Cross-species amplification of microsatellites in crocodilians: assessment and applications for the future

Microsatellite DNA loci have emerged as the dominant genetic tool for addressing questions associated with genetic diversity in many wildlife species, including crocodilians. Despite their usefulness, their isolation and development can be costly, as well as labour intensive, limiting their wider use in many crocodilian species. In this study, we investigate the cross-species amplification success of 82 existing microsatellites previously isolated for the saltwater crocodile (Crocodylus porosus) in 18 non-target crocodilian species; Alligator sinensis, Caiman crocodylus, Caiman latirostris, Caiman yacare, Melanosuchus niger, Paleosuchus palpebrosus, Crocodylus acutus, Mecistops cataphractus, Crocodylus intermedius, Crocodylus johnstoni, Crocodylus mindorensis, Crocodylus moreletii, Crocodylus niloticus, Crocodylus novaeguineae, Crocodylus palustis, Crocodylus rhombifer, Crocodylus siamensis, and Osteolaemus tetraspis. Our results show a high level of microsatellites cross-amplification making available polymorphic markers for a range of crocodilian species previously lacking informative genetic markers.     

Molecular identification of crocodile species using novel primers for forensic analysis

All crocodilians are under varying degrees of threat due to over exploitation and these species have been listed in Appendix I or II of CITES. The lack of molecular techniques for the identification of confiscated samples makes it difficult to enforce the law. Conclusive forensic identification of species requires a complete gene sequence which is difficult in case of degraded samples. We have developed two novel sets of primers to amplify two partial cytochrome b gene sequences of six crocodile species i.e. Crocodylus palustris, Crocodylus porosus, Crocodylus siamensis, Crocodylus niloticus, Gavialis gangeticus and Caiman crocodilus. These partial sequences were edited to give a complete cyt b gene sequence, which can be used as an effective tool for forensic authentication of crocodile species. A phylogeny of crocodile species was reconstructed using these sequences. The described primers hold great promise in forensic identification of crocodile species, which can aid in the effective enforcement of law and conservation of these ancient species.     

Differences in renal-cloacal function between Crocodylus porosus and Alligator mississippiensis have implications for crocodilian evolution

Major electrolytes and nitrogenous excretory products were analysed in the blood plasma, ureteral urine and cloacal urine of juvenile Alligator mississippiensis and Crocodylus porosus in fresh and hypoosmotic salt water (206 mosmol · l⁻¹). Both species coped well with saline water, showing little (Alligator) or no (Crocodylus) change in plasma composition. Comparisons of renal-cloacal function point to major differences in their osmoregulatory physiology. The cloaca of C. porosus is a very active osmoregulatory organ in salt and fresh water, contributing to water conservation and NaCl excretion through the lingual salt glands. In contrast, the cloaca of Alligator has little impact on the composition of excreted urine. It seems likely that A.␣mississippiensis is largely constrained to a renal response to osmotic and ionic stress while C. porosus is able to call on a more complex mix of renal response, post-renal modification of urine in the cloaca, and excretion of excess NaCl through the salt glands. The results support the idea that there are deep-seated differences in the osmoregulatory physiology of alligatorids and crocodylids (Eusuchia), an understanding of which should provide valuable insights into their evolution and zoogeography. Accepted: 7 September 1996     

Osmoregulation by the broad-snouted caiman, Caiman latirostris, in estuarine habitat in southern Brazil

The broad-snouted caiman Caiman latirostris, of South America mostly frequents freshwater but occurs also in estuaries. Nothing of substance is known of its osmoregulatory physiology but, in the light of accumulating evidence that alligatorids lack specialised adaptations for life in hyperosmotic waters, we anticipated its physiology would be more similar to that of Alligator mississippiensis than the euryhaline Crocodylus porosus, which has both lingual salt glands and a more complex renal:cloacal system. This proved to be the case. Caiman captured in estuaries of the Ilha do Cardoso in southern Brazil were effective hypo-osmotic osmoregulators in salinities of 0–24 ppt (seawater = 35 ppt). Plasma osmolarity, sodium and chloride were similar to those in other crocodilians and not influenced by salinity. Plasma urea was low and did not vary with salinity. We found no evidence of lingual or other salt glands. Urinary electrolyte concentrations varied considerably with salinity and in ways reminiscent of A. mississippiensis but very different from C. porosus. Ca. latirostris dehydrated in seawater more rapidly than C. porosus and had substantially higher integumental permeability to water. Caiman did not drink seawater but rehydrated rapidly when returned to freshwater (FW). We found small caiman (<500 g) only in very low salinities (<3 ppt) and larger caiman closer to the sea. We postulate that medium to large Ca. latirostris can take advantage of the feeding opportunities presented by the estuarine mangal despite lacking the physiological specialisations of crocodylids. Two individuals which we re-sighted by chance had travelled at least 600 m in 2–3 days, showing that every caiman we captured or saw was within easy reach of FW. Most likely their habitation of the estuary and its mangal is achieved through a combination of low surface area:volume ratio, relatively impermeable skin, and periodic access to FW. Accepted: 11 May 1998     

A re-evaluation of aff. Megaloolithidae eggshell fragments from the uppermost Cretaceous of the Pyrenees and implications for crocodylomorph eggshell structure

The Upper Cretaceous outcrops of the Pyrenees yield one of the most extensive and continuous records of paleoological remains anywhere in the world. Most of eggs and eggshells have been referred to the oofamily Megaloolithidae. In this study, we present a revision of eggshell fragments from the Blasi 2 locality, lattermost Maastrichtian in age, previously assigned to aff. Megaloolithidae. The presence of a blocky extinction pattern and basal knobs supports a crocodilian affinity of these materials. We classify them as Krokolithidae indet. Three structural layers can be recognised in the Blasi 2 eggshells, a feature that is shared with other recent eggshells (e.g. Crocodylus porosus and Crocodylus niloticus) and fossil crocodylomorph eggshells (Krokolitheswilsoni), which were previously described as single layered. The new proposed affinity of the Blasi 2 eggshells reduces the Megaloolithidae oodiversity of the last few million years of the Cretaceous in the Pyrenees to only two valid ootaxa, Megaloolithusmamillare and Megaloolithusbaghensis. The lack of more complete material precludes the erection of new ootaxa based on the Blasi 2 material.     

Crocodile swim tracks from the latest Cretaceous of Europe

Recently discovered evidence of tracks in the continental beds of the Late Cretaceous Tremp Formation in the southern Pyrenees (NE Iberian Peninsula) has been identified as scratch marks made by buoyant crocodiles. The tracks are preserved in two distinct environments and substrates (marly limestones originating in a littoral mud flat and fine‐grained sandstones deposited in fluvial settings). Most of the crocodylian traces are ascribed to ichnogenus Characichnos, whereas a single plantigrade pes track is assigned to ichnogenus cf. Crocodylopodus. The crocodylian swim traces (Characichnos ichnofacies) found in the early and late Maastrichtian co‐occur with Brontopodus ichnofacies attributable to terrestrial tetrapods (titanosaur sauropods, cf. Brontopodus ichnogenus; and hadrosaurid ornithopods, Hadrosauropodus ichnogenus). Analysis of the tracks allows the interpretation of palaeoenvironmental settings and track production. Thus, in lagoonal environments, swim tracks of crocodylians were produced during the rise of the water level in successive tide cycles; in fluvial settings, the swim traces of crocodylians were produced within the channel at the low‐water stage. To date, there are no reports of Late Cretaceous crocodylian tracks in Europe, and the studied evidence represents the first and youngest track record of the group in the latest part of the Cretaceous (C29r) in this continent and probably in the world.     

Osmoregulation in Crocodilians

Recent crocodilians live primarily in freshwater habitats. Howevertwo species (Crocodylus acutus and C. porosus) are estuarinespecialists; two others (C. niloticus and C. johnstoni) thatare primarily found in fresh water, have estuarine populations.Routes of uptake of water and sodium include drinking, feedingand associated incidental drinking, and integumental and buccaldiffusion. Routes of loss include faeces-cloacal fluid, lingualsalt glands, integumental and buccal diffusion, and respiratoryloss. The least understood route of salt and water exchangeis that of the oral and buccal epithelia, which are much morepermeable to water and sodium than the general integument. Thefreshwater alligator (Alligator mississippiensis) osmoregulatesin a manner typical for an amphibious reptile. Body sodium turnoveris low and the general integument is quite low in permeabilityto sodium. Water turnover is more rapid (in terms of molar exchange)but still relatively low for an aquatic reptile. Most waterexchange occurs across the integument and buccal epithelia.The presence of lingual salt glands in freshwater crocodiliansremains enigmatic, as does the failure of these exocrine glandsin estuarine species to respond to saline loading. Secretiondoes occur after injection of the parasympathetic stimulantmethacholine chloride. The "salt water crocodile" (C. porosus)possesses a suite of osmoregulatory adaptations similar to thosefound in other estuarine reptiles. Water and sodium balanceare maintained primarily by an extremely low general permeabilityto sodium, by economies in water loss, and by excretion of excesssodium by the lingual salt glands. Further work is needed toexamine newly hatched C. porosus, and the possibility of ontogeneticchange in lingual gland function in C. acutus. The importanceof incidental drinking of sea water during feeding (recentlydiscovered in turtles) needs to be evaluated in crocodilians.The use of osmoregulatory data in interpretation of the evolutionaryhistory of the genus Crocodylus needs to be viewed with caution.The hypothesis that all species of Crocodylus originated fromthe transoceanic migration of a saline-tolerant form may notbe the most parsimonious explanation.     

A diagnosis of crocodile feeding traces on larger mammal bone, with fossil examples from the Plio-Pleistocene Olduvai Basin, Tanzania

Neotaphonomic studies have determined the patterns of bone damage created by larger mammalian carnivores when consuming mammalian carcasses. Typically, mammalian carnivores gnaw and break bones to various degrees in order to access marrow, grease, and brain tissue. In contrast, crocodiles attempt to swallow whole parts of mammal carcasses, inflicting in the process tooth marks and other feeding traces on some of the bones they are unable to ingest. Although crocodiles are major predators of larger mammals along the margins of protected tropical rivers and lakes, their feeding traces on bone have received little systematic attention in neotaphonomic research. We present diagnostic characteristics of Crocodylus niloticus damage to uningested mammal bones resulting from a series of controlled observations of captive crocodile feeding. The resulting bone assemblages are composed of primarily complete elements from articulating units, some of which bear an extremely high density of shallow to deep, transversely to obliquely oriented tooth scores over often large areas of the bone, along with shallow to deep pits and punctures. Some of the tooth marks (bisected pits and punctures, hook scores) have a distinctive morphology we have not observed to be produced by mammalian carnivores. The assemblages are also characterized by the retention of both low- and high-density bone portions, an absence of gross gnawing, and minimal fragmentation. Together, the damage characteristics associated with feeding by crocodiles are highly distinctive from those produced by mammalian carnivores. Modern surface bone assemblages along the Grumeti River in Tanzania's Serengeti National Park contain a mixture of specimens bearing damage characteristic of crocodiles and mammalian carnivores. Comparison of Plio-Pleistocene fossil bones from Olduvai Gorge, Tanzania, to bones damaged by captive and free-ranging Nile crocodiles reveals direct evidence of fossil crocodilian feeding from larger mammal bones associated with Oldowan stone artifacts.     

Molecular assessment of the genetic integrity,distinctiveness and phylogeographic context of the Saltwater crocodile (<Emphasis Type="Italic">Crocodylus porosus</Emphasis>) on Palau

The saltwater crocodile (Crocodylus porosus) is the largest and most broadly distributed crocodilian species, and thus is of special conservation and economic interest. Similar to other parts of its range throughout the Indo-Pacific, C. porosus distributed in the Republic of Palau have experienced a severe population decline over the past century primarily due to commercial hunting and eradication campaigns. In addition, several thousand crocodiles of undocumented species and origin were imported into Palau during the 1930’s for commercial farming purposes, potentially polluting the gene pool of the endemic saltwater crocodiles. Analysis of 39 individuals collected throughout the Republic of Palau revealed a single mitochondrial DNA control region haplotype shared by populations sampled in Sulawesi, Borneo and Australia. The mtDNA results, in combination with microsatellite genotypic data at six loci, detected no evidence for inter-specific hybridization between endemic Palauan C. porosus and potentially introduced Crocodylus species. There was no evidence for a genetic bottleneck in the Palauan population, however an excess of rare alleles was identified, indirectly suggesting a recent history of admixture potentially linked to introductions of non-native C. porosus. Following from these findings, Palauan C. porosus should be included in the single ESU previously established for all saltwater crocodiles given the recovery of a fixed, but geographically widespread haplotype. Although Palauan C. porosus exhibited significant genetic differentiation relative to all other sampled populations, it’s delineation as a distinct management unit is precluded at the present time by evidence that the genetic integrity of the population may have been compromised by the introduction of non-native saltwater crocodiles.     

Renivermis crocodyli (Digenea: Exotidendriidae) n. g., n. sp. from the kidneys of the saltwater crocodileCrocodylus porosus in Australia

Renivermis crocodyli, n. g., n. sp., is described from the kidneys of the saltwater crocodileCrocodylus porosus Schneider from northern Australia. The genusRenivermis is grouped withExotidendrium Mehra, 1935 andSimhatrema Chattopadhyaya, 1970 in the family Exotidendriidae Mehra, 1935. An amended diagnosis of the family and a key to its genera are given.     

Exhaustive exercise training enhances aerobic capacity in American alligator (<Emphasis Type="Italic">Alligator mississippiensis</Emphasis>)

The oxygen transport system in mammals is extensively remodelled in response to repeated bouts of activity, but many reptiles appear to be ‘metabolically inflexible’ in response to exercise training. A recent report showed that estuarine crocodiles (Crocodylus porosus) increase their maximum metabolic rate in response to exhaustive treadmill training, and in the present study, we confirm this response in another crocodilian, American alligator (Alligator mississippiensis). We further specify the nature of the crocodilian training response by analysing effects of training on aerobic [citrate synthase (CS)] and anaerobic [lactate dehydrogenase (LDH)] enzyme activities in selected skeletal muscles, ventricular and skeletal muscle masses and haematocrit. Compared to sedentary control animals, alligators regularly trained for 15 months on a treadmill (run group) or in a flume (swim group) exhibited peak oxygen consumption rates higher by 27 and 16%, respectively. Run and swim exercise training significantly increased ventricular mass (~11%) and haematocrit (~11%), but not the mass of skeletal muscles. However, exercise training did not alter CS or LDH activities of skeletal muscles. Similar to mammals, alligators respond to exercise training by increasing convective oxygen transport mechanisms, specifically heart size (potentially greater stroke volume) and haematocrit (increased oxygen carrying-capacity of the blood). Unlike mammals, but similar to squamate reptiles, alligators do not also increase citrate synthase activity of the skeletal muscles in response to exercise.     

New haplotype of the complete mitochondrial genome of <Emphasis Type="Italic">Crocodylus siamensis</Emphasis> and its species-specific DNA markers: distinguishing <Emphasis Type="Italic">C. siamensis</Emphasis> from <Emphasis Type="Italic">C. porosus</Emphasis> in Thailand

Based on molecular phylogeny of available complete mitochondrial DNA (mtDNA) genome sequences reveals that Crocodylus siamensis and C. porosus are closely related species. Yet, the sequence divergence of their mtDNA showed only a few values under conspecific level. In this study, a new haplotype (haplotype2, EF581859) of the complete mtDNA genome of Siamese crocodile (C. siamensis) was determined. The genome organization, which appeared to be highly similar to haplotype1 (DQ353946) mtDNA genome of C. siamensis, was 16,814 bp in length. However, the sequence divergence between the two genomes differed by around 7–10 and 0.7–2.1% for the haplotype1 between C. siamensis and C. porosus (AJ810453). These results were consistent with the phylogenetic relationship among the three genomes, suggesting that C. siamensis haplotype1 mtDNA genome might be the hybrid or the intraspecific variation of C. porosus. On the other hand, our specimen was found to be a true C. siamensis. Simultaneously, the seven species-specific DNA markers designed based on the distinctive site between haplotype2 mtDNA sequences of C. siamensis and haplotype1 mtDNA sequence of C. siamensis–C. porosus were successfully used to distinguish C. siamensis from C. porosus. These effective markers could be used primarily for rapid and accurate species identification in population, ecology and conservation studies.     

Crocodylian Snouts in Space and Time: Phylogenetic Approaches Toward Adaptive Radiation

Recent phylogenetic analyses of fossil and living crocodyliansallow us to compare the taxonomic, geographic, and temporaldistributions of morphological features, such as snout shapes.A few basic snout morphotypes—generalized, blunt, slender,deep, and excessively broad ("duck-faced")—occur multipletimes in distantly-related lineages. Some clades—especiallythose found in the Northern Hemisphere or with minimum originationdates in the Cretaceous or lower Tertiary—are morphologicallyuniform, but geographically widespread; crocodylian faunas ofthe early Tertiary tend to be composite, with sympatric taxabeing distantly related, and similar-looking taxa on differentcontinents being close relatives. In contrast, crocodylian faunasof the later Tertiary tend to be more endemic, with local adaptiveradiations occurring in Africa and Australia containing membersof most basic snout shapes. Endemic radiations in Africa andAustralia have largely been replaced by Crocodylus, which canbe divided into subclades that may individually represent endemicadaptive radiations.     

Trackways of the American Crocodile (Crocodylus acutus) in Northwestern Costa Rica: Implications for Crocodylian Ichnology

We documented trackways of free-living Crocodylus acutus on beaches at the mouths of Tamarindo and Ventanas estuaries, Costa Rica. Our crocodiles had estimated total lengths of 1–3 meters or more. Manus prints have five digits, with digits I–III bearing claw marks. Pes prints have four digits, with claw marks on digits I–III. The pes is plantigrade. Claws generally dig into the substrate. Apart from claw marks, digit I and the heel of the pes are usually the most deeply impressed parts of footprints. Trackways are wide-gauge. Pes prints are usually positioned just behind ipsilateral manus prints of the same set and may overlap them. Manus and pes prints angle slightly outward with respect to the crocodile's direction of movement. Claw-bearing digits of both the manus and pes may create curved, concave-toward-the-midline drag marks as the autopodium is protracted. The tail mark varies in depth and clarity, and in shape from nearly linear to markedly sinuous. Sometimes the tail mark hugs the trackway midline, but sometimes it is closer to, or even cuts across, prints of one side. American crocodile footprints and trackways are similar to those observed in other extant crocodylian species, indicating substantial trackway conservatism across the group.     

First European evidence for transcontinental dispersal of Crocodylus (late Neogene of southern Italy)

It is generally assumed that the Neogene crocodylian fauna of Europe has been represented only by brevirostrine alligatoroid Diplocynodon and longirostrine false gharials ( Gavialosuchus and/or Tomistoma ), which became extinct prior to 6 Mya. Although several lines of evidence suggest that Crocodylus originated in Africa during the Miocene and then promptly dispersed to other continents, the occurrence of this genus in Europe has never been rigorously proven and the traditional palaeontological approach failed to identify a monophyletic group of fossil Crocodylus (simply leading to a proliferation of extinct taxa). The new remains reported here, from an endemic insular fauna from southern Italy, Late Messinian to earliest Pliocene in age (5–6 million years old), represent the youngest European crocodylian, and allow, for the first time in a phylogenetic context, an unambiguous demonstration that Crocodylus dispersed into Europe, possibly during the Tortonian. If the peculiar morphology of the medial maxillary edge is interpreted as evidence for a medial dorsal boss, the southern Italian Crocodylus could be related to C. checchiai from the late Neogene of Libya. The presence of this African immigrant in Europe confirms the role of climate change for faunal dispersal and island colonization.  © 2007 The Linnean Society of London, Zoological Journal of the Linnean Society , 2007, 149 , 293–307.     

Echinoid test damage by a stingray predator

Stingrays (Order: Myliobatiformes) are well‐known predators that feed on a variety of marine species including crustaceans, teleosts, molluscs and annelids. Here, a predatory attack by a stingray on the large spatangoid echinoid Meoma ventricosa (Lamarck) is reported from the shallow‐water carbonate platform of San Salvador, Bahamas. A single stingray was observed feeding on a large adult specimen of M. ventricosa near Sand Dollar Beach, San Salvador Island. The partially crushed and partially eviscerated test of the attacked echinoid was collected from the feeding site. In addition, a crushed test of a freshly killed, smaller irregular clypeasteroid echinoid Leodia sexiesperforata (Leske) was also recovered at the same site. In both cases, the test was broken across the test plates and not along plate boundaries. Meoma ventricosa showed major plate loss on the oral side; however, bite traces and spine loss were not observed in the damaged areas. Similarly, L. sexiesperforata showed no evidence of spine removal; however, bite marks were identifiable. The observed test damage in both taxa is morphologically distinct and could be potentially identifiable in the fossil record.     

Effects of Incubation Temperature on Crocodiles and the Evolution of Reptilian Oviparity

Crocodylus porosus is a mound-nesting crocodilian in which incubationtemperature influences the rate of embryonic development, theprobability that embryos will survive to hatching, post-hatchinggrowth rates and the probability of hatchlings surviving to2 yr of age. Similar responses have been described in Alligatormississippiensis (Joanen et al., 1987) and C. niloticus (Hutton,1987), and they reflect a suite of "non-sexual" effects of incubationtemperature. Temperature-dependent sex determination allocatessex on the basis of these "non-sexual" effects. In C. porosus,it results in maleness being assigned to embryos with high probabilitiesof surviving and good potential for post-hatching growth. Withinthe limits of survival, effects of the moisture environmenton embryological development rate and hatchling fitness seemminor relative to those of the temperature environment. Reptilian orders have either obligate oviparity (chelonians,crocodilians and rhynchocephalians) or facultative oviparity(squamates), depending on the extent of embryonic developmentwithin the oviducts. The distinction is equally one betweenembryos which are buffered from thermal effects within a female'sbody (facultative oviparity) and those that are not (obligateoviparity). Facultative oviparity and internal thermal bufferingmay be the primitive condition within the Class Reptilia, andthe "shell-less" eggs of extant squamates may reflect the originalamniote egg. Obligate oviparity, which also exists in birds,appears to have been a specialized development, and is a blindend in the evolution of viviparity among vertebrates. The significanceof thermal buffering being lost in obligate oviparous reptilesremains unclear.     

A time-calibrated species tree of Crocodylia reveals a recent radiation of the true crocodiles

True crocodiles (Crocodylus) are the most broadly distributed, ecologically diverse, and species-rich crocodylian genus, comprising about half of extant crocodylian diversity and exhibiting a circumtropical distribution. Crocodylus traditionally has been viewed as an ancient group of morphologically conserved species that originated in Africa prior to continental breakup. In this study, these long-held notions about the temporal and geographic origin of Crocodylus are tested using DNA sequence data of 10 loci from 76 individuals representing all 23 crocodylian species. I infer a time-calibrated species tree of all Crocodylia and estimate the spatial pattern of diversification within Crocodylus. For the first time, a fully resolved phylogenetic estimate of all Crocodylia is well-supported. The results overturn traditional views of the evolution of Crocodylus by demonstrating that the true crocodiles are not "living-fossils" that originated in Africa. Rather, Crocodylus originated from an ancestor in the tropics of the Late Miocene Indo-Pacific, and rapidly radiated and dispersed around the globe during a period marked by mass extinctions of fellow crocodylians. The findings also reveal more diversity within the genus than is recognized by current taxonomy.