Unraveling Polyphyly in Flourensia (Asteraceae, Heliantheae, Enceliinae) and the Establishment of a New Genus Austroflourensia

Abstract The monophyly of Flourensia was examined for the first time by sequencing the nuclear ITS and plastid psbA- trnH regions in 18 species of the genus, analyzing them along with representatives of the remaining genera of subtribe Enceliinae. Results showed strong evidence for the polyphyly of Flourensia identifying two well-supported groups: Flourensia, a clade from North America including the type F. laurifolia, and another clade, here designed as the new genus Austroflourensia, containing the South American species. Austroflourensia is related to the other four genera composing the subtribe Enceliinae, whereas Flourensia s.s. is sister to all of them. Austroflourensia can be mainly distinguished by having a shrubby or subshrubby habit, capitula always radiate usually arranged in weakly cymose-corymbose capitulescences, phyllaries 2-3-seriate, and disc corollas shortly dentate. This paper proposes twelve new combinations to accommodate species previously described in the genus Flourensia and provides emended descriptions of Flourensia and the new genus Austroflourensia. The illustration of the type of the new genus, a distribution map, and a key to the genera of Enceliinae are also provided.

Flourensia is the largest genus of subtribe Enceliinae (Panero 2005(Panero , 2007) ) with an amphitropical disjunct distribution in arid to semi-arid regions of North and South America.Dillon (1984) published a complete monograph of Flourensia in which 31 species of resinous shrubs or small trees were recognized; subsequently one additional species from Peru was described (Dillon 1986).Our recent taxonomic work on South American species of the genus reduced the number to 25 (Ospina et al. 2018).Dillon (1984) divided Flourensia into North and South American species, with the North American group including the type species F. laurifolia DC.Although these species were not formally grouped into infrageneric categories and no diagnostic characters were noted to separate these two groups, he suggested that Flourensia, as many genera of the Heliantheae of the New World, had its origin in North America with early incursions into South America.
The genus is considered as a probably monophyletic group supported by the SUSANA E. FREIRE et al.
The main objective of this paper is to perform a molecular phylogenetic study to test the monophyly of the genus Flourensia.Additionally, we will analyze the morphological features of the group and related genera to determine diagnostic characters for the natural groups obtained in the molecular phylogeny.

Taxon sampling
The plastid psbA-trnH and nuclear ITS markers were sequenced for 18 species of Flourensia, eight from South America and ten from North America, for a total of 17 ITS and 12 psbA-trnH new sequences for the genus.We also sequenced the ITS and the psbA-trnH of Geraea canescens Torr.& A. Gray and Enceliopsis argophylla (D.C.Eaton) A. Nelson.We produced 33 new sequences for this study.We also included in the data matrix representatives of the Enceliinae available in GenBank including 17 sequences of Encelia, four of Enceliopsis, one of Geraea, and two sequences of Helianthella.To serve as outgroups in the analyses, we included in the data matrix several representatives of other subtribes of the Heliantheae related to Enceliinae (Panero 2007, Schilling & Panero 2011)

DNA extraction and sequencing
Samples were either collected in the field and leaves dried in silica gel or leaf fragments were removed from herbarium specimens.Total DNA was extracted using the modified CTAB protocol of Doyle & Doyle (1987).The DNeasy plant mini kit (Qiagen, Hilden, Germany) was used to extract DNA from herbarium specimens.The plastid psbA-trnH and the nuclear ITS marker were amplified using PCR.For psbA-trnH, the primers designed by Hamilton (1999) were used; the ITS region was amplified using the ITS4 and ITS5 universal primers.The PCR reactions were performed in 25 µl of final volume with 50-100 ng of template DNA, 0.2 µM of each primer, 25 µM of dNTPs, 5 µM MgCl2, buffer 10x, and 1.5 units of Taq polymerase (Invitrogen, Brazil).The reaction conditions were: an initial period of denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 48°C for psbA-trnH and 52°C for ITS for 1 min, extension at 72°C for 1 min, 30 s, and a final extension at 72°C for 6 min.Products were run on 1% TBE agarose gel and stained with SybrSafe (Invitrogen).Sequencing reactions were performed by Macrogen, Inc. (Seoul, Korea).Editing and manual alignment of the sequences were performed by BioEdit 5.0.9 (Hall 1999).

Data analysis
The nuclear and plastid matrix were initially analyzed separately using parsimony.The two consensus trees obtained in the separate analyses were assessed for congruence.Because the plastid tree was mainly unresolved and did not contradict the nuclear topology, both matrices were assembled in a combined matrix (Supplementary Material Appendix SI) and a simultaneous analysis (Nixon & Carpenter 1996) was carried out.The matrices and the analyses can be found in TreeBase: S26566 (nuclear matrix, separated analysis), S26567 (plastid matrix, separated analysis), and S26568 (combined matrix, simultaneous analysis).Additionally, the nuclear matrix was analyzed under Bayesian Inference (TreeBase: S26572) to assess congruence between the results of both methods.
Parsimony analyses were carried out using TNT version 1.1 (Goloboff et al. 2008).Uninformative characters were not used in parsimony searches.Informative indels for the ingroup were coded as Present/Absent (see Table I and matrices in Appendix SI).The search strategy involved 1000 replicates, each of which generated a Wagner tree using a random addition sequence of taxa from the data matrix, swapping the initial tree with TBR (tree bisection and reconnection) and retaining a maximum of 10 trees in each replicate.Subsequently, all optimal trees were swapped using TBR, holding a maximum of 10,000 trees.A strict consensus tree was generated from the most parsimonious trees.Branch support were estimated with Bootstrap (Felsenstein 1985) using a total of 10,000 replicates.Each replicate was analyzed using 10 Wagner trees as a starting point followed by TBR branch swapping, saving only one tree per replicate.Bootstrap values (BS) over 50 % are reported.
The Bayesian analysis was conducted with BEAST v1.10.4 package (Drummond et al. 2012).Parameters in BEAUti were set as follows: GTR+I+Γ as the nucleotide substitution model, site-rate heterogeneity modeled with four gamma categories, estimated base frequencies, a lognormal uncorrelated relaxed clock, a Yule process as tree prior, and default values for all other operators.Two independent runs, each using four Markov chains (one cold and three hot) of 10 million generations, were sampled every 1000 generations.To identify when the analyses had reached stationarity, we checked the output files for convergence and effective sample size (ESS) > 200 with Tracer v1.6 (Rambaut et al. 2014).Based on this convergence diagnostic, the first 2500 sampled trees were discarded as burnin from each analysis using TreeAnnotator 1. 7.1 (Drummond et al. 2012).Trees of the two runs were combined using LogCombiner 1.8.4 and the Maximum Credibility Tree was displayed in FigTree 1.3.1 (Rambaut 2009).Statistical support was determined by assessing the Bayesian posterior probabilities (PP).

RESULTS
The nuclear matrix had 50 taxa and 548 base positions, 165 of which were informative, also four informative indels were coded for the parsimony analysis (Table I).Parsimony analysis of ITS sequences yielded 576 most parsimonious trees of 465 steps.The strict consensus tree is shown as Supplementary Material -Figure S1.
The Bayesian analysis of the ITS sequences yielded a Maximum Credibility Tree displaying a topology congruent with that obtained in the parsimony analysis; the Maximum Credibility Tree is shown in Figure S2.
The plastid matrix was composed of 36 taxa and 511 characters, 63 of these being informative; also, seven informative indels were coded (Table I).Parsimony analysis of psbA-trnH sequences resulted in four trees 87 steps long.The strict consensus tree (data not shown) was poorly resolved (due to the low variability among the sequences).However, the topology obtained did not contradict the ITS consensus tree, so both matrices were combined and analyzed in a simultaneous analysis.
The combined matrix (ITS + psbA-trnH) had 1070 characters and 51 taxa.The simultaneous analysis yielded 832 most parsimonious trees 559 steps long.The strict consensus tree obtained from the combined sequences is shown in Figure 1.The topology obtained in the simultaneous analysis (Fig. 1) was mostly congruent with those obtained from the analyses of the ITS matrix (Figure S1, S2).
All the analyses displayed, ITS (under Parsimony and Bayesian) and the simultaneous analysis, strongly rejected the monophyly of Flourensia (Fig. 1).The two well-supported and independent lineages of Flourensia s.l. were Flourensia s.s., grouping the North American Flourensia species, including the type F. laurifolia, (BS = 89%, PP = 1, and BS = 87%), and another clade containing all the South American species of Flourensia (BS = 100%, PP = 1, and BS = 99%).In addition, two indels (at positions 147-148 and 479-480 of the ITS alignment) support the differentiation of these two lineages.
The South American Flourensia clade is in a strongly supported group (BS = 99%, PP = 1, and BS = 99%) sister to the remaining North American genera of Enceliinae, i.e.Helianthella, Geraea, Enceliopsis, and Encelia.Within this clade the relationships between the genera remain unresolved or with low support.Flourensia s.s. is the sister group of the entire clade including the South American Flourensia and the remaining genera of Enceliinae.These two sister clades constitute the subtribe Enceliinae and is recovered as monophyletic with high support (BS = 100, PP = 1, and BS = 99%).
Within the South American Flourensia clade, interspecific relationships are mostly unresolved or resolved with low support.Our analyses showed some discrepancies: species whose relationships were unresolved in the ITS analyses, were resolved in the combined analysis (but with low support), whereas other species whose relationships were resolved in the ITS analyses, were unresolved in the combined analysis.
The morphological features recorded and observed for Flourensia s.s., the South American clade, and the remaining genera of the subtribe Enceliinae are summarized in Table II.

Molecular phylogenetic analysis
The molecular phylogenetic analyses performed here included 60% of the species of Flourensia, most of them sampled for the first time.The results obtained confirm Flourensia as polyphyletic with respect to the remaining genera of Enceliinae.Our results showing two lineages of Flourensia s.l., i.e.Flourensia s.s. and the South American clade, agree with those of Dillon (1984) in supporting the subdivision of Flourensia into two main geographical groups.

North American clade: Flourensia
From a total of thirteen North American Flourensia species, ten were analyzed in this study, including the type F. laurifolia.All these species form a strongly supported clade, redefined here as the genus Flourensia, which is sister to all the remaining genera of the Enceliinae.They are diagnosed by the combination of the following morphological characters: shrubs or small trees with discoid or radiate capitula, phyllaries in 2-4(-5) series, usually solitary capitula or weakly cymose 2-5-headed, and disc corollas shallowly to deeply dentate.Within Flourensia, the two internal clades identified show low support and scarcely contrasting morphologies.
The "microphylla subclade" comprises three shrubby species, F. microphylla, F. pulcherrima, Here, we define the South American clade as the new genus Austroflourensia.Austroflourensia is diagnosed by the combination of the following morphological characters: shrubs or subshrubs with radiate capitula, phyllaries in 2-3 series, solitary capitula or weakly cymose capitulescences, and disc corollas shortly dentate.Within this clade, the combined analysis shows five species in a basal polytomy and the remaining three species in an unsupported subclade (Fig. 1).The members of this subclade, i.e.F. hirta, F. fiebrigii and F. blakeana were recognized as three species sensu Dillon (1984) or a single polymorphic species sensu Ospina et al. (2018), the last emphasizing the lack of differentiation in foliar, floral and capitulescence morphology.In the Taxonomic Treatment section, we follow Ospina et al. 2018.
The molecular markers used in this study were not sufficient to clarify the relationships within Austroflourensia or within Flourensia, because of the low variability displayed.Future studies using more variable markers would be useful to identify the interspecific relationships within the two groups.
Austroflourensia is sister to Helianthella (Fig. 1), although with low support (less than 50%).Both share their glabrous leaves and solitary to corymbose capitula.However, Austroflourensia mainly differs from Helianthella by its shrub or subshrubby habit, foliage with resin, yellow florets and pappose achenes not winged (Table II).Regarding intergeneric relationships of Austroflourensia with the remaining genera of Enceliinae, this genus shares with Encelia, Enceliopsis, and Geraea many morphological characters, e.g.radiate capitula, neuter ray florets, phyllaries 2-3-seriate (Table II).However, Austroflourensia species differ mainly from the mentioned genera by their shrub or subshrubby habit, foliage with resin, achenes slightly compressed, and the absence of ray florets with 3-lobed corollas (Table II).
U n f o r t u n a t e l y, t h e p h y l o g e n e t i c relationships between Austroflourensia and the remaining North American genera of Enceliinae were not resolved by the molecular markers used here.
Although Austroflourensia is vegetatively and reproductively similar to Flourensia in their leaves (e.g.shape, size, pubescence), ray and disc florets (e.g.sex, number, disc corolla color), achenes, pappus, and all are resinous plants, they can be mainly differentiated by their shrubby or subshrubby habit (vs.shrubs or small trees), capitula always radiate (vs.radiate or discoid), usually arranged in weakly cymose-corymbose 2-8-headed capitulescences (vs.usually solitary or weakly cymose 2-5-headed capitulescences), phyllaries 2-3-seriate (vs.2-4(-5)-seriate), and disc corollas shortly dentate (vs.shallowly to deeply dentate) (Table II).(2023) 95(2) e20191135 8 | 17 We agree with Dillon (1984) that the North American species of Flourensia have greater morphological divergence compared to the South American Flourensia species.For example, phyllaries imbricate or subequal, equaling or overtopping the disc florets (vs.usually subequal to equal and equaling the disc florets), disc corollas shallowly to deeply 5-lobed (vs.shallowly 5-lobed), achenes from densely villoussericeous to glabrous (vs.villous-sericeous to glabrescent) (Figure 2).Dillon (1984) suggested a North American origin for Flourensia s.l.based on the greater morphological divergence among North American taxa; this assumption agrees with our phylogenetic results in the sense that there is higher resolution of the North American species as compared to the South American taxa and the sister position of the former (Fig. 1).
We hypothesize that the low sequence divergence and the few morphological d i f fe re n ce s b e t w e e n Flo u re n s i a a n d Austroflourensia are probably the result of a rapid and recent diversification of their species in the arid and semiarid regions of both hemispheres.The Enceliinae have diversified in many areas of North America since the ancestor of Austroflourensia dispersed to South America.For example, species of Geraea, Enceliopsis and Helianthella have adapted to a myriad of habitats including the interior deserts and the chaparral area of the Californian Mediterranean region and the deserts of the western United States.Some species of Helianthella are endemic to the Chihuahuan desert and the conifer and coniferoak forests of northern Mexico, the western United States and western Canada.
In South America, the species of Austroflourensia have radiated in the inter-Andean valleys of the High Monte ecoregion (sensu Olson et al. 2001) that hold some of the most arid environments found in the Southern Cone of South America (Abraham et al. 2009).However, this expansion has not resulted in differences in growth forms.Sorting out the phylogeny and divergence time of the Austroflourensia clade would provide valuable information on the assembling of the arid adapted flora of the Southern Cone.
We recognize that additional multilocus analyses will be necessary to confirm and expand the results obtained here.For now, this study represents the first molecular phylogeny of Flourensia, and it strongly supports the polyphyly of the genus and the transfer of the South American species to a new genus, Austroflourensia.
Distribution and habitat.The genus is found on dry, limestone slopes and rocky soils, mainly restricted to Mexico, only two species (F.cernua and F. pringlei) extend into the southwestern United States, inhabiting the Chihuahuan Desert region and the tropical deciduous forests of eastern and southern Mexico, between 400-2100 m. (Figure 3).
Etymology.The name Austroflourensia combines its southern distribution (from Latin australis=southern) with the genus name Flourensia.
Distribution and habitat.The genus is found on dry, rocky slopes in the Andes of Peru, Bolivia, Chile, and northern Argentina (500-4000 m) to southern Argentina in the Patagonian steppe (250-360 m). Figure 3.
Twelve South American species: including one species each of Ambrosia L. (Ambrosiinae), Chromolepis Benth.(Chromolepidinae), Calanticaria (B.L. Rob.& Greenm.)E.E.Schill.& Panero and Simsia Pers.(Helianthinae), Acmella Rich.ex Pers.and Spilanthes Jacq.(Spilanthinae) and Zaluzania Pers.(Zaluzaniinae).After adding the outgroup species, the data matrix contained 51 taxa.Chromolepis heterophylla Benth.was chosen to root the trees.The detail of all species included in our analyses and their GenBank numbers are listed in the Appendix; the GenBank Accessions of the new sequences (MK417525 -MK417557) and their voucher information are highlighted in bold.

Table I .
Positions in the alignment of the informative indels coded for each marker.
POLYPHYLY IN Flourensia (ASTERACEAE) AND A NEW GENUSAn Acad Bras Cienc

Table II .
Diagnostic characters of the two lineages of Flourensia s.l. and the remaining genera of the subtribe Enceliinae.