Chloroplast variation is incongruent with classification of the Australian bloodwood eucalypts (genus Corymbia, family Myrtaceae)
Chloroplast variation is incongruent with classification of the Australian bloodwood eucalypts (genus Corymbia, family Myrtaceae)
Tanja M. Schuster 0 1
Sabrina D. Setaro 1
Josquin F. G. Tibbits 1
Erin L. Batty 0 1
Rachael M. Fowler 0 1
Todd G. B. McLay 0 1
Stephen Wilcox 1
Peter K. Ades 1
Michael J. Bayly 0 1
0 School of BioSciences, The University of Melbourne , Parkville, VIC , Australia , 2 National Herbarium of Victoria, Royal Botanic Gardens Victoria , Birdwood Avenue, South Yarra, VIC , Australia , 3 Department of Biology, Wake Forest University , Winston-Salem, NC , United States of America, 4 Department of Economic Development , Jobs, Transport and Resources , AgriBiosciences Centre, La Trobe University , Bundoora, VIC , Australia , 5 Genomics Hub , The Walter and Eliza Hall Institute of Medical Research , 1G Royal Parade, Parkville, Melbourne, VIC , Australia , 6 School of Ecosystem and Forest Sciences, The University of Melbourne , Parkville, Melbourne, VIC , Australia
1 Editor: Tzen-Yuh Chiang, National Cheng Kung University , TAIWAN
Previous molecular phylogenetic analyses have resolved the Australian bloodwood eucalypt genus Corymbia (~100 species) as either monophyletic or paraphyletic with respect to Angophora (9±10 species). Here we assess relationships of Corymbia and Angophora using a large dataset of chloroplast DNA sequences (121,016 base pairs; from 90 accessions representing 55 Corymbia and 8 Angophora species, plus 33 accessions of related genera), skimmed from high throughput sequencing of genomic DNA, and compare results with new analyses of nuclear ITS sequences (119 accessions) from previous studies. Maximum likelihood and maximum parsimony analyses of cpDNA resolve well supported trees with most nodes having >95% bootstrap support. These trees strongly reject monophyly of Corymbia, its two subgenera (Corymbia and Blakella), most taxonomic sections (Abbreviatae, Maculatae, Naviculares, Septentrionales), and several species. ITS trees weakly indicate paraphyly of Corymbia (bootstrap support <50% for maximum likelihood, and 71% for parsimony), but are highly incongruent with the cpDNA analyses, in that they support monophyly of both subgenera and some taxonomic sections of Corymbia. The striking incongruence between cpDNA trees and both morphological taxonomy and ITS trees is attributed largely to chloroplast introgression between taxa, because of geographic sharing of chloroplast clades across taxonomic groups. Such introgression has been widely inferred in studies of the related genus Eucalyptus. This is the first report of its likely prevalence in Corymbia and Angophora, but this is consistent with previous morphological inferences of hybridisation between species. Our findings (based on continent-wide sampling) highlight a need for more focussed studies to assess the extent of hybridisation and introgression in the evolutionary history of these genera, and that critical testing of the classification of Corymbia and Angophora requires additional sequence data from nuclear genomes.
Data Availability Statement: DNA sequences are
available in GenBank (https://www.ncbi.nlm.nih.
gov/genbank/), with accession numbers as listed in
Table 1. DNA alignments and other files are
provided as Supplementary Material.
Funding: This research was funded by The
Hermon Slade Foundation (grant HSF14-09 to TMS
and JFGT; http://www.hermonslade.org.au), The
Bjarne K. Dahl Trust (grants in 2014 and 2015 to
MJB and PKA; https://dahltrust.org.au), and The
University of Melbourne Botany Foundation (TMS
support funds; http://science.unimelb.edu.au/
funders had no role in study design, data collection
and analysis, decision to publish, or preparation of
The bloodwood eucalypts are sclerophyllous trees (c. 100 species) [
], currently classified in
the genus Corymbia K.D.Hill & L.A.S.Johnson, which was taxonomically segregated from the
genus Eucalyptus L'Her. in 1995 [
]. Bloodwoods include several morphologically distinct
groups that have been formally or informally classified at a range of taxonomic levels (e.g.
]), and are here identified (following Parra-Osorio et al. ) as the red bloodwoods
(subg. Corymbia), yellow bloodwoods (subg. Blakella sect. Naviculares), ghost gums or
paperfruited bloodwoods (subg. Blakella sect. Abbreviatae), spotted gums (subg. Blakella sect.
Maculatae), and cadaghi (monotypic subg. Blakella sect. Torellianae). These groups occur primarily
in northern or eastern Australia (Fig 1), where they are well-represented in monsoonal,
tropical savannahs, and there are two small groups of red bloodwoods restricted to the south-west
and south-east of Australia (sections Calophyllae and Corymbia, respectively) in areas with
Mediterranean, temperate climates. Bloodwoods are dominant trees in many of the areas in
which they occur (Fig 2) and are thus ecologically important ; they also have a history of
traditional and modern uses, and some species are widely grown for timber, for pulp, or as
ornamental trees [
]. Traditional uses include kino (hardened sap), which is blood-red and
confers the groups' common name, utilised for art and medicinal purposes [
], and C.
citriodora yields essential oils used as insect repellent and that have antimicrobial and
antifungal properties [
]. Corymbia is part of the "eucalypt group"  (tribe Eucalypteae) 
that also includes the sclerophyll genera Eucalyptus (>665 species) [
] and Angophora (9±10
], and the rainforest genera Arillastrum (1 species) , Stockwellia (1 species)
], Allosyncarpia (1 species) [
], and Eucalyptopsis (2 species) [
The taxonomic splitting of Corymbia from Eucalyptus was contentious (e.g. [
the key motivation for the separation of Corymbia being that the bloodwoods, on the basis of
both morphological analyses [
] and early molecular analyses [
], were more closely
related to Angophora than to Eucalyptus. That relationship has been unequivocally supported
by all subsequent molecular phylogenetic analyses of the group (e.g. [
is supported by some morphological characters, including patterns of leaf venation, features of
trichomes, and the presence of oil ducts in the pith of branches [
]. There are,
nonetheless, clear differences in some macro-morphological features between the two groups that have
led to the longstanding treatment of Angophora as a separate genus from the bloodwoods
(whether placed in Eucalyptus or treated as Corymbia) by almost all authors (e.g., [
]) since Angophora was first described in 1797 . The most notable differences between
the groups are in the flowers, which in Angophora have free sepals and petals, in contrast to the
calyptrate/operculate perianth of Corymbia (Fig 2). Despite such morphological differences,
molecular phylogenetic analyses have presented conflicting signals regarding monophyly of
Corymbia , with some resolving the genus as monophyletic (e.g. [
]), while others
resolve it as paraphyletic, with Angophora nested within it [
Most phylogenetic analyses assessing the relationships of bloodwoods to other eucalypts
have employed few DNA markers generated by conventional Sanger sequencing methods (e.g.
]). The use of High-Throughput Sequencing (HTS) methods, which can
generate larger volumes of sequence data, are only just beginning to be used in eucalypt studies
(e.g. ). Partly as a result of the small size of most molecular datasets, some key relationships
have typically been poorly supported, including that of the bloodwoods to Angophora. For
example, Maximum Parsimony (MP) bootstrap support values indicating paraphyly of Corymbia
have generally been in the range of 51±93% for clades showing Angophora nested in Corymbia
], and those for a monophyletic Corymbia have ranged from 78±100% [
An exception is a recent study that used analyses of whole chloroplast (cp) genomes [
2 / 28
Fig 1. Distribution of infrageneric groups in Corymbia (adapted from [
] and following the classification of [
Colour coding of groups matches that used in other figures, i.e., taxonomic sections of Corymbia sensu Parra-Osorio
et al. [
which showed strong support for Corymbia as paraphyletic with respect to Angophora
(parsimony bootstrap support and Bayesian posterior probability both 100%), with subg. Blakella
being more closely related to Angophora than to subg. Corymbia. However, that study, despite
3 / 28
Fig 2. Photographs of Corymbia and Angophora. (A) Woodland dominated by Corymbia cliftoniana (a red
bloodwood, sect. Septentrionales), near Victoria River, Northern Territory; (B) woodland dominated by Corymbia
grandifolia (a ghost gum, sect. Abbreviatae), near Daly Waters, Northern Territory; (C) partially opened flower bud of
Corymbia ficifolia (sect. Calophyllae), showing operculate perianth (O) separating from hypanthium; (D) open flower
of Angophora floribunda showing perianth composed of five, free sepals (S) and five, free petals (P).
using a large amount of sequence data, included only one sample each of the red bloodwoods,
yellow bloodwoods, ghost gums and spotted gums, and it was unclear whether the result was
an artefact of sparse taxon sampling.
In this study, we assess relationships of bloodwood eucalypts, including those among
species, series, sections and subgenera of Corymbia, and those of Corymbia to Angophora. We
expand on the sampling of Bayly et al. [
] to include the largest sample of species of
Angophora and Corymbia in any molecular study using HTS data to date (Table 1). We specifically
address questions relating to the evolutionary history of this large and ecologically and
economically important group and test the current taxonomic classification, especially at the
ranks of genus and subgenus. We use both chloroplast genome derived sequences and
combine sequences of nuclear ribosomal internal transcribed spacer (ITS) regions from previous
studies for separate phylogenetic analyses using Maximum Likelihood (ML) and MP methods,
to provide assessment of phylogenetic signal from both nuclear and chloroplast markers.
4 / 28
MJB2501, MELU 114300
MJB2503, MELU 114302
NT, Stuart Highway, turnoff to
NT, 16.7 km N Renner Springs,
DN2542, AD 163675
QLD, Cherwell Range
MJB2237, MELU 108352
VIC, PFP Arboretum [
DN2752, AD 163641
C. flavescens K.D.Hill & L.A.S.Johnson
C. foelscheana (F.Muell.) K.D.Hill & L.A.S. Corymbia
C. grandifolia (R.Br. ex Benth.) K.D.Hill & Blakella
C. gummifera (Gaertn.) K.D.Hill & L.A.S.
C. haematoxylon (Maiden) K.D.Hill & L.
C. hamersleyana (D.J.Carr & S.G.M.Carr)
K.D.Hill & L.A.S.Johnson
C. henryi (S.T.Blake) K.D.Hill & L.A.S.
C. intermedia (R.T.Baker) K.D.Hill & L.A. Corymbia
C. jacobsiana (Blakely) K.D.Hill & L.A.S.
C. leichhardtii (F.M.Bailey) K.D.Hill & L.
C. lenziana (D.J.Carr & S.G.M.Carr) K.D.
Hill & L.A.S.Johnson
C. ligans K.D.Hill & L.A.S.Johnson
C. maculata (Hook.) K.D.Hill & L.A.S.
C. opaca (D.J.Carr & S.G.M.Carr) K.D.
Hill & L.A.S.Johnson
C. peltata (Benth.) K.D.Hill & L.A.S.
C. petalophylla (Brooker & A.R.Bean) K.
D.Hill & L.A.S.Johnson
C. plena K.D.Hill & L.A.S.Johnson
C. polysciada (F.Muell.) K.D.Hill & L.A.S.
C. pachycarpa K.D.Hill & L.A.S.Johnson
7 / 28
Classification of subgenera and sections follows [
]; classification of series follows the informal classification of [
], where consistent with the higher-level groupings.
CAPO = Carlos A. Parra-Osorio, DN = Dean Nicolle, GB = Gillian Brown, KLW = Karen L. Wilson, MJB = Michael J. Bayly, NG = Neil Gibson, TMS = Tanja M.
Schuster. Herbarium codes follow Index Herbariorum: AD = State Herbarium of South Australia, HO = Tasmanian Herbarium, MEL = National Herbarium of
Victoria, MELU = University of Melbourne Herbarium, NSW = National Herbarium of New South Wales, PERTH = Western Australian Herbarium. Provenance data:
BG = botanic garden, NSW = New South Wales (Australia), NT = Northern Territory (Australia), QLD = Queensland (Australia), TAS = Tasmania (Australia),
VIC = Victoria (Australia), WA = Western Australia (Australia). Further abbreviations are PFP = Peter Francis Points Arboretum, Coleraine, Victoria; RBGV = Royal
Botanic Garden Victoria, and UoM = The University of Melbourne (, followed by the respective campus).
i Collected as A. euryphylla (G.J.Leach) L.A.S.Johnson & K.D.Hill, a name that is synonymised with A. costata subsp. euryphylla in the current Australian Plant Census
ii Collected as Corymbia variegata (F.Muell.) K.D.Hill & L.A.S.Johnson, a name that is synonymised with C. citriodora in the current APC.
iii Collected as Corymbia capricornia (D.J.Carr & S.G.M.Carr) K.D.Hill & L.A.S.Johnson, a name that is synonymised with C. dichromophloia in the current APC.
iv Collected as Corymbia rubens K.D.Hill & L.A.S.Johnson, a name that is synonymised with C. dichromophloia in the current APC.
v Collected as Corymbia semiclara K.D.Hill & L.A.S.Johnson, a name that is synonymised with C. hamersleyana in the current APC.
vi Collected as Corymbia trachyphloia subsp. amphistomatica K.D.Hill & L.A.S.Johnson, a name that is synonymised with C. trachyphloia in the current APC.
Specifically these methods are used to test the hypotheses that 1) Corymbia is monophyletic
and 2) the currently recognised subgenera [
] are monophyletic.
Materials and methods
Taxon sampling for chloroplast DNA study
Samples and sequences of chloroplast DNA used in this study are listed in Table 1. Names of
species and infraspecific taxa generally follow the Australian Plant Census [
], and authorities
are only given in the text for species names not listed in Table 1. Taxonomic works used to
9 / 28
identify samples were [
]. For Corymbia, our sampling included 55 of the 97 species
accepted by the Council of Heads of Australasian Herbaria (CHAH) [
], including all of the
subgenera and sections recognised in the classification of [
], and with 19 species represented
by at least two accessions. Sampling for Angophora included eight of the ten species recognised
by CHAH [
], four of which were represented by at least two accessions. We also included
sequences for 31 species of Eucalyptus and outgroups Allosyncarpia and Stockwellia from [
In total, the analysis included 123 accessions, of which 84 were newly sequenced for this study.
DNA isolation from silica dried leaves
For the cpDNA study, total genomic DNA (gDNA) was extracted from ca. 80 mg of recently
collected leaf tissue (no older than one year) using a modified CTAB DNA extraction protocol
]. Older silica-dried collections were difficult to extract suitable DNA from, probably
due to chemical DNA degradation in this plant group rich in secondary metabolites. The
CTAB lysing buffer (2% w/v cetyltrimethylammonium bromide (CTAB), 2% w/v
polyvinylpyrrolidone 40,000 (PVP±40), 1.4 M NaCl, 20 mM EDTA, 100 mM Tris±HCl pH 8.0) was
modified by addition of 0.6% v/v each of 2±mercaptoethanol, RNase A, and proteinase K per
sample. Further modifications to the CTAB extraction protocol included a sucrose/Tris/EDTA
(STE) wash (8% w/v sucrose, 1 M Tris±HCl pH 7.0, 0.5 M EDTA) before lysis using 1 mL of
STE per 80 mg of ground plant tissue . The STE solution was discarded after centrifugation
at 5,000 rpm for 10 min, and the pellet suspended in 700 μL of preheated (65ÊC) CTAB lysate
buffer. After adding 110 μL bovine serum albumin (BSA)/NaCl (1:10, 4% BSA:5 M NaCl) to
each sample, they were left to incubate for ca. 16 hrs at 60ÊC. Two 2/3 volume chloroform
extractions were done, centrifuging for 10 min at 14,800 rpm for the first and then 8 min at the
same speed for the second. DNA was precipitated with 2/3 volume of 100% isopropanol (room
temperature). After 30±60 min incubation at room temperature, the DNA was centrifuged
into a pellet at 14,800 rpm for 15 min and washed twice with 70% ethanol after discarding the
isopropanol. DNA was resuspended in 100 μL TE pH 8.0 (10 mM Tris±HCl:1 mM EDTA pH
8.0) after leaving the pellet to dry overnight to allow all of the ethanol to evaporate. DNA
quantity and quality were checked with Nanodrop 2000 (NanoDrop Products) and Qubit 2.0
fluorometer (Invitrogen) instruments and visualised by electrophoresis (1.5% agarose gel) with
DNA library construction and sequencing
This section details a relatively cost-effective library preparation protocol at ca. AUD 35 per
sample using no proprietary kits. All reagents are from New England BioLabs (NEB) if not
stated otherwise. Immediately before sonication, a DNA aliquot was washed with ethanol/
sodium acetate (5.5:1, 100% ethanol:2.4M NaAc) at a 1:4.7 DNA:wash solution volume, and
then centrifuged at 14,800 rpm for 10 min. After discarding the wash solution, the resulting
pellet was washed with 70% ethanol and resuspended in 100 μL 1 M Tris±HCl pH 8.0. DNA
was quantified with a Qubit 2.0 (Invitrogen), and an aliquot of 3 μg of gDNA per sample was
brought to 115 μL with ultrapure H2O. The DNA was sonicated for 50 sec with a S220
Focused-ultrasonicator (Covaris) set to 6±8ÊC, 120W peak incident power, 200 cycles per
burst, and on duty cycle 5%, aiming for 800 bp mean fragment size.
The sonicated samples (100 μL each) were cleaned using Serapure SPRI beads [
] at a
0.6:1.0 beads:sample ratio to remove short fragments (<300 bp) by incubating this mixture for
20 min at room temperature, immobilising beads on a 96S super magnet plate (Alpaqua) for
15 min, discarding the supernatant and washing with 170 μL 80% ethanol, and then leaving
the magnet-trapped beads to air dry for 2 min.
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NEBNext End Repair Module produced blunt ends on the fragmented DNA by eluting the
DNA from the magnet-trapped beads and incubating with 2.0 μL 10 × reaction buffer, 0.4 μL
enzyme mix, and 17.6 μL ultrapure H2O per sample at 20ÊC for 60 min. Samples were again
purified using the Serapure SPRI beads by adding 50 μL PEG:NaCl (20% PEG w/v:5 M NaCl)
and 50 μL 100% isopropanol to each sample, incubating this for 15 min at room temperature,
and washed using 80% ethanol as in the above steps. Then dA tails were attached to the
fragments using 0.5 μL 10 mM deoxyadenosine 5'-triphosphate, 0.4 μL Klenow Fragment, 2.0 μL
NEB 10 × Buffer 2, and 17.1 μL ultrapure H2O per sample to elute the DNA and incubated at
37ÊC for 60 min followed by 65ÊC for 20 min. Per sample, 2 μL of 25 μM multiplex hairpin
adaptors (top plus bottom strands) in 10 mM Tris±HCl pH 8.0 [
] were ligated to the DNA
fragments with 0.25 μL10 mM ATP, 0.4 μL T4 DNA ligase, 0.8 μL T4 DNA ligase buffer, and
6.55 μL ultrapure H2O at 12ÊC overnight and then 10 min of 65ÊC to stop the reaction.
Exonuclease digestion degraded all non-competent molecules with 0.5 μL 10 × NEB Buffer I, 0.25 μL
Lambda Exonuclease, 0.25 μL Exonuclease I, and 4.0 μL ultrapure H2O per sample at 37ÊC for
2 hrs, and 80ÊC for 20 min. After the digestion, samples were cleaned on the magnetic plate as
before, but using 80 μL 20% PEG:5M NaCl and 80 μL 100% isoproplanol per sample. Samples
were eluted with 60 μL 10 mM Tris±HCl pH 8.0 and stored at 4ÊC until q-PCR titration, after
which the libraries were kept at -20ÊC for long term storage.
For titration, a 20 μL q-PCR reaction, using 5 μL of each 800 bp library as template,
0.5 μL 10× SYBR Green (Thermo Fisher Scientific), 10.0 μL Kapa HiFi HotStart ReadyMix
(KAPA Biosystems), 2.5 μL ultrapure H2O, and 1.0 μL each of 5 μM TRUESEQ (Illumina)
compatible PE primers [
] was run to 20 cycles on a BioRad CFX q-PCR machine. Settings
for the q-PCR were 30 sec of 98ÊC for denaturation and 20 cycles of 98ÊC for 10 sec, 67ÊC for
30 sec, and 72ÊC for 30 sec. Once the sample appropriate cycle number was determined from
this q-PCR, a 40 μL reaction and including sample-specific PE primers including indexing
barcodes to allow pooling of multiple samples per sequencing was run using the same settings as
Samples were pooled and quality checked either with an Agilent Bioanalyser DNA1000
chip system (Agilent) for HiSeq 1500 (Illumina) sequencing or a 2200 Tape Station using the
D1000 kit (Agilent) and Qubit 3.0 (Invitrogen) for sequencing on a NextSeq 500 machine
(Illumina). A 250 cycle (2 × 125 paired end reads) kit (Illumina) was used for the former or a 300
cycle (2 × 150 paired end reads) kit (Illumina) for the latter sequencer.
Sequence trimming, quality control, read mapping and chloroplast sequence assembly
Base calling and quality filtering was done with Illumina pipeline software (v.1.7 or later) and
samples were pre-processed with custom scripts at the Walter and Eliza Hall Institute of
Medical Research (WEHI) sequencing facility.
The new sequences were assessed, trimmed and assembled with CLC Genomics
Workbench v. 9.5.1 and 9.5.2 (Qiagen) and the CLC Workflow is available as supplementary
material (S1 File). Paired-end reads were paired and reads shorter than 15 or longer than 1000 bp
were discarded. Reads below PHRED score 20 were also discarded. The fraction of low quality
bases that were allowed in a read was 5%.
The quality-filtered and paired reads were mapped against a reference chloroplast genome
(Eucalyptus globulus, GenBank accession: NC_008115.1). Sequence coverage was generally
sufficient to unambiguously assemble most of the chloroplast genome for each sample, but all
genomes included some regions with low coverage of mapped reads. A consensus sequence of
the mapped assembly was created by removing regions with low coverage and inserting
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ambiguity codes for bases with more than one possible nucleotide using a threshold of 50%
and `maximum number of ambiguous nucleotides allowed after trimming = 2'.
Sequence alignment and phylogenetic analyses of chloroplast DNA
All newly generated chloroplast sequences included in the study were aligned with MAFFT
] and the fast and progressive method FFT-NS-2, suitable for large alignments.
One inverted repeat region (IRa) was excluded from the alignment. The alignment was viewed
with SeaView v.4.6 [
] or Mesquite v.3.10 [
] and subsequently processed with GBLOCKS
] using default parameters, which stringently trims alignments allowing no gaps.
Hence, the final dataset only included regions with sequence coverage for all samples, and all
indels were removed.
We used jModelTest v.2.1.10 [
] and the AIC and BIC criteria, to estimate the model of
nucleotide substitution that best fits the chloroplast data. The maximum likelihood analysis
was done with Standard RAxML v.8.2.8  with 1000 rapid bootstrap inferences and a
thorough ML search under the GAMMA model of rate heterogeneity. The maximum parsimony
analysis was done with PAUP v.4.0a151 [
] with the following settings: all characters were
treated as unordered and of equal weight. Heuristic searches employed
tree-bisection-reconnection branch swapping and 1000 replicates of random stepwise additions. The number of
bootstrap replicates was 1000 with one tree held at each step. Trees were viewed and exported
for rendering in FigTree v.1.4.3 [
Analysis of nuclear ribosomal DNA
For comparison with the cpDNA phylogeny, we combined sequences of the ITS regions of
nuclear ribosomal DNA (nrDNA) from previous studies [
] for phylogenetic
analyses. Separate analyses of these nrDNA sequences have not been presented in previous
studies, with most including only a small number of Corymbia samples or, in the case of the
largest study to date [
], also combining a subset of these sequences with cpDNA markers
in analyses of a concatenated dataset. Our dataset included 66 accessions of Corymbia
(representing all taxonomic sections), 15 of Angophora (9 of 10 species), 31 of Eucalyptus (the
same species as in the cpDNA dataset, representing major lineages), two of Stockwellia, one
of Eucalyptopsis, three of Allosyncarpia, and one of Arillastrum (used as outgroup). Partial
sequences, or those identified as spacers associated with pseudogenes using established criteria
], were excluded from analyses. We used existing nrDNA sequences for analyses,
rather than assembling novel sequences from our current genomic data for Corymbia, because
of the presence of substantial within-genome variation in our samples (in line with previous
]), and associated difficulties in separating and assembling sequences of the
various paralogues/alleles, which is a challenging task worthy of separate investigation and
discussion. The nrITS sequences from GenBank were aligned using Geneious v.9.1.7 . Model
testing, ML and MP analyses were conducted as outlined above with the addition that gaps
present in the nrDNA alignment were treated as missing data in the MP analysis.
Analysis of chloroplast DNA
GBLOCKS eliminated 37% of the MAFFT alignment, resulting in 121,016 characters and
10,847 distinct alignment patterns in the final alignment (see supplementary material S2 File).
Both, AIC and BIC from jModelTest indicated the General Time Reversible model using
gamma and invariant sites (GTR+I+G) as the best fit. Final ML Optimization Likelihood was
12 / 28
-254343.104231. The maximum parsimony analysis had 3771 parsimony informative
characters, 4116 variable but uninformative characters, and resulted in 12 trees with length = 10413,
consistency index = 0.81, retention index = 0.96. Topologies of the ML and MP trees were
similar (Fig 3), and most nodes had 85±100% bootstrap support (BS) for both ML and MP
analyses, with the backbone, in particular, well supported.
Rooting the trees with Allosyncarpia and Stockwellia recovered relationships of a
monophyletic Eucalyptus as sister to Angophora + Corymbia. Eucalyptus is composed of three clades
corresponding to `Eudesmids' (subg. Eudesmia, represented by E. erythrocorys) subtending a
wellsupported clade of `Monocalypts' sensu [
] (including representatives of subg. Acerosae [E.
curtisii], subg. Idiogenes [E. cloeziana], and subg. Eucalyptus [remainder of that clade]), which
were sister to a `Symphyomyrt' clade (including members of subg. Alveolata [E. microcorys],
subg. Cruciformes [E. guilfoylei], and subg. Minutifrucuts [E. deglupta] nested in subg.
Symphyomyrtus [remainder of that clade]) that was moderately supported with BS ML/MP 76/
Corymbia is paraphyletic because of the inclusion of Angophora in one Corymbia clade
(clade A; Fig 3). Furthermore, subgenera Blakella and Corymbia, most non-monotypic sections
(Abbreviatae, Maculatae, Naviculares, Septentrionales), and several species including more
than one accession here are not monophyletic. In addition to Angophora, clade A includes a
basal grade of a few species of red bloodwoods from southern Australia that do not group with
all other red bloodwoods in clade B. The red bloodwoods in clade A include C. gummifera
(monotypic sect. Corymbia) from south-eastern Australia and C. calophylla, C. ficifolia, and C.
haematoxylon that correspond to sect. Calophyllae from south-western Western Australia (Fig
1). Clade A also includes the yellow bloodwoods (sect. Naviculares), spotted gums (sect.
Maculatae), cadagi or C. torelliana (monotypic sect. Torellianae), and two ghost gums (sect.
Abbreviatae). Although the latter all form a clade, spotted gum and yellow bloodwood species are
interdigitated and taxonomic sections based on morphology do not form groups here. The
ghost gums sensu Parra-Osorio et al. [
] are polyphyletic, because clade B also includes two
separate clades of sect. Abbreviatae. In addition, clade B contains most of the red bloodwood
species (sect. Septentrionales), in which the ghost gums are embedded.
Of 23 species of Angophora and Corymbia represented in the dataset by two or more
samples, only four species (A. melanoxylon, C. eximia, C. grandifolia, and C. gummifera) are
resolved as monophyletic, whereas most are indicated as paraphyletic or polyphyletic, and two
species (C. trachyphloia and C. aparrerinja) have accessions split between the two major
ingroup clades (clades A and B). This widespread incongruence between morphological and
chloroplast data likely points to a complex evolutionary history in this group. In conclusion,
both hypotheses to be tested, 1) that Corymbia is monophyletic and 2) that the currently
recognised subgenera of Corymbia are monophyletic, are not supported based on the chloroplast
Some phylogenetic signal in the cpDNA data is geographic, and Fig 4 (for clade A) and Fig
5 (for clade B) illustrate the proximity of accession localities that form subclades within the
major two ingroup clades. For example, Fig 4B shows the geographic proximity of accessions
included in clade A1 (Fig 3), which is composed of members of sections Maculatae and
Naviculares, and Fig 5 shows geographic groups within clade B that each include a mix of species
from different taxonomic series (see Table 1) within sections Septentrionales and Abbreviatae.
Analysis of nuclear ribosomal DNA
The nrDNA dataset included 663 aligned bases and 337 distinct alignment patterns (see
supplementary material S3 File). AIC from jModelTest indicated a General Time Reversible
13 / 28
Fig 3. Phylogeny resulting from cpDNA analyses. Best-scoring maximum likelihood tree from a RAxML analysis (final ML
optimization likelihood of -254343.104231) of a cpDNA dataset (121,016 base pairs, 10,847 distinct alignment patterns, 123
accessions) of eucalypts. Names of Corymbia species are colour-coded by taxonomic section as indicated. Labelling to the right of
the tree indicates the outgroup (`OG'), major groups of Eucalyptus, including subg. Eudesmia (`Eudesmids'), the symphyomyrt clade
(`Symph'; including subgenera Alveolata, Cruciformes Minutifructus and Syphyomyrtus), and the monocalypt clade (`Mono';
including subgenera Acerosae, Eucalyptus and Idiogenes), as well as subclades of Corymbia clustering by geographic proximity (A1±
A3 and B1±B6) referred to in the text and Fig 4 (for clade A) and Fig 5 (for clade B). Species names of species represented by
multiple accessions are followed by collection number for newly generated sequences or GenBank accession number (NC) for data
generated for previous studies. Bootstrap support values are shown as percent maximum likelihood/maximum parsimony (MP
mapped onto ML tree) with weighted edges indicating 100% support for both ML and MP. Support values <50% are omitted or
dashed when the alternate analysis method had 50% support.
model using gamma distribution of rates and a proportion of invariant sites (GTR+I+G), used
for analysis here, and BIC indicated GTR+G as the best fit. Final ML optimization likelihood
was -4709.258464. The MP analysis included 170 parsimony informative characters and the
alignment had 90 variable but uninformative characters. Maximum parsimony analysis
resulted in 1610 trees with length = 644, consistency index = 0.55, and retention index = 0.90.
Topologies of the ML and MP trees were similar, and the ML tree is shown here, with MP
bootstrap support values mapped onto it (Fig 6).
Analyses strongly supported the monophyly of Eucalyptus and of Corymbia + Angophora
(both with ML/MP BS of 100%). Relationships within Eucalyptus were similar to those in the
cpDNA tree, in that they resolved the main `Monocalypt' and `Symphyomyrt' clades,
subtended by subg. Eudesmia (E. erythrocorys), although the position of monotypic subg. Acerosae
(E. curtisii) was not resolved with support.
Corymbia was resolved as paraphyletic with respect to Angophora, and that relationship
received weak to moderate support (BS of<50% for ML and 71% for MP). Within Corymbia,
there was support for the monophyly of subg. Corymbia (ML/MP BS of 74/75%) and subg.
Blakella (BS 99/98%). Monophyly was also supported for Corymbia sections Maculatae (BS 75/
83%) and Naviculares (BS 90/86%). Relationships of other Corymbia sections represented by
more than one species were generally poorly supported, i.e.: sect. Septentrionales was resolved
as paraphyletic, but with < 50% ML or MP BS; sect. Calophyllae was resolved as paraphyletic,
on account of placement of C. gummifera (monotypic sect. Corymbia) with two samples of C.
ficifolia with weak support (BS 60/58%); sect. Abbreviatae was resolved as paraphyletic with
respect to monotypic sect. Torellianae, but only in the ML tree and with <50% bootstrap
support. In conclusion, support was mixed for the hypotheses being tested here, 1) that Corymbia
is monophyletic and 2) that the currently recognised subgenera of Corymbia are monophyletic.
For hypothesis 1), the data are largely equivocal, there being only weak support for the nesting
of Angophora in Corymbia (<50% BS for ML and 71% for MP); for hypothesis 2), the current
subgeneric classification of Corymbia was moderately to strongly supported.
The relationships within Eucalyptus generally confirm those of previous HTS studies [
therefore, our results for relationships among Eucalyptus will not be discussed further as our
focus here is on Corymbia and Angophora.
Why are cpDNA relationships incongruent with nrDNA relationships and
infrageneric classification of Corymbia?
A key result of the current study is that cpDNA relationships in Corymbia (Fig 3) are largely
incongruent with the current circumscriptions of subgenera and sections, and with
relationships inferred based on nrDNA (Fig 6). Such incongruence could occur if: A) current
15 / 28
Fig 4. Distribution of samples placed in clade A in the cpDNA phylogeny (Fig 3). Colour coding of groups matches that used
in other figures. Accession details are shown for four samples mentioned in the text.
infrageneric groups are poorly defined and in need of taxonomic revision; B) the nrDNA gene
tree does not accurately reflect phylogenetic relationships, e.g., as a result of mixing of
orthologous and paralogous copies of this multi-copy cistron [
]; C) the cpDNA gene tree does
not accurately reflect the phylogenetic relationships of taxa, e.g., as a result of processes such as
incomplete lineage sorting [
] or chloroplast capture resulting from hybridisation and
]. We infer that the observed incongruence is consistent with the last
explanation and, in particular, points to historical hybridisation and cpDNA introgression
between lineages, as outlined below.
In conflict with the cpDNA gene tree, evidence for the monophyly of major infrageneric
groups in Corymbia (subgenera and sections) comes from a general concordance between
phylogenies based on nrDNA sequences (e.g., [
]) and morphologically defined infrageneric
groups. For instance, the analysis of nrDNA presented here (Fig 6), based on ITS sequences,
supports monophyly of the two subgenera (subg. Corymbia and subg. Blakella), the yellow
bloodwoods (sect. Naviculares) and the spotted gums (sect. Maculatae), and it does not
strongly contradict the monophyly of the ghost gums (sect. Abbreviatae). Most of these groups
have historically been recognised on morphological grounds, although at varying taxonomic
levels (e.g., [
]). Such concordance, from independent data sources, provides support for
the notion that, on the whole, these taxa represent phylogenetic groups. Thus, it is striking
that, among the molecular phylogenetic studies of Corymbia, it is only those including
chloroplast data that show strongly supported nodes in conflict with the recognition of these groups
(current study and [
]). Understanding the reasons for this conflict is central to gaining
insight into the evolutionary history of Corymbia, and to properly testing its classification.
Chloroplast capture and incomplete lineage sorting are two processes commonly inferred
to account for incongruence in plants between chloroplast DNA relationships and nuclear
DNA phylogenies/morphological taxonomy. The relative importance of these processes can be
difficult to infer or disentangle [
], but some clues can come from knowledge of the
reproductive biology of the plants and of geographic patterns of DNA sequence variation. In terms
of reproductive biology, a capacity to hybridise and interbreed is a necessary pre-requisite for
the transfer of chloroplasts between lineages. In terms of geography, introgression necessarily
occurs at particular locations, and can lead to geographic clustering in the sharing of related
chloroplast sequences between species [
]. In contrast, such geographic clustering might not
be expected in cases where incongruence with taxonomy results from incomplete lineage
sorting of chloroplast genomes (e.g. [
Although incomplete lineage sorting cannot be excluded as an explanation for aspects of
cpDNA relationships in Corymbia, it seems likely from the reproductive biology of these trees
(and that of other eucalypt genera), together with geographic patterns of cpDNA variation,
that the observed patterns are largely consistent with a history of hybridisation and
introgression. In terms of reproductive compatibility, pre-zygotic barriers to reproduction have been
reported between some Corymbia species [
], but all members of Corymbia investigated so
far have the same chromosome number (2n = 22; [
]). Both morphological variation and
experimental crosses [
] provide evidence of substantial potential for hybridisation
between species classified in different series, sections, and subgenera (summarised in Fig 7).
Given this capacity for hybridisation across infrageneric groups, it seems likely that the
taxonomic incongruence of cpDNA relationships in Corymbia could reflect similar processes to
those seen in the better studied Eucalyptus. In Eucalyptus, such incongruence is clearly evident,
17 / 28
Fig 5. Distribution of samples placed in clade B in the cpDNA phylogeny (Fig 3). (A) Samples classified in sect. Septentrionales; (B)
samples classified in sect. Abbreviatae, with inset in lower right showing detail for area outlined by dashed rectangle. Details are shown
for some clades, species, and accessions mentioned in the text. Colour coding of groups matches that used in other figures.
PLOS ONE | https://doi.org/10.1371/journal.pone.0195034
18 / 28
Fig 6. Phylogeny resulting from nrDNA analyses. Best-scoring maximum likelihood tree from a RAxML analysis (final ML
optimization likelihood of -4709.258464) of a nrITS dataset (663 base pairs, 337 distinct alignment patterns, 119 accessions) of
eucalypts. The outgroup (OG), major groups in Eucalyptus (`Eudesmids', `Monocalypts' [Mono], and `Symphyomyrts' [Symph]),
subgenera and infrageneric groups of Corymbia are indicated with bars or colouring scheme (see textbox) on the tree. Species
names are followed by GenBank accession numbers. Bootstrap support values are shown as percent maximum likelihood/
maximum parsimony (MP mapped onto ML tree) with weighted edges indicating 100% support for both ML and MP. Support
values <50% are omitted or dashed when the alternate analysis method had 50% support. i Identified in GenBank (GB) as
Angophora exul K.D.Hill, a name that is synonymised with A. bakeri in the current Australian Plant Census (APC) [
]. ii Identified
in GB as Angophora euryphylla (G.J.Leach) L.A.S.Johnson & K.D.Hill, a name that is synonymised with A. costata subsp. euryphylla
in the current APC. iii Identified in GB as Corymbia variegata (F.Muell.) K.D.Hill & L.A.S.Johnson, a name that is synonymised
with C. citriodora in the current APC. iv Identified in GB as Corymbia dolichocarpa (D.J.Carr & S.G.M.Carr) K.D.Hill & L.A.S.
Johnson, a name that is synonymised with C. clarksoniana in the current APC. v Identified in GB as Corymbia dampieri (D.J.Carr
& S.G.M.Carr) K.D.Hill & L.A.S.Johnson, a name that is synonymised with C. greeniana in the current APC. vi Identified in GB as
Corymbia dimorpha (Brooker & A.R.Bean) K.D.Hill & L.A.S.Johnson, a name that is synonymised with C. peltata in the current
APC. vii Identified in GB as Corymbia catenaria K.D.Hill & L.A.S.Johnson, a name that is synonymised with C. watsoniana subsp.
capillata in the current APC.
with chloroplast variation commonly reflecting geography, rather than taxonomy, and
widespread regional introgression of cpDNA between species, series, and sections is regularly
Fig 7. Summary of hybridisation between infrageneric groups of Corymbia inferred by previous studies [
Arrows connect taxa with inferred hybrids. (A) Inferred intersectional hybrids; (B) interseries hybrids in subg.
Corymbia; (C) interseries hybrids in subg. Blakella.
20 / 28
Geographic patterns in Corymbia observed here (Figs 4 and 5) provide support for a history
of cpDNA introgression between species, in the form of geographically clustered cpDNA
clades shared among taxa that would be considered distinct lineages based on morphological
or nrDNA evidence. Such patterns are consistent with cpDNA introgression between species
across a range of morphological infrageneric groups.
Some of the clearest geographic patterns in our dataset relate to geographic cpDNA clades
shared among species classified, on the basis of morphology [
], in different taxonomic series
(Table 1) within the same section. For example, within sect. Septentrionales three geographic
clades in north Queensland each contain a mixture of species placed in different taxonomic
series, i.e., clade B1 (containing members of ser. Arenariae, Polycarpae, and Dichromophloiae;
Fig 3), clade B2 (containing members of ser. Rhodopes and Abergianae), and clade B3
(containing members of ser. Trachyphloiae and Polycarpae). Likewise, clade B5 (including members of
ser. Dichromophloiae and ser. Ferrugineae) is geographically clustered in the Mid-West region
of Western Australia. Apart from the monotypic ser. Abergianae, series with species falling in
clades B1, B2, B3, and B5 are also distributed across other clades in the phylogeny and hence
not monophyletic (e.g., C. dichromophloia, C. hamersleyana, and C. trachyphloia). Similar
patterns are also seen in clades of sect. Abbreviatae, where clade B4 includes intermixed
representatives of series Polysciadae and Papuanae, and clade B6 includes intermixed members of ser.
Grandifoliae and Asperae. Interestingly, in clade B4, the morphologically distinctive species C.
polysciada (ser. Polysciadae) is strongly supported as polyphyletic, with the two accessions each
having cpDNA haplotypes more closely related to those of closely occurring species from ser.
Papuanae (compare Fig 3 and inset on Fig 5B). Although each of these morphologically
defined series present in clades B1±B6 cannot be assumed a priori to represent monophyletic
groups (e.g., there is no nuclear genetic evidence to support their monophyly), the presence of
geographically distinct chloroplast clades found across morphologically distinctive taxa
suggests a history of local chloroplast introgression between lineages, much as seen in Eucalyptus.
Geographic patterns in cpDNA clades shared across different taxonomic sections or
subgenera are less clear than those among series, but geographic links can still be discerned. For
instance, clade A1 (Fig 3) although ranging from north Queensland to southern New South
Wales in eastern Australia, has a cluster of samples from south-east Queensland including one
of C. citriodora (sect. Maculatae) that groups in the cpDNA gene tree as sister to a clade
including a sample of C. leichhardtii (sect. Naviculares) that occurs nearby (Fig 4B). Similarly, clade
A2, ranging from northern New South Wales to mid-east Queensland (Fig 4C), includes, in
reasonably close geographic proximity, members of sect. Maculatae, together with C. torelliana
of monotypic sect. Torellianae. However, the representative of sect. Naviculares in this clade is
geographically more distant, and the representative of sect. Abbreviate could not be mapped
because the sample is of unknown provenance. At least the geographic and cpDNA association
of C. torelliana and the three samples of sect. Maculatae is consistent with known capacity for
these groups to interbreed [
]. Again, these groups (although not these species) have
previously been reported to hybridise (; Fig 7).
A striking feature of the cpDNA tree is placement of two clades of ghost gums, subg.
Blakella sect. Abbreviatae (clades B4 and B6; Fig 3), within the large clade B that otherwise
includes almost all samples of the red bloodwood group subg. Corymbia sect. Septentrionales.
This contrasts with phylogenetic analyses of nrDNA sequences (Fig 6 and [
which sect. Abbreviatae groups with other members of subg. Blakella. The geographic
clustering of clades B4 and B6, and their distribution in areas where members of sect. Septentrionales
are common, especially in the Northern Territory, is consistent with these clades reflecting
historical chloroplast transfer from red bloodwoods to ghost gums, e.g., potentially two distinct
events of chloroplast transfer, in each case with a red bloodwood as the initial maternal parent.
21 / 28
Hybridisation between these groups has not, to our knowledge, been reported (e.g. Fig 7;
It is worth noting that taxon sampling for the current study was designed primarily to
sample across the major taxonomic groups of Corymbia, to assess their relationships on the basis
of chloroplast sequences, and it was not designed specifically to assess geographic patterns of
chloroplast variation. As such, there are substantial geographic distances between many
samples, and the spread of samples is quite unbalanced, e.g., with only one sample of sect.
Abbreviatae from eastern Australia (C. tessellaris, of unknown wild provenance), and none from the
north-west of Western Australia. The inferences here of historical chloroplast introgression
between major taxonomic groups, especially subgenera and some sections, are consistent with
the observed patterns of cpDNA variation, and knowledge of bloodwood reproductive biology,
but remain speculative. Our study provides insight into the potential importance of this
process in the evolutionary history of Corymbia, but more detailed studies using fine-scale
geographic sampling, including multiple replicates of species, are necessary both to properly test
for the presence of chloroplast introgression, and to more fully appreciate its significance and
any accompanying patterns of nuclear gene flow.
Chloroplast DNA relationships in Angophora
The genus Angophora was strongly supported as monophyletic, as universally found in all
molecular phylogenetic studies of eucalypts that have sampled two or more Angophora species
]). Most previous studies have included only a small number of exemplars
from Angophora and thus both species limits, which differ between some treatments [
], and the proposed infrageneric classification , have not been critically tested by
molecular data. Our cpDNA study included multiple accessions of four species, three of which
were strongly indicated as paraphyletic or polyphyletic (Fig 3); the one species that was
resolved as monophyletic, A. melanoxylon, was represented by only two samples from the
same geographic area, near St George in south-east Queensland. The sample set here is small
(15 samples from 8 species), and was not collected to assess geographic variation within/
between taxa, but it seems reasonable that taxonomic incongruence with cpDNA variation in
Angophora, as in Eucalyptus and Corymbia (see above) reflects, at least in part, a history of
cpDNA introgression between species. Consistent with this is the resolution of A. subvelutina
as paraphyletic (in particular, with one sample shown as sister to a closely co-occurring sample
of A. bakeri), and the polyphyly of A. floribunda, in which the northern-most sample of known
provenance (TMS14±33; Table 1) falls in a clade of other samples from nearby areas, and is
well separated in the phylogeny from the southernmost sample (MJB 2471). An influence of
chloroplast introgression on this gene tree would be consistent with the observation of Leach
] that hybridisation between species of Angophora ". . . has been observed in virtually all
combinations that are geographically or ecologically conceivable". As with Corymbia,
finescale studies of cpDNA variation could be used to test for both the presence and extent of
cpDNA introgression amongst Angophora species.
Implications for genus-level taxonomy
A primary aim of this study was to use HTS chloroplast data, from a broad sample of
infrageneric groups, to test the monophyly of the bloodwood genus Corymbia as currently
circumscribed. The inferred chloroplast relationships strongly support nesting of Angophora in
Corymbia thus making it paraphyletic (Fig 3), as also shown, with less support, in previous
cpDNA studies using either more limited taxon sampling or more limited sampling of the
chloroplast genome [
]. However, given the clear incongruence between the
22 / 28
cpDNA gene tree and taxonomic boundaries that are otherwise supported by both
morphological characters and analyses of nrDNA, and given the likely influence of historical cpDNA
introgression between lineages (discussed above), the cpDNA data, on their own, do not
provide a sound basis for assessing generic limits in this group. It is worth noting that it seems
unlikely that the close cpDNA relationship of Angophora to some groups of Corymbia could be
directly attributed to cpDNA introgression (at least not recently), because
Corymbia±Angophora hybrids have not been reported (e.g. [
]), despite common co-occurrence of
species  and attempts at artificial crosses (e.g. [
To make sound taxonomic decisions, especially regarding the limits of genera and
subgenera, among the bloodwoods and their relatives, better knowledge of relationships based on
nuclear DNA sequences is essential. Analyses of nuclear DNA datasets have so far been limited
to the ITS, ETS and 5S regions of nrDNA [
] and a small number of
microsatellite markers , and have given mixed support for the monophyly/paraphyly of Corymbia.
The nrDNA analyses here (Fig 6), for instance, using only ITS data, show 71% BS for the
nesting of Angophora in Corymbia in the MP analysis but <50% support in the ML analysis,
leaving open the possibility that Angophora might be sister to a monophyletic Corymbia. More
thorough assessment of relationships will require analysis of more substantial datasets, for
which there are now good prospects using HTS methods [
Even if Angophora proves to be nested in Corymbia based on nuclear data, placing them
together in one genus might not be the best taxonomic solution for this group. Instead, raising
one or more of the infrageneric groups of Corymbia to genus rank might be a better solution
for recognising monophyletic, morphologically diagnosable (less heterogeneous) groups and
minimising taxonomic upheaval (number of name changes). Adopting such a solution would
first require clear understanding of the relationships of the bloodwood lineages to each other
and to Angophora, informed by nuclear data, as well as the chloroplast data presented here. In
the interim, and in the absence of other strongly contradictory evidence, we support continued
recognition of both Angophora and Corymbia and the infrageneric groups of Corymbia as
currently defined [
]. This is because there is support for most of these groups based on nrDNA
data and morphology, and because name changes that are not soundly based, or might
subsequently need revision in the face of stronger evidence, would cause major taxonomic
instability in these economically important groups.
S1 File. Workflow used in CLC Genomics Workbench v. 9.5.1 and 9.5.2 (Qiagen). Quality
control and assembly of HTS chloroplast DNA sequences (Excel file).
S2 File. Alignment of chloroplast data. Alignment (121,016 base pairs) of chloroplast
sequences for 123 accessions of eucalypts (phylip file format).
S3 File. Alignment of nuclear ribosomal data. Alignment (663 base pairs) of nrITS sequences
for 119 accessions of eucalypts (phylip file format).
We thank Frances Bayly, Gill Brown (BRI), Alison Kellow (La Trobe University), Pauline
Ladiges (The University of Melbourne), and Karen Wilson (NSW), who either provided
material or assisted with field work, and Claudine Griffin-Beale for helping to locate some
23 / 28
Angophora species. We are grateful to Neil Gibson (WA) for collecting Corymbia during the
ABRS Katjarra Region Bushblitz. In particular, we thank Dean Nicolle for access to the
impressive living collection of eucalypts at Currency Creek Arboretum. We gratefully acknowledge
Alex Johnson and Julien Bonneau for access to equipment (The University of Melbourne,
Plant Nutrition Lab), Liz Milla (Walter and Eliza Hall Institute of Medical Research) for HTS
data pre-processing, and Heroen Verbruggen (The University of Melbourne, Algae Lab) and
Wake Forest University for computational resources. Will Neal and Harvey Orel assisted with
the mounting and databasing of voucher specimens. Plant collecting permits were provided by
the Victorian Department of Sustainability and Environment, New South Wales National
Parks and Wildlife Service, and Northern Territory Parks and Wildlife Commission.
Conceptualization: Tanja M. Schuster, Josquin F. G. Tibbits, Peter K. Ades, Michael J. Bayly.
Data curation: Tanja M. Schuster.
Formal analysis: Tanja M. Schuster, Sabrina D. Setaro.
Funding acquisition: Tanja M. Schuster, Josquin F. G. Tibbits, Peter K. Ades, Michael J.
Investigation: Tanja M. Schuster, Erin L. Batty, Stephen Wilcox.
Methodology: Tanja M. Schuster, Josquin F. G. Tibbits, Rachael M. Fowler, Todd G. B.
McLay, Michael J. Bayly.
Project administration: Tanja M. Schuster.
Resources: Tanja M. Schuster, Michael J. Bayly.
Supervision: Michael J. Bayly.
Validation: Tanja M. Schuster, Sabrina D. Setaro, Michael J. Bayly.
Visualization: Tanja M. Schuster, Michael J. Bayly.
Writing ± original draft: Tanja M. Schuster, Sabrina D. Setaro, Michael J. Bayly.
Writing ± review & editing: Tanja M. Schuster, Sabrina D. Setaro, Josquin F. G. Tibbits, Erin
L. Batty, Rachael M. Fowler, Todd G. B. McLay, Stephen Wilcox, Peter K. Ades, Michael J.
24 / 28
Williams JE, Woinarski JCZ (1997) Eucalypt Ecology: Individuals to Ecosystems. Cambridge
Cambridge University Press.
25 / 28
Cavanilles A (1797) Icones et Descriptiones Plantarum, IV. Madrid: Regia Typographia.
26 / 28
27 / 28
1. CHAH ( 2017 ) Australian plant census [online]. Council of Heads of Australasian Herbaria . Available from: https://biodiversity.org.au/nsl/services/apc.
2. Hill KD , Johnson LA ( 1995 ) Systematic studies in the eucalypts 7. A revision of the bloodwoods, genus Corymbia (Myrtaceae) . Telopea 6 : 185 ± 504 .
3. Pryor LD , Johnson LA ( 1971 ) A classification of the eucalypts . Canberra: Australian National University Press.
4. Brooker MIH ( 2000 ) A new classification of the genus Eucalyptus L'Her. (Myrtaceae) . Aust Syst Bot 13 : 79 ± 148 .
5. Nicolle D ( 2015 ) Classification of the eucalypts . version 2 . Available from: http://www.dn.com.au/ Classification-Of-The-Eucalypts.pdf.
6. Parra O C , Bayly MJ , Drinnan A , Udovicic F , Ladiges P ( 2009 ) Phylogeny, major clades and infrageneric classification of Corymbia (Myrtaceae), based on nuclear ribosomal DNA and morphology . Aust Syst Bot 22 : 384 ± 399 .
8. Chippendale GM ( 1988 ) Flora of Australia . Vol. 19 . Canberra: Australian Government Publishing Service.
9. Boland DJ , Brooker MIH , Chippendale G , Hall N , Hyland B , et al. ( 2006 ) Forest trees of Australia. Melbourne: CSIRO publishing .
10. MacPherson J ( 1939 ) The Eucalyptus in the Daily Life and Medical Practice of the Australian Aborigines . Mankind 2 : 175 ± 180 .
11. Packer J , Brouwer N , Harrington D , Gaikwad J , Heron R , et al. ( 2012 ) An ethnobotanical study of medicinal plants used by the Yaegl Aboriginal community in northern New South Wales, Australia . J Ethnopharmacol 139 : 244 ± 255 . https://doi.org/10.1016/j.jep. 2011 . 11 .008 PMID: 22101358
12. Reid EJ , Betts TJ ( 1979 ) Records of Western Australian Plants Used by Aboriginals as Medicinal Agents . Planta Med 36 : 164 ± 173 . https://doi.org/10.1055/s-0028-1097257 PMID: 461569
13. Batish DR , Singh HP , Kohli RK , Kaur S ( 2008 ) Eucalyptus essential oil as a natural pesticide . For Ecol Manage 256 : 2166 ± 2174 .
14. Low D , Rawal BD , Griffin WJ ( 1974 ) Antibacterial action of the essential oils of some Australian Myrtaceae with special references to the activity of chromatographic fractions of oil of Eucalyptus citriodora . Planta Med 26 : 184 ± 189 . https://doi.org/10.1055/s-0028-1097987 PMID: 4212944
15. Ramezani H , Singh HP , Batish DR , Kohli RK ( 2002 ) Antifungal activity of the volatile oil of Eucalyptus citriodora . Fitoterapia 73 : 261 ± 262 . PMID: 12048022
16. Ladiges PY , Udovicic F , Nelson G ( 2003 ) Australian biogeographical connections and the phylogeny of large genera in the plant family Myrtaceae . J Biogeogr 30 : 989 ± 998 .
Wilson PG , O 'Brien M , Heslewood M , Quinn C ( 2005 ) Relationships within Myrtaceae sensu lato based on a matK phylogeny . Pl Syst Evol 251 : 3± 19 .
18. Slee AV , Connors J , Brooker MIH , Duffy SM , West JG ( 2006 ) EUCLID Eucalypts of Australia. CD ROM. Centre for Plant Biodiversity Research . Melbourne: CSIRO Publishing.
19. Dawson JW ( 1970 ) Pacific capsular Myrtaceae 1. Reproductive morphology of Arillastrum gummiferum Panch. ex Baillon (New Caledonia) . Blumea 18 : 431 ± 440 .
20. Carr DJ , Carr S , Hyland B , Wilson PG , Ladiges PY ( 2002 ) Stockwellia quadrifida (Myrtaceae), a new Australian genus and species in the eucalypt group . Bot J Linn Soc 139 : 415 ± 421 .
21. Blake S ( 1977 ) Allosyncarpia ternata, a new genus and species of Myrtaceae subfamily Leptospermoideae from northern Australia . Austrobaileya: 43 ± 46 .
22. Craven L ( 1990 ) One new species each in Acmena and Eucalyptopsis and a new name in Lindsayomyrtus (all Myrtaceae) . Aust Syst Bot 3 : 727 ± 732 .
23. Ladiges PY , Udovicic F ( 2000 ) Comment on a new classification of the eucalypts . Aust Syst Bot 13 : 149 ± 152 .
24. Ladiges PY , Udovicic F , Drinnan AN ( 1995 ) Eucalypt phylogeny±molecules and morphology . Aust Syst Bot 8 : 483 ± 497 .
25. Udovicic F , McFadden G , Ladiges P ( 1995 ) Phylogeny of Eucalyptus and Angophora based on 5S rDNA spacer sequence data . Mol Phyl Evol 4 : 247 ± 256 .
26. Udovicic F , Ladiges PY ( 2000 ) Informativeness of nuclear and chloroplast DNA relationships of the eucalypt and related genera (Myrtaceae) . Kew Bull 55 : 633 ± 645 .
27. Parra O C , Bayly MJ , Udovicic F , Ladiges PY ( 2006 ) ETS sequences support the monophyly of the eucalypt genus Corymbia (Myrtaceae) . Taxon 55 : 653 ± 663 .
Whittock S , Steane D , Vaillancourt R , Potts B ( 2003 ) Molecular evidence shows that the tropical boxes (Eucalyptus subgenus Minutifructus) are over-ranked . Trans R Soc S Aust 1217 : 27 ± 32 .
29. Steane DA , McKinnon GE , Vaillancourt RE , Potts BM ( 1999 ) ITS sequence data resolve higher level relationships among the eucalypts . Mol Phyl Evol 12 : 215 ± 223 .
30. Steane DA , Nicolle D , McKinnon GE , Vaillancourt RE , Potts BM ( 2002 ) Higher-level relationships among the eucalypts are resolved by ITS-sequence data . Aust Syst Bot 15 : 49 ± 62 .
31. GonzaÂlez-Orozco CE , Pollock LJ , Thornhill AH , Mishler BD , Knerr N , et al. ( 2016 ) Phylogenetic approaches reveal biodiversity threats under climate change . Nat Clim Change 6 : 1110 ± 1114 .
32. Ladiges P ( 1984 ) A comparative study of Trichomes in Angophora Cav. and Eucalyptus±a question of homology . Aust J Bot 32 : 561 ± 574 .
33. Bentham G ( 1866 ) Flora Australiensis , III. London: Lovell Reeve.
34. Maiden JH ( 1909 ) A critical revision of the genus Eucalyptus . Sydney: Gullick.
35. Blakely WF ( 1965 ) A Key to the Eucalypts . Canberra: Forestry and Timber Bureau .
37. Bayly MJ ( 2016 ) Phylogenetic studies of eucalypts: fossils, morphology and genomes . Proc R Soc Vic 128 : 12 ± 24 .
38. Ochieng JW , Henry RJ , Baverstock PR , Steane DA , Shepherd M ( 2007 ) Nuclear ribosomal pseudogenes resolve a corroborated monophyly of the eucalypt genus Corymbia despite misleading hypotheses at functional ITS paralogs . Mol Phyl Evol 44 : 752 ± 764 .
39. Ochieng JW , Steane DA , Ladiges PY , Baverstock PR , Henry RJ , et al. ( 2007 ) Microsatellites retain phylogenetic signals across genera in eucalypts (Myrtaceae) . Genet Mol Biol 30 : 1125 ± 1134 .
40. Parra-O C ( 2009 ) Chapter 6 , Chloroplast DNA ( cpDNA ). In: Parra-O C, editor. A phylogenetic analysis of the bloodwood eucalypts (Myrtaceae): PhD Thesis , The University of Melbourne.
41. Bayly MJ , Rigault P , Spokevicius A , Ladiges PY , Ades PK , et al. ( 2013 ) Chloroplast genome analysis of Australian eucalypts±Eucalyptus, Corymbia, Angophora, Allosyncarpia and Stockwellia (Myrtaceae) . Mol Phyl Evol 69 : 704 ± 716 .
42. Steane DA ( 2005 ) Complete nucleotide sequence of the chloroplast genome from the Tasmanian blue gum, Eucalyptus globulus (Myrtaceae) . DNA Res 12 : 215 ± 220 . https://doi.org/10.1093/dnares/dsi006 PMID: 16303753
43. Paiva JA , Prat E , Vautrin S , Santos MD , San-Clemente H , et al. ( 2011 ) Advancing Eucalyptus genomics: identification and sequencing of lignin biosynthesis genes from deep-coverage BAC libraries . BMC Genomics 12 : 137 . https://doi.org/10.1186/ 1471 -2164-12-137 PMID: 21375742
44. Doyle JJ , Doyle JL ( 1987 ) A rapid isolation procedure for small quantities of fresh leaf material . Phytochem Bull 19 : 11 ± 15 .
45. McLay TG ( 2017 ) High quality DNA extraction protocol from recalcitrant plant tissues . protocols.io. Available from: https://www.protocols. io/view/high-quality-dna-extraction-protocol-from-recalciti8jchun?more.
46. Shepherd LD , McLay TG ( 2011 ) Two micro-scale protocols for the isolation of DNA from polysaccharide-rich plant tissue . J Plant Res 124 : 311 ± 314 . https://doi.org/10.1007/s10265-010 -0379-5 PMID: 20927638
47. Rohland N , Reich D ( 2012 ) Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture . Genome Res 22 : 939 ± 946 . https://doi.org/10.1101/gr.128124.111 PMID: 22267522
48. Katoh K , Standley DM ( 2013 ) MAFFT multiple sequence alignment software version 7: improvements in performance and usability . Mol Biol Evol 30 : 772 ± 780 . https://doi.org/10.1093/molbev/mst010 PMID: 23329690
49. Gouy M , Guindon S , Gascuel O ( 2009 ) SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building . Mol Biol Evol 27 : 221 ± 224 . https://doi.org/10.1093/ molbev/msp259 PMID: 19854763
50. Maddison WP , Maddison DR ( 2013 ) Mesquite version 3.1: a modular system for evolutionary analysis . http://mequiteproject.org/.
51. Castresana J ( 2000 ) Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis . Mol Biol Evol 17 : 540 ± 552 . https://doi.org/10.1093/oxfordjournals.molbev. a026334 PMID: 10742046
52. Darriba D , Taboada GL , Doallo R , Posada D ( 2012 ) jModelTest 2: more models, new heuristics and parallel computing . Nat Methods 9 : 772 ± 772 .
53. Guindon S , Gascuel O ( 2003 ) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood . Syst Biol 52 : 696 ± 704 . PMID: 14530136
54. Stamatakis A ( 2014 ) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies . Bioinformatics 30 : 1312± 1313 . https://doi.org/10.1093/bioinformatics/btu033 PMID: 24451623
55. Swofford D ( 2002 ) PAUP* . Phylogenetic Analysis Using Parsimony (* and Other Methods) . 4 ed: Sinauer Associates, Sunderland, Massachusetts.
56. Rambaut A ( 2016 ) FigTree Tree Figure Drawing Tool version 1.4.3 . http://tree.bio.ed.ac.uk/software/ figtree/.
57. Gibbs AK , Udovicic F , Drinnan AN , Ladiges PY ( 2009 ) Phylogeny and classification of Eucalyptus subgenus Eudesmia (Myrtaceae) based on nuclear ribosomal DNA, chloroplast DNA and morphology . Aust Syst Bot 22 : 158 ± 179 .
58. Ladiges PY , Bayly M , Nelson G ( 2010 ) East-west continental vicariance in Eucalyptus subgenus Eucalyptus . In: Williams D , Knapp S , editors. Beyond Cladistics. The Branching of a Paradigm . Berkeley: University of California Press. pp. 267 ± 302 .
59. Bayly MJ , Ladiges PY ( 2007 ) Divergent paralogues of ribosomal DNA in eucalypts (Myrtaceae) . Mol Phyl Evol 44 : 346 ± 356 .
60. Bayly MJ , Udovicic F , Gibbs AK , Ladiges PY ( 2008 ) Ribosomal DNA pseudogenes are widespread in the eucalypt group (Myrtaceae): implications for phylogenetic analysis . Cladistics 24 : 131 ± 146 .
61. Burke JM , Bayly MJ , Adams PB , Ladiges PY ( 2008 ) Molecular phylogenetic analysis of Dendrobium (Orchidaceae), with emphasis on the Australian section Dendrocoryne, and implications for generic classification . Aust Syst Bot 21 : 1± 14 .
62. Holmes GD , Downing TL , James EA , Blacket MJ , Hoffmann AA , et al. ( 2014 ) Phylogeny of the holly grevilleas (Proteaceae) based on nuclear ribosomal and chloroplast DNA . Aust Syst Bot 27 : 56 ± 77 .
63. Kearse M , Moir R , Wilson A , Stones-Havas S , Cheung M , et al. ( 2012 ) Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data . Bioinformatics 28 : 1647± 1649 . https://doi.org/10.1093/bioinformatics/bts199 PMID: 22543367
64. Bailey CD , Carr TG , Harris SA , Hughes CE ( 2003 ) Characterization of angiosperm nrDNA polymorphism, paralogy, and pseudogenes . Mol Phyl Evol 29 : 435 ± 455 .
65. Mayol M , RosselloÂ JA ( 2001 ) Why nuclear ribosomal DNA spacers (ITS) tell different stories in Quercus . Mol Phyl Evol 19 : 167 ± 176 .
66. Baum DA , Smith SD ( 2013 ) Tree thinking: an introduction to phylogenetic biology . Greenwood Village , Colarado: Roberts and Company.
67. Doyle JJ ( 1992 ) Gene trees and species trees: molecular systematics as one-character taxonomy . Syst Bot : 144 ± 163 .
68. Maddison WP ( 1997 ) Gene trees in species trees . Syst Biol 46 : 523 ± 536 .
69. Tsitrone A , Kirkpatrick M , Levin DA , Morgan M ( 2003 ) A model for chloroplast capture . Evolution 57 : 1776 ± 1782 . PMID: 14503619
70. Rieseberg LH , Soltis D ( 1991 ) Phylogenetic consequences of cytoplasmic gene flow in plants . Evol Trends Pl 5 : 65 ± 84 .
71. McKinnon GE , Jordan GJ , Vaillancourt RE , Steane DA , Potts BM ( 2004 ) Glacial refugia and reticulate evolution: the case of the Tasmanian eucalypts . Philos Trans R Soc Lond, B, Biol Sci 359 : 275 ± 284 . https://doi.org/10.1098/rstb. 2003 .1391 PMID: 15101583
72. Meudt HM , Bayly MJ ( 2008 ) Phylogeographic patterns in the Australasian genus Chionohebe (Veronica s .l., Plantaginaceae) based on AFLP and chloroplast DNA sequences . Mol Phyl Evol 47 : 319 ± 338 .
73. Dickinson GR , Lee DJ , Wallace HM ( 2012 ) The influence of pre-and post-zygotic barriers on interspecific Corymbia hybridization . Ann Bot 109 : 1215 ± 1226 . https://doi.org/10.1093/aob/mcs050 PMID: 22419764
74. Grattapaglia D , Vaillancourt RE , Shepherd M , Thumma BR , Foley W , et al. ( 2012 ) Progress in Myrtaceae genetics and genomics: Eucalyptus as the pivotal genus . Tree Genetics & Genomes 8 : 463 ± 508 .
75. Oudjehih B , Abdellah B ( 2006 ) Chromosome numbers of the 59 species of Eucalyptus L'Herit. (Myrtaceae) . Caryologia 59 : 207 ± 212 .
76. Barbour R , Crawford AC , Henson M , Lee DJ , Potts B , et al. ( 2008 ) The risk of pollen-mediated gene flow from exotic Corymbia plantations into native Corymbia populations in Australia . For Ecol Manage 256 : 1± 19 .
77. Griffin A , Burgess I , Wolf L ( 1988 ) Patterns of natural and manipulated hybridisation in the genus Eucalyptus L'HeÂr.±a review . Aust J Bot 36 : 41 ± 66 .
78. Jackson H , Steane D , Potts B , Vaillancourt R ( 1999 ) Chloroplast DNA evidence for reticulate evolution in Eucalyptus (Myrtaceae) . Mol Ecol 8 : 739 ± 751 .
79. McKinnon G , Smith J , Potts B ( 2010 ) Recurrent nuclear DNA introgression accompanies chloroplast DNA exchange between two eucalypt species . Mol Ecol 19 : 1367 ± 1380 . https://doi.org/10.1111/j. 1365 - 294X . 2010 . 04579 . x PMID : 20298471
80. McKinnon GE , Steane DA , Potts BM , Vaillancourt RE ( 1999 ) Incongruence between chloroplast and species phylogenies in Eucalyptus subgenus Monocalyptus (Myrtaceae) . Am J Bot 86 : 1038 ± 1046 . PMID: 10406727
81. McKinnon GE , Vaillancourt RE , Jackson HD , Potts BM ( 2001 ) Chloroplast sharing in the Tasmanian eucalypts . Evolution 55 : 703 ± 711 . PMID: 11392388
82. Steane D , Byrne M , Vaillancourt R , Potts B ( 1998 ) Chloroplast DNA polymorphism signals complex interspecific interactions in Eucalyptus (Myrtaceae) . Aust Syst Bot 11 : 25 ± 40 .
83. Nevill PG , DespreÂs T , Bayly MJ , Bossinger G , Ades PK ( 2014 ) Shared phylogeographic patterns and widespread chloroplast haplotype sharing in Eucalyptus species with different ecological tolerances . Tree Genetics & Genomes 10 : 1079 ± 1092 .
84. Pollock LJ , Bayly MJ , Nevill PG , Vesk PA ( 2013 ) Chloroplast DNA diversity associated with protected slopes and valleys for hybridizing Eucalyptus species on isolated ranges in south-eastern Australia . J Biogeogr 40 : 155 ± 167 .
85. Pollock LJ , Bayly MJ , Vesk PA ( 2015 ) The roles of ecological and evolutionary processes in plant community assembly: the environment, hybridization, and introgression influence co-occurrence of Eucalyptus . Am Nat 185 : 784 ± 796 . https://doi.org/10.1086/680983 PMID: 25996863
86. Leach GJ ( 1986 ) A revision of the genus Angophora (Myrtaceae) . Telopea 2 : 749 ± 779 .
87. Thiele K , Ladiges PY ( 1988 ) A cladistic analysis of Angophora Cav. (Myrtaceae) . Cladistics 4 : 23 ± 42 .