Effects of aerial hypoxia and temperature on pulmonary breathing pattern and gas exchange in the South American lungfish, Lepidosiren paradoxa.

The South American lungfish Lepidosiren paradoxa is an obligatory air-breathing fish possessing well-developed bilateral lungs, and undergoing seasonal changes in its habitat, including temperature changes. In the present study we aimed to evaluate gas exchange and pulmonary breathing pattern in L. paradoxa at different temperatures (25 and 30°C) and different inspired O2 levels (21, 12, 10, and 7%). Normoxic breathing pattern consisted of isolated ventilatory cycles composed of an expiration followed by 2.4±0.2 buccal inspirations. Both expiratory and inspiratory tidal volumes reached a maximum of about 35mlkg-1, indicating that L. paradoxa is able to exchange nearly all of its lung air in a single ventilatory cycle. At both temperatures, hypoxia caused a significant increase in pulmonary ventilation (V̇E), mainly due to an increase in respiratory frequency. Durations of the ventilatory cycle and expiratory and inspiratory tidal volumes were not significantly affected by hypoxia. Expiratory time (but not inspiratory) was significantly shorter at 30°C and at all O2 levels. While a small change in oxygen consumption (V̇O2) could be noticed, the carbon dioxide release (V̇CO2, P=0.0003) and air convection requirement (V̇E/V̇O2, P=0.0001) were significantly affected by hypoxia (7% O2) at both temperatures, when compared to normoxia, and pulmonary diffusion capacity increased about four-fold due to hypoxic exposure. These data highlight important features of the respiratory system of L. paradoxa, capable of matching O2 demand and supply under different environmental change, as well as help to understand the evolution of air breathing in lungfish.

Acute effects of temperature and hypercarbia on cutaneous and branchial gas exchange in the South American lungfish, Lepidosiren paradoxa.

The South American lungfish, Lepidosiren paradoxa inhabits seasonal environments in the Central Amazon and Paraná-Paraguay basins that undergo significant oscillations in temperature throughout the year. They rely on different gas exchange organs, such as gills and skin for aquatic gas exchange while their truly bilateral lungs are responsible for aerial gas exchange; however, there are no data available on the individual contributions of the skin and the gills to total aquatic gas exchange in L. paradoxa. Thus, in the present study we quantify the relative contributions of skin and gills on total aquatic gas exchange during warm (35°C) and cold exposure (20°C) in addition to the effects of aerial and aquatic hypercarbia on aquatic gas exchange and gill ventilation rate (fG; 25°C), respectively. Elevated temperature (35°C) caused a significant increase in the contribution of cutaneous (from 0.61±0.13 to 1.34±0.26ml. STPD.h-1kg-1) and branchial (from 0.54±0.17 to 1.73±0.53ml. STPD.h-1kg-1) gas exchange for V̇CO2 relative to the lower temperature (20°C), while V̇O2 remained relatively unchanged. L. paradoxa exhibited a greater branchial contribution in relation to total aquatic gas exchange at lower temperatures (20 and 25°C) for oxygen uptake. Aerial hypercarbia decreased branchial V̇O2 whereas branchial V̇CO2 was significantly increased. Progressive increases in aquatic hypercarbia did not affect fG. This response is in contrast to increases in pulmonary ventilation that may offset any increase in arterial partial pressure of CO2 owing to CO2 loading through the animals' branchial surface. Thus, despite their reduced contribution to total gas exchange, cutaneous and branchial gas exchange in L. paradoxa can be significantly affected by temperature and aerial hypercarbia.

Cloning and expression of the epithelial sodium channel and its role in osmoregulation of aquatic and estivating African lungfish Protopterus annectens.

The epithelial sodium channel (ENaC) is a sodium (Na(+))-selective aldosterone-stimulated ion channel involved in Na(+) transport homeostasis of tetrapods. We examined full-length cDNA sequences and tissue distributions of ENaCα, ENaCß, and ENaCγ subunits in the African lungfish Protopterus annectens. Protopterus ENaC (pENaC) comprises 3 subunits: pENaCα, pENaCß, and pENaCγ. pENaCα, pENaCß, and pENaCγ subunits are closely related to α, ß, and γ subunits of the Australian lungfish Neoceratodus forsteri ENaC (nENaC), respectively. Three ENaC subunit mRNAs were highly expressed in the gills and moderately expressed in the kidney and rectum of P. annectens. During estivation for 2-4weeks and 2-3months, plasma Na(+) concentration was relatively stable, but plasma urea concentration significantly increased in comparison with the control fish kept in a freshwater environment. Plasma aldosterone concentration and mRNA expression of the ENaCα subunit gradually and significantly decreased in the gills and kidney after 2months of estivation. Thus, aldosterone-dependent Na(+) absorption via ENaC probably exists in the epithelial cells of osmoregulatory organs of lungfish kept in fresh water, whereas plasma Na(+) concentration may be maintained by a mechanism independent of aldosterone-ENaC axis during estivation in lungfish.

The West African lungfish secretes a living cocoon during aestivation with uncertain antimicrobial function.

One of the most exceptional adaptations to extreme drought is found in the sister group to tetrapods, the lungfishes (Dipnoi), which can aestivate inside a mucus cocoon for multiple years at reduced metabolic rates with complete cessation of ingestion and excretion. However, the function of the cocoon tissue is not fully understood. Here we developed a new more natural laboratory protocol for inducing aestivation in the West African lungfish, Protopterus annectens, and investigated the structure and function of the cocoon. We used electron microscopy and imaging of live tissue-stains to confirm that the inner and outer layers of the paper-thin cocoon are composed primarily of living cells. However, we also repeatedly observed extensive bacterial and fungal growth covering the cocoon and found no evidence of anti-microbial activity in vitro against E. coli for the cocoon tissue in this species. This classroom discovery-based research, performed during a course-based undergraduate research experience course (CURE), provides a robust laboratory protocol for investigating aestivation and calls into the question the function of this bizarre vertebrate adaptation.

Distribution of recesses in the olfactory organ of African lungfish Protopterus aethiopicus.

In the olfactory organ of lungfish, recesses at the bases of lamellae comprise sensory and nonsensory epithelia. The sensory epithelium of the recesses, the recess epithelium, is distinguished from the olfactory epithelium covering the lamella by the absence of ciliated olfactory receptor cells. Therefore, it has been suggested that the recess epithelium is a primordium of the vomeronasal organ of tetrapods. However, developmental changes in the number and distribution of recesses in the olfactory organ of lungfish were unknown. We examined four Protopterus aethiopicus specimens of body lengths 215-800 mm to determine the localization of recesses in their olfactory organs. Histological examination showed recesses at the bases of lamellae in all individuals examined. The recesses were localized mainly in the medial and caudal parts of the olfactory organs, especially in juveniles. Compared to smaller fish, larger fish had a larger number of recesses, distributed more broadly in their olfactory organs. Significance of the recess localization and its relationship to the function of lungfish olfactory organ warrants further investigation.

Morphometric analysis of lungfish endocasts elucidates early dipnoan palaeoneurological evolution.

The lobe-finned fish, lungfish (Dipnoi, Sarcoptergii), have persisted for ~400 million years from the Devonian Period to present day. The evolution of their dermal skull and dentition is relatively well understood, but this is not the case for the central nervous system. While the brain has poor preservation potential and is not currently known in any fossil lungfish, substantial indirect information about it and associated structures (e.g. labyrinths) can be obtained from the cranial endocast. However, before the recent development of X-ray tomography as a palaeontological tool, these endocasts could not be studied non-destructively, and few detailed studies were undertaken. Here, we describe and illustrate the endocasts of six Palaeozoic lungfish from tomographic scans. We combine these with six previously described digital lungfish endocasts (4 fossil and 2 recent taxa) into a 12-taxon dataset for multivariate morphometric analysis using 17 variables. We find that the olfactory region is more highly plastic than the hindbrain, and undergoes significant elongation in several taxa. Further, while the semicircular canals covary as an integrated module, the utriculus and sacculus vary independently of each other. Functional interpretation suggests that olfaction has remained a dominant sense throughout lungfish evolution, and changes in the labyrinth may potentially reflect a change from nektonic to near-shore environmental niches. Phylogenetic implications show that endocranial form fails to support monophyly of the 'chirodipterids'. Those with elongated crania similarly fail to form a distinct clade, suggesting these two paraphyletic groups have converged towards either head elongation or truncation driven by non-phylogenetic constraints.

A diverse Late Cretaceous vertebrate tracksite from the Winton Formation of Queensland, Australia.

The Upper Cretaceous 'upper' Winton Formation of Queensland, Australia is world famous for hosting Dinosaur Stampede National Monument at Lark Quarry Conservation Park, a somewhat controversial tracksite that preserves thousands of tridactyl dinosaur tracks attributed to ornithopods and theropods. Herein, we describe the Snake Creek Tracksite, a new vertebrate ichnoassemblage from the 'upper' Winton Formation, originally situated on Karoola Station but now relocated to the Australian Age of Dinosaurs Museum of Natural History. This site preserves the first sauropod tracks reported from eastern Australia, a small number of theropod and ornithopod tracks, the first fossilised crocodyliform and ?turtle tracks reported from Australia, and possible lungfish and actinopterygian feeding traces. The sauropod trackways are wide-gauge, with manus tracks bearing an ungual impression on digit I, and anteriorly tapered pes tracks with straight or concave forward posterior margins. These tracks support the hypothesis that at least one sauropod taxon from the 'upper' Winton Formation retained a pollex claw (previously hypothesised for Diamantinasaurus matildae based on body fossils). Many of the crocodyliform trackways indicate underwater walking. The Snake Creek Tracksite reconciles the sauropod-, crocodyliform-, turtle-, and lungfish-dominated body fossil record of the 'upper' Winton Formation with its heretofore ornithopod- and theropod-dominated ichnofossil record.

A high latitude Devonian lungfish, from the Famennian of South Africa.

New fossil lungfish remains comprising two parasphenoids, tooth plates and scales from the Famennian Witpoort Formation of South Africa are described. From the parasphenoid material, which bears similarity to Oervigia and Sagenodus but is nevertheless unique, a new genus, Isityumzi mlomomde gen. et sp. nov. is erected. Tooth plates and scales from the same locality may be conspecific but are not yet assigned until further material becomes available. The tooth plates closely resemble those of some taxa in the Carboniferous genus Ctenodus. The new taxon is significant as only the second Devonian lungfish described from the African continent, and for hailing from the high-latitude (polar) Waterloo Farm environment situated close to 70° south during the Famennian.

The cranial endocast of the Upper Devonian dipnoan 'Chirodipterus' australis.

One of the first endocasts of a dipnoan (lungfish) to be realised was that of the Upper Devonian taxon Chirodipterus australis. This early interpretation was based on observations of the shape of the cranial cavity alone and was not based on a natural cast or 'steinkern' nor from serial sectioning. The validity of this reconstruction is therefore questionable and continued reference to and use of this interpretation in analyses of sarcopterygian cranial evolution runs the risk of propagation of error. Here we present a new detailed anatomical description of the endocast of 'Chirodipterus' australis from the Upper Devonian Gogo Formation of Western Australia, known for exceptional 3D preservation which enables fine-scale scrutiny of endocranial anatomy. We show that it exhibits a suite of characters more typical of Lower and Middle Devonian dipnoan taxa. Notably, the small utricular recess is unexpected for a taxon of this age, whereas the ventral expansion of the telencephalon is more typical of more derived taxa. The presence of such 'primitive' characters in 'C.' australis supports its relatively basal position as demonstrated in the most recent phylogenies of Devonian Dipnoi.

Cephalic muscle development in the Australian lungfish, Neoceratodus forsteri.

Lungfishes are the extant sister group of tetrapods. As such, they are important for the study of evolutionary processes involved in the water to land transition of vertebrates. The evolution of a true neck, that is, the complete separation of the pectoral girdle from the cranium, is one of the most intriguing morphological transitions known among vertebrates. Other salient changes involve new adaptations for terrestrial feeding, which involves both the cranium and its associated musculature. Historically, the cranium has been extensively investigated, but the development of the cranial muscles much less so. Here, we present a detailed study of cephalic muscle development in the Australian lungfish, Neoceratodus forsteri, which is considered to be the sister taxon to all other extant lungfishes. Neoceratodus shows several developmental patterns previously described in other taxa; the tendency of muscles to develop from anterior to posterior, from their region of origin toward insertion, and from lateral to ventral/medial (outside-in), at least in the branchial arches. The m.protractor pectoralis appears to develop as an extension of the most posterior m.levatores arcuum branchialium, supporting the hypothesis that the m.cucullaris and its derivatives (protractor pectoralis, levatores arcuum branchialium) are branchial muscles. We present a new hypothesis regarding the homology of the ventral branchial arch muscles (subarcualis recti and obliqui, transversi ventrales) in lungfishes and amphibians. Moreover, the morphology and development of the cephalic muscles confirms that extant lungfishes are neotenic and have been strongly influenced via paedomorphosis during their evolutionary history.

Specimen-level phylogenetics in paleontology using the Fossilized Birth-Death model with sampled ancestors.

Bayesian phylogenetic methods integrating simultaneously morphological and stratigraphic information have been applied increasingly among paleontologists. Most of these studies have used Bayesian methods as an alternative to the widely-used parsimony analysis, to infer macroevolutionary patterns and relationships among species-level or higher taxa. Among recently introduced Bayesian methodologies, the Fossilized Birth-Death (FBD) model allows incorporation of hypotheses on ancestor-descendant relationships in phylogenetic analyses including fossil taxa. Here, the FBD model is used to infer the relationships among an ingroup formed exclusively by fossil individuals, i.e., dipnoan tooth plates from four localities in the Ain el Guettar Formation of Tunisia. Previous analyses of this sample compared the results of phylogenetic analysis using parsimony with stratigraphic methods, inferred a high diversity (five or more genera) in the Ain el Guettar Formation, and interpreted it as an artifact inflated by depositional factors. In the analysis performed here, the uncertainty on the chronostratigraphic relationships among the specimens was included among the prior settings. The results of the analysis confirm the referral of most of the specimens to the taxa Asiatoceratodus, Equinoxiodus, Lavocatodus and Neoceratodus, but reject those to Ceratodus and Ferganoceratodus. The resulting phylogeny constrained the evolution of the Tunisian sample exclusively in the Early Cretaceous, contrasting with the previous scenario inferred by the stratigraphically-calibrated topology resulting from parsimony analysis. The phylogenetic framework also suggests that (1) the sampled localities are laterally equivalent, (2) but three localities are restricted to the youngest part of the section; both results are in agreement with previous stratigraphic analyses of these localities. The FBD model of specimen-level units provides a novel tool for phylogenetic inference among fossils but also for independent tests of stratigraphic scenarios.

The cranial endocast of Dipnorhynchus sussmilchi (Sarcopterygii: Dipnoi) and the interrelationships of stem-group lungfishes.

The first virtual cranial endocast of a lungfish from the Early Devonian, Dipnorhynchus sussmilchi, is described. Dipnorhynchus, only the fourth Devonian lungfish for which a near complete cranial endocast is known, is a key taxon for clarifying primitive character states within the group. A ventrally-expanded telencephalic cavity is present in the endocast of Dipnorhynchus demonstrating that this is the primitive state for "true" Dipnoi. Dipnorhynchus also possesses a utricular recess differentiated from the sacculolagenar pouch like that seen in stratigraphically younger lungfish (Dipterus, Chirodipterus, Rhinodipterus), but absent from the dipnomorph Youngolepis. We do not find separate pineal and para-pineal canals in contrast to a reconstruction from previous authors. We conduct the first phylogenetic analysis of Dipnoi based purely on endocast characters, which supports a basal placement of Dipnorhynchus within the dipnoan stem group, in agreement with recent analyses. Our analysis demonstrates the value of endocast characters for inferring phylogenetic relationships.

Permian-Triassic Osteichthyes (bony fishes): diversity dynamics and body size evolution.

The Permian and Triassic were key time intervals in the history of life on Earth. Both periods are marked by a series of biotic crises including the most catastrophic of such events, the end-Permian mass extinction, which eventually led to a major turnover from typical Palaeozoic faunas and floras to those that are emblematic for the Mesozoic and Cenozoic. Here we review patterns in Permian-Triassic bony fishes, a group whose evolutionary dynamics are understudied. Based on data from primary literature, we analyse changes in their taxonomic diversity and body size (as a proxy for trophic position) and explore their response to Permian-Triassic events. Diversity and body size are investigated separately for different groups of Osteichthyes (Dipnoi, Actinistia, 'Palaeopterygii', 'Subholostei', Holostei, Teleosteomorpha), within the marine and freshwater realms and on a global scale (total diversity) as well as across palaeolatitudinal belts. Diversity is also measured for different palaeogeographical provinces. Our results suggest a general trend from low osteichthyan diversity in the Permian to higher levels in the Triassic. Diversity dynamics in the Permian are marked by a decline in freshwater taxa during the Cisuralian. An extinction event during the end-Guadalupian crisis is not evident from our data, but 'palaeopterygians' experienced a significant body size increase across the Guadalupian-Lopingian boundary and these fishes upheld their position as large, top predators from the Late Permian to the Late Triassic. Elevated turnover rates are documented at the Permian-Triassic boundary, and two distinct diversification events are noted in the wake of this biotic crisis, a first one during the Early Triassic (dipnoans, actinistians, 'palaeopterygians', 'subholosteans') and a second one during the Middle Triassic ('subholosteans', neopterygians). The origination of new, small taxa predominantly among these groups during the Middle Triassic event caused a significant reduction in osteichthyan body size. Neopterygii, the clade that encompasses the vast majority of extant fishes, underwent another diversification phase in the Late Triassic. The Triassic radiation of Osteichthyes, predominantly of Actinopterygii, which only occurred after severe extinctions among Chondrichthyes during the Middle-Late Permian, resulted in a profound change within global fish communities, from chondrichthyan-rich faunas of the Permo-Carboniferous to typical Mesozoic and Cenozoic associations dominated by actinopterygians. This turnover was not sudden but followed a stepwise pattern, with leaps during extinction events.