Molecular phylogenetic relationships of two extinct potoroid marsupials, Potorous platyops and Caloprymnus campestris (Potoroinae: Marsupialia)

MOLECULAR PHYLOGENETICS AND EVOLUTION Molecular Phylogenetics and Evolution 31 (2004) 476–485 www.elsevier.com/locate/ympev

Molecular phylogenetic relationships of two extinct potoroid marsupials, Potorous platyops and Caloprymnus campestris (Potoroinae: Marsupialia) M. Westerman,a,* S. Loke,a and M.S. Springerb a b

Genetics Department, LaTrobe University, Bundoora, Vic. 3086, Australia Biology Department, University of California, Riverside, CA 92521, USA Received 6 March 2003; revised 4 August 2003

Abstract Complete 12S rRNA and partial cytochrome b (cytb) gene sequences have been obtained from museum samples of two recently extinct potoroids—Potorous platyops and Caloprymnus campestris. Phylogenetic analyses based on these mitochondrial DNA sequences suggest that the broad-faced potoroo (P. platyops) was a close relative of the recently discovered Potorous longipes and the recently re-discovered Potorous gilberti. Although the extinct desert rat-kangaroo (C. campestris) was clearly resolved as a member of the subfamily Potoroinae, its precise relationships vis a vis other living potoroines are unclear. We confirmed that the rufous ratkangaroo (Aepyprymnus rufescens) is sister to all living Bettongia species, but the molecular data provide no support for a sister relationship between A. rufescens and C. campestris as suggested by Flannery (1989) on the basis of four shared morphological characters. Molecular dating analyses suggest that the initial radiation of potoroinae seems to have occurred soon after its origin in the early Miocene. Within Potoroinae, C. campestris diverged from other taxa approximately 16 million years ago. P. platyops diverged from P. longipes + P. gilberti approximately 14–15 million years ago. Ó 2003 Elsevier Inc. All rights reserved.

1. Introduction Kangaroos and their relatives are thought to have evolved from an arboreal, possum-like ancestor about 40–50 million years ago (mya) (Flannery and Archer, 1987; Kirsch et al., 1997; Springer and Kirsch, 1991) although unequivocal macropodoid fossils do not appear before the late Oligocene-early Miocene (
25 mya) (Woodburne et al., 1993). The phylogeny of macropodoids has been extensively reviewed (Flannery, 1989; Cooke and Kear in Archer et al., 1999) and eight subfamilies are currently recognized. Four of these are now extinct (Palaeopotoroinae, Propleopinae, Balbarinae, and Bulungamyinae); two others (Hypsiprymnodontinae and Sthenurinae) are each represented by a single living species, whilst two (Macropodinae and Potoroinae) each have a number of living genera and species.

* Corresponding author. Fax: +61-3-94792480. E-mail address: [email protected] (M. Westerman).

1055-7903/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2003.08.006

The Potoroinae, often referred to as rat-kangaroos, are recognizable by several dental and other characters (Burk and Springer, 2000; Flannery, 1989; Szalay, 1994) and diverged from the macropodines approximately 25 mya (Westerman et al., 2002), though as noted by Prideaux (1999), pre-quaternary records are quite meagre. Although this group was once a conspicuous element of many Miocene faunas (Archer et al., 1991; Flannery, 1989) it had become relatively insignificant by the end of this period. Some monotypic genera are known only from very limited remains. Of the modern genera, only Bettongia is known from the late Tertiary (see Prideaux (1999) for references). European settlement of Australia seems to have further exacerbated the decline of Potoroinae, especially following major alterations to the landscape and the introduction of both predators (foxes and cats) and competitors (ungulates and rabbits). There have been massive range contractions for most species with only three modern ones remaining relatively common (see Table 1). Most of the other species are relatively rare, especially on the

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477

Table 1 Status of modern potoroines (after Flannery, 1989) Taxon

Tribe Potoroini Potorous gilberti

Common name

Status

Genbank submission number 12S rRNA

Cytochrome b AY237230 AY237231 AY237232 AY237233 AY237248 AY237247 AY237234 AY237235

GilbertÕs potoroo

Rare* 1994

AY245616

Potorous longipes

Long-footed potoroo

Rare

AF028000

Potorous platyops Potorous tridactylus

Broad-faced potoroo Long-nosed potoroo

Extinct** 1875 Common

AY245621 AY245617

Tribe Bettongini Aepyprymnus rufescens Caloprymnus campestris Bettongia gaimardi Bettongia lesueur

Rufous rat-kangaroo Desert rat-kangaroo Tasmanian bettong Burrowing bettong

Common Extinct** 1935 Commona Vulnerable

AF027999 AY245615 AY245619 AY245620

Bettongia penicillata

Brush-tailed bettong

Rare

AF027998

Bettongia tropica

Northern bettong

Rare

AY245618

AY237243 AY237246 AY237244 AY237240 AY237241 AY237242 AY237238 AY237239 AY237236 AY237237

Asterisk indicates date rediscovered, double asterisk date last reported. a Mainland subspecies extinct.

mainland, and two are now extinct (see Seebeck et al. (1989) for modern and previous distributions). The phylogenetic relationships of modern potoroines have been little studied and are not clear. FlanneryÕs (1989) comprehensive review, based mainly on morphological characters, suggested that the monophyletic Potoroinae comprised two tribes—Potoroini and Bettongini1 which had been separate for a considerable time period and were probably separate by the Miocene. Case (in Westerman et al., 2002) suggests that the divergence between the Potoroini and Bettongini occurred approximately 15 mya. Whilst Potoroini comprises only one genus—Potorous, Bettongini is usually regarded as encompassing three genera Bettongia, Aepyprymnus, and Caloprymnus. The latter two genera, each with a single constituent species, are regarded as sister taxa based on four shared characters (Flannery, 1989, p. 20). Little molecular work has been done on the interrelationships of potoroines and most of this has included few representative species. Usually only Aepyprymnus rufescens (the rufous rat-kangaroo) and perhaps a single species of Potorous or Bettongia are included. Baverstock et al.Õs (1989) MC0 F results failed to resolve any relationships amongst the three potoroine species used, precise relationships being dependent upon what taxa were used as their outgroup. Burk et al. (1998), using mitochondrial DNA sequences, showed only moderate 1

Some of the taxa regarded as potoroines by Flannery (Bettongia moyesi, Wakiewakie lawsoni) are now thought to be bulungamyines (see Cooke and Kear in Archer et al., 1999, p. 28).

support for a monophyletic Potoroinae, though there was strong support for Bettongia + Aepyprymnus—a finding confirmed by a larger dataset including nuclear gene sequences (Burk and Springer, 2000). Other studies have usually been more concerned with the taxonomic status of particular potoroine species. Johnston and Sharman (1977) used allozymes and chromosomes to support the recognition of Potorous longipes as a new species; Pope et al. (2000) used mitochondrial d-loop sequences to demonstrate that Bettongia penicillata tropica warranted its specific status and Sinclair and Westerman (1997) used allozymes and partial cytochrome b (cytb) sequences to show that the newly rediscovered Potorous gilberti was genetically distinct from Potorous tridactylus. Little is known about the phylogenetic relationships of the two recently extinct potoroines. The desert rat-kangaroo, Caloprymnus campestris, is a bettong-like animal first reported in 1841 from the gibber plains and loamy flats of north-eastern South Australia and south-western Queensland. It was subsequently not recorded again for a hundred years and has not been seen since 1935 (Carr and Robinson, 1997). The broad-faced potoroo, Potorous platyops, was first recorded from the southwest of Western Australia by John Gilbert in 1839. Only 12 specimens were ever obtained before it was last seen in 1875 (Flannery and Schouten, 2001, p. 57), though sub-fossils suggest that it had an extensive coastal distribution in Western Australia from the South Australian border region to North-West Cape. The precise phylogenetic relationships of these two species are unknown.

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Spirit-preserved specimens of these two species are held in the National Museum of Victoria and material was made available to us to attempt DNA extractions for DNA sequencing studies. Since this proved successful, we decided to investigate the molecular relationships of all living potoroines using full-length sequences of the mitochondrial 12S rRNA gene together with partial sequences of cytochrome b.

2. Materials and methods DNA samples were obtained from the extinct taxa C. campestris (Museum of Victoria male C8981 liver) and P. platyops (MoV skin C6770 collected in 1874) as well as from the living taxa P. tridactylus, B. tropica, Bettongia gaimardi, and Bettongia lesueur. 12S rRNA sequences for A. rufescens (AF027999), Bettongia penicillata (AF028000), and P. longipes (AF027998) were obtained from Genbank, as were cytochrome b and 12S rRNA sequences for a number of outgroup taxa. These latter included several macropodines Macropus eugenii (AY237226, AY245606);2 M. rufogriseus (AY237227, AY245607); M giganteus (AF187885, AY099267); M. parryi (AY237229, AF187887); M. robustus (Y10524); Hypsiprymnodon moschatus (AY237245, AF027997), and the brush-tail possums Trichosurus vulpecula (AF031823, AF152857); and Trichosurus caninus (AF152858, AF152861). Mitochondrial 12S rRNA gene sequences were obtained as outlined in Burk et al. (1998) and Burk and Springer (2000), using a series of primers to amplify short fragments from the museum samples. Cytochrome b sequences were obtained as outlined in Sinclair and Westerman (1997). Our primary data set consisted of complete 12S rRNA + partial cytochrome b sequences for representatives of each of 18 species (16 macropodoids; two phalangerids). Data were combined after performing partition homogeneity tests (Farris et al., 1995) as implemented in PAUP* 4.0b (Swofford, 2002). To gain a better understanding of relationships between species of Bettongia and Potorous, we also examined a taxonomically expanded dataset of partial cytochrome b sequences for 29 taxa (27 macropodoids; two phalangerids) that included two or three exemplars of most potoroines, wherever possible choosing animals from geographically different parts of the species range. PAUP* 4.0b10 (Swofford, 2002) was used to perform maximum parsimony (MP), neighbor-joining (NJ), maximum-likelihood (ML), and quartet puzzling (QP) analyses. NJ and ML analyses used the GTR + I + C model of sequence evolution suggested by the Akaike Information Criterion of Modeltest 3.06 (Posada and 2 Genbank Accession Numbers for cytochrome b and 12S rRNA gene sequences, respectively.

Crandall, 1998). Two-tailed Kishino and Hasegawa (1989) tests with RELL optimization and 1000 bootstrap pseudoreplicates (K–H) were used to test a priori hypotheses that Caloprymnus is a member of the tribe Bettongini, that Aepyprymnus is sister to Caloprymnus (Flannery, 1989; Flannery and Archer, 1987), and that Potoroinae is monophyletic (Flannery, 1989; Baverstock et al., 1989). Posterior probabilities were calculated using the program MrBayes 2.01 (Huelsenbeck and Rondquist, 2001), employing random starting trees and a GTR + C + I model of sequence evolution. We performed two different runs with MrBayes to assess whether or not chains were converging on the same posterior probability distribution. The estbranches and divtime5b programs of Thorne et al. (1998) and Kishino et al. (2001) were used to estimate branch lengths and divergence times, respectively, for the combined 12S rRNA + cytochrome b data set. In estimating branch lengths, we used topological relationships shown in Fig. 1 with the two phalangerids as the outgroup taxa. We employed the F84 model of sequence evolution (Swofford et al., 1996) with an allowance for a C distribution of rates. This is currently the most complex model of evolution for DNA sequences implemented in Thorne et al.Õs (1998) program. Base frequencies, the transition/transversion parameter, and the shape parameter (a) of the C distribution were estimated with PAUP 4.0b10 (Swofford, 2002). The divtime5b program uses a Markov chain Monte Carlo approach to estimate posterior probabilities of divergence times. Chains were run for one million generations after a burn-in of 100,000 generations to achieve a stationary sample from the posterior probability distribution; samples were taken from the Markov chain every 100 generations. We used ‘‘semi-random’’ starting states for the Markov chain. We performed two separate analyses to check for convergence. Application of the divtime5b program requires a value for the mean of the prior distribution for the time separating the ingroup root (i.e., Hypsiprymnodon to other macropodoids) from the present (rttm). We used two different values: first,
35 million years following the molecular clock estimates of Burk and Springer (2000) and second,
25 million years based on the age of the oldest potoroine from the Etadunna Formation (see Burk and Springer, 2000). Other settings for divtime5b were as follows: rttmsd ¼ 0.5  rttm, where rttmsd is the standard deviation of rttm; rtrate ¼ X/rttm, where rtrate is the mean of prior distribution for the rate at the root node and X is the median amount of evolution from the ingroup root to the ingroup tips; rtratesd ¼ 0.5  rtrate, where rtratesd is the standard deviation of rtrate; rttm  brownmean ¼ 1, where brownmean is the mean of the prior distribution for the autocorrelation parameter (u); brownsd ¼ brownmean, where brownsd is the standard deviation of the prior distribution for u,

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Fig. 1. Maximum likelihood phylograms of (A) combined 12S rRNA and cytochrome b sequences (GTR + C + I model;  ln likelihood ¼ 6688.316, I ¼ 0:2724, C ¼ 0:3036) based on 1374 base pairs of mitochondrial DNA and (B) 407bp cytochrome b gene (GTR+ C + I model;  ln likelihood ¼ 2609.8922, C ¼ 1:2334, I ¼ 0:4292) for multiple exemplars of each potoroine species. ML Bootstrap support values are shown adjacent to the nodes for those clades supported above 50%, bootstraps for other analyses are given in Table 2.

and minab ¼ 0.5, 1.0 (most analyses), or 1.5, where minab sets the prior for the times of the interior nodes given the time of the root (values of minab > 1 cause internal node times ‘‘repel’’ each other; values of minab < 1 cause internal node times to ‘‘attract’’ one another). The Kishino et al. (2001) method allows for multiple constraints on divergence times. We used the following minima and maxima a. A minimum date of 23 mya for the split between the Macropodinae and the Potoroinae, as this date approximates the estimated age of the middle faunal zone (Ngapakaldi Local Fauna or faunal zone C) of the Etadunna Formation (Woodburne et al., 1993). This faunal zone is the oldest to include both potoroine (Purtia mosaicus, Case, 1984) and macropodine (Nambaroo species A and B, Genus P; Woodburne et al., 1993) kangaroos. b. We used a minimum of 15 million years for the split between the long-footed potoroo and other extant potoroines. The first appearance of potoroos is from the Bullock Creek Local Fauna from the Northern Territory (L. Schwartz, pers. commun. to Judd A.

Case in Westerman et al., 2002) and these deposits are considered as equivalent to Riversleigh System C units (Cooke, 1997). We also imposed a maximum of 23 million years for the split between the longfooted potoroo and other living potoroines based on the plesiomorphic characteristics of earlier potoroines including fossils from the Etadunna Formation (Woodburne et al., 1993). c. We used a maximum of 8 million years for the base of Macropus based on the observation that both the Alcoota and Ongeva local faunas lack more derived macropodines such as Macropus. In addition to analyses that employed all four constraints, we also performed analyses that relaxed individual constraints, (1) both minima or (2) both maxima.

3. Results Although partition homogeneity tests (Farris et al., 1995) implemented in PAUP* suggested that the cytb and 12S datasets might be heterogeneous (P ¼ 0:01) there was no difference between the 12S and cytb trees

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for any of the nodes above 90%. The two datasets were therefore combined. The ML tree is shown in Fig. 1A. The macropodid subfamilies Macropodinae and Potoroinae are clearly resolved (Macropodinae 99%, Potoroinae 76%), but there is little obvious sub-structure in the Potoroinae. The only nodes with even moderate bootstrap support are Bettongia (100% with ML, NJ, MP, and 99% QP) and Bettongia + Aepyprymnus (68% ML, 87% NJ, 66% MP, and 91% QP). Neither of FlanneryÔs tribes Bettongini or Potoroini is apparent in this phylogram, with Caloprymnus placed along with all the Potorous species. Potoroini is moderately well supported (89% bootstrap support) in NJ analyses of the combined data as well as in the QP analyses of the 12S rRNA and cytb genes analyzed separately (66% and 82% respectively, data not shown). The only other ‘‘support’’ for this tribe was from NJ (74%) and, weakly, from ML (55%) and MP (62%) for the cytochrome b gene (see Table 2). Bettongini, at least in the sense of a clade including Bettongia, Aepyprymnus, and Caloprymnus, finds support only from QP (66%) for the cytb dataset. When C. campestris was constrained to be sister to Bettongia + Aepyprymnus, the resulting tree for the combined data was not significantly longer than the best one (K–H diff  ln L ¼ 3:9871, P ¼ 0:299). However constraining C. campestris to be sister to Aepyprymnus (Flannery, 1989) results in a significantly worse fit to the data (K–H diff  ln L ¼ 14:97824, P ¼ 0:03 ). Genetic relationships within the genus Potorous seem clear, with P. tridactylus being sister to the other three species. In Fig. 1A, Aepyprymnus is sister to the genus Bettongia. To examine whether inclusion of additional exemplars of most potoroine species might clarify inter-specific relationships for Potorous and Bettongia, we used partial cytb sequences from animals drawn, wherever possible, from geographically distant parts of each species range. The results in Fig. 1B show that Potoroini now has consistent, though only moderate support (55% ML, 74% NJ, 62% MP, 60% QP—Table 2). The two sub-

species of P. tridactylus are genetically quite distinct, with an uncorrected percent sequence divergence (p) of 3.44% (data not shown) and represent the most divergent taxa in the genus. However, interrelationships between P. platyops, P. longipes, and P. gilberti are still not resolved by inclusion of the extra partial cytb data probably due to the short sequence obtained from the extinct P. platyops sample (407 bp), although these three species do form a reasonably well-resolved clade (63% ML, 88% NJ, 66% MP, and 57% QP) distinct from P. tridactylus. Within the genus Bettongia, once again B. lesueur is clearly delineated as the most genetically divergent species but the interrelationships of the others are not clarified. The Tasmanian bettong, B. gaimardi, is only well resolved from B. tropica and B. penicillata in MP analyses (83%). These two latter, which appear to be sister species with an uncorrected sequence divergence of 2.95%, have sometimes been regarded as subspecies of the Brush-tailed bettong. Results of analyses of the combined 12S rRNA and cytb data with MrBayes are summarized in Table 2. Independent runs starting with different random trees converged on nearly equivalent posterior probability distributions. Bayesian posterior probabilities were often higher than likelihood bootstrap percentages. Posterior probabilities for Macropodidae, Macropodinae, and Potoroinae were 1.00. Within Potoroinae, Bettongia monophyly, Bettongia + Aepyprymnus, and an association of P. platyops with P. gilberti and P. longipes were also strongly supported. In contrast to the ML bootstrap tree, C. campestris was the sister-taxon to other potoroines rather than the sister-taxon to P. tridactylus. However, the posterior probability for all potoroines to the exclusion of C. campestris was only 0.54 in two independent analyses. Other than this difference, Bayesian results are congruent with the ML tree under the same model of sequence evolution. Table 3 shows the results of Bayesian analyses with divtime5b that included all of the constraints listed in

Table 2 Bootstrap support values for the nodes shown in Figs. 1A and B Clade

Macropodinae Potoroinae Bettongia Bettongia + Aepyprymnus Potorini P. platyops + (P. gilberti and P. longipes)

Analytical method Parsimony

Tv Parsimony

NJ

ML

QP

MrBayes

a

b

a

b

a

b

a

b

a

b

a

b

99 51 100 60 <50 57

96 <50 76 <50 62 66

99 68 100 66 <50 <50

84 <50 94 <50 60 60

100 <50 100 87 89 73

98 53 84 <50 74 88

99 76 100 68 <50 73

88 86 92 74 55 63

95 <50 99 91 58 75

84 <50 86 66 60 57

1.00 1.00 1.00 0.99 0.52 0.95

1.00 0.65 0.98 <0.50 0.99 0.98

All values based on 1000 pseudoreplicates for MP, QP, and NJ analyses, 200 pseudoreplicates for ML. Markov chains were run for 500,000 generations with a burn-in of 50,000 generations using MrBayes to generate posterior probabilities.

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Table 3 Divergence time estimates with different priorsa Split

Base of Macropodoidea Base of Macropodinae Base of Macropus Base of Potoroinae Aepyprymnus to Bettongia Base of Bettongia Base of Potoroini P. platyops to P. gilberti + P. longipes C. campestris to P. tridactylus a

Settings for priors IR ¼ 35 my; minab ¼ 1.0; first run

IR ¼ 35 my; minab ¼ 1.0; second run

IR ¼ 35 my; minab ¼ 0.5

IR ¼ 35 my; minab ¼ 1.5

IR ¼ 25 my; minab ¼ 1.0

37.0 23.8 7.6 20.5 16.1 8.3 17.3 14.5

36.9 23.9 7.6 20.5 16.0 8.2 17.3 14.6

38.1 23.9 7.6 20.6 16.2 8.1 17.5 15.0

36.0 23.8 7.7 20.4 16.0 8.4 17.2 14.3

35.7 23.7 7.6 20.5 16.1 8.3 17.4 14.6

(28.9–47.5) (23.0–25.9) (6.7–8.0) (17.0–22.9) (11.8–20.4) (5.1–12.1) (13.7–20.6) (10.9–18.2)

16.1 (12.4–19.7)

(38.7–47.2) (23.0–25.9) (6.7–8.0) (16.8–22.8) (11.5–20.4) (5.1–12.1) (13.7–20.7) (10.9–18.3)

16.1 (12.4–19.6)

(29.5–49.4) (23.0–26.0) (6.6–8.0) (16.8–22.9) (11.4–21.2) (4.9–11.9) (13.8–21.0) (11.1–18.9)

16.6 (12.8–20.4)

(28.3–45.8) (23.0–25.9) (6.8–8.0) (17.1–22.8) (11.8–20.1) (5.4–12.3) (13.8–20.4) (10.7–17.9)

15.7 (12.2–19.1)

(27.9–45.0) (23.0–25.7) (6.7–8.0) (17.0–22.9) (11.8–20.5) (5.1–12.2) (13.8–20.7) (10.9–18.3)

16.2 (12.5–19.7)

95% credibility intervals are given in parentheses; dates are in millions of years; IR, mean of the prior for the ingroup root.

16, 8, 17, and 16–17 million years. The split between P. platyops and P. gilberti + P. longipes was estimated at
14–15 million years. Analyses that relaxed one or more fossil constraints are shown in Table 4. In comparison to analyses that included all four constraints, these analyses yielded estimates of divergence times that exhibit considerably more variation. Most striking is the contrast between analyses that relaxed both minima and both maxima. In the former case, the base of Macropodoidea was
21 million years, the split between Macropodinae and Potoroinae was
13 million years, and that between Bettongini to Potoroini was
11 million years. In the latter case, the base of Macropodoidea was
45 million years, Macropodinae to Potoroinae was
32 million years, and Bettongini to Potoroini was
27 million years. Other nodes show similar variation. Among individual constraints, relaxing the minimum of 23 million years for the macropodine–potoroine split had the

Section 2 (i.e., two minima and two maxima). When all fossil constraints were included, estimates of divergence times were in good agreement. Thus, changing the mean prior for the ingroup root from 35 to 25 million years had minimal impact and resulted in estimates that differed by 6 1.3 million years relative to estimates that used the 35 million year prior. Allowing the minab setting to range from 0.5 to 1.5, which increases the attraction between internal nodes, results in estimates that differed by 6 2.1 million years. Point estimates for the base of Macropodoidea (node A, Fig. 1A) were in the range of
36 to 38 million years, with 95% credibility intervals that range from
28 to
49 million years (Table 3). The split between Potoroinae and Macropodinae (node B, Fig. 1A) was estimated at
24 million years. Within Potoroinae, approximate estimates for Bettongini to Potoroini (node C, Fig. 1A), Aepyprymnus to Bettongia, the base of Bettongia, the base of Potoroini, and Caloprymnus to P. tridactylus were 20–21, Table 4 Divergence time estimates when different constraints are omitted Split

Base of Macropodoidea Base of Macropodinae Base of Macropus Base of Potoroinae Aepyprymnus to Bettongia Base of Bettongia Base of Potoroini P. platyops to P. gilberti + P. longipes C. campestris to P. tridactylus

Relaxed constraints Potoroine– macropodine minimum of 23 million years

Potoroini– Bettongini minimum of 15 million years

Potoroini– Bettongini maximum of 23 million years

Base of Macropus Both minima maximum of 8 million years

Both maxima

27.6 18.0 7.4 16.4 14.5 7.7 12.5 10.3

34.8 23.7 7.8 20.4 17.9 9.5 15.5 12.8

34.9 23.7 7.7 20.6 18.1 9.6 15.6 12.9

37.4 24.8 10.8 21.3 18.8 10.3 16.6 13.8

44.7 31.9 18.6 26.6 21.0 11.0 22.6 18.9

(21.8–34.1) (15.7–21.3) (6.3–8.0) (15.0–19.3) (11.5–17.7) (5.7–10.0) (10.5–15.0) (8.3–12.7)

11.5 (9.3–14.0)

(28.6–41.4) (23.0–25.4) (7.1–8.0) (17.7–22.7) (14.1–21.3) (7.1–12.0) (13.2–18.0) (10.5–15.3)

14.3 (11.7–17.0)

(28.5–41.8) (23.0–25.7) (7.1–8.0) (17.8–23.5) (14.2–21.7) (7.2–12.1) (13.3–18.2) (10.5–15.5)

14.4 (11.7–17.3)

(30.0–45.8) (23.1–28.3) (8.8–13.2) (18.9–22.9) (14.9–22.0) (7.6–13.3) (14.0–19.2) (11.1–16.6)

15.3 (12.4–18.2)

20.5 13.3 6.6 11.2 8.7 4.5 9.4 7.8

(12.6–30.5) (8.3–18.8) (4.1–7.9) (6.5–16.7) (4.8–13.8) (2.2–7.6) (5.4–14.2) (4.4–12.1)

8.8 (5.0–13.4)

(30.4–72.7) (23.3–53.0) (11.1–33.8) (17.8–45.0) (12.7–36.5) (5.7–20.9) (14.7–38.7) (11.6–33.0)

21.2 (13.5–36.6)

* Ninety five percent credibility intervals are given in parentheses; dates are in millions of years; all analyses assumed a mean prior of 35 million years for the ingroup root and set minab at 1.0.

M. Westerman et al. / Molecular Phylogenetics and Evolution 31 (2004) 476–485

— — —

0.1064 0.1646 0.1843 0.2015

— —

0.1327 0.0884 0.1597 0.1892 0.1941

0.0958 0.1474 0.1210 0.1843 0.1892 0.1917

— —

0.0958 0.0393 0.1450 0.0752 0.1474 0.1892 0.1941

0.0295 0.0983 0.0393 0.1376 0.0855 0.1548 0.1843 0.1941

— —

0.1253 0.1302 0.1401 0.1351 0.1376 0.1029 0.1425 0.1671 0.1712

0.0835 0.1143 0.1130 0.1204 0.1192 0.1315 0.0932 0.1609 0.1622 0.1683

— —

0.1130 0.0860 0.1353 0.1450 0.1524 0.1500 0.1426 0.1232 0.1573 0.1622 0.1745

0.0958 0.0983 0.0835 0.1450 0.1548 0.1769 0.1450 0.1401 0.1322 0.1720 0.1843 0.1917

— —

0.1401 0.1425 0.1204 0.1278 0.1351 0.1351 0.1351 0.1425 0.1548 0.1242 0.1450 0.1670 0.1769

0.0516 0.1401 0.1425 0.1179 0.1277 0.1376 0.1425 0.1401 0.1450 0.1573 0.1142 0.1548 0.1597 0.1720

— —

0.1069 0.1007 0.1646 0.1646 0.1425 0.1487 0.1744 0.1671 0.1597 0.1695 0.1548 0.1531 0.1720 0.1794 0.1917

0.0975 0.0737 0.0786 0.1499 0.1352 0.1241 0.1401 0.1376 0.1425 0.1450 0.1401 0.1499 0.1336 0.1499 0.1745 0.1769

—

M. eugenii M. giganteus M. rufogriseus M. parryi M. robustus P. gilberti P. longipes P. tridactylus P. platyops B. tropica B. penicillata B. lesueur B. gaimardi A. rufescens C. campestris H. moschatus T. vulpecula T. caninus 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

0.1106 0.1106 0.0983 0.0885 0.1720 0.1720 0.1364 0.1499 0.1720 0.1622 0.1597 0.1646 0.1548 0.1466 0.1671 0.1622 0.1892

—

0.1745 0.1794

16 14 13 12 11 10 9 8 7 6 5 4 3 2 1 Species

Table 5 Uncorrected p-values for the cytochrome b gene sequences

Although all analyses of the mitochondrial DNA sequences have consistently shown a separation of the two subfamilies of macropodids, only the Macropodinae consistently received strong bootstrap support. There was no unambiguous support for a Potorinae sensu Flannery (1989) with its two monophyletic tribes Potoroini and Bettongini. Although up to five species of Potorous had been recognized prior to 1888, more recently this had been reduced to just two—P. platyops and P. tridactylus, the latter with two subspecies—P. t. tridactylus on the Australian mainland and P. t. apicalis on the Bass Strait Islands and Tasmania. In 1980, a new species, P. longipes, was discovered in eastern Victoria by Seebeck and Johnston with a subsequent range extension. This discovery was followed in 1996 by the rediscovery of P. gilberti at Two Peoples Bay in Western Australia (Sinclair et al., 1996). Limited molecular and chromosomal studies (Seebeck and Johnston, 1980; Sinclair and Westerman, 1997) confirmed that P. gilberti and P. longipes were indeed genetically distinct from P. tridactylus. The mitochondrial sequences reported above suggest that Potorous lies outside of the clade containing Bettongia and Aepyprymnus and that whilst there is considerable genetic divergence between constituent species of this genus, their precise interrelationships are still not clear, indeed even the monophyly of this genus is not strongly supported. There is some suggestion from the ML and NJ trees that P. tridactylus may be, genetically, the most divergent species in the genus. This was also suggested when sequences from multiple exemplars of each species of potoroo covering most of the known species ranges were included for analysis. Thus it would seem that P. gilberti and P. longipes are genetically slightly more closely related to one another than either is to the extinct species, P. platyops. Given that P. tridactylus and P. gilberti have very similar karyotypes (2N ¼ 13#; 12$) with virtual G-band identity (Sinclair et al., 2000), the mtDNA sequence data suggest that the highly divergent karyotype seen in P. longipes (2N ¼ 24) must be one derived via a number of chromosomal fissions, and not ‘‘ancestral’’ as suggested by Johnston et al. (1984). The levels of sequence divergences between Potorous species are very large (see Table 5)—even the two subspecies of P. tridactylus differ by 3.4% (uncorrected

15

4. Discussion

0.0860

17

—

18

greatest impact on estimated divergence times (Table 4). Although relaxing the maximum constraint for the base of Macropus (node D, Fig. 1A) had minimal impact on most nodes, it did result in earlier estimates of divergence times within Macropus (not shown) and at the base of Macropus (Table 4).

0.1218 0.1621 0.1729

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p-value, data not shown). Among the divergence time estimates that we report, we have most confidence in those that employed the full assemblage of constraints. Analyses that omitted both minimum constraints and both maximum constraints resulted in dates that appear too young and too old respectively. Given a macropodine–potoroine divergence some 24 mya (see above), the genetic data suggest an initial radiation of Potoroines occurred soon after this, in the early Miocene, about 21 mya. This initial Potoroine radiation into separate Potorous and Aepyprymnus + Bettongia lineages led to their becoming a conspicuous element of many Miocene Faunas (see Archer et al., 1991), but they had become relatively less so by the end of this period when the Macropodines began to radiate. Subsequent radiation of the genus Potorous took place about 17.5 mya in the early-middle Miocene with the separation of P. tridactylus from the common ancestor of the other three species. This was followed by the divergence of P. platyops from the common ancestor of P. longipes and P.gilberti.—All these divergences being considerably earlier than the initial radiation of the genus Bettongia (see below), and well before the oldest known potoroo species fossil (a P3 from the early Pleistocene of Western Australia). Even the divergence between the mainland Australian and Tasmanian subspecies of P. tridactylus is apparently an old one (
2 mya), considerably predating the latest separation of Tasmania by the rising sea levels of Bass Strait about 12,000 years ago. Monophyly of the genus Bettongia was strongly supported by the combined mitochondrial gene sequence data. This data also clearly confirmed the inclusion of the rufous rat-kangaroo, A. rufescens, as sister to Bettongia as suggested by Flannery (1989), Flannery and Archer (1987), and Prideaux (1999). This particular relationship has also been supported by other mitochondrial (16S rRNA) and nuclear (Protamine P1) gene sequence data (Burk et al., 1998; Burk and Springer, 2000), but only equivocally by albumin MC0 F data (Baverstock et al., 1989). The phylogenetic relationships of the extinct desert rat-kangaroo, C. campestris, are, in contrast, much less clear. The morphological evidence is somewhat contentious, but seems to support a slightly greater affinity of Caloprymnus with Bettongia and Aepyprymnus (Flannery, 1989) than with Potorous (Bensley, 1903). Molecular evidence supporting the inclusion of Caloprymnus in a tribe Bettongini is consistent with these three genera sharing a number of morphological characters including possession of a post-glenoid process, fusion of pedal digital pads into a single entity, mastoid process of the periotic projecting markedly and discreteness of the ectoptympanic process of the periotic. There was, however, no molecular support for a Bettongini sensu Flannery (1989) in which Caloprymnus and Aepyprymnus are sister species. This latter relationship was suggested on the basis of four shared

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characters—medially rotated occlusal crest of I3 ; welldeveloped post-hypocristid; foreshortened premaxilla relative to all other potoroids and shortened P3 -canine diastema, though it was noted (Flannery, 1989, p. 21) that some of these characters might be homoplasious and/or related to dietary specializations. User trees predicated on the sister relationship of Aepyprymnus and Caloprymnus within Bettongini were consistently significantly worse fits to the data than others. Such a result would suggest that the four presumed synapomorphies identified by Flannery as ‘‘. . .convincing evidence of monophyly.’’ (of Aepyprymnus and Caloprymnus) are not so. Prideaux (1999) has also argued on morphological grounds that Aepyprymnus, Caloprymnus, and the extinct genus Millyowi are members of a lineage that had as sister taxon the extinct Borungaboodie hatcheri of southwestern Australia. He also noted that B. hatcheri is more derived than Bettongia and that rotation of the occlusal crest of I3 is also seen in B. lesueur and B. penicillata. In his more detailed morphological analysis of the genus Bettongia, Flannery (1989, p. 23) noted that ‘‘. . .the inter-relationships of the species. . . are not greatly illuminated by this study. However, there is some evidence that B. lesueur represents the most primitive branch. . ..’’ This suggestion, which contradicted earlier studies that B. penicillata (Bensley, 1903) or B. gaimardi (Tate, 1948) were the most plesiomorphic members of the genus, is borne out by the mitochondrial sequence data. Whilst the genetic interrelationships of the remaining three Bettongia species are not clear-cut, the genetic data suggest a possibly slightly closer relationship between B. penicillata and the northern bettong, B. tropica, than either of these with the Tasmanian bettong, B. gaimardi. This relationship is consistent with the fact that some previous authorities have regarded B. tropica as simply a subspecies of Brush-tailed bettong— Bettongia penicillata tropica. However, the observed size of the uncorrected genetic sequence divergence (2.95%, Table 5) between B. tropica and B. penicillata supports their recognition as distinct species as this value is consistent with those seen between other bettongia species (see Table 5). Such a recognition is consistent with the observations of Pope et al. (2000) on mitochondrial d-loop and microsatellite differences between these two species. As noted above, genetic estimates of divergences suggest an early divergence between bettongin taxa occurred in the middle to late Miocene, with estimates for the divergence of Bettongia from Caloprymnus and Aepyprymnus ranging between 15 and 18 mya. Aepyprymnus is estimated to have diverged from Caloprymnus at about the same time. Radiations within the genus Bettongia began in the middle to late Miocene about
8.3 mya, leading to the emergence of B. lesueur. Subsequent divergences within the genus were not until the

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early Pliocene (
5 mya), at about the time of increasing global ice volume and increasing dryness, both of which had profound environmental effects worldwide (White, 1994). These effects included the establishment of many dryland vegetation types and the advent of winter rainfall-summer dry climate regimes in southern parts of Australia. The suggested divergence dates are somewhat later than the time proposed by Prideaux (1999), who noted that, as only one strong synapomorphy unites species of Bettongia, the genus probably diverged close to the base of the bettongin radiation. The molecular data suggest that the separation of B. lesueur took place considerably earlier than the radiation of the other species in the genus. The inability of the mitochondrial sequence data to identify either a clearly monophyletic Potoroinae sensu Flannery (1989), or clearly monophyletic constituent tribes (Potoroini and Bettongini), together with the recent suggestion that other taxa included in the subfamily by Flannery (e.g., B. moyesi and W. lawsoni) may, in fact, not be potoroines (see Archer et al., 1999, p. 28), would suggest the urgent need for a reworking of the taxonomy of this group of macropodids.

Acknowledgments We would like to thank a number of people for the kind donation of samples. Dr. Joan Dixon of the Museum of Victoria made available material from Potorous platyops and Caloprymnus campestris, Dr. Lisa Pope gave us the B. tropica samples and Dr. Elizabeth Sinclair donated the P. gilberti DNA.

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