Parallel patterns of genetic diversity and structure in circumboreal species of the Sphagnum capillifolium complex.

PREMISE: Shared geographical patterns of population genetic variation among related species is a powerful means to identify the historical events that drive diversification. The Sphagnum capillifolium complex is a group of closely related peat mosses within the Sphagnum subgenus Acutifolia and contains several circumboreal species whose ranges encompass both glaciated and unglaciated regions across the northern hemisphere. In this paper, we (1) inferred the phylogeny of subg. Acutifolia and (2) investigated patterns of population structure and genetic diversity among five circumboreal species within the S. capillifolium complex. METHODS: We generated RAD sequencing data from most species of the subg. Acutifolia and samples from across the distribution ranges of circumboreal species within the S. capillifolium complex. RESULTS: We resolved at least 14 phylogenetic clusters within the S. capillifolium complex. Five circumboreal species show some common patterns: One population system comprises plants in eastern North America and Europe, and another comprises plants in the Pacific Northwest or around the Beringian and Arctic regions. Alaska appears to be a hotspot for genetic admixture, genetic diversity, and sometimes endemic subclades. CONCLUSIONS: Our results support the hypothesis that populations of five circumboreal species within the S. capillifolium complex survived in multiple refugia during the last glacial maximum. Long-distance dispersal out of refugia, population bottlenecks, and possible adaptations to conditions unique to each refugium could have contributed to current geographic patterns. These results indicate the important role of historical events in shaping the complex population structure of plants with broad distribution ranges.

Short stories from Sphagnum of rare species, taxonomy, and speciation.

Conserving species and their genetic variation are a global priority to safeguard evolutionary potential in a rapidly changing world. Species are fundamental units in research and nature management, but taxonomic work is increasingly undermined. Increasing knowledge on the species genetic diversity would aid in prioritizing conservation efforts. Sphagnum is a diverse, well-known bryophyte genus, which makes the genus suited to study speciation and cryptic variation. The species share specific characteristics and can be difficult to separate in the field. By combining molecular data with thorough morphological examination, new species have recently been discovered. Still, there are taxonomic uncertainties, even for species assessed on the IUCN Red List of threatened species. Here, we use molecular data to examine three rare species within the subgenus Acutifolia described based on morphological characters. All species have narrow distributions and limited dispersability. First, we confirm the genetic origin of S. skyense. Second, we show that S. venustum is a haploid species genetically distinct from morphologically similar species. Lastly, S. nitidulum was found to have a distinct haplotype, but cannot be genetically separated from other red Acutifolia species. We also found high genetic variation within red Acutifolia specimens, indicating the need of further morphological examination and possibly taxonomic revision. Until then, our results have shown that genetic data can aid in prioritizing targets of conservation efforts when taxonomy is unresolved. All three taxa should be further searched for by field biologists to increase knowledge about their distribution ranges.

A regionally coherent ecological fingerprint of climate change, evidenced from natural history collections.

Climate change has dramatic impacts on ecological systems, affecting a range of ecological factors including phenology, species abundance, diversity, and distribution. The breadth of climate change impacts on ecological systems leads to the occurrence of fingerprints of climate change. However, climate fingerprints are usually identified across broad geographical scales and are potentially influenced by publication biases. In this study, we used natural history collections spanning over 250 years, to quantify a range of ecological responses to climate change, including phenology, abundance, diversity, and distributions, across a range of taxa, including vertebrates, invertebrates, plants, and fungi, within a single region, Central Norway. We tested the hypotheses that ecological responses to climate change are apparent and coherent at a regional scale, that longer time series show stronger trends over time and in relation to temperature, and that ecological responses change in trajectory at the same time as shifts in temperature. We identified a clear regional coherence in climate signal, with decreasing abundances of limnic zooplankton (on average by 7691 individuals m-3 °C-1) and boreal forest breeding birds (on average by 1.94 territories km-2 °C-1), and earlier plant flowering phenology (on average 2 days °C-1) for every degree of temperature increase. In contrast, regional-scale species distributions and species diversity were largely stable. Surprisingly, the effect size of ecological response did not increase with study duration, and shifts in responses did not occur at the same time as shifts in temperature. This may be as the long-term studies include both periods of warming and temperature stability, and that ecological responses lag behind warming. Our findings demonstrate a regional climate fingerprint across a long timescale. We contend that natural history collections provide a unique window on a broad spectrum of ecological responses at timescales beyond most ecological monitoring programs. Natural history collections are thus an essential source for long-term ecological research.

Phylogenomic structure and speciation in an emerging model: the Sphagnum magellanicum complex (Bryophyta).

Sphagnum magellanicum is one of two Sphagnum species for which a reference-quality genome exists to facilitate research in ecological genomics. Phylogenetic and comparative genomic analyses were conducted based on resequencing data from 48 samples and RADseq analyses based on 187 samples. We report herein that there are four clades/species within the S. magellanicum complex in eastern North America and that the reference genome belongs to Sphagnum divinum. The species exhibit tens of thousands (RADseq) to millions (resequencing) of fixed nucleotide differences. Two species, however, referred to informally as S. diabolicum and S. magni because they have not been formally described, are differentiated by only 100 (RADseq) to 1000 (resequencing) of differences. Introgression among species in the complex is demonstrated using D-statistics and f4 ratios. One ecologically important functional trait, tissue decomposability, which underlies peat (carbon) accumulation, does not differ between segregates in the S. magellanicum complex, although previous research showed that many closely related Sphagnum species have evolved differences in decomposability/carbon sequestration. Phylogenetic resolution and more accurate species delimitation in the S. magellanicum complex substantially increase the value of this group for studying the early evolutionary stages of climate adaptation and ecological evolution more broadly.

Extensive Genome-Wide Phylogenetic Discordance Is Due to Incomplete Lineage Sorting and Not Ongoing Introgression in a Rapidly Radiated Bryophyte Genus.

The relative importance of introgression for diversification has long been a highly disputed topic in speciation research and remains an open question despite the great attention it has received over the past decade. Gene flow leaves traces in the genome similar to those created by incomplete lineage sorting (ILS), and identification and quantification of gene flow in the presence of ILS is challenging and requires knowledge about the true phylogenetic relationship among the species. We use whole nuclear, plastid, and organellar genomes from 12 species in the rapidly radiated, ecologically diverse, actively hybridizing genus of peatmoss (Sphagnum) to reconstruct the species phylogeny and quantify introgression using a suite of phylogenomic methods. We found extensive phylogenetic discordance among nuclear and organellar phylogenies, as well as across the nuclear genome and the nodes in the species tree, best explained by extensive ILS following the rapid radiation of the genus rather than by postspeciation introgression. Our analyses support the idea of ancient introgression among the ancestral lineages followed by ILS, whereas recent gene flow among the species is highly restricted despite widespread interspecific hybridization known in the group. Our results contribute to phylogenomic understanding of how speciation proceeds in rapidly radiated, actively hybridizing species groups, and demonstrate that employing a combination of diverse phylogenomic methods can facilitate untangling complex phylogenetic patterns created by ILS and introgression.

Phylogenetic structure in the Sphagnum recurvum complex (Bryophyta) in relation to taxonomy and geography.

PREMISE: The Sphagnum recurvum complex comprises a group of closely related peat mosses that are dominant components of many northern wetland ecosystems. Taxonomic hypotheses for the group range from interpreting the whole complex as one polymorphic species to distinguishing 6-10 species. The complex occurs throughout the Northern Hemisphere, and some of the putative species have intercontinental ranges. Our goals were to delimit the complex and assess its phylogenetic structure in relation to morphologically defined species and intercontinental geography. METHODS: RADseq analyses were applied to a sample of 384 collections from Europe, North America, and Asia. The data were subjected to maximum likelihood phylogenetic analyses and analyses of genetic structure using the software STRUCTURE and multivariate ordination approaches. RESULTS: The S. recurvum complex includes S. angustifolium, S. fallax, S. flexuosum, S. pacificum, and S. recurvum as clades with little evidence of admixture. We also resolved an unnamed clade that is referred to here as S. "pseudopacificum." We confirm that S. balticum and S. obtusum are nested within the complex. Species with bluntly acute to obtuse stem leaf apices are sister to those with acute to apiculate leaves. Most of the species exhibit some differentiation between intraspecific population systems disjunct on different continents. CONCLUSIONS: We recognize seven species in the amended S. recurvum complex, including S. balticum and S. obtusum, in addition to the informal clade S. "pseudopacificum." Although we detected some geographically correlated phylogenetic structure within widespread morphospecies, our RADseq data support the interpretation that these species have intercontinental geographic ranges.

Can mowing restore boreal rich-fen vegetation in the face of climate change?

Low-frequency mowing has been proposed to be an effective strategy for the restoration and management of boreal fens after abandonment of traditional haymaking. This study investigates how mowing affects long-term vegetation change in both oceanic and continental boreal rich-fen vegetation. This will allow evaluation of the effectiveness of mowing as a management and restoration tool in this ecosystem in the face of climate change. At two nature reserves in Central Norway (Tågdalen, 63° 03' N, 9° 05 E, oceanic climate and Sølendet, 62° 40' N, 11° 50' E, continental climate), we used permanent plot data from the two sites to compare plant species composition from the late 1960s to the early 1980s with that recorded in 2012-2015 in abandoned and mown fens. Changes in species composition and frequency were analysed by multivariate and univariate methods in relation to environmental variables and modelled climate and groundwater data. Mowing resulted in a decline in shrub and Molinia caerulea cover at the continental and oceanic sites respectively, and the total cover of specialist fen species had increased to a significantly greater extent in the mown plots than the unmown at the continental site. However, mowing did not have an effect on the cover of specialist bryophyte species, and some specialist species declined regardless of mowing treatment. Temperature sums had increased at both sites, but precipitation had not changed significantly. Mowing was shown to be the most important determinant of plant community composition at both sites, with local environmental conditions being of secondary importance. In conclusion, the abandonment of traditional management practices results in the loss of characteristic fen species. In order to encourage the restoration of typical rich-fen vegetation, particularly in oceanic areas, additional management measures, such as more intensive mowing, may be required.

Contrasting spatial, temporal and environmental patterns in observation and specimen based species occurrence data.

Species occurrence data records the location and time of an encounter with a species, and is valuable for many aspects of ecological and evolutionary analyses. A key distinction within species occurrence data is between (1) collected and preserved specimens that can be taxonomically validated (i.e., natural history collections), and (2) observations, which are more error prone but richer in terms of number and spread of observations. In this study we analyse the distribution in temporal, spatial, taxonomic and environmental coverage of specimen- and observation based species occurrence data for land plants in Norway, a region with strong climatic and human population density gradients. Of 4.8 million species occurrence records, the majority (78%) were observations. However, there was a greater species richness in the specimen record (N = 4691) than in the observation record (N = 3193) and most species were recorded more as specimens than observations. Specimen data was on average older, and collected later during the year. Both record types were highly influenced by a small number of prolific contributors. The species most highly represented in the observation data set were widespread or invasive, while in the specimen records, taxonomically challenging species were overrepresented. Species occurrence records were unevenly spatially distributed. Both specimen and observation records were concentrated in regions of Norway with high human population density and with high temperatures and precipitation, but in different regions within Norway. Observation and specimen records thus differ in taxonomic, temporal, spatial and environmental coverage for a well-sampled group and study region, potentially influencing the ecological inferences made from studies utilizing species occurrence data. The distribution of observation data dominates the dataset, so inferences of species diversity and distributions do not correspond to the evolutionary or physiological knowledge of species, which is based on specimen data. We make recommendations for users of biodiversity data, and collectors to better exploit the complementary strengths of these distinct biodiversity data types.

Divergent evolution and niche differentiation within the common peatmoss Sphagnum magellanicum.

PREMISE OF THE STUDY: Populations with phenotypic polymorphism in discrete characters may be good models for investigating genome evolution and speciation. Sphagnum magellanicum Brid. is found throughout the northern hemisphere, and despite considerable variation in morphological characters, it is considered one of the least taxonomically controversial peatmoss species. We have observed two main morphs of the species associated with different microhabitats. Here we investigated the genomic and environmental basis of this intraspecific morphological variation. METHODS: We conducted transplant and common garden experiments to test whether the two morphs are genetically differentiated. We then used RAD-sequencing to quantify the genomic divergence between the morphs and approximate Bayesian computation (ABC) to infer the most likely demographic scenario explaining the genome-wide differentiation of the two morphs. KEY RESULTS: We found that genomic differentiation between the two morphs is unexpectedly high and that several of the differentiated morphological characters have a genetic basis. Using simulation approaches, we found support for a scenario of ancient divergence followed by recent secondary contact. CONCLUSIONS: We show that the two morphs represent the two main genetic clusters previously found worldwide. Our results demonstrate that relatively minor morphological differentiation in a presumed phenotypically plastic peatmoss may be associated with massive divergence across the genome.

Spatial Genetic Structure of the Abundant and Widespread Peatmoss Sphagnum magellanicum Brid.

Spore-producing organisms have small dispersal units enabling them to become widespread across continents. However, barriers to gene flow and cryptic speciation may exist. The common, haploid peatmoss Sphagnum magellanicum occurs in both the Northern and Southern hemisphere, and is commonly used as a model in studies of peatland ecology and peatmoss physiology. Even though it will likely act as a rich source in functional genomics studies in years to come, surprisingly little is known about levels of genetic variability and structuring in this species. Here, we assess for the first time how genetic variation in S. magellanicum is spatially structured across its full distribution range (Northern Hemisphere and South America). The morphologically similar species S. alaskense was included for comparison. In total, 195 plants were genotyped at 15 microsatellite loci. Sequences from two plastid loci (trnG and trnL) were obtained from 30 samples. Our results show that S. alaskense and almost all plants of S. magellanicum in the northern Pacific area are diploids and share the same gene pool. Haploid plants occur in South America, Europe, eastern North America, western North America, and southern Asia, and five genetically differentiated groups with different distribution ranges were found. Our results indicate that S. magellanicum consists of several distinct genetic groups, seemingly with little or no gene flow among them. Noteworthy, the geographical separation of diploids and haploids is strikingly similar to patterns found within other haploid Sphagnum species spanning the Northern Hemisphere. Our results confirm a genetic division between the Beringian and the Atlantic that seems to be a general pattern in Sphagnum taxa. The pattern of strong genetic population structuring throughout the distribution range of morphologically similar plants need to be considered in future functional genomic studies of S. magellanicum.

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.

Restricted variation in plant barcoding markers limits identification in closely related bryophyte species.

Species-level identification and delimitation of bryophytes using the proposed general barcode markers for land plants has been challenging. Bryophyta (mosses) is the second most species-rich group of land plants after angiosperms, and it is thus of great importance to find useful barcoding regions also for this group of plants. We investigated how the plastid regions atpF-atpH, rbcL and trnH-psbA and the nuclear ITS2 region performed as barcode markers on closely related bryophyte taxa of selected moss (Bartramia, Distichium, Fissidens, Meesia and Syntrichia) and liverwort (Blepharostoma) genera from boreal and arctic regions. We also evaluated how sequencing success of herbarium specimens is related to length of the sequenced fragment, specimen age and taxonomic group. Sequencing success was higher for shorter fragments and younger herbarium specimens, but was lower than expected in the genera Distichium and Fissidens, indicating imperfect universality of the primers used. None of the studied DNA barcode regions showed a consistent barcode gap across the studied genera. As a single locus, the region atpF-atpH performed slightly better than rbcL and ITS2 and much better than trnH-psbA in terms of grouping conspecific sequences in monophyletic groups. This marker also gave a higher percentage of correct hits when conducting blast searches on a local database of identified sequences. Concatenated data sets of two and three markers grouped more conspecific sequences in monophyletic groups, but the improvement was not great compared with atpF-atpH alone. A discussion of recent studies testing barcode regions for bryophytes is given. We conclude that atpF-atpH, rbcL and ITS2 are to be the most promising barcode markers for mosses.

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.

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.

The rare peat moss Sphagnum wulfianum (Sphagnaceae) did not survive the last glacial period in northern European refugia.

PREMISE OF THE STUDY: Organisms may survive unfavorable conditions either by moving to more favorable areas by means of dispersal or by adapting to stressful environments. Pleistocene glacial periods represent extremely unfavorable conditions for the majority of life forms, especially sessile organisms. Many studies have revealed placements of refugial areas and postglacial colonization patterns of seed plants, but little is still known about areas of long-term survival and historical migration routes of bryophytes. Given overall differences in stress tolerance between seed plants and bryophytes, it is of interest to know whether bryophytes have survived periods of extreme climatic conditions better then seed plants in northern areas. METHODS: The haploid and rarely spore-producing peat moss Sphagnum wulfianum is mostly found in areas that were covered by ice during the last glacial maximum. Twelve microsatellite markers were amplified from 43 populations (367 shoots) of this species, and data were analyzed using population genetic diversity statistics, Bayesian clustering methods, and coalescence-based inference tools to estimate historical and demographic parameters. KEY RESULTS: Genetic diversity within populations was low, but populations were highly differentiated, with two main genetic clusters being recognized. CONCLUSION: The two main genetic groups have diverged quite recently in the Holocene, and the pattern of genetic variability and structuring gives no support for survival in Scandinavian refugia during the last glacial period in this species. The dispersal ability of this plant thus seems surprisingly high despite its infrequent spore production.

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.

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.

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.