Shades of white: The Petunia long corolla tube clade evolutionary history

Abstract

Delimiting species is challenging in recently diverged species, and adaptive radiation is fundamental to understanding the evolutionary processes because it requires multiple ecological opportunities associated with adaptation to biotic and abiotic environments. The young Petunia genus (Solanaceae) is an excellent opportunity to study speciation because of its association with pollinators and unique microenvironments. This study evaluated the phylogenetic relationships among a Petunia clade species with different floral syndromes that inhabit several environments. We based our work on multiple individuals per lineage and employed nuclear and plastid phylogenetic markers and nuclear microsatellites. The phylogenetic tree revealed two main groups regarding the elevation of the distribution range, whereas microsatellites showed high polymorphism-sharing splitting lineages into three clusters. Isolation by distance, migration followed by new environment colonization, and shifts in floral syndrome were the motors for lineage differentiation, including infraspecific structuring, which suggests the need for taxonomic revision in the genus.

Keywords:
Solanaceae; genetic variability; speciation; evolutionary relationships

Introduction

Adaptive radiation plays a fundamental role in our understanding of the evolutionary process, and it is frequently accepted that adaptive radiation requires multiple ecological opportunities associated with adaptation to biotic and abiotic environments (Gillespie et al., 2020). Criteria such as common ancestry, phenotype-driver selector, and rapid speciation have been proposed to identify adaptive radiation (Schluter, 2009). However, some authors consider it challenging to prove for most studies (Gillespie et al., 2020). Delimiting species is difficult in recently derived species because of the short time interval since speciation could not be enough to accumulate genetic differentiation (e.g., Knowles and Carstens, 2007).

The genus Petunia (Solanaceae) encompasses 17 wild species distributed in southern South America (Greppi et al., 2019) and one of the most important ornamental plants, P. hybrida. Divided into two main clades based on molecular phylogenetic analysis (Reck-Kortmann et al., 2014), the genus has 14 bee-pollinated species that share several morphological traits, especially the corolla tube length, which is short, and the bluish pollen. Three other species display long corolla tubes and yellow pollen and are more variable in attracting different pollinators (Stehmann et al., 2009; Fregonezi et al., 2013). The ornamental species P. hybrida is considered a perfect supermodel for genetic and physiological studies (Vandenbussche et al., 2016), and the wild species might be excellent models for understanding the evolutionary process for young groups. The clades diverged ca. 2.8 Mya (Särkinen et al., 2013), and species in the short corolla clade colonized highland grasslands, diversifying ca. 1.0 Mya (Lorenz-Lemke et al., 2010).

The topology of Petunia phylogenetic trees profoundly changes when different molecular markers are considered. When based only on plastid markers, species are preferentially grouped according to their distribution in highlands (elevation up to 500 m above the sea level - a.s.l.) or lowlands (below 500 m high), respectively (Ando et al., 2005; Lorenz-Lemke et al., 2010). When the relationships are recovered based on only nuclear markers or combining nuclear and plastid sequences, the clades’ composition is supported by the corolla tube length, with the terminals’ position varying among gene trees (Chen et al., 2007; Kriedt et al., 2014; Reck-Kortmann et al., 2014; Segatto et al., 2016).

The species in the short corolla tube clade (ST) share several morphological and ecological traits, and often it is difficult to distinguish them based only on morphology (Longo et al., 2014). The extensive genetic polymorphism sharing and some variable traits have promoted changes in the taxonomic classification of this group over time (Segatto et al., 2017). In the long corolla tube group (LT), the species are identified based on the corolla color (Stehmann et al., 2009), and no doubt has been put on their identity.

The diversification in each clade has been attributed to different main drivers. For species in the ST, especially those occupying higher elevations (ca. 900 m a.s.l. or more), it has been proposed an allopatric speciation, strongly influenced by climate changes during the late Pleistocene (Lorenz-Lemke et al., 2010; Barros et al., 2015, 2020). Pleistocene effects were also implicated in the intraspecific diversification of some species (Backes et al., 2019; Souza et al., 2022; Soares et al., 2023). Additionally, for ST lowland species (elevation < 500 m), ecological factors and geomorphology were the most important features, even when the species are parapatric (Ramos-Fregonezi et al., 2015; Segatto et al., 2017). The LT species show morphological traits associated with distinct floral syndromes, and the interaction with different pollinators is described as the main driver for diversification (Fregonezi et al., 2013).

The LT clade encompasses the species P. axillaris, divided into three subspecies [P. axillaris subsp. axillaris; P. axillaris subsp. parodii, and P. axillaris subsp. subandina - (hereafter shortly P. axillaris, P. parodii, and P. subandina, respectively)], P. exserta, P. secreta, and P. occidentalis. The P. axillaris subspecies display white flowers that are moth-pollinated (Ando et al., 1995; Venail et al., 2010); the bright red color and flower morphology of P. exserta attract hummingbirds (Stehmann et al., 2009); P. secreta shows pink corollas and is a bee-pollinated species (Rodrigues et al., 2018). The morphology of P. occidentalis corresponds to the melitophilous floral syndrome. However, no systematic pollination studies have been conducted with this taxon, and its effective pollinator is still unknown.

Each taxon in LT shows different patterns of genetic structure throughout the geographic range (Segatto et al., 2014; Turchetto et al., 2014a,b, 2016; Giudicelli et al., 2022) and a complex process of intraspecific diversification emerges: P. parodii shows three main lineages, geographically structured (Chaco, Pampa-Brazil, and Pampa-Uruguay; Giudicelli et al., 2022); P. exserta revealed two lineages with slight morphological variation and distribution (P. exserta E1 and P. exserta E2), each one occurring in a different rock formation in Serra do Sudeste; and P. secreta that would have two main genetic lineages (Turchetto et al., 2016), more distinct from each other than canonical P. secreta is from P. axillaris (here treated as P. secreta and P. sp1, respectively). An unnamed taxon (P. sp3) occurs close to P. secreta and P. exserta E1.

All taxa in LT have high levels of genetic polymorphism sharing (Kulcheski et al., 2006; Fregonezi et al., 2013; Reck-Kortmann et al., 2014; Turchetto et al., 2016), and interspecific hybridization has been observed among them (Lorenz-Lemke et al., 2006; Segatto et al., 2014; Turchetto et al., 2015, 2019a, b; Giudicelli et al., 2019; Teixeira et al., 2019; Schnitzler et al., 2020; Caballero-Villalobos et al., 2021). Intraspecific morphological diversity was also observed (Turchetto et al., 2016; Giudicelli et al., 2019; Teixeira et al., 2020), even in taxa that did not display differentiated genetic lineages as P. axillaris (Turchetto et al., 2014b), which has a morphotype from coastal (A1) and another from inland (A2) distribution.

Except for phylogenetic analyses, the LT taxa were not evaluated together based on their intra and interspecific genetic diversity. Thus, we aimed to (i) determine the phylogenetic relationships among taxa and intraspecific lineages in the long corolla tube clade of Petunia based on phylogenetic informative markers; (ii) compare the intraspecific genetic diversity among the LT taxa based on nuclear microsatellites; and (iii) identify any diversification process in course among LT lineages. We based our study on the cohesive species concept proposed by Templeton (1989) and as treated in Haselhorst et al. (2019).

Material and Methods

Phylogenetic approach

We collected young and healthy leaves from multiple individuals of each LT lineage (Figure 1), except for P. occidentalis, for which we used an herbarium-derived sample (^{Table S1}). We extracted the total DNA using the CTAB (cetyl-trimethyl ammonium bromide)-based method (Roy et al., 1992), evaluated DNA quality in a NanoDrop DN 1000 spectrophotometer (Thermo Fischer Scientific Co., Waltham, USA), and estimated the quantity using a Qubit fluorometer (Thermo Fischer).

Figure 1 -
Representative individuals of each analyzed Petunia lineage. (A) P. subandina; (B) P. exserta E2; (C) P. sp3; (D) P. axillaris A2; (E) P. sp1; (F) P. secreta; (G) P. occidentalis; (H) P. axillaris A1; (I) P. parodii; (J) P. exserta E1.

We amplified seven nuclear regions and five plastid DNA markers through PCR reactions using previously described primers and protocols (^{Table S2}). We included once-obtained sequences (Reck-Kortmann et al., 2014) for some samples. We used two Calibrachoa species (Mäder and Freitas, 2019) and P. integrifolia representing the ST (Reck-Kortmann et al., 2014) as outgroups. Amplicons were purified using a polyethylene glycol method (Dunn and Blattner, 1987) and sequenced in an ABI 3730XL (Thermo Fischer Sci.) sequencer.

We assembled and edited sequences using Chromas v.2.0 software (Technelysium, Helensvale, Australia) and prepared alignments per DNA marker using Muscle in MEGA X (Kumar et al., 2018) and concatenated them to the phylogenetic analyses. We manually edited the alignments when necessary and coded contiguous insertion/deletion (indels) events involving more than one base pair (bp) as one mutational event (Simmons and Ochoterena, 2000). We did not include ambiguous sites (more than one pick in the chromatogram) from nuclear markers in the final matrix (Mäder et al., 2010). One representative of each different sequence was deposited at GenBank (^{Table S3}). We also used MEGA to estimate genetic diversity per marker (Table 1).

Table 1 -
Genetic diversity per marker used to obtain the phylogenetic tree for Petunia long corolla tube clade.

Genetic Marker	Alignment length	Variable sites (%)	PI sites (%)	Evolutionary Model*
trnH-psbA 1	424	20 (4.7)	9 (2.1)	HKY+G
trnS-trnG 1	652	15 (2.3)	9 (1.4)	HKY+G
rps12-rpl20 1	777	26 (3.4)	13 (1.7)	HKY+G
trnL-rpl32 1	976	25 (2.6)	13 (1.3)	HKY+G
matK	857	21 (2.5)	13 (1.5)	GTR+I
cpDNA Total	3,686	107 (2.9)	57 (1.6)	-
ITS	536	55 (10.3)	29 (5.4)	GTR+G
Hf1	1,395	50 (3.6)	24 (1.7)	GTR+G
PolA1	987	25 (2.5)	9 (0.9)	HKY+G
G3pdh	544	28 (5.2)	15 (2.8)	HKY+G
PID3C4	209	10 (4.8)	6 (2.9)	HKY+G
WOX4	231	43 (18.6)	32 (13.9)	HKY+G
WUS	206	31 (15.1)	18 (8.7)	HKY
nuDNA Total	4,108	242 (5.9)	133 (3.2)	-
Total	7,794	349 (4.5)	190 (2.4)

• PI - parsimoniously informative sites; *Best substitution model estimated with jModelTest based on Akaike information criterion; ¹concatenated sequences

To estimate the evolutionary relationships among taxa and lineages, we used a Bayesian inference (BI) as implemented in BEAST v.1.10 (Suchard et al., 2018), assessing the tree support with posterior probability (PP) with 10⁷ chains. We estimated the best substitution model and gamma rate heterogeneity using jModelTest v.3.06 (Darriba et al., 2012) based on the Akaike information criterion (AIC) for each nuclear marker, matK gene, and combined intergenic plastid spacers, respectively (Table 1). We conducted BI analysis under the Yule process and two independent runs of 10 million generations, sampling every 1000 generations. We assessed Markov chain Monte Carlo (MCMC) convergence by examining effective sample size values (ESS > 200) and likelihood plots in Tracer v.1.7 (Rambaut et al., 2018). We discarded the initial 25% of trees as burn-in and summarized the remaining trees to generate a maximum clade credibility tree using TreeAnnotator v.1.7.5 (Suchard et al., 2018) visualized with FigTree v.1.4.1 (http://tree.bio.ed.ac.uk/software/figtree/). PP ≥ 0.90 values were considered to represent strong support.

Intraspecific variability

To estimate the intraspecific diversity, we amplified seven nuclear microsatellite loci (^{Table S4}) for all taxa (except P. occidentalis), including individuals throughout the entire geographic distribution of each lineage, proportional to population density. We genotyped 10 P. axillaris A1, 63 P. axillaris A2, 13 P. exserta E1, 82 P. exserta E2, 50 P. secreta, 39 P. parodii, 23 P. subandina, 23 P. sp1, and 11 P. sp3. We visualized and scored the alleles with GeneMarker v.1.97 software (Softgenetics LLC, State College, USA) and used Micro-Checker (van Oosterhout et al., 2004) software to identify possible null alleles, significant allele dropout, and scoring errors due to stutter peaks.

We used the FSTAT v.2.9.3.2 software (Goudet, 1995) to evaluate the number of alleles per locus (A) and Nei’s unbiased gene diversity (GD; Nei, 1987). Additionally, we used AZDE (Szpiech et al., 2008) to estimate allelic richness (AR) and number of private alleles (PA) through rarefaction, as sample sizes vary among lineages.

We conducted a discriminant analysis of principal components (DAPC; Jombart et al., 2010) employed in the R program for Statistical Computing v.3.3.2 (R Core Team, 2020) to explore genetic groups. The lowest Bayesian information criterion (BIC) in DAPC was used to assess the best number of groups, and we did not include taxonomic and geographic prior information.

Results

Evolutionary relationships

We obtained a data matrix with 7,794 characters based on the DNA markers, from which ~5% were variable, and ~3% were parsimoniously informative. Nuclear regions were more variable and informative than plastid markers (Table 1). The BI analysis (Figure 2A) split the species with long corolla tubes in two main clades, mainly based on elevation: clade I, species distributed in elevations higher than 700 m a.s.l (P. subandina and P. occidentalis), and clade II, species found at less than 700 m a.s.l (remain lineages). Clade II also could be divided into two subclades, IIA encompassing P. secreta, P. sp1, P. sp3, and the inland lineage of P. axillaris. In subclade IIB, we found coastal P. axillaris lineage, two P. exserta lineages, and P. parodii. These ten lineages were well supported (PP ≥ 0.90), except for the P. parodii positioning (PP < 0.90). The separation between Petunia LT and ST clades was confirmed.

Figure 2 -
Evolutionary relationships among Petunia long corolla tube clade. (A) Bayesian inference phylogenetic tree including plastid and nuclear sequences. Each branch represents collapsed individuals with identical sequences. (B) Cartesian plane obtained in DAPC analysis based on nuclear microsatellites (best K = 3). Colors indicate clusters: red, cluster 1; green, cluster 2; and blue, cluster 3. Cluster composition in lineages and individual numbers follow ^{Table S5}.

Intraspecific variability

Considering the seven SSR loci, all individuals exhibited a maximum of two alleles per locus, as expected for diploid species, and the sizes of the alleles were compatible with the repetition for each locus. All loci were polymorphic among lineages. The most variable lineage was P. axillaris A1, considering AR and GD indices, whereas the least variable was P. sp1. The highest number of private alleles (PA) was observed in P. subandina, whereas P. exserta E1 has the lowest (Table 2).

Table 2 -
Median values for genetic diversity indices observed in Petunia long corolla tube lineages based on seven nuclear microsatellites

Lineages	N	A	AR	PA	GD
P. axillaris A1	10	6.3	6.3	0.68	0.74
P. axillaris A2	63	8.1	5.5	0.25	0.70
P. exserta E1	13	3.7	3.5	0.06	0.54
P. exserta E2	82	6.7	4.5	0.59	0.57
P. secreta	50	7.6	5.2	0.43	0.62
P. parodii	39	6.3	4.7	0.37	0.64
P. subandina	23	4.9	4.3	0.70	0.69
P. sp1	23	4.1	3.2	0.38	0.43
P. sp3	11	4.7	4.7	0.11	0.67

• N - number of analyzed individuals; A - total number of alleles per species; AR - allele richness; PA - number of private alleles; GD - gene diversity.

The DAPC analysis (Figure 2B), including all individuals and microsatellite loci, revealed the most probable K = 3 groups. Individuals of most lineages were distributed in two or three groups, except P. sp1, from which all individuals belonged to the first cluster. Approximately 50% of P. subandina, P. parodii, and P. axillaris A2 samples composed the first cluster. The second cluster encompassed most P. exserta E1 and E2, and P. sp3 individuals, whereas all lineages had representatives in group 3, except P. sp1. Most P. secreta and P. axillaris A1 belonged to the third group (^{Table S5}). The polymorphism sharing based on microsatellite alleles did not replicate the evolutionary relationships among species. Groups were homogeneous with low superimposition in the Cartesian plane.

Discussion

Here, we investigated the evolutionary relationships among the Petunia long corolla tube species employing a phylogenetic approach and intraspecific genetic variability. The taxa in the LT clade display marked differentiation in floral traits associated with pollinator attraction (Stehmann et al., 2009), and plant-pollinator interaction was proposed as the main speciation driver in the group (Fregonezi et al., 2013). Despite attracting different pollinators, several hybrid populations are found (e.g., Turchetto et al., 2019b; Giudicelli et al., 2019).

Our results revealed unexpected relationships regarding previous studies (e.g., Reck-Kortmann et al., 2014). On the other hand, the present work is the first to include multiple samples and intraspecific lineages throughout their entire geographic distribution. In the Petunia genus, geographic isolation is often implicated in population structure and reproductive isolation (e.g., Giudicelli et al., 2022; Guzmán et al., 2022). Moreover, local adaptation and microenvironmental conditions keep species limits (e.g., Segatto et al., 2017; Caballero-Villalobos et al., 2021), contributing to differentiation (Fregonezi et al., 2013; Pezzi et al., 2022).

The phylogenetic tree and SSR-based analyses were not entirely congruent. Phylogenetic markers indicated with full support the split between high elevation-distributed species (P. occidentalis and P. subandina) and the lowland species (remaining lineages, all distributed at < 500 m a.s.l.), whereas SSR profiles formed three groups that did not reflect phylogenetic clades and subclades. SSR-based group 2 encompassed all P. exserta individuals, independently of their occurrence area, most P. sp3, one P. secreta from the same region than P. sp3, and one P. axillaris A2 sampled close to P. exserta. Petunia exserta occupies the subclade IIB in the tree, whereas the remaining lineages from group 2 form the subclade IIA. In turn, groups 1 and 3 clustered individuals of all lineages in different proportions (except for P. sp1, which integrates only group 1): P. axillaris A2, P. parodii, and P. subandina were equally distributed between groups 1 and 3, whereas P. axillaris A1 and P. secreta mainly integrated the group 3. The lineages P. axillaris A1 and P. secreta were not closely related in the phylogenetic tree, occupying different subclades despite the high similarity in their SSR profiles. The geographic distribution of P. axillaris A1 is on the southern Atlantic coast, predominantly in Uruguay (Turchetto et al., 2014a), whereas P. secreta is endemic to Serra do Sudeste in Rio Grande do Sul (Stehmann and Semir, 2005).

Almost all phylogenetic analyses including the LT taxa (Ando et al., 2005; Kriedt et al., 2014; Reck-Kortmann et al., 2014; Segatto et al., 2016) placed P. subandina and P. occidentalis as sister species (but also see Chen et al., 2007), despite the first displays long corolla tube and yellow pollen as the remaining species in the LT, whereas P. occidentalis shows a short corolla tube and bluish pollen as all species in the ST. Regarding the geographical distribution, P. occidentalis is restricted to the sub-Andean region, in elevation up to 900 m, and isolated from the other Petunia species by the Chaco (Tsukamoto et al., 1998); the remaining species in LT are found in grasslands in Chaco or Pampa (Stehmann et al., 2009), in open rocky ground areas and roadside slopes, except for P. subandina, which occurs only in the sub-Andean mountains (Ando, 1996). The taxa P. axillaris, P. exserta, and P. secreta occur in sympatry in Brazil. However, P. axillaris is widely distributed in the Uruguayan Pampa, whereas the other two species are narrowly endemic to rocky formations in southern Brazil. The P. parodii can be found in Chaco (Argentina) and Pampa (southern Brazil, Uruguay, and Argentina), where the plants grow disjunct from P. axillaris. Except for P. subandina and P. occidentalis, the species in LT are distributed from zero to less than 500 m a.s.l., occupying areas proposed as ancestral for the Petunia genus (Reck-Kortmann et al., 2014).

The most surprising result was the divergence between P. axillaris interspecific lineages A1 and A2. According to the phylogenetic markers, this taxon was paraphyletic. Previous works (Turchetto et al., 2014a, b) support the separation found here among P. axillaris, P. parodii, and P. subandina, indicating they should be treated as independent evolutionary units and not only as subspecies. Although P. axillaris, P. parodii, and P. subandina shared several plastid haplotypes and no genetic-based intraspecific groups have been found (Turchetto et al., 2014a), morphologic floral traits revealed that P. axillaris can be divided in two groups that correspond to coastal (A1 in the present work) and inland (A2) populations. In the same way, ecological features pointed to the same P. axillaris subgroups and three groups in P. parodii (Chaco, Pampa-Brazil, and Pampa-Uruguay), which were not perceived based on morphologic analysis. The P. parodii subdivision was not confirmed here in the phylogenetic tree and SSR, but it was also identified using a sizeable genomic evaluation (Giudicelli et al., 2022).

It is widely accepted that ecological divergence due to habitat differences plays an essential role in lineage differentiation (e.g., Foster et al., 2007), notably regarding to adaptation to extreme environments such as coastal areas (e.g., Lowry et al., 2008) that are often reflected in morphological traits in addition to genetic markers. Significant morphologic differences were already observed comparing P. axillaris inland populations in Brazil with coastal populations from Uruguay, whereas P. parodii Brazilian populations were not different from those collected in Uruguay (Kokubun et al., 2006). Such differences or their absence followed taxa’s self(in)-compatibility system.

The polymorphism sharing between some lineages in the Petunia LT can be explained by introgression due to hybrid populations’ high frequency and stability (e.g., Schnitzler et al., 2020), whereas others are based on shared ancestry. Hybridization could be discarded because of the long distance between populations, such as P. exserta and P. axillaris A1 or P. subandina and all others, as the distance between populations exceeds 1 km, which is the maximum estimated distance for pollen dispersal (e.g., Turchetto et al., 2015, 2022; Rodrigues et al., 2019). Moreover, seed dispersal in Petunia is very limited, with seeds falling close to the mother plant by autochory (Stehmann et al., 2009).

The evolutionary relationships and polymorphism-sharing in the Petunia long corolla tube clade could be explained based on the migration routes (Figure 3) from an albino ancestor (Wijsman, 1983), which originated in lowland (Reck-Kortmann et al., 2014), ca. 2.8 Mya (Särkinen et al., 2013), with subsequent diversification after colonized new environments or under pollinator selection (Fregonezi et al., 2013). The albino lineage arose from the anthocyanin 2 (AN2) gene inactivation. The AN2 is active in the species of the ST clade and responsible for the pink color (Quattrocchio et al., 1999), the critical morphologic trait to attract bees. The ST species probably represent the genus ancestor, which appeared in lowlands in southern South America, likely in the Pampa (Reck-Kortmann et al., 2014). The genus diverged from the sister group ca. 8.0 Mya (Särkinen et al., 2013).

Figure 3 -
Putative migratory routes and diversification for Petunia corolla tube clade species.

The first step in LT clade differentiation was the highlands’ colonization, which also explains the presence of P. occidentalis in the clade despite its several morphological traits in common with ST species. Petunia occidentalis could represent an incomplete lineage sorting in the highland LT clade, sister of the albino P. subandina. The albino lineage would expand its distribution towards the southern South American grasslands as the Pleistocene climate changes allowed. The albino lineage colonized the Chaco, migrating to the north, and Pampa, growing to the south and east, nowadays represented by P. parodii (Giudicelli et al., 2022) and its parapatric lineage P. axillaris A2.

These last two lineages, P. parodii and P. axillaris A2, could have given rise to the colored lineages in the clade as they advanced colonizing new environments. The albino P. parodii and P. axillaris A1 and the red-flowered P. exserta share several polymorphisms (e.g., Segatto et al., 2014; Li et al., 2023), despite currently not being found close. Mainly regarding P. exserta, this species inhabits a very particular microenvironment, inside small caves where plants grow protected from direct sunlight and rain (Stehmann et al., 2009; Segatto et al., 2014), an inhospitable environment for other Petunia species. The two P. exserta lineages (E1 and E2) differ mainly in flower color hue (Figure 1) and distribution as each inhabits a different rock formation. Petunia exserta E2 is sympatric to some P. axillaris A2 populations, whereas P. exserta E1 occurs in the same formation as P. secreta. The red color of P. exserta petals is reached through a complex gene interaction that begins with a moderate upregulation and shifts in tissue specificity of the Deep Purple gene that restores anthocyanin biosynthesis (Berardi et al., 2021). P. exserta retains the same nonfunctional AN2 copy present in P. axillaris.

The pink-flowered P. secreta and P. sp1 differ from P. axillaris only based on the flower color (Stehmann and Semir, 2005), and this difference is due to the regain in AN2 gene function (Esfeld et al., 2018). Petunia secreta and P. sp1 occur in the same geographic area as P. axillaris A2. Still, whereas P. sp1 occupies a similar environment closely distributed to P. axillaris, P. secreta is found ca. 40 Km away from the closest P. axillaris A2 population and in an entirely diverse microenvironment (Turchetto et al., 2016; Rodrigues et al., 2019). Petunia sp3 is the P. secreta sister lineage, despite being morphologically similar to P. exserta, mainly regarding the exserted styles and anthers (Figure 1). Indeed, P. sp3, P. secreta, and P. exserta E1 are endemic to the same rock formation. Still, whereas P. exserta E1 occupies shaded locations, P. secreta and P. sp3 individuals grow in sunny places. Our results did not discard a hybrid status for P. sp3.

In conclusion, we described the evolutionary relationships among the Petunia long corolla tube clade due to ancestral geographic expansion with local adaptation and pollinator interaction as the vital diversification drivers. Structuring in LT lineages depends on isolation by distance, and high polymorphism-sharing is due to a common ancestor and rapid adaptive radiation.

Acknowledgements

Authors thank to Dr. João R. Stehmann for plant identification and Miss Luisa Bonatto for help with figures 1 and 2 edition. This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Programa de Pós-Graduação em Genética e Biologia Molecular da Universidade Federal do Rio Grande do Sul (PPGBM-UFRGS).

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Internet Resources

• FigTree v. 1.4.1, 1, http://tree.bio.ed.ac.uk/software/figtree/ (accessed 10 September 2023).
  » http://tree.bio.ed.ac.uk/software/figtree/

Supplementary material

The following online material is available for this article:

Table S1 - Sampling information for Petunia long corolla tube clade and outgroups. Table S2 - Genetic markers that were used to obtain the phylogenetic tree for the Petunia long corolla tube clade. Table S3 - GenBank numbers for phylogenetic markers. Table S4 - Nuclear microsatellite markers used to genotype Petunia long corolla tube clade. Table S5 - Number of individuals per lineage per DAPC group (best K = 3).

Edited by

• Associate Editor:
  Felipe dos Santos Maraschin

Publication Dates

• Publication in this collection
  12 Feb 2024
• Date of issue
  2024

History

• Received
  26 Sept 2023
• Accepted
  21 Dec 2023