Phalangeal Elements Research Articles

Single-cell mapping of regenerative and fibrotic healing responses after musculoskeletal injury.

SummaryAfter injury, a cascade of events repairs the damaged tissue, including expansion and differentiation of the progenitor pool and redeposition of matrix. To guide future wound regeneration strategies, we compared single-cell sequencing of regenerative (third phalangeal element [P3]) and fibrotic (second phalangeal element [P2]) digit tip amputation (DTA) models as well as traumatic heterotopic ossification (HO; aberrant). Analyses point to a common initial response to injury, including expansion of progenitors, redeposition of matrix, and activation of transforming growth factor β (TGF-β) and WNT pathways. Surprisingly, fibrotic P2 DTA showed greater transcriptional similarity to HO than to regenerative P3 DTA, suggesting that gene expression more strongly correlates with healing outcome than with injury type or cell origin. Differential analysis and immunostaining revealed altered activation of inflammatory pathways, such as the complement pathway, in the progenitor cells. These data suggests that common pathways are activated in response to damage but are fine tuned within each injury. Modulating these pathways may shift the balance toward regenerative outcomes.

Mouse Digit Tip Regeneration Is Mechanical Load Dependent.

Amputation of the mouse digit tip results in blastema-mediated regeneration. In this model, new bone regenerates de novo to lengthen the amputated stump bone, resulting in a functional replacement of the terminal phalangeal element along with associated non-skeletal tissues. Physiological examples of bone repair, such as distraction osteogenesis and fracture repair, are well known to require mechanical loading. However, the role of mechanical loading during mammalian digit tip regeneration is unknown. In this study, we demonstrate that reducing mechanical loading inhibits blastema formation by attenuating bone resorption and wound closure, resulting in the complete inhibition of digit regeneration. Mechanical unloading effects on wound healing and regeneration are completely reversible when mechanical loading is restored. Mechanical unloading after blastema formation results in a reduced rate of de novo bone formation, demonstrating mechanical load dependence of the bone regenerative response. Moreover, enhancing the wound-healing response of mechanically unloaded digits with the cyanoacrylate tissue adhesive Dermabond improves wound closure and partially rescues digit tip regeneration. Taken together, these results demonstrate that mammalian digit tip regeneration is mechanical load-dependent. Given that human fingertip regeneration shares many characteristics with the mouse digit tip, these results identify mechanical load as a previously unappreciated requirement for de novo bone regeneration in humans. © 2021 American Society for Bone and Mineral Research (ASBMR).

Novel splice mutation in LRP4 causes severe type of Cenani-Lenz syndactyly syndrome with oro-facial and skeletal symptoms

Cenani-Lenz syndactyly syndrome (CLSS; MIM-212780) is a rare autosomal recessive limb malformation characterized by complete osseous fusion of all fingers and toes, disorganization of phalangeal elements and severe shortening of the radius and ulna. It is occasionally associated with renal hypoplasia, oro-facial defects, scoliosis of the thoracic spine, hearing loss, and genital anomalies. Here we describe a consanguineous Pakistani kindred with a severe form of CLSS characterized by complete syndactyly and disorganization of fingers, oligo-syndactyly of toes, shortening of limbs, frontal bossing, and hypoplasia/agenesis of left kidney. The affected individuals were additionally presented with short stature, cleft-lip and hypoplastic shoulder joint with restricted upper limb movement. A novel splice variant in LRP4 (c.316+1G > A) segregated with the phenotype in a five generations family. The mutation is predicted to add 29 non-native amino acids with a premature termination, resulting in approximately 90% length reduction of the wild-type transcript. These findings not only further expand the phenotypic variability of CLSS but also indicate that early truncated and loss-of-function mutations in LRP4 lead to a more severe CLSS phenotype.

Analogous cellular contribution and healing mechanisms following digit amputation and phalangeal fracture in mice.

Regeneration of amputated structures is severely limited in humans and mice, with complete regeneration restricted to the distal portion of the terminal phalanx (P3). Here, we investigate the dynamic tissue repair response of the second phalangeal element (P2) post amputation in the adult mouse, and show that the repair response of the amputated bone is similar to the proximal P2 bone fragment in fracture healing. The regeneration‐incompetent P2 amputation response is characterized by periosteal endochondral ossification resulting in the deposition of new trabecular bone, corresponding to a significant increase in bone volume; however, this response is not associated with bone lengthening. We show that cells of the periosteum respond to amputation and fracture by contributing both chondrocytes and osteoblasts to the endochondral ossification response. Based on our studies, we suggest that the amputation response represents an attempt at regeneration that ultimately fails due to the lack of a distal organizing influence that is present in fracture healing.

A new cursorial hyena from Tibet, and analysis of biostratigraphy, paleozoogeography, and dental morphology of Chasmaporthetes (Mammalia, Carnivora)

ABSTRACT Recent field work in the late Cenozoic Zanda Basin in southwestern Tibetan Plateau has provided new fossil evidence of vertebrate faunas spanning the late Miocene to Pleistocene, which represents new occurrences hitherto unknown in that region of Asia. In this paper we describe a new species of the cursorial hyaenid Chasmaporthetes, C. gangsriensis, sp. nov., from the Zanda Basin. Chasmaporthetes gangsriensis is smaller than other Plio-Pleistocene Eurasian records of the genus, and retains relatively wide premolars that are underdifferentiated in size. The m1 talonid has a trenchant hypoconid typical of Chasmaporthetes, but with a trigonid length-to-width ratio lower than all specimens referred to the genus. Metatarsal and phalangeal elements referred to C. gangsriensis are long and gracile, indicating cursorial abilities typical of Chasmaporthetes. With an age of early Pliocene (4.89–4.08 Ma), C. gangsriensis is morphologically the most basal Pliocene Chasmaporthetes in China, and is consistent with the ‘out of Tibet’ hypothesis for some Pleistocene megafauna. An analysis of nasal bone morphology revealed large intraspecific variation in extant spotted hyenas, showing that it is not a reliable criterion for species diagnosis in Chasmaporthetes. An evaluation of the biostratigraphic relationships among Asian and North American occurrences of Chasmaporthetes indicates that the genus first dispersed into the New World during the early Blancan North American land mammal age (NALMA) with likely ancestry close to the heterogeneous sample of C. lunensis in Eurasia. The possibility of a second dispersal is indicated by a mixed sample of specimens with significantly smaller p4/m1 length ratio than other Chasmaporthetes. SUPPLEMENTAL DATA—Supplemental materials are available for this article for free at www.tandfonline.com/UJVP

Increased oxygen enhances bone degradation and promotes bone regeneration after digit amputation in mice

In 2005, approximately 1.6 million persons in the United States were living with the loss of a limb. That number is projected to increase to 3.6 million by 2050. While full limb replacement is not yet a possibility, prosthetic limb replacement is common amongst amputees, and the ability to control bone regeneration is highly favorable for comfortable fit. The capacity to regenerate bone post‐amputation is limited to only the distal 1/3 of the third phalangeal element (P3) of rodents, monkeys, and humans. Regeneration in adult mice is defined as three distinct steps: 1) wound healing and bone degradation, 2) formation of the blastema, and 3) differentiation of the blastema into bone and tissue. Our hypothesis is that P3 regeneration is facilitated by temporally specific fluctuations in oxygen. The objective of this study is to apply hyperbaric oxygen treatment (HBOT) to our well‐characterized P3 regenerative model to evaluate the effects of increased oxygen during P3 regeneration. Our data show that digits treated with HBOT immediately after amputation display elevated levels of bone degradation. These digits are then able to fully regenerate P3, demonstrating an expansion of regenerative capacity to the proximal P3 bone stump. Our findings strongly support that oxygen is able to act as a primary signaling event to influence bone regeneration in vivo. Research funded by grant 1F32HD071763–01A1 from the NIH

Modulating the Physical Microenvironment to Study Regenerative Processes In Vitro Using Cells from Mouse Phalangeal Elements

Epimorphic regeneration in humans of complex multitissue structures is primarily limited to the digit tip. In a comparable mouse model, the response is level-specific in that regeneration occurs after amputation at the distal end of the terminal phalanx, but not more proximally. Recent isolation of stromal cells from CD1 murine phalangeal elements two and three (P2 and P3) allow for comparative studies of cells prevalent at the amputation plane of a more proximal region (considered nonregenerative) and a more distal region (considered regenerative), respectively. This study used adherent, suspension, and collagen gel cultures to investigate cellular processes relevant to the initial response to injury. Overall, P2 cells were both more migratory and able to compact collagen gels to a greater extent compared to P3 cells. This observed increased capacity of P2 cells to generate traction forces was likely related to the higher expression of key cytoskeletal proteins (e.g., microfilament, nonkeratin intermediate filaments, and microtubules) compared to P3 cells. In contrast, P3 cells were found to be more proliferative than P2 cells under all three culture conditions and to have higher expression of keratin proteins. In addition, when cultured in suspension rather than on adherent surfaces, P3 cells were both more proliferative and had greater gene expression for matrix proteins. Together these results add to the known inherent differences in these stromal cells by characterizing responses to the physical microenvironment. Further, while compaction by P2 cells confirm that collagen gels is a useful model to study wound healing, the response of P3 cells indicate that suspension culture, in which cell-cell interactions dominate like in the blastema, may be better suited to study regeneration. Therefore, this study can help develop clinical strategies for promoting regeneration through increased understanding in the properties of cells involved in endogenous repair as well as informed selection of useful in vitro models.

Connective tissue fibroblast properties are position-dependent during mouse digit tip regeneration.

A key factor that contributes to the regenerative ability of regeneration-competent animals such as the salamander is their use of innate positional cues that guide the regeneration process. The limbs of mammals has severe regenerative limitations, however the distal most portion of the terminal phalange is regeneration competent. This regenerative ability of the adult mouse digit is level dependent: amputation through the distal half of the terminal phalanx (P3) leads to successful regeneration, whereas amputation through a more proximal location, e.g. the subterminal phalangeal element (P2), fails to regenerate. Do the connective tissue cells of the mammalian digit play a role similar to that of the salamander limb in controlling the regenerative response? To begin to address this question, we isolated and cultured cells of the connective tissue surrounding the phalangeal bones of regeneration competent (P3) and incompetent (P2) levels. Despite their close proximity and localization, these cells show very distinctive profiles when characterized in vitro and in vivo. In vitro studies comparing their proliferation and position-specific interactions reveal that cells isolated from the P3 and P2 are both capable of organizing and differentiating epithelial progenitors, but with different outcomes. The difference in interactions are further characterized with three-dimension cultures, in which P3 regenerative cells are shown to lack a contractile response that is seen in other fibroblast cultures, including the P2 cultures. In in vivo engraftment studies, the difference between these two cell lines is made more apparent. While both P2 and P3 cells participated in the regeneration of the terminal phalanx, their survival and proliferative indices were distinct, thus suggesting a key difference in their ability to interact within a regeneration permissive environment. These studies are the first to demonstrate distinct positional characteristics of connective tissue cells that are associated with their regenerative capabilities.

BMP2 induces segment-specific skeletal regeneration from digit and limb amputations by establishing a new endochondral ossification center

Bone morphogenetic proteins (BMPs) are required for bone development, the repair of damage skeletal tissue, and the regeneration of the mouse digit tip. Previously we showed that BMP treatment can induce a regeneration response in mouse digits amputated at a proximal level of the terminal phalangeal element (P3) (Yu et al., 2010). In this study, we show that the regeneration-inductive ability of BMP2 extends to amputations at the level of the second phalangeal element (P2) of neonatal digits, and the hindlimb of adult limbs. In these models the induced regenerative response is restricted in a segment-specific manner, thus amputated skeletal elements regenerate distally patterned skeletal structures but does not form joints or more distal skeletal elements. Studies on P2 amputations indicate that BMP2-induced regeneration is associated with a localized proliferative response and the transient expression of established digit blastema marker genes. This is followed by the formation of a new endochondral ossification center at the distal end of the bone stump. The endochondral ossification center contains proliferating chondrocytes that establish a distal proliferative zone and differentiate proximally into hypertrophic chondrocytes. Skeletal regeneration occurs from proximal to distal with the appearance of osteoblasts that differentiate in continuity with the amputated stump. Using the polarity of the endochondral ossification centers induced by BMP2 at two different amputation levels, we show that BMP2 activates a level-dependent regenerative response indicative of a positional information network. In summary, our studies provide evidence that BMP2 induces the regeneration of mammalian limb structures by stimulating a new endochondral ossification center that utilizes an existing network of positional information to regulate patterning during skeletal regeneration.

The role of oxygen in bone regeneration

One in every 200 people in the U.S. will suffer an amputation. Amputation has protracted effects on vocational outcome, individual productivity, and personal independence far beyond the initial trauma. Amputation of the digit tip within the terminal phalangeal bone of rodents, monkeys, and humans results in near‐perfect regeneration from a cell population called the blastema, which has been well characterized in both adult and neonatal mice. However, it remains unclear why amputations within the more proximal phalangeal elements, or within limbs, fail to produce the same regenerative result. The objective of this study is to gain insight into the role of oxygen in bone regeneration by manipulating oxygen to alter the regenerative process. Using the murine amputation model, we have developed a novel multi‐tissue in vitro slice culture model that is used with mixed gas to control oxygen tension in vitro. To our knowledge, this is the first time such a model has been used in digit regeneration and shows promise as a model for studies involving bone remodeling. Using this model we have determined that 3% oxygen delays bone redifferentiation while 20% oxygen promotes redifferentiation into bone. Incubation of the blastema at 3% oxygen, prior to incubation at 20% oxygen, further accelerated the rate of bone formation. Our results show that the use of sequential incubation in 3% and 20% oxygen can be used to accelerate the regenerative process of bone when used at the correct temporal interval during the regenerative process. Research enabled by this work will lead to a defined mechanistic understanding of oxygen's role in bone regeneration and identify therapeutic targets to enhance regenerative capacity.

The postcranial anatomy of Suminia getmanovi (Synapsida: Anomodontia), the earliest known arboreal tetrapod

The basal anomodont Suminia getmanovi Ivakhnenko, 1994 from the late Palaeozoic of Russia is highly specialized in its masticatory apparatus, and has been suggested to represent the earliest arboreal tetrapod in the fossil record. Its postcranial anatomy is described in detail for the first time, revealing a large number of autapomorphies for this small herbivore. These include a reduced number of presacral and therein dorsal vertebrae, an elongate neck, a long and possibly prehensile tail, a procoracoid with a notch at its ventromedial margin rather than a foramen, an iliac blade with a robust ridge at its anteromedial edge, a pubis with a puboischiadic fenestra and separate pubic foramen, and elongate limbs. Additional autapomorphic characters are displayed in the autopodium, which comprises about 40% of the entire limb length. These features include an enlarged, phalangiform distal carpal 1 and tarsal 1, a short and robust first metacarpal, a crescent-shaped distal tarsal 4, and elongate penultimate phalangeal elements. The phylogenetic relationships of basal anomodonts are revisited using an expanded data set, with the addition of key taxa and several postcranial characters. Unlike dicynodonts, Suminia retained the plesiomorphic phalangeal formula for amniotes of 2-3-4-5-3 (manus) and 2-3-4-5-4 (pes). This pattern is achieved by the retention of disc-like phalangeal elements between the proximal and penultimate phalanges in digits III, IV (manus and pes), and V (pes only). In light of the new material, Suminia can be recognized as the most complete basal anomodont, offering new insights into the early evolution of the group. © 2011 The Linnean Society of London, Zoological Journal of the Linnean Society, 2011, 162, 661–698.

Embryonic skeletal anatomy of the sauropodomorph dinosaur Massospondylus from the Lower Jurassic of South Africa

ABSTRACT Two embryonic skeletons preserved inside thin-shelled eggs of a partially preserved clutch from the Upper Elliot Formation (Lower Jurassic) of South Africa have been attributed to the sauropodomorph dinosaur Massospondylus carinatus. A virtually complete skeleton is exposed in right lateral view, with the slightly telescoped skull and several cervical vertebrae extending beyond the eggshell. A second, partial skeleton has a skull preserved in dorsal view. The embryos have proportionately very large skulls, with the broad skull table formed by wide parietals and frontals. The wide posterolateral wing of the frontal separates the postorbital from contact with the parietal. The embryos have short rather than elongated cervical vertebrae, with tall rather than low neural arches. The large forelimbs are only slightly shorter than the hind limbs, which suggests an obligatory quadrupedal posture for the hatchlings. This pattern may represent an ontogenetic constraint related to the large size of the head and horizontally oriented neck. Similarities between the embryonic and post-hatchling specimens include the slenderness of the lower jaw and slight ventral curvature of the symphyseal portion of the dentary, the large supraorbital process of the prefrontal, and the tall antorbital and infratemporal fenestrae. There are 10 cervical, 14 dorsal, and three sacral vertebrae. The large distal claw-bearing phalanx of manual digit 1 is longer than any other phalangeal element of either manus or pes. The embryos of Massospondylus carinatus represent the oldest dinosaurian embryos known to date.

Paedomorphic Ossification in Porpoises with an Emphasis on the Vaquita (<I>Phocoena sinus</I>)

Heterochrony, the change in timing of developmental processes, is thought to be a key process shaping the numerous limb morphologies of tetrapods. Through a delayed offset in digit development, all cetaceans (i.e., whales, dolphins, and porpoises) have evolved supernumary phalanges (hyperphalangy). Moreover, some toothed cetaceans further alter digital morphologies by delayed endochondral and perichondral ossification of individual elements. In the harbor porpoise (Phocoena phocoena), these paedomorphic patterns have created poorly ossified phalangeal elements. However, no studies have addressed this morphology in other porpoise taxa. This study documents the timing of carpal and digital epiphyseal ossification in the poorly studied vaquita (Phocoena sinus) based on radiographs (n = 18) of known-age specimens. Patterns of vaquita manus ossification were compared between other porpoise and delphinid taxa. Adult vaquitas are paedomorphic in carpal, metacarpal, and digital development as they maintain a juvenile ossification pattern relative to that of other porpoise species of equivalent ages. Vaquitas also ossify fewer carpal elements as compared to other porpoise and some delphinid cetaceans, and ossification arrests relative to that of the harbor porpoise. Vaquitas also display sexual dimorphism as females reach a greater body size and display more ossified elements in the manus relative to their paedomorphic male cohorts.

Fgf-Dependent Etv4/5 Activity Is Required for Posterior Restriction of Sonic hedgehog and Promoting Outgrowth of the Vertebrate Limb

Crosstalk between the fibroblast growth factor (FGF) and Sonic Hedgehog (Shh) pathways is critical for proper patterning and growth of the developing limb bud. Here, we show that FGF-dependent activation of the ETS transcription factors Etv4 and Etv5 contributes to proximal-distal limb outgrowth. Surprisingly, blockage of Etv activity in early distal mesenchyme also resulted in ectopic, anterior expansion of Shh, leading to a polydactylous phenotype. These data indicate an unexpected function for an FGF/Etv pathway in anterior-posterior patterning. FGF activity in the limb is not only responsible for maintaining posterior-specific Shh expression, but it also acts via Etvs to prevent inappropriate anterior expansion of Shh. This study identifies another level of genetic interaction between the orthogonal axes during limb development.

Brachy–syndactyly caused by loss of Sfrp2 function

Wnt signaling pathways are regulated both at the intracellular and extracellular levels. During embryogenesis, the in vivo effects of the secreted frizzled-related protein (Sfrp) family of Wnt inhibitors are poorly understood. Here, we show that inactivation of Sfrp2 results in subtle limb defects in mice with mesomelic shortening and consistent shortening of all autopodal elements that is clinically manifested as brachydactyly. In addition, there is soft-tissue syndactyly of the hindlimb. The brachydactyly is caused by decreased chondrocyte proliferation and delayed differentiation in distal limb chondrogenic elements. These data suggest that Sfrp2 can regulate both chondrogenesis and regression of interdigital mesenchyme in distal limb. Sfrp2 can also repress canonical Wnt signaling by Wnt1, Wnt9a, and Wnt4 in vitro. Sfrp2-/- and TOPGAL/Sfrp2-/- mice have a mild increase in beta-catenin and beta-galactosidase staining, respectively, in some phalangeal elements. This however does not exclude a potential concurrent effect on non-canonical Wnt signaling in the growth plate. In combination with what is known about BMP and Wnt signaling in human brachydactylies, our data establish a critical role for Sfrp2 in proper distal limb formation and suggest SFPR2 could be a novel candidate gene for human brachy-syndactyly defects.

Evolution of hyperphalangy and digit reduction in the cetacean manus

Cetaceans (whales, dolphins, and porpoises) have a soft tissue flipper that encases most of the forelimb, and elongated digits with an increased number of phalanges (hyperphalangy). In addition, some cetaceans exhibit a reduction in digit number. Although toothed cetaceans (odontocetes) are pentadactylous, most baleen whales (mysticetes) are tetradactylous and also lack a metacarpal. This study conducts a survey of cetacean metacarpal and phalangeal morphologies, traces the evolution of hyperphalangy in a phylogenetic context, optimizes characters onto previously published cetacean phylogenies, and tests various digit loss hypotheses. Dissections were performed on 16 cetacean flippers representing 10 species (8 mysticetes, 2 odontocetes). Phalangeal count data were derived from forelimb radiographs (36 odontocetes, 5 mysticetes), osteological specimens of articulated forelimbs (8 mysticetes), and were supplemented with published counts. Modal phalangeal counts were coded as ordered and unpolarized characters and optimized onto two known cetacean phylogenies. Results indicate that digital ray I is reduced in many cetaceans (except Globicephala) and all elements of digital ray I were lost in tetradactylous mysticetes. Fossil evidence indicates this ray may have been lost approximately 14 Ma. Most odontocetes also reduce the number of phalangeal elements in digit V, while mysticetes typically retain the plesiomorphic condition of three phalanges. Results from modal phalangeal counts show the greatest degree of hyperphalangy in digits II and III in odontocetes and digits III and IV in mysticetes. Fossil evidence indicates cetacean hyperphalangy evolved by at least 7-8 Ma. Digit loss and digit positioning may underlie disparate flipper shapes, with narrow, elongate flippers facilitating fast swimming and broad flippers aiding slow turns. Hyperphalangy may help distribute leading edge forces, and multiple interphalangeal joints may smooth leading edge flipper contour.

First well-established track-trackmaker association of paleozoic tetrapods based on Ichniotherium trackways and diadectid skeletons from the Lower Permian of Germany

ABSTRACT As a single stratigraphic source and site of high-fidelity vertebrate trackways and superbly preserved skeletons, the Lower Permian Tambach Formation, lowermost unit of the Upper Rotliegend, of the Bromacker locality in the middle part of the Thuringian Forest near Gotha, central Germany, provides a unique opportunity of matching late Paleozoic trackways with their trackmakers. Here the track-trackmaker association is firmly established between two species of the ichnogenus Ichniotherium, Ichniotherium cottae and Ichniotherium sphaerodactylum, and the skeletal fossils of the closely related diadectids Diadectes absitus and Orobates pabsti, respectively. These are the first well-documented species-level identifications of the trackmakers of Paleozoic trackways. The Ichniotherium ichnospecies are principally separated by the relative lengths of the digits of the pes imprint and the degree of overstepping of the pes and manus imprints. Both characters are shown to be clearly due to differences in the number and lengths of phalangeal elements and the number of presacral vertebrae of the diadectid species. The unique methods employed here in establishing the track-trackmaker associations provide not only an innovative data source for studying the evolutionary biology, paleobiogeography, and locomotor behaviour of the trackmakers, but also a valuable methodology for evaluating taxonomic concepts in vertebrate ichnology.

Evidence at hand: Diversity, functional implications, and locomotor prediction in intrinsic hand proportions of diprotodontian marsupials

Knowledge about the diversity, locomotor adaptations, and evolution of the marsupial forelimb is limited, resulting in an underrepresentation of marsupials in comparative anatomical literature on mammalian forelimb anatomy. This study investigated hand proportions in the diverse marsupial order Diprotodontia. Fifty-two measurements of 95 specimens representing 47 species, as well as 6 non-diprotodontian specimens, were explored using principal components analysis (PCA). Bootstrapping was used to assess the reliability of the loadings. Phylogenetically independent contrasts and phylogenetic ANOVA were used to test for correlation with size and functional adaptation of forelimbs for locomotor habit, scored as arboreal vs. terrestrial. Analysis of first principal component (PC1) scores revealed significant differences between arboreal and terrestrial species, and was related to relative slenderness of their phalangeal elements. Both locomotor groups displayed allometry along PC1 scores, but with different intercepts such that PC1 discriminated between the two locomotor habits almost completely. PC2 separated some higher-level clades and burrowing species. Analysis of locomotor predictors commonly applied by palaeontologists indicates that ratios between proximal and intermediate phalanges were unsuitable as predictors of arboreality/terrestriality, but the phalangeal index was more effective. From PCA results, a phalangeal slenderness ratio was developed which proved to be a useful discriminator, suggesting that a single unallocated phalanx can be used for an impression of locomotor mode in fossils. Most Diprotodontia are laterally paraxonic or ectaxonic, with the exception of digging species whose hands are medially paraxonic. Our results complement those of studies on placental mammals, suggesting that the demands of arboreality, terrestriality, or frequent digging on intrinsic hand proportions are met with similar anatomical adaptations in marsupials.

Reconsideration of skeletal development of Chelydra serpentina (Reptilia: Testudinata: Chelydridae): evidence for intraspecific variation

Patterns of formation and sequence of ossification of the entire skeleton are described for the snapping turtle Chelydra serpentina, based on new and previously published data. Differences in the sequences of ossification events are described and demonstrate considerable intraspecific variation in these developmental data. The chondrocranium of a late developmental-stage specimen is described and illustrated as baseline data for comparative studies. Patterns of formation and chondrification of forelimb and hind limb elements are generally consistent with those of previous studies; however, conspicuous differences in the patterns of ossification are observed among metapodial and phalangeal elements. In the cranium, patterns of ossification of dermal elements are generally more variable than are those of endochondral elements of the braincase, and as in previous studies, endochondral elements ossify after dermal elements. Documented intraspecific variation in sequence and timing of formation, chondrification, and ossification of skeletal elements should serve as a caveat for those conducting studies of phylogeny, heterochrony, and evolution with these data. Causes of natural and induced variation are discussed briefly.

A NEW SPECIES OF ELEUTHERODACTYLUS (ANURA: LEPTODACTYLIDAE) FROM THE NORTHWESTERN LOWLANDS OF ECUADOR

A new species of Eleutherodactylus is described from the lowlands of northwestern Ecuador. The new species is a small frog (snout–vent length <23 mm) characterized by: expanded and spadate disc cover on some fingers (usually III and IV) and most toes (usually III–V); bifid palmar tubercle; and brown dorsum with dark brown W-shaped scapular mark. A detailed osteological description of the new species is provided. Some of the osteological characters include relatively large nasals, small sphenethmoid, eight procoelous presacral vertebrae, manus with six carpal elements, and pes with four tarsal elements. A small portion of the frontoparietal fontanelle is exposed between the frontoparietals. Although the new species is small, there is no loss of skull bones and phalangeal elements.