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Edwards et al.

, 2018; Australian Systematic Botany; 31(5-6):495-503

Species limits and cryptic biogeographic structure in a widespread complex of Australian monsoon tropics trees
(broad-leaf paperbarks: Melaleuca, Myrtaceae)

Robert D. EdwardsA, Michael D. CrispB and Lyn G. CookC.

US National Herbarium, Department of Botany, National Museum of Natural History, Smithsonian Institution, 10th St.
& Constitution Ave. NW, Washington, DC 20013-7012, USA.
Evolution, Ecology and Genetics, Research School of Biology, The Australian National University, Banks Building,
Daley Road, Acton, ACT 2601, Australia.
The University of Queensland, School of Biological Sciences, Goddard Building, Mansfield Place, St Lucia, Brisbane,
Qld 4072, Australia.
Corresponding author. Email:

Abstract. The Australian monsoon tropics are currently dominated by savanna and tropical woodland biomes that have arisen in
response to a cooling and drying trend within the past ~3 million years. It is expected that organisms well adapted to these conditions
have expanded into available habitats, leading to the differentiation of populations and species across this landscape, a process that
could be magnified by the presence of several biogeographic barriers. The broad-leaved paperbark (Melaleuca leucadendra (L.) L.)
complex is one such group of plants, with 14 poorly morphologically differentiated species occupying large overlapping distributions
across the region, and across several recognised biogeographic barriers. Using phylogenetic and network analyses of nuclear and
plastid sequences, we tested species limits among currently described species within the complex and for phylogeographic structure
within species across seven of these barriers. Overall, our data suggested patterns of differentiation among species consistent with the
early to middle stages of incomplete lineage sorting, and evidence for an idiosyncratic cryptic response of species to biogeographic
barriers. Unexpectedly, we found a deep molecular split across all species, broadly coinciding with the northern part of the Great
Dividing Range, a feature not typically considered to be a barrier to dispersal. Our study has offered one of the first insights into the
dynamics within and among widespread species across the north of Australia, suggesting considerably more geographic structure than
was previously recognised.


R. D. Edwards et al.

Cryptic structure in broadleaf paperbarks

Additional keywords: barriers, biogeography, cryptic divergence, melaleuca, reticulation, non-monophyly, the Great Dividing

The Melaleuca leucadendra (L.) L. (broad-leaved paperbark) complex is an important component of the tropical and
subtropical Australian flora, particularly in wetlands of high conservation significance (Blake 1968, Franklin et al. 2007).
As currently recognised, 14 species (Table S1, available as Supplementary material to this paper) are distributed almost
continuously across seasonally wet–dry savanna habitat (Fig. 1A). Seven (M. argentea W.Fitzg., M. cajuputi Powell, M.

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dealbata S.T.Blake, M. leucadendra, M. nervosa (Lindl.) Cheel, M. quinquenervia (Cav.) S.T.Blake and M. viridiflora
Sol. ex Gaertn.) have large ranges, each covering over 4000 km in wide arcs across the tropical north of the continent.
Melaleuca triumphalis Craven has the smallest range, being restricted to an ~150-km length of cliff lines in the Victoria
River Gorge and associated gorges in the Northern Territory (Fig. 1A; Craven 1998; Crase et al. 2006). None of the
species is allopatric across its entire range, and only the following three naturally occur alone to any considerable extent
(Fig. 1A): M. quinquenervia in the southern half of the eastern Australian coast and in New Caledonia; M. argentea with a
disjunct population in the Pilbara region of Western Australia; and M. cajuputi, found in isolation from Timor northward
into south-eastern Asia. The distributions of all species (bar M. triumphalis) overlap on Cape York Peninsula. At a local
scale, various combinations of species may be found in sympatry (Franklin et al. 2007; L. Cook, M. Crisp, R. Edwards,
pers. obs.) with localised segregation of morphotypes across water-availability gradients (Bowman and Minchin 1987;
Franklin et al. 2007; L. Cook, M. Crisp, R. Edwards, pers. obs.). These habitats are typically associated with poor sandy
soils on sites that range from permanently submerged to seasonally dry (Bowman and Minchin 1987), with several species
also tolerating highly saline conditions (Woodall 1982; Hofstetter 1991; Tran et al. 2013).

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Fig. 1. A. Distributions of the 13 species considered in the study. B. Seven biogeographic barriers across which genetic structure
was tested: a. Great Sandy Desert, b. Ord Basin, c. Daly River Plains, d. Arnhem Land Plateau, e. Carpentaria Basin, f. Normany
Basin, g. Townsville Lowlands–Paluma Range (from Edwards et al. 2017).

Members of the complex range in habit from 2.5 to 25 m in height, typically with long, flat, broad leaves, often falcate,
and trunks covered in layers of thin-papery to fibrous subpapery bark (giving the group its common name). There are
several exceptions, including the following: leaves are short in M. arcana S.T.Blake, narrower in M. saligna Schauer and
M. stenostachya S.T.Blake, and the bark is hard and fissured in M. clarksonii Barlow (Table S1). However, morphologies
within species are highly variable and represent a nearly continuous and intergrading distribution across the complex
(Cook et al. 2008). Flowers have a ring of filamentous stamens that are held in elongated spikes, lending them a fluffy
bottlebrush-like appearance typical of the genus. Flowers are generally cream to white, although those of M. viridiflora
range from pale cream to bright green and, occasionally, red. Melaleuca nervosa is also known to have a rare, red-
flowered form (Holliday 2004). The simple and generalist nature of the flowers with exposed stamens and stigma suggest
no specialised pollination syndrome and all species produce nectar that can range from light with little odour to sticky and
pungent, with wide variation even within populations (Franklin and Noske 2000; Woinarski et al. 2000; L. Cook, M.
Crisp, R. Edwards, pers. obs.). Birds, insects, possums and fruit bats have been observed feeding on various species
(Geary and Woodall 1990; Hofstetter 1991; Woinarski and Braithwaite 1993; Woinarski et al. 2000; Barclay 2002; L.
Cook, M. Crisp, R. Edwards, pers. obs.) and are presumed to share the dispersal of pollen, which is granular, sticky and
apparently ill-suited for wind dispersal. Movement of seed is highly localised (Meskimen 1962; Woodall 1978, 1982;
Browder and Schroeder 1981), and with water as the primary means of dispersal (Hartman 1999), there is evidence for
considerable isolation between some drainage systems (Chong 2008) and the potential for asymmetrical geographical
patterns of gene flow (Edwards et al. 2013).

Members of the M. leucadendra complex form a distinct clade within the larger Melaleuca genus, sister to M.
acacioides F.Muell. (Edwards et al. 2010), with limited molecular data showing reticulate relationships among species
(Cook et al. 2008). With there being no evidence for polyploidy in M. leucadendra or M. quinquenervia (Brighton and
Ferguson 1976), it is likely that either introgression or incomplete lineage sorting (ILS) is obscuring species boundaries
(Cook et al. 2008). A recent rapid radiation is consistent with the likely expansion of savanna habitat during increased
aridification ~4–2 million years ago (Fujioka 2005), with estimated crown ages for the complex of 3.6 million years and
6.7 million years (Cook et al. 2008). Sampling for these studies has been limited both taxonomically and geographically,
and it is necessary to establish to what extent future studies should account for biogeographic signal when considering
evolutionary histories.

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We used sampling across the distributions of 13 of the 14 species to (1) assess the utility of nuclear and chloroplast loci
in defining species boundaries and (2) look for signatures of geographic structure within the complex, specifically the
possibility of limited geneflow where species span previously identified biogeographic barriers (Fig. 1C; Edwards et al.

Materials and methods

Sampling, DNA extraction, amplification and sequencing

Field-collected silica gel-preserved leaf material was supplemented by herbarium specimens to provide a broad
geographic sampling across all species in proportion to the distribution of each (Table S2, available as Supplementary
Material to this paper; M. arcana is under-sampled and M. ferruginea Craven & Cowie was described after data collection
and is not included; Craven and Cowie 2013). Efforts were made to collect from specimens growing in sympatry, where
possible, so as to limit any genetic sorting bias from locally isolated populations. Genomic DNA was extracted using a
CTAB and chloroform extraction (Doyle and Doyle 1987). One chloroplast (psbA–rpl2) and three nuclear (internal
transcribed spacer (ITS), LFY (LEAFY meristem identity gene) and P5 (EPSP synthase)) DNA regions were amplified
using the primers shown in Table S3 (available as Supplementary Material to this paper). All amplifications were
performed using 0.2 µL (1 unit) of Mango Taq (Bioline, Sydney, NSW, Australia) with 1.5 µL of 50 mM MgCl2, 5 µL of
5× buffer, 2 µL of 2 mM dNTP, 0.5 µL of 10 µM each of forward and reverse primer and 14 µL of nuclease-free water
and 2 µL of DNA in a final volume of ~25 µL. A standard polymerase chain-reaction (PCR) cycling protocol was
followed with an annealing temperature of 55°C. The length of amplified PCR products was checked against a 1-kb plus
ladder (Invitrogen, Carlsbad, CA, USA) on a 1% agarose gel and visualised with ethidium bromide. PCR products were
cleaned using ammonium acetate with ethanol precipitation and sequenced by Macrogen (Seoul, Republic of Korea) by
using Sanger sequencing. Where the PCR product was weak, both directions were sequenced, allowing fragment
concatenation to construct full-length sequences, and to avoid false base-calls between a weak product and background
noise. DNA sequences were edited using CodonCode Aligner (CodonCode Corporation, Dedham, MA, USA) and aligned
by eye in Se-Al 2.0a11 (Se-Al; A. Rambaut, distributed by the author at Indels were
coded as binary unordered characters. Early sequencing of the LFY region returned a high proportion of multiple single
nucleotide polymorphisms (SNPs) within individuals. Because of the difficulty of estimating copy number in such
situations, a restriction site A/C SNP was identified and further samples were amplified as above, incubated with Hinfl
enzyme (New England Biolabs) as per manufacturer’s instructions, run on a 2% agarose gel against a 500-bp ladder and
presence or absence of bands for no digestion (Homozygote C), partial digestion (heterozygote) and complete digestion
(Homozygote A) was scored by eye. Bands for each outcome were easily distinguishable.

For the nuclear loci, monomorphic sequences or sequences with a single SNP were manually split into two constituent
alleles. Length-polymorphic sequences were separated by eye during initial sequence editing. Alleles from the remaining
multiply-polymorphic sequences were estimated using a nested phasing approach in the Bayesian statistical package
PHASE2.1.1 (Stephens and Donnelly 2003). Input files generated from sequence data using SeqPhase (Flot 2010) were
submitted to an initial PHASE run, with resolved alleles designated as ‘phase known’ and the program re-run. This
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process was repeated until all polymorphisms were resolved, or until there were no further improvements of phase
estimation. Default parameters for all runs were modified by removing the constraint of stepwise mutation (-d1),
increasing the number of iterations of the final run by a factor of 10 (-×10) and automatically running the algorithm 10
times, reporting the run with the best average values (-×10). Unambiguous alleles, including those from clones generated
in a previous study (Cook et al. 2008), were included in the analysis to help allele prediction.

Species boundaries and genetic structure

Relationships among species were examined using the three sequenced individual gene regions (ITS, psbA–rpl2, and
P5). Because copy-number variation in the ITS region was expected (Cook et al. 2008) and the copy number may be
underestimated by PHASE estimation of haplotypes, a reduced dataset of monomorphic sequences and those with only a
single SNP was also analysed. Population-genetic analysis of combined haploid, diploid and SNP data is problematic,
limiting our ability to use coalescence methods to test for natural genetic groupings; similarly, the number of pair-wise
species comparisons that would be required to test between all members of the group also makes statistical analysis (e.g.
FST) prone to false positives, with loss of power under correction. To explore patterns of genetic structure among species
for each locus, we generated maximum-likelihood (ML) phylogenies using RAxML (ver. 2.2.3; Stamatakis 2006,
distributed by the author at through the online portal
(; Nov-Dec 2012), by using default parameters and a gamma model of rate
heterogeneity partitioned by gene and codon. Maximum-likelihood phylogenies were estimated for cpDNA and ITS only,
with lack of congruence between these loci arguing against concatenation. Strictly tree-like relationships among species
were not expected; therefore, statistical parsimony allelic networks were also constructed for each marker in the program
TCS (Templeton, Crandall and Sing; Clement et al. 2000) to visualise reticulation or clustering among species.
Haplotypes were coloured by species to explore relationships, and transposed onto maps to visualise geographical
patterns. To test for signatures of isolation across putative barriers, Analysis of Molecular Variance (AMOVAs) were run
using Arlequin (ver. 3.5.1; Excoffier and Lischer 2010, distributed by the authors at for species with sufficient population sampling on either
side of the barriers. The power of AMOVA is adequate only when seven or more populations are included (Fitzpatrick
2009), meaning that sufficient sampling was available only for 12 tests (six species across seven putative barriers, Table
1). Of these, one test was between populations that are physically disjunct (M. argentea across the Great Sandy Desert),
whereas the remaining 10 barrier tests were within distributions that are apparently unbroken across the barriers in

Effort was made to obtain ITS, psbA–rpl2 (sequences) and LFY (SNP) for each sample; however, amplification of all
three was not always possible despite multiple extractions, experimentation with less specific PCR protocols, variation in
MgCl2 and template concentrations, and the inclusion of polyethylene glycol or dimethyl sulfoxide to reduce inhibiting
agents and non-specific binding (Bachmann et al. 1990). Failures were greater, but not limited to, older herbarium
samples, with no apparent systematic bias in species. Of the ~400 samples extracted, 223 returned at least one amplified
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gene, which, combined with 23 individuals from GenBank, resulted in a total of 246 individuals (outgroup n = 8). Of
these, ITS was sequenced for 186 individuals, with 50 additional sequences from 22 individuals being retrieved from
GenBank (outgroup n = 8). Only three sequences were non-polymorphic with separation of length-polymorphisms and
phasing revealing 190 bi-allelic individuals, seven tri-allelic individuals, and 12 individuals with four alleles, for a total of
259 phased sequences. For psbA–rpl2, 180 individuals were sequenced with the addition of 23 GenBank sequences
(outgroup n = 7), and 27 individuals were sequenced for the P5 locus, resulting in 51 phased sequences (outgroup n = 0).
For the LFY SNP, 134 individuals were scored (Table S2).

Species boundaries
Maximum-likelihood phylogenies returned for all three sequenced loci confirmed a lack of monophyly of species, but
with some clustering of haplotypes by species (Fig. S1–S3, available as Supplementary Material to this paper). When
polymorphic sequences were excluded, the results were qualitatively no different from those including phased
polymorphic data. Only eight individuals (M. clarksonii: Cumming24362; M. cajuputi: MC10273, LGC00771; M.
nervosa: MC10227, RDE288; M. stenostachya: Wannan2342; M. quinquenervia: RDE219; M. viridiflora: GB8) had
highly divergent ITS alleles, which may suggest deep paralogy of ITS copies, leading to ‘mirroring’ of clades (Koonin
2005). High levels of incongruence between topologies for all three loci precluded concatenation, and with there being
strong evidence for reticulate evolution, relationships were assessed using haplotype networks. Networks generated from
each of the three sequenced markers showed high levels of reticulation and interspersion of haplotypes from different
species (Figs 2A, S3 available as Supplementary Material to this paper). However, there were consistent patterns across
loci, namely, the sharing of frequent internal haplotypes by multiple species, surrounded by low-frequency external
haplotypes unique to a single species, typical of incomplete lineage sorting (Omland et al. 2006). There were also distinct
clusters of haplotypes dominated by one or few species, suggesting a greater reduction of geneflow between some species
and groups of species. Of particular note in the nDNA network is the almost exclusive association of M. fluviatilis Barlow
and M. leucadendra with each other and M. argentea, the limitation of M. nervosa to only two clusters, and M. dealbata
to a single cluster. Melaleuca quinquenervia shared haplotypes with only three species, and although being widespread
throughout the network, M. cajuputi shared with only two other species. Similar patterns are less pronounced in the
cpDNA network, but partitioning of genotypes by subsets of taxa was also apparent.

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Vietnam New Guinea



New Caledonia

M. arcana
M. argentea
M. cajuputi
M. clarksonii
M. dealbata
M. fluviatilis
M. leucadendra
rM. nervosa
M. quinquenervia
M. saligna
M. stenostachya
M. triumphalis
M. viridiflora

New Guinea



New Guinea
at D


New Caledonia

E Clade
W Clade
M. arcana
M. argentea
M. cajuputi
M. clarksonii
M. dealbata
M. fluviatilis
M. leucadendra
rM. nervosa
M. quinquenervia
M. saligna
M. stenostachya
M. triumphalis
M. viridiflora Page 8 of 16
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Fig. 2. A. Sampling localities and statistical parsimony allelic network for the internal transcribed spacer (ITS; nrDNA) locus.
Localities and haplotypes are coloured by species, with circles being proportional to the number of individuals. Red arrow indicates
root (outgroups not shown). B. Distribution of LFY (nDNA) single-nucleotide polymorphisms (SNP: A (blue), C (red), or
polymorphic A/C (green)) across all individuals sampled. C. Statistical parsimony allelic network for psbA–rpl2 (cpDNA) coloured
by species, with the two main haplogroups being outlined in blue (W clade) and red (E clade). Red arrows indicate equally likely
rooting points. Sample localites are located on the map with membership to either E or W clade indicated by colour. The Great
Dividing Range is indicated with a dashed line.

Gene flow across biogeographic barriers

Estimates of genetic differentiation among populations separated by putative biogeographic barriers found low but
significant signals of isolation in nDNA but not cpDNA (Table 1), with one exception, namely, both genomes showed
very strong and significant differentiation among populations of M. argentea across the Great Sandy Desert (cpDNA: FST
= 1.00, P = 0.00; nDNA: FST = 0.22, P = 0.001).

Table 1. Analyses of Molecular Variance (AMOVAs) among populations of six species either side of putative
biogeographic barriers within the Australian monsoon tropics for cpDNA (psbA–rpl2) and nrDNA (internal
transcribed spacer, ITS)
Significant values are denoted by asterisks (*)

Species Barrier cpDNA nrDNA

Melaleuca Great Sandy Desert Fst = 1.00 Fst = 0.22314

P = 0.00* P = 0.00098*
Ord Basin Fst = 0.000 Fst = 0.01211
P = 1.000 P = 0.42326
Arnhem Land Plateau Fst = 0.13893 Fst = 0.07907
P = 0.12219 P = 0.00489*
Carpentaria Basin Fst = 0.09098 Fst = 0.02869
P = 0.23167 P = 0.05865
M. cajuputi Daly River Plains Fst = 0.00 Fst = 0.26987
P = 1.00 P = 0.05083
Normanby Basin Fst = –0.03860 Fst = 0.12941
P = 0.69697 P = 0.03226*
M. fluviatilis Townsville Lowlands Fst = 0.12308 Fst = 0.23292
P = 0.24242 P = 0.00098*
M. nervosa Townsville Lowlands Fst = 0.31313 Fst = 0.00710
P = 0.2434 P = 0.54350
M. stenostachya Normanby Basin Fst = –0.04281 Fst = –0.04348
P = 0.57283 P = 1.00
M. viridiflora Daly River Plains Fst = 0.00 Fst = –0.02909
P = 1.00 P = 0.74096
Normanby Basin Fst = 0.46400 Fst = 0.15036
P = 0.13196 P = 0.01662*

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There was no evidence for a significant reduction of admixture in the chloroplast; however, the cpDNA phylogeny did
recover two moderately supported but distinct clades not present in either sequenced nuclear locus (Fig. S1). This split
was even more apparent when presented as a network (Fig. 2C). This divergence showed a distinct geographic structure,
with haplotypes from one of the two clades (E clade) being sorted on the eastern half of Cape York Peninsula and south
down the eastern margin of the continent. Several species with ranges restricted to this area possessed haplotypes only
within this clade (i.e. Melaleuca arcana, M. clarksonii, M. quinquenervia, M. saligna and M. stenostachya). Structure was
also found in species that extend more broadly across the Australian monsoon tropics (AMT), namely M. cajuputi, M.
leucadendra, M. nervosa and M. viridiflora, M. dealbata and M. cajuputi, with individuals from these species possessing
E clade haplotypes in the east and haplotypes from W clade in populations to the west, with the exception of a scattering
of individuals with E clade haplotypes extending across the Gulf of Carpentaria into the north of the Northern Territory.
Although neither sequenced nuclear locus showed strong geographic structure, scoring of a single SNP from the LFY
nuclear locus found a pattern of east–west differentiation similar to that of the cpDNA, with individuals polymorphic for
the SNP being restricted to northern Cape York Peninsula and the south-east of the entire complex range (Fig. 2B).


Species boundaries
Our data tentatively supported recognised species boundaries across the M. leucadendra species complex (Craven and
Lepschi 1999), despite considerable reticulation and discordant chloroplast and nuclear signals. Patterns of haplotype
sharing between species are indicative of early (cpDNA) to middle (nDNA) stages of incomplete lineage sorting, rather
than of high rates of ongoing introgression (Omland et al. 2006), where large internal haplotypes represent retained shared
ancestry, and private external (putatively derived alleles) indicate recent divergence between species in the absence of
geneflow. Coalescence theory predicts that it can take several million years for newly isolated species to achieve full
reciprocal monophyly, and that haplotypes for a given locus will be intermixed within a network during this period. This
could be extended by the extremely large population sizes of these species, and long, multiply-overlapping, generations. A
scenario of differentiation with ILS fits the estimated recent timing for the emergence of the complex 3.5–5.5 million
years ago (Cook et al. 2008). Sharing and exclusion of haplotype clusters among certain species and groups of species
suggests a complex hierarchy of dissociation among species through time, with particular species groups appearing to
have maintained more recent connectivity (e.g. M. argentea, M. fluviatilis and M. leucadendra). Further work using many
loci and more sensitive analyses will be required to disentangle these relationships.

Divergence across putative barriers

Despite most species being more or less continuously distributed within their ranges across the study region, signals of
reduced gene flow were found for 5 of the 11 tests of differentiation between populations across recognised biogeographic
barriers. In all but one of these cases, significant differentiation was identified only in the nuclear data, being consistent
with slower mutation and coalescent rates in chloroplast markers (Birky et al. 1989; Galtier et al. 2009). The only instance
where both nDNA and cpDNA supported isolation was found between populations of M. argentea split across the Great

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Sandy Desert, which is one of few distinct physical gaps that exist within the distribution of a species within the complex.
This appears to be a case of allopatric divergence, with differentiation approaching between-species level (Hartl and Clark
1997; cpDNA: FST = 1.00, P = 0.00; nDNA: FST = 0.22, P = 0.001), and is consistent with lack of surface water limiting
not only the presence of individuals within this region, but the water-borne dispersal of seed. Morphological and
molecular work to better estimate the degree and age of differentiation among these populations, and subsequent
taxonomic treatment, is ongoing. Half of the remaining tests for differentiation identified isolation among populations,
despite apparently continuous distributions across barriers (i.e. cryptic divergence), with M. fluviatilis and M. viridiflora
both showing moderate reductions in gene flow across the Normanby Basin and the former also across the Townsville
Lowlands–Paluma Range barrier (Table 1). This suggests either past vicariance with subsequent re-expansion, or ongoing
filtering of gene flow by reduced pollen and seed dispersal across these areas. Porous barriers to gene flow fit best with
our findings of idiosyncratic responses to the same barriers by different members of the complex, and have been identified
in several arid-adapted birds across other barriers in the AMT (Jennings and Edwards 2005; Lee & Edwards, 2008; Toon
et al. 2010) but have not been previously recognised in plants in this biome. It should be noted that there are several
further putative biogeographic barriers within the AMT (Fig. 1B) that may also be contributing to reduced geneflow
between populations; however, further sampling is required to test these.

Deeper cryptic divergence

Although nuclear (ITS) data showed traces of reduced geneflow across several biogeographic barriers, this is not
evident in the cpDNA. Instead, networks for the psbA–rpl2 locus showed a single deep divergence separating two large
clades, considerably stronger than any signal of divergence across expected biogeographic barriers, or between putative
species. The split has a strong geographic signal with the separation of haplogroups east to west. Division runs
approximately down the Great Dividing Range from Cape York Peninsula and south along the east of the continent
(although a small number of outliers from the otherwise eastern clade are found to the west in the Northern Territory).
This pattern is supported by the segregation of LFY haplotypes (nDNA) along a similar east–west axis (Fig. 2B).
Although the Great Dividing Range (GDR) is not generally recognised as a significant barrier to dispersal (Ford 1986,
1987), divergence between populations of aquatic animals with a high fidelity to watersheds and drainage systems has
been suggested (McGlashan and Hughes 2001). Divergence between a pair of narrowly overlapping species within this
complex (M. argentea and M. fluviatilis) has previously been investigated, with differentiation in allopatry across the
GDR being implicated in their speciation, followed by secondary contact (Edwards et al. 2013); however, a similar signal
within the entire complex was not expected. Most notably, eastern haplogroups are not limited to species only found on
the eastern seaboard (i.e. M. arcana, M. clarksonii, M. quinquenervia, M. stenostachya and M. saligna), but are also
represented in species that span the entire AMT (i.e. M. cajuputi, M. dealbata, M. leucadendra, M. nervosa and M.
viridiflora). Geographic concordance of cpDNA divergence across all species is indicative of a concerted process such as
the presence of a physical barrier to dispersal either to a common ancestral population before species divergence, or across
multiple species subsequent to their separation. Differentiating between these two scenarios in a complex with widespread
ILS is challenging and requires further data. The lack of congruence between the full-sequence nDNA and cpDNA also

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suggests that there may be an asymmetrical response to physical isolation between the less mobile cpDNA, which is
dispersed by seed alone (resulting in a deep divergence), and more mobile nuclear genes, which are dispersed by both
seed and pollen (little signal of separation). This could have resulted in the isolation of the chloroplast genome, but with
maintenance of species identity by the uninterrupted flow of nuclear DNA. Alternatively, a historical barrier along the
GDR could have resulted in an east–west split in both nuclear and chloroplast genomes (explaining the restriction of
several species to the east and some signal of differentiation at the nuclear LFY locus). Subsequent relaxation of barriers
would have allowed re-homogenisation of both genomes, a process more rapidly reflected in the mobile nDNA, with
greater residual geographic signal being captured and preserved in the chloroplast genome, similar to ‘chloroplast
capture’, as has been documented in other members of the Myrtaceae family (Steane et al. 1998). Recent admixture could
explain the imperfect geographical division between the E and W clades, especially those members of the E clade present
in the central-north of the continent. An alternative explanation for these outliers is via land connections during glacial
maxima linking Cape York Peninsula, New Guinea and the Northern Territory (van der Kaars 1991; Schidelko et al.
2012), which, in theory, could allow dispersal to skirt the northern end of the GDR. However, evidence to date suggests
that even with the exposure of potential land bridges across the Arafura Shelf, unsuitable habitat and the presence of Lake
Carpentaria has continued to restrict dispersal of species across this region (Kearns et al. 2011).

Our data confirmed complicated signals of reticulation across all species in the M. leucadendra group, with patterns of
genetic differentiation being more consistent with the early to middle stages of ILS than with geneflow by hybridisation.
Straightforward interpretation is complicated by underlying signals of geographic isolation within and among species,
which are differentially reflected in genomes with contrasting mobility. Nuclear and chloroplast sequence data, as well as
SNPs, all show utility in exploring both species boundaries and biogeographic history at different levels. Although
traditional population-genetic methods such as microsatellites typically provide greater sensitivity for identifying recent
divergence events, untangling the likely nested evolutionary history of this group will be best tackled using large numbers
of sequenced loci. Whole genome and transcriptome resources exist for Melaleuca and Eucalyptus L’Herit., with a library
of several hundred loci, which are informative across a range of phylogenetic depths, currently in development (Choi et
al., in review). Our results also highlighted that cryptic signatures of reduced geneflow exist even within seemingly large
and uniform species’ distributions, and may hold important information for reconstructing the biogeographic history of
the continent.

Conflicts of interest
The authors declare that they have no conflicts of interest.

We thank the late Lyn Craven for access to herbarium material and inspiration, and Anna Kearns and other members of the Crisp and
Cook laboratories for valuable feedback and support. Plant material was collected under permits held by Mike Crisp. Robert Edwards.

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was supported by a Postgraduate Research Scholarship from The University of Queensland and Lyn Cook and Mike Crisp were
supported by Australian Research Council Discovery Grants


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