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Research Article
Relationships among Calibrachoa, Fabiana and Petunia (Petunieae tribe, Solanaceae) and a new generic placement of Argentinean endemic Petunia patagonica
expand article infoAlejandrina Alaria‡, John H. Chau§|, Richard G. Olmstead§, Iris E. Peralta‡
‡ National University of Cuyo, Mendoza, Argentina
§ University of Washington, Seattle, United States of America
| University of Johannesburg, Johannesburg, South Africa
¶ IADIZA CCT CONICET, Mendoza, Argentina

Corresponding author: Iris E. Peralta ( iperalta@fca.uncu.edu.ar )

Academic editor: Leandro Giacomin
© 2022 Alejandrina Alaria, John H. Chau, Richard G. Olmstead, Iris E. Peralta.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Alaria A, Chau JH, Olmstead RG, Peralta IE (2022) Relationships among Calibrachoa, Fabiana and Petunia (Petunieae tribe, Solanaceae) and a new generic placement of Argentinean endemic Petunia patagonica. PhytoKeys 194: 75-93. https://doi.org/10.3897/phytokeys.194.68404
Open Access

Abstract

Calibrachoa Cerv., Fabiana Ruiz & Pav., and Petunia Juss. form a clade within tribe Petunieae (Solanaceae). Phylogenetic studies of Petunieae, either as part of a family-wide analysis or focusing on the genera Calibrachoa and Petunia, have either left Fabiana unsampled or included only a single species. These studies have found conflicting relationships among the three genera with all three possible topologies obtained. Petunia patagonica (Speg.) Millán, originally described in the genus Nierembergia Ruiz & Pav., is morphologically distinct within Petunia and geographically disjunct from other members of the genus. For the first time, in this study we include multiple species of Fabiana, Calibrachoa, and Petunia, including P. patagonica. Using three chloroplast DNA regions and the nuclear gene GBSSI, or “waxy,” our results provide strong support for a sister group relationship between Calibrachoa and Fabiana and for the placement of P. patagonica within Fabiana. Since there is already a species Fabiana patagonica Speg., we provide the new name Fabiana australis Alaria nom. nov. to replace Petunia patagonica.

Keywords

Fabiana australis, Patagonia, Petunia patagonica, Petunieae, phylogeny

Introduction

Solanaceae Juss. are one of the most important families among Angiosperms not only for their fundamental contribution to the human diet but also for their diversity and ecological functions in many ecosystems wordwide, especially in the Neotropics (Olmstead 2013). Solanaceae are a family of approximately 90 genera and ca. 2700–2800 species (Olmstead and Bohs 2007; Olmstead et al. 2008, but see Knapp et al. 2004 for a higher estimate). Molecular phylogenetic studies in Solanaceae, initially based on chloroplast DNA sequence data (Olmstead and Palmer 1992; Spooner et al. 1993; Olmstead and Sweere 1994; Olmstead et al. 1999; Gemeinholzer and Wink 2001; Santiago-Valentin and Olmstead 2003; Clarkson et al. 2004; Bohs 2005; Levin et al. 2005, 2006; Weese and Bohs 2007; Olmstead et al. 2008), and more recently using a combination of chloroplast and nuclear loci and with nearly all genera and over 1,000 species sampled, have produced a robust dated molecular phylogeny (Goldberg et al. 2010; Särkinen et al. 2013; Ng and Smith 2015; Dupin et al. 2016).

Molecular phylogenetic studies unraveled relationships that were not consistent with traditional classification of the family and split up tribe Nicotianeae and resurrected tribe Petunieae to include most of the former Nicotianeae, excluding Nicotiana L., but including Brunfelsia L. (Olmstead et al. 1999, 2008; Olmstead and Bohs 2007). These results showed that the genera Petunia Juss., Calibrachoa Cerv., and Fabiana Ruiz & Pav. form a strongly supported clade and provided weak evidence suggesting that Fabiana is sister to Calibrachoa and that together they are sister to Petunia. The tribe Petuniae has been subject to several phylogenetic studies, with most of the focus on Petunia and Calibrachoa (Ando et al. 2005; Kulcheski et al. 2006; Stehmann et al. 2009; Fregonezi et al. 2012, 2013; Reck-Kortmann et al. 2014, 2015; Mäder and Freitas 2019). Few of these studies have included representatives of Fabiana, and when they do, relationships among the three genera sometimes do not agree (e.g., Särkinen et al. 2013; Reck-Kortmann et al. 2015; Mäder and Freitas 2019). Also, the rare species Petunia patagonica (Speg.) Millán, which is morphologically distinct and geographically disjunct from other Petunia species, has an uncertain placement in Petunieae (Stehmann and Greppi 2013; Reck-Kortmann et al. 2015).

Fabiana is endemic to South America, distributed in southern Peru, Bolivia, Chile, and Argentina, with 15 species of shrubs adapted to the high Andean deserts of Puna, Prepuna, Monte, and Patagonia, growing from sea level to 4900 m elevation in sandy, rocky soils of very low fertility, low organic matter and variable salt content (Barboza and Hunziker 1993; Alaria and Peralta 2013; Alaria 2015). Fabiana species have characteristic morphological adaptations: leaflessness or small leaves, photosynthetic and resinous stems, and flat or cushion growth habits (Alaria 2015), and they play an important ecological role as codominant shrubs in some plant communities. Petunia patagonica is restricted to the Patagonian region of Argentina, and its identity is controversial; it was first described in Nierembergia Ruiz & Pav. by Spegazzini (1897) and subsequently transferred to Petunia by Millán (1941).

In this study we explore the relationships among these three genera using sequences of the plastid trnS-trnG, trnL-trnF and psbA-trnH regions and partial sequences of the nuclear GBSSI or “waxy” gene, providing the first evidence of relationships among species of Fabiana and resolving the systematic position of the enigmatic species Petunia patagonica.

Materials and methods

Taxon sampling

Leaf samples were obtained from fresh material collected in the natural habitat of species or from cultivated plants, and preserved in silica gel. Data, including collecting site, voucher, and herbarium where the voucher has been deposited, are indicated in Appendix 1 for specimens of tribe Petunieae: Bouchetia Dunal (1 species), Brunfelsia (1), Calibrachoa (4), Fabiana (9), Nierembergia (1), and Petunia (8, including P. patagonica), and outgroup taxa in the genera Benthamiella Speg. (1 species), Nicotiana (3), Pantacantha Speg. (1), and Solanum L. (1).

Additional specimens were analyzed for morphological traits, mainly species of Calibrachoa, Fabiana, Nierembergia, and Petunia in the Argentinean herbaria: BAB, CORD, CTES, LP, LIL, MEN, MERL, and SI, as well as in herbaria in Bolivia: LPB and HSB; Chile: LS; Perú: USM; and England: K (all acronyms are in accordance to Index Herbariorum; http://sweetgum.nybg.org/science/ih/). Specimens of Petunia patagonica were examined and are cited after the species description in the Discussion.

DNA amplification and sequencing

DNA extraction was performed using the Qiagen DNeasy Plant Mini Kit (http://www.qiagen.com). Three regions of chloroplast DNA were amplified. For the trnL-trnF region, the primers and amplification conditions of Taberlet et al. (1991) were used. This region was included in the phylogenetic analysis of Olmstead et al. (2008), where it provided essential characters for their classification of Solanaceae. Two other plastid fragments were selected for being more variable molecular markers in Calibrachoa (Fregonezi et al. 2012) and Petunia (Lorenz-Lemke et al. 2006; Kulcheski et al. 2006): the trnS-trnG region and the psbA-trnH region. For the trnS-trnG region, the primers and amplification conditions of Hamilton (1999) were used, and for the psbA-trnH region, the primers and amplification conditions of Sang et al. (1997) were used. The nuclear gene GBSSI or “waxy”, which has a single copy in tomato (Solanum lycopersicum L.) and diploid potato varieties (Solanum tuberosum L.) (van der Leij et al. 1991), was selected because it is an informative marker for phylogenetic analyses in Solanaceae (Peralta and Spooner 2001; Walsh and Hoot 2001; Levin and Miller 2005; Yuan et al. 2006; Spooner et al. 2008; Levin et al. 2011; Särkinen et al. 2013). The first waxy sequences of Fabiana specimens were obtained with primers initially designed for Solanum lycopersicum (Peralta and Spooner 2001), but new Petunieae-specific primers were generated: WAXY 5´ GTGGGTACTGAGGTTGGTCCTT and WAXY 3´ GGGCTCACTGTAACCACCCTAT, improving amplification of representative samples. Tomato and potato specimens were also amplified for waxy as controls for the expected fragment length, using a known molecular mass marker (Lambda/EcoRI-HindIII Marker). Purified PCR products were sequenced using standard Sanger sequencing methods at the Biogenomics Unit of the Institute of Biotechnology of the National Institute of Agricultural Technology (INTA Castelar), Buenos Aires, Argentina.

Editing and aligning of chloroplast and nuclear sequences

The sequences were manually edited with PROcessor of SEQuences (PROSEQ) version 2.9 (Filatov 2002). Alignment was performed in BioEdit version 5.0.6. (Hall 2004), first using ClustalW and then adjusted by manual alignment. Waxy alignments were made by comparing sequences with the gene in Solanum tuberosum (GenBank accession X83220). Chloroplast fragments were aligned with Petunia axillaris sequences for trnS-trnG (JF918370), trnL-trnF (AY098702), and psbA-trnH (DQ225610).

Phylogenetic analysis

We created three datasets for phylogenetic analyses. One dataset consisted of the three plastid loci for 21 species. A second dataset comprising 20 samples consisted of the nuclear waxy sequences. The third dataset comprising 18 samples consisted of the three plastid loci and the nuclear waxy gene concatenated for each species for which sequences for waxy and at least two of the three plastid loci were available. For each locus, we compared nucleotide substitution models using the Akaike Information Criterion from analyses in jModeltest 2.1.4 (Guindon and Gascuel 2003; Darriba et al. 2012) and chose an appropriate model within the 95% confidence interval. Phylogenetic analyses were performed using two different inference methods, maximum likelihood (ML) and Bayesian. In analyses with a concatenated sequence dataset, each locus was treated as a separate partition, and the GTR + gamma model of nucleotide substitution was used for each partition. In analyses with just waxy, the HKY + gamma model was chosen. We performed ML analyses using GARLI 2.0 (Zwickl 2006). We executed four replicates of each full search, and used a generation threshold for termination of 20,000 and score threshold for termination of 0.001. Default settings were used for all other parameters. We additionally performed bootstrap searches using a generation threshold for termination of 10,000. For concatenated datasets, 500 bootstrap replicates were done, and for the waxy dataset, 1,000 bootstrap replicates were done. Bayesian analyses were done in MrBayes 3.2.1 (Ronquist et al. 2012) with two runs with four chains each. For concatenated datasets, analyses were run for 10,000,000 generations, sampling every 1,000 generations. For the waxy dataset, analyses were run for 5,000,000 generations with a sampling frequency of 500 generations. We determined that convergence was attained when the average standard deviation of split frequencies was <0.05 and the estimated sample size of parameters was >200 in Tracer 1.5 (Rambaut and Drummond 2009). Majority-rule consensus trees were constructed after discarding the initial 25% of samples as burn-in.

Results

The analyses of concatenated chloroplast sequences (Fig. 1) confirmed monophyly of the genera Calibrachoa, Fabiana, and Petunia, with the exclusion of P. patagonica (see below) and the phylogenetic relationships among the three genera was the same found by Olmstead et al. (2008) with Petunia sister to Fabiana and Calibrachoa, and the last two sister to each other. With limited sampling in the rest of tribe Petunieae and outgroups, Nierembergia is weakly supported as sister to Nicotiana. The analysis also strongly supports the placement of Petunia patagonica within Fabiana.

Figure 1. 

Chloroplast DNA tree of 17 Petunieae species and four outgroups. Phylogeny based on maximum likelihood analysis of concatenated trnS-trnG, trnL-trnF, and psbA-trnH chloroplast fragments. Maximum likelihood bootstrap values and Bayesian posterior probabilities shown at nodes.

Waxy amplifications always showed a single band for a fragment of similar size to the one in potato and tomato controls. Although waxy gene copy number is unknown in Fabiana, Calibrachoa, and Petunia, it is expected to be a single copy and orthologous in the analyzed taxa, as has been demonstrated in other diploid Solanaceae species (van der Leij et al. 1991). The waxy phylogeny of 20 taxa (Fig. 2) is consistent with the chloroplast DNA analyses in recovering the monophyly of Calibrachoa, Fabiana, and Petunia, and the same relationships among them. Similarly, Petunia patagonica is included within Fabiana. With strong support, Nierembergia is resolved as sister to Bouchetia.

Figure 2. 

Nuclear waxy tree of 20 Petunieae species. Phylogeny based on maximum likelihood analysis of the nuclear waxy gene. Maximum likelihood bootstrap values and Bayesian posterior probabilities shown at nodes.

The results of the analyses of the concatenated sequences of chloroplast loci and waxy combined also recovered the same major relationships (Fig. 3). Monophyly of Fabiana with Petunia patagonica nested within it is strongly supported (posterior probability = 1.0). In these analyses, P. patagonica is weakly supported as sister to Fabiana imbricata Ruiz and Pav. Additionally, Fabiana is resolved as sister to Calibrachoa with strong support, and Petunia is sister to the clade comprising Fabiana and Calibrachoa. Nierembergia forms a clade with the remaining representatives of Petunieae with strong support.

Figure 3. 

Combined chloroplast and nuclear DNA tree of 14 Petunieae species and four outgroups. Phylogeny based on maximum likelihood analysis of chloroplast fragments trnS-trnG, trnL-trnF and psbA-trnH and the nuclear waxy gene concatenated. Maximum likelihood bootstrap values and Bayesian posterior probabilities shown at nodes.

Discussion

Sequences of three chloroplast markers and the nuclear gene waxy were informative for the inference of phylogenetic relationships among Calibrachoa, Fabiana, and Petunia in tribe Petunieae. Both the analyses of the combined chloroplast regions and the nuclear gene waxy corroborated Calibrachoa as sister to Fabiana with strong support, as obtained by Olmstead et al. (2008). A few studies of Petunieae with a single species of Fabiana sampled obtained results with Fabiana either sister to Petunia plus Calibrachoa (Särkinen et al. 2013) or Petunia (Reck-Kortmann et al. 2015; Mäder and Freitas 2019), in contrast to our results. All of our analyses also found Petunia patagonica to be nested within Fabiana. This result is consistent with that of Reck-Kortmann et al. (2015), who found P. patagonica sister to F. imbricata and the combined clade sister to the rest of Petunia; but with only a single representative of Fabiana, that study was not able to reveal conclusively the placement of P. patagonica within Fabiana. Considering these phylogenetic results as well as shared morphological characteristics, geographical distribution, and chromosomal number between Fabiana and Petunia patagonica (Reck-Kortmann et al. 2015), the transfer of Petunia patagonica to the genus Fabiana is strongly supported.

Circumscription of Fabiana and transfer of Petunia patagonica to Fabiana

This study resolves the phylogenetic position of Petunia patagonica, an enigmatic species of controversial generic affinity. Spegazzini (1897) originally described this species in the genus Nierembergia based on a specimen collected in Gulf Saint George, Argentina. The corolla and androecium resemble other Nierembergia species, with a narrow base to the corolla tube that expands distally. Subsequently, in a review of Nierembergia, Millán (1941) transferred the species to Petunia, considering its floral characters as closer to this genus. The species has several characteristics that differentiate it from all other Petunia species and has been considered an outlier in the genus based on its geographic distribution and chromosome number (Stehmann and Greppi 2013; Reck-Kortmann et al. 2015). The species was included in a molecular phylogenetic study with several species of Petunia, two species of Calibrachoa, but only one species of Fabiana and was found in a small clade with F. imbricata, which was sister to a clade comprising the other species of Petunia (Reck-Kortmann et al. 2015).

Traditional classifications of Petunia patagonica have relied primarily on morphology, but with the insight gained from molecular phylogenetic studies, we can see that taxonomists weighed different floral traits in assigning the species first to Nierembergia and then to Petunia, while overlooking the similarities with Fabiana, including the resinous stems and dorsifixed anthers. Other characteristics, such as the chromosome number of Petunia patagonica (n = 9), match those found in Fabiana species (Acosta et al. 2006). The particular distribution of this species in southern Patagonia (Fig. 4) and the environment where it grows, are similar to those of other Fabiana species (e.g., F. imbricata, F. foliosa (Speg.) S.C.Arroyo and F. nana (Speg.) S.C.Arroyo). The results of the molecular phylogenetic analyses obtained in this work also support its transfer to the genus Fabiana.

Figure 4. 

Geographic distribution of the genera Fabiana, Calibrachoa, Petunia, and Nierembregia, and the species Fabiana australis Alaria. Flowers and seeds of representative species: Fabiana patagonica Speg (first flower scale: 2.5 mm, second flower scale: 5 mm, seed scale: 0.5 mm, surface details magnifications 600× and 1,500×); Calibrachoa parviflora (Juss.) D’Arcy (first flower scale: 2.5 mm, second flower scale: 5 mm, seed scale: 0.5mm, surface details magnifications 500× and 1,500×); Petunia axillaris (Lam.) Britton, Sterns & Poggenb. (first flower scale= 5mm, second flower scale: 10mm, seed scale: 0.5mm, surface details magnifications 600× and 1,500×); Nierembergia pulchella Gillies ex Miers (first flower scale: 5mm, second flower scale: 10mm, seed scale: 0.5mm, surface details magnifications 600× and 1,500×). Photograph IBODA, Flora Argentina database.

Fabiana australis Alaria, nom. nov.

urn:lsid:ipni.org:names:77297063-1
Figures 4, 5

Petunia patagonica (Speg.) Millán. Darwiniana 5: 544 1941.

Basionym

Nierembergia patagonica Speg. Revista Fac. Agron. Univ. Nac. La Plata 3: 357. 1897, non Fabiana patagonica Speg. (1897). Type: Argentina. Prov. Santa Cruz, Golfo de San Jorge, C. Ameghino, Febr. 1896, “in campis aridis glariosis” (holotype: LP 006658!).

Description

Densely branched shrubs forming compact cushions of approximately 50 cm tall and up to 2.5 (-4) m in diameter; stems erect, leafy, glandular-pubescent, resinous. Leaves alternate but apparently verticillated by shortening of the internodes, sessile, fleshy, glandular; linear, elliptical or obovate, blade 3–4 (5) mm long by 1–2 mm wide. Flowers terminal, solitary, erect; flowering pedicels of 4–6 mm. Calyx tubular or slightly campanulate, (8–) 11–12 mm long, externally with dense glandulous indumentum, internally with scattered glandulous pubescence, short and broadly triangular lobes, 2–3 mm long by 2–3.5 mm wide, almost as long as wide. Corolla yellow with marked violet nerves, but also with color variation from light purple to deep violet, infundibuliform, 20–25 (–30) mm long, externally glandular, broad triangular lobes 2.5–5 mm long by 6–10 mm wide. Stamens heterodynamous, adhered to the middle third of the corolla, about 9 mm from the base of the corolla; filaments 2 short and 3 long, somewhat geniculate at the point of insertion with short glandular hairs scattered at the base; ellipsoid anthers of 1–1.5 mm. Ovary obovoid, 2.5–3 mm long, 1.2–1.5 mm wide, with nectariferous disc surrounding the base, 10–15 mm long style, truncated stigma, shallowly split. Capsule ovoid, 5–9 mm long by 3–5.5 mm wide. Seeds numerous, polyhedrical in shape, 2–2.5 mm long.

Figure 5. 

Fabiana australis Alaria. A plant B flowering branch C flower D corolla deployed showing gynoecium and stamens of different length E stigma F anthers G capsule H capsule showing seeds, I capsule valve J seeds. Scale bars: 10 mm (A); 4 mm (B); 5 mm (C); 2.5 mm (D); 1 mm (E); 0.5 mm (F); 2 mm (G, H). Illustration by Cecilia Scoones.

Common name

“Mogote” meaning mound shape (Santa Cruz: Arroyo 1999)

Geographical distribution and habitat

Endemic to Patagonian Argentina, in the provinces of Chubut and Santa Cruz, from 90 to 700 m elevation, inhabiting dry and cold environments, on stony, sandy soils, sometimes rich in silt and clay, poor in organic matter. It forms large populations of cushion shrubs with numerous showy flowers (Fig. 6).

Figure 6. 

Fabiana australis Alaria. Plants habit and flower details (Zuloaga FO 13991, SI). Photograph IBODA, Flora Argentina database.

Taxonomic notes

The epithet autralis was selected based on the restricted distribution of this species in southern Argentina. It is not possible to use patagonica as a specific epithet in Fabiana, because it is already in use in Fabiana patagonica Speg. Fabiana australis is one of the southernmost species of the genus. It shares with F. foliosa and F. nana a similar habit forming dense cushions in dry, cold, and poor soils of Patagonian Argentina.

Representative specimens examined

Argentina: Santa Cruz: Dpto. Corpen Aike, G.E. Barboza 3706 (CORD); O. Boelcke 16264 (BAB); A.A. Cocucci 3684 & 3723 (CORD); M.N. Correa 6527 (BAB); R.H. Fortunato 7492 (BAB); C.A. O’Donell 3794 (CORD00015699). Dpto. Deseado, L.M. Bernardello & M.R. Figueroa Romero 335 (CORD00015696!); O. Boelcke, 12214 (BAB); A.A. Cocucci 4175 (CORD); M.N. Correa 2644 & 6697 (BAB); B.E Leuenberger 4100 (B: D-14191 Berlin); M.C. Romanczuk 989 (UEC); F.B. Vervoorst 5658 (CORD 00015700!). Dpto. Güer Aike, A. Soriano 5062 (BAB). Dpto. Lago Argentino, G.E. Barboza 3732 (CORD); A.A. Cocucci 471 (CORD 00015694!); R.H. Fortunato 4967 (BAB, ARIZ, NY, HRP); C. Guerrido 785 (SI). Dpto. Magallanes, G.E. Barboza 3704 (CORD); O. Boelcke 15394 (BAB); Iter Patagonicum 762 L. Hauman & C.M. Hicken (SI); B.E. Leuenberger & S. Arroyo 3710 (CORD 00015698!). Dpto. Río Chico, M.N. Correa & E.G. Nicora 3517 (BAB; CORD 00015697!); G.E. Barboza 3696 (CORD); G.E. Barboza 3746 (CORD; SI 063988!); G.E. Barboza 3748 (CORD); O. Boelcke 12810 (BAB); J.M. Bonifacino 2986 (SI); C.M. Hicken 10245 (SI); F.O. Zuloaga 13978 & 13991 (SI). Valle del Río Santa Cruz C. Burmeister s.n. & 95 (SI); M. Gentili 330 (BAB); J. Koslowsky 122 (CORD 00015695!). Without locality A. Donat 206 (SI); P.K.H. Dusén 5496 (SI); E. Molina Massey 31 (SI); Tessleff 5496 (SI). Chubut: Dpto. Futaleufú, A.A. Cocucci 3997 (CORD 00022097!); Dpto. Paso de Indios, S.C. Arroyo 208 (BAB, LIL, K); Dpto. Sarmiento, A. Alaria 321 (MERL). Dpto. Languiñeo, A. Alaria 324 (MEN).

Taxonomic characters differentiating Calibrachoa, Fabiana, Petunia, and Nierembregia are described in the following key. Geographic distribution of the four genera and Petunia patagonica, as well as photographs of flowers and seeds of representative species of each genus, are illustrated in Figure 4.

Key to genera

1 Resinous shrubs to camephytes, stems densely leafy to partially foliated and even aphyllous; reduced membranaceous, slightly fleshy or leathery leaves. Dorsifixed anthers, usually elongated Fabiana
– Non resinous, annual or perennial herbs, rarely subshrubs; leafy stems, developed membranaceous to fleshy leaves. Ventrifixed anthers with different shapes: reniform, globose, or ovate 2
2 Hypocrateriform corolla with narrow and cylindrical tube. Androecium with 5 fertile stamens equal in length or heterodynamous, generally with 2 longer and 3 shorter stamens, adnate at the top edge of the corolla tube and generally connivent around the style; wide stigma usually tightly arranged between the anthers; staminal filaments and style apex usually straight. Nectary absent. Polyhedral seed, straight embryo Nierembergia
– Infundibuliform to campanulate, rarely hypocrateriform, corolla with wide tube. Androecium with 5 fertile heterodynamous stamens, generally with 2 longer, 2 medium length, and one shorter stamen, or 4 subequal and one shorter stamen, adnate at the top edge of the corolla tube but rarely connivent around the style; narrow stigma, staminal filaments and apex style usually curved. Nectary present. Ellipsoid, round, or reniform seed, straight or slightly curved embryo 3
3 Corolla with reciprocative aestivation, the induplicated anterior lobe covering the other four conduplicated lobes, or rarely imbricate aestivation; calyx usually divided nearly to the middle, lobes narrowing towards the apex; seed episperm with straight anticlinal cell walls Calibrachoa
– Corolla with imbricate aestivation; deeply lobed calyx, lobes linear or spatulate, widening towards the apex; seed episperm with wavy anticlinal cell walls Petunia

Acknowledgements

The authors thank Gloria Barboza and Sandra Knapp for their taxonomic insights into tribe Petunieae, Cecilia Scoones for botanical illustrations, and Cinthia Costa for map and photograph designs. This study was supported by CONICET and SIIP - UNCuyo awards. The authors have declared that no competing interests exist.

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Appendix 1

Voucher and locality information for specimens of species collected for this study and GenBank accession numbers for sequences used, with those generated for this study in bold.

Table A1.
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XLSX
Species Voucher Location Coordinates trnL-trnF trnS-trnG psbA-trnH waxy
Petunieae Tribe
Bouchetia erecta DC ex Dunal D’Arcy 18213 MO Mexico – – – – OK120263
Brunfelsia americana L. no voucher Cultivated. USA, Matthaei Bot Gard – – – – OK166947
Calibrachoa humilis (R.E.Fr.) Stehmann & Semir no voucher Cultivated. Argentina, FCA, UNCuyo 33°00'28.2"S, 68°52'16.4"W MZ855907 OK120228 MZ855925 OK120243
Calibrachoa parviflora (Juss) D´Arcy Alaria 432 MERL Cultivated. Argentina, FCA, UNCuyo 33°00'28.2"S, 68°52'16.4"W MZ855908 OK120229 MZ855926 OK120244
Calibrachoa missionica Stehmann & Semir no voucher Cultivated. Argentina, FCA, UNCuyo 33°00'28.2"S, 68°52'16.4"W MZ855909 OK120230 MZ855927 –
Calibrachoa thymifolia (A.St.-Hil.) Stehmann & Semir no voucher Cultivated. Argentina, FCA, UNCuyo 33°00'28.2"S, 68°52'16.4"W MZ855910 – MZ855928 OK120245
Fabiana bryoides Phil. Alaria 444 MERL Argentina, Jujuy 22°31'47.8"S, 66°18'45.4"W MZ855911 OK120231 MZ855929 _____
Fabiana densa J. Rémy Alaria 365 MERL Bolivia, Potosi 19°52'48.2"S, 65°40'44.4"W MZ855912 OK120232 MZ855930 OK120246
Fabiana denudata Miers Alaria 356 MERL Argentina, Mendoza 32°29'16.3"S, 69°05'07.5"W MZ855913 OK120233 MZ855931 OK120247
Fabiana foliosa (Speg.) S.C.Arroyo Barboza 3760 CORD Argentina, Santa Cruz 47°20'09"S, 70°59'05"W – – MZ855932 OK120248
Fabiana imbricata Ruiz & Pav. Alaria 397 MERL Argentina, Mendoza 35°51'451"S, 69°48'27.5"W MZ855914 OK120234 MZ855933 OK120249
Fabiana nana (Speg.) S.C.Arroyo Alaria 316 MERL Argentina, Chubut 45°47'44.5"S, 69°04'56.7"W MZ855915 OK120235 MZ855934 OK120250
Fabiana patagonica Speg. Alaria 359 MERL Argentina Jujuy 22°57'30"S, 65°25'39"W MZ855916 OK120236 MZ855935 OK120251
Fabiana peckii Niederl. Alaria 403 MERL Argentina, Mendoza 34°31'55.2"S, 68°28'14.7"W MZ855917 OK120237 MZ855936 OK120252
Fabiana punensis S.C.Arroyo Alaria 048 MERL Argentina, Tucumán 26°38'39.5"S, 65°49'12.5"W MZ855918 OK120238 MZ855937 OK120253
Nierembergia scoparia Sendtn. Alaria 431 MERL Cultivated. Argentina, FCA, UNCuyo 33°00'28.2"S, 68°52'16.4"W MZ855920 OK120240 MZ855939 OK120255
Petunia altiplana T. Ando & Hashim – – – AY772868 DQ792185 DQ791917 –
Petunia axillaris (Lam.) Britton, Sterns & Poggenb. Alaria 430 MERL Argentina, Mendoza 32°58'45.7"S, 68°58'8"W AY098702 JF918370 DQ225610 OK120258
Petunia exserta Stehmann Chau 312 WTU Cultivated. USA, University of Washington – – – – OK120259
Petunia inflata R.E. Fr. Olmstead S-62 WTU Cultivated. USA, seed from Birmingham seed collection – – – – OK120260
Petunia integrifolia (Hook.) Schinz & Tell. Chau 311 WTU Cultivated. USA, University of Washington – AY772873 JN565848 DQ208151 OK120261
Petunia nyctaginiflora Juss. Olmstead S-63 WTU Cultivated. USA, seed from Birmingham seed collection – – – – OK120262
Petunia patagonica (Speg.) Millán Alaria 321 MERL Argentina, Chubut 45°56'16.8"S, 69°09'12.4"W MZ855919 OK120239 MZ855938 OK120254
Petunia scheideana L.B. Sm. & Downs – – – AY772870 DQ792448 DQ792149 –
Outgroups
Benthamiella pycnophylloides Speg Barboza 3688 CORD Argentina, Santa Cruz 46°57'2"S, 67°22'24.6"W MZ855921 OK120241 MZ855940 –
Nicotiana attenuata Torr. Ex S. Watson – – – AY098697 AJ584953 MG182422 KR083023
Nicotiana longiflora Cav. Alaria 437 MERL Argentina, Mendoza 32°59'47.7"S, 68°56'1.5"W MZ855923 AJ584951 MZ855942 OK120256
Nicotiana noctiflora Hook. Alaria 438 MERL Argentina, Mendoza 32°59'49.4"S, 68°55'56.6"W MZ855924 AJ584975 GQ248352 OK120257
Pantacantha ameghinoi Speg. Barboza 3775 CORD Argentina, Neuquén 38°52'0"S, 70°34'36.2"W MZ855922 OK120242 MZ855941 –
Solanum lycopersicum L. – – – KY887587 HQ856092 KY887587 DQ169036
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