This phylogenetic analysis aims to resolve: (1) the relationships of Australian fossil penguins to each other and other taxa; (2) the timing of the arrival of crown group penguins in Australia; and (3) identify any biogeographic implications of Australian fossil penguin relationships, specifically whether the Australian penguin fauna is the result of a single dispersal event to Australia or represents independent dispersals throughout the Cenozoic.

Strict consensus of: A, 194 most parsimonious trees with an unconstrained search; and B, 219 most parsimonious trees with crown Sphenisciformes constrained (denoted by the yellow star) in a constrained search. Standard bootstrap (1000 replicates) absolute frequency values are in bold above their respective nodes and symmetric resampling (1000 replicates) frequency difference values are in regular font below their respective nodes. Best score reached was 5377 for both phylogenies.

The total number of operational taxonomic units (OTUs) included in the data matrix is 76: 61 penguin taxa (all 19 extant species, 39 fossil species and three unnamed specimens) and 15 outgroup taxa (two Gaviiformes and 13 Procellariiformes). In all analyses, the trees were rooted to the Gaviiformes following the procedure of Ksepka et al. [ 2 ]. This study analyses 245 morphological characters (26 pertaining to the humerus directly) plus 8145 DNA characters from the 12S, 16S, COI, cytochrome b and RAG-1 genes: a total matrix of 8390 characters, adapted from Ksepka et al. [ 2 ]. Their matrix was not modified apart from the addition of the Australian fossil taxa. Phylogenetic analyses were performed using TNT 1.1 (no taxon limit) [ 42 ], using a new technology search strategy using 10 000 additional replicates with sectorial and tree fusing options checked. Characters were equally weighted and any zero-length branches were collapsed. The consistency and retention indices were applied to the strict consensus tree in order to determine the degree of homoplasy (analogous traits) and amount of character state similarity across taxa which is interpretable as synapomorphy respectively [ 43 ]. Both standard bootstrapping and symmetric resampling (33%) analyses were run for 1000 replicates each, with the values being represented by absolute frequencies and frequency differences respectively. The initial analysis found that the inclusion of Pseudaptenodytes macraei resulted in the paraphyly of extant Pygoscelis, a clade that is otherwise well supported by numerous molecular [ 44 , 45 ] and morphological [ 23 , 39 , 46 , 47 ] analyses. We therefore ran a constrained analysis with the crown clade constrained using the same parameters as above to resolve this issue. Both phylogenies are shown in Fig 4 . The following results and discussion are based on the phylogeny obtained in the constrained analysis ( Fig 4B ).The phylogenetic matrix file can be found in the supporting information ( S1 File ).

Results

The parsimony analysis resulted in 194 (unconstrained) and 219 (constrained) most parsimonious trees (MPTs) with a best score of 5377; consistency index (CI) of 0.536; and a retention index (RI) of 0.698. The CI and RI values were the same in both analyses. Specific results pertaining to Australian OTUs are discussed below.

Pachydyptes simpsoni: In our analysis Pachydyptes simpsoni is resolved in a polytomy (Fig 4) with Marambiornis exilis, Mesetaornis polaris, Delphinornis spp., SAM P7158, SAM P10863, Anthropornis spp., Palaeeudyptes spp., Inkayacu paracasensis, Icadyptes salasi, Perudyptes devriesi, Pachydyptes ponderosus and Kairuku spp. This position does not reveal a most parsimonious ancestral area for P. simpsoni. However, a relationship between P. simpsoni and the Seymour Island taxa has been hypothesised by Acosta Hospitaleche et al. [48], wherein stem taxa dispersed from the Weddellian Province [49] to Australia (and South America) when there was a shallow, warm ocean current (the “Weddellian Current” of Acosta Hospitaleche et al. [48]) during the early Eocene. Furthermore, reanalysis of Antarctic material [4] has shown that the late Eocene Anthropornis also possessed the same derived morphology of the carpometacarpus. This is significant as P. simpsoni was referred to Anthropornis nordenskjoeldi by Jenkins [50] and could be interpreted as supporting the contention that P. simpsoni is a member of the genus Anthropornis. This may be an artefact of the poorly preserved holotype material of P. simpsoni, or perhaps P. simpsoni is not a species of Anthropornis, with the shared derived morphology of the carpometacarpus being convergent. Furthermore, the preserved material of the P. simpsoni holotype contains elements that are poorly known in other late Eocene taxa (e.g. radius, carpometacarpus), which hinders comparisons and clarification of relationships.

SAM P7158 (cf. Palaeeudyptes): SAM P7158 was initially designated as Palaeeudyptes cf. antarcticus in previous studies [51,52]. More recent work has classified SAM P7158 as cf. Palaeeudyptes [53] based on comparable Antarctic taxa given that P. antarcticus is known only from a tarsometatarsus [54] and therefore is not comparable to other elements. SAM P7158 occupies same polytomy as P. simpsoni.

SAM P10863 (Sphenisciformes gen. et. sp. indet.): SAM P10863 was initially referred to Spheniscidae gen. et. sp. indet. by Simpson [12]. SAM P10863 differs from Pachydyptes and Icadyptes by having a shaft that is more slender. SAM P10863 differs from Anthropornis by having a shaft that is less sigmoid in dorsal view. SAM P10863 differs from Palaeeudyptes klekowskii by having a humeral head that is relatively larger and more rhomboid. SAM P10863 differs from Anthropodyptes gilli by having a smaller fossa pneumotricipitalis SAM P10863 differs from Kairuku by having a more distally extended impressio insertii m. supracoracoideus, more curved cranial edge of the humeral shaft and different angle of the sulcus formed from the contiguous sulcus ligamentum transversus and incisura capitis. SAM P10863 is smaller in length than K. grebneffi but is almost identical in size to K. waitaki. SAM P10863 is designated here as Sphenisciformes gen et. sp. indet.

Anthropodyptes gilli: Anthropodyptes gilli is positioned one node basal to a polytomy consisting of Duntroonornis parvus, Archaeospheniscus spp. and Paraptenodytes antarcticus. In terms of humeral morphology, A. gilli is by far the most archaic known taxon from the Miocene. It is similar in morphology to Kairuku; differing mainly in the lack of the elongate depression near the caudal margin of the ventral face of the shaft (164: 0) and also the oblique angle of the cranial margin proximal to the preaxial angle. Other early Miocene taxa outside Australia are more derived morphologically than A. gilli, possessing a larger angle between the main axis of the humeral shaft and the tangent of the ulnar and radial condyles. Anthropodyptes gilli, on the other hand, retains the plesiomorphic humeral morphology typical of Paleogene taxa.

NMV P221273 (Sphenisciformes gen. et. sp. indet).: NMV P221273 is placed in a polytomy with Palaeospheniscus spp., Eretiscus tonnii, and Pseudaptenodytes macraei one node basal to crown Sphenisciformes. In terms of humeral morphology, only one unambiguous morphological character supports exclusion of this cluster of taxa from crown Sphenisciformes (158: 1, impressio insertii m. supracoracoideus and insertii m. latissimus dorsi separated by a moderate gap). Another ambiguous character (150: 0, lacking a pit for ligament insertion adjacent to the humeral head (Aptenodytes patagonicus is variable in this character)) also supports this position. As noted above, NMV P221273 shares with M. novaezealandiae a similar morphology of the fossa pneumotricipitalis, hinting at an affinity. The lack of comparative figures showing this feature in the literature prevents further investigation at present.

Pseudaptenodytes macraei: This is a diagnosable taxon possessing at least two clear autapomorphies: a flattened elliptical ventral portion of the fossa tricipitalis and a curved margo cranialis lacking a preaxial angle [15]. As noted above, P. macraei is placed one node basal to crown Sphenisciformes in the phylogenetic analysis. Based on its morphological features it is unlikely to be a crown spheniscid, with the caveat that more complete referred specimens are needed to more accurately resolve the relationships of this unusual species.

Crown Sphenisciformes: This analysis recovers one Australian OTU as a crown group penguin: the living species Eudyptula minor. Its phylogenetic position is generally consistent with previous studies [2,39].

One other named Australian crown sphenisciform, Tasidyptes hunteri was not included in the analysis. This subfossil species, dated to 760 ± 70 ybp [16], is comprised of an adult synsacrum (holotype), a tarsometatarsus (paratype), a juvenile synsacrum and a coracoid (referred material). These elements are from different ontogenetic stages and were found in multiple stratigraphic layers of an aboriginal midden, meaning that the paratype and referred material are not strictly comparable with the holotype. T. hunteri differs from Eudyptula and Megadyptes by having: a caudal part of the synsacrum with relatively broader fused vertebrae and long slender lateral processes; and the lateral foramen vasculare proximale situated more distal than the medial foramen vasculare proximale on the plantar surface of the tarsometatarsus [16]. Furthermore, the coracoid and tarsometatarsus are indistinguishable from Eudyptes, although the long slender lateral processes of the holotype synsacrum may be a diagnostic character [53]. We therefore recommend restricting the hypodigm of this taxon to the holotype synsacrum only.