Insights into mammalian TE diversity through the curation of 248 genome assemblies.

We examined transposable element (TE) content of 248 placental mammal genome assemblies, the largest de novo TE curation effort in eukaryotes to date. We found that although mammals resemble one another in total TE content and diversity, they show substantial differences with regard to recent TE accumulation. This includes multiple recent expansion and quiescence events across the mammalian tree. Young TEs, particularly long interspersed elements, drive increases in genome size, whereas DNA transposons are associated with smaller genomes. Mammals tend to accumulate only a few types of TEs at any given time, with one TE type dominating. We also found association between dietary habit and the presence of DNA transposon invasions. These detailed annotations will serve as a benchmark for future comparative TE analyses among placental mammals.

Conservation status and regional habitat priorities for the Orinoco crocodile: Past, present, and future.

Conservation of large predator species has historically been a challenge because they often overlap in resource utilization with humans; furthermore, there is a general lack of in-depth knowledge of their ecology and natural history. We assessed the conservation status of the Orinoco crocodile (Crocodylus intermedius), defining regional habitat priorities/crocodile conservation units (RHP/CCU) and regional research priorities (RRP) for this species. We also estimated a species distribution model (SDM) to define current suitable areas where the species might inhabit and/or that might be successfully colonized. The SDM area obtained with a suitable habitat probability ≥ 0.5 was 23,621 km2. Out of 2,562 km2 are included within protected areas in both Colombia (1,643 km2) and Venezuela (919 km2), which represents only 10.8% of C. intermedius' potential range. Areas such as Laguna de Chigüichigüe (flood plain lagoon) exhibited an increase in population abundance. In contrast, localities such as the Cojedes and Manapire Rivers reported a significant reduction in relative abundance values. In Colombia, disparity in previous survey methods prevented accurate estimation of population trends. Only one study in this country described an increase over a 13 years span in the Ele, Lipa, and Cravo Norte River populations based on nest surveys. We defined 34 critical areas (16 in Colombia, 17 in Venezuela, and one covering both countries) where we need to preserve/research/monitor and/or generate management actions, 10 RHP/CCU (six from Venezuela and four from Colombia) and 24 RRP (11 from Venezuela, 12 from Colombia, and one in both countries). Caño Guaritico (Creek) and the Capanaparo River in Venezuela and the Ele, Lipa, Cravo Norte River System and the Guayabero River in Colombia were defined as areas with the most optimal conditions for long-term preservation and maintenance of C. intermedius populations. We conclude that the conservation status of this species is still critical, which implies the necessity to increase efforts to recover the species, especially in Colombia, to guarantee its survival as a structural and functional component of the ecosystems it inhabits.

Spatial Ecology of the American Crocodile in a Tropical Pacific Island in Central America.

Conservation of large predators has long been a challenge for biologists due to the limited information we have about their ecology, generally low numbers in the wild, large home ranges and the continuous expansion of human settlements. The American crocodile (Crocodylus acutus) is a typical apex predator, that has suffered from all of these characteristic problems, especially the latter one. Humans have had a major impact on the recovery of this species throughout its range, even though most of the countries it inhabits have banned hunting. The last decade has made it clear that in order to implement sound conservation and management programs, we must increase our understanding of crocodile spatial ecology. However, in only two countries where American crocodiles have telemetry studies even been published. Herein we have characterized the spatial ecology of C. acutus on Coiba Island, Panama, by radio-tracking (VHF transmitters) 24 individuals between 2010 and 2013, to determine movement patterns, home range, and habitat use. We have then compared our findings with those of previous studies to develop the most comprehensive assessment of American crocodile spatial ecology to date. Females showed a higher average movement distance (AMD) than males; similarly, adults showed a higher AMD than sub-adults and juveniles. However, males exhibited larger home ranges than females, and concomitantly sub-adults had larger home ranges than juveniles, hatchlings, and adults. There was an obvious relationship between seasonal precipitation and AMD, with increased AMD in the dry and "low-wet" seasons, and reduced AMD during the "true" wet season. We found disaggregate distributions according to age groups throughout the 9 habitat types in the study area; adults and hatchlings inhabited fewer habitat types than juveniles and sub-adults. These sex- and age-group discrepancies in movement and habitat choice are likely due to the influences of reproductive biology and Coiba's precipitation cycle. Juveniles also showed distinct movement patterns and home ranges; however, with sexual maturation and development, these behaviors became more characteristic of adults and sub-adults. Ours is one of a very small number of studies that will allow future management and conservation planning to be based on the comprehensive integration of the spatial ecology of a Neotropical crocodylian apex predator.

Sequencing three crocodilian genomes to illuminate the evolution of archosaurs and amniotes.

The International Crocodilian Genomes Working Group (ICGWG) will sequence and assemble the American alligator (Alligator mississippiensis), saltwater crocodile (Crocodylus porosus) and Indian gharial (Gavialis gangeticus) genomes. The status of these projects and our planned analyses are described.

A new horned crocodile from the Plio-Pleistocene hominid sites at Olduvai Gorge, Tanzania.

BACKGROUND: The fossil record reveals surprising crocodile diversity in the Neogene of Africa, but relationships with their living relatives and the biogeographic origins of the modern African crocodylian fauna are poorly understood. A Plio-Pleistocene crocodile from Olduvai Gorge, Tanzania, represents a new extinct species and shows that high crocodylian diversity in Africa persisted after the Miocene. It had prominent triangular "horns" over the ears and a relatively deep snout, these resemble those of the recently extinct Malagasy crocodile Voay robustus, but the new species lacks features found among osteolaemines and shares derived similarities with living species of Crocodylus. METHODOLOGY/PRINCIPAL FINDINGS: The holotype consists of a partial skull and skeleton and was collected on the surface between two tuffs dated to approximately 1.84 million years (Ma), in the same interval near the type localities for the hominids Homo habilis and Australopithecus boisei. It was compared with previously-collected material from Olduvai Gorge referable to the same species. Phylogenetic analysis places the new form within or adjacent to crown Crocodylus. CONCLUSIONS/SIGNIFICANCE: The new crocodile species was the largest predator encountered by our ancestors at Olduvai Gorge, as indicated by hominid specimens preserving crocodile bite marks from these sites. The new species also reinforces the emerging view of high crocodylian diversity throughout the Neogene, and it represents one of the few extinct species referable to crown genus Crocodylus.

Evidence of multiple paternity in Morelet's Crocodile (Crocodylus moreletii) in Belize, CA, inferred from microsatellite markers.

Microsatellite data were generated from hatchlings collected from ten nests of Morelet's Crocodile (Crocodylus moreletii) from New River Lagoon and Gold Button Lagoon in Belize to test for evidence of multiple paternity. Nine microsatellite loci were genotyped for 188 individuals from the 10 nests, alongside 42 nonhatchlings from Gold Button Lagoon. Then mitochondrial control region sequences were generated for the nonhatchlings and for one individual from each nest to test for presence of C. acutus-like haplotypes. Analyses of five of the nine microsatellite loci revealed evidence that progeny from five of the ten nests were sired by at least two males. These data suggest the presence of multiple paternity as a mating strategy in the true crocodiles. This information may be useful in the application of conservation and management techniques to the 12 species in this genus, most of which are threatened or endangered.

Mitochondrial DNA phylogeography of Caiman crocodilus in Mesoamerica and South America.

The Neotropical crocodylian species, Caiman crocodilus, is widely distributed through Mesoamerica, northern South America, and the Amazon basin. Four subspecies are recognized within C. crocodilus, suggesting some geographic variation in morphology. In this study, we utilized mitochondrial DNA (mtDNA) sequence data from 45 individuals of C. crocodilus throughout its range to infer its evolutionary history and population structure, as well as to evaluate genealogical support for subspecies and their geographic distributions. Our molecular phylogenetic results identified five mtDNA haplotype clades with a mean sequence divergence of 3.4%, indicating considerable evolutionary independence among phylogeographic lineages. Our results were also broadly consistent with current subspecific taxonomy, with some important additional findings. First, we found substantial genetic structuring within C. c. fuscus from southern Mesoamerica. Second, though we confirmed the existence of a widespread Amazonian clade, we also discovered a cryptic and divergent mtDNA lineage that was indistinguishable from C. c. crocodilus based on external morphology. Third, we confirm the status of C. c. chiapasius as a distinct evolutionary lineage, and provide evidence that C. c. fuscus may be moving northward and hybridizing with C. c. chiapasius in northern Mesoamerica. Finally, our results parallel previous phylogeographic studies of other organisms that have demonstrated significant genetic structure over shorter geographic distances in Mesoamerica compared with Amazonia. We support conservation efforts for all five independent lineages within C. crocodilus, and highlight the subspecies C. c. chiapasius as a unit of particular conservation concern.

Hybridization between Crocodylus acutus and Crocodylus moreletii in the Yucatan Peninsula: II. Evidence from microsatellites.

Detecting and quantifying hybridization between endangered or threatened taxa can provide valuable information with regards to conservation and management strategies. Hybridization between members of the genus Crocodylus has been known to occur in captivity and in some wild populations. We tested for hybridization among wild populations of American crocodile (C. acutus) and Morelet's crocodile (C. moreletii) in the Yucatan Peninsula by comparing Bayesian assignment tests, based on microsatellite data, to mitochondrial and morphological assignments. Skin clips from 83 individuals were taken for genetic identification, and a total of 32 individuals (38.6%) exhibited some evidence of hybridization by combined morphological, mitochondrial and microsatellite analyses. The majority of hybrids were classified as F(2) hybrids and backcrosses to C. moreletii. Most of the introgression occurs in two national biosphere reserves located on the northern and eastern coasts of the Yucatan Peninsula. Preliminary tests did not find a significant decrease in hybridity across three life stages, thus far indicating a low level of selection against hybrids. Model-based analyses on multilocus genotypes of pure individuals returned little geographic partitioning in both C. acutus and C. moreletii.

Genetic characterization of captive Cuban crocodiles (Crocodylus rhombifer) and evidence of hybridization with the American crocodile (Crocodylus acutus).

There is a surprising lack of genetic data for the Cuban crocodile (Crocodylus rhombifer), especially given its status as a critically endangered species. Samples from captive individuals were used to genetically characterize this species in comparison with other New World crocodilians. Partial mitochondrial sequence data were generated from cyt-b (843 bp) and the tRNA(Pro)- tRNA(Phe)-D-loop region (442 bp). Phylogenetic analyses were performed by generating maximum parsimony, maximum likelihood, and Bayesian-based topologies. In addition, in an effort to identify species-specific alleles, ten polymorphic microsatellite loci were genotyped. Distance and model-based clustering analyses were performed on microsatellite data, in addition to a model-based assignment of hybrid types. Both mitochondrial and nuclear markers identified two distinct C. rhombifer genetic sub-clades (alpha and beta); and microsatellite analyses revealed that most admixed individuals were F(2) hybrids between C. rhombifer-alpha and the American crocodile (C. acutus). All individuals in the C. rhombifer-beta group were morphologically identified as C. acutus and formed a distinct genetic assemblage. J. Exp. Zool. 309A:649-660, 2008. (c) 2008 Wiley-Liss, Inc.

Hybridization between Crocodylus acutus and Crocodylus moreletii in the Yucatan Peninsula: I. Evidence from mitochondrial DNA and morphology.

The American crocodile (Crocodylus acutus) and the Morelet's crocodile (C. moreletii) are broadly sympatric in Belize and Mexico. The presence of morphologically anomalous individuals in the overlapping range area suggests possible hybridization between these species. Analysis of 477 base pairs of the mitochondrial tRNA(Pro)-tRNA(Phe)-Dloop region revealed the presence of pure C. acutus (N=43) and C. moreletii (N=56), as well as a high proportion of interspecific hybrids (N=17, 14.6%) in the Yucatan Peninsula, Mexico. Although all individuals could be assigned to one species or other based on phenotypic characters, some had been characterized as potential hybrids in the field by anomalous scale counts. The hybridization zone lies along the area of sympatry between C. acutus and C. moreletii investigated in this study, but extends further inland if hybrid localities from Belize are included. Hybridization in the Yucatan Peninsula is bidirectional, which indicates considerably more genetic contact between these species than previously recognized, and is probably more detrimental to the genetic integrity of smaller C. acutus populations. A more intensive study of the pattern of hybridization is warranted and supports continued classification of C. acutus as a critically threatened species in the Yucatan Peninsula.

Plasma vitellogenin in Morelet's crocodiles from contaminated habitats in northern Belize.

Vitellogenin induction has been widely used as a biomarker of endocrine disruption in wildlife, but few studies have investigated its use in wild reptiles living in contaminated habitats. This study examined vitellogenin induction in Morelet's crocodiles (Crocodylus moreletii) from wetlands in northern Belize contaminated with organochlorine (OC) pesticides. Vitellogenin was measured in 381 crocodile plasma samples using a vitellogenin ELISA previously developed for this species. Vitellogenin was detected in nine samples, all from adult females sampled during the breeding season. Males and juvenile females did not contain detectable levels of vitellogenin; however, many of these animals contained OC pesticides in their caudal scutes, confirming contaminant exposure. The lack of a vitellogenic response in these animals may be attributable to several factors related to the timing and magnitude of exposure to endocrine-disrupting chemicals and should not be interpreted as an absence of other contaminant-induced biological responses.

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.

Are crocodiles really monophyletic?--Evidence for subdivisions from sequence and morphological data.

Recently, the phylogenetic placement of the African slender snouted crocodile, Crocodylus cataphractus, has come under scrutiny and herein we address this issue using molecular and morphological techniques. Although it is often recognized as being a "basal" form, morphological studies have traditionally placed C. cataphractus within the genus Crocodylus, while molecular studies have suggested that C. cataphractus is very distinct from other Crocodylus. To address the relationship of this species to its congeners we have sequenced portions of two nuclear genes (C-mos 302bp and ODC 294bp), and two mitochondrial genes (ND6-tRNA(glu)-cytB 347bp and control region 457bp). Analyses of these molecular datasets, both as individual gene sequences and as concatenated sequences, support the hypothesis that C. cataphractus is not a member of Crocodylus or Osteolaemus. Examination of 165 morphological characters supports and strengthens our resurrection of an historic genus, Mecistops (Gray 1844) for cataphractus.

Development of an enzyme-linked immunosorbent assay for vitellogenin of Morelet's crocodile (Crocodylus moreletii).

The purpose of this study was to develop an immunoassay for vitellogenin in Morelet's crocodile (Crocodylus moreletii). Blood was collected from wild-caught crocodiles in Belize. Plasma samples from adult females taken during the breeding season were used for vitellogenin purification and samples from adult males were used for comparison. No differences were detected between males and females for plasma total protein concentration, as measured by Coomassie assay. However, denaturing polyacrylamide gel electrophoresis (SDS-PAGE) revealed that female plasma contained a 210-kDa protein, presumably the vitellogenin monomer, that was absent in adult male plasma. The identity of the putative vitellogenin was confirmed by its cross-reactivity in Western blots with a vitellogenin antiserum that was generated against a conserved vitellogenin peptide sequence. Crocodile vitellogenin was purified by two successive rounds of DEAE chromatography. The purified protein had an apparent molecular mass of 450 kDa, as determined by gel filtration chromatography, and 210 kDa on SDS-PAGE. An indirect enzyme-linked immunosorbent assay (ELISA) was then developed for C. moreletii vitellogenin. The detection limit of the assay was 20.0 ng/mL. The intra- and inter-assay coefficients of variation were 5.3% and 9.8%, respectively. The recovery of vitellogenin diluted into male plasma was 94.7%. The ELISA assay revealed that vitellogenin levels of adult female plasma during the breeding season ranged from 1.8 to 3.1 mg/mL with a mean of 2.5+/-0.25 mg/mL. No vitellogenin was detected in adult male plasma. Induction of vitellogenin in Morelet's crocodile may be a useful model system for field studies of crocodile reproduction and for investigations of endocrine disruption in this species.

Chompy: an infestation of MITE-like repetitive elements in the crocodilian genome.

Interspersed repeats are a major component of most eukaryotic genomes and have an impact on genome size and stability, but the repetitive element landscape of crocodilian genomes has not yet been fully investigated. In this report, we provide the first detailed characterization of an interspersed repeat element in any crocodilian genome. Chompy is a putative miniature inverted-repeat transposable element (MITE) family initially recovered from the genome of Alligator mississippiensis (American alligator) but also present in the genomes of Crocodylus moreletii (Morelet's crocodile) and Gavialis gangeticus (Indian gharial). The element has all of the hallmarks of MITEs including terminal inverted repeats, possible target site duplications, and a tendency to form secondary structures. We estimate the copy number in the alligator genome to be approximately 46,000 copies. As a result of their size and unique properties, Chompy elements may provide a useful source of genomic variation for crocodilian comparative genomics.

Members of a new group of chitinase-like genes are expressed preferentially in cotton cells with secondary walls.

Two homologous cotton (Gossypium hirsutum L.) genes, GhCTL1 and GhCTL2, encode members of a new group of chitinase-like proteins (called the GhCTL group) that includes other proteins from two cotton species, Arabidopsis, rice, and pea. Members of the GhCTL group are assigned to family GH19 glycoside hydrolases along with numerous authentic chitinases (http://afmb.cnrs-mrs.fr/CAZY/index.html), but the proteins have novel consensus sequences in two regions that are essential for chitinase activity and that were previously thought to be conserved. Maximum parsimony phylogenetic analyses, as well as Neighbor-Joining distance analyses, of numerous chitinases confirmed that the GhCTL group is distinct. A molecular model of GhCTL2 (based on the three-dimensional structure of a barley chitinase) had changes in the catalytic site that are likely to abolish catalytic activity while retaining potential to bind chitin oligosaccharides. RNA blot analysis showed that members of the GhCTL group had preferential expression during secondary wall deposition in cotton lint fiber. Cotton transformed with a fusion of the GhCTL2 promoter to the beta -d-glucuronidase gene showed preferential reporter gene activity in numerous cells during secondary wall deposition. Together with evidence from other researchers that mutants in an Arabidopsis gene within the GhCTL group are cellulose-deficient with phenotypes indicative of altered primary cell walls, these data suggest that members of the GhCTL group of chitinase-like proteins are essential for cellulose synthesis in primary and secondary cell walls. However, the mechanism by which they act is more likely to involve binding of chitin oligosaccharides than catalysis.

Repetitive sequences in the crocodilian mitochondrial control region: poly-A sequences and heteroplasmic tandem repeats.

Heteroplasmic tandem repeats in the mitochondrial control region have been documented in a wide variety of vertebrate species. We have examined the control region from 11 species in the family Crocodylidae and identified two different types of heteroplasmic repetitive sequences in the conserved sequence block (CSB) domain-an extensive poly-A tract that appears to be involved in the formation of secondary structure and a series of tandem repeats located downstream ranging from approximately 50 to approximately 80 bp in length. We describe this portion of the crocodylian control region in detail and focus on members of the family Crocodylidae. We then address the origins of the tandemly repeated sequences in this family and suggest hypotheses to explain possible mechanisms of expansion/contraction of the sequences. We have also examined control region sequences from Alligator and Caiman and offer hypotheses for the origin of tandem repeats found in those taxa. Finally, we present a brief analysis of intraindividual and interindividual haplotype variation by examining representatives of Morelet's crocodile (Crocodylus moreletii).

Studies on the molecular evolution of the Crocodylia: footprints in the sands of time.

A reasonably large number of studies focusing on the molecular evolution of crocodilians have been completed during the past 100 years. Proteins were initially studied before DNA was known to carry the genetic information of cells and organisms, and were subsequently studied to infer changes at the DNA level. More recently, studies on the DNA itself have been completed. We have had the pleasure of taking part in or facilitating many studies conducted over the past 50 years, especially several of the earliest studies done using newly developed molecular techniques. We provide a review of the molecular genetic studies on crocodilians, summarizing the findings of these studies as well as the context in which they were undertaken. This review is a personal look at the history of molecular studies on the evolutionary biology of crocodilians. Our excuse for this focus is that our professors, our students and we have had the opportunity to be among the first to apply many new techniques to studies of crocodilians since 1950, when one of us (HCD) was a graduate student of Roland Coulson and Tom Hernandez. Although we will review much of the material in this subject area, we do not claim that it is complete. Instead, we focus our presentation on work in which we have participated or with which we are particularly familiar. We especially focus on materials relevant to the research presented at the 2(nd) International Crocodilian DNA Workshop, 7-9 November, 2001, at the San Diego Zoo. Thus, the following review also stands as a tribute to our mentors, students, and colleagues.