Introduction

Arid zone marsupials are icons of Australia and have an inferred evolutionary history that extends back over some ~ 15 Ma1. Nevertheless, the precise divergence timings of the major extant clades are ambiguous, as are the possible drivers behind their adaptive radiations2,3,4,5,6,7,8,9,10,11,12,13.

Macropodoids (Macropodiformes: Macropodoidea)—the group encompassing living kangaroos, wallaroos, wallabies, pademelons and tree-kangaroos (Macropodidae), bettongs and potoroos (Potoroidae), the Musky rat-kangaroo (Hypsiprymnodon moschatus: Hypsyprymnodontidae), and their stem antecedents14—incorporate some of the most distinctive Australian arid zone marsupials, as epitomised by the famous Red kangaroo, Osphranter rufus15. The well-documented fossil record of this and other ‘true kangaroos’ (Macropodini) has been used to correlate arid zone macropodoid evolution with the expansion of intracontinental grasslands during the Pliocene and Pleistocene, from ~ 3–4 Ma3,9,12. By contrast, the contemporary diversification of xeric-adapted bettongs is often overlooked, but has considerable significance because it includes the only example of an exclusively desert-inhabiting macropodoid, the Desert ‘rat-kangaroo’, which is alternatively referred to as the “Oolacunta”16 or Ngudlukanta17, Caloprymnus campestris (Fig. 1A).

Figure 1
figure 1

(A) Painting of Caloprymnus campestris as illustrated by Gould81 (image in public domain). (B) Estimated historical distribution of C. campestris (grey shaded area) and localities from which specimens were collected: (1) Koonchera; (2) Ooroowillanie; (3) Mulka; (4) Killalpaninna (based on data from Google Maps and OZCAM Online Zoological Collections of Australian Museums: https://ozcam.org.au/). (C) Sturt Stony Desert gibber plain habitat of C. campestris showing a ‘jump-up’ escarpment and ephemeral drainage channel lined by riparian vegetation in the distance (photograph reproduced with permission from Michael Letnic, University of New South Wales). (D) Preserved skin of Caloprymnus campestris (Australian Museum, Sydney [AM] M21674) from Killalpaninna in northeastern South Australia22 (photograph reproduced with permission from Mark Eldridge, AM).

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The first scientific specimens of C. campestris were collected from northeastern South Australia (Fig. 1B) in 1842, with three preserved examples subsequently shipped to London for study18. These were dubbed ‘Bettongia’ campestris by Gould19, although Thomas20 later recognised ‘B.’ campestris as morphologically distinct from Bettongia, and thus established a separate genus, Caloprymnus. No further sightings of C. campestris were reported after this initial description, and the species was assumed to be extinct for some 90 years until Finlayson16,21 announced the “Rediscovery of Caloprymnus campestris” in 1931–1932, from the remote Kooncheera Dune17 region in the Sturt Stony Desert of far northeastern South Australia (Fig. 1C). Since then, only a skin recovered sometime between 1902 and 1905 (Fig. 1D) has been reidentified22, and various unsubstantiated live sightings made17,23,24, with the most recent in 201124 and 201317 prompting unsuccessful surveys for the species in 2018 and 201917. Caloprymnus campestris has otherwise been classified as Extinct by the IUCN (https://www.iucnredlist.org/) since 1994, with the probable cause being over-predation by feral dogs, cats and foxes25.

At latest count, only 25 specimens of C. campestris are catalogued in museums worldwide22. This dearth of research material has led to uncertainty about potoroid interrelationships26, as well as the concomitant chronicle of their arid zone evolution. Here, we therefore analyse the first complete mitochondrial (mt) genome of C. campestris, which augments the 12S rRNA (AY245615) and partial cytochrome b (AY237246) gene sequences27 already available from GenBank (https://www.ncbi.nlm.nih.gov/genbank/). Our novel dataset is used to construct a comprehensive phylogeny of crown potoroid species and subspecies within Macropodoidea. We also apply molecular clock calibrated ancestral range estimations to infer both the timing and context of macropodoid habitat change over the last ~ 25 Ma.

Materials and methods

Samples and sequencing

We obtained non-formalin-fixed liver samples from a male Caloprymnus campestris (Museums Victoria, Melbourne, Australia [NMV] C8981) that was collected in 1834 from Mulka cattle station in northeastern South Australia (Fig. 1B). Our DNA extraction, PCR amplifications, sequencing and alignment procedures followed Westerman et al.27,28. Whole genome libraries were prepared with the Nextera DNA flex library kit (Illumina, CA), incorporating 50 ng of input DNA per sample. Sequencing was performed on the Illumina MiSeq platform using 2 × 300 bp V3 chemistry to generate 4,445,476 read pairs and 1.55 Gb total sequence data. Raw reads were trimmed for adapters and quality using Trimmomatic 0.3629 (sliding window = 4:15; leading = 3; trailing = 3), and then assembled via genome skimming with IDBA-UD 1.1.130 (mink = 20; maxk = 300; min_contig = 500); this yielded an average depth-of-coverage of 76.7x (median = 70x; minimum = 14x; maximum = 311x) and insert length of 109.9 bp. The resulting C. campestris mitogenome (A = 34%; C = 24.1%; G = 12.1%; T = 29.8%) was annotated using the MITOS webserver31 with start-stop positions for protein coding genes manually curated using blastp homologies extracted from the NCBI non-redundant (nr) database.

Phylogenetic and molecular clock analyses

Phylogenetic relationships within Macropodoidea were examined using a mitogenome dataset including representatives of all potoroid species, together with Hypsiprymnodon moschatus and multiple species-level exemplars for selected macropodid genera (see Supplementary Table S1). The Northern common cuscus, Phalanger orientalis (Phalangeridae), and Western pygmy possum, Cercartetus concinnus (Burramyidae), were added as non-macropodoid outgroups. To accommodate for recognised gene incongruence32, we then compared these results with analyses of nuclear (n), and combined mitogenome/mtDNA/nDNA sequence datasets derived from GenBank, which integrated an expanded taxon sample of all potoroid species and subspecies (see Supplementary Tables S1 and S2). The mitogenomes were treated as a single partition, or alternatively sub-partitioned into 12S/16S rRNA stems and loops, pooled 1st, 2nd and 3rd protein codon positions, and 3rd codon positions with RY coding to allow for heterogeneity and saturation. A General Time Reversible gene partition model, gamma distribution and variable site proportions were determined using jModelTest33 (Supplementary Table S3).

Tree building employed Maximum likelihood and Bayesian methods implemented in RAxML 7.2.834, MrBayes 3.2.735 and BEAST 2.2.136 with node support calculations based on 1000 bootstrap pseudoreplicates (%) and Bayesian Posterior Probabilities (BPP), respectively. Maximum likelihood used a GTR + I + Γ partition model, while non-dated Bayesian MCMC analyses were run for 6 × 106 generations with a sample frequency of 1000, eight chains, default temperature of 0.2, and burn-in fixed at 6 × 104. Time-trees were constructed in BEAST 2.2.136 with relaxed clocks and the minimum–maximum node age constraints listed in the Supplementary Information. Up to 95% of the normal prior distributions were assigned to the interval between minimum and maximum, with 2.5% to each tail. Gamma priors (shape = 1; scale = 1) were assigned to the “ucld.mean” parameter for each partition. MCMC analyses were run for 65 × 106 generations with a burn-in of 10 × 106 generations and sampling every 10 × 103 generations. ESS values were > 200 for all estimated parameters. TreeAnnotator 2.2.1 (https://www.beast2.org/treeannotator/) was used to summarise the tree sample with mean node heights.

Ancestral area analyses

Distributional areas were optimised onto the time-calibrated BEAST consensus tree and analysed using the R package BioGeoBEARS37 to compare alternative biogeographical range models, and a Bayesian Binary MCMC (BBM) approach38,39 to reconstruct ancestral ranges in RASP 440. Area codes (Supplementary Table S4) followed standard units6 but were refined to represent a generalised vegetation map41: A = humid forest (rainforest and/or ‘wet’ sclerophyll dominant) prevalent throughout eastern coastal Australia, western Tasmania and New Guinea; B = woodland (‘dry’ sclerophyll dominant) prevalent throughout northern, eastern and southwestern inland Australia and northeastern Tasmania; C = shrubland (Acacia and chenopodiaceous shrubland dominant) prevalent throughout central and central-western Australia; and D = grassland-desert (arid grasslands and/or desert dominant) prevalent in central and central-northwestern Australia. The maximum number of ancestral areas was restricted to three because this equalled the maximum number of areas occupied by our terminal taxa at any given node.

BioGeoBEARS comparisons proceeded with likelihood ratio testing of ‘Jumping dispersal events (+ J)’, which have been considered inappropriate for dispersal-extinction-cladogenesis (DEC) models42. However, the three parameter Bayesian inference of historical biogeography for discrete areas (BAYAREALIKE) + J model (P = 0.0006) received overwhelmingly highest support (AICc = 199.6; AICc_wt = 0.98) for conferring best statistical likelihood on our data (Supplementary Table S5). Finally, we accommodated for connectivity by designating a dispersal multiplier of ‘1’ for adjacent areas (A-B-C)41 versus non-adjacent areas (A-D)41, which were assigned a value of ‘0.5’.

Our BBM analyses utilised 10 MCMC chains with default temperature 0.1, and run over 5 × 106 generations with sampling frequency and burn-in fixed at 1000. Model settings included ‘Gamma(+ G)’ for among-site rate variation, and ‘Fixed (JC)’ for state frequencies.

Results and discussion

The Caloprymnus campestris mitogenome (16,866 bp) is ordered with 13 protein-coding genes, two ribosomal (r)RNA genes, 21 transfer (t)RNAs, and a non-coding AT-rich control region, which follows the typical configuration for marsupials43,44. The tRNAs are arranged around the origin of the L strand (A-C-W-OL-N-Y) and intersected between the NADH2 and COX1 genes. Substitution of the anticodon GCC for tRNAASP (trnD) is also consistent with RNA-editing45.

Maximum likelihood and Bayesian analyses of our mitogenome dataset produce unanimous resolution of Macropodoidea with Potoroidae as the sister to Macropodidae (Supplementary Figures S1–S6). This pivotal higher-level grouping accords with other crown macropodoid phylogenies12,46,47,48,49, and warrants a new taxonomic definition50, which we coin as Macropodia, new clade, herein (Table 1; Supplementary Information). Bootstrap and BPP support is > 90% for almost all constituent nodes except those uniting: (1) the extinct short-faced kangaroo, Simosthenurus occidentalis, with the Banded hare-wallaby, Lagostrophus fasciatus, as basally branching macropodids (partitioned/non-partitioned bootstrap = 58/63%; MrBayes partitioned/non-partitioned BPP = 0.54/0.56; BEAST partitioned/non-partitioned BPP = 1/1); (2) the Quokka, Setonix brachyurus, with other macropodines (bootstrap = 48/60%; MrBayes BPP = 1/1; BEAST BPP = 0.63/0.72); (3) grey kangaroos in the genus Macropus with Osphranter rufus and brush wallabies representing the genus Notamacropus (bootstrap = 80/64%; MrBayes BPP = 0.99/1; BEAST BPP = 0.99/0.96); and (4) O. rufus with Notamacropus (bootstrap = 58/55%; MrBayes BPP = 0.81/1; BEAST BPP = 0.72/0.76). As found by previous studies5,12,27,46,47,48,49,50,51, Potoroinae comprises potoroos within the genus Potorous and is distinguished from its sister clade, which we designate Bettonginae52 to include the Rufous bettong, Aepyprymnus rufescens, as the basally branching sister to C. campestris and the species of Bettongia (Table 1). Alternative monophyly of C. campestris with either A. rufescens53,54, or the species of Potorous27,50 were tested using topological constraints in PAUP* 4.0b1055 (Supplementary Table S6), but decisively rejected (P < 0.0001***). Taxonomically, therefore, we conclude that the original classification of Gould’s Desert ‘bettong’19 as generically consistent with Bettongia is feasible, but defer any formal nomenclatural amendment pending a detailed morphological re-evaluation.

Table 1 Phylogenetic definitions for Macropodiformes, including Macropodia, new clade, and other selected constituent subclades.
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Our maximum likelihood, Bayesian and time-tree analyses of the nDNA (Supplementary Figures S7–S9) and combined mitogenome/mtDNA/nDNA datasets (Fig. 2; Supplementary Figures S10–S12) yield broadly compatible topologies, with the basal divergence of potoroids and macropodids, and subsequent split between potoroines and bettongines both occurring from the latest Oligocene to earliest-middle Miocene (Table 2; Supplementary Table S7). Notably, this concurs with divergence times derived using different dating methods and constraints12,46,47,48,49,50,56. Furthermore, while our BioGeoBEARS and BBM ancestral range estimations correlate the latest Eocene (or mid-Eocene using nDNA: Supplementary Table S7) to late Oligocene emergence of crown macropodoids with predominantly humid forest habitats (> 50% probability values from BAYAREALIKE + J [A] = 65.76%; BBM [A] = 61.31%: Supplementary Tables S8 S8 and S9), the initial radiation of potoroids (BAYAREALIKE + J [B/A] = 45.42/25.55%; BBM [B/AB] = 42.62/28.9%), together with the macropodid subclades Sthenurinae (BAYAREALIKE + J [B] = 82.31%; BBM [B] = 66.1%) and Lagostrophinae + Macropodinae (BAYAREALIKE + J [B] = 70.45%; BBM [B/BC] = 41.79/27.29%) are coordinated with earlier Miocene dispersals into woodland dominated mosaics (Fig. 2; Supplementary Tables S8–S11; Supplementary Figures S13 and S14). These potentially included ‘mallee-like’57 sclerophyll communities, which propagated throughout central Australia from the early to middle Miocene41.

Figure 2
figure 2

Time calibrated phylogeny of crown Macropodoidea (filled black diamond) showing divergence of Caloprymnus campestris (bold type) within Bettonginae (black open circle), and correlated against a schematic of changing palaeohabitats across the late Oligocene–Holocene interval (modified from Kear et al.6 and Den Boer et al.82). Topology is based on the partitioned mitogenome/mtDNA/nDNA dataset. Bayesian posterior probability (< 1.0) and bootstrap (< 100%) support values (regular type) derived using BEAST 2.2.136/MrBayes 3.2.735/RAxML 7.2.834 are indicated at relevant nodes. Branch colours denote major clades: Hypsiprymnodontidae (purple); Macropodia, new clade (burgundy); Potoroidae (pink); Potoroinae (orange); Bettonginae (ochre); Macropodidae (red); Sthenurinae (green); Lagostrophinae (yellow); Macropodinae (light blue); Dorcopsini (grey) Dendrolagini (brown); Macropodini (dark blue). *Extinct taxa. See Table 2 for node number references (bold type) and the Supplementary Information for other analyses. Graphics produced with Adobe CC2021 by B.P.K.

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Table 2 Estimated divergence times (Ma) with confidence intervals for crown macropodoid clades based on the partitioned mitogenome/mtDNA/nDNA dataset.
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The globally recognised58 middle to late Miocene climatic transition from equable to increasingly cool, dry conditions41,59 coincides with potoroine speciations into mesic environments throughout southern Australia27,56. These are tracked by our BioGeoBEARS and BBM estimates, which infer occupation of primarily woodland and forest habitats after the earliest-late Miocene (Supplementary Tables S7–S9; Supplementary Figures S13 and S14). This is concurrent with the incipient desertification of inland Australia60, which may have promoted genetic segregation of the extinct Broad-faced potoroo, Potorous platyops, from Gilbert’s potoroo, Potorous gilbertii, in central-southern61 and southwestern Australia (BAYAREALIKE + J [B] = 79.64%; BBM [B/AB] = 45.87/44.42%), versus the Long-nosed potoroo, Potorous tridactylus (BAYAREALIKE + J [AB] = 77.96%; BBM [AB] = 94.82%), and basally branching Long-footed potoroo, Potorous longipes, in southeastern Australia56. Additionally, we show that regional subspecies distinctions within P. tridactylus were completed by the latest Pliocene to mid-Pleistocene (Table 2; Supplementary Table S7). Curiously, though, Cyt b K2P variation (Supplementary Table S12) implies substantially less genetic difference between the Tasmanian P. tridactylus apicalis and northeastern mainland P. tridactylus tridactylus (1.93%), in comparison to the southeastern mainland P. tridactylus trisulcatus (4.21%). Indeed, these values approximate those contrasting P. tridactylus tridactylus/P. tridactylus trisulcatus with P. gilbertii (2.69/5%), P. platyops (4.1/5%), and P. longipes (5.84/5.69%), supporting inferences of cryptic taxa56, but in our opinion, only up to species-level.

Despite the currently limited DNA sequence coverage for the extinct Finlayson’s62 Desert bettong, Bettongia anhydra63, we derive unequivocal support (Fig. 2; Supplementary Figures S1–S12) for the monophyly of Bettongia spp. (bootstrap =  > 90%; BPP = 1), together with close relationships between the woodland-forest dwelling Eastern bettong, Bettongia gaimardi, Northern bettong, Bettongia tropica, and Brush-tailed bettong, Bettongia penicillata penicillata (bootstrap =  > 99%; BPP = 1). Only a few hundred Cyt b (or control region) nucleotides are available for the Woylie, Bettongia penicillata ogilbyi64. Nevertheless, our BioGeoBEARS and BBM estimates suggest a latest middle to probably late Miocene divergence of B. anhydra (BAYAREALIKE + J [CD] = 98.54%; BBM [CD] = 55.06%) and the Boodie, Bettongia lesueur, (BAYAREALIKE + J [CD] = 98.12%; BBM [BCD] = 81.72%) in xeromorphic habitats (Table 2; Supplementary Tables S7–S9; Supplementary Figures S13 and S14), followed by Pliocene to as recent as mid-Pleistocene radiations of B. gaimardi (BAYAREALIKE + J [CD] = 90.65%; BBM [BCD/BC] = 27.79/23.75%) and B. tropica + B. penicillata subsp. (BAYAREALIKE + J [CD] = 79.96.12%; BBM [BCD/BC] = 28.32/21.98%) coupled with increasing habitat variegation41. We correlate this with vicariant ‘reversions’5 into eucalypt woodlands and forests65,66,67 (Supplementary Tables S10 and S11), which contracted and fragmented with intensifying aridification over the Pliocene–Pleistocene interval68.

Bettongia is karyotypically conservative, retaining the 2n = 22 chromosomal number of most macropodoids69,70. Conversely, chromosomal fission in P. longipes has produced 2n = 24, while fusions (and inversions) in P. tridactylus and P. gilbertii manifest unusual reductions to 2n = 12♀, 13♂71. Aepyprymnus rufescens, on the other hand, exhibits a unique karyotypic increase to 2n = 32, which is the highest for any marsupial71, and presumably reflects its independent evolution since the later-early to early-late Miocene (nDNA favouring a younger later-middle to early-late Miocene range: Table 2; Supplementary Table S7). Although the chromosomal arrangement of C. campestris is unknown, our robustly supported (bootstrap =  > 90%; BPP = 1) earliest-middle to early-late Miocene split from Bettongia (Table 2; Supplementary Table S7) suggests a similarly protracted ancestry, yet with genetic differentiation that approaches intrageneric levels within Bettongia spp. (Cyt b K2P variation being as little as 6.91% compared to B. penicillata: Supplementary Table S12). Significantly, our BioGeoBEARS (BAYAREALIKE + J [CD] = 87.73%) and BBM ([CD] = 53.41%) estimates correlate the C. campestris-Bettongia divergence with a seminal invasion of xeric environments (Supplementary Tables S8–S12; Supplementary Figures S13 and S14), perhaps incorporating arid chenopod shrublands that spread across central Australia from the middle to late Miocene41,57,60. The coeval radiation of macropodines is otherwise linked to predominantly woodland and forest settings (Table 2; Supplementary Tables S8–S12; Supplementary Figures S13 and S14). This includes dorcopsins (BAYAREALIKE + J [B] = 54.21%, BBM [AB/B] = 38.93/25.2%) and dendrolagins (BAYAREALIKE + J [B/A] = 49.35/34.51%, BBM [AB/ABC] = 47.93/26.27%) diverging coincident with uplift of the New Guinean landmass3,72,73, and macropodins which initially diversified in woodland habitats (BAYAREALIKE + J [B] = 95.79%; BBM [B] = 77.19%), but subsequently expanded into open shrublands and eventually grasslands (e.g., Osphranter rufus: BAYAREALIKE + J [B] = 51.33%, BBM [BC/BCD] = 31.62/24.84%) after the late Miocene to as recent as Pliocene to mid-Pleistocene (Table 2; Supplementary Table S7), thereby presaging the modern prevalence of grazing kangaroos9.

Conclusions

Our characterisation of the complete mitogenome for Caloprymnus campestris provides an ecological diversification timescale for bettongs and potoroos within the context of crown macropodoid evolution. Most importantly, we show that the unambiguously monophyletic C. campestris-Bettongia lineage probably originated with the onset of increasingly arid intracontinental climates during the middle to late Miocene41,57,58,59,60,74, corresponding with the deepest divergences of Australia’s arid zone biota around ~ 15 Ma1. This contrasts with the largely late Miocene to Pleistocene radiation of kangaroos, whose abundance in modern arid zone habitats has been attributed to grazing adaptations and the spread of grasslands during the Pliocene and Pleistocene3,9,12. Clearly, therefore, the appearances of Australia’s distinctive arid zone macropodoids were staged over some ~ 3–6 Ma (based on minimum–maximum confidence interval differences for C. campestris versus Osphranter rufus: Table 2), and likely occurred in response to a complex interplay of abiotic and biotic drivers involving both climate and vegetation change.

Unfortunately, little is known about the biology of C. campestris or other extinct ‘Desert bettongs’, such as Bettongia anhydra63, and the Nullarbor dwarf bettong75, Bettongia pusilla76. Nonetheless, early eye-witness reports state that C. campestris inhabited sparsely vegetated gibber plains16. The diet of C. campestris is also uncertain23, but might have been varied16,23 similar to the extant arid zone Bettongia lesueur77 and Bettongia penicillata64, which consume a range of plant matter, fungi and insects78,79. Caloprymnus campestris was thus probably an important ‘ecosystem engineer’63 whose tragic loss is compounded by dramatic range reductions and the Near Threatened (Bettongia gaimardi, B. lesueur, Potorous tridactylus), Vulnerable (Potorous longipes), Endangered (Bettongia tropica), Critically Endangered (B. penicillata, Potorous gilbertii), or Extinct (B. anhydra, C. campestris, Potorous platyops) IUCN Red listings (https://www.iucnredlist.org/) for 10 out of the 11 named non-fossil crown potoroids. The extinction susceptibility of C. campestris was presumably exacerbated by its limited distribution (only four recognised collection22, and 13 potential sighting localities17 within a ~ 350 km radius) and desert specialisation, which when coupled with habitat modification and the introduction of exotic species via European pastoralism80, underscores the extreme conservation sensitivity of Australia’s unique arid zone marsupials and the urgent need to document their now dwindling multi-million-year evolutionary histories.

Ethical approval and informed consent

No live animal subjects were used for experiments in this study. All extinct animal tissues were obtained and their use approved by the La Trobe University Animal Ethics Committee (AEC). All experiments were performed in accordance with institutional guidelines and regulations.