Insights into the phylogeny and evolutionary history of ...

Denham & al. • Phylogeny and evolutionary history of Calyceraceae

TAXON 65 (6) • December 2016: 1328–1344

Insights into the phylogeny and evolutionary history of Calyceraceae Silvia S. Denham,1,2 Lucio Zavala-Gallo,1 Leigh A. Johnson3 & Raúl E. Pozner1 1 Instituto de Botánica Darwinion (Consejo Nacional de Investigaciones Científicas y Técnicas, Academia Nacional de Ciencias Exactas, Físicas y Naturales), casilla de correo 22, B1642HYD San Isidro, Buenos Aires, Argentina 2 Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, Avenida 122 y 60, La Plata, Buenos Aires, Argentina 3 Department of Biology and M. L. Bean Life Science Museum, 4102 LSB, Brigham Young University, Provo, Utah 84602, U.S.A. Authors for correspondence: Silvia S. Denham, [email protected]; Raúl E. Pozner, [email protected] ORCID SSD, http://orcid.org/0000-0002-4727-504x; LAH, http://orcid.org/0000-0002-1026-0944; REP, http://orcid.org/0000-0002-1467-1441 DOI https://doi.org/10.12705/656.7 Abstract Calyceraceae is a small family with six traditionally recognized genera and 47 species from southern South America. Most species grow along the Andes (of both Argentina and Chile) and in arid regions of the Patagonian steppe. This family belongs to the well-supported MGCA clade within Asterales, which includes Menyanthaceae + Goodeniaceae + Calyceraceae + Asteraceae. Calyceraceae is monophyletic and sister to Asteraceae, one of the five largest families of angiosperms. Although Calyceraceae is clearly distinct as a family, its genera are not, and taxonomic revisionary effort has confirmed the lack of sharp boundaries among genera. We performed a phylogenetic analysis of Calyceraceae with a broad taxon sampling (41 of 47 species), and with sequence data from multiple regions from the nuclear (ITS) and plastid genomes (ycg6-psbM, psbM-trnD, trnS-trnG, trnH-psbA, trnD-trnT) using maximum parsimony and Bayesian approaches. We aimed at identifying monophylectic groups, their putative morphological synapomorphies and their geographical distribution; we also estimated divergence times and examined chromosomes numbers in an evolutionary context. We obtained well-resolved and strongly supported phylogenies that show Calyceraceae to be divided into two major clades with geographically structured subclades within each. Our results indicate that an early split within Calyceraceae occurred about 27.4 Ma, probably related to differential changes in chromosome numbers, which allowed the two lineages to evolve in sympatry. We found that major natural subgroups diverged 15–12 Ma, following the Early-Miocene South Andes construction stage. Finally, the diversification of the extant species is probably associated to Andean orogeny and climate changes in the last 5–4 Myr. We recovered Acicarpha as monophyletic, while the remaining traditionally recognized genera of Calyceraceae are para- or polyphyletic. Most species of Moschopis are included in the Glutinose group, but M. monocephala is more closely related to some Calycera species. Calycera is divided into two clades: the Calycera group and the Pilose group. All species of Nastanthus are placed in a well-supported main group with species of Gamocarpha and Boopis. Gamocarpha could be monophyletic after exclusion of G. dentata and G. angustifolia, but is nested within Nastanthus and Boopis species. Boopis is clearly polyphyletic with its species distributed in all main groups. Keywords Andean clades; divergence times; molecular phylogeny; morphology; poly-paraphyletic genera; South America Supplementary Material Electronic Supplement (Figs. S1, S2) and DNA sequence alignment are available in the Supplementary Data section of the online version of this article at http://www.ingentaconnect.com/content/iapt/tax

INTRODUCTION Calyceraceae is a small family with 47 species in southern South America (Zavala-Gallo, 2013). Most of its species grow in arid environments along the southernmost Andes up to Bolivia and the Patagonian steppe in Argentina and Chile. A few species grow in mesophytic conditions in northern and central Argentina, and Uruguay, reaching the Atlantic sea shore, with four species growing in southern Brazil. One species is endemic to the Malvinas (Falkland) Islands. The family comprises small caulescent or rosulate herbs, or sometimes subwoody plants, and are annual or perennial. Most species are geophytes or hemicryptophytes, although a few are chamaephytes or therophytes. The family belongs to the MGCA clade within Aster ales. The MGCA clade is very well supported and includes

Menyanthaceae + Goodeniaceae + Calyceraceae + Asteraceae (Lundberg & Bremer, 2003). Calyceraceae is sister to the Aster aceae (Lundberg, 2009), one of the five largest families of angiosperms. Synapomorphies of the subclade Calyceraceae + Asteraceae are tiny flowers crowded in head-like inflorescences surrounded by involucral bracts, anthers connate in a ring that surrounds the style, introrse anther dehiscence and secondary pollen presentation, pollen spinulate or smooth with intercolpar concavities, 1-locular ovary with one ovule, and cypselae with a persistent, modified calyx. Calyceraceae differs from Asteraceae by the apical pendulous ovule (basal, erect in Asteraceae), punctiform or capitate stigma (split in two branches in Asteraceae, secondarily undivided in some taxa), and a more or less developed staminal tube (very rare in Asteraceae). The basic inflorescence structure of Calyceraceae

Received: 18 Jan 2016 | returned for (first) revision: 21 Apr 2016 | (last) revision received: 30 Aug 2016 | accepted: 1 Sep 2016 || publication date(s): online fast track, 6 Dec 2016; in print and online issues, 22 Dec 2016 || © International Association for Plant Taxonomy (IAPT) 2016

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is a cephalioid (a very condensed botryoid/thyrsoid), from which the Asteraceae capitulum could have arisen (Pozner & al., 2012). Recently, Katinas & al. (2016) observed bilobed stigmas in Acicarpha tribuloides Juss., Boopis multicaulis Phil. and Nastanthus scapigerus (J.Rémy) Miers, and suggested that stigmatic papillae show a temporal differentiation in Calycer aceae, being non-receptive or receptive in the male or female floral phase, respectively, while Asteraceae have a clear differentiation in the position of receptive (inner) and non-receptive (outer) papillae in the stigma. All previous phylogenetic analyses have shown Calycer aceae as a monophyletic family (Gustafsson & Bremer, 1995; Lundberg & Bremer, 2003; Lundberg, 2009; Pozner & al., 2012). Based on systematic treatments by Pontiroli (1963), Chiapella (1999a, b), Galvão Magenta & Pirani (2002), or Zanotti & Pozner (2008), six genera have been traditionally distinguished within Calyceraceae: Acicarpha Juss. (5 species from Argentina, Bolivia, Brazil, and Uruguay); Boopis Juss. (13 species from Argentina and Chile); Calycera Cav. (14 species from Argentina, Bolivia, and Chile); Gamocarpha DC. (6 species from Argentina and Chile); Moschopis Phil. (7 species from Argentina and Chile) and Nastanthus Phil. (9 species from Argentina and Chile). Although Calyceraceae is clearly distinct as a family, its genera are not. Problems in interpreting morphology and structural variation in taxa with a wide geographic distribution have blurred boundaries among genera (i.e., Hellwig, 2007; Zavala-Gallo, 2013). Reitz (1988) was one of the first authors to suggest synonymy of Boopis, Gamocarpha and Nastanthus due to poor morphological differentiation. The palynological results published by Hansen (1992) showed at least two main pollen morphologies in the family, which are not uniform within genera (DeVore & al., 2007). According to Carlquist & DeVore (1998), the diversity of wood anatomy in Calyceraceae (14 species of five genera sampled) suggests adaptation to particular ecological conditions, with little phylogenetic information. Characters of fruit morphology have been used to delimit genera of Calyceraceae; however, Zanotti & Pozner (2007) did not find enough morphological or anatomical evidence to distinguish Boopis from Nastanthus, in agreement with Hellwig (2007), who suggested synonymy of Nastanthus, Boopis and Moschopis. More recently, Zavala-Gallo & al. (2010) updated the taxonomy of Nastanthus, delimited species, and addressed nomenclatural issues in this genus. A taxonomic revision, together with a detailed study of morphology and a critical analysis of Calycera species boundaries was performed by Denham & al. (2014). The most complete and comprehensive taxonomic revision of species of Calyceraceae was accomplished by Zavala-Gallo (2013), who clearly defined 47 species distributed among the six traditional genera which is followed hereafter. This revisionary effort resolved species boundaries and resulted in reliable terminals for phylogenetic studies, but these new data also reiterated the major taxonomical problem in the family: the lack of sharp boundaries among genera. The only attempt to present a hypothesis about evolutionary relationships or monophyletic groups within Calyceraceae was made by Zavala-Gallo (2013). His exploratory phylogenetic analysis

included 32 species of Calyceraceae, two cpDNA markers and 37 morphological characters. He recovered Acicarpha as monophyletic and all remaining genera as poly- or paraphyletic, in a cladogram with very low or no branch support. Calyceraceae needs a reevaluation of generic boundaries within a phylogenetic context to establish a natural classification and develop hypotheses of morphological evolution. In pursuit of these goals, this paper presents the first phylogenetic hypothesis of Calyceraceae with nearly complete taxonomic sampling based on five plastid and one nuclear DNA regions. We aimed at identifying monophyletic groups, their putative morphological synapomorphies and their geographical distribution; we also estimated divergence times and examined chromosomes numbers in an evolutionary context. We question the traditional generic circumscriptions and comment on taxa that need further investigation.

MATERIALS AND METHODS Examined material. — We studied collections of Calycer aceae housed at BAA, BAB, CONC, CORD, CTES, LIL, LP, MERL, SGO, and SI, complemented by observations on material obtained in field work. Maps were based on the examined material (Zavala-Gallo, 2013), and we used the biogeographic scheme of Cabrera & Willink (1973) for descriptions of geographic distribution. Morphology. — Interpretations of morphological characters used in clade descriptions were based on: Troll (1969) for the general structure of the plant; Erbar (1993), Denham & al. (2014) with modifications according to Leins & Erbar (2010) for flower morphology; and Pozner & al. (2012) and Pozner & al. (in prep.) for inflorescence structure. Precise description of flower morphology (Fig. 1) becomes particularly relevant for the description of some groups. The perianth in Calycer aceae consists of calyx, stamen-corolla tube (= hypanthium in Denham & al., 2014), and corolla (including tube and lobes, Fig. 1). The junction between the stamen-corolla tube and the corolla tube also includes the staminal tube insertion and nectar glands, all integrated into a cup or funnel-shaped structure: the nectarial region. Finally, because the stamen-corolla tube may elongate during anthesis together with the style, proportions among different parts of the perianth were based on fully elongated flowers with fully elongated style. Taxon sampling for phylogenetic analyses. — Ninetyseven specimens of Calyceraceae were sampled, representing 41 of 47 species (= 46 of 52 species and varieties). All six commonly recognized genera were sampled, with Acicarpha, Gamocarpha, and Moschopis sampled completely. No material for DNA extraction was available from Boopis bupleuroides (Less.) C.A.Müll., B. castilloni (Hicken) Pontiroli, B. itatiaiae Dusén, B. jürgensii Pilg., Calycera eryngioides J.Rémy, and Nastanthus falklandicus D.M.Moore. Relationships among families in the MGCA clade have been previously established by Gustafsson & Bremer (1995), Lundberg & Bremer (2003) and Lundberg (2009), and the phylogenetic structure (Menyanthaceae (Goodeniaceae (Calyceraceae,

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Asteraceae))) was used to select outgroup representatives. DNA sequences (mainly from GenBank) were added for Asteraceae representatives: Barnadesia lehmannii Hieron. var. lehmannii, Barnadesia lehmannii var. villosa (I.C.Chung) Urtubey, Dasyphyllum argenteum Kunth, Dasyphyllum brevispinum Sagást. & M.O.Dillon, Fulcaldea laurifolia (Bonpl.) Poir., and Nassauvia glomerulosa (Lag. ex Lindl.) D.Don (two vouchers). Scaevola gaudichaudiana Cham. (Goodeniaceae) was used to root the trees. Detailed information for taxa included in molecular analyses is given in Appendix 1, including voucher information and accession numbers of new sequences and of those obtained from GenBank. DNA extraction, amplification, and sequencing. — DNA was isolated primarily from field-collected, silica-dried leaf tissue, although some samples were obtained from herbarium specimens. Total genomic DNA was extracted using the CTAB protocol by Doyle & Doyle (1987) or using Qiagen DNeasy kits (Qiagen, Valencia, California, U.S.A.) following the manufacturer’s protocol. Five plastid regions (ycg6-psbM, psbM-trnD, trnS-trnG, trnH-psbA, trnD-trnT) and the nuclear ribosomal intergenic region ITS (including spacer ITS-1, the 5.8S subunit, and spacer ITS-2) were amplified by polymerase chain reaction and sequenced for each taxon. Primers used for amplification and sequencing are listed in Table 1. Two primers were newly designed.

Fig. 1. Flower morphology in Calyceraceae (regions labeled only on diagram B). A, Flower with long stamen-corolla tube, short corolla tube, and short staminal tube (e.g., Calycera herbacea). B, Flower with short stamen-corolla tube, long corolla tube, and long staminal tube (e.g., Moschopis caleofuensis). Abbreviations: a, anthers; cl, corolla lobes; ct, corolla tube; n, nectarial region; o, ovary; s, sepal; sct, stamen-corolla tube; st, staminal tube.

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Amplification reactions were performed in a 25 µl final volume, with 50–100 ng of template DNA (2.5 µl of pure or diluted 1/10 template), 1.2 µl (10 mM) of forward and reverse primers, 0.25 µl (2.5 mM) dNTPs, 2.5 µl MgCl2, 2.5 µl buffer, 0.3 µl of 5 units/µl Taq polymerase (Invitrogen, Sao Paulo, Brazil), and water. The reaction conditions were: denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 52°C–56°C for 1 min, and extension at 72°C for 1 min; final extension at 72°C for 6 min. A negative control with no template was included for each series of amplifications to detect contaminations. PCR products were run out on a 1% TBE agarose gel stained with SYBR Safe DNA gel stain (Invitrogen) and visualized in a blue light transilluminator. Automated sequencing was performed by Macrogen (Seoul, South Korea). Phylogenetic analysis. — Chloroplast sequence editing, assembly, and alignment were performed with BioEdit v.5.0.9 (Hall, 1999). Initial ITS sequence alignments were performed with MAFFT v.7 (available at http://align.bmr.kyushu-u.ac.jp/ mafft/software/) using default parameters, and manually refined with BioEdit. Three matrices were constructed: the “plastid matrix” combining the five plastid regions, the “nuclear matrix” including ITS sequences, and (in the absence of supported conflict between the resulting gene trees) the “total evidence matrix” combining the plastid and nuclear matrices. When amplification failed either in part or for the complete sequence for a given region, positions were coded as missing data in the separate and combined matrices. Gaps were treated as missing data in the analyses but, for plastid markers, informative and unambiguous gaps were coded as binary characters using simple indel coding (Simmons & Ochoterena, 2000) implemented in FastGap v.1.2 (Borchsenius, 2009) and added to the plastid matrix for the maximum parsimony analyses only. Phylogenetic analyses were performed using maximum parsimony and Bayesian inference approaches. Maximum parsimony analyses of the three matrices were performed using TNT v.1.1 (Goloboff & al., 2003). All characters were considered to be unordered and were equally weighted. We confirmed that monophyly of Calyceraceae was achieved with our partitioned data in preliminary searches. Accordingly, the command “force” was used to constrain monophyly of Calyceraceae to optimize searches and to reduce the computational time of the resampling analyses (Jackknife). The search strategy consisted of heuristic searches with 10,000 replications of random addition followed by TBR branch swapping, and retaining 10 trees per replication, keeping a maximum of 10,000 trees in memory. Thereafter, a new search with TBR branch swapping was performed using the shortest trees saved in memory. Branches of ambiguous length were collapsed, according to collapsing rule 1. A strict consensus tree was generated from the most parsimonious trees. Branch support was calculated using a Jackknife (JK) analysis (Farris & al., 1996) with a character removal probability of 36% in each of 8000 replicates employing heuristic searches with five random-addition replications, TBR branch-swapping, and one tree saved per replicate. Strict consensus trees of the most parsimonious trees obtained from

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the nuclear and plastid matrices were compared to evaluate congruence. Topologies were considered incongruent when clades in conflict are well supported (i.e., Jackknife values > 90). Prior to the Bayesian analysis, the optimal model of nucleotide substitution for each partition was investigated using the Akaike information criterion (AIC) as implemented in jModelTest v.2.1.1 (Darriba & al., 2012); optimal models for each partition are shown in Table 2. A Bayesian inference analysis with the total evidence matrix was performed with MrBayes v.3.2 (Ronquist & al., 2012) with the following settings: GTR + I + G for trnH-psbA, GTR + G for trnS-trnG, psbM-trnD and ITS, HKY + G for ycg6-psbM, and HKY + I + G for trnD-trnT ; priors on state frequencies, rates and shape of the gamma distribution were estimated automatically from the data (default priors in MrBayes) assuming no prior knowledge about their values. Two independent runs of 5 million generations were sampled every 1000th generation. The first 25% of the tree samples from the cold chain were discarded as burn-in. The average standard deviation of split frequencies at the end of the run, the potential

scale reduction factor (PSRF) and the estimated sample size (ESS) were used to check convergence and adequate sample size. The remaining trees from both runs were combined in a 50% majority-rule consensus. FigTree v.1.4.2 (available at http://tree.bio.ed.ac.uk/) was used to display the consensus topology and posterior probability values (PP). Clade ages estimation. — Divergence time estimation analyses were performed using the program BEAST v.2.4.1 (available at http://beast2.org/). We used a reduced matrix with only one representative per species within Calyceraceae (41 terminals) with the total evidence matrix. The same substitution models as used in the Bayesian analysis were used (unlinked site models). The molecular clock model was defined as relaxed, uncorrelated Lognormal (allowing independent rates across branches and without a priori correlation between rates of lineages and their ancestors). A Birth-Death model was used as tree prior. Two fossil records were used as calibration points. The first calibration point was Psilatricolporites protrudens Palazzesi & Barreda, a fossil pollen grain dated to

Table 1. Primers used for amplification and sequencing.

Region

Primer

Primer sequence from the 5′ end

Reference

ycg6-psbM

ycf6F

ATG GAT ATA GTA AGT CTY GCT TGG GC

Shaw & al., 2005

psbMR

ATG GAA GTA AAT ATT CTY GCA TTT TT GCT

Shaw & al., 2005

cp7 iF

GTG CAT TTA CKG CTT GTT TTC

newly designed

cp7 iR

ACT TCT GTT AAT GGC TCA ATC

newly designed

psbMF

AGC AAT AAA TGC RAG AAT ATT TAC TTC CAT

Shaw & al., 2005

trnDGUCR

GGG ATT GTA GYT CAA TTG GT

Shaw & al., 2005

psbM-trnD trnS-trnG trnH-psbA trnD-trnT

ITS

trnSGCU

AGA TAG GGA TTC GAA CCC TCG GT

Shaw & al., 2005

5′trnG2S

TTT TAC CAC TAA ACT ATA CCC GC

Shaw & al., 2005

trnHGUG

CGC GCA TGG TGG ATT CAC AAT CC

Tate & Simpson, 2003

psbA

GTT ATG CAT GAA CGT AAT GCT C

Sang & al., 1997

trnDGUCF

ACC AAT TGA ACT ACA ATC CC

Demesure & al., 1995

trnTGGU

CTA CCA CTG AGT TAA AAG GG

Demesure & al., 1995

trnEUUC

AGG ACA TCT CTC TTT CAA GGA G

Shaw & al., 2005

trnYGUA

CCG AGC TGG ATT TGA ACC A

Shaw & al., 2005

ITS5

GGA AGT AAA AGT CGT AAC AAG G

White & al., 1990

ITS4

TCC TCC GCT TAT TGA TAT GC

White & al., 1990

Table 2. Summary information for the data matrices and phylogenetic analyses.

ycg6-psbM

psbM-trnD

trnS-trnG

trnH-psbA

trnD-trnT

Plastid combined

ITS

Plastid and nuclear combined

Sequences

99

79

96

99

95

105

85

105

Aligned characters

833

680

900

590

968

3971

662

4633

Informative characters within Calyceraceae

53

57

42

102

134

388

179

567

Coded indels

13

5

22

19

26

85

0

85

Model selected by AIC

TPMuf + G

TVM + G

TVM + G

TVM + I + G

TPM1uf + I + G

Number of MPTs (steps)

GTR + G >10,000 (1262) 144 (625)

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600 (1925)

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approximately 21–22 Ma (Palazzesi & al., 2010) which can be attributed to Calyceraceae, particularly to the “Gamocarpha” morphological type (sensu DeVore & al., 2007), usually present in Gamocarpha and Nastanthus species, i.e.: no intercolpar concavities, no intercolpar ledges, no ectexine bridge over the pores. This fossil was assigned to the Gamocarpha-Nastanthus node in Panero & Crozier (2016), who included a very restricted sample of Calyceraceae, with only four species: Acicarpha spathulata R.Br., Calycera crassifolia (Miers) Hicken, Gamocarpha alpina (Poepp. ex Less.) H.V.Hansen, and Nastanthus caespitosus (Phil.) Reiche. Preliminary mapping of published palynological data on our phylogenetic trees revealed that the “Gamocarpha” pollen type occurs in both major clades: in Boopis anthemoides Juss. (Palazzesi & al., 2010: pl. II) and Moschopis sp. (DeVore & al., 2007) in one major clade, and in Gamocarpha and Nastanthus in the other major clade (Hansen, 1992; DeVore & al., 2007). Additionally, B. anthemoides, species of Nastanthus, Gamocarpha and Moschopis all are currently distributed in Chubut, in Patagónica province, where the fossil was discovered. Consequently, we used Psilatricolporites protrudens to calibrate the minimum crown group age of the entire Calyceraceae. The second calibration point is another fossil pollen grain, Quilembaypollis gamerroi Palazzesi & Barreda, assigned to subfamily Barnadesioideae, Asteraceae, and dated to 23 Ma. Exponential prior distributions with hard minimum bounds, appropriate for fossil calibrations (Pirie & Doyle, 2012), were used in both cases. Parameters were set as follows: offset of 22 Myr for Psilatricolporites protrudens, offset of 23 Myr for Quilembaypollis gamerroi, and means of 3 for both calibrations to allow for some probability of an earlier divergence than the appearance of the fossils. Three independent MCMC runs were performed at the CIPRES Science Gateway (Miller & al., 2010); each chain ran for 70 million generations and trees and parameters were sampled every 7000 generations. Convergence of each MCMC analysis was assessed using the program Tracer v.1.6 (available at http://beast.bio.ed.ac.uk/), and ESS >> 200 obtained with the 70 millions generations were considered optimal for convergence and stationarity. All sampled trees were combined using LogCombiner v2.4.1 where the first 50% of the trees in each file was discarded. A maximum clade credibility tree was constructed in TreeAnnotator v.2.4.1. This tree and its annotations were visualized in the program FigTree v.1.4.2 .

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of the most parsimonious trees from the plastid and nuclear partitions were compared. Summary representations of the strict consensus of most parsimonious trees obtained from the separate nuclear and plastid matrices are shown in Fig. 2 (and Electr. Suppl.: Figs. S1, S2). The position of Boopis gracilis Phil. differs in the two topologies but involves branches without Jackknife support in either tree (Fig. 2). Calycera sessiliflora Phil. is placed in a polytomy by ITS data, but the species is included in a weakly supported group (Jackknife