Patterns of differentiation and hybridization in North American wolflike canids, revealed by analysis of microsatellite loci

Genetic divergence and gene flow among closely related populations are difficult to measure because mutation rates of most nuclear loci are so low that new mutations have not had sufficient time to appear and become fixed. Microsatellite loci are repeat arrays of simple sequences that have high mutation rates and are abundant in the eukaryotic genome. Large population samples can be screened for variation by using the polymerase chain reaction and polyacrylamide gel electrophoresis to separate alleles. We analyzed 10 microsatellite loci to quantify genetic differentiation and hybridization in three species of North American wolflike canids. We expected to find a pattern of genetic differentiation by distance to exist among wolflike canid populations, because of the finite dispersal distances of individuals. Moreover, we predicted that, because wolflike canids are highly mobile, hybrid zones may be more extensive and show substantial changes in allele frequency, relative to nonhybridizing populations. We demonstrate that wolves and coyotes do not show a pattern of genetic differentiation by distance. Genetic subdivision in coyotes, as measured by theta and Gst, is not significantly different from zero, reflecting persistent gene flow among newly established populations. However, gray wolves show significant subdivision that may be either due to drift in past Ice Age refugia populations or a result of other causes. Finally, in areas where gray wolves and coyotes hybridize, allele frequencies of gray wolves are affected, but those of coyotes are not. Past hybridization between the two species in the south-central United States may account for the origin of the red wolf.      

Female marsh wrens do not provide evidence of anatomical specializations of song nuclei for perception of male song

In songbirds the forebrain nuclei HVC (high vocal center) and RA (robust nucleus of the archistriatum) are larger in individuals or species that produce larger song repertoires, but the extent to which the size of these nuclei reflects a need for either producing or perceiving large repertoires is unknown. We, therefore, tested the hypothesis that species differences in the size of song nuclei reflect a commitment of “brain space” to the perceptual processing of conspecific song. The two species of marsh wren (Cistothorus palustris western and eastern) provide a good test case. Western males produce larger song repertoires, and have larger HVC and RA than do eastern males. Female marsh wrens do not sing, and if they use their song nuclei to assess conspecific male song repertoires, then we predicted that measurable cellular and nuclear parameters of HVC and RA would be greater in western than eastern female wrens. For males we confirmed that the volumes of HVC and RA, and cellular parameters of HVC, are greater in western than in eastern birds. These nuclei were also considerably larger in males than in conspecific females. Western and eastern female wrens, however, did not differ in any measured parameters of HVC or RA. Females of these wren species thus do not provide any direct evidence of anatomical specializations of song nuclei for the perceptual processing of conspecific male song. 1994 John Wiley & Sons, Inc.     

A gas-liquid-chromatographic procedure for separating a wide range of metabolites occuring in urine or tissue extracts

1. A gas–liquid-chromatographic procedure is described which permits separation and identification on the same chromatogram of a wide range of substances occurring in urine or tissue extracts. The method uses hydrogen flame ionization, which detects organic compounds whether free or conjugated with no requirement for specific reactive groups. 2. For chromatography, carboxyl groups are quantitatively converted into methyl esters or trimethylsilyl esters. Phenolic, alcoholic and potential enolic groups are converted into trimethylsilyl ethers. Separations are carried out on a 6ft. column of either 10% F-60 (a polysiloxane) or 1% F-60, temperature programming at 2°/min. being used over such part of the temperature range 30°–260° as is required. Propionyl derivatives of hydroxy compounds can also be used, but only on a non-quantitative basis. Derivatives and columns have been selected for optimum range of usefulness when large numbers of samples are examined by using automated gas chromatography. 3. The method is applicable to: fatty acids above butyric acid; di- and tri-carboxylic acids; hydroxy acids and keto acids; polyhydroxy and alicyclic compounds such as glycerol, inositol, quinic acid, shikimic acid, ascorbic acid and sugar alcohols; aromatic hydroxy and acidic compounds, both benzenoid and indolic; sesquiterpenes; steroids; glycine conjugates; mercapturic acids; glucuronides. It is not satisfactory for sulphate conjugates, iminazoles or polypeptides. 4. Methylene units provide an accurate and reproducible parameter for characterizing peak position. Methylene unit values are reported for a large variety of substances occurring in, or related to those occurring in, urine and tissue extracts. 5. The nature of derivatives was confirmed by combining gas chromatography with mass spectrometry. Combined gas chromatography–mass spectrometry gives a diagnostic tool of great power in the evaluation of metabolic patterns, and various uses are discussed.     

Breeding and management of the common trumpeter (Psophia crepitans)

The breeding biology and management of three wild-caught adult Common trumpeters (one male and two females) was documented at the Woodland Park Zoological Gardens, Seattle, from April, 1984 to August, 1986. A total of 27 eggs were laid, and eight young survived to fledging. Both sexes exhibited crane-like dances during courtship, but the male appeared to perform these behaviors more frequently and with greater intensity than the females. Courtship feeding and allo-preening also occurred. Nests consisted of simple scrapes on the ground, but the birds had no opportunity to rest in trees or other elevated sites. Clutches contained two or three eggs, and the incubation period was aproximately 28 days. Although the breeding pair was generally aggressive toward the second female, all three adults participated in incubation and in caring for the young. Parental behavior consisted of brooding, allopreening, and feeding. The male preened and fed one chick significantly more often than either of the females. Trumpeter chicks were highly precocial, but grew relatively slowly, reaching 50% of adult weight by 45 to 50 days of age. Trumpeters are difficult to maintain and breed in captivity and appear to be susceptible to mycotic diseases, such as aspergillosis. Changes in the social composition of captive groups may result in improved breeding.     

Age dependent changes of a biochemical rhythm--the glycolytic oscillator of the blowfly Phormia terraenovae

1. By monitoring changes of fluorescence of NADH the frequencies, amplitudes and maximum slopes of the glycolytic oscillator of Phormia were analyzed in 5, 9, 15 and 21-day-old male flies. 2. In order to evaluate the possible existence of circadian rhythms within the oscillatory system, all determinations were repeated eight times/day. 3. In addition, the activities of three key enzymes of glycolysis, PFK, GAPDH and PK, which are central to the glycolytic oscillator were measured with respect to age and day time. 4. With increasing age the amplitudes of oscillations increased together with the maximum slopes of the oscillatory waves. The frequency appeared to be independent of age. 5. Variations of enzyme activities over the day indicated an age dependent circadian rhythm which, due to the simultaneous activity changes of the three measured enzymes, was not reflected in the whole oscillatory system. 6. The results suggest that modifications in the allosteric regulation of enzymes are responsible for the age dependent changes of the glycolytic oscillator.     

A novel approach to measuring heat flux in swimming animals

We present a design for long-term or removable attachment of heat flux sensors (HFSs) to stationary or swimming animals in water that enables collection of heat flux data on both captive and free-ranging pinnipeds. HFSs were modified to allow for independent, continuous, and long-term or removable attachment to study animals. The design was tested for effects of HFSs and the attachment mechanism on resultant heat flux. Effects were insulative and consistent across water temperatures and flow speeds, resulting in a correction factor of 3.42. This correction factor was applied to all measurements of heat flux from animal experiments to account for the thermal resistance of HFSs and insulative effects of the attachment mechanism. Heat flux and skin temperature data were collected from two captive Steller sea lions (Eumetopias jubatus) as they swam in a large habitat tank over time periods ranging from approximately 4 to 9 min. Of the 72 HFSs deployed using the attachment mechanism, data were successfully retrieved from 70. The HFS attachment mechanism was also used on two wild free-ranging Weddell seals (Leptonychotes weddellii) off Ross Island, Antarctica, for up to 7 days. Heat flux data were retrieved from all eight sensors deployed. These results, along with those from Steller sea lions, suggest that HFSs can be deployed with success on captive and wild animals using the designed attachment mechanism.     

In silico single strand melting curve: a new approach to identify nucleic acid polymorphisms in Totiviridae

Background

The PCR technique and its variations have been increasingly used in the clinical laboratory and recent advances in this field generated new higher resolution techniques based on nucleic acid denaturation dynamics. The principle of these new molecular tools is based on the comparison of melting profiles, after denaturation of a DNA double strand. Until now, the secondary structure of single-stranded nucleic acids has not been exploited to develop identification systems based on PCR. To test the potential of single-strand RNA denaturation as a new alternative to detect specific nucleic acid variations, sequences from viruses of the Totiviridae family were compared using a new in silico melting curve approach. This family comprises double-stranded RNA virus, with a genome constituted by two ORFs, ORF1 and ORF2, which encodes the capsid/RNA binding proteins and an RNA-dependent RNA polymerase (RdRp), respectively.

Results

A phylogenetic tree based on RdRp amino acid sequences was constructed, and eight monophyletic groups were defined. Alignments of RdRp RNA sequences from each group were screened to identify RNA regions with conserved secondary structure. One region in the second half of ORF2 was identified and individually modeled using the RNAfold tool. Afterwards, each DNA or RNA sequence was denatured in silico using the softwares MELTSIM and RNAheat that generate melting curves considering the denaturation of a double stranded DNA and single stranded RNA, respectively. The same groups identified in the RdRp phylogenetic tree were retrieved by a clustering analysis of the melting curves data obtained from RNAheat. Moreover, the same approach was used to successfully discriminate different variants of Trichomonas vaginalis virus, which was not possible by the visual comparison of the double stranded melting curves generated by MELTSIM.

Conclusion

In silico analysis indicate that ssRNA melting curves are more informative than dsDNA melting curves. Furthermore, conserved RNA structures may be determined from analysis of individuals that are phylogenetically related, and these regions may be used to support the reconstitution of their phylogenetic groups. These findings are a robust basis for the development of in vitro systems to ssRNA melting curves detection.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-243) contains supplementary material, which is available to authorized users.     

Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap

Mass accuracy is a key parameter of mass spectrometric performance. TOF instruments can reach low parts per million, and FT-ICR instruments are capable of even greater accuracy provided ion numbers are well controlled. Here we demonstrate sub-ppm mass accuracy on a linear ion trap coupled via a radio frequency-only storage trap (C-trap) to the orbitrap mass spectrometer (LTQ Orbitrap). Prior to acquisition of a spectrum, a background ion originating from ambient air is first transferred to the C-trap. Ions forming the MS or MS(n) spectrum are then added to this species, and all ions are injected into the orbitrap for analysis. Real time recalibration on the "lock mass" by corrections of mass shift removes mass error associated with calibration of the mass scale. The remaining mass error is mainly due to imperfect peaks caused by weak signals and is addressed by averaging the mass measurement over the LC peak, weighted by signal intensity. For peptide database searches in proteomics, we introduce a variable mass tolerance and achieve average absolute mass deviations of 0.48 ppm (standard deviation 0.38 ppm) and maximal deviations of less than 2 ppm. For tandem mass spectra we demonstrate similarly high mass accuracy and discuss its impact on database searching. High and routine mass accuracy in a compact instrument will dramatically improve certainty of peptide and small molecule identification.