wu-2013

Full text

Molecular phylogeny of the nettle family (Urticaceae) inferred from
multiple loci of three genomes and extensive generic sampling
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, China
b
Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China
c
Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK
d
Department of Life Sciences, Natural History Museum, London SW7 5BD, UK
e
Institute of Molecular Plant Sciences, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3JH, UK
f
Royal Botanic Garden Edinburgh, 20A Inverleith Row, Edinburgh, EH3 5LR, Scotland, UK
g
University of Chinese Academy of Sciences, Beijing 100049, China
article info
Article history:
Received 6 March 2013
Revised 26 June 2013
Accepted 27 June 2013
Available online 9 July 2013
Keywords:
Molecular phylogeny
Urticaceae
Phylogenetic trees
Three genomes
Cecropiaceae
infra-familial relationships
abstract
Urticaceae is one of the larger Angiosperm families, but relationships within it remain poorly known. This
study 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 two
nuclear (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. Major
results 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-supported
clades; (4) previously erected tribes or subfamilies were broadly supported, with some additions and
alterations; (5) the monophyly of many genera was supported, whereas Boehmeria, Pellionia, Pouzolzia
and 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 clarify
that 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.
Ó2013 Elsevier Inc. All rights reserved.
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 the
largest 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 life
forms, 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 orthotropous
ovule, having filaments inflexed in bud, and some species armed
with distinctive stinging hairs (Friis, 1989, 1993; Wang and Chen,
1995; Chen et al., 2003). Economically important genera include
many (e.g. Boehmeria and Girardinia), whose stem fiber is of high
quality and therefore used to make cloth, fishing nets, ropes and
some industrial materials (Singh and Shrestha, 1988; Angelini
et al., 2000; Bodros and Baley, 2008). Recently, the medicinal usage
of some taxa within Urticaceae has been increasingly studied (e.g.
Gulcin et al., 2004; Piccinelli et al., 2005; Momo et al., 2006; Luo
et al., 2011). Furthermore, some genera, including Elatostema,Pilea
and Pellionia, are dominant and ecologically important elements of
the forest floor vegetation in subtropical forests (Wang and Chen,
1995; Chen et al., 2003). A full understanding of relationships
within this family would help to understand how these ecological
assemblages evolved, and also to detect potential new sources of
medicinal compounds.
The infra-familial classification of Urticaceae has been contro-
versial for more than one century. Gaudichaud (1830) treated
members of Urticaceae, Cecropiaceae, Moraceae and Cannabina-
ceae as a single family, and divided those now in Urticaceae into
1055-7903/$ - see front matter Ó2013 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: dzl@mail.kib.ac.cn (D.-Z. Li).
Molecular Phylogenetics and Evolution 69 (2013) 814–827
Contents lists available at SciVerse ScienceDirect
Molecular Phylogenetics and Evolution
journal homepage: www.elsevier.com/locate/ympev

five tribes named Urereae, Elatostemeae, Boehmerieae, Pari-
etarieae, and Forskalieae (Table 1). Weddell (1854, 1856, 1869)
separated Moraceae and Cannabaceae from Urticaceae, dividing
the 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’s
classifications, calling the five tribes Urticeae, Lecantheae, Boeh-
merieae, Parietarieae and Forsskaoleae (Table 1). However, he
questioned 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 subfamilies
within Urticaceae: Urticoideae, Lecanthoideae, and Boehmerioi-
deae; these largely correspond with Urticeae, Lecantheae, and
Boehmerieae–Parietarieae–Forsskaoleae, respectively (Friis,
1993), except that Touchardia was transferred to Lecanthoideae.
Additionally, six tribes under these subfamilies were proposed:
Urticeae, Leacantheae, Touchardieae, Boehmerieae, Forsskaoleae,
Parietarieae (Table 1).
The genera Cecropia and Coussapoa formed the tribe Cecropieae
of Gaudichaud’s classification (Gaudichaud, 1830), which with
Musanga,Poikilospermum, Pourouma and Myrianthus as Conoceph-
aleae were considered by Bentham and Hooker (Bentham and
Hooker, 1880) to belong in the equivalent of modern Moraceae.
However, Weddell (1869) placed Poikilospermum in Urticaceae;
Chew (1963) also moved Cecropia,Musanga and Coussapoa into
Urticaceae; and Corner (1962) considered all six to belong to
Urticaceae. Berg (1978) raised Cecropia,Musanga,Coussapoa,
Poikilospermum, Pourouma and Myrianthus together to family level
as Cecropiaceae, considering it distinct from Urticaceae based on
filaments that are mostly straight in the bud, and the absence of
Fig. 1. (Part A + Part B) Phylogenetic tree produced by Bayesian Inference (BI) analysis based on the matrix with plastic, mitochondrial, and nuclear datasets combined,
considering those clades with bootstrap BP P50%, and posterior probability PP P0.6, respectively. Clades are referred by numbers in the box; Numbers associated with
branches (ML_BS/MP_BS/BI_PP) are assessed by Maximum Likelihood Bootstrap (ML_BS), Maximum Parsimony Bootstrap (MP_BS) and Bayesian posterior (BI_PP). ‘‘–’’ shows
no support, ‘‘

’’ means fully support (100%/1.00). Numbers following species name denote the lab codes.
Z.-Y. Wu et al. / Molecular Phylogenetics and Evolution 69 (2013) 814–827 815

any tendencies towards a herbaceous habit. His treatment has
been 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 far
the phylogenetic relationships between these two families have
not 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 is
always 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 to
elucidate relationships within Urticaceae. Recent systematic stud-
ies on this family have so far mostly been based on morphological
characters (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 chloroplast
loci (rbcL and trnL–trnF), their results indicated that Cecropia-
ceae was not distinct from Urticaceae, and resolved three
lineages within Urticaceae, i.e. (1) Boehmeriea-Cecropieae–For-
sskaoleeae–Parietarieae, (2) Urticeae and (3) Elatostemeae. This
was broadly consistent with previous molecular phylogenetic
analyses (Sytsma et al., 2002; Datwyler and Weiblen, 2004;
Zerega et al., 2005; Monro, 2006), but their phylogeny had much
incongruence with previous morphological classifications. How-
ever, due to the limitations of sampling and regions, the
relationships and circumscription of many clades, and the gen-
era within them, remained poorly-resolved. Therefore, a better
resolved phylogenetic framework for the family is urgently
needed, which would require more extensive sampling of both
taxa and the genomes.
Fig. 1 (continued)
816 Z.-Y. Wu et al. / Molecular Phylogenetics and Evolution 69 (2013) 814–827

In the present study, to generate a phylogenetic hypothesis of
generic and tribal relationships within Urticaceae, we used the
most comprehensive taxon sampling to date, encompassing 47 of
the 54 recognized genera within Urticaceae, including four of the
six 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 three
analytical 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 in
the light of molecular data; (5) test the monophyly of some larger
genera; 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; in
some cases two individuals per species were examined. According
to the list of Urticaceae (Stevens, 2012), we sampled 169 acces-
sions of 122 species from 47 genera of Urticaceae, representing
87% of the recognized genera. These included four genera some-
times separated as Cecropiaceae, and Pellionia, which is usually
recognized as a subgenus of Elatostema (Schröter and Winkler,
1935, 1936; Hallier, 1986). Based on previous molecular analyses
on Rosales (Zhang et al., 2011), three species from Moraceae and
Ulmaceae were included for rooting purposes. The complete list
of sample numbers, collection localities, voucher specimens num-
ber and GenBank accession numbers is given in Table 2. Voucher
specimens of these samples are deposited at KUN, BM, K, PE, G or
C(Table 2).
Regarding selection of DNA regions, according to the ‘‘bottom up-
top down’’ approach (Wiens et al., 2005), variable and fast-evolving
loci are more useful in resolving phylogenetic relationships at lower
taxonomic levels, and hence for an entire study group, whereas
slow-evolving loci are more effective at higher levels, and hence
may be more appropriate when a few species per genus are sampled.
We selected trnL–trnF, rpll4–rps8–infA–rpl36, matK, rbcL, and ITS, all
of which have been used extensively to resolve relationships at the
genus level (Milne, 2004; Couvreur et al., 2010; Liu et al., 2011,
2013; Nürk et al., 2013). To increase resolution at higher taxonomic
levels, we included 18S and matR, which are usually considered as
slow-evolving loci (Soltis et al., 2000; Zhu et al., 2007; Chen et al.,
2012). Therefore, only one or two species per genus were examined
using these two loci. Primers for amplification and sequencing are
provided 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 or
fresh leaves, with some from herbarium material supplied by BM
and K. All material was extracted using the CTAB method (Doyle
and Doyle, 1987) with minor modifications. PCR was conducted
in a total volume of 25
l
L containing 2.5
l
L10PCR buffer,
2.5
l
L MgCl
2
(25 mM), 2.0
l
L dNTP mixture (2.5 mM), 0.75
l
L each
primer (10
l
M), 0.125
l
L Taq polymerase (5 U/
l
L) (TaKaRa, Dalian,
China), 1–2
l
L template DNA (containing 5–50 ng genomic DNA),
and finally distilled deionized water to give a final volume of
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 morphological
diversity in Clade I; (C) Pipturus arborescens; (D) Boehmeria tricuspis; (E) Debregeasia longifolia; (F) B. nivea var. tenacissima; (G) Chamabainia cuspidata; (H) Archiboehmeria
atrata; (I) Oreocnide frutescens subsp. frutescens; (J–M) examples of morphological diversity in Clade II; (J) E. densistriolatum; (K) L. pileoides; (L) E. tenuicaudatum; (M) Pellionia
radicans; (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.
Z.-Y. Wu et al. / Molecular Phylogenetics and Evolution 69 (2013) 814–827 817

Table 1
Currently 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 in
genus
No. of spp.
sampled
Total accessions
sampled
Gaudichaud
(1830)
Weddell
(1869)
a
Friis (1993) Kravtsova
(2009)
Urtica Urereae Urereae Urticeae Urticeae 3A Subcosmopolitan 80 9 13
Hesperocnide – Urereae Urticeae Urticeae 3A California, Hawaii Is. 2 1 1
Nanocnide – Urereae
b
Urticeae Urticeae 3B Warm parts of East Asia 2 2 4
Obetia – Urereae Urticeae Urticeae 3F C & S Africa; Madagascar,
Mascarenes
711
Laportea Urereae Urereae Urticeae Urticeae 3E E Asia, eastern N America 21 1 2
Discocnide – – Urticeae Urticeae 3C Mexico, Guatemala 1 1 1
Girardinia Urereae Urereae Urticeae Urticeae 3D Africa, E Asia 2 1 6
Dendrocnide – – Urticeae Urticeae 3C Indomalayan region, W Pacific
Islands
37 3 4
Urera Urereae Urereae Urticeae Urticeae 3F Tropical Africa & America, 35 7 8
Madagascar, Hawaii
Gyrotaenia – Urereae Urticeae Urticeae 2D West Indies 4 3 3
Elatostema Elatostemeae Procrideae
c
Lecantheae
d
Lecantheae 2B Warm parts of old world 300 11 12
Meniscogyne – – Lecantheae
d
– 1A SE Asia (Indo-China) 2 0 0
Procris Elatostemeae Procrideae Lecantheae
d
Lecantheae 2C Warm and tropical parts of old
world
20 1 2
Pilea Elatostemeae Procrideae
c
Lecantheae
d
Lecantheae 2F Old and New world tropics 250 12 14
Sarcopilea – – Lecantheae
d
– 2F Hespaniola 1 1 1
Lecanthus – Procrideae
c
Lecantheae
4
Lecantheae 2E Africa, S Asia and Oceania 1 2 4
Petelotiella – – Lecantheae
d
– – NE Vietname 1 0 0
Pellionia Elatostemeae Procrideae
b
Elatostemeae
e
Lecantheae 2A/2C Tropical & subtropical Asia,
Pacific islands
60 5 5
Aboriella – – Elatostemeae
f
– – East Himalayas 1 0 0
Boehmeria Boehmerieae Boehmerieae Boehmerieae Boehmerieae 1A1/
3/7
Pantropical, N warm
temperate regions
80 14 23
Chamabainia – Boehmerieae Boehmerieae Boehmerieae 1A4 China & Indonesia to India 1 or 2 1 2
Pouzolzia Parietarieae Boehmerieae Boehmerieae Boehmerieae 1A5/
1A7
Pantropical 70 5 8
Gonostegia – – Boehmerieae – 1A6 SE Asia and N Australia 5 2 3
Neodistemon – – Boehmerieae Boehmerieae 1A7 Indomalesia: India to
Philippines
111
Cypholophus – Boehmerieae Boehmerieae Boehmerieae – Malesia, W Pacific Islands 15 0 0
Sarcochlamys – Boehmerieae Boehmerieae Boehmerieae 1A3 Himalayas and Indo-china 1 1 1
Touchardia – Boehmerieae
c
Boehmerieae Touchardieae 3F Hawaii 1 or 2 1 1
Neraudia Boehmerieae Boehmerieae Boehmerieae Boehmerieae 1A8 Hawaii 5 2 2
Piptuirus – Boehmerieae Boehmerieae Boehmerieae 1A9 Malaysia to N Australia &
Polynesia
30 3 4
Nothocnide – – Boehmerieae Boehmerieae 1A9 From Indonesia to Melanesia 4 1 1
Oreocnide – – Boehmerieae Boehmerieae 1B From Sri Lanka to Japan 15 1 3
Debregeasia – Boehmerieae Boehmerieae Boehmerieae 1A2 Malesia, Indo-China, Arabia,
NE Africa
459
Astrothalamus – – Boehmerieae Boehmerieae 1A3 Indonesia, Philippines 1 1 1
Leucosyke – Boehmerieae Boehmerieae Boehmerieae 4B SE Asia, Indonesia, S Polynesia 35 1 2
Gibbsia – – Boehmerieae Boehmerieae – Western New Guinea 2 0 0
Phenax – Boehmerieae Boehmerieae Boehmerieae 1E Tropical and S America 12 1 1
Maoutia – Boehmerieae Boehmerieae Boehmerieae 4B N India, Indo-China, Malesia,
Polynesia
15 1 1
Myriocarpa – Boehmerieae Boehmerieae Boehmerieae 2D Tropical and South America 18 2 2
Archiboehmeria – – Boehmerieae
e
Boehmerieae 1A3 S China and N Indo-China 1 1 2
Gesnouinia Parietarieae Parietarieae Parietarieae Parietarieae 1D Canary Islands 2 1 1
Hemistylus – Parietarieae Parietarieae Boehmerieae 1A5 Tropical south & central
America
411
Parietaria Parietarieae Parietarieae Parietarieae Parietarieae 1D Subcosmopolitan 20 2 2
Soleirolia Parietarieae – Parietarieae Parietarieae 1D West Mediterranean 1 1 1
Rousselia Parietarieae Parietarieae Parietarieae Boehmerieae 1A5 C America, Colombia, West
Indies
311
Forsskaolea Forskalieae Forskohleae Forsskaoleae Forsskaoleae 1C Macaronesia, Africa, S Spain,
India
612
Droguetia Forskalieae Forskohleae Forsskaoleae Forsskaoleae 1C E & S Africa, Madagascar, 7 2 3
Mascarenes, S India, Java
Australina Forskalieae Forskohleae Forsskaoleae Forsskaoleae 1C E Africa, SE Australia, New
Zealand
211
Didymodoxa – Forskohleae Forsskaoleae Forsskaoleae 1C E & S Africa 2 1 1
Poikilospermum – Cecropiaceae Cecropiaceae 3F Tropical Asia 20 2 3
Pourouma – Cecropiaceae Cecropiaceae – Tropical America >50 0 0
Myrianthus – Cecropiaceae Cecropiaceae 4A Tropical Africa 7 1 1
818 Z.-Y. Wu et al. / Molecular Phylogenetics and Evolution 69 (2013) 814–827

25
l
L. 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 for
ITS), 80 s at 72 °C and a final extension at 72 °C for 10 min. The
PCR parameters for 18S were as follows: a 97 °C initial hot start
for 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 at
95 °C for 3 min, 16 cycles of 20 s at 94 °C, 40 s at 65 °C, and degrade
1°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 of
5 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. The
purified PCR products were directly used for cycle sequencing with
the ABI Prism BigDye Terminator Cycle Sequencing Kit (Applied
Biosystems, Foster City, California, USA) following the manufac-
turer’s recommendations. Both strands of the resulting products
were 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 the
alignments of cleaned sequences were carried out by GENEIOUS
v4.8.4 (Drummond et al., 2009) with additional manual refine-
ments where necessary. Phylogenetic analyses were conducted
for single DNA regions and the concatenated datasets of the nucle-
ar, chloroplast, organelle and the two slow-evolving loci separately
using three methods of phylogenetic analysis, namely maximum
likelihood (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. 1000
bootstrap replicates were conducted with a rapid bootstrapping
and subsequent ML search. The MP analyses were carried out using
PAUP v4.0b10 (Swofford, 2003). Heuristic searches were per-
formed with 1000 random addition replicates followed by tree
bisection–reconnection branch swapping, with zero-length
branches collapsed. All character states were treated as unordered
and equally weighted, with gaps treated as missing data. Topolog-
ical robustness was assessed by 1000 bootstrap replicates with the
same settings as for heuristic searches. Bayesian inference (BI) of
phylogeny was done using MRBAYES v3.1.2 (Ronquist and
Huelsenbeck, 2003) with default priors. The model of best fit for
the 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 and
three 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 were
sampled every 100 generations, estimating all parameters during
the analysis. A partitioned Bayesian analysis of the combined data-
sets was implemented by applying the previously determined
models to each data partition. Majority rule (>50%) consensus trees
were constructed after removing the burn-in period samples (the
first 25% of sampled trees). The posterior probability (PP) value
of each topological bipartition was calculated as the frequency of
that bipartition across all sampled trees.
3. Results
3.1. Analysis of nuclear sequence data
The sequence characteristics and the best-fit model determined
by MODELTEST for the all of the datasets are given in Table 3. The
tree generated by 18S (Supplementary Fig. S1) was largely consis-
tent with that of ITS (Supplementary Fig. S2). The 18S analysis did
not resolve one of the three major clades discussed below (Clade I),
which was present in all other analyses. When two datasets were
combined, 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 (Supplementary
Fig. 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 differences
between them that had meaningful statistical support, and none
of the conflicting relationships had bootstrap support of >90%.
Therefore the four regions were analyzed together.
For the combined cpDNA dataset, the numbers of variable and
parsimony informative sites, tree statistics for the MP analysis
and the best-fit model determined by MODELTEST are presented
in Table 3. Phylogenetic analyses of the concatenated sequences
using MP, ML and BI produced similar topologies (Supplementary
Table S1; Fig. S6).
3.3. Analysis of combined genome data
Reduced taxon sets were examined using two slow-evolving
genes, mitochondrial matR and nuclear 18S. A visual check
Table 1 (continued)
Genus Tribal classification according to: Clade Geographic range No. of species in
genus
No. of spp.
sampled
Total accessions
sampled
Gaudichaud
(1830)
Weddell
(1869)
a
Friis (1993) Kravtsova
(2009)
Cecropia Cecropieae Cecropiaceae Cecropiaceae 4A Tropical America 70–80 2 2
Coussapoa Cecropieae Cecropiaceae Cecropiaceae 4A Tropical America >50 1 1
Musanga – 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 placed
in 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);inWeddell (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).
Z.-Y. Wu et al. / Molecular Phylogenetics and Evolution 69 (2013) 814–827 819

Table 2
Plant materials examined, its provenance, and GenBank accession numbers for all DNA regions successfully sequenced.
Accession Herbarium Taxon
a
Voucher Country GenBank accession numbers for each DNA region
ITS trnL–trnFrbcLrpl14–rps8–
infA–rpl36
matK 18S matR
A1 KUN Archiboehmeria atrata WuZY-09469 Guangxi, China KF137798 KF138269 KF138106 KF138434 KF137946 KF137743 KF138078
A2 KUN Archiboehmeria atrata WuZY-09414 Guangxi, China KF137799 KF138270 KF138107 KF138435 KF137947
23592 K Astrothalamus reticulatus Argent et al.
981987
Sabah, Malaysia KF137800 KF138271 KF138108 KF137744
23601 K Australina flaccida Friis, I. et al.
12293
Gonder,
Ethiopia
KF137801 KF138272 KF138109 KF138436 KF137745
B2 KUN Boehmeria clidemioides
b
WuZY-10124 Yunnan, China KF137803 KF138274 KF138111 KF138438 KF137949 KF137746 KF138081
B15 KUN Boehmeria clidemioides
b
Liuj-10675 Fujian, Chian KF137802 KF138273 KF138110 KF138437 KF137948
B16 KUN Boehmeria clidemioides
var. diffusa
WuZY-09291 Yunnan, China KF137804 KF138275 KF138112 KF138439 KF137950
B52 KUN Boehmeria densiflora Yi20111199 Taiwan, China KF137805 KF138276 KF138113
B53 KUN Boehmeria densiflora WuZY-2012450 Lanyu, Taiwan,
China
KF137806 KF138277 KF138114 KF137951
B5 KUN Boehmeria glomerulifera GBOWS990 Yunnan, China KF137807 KF138278 KF138115 KF138440
B47 KUN Boehmeria japonica 100024 Hawaii, USA KF137808 KF138279 KF138116
B20 KUN Boehmeria longispica Liuj-10722 Zhejiang, China KF137809 KF138280 KF138117 KF138441 KF137952
B24 KUN Boehmeria macrophylla
b
WuZY-10060 Yunnan, China KF137810 KF138281 KF138118 KF138442 KF137953 KF137747 KF138079
B28 KUN Boehmeria macrophylla
b
WuZY-09196 Yunnan, China KF137811 KF138282 KF138119 KF138443 KF137954
B21 KUN Boehmeria macrophylla
var. rotundifolia
Liuj-10629 Yunnan, China KF137812 KF138283 KF138120 KF138444 KF137955
B26 KUN Boehmeria macrophylla
var. 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 nivea
b
Liuj-10645 Fujian, China KF137815 KF138286 KF138123 KF138447 KF137958 KF137748 KF138077
B33 KUN Boehmeria penduliflora WuZY-09460 Guangxi, China KF137816 KF138287 KF138124 KF138448 KF137959
B45 KUN Boehmeria rugulosa LiDZ1071 Near
Kathmandu,
Nepal
KF137817 KF138288 KF138125 KF138449 KF137960
B46 KUN Boehmeria sp. RC1555 Mayagdi, Nepal KF137818 KF138289 KF138126 KF138450 KF137961
B9 KUN Boehmeria spicata Liuj-10743 Zhejiang, China KF137819 KF138290 KF138127 KF138451 KF137962
B38 KUN Boehmeria tomentosa WuZY-09011 Yunnan, China KF137820 KF138291 KF138128 KF138452 KF137963
B39 KUN Boehmeria tricuspis WuZY-09430 Guangxi, China KF137821 KF138292 KF138129 KF137964
B12 KUN Boehmeria umbrosa WuZY-10336 Yunnan,hina KF137822 KF138293 KF138130 KF138453 KF137965
B40 KUN Boehmeria umbrosa WuZY-09465 Guangxi, China KF137823 KF138294 KF138131 KF138454
B1 KUN Boehmeria zollingerianavar.
blinii
LiDZ1084 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 KF137967
C1 KUN Chamabainia cuspidata WuZY-10086 Yunnan, China KF137827 KF138299 KF138136 KF138457 KF137969 KF137751 KF138080
C2 KUN Chamabainia cuspidata WuZY-09071 Yunnan, China KF137828 KF138300 KF138137 KF138458 KF137970
386A BM Coussapoa parvifolia Monro et al.
6833
Costa Rica KF138301 KF138459
De19 KUN Debregeasia elliptica WuZY-10134 Yunnan, China F137829 KF138302 KF138138 KF138460 KF137971
De7 KUN Debregeasia elliptica WuZY-10061 Yunnan, China KF137830 KF138303 KF138139 KF138461 KF137972
De10 KUN Debregeasia longifolia WuZY-09471 Guangxi, China KF137831 KF138304 KF138140 KF138462 KF137973 KF137752 KF138104
De9 KUN Debregeasia longifolia Liuj-10626 Yunnan, China KF137832 KF138305 KF138141 KF138463 KF137974
De13 KUN Debregeasia orientalis GLM-07-659 Yunnan, China KF137833 KF138306 KF138142 KF138464 KF137975
De15 KUN Debregeasia orientalis 81482 Xizang, China KF137834 KF138307 KF138143 KF138465 KF137976
De17 KUN Debregeasia saeneb 81107 Xizang, China KF137835 KF138308 KF138144 KF138466 KF137977
De25 KUN Debregeasia sp. RC1556 Mayagdi, Nepal KF137836 KF138309 KF138145 KF138467 KF137978 KF137753
De5 KUN Debregeasia squamata WuZY-09204 Yunnan, China KF137837 KF138310 KF138146 KF138468 KF137979
D2 KUN Dendrocnide meyeniana Yi20111184 Taiwan, China KF137838 KF138311 KF138147 KF138469 KF137980
D1 KUN Dendrocnide sinuata WuZY-09238 Yunnan, China KF137839 KF138312 KF138148 KF138470 KF137981 KF137754 KF138096
W1 KUN Dendrocnide sp. WuZY-09035 Yunnan, China KF137840 KF138313 KF138149 KF138471 KF137982
D5 KUN Dendrocnide urentissima WuZY-09211 Yunnan, China KF137841 KF138314 KF138150 KF138472
23599 K Didymodoxa caffra Abdallah et al.
96/92
Mkomazi,
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 & Styles
2426
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 KF138090
E2 KUN Elatostema atropurpureum GBOWS298 Yunnan, China KF137847 KF138321 KF138157 KF138479 KF137987
E3 KUN Elatostema crytandrifolium
b
WuZY-09231 Yunnan, China KF137848 KF138322 KF138158 KF138480 KF137988
E4 KUN Elatostema cuspidatum
b
WuZY-09253 Yunnan, China KF137849 KF138323 KF138159 KF138481 KF137989 KF137759 KF138091
E9 KUN Elatostema densistriolatum WuZY-10219 Yunnan, China KF137850 KF138324 KF138160 KF138482 KF137990
820 Z.-Y. Wu et al. / Molecular Phylogenetics and Evolution 69 (2013) 814–827

Table 2 (continued)
Accession Herbarium Taxon
a
Voucher Country GenBank accession numbers for each DNA region
ITS trnL–trnFrbcLrpl14–rps8–
infA–rpl36
matK 18S matR
E6 KUN Elatostema longibracteatum GBOWS148 Yunnan, China KF137851 KF138325 KF138161 KF138483 KF137991
E7 KUN Elatostema parvum
b
WuZY-09214 Yunnan, China KF137852 KF138326 KF138162 KF138484 KF137992
E8 KUN Elatostema petelotii GBOWS342 Yunnan, China KF137853 KF138327 KF138163 KF138485 KF137993
E13 KUN Elatostema sp. RC1700 Panchthar,
Nepal
KF137854 KF138328 KF138164 KF138486 KF137994
E10 KUN Elatostema stewardii Liuj-10729 Zhejiang, China KF137855 KF138329 KF138165 KF138487 KF137995
E11 KUN Elatostema
subtrichotomum
b
GBOWS043 Yunnan, China KF137856 KF138330 KF138166 KF138488 KF137996
E12 KUN Elatostema tenuicaudatum
b
GBOWS509 Yunnan, China KF137857 KF138167 KF138489 KF137997
6515 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 diversifolia
c
GLM-103093 Taiwan, China KF138336 KF138173 KF138495 KF138001
G9 KUN Girardinia diversifolia
c
Liuj-10776 Zhejiang, China KF137863 KF138337 KF138174 KF138496 KF138002 KF137763 KF138095
G16 KUN Girardinia diversifolia
subsp. suborbiculata
WuZY-10011 Beijing, China KF138338 KF138175 KF138497 KF138003
G17 KUN Girardinia diversifolia
subsp. suborbiculata
WuZY-10014 Beijing, China KF138339 KF138176 KF138498 KF138004
G19 KUN Girardinia diversifolia
subsp. triloba
WuZY-10089 Yunnan, China KF137864 KF138340 KF138177 KF138499 KF138005
G6 KUN Girardinia diversifolia
subsp. triloba
WuZY-10229 Yunnan, China KF138341 KF138178 KF138500 KF138006
Go3 KUN Gonostegia hirta WuZY-09478 Guangxi, China KF137865 KF138342 KF138179 KF138007
Go1 KUN Gonostegia parvifolia Liuj-10667 Fujian, China KF137866 KF138343 KF138180 KF138501 KF138008 KF137764 KF138103
Go4 KUN Gonostegia parvifolia Liuj-10649 Fujian, China KF137867 KF138344 KF138181 KF138502 KF138009
475A BM Gyrotaenia crassifolia Whitefoord
4139
Dominica KF138010
473A K Gyrotaenia microcarpa Breedlove 68859 Mexico KF138345 KF138011
23567 K Gyrotaenia spicata Acevedo-
Rodriguez, 9614
Manchester,
Jamaica
KF137765
23597 K Hemistylus macrostachya Pittier 11788 Curricuti,
Venezuela
KF137868 KF138346 KF138182 KF138503 KF137766
331A G Hesperocnide tenella Howell 52355 USA KF138504
L3 KUN Laportea bulbifera 10CS2513 Shanxi, China KF138348 KF138184 KF138506 KF138014
L5 KUN Laportea bulbifera WuZY-09423 Guangxi, China KF137870 KF138349 KF138185 KF138507 KF138015
Le1 KUN Lecanthus peduncularis Liuj-10607 Yunnan, China KF137871 KF138350 KF138186 KF138508 KF138016 KF137768
Le3 KUN Lecanthus peduncularis 81148 Xizang, China KF137872 KF138351 KF138187 KF138509
Le2 KUN Lecanthus petelotii var.
corniculata
81066 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 KF138013
23604 K Myrianthus preussii Bogner 672 Kagala, Gabon KF137769
C2A BM Myriocarpa cordata Monro 4630 Panama KF137877 KF138357 KF138193 KF138512 KF137770
370A BM Myriocarpa obovata Monro & Penn
6534
Belize KF137878 KF138358 KF138513 KF138021 KF137771 KF138084
N1 KUN Nanocnide japonica Liuj-10735 Zhejiang, China KF137879 KF138359 KF138194 KF138514 KF138022 KF137772
N4 KUN Nanocnide japonica Liuj-10749 Zhejiang, China KF137880 KF138360 KF138195 KF138515 KF138023
N5 KUN Nanocnide lobata Liuj-10701 Zhejiang, China KF137881 KF138361 KF138196 KF138516 KF138024
N6 KUN Nanocnide lobata Liuj-10799 Zhejiang, China KF137882 KF138362 KF138197 KF138517 KF138025 KF137773
279A K Neodistemon indicum Larsen et al.
2669
Thailand KF138363 KF138198 KF138026 KF137774
Ne2 KUN Neraudiakauaiensis 678003 Hawaii, USA KF137883 KF138364 KF138199 KF138027 KF137775 KF138085
Ne1 KUN Neraudia melastomifolia 90861 Hawaii, USA KF137884 KF138365 KF138200 KF138518 KF138028
23585 K Nothocnide mollissima Beaman 8882 Sabah, Malaysia KF137885 KF138366 KF138201 KF138519 KF137776
28719 K Obetia tenax Botha 9 Limpopo, S
Africa
KF137886 KF138367 KF138202 KF138520 KF137777
O2 KUN Oreocnide frutescens
c
Liuj-10623 Yunnan, China KF137887 KF138368 KF138203 KF138521 KF138029 KF137778 KF138102
O8 KUN Oreocnide frutescens
c
WuZY-09428 Yunnan, China KF137888 KF138369 KF138204 KF138522 KF138030
O12 KUN Oreocnide frutescens subsp.
occidentalis
WuZY-09260 Yunnan, China KF138370 KF138205 KF138523 KF138031
11077 K Parietaria judaica Fay MFF185 England, UK KF138371 KF138206 KF138524 KF137779
Pa1 KUN Parietaria micrantha WuZY-10373 Yunnan, China KF138372 KF138207 KF138525 KF137780 KF138094
(continued on next page)
Z.-Y. Wu et al. / Molecular Phylogenetics and Evolution 69 (2013) 814–827 821

Table 2 (continued)
Accession Herbarium Taxon
a
Voucher Country GenBank accession numbers for each DNA region
ITS trnL–trnFrbcLrpl14–rps8–
infA–rpl36
matK 18S matR
Pe1 KUN Pellionia macrophylla GBOWS1374 Yunnan, China KF137889 KF138373 KF138208 KF138526 KF138032
Pe2 KUN Pellionia paucidentata
b
GBOWS130 Yunnan, China KF137890 KF138374 KF138209 KF138527 KF138033 KF137781
Pe3 KUN Pellionia radicans GBOWS962 Yunnan, China KF137891 KF138375 KF138210 KF138528 KF138034
Pe4 KUN Pellionia repens WuZY-09159 Yunnan, China KF137892 KF138376 KF138211 KF138529 KF138035 KF137782 KF138089
Pe5 KUN Pellionia tsoongii WuZY-09495 Guangxi, China KF137893 KF138377 KF138212 KF138530 KF138036
378A BM Phenax mexicanus Breedlove &
Smith 21717
Mexico KF138378
P1 KUN Pilea angulata subsp.
petiplaris
81352 Xizang, China KF137894 KF138379 KF138213 KF138531 KF138037 KF137783 KF138092
P3 KUN Pilea cavaleriei
b
LiDZ1080 Guizhou, China KF137895 KF138380 KF138214 KF138532 KF138038
P11 KUN Pilea insolens STET1838 Xizang, China KF137896 KF138381 KF138215 KF138533 KF138039
P5 KUN Pilea longipedunculata WuZY-09199 Yunnan, China KF137897 KF138382 KF138216 KF138534 KF137784 KF138093
P6 KUN Pilea martinii WuZY-09107 Yunnan, China KF137898 KF138383 KF138217 KF138535 KF138040
P20 KUN Pilea melastomoides WuZY-09463 Guangxi, China KF137899 KF138384 KF138218 KF138536 KF138041
P21 KUN Pilea microphylla WuZY-09174 Yunnan, China KF137900 KF138385 KF138219 KF138537 KF138042
P22 KUN Pilea microphylla WuZY-09399 Guangxi, China KF137901 KF138386 KF138220 KF138538 KF138043
P9 KUN Pilea oxyodon STET2663 Xizang, China KF137902 KF138387 KF138221 KF138539 KF138044
P24 KUN Pilea plantaniflora LiDZ1081 Guizhou, China KF137903 KF138222 KF138540 KF138045
P10 KUN Pilea pumila
b
WuZY-09335 Yunnan, China KF137904 KF138388 KF138223 KF138541 KF138046
P26 KUN Pilea sinofasciata Liuj-10605 Yunnan, China KF137905 KF138389 KF138224 KF138542 KF138047
P12 KUN Pilea sp. RC1554 Myagdi, Nepal KF137906 KF138390 KF138225 KF138543 KF138048
P29 KUN Pilea verrucossa
b
Liuj-09527 Yunnan, China KF137907 KF138391 KF138226 KF138544 KF138049
Pip1 PE Pipturus arborescens 11879 Taiwan, China KF137908 KF138392 KF138227 KF138545 KF137785
Pip7 KUN Pipturus arborescens WuZY-2012437 Lanyu, Taiwan,
China
KF137909 KF138393 KF138228 KF138050
Pip5 KUN Pipturus kauaiensis 90441 Hawaii, USA KF137910 KF138394 KF138229 KF138546 KF138051
Pip6 KUN Pipturus ruber 80829 Hawaii, USA KF137911 KF138395 KF138230 KF138547 KF138052
Pi1 KUN Poikilospermum
lanceolatum
WuZY-09235 Yunnan, China KF137912 KF138396 KF138231 KF138548 KF138053 KF137786 KF138097
Pi2 KUN Poikilospermum suaveolens GBOWS736 Yunnan, China KF137913 KF138397 KF138232 KF138549
Pi3 KUN Poikilospermum suaveolens WuZY-09160 Yunnan, China KF137914 KF138398 KF138233 KF138550 KF138054
Po5 KUN Pouzolzia argenteonitida STET1914 Xizang, China KF137915 KF138399 KF138234 KF138551
282A K Pouzolzia guineensis Bidgood et al.
3008
Tanzania 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 sanguinea
b
WuZY-09483 Guangxi, China KF137918 KF138403 KF138238 KF138555 KF138057
Po9 KUN Pouzolzia sp. RC1682 Panchthar,
Nepal
KF137919 KF138404 KF138239 KF138556 KF138058
Po4 KUN Pouzolzia zeylanica
b
WuZY-10167 Yunnan, China KF137920 KF138405 KF138240 KF138557 KF138059
Po7 KUN Pouzolzia zeylanica
b
Liuj-10686 Zhejiang, China KF137921 KF138406 KF138241 KF138558 KF138060 KF138101
Pr1 KUN Procris wightiana WuZY-10090 Yunnan, China KF137922 KF138407 KF138242 KF138559 KF138061
Pr2 KUN Procris wightiana WuZY-10230 Yunnan, China KF137923 KF138408 KF138243 KF138560 KF138062 KF137788 KF138088
23596 K Rousselia humulis Ekman 4973 Hispaniola,
Haiti
KF137789
S1 KUN Sarcochlamys pulcherrima STET2106 Xizang, China KF137924 KF138409 KF138244 KF138561 KF137790 KF138105
302A BM Sarcopilea domingensis Monro 5408 Dominican
Republic
KF137925 KF138410 KF138245 KF138562 KF137791 KF138076
312A BM Soleirolia soleirolii Monro in cult. Cult. In UK KF137926 KF138411 KF138246 KF138563 KF138063
T1 KUN Touchardia latifolia Jffrey201101 Hawaii, USA KF137927 KF138412 KF138247 KF138564 KF137792
C11A BM Urera alceifolia Monro 4346 Bocas del Toro,
Panama
KF138413 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 KF137794
377A C Urera hypselodendron Friis et al. 4125 Ethiopia KF138417 KF138252 KF138569 KF138066
313A BM Urera lianoides Solano 6825 Costa Rica KF138418 KF138253 KF138570
L2 KUN Urera sp. WangH-201001 South Africa KF137931 KF138419 KF138254 KF138571 KF137795 KF138098
374A C Urera trinervis Friis et al. 3920 Ethiopia KF137932 KF138420 KF138255 KF138572
U1 KUN Urtica angustifolia 10CS2322 Inner Mongolia,
China
KF137933 KF138421 KF138256 KF138573 KF138067 KF137796
U2 KUN Urtica ardens 81152 Xizang, China KF137934 KF138422 KF138257 KF138574
U3 KUN Urtica atrichocaulis WuZY-10358 Yunnan, China KF137935 KF138423 KF138258 KF138575 KF138068
U21 KUN Urtica dioica BROWP135 Edinburgh, UK KF137936 KF138424 KF138259 KF138576
U4 KUN Urtica fissa WuZY-10378 Yunnan, China KF137937 KF138425 KF138260 KF138577 KF138069
U14 KUN Urtica hyperborea 80966 Xizang, China KF137938 KF138426 KF138261 KF138578 KF138070
U5 KUN Urtica hyperborea 81200 Xizang, China KF137939 KF138427 KF138262 KF138579 KF138071
U7 KUN Urtica mairei WuZY-09354 Yunnan, China KF137940 KF138428 KF138263 KF138580 KF137797
U18 KUN Urtica sp. Lixinhui-1102 Kenya, Africa KF137941 KF138429 KF138264 KF138581 KF138072
822 Z.-Y. Wu et al. / Molecular Phylogenetics and Evolution 69 (2013) 814–827

indicates no topological contradictions with significant bootstrap
support congruence between these two datasets; therefore, these
regions were combined in our analyses (Supplementary Fig. S5).
Compared to either gene individually, the combined data produced
stronger support for basal relationships within Urticaceae, with
maximum support (100/100/1, ML/MP/BI, the same order thereaf-
ter) for a sister relationship between Clades II and III, and for the
monophyly of Urticaceae.
Preparing to combine all data, we first compared combined
cpDNA data with that for mitochondrial matR. Finding no incon-
gruences that had strong bootstrap support, we combined these
datasets to give an organelle phylogeny (Supplementary Fig. S7).
Comparing this with the combined nuclear DNA (Supplementary
Fig. 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 not
within genera), and always had weak bootstrap support (Supple-
mentary material, Table S1). Therefore, all data were combined
into, and analyzed as, a single matrix. MP, ML and BI analyses of
this dataset not only yielded mostly congruent topologies for the
combined datasets from the nuclear and organelle genomes within
Urticaceae, but also resulted in greater resolution as compared to
the separate analyses (Fig. 1). The relationships described in the
next 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 a
monophyletic clade with maximum support in the combined,
cpDNA and 18S topologies, but fairly strong support also from
ITS (92/–/1.00) and matR (80/64/0.79). Cecropiaceae was split
across 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% supports
in all analyses for combined DNA, whereas Clade I and IV had no
support 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 another
with maximum support; the same was true of Clades II and III. For
convenience, Clades I, II, III and IV were subdivided into named
subclades, 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 groupings
thereof, across all datasets and analyses are given in Supplemen-
tary material, Table S1.
Of the genera examined for which more than one species was
sampled, only three were split across more than one subclade,
and were hence polyphyletic. These were Boehmeria (subclades
1A1, 1A3 and 1A7), Pouzolzia (subclades 1A5 and 1A7), and Pellionia
(subclades 2A and 2C). In addition, four more genera were strongly
supported 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; subclade
2F) 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 successively
sister 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 analysis
than in any other, and disappears altogether when 18S data is com-
bined with ITS, indicating some incongruence. Clade 1D comprised
Didymodoxa 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 that
included cpDNA (Supplementary material, Table S1). In the other
part of Clade 1A, the genus Pouzolzia was split between subclade
1A5 (with Rousselia,Hemistylus,Neodistemon) and 1A7 (with
Boehmeria rugulosa). Subclade 1A7 formed a larger clade with
1A8 (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 of
1A5 + 1A6 + 1A7 + 1A8 + 1A9 had maximum support from all anal-
yses that included cpDNA, and maximum Bayesian support across
every dataset.
Within Clade II, Clades 2E (Lecanthus) and 2F (Pilea +Sarcopi-
lea) each had maximum support; they were sister to one another
with strong support from all datasets except ITS. Of two clades
containing Pellionia species, 2A (P. paucidentata,P. radicans,P.
macrophylla) had maximum support from almost all datasets
Table 2 (continued)
Accession Herbarium Taxon
a
Voucher Country GenBank accession numbers for each DNA region
ITS trnL–trnFrbcLrpl14–rps8–
infA–rpl36
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 KF138074
U17 KUN Urtica zayuensis WuZY-10361 Yunnan, China KF137945 KF138433 KF138268 KF138585 KF138075
Outgroups
ULM2 KUN Celtis kunmingensis WZY-1102 Yunnan, China KF137826 KF138298 KF138135 KF138456 KF137968 KF137750 KF138087
F1 KUN Fatoua villosa Liuj-10688 Zhejiang, China KF137858 KF138331 KF138168 KF138490 KF137998 KF137760 KF138082
F2 KUN Fatoua villosa GLM-103136 Taiwan, China KF137859 KF138332 KF138169 KF138491 KF137999
H1 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.
Z.-Y. Wu et al. / Molecular Phylogenetics and Evolution 69 (2013) 814–827 823

and analyses, and was strongly supported by all as sister to subc-
lade 2B (Elatostema). These in turn were sister in all datasets to
subclade 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 weak
for relationships within the clade, and for its sister relationship
to 2E + 2F (68/–/0.66).
Clade III comprised six subclades, all of which had maximum
support from the combined dataset, and all but one of which were
strongly supported across all datasets. The exception, subclade 3E
(Laportea), was not sampled for matR or 18S, but had maximum
support from each of ITS and cpDNA. Subclade 3F had maximum
support and contained Urera, within which were nested Touchar-
dia,Poikilospermum and Obetia; the presence of Poikilospermum in
this 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 monophyly
for all genera involved except for Urtica (paraphyletic with respect
to 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 three
genera previously assigned to Cecropiaceae (Cecropia,Myrianthus
and Coussapoa), whereas 4B (100/88/1) contained Leucosyke and
Maoutia.
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 examined
supported this relationship, but combining them all into a single
dataset improved the resolution and support for relationships
within Urticaceae. Furthermore, there was little incongruence be-
tween datasets, and that which there was tended to involve
slow-evolving loci for which fewer taxa were sampled, and hence
could reflect undersampling or lack of phylogenetic signal. There
were no apparent cases of discordance that might reflect reticulate
evolution. Therefore, the discussion that follows is largely based on
the results from the combined dataset.
As in previous molecular phylogenies (Hadiah et al., 2008; see
also Sytsma et al., 2002; Datwyler and Weiblen, 2004; Zerega
et al., 2005; Monro, 2006), Cecropiaceae was biphyletic and both
lineages were nested within Urticaceae, indicating that it should
not be recognized as a distinct family. However, Urticaceae plus
Cecropiaceae comprised four well supported clades, the smallest
of which (Clade IV) comprised three genera previously assigned
to Cecropiaceae, i.e. Cecropia,Coussapoa, and Myrianthus, plus
two 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 Urticeae
of Friis (1993) or Urticoideae of Kravtsova (2009). In each case
there were exceptions, as detailed below.
Clade I contained, with few exceptions, members of Friis’ (1993)
other three tribes Forsskaoleae, Parietarieae and Boehmerieae; it
therefore 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 example
the sister family Moraceae has two stigmas and its ovules are
either laterally or apically fixed, and never orthotropous. Con-
versely, characters such as the difference between inflexed and
straight stamens may be over-emphasized in delimiting taxa at
the rank of family.
4.2. Relationships of genera previously assigned to Cecropiaceae
The recognition of Cecropiaceae was based on morphological
characters such as basal/subbasal and orthotropous/suborthotropous
ovules, stamens filaments being mostly straight in the bud and not
bending 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; Hadiahet al., 2008). As with a previousmolecular
study (Hadiah et al., 2008), our data indicated that Cecropiaceae was
neither distinct from Urticaceae nor monophyletic.
Of the four Cecropiaceae genera examined, Poikilospermum is
not 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 after
antithesis (Chew, 1963). Conversely, it resembles Urera in its elon-
gated cystoliths (Friis, 1993), and like some Urera species it is a
woody climber. It is the only Asian genus in Clade 3F, although
the whole clade is tropical; this indicates that it might be an Asian
vicariant of Urera.IfPoikilospermum is excluded, the remaining
members of Cecropiaceae may be a natural group, as the three gen-
era examined (Cecropia,Coussapoa,Myrianthus) formed a subclade
within Clade IV, sister to Leucosyke and Maoutia of Urticaceae and
relatively basal within the family. Nesting of this within Urticaceae
precludes family status of the subclade but tribal status might be
appropriate (see Section 4.6 below).
Table 3
Characteristics of datasets.
Information ITS 18S matRtrnL–
trnF
rpll4–rps8–infA–
rpl36
matKrbcL 2nrDNA 4cp DNA 5organelle
DNA
18S + matR Combined
7DNA
No. ingroups 144 52 27 161 148 126 159 151 166 166 53 169
No. outgroups 4 3 3 4 4 4 4444 3 4
Aligned length 991 1733 1988 1735 1956 966 750 2724 5407 7395 3721 10119
No. variable characters 770 207 167 896 1119 587 210 977 2812 2979 374 3956
No. parsimony-informative
characters
654 143 49 657 870 479 165 797 2171 2220 192 3017
Tree length 7262 520 180 2450 3128 1744 608 7716 8145 8333 671 16125
Consistency index (CI) 0.251 0.515 0.95 0.576 0.56 0.527 0.447 0.271 0.534 0.543 0.654 0.41
Retention index (RI) 0.703 0.741 0.944 0.888 0.888 0.884 0.865 0.709 0.878 0.879 0.796 0.812
Rescaled 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 0.333
Model 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
824 Z.-Y. Wu et al. / Molecular Phylogenetics and Evolution 69 (2013) 814–827

4.3. Phylogenetic relationships within Clade I
Clade I was strongly supported as monophyletic; it comprised
all 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), Touchardia
in Clade III and Myriocarpa in Clade II. Touchardia and Myriocarpa
differ 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 the
leaves (Friis, 1989). Furthermore, female tepals are free in Touchar-
dia and absent in Myriocarpa, whereas Boemerieae perianths are
typically tubular. Hence morphological evidence corroborates
molecular in showing that neither belongs in Boehmerieae.
Clade I comprised five subclades among which relationships are
strongly but not unequivocally supported. Clade 1D comprised all
genera of Forsskaoleae examined, making this tribe monophyletic
in our analysis, consistent with Hadiah et al. (2008). Hence the
tribe’s defining character of male flowers with only one stamen
is probably a genuine apomorphy. Within Forsskaoleae, however,
Australina is nested within Droguetia, requiring the former to be
sunk or the latter split to achieve generic monophyly.
Sister to Clade 1D is Clade 1C, again consistent with Hadiah
et al. (2008), Clade 1C comprised three genera of Parietarieae,
among which the shrubby island endemic Gesnouinia and the
tiny-leaved creeper Soleirolia are nested within Parietaria. Hence
generic limits here need reexamination. The other two genera of
Parietarieae, Rousselia and Hemistylus are placed in Clade 1A5 with
Pouzolzia, 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 with
an involucre of two large bracts. Indeed, Gaudichaud (1830) placed
all three in Parietarieae. Moreover, Rousselia and Hemistylus are
linked to Pouzolzia and Neodistemon by pericarp structure, a similar
form of heterocarpy, and the presence of dense two-fruited involu-
crate infructescence (Kravtsova, 2009), providing synapomorphies
for subclade 1A5 and supporting the placement of Rousselia and
Hemistylus in Boehmerieae. Meanwhile, absence of stipules pro-
vides a synapomorphy for a reduced Parietarieae comprising only
Clade 1C (Gesnouinia,Solierola and Parietaria).
Subclades 1A, 1B and 1E correspond approximately to Friis’
(1993) Boehmerieae. Although the monophyly of 1A + 1B + 1E is
only modestly supported, the shared morphological characters of
Boehmerieae also support this relationship, despite several excep-
tions both within and outside the clade. Basal in most datasets and
analyses was Clade 1B, containing Oreocnide, whose stigmas are
either peltate with long ciliate margin or penicillate-capitate, a
character 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 be
considered as a synapomorphy of Oreocnide. Like Phenax (Clade
1E), Oreocnide falls outside the main clade of Boehmerieae (Clade
1A) 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 phylogenetic
affinities to be settled (Friis, 1993). Our data places it firmly within
Boehmerieae, 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 morphological
synapomorphies for this group, although most genera have free
or united female perianth, and bracts never united into an involu-
cre. Of the larger genera within Clade 1A, consistent with Hadiah
et al. (2008),Debregeasia appeared to be monophyletic, whereas
Boehmeria and Pouzolzia were not. Boehmeria was split across three
strongly supported clades (1A3, 1A5 and 1A7) and hence is almost
certainly not a natural group. Pouzolzia was split between
subclades 1A5 and 1A7, in neither of which did the Pouzolzia spe-
cies form a natural group. No morphological characters are known
that distinguish the members of Boehmeria or Pouzolzia in one
subclade from those in another. This means that the morphological
characters defining both genera may be plesiomorphic within
Clade 1A, and leaving no good solution for how to make either
genus monophyletic. Possibly a more intensive sampling might
help 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. Differences
between Myriocarpa and Boehmerieae are noted above, but it also
shares with Lecantheae elongated-linear cystoliths, indicating
that the importance of this character may be greater than previ-
ously supposed (Friis, 1993). Gyrotaenia’s current placement is in
Urticeae (Friis, 1993) despite it its absent stinging hairs, penicil-
late-capitate stigma, and punctiform and linear cystoliths; hence
again morphology does not contradict an alternative placement
in Lecantheae. These two genera together form a well-supported
subclade (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 are
truly distinct, or if fleshy peduncles evolved multiple times in this
clade.
Clade II includes the two largest genera in Urticaceae, Pilea and
Elatostema. Of these, Elatostema is monophyletic, whereas Pilea is
only monophyletic if Sarcopilea is included within it. Hence the
only species of Sarcopilea may be better regarded as merely an
aberrant member of Pilea. Sister to Pilea is a monophyletic Lecan-
thus, supporting the recognition of these as separate genera, as is
also 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 more
than 200 of its ca. 500 species recorded from China, including
many local endemics (Wang, 2012). However, generic limits
involving Elatostema,Pellionia and Procris have been controver-
sial. Schröter and Winkler (1935, 1936) considered Pellionia as
a subgenus within Elatostema, whereas Hallier (1986) considered
both Pellionia and Procris to be subgenera within Elatostema.
However, Wang (1980a,b, 1995, 2012) supported recognizing
Elatostema as distinct from Pellionia on the basis of its perianth
lobes of female flowers being much shorter than the ovary or
strongly reduced, and not corniculate at apex. Friis (1993) was
uncertain about the monophyly or affinities of Pellionia. Previous
molecular analysis based on one DNA region (trnL–F) (Hadiah
et al., 2003) also showed Procris nested within a subset of Pellio-
nia and did not conflict the monophyly of Elatostema excluding
Pellionia. 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 more
intensive taxon sampling is needed to resolve generic limits in
Pellionia and Procris.
4.5. Phylogenetic relationships within Clade III
The strongly supported Clade III comprises 11 genera, which is
largely consistent with Friis’ (1993) tribe Urticeae, except that
Gyrotaenia was excluded and Touchardia and Poikilospermum were
included. Touchardia has no obvious morphological links to Urti-
ceae but is nested within Urera, as might be Poikilospermum and
Z.-Y. Wu et al. / Molecular Phylogenetics and Evolution 69 (2013) 814–827 825

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 monophyletic
genus each (Nanocnide,Laportea,Girardinia), one contains two
sister genera (Dendrocnide with Discocnide), and the last comprises
Urtica with Hesperocnide nested within it; however only one DNA
region 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 that
closely encloses the ovary, and its sessile inflorescences with
many-flowered glomerules or cymules (Friis, 1993).
A sister relationship between Urtica and Nanocnide is strongly
supported, and there is weaker support for these forming a clade
with Girardinia,Dendrocnide and Discocnide. This group of genera
are all characterized by stinging hairs (see Fig. 2N–P). Outside this
group fall Laportea and Clade 3F, whose relationships to it and to
one another within Clade III are not supported. Furthermore, only
one Laportea species was sampled in this study, so the monophyly
and relationships of this genus require further investigation.
4.6. Phylogenetic relationships within Clade IV
Clade IV comprises two subclades. Subclade 4A has fairly strong
support, and comprises of three genera from Cecropiaceae: Cecro-
pia,Coussapoa and Myrianthus. Based on morphology, the unsam-
pled Pourouma and Musanga of Cecropiaceae presumably also
belong here.
Subclade 4B includes the one sampled species each of Maoutia
and 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 united
with Gibbsia (not sampled in our study) by Mabberley (2008).
Hence our detection of a close relationship between Maoutia and
Leucosyke fits their morphology.
Because Clades I, II and III each comprise one or more distinct
tribes, it seems appropriate to recognize Clade IV at a similar tax-
onomic level. However, there are no uniting characters between
subclades 4A and 4B. Subclade 4A is defined by the characters of
Cecropiaceae 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 morphologically
very similar to Boehmerieae, so treating Clade 4A as a tribe would
either create a paraphyletic Boehmerieae or necessitate a new tribe
(Clade 4B) with no known morphological distinctions from the
existing Boehmerieae.
5. Conclusions
Our study provides the most comprehensive phylogeny so far of
Urticacae, both for taxon sampling and DNA regions examined; it
indicates that the family is monophyletic if Cecropiaceae is merged
with it. Most clades were well-supported, and some large genera
were clearly monophyletic, but a few instances of paraphyly were
detected 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 phylogenetic
understanding of the family overall, especially at generic level,
remains incomplete. Relationships between some subclades
remained poorly supported, though this does not hamper
classification.
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 creates
relatively few taxonomic problems or morphological anomalies.
Cecropiaceae (excluding Poikilospermum) may be best treated as
a tribe within Urticaceae, but this requires that a new tribe is
erected 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 an
altered Boehmerieae, making seven tribes in total. Treating Clades
I, II and III as subfamilies, as per Kravtsova (2009), fit the molecular
data 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, but
it 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 Boehmeria
and Urera, and evolutionary studies of aberrant genera such as Poi-
kilospermum and Touchardia. The current phylogeny provides a
firm starting point for all such work.
Acknowledgments
We are deeply indebted to Profs. Wen-Tsai Wang and Chia-Jui
Chen (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-Dong
Zhang, Jie Cai, Lu Lu, Wei Jiang, Ram C. Poudel, Yu-Xiao Zhang
(all KUN), Tsai-Wen Hsu (TAIE), and Jeffrey Boutain (University of
Hawaii) for their kind help with sample collection and technical
assistance in the lab. We are grateful to the National Tropical
Botanical Garden of Hawaii for providing some molecular samples
for this study. We are particularly grateful to Royal Botanic Gar-
dens, Kew for providing kindly DNA samples of some taxa. The
authors also thank both anonymous reviewers for their valuable
comments on the manuscript. The present study was supported
by a Keynote Project of National Natural Science Foundation of Chi-
na (Grant No. 40830209), and the Research Fund of the Natural
History Museum, London and of the Systematics Association and
Linnean Society of London. Zeng-Yuan Wu was supported by the
China Scholarship Council for 1 year study at the Natural History
Museum, and the Royal Botanic Gardens, Kew.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ympev.2013.06.
022.
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