Jan Douda

Higher genetic diversity in recolonized areas than in refugia of Alnus glutinosa triggered by continent‐wide lineage admixture

Genetic admixture is supposed to be an important trigger of species expansions because it can create the potential for selection of genotypes suitable for new climatic conditions. Up until now, however, no continent‐wide population genetic study has performed a detailed reconstruction of admixture events during natural species expansions. To fill this gap, we analysed the postglacial history of Alnus glutinosa, a keystone species of European swamp habitats, across its entire distribution range using two molecular markers, cpDNA and nuclear microsatellites. CpDNA revealed multiple southern refugia located in the Iberian, Apennine, Balkan and Anatolian Peninsulas, Corsica and North Africa. Analysis of microsatellites variation revealed three main directions of postglacial expansion: (i) from the northern part of the Iberian Peninsula to Western and Central Europe and subsequently to the British Isles, (ii) from the Apennine Peninsula to the Alps and (iii) from the eastern part of the Balkan Peninsula to the Carpathians followed by expansion towards the Northern European plains. This challenges the classical paradigm that most European populations originated from refugial areas in the Carpathians. It has been shown that colonizing lineages have met several times and formed secondary contact zones with unexpectedly high population genetic diversity in Central Europe and Scandinavia. On the contrary, limited genetic admixture in southern refugial areas of A. glutinosa renders rear‐edge populations in the Mediterranean region more vulnerable to extinction due to climate change.

Migration Patterns of Subgenus Alnus in Europe since the Last Glacial Maximum: A Systematic Review

Background/Aims Recently, new palaeoecological records supported by molecular analyses and palaeodistributional modelling have provided more comprehensive insights into plant behaviour during the last Quaternary cycle. We reviewed the migration history of species of subgenus Alnus during the last 50,000 years in Europe with a focus on (1) a general revision of Alnus history since the Last Glacial Maximum (LGM), (2) evidence of northern refugia of Alnus populations during the LGM and (3) the specific history of Alnus in particular European regions. Methodology We determined changes in Alnus distribution on the basis of 811 and 68 radiocarbon-dated pollen and macrofossil sites, respectively. We compiled data from the European Pollen Database, the Czech Quaternary Palynological Database, the Eurasian Macrofossil Database and additional literature. Pollen percentage thresholds indicating expansions or retreats were used to describe patterns of past Alnus occurrence. Principal Findings An expansion of Alnus during the Late Glacial and early Holocene periods supports the presence of alders during the LGM in southern peninsulas and northerly areas in western Europe, the foothills of the Alps, the Carpathians and northeastern Europe. After glaciers withdrew, the ice-free area of Europe was likely colonized from several regional refugia; the deglaciated area of Scandinavia was likely colonized from a single refugium in northeastern Europe. In the more northerly parts of Europe, we found a scale-dependent pattern of Alnus expansion characterised by a synchronous increase of Alnus within individual regions, though with regional differences in the times of the expansion. In southern peninsulas, the Alps and the Carpathians, by contrast, it seems that Alnus expanded differently at individual sites rather than synchronously in whole regions. Conclusions Our synthesis supports the idea that northern LGM populations were important sources of postglacial Alnus expansion. The delayed Alnus expansion apparent in some regions was likely a result of environmental limitations.

Development of alder carr after the abandonment of wet grasslands during the last 70 years

Abstract• The secondary succession of wet grasslands to communities of alder carr dominated by Alnus glutinosa was recorded in different parts of Europe during the 20th century. However, knowledge of such development of alder carr remains insufficient.• The development of alder carr was reconstructed at five sites in the Czech Republic, using historical aerial photographs and methods of dendrochronology. The aims were to investigate the succession from wet grasslands to alder carr at sites previously intensively managed for agriculture and to find out the role of fluctuations in the groundwater table, caused by artificial drainage channels, in the observed stand dynamics and tree growth.• The spread of forest (i.e., an increase in forest cover) predominated until the 1970s at all sites. This trend was disrupted by a large-scale dieback of forest stands in four of the five sites after the 1970s, followed by an increase in patch heterogeneity, as indicated by landscape metrics. The radial growth increment in Alnus glutinosa has been affected predominately by local environmental factors, probably including the changing degree of waterlogging. Forest dieback was presumably connected with a lesser extent of drainage channels.• Our results indicate that observed successional pathways at sites of alder carr were probably caused by local changes in the groundwater table.Résumé• La succession secondaire des prairies humides en communautés de taillis d’aunes dominées par Al-nus glutinosa a été enregistrée dans différentes parties de l’Europe au cours du 20e siècle. Toutefois, la connaissance du développement de taillis d’aunes reste insuffisante.• Le développement de taillis d’aunes a été reconstruit dans cinq sites en République Tchèque, à l’aide de photographies aériennes historiques et des méthodologies de la dendrochronologie. Les objectifs étaient d’étudier la succession de prairies humides en taillis d’aunes sur des sites précédemment exploités intensivement par l’agriculture et de découvrir le rôle des fluctuations de la nappe phréatique, causées par le drainage artificiel des canaux, et d’observer la dynamique des peuplements et la croissance des arbres.• La progression de la forêt (c’est-à-dire, l’augmentation du couvert forestier) a prédominé jusque dans les années 1970 dans tous les sites. Cette tendance a été perturbée par un vaste dépérissement des peuplements forestiers dans quatre des cinq sites après les années 1970, suivie par une augmentation de l’hétérogénéité des bouquets d’arbres, comme indiqué par les paramètres du paysage. L’augmentation de la croissance radiale de Alnus glutinosa a été affectée principalement par les facteurs environnementaux, y compris sans doute l’évolution du degré d’engorgement. Le dépérissement des forêts est probablement lié à une moindre importance des canaux de drainage.• Nos résultats indiquent que les voies de succession observées sur les sites des taillis d’aunes ont probablement été causées par des changements locaux dans la nappe phréatique.

Flow cytometry, microsatellites and niche models reveal the origins and geographical structure of Alnus glutinosa populations in Europe

BACKGROUND AND AIMS Polyploidy in plants has been studied extensively. In many groups, two or more cytotypes represent separate biological entities with distinct distributions, histories and ecology. This study examines the distribution and origins of cytotypes of Alnus glutinosa in Europe, North Africa and western Asia. METHODS A combined approach was used involving flow cytometry and microsatellite analysis of 12 loci in 2200 plants from 209 populations combined with species distribution modelling using MIROC and CCSM climatic models, in order to analyse (1) ploidy and genetic variation, (2) the origin of tetraploid A. glutinosa, considering A. incana as a putative parent, and (3) past distributions of the species. KEY RESULTS The occurrence of tetraploid populations of A. glutinosa in Europe is determined for the first time. The distribution of tetraploids is far from random, forming two geographically well-delimited clusters located in the Iberian Peninsula and the Dinaric Alps. Based on microsatellite analysis, both tetraploid clusters are probably of autopolyploid origin, with no indication that A. incana was involved in their evolutionary history. A projection of the MIROC distribution model into the Last Glacial Maximum (LGM) showed that (1) populations occurring in the Iberian Peninsula and North Africa were probably interconnected during the LGM and (2) populations occurring in the Dinaric Alps did not exist throughout the last glacial periods, having retreated southwards into lowland areas of the Balkan Peninsula. CONCLUSIONS Newly discovered tetraploid populations are situated in the putative main glacial refugia, and neither of them was likely to have been involved in the colonization of central and northern Europe after glacial withdrawal. This could mean that neither the Iberian Peninsula nor the western part of the Balkan Peninsula served as effective refugial areas for northward post-glacial expansion of A. glutinosa.

Allopolyploid Origin of Chenopodium album s. str. (Chenopodiaceae): A Molecular and Cytogenetic Insight.

Reticulate evolution is characterized by occasional hybridization between two species, creating a network of closely related taxa below and at the species level. In the present research, we aimed to verify the hypothesis of the allopolyploid origin of hexaploid C. album s. str., identify its putative parents and estimate the frequency of allopolyploidization events. We sampled 122 individuals of the C. album aggregate, covering most of its distribution range in Eurasia. Our samples included putative progenitors of C. album s. str. of both ploidy levels, i.e. diploids (C. ficifolium, C. suecicum) and tetraploids (C. striatiforme, C. strictum). To fulfil these objectives, we analysed sequence variation in the nrDNA ITS region and the rpl32-trnL intergenic spacer of cpDNA and performed genomic in-situ hybridization (GISH). Our study confirms the allohexaploid origin of C. album s. str. Analysis of cpDNA revealed tetraploids as the maternal species. In most accessions of hexaploid C. album s. str., ITS sequences were completely or nearly completely homogenized towards the tetraploid maternal ribotype; a tetraploid species therefore served as one genome donor. GISH revealed a strong hybridization signal on the same eighteen chromosomes of C. album s. str. with both diploid species C. ficifolium and C. suecicum. The second genome donor was therefore a diploid species. Moreover, some individuals with completely unhomogenized ITS sequences were found. Thus, hexaploid individuals of C. album s. str. with ITS sequences homogenized to different degrees may represent hybrids of different ages. This proves the existence of at least two different allopolyploid lineages, indicating a polyphyletic origin of C. album s. str.

Cross-amplification and multiplexing of SSR markers for Alnus glutinosa and A. incana

We investigated 39 previously developed Betula, Alnus, and Corylus simple sequence repeat (SSR) markers for their utility in the cross-generic amplification of two European alder species, i.e., Alnus glutinosa and A. incana. Of these markers, ten loci had successful amplification within Alnus species. Finally, we designed two multiplexes composed of eight and nine loci for A. glutinosa and A. incana, respectively. Multiplexes were tested on 100 samples from five different populations of each species across Europe. The majority of loci had a relatively high genetic diversity, were in Hardy–Weinberg equilibrium, and showed low error rates and low occurrence of null alleles. By comparing sequences of source species and both Alnus species, we concluded that repeat motifs of five of these ten loci differed from those described for the source species. These differences represent mainly the modifications of the original motifs and affected compound or interrupted repeats as well as pure ones. The repeat motifs of three loci of the two alder species also differed. These mutations could lead to erroneous estimates of allele homology, because alleles with identical lengths will not have the same number of repeat units. Hence, before using microsatellite markers in studies comparing two or more species, they should be carefully examined and sequenced to ensure that allele homology is really stable and not affected by various inserts that change the sequence.

Vegetative sprouting as an additional pathway for a seed size-number trade-off: a field-parameterised simulation approach

Studies of perennial plants generally search for a seed size vs. seed number trade-off. Surprisingly, the fact that perennials may replace an investment in large seeds by the allocation to vegetative propagation has not yet been investigated as an additional pathway enabling species coexistence. We focused on the mechanisms of coexistence in Carex elata and C. elongata, two co-occurring clonal sedges dominant in European swamp alder forests. We asked the following questions: i) Is the number of germinated seeds a better predictor of species coexistence than the total number of seeds? ii) What recruitment conditions and competition rules determine vegetative sprouting to be an alternative to large, competitively superior seeds? We measured several species functional traits related to the colonisation and fitness of perennials. To examine the competitive hierarchy between species and microsite species preferences, we analysed the effects of environmental factors and plant densities on fitness-related traits using Structural Equation Modelling (SEM). Then, using a series of spatially explicit simulations partly parameterised based on the field measurement, we evaluated the importance of seed and ramet propagation and recruitment conditions for long-term species coexistence. SEM indicated a competitive hierarchy and a large overlap in microsite preferences between species. As a response to our initial questions we found that: i) Only differences in the numbers of germinated seeds, allowed the two species to coexist. If we consider only differences in the total number of seeds, the superior competitor (Carex elata) outcompeted the inferior competitor (C. elongata) in all scenarios. This is because the former produced about three-times as many seeds as the latter. ii) We show that vegetative sprouting represents an additional pathway for the seed size-number trade-off when the competitive superiority of species is attributed to vegetative propagation. This is another way that a species deals with the omnipresent seeds of other species. Taken together, our study demonstrates that differences in seed performance, coupled with differences in vegetative propagation related to competitive ability, are an additional mechanism allowing the coexistence of perennial plants.

The complexity underlying invasiveness precludes the identification of invasive traits: A comparative study of invasive and non-invasive heterocarpic Atriplex congeners

Heterocarpy enables species to effectively spread under unfavourable conditions by producing two or more types of fruit differing in ecological characteristics. Although it is frequent in annuals occupying disturbed habitats that are vulnerable to invasion, there is still a lack of congeneric studies addressing the importance of heterocarpy for species invasion success. We compared two pairs of heterocarpic Atriplex species, each of them comprising one invasive and one non-invasive non-native congener. In two common garden experiments, we (i) simulated the influence of different levels of nutrients and population density on plants grown from different types of fruits and examined several traits that are generally positively associated with invasion success, and (ii) grew plants in a replacement series experiment to evaluate resource partitioning between them and to compare their competitive ability. We found that specific functional traits or competitiveness of species cannot explain the invasiveness of Atriplex species, indicating that species invasiveness involves more complex interactions of traits that are important only in certain ecological contexts, i.e. in specific environmental conditions and only some habitats. Interestingly, species trait differences related to invasion success were found between plants growing from the ecologically most contrasting fruit types. We suggest that fruit types differing in ecological behaviour may be essential in the process of invasion or in the general spreading of heterocarpic species, as they either the maximize population growth (type C fruit) or enhance the chance of survival of new populations (type A fruit). Congeners offer the best available methodical framework for comparing traits among phylogenetically closely related invasive and non-invasive species. However, as indicated by our results, this approach is unlikely to reveal invasive traits because of the complexity underlying invasiveness.

Germination strategies of two dominant Carex species in a swamp alder forest: implications for restoration

Abstract Germination strategy is an essential mechanism that determines plant survival in previously established populations or newly colonised sites. Carex is a group of species that has shown difficulties to germinate experimentally and also many of them failed in order to use in restoration projects. Our aim was to determine whether Carex elata and C. elongata that dominate in vegetation of Central European swamps differ in their germination strategy. We conducted germination experiments with stratified and unstratified seeds of both species to determine: 1) if they are able to germinate fresh, 2) if they exhibit a cyclic dormancy pattern, and 3) if they will germinate from a seed bank. We demonstrate fresh seed germination and no evidence of cyclic dormancy in either species. Stratification did not enhance final germination but it did accelerate germination. Seed bank seeds of both species germinate sparsely. We demonstrate that these coexisting Carex species differ with respect to final germination. The higher germination percentages of the fresh seeds compared to buried and seed bank seeds of both species probably reflect adaptation to fluctuating water-level conditions. In summary, these findings support a strategy of fresh germination in a highly-variable environment. Our study indicates that both C. elata and C. elongata are suitable for restoration projects. Successful establishment and revegetation with C. elongata may result simply from sowing fresh seeds. In contrast, seed sowing, combined with vegetatively produced seedling transplants is essential for the successful restoration of C. elata.

Hybridization and polyploidization within the Chenopodium album aggregate analysed by means of cytological and molecular markers

Hybridization and polyploidization represent an important speciation mechanism in the diploid-polyploid complex of the Chenopodium album aggregate. In the present study we successfully reconstructed the evolutionary histories of the majority of Eurasian representatives of the C. album aggregate, resulting in the most comprehensive phylogenetic analysis of this taxonomically intricate group of species to date. We applied a combination of classical karyology for precise chromosome number determination, genomic in-situ hybridization for the determination of genomic composition, flow cytometry for the estimation of genome size and sequencing of plastid (cpDNA) and nuclear (ribosomal internal transcribed spacer - ITS and the introns of the FLOWERING LOCUS T LIKE genes - FTL) markers for a phylogenetic reconstruction and the identification of parental genomes in polyploid taxa. The FTL markers identified eight well supported evolutionary lineages. Five of them include at least one diploid species, and the remaining three comprise solely the subgenomes of polyploids that probably represent extinct or unknown diploid taxa. The existence of eight basic diploid lineages explains the origin of seven Eurasian polyploid groups and brings evidence of a nearly unlimited number of subgenomic combinations. The supposed promiscuity generated new species wherever different diploid lineages met each other and gave rise to tetraploid species or whenever they met other tetraploid species to produce hexaploid species throughout their evolutionary history. Finally, we unravelled a surprisingly simple scheme of polyploid species formation within the C. album aggregate. We determined seven groups of polyploid species differing in their origin in either Eurasia or Africa and convincingly demonstrated that (1) all Chenopodium polyploid species under study are of allopolyploid origin, (2) there are eight major monophyletic evolutionary lineages represented by extant or extinct/unknown diploid taxa, (3) those monophyletic lineages represent individual subgenomes, (4) hybridization among the lineages created seven subgenomic combinations of polyploid taxa, (5) taxa represented by particular subgenome combinations were further subjected to diversification, and (6) the majority of species are relatively young, not exceeding the age of the Quaternary period.