Kristian Hassel

New environmental metabarcodes for analysing soil DNA: potential for studying past and present ecosystems

Metabarcoding approaches use total and typically degraded DNA from environmental samples to analyse biotic assemblages and can potentially be carried out for any kinds of organisms in an ecosystem. These analyses rely on specific markers, here called metabarcodes, which should be optimized for taxonomic resolution, minimal bias in amplification of the target organism group and short sequence length. Using bioinformatic tools, we developed metabarcodes for several groups of organisms: fungi, bryophytes, enchytraeids, beetles and birds. The ability of these metabarcodes to amplify the target groups was systematically evaluated by (i) in silico PCRs using all standard sequences in the EMBL public database as templates, (ii) in vitro PCRs of DNA extracts from surface soil samples from a site in Varanger, northern Norway and (iii) in vitro PCRs of DNA extracts from permanently frozen sediment samples of late‐Pleistocene age (∼16 000–50 000 years bp) from two Siberian sites, Duvanny Yar and Main River. Comparison of the results from the in silico PCR with those obtained in vitro showed that the in silico approach offered a reliable estimate of the suitability of a marker. All target groups were detected in the environmental DNA, but we found large variation in the level of detection among the groups and between modern and ancient samples. Success rates for the Pleistocene samples were highest for fungal DNA, whereas bryophyte, beetle and bird sequences could also be retrieved, but to a much lesser degree. The metabarcoding approach has considerable potential for biodiversity screening of modern samples and also as a palaeoecological tool.

Genetic variation and structure in the expanding moss Pogonatum dentatum (Polytrichaceae) in its area of origin and in a recently colonized area

Genetic variation in the expanding moss species Pogonatum dentatum was studied using intersimple sequence repeat (ISSR) markers. The genetic consequences of range expansion were studied by comparing source populations in a mountain area with populations from a recently colonized lowland area in Sweden. Indices of genetic variation show slightly lower number of alleles per locus in the lowlands and a similar gene diversity in both areas. Three of four lowland populations had evidence of a recently passed bottleneck. Considerably higher haplotype diversity was found in the recently colonized lowlands compared to source populations in the mountains. Patterns of allelic diversity suggest that P. dentatum experiences loss of genetic variation through founder effects and genetic drift when expanding its distribution range. Higher haplotypic diversity, less linkage disequilibrium, and fewer compatible loci indicate that sexual recombination is relatively more important in the lowlands compared to the mountains. A likely explanation is higher success of establishment from spores in the lowlands, while clonal propagation predominates in the mountains. Less genetic differentiation among lowland populations indicates more gene flow in the lowland area, involving more spores and/or fragments moving among populations.

Genetic structure of the endangered peat moss Sphagnum angermanicum in Sweden : a result of historic or contemporary processes?

Abstract Genetic structure and diversity were studied in the endangered peatmoss Sphagnum angermanicum Melin to assess its conservation status. In total, 128 shoots from eleven populations throughout its Swedish distribution were analyzed. Among these shoots 28 haplotypes were identified by 19 ISSR loci. The most common haplotype (50% of the sampled shoots) occurred in all populations. The level of gene diversity over loci was intermediate compared to records from most other bryophytes. There was no genetic isolation between the populations and most of the genetic variation was found within populations. This implies either that the populations have originated from only a few common individuals or a high gene flow between populations. A relict population model is suggested to explain the observed pattern. Just after the last glaciation, S. angermanicum may have expanded its range when suitable habitat became available after the glacial ice retreated. Since then, habitats have vanished or fragmented and today only a few relict populations exist. This dioicous species has only once been reported with sporophytes in Scandinavia in modern time, but according to the genetic data, both the low level of linkage among loci and the estimated rate of recombination show evidence of sexual reproduction. However, reproduction may have been more frequent in the past. Based on the current knowledge of the species habitat requirements, life history, and the small population sizes, we conclude that the species will have an uncertain future in Sweden.

NORTH AMERICAN ORIGIN AND RECENT EUROPEAN ESTABLISHMENTS OF THE AMPHI-ATLANTIC PEAT MOSS SPHAGNUM ANGERMANICUM

Genetic and morphological similarity between populations separated by large distances may be caused by frequent long‐distance dispersal or retained ancestral polymorphism. The frequent lack of differentiation between disjunct conspecific moss populations on different continents has traditionally been explained by the latter model, and has been cited as evidence that many or most moss species are extremely ancient and slowly diverging. We have studied intercontinental differentiation in the amphi‐Atlantic peat moss Sphagnum angermanicum using 23 microsatellite markers. Two major genetic clusters are found, both of which occur throughout the distributional range. Patterns of genetic structuring and overall migration patterns suggest that the species probably originated in North America, and seems to have been established twice in Northern Europe during the past 40,000 years. We conclude that similarity between S. angermanicum populations on different continents is not the result of ancient vicariance and subsequent stasis. Rather, the observed pattern can be explained by multiple long‐distance dispersal over limited evolutionary time. The genetic similarity can also partly be explained by incomplete lineage sorting, but this appears to be caused by the short time since separation. Our study adds to a growing body of evidence suggesting that Sphagnum, constituting a significant part of northern hemisphere biodiversity, may be more evolutionary dynamic than previously assumed.

Interactions between sheep, rodents, graminoids, and bryophytes in an oceanic alpine ecosystem of low productivity

ABSTRACT Large herbivores directly affect plant communities in alpine ecosystems. In addition, they may compete with, or facilitate foraging by, small herbivores and also cause strong indirect effects on plants. We used an exclosure experiment to examine short-term (5-y) effects of cessation of sheep grazing on rodent grazing and plant communities in an oceanic alpine environment of low productivity with a long history of heavy sheep grazing. Exclusion of sheep significantly impacted plant communities. Vascular plant height increased, but Deschampsia flexuosa was the only vascular plant species that increased in sheep exclosures. Changes in the frequency of graminoids, herbs, and dwarf shrubs in exclosed plots were not related to cessation of grazing, but 6 bryophyte species significantly increased or decreased in response to exclusion of sheep. The absence of large grazers thus brought about a change in the species composition in favour of successional bryophytes and the preferred fodder plant. Neither vascular plant nor bryophyte species richness, nor the total cover of bryophytes and lichens, were affected. Cessation of sheep grazing reduced the level of rodent grazing. Rodent grazing correlated with changes in plant communities that led to reduced height and cover of vascular plants, reduced cryptogam cover, and reduced frequencies of 3 bryophyte species. A strong correlation between sheep fodder value index and rodent grazing indirectly indicated additive herbivory. In addition, some of the rodent effects were compensatory; e.g., Nardus stricta, which is not grazed by sheep, was significantly reduced by rodents. Our study points to a more central role of facilitation in structuring herbivore assemblages in the short term, with direct implications for the joint effects of large and small herbivores on the cover and frequency of graminoids and bryophytes.

Highly overlapping winter diet in two sympatric lemming species revealed by DNA metabarcoding

Sympatric species are expected to minimize competition by partitioning resources, especially when these are limited. Herbivores inhabiting the High Arctic in winter are a prime example of a situation where food availability is anticipated to be low, and thus reduced diet overlap is expected. We present here the first assessment of diet overlap of high arctic lemmings during winter based on DNA metabarcoding of feces. In contrast to previous analyses based on microhistology, we found that the diets of both collared (Dicrostonyx groenlandicus) and brown lemmings (Lemmus trimucronatus) on Bylot Island were dominated by Salix while mosses, which were significantly consumed only by the brown lemming, were a relatively minor food item. The most abundant plant taxon, Cassiope tetragona, which alone composes more than 50% of the available plant biomass, was not detected in feces and can thus be considered to be non-food. Most plant taxa that were identified as food items were consumed in proportion to their availability and none were clearly selected for. The resulting high diet overlap, together with a lack of habitat segregation, indicates a high potential for resource competition between the two lemming species. However, Salix is abundant in the winter habitats of lemmings on Bylot Island and the non-Salix portion of the diets differed between the two species. Also, lemming grazing impact on vegetation during winter in the study area is negligible. Hence, it seems likely that the high potential for resource competition predicted between these two species did not translate into actual competition. This illustrates that even in environments with low primary productivity food resources do not necessarily generate strong competition among herbivores.

Shedding new light on the diet of Norwegian lemmings: DNA metabarcoding of stomach content

Lemmings are key herbivores in many arctic food webs, and their population dynamics have major impacts on the functioning of tundra systems. However, current knowledge of lemming diet is limited, hampering evaluation of lemming–vegetation interactions. This lack of knowledge is mainly due to methodological challenges, as previously used microhistological methods result in large proportions of poorly resolved plant taxa. We analyzed diets of Norwegian lemmings (Lemmus lemmus) in three different habitats using a new method, DNA metabarcoding of stomach contents. To achieve detailed information on ingested vascular plants, bryophytes, and fungi, we amplified short fragments of chloroplast DNA (for plants; P6 loop of the trnL intron) and nuclear ribosomal DNA (for fungi; ITS1-region). Our results revealed that lemming diets were dominated by grasses, mainly Avenella flexuosa, and mosses, mainly Dicranum spp., but that a variety of other food items were also eaten. Vascular plant composition of the diets differed between heath, meadow, and wetland habitats, whereas bryophyte composition did not. Also, a variety of fungal taxa were retrieved, but as most of the identified taxa belong to micromycetes, they were unlikely to be consumed as food. The role of fungi in the diet of lemmings remains to be investigated. We suggest that there may be substantial variation between habitats and regions in lemming diet.

High genetic diversity in a remote island population system: sans sex

It has been proposed that long-distance dispersal of mosses to the Hawaiian Islands rarely occurs and that the Hawaiian population of the allopolyploid peat moss Sphagnum palustre probably resulted from a single dispersal event. Here, we used microsatellites to investigate whether the Hawaiian population of the dioicous S. palustre had a single founder and to compare its genetic diversity to that found in populations of S. palustre in other regions. The genetic diversity of the Hawaiian population is comparable to that of larger population systems. Several lines of evidence, including a lack of sporophytes and an apparently restricted natural distribution, suggest that sexual reproduction is absent in the Hawaiian plants. In addition, all samples of Hawaiian S. palustre share a genetic trait rare in other populations. Time to most recent ancestor (TMRCA) analysis indicates that the Hawaiian population was probably founded 49-51 kyr ago. It appears that all Hawaiian plants of S. palustre descend from a single founder via vegetative propagation. The long-term viability of this clonal population coupled with the development of significant genetic diversity suggests that vegetative propagation in a moss does not necessarily preclude evolutionary success in the long term.

Molecular Species Delimitation in the Racomitrium canescens Complex (Grimmiaceae) and Implications for DNA Barcoding of Species Complexes in Mosses

In bryophytes a morphological species concept is still most commonly employed, but delimitation of closely related species based on morphological characters is often difficult. Here we test morphological species circumscriptions in a species complex of the moss genus Racomitrium, the R. canescens complex, based on variable DNA sequence markers from the plastid (rps4-trnT-trnL region) and nuclear (nrITS) genomes. The extensive morphological variability within the complex has led to different opinions about the number of species and intraspecific taxa to be distinguished. Molecular phylogenetic reconstructions allowed to clearly distinguish all eight currently recognised species of the complex plus a ninth species that was inferred to belong to the complex in earlier molecular analyses. The taxonomic significance of intraspecific sequence variation is discussed. The present molecular data do not support the division of the R. canescens complex into two groups of species (subsections or sections). Most morphological characters, albeit being in part difficult to apply, are reliable for species identification in the R. canescens complex. However, misidentification of collections that were morphologically intermediate between species questioned the suitability of leaf shape as diagnostic character. Four partitions of the molecular markers (rps4-trnT, trnT-trnL, ITS1, ITS2) that could potentially be used for molecular species identification (DNA barcoding) performed almost equally well concerning amplification and sequencing success. Of these, ITS1 provided the highest species discrimination capacity and should be considered as a DNA barcoding marker for mosses, especially in complexes of closely related species. Molecular species identification should be complemented by redefining morphological characters, to develop a set of easy-to-use molecular and non-molecular identification tools for improving biodiversity assessments and ecological research including mosses.

The Role of Life History Traits for Bryophyte Community Patterns in Two Contrasting Alpine Regions

Abstract Bryophytes are an important plant group with long-term dominance in alpine habitats. The alpine environment is however severe, and may restrict variation in bryophyte life history. This study examines the relationship between frequencies of bryophyte species and their life history traits between and within two alpine environments that differ in important environmental variables identified for bryophytes: 1) an oceanic region, with humid acidic soils (Setesdal) and 2) a sub-continental region, with more dry, productive, base-rich soils (Hol). A comparison between regions showed a higher frequency of base-demanding bryophytes in the sub-continental region. Sexual reproduction was more common among bryophytes in the oceanic region and these species also had higher indicator values for oceanic conditions and temperature as expected. Several life history traits were strongly correlated with bryophyte frequencies in the oceanic region—local species abundance increased in relation to shoot longevity, dioicism (mainly vegetative reproduction), and high humidity indicator values, but decreased for species with asexual propagules (gemmae). No traits were correlated with bryophyte frequencies in the sub-continental region. The importance of life history traits in the oceanic region indicates that severe environmental factors override bryophyte community heterogeneity only in the oceanic, acidic region. A possible explanation for higher environmental stress favoring tolerant long-lived species with vegetative reproduction could be higher grazing pressure, which is also reflected in significantly lower cryptogam coverage and correspondingly larger area of open soil in the oceanic region.