Molecular phylogeny of the nettle family (Urticaceae) inferred ...

Molecular Phylogenetics and Evolution 69 (2013) 814–827

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Molecular Phylogenetics and Evolution

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Molecular phylogeny of the nettle family (Urticaceae) inferred frommultiple loci of three genomes and extensive generic sampling

1055-7903/$ - see front matter � 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.ympev.2013.06.022

⇑ Corresponding author. Address: 132 Lanhei Road, Kunming 650201, Yunnan,China. Fax: +86 871 6521 7791.

E-mail address: [email protected] (D.-Z. Li).

Zeng-Yuan Wu a,b,g, Alex K. Monro c,d, Richard I. Milne e,f, Hong Wang a, Ting-Shuang Yi b, Jie Liu a,b,De-Zhu Li a,b,⇑a Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Chinab Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Chinac Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UKd Department of Life Sciences, Natural History Museum, London SW7 5BD, UKe Institute of Molecular Plant Sciences, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3JH, UKf Royal Botanic Garden Edinburgh, 20A Inverleith Row, Edinburgh, EH3 5LR, Scotland, UKg University of Chinese Academy of Sciences, Beijing 100049, China

a r t i c l e i n f o

Article history:Received 6 March 2013Revised 26 June 2013Accepted 27 June 2013Available online 9 July 2013

Keywords:Molecular phylogenyUrticaceaePhylogenetic treesThree genomesCecropiaceaeinfra-familial relationships

a b s t r a c t

Urticaceae is one of the larger Angiosperm families, but relationships within it remain poorly known. Thisstudy presents the first densely sampled molecular phylogeny of Urticaceae, using maximum likelihood(ML), maximum parsimony (MP) and Bayesian inference (BI) to analyze the DNA sequence data from twonuclear (ITS and 18S), four chloroplast (matK, rbcL, rpll4–rps8–infA–rpl36, trnL–trnF) and one mitochon-drial (matR) loci. We sampled 169 accessions representing 122 species, representing 47 of the 54 recog-nized genera within Urticaceae, including four of the six sometimes separated as Cecropiaceae. Majorresults included: (1) Urticaceae including Cecropiaceae was monophyletic; (2) Cecropiaceae was biphy-letic, with both lineages nested within Urticaceae; (3) Urticaceae can be divided into four well-supportedclades; (4) previously erected tribes or subfamilies were broadly supported, with some additions andalterations; (5) the monophyly of many genera was supported, whereas Boehmeria, Pellionia, Pouzolziaand Urera were clearly polyphyletic, while Urtica and Pilea each had a small genus nested within them;(6) relationships between genera were clarified, mostly with substantial support. These results clarifythat some morphological characters have been overstated and others understated in previous classifica-tions of the family, and provide a strong foundation for future studies on biogeography, character evolu-tion, and circumscription of difficult genera.

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1. Introduction

The family Urticaceae Jussieu consists of approximately 54 gen-era with more than 2000 species, and is widely distributed in trop-ical regions but less common in temperate regions, with by far thelargest concentration of genera and species in tropical Asia (Friis,1989; Takhtajan, 2009; Stevens, 2012). Urticaceae shows remark-able morphological diversity, and its members exhibit various lifeforms, ranging from herbs, shrubs, to lianas or small trees. Urtica-ceae sensu stricto (i.e. excluding Cecropiaceae; see below) is charac-terized by the pistil with a single stigma and a basal orthotropousovule, having filaments inflexed in bud, and some species armedwith distinctive stinging hairs (Friis, 1989, 1993; Wang and Chen,

1995; Chen et al., 2003). Economically important genera includemany (e.g. Boehmeria and Girardinia), whose stem fiber is of highquality and therefore used to make cloth, fishing nets, ropes andsome industrial materials (Singh and Shrestha, 1988; Angeliniet al., 2000; Bodros and Baley, 2008). Recently, the medicinal usageof some taxa within Urticaceae has been increasingly studied (e.g.Gulcin et al., 2004; Piccinelli et al., 2005; Momo et al., 2006; Luoet al., 2011). Furthermore, some genera, including Elatostema, Pileaand Pellionia, are dominant and ecologically important elements ofthe forest floor vegetation in subtropical forests (Wang and Chen,1995; Chen et al., 2003). A full understanding of relationshipswithin this family would help to understand how these ecologicalassemblages evolved, and also to detect potential new sources ofmedicinal compounds.

The infra-familial classification of Urticaceae has been contro-versial for more than one century. Gaudichaud (1830) treatedmembers of Urticaceae, Cecropiaceae, Moraceae and Cannabina-ceae as a single family, and divided those now in Urticaceae into

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five tribes named Urereae, Elatostemeae, Boehmerieae, Pari-etarieae, and Forskalieae (Table 1). Weddell (1854, 1856, 1869)separated Moraceae and Cannabaceae from Urticaceae, dividingthe remaining Urticaceae into tribes similar to those of Gaudichaud(1830), i.e. Urereae, Procrideae, Boehmerieae, Parietarieae, and For-skohleae (Table 1). Friis (1989, 1993) largely followed Weddell’sclassifications, calling the five tribes Urticeae, Lecantheae, Boeh-merieae, Parietarieae and Forsskaoleae (Table 1). However, hequestioned the circumscription of the last three, and suggested fur-ther taxonomic work should be carried out. Based on morphologi-cal characters of fruit, Kravtsova (2009) proposed three subfamilieswithin Urticaceae: Urticoideae, Lecanthoideae, and Boehmerioi-deae; these largely correspond with Urticeae, Lecantheae, andBoehmerieae–Parietarieae–Forsskaoleae, respectively (Friis,1993), except that Touchardia was transferred to Lecanthoideae.

Fig. 1. (Part A + Part B) Phylogenetic tree produced by Bayesian Inference (BI) analysisconsidering those clades with bootstrap BP P 50%, and posterior probability PP P 0.6,branches (ML_BS/MP_BS/BI_PP) are assessed by Maximum Likelihood Bootstrap (ML_BS)no support, ‘‘�’’ means fully support (100%/1.00). Numbers following species name deno

Additionally, six tribes under these subfamilies were proposed:Urticeae, Leacantheae, Touchardieae, Boehmerieae, Forsskaoleae,Parietarieae (Table 1).

The genera Cecropia and Coussapoa formed the tribe Cecropieaeof Gaudichaud’s classification (Gaudichaud, 1830), which withMusanga, Poikilospermum, Pourouma and Myrianthus as Conoceph-aleae were considered by Bentham and Hooker (Bentham andHooker, 1880) to belong in the equivalent of modern Moraceae.However, Weddell (1869) placed Poikilospermum in Urticaceae;Chew (1963) also moved Cecropia, Musanga and Coussapoa intoUrticaceae; and Corner (1962) considered all six to belong toUrticaceae. Berg (1978) raised Cecropia, Musanga, Coussapoa,Poikilospermum, Pourouma and Myrianthus together to family levelas Cecropiaceae, considering it distinct from Urticaceae based onfilaments that are mostly straight in the bud, and the absence of

based on the matrix with plastic, mitochondrial, and nuclear datasets combined,respectively. Clades are referred by numbers in the box; Numbers associated with, Maximum Parsimony Bootstrap (MP_BS) and Bayesian posterior (BI_PP). ‘‘–’’ showste the lab codes.

Fig. 1 (continued)

816 Z.-Y. Wu et al. / Molecular Phylogenetics and Evolution 69 (2013) 814–827

any tendencies towards a herbaceous habit. His treatment hasbeen challenged based on wood anatomy (Bonsen and Ter Welle,1983, 1984) and molecular data (Hadiah et al., 2003, 2008; Datwy-ler and Weiblen, 2004; Zerega et al., 2005; Monro, 2006), and so farthe phylogenetic relationships between these two families havenot been fully addressed.

Historically, most of the diagnostic morphological characters(e.g. stigma, infloresences, stipules, achenes, etc.) used in classifica-tions of Urticaceae are of such small size that microscopic work isalways necessary to obtain accurate identifications of species. This,combined with high species diversity and the absence of a well-sampled phylogeny, has been challenging to previous efforts toelucidate relationships within Urticaceae. Recent systematic stud-ies on this family have so far mostly been based on morphologicalcharacters (Friis, 1989, 1993; Wilmot-Dear and Friis, 1996, 2006;Kravtsova, 2009; Wilmot-Dear, 2009; Wilmot-Dear and Friis,2011; Wu et al., 2011a,b; Wang, 2012; Wu et al., 2012).

Although several molecular phylogenetic studies have in-cluded Urticaceae, these were either focused on order- or

family-level relationships (Sytsma et al., 2002; Zhang et al.,2011), or have focused only on subgroups within the family(Hadiah et al., 2003; Monro, 2006). Only Hadiah et al. (2008)have so far addressed molecular relationships within the family,examining 63 species from 25 genera using two chloroplastloci (rbcL and trnL–trnF), their results indicated that Cecropia-ceae was not distinct from Urticaceae, and resolved threelineages within Urticaceae, i.e. (1) Boehmeriea-Cecropieae–For-sskaoleeae–Parietarieae, (2) Urticeae and (3) Elatostemeae. Thiswas broadly consistent with previous molecular phylogeneticanalyses (Sytsma et al., 2002; Datwyler and Weiblen, 2004;Zerega et al., 2005; Monro, 2006), but their phylogeny had muchincongruence with previous morphological classifications. How-ever, due to the limitations of sampling and regions, therelationships and circumscription of many clades, and the gen-era within them, remained poorly-resolved. Therefore, a betterresolved phylogenetic framework for the family is urgentlyneeded, which would require more extensive sampling of bothtaxa and the genomes.

A B B C D E

F G H I J

K L M N

O P Q R

Fig. 2. Morphological diversity of Urticaceae. (A–B) Inflexed filaments in Urticaceae; (A) Elatostema obtusum; (B) Lecanthus pileoides; (C–I) examples of morphologicaldiversity in Clade I; (C) Pipturus arborescens; (D) Boehmeria tricuspis; (E) Debregeasia longifolia; (F) B. nivea var. tenacissima; (G) Chamabainia cuspidata; (H) Archiboehmeriaatrata; (I) Oreocnide frutescens subsp. frutescens; (J–M) examples of morphological diversity in Clade II; (J) E. densistriolatum; (K) L. pileoides; (L) E. tenuicaudatum; (M) Pellioniaradicans; (N–P) stinging hairs and punctate cystoliths in Clade III; (N) Urtica macrorrhiza; (O) Laportea cuspidata; (P) U. fissa; (Q–R) examples of inflorescences in Clade IV; (Q)Leucosyke quadrinervia; (R) Maoutia setosa. (B, F, and M) were photographed by T. Zhang, the others were photographed by Z.Y. Wu.


In the present study, to generate a phylogenetic hypothesis ofgeneric and tribal relationships within Urticaceae, we used themost comprehensive taxon sampling to date, encompassing 47 ofthe 54 recognized genera within Urticaceae, including four of thesix sometimes separated as Cecropiaceae. We conducted phyloge-netic analyses based on the DNA sequences of two nuclear loci,four chloroplast regions, and one mitochondrial gene, using threeanalytical methods (ML, MP, and BI). Our main aims were to (1)test the monophyly of Urticaceae; (2) evaluate the status of Cecro-piaceae; (3) determine phylogenetic relationships within Urtica-ceae; (4) evaluate existing tribal and subfamily classifications inthe light of molecular data; (5) test the monophyly of some largergenera; and (6) provide insight into the usefulness of certain mor-phological characters in defining groups within the family.

2. Materials and methods

2.1. Taxon sampling and molecular loci

Our sampling attempted to maximize the taxonomic and geo-graphic coverage of each recognized genus within Urticaceae; insome cases two individuals per species were examined. Accordingto the list of Urticaceae (Stevens, 2012), we sampled 169 acces-sions of 122 species from 47 genera of Urticaceae, representing87% of the recognized genera. These included four genera some-times separated as Cecropiaceae, and Pellionia, which is usuallyrecognized as a subgenus of Elatostema (Schröter and Winkler,1935, 1936; Hallier, 1986). Based on previous molecular analyseson Rosales (Zhang et al., 2011), three species from Moraceae andUlmaceae were included for rooting purposes. The complete listof sample numbers, collection localities, voucher specimens num-

ber and GenBank accession numbers is given in Table 2. Voucherspecimens of these samples are deposited at KUN, BM, K, PE, G orC (Table 2).

Regarding selection of DNA regions, according to the ‘‘bottom up-top down’’ approach (Wiens et al., 2005), variable and fast-evolvingloci are more useful in resolving phylogenetic relationships at lowertaxonomic levels, and hence for an entire study group, whereasslow-evolving loci are more effective at higher levels, and hencemay be more appropriate when a few species per genus are sampled.We selected trnL–trnF, rpll4–rps8–infA–rpl36, matK, rbcL, and ITS, allof which have been used extensively to resolve relationships at thegenus level (Milne, 2004; Couvreur et al., 2010; Liu et al., 2011,2013; Nürk et al., 2013). To increase resolution at higher taxonomiclevels, we included 18S and matR, which are usually considered asslow-evolving loci (Soltis et al., 2000; Zhu et al., 2007; Chen et al.,2012). Therefore, only one or two species per genus were examinedusing these two loci. Primers for amplification and sequencing areprovided in Supplementary Table S2. For each of DNA loci, all se-quences were newly generated in this study.

2.2. DNA isolation, PCR amplification and sequencing

Total genomic DNA was mostly isolated from silica–gel dried orfresh leaves, with some from herbarium material supplied by BMand K. All material was extracted using the CTAB method (Doyleand Doyle, 1987) with minor modifications. PCR was conductedin a total volume of 25 lL containing 2.5 lL 10� PCR buffer,2.5 lL MgCl2 (25 mM), 2.0 lL dNTP mixture (2.5 mM), 0.75 lL eachprimer (10 lM), 0.125 lL Taq polymerase (5 U/lL) (TaKaRa, Dalian,China), 1–2 lL template DNA (containing 5–50 ng genomic DNA),and finally distilled deionized water to give a final volume of

Table 1Currently recognized genera of Urticaceae, their tribal classifications, clade membership, geographic range, species diversity and sampling for this study.

Genus Tribal classification according to: Clade Geographic range No. of species ingenus

No. of spp.sampled

Total accessionssampled

Gaudichaud(1830)

Weddell(1869)a

Friis (1993) Kravtsova(2009)

Urtica Urereae Urereae Urticeae Urticeae 3A Subcosmopolitan 80 9 13Hesperocnide – Urereae Urticeae Urticeae 3A California, Hawaii Is. 2 1 1Nanocnide – Urereaeb Urticeae Urticeae 3B Warm parts of East Asia 2 2 4Obetia – Urereae Urticeae Urticeae 3F C & S Africa; Madagascar,

Mascarenes7 1 1

Laportea Urereae Urereae Urticeae Urticeae 3E E Asia, eastern N America 21 1 2Discocnide – – Urticeae Urticeae 3C Mexico, Guatemala 1 1 1Girardinia Urereae Urereae Urticeae Urticeae 3D Africa, E Asia 2 1 6Dendrocnide – – Urticeae Urticeae 3C Indomalayan region, W Pacific

Islands37 3 4

Urera Urereae Urereae Urticeae Urticeae 3F Tropical Africa & America, 35 7 8Madagascar, Hawaii

Gyrotaenia – Urereae Urticeae Urticeae 2D West Indies 4 3 3Elatostema Elatostemeae Procrideaec Lecantheaed Lecantheae 2B Warm parts of old world 300 11 12Meniscogyne – – Lecantheaed – 1A SE Asia (Indo-China) 2 0 0Procris Elatostemeae Procrideae Lecantheaed Lecantheae 2C Warm and tropical parts of old

world20 1 2

Pilea Elatostemeae Procrideaec Lecantheaed Lecantheae 2F Old and New world tropics 250 12 14Sarcopilea – – Lecantheaed – 2F Hespaniola 1 1 1Lecanthus – Procrideaec Lecantheae4 Lecantheae 2E Africa, S Asia and Oceania 1 2 4Petelotiella – – Lecantheaed – – NE Vietname 1 0 0Pellionia Elatostemeae Procrideaeb Elatostemeaee Lecantheae 2A/2C Tropical & subtropical Asia,

Pacific islands60 5 5

Aboriella – – Elatostemeaef – – East Himalayas 1 0 0Boehmeria Boehmerieae Boehmerieae Boehmerieae Boehmerieae 1A1/

3/7Pantropical, N warmtemperate regions

80 14 23

Chamabainia – Boehmerieae Boehmerieae Boehmerieae 1A4 China & Indonesia to India 1 or 2 1 2Pouzolzia Parietarieae Boehmerieae Boehmerieae Boehmerieae 1A5/

1A7Pantropical 70 5 8

Gonostegia – – Boehmerieae – 1A6 SE Asia and N Australia 5 2 3Neodistemon – – Boehmerieae Boehmerieae 1A7 Indomalesia: India to

Philippines1 1 1

Cypholophus – Boehmerieae Boehmerieae Boehmerieae – Malesia, W Pacific Islands 15 0 0Sarcochlamys – Boehmerieae Boehmerieae Boehmerieae 1A3 Himalayas and Indo-china 1 1 1Touchardia – Boehmerieaec Boehmerieae Touchardieae 3F Hawaii 1 or 2 1 1Neraudia Boehmerieae Boehmerieae Boehmerieae Boehmerieae 1A8 Hawaii 5 2 2Piptuirus – Boehmerieae Boehmerieae Boehmerieae 1A9 Malaysia to N Australia &

Polynesia30 3 4

Nothocnide – – Boehmerieae Boehmerieae 1A9 From Indonesia to Melanesia 4 1 1Oreocnide – – Boehmerieae Boehmerieae 1B From Sri Lanka to Japan 15 1 3Debregeasia – Boehmerieae Boehmerieae Boehmerieae 1A2 Malesia, Indo-China, Arabia,

NE Africa4 5 9

Astrothalamus – – Boehmerieae Boehmerieae 1A3 Indonesia, Philippines 1 1 1Leucosyke – Boehmerieae Boehmerieae Boehmerieae 4B SE Asia, Indonesia, S Polynesia 35 1 2Gibbsia – – Boehmerieae Boehmerieae – Western New Guinea 2 0 0Phenax – Boehmerieae Boehmerieae Boehmerieae 1E Tropical and S America 12 1 1Maoutia – Boehmerieae Boehmerieae Boehmerieae 4B N India, Indo-China, Malesia,

Polynesia15 1 1

Myriocarpa – Boehmerieae Boehmerieae Boehmerieae 2D Tropical and South America 18 2 2Archiboehmeria – – Boehmerieaee Boehmerieae 1A3 S China and N Indo-China 1 1 2Gesnouinia Parietarieae Parietarieae Parietarieae Parietarieae 1D Canary Islands 2 1 1Hemistylus – Parietarieae Parietarieae Boehmerieae 1A5 Tropical south & central

America4 1 1

Parietaria Parietarieae Parietarieae Parietarieae Parietarieae 1D Subcosmopolitan 20 2 2Soleirolia Parietarieae – Parietarieae Parietarieae 1D West Mediterranean 1 1 1Rousselia Parietarieae Parietarieae Parietarieae Boehmerieae 1A5 C America, Colombia, West

Indies3 1 1

Forsskaolea Forskalieae Forskohleae Forsskaoleae Forsskaoleae 1C Macaronesia, Africa, S Spain,India

6 1 2

Droguetia Forskalieae Forskohleae Forsskaoleae Forsskaoleae 1C E & S Africa, Madagascar, 7 2 3Mascarenes, S India, Java

Australina Forskalieae Forskohleae Forsskaoleae Forsskaoleae 1C E Africa, SE Australia, NewZealand

2 1 1

Didymodoxa – Forskohleae Forsskaoleae Forsskaoleae 1C E & S Africa 2 1 1Poikilospermum – Cecropiaceae Cecropiaceae 3F Tropical Asia 20 2 3Pourouma – Cecropiaceae Cecropiaceae – Tropical America >50 0 0Myrianthus – Cecropiaceae Cecropiaceae 4A Tropical Africa 7 1 1


Table 1 (continued)

Genus Tribal classification according to: Clade Geographic range No. of species ingenus

No. of spp.sampled

Total accessionssampled

Gaudichaud(1830)

Weddell(1869)a

Friis (1993) Kravtsova(2009)

Cecropia Cecropieae Cecropiaceae Cecropiaceae 4A Tropical America 70–80 2 2Coussapoa Cecropieae Cecropiaceae Cecropiaceae 4A Tropical America >50 1 1Musanga – Cecropiaceae Cecropiaceae – Tropical Africa 2 0 0

a Two earlier classifications by Weddell (1854, 1856) exist. Unless indicated otherwise via footnotes, the genus was either not recognized in earlier classifications or placedin the same tribe as for the Weddell (1869) classification shown here. Weddell (1869)’s classification further subdivided Boehmerieae into subtribes Euboehmereieae(Boehmeria, Chamabainia, Pouzolzia, Memorialis), Sarcochlamydeae (Cypholophus, Neraudia, Laurea, Sarcochlamys, Touchardia, Poikilospermum), Villebruneae (Pipturus, Vil-lebrunea, Debregeasia), and Maoutieae (Leucosyke, Maoutia, Myriocarpa, Phenax). Likewise, Forskohleae comprised subtribes Euforskohleae (Forsskaolea, Droguetia) and Aus-tralineae (Australiana, Didymodoxa, Distemon).

b Procrideae in Weddell (1856); not in Weddell (1854).c Lecantheae in Weddell (1854); in Weddell (1856) same as Weddell (1869).d The tribe Lecantheae in Friis (1993) was called Elatostemeae in Friis (1989).e The tribe given is from Friis (1989); genus was treated as imperfectly known in Friis (1993).f Aboriella was not recognized in Friis (1993) but was placed in Elatostemeae in Friis (1989).


25 lL. The PCR profiles for trnL–trnF, rpll4–rps8–infA–rpl36, matK,rbcL, ITS included an initial denaturation step at 94 �C for 1 min,followed by 30 cycles of 50 s at 94 �C, 1 min at 52 �C (55 �C forITS), 80 s at 72 �C and a final extension at 72 �C for 10 min. ThePCR parameters for 18S were as follows: a 97 �C initial hot startfor 4 min, followed by 35 cycles of 94 �C for 30 s, 52 �C for 30 s,72 �C for 70 s, finished with an extension step of 7 min at 72 �C.The PCR conditions for matR were below: initial denaturation at95 �C for 3 min, 16 cycles of 20 s at 94 �C, 40 s at 65 �C, and degrade1 �C per cycle, 90 s at 72 �C. Additionally, 20 cycles of 20 s at 94 �C,40 s at 50 �C, 90 s at 72 �C, and with a final extension period of5 min at 72 �C. PCR Products were checked on 1% agarose gels be-fore being purified using the DNA purification kit (Sangon Inc.,Shanghai, China) according to the manufacturer’s protocol. Thepurified PCR products were directly used for cycle sequencing withthe ABI Prism BigDye Terminator Cycle Sequencing Kit (AppliedBiosystems, Foster City, California, USA) following the manufac-turer’s recommendations. Both strands of the resulting productswere sequenced by using an ABI 3730xl automated sequencer (Ap-plied Biosystems).

2.3. Phylogenetic analysis

The raw sequences were assembled and edited with SEQUEN-CHER v4.1.4 (Gene Codes, Ann Arbor, Michigan, USA), and thealignments of cleaned sequences were carried out by GENEIOUSv4.8.4 (Drummond et al., 2009) with additional manual refine-ments where necessary. Phylogenetic analyses were conductedfor single DNA regions and the concatenated datasets of the nucle-ar, chloroplast, organelle and the two slow-evolving loci separatelyusing three methods of phylogenetic analysis, namely maximumlikelihood (ML), maximum parsimony (MP) and Bayesian inference(BI). Maximum likelihood analysis was performed in RAxML v7.0.4(Stamatakis et al., 2008); available at http://phylobench.vital-it.ch/raxml-bb/, under the Gamma model of rate heterogeneity. 1000bootstrap replicates were conducted with a rapid bootstrappingand subsequent ML search. The MP analyses were carried out usingPAUP v4.0b10 (Swofford, 2003). Heuristic searches were per-formed with 1000 random addition replicates followed by treebisection–reconnection branch swapping, with zero-lengthbranches collapsed. All character states were treated as unorderedand equally weighted, with gaps treated as missing data. Topolog-ical robustness was assessed by 1000 bootstrap replicates with thesame settings as for heuristic searches. Bayesian inference (BI) ofphylogeny was done using MRBAYES v3.1.2 (Ronquist andHuelsenbeck, 2003) with default priors. The model of best fit forthe single regions as well as combined dataset were determined

by the Akaike information criterion (AIC) in MODELTEST v3.7 (Po-sada and Crandall, 1998; Posada and Buckley, 2004). One cold andthree incrementally heated Markov chain Monte Carlo (MCMC)chains were run for three or five million generations until the aver-age deviation of split frequencies fell well below 0.01. Trees weresampled every 100 generations, estimating all parameters duringthe analysis. A partitioned Bayesian analysis of the combined data-sets was implemented by applying the previously determinedmodels to each data partition. Majority rule (>50%) consensus treeswere constructed after removing the burn-in period samples (thefirst 25% of sampled trees). The posterior probability (PP) valueof each topological bipartition was calculated as the frequency ofthat bipartition across all sampled trees.

3. Results

3.1. Analysis of nuclear sequence data

The sequence characteristics and the best-fit model determinedby MODELTEST for the all of the datasets are given in Table 3. Thetree generated by 18S (Supplementary Fig. S1) was largely consis-tent with that of ITS (Supplementary Fig. S2). The 18S analysis didnot resolve one of the three major clades discussed below (Clade I),which was present in all other analyses. When two datasets werecombined, three analysis methods of ML, MP and BI produced lar-gely identical topology with both resolution and support values in-creased relative to either individual dataset (SupplementaryFig. S4).

3.2. Analysis of chloroplast sequence data

A visual check of phylogenetic trees from the four regions, gen-erated by each of ML, MP and BI analysis, revealed no differencesbetween them that had meaningful statistical support, and noneof the conflicting relationships had bootstrap support of >90%.Therefore the four regions were analyzed together.

For the combined cpDNA dataset, the numbers of variable andparsimony informative sites, tree statistics for the MP analysisand the best-fit model determined by MODELTEST are presentedin Table 3. Phylogenetic analyses of the concatenated sequencesusing MP, ML and BI produced similar topologies (SupplementaryTable S1; Fig. S6).

3.3. Analysis of combined genome data

Reduced taxon sets were examined using two slow-evolvinggenes, mitochondrial matR and nuclear 18S. A visual check

http://phylobench.vital-it.ch/raxml-bb/

http://phylobench.vital-it.ch/raxml-bb/

Table 2Plant materials examined, its provenance, and GenBank accession numbers for all DNA regions successfully sequenced.

Accession Herbarium Taxona Voucher Country GenBank accession numbers for each DNA region

ITS trnL–trnF rbcL rpl14–rps8–infA–rpl36

matK 18S matR

A1 KUN Archiboehmeria atrata WuZY-09469 Guangxi, China KF137798 KF138269 KF138106 KF138434 KF137946 KF137743 KF138078A2 KUN Archiboehmeria atrata WuZY-09414 Guangxi, China KF137799 KF138270 KF138107 KF138435 KF13794723592 K Astrothalamus reticulatus Argent et al.

981987Sabah, Malaysia KF137800 KF138271 KF138108 KF137744

23601 K Australina flaccida Friis, I. et al.12293

Gonder,Ethiopia

KF137801 KF138272 KF138109 KF138436 KF137745

B2 KUN Boehmeria clidemioidesb WuZY-10124 Yunnan, China KF137803 KF138274 KF138111 KF138438 KF137949 KF137746 KF138081B15 KUN Boehmeria clidemioidesb Liuj-10675 Fujian, Chian KF137802 KF138273 KF138110 KF138437 KF137948B16 KUN Boehmeria clidemioides

var. diffusaWuZY-09291 Yunnan, China KF137804 KF138275 KF138112 KF138439 KF137950

B52 KUN Boehmeria densiflora Yi20111199 Taiwan, China KF137805 KF138276 KF138113B53 KUN Boehmeria densiflora WuZY-2012450 Lanyu, Taiwan,

ChinaKF137806 KF138277 KF138114 KF137951

B5 KUN Boehmeria glomerulifera GBOWS990 Yunnan, China KF137807 KF138278 KF138115 KF138440B47 KUN Boehmeria japonica 100024 Hawaii, USA KF137808 KF138279 KF138116B20 KUN Boehmeria longispica Liuj-10722 Zhejiang, China KF137809 KF138280 KF138117 KF138441 KF137952B24 KUN Boehmeria macrophyllab WuZY-10060 Yunnan, China KF137810 KF138281 KF138118 KF138442 KF137953 KF137747 KF138079B28 KUN Boehmeria macrophyllab WuZY-09196 Yunnan, China KF137811 KF138282 KF138119 KF138443 KF137954B21 KUN Boehmeria macrophylla

var. rotundifoliaLiuj-10629 Yunnan, China KF137812 KF138283 KF138120 KF138444 KF137955

B26 KUN Boehmeria macrophyllavar. rotundifolia

WuZY-09468 Guangxi, China KF137813 KF138284 KF138121 KF138445 KF137956

B32 KUN Boehmeria nivea var.nipononivea

Liuj-10679 Zhejiang, China KF137814 KF138285 KF138122 KF138446 KF137957

B6 KUN Boehmeria niveab Liuj-10645 Fujian, China KF137815 KF138286 KF138123 KF138447 KF137958 KF137748 KF138077B33 KUN Boehmeria penduliflora WuZY-09460 Guangxi, China KF137816 KF138287 KF138124 KF138448 KF137959B45 KUN Boehmeria rugulosa LiDZ1071 Near

Kathmandu,Nepal

KF137817 KF138288 KF138125 KF138449 KF137960

B46 KUN Boehmeria sp. RC1555 Mayagdi, Nepal KF137818 KF138289 KF138126 KF138450 KF137961B9 KUN Boehmeria spicata Liuj-10743 Zhejiang, China KF137819 KF138290 KF138127 KF138451 KF137962B38 KUN Boehmeria tomentosa WuZY-09011 Yunnan, China KF137820 KF138291 KF138128 KF138452 KF137963B39 KUN Boehmeria tricuspis WuZY-09430 Guangxi, China KF137821 KF138292 KF138129 KF137964B12 KUN Boehmeria umbrosa WuZY-10336 Yunnan,hina KF137822 KF138293 KF138130 KF138453 KF137965B40 KUN Boehmeria umbrosa WuZY-09465 Guangxi, China KF137823 KF138294 KF138131 KF138454B1 KUN Boehmeria zollingerianavar.

bliniiLiDZ1084 Guizhou, China KF137824 KF138295 KF138132 KF137966

23606 K Cecropia ficifolia Berg et al.P18418

Rio Aripuana,Brazil

KF137825 KF138296 KF138133 KF137749

162A BM Cecropia obtusifolia Monro 3767 El Salvador KF138297 KF138134 KF138455 KF137967C1 KUN Chamabainia cuspidata WuZY-10086 Yunnan, China KF137827 KF138299 KF138136 KF138457 KF137969 KF137751 KF138080C2 KUN Chamabainia cuspidata WuZY-09071 Yunnan, China KF137828 KF138300 KF138137 KF138458 KF137970386A BM Coussapoa parvifolia Monro et al.

6833Costa Rica KF138301 KF138459

De19 KUN Debregeasia elliptica WuZY-10134 Yunnan, China F137829 KF138302 KF138138 KF138460 KF137971De7 KUN Debregeasia elliptica WuZY-10061 Yunnan, China KF137830 KF138303 KF138139 KF138461 KF137972De10 KUN Debregeasia longifolia WuZY-09471 Guangxi, China KF137831 KF138304 KF138140 KF138462 KF137973 KF137752 KF138104De9 KUN Debregeasia longifolia Liuj-10626 Yunnan, China KF137832 KF138305 KF138141 KF138463 KF137974De13 KUN Debregeasia orientalis GLM-07-659 Yunnan, China KF137833 KF138306 KF138142 KF138464 KF137975De15 KUN Debregeasia orientalis 81482 Xizang, China KF137834 KF138307 KF138143 KF138465 KF137976De17 KUN Debregeasia saeneb 81107 Xizang, China KF137835 KF138308 KF138144 KF138466 KF137977De25 KUN Debregeasia sp. RC1556 Mayagdi, Nepal KF137836 KF138309 KF138145 KF138467 KF137978 KF137753De5 KUN Debregeasia squamata WuZY-09204 Yunnan, China KF137837 KF138310 KF138146 KF138468 KF137979D2 KUN Dendrocnide meyeniana Yi20111184 Taiwan, China KF137838 KF138311 KF138147 KF138469 KF137980D1 KUN Dendrocnide sinuata WuZY-09238 Yunnan, China KF137839 KF138312 KF138148 KF138470 KF137981 KF137754 KF138096W1 KUN Dendrocnide sp. WuZY-09035 Yunnan, China KF137840 KF138313 KF138149 KF138471 KF137982D5 KUN Dendrocnide urentissima WuZY-09211 Yunnan, China KF137841 KF138314 KF138150 KF13847223599 K Didymodoxa caffra Abdallah et al.

96/92Mkomazi,Tanzania

KF138315 KF138151 KF138473 KF137755

167 A BM Discocnide mexicana Gereau et al.2205

Mexico KF137842 KF138316 KF138152 KF138474 KF137983

28892 K Droguetia ambigua Styles & Styles2426

Kwazulu-Natal,S Africa

KF137843 KF138317 KF138153 KF138475 KF137756

Dr1 KUN Droguetia iner subsp.urticoides

Liuj-10621 Yunnan, China KF137844 KF138318 KF138154 KF138476 KF137984 KF137757

Dr4 KUN Droguetia iner subsp.urticoides

LiDZ1106 Yunnan, China KF137845 KF138319 KF138155 KF138477 KF137985

E1 KUN Elatostema albopilosum GBOWS1075 Yunnan, China KF137846 KF138320 KF138156 KF138478 KF137986 KF137758 KF138090E2 KUN Elatostema atropurpureum GBOWS298 Yunnan, China KF137847 KF138321 KF138157 KF138479 KF137987E3 KUN Elatostema crytandrifoliumb WuZY-09231 Yunnan, China KF137848 KF138322 KF138158 KF138480 KF137988E4 KUN Elatostema cuspidatumb WuZY-09253 Yunnan, China KF137849 KF138323 KF138159 KF138481 KF137989 KF137759 KF138091E9 KUN Elatostema densistriolatum WuZY-10219 Yunnan, China KF137850 KF138324 KF138160 KF138482 KF137990


Table 2 (continued)



matK 18S matR

E6 KUN Elatostema longibracteatum GBOWS148 Yunnan, China KF137851 KF138325 KF138161 KF138483 KF137991E7 KUN Elatostema parvumb WuZY-09214 Yunnan, China KF137852 KF138326 KF138162 KF138484 KF137992E8 KUN Elatostema petelotii GBOWS342 Yunnan, China KF137853 KF138327 KF138163 KF138485 KF137993E13 KUN Elatostema sp. RC1700 Panchthar,

NepalKF137854 KF138328 KF138164 KF138486 KF137994

E10 KUN Elatostema stewardii Liuj-10729 Zhejiang, China KF137855 KF138329 KF138165 KF138487 KF137995E11 KUN Elatostema

subtrichotomumbGBOWS043 Yunnan, China KF137856 KF138330 KF138166 KF138488 KF137996

E12 KUN Elatostema tenuicaudatumb GBOWS509 Yunnan, China KF137857 KF138167 KF138489 KF1379976515 K Forsskaolea angustifolia Chase 6515 Canary Is

(Spain)KF137861 KF138334 KF138171 KF138493 KF137762 KF138100

16132 K Forsskaolea angustifolia Chase 16132 Canary Is(Spain)

KF137860 KF138333 KF138170 KF138492 KF137761 KF138099

177A BM Gesnouinia arborea C. Evrard 12088 Canary Is(Spain)

KF137862 KF138335 KF138172 KF138494 KF138000

G31 KUN Girardinia diversifoliac GLM-103093 Taiwan, China KF138336 KF138173 KF138495 KF138001G9 KUN Girardinia diversifoliac Liuj-10776 Zhejiang, China KF137863 KF138337 KF138174 KF138496 KF138002 KF137763 KF138095G16 KUN Girardinia diversifolia

subsp. suborbiculataWuZY-10011 Beijing, China KF138338 KF138175 KF138497 KF138003

G17 KUN Girardinia diversifoliasubsp. suborbiculata

WuZY-10014 Beijing, China KF138339 KF138176 KF138498 KF138004

G19 KUN Girardinia diversifoliasubsp. triloba

WuZY-10089 Yunnan, China KF137864 KF138340 KF138177 KF138499 KF138005

G6 KUN Girardinia diversifoliasubsp. triloba

WuZY-10229 Yunnan, China KF138341 KF138178 KF138500 KF138006

Go3 KUN Gonostegia hirta WuZY-09478 Guangxi, China KF137865 KF138342 KF138179 KF138007Go1 KUN Gonostegia parvifolia Liuj-10667 Fujian, China KF137866 KF138343 KF138180 KF138501 KF138008 KF137764 KF138103Go4 KUN Gonostegia parvifolia Liuj-10649 Fujian, China KF137867 KF138344 KF138181 KF138502 KF138009475A BM Gyrotaenia crassifolia Whitefoord

4139Dominica KF138010

473A K Gyrotaenia microcarpa Breedlove 68859 Mexico KF138345 KF13801123567 K Gyrotaenia spicata Acevedo-

Rodriguez, 9614Manchester,Jamaica

KF137765

23597 K Hemistylus macrostachya Pittier 11788 Curricuti,Venezuela

KF137868 KF138346 KF138182 KF138503 KF137766

331A G Hesperocnide tenella Howell 52355 USA KF138504L3 KUN Laportea bulbifera 10CS2513 Shanxi, China KF138348 KF138184 KF138506 KF138014L5 KUN Laportea bulbifera WuZY-09423 Guangxi, China KF137870 KF138349 KF138185 KF138507 KF138015Le1 KUN Lecanthus peduncularis Liuj-10607 Yunnan, China KF137871 KF138350 KF138186 KF138508 KF138016 KF137768Le3 KUN Lecanthus peduncularis 81148 Xizang, China KF137872 KF138351 KF138187 KF138509Le2 KUN Lecanthus petelotii var.

corniculata81066 Xizang, China KF137873 KF138352 KF138188 KF138510 KF138017

Le4 KUN Lecanthus petelotii var.corniculata

WuZY-10374 Yunnan, China KF137874 KF138353 KF138189 KF138511 KF138018

Leu3 KUN Leucosyke quadrinervia WuZY-2012438 Lanyu, Taiwan,China

KF137875 KF138354 KF138190

Leu4 KUN Leucosyke quadrinervia WuZY-2012460 Lanyu, Taiwan,China

KF137876 KF138355 KF138191 KF138019

M2 KUN Maoutia setosa WuZY-2012439 Lanyu, Taiwan,China

KF138356 KF138192 KF138020

154A BM Indet. Kerr 315 Siam KF13801323604 K Myrianthus preussii Bogner 672 Kagala, Gabon KF137769C2A BM Myriocarpa cordata Monro 4630 Panama KF137877 KF138357 KF138193 KF138512 KF137770370A BM Myriocarpa obovata Monro & Penn

6534Belize KF137878 KF138358 KF138513 KF138021 KF137771 KF138084

N1 KUN Nanocnide japonica Liuj-10735 Zhejiang, China KF137879 KF138359 KF138194 KF138514 KF138022 KF137772N4 KUN Nanocnide japonica Liuj-10749 Zhejiang, China KF137880 KF138360 KF138195 KF138515 KF138023N5 KUN Nanocnide lobata Liuj-10701 Zhejiang, China KF137881 KF138361 KF138196 KF138516 KF138024N6 KUN Nanocnide lobata Liuj-10799 Zhejiang, China KF137882 KF138362 KF138197 KF138517 KF138025 KF137773279A K Neodistemon indicum Larsen et al.

2669Thailand KF138363 KF138198 KF138026 KF137774

Ne2 KUN Neraudiakauaiensis 678003 Hawaii, USA KF137883 KF138364 KF138199 KF138027 KF137775 KF138085Ne1 KUN Neraudia melastomifolia 90861 Hawaii, USA KF137884 KF138365 KF138200 KF138518 KF13802823585 K Nothocnide mollissima Beaman 8882 Sabah, Malaysia KF137885 KF138366 KF138201 KF138519 KF13777628719 K Obetia tenax Botha 9 Limpopo, S

AfricaKF137886 KF138367 KF138202 KF138520 KF137777

O2 KUN Oreocnide frutescensc Liuj-10623 Yunnan, China KF137887 KF138368 KF138203 KF138521 KF138029 KF137778 KF138102O8 KUN Oreocnide frutescensc WuZY-09428 Yunnan, China KF137888 KF138369 KF138204 KF138522 KF138030O12 KUN Oreocnide frutescens subsp.

occidentalisWuZY-09260 Yunnan, China KF138370 KF138205 KF138523 KF138031

11077 K Parietaria judaica Fay MFF185 England, UK KF138371 KF138206 KF138524 KF137779Pa1 KUN Parietaria micrantha WuZY-10373 Yunnan, China KF138372 KF138207 KF138525 KF137780 KF138094

(continued on next page)


Table 2 (continued)



matK 18S matR

Pe1 KUN Pellionia macrophylla GBOWS1374 Yunnan, China KF137889 KF138373 KF138208 KF138526 KF138032Pe2 KUN Pellionia paucidentatab GBOWS130 Yunnan, China KF137890 KF138374 KF138209 KF138527 KF138033 KF137781Pe3 KUN Pellionia radicans GBOWS962 Yunnan, China KF137891 KF138375 KF138210 KF138528 KF138034Pe4 KUN Pellionia repens WuZY-09159 Yunnan, China KF137892 KF138376 KF138211 KF138529 KF138035 KF137782 KF138089Pe5 KUN Pellionia tsoongii WuZY-09495 Guangxi, China KF137893 KF138377 KF138212 KF138530 KF138036378A BM Phenax mexicanus Breedlove &

Smith 21717Mexico KF138378

P1 KUN Pilea angulata subsp.petiplaris

81352 Xizang, China KF137894 KF138379 KF138213 KF138531 KF138037 KF137783 KF138092

P3 KUN Pilea cavalerieib LiDZ1080 Guizhou, China KF137895 KF138380 KF138214 KF138532 KF138038P11 KUN Pilea insolens STET1838 Xizang, China KF137896 KF138381 KF138215 KF138533 KF138039P5 KUN Pilea longipedunculata WuZY-09199 Yunnan, China KF137897 KF138382 KF138216 KF138534 KF137784 KF138093P6 KUN Pilea martinii WuZY-09107 Yunnan, China KF137898 KF138383 KF138217 KF138535 KF138040P20 KUN Pilea melastomoides WuZY-09463 Guangxi, China KF137899 KF138384 KF138218 KF138536 KF138041P21 KUN Pilea microphylla WuZY-09174 Yunnan, China KF137900 KF138385 KF138219 KF138537 KF138042P22 KUN Pilea microphylla WuZY-09399 Guangxi, China KF137901 KF138386 KF138220 KF138538 KF138043P9 KUN Pilea oxyodon STET2663 Xizang, China KF137902 KF138387 KF138221 KF138539 KF138044P24 KUN Pilea plantaniflora LiDZ1081 Guizhou, China KF137903 KF138222 KF138540 KF138045P10 KUN Pilea pumilab WuZY-09335 Yunnan, China KF137904 KF138388 KF138223 KF138541 KF138046P26 KUN Pilea sinofasciata Liuj-10605 Yunnan, China KF137905 KF138389 KF138224 KF138542 KF138047P12 KUN Pilea sp. RC1554 Myagdi, Nepal KF137906 KF138390 KF138225 KF138543 KF138048P29 KUN Pilea verrucossab Liuj-09527 Yunnan, China KF137907 KF138391 KF138226 KF138544 KF138049Pip1 PE Pipturus arborescens 11879 Taiwan, China KF137908 KF138392 KF138227 KF138545 KF137785Pip7 KUN Pipturus arborescens WuZY-2012437 Lanyu, Taiwan,

ChinaKF137909 KF138393 KF138228 KF138050

Pip5 KUN Pipturus kauaiensis 90441 Hawaii, USA KF137910 KF138394 KF138229 KF138546 KF138051Pip6 KUN Pipturus ruber 80829 Hawaii, USA KF137911 KF138395 KF138230 KF138547 KF138052Pi1 KUN Poikilospermum

lanceolatumWuZY-09235 Yunnan, China KF137912 KF138396 KF138231 KF138548 KF138053 KF137786 KF138097

Pi2 KUN Poikilospermum suaveolens GBOWS736 Yunnan, China KF137913 KF138397 KF138232 KF138549Pi3 KUN Poikilospermum suaveolens WuZY-09160 Yunnan, China KF137914 KF138398 KF138233 KF138550 KF138054Po5 KUN Pouzolzia argenteonitida STET1914 Xizang, China KF137915 KF138399 KF138234 KF138551282A K Pouzolzia guineensis Bidgood et al.

3008Tanzania KF138400 KF138235 KF138552 KF138055

288A K Pouzolzia mixta Lovett &Congdon 2945

Tanzania KF137916 KF138401 KF138236 KF138553

Po2 KUN Pouzolzia sanguinea var.elegans

STET2322 Xizang, China KF137917 KF138402 KF138237 KF138554 KF138056 KF137787 KF138086

Po6 KUN Pouzolzia sanguineab WuZY-09483 Guangxi, China KF137918 KF138403 KF138238 KF138555 KF138057Po9 KUN Pouzolzia sp. RC1682 Panchthar,

NepalKF137919 KF138404 KF138239 KF138556 KF138058

Po4 KUN Pouzolzia zeylanicab WuZY-10167 Yunnan, China KF137920 KF138405 KF138240 KF138557 KF138059Po7 KUN Pouzolzia zeylanicab Liuj-10686 Zhejiang, China KF137921 KF138406 KF138241 KF138558 KF138060 KF138101Pr1 KUN Procris wightiana WuZY-10090 Yunnan, China KF137922 KF138407 KF138242 KF138559 KF138061Pr2 KUN Procris wightiana WuZY-10230 Yunnan, China KF137923 KF138408 KF138243 KF138560 KF138062 KF137788 KF13808823596 K Rousselia humulis Ekman 4973 Hispaniola,

HaitiKF137789

S1 KUN Sarcochlamys pulcherrima STET2106 Xizang, China KF137924 KF138409 KF138244 KF138561 KF137790 KF138105302A BM Sarcopilea domingensis Monro 5408 Dominican

RepublicKF137925 KF138410 KF138245 KF138562 KF137791 KF138076

312A BM Soleirolia soleirolii Monro in cult. Cult. In UK KF137926 KF138411 KF138246 KF138563 KF138063T1 KUN Touchardia latifolia Jffrey201101 Hawaii, USA KF137927 KF138412 KF138247 KF138564 KF137792C11A BM Urera alceifolia Monro 4346 Bocas del Toro,

PanamaKF138413 KF138248 KF138565 KF138064

C4A BM Urera baccifera Monro 4663 Bocas del Toro,Panama

KF137928 KF138414 KF138249 KF138566 KF138065

23561 K Urera caracasana Wood 8834 Chuquisaa,Bolivia

KF137929 KF138415 KF138250 KF138567 KF137793

Ur1 KUN Urera glabra 100694 Hawaii, USA KF137930 KF138416 KF138251 KF138568 KF137794377A C Urera hypselodendron Friis et al. 4125 Ethiopia KF138417 KF138252 KF138569 KF138066313A BM Urera lianoides Solano 6825 Costa Rica KF138418 KF138253 KF138570L2 KUN Urera sp. WangH-201001 South Africa KF137931 KF138419 KF138254 KF138571 KF137795 KF138098374A C Urera trinervis Friis et al. 3920 Ethiopia KF137932 KF138420 KF138255 KF138572U1 KUN Urtica angustifolia 10CS2322 Inner Mongolia,

ChinaKF137933 KF138421 KF138256 KF138573 KF138067 KF137796

U2 KUN Urtica ardens 81152 Xizang, China KF137934 KF138422 KF138257 KF138574U3 KUN Urtica atrichocaulis WuZY-10358 Yunnan, China KF137935 KF138423 KF138258 KF138575 KF138068U21 KUN Urtica dioica BROWP135 Edinburgh, UK KF137936 KF138424 KF138259 KF138576U4 KUN Urtica fissa WuZY-10378 Yunnan, China KF137937 KF138425 KF138260 KF138577 KF138069U14 KUN Urtica hyperborea 80966 Xizang, China KF137938 KF138426 KF138261 KF138578 KF138070U5 KUN Urtica hyperborea 81200 Xizang, China KF137939 KF138427 KF138262 KF138579 KF138071U7 KUN Urtica mairei WuZY-09354 Yunnan, China KF137940 KF138428 KF138263 KF138580 KF137797U18 KUN Urtica sp. Lixinhui-1102 Kenya, Africa KF137941 KF138429 KF138264 KF138581 KF138072


Table 2 (continued)



matK 18S matR

U19 KUN Urtica sp. LL-2011 Melbourne,Australia

KF137942 KF138430 KF138265 KF138582

U10 KUN Urtica triangularis subsp.pinnatifida

80860 Xizang, China KF137943 KF138431 KF138266 KF138583 KF138073

U11 KUN Urtica zayuensis WuZY-10133 Yunnan, China KF137944 KF138432 KF138267 KF138584 KF138074U17 KUN Urtica zayuensis WuZY-10361 Yunnan, China KF137945 KF138433 KF138268 KF138585 KF138075OutgroupsULM2 KUN Celtis kunmingensis WZY-1102 Yunnan, China KF137826 KF138298 KF138135 KF138456 KF137968 KF137750 KF138087F1 KUN Fatoua villosa Liuj-10688 Zhejiang, China KF137858 KF138331 KF138168 KF138490 KF137998 KF137760 KF138082F2 KUN Fatoua villosa GLM-103136 Taiwan, China KF137859 KF138332 KF138169 KF138491 KF137999H1 KUN Humulus scandense Liuj-10681 Zhejiang, China KF137869 KF138347 KF138183 KF138505 KF138012 KF137767 KF138083

a Names follow Friis (1993) and Chen et al. (2003).b This accession was the type variety for this species.c This accession was the type subspecies for this species.


indicates no topological contradictions with significant bootstrapsupport congruence between these two datasets; therefore, theseregions were combined in our analyses (Supplementary Fig. S5).Compared to either gene individually, the combined data producedstronger support for basal relationships within Urticaceae, withmaximum support (100/100/1, ML/MP/BI, the same order thereaf-ter) for a sister relationship between Clades II and III, and for themonophyly of Urticaceae.

Preparing to combine all data, we first compared combinedcpDNA data with that for mitochondrial matR. Finding no incon-gruences that had strong bootstrap support, we combined thesedatasets to give an organelle phylogeny (Supplementary Fig. S7).Comparing this with the combined nuclear DNA (SupplementaryFig. S4) tree, there were some discrepancies between their topolo-gies, summarized below. However, these generally concerned rela-tionships between and among small subclades (hence between notwithin genera), and always had weak bootstrap support (Supple-mentary material, Table S1). Therefore, all data were combinedinto, and analyzed as, a single matrix. MP, ML and BI analyses ofthis dataset not only yielded mostly congruent topologies for thecombined datasets from the nuclear and organelle genomes withinUrticaceae, but also resulted in greater resolution as compared tothe separate analyses (Fig. 1). The relationships described in thenext section are from the combined tree unless otherwise stated.

3.4. Summary of generic relationships according to the phylogeny

All species examined of Urticaceae and Cecropiaceae formed amonophyletic clade with maximum support in the combined,cpDNA and 18S topologies, but fairly strong support also fromITS (92/–/1.00) and matR (80/64/0.79). Cecropiaceae was splitacross two clades within Urticaceae. The ingroup can be subdi-vided into four major clades, here termed I, II, III and IV, and de-scribed in detail below. Clades II and III had at least 85% supportsin all analyses for combined DNA, whereas Clade I and IV had nosupport in the 2 nrDNA combined analysis. Support for Clades II,III and IV was similar across datasets, however Clade I was not sup-ported by ITS or 18S data. Clades I and IV were sister to one anotherwith maximum support; the same was true of Clades II and III. Forconvenience, Clades I, II, III and IV were subdivided into namedsubclades, and the largest subclade of Clade I (Clade 1A) was fur-ther divided into nine smaller subclades. Subclades were chosen,as far as possible, to contain a single genus and/or have strong sta-tistical support. Support values for all subclades, and groupingsthereof, across all datasets and analyses are given in Supplemen-tary material, Table S1.

Of the genera examined for which more than one species wassampled, only three were split across more than one subclade,and were hence polyphyletic. These were Boehmeria (subclades1A1, 1A3 and 1A7), Pouzolzia (subclades 1A5 and 1A7), and Pellionia(subclades 2A and 2C). In addition, four more genera were stronglysupported as paraphyletic with respect to those indicated in brack-ets; these were Parietaria (Gesnouinia and Soleirolia Clade 1C),Droguetia (Australina, part of Clade 1D), Pilea (Sarcopilea; subclade2F) and Urtica (Hesperocnide; subclade 3A). Paraphyly of Urtica,however, was based on a single cpDNA region (rpl14–rps8–infA–rpl36) successfully sequenced for Hesperocnide. The clade of Urera(subclade 3F) likewise had Poikilospermum, Touchardia, and possi-bly Obetia nested within it. The same was indicated, but with weak-er support, for Gyrotaenia (Myriocarpa, Clade 2D).

Clade I comprised a large subclade (1A) which was successivelysister to Phenax (1E), and Oreocnide (1B), although both these rela-tionships had moderate support, and only from the cpDNA dataset(Supplementary material, Table S1). The remaining two subclades(1C and 1D) were sister to each other with relatively low support(56/–/0.73); however this support was higher in the 18S analysisthan in any other, and disappears altogether when 18S data is com-bined with ITS, indicating some incongruence. Clade 1D comprisedDidymodoxa and Forsskaolea as successive sister groups to Drogue-tia plus Australina.

Within Clade 1A, a monophyletic Debregeasia (subclade 1A2)was sister to subclade 1A3, within which Astrothalamus was sup-ported as sister to a poorly resolved clade of Boehmeria, Archi-boehmeria and Sarcochlamys species by all datasets except 18S.These two clades were well-supported as sister to Clade 1A1(Boehmeria), though support for this came only from datasets thatincluded cpDNA (Supplementary material, Table S1). In the otherpart of Clade 1A, the genus Pouzolzia was split between subclade1A5 (with Rousselia, Hemistylus, Neodistemon) and 1A7 (withBoehmeria rugulosa). Subclade 1A7 formed a larger clade with1A8 (Neraudia) and 1A9 (Nothocnide and Pipturus) that was sup-ported by all datasets except 18S (Supplementary material,Table S1). Subclade 1A5 was sister to 1A6 (Gonostegia), with sup-port from all datasets; likewise the grouping of1A5 + 1A6 + 1A7 + 1A8 + 1A9 had maximum support from all anal-yses that included cpDNA, and maximum Bayesian support acrossevery dataset.

Within Clade II, Clades 2E (Lecanthus) and 2F (Pilea + Sarcopi-lea) each had maximum support; they were sister to one anotherwith strong support from all datasets except ITS. Of two cladescontaining Pellionia species, 2A (P. paucidentata, P. radicans, P.macrophylla) had maximum support from almost all datasets

Table 3Characteristics of datasets.

Information ITS 18S matR trnL–trnF

rpll4–rps8–infA–rpl36

matK rbcL 2nrDNA 4cp DNA 5organelleDNA

18S + matR Combined7DNA

No. ingroups 144 52 27 161 148 126 159 151 166 166 53 169No. outgroups 4 3 3 4 4 4 4 4 4 4 3 4Aligned length 991 1733 1988 1735 1956 966 750 2724 5407 7395 3721 10119No. variable characters 770 207 167 896 1119 587 210 977 2812 2979 374 3956No. parsimony-informative

characters654 143 49 657 870 479 165 797 2171 2220 192 3017

Tree length 7262 520 180 2450 3128 1744 608 7716 8145 8333 671 16125Consistency index (CI) 0.251 0.515 0.95 0.576 0.56 0.527 0.447 0.271 0.534 0.543 0.654 0.41Retention index (RI) 0.703 0.741 0.944 0.888 0.888 0.884 0.865 0.709 0.878 0.879 0.796 0.812Rescaled consistency index (RC) 0.177 0.382 0.897 0.511 0.479 0.466 0.387 0.192 0.469 0.477 0.521 0.333Model GTR+I+G GTR+I+G TVM+G GTR+I+G GTR+I+G GTR+G GTR+I+G GTR+I+G GTR+I+G GTR+I+G GTR+I+G GTR+I+G


and analyses, and was strongly supported by all as sister to subc-lade 2B (Elatostema). These in turn were sister in all datasets tosubclade 2C, which contained P. tsoongii and P. repens as succes-sive sisters to Procris, although cpDNA data did not support subc-lade 2C. The remaining subclade, 2D (Gyrotaenia + Myriocarpa)was strongly supported in all analyses, but support was weakfor relationships within the clade, and for its sister relationshipto 2E + 2F (68/–/0.66).

Clade III comprised six subclades, all of which had maximumsupport from the combined dataset, and all but one of which werestrongly supported across all datasets. The exception, subclade 3E(Laportea), was not sampled for matR or 18S, but had maximumsupport from each of ITS and cpDNA. Subclade 3F had maximumsupport and contained Urera, within which were nested Touchar-dia, Poikilospermum and Obetia; the presence of Poikilospermum inthis clade means that Cecropiaceae is not a monophyletic group.Subclades 3A (Urtica plus Hesperocnide), 3B (Nanocnide), 3C (Dend-rocnide plus Discocnide) and 3D (Girardinia) supported monophylyfor all genera involved except for Urtica (paraphyletic with respectto Hesperocnide) and the monotypic Discocnide. As sister to one an-other, Clades 3A and 3B had maximum support, but otherwise rela-tionships among these clades were not supported or resolved.

The final clade, IV, comprised two subclades, each strongly sup-ported as monophyletic. Of these, 4A (80/–/0.86) comprised threegenera previously assigned to Cecropiaceae (Cecropia, Myrianthusand Coussapoa), whereas 4B (100/88/1) contained Leucosyke andMaoutia.

4. Discussion

4.1. Monophyly of Urticaceae

Our results strongly supported Urticaceae sens. lat. (i.e. includ-ing Cecropiaceae) as a monophyletic group. Every locus examinedsupported this relationship, but combining them all into a singledataset improved the resolution and support for relationshipswithin Urticaceae. Furthermore, there was little incongruence be-tween datasets, and that which there was tended to involveslow-evolving loci for which fewer taxa were sampled, and hencecould reflect undersampling or lack of phylogenetic signal. Therewere no apparent cases of discordance that might reflect reticulateevolution. Therefore, the discussion that follows is largely based onthe results from the combined dataset.

As in previous molecular phylogenies (Hadiah et al., 2008; seealso Sytsma et al., 2002; Datwyler and Weiblen, 2004; Zeregaet al., 2005; Monro, 2006), Cecropiaceae was biphyletic and bothlineages were nested within Urticaceae, indicating that it shouldnot be recognized as a distinct family. However, Urticaceae plusCecropiaceae comprised four well supported clades, the smallest

of which (Clade IV) comprised three genera previously assignedto Cecropiaceae, i.e. Cecropia, Coussapoa, and Myrianthus, plustwo genera from Urticaceae.

Clade II corresponded approximately to Lecantheae or Lecan-thoideae of the classifications of Friis (1993) and Kravtsova(2009). Likewise, Clade III corresponded approximately to Urticeaeof Friis (1993) or Urticoideae of Kravtsova (2009). In each casethere were exceptions, as detailed below.

Clade I contained, with few exceptions, members of Friis’ (1993)other three tribes Forsskaoleae, Parietarieae and Boehmerieae; ittherefore corresponded closely with Kravtsova’s (2009) third sub-family, Boehmerioideae.

Therefore, we infer that the pistil with a single stigma and a ba-sal or subbasal orthotropous ovule could be considered as impor-tant diagnostic characteristics of Urticaceae sens. lat., for examplethe sister family Moraceae has two stigmas and its ovules areeither laterally or apically fixed, and never orthotropous. Con-versely, characters such as the difference between inflexed andstraight stamens may be over-emphasized in delimiting taxa atthe rank of family.

4.2. Relationships of genera previously assigned to Cecropiaceae

The recognition of Cecropiaceae was based on morphologicalcharacters such as basal/subbasal and orthotropous/suborthotropousovules, stamens filaments being mostly straight in the bud and notbending outward elastically, and plants never tending towards a her-baceous habit (Berg, 1978). However, this family was always contro-versial (Bonsen and Ter Welle, 1983, 1984; Datwyler and Weiblen,2004; Monro, 2006; Hadiah et al., 2008). As with a previous molecularstudy (Hadiah et al., 2008), our data indicated that Cecropiaceae wasneither distinct from Urticaceae nor monophyletic.

Of the four Cecropiaceae genera examined, Poikilospermum isnot closely related to Cecropia, Coussapoa, and Myrianthus, and in-stead is nested within subclade 3F, confirming Hadiah et al.’s(2008) finding of a close relationship to Urera. Any characters link-ing it to other Cecropiaceae would appear to be homoplasious. Un-like these, the stamens of Poikilospermum straighten only afterantithesis (Chew, 1963). Conversely, it resembles Urera in its elon-gated cystoliths (Friis, 1993), and like some Urera species it is awoody climber. It is the only Asian genus in Clade 3F, althoughthe whole clade is tropical; this indicates that it might be an Asianvicariant of Urera. If Poikilospermum is excluded, the remainingmembers of Cecropiaceae may be a natural group, as the three gen-era examined (Cecropia, Coussapoa, Myrianthus) formed a subcladewithin Clade IV, sister to Leucosyke and Maoutia of Urticaceae andrelatively basal within the family. Nesting of this within Urticaceaeprecludes family status of the subclade but tribal status might beappropriate (see Section 4.6 below).


4.3. Phylogenetic relationships within Clade I

Clade I was strongly supported as monophyletic; it comprisedall species examined of Friis’ (1993) Parietarieae and Forsskaoleae,and all but four genera of Friis’ (1993) Boehmerieae. Of those four,Maoutia and Leucosyke fall within Clade IV (see below), Touchardiain Clade III and Myriocarpa in Clade II. Touchardia and Myriocarpadiffer from other Boehmerieae in presence of unlignified elements(Bonsen and Ter Welle, 1984), presence of elongated-linear cysto-liths, and absence of arachnoid tomentum on the underside of theleaves (Friis, 1989). Furthermore, female tepals are free in Touchar-dia and absent in Myriocarpa, whereas Boemerieae perianths aretypically tubular. Hence morphological evidence corroboratesmolecular in showing that neither belongs in Boehmerieae.

Clade I comprised five subclades among which relationships arestrongly but not unequivocally supported. Clade 1D comprised allgenera of Forsskaoleae examined, making this tribe monophyleticin our analysis, consistent with Hadiah et al. (2008). Hence thetribe’s defining character of male flowers with only one stamenis probably a genuine apomorphy. Within Forsskaoleae, however,Australina is nested within Droguetia, requiring the former to besunk or the latter split to achieve generic monophyly.

Sister to Clade 1D is Clade 1C, again consistent with Hadiahet al. (2008), Clade 1C comprised three genera of Parietarieae,among which the shrubby island endemic Gesnouinia and thetiny-leaved creeper Soleirolia are nested within Parietaria. Hencegeneric limits here need reexamination. The other two genera ofParietarieae, Rousselia and Hemistylus are placed in Clade 1A5 withPouzolzia, making Parietarieae biphyletic. Morphologically, Rouss-elia and Hemistylus are linked to Pouzolzia by having free stipules,similar leaf morphology and paired female flowers in leaf axil withan involucre of two large bracts. Indeed, Gaudichaud (1830) placedall three in Parietarieae. Moreover, Rousselia and Hemistylus arelinked to Pouzolzia and Neodistemon by pericarp structure, a similarform of heterocarpy, and the presence of dense two-fruited involu-crate infructescence (Kravtsova, 2009), providing synapomorphiesfor subclade 1A5 and supporting the placement of Rousselia andHemistylus in Boehmerieae. Meanwhile, absence of stipules pro-vides a synapomorphy for a reduced Parietarieae comprising onlyClade 1C (Gesnouinia, Solierola and Parietaria).

Subclades 1A, 1B and 1E correspond approximately to Friis’(1993) Boehmerieae. Although the monophyly of 1A + 1B + 1E isonly modestly supported, the shared morphological characters ofBoehmerieae also support this relationship, despite several excep-tions both within and outside the clade. Basal in most datasets andanalyses was Clade 1B, containing Oreocnide, whose stigmas areeither peltate with long ciliate margin or penicillate-capitate, acharacter otherwise seen in Urticaceae only in Gibbsia (Chen,1985), and occasionally occur in some Debregeasia and Urera spe-cies. Furthermore, the individual fleshy receptacle for each fruit(Wang and Chen, 1995; Chen et al., 2003) (see Fig. 2I) also can beconsidered as a synapomorphy of Oreocnide. Like Phenax (Clade1E), Oreocnide falls outside the main clade of Boehmerieae (Clade1A) and could therefore be treated as a separate tribe of its own.

Archiboehmeria is characterized by a unique lingulate stigma(Chen, 1980), but as yet is too poorly known for its phylogeneticaffinities to be settled (Friis, 1993). Our data places it firmly withinBoehmerieae, close to Sarcochlamys and some species of Boehmeria.

Treating subclades 1A + 1B + 1E as a revised Boehmerieae there-fore fits the molecular data. There are no known morphologicalsynapomorphies for this group, although most genera have freeor united female perianth, and bracts never united into an involu-cre. Of the larger genera within Clade 1A, consistent with Hadiahet al. (2008), Debregeasia appeared to be monophyletic, whereasBoehmeria and Pouzolzia were not. Boehmeria was split across threestrongly supported clades (1A3, 1A5 and 1A7) and hence is almost

certainly not a natural group. Pouzolzia was split betweensubclades 1A5 and 1A7, in neither of which did the Pouzolzia spe-cies form a natural group. No morphological characters are knownthat distinguish the members of Boehmeria or Pouzolzia in onesubclade from those in another. This means that the morphologicalcharacters defining both genera may be plesiomorphic withinClade 1A, and leaving no good solution for how to make eithergenus monophyletic. Possibly a more intensive sampling mighthelp with defining these clades in future.

4.4. Phylogenetic relationships within Clade II

Clade II mostly corresponded to Friis’ (1993) Lecantheae, ex-cept that Myriocarpa and Gyrotaenia were included. Differencesbetween Myriocarpa and Boehmerieae are noted above, but it alsoshares with Lecantheae elongated-linear cystoliths, indicatingthat the importance of this character may be greater than previ-ously supposed (Friis, 1993). Gyrotaenia’s current placement is inUrticeae (Friis, 1993) despite it its absent stinging hairs, penicil-late-capitate stigma, and punctiform and linear cystoliths; henceagain morphology does not contradict an alternative placementin Lecantheae. These two genera together form a well-supportedsubclade (1D) within which neither is resolved as monophyletic,despite the apparent synapomorphy of fleshy peduncles in Gyro-taenia. More data is required to test whether these genera aretruly distinct, or if fleshy peduncles evolved multiple times in thisclade.

Clade II includes the two largest genera in Urticaceae, Pilea andElatostema. Of these, Elatostema is monophyletic, whereas Pilea isonly monophyletic if Sarcopilea is included within it. Hence theonly species of Sarcopilea may be better regarded as merely anaberrant member of Pilea. Sister to Pilea is a monophyletic Lecan-thus, supporting the recognition of these as separate genera, as isalso indicated by differences in achene characters (Chen et al.,2003).

Elatostema is widespread across in tropical and subtropical re-gions of Africa, Asia, and Oceania (Wang, 1995, 2012), with morethan 200 of its ca. 500 species recorded from China, includingmany local endemics (Wang, 2012). However, generic limitsinvolving Elatostema, Pellionia and Procris have been controver-sial. Schröter and Winkler (1935, 1936) considered Pellionia asa subgenus within Elatostema, whereas Hallier (1986) consideredboth Pellionia and Procris to be subgenera within Elatostema.However, Wang (1980a,b, 1995, 2012) supported recognizingElatostema as distinct from Pellionia on the basis of its perianthlobes of female flowers being much shorter than the ovary orstrongly reduced, and not corniculate at apex. Friis (1993) wasuncertain about the monophyly or affinities of Pellionia. Previousmolecular analysis based on one DNA region (trnL–F) (Hadiahet al., 2003) also showed Procris nested within a subset of Pellio-nia and did not conflict the monophyly of Elatostema excludingPellionia. Our data strongly supported the monophyly of Elatoste-ma, whereas Pellionia was split between two subclades (2A, 2C),one of which also had Procris nested within it. Our work there-fore supports recognition of Elatostema and Procris but moreintensive taxon sampling is needed to resolve generic limits inPellionia and Procris.

4.5. Phylogenetic relationships within Clade III

The strongly supported Clade III comprises 11 genera, which islargely consistent with Friis’ (1993) tribe Urticeae, except thatGyrotaenia was excluded and Touchardia and Poikilospermum wereincluded. Touchardia has no obvious morphological links to Urti-ceae but is nested within Urera, as might be Poikilospermum and


Obetia though support is lacking. Further research on generic rela-tionships in this morphologically variable Clade (3F) is needed.

Of the other five subclades, three comprise one monophyleticgenus each (Nanocnide, Laportea, Girardinia), one contains twosister genera (Dendrocnide with Discocnide), and the last comprisesUrtica with Hesperocnide nested within it; however only one DNAregion was successfully sampled for Hesperocnide (rpl14–rps8–infA–rpl36) so this conclusion must be viewed as tentative.Hesperocnide differs from Urtica in its tubular female perianth thatclosely encloses the ovary, and its sessile inflorescences withmany-flowered glomerules or cymules (Friis, 1993).

A sister relationship between Urtica and Nanocnide is stronglysupported, and there is weaker support for these forming a cladewith Girardinia, Dendrocnide and Discocnide. This group of generaare all characterized by stinging hairs (see Fig. 2N–P). Outside thisgroup fall Laportea and Clade 3F, whose relationships to it and toone another within Clade III are not supported. Furthermore, onlyone Laportea species was sampled in this study, so the monophylyand relationships of this genus require further investigation.

4.6. Phylogenetic relationships within Clade IV

Clade IV comprises two subclades. Subclade 4A has fairly strongsupport, and comprises of three genera from Cecropiaceae: Cecro-pia, Coussapoa and Myrianthus. Based on morphology, the unsam-pled Pourouma and Musanga of Cecropiaceae presumably alsobelong here.

Subclade 4B includes the one sampled species each of Maoutiaand Leucosyke, both of which were previously placed in Boehmer-ieae based on no or very short female perianth (Weddell, 1856;Friis, 1989, 1993). Morphologically, the distinctions between Leu-cosyke and Maoutia have always been vague, and both were unitedwith Gibbsia (not sampled in our study) by Mabberley (2008).Hence our detection of a close relationship between Maoutia andLeucosyke fits their morphology.

Because Clades I, II and III each comprise one or more distincttribes, it seems appropriate to recognize Clade IV at a similar tax-onomic level. However, there are no uniting characters betweensubclades 4A and 4B. Subclade 4A is defined by the characters ofCecropiaceae excluding Poikilospermum, such as straight stamens,woody habit and no distinctive cystoliths. It could therefore be rec-ognized as tribe Cecropieae. However, Clade 4B is morphologicallyvery similar to Boehmerieae, so treating Clade 4A as a tribe wouldeither create a paraphyletic Boehmerieae or necessitate a new tribe(Clade 4B) with no known morphological distinctions from theexisting Boehmerieae.

5. Conclusions

Our study provides the most comprehensive phylogeny so far ofUrticacae, both for taxon sampling and DNA regions examined; itindicates that the family is monophyletic if Cecropiaceae is mergedwith it. Most clades were well-supported, and some large generawere clearly monophyletic, but a few instances of paraphyly weredetected and clusters of closely related genera exist where exten-sive taxon sampling may be necessary to elucidate their relation-ships and meaningful generic limits. Moreover, as only 122 of�2500 Urticaeae species were examined, therefore phylogeneticunderstanding of the family overall, especially at generic level,remains incomplete. Relationships between some subcladesremained poorly supported, though this does not hamperclassification.

Broadly speaking, the classifications of Friis (1993) and Kravts-ova (2009) were both generally supported, subject to the transfer-ring of certain genera between tribes to match clade membership.

Earlier classifications by Gaudichaud (1830) and Weddell (1854,1856, 1869) were likewise broadly supported at the tribal level.Treating Clades II and III as a single tribe or subfamily each createsrelatively few taxonomic problems or morphological anomalies.Cecropiaceae (excluding Poikilospermum) may be best treated asa tribe within Urticaceae, but this requires that a new tribe iserected containing Leucosyke, Maoutia and probably Gibbsia, to pre-serve a monophyletic Boehmerieae. Within Clade I could be recog-nized an unchanged Forsskaoleae, a reduced Parietarieae and analtered Boehmerieae, making seven tribes in total. Treating CladesI, II and III as subfamilies, as per Kravtsova (2009), fit the moleculardata but logically requires that Clade IV is treated as a fourth sub-family, which would contain two very dissimilar clades. Alterna-tively, Clades I and IV could be treated as a single subfamily, butit would be very morphologically diverse.

Useful further work on the family would therefore include fur-ther expanding taxon and DNA region sampling for the family,molecular dating and biogeographic inference, focused examina-tions of apparently non-monophyletic genera such as Boehmeriaand Urera, and evolutionary studies of aberrant genera such as Poi-kilospermum and Touchardia. The current phylogeny provides afirm starting point for all such work.

Acknowledgments

We are deeply indebted to Profs. Wen-Tsai Wang and Chia-JuiChen (both PE) for their great help for specimen identification,encouragements and some constructive suggestions on the manu-script. We are also very grateful to Profs. Ib Friis (SNM) and Chris-tine Melanie Wilmot-Dear (K) for their expert advice on the results.We thank particularly Lian-Ming Gao, Jun-Bo Yang, Shu-DongZhang, Jie Cai, Lu Lu, Wei Jiang, Ram C. Poudel, Yu-Xiao Zhang(all KUN), Tsai-Wen Hsu (TAIE), and Jeffrey Boutain (University ofHawaii) for their kind help with sample collection and technicalassistance in the lab. We are grateful to the National TropicalBotanical Garden of Hawaii for providing some molecular samplesfor this study. We are particularly grateful to Royal Botanic Gar-dens, Kew for providing kindly DNA samples of some taxa. Theauthors also thank both anonymous reviewers for their valuablecomments on the manuscript. The present study was supportedby a Keynote Project of National Natural Science Foundation of Chi-na (Grant No. 40830209), and the Research Fund of the NaturalHistory Museum, London and of the Systematics Association andLinnean Society of London. Zeng-Yuan Wu was supported by theChina Scholarship Council for 1 year study at the Natural HistoryMuseum, and the Royal Botanic Gardens, Kew.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ympev.2013.06.022.

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Molecular phylogeny of the nettle family (Urticaceae) inferred ...

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