Annals of Botany Page 1 of 124 doi:10.1093/aob/mcq258, available online at www.aob.oxfordjournals.org Nuclear DNA amounts in angiosperms: targets, trends and tomorrow M. D. Bennett* and I. J. Leitch Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK * For correspondence. E-mail: [email protected] Received: 25 August 2010 Returned for revision: 18 October 2010 Accepted: 24 November 2010 CONTENTS INTRODUCTION 2 Extending the range of genome sizes encountered in angiosperms 3 The need for reference lists 4 TARGETS IN GENOME SIZE RESEARCH 4 Downloaded from Meeting targets for species representation 4 Progress towards targets for familial representation 5 Improved systematic representation for genera 6 Improved representation of other groups 6 TRENDS IN TECHNIQUES USED TO ESTIMATE GENOME SIZE 7 The rise in flow cytometry as the technique of choice for genome size estimations 7 http://aob.oxfordjournals.org/ Development of different isolation buffers for flow cytometry 7 The application of flow cytometry to plant systematics 8 Recent developments in the application of flow cytometry to genome size studies 8 (i) The use of seeds 8 (ii) Ease of access to methodological data 8 (iii) New equipment 8 Are there any new techniques for estimating genome size on the horizon? 9 (i) Can real time PCR be used for estimating plant genome sizes? 9 (ii) Will ‘complete’ genome sequencing give useable genome size estimates? 9 TOMORROW 13 at NIH Library on December 30, 2015 Uncovering and collating genome size data from diverse published sources 14 Screening ex situ and in situ collections as sources of target taxa 15 DEDICATION 15 LITERATURE CITED 16 APPENDIX 19 Notes to the Appendix 19 Original references for DNA values 121 † Background and Aims The amount of DNA in an unreplicated gametic chromosome complement is known as the C-value and is a key biodiversity character of fundamental significance with many practical and predictive uses. Since 1976, Bennett and colleagues have assembled eight compilations of angiosperm C-values for reference pur- poses and subsequently these have been pooled into the Angiosperm DNA C-values Database (http://data.kew.org/ cvalues/). Since the last compilation was published in 2005, a large amount of data on angiosperm genome size has been published. It is therefore timely to bring these data together into a ninth compilation of DNA amounts. † Scope The present work lists DNA C-values for 2221 species from 151 original sources (including first values for 1860 species not listed in previous compilations). Combining these data with those published previously shows that C-values are now available for 6287 angiosperm species. † Key Findings Analysis of the dataset, which is by far the largest of the nine compilations published since 1976, shows that angiosperm C-values are now being generated at the highest rate since the first genome sizes were esti- mated in the 1950s. The compilation includes new record holders for the smallest (1C ¼ 0.0648 pg in Genlisea mar- garetae) and largest (1C ¼ 152.23 pg in Paris japonica) genome sizes so far reported, extending the range encountered in angiosperms to nearly 2400-fold. A review of progress in meeting targets set at the Plant Genome Size meetings shows that although representation for genera, geographical regions and some plant life forms (e.g. island floras and parasitic plants) has improved, progress to increase familial representation is still slow. In terms of technique it is now clear that flow cytometry is soon likely to become the only method available for plant genome size estimations. Fortunately, this has been accompanied by numerous careful studies to improve the quality of data generated using this technique (e.g. design of new buffers, increased awareness and understanding of problems caused by cytosolic inhibitors). It is also clear that although the speed of DNA sequencing continues to rise dramatically with the advent of next-generation and third-generation sequencing technologies, ‘complete genome sequencing’ projects are still unable to generate accurate plant genome size estimates. Key words: DNA C-value, nuclear genome size, Plant DNA C-values Database, flow cytometry. # The Author 2011. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: [email protected] Page 2 of 124 Bennett & Leitch – Nuclear DNA amounts in angiosperms INTRODUCTION amount in the unreplicated gametic nuclear chromosome complement [defined by Swift (1950) as the 1C-value) of It has been possible to estimate the amount of DNA in plant different taxa. [Recently this was defined as the holoploid and animal nuclei using various methods for over 60 years. genome size by Greilhuber et al. (2005).] Following the discovery of the key role of DNA in biology Easy access to data for these key characters is clearly an in 1953 (Watson and Crick, 1953) such research has increased important facility. Yet in practice it has often been difficult in each successive decade, and this trend has further intensified to know whether a C-value exists for a particular taxon, and in the 21st century. These decades saw the loss of many who if so, where to find it, especially as estimates are widely scat- played key roles in founding and developing this interest tered in the literature or even unpublished. Although small lists [including Hewson Swift (1920–2004), John McLeish of nuclear DNA amounts were published in reviews and (1929–1971), Arnold Sparrow (1914–1976) and Hugh Rees research papers as genome size data started to accumulate in (1923–2009)]. Yet until now the remarkable birth and the 1960s and 1970s, the first large compilation of DNA explosion of genome size data in ‘the DNA age’ occurred in amounts for angiosperms, compiled primarily as a reference the scientific lifetime of some individuals who witnessed or source, was published in 1976 with data for 753 species experienced the entire development of this field. However, from 54 sources (Bennett and Smith, 1976). This noted an the death of Jim Price (1944–2005 – Arnold Sparrow’s post- intention to publish supplementary compilations for reference Downloaded from doctoral student) in 2004, and the retirement of the first author purposes at intervals and seven followed (see Table 1). (Mike Bennett – Hugh Rees’s postdoctoral student) in 2006 Increasing accessibility and ease of access to nuclear DNA and Johann Greilhuber in 2010 shows that an historic change amounts was made possible through pooling of data in the has begun as the baton passes to generations unborn at the published reference compilations into one electronic database start of the DNA age, whose knowledge of early genome available on the internet. The Angiosperm DNA C-values size research reflects written accounts alone, with no further http://aob.oxfordjournals.org/ Database (release 1), which collated all the data from the recourse to supplementary first-hand sources. Happily, recent first five published compilations (Table 1), went live in April trends noted in this review show that tomorrow’s studies of 1997. After several updates, a major new release (3.1) of nuclear DNA amounts in angiosperms rest with an enthusiastic angiosperm data was combined with databases for gymnos- new cohort of practitioners focused on carefully improving perms, pteridophytes and bryophytes, into the Plant DNA techniques and data representation to better understand the C-values Database (release 1.0) in 2001. This database was origin, extent and effects of variation in plant genome size. further expanded in 2004 (release 3.0) to incorporate not As noted by Bennett and Leitch (2005a), ‘Nuclear DNA only first values for 628 species not previously listed in the amount and genome size (C-value) are important biodiversity Angiosperm DNA C-values Database, but also, for the first characters, whose study provides a strong unifying element in at NIH Library on December 30, 2015 time, DNA amounts for 253 algal species. An additional biology with practical and predictive uses’. Of all the major update of the Plant DNA C-values Database (release 4.0), taxonomic groups studied, Bennett and Leitch (2005a) noted adding data for over 300 angiosperm species (from that angiosperms were probably the most intensively studied. Zonneveld et al., 2005), went live in 2005. Release 5.0of With published prime C-values for over 6280 species now the Plant DNA C-values Database (December 2010) incorpor- available (Table 1), they remain so today. Indeed, work on ates all the data presented in the Appendix here, and brings the plants has played a leading part in the research on the DNA TABLE 1. Total numbers of first estimates for angiosperm species and of original references from which they come in nine compilations of nuclear genome size estimates collated for reference purposes since 1976 No.. of first estimates for No. of prime estimates for species No. of original No. of first estimates per ‘species’ in abstract of in the Plant DNA C-values Database references in original reference (column Original compilation compilation (release 5.0) compilation 2/4) 1. Bennett and Smith (1976) 753 596† 54 13.9 2. Bennett et al. (1982) 240 195† 53 4.5 3. Bennett and Smith (1991) 588 552† 56 10.5 4. Bennett and Leitch (1995) 899 868 † 106 8.5 5. Bennett and Leitch (1997) 471 481† 37 12.7 6. Bennett et al. (2000) 691 686† 71 9.7 7. Bennett and Leitch (2005a) 628 636† 88 7.7 8. Zonneveld et al. (2005) 308 308 1 308.0 9. Present work 1860 1974† 151 13.1 Total 6438* 6287* 617 – * The total number of taxa in the Plant DNA C-values Database (i.e. 6287) is lower by 151 than the total for paper compilations (i.e.