Molecular data from contemporary and historical collections reveal a complex story of cryptic diversification in the Varanus (Polydaedalus) niloticus Species Group.

Previous studies of color pattern, tongue pigmentation, and scale counts have been used to distinguish two species of semiaquatic varanids in Africa, but these findings have yet to be tested with molecular data. The Varanus (Polydaedalus) niloticus Species Group is comprised of the Nile monitor (V. niloticus) and the Ornate monitor (V. ornatus). Due to the high rate of exploitation of both species for bushmeat, the leather industry, and the pet trade, a clear understanding of the taxonomy and genetic partitioning is necessary for effective management. Here we utilize a multilocus approach, consisting of mitochondrial and nuclear markers, totaling 4251 bp, as well as microsatellite loci to assess the taxonomic validity and intraspecific evolutionary patterns within the V. niloticus Species Group. By incorporating historical specimens from museum collections as well as contemporary samples, we obtained range-wide coverage for both species across Africa. Concordant results from various approaches all suggest that V. ornatus does not represent a distinct monophyletic group. Our analyses recovered three genetic clades within V. niloticus, representing western, northern, and southern lineages. The western clade was found to diverge first, around 7.7 mya (95% HPD: 4.6-11.0 mya) and exhibits 8.4% and 8.7% uncorrected sequence divergence between the northern and southern V. niloticus clades, respectively. This geographically separate lineage corresponds to previous descriptions of Tupinambis stellatusDaudin (1802). These findings not only call for taxonomic revision of this species group, but also shed light on the biogeographic history of Africa as well as aid in the management planning of varanids and other co-distributed African species.

Fine-scale genetic analysis of the exploited Nile monitor (Varanus niloticus) in Sahelian Africa.

BACKGROUND: Overexploitation of wildlife populations results in direct consequences, such as extinction and local extirpation, as well as indirect effects including genetic diversity loss and changes in genetic structure. A clear understanding of the underlying genetic patterns of harvested species is necessary for sustainable management. The Nile monitor (Varanus niloticus) is a commercially valuable species in the international leather industry, with the highest levels of exploitation concentrated throughout Sahelian Africa. In this study, we examined the fine-scale genetic patterns of V. niloticus populations in the Sahel, with the expectation that the genetic structure would correspond to riverine drainage basins. The analyses were based on genotypes at 11 microsatellite loci for 318 individuals, spanning three separate watersheds throughout the Sahel. RESULTS: Our analyses identified four genetic clusters throughout the region, one of which (the westernmost population) exhibited very high levels of genetic differentiation (FST = 0.47). Contrary to our expectation, the largest genetic break occurred within a single watershed, the Niger basin, rather than between watersheds. However, other localities displayed evidence of reduced gene flow between watershed boundaries. Across methods, the westernmost population exhibited lower estimates of N e as well as lower levels of genetic diversity compared to the other inferred populations. While we did not detect evidence for recent population bottlenecks, our analyses indicated historic population declines around 1,000-1,800 years ago. CONCLUSION: We found that the underlying genetic structure of Varanus niloticus across Sahelian Africa reflects historic as well as present-day patterns of riverine drainages. The high degree of differentiation found for the westernmost population indicates the presence of a separate lineage, and should be taken into consideration when setting harvest limits. The historic population decline for two of the populations corresponds to a drastic expansion of an ancient human civilization in the region, suggesting that human exploitation of V. niloticus has a longer history than previously thought.

Gradual adaptation of bone structure to aquatic lifestyle in extinct sloths from Peru.

Non-pathological densification (osteosclerosis) and swelling (pachyostosis) of bones are the main modifications affecting the skeleton of land vertebrates (tetrapods) that returned to water. However, a precise temporal calibration of the acquisition of such adaptations is still wanting. Here, we assess the timing of such acquisition using the aquatic sloth Thalassocnus, from the Neogene of the Pisco Formation, Peru. This genus is represented by five species occurring in successive vertebrate-bearing horizons of distinct ages. It yields the most detailed data about the gradual acquisition of aquatic adaptations among tetrapods, in displaying increasing osteosclerosis and pachyostosis through time. Such modifications, reflecting a shift in the habitat from terrestrial to aquatic, occurred over a short geological time span (ca 4 Myr). Otherwise, the bones of terrestrial pilosans (sloths and anteaters) are much more compact than the mean mammalian condition, which suggests that the osteosclerosis of Thalassocnus may represent an exaptation.

A survey of osteoderm histology and ornamentation among Crocodylomorpha: A new proxy to infer lifestyle?

Osteoderms of eight extant and extinct species of crocodylomorphs are studied histologically and morphologically. Most osteoderms display the typical "crocodilian" structure with a woven-fibered matrix surrounded by an upper and a lower parallel fibered matrix. The dorsal ornamentation of those specimens consists of a pit-and-ridge structure, with corresponding remodeling mechanisms. However, an osteoderm of Iberosuchus, studied here for the first time, differs in being nearly devoid of ornamentation; moreover, it shows strong bundles of straight Sharpey's fibers perpendicular to the surface in its lateral and dorsal walls, along with a rough plywood-like structure in its basal plate. This suggests that this osteoderm was more deeply anchored within the dermis than the other osteoderms studied hitherto. This peculiar structure might have been linked to a terrestrial ecology and a specific thermoregulation strategy. Some other notosuchians in our sample do not exhibit ornamentation on their osteoderms, as opposed to neosuchians. Considering current interpretations of osteoderm function(s) in crocodilians, our observations are discussed in reference to possible ecophysiological peculiarities of Notosuchia in general, and Iberosuchus in particular.

Hypersalinity drives convergent bone mass increases in Miocene marine mammals from the Paratethys.

Pachyosteosclerosis-a condition that creates dense, bulky bones-often characterizes the early evolution of secondarily aquatic tetrapods like whales and dolphins1-3 but then usually fades away as swimming efficiency increases.4 Here, we document a remarkable reversal of this pattern, namely the convergent re-emergence of bone densification in Miocene seals, dolphins, and whales from the epicontinental Paratethys Sea of eastern Europe and central Asia. This phenomenon was driven by imbalanced remodeling and inhibited resorption of primary trabeculae and coincided with hypersaline conditions-the Badenian salinity crisis-that affected the Central Paratethys between 13.8 and 13.4 Ma.5 Dense bones acting as ballast would have facilitated efficient swimming in the denser and more buoyant water and hence were likely adaptive in this setting. From the Central Paratethys, pachyosteosclerosis subsequently spread eastward, where it became a defining feature of the endemic late Miocene whale assemblage.6,7.

Vertebral microanatomy in squamates: structure, growth and ecological correlates.

The histological study of vertebrae in extant squamates shows that the internal vertebral structure in this group differs from that of other tetrapods. Squamate vertebrae are lightly built and basically composed of two roughly concentric osseous tubes--one surrounding the neural canal and the other constituting the peripheral cortex of the vertebra--connected by few thin trabeculae. This structure, which characteristically evokes that of a tubular bone, results from a peculiar remodelling process characterised by an imbalance between local bone resorption and redeposition; in both periosteal and endosteo-endochondral territories, bone is extensively resorbed but not reconstructed in the same proportion by secondary deposits. This process is particularly intense in the deep region of the centrum, where originally compact cortices are made cancellous, and where the endochondral spongiosa is very loose. This remodelling process starts at an early stage of development and remains active throughout subsequent growth. The growth of squamate centra is also strongly asymmetrical, with the posterior (condylar) part growing much faster than the anterior (cotylar) part. Preliminary analyses testing for associations between vertebral structure and habitat use suggest that vertebrae of fossorial taxa are denser than those of terrestrial taxa, those in aquatic taxa being of intermediate density. However, phylogenetically informed analyses do not corroborate these findings, thus suggesting a strong phylogenetic signal in the data. As our analyses demonstrate that vertebrae in snakes are generally denser than those of lizards sensu stricto, this may drive the presence of a phylogenetic signal in the data. More comprehensive sampling of fossorial and aquatic lizards is clearly needed to more rigorously evaluate these patterns.

Bone vascular supply in monitor lizards (Squamata: Varanidae): influence of size, growth, and phylogeny.

Bone vascular canals occur irregularly in tetrapods; however, the reason why a species has or lacks bone canals remains poorly understood. Basically, this feature could depend on phylogenetic history, or result from diverse causes, especially cortical accretion rate. The Varanidae, a monophyletic clade that includes species with impressive size differences but similar morphologies, is an excellent model for this question. Cortical vascularization was studied in 20 monitor species, on three bones (femur, fibula, and tibia) that differ in their shaft diameters, and in the absolute growth speed of their diaphyseal cortices. In all species smaller than 398 mm SVL (133-397 mm in sample), bone cortices lack vascular canals, whereas all larger species (460-1,170 mm in sample) display canals. The size 398-460 mm SVL is thus a threshold for the appearance of the canals. The distribution of vascular and avascular bone tissues among species does not precisely reflect phylogenetic relationships. When present, vascular canals always occur in the femur and tibia, but are less frequent, sparser, and thinner in the fibula. Vascular density increases linearly with specific size but decreases exponentially during individual growth. In most species, canal orientation varies between individuals and is diverse in a single section. No clear relationship exists between canal orientation and vascular density. These results suggest that: a) the occurrence and density of bone vascular canals are basically dependant on specific size, not phylogenetic relationships; b) vascular density reflects the absolute growth rates of bone cortices; c) the orientation of vascular canals is a variable feature independent of phylogeny or growth rate.

Structure and growth pattern of the bizarre hemispheric prominence on the rostrum of the fossil beaked whale Globicetus hiberus (Mammalia, Cetacea, Ziphiidae).

The rostrum of most ziphiids (beaked whales) displays bizarre swollen regions, accompanied with extreme hypermineralisation and an alteration of the collagenous mesh of the bone. The functional significance of this specialization remains obscure. With the voluminous and dense hemispheric excrescence protruding from the premaxillae, the recently described fossil ziphiid Globicetus hiberus is the most spectacular case. This study describes the histological structure and interprets the growth pattern of this unique feature. Histologically, the prominence in Globicetus is made up of an atypical fibro-lamellar complex displaying an irregular laminar organization and extreme compactness (osteosclerosis). Its development is suggested to have resulted from a protraction of periosteal accretion over the premaxillae, long after the end of somatic growth. Complex shifts in the geometry of this tissue are likely to have occurred during its accretion and no indication of Haversian remodeling could be found. X-ray diffraction and Raman spectroscopy indicate that the bone matrix in the premaxillary prominence of Globicetus closely resembles that of the rostrum of the extant beaked whale Mesoplodon densirostris: apatite crystals are of common size and strongly oriented, but the collagenous meshwork within bone matrix seems to be extremely sparse. These morphological and structural data are discussed in the light of functional interpretations proposed for the highly unusual and diverse ziphiid rostrum. J. Morphol. 277:1292-1308, 2016. © 2016 Wiley Periodicals, Inc.

Comparative data on the differentiation and growth of bone ornamentation in gnathostomes (Chordata: Vertebrata).

Bone ornamentation, in the form of rounded pits framed by a network of ridges, is a frequent feature among a great diversity of gnathostome taxa. However, the basic osteogenic processes controlling the differentiation and development of these reliefs remain controversial. The present study is a broad comparative survey of this question with the classical methods used in hard tissue histology and paleohistology. Distinct processes, unevenly distributed among taxa, are involved in the creation and growth of pits and ridges. The simplest one is mere differential growth between pit bottom (slow growth) and ridge top (faster growth). The involvement of several complex remodeling processes, with the local succession of resorption and reconstruction cycles, is frequent and occurs in all major gnathostome clades. Some broad, inclusive clades (e.g., Temnospondyli) display consistency in the mechanisms controlling ornamentation, whereas other clades (e.g., Actinopterygii) are characterized by the diversity of the mechanisms involved. If osteogenic mechanisms are taken into account, bone ornamentation should be considered as a character extremely prone to homoplasy. Maximum likelihood (ML) optimizations reveal that the plesiomorphic mechanism creating ornamentation is differential apposition rate over pits (slow growth) and ridges (faster growth). In some taxas e.g., temnospondyls vs lissamphibians or pseudosuchians, bone ornamentation is likely to be a homoplastic feature due to a convergence process driven by similar selective pressures. ML models of character evolution suggest that the presence of resorption in the development of ornamentation may be selectively advantageous, although support for this conclusion is only moderate.

Body location and tail regeneration effects on osteoderms morphology-are they useful tools for systematic, paleontology, and skeletochronology in diploglossine lizards (squamata, anguidae)?

Although diploglossine osteoderms were mentioned in several systematic and paleontological studies, their morphological variability in single specimens or within species remains paradoxically undescribed. This is mainly the effect of the lack of attention paid hitherto to the morphological and histological characteristics of the tail osteoderms. This study demonstrated that a previously undescribed morphological variability exists in these osteoderms, especially in those resulting from tail regeneration. Indeed, regenerated osteoderms display a plesiomorphic anguid morphology that was previously considered to be absent in Diploglossinae. We also provide the first histological description of diploglossine osteoderms and new information about the obvious differences in growth dynamic between regenerated and nonregenerated osteoderms. These new data raise questions about the usefulness of diploglossine osteoderms in systematic, paleontological, and skeletochronological studies. Our study shows that the exact position on the trunk or on a regenerated or nonregenerated tail of each studied osteoderm must be known in order to avoid mistakes related to their important interspecies and intraspecies variability.

A quantitative assessment of bone area increase due to ornamentation in the Crocodylia.

Bone ornamentation, in the form of highly repetitive motives created by pits and ridges, is a frequent feature on vertebrate skull roofs and osteoderms. The functional significance of this character remains a matter of controversy and speculation. The many diverging hypotheses proposed to explain it all share a common logical prerequisite: bone ornamentation should increase significantly the surface area of the bones that bear it. In order to test this assumption in the Crocodylia, we developed a method for quantifying the gain in area due to ornamentation using a three-dimensional-surface scanner. On crocodylian osteoderms, the gain in area can be up to 40%, and on the cranial table, it ranges between 10 and 32% in adult specimens (in both cases, it shows substantial differences between the adults of the various species included in the sample). Area gain on the snout is lesser (0-20% in adults), and more variable between species. In general, bone ornamentation is less pronounced, and results in fewer area gains in juvenile specimens. The main morphometric results yielded by this study are discussed in reference to the few comparative data available hitherto, and to the functional interpretations proposed by previous authors.

Inner architecture of vertebral centra in terrestrial and aquatic mammals: a two-dimensional comparative study.

Inner vertebral architecture is poorly known, except in human and laboratory animals. In order to document this topic at a broad comparative level, a 2D-histomorphometric study of vertebral centra was conducted in a sample of 98 therian mammal species, spanning most of the size range and representing the main locomotor adaptations known in therian taxa. Eleven variables relative to the development and geometry of trabecular networks were extracted from CT scan mid-sagittal sections. Phylogeny-informed statistical tests were used to reveal the respective influences of phylogeny, size, and locomotion adaptations on mammalian vertebral structure. The use of random taxon reshuffling and squared change parsimony reveals that 9 of the 11 characteristics (the two exceptions are total sectional area and structural polarization) contain a phylogenetic signal. Linear discriminant analyses suggest that the sampled taxa can be arranged into three categories with respect to locomotion mode: a) terrestrial + flying + digging + amphibious forms, b) coastal oscillatory aquatic taxa, and c) pelagic oscillatory aquatic forms represented by oceanic cetaceans. Pairwise comparison tests and linear regressions show that, when specific size increases, the length of trabecular network (Tt.Tb.Le), as well as trabecular proliferation in total sections (Pr.Tb.Tt), increase with positive allometry. This process occurs in all locomotion categories but is particularly pronounced in pelagic oscillators. Conversely, mean trabecular width has a lesser increase with size in pelagic oscillators. Trabecular orientation is not influenced by size. All tests were corrected for multiple testing. By using six structural variables or indices, locomotion mode can be predicted with a 97.4% success rate for terrestrial forms, 66.7% for coastal oscillatory, and 81.3% for pelagic oscillatory. The possible functional meaning of these results and their potential use for paleobiological inference of locomotion in extinct taxa are discussed.

Structure and growth pattern of pseudoteeth in Pelagornis mauretanicus (Aves, Odontopterygiformes, Pelagornithidae).

The extinct Odontopterygiformes are the sole birds known to possess strong and sharp bony pseudoteeth, the shape and location of which are closely mimetic of real teeth. The structure of the pseudoteeth is investigated here in a late Pliocene/early Pleistocene species, Pelagornis mauretanicus, using X-ray microtomography and thin sections. The results are interpreted with regard to the pseudotooth mode of growth, and have implications concerning aspects of Pelagornis ecology. The larger pseudoteeth are hollow and approximately cone-shaped, and the smaller ones are rostro-caudally constricted. The walls of pseudoteeth are composed of bone tissue of the fibro-lamellar type, which is intensively remodeled by Haversian substitution. The jaw bones display the same structure as the pseudoteeth, but their vascular canals are oriented parallel to the long axis of the bones, whereas they are perpendicular to this direction in the pseudoteeth. There is no hiatus or evidence of a fusion between the pseudoteeth and the jaw bones. Two possible models for pseudotooth growth are derived from the histological data. The most plausible model is that pseudotooth growth began after the completion of jaw bone growth, as a simple local protraction of periosteal osteogenic activity. Pseudotooth development thus occurred relatively late during ontogeny. The highly vascularized structure and the relative abundance of parallel-fibered bone tissue in the pseudoteeth suggest poor mechanical capabilities. The pseudoteeth were most likely covered and protected by the hardened, keratinized rhamphotheca in the adult during life. The late development of the pseudoteeth would involve a similarly late and/or partial hardening of the rhamphotheca, as displayed by extant Anseriformes, Apterygiformes and some Charadriiformes. This would add support to the hypothesis of a close phylogenetic relationship between Odontopterygiformes and Anseriformes. The late maturation of the Pelagornis feeding apparatus, and hence the delayed capability for efficient prey catching, suggests that Pelagornis was altricial.

Experimental exposure of juvenile savannah monitors (Varanus exanthematicus) to an environmentally relevant mixture of three contaminants: effects and accumulation in tissues.

Using varanids as indicators of pollution in African continental wetlands was previously proposed. The present study aimed at understanding experimentally how monitors absorb and accumulate pollutants and how they are affected. The relevance of non-destructive sampling was also evaluated. Savannah monitors (Varanus exanthematicus) were orally exposed during 6 months to a mixture of lead, 4,4'-dichlorodiphenyltrichloroethane (4,4'-DDT) and chlorpyrifos-ethyl (CPF) or to the vehicle only. Proportionally to their mass, exposed monitors received the same dose: 20 then 10 mg lead kg(-1), 2 then 0.5 mg CPF kg(-1) and 4 mg 4,4'-DDT kg(-1). Individuals surviving contamination were euthanized after 4 or 6 months of experiment. Tissues were analysed for lead by atomic absorption spectrophotometry and for DDT and CPF by gas chromatography. Exposed monitors absorbed all three pollutants but only lead (essentially in bone, tail tips and phalanxes) and 4,4'-DDT plus its main metabolites (essentially in fat and liver) accumulated. CPF killed ten individuals. Clear correlations occurred between the total quantity of lead or 4,4'-DDT administered and concentrations in tissues. Tail tips and skin samples are recommended non-destructive indicators for lead and organochlorine pesticides contamination, respectively. This work confirms that monitors can be used as relevant indicators of environmental pollution by lead and organochlorine pesticides. Although varanids withstand heavy lead and DDT contamination, our results suggest that CPF can be lethal at very low doses to the herpetofauna and emphasize the importance of considering all taxa in impact assessment studies, including reptiles.

Assessing environmental contamination around obsolete pesticide stockpiles in West Africa: using the Nile monitor (Varanus niloticus) as a sentinel species.

Environmental contamination caused by obsolete pesticide stocks was assessed using the Nile monitor (Varanus niloticus) as a sentinel species. Organochlorines and organophosphates were quantified by gas chromatography in abdominal fat and the liver, respectively. Results were compared to those obtained from three other sites, characterized by different histories of contamination. None of the previously stocked pesticides were recovered. Low to moderate levels of 4,4'-dichlorodiphenyldichloroethylene (4,4'-DDE) were quantified in monitors from all sites. Malathion and 4,4'-dichlorodiphenyldichloroethane (4,4'-DDD) also were detected sporadically. Interindividual variability was substantial. Correlations between pesticide loads and individual characteristics were considered. The nondetection of previously stocked pesticides in the monitors' tissues, their contamination by other pesticides, and the value of V. niloticus as a monitoring tool for environmental contamination are discussed. The results indicate a situation of low concern and draw attention to the importance of local conditions in determining environmental dangers associated with potential pollution sources.

The Nile monitor (Varanus niloticus; Squamata: Varanidae) as a sentinel species for lead and cadmium contamination in sub-Saharan wetlands.

Wetland pollution is a matter of concern in sub-Saharan Africa. Though regularly exploited, the Nile monitor (Varanus niloticus), a large amphibious lizard, is not threatened. This work aims at assessing the value of this varanid as a sentinel species in surveys of environmental contamination by metals. Lead and cadmium quantifications were performed by graphite furnace-atomic absorption spectrophotometry in bone, intestine, kidney, liver and muscle in 71 monitors from three unevenly polluted sites in Mali and Niger, plus a reference site. The effects of sex, size and fat reserves as well as factors related to the sampling strategy (tissue sampled, sampling site) were studied with a mixed linear model. Metal contamination is moderate at the four sites but clear differences nevertheless occur. Lead levels are generally maximal in bone, with a gender-independent median value 320ng.g(-1). Median cadmium concentrations never exceed 70.2ng.g(-1) in females (kidney) and 57.5ng.g(-1) in males (intestine). Such levels should have no detrimental effects on the monitors. Lead and cadmium levels in muscles are generally below 200 and 20ng.g(-1), respectively, and should provoke no health hazard to occasional consumers of monitor meat. Metal organotropisms are consistent with those observed in other studies about Squamates: for lead: bone>[kidney, intestine, liver]>muscle in males and [bone, kidney]>[intestine, liver]>muscle in females; for cadmium: [liver, intestine, kidney]>[bone, muscle] for both genders. Females are more contaminated, especially in their kidneys. In this tissue, median values in ng.g(-1) are 129.7 and 344.0 for lead and 43.0 and 70.2 for cadmium, for males and females, respectively. Nile monitors can reveal subtle differences in local pollution by metals; moreover, the spatial resolution of the pollution indication that they give seems to be very sharp. The practical relevance of this new tool is thus validated.