Hindlimb bones texture through postnatal ages of Rhea americana (Aves: Palaeognathae).

The bone texture of Rhea americana was evaluated through the examination of a postnatal ontogenetic series. The hind limb bone surfaces of specimens of one, three and five months old, and adults were compared to characterize each stage according to the morphological features generated by their differential ossification. The results suggest a similar process of tissue maturation concerning neognathous birds, although with some differences. A spongy or striated surface with abundant pores in the femur and longitudinal grooves in the tibiotarsus and tarsometatarsus characterizes chicks. Vascularity decreases, and the bone texture gradually changes acquiring a more homogeneous surface, to eventually reach the smooth appearance of adult bones. The establishment of particular textures corresponding to different bones and ontogenetic stages permits the accurate interpretation of remains in ecological, archaeological and paleontological contexts in which bones are fragmented and/or incomplete.

The feeding apparatus of Rhea americana (Aves, Palaeognathae): Jaw myology and ontogenetic allometry.

In birds, the jaw musculature is a crucial adaptive feature involved in feeding. The morphological traits and postnatal growth patterns of jaw muscles constitute a useful proxy to interpret feeding function and ecology. This study aims to describe the jaw muscles of Rhea americana and explore their postnatal growth pattern. A total of 20 specimens of R. americana representing four ontogenetic stages were studied. Jaw muscles were described, weighed and their proportions with respect to body mass were calculated. Linear regression analysis was used to characterize ontogenetic scaling patterns. The morphological patterns of jaw muscles were characterized by their simplicity: bellies with few or no subdivisions and similar to those described for other flightless paleognathous birds. In all stages, the muscles pterygoideus lateralis, depressor mandibulae, and pseudotemporalis had the greatest mass values. The proportion of total jaw muscle mass decreased with age from 0.22% in 1-month-old chicks to 0.05% in adults. Linear regression analysis showed that all muscles scaled with negative allometry with respect to body mass. The progressive decrease of jaw muscle mass relative to body mass in adults could be related to the generation of less force, which is in accordance with the herbivorous diet of adults. In contrast, the diet of rhea chicks includes a large proportion of insects thus, this greater muscle proportion could be associated with the ability to generate more force, thus providing better abilities to grasp and hold more mobile prey.

3D atlas of tinamou (Neornithes: Tinamidae) pectoral morphology: Implications for reconstructing the ancestral neornithine flight apparatus.

Palaeognathae, the extant avian clade comprising the flightless ratites and flight-capable tinamous (Tinamidae), is the sister group to all other living birds, and recent phylogenetic studies illustrate that tinamous are phylogenetically nested within a paraphyletic assemblage of ratites. As the only extant palaeognaths that have retained the ability to fly, tinamous may provide key information on the nature of the flight apparatus of ancestral crown palaeognaths-and, in turn, crown birds-as well as insight into convergent modifications to the wing apparatus among extant ratite lineages. To reveal new information about the musculoskeletal anatomy of tinamous and facilitate development of computational biomechanical models of tinamou wing function, we generated a three-dimensional musculoskeletal model of the flight apparatus of the extant Andean tinamou (Nothoprocta pentlandii) using diffusible iodine-based contrast-enhanced computed tomography (diceCT). Origins and insertions of the pectoral flight musculature of N. pentlandii are generally consistent with those of other extant volant birds specialized for burst flight, and the entire suite of presumed ancestral neornithine flight muscles are present in N. pentlandii with the exception of the biceps slip. The pectoralis and supracoracoideus muscles are robust, similar to the condition in other extant burst-flying birds such as many extant Galliformes. Contrary to the condition in most extant Neognathae (the sister clade to Palaeognathae), the insertion of the pronator superficialis has a greater distal extent than the pronator profundus, although most other anatomical observations are broadly consistent with the conditions observed in extant neognaths. This work will help form a basis for future comparative studies of the avian musculoskeletal system, with implications for reconstructing the flight apparatus of ancestral crown birds and clarifying musculoskeletal modifications underlying the convergent origins of ratite flightlessness.

Effect of Different Types of Sequence Data on Palaeognath Phylogeny.

Palaeognathae consists of five groups of extant species: flighted tinamous (1) and four flightless groups: kiwi (2), cassowaries and emu (3), rheas (4), and ostriches (5). Molecular studies supported the groupings of extinct moas with tinamous and elephant birds with kiwi as well as ostriches as the group that diverged first among the five groups. However, phylogenetic relationships among the five groups are still controversial. Previous studies showed extensive heterogeneity in estimated gene tree topologies from conserved nonexonic elements, introns, and ultraconserved elements. Using the noncoding loci together with protein-coding loci, this study investigated the factors that affected gene tree estimation error and the relationships among the five groups. Using closely related ostrich rather than distantly related chicken as the outgroup, concatenated and gene tree-based approaches supported rheas as the group that diverged first among groups (1)-(4). Whereas gene tree estimation error increased using loci with low sequence divergence and short length, topological bias in estimated trees occurred using loci with high sequence divergence and/or nucleotide composition bias and heterogeneity, which more occurred in trees estimated from coding loci than noncoding loci. Regarding the relationships of (1)-(4), the site patterns by parsimony criterion appeared less susceptible to the bias than tree construction assuming stationary time-homogeneous model and suggested the clustering of kiwi and cassowaries and emu the most likely with ∼40% support rather than the clustering of kiwi and rheas and that of kiwi and tinamous with 30% support each.

The selfish preen: absence of allopreening in Palaeognathae and its socio-cognitive implications.

Preening behaviours are widespread in extant birds. While most birds appear to autopreen (self-directed preening), allopreening (preening directed at conspecifics) seems to have emerged only in certain species, but across many families. Allopreening has been hypothesised to reinforce mutual relationships and cooperation between individuals, and to underpin various socio-cognitive abilities. Palaeognathae is a bird group exhibiting neurocognitively plesiomorphic traits compared to other birds. They share many features with non-avian paravian dinosaurs and are thus important for the study of cognitive evolution in birds. Despite this, and the important correlation of allopreening with many complicated social behaviours, allopreening has not been systematically studied in Palaeognathae. Therefore, we examined the preening behaviours in four species of palaeognaths: common ostriches (Struthio camelus), greater rheas (Rhea americana), emus (Dromaius novaehollandiae), and elegant crested tinamous (Eudromia elegans). We compared findings with common ravens (Corvus corax), a neognath species known for its allopreening and complex social cognition. We found autopreening, but no allopreening, in the palaeognath species, while both autopreening and allopreening was found in common ravens. The absence of allopreening in Palaeognathae suggests an emergence of this behaviour within Neognathae. We contextualise our results in relation to the socio-cognitive underpinnings of allopreening and its implications for the understanding of the evolution of socio-cognitive abilities in non-avian paravian dinosaurs and early birds.

Molecular characterisation and additional morphological descriptions of Eimeria spp. (Apicomplexa: Eimeriidae) from brown kiwi (Apteryx mantelli Bartlett).

Brown kiwi (Apteryx mantelli Bartlett), a ratite endemic to New Zealand, is currently listed as "Vulnerable" under the IUCN classification system due to predation by introduced mammals. Operation Nest Egg (ONE) raises chicks and juveniles in predator-proof enclosures until they are large enough to defend themselves. These facilities experience an environmental accumulation of coccidial oocysts, which leads to severe morbidity and mortality of these kiwi. Four species of coccidia have been morphologically described from sporulated oocysts with additional opportunistic descriptions of endogenous stages. This research continues the morphological descriptions of these species of Eimeria with an additional novel morphotype also morphologically described. It also provides the first genetic characterisation targeting the mitochondrial cytochrome c oxidase I (COI) gene. Based on these findings, it was determined there are at least five morphotypes of Eimeria that infect brown kiwi and co-infections are common at the ONE facilities surveyed. The COI amplicon targeted for this study was sufficient to provide differentiation from other members of this genus. Sanger sequencing yielded ambiguous bases, indicating the need for more in-depth sequencing.

Cardiac morphology of North Island brown kiwi (Apteryx mantelli).

AIMS: To investigate the cardiac anatomy of North Island brown kiwi (Apteryx mantelli) through heart morphometric parameters measured at post-mortem examination. METHODS: Morphometric cardiac parameters were established at post-mortem examination of 20 North Island brown kiwi. Birds were classified by gender and age (chicks vs. adults). Measurements included: body mass, heart mass, sternal length, midpoint thickness of left ventricular free wall, midpoint thickness of right ventricular free wall and ratios of heart mass to body mass, left ventricular length to sternal length, right ventricular length to sternal length, length of left ventricle to right ventricle, interventricular septal thickness relative to the sternal length and interventricular septal thickness relative to the left ventricular length. Unadjusted estimates of the median difference and their 95% CI were then reported at each age and sex for all the cardiac morphometric parameters and their ratios. RESULTS: The small sample size led to wide 95% CI for the median difference between gender and age for the cardiac morphometric measurements. Nevertheless, between adult female and male kiwi, the estimated population median differences for heart mass (2.2 (95% CI = -2.9-5.6) g), length (1.2 (95% CI = -2.2-5.6) mm), width (6.1 (95% CI = -1.0-8.2) mm), left ventricular free wall length (5.5 (95% CI = -0.5-8.8) mm) and right ventricular free wall length (2.6 (95% CI = -3.7-6.9) mm) were established. In adult North Island brown kiwi, the heart mass is 0.8 (95% CI = 0.7-0.8)% of the body mass. CONCLUSIONS: The precision of the differences noted in heart measurements recorded between male and female kiwi at each age was limited by the low sample size available for this study. This led to wide CI and an inability to adjust differences observed for gender by differences in other confounders such as body size. With this caveat, there is weak evidence that adult female kiwi have a larger heart size and mass than the adult males. CLINICAL RELEVANCE: These results can be used to improve the diagnosis of cardiac disease in kiwi at post-mortem examination and aid in interpretation of the results of echocardiography in live birds for the antemortem diagnosis of cardiac disorders.

Causes of mortality of kiwi (Apteryx spp.) in New Zealand: a retrospective analysis of post-mortem records, 2010-2020.

AIMS: To examine and assess causes of mortality of kiwi (Apteryx spp.) submitted to Massey University between 2010 and 2020 across the five recognised species according to location, age group and captivity status in New Zealand. METHODS: Post-mortem reports were obtained from the Massey University/Te Kunenga ki Purehuroa School of Veterinary Science/Wildbase Pathology Register. Inclusion criteria were all species of kiwi with a date of post-mortem examination between August 2010 and August 2020. Data from each report was exported, categorised and compared using Microsoft Excel. RESULTS: Of a total of 1,005 post-mortem reports, there were 766 North Island brown kiwi (NIBK; A. mantelli), 83 tokoeka (A. australis), 73 rowi (A. rowi), 49 great spotted kiwi (A. haastii), and 34 little spotted kiwi (A. owenii). This comprised 19 eggs/embryos, 125 neonatal, 473 juvenile, 153 subadult, and 235 adult kiwi. There were 615 kiwi from wild populations, 148 from sanctuary populations, 238 from captivity, and four from unspecified locations. The leading cause of death was trauma, affecting 322 (32.0 (95% CI = 29.2-35.0)%) kiwi including 289 (37.3 (95% CI = 26.0-31.7)%) NIBK. Nearly half of these died from predation by mustelids, with losses recorded from neonates to adults and clustered in the central to southern North Island. Predation by dogs was the second most common cause of death, killing 84 (8.4 (95% CI = 6.7-10.2)%) kiwi, of which 65.5% came from the northern districts of the North Island. Non-infectious disease killed 214 (21 (95% CI = 18.8-24.0)%) kiwi, and included developmental deformities, gastrointestinal foreign bodies and predator trap injuries. Infectious disease killed 181 (18.0 (95% CI = 15.7-20.5)%) kiwi and the proportion decreased with age, with common diagnoses including coccidiosis, bacterial septicaemia, avian malaria, and fungal respiratory disease. Starvation affected 42 (4.2 (95% CI = 3.0-5.6)%) kiwi, comprised of mainly neonatal or juvenile individuals from wild or sanctuary populations, with a higher percentage seen in tokoeka (11/83; 13.3%) compared to other species (min 0%, max 5.9%). The cause of death was undetermined in 246 (24.5 (95% CI = 21.8-27.3)%) cases, which was most often due to poor preservation of remains. This included 33/73 (46%) rowi and 32/83 (39%) tokoeka, and affected mainly birds from sanctuary and wild populations. CONCLUSIONS: This study enhances our understanding of causes of mortality in captive, wild and sanctuary populations of all kiwi species and age groups within contemporary New Zealand.

Carcass and meat quality of red-winged tinamou (Rhynchotus rufescens) selected for muscle growth.

The aim of the present study was to evaluate the efficiency of selection for body growth and its association with carcass and meat quality traits in the red-winged tinamou. Two experimental groups were selected based on the selection index: selection group with a higher index (TinamouS) and commercial group with a lower index (TinamouC). Weight at 180 days and slaughter weight were significantly higher (p < 0.05) in the TinamouS group, as were hot carcass weight, skinless breast weight, wing weight, and thigh and drumstick weight. The meat quality traits or sensory attributes did not differ significantly (p > 0.05) between groups. A significant positive correlation (0.59; p < 0.05) was found between shear force and chewability and a significant negative correlation (- 0.59; p < 0.05) between aroma intensity and strange aroma. The present study suggests that the selection index promotes greater body growth and preserves meat quality and sensory traits in red-winged tinamou.

Genomic insights into the evolutionary relationships and demographic history of kiwi.

Kiwi are a unique and emblematic group of birds endemic to New Zealand. Deep-time evolutionary relationships among the five extant kiwi species have been difficult to resolve, in part due to the absence of pre-Quaternary fossils to inform speciation events. Here, we utilise single representative nuclear genomes of all five extant kiwi species (great spotted kiwi, little spotted kiwi, Okarito brown kiwi, North Island brown kiwi, and southern brown kiwi) and investigate their evolutionary histories with phylogenomic, genetic diversity, and deep-time (past million years) demographic analyses. We uncover relatively low levels of gene-tree phylogenetic discordance across the genomes, suggesting clear distinction between species. However, we also find indications of post-divergence gene flow, concordant with recent reports of interspecific hybrids. The four species for which unbiased levels of genetic diversity could be calculated, due to the availability of reference assemblies (all species except the southern brown kiwi), show relatively low levels of genetic diversity, which we suggest reflects a combination of older environmental as well as more recent anthropogenic influence. In addition, we suggest hypotheses regarding the impact of known past environmental events, such as volcanic eruptions and glacial periods, on the similarities and differences observed in the demographic histories of the five kiwi species over the past million years.

Osteohistological description of ostrich and emu long bones, with comments on markers of growth.

Ostriches and emus are among the largest extant birds and are frequently used as modern analogs for the growth dynamics of non-avian theropod dinosaurs. These ratites quickly reach adult size in under 1 year, and as such do not typically exhibit annually deposited growth marks. Growth marks, commonly classified as annuli or lines of arrested growth (LAGs), represent reduced or halted osteogenesis, respectively, and their presence demonstrates varying degrees of developmental plasticity. Growth marks have not yet been reported from ostriches and emus, prompting authors to suggest that they have lost the plasticity required to deposit them. Here we observe the hind limb bone histology of three captive juvenile emus and one captive adult ostrich. Two of the three juvenile emus exhibit typical bone histology but the third emu, a 4.5-month-old juvenile, exhibits a regional arc of avascular tissue, which we interpret as a growth mark. As this mark is not present in the other two emus from the same cohort and it co-occurs with a contralateral broken fibula, we suggest variable biomechanical load as a potential cause. The ostrich exhibits a complete ring of avascular, hypermineralized bone with sparse, flattened osteocyte lacunae. We identify this as an annulus and interpret it as slowing of growth. In the absence of other growth marks and lacking the animal's life history, the timing and cause of this ostrich's reduced growth are unclear. Even so, these findings demonstrate that both taxa retain the ancestral developmental plasticity required to temporarily slow growth. We also discuss the potential challenges of identifying growth marks using incomplete population data sets and partial cortical sampling.

An updated look at the mating system, parental care and androgen seasonal variations in ratites.

Androgens modulate multiple key aspects of male reproduction, from morphology to mating behavior. Across animals the seasonal patterns of androgens are tightly linked to many of the species' life-history traits and their evolution. One popular framework to address this issue has been the Challenge Hypothesis, which proposed a testosterone-mediated trade-off between mating and parental care in males. Given the lack of empirical support, especially in birds, this hypothesis has been recently revisited (Challenge Hypothesis 2.0), integrating aspects such as male-female interactions and the diversity of reproductive systems in birds. Ratites constitute the most basal avian group (Palaeognathae: ratites together with Tinamiformes) and have certain characteristics that make them unique. They are flightless and generally have promiscuous mating systems with communal nests and male-only parental care (nest building, incubation and chick rearing). Furthermore, male testosterone concentrations remain high during the entire parental care period. Here we review the reproductive biology of ratites, integrating information on seasonal variations in parental care, social interactions and androgen levels across the group, in light of the Challenge Hypotheses and the Challenge Hypothesis 2.0 (there are no seasonal hormonal data for Tinamiformes, therefore they are not included in this review). We also discuss the constraints that could explain the lack of experimental approaches in behavioral endocrinology across ratites. I hope this review will motivate further research on this basal group of birds and further our understanding of the evolution of the mechanisms in the endocrine system that underly reproductive behavior across birds.

The Length Polymorphism of the 9th Intron in the Avian CHD1 Gene Allows Sex Determination in Some Species of Palaeognathae.

In palaeognathous birds, several PCR-based methods and a range of genes and unknown genomic regions have been studied for the determination of sex. Many of these methods have proven to be unreliable, complex, expensive, and time-consuming. Even the most widely used PCR markers for sex typing in birds, the selected introns of the highly conserved CHD1 gene (primers P2/P8, 1237L/1272H, and 2550F/2718R), have rarely been effective in palaeognathous birds. In this study we used eight species of Palaeognathae to test three PCR markers: CHD1i9 (CHD1 gene intron 9) and NIPBLi16 (NIPBL gene intron 16) that performed properly as Psittaciformes sex differentiation markers, but have not yet been tested in Palaeognathae, as well as the CHD1iA intron (CHD1 gene intron 16), which so far has not been used effectively to sex palaeognathous birds. The results of our research indicate that the CHD1i9 marker effectively differentiates sex in four of the eight species we studied. In Rhea americana, Eudromia elegans, and Tinamus solitarius, the electrophoretic patterns of the amplicons obtained clearly indicate the sex of tested individuals, whereas in Crypturellus tataupa, sexing is possible based on poorly visible female specific bands. Additionally, we present and discuss the results of our in silico investigation on the applicability of CHD1i9 to sex other Palaeognathae that were not tested in this study.

Theoretical and Practical Considerations when using Retroelement Insertions to Estimate Species Trees in the Anomaly Zone.

A potential shortcoming of concatenation methods for species tree estimation is their failure to account for incomplete lineage sorting. Coalescent methods address this problem but make various assumptions that, if violated, can result in worse performance than concatenation. Given the challenges of analyzing DNA sequences with both concatenation and coalescent methods, retroelement insertions (RIs) have emerged as powerful phylogenomic markers for species tree estimation. Here, we show that two recently proposed quartet-based methods, SDPquartets and ASTRAL_BP, are statistically consistent estimators of the unrooted species tree topology under the coalescent when RIs follow a neutral infinite-sites model of mutation and the expected number of new RIs per generation is constant across the species tree. The accuracy of these (and other) methods for inferring species trees from RIs has yet to be assessed on simulated data sets, where the true species tree topology is known. Therefore, we evaluated eight methods given RIs simulated from four model species trees, all of which have short branches and at least three of which are in the anomaly zone. In our simulation study, ASTRAL_BP and SDPquartets always recovered the correct species tree topology when given a sufficiently large number of RIs, as predicted. A distance-based method (ASTRID_BP) and Dollo parsimony also performed well in recovering the species tree topology. In contrast, unordered, polymorphism, and Camin-Sokal parsimony (as well as an approach based on MDC) typically fail to recover the correct species tree topology in anomaly zone situations with more than four ingroup taxa. Of the methods studied, only ASTRAL_BP automatically estimates internal branch lengths (in coalescent units) and support values (i.e., local posterior probabilities). We examined the accuracy of branch length estimation, finding that estimated lengths were accurate for short branches but upwardly biased otherwise. This led us to derive the maximum likelihood (branch length) estimate for when RIs are given as input instead of binary gene trees; this corrected formula produced accurate estimates of branch lengths in our simulation study provided that a sufficiently large number of RIs were given as input. Lastly, we evaluated the impact of data quantity on species tree estimation by repeating the above experiments with input sizes varying from 100 to 100,000 parsimony-informative RIs. We found that, when given just 1000 parsimony-informative RIs as input, ASTRAL_BP successfully reconstructed major clades (i.e., clades separated by branches $>0.3$ coalescent units) with high support and identified rapid radiations (i.e., shorter connected branches), although not their precise branching order. The local posterior probability was effective for controlling false positive branches in these scenarios. [Coalescence; incomplete lineage sorting; Laurasiatheria; Palaeognathae; parsimony; polymorphism parsimony; retroelement insertions; species trees; transposon.].

Phylogeny and sex chromosome evolution of Palaeognathae.

Many paleognaths (ratites and tinamous) have a pair of homomorphic ZW sex chromosomes in contrast to the highly differentiated sex chromosomes of most other birds. To understand the evolutionary causes for the different tempos of sex chromosome evolution, we produced female genomes of 12 paleognathous species and reconstructed the phylogeny and the evolutionary history of paleognathous sex chromosomes. We uncovered that Palaeognathae sex chromosomes had undergone stepwise recombination suppression and formed a pattern of "evolutionary strata". Nine of the 15 studied species' sex chromosomes have maintained homologous recombination in their long pseudoautosomal regions extending more than half of the entire chromosome length. We found that in the older strata, the W chromosome suffered more serious functional gene loss. Their homologous Z-linked regions, compared with other genomic regions, have produced an excess of species-specific autosomal duplicated genes that evolved female-specific expression, in contrast to their broadly expressed progenitors. We speculate such "defeminization" of Z chromosome with underrepresentation of female-biased genes and slow divergence of sex chromosomes of paleognaths might be related to their distinctive mode of sexual selection targeting females rather than males, which evolved in their common ancestors.

Analysis of Sex Chromosome Evolution in the Clade Palaeognathae from Phased Genome Assembly.

Birds in the clade Palaeognathae, excluding Tinamiformes, have morphologically conserved karyotypes and less differentiated ZW sex chromosomes compared with those of other birds. In particular, the sex chromosomes of the ostrich and emu have exceptionally large recombining pseudoautosomal regions (PARs), whereas non-PARs are classified into two strata according to the date of their origins: stratum 0 and stratum 1 (S1). However, the construction and analysis of the genome sequences in these regions in the clade Palaeognathae can be challenging because assembling the S1 region is difficult owing to low sequence diversity between gametologs (Z-linked and W-linked sequences). We addressed this issue by applying the Platanus-allee assembler and successfully constructed the haplotype-resolved (phased) assembly for female emu, cassowary, and ostrich using only sequence read data derived from the Illumina platform. Comparative genomic and phylogenetic analyses based on assembled Z-linked and W-linked sequences confirmed that the S1 region of emu and cassowary formed in their common ancestor. Moreover, the interspersed repetitive sequence landscapes in the S1 regions of female emu showed an expansion of younger repetitive elements in the W-linked S1 region, suggesting an interruption in homologous recombination in the S1 region. These results provide novel insights into the trajectory of sex chromosome evolution in the clade Palaeognathae and suggest that the Illumina-based phased assembly method is an effective approach for elucidating the evolutionary process underlying the transition from homomorphic to differentiated sex chromosomes.

Late Pleistocene/Early Holocene sites in the montane forests of New Guinea yield early record of cassowary hunting and egg harvesting.

How early human foragers impacted insular forests is a topic with implications across multiple disciplines, including resource management. Paradoxically, terminal Pleistocene and Early Holocene impacts of foraging communities have been characterized as both extreme-as in debates over human-driven faunal extinctions-and minimal compared to later landscape transformations by farmers and herders. We investigated how rainforest hunter-gatherers managed resources in montane New Guinea and present some of the earliest documentation of Late Pleistocene through mid-Holocene exploitation of cassowaries (Aves: Casuariidae). Worldwide, most insular ratites were extirpated by the Late Holocene, following human arrivals, including elephant birds of Madagascar (Aepyornithidae) and moa of Aotearoa/New Zealand (Dinornithiformes)-icons of anthropogenic island devastation. Cassowaries are exceptional, however, with populations persisting in New Guinea and Australia. Little is known of past human exploitation and what factors contributed to their survival. We present a method for inferring past human interaction with mega-avifauna via analysis of microstructural features of archaeological eggshell. We then contextualize cassowary hunting and egg harvesting by montane foragers and discuss the implications of human exploitation. Our data suggest cassowary egg harvesting may have been more common than the harvesting of adults. Furthermore, our analysis of cassowary eggshell microstructural variation reveals a distinct pattern of harvesting eggs in late ontogenetic stages. Harvesting eggs in later stages of embryonic growth may reflect human dietary preferences and foraging seasonality, but the observed pattern also supports the possibility that-as early as the Late Pleistocene-people were collecting eggs in order to hatch and rear cassowary chicks.

The Anti-Inflammatory Effect of a γ-Lactone Isolated from Ostrich Oil of Struthio camelus (Ratite) and Its Formulated Nano-Emulsion in Formalin-Induced Paw Edema.

The ostrich oil of Struthio camelus (Ratite) found uses in folk medicine as an anti-inflammatory in eczema and contact dermatitis. The anti-inflammatory effect of a γ-lactone (5-hexyl-3H-furan-2-one) isolated from ostrich oil and its formulated nano-emulsion in formalin-induced paw edema was investigated in this study. Ostrich oil was saponified using a standard procedure; the aqueous residue was fractionated, purified, and characterized as γ-lactone (5-hexyl-3H-furan-2-one) through the interpretation of IR, NMR, and MS analyses. The γ-lactone was formulated as nano-emulsion using methylcellulose (MC) for oral solubilized form. The γ-lactone methylcellulose nanoparticles (γ-lactone-MC-NPs) were characterized for their size, shape, and encapsulation efficiency with a uniform size of 300 nm and 59.9% drug content. The γ-lactone was applied topically, while the formulated nanoparticles (NPs) were administered orally to rats. A non-steroidal anti-inflammatory drug (diclofenac gel) was used as a reference drug for topical use and ibuprofen suspension for oral administration. Edema was measured using the plethysmograph method. Both γ-lactone and γ-lactone-MC-NPs showed reduction of formalin-induced paw edema in rats and proved to be better than the reference drugs; diclofenac gel and ibuprofen emulsion. Histological examination of the skin tissue revealed increased skin thickness with subepidermal edema and mixed inflammatory cellular infiltration, which were significantly reduced by the γ-lactone compared to the positive control (p-value = 0.00013). Diuretic and toxicity studies of oral γ-lactone-MC-NPs were performed. No diuretic activity was observed. However, lethargy, drowsiness, and refusal to feeding observed may limit its oral administration.

Computational modelling of muscle fibre operating ranges in the hindlimb of a small ground bird (Eudromia elegans), with implications for modelling locomotion in extinct species.

The arrangement and physiology of muscle fibres can strongly influence musculoskeletal function and whole-organismal performance. However, experimental investigation of muscle function during in vivo activity is typically limited to relatively few muscles in a given system. Computational models and simulations of the musculoskeletal system can partly overcome these limitations, by exploring the dynamics of muscles, tendons and other tissues in a robust and quantitative fashion. Here, a high-fidelity, 26-degree-of-freedom musculoskeletal model was developed of the hindlimb of a small ground bird, the elegant-crested tinamou (Eudromia elegans, ~550 g), including all the major muscles of the limb (36 actuators per leg). The model was integrated with biplanar fluoroscopy (XROMM) and forceplate data for walking and running, where dynamic optimization was used to estimate muscle excitations and fibre length changes throughout both gaits. Following this, a series of static simulations over the total range of physiological limb postures were performed, to circumscribe the bounds of possible variation in fibre length. During gait, fibre lengths for all muscles remained between 0.5 to 1.21 times optimal fibre length, but operated mostly on the ascending limb and plateau of the active force-length curve, a result that parallels previous experimental findings for birds, humans and other species. However, the ranges of fibre length varied considerably among individual muscles, especially when considered across the total possible range of joint excursion. Net length change of muscle-tendon units was mostly less than optimal fibre length, sometimes markedly so, suggesting that approaches that use muscle-tendon length change to estimate optimal fibre length in extinct species are likely underestimating this important parameter for many muscles. The results of this study clarify and broaden understanding of muscle function in extant animals, and can help refine approaches used to study extinct species.

On the validity and taxonomic status of emCrypturellus/em emobsoletus/em emgriseiventris/em (Salvadori, 1895) and emC. o. hypochraceus/em (Miranda-Ribeiro, 1938) (Aves, Tinamidae).

The nine currently recognized subspecies in the Brown Tinamou (Crypturellus obsoletus) complex are disjunctly widespread in South America, and at least three of them occur in Brazil. Morphological diagnosis of most of these taxa is imprecise, in contrast with consistent vocal differences described in the literature. We conducted a taxonomic review of two Amazonian taxa, C. o. griseiventris and C. o. hypochraceus, using morphological, morphometric, and vocal characters. Our results indicate that C. o. hypochraceus (Miranda-Ribeiro, 1938) is a junior synonym of C. o. griseiventris (Salvadori, 1895), and that Crypturellus griseiventris (Salvadori, 1895) must be treated as a full species, based on unique and fully diagnosable plumage and vocal patterns.