DOI: 10.5061/dryad.905qfttjz
title: Major biogeographic barriers in eastern Australia have shaped population structure of widely distributed Eucalyptus moluccana and its four putative subspecies

description: We have investigated the impact of recognized biogeographic barriers on
 genetic differentiation of grey box (Eucalyptus moluccana), a common and
 widespread tree species of the family Myrtaceae in eastern Australian
 woodlands, and its previously proposed four subspecies moluccana,
 pedicellata, queenslandica and crassifolia. A range of phylogeographic
 analyses were conducted to examine the population genetic differentiation
 and subspecies genetic structure in E. moluccana in relation to
 biogeographic barriers. Slow evolving markers uncovering long term
 processes (chloroplast DNA) were used to generate a haplotype network and
 infer phylogeographic barriers. Additionally, fast evolving, hypervariable
 markers (microsatellites) were used to estimate demographic processes and
 genetic structure among five geographic regions (29 populations) across
 the entire distribution of E. moluccana. Morphological features of
 seedlings, such as leaf and stem traits were assessed to evaluate
 population clusters and test differentiation of the putative subspecies.
 Haplotype network analysis revealed twenty chloroplast haplotypes with a
 main haplotype in a central position shared by individuals belonging to
 the regions containing the four putative subspecies. Microsatellite
 analysis detected genetic structure between Queensland (QLD) and New South
 Wales (NSW) populations consistent with the McPherson Range barrier, an
 east-west spur of the Great Dividing Range. Substructure was detected
 within QLD and NSW in line with other barriers in eastern Australia. The
 morphological analyses supported differentiation between QLD and NSW
 populations, with no difference within QLD, yet some differentiation
 within NSW populations. Our molecular and morphological analyses provide
 evidence that several geographic barriers in eastern Australia, including
 the Burdekin Gap and the McPherson Range have contributed to the genetic
 structure of E. moluccana. Genetic differentiation among E. moluccana
 populations supports the recognition of some but not all the four
 previously proposed subspecies, with crassifolia being the most
 differentiated.
description: This dataset contains psbA-trnH sequences of the chloroplast of
 several eucalypts. The
 intergenic region <i>psbA-trnH</i> is among the most variable
 regions of the chloroplast in <i>E. moluccana</i>
 (Flores-Renteria <i>et al</i>. 2017). Sequences for this
 chloroplast intergenic region were obtained using the methods of
 Flores-Renteria <i>et al</i>. (2017). Additionally, sequences
 from a subset of seedlings already available were included
 (accession numbers KY596186 – KY596665). DNA of the
 chloroplast sequences were aligned using the multiple progressive
 alignment procedure of MUSCLE (Edgar, 2004), and manual corrections were
 done around indels or microsatellites. Minimum spanning haplotype
 networks (Bandelt <i>et al</i>., 1999) were constructed using
 the program PopART (Leigh and Bryant, 2015) to better
 visualize non-bifurcating relationships (multifurcations and
 reticulations) in chloroplast haplotypes (Posada and Crandall, 2001).
 DNAsp software was used to create the haplotype data file (Librado and
 Rozas, 2009). PopART software was used to infer the haplotype network by
 region (http://popart.otago.ac.nz).
 A second file
 contains the dataset of nuclear microsatellites. Ten
 microsatellite loci of <i>E. moluccana </i>were amplified in a
 multiplex design<i>. </i>Each specific forward primer was
 linked with one of the 5′ universal primer sequence tails and
 fluorescence-labeled (Table S2, Flores-Renteria <i>et
 al</i>. 2011, 2013). PCR reactions were carried out
 using QIAGEN Multiplex PCR Kit following manufacturer’s instructions, and
 amplicons were sized as in Flores-Renteria and Krohn
 (2013).