A Nearly Complete Juvenile Skull of the Marsupial Sparassocynus derivatus from the Pliocene of Argentina, the Affinities of “Sparassocynids”, and the Diversification of Opossums (Marsupialia; Didelphimorphia; Didelphidae)

Definition

We propose a stem-based phylogenetic definition of Didelphimorphia, namely the most inclusive clade containing Didelphis marsupialis, but not Caenolestes fuliginosus or Phalanger orientalis.

DIDELPHIDAE GRAY, 1821

SPARASSOCYNINI REIG, 1958b, new rank

Revised Diagnosis

Sparassocynins differ from other known didelphids in the following combination of probable apomorphic features (most of which can currently only be assessed in Sparassocynus): i1–2 without a distinct lingual cusp or heel (also seen in didelphins); molar dentition strongly carnassialized, with postmetacrista longer, paracone and protocone smaller, protoconid taller, and talonid narrower (with the m3 hypoconid lingually placed relative to the protoconid) than in most other known didelphids; rostrum proportionally shorter and posterior braincase proportionally wider than in other known didelphids; premaxillary rostral process sensu Voss and Jansa (2003, 2009) absent; maxillopalatine fenestrae present but small (not extending anteriorly beyond the midpoint of m1 or posteriorly beyond the posterior margin of m2) and well separated mediolaterally; posterior palatal margin (postpalatal torus) with distinct “corners” (as in all known didelphids except Caluromys, Caluromysiops, and Glironia); lacrimal with a distinct tubercle (the orbital margin of the lacrimal is smoothly rounded in all other known didelphids); maxilla and alisphenoid in contact in the ventral floor of the orbit (also seen in Lutreolina, Monodelphis, and Thylatheridium); midfrontal suture fused in subadults (also seen in Chironectes, Didelphis, Lutreolina, and Philander); postorbital processes well developed, becoming enormous and hornlike (larger than in any other known didelphids) in adult specimens; prominent squamosal epitympanic sinus present lateral to the epitympanic recess (also seen in Caluromysiops); hypotympanic sinus enormously expanded both ventrally and dorsally, lateral to endocranial cavity; alisphenoid tympanic process very large, forming ventrally expanded hemisphere, with posterior margin separated from the rostral tympanic process of the petrosal by a narrow gap; after exiting the endocranial cavity, the mandibular division of trigeminal nerve is enclosed in a bony canal in the medial wall of the hypotympanic sinus; transverse canal foramen and foramen ovale open within shared depression; rostral tympanic process of the petrosal extends the length of the promontorium, forming a triangular lamina the apex of which points laterally; rostral and caudal tympanic processes in contact but unfused; ectotympanic is a mediolaterally very broad half-cylinder, with a thickened lateral edge.

SPARASSOCYNUS MERCERAT, 1898

SPARASSOCYNUS DERIVATUS REIG and SIMPSON, 1972

Locality and Horizon

MMP M-5292 (the focus of this paper) is from Playa Las Palomas, near Mar del Plata, Buenos Aires Province, Argentina (Fig. 1). The specimen was collected from a paleoburrow deposit in the lower part of paleosol P3 of the “Aloformación Playa San Carlos” (APSC) of the Chapadmalal Formation (Fig. 2; Isla et al. 2015). The age of the Chapadmalal Formation is approximately 5–3 MYA (= Chapadmalalan SALMA; Schultz et al. 1998; Woodburne 2010), with the APSC comprising the oldest levels (Fig. 2; Isla et al. 2015). The other S. derivatus specimens examined here are also from the Chapadmalal Formation (Fig. 2). Locality information for MMP S-172 (the holotype), MMP S-S-339, S-571 and S-678 is given in Reig and Simpson (1972). MMP M-1341 is from the Playa La Estafeta locality, “alocapa 5” of the “Aloformación Playa Los Lobos” (APLL). A Plio-Pleistocene stratigraphic profile for Mar del Plata region, including the Chapadmalal Formation and indicating the levels where these S. derivatus specimens were collected, is shown in Fig. 2.

Identity

MMP M-5292 is clearly not Sparassocynus maimarai, due to its very small entoconids, and the lack of a lingual cingulid extending between the paraconid and metaconid on m1–3 (Fig. 3b; Abello et al. 2015). The P1 and P2 both have a posterolingual cuspule (Fig. 3a), suggesting that it represents S. derivatus, rather than S. bahiai (see Reig and Simpson 1972).

Cranial Morphology and Phylogenetic Affinities of Sparassocynus and Hesperocynus

Fusion of the interparietal with the supraoccipital, which is clearly present in the Sparassocynus derivatus specimen described here (MMP M-5292), is a distinctive cranial feature that we have observed only in didelphids (see Nesslinger 1956; Clark and Smith 1993; Voss and Jansa 2009) and dactylopsiline petaurids (pers. obv.) among metatherians. Dactylopsilines are diprotodontian marsupials, and are otherwise radically different from Sparassocynus in terms of their craniodental anatomy. Its presence in Sparassocynus seems strong evidence that this taxon is member of Didelphimorphia sensu stricto. The presphenoid of Sparassocynus is also exposed in the roof of the nasopharyngeal orifice, above the posterior palatal margin, as in extant didelphids, but unlike the non-didelphids examined by Voss and Jansa (2009), namely caenolestids, dasyurids, and peramelids, in which it is concealed by the vomer.

Other probable apomorphies present in Sparassocynus suggest that it falls within crown-clade Didelphidae. These include the presence of a posterior palate with distinct corners, which is a synapomorphy of Hyladelphinae+Didelphinae, and contact between the maxilla and alisphenoid, which is seen in Monodelphis and Lutreolina but no other extant didelphids (Voss and Jansa 2003, 2009); among other metatherians, we have seen this latter feature only in some diprotodontians. Sparassocynus shares with didelphins the absence of a lingual cusp or heel on the lower incisors, and also fusion of the midfrontal suture, while Sparassocynus and Hesperocynus resemble Chacodelphys, Lestodelphys, Lutreolina, and Monodelphis in that the hypoconid is lingual to the protoconid, and many extant didelphids share with Sparossocynus the presence of only two distinct cuspids on the trigonid of dp3; however, all of these latter apomorphic features are also seen in some non-didelphid metatherians (Voss and Jansa 2009; pers. obv.).

The auditory region of Sparassocynus differs radically from those of all extant didelphids, as has been discussed by many previous authors (e.g., Reig 1958b; Reig and Simpson 1972; Simpson 1972, 1974; Archer 1976a; Forasiepi et al. 2009): most obviously, it is far more inflated in Sparassocynus than in any extant didelphid, with the hypotympanic sinus extending far dorsally either side of the endocranial cavity (see “Description” above). This morphology is evidently also present, although not quite as well-developed, in Hesperocynus dolgopolae (see Simpson 1974; Forasiepi et al. 2009). Species of another carnivorously-adapted Neogene didelphimorphian genus from Argentina, Hyperdidelphys, also have hypotympanic sinuses that are larger than any extant didelphid: they are moderately large in the Huayquerian species H. pattersoni, and reach an extreme in the Chapadmalalan species H. dimartinoi (see Goin and Pardiñas 1996; Goin et al. 2016: fig. 5.5). Voss and Jansa (2009: 100) reported that Hyperdidelphys is a didelphine, while Goin and Pardiñas (1996) concluded that it is probably most closely related to Lutreolina among extant didelphids. Regardless, it does not appear to be closely related to Sparassocynus or Hesperocynus. An undescribed specimen (MMP 2598-M) of Thylophorops chapadmalensis (a probable didelphin; Voss and Jansa 2009) from the Chapadmalal Formation shows that this taxon also has auditory bullae that are larger than those of any extant didelphid (R. S. Voss, pers. comm.). Thus, it seems that, during the Neogene, multiple didelphid lineages independently evolved hypotympanic sinuses that are markedly larger than those of extant didelphids.

Another major difference between Sparassocynus and most extant didelphids is the presence of a well-developed epitympanic sinus (see “Materials and Methods: Anatomical Terminology”) in the squamosal, posterolateral to the epitympanic recess. The entrance of this sinus was probably covered by the pars flaccida of the tympanum in life, as in dasyuromorphians and in those peramelemorphians in which the sinus is present (Archer 1976a). Based on available material, it is uncertain whether or not a similar sinus was present in Hesperocynus. Most, but not all, extant Australian marsupials have a well-developed squamosal epitympanic sinus posterolateral to the epitympanic recess, and this feature may even represent a synapomorphy of the extant Australian radiation (= Eomarsupialia sensu Beck et al. 2014; Marshall et al. 1990; but see Godthelp et al. 1999); regardless, its presence in Sparassocynus is almost certainly homoplastic.

There is disagreement in the literature as to whether any extant didelphids have a structure that can reasonably be described as a squamosal epitympanic sinus (Archer 1976a, 1982; Wible 1990; Voss and Jansa 2009). We agree with Archer (1976a: 304), who reported that “[i]n some didelphids (e.g., Metachirus) there is a depression in the squamosal which is clearly the homologue of this sinus” (see also Archer 1982: 461; Reig et al. 1987: figs. 39–44), and incompletely cleaned didelphid crania (e.g., AMNH M-212909, a specimen of Chironectes minimus) suggest that, where present, this depression may be covered by the pars flaccida; however, if present at all, this depression is usually very weakly developed. The sole exception to this among extant didelphids is Caluromysiops irrupta, in which a prominent, well-excavated epitympanic sinus is present within the squamosal, posterolateral to the epitympanic recess (see Reig et al. 1987: fig. 44c). The presence of an epitympanic sinus in Caluromyisops was also noted by Wible (1990: 200), who referred to it as a “suprameatal fossa,” following Segall (1943; see “Materials and Methods: Anatomical Terminology” above).

Reig and Simpson (1972) noted several similarities between the auditory region of Sparassocynus and that of dasyurids, especially Dasyuroides and Dasycercus, which have particularly inflated auditory sinuses. However, there are some key differences, notably in that the rostral and caudal tympanic processes of the petrosal are closely approximated, but still separated by a distinct gap, in Sparassocynus, whereas in all extant dasyurids these processes are fused, forming a “petrosal plate” sensu MacPhee (1981; Archer 1976a; Wroe 1999; Sánchez-Villagra and Wible 2002; Ladevèze et al. 2008; Voss and Jansa 2009).

A distinctive apomorphy of Sparassocynus (and apparently also Hesperocynus; Forasiepi et al. 2009) is the path of the mandibular division of the trigeminal nerve (V₃) after it leaves the endocranial cavity, namely within a bony canal in the medial wall of the hypotympanic sinus, formed by the alisphenoid (Fig. 9); it has no close counterpart in either extant didelphids or dasyurids, in which this nerve is either unenclosed after it exits the endocranium, or is enclosed by a simple strut or lamina of the alisphenoid tympanic process (Wroe 1997; Voss and Jansa 2003, 2009). In fact, the closest resemblance within metatherians is seen in phascolarctids (the extant koala and fossil relatives), although here the mandibular division of the trigeminal nerve is enclosed within a bony canal in the roof of the hypotympanic sinus, rather than in the medial wall (Aplin 1987, 1990; Louys et al. 2009). The condition seen in Sparassocynus and Hesperocynus may represent a plausible structural (although clearly not phylogenetic) ancestral morphology for the phascolarctid condition: progressive medial expansion of the hypotympanic sinus first leads to the mandibular division of the trigeminal nerve being incorporated into the medial wall of the sinus (the condition in Sparassocynus and Hesperocynus), with further medial expansion leading to the development of a nerve canal in the roof of the sinus (the phascolarctid condition).

In summary, qualitative interpretation of the known morphology of Sparassocynus and Hesperocynus suggests that they are probably members (albeit highly autapomorphic ones) of the didelphimorphian crown-clade, i.e., Didelphidae. This conclusion is confirmed by our Bayesian undated and dated total evidence phylogenetic analyses, both of which strongly support monophyly of Didelphidae with Sparassocynus and Hesperocynus within this clade (BPP = 1.00), with these two genera forming a strongly-supported clade with the extant genus Monodelphis (BPP = 1.00), within the subfamily Didelphinae (BPP = 1.00). Interparietal-supraoccipital fusion was not recovered as a synapomorphy of Didelphidae in our analyses, because the condition in caenolestids Caenolestes and Rhyncholestes (used as two of the seven outgroup taxa here) is unknown (Voss and Jansa 2009: 36). However, exposure of the presphenoid in the roof of the nasopharyngeal orifice above the posterior palatal margin (present in Sparassocynus) was found to be an unambiguous synapomorphy of Didelphidae, and presence of a posterior palate with corners (also present in Sparassocynus) was an unambiguous synapomorphy for Hyladelphinae+Didelphinae (Electronic Supplementary Material, Table S2).

Based on these results, recognition of the distinct family Sparassocynidae is unjustified; instead, we argue that, within the taxonomic framework proposed by Voss and Jansa (2009), they are most appropriately classified as a tribe, Sparassocynini, within Didelphidae. However, as already noted (see “Materials and Methods: Systematics” above), this would also require raising the Monodelphis and possibly also the Tlacuatzin lineages to tribal rank, as Monodelphini and Tlacuatzini, respectively.

Differences between Undated and Dated Bayesian Phylogenetic Analyses

The position of Sparassocynus and Hesperocynus differs markedly between the undated and dated phylogenetic analyses: they form a clade with Monodelphis peruviana in the undated analysis, but are placed as sister to a monophyletic Monodelphis in the dated analysis. Each of these arrangements is supported by a single unambiguous synapomorphy, but support for the Sparassocynus + Hesperocynus + M. peruviana clade found in the undated analysis is much lower (BPP = 0.5) than that for the Monodelphis clade found in the dated analysis (BPP = 0.87). The difference in topology between the two analyses seems to be related to the highly autapomorphic morphology of sparassocynins, as indicated by their very long branch in the undated analysis (Fig. 15). Clock models (whether for molecular, morphological, or total evidence data) assume that evolutionary change is proportional to time (Zuckerkandl and Pauling 1965; Ronquist et al. 2012a). Relaxed clock models allow evolutionary rate to vary somewhat between branches, but they still tend to disfavor extreme rate variation (Beck and Lee 2014; Beaulieu et al. 2015; Phillips and Fruciano 2018). Presumably, the extreme morphological rate variation implied by the undated topology (specifically, the much higher rate along the Sparassocynus + Hesperocynus branch than anywhere else in the tree; Fig. 17) is too high to be accommodated by the IGR clock model in the dated analysis, and so the Sparassocynus + Hesperocynus clade is shifted deeper within the tree, yet still closely related to Monodelphis, with which it shares numerous synapomorphies (Electronic Supplementary Material, Table S2).

Our results agree with other recent studies that have found that dated Bayesian analyses of datasets that include fossil taxa (whether as tip-dating, or, as here, in combined tip-and-node dating) can result in major topological differences relative to undated analyses (King et al. 2017; Turner et al. 2017; Lee and Yates 2018). In cases where dated and undated analyses show major topological differences, whether one should be consistently preferred over the other is currently an open question. However, if there is a broad relationship between time and morphological change (as there undoubtedly is for time and molecular change), then, ceteris paribus, we might expect dated analyses to result in more accurate phylogenies than their undated equivalents (see also Lee and Yates 2018). Some support for this assumption is provided by simulation studies by King et al. (2017), who found that Bayesian tip-dating outperformed (undated) parsimony; however, they did not investigate the performance of undated Bayesian analysis.

Having said that, there may be periods of time (perhaps very short) where rates of morphological evolution might be much higher than at other times, for example in the aftermath of a mass extinction event, when lineages evolve rapidly to fill newly vacant ecological niches. In such cases, current clock models might be too restrictive, overly “smoothing” this extreme rate variation (see e.g., Beck and Lee 2014; Beaulieu et al. 2015; Phillips and Fruciano 2018); future clock models might incorporate Lévy processes (see also Waddell 2008), which can allow sudden evolutionary “jumps” and which have already been used to model trait evolution (Landis et al. 2013; Duchen et al. 2017). Further studies are clearly needed to investigate the behavior of clock models with morphological and total evidence data (Parins-Fukuchi and Brown 2017). In the meantime, for datasets that include fossil taxa, we recommend that authors should carry out both undated and dated analyses, to determine whether they result in very different topologies.

Divergence Dates and Diversification of Didelphimorphia

Our estimated dates for divergences within Didelphidae are broadly similar to those of Jansa et al. (2014; see Table 1), but considerably younger than those of several other molecular clock studies (Steiner et al. 2005; Meredith et al. 2008, 2011; Mitchell et al. 2014; Vilela et al. 2015; Dias and Perini 2018). Our use of a total evidence dataset and tip-and-node dating (rather than a molecular-only dataset with node dating) does not seem to be the major driver for the younger dates, because repeating our dated analysis using molecular data only resulted in very similar estimates (Table 1). Our divergence estimates for the molecular-only analysis were also almost identical regardless of whether a fossilized birth-death or a Yule (= uniform) prior on tree shape was used (Table 1), suggesting that the tree prior does not explain our younger dates either (contra the findings of Condamine et al. 2015; Matzke and Wright 2016; Zhang et al. 2016). Instead, they are presumably due to our choice of fossil calibrations, and also differences in clock models and programs between analyses (see Inoue et al. 2010; Barba-Montoya et al. 2017). Pending a detailed exploration of these discrepancies, the divergence dates presented here should be viewed as tentative. However, they match well with Goin’s (1991, 1995) proposal that extant didelphids represent a comparatively young, Neogene radiation. We use our dated phylogeny, in combination with additional fossil and paleoenvironmental evidence, to propose a revised scenario for the diversification of Didelphimorphia.

Similar to Jansa et al. (2014), we find evidence of a long branch between the divergence of Didelphimorphia from other marsupials (59.8 MYA; 95% HPD: 48.1–72.9 MYA) and the first diversification within Didelphidae (24.3 MYA; 95% HPD: 19.0–30.5 MYA). This implies either extensive extinction within Didelphimorphia, with the total loss of early-branching lineages, or (less likely) that there are additional, deep-diverging didelphimorphian lineages that are still extant but have yet to be discovered. Assuming the former explanation is correct, then an obvious potential driver of extinction within Didelphimorphia is the major drop in global temperatures and associated environmental change that occurred at the Eocene-Oligocene boundary (Hansen et al. 2013). This event coincided with extensive turnover in South American mammal faunas, with the loss of many marsupialiform lineages (Goin et al. 2010a, 2012, 2016, in press). Competition from platyrrhine primates and caviomorph rodents, both of which appear to have reached South America during the middle-to-late Eocene (Antoine et al. 2011; Bond et al. 2015; Boivin et al. 2017), could potentially also have played a role, particularly if the extinct didelphimorphian lineages included forms that were more specialized for frugivorous or herbivorous diets than are extant didelphids (Gardner 2008). However, our understanding of the early diversification dynamics of Didelphimorphia is hampered by the difficulty in unequivocally distinguishing didelphimorphians from other dentally plesiomorphic crown- and stem-marsupials, particularly when only isolated dental remains are available (Voss and Jansa 2009).

We date the origin of Didelphidae to the Oligocene or early Miocene (24.3 MYA; 95% HPD: 19.0–30.5 MYA; Table 1). Goin et al. (2007a) identified the oldest known putative didelphids based on isolated dental specimens from Level C of the Lower Fossil Zone of the Colhue-Huapi Member of the Sarmiento Formation in southern Argentina. This is the type locality of the Colhuehuapian SALMA and is estimated to span 21.0–20.1 MYA (Dunn et al. 2012). The specimens include a small taxon that Goin et al. (2007a) suggested, based on its very small entoconid, may be a plesiomorphic relative of Monodelphis and Thylatheridium (i.e., a member of Monodelphini). However, it should be noted that reduction of the entoconid is homoplastic within Didelphidae: among extant didelphids, a small entoconid is also seen in Chacodelphys (Voss et al. 2004; Voss and Jansa 2009), which is a member of Thylamyini, not Monodelphini (Díaz-Nieto et al. 2016). Goin et al. (2007a) also identified a second Colhue-Huapi taxon as a possible caluromyine. These Colhue-Huapi specimens largely postdate our estimate for the origin of Didelphidae, and so could represent very early representatives of the family. However, our point estimates for the vast majority of divergences within Didelphidae (including the origin of both Monodelphini and Thylamyini) are younger than the Colhuehuapian. In any case, the referral of these highly fragmentary remains to Didelphidae is uncertain; the putative caluromyine, for example, may in fact be a microbiotherian (Goin et al. 2007a).

It is striking that didelphids have not been reported from the Pinturan (~19–18 MYA; Cuitiño et al. 2016) or Santacrucian (~18–15.6 MYA, including the “Friasian” and “Colloncuran”; Cuitiño et al. 2016) faunas of southern South America, even though the latter in particular are extremely rich in fossil mammals (Vizcaíno et al. 2012). Instead, the dominant small-to-medium-bodied insectivorous-omnivorous mammals in these faunas are microbiotherians and paucituberculatans, with sparassodonts filling carnivorous niches (Marshall 1990; Bown and Fleagle 1993; Dumont et al. 2000; Croft 2007: appendix; Goin et al. 2010b; Abello et al. 2012; Chornogubsky and Kramarz 2012; Prevosti et al. 2012). Assuming that Didelphidae had indeed originated prior to the Pinturan, as suggested by our divergence dates, then the family was presumably restricted to lower latitudes at this time. However, the fossil record of mammals in northern South America is much poorer than that of the southern part of the continent (Goin et al. 2016), and so our knowledge of this key period in didelphid evolution is limited. Antoine et al. (2016, 2017) reported representatives of Didelphimorphia (sensu stricto) from the early Miocene (Colhuehuapian-Santacrucian; ~20–17 MYA) CTA-63 site at Contamana, Peru. However, it is unclear whether these represent didelphids or not, with Antoine et al. (2017: 9) referring to them as being of “uncertain affinities.” Nevertheless, our divergence dates suggest that they could potentially belong to the deepest lineages within Didelphidae.

Fossil didelphids from the middle Miocene (Laventan SALMA; ~15.6–11.6 MYA) of Colombia and Peru include the oldest putative representatives of the extant genera Marmosa and Thylamys (Marshall 1976; Goin 1997a; Antoine et al. 2013, 2016, 2017). Based on our estimated divergence dates, the Marmosa lineage (= Marmosini) had probably originated by this time (16.2 MYA; 95% HPD: 12.6–20.1 MYA), but the Thylamys lineage had not; in fact, our estimate for the split between Lestodelphys and Thylamys is in the latest Miocene or Pliocene (4.6 MYA; 95% HPD: 3.0–6.6 MYA). If this date is broadly accurate, then the middle Miocene “Thylamys” species cannot belong to this genus. Among other putative didelphid material from the Laventan of Colombia, Goin (1997a: 194–195) briefly discussed a partial skull (lGM KU-IV-1) that apparently represents a new didelphid genus, and tentatively referred to this taxon some additional isolated dental remains that show carnivorous adaptations (e.g., a strongly developed paracristid). In an earlier paper, Goin (1995) suggested that this taxon may be related to sparassocynins; however, the skull is still undescribed, and so its affinities remain uncertain.

Several fossil didelphids have been described from the lowest levels of the Ituzaingó Formation (= the “Mesopotamiense” or “Conglomerado Osífero”) of Entre Rios Province, northeast Argentina (Ameghino 1898, 1899, 1900; Reig 1955, 1957b, 1958b; Marshall 1977c; Cione et al. 2000; Goin et al. 2013). They include two putative representatives of extant genera, namely Philander entrerianus and Chironectes sp. A third taxon, Zygolestes pararensis, has been proposed to be closely related to the extant Gracilinanus (Goin 1997b; Goin et al. 2000, 2013). However, none of these proposed relationships have been tested via formal phylogenetic analysis. The age of the “Mesopotamiense” has been controversial: several authors have suggested that it may represent a mixed fauna (Marshall 1977c; Goin 1997b; Cione et al. 2000), but this interpretation now appears to be incorrect (Goin et al. 2013). A radiometric date of 9.47 MYA from the underlying Paraná Formation provides a maximum age for this fauna (Pérez 2013), but there is debate (e.g., Cione et al. 2000; Brandoni 2013; Brunetto et al. 2013; Carrillo et al. 2014) as to whether it falls within the Chasicoan (~10–8.7 MYA; Zarate et al. 2007) or Huayquerian SALMA. In either case, Philander entrerianus would seem to predate our estimate for the age of the Didelphis-Philander split (4.1 MYA; 95% HPD: 3.3–5.4 MYA). However, we note here that Didelphis and Philander are craniodentally very similar (Voss and Jansa 2009), with the major difference being that the skull of Philander is somewhat smaller and less robust (Voss et al. 2018: 26); P. entrerianus is currently known only from a single lower molar (the holotype) and a partial right mandible (Goin et al. 2013), and so it cannot be assessed for cranial features.

The oldest putative Didelphis species, D. solimoensis from the Huayquerian Acre local fauna in western Brazil (Cozzuol et al. 2006; Antoine et al. 2017), also predates our estimate for the Didelphis-Philander split. However, like P. entrerianus, D. solimoensis is known from extremely limited remains, specifically a single partial right mandible (Cozzuol et al. 2006). Jansa et al. (2014: supporting information) considered the oldest fossil taxon to preserve diagnostic synapomorphies of Didelphis to be D. brachyodonta from the Chapadmalal Formation (Chapadmalalan SALMA; see below). At least three other didelphids are known from the Acre local fauna. Goin and de los Reyes (2011) described Lutreolina materdei, the oldest putative representative of this genus, based on a single lower molar; the Huayquerian age of L. materdei is approximately congruent with our estimate for the age of the Lutreolina lineage (5.5 MYA; 95% HPD: 4.1–7.4 MYA). Czaplewski (1996) described, but did not name, a further two taxa (a larger form resembling Hyperdidelphys and Thylamys, and a smaller form that might represent a marmosin) from the Acre local fauna, also based on isolated molars.

Turning now to the record from southern South America, Reig (1957a; see also Marshall 1976) identified a partial edentulous mandibular ramus from the Arroyo Chasicó Formation (type locality for the Chasicoan SALMA) in central Argentina as cf. Monodelphis or Thylatheridium. More recently, however, Croft (2007:303) referred to this specimen as an “indeterminate didelphid, probably a didelphine,” while Engelman et al. (2016: 27) considered it to be “an indeterminate didelphoid.” Given its incompleteness, relatively little can be said about this specimen. Goin (1995: 170) briefly mentioned other Chasicoan-aged remains from the Pampean Region that he concluded were referable to Sparassocynini, but these have not been mentioned in subsequent publications. Didelphids have otherwise not been described from the middle Miocene (= Laventan, Mayoan, and Chasicoan SALMAs) of southern South America. This may be due to a paucity of fossil-bearing sites (Engelman et al. 2016; Croft et al. 2018). However, Villafañe et al. (2008) reported the discovery of a rich fossil fauna (comprising >600 specimens) from the “El Petiso” site in Chubut Province, southern Argentina, which appears to be middle Miocene (perhaps Laventan or slightly earlier) in age; strikingly, although marsupialiforms (including insectivorous-omnivorous palaeothentids and carnivorous hathliacynids) are present at “El Petiso”, didelphids appear to be absent (Villafañe et al. 2008; M. A. Abello, pers. comm.).

By contrast, multiple didelphid taxa have been reported from various Huayquerian sites in southern South America. They include two putative Thylamys species (T. pinei and T. zettii; Goin 1997c; Goin et al. 2000), although, like the Laventan “Thylamys,” these taxa predate our estimate for the divergence between Thylamys and Lestodelphys (see above). Also present are the oldest known sparassocynin, Hesperocynus dolgopolae, and another extinct, carnivorously-adapted didelphid, Hyperdidelphys pattersoni (Goin and Pardiñas 1996; Forasiepi et al. 2009). A third carnivorous didelphid from the Huayqerian, “Thylatheridium” hudsoni, may also be a sparassocynin (Goin and Montalvo 1988; Forasiepi et al. 2009); if so, it is not closely related to the younger (Chapadmalan) Thylatheridium cristatum and T. pascuali, which appear to be very close to (if not within) Monodelphis (Reig 1958a; Goin and Rey 1997; Voss and Jansa 2009). Two other specimens from the Cerro Azul Formation – a partial skull originally referred to Hesperocynus dolgopolae (GHUNLPam 5119) and a right mandibular fragment identified as a probable sparassocynid (GHUNLPam 8334; Goin et al. 2000) – may represent additional carnivorously-adapted didelphid taxa (Forasiepi et al. 2009).

The appearance in the fossil record of several carnivorously-adapted didelphid lineages during the Huayquerian seems to be part of a broader turnover in the South American mammal fauna, which appears to have occurred at some point between the Santacrucian-Laventan and Huayquerian (Engelman and Croft 2014; Goin et al. 2016). The precise timing and extent of this turnover is still unclear due to a comparative lack of fossil sites for this interval, and there may have been latitudinal differences in terms of faunal change (Engelman et al. 2016). However, among marsupialiforms, there is evidence for major declines in the insectivorous-omnivorous paucituberculatans (with only the family Caenolestidae surviving; Abello 2013: Fig. 8) and microbiotherians (possibly with only the lineage leading to the extant species Dromiciops gliroides persisting; Goin and Abello 2013: fig. 6), and also in the carnivorously-adapted sparassodonts (from more than ten genera in the Santacrucian to five in the Huayquerian; Croft et al. 2018; Prevosti and Forasiepi 2018).

The cause(s) of this faunal turnover continue to be debated. One potential extrinsic driver is the major fall in global temperatures (the middle Miocene Climatic Transition, ~14 MYA) that followed the middle Miocene Climatic Optimum (~15–17 MYA). Another is the spread of drier, more open habitats in South America, probably partly the result of the drop in temperatures and partly the result of Andean uplift (Strömberg 2011; Palazzesi and Barreda 2012; Strömberg et al. 2013; Engelman et al. 2016; Goin et al. 2016). This culminated in the grassland-dominated habitats characteristic of the “Age of the Southern Plains” (““Edad de las Planicies Australes”), which developed following the regression of the Paranean Sea, ~10 MYA (Pascual and Bondesio 1982; Pascual et al. 1996; Ortiz Jaureguizar 1998; Ortiz-Jaureguizar and Cladera 2006). Potential intrinsic drivers, meanwhile, include competition – for example, between paucituberculatans and microbiotherians and insectivorous-omnivorous didelphids, and between sparassodonts and carnivorous didelphids –, the arrival of novel predators (such as pitvipers and procyonid carnivorans) from North America, or other, unspecified biotic interactions (Prevosti et al. 2013; Engelman and Croft 2014; Jansa et al. 2014; Zimicz 2014; Engelman et al. 2016; López-Aguirre et al. 2017; Prevosti and Forasiepi 2018). Confidently resolving the relative importance of these different factors to the changes in marsupialiform diversity observed in South America during the Miocene will require improvements in the fossil record, refinements in the dating of fossil sites, and methods that can specifically compare the relative importance of extrinsic and intrinsic drivers (see Silvestro et al. 2015).

Focusing specifically on carnivorous marsupialiforms, a key question has been whether or not the appearance of carnivorously-adapted didelphids in the Huayquerian is related to a decline in sparassodonts prior to this (e.g., Patterson and Pascual 1968; Marshall 1977b, 1978; Goin 1989, 1995; Alberdi et al. 1995; Pascual et al. 1996; Ortiz Jaureguizar 1998, 2001; Forasiepi et al. 2007; Prevosti et al. 2013; Engelman and Croft 2014; Zimicz 2014; Engelman et al. 2016; López-Aguirre et al. 2017; Prevosti and Forasiepi 2018). Some authors have argued that carnivorously-adapted didelphids may have replaced hathliacynids ecologically (e.g., Patterson and Pascual 1968; Marshall 1977b, 1978; Goin 1989; Ortiz Jaureguizar 1998, 2001; Forasiepi et al. 2007). However, Zimicz (2014) and Croft et al. (2018) both concluded that small sparassodonts (e.g., the smaller hathliacynids) are unlikely to have represented ecological competitors of carnivorously-adapted didelphids (including sparassocynins), based principally on the results of morphometric studies of tooth shape in which sparassodonts and didelphids occupy largely non-overlapping morphospaces (see also Prevosti et al. 2013; Maga and Beck 2017: Fig. 36). However, it is likely that these results are strongly influenced by phylogenetic factors, as we discuss in detail here.

Zimicz (2014) found that carnivorously-adapted didelphids and hathliacynids could be distinguished based on Relative Grinding Area (RGA; = square root of area of talonid/total length of molar), which is consistently larger in didelphids, and Relative Blade Length (RBL; = length of trigonid/total length of molar), which is consistently larger in hathliacynids. However, Zimicz (2014) calculated both of these metrics based on the last lower molar (m4) only, and the m4 of hathliacynids and other sparassodonts is particularly specialized, with a greatly reduced talonid. We agree with Croft et al. (2018: supplementary) that “measuring RGA in sparassodonts using only m4 likely underestimates the total grinding area of the tooth row in many cases.” The highly reduced m4 talonid in hathliacynids also results in very large values for RBL. When RGA is calculated based on m1–4 combined (Croft et al. 2018: character 16), values for didelphids (state 4, RGA = 0.25–0.43) are broadly similar to those for hathliacynids (states 4 and 5, RGA = 0.167–0.43).

The major difference between carnivorously-adapted didelphids and hathliacynids in the analysis of Croft et al. (2018) concerns character 12, namely the “angle of the lower carnassial trigonid cusps in occlusal view,” which was again scored based on m4; the didelphids (including Sparassocynus) have an angle of 40–80° (state 3), whereas most sparassodonts (including all hathliacynids) entirely lack a metaconid on m4, and so were assigned a separate state based on their non-comparable morphology (state 6). Loss of the metaconid on m1–4 appears to be a synapomorphy of Hathliacynidae+Borhyaenoidea within Sparassodonta, in which case it must have been lost >40 MYA (Forasiepi 2009: fig. 56). Reduction of the metaconid is a characteristic carnivorous adaptation, but total loss of this cusp on all molars, as in Hathliacynidae+Borhyaenoidea, is usually only seen in specialized hypercarnivorous forms (Muizon and Lange-Badré 1997; Solé and Ladevèze 2017). However, hathliacynids otherwise show features that suggest that they were less carnivorously-specialized than borhyaenoids, such as less carnassialized molars with larger RGA (Prevosti et al. 2012, 2013; Zimicz 2014; Croft et al. 2018: characters 8, 9 and 16; Prevosti and Forasiepi 2018), and comparatively longer and more slender mandibles (Echarri et al. 2017). The absence of the metaconid in hathliacynids may therefore simply reflect a phylogenetic constraint inherited from hypercarnivorous ancestors, and not necessarily an indicator of a more carnivorous diet than that of the carnivorously-adapted didelphids. Indeed, Solé and Ladevèze (2017) observed that in carnivorous mammals the metaconid is only rarely reacquired after having been lost (although we note that the metaconid was apparently reacquired on m2–4 by at least two borhyaenoid lineages; Forasiepi et al. 2014: online supplementary material fig. 5). Echarri et al.’s (2017) analysis of mandible shape suggested that most hathylacynids were probably omnivores or mesocarnivores, perhaps ecologically similar to extant Dasyurus spp., in contrast to analyses based on tooth shape, which indicate that hathliacynids were probably all hypercarnivores (Prevosti et al. 2012, 2013; Zimicz 2014; Croft et al. 2018; Prevosti and Forasiepi 2018).

Of the remaining characters used by Croft et al. (2018), all of the states found in hathliacynids were also observed in at least one didelphid taxon, and vice versa. Based on their similar body size (<1.5 kg) and molars with a similar degree of “carnassialization,” we therefore consider it plausible that the smallest hathliacynids (Notictis, Perathereutes, and Pseudonotictis) and the most carnivorously-adapted didelphids (sparassocynins, Hyperdidelphys, Lutreolina, and Thylatheridium) represent broad ecological equivalents, contra Zimicz (2014) and Croft et al. (2018) but congruent with the findings of Echarri et al. (2017). Even if this hypothesis is correct, however, this does not necessarily indicate that the decline and ultimate extinction of hathliacynids was due to direct competition from didelphids. Carnivorously-adapted didelphids do not appear to have become widespread until the Huayquerian, by which time sparassodont diversity appears to have already declined (Prevosti et al. 2013; Zimicz 2014; Croft et al. 2018: fig. 2a; Prevosti and Forasiepi 2018), although this may be at least partly the result of sampling biases, in particular the excellent record of sparassodonts in the Santa Cruz Formation and the relative lack of fossil-bearing sites from the middle Miocene (see above). Furthermore, small-bodied (<3 kg) hathliacynids (Borhyaenidium, Notocynus, Notictis) coexisted with carnivorously-adapted didelphids during the Huayquerian and Chapadmalan, a timespan of several million years (Prevosti et al. 2013; Zimicz 2014; Prevosti and Forasiepi 2018).

Based on an analysis of diversification of extant didelphids, Jansa et al. (2014) found evidence of a probable mass-extinction event affecting multiple didelphid lineages during the early-to-middle Miocene. We found evidence of a similar pattern when only extant didelphid lineages are considered, namely a period of low diversification 15.0–10.3 MYA (95% HPD: 18.9–7.9 MYA; Figs. 12 and 14). Jansa et al. (2014) identified two possible causes for this: the existence of the Pebas lacustrine system in western Amazonia 23–8 MYA (reaching its maximum extent 10–14 MYA), or the arrival of new predators (including procyonid carnivorans and pitvipers) from North America, which may have begun in the middle Miocene (Jansa et al. 2014: 692). Jansa et al. (2014: 691) specifically ruled out Miocene climate change and tectonic activity (e.g., Andean uplift) as potential explanations, because molecular phylogenies of a range of other Neotropical organisms do not indicate a Miocene extinction event (Ramirez et al. 2010; Derryberry et al. 2011; Eiserhardt et al. 2011). However, diversification rates that are inferred from extant lineages only can often be misleading (Rabosky 2010, 2016; Marshall 2017; Mitchell et al. 2018), and (as discussed above) the mammalian fossil record of South America clearly does suggest a major faunal turnover between the Santacrucian and the Huayquerian (Pascual et al. 1996; Engelman and Croft 2014; Engelman et al. 2016; Goin et al. 2016). In any case, our phylogenetic results suggest an alternative interpretation, showing the importance of incorporating evidence from the fossil record into studies of diversification.

Specifically, our estimate for the origin of Sparassocynini (10.5 MYA; 95% HPD: 7.2–16.0 MYA) falls within the period of zero diversification observed when only extant didelphid lineages are considered. Although we have not tested its phylogenetic affinities here, Hyperdidelphys represents another carnivorous didelphid lineage that does not appear to be closely related to Sparassocynini (Goin and Pardiñas 1996; Voss and Jansa 2009), and which must have originated prior to the Huayquerian, i.e., potentially within the interval of zero diversification observed in extant didelphids. Thus, sparassocynins and Hyperdidelphys may represent two “missing” didelphid lineages. Other Huayquerian-aged didelphid remains represent possible additional carnivorously-adapted lineages that similarly may have originated within this period (Goin et al. 2000; Forasiepi et al. 2009). Sparassocynins and Hyperdidelphys both survived into the Pliocene, and so their extinction cannot be explained by any events that took place in the Miocene. Furthermore, all known sparassocynin and Hyperdidelphys fossils are known from outside Amazonia, suggesting that the spread of the Pebas lake system, and concomitant loss of terrestrial habitat in this region, is unlikely to have affected their diversification. Thus, neither of Jansa et al.’s (2014) proposed explanations for the apparent “pause” in diversification seen in extant didelphids plausibly accounts for the extinction of sparassocynins and Hyperdidelphys.

Instead, we propose a revised scenario. Sparassocynins and Hyperdidelphys show greater dental specializations for carnivory than all living didelphids except Lutreolina (see Reig and Simpson 1972; Voss and Jansa 2003: Fig. 12), and we suggest that these two carnivorously-adapted lineages (and possibly others) originated during the middle-to-late Miocene, perhaps in connection with the decline in sparassodont diversity seen at this time. As already noted, sparassocynins and Hyperdidelphys also exhibit hypotympanic sinuses that are larger than those of all extant didelphids. Inflation of the middle ear cavity results in improved low-frequency hearing, and in small-to-medium-sized mammals is usually associated with more open habitats (see Mason 2016a, 2016b and references therein). Pollen evidence suggests that open-habitat ecosystems developed in southern South America within the last 10 Ma, i.e., from the Huayquerian onwards (Palazzesi and Barreda 2012), which coincides with the appearance in the fossil record of these didelphid lineages with comparatively inflated hypotympanic sinuses. These carnivorous, open-habitat-adapted lineages then went extinct at some point after the Chapadmalalan, possibly due to competition from placental mustelids, which first appear in the South American fossil record during the Vorohuean subage of the Marplatan (~2.9–2.7 MYA; Prevosti and Pardiñas 2009; Prevosti et al. 2013; Prevosti and Forasiepi 2018). Another possible driver of their extinction is a major reduction in the diversity of small, burrowing caviomorph rodents (potential prey items for carnivorous didelphids) observed after the Chapadmalalan (Ortiz Jaureguizar et al. 1995, 2012). This post-Chapadmalalan reduction in caviomorph diversity is indicated both by changes in the taxonomic composition of fossil small mammal assemblages (Ortiz Jaureguizar et al. 2012), and by a marked reduction in the frequency of paleoburrows (many of which were undoubtedly made by small caviomorphs; Genise 1989; Cenizo et al. 2015) between the Chapadmalalan and Barrancalobian (Ortiz Jaureguizar et al. 1995). This reduction in caviomorph diversity may have been due to the development of increasingly cool and arid conditions (Ortiz Jaureguizar et al. 1995), the arrival of predatory mustelids, competition from sigmodontine rodents, or a combination of these factors. Whatever the precise cause, we propose that the middle-to-late Miocene period of low diversification observed in extant didelphids is not due to a extinction event at this time (as was proposed by Jansa et al. 2014), but rather due to the later (Pliocene) extinction of carnivorous, open-habitat-adapted lineages that originated in middle-to-late Miocene.

As noted by Engelman et al. (2016: 25–27), there appear to be striking parallels between the evolution of South American and Australian marsupial carnivores during the Neogene. On both landmasses, the middle-to-late Miocene saw a decline in the previously dominant carnivorous clade (Sparassodonta in South America, Thylacinidae in Australia), with this decline approximately coinciding with the diversification of carnivorously-adapted members of a different clade (sparassocynins, Hyperdidelphys, and possibly other didelphid lineages in South America, dasyurids in Australia). In both cases, this coincides with evidence for the development of more open habitats, and the clades showing increased diversification exhibit auditory adaptations suitable for such environments (Wroe 1996; Black et al. 2012; Engelman et al. 2016; Kealy and Beck 2017). In South America, most of the carnivorously-adapted didelphids went extinct after the Chapadmalalan, which we suggest may have been due to competition from placental invaders, such as mustelids (as previously proposed by Marshall 1977b, 1978; Goin 1989; Ortiz Jaureguizar 2001; Prevosti et al. 2013: 13). In Australia, meanwhile, dasyurids have suffered recent extreme range reductions, with many species now critically endangered, due (at least in part) to the introduction of placental carnivorans, namely dogs, foxes, and cats (Jones et al. 2003; Wilson et al. 2003).