Karol Krak

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.

Effects of Inoculum Additions in the Presence of a Preestablished Arbuscular Mycorrhizal Fungal Community

ABSTRACT Communities of arbuscular mycorrhizal fungi (AMF) are crucial for promoting plant productivity in most terrestrial systems, including anthropogenically managed ecosystems. Application of AMF inocula has therefore become a widespread practice. It is, however, pertinent to understand the mechanisms that govern AMF community composition and their performance in order to design successful manipulations. Here we assess whether the composition and plant growth-promotional effects of a synthetic AMF community can be altered by inoculum additions of the isolates forming the community. This was determined by following the effects of three AMF isolates, each inoculated in two propagule densities into a preestablished AMF community. Fungal abundance in roots and plant growth were evaluated in three sequential harvests. We found a transient positive response in AMF abundance to the intraspecific inoculation only in the competitively weakest isolate. The other two isolates responded negatively to intra- and interspecific inoculations, and in some cases plant growth was also reduced. Our results suggest that increasing the AMF density may lead to increased competition among fungi and a trade-off with their ability to promote plant productivity. This is a key ecological aspect to consider when introducing AMF into soils.

Trichomes in the tribe Lactuceae (Asteraceae) — taxonomic implications

Although the infratribal classification of the Lactuceae has been the focus of several studies, relationships between the genera are not yet fully resolved. While trichome characters have been shown to be an important tool in taxa delimitation in many plant families, a detailed study on trichomes within the Lactuceae was still lacking. We assess here how trichome types can contribute to the clarification of generic relationships in this tribe. Trichome diversity was investigated on stems and leaves of 135 representatives of 53 genera by light and scanning electron microscopy. According to their morphology and anatomy, trichomes were classified into eight types and several subtypes. Phenetic analysis based on the presence or absence of particular trichome types was performed. The previously supposed monophyly of exclusively American genera and affinities between several closely related genera were confirmed. A new circumscription of the subtribe Hieraciinae is proposed, in accordance with recent molecular data. According to the present study, this subtribe is characterized by a unique combination of two trichome types and comprises the following genera: Andryala, Hieracium, Hispidella, Pilosella, Stenotheca. The previously included genera Arnoseris, Hololeion and Tolpis do not belong to this assemblage and their removal from the Hieraciinae should be reconsidered.

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.

Intraradical Dynamics of Two Coexisting Isolates of the Arbuscular Mycorrhizal Fungus Glomus intraradices Sensu Lato as Estimated by Real-Time PCR of Mitochondrial DNA

ABSTRACT Real-time PCR in nuclear ribosomal DNA (nrDNA) is becoming a well-established tool for the quantification of arbuscular mycorrhizal (AM) fungi, but this genomic region does not allow the specific amplification of closely related genotypes. The large subunit of mitochondrial DNA (mtDNA) has a higher-resolution power, but mtDNA-based quantification has not been previously explored in AM fungi. We applied real-time PCR assays targeting the large subunit of mtDNA to monitor the DNA dynamics of two isolates of Glomus intraradices sensu lato coexisting in the roots of medic (Medicago sativa). The mtDNA-based quantification was compared to quantification in nrDNA. The ratio of copy numbers determined by the nrDNA- and mtDNA-based assays consistently differed between the two isolates. Within an isolate, copy numbers of the nuclear and the mitochondrial genes were closely correlated. The two quantification approaches revealed similar trends in the dynamics of both isolates, depending on whether they were inoculated alone or together. After 12 weeks of cultivation, competition between the two isolates was observed as a decrease in the mtDNA copy numbers of one of them. The coexistence of two closely related isolates, which cannot be discriminated by nrDNA-based assays, was thus identified as a factor influencing the dynamics of AM fungal DNA in roots. Taken together, the results of this study show that real-time PCR assays targeted to the large subunit of mtDNA may become useful tools for the study of coexisting AM fungi.

Multiple hybridization events in Cardamine (Brassicaceae) during the last 150 years: revisiting a textbook example of neoallopolyploidy

BACKGROUND AND AIMS Recently formed allopolyploid species represent excellent subjects for exploring early stages of polyploid evolution. The hexaploid Cardamine schulzii was regarded as one of the few nascent allopolyploid species formed within the past ∼150 years that presumably arose by autopolyploidization of a triploid hybrid, C. × insueta; however, the most recent investigations have shown that it is a trigenomic hybrid. The aims of this study were to explore the efficiency of progenitor-specific microsatellite markers in detecting the hybrid origins and genome composition of these two allopolyploids, to estimate the frequency of polyploid formation events, and to outline their evolutionary potential for long-term persistence and speciation. METHODS Flow-cytometric ploidy-level screening and genotyping by progenitor-specific microsatellite markers (20 microsatellite loci) were carried out on samples focused on hybridizing populations at Urnerboden, Switzerland, but also including comparative material of the parental species from other sites in the Alps and more distant areas. KEY RESULTS It was confirmed that hybridization between the diploids C. amara and C. rivularis auct. gave rise to triploid C. × insueta, and it is inferred that this has occurred repeatedly. Evidence is provided that C. schulzii comprises three parental genomes and supports its origin from hybridization events between C. × insueta and the locally co-occurring hypotetraploid C. pratensis, leading to two cytotypes of C. schulzii: hypopentaploid and hypohexaploid. Each cytotype of C. schulzii is genetically uniform, suggesting their single origins. CONCLUSIONS Persistence of C. schulzii has presumably been achieved only by perennial growth and clonal reproduction. This contrasts with C. × insueta, in which multiple origins and occasional sexual reproduction have generated sufficient genetic variation for long-term survival and evolutionary success. This study illustrates a complex case of recurrent hybridization and polyploidization events, and highlights the role of triploids that promoted the origin of trigenomic hybrids.

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.

Development of novel low-copy nuclear markers for Hieraciinae (Asteraceae) and their perspective for other tribes

PREMISE OF THE STUDY The development of three low-copy nuclear markers for low taxonomic level phylogenies in Asteraceae with emphasis on the subtribe Hieraciinae is reported. METHODS AND RESULTS Marker candidates were selected by comparing a Lactuca complementary DNA (cDNA) library with public DNA sequence databases. Interspecific variation and phylogenetic signal of the selected genes were investigated for diploid taxa from the subtribe Hieraciinae and compared to a reference phylogeny. Their ability to cross-amplify was assessed for other Asteraceae tribes. All three markers had higher variation (2.1-4.5 times) than the internal transcribed spacer (ITS) in Hieraciinae. Cross-amplification was successful in at least seven other tribes of the Asteraceae. Only three cases indicating the presence of paralogs or pseudogenes were detected. CONCLUSIONS The results demonstrate the potential of these markers for phylogeny reconstruction in the Hieraciinae as well as in other Asteraceae tribes, especially for very closely related species.