Biogeographical, ecological, and phylogenetic analyses clarifying the evolutionary history of Calibrachoa in South American grasslands

Molecular Phylogenetics and Evolution 141 (2019) 106614

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Biogeographical, ecological, and phylogenetic analyses clarifying the evolutionary history of Calibrachoa in South American grasslands Geraldo Mäder, Loreta B. Freitas

T

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Laboratory of Molecular Evolution, Department of Genetics, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS 91501-970, Brazil

A R T I C LE I N FO

A B S T R A C T

Keywords: Phylogeny Ecological niche modeling (ENM) Multigenic Petunia Multilocus

Calibrachoa is a charismatic South American genus of Solanaceae, closely related to Petunia, which encompasses approximately 30 species. Studies that were based solely on plastid molecular markers indicated the monophyly of the genus and distributed its species in two subgenera; to date no phylogeny has included a broad morphological variants and nuclear markers. Here, we present a phylogenetic analysis based on eight plastid and eight nuclear markers that cover the most extensive geographic distribution for the genus. We use this phylogeny to infer the biogeographic history of the genus and to understand the primary drivers for species diversification. Our results yield a fully supported tree where monophyly is confirmed to genus and subgenera. The species of Stimomphis subgenus that were previously considered uncertain, here emerge in four highly supported clades. The hypothesis of niche conservatism is confirmed, and adaptive radiation explains the species diversification. The lowlands are the most likely ancestral area of the genus, subgenera, and two clades of Stimomphis subgenus. Our results constitute an excellent starting point for further evolutionary and taxonomic studies and explain several uncertain evolutionary relationships in the group and the evolution of their distribution.

1. Introduction The modern biodiversity patterns on Earth resulted from a long and complex history of evolutionary trends mediated by ecological processes and governed by external environmental forces (Rull, 2011). Speciation is the most critical process for the generation of biodiversity (de Queiroz, 1998) and ecological, geological, and climatic factors are the most important factors that influence the diversification process and may result in speciation because they modify the landscapes in which organisms live (Nelson and Platnick, 1981). Any species is characterized not only by a distinct group of genes but also by a distinct set of ecological characteristics to which it is adapted. Therefore, analyses of ecological niche conservatism are necessary to understand the ecological context related to speciation (Warren et al., 2008). In this sense, phylogenetic niche conservatism refers to the tendency of closely related lineages occupying similar environmental spaces through events of speciation over evolutionary time, whereas most differentiated lineages may occupy most ecologically distinct environments (Pyron et al., 2015). Divergent and ecologically mediated selection promotes lineage separation, and in this case, distinct lineages can experience shifts in different directions and track changing environments (Pyron et al., 2015). In the Neotropical region, the isolation by distance (allopatric ⁎

speciation) during the glacial and interglacial periods in the Pleistocene has been proposed as the primary driver of plant speciation in the subtropical highland grasslands (SHG) of South America (Behling and Pillar, 2007; Lorenz-Lemke et al., 2010; Iganci et al., 2011; Barros et al., 2015). When the forest expanded to the south during the warmer periods (interglacial), it isolated grassland populations, thus disrupting gene flow and promoting speciation. Adaptive radiation is proposed as an explanation for the high diversification presented by several plant groups in some regions, especially on islands (e.g., Hou et al., 2011; Rowe et al., 2011), but it has also likely occurred in areas that have experienced rapid climate or geological changes (Hughes and Eastwood, 2006). Less is known about the speciation of lowland species in southern South America, specifically in the Pampas and Chaco. Unfortunately, only a few studies have examined speciation in this region (Fregonezi et al., 2013; Turchetto-Zolet et al., 2013). Grassland biomes constitute a significant component of global biodiversity, and several of these grasslands harbor high levels of endemism (Safford, 2007; Iganci et al., 2011; Fregonezi et al., 2013). In this region, the Andean Cordillera strongly influenced evolutionary processes in both highland and lowland flora (Antonelli et al., 2009). Although the highland plateaus in southern Brazil are lower than the highest altitudes in the Andean Cordillera, an Andean origin is attributed to several plant groups inhabiting the Brazilian highland grasslands (Safford, 1999; Sklenář et al.,

Corresponding author: Department of Genetics, UFRGS, P.O. Box 15053, Porto Alegre, RS 91501-970, Brazil. E-mail address: [email protected] (L.B. Freitas).

https://doi.org/10.1016/j.ympev.2019.106614 Received 10 July 2019; Received in revised form 9 September 2019; Accepted 9 September 2019 Available online 10 September 2019 1055-7903/ © 2019 Elsevier Inc. All rights reserved.

Molecular Phylogenetics and Evolution 141 (2019) 106614

G. Mäder and L.B. Freitas

Calibrachoa species based on sequences of eight nuclear and eight plastid regions. We then used the resulting phylogenetic hypothesis to reconstruct the biogeographic history of Calibrachoa species. We further used ecological niche modeling (ENM) and niche-overlapping tests to verify whether the species distribution and niche specificities are linked and if it is possible to observe niche conservatism in the Calibrachoa genus. This study is the first Calibrachoa phylogenetic analysis that includes nuclear data.

2011; Iganci et al., 2013); moreover, some of the Andean biomes and the niches from the SHG have similar temperature and humidity conditions (Safford, 1999). South American grasslands harbor particular floras with a high level of endemism. However, the same attention is not afforded to the biodiversity from this open vegetation as is provided to the forest vegetation (Safford, 2007; Overbeck et al., 2007; Iganci et al., 2011; Barros et al., 2015). The species complexes that originated from these unstable open areas are of particular interest for evolutionary studies, as they represent ongoing speciation and often include rare taxa (Lorenz-Lemke et al., 2010; Turchetto-Zolet et al., 2013; Barros et al., 2015). Calibrachoa is a Neotropical genus of Solanaceae that belongs to the clade Petunieae (Olmstead, 2013), encompassing two subgenera, Calibrachoa and Stimomphis, and 26 species that are found in open areas in southern South America, with a predominantly subtropical distribution and occurring most densely in southern Brazil, northeast Argentina, and the entirety of Uruguay (Fregonezi et al., 2012; Greppi et al., 2013). The species diversification for Calibrachoa from the highlands is attributed to an allopatric scenario under the influence of environmental discontinuities within the SHG region (Fregonezi et al., 2013). In the Pampas region, the adaptive evolution to different pollinators appears as a critical driver of speciation in both Calibrachoa and Petunia (Fregonezi et al., 2013). Considering the phylogenetic structure of the genera in the Petunieae clade and their geographical distribution, it has been suggested that this clade has ancestors with Andean origin (Olmstead et al., 2008). According to Särkinen et al. (2013), most Petunieae genera have recently diversified, and the most recent common ancestor of Calibrachoa would have diversified ~5 Mya (million years before present). The Calibrachoa genus recently colonized the current geographical range and displays fast morphological radiation, with higher species diversification during the Pleistocene (Mäder et al., 2013). From the southern limit, the genus is widely distributed in the Pampas where rocky and shallow soils support grasslands that extend to the SHG (Fregonezi et al., 2013); moreover, some species also occur in the tropical highland grasslands (THG) and Chaco. The vast majority of the Calibrachoa species has a continuous distribution pattern restricted to specific geographic regions, which appear to be associated with particular environmental conditions. Several species of Calibrachoa are considered endemic and occur in few and small populations (Fregonezi et al., 2012). An exception into the genus is Calibrachoa parviflora, which is an autogamous species that also occurs in North America and Europe (Fregonezi et al., 2012). Calibrachoa parviflora and C. pygmaea have a unique seed morphology and nuclear DNA content, being both compatible to each other and incompatible with the other Calibrachoa species (Ando et al., 2005). The evolutionary history of Calibrachoa, as investigated using molecular tools, reveals short genetic distances between species, with consequent poorly resolved phylogenetic trees, and recent diversification (Ando et al., 2005; Fregonezi et al., 2012; Fregonezi et al., 2013). Despite low support, different analyses have confirmed the genus monophyly. Phylogenetic trees based on restriction fragment length polymorphisms of plastid DNA (cpDNA-RFLPs) have revealed two highly supported clades, one including only C. parviflora and C. pygmaea and another grouping of the remaining species (Ando et al., 2005). Analyses based on plastid sequences have confirmed these two clades and a taxonomic treatment proposed two subgenera, subgenus Calibrachoa including C. pygmaea and C. parviflora, and subgenus Stimomphis for the other species group (Fregonezi et al., 2012). Despite good resolution at the subgenus level, the relationships among species of subgenus Stimomphis remain poorly resolved and a multilocus approach, including plastid and nuclear markers, can offer better resolution, as in Petunia (Reck-Kortmann et al., 2014). The use of a large number of DNA fragments can provide better phylogenetic resolution, allowing the determination of previously unidentified relationships (López-Fernández et al., 2010; Rowe et al., 2011). In this study, we recovered the phylogenetic relationships among

2. Materials and methods 2.1. Taxon sampling and DNA extraction We included 25 of the 26 currently recognized (Greppi et al., 2013) Calibrachoa species, identified according to Fregonezi et al. (2012). We deposited vouchers for each taxon at the BHCB herbarium (Universidade Federal de Minas Gerais, Belo Horizonte, Brazil). We also included samples of one not formally described taxon (C. excellens subsp. atropurpurea) and two other nonidentified putative taxa (Calibrachoa sp.1 and Calibrachoa sp.2) to evaluate their evolutionary relationships. Furthermore, we used samples of Petunia axillaris, P. integrifolia, Fabiana imbricata, and Nierembergia riograndensis as outgroups. We preferably collected samples in the same (or at least as close to as possible) location type, and all sampled individuals exhibited morphology as reported in the original species description. The geographic coordinates were obtained using the Global Positioning System (GPS) and are included, together voucher information and taxa authorities, in Supplementary Table S1. We extracted the total genomic DNA from silica-dried leaves following a CTAB (cetyl-trimethyl ammonium bromide)-based method (Roy et al., 1992). 2.2. Amplification, sequencing and alignment We sequenced eight plastid (cpDNA) and eight nuclear (nDNA) segments (Table 1). For PCR reactions of nuclear markers, we added 10% of dimethyl sulfoxide (DMSO, Merck & Co., Kenilworth, USA). The PCR products obtained from plastid markers and ITS were purified using 20% polyethyleneglycol (Thermo Fischer Sci., Waltham, USA) according to the method of Dunn and Blattner (1987), whereas for the remaining nuclear markers we used the QIAquick kit (Qiagen Co., Hilden, GE). All PCR products were sequenced in an ABI 3730XL Table 1 PCR conditions and primer references. Marker

Ta (°C)

Cycles

Extension (min)

MgCl2 (mM)

References

trnH-psbA psbB-psbH trnS-trnG trnL-trnF

52 54 54 54

35 32 32 32

1.5 1.5 1.5 1.5

2 2 2 2

trnL intron

54

32

1.5

2

matK

54

32

1.5

2

rps12-rpl20 rpl32-trnL ITS

54 54 55

32 32 35

1.5 1.5 1

2 2 2.5

PolA1 Hf1b

57 58

40 40

1.5 1.5

2.5 2.5

WUS WOX1 WOX4 SOE EVG

57 57 57 54 55

35 35 35 45 45

1 1 1 0.5 0.5

2.5 2.5 2.5 2.5 2.5

Sang et al. (1997) Hamilton (1999) Hamilton (1999) Taberlet et al. (1991) Taberlet et al. (1991) Johnson and Soltis (1994) Shaw et al. (2005) Shaw et al. (2007) Desfeux and Lejeune (1996) Zhang et al. (2008) Reck-Kortmann et al. (2014) Segatto et al. (2016) Segatto et al. (2016) Segatto et al. (2016) Segatto et al. (2016) Segatto et al. (2016)

Ta = annealing temperature. 2

Molecular Phylogenetics and Evolution 141 (2019) 106614

G. Mäder and L.B. Freitas

visualized with FigTree 1.4.1 (http://tree.bio.ed.ac.uk/software/ figtree/); posterior probabilities (PP) values ≥ 0.95 were considered to represent strong support. For the ML analysis, we performed the phylogenetic reconstruction with the same nucleotide substitution models used in BI and 1000 nonparametric regular bootstraps using the CIPRES Science Gateway (Miller et al., 2010). Bootstrap support values were interpreted as indicating weak (50–70%), moderate (71–80%), and strong support (81–100%).

(Thermo Fischer Sci.) sequencer on demand. We also included previously published sequences for some Calibrachoa species and outgroups (Table S1). We assembled and edited the sequences using the software CHROMAS 2.0 (Technelysium, Helensvale, Australia) and prepared the alignments per molecular marker in MEGA 7 (Kumar et al., 2016). We manually edited the alignments when it was 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 of nuclear markers in the final matrix (Mäder et al., 2010).

2.4. Ancestral area reconstructions Calibrachoa species present clear patterns of distribution on the elevation gradient occupied by the genus (Fig. 1; Fig. S4). This pattern was employed to identify three main areas that were used for the biogeographic reconstructions: (A) highland grasslands (populations distributed above 1000 m in elevation); (B) plateau grasslands (populations distributed between 500 and 1000 m); and (C) lowlands (populations distributed below 500 m). For each Calibrachoa species, we recovered the occurrence data from the Global Biodiversity Information Facility (GBIF; https://www.gbif.org/species/2928904) and SpeciesLink (http://splink.cria.org.br/), supplemented with records from field collections (Table S2; Fig. S4). We carefully verified and retained only those records that were confirmed by Solanaceae specialists and included the global positioning system coordinates and detailed localization. We used statistical dispersal–vicariance analysis (S-DIVA; Yu et al., 2010) and Bayesian binary Markov (BBM) chain Monte Carlo analysis as implemented in RASP 3.2 (Yu et al., 2015) to reconstruct the Calibrachoa ancestral ranges on the maximum clade credibility tree obtained through BI analysis (without outgroups). To account for uncertainties in tree topologies, the frequencies of ancestral ranges at nodes were averaged for all post-burn-in trees sampled in the BI analysis. The analyses were implemented using default parameters in RASP. For BBM analysis, two MCMC chains were run simultaneously for 500,000 generations. The state was sampled every 1000 generations.

2.3. Genetic diversity and phylogenetic analysis We estimated the genetic variability among taxa based on parsimony informative (PI) and polymorphic sites (PS) using DnaSP 5 (Librado and Rozas, 2009), and determined the preliminary phylogenetic relationships of the multilocus cpDNA and nDNA datasets through Bayesian inference (BI) as implemented in BEAST 1.10 (Suchard et al., 2018) and maximum likelihood (ML) analysis performed in RAxML 8.2.12 (Stamatakis, 2014). We assessed the tree support with posterior probabilities (PP) with 107 chains and a bootstrap (BS) analysis with 1 000 replicates for BI and ML, respectively. We visually assessed the tree congruence (cpDNA × nDNA) comparing the topologies according to BI and ML (Figs. S1–S3). As no conflict between the two topologies was observed related to well-supported branches in both methods (i.e., ML BS > 70% and BI PP > 0.95; Daniels and Klaus, 2018; Niu et al., 2018), we combined the datasets for further analyses. We selected the best substitution model and gamma rate heterogeneity using jModelTest 3.06 (Darriba et al., 2012) based on Akaike information criterion (AIC; Table 2) independently for each nuclear marker, the cpDNA matK gene and trnL intron, and the combined intergenic plastid spacer sequences. We conducted BI analysis under the Yule process and two independent runs of 10 million generations, with 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 1.6 (Rambaut and Drummond, 2013). We discarded the initial 25% of trees as burn-in, and the remaining trees were summarized to generate a maximum clade credibility tree using TreeAnnotator 1.7.5 (Suchard et al., 2018)

2.5. Ecological niche modeling and niche overlapping For ENM and niche overlapping, we used the same data set employed to estimate the ancestral range. Distributional records were entered in MAXENT 3.2.2 to infer the lineage distribution modes with the maximum entropy algorithm (Phillips et al., 2006). The number of occurrences used for the four lineages in Stimomphis subgenus (see Results) was 34, 21, 76, and 121, respectively for clades A to D. These ecological analyses were not performed for the Calibrachoa subgenus because it presents only two species, one of which (C. parviflora) is considered a subspontaneous and ruderal species that occurs in North America and Europe; the number of positive records (presence) has a severe impact on distribution models. In this study, the positive records of the number per species were proportional to each species range. As most Calibrachoa species are known to occur in few localities and very few species are considered widely distributed, we opted to reconstruct whole clade niches instead (A-D). Explanatory variables included a set of bioclimatic layers at 30 arc-second resolution obtained from the WorldClim website (Hijmans et al., 2005). We selected this fine-scale resolution because it provides better predictions for fixed or only locally mobile organisms, such as plants (Guisan and Thuiller, 2005) and to account for the heterogeneity of highly variable relief environments as observed in South America (Leal et al., 2016). To avoid highly correlated variables in our dataset, we calculated the pairwise Pearson correlation coefficients, including the 19 bioclimatic layers, in ENMTools 1.4 (Warren et al., 2010). We identified variable pairs with R > 0.75 and discarded those that presented the lowest importance to obtain the best model in a preliminary run (Peterson, 2007). Based on this run result, the eight retained variables per lineage were: clade A (BIO2 = mean diurnal range;

Table 2 Genetic variability per molecular marker. Marker

Length

PS (%)

PI (%)

Missing

Model

trnH-psbA psbb-psbh trnS-trnG trnL-trnF trnL intron matK rps12-rpl20 rpl32-trnL

422 747 747 360 478 849 785 1020

48 (11.4) 37 (5.0) 25 (3.3) 43 (11.9) 26 (5.4) 77 (9.1) 56 (7.1) 137 (13.4)

15 (3.6) 1 (0.1) 13 (1.7) 6 (1.7) 3 (0.6) 19 (2.4) 19 (2.4) 25 (2.5)

3 6.1 3.7 0 0 0.8 0 0

GTR+G GTR+G GTR+G GTR+G GTR GTR+I GTR+G GTR+G

cpDNA ITS PolA1 Hf1b WUS WOX1 WOX4 SOE EVG

5408 600 695 778 512 640 652 639 500

449 (8.3) 135 (22.5) 42 (6.0) 77 (9.9) 42 (8.2) 84 (13.1) 61 (9.4) 33 (5.2) 51 (10.2)

101 (1.9) 56 (9.3) 13 (1.9) 26 (3.3) 16 (3.1) 35 (5.5) 35 (5.4) 16 (2.5) 31 (6.2)

1.6 0.3 4.9 10.4 3.6 18.7 4.8 12.2 27.3

GTR+G HKY+G GTR+G HKY HKY+G HKY+G GTR+G HKY

nDNA

5016

525 (10.5)

228 (4.6)

10

TOTAL

10,424

974 (9.3)

329 (3.2)

5.6

Length - alignment length (base pairs); PS - polymorphic sites; PI - parsimonyinformative sites; Missing - percentage of missing data; Model - substitution model used in phylogenetic analyzes (BI and ML). 3

Molecular Phylogenetics and Evolution 141 (2019) 106614

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Fig. 1. (a) Geographical distribution of the main Calibrachoa subgenus Stimomphis clades and C. pygmaea (C. parviflora was not included due to its widespread distribution); (b) C. pygmaea (photo J.R. Stehmann); (c) C. missionica (photo J. N. Fregonezi); (d) Calibrachoa sp.1 (photo G. C. Giudicelli); (e) Calibrachoa sp.2 (photo G. C. Giudicelli); (f) C. excellens subsp. atropurpurea (photo G. Mäder); (g) C. linoides subsp. furcata (photo J. N. Fregonezi); (h) C. sendtneriana (photo M. C. Teixeira); (i) C. heterophylla (photo J. R. Stehmann).

whether pairs of clade models were more different than would be expected given the underlying environmental differences between the areas in which they occur. This test assumes that the ENM probabilities produced considering two populations are identical (equivalent) if sampling is unbiased concerning the species’ environmental tolerances (Warren et al., 2010). The hypothesis of niche identity is rejected when the observed D value is significantly lower than the values expected from pseudoreplicates (Warren et al., 2008, 2010). We assessed the significance of differences in Schoener's D metric from the null expectation (one-tailed) counting the number of bootstrap replicates with lower values than the observed D index. To graphically visualize niche overlap, the grid files obtained from the ecological niche modeling for each clade were merged pairwise in the DIVA-GIS considering suitability values between 0.2 and 1.

BIO3 = Isothermality; BIO4 = temperature seasonality; BIO5 = max temperature of warmest month; BIO8 = mean temperature of wettest quarter; BIO14 = precipitation of driest month; BIO15 = precipitation seasonality; and BIO17 = precipitation of driest quarter); clade B (BIO3, BIO4, BIO5, BIO8, BIO9 = mean temperature of driest quarter; BIO10 = mean temperature of warmest quarter; BIO14; and BIO15); clade C (BIO3; BIO4; BIO5; BIO8; BIO14; BIO15; BIO17; and BIO19 = precipitation of coldest quarter); and clade D (BIO3; BIO4; BIO5; BIO8; BIO11 = mean temperature of coldest quarter; BIO14; BIO15; and BIO17). We ran ten iterations per prediction model using 10% random records to test each run with a subsample option per replicated run type. The quality of the models was evaluated based on the area under the curve (AUC) and true skill statistic (TSS; Allouche et al., 2006) scores. AUC values > 0.75 reflect good prediction, whereas values < 0.5 suggest poor prediction (Pearce and Ferrier, 2000). TSS values range between −1 and 1: TSS < 0 indicates that the model does not predict the known localities better than random choice; TSS = 1 indicates a perfect discrimination power for the model between known localities and geographical background; and TSS values ranging between 0.4 and 0.8 are indicative of good performance for the model (Landis and Koch, 1977; Fielding and Bell, 1997). For the TSS, the threshold value was the maximum sum of the sensitivity and specificity of the analyzed data. Bioclimatic layers from latitudes 10 to 40°S and longitudes 35 to 65°W were cropped using DIVA-GIS 7.5 (http://www.diva-gis.org). The resulting geographical area includes the entire Stimomphis subgenus distribution (Fig. 1) and the predicted distribution in the analysis including the 19 bioclimatic variables We subsequently quantified the climate niche overlap among the four observed clades according to Schoener's D index (Schoener, 1970), which varies from 0 (no overlap) to 1 (complete overlap) using ENMTools. It has been argued that the ecological interpretation of this index suggests that suitability scores generated by MaxEnt are relatively proportional to species abundance (Warren et al., 2010). We applied a randomization test (Warren et al., 2008), the identity test, to explore

3. Results 3.1. Alignment characterization This study reports the first phylogeny utilizing a large number of nuclear and plastid markers for Calibrachoa. The complete sequence data matrix consisted of 33 accessions, including outgroups, and 10,424 characters, from which 974 (9.3%) were polymorphic and 329 (3.2%) were parsimoniously informative. The plastid matrix (cpDNA) consisted of 3135 characters with the PI mean = 1.9% (ranging from 0.8% in psbB-psbH to 3.6% in trnH-psbA regions). The nuclear markers were more parsimoniously informative (PI mean = 4.6%, with 1.9% in PolA1 and 9.3% in ITS). See Table 2 for more information about the individual sequences. The final alignment consisted of only 5.6% empty cells. We deposited all sequences in GenBank (Table S1). 3.2. Phylogenetic reconstructions Phylogenetic reconstruction based on total evidence using BI and ML approaches revealed similar topologies (Fig. 2; Figs. S1–S3) and 4

Molecular Phylogenetics and Evolution 141 (2019) 106614

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Fig. 2. Bayesian inference of Calibrachoa species tree based on combined plastid and nuclear sequences. Posterior probabilities > 0.95 are shown close to the nodes. The vertical bars to the right of the tree indicate the main clades identified in the analysis. * Most recent common ancestor of Calibrachoa species.

13 taxa, occurs preferably at reduced altitudes, below 500 m above sea level, in the Pampas and humid Chaco ecoregions (27–35°S latitude). The four main clades of Stimomphis subgenus exhibited a strong association between species’ phylogenetic position (Fig. 2) and geographical distribution (Fig. 1; Fig. S4). In clade D, three of the 13 taxa are not formally recognized (Fig. 2): Calibrachoa excellens subsp. atropurpurea, which occurs exclusively in the Pampean lowlands (Fig. S4 A) and is deeply morphologically and evolutionarily divergent from C. excellens, which has an extensive distribution, occurring abundantly in the SHG (Fig. S4 B), and is integrated into clade C. Calibrachoa excellens has a uniform pink corolla, whereas C. excellens subsp. atropurpurea displays a central dark ring similar to C. heterophylla; Calibrachoa sp.1, which is the sister species of C. missionica, shows a white corolla while the C. missionica corolla is purple; and Calibrachoa sp.2 that is grouped with C. excellens subsp. atropurpurea (Fig. 2) despite being a shrub higher than 1 m and having a uniform corolla color. These two putative new taxa (Calibrachoa sp.1 and Calibrachoa sp.2; Fig. 1D and E, respectively), as well as C. excellens subsp. atropurpurea, exhibit high morphological differentiation compared to other Calibrachoa species. Moreover, Calibrachoa linoides subsp. furcata, despite being morphologically very similar to C. linoides subsp. linoides, exhibited high genetic divergence and integrated distinct clade (clade D). All these lineages deserve detailed taxonomical treatment that is already in process.

confirmed Calibrachoa to be a monophyletic group. Despite the nuclear data matrix is more informative (PS and PI) than cpDNA data, the support values in the BI and ML trees were proportionally lower when compared to plastid-based trees, especially considering terminal branches. This lower support is probably due to most nuclear variation was observed between main groups (between in- and outgroups and between the Calibrachoa subgenera). To test this assumption, we estimated the percentage of PS and PI within Stimomphis subgenus; which indicated that the plastid data were more informative to investigate the relationships between closely related species (plastid matrix, PS = 1.74% and PI = 0.60; nuclear matrix, PS = 1.68% and PI = 0.37). The description below refers to the combined trees (Fig. 2; Fig. S3). Two main groups with high support (PP = 1.0; BS = 99) resulted from these analyses and corresponded to Calibrachoa (C. parviflora and C. pygmaea) and Stimomphis (remaining species) subgenera, respectively. In addition to the division of Calibrachoa in two subgenera, the combined tree clearly indicated that species of Stimomphis subgenus are grouped in four main clades. Clade A, comprised of five species, is primarily distributed from 24 to 27°S latitude in the SHG. Clade B is composed of six species: four endemics from the SHG between 25 and 28°S latitude; C. elegans, which inhabits the THG, where it is isolated from other species in the northern boundary of the Stimomphis subgenus distribution (20–21°S); and C. linoides, which presents a broader distribution, occurring in the SHG, THG, and grasslands between 500 and 1000 m in elevation. Clade C encompassed three species: C. sellowiana, which occurs in the SHG (25–30°S); C. excellens, found in the SHG and in the Pampas; and C. heterophylla, a species whose distribution is predominantly in the South Atlantic Coastal Plain, where it inhabits dunes and sandy grasslands at sea level. Finally, clade D, comprised of

3.3. Ancestral area reconstruction We inferred the Calibrachoa biogeographic history through the reconstruction of ancestral geographic distributions with a BBM, prioritizing how clades and species occupy distinct elevations and from 5

Molecular Phylogenetics and Evolution 141 (2019) 106614

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Fig. 3. Ancestral area reconstructions for selected nodes based on Bayesian Binary Markov chain Monte Carlo (BBM) = I: 99.18A, 0.56AB, 0.25AC; II: 99.33A, 1.32AC, 0.19C; III: 99.58 A, 0.25 AB, 0.16 AC; IV: 62.45 C, 24.4 A, 11.74 AC; V: 80.6 C, 9.1B C, 8.04 AC; VI: 94.75 C, 2.97 AC, 1.46 BC; VII: 83.68 C, 8.07 A, 6.24 AC; VIII: 99.53 C, 0.31 BC, 0.16 AC; IX: 75.39 C, 10.96 A, 9.05 BC. Current distributions are indicated before the species names. The map shows the geographical extents of the three areas considered in the analysis. * C. parviflora was not included due to its widespread distribution.

lineage established over different ecological areas, with contact areas located primarily on the border of each range. Schoener’s D was low in comparison among the ENMs for all clades (0.15–0.47). All observed niche-overlapping values were significantly smaller (P < 0.05) than those for the null models in the niche identity tests. The most similar niches were observed between clades A and B, as indicated by the highest values of niche overlapping (D: 0.47). Clade D exhibited the lowest overlapping (D: 0.15–0.30) with the other clades (Fig. 5; Table 3).

which environment their ancestors would have emerged. S-DIVA analysis (Fig. 3) identified lowland regions (83.68%) as the most likely ancestral area for the most recent common ancestor (MRCA) for the genus Calibrachoa. The subgenera (Calibrachoa and Stimomphis) also had their MRCAs in the lowlands (75.4% and 62.5%, respectively). The four main clades observed for species of Stimomphis subgenus had their respective ancestors in different regions (Fig. 3): clades A and B, as well as their respective MCRAs, have originated in open areas at elevations above 1000 m (probabilities of these predictions 99.2%, 99.6%, and 99.3%, respectively), whereas clades C and D and their MCRAs appeared in the lowlands (80.6%, 99.5%, and 94.8, respectively).

4. Discussion

3.4. Ecological lineage distributions

4.1. Phylogenetic relationships

Variables with the most important relative contribution to the main models (Fig. 4) were precipitation of driest month (BIO14) for clades A, C, and D, and max temperature of warmest month (BIO5) for clade B. All models exhibited high predictive power, with good to very good AUC average values. The AUC values for the replicate runs had a mean ( ± SD – standard deviation) of 0.973 ( ± 0.012; clade A), 0.988 ( ± 0.005; clade B), 0.980 ( ± 0.007; clade C), and 0.962 ( ± 0.006; clade D). Establishing the threshold probability for the predicted presence of each clade of Stimomphis subgenus (A, B, C, and D) using TSS resulted in a mean ( ± SD) proportion of correctly classified training observations of 0.69 ± 0.03, 0.76 ± 0.02, 0.77 ± 0.09, and 0.68 ± 0.07, respectively. The distribution models indicated that each

The phylogenetic tree is overall well resolved and well supported and is a significant improvement to previously published phylogenies only based on a few plastid loci (Ando et al., 2005; Fregonezi et al., 2012; Fregonezi et al., 2013). The monophyly of the Calibrachoa genus, as well as the two subgenera Calibrachoa and Stimomphis (Fregonezi et al., 2012), was confirmed and fully supported. Moreover, this more comprehensive genome coverture allowed for a better understanding of the evolutionary relationships among the species of Stimomphis subgenus. Some low resolution in terminal branches is expected in groups that have recently experienced diversification (e.g., Richardson et al., 2001; Carstens and Knowles, 2007; Alarcón et al., 2012), such as Calibrachoa genus, whose origin is recent (< 4 Mya; Särkinen et al., 2013), followed by rapid morphological divergence (Fregonezi et al., 2013). In addition, this low resolution is evidenced, especially in clade D that corresponds to the lowlands region where the most significant species richness is observed in Calibrachoa (Fregonezi et al., 2013), and at least partially in clade C for which, due to Pleistocene effects, several areas only recently were made available (e.g., Mäder et al., 2013). Closely related taxa frequently exhibit morphological similarities and low levels of genetic differentiation due to their evolutionary proximity (Zhang et al., 2019). In such cases, the partial or full reduction of the gene exchange between newly established and ancestral populations could allow the independent evolution and differentiation of their gene pools over time, without necessarily leading to high morphological differentiation. Environmental heterogeneity is

Fig. 4. Relative contribution of the environmental variables to the Ecological Niche Models (ENMs). Precipitation of driest month (PrDM), max temperature of warmest month (MaTWa), precipitation seasonality (PrSea) isothermality (Isoth), mean temperature of wettest quarter (MeTWe), temperature seasonality (TeSea). 6

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Fig. 5. Ecological niche models (ENM) and niche overlapping among the clades of Stimomphis subgenus. (A–D) ENMs for the clades A to D, respectively. (E–J) Niche overlapping considering the suitability of 0.2–1, D = values of Schoener's D index for niche overlapping.

the Pampas and Chaco ecoregions (Pezza and Ambrizzi, 2005; Barros et al., 2015). The ancestor of clades A and B would have colonized the SHG and subsequently diversified through adaptive radiation. Groups that experience adaptive radiation, as the tribe Petunieae, typically exhibit rapid morphological evolution and may present little genetic divergence between species (Ando et al., 2005, Reck-Kortmann et al., 2014). Later and independently, secondary migration of the ancestors of the subclade [(C. sellowiana / C. excellens) C. linoides subsp. furcata] that probably originated in the lowlands would also reach the highlands (see Fig. 3). Similar to the Calibrachoa genus, the most Petunia species are found in the Pampas region at low latitudes; nonetheless, other colonization and diversification processes have been proposed to different regions that have different ecological conditions, including the SHG (Fregonezi et al., 2013; Reck-Kortmann et al., 2014). For plants, radiations have occurred in many different geographical and ecological settings and involve several distinct groups. Steep changes in numerous features of the physical environment, such as temperature, atmospheric pressure, moisture, sunshine hours, ultraviolet radiation, wind, season length, and geology characterize the altitudinal gradients. In turn, different elevations influence aspects of the biotic environment, for example, number and type of pollinator, herbivores, and competitors present in each altitudinal stratum. Although some plant species can spread to a wide range of elevations due to their adaptation to ecological condition shifts, most species restrict their distribution to narrower altitude ranges (Abbott and Brennan, 2014). The lowland clade D may also has diversified as it has expanded into new environments through adaptive radiation because the region where the species occur is geomorphologically complex and includes several areas that emerged under different tectonic and paleogeographic contexts (Turchetto et al., 2014; Moreno et al., 2018). In addition to the diversification through environmental adaptations and

Table 3 Niche overlapping (Schoener's D) and Identity Test for main clades of Stimomphis subgenus. Statistics

A-B

A-C

A-D

B-C

B-D

C-D

Schoener's D Null model

0.47

0.35

0.20

0.43

0.15

0.30

Mean SD Minimum Maximum P-value

0.70 0.05 0.62 0.77 < 0.01

0.79 0.02 0.76 0.81 < 0.01

0.74 0.03 0.71 0.77 < 0.01

0.75 0.02 0.73 0.78 < 0.01

0.83 0.02 0.80 0.85 < 0.01

0.82 0.03 0.78 0.85 < 0.01

SD: Standard deviation.

considered a trigger for speciation processes, which may have occurred without promoting profound morphological shifts, as is the case with Petunia integrifolia complex (Segatto et al., 2017), which is from the same region where clade D species preferentially occur. This high morphological similarity can lead to some sampling gaps that, by its turn, may promote some noise into phylogenetic relationships identification due to terminals or absence of a sister group. 4.2. Biogeographic considerations According to the ancestral geographical reconstruction, Calibrachoa genus and its two subgenera originated in the lowlands. Our results also indicated that the four main clades of Stimomphis subgenus are associated with different environments that are under the influence of diverse climate conditions whose changes modified the composition of the natural landscape. The climate in the SHG is currently subject to seasonal low temperatures, including frosts and snowfalls that make these environments very distinct compared with the lowland fields from 7

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overlapping indicated that the four clades occupy different niches. The very restricted distribution of several taxa prevents a more detailed scenario on niche divergence to be achieved within each of the clades. According to Shoener's D index, the most extensive niche overlapping occurs between clades A and B, which should be expected because these clades are closely related and all species within both are found in the highlands. Comparisons with clade C exhibited intermediary D values likely because the species in this clade occur in very distinct environments (C. excellens with wide distribution, C. sellowiana in the highlands, and C. heterophylla in the coastal lowlands). Accordingly, comparisons with clade D were those that resulted in lower Shoener's D values probably because the species in this clade (except C. linoides subsp. furcata) exclusively inhabit the Pampas and Chaco lowlands where the climatic variables differ significantly from the highland regions. We observed a higher niche similarity between taxa more closely related in the phylogeny, which is in agreement with the hypothesis of niche conservatism (Pyron et al., 2015). The clades of Stimomphis subgenus exhibited particular identities with their environmental requirements. Furthermore, clades A and B, which are closely related in the phylogeny, have the largest overlapping niche, whereas clade D seems to be the most distinct. A clumped phylogenetic distribution of taxa indicates that habitat use is a conserved trait within the pool of species in the community and that phenotypic attraction dominates over repulsion (Webb et al., 2002). From this perspective, phylogenetic and ecological approaches are essential to understanding the evolutionary and historical determinants of ecological processes that shape species distribution and the biological communities’ structure in South America’s subtropical grasslands. Nonetheless, to comprehensively assess whose ranges are strongly constrained by temperature and precipitation, we must consider not only climatic information of their distributions but also integrate information on species dispersal mechanisms, biological interactions, and barriers for dispersion (Aguirre-Gutiérrez et al., 2015). Therefore, our results support divergence due to allopatry (geographic isolation), followed by ecological specialization linked to ecological gradients as proposed for other species (Smith et al., 2001; Ortega-Andrade et al., 2015), thus driving diversity and species diversification in Calibrachoa, especially the Stimomphis subgenus.

geographic isolation, the differentiation of the Calibrachoa species may have been shaped under the influence of pollinators’ interaction (Fregonezi et al., 2013). During the Late Miocene and Pliocene (ca. 11–3 Mya), widespread and varied plains succeeded a similar widespread flood by the Paranean Sea surface. These changes opened new habitats in the lowlands of southern South America and led to the current configuration of the subtropical grasslands (Donato et al., 2003). Due to the recent divergence of Calibrachoa (ca. 4 Mya; Särkinen et al., 2013), we can infer that Quaternary climate dynamics have had an important influence on the ancestral distribution patterns in the lowlands primarily because they have often modified the availability of habitats due to changes in sea level. During this period, there were strong temperature decreases that characterized Pliocene-Quaternary climate transitions, processes that promoted the expansion of grasslands, thus allowing the Calibrachoa species to occupy their current geographical distribution (Behling and Pillar, 2007; Mäder et al., 2013). This scenario allows us to assume that the current Pampas and Chaco regions provided suitable environments for Calibrachoa species in their early diversification stages. The geographical and ecological ubiquity of radiations, the multiepisodic nature of plant diversification, and the diversity and complexity of triggers of diversification explain the intrinsic evolutionary liability and ability of flowering plants to repeatedly reinvent themselves using diverse trait innovations and exploiting diverse opportunities, which have perhaps been key to the progressive radiation of angiosperms (Crepet and Niklas, 2009). Our results contribute to the understanding of the relative importance of specific factors involved in the biogeographical and diversification history of Neotropical plant groups. 4.3. Ecological divergence restricts the geographic range of distinct clades The results of ENM indicated that lineages of Stimomphis subgenus present relatively similar environmental preferences, which is probably due to ancestral habitat sharing (Couvreur et al., 2011). In clades A, B, and C, the contribution of environmental variables was similar, where the maximum temperature of the warmest month and precipitation of the driest month were the most important to predicting the best model for each clade. The summer temperatures in occurrence regions for these groups of species do not reach high levels because the elevation (> 1000 m above sea level) or maritime influence (in the case if C. heterophylla) can reduce the temperatures compared with other environments at the same latitude. Similarly, precipitation during the driest month in these regions is high, with environments being quite humid throughout the year (Hasenack and Ferraro, 1989; Iganci et al., 2011; Silva-Arias et al., 2017). For clade B, precipitation of the driest month was proportionally less critical because the distribution of the species in this clade varies significantly with latitude. Species of this clade occur in different conditions regarding this climatic variable, especially C. elegans and C. linoides subsp. linoides, which occur in regions of reduced precipitation during the driest month. However, all species of clade B occur at altitudes above 1000 m, where maximum temperatures during the warmest month do not reach high values regardless of latitude. In clade D, the maximum temperature during the warmest month is less important because, in the species distribution region, temperature seasonality has a more critical role in the environmental characterization. In this region, seasonality is more pronounced, with warmer summers compared with the same season in the highland and plateau grasslands, and cold winters. However, the region where clade D occurs has similar levels of precipitation along the year with low seasonal differentiation concerning to this variable, and it also exhibited only relative importance to the species’ predicted model. The four clades of Stimomphis subgenus occur in different geographic areas and seem to have undergone niche differentiation. It is possible that a different set of variables within their environmental niche may have limited the clades distribution. Overall, tests for niche

5. Conclusion In the most recent comprehensive sampling and inclusive genomic coverture of Calibrachoa species, we confirmed the monophyletic nature of the genus and subgenera and clarified the main evolutionary relationships among terminals. The Calibrachoa species have evolved under the influence of adaptive radiation and in a strong manner of niche conservatism; thus, this charismatic genus can serve as a model to explain the evolutionary patterns that modeled grasslands in the highlands, Pampas, and Chaco regions under the influence of Pleistocene climatic changes. Future studies are necessary to solve some inconclusive terminal positions and species' delimitation approaches, including analyzing a more extensive number of individuals per taxon, primarily for those occurring in the lowlands; wider genome coverture seems the best strategy to accomplish this objective. Moreover, additional morphotypes deserve a deep taxonomic review. Declaration of Competing Interest The authors declare no competing financial interests. Acknowledgments We thank J.N. Fregonezi, C. Turchetto, G.C. Giudicelli, and A. Backes for help with collections, and J.R. Stehmann for help with plant identification. We are also grateful to colleagues that provided photos, 8

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some of whom appear in Fig. 1. This project was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). G.M. was supported by PNPD-CAPES/PPGBot, UFRGS.

Hughes, C., Eastwood, R., 2006. Island radiation on a continental scale: exceptional rates of plant diversification after uplift of the Andes. Proc. Natl. Acad. Sci. USA 103, 10334–10339. Iganci, J.R., Heiden, G., Miotto, S.T.S., Pennington, R.T., 2011. Campos de Cima da Serra: the Brazilian Subtropical Highland Grasslands show an unexpected level of plant endemism. Bot. J. Linn. Soc. 167, 378–393. Iganci, J.R.V., Miotto, S.T.S., Souza-Chies, T.T., Särkinen, T.E., Simpson, B.B., Simon, M.F., Pennington, R.T., 2013. Diversification history of Adesmia ser Psoraleoides (Leguminosae): evolutionary processes and the colonization of the southern Brazilian highland grasslands. S. Afr. J. Bot. 89, 257–264. Johnson, L.A., Soltis, D.E., 1994. matK DNA sequences and phylogenetic reconstruction in Saxifragaceae s. str. Syst. Bot. 19, 143–156. Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870–1874. Landis, J.R., Koch, G.G., 1977. The measurement of observer agreement for categorical data. Biometrics 33, 159–174. Leal, B.S.S., Palma-Silva, C., Pinheiro, F., 2016. Phylogeographic studies depict the role of space and time scales of plant speciation in a highly diverse Neotropical region. Crit. Rev. Plant Sci. 35, 215–230. Librado, P., Rozas, J., 2009. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25, 1451–1452. López-Fernández, H., Winemiller, K.O., Honeycutt, R.L., 2010. Multilocus phylogeny and rapid radiations in Neotropical cichlid fishes (Perciformes: Cichlidae: Cichlinae). Mol. Phylogenet. Evol. 55, 1070–1086. Lorenz-Lemke, A.P., Togni, P.D., Mäder, G., Kriedt, R.A., Stehmann, J.R., Salzano, F.M., Bonatto, S.L., Freitas, L.B., 2010. Diversification of plant species in a subtropical region of eastern South American highlands: a phylogeographic perspective on native Petunia (Solanaceae). Mol. Ecol. 19, 5240–5251. Mäder, G., Zamberlan, P.M., Fagundes, N.J., Magnus, T., Salzano, F.M., Bonatto, S.L., Freitas, L.B., 2010. The use and limits of ITS data in the analysis of intraspecific variation in Passiflora L. (Passifloraceae). Genet. Mol. Biol. 33, 99–108. Mäder, G., Fregonezi, J.N., Lorenz-Lemke, A.P., Bonatto, S.L., Freitas, L.B., 2013. Geological and climatic changes in quaternary shaped the evolutionary history of Calibrachoa heterophylla, an endemic South Atlantic species of petunia. BMC Evol. Biol. 13, 178. Miller, M.A., Pfeiffer, W., Schwartz, T., 2010. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. Gateway Comput. Environ. Workshop 1, 1–8. Moreno, E.M.S., Freitas, L.B., Speranza, P.R., Solís-Neffa, V.G., 2018. Impact of Pleistocene geoclimatic events on the genetic structure in mid-latitude South American plants: insights from the phylogeography of Turnera sidoides complex (Passifloraceae, Turneroideae). Bot. J. Linn. Soc. 188, 377–390. Nelson, G., Platnick, N.I., 1981. Systematics and Biogeography. Harcourt, Brace and World. Niu, Y.-T., Jabbour, F., Barret, R.L., Ye, J.-F., Zhang, Z.-Z., Lu, K.-Q., Lu, L.-M., Chen, Z.D., 2018. Combining complete chloroplast genome sequences with target loci data and morphology to resolve species limits in Triplostegia (Caprifoliaceae). Mol. Phylogenet. Evol. 129, 15–26. Olmstead, R.G., Bohs, L., Migid, H.A., Santiago-Valentin, E., Garcia, V.F., Collier, S.M., 2008. A molecular phylogeny of the Solanaceae. Taxon 57, 1159–1181. Olmstead, R.G., 2013. Phylogeny and biogeography in Solanaceae, Verbenaceae and Bignoniaceae: a comparison of continental and intercontinental diversification patterns. Bot. J. Linn. Soc. 171, 80–102. Ortega-Andrade, H.M., Rojas-Soto, O.R., Valencia, J.H., Monteros, A.E., Morrone, J.J., Ron, S.R., Cannatella, D.C., 2015. Insights from integrative systematics reveal cryptic diversity in Pristimantis frogs (Anura: Craugastoridae) from the upper Amazon Basin. PLoS ONE 10, 1–43. Overbeck, G.E., Müller, S.C., Fidelis, A., Pfadenhauer, J., Pillar, V.D., Blanco, C.C., Boldrini, I.I., Both, R., Forneck, E.D., 2007. Brazil’s neglected biome: the South Brazilian Campos. Perspect. Plant Ecol. Evol. Syst. 9, 101–116. Pearce, J., Ferrier, S., 2000. Evaluating the predictive performance of habitat models developed using logistic regression. Ecol. Model. 133, 225–245. Peterson, A.T., 2007. Why not, why where: the need for more complex models of simpler environmental spaces. Ecol. Model. 203, 527–530. Pezza, A.B., Ambrizzi, T., 2005. Cold waves in South America and freezing temperatures in São Paulo: historical background (1888–2003) and case studies of cyclone and anticyclone tracks. Rev. Bras. Meteorol. 20, 141–158. Phillips, S.J., Anderson, R.P., Schapire, R.E., 2006. Maximum entropy modeling of species geographic distributions. Ecol. Model. 190, 231–259. Pyron, R.A., Costa, G.C., Patten, M.A., Burbrink, F.T., 2015. Phylogenetic niche Conservatism and the evolutionary basis of ecological speciation. Biol. Rev. Camb. Philos. Soc. 90, 1248–1262. de Queiroz, K., 1998. The general lineage concept of species, species criteria and the process of speciation: a conceptual unification and terminological recommendations. In: Howard, D.J., Berlocher, S.H. (Eds.), Endless Forms: Species and Speciation. Oxford University Press, pp. 57–75. Rambaut, A., Drummond, A., 2013. Tracer. Version 1.6. http://beast.bio.ed.ac.uk/tracer. Reck-Kortmann, M., Silva-Arias, G.A., Segatto, A.L.A., Mäder, G., Bonatto, S.L., Freitas, L.B., 2014. Multilocus phylogeny reconstruction: new insights into the evolutionary history of the genus Petunia. Mol. Phylogenet. Evol. 81, 19–28. Richardson, J.E., Pennington, R.T., Pennington, T.D., Hollingsworth, P.M., 2001. Rapid diversification of a species-rich genus of Neotropical rain forest trees. Science 293, 2242–2245. Rowe, K.C., Aplin, K.P., Baverstock, P.R., Moritz, C., 2011. Recent and rapid speciation with limited morphological disparity in the genus Rattus. Syst. Biol. 60, 188–203. Roy, A., Frascaria, N., MacKay, J., Bousquet, J., 1992. Segregating random amplified Polymorphic DNAs (RAPDs) in Betula alleghaniensis. Theor. Appl. Genet. 85, 173–180.

Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ympev.2019.106614. References Abbott, R.J., Brennan, A.C., 2014. Altitudinal gradients, plant hybrid zones and evolutionary novelty. Philos. Trans. R. Soc. B. 369, 20130346. Aguirre-Gutiérrez, J., Serna-Chavez, H.M., Villalobos-Arambula, A.R., Pérez de la Rosa, J.A., Raes, N., 2015. Similar but not equivalent: ecological niche comparison across closely–related Mexican white pines. Divers. Distrib. 21, 245–257. Alarcón, M., Vargas, P., Sáez, L., Molero, J., Aldasoro, J.J., 2012. Genetic diversity of mountain plants: Two migration episodes of Mediterranean Erodium (Geraniaceae). Mol. Phylogenet. Evol. 63, 866–876. Allouche, O., Tsoar, A., Kadmon, R., 2006. Assessing the accuracy of species distribution models: prevalence, kappa, and the true skill statistic (TSS). J. Appl. Ecol. 43, 1223–1232. Ando, T., Kokubun, H., Watanabe, H., Tanaka, N., Yukawa, T., Hashimoto, G., Marchesi, E., Suaréz, E., Basualdo, I.L., 2005. Phylogenetic analysis of Petunia sensu Jussieu (Solanaceae) using chloroplast DNA RFLP. Ann. Bot. 96, 289–297. Antonelli, A., Nylander, J.A.A., Persson, C., Sanmartín, I., 2009. Tracing the impact of the Andean uplift on Neotropical plant evolution. Proc. Natl. Acad. Sci. USA 106, 9749–9754. Barros, M.J.F., Silva-Arias, G.A., Fregonezi, J.N., Turchetto-Zolet, A.C., Iganci, J.R.V., Diniz-Filho, J.A.F., Freitas, L.B., 2015. Environmental drivers of diversity in Subtropical Highland Grasslands. Perspect. Plant Ecol. Evol. Syst. 17, 360–368. Behling, H., Pillar, V.D.P., 2007. Late Quaternary vegetation, biodiversity and fire dynamics on the southern Brazilian highland and their implication for conservation and management of modern Araucaria forest and grassland ecosystems. Philos. Trans. R. Soc. B Biol. Sci. 362, 243–251. Carstens, B.C., Knowles, L.L., 2007. Shifting distributions and speciation: species divergence during rapid climate change. Mol. Ecol. 16, 619–627. Couvreur, T.L.P., Porter-Morgan, H., Wieringa, J.J., Chatrou, L.W., 2011. Little ecological divergence associated with speciation in two African rain forest tree genera. BMC Evol. Biol. 11, 296. Crepet, W.L., Niklas, K.J., 2009. Darwin’s second “abominable mystery”: Why are there so many angiosperm species? Am. J. Bot. 96, 366–381. Daniels, S.R., Klaus, S., 2018. Divergent evolutionary origins and biogeographic histories of two freshwater crabs (Brachyura: Potamonautes) on the West African conveyer belt islands of São Tomé and Príncipe. Mol. Phylogenet. Evol. 127, 119–128. Darriba, D., Taboada, G.L., Doallo, R., Posada, D., 2012. jModelTest 2: more models, new heuristics and parallel computing. Nat. Methods 9, 772. Desfeux, C., Lejeune, B., 1996. Systematics of Euromediterranean Silene (Caryophyllaceae): evidence from a phylogenetic analysis using ITS sequence. Comptes. Rendus. Acad. Sci. 319, 351–358. Donato, M., Posadas, P., Miranda-Esquivel, D.R., Jaureguizar, E.O., Cladera, G., 2003. Historical biogeography of the Andean region: evidence from Listroderina (Coleoptera: Curculionidae: Rhytirrhinini) in the context of the South American geobiotic scenario. Biol. J. Linn. Soc. 80, 339–352. Dunn, I.S., Blattner, F.R., 1987. Charons 36–40: multi-enzyme, high capacity, recombination deficient replacement vectors with polylinkers and polystuffers. Nucleic Acids Res. 15, 2677–2698. Fielding, A.H., Bell, J.F., 1997. A review of methods for the assessment of prediction errors in conservation presence/absence models. Environ. Conserv. 24, 38–49. Fregonezi, J.N., Freitas, L.B., Bonatto, S.L., Semir, J., Stehmann, J.R., 2012. Infrageneric classification of Calibrachoa (Solanaceae) based on morphological and molecular evidence. Taxon 61, 120–130. Fregonezi, J.N., Turchetto, C., Bonatto, S.L., Freitas, L.B., 2013. Biogeographical history and diversification of Petunia and Calibrachoa (Solanaceae) in the Neotropical Pampas grassland. Bot. J. Linn. Soc. 171, 140–153. Greppi, J.A., Hagiwara, J.C., Stehmann, J.R., 2013. Novedades en Calibrachoa (Solanaceae) y notas taxonómicas sobre el género para la Argentina. Darwiniana 1, 173–187. Guisan, A., Thuiller, W., 2005. Predicting species distribution: offering more than simple habitat models. Ecol. Lett. 8, 993–1009. Hamilton, M.B., 1999. Four primers pairs for the amplification of chloroplast intergenic regions with intraspecific variation. Mol. Ecol. 8, 513–525. Hasenack, H., Ferraro, L.W., 1989. Considerações sobre o clima da região de Tramandaí. Pesquisas 22, 53–70. Hijmans, R.J., Cameron, S.E., Parra, J.L., Jones, P.G., Jarvis, A., 2005. Very high resolution interpolated climate surfaces for global land areas. Int. J. Climatol. 25, 1965–1978. Hou, Z., Sket, B., Fišer, C., Li, S., 2011. Eocene habitat shift from saline to freshwater promoted Tethyan amphipod diversification. Proc. Natl. Acad. Sci. 108, 14533–14538.

9

Molecular Phylogenetics and Evolution 141 (2019) 106614

G. Mäder and L.B. Freitas

Smith, T.B., Schneider, C.J., Holder, K., 2001. Refugial isolation versus ecological gradients. Genetica 112–113, 383–398. Stamatakis, A., 2014. RaxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30, 1312–1313. Suchard, M.A., Lemey, P., Baele, G., Ayres, D.L., Drummond, A.J., Rambaut, A., 2018. Bayesian phylogenetic and phylodynamic data integration using BEAST 1.10. Virus Evol. 4, vey016. Taberlet, P., Gielly, L., Pautou, G., Bouvet, J., 1991. Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Mol. Biol. 17, 1105–1109. Turchetto, C., Segatto, A.L.A., Telles, M.P.C., Diniz-Filho, J.A.F., Freitas, L.B., 2014. Infraspecific classification reflects genetic differentiation in the widespread Petunia axillaris complex: a comparison among morphological, ecological, and genetic patterns of geographic variation. Perspect. Plant Ecol. Evol. Syst. 16, 75–82. Turchetto-Zolet, A.C., Pinheiro, F., Salgueiro, F., Palma-Silva, C., 2013. Phylogeographical patterns shed light on evolutionary process in South America. Mol. Ecol. 22, 1193–1213. Warren, D.L., Glor, R.E., Turelli, M., 2008. Environmental niche equivalency versus conservatism: quantitative approaches to niche evolution. Evolution 62, 2868–2883. Warren, D.L., Glor, R.E., Turelli, M., 2010. ENMTools: a toolbox for comparative studies of environmental niche models. Ecography 33, 607–611. Webb, C.O., Ackerly, D.D., McPeek, M.A., Donoghue, M.J., 2002. Phylogenies and community ecology. Annu. Rev. Ecol. Syst. 33, 475–505. Yu, Y., Harris, A.J., He, X., 2010. S-DIVA (Statistical Dispersal-Vicariance Analysis): a tool for inferring biogeographic histories. Mol. Phylogenet. Evol. 56, 848–850. Yu, Y., Harris, A.J., Blair, C., He, X., 2015. RASP (Reconstruct Ancestral State in Phylogenies): a tool for historical. Mol. Phylogenet. Evol. 87, 46–49. Zhang, L., Lu, N.T., Zhou, X.-M., Chen, D.-K., Knapp, R., Zhou, L., Guo, L., Luong, T.T., Sun, H., Gao, X.-F., Zhang, L.-B., 2019. A plastid phylogeny of the Old World fern genus Leptochilus (Polypodiaceae): Implications for cryptic speciation and progressive colonization from lower to higher latitudes. Mol. Phylogenet. Evol. 134, 311–322. Zhang, X., Takahashi, H., Nakamura, I., Mii, M., 2008. Molecular discrimination among taxa of Petunia axillaris complex and P. integrifolia complex based on PolA1 sequence analysis. Breed. Sci. 58, 71–75.

Rull, V., 2011. Neotropical biodiversity: timing and potential drivers. Trends Ecol. Evol. 26, 508–513. Safford, H.D., 1999. Brazilian Páramos I. An introduction to the physical environment and vegetation of the campos de altitude. J. Biogeogr. 26, 693–712. Safford, H.D., 2007. Brazilian Páramos IV Phytogeography of the campos de altitude. J. Biogeogr. 34, 1701–1722. Sang, T., Crawford, D.J., Stuessy, T.F., 1997. Chloroplast DNA phylogeny, reticulate evolution, and biogeography of Peonia (Peoniaceae). Am. J. Bot. 84, 1120–1136. Särkinen, T., Bohs, L., Olmstead, R.G., Knapp, S., 2013. A phylogenetic framework for evolutionary study of the nightshades (Solanaceae): a dated 1000-tip tree. BMC Evol. Biol. 13, 214. Schoener, T.W., 1970. Nonsynchronous spatial overlap of lizards in patchy habitats. Ecology 51, 408–418. Segatto, A.L.A., Thompson, C.E., Freitas, L.B., 2016. Contribution of WUSCHEL-related homeobox (WOX) genes to identify the phylogenetic relationships among Petunia species. Genet. Mol. Biol. 39, 658–664. Segatto, A.L.A., Reck-Kortmann, M., Turchetto, C., Freitas, L.B., 2017. Multiple markers, niche modelling, and bioregions analyses to evaluate the genetic diversity of a plant species complex. BMC Evol. Biol. 17, 234. Shaw, J., Lickey, E.B., Beck, J.T., Farmer, S.B., Liu, W., Miller, J., Siripun, K.C., Winder, C.T., Schilling, E.E., Small, R.L., 2005. The tortoise and the hare II: Relative utility of 21 noncoding chloroplast DNA sequences for phylogenetic analysis. Am. J. Bot. 92, 142–166. Shaw, J., Lickey, E.B., Shilling, E.E., Small, R.L., 2007. Comparison of whole chloroplast genome sequences to choose noncoding regions for phylogenetic studies in angiosperms: The tortoise and the hare III. Am. J. Bot. 94, 275–288. Silva-Arias, G.A., Reck-Kortmann, M., Carstens, B.C., Hasenack, H., Bonatto, S.L., Freitas, L.B., 2017. From inland to the coast: Spatial and environmental signatures on the genetic diversity in the colonization of the South Atlantic Coastal Plain. Perspect. Plant Ecol. Evol. Syst. 28, 47–57. Simmons, M.P., Ochoterena, H., 2000. Gaps as characters in sequence-based phylogenetic analyses. Syst. Biol. 49, 369–381. Sklenář, P., Duˇsková, E., Balslev, H., 2011. Tropical and Temperate: evolutionary history of Páramo flora. Bot. Rev. 77, 71–108.

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