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Targeted Illumina sequencing of nitrogenase reductase (nifH) gene fragments and analyses of pair-end reads through a modified QIIME pipeline were used to assess the diversity of the actinomyceteous genus Frankia in three soils. Soils were vegetated with host or non-host plants, and included locations in Illinois (ABA, host), Colorado (CoMt, non-host), and Wisconsin (FMWI, non-host). After filtering, seven unique sequences were recovered for soil ABA, six for CoMt, and four sequences for FMWI. These sequences were included in a Bayesian topology anchored by published sequence data from pure cultures of Frankia. Sequences from all three soils showed affinities to Frankia strains from both the Alnus and Elaeagnus host infection groups. Reads representing Casuarina-infective strains were not detected. Four sequences from soil CoMt and five sequences from soil ABA did not cluster, at 97% similarity, into a shared OTU that contained a cultured relative. These results demonstrate that targeted Illumina sequencing provides an efficient and economical method for assessing haplotype diversity of ecofunctional genes (e.g. nifH) at the genus level in microorganisms that perform important ecosystem functions., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Freshwater turtles are being exploited for meat, eggs, traditional medicine, and pet trade. As a response, turtle farming became a booming aquaculture industry in the past two decades, specifically in the southeastern states of the United States of America (US) and across Southeast Asia. However, US turtle farms are currently producing turtles only for the pet trade while commercial trappers remain focused on catching the largest individuals from the wild. In our analyses we have created a biological and economic model that describes farming operations on a representative turtle farm in Louisiana. We first modeled current production of hatchling and yearling red-eared slider turtles (Trachemys scripta elegans) (i.e., traditional farming) for foreign and domestic pet markets, respectively. We tested the possibility of harvesting adult turtles from the breeding stock for sale to meat markets to enable alternative markets for the farmers, while decreasing the continued pressures on wild populations (i.e., non-traditional farming). Our economic model required current profit requirements of ~$13/turtle or ~$20.31/kg of meat from non-traditional farming in order to acquire the same profit as traditional farming, a value which currently exceeds market values of red-eared sliders. However, increasing competition with Asian turtle farms and decreasing hatchling prices may force the shift in the US toward producing turtles for meat markets. In addition, our model can be modified and applied to more desirable species on the meat market once more knowledge is acquired about species life histories and space requirements under farmed conditions.

Unregulated commercial harvest remains a major threat for turtles across the globe. Due to continuing demand from Asian markets, a significant number of turtles are exported from the United States of America (US). Beginning in 2007, several southeastern states in the US implemented restrictions on the commercial harvest of turtles, in order to address the unsustainable take. We have summarized freshwater turtle exports from the US between 2002 and 2012 and demonstrated that the magnitude of turtle exports from the US remained high although the exports decreased throughout the decade. Louisiana and California were the major exporters. The majority of exports were captive bred, and from two genera, Pseudemys and Trachemys. We review the changes over the decade and speculate that the increase in export of wild turtles out of Louisiana after 2007 could be a consequence of strict regulations in surrounding states (e.g., Alabama, Florida). We suggest that if wild turtle protection is a goal for conservation efforts, then these states should work together to develop comprehensive regulation reforms pertaining to the harvest of wild turtles.

Tropical forest canopies are among the most species-rich terrestrial habitats on earth and one of the remaining relatively unexplored biotic frontiers. Epiphytic bromeliads provide microhabitat for a high diversity of organisms in tropical forest canopies and are considered a keystone resource. A number of amphibians inhabit these phytotelmata, yet their ecological role and status in forest canopies remains unknown. For this study, anurans were collected from an upper canopy tank bromeliad (Aechmea zebrina) at ∼20-45 m (x¯ = 33 m) above the forest floor. Bromeliads were sampled from trees located near trails in undisturbed primary rainforest and oil access roads in the Yasuní Biosphere Reserve of Amazonian Ecuador. We collected 95 anurans representing 10 species from 160 bromeliads in 32 trees. We used generalized linear mixed models to assess the effects of disturbance and habitat factors on the occupancy and abundance of anurans collected. Bromeliads in forest along oil roads had a lower occupancy and abundance of anurans than those in undisturbed forest, a somewhat unexpected result due to the intactness and quality of forest adjacent to the roads. Recorded habitat variables had no relationship with occupancy or abundance of anurans, and did not differ significantly between treatments. Our findings reveal that even the minimal footprint of natural resource extraction operations, primarily roads, in rainforest environments can have significant negative impacts on the unique upper canopy anuran community. Based on these results, we recommend that natural resource development treat rainforest habitat as an offshore system where roads are not used, employ industry best practice guidelines, and current access roads be protected from colonization and further deforestation.

The potential of fish to transfer salmonellae from heterogeneous aquatic biofilms into feces was assessed in controlled aquarium studies with Suckermouth Catfish Hypostomus plecostomus and with biofilms inoculated with salmonellae. Neither the presence of catfish nor inoculation with salmonellae had detectable effects on the abundance of the microbial community. Densities of the microbial community were about 10(5) cells/mL in the water during a 1-week period, whereas densities of the microbial community increased 10-fold (10(6) to 10(7) cells/mg) in catfish feces during the same period. Salmonellae were detected by both quantitative polymerase chain reaction (qPCR) and situ hybridization in water samples immediately after inoculation, in numbers of about 10(4) cells/mL, representing up to 20% of the cells of the microbial community. Numbers decreased by three orders of magnitude within the first 3 d of the study, which represented only 0.01% of the community, and became undetectable after day 5. In catfish feces, numbers of Salmonella initially increased to up to 6% of the cells of the community but then declined. These results suggest that Salmonella are not biomagnified during gut passage, and thus, fish only provide a means for the translocation of this pathogen.

The diversity of Salmonella was analysed in biofilm and water samples from the spring and slough arms of Spring Lake, the headwaters of the San Marcos River, Texas, during the drought of 2011, with only one potential run-off event at the beginning of the study. Salmonellae were detected in semiselective enrichment cultures by end-point PCR during the entire sampling period of (11 sampling events during 2 months). From the spring arm site, 73% of the biofilms and 41% of the water samples were positive for salmonellae, while only 9% of the biofilms and 23% of the water samples were positive from the slough arm site. Salmonellae could be isolated from all positive samples, with higher diversity in biofilms compared with water samples and more strains obtained from the spring arm than from the slough arm. Differences between sites were generally caused by less frequently detected isolates, while the majority of isolates that were present in both biofilms and water from both sites was represented by three strains only. Quantification attempts by quantitative PCR directly in samples without prior enrichment did not result in a reliable detection of salmonellae, suggesting that numbers in all samples were below the detection limit., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Members of the bacterial genus Salmonella are recognized worldwide as major zoonotic pathogens often found to persist in non-enteric environments including heterogeneous aquatic biofilms. In this study, Salmonella isolates that had been detected repeatedly over time in aquatic biofilms at different sites in Spring Lake, San Marcos, Texas, were identified as serovars Give, Thompson, Newport and -:z10:z39. Pathogenicity results from feeding studies with the nematode Caenorhabditis elegans as host confirmed that these strains were pathogenic, with Salmonella-fed C. elegans dying faster (mean survival time between 3 and 4 days) than controls, i.e., Escherichia coli-fed C. elegans (mean survival time of 9.5 days). Cells of these isolates inoculated into water at a density of up to 10(6) ml(-1) water declined numerically by 3 orders of magnitude within 2 days, reaching the detection limit of our quantitative polymerase chain reaction (qPCR)-based quantification technique (i.e., 10(3) cells ml(-1)). Similar patterns were obtained for cells in heterogeneous aquatic biofilms developed on tiles and originally free of Salmonella that were kept in the inoculated water. Cell numbers increased during the first days to more than 10(7) cells cm(-2), and then declined over time. Ten-fold higher cell numbers of Salmonella inoculated into water or into biofilm resulted in similar patterns of population dynamics, though cells in biofilms remained detectable with numbers around 10(4) cells cm(-2) after 4 weeks. Independent of detectability by qPCR, samples of all treatments harbored viable salmonellae that resembled the inoculated isolates after 4 weeks of incubation. These results demonstrate that pathogenic salmonellae were isolated from heterogeneous aquatic biofilms and that they could persist and stay viable in such biofilms in high numbers for some time.

Multiple studies have addressed the mechanisms maintaining polymorphism within a population. However, several examples exist where species inhabiting diverse habitats exhibit local population-specific polymorphism. Numerous explanations have been proposed for the maintenance of geographic variation in color patterns. For example, spatial variation in patterns of selection or limited gene flow can cause entire populations to become fixed for a single morph, resulting in separate populations of the same species exhibiting separate and distinct color morphs. The mottled rock rattlesnake (Crotalus lepidus lepidus) is a montane species that exhibits among-population color polymorphism that correlates with substrate color. Habitat substrate in the eastern part of its range is composed primarily of light colored limestone and snakes have light dorsal coloration, whereas in the western region the substrate is primarily dark and snakes exhibit dark dorsal coloration. We hypothesized that predation on high contrast color and blotched patterns maintain these distinct color morphs. To test this we performed a predation experiment in the wild by deploying model snakes at 12 sites evenly distributed within each of the two regions where the different morphs are found. We employed a 2×2 factorial design that included two color and two blotched treatments. Our results showed that models contrasting with substrate coloration suffered significantly more avian attacks relative to models mimicking substrates. Predation attempts on blotched models were similar in each substrate type. These results support the hypothesis that color pattern is maintained by selective predation.

Salmonellae are pathogenic bacteria often detected in waters impacted by human or animal wastes. In order to assess the fate of salmonellae in supposedly pristine environments, water and natural biofilm samples along with snails (Tarebia granifera) and crayfish (Procambarus clarkia) were collected before and up to 7 days following four precipitation events from sites within the headwater springs of Spring Lake, San Marcos, TX. The samples were analyzed for the presence of salmonellae by polymerase chain reaction (PCR) after semi-selective enrichment. Salmonellae were detected in one water sample directly after precipitation only, while detection in ten biofilm and two crayfish samples was not related to precipitation. Salmonellae were not detected in snails. Characterization of isolates by rep-PCR revealed shared profiles in water and biofilm samples, biofilm and crayfish samples, and biofilm samples collected 23 days apart. These results suggest that salmonellae are infrequently washed into this aquatic ecosystem during precipitation runoff and can potentially take up residency in biofilms which can help facilitate subsequent long-term persistence and eventual transfer through the food chain.

In previous work, we have shown that light-adaptive pigment granule dispersion can be induced in vitro by treating retinal pigment epithelium (RPE) isolated from bluegill retina with acetylcholine or its analog carbachol and that these agents act through muscarinic receptors to induce pigment granule dispersion. RPE is a monolayer of tissue found between the neural retina and the choroid. In fish, RPE has long apical projections enmeshed with the distal part of photoreceptors, reaching down to the level of their nuclei. The RPE disperses melanin pigment granules into the apical projections to shield light-sensitive photoreceptor outer segments from photobleaching when fish are under bright-light conditions. During development, RPE begin to respond to light at 5days post-fertilization, raising the question of whether responsiveness is correlated to receptor expression. Here, we isolate, clone and sequence chrm-odd receptor genes in zebrafish, characterize them phylogenetically and observe their expression in the eyes of the zebrafish at different developmental stages using RT-PCR and immunofluorescence microscopy. We find that zebrafish express six unique chrm-odd receptor subtypes: chrm1a, chrm1b, chrm3a, chrm3b, chrm5a and chrm5b - and these receptors are differentially expressed during development. Our phylogenetic analysis confirms the assignments of chrm1b and chrm5b, isolated here, as well as other muscarinic receptor genes and their duplicates and suggests previously described muscarinic receptors may need to be reclassified. Differences between the expression patterns of ostensibly duplicated genes raise the possibility that subtle differences between the duplicates may enable refined regulation of specific developmental events., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The diversity and distribution of salmonellae in freshwater biofilms were analyzed at a fine scale (i.e. in 20 locations from a 324 cm(2) area) for two sites in San Marcos, TX. A concrete storm water overflow channel (City Park) was sampled 4 times and a concrete surface in the spring-fed headwaters of the San Marcos River (Spring Lake) 5 times between April and September 2009, and each biofilm sample analyzed by a combination of traditional enrichment methods and molecular techniques. PCR detection of the invA gene, that encodes a protein of a type III secretion system present in salmonellae, after semi-selective enrichment of salmonellae was achieved in biofilms from all 20 locations at the City Park site, with locations generally being positive 2-3 times out of 4 sampling times for a total of 59% positive samples. InvA gene fragment detection in biofilms was less frequent for the 5 sampling times and 20 locations from the Spring Lake site (18% of all samples), with 1 sampling time being entirely negative and 8 locations remaining negative throughout the study. Rep-PCR fingerprinting of 491 Salmonella isolates obtained from both sites resulted in 30 distinct profiles, with 26 and 7 profiles retrieved from City Park and Spring Lake samples, respectively, and thus with 3 profiles present at both sites, and multiple strains frequently obtained from single locations at both sites. The composition of Salmonella strains in the area analyzed changed in time with large differences between early (April, June) and late sampling times (September) within and among sites, except for one strain (S12) that was present at almost all sampling times at both sites, though often at different locations within the area analyzed. These results demonstrate the presence of salmonellae in natural biofilms and a significant micro-heterogeneity with differences in diversity and persistence of salmonellae., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Effective management of wetland quantity and quality is crucial for effective conservation of declining amphibian populations. In particular, frogs and toads that employ aggregative breeding strategies may suffer negative population impacts in response to changes in availability of aquatic breeding habitat, including overabundance of suitable habitat, if density of conspecifics attending aggregations is positively correlated with reproductive success. Here we document such a positive relationship, potentially the first example of a component Allee effect in an anuran, in the critically endangered Houston toad (Bufo houstonensis). We assessed the relationship between mean yearly chorus size and reproductive success of males at the pond level using an information theoretic model selection approach and a two-sample t-test. The chosen model contained the single variable of mean yearly chorus size to predict probability of reproduction, as selected using the Akaike Information Criterion corrected for small sample size and Akaike weight. Mean chorus sizes were significantly higher among ponds exhibiting evidence of reproduction than in those that showed no evidence of reproduction. Our results suggest that chorusing alone is a poor proxy for inference of population stability and highlight a need for reassessment of widely-used amphibian monitoring protocols. Further, amphibian conservation efforts should account for potential Allee effects in order to optimize benefits and avoid underestimating critical population thresholds, particularly in species exhibiting rapid population declines.

A nested PCR protocol was used to analyze five endemic salamander species from Central Texas for the presence of the emerging pathogen, chytrid fungus (Batrachochytrium dendrobatidis). Chytrid fungus was detected from samples of each of the five species sampled: with low abundance, in the Texas salamander (Eurycea neotenes) (1 positive out of 16 individuals tested; 1/16), the Blanco River Springs salamander (E. pterophila) (1/20), the threatened San Marcos salamander (E. nana) (1/17), and the endangered Barton Springs salamander (E. sosorum) (1/7); much higher abundance was obtained for the Jollyville Plateau salamander (E. tonkawae) (6/14), which has recently been petitioned for addition to the USA endangered species list. With one exception, sequences of PCR products were identical to the 5.8S rRNA gene, and nearly so for the flanking internal transcribed spacer (ITS) regions of B. dendrobatidis which confirmed the detection of chytrid fungus, and thus demonstrated the presence of this pathogen in populations of endangered species in Central Texas. These confirmations were obtained from nonconsumptive tail clippings which confirms the applicability of historically collected samples from other studies in the examination of the fungus across time.

Sediments and water from the spring and slough arm of Spring Lake, the pristine headwaters of the San Marcos River, Texas, were analyzed for Salmonellae by culture and molecular techniques before and after three major precipitation events, each with intermediate dry periods. Polymerase chain reaction (PCR)-assisted analyses of enrichment cultures detected Salmonellae in samples after all three precipitation events, but failed to detect them immediately prior to the rainfall events. Detection among individual locations differed with respect to the precipitation event analyzed, and strains isolated were highly variable with respect to serovars. These results demonstrate that rainwater associated effects, most likely surface runoff, provide an avenue for short-term pollution of aquatic systems with Salmonellae that do not, however, appear to establish for the long-term in water nor sediments.

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.

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., ((c) 2008 Wiley-Liss, Inc.)

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.

Sediment, water, and fish gut samples taken at three sites near the headwaters of the San Marcos River, Texas, were analyzed for salmonellae Salmonella spp. by culture and molecular techniques. While enrichment cultures from sediment and water samples from the two uppermost sites were negative for salmonellae in polymerase chain reaction analyses, both sediment and water samples were positive at the downstream site. At all sites, salmonellae were present in the guts of different fishes (e.g., largemouth bass Micropterus salmoides, channel catfish Ictalurus punctatus, common carp Cyprinus carpio, and suckermouth catfish Hypostomus plecostomus). The highest percentage of detection (33% of analyzed fish) occurred at the downstream site, whereas detection percentages at the upper two sites were 18% and 17%. Detection of salmonellae was usually limited to one segment of the gut (i.e., upper or lower part). Serovars were highly variable among individuals and differed between the upper and lower gut in the only individual (a common carp) that had salmonellae in both gut segments. In situ hybridization demonstrated that salmonellae were normally associated with particulate material in the gut and occurred in highly variable numbers ranging from an occasional organism to a majority of the gut microbe population. These results demonstrate the presence of different serovars of potentially human pathogenic salmonellae among four ecologically distinct fishes within natural environments. They also suggest that salmonellae are not components of the indigenous microbial community in fish intestines but rather are ingested with particulate material.

Sediments and water from the slough arm of Spring Lake, the headwaters of the San Marcos River, Texas, USA, as well as swabs from biofilms on carapaces and from the cloacae of 18 common musk turtles (Sternotherus odoratus), 21 red-eared sliders (Trachemys scripta elegans), nine Texas river cooters (Pseudemys texana), one snapping turtle (Chelydra serpentina serpentina), and three Guadalupe spiny soft-shell turtles (Apalone spinifera guadalupensis), caught at the same site, were analyzed for salmonellae by culture and molecular techniques. Although enrichment cultures from sediment and water samples were negative for salmonellae in polymerase chain reaction (PCR)-based analyses, this technique detected salmonellae in the enrichments from both carapaces and cloacae of 11 musk turtles (61%), eight red-eared sliders (38%), and the snapping turtle. Salmonellae could also be detected in the enrichments from the carapaces of two additional red-eared sliders and two Texas river cooters; the remaining samples were negative. Further characterization of isolates obtained from the enrichment cultures of seven selected individuals that represented all turtle species with salmonellae confirmed the presence of Salmonella enterica subspecies enterica, with serovars Rubislaw, Newport, Gaminara, and Thompson identified. These results demonstrate the presence of different strains of potentially human pathogenic salmonellae naturally occurring on several turtle species with different life histories even within supposedly pristine environments.

Using a novel library of 5637 expressed sequence tags (ESTs) from the brain tissue of the Asian seabass (Lates calcarifer), we first characterized the brain transcriptome for this economically important species. The ESTs generated from the brain of L. calcarifer yielded 2410 unique transcripts (UTs) which comprise of 982 consensi and 1428 singletons. Based on database similarity, 1005 UTs (41.7%) can be assigned putative functions and were grouped into 12 functional categories related to the brain function. Amongst others, we have identified genes that are putatively involved in energy metabolism, ion pumps and channels, synapse related genes, neurotransmitter and its receptors, stress induced genes and hormone related genes. Subsequently we selected a putative preprocGnRH-II precursor for further characterization. The complete cDNA sequence of the gene obtained was found to code for an 85-amino acid polypeptide that significantly matched preprocGnRH-II precursor sequences from other vertebrates, and possesses structural characteristics that are similar to that of other species, consisting of a signal peptide (23 residues), a GnRH decapeptide (10 residues), an amidation/proteolytic-processing signal (glycine-lysine-argine) and a GnRH associated peptide (GAP) (49 residues). Phylogenetic analysis showed that this putative L. calcarifer preprocGnRH-II sequence is a member of the subcohort Euteleostei and divergent from the sequences of the subcohort Otocephalan. These findings provide compelling evidence that the putative L. calcarifer preprocGnRH-II precursor obtained in this study is orthologous to that of other vertebrates. The functional prediction of this preprocGnRH-II precursor sequence through in silico analyses emphasizes the effectiveness of the EST approach in gene identification in L. calcarifer.

The systematic relationships of snakes in the Lampropeltis mexicana complex (L. mexicana, L. alterna, and L. ruthveni) are poorly known despite several taxonomic studies over the last 80 years. Mitochondrial DNA sequences were used to infer the phylogeny of the L. mexicana complex. At least one representative sample from the nine currently recognized species of Lampropeltis was sequenced. Our results suggest that a deep basal split resulted in the divergence of two groups of Lampropeltis, with one group occupying the upland areas of western United States and most of western and central Mexico, and the other northeastern Mexico and the lowland areas of the southern United States. Results also revealed that the L. mexicana complex and Lampropeltis triangulum are polyphyletic, with taxa from both groups nested together in deeply divergent northern and southern clades. These results are incongruent with previous hypotheses of phylogenetic relationships based on morphology, and suggest that morphological characters shared among the various tri-colored Lampropeltis (e.g., hemipenal structure and tri-colored pattern) may be difficult to interpret phylogenetically.

Sediments and water from the pristine headwaters of the San Marcos River, Texas, USA, as well as swabs from biofilms on the carapace and from the cloacae of 17 musk turtles (Sternotherus odoratus) and one snapping turtle (Chelydra serpentina serpentina) caught at the same site, were analysed for salmonellae by culture and molecular techniques. Whereas enrichment cultures from sediment and water samples were negative for salmonellae in PCR- and in situ hybridization-based analyses, both techniques detected salmonellae after enrichments from both carapace and cloacae of nine (i.e. of 53%) musk turtles. Further characterization of 10 isolates obtained from the enrichment cultures of four selected individuals and confirmed as salmonellae by PCR analysis was achieved by fingerprinting techniques (rep-PCR). The results show differences between individuals and, in one case, variation among isolates from a single individual. All isolates from two individuals displayed identical profiles. These profiles were different from those obtained from the isolates of the third individual, which were, themselves, also identical for all isolates. Salmonellae were much more diverse in samples from the carapace of the last individual with five different rep-PCR profiles retrieved. Serotyping of seven isolates representative for each rep-PCR profile identified all isolates as representing Salmonella enterica subspecies enterica serotype Rubislaw, which demonstrates the presence of different strains of potentially human pathogenic salmonellae naturally occurring on turtles even within pristine environments. The frequent detection of these organisms in biofilms on the carapace opens the door for speculations on the role of this habitat as a reservoir for salmonellae, and on potential implications for turtles acting as a dispersal vector.

Dialysis tubing containing spent culture media, when placed in a lake, was colonized by a low diversity of bacteria, whereas abiotic controls had considerable diversity. Changes were seen in the presence and absence of acylated homoserine lactones, suggesting that these molecules and other factors may influence adherent-population composition.

The painted turtle, Chrysemys picta, is currently recognized as a continentally distributed polytypic species, ranging across North America from southern Canada to extreme northern Mexico. We analyzed variation in the rapidly evolving mitochondrial control region (CR) in 241 turtles from 117 localities across this range to examine whether the painted turtle represents a continentally distributed species based on molecular analysis. We found strong support for the novel hypothesis that C. p. dorsalis is the sister group to all remaining Chrysemys, with the remaining Chrysemys falling into a single, extremely wide-ranging and genetically undifferentiated species. Given our goal of an evolutionarily accurate taxonomy, we propose that two evolutionary lineages be recognized as species within Chrysemys: C. dorsalis (Agassiz 1857) in the southern Mississippi drainage region, and C. picta (Schneider 1783) from the rest of the range of the genus. Neither molecular nor recent morphological analyses argue for the hybrid origin of C. p. marginata as previously proposed. Within C. picta, we find evidence of at least two independent range expansions into previously glaciated regions of North America, one into New England and the other into the upper Midwest. We further find evidence of a massive extinction/recolonization event across the Great Plains/Rocky Mountain region encompassing over half the continental United States. The timing and extent of this colonization is consistent with a recently proposed regional aridification as the Laurentide ice sheets receded approximately 14,000 years ago, and we tentatively propose this paleoclimatological event as a major factor shaping genetic variation in Chrysemys.

The family Lemuridae includes four genera: Eulemur, Hapalemur, Lemur,Varecia. Taxonomy and phylogenetic relationships between L. catta, Eulemur and Hapalemur, and of Varecia to these other lemurids, continue to be hotly debated. Nodal relationships among the five Eulemur species also remain contentious. A mitochondrial DNA sequence dataset from the ND 3, ND 4 L, ND 4 genes and five tRNAs (Gly, Arg, His, Ser, Leu) was generated to try to clarify phylogenetic relationships w ithin the Lemuridae. Samples (n=39) from all ten lemurid species were collected and analysed. Three Daubentonia madagascariensis were included as outgroup taxa. The approximately 2400 bp sequences were analysed using maximum parsimony, neighbor-joining and maximum likelihood methods. The results support monophyly of Eulemur, a basal divergence of Varecia, and a sister-group relationship for Lemur/Hapalemur. Based on tree topology, bootstrap values, and pairwise distance comparisons, we conclude thatVarecia and Eulemur both represent distinct genera separate from L. catta. H. griseus andH. aureus form a clade with strong support, but the sequence data do not permit robust resolution of the trichotomy involving H. simus, H. aureus/H. griseus and L. catta. Within Eulemur there is strong support for a clade containing E. fulvus, E. mongoz and E. rubriventer. However, analyses failed to clearly resolve relationships among those three species or with the more distantly related E. coronatus and E. macaco. Our sequencing data support the current subspecific status of E.m. macaco and E.m. flavifrons, and that of V.v. variegata and V.v. rubra. However, tree topology and relatively large genetic distances among individual V.v. variegata indicate that there may be more phylogenetic structure within this taxon than is indicated by current taxonomy.

Historical biogeography is dominated by vicariance methods that search for a congruent pattern of fragmentation of ancestral distributions produced by shared Earth history. A focus of vicariant studies has been austral area relationships and the break-up of the supercontinent Gondwana. Chameleons are one of the few extant terrestrial vertebrates thought to have biogeographic patterns that are congruent with the Gondwanan break-up of Madagascar and Africa. Here we show, using molecular and morphological evidence for 52 chameleon taxa, support for a phylogeny and area cladogram that does not fit a simple vicariant history. Oceanic dispersal--not Gondwanan break-up--facilitated species radiation, and the most parsimonious biogeographic hypothesis supports a Madagascan origin for chameleons, with multiple 'out-of-Madagascar' dispersal events to Africa, the Seychelles, the Comoros archipelago, and possibly Reunion Island. Although dispersal is evident in other Indian Ocean terrestrial animal groups, our study finds substantial out-of-Madagascar species radiation, and further highlights the importance of oceanic dispersal as a potential precursor for speciation.

Cheirogaleidae currently comprises five genera whose relationships remain contentious. The taxonomic status and phylogenetic position of both Mirza coquereli and Allocebus trichotis are still unclear. The taxonomic status of the recently discovered Microcebus ravelobensis (a sympatric sibling species of Microcebus murinus) and its phylogenetic position also require further examination. A approximately 2.4-kb mitochondrial DNA sequence including part of the COIII gene, complete ND3, ND4L, and ND4 genes, and 5 tRNAs was used to clarify relationships among cheirogaleids. Mirza and Microcebus form a clade representing the sister group of Allocebus, with a clade containing Cheirogaleus major and Cheirogaleus medius diverging first. M. ravelobensis and Microcebus rufus form a subclade within Microcebus, with M. murinus as its sister group. The molecular data support the generic status of Mirza coquereli and species-level divergence of M. ravelobensis. Furthermore, "M. rufus" may well represent more than one species., (Copyright 2001 Academic Press.)

The sifakas (Propithecus) include three species containing up to 10 described subspecies, whose evolutionary relationships remain contentious. In particular, it is unclear whether P. verreauxi deckeni and P.v. coronatus populations are differentiated at the subspecific level. Furthermore, the taxonomic status of the recently discovered P. tattersalli and its phylogenetic position also require further examination. About 2,400 bp of mitochondrial DNA sequence data from part of the COIII gene, together with complete genes for ND3, ND4L, ND4, and five tRNAs, were used to clarify relationships among Propithecus species and subspecies. All analyses group Avahi as the sister group to all sifakas. P. diadema is placed as a sister group to all other Propithecus. Among the remaining sifakas, one subclade is formed by Puv. coquereli and P. tattersalli, while P.v. verreauxi, P.v. deckeni, and P.v. coronatus form the second subclade. All analyses fail to resolve P.vu. coronatus and P.v. deckeni into separate monophyletic lineages. Based on pairwise distance comparisons and tree topology, we conclude that P. tattersalli does not represent a distinct species and that P.v. deckeni and P.v. coronatus do not deserve subspecific rank. On the other hand, our analyses indicate that P.v. coquereli may well represent a separate species.

The brown lemurs (Eulemur fulvus) include seven subspecies, whose evolutionary relationships remain contentious. In particular, it is unclear whether the Malagasy and Comorian E. f. fulvus populations are differentiated at the subspecific level (E. f. mayottensis). Furthermore, it has been suggested that E. f. collaris and E. f. albocollaris are separate species. Analyses of approximately 2400 bp of mitochondrial DNA sequence data from part of the COIII gene, together with complete genes for ND3, ND4L, and ND4 and 5 tRNAs, resolved 34 E. fulvus specimens into six clades: ((albocollaris, collaris) (rufus (rufus (fulvus/mayottensis (albifrons/fulvus/sanfordi))))). Based on the information available and our analyses we conclude that E. f. albocollaris and E. f. collaris do not represent species distinct from E. fulvus and that Comorian brown lemurs do not deserve subspecific rank. No genetic differentiation was detected between E. f. albifrons and E. f. sanfordi; on the other hand, there are obviously two separate lineages of E. f. rufus., (Copyright 2000 Academic Press.)

The New World monkeys are divided into two main groups, Callitrichidae and Cebidae. Callimico goeldii shares traits with both the Cebidae and the Callitrichidae. Recent morphological phyletic studies generally place Callimico as the most basal member of the Callitrichidae. In contrast, genetic studies (immunological, restriction fragment, and sequence data) have consistently placed Callimico somewhere within the Callitrichidae, not basal to this clade. A DNA sequence data set from the terminal 236 codons of the mitochondrial ND4 gene and the tRNA(His), tRNA(Ser), and tRNA(Leu) genes was generated to clarify the position of Callimico. The sequences of 887 base pairs were analyzed by maximum-parsimony, neighbor-joining, and maximum-likelihood methods. The results of these various methods are generally congruent and place Callimico within the Callitrichidae between the marmosets (Callithrix and Cebuella) and the tamarins (Saguinus and Leontopithecus). Combined analyses of all suitable nuclear and mitochondrial gene sequences confirm the position of Callimico between the marmosets and the tamarins. As available molecular evidence indicates that Callimico is more closely related to the marmosets than to the tamarins, a reconsideration of the morphological evidence in light of the consensus tree from DNA sequence analyses is warranted. The marmosets and tamarins share four morphological characters (loss of the third molar, loss of the hypocone, reduced body size, reproductive twinning). Dwarfism may have evolved repeatedly among the Callitrichidae. It is well-known that the loss of a character can occur many times independently. The reproduction of marmosets and tamarins is extremely specialized and it is difficult to imagine that this complex and unique twinning system evolved separately in marmosets and tamarins. However, it is possible that a secondary reversal to single offspring took place in Callimico.

Snakes represent one of the most ubiquitous and successful groups of terrestrial vertebrates; however, many aspects of their evolutionary relationships remain uncertain. Previous research, which utilized morphological and immunological data, has not resolved the origin of snakes or clearly delineated the relationships between snakes and other lizards. A DNA sequence data set from the mitochondrial ND4 gene and the histidine, serine, and leucine tRNAs has been generated for use in the examination of these relationships. Parsimony analyses employing multiple outgroups resolve snakes within the lizard clade. Varanus is the sister group to the snakes in 81% of bootstrap replications using Bos as the outgroup specified, 85% using Trachemys, 57% using Alligator, and 80% using all three outgroups. The primitive, fossorial snake genera Leptotyphlops and Typhlops are basal to the other snakes and provide tentative molecular evidence in support of a fossorial or subfossorial origin of limblessness for the suborder Serpentes.