Evidence for placing the false gharial (Tomistoma schlegelii) into the family Gavialidae: inferences from nuclear gene sequences

The extant crocodylians comprise 23 species divided among three families, Alligatoridae, Crocodylidae, and Gavialidae. Currently, based on morphological data sets, Tomistoma schlegelii (false gharial) is placed within the family Crocodylidae. Molecular data sets consistently support a sister-taxon relationship of T. schlegelii with Gavialis gangeticus (Indian Gharial), which is the sole species in Gavialidae. To elucidate the placement of T. schlegelii within the extant crocodylians, we have sequenced 352bp of the dentin matrix protein 1 (DMP1) nuclear gene in 30 individuals and 424bp of the nuclear gene C-mos in 74 individuals. Molecular analysis of the DMP1 data set indicates that it is highly conserved within the Crocodylia. Of special note is a seven base-pair indel (GTGCTTT) shared by T. schlegelii and G. gangeticus, that is absent in the genus Crocodylus, Osteolaemus, and Mecistops. To date, C-mos is the largest molecular data set analyzed for any crocodylian study including multiple samples from all representatives of the eight extant genera. Analysis of these molecular data sets, both as individual gene sequences and concatenated sequences, support the hypothesis that T. schlegelii should be placed within the family Gavialidae.     

The crocodilian mitochondrial control region: general structure,conserved sequences,and evolutionary implications

We present the first comprehensive analysis of the crocodilian control region. We have analyzed sequences from all three families of Crocodylia (Crocodylidae, Gavialidae, Alligatoridae), incorporating all genera except Paleosuchus and Melanosuchus. Within the control region of other vertebrates, several sequence motifs and their order appear to be conserved. Herein, we compare aligned crocodilian D-loop sequences to homologous sequences from other vertebrates ranging from fish to birds. Among other findings, we have discovered that while domain I tends to be shorter than the same region in mammals and birds, it contains sequences similar in structure to both the goose-hairpin and termination associated sequences (TAS). Domain II is highly conservative with regard to size among the taxa examined and contains several of the conserved sequence boxes characterized in other vertebrates. Domain III contains several interesting sequence motifs including tandemly repeated sequences, a long poly-A region in the Crocodylidae, and possible bidirection promoter sequences.     

Extended mitogenomic phylogenetic analyses yield new insight into crocodylian evolution and their survival of the Cretaceous-Tertiary boundary

The mitochondrial genomes of the dwarf crocodile, Osteolaemus tetraspis, and two species of dwarf caimans, the smooth-fronted caiman, Paleosuchus trigonatus, and Cuvier's dwarf caiman, Paleosuchus palpebrosus, were sequenced and included in a mitogenomic phylogenetic study. The phylogenetic analyses, which included a total of ten crocodylian species, yielded strong support to a basal split between Crocodylidae and Alligatoridae. Osteolaemus fell within the Crocodylidae as the sister group to Crocodylus. Gavialis and Tomistoma, which joined on a common branch, constituted a sister group to Crocodylus/Osteolaemus. This suggests that extant crocodylians are organized in two families: Alligatoridae and Crocodylidae. Within the Alligatoridae there was a basal split between Alligator and a branch that contained Paleosuchus and Caiman. The analyses also provided molecular estimates of various divergences applying recently established crocodylian and outgroup fossil calibration points. Molecular estimates based on amino acid data placed the divergence between Crocodylidae and Alligatoridae at 97-103 million years ago and that between Alligator and Caiman/Paleosuchus at 65-72 million years ago. Other crocodilian divergences were placed after the Cretaceous-Tertiary boundary. Thus, according to the molecular estimates, three extant crocodylian lineages have their roots in the Cretaceous. Considering the crocodylian diversification in the Cretaceous the molecular datings suggest that the extinction of the dinosaurs was also to some extent paralleled in the crocodylian evolution. However, for whatever reason, some crocodylian lineages survived into the Tertiary.     

Crocodilian evolution: insights from immunological data.

The quantitative immunological technique of microcomplement fixation was used to examine serum albumin evolution among members of the order Crocodylia. The cross-reactivity of the albumin antisera and antigens employed in this study had been examined previously using the qualitative technique of immunodiffusion. The phylogenetic conclusions derived from these two data sets are highly congruent, including support of the families Alligatoridae and Crocodylidae, with the placement of Gavialis as the sister taxon of Tomistoma. Both methods provide similar information on the relative amounts of amino acid sequence divergence between albumin molecules; however, the data obtained from microcomplement fixation comparisons are more discriminating than those derived from immunodiffusion. The estimated divergence times within the Crocodylia derived from the fossil record are examined in light of divergence times predicted by the microcomplement fixation-based albumin clock. The traditional phylogenetic placement of Gavialis outside the remaining extant crocodilians is inconsistent with all molecular data sets and we suggest that a careful reexamination of both the extant and the fossil morphological data is warranted.     

Crocodilian phylogeny inferred from twelve mitochondrial protein-coding genes, with new complete mitochondrial genomic sequences for Crocodylus acutus and Crocodylus novaeguineae

We report complete mitochondrial genomic sequences for Crocodylus acutus and Crocodylus novaeguineae, whose gene orders match those of other crocodilians. Phylogenetic analyses based on the sequences of 12 mitochondrial protein-coding genes support monophyly of two crocodilian taxonomic families, Alligatoridae (genera Alligator, Caiman, and Paleosuchus) and Crocodylidae (genera Crocodylus, Gavialis, Mecistops, Osteolaemus, and Tomistoma). Our results are consistent with monophyly of all crocodilian genera. Within Alligatoridae, genus Alligator is the sister taxon of a clade comprising Caiman and Paleosuchus. Within Crocodylidae, the basal phylogenetic split separates a clade comprising Gavialis and Tomistoma from a clade comprising Crocodylus, Mecistops, and Osteolaemus. Mecistops and Osteolaemus form the sister taxon to Crocodylus. Within Crocodylus, we sampled five Indopacific species, whose phylogenetic ordering is ((C. mindorensis, C. novaeguineae), (C. porosus, (C. siamensis, C. palustris))). The African species C. niloticus and New World species C. acutus form the sister taxon to the Indopacific species, although our sampling lacks three other New World species and an Australian species of Crocodylus.     

An Expanded Combined Evidence Approach to the Gavialis Problem Using Geometric Morphometric Data from Crocodylian Braincases and Eustachian Systems

The phylogenetic position of the Indian gharial (Gavialis gangeticus) is disputed - morphological characters place Gavialis as the sister to all other extant crocodylians, whereas molecular and combined analyses find Gavialis and the false gharial (Tomistoma schlegelii) to be sister taxa. Geometric morphometric techniques have only begun to be applied to this issue, but most of these studies have focused on the exterior of the skull. The braincase has provided useful phylogenetic information for basal crurotarsans, but has not been explored for the crown group. The Eustachian system is thought to vary phylogenetically in Crocodylia, but has not been analytically tested. To determine if gross morphology of the crocodylian braincase proves informative to the relationships of Gavialis and Tomistoma, we used two- and three-dimensional geometric morphometric approaches. Internal braincase images were obtained using high-resolution computerized tomography scans. A principal components analysis identified that the first component axis was primarily associated with size and did not show groupings that divide the specimens by phylogenetic affinity. Sliding semi-landmarks and a relative warp analysis indicate that a unique Eustachian morphology separates Gavialis from other extant members of Crocodylia. Ontogenetic expansion of the braincase results in a more dorsoventrally elongate median Eustachian canal. Changes in the shape of the Eustachian system do provide phylogenetic distinctions between major crocodylian clades. Each morphometric dataset, consisting of continuous morphological characters, was added independently to a combined cladistic analysis of discrete morphological and molecular characters. The braincase data alone produced a clade that included crocodylids and Gavialis, whereas the Eustachian data resulted in Gavialis being considered a basally divergent lineage. When each morphometric dataset was used in a combined analysis with discrete morphological and molecular characters, it generated a tree that matched the topology of the molecular phylogeny of Crocodylia.     

True and false gharials: a nuclear gene phylogeny of crocodylia

The phylogeny of Crocodylia offers an unusual twist on the usual molecules versus morphology story. The true gharial (Gavialis gangeticus) and the false gharial (Tomistoma schlegelii), as their common names imply, have appeared in all cladistic morphological analyses as distantly related species, convergent upon a similar morphology. In contrast, all previous molecular studies have shown them to be sister taxa. We present the first phylogenetic study of Crocodylia using a nuclear gene. We cloned and sequenced the c-myc proto-oncogene from Alligator mississippiensis to facilitate primer design and then sequenced an 1,100-base pair fragment that includes both coding and noncoding regions and informative indels for one species in each extant crocodylian genus and six avian outgroups. Phylogenetic analyses using parsimony, maximum likelihood, and Bayesian inference all strongly agreed on the same tree, which is identical to the tree found in previous molecular analyses: Gavialis and Tomistoma are sister taxa and together are the sister group of Crocodylidae. Kishino-Hasegawa tests rejected the morphological tree in favor of the molecular tree. We excluded long-branch attraction and variation in base composition among taxa as explanations for this topology. To explore the causes of discrepancy between molecular and morphological estimates of crocodylian phylogeny, we examined puzzling features of the morphological data using a priori partitions of the data based on anatomical regions and investigated the effects of different coding schemes for two obvious morphological similarities of the two gharials.     

Evolution of MHC class I in the Order Crocodylia

The major histocompatibility complex (MHC) is a dynamic genomic region with an essential role in the adaptive immunity of jawed vertebrates. The evolution of the MHC has been dominated by gene duplication and gene loss, commonly known as the birth-and-death process. Evolutionary studies of the MHC have mostly focused on model species. However, the investigation of this region in non-avian reptiles is still in its infancy. To provide insights into the evolutionary mechanisms that have shaped the diversity of this region in the Order Crocodylia, we investigated MHC class I exon 3, intron 3, and exon 4 across 20 species of the families Alligatoridae and Crocodilidae. We generated 124 DNA sequences and identified 31 putative functional variants as well as 14 null variants. Phylogenetic analyses revealed three gene groups, all of which were present in Crocodilidae but only one in Alligatoridae. Within these groups, variants generally appear to cluster at the genus or family level rather than in species-specific groups. In addition, we found variation in gene copy number and some indication of interlocus recombination. These results suggest that MHC class I in Crocodylia underwent independent events of gene duplication, particularly in Crocodilidae. These findings enhance our understanding of MHC class I evolution and provide a preliminary framework for comparative studies of other non-avian reptiles as well as diversity assessment within Crocodylia.     

湾鳄(Crocodylus porosus)线粒体基因组全序列结构分析与鳄类系统发生

该文测序了湾鳄的线粒体基因组全序列,全长为16,917bp。湾鳄mtDNA结构与其他脊椎动物相似,由22个tRNA,2个rRNA和13个蛋白质编码基因及1个非编码的控制区(D-loop)所组成。除NADH6和tRNAGln、tRNAAla、tRNAAsn、tRNACys、tRNATyr、tRNASer(UCN)、tRNAGlu、tRNAPro在L-链上编码之外,其余基因均在H-链编码。基因排列顺序与已测序的鳄类一致,这显示了鳄类线粒体基因排列顺序上的保守性。但鳄类线粒体基因排列顺序与脊椎动物的典型排列方式相比,有较大的差异,尤其是tRNAPhe基因的重排、tRNASer-tRNAHis-tRNALeu基因族的排列方式等。湾鳄mtDNA和已测序的鳄类一样,缺失轻链复制起始点(OLR)。基于17种鳄mtDNA控制区保守区,采用PAUP4.0最大简约法(Maximumparsimony,MP)构建MP树,邻接法(Neighbor-joiningmethod,NJ)构建NJ树,结果显示:食鱼鳄(Gavialisgangeticus)和假食鱼鳄(Tomistomaschlegelii)聚为一支后再与鳄科(Crocodylidae)的其他物种形成姐妹群,这与基于食鱼鳄和假食鱼鳄的线粒体全序列的分析结果一致,支持将食鱼鳄并入鳄科的观点。结果还支持非洲窄吻鳄(Crocodyluscataphractus)与鳄属(Crocodylus)构成姐妹群,可以单独划分为属的观点。     

Historical Zoogeography of the Eusuchian Crocodilians: A Physiological Perspective

The historical zoogeography of eusuchian crocodilians has rarelybeen reviewed in any detail and yet is of increasing interestto students of crocodilian biology as large amounts of comparativeinformation on a wide range of species come to hand. Previousinterpretations of crocodilian zoogeography have been basedon one or another of two assumptions–that the major continentalland masses have remained more or less fixed in position, andthat the eusuchians have had only very limited powers of dispersalacross marine barriers. Both of these assumptions are inappropriatein light of our present knowledge of continental drift and crocodilianphysiology. In this paper we attempt a reinterpretation of eusuchian zoogeographybased on new information on their systematic relationships,physiological capacity for marine dispersal, and fossil history.We postulate that anatomical and physiological adaptations toa marine existence have played an important role in eusuchianhistory. We propose that Gavialis and Tomistoma, now restrictedto fresh waters, may have been derived secondarily from ancestorsadapted to salt water. In the case of Tomistoma, similaritiesin lingual gland and buccal cavity anatomy to the true crocodiles(Crocodylus and Osteolaemus) suggest that marine adaptationspredated the divergence of tomistomine and crocodyline stocks.The buccal morphology of Gavialis suggests it also has a marineancestry. Its systematic affinities are uncertain, lying perhapswith Tomistoma or, on other interpretations, with the Mesosuchia.In both cases, the fossil record is not inconsistent with thispossibility. Palaeontological information now available is inadequate toreconstruct the evolutionary history of the Eusuchia in detail.However, saltwater adapted eusuchians are more common in thefossil record than is widely recognized and the likelihood ofdispersal across marine "barriers" by non-alligatorid crocodilianscannot be ignored.     

Patterns of morphospace occupation and mechanical performance in extant crocodilian skulls: A combined geometric morphometric and finite element modeling approach

Extant and fossil crocodilians have long been divided into taxonomic and/or ecological groups based on broad patterns of skull shape, particularly the relative length and width of the snout. However, these patterns have not been quantitatively analyzed in detail, and their biomechanical and functional implications are similarly understudied. Here, we use geometric morphometrics and finite element analysis to explore the patterns of variation in crocodilian skull morphology and the functional implications of those patterns. Our results indicate that skull shape variation in extant crocodiles is much more complex than previously recognized. Differences in snout length and width are the main components of shape variation, but these differences are correlated with changes in other regions of the skull. Additionally, there is considerable disparity within general classes such as longirostrine and brevirostrine forms. For example, Gavialis and Tomistoma occupy different parts of morphospace implying a significant difference in skull shape, despite the fact that both are traditionally considered longirostrine. Skull length and width also strongly influence the mechanical performance of the skull; long and narrow morphotypes (e.g., Tomistoma) experience the highest amount of stress during biting, whereas short and broad morphotypes (e.g., Caiman latirostris) experience the least amount of stress. Biomechanical stress and the hydrodynamic properties of the skull show a strong relationship with the distribution of crocodilians in skull morphospace, whereas phylogeny and biogeography show weak or no correlation. Therefore, ecological specializations related to feeding and foraging likely have the greatest influence on crocodilian skull shape. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

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.     

Pectoral girdle and forelimb variation in extant Crocodylia: the coracoid–humerus pair as an evolutionary module

To date, all statements about evolutionary morphological transformation in Crocodylia have essentially been based on qualitative observations. In the present study, we assessed the morphological variation and covariation (integration) between the scapula, coracoid, humerus, radius, and ulna of 15 species of Crocodylidae, Alligatoridae, and Gavialis + Tomistoma using three‐dimensional geometric morphometrics. The results obtained reveal that the variation of elements within species (intraspecific) is large. However, despite this variability, variation across species (interspecific) is mainly concentrated in two dimensions where the disparity is constrained: ‘robusticity’ and ‘twist’ (forelimbs) and ‘robusticity’ and ‘flexion’ (pectoral girdle). Robusticity (first dimension of variation) embodies a set of correlated geometrical features such as the broadening of the girdle heads and blades, or the enlargement of proximal and distal bone ends. The twist is related to the proximal and/or distal epiphyses in the forelimb elements, and flexion of the scapula and coracoid blades comprises the second dimension of variation. In all crocodylians, forelimb integration is characterized by the strong correlations of a humerus–ulna–radius triad and by a radius–ulna pair, thus forming a tight forelimb module. Unexpectedly, we found that the humerus and coracoid form the most integrated pair, whereas the scapula is a more variable and relatively independent element. The integration pattern of the humerus–coracoid pair distinguishes a relatively robust configuration in alligatorids from that of the remainder groups. The patterns of variation and integration shared by all the analyzed species have been interpreted as an inherited factor, suggesting that developmental and functional requirements would have interacted in the acquisition of a semi‐aquatic and versatile locomotion at the Crocodylia node at least 65 Mya. Our findings highlight the need to incorporate the humerus–coracoid pair in biodynamic and biomechanical studies. © 2012 The Linnean Society of London     

Morphology, fossils, divergence timing, and the phylogenetic relationships of Gavialis

Although morphological data have historically favored a basal position for the Indian gharial (Gavialis gangeticus) within Crocodylia and a Mesozoic divergence between Gavialis and all other crocodylians, several recent molecular data sets have argued for a sister-group relationship between Gavialis and the Indonesian false gharial (Tomistoma schlegelii) and a divergence between them no earlier than the Late Tertiary. Fossils were added to a matrix of 164 discrete morphological characters and subjected to parsimony analysis. When morphology was analyzed alone, Gavialis was the sister taxon of all other extant crocodylians whether or not fossil ingroup taxa were included, and a sister-group relationship between Gavialis and Tomistoma was significantly less parsimonious. In combination with published sequence and restriction site fragment data, Gavialis was the sister taxon of all other living crocodylians, but the position of Tomistoma depended on the inclusion of fossil ingroup taxa; with or without fossils, preferred morphological and molecular topologies were not significantly different. Fossils closer to Gavialis than to Tomistoma can be recognized in the Late Cretaceous, and fossil relatives of Tomistoma are known from the basal Eocene, strongly indicating a divergence long before the Late Tertiary. Comparison of minimum divergence time from the fossil record with different measures of molecular distance indicates evolutionary rate heterogeneity within Crocodylia. Fossils strongly contradict a post-Oligocene divergence between Gavialis and any other living crocodylian, but the phylogenetic placement of Gavialis is best viewed as unresolved.     

Dinosaurs: a field guide/The Princeton field guide to dinosaurs

Eggshells from the three extant crocodilian species Crocodylus mindorensis (Philippine Crocodile), Paleosuchus palpebrosus (Cuvier's Smooth-fronted Caiman or Musky Caiman) and Alligator mississippiensis (American Alligator or Common Alligator) were prepared for thin section and scanning electron microscope analyses and are described in order to improve the knowledge on crocodilian eggs anatomy and microstructure, and to find new apomorphies that can be used for identification. Both extant and fossil crocodilian eggs present an ornamentation that vary as anastomo-, ramo- or the here newly described rugosocavate type. The angusticaniculate pore system is a shared character for Crocodylomorpha eggshells and some dinosaurian and avian groups. Previously reported signs of incubated crocodilian eggs were found also on our only fertilised and hatched egg. Paleosuchus palpebrosus presents unique organization and morphology of the three eggshell layers, with a relatively thin middle layer characterised by dense and compact tabular microstructure.     

A bacterial artificial chromosome library for the Australian saltwater crocodile (<Emphasis Type="Italic">Crocodylus porosus</Emphasis>) and its utilization in gene isolation and genome characterization

Background

Crocodilians (Order Crocodylia) are an ancient vertebrate group of tremendous ecological, social, and evolutionary importance. They are the only extant reptilian members of Archosauria, a monophyletic group that also includes birds, dinosaurs, and pterosaurs. Consequently, crocodilian genomes represent a gateway through which the molecular evolution of avian lineages can be explored. To facilitate comparative genomics within Crocodylia and between crocodilians and other archosaurs, we have constructed a bacterial artificial chromosome (BAC) library for the Australian saltwater crocodile, Crocodylus porosus. This is the first BAC library for a crocodile and only the second BAC resource for a crocodilian.

Results

The C. porosus BAC library consists of 101,760 individually archived clones stored in 384-well microtiter plates. Not I digestion of random clones indicates an average insert size of 102 kb. Based on a genome size estimate of 2778 Mb, the library affords 3.7 fold (3.7×) coverage of the C. porosus genome. To investigate the utility of the library in studying sequence distribution, probes derived from CR1a and CR1b, two crocodilian CR1-like retrotransposon subfamilies, were hybridized to C. porosus macroarrays. The results indicate that there are a minimum of 20,000 CR1a/b elements in C. porosus and that their distribution throughout the genome is decidedly non-random. To demonstrate the utility of the library in gene isolation, we probed the C. porosus macroarrays with an overgo designed from a C-mos (oocyte maturation factor) partial cDNA. A BAC containing C-mos was identified and the C-mos locus was sequenced. Nucleotide and amino acid sequence alignment of the C. porosus C-mos coding sequence with avian and reptilian C-mos orthologs reveals greater sequence similarity between C. porosus and birds (specifically chicken and zebra finch) than between C. porosus and squamates (green anole).

Conclusion

We have demonstrated the utility of the Crocodylus porosus BAC library as a tool in genomics research. The BAC library should expedite complete genome sequencing of C. porosus and facilitate detailed analysis of genome evolution within Crocodylia and between crocodilians and diverse amniote lineages including birds, mammals, and other non-avian reptiles.

     

A new Indo-Malayan member of the Stenostiridae (Aves: Passeriformes) revealed by multilocus sequence data: Biogeographical implications for a morphologically diverse clade of flycatchers

Recent molecular studies on passerine birds have highlighted numerous discrepancies between traditional classification and the phylogenetic relationships recovered from sequence data. Among the traditional families that were shown to be highly polyphyletic are the Muscicapidae Old World flycatcher. This family formerly included all Old World passerines that forage on small insects by performing short sallies from a perch. Genera previously allocated to the Muscicapidae are now thought to belong to at least seven unrelated lineages. While the peculiarity of most of these lineages has been previously recognized by Linnean classification, usually at the rank of families, one, the so-called Stenostiridae, a clade comprising three Afrotropical and Indo-Malayan genera, has only recently been discovered. Here, we address in greater detail the phylogenetic relationships and biogeographic history of the Stenostiridae using a combination of mitochondrial and nuclear data. Our analyses revealed that one species, Rhipidura hypoxantha, previously attributed to the Rhipiduridae (fantails), is in fact a member of the Stenostiridae radiation and sister to the South African endemic genus Stenostira (Fairy Flycatcher). Our dating analyses, performed in a relative-time framework, suggest that the splits between Stenostira/R. hypoxantha and Culicicapa/Elminia occurred synchronously. Given that the Stenostiridae assemblage has been consistently recovered by independent studies, we clarify its taxonomic validity under the rules of the International Code of Zoological Nomenclature.     

Italian Cenozoic crocodilians: taxa, timing and palaeobiogeographic implications

Crocodylian remains are collected in 39 fossil-bearing localities but only in seven localities specimens with reliable taxonomic attributions, at least to genus level have been collected. Three species have been reported from the early Lutetian Purga di Bolca site: Pristichampsus cf. Pristichampsus rollinati, Asiatosuchus sp., Hassiacosuchus sp. (=Allognathosuchus sp.). The three crocodilians discovered at Purga di Bolca have been reported also from Geiseltal and Messel (Middle Eocene, Germany). Bolca at that time was part of a Tethysian archipelago and no mammals have been found there till now. Crocodilians and turtles clearly arrived from the European mainland across a marine water barrier. Among the other fossiliferous localities of Veneto, very interesting is the Monte Zuello site, of late Middle Eocene age, yielding a longirostrine crocodilian, Megadontosuchus arduini, a tomistomine species. Tomistomines are known in contemporaneous sediments of both Europe and Africa, but the European forms Dollosuchus and Kentisuchus seem the closest taxa. Remains of Oligocene age have been collected in Veneto and Liguria, but the fossils discovered in the second region are teeth or fragmented bones. The fossil crocodilians of Monteviale (Veneto), of Early Oligocene age, have been assigned to two species but they have been recently all identified as Diplocynodon ratelii, known from several European sites of Late Eocene, Oligocene and Miocene age. This species arrived in the Monteviale area from the European mainland across a narrow sea. Several crocodilian fossils of Miocene age are very fragmentary or represented by isolated teeth. In the Middle and Late Miocene of Sardinia, a well-established species, Tomistoma calaritanum is present. Remains of Tomistoma of the same age have been reported in some localities in Tuscany, Apulia, Sicily and Malta. In the Mediterranean area, the genus is known from European and African sites (of older age). The colonisation of Europe by this genus is the result of a dispersion from Africa (or less probably from Asia). During Late Miocene Sardinia and Tuscany belong to the same palaeobioprovince characterized by the Oreopithecus-Maremmia fauna. In Tuscany, a crocodilian identified as Crocodylus bambolii is present in the late Miocene site of Monte Bamboli. If the generic attribution of this form is correct, its ancestors must have arrived from Africa. Another fossil assemblage of Late Miocene age characterizes the Apulia-Abruzzi palaeobioprovince (Hoplitomeryx-Microtia fauna) and testifies complete isolation between the two palaeobioprovinces. In this last area, remains of Crocodylus sp. have been collected in coastal sandstones at Scontrone (Abruzzi) and in several fissure fillings of Gargano of slightly younger age. The ancestors of this species arrived from Africa while no African elements are present among the mammalian fauna. The dispersion of the genus Crocodylus in the Italian palaeoislands may have taken place once, with allopatric differentiation of the two populations (Tuscany-Sardinia and Apulia-Abruzzi) or twice with independent colonisation of each area.     

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.