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Journal of Mammalian Evolution, Vol. 6, No. 1, 1999

DNA Sequence Evidence for Placement of the Ground Cuscus, Phalanger gymnotis, in the Tribe Phalangerini (Marsupialia: Phalangeridae) Aaron T. Hamilton1,2 and Mark S. Springer1

A region of mitochondrial DNA, including the 3' end of tRNA phenylalanine, the complete 12S rRNA and tRNA valine genes, and the 5' end of 16S rRNA, was sequenced for four phalangerids and one burramyid; additional marsupial sequences were extracted from GenBank. Parsimony, minimum evolution, and maximum likelihood analyses show that the ground cuscus, Phalanger gymnotis, groups with the tribe Phalangerini, not with the tribe Trichosurini as had been suggested on the basis of certain morphological characters. This result is in agreement with an earlier study using DNA hybridization and is supported by some morphological evidence as well. We conclude that the character states that link the ground cuscus with the Trichosurini are the result of convergence, and therefore the placement of several other species in the trichosurin genus Strigocuscus based on the same characters should be reconsidered. The hypothesized close relationship of two fossil taxa, Strigocuscus reidi and S. notialis, to Phalanger gymnotis is also questionable because the fossils do not share morphological synapomorphies that link the ground cuscus to the Phalangerini. KEY WORDS: Phalangeridae; marsupials; 12S rRNA; phylogeny; cuscus.

INTRODUCTION The marsupial family Phalangeridae includes brushtail possums (Trichosurus), scalytailed possums (Wyulda), and cuscuses (Ailurops, Phalanger, Spilocuscus, Strigocuscus). Phalangerids are found on Australia, New Guinea, and surrounding islands, with the present center of diversity on New Guinea. Evolutionary relationships among phalangerids have been studied using both morphological characters and molecular techniques (Tate, 1945; George, 1982, 1987; Flannery et al., 1987a; Springer et al., 1990). Aside from the plesiomorphic Ailurops ursinus, which is placed in a separate subfamily Ailuropinae, there are two main lineages recognized within the subfamily Phalangerinae, the trichosurins (tribe Trichosurini, containing Trichosurus, Wyulda, and sometimes Strigocuscus) and the phalangerins (tribe Phalangerini, grouping the genera Phalanger and Spilocuscus) (Flannery et al., 1987a; Norris, 1994). However, the relationship of the ground cuscus (Phalanger gymnotis) to these two clades has been debated (Flannery et al., 1987a; 1Department 2To

of Biology, University of California, Riverside, California 92521. whom correspondence should be addressed. email: [email protected]

1 1064-7554/99/0300-0001S16.00/0 © 1999 Plenum Publishing Corporation

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Hamilton and Springer

Springer et al., 1990; Norris, 1994). Because of the proposed morphological links between this species and some of the most important fossil phalangerids, resolving the phylogenetic relationships of P. gymnotis is important for understanding the evolutionary history of this group (Flannery et al., 1987a; Springer et al., 1990). The ground cuscus was originally classified in one of several species groups within the genus Phalanger. Tate (1945) grouped Phalanger gymnotis with P. orientalis/P. lullulae, P. vestitus, and P. celebensis. George (1982) placed P. gymnotis in a species group with P. celebensis and P. ornatus, split from the P. orientalis/P. lullulae group. George (1982, 1987) used the generic names Spilocuscus and Strigocuscus to split species groups from the genus Phalanger. In his updated arrangement (George, 1987), Strigocuscus celebensis (possibly more closely related to Trichosurus than to the phalangerins) was separated at the generic level from Phalanger gymnotis. It should be noted that the Papuan and Aru islands populations of the ground cuscus were occasionally referred to with different specific names, the former termed P. leucippus (Tate, 1945; George, 1987). Flannery et al. (1987a) revised the systematics of the Phalangeridae after cladistic analysis of a set of 35 morphological characters. The most likely phylogenetic hypothesis, based on six putative synapomorphies, grouped P. gymnotis with the trichosurins. Flannery et al. (1987a) also suggested an alternate hypothesis in which P. gymnotis was basal to the phalangerins, but concluded that this arrangement was unlikely because of increased homoplasy, especially when fossil taxa were considered. On the strength of their analysis, Flannery et al. (1987a) reclassified the ground cuscus as Strigocuscus gymnotis, placing it with S. celebensis, S. ornatus, and 5. mimicus in the tribe Trichosurini. In the scheme of Flannery et al. (1987a), Strigocuscus is a paraphyletic assemblage at the base of Trichosurini. This new classification was called into question when DNA hybridization data (Springer et al., 1990) supported the grouping of Phalanger gymnotis with P. orientalis and P. vestitus, to the exclusion of Spilocuscus. This arrangement, upheld by FITCH and KITSCH average-consensus jackknife analyses (Kirsch et al., 1997), placed P. gymnotis within the phalangerin clade rather than with Trichosurus. In consequence of this finding, Springer et al. (1990) proposed a less parsimonious interpretation of the morphological data of Flannery et al. (1987a) than advocated by those authors. Recently, Flannery (1994, 1995) returned the ground cuscus to the genus Phalanger in agreement with Springer et al. (1990). Other studies have addressed the phylogenetic placement of P. gymnotis. Hayman and Martin (1974) found that Phalanger gymnotis and Phalanger vestitus had 2n = 14 chromosomes, while in three Trichosurus species the 2n number was 20. Baverstock (1984) presented data from microcomplement fixation studies on albumin that suggested that P. gymnotis and P. carmelitae were more closely related to Trichosurus than to other species of Phalanger. In contrast, Colgan et al.'s (1993) electrophoretic study suggested that P. gymnotis is more closely related to P. orientalis than to Trichosurus. However, lack of a non-phalangerid outgroup complicates the interpretation of both the albumin and electrophoretic results, especially if rates of evolution have been unequal in different lineages (Baverstock, 1984). Norris (1994) suggested that the ground cuscus should not be classified within Strigocuscus based on a study of phalangerid periotic bones. Norris' (1994) results matched the classification of George (1987), which placed only one species (S. celebensis) in the tri-

Placement of the Cuscus in the Phalangerini

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chosurin genus Strigocuscus and retained the ground cuscus in the genus Phalanger. This scheme is consistent with the DNA hybridization data of Springer et al. (1990), allowing that the DNA hybridization data set did not include S. celebensis. Also, Norris (1994) excluded P. gymnotis as well as Spilocuscus from a clade containing P. orientalis and other "derived" phalangerins, whereas DNA hybridization (Springer et al., 1990) suggested that P. gymnotis is closely related to other species of Phalanger. The placement of Phalanger gymnotis has implications for the interpretation of the phalangerid fossil record. The described fossils from this family include two pairs of species from the Miocene and Pliocene in Australia, respectively, with each pair including a specimen from the genus Trichosurus and a form from the genus Strigocuscus, so named because of apparent similarity to the ground cuscus [which is referred to as Strigocuscus gymnotis by Flannery and Archer (1987) and Flannery et al. (1987a,b)]. If P. gymnotis is a trichosurin, then two parallel trichosurin lineages are evident since the mid-Miocene, while there is a notable absence of a fossil record for the phalangerin lineage (Phalanger and Spilocuscus). If P. gymnotis is more closely related to the phalangerins, then the relationships of the fossil Strigocuscus species would have to be reexamined. In the present study, we examine the phylogenetic position of Phalanger gymnotis using mitochondrial rRNA and tRNA gene sequences. We also discuss the implications of our results for understanding the evolution of key morphological characters in phalangerid systematics. MATERIALS AND METHODS DNA Extraction, Amplification, and Sequencing DNA was extracted from tissues of the following taxa using the procedure of Kirsch et al. (1990): Phalanger gymnotis (ground cuscus), Phalanger lullulae (Woodlark Island cuscus), Spilocuscus maculatus (spotted cuscus; 2 specimens), Spilocuscus rufoniger (black-spotted cuscus), and Burramys parvus (mountain pygmy possum). The primers 12G and 12C (Springer et al., 1995) were used to amplify a 1.18 kb region of the mitochondrial genome that includes the 3' end of tRNA phenylalanine, all of 12S rRNA and tRNA valine, and the 5' end of 16S rRNA. PCR products were cloned and sequenced as described elsewhere (Springer et al., 1995). Accession numbers for the new sequences are AF108218-AF108223. Collector information is provided in the GenBank files. Sequences for Phalanger orientalis (common or gray cuscus; U33496), Trichosurus vulpecula (common brushtail possum; AF031823), Macropus robustus (wallaroo; Y10524), and Vombatus ursinus (common wombat; U61078) were extracted from GenBank. The nonphalangerid outgroups (Burramys, Macropus, Vombatus) are representative of the diprotodontian families Burramyidae, Macropodidae, and Vombatidae. Among these, both burramyids and macropodids have been hypothesized as the sister taxon to the Phalangeridae (Pearson, 1950; Archer, 1984; Springer and Kirsch, 1991). Sequence Alignment and Phylogenetic Analyses Sequences were aligned using CLUSTAL V (Higgins and Sharp, 1988). Secondary structure models for 12S rRNA (Springer and Douzery, 1996), tRNA phenylalanine,

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tRNA valine (Anderson et al., 1982), and 16S rRNA (DeRijk et al., 1994) were used to refine alignments and to divide sequences into unpaired (loop) and paired (stem) regions. Regions where the alignment was ambiguous because of complex indels were excluded from phylogenetic analyses (Swofford et al., 1996). This resulted in a data set that included 1026 aligned nucleotide positions. The aligned sequences are available from the authors on request ([email protected]). Phylogenetic analyses were performed using PAUP* (Swofford, 1998). Parsimony, minimum evolution (Rzhetsky and Nei, 1992), and maximum likelihood (Felsenstein, 1981) methods were used to estimate the phylogeny from the sequence data. Searches were exhaustive for parsimony and heuristic for minimum evolution and maximum likelihood. Clade support was assessed using the bootstrap procedure (Felsenstein, 1985) with 500 replications. For the initial parsimony analysis, all characters were weighted equally; further analyses were conducted with stems downweighted relative to loops [weights, 0.62 vs. 1.0 (Springer et al., 1995; Burk et al., 1998)] and with transitions excluded in favor of transversions. Decay indices (Bremer, 1988) were calculated for both unweighted and weighted parsimony trees. The minimum evolution trees were constructed using the logdet/paralinear and maximum-likelihood distance corrections, the latter with both 2:1 and estimated transition: transversion ratios. Maximum-likelihood trees were obtained using the Hasegawa et al. (1985) model of sequence evolution (HKY85) with (1) a 2:1 transitionto-transversion ratio and (2) a maximum-likelihood estimate of the transition to transversion ratio, a gamma distribution of rates among sites (Yang, 1996), and an allowance for invariant sites. In the latter case, values that were obtained for the best tree in a heuristic search with the original data (ts: tv = 4.497, I = 0.599, a = 0.5294) were used in a subsequent bootstrap resampling analysis. A priori hypotheses for the placement of Phalanger gymnotis were tested using the Kishino and Hasegawa (1989), Templeton (1983), and winning-sites (Prager and Wilson, 1988) tests with parsimony and the Kishino-Hasegawa test with maximum likelihood. For all three methods of tree estimation, analyses were also run using only the "stem" and "loop" partitions of the data. The stem and loop partitions included 548 and 478 bp, respectively. Divergence times were estimated using Tamura-Nei transversion distances for 12S rRNA as outlined by Springer (1997) and Springer et al. (1997a).

RESULTS Parsimony An exhaustive maximum parsimony search with uniform character weighting resulted in a single best tree (302 steps) (not shown). A 500-replicate bootstrap generated the tree shown in Fig. 1. Phalangerids were monophyletic, but only at the 52% bootstrap support level (phalangerid monophyly required one extra step relative to the most parsimonious tree; see Table I). There was strong support (bootstrap value of 99%) for the grouping of Phalanger gymnotis with the phalangerin clade (tribe Phalangerini, including Phalanger orientalis, P. lullulae, Spilocuscus maculatus, and S. rufoniger) to the exclusion of Trichosurus and the outgroups. There was also strong support (93%) for the monophyly of Spilocuscus. Parsimony analyses conducted using other weighting schemes


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Fig. 1. Bootstrap tree for seven phalangerid and three outgroup sequences (500 replications) based on parsimony with uniform weights.

also showed high bootstrap support (98% with stems downweighted, 90% for transversion parsimony) for placing P. gymnotis within the Phalangerini (Table I). The best unweighted parsimony tree matched the DNA hybridization hypothesis grouping P. gymnotis with other species of Phalanger. To test this result against the alternative hypotheses, this tree was compared to trees in which P. gymnotis was constrained to group with Trichosurus or constrained to appear as the sister group to the Phalanger + Spilocuscus clade. Kishino-Hasegawa, Templeton, and winning-sites tests did not indicate a significant difference between the best tree and the alternate trees, except under transversions-only parsimony, where four of seven trees generated with P. gymnotis as a trichosurin were significantly worse than the best tree in the Templeton and KH tests (averaged values in Table II). Decay indices of 6, 4.86, and 4, respectively, were found for the Phalangerini under unweighted parsimony, parsimony with stems downweighted, and transversion parsimony (Table I).

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Table II. Significance Levels of Templeton, Winning-Sites, and Kishino-Hasegawa Tests for Parsimony Treesa Test Constraint P. gymnotis + Trichosurus (vs. best tree) Parsimony, unweighted Parsimony, stems at 0.62 Transversion parsimony (7 trees) P. gymnotis as sister group to other phalangerins (vs. best tree) Parsimony, unweighted (2 trees) Parsimony, stems at 0.62 (2 trees) Transversion parsimony

Templeton

Winning-sites

KH-P

0.0947 0.2701 0.0397*,a

0.1360 0.1360 0.0892a

0.0947 0.1484 0.0396*,a

0.7316a 0.8993a 0.3173

1.0000a 1.0000a 1.0000

0.7317a 0.9247a 0.3175

aAverage

P values in cases where there were two or more equally parsimonious trees. *Significant differences at P < 0.05.

Minimum Evolution Figure 2 shows a minimum evolution bootstrap tree calculated using a maximum likelihood-based distance correction, with an assumed 2:1 transition: transversion ratio. As in the parsimony analysis, bootstrap support for the union of P. gymnotis with the phalangerin clade was strong (100%). The same level of support was reached when using a maximum likelihood estimate for the transition: transversion ratio and when applying the logdet/paralinear distance correction (Table I). In comparison to parsimony bootstrap trees, minimum evolution trees indicated greater support (87-90%) for joining P. gymnotis as the sister taxon to P. orientalis plus P. lullulae (Table I). Spilocuscus monophyly was supported at or above 96% with different distance corrections (Table I). Bootstrap values for phalangerid monophyly were lower (44-65%) (Table I).

Maximum Likelihood Figure 3 shows a bootstrap tree generated under the HKY85 model with a 2:1 transition: transversion ratio. The maximum likelihood tree agreed with the parsimony and minimum evolution analyses in associating P. gymnotis with the phalangerin clade (99% bootstrap value). When estimated values for the transition: transversion ratio (4.497), I (0.599), and a (0.5294) were incorporated into the maximum likelihood analysis, the resulting tree supported P. gymnotis as a phalangerin (bootstrap value of 99%) and showed increased support for linking P. gymnotis with P. orientalis and P. lullulae (78 vs. 60%) and for phalangerid monophyly (83 vs. 69%) but less support for Spilocuscus monophyly (77 vs. 91%). Kishino-Hasegawa tests comparing the best heuristic ML tree with the alternative constrained trees indicated that the best tree was significantly better than any tree in which P. gymnotis is constrained to group with Trichosurus (Table III). However, the best tree was not significantly better than a tree in which P. gymnotis was the sister group to Phalanger + Spilocuscus instead of P. orientalis plus P. lullulae (Table III).

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Fig. 2. Bootstrap tree for seven phalangerid and three outgroup sequences (500 replications) based on a minimum evolution criterion, using the ML distance correction with an assumed 2: 1 transition-to-transversion ratio.

Stem and Loop Regions For all three tree-generation methods, the stem and loop partitions both supported the grouping of P. gymnotis with the phalangerins rather than with Trichosurus (trees not shown). With stems, bootstrap support ranged from 83% to 94%; bootstrap support based on loops, in turn, ranged from 88% to 96%. Kishino–Hasegawa tests (maximum likelihood) rejected an association of P. gymnotis with Trichosurus when loops only were considered, but there was no significant difference between the trees when stems only were analyzed (Table III). Divergence Time Estimates Divergence times estimated from Tamura–Nei transversion distances were 17.3 million years ago for the phalangerins (including P. gymnotis) vs. Trichosurus, 4.7 million


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Fig. 3. Bootstrap tree for seven phalangerid and three outgroup sequences (500 replications) based on likelihood, using the HKY85 model of sequence evolution with a 2: 1 transition-to-transversion ratio.

years for the split between Phalanger (including P. gymnotis) and Spilocuscus, and 4.3 million years ago for the P. gymnotis—P. orientalis split (see Table IV). DISCUSSION Systematics of the Phalangeridae and the Evolutionary Relationships of Phalanger gymnotis The mitochondrial gene sequence data show weak support for phalangerid monophyly. In contrast, DNA hybridization data provide robust support for a monophyletic Phalangeridae, as both bootstrapping (sensu Krajewski and Dickerman, 1990) and jackknifing (sensu Lapointe et al., 1994) validate this clade (Kirsch et al., 1997; Springer et

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Table III. Significance Levels of Kishino-Hasegawa Tests for Maximum-Likelihood Treesa KH-ML test

Constraint P. gymnotis + Trichosurus (vs. best tree) ML, HKY85, Ti:Tv = 2:l ML, parameters estimated from tree ML, partitioned (stems vs. loops) ML, loops only ML, stems only P. gymnotis as sister group to other phalangerins (vs. best tree) ML, HKY85, Ti:Tv = 2:1 ML, parameters estimated from tree ML, partitioned (stems vs. loops) ML, loops only ML, stems only

0.0146* 0.0262* 0.0112* 0.0393* 0.1202 0.693 0.4048 0.5141 1.000 0.3881

*Significant differences at P < 0.05.

al., 1997b). The monophyly of Spilocuscus and the tribe Phalangerini, respectively, are strongly supported by mitochondrial sequence data (here) and DNA hybridization (Kirsch et al., 1997; Springer et al., 1997b). The hypothesis that Phalanger gymnotis belongs to the Phalangerini instead of the Trichosurini is supported. Bootstrap values for the Phalangerini+P. gymnotis clade are high, and under the maximum likelihood criterion the Kishino-Hasegawa test showed that the best tree, linking P. gymnotis with other phalangerins, is significantly better than the alternative tree constraining P. gymnotis to group with Trichosurus. The relationships of P. gymnotis to other species within the Phalangerini are less clear, although it and the Woodlark Island cuscus, Phalanger lullulae, may both belong in a clade with Phalanger orientalis to the exclusion of Spilocuscus. The mitochondrial sequence data agree with an earlier DNA hybridization study (Springer et al., 1990) in supporting the association of Phalanger gymnotis with the phalangerins instead of with Trichosurus. This conclusion conflicts with the taxonomic arrangement proposed by Flannery et al. (1987a); however, it is in agreement with the analyses of George (1987) and Norris (1994) and the revised taxonomy used by Flan-

Table IV. Divergence Times (MYA) Estimated from Molecular Dataa Divergence time Taxa compared Trichosurini vs. Phalangerini Spilocuscus vs. Phalanger Phalanger orientalis vs. P. gymnotis Spilocuscus rufoniger vs. S. maculatus aDNA

1997).

DNA hybridization

12S rRNA transversions

19.9 11.9

17.3

7.4 3.2

4.7 4.3 2.1

hybridization values from Kirsch et al. (1997); 12S rRNA values are with XR adjustment (Springer,


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Table V. Key Morphological Characters Used to Classify Phalangeridsa Morphological character (derived state is listed) 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

(F11) Lachrymal is retracted from face (F12) Rostrum is narrowed (F13) The ventral rim of the orbit is visible from below (F14) Ectotympanic is excluded from anterior of postglenoid process (F15) P3 is at oblique angle to the molar row (F16) P3 has at least four cuspules (F18) P3 very large (F25) M2 preprotocrista does not contact parastyle (F27) I3-C1 diastema lost (F28) Molars complexly crenulated (F34) I3 is extremely small (N4) Small internal auditory meatus (N5) Mesial expansion of crista petrosa (N6) Reduction of crista promontorii medioventralis (N9) Reduction of promontorium (N10) Recessus mesotympanicus curved (N12) Incudal fossa enclosed by crista facialis petrosi (N14) Caudal rim of subarcuate fossa expanded (N15) Sigmoid sinus runs lateral to margin of subarcuate fossa Separation of striated pads on plantar surface of the pes

a(FNo.),

character from Flannery et al. (1987a); derived character states defined relative to Ailurops ursinus, the most plesiomorphic phalangerid. (NNo.), character from Norris (1994); nonphalangerid outgroups used to define derived character states. Character states for character 20 were inferred from Flannery et al. (1987a).

nery (1994, 1995). The available chromosome number information (Hayman and Martin, 1974) is consistent with this systematic arrangement. The one molecular study indicating that P. gymnotis might be a trichosurin was that of Baverstock (1984). The discrepancy between this result and the other molecular studies may be explained by Baverstock's alternative suggestion, that there is high rate variation in albumin evolution between different phalangerid lineages. Flannery et al. (1987a) rejected the proposed hypothesis that P. gymnotis was a phalangerin because six morphological characters united P. gymnotis with Trichosurus, whereas only three united P. gymnotis with phalagerins (Table V and Table VI). Springer et al. (1990) argued against strict adherence to the most parsimonious hypothesis based on morphology because of the conflict with DNA hybridization data that linked P. gymnotis with the phalangerin clade. A phalangerin affinity for P. gymnotis requires that the six morphological character states shared by Phalanger gymnotis and Trichosurus (characters 1-6, Tables V and VI) are the result of homoplasy instead of being shared derived traits (Springer et al., 1990). For a few of the morphological characters, there may be enough information to argue for such an interpretation, or to question phylogenetic utility because of high levels of variability. Character 5 (P3 at an oblique angle to the molar row; see Table V) is also found in some individuals of P. orientalis and P. carmelitae (Flannery et al., 1987a), and exhibits convergence whether or not P. gymnotis is included within the Phalangerini. Character 4 (ectotympanic is excluded from anterior of postglenoid process) is age dependent in P.

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gymnotis, the trichosurin state not appearing in juveniles; Flannery et al. (1987a) note that examination of specimens at different stages of development would be useful in assessing such characters, but that such sets of specimens are often unavailable. Additional morphological characters were surveyed by Norris (1994), although not for an identical set of species. In that study, periotic bone characters 14, 15, 16, 17, and 19 (see Table V) defined the tribe Phalangerini, including P. gymnotis. Character 18 defined "derived Phalanger," as opposed to Spilocuscus and P. gymnotis. Another phalangerin synapomorphy, complexly crenulated tooth enamel (character 10), was not listed as a feature of the ground cuscus by Flannery et al. (1987a). However, P. gymnotis apparently has more visible crenulation than the fossil species Strigocuscus reidi, which was listed as an argument against including S. reidi as a phalangerin, even if P. gymnotis is retained in Phalangerini (Flannery et al., 1987a; Flannery and Archer, 1987). Status of the Other Species Assigned to the Genus Strigocuscus Because the other species assigned to Strigocuscus by Flannery et al. (1987a) are also linked to the trichosurin tribe by the same character states that were used to rename P. gymnotis, their classification is also in doubt. The genus Strigocuscus of Flannery et al. (1987a) is not monophyletic; there are no synapomorphies that are distinct from the traits that define the tribe Trichosurini. Of the four extant species listed, the ground cuscus shares more character states with Trichosurus than do any of the other Strigocuscus species. Strigocuscus mimicus shares characters 1-3 and 5 with Trichosurus, as well as putative phalangerin synapomorphies (characters 8, 9, and 11) with P. gymnotis (Table VI). This similarity may indicate that S. mimicus is also a phalangerin, perhaps related to P. gymnotis, depending on how the above "trichosurin-convergent" traits evolved in these phalangerins. The status of Strigocuscus ornatus is less clear, as several character states for key characters (4, 6, 9, 11) remain unknown (Flannery et al., 1987a); Norris (1994) listed the species as one of the "derived Phalanger" clade based on periotic character 18. Strigocuscus celebensis lacks the character states that the ground cuscus and S. mimicus share with the phalangerins. S. celebensis apparently shares a derived character state of the toe-pads (separated striated surfaces) with Trichosurus (Flannery et al., 1987a). In contrast, striations cover the entire plantar surface of toe-pads in a set of species that includes P. gymnotis and other phalangerins and, also, Ailurops ursinus (Character 20, Table VI). Furthermore, S. celebensis may be linked to Trichosurus by one periotic bone character [No. 13, mesial expansion of crista petrosa (Norris, 1994)]. Character 12 (small internal auditory meatus) is also shared by the two taxa, but it appears also in Macropus and Bettongia and may be a plesiomorphic trait. S. celebensis was considered a trichosurin by George (1982, 1987) as well; it was not included in any of the molecular studies. Possible Relationships of the Fossil Species of Strigocuscus The conclusion that Phalanger gymnotis is a phalangerin affects the interpretation of the fossil record for the Phalangeridae. The fossil record includes pairs of species representing both Trichosurus and Strigocuscus in mid-Miocene and early Pliocene Australia (Flannery et al., 1987b; Flannery and Archer, 1987; Springer et al., 1990). One fossil

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species, Strigocuscus reidi from the Miocene, shares a possible synapomorphy with Phalanger gymnotis, P3 hypertrophy. This trait and phenetic similarity led to the hypothesis that S. reidi was closely related to or perhaps ancestral to P. gymnotis (Flannery et al., 1987a; Flannery and Archer, 1987). The second fossil species, Strigocuscus notialis from the Pliocene, lacked P3 hypertrophy but was otherwise similar to S. reidi; its relationship to P. gymnotis was not as clear (Flannery et al., 1987b). The hypothesis about the evolutionary significance of S. reidi was tied to the classification of P. gymnotis as a trichosurin (Flannery et al., 1987a; Flannery and Archer, 1987). The lack of some "phalangerin" characters in the fossils therefore supported the conclusion that those character states had evolved independently in phalangerins and P. gymnotis (Flannery et al., 1987a; Flannery and Archer, 1987). However, inclusion of P. gymnotis in the Phalangerini forces us to consider other interpretations of the group's evolutionary history. One possibility that has been suggested (Springer et al., 1990) is that the fossil species of Strigocuscus may be considered phalangerins due to their apparent link with P. gymnotis. A difficulty with this hypothesis is that Strigocuscus reidi lacks morphological traits (Nos. 8, 9) that are considered shared derived character states for the Phalangerini (Flannery et al., 1987a; Flannery and Archer, 1987). However, one of these characters does show an intermediate condition (i.e., reduction in the I3-C1 diastema) in S. reidi. Flannery et al. (1987a) proposed that, if P. gymnotis were a phalangerin and not a trichosurin, then it would be a sister group to a clade including Spilocuscus and other members of Phalanger. Hypothesizing that the ground cuscus is basal to the other phalangerins would allow an animal similar to 5. reidi to be the ancestor of not only P. gymnotis but also the rest of the phalangerin clade. This could explain why this fossil form lacks the phalangerin synapomorphies while keeping it as a possible ancestor of P. gymnotis (Flannery et al., 1987a) and other phalangerins. The crenulation of tooth enamel in P. gymnotis may not be as complex as that of other phalangerins but is apparently visibly greater than in the two fossil species (Flannery et al., 1987a; Flannery and Archer, 1987). Among phalangerins, P. orientalis has less crenulation than the montane species such as P. carmelitae, P. interpositus, and P. vestitus (Flannery et al., 1987a), indicating a possible trend of increasing crenulation. Also, as mentioned above, Strigocuscus reidi shows a reduction in the I3-C1 diastema compared to Trichosurus (Flannery and Archer 1987). Loss of this diastema (character 9) is one of the phalangerin synapomorphies that is present in P. gymnotis. Both DNA hybridization and mitochondrial sequences place the ground cuscus closer to other members of Phalanger than is Spilocuscus. This arrangement, although not well supported by bootstrap or significance tests with sequence data, presents a problem to the hypothesis that P. gymnotis represents a transition between a S. reidi-like ancestor and derived phalangerins. If the other members of Phalanger are more closely related to the ground cuscus than they are to Spilocuscus, characters shared by the genera Spilocuscus and Phalanger, but not P. gymnotis, would have to be convergent, or else lost in P. gymnotis. The multiple convergent characters linking P. gymnotis and Trichosurus may have arisen in P. gymnotis after it diverged from Spilocuscus and other species of Phalanger—which may have occurred late in the lineage's history if the close relationship between P. gymnotis and species such as P. orientalis indicated by the molecular data is upheld in future studies. Molecular estimates of divergence times have implications for the hypothesis that P.


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gymnotis and S. reidi are closely related. Molecular data suggest that P. gymnotis diverged from other Phalanger species