An optimized method for the extraction of ancient eukaryote DNA from marine sediments

Marine sedimentary ancient DNA (sedaDNA) provides a powerful means to reconstruct marine palaeo‐communities across the food web. However, currently there are few optimized sedaDNA extraction protocols available to maximize the yield of small DNA fragments typical of ancient DNA (aDNA) across a broad diversity of eukaryotes. We compared seven combinations of sedaDNA extraction treatments and sequencing library preparations using marine sediments collected at a water depth of 104 m off Maria Island, Tasmania, in 2018. These seven methods contrasted frozen versus refrigerated sediment, bead‐beating induced cell lysis versus ethylenediaminetetraacetic acid (EDTA) incubation, DNA binding in silica spin columns versus in silica‐solution, diluted versus undiluted DNA in shotgun library preparations to test potential inhibition issues during amplification steps, and size‐selection of low molecular‐weight (LMW) DNA to increase the extraction efficiency of sedaDNA. Maximum efficiency was obtained from frozen sediments subjected to a combination of EDTA incubation and bead‐beating, DNA binding in silica‐solution, and undiluted DNA in shotgun libraries, across 45 marine eukaryotic taxa. We present an optimized extraction protocol integrating these steps, with an optional post‐library LMW size‐selection step to retain DNA fragments of ≤500 base pairs. We also describe a stringent bioinformatic filtering approach for metagenomic data and provide a comprehensive list of contaminants as a reference for future sedaDNA studies. The new extraction and data‐processing protocol should improve quantitative paleo‐monitoring of eukaryotes from marine sediments, as well as other studies relying on the detection of highly fragmented and degraded eukaryote DNA in sediments.     

Arctic shrub colonization lagged peak postglacial warmth: Molecular evidence in lake sediment from Arctic Canada

Arctic shrubification is an observable consequence of climate change, already resulting in ecological shifts and global‐scale climate feedbacks including changes in land surface albedo and enhanced evapotranspiration. However, the rate at which shrubs can colonize previously glaciated terrain in a warming world is largely unknown. Reconstructions of past vegetation dynamics in conjunction with climate records can provide critical insights into shrubification rates and controls on plant migration, but paleoenvironmental reconstructions based on pollen may be biased by the influx of exotic pollen to tundra settings. Here, we reconstruct past plant communities using sedimentary ancient DNA (sedaDNA), which has a more local source area than pollen. We additionally reconstruct past temperature variability using bacterial cell membrane lipids (branched glycerol dialkyl glycerol tetraethers) and an aquatic productivity indicator (biogenic silica) to evaluate the relative timing of postglacial ecological and climate changes at a lake on southern Baffin Island, Arctic Canada. The sedaDNA record tightly constrains the colonization of dwarf birch (Betula, a thermophilous shrub) to 5.9 ± 0.1 ka, ~3 ka after local deglaciation as determined by cosmogenic ¹⁰Be moraine dating and >2 ka later than Betula pollen is recorded in nearby lake sediment. We then assess the paleovegetation history within the context of summer temperature and find that paleotemperatures were highest prior to 6.3 ka, followed by cooling in the centuries preceding Betula establishment. Together, these molecular proxies reveal that Betula colonization lagged peak summer temperatures, suggesting that inefficient dispersal, rather than climate, may have limited Arctic shrub migration in this region. In addition, these data suggest that pollen‐based climate reconstructions from high latitudes, which rely heavily on the presence and abundance of pollen from thermophilous taxa like Betula, can be compromised by both exotic pollen fluxes and vegetation migration lags.     

The mummy returns… and sheds new light on old questions

Whether as the ancient Egyptian crocodile‐god Sobek, a terrifying predator of African waterways, or simply as a premium handbag leather, the Nile crocodile (Crocodylus niloticus) has long held the fascination of mankind. Despite 200 years of study, however, uncertainty remains as to its taxonomy. While resolving such issues are key to understanding the origins and biogeography of the so‐called true crocodiles of genus Crocodylus, given widespread ongoing range contraction, such issues are paramount for design of future conservation strategies. In this issue of Molecular Ecology, Hekkala et al. (2011) apply analysis of modern, historic and ancient DNA (aDNA) to the questions, with far‐reaching implications. First they demonstrate that, as currently described, the Nile crocodile is paraphyletic, with individuals from the east and western clades separated by a number of New World crocodile species. The consequences of this finding are as important for conservation efforts as for their impact on crocodile taxonomy. Furthermore, they strike at the heart of the long‐standing debate over whether aDNA analysis of ancient Egyptian mummies is scientifically sound.     

Paleo‐metagenomics of North American fossil packrat middens: Past biodiversity revealed by ancient DNA

Fossil rodent middens are powerful tools in paleoecology. In arid parts of western North America, packrat (Neotoma spp.) middens preserve plant and animal remains for tens of thousands of years. Midden contents are so well preserved that fragments of endogenous ancient DNA (aDNA) can be extracted and analyzed across millennia. Here, we explore the use of shotgun metagenomics to study the aDNA obtained from packrat middens up to 32,000 C¹⁴ years old. Eleven Illumina HiSeq 2500 libraries were successfully sequenced, and between 0.11% and 6.7% of reads were classified using Centrifuge against the NCBI “nt” database. Eukaryotic taxa identified belonged primarily to vascular plants with smaller proportions mapping to ascomycete fungi, arthropods, chordates, and nematodes. Plant taxonomic diversity in the middens is shown to change through time and tracks changes in assemblages determined by morphological examination of the plant remains. Amplicon sequencing of ITS2 and rbcL provided minimal data for some middens, but failed at amplifying the highly fragmented DNA present in others. With repeated sampling and deep sequencing, analysis of packrat midden aDNA from well‐preserved midden material can provide highly detailed characterizations of past communities of plants, animals, bacteria, and fungi present as trace DNA fossils. The prospects for gaining more paleoecological insights from aDNA for rodent middens will continue to improve with optimization of laboratory methods, decreasing sequencing costs, and increasing computational power.     

Multiscale climate change impacts on plant diversity in the Atacama Desert

Comprehending ecological dynamics requires not only knowledge of modern communities but also detailed reconstructions of ecosystem history. Ancient DNA (aDNA) metabarcoding allows biodiversity responses to major climatic change to be explored at different spatial and temporal scales. We extracted aDNA preserved in fossil rodent middens to reconstruct late Quaternary vegetation dynamics in the hyperarid Atacama Desert. By comparing our paleo‐informed millennial record with contemporary observations of interannual variations in diversity, we show local plant communities behave differentially at different timescales. In the interannual (years to decades) time frame, only annual herbaceous expand and contract their distributional ranges (emerging from persistent seed banks) in response to precipitation, whereas perennials distribution appears to be extraordinarily resilient. In contrast, at longer timescales (thousands of years) many perennial species were displaced up to 1,000 m downslope during pluvial events. Given ongoing and future natural and anthropogenically induced climate change, our results not only provide baselines for vegetation in the Atacama Desert, but also help to inform how these and other high mountain plant communities may respond to fluctuations of climate in the future.     

Plant and soil effects on bacterial communities associated with Miscanthus × giganteus rhizosphere and rhizomes

Bacterial assemblages, especially diazotroph assemblages residing in the rhizomes and the rhizosphere soil of Miscanthus × giganteus, contribute to plant growth and nitrogen use efficiency. However, the composition of these microbial communities has not been adequately explored nor have the potential ecological drivers for these communities been sufficiently studied. This knowledge is needed for understanding and potentially improving M. × giganteus – microbe interactions, and further enhancing sustainability of M. × giganteus production. In this study, cultivated M. × giganteus from four sites in Illinois, Kentucky, Nebraska, and New Jersey were collected to examine the relative influences of soil conditions and plant compartments on assembly of the M. × giganteus‐associated microbiome. Automated ribosomal intergenic spacer (ARISA) and terminal restriction fragment length polymorphism (T‐RFLP) targeting the nifH gene were applied to examine the total bacterial communities and diazotroph assemblages that reside in the rhizomes and the rhizosphere. Distinct microbial assemblages were detected in the endophytic and rhizosphere compartments. Site soil conditions had strong correlation with both total bacterial and diazotroph assemblages, but in different ways. Nitrogen treatments showed no significant effect on the composition of diazotroph assemblages in most sites. Endophytic compartments of different M. × giganteus plants tended to harbor similar microbial communities across all sites, whereas the rhizosphere soil of different plant tended to harbor diverse microbial assemblages that were distinct among sites. These observations offer insight into better understanding of the associative interactions between M. × giganteus and diazotrophs, and how this relationship is influenced by agronomic and edaphic factors.     

An exotic invader drives the evolution of plant traits that determine mycorrhizal fungal diversity in a native competitor

The symbiosis between land plants and arbuscular mycorrhizal fungi (AMF) is one of the most widespread and ancient mutualisms on the planet. However, relatively little is known about the evolution of these symbiotic plant–fungal interactions in natural communities. In this study, we investigated the symbiotic AMF communities of populations of the native plant species Pilea pumila (Urticaceae) with varying histories of coexistence with a nonmycorrhizal invasive species, Alliaria petiolata (Brassicaceae), known to affect mycorrhizal communities. We found that native populations of P. pumila with a long history of coexistence with the invasive species developed more diverse symbiotic AMF communities. This effect was strongest when A. petiolata plants were actively growing with the natives, and in soils with the longest history of A. petiolata growth. These results suggest that despite the ancient and widespread nature of the plant–AMF symbiosis, the plant traits responsible for symbiotic preferences can, nevertheless, evolve rapidly in response to environmental changes.     

Ancient DNA sheds new light on the Svalbard foraminiferal fossil record of the last millennium

Recent palaeogenetic studies have demonstrated the occurrence of preserved ancient DNA (aDNA) in various types of fossilised material. Environmental aDNA sequences assigned to modern species have been recovered from marine sediments dating to the Pleistocene. However, the match between the aDNA and the fossil record still needs to be evaluated for the environmental DNA approaches to be fully exploited. Here, we focus on foraminifera in sediments up to one thousand years old retrieved from the Hornsund fjord (Svalbard). We compared the diversity of foraminiferal microfossil assemblages with the diversity of aDNA sequenced from subsurface sediment samples using both cloning and high‐throughput sequencing (HTS). Our study shows that 57% of the species archived in the fossil record were also detected in the aDNA data. However, the relative abundance of aDNA sequence reads and fossil specimens differed considerably. We also found a limited match between the stratigraphic occurrence of some fossil species and their aDNA sequences, especially in the case of rare taxa. The aDNA data comprised a high proportion of non‐fossilised monothalamous species, which are known to dominate in modern foraminiferal communities of the Svalbard region. Our results confirm the relevance of HTS for studying past micro‐eukaryotic diversity and provide insight into its ability to reflect fossil assemblages. Palaeogenetic studies including aDNA analyses of non‐fossilised groups expand the range of palaeoceanographical proxies and therefore may increase the accuracy of palaeoenvironmental reconstructions.     

Note on using nuclear 28S rDNA for sequencing ancient and strongly degraded insect DNA

A protocol using insect specimens or parts thereof allows for sequencing of sections of nuclear 28S rDNA. In the present note it is demonstrated that this protocol can readily be applied to strongly degraded DNA (ancient, fixed or contaminated). Primers that are specifically designed to discriminate against human DNA but also other non‐arthropod species are tested on a range of species covering all insect groups (59 insect species from all 33 orders). Additionally, the samples represent a selection of various, mostly DNA‐degrading, preservation methods, including the most common fixatives used for morphological investigations and for long‐term storage in collections. Successful amplification was possible for all tested samples including ca. 200 year‐old dried museum specimens as well as for over 4000 year‐old fossil insects embedded in copal. When the NCBI database contained information on the tested species an unambiguous taxonomic discrimination was possible. This approach is based on a standardized protocol that guarantees easy application. This note presents primer pairs for 28S rDNA that can be a useful tool for ancient DNA (aDNA) research.     

Vegetation of Sphagnum bogs in highly disturbed landscapes: relative influence of abiotic and anthropogenic factors

Question: Has the vegetation of Sphagnum bogs been affected by more than 200 years of human activities? Location: Bas‐Saint‐Laurent region, southeastern Québec, Canada. Methods: Data (species assemblages, abiotic and spatio‐historical variables) were collected in 16 bogs ranging from 2 to 189 ha, and incorporated in a geographical information system. Major gradients in vegetation composition were identified using DCA. CCA was used to relate vegetation gradients to abiotic and spatio‐historical variables. Results: A clear segregation of species assemblages was observed, from open and undisturbed bogs to forested and highly disturbed sites. Among abiotic factors, tree basal area, water table level and peat thickness had a significant influence on plant species composition. Among spatio‐historical factors, disturbance level, area loss and fire were the most influential factors. Variance partitioning between these groups of factors suggests that spatio‐historical factors had a major influence on peatlands, representing 22% of the variation observed in the plant species assemblages while abiotic factors represent only 17% of the variation. Conclusions: The results highlight the influence of agricultural and other anthropogenic activities on plant assemblages and suggest that even wetlands apparently resistant to disturbances, such as peatlands, can be severely affected by anthropogenic factors. Plant species assemblages of ombrotrophic peatlands of the Bas‐Saint‐Laurent region were, and still are, largely influenced by human activities.     

Ecosystem functioning impacts of the invasive seaweed Sargassum muticum (Fucales,Phaeophyceae)

Ongoing changes in natural diversity due to anthropogenic activities can alter ecosystem functioning. Particular attention has been given to research on biodiversity loss and how those changes can affect the functioning of ecosystems, and, by extension, human welfare. Few studies, however, have addressed how increased diversity due to establishment of nonindigenous species (NIS) may affect ecosystem function in the recipient communities. Marine algae have a highly important role in sustaining nearshore marine ecosystems and are considered a significant component of marine bioinvasions. Here, we examined the patterns of respiration and light‐use efficiency across macroalgal assemblages with different levels of species richness and evenness. Additionally, we compared our results between native and invaded macroalgal assemblages, using the invasive brown macroalga Sargassum muticum (Yendo) Fensholt as a model species. Results showed that the presence of the invader increased the rates of respiration and production, most likely as a result of the high biomass of the invader. This effect disappeared when S. muticum lost most of its biomass after senescence. Moreover, predictability–diversity relationships of macroalgal assemblages varied between native and invaded assemblages. Hence, the introduction of high‐impact invasive species may trigger major changes in ecosystem functioning. The impact of S. muticum may be related to its greater biomass in the invaded assemblages, although species interactions and seasonality influenced the magnitude of the impact.     

Ancient pathogen DNA in human teeth and petrous bones

Recent ancient DNA (aDNA) studies of human pathogens have provided invaluable insights into their evolutionary history and prevalence in space and time. Most of these studies were based on DNA extracted from teeth or postcranial bones. In contrast, no pathogen DNA has been reported from the petrous bone which has become the most desired skeletal element in ancient DNA research due to its high endogenous DNA content. To compare the potential for pathogenic aDNA retrieval from teeth and petrous bones, we sampled these elements from five ancient skeletons, previously shown to be carrying Yersinia pestis. Based on shotgun sequencing data, four of these five plague victims showed clearly detectable levels of Y. pestis DNA in the teeth, whereas all the petrous bones failed to produce Y. pestis DNA above baseline levels. A broader comparative metagenomic analysis of teeth and petrous bones from 10 historical skeletons corroborated these results, showing a much higher microbial diversity in teeth than petrous bones, including pathogenic and oral microbial taxa. Our results imply that although petrous bones are highly valuable for ancient genomic analyses as an excellent source of endogenous DNA, the metagenomic potential of these dense skeletal elements is highly limited. This trade‐off must be considered when designing the sampling strategy for an aDNA project.     

Nitrogen‐dependent bacterial community shifts in root,rhizome and rhizosphere of nutrient‐efficient Miscanthus x giganteus from long‐term field trials

The perennial energy crop Miscanthus × giganteus is recognized for its extraordinary nitrogen‐use efficiency. While the remobilization of nitrogen (N) to the rhizome after the growth phase contributes to this efficiency, the plant‐associated microbiome might also contribute, as N‐fixing bacterial species had been isolated from this grass. Here, we studied established Miscanthus × giganteus plots in southern Germany that either received 80 kg N ha^?1 a^?1 or that were not N‐fertilized for 14 years. The bacterial communities of the bulk soil, rhizosphere, roots and rhizomes were analysed. Major differences were encountered between plant‐associated fractions. Nitrogen had little effect on soil communities. The roots and rhizomes showed less microbial diversity than soil fractions. In these compartments, Actinobacteria and N‐fixing symbiosis‐associated Proteobacteria depended on N. Intriguingly, N₂‐fixing‐related bacterial families were enriched in the rhizomes in long‐term zero N plots, while denitrifier‐related families were depleted. These findings point to the rhizome as a potentially interesting plant organ for N fixation and demonstrate long‐term differences in the organ‐specific bacterial communities associated with different N supply, which are mainly shaped by the plant.     

Long‐term phosphite application maintains species assemblages,richness and structure of plant communities invaded by Phytophthora cinnamomi

The impact of the plant pathogen Phytophthora cinnamomi and the fungicide phosphite on species assemblages, richness, abundance and vegetation structure was quantified at three sites in Kwongkan communities in the Southwest Australian Floristic Region. Healthy and diseased vegetation treated with phosphite over 7–16 years was compared with non‐treated healthy and diseased vegetation. After site differences, disease had the greatest effect on species assemblages, species richness and richness within families. Disease significantly reduced cover in the upper and lower shrub layers and increased sedge and bare ground cover. Seventeen of 21 species assessed from the families Ericaceae, Fabaceae, Myrtaceae and Proteaceae were significantly less abundant in non‐treated diseased vegetation. In diseased habitats, phosphite treatment significantly reduced the loss of shrub cover and reduced bare ground and sedge cover. In multivariate analysis of species assemblages, phosphite‐treated diseased plots grouped more closely with healthy plots. Seven of 17 susceptible species were significantly more abundant in phosphite‐treated diseased plots compared with diseased non‐treated plots. The abundance of seven of 10 Phytophthora‐susceptible species was significantly higher along transects in phosphite‐treated vegetation. Comparison of the floristics of healthy non‐treated with healthy‐treated plots showed no significant differences in species assemblages. Of 21 species assessed, three increased in abundance and only one decreased significantly in phosphite‐treated healthy plots. In three Kwongkan communities of the SWAFR, P. cinnamomi had a profound impact on species assemblages, richness, abundance and vegetation structure. There was no evidence of adverse effects of phosphite treatment on phosphorus‐sensitive species, even after fire. Treatment with phosphite enhanced the survival of key susceptible species and mitigated disease‐mediated changes in vegetation structure. In the absence of alternative methods of control in native communities, phosphite will continue to play an important role in the protection of high priority species and communities at risk of extinction due to P. cinnamomi.     

Climatically driven changes in primary production propagate through trophic levels

Climate and land‐use change are the major drivers of global biodiversity loss. Their effects are particularly acute for wide‐ranging consumers, but little is known about how these factors interact to affect the abundance of large carnivores and their herbivore prey. We analyzed population densities of a primary and secondary consumer (mule deer, Odocoileus hemionus, and mountain lion, Puma concolor) across a climatic gradient in western North America by combining satellite‐based maps of plant productivity with estimates of animal abundance and foraging area derived from Global Positioning Systems telemetry data (GPS). Mule deer density exhibited a positive, linear relationship with plant productivity (r² = 0.58), varying by a factor of 18 across the climate‐vegetation gradient (range: 38–697 individuals/100 km²). Mountain lion home range size decreased in response to increasing primary productivity and consequent changes in the abundance of their herbivore prey (range: 20–450 km²). This pattern resulted in a strong, positive association between plant productivity and mountain lion density (r² = 0.67). Despite varying densities, the ratio of prey to predator remained constant across the climatic gradient (mean ± SE = 363 ± 29 mule deer/mountain lion), suggesting that the determinacy of the effect of primary productivity on consumer density was conserved across trophic levels. As droughts and longer term climate changes reduce the suitability of marginal habitats, consumer home ranges will expand in order for individuals to meet basic nutritional requirements. These changes portend decreases in the abundance of large‐bodied, wide‐ranging wildlife through climatically driven reductions in carrying capacity, as well as increased human–wildlife interactions stemming from anthropogenic land use and habitat fragmentation.     

Introduced plants on Kilimanjaro: tourism and its impact

Kilimanjaro, a world heritage site and an icon of global change, not only suffers from climatic alterations but also is undergoing a drastic socio-economic upheaval. A strong increase of tourism enhances the risk of introducing alien plant species in particular in the upper zones of Kilimanjaro. One such species is Poa annua L., a cosmopolitan weed of European origin on roadsides and pastures. The aim of this study is to document its distribution, the speed of its propagation and risks for the indigenous vegetation of Kilimanjaro, and to compare the findings with other introduced species on this mountain. Based on a complete survey of the vegetation of Kilimanjaro with about 1,500 vegetation plots, plant communities invaded by Poa annua are determined. As with most of the other neophytes on Kilimanjaro, Poa annua invades only anthropogenic vegetation but not undisturbed natural vegetation. Similar to the situation in middle Europe, this neophyte is on Kilimanjaro a constituent of the vegetation of trampled ground, occurring between about 1,600 and 4,000 m asl along climbing routes or their vicinity. On a newly opened climbing route a rapid invasion (5.6 km in 3 months) was observed, which makes it likely that Poa annua spread on Kilimanjaro during the last 30 years in parallel to the increase of the climbing tourism. Although Poa annua is still in the stage of propagation, an invasion of natural vegetation types seems to be unlikely.     

A deep dig––hindsight on Holocene vegetation composition from ancient environmental DNA

Want a glimpse at past vegetation? Studying pollen and other plant remains, which are preserved for example in lake sediments or mires for thousands of years, allows us to document regional occurrences of plant species over radiocarbon‐dated time series. Such vegetation reconstructions derived from optical analyses of fossil samples are inherently incomplete because they only comprise taxa that contribute sufficient amounts of pollen, spores, macrofossil or other evidences. To complement optical analyses for paleoecological inference, molecular markers applied to ancient DNA (aDNA) may help in disclosing information hitherto inaccessible to biologists. Parducci et al. (2013) targeted aDNA from sediment cores of two lakes in the Scandes Mountains with generic primers in a meta‐barcoding approach. When compared to palynological records from the same cores, respective taxon lists show remarkable differences in their compositions, but also in quantitative representation and in taxonomic resolution similar to a previous study (Jørgensen et al. 2012). While not free of assumptions that need critical and robust testing, notably the question of possible contamination, this study provides thrilling prospects to improve our knowledge about past vegetation composition, but also other organismic groups, stored as a biological treasure in the ground.     

Do abandoned nests of leaf‐cutting ants enhance plant recruitment in the Atlantic Forest?

The role played by abandoned nests of leaf‐cutting ants (Atta spp.) as a small‐scale disturbance regime that affects plant recruitment, species coexistence and forest regeneration remains poorly investigated. Here we examine whether abandoned nests of Atta cephalotes serve as regeneration niches and operate as particular plant recruitment habitats, favouring forest regeneration after ant activities cease and leading to the establishment of taxonomically/ecologically distinct plant assemblages. Soil properties, canopy openness, light availability and regenerating plant assemblages were evaluated across 18 nests and adjacent control plots in a large remnant of Atlantic Forest in north‐east Brazil from December 2004 to December 2005. Surprisingly, nests and control plots exhibited very similar light environments irrespective of nest age, but nest soils exhibited substantial reductions in carbon content (1.45 ± 0.24 vs. 1.79 ± 0.13%) and organic matter (2.50 ± 0.41 vs. 3.08 ± 0.23%), and proved to be much more resistant to penetration (30.57 ± 6.08 vs. 39.48 ± 7.53 mm). Functional signature of regenerating plant assemblages exhibited little variation across both habitat types, as they were dominated by pioneer, small‐seeded and vertebrate‐dispersed species. However, abandoned nests exhibited less dense, impoverished and more homogeneous regenerating plant assemblages at local and landscape scale; they clearly lacked nest‐dependent plant species and represented floristic subsets of the flora inhabiting the undisturbed forest. This recruitment bottleneck was transient in the long term because nest‐related effects ameliorated in older nests. Our results suggest that, unlike treefall gaps, abandoned nests represent temporary (relatively long‐lasting) islands of unsuitable substrate that reduce plant recruitment, retard forest regeneration, and fail in providing a special regeneration niche able to promote species coexistence and plant diversity.     

Land‐use legacies and present fire regimes interact to mediate herbivory by altering the neighboring plant community

Past and present human activities, such as historic agriculture and fire suppression, are widespread and can create depauperate plant communities. Although many studies show that herbivory on focal plants depends on the density of herbivores or the composition of the surrounding plant community, it is unclear whether anthropogenic changes to plant communities alter herbivory. We tested the hypothesis that human activities that alter the plant community lead to subsequent changes in herbivory. At 20 sites distributed across 80 300 hectares, we conducted a field experiment that manipulated insect herbivore access (full exclosures and pseudo‐exclosures) to four focal plant species in longleaf pine woodlands with different land‐use histories (post‐agricultural sites or non‐agricultural sites) and degrees of fire frequency (frequent and infrequent). Plant cover, particularly herbaceous cover, was lower in post‐agricultural and fire suppressed woodlands. Density of the dominant insect herbivore at our site (grasshoppers) was positively related to plant cover. Herbivore access reduced biomass of the palatable forb Solidago odora in frequently burned post‐agricultural sites and in infrequently burned non‐agricultural woodlands and increased mortality of another forb (Pityopsis graminifolia), but did not affect two other less palatable species (Schizachyrium scoparium and Tephrosia virginiana). Herbivory on S. odora exhibited a hump‐shaped response to plant cover, with low herbivory at low and high levels of plant cover. Herbivore density had a weak negative effect on herbivory. These findings suggest that changes in plant cover related to past and present human activities can modify damage rates on focal S. odora plants by altering grasshopper foraging behavior rather than by altering local grasshopper density. The resulting changes in herbivory may have the potential to limit natural recovery or restoration efforts by reducing the establishment or performance of palatable plant species.     

Application of ancient DNA to the reconstruction of past microbial assemblages and for the detection of toxic cyanobacteria in subtropical freshwater ecosystems

Ancient DNA (aDNA) analysis of lake sediments is a promising tool for detecting shifts in past microbial assemblages in response to changing environmental conditions. We examined sediment core samples from subtropical, freshwater Laguna Blanca (Uruguay), which has been severely affected by cultural eutrophication since 1960 and where cyanobacterial blooms, particularly those of the saxitoxin‐producer Cylindrospermopsis raciborskii, have been reported since the 1990s. Samples corresponding to ~1846, 1852, 2000 and 2007 AD were selected to perform denaturing gradient gel electrophoresis (DGGE) analysis of the 16S–23S rRNA intergenic transcribed spacer (ribosomal ITS) to compare their prokaryotic assemblage composition. Each stratum showed different ITS patterns, but the composition of 21st century samples was clearly different than those of mid‐19th century. This compositional change was correlated with shifts in sediment organic matter and chlorophyll a content, which were significantly higher in recent samples. The presence of saxitoxin‐producing cyanobacteria was addressed by quantitative real‐time PCR of the sxtU gene involved in toxin biosynthesis. This gene was present only in recent samples, for which clone libraries and ITS sequencing indicated the presence of Cyanobacteria. Phylogenetic analyses identified C. raciborskii only in the 2000 sample, shortly after several years when blooms were recorded in the lake. These data suggest the utility of aDNA for the reconstruction of microbial assemblage shifts in subtropical lakes, at least on centennial scales. The application of aDNA analysis to genes involved in cyanotoxin synthesis extends the applicability of molecular techniques in palaeolimnological studies to include key microbial community characteristics of great scientific and social interest.