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.

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.

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.].

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.

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.

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.

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.

Symmetry breaking in the embryonic skin triggers directional and sequential plumage patterning.

The development of an organism involves the formation of patterns from initially homogeneous surfaces in a reproducible manner. Simulations of various theoretical models recapitulate final states of natural patterns, yet drawing testable hypotheses from those often remains difficult. Consequently, little is known about pattern-forming events. Here, we surveyed plumage patterns and their emergence in Galliformes, ratites, passerines, and penguins, together representing the three major taxa of the avian phylogeny, and built a unified model that not only reproduces final patterns but also intrinsically generates shared and varying directionality, sequence, and duration of patterning. We used in vivo and ex vivo experiments to test its parameter-based predictions. We showed that directional and sequential pattern progression depends on a species-specific prepattern: an initial break in surface symmetry launches a travelling front of sharply defined, oriented domains with self-organising capacity. This front propagates through the timely transfer of increased cell density mediated by cell proliferation, which controls overall patterning duration. These results show that universal mechanisms combining prepatterning and self-organisation govern the timely emergence of the plumage pattern in birds.

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.

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.

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.

Epidemiologic significance of Toxoplasma gondii infections in turkeys, ducks, ratites and other wild birds: 2009-2020.

Toxoplasma gondii infections are common in humans and animals worldwide. Wild and domestic avian species are important in the epidemiology of T. gondii infections because felids prey on them and excrete millions of oocysts in the environment, disseminating the infection. Herbivorous birds are also excellent sentinels of environmental contamination with T. gondii oocysts because they feed on the ground. Toxoplasma gondii infections in birds of prey reflect infections in intermediate hosts. Humans can become infected by consuming undercooked avian tissues. Here, the authors reviewed prevalence, persistence of infection, clinical disease, epidemiology and genetic diversity of T. gondii strains isolated from turkeys, geese, ducks, ratites and avian species (excluding chickens) worldwide 2009-2020. Genetic diversity of 102 T. gondii DNA samples isolated worldwide is discussed. The role of migratory birds in dissemination of T. gondii infection is discussed.

Coprolites reveal ecological interactions lost with the extinction of New Zealand birds.

Over the past 50,000 y, biotic extinctions and declines have left a legacy of vacant niches and broken ecological interactions across global terrestrial ecosystems. Reconstructing the natural, unmodified ecosystems that preceded these events relies on high-resolution analyses of paleoecological deposits. Coprolites are a source of uniquely detailed information about trophic interactions and the behaviors, gut parasite communities, and microbiotas of prehistoric animal species. Such insights are critical for understanding the legacy effects of extinctions on ecosystems, and can help guide contemporary conservation and ecosystem restoration efforts. Here we use high-throughput sequencing (HTS) of ancient eukaryotic DNA from coprolites to reconstruct aspects of the biology and ecology of four species of extinct moa and the critically endangered kakapo parrot from New Zealand (NZ). Importantly, we provide evidence that moa and prehistoric kakapo consumed ectomycorrhizal fungi, suggesting these birds played a role in dispersing fungi that are key to NZ's natural forest ecosystems. We also provide the first DNA-based evidence that moa frequently supplemented their broad diets with ferns and mosses. Finally, we also find parasite taxa that provide insight into moa behavior, and present data supporting the hypothesis of coextinction between moa and several parasite species. Our study demonstrates that HTS sequencing of coprolites provides a powerful tool for resolving key aspects of ancient ecosystems and may rapidly provide information not obtainable by conventional paleoecological techniques, such as fossil analyses.

Ratite oils for local transdermal therapy of 4-OH tamoxifen: development, characterization, and ex vivo evaluation.

The anti-inflammatory property of ratite oils as well as its ability to act as a penetration enhancer makes it an ideal agent to be used in transdermal formulations. The present study aims to develop an effective transfersomal delivery of 4-hydroxytamoxifen (4-OHT), an anti-cancer drug, using ratite oil as a carrier agent for the treatment of breast cancer (BC). The 4-OHT transfersomes were prepared with and without ratite oils using soy phosphatidylcholine and three different edge activators (EAs) in five different molar ratios using the rotary evaporation-ultrasonication method. Optimal transfersome formulations were selected using physical-chemical characterization and ex vivo studies. Results from physical-chemical characterization of the developed formulations found sodium taurocholate to be the most suitable EA, which recorded highest entrapment efficiency of 95.1 ± 2.70% with 85:15, (w/w) and lowest vesicle size of 82.3 ± 0.02 nm with 75:25, (w/w) molar ratios. TEM and DSC studies showed that the vesicles were readily identified and present in a nearly perfect spherical shape. In addition, formulations with emu oil had better stability than formulations with ostrich oil. Physical stability studies at 4 °C showed that ratite oil transfersomes were stable up to 4 weeks, while transfersomes without ratite oils were stable for 8 weeks. Ex vivo permeability studies using porcine skin concluded that 4-OHT transfersomal formulations with (85:15, w/w) without emu oil have the potential to be used in transdermal delivery approach to enhance permeation of 4-OHT, which may be beneficial in the treatment of BC.

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.

New view on the organization and evolution of Palaeognathae mitogenomes poses the question on the ancestral gene rearrangement in Aves.

BACKGROUND: Bird mitogenomes differ from other vertebrates in gene rearrangement. The most common avian gene order, identified first in Gallus gallus, is considered ancestral for all Aves. However, other rearrangements including a duplicated control region and neighboring genes have been reported in many representatives of avian orders. The repeated regions can be easily overlooked due to inappropriate DNA amplification or genome sequencing. This raises a question about the actual prevalence of mitogenomic duplications and the validity of the current view on the avian mitogenome evolution. In this context, Palaeognathae is especially interesting because is sister to all other living birds, i.e. Neognathae. So far, a unique duplicated region has been found in one palaeognath mitogenome, that of Eudromia elegans. RESULTS: Therefore, we applied an appropriate PCR strategy to look for omitted duplications in other palaeognaths. The analyses revealed the duplicated control regions with adjacent genes in Crypturellus, Rhea and Struthio as well as ND6 pseudogene in three moas. The copies are very similar and were subjected to concerted evolution. Mapping the presence and absence of duplication onto the Palaeognathae phylogeny indicates that the duplication was an ancestral state for this avian group. This feature was inherited by early diverged lineages and lost two times in others. Comparison of incongruent phylogenetic trees based on mitochondrial and nuclear sequences showed that two variants of mitogenomes could exist in the evolution of palaeognaths. Data collected for other avian mitogenomes revealed that the last common ancestor of all birds and early diverging lineages of Neoaves could also possess the mitogenomic duplication. CONCLUSIONS: The duplicated control regions with adjacent genes are more common in avian mitochondrial genomes than it was previously thought. These two regions could increase effectiveness of replication and transcription as well as the number of replicating mitogenomes per organelle. In consequence, energy production by mitochondria may be also more efficient. However, further physiological and molecular analyses are necessary to assess the potential selective advantages of the mitogenome duplications.

Extensive in situ radiation of feather lice on tinamous.

Tinamous host the highest generic diversity of lice of any group of birds, as well as hosting representatives of all four avian feather louse ecomorphs. Although the generic diversity of tinamou feather lice is well documented, few attempts have been made to reconstruct the phylogenetic relationships among these lice. To test whether tinamou feather lice form a monophyletic group as a whole, we used whole-genome sequencing to estimate a higher-level phylogeny of tinamou feather lice, together with a broad diversity of other avian feather louse groups. In total, we analysed sequences from over 1000 genes for 48 genera of avian lice using both concatenated and coalescent approaches to estimate the phylogeny of this diverse group of avian feather lice. Although the body louse ecomorph of tinamou feather lice formed a monophyletic group, they did not strictly form a monophyletic group together with the other three ecomorphs of tinamou feather lice. In particular, a clade comprised of several feather louse genera, mainly from South America, is nested phylogenetically within tinamou lice, which also have their main centre of diversity in South America. These results suggest in situ radiation of these parasites in South America.

Sequence comparisons of cytochrome P450 aromatases from Australian animals predict differences in enzymatic activity and/or efficiency†.

Aromatase (P450arom, CYP19A1) is the terminal enzyme in the synthesis of the steroid hormone family of estrogens. Not surprisingly, this enzyme has structural similarities between the limited number of species studied thus far. This study examined the structure of aromatases from four diverse Australian species including a marsupial (tammar wallaby; Macropus eugenii), monotreme (platypus; Ornithorhynchus anatinus), ratite (emu; Dromaius novaehollandiae) and lizard (bearded dragon; Pogona vitticeps). We successfully built homology models for each species, using the only crystallographically determined structure available, human aromatase. The amino acid sequences showed high amino acid sequence identity to the human aromatase: wallaby 81%, platypus 73%, emu 75% and bearded dragon at 74%. The overall structure was highly conserved among the five species, although there were non-secondary structures (loops and bends) that were variable and flexible that may result in some differences in catalytic activity. At the N-terminal regions, there were deletions and variations that suggest that functional distinctions may be found. We found that the active sites of all these proteins were identical, except for a slight variation in the emu. The electrostatic potential across the surfaces of these aromatases highlighted likely variations to the protein-protein interactions of these enzymes with both redox partner cytochrome P450 reductase and possibly homodimerization in the case of the platypus, which has been postulated for the human aromatase enzyme. Given the high natural selection pressures on reproductive strategies, the relatively high degree of conservation of aromatase sequence and structure across species suggests that there is biochemically very little scope for changes to have evolved without the loss of enzyme activity.

Whole-Genome Analyses Resolve the Phylogeny of Flightless Birds (Palaeognathae) in the Presence of an Empirical Anomaly Zone.

Palaeognathae represent one of the two basal lineages in modern birds, and comprise the volant (flighted) tinamous and the flightless ratites. Resolving palaeognath phylogenetic relationships has historically proved difficult, and short internal branches separating major palaeognath lineages in previous molecular phylogenies suggest that extensive incomplete lineage sorting (ILS) might have accompanied a rapid ancient divergence. Here, we investigate palaeognath relationships using genome-wide data sets of three types of noncoding nuclear markers, together totaling 20,850 loci and over 41 million base pairs of aligned sequence data. We recover a fully resolved topology placing rheas as the sister to kiwi and emu + cassowary that is congruent across marker types for two species tree methods (MP-EST and ASTRAL-II). This topology is corroborated by patterns of insertions for 4274 CR1 retroelements identified from multispecies whole-genome screening, and is robustly supported by phylogenomic subsampling analyses, with MP-EST demonstrating particularly consistent performance across subsampling replicates as compared to ASTRAL. In contrast, analyses of concatenated data supermatrices recover rheas as the sister to all other nonostrich palaeognaths, an alternative that lacks retroelement support and shows inconsistent behavior under subsampling approaches. While statistically supporting the species tree topology, conflicting patterns of retroelement insertions also occur and imply high amounts of ILS across short successive internal branches, consistent with observed patterns of gene tree heterogeneity. Coalescent simulations and topology tests indicate that the majority of observed topological incongruence among gene trees is consistent with coalescent variation rather than arising from gene tree estimation error alone, and estimated branch lengths for short successive internodes in the inferred species tree fall within the theoretical range encompassing the anomaly zone. Distributions of empirical gene trees confirm that the most common gene tree topology for each marker type differs from the species tree, signifying the existence of an empirical anomaly zone in palaeognaths.