Evolution of the patellar sesamoid bone in mammals

Mesozoic pre-therian and stem-therian mammals

The earliest mammals as widely construed include Sinoconodon, the Morganucodonta and Docodonta. These were mostly small, probably insectivorous animals, that appear to have lacked a patella, although it is unclear whether the known specimens contain sufficient postcranial material or are from verified adults, to allow for definitive conclusions. The absence of a clear patella in two stunningly preserved docodonts (the scansorial [climbing-adapted] Agilodocodon and fossorial [digging-adapted] Docofossor) lends credence to the conclusion that it was generally absent in early mammaliaforms (Luo et al., 2015b; Meng et al., 2015). There is convincingly strong evidence of absence of a bony patella in earlier pre-mammals in lineages dating from the divergence of Synapsida and Sauropsida/Reptilia (∼320 Mya), including the early “pelycosaurs”, therapsids and cynodonts (Kemp, 2005).

Australosphenida, the clade containing and thus ancestral to extant Monotremata, diverged from other mammals extremely early, possibly in the mid-Jurassic (Kielan-Jaworowska, Cifelli & Luo, 2004). There is little postcranial material for any extinct members of this lineage however, and no hindlimbs (Kemp, 2005). The patella in crown clade monotremes is discussed below.

Fruitafossor, from the late Jurassic (150 Mya), diverged after the Australosphenida (Luo & Wible, 2005). Its relationship to other early mammals is complicated by its mixture of characters in the molar teeth, middle ear and elsewhere. Fruitafossor is described as lacking a patella, and it is proposed to have had a fossorial lifestyle.

The Eutriconodonta were found abundantly across the world from the middle Jurassic to early Cretaceous periods (Kielan-Jaworowska, Cifelli & Luo, 2004). Among eutriconodonts, a poorly developed patellar groove on the distal femur is found but an ossified patella is absent.

The Allotheria were an extremely successful and widely dispersed group of mammals, among which the best understood are the multituberculates (Kielan-Jaworowska, Cifelli & Luo, 2004; Wilson et al., 2012). Generally Allotheria are found from the late Triassic to the Eocene; thus this group spanned the heyday of the non-avian dinosaurs and survived the K–Pg extinction (Kielan-Jaworowska, Cifelli & Luo, 2004). Multituberculates were predominantly small animals, either herbivorous or omnivorous (Kielan-Jaworowska, Cifelli & Luo, 2004). A patella is noted for the nearly complete multituberculate Ptilodus, a proposed scansorial animal from the early Cenozoic. A patella is also present in the Cretaceous multituberculate Chulsanbaatar. It is unclear whether a patella is typical of all members of the multituberculate group and is under-reported due to lack of hindlimb material for most group members, or whether it occurs only among selected species, although the former seems more plausible. A patella is not reported, however, for the early Jurassic Rugosodon, a proposed multituberculate specimen with one relatively intact knee joint (Yuan et al., 2013), so it is conceivable that an ossified patella evolved later within the Allotheria (Fig. 5).

Specimens of the diverse group “Haramiyida” are mostly restricted to cranial material, and the relationship of this ancient group to other Allotheria and Mammaliaformes has been controversial (Butler, 2000; Kielan-Jaworowska, Cifelli & Luo, 2004; Rose, 2006). However, several recently described more complete haramiyid specimens from the Jurassic with at least one preserved knee joint lack a patella (Bi et al., 2014; Zheng et al., 2013; Zhou et al., 2013). These new specimens have been interpreted to support an Allotheria clade including a paraphyletic “Haramiyida” (but a valid clade Euharamyida including many “haramiyid” taxa) and Multituberculata (Fig. 5), although new analyses of a key specimen of Haramiyavia concluded that the haramiyids and multituberculates were not closely related (Luo et al., 2015a). The inclusion of the “Euharamiyida” in Allotheria pushes the divergence date of the group significantly earlier into the late Triassic, whereas multituberculates themselves appear only in the middle to late Jurassic. Final resolution of this controversy will undoubtedly require additional fossil material.

Symmetrodonta were a group of diverse, small mammals widely distributed in time from the late Triassic to the late Cretaceous (Kielan-Jaworowska, Cifelli & Luo, 2004). In the subgroup of spalacotheroids, a patella is reported for one fairly complete specimen (Zhangheotherium) but not for another (Akidolestes) (Chen & Luo, 2012; Luo & Ji, 2005) (these two specimens are coded oppositely in character matrices in some subsequent publications Bi et al., 2014; Zhou et al., 2013, probably in error); a patella seems absent in Maotherium.

Eupantotheria was a diverse group found commonly from the mid-Jurassic to the early Cretaceous (Kielan-Jaworowska, Cifelli & Luo, 2004). The patella is reported as absent in both an early European specimen (Henkelotherium, late Jurassic) and a later South American specimen (Vincelestes, early Cretaceous) (Fig. 5). The large group of dryolestid Eupantotheria possibly survived past the K–Pg boundary, and have an unknown patellar status.

The tribotherians were the earliest-diverging group to share key molar features with the therians. However, no postcranial specimens have been reported; thus, nothing is known of their patellar morphology (Kielan-Jaworowska, Cifelli & Luo, 2004).

The single specimen of Juramaia from the Jurassic (∼160 Mya) unfortunately lacks hindlimb material; therefore, its patellar status is unknown. Based on its forelimb, Juramaia is proposed to have been scansorial or possibly arboreal (Luo et al., 2011). The later specimen of Eomaia from the early Cretaceous includes all limb elements, and is described with a patella (Ji et al., 2002). Based on limb and foot features, Eomaia was probably scansorial or arboreal. In the original publication, Eomaia was described as the earliest eutherian mammal (Fig. 5), however a more recent and much more extensive analysis confidently placed Eomaia prior to the eutherian/metatherian divergence (O’Leary et al., 2013) and thus at least as a stem member of the clade Theria (see Fig. S4). Eomaia (and presumably Juramaia) postdate the divergence of the Symmetrodonta, but their positions relative to the Eupantotheria remain to be determined, as does any close relationship between these two key taxa. Lacking a better alternative, here we refer to these taxa as “Theria”, and in Fig. 5 versus Fig. S4, consider the consequences of Eomaia’s phylogenetic position on our conclusions.

In surveying, the available data mapped onto our composite phylogeny (Fig. 5; Fig. S4), it becomes evident that an ossified patella evolved multiple times (at least four) along the mammalian stem lineages during the Mesozoic era, whether using parsimony or maximum likelihood optimisation methods: at some highly uncertain time in the long mammalian lineage that led to Monotremata, in multituberculates/Allotheria, in Zhangheotherium or a direct ancestor, and likely twice (or between one to three times, depending on the placement of Eomaia; see Fig. 5 and Fig. S4) in the clade containing Eomaia and Theria (Metatheria and Eutheria). This result remained the same if the Euharamiyida were not included with multituberculates but pre-dated crown Mammalia, as suggested by some recent studies (e.g. Luo et al., 2015a).

Mesozoic Metatheria and Eutheria

The two major extant mammalian groups, the Metatheria and Eutheria (together forming the clade Theria), diverged as early as the Jurassic (Fig. 5). The earliest fossil identified as stem metatherian, Sinodelphys, dates from the early Cretaceous of China (125 Mya, approximately contemporary to Eomaia) and lacks a patella (Luo et al., 2003). A patella also seems absent in the less complete Cretaceous stem metatherian Asiatherium (Szalay & Trofimov, 1996).

The earliest known occurrences of the patella in definitive stem eutherians (Figs. 5 and 7) were in the late Cretaceous Ukhaatherium (Horovitz, 2003), a relatively unspecialized form, and in Zalambdalestes (Wible, Rougier & Novacek, 2005), a more specialized taxon sometimes described as resembling later lagomorphs (Rose, 2006). Patellar status at the crown group node for Theria (plus Eomaia) remains ambiguous (Figs. 5 and 6; Fig. S4), as we consider below.

Cenozoic Monotremata

The origins of the Monotremata (egg-laying mammals) are poorly understood. They are considered extant members of the clade Australosphenida (the alternative term Prototheria has been superseded), and hence with early roots in the Mesozoic. Molecular studies based on the sequenced genome of the platypus corroborate the long held interpretation that the monotremes diverged prior to the metatherian/eutherian split, consistent with proposed fossil-based phylogenies (Warren et al., 2008). Unfortunately, there are almost no reported hindlimb specimens of any extinct monotreme (including probable early monotreme fossils found in South America; Musser, 2003), with the exception of the Pleistocene Zaglossus (echidna) from Australia and New Guinea (which may be the same as the extant species of that name). Unfortunately, although fossil Zaglossus hindlimb elements exist, including an articulated knee, neither presence nor absence of the patella has been reported (Murray, 1984). The extant monotremes, the platypus (Ornithorhynchus anatinus) and the echidnas (Tachyglossidae, two genera Zaglossus and Tachyglossus; four known species) all have substantial patellae (see Figs. 4A–4D) (Herzmark, 1938; Rowe, 1988). It is unclear when the two extant monotreme genera diverged, although a date early in the Cretaceous has been proposed (Rowe et al., 2008), and it is impossible for now to date the appearance of the patella in the monotreme lineage. Regardless, an ossified patella is homologous for this crown clade (Fig. 5), and alternative phylogenetic topologies did not change the general pattern of patellar evolution (Fig. S4).

Cenozoic Metatheria

All extant Metatheria are within the subgroup of Marsupialia, however non-marsupials did exist earlier during the Cenozoic. As documented in the pioneering study of sesamoids in Marsupialia by Reese et al. (2001), an ossified patella seems to be absent in the great majority of extant marsupial species, both from Australia and the Americas (Flores, 2009; Herzmark, 1938; Holladay et al., 1990; Reese et al., 2001; Rose, 2006; Rowe, 1988), including the sole surviving North American marsupial, the opossum Didelphis virginiana (Figs. 4E and 4F). Many marsupials have other sesamoid bones in the knee region (e.g. the parafibula, lateral sesamoid or “sesamoid bone of Vesalli”; Fig. 1), as well as a fibrocartilaginous “patelloid”, which may to some degree serve the mechanical function of a bony patella (Reese et al., 2001). However, the mechanics of a fibrous or bony patella remain essentially unstudied (to our knowledge) in non-placental mammals, so this is simply speculation. Studies have claimed some association between reduction of the patella in many marsupials and locomotor style or ecology (Holladay et al., 1990; Reese et al., 2001), but these deserve testing with more detailed sampling across phylogeny and ontogeny.

Nonetheless, an ossified patella is found in a small number of extant marsupial species among otherwise divergent clades, both from Australia: at least several Peramelidae or bandicoots, and the two marsupial mole species of Notoryctes); and from South America: Tarsipes, a honey possum; and several, and possibly all, Caenolestidae or shrew opossums (see Fig. 6: note collapse of several large clades in terms of total number of species, in which no species have been shown to possess a bony patella; Table S1).

Possibly uniquely among crown clade marsupials, bandicoots also possess a chorioallantois fused to the uterine epithelium (i.e. a true placenta) (Freyer, Zeller & Renfree, 2003; Padykula & Taylor, 1976), which combined with an osseous patella led to the initial suggestion that they might actually be eutherians (Reese et al., 2001). However, more recent molecular and fossil-based phylogenetic studies provide no support for that hypothesis of eutherian bandicoots (Asher, Horovitz & Sanchez-Villagra, 2004; Meredith, Westerman & Springer, 2008; Sánchez-Villagra et al., 2007; Westerman et al., 2012). Bandicoots clearly are metatherians, and their chorioallantois is thus a convergently evolved trait rather than plesiomorphic. It remains to be determined whether an ossified patella is present in all or only some bandicoots, as so far it is only reported in the Peramelinae of dry or temperature forests of Australia, not yet in the Peroryctinae of tropical rainforests of New Guinea, or the more distantly related bilbies (Groves & Flannery, 1990; Meredith, Westerman & Springer, 2008; Westerman et al., 2012). Similarly, a comprehensive study of the Caenolestidae remains to be performed, much as a more thorough study of the major marsupial clade Diprotodontia (wombats, kangaroos and kin) is needed.

Not surprisingly given the absence of a bony patella in most extant marsupials, any evidence of a patella is absent in the early Cenozoic Metatheria Pucadelphys, Mayulestes, and the later Herpetotherium. Unexpectedly, a bony patella is reliably reported in the Borhyaenoidea, an unusual group of dog-like carnivorous South American marsupials found from the Palaeocene through the Miocene (Argot, 2002, 2003a, 2003b, 2003c, 2004; Argot & Babot, 2011; de Muizon, Cifelli & Paz, 1997). Patellar status in some members of Borhyaenoidea (e.g. Borhyaena itself and Lycopsis Argot, 2004), and in the more inclusive group Sparassodonta, is uncertain due to the incomplete state of specimens. Szalay & Sargis (2001) noted other enigmatic fossil patellae from the Palaeocene of Brazil that they assigned to Metatheria, but the phylogenetic relationships of those fragmentary remains are unclear and no patellae were shown. However, no ossified patella is reported in extant or recent carnivorous marsupials such as Thylacinus.

Two related, pernicious problems remain for interpreting the evolution of the patella in Metatheria that may have ramifications for all of Mammalia/Mammaliaformes. First, Szalay & Sargis (2001; pp. 164–165) reported the presence of an ossified patella in older individuals of Didelphis virginiana in their study of an ontogenetic series from this species. They stated (p. 165) “In older individuals there is occasionally an elongated and small sesamoid ossification within the tendon of the quadriceps femoris where it crosses the knee joint when the knee is flexed”. However, this observation was not documented with illustrations or photographs (especially tissue histology or x-rays) and hence remains a tantalizing anecdote. Similarly, Owen (1866) commented that some marsupials had no ossifications in their patellar tendon but others had “only a few irregular specks of ossification” and a “distinct but small bony patella in the Macropus Bennettii”. In contrast, Reese et al. (2001) and Holladay et al. (1990), respectively sampled 61 specimens (∼39 adults) from 30 species of marsupials and three macropodid specimens (of unknown maturity), documenting no ossified patellae except as noted in bandicoots, and their studies used clear methods for identifying ossified tissues. It remains possible that patellar ossification occurs variably in older individuals among Metatheria, which would help explain its patchy description in known taxa.

If the latter situation is the case (i.e. the literature is unclear about patellar ossification in marsupials because they have more inherent variability), then it relates to a second problem, a cladistic one of character coding and transformational homology (sensu Brower & Schawaroch, 1996; Pinna, 1991). Should character states of the patella in metatherians, or even all mammals and their kin, be coded as an ordered transformational series such as absent (0), fibrocartilaginous (1) or ossified (2), or as an unordered series (i.e. should evolutionary steps be required to go from 0–1–2 as two steps, or unordered allowing 0–2 transformations as one step)? We chose the unordered character option by default for all crown group mammals, but where relevant explain how an ordered option changed (or did not change) our results. An endochondral ossification of the bony patella is certain, but a fibrocartilaginous or otherwise soft tissue composition of the patella (coded as state 1) in adults is not unambiguously the necessary (i.e. ordered) evolutionary precursor character state-to-state 2 (ossified patella in adults). The solution to both of these problems lies in more developmental data for the patella (bony and otherwise) in diverse mammalian species, in addition to more scrutiny of the adult morphology in extant and fossil Mammalia (especially Metatheria).

As noted briefly in the Introduction, many marsupials have a primarily fibrocartilaginous patelloid in place of an ossified patella and some other mammals may have a “suprapatella”. The developmental and evolutionary relationships of these structures remain somewhat unclear, particularly as some marsupials with an ossified patella (e.g. bandicoots) also possess a patelloid (Reese et al., 2001), suggesting that the patelloid is not developmentally equivalent to the patella in marsupials (Vickaryous & Olson, 2007). If so, this would indicate independent evolutionary histories of these two structures. Further work is required to clarify the relationships of the patelloid and suprapatella at least in extant taxa, before definitive evolutionary trajectories can be inferred. We reiterate that, just because a patella-like structure is not ossified, that does not mean it is a distinct organ deserving a new name and different homology as a phylogenetic character—although it may be a distinct state of the character “patella”. However, either of these two possibilities needs careful testing particularly for Metatheria.

A non-osseous patelloid/suprapatella is also found in several closely related modern placental clades that lie far from the base of Eutheria (Fig. 7), suggesting that these represent independent acquisitions. We have not attempted to explicitly reconstruct the evolution of the patelloid in Eutheria. Lewis (1958) and Broome & Houghton (1989) speculated that the mammalian patelloid might be a precursor to the tibial epiphysis (Broome & Houghton, 1989; Lewis, 1958)—a so-called “traction epiphysis” (Vickaryous & Olson, 2007). Yet considering that the patelloid evolved after the tibial tuberosity (and proximal tibial epiphysis as well as distal femoral epiphysis; Carter, Mikić & Padian, 1998) of mammals, not before it, and lies proximal rather than distal to the patella, we reject this hypothesis. More study of the evolution of mammaliaform long bone epiphyses, however, is warranted to strongly and more generally test for associations between any epiphyses and sesamoids. Furthermore, this same phylogenetic evidence indicates that the patelloid in Euarchontoglires, some Carnivora and bandicoots is not ancestrally associated with leaping or other behaviours (e.g. Jungers, Jouffroy & Stern, 1980). As Walji & Fasana (1983) caution, the ancestral mechanical environment of the patelloid/suprapatella and its roles in different behaviours remain unclear, although it does seem to be associated with knee hyperflexion, like a typical fibrocartilaginous “wrap-around” tendon (e.g. Ralphs, Benjamin & Thornett, 1991; Alexander & Dimery, 1985).

Our unordered parsimony reconstruction (Fig. 6) indicated that an ossified patella was absent in the ancestor of Metatheria, then evolved in the ancestor of Sparassodonta and Marsupialia. The bony patella may have been lost in the basal lineages of Marsupialia (reconstructed state here was equally parsimonious between an ossified and fibrocartilaginous patella), with subsequent re-acquisition in certain groups (Tarsipedidae, possibly Notoryctidae, Thylacomyidae + Peramelidae and Tarsipedidae) (Fig. 6). Ordered parsimony reconstruction resulted in subtle differences; making some nodes less ambiguous (i.e. state 1 [patelloid present] within basal Marsupialia) and others more ambiguous (such as the ancestor of Sparassodonta and Marsupialia, which became equally parsimonious between states 1 and 2). In contrast, maximum likelihood reconstruction indicated a single origin of the osseous patella in Metatheria (Fig. 6), with reduction to a fibrocartilage patelloid (in Didelphidae and the clade containing Pseudocheiridae + Vombatidae) and re-acquisition of a bony patella (in Tarsipedidae) marginally more likely than multiple instances of ossified patella evolution. Because presence of a patelloid has not been clearly excluded in some extant marsupials (e.g. Petauridae, Acrobatidae) and is unlikely to be fossilized, its reconstruction must be treated carefully. Finally, alternative placement of Microbiotheriidae did not drastically alter our evolutionary reconstructions (Fig. S5), aside from making a single origin of the ossified patella slightly more likely. Overall, we caution that inferences about the evolutionary history of the patella in Metatheria must remain tentative until further data become available.

Cenozoic Eutheria

The Placentalia include all extant Eutheria as well as some fossil stem taxa (Fig. 7). Although there is some fossil evidence for placentals pre-dating the K–Pg event (Archibald et al., 2011), as well as substantial molecular dating consistent with an older placental radiation, the timing of the placental radiation remains highly controversial. However, our major conclusions about patellar evolution in placentals are not dependent on how this controversy is ultimately resolved, as a recent large-scale phylogenetic analysis convincingly established the presence of an osseous patella as a derived character state in the ancestral placental irrespective of its true date of divergence (O’Leary et al., 2013).

Fossil evidence supports the presence of the bony patella in essentially all Cenozoic placental groups (Fig. 7; also see Table S1 and Figs. S1–S4, with citations therein). Specimens with sufficient hindlimb material to make a determination of patellar status are rare in the early Cenozoic Palaeogene period (∼66–23 Mya), but Palaeocene groups in which an ossified patella has been reported include the Taeniodonta (small to medium sized fossorial animals), Pantodonta (early herbivores), Palaeanodonta (small, possible insectivores; perhaps related to pangolins), “Condylarthra” (a diverse assemblage of putatively related taxa, probably polyphyletic, including both herbivores and carnivores, many of which may be stem members of subclades within the placental crown group) and the Plesiadapiformes, a sister group to crown clade primates (and possibly members of the clade Primates as well) (Bloch & Boyer, 2007; Silcox, 2007). In general, the evolutionary relationships between Palaeocene taxa and more recent placentals remain enigmatic.

Eocene placentals include examples whose close relationships to modern groups are well accepted. Among Eocene groups (Fig. 7; Table S1), an osseous patella has been reported in older, extinct groups such as “Condylarthra”, Creodonta (carnivores), Mesonychia (carnivorous/omnivorous artiodactyls or cetartiodactyls), Dinocerata (large hippo/equid-like herbivores), Brontotheriidae (large rhino-like herbivores), and Notoungulata (diverse South American hoofed herbivores; probably related to Afrotheria) (O’Leary et al., 2013), as well as in extinct species (in parentheses, see Table S1 for citations) recognized as stem members of several extant groups: Glires (Rhombomylus), Perissodactyla (Propalaotherium), early Sirenia retaining hindlimbs (Pesoziren, Protosiren), Proboscidea (Numidotherium, Moeritherium, Barytherium), Rodentia (the horse-sized Pseudotomus, Paramys), Pholidota (Eomanis), Artiodactyla (Gervachoerus), early Cetacea retaining hindlimbs (Maiacetus) and Chiroptera (Icaronycteris, Tachypteron). A bony patella is also reported for several Eocene primates, including the lemur-like Notharctidae (Northarctus) and the tarsier-like Omomys and Archicebus, in addition to the enigmatic primate Darwinius.

Despite an extensive literature search, we found no reports attesting to the presence of an osseous patella in certain widely cited Palaeocene and Eocene species, including: Protungulatum, frequently cited as the earliest true placental; Miacis, Vulpavus, Viverravus and Didymictis, which were stem Carnivora (Gregory, 1920; Heinrich & Houde, 2006; Heinrich & Rose, 1995, 1997; Samuels, Meachen & Sakai, 2013); Pakicetus, a fully quadrupedal early cetacean (though sometimes reconstructed with a bony patella as in Fig. 7 and Figs. S1M and S1N) (Thewissen et al., 2001); Leptictis, possibly related to crown clade lagomorphs (Rose, 1999); Sinopa, a creodont (Matthew, 1906); and the early primates Adapis, Leptadapis, Teilhardina, and Cantius (Dagosto, 1983; Gebo et al., 2012; Gebo, Smith & Dagosto, 2012; Rose & Walker, 1985; Schlosser, 1887; Szalay, Tattersall & Decker, 1975). There is no reason to expect that a bony patella is missing in these species. These absences are more likely due to incompleteness of the fossil record and/or literature descriptions and images. Moreover, the massive collections of Eocene specimens from the Messel and Green River lagerstätten in Germany and Wyoming have not yet been fully described (Grande, 1984; Schaal & Ziegler, 1992). There are many examples of an ossified patella in specimens from extant placental groups across the more recent Miocene, Oligocene, Pliocene and Pleistocene, but a comprehensive search of the literature for those geologic epochs was deemed redundant for our major conclusions.

Based on fossil/morphological evidence plus extensive genomic DNA sequencing, there is a consensus that crown clade placentals can be historically and geographically defined by four major groups: Xenarthra, Afrotheria, Euarchontoglires (further divided into Euarchonta; featuring Primates; and Glires) and Laurasiatheria (Rose, 2006). These in turn may be resolved, with somewhat less consensus, into 19 crown clade “orders” (Fig. 7) (O’Leary et al., 2013). In two of these orders, the afrotherian clade Sirenia and the cetacean branch of (Cet)artiodactyla (laurasiatherian clade), extant members have extensively reduced or absent hindlimbs and thus lack skeletal knee structures, including an osseous patella. In contrast, the bony patella is retained among the aquatic seals and sea lions in Carnivora, although unlike Sirenia and Cetacea these animals still display some terrestrial habits and thus presumably still employ the gearing mechanism that the patella is involved in at the knee. An ossified patella is documented as present in at least some members of all other 17 placental “orders” (e.g. Figs. 4G, 4H and 7; Figs. S1–S3; Table S1) (de Panafieu & Gries, 2007; De Vriese, 1909; Dye, 1987; Herzmark, 1938; Lessertisseur & Saban, 1867; Rose, 2006).

The evolution of the Cetacea presents an interesting scenario regarding patellar evolution (Fig. 7). Cetaceans evolved from a common ancestor with other (cet)artiodactyls (Spaulding, O’Leary & Gatesy, 2009; Thewissen et al., 2007). Early artiodactyls (including cetaceans), such as Diacodexis and Indohyus, shared morphological similarities with both extant groups of Cetacea (toothed and baleen whales) and yet retained an osseous patella (Rose, 1982; Thewissen et al., 2007), much as stem Sirenia did (Domning, 2001; Zalmout, 2008). Patellar status in Pakicetus, a presumptive early cetacean with full hindlimbs, remains uncertain based on the primary literature, but presence is likely considering the presence of a bony patella in its closest relatives. Rodhocetus and Ambulocetus, probably semi-aquatic early cetaceans, still had large hindlimbs and ossified patellae (Madar, Thewissen & Hussain, 2002). The pelvis and hindlimbs are greatly reduced in the later cetaceans Dorudon and Basilosaurus, but a bony patella is still present in these animals (Gingerich, Smith & Simons, 1990; Uhen, 2004). It is not clear exactly when the patella was lost altogether in later cetaceans with increasingly reduced hindlimbs.

Bats present another interesting case of patellar evolution (Fig. 7; Table S1). An osseous patella is generally present in bats (Pearson & Davin, 1921b). A bony patella is also reported in a well-preserved hindlimb of an early Eocene bat, Icaronycteris, of intermediate form but proposed to be a microchiropteran (Jepsen, 1966). However, in studies of multiple genera of modern bats including members from both of the major subgroups Megachiroptera and Microchiroptera (which is possibly paraphyletic), a bony patella was noted as absent in four species of the megachiropteran Pteropus (flying foxes of various sizes), and a few individual species of Cephalotes, Epomophorus and Vespertilio (De Vriese, 1909; Lessertisseur & Saban, 1867; Smith, Holladay & Smith, 1995). No obvious lifestyle distinction was noted for the Pteropus genus as compared to many other bats, hence the loss of the ossified patella in members of this particular subgroup (and others) remains mysterious. In general, bat hindlimbs are highly derived, adapted to hanging and pulling rather than pushing. A few bats such as the vampire bats are actively quadrupedal (Adams & Thibault, 2000; Riskin & Hermanson, 2005). Bat hindlimbs are articulated in abduction, so that the knee faces dorsally; as in the original ancestral orientation for Tetrapoda (Fig. 2) (Neuweiler, 2000; Schutt & Simmons, 2006). There remains a need for a comprehensive study of the patella in bats (Smith, Holladay & Smith, 1995 only studied 31 specimens of 13 species), but this is challenging due to the existence of >900 extant bat species (Jones et al., 2002). The microstructure of the “patelloid” in Pteropus is generally similar to that in many marsupials (e.g. deep layer of fibrocartilage; superficial layer of dense connective tissue contiguous with the quadriceps/patellar tendon) (Smith, Holladay & Smith, 1995). This also raises the question of whether the patella only ossifies later in adulthood in Pteropus, rather than not ossifying at all.

General evolutionary patterns and ambiguities

Considering the above distributions of patellar presence/absence in Mammalia (Figs. 5–7; Figs. S4 and S5) and our data matrix (Table S1), the simplest interpretation of the evolutionary record of the patella in mammals (by parsimony and maximum likelihood mapping of presence/absence) is that this structure arose (i.e. ossified) independently at least four times (but possibly up to six), mostly during the Mesozoic era: (1) in Australosphenida ancestral to modern monotremes; (2) in Multituberculata (later than Rugosodon); (3) in Symmetrodonta (specifically in Spalacotheroidea that were ancestral to Zhangheotherium but not Akidolestes); (4–6) in early Theria (including Eutheria, Metatheria, Eomaia and related stem groups; depending on topology between one and three times in this clade). Conceivably, a single common patelloid precursor may pre-date the origins of the bony patellae, or the bony patella may have arisen fewer times and undergone loss (and re-gain) in some lineages, similarly to the pattern in Metatheria. Each of these scenarios remain difficult to test purely with fossil evidence, however, due to the typical lack of preservation of cartilaginous or fibrous structures.

Once the bony patella evolved in Eutheria, it was highly conservative in its presence (Fig. 7). There are very few examples of fossil or extant Eutheria in which the hindlimb remains intact but the patella is unossified in adults (e.g. Pteropus). A caveat is that many fossil specimens are not sufficiently complete for a definitive rejection of patellar ossification in those taxa. Still, the evolutionary stability of the osseous patella in Eutheria stands in contrast to its general variability across mammals, and suggests some conserved functional requirement and/or ontogenetic mechanism that remains to be determined.

Although an ossified patella is absent in the majority of Metatheria, it is reported in several groups (Fig. 6; Fig. S5). This likely represents some loss and regain(s) of the early metatherian bony patella. Importantly, in this case the presence of a fibrocartilaginous “patelloid” in most marsupials shows a clear evolutionary polarity from an ossified patella to a non-ossified patelloid, and back again in the case of the secondary gain of ossification, in each case within Metatheria (Reese et al., 2001). This “patella to patelloid” transition suggests the reverse may also be possible—that a soft tissue patelloid may represent the evolutionary precursor to an ossified patella—but it has yet to be clearly documented. There is no obvious lifestyle or biomechanical correlate among all four groups of osseous patella-bearing Metatheria: the notoryctid moles are underground burrowers, and bandicoots may dig for insects, but Tarsipes is a nectar feeder and the borhyaenoids/sparassodonts were largely terrestrial carnivores. In contrast, other Australasian carnivorous marsupials including the recently extinct thylacine, and the extant quoll, numbat and Tasmanian devil are not reported to have a bony patella.

The large size of the patella in the monotreme platypus might be related to its aquatic (and partly fossorial) lifestyle. The other monotremes, the echidnas, also burrow and the long-beaked species (Zaglossus) lives in underground dens—further suggesting an association between fossorial habits and the presence or enlargement of a bony patella in Monotremata, as well as in some fossil Mammaliaformes (multituberculates?) but curiously not in other fossorial stem taxa (e.g. the docodont Docofossor). Reduction of the patella in the Cetacea and Sirenia is not intrinsically correlated with their aquatic lifestyle, but with the reduction of the hindlimbs as part of their particular adaptations. Elsewhere in groups with aquatic adaptations, for example in various diving birds, an unusually large patella is found. It seems premature to weave detailed scenarios around the high degree of convergent evolution of the osseous patella in mammals until the biomechanical function and genomic control of the patella are better understood, and improved phylogenetic sampling improves resolution of when it evolved in particular lineages.

Patellar developmental genetics

Molecular phylogenomics provides a potential independent or synergistic approach to resolving issues of patellar evolution. If specific genomic sequence signatures could be associated with patellar status, then comparison of the genomes of the various extant but widely separated groups with a bony patella might indicate whether these represent convergence events or a common ancestral event (i.e. identified via shared evolutionarily transmitted genetic markers required for patellar development). For example, it has recently been shown that the ability to taste sweet carbohydrates in hummingbirds represents a trait convergence. Hummingbirds diverged from the insectivorous swifts, in which the sweet taste receptor is inactivated by mutations in the receptor coding gene. In hummingbirds, the ability to taste sweet has been re-acquired, apparently through molecular adaptation of the umami receptor to detect sweet molecules (Baldwin et al., 2014). It would be helpful to understand the (developmental) genetics of the patella as a step toward the identification of such sequence signatures. Developmental genetic studies in two mammals, humans and mice, have identified genes required for correct patellar specification. The known functions of some of these genes are informative regarding their requirements.

There are currently approximately 12 human genetic disorders with identified molecular bases that regularly include abnormal, reduced or absent patellae (hypoplasia or aplasia) as an important aspect of the phenotype (reviewed by Bongers et al. (2005), see also Warman et al. (2011) and Table S2 for details). There are also several genes whose genetics in mice indicates relevance to patellar development at least in rodents. A detailed discussion of all these syndromes and genes is beyond the scope of this study. However, the known patella-related genes can be broadly organized according to three major developmental processes: limb specification and pattern formation (transcription factors such as LMX1B, TBX4, PITX1 and mouse Hoxaaccdd-11, SOX11 and signalling factor WNT7A); bone development, biochemistry and regulation (GDF5, CHRNG, SLC26A2, COL9A2 and AKT1); and genes involved in DNA replication and chromatin (ORC1, ORC4, ORC6, CDT1, CDC6, GMNN, CDC45, RECQL4, KAT6B and ESCO2). Of these, the genes of replication and chromatin are the most unexpected, and potentially of the most interest for evolutionary studies. Patellar ossification may be dependent on the timing of DNA replication in particular cells, or else may be affected by aberrant gene regulation resulting from mutations in replication and chromatin factors. In either case, the target genes mis-regulated in these syndromes, if they can be identified, may provide useful evolutionary markers to distinguish convergent from homologous patellar status.

Developmental studies in mouse or chick embryos, sometimes with induced paralysis, document the additional importance of local environmental factors in patellar ontogenesis (Hosseini & Hogg, 1991; Mikic et al., 2000; Nowlan et al., 2010a, 2010b; Osborne et al., 2002; Rot-Nikcevic et al., 2006). Similarly, embryonic development and hindlimb activity in the case of particular marsupials may be important in understanding the diversity of patellar states in this group. A better understanding of these environmental processes will also be helpful to disentangle genomic versus epigenomic regulation of patellar development, and hence evolution.

How “the mammalian patella” evolved

The widespread, repeated evolution of the bony patella across evolution argues for an important role in locomotor biomechanics. In animals lacking an ossified patella (e.g. Lissamphibia, Testudines, Crocodylia; as well as many extinct lineages of tetrapods), the consequences of this ancestral absence for hindlimb function remain mostly unstudied. This mystery is striking, in particular, within Mammalia where most marsupials lack an ossified patella, as did numerous fossil stem-mammals, despite seeming to share common ecological niches and the associated locomotor requirements. This sporadic occurrence in marsupials and stem mammals contrasts with its near universality and evolutionary stability in the Eutheria as noted above.

The exact number of independent origins of a bony patella among mammals remains unclear, but we have estimated at least four convergent episodes inside Mammaliaformes, and several instances of patellar “loss” (with apparent re-gain in some marsupials). The pattern of acquisition and loss will require revisiting as new fossil material is discovered, as our evolutionary reconstructions are dependent on single specimens for many ancient taxa. Moreover, patellar status has not been verified for all >5,000 eutherian and >330 metatherian species (Wilson & Reeder, 2005), so it is possible that additional placental species (other than the fully aquatic forms) may be found lacking, or marsupials having, a bony patella. A recent evolutionary study documented many apparently independent evolutionary origins of the caecal appendix in mammals; thus the convergent evolution of unusual anatomical structures like the osseous patella has precedent (Smith et al., 2013). Similarly, blue colouration among tarantula spiders apparently involved at least eight independent evolutionary acquisitions, among different microscopic anatomical structures affecting spectral reflectance and hence general external colour (Hsiung et al., 2015). A better understanding of the genomic signatures required for development of such novel structures should be very helpful to deconstruct the observed complex patterns of evolution, distinguishing between convergent evolution (homoplasy) and shared inheritance (synapomorphy/homology).

Given that the patella evolved, and was also lost, multiple times in mammals and other Tetrapoda (Fig. 3), one thing is clear. Much as we have referred to “the patella” throughout this study, there is no such thing—perhaps not even a single “mammalian patella”. The story of patellar evolution is one of many (bony) patellae; a story of diverse evolutionary origins as well as forms, functions, ontogenies and perhaps even diverse underlying genetics. Mottershead (1988) wondered if the patella is “not typical of its kind” for a sesamoid bone (Mottershead, 1988). Yet even patellae are not necessarily typical for patellae, let alone other sesamoids—there are double or fatty patellae in some birds (Regnault, Pitsillides & Hutchinson, 2014), proximal suprapatellae and/or fibrocartilaginous patelloids in many marsupials, no ossified (or even other forms of) patellae in many species, and even amongst those animals that have patellae, there are numerous shapes and sizes of patellae (Fig. 4; Figs. S1–S3), suggesting still-unappreciated lifestyle constraints in patellar (and knee joint) mechanics.

While we have provisionally used the terms “patelloid” and “suprapatella” for non-ossified tissues near where the patella is or might be found, the validity of these terms needs further inspection in a broader context. Certainly, patellae exist in non-ossified forms in younger animals before endochondral ossification completes, and where such ossification does not initiate at all during ontogeny it may be best to apply the term “patella” to such tissues rather than invoke new terms for the same organ that simply underwent different tissue development; as above, a case of divergent character state transformation rather than distinct characters (i.e. new organs). This is not simply a semantic issue as the implications for evolutionary novelty, adaptation and “evo-devo” of patella-like structures will depend on the decisions made about homology of these traits in organisms, and how those decisions are communicated by the choice of anatomical terminology.

Future prospects

Our discussion of patellar evolution in Mammalia has identified several areas where key questions remain unresolved, in addition to uncertainties about the amount of convergence/parallel evolution in origins of the osseous patella and about specific roles of (and interactions between) genetic/developmental factors in bony patellar formation/loss. Considering that mechanical loads are known to play an important role in the development of sesamoid bones (in particular in early ontogeny), studies linking these loads to genetic/developmental control as well as broad evolutionary patterns could prove very insightful, especially in explaining the seemingly large amount of patellar homoplasy in mammalian evolution. Mammals may be less sensitive (i.e. more genetically assimilated e.g. Vickaryous & Olson, 2007) than birds in terms of the relative influence of mechanical loads on bone (including sesamoid) ontogeny (Nowlan et al., 2010b)—this idea deserves better testing as insight into load-based influences improves. Furthermore, indications that some bones within an organism may be more responsive to their loading regime (Nowlan et al., 2010a) may be of great relevance to interpreting patellar biology and evolution, but at present strong inferences cannot be drawn about how variable the patella’s responsiveness to mechanics is within or among organisms. There is clearly much room for further study of the patellae of mammals and other tetrapods, and here we have noted directions in which these might most beneficially be directed.