molecular phylogenetics of alternanthera (gomphrenoideae

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2012

Molecular Phylogenetics of Alternanthera(Gomphrenoideae, Amaranthaceae): Resolving aComplex Taxonomic History Caused by DifferentInterpretations of Morphological Characters in aLineage with C4 and C3-C4 Intermediate SpeciesIvonne Sánchez-Del Pino

Timothy J. MotleyOld Dominion University

Thomas Borsch

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Repository CitationSánchez-Del Pino, Ivonne; Motley, Timothy J.; and Borsch, Thomas, "Molecular Phylogenetics of Alternanthera (Gomphrenoideae,Amaranthaceae): Resolving a Complex Taxonomic History Caused by Different Interpretations of Morphological Characters in aLineage with C4 and C3-C4 Intermediate Species" (2012). Biological Sciences Faculty Publications. 267.https://digitalcommons.odu.edu/biology_fac_pubs/267

Original Publication CitationSanchez-Del Pino, I., Motley, T. J., & Borsch, T. (2012). Molecular phylogenetics of Alternanthera (gomphrenoideae, amaranthaceae):Resolving a complex taxonomic history caused by different interpretations of morphological characters in a lineage with C4 and C3-C4intermediate species. Botanical Journal of the Linnean Society, 169(3), 493-517. doi:10.1111/j.1095-8339.2012.01248.x

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Molecular phylogenetics of Alternanthera(Gomphrenoideae, Amaranthaceae): resolving a complextaxonomic history caused by different interpretations ofmorphological characters in a lineage with C4 and C3–C4

intermediate species

IVONNE SÁNCHEZ-DEL PINO1*, TIMOTHY J. MOTLEY2 and THOMAS BORSCH3

1The Lewis B. and Dorothy Cullman Program for Molecular Systematics Studies, The New YorkBotanical Garden, 200th St. and Southern Blvd., Bronx, NY 10458-5126, USA2Old Dominion University, Department of Biological Sciences, 110 Mills Godwin Building/45th St.,Norfolk, VA 23529-0266, USA3Botanischer Garten und Botanisches Museum Berlin-Dahlem und Institut für Biologie, DahlemCentre of Plant Sciences, Freie Universität Berlin, Königin Luise-Straße 6-8, 14195 Berlin, Germany

Received 27 September 2011; revised 1 February 2012; accepted for publication 15 February 2012

Alternanthera (Amaranthaceae) is a diverse genus (80–200 species) largely restricted to the American Tropics. WithPedersenia and Tidestromia, it makes up the ‘Alternantheroid clade’ in Gomphrenoideae. Parsimony and Bayesiananalyses of nucleotide sequences of nuclear (ITS) and plastid (rpl16, trnL-F) and morphological characters identifythat the capitate stigma of Alternanthera is a synapomorpy within the Alternantheroids. Within Alternanthera, twomajor clades were resolved, both of which were marked by otherwise homoplasious characters of the gynoecium:Clade A [99% jackknife (JK); 1.0 posterior probability (PP)] with nine species and Clade B (60% JK; 0.98 PP) with22 species. Four subclades (B1–B4), strongly supported statistically, were identified in Clade B. Previous subge-neric classifications of Alternanthera appear artificial in light of our new molecular phylogenetic analyses. Mostmajor lineages are congruently resolved by nuclear and plastid data but some incongruence between the nrITS andplastid phylogenetic trees suggests hybridization may have played a role in the rampant speciation in Alternan-thera. Whereas C4 photosynthesis appears to have evolved in a single clade, the position of A. littoralis var.maritima (C3) in this clade may be explained by hybrid speciation rather than a reversal from C4 to C3. All C3–C4

intermediates belong to a different clade that also contains C3 species, but species limits, including the widelystudied A. tenella, are unclear. The clade including A. tenella and A. halimifolia contains most of the speciesendemic to the Galápagos whereas A. nesiotes, also endemic to the islands, is nested among widespread Americantaxa. This suggests that the Galápagos radiation of Alternanthera may have arisen from at least two independentcolonization events followed by a subsequent radiation in the former lineage. © 2012 The Linnean Society ofLondon, Botanical Journal of the Linnean Society, 2012, 169, 493–517.

ADDITIONAL KEYWORDS: Caryophyllales – classification systems – Galápagos – gene trees – Neotropics– photosynthetic pathways – reconstructing character evolution – reticulate evolution.

INTRODUCTION

Alternanthera Forssk. is the second largest genus insubfamily Gomphrenoideae of Amaranthaceae (Elias-son, 1990; Townsend, 1993). The highest diversity isfound in South America (Mears, 1977), but many

*Corresponding author. Current address: Centro deInvestigación Científica de Yucatán, A. C. Calle 43 no. 130Col. Chuburná de Hidalgo, CP. 97200, Mérida, Yucatán,México. E-mail: [email protected]

Botanical Journal of the Linnean Society, 2012, 169, 493–517. With 3 figures

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species also occur in the Caribbean, Central Americaand Mexico. About 20 new taxa were rather recentlydescribed from Argentina, Paraguay and Brazil (Ped-ersen, 1997, 2000). Estimates of species numbers indifferent treatments and by different authors varyfrom 80 (Mears, 1977) to over 100 (Townsend, 1993;Borsch, 2001) or 200 (Robertson, 1981; Eliasson,1987, 1990; Siqueira, 2004). These considerably devi-ating numbers are largely the result of differingpoints of view on species limits in alpha-taxonomictreatments and indicate the need for more thoroughstudies including molecular approaches in Alternan-thera. Thirteen indigenous Alternanthera spp. occuron the Galápagos Islands (nine endemic) making itone of the most species-rich genera on the archipelago(Eliasson, 1988, 2004). Several species are native tothe Old World, occurring in Africa, Asia and Australia(Robertson, 1981), whereas a few others were intro-duced from the New World and have now becomeinvasive weeds (e.g. A. caracasana Kunth, A. parony-chioides A.St-Hil., A pungens Kunth and A. sessilis(L.) DC.; Robertson, 1981; Eliasson, 1987). Economi-cally, A. bettzichiana (Regel) Voss is commonly usedas an ornamental for its colourful foliage (Robertson,1981; Eliasson, 1987); A. tenella Colla is reportedlyused in Brazil as an anti-inflammatory remedy(Guerra et al., 2003); and A. repens (L.) Link(= A. pungens) is used in Mexico to treat gastrointes-tinal infections because of its tested antiprotozoalactivity (Tapia-Pérez et al., 2003).

Alternanthera spp. are annual or perennial herbs,shrubs, small trees or rarely vines (Robertson, 1981;Borsch, 2001). They are characterized by subgloboseto short-cylindrical inflorescences with dense solitaryflowers, the stamens basally united in a cup andalternating with long, laciniate, small triangular orrarely obsolete pseudostaminodia and capitatestigmas (Eliasson, 1987; Townsend, 1993).

Alternanthera has long been of interest to physiolo-gists because of the occurrence of C3–C4 intermediateand C4 species (Devi, Rajagopalan & Raghavendra,1995; Chinthapalli et al., 2000; Gowik et al., 2006;Sage et al., 2007). Whereas Sage et al. (2007) providedd13C carbon isotope values for a large number ofAlternanthera spp., they included only three out of atleast 17 C4 species in their reconstruction of theevolution of photosynthetic pathways in Amaran-thaceae. It was evident that C4 photosynthesis isderived within Alternanthera, but so far there are nohypotheses on the origin of A. tenella, which as aC3–C4 intermediate is comparatively studied with theC4 A. pungens (e.g. Gowik et al., 2006).

Apart from considerable morphological variationamong populations of many species and high pheno-typic plasticity that has led to the description of manyinfraspecific taxa such as forms, varieties and sub-

species in Alternanthera (e.g. Moquin-Tandon, 1849;Pedersen, 1967, 1990), generic concepts have shiftedconsiderably in treatments published during the pasttwo centuries. This was due to the fact that differentauthors gave different emphasis to individual mor-phological characters and treated them as diagnosticwithout insight into possible homoplasy. As a result,there is an enormous number of names that mayexceed our conservative estimates of species diversityby three or four times, and complicates the study ofAlternanthera. Mears (1977) attempted to clarify typi-fication issues and proposed various lectotypes of ninewidespread species of Alternanthera, but his plannedrevision of the genus was never carried out, and themost comprehensive taxonomic treatment of Alter-nanthera is that of Moquin-Tandon (1849).

The generic concept employed by Moquin-Tandon(1849) was rather broad, including Pedersenia Holub(= Trommsdorffia Mart.), whereas Martius (1826) andEndlicher (1836–1840) recognized individual parts ofAlternanthera as distinct genera. Alternanthera ascurrently widely accepted goes back to Schinz (1893),later adopted by Townsend (1993) who circumscribedthe genus like Moquin-Tandon (1849) but excludedPedersenia (for an overview of classification systemssee Table 1).

The phylogenetic position of Alternanthera in Gom-phrenoideae has been demonstrated in recent phylo-genetic studies of Amaranthaceae (Müller & Borsch,2005; Sánchez-del Pino, Borsch & Motley, 2009).Thus, far the best sampling included only 13 Alter-nanthera spp. (Sánchez-del Pino et al., 2009) usingtrnL-F and rpl16 sequence data. They inferred aplastid tree for Gomphrenoideae and provided strongevidence [93% jackknife (JK); 1.0 posterior probablity(PP)] for an ‘Alternantheroid clade’ that includesAlternanthera (99% JK; 1.0 PP) as sister to a cladecomprising Pedersenia (= Trommsdorffia) and Tide-stromia Standl. (= Cladothrix Nutt. ex Moq.; Sánchez-del Pino et al., 2009). A matK/trnK analysis focusingon Pedersenia also indicated the monophyly of Alter-nanthera but did not resolve an ‘Alternantheroidclade’, indicating a position of Pedersenia sister to alineage formed by Pfaffia Mart. and relatives plusGomphrena L. and relatives, all together in a poly-tomy with Alternanthera and Tidestromia (Borsch,Ortuño & Nee, 2011).

The goal of the present study is to provide a firstinsight into phylogenetic relationships in the genusAlternanthera and to provide a comprehensive over-view on the complex history of classification as a basisfor future taxonomic treatments reflecting naturalentities. To test for reticulate patterns in evolution ofspecies diversity, trees from the nuclear ribosomalinternal transcribed spacer (nrITS) region were com-pared with trees inferred from data sets of the highly

494 I. SÁNCHEZ-DEL PINO ET AL.

© 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 169, 493–517


Tab

le1.

Trad

itio

nal

clas

sifi

cati

ons

prop

osed

for

the

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us

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ern

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era

and

rela

ted

gen

era

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tiu

s(1

826)

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dlic

her

(183

6–18

40)

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uin

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don

(184

9)B

enth

am&

Hoo

ker

(188

0)S

chin

z(1

893)

Sch

inz

(193

4)To

wn

sen

d(1

993)

Alt

ern

anth

era

Alt

ern

anth

era

Alt

ern

anth

era

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ern

anth

era

Alt

ern

anth

era

Alt

ern

anth

era

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anth

era

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bgen

us

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ther

aS

ecti

onTr

omm

sdor

ffia

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ine*

Ped

erse

nia

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esin

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assi

era

Sec

tion

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sier

aS

ecti

onA

llag

anth

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Sec

tion

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agan

ther

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ecti

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anth

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Sec

tion

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ith

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rix

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nd

esia

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MOLECULAR PHYLOGENETICS OF ALTERNANTHERA 495



performing non-coding plastid regions trnL-F andrpl16. Molecular phylogenetic hypotheses were thenused to reconstruct the evolution of selected morpho-logical characters considered diagnostic in previousclassification systems and to obtain first insight intothe evolution of photosynthetic pathways and bio-geography in Alternanthera.

MATERIAL AND METHODSTAXON SAMPLING

The ingroup contains 33 samples representing a totalof 31 out of the c. 80–200 described Alternantheraspp. (Eliasson, 1987; Townsend, 1993; Appendix 1).The sampling approach was designed to include allpreviously described sections (Table 1) and was alsoguided by the comprehensive taxonomic treatment ofMoquin-Tandon (1849) to cover morphological diver-sity. To account for possible reticulate speciation andincomplete lineage sorting in a group with unreliablespecies classification, the same individuals were usedfor each data set, with the exceptions of Tidestromiacarnosa (Steyerm.) I.M.Johnst. and T. lanuginosa(Nutt.) Standl., both outgroup taxa for which twoindividuals each were used (Appendix 1). Vouchersare deposited at B, GB, MEXU and NY (Appendix 1).

Outgroup taxa included Pedersenia cardenasii(Standl.) Holub, Pedersenia cf. hassleriana (Mart.)Holub, Tidestromia carnosa, T. lanuginosa and T. val-desiana Sanch. Pino & Flores Olv., and were selectedbased on recent molecular analyses of trnL-F andrpl16 data for Gomphrenoideae (Sánchez-del Pinoet al., 2009). Parsimony reconstructions that requireda single taxon as the outgroup (and placing of theroot) used T. valdesiana for this purpose.

MORPHOLOGY

The data matrix consisted of 11 characters. All char-acters were treated as unordered (non-additive) andequally weighted. Morphological characters andstates are given in Appendices 2 and 3 and wereoptimized using Winclada.

C4 AND C3–C4 PHOTOSYNTHESIS

To obtain insight into the phylogenetic distribution ofcurrently known types of photosynthetic pathways inAlternanthera, data were taken from publishedworks. The major source for isotope and, with somelimitations, anatomical data was Sage et al. (2007)who examined a large number of Alternanthera spp.Two taxa [A. tenella and A. ficoidea (L.) P.Beauv., butsee discussion below] were considered as C3–C4 inter-mediate species based on Devi et al. (1995). The threedifferent kinds of photosynthetic pathways were

coded as unordered states and optimized using Win-clada on the respective gene trees (the plastid DNAtree is presented here in Fig. 1) to estimate thenumber and position of C4 and intermediate lineagesin Alternanthera.

DNA EXTRACTION, AMPLIFICATION AND SEQUENCING

Total genomic DNA was isolated from leaf tissue,dried in silica gel or taken from herbarium specimens.DNA extraction followed the Qiagen Plant DNeasy(Qiagen Inc.) manufacturer’s protocol and FastPrepTM method (Qbiogen Inc.) or used a modifiedCTAB protocol for silica-dried samples (Borsch et al.,2003). DNA extraction from herbarium materialincluded 30 mL of b-mercaptoethanol and 30 mL ofhighly purified proteinase K solution (Roche) added tothe recommended 400 mL of AP1 lysis buffer withconstant mixing and incubation at 42 °C for 12–24 hbased on the methods of Motley, Wurdack & Delprete(2005).

The polymerase chain reaction (PCR) was per-formed with 25-mL reactions containing 1 ¥ Taq bufferwith 1.5 mM MgCl2, 1 mM dNTP mix (2.5 mM each),0.4 mM of each primer, 1 U Taq DNA polymerase(Qiagen) and 1 mL of DNA template. To improveamplification, bovine serum albumin (0.25 mg mL-1),dimthyl sulphoxide (10%) or betaine (1 M) wereadded. Ex TaqTM DNA polymerase (hot-start version;Takara Mirus Bio) was used to amplify difficultsamples. Amplification and cycle sequencing reactionswere run on a Gene Amp PCR system 9600 (AppliedBiosystems). Double-stranded DNA templates wereamplified for two plastid regions (rpl16, trnL-F) andnuclear ITS. All PCR and cycle sequencing reactionswere run on a Gene Amp PCR system 9600 (AppliedBiosystems). Amplification and sequencing of thetrnL-F region was carried out using primers c and f ofTaberlet et al. (1991), sometimes complemented withthe internal universal forward sequencing primertrnL460F (Worberg et al., 2007) to produce reads ofthe trnL-F spacer. The rpl16 intron was amplifiedusing primers designed by Asmussen (1999) andanother primer based on the reverse complement ofrp116-584R (5′-TTCATTGGGTGGGAGGCGGAA-3′)was designed at NYBG. Two primers, forward (5′-CCTTATCATTAGAGGAAGGAG-3′) and reverse (5′-ATGCTTAAAYTCAGCGGGT-3′; modified from Whiteet al., 1990; Baldwin et al., 1995), were used toamplify the ITS region. The PCR conditions for ampli-fications of the trnL-F region were: one cycle at 97 °Cfor 2 min; 30 cycles each at 94 °C for 1 min, 48 °C for2 min and 72 °C for 2 min; and one cycle at 72 °C for16 min, hold at 4 °C. PCR conditions for amplifica-tions of the rpl16 intron were: one cycle at 94 °C for3 min; 30 cycles each at 93 °C for 1 min, 55 °C for




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1 min and 72 °C for 1.5 min; and one cycle at 72 °C for5 min, hold at 4 °C. The temperature profiles for ITSamplifications were: one cycle at 97 °C for 50 s, 30cycles each at 97 °C for 50 s, 53 °C for 50 s and 72 °Cfor 1 min 50 s, and a final extension at 72 °C for7 min.

Amplified products were purified with the QIAquickPCR Purification Kit (Qiagen Inc.). Purified productswere cycle sequenced with dye terminator ABI PrismReady reaction mix v.3.1 (Applied Biosystems).Sequences were electrophoresed on an ABI 377XLDNA automated sequencer.

SEQUENCE ALIGNMENT AND INDEL CODING

Sequences were edited in Sequencher version 4.1 forPC (Gene Codes). Edited sequences were automati-cally pre-aligned with Clustal X v. 2.0.12 (Larkinet al., 2007) using default settings. The alignmentswere then adjusted by eye using the BioEditSequence Alignment Editor v 7.0.9 (Hall, 1999) andPhyDe version 0.9971 (Müller et al., 2010). The align-ment method followed the criteria for homologyassessment suggested by Borsch et al. (2003). Thesecriteria account for microstructural changes involvingfrom one to many nucleotides in a single mutationalstep. Hotspots (Borsch et al., 2003) or regions withuncertain primary homology were excluded in phylo-genetic analyses (the number of regions and totalnumber of positions excluded in the analyses areindicated below and in Table 3). Substitutions withinrepeats were coded with ambiguity codes. Gaps werecoded as binary characters using the ‘simple gapcoding’ method (Simmons & Ochoterena, 2000). Theprogram SeqState version 1.4.1 (Müller, 2005) wasemployed to score indels automatically.

PHYLOGENETIC ANALYSIS

Four data sets were prepared for the phylogeneticanalyses and the number of aligned positions andparsimonious informative characters are given inTable 2. Specimen details for the data sets of trnL-F,rpl16 (each 38 terminals) and ITS (37 terminals) arelisted in Appendix 1. Data sets were analysed inde-pendently for each of the three genomic partitionsand simultaneous analyses were conducted for thecombined data sets of plastid regions.

PARSIMONY ANALYSIS

Constant invariable characters were deactivated.Heuristic parsimony analyses were conducted usingNona (Goloboff, 1993) spawned by Winclada (Nixon,1999). TBR branch swapping on Wagner trees wereconducted from 10 000 random taxon addition

sequences with 10 trees held in memory for each ofthe replicate initiations expanding the memory to100 000 for further TBR (h 100 000; mult* 10 000;ho/10).

JK branching support was calculated by Nonausing Winclada with 10 000 replications with 100search replications and 10 trees held in memory withthe next parameters (mult*100; ho/10; max*). In thispaper JK values are described as high (85–100%),moderate (75–84%) or low (� 74%).

BAYESIAN ANALYSIS

Bayesian analysis was conducted with the programMrBayes v3.1.2 (Ronquist & Huelsenbeck, 2003).Modeltest 3.7 (Posada & Crandall, 1998) was used toselect the best-fit model of nucleotide substitution forthe present data based on the Akaike InformationCriterion (AIC; Akaike, 1974) and analyses were per-formed for five structurally and functionally differentgenomic regions. Data were partitioned into the rpl16intron (group II), rpl16 exon, trnL intron (group I),trnL-F spacer and ITS with an individual modelassigned to each partition. The TVM+G model ofsubstitution was selected for the rpl16 intron andtrnL-F spacer data sets, TIM+G for the rpl16 exon,K81uf+I+G for the trnL intron and the SYM+I+Gmodel for the ITS partition. For the simultaneousanalysis of plastid markers, MrBayes was run for5000 000 generations. PP distributions of trees andbranch lengths were obtained using the MarkovChain Monte Carlo (MCMC) method (Metropoliset al., 1953; Hastings, 1970) and transferred on theresulting phylogenetic trees. Four chains were runwith a temperature setting of 0.2. Chains weresampled every 100 generations after burn in, whichwas set at 5000 generations when PP reached stablelog likelihood values of the model and tree. Startingtrees for two independent runs were randomlyselected. A majority rule consensus tree was thenobtained using all saved trees.

CONGRUENCE OF DATA SETS

The Incongruence Length Difference (ILD) test(Farris et al., 1995) assesses character conflictbetween data sets and calculates the significance ofthat conflict. The null hypothesis is that conflictbetween data sets is no greater than conflict amongrandom partitions of the combined data set. The useof various data types provides phylogenetic informa-tion that can either converge towards the same phy-logenetic tree or show discrepancies leading toconflicting conclusions (Darlu & Lecointre, 2002).Darlu & Lecointre (2002), Lee (2001) and van derNiet & Linder (2008) suggested that the ILD test is




Tab

le2.

Seq

uen

ceva

riat

ion

amon

gth

etr

nL

-F,

rpl1

6an

dIT

Sre

gion

s

Mol

ecu

lar

mar

ker

Nc

Len

gth

ran

geM

ean

len

gth

SD

%di

v.(r

ange

)ts

/tv

rati

o%

var.

%in

f.(c

har

.in

f.)

%G

CIn

.in

f.in

.

trn

L-F

(tot

al)

1202

626–

1059

880.

053

77.7

233.

014

(0.0

00–5

.288

)0.

715

(0.0

00–5

.000

)14

.143

8.07

31.0

64tr

nL

-in

tron

754

391–

655

623.

421

49.8

733.

08(0

.000

–5.4

24)

0.54

6(0

.000

–3.0

00)

16.4

468.

886

27.7

31tr

nL

-spa

cer

398

162–

371

206.

711

60.5

763.

323

(0.0

00–8

.889

)1.

979

(0.0

00–8

.000

)11

.307

7.28

637

.7tr

nL

-F(e

xclu

din

gh

otsp

ots)

1084

582–

964

809.

079

72.9

242.

969

(0.0

00–5

.007

)0.

729

(0.0

00–4

.000

)13

.653

8.02

632

.929

6631

trn

L-i

ntr

on66

335

3–59

856

4.31

645

.475

2.99

6(0

.000

–5.3

51)

0.55

2(0

.000

–2.0

00)

15.6

868.

597

(57)

29.4

1146

22tr

nL

-spa

cer

370

149–

348

194.

842

58.4

093.

421

(0.0

00–8

.876

)1.

951

(0.0

00–8

.000

)11

.622

7.83

8(3

0)39

.972

189

rpl1

6(t

otal

)13

9894

3–11

701,

120.

9038

.471

3.82

(0.0

00–7

.192

)0.

727

(0.0

00–3

.500

)20

.529

12.3

0329

.99

rpl1

6-in

tron

1258

803–

1030

981.

579

37.3

453.

984

(0.0

00–7

.200

)0.

66(0

.000

–3.0

00)

21.0

6512

.719

28.5

1rp

l16-

exon

140

115–

140

138.

316

3.86

72.

748

(0.0

00–7

.914

)1.

567

(0.0

00–5

.000

)15

.714

8.57

1(1

2)39

.96

10

rpl1

6(e

xclu

din

gh

otsp

ots)

1238

894–

1085

1,04

5.08

34.7

263.

492

(0.0

00–7

.003

)0.

788

(0.0

00–5

.000

)16

.64

9.93

531

.47

9544

rpl1

6-in

tron

1097

754–

945

905.

763

33.5

053.

613

(0.0

00–6

.980

)0.

708

(0.0

00–5

.000

)16

.773

10.1

19(1

11)

30.0

994

44IT

S(t

otal

)64

558

4–61

159

6.43

24.

979

15.3

61(0

.000

–27.

087)

1.10

2(0

.000

–4.5

00)

48.6

8238

.605

55.6

94IT

S1

251

213–

231

225.

568

2.65

621

.245

(0.0

00–3

6.69

7)1.

159

(0.0

00–4

.000

)64

.542

52.5

954

.679

5.8S

163

162–

163

162.

973

0.16

21.

826

(0.0

00–4

.908

)2.

224

(0.0

00–6

.000

)9.

816

5.52

1(9

)54

.909

10

ITS

223

219

6–21

920

7.89

23.

5419

.767

(0.0

00–3

7.07

3)1.

02(0

.000

–11.

000)

58.6

2146

.121

57.3

97IT

S(e

xclu

din

gh

otsp

ots)

624

566–

595

579.

703

4.94

814

.911

(0.0

00–2

6.74

8)1.

143

(0.0

00–4

.200

)47

.596

37.5

55.7

3564

26

ITS

123

920

2–22

321

6.18

92.

958

21.4

01(0

.000

–37.

143)

1.19

8(0

.000

–5.0

00)

64.4

3552

.72

(128

)54

.163

3519

ITS

222

218

9–21

120

0.54

13.

4318

.74

(0.0

00–3

6.18

1)1.

076

(0.0

00–1

0.00

0)57

.207

44.5

95(9

9)58

.128

7

Nc,

nu

mbe

rof

char

acte

rs;S

D,s

tan

dard

devi

atio

nof

mea

nle

ngt

h;%

div.

(ran

ge),

perc

enta

geof

pair

wis

ese

quen

cedi

stan

ce(u

nco

rrec

ted

pdi

stan

ce,o

vera

llm

ean

s,lo

wes

tan

dh

igh

est

valu

esin

pare

nth

eses

);ts

/tv

rati

o,ra

tio

oftr

ansi

tion

sto

tran

sver

sion

s;%

var.

,pe

rcen

tage

ofva

riab

lepo

siti

ons;

%in

f.(c

har

.in

f.),

perc

enta

geof

pars

imon

y-in

form

ativ

epo

siti

ons

(nu

mbe

rof

pote

nti

ally

pars

imon

y-in

form

ativ

ech

arac

ters

inpa

ren

thes

es);

%G

C,

GC

con

ten

t;In

.,n

um

ber

ofin

dels

;in

f.in

.,n

um

ber

ofin

form

ativ

ein

dels

.




still suitable as an explanatory method to detectsignificant incongruence and it produces more accu-rate results than other tests.

In this study, congruence among data sets wasevaluated observing topological congruence (Figs 1,3). In addition, quantitative congruence among datasets was tested with the ILD test. The incongruencetest value was calculated using Nona in Winclada.One thousand replications were executed with 10searches per replication holding 10 trees for eachsearch and holding 100 trees with the next param-eters (100 replications, 10 mult per rep; holding 10trees per mult; hold 100 trees for ‘hold*’). Uninforma-tive characters were removed from the analysesbefore running the ILD test (Lee, 2001).

RESULTSSEQUENCE VARIABILITY OF THE TRNL-F REGION

Statistical values of the sequences included in themolecular matrices are summarized in Table 2. Thetotal length of the trnL-F sequence comprised 1202 bppositions. The trnL-F spacer was more variable inrange (162–371 bp), in variable sites (45 characters,11.3%), in transitions/transversions (ts/tv) ratio andin GC percentage than the trnL intron (Table 2).However, the trnL intron has 40 more potentiallyphylogenetically informative characters, 57 nucle-otides (46% of the proportion of the variable sites) and22 indels (70% of the total number of indels). Thesevalues may be affected by a large indel (ranging from169 to 178 bp) in the trnL-F spacer, which was pre-viously proposed as a potential synapomorphic char-acter for Alternanthera (Sánchez-del Pino et al.,2009). Six hotspot regions in the trnL intron and twoin the trnL-F spacer were identified (Table 3). Exclud-ing the 5′trnL exon and hotspots, the aligned matrix

length of the trnL-F sequence included a total of1084 bp positions.

SEQUENCE VARIABILITY OF THE RPL16 REGION

The total length of rpl16 comprised 1398 charactersand the rpl16 intron included 155 potentiallyparsimony-informative sites (Table 2). Of these, 111are point mutations (41.8% of the proportion of thevariable sites) and 44 are indels (100%), whereas therpl16 exon contained only 12 bp that were potentiallyparsimony-informative characters (54.5% of the pro-portion of the variable sites). Six hotspots were foundin the rpl16 intron (Table 3). The aligned length of therpl16 comprised a total of 1238 bp positions (exclud-ing hotspots).

SEQUENCE VARIABILITY OF THE ITS REGION

The total length of the ITS sequence (ITS1+5.8S+ITS2) is 645 bp (Table 2). From two species (A. fla-vicoma, A. pubiflora) no clean sequences could beobtained from herbarium material, so they could notbe included in the analysis. Compared with ITS2,the ITS1 spacer had a higher ts/tv ratio, a higherpercentage of variable sites and possessed 41 morepotentially parsimony-informative characters. TheITS1 ranged from 213 to 231 bp, of which 163 bpwere variable (65%), and 128 nucleotides (78.5% ofthe proportion of the variable sites) and 19 indelswere identified as potentially parsimony-informativecharacters (Table 2). The ITS2 region ranged from196 to 219 bp, of which 137 were variable (59%) and99 nucleotides (72.2% of the proportion of the vari-able sites) and seven indels were found to be poten-tially phylogenetically informative. Low divergencewas found in the 5.8S gene with only nine poten-tially parsimony-informative characters (represent-

Table 3. Positions of hotspots and exons in trnL-F, rpl16 and ITS regions

trnL-F region rpl16 region ITS

trnL 5′ exon 1–12 rpl16 intron ITS 1trnL intronH1. 73–79 poly A H1. 166–191 poly T and G H1. 54–65H2. 122–128 poly A H2. 231–272 poly AH3. 157–174 poly A H3. 307–323 poly AH4. 343–346 poly A H4. 751–770 poly TH5. 399–416 poly A H5. 880–893 poly AH6. 509–533 poly A and T H6. 1086–1127 poly A and TtrnL 3′ exon 755–804 ITS2

H2. 436–445H7. 840–854 poly TH8. 1079–1091 poly T




ing 55.2% of the variable sites). One hotspot regionwas observed in each of the ITS1 and ITS2 spacers,respectively. The total aligned length of ITS included624 positions (excluding hotspots; Table 3). Aboutone-third of the ITS sequences showed polymorphicsites that hint to introgression or hybridization(divergent paralogues; Table 4). This was by farstrongest in the sequence of A. kurtzii (AC617) with11 polymorphic sites.

TREES OBTAINED FROM PLASTID TRNL-F AND

RPL16 SEQUENCES

Parsimony analysis of the combined plastid trnL-Fand rpl16 data yielded a single most-parsimonioustree (MPT) with a length of 476 steps (CI = 0.70,RI = 0.89; Fig. 1). Both maximum-parsimony (MP)and Bayesian analyses of the plastid DNA data setresolved a strongly supported monophyletic Alternan-thera (99% JK, 1.0 PP) and revealed two major cladesin Alternanthera.

Clade A (Node 6 in Figs 1, 2). Apart from A. macbri-dei Standl., this highly supported (99% JK, 1.0 PP;Fig. 1) clade includes species with long, simple orcompound pedunculate inflorescences. All nine specieshave globose stigmas with distinctive carpel demarca-tions [except for A. lanceolata (Benth.) Schinz; charac-ter 9(1); Appendix 2]. The species forming Clade A arepredominantly distributed in South America withseveral extending to Central America [A. brasiliana(L.) Kuntze, A. pubiflora Kuntze] and Florida (A. fla-vescens Kunth; Figs 1, 2).

Clade B (Node 1 in Figs 1, 2). This clade includesspecies that have entire stigmas without distinctivecarpel demarcations [character 9(1); Appendix 2],except for A. crucis Bold. and A. pungens. This clade

is weakly supported in both plastid (Fig. 1; 60% JK,0.98 PP) and includes four well-supported subclades(nodes 2–5). Most of the species included in this clade[subclades B2–B4 (57% JK, 0.94 PP); Fig. 1] havesessile, axillary inflorescences either solitary orgrouped into two- to five-flowered spikes. However,species with pedunculate inflorescences or bothsessile and pedunculate inflorescences also occur inthis clade [subclade B1 and A. philoxeroides (Mart.)Griseb.].

Within Clade B, there are four subclades (CladesB1–B4).

Clade B1 (Node 5; 99% JK, 1.0 PP) consists of fourspecies [A. geniculata Urb., A. laguroides (Standl.)Standl., A. olivacea Urb. and A. serpyllifolia Urb.;Figs 1, 2] which have an erect habit, flowers usuallyarranged along a rachis to form slender spicate inflo-rescences, and are predominately distributed inCentral America and the Caribbean islands. However,A. laguroides differs in having a more globose spicateinflorescence and a distribution restricted to CentralAmerica.

Clade B2 (Node 4; 100% JK, 1.0 PP) comprises twospecies (A. obovata Millsp. and A. philoxeroides;Figs 1, 2), which are procumbent herbs and possesssessile, globose or cylindrical inflorescences. Thesespecies also share a preference for aquatic environ-ments. The latter species is widespread, being nativein the New World and invasive in the Palaeotropics(Mears, 1977).

Clade B3 (Node 3; 97% JK, 1.0 PP) includes sevenspecies [A. caracasana, A. chacoënsis Morong exMorong & Britton, A. littoralis Beauv. ex Moq. var.maritima (Mart.) Pedersen, A. microphylla R.E.Fr.,A. nesiotes I.M.Johnst., A. paronychioides and A. pun-gens; Figs 1, 2] that are procumbent plants andmostly have sessile, globose or cylindrical inflores-cences similar to species in Clade B2. Two species,A. caracasana and A. pungens, are widespreadthroughout the Neotropics and invasive in the OldWorld. One variety of A. littoralis P.Beauv. occursalong the east coasts of tropical America and threevarieties in the west coast of tropical Africa (Peder-sen, 1990). Alternanthera microphylla is an endemicto the Prepuna of the Andes.

Clade B4 (Node 2; 99% JK, 1.0 PP) consists of ninespecies [A. crucis, A. filifolia (Hook.f.) J.T.Howell,A. flavicoma (Andersson) J.T.Howell, A. galapagensis(Stewart) J.T.Howell, A. halimifolia Standl. ex Pittier,A. kurtzii Schinz ex Pedersen, A. snodgrassii (B. L.Rob.) J.T.Howell, A. tenella and A. vestita (Andersson)J.T.Howell; Figs 1, 2). Except for A. tenella andA. crucis, which have a herbaceous habit, the speciesin Clade B4 are shrubby. Several of these taxa aredistributed in the Galápagos Islands and some areindigenous to Central America and South America.

Table 4. Taxa with polymorphic nucleotide sites andlength polymorphisms in ITS

Taxon ITS1 5.8S ITS2 NPST

A. altacruzensis 1 0 0 1A. brasiliana 3 0 1 4A. caracasana 0 0 1 1A. elongata 0 0 1 1A. filifolia 1 0 0 1A. flavescens 1 0 2 3A. galapagensis 0 1 0 1A. kurtzii 5 0 6 11A. laguroides 1 0 0 1A. littoralis var. maritima 0 1 0 1A. tenella_01 2 0 0 2A. tenella_02 1 0 2 3

NPST, total number of polymorphic sites.




MORPHOLOGICAL CHARACTER MAPPING

The single MPT resulting from the plastid DNA datashows character state transformations of 11 morpho-logical characters listed in Appendix 2. The morpho-logical characters resolved as either homologous orhomoplasious for this analysis are depicted inFigure 1.

DISTRIBUTION OF PHOTOSYNTHETIC PATHWAYS

The plesiomorphic condition is represented by C3 pho-tosynthesis. C4 photosynthesis occurs in one lineagerepresented by widespread species (Clade B3; Fig. 1),except for Alternanthera littoralis var. maritima(= A. maritima), which is C3. Two intermediate C3–C4

species, A. crucis and A. tenella, are found in CladeB4.

TREES OBTAINED FROM NUCLEAR ITS SEQUENCES

Parsimony analysis of the nuclear ITS data resultedin three MPTs of 788 steps in length (CI = 0.55,RI = 0.80). The strict consensus tree of the ITS data(L = 789, CI = 0.55, RI = 0.80; Fig. 3) shows a topologylargely consistent with that from the analysis ofplastid data albeit with lower support for majorclades. However, some individual species show differ-ent placement as compared with the plastid DNAtree. The ITS tree congruently reveals the two majorclades, Clades A (Node 6) and B (Node 1), andstrongly supports the monophyly of Alternanthera(99% JK). In general, the same four subclades (B1–B4; Nodes 2–5) are resolved (with the exception of theplacement of A. littoralis var. maritima).

A comparison between MP and Bayesian analyses(Fig. 3) of the ITS dataset indicates some topological

Alternanthera crucisA. halimifoliaA. vestita

A. snodgrassiiA. galapagensisA. flavicomaA. filifolia

A. tenella_02A. tenella_01A. kurtzii

A. chacoënsisA. paronychioides

A. nesiotes

A. caracasanaA. pungens

A. littoralis var. maritimaA. microphylla

A. obovataA. philoxeroides

A. olivaceaA. geniculata

A. serpyllifoliaA. laguroides

A. elongata_01A. elongata_02

A. pubiflora

A. porrigensA. lanceolataA. macbridei

A. flavaA. flavescens

A. brasilianaA. altacruzensis

Pedersenia cardenasiiP. cf. hassleriana

SA, Galápagos and Caribbean

Galápagos (endemics)

SA and Galápagos

CA to SA

NA to SA (widespread)

Galápagos (endemic)


SA


Caribbean

CA

CA to SA

Caribbean

Topology Taxa Distribution

SA

Tidestromia carnosa

T. lanuginosaT. valdesiana

0.01

B4

B3

B2

B1

B

A

1

2

4

5

6

7

3

Outgroup

8

9

Figure 2. Fifty per cent majority-rule tree from the Bayesian analysis of combined trnL-F and rpl16 data withdistributions of Alternanthera taxa. NA, North America; SA, South America; CA, Central America. The two major clades(A and B) and the four subclades of B (B1–B4) are also denoted on the tree.




III

0.1

0.5

4

0.9

8

0.6

6

0.9

9

0.7

6

1.0

0.8

2

1.0

1.0 1

.0

1.0 1

.0

A. obova

ta

A. philo

xero

ides

A. fla

vesc

ens

A. bra

silia

na

A. alta

cruze

nsi

s

A. fla

va

A. porr

igens

Peders

enia

card

enasi

i

P. cf

. hass

leriana

Tid

est

rom

ia c

arn

osa

T. la

nugin

osa

T. va

ldesi

ana

0.8

0

Alte

rnanth

era

gala

pagensi

sA

. fil

ifolia

A. ve

stita

A. sn

odgra

ssii

A. lit

tora

lis v

ar.

maritim

a

A. cr

uci

s

A. halim

ifolia

2

A. ku

rtzi

i

A. te

nella

_01

A. te

nella

_02

A. pungens_

01

A. pungens_

02

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

A. ca

raca

sana

A. m

icro

phyl

la

A. nesi

ote

s

A. paro

nyc

hio

ides

A. ch

aco

ënsi

s

A. oliv

ace

a

A. genic

ula

ta

A. se

rpyl

lifolia

A. la

guro

ides

A. elo

ngata

_02

A. la

nce

ola

ta

A. elo

ngata

_01

A. m

acb

ridei

8

95

4

0.8

5

7

31

0.8

9

0.8

1

1.0 1

.0

1.0

0.7

56

97

21

36 17

27

96

97

80

98

99

100

100

63

96

99

96

40

88

38

90

99

99 5

738

99

98

100

100

99

100

Alte

rnanth

era

fili

folia

A. gala

pagensi

s

A. sn

odgra

ssii

A. ve

stita

A. halim

ifolia

A. cr

uci

s

A. lit

tora

lis v

ar.

maritim

a

A. te

nella

_02

A. ku

rtzi

i

A. te

nella

_01

A. m

icro

phyl

la

A. ca

raca

sana

A. pungens_

02

A. nesi

ote

s

A. ch

aco

ënsi

s

A. paro

nyc

hio

ides

A. obova

ta

A. philo

xero

ides

A. la

guro

ides

A. se

rpyl

lifolia

A. genic

ula

ta

A. oliv

ace

a

A. fla

vesc

ens

A. bra

silia

na

A. alta

cruze

nsi

s

A. fla

va

A. m

acb

ridei

A. porr

igens

A. la

nce

ola

ta

A. elo

ngata

_01

A. elo

ngata

_02

Peders

enia

card

enasi

i

P. cf

. hass

leriana

Tid

est

rom

ia c

arn

osa

T. la

nugin

osa

T. va

ldesi

ana

A. pungens

_01

B4 B

3

B2

B

B1

A

61

2

7

3

4

5

1

8

9

6

100

Fig

ure

3.I,

the

stri

ctco

nse

nsu

str

ees

of3

MP

Ts(L

=78

9st

eps,

CI

=0.

55,

RI

=0.

80)

resu

ltin

gfr

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inconsistency in weakly supported parts of the tree.Thus, a clear nuclear-based hypothesis on the rela-tionships of the four subclades (B1–B4) and regardingspecies relationships within Clade A is not possibleusing ITS alone.

COMPARISONS BETWEEN PLASTID DNA AND ITS

DATA SETS

All analyses indicated the monophyly of the genusAlternanthera. However, there was an area of incon-sistency between the two genomic data sets. Theinconsistency mentioned above was the position ofA. littoralis var. maritima. The plastid DNA dataplaced A. littoralis var. maritima with six otherspecies in Clade B3 (Node 3, 97% JK; Fig. 1), whichincludes A. caracasana, A. chacoënsis, A. microphylla,A. nesiotes, A. paronychioides and A. pungens, but theITS data resolved A. littoralis var. maritima sister toA. crucis, in Clade B4 (Node 2, 96% JK; Fig. 3).

INCONGRUENCE TEST

Comparison of plastid DNA and ITS (Figs 1, 3) treessuggested some conflicts among the data partitionsfrom the nuclear and plastid genome compartments.The ILD test was conducted to evaluate if there wassignificant conflict in signal between plastid DNA andITS, and between plastid DNA and morphology data.Incongruency was significant (P = 0.0099), so a com-bined analysis of the three molecular data sets andmorphology (Appendix 2) was not performed in thisstudy.

DISCUSSIONPHYLOGENETIC RELATIONSHIPS IN ALTERNANTHERA

The use of nrITS, morphology and plastid DNA(trnL-F, rpl16) confirmed previous hypotheses that thegenus Alternanthera (100% JK) is monophyletic(Müller & Borsch, 2005; Sánchez-del Pino et al., 2009).Contrary to the widely accepted concept of Alternan-thera developed by Schinz (1893), previous classifica-tion systems recognizing several independent generasuch as Brandesia, Bucholzia, Mogiphanes or Telan-thera (Table 1) are thus not supported by phylogeneticdata. This study indicates that Alternanthera com-prises two major clades (Clades A and B), which arealso supported by gynoecium characters (Figs 1–3).

Clade A (Node 6; Figs 1–3): Most of the speciesincluded in this clade were historically classified in(genus or section) Brandesia, and a few in Bucholziaand Mogiphanes by several authors (Martius, 1826;Endlicher, 1836–1840; Moquin-Tandon, 1849; Schinz,1934). Among these species, some authors (Martius,

1826; Moquin-Tandon, 1849) placed A. flavescensKunth in (genus or section) Mogiphanes and A. bra-siliana was assigned in sections Bucholzia/Mogiphanes (Moquin-Tandon, 1849). These twospecies form a strongly supported clade (100% JK, 1.0PP; Fig. 1) and both have pedicellate flowers, ahomoplasious character [character 4(1); Appendix 2].Pedicellate flowers were a key character in the diag-nosis of the genus Mogiphanes as proposed byMartius (1826), and later adopted as a section byEndlicher (1836–1840) and Schinz (1934). However,the character occurs several times in the evolution ofAlternanthera such that it is not a synapomorphy andtherefore Mogiphanes is not a well-defined taxon.

The remaining species of Clade A are included in awell-supported subclade (Node 9; 100% JK, 1.0 PP) ofspecies placed in section Brandesia following Moquin-Tandon’s (1849) and Schinz’s (1934) systems. Thissubclade includes A. elongata (Willd.) Schinz, A. flava(L.) Mears, A. lanceolata, A. macbridei and A. porri-gens Kuntze supported by the homoplasious characterof long style [character 6(0); Appendix 2] with a rever-sal condition for A. pubiflora, which is supported bythe presence of short styles [character 6(1);Appendix 2].

Alternanthera altacruzensis Suess., formally placedin section Bucholzia by Suessenguth (1950), is sisterto the species belonging to sections Mogiphanes andBrandesia. It shares morphological characters withboth clades and differs mainly in its inflorescencetype. It has three long pedunculate heads originatingfrom a single axis, whereas all other speciesin the clade have more or less branched thyrsoidsynflorescences.

Clade B1 (Node 5; Figs 1–3): This clade (99% JK, 1.0PP; Fig. 1) includes four species. Two, A. serpyllifoliaand A. geniculata, were formerly assigned to Bran-desia by several authors (Martius, 1826; Endlicher,1836–1840; Moquin-Tandon, 1849; Moore, 1895;Schinz, 1934; Fig. 1). Alternanthera serpyllifolia,A. geniculata, A. olivacea and A. costaricensis (thelast-named species was not sampled here) wereplaced in a separate genus ‘Jamesbondia’ by A. J.Mears (unpubl. data). However, other than annotat-ing many herbarium specimens with this invalidname, his proposal was never published. Neverthe-less, molecular data resolved a strongly supported‘Jamesbondia’ clade (97% JK, 1.0 PP; Fig. 1), whichwas sister to A. laguroides. The ‘Jamesbondia’ taxaare suffrutescent, procumbent or prostrate perenni-als (Standley, 1917). Personal observations of her-barium material suggested that this group of speciesshare the presence of long-cylindrical inflorescences,which are different from globose inflorescences (thatcharacterize all the other Alternanthera spp.) in




which flowers emerge from a common point[homoplasious character 8(1); Appendix 2]. Amongthese ‘Jamesbondia’ spp., A. geniculata has stipitateflowers [homoplasious character 4(1); Appendix 2],whereas A. olivacea has sessile flowers. Both havelong styles, small stigmas and ligulate, laciniatepseudostaminodia. Their closely related sisterspecies, A. serpyllifolia, differs by having bracts,bracteoles, and tepals with thick midnerves, pistilswith short styles and flowers lacking pseudostami-nodia. Alternanthera laguroides is sister to thesethree species and shares with them the presence ofa long styled pistil and globose, long papillate glan-dulose stigmas.

Clade B2 (Node 4; Figs 1–3): A. philoxeroides andA. obovata, both formerly placed in section Bucholzia(Moquin-Tandon, 1849; Schinz, 1934; Fig. 1), form astrongly supported clade (100% JK, 1.0 PP; Fig. 1).They share the presence of glabrous tepals [homopla-sious character 1(0); Appendix 2] and are character-ized by a procumbent perennial habit, obovate leavesand mostly sessile, globose or cylindrical–globoseinflorescences. Alternanthera obovata further has twooverall homoplasious floral features, i.e. pedicellateflowers [character 4(1); Appendix 2] and crenate pseu-dostaminodia [character 7(1); Appendix 2]. Alternan-thera philoxeroides is an aquatic or subaquatic(Mears, 1977) perennial herb with ascending ordecumbent stems (Standley, 1937; Duke, 1961),whereas A. obovata grows in aquatic environments (I.Sánchez-del Pino, pers. observ.), and can be eitherprostrate or decumbent herbs (Standley, 1917).

Clade B3 (Node 3; Figs 1–3): The species in this cladehave been placed in sections Bucholzia and in Alla-ganthera (Martius, 1826; Endlicher, 1836–1840;Moquin-Tandon, 1849; Schinz, 1934; Fig. 1). The cladeincludes A. microphylla as sister to the rest of thespecies A. caracasana, A. chacoënsis, A. littoralis var.maritima, A. nesiotes, A. paronychioides and A. pun-gens based in the combined plastid DNA tree. Alter-nanthera littoralis var. maritima is a prostrateperennial with succulent, ovoid leaves and regularlyapically fimbriate pseudostaminodia (Mears, 1977)and occurs along the Atlantic coast of Africa andTropical America (Pedersen, 1990). Alternanthera lit-toralis var. maritima is characterized by two homopla-sious features: absence of trichomes on tepals[character 1(0); Appendix 2] and presence of pedicel-late flowers [character 4(1); Appendix 2]. In the plastidDNA tree it diverges second after A. microphylla inclade B3 (Fig. 1). Alternatively, in the nrITS tree(Fig. 3) A. littoralis var. maritima is nested in sub-clade B4 sister to A. crucis. Alternanthera littoralis

var. maritima may be of hybrid origin as will bediscussed in more detail below.

Alternanthera nesiotes is a subshrub (Jørgensen,1999) species from the Galápagos islands and isclosely related to A. chacoënsis and A. paronychioides,which are prostrate perennial herbs (Figs 1–3). Alter-nanthera caracasana and A. pungens are sisterspecies, which share many morphological character-istics. The close relationship between these specieswas noted by others (e.g. Standley, 1917; Eliasson,1987). Eliasson (1987) distinguished A. caracasanafrom A. pungens by its shorter tepals with almostnon-pungent tips and proportionally narrower leaves.After further evaluation of morphological characters,it was determined that the characters distinguishingthe species are the apex of the tepals and bracteolesand tepal and leaf size (Sánchez-del Pino, FloresOlvera & Valdés, 1999). Alternanthera pungens has alonger bracteole midrib and the tepals have longpungent tips, whereas A. caracasana has bracteolesand tepals with acute to apiculate apices. Alternan-thera caracasana and A. pungens (92% JK) groupedtogether in this study based on one homoplasiouscharacter: midrib of bracteoles not prominent [char-acter 3(0), Appendix 2]. Other sister species (99% JK,1.0 PP) in the subclade, A. chacoënsis and A. parony-chioides, have been variously treated by authors inthe past. Pedersen (1967) described six varieties ofA. paronychioides, including A. paronychioides var.chacoënsis. Mears (1977) treated A. chacoënsis as asynonym of A. paronychioides, but Pedersen (1990)retained A. chacoënsis as a species. To clarify specieslimits further, a geographically representative sam-pling of populations from both entities will be needed.

Clade B4 (Node 2; Figs 1–3): Most of the speciesincluded in this highly supported subclade (99% JK,1.0 PP; Fig. 1) were referred to Bucholzia by severalauthors (Martius, 1826; Endlicher, 1836–1840;Moquin-Tandon, 1849; Schinz, 1934; Fig. 1). Thesingle MPT from the plastid DNA data sets showsthat A. crucis, A. filifolia, A. flavicoma, A. halimifolia,A. snodgrassii and A. vestita were grouped together ina weakly supported clade (62% JK). Although Alter-nanthera filifolia, A. flavicoma and A. vestita sharemany morphological characters, such as inflorescencetypes and several floral characters, they can be iden-tified based on leaf shape. The latter has elliptic-lanceolate, oblanceolate or narrowly obovate leavesand the former two species have narrowly linearlanceolate leaves (Eliasson, 1971). Alternanthera fili-folia is a highly variable species with numerousinfraspecific taxa (Howell, 1933; Eliasson, 1971).Howell (1933) stated that A. filifolia might be apubescent variant of A. flavicoma and noted it wasnot easy to distinguish the two species. Along the




same lines, Mears’s annotations on herbarium speci-mens show that he considered A. flavicoma to be asubspecies or form of A. filifolia, but a nomenclaturalchange was never published. Later, Eliasson (1990)mentioned that some species closely related to A. fili-folia, such as A. flavicoma, represent branches of thesame evolutionary tree and could perhaps be accom-modated as subspecies of A. filifolia. The sisterspecies to this unresolved clade is A. snodgrassii,which shares their shrubby habit. Howell (1933) andEliasson (1971) suggested that A. snodgrassii isclosely related to A. vestita. These authors mentionedthat both species differ mainly in trichome type:A. snodgrassii has simple and A. vestita has stellatetrichomes. However, the type of trichomes is the samefor both species based on our observations.

Alternanthera halimifolia is a perennial with stemseither spreading and rooting at the nodes, or ascend-ing and forming bushes up to 1 m in height (Eliasson,1971), and is sister to the shrubby species endemic(A. filifolia, A. flavicoma and A. snodgrassii) andindigenous (A. vestita) to the Galápagos. Leaf size wasthought to be an important taxonomic character thatcharacterized A. halimifolia. Eliasson (1971) men-tioned that differences in leaf size seem to be in directresponse to the environment. Howell (1933), based onthis morphological variation, considered the Galápa-gos population to be a subspecies of the populationsfrom Pacific coast of South America. In the presentstudy, our results based on polytomy suggest that it ispossible to recognize only one species. However, theidentity of A. halimifolia is not entirely clear andtreatments vary from treating it as an endemic fromthe Lomas of Peru (Borsch, 1993) to a widespreadspecies in many parts of the Neotropics.

Alternanthera crucis, which is part of the clade ofthe five bushy species from the Galápagos, shares thecharacter leaf-type with A. halimifolia. In fact, Duke(1961) considered A. crucis as a synonym of A. hal-imifolia. However, in this study two homoplasiouscharacters distinct from A. halimifolia supportA. crucis: long styles [character 6(0); Appendix 2;Fig. 1] and stigmatic surface with distinctive carpeldemarcations [character 9(0); Appendix 2; Fig. 1].

Alternanthera galapagensis is the closely relatedsister species to A. crucis and the five bushy speciesfrom the Galápagos. Alternanthera galapagensis issupported by the homoplasious character pedicellateflower insertion [character 4(1); Fig. 1]. This species isa low-growing shrub endemic to the Galápagos withsemi-succulent, glaucous leaves (Eliasson, 1990).Alternanthera tenella is sister taxon to the speciesfrom the Galápagos and A. crucis. Alternantheratenella and A. crucis differ from all the Galápagosspecies by being herbaceous rather than woody. Thissuggests that Alternanthera is another classic case of

derived secondary woodiness associated with insularspecies (Carlquist, 1962, 1974, 2010a, b).

INCONGRUENCE OF PLASTID AND NUCLEAR DATA AND

POSSIBLE EVIDENCE FOR RETICULATE EVOLUTION

Comparisons between tree topologies show a conflictwith regard to the position of four species in theplastid and nuclear trees (Figs 1, 3). The variableposition of A. littoralis var. maritima in trees derivedfrom biparentally and maternally inherited markerssuggests a possible hybrid origin.

Hybridization has often been favoured as a mecha-nism to explain tree incongruence in plants, andincongruence between plastid and nuclear data isoften attributed to introgression and hybrid specia-tion (Baldwin et al., 1995; Morrell & Rieseberg, 1998;Widmer & Baltisberger, 1999; Hamzeh & Dayanan-dan, 2004; Kim & Donoghue, 2008). Similar incongru-ent results have been demonstrated in the closelyrelated genus Tidestromia. Chromosome evidencerelated to number and the form of meiotic divisionindicated hybridization in some members of Tidestro-mia (Sánchez-del Pino & Motley, 2010). It is interest-ing to note that Turner (1994) stated that althoughtwo-thirds of the genera of the Amaranthaceaeremain to be counted, polyploidy appears to becommon in the family. He also suggested that dysp-loidy (7 ← 8 → 9, 10, 11) and amphiploidy (17 = 8 + 9)play a role in Amaranthaceae and in the entire orderCaryophyllales.

Other species, such as A. altacruzensis, A. gal-apagensis and A. tenella, have inconsistent place-ments in the plastid and nuclear trees (Figs 1, 3).However, these minor incongruences are confined to asingle clade in which branch support is low, anddifferences are basically related to sister relationshipsamong species in the same clades. These results maynot be related to introgression, but possibly to inad-equate characters, stochastic errors, horizontal genetransfer, lineage sorting or heterogeneous rates ofmolecular evolution (Baldwin et al., 1995; Kim &Donoghue, 2008). Therefore, our results are not con-clusive with regard to reticulate patterns. ITS datawill have to be compared with other nuclear markersto get better understanding of species relationships.

EVOLUTION OF DIAGNOSTIC MORPHOLOGICAL

CHARACTERS OF ALTERNANTHERA AND ITS

MAJOR LINEAGES

Diagnostic characters at the generic and sectionalranks include several vegetative characteristics andmany floral structures (Martius, 1826; Endlicher,1836–1840). The characteristic features of Alternan-thera have changed along with its taxonomic history




such that after a re-evaluation, Eliasson (1987) sug-gested that the genus is a natural group character-ized by a combination of floral and pollencharacteristics.

Here we corroborate Eliasson’s proposal that theonly morphological character that supports themonophyly of Alternanthera is the synapomorphyrelated to stigma form. Our data indicate that prob-ably the only useful characters to recognize majorclades in Alternanthera are related to pistil charac-ters and inflorescence type. Several species observedin this study that form the major Clade A weredescribed by Moquin-Tandon (1849) as havingbilobed, inconspicuously subbilobed or capitatestigmas. Detailed observations of the sampling usedin this study suggested that this characteristic isrelated to a stigma with distinctive carpel demarca-tions (excluding A. lanceolata; character 9, Appen-dix 2), whereas the species in Clade B have stigmaslacking distinctive carpel demarcations (excludingA. crucis and A. pungens). Although this attributeresolved as homoplasious, features of the gynoeciumshould be studied carefully with scanning electronmicroscopy to examine their potential importance inthe taxonomy and classification of Alternanthera (seealso Appendix 2; Fig. 2).

ALTERNANTHERA AND MONOPHYLETIC

INFRAGENERIC ENTITIES

The circumscription of Alternanthera has varied con-siderably (Table 1) over time among different authors.Taxonomic problems in Alternanthera began with thedesignation of the type. Forsskål (1775) proposed thegenus Alternanthera in the Flora Aegyptiaco Arabicawithout mentioning the species type on the page onwhich the genus was described. However, a singlespecies name is mentioned for the genus among thelist of Triandra on page LIX as Alternanthera achy-ranthes. Mears (1977) mentioned that Lamarckvalidly published the identity of the type species ofAlternanthera in 1753. Then, the type species of Alter-nanthera was designated as A. sessilis (L.) DC., whichhas the basionym Gomphrena sessilis L. (Melville,1958; Mears, 1977). The situation was complicated byan incorrect designation of the type species of Achy-ranthes L. by Standley (1915). Mears (1977) laterexplained that for many years it was thought thatAlternanthera Forsskål was based on Achyranthesrepens L. Then, Standley (1915) considered Achyran-thes repens to be the type species of Achyranthes andhe placed most of the species of Alternanthera inAchyranthes while transferring the species of Achy-ranthes to Centrostachys Wallich (Bullock, 1957;Melville, 1958; Mears, 1977; Robertson, 2003). Stand-ley’s (1915) circumscription of Alternanthera includ-

ing Achyranthes was so artificial that mosttaxonomists never adopted it. The widely acceptedclassification system of Amaranthaceae by Schinz(1893, 1934), later refined by Townsend (1993), placedAchyranthes in subfamily Amaranthoideae whereasAlternanthera was located in subfamily Gom-phrenoideae. The distant positions of both genera alsoappear in recent molecular phylogenetic analyses ofAmaranthaceae (Müller & Borsch, 2005; Sánchez-delPino et al., 2009).

Many early classifications recognized segregategenera. The first classification for Alternanthera andrelated genera (Brandesia, Bucholzia and Mogiph-anes) was proposed by Martius (1826). Later, severalauthors (Endlicher, 1836–1840; Moquin-Tandon,1849; Bentham & Hooker, 1880) recognized Alternan-thera and Telanthera R.Br. as different genera,whereas others treated these groups as subgenera(Schinz, 1934) and others still recognized them assections of Alternanthera. Endlicher (1836–1840) rec-ognized three sections (Bucholzia Mart., BrandesiaMart. and Mogiphanes Mart.) within Telanthera.Moquin-Tandon (1849) accepted Endlicher’s (1836–1840) subgeneric classification of Telanthera and pro-posed four sections in Alternanthera (TrommsdorffiaMart., Dassiera Moq., Allaganthera Mart. and Clado-thrix Nutt.). Bentham & Hooker (1880) followedMoquin-Tandon’s circumscription but differed in thatthey recognized only two of the three sections inTelanthera (Bucholzia and Brandesia) and two inAlternanthera (Allaganthera as already proposed byMoquin-Tandon and a new section Lithophila).

The classifications proposed for Alternanthera byMartius (1826) and Endlicher (1836–1840) primarilyused pseudostaminodium shape and flower pedicels(present or absent) along with other several flowermorphology to define generic or infrageneric units.Moquin-Tandon (1849) recognized infrageneric taxabased on sexual expression, stem habit, inflores-cence type, stamen number and fusion, stigmashape, tepal features, style size and pseudostamino-dium shape. Schinz (1934) used the charactersstamen filaments and pseudostaminodia shape ashis diagnostic units.

Martius (1826) first described the diagnostic char-acters of three genera (Mogiphanes, Brandesia andBucholzia) but it was Moquin-Tandon (1849) whopublished an extensive list of Alternanthera spp.following the diagnosis of Endlicher’s sections. It isimportant to emphasize that species described afterMoquin-Tandon’s (1849) classification are includedin this study and have no sectional designationin Figure 1. However, information about sectionaldesignation for most of the species included inthis sampling was obtained from the originaldescriptions.




The analyses with plastid DNA (trnL-F, rpl16) andITS used in this study confirm the findings ofSánchez-del Pino et al. (2009) on the monophyly ofAlternanthera (100% JK, 1.0 PP). Nevertheless, itindicated that the subgeneric classifications for Alter-nanthera proposed in the past by Martius (1826),Endlicher (1836–1840), Moquin-Tandon (1849) andSchinz (1934; Fig. 1) do not reflect monophyleticgroups. Some sections previously recognized for Alter-nanthera were elevated to the generic level [e.g.Lithophila Swartz (= Section Lithophila) and Cladot-hrix (= Section Cladothrix)], whereas some formerAlternanthera spp. (= Section Trommsdorffia) weretransferred to Iresine P.Browne (Endlicher, 1836–1840; Moquin-Tandon, 1849; Bentham & Hooker,1880; Schinz, 1893, 1934; Townsend, 1993). Townsend(1993) adopted this concept. Despite the considerablevariation of Alternanthera spp. in life form, Eliasson(1987) argued that the genus in the latter circum-scription seems to be a natural taxon characterized bycapitate stigmas and a dodecahedral pollen form. Wefound that in addition to some floral characteristics(stigmatic characteristics resolved relevant clades inthis study), the inflorescences types and life forms areimportant features in the taxonomy of the genus.

Our results suggest that two major clades can berecognized within Alternanthera based on stigmasurface, bracteole form and also inflorescence type.Within Clade B, Subclades B3 and B4 (Node 7, Fig. 1)are supported by the synapomorphy bracteoles withmidnerve prominently keeled [character 3(1); Appen-dix 2]. Bracteoles in some taxa become stronglycurved and boat-shaped. This character was used byMartius (1826) to distinguish Bucholzia (includingspecies with concave bracteoles) from Mogiphanesand Brandesia (including species with carinatebracteoles). However, in general molecular data dolend some support to Martius’s classification. Muchfurther study and sampling will be needed before anew infrageneric classification can be proposed.

This study did find that the species assigned by A.J. Mears (unpubl. data) to the invalid genus ‘James-bondia’ form a monophyletic group and share somecommon morphological characters and a similar dis-tribution. However, species of ‘Jamesbondia’ arenested within Alternanthera based on the treesobtained from using plastid (trnL-F, rpl16), nuclear(ITS) and morphology data and perhaps will be auseful subgeneric lineage.

EVOLUTION OF C4 AND C3–C4 PHOTOSYNTHESIS

IN ALTERNANTHERA

The clade including Amaranthaceae sensu stricto andChenopodiaceae [together treated as Amaranthaceaein APG III 2009; however, in light of ongoing multi-

gene studies (T. Borsch et al. pers. observ.) of thegroup the authors prefer to retain the family nameChenopodiaceae in addition to Amaranthaceae] arethe major lineage with C4 species in eudicots(Kadereit et al., 2003; Sage, Christin & Edwards,2011). Phylogenetic analyses in the Amaranthaceaeand allies (e.g. Kadereit et al., 2003; Müller &Borsch, 2005; Akhani, Edwards & Roalson, 2007;Sage et al., 2007) and character state mapping indi-cated no fewer than 16 independent C4 lineages.Alternanthera is one of those but was only repre-sented by a few species in phylogenetic reconstruc-tions (Sage et al., 2007). Much earlier, severalauthors had examined C4 and C4–C3 intermediatespecies in Alternanthera with the aim of understand-ing the molecular basis and evolution of photosyn-thetic pathways (Rajendrudu, Prasad & Rama Das,1986; Devi et al., 1995; Chinthapalli et al., 2000;Gowik et al., 2006). The most extensive survey usingd13C values was that of Sage et al. (2007) who analy-sed 87 Alternanthera spp. and found that 17 (19.5%)had C4 metabolism. The carbon isotope ratios of thethree previously identified C3–C4 intermediate taxawere in the same range as the C3 species.

Our results depict all C4 species in Clade B3(Figs 1, 3) with several of the species widely distrib-uted in tropical and subtropical America, whereasA. microphylla is an endemic of dry chaparral vegeta-tion at high elevations in the central Andes (Borsch,Ortuño & Nee, in press). Alternanthera littoralis var.maritima as a member of this clade shows the C3

pathway. Alternanthera littoralis (= A. maritima)grows in moist dunes, with three varieties consideredto be vicariant along the coasts of the Caribbean,South America and western tropical Africa, respec-tively (Pedersen, 1990), and has succulent leaveswithout Kranz anatomy (T. Borsch, pers. observ.). IfC4 photosynthesis is coded as an individual unorderedcharacter state as in our study, a reversal from a C4

ancestor of Clade B3 to C3 photosynthesis in A. lit-toralis requires the same number of steps as anindependent acquisition of C4 photosynthesis inA. microphylla, if optimized on the plastid DNA tree.Losses of C4 appear to be rare based on the optimi-zation of C4 photosynthesis as a character state onphylogenetic trees, but it is so far unclear if there isa mechanism that would explain a reversal from C4 toC3 (Christin, Freckleton & Osborne, 2010). However,further taxon sampling is also needed to confirm theposition of A. microphylla, which might in fact beresolved in the same clade but further away once allspecies are included, or ancient close C3 relativesmight today be extinct. When the nuclear ITS tree isused to reconstruct character evolution, there isclearly only one C4 origin in Alternanthera, in sub-clade B3 (see Fig. 3). Because incongruence of nuclear




and plastid trees with respect to the position of A. lit-toralis points to reticulate evolution, it may even bepossible that the species arose through hybridizationof a C3 and a C4 ancestor, with the C3 ancestor as thematernal parent.

According to the current literature (summarized inSage et al., 2007), three species are considered C3–C4

intermediates: A. crucis, A. ficoidea and A. tenella.Alternanthera tenella was considered to be wide-spread at relatively low elevations through manyparts of the Neotropics (Borsch, 2001), and in light ofour trees does not appear to be monophyletic. Unfor-tunately, our samples of A. tenella were not includedin any physiological study, so their precise photosyn-thetic pathway is not known. However, it is alsounclear to which genotype the material cultivated inIndia belongs, which was the source for currentlyavailable physiological and biochemical data. A purecorrelation of such primary research data to our indi-viduals here identified as A. tenella is in such a situ-ation rather speculative. Alternanthera crucis (aspecies considered to be endemic to the Caribbean) isa close relative to our samples of A. tenella but so isthe plant included here from Peru to represent A. hal-imifolia (the species is considered to be C3; Sage et al.,2007). The case of A. ficoidea [‘A. ficoides (L.) R.Br.’ inChinthapalli et al., 2000], however, cannot be dis-cussed further because the plants used in physiologi-cal studies may have been misidentified. The nameA. ficoidea has indeed been the source of profoundconfusion (often with A. tenella) but could clearly beshown to be a synonym of A. paronychioides (Mears,1977; Eliasson, 1987; Borsch, 2001), a distantlyrelated C4 species. A lack of resolution and support inour trees based on the current limited taxon andcharacter sampling allows no firm conclusions aboutevolutionary patterns of the C3–C4 intermediates andthe morphologically closely allied C3 taxa. Neverthe-less, based on the results of this study it appears thatthe evolution of photosynthesis in Alternanthera doesnot exhibit a stepwise development from C3–C4 inter-mediates to C4. However, A. kurtzii, which is thesister group, is most likely of hybrid origin (parentaltaxa are unknown); thus, the more derived position ofthe putative C3–C4 intermediate taxa in the plastidtree may not depict the true origin of this photosyn-thetic pathway if a C3 species is the paternal parentand C3 is dominant. The survey of Sage et al. (2011)indicates that the C3–C4 intermediate type does notoften occur in immediate relatives, suggesting that itoften evolves independently from C4 in other lineagesof angiosperms including Alternanthera.

IMPLICATIONS FOR BIOGEOGRAPHY

Although distribution patterns in South and CentralAmerica, Mexico and the Caribbean are complex and

biogeographical results will probably be stronglyinfluenced by denser taxon sampling, the phyloge-netic trees for Alternanthera suggest some specificbiogeographical scenarios. One regards the coloniza-tion of the Galápagos Islands and the subsequentradiation of species. Many colonizers in the Galápagosappear to be from weedy ancestral species that couldsucceed in the islands by inhabiting varied and dis-turbed environments. Several weedy Alternantheraspp. have been reported in the Galápagos Islands(A. caracasana, A. lanceolata and A. sessilis; Eliasson,1990) and nine are considered endemic to the Galápa-gos (Jørgensen, 1999; Eliasson, 2004). In this study,the phylogenetic trees resolved the strongly sup-ported Clade B4 (96–99% JK, 1.0 PP; Figs 1, 3),including six species that are either endemic orindigenous to the Galápagos Islands; the Galápagosendemics are A. filifolia, A. flavicoma, A. galapagensisand A. snodgrassii in Clade B4 and A. nesiotes inClade B3. Alternanthera halimifolia, A. tenella andA. vestita are species that also occur in South America(in Subclade B4).

The most parsimonious hypothesis based on opti-mizing distributions of the species for the origin ofGalápagos Alternanthera spp. based on our data sug-gests two independent introductions to the GalápagosIslands and two back migrations to the mainland.Because of the poor branch support in Clade B4, thegeographical optimization in the clade is limited. Infact, Carlquist (1974) indicated that Galápagos Alter-nanthera spp. most probably represented two or pos-sibly more introductions whereas Eliasson (2004)proposed, based on morphological features, that theendemic species could be traced back to two or pos-sibly three successful colonization events.

In this study, the phylogenetic trees using plastidDNA and ITS suggest that it is possible to hypoth-esize a single introduction from an A. kurtzii- orA. tenella-like ancestor in Clade B. Alternantheratenella occurs from southern Mexico through CentralAmerica and the Caribbean Islands to Bolivia andsouthern Brazil (Burger, 1983) and A. kurtzii occursfrom Bolivia to Brazil (Pedersen, 1967). Alternantheraflavicoma, A. filifolia, A. galapagensis and A. sn-odgrassii, which are endemics to the Galápagos, andtwo more widespread species (A. halimifolia andA. vestita) that occur in the Galápagos (Eliasson,1971; Fournet, 2002; DeFilipps & Maina, 2003) prob-ably share a common ancestor with this lineage.Therefore, if A. halimifolia and A. vestita are derivedfrom these Galápagos endemics it would mean thatthere have been two back migrations to the mainland.The alternative is that A. halimifolia and A. vestitaeach represent separate introductions or that one orthe other may have given rise to the remaining taxain the radiation (Fig. 2).




The second introduction is an A. nesiotes ancestor, aspecies endemic to the Galápagos Islands (Jørgensen,1999). Because A. nesiotes is resolved in a subcladeconsisting of species that are distributed throughoutthe Americas (Clade B3; Fig. 2), it must be a separatedistinct introduction to the archipelago.

This study suggests that it might be possible forGalápagos Alternanthera spp. to have affinities withtaxa occurring in Chile, Peru and Mexico. Eliasson(1985, 1990, 2004) already hypothesized that two orthree Galápagos Alternanthera spp. are morphologi-cally more similar to plants from Chile and southernPeru than to species from the geographically closerEcuador. Galápagos Alternanthera spp. must be theresult of long-distance dispersal. It is known that theorganisms best adapted for long-distance dispersal areweedy plants (Carlquist, 1965). In addition, the esti-mated age for Amaranthaceae ranges from 83 Ma(Magallón, Crane & Herendeen, 1999) to 104–111 Ma(Wikström, Savolainen & Chase, 2001). The family istherefore much older than the relatively youngGalápagos Islands (3–4 Ma; McMullen, 1987), whichare volcanic in origin and have never been in contactwith the continental mainland. Amaranthaceae is thesixth largest family of vascular plants in the Galápa-gos Islands and is represented by 29 species in sevengenera (Stewart, 1911; Eliasson, 1990). This suggeststhat the rate of speciation in the group was fast or thatthere were multiple introductions of the family to thearchipelago. The closest relatives of the Galápagosflora appear to have affinities with South America, andto a lesser extent with Mexico and Central America,and only occasionally with the Caribbean Islands(Carlquist, 1965). Porter (1984) confirmed this andpinpointed many colonizers from South America, inparticular the Andean region. For flowering plants, heindicated that birds and wind are the main dispersers.Eliasson (2004) suggested that species of subfamilyGomphrenoideae, which is strongly established on theSouth American mainland, were probably transportedto the Galápagos, most likely by birds.

Regarding species of Central America and the Car-ibbean, Clade B1 (Fig. 2) only includes species dis-tributed in that area. The islands of the West Indiesextend 200 km south of North America (Florida), eastof Central America and South America to Venezuela(Fritsch & McDowell, 2003). Although all of theseareas are close to the Caribbean islands they were notconnected to the continents when Caribbean florasand faunas were being established (Carlquist, 1974).Long-distance dispersal is the predominant biogeo-graphical explanation for groups (e.g. Rubiaceae) inthe Caribbean islands (Fritsch & McDowell, 2003).Alternanthera laguroides from Central America issister to the Caribbean species. Alternanthera serpyl-lifolia, A. olivacea and A. geniculata occur in the Car-

ibbean islands (Standley, 1917), and only A. olivaceahas been collected outside the islands in Brazil, Ven-ezuela, Costa Rica, Nicaragua and Panama (Burger,1983). It seems likely that there was one long-distance dispersal introduction to the Caribbeanislands with affinities to Central America followed byradiation and dispersal throughout the Caribbean.Because A. olivacea is nested within the Caribbeanspecies, its existence in Central and South Americaprobably reflects migration to the continent.

CONCLUSIONS AND FUTURE WORK

The present study provides a comprehensive picture ofthe overall relationships in Alternanthera (Amaran-thaceae, Gomphrenoideae). Our data establish itsmonophyly and identify several major lineages in thegenus, but future work should focus on increased taxonsampling. We have aimed at representing all previ-ously recognized taxonomic entities above the specieslevel and covering morphologically deviating species(e.g. those provisionally placed in ‘Jamesbondia’) andare therefore content to provide a solid first picture onthe relationships and evolution of this large Neotropi-cal genus. However, available recent studies of Alter-nanthera were limited, either regionally (in the contextof treatments for national floras) and in the analysis ofonly gross morphological characters.

A truly integrative molecular approach with adense sampling of populations across the possiblerange of species will therefore be essential to delimitspecies in Alternanthera and to assess the diversity inthis genus robustly. The situation may be complicatedby introgression and hybridization, underlining theneed for generating a spectrum of molecular data onwell-documented individual plants that will at thesame time be analysed morphologically and anatomi-cally. Such work is underway to set the base for amodern monograph of Alternanthera for Flora Neo-tropica (I. Sánchez-del Pino, L. Senna, T. Borsch,pers. comm.).

For a better understanding of the evolution of pho-tosynthetic pathways, it will be important to clearlydocument the plant materials studied biochemicallyand physiologically. With the lack of a propertaxonomic treatment, especially for subclade B4(A. tenella and relatives), the evolutionary position ofplant individuals should be determined by generatingsequence data from markers otherwise used in phy-logenetic analyses, ideally by studying wild materialfrom documented origins and by keeping voucherspecimens that can later be analysed for features ofthe phenotype. In this way it may be possible to linkthe origin of C3–C4 intermediate types with certainspeciation processes in Alternanthera.




ACKNOWLEDGEMENTS

We thank Zachary Rogers (MO) who greatly helped toimprove the English text, Joseph Rachlin (LehmanCollege, CUNY), Henrik Nilsson (GB) and two anony-mous reviewers for valuable comments on the manu-script, and Uno H. Eliasson (GB) for access to hismaterial of Alternanthera from the GalápagosIslands. We are very grateful to Damon Little (NYBG)for assistance in obtaining most of the ITS sequencesof Alternanthera. Bettina Giesicke carried out thelaboratory work for the samples sequenced in Berlin.We are also grateful to the curators of the herbaria F,GB, S, MEXU, MO and NY for the loan of material.Teresa Ortuño (La Paz) helped during fieldwork ofT.B. in Bolivia. Samples from Cuba were collectedwith financial support of the Programa Flora de Cubaof the Botanischer Garten und Botanisches MuseumBerlin-Dahlem. Luisa Senna (Feira de Santana) andHossein Akhani (Tehran) provided helpful commentson this manuscript. This study was supported bythe Consejo Nacional de Ciencia y Tecnología(CONACYT) as part of the PhD scholarship for I.S.P.(149785), a grant from the American Society of PlantTaxonomy to I.S.P., a grant from Ciencia BásicaCONACYT for I.S.P. (106994) and a stipend from TheLewis B and Dorothy Cullman Foundation. The Deut-sche Forschungsgemeinschaft is acknowledged forgrants BO 1815/1-1 to 1–4 to T.B.

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APPENDIX 1

Taxon sampling and voucher information. Species name, vouchers information from the NYBG (DNA bankaccessions starting with ISP) and B (DNA bank accessions starting with AC) databases, DNA bank accession,and GenBank accession numbers for taxa used in this study. NA (sample not able to be amplified1 or used forthe analyses2)

Taxon Voucher specimenDNA bankaccession trnL-F rpl16

ITS(ITS1/ITS2)

Alternanthera altacruzensisSuess.

Bolivia, Nee & Vargas 43479(NY)

ISP127 EF688732 EF688659 JQ403572

Alternanthera brasiliana (L.)Kuntze

Bolivia, Gonzales 147 (NY) ISP116 JQ315137 JQ403544 JQ403565

Alternanthera caracasanaKunth

Mexico, Sánchez-del Pinoet al. 20 (MEXU)

ISP64 EF688733 EF688662 JQ403581

Alternanthera chacoënsisMorong ex Morong & Britton

Bolivia, Nee & Coimbra40161 (NY)

ISP182 JQ315138 JQ403550 JQ403578

Alternanthera crucis Bold. Puerto Rico, Taylor 9531&Lodge (NY)

ISP181 EF688735 EF688663 JQ403577

(01) Alternanthera elongata(Willd.) Schinz

Bolivia, Beck 11078 (LPB,NY)

ISP171 EF688736 EF688664 JQ403576

(02) Alternanthera elongata(Willd.) Schinz

Bolivia, Borsch & Ortuño3617 (B, LPB)

AC618 JQ315139 JQ403534 JQ403555

Alternanthera filifolia (Hook.f.)J.T.Howell

Ecuador (Galápagos),Eliasson & Eliasson 1668(GB)

ISP106 JQ315140 JQ403539 JQ403560

Alternanthera flava (L.) Mears Mexico, Nee & Taylor 28763(NY)

ISP117 EF688737 EF688665 JQ403566

Alternanthera flavescens Kunth Mexico, Martínez s.n. (NY) ISP118 EF688738 EF688666 JQ403567Alternanthera flavicoma

(Andersson) J.T.HowellEcuador (Galápagos),

Eliasson & Eliasson 1888(GB)

ISP111 JQ315141 JQ403540 NA1

Alternanthera galapagensis(Stewart) J.T.Howell


ISP112 EF688739 EF688667 JQ403561

Alternanthera geniculata Urb. Dominican Republic, Alain &Liogier 26490 (NY)

ISP102 JQ315142 JQ403537 JQ403558

Alternanthera halimifoliaStandl. ex Pittier

Peru, FLSP2171 (NY) ISP199 EF688740 EF688668 JQ403579

Alternanthera kurtzii Schinz exPedersen


AC617 JQ315143 JQ403533 JQ403554

Alternanthera laguroides(Standl.) Standl.

Costa Rica, Taylor 17394(NY)

ISP152 EF688741 EF688669 EU567664

Alternanthera lanceolata(Benth.) Schinz

Costa Rica, Barringer et al.2270 (NY)

ISP119 JQ315144 JQ403545 JQ403568

Alternanthera littoralis Beauv.ex Moq. var. maritima (Mart.)Pedersen

Bahamas Islands, Correll &Popenoe 45459 (NY)

ISP129 JQ315145 JQ403548 JQ403574

Alternanthera macbridei Standl. Peru, Cowan et al., 4276(NY)

ISP90 JQ315146 JQ403552 JQ403582

Alternanthera microphyllaR.E.Fr.


AC619 JQ315147 JQ403535 JQ403556

Alternanthera nesiotesI.M.Johnst.


ISP113 JQ315148 JQ403541 JQ403562

Alternanthera obovata Millsp. Mexico, Ventura 1314 (NY) ISP164 JQ315149 JQ403549 JQ403575




APPENDIX 1 Continued

Taxon Voucher specimenDNA bankaccession trnL-F rpl16

ITS(ITS1/ITS2)

Alternanthera olivacea Urb. Brazil, Van Proosdij 1105(NY)

ISP128 EF688744 EF688672 JQ403573

Alternanthera paronychioidesA.St.-Hil.

USA, Thomas 1141179 (NY) ISP2 JQ315150 JQ403551 JQ403580

Alternanthera philoxeroides(Mart.) Griseb.

USA, Thomas and Amason142585 (NY)

ISP121 EF688745 EF688673 JQ403569

Alternanthera porrigens Kuntze Peru, Weigend et al. 544 (NY) ISP122 JQ315151 JQ403546 JQ403570Alternanthera pubiflora Kuntze Panama, Burch et al. 1176

(NY)ISP123 JQ315152 JQ403547 NA1

Alternanthera pungens Kunth Brazil, Agra et al. 2084 (NY) ISP125 EF688746 EF688674 JQ403571Alternanthera pungens USA, Borsch, Pratt & Müller

3449 (B, ISC)AC061 NA2 NA2 JQ403553

Alternanthera serpyllifolia Urb. Dominican Republic, Alain &Liogier 11185 (NY)

ISP104 JQ315153 JQ403538 JQ403559

Alternanthera snodgrassii(B.L.Rob.) J.T.Howell


ISP114 JQ315154 JQ403542 JQ403563

(01) Alternanthera tenella Colla Brazil, Nee 42581 (NY) ISP119 EF688747 EF688675 EU567665(02) Alternanthera tenella Colla Cuba, Borsch 3951 (B,

HAJB)AC620 JQ315155 JQ403536 JQ403557

Alternanthera vestita(Andersson) J.T.Howell


ISP115 JQ315156 JQ403543 JQ403564

Pedersenia cardenasii (Standl.)Holub


ISP187 EF688782 EF688712 EU567666

Pedersenia cf. hassleriana(Mart.) Holub

Bolivia, Borsch & Ibisch3532 (B, LPB)

ISP188 EF688783 EF688713 EU567667

Tidestromia carnosa (Steyerm.)I.M.Johnst.

Mexico, Flores et al. HF02-22 (MEXU)

ISP37 EF688789 EF688720 EU567668

Mexico, Sánchez-del Pinoet al. 70 (MEXU)

ISP14

Tidestromia lanuginosa (Nutt.)Standl.


ISP30-31 EF688791 EF688722 EU567670


ISP36

Tidestromia valdesiana Sánch.Pino & Flores Olv.


ISP35 EF688796 EF688726 EU567675

APPENDIX 2

Characters and character states used in the study.1. Tepal trichomes on tepals. 0 = absent, 1 = present. Trichomes on tepals are present in most of the taxa

and absent in Pedersenia hassleriana, Alternanthera littoralis var. maritima, A. obovata, A. philoxeroides anda few representatives of A. paronychioides.

2. Kind of tepal trichomes form on tepals. 0 = simple, 1 = dendritic, 2 = barbed. Type of trichomes wascharacterized by Sánchez-del Pino & Flores Olvera (2006). This study follows the same descriptions. The simpleform was the most common for the sampling. Dendritic trichomes are present in Tidestromia carnosa, T.lanuginosa, Alternanthera altacruzensis, A. kurtzii, A. crucis, A. brasiliana, A. flavescens, A. flavicoma, A.




galapagensis and A. halimifolia. Barbed trichomes are present in A. caracasana, A. pungens and T. valdesiana.The character is coded as not applicable in Pedersenia hassleriana, A. littoralis var. maritima, A. obovata andA. philoxeroides.

3. Bracteoles midnerve shape. 0 = not keeled, 1 = prominently keeled. Midrib of bracteoles in sometaxa is sharply keeled and became strongly curved, having a boat-shaped form. Prominent midribs of bracteolesare present in Alternanthera filifolia, A. flavicoma, A. galapagensis, A. halimifolia, A. littoralis var. maritima,A. nesiotes, A. paronychioides, A. chacoënsis, A. kurtzii, A. crucis, A. snodgrassii, A. tenella and A. vestita. Theremaining species have a distinct midrib but never prominent so that bracteoles are flattened or convex. Thischaracter is not applicable in A. brasiliana.

4. Flower insertion. 0 = sessile, 1 = pedicellate. Flowers in Alternanthera can be sessile to short-pedicellate within the bracteoles. Previous authors (Martius, 1826; Endlicher, 1836–1840; Moquin-Tandon,1849; Bentham & Hooker, 1880; Schinz, 1934) recognized the presence of flowers stipitated with short andsulcate pedicels as diagnostic of section Mogiphanes. However, this character is shared for species of Alternan-thera placed in other sections, suggesting that the character is not diagnostic of sections within Alternanthera.Therefore, pedicellate flowers are present in A. brasiliana, A. flavescens, A. galapagensis, A. geniculata, A.macbridei, A. littoralis var. maritima and A. obovata. The remaining species have sessile flowers.

5. Inflorescence insertion. 0 = sessile, 1 = pedunculate. Inflorescence architecture helps to characterizetwo main groups within Alternanthera. There is a group with spikes sessile and mostly axillary. This groupcorresponds to A. caracasana, A. chacoënsis, A. filifolia, A. flavicoma, A. galapagensis, A. geniculata, A.halimifolia, A. macbridei, A. littoralis var. maritima, A. kurtzii, A. crucis, A. nesiotes, A. obovata, A. parony-chioides, A. pungens, A. serpyllifolia, A. snodgrassii, A. tenella, A. vestita and Tidestromia. Species that haveinflorescences either pedunculate or sessile are common in A. laguroides, A. olivacea, A. porrigens and A.pubiflora. The remaining species have spikes pedunculate, arranged in synflorescences of thyrsoid paracladiafollowing correct terminology according to Acosta et al. (2009).

Inflorescences either sessile or pedunculate has been a character used along the infrageneric classification ofAlternanthera proposed in the past by several authors (Martius, 1826; Endlicher, 1836–1840; Moquin-Tandon,1849; Bentham & Hooker, 1880; Schinz, 1934) and the character is still useful to recognize groups within thegenus.

6. Style length. 0 = long, 1 = short. Size of styles has been an important character at section level basedon Schinz (1934). Long styles are present in A. kurtzii, A. crucis, A. elongata, A. flava, A. geniculata,A.laguroides, A. macbridei, A. lanceolata, A. obovata, A. olivacea, A. philoxeroides, A. porrigens, A. serpyllifolia,A. tenella and Tidestromia valdesiana. Short styles occur in the remaining species.

7. Pseudostaminodial margin. 0 = laciniate, 1 = crenate. The diagnostic character for Alternanthera isthe presence of laciniate pseudostaminodia. However, few species within Alternanthera have crenate pseudos-taminodia and shorter than the common form in the genus as occurs in A. altacruzensis, A. caracasana, A.chacoënsis, A. obovata, A. paronychioides, A. pungens, Pedersenia, Tidestromia lanuginosa and T. carnosa. Thischaracter is inapplicable in A. serpyllifolia and T. valdesiana because the structure is lacking in these taxa.

8. Flower arrangement along rachis. 0 = dense, 1 = loose. Inflorescence units of Alternanthera areflowers crowded along the rachis in globose spikes (dense) whereas other taxa have few flowers arranged alongthe rachis forming slender spikes (loose). The latter is common in A. geniculata, A. olivacea and A. serpyllifoliaas well as the outgroup taxa Pedersenia hassleriana. This is inapplicable in Tidestromia because it is adichasium.

9. Stigmatic surface. 0 = with distinctive carpel demarcations, 1 = without distinctive carpeldemarcations. Some taxonomic descriptions at species (Moquin-Tandon, 1849) and section level (Schinz, 1934)suggested that some groups within Alternanthera have capitate stigma whereas others have bilobed or obscurebilobed stigmas. Observations suggested that some stigmas have small stigmatic surface and dense hairyglandulous stigmatic area that make stigmas look hairy. Other taxa have a larger stigmatic surface that doesnot look hairy and carpel lines are visible so that stigmas seem bilobed or more divided. Taxa that havedistinctive carpel demarcations in the stigma surface are A. altacruzensis, A. brasiliana, A. crucis, A. elongata,A. flava, A. flavescens, A. macbridei, A. porrigens, A. pubiflora, A. pungens and the outgroup taxa Pederseniaand Tidestromia.

10. Stigma form. 0 = capitate; 1 = bilobed. Stigma bilobed is the common form in most of the outgrouptaxa such as Tidestromia and Pedersenia. Species of Alternanthera have a capitate stigma which never has twoevident deeply lobes as in the outgroup taxa.

11. Pollen exine surface. 0 = psilate, 1 = ornamented. Psilate pollen is common in Tidestromia whereasthe remaining taxa have ornamented pollen.




APPENDIX 3

Morphological data matrix of Alternanthera and outgroups (OG). Characters and coding are detailed inAppendix 2. ‘?’ represents missing data.

Character states

Taxon 01 02 03 04 05 06 07 08 09 10 11

Alternanthera altacruzensis 1 1 0 0 1 1 1 0 0 0 1Alternanthera brasiliana 1 1 – 1 1 1 0 0 0 0 1Alternanthera caracasana 1 2 0 0 0 1 1 0 1 0 1Alternanthera chacoënsis 1 0 1 0 0 1 1 0 1 0 1Alternanthera crucis 1 1 1 0 0 0 0 0 0 0 1(01) Alternanthera elongata 1 0 0 0 1 0 0 0 0 0 1(02) Alternanthera elongata 1 0 0 0 1 0 0 0 0 0 1Alternanthera filifolia 1 0 1 0 0 1 0 0 1 0 1Alternanthera flava 1 0 0 0 1 0 0 0 0 0 1Alternanthera flavescens 1 1 0 1 1 1 0 0 0 0 1Alternanthera flavicoma 1 1 1 0 0 1 0 0 1 0 1Alternanthera galapagensis 1 1 1 1 0 1 0 0 1 0 1Alternanthera geniculata 1 0 0 1 0 0 0 1 1 0 1Alternanthera halimifolia 1 1 1 0 0 1 0 0 1 0 1Alternanthera kurtzii 1 1 1 0 0 0 0 0 1 0 1Alternanthera laguroides 1 0 0 0 0,1 0 0 0 1 0 1Alternanthera lanceolata 1 0 0 0 1 0 0 0 1 0 1Alternanthera littoralis var. maritima 0 – 1 1 0 1 0 0 1 0 1Alternanthera macbridei 1 0 0 1 0 0 0 0 0 0 1Alternanthera microphylla ? ? ? ? ? ? ? ? ? ? 1Alternanthera nesiotes 1 0 1 0 0 1 0 0 1 0 1Alternanthera obovata 0 – 0 1 0 0 1 0 1 0 1Alternanthera olivacea 1 0 0 0 0,1 0 0 1 1 0 1Alternanthera paronychioides 0,1 0 1 0 0 1 1 0 1 0 1Alternanthera philoxeroides 0 – 0 0 1 0 0 0 1 0 1Alternanthera porrigens 1 0 0 0 0,1 0 0 0 0 0 1Alternanthera pubiflora 1 0 0 0 0,1 1 0 0 0 0 1Alternanthera pungens 1 2 0 0 0 1 1 0 0 0 1Alternanthera serpyllifolia 1 0 0 0 0 0 – 1 1 0 1Alternanthera snodgrassii 1 0 1 0 0 1 0 0 1 0 1(01) Alternanthera tenella 1 0 1 0 0 0 0 0 1 0 1(02) Alternanthera tenella 1 0 1 0 0 0 0 0 1 0 1Alternanthera vestita 1 0 1 0 0 1 0 0 1 0 1Pedersenia cardenasii (OG) 1 0 0 0 1 1 1 0 0 1 1Pedersenia cf. hassleriana (OG) 0 – 0 0 1 1 1 1 0 1 1Tidestromia carnosa (OG) 1 1 0 0 0 1 1 – 0 1 0Tidestromia lanuginosa (OG) 1 1 0 0 0 1 1 – 0 1 0Tidestromia valdesiana (OG) 1 2 0 0 0 0 – – 0 1 0




molecular phylogenetics of alternanthera (gomphrenoideae

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