and Amaranthus caudatus L. using micromorphology and AFLP analysis: an application in germplasm identification. Genet Resour Crop Ev 53:1625–1633CrossRefGoogle Scholar
  15. Cuénoud P, Savolainen V, Chatrou LW, Powell M, Grayer RJ, Chase MW (2002) Molecular phylogenetics of Caryophyllales based on nuclear 18S rDNA and plastid rbcL, atpB, and matK DNA sequences. Am J Bot 89:132–144CrossRefPubMedGoogle Scholar
  16. Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: more models, new heuristics and parallel computing. Nat Methods 9:772–772CrossRefPubMedPubMedCentralGoogle Scholar
  17. Das S (2011) Systematics and taxonomic delimitation of vegetable, grain and weed amaranths: a morphological and biochemical approach. Genet Resour Crop Evol 59:289–303CrossRefGoogle Scholar
  18. Davis CC, Xi Z, Mathews S (2014) Plastid phylogenomics and green plant phylogeny: almost full circle but not quite there. BMC Biol 12:11–15CrossRefPubMedPubMedCentralGoogle Scholar
  19. Dong W, Xu C, Cheng T, Lin K, Zhou S (2013) Sequencing angiosperm plastid genomes made easy: a complete set of universal primers and a case study on the phylogeny of Saxifragales. GenBiol Evol 5:989–997Google Scholar
  20. Dong W, Liu H, Xu C, Zuo Y, Chen Z, Zhou S (2014) A chloroplast genomic strategy for designing taxon specific DNA mini-barcodes: a case study on ginsengs. BMC Genet 15:138CrossRefPubMedPubMedCentralGoogle Scholar
  21. Dong W, Xu C, Li C, Sun J, Zuo Y, Shi S, Cheng T, Guo J, Zhou S (2015))ycf1, the most promising plastid DNA barcode of land plants. Sci Rep 5:8348CrossRefPubMedPubMedCentralGoogle Scholar
  22. Ebert AW (2014) Potential of underutilized traditional vegetables and legume crops to contribute to food and nutritional security, income and more sustainable production systems. Sust 6:319–335CrossRefGoogle Scholar
  23. Gerrano AS, van Rensburg WSJ, Adebola PO (2015) Genetic diversity of Amaranthus species in South Africa. S. Afr J Plant Soil 32:39–46CrossRefGoogle Scholar
  24. Gudu S, Gupta V (1988) Male-sterility in the grain amaranth (Amaranthus hypochondriacus ex-Nepal) variety Jumla. Euphytica 37:23–26CrossRefGoogle Scholar
  25. Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52:696–704CrossRefPubMedGoogle Scholar
  26. Hackett SJ, Kimball RT, Reddy S, Bowie RC, Braun EL, Braun MJ, Chojnowski JL, Cox WA, Han K-L, Harshman J (2008) A phylogenomic study of birds reveals their evolutionary history. Science 320:1763–1768CrossRefPubMedGoogle Scholar
  27. Hollingsworth PM, Graham SW, Little DP (2011) Choosing and using a plant DNA barcode. PLoS ONE 6:e19254CrossRefPubMedPubMedCentralGoogle Scholar
  28. Hollingsworth PM, Li D-Z, van der Bank M, Twyford AD (2016) Telling plant species apart with DNA: from barcodes to genomes. Phil Trans R Soc B 371:20150338CrossRefPubMedPubMedCentralGoogle Scholar
  29. Huang H, Shi C, Liu Y, Mao S-Y, Gao L-Z (2014) Thirteen Camellia chloroplast genome sequences determined by high-throughput sequencing: genome structure and phylogenetic relationships. BMC Evol Biol 14:151–168CrossRefPubMedPubMedCentralGoogle Scholar
  30. Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17:754–755CrossRefPubMedGoogle Scholar
  31. Kavita P, Gandhi P (2017) Rediscovering the therapeutic potential of Amaranthus species: a review. Egypt J Basic Appl Sci 4:196–205CrossRefGoogle Scholar
  32. Kim JS, Kim JH (2013) Comparative genome analysis and phylogenetic relationship of order Liliales insight from the complete plastid genome sequences of two Lilies (Lilium longiflorum and Alstroemeria aurea). PLoS One 8:e68180CrossRefPubMedPubMedCentralGoogle Scholar
  33. Kress WJ, Erickson DL (2007) A two-locus global DNA barcode for land plants: the coding rbcL gene complements the non-coding trnH-psbA spacer region. PLoS ONE 2:e508CrossRefPubMedPubMedCentralGoogle Scholar
  34. Kuzoff RK, Gasser CS (2000) Recent progress in reconstructing angiosperm phylogeny. Trends Plant Sci 5:330–336CrossRefPubMedGoogle Scholar
  35. Lahaye R, Van der Bank M, Bogarin D, Warner J, Pupulin F, Gigot G, Maurin O, Duthoit S, Barraclough TG, Savolainen V (2008) DNA barcoding the floras of biodiversity hotspots. PNAS 105:2923–2928CrossRefPubMedPubMedCentralGoogle Scholar
  36. Li H, Cao H, Cai Y-F, Wang J-H, Qu S-P, Huang X-Q (2014) The complete chloroplast genome sequence of sugar beet (Beta vulgaris ssp. vulgaris). Mitochondrial DNA 25:209–211CrossRefPubMedGoogle Scholar
  37. Lightfoot DJ, Jarvis DE, Ramaraj T, Lee R, Jellen EN, Maughan PJ (2017) Single-molecule sequencing and Hi-C-based proximity guided assembly of amaranth (Amaranthus hypochondriacus) chromosomes provide insights into genome evolution. BMC Biol 15:74CrossRefPubMedPubMedCentralGoogle Scholar
  38. Liu Y, Huo N, Dong L, Wang Y, Zhang S, Young HA, Feng X, Gu YQ (2013) Complete chloroplast genome sequences of Mongolia medicine Artemisia frigida and phylogenetic relationships with other plants. PLoS ONE 8:e57533CrossRefPubMedPubMedCentralGoogle Scholar
  39. Lubbe E, Rodda N (2016) Effects of greywater irrigation on germination, growth and photosynthetic characteristics in selected African leafy vegetables. Water SA 42:203–212CrossRefGoogle Scholar
  40. Ma P-F, Zhang Y-X, Zeng C-X, Guo Z-H, Li D-Z (2014) Chloroplast phylogenomic analyses resolve deep-level relationships of an intractable bamboo tribe Arundinarieae (Poaceae). Syst Biol 63:933–950CrossRefPubMedGoogle Scholar
  41. Mallory MA, Hall RV, McNabb AR, Pratt DB, Jellen EN, Maughan PJ (2008) Development and characterization of microsatellite markers for the grain amaranths. Crop Sci 48:1098–1106CrossRefGoogle Scholar
  42. Mandal N, Das P (2002) Intra-and interspecific genetic diversity in grain Amaranthus using random amplified polymorphic DNA markers. Plant Tissue Cult 12:49–56Google Scholar
  43. Maughan PJ, Yourstone SM, Jellen EN, Udall JA (2009) SNP discovery via genomic reduction, barcoding, and 454-pyrosequencing in amaranth. The Plant Gen J 2:260–270CrossRefGoogle Scholar
  44. Mlakar SG, Turinek M, Jakop M, Bavec M, Bavec F (2010) Grain amaranth as an alternative and perspective crop in temperate climate. J Geogr 5:135–145Google Scholar
  45. Mnkeni A, Masika P, Maphaha M (2007) Nutritional quality of vegetable and seed from different accessions of Amaranthus in South Africa. Water SA 33:377–380Google Scholar
  46. Mosyakin SL, Robertson KR (1996) New infrageneric taxa and combinations in Amaranthus (Amaranthaceae). A Bot Fennici 33:275–281Google Scholar
  47. Nikiforova SV, Cavalieri D, Velasco R, Goremykin V (2013) Phylogenetic analysis of 47 chloroplast genomes clarifies the contribution of wild species to the domesticated apple maternal line. Mol Biol Evol 30:1751–1760CrossRefPubMedGoogle Scholar
  48. Nock CJ, Waters DL, Edwards MA, Bowen SG, Rice N, Cordeiro GM, Henry RJ (2011) Chloroplast genome sequences from total DNA for plant identification. Plant Biotechnol J 9:328–333CrossRefPubMedGoogle Scholar
  49. Panero JL, Funk V (2008) The value of sampling anomalous taxa in phylogenetic studies: major clades of the Asteraceae revealed. Mol Phylogenet Evol 47:757–782CrossRefPubMedGoogle Scholar
  50. Parks M, Cronn R, Liston A (2009) Increasing phylogenetic resolution at low taxonomic levels using massively parallel sequencing of chloroplast genomes. BMC Biol 7:84CrossRefPubMedPubMedCentralGoogle Scholar
  51. Patil SM, Rane NR, Adsul AA, Gholave AR, Yadav SR, Jadhav JP, Govindwar SP (2016) Study of molecular genetic diversity and evolutionary history of medicinally important endangered genus Chlorophytum using DNA barcodes. Biochem Syst Ecol 65:245–252CrossRefGoogle Scholar
  52. Poliseno L, Salmena L, Zhang J, Carver B, Haveman WJ, Pandolfi PP (2010) A coding-independent function of gene and pseudogene mRNAs regulates tumour biology. Nature 465:1033–1038CrossRefPubMedPubMedCentralGoogle Scholar
  53. Puigbò P, Garcia-Vallvé S, McInerney JO (2007) TOPD/FMTS: a new software to compare phylogenetic trees. Bioinformatics 23:1556–1558CrossRefPubMedGoogle Scholar
  54. Raju M, Varakumar S, Lakshminarayana R, Krishnakantha T, Baskaran V (2007) Carotenoid composition and vitamin A activity of medicinally important green leafy vegetables. Food Chem 101:1598–1605CrossRefGoogle Scholar
  55. Rastogi A, Shukla S (2013) Amaranth: a new millennium crop of nutraceutical values. Crit Rev Food Sci Nutr 53:109–125CrossRefPubMedGoogle Scholar
  56. Sangeetha RK, Baskaran V (2010) Carotenoid composition and retinol equivalent in plants of nutritional and medicinal importance: efficacy of β-carotene from Chenopodium album in retinol-deficient rats. Food Chem 119:1584–1590CrossRefGoogle Scholar
  57. Sato S, Nakamura Y, Kaneko T, Asamizu E, Tabata S (1999) Complete structure of the chloroplast genome of Arabidopsis thaliana. DNA Res 6:283–290CrossRefPubMedGoogle Scholar
  58. Sauer JD (1967) The grain amaranths and their relatives: a revised taxonomic and geographic survey. Ann Mo Bot Gard 54:103–137CrossRefGoogle Scholar
  59. Schmitz-Linneweber C, Maier RM, Alcaraz J-P, Cottet A, Herrmann RG, Mache R (2001) The plastid chromosome of spinach (Spinacia oleracea): complete nucleotide sequence and gene organization. Plant Mol Biol 45:307–315CrossRefPubMedGoogle Scholar
  60. Shaw J, Shafer HL, Leonard OR, Kovach MJ, Schorr M, Morris AB (2014) Chloroplast DNA sequence utility for the lowest phylogenetic and phylogeographic inferences in angiosperms: the tortoise and the hare IV. AmJBot 101:1987–2004Google Scholar
  61. Shinozaki K, Ohme M, Tanaka M, Wakasugi T, Hayashida N, Matsubayashi T, Zaita N, Chunwongse J, Obokata J, Yamaguchi-Shinozaki K (1986) The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression. The EMBO J 5:2043–2049PubMedGoogle Scholar
  62. Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Söding J (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7:539–545CrossRefPubMedPubMedCentralGoogle Scholar
  63. Soltis PS, Soltis DE, Chase MW (1999) Angiosperm phylogeny inferred from multiple genes as a tool for comparative biology. Nature 402:402–404CrossRefPubMedGoogle Scholar
  64. Srivastava R (2017) An updated review on phyto-pharmacological and pharmacognostical profile of Amaranthus tricolor: A herb of nutraceutical potentials. Pharma Innov J 6:127–129Google Scholar
  65. Stamatakis A (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 22:2688–2690CrossRefGoogle Scholar
  66. Stetter MG, Schmid KJ (2017) Analysis of phylogenetic relationships and genome size evolution of the Amaranthus genus using GBS indicates the ancestors of an ancient crop. Mol Phylogenet Evol 109:80–92CrossRefPubMedGoogle Scholar
  67. Stetter MG, Müller T, Schmid KJ (2017) Genomic and phenotypic evidence for an incomplete domestication of South American grain amaranth (Amaranthus caudatus). Mol Ecol 26:871–886CrossRefPubMedGoogle Scholar
  68. Straub SC, Parks M, Weitemier K, Fishbein M, Cronn RC, Liston A (2012) Navigating the tip of the genomic iceberg: next-generation sequencing for plant systematics. Am J Bot 99:349–364CrossRefPubMedGoogle Scholar
  69. Stull GW, Moore MJ, Mandala VS, Douglas NA, Kates HR, Qi X, Brockington SF, Soltis PS, Soltis DE, Gitzendanner MA (2013) A targeted enrichment strategy for massively parallel sequencing of angiosperm plastid genomes. Appl Plant Sci 1:1200497CrossRefGoogle Scholar
  70. Sugiura M (1992) The chloroplast genome. In: Schilperoort RA, Dure L (eds) 10 Years plant molecular biology. Springer, Dordrecht, pp 149–168CrossRefGoogle Scholar
  71. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739CrossRefPubMedPubMedCentralGoogle Scholar
  72. Timofte I, Timofte N, Brega V (2009) Development of bioenergy in Moldova. Problemele Energeticii Regionale 2:1–12Google Scholar
  73. Van Rensburg WJ, Van Averbeke W, Slabbert R, Faber M, Van Jaarsveld P, Van Heerden I, Wenhold F, Oelofse A (2007) African leafy vegetables in South Africa. Water SA 33:317–326Google Scholar
  74. Venskutonis PR, Kraujalis P (2013) Nutritional components of amaranth seeds and vegetables: a review on composition, properties, and uses. Comp Rev Food Sci Food Saf 12:381–412CrossRefGoogle Scholar
  75. Waselkov K (2013) Population Genetics and Phylogenetic Context of Weed Evolution in the Genus Amaranthus: Amaranthaceae. PhD Thesis, University of WashingtonGoogle Scholar
  76. Wassom JJ, Tranel PJ (2005) Amplified Fragment Length Polymorphism-Based genetic relationships among weedy Amaranthus species. J Hered 96:410–416CrossRefPubMedGoogle Scholar
  77. Williams AV, Miller JT, Small I, Nevill PG, Boykin LM (2016) Integration of complete chloroplast genome sequences with small amplicon datasets improves phylogenetic resolution in Acacia. Mol Phylogenet Evol 96:1–8CrossRefPubMedGoogle Scholar
  78. Xu F, Sun M (2001) Comparative analysis of phylogenetic relationships of grain amaranths and their wild relatives (Amaranthus; Amaranthaceae) using internal transcribed spacer, amplified fragment length polymorphism, and double-primer fluorescent intersimple sequence repeat markers. Mol Phylogenet Evol 21:372–387CrossRefPubMedGoogle Scholar
  79. Yang J-B, Yang S-X, Li H-T, Yang J, Li D-Z (2013) Comparative chloroplast genomes of Camellia species. PLoS One 8:e73053CrossRefPubMedPubMedCentralGoogle Scholar
  80. Zhang YJ, Ma PF, Li DZ (2011) High-throughput sequencing of six bamboo chloroplast genomes: phylogenetic implications for temperate woody bamboos (Poaceae: Bambusoideae). PLoS ONE 6:e20596CrossRefPubMedPubMedCentralGoogle Scholar
  81. Zhang T, Fang Y, Wang X, Deng X, Zhang X, Hu S, Yu J (2012) The complete chloroplast and mitochondrial genome sequences of Boea hygrometrica: insights into the evolution of plant organellar genomes. PLoS ONE 7:e30531CrossRefPubMedPubMedCentralGoogle Scholar

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Authors and Affiliations

  1. 1.Biotechnology PlatformAgricultural Research Council, OnderstepoortPretoriaSouth Africa
  2. 2.Department of Plant and Soil Sciences, Forestry and Agricultural Biotechnology Institute (FABI)University of PretoriaHatfieldSouth Africa
  3. 3.International Crops Research Institute for the Semi-Arid TropicsNairobiKenya
  4. 4.Department of Genetics, Forestry and Agricultural Biotechnology Institute (FABI)University of PretoriaHatfieldSouth Africa
  5. 5.Department of Life and Consumer Sciences, College of Agricultural and Environmental SciencesUniversity of South AfricaFloridaSouth Africa

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