Molecular Phylogenetics and Evolution 67 (2013) 404–413

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Molecular Phylogenetics and Evolution
journal homepage: www.elsevier.com/locate/ympev

Higher level phylogenetic relationships within the bamboos (Poaceae:
Bambusoideae) based on five plastid markers
Scot A. Kelchner ⇑, Bamboo Phylogeny Group 1
Department of Biological Sciences, Campus Stop 8007, Idaho State University, Pocatello, ID 83209-8007, USAa r t i c l e

i n f oArticle history:
Received 2 July 2012
Revised 14 November 2012
Accepted 6 February 2013
Available online 20 February 2013
Keywords:
BambooPlastid phylogeny
Exploratory data analysis
Noncoding DNA
ndhF
Microstructural charactersa b s t r a c t
Bamboos are large perennial grasses of temperate and tropical forests worldwide. Two general growthforms exist: the economically and ecologically important woody bamboos (tribes Arundinarieae andBambuseae), and the understory herbaceous bamboos (tribe Olyreae). Evolutionary relationships amongthe 1400 + described species have been difficult to resolve with confidence. Comparative analysis of bamboo plastid (chloroplast) DNA has revealed three to five major lineages that show distinct biogeographic
distributions. Taxon sampling across tribes and subtribes has been incomplete and most published data
sets include a relatively small number of nucleotide characters. Branching order among lineages is often
poorly supported, and in more than one study herbaceous bamboos form a clade within the woody bamboos. In this paper, the Bamboo Phylogeny Group presents the most complete phylogeny estimation todate of bamboo tribes and subtribes using 6.7 kb of coding and noncoding sequence data and 37 microstructural characters from the chloroplast genome. Quality of data is assessed, as is the possibility of long
branch attraction, the degree of character conflict at key nodes in the tree, and the legitimacy of threealternative hypotheses of relationship. Four major plastid lineages are recognized: temperate woody,
paleotropical woody, neotropical woody, and herbaceous bamboos. Woody bamboos are resolved asparaphyletic with respect to Olyreae but SH tests cannot reject monophyly of woody species(Arundinarieae + Bambuseae).
Published by Elsevier Inc.

1. Introduction
Bamboos are remarkably robust forest grasses that number
more than 1400 described species in 115 genera (Bamboo Phylogeny Group [BPG], 2012). Most familiar and useful are those with‘‘woody’’ (lignified) stems that belong to the tribes Arundinarieae(temperate woody bamboos) and Bambuseae (tropical woody
bamboos). The roughly 1300 woody species often play critical rolesin the ecology of their forest habitats and have long been of economic importance to humans (see McClure, 1966; Judziewicz
et al., 1999). Less popularly known are the herbaceous bamboos(tribe Olyreae), a lineage of about 120 non-woody understory species found in tropical forests, predominantly in the New World. Together the three tribes constitute the grass subfamily
Bambusoideae, members of which share the unique character ofasymmetrically invaginated arm cells in the chlorenchyma (Zhang⇑ Fax: +1 208 282 4570.

E-mail address: kelchner@isu.edu
Bamboo Phylogeny Group members: Lynn Clark, Gilberto Cortés, Reyjane P.
Oliveira, Soejatmi Dransfield, Tarciso Filgueiras, Amanda E. Fisher, Gerald F. Guala,
Trevor Hodkinson, Emmet Judziewicz, M. Kumar, De-Zhu Li, Ximena Londoño, M.
Teresa Mejia, Ana Paula Santos-Gonçalves, Chris Stapleton, Sarawood Sungkaew,
Jimmy Triplett, Elizabeth Widjaja, Khoon Meng Wong, Nian-He Xia.
11055-7903/$ - see front matter Published by Elsevier Inc.
http://dx.doi.org/10.1016/j.ympev.2013.02.005

and Clark, 2000). Most species also possess fusoid cells in themesophyll, papillae on at least the abaxial leaf epidermis, and abroad leaf blade with a basal constriction called a pseudopetiole(GPWG, 2001; Judziewicz and Clark, 2007; BPG, 2012).
Members of Bambusoideae are found from sea level to over
4000 m in mainly forest and high montane grassland habitats
worldwide, except for Europe and Antarctica (Fig. 1). At least 40%of species (including many awaiting formal description) are endemic to the Americas. In both the Americas and Asia, several taxa
can reach gigantic proportions: plant height can be to 40 m andclonal individuals can occupy many hectares (Judziewicz et al.,1999). Famously, woody bamboos can take between 7 and120 years to flower, often in gregarious events that lead to massseed production and an accompanying dieback of parent plants.
Because of this rarity of flowering, woody bamboos are usuallyencountered in their vegetative stage and identification of species
can be difficult for a non-specialist.
Bambusoideae is one of three subfamilies in the BEP clade ofgrass family Poaceae (GPWG, 2001; GPWG II, 2012). Although each
is monophyletic in molecular analyses, branching order of the BEP
subfamilies has been difficult to resolve. Several recent analyses(e.g., Bouchenak-Khelladi et al., 2008; Davis and Soreng, 2010;
GPWG II, 2012; Wu and Ge, 2012) support a sister relationship between Bambusoideae (B) and Pooideae (P; the bluegrasses), with

S.A. Kelchner, Bamboo Phylogeny Group / Molecular Phylogenetics and Evolution 67 (2013) 404–413

405

Fig. 1. World distribution of bamboos (Poaceae: Bambusoideae).

the B + P clade being sister to Ehrhartoideae (E; the rices). Branching order of the three clades varies across studies, possibly as afunction of outgroup sampling.
Within Bambusoideae, three to five major lineages are recovered by comparative DNA sequence analyses (Clark et al., 1995,
2007; Kelchner and Clark, 1997; Zhang and Clark, 2000; Bouchenak-Khelladi et al., 2008; Sungkaew et al., 2009) despite the often
marked dissimilarity of taxa and genetic loci sampled. Temperate
woody bamboos form the most robust of these lineages, appearing
as an uninterrupted branch in molecular phylogenies that suggests
a long evolutionary history followed by recent and rapid radiation(Hodkinson et al., 2010). Herbaceous bamboos are also clearlymonophyletic and at many loci show rates of sequence evolution
much higher than those of other bamboos and more similar to
grasses in general (Gaut et al., 1997). The tropical woody bamboos
form between one and three main lineages; they are most commonly resolved as two (paleotropical woody; neotropical woody)or three (paleotropical woody; Arthrostylidiinae + Guaduinae;
Chusqueinae). Rate of sequence evolution in subtribe Chusqueinaealso appears to be accelerated, though to a lesser degree than theherbaceous lineage.
Since the first comparative DNA sequence analysis of bamboos by
Kelchner and Clark (1997), more than 20 molecular phylogenetic
analyses have been published on taxonomic subsets of Bambusoideae (reviewed by BPG (2012)). In that time, several phenomenathat complicate phylogeny estimation have been observed in mostbamboo nuclear and plastid (chloroplast) DNA data sets. These include strong heterogeneity of sequence evolution rates among lineages, poor support for internodes that separate major clades, long
branch attraction that can affect both ingroup and outgroup topology, lack of strict congruence among trees from different studies(or data sets from different genomic compartments), and problems
with attaining supported resolution in certain lineages such as thetemperate woody and paleotropical woody clades.
Molecular phylogeny estimation in bamboos is thus widely considered to be a difficult problem. Maximum parsimony has been
the most common optimization criterion used for tree selection
in bamboo molecular systematics, yet it is well known that parsimony can mislead phylogeny estimation when the above phenomena are a feature of the data set and its analysis. Recent studies
have used Bayesian inference together with parsimony (e.g., Yanget al., 2007; Peng et al., 2008; Sungkaew et al., 2009; Triplett andClark, 2010), which at least provides an opportunity to test robustness of a phylogeny estimation to certain changes in the model ofcharacter evolution employed. Frequently the two analyticalframeworks do not produce the same topology (e.g., Yang et al.,2007; Peng et al., 2008), suggesting that the problematic issues
listed here for bamboo molecular studies can indeed affect the estimate of bamboo phylogeny.
In response to the challenge of generating a well sampled, datarich, and carefully tested molecular phylogeny of Bambusoideae,
the Bamboo Phylogeny Group was formed to better coordinate efforts among many of the world’s bamboo systematists (BPG, 2006).
Molecular studies had already suggested that a taxonomic reorganization of bamboos was inevitable; BPG members sought to alignthat reclassification to a strongly supported estimate of phylogeny.
The endeavor was to begin with a phylogeny estimation of chloroplast genomes (plastomes) in bamboos because the task could bereadily addressed with existing collections of silica-gel-dried leaf
tissue from which sequence data of chloroplast loci are easily obtained. Taxon sampling was designed to include all subtribes ofbamboos, and DNA sequences would be taken from five plastomeloci. Phylogenetic analyses were to employ several available tests
that determine data quality, tree stability, and whether bias and error were negatively affecting the phylogeny estimation.
In this paper, the Bamboo Phylogeny Group presents its first rigorously tested plastome phylogeny estimation. The estimate represents the breadth of taxonomic, geographic, and morphologicaldiversity in the subfamily Bambusoideae. Aims of this study include (i) improvement of resolution and support for commonly
recognized lineages, (ii) identification of previously unknown relationships, (iii) establishment of branching order among major
clades, (iv) stabilization of the plastid phylogeny estimation, and(v) identification of remaining areas of concern in the topology thatmight hinder further progress in deciphering the biogeography,
history, and evolution of bamboos.

2. Material and methods
2.1. Taxon sampling
A total of 40 species, including 33 ingroup (Bambusoideae) andseven outgroup (Ehrhartoideae, Pharoideae, Pooideae) taxa, was406

S.A. Kelchner, Bamboo Phylogeny Group / Molecular Phylogenetics and Evolution 67 (2013) 404–413

analyzed (Table 1). Ingroup species were chosen to represent thethree major lineages of bamboos now recognized as tribes (Arundinarieae, Bambuseae and Olyreae), and each of the currently or, inthe case of Arundinarieae, previously recognized, subtribes of Bambusoideae (BPG, 2012). With the exceptions of Parianinae, Racemobambosinae and the monotypic Buergersiochloinae, each subtribe
was represented by two to four species. Neurolepis taxa are treatedhere as Chusquea after Fisher et al. (2009). In addition to representing taxonomic diversity, the particular species sampled in thisstudy were also chosen to represent as much morphological diversity as possible, given samples available at the time. The seven outgroup taxa consisted of three species each from BEP clade
subfamilies Pooideae and Ehrhartoideae (including a sister taxon
to the main lineage of each subfamily), and one species from the
Pharoideae, an early diverging Poaceae lineage that is sister tomost of the remaining grasses (GPWG, 2001; GPWG II, 2012). Leaf
material was collected in the field or from greenhouse-grown specimens and silica-gel dried tissue (Chase and Hills, 1991) except for
Oryza sativa and Triticum sativum for which sequences were downloaded from GenBank (Table 1).
2.2. DNA sequencing and alignment
Five plastid DNA regions were used in this analysis: one gene
(ndhF), two group II introns (rpl16 intron, rps16 intron), and twointergenic spacers (trnD–trnT, trnT–trnL). Total genomic DNA
extractions were performed with Qiagen DNeasy Plant Mini Kits
(Qiagen, Valencia, USA) on vouchered silica gel-dried leaf tissue.
Isolations were cleaned using Qiagen PCR Purification Columns
and then quantified with fluorometry. Target regions were PCRamplified in the laboratories of Clark and Kelchner. Each automated sequence file was reviewed for base-calling errors, poor sequence reads, and multiple peaks. Unusual sequences were verified
by conducting a second DNA isolation, PCR amplification, and sequence from the original vouchered leaf materials. BLAST searches
were used to confirm probable homology of each target sequence.
Regions were aligned individually by process partition using acriterion-based manual methodology that infers secondary structures and mutational mechanisms to then inform the positioningof gaps (Kelchner and Clark, 1997; Kelchner, 2000). Specific criteria
for choosing insertions, deletions, and hairpin inversions (Kelchnerand Wendel, 1996) as scored microstructural characters followed
Kelchner (2000) and Löhne and Borsch (2005). Gap placements inthe combined alignment are staggered to reflect hypothesizedindependent insertion and deletion events (Morrison, 2006,2009). This approach allowed us to recover nucleotide substitution
characters within length mutations that had reasonable biologicalevidence of homology. Insertions and deletions that involvedmononucleotide repeats were not coded as microstructuralcharacters.
2.3. Data quality and model selection
Ambiguous characters in each alignment were included for
pairwise characterization of sequence structure (base frequency
and sequence length comparisons) but were excluded prior to phylogenetic analyses of individual data sets following Morgan and
Kelchner (2010). In the combined data set, ambiguous regions
were deleted entirely from the matrix prior to phylogeny estimation for the purposes of decreasing the overall quantity of missing
data and for facilitating maximum likelihood and network analyses
across multiple software platforms.
Base composition homogeneity was assessed for individual andcombined data sets by the chi-squared test of base frequency
inequalities implemented in PAUP! 4.0b10 (Swofford, 2002). PAUP!
was also used to measure variation in uncorrected pairwise

Table 1
Taxon samples used in this study, with classification and voucher information provided.
Subtribe nomenclature, when previously contradictory, follows that of BPG (2012).
Ingroup taxa
Tribe Arundinarieae
Ampelocalamus scandens J.R. Xue and W.D.
Li
Arundinaria gigantea (Walter) Muhl.
Chimonobambusa marmorea (Mitford)
MakinoChimonocalamus pallens J.R. Xue and T.P. Yi
Phyllostachys bambusoides Siebold and
Zucc.
Pleioblastus argenteo-striatus (Regel) Nakai
Sasa veitchii (Carriere) RehderShibataea kumasaca (Zoll. ex Steud.)
MakinoThamnocalamus spathiflorus (Trin.) Munro
Tribe Bambuseae
Subtribe Arthrostylidiinae
Atractantha radiata McClure
Glaziophyton mirabile Franch.
Rhipidocladum pittieri (Hack.) McClure
Subtribe Bambusinae
Bambusa vulgaris Schrad. ex. J.C. Wendl.
Cyrtochloa luzonica (Gamble) S. Dransf.
Neololeba atra (Lindl.) Widjaja
Oxytenanthera abyssinica (A. Rich.) Munro
Temochloa liliana S. Dransf.
Subtribe Chusqueinae
Chusquea bambusoides (Raddi) Hack.
Chusquea elata (Kunth) L.G. Clark(=Neurolepis elata)
Chusquea scandens Kunth
Chusquea spectabilis L.G. Clark (=Neurolepis
aperta)Subtribe Guadinae
Apoclada simplex McClure and L.B. Sm.
Guadua angustifolia Kunth
Otatea acuminata (Munro) C. Calderon andSoderstr.
Subtribe Hickeliinae
Hickelia madagascariensis A. Camus
Nastus borbonicus J.F. Gmel.
Subtribe Melocanninae
Cephalostachyum pergracile (Munro) R.B.
Majumdar
Melocanna baccifera (Roxb.) Kurz
Subtribe Racemobambosinae
Racemobambos hepburnii S. Dransf.
Tribe Olyreae
Subtribe Buergersiochloinae
Buergersiochloa bambusoides Pilg.
Subtribe Olyrinae
Olyra latifolia L.
Sucrea maculata Soderstr.
Subtribe Parianineae
Pariana radiciflora Doell in Martius
Outgroup taxa
Ehrhartoideae
Ehrharta dura Nees ex Trin.
Oryza sativa L.
Streptogyna americana C.E. Hubb.

(LC 1291)(JT 197)(JT 69)(JT238)(LC 1289, JT 121)
(JT 66)(JT 126, LC 1325)(LC 1290)(LC 1319)(ASG 599, AMC 4362)(LSS 1066)(LC 1349, LC and WZ 1349)(GSK 666)(SD 1323)(LC and JT 1663)(LC and JT1664)(SD 1494)(LC 1029)(LC and PA1409)(LC and XL 1235)(LC 919)(LC and WO 1027)(LC and XL 931)
(LC and WZ 1348, LC et al.
1312)(SD 1290, SD 1292)(LC and SD 1656)(WZ 8400635, SD 1435)(XL and LC 930)
(WKM2891)(SD 1365, SD 1382)(XL and LC911)(LC and WZ 1345)(LC and WZ 1344)(NB 1118)(GenBankNC 0013201)(RP and GD 12310, GSK
657)

Pharoideae
Pharus latifolius L.

(LC 1302)

Pooideae
Brachyelytrum erectum (Schreb.) P. Beauv.
Diarrhena obovata (Gleason) Brandenberg
Triticum aestivum L.

(JT 199)(LC and WZ 1216, JT 290)
(GenBankNC 0027621)

Voucher abbreviations: AMC – Andre Mauricio Carvalho; ASG – Ana Paula SantosGonçalves; GD –Gerrit Davidse; GSK – Gabriel Sanchez-Ken; JT – Jimmy Triplett; LC– Lynn Clark; LSS – Luis Sergio Sarahyba; NB – Nigel Barker; PA – Patricio Asimbaya;
RP – Richard Pohl; SD – Soejatmi Dransfield; WKM – Wong Khoon Meng; WO –Walter de Oliveira; WZ – Weiping Zhang; XL – Ximena Londoño.

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distances among sequences and the number of potentially informative substitutions observed in each alignment matrix. Charactersaturation was assessed by plotting uncorrected pairwise distances
against model-corrected distances (Philippe et al., 1994), a methodthat can be use for noncoding DNA because it does not rely on codon position. Model-corrected distances were produced using a
best-fit model for the combined data set (discussed below).
For phylogeny estimations using probability frameworks (Section 2.4), a model of character evolution had to be specified. Boththe hierarchical likelihood ratio test (hLRT) and the Akaike Information Criterion (AIC) were used to ascertain model adequacy
among the 56 models available for comparison in ModelTest (Posada and Crandall, 1998). The hLRT method might be biased due toits hierarchical approach (Kelchner and Thomas, 2007), but all
hLRT results were compared to models that show reasonable evidence of being ‘‘best fit’’ by AIC (i.e., those models that have AIC
differences of D 6 2; the rationale follows Posada and Buckley(2004) and Kelchner (2009)).
Conflict among character state changes in the data was evaluated in two ways. First, the data were visualized with a neighbor-net analysis using the program SplitsTree 4 (Huson andBryant, 2006). Networks of uncorrected and model-corrected distances were surveyed to identify sequences involved in large reticulations that indicate character conflict (Morrison, 2005, 2010).
Second, nonparametric bootstrap analyses were performed on eachdata set using maximum likelihood in PhyML 3 (Guindon and
Gascuel, 2003). The best-fit model was set for each data partition
with all parameter values estimated in 1000 pseudoreplicates. Results were surveyed for bootstrap-supported incongruence among
trees recovered for each sequence region. In one case (rpl16 intron),
the Shimodaira–Hasegawa test (SH test, Shimodaira and Hasegawa(1999)) was used to determine if an observed incongruence withbootstrap support of 80 was significantly different to the combined
nucleotide phylogeny estimation when given the rpl16 intron data
and three different models of character evolution. The SH test was
performed with PAUP! using RELL and 10,000 pseudoreplicates.
2.4. Tree estimation
Three analytical frameworks were applied to establish whether
the phylogeny estimation for Bambusoideae plastomes was robust
to reasonable changes in assumptions about character evolution(Penny et al., 1992; Kelchner and Thomas, 2007). Two of the frameworks, maximum parsimony (MP) and Bayesian inference (BI),could be applied to both nucleotide and microstructural characters.
The third framework, maximum likelihood (ML), is most efficiently
used for phylogeny estimation from nucleotide data. Therefore,
only nucleotide characters were used for topology comparisonsthat evaluated robustness across frameworks. In the estimations
produced by MP and BI from combined data, scored microstructural changes were included at the end of the alignment matrix
as 0/1 standard characters.
Equal weight MP analyses (sensu Fitch, 1971) were conductedin PAUP! using a rapid, suboptimal search strategy in which 1000
replicates of random taxon-addition starting trees produced a poolof 100,000 topologies from which a secondary TBR search was
swapped to completion and a strict consensus of the most parsimonious trees was produced. This strategy provided a computationally tractable way to perform nonparametric MP bootstrap
analyses using PAUP! (1000 pseudoreplicates). The most parsimonious topology was not sought for or required by our assessment ofrobust signal across analytical frameworks.
ML analysis was performed in PAUP! when the aim was to obtain an optimal (i.e., maximized likelihood estimate, or MLE) topology for the individual and combined data sets. The program PhyML
was used for nonparametric ML bootstrapping (10,000

407

pseudoreplicates). In both programs, the model was designatedto be one of the best fit candidate models for the data set, withmodel parameter values estimated during analysis and microstructural characters excluded. Each PAUP! MLE search was conducted
twice, and MLE topologies were used primarily for SH testing ofalternative hypotheses. Although PhyML might not be an optimal
search algorithm for MLE topologies, it is computationally inexpensive and is likely to be conservative when used for nonparametric bootstrapping: if some pseudoreplicates fail to produce
truly optimal estimates, then the spurious topological conflict overten thousand repetitions would slightly lower bootstrap support
values.
BI was conducted with MrBayes 3.1 (Huelsenbeck and Ronquist,
2001) using a partitioned GTR + I + G model for reasons outlined by
Huelsenbeck and Rannala (2004) with all parameter values estimated during analysis. A Dirichlet prior was used for base frequencies and the rate matrix. A uniform prior was used for the shapeparameter (a), proportion of invariable sites (I), and topology.
Branch lengths were unconstrained. Partitions were designatedfor each data set and for the microstructural characters and allparameters were unlinked across partitions. Four separate analyses
were performed with four runs of four chains each that continued
for 20 million generations. Posterior probabilities were calculatedusing the final 10 million generations in each run, with sampling
every 100 generations; this cut-off value was computationally efficient and it conservatively satisfied a rough convergence criterion
that average standard deviation of split frequencies should stabilize below 0.01.
2.5. Rooting and potential long branch attraction
Estimated phylogenies were rooted with Pharus latifolius. P.
latifolius is, however, one of many long branches in this study that
attaches to a short internode in each tree. To survey for potential
error caused by long branch attraction (LBA) in our analyses, taxon
removal experiments were performed in PAUP! with combined
nucleotide data using ML (model GTR + I + G). The following taxon
removals were tested individually: (i) Olyreae; (ii) all outgrouptaxa except Streptogyna americana; (iii) Pooideae; (iv) Triticum aestivum; (v) Ehrhartoideae; (vi) Oryza sativa; (vii) Chusqueineae; and(viii) Streptogyna americana. The MLE topology from each analysis
was checked for changes in relationship among remaining taxa
when rooted with P. latifolius. The removal of Streptogyna americana also included a nonparametric ML bootstrap analysis (1000
pseudoreplicates) because of its high level of character conflict at
key nodes in the SplitsTree neighbor-net graph.
2.6. Testing of alternative hypotheses
Three ML constraint analyses of combined nucleotide data wereconducted with PAUP!. In the first analysis, all woody bamboos(Arundinarieae and Bambuseae) were required to form a single
clade that was sister to herbaceous bamboos (Olyreae). In the second analysis, Ehrhartoideae was constrained to be sister to Bambusoideae. In the third analysis, Chusqueinae was forced to be sister
to a clade of all other tropical woody bamboos (Paleotropical + Arthrostylidiinae + Guaduinae). A fourth analysis involved
the incongruent topology from the rpl16 intron data set. Constraint
MLE topologies were tested individually against the unconstrained
MLE topology of combined nucleotide data in a two–tree SH test
(sensu Buckley et al., 2001) that maintains excellent type 1 error
control yet avoids the conservative behavior the test shows in multiple tree comparisons (Shimodaira, 2002); in this form, the test reduces to a KH test with correction for a posteriori topologies(Shimodaira and Hasegawa, 1999; Goldman et al., 2000). All SH
tests were performed with PAUP! and used 10,000 RELL replicates.

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Because SH test results can vary with model choice (Buckley et al.,2001), each test was performed three times using the models JC,
HKY + G, and GTR + I + G.
3. Results
3.1. Sequence alignments and data quality
Sequence recovery from the five plastid regions was generally
straightforward. PCR amplifications resulted in single bands andthe sequence files showed no problematic double peaks. Of the
GenBank sequences downloaded for the analysis, the rps16 intron
in T. aestivum appeared to be inaccurately labeled (see below, thissection) and was scored as missing data in the combined analyses.
GenBank accession numbers for sequences generated by this study
are KC020491–KC020545, and KC020547–KC020602; a table of
GenBank accession numbers by sequence region and taxon is included in the online Supplementary material.
Alignment length (number of characters) by region was 2120
for ndhF, 1364 for rpl16 intron, 1006 for rps16 intron, 1465 for
trnD–trnT spacer, and 1066 for trnT–trnL spacer. Percent informative characters for all regions was between 10% and 11% except
for the rps16 intron (6.96%). The combined alignment with ambiguous regions removed was 6657 nucleotide characters with 10.03%
informative characters. Microstructural changes for which reasonable estimation of homology could be made totaled 37: two in ndhF
(including one hairpin inversion), 13 in rpl16 intron (including one
hairpin inversion), three in rps16 intron, nine in trnD–trnT spacer(including one hairpin inversion, with an additional hairpin inversion located in a deleted ambiguous region), and ten in trnT–trnL. A
list of character types and scoring across taxa is included in Supplementary material.
None of the alignments showed significant differences in basecomposition among taxa. Saturation plots indicated slight deviation when model correction was applied, but the deviation was
not strong (Supplementary material) and there was no obvious
plateau to indicate problematic saturation of the noncoding data
(e.g., Simon et al., 2009). Neighbor-net analysis of uncorrected
and model-corrected pairwise distances showed considerable
character conflict among outgroup sequences, particularly in relation to S. americana and the herbaceous bamboo lineage Olyreae.
The network also allowed for the identification of an incorrect sequence for the rps16 intron in T. aestivum, which was subsequently
removed from the alignments. A table of data set characteristics is
included in Supplementary material.
Phylogeny estimations of individual data sets showed no well
supported incongruence among region signal except in the rpl16
intron for the clade Rhipidocladum pittieri + Glaziophyton mirabile.
However, an SH test showed no significant difference between thisarrangement and the combined data topology when given rpl16 intron data and three models of character evolution (Supplementarymaterials). The clade is also not present in the MLE topology ofrpl16 intron data produced by PAUP!. These observations, as wellas the general lack of character conflict among ingroup sequencesin the neighbor-net analysis and the relatively low bootstrap values on incongruent nodes from each region, suggested that thesame tree-like signal was present among data sets and that combination of data was acceptable.

TVM + G or TVM + I + G for all data sets. The combined matrix
had both GTR + I + G and TVM + I + G as potentially best-fit using
the same AIC and hLRT criteria. ML analyses of each data set, therefore, used the following models: GTR + I + G for ndhF, GTR + G for
rpl16 intron, GTR + G for rps16 intron, GTR + G for trnD–trnT spacer,
GTR + G for trnT–trnL spacer, and GTR + I + G for combined nucleotide data.
ML bootstrap topologies for each region, with minor incongruences identified, are presented in Supplementary material.
Although the percent of informative characters is nearly equal for
all regions except rps16 intron, ndhF gave good resolution throughout its tree whereas noncoding regions provided improved resolution at mid-levels of divergence (e.g., among the four major
lineages of Bambusoideae). The position of Olyreae is dependent
on noncoding DNA data in this study.
The ndhF phylogeny estimation was congruent with the topology of the combined nucleotide analysis. Each noncoding region,
however, showed between two and five minor incongruences. All
but one of these had low bootstrap support (between 50 and 69)
on generally very short branches. The case of the rpl16 intronincongruence with a bootstrap value of 80 is discussed above
and was ruled out as being significant conflicting signal. Rps16 intron showed the most incongruence (five nodes) but also had theleast number of potentially informative characters and the least
number of resolved nodes with bootstrap support of P80. Similarly supported nodes in other data sets totaled 15 in ndhF, 13 in
rpl16 intron, 15 in trnD–trnT, and only nine in trnT–trnL despite
its 10.88% of potentially informative characters. Microstructuralcharacters resolved 14 nodes in the MP consensus tree of 641 mostparsimonious topologies (Supplementary material) with the single
incongruence of Atractantha radiata + R. pittieri; the tree has alength of 83, a consistency index of 0.446, a homoplasy index of
0.554, and a retention index of 0.711.
The independent phylogeny estimations of rps16 intron, trnD–
trnT, and trnT–trnL data place Olyreae within Bambusoideae, but
ndhF and rpl16 intron data recover only a polytomy. Rpl16 intron
and trnT–trnL place Ehrhartoideae sister to Bambusoideae withlow bootstrap support (69 and 57, respectively). This differs fromndhF, trnD–trnT and the combined data which place Pooideae assister to Bambusoideae with ML bootstrap support below 67. Bootstrapping of rps16 intron data produced a polytomy involving thesethree lineages.
Phylogenetic analysis of combined nucleotide characters produced a congruent topology across MP, ML and BI frameworks(Fig. 2a). The topology was robust to all models of character evolution trialed. Four main lineages are present within Bambusoideae:
the Paleotropical Woody clade (subtribes Bambusinae, Racemobambosinae, Hickeliinae, Melocanninae), the Neotropical Woody
clade (subtribes Chusqueinae, Arthrostylidiinae, Guaduinae), theHerbaceous clade (subtribes Buergersiochloinae, Olyrinae, Parianinae), and the Temperate Woody clade (tribe Arundinarieae). Pooideae is weakly supported as sister to Bambusoideae (ML bootstrapof 67, posterior probability of 0.94). Twenty-seven nodes wererecovered with ML bootstrap support of P80 although five nodes
within the Paleotropical Woody clade and the Temperate Woody
clade show either very low bootstrap support or low posterior
probabilities. MP and BI analyses that included microstructuralcharacters with the combined nucleotide data gave the same topology as Fig. 2a (see Supplementary materials).

3.2. Phylogeny estimations
3.3. LBA and alternative hypothesis testing
Models selected for each data set, together with relevant AIC
information, are presented in Supplementary material. Each region
had GTR + G or GTR + I + G as one of its best-fit models from themodel candidate pool according to the criterion of AIC differences
D 6 2. Hierarchical likelihood ratio test results were either

Taxon removal experiments to survey for long branch attraction
resulted in no changes to ingroup or outgroup topologies. MLE
topology estimates from each taxon removal data set show only
one minor difference: the position of Racemobambos hepburnii

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409

Fig. 2. Phylogeny estimation and geographic distribution of major bamboo lineages based upon plastid (chloroplast) DNA sequences from five loci: ndhF, rpl16 intron, rps16
intron, trnD–trnT intergenic spacer, and trnL–trnF intergenic spacer. (A) Consensus posterior topology of the partitioned BI analysis of combined nucleotide andmicrostructural characters. The topology is congruent with the MP strict consensus topology of the same data set and the ML topology of nucleotide data alone. Posterior
probabilities are shown above branches. ML bootstrap values are listed first below each branch, followed by MP bootstrap values. Paired dashes indicate support values below
50 (bootstraps) or 0.5 (posterior probabilities). The ML estimate of topology shows additional resolution (Fig. 4). (B) Subtribe relationships and geographic distributions of thefour major lineages of bamboos recovered in this study. Clade order follows that of (A). Question mark in the Herbaceous diagram represents uncertainty about the native
status of Olyra latifolia in Africa and Madagascar.

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usually collapses into a polytomy with other members of the
Paleotropical Woody clade (see Supplementary materials). Pooideae remains sister to Bambusoideae in all taxon removal experiments which included members of that lineage, when P. latifolius
is used to root each topology.
Two–tree SH tests of three alternative hypotheses of bamboo
and outgroup relationships failed to reject any alternative constraint MLE topology for each of three character evolution models(attained significance p = 0.05). A table of SH test results can befound in Supplementary materials.
4. Discussion
Bamboos have long been a problematic plant group to examinewith molecular phylogenetic techniques. Rate heterogeneity
among lineages, lack of sequence variation in certain genera andsubtribes, and short internodes deep within the topology have prevented consistent and well supported resolution of branching order among and within major bamboo clades. Despite thedifficulty of comparative sequence analysis in Bambusoideae, wehave been able to use 6.7 kb of high quality nucleotide data and37 microstructural changes to produce a robust and rigorously
tested phylogeny estimation for bamboo chloroplasts. Nuclear data
will be required before an organismal phylogeny of bamboos canbe established with greatest confidence, but the generation of areliable chloroplast phylogeny is an essential step in that process.
We therefore present an evaluation of our phylogeny estimation,
its strengths and weaknesses, and corresponding evidence frompublished literature that allow us to assess whether this topology
should be considered a best estimate of higher level relationships
among bamboo plastid genomes.
4.1. Strengths of the phylogeny estimation
A worldwide effort of Bamboo Phylogeny Group members to attain leaf tissue samples for this study has improved upon previous
efforts of taxon sampling and vouchering of materials for bamboo
molecular analysis. The phylogeny estimation represents the greatest breadth to date of bamboo taxonomic, morphological and geographic diversity to be included in a single analysis. The estimate isderived from data representing multiple process partitions in theplastid genome, including one fast-evolving gene, two group II introns, and two intergenic spacers. Congruence of supported tree
resolution among each of those partitions indicates that the datacontains one dominant signal. Phylogeny estimation from microstructural characters mirrors the estimation derived from nucleotide characters. These observations suggest that contradictions in
the data to this dominant signal are minor and are likely due to
conflict among homoplastic characters.
Potential bias in the phylogeny estimation was not detectedwith available tests for error. There is no evidence of base composition inequalities among sequences and lineages, and no problematic site saturation. The topology is robust to moderate changes in
the model of character evolution (sensu Kelchner and Thomas,
2007) including the analytical framework (MP, ML, BI). Taxon removal experiments cause no alteration of ingroup topology thatwould indicate the misleading effect of long branch attraction.
With nearly 6.7 kb of nucleotide data showing 10–11% sequence variation among taxa, posterior probabilities and bootstrap
values are high on most nodes of the Bambusoideae phylogeny
estimation. This observation indicates that more sequence datafrom the chloroplast genome in these specimens is likely to show
the same branching order among well supported clades. Further
sequencing of chloroplast regions, if bias and poor data quality
are minimal, is therefore unlikely to alter our estimation ofhigher-level plastid relationships among the bamboos. An exception might be in the resolution of monophyly of neotropical bamboos (Section 4.2). Additional independent sequencing efforts
have, encouragingly, provided corroborative evidence of these results (Kelchner et al., in prep.).
4.2. Weaker nodes of the tree
Despite many positive signs that our topology is robust and well
supported, there remain a few areas of concern in the chloroplast
phylogeny estimation that warrant further consideration. A resultthat justifies caution is the failure to reject alternative hypothesesof relationships in the three cases tested. Although the SH test canbe unnecessarily conservative when several topologies are compared at once, the test is proper to use when the number of candidate topologies is very small and it does give better type 1 error
control than the AU test (Shimodaira, 2002). In our case only twotrees were compared in each test, conditions under which the SH
test reduces to a KH test (Kishino and Hasegawa, 1989) with correction for the comparison of a posteriori topologies.
One possible explanation for the failure to reject alternative
hypotheses can be found in the neighbor-net network of gap-free,model-corrected sequence data (Fig. 3) and in the branch lengthsof the MLE phylogram of combined nucleotide data (Fig. 4). Substantial character conflict exists in the network, represented in
the diagram as boxes outlining alternative edges of character support. The most important ingroup relationship affected by this conflict is the edge between temperate woody bamboos andherbaceous bamboos, a part of the network to which all outgroupsequences join. Although this character conflict might arise simply
from stochastic parallelism and reversal of nucleotide changes, it isproblematic enough that bootstrap-supported resolution still represents indecisive signal in SH tests. The conflict allows for a possibility that outgroup sequences are misrooting to the temperateclade sequences, which produces an apparent paraphyly of thewoody bamboos that is due to analytical error and not evolutionary history.
A second problem spot is the monophyly of Neotropical woody
bamboos, a relationship that is likely to be unstable due to a paucity of unique character variation at the node. Again the issue ispresent despite bootstrap values of 73 (ML) and 75 (MP), and posterior probability (1.00) for the clade (Fig. 2). The branch is comparatively short in the MLE phylogram of combined nucleotide
data (Fig. 4) and involves a clade (Chusqueinae) with faster thannormal rates of sequence evolution for woody bamboo chloroplasts. An SH test failed to reject the possibility that Neotropical
woody bamboos are paraphyletic with respect to the Paleotropical
Woody clade, which allows for a reasonable alternative hypothesisthat Chusqueinae is sister to a lineage that includes all remaining
tropical woody bamboos.
Although Melocanninae is supported as sister to the remaining
paleotropical woody bamboos (bootstraps 96 ML, 96 MP; posterior
probability 1.00), the network analysis (Fig. 3) shows character
conflict from R. hepburnii that likely prevents supported resolutionof branching order within the paleotropical woody lineage. The final problem area is already well known to bamboo phylogeneticists: a lack of sufficient resolution to clarify relationships within
the temperate woody clade. Encouragingly, the absence of a clear
bifurcation pattern here is not due to character conflict (Fig. 3)
but rather an absence of observed character state variation in thesequence data. Adding further high quality sequences from additional chloroplast loci therefore holds considerable promise in
the eventual resolution of major lineages within this clade (e.g.,
Triplett and Clark, 2010; Zheng et al., 2010; Zhang et al., 2011),
although if the radiation is recent enough the bamboos in the temperate clade might still fail to exhibit clear signs of lineage sorting.

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411

Fig. 3. A data-display network of combined nucleotide sequences. The neighbor-net method is used here to explore data conflict and not to estimate phylogeny. For visualclarity, taxon names have been abbreviated as the first four letters of the genus name except in the following cases: chuB (Chusquea bambusoides), chuS (Chusquea scandens),chuA (Chusquea spectabilis), chuE (Chusquea elata), chib (Chimonobambusa marmorea) and chic (Chimonocalamus pallens). The shaded arrow shows a network location that
corresponds to the question of monophyly in neotropical woody bamboos. The white arrow indicates the joining location of outgroup sequences to the long edges between
the herbaceous and temperate woody bamboo groups.

Fig. 4. Maximized likelihood estimate of topology based upon combined nucleotide data and a best-fit model (GTR + I + G). Nodes of persistent difficulty are (1) paraphyly of
woody bamboos with respect to the herbaceous lineage, (2) monophyly of neotropical woody bamboos, (3) monophyly of Bambusinae sensu stricto, and (4) the long branchleading to a probable rapid diversification of temperate woody bamboos.

Despite the robustness of the ingroup topology to long branch
attraction experiments, several long branches still exist in thephylogeny estimation that might not improve with additional taxon sampling. The branch leading to the radiation of Temperate

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Woody bamboos, for example, could remain uninterrupted: newly
sequenced members continue to show strong sequence similarity
to all other species in the clade (Triplett and Clark, 2010; Zheng
et al., 2010; Hodkinson et al., 2010). Similarly, the sister relationship of Buergersiochloa bambusoides to all other herbaceous bamboos will probably remain the earliest split in that lineage: theremaining unsampled species taxonomically fall within the Parianinae and Olyrinae clades further up that branch. So far, outgrouptaxa have proven to be relatively distant in sequence similarity andwill continue to cause problematic character conflict when joining
the long branch between herbaceous and temperate woody clades
in unrooted topologies and networks.
4.3. Congruence with other studies
Several features of our topology are worth comparison withpublished literature on bamboo phylogenetics. Presented here isa short discussion of congruence and inconsistencies between
our phylogeny estimation and those published in other studies
for the purpose of evaluating our topology. A more complete review of bamboo molecular phylogenetics literature is presented
in an accompanying classification paper by the Bamboo Phylogeny
Group (BPG, 2012).
Four major bamboo lineages were recovered that show biogeographic and morphological distinction (Fig. 2). The finding is consistent with two other recent studies that have comparable taxon
and marker sampling (Bouchenak-Khelladi et al., 2008; Sungkaew
et al., 2009). The Temperate Woody clade has long been distinct inmolecular analyses. Early evidence came from plastid RFLPs(Watanabe et al., 1994; Kobayashi, 1997), followed by comparative
DNA sequence analysis (Kelchner and Clark, 1997; Zhang and
Clark, 2000; Bouchenak-Khelladi et al., 2008; Peng et al., 2008
and several subsequent studies). Lack of resolution within theTemperate Woody clade is common to all chloroplast DNA studies,including the present one.
The Herbaceous clade has also been recognized for more than adecade, both in chloroplast and nuclear DNA studies (e.g., Clark
et al., 1995; Kelchner and Clark, 1997; Mathews et al., 2000; Bouchenak-Khelladi et al., 2008; Sungkaew et al., 2009). A Paleotropical Woody clade first appeared in Kelchner and Clark (1997) andwas supported with additional taxon sampling in Zhang and Clark(2000). This clade has since been recovered in the well sampledstudies of Bouchenak-Khelladi et al. (2008) and Sungkaew et al.
(2009). A novel result of the present study is the well-supported
inclusion of Hickeliinae, represented by two Malagasy species, in
the Paleotropical Woody clade. The subtribe forms a trichotomy
with Racemobambosinae and the Bambusinae. Although Clark
et al. (2007) included a more extensive sampling of Hickeliinae
in their analysis, the subtribe was only weakly associated withthe other paleotropical woody bamboos.
A tropical woody clade (Paleotropical + Neotropical Woody
bamboos) is well supported in both Bouchenak-Khelladi et al.
(2008) and Sungkaew et al. (2009), with only slightly less support
in our analysis. More problematic has been a confirmation of the
Neotropical Woody clade. Previous to this study, the only published molecular evidence of a Neotropical Woody clade was a preliminary rpl16 intron analysis by Kelchner and Clark (1997) and amore taxon and data rich analysis by Sungkaew et al. (2009). We
note that bootstrap support for this clade in both Sungkaew et al.
(2009) and the present study is moderate at best, that problematiccharacter conflict exists at these nodes in the network analyses,
and that possible paraphyly of Neotropical woody bamboos withrespect to the Paleotropical Woody clade cannot be rejected withSH tests.
As in Bouchenak-Khelladi et al. (2008), Sungkaew et al. (2009),
GPWG II (2012) and Kelchner et al. (in prep) our tree suggests

paraphyly of plastids in woody bamboos. The topology, however,is incongruent with Mathews et al.’s (2000) phylogeny estimationof nuclear phyB sequences in grasses, the only published nuclear
data set to include taxa from all three woody and herbaceous bamboo tribes. Although the phyB analysis was focused on tribal relationships in Poaceae, a bamboo clade was recovered with awoody lineage sister to an herbaceous lineage, as predicted by
the traditional classification scheme of two Bambusoideae tribes.
Only the herbaceous clade had bootstrap support. SH tests of ourplastid data fail to reject this alternative arrangement of taxa,
which suggests that the potential incongruence of nuclear signal
with most plastid studies could be due to analytical error fromthe accumulation of homoplasious characters in bamboo plastid
lineages. Alternatively, it is also possible that the incongruence ishistorically accurate and represents an echo of early hybridization
events among ancestral populations of diverging herbaceous andwoody bamboos. Until more data is available from the nuclear genome and directed morphological analyses, commitment to a stanceof paraphyly in woody bamboos is inadvisable.
4.4. Summary and future directions
In this paper, the Bamboo Phylogeny Group presents a rigorously tested plastid phylogeny estimation of branching orderamong the world’s major bamboo lineages. The tree is well resolved, most branches have high support values, and all tribes
and subtribes of bamboos are adequately positioned within the
topology. Corroboration of the tree by an independent study (Kelchner et al., in prep.) and congruence of major lineages with previous molecular phylogeny estimations suggests that we have
converged upon a predominant plastid signal for Bambusoideae.
Four main plastid lineages are recognized that correspond to
distinct biogeographic categories: paleotropical woody, neotropical woody, temperate woody, and herbaceous bamboos. The subtribe Hickeliinae is robustly placed in a trichotomy with
Racemobambosinae and Bambusinae that is sister to Melocanninae
in the paleotropical woody clade. Chusqueinae is sister to theremaining neotropical woody bamboos, and Buergersiochloinae issister to the remaining herbaceous subtribes. Resolution withintribe Arundinarieae is weak because of limited sequence variation
among sampled taxa.
Monophyly of the woody bamboos remains plausible because ofcharacter conflict in the plastid data and the possible incongruenceof plastid signal with a phylogeny estimation based on nuclear
DNA sequences. What is evident, however, is that a deep split exists in the woody bamboo plastid phylogeny between tropical species and temperate ones. By classifying the two woody lineages as
separate tribes (Arundinarieae and Bambuseae), the Bamboo Phylogeny Group can provide a stable system for bamboo taxonomy
that is resilient to the possibility of monophyly of woody bamboo
nuclear genomes (BPG, 2012).
A thorough reconstruction of organismal phylogeny for bamboos will require morphological and nuclear data to be evaluatedin addition to the chloroplast sequences presented here. A detailed
morphological analysis of bamboo species is currently being prepared by members of the BPG. Ongoing efforts in bamboo plastid
comparisons are focusing on the addition of many more taxa inmost subtribe lineages (BPG, in prep.) and on whole plastome sequences (Zhang et al., 2011) to generate additional resolution
within each of the four major bamboo clades. This is likely to reinforce and enhance our knowledge of subtribal plastid relationships
over the next few years. Sequencing of low-copy nuclear loci is also
underway that will offer critical evidence for an accurate reconstruction of bamboo evolutionary history. These data sets, whenavailable, should provide a robust framework for advanced testingof morphological and molecular character evolution, biogeographic

S.A. Kelchner, Bamboo Phylogeny Group / Molecular Phylogenetics and Evolution 67 (2013) 404–413

hypotheses, and discernment of evolutionary steps in the development of modern day Bambusoideae.
Acknowledgments
Chloroplast DNA sequences were produced in the laboratories
of Lynn Clark and Scot Kelchner with the assistance of Jimmy Triplett, Amanda Fisher, Chi-Sing Ho, Idaho State University’s Molecular Research Core Facility, and the DNA Facility of the Iowa State
University Office of Biotechnology. Funding for the Project wasprovided by National Science Foundation awards DEB-0515828
to Scot Kelchner and DEB-0515712 and DEB-9806877 to Lynn
Clark. Field work for temperate bamboo collections was supported
in part by National Geographic Society Grant 7336-02 to Lynn
Clark and De-Zhu Li.

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.
02.005.
References
Bamboo Phylogeny Group, 2006. The bamboo phylogeny project. Bamboo 27, 11–14.
Bamboo Phylogeny Group, 2012. An updated tribal and subtribal classification of
the bamboos (Poaceae: Bambusoideae). In: Proceedings of the 9th World
Bamboo Congress, 10–12 April 2012, Antwerp, Belgium, pp. 3–27.
Bouchenak-Khelladi, Y., Salamin, N., Savolainen, V., Forest, F., van der Bank, M.,
Chase, M.W., Hodkinson, T.R., 2008. Large multi-gene phylogenetic trees of thegrasses (Poaceae): progress towards complete tribal and generic level sampling.
Mol. Phylogenet. Evol. 47, 488–505.
Buckley, T.R., Simon, C., Shimodaira, H., Chambers, G.K., 2001. Evaluating
hypotheses on the origin and evolution of the New Zealand alpine cicadas(Maoricicada) using multiple-comparison tests of tree topology. Mol. Biol. Evol.
18, 223–234.
Chase, M.W., Hills, H.H., 1991. Silica gel: an ideal material for field preservation ofleaf samples for DNA studies. Taxon 40, 215–220.
Clark, L.G., Dransfield, S., Triplett, J., Sánchez-Ken, J.G., 2007. Phylogenetic
relationships among the one-flowered, determinate genera of Bambuseae(Poaceae: Bambusoideae). Aliso 23, 315–332.
Clark, L.G., Zhang, W., Wendel, J.F., 1995. A phylogeny of the grass family (Poaceae)
based on ndhF sequence data. Syst. Bot. 20, 436–460.
Davis, J.I., Soreng, R.J., 2010. Migration of endpoints of two genes relative toboundaries between regions of the plastid genome in the grass family(Poaceae). Am. J. Bot. 97, 874–892.
Fisher, A.E., Triplett, J.K., Ho, C.S., Schiller, A.D., Oltrogge, K.A., Schroder, E.S.,
Kelchner, S.A., Clark, L.G., 2009. Paraphyly in the bamboo subtribe Chusqueinae(Poaceae: Bambusoideae) and a revised infrageneric classification for Chusquea.
Syst. Bot. 34, 673–683.
Fitch, W.M., 1971. Toward defining the course of evolution: minimum change for a
specific tree topology. Syst. Zool. 20, 406–416.
Gaut, B.S., Clark, L.G., Wendel, J.F., Muse, S.V., 1997. Comparisons of the molecular
evolutionary process at rbcL and ndhF in the grass family (Poaceae). Mol. Biol.
Evol. 14, 769–777.
Goldman, N., Anderson, J.P., Rodrigo, A.G., 2000. Likelihood-based tests of topologies
in phylogenetics. Syst. Biol. 49, 652–670.
Grass Phylogeny Working Group [GPWG], 2001. Phylogeny and subfamilial
classification of the grasses (Poaceae). Ann. Missouri Bot. Gard. 88, 373–457.
Grass Phylogeny Working Group II [GPWG II], 2012. New grass phylogeny resolves
deep evolutionary relationships and discovers C4 origins. New Phytol. 193, 304–
312.
Guindon, S., Gascuel, O., 2003. A simple, fast, and accurate algorithm to estimate
large phylogenies by maximum likelihood. Syst. Biol. 52, 696–704.
Hodkinson, T.R., Chonghaile, G.N., Sungkaew, S., Chase, M.W., Salamin, N., Stapleton,
C.M.A., 2010. Phylogenetic analyses of plastid and nuclear DNA sequencesindicate a rapid late Miocene radiation of the temperate bamboo tribe
Arundinarieae (Poaceae, Bambusoideae). Plant Ecol. Divers. 3, 109–120.
Huelsenbeck, J.P., Rannala, B., 2004. Frequentist properties of Bayesian posterior
probabilities of phylogenetic trees under simple and complex substitution
models. Syst. Biol. 53, 904–913.
Huelsenbeck, J.P., Ronquist, F., 2001. MrBayes: Bayesian inference of phylogeny.
Biometrics 17, 754–755.
Huson, D.H., Bryant, D., 2006. Application of phylogenetic networks in evolutionary
studies. Mol. Biol. Evol. 23, 254–267.

413

Judziewicz, E.J., Clark, L.G., 2007. Classification and biogeography of New World
grasses: Anomochlooideae, Pharoideae, Ehrhartoideae, and Bambusoideae.
Aliso 23, 303–314.
Judziewicz, E.J., Clark, L.G., Londoño, X., Stern, M.J., 1999. American Bamboos.
Smithsonian Institution Press, Washington, DC.
Kelchner, S.A., 2000. The evolution of non-coding chloroplast DNA and its
application in plant systematics. Ann. Missouri Bot. Gard. 87, 482–498.
Kelchner, S.A., 2009. Phylogenetic models and model selection for noncoding DNA.
Plant Syst. Evol. 282, 109–126.
Kelchner, S.A., Clark, L.G., 1997. Molecular evolution and phylogenetic utility of therpl16 intron in Chusquea and the Bambusoideae (Poaceae). Mol. Phylogenet.
Evol. 8, 385–397.
Kelchner, S.A., Thomas, M.A., 2007. Model use in phylogenetics: nine key questions.
Trends Ecol. Evol. 22, 87–94.
Kelchner, S.A., Wendel, J.F., 1996. Hairpins create minute inversions in non-coding
regions of chloroplast DNA. Curr. Genet. 30, 259–262.
Kishino, H., Hasegawa, M., 1989. Evaluation of the maximum likelihood estimate of
the evolutionary tree topologies from DNA sequence data, and the branchingorder in Hominoidea. J. Mol. Evol. 29, 170–179.
Kobayashi, M., 1997. Phylogeny of world bamboos analysed by restriction fragment
length polymorphisms of chloroplast DNA. In: Chapman, G.P. (Ed.), The
Bamboos. Academic Press, London, England, pp. 227–234.
Löhne, C., Borsch, T., 2005. Molecular evolution and phylogenetic utility of the petDgroup II intron: a case study in basal angiosperms. Mol. Biol. Evol. 22, 317–332.
Mathews, S., Tsai, R.C., Kellogg, E.A., 2000. Phylogenetic structure in the grass family(Poaceae): evidence from the nuclear gene phytochrome B. Am. J. Bot. 87, 96–
107.
McClure, F.M., 1966. The Bamboos: A Fresh Perspective. Harvard University Press,
Cambridge, Massachusetts.
Morgan, M.J., Kelchner, S.A., 2010. Inference of molecular homology and sequencealignment by direct optimization. Mol. Phylogenet. Evol. 56, 305–311.
Morrison, D.A., 2005. Networks in phylogenetic analysis: new tools for population
biology. Int. J. Parasitol. 35, 567–582.
Morrison, D.A., 2006. Multiple sequence alignment for phylogenetic purposes. Aust.
Syst. Bot. 19, 479–539.
Morrison, D.A., 2009. A framework for phylogenetic sequence alignment. Plant Syst.
Evol. 282, 127–149.
Morrison, D.A., 2010. Using data-display networks for exploratory data analysis in
phylogenetic studies. Mol. Biol. Evol. 27, 1044–1057.
Peng, S., Yang, H.-Q., Li, D.-Z., 2008. Highly heterogeneous generic delimitation
within the temperate bamboo clade (Poaceae: Bambusoideae): evidence fromGBSSI and ITS sequences. Taxon 57, 799–810.
Penny, D., Hendy, M.D., Steel, M.A., 1992. Progress with methods for constructing
evolutionary trees. Trends Ecol. Evol. 7, 73–79.
Philippe, H., Sorhannus, U., Baroin, A., Perasso, R., Gasse, F., Adoutt, A., 1994.
Comparison of molecular and paleontological data in diatoms suggests a major
gap in the fossil record. J. Evol. Biol. 7, 247–265.
Posada, D., Buckley, T.R., 2004. Model selection and model averaging in
phylogenetics: advantages of Akaike information criterion and Bayesian
approaches over likelihood ratio tests. Syst. Biol. 53, 793–808.
Posada, D., Crandall, K.A., 1998. ModelTest: testing the model of DNA substitution.
Bioinformatics 14, 817–818.
Shimodaira, H., 2002. An approximately unbiased test of phylogenetic tree
selection. Syst. Biol. 51, 492–508.
Shimodaira, H., Hasegawa, M., 1999. Multiple comparisons of log-likelihoods withapplications to phylogenetic inference. Mol. Biol. Evol. 16, 1114–1116.
Simon, D.M., Kelchner, S.A., Zimmerly, S., 2009. A broadscale phylogenetic analysis
of group II intron RNAs and intron-encoded reverse transcriptases. Mol. Biol.
Evol. 26, 2795–2808.
Sungkaew, S., Stapleton, S.M.A., Salamin, N., Hodkinson, T.R., 2009. Non-monophylyof the woody bamboos (Bambuseae; Poaceae): a multi-gene region
phylogenetic analysis of Bambusoideae s.s. J. Plant Res. 122, 95–108.
Swofford, D.L., 2002. PAUP! Phylogenetic Analysis using Parsimony (!and othermethods). Sinauer Associates, Sunderland, Massachusetts.
Triplett, J.K., Clark, L.G., 2010. Phylogeny of the temperate woody bamboos(Poaceae: Bambusoideae) with an emphasis on Arundinaria and allies. Syst.
Bot. 35, 102–120.
Watanabe, M., Ito, M., Kurita, S., 1994. Chloroplast DNA phylogeny of Asianbamboos (Bambusoideae, Poaceae) and its systematic implication. J. Plant Res.
107, 253–261.
Wu, Z.-Q., Ge, S., 2012. The phylogeny of the BEP clade in grasses revisited: evidence
from the whole-genome sequences of chloroplasts. Mol. Phylogenet. Evol. 62,
573–578.
Yang, H.-Q., Peng, S., Li, D.-Z., 2007. Generic delimitations of Schizostachyum and its
allies (Gramineae: Bambusoideae) inferred from GBSSI and trnL-F sequencephylogenies. Taxon 56, 45–54.
Zhang, W.P., Clark, L.G., 2000. Phylogeny and classification of the Bambusoideae(Poaceae). In: Jacobs, S.W.L., Everett, J. (Eds.), Grasses: Systematics andEvolution. CSIRO, Melbourne, Australia, pp. 35–42.
Zhang, Y.-J., Ma, P.-F., Li, D.-Z., 2011. High-throughput sequencing of six bamboo
chloroplast genomes: phylogenetic implications for temperate woody bamboos(Poaceae: Bambusoideae). PLoS ONE 6, e20596.
Zheng, C.-Z., Zhang, Y.-X., Triplett, J.K., Yang, J.-B., Li, D.-Z., 2010. Large multi-locus
plastid phylogeny of the tribe Arundinarieae (Poaceae: Bambusoideae) reveals
ten major lineages and low rate of molecular divergence. Mol. Phylogenet. Evol.
56, 821–839.