Using a nitrogen and oxygen isotopic approach to identify nitrate sources and cycling in the Wei River of northwestern China

The Wei River is the largest tributary of the Yellow River in China. To understand the sources and cycling of nitrate in the Wei River, we determined the concentrations and nitrogen and oxygen isotopic values of nitrate from water samples. Our results revealed that NO₃^?-N dominated the inorganic N and ranged from 0.1 to 8.8 mg/L (averaging 3.3 mg/L). Although this NO₃^?-N concentration does not exceed the World Health Organization's drinking water standard of 10 mg/L, the NO₃^?-N content of most water samples exceeded 3 mg/L, indicating poor water quality. The NO₃^?-N concentrations and δ¹⁵N-NO₃^? values demonstrate that there are significant differences in the spatial distribution of nitrogen between the tributaries and the main stream of the Wei River. In addition, a negative linear relationship (r² = 0.63) between NO₃^?-N concentrations and δ¹⁸O-NO₃^? values suggests mixing between two distinct sources (fertilizer and manure or sewage). Furthermore, we infer that the main source of nitrate is not manure or sewage itself, but rather the nitrification of NH₄⁺ in manure and sewage. Finally, no obvious denitrification processes were observed. These results expand our understanding of sewage as a major source of nitrate to the Wei River, emphasizing the role of nitrification.     

Temporal trends in genetic data and effective population size support efficacy of management practices in critically endangered dusky gopher frogs (Lithobates sevosus)

Monitoring temporal changes in population genetic diversity and effective population size can provide vital information on future viability. The dusky gopher frog, Lithobates sevosus, is a critically endangered species found only in coastal Mississippi, with low genetic variability as a consequence of isolation and population size reduction. Conservation management practices have been implemented, but their efficacy has not been addressed. We genotyped individuals collected 1997–2014 to determine temporal trends in population genetic variation, structure, and effective size. Observed and expected heterozygosity and allelic richness revealed temporally stable, but low, levels of genetic variation. Positive levels of inbreeding were found in each year. There was weak genetic structure among years, which can be attributed to increased effects of genetic drift and inbreeding in small populations. L. sevosus exhibited an increase in effective population size, and currently has an estimated effective size of 33.0–58.6 individuals, which is approximately half the census size. This large ratio could possibly be explained by genetic compensation. We found that management practices have been effective at maintaining and improving effective size and genetic diversity, but that additional strategies need to be implemented to enhance viability of the species.     

Using predictive modelling to guide the conservation of a critically endangered coastal wetland amphibian

Amphibians are the most threatened Class of vertebrate, with wetland-associated anurans in particular suffering high levels of habitat loss. We used predictive modelling to better understand the distribution of a critically endangered South African endemic (Hyperolius pickersgilli) and to guide conservation action. MaxEnt distribution models were produced based on limited occurrence data. Predicted localities with probability of occurrence ≥60% were surveyed. Ten new sub-populations were discovered. The mean probability of occurrence for the species at wetlands where it was detected was greater than that at wetlands where it was not detected or absent. In addition, 17 known historical localities were re-visited and the species deemed absent at 8 of these. The total number of localities at which the species is now known to occur is 18, which is an increase in the known extant sub-populations of six. We recalculate the area of occupancy and extent of occurrence for the species as 108 km² and 2081.5 km², respectively; both increases on previous estimates. Implications of these changes on the IUCN Red List status of H. pickersgilli are discussed. A friction map was created to identify possible linkages between sub-populations, which can be used to guide habitat restoration and population repatriation. Given the degree of isolation of subpopulations and the potentially severe threats to most of these, urgent conservation action for H. pickersgilli remains crucial. This study provides a method for use in conservation planning for wetland-breeding amphibians in eastern coastal regions of Africa and elsewhere.     

Fine-scale spatial genetic structure and gene flow in a small, fragmented population of Sinojackia rehderiana (Styracaceae), an endangered tree species endemic to China

Populations of Sinojackia rehderiana are highly threatened and have small and scattered distribution due to habitat fragmentation and human activities. Understanding changes in genetic diversity, the fine-scale spatial genetic structure (SGS) at different life stages and gene flow of S. rehderiana is critical for developing successful conservation strategies for fragmented populations of this endangered species. In this study, 208 adults, 114 juveniles and 136 seedlings in a 50 × 100-m transect within an old-growth forest were mapped and genotyped using eight microsatellite makers to investigate the genetic diversity and SGS of this species. No significant differences in genetic diversity among different life-history stages were found. However, a significant heterozygote deficiency in adults and seedlings may result from substantial biparental inbreeding. Significant fine-scale spatial structure was found in different life-history stages within 19 m, suggesting that seed dispersal mainly occurred near a mother tree. Both historical and contemporary estimates of gene flow (13.06 and 16.77 m) indicated short-distance gene dispersal in isolated populations of S. rehderiana. The consistent spatial structure revealed in different life stages is most likely the result of limited gene flow. Our results have important implications for conservation of extant populations of S. rehderiana. Measures for promoting pollen flow should be taken for in situ conservation. The presence of a SGS in fragmented populations implies that seeds for ex situ conservation should be collected from trees at least 19-m apart to reduce genetic similarity between neighbouring individuals.     

Using eggshell membranes as a non-invasive tool to investigate the source of nutrients in avian eggs

Development of minimally invasive techniques to collect nutritional information from free-living birds is desirable for both ethical and conservation reasons. Here, we explore the utility of waterfowl eggshell membranes to determine the nutrient source of egg formation by using stable isotope ratios. We compared δ¹³C and δ¹⁵N of membranes from complete king eider (Somateria spectabilis) eggs to membranes of hatched or depredated eggs of the same clutch remaining after incubation. Despite large variation among membranes (δ¹³C: −26 to −14‰) we found a highly predictable relationship between δ¹³C of complete egg membranes and remaining (hatched or depredated) membranes from the same clutch. We did not find a consistent change in either δ¹³C or δ¹⁵N of eggshell membranes during incubation. We suggest that isotope ratios of membranes can be used to determine the source of exogenous nutrients for egg production in income breeders, and that membranes may offer a clutch-specific reference point for dietary nutrients (‘income endpoint’) in isotopic mixing models quantifying nutrient allocation in capital or mixed-strategy breeders.     

Using genotype simulations and Bayesian analyses to identify individuals of hybrid origin in Australian bass: lessons for fisheries management

Simulated genotypes, Bayesian analyses and molecular genetic data were used to detect individuals of hybrid origin and hybrid introgression between the Australian bass ( Macquaria novemaculeata ), a species extensively stocked in Australia, and estuary perch ( Macquaria colonorum ). Based on this analytical framework, 93% of the hybrids up to three generations later could be distinguished from the Australian bass. Individuals of hybrid origin were identified in all three rivers sampled. In addition, this study verified the fertility of hybrids between Australian bass and estuary perch as determined through genomic introgression. This study exemplifies an analytical procedure that has implications for identifying suitable individuals for use in breeding and restocking programmes for other species.     

Using connectivity to identify climatic drivers of local adaptation: a response to Macdonald et al.

Macdonald et al. (Ecol. Lett., 21, 2018, 207–216) proposed an analytical framework for identifying evolutionary processes underlying trait‐environment relationships observed in natural populations. Here, we propose an expanded and refined framework based on simulations and bootstrap‐based approaches, and we elaborate on an important statistical caveat common to most datasets.     

Genetic diversity of Sinojackia dolichocarpa (Styracaceae), a species endangered and endemic to China,detected by inter-simple sequence repeat (ISSR)

Sinojackia dolichocarpa, a species endangered and endemic to China, is distributed only in the regional area of Shimen and Sangzhi in Hunan Province. Inter-simple sequence repeat (ISSR) markers were used to investigate the genetic diversity within and among the four natural populations of S. dolichocarpa. Leaf samples were collected from 84 individuals. Thirteen ISSR primers selected from 80 primers gave rise to 137 discernible DNA bands of which 100 (72.99%) were polymorphic. On average each primer gave rise to 10.5 bands including 7.7 bands with polymorphic profile. At the species level, high genetic diversity was detected (PPB: 72.99%; H_E: 0.2255; Ho: 0.3453). However, relatively low genetic diversity existed within populations. Population in Maozhuhe (MZH) exhibits the greatest level of variability (PPB: 40.38%, H_E: 0.1566, Ho: 0.2330), whereas the population in Jingguanmen (JGM) finds its own variability at the lowest level (PPB: 30.66%; H_E: 0.1078; Ho: 0.1601). A high level of genetic differentiation among populations was revealed by Nei's gene diversity statistics (45.30%), Shannon's information measure (45.24%) and analysis of molecular variance (AMOVA) (52.88%). The main factors responsible for the high level of differentiation among populations are probably related to the selfing reproductive system and the isolation of populations. The strong genetic differentiation among populations indicates that the management for the conservation of genetic variability in S. dolichocarpa should aim to preserve every population.     

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.     

Using a strategy based on the concept of convergent evolution to identify residue substitutions responsible for thermal adaptation

Factors that are related to thermostability of proteins have been extensively studied in recent years, especially by comparing thermophiles and mesophiles. However, most of them are global characters. It is still not clear how to identify specific residues or fragments which may be more relevant to protein thermostability. Moreover, some of the differences among the thermophiles and mesophiles may be due to phylogenetic differences instead of thermal adaptation. To resolve these problems, I adopted a strategy to identify residue substitutions evolved convergently in thermophiles or mesophiles. These residues may therefore be responsible for thermal adaptation. Four classes of genomes were utilized in this study, including thermophilic archaea, mesophilic archaea, thermophilic bacteria, and mesophilic bacteria. For most clusters of orthologous groups (COGs) with sequences from all of these four classes of genomes, I can identify specific residues or fragments that may potentially be responsible for thermal adaptation. Functional or structural constraints (represented as sequence conservation) were suggested to have higher impact on thermal adaptation than secondary structure or solvent accessibility does. I further compared thermophilic archaea and mesophilic bacteria, and found that the most diverged fragments may not necessarily correspond to the thermostability-determining ones. The usual approach to compare thermophiles and mesophiles without considering phylogenetic relationships may roughly identify sequence features contributing to thermostability; however, to specifically identify residue substitutions responsible for thermal adaptation, one should take sequence evolution into consideration.