Plant material and DNA sequencing

DNA sequences for the Nicotiana species shown in Table S2 were obtained from GenBank ( www.ncbi.nlm.nih.gov/genbank). Although N. suaveolens Lehm. and N. exigua H.-M.Wheeler are considered conspecific (Horton 1981), the sequences lodged in GenBank under these names for matK, ITS and the two GS paralogs are not identical, and thus the original species names were retained and have been treated here as independent taxa. The voucher material for these sequences should be re-examined to confirm their identities.

Twelve species of Nicotiana, including the undescribed N. sp. Corunna (D.E.Symon 17088) and N. burbidgeae, were propagated for at least two generations, confirmed to have the morphological features diagnostic of the taxa, and used for DNA extraction. Total genomic DNA was extracted from leaf tissues using the CTAB method (Clarke 2009) and amplified with a high-fidelity Taq polymerase and the primers described in Table S1 of the   Supplementary material (SB20025_AC.pdf), using a touchdown cycling technique and annealing temperatures of 50–55°C. For species without existing sequence information, the nuclear DNA regions amplified were the internal transcribed spacer of rRNA (ITS), the long and short forms of the chloroplast-expressed glutamine synthetase (GSL and GSS respectively; Clarkson et al. 2010), RNA-dependent RNA polymerase 1 (RDR1; Bally et al. 2015) and alcohol dehydrogenase C locus (ADHC); and the plastid region amplified was maturase K (matK). Because nuclear and chloroplast DNA sequences were not available for N. sp. Corunna (D.E.Symon 17088) or N. burbidgeae, regions of ribosomal ITS and matK from these accessions, and also from N. benthamiana, were amplified and sequenced (Table S2). Cycle sequence reactions, performed with BigDye Terminator (ver. 3.1, Applied Biosystems) at suggested cycling conditions, were purified with an ethanol and EDTA precipitation. After purification, the amplified fragments were run on a Life Technologies 3500 Genetic Analyser at the Central Analytical Research Facility Genomics Laboratory at the Queensland University of Technology. Analysis of output chromatograms and further preliminary sequence editing was conducted using Geneious (ver. R11, Biomatters, New Zealand, see  www.geneious.com/; Kearse et al. 2012).

Sequence alignment and phylogenetic analyses

Gene sequences were aligned (Fig. S1 of the   Supplementary material (SB20025_AC.pdf)) using MUSCLE (ver. 3.8.31, see  http://www.drive5.com/muscle/; Edgar 2004), followed by manual adjustments in BioEdit (ver. 7.2.6, see  https://bioedit.software.informer.com/). For concatenation, the sequences were appended in the following order: ITS, matK, GSL, GSS, RDR1 and ADHC. The Akaike information criteria in ModelFinder in IQ-TREE (ver. 1.5.4, see  http://www.iqtree.org/; Kalyaanamoorthy et al. 2017) and jModelTest2 (ver. 2.1.10, see  https://github.com/ddarriba/jmodeltest2/; Darriba et al. 2012) were used to estimate the best-fit substitution models. Model selection and the parameters used are described in Table S3. Phylogenetic analyses of individual and concatenated gene sequences were based on maximum likelihood implemented by a rapid and effective stochastic algorithm in IQ-TREE (Trifinopoulos et al. 2016) including partition files. Additional phylogenetic trees for concatenated data of all six genes were built through Bayesian inference as implemented in MrBayes (ver. 3.2.6, see  https://github.com/NBISweden/MrBayes/; Ronquist et al. 2012) including partition files. The final consensus trees were displayed using FigTree (ver. 1.4.3, see  https://github.com/rambaut/figtree/releases/tag/release_1_3/).

Morphological assessment of flowers and phenetic analysis

Marks et al. (2011a) reported measurements of 21 floral character states from a range of N. section Suaveolentes taxa, with each measurement being based on a minimum of 10 biological replicates. Measurements for selected taxa were extracted from those reported in this paper and data matrices of floral characters were subject to principal-component analysis (PCA) using R (ver. 3.5.0, R Foundation for Statistical Computing, Vienna, Austria) and the package factoextra (ver. 1.0.3, see  https://CRAN.R-project.org/package=factoextra/).

Phylogenetic analyses

A maximum-likelihood analysis (Fig. 1) was performed using the concatenated ITS and matK sequences for N. burbidgeae, N. benthamiana and N. sp. Corunna (D.E.Symon 17088), and equivalent sequences from other Nicotiana taxa and from the Australian genus Anthocercis Labill. (Solanaceae: Anthocercideae) as the designated outgroup (Clarkson et al. 2010). The trees produced with ITS or matK sequences alone are shown in Fig. S1 and S2. For the concatenated tree, the total number of nucleotides used was 2213, of which 406 were variable. Nicotiana formed a well supported clade made up of several lineages and the branching pattern shown in Fig. 1 was consistent with previous analyses of this genus (e.g. see Clarkson et al. 2010). The branching pattern of the ITS tree was like that of the concatenated tree but the matK tree had fewer resolved nodes. All trees placed members of N. section Suaveolentes in a well supported lineage that included N. benthamiana, N. burbidgeae and N. sp. Corunna (D.E.Symon 17088). Nicotiana benthamiana was sister to N. excelsior (J.M.Black) J.M.Black (bootstrap support (BS) = 100%) and N. burbidgeae along with N. umbratica were on early diverging branches among Australian representatives of the section (Fig. 1). All trees showed that N. sp. Corunna (D.E.Symon 17088) and N. goodspeedii were on separate branches, as were N. suaveolens and its synonym N. exigua.

Fig. 1.

Phylogeny of Nicotiana species and the N. sp. Corunna (D.E.Symon 17088) accession inferred from maximum-likelihood analysis of combined ITS and matK sequences. Bootstrap support values >70% are shown above branches. Branches with ≤70% bootstrap support values are annotated with an en-dash (–). Sections of Nicotiana are shown on the right-hand side corresponding to the shaded boxes.

To further confirm N. sp. Corunna (D.E.Symon 17088) as a distinct species, additional nuclear sequences were obtained. Species in the allopolyploid N. section Suaveolentes retain both parental copies of the nuclear-encoded, chloroplast-expressed glutamine synthetase, and the paralogs, called GSL and GSS, have previously been used to determine phylogenetic relationships (Clarkson et al. 2010). As well as N. sp. Corunna (D.E.Symon 17088), GSL and GSS sequences were obtained for N. burbidgeae, N. benthamiana and N. rosulata (S.Moore) Domin and added to the existing N. section Suaveolentes sequence dataset (Table S2). Regions of a further two genes, RDR1 and ADHC, were either sequenced from these species or retrieved from GenBank. Most species in N. section Suaveolentes appear to have retained only one of the parental copies of these genes (Kelly et al. 2013; Bally et al. 2015, 2018). Consistent with this, there were no polymorphisms observed in the amplified ADHC and RDR1 products for any accession. The sequences of all six gene regions (ITS, matK, GSL, GSS, RDR1 and ADHC) were used to generate phylogenetic trees for each individual gene region (Fig. S1–S6) and for a concatenation of all six (Fig. 2); their counterpart sequences from Anthocercis gracilis Benth., Anthocercis sylvicola T.D.Macfarl. & Ward.-Johnson, Symonanthus bancroftii (F.Muell.) Haegi or N. tabacum L. and N. sylvestris Speg. were used as outgroups, depending on availability. As previously encountered in phylogenetic trees of members of the N. section Suaveolentes using different sequence datasets (Clarkson et al. 2010; Marks et al. 2011a), the patterns from the different gene-region sequences had several conflicting branches. Nevertheless, in each tree, with one exception, N. sp. Corunna (D.E.Symon 17088) was distinct from other members of N. section Suaveolentes. On the basis of the ADHC sequences N. sp. Corunna (D.E.Symon 17088) was not distinct from N.burbidgeae. Using the tree generated from the concatenated sequences, among N. section Suaveolentes, the African species N. africana Merxm. and the New Caledonian species N. fragrans Hook. were sister to a well supported clade that contained all the Australian members. Most nodes in the Australian Suaveolentes clade were poorly supported and only two clades, being composed of more derived species, were well resolved. Species in these two groups generally have fewer chromosomes than do other members of the section. In Fig. 2, these clades are labelled A (six species; BS = 81%, posterior probability (PP) = 0.99) and B (4 species; BS = 99%, PP = 1). Clade A and Clade B are well supported as sisters (BS = 100%, PP = 1) in the tree. Nicotiana sp. Corunna (D.E.Symon 17088) forms a clade with N. goodspeedii, N. maritima, N. exigua, N. amplexicaulis N.T.Burb. and N. suaveolens. Although both are placed in Clade A, N. suaveolens and its synonym N. exigua do not cluster together. Clade B contains N. excelsior, N. rosulata, N. rotundifolia Lindl., and N. velutina H.-M.Wheeler.

Fig. 2.

Relationships of taxa in Nicotiana section Suaveolentes and relative position of the N. sp. Corunna (D.E.Symon 17088) accession. A. PCA biplot of flower measurements collected from selected Nicotiana species. The 95% confidence ellipses indicate the true population mean of the bivariate distribution for groups. Vector arrows represent the variable correlation plot. B. Summary phylogenetic maximum-likelihood and Bayesian-inference tree from combined ITS, matK, GSS, GSL, RDR1 and ADHC sequences with support values (bootstrap support and posterior probability values >70% or 0.7 respectively) are shown above branches. Branches with ≤70% /0.7 support value are annotated with an en-dash (–). Chromosome number is indicated in parentheses for each taxon.

Phenetic analysis of floral characters

Because DNA-based phylogenies pointed to N. sp. Corunna (D.E.Symon 17088) being a distinct species, a phenetic analysis of flowers was performed to obtain further evidence for it being a new species and to find characters that could be potentially useful in its identification. A PCA biplot (Fig. 2, inset) was constructed using N. sp. Corunna (D.E.Symon 17088) and the three species (N. goodspeedii, N. maritima and N. velutina) that overlap its geographic distribution (Fig. 3) and with its sequence-based cladistic sister, N. amplexicaulis. Nicotiana sp. Corunna (D.E.Symon 17088) flower characters formed a cluster that was well separated from N. goodspeedii, N. velutina and N. amplexicaulis, and predominantly separated from N. maritima. Nicotiana sp. Corunna (D.E.Symon 17088) was readily distinguished from N. goodspeedii, by its shorter stamens, smaller floral tubes and calyx, and thinner corollas; it was distinguishable from N. velutina by its shorter calyx length and narrower corolla limb diameter. A PCA that uses all the species described in Marks et al. (2011a) is shown in Fig. S7.

Fig. 3.

Nicotiana paulineana showing phyllotaxis, leaf form, flower structure, capsule and seed. Photograph inset: flower showing deeply cleft corolla lobes and approach herkogamy.

Nicotiana paulineana Newbigin & P.M.Waterh., sp. nov.

Type: Cultivated. Victoria. Melbourne University, Parkville, glasshouse on Natural Philosophy Building, 25 June 2007, C.E.Marks 299 (holo: MELU D106463!).

Plants glabrous, erect, annual, 0.4–1 m high. Initially single-stemmed, commonly developing several branched stems. Seedlings with cotyledons 6–7 mm long. Basal leaves rosulate, petiolate, attenuate, 10–30 cm long. Eglandular trichomes with one single gland cell on the calyx. Panicles loosely decompound, major branching long, rapidly ascendant. Calyx 5–7 mm long, appressed to tube. Corolla slightly zygomorphic, lobes emarginate, spreading; corolla limb lobes pure white inside, with a green to yellow vein running down the back of each lobe; floral tube broadening above calyx, 15–25 mm long exclusive of limb, 2–3 mm wide, often purplish. Stamens all included, 4 anthers at or close to one level, near mouth of corolla, anther of 5th stamen 2–3 mm lower; filaments inserted in lower half of corolla. Capsule not constricted or thickened, no seeds retained. Seeds reinform, sinuous seeds testa, 0.9 mm long, brown. Chromosome number 16 pairs.