Conservation genetics of Neotropical pollinators revisited: microsatellite analysis suggests that diploid males are rare in orchid bees

Allozyme analyses have suggested that Neotropical orchid bee (Euglossini) pollinators are vulnerable because of putative high frequencies of diploid males, a result of loss of sex allele diversity in small hymenopteran populations with single locus complementary sex determination. Our analysis of 1010 males from 27 species of euglossine bees sampled across the Neotropics at 2-11 polymorphic microsatellite loci revealed only 5 diploid males at an overall frequency of 0.005 (95% CIs 0.002-0.010); errors through genetic non-detection of diploid males were likely small. In contrast to allozyme-based studies, we detected very weak or insignificant population genetic structure, even for a pair of populations >500 km apart, possibly accounting for low diploid male frequencies. Technical flaws in previous allozyme-based analyses have probably led to considerable overestimation of diploid male production in orchid bees. Other factors may have a more immediate impact on population persistence than the genetic load imposed by diploid males on these important Neotropical pollinators.

Quaternary pollen analysis: recent progress in palaeoecology and palaeoclimatology

During the last decade Quaternary pollen analysis has developed towards improved pollen-taxonomical precision, automated pollen identification and more rigorous definition of pollen assemblage zones. There have been significant efforts to model the spatial representation of pollen records in lake sediments which is important for more precise interpretation of the pollen records in terms of past vegetation patterns. We review the difficulties in matching modelled post-glacial plant migration patterns with pollen-based palaeorecords and discuss the potential of DNA analysis of pollen to investigate the ancestry and past migration pathways of the plants. In population ecology there has been an acceleration of the widely advocated conceptual advance of pollen-analytical research from vaguely defined ‘environmental reconstructions’ towards investigating more precisely defined ecological problems aligned with the current ecological theories. Examples of such research have included an increasing number of investigations about the ecological impacts of past disturbances, often integrating pollen records with other palaeoecological data. Such an approach has also been applied to incorporate a time perspective to the questions of ecosystem restoration, nature conservation and forest management. New lines of research are the use of pollen analysis to study long-term patterns of vegetation diversity, such as the role of glacial-age vegetation fragmentation as a cause of Amazonian rain forest diversity, and to investigate links between pollen richness and past plant diversity. Palaeoclimatological use of pollen records has become more quantitative and has included more precise and rigorous testing of pollen-climate calibration models with modern climate data. These tests show the approximate nature of the models and warn against a too straightforward climatic interpretation of the small-scale variation in reconstructions. Pollenbased climate reconstructions over the Late Glacial–early Holocene boundary have indicated that pollen-stratigraphical changes have been rapid with no evidence for response lags. This does not rule out the possibility of migrational disequilibrium, however, as the rapid changes may be mostly due to nonmigrational responses of existing vegetation. It is therefore difficult to assess whether the amplitude of reconstructed climate change reflects real climate change. Other outstanding problems remain the obscure relationship of pollen production and climate, the role of human impact and other nonclimatic factors, and nonanalogue situations.

The importance of reproductive strategies in population genetic approaches to conservation: an example from the marine angiosperm genus Zostera

Knowledge of the levels of genetic diversity maintained in natural populations can play a central role in conservation programmes, particularly in threatened habitats or species. Fluctuations in population size can lead to loss of variation and, consequently, increase the risk of extinction. We have examined whether such a genetic bottleneck has occurred in populations of two species in the seagrass genus Zostera, which are believed to have been affected by an outbreak of wasting disease at the start of the last century. A test for heterozygote excess at five nuclear microsatellite loci did not suggest the occurrence of a genetic bottleneck, but analysis of seven chloroplast microsatellite loci and sequence data from two regions did suggest a bottleneck in the chloroplast genome. Extremely low levels of between-population diversity suggest that all subpopulations can be treated as a single management unit for each species. Comparable levels of nuclear genetic diversity were found in the three populations of the primarily sexual Zostera marina var. angustifolia studied but a wider range of within-population diversity was found in Zostera noltii, which displays both. sexual and vegetative reproductive strategies. This may be due to an increase in sexual recruitment due to localised fresh water inflow into the study site near to the most diverse population. Such populations should be prioritised as source material for any replanting or remediation due to natural or anthropogenic loss of Zostera beds in the area.

Genetic differentiation of Euterpe edulis Mart. populations estimated by AFLP analysis

Heart-of-palm (Euterpe edulis Mart.) is a wild palm with a wide distribution throughout the Atlantic Rainforest. Populations of E. edulis represent important renewable natural resources but are currently under threat from predatory exploitation. Furthermore, because the species is indigenous to the Atlantic Rainforest, which is located in the most economically developed and populated region of Brazil, social and economic pressures have devastated heart-of-palm forests. In order to estimate the partitioning of genetic variation of endangered E. edulis populations, 429 AFLP markers were used to analyse 150 plants representing 11 populations of the species distribution range. Analysis of the genetic structure of populations carried out using analysis of molecular variance (AMOVA) revealed moderate genetic variation within populations (57.4%). Genetic differentiation between populations (F-ST = 0.426) was positively correlated with geographical distance. These results could be explained by the historical fragmentation of the Atlantic coastal region, together with the life cycle and mating system The data obtained in this work should have important implications for conservation and future breeding programmes of E. edulis.

Comparative genomic analysis reveals species-dependent complexities that explain difficulties with microsatellite marker development in molluscs

Reliable population DNA molecular markers are difficult to develop for molluscs, the reasons for which are largely unknown. Identical protocols for microsatellite marker development were implemented in three gastropods. Success rates were lower for Gibbula cineraria compared to Littorina littorea and L. saxatilis. Comparative genomic analysis of 47.2?kb of microsatellite containing sequences (MCS) revealed a high incidence of cryptic repetitive DNA in their flanking regions. The majority of these were novel, and could be grouped into DNA families based upon sequence similarities. Significant inter-specific variation in abundance of cryptic repetitive DNA and DNA families was observed. Repbase scans show that a large proportion of cryptic repetitive DNA was identified as transposable elements (TEs). We argue that a large number of TEs and their transpositional activity may be linked to differential rates of DNA multiplication and recombination. This is likely to be an important factor explaining inter-specific variation in genome stability and hence microsatellite marker development success rates. Gastropods also differed significantly in the type of TEs classes (autonomous vs non-autonomous) observed. We propose that dissimilar transpositional mechanisms differentiate the TE classes in terms of their propensity for transposition, fixation and/or silencing. Consequently, the phylogenetic conservation of non-autonomous TEs, such as CvA, suggests that dispersal of these elements may have behaved as microsatellite-inducing elements. Results seem to indicate that, compared to autonomous, non-autonomous TEs maybe have a more active role in genome rearrangement processes. The implications of the findings for genomic rearrangement, stability and marker development are discussed.

Aligning conservation priorities across taxa in Madagascar with high-resolution planning tools

Globally, priority areas for biodiversity are relatively well known, yet few detailed plans exist to direct conservation action within them, despite urgent need. Madagascar, like other globally recognized biodiversity hot spots, has complex spatial patterns of endemism that differ among taxonomic groups, creating challenges for the selection of within-country priorities. We show, in an analysis of wide taxonomic and geographic breadth and high spatial resolution, that multitaxonomic rather than single-taxon approaches are critical for identifying areas likely to promote the persistence of most species. Our conservation prioritization, facilitated by newly available techniques, identifies optimal expansion sites for the Madagascar government's current goal of tripling the land area under protection. Our findings further suggest that high-resolution multitaxonomic approaches to prioritization may be necessary to ensure protection for biodiversity in other global hot spots.

Microsatellite standardization and evaluation of genotyping error in a large multi-partner research programme for conservation of Atlantic salmon (Salmo salar L.).

Microsatellite genotyping is a common DNA characterization technique in population, ecological and evolutionary genetics research. Since different alleles are sized relative to internal size-standards, different laboratories must calibrate and standardize allelic designations when exchanging data. This interchange of microsatellite data can often prove problematic. Here, 16 microsatellite loci were calibrated and standardized for the Atlantic salmon, Salmo salar, across 12 laboratories. Although inconsistencies were observed, particularly due to differences between migration of DNA fragments and actual allelic size ('size shifts'), inter-laboratory calibration was successful. Standardization also allowed an assessment of the degree and partitioning of genotyping error. Notably, the global allelic error rate was reduced from 0.05 ± 0.01 prior to calibration to 0.01 ± 0.002 post-calibration. Most errors were found to occur during analysis (i.e. when size-calling alleles; the mean proportion of all errors that were analytical errors across loci was 0.58 after calibration). No evidence was found of an association between the degree of error and allelic size range of a locus, number of alleles, nor repeat type, nor was there evidence that genotyping errors were more prevalent when a laboratory analyzed samples outside of the usual geographic area they encounter. The microsatellite calibration between laboratories presented here will be especially important for genetic assignment of marine-caught Atlantic salmon, enabling analysis of marine mortality, a major factor in the observed declines of this highly valued species.

Towards a strategy for the conservation of the freshwater pearl mussel Margaritifera margaritifera in County Donegal, Ireland

This study attempts to identify the habitat requirements of the pearl mussel Margaritifea margaritifera in County Donegal, in north west Ireland, an area with little urban, industrial or intensive agricultural development. No mussels occur in rivers where calcium and conductivity levels are high or where the substratum is predominantly bedrock or fine sediment but it was not possible to distinguish clearly between mussel and non-mussel sites on the basis of ordination analysis. However, rivers which still support mussels and rivers with historical records of mussels are loosely grouped. Rivers which formerly supported mussels but lack living M. margaritifera appear to have suitable habitat for mussels; pearl fishing is the most likely reason for the extinction of these mussel populations. Where population densities are high, for example in locations on the rivers Eske, Clady and Owenea, conservation may necessitate the establishment of reserves. The prospect for the successful reintroduction of mussels into former mussel rivers such as the Finn and Eany Water, where suitable habitat exists and water quality is high, is very good.

Novel Analysis of Oceanic Surface Water Metagenomes Suggests Importance of Polyphosphate Metabolism in Oligotrophic Environments

Polyphosphate is a ubiquitous linear homopolymer of phosphate residues linked by high-energy bonds similar to those found in ATP. It has been associated with many processes including pathogenicity, DNA uptake and multiple stress responses across all domains. Bacteria have also been shown to use polyphosphate as a way to store phosphate when transferred from phosphate-limited to phosphate-rich media - a process exploited in wastewater treatment and other environmental contaminant remediation. Despite this, there has, to date, been little research into the role of polyphosphate in the survival of marine bacterioplankton in oligotrophic environments. The three main proteins involved in polyphosphate metabolism, Ppk1, Ppk2 and Ppx are multi-domain and have differential inter-domain and inter-gene conservation, making unbiased analysis of relative abundance in metagenomic datasets difficult. This paper describes the development of a novel Isofunctional Homolog Annotation Tool (IHAT) to detect homologs of genes with a broad range of conservation without bias of traditional expect-value cutoffs. IHAT analysis of the Global Ocean Sampling (GOS) dataset revealed that genes associated with polyphosphate metabolism are more abundant in environments where available phosphate is limited, suggesting an important role for polyphosphate metabolism in marine oligotrophs.

Functional analysis of the large periplasmic loop of the Escherichia coli K-12 WaaL O-antigen ligase

WaaL is a membrane enzyme implicated in ligating undecaprenyl-diphosphate (Und-PP)-linked O antigen to lipid A-core oligosaccharide. We determined the periplasmic location of a large (EL5) and small (EL4) adjacent loops in the Escherichia coli K-12 WaaL. Structural models of the EL5 from the K-12, R1 and R4 E. coli ligases were generated by molecular dynamics. Despite the poor amino acid sequence conservation among these proteins, the models afforded similar folds consisting of two pairs of almost perpendicular alpha-helices. One alpha-helix in each pair contributes a histidine and an arginine facing each other, which are highly conserved in WaaL homologues. Mutations in either residue rendered WaaL non-functional, since mutant proteins were unable to restore O antigen surface expression. Replacements of residues located away from the putative catalytic centre and non-conserved residues within the centre itself did not affect ligation. Furthermore, replacing a highly conserved arginine in EL4 with various amino acids inactivates WaaL function, but functionality reappears when the positive charge is restored by a replacement with lysine. These results lead us to propose that the conserved amino acids in the two adjacent periplasmic loops could interact with Und-PP, which is the common component in all WaaL substrates.

Proteomics and phylogenetic analysis of the cathepsin L protease family of the helminth pathogen Fasciola hepatica:Expansion of a repertoire of virulence-associated factors

Cathepsin L proteases secreted by the helminth pathogen Fasciola hepatica have functions in parasite virulence including tissue invasion and suppression of host immune responses. Using proteomics methods alongside phylogenetic studies we characterized the profile of cathepsin L proteases secreted by adult F. hepatica and hence identified those involved in host-pathogen interaction. Phylogenetic analyses showed that the Fasciola cathepsin L gene family expanded by a series of gene duplications followed by divergence that gave rise to three clades associated with mature adult worms (Clades 1, 2, and 5) and two clades specific to infective juvenile stages (Clades 3 and 4). Consistent with these observations our proteomics studies identified representatives from Clades 1, 2, and 5 but not from Clades 3 and 4 in adult F. hepatica secretory products. Clades 1 and 2 account for 67.39 and 27.63% of total secreted cathepsin Ls, respectively, suggesting that their expansion was positively driven and that these proteases are most critical for parasite survival and adaptation. Sequence comparison studies revealed that the expansion of cathepsin Ls by gene duplication was followed by residue changes in the S2 pocket of the active site. Our biochemical studies showed that these changes result in alterations in substrate binding and suggested that the divergence of the cathepsin L family produced a repertoire of enzymes with overlapping and complementary substrate specificities that could cleave host macromolecules more efficiently. Although the cathepsin Ls are produced as zymogens containing a prosegment and mature domain, all secreted enzymes identified by MS were processed to mature active enzymes. The prosegment region was highly conserved between the clades except at the boundary of prosegment and mature enzyme. Despite the lack of conservation at this section, sites for exogenous cleavage by asparaginyl endopeptidases and a Leu-Ser[downward arrow]His motif for autocatalytic cleavage by cathepsin Ls were preserved.

Conservation genetics of Ireland's sole population of the River water crowfoot (Ranunculus fluitans Lam.)

Populations of many freshwater species are becoming increasingly threatened as a result of a wide range of anthropogenically mediated factors. In the present study, we wanted to assess levels and patterns of genetic diversity in Ireland's sole population of the River water crowfoot (Ranunculus fluitans), which is restricted to a 12 km stretch of a single river, to assist the formation of conservation strategies. Analysis using amplified fragment length polymorphism (AFLP) indicated comparable levels of genetic diversity to those exhibited by a more extensive population of the species in England, and revealed no evidence of clonal reproduction. Allele-specific PCR analysis of five nuclear single nucleotide polymorphisms (SNPs) indicated no evidence of hybridization with its more abundant congener Ranunculus penicillatus, despite previous anecdotal reports of the occurrence of hybrids. Although the population currently exhibits healthy levels of genetic diversity and is not at risk of genetic assimilation via hybridization with R. penicillatus, it still remains vulnerable to other factors such as stochastic events and invasive species. © 2013 Elsevier B.V. All rights reserved.

Population enumeration and assessing conservation status in a widespread amphibian: A case study of Rana temporaria in Ireland

Global amphibian declines are a major element of the current biodiversity crisis. Monitoring changes in the distribution and abundance of target species is a basic component in conservation decision making and requires robust and repeatable sampling. For EU member states, surveillance of designated species, including the common frog Rana temporaria, is a formal requirement of the 'EC Habitats & Species Directive'. We deployed established methods for estimating frog population density at local water bodies and extrapolated these to the national and ecoregion scale. Spawn occurred at 49.4% of water bodies and 70.1% of independent 500-m survey squares. Using spawn mat area, we estimated the number of adult breeding females and subsequently the total population assuming a sex ratio of 1:1. A negative binomial model suggested that mean frog density was 23.5 frogsha [95% confidence interval (CI) 14.9-44.0] equating to 196M frogs (95%CI 124M-367M) throughout Ireland. A total of 86% of frogs bred in drainage ditches, which were a notably common feature of the landscape. The recorded distribution of the species did not change significantly between the last Article 17 reporting period (1993-2006) and the current period (2007-2011) throughout the Republic of Ireland. Recording effort was markedly lower in Northern Ireland, which led to an apparent decline in the recorded distribution. We highlight the need to coordinate biological surveys between adjacent political jurisdictions that share a common ecoregion to avoid apparent disparities in the quality of distributional information. Power analysis suggested that a reduced sample of 40-50 survey squares is sufficient to detect a 30% decline (consistent with the International Union for Conservation of Nature Category of 'Vulnerable') at 80% power providing guidance for minimizing future survey effort. Our results provin assessments for R. temporaria and other clump-spawning amphibians. 2013 The Zoological Society of London.

Restrospective genetic monitoring of the threatened Yellow marsh saxifrage (Saxifraga hirculus) reveals genetic erosion but provides valuable information for conservation strategies

Aim: Retrospective genetic monitoring, comparing genetic diversity of extant populations with historical samples, can provide valuable and often unique insights into evolutionary processes informing conservation strategies. The Yellow marsh saxifrage (Saxifraga hirculus) is listed as ‘critically endangered’ in Ireland with only two extant populations. We quantified genetic changes over time and identified genotypes in extant populations that could be used as founders for reintroductions to sites where the species is extinct.

Location: Ireland.

Methods: Samples were obtained from both locations where the species is currently found, including the most threatened site at the Garron Plateau, Co. Antrim, which held only 13 individuals during 2011. Herbarium samples covering the period from 1886 to 1957 were obtained including plants from the same area as the most threatened population, as well as three extinct populations. In total, 422 individuals (319 present-day and 103 historical) were genotyped at six microsatellite loci. Species distribution modelling was used to identify areas of potentially suitable habitat for reintroductions.

Results: Level of phenotypic diversity within the most threatened population was significantly lower in the present-day compared with historical samples but levels of observed heterozygosity and number of alleles, whilst reduced, did not differ significantly. However, Bayesian clustering analysis suggested gradual lineage replacement over time. All three measures of genetic diversity were generally lower at the most threatened population compared with the more substantial extant populations in Co. Mayo. Species distribution modelling suggested that habitat at one site where the species is extinct may be suitable for reintroduction.

Main conclusions: The dominant genetic lineage in the most threatened population is rare elsewhere; thus, care needs to be taken when formulating any potential reintroduction programme. Our findings highlight both the need for genetic monitoring of threatened populations, but also for its swift implementation before levels of diversity become critically low.

Spatial and Temporal Changes in Soil Organic Carbon Assessment using an Integrated Geostatistical and Compositional Data Analysis Approach

The Irish and UK governments, along with other countries, have made a commitment to limit the concentrations of greenhouse gases in the atmosphere by reducing emissions from the burning of fossil fuels. This can be achieved (in part) through increasing the sequestration of CO2 from the atmosphere including monitoring the amount stored in vegetation and soils. A large proportion of soil carbon is held within peat due to the relatively high carbon density of peat and organic-rich soils. This is particularly important for a country such as Ireland, where some 16% of the land surface is covered by peat. For Northern Ireland, it has been estimated that the total amount of carbon stored in vegetation is 4.4Mt compared to 386Mt stored within peat and soils. As a result it has become increasingly important to measure and monitor changes in stores of carbon in soils. The conservation and restoration of peat covered areas, although ongoing for many years, has become increasingly important. This is summed up in current EU policy outlined by the European Commission (2012) which seeks to assess the relative contributions of the different inputs and outputs of organic carbon and organic matter to and from soil. Results are presented from the EU-funded Tellus Border Soil Carbon Project (2011 to 2013) which aimed to improve current estimates of carbon in soil and peat across Northern Ireland and the bordering counties of the Republic of Ireland.
Historical reports and previous surveys provide baseline data. To monitor change in peat depth and soil organic carbon, these historical data are integrated with more recently acquired airborne geophysical (radiometric) data and ground-based geochemical data generated by two surveys, the Tellus Project (2004-2007: covering Northern Ireland) and the EU-funded Tellus Border project (2011-2013) covering the six bordering counties of the Republic of Ireland, Donegal, Sligo, Leitrim, Cavan, Monaghan and Louth. The concept being applied is that saturated organic-rich soil and peat attenuate gamma-radiation from underlying soils and rocks. This research uses the degree of spatial correlation (coregionalization) between peat depth, soil organic carbon (SOC) and the attenuation of the radiometric signal to update a limited sampling regime of ground-based measurements with remotely acquired data. To comply with the compositional nature of the SOC data (perturbations of loss on ignition [LOI] data), a compositional data analysis approach is investigated. Contemporaneous ground-based measurements allow corroboration for the updated mapped outputs. This provides a methodology that can be used to improve estimates of soil carbon with minimal impact to sensitive habitats (like peat bogs), but with maximum output of data and knowledge.