DNA phylogeny of the marsupial wolf resolved.

The phylogenetic position of the recently extinct marsupial ''wolf'', or thylacine (Thylacinus cynocephalus), has been a source of contention in mammalian systematics for nearly a century. Thylacines were endemic to Australasia, but possessed striking anatomical similarities to Oligo-Miocene borhyaenid marsupials of South America. At issue has been whether these features are indicative of common ancestry or convergent adaptation to carnivory. Recent morphological studies have supported both conclusions. Although current marsupial classifications group thylacines with Australian dasyuromorphians, this putative clade is characterized by mostly primitive morphological features. Attempts to determine thylacine affinities with ancient protein and DNA analyses have supported, but not resolved, a dasyuromorphian placement. We report 1546 bp of mitochondrial DNA sequence (from cytochrome b and 12S rRNA genes) and 841 bp of nuclear protamine gene sequence from the thylacine and representatives of all or most other marsupial orders. Phylogenetic analysis of these sequences shows unambiguously that thylacines are members of Dasyuromorphia, and suggests a late Oligocene or very early Miocene divergence of familial lineages.     

DNA Sequence Analysis of Familial Relationships Among Dasyuromorphian Marsupials

Although modern morphological and molecular analyses support the monophyly of the Australasian marsupial order Dasyuromorphia, there is much less certainty about relationships among its constituent families (Dasyuridae, Myrmecobiidae, and Thylacinidae). While most authors regard Dasyuridae as monophyletic, a few have suggested that thylacines, numbats, or both have their closest relatives among dasyurids. Recent morphocladistic studies have identified several basicranial characters as putative synapomorphies of dasyurids, but no features that clearly implicate thylacinids, myrmecobiids, or both, as the sister group of Dasyuridae. Only two previous DNA studies have included both thylacine and numbat sequences along with dasyurids, and neither provided strong resolution of interfamilial relationships. In this study, we report a more thorough analytical treatment of complete cytochrome b, 12S rRNA, and protamine P1 gene sequences from dasyuromorphians than has heretofore been attempted. Our results concur with previous morphological studies in showing that Dasyuridae is monophyletic and with immunological findings that thylacinids and dasyurids are sister groups, apart from myrmecobiids. However, the level of support for nodes is highly dependent on the method of phylogenetic analysis employed. Our results also suggest that partitioning of sequence data sets to account for substitutional heterogeneity within and among genes does not necessarily lead to a major reduction in the precision of estimated phylogenies.     

Immunological Insights into the Life and Times of the Extinct Tasmanian Tiger (Thylacinus cynocephalus)

The thylacine (Thylacinus cynocephalus) was Australia’s largest marsupial carnivore until its extinction within the last century. There remains considerable interest and debate regarding the biology of this species. Studies of thylacine biology are now limited to preserved specimens, and parts thereof, as well as written historical accounts of its biology. This study describes the development of the immune tissues of a pouch young thylacine, one of only eleven in existence, and the only specimen to be histologically sectioned. The appearance of the immune tissue of the developing pouch young thylacine is compared to the immune tissues of extant marsupials, providing insights into the immunity, biology and ecology of the extinct thylacine.     

Computer simulation of feeding behaviour in the thylacine and dingo as a novel test for convergence and niche overlap

The extinct marsupial thylacine (Thylacinus cynocephalus) and placental grey wolf (Canis lupus) are commonly presented as an iconic example of convergence. However, various analyses suggest distinctly different behaviours and specialization towards either relatively small or large prey in the thylacine, bringing the degree of apparent convergence into question. Here we apply a powerful engineering tool, three-dimensional finite element analysis incorporating multiple material properties for bone, to examine mechanical similarity and niche overlap in the thylacine and the wolf subspecies implicated in its extinction from mainland Australia, Canis lupus dingo. Comparisons of stress distributions not only reveal considerable similarity, but also informative differences. The thylacine's mandible performs relatively poorly where only the actions of the jaw muscles are considered, although this must be considered in the light of relatively high bite forces. Stresses are high in the posterior of the thylacine's cranium under loads that simulate struggling prey. We conclude that relative prey size may have been comparable where both species acted as solitary predators, but that the dingo is better adapted to withstand the high extrinsic loads likely to accompany social hunting of relatively large prey. It is probable that there was considerable ecological overlap. As a large mammalian hypercarnivore adapted to taking small-medium sized prey, the thylacine may have been particularly vulnerable to disturbance.     

Limited genetic diversity preceded extinction of the Tasmanian tiger

The Tasmanian tiger or thylacine was the largest carnivorous marsupial when Europeans first reached Australia. Sadly, the last known thylacine died in captivity in 1936. A recent analysis of the genome of the closely related and extant Tasmanian devil demonstrated limited genetic diversity between individuals. While a similar lack of diversity has been reported for the thylacine, this analysis was based on just two individuals. Here we report the sequencing of an additional 12 museum-archived specimens collected between 102 and 159 years ago. We examined a portion of the mitochondrial DNA hyper-variable control region and determined that all sequences were on average 99.5% identical at the nucleotide level. As a measure of accuracy we also sequenced mitochondrial DNA from a mother and two offspring. As expected, these samples were found to be 100% identical, validating our methods. We also used 454 sequencing to reconstruct 2.1 kilobases of the mitochondrial genome, which shared 99.91% identity with the two complete thylacine mitochondrial genomes published previously. Our thylacine genomic data also contained three highly divergent putative nuclear mitochondrial sequences, which grouped phylogenetically with the published thylacine mitochondrial homologs but contained 100-fold more polymorphisms than the conserved fragments. Together, our data suggest that the thylacine population in Tasmania had limited genetic diversity prior to its extinction, possibly as a result of their geographic isolation from mainland Australia approximately 10,000 years ago.     

Molecular evidence for the pattern and timing of cladogenesis in dasyurid marsupials

Recent molecular studies have provided estimates of phylogeny for nearly all living and recently extinct species in the Order Dasyuromorphia, the dominant clade of insectivorous‐carnivorous marsupials in Australasia. We review these studies along with morphology‐based ones, and present an analysis of all cytochrome b, 12S rRNA, and protamine Pl gene sequences available. In light of these results, we provide a revised suprageneric classification and assess the implications of molecular and paleontological data for dasyurid cladogenesis. Molecular results divide extant dasyurids (Dasyuridae) into four major clades apart from the numbat (Myrmecobiidae) and thylacines (Thylacinidae). We recognize these clades as tribes Dasyurini (Dasyurus, Phascolosorex, and allied genera) and Phascogalini (Antechinus, Murexia, Phascogale) in the Subfamily Dasyurinae, and tribes Sminthopsini (Sminthopsis, Ningaui, Antechinomys) and Planigalini (Planigale) in the Subfamily Sminthopsinae. Each tribe shows a basal radiation of lineages corresponding to genera or species groups. Our results concur with the most recent previous synthesis of dasyurid phylogeny in many respects, but subsumption of New Guinean ‘phascolosoricines’ and ‘muricines’ within Dasyurini and Phascogalini, respectively, constitute significant differences. In particular, the sister‐pairing of ‘phascolosoricines’ with a Dasyurus‐Sarcophilus clade implied by molecular data is difficult to reconcile with anatomy. Divergence rates of mitochondrial sequences are calibrated approximately by comparing thylacine‐to‐dasyurid distances with the age of the oldest thylacinid (Badjcinus, latest Oligocene). Estimated cladogenic dates suggest that extant subfamilies shared a common ancestor around 24 Mya and that major radiations began late in the mid‐Miocene, consistent with the results of previous paleontological studies. The late‐middle and late Miocene corresponds to an episode of faunal turnover in Australian marsupials (including the decline of thylacinid and bandicoot genera, as well as the rise of dasyurids) and to a time when uplift of the New Guinean highlands accelerated the transition from rainforest to drier habitats. Our findings are consistent with the hypothesis that continent‐wide climate changes modulated macroevolution across these independent marsupial clades.     

The predatory behaviour of the thylacine: Tasmanian tiger or marsupial wolf?

The extinct thylacine (Thylacinus cynocephalus) and the extant grey wolf (Canis lupus) are textbook examples of convergence between marsupials and placentals. Craniodental studies confirm the thylacine's carnivorous diet, but little attention has been paid to its postcranial skeleton, which would confirm or refute rare eyewitness reports of a more ambushing predatory mode than the pack-hunting pursuit mode of wolves and other large canids. Here we show that thylacines had the elbow morphology typical of an ambush predator, and propose that the 'Tasmanian tiger' vernacular name might be more apt than the 'marsupial wolf'. The 'niche overlap hypothesis' with dingoes (Canis lupus dingo) as a main cause of thylacine extinction in mainland Australia is discussed in the light of this new information.     

Bite club: comparative bite force in big biting mammals and the prediction of predatory behaviour in fossil taxa

We provide the first predictions of bite force (BS) in a wide sample of living and fossil mammalian predators. To compare between taxa, we calculated an estimated bite force quotient (BFQ) as the residual of BS regressed on body mass. Estimated BS adjusted for body mass was higher for marsupials than placentals and the Tasmanian devil (Sarcophilus harrisii) had the highest relative BS among extant taxa. The highest overall BS was in two extinct marsupial lions. BFQ in hyaenas were similar to those of related, non-osteophagous taxa challenging the common assumption that osteophagy necessitates extreme jaw muscle forces. High BFQ in living carnivores was associated with greater maximal prey size and hypercarnivory. For fossil taxa anatomically similar to living relatives, BFQ can be directly compared, and high values in the dire wolf (Canis dirus) and thylacine (Thylacinus cynocephalus) suggest that they took relatively large prey. Direct inference may not be appropriate where morphologies depart widely from biomechanical models evident in living predators and must be considered together with evidence from other morphological indicators. Relatively low BFQ values in two extinct carnivores with morphologies not represented among extant species, the sabrecat, Smilodon fatalis, and marsupial sabretooth, Thylacosmilus atrox, support arguments that their killing techniques also differed from extant species and are consistent with 'canine-shear bite' and 'stabbing' models, respectively. Extremely high BFQ in the marsupial lion, Thylacoleo carnifex, indicates that it filled a large-prey hunting niche.     

Virtual Reconstruction and Prey Size Preference in the Mid Cenozoic Thylacinid,Nimbacinus dicksoni (Thylacinidae,Marsupialia)

Thylacinidae is an extinct family of Australian and New Guinean marsupial carnivores, comprizing 12 known species, the oldest of which are late Oligocene (∼24 Ma) in age. Except for the recently extinct thylacine (Thylacinus cynocephalus), most are known from fragmentary craniodental material only, limiting the scope of biomechanical and ecological studies. However, a particularly well-preserved skull of the fossil species Nimbacinus dicksoni, has been recovered from middle Miocene (∼16-11.6 Ma) deposits in the Riversleigh World Heritage Area, northwestern Queensland. Here, we ask whether N. dicksoni was more similar to its recently extinct relative or to several large living marsupials in a key aspect of feeding ecology, i.e., was N. dicksoni a relatively small or large prey specialist. To address this question we have digitally reconstructed its skull and applied three-dimensional Finite Element Analysis to compare its mechanical performance with that of three extant marsupial carnivores and T. cynocephalus. Under loadings adjusted for differences in size that simulated forces generated by both jaw closing musculature and struggling prey, we found that stress distributions and magnitudes in the skull of N. dicksoni were more similar to those of the living spotted-tailed quoll (Dasyurus maculatus) than to its recently extinct relative. Considering the Finite Element Analysis results and dental morphology, we predict that N. dicksoni likely occupied a broadly similar ecological niche to that of D. maculatus, and was likely capable of hunting vertebrate prey that may have exceeded its own body mass.     

Foraging behavior of three Tasmanian macropodid marsupials in response to present and historical predation threat

It is often essential to understand historical selection regimes to explain current traits. We studied antipredator behavior of three Tasmanian macropodid marsupials – Forester kangaroos Macropus giganteus , Bennett's wallabies M. rufogriseus , and Tasmanian pademelons Thylogale billardierii – to understand how antipredator behavior functions in a relatively intact predator community. We also compared behavior of the kangaroos and wallabies on a predator-free island where they were translocated from mainland Tasmania 30 yr ago. Both species allowed humans to get closer to them on the predator-free island; a finding consistent with a reduced risk of predation on the island. Neither kangaroos, nor wallabies, exhibited group size effects – they did not modify time allocated to foraging or antipredator vigilance as a function of group size at either site. Nor did overall time allocation vary in any consistent way. In contrast, mainland Australian sibling-species of Forester kangaroos and Bennett's wallabies have both been reported to have group size effects. It is possible either that the extinction of the thylacine Thylacinus cynocephalus in the last century has led to an evolutionary loss of group size effects and other antipredator behavior, or that thylacines were never that important a predator on Tasmanian subspecies. In contrast, Tasmanian pademelons studied on the Tasmanian mainland modified time allocation as a function of group size suggesting that they perceived safety in numbers. Pademelons, because of their body size, are relatively more vulnerable than larger-bodied macropodids to the rich community of marsupial carnivores in Tasmania, and used a mix of social and individual strategies to manage predation risk.     

Could direct killing by larger dingoes have caused the extinction of the thylacine from mainland Australia?

Invasive predators can impose strong selection pressure on species that evolved in their absence and drive species to extinction. Interactions between coexisting predators may be particularly strong, as larger predators frequently kill smaller predators and suppress their abundances. Until 3500 years ago the marsupial thylacine was Australia's largest predator. It became extinct from the mainland soon after the arrival of a morphologically convergent placental predator, the dingo, but persisted in the absence of dingoes on the island of Tasmania until the 20th century. As Tasmanian thylacines were larger than dingoes, it has been argued that dingoes were unlikely to have caused the extinction of mainland thylacines because larger predators are rarely killed by smaller predators. By comparing Holocene specimens from the same regions of mainland Australia, we show that dingoes were similarly sized to male thylacines but considerably larger than female thylacines. Female thylacines would have been vulnerable to killing by dingoes. Such killing could have depressed the reproductive output of thylacine populations. Our results support the hypothesis that direct killing by larger dingoes drove thylacines to extinction on mainland Australia. However, attributing the extinction of the thylacine to just one cause is problematic because the arrival of dingoes coincided with another the potential extinction driver, the intensification of the human economy.     

Phylogenetic relationships of dasyuromorphian marsupials revisited

We reassessed the phylogenetic relationships of dasyuromorphians using a large molecular database comprising previously published and new sequences for both nuclear (nDNA) and mitochondrial (mtDNA) genes from the numbat (Myrmecobius fasciatus), most living species of Dasyuridae, and the recently extinct marsupial wolf, Thylacinus cynocephalus. Our molecular tree suggests that Thylacinidae is sister to Myrmecobiidae + Dasyuridae. We show robust support for the dasyurid intrafamilial classification proposed by Krajewski & Westerman as well as for placement of most dasyurid genera, which suggests substantial homoplasy amongst craniodental characters presently used to generate morphology‐based taxonomies. Molecular dating with relaxed molecular clocks suggests that dasyuromorphian cladogenesis began in the Eocene, and that all three dasyuromorphian families originated prior to the end of this epoch. Radiation within Thylacinidae and Dasyuridae had occurred by the middle to late Oligocene, consistent with recognition of primitive thylacinids (e.g. Badjcinus turnbulli) in the later Oligocene and of putative dasyurids (e.g. Barinya wangala) by the early Miocene. We propose that all four extant dasyurid tribes were in existence by the early Miocene and that most modern dasyurid genera/species were established before the later Miocene. This is in marked contrast to the popularly accepted advocation of their origins in the latest Miocene–early Pliocene. © 2015 The Linnean Society of London     

Microsatellites for the Tasmanian devil (Sarcophilus laniarius)

The Tasmanian devil (Sarcophilus laniarius), a medium‐sized predator/scavenger, is the largest member of the short‐lived carnivorous marsupial Family Dasyuridae. Now restricted to Tasmania, populations are impacted by habitat clearance and anthropogenic mortality and genetic studies could be of value in informing levels of genetic diversity, mating system, dispersal and the effects of natural and anthropogenic landscape features on gene flow. Microsatellite markers were isolated from a partial, size‐selected genomic library that was enriched for microsatellite sequences. Primer pairs were developed for 11 polymorphic dinucleotide microsatellite loci that conform with Hardy–Weinberg equilibrium and reveal moderate genetic variability across the species range.     

Phylogenetic studies of marsupials based on phosphoglycerate kinase DNA sequences

Phosphoglycerate kinase sequences were obtained for 313 aligned bases of 41 individuals from 39 marsupial species. In contrast to previous molecular analyses, the relationships suggested by these data show a high level of congruence with morphologically defined orders and families. Four main monophyletic lineages are recognizable. These are the monogeneric orders Microbiotheria (Dromiciops australis) and Notoryctemorphia (Notoryctes typhlops), a grouping of the American orders Didelphimorphia and Paucituberculata, and the Australasian species other than N. typhlops. Within the Australasian lineage, there are again four main monophyletic groups; the Dasyuridae, two peramelemorph (bandicoot) lineages (one comprised of pseudogene sequences) and the Diprotodontia. This topology is not greatly affected by the exclusion of pseudogenes except that a clade of syndactylous species (Peramelemorphia plus Diprotodontia) is recovered. Two other peramelemorph pseudogenes have inserts of about 1 kb with high levels of similarity to LINE 1 elements. The Diprotodontia is notable for its relative lack of intersequence variation in comparison to the Dasyuromorphia.     

The evolution of protamine P1 genes in dasyurid marsupials

We report the complete DNA sequences of the protamine P1 gene and flanking regions for 13 species of the marsupial family Dasyuridae. The structure of the protamine locus is conserved in dasyurids and consists of two exons (of lengths 142–151 and 47 bp) separated by an intron (208–240 bp). A key feature of the dasyund intron is a 38–40 by duplication found in all species examined to date. This duplication apparently predates the radiation of modern dasyurid lineages and may be homologous to a similar feature in the marsupial mole (Notoryctes). Sequences from a species of Planigale demonstrate that this genus is unique among marsupials in possessing cysteine residues in its protamine P1 molecules. Cysteines may provide enhanced chemical stability for condensed sperm nuclei, a physiological feature that would converge on the common eutherian pattern. Phylogenetic analysis of the protamine genes yields a tree that is largely congruent with previous molecular systematic studies in two areas: (1) There are three main dasyurid lineages corresponding to the Sminthopsinae, Dasyurinae, and Phascogalinae; (2) Dasyurinae and Phascogalinae are sister groups. This study is the first estimate of dasyurid relationships based on a nuclear DNA sequence. Correspondence to: J.D. Retief     

Insular tammar wallabies (Macropus eugenii) respond to visual but not acoustic cues from predators

We studied the way in which a population of tammar wallabies(Macropus eugenii), which have been isolated from mammalianpredators since the last ice age, responded to the sight andsound of historical and ontogenetically and evolutionarilynovel predators. Tammars were shown a range of visual stimuli,including taxidermic mounts of two evolutionarily novel predators,a red fox (Vulpes vulpes) and a cat (Felis catus), and a modelof an extinct predator, the thylacine (Thylacinus cynocephalus).Controls were a conspecific, the cart on which all mounts werepresented, and blank trials in which spontaneous change in behaviorwas measured. We played back recorded sounds to characterizeresponses to acoustic cues from predators and to a putativeconspecific antipredator signal. Treatments included the howlsof dingoes (Canis lupus dingo), an evolutionarily novel predator;calls of a wedge-tailed eagle (Aquila audax), a historicaland current predator; and wallaby foot thumps. Controls werethe song of an Australian magpie (Gymnorhina tibicen) and ablank trial. After seeing a fox, wallabies thumped their hindfeet in alarm, suppressed foraging, and increased looking.The sight of a cat similarly suppressed foraging and increasedlooking. The sounds of predators did not influence responsiveness,but wallabies foraged less and looked more after thump playbacks.Our results suggest that tammars respond to the sight, butnot the sounds, of predators. In contrast, the response to footthumps demonstrates that this particular sound functions asan antipredator signal. We suggest that responsiveness to visualcues has been preserved under relaxed selection because predatormorphology is convergent, but vocalizations are not.     

Spatial organization of the arboreal carnivorous marsupial Phascogale tapoatafa

Phascogale tapoatafa is a squirrel-sized carnivorous marsupial (Dasyuridae) found in dry eucalyptus forests of Australia. Radio-tracking and live-trapping studies at three sites in Victoria indicated that population densities were typically low, with females occupying home ranges averaging 41 ha (minimum convex polygon method) that were intrasexually exclusive. Male home ranges (mean 106 ha) overlapped extensively with females and other males, and expanded during the short breeding season to an average length of 2.7 km. When presented with the faeces of unfamiliar conspecifics, both female and male P. tapoatafa investigated the faeces of females significantly longer than those of males, suggesting that the exclusive home ranges of females are reinforced by olfactory communication, and that males are less responsive to the presence of potential rivals than to potential mates. Both sexes foraged throughout their home range, but the core area of intensive use was less than one-third of the total area traversed. The spatial organization of P. tapoatafa differs markedly from that of the closely related species Antechinus stuartii , but is similar to that of the only other extensively studied carnivorous marsupial, Dasyurus geoffroii.     

An Analysis of Marsupial Interordinal Relationships Based on 12S rRNA, tRNA Valine, 16S rRNA, and Cytochrome b Sequences

The basal split among living marsupials is traditionally placed between the cohorts Ameridelphiaand Australidelphia. Ameridelphia includes all American forms excepting the South AmericanDramicuipx gliroidex (Order Microbiotheria). Australidelphia includes all Australasian taxaplus Dromiciops glinmles. DNA data support Eometatheria Dromiciaps + Diprotodontia +Dasyuromorphia + Notoryctemorphia) but do not resolve the position of bandicoots, whetherwith other australidelphians or with ameridelphians. Also, the most robust molecular trees (DNAhybridization, multigene studies) exhibit minimal branch subdivision and raise the possibility ofartit'actual associations owing to long branch attraction. We analyzed data sets that consistedof complete sequences tor four niitochondrial genes (cytochrome b, 12S rRNA, tRNA valine,16S rRNA). One data set included 14 marsupial taxa. A second data set included 14 marsupialsas well as outgroup sequences (one monolreme; 20 placentals). Phylogenetic analyses includedparsimony, minimum evolution, maximum likelihood, and quartet puzzling. When phylogeneticanalyses were restricted to just the marsupial sequences, there was 75 to 96% boostrap supportfor the separation of Ameridelphia versus Australidelphia. This suggests that either one orboth of these groups are monophyletic. Also, there was 71 to 98% bootstrap support for theseparation of Eometatheria versus Ameridelphia + Peramelina. Nonmonophyly of several a prioriclades was accepted by at least some statistical tests including the following: Diprotodontia+ Peramelina, Notoryctemorphia + Peramelina, Diprotodonlia + Notoryctemorphia, and themonophyly of Australasian marsupials. With the inclusion of outgroup sequences, there wasreduced bootstrap support for associations among marsupial orders and statistical tests failed toreject all interordinal associations that were tested.