A comparison of the efficiency of DNA barcoding regions in a small and a large genus

Abstract

English: Clivia (family Amaryllidaceae; tribe Haemantheae) with its six species is closest related to the genus Cryptostephanus. These genera share the highest chromosome numbers (2n = 22 & 24 respectively) in the tribe and has similar 2C values. Evolution in Clivia correlates with geographical distribution from west to east and genome size from small to large. The tree, character and distance-based analyses are effective in the identification process of three Clivia species. Clivia mirabilis, C. nobilis, C. miniata and C. caulescens / C. × nimbicola can be identified with DNA-barcoding. The best barcoding regions regarding discriminatory power are the trnL-F chloroplast region, the matK chloroplast gene and the ITS2 nuclear spacer region. A single DNA-barcoding locus is insufficient to be used in Clivia barcoding. Hybridization events in Clivia may lead to false positive or false negative identifications. Analysis of an unknown sample resulting in a C. miniata, C. gardenii or C. robusta identification should need further analysis on additional data (e.g. morphology, distribution) to confirm the results. All the C. × nimbicola natural hybrids included in this study shared the chloroplast DNA of C. caulescens. Alternative methods should be developed to effectively distinguish and identify hybrid species. The genus Lachenalia has 133 species and shows extensive morphological variation and exceptional diverse chromosome numbers (x = 5, 6, 7, 8, 9, 10, 11, 12, 13 and 15). Lachenalia might have evolved from a common ancestor and the two largest basic chromosome number groups, x = 7 and 8 have evolved from a common predecessor. It seems as if the higher basic numbers (x = 9, 10, 11 and 13) evolved independently from the lower numbers. Several speciation events were involved in the evolution of Lachenalia, resulting in the morphological and chromosomal diversity. Many of the species with the same basic chromosome numbers share a common ancestor, and it is expected that there may be incomplete lineage sorting in some species resulting in non-specific DNA-barcodes. The tree-based and character-based analyses are effective methods to identify all the focus species (L. unifolia, L. bifolia, L. punctata and L. mediana) in this study. The matK, trnL-F and ITS2 regions results in the positive identification of an unknown specimen (tree-based analyses). SNP analyses can be used in the analyses of matK and atpH-I. The combination of atpH-I + trnL-F and trnL-F + ITS2 will effectively distinguish an unknown sample of the focus species. Due to the large size of the genus, a two to three DNA-barcode locus is preferred over a single DNA-barcoding locus. The nuclear ITS2 gene region has to be included as a DNA-barcode to detect hybrid species and plastid capture. The chloroplast trnL-F region together with the nuclear ITS2 region can be universally used for barcoding the small genus Clivia and the large genus Lachenalia. Both these regions require an third region (or more in the case of Lachenalia) to be effective for identification of species. Ancient hybridization, introgression and incomplete lineage sorting occurs in both genera. Therefore, certain species will not be effectively identified based on barcodes alone. There are some small differences between the DNA-barcoding of the small (Clivia) and the large genus (Lachenalia). However, the similarities between the genera are that identifications in both genera are influenced by the degree of hybridization (ancient or recent) and the time of divergence (thus incomplete lineage sorting). The classification of Clivia and Lachenalia must be properly resolved before barcodes can be implemented for the species-level identification of these genera.

Afrikaans: Clivia (Familie Amaryllidaceae; Tribus Haemantheae), met sy ses spesies, is die naaste verwant aan die genus Cryptostephanus en deel die hoogste chromosoomgetalle (2n = 22 & 24 onderskeidelik) in die tribus. Beide genera het soortgelyke 2C waardes. Evolusie in Clivia stem ooreen met ʼn geografiese verspreiding van wes na oos en genoom grootte van klein tot groot. Die kladogram-, karakter- en afstandsgebaseerde ontledings is effektief in die identifiseringsproses van drie Clivia-spesies. Clivia mirabilis, C. nobilis, C. miniata en C. caulescens/C. × nimbicola kan geïdentifiseer word met DNA-strepieskodes. Die beste strepieskode (beste onderskeidingsvermoë) word met die trnL-F en die matK gebiede in die chloroplaste verkry, asook die kern se ITS2 streek. 'n Enkele DNA-strepieskode lokus is onvoldoende om tussen Clivia spesies te onderskei. Verbastering in Clivia kan lei tot vals positiewe of negatiewe identifikasies. Ontleding van 'n onbekende monster wat in 'n C. miniata, C. gardenii of C. robusta identifikasie eindig, verg verdere ontleding van die bykomende data (bv. morfologie, verspreiding) om die resultate te bevestig. Al die C. × nimbicola natuurlike kruisings in hierdie studie stem ooreen met die chloroplaste DNA van C. caulescens. Alternatiewe metodes moet ontwikkel word om baster spesies te identifiseer. Die genus Lachenalia het 133 spesies en toon uitgebreide morfologiese variasie en uitsonderlik diverse chromosoomgetalle (x = 5, 6, 7, 8, 9, 10, 11, 12, 13 en 15 ). Lachenalia kon ontwikkel het uit 'n gemeenskaplike voorouer en die twee grootste groepe met basiese chromosoomgetalle van x = 7 en 8 het moontlik ontwikkel uit 'n gemeenskaplike voorvader. Dit lyk asof die hoër basiese getalle (x = 9, 10, 11 en 13) onafhanklik ontwikkel het uit laer getalle. Verskeie spesiasieprosesse was betrokke by die evolusie van Lachenalia en het gelei tot die morfologiese en chromosomale diversiteit. Baie van die spesies met dieselfde basiese chromosoomgetalle deel 'n gemeenskaplike voorouer, en dit word verwag dat daar dalk onvolledige lyne in sommige spesies kan voorkom wat lei tot nie-spesifieke DNA-strepieskodes. Die kladogram- en karakter-gebaseerde ontledings is effektiewe metodes om al die fokus spesies (L. unifolia, L. bifolia, L. punctata en L. mediana) in hierdie studie te identifiseer. Die matK, trnL-F en ITS2 streke lewer ʼn positiewe identifikasie van ’n onbekende eksemplaar (kladogram-gebaseerde ontledings). SNP ontledings kan gebruik word in die ontleding van matK en atpH-I. Die kombinasie van atpH-I + trnL-F asook trnL-F + ITS2 sal effektief kan onderskei indien ’n onbekende monster van die fokus spesies bestudeer word. As gevolg van die omvang van die genus, is ʼn 2-3 DNA-strepieskode lokus verkieslik bo ʼn enkele DNA-strepieskode lokus. Die kern ITS2 streek moet as ʼn DNA-strepieskode ingesluit word om basterspesies en introgressie waar te neem. Die chloroplast trnL-F streek saam met die kern ITS2 streek kan universeel gebruik word vir as DNA-strepieskode in beide die klein genus Clivia en die groot genus Lachenalia. Albei streke vereis egter dat ’n derde streek (of selfs meer in die geval van Lachenalia) nodig is vir die identifisering van spesies. Oerverbastering, introgressie en onvolledige geslagslyn sortering vind plaas in beide genera. Derhalwe sal sekere spesies nie effektief geïdentifiseer kan word op grond van slegs strepieskodes nie. Daar is ’n paar klein verskille tussen die DNA-strepieskode van die klein (Clivia) en die groot genus (Lachenalia). Maar die ooreenkomste tussen die genera is dat die identifisering in beide genera deur die graad van verbastering (oer of onlangse) en die tyd van divergensie (dus onvolledig geslagslyn sortering) beïnvloed word. Die klassifikasie van Clivia en Lachenalia moet behoorlik uitsorteer word voordat strepieskodes vir spesie-vlak identifikasie van hierdie genera geïmplementeer kan word.