Inferring Kangaroo Phylogeny from Incongruent Nuclear and Mitochondrial Genes

The marsupial genus Macropus includes three subgenera, the familiar large grazing kangaroos and wallaroos of M. (Macropus) and M. (Osphranter), as well as the smaller mixed grazing/browsing wallabies of M. (Notamacropus). A recent study of five concatenated nuclear genes recommended subsuming the predominantly browsing Wallabia bicolor (swamp wallaby) into Macropus. To further examine this proposal we sequenced partial mitochondrial genomes for kangaroos and wallabies. These sequences strongly favour the morphological placement of W. bicolor as sister to Macropus, although place M. irma (black-gloved wallaby) within M. (Osphranter) rather than as expected, with M. (Notamacropus). Species tree estimation from separately analysed mitochondrial and nuclear genes favours retaining Macropus and Wallabia as separate genera. A simulation study finds that incomplete lineage sorting among nuclear genes is a plausible explanation for incongruence with the mitochondrial placement of W. bicolor, while mitochondrial introgression from a wallaroo into M. irma is the deepest such event identified in marsupials. Similar such coalescent simulations for interpreting gene tree conflicts will increase in both relevance and statistical power as species-level phylogenetics enters the genomic age. Ecological considerations in turn, hint at a role for selection in accelerating the fixation of introgressed or incompletely sorted loci. More generally the inclusion of the mitochondrial sequences substantially enhanced phylogenetic resolution. However, we caution that the evolutionary dynamics that enhance mitochondria as speciation indicators in the presence of incomplete lineage sorting may also render them especially susceptible to introgression.This work has been supported by Australian Research Council grants to MJP (DP07745015) and MB (FT0991741). The website for the funder is www.arc.gov.au. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Thorough assessment of DNA preservation from fossil bone and sediments excavated from a late Pleistocenee-Holocene cave deposit on Kangaroo Island, South Australia

Fossils and sediments preserved in caves are an excellent source of information for investigating impacts of past environmental changes on biodiversity. Until recently studies have relied on morphology-based palaeontological approaches, but recent advances in molecular analytical methods offer excellent potential for extracting a greater array of biological information from these sites. This study presents a thorough assessment of DNA preservation from late Pleistocene-Holocene vertebrate fossils and sediments from Kelly Hill Cave Kangaroo Island, South Australia. Using a combination of extraction techniques and sequencing technologies, ancient DNA was characterised from over 70 bones and 20 sediment samples from 15 stratigraphic layers ranging in age from >20 ka to ~6.8 ka. A combination of primers targeting marsupial and placental mammals, reptiles and two universal plant primers were used to reveal genetic biodiversity for comparison with the mainland and with the morphological fossil record for Kelly Hill Cave. We demonstrate that Kelly Hill Cave has excellent long-term DNA preservation, back to at least 20 ka. This contrasts with the majority of Australian cave sites thus far explored for ancient DNA preservation, and highlights the great promise Kangaroo Island caves hold for yielding the hitherto-elusive DNA of extinct Australian Pleistocene species

Using ancient DNA to investigate extinction, extirpation and past biodiversity of Australian macropods

The field of ancient DNA (aDNA) involves the isolation and retrieval of trace amounts of degraded DNA from a variety of substrates including fossils, sediments and historical material. The fragmentary nature of aDNA necessitates the use of methods with the ability to capture and amplify short segments of DNA. Collectively aDNA studies have made significant and unique contributions to a wide field of research including conservation, population genetics, taxonomy and phylogeny. The primary aim of this thesis research is to explore the utility of aDNA techniques to study extirpation, extinction and past biodiversity of Australian macropods. Using a combination of historical, Holocene and Pleistocene aged fossils, this research will attempt to investigate what ancient mitochondrial DNA (mtDNA) can add to our knowledge of Australia’s macropods. Traditional aDNA techniques have largely been used to isolate mtDNA from single fossil samples - an example of this approach is shown in Chapter Two where a wellpreserved wallaby fossil bone from Depuch Island (Western Australia) was studied. The ancient mtDNA (cytochrome b and control region) data produced strong phylogenetic signal and shows that the Depuch Island rock-wallaby specimen is most similar to the mainland Petrogale lateralis lateralis. This finding has conservation implications for ongoing rehabilitation and translocation efforts in the Pilbara region. Chapter Three of this thesis also uses mitochondrial aDNA techniques, to explore questions regarding interrelationships and former distribution of a macropod species complex; Bettongia spp. Cytochrome b and control region data retrieved from 88 historical samples, along with ~214 already sequenced samples, place the most recent common ancestor of the brush-tailed bettongs at c. 2.5 Myr. Ancient mtDNA is suggestive of connectivity between what are now highly fragmented populations, a result that has implications for how critically endangered brush-tailed bettongs should be managed. Ancient DNA analyses and DNA sequencing technology have evolved over recent years and during the course of this study. Therefore in keeping up with the latest high-throughput sequencing (HTS) technology, aDNA analyses in ~70 bones and 20 sediment samples excavated from a Late Pleistocene–Holocene cave deposit on Kangaroo Island, South Australia was undertaken. Samples were selected from 15 stratigraphic layers, ranging in age from >20 ka to ~6.8 ka. The successful retrieval of bona fide aDNA sequences, back to at least 20 ka, demonstrates excellent longterm DNA preservation at the site. All unidentified bones that were screened revealed a number of taxa from the assemblage including, Macropus, Onychogalea, Potorous, Bettongia, Dasyurus, Rattus and Notechis. The results from this study add significant value to the late Pleistocene-mid-Holocene paleontological record, detailing the past diversity of flora and fauna on Kangaroo Island. Lastly, Chapter 5 introduces the latest molecular techniques in capturing and enriching highly fragmented aDNA bone from four sites across Australia. Ancient DNA extractions techniques, targeting ultra-short DNA fragments, were employed in an attempt to obtain Pleistocene-aged material. The warm conditions, a factor common in Australian caves, are not conductive to long-term DNA preservation at many sites. Shotgun sequencing was only successful on six bone samples (including one incisor) from a total of 25 samples that were screened. Three samples were successfully captured and enriched for endogenous DNA; one bettong sample generated 89.6% of a mtDNA genome with 5.4X coverage. Overall, the decay rate of DNA and preservation across all four sites was high, and extremely degraded, with an average fragment length between 47 bp and 57 bp. These data demonstrate that recovery of Pleistocene-aged aDNA from warm climate sites across Australia will remain a challenge and that better ways to screen and predict DNA survival are needed. This thesis presents a combination of work from multiple sites across Australia using a range of aDNA techniques and sequencing technologies that have evolved over the tenure of this thesis. Collectively, this body of work has demonstrated the value of integrating aDNA data into modern-day conservation decision-making and has contributed to a wider understanding of Australian macropods both past and present

Individual nuclear gene –ln<i>L</i> differences and SH test results.

<p>ML placements in bold.</p><p>To ensure relevance of the individual gene results to the overall nuclear phylogeny, the relative positions of the outgroups and placements within <i>M. (Macropus)</i>, <i>M. (Osphranter)</i> and core-<i>Notamacropus</i> were fixed (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057745#pone-0057745-g002" target="_blank">Figure 2</a>).</p

Macropodoid divergence time estimates in millions of years before present.

<p>(A) BEAST analysis of Mt₁₆, (B) average of four BEAST analyses on the five nuclear gene concatenate from Meredith et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057745#pone.0057745-Meredith1" target="_blank">[7]</a> and (C) *BEAST species tree analysis of MtNuc₁₆.</p

Macropodid clade support from datasets simulated under coalescence.

<p>(A) Simulation workflow. (B) Mean number of the five nuclear genes supporting each clade in maximum likelihood analyses of 200 simulations of the combined data *BEAST species tree for <i>N</i>_e values of 1,000 (triangle), 10,000 (open circle), 100,000 (square) and 1,000,000 (filled circle). For comparison, the grey bars show the number of genes supporting each clade on the observed data. (C) Percentage of ML analyses supporting each clade among 200 mtDNA simulations on the nuclear-only *BEAST species tree for <i>N</i>_e values set to mitochondrial equivalency for the same populations (one quarter of the corresponding nuclear values). Abbreviations: <i>Lagor</i>.; <i>Lagorchestes</i>, <i>Wall</i>.; <i>Wallabia</i>, <i>M. (Notamac.)</i>; <i>M. (Notamacropus)</i>, <i>M. (Osphran.)</i>; <i>M. (Osphranter)</i>.</p

Approximately unbiased (AU) test results.

<p>Nuclear sequences are partitioned into protein codon positions and mitochondrial sequences are partitioned into protein codon positions and RNA stems and loops.</p><p>Comparisons (A) – (D) employ Mt₁₆ and Nuc₁₇. Comparison (E) employs Mt₁₇.</p> ˆ<p>Allowing <i>W. bicolor</i> and <i>M. irma</i> to float unconstrained on the tree</p>#<p>Not including <i>M. irma</i>, which is favoured as sister to <i>M. robustus</i> on the mt data.</p

Phylogenetic relationships of <i>Wallabia</i> and the three <i>Macropus</i> subgenera, <i>M. (Macropus)</i>, <i>M. (Osphranter)</i> and <i>M. (Notamacropus)</i>.

<p>(A) The supertree of Cardillo et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057745#pone.0057745-Cardillo1" target="_blank">[6]</a> summarizing previous molecular and morphological phylogenies and (B) Meredith et al.'s <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057745#pone.0057745-Meredith1" target="_blank">[7]</a> evolutionary timescale (ave. of four BEAST analyses), showing the 2–2.4 Ma duration divergence cluster. Both trees are modified to include only the taxa sampled in the present study. Dendrolagini was not recovered by Cardillo et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057745#pone.0057745-Cardillo1" target="_blank">[6]</a>, however its inclusion in the summary tree is warranted on subsequent strong evidence from morphology <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057745#pone.0057745-Prideaux1" target="_blank">[2]</a> and all recent molecular analyses. Photos include (from the top) <i>W. bicolor</i>, <i>M. rufogriseus</i> (left), <i>M. irma</i> (right), <i>M. rufus and M. giganteus</i>. Photo credits – Matt Phillips, except <i>M. irma</i> (Ric Dawson) and <i>M. rufus</i> (Daniel Hoops).</p

Phylogenetic analysis of kangaroos and wallabies.

<p>Maximum likelihood phylogenies inferred from the (A) mitochondrial (Mt₁₆) and (B) nuclear (Nuc₁₇) concatenated datasets, with RAxML bootstrap values (BP_ML) above branches and MrBayes Bayesian posterior probabilities (BPP) below branches. The mt placement of <i>M. dorsalis</i> is derived from the reduced-length Mt₁₇ and the mt placements of <i>M. antilopinus</i> and <i>W. bicolor</i> (NSW, New South Wales) are derived from the <i>Cytb</i>₁₈ alignment. Support for grouping <i>M. eugenii</i> and <i>M. agilis</i> increases (BP_ML = 88; BPP = 0.98) for Mt₁₆, which excludes <i>M. dorsalis</i>, but increases sequence length. Asterisks indicate full support. Clades including members of <i>Macropus</i> are shaded.</p

A molecular and morphometric assessment of the systematics of the Macropus complex clarifies the tempo and mode of kangaroo evolution

Kangaroos and wallabies of the Macropus complex include the largest extant marsupials and hopping mammals. They have traditionally been divided among the genus Macropus (with three subgenera: Macropus, Osphranter and Notamacropus) and the monotypic swamp wallaby, Wallabia bicolor. Recent retrotransposon and genome-scale phylogenetic analyses clarify the placement of Wallabia as sister to Notamacropus, with Osphranter and Macropus branching successively deeper. In view of the traditional Macropus concept being paraphyletic, we undertake to resolve the species-level phylogeny and genus-level taxonomy of the Macropus complex. For the first time, we include nuclear and mitochondrial DNA covering all extant species, and the first DNA sequences from the extinct Toolache wallaby (Notamacropus greyi), which we find groups with the black-gloved wallaby (Notamacropus irma). Morphological variation was examined using geometric morphometric methods on three-dimensional surface-scanned skulls. Wallabia skull shape fell close to Notamacropus (or Thylogale when controlling for allometry). We recommend the subgenera Macropus, Osphranter and Notamacropus be elevated to genera, alongside Wallabia, based on comparisons with other established macropodine genera for cranial disparity, ecology and molecular divergence. Our time tree estimates that all four ‘Macropus’ genera diverged close to the Miocene–Pliocene boundary (~6–5 Mya), then diversified coincident with Pliocene expansion of grasslands in Australia