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.     

Identification of purebred Crocodylus siamensis for reintroduction in Vietnam

Crocodylus siamensis, the Siamese crocodile, is a critically endangered species of freshwater crocodile previously distributed throughout much of SE Asia. Recovery plans call for reintroductions to the wild using founder individuals currently in captivity, mostly in commercial crocodile farms. On many farms C. siamensis has been intentionally hybridised with either Cuban crocodiles, C. rhombifer, or the estuarine crocodile, C. porosus, and hybrids may be difficult to distinguish morphologically. We report on the combined use of microsatellite and mtDNA genetic markers to determine the species status of potential founder individuals for reintroduction of C. siamensis. Genetic markers were used to characterise 103 captive and wild-caught individuals of C. siamensis, C. rhombifer and C. porosus in Vietnam and to distinguish purebred versus hybrid individuals. Although the microsatellite loci used had some overlap of allele sizes among species, assignment tests allowed differentiation. Four hybrids were identified, two of which had not been recognised morphologically as hybrids, and one of these was thought to be a C. siamensis suitable for reintroduction. Ten of the identified purebred C. siamensis were subsequently released into Cat Tien National Park in southern Vietnam.     

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 and morphological evidence of a geographically widespread hybrid zone between two crocodile species,Crocodylus acutus and Crocodylus moreletii

Hybrid zones represent natural laboratories to study gene flow, divergence and the nature of species boundaries between closely related taxa. We evaluated the level and extent of hybridization between Crocodylus moreletii and Crocodylus acutus using genetic and morphological data on 300 crocodiles from 65 localities. To our knowledge, this is the first genetic study that includes the entire historic range and sympatric zone of the two species. Contrary to expectations, Bayesian admixture proportions and maximum‐likelihood estimates of hybrid indexes revealed that most sampled crocodiles were admixed and that the hybrid zone is geographically extensive, extending well beyond their historical region of sympatry. We identified a few geographically isolated, nonadmixed populations of both parental species. Hybrids do not appear to be F₁s or recent backcrosses, but rather are more likely later‐generation hybrids, suggesting that hybridization has been going on for several to many generations and is mostly the result of natural processes. Crocodylus moreletii is not the sister species of C. acutus, suggesting that the hybrid zone formed from secondary contact rather than primary divergence. Nonadmixed individuals from the two species were distinguishable based on morphological characters, whereas hybrids had a complex mosaic of morphological characters that hinders identification in the wild. Very few nonadmixed C. acutus and C. moreletii populations exist in the wild. Consequently, the last nonadmixed C. moreletii populations have become critically endangered. Indeed, not only the parental species but also the naturally occurring hybrids should be considered for their potential conservation value.     

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.     

Genetic evidence of hybridization between the critically endangered Cuban crocodile and the American crocodile: implications for population history and in situ/ex situ conservation

Inter-specific hybridization may be especially detrimental when one species is extremely rare and the other is abundant owing to the potential for genetic swamping. The Cuban crocodile (Crocodylus rhombifer) is a critically endangered island endemic largely restricted to Zapata Swamp, where it is sympatric with the widespread American crocodile (C. acutus). An on-island, C. rhombifer captive breeding program is underway with the goals of maintaining taxonomic integrity and providing a source of individuals for reintroduction, but its conservation value is limited by lack of genetic information. Here we collected mtDNA haplotypic and nuclear genotypic data from wild and captive C. rhombifer and C. acutus in Cuba to: (1) investigate the degree of inter-specific hybridization in natural (in situ) and captive (ex situ) populations; (2) quantify the extent, distribution and in situ representation of genetic variation ex situ; and (3) reconstruct founder relatedness to inform management. We found high levels of hybridization in the wild (49.1%) and captivity (16.1%), and additional evidence for a cryptic lineage of C. acutus in the Antilles. We detected marginally higher observed heterozygosity and allelic diversity ex situ relative to the wild population, with captive C. rhombifer exhibiting over twice the frequency of private alleles. Although mean relatedness was high in captivity, we identified 37 genetically important individuals that possessed individual mean kinship (MK) values lower than the population MK. Overall, these results will guide long-term conservation management of Cuban crocodiles for maintaining the genetic integrity and viability of this species of high global conservation value.     

Molecular phylogeny and phylogeography of the Cuban cave-fishes of the genus Lucifuga: evidence for cryptic allopatric diversity

Underground environments are increasingly recognized as reservoirs of faunal diversity. Extreme environmental conditions and limited dispersal ability of underground organisms have been acknowledged as important factors promoting divergence between species and conspecific populations. However, in many instances, there is no correlation between genetic divergence and morphological differentiation. Lucifuga Poey is a stygobiotic fish genus that lives in Cuban and Bahamian caves. In Cuba, it offers a unique opportunity to study the influence of habitat fragmentation on the genetic divergence of stygobiotic species and populations. The genus includes four species and one morphological variant that have contrasting geographical distributions. In this study, we first performed a molecular phylogenetic analysis of the Lucifuga Cuban species using mitochondrial and nuclear markers. The mitochondrial phylogeny revealed three deeply divergent clades that were supported by nuclear and morphological characters. Within two of these main clades, we identified five lineages that are candidate cryptic species and a taxonomical synonymy between Lucifuga subterranea and Lucifuga teresinarum. Secondly, phylogeographic analysis using a fragment of the cytochrome b gene was performed for Lucifuga dentata, the most widely distributed species. We found strong geographical organization of the haplotype clades at different geographic scales that can be explained by episodes of dispersal and population expansion followed by population fragmentation and restricted gene flow. At a larger temporal scale, these processes could also explain the diversification and the distribution of the different species.     

Weak premating isolation between Clitarchus stick insect species despite divergent male and female genital morphology

Documenting natural hybrid systems builds our understanding of mate choice, reproductive isolation and speciation. The stick insect species Clitarchus hookeri and C. tepaki differ in their genital morphology and hybridize along a narrow peninsula in northern New Zealand. We utilize three lines of evidence to understand the role of premating isolation and species boundaries: (a) genetic differentiation using microsatellites and mitochondrial DNA; (b) variation in 3D surface topology of male claspers and 2D morphometrics of female opercular organs; and (c) behavioural reproductive isolation among parental and hybrid populations through mating crosses. The genetic data show introgression between the parental species and formation of a genetically variable hybrid swarm. Similarly, the male and female morphometric data show genital divergence between the parental species as well as increased variation within the hybrid populations. This genital divergence has not resulted in reproductive isolation between species, instead weak perimating isolation has enabled the formation of a hybrid swarm. Behavioural analysis demonstrates that the entire mating process influences the degree of reproductive isolation between species undergoing secondary contact. Mechanical isolation may appear strong, whereas perimating isolation is weak.     

Systematics and conservation of the hook-billed kite including the island taxa from Cuba and Grenada

Taxonomic uncertainties within the genus Chondrohierax stem from the high degree of variation in bill size and plumage coloration throughout the geographic range of the single recognized species, hook-billed kite Chondrohierax uncinatus . These uncertainties impede conservation efforts as local populations have declined throughout much of its geographic range from the Neotropics in Central America to northern Argentina and Paraguay, including two island populations on Cuba and Grenada, and it is not known whether barriers to dispersal exist between any of these areas. Here, we present mitochondrial DNA (mtDNA; cytochrome B and NADH dehydrogenase subunit 2) phylogenetic analyses of Chondrohierax , with particular emphasis on the two island taxa (from Cuba, Chondrohierax uncinatus wilsonii and from Grenada, Chondrohierax uncinatus mirus ). The mtDNA phylogenetic results suggest that hook-billed kites on both islands are unique; however, the Cuban kite has much greater divergence estimates (1.8–2.0% corrected sequence divergence) when compared with the mainland populations than does the Grenada hook-billed kite (0.1–0.3%). Our findings support species status for the Cuban form, as Chondrohierax wilsonii , and subspecific status for the Grenada form. For mainland taxa, we do not find support for the currently recognized subspecies Chondrohierax uncinatus aquilonis in western Mexico, but we do find evidence for a genetic subdivision between populations in Central and South America, a difference previously unsuspected. The results of this study help identify conservation priorities associated with these unique Neotropical raptors. This information is of immediate interest because the Cuban kite has not been reliably seen since 1992, and <50 hook-billed kites currently inhabit Grenada.     

Molecular and morphological support for a Florida origin of the Cuban oak

Aim The origins of the Cuban biota are of long‐standing interest in biogeography, and the source of a small live oak (Quercus series Virentes) population on Cuba remains unresolved. Based on morphological evidence, previous authors have hypothesized a Florida origin from either Q. geminata or Q. virginiana or both; a Mexican origin from Q. oleoides; or a hybrid origin from both sources. We use molecular data and taxonomically informative leaf morphology to identify the source species and timing of colonization. Location Cuba, Central America, Mexico and the south‐eastern United States. Methods  We collected representative samples of Cuban oaks and each putative source species and genotyped each sample at 12 nuclear microsatellites and two chloroplast DNA sequences. We estimated population structure using a Bayesian clustering analysis and F‐statistics, pairwise migration rates among taxa, and divergence time using an isolation‐with‐migration model. We measured seven leaf traits and conducted an analysis of similarity (ANOSIM) to determine which putative source species was most similar to Cuban oaks. Results Cuban oak contains one chloroplast DNA haplotype, which is common in southern Florida. Bayesian clustering analysis of microsatellites revealed that the Cuban oak forms a distinct and pure population cluster, and F‐statistics showed that Cuban oaks are differentiated least from Q. virginiana and most from Q. geminata. Migration rates were highest out of Cuba to Q. oleoides. Molecular diversity, the ratio of allelic richness to allele size range, and effective population size of the Cuban oak were relatively low, suggesting a founder effect. Divergence time estimates fell entirely within the Pleistocene (628–6 ka), considering a range of mutation rates and generation times. Cuban oaks were morphologically most similar to Q. virginiana and least similar to Q. geminata. Main conclusions Molecular and morphological data support a Pleistocene dispersal of Q. virginiana from Florida to Cuba, followed by isolation and divergence, then limited dispersal and introgression from Cuba to Q. oleoides in Central America. Birds could have dispersed acorns to Cuba during a glacial period when sea levels were low. These results highlight the varied origin of the Cuban biota and the possible role of Pleistocene glaciations in the establishment of temperate taxa in the tropics.     

Molecules and morphology: evidence for cryptic hybridization in African Hyalomma (Acari: Ixodidae)

The role of natural hybridization and introgression as part of the evolutionary process is of increasing interest to zoologists, particularly as more examples of gene exchange among species are identified. We present mitochondrial and nuclear sequence data for Hyalomma dromedarii, Hyalomma truncatum, and Hyalomma marginatum rufipes (Acari: Ixodidae) collected from one-humped camels in Ethiopia. These species are well differentiated morphologically and genetically; sequence data from the mitochondrial DNA (mtDNA) cytochrome oxidase I gene indicates 10-14% divergence between the species. However, incongruence between morphology and the mtDNA phylogeny was observed, with multiple individuals of H. dromedarii and H. truncatum present on the same mtDNA lineage as H. marginatum rufipes. Thus, individuals with morphology of H. dromedarii and H. truncatum are indistinguishable from H. marginatum rufipes on the basis of mtDNA. Multiple copies of ITS-2 were subsequently cloned and sequenced for a subset of individuals from the mtDNA phylogeny, representing both 'normal' and 'putative hybrid' individuals. Very low sequence divergence (0.3%) was observed within 'normal' individuals of both H. dromedarii and H. truncatum relative to the 'putative hybrid' individuals (6 and 2.7%, respectively). The pattern of intra-individual variation in ITS-2 within 'putative hybrid' individuals, particularly in H. dromedarii, strongly suggests that gene flow has occurred among these Hyalomma species, but no indication of this is given by the morphology of the individuals.     

Alpha-2-macroglobulin-like protease inhibitor from the egg white of cuban crocodile (Crocodylus rhombifer)

A high molecular weight protease inhibitor was purified from the egg white of Cuban crocodile (Crocodylus rhombifer). It inhibited the casein hydrolyzing activity of trypsin, subtilisin and papain. Its native molecular weight was 730,000 and it consisted of four subunits of equal molecular weight, each pair of which were disulfide bonded. The amino acid composition, circular dichroic spectrum and electron micrographs of this protein are also presented. Upon incubation with trypsin this protein yielded a fragment of Mr = 80,000, similar in size to the one known to originate from alpha 2-macroglobulin under the same conditions. The molecular parameters of this protein and the broad inhibitory activity towards thiol and serine proteases with different substrate specificities suggest that it is a protein closely related to alpha 2-macroglobulin in mammalian serum. From its native molecular weight and amino acid composition we believe that this protein is also a reptilian counterpart of the avian ovomacroglobulin described by Miller and Feeney (3).     

Hybridization at an ecotone: ecological and genetic barriers between three Iberian vipers

The formation of stable genetic boundaries between emerging species is often diagnosed by reduced hybrid fitness relative to parental taxa. This reduced fitness can arise from endogenous and/or exogenous barriers to gene flow. Although detecting exogenous barriers in nature is difficult, we can estimate the role of ecological divergence in driving species boundaries by integrating molecular and ecological niche modelling tools. Here, we focus on a three‐way secondary contact zone between three viper species (Vipera aspis, V. latastei and V. seoanei) to test for the contribution of ecological divergence to the development of reproductive barriers at several species traits (morphology, nuclear DNA and mitochondrial DNA). Both the nuclear and mitochondrial data show that all taxa are genetically distinct and that the sister species V. aspis and V. latastei hybridize frequently and backcross over several generations. We find that the three taxa have diverged ecologically and meet at a hybrid zone coincident with a steep ecotone between the Atlantic and Mediterranean biogeographical provinces. Integrating landscape and genetic approaches, we show that hybridization is spatially restricted to habitats that are suboptimal for parental taxa. Together, these results suggest that niche separation and adaptation to an ecological gradient confer an important barrier to gene flow among taxa that have not achieved complete reproductive isolation.     

Molecular phylogeny of an endemic radiation of Cuban toads (Bufonidae: Peltophryne) based on mitochondrial and nuclear genes

Aim In this study we present a molecular phylogenetic and phylogeographical analysis of Peltophryne (Anura: Bufonidae), an endemic genus of Antillean toads, to investigate the spatial and temporal origins of the genus, with particular focus on the eight Cuban species. Location Greater Antilles, with extensive sampling of the Cuban archipelago. Methods We obtained DNA sequence data from two mitochondrial genes, cytochrome c oxidase subunit I (COI) and ribosomal RNA (16S), for 124 toads representing all eight Cuban species, and combined this with published data from Hispaniola (one of three species) and Puerto Rico (one of one species) to establish a molecular phylogeny for Peltophryne. In addition, we explored the phylogeographical structure of widespread Cuban species. For a subset of 42 toads we also obtained DNA sequence data from two nuclear genes, recombination activator‐1 (RAG‐1) and chemokine receptor 4 (CXCR‐4). We combined our molecular data with published DNA sequences from a global sample of bufonid toads to place the spatial and temporal origins of Peltophryne in the Caribbean within a fuller geographical and phylogenetic context. Results All phylogenetic analyses supported the monophyly of West Indian toads. The ancestor of Peltophyrne diverged from its mainland source around the Eocene–Oligocene boundary, with a subsequent radiation across the Caribbean islands taking place during the Miocene. Cuban species are monophyletic with a basal split in the early–middle Miocene that separates extant small‐bodied from large‐bodied species. Extensive mitochondrial DNA (mtDNA) sampling within widespread Cuban species revealed contrasting phylogeographical patterns. Peltophryne taladai and P. empusa showed deeply divergent lineages, whereas no geographical structure was observed in the widespread P. peltocephala. Main conclusions Our timeline for Peltophryne diversification is consistent with a biogeographical model requiring no long‐distance overwater dispersal. Although confidence intervals on divergence time estimates are wide, the stem age of Peltophyrne coincides with the hypothesized GAARlandia landspan or archipelago, which may have connected South America briefly with the Antilles. The ages of Peltophryne for Puerto Rico, Hispaniola and Cuba are consistent with a recently proposed vicariance scenario for the region. Our molecular results support the recognition of all eight species in Cuba, and provide evidence of possible cryptic species.     

Partial island submergence and speciation in an adaptive radiation: a multilocus analysis of the Cuban green anoles

Sympatric speciation is often proposed to account for species-rich adaptive radiations within lakes or islands, where barriers to gene flow or dispersal may be lacking. However, allopatric speciation may also occur in such situations, especially when ranges are fragmented by fluctuating water levels. We test the hypothesis that Miocene fragmentation of Cuba into three palaeo-archipelagos accompanied species-level divergence in the adaptive radiation of West Indian Anolis lizards. Analysis of morphology, mitochondrial DNA (mt DNA) and nuclear DNA in the Cuban green anoles (carolinensis subgroup) strongly supports three pre dictions made by this hypothesis. First, three geographical sets of populations, whose ranges correspond with palaeo-archipelago boundaries, are distinct and warrant recognition as independent evolutionary lineages or species. Coalescence of nuclear sequence fragments sampled from these species and the large divergences observed between their mtDNA haplotypes suggest separation prior to the subsequent unification of Cuba ca. 5 Myr ago. Second, molecular phylogenetic relationships among these species reflect historical geographical relationships rather than morphological similarity. Third, all three species remain distinct despite extensive geographical contact subsequent to island unification, occasional hybridization and introgression of mtDNA haplotypes. Allopatric speciation initiated during partial island submergence may play an important role in speciation during the adaptive radiation of Anolis lizards.     

Mitochondrial DNA analysis reveals hidden genetic diversity in captive populations of the threatened American crocodile (Crocodylus acutus) in Colombia

Identification of units within species worthy of separate management consideration is an important area within conservation. Mitochondrial DNA (mtDNA) surveys can potentially contribute to this by identifying phylogenetic and population structure below the species level. The American crocodile (Crocodylus acutus) is broadly distributed throughout the Neotropics. Its numbers have been reduced severely with the species threatened throughout much of its distribution. In Colombia, the release of individuals from commercial captive populations has emerged as a possible conservation strategy that could contribute to species recovery. However, no studies have addressed levels of genetic differentiation or diversity within C. acutus in Colombia, thus complicating conservation and management decisions. Here, sequence variation was studied in mtDNA cytochrome b and cytochrome oxidase I gene sequences in three Colombian captive populations of C. acutus. Two distinct lineages were identified: C. acutus‐I, corresponding to haplotypes from Colombia and closely related Central American haplotypes; and C. acutus‐II, corresponding to all remaining haplotypes from Colombia. Comparison with findings from other studies indicates the presence of a single “northern” lineage (corresponding to C. acutus‐I) distributed from North America (southern Florida), through Central America and into northern South America. The absence of C. acutus‐II haplotypes from North and Central America indicates that the C. acutus‐II lineage probably represents a separate South American lineage. There appears to be sufficient divergence between lineages to suggest that they could represent two distinct evolutionary units. We suggest that this differentiation needs to be recognized for conservation purposes because it clearly contributes to the overall genetic diversity of the species. All Colombian captive populations included in this study contained a mixture of representatives of both lineages. As such, we recommend against the use of captive‐bred individuals for conservation strategies until further genetic information is available.     

Wolbachia infection and dramatic intraspecific mitochondrial DNA divergence in a fig wasp

Mitochondria and Wolbachia are maternally inherited genomes that exhibit strong linkage disequilibrium in many organisms. We surveyed Wolbachia infections in 187 specimens of the fig wasp species, Ceratosolen solmsi, and found an infection prevalence of 89.3%. DNA sequencing of 20 individuals each from Wolbachia-infected and -uninfected subpopulations revealed extreme mtDNA divergence (up to 9.2% and 15.3% in CO1 and cytochrome b, respectively) between infected and uninfected wasps. Further, mtDNA diversity was significantly reduced within the infected group. Our sequencing of a large part of the mitochondrial genome from both Wolbachia-infected and -uninfected individuals revealed that high sequence divergence is common throughout the mitochondrial genome. These patterns suggest a partial selective sweep of mitochondria subsequent to the introduction of Wolbachia into C. solsmi, by hybrid introgression from a related species.     

Low Levels of Nucleotide Diversity in Crocodylus moreletii and Evidence of Hybridization with C. acutus

Examinations of both population genetic structure and the processes that lead to such structure in crocodilians have been initiated in several species in response to a call by the IUCN Crocodile Specialist Group. A recent study used microsatellite markers to characterize Morelet's crocodile (Crocodylus moreletii) populations in north-central Belize and presented evidence for isolation by distance. To further investigate this hypothesis, we sequenced a portion of the mitochondrial control region for representative animals after including samples from additional locales in Belize, Guatemala and Mexico. While there is limited evidence of subdivision involving other locales, we found that most of the differentiation among populations of C. moreletiican be attributed to animals collected from a single locale in Belize, Banana Bank Lagoon. Furthermore, mitochondrial DNA sequence analysis showed that animals from this and certain other locales display a haplotype characteristic of the American crocodile, C. acutus, rather than C. moreletii. We interpret this as evidence of hybridization between the two species and comment on how these new data have influenced our interpretation of previous findings. We also find very low levels of nucleotide diversity in C. moreletiihaplotypes and provide evidence for a low rate of substitution in the crocodilian mitochondrial control region. Finally, the conservation implications of these findings are discussed.     

DNA barcoding of Cuban freshwater fishes: evidence for cryptic species and taxonomic conflicts

Despite ongoing efforts to protect species and ecosystems in Cuba, habitat degradation, overuse and introduction of alien species have posed serious challenges to native freshwater fish species. In spite of the accumulated knowledge on the systematics of this freshwater ichthyofauna, recent results suggested that we are far from having a complete picture of the Cuban freshwater fish diversity. It is estimated that 40% of freshwater Cuban fish are endemic; however, this number may be even higher. Partial sequences (652 bp) of the mitochondrial gene COI (cytochrome c oxidase subunit I) were used to barcode 126 individuals, representing 27 taxonomically recognized species in 17 genera and 10 families. Analysis was based on Kimura 2-parameter genetic distances, and for four genera a character-based analysis (population aggregation analysis) was also used. The mean conspecific, congeneric and confamiliar genetic distances were 0.6%, 9.1% and 20.2% respectively. Molecular species identification was in concordance with current taxonomical classification in 96.4% of cases, and based on the neighbour-joining trees, in all but one instance, members of a given genera clustered within the same clade. Within the genus Gambusia, genetic divergence analysis suggests that there may be at least four cryptic species. In contrast, low genetic divergence and a lack of diagnostic sites suggest that Rivulus insulaepinorum may be conspecific with Rivulus cylindraceus. Distance and character-based analysis were completely concordant, suggesting that they complement species identification. Overall, the results evidenced the usefulness of the DNA barcodes for cataloguing Cuban freshwater fish species and for identifying those groups that deserve further taxonomic attention.