The potential of multispectral imaging flow cytometry for environmental monitoring.

Environmental monitoring involves the quantification of microscopic cells and particles such as algae, plant cells, pollen, or fungal spores. Traditional methods using conventional microscopy require expert knowledge, are time-intensive and not well-suited for automated high throughput. Multispectral imaging flow cytometry (MIFC) allows measurement of up to 5000 particles per second from a fluid suspension and can simultaneously capture up to 12 images of every single particle for brightfield and different spectral ranges, with up to 60x magnification. The high throughput of MIFC has high potential for increasing the amount and accuracy of environmental monitoring, such as for plant-pollinator interactions, fossil samples, air, water or food quality that currently rely on manual microscopic methods. Automated recognition of particles and cells is also possible, when MIFC is combined with deep-learning computational techniques. Furthermore, various fluorescence dyes can be used to stain specific parts of the cell to highlight physiological and chemical features including: vitality of pollen or algae, allergen content of individual pollen, surface chemical composition (carbohydrate coating) of cells, DNA- or enzyme-activity staining. Here, we outline the great potential for MIFC in environmental research for a variety of research fields and focal organisms. In addition, we provide best practice recommendations.

Plankton community changes during the last 124 000 years in the subarctic Bering Sea derived from sedimentary ancient DNA.

Current global warming results in rising sea-water temperatures, and the loss of sea ice in Arctic and subarctic oceans impacts the community composition of primary producers with cascading effects on the food web and potentially on carbon export rates. This study analyzes metagenomic shotgun and diatom rbcL amplicon sequencing data from sedimentary ancient DNA of the subarctic western Bering Sea that records phyto- and zooplankton community changes over the last glacial-interglacial cycles, including the last interglacial period (Eemian). Our data show that interglacial and glacial plankton communities differ, with distinct Eemian and Holocene plankton communities. The generally warm Holocene period is dominated by picosized cyanobacteria and bacteria-feeding heterotrophic protists, while the Eemian period is dominated by eukaryotic picosized chlorophytes and Triparmaceae. By contrast, the glacial period is characterized by microsized phototrophic protists, including sea ice-associated diatoms in the family Bacillariaceae and co-occurring diatom-feeding crustaceous zooplankton. Our deep-time record of plankton community changes reveals a long-term decrease in phytoplankton cell size coeval with increasing temperatures, resembling community changes in the currently warming Bering Sea. The phytoplankton community in the warmer-than-present Eemian period is distinct from modern communities and limits the use of the Eemian as an analog for future climate scenarios. However, under enhanced future warming, the expected shift toward the dominance of small-sized phytoplankton and heterotrophic protists might result in an increased productivity, whereas the community's potential of carbon export will be decreased, thereby weakening the subarctic Bering Sea's function as an effective carbon sink.

Tibetan terrestrial and aquatic ecosystems collapsed with cryosphere loss inferred from sedimentary ancient metagenomics.

Glacier and permafrost shrinkage and land-use intensification threaten mountain wildlife and affect nature conservation strategies. Here, we present paleometagenomic records of terrestrial and aquatic taxa from the southeastern Tibetan Plateau covering the last 18,000 years to help understand the complex alpine ecosystem dynamics. We infer that steppe-meadow became woodland at 14 ka (cal BP) controlled by cryosphere loss, further driving a herbivore change from wild yak to deer. These findings weaken the hypothesis of top-down control by large herbivores in the terrestrial ecosystem. We find a turnover in the aquatic communities at 14 ka, transitioning from glacier-related (blue-green) algae to abundant nonglacier-preferring picocyanobacteria, macrophytes, fish, and otters. There is no evidence for substantial effects of livestock herding in either ecosystem. Using network analysis, we assess the stress-gradient hypothesis and reveal that root hemiparasitic and cushion plants are keystone taxa. With ongoing cryosphere loss, the protection of their habitats is likely to be of conservation benefit on the Tibetan Plateau.

Mitochondrial genomes of Pleistocene megafauna retrieved from recent sediment layers of two Siberian lakes.

Ancient environmental DNA (aeDNA) from lake sediments has yielded remarkable insights for the reconstruction of past ecosystems, including suggestions of late survival of extinct species. However, translocation and lateral inflow of DNA in sediments can potentially distort the stratigraphic signal of the DNA. Using three different approaches on two short lake sediment cores of the Yamal peninsula, West Siberia, with ages spanning only the past hundreds of years, we detect DNA and identified mitochondrial genomes of multiple mammoth and woolly rhinoceros individuals-both species that have been extinct for thousands of years on the mainland. The occurrence of clearly identifiable aeDNA of extinct Pleistocene megafauna (e.g. >400 K reads in one core) throughout these two short subsurface cores, along with specificities of sedimentology and dating, confirm that processes acting on regional scales, such as extensive permafrost thawing, can influence the aeDNA record and should be accounted for in aeDNA paleoecology.

Marine ecosystem shifts with deglacial sea-ice loss inferred from ancient DNA shotgun sequencing.

Sea ice is a key factor for the functioning and services provided by polar marine ecosystems. However, ecosystem responses to sea-ice loss are largely unknown because time-series data are lacking. Here, we use shotgun metagenomics of marine sedimentary ancient DNA off Kamchatka (Western Bering Sea) covering the last ~20,000 years. We traced shifts from a sea ice-adapted late-glacial ecosystem, characterized by diatoms, copepods, and codfish to an ice-free Holocene characterized by cyanobacteria, salmon, and herring. By providing information about marine ecosystem dynamics across a broad taxonomic spectrum, our data show that ancient DNA will be an important new tool in identifying long-term ecosystem responses to climate transitions for improvements of ocean and cryosphere risk assessments. We conclude that continuing sea-ice decline on the northern Bering Sea shelf might impact on carbon export and disrupt benthic food supply and could allow for a northward expansion of salmon and Pacific herring.

Early human impact on lake cyanobacteria revealed by a Holocene record of sedimentary ancient DNA.

Sedimentary DNA-based studies revealed the effects of human activity on lake cyanobacteria communities over the last centuries, yet we continue to lack information over longer timescales. Here, we apply high-resolution molecular analyses on sedimentary ancient DNA to reconstruct the history of cyanobacteria throughout the Holocene in a lake in north-eastern Germany. We find a substantial increase in cyanobacteria abundance coinciding with deforestation during the early Bronze Age around 4000 years ago, suggesting increased nutrient supply to the lake by local communities settling on the lakeshore. The next substantial human-driven increase in cyanobacteria abundance occurred only about a century ago due to intensified agricultural fertilisation which caused the dominance of potentially toxic taxa (e.g., Aphanizomenon). Our study provides evidence that humans began to locally impact lake ecology much earlier than previously assumed. Consequently, managing aquatic systems today requires awareness of the legacy of human influence dating back potentially several millennia.

Hidden diversity in diatoms of Kenyan Lake Naivasha: a genetic approach detects temporal variation.

This study provides insights into the morphological and genetic diversity in diatoms occurring in core sediments from tropical lakes in Kenya. We developed a genetic survey technique specific for diatoms utilizing a short region (76-67 bp) of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene as genetic barcode. Our analyses (i) validated the use of rbcL as a barcoding marker for diatoms, applied to sediment samples, (ii) showed a significant correlation between the results obtained by morphological and molecular data and (iii) indicated temporal variation in diatom assemblages on the inter- and intra-specific level. Diatom assemblages from a short core from Lake Naivasha show a drastic shift over the last 200 years, as littoral species (e.g. Navicula) are replaced by more planktonic ones (e.g. Aulacoseira). Within that same period, we detected periodic changes in the respective frequencies of distinct haplotype groups of Navicula, which coincide with wet and dry periods of Lake Naivasha between 1820 and 1938 AD. Our genetic analyses on historical lake sediments revealed inter- and intra-specific variation in diatoms, which is partially hidden behind single morphotypes. The occurrence of particular genetic lineages is probably correlated with environmental factors.

Larix species range dynamics in Siberia since the Last Glacial captured from sedimentary ancient DNA.

Climate change is expected to cause major shifts in boreal forests which are in vast areas of Siberia dominated by two species of the deciduous needle tree larch (Larix). The species differ markedly in their ecosystem functions, thus shifts in their respective ranges are of global relevance. However, drivers of species distribution are not well understood, in part because paleoecological data at species level are lacking. This study tracks Larix species distribution in time and space using target enrichment on sedimentary ancient DNA extracts from eight lakes across Siberia. We discovered that Larix sibirica, presently dominating in western Siberia, likely migrated to its northern distribution area only in the Holocene at around 10,000 years before present (ka BP), and had a much wider eastern distribution around 33 ka BP. Samples dated to the Last Glacial Maximum (around 21 ka BP), consistently show genotypes of L. gmelinii. Our results suggest climate as a strong determinant of species distribution in Larix and provide temporal and spatial data for species projection in a changing climate.

Holocene chloroplast genetic variation of shrubs (Alnus alnobetula, Betula nana, Salix sp.) at the siberian tundra-taiga ecotone inferred from modern chloroplast genome assembly and sedimentary ancient DNA analyses.

Climate warming alters plant composition and population dynamics of arctic ecosystems. In particular, an increase in relative abundance and cover of deciduous shrub species (shrubification) has been recorded. We inferred genetic variation of common shrub species (Alnus alnobetula, Betula nana, Salix sp.) through time. Chloroplast genomes were assembled from modern plants (n = 15) from the Siberian forest-tundra ecotone. Sedimentary ancient DNA (sedaDNA; n = 4) was retrieved from a lake on the southern Taymyr Peninsula and analyzed by metagenomics shotgun sequencing and a hybridization capture approach. For A. alnobetula, analyses of modern DNA showed low intraspecies genetic variability and a clear geographical structure in haplotype distribution. In contrast, B. nana showed high intraspecies genetic diversity and weak geographical structure. Analyses of sedaDNA revealed a decreasing relative abundance of Alnus since 5,400 cal yr BP, whereas Betula and Salix increased. A comparison between genetic variations identified in modern DNA and sedaDNA showed that Alnus variants were maintained over the last 6,700 years in the Taymyr region. In accordance with modern individuals, the variants retrieved from Betula and Salix sedaDNA showed higher genetic diversity. The success of the hybridization capture in retrieving diverged sequences demonstrates the high potential for future studies of plant biodiversity as well as specific genetic variation on ancient DNA from lake sediments. Overall, our results suggest that shrubification has species-specific trajectories. The low genetic diversity in A. alnobetula suggests a local population recruitment and growth response of the already present communities, whereas the higher genetic variability and lack of geographical structure in B. nana may indicate a recruitment from different populations due to more efficient seed dispersal, increasing the genetic connectivity over long distances.

Hybridization capture of larch (Larix Mill.) chloroplast genomes from sedimentary ancient DNA reveals past changes of Siberian forest.

Siberian larch (Larix Mill.) forests dominate vast areas of northern Russia and contribute important ecosystem services to the world. It is important to understand the past dynamics of larches in order to predict their likely response to a changing climate in the future. Sedimentary ancient DNA extracted from lake sediment cores can serve as archives to study past vegetation. However, the traditional method of studying sedimentary ancient DNA-metabarcoding-focuses on small fragments, which cannot resolve Larix to species level nor allow a detailed study of population dynamics. Here, we use shotgun sequencing and hybridization capture with long-range PCR-generated baits covering the complete Larix chloroplast genome to study Larix populations from a sediment core reaching back to 6700 years from the Taymyr region in northern Siberia. In comparison with shotgun sequencing, hybridization capture results in an increase in taxonomically classified reads by several orders of magnitude and the recovery of complete chloroplast genomes of Larix. Variation in the chloroplast reads corroborates an invasion of Larix gmelinii into the range of Larix sibirica before 6700 years ago. Since then, both species have been present at the site, although larch populations have decreased with only a few trees remaining in what was once a forested area. This study demonstrates for the first time that hybridization capture applied directly to ancient DNA of plants extracted from lake sediments can provide genome-scale information and is a viable tool for studying past genomic changes in populations of single species, irrespective of a preservation as macrofossil.

Sedimentary ancient DNA reveals a threat of warming-induced alpine habitat loss to Tibetan Plateau plant diversity.

Studies along elevational gradients worldwide usually find the highest plant taxa richness in mid-elevation forest belts. Hence, an increase in upper elevation diversity is expected in the course of warming-related treeline rise. Here, we use a time-series approach to infer past taxa richness from sedimentary ancient DNA from the south-eastern Tibetan Plateau over the last ~18,000 years. We find the highest total plant taxa richness during the cool phase after glacier retreat when the area contained extensive and diverse alpine habitats (14-10 ka); followed by a decline when forests expanded during the warm early- to mid-Holocene (10-3.6 ka). Livestock grazing since 3.6 ka promoted plant taxa richness only weakly. Based on these inferred dependencies, our simulation yields a substantive decrease in plant taxa richness in response to warming-related alpine habitat loss over the next centuries. Accordingly, efforts of Tibetan biodiversity conservation should include conclusions from palaeoecological evidence.

Lake-depth related pattern of genetic and morphological diatom diversity in boreal Lake Bolshoe Toko, Eastern Siberia.

Large, old and heterogenous lake systems are valuable sources of biodiversity. The analysis of current spatial variability within such lakes increases our understanding of the origin and establishment of biodiversity. The environmental sensitivity and the high taxonomic richness of diatoms make them ideal organisms to investigate intra-lake variability. We investigated modern intra-lake diatom diversity in the large and old sub-arctic Lake Bolshoe Toko in Siberia. Our study uses diatom-specific metabarcoding, applying a short rbcL marker combined with next-generation sequencing and morphological identification to analyse the diatom diversity in modern sediment samples of 17 intra-lake sites. We analysed abundance-based compositional taxonomic diversity and generic phylogenetic diversity to investigate the relationship of diatom diversity changes with water depth. The two approaches show differences in taxonomic identification and alpha diversity, revealing a generally higher diversity with the genetic approach. With respect to beta diversity and ordination analyses, both approaches result in similar patterns. Water depth or related lake environmental conditions are significant factors influencing intra-lake diatom patterns, showing many significant negative correlations between alpha and beta diversity and water depth. Further, one near-shore and two lagoon lake sites characterized by low (0-10m) and medium (10-30m) water depth are unusual with unique taxonomic compositions. At deeper (>30m) water sites we identified strongest phylogenetic clustering in Aulacoseira, but generally much less in Staurosira, which supports that water depth is a strong environmental filter on the Aulacoseira communities. Our study demonstrates the utility of combining analyses of genetic and morphological as well as phylogenetic diversity to decipher compositional and generic phylogenetic patterns, which are relevant in understanding intra-lake heterogeneity as a source of biodiversity in the sub-arctic glacial Lake Bolshoe Toko.

Plant diversity in sedimentary DNA obtained from high-latitude (Siberia) and high-elevation lakes (China).

BACKGROUND: Plant diversity in the Arctic and at high altitudes strongly depends on and rebounds to climatic and environmental variability and is nowadays tremendously impacted by recent climate warming. Therefore, past changes in plant diversity in the high Arctic and high-altitude regions are used to infer climatic and environmental changes through time and allow future predictions. Sedimentary DNA (sedDNA) is an established proxy for the detection of local plant diversity in lake sediments, but still relationships between environmental conditions and preservation of the plant sedDNA proxy are far from being fully understood. Studying modern relationships between environmental conditions and plant sedDNA will improve our understanding under which conditions sedDNA is well-preserved helping to a.) evaluate suitable localities for sedDNA approaches, b.) provide analogues for preservation conditions and c.) conduct reconstruction of plant diversity and climate change. This study investigates modern plant diversity applying a plant-specific metabarcoding approach on sedimentary DNA of surface sediment samples from 262 lake localities covering a large geographical, climatic and ecological gradient. Latitude ranges between 25°N and 73°N and longitude between 81°E and 161°E, including lowland lakes and elevated lakes up to 5168 m a.s.l. Further, our sampling localities cover a climatic gradient ranging in mean annual temperature between -15°C and +18°C and in mean annual precipitation between 36- and 935 mm. The localities in Siberia span over a large vegetational gradient including tundra, open woodland and boreal forest. Lake localities in China include alpine meadow, shrub, forest and steppe and also cultivated areas. The assessment of plant diversity in the underlying dataset was conducted by a specific plant metabarcoding approach. NEW INFORMATION: We provide a large dataset of genetic plant diversity retrieved from surface sedimentary DNA from lakes in Siberia and China spanning over a large environmental gradient. Our dataset encompasses sedDNA sequence data of 259 surface lake sediments and three soil samples originating from Siberian and Chinese lakes. We used the established chloroplastidal P6 loop trnL marker for plant diversity assessment. The merged, filtered and assigned dataset includes 15,692,944 read counts resulting in 623 unique plant DNA sequence types which have a 100% match to either the EMBL or to the specific Arctic plant reference database. The underlying dataset includes a taxonomic list of identified plants and results from PCR replicates, as well as extraction blanks (BLANKs) and PCR negative controls (NTCs), which were run along with the investigated lake samples. This collection of plant metabarcoding data from modern lake sediments is still ongoing and additional data will be released in the future.

Sampling and Extraction of Ancient DNA from Sediments.

Environmental DNA preserved in sediments is rapidly gaining importance as a tool in paleoecology. Sampling procedures for sedimentary ancient DNA (sedaDNA) have to be well planned to ensure clean subsampling of the inside of sediment cores and avoid introducing contamination. Additionally, ancient DNA extraction protocols may need to be optimized for the recovery of DNA from sediments, which may contain inhibitors. Here we describe procedures for subsampling both nonfrozen and frozen sediment cores, and we describe an efficient method for ancient DNA extraction from such samples.

Chloroplast and mitochondrial genetic variation of larches at the Siberian tundra-taiga ecotone revealed by de novo assembly.

Larix populations at the tundra-taiga ecotone in northern Siberia are highly under-represented in population genetic studies, possibly due to the remoteness of these regions that can only be accessed at extraordinary expense. The genetic signatures of populations in these boundary regions are therefore largely unknown. We aim to generate organelle reference genomes for the detection of single nucleotide polymorphisms (SNPs) that can be used for paleogenetic studies. We present 19 complete chloroplast genomes and mitochondrial genomic sequences of larches from the southern lowlands of the Taymyr Peninsula (northernmost range of Larix gmelinii (Rupr.) Kuzen.), the lower Omoloy River, and the lower Kolyma River (both in the range of Larix cajanderi Mayr). The genomic data reveal 84 chloroplast SNPs and 213 putatively mitochondrial SNPs. Parsimony-based chloroplast haplotype networks show no spatial structure of individuals from different geographic origins, while the mitochondrial haplotype network shows at least a slight spatial structure with haplotypes from the Omoloy and Kolyma populations being more closely related to each other than to most of the haplotypes from the Taymyr populations. Whole genome alignments with publicly available complete chloroplast genomes of different Larix species show that among official plant barcodes only the rcbL gene contains sufficient polymorphisms, but has to be sequenced completely to distinguish the different provenances. We provide 8 novel mitochondrial SNPs that are putatively diagnostic for the separation of L. gmelinii and L. cajanderi, while 4 chloroplast SNPs have the potential to distinguish the L. gmelinii/L. cajanderi group from other Larix species. Our organelle references can be used for a targeted primer and probe design allowing the generation of short amplicons. This is particularly important with regard to future investigations of, for example, the biogeographic history of Larix by screening ancient sedimentary DNA of Larix.

Temporal and spatial patterns of mitochondrial haplotype and species distributions in Siberian larches inferred from ancient environmental DNA and modeling.

Changes in species' distributions are classically projected based on their climate envelopes. For Siberian forests, which have a tremendous significance for vegetation-climate feedbacks, this implies future shifts of each of the forest-forming larch (Larix) species to the north-east. However, in addition to abiotic factors, reliable projections must assess the role of historical biogeography and biotic interactions. Here, we use sedimentary ancient DNA and individual-based modelling to investigate the distribution of larch species and mitochondrial haplotypes through space and time across the treeline ecotone on the southern Taymyr peninsula, which at the same time presents a boundary area of two larch species. We find spatial and temporal patterns, which suggest that forest density is the most influential driver determining the precise distribution of species and mitochondrial haplotypes. This suggests a strong influence of competition on the species' range shifts. These findings imply possible climate change outcomes that are directly opposed to projections based purely on climate envelopes. Investigations of such fine-scale processes of biodiversity change through time are possible using paleoenvironmental DNA, which is available much more readily than visible fossils and can provide information at a level of resolution that is not reached in classical palaeoecology.

A comparison of sedimentary DNA and pollen from lake sediments in recording vegetation composition at the Siberian treeline.

Reliable information on past and present vegetation is important to project future changes, especially for rapidly transitioning areas such as the boreal treeline. To study past vegetation, pollen analysis is common, while current vegetation is usually assessed by field surveys. Application of detailed sedimentary DNA (sedDNA) records has the potential to enhance our understanding of vegetation changes, but studies systematically investigating the power of this proxy are rare to date. This study compares sedDNA metabarcoding and pollen records from surface sediments of 31 lakes along a north-south gradient of increasing forest cover in northern Siberia (Taymyr peninsula) with data from field surveys in the surroundings of the lakes. sedDNA metabarcoding recorded 114 plant taxa, about half of them to species level, while pollen analyses identified 43 taxa, both exceeding the 31 taxa found by vegetation field surveys. Increasing Larix percentages from north to south were consistently recorded by all three methods and principal component analyses based on percentage data of vegetation surveys and DNA sequences separated tundra from forested sites. Comparisons of the ordinations using procrustes and protest analyses show a significant fit among all compared pairs of records. Despite similarities of sedDNA and pollen records, certain idiosyncrasies, such as high percentages of Alnus and Betula in all pollen and high percentages of Salix in all sedDNA spectra, are observable. Our results from the tundra to single-tree tundra transition zone show that sedDNA analyses perform better than pollen in recording site-specific richness (i.e., presence/absence of taxa in the vicinity of the lake) and perform as well as pollen in tracing vegetation composition.

The History of Tree and Shrub Taxa on Bol'shoy Lyakhovsky Island (New Siberian Archipelago) since the Last Interglacial Uncovered by Sedimentary Ancient DNA and Pollen Data.

Ecosystem boundaries, such as the Arctic-Boreal treeline, are strongly coupled with climate and were spatially highly dynamic during past glacial-interglacial cycles. Only a few studies cover vegetation changes since the last interglacial, as most of the former landscapes are inundated and difficult to access. Using pollen analysis and sedimentary ancient DNA (sedaDNA) metabarcoding, we reveal vegetation changes on Bol'shoy Lyakhovsky Island since the last interglacial from permafrost sediments. Last interglacial samples depict high levels of floral diversity with the presence of trees (Larix, Picea, Populus) and shrubs (Alnus, Betula, Ribes, Cornus, Saliceae) on the currently treeless island. After the Last Glacial Maximum, Larix re-colonised the island but disappeared along with most shrub taxa. This was probably caused by Holocene sea-level rise, which led to increased oceanic conditions on the island. Additionally, we applied two newly developed larch-specific chloroplast markers to evaluate their potential for tracking past population dynamics from environmental samples. The novel markers were successfully re-sequenced and exhibited two variants of each marker in last interglacial samples. SedaDNA can track vegetation changes as well as genetic changes across geographic space through time and can improve our understanding of past processes that shape modern patterns.

Genetic data from algae sedimentary DNA reflect the influence of environment over geography.

Genetic investigations on eukaryotic plankton confirmed the existence of modern biogeographic patterns, but analyses of palaeoecological data exploring the temporal variability of these patterns have rarely been presented. Ancient sedimentary DNA proved suitable for investigations of past assemblage turnover in the course of environmental change, but genetic relatedness of the identified lineages has not yet been undertaken. Here, we investigate the relatedness of diatom lineages in Siberian lakes along environmental gradients (i.e. across treeline transects), over geographic distance and through time (i.e. the last 7000 years) using modern and ancient sedimentary DNA. Our results indicate that closely-related Staurosira lineages occur in similar environments and less-related lineages in dissimilar environments, in our case different vegetation and co-varying climatic and limnic variables across treeline transects. Thus our study reveals that environmental conditions rather than geographic distance is reflected by diatom-relatedness patterns in space and time. We tentatively speculate that the detected relatedness pattern in Staurosira across the treeline could be a result of adaptation to diverse environmental conditions across the arctic boreal treeline, however, a geographically-driven divergence and subsequent repopulation of ecologically different habitats might also be a potential explanation for the observed pattern.

Molecular profiling of diatom assemblages in tropical lake sediments using taxon-specific PCR and Denaturing High-Performance Liquid Chromatography (PCR-DHPLC).

Here we present a protocol to genetically detect diatoms in sediments of the Kenyan tropical Lake Naivasha, based on taxon-specific PCR amplification of short fragments (approximately 100 bp) of the small subunit ribosomal (SSU) gene and subsequent separation of species-specific PCR products by PCR-based denaturing high-performance liquid chromatography (DHPLC). An evaluation of amplicons differing in primer specificity to diatoms and length of the fragments amplified demonstrated that the number of different diatom sequence types detected after cloning of the PCR products critically depended on the specificity of the primers to diatoms and the length of the amplified fragments whereby shorter fragments yielded more species of diatoms. The DHPLC was able to discriminate between very short amplicons based on the sequence difference, even if the fragments were of identical length and if the amplicons differed only in a small number of nucleotides. Generally, the method identified the dominant sequence types from mixed amplifications. A comparison with microscopic analysis of the sediment samples revealed that the sequence types identified in the molecular assessment corresponded well with the most dominant species. In summary, the PCR-based DHPLC protocol offers a fast, reliable and cost-efficient possibility to study DNA from sediments and other environmental samples with unknown organismic content, even for very short DNA fragments.