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

Morphological and molecular evidence supports specific recognition of the recently extinct Bettongia anhydra (Marsupialia: Macropodidae)

In 1933, geologist and explorer Michael Terry collected the skull of a small macropodid captured by members of his party near Lake Mackay, western Northern Territory. In 1957, this skull was described as the sole exemplar of a distinct subspecies, Bettongia penicillata anhydra, but was later synonymized with B. lesueur and thereafter all but forgotten. We use a combination of craniodental morphology and ancient mitochondrial DNA to confirm that the Lake Mackay specimen is taxonomically distinct from all other species of Bettongia and recognize an additional specimen from a Western Australian Holocene fossil accumulation. B. anhydra is morphologically and genetically most similar to B. lesueur but differs in premolar shape, rostrum length, dentary proportions, and molar size gradient. In addition, it has a substantial mitochondrial cytochrome b pairwise distance of 9.6–12% relative to all other bettongs. The elevation of this recently extinct bettong to species status indicates that Australia’s mammal extinction record over the past 2 centuries is even worse than currently accepted. Like other bettongs, B. anhydra probably excavated much of its food and may have performed valuable ecological services that improved soil structure and water infiltration and retention, as well as playing an important role in the dispersal of seeds and mycorrhizal fungal spores. All extant species of Bettongia have experienced extensive range contractions since European colonization and some now persist only on island refugia. The near total loss of these ecosystem engineers from the Australian landscape has far-reaching ecological implications

Genetic diversity loss in a biodiversity hotspot: ancient DNA quantifies genetic decline and former connectivity in a critically endangered marsupial

The extent of genetic diversity loss and former connectivity between fragmented populations are often unknown factors when studying endangered species. While genetic techniques are commonly applied in extant populations to assess temporal and spatial demographic changes, it is no substitute for directly measuring past diversity using ancient DNA (aDNA). We analysed both mitochondrial DNA (mtDNA) and nuclear microsatellite loci from 64 historical fossil and skin samples of the critically endangered Western Australian woylie (Bettongia penicillata ogilbyi), and compared them with 231 (n = 152 for mtDNA) modern samples. In modern woylie populations 15 mitochondrial control region (CR) haplotypes were identified. Interestingly, mtDNA CR data from only 29 historical samples demonstrated 15 previously unknown haplotypes and detected an extinct divergent clade. Through modelling, we estimated the loss of CR mtDNA diversity to be between 46% and 91% and estimated this to have occurred in the past 2000-4000 years in association with a dramatic population decline. In addition, we obtained near-complete 11-loci microsatellite profiles from 21 historical samples. In agreement with the mtDNA data, a number of 'new' microsatellite alleles was only detected in the historical populations despite extensive modern sampling, indicating a nuclear genetic diversity loss >20%. Calculations of genetic diversity (heterozygosity and allelic rarefaction) showed that these were significantly higher in the past and that there was a high degree of gene flow across the woylie's historical range. These findings have an immediate impact on how the extant populations are managed and we recommend the implementation of an assisted migration programme to prevent further loss of genetic diversity. Our study demonstrates the value of integrating aDNA data into current-day conservation strategies

The taxonomy, phylogeny and distribution of lampreys

The lampreys (Petromyzontiformes), one of the two surviving groups of agnathan (jawless) vertebrates, currently consist of 41 recognized species. This group has an antitropical distribution, with the 37 species of Northern Hemisphere lampreys assigned to the Petromyzontidae, whereas the four species of Southern Hemisphere lampreys are separated into either the Geotriidae (one species) or Mordaciidae (three species). All lamprey species have a blind and microphagous, burrowing larva (ammocoete), which spends a number of years in the soft sediment of creeks and rivers, after which it undergoes a radical metamorphosis. Eighteen lamprey species then embark on an adult parasitic phase (nine at sea and nine in fresh water) during which they increase markedly in size, whereas the other 23 species do not feed as adults and remain in fresh water. On the basis of morphology, 17 of the 23 non-parasitic species each evolved from a particular parasitic species whose descendants are still represented in the contemporary fauna. The remaining six non-parasitic species, the so-called “southern relict” species, have no obvious potential ancestral parasitic species, implying they have diverged markedly from their parasitic ancestor or that the parasitic ancestor is now extinct. Many of the main taxonomic characteristics reside in features that are associated with parasitic feeding, for example, the type and arrangement of the teeth on the suctorial disc and tongue-like piston. The phylogenetic relationships, derived by maximum parsimony analyses of morphological and anatomical data for the 18 parasitic species, were similar in most respects to those obtained by subjecting molecular data (cytochrome b mitochondrial DNA sequence data) for those species to Bayesian analyses. However, in contrast to the results of morphological analyses, the genera Eudontomyzon and Lampetra were not monophyletic when using molecular analyses. When non-parasitic species were included in the molecular analyses, some of the six relict non-parasitic species formed clades with parasitic species which, from their morphology, had been allocated by taxonomists to different genera. More genes, and particularly nuclear genes, should be used to help resolve the basis for these differences between the morphological and molecular phylogenies

Scrapheap Challenge: A novel bulk-bone metabarcoding method to investigate ancient DNA in faunal assemblages

Highly fragmented and morphologically indistinct fossil bone is common in archaeological and paleontological deposits but unfortunately it is of little use in compiling faunal assemblages. The development of a cost-effective methodology to taxonomically identify bulk bone is therefore a key challenge. Here, an ancient DNA methodology using high-throughput sequencing is developed to survey and analyse thousands of archaeological bones from southwest Australia. Fossils were collectively ground together depending on which of fifteen stratigraphical layers they were excavated from. By generating fifteen synthetic blends of bulk bone powder, each corresponding to a chronologically distinct layer, samples could be collectively analysed in an efficient manner. A diverse range of taxa, including endemic, extirpated and hitherto unrecorded taxa, dating back to c.46,000 years BP was characterized. The method is a novel, cost-effective use for unidentifiable bone fragments and a powerful molecular tool for surveying fossils that otherwise end up on the taxonomic “scrapheap”

Ancient DNA reveals complexity in the evolutionary history and taxonomy of the endangered Australian brush-tailed bettongs (Bettongia: Marsupialia: Macropodidae: Potoroinae)

The three surviving ‘brush-tailed’ bettong species—Bettongia gaimardi (Tasmania), B. tropica (Queensland) and B. penicillata (Western Australia), are all classified as threatened or endangered. These macropodids are prolific diggers and are recognised as important ‘ecosystem engineers’ that improve soil quality and increase seed germination success. However, a combination of introduced predators, habitat loss and disease has seen populations become increasingly fragmented and census numbers decline. Robust phylogenies are vital to conservation management, but the extent of extirpation and fragmentation in brush-tailed bettongs is such that a phylogeny based upon modern samples alone may provide a misleading picture of former connectivity, genetic diversity and species boundaries. Using ancient DNA isolated from fossil bones and museum skins, we genotyped two mitochondrial DNA (mtDNA) genes: cytochrome b (266 bp) and control region (356 bp). These ancient DNA data were combined with a pre-existing modern DNA data set on the historically broadly distributed brush-tailed bettongs (~300 samples total), to investigate their phylogenetic relationships. Molecular dating estimates the most recent common ancestor of these bettongs occurred c. 2.5 Ma (million years ago), which suggests that increasing aridity likely shaped their modern-day distribution. Analyses of the concatenated mtDNA sequences of all brush-tailed bettongs generated five distinct and well-supported clades including: a highly divergent Nullarbor form (Clade I), B. tropica (Clade II), B. penicillata (Clades III and V), and B. gaimardi (Clade IV). The generated phylogeny does not reflect current taxonomy and the question remains outstanding of whether the brush-tailed bettongs consisted of several species, or a single widespread species. The use of nuclear DNA markers (single nucleotide polymorphisms and/or short tandem repeats) will be needed to better inform decisions about historical connectivity and the appropriateness of ongoing conservation measures such as translocations and captive breeding

The identity of the Depuch Island rock-wallaby revealed through ancient DNA

Ancient DNA is becoming increasingly recognised as a tool in conservation biology to audit past biodiversity. The widespread loss of Australian biodiversity, especially endemic mammal populations, is of critical concern. An extreme example occurred on Depuch Island, situated off the north-west coast of Western Australia, where an unidentified species of rock-wallaby (Petrogale sp.) became extinct as a result of predation by red foxes. Two potential candidate species, Petrogale lateralis and P. rothschildi, both have ranges adjacent to Depuch Island, making identification based on geography difficult. A museum bone (one of the only surviving Depuch Island specimens) was subjected to standard ancient DNA analyses and procedures. Mitochondrial DNA cytochrome b and hypervariable control region were targeted for species identification. Ancient DNA was successfully recovered from the bone: 200 base pairs (bp) of control region and 975bp of the cytochrome b gene. Bayesian phylogenetic analyses were employed to model the Depuch Island rock-wallaby DNA sequences together with sequences of other rock-wallaby taxa from GenBank. Evidence suggests that of the two Petrogale lateralis subspecies proposed to have inhabited Depuch Island, Petrogale lateralis lateralis was identified as the most likely. The identification of the Depuch Island rock-wallaby population may assist in the reintroduction of an insurance population of Petrogale lateralis lateralis, which is becoming increasingly threatened on mainland Australia

Inferring Kangaroo Phylogeny from Incongruent Nuclearand 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 razing/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