Karyotypic variation in Nicotiana section Suaveolentes
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- Marks, C.E., Ladiges, P.Y. & Newbigin, E. Genet Resour Crop Evol (2011) 58: 797. doi:10.1007/s10722-011-9724-3
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Nicotiana section Suaveolentes (Solanaceae) currently includes 28 species and subspecies that are endemic to Australasia and the South Pacific and one African species, N. africana. The section is monophyletic and of allotetraploid origin, but relationships among the species in it and its diploid progenitors are poorly understood. Here we report chromosome numbers for 20 of the 29 taxa from the Suaveolentes, including a count for one recently proposed species for which no number has previously been available. Many of the published chromosome numbers for the Suaveolentes are confirmed in this study. However, six counts were different from the published numbers including n = 15 for N. maritima and N. suaveolens, which is a new chromosome number for the genus. Nicotiana goodspeedii and N. rotundifolia were n = 16, and the same number was found in the suggested species N. sp. ‘Corunna’. Nicotiana suaveolens contains polyploid races of n = 32 and here we report the probable existence of an n = 31 race as well. Karyotypic variation within species and within the section is apparently much greater than previously thought and further investigation is warranted.
KeywordsChromosome countsKaryotypic variationNicotianaSuaveolentes
The genus Nicotiana L., best known for cultivated tobacco, N. tabacum L., currently includes 76 species that are divided into 13 sections (Knapp et al. 2004). Although many nicotianas are found in South America, the largest section in the genus, section Suaveolentes Goodsp. with 29 species and subspecies, represents a significant radiation outside of South America. Most of the species in the Suaveolentes are endemic to Australia, with one of the eastern Australian species, N. forsteri, also occurring on Lord Howe Island and New Caledonia. In addition, the section includes two South Pacific taxa (N. fragrans and N. fatuhivensis) and a single species from Africa (N. africana).
Although Suaveolentes is accepted as a monophyletic group of allopolyploid origin, the taxonomic relationships of its members are not as well resolved as those of other sections in Nicotiana (Knapp et al. 2004). Plants in this group have few distinctive morphological features, being mostly ephemeral, white-flowered herbs; molecular phylogenies are characterized by few well-supported nodes and often show conflicting patterns between different DNA data sets (Aoki and Ito 2000; Chase et al. 2003; Clarkson et al. 2004; Clarkson et al. 2010). Allopolyploidy and hybridization between the species in this section are often cited as reasons why determining their relationships has been difficult (Goodspeed 1954; Chase et al. 2003).
Chromosome numbers are useful characters in systematic studies because ploidy changes are often associated with speciation events. The base chromosome number (x) for Nicotiana is 12 and the genus is currently divided into eight diploid sections, where n = 9, 10 or 12, and five allopolyploid sections, where n = 24 (Olmstead et al. 1999). Section Suaveolentes is the sole exception to this pattern because of its wide range of chromosome numbers. Haploid chromosome numbers reported for the section are n = 16, 18, 19, 20, 21, 22, 23 and 24, with only n = 17 never having been recorded within this range. Chromosome numbers for the section are interpreted as the result of aneuploid and/or dysploid reduction beginning with an allotetraploid ancestor with n = 24 (Narayan 1987; Clarkson et al. 2004).
Chromosome numbers found in this study and numbers previously published for all current Suaveolentes taxa
Seed lot (MELU voucher number)
Previous number (Reference)
Nicotiana africana Merxm.
n = 23
n = 23 (Merxmüller and Buttler 1975)
Nicotiana amplexicaulis N. Burb.
n = 18
n = 18 (Burbidge 1960)
Nicotiana benthamiana Domin
n = 19
Nicotiana burbidgeae Symon
n = 21
n = 21 (Symon 1984)
Nicotiana cavicola N. Burb.
Nicotiana excelsior (J. Black) J. Black
n = 19
Nicotiana forsteri Roem. et Schult.
n = 24
Nicotiana fatuhivensis F. Br.
Nicotiana fragrans Hook.
Nicotiana goodspeedii H. Wheeler
n = 16
Nicotiana gossei Domin
n = 18
Nicotiana heterantha Symon et Kenneally
n = 24
n = 24 (Symon and Kenneally 1994)
Nicotiana maritima H. Wheeler
n = 15
Nicotiana megalosiphon Van Heurck et Mull. Arg. ssp. megalosiphon
n = 20
SL1, SL16 (D106518, D106517)
Nicotiana megalosiphon Van Heurck et Mull. Arg. ssp. sessifolia P. Horton
Nicotiana monoschizocarpa (P. Horton) Symon et Lepschi
n = 24
n = 24 (Horton 1981)
Nicotiana occidentalis H. Wheeler ssp. hesperis (N. Burb.) P. Horton
n = 21 (Burbidge 1960)
Nicotiana occidentalis H. Wheeler ssp. obliqua N. Burb.
n = 21
SL17, SL32 (D106540, D106541)
n = 21 (Burbidge 1960)
Nicotiana occidentalis H. Wheeler ssp. occidentalis
Nicotiana rosulata (S. Moore) Domin ssp. ingulba (J. Black) P. Horton
n = 20 (Goodspeed 1954)
Nicotiana rosulata (S. Moore) Domin ssp. rosulata
n = 20 (Goodspeed 1954)
Nicotiana rotundifolia Lindley
n = 16
Nicotiana simulans N. Burb.
n = 20
n = 20 (Burbidge 1960)
Nicotiana sp. ‘Corunna’ Symon 17088
n = 16
Nicotiana suaveolens Lehm.
n = 15
Nicotiana truncata Symon
n = 18
n = 18 (Symon 1998)
Nicotiana umbratica N. Burb.
n = 23 (Burbidge 1960)
Nicotiana velutina H. Wheeler
n = 16
n = 16 (Goodspeed 1954)
Nicotiana wuttkei J. Clarkson et Symon
Prob. Nicotiana suaveolens
n = 31
Prob. Nicotiana rosulata ssp. ingulba
n = 32
As part of a study of taxonomic relationships within Suaveolentes, we determined chromosome numbers for 20 of the taxa in the section using plants of known identity. We also determined chromosome numbers for two problematic taxa. Here we report examples of chromosome counts that are new to the section and provide a count for the suggested new species N. sp. ‘Corunna’.
Materials and methods
Seed sources for each taxon used in this study are given in Marks et al. (2011) and voucher specimens (lodged at the University of Melbourne herbarium) are listed in Table 1. Seeds were germinated on seedling mix under glasshouse conditions and transferred to 20 L hydroponics tanks when seedlings had three or more non-cotyledon leaves. Plants were suspended in rock wool inside large pipette tips with the tips cut off. Ionic Grow nutrient solution (Growth Technology, Australia) was used at the recommended rates (100 mL/20 L for seedlings, up to 150 mL/20 L for mature plants) and was changed once a week. Tanks were aerated and additional lamps were used over winter.
Roots were harvested when at least 10 cm long and growing vigorously. The terminal 1 cm of root was collected directly into a saturated solution of paradichlorobenzene at room temp in 2 mL disposable tubes and left overnight at 4°C. The following day roots were rinsed twice in water and then placed in fixative (freshly made ethanol: glacial acetic acid 3:1; v:v) and left for 4–24 h at room temperature. Roots were transferred to 70% ethanol for storage at −20°C.
On the day when counts were to be made, roots were hydrolysed in 1 M HCl at 60°C for 8 min, rinsed in water and placed in 45% acetic acid. Approximately three roots were selected, placed on a slide, and the tips located under the dissecting microscope. The top 2 mm of the root tip was excised and the rest discarded.
Formic-lactic-propionic Orcein stain was prepared according to Jackson (1973). Root tips were macerated in a large drop of FLP Orcein using a metal rod and gentle heat from a spirit burner. Slides were observed at ×10 magnification using an Olympus BH-2 to identify cells with condensed chromosomes. Cells were viewed at ×400 and representative cells photographed and counted at ×1,000 using an oil emersion lens. Counts were made from at least five cells for each taxon, and for some taxa plants grown from two or more seed lots were examined.
Results and discussion
Chromosome counts were obtained for 20 of the 29 taxa in section Suaveolentes and from a proposed new species for which no number had previously been available. Additionally, counts were obtained from two problematic taxa. Table 1 lists all the available chromosome counts for this section, including those from this study.
Nicotiana chromosomes are relatively small (2–5.5 μm) with well-defined centromeres, with one to three pairs typically having a well-defined secondary constriction as well (Goodspeed 1954). Wheeler (1945) reported that the thirteen Suaveolentes species examined all had chromosomes with a mix of median, submedian and subterminally placed centromeres. All species observed in this study had chromosomes that conformed to this general description although no records were made of the placement of centromeres or the presence of secondary constrictions.
Chromosome numbers were confirmed for 14 taxa. Mitotic cells of N. velutina have 32 chromosomes or n = 16. Nicotiana amplexicaulis, N. gossei and N. truncata have 36 chromosomes, or n = 18. Nicotiana benthamiana and N. excelsior have 38 chromosomes or n = 19. Nicotiana simulans and two different seed lots of N. megalosiphon ssp. megalosiphon have 40 chromosomes or n = 20. Nicotiana burbidgeae and three different seed lots of N. occidentalis ssp. obliqua have 42 chromosomes or n = 21. Nicotiana africana has 46 chromosomes or n = 23 and N. forsteri, N. heterantha and N. monoschizocarpa have 48 chromosomes or n = 24.
The proposed new taxon N. sp. ‘Corunna’ had 32 chromosomes or n = 16 (Fig. 1c), which is consistent with the suggested close association between it and N. goodspeedii (Marks et al. 2011), which also has n = 16.
Plants grown from seed from two seed lots had unusual chromosome numbers for this section. One seed lot was supplied as N. occidentalis ssp. hesperis but was provisionally identified as N. suaveolens, although it matched the description of N. suaveolens less closely than other accessions (Marks 2010). Plants from this seed lot (SL15) had 62 chromosomes or n = 31 (Fig. 1g), a number not previously reported for the section and not fitting with published numbers for N. suaveolens (n = 16 or n = 32) or N. occidentalis ssp. hesperis (n = 21). A second seed lot (SL18) was supplied as N. rosulata ssp. ingulba and was from seed collected within the range of this subspecies. This identification was provisionally confirmed but recognized as problematic (Marks 2010). Plants from this seed lot had 64 chromosomes or n = 32 (Fig. 1f), a number different from the published n = 20 for N. rosulata ssp. ingulba. Both accessions were recognized as taxonomically problematic and were either unusual members of their species or not well classified by the current species key (Horton 1981; Purdie et al. 1982).
The observed differences in karyotype indicate the existence of distinct cytotypes within some members of the Suaveolentes. Cytotypes are surprisingly common in plant populations (de Lange et al. 2008) and examples of different ploidy levels within morphologically cohesive species include Scilla (Liliaceae; Vaughan et al. 1997), Crassula (Crassulaceae; de Lange et al. 2008), Rutidosis (Asteraceae; Murray and Young 2001), Phragmites (Poaceae; Connor et al. 1998) and Chaenactis (Asteraceae; Mooring 1980). Because differences in the chromosome numbers for N. maritima (n = 15, 16), N. suaveolens (n = 15, 16, 31, 32), N. goodspeedii (n = 16, 20), N. rotundifolia (n = 16, 22), N. rosulata ssp. ingulba (n = 20, 32) and N. cavicola (n = 20, 23) could be due to the existence of distinct cytotypes within these species, a more extensive, population-level survey of karyotypes in the Suaveolentes is recommended.
Although members of the Nicotiana section Suaveolentes are all allopolyploids arising from a single polyploid event estimated to have occurred over 10 million years ago (Leitch et al. 2008), only four of the species in the section (N. forsteri, N. fragrans, N. heterantha and N. monoschizocarpa) retain the original n = 24 karyotype (Table 1), with all other chromosome numbers representing the consequences of repeated aneuploid and/or dysploid reductions. Consistent with this, it appears that species with a chromosome count of n = 24 or 23 are all early branching lineages within the section (Marks et al. 2011). Further, those taxa with reduced chromosome numbers can be placed into one of two clades: the ‘N. simulans clade’ (containing all taxa with n = 20) and the ‘N. suaeveolens clade’ (containing taxa with n = 19, 18, 16 or 15). Although phylogenetic relationships generally cannot be inferred from chromosome numbers alone, in this instance karyotypic variation seems to reflect phylogenetic divergence, being best explained by repeated dysploidy and/or aneuploidy reductions from the original n = 24 karyotype.
CM acknowledges the receipt of an Australian Postgraduate Award and a Hansjörg Eichler Scientific Research Award from the Australian Systematic Botany Society, and student travel bursaries from the Australian Biological Resources Study, the School of Botany Foundation and the University of Melbourne. Dr Brian Murray (University of Auckland) is thanked for his help and advice with cytology.