Bioprospecting the American Alligator Peptidome for antiviral peptides against Venezuelan equine encephalitis virus.

The innate immune protection provided by cationic antimicrobial peptides (CAMPs) has been shown to extend to antiviral activity, with putative mechanisms of action including direct interaction with host cells or pathogen membranes. The lack of therapeutics available for the treatment of viruses such as Venezuelan equine encephalitis virus (VEEV) underscores the urgency of novel strategies for antiviral discovery. American alligator plasma has been shown to exhibit strong in vitro antibacterial activity, and functionalized hydrogel particles have been successfully employed for the identification of specific CAMPs from alligator plasma. Here, a novel bait strategy in which particles were encapsulated in membranes from either healthy or VEEV-infected cells was implemented to identify peptides preferentially targeting infected cells for subsequent evaluation of antiviral activity. Statistical analysis of peptide identification results was used to select five candidate peptides for testing, of which one exhibited a dose-dependent inhibition of VEEV and also significantly inhibited infectious titers. Results suggest our bioprospecting strategy provides a versatile platform that may be adapted for antiviral peptide identification from complex biological samples.

Developmental origins of the crocodylian skull table and platyrostral face.

The dorsoventrally flattened skull typifies extant Crocodylia perhaps more than any other anatomical feature and is generally considered an adaptation for semi-aquatic feeding. Although the evolutionary origins of caniofacial flattening have been extensively studied, the developmental origins have yet to be explored. To understand how the skull table and platyrostral snout develop, we quantified embryonic development and post-hatching growth (ontogeny) of the crocodylian skull in lateral view using geometric morphometrics. Our dataset (n = 103) includes all but one extant genus and all of the major ecomorphs, including the extremely slender-snouted Gavialis and Tomistoma. Our analysis reveals that the embryonic development of the flattened skull is remarkably similar across ecomorphs, including the presence of a conserved initial embryonic skull shape, similar to prior analysis of dorsal snout shape. Although differences during posthatching ontogeny are recovered among ecomorphs, embryonic patterns are not distinct, revealing an important shift in developmental rate near hatching. In particular, the flattened skull table is achieved by the end of embryonic development with no changes after hatching. Further, the rotation of skull roof and facial bones during development is critical for the stereotypical flatness of the crocodylian skull. Our results suggest selection on hatchling performance and constraints on embryonic skull shape may have been important in this pattern of developmental conservation. The appearance of aspects of cranial flatness among Jurassic stem crocodylians suggests key aspects of these cranial developmental patterns may have been conserved for over 200 million years.

Use of continuous cranial shape variation in the identification of divergent crocodile species of the genus Mecistops.

The discovery of cryptic biodiversity has blossomed under the advancements of genetic techniques, but species identification via morphology remains crucial to effective conservation efforts. In this study, we tested the use of continuous cranial shape variation in distinguishing the two living species of Mecistops: the West African slender-snouted crocodile (M. cataphractus) and the Central African slender-snouted crocodile (M. leptorhynchus). Using a combination of geometric morphometric characters and ratios of linear measurements, we identified statistically significant variation in cranial bone and overall skull shape of mature individuals that corroborates existing molecular and discrete morphological evidence for two distinct, extant species within Mecistops. Specifically, variation in the shape of the nasal appears particularly diagnostic, while ratios involving metrics of snout length to snout width at the premaxillary notch offer distinguishing features easily measured in the field. Because of the complementary results and applications of the morphometric and cranial ratio analyses, we argue that both methodologies remain relevant to species identification. Moreover, we recommend continued cooperation between geneticists and morphologists in diagnosing species of conservation concern.

High levels of population genetic differentiation in the American crocodile (Crocodylus acutus).

The American crocodile (Crocodylus acutus) is a widely distributed species across coastal and brackish areas of the Neotropical region of the Americas and the Greater Antilles. Available information on patterns of genetic differentiation in C. acutus shows a complex structuring influenced by interspecific interactions (mainly hybridization) and anthropogenic actions (mostly historical hunting, recent poaching, habitat loss and fragmentation, and unintentional translocation of individuals). In this study, we used data on mitochondrial DNA control region and 11 nuclear polymorphic microsatellite loci to assess the degree of population structure of C. acutus in South America, North America, Central America and the Greater Antilles. We used traditional genetic differentiation indices, Bayesian clustering and multivariate methods to create a more comprehensive picture of the genetic relationships within the species across its range. Analyses of mtDNA and microsatellite loci show evidence of a strong population genetic structure in the American crocodile, with unique populations in each sampling locality. Our results support previous findings showing large degrees of genetic differentiation between the continental and the Greater Antillean C. acutus. We report three new haplotypes unique to Venezuela, which are considerably less distant from the Central and North American haplotypes than to the Greater Antillean ones. Our findings reveal genetic population differentiation between Cuban and Jamaican C. acutus and offer the first evidence of strong genetic differentiation among the populations of Greater Antillean C. acutus.

The Frontoparietal Fossa and Dorsotemporal Fenestra of Archosaurs and Their Significance for Interpretations of Vascular and Muscular Anatomy in Dinosaurs.

The attachments of jaw muscles are typically implicated in the evolution and shape of the dorsotemporal fenestra on the skull roof of amniotes. However, the dorsotemporal fenestrae of many archosaurian reptiles possess smooth excavations rostral and dorsal to the dorsotemporal fossa which closely neighbors the dorsotemporal fenestra and jaw muscle attachments. Previous research has typically identified this region, here termed the frontoparietal fossa, to also have attachment surfaces for jaw-closing muscles. However, numerous observations of extant and extinct archosaurs described here suggest that other tissues are instead responsible for the size and shape of the frontoparietal fossa. This study reviewed the anatomical evidence that support soft-tissue hypotheses of the frontoparietal fossa and its phylogenetic distribution among sauropsids. Soft-tissue hypotheses (i.e., muscle, pneumatic sinus, vascular tissues) were analyzed using anatomical, imaging and in vivo thermography techniques within a phylogenetic framework using extant and extinct taxa to determine the inferential power underlying the reconstruction of the soft tissues in the skull roofs of dinosaurs, pseudosuchians, and other reptiles. Relevant anatomical features argue for rejection of the default hypothesis-that the fossa was muscular-due to a complete lack of osteological correlates reflective of muscle attachment. The most-supported inference of soft tissues is that the frontoparietal fossa contained a large vascular structure and adipose tissue. Despite the large sizes and diverse morphologies of these fossae found among dinosaur taxa, these data suggest that non-avian dinosaurs had the anatomical foundation to support physiologically significant vascular devices and/or vascular integumentary structures on their skull roofs. Anat Rec, 303:1060-1074, 2020. © 2019 Wiley Periodicals, Inc.

Divergent evolution of terrestrial locomotor abilities in extant Crocodylia.

Extant Crocodylia are exceptional because they employ almost the full range of quadrupedal footfall patterns ("gaits") used by mammals; including asymmetrical gaits such as galloping and bounding. Perhaps this capacity evolved in stem Crocodylomorpha, during the Triassic when taxa were smaller, terrestrial, and long-legged. However, confusion about which Crocodylia use asymmetrical gaits and why persists, impeding reconstructions of locomotor evolution. Our experimental gait analysis of locomotor kinematics across 42 individuals from 15 species of Crocodylia obtained 184 data points for a wide velocity range (0.15-4.35 ms-1). Our results suggest either that asymmetrical gaits are ancestral for Crocodylia and lost in the alligator lineage, or that asymmetrical gaits evolved within Crocodylia at the base of the crocodile line. Regardless, we recorded usage of asymmetrical gaits in 7 species of Crocodyloidea (crocodiles); including novel documentation of these behaviours in 5 species (3 critically endangered). Larger Crocodylia use relatively less extreme gait kinematics consistent with steeply decreasing athletic ability with size. We found differences between asymmetrical and symmetrical gaits in Crocodylia: asymmetrical gaits involved greater size-normalized stride frequencies and smaller duty factors (relative ground contact times), consistent with increased mechanical demands. Remarkably, these gaits did not differ in maximal velocities obtained: whether in Alligatoroidea or Crocodyloidea, trotting or bounding achieved similar velocities, revealing that the alligator lineage is capable of hitherto unappreciated extreme locomotor performance despite a lack of asymmetrical gait usage. Hence asymmetrical gaits have benefits other than velocity capacity that explain their prevalence in Crocodyloidea and absence in Alligatoroidea-and their broader evolution.

The Komodo dragon (Varanus komodoensis) genome and identification of innate immunity genes and clusters.

BACKGROUND: We report the sequencing, assembly and analysis of the genome of the Komodo dragon (Varanus komodoensis), the largest extant lizard, with a focus on antimicrobial host-defense peptides. The Komodo dragon diet includes carrion, and a complex milieu of bacteria, including potentially pathogenic strains, has been detected in the saliva of wild dragons. They appear to be unaffected, suggesting that dragons have robust defenses against infection. While little information is available regarding the molecular biology of reptile immunity, it is believed that innate immunity, which employs antimicrobial host-defense peptides including defensins and cathelicidins, plays a more prominent role in reptile immunity than it does in mammals. . RESULTS: High molecular weight genomic DNA was extracted from Komodo dragon blood cells. Subsequent sequencing and assembly of the genome from the collected DNA yielded a genome size of 1.6 Gb with 45x coverage, and the identification of 17,213 predicted genes. Through further analyses of the genome, we identified genes and gene-clusters corresponding to antimicrobial host-defense peptide genes. Multiple ß-defensin-related gene clusters were identified, as well as a cluster of potential Komodo dragon ovodefensin genes located in close proximity to a cluster of Komodo dragon ß-defensin genes. In addition to these defensins, multiple cathelicidin-like genes were also identified in the genome. Overall, 66 ß-defensin genes, six ovodefensin genes and three cathelicidin genes were identified in the Komodo dragon genome. CONCLUSIONS: Genes with important roles in host-defense and innate immunity were identified in this newly sequenced Komodo dragon genome, suggesting that these organisms have a robust innate immune system. Specifically, multiple Komodo antimicrobial peptide genes were identified. Importantly, many of the antimicrobial peptide genes were found in gene clusters. We found that these innate immunity genes are conserved among reptiles, and the organization is similar to that seen in other avian and reptilian species. Having the genome of this important squamate will allow researchers to learn more about reptilian gene families and will be a valuable resource for researchers studying the evolution and biology of the endangered Komodo dragon.

Heterochronic shifts and conserved embryonic shape underlie crocodylian craniofacial disparity and convergence.

The distinctive anatomy of the crocodylian skull is intimately linked with dietary ecology, resulting in repeated convergence on blunt- and slender-snouted ecomorphs. These evolutionary shifts depend upon modifications of the developmental processes which direct growth and morphogenesis. Here we examine the evolution of cranial ontogenetic trajectories to shed light on the mechanisms underlying convergent snout evolution. We use geometric morphometrics to quantify skeletogenesis in an evolutionary context and reconstruct ancestral patterns of ontogenetic allometry to understand the developmental drivers of craniofacial diversity within Crocodylia. Our analyses uncovered a conserved embryonic region of morphospace (CER) shared by all non-gavialid crocodylians regardless of their eventual adult ecomorph. This observation suggests the presence of conserved developmental processes during early development (before Ferguson stage 20) across most of Crocodylia. Ancestral state reconstruction of ontogenetic trajectories revealed heterochrony, developmental constraint, and developmental systems drift have all played essential roles in the evolution of ecomorphs. Based on these observations, we conclude that two separate, but interconnected, developmental programmes controlling craniofacial morphogenesis and growth enabled the evolutionary plasticity of skull shape in crocodylians.

Crocodylian Head Width Allometry and Phylogenetic Prediction of Body Size in Extinct Crocodyliforms.

Body size and body-size shifts broadly impact life-history parameters of all animals, which has made accurate body-size estimates for extinct taxa an important component of understanding their paleobiology. Among extinct crocodylians and their precursors (e.g., suchians), several methods have been developed to predict body size from suites of hard-tissue proxies. Nevertheless, many have limited applications due to the disparity of some major suchian groups and biases in the fossil record. Here, we test the utility of head width (HW) as a broadly applicable body-size estimator in living and fossil suchians. We use a dataset of sexually mature male and female individuals (n = 76) from a comprehensive sample of extant suchian species encompassing nearly all known taxa (n = 22) to develop a Bayesian phylogenetic model for predicting three conventional metrics for size: body mass, snout-vent length, and total length. We then use the model to estimate size parameters for a select series of extinct suchians with known phylogenetic affinity (Montsechosuchus, Diplocynodon, and Sarcosuchus). We then compare our results to sizes reported in the literature to exemplify the utility of our approach for a broad array of fossil suchians. Our results show that HW is highly correlated with all other metrics (all R 2≥0.85) and is commensurate with femoral dimensions for its reliably as a body-size predictor. We provide the R code in order to enable other researchers to employ the model in their own research.

Alometría del Ancho de la Cabeza de Cocodrilo y Predicción Filogenética del Tamaño Corporal en Cocodrilos Extintos (Crocodylian Head Width Allometry and Phylogenetic Prediction of Body Size in Extinct Crocodyliforms)El tamaño corporal y los cambios de tamaño corporal afectan ampliamente los parámetros de la historia de vida de todos los animales, lo que ha hecho que las estimaciones precisas del tamaño corporal de los taxones extintos sean un componente importante para comprender su paleobiología. Entre los crocodilianos extintos y sus precursores (por ejemplo, los suquios), se han desarrollado varios métodos para predecir el tamaño corporal a partir de conjuntos de indicadores de tejido duro. Sin embargo, muchos tienen aplicaciones limitadas debido a la disparidad de algunos grupos importantes de crocodiliformes y sesgos en el registro fósil. Aquí, probamos la utilidad del ancho de la cabeza como un estimador de tamaño corporal ampliamente aplicable en crocodiliformes vivos y fósiles. Utilizamos un conjunto de datos de individuos machos y hembras sexualmente maduros (n=76) de una muestra exhaustiva de especies existentes de cocodrilos que abarcan casi todos los taxones conocidos (n=22) para desarrollar un modelo filogenético bayesiano y predecir tres métricas convencionales para el tamaño: masa corporal, longitud del orificio de ventilación y longitud total. Luego usamos el modelo para estimar los parámetros de tamaño para una serie selecta de crocodiliformes extintos con afinidad filogenética conocida (Montsechosuchus, Diplocynodon, y Sarcosuchus). Luego comparamos nuestros resultados con los tamaños reportados en la literatura para demostrar la utilidad de nuestro enfoque en una gama amplia de tales fósiles. Nuestros resultados muestran que el ancho de la cabeza está altamente correlacionado con todas las otras métricas (todo R 2≥0.85) y es conmensurable a las dimensiones femorales debido a su confiabilidad como predictor del tamaño corporal. Proporcionamos el código R para permitir que otros investigadores empleen el modelo en su propia investigación.Translated to Spanish by C.A. Alfonso (calfonsoc@vt.edu).

Systematic revision of the living African Slender-snouted Crocodiles (Mecistops Gray, 1844).

Molecular and morphological evidence has shown that the African slender-snouted, or sharp-nosed, crocodile Mecistops cataphractus (Cuvier, 1824) is comprised of two superficially cryptic species: one endemic to West Africa and the other endemic to Central Africa. Our ability to characterize the two species is compromised by the complicated taxonomic history of the lineage and overlapping ranges of variation in distinguishing morphological features. The name M. cataphractus was evidently originally based on West African material, but the holotype is now lost. Although types exist for other names based on the West African form, the name M. cataphractus is sufficiently entrenched in the literature, and other names sufficiently obscure, to justify retypification. Here, we designate a neotype for M. cataphractus and restrict it to West Africa. We resurrect M. leptorhynchus as a valid species from Central Africa and identify exemplary referred specimens that, collectively, overcome the obscurity and diagnostic limits of the extant holotype. We additionally indicate suitable neotype material in the event the holotype is lost, destroyed, or otherwise needing replacement, and we rectify the previously erroneous type locality designation. We provide a revised diagnosis for crown Mecistops, and revise and update previous descriptions of the two living species, including providing both more complete descriptions and discussion of diagnostic characters. Finally, we provide considerable discussion of the current state of knowledge of these species' ecology, natural history, and distribution.

Formants provide honest acoustic cues to body size in American alligators.

In many vertebrates, acoustic cues to body size are encoded in resonance frequencies of the vocal tract ("formants"), rather than in the rate of tissue vibration in the sound source ("pitch"). Anatomical constraints on the vocal tract's size render formants honest cues to size in many bird and mammal species, but it is not clear whether this correlation evolved convergently in these two clades, or whether it is widespread among amniotes (mammals, birds, and non-avian reptiles). We investigated the potential for honest acoustic cues in the bellows of adult American alligators and found that formant spacing provided highly reliable cues to body size, while presumed correlates of the source signal did not. These findings held true for both sexes and for all bellows whether produced in or out of water. Because birds and crocodilians are the last extant Archosaurians and share common ancestry with all extinct dinosaurs, our findings support the hypothesis that dinosaurs used formants as cues to body size. The description of formants as honest signals in a non-avian reptile combined with previous evidence from birds and mammals strongly suggests that the principle of honest signalling via vocal tract resonances may be a broadly shared trait among amniotes.

Presence of Breeding Birds Improves Body Condition for a Crocodilian Nest Protector.

Ecological associations where one species enhances habitat for another nearby species (facilitations) shape fundamental community dynamics and can promote niche expansion, thereby influencing how and where species persist and coexist. For the many breeding birds facing high nest-predation pressure, enemy-free space can be gained by nesting near more formidable animals for physical protection. While the benefits to protected species seem well documented, very few studies have explored whether and how protector species are affected by nest protection associations. Long-legged wading birds (Pelecaniformes and Ciconiiformes) actively choose nesting sites above resident American alligators (Alligator mississippiensis), apparently to take advantage of the protection from mammalian nest predators that alligator presence offers. Previous research has shown that wading bird nesting colonies could provide substantial food for alligators in the form of dropped chicks. We compared alligator body condition in similar habitat with and without wading bird nesting colonies present. Alligator morphometric body condition indices were significantly higher in colony than in non-colony locations, an effect that was statistically independent of a range of environmental variables. Since colonially nesting birds and crocodilians co-occur in many tropical and subtropical wetlands, our results highlight a potentially widespread keystone process between two ecologically important species-groups. These findings suggest the interaction is highly beneficial for both groups of actors, and illustrate how selective pressures may have acted to form and reinforce a strongly positive ecological interaction.

Large Scale Discovery and De Novo-Assisted Sequencing of Cationic Antimicrobial Peptides (CAMPs) by Microparticle Capture and Electron-Transfer Dissociation (ETD) Mass Spectrometry.

The identification and sequencing of novel cationic antimicrobial peptides (CAMPs) have proven challenging due to the limitations associated with traditional proteomics methods and difficulties sequencing peptides present in complex biomolecular mixtures. We present here a process for large-scale identification and de novo-assisted sequencing of newly discovered CAMPs using microparticle capture followed by tandem mass spectrometry equipped with electron-transfer dissociation (ETD). This process was initially evaluated and verified using known CAMPs with varying physicochemical properties. The effective parameters were then applied in the analysis of a complex mixture of peptides harvested from American alligator plasma using custom-made (Bioprospector) functionalized hydrogel particles. Here, we report the successful sequencing process for CAMPs that has led to the identification of 340 unique peptides and the discovery of five novel CAMPs from American alligator plasma.

Bioprospecting the American alligator (Alligator mississippiensis) host defense peptidome.

Cationic antimicrobial peptides and their therapeutic potential have garnered growing interest because of the proliferation of bacterial resistance. However, the discovery of new antimicrobial peptides from animals has proven challenging due to the limitations associated with conventional biochemical purification and difficulties in predicting active peptides from genomic sequences, if known. As an example, no antimicrobial peptides have been identified from the American alligator, Alligator mississippiensis, although their serum is antimicrobial. We have developed a novel approach for the discovery of new antimicrobial peptides from these animals, one that capitalizes on their fundamental and conserved physico-chemical properties. This sample-agnostic process employs custom-made functionalized hydrogel microparticles to harvest cationic peptides from biological samples, followed by de novo sequencing of captured peptides, eliminating the need to isolate individual peptides. After evaluation of the peptide sequences using a combination of rational and web-based bioinformatic analyses, forty-five potential antimicrobial peptides were identified, and eight of these peptides were selected to be chemically synthesized and evaluated. The successful identification of multiple novel peptides, exhibiting antibacterial properties, from Alligator mississippiensis plasma demonstrates the potential of this innovative discovery process in identifying potential new host defense peptides.

Multiple lineages of ancient CR1 retroposons shaped the early genome evolution of amniotes.

Chicken repeat 1 (CR1) retroposons are long interspersed elements (LINEs) that are ubiquitous within amniote genomes and constitute the most abundant family of transposed elements in birds, crocodilians, turtles, and snakes. They are also present in mammalian genomes, where they reside as numerous relics of ancient retroposition events. Yet, despite their relevance for understanding amniote genome evolution, the diversity and evolution of CR1 elements has never been studied on an amniote-wide level. We reconstruct the temporal and quantitative activity of CR1 subfamilies via presence/absence analyses across crocodilian phylogeny and comparative analyses of 12 crocodilian genomes, revealing relative genomic stasis of retroposition during genome evolution of extant Crocodylia. Our large-scale phylogenetic analysis of amniote CR1 subfamilies suggests the presence of at least seven ancient CR1 lineages in the amniote ancestor; and amniote-wide analyses of CR1 successions and quantities reveal differential retention (presence of ancient relics or recent activity) of these CR1 lineages across amniote genome evolution. Interestingly, birds and lepidosaurs retained the fewest ancient CR1 lineages among amniotes and also exhibit smaller genome sizes. Our study is the first to analyze CR1 evolution in a genome-wide and amniote-wide context and the data strongly suggest that the ancestral amniote genome contained myriad CR1 elements from multiple ancient lineages, and remnants of these are still detectable in the relatively stable genomes of crocodilians and turtles. Early mammalian genome evolution was thus characterized by a drastic shift from CR1 prevalence to dominance and hyperactivity of L2 LINEs in monotremes and L1 LINEs in therians.

Rigorous approaches to species delimitation have significant implications for African crocodilian systematics and conservation.

Accurate species delimitation is a central assumption of biology that, in groups such as the Crocodylia, is often hindered by highly conserved morphology and frequent introgression. In Africa, crocodilian systematics has been hampered by complex regional biogeography and confounded taxonomic history. We used rigorous molecular and morphological species delimitation methods to test the hypothesis that the slender-snouted crocodile (Mecistops cataphractus) is composed of multiple species corresponding to the Congolian and Guinean biogeographic zones. Speciation probability was assessed by using 11 mitochondrial and nuclear genes, and cranial morphology for over 100 specimens, representing the full geographical extent of the species distribution. Molecular Bayesian and phylogenetic species delimitation showed unanimous support for two Mecistops species isolated to the Upper Guinean and Congo (including Lower Guinean) biomes that were supported by 13 cranial characters capable of unambiguously diagnosing each species. Fossil-calibrated phylogenetic reconstruction estimated that the species split ± 6.5-7.5 Ma, which is congruent with intraspecies divergence within the sympatric crocodile genus Osteolaemus and the formation of the Cameroon Volcanic Line. Our results underscore the necessity of comprehensive phylogeographic analyses within currently recognized taxa to detect cryptic species within the Crocodylia. We recommend that the community of crocodilian researchers reconsider the conceptualization of crocodilian species especially in the light of the conservation ramifications for this economically and ecologically important group.

Insights into the ecology and evolutionary success of crocodilians revealed through bite-force and tooth-pressure experimentation.

BACKGROUND: Crocodilians have dominated predatory niches at the water-land interface for over 85 million years. Like their ancestors, living species show substantial variation in their jaw proportions, dental form and body size. These differences are often assumed to reflect anatomical specialization related to feeding and niche occupation, but quantified data are scant. How these factors relate to biomechanical performance during feeding and their relevance to crocodilian evolutionary success are not known. METHODOLOGY/PRINCIPAL FINDINGS: We measured adult bite forces and tooth pressures in all 23 extant crocodilian species and analyzed the results in ecological and phylogenetic contexts. We demonstrate that these reptiles generate the highest bite forces and tooth pressures known for any living animals. Bite forces strongly correlate with body size, and size changes are a major mechanism of feeding evolution in this group. Jaw shape demonstrates surprisingly little correlation to bite force and pressures. Bite forces can now be predicted in fossil crocodilians using the regression equations generated in this research. CONCLUSIONS/SIGNIFICANCE: Critical to crocodilian long-term success was the evolution of a high bite-force generating musculo-skeletal architecture. Once achieved, the relative force capacities of this system went essentially unmodified throughout subsequent diversification. Rampant changes in body size and concurrent changes in bite force served as a mechanism to allow access to differing prey types and sizes. Further access to the diversity of near-shore prey was gained primarily through changes in tooth pressure via the evolution of dental form and distributions of the teeth within the jaws. Rostral proportions changed substantially throughout crocodilian evolution, but not in correspondence with bite forces. The biomechanical and ecological ramifications of such changes need further examination.

An ancient icon reveals new mysteries: mummy DNA resurrects a cryptic species within the Nile crocodile.

The Nile crocodile (Crocodylus niloticus) is an ancient icon of both cultural and scientific interest. The species is emblematic of the great civilizations of the Nile River valley and serves as a model for international wildlife conservation. Despite its familiarity, a centuries-long dispute over the taxonomic status of the Nile crocodile remains unresolved. This dispute not only confounds our understanding of the origins and biogeography of the 'true crocodiles' of the crown genus Crocodylus, but also complicates conservation and management of this commercially valuable species. We have taken a total evidence approach involving phylogenetic analysis of mitochondrial and nuclear markers, as well as karyotype analysis of chromosome number and structure, to assess the monophyletic status of the Nile crocodile. Samples were collected from throughout Africa, covering all major bioregions. We also utilized specimens from museum collections, including mummified crocodiles from the ancient Egyptian temples at Thebes and the Grottes de Samoun, to reconstruct the genetic profiles of extirpated populations. Our analyses reveal a cryptic evolutionary lineage within the Nile crocodile that elucidates the biogeographic history of the genus and clarifies long-standing arguments over the species' taxonomic identity and conservation status. An examination of crocodile mummy haplotypes indicates that the cryptic lineage corresponds to an earlier description of C. suchus and suggests that both African Crocodylus lineages historically inhabited the Nile River. Recent survey efforts indicate that C. suchus is declining or extirpated throughout much of its distribution. Without proper recognition of this cryptic species, current sustainable use-based management policies for the Nile crocodile may do more harm than good.

Comparisons of innate immune activity of all known living crocodylian species.

Serum samples from all twenty-three known living members of the Crocodylia were tested for antibacterial activity against eight bacterial species. These data were used to generate an immune profile for each crocodylian species. Statistical analyses revealed that the three living lineages of crocodylians, Alligatoroidea, Crocodyloidea, and Gavialoidea, were distinguishable by their immunological activities. For instance, species within the Alligatoroidea and Crocodyloidea exhibited remarkable immune activity similarities to others in their own lineages. Comparisons of the members of the different lineages, however, revealed substantial differences in immune profiles. Furthermore, species that are in the same genus were shown to exhibit more immune similarities to each other than to members of other genera within the same family. Finally, our immunological analyses reveal that Tomistoma schlegelii aligns more closely with the Gavialoidea than the Crocodyloidea.

Pharmacokinetics of enrofloxacin after single-dose oral and intravenous administration in the American alligator (Alligator mississippiensis).

The pharmacokinetics of enrofloxacin administered orally and i.v. to American alligators (Alligator mississippiensis) at 5 mg/kg was determined. Plasma levels of enrofloxacin and its metabolite ciprofloxacin were measured using high-performance liquid chromatography and the resulting concentration versus time curve analyzed using compartmental modeling techniques for the i.v. data and noncompartmental modeling techniques for the oral data. A two-compartment model best represented the i.v. data. Intravenous administration of enrofloxacin resulted in an extrapolated mean plasma concentration of 4.19 +/- 4.23 microg/ml at time zero, with average plasma drug levels remaining above 1.0 microg/ml for an average of 36 hr. Plasma volume of distribution for i.v. enrofloxacin was 1.88 +/- 0.96 L/kg, with a harmonic mean elimination half-life of 21.05 hr and mean total body clearance rate of 0.047 +/- 0.021 L/hr/kg. Plasma levels of p.o. enrofloxacin remained below 1.0 microg/ml in all test animals, and average concentrations ranged from 0.08 to 0.50 microg/ml throughout the sampling period. Oral administration of enrofloxacin achieved a mean maximum plasma concentration of 0.50 +/- 0.27 microg/ml at 55 +/- 29 hr after administration, with a harmonic mean terminal elimination half-life of 77.73 hr. Minimal levels of ciprofloxacin were detected after both oral and i.v. enrofloxacin administration, with concentrations below minimum inhibitory concentrations for most susceptible organisms. On the basis of the results of this study, enrofloxacin administered to American alligators at 5 mg/kg i.v. q 36 hr is expected to maintain plasma concentrations that approximate the minimum inhibitory concentration for susceptible organisms (0.5 microg/ml). Enrofloxacin administered to American alligators at 5 mg/kg p.o. is not expected to achieve minimum inhibitory values for susceptible organisms.