Abstract

The evolutionary significance of the c. 1000‐fold range of DNA C‐values in angiosperms (1C = c. 0.1–127.4 pg) has often attracted interest. A recent analysis, which superimposed available C‐value data onto the angiosperm phylogeny, that placed Ceratophyllaceae as the most basal angiosperm family led to the conclusion that ancestral angiosperms were characterized by small genomes (defined as 1C £ 3.5 pg). However, with the recent increase in DNA sequence data and large‐scale phylogenetic analyses, strong support is now provided for Amborellaceae and/or Nymphaeaceae as the most basal angiosperm families, followed by Austrobaileyales (comprising Schisandraceae, Trimeniaceae and Austrobaileyaceae). Together these five families comprise the ANITA grade. The remaining basal angiosperm families (Ceratophyllaceae, Chloranthaceae and magnoliids), together with monocotyledons and eudicotyledons, form a strongly supported clade. A survey showed that C‐value data were scarce in the basal angiosperm families, especially the ANITA grade. The present paper addresses these phylogenetic gaps by providing C‐value estimates for each family in ANITA, together with C‐values for species in Chloranthaceae, Ceratophyllaceae and a previously unrepresented family in the magnoliids, the Winteraceae.

Introduction

The amount of DNA in the unreplicated nuclear genome of an individual is referred to as its C‐value (Swift, 1950) and in angiosperms this has been shown to vary over 1000‐fold, ranging from c. 0.10 pg in Aesculus hippocastanum L. to 127.4 pg in Fritillaria assyriaca Baker (Bennett & Smith, 1976, 1991). Knowledge of C‐values has continued to increase, with data now available for over 3500 different species corresponding to c. 1.4% of the angiosperm flora (Bennett & Leitch, 2001; Hanson,et al., 2001a,b).

The origin and significance of this huge variation in C‐values has often attracted interest (Stebbins, 1976; Cavalier‐Smith, 1985a; Bennett, 1987, 1998) and there is general agreement that the differences can be attributed largely to changes in the proportion of non‐coding, repetitive DNA sequences such as transposable elements (Flavell, 1988; Kubis, Schmidt & Heslop‐Harrison, 1998) and the extent of genome duplication (Bennetzen, 2000; Sankoff, 2001). Indeed, recent studies are beginning to shed light on the mechanisms which can lead to both increases and decreases in DNA amount (Vicient et al., 1999; Kirik, Salomon & Puchta, 2000; Bensasson et al., 2001; Petrov, 2001).

In addition to this new understanding of how C‐values may change, the evolutionary significance of the huge range of C‐values has started to be evaluated. While earlier studies were sometimes flawed by the lack of a rigorous phylogenetic framework to underpin the analyses of the data (Stebbins, 1976; Price, 1988; Ohri, 1998), in more recent years evolutionary relationships between different families are increasingly being resolved through large‐scale analyses. In 1998, Leitch, Chase & Bennett (1998) superimposed available C‐value data (Bennett, Cox & Leitch, 1998) onto the angiosperm phylogenetic tree of Chase et al. (1993), which was based on DNA sequence information together with 252 non‐molecular characters for 499 angiosperm species. Leitch et al. (1998) concluded that ancestral angiosperms were characterized by the possession of small genomes (defined as 1C £ 3.5 pg).

Since the analysis of Leitch et al. (1998), further DNA sequence data have increasingly resolved relationships between families and, in particular, amongst the earliest, most basal angiosperms (Qiu et al., 1999, 2000; Zanis et al., 2002). In contrast to the tree used by Leitch et al. (1998) that placed Ceratophyllaceae at the root of angiosperms, these recent studies have provided strong support for Amborellaceae and/or Nymphaeaceae as the most basal families that are sister to all other angiosperms (Zanis et al., 2002). The data have also revealed that the next diverging lineage is Austrobaileyales comprising Austrobaileyaceae, Trimeniaceae, and Schisandraceae (Angiosperm Phylogeny Group II, 2002). These five families have been described as the ANITA grade (Qiu et al., 1999). Initially, ANITA was defined as containing Amborellaceae, Nymphaeaceae and Illiciales‐Trimeniaceae‐Austrobaileyaceae (Qiu et al., 1999). Subsequently, changes in taxonomy have led to a revision in the circumscription of some families (Angiosperm Phylogeny Group II, 2002). In the context of this work, Schisandraceae has been newly circumscribed to include Illiciaceae, therefore families comprising ANITA are now recognized as Amborellaceae, Nymphaeaceae, Trimeniaceae, Schisandraceae and Austrobaileyaceae. The present paper follows these new circumscriptions. All angiosperms outside ANITA form a strongly supported clade comprising the other basal angiosperms (Ceratophyllaceae, Chloranthaceae and magnoliids), together with the monocotyledons and eudicotyledons.

The analysis of Leitch et al. (1998) included only three C‐values in the ANITA grade, no data for Ceratophyllaceae nor Chloranthaceae, and only 29 values in the magnoliids. The present paper aims to address these phylogenetic gaps by providing new C‐value estimates for representatives of seven families, thereby completing familial coverage in ANITA, by providing C‐value estimates for Chloranthaceae and Ceratophyllaceae, and adding an estimate for a previously unrepresented family in the magnoliid clade, the Winteraceae.

Material and methods

Plant material

Taxonomic details of the seven species studied are given in Table 1, together with distribution data. Representative vouchers are lodged at the Royal Botanic Gardens, Kew Herbarium.

Table 1.

The seven angiosperm taxa studied in the present work, together with their distribution, cytology number, method used to estimate C‐value and calibration standard used

Taxon Distribution of genus Cytology. no. Method used to estimate C‐value* Calibration standard used† 
Amborella trichopoda Baill. New Caledonia, USA 01–174 FC: PI Oryza 
Austrobaileyales     
Austrobaileya scandens C.T. White Queensland, Australia 02–76 FC: PI Pisum 
Piptocalyx moorei Oliver Central Malaysia to Marquesas & Samoa 02–133 FC: PI Pisum 
Schisandra rubriflora‡ East Asia and East and North America 02–78 FC: PI Pisum 
Ceratophyllum demersum L. Widespread 02–118 Fe Vigna 
Chloranthus spicatus Mak. Indonesia to Malaysia & E. Asia 01–215 FC: PI Pisum 
MAGNOLIIDS     
Canellales     
Drimys vickeriana A.C. Smith Trop. America, central Malaysia to Tahiti 02–70 FC: PI Oryza 
Taxon Distribution of genus Cytology. no. Method used to estimate C‐value* Calibration standard used† 
Amborella trichopoda Baill. New Caledonia, USA 01–174 FC: PI Oryza 
Austrobaileyales     
Austrobaileya scandens C.T. White Queensland, Australia 02–76 FC: PI Pisum 
Piptocalyx moorei Oliver Central Malaysia to Marquesas & Samoa 02–133 FC: PI Pisum 
Schisandra rubriflora‡ East Asia and East and North America 02–78 FC: PI Pisum 
Ceratophyllum demersum L. Widespread 02–118 Fe Vigna 
Chloranthus spicatus Mak. Indonesia to Malaysia & E. Asia 01–215 FC: PI Pisum 
MAGNOLIIDS     
Canellales     
Drimys vickeriana A.C. Smith Trop. America, central Malaysia to Tahiti 02–70 FC: PI Oryza 
*

Fe, Feulgen microdensitometry; FC: PI, flow cytometry using the fluorochrome propidium iodide.

Vigna=Vigna radiata cv. Berken, 4C = 2.12 pg; Oryza=Oryza sativa cv. IR36, 4C = 2.02 pg; Pisum=Pisum sativum cv. Minerva Maple, 4C = 19.46 pg.

Authority unknown or unclear to the present authors.

Estimation of dna amounts

Either flow cytometry (Obermayer et al., 2002) or Feulgen microdensitometry (following methods described in Hanson et al., 2001a) was used to estimate the DNA C‐values. Details of the calibration standard and method used for each species are given in Table 1.

Chromosome counts

Where possible, chromosome counts were obtained from root tip squashes prepared using a standard Feulgen‐stained squash technique as described previously in Hanson et al. (2001a). However, in many cases this was not possible, as only leaf material was available for analysis by flow cytometry.

Results and discussion

Cvalues in the most basal angiosperm lineagethe anita grade

Table 2 lists DNA C‐value estimates for four species in the ANITA grade, including representatives of Amborellaceae, Austrobaileyaceae and Trimeniaceae in which C‐values were previously unknown. Thus a C‐value estimate is now available for at least one species in all five families comprising ANITA.

Table 2.

DNA C‐values for seven angiosperm species studied together with chromosome numbers (2n) obtained in the present work and counts taken from Goldblatt & Johnson (2002)

Taxon Family* 2n Published 2n DNA amount 1C (Mbp)† 1C (pg) 2C (pg) 4C (pg) ± SD 
Amborella trichopoda Amborellaceae – 26 870 0.89 1.78 3.55 ± 0.02 
Austrobaileyales        
Austrobaileya scandens Austrobaileyaceae – 44 9327 9.52 19.04 38.07 ± 0.78 
Piptocalyx moorei Trimeniaceae – 16 3998 4.08 8.17 16.33 ± 0.11 
Schisandra rubriflora Schisandraceae – 28‡ 8938 9.12 18.24 36.48 ± 0.38 
Ceratophyllum demersum Ceratophyllaceae c. 70 24, 48 674 0.69 1.38 2.75 ± 0.26 
Chloranthus spicatus Chloranthaceae 30 30 3528 3.60 7.20 14.39 ± 0.29 
MAGNOLIIDS        
Canellales        
Drimys vickeriana Winteraceae – 90–91‡ 1104 1.13 2.26 4.51 ± 0.06 
Taxon Family* 2n Published 2n DNA amount 1C (Mbp)† 1C (pg) 2C (pg) 4C (pg) ± SD 
Amborella trichopoda Amborellaceae – 26 870 0.89 1.78 3.55 ± 0.02 
Austrobaileyales        
Austrobaileya scandens Austrobaileyaceae – 44 9327 9.52 19.04 38.07 ± 0.78 
Piptocalyx moorei Trimeniaceae – 16 3998 4.08 8.17 16.33 ± 0.11 
Schisandra rubriflora Schisandraceae – 28‡ 8938 9.12 18.24 36.48 ± 0.38 
Ceratophyllum demersum Ceratophyllaceae c. 70 24, 48 674 0.69 1.38 2.75 ± 0.26 
Chloranthus spicatus Chloranthaceae 30 30 3528 3.60 7.20 14.39 ± 0.29 
MAGNOLIIDS        
Canellales        
Drimys vickeriana Winteraceae – 90–91‡ 1104 1.13 2.26 4.51 ± 0.06 
*

Family nomenclature follows Angiosperm Phylogeny Group II (2002).

1 pg = 980 megabase pairs, Mbp (Cavalier‐Smith, 1985b).

Where no chromosome count is known for the species studied, counts for other species in the same genus are given and marked with an asterisk.

The smallest C‐value estimated in ANITA was 1C = 0.89 pg for Amborella trichopoda in Amborellaceae, a monotypic family. Interestingly, it is similar to C‐values reported previously for two species in Nymphaeaceae; Nymphaea caerula, 1C = 0.6 pg (Bharathan, Lambert & Galbraith, 1994) and N. lotus, 1C = 1.1 pg (Bennett & Leitch, 1995). Thus the two most basal angiosperm families (i.e. Amborellaceae and Nymphaeaceae) both have very small C‐values (Leitch et al., 1998). Although no chromosome counts are available, the genome size for these species in the most basal angiosperm families cannot be greater than the 1C‐values listed above because an estimated genome size is calculated from the 2C‐value divided by ploidy level. If the material studied was polyploid rather than diploid, this would result in even smaller genome sizes in these species. Thus while further work will seek to obtain chromosome counts for the material studied, the C‐value data given here are still informative.

Three new C‐value estimates are reported for species in Austrobaileyales (comprising Austrobaileyaceae, Trimeniaceae and Schisandraceae). Those for Austrobaileya scandens (Austrobaileyaceae) of 1C = 9.52 pg and Schisandra rubriflora (Schisandraceae; 1C = 9.12 pg) are similar to estimates for three other Schisandraceae species reported recently by Morawetz and Samuel (pers. comm., listed in Bennett, Bhandol & Leitch (2000), i.e. Kadsura longespicata, 1C = 8.9 pg, K. japonica, 1C = 8.1 pg, and K. coccinea, 1C = 7.4 pg). In contrast, the 1C‐value of 4.08 pg for Piptocalyx moorei (Trimeniaceae) is more similar to the estimate of 1C = 3.4 pg for Illicium anisatum (Schisandraceae) reported by Nagl, Habermann & Fusenig (1977). Overall, the C‐value estimates in Austrobaileyales ranged nearly three‐fold, from 1C = 3.4 to 9.52 pg.

Cvalues in ceratophyllaceae and chloranthaceae

The phylogenetic affinities of Ceratophyllaceae and Chloranthaceae still await determination and are currently controversial (reviewed in Qiu et al., 2000). Some analyses have suggested that Ceratophyllaceae might be sister to the monocotyledons. It is therefore of interest that the small value of 1C = 0.69 pg reported here for Ceratophyllum demersum is similar to those for the two species comprising Acoraceae (Bharathan et al., 1994), the most basal family in the monocotyledons (Chase et al., 2000). Further, the chromosome number obtained for the material studied (2n = c. 70) suggests that it is hexaploid with an unreplicated genome size of 1C = 0.23 pg (the 2C‐value divided by ploidy level), as previous counts have included 2n = 24 and 48 for C. demersum and 2n = 72 for the related species C. submersum (Les, 1988).

In Chloranthaceae, the C‐value estimate of 3.6 pg for Chloranthus spicatus reported here is similar to previous reports for two other Chloranthaceae species: 1C = 2.9 pg for Chloranthus officinalis and 1C = 4.4 pg for Sarcandra glabra (Bennett et al., 2000). The chromosome number of 2n = 30 obtained for C. spicatus agrees with previous reports for the species and is considered to be diploid (Song, 2000).

Together the 14 C‐values now available for these basal angiosperms range 16‐fold, from 1C = 0.6 pg in Nymphaea caerula (Nymphaeaceae) to 9.52 pg in Austrobaileya scandens (Austrobaileyaceae). This range falls towards the lowest 14% of the c. 1000‐fold range encountered in angiosperms (see Introduction).

Cvalues in magnoliids

Our estimate of 1C = 1.13 pg for Drimys vickeriana is the first value reported for the magnoliid family Winteraceae. It is only a fifth of the estimate of 1C = 5.8 pg for Canella winterana which is in the sister family Canellaceae. Together Canellaceae and Winteraceae comprise Canellales (Qiu et al., 2000), one of the four higher orders of the magnoliid clade, the other three being Laurales, Magnoliales and Piperales (Qiu et al., 2000). Since the analysis by Leitch et al. (1998), the number of C‐values for magnoliids has doubled, with data now available for 60 species and ranging 14.5‐fold, from 1C = 0.4 pg in Friesodielsia obovata to 5.8 pg in Canella winterana (Bennett & Leitch, 2001). The C‐value reported for Drimys vickeriana therefore falls within this range.

The future

New studies, particularly those targeting systematic gaps in the data, have contributed significantly to an increase in the phylogenetic coverage of C‐value data. For example, two papers by Hanson et al. (2001a,b) have increased familial representation of data from 34% to 44.8%. With the new data presented in the current paper which have filled all familial gaps in basal angiosperm families, and with an increasingly robust angiosperm phylogeny now available, it is timely to repeat and enlarge the large‐scale phylogenetic analysis of C‐values by Leitch et al. (1998) to provide a deeper insight into the evolution of DNA amounts in angiosperms.

Acknowledgement

The authors thank Iain Dawson of the Australian National Botanic Gardens, Canberra for kindly providing the material of Piptocalyx moorei used in this study.

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