Andreas D. Drouzas

Molecular‐ and pollen‐based vegetation analysis in lake sediments from central Scandinavia

Plant and animal biodiversity can be studied by obtaining DNA directly from the environment. This new approach in combination with the use of generic barcoding primers (metabarcoding) has been suggested as complementary or alternative to traditional biodiversity monitoring in ancient soil sediments. However, the extent to which metabarcoding truly reflects plant composition remains unclear, as does its power to identify species with no pollen or macrofossil evidence. Here, we compared pollen‐based and metabarcoding approaches to explore the Holocene plant composition around two lakes in central Scandinavia. At one site, we also compared barcoding results with those obtained in earlier studies with species‐specific primers. The pollen analyses revealed a larger number of taxa (46), of which the majority (78%) was not identified by metabarcoding. The metabarcoding identified 14 taxa (MTUs), but allowed identification to a lower taxonomical level. The combined analyses identified 52 taxa. The barcoding primers may favour amplification of certain taxa, as they did not detect taxa previously identified with species‐specific primers. Taphonomy and selectiveness of the primers are likely the major factors influencing these results. We conclude that metabarcoding from lake sediments provides a complementary, but not an alternative, tool to pollen analysis for investigating past flora. In the absence of other fossil evidence, metabarcoding gives a local and important signal from the vegetation, but the resulting assemblages show limited capacity to detect all taxa, regardless of their abundance around the lake. We suggest that metabarcoding is followed by pollen analysis and the use of species‐specific primers to provide the most comprehensive signal from the environment.

Allozyme differentiation in the Mediterranean firs (Abies, Pinaceae). A first comparative study with phylogenetic implications

Nineteen natural Mediterranean fir populations, belonging to eight species and to one natural hybrid (A. ×borisii-regis), were investigated by starch and polyacrylamide gel electrophoresis. A total of 31 alleles was scored at eight loci (IDH-B, ACP-A, PER-B, 6PGD-A, 6PGD-B, MNR-B, PGI-B, PGM-A. Great variation was observed in the heterozygosity among the population studied and ranged from 0.010 (A. pinsapo) to 0.328 (A. cephalonica). The interpopulation genetic diversity was about 26% of the total genetic diversity. From the dendrogram, new phylogenetic relationships were revealed. High affinity was observed between the Calabrian fir population and the one from north-west Greece as well as betweenA. equi-trojani grown in Asia Minor and the southern Greek populations. Species specific alleles were found inA. cilicica. From the findings of the present work, a new hypothesis concerning the taxonomy, distribution and evolution ofAbies species in the Balkan Peninsula is supported.

Local Adaptation in European Firs Assessed through Extensive Sampling across Altitudinal Gradients in Southern Europe

Background Local adaptation is a key driver of phenotypic and genetic divergence at loci responsible for adaptive traits variations in forest tree populations. Its experimental assessment requires rigorous sampling strategies such as those involving population pairs replicated across broad spatial scales. Methods A hierarchical Bayesian model of selection (HBM) that explicitly considers both the replication of the environmental contrast and the hierarchical genetic structure among replicated study sites is introduced. Its power was assessed through simulations and compared to classical ‘within-site’ approaches (FDIST, BAYESCAN) and a simplified, within-site, version of the model introduced here (SBM). Results HBM demonstrates that hierarchical approaches are very powerful to detect replicated patterns of adaptive divergence with low false-discovery (FDR) and false-non-discovery (FNR) rates compared to the analysis of different sites separately through within-site approaches. The hypothesis of local adaptation to altitude was further addressed by analyzing replicated Abies alba population pairs (low and high elevations) across the species’ southern distribution range, where the effects of climatic selection are expected to be the strongest. For comparison, a single population pair from the closely related species A. cephalonica was also analyzed. The hierarchical model did not detect any pattern of adaptive divergence to altitude replicated in the different study sites. Instead, idiosyncratic patterns of local adaptation among sites were detected by within-site approaches. Conclusion Hierarchical approaches may miss idiosyncratic patterns of adaptation among sites, and we strongly recommend the use of both hierarchical (multi-site) and classical (within-site) approaches when addressing the question of adaptation across broad spatial scales.

Genetic insights into the hybrid origin of Abies × borisii-regis Mattf.

Abies × borisii-regis Mattf. (King Boris fir) is a taxon endemic to the southern Balkan Peninsula, described as a hybrid between the widespread A. alba Mill. (Silver fir) and the Greek endemic A. cephalonica Loud (Greek fir). Even though A. × borisii-regis has attracted much research attention in the past, its origin, geographical distribution and taxonomic status are not fully elucidated and molecular evidence for hybridization is missing. To shed more light on this issue, we analyzed representative populations from these three Abies taxa using paternally inherited (chloroplast) and maternally inherited (mitochondrial) DNA markers. Both Silver and Greek fir could be clearly distinguished using mitochondrial markers, while we observed a mixture of maternal lineages in the A. × borisii-regis populations. In contrast, using chloroplast markers, we could not identify species-specific haplotypes, but a neighbor-joining analysis of population genetic distances revealed two separate clusters for the Silver fir and the Greek fir, while the A. × borisii-regis populations were placed in intermediate positions. Our results are in agreement with the hypothesis that the A. × borisii-regis populations investigated are a result of hybridization between A. cephalonica and A. alba.

The European Paleoendemic Haberlea rhodopensis (Gesneriaceae) Has an Oligocene Origin and a Pleistocene Diversification and Occurs in a Long-Persisting Refugial Area in Southeastern Europe

Premise of research. Even though the Balkan Peninsula is a biodiversity hot spot in southeastern Europe harboring many endemic plants, very little is known about the temporal extent of the phylogeographic history and the contemporary genetic diversity of the endemics there. Haberlea rhodopensis is one of the European Gesneriaceae species occurring in this region and represents appropriate study material to address these questions. Methodology. We generated fossil-dated molecular phylogenies (atpB-rbcL, trnH-psbA, trnLF, ITS) across the Lamiales to determine the origin and age of H. rhodopensis and conducted phylogeographic (trnH-psbA, ITS) and population genetic (ISSRs) analyses on 17 populations from Bulgaria and Greece, covering the entire distribution range of the species, to investigate their biogeographic history, present-day genetic diversity, and differentiation levels. Pivotal results. The European Gesneriaceae genera have a Tertiary origin in the early Oligocene, while the Haberlea lineage emerged in the late Oligocene. Extinctions appear to have marked the history of the genus for a long period of time, and the extant populations diverged in the late Pleistocene. A significant differentiation was apparent between populations from Bulgaria, that is, the Balkan Mountains in the north and the Bulgarian side of the Rhodopi massif in the south, but there was an even stronger differentiation between the latter and populations from Greece. This might be explained by the Rhodopi massif representing a barrier to gene flow, enforced during the Last Glacial Maximum, during which populations descended on opposing north- and south-facing mountain slopes. Conclusions. Haberlea represents an ancient lineage with recent diversification. The extant populations are of recent origin and indicate glacial refugial areas in Bulgaria and Greece. This study sheds light on historic and current phylogenetic and phylogeographic events that shaped the flora of the Balkan Peninsula, an area that has long been recognized for its species diversity and richness. Our data suggest that it may have acted as a persistent refugial area in southeast Europe since the mid-Tertiary.

Demography and mating system shape the genome-wide impact of purifying selection in Arabis alpina

Significance Intermediate outcrossing rates are theoretically predicted to maintain effective selection against harmful alleles, but few studies have empirically tested this prediction with the use of genomic data. We used whole-genome resequencing data from alpine rock-cress to study how genetic variation and purifying selection vary with mating system. We find that populations with intermediate outcrossing rates have similar levels of genetic diversity as outcrossing populations, and that purifying selection against harmful alleles is efficient in mixed-mating populations. In contrast, self-fertilizing populations from Scandinavia have strongly reduced genetic diversity and accumulate harmful mutations, likely as a result of demographic effects of postglacial colonization. Our results suggest that mixed-mating populations can avoid some of the negative evolutionary consequences of high self-fertilization rates. Plant mating systems have profound effects on levels and structuring of genetic variation and can affect the impact of natural selection. Although theory predicts that intermediate outcrossing rates may allow plants to prevent accumulation of deleterious alleles, few studies have empirically tested this prediction using genomic data. Here, we study the effect of mating system on purifying selection by conducting population-genomic analyses on whole-genome resequencing data from 38 European individuals of the arctic-alpine crucifer Arabis alpina. We find that outcrossing and mixed-mating populations maintain genetic diversity at similar levels, whereas highly self-fertilizing Scandinavian A. alpina show a strong reduction in genetic diversity, most likely as a result of a postglacial colonization bottleneck. We further find evidence for accumulation of genetic load in highly self-fertilizing populations, whereas the genome-wide impact of purifying selection does not differ greatly between mixed-mating and outcrossing populations. Our results demonstrate that intermediate levels of outcrossing may allow efficient selection against harmful alleles, whereas demographic effects can be important for relaxed purifying selection in highly selfing populations. Thus, mating system and demography shape the impact of purifying selection on genomic variation in A. alpina. These results are important for an improved understanding of the evolutionary consequences of mating system variation and the maintenance of mixed-mating strategies.

Chloroplast DNA variation in Epipactis atrorubens populations from northern Greece

Abstract Epipactis Zinn. is a highly diverse genus, with numerous taxa naturally occurring throughout Europe. While their morphological variability has resulted in contradictory opinions about their taxonomy, the knowledge of the genetic variation of many of its species is limited. Such an example is E. atrorubens in the Balkan Peninsula. In this work, the cpDNA variation of seven E. atrorubens populations from northern Greece and of one population from Germany has been investigated by means of PCR-RFLP markers. Two regions of the cpDNA were studied (trnH-psbA, trnL intron) and four PCR-RFLP markers were employed (trnH-psbA/HaeIII, trnH-psbA/HinfI, trnH-psbA/VspI, trnL/MboI), which have revealed variation in other Epipactis species. The results demonstrate limited genetic variability in the studied populations, as only one haplotype was present in all the populations (both Greek and German), with the exception of Mt. Menikion (Greece) where a second haplotype was also recorded. This limited variation may be attributed to the properties of the genetic markers involved, to the conserved status of the two cpDNA regions in this species or to factors related to the biology and evolutionary history of E. atrorubens. The presence of a second haplotype only in Mt. Menikion could be attributed to a hybridisation event in the past and/or to the potential existence of a glacial refugium on this mountain.