Miklós Bálint

Patterns of population structure in two closely related, partially sympatric caddisflies in Eastern Europe: historic introgression, limited dispersal, and cryptic diversity1

Abstract We used 2 caddisflies, Drusus discolor and Drusus romanicus, to test explicitly whether closely related species that occupy similar niches and occur in partial sympatry maintain comparable population structure and share a similar population history. We used mitochondrial sequence data to analyze and compare the population structure and the phylogeography of 105 specimens of D. discolor and 74 individuals of D. romanicus collected in southeastern Europe. We examined the relationship between both species with phylogenetic inference and coalescent modeling and used the results to assign larvae to species. We were able to unambiguously assign larvae to species level based on clearly defined association criteria within a phylogenetic analysis of all specimens. The species were closely related and not reciprocally monophyletic in our haplotype phylogeny. One D. romanicus haplotype from the Bucegi Mountains was nested within D. discolor, a result that suggests isolation with migration, introgression, or incomplete lineage sorting between the 2 species. For each species, we examined population genetic structure with median joining networks, analysis of molecular variance (AMOVA), exact tests of population differentiation, and Mantel tests of isolation by distance. We used tests for selective neutrality (Tajimas D, Fus F) to infer potential population growth and expansion. Species differed in their genetic population structure. Drusus discolor had haplotype overlap among several mountain ranges in the study region. No D. romanicus haplotypes were shared among any regions examined, and levels of divergence between haplotype clades exceeded those of D. discolor by a factor of up to 2.1. The different degree of population differentiation and divergence of both species probably reflects different Pliocene/Pleistocene population histories and might be related to differences in dispersal capabilities or competitive exclusion of D. romanicus by D. discolor in the mountain ranges north and west of the Western Carpathians. Based on our results, we discuss the importance of the Carpathian Mountains and Bulgarian highlands as Pliocene/Pleistocene refugia and centers of diversification.

Meta‐analysis of deep‐sequenced fungal communities indicates limited taxon sharing between studies and the presence of biogeographic patterns

High-throughput amplicon sequencing gives new insights into fungal community ecology. Massively generated molecular data lead to the discovery of vast fungal diversity. However, it is unclear to what extent operational taxonomic units (OTUs) overlap among independent studies, because no comparative studies exist. We compared fungal diversity based on the internal transcribed spacer (ITS1) region among 10 published studies. Starting from the raw 454 pyrosequencing data, we used a uniform pipeline to prune the reads. We investigated fungal richness and taxonomic composition among phyllosphere and soil fungal communities, as well as biogeographic signals in the data. We did not find globally distributed OTUs, even when comparing fungal communities from similar habitats (phyllosphere or soil). This suggests that high local fungal diversity scales up to high global diversity. The most OTU-rich classes in the phyllosphere were Dothideomycetes (21%) and Sordariomycetes (14%), and in the soil were Sordariomycetes (13%) and Agaricomycetes (12%). The richness estimates suggest the presence of undiscovered fungal diversity even in deeply sequenced study systems. The small number of OTUs shared among studies indicates that globally distributed taxa and habitat generalists may be rare. Latitudinal diversity decline and distance decay relationships suggest the presence of biogeographic patterns similar to those in plants and animals.

Millions of reads, thousands of taxa: microbial community structure and associations analyzed via marker genes

With high-throughput sequencing (HTS), we are able to explore the hidden world of microscopic organisms to an unpre-cedented level. The fast development of molecular technology and statistical methods means that microbial ecologists must keep their toolkits updated. Here, we review and evaluate some of the more widely adopted and emerging techniques for analysis of diversity and community composition, and the inference of species interactions from co-occurrence data generated by HTS of marker genes. We emphasize the importance of observational biases and statistical properties of the data and methods. The aim of the review is to critically discuss the advantages and disadvantages of established and emerging statistical methods, and to contribute to the integration of HTS-based marker gene data into community ecology.

An Illumina metabarcoding pipeline for fungi

High-throughput metabarcoding studies on fungi and other eukaryotic microorganisms are rapidly becoming more frequent and more complex, requiring researchers to handle ever increasing amounts of raw sequence data. Here, we provide a flexible pipeline for pruning and analyzing fungal barcode (ITS rDNA) data generated as paired-end reads on Illumina MiSeq sequencers. The pipeline presented includes specific steps fine-tuned for ITS, that are mostly missing from pipelines developed for prokaryotes. It (1) employs state of the art programs and follows best practices in fungal high-throughput metabarcoding; (2) consists of modules and scripts easily modifiable by the user to ensure maximum flexibility with regard to specific needs of a project or future methodological developments; and (3) is straightforward to use, also in classroom settings. We provide detailed descriptions and revision techniques for each step, thus giving the user maximum control over data treatment and avoiding a black-box approach. Employing this pipeline will improve and speed up the tedious and error-prone process of cleaning fungal Illumina metabarcoding data.

Relocation, high-latitude warming and host genetic identity shape the foliar fungal microbiome of poplars

Micro‐organisms associated with plants and animals affect host fitness, shape community structure and influence ecosystem properties. Climate change is expected to influence microbial communities, but their reactions are not well understood. Host‐associated micro‐organisms are influenced by the climate reactions of their hosts, which may undergo range shifts due to climatic niche tracking, or may be actively relocated to mitigate the effects of climate change. We used a common‐garden experiment and rDNA metabarcoding to examine the effect of host relocation and high‐latitude warming on the complex fungal endophytic microbiome associated with leaves of an ecologically dominant boreal forest tree (Populus balsamifera L.). We also considered the potential effects of poplar genetic identity in defining the reactions of the microbiome to the treatments. The relocation of hosts to the north increased the diversity of the microbiome and influenced its structure, with results indicating enemy release from plausible pathogens. High‐latitude warming decreased microbiome diversity in comparison with natural northern conditions. The warming also caused structural changes, which made the fungal communities distinct in comparison with both low‐latitude and high‐latitude natural communities, and increased the abundance of plausible pathogens. The reactions of the microbiome to relocation and warming were strongly dependent on host genetic identity. This suggests that climate change effects on host–microbiome systems may be mediated by the interaction of environmental factors and the population genetic processes of the hosts.

Population dynamics and genetic changes of Picea abies in the South Carpathians revealed by pollen and ancient DNA analyses

BackgroundStudies on allele length polymorphism designate several glacial refugia for Norway spruce (Picea abies) in the South Carpathian Mountains, but infer only limited expansion from these refugia after the last glaciation. To better understand the genetic dynamics of a South Carpathian spruce lineage, we compared ancient DNA from 10,700 and 11,000-year-old spruce pollen and macrofossils retrieved from Holocene lake sediment in the Retezat Mountains with DNA extracted from extant material from the same site. We used eight primer pairs that amplified short and variable regions of the spruce cpDNA. In addition, from the same lake sediment we obtained a 15,000-years-long pollen accumulation rate (PAR) record for spruce that helped us to infer changes in population size at this site.ResultsWe obtained successful amplifications for Norway spruce from 17 out of 462 pollen grains tested, while the macrofossil material provided 22 DNA sequences. Two fossil sequences were found to be unique to the ancient material. Population genetic statistics showed higher genetic diversity in the ancient individuals compared to the extant ones. Similarly, statistically significant Ks and Kst values showed a considerable level of differentiation between extant and ancient populations at the same loci.Lateglacial and Holocene PAR values suggested that population size of the ancient population was small, in the range of 1/10 or 1/5 of the extant population. PAR analysis also detected two periods of rapid population growths (from ca. 11,100 and 3900 calibrated years before present (cal yr BP)) and three bottlenecks (around 9180, 7200 and 2200 cal yr BP), likely triggered by climatic change and human impact.ConclusionOur results suggest that the paternal lineages observed today in the Retezat Mountains persisted at this site at least since the early Holocene. Combination of the results from the genetic and the PAR analyses furthermore suggests that the higher level of genetic variation found in the ancient populations and the loss of ancient allele types detected in the extant individuals were likely due to the repeated bottlenecks during the Holocene; however our limited sample size did not allow us to exclude sampling effect.This study demonstrates how past population size changes inferred from PAR records can be efficiently used in combination with ancient DNA studies. The joint application of palaeoecological and population genetics analyses proved to be a powerful tool to understand the influence of past population demographic changes on the haplotype diversity and genetic composition of forest tree species.

Genetic population structure of an autumn-emerging caddisfly with inherently low dispersal capacity and insights into its phylogeography

Abstract We used ecological information and current and historic species distribution models for the last glacial maximum (LGM) to develop hypotheses regarding the Pleistocene history of the montane, autumn-emerging caddisfly Chaetopterygopsis maclachlani. We used mitochondrial sequence data from the cytochrome oxidase I (mtCOI) gene of 282 specimens from populations across Europe to analyze the population structure and phylogeography of C. maclachlani and to test our hypotheses. We examined the population genetic structure with median-joining networks, analysis of molecular variance (AMOVA), exact tests of population differentiation, and Mantel tests of isolation by distance. Furthermore, we used tests for selective neutrality (Tajimas D, Fus FS) to infer potential population growth and expansion and Bayesian Skyline Plots to analyze past population dynamics. We found strong population structure with 47 different haplotypes that could be separated into a southeastern and a western clade. The western clade seems to have survived at least the LGM in an extra-Mediterranean refugium, independent of the southeastern clade. Within the 2 main clades, we found haplotype overlap between mountain ranges and a high proportion of private haplotypes (89%). As predicted for a montane species with very limited adult dispersal capabilities, many regions and populations are currently isolated and appear to be diversifying into independent lineages. We discuss speciation within the Chaetopterygini and the diversification of C. maclachlani in particular.

The Carpathians as a Major Diversity Hotspot in Europe

The Carpathians are one of the major mountain ranges of Europe, but still one of its least studied regions. It is increasingly recognized that they played a major role in the formation and Pleistocene survival of numerous continental, arctic, and arctic–alpine taxa. Many endemic taxa have been described from these mountains. The number of phylogeographic/phylogenetic studies covering at least partially the Carpathians is also increasing. These studies reveal unevenly distributed genetic and taxonomic diversity. In this work, we analyse population genetic structures in the Carpathians revealed by case studies on aquatic insects, comparing them to existing literature data on plants, butterflies, vertebrates, and the distribution of several microendemics. The distribution of molecular lineages and/or microendemics show strong biogeographic structures within the Carpathians. The overlap between the distribution barriers of microendemics and intraspecific molecular lineages suggests that isolation of populations among the major Carpathian ranges (Western Carpathians, Eastern Carpathians, Apuseni Mts, Southern Carpathians, Banat and Serbian Carpathians) played a major role in promoting Carpathian diversity.

Misperceived invasion: the Lusitanian slug (Arion lusitanicus auct. non-Mabille or Arion vulgaris Moquin-Tandon 1855) is native to Central Europe.

The Lusitanian slug, presumed to be native to south‐west Europe, was ranked among the 100 worst invading species in Central Europe. However, from the very beginning of its recognition in the presumed invasion area, there was little evidence that the species was actually anthropogenically introduced. We investigated the invasive status of the species by comparing specific predictions on the population genetic structure in the invasion area with the pattern actually found. In a DNA‐taxonomy approach, the species could not be found in its presumed native range. Using statistical phylogeographic techniques on a mitochondrial (COI) and nuclear (ZF) marker and species distribution modelling, we could show that the species is with very high probability not an invasor, but native to Central Europe. The study underlines the value of statistical phylogeography in rigorously testing hypotheses on the dynamics of biological invasions.

Methodological framework for projecting the potential loss of intraspecific genetic diversity due to global climate change

BackgroundWhile research on the impact of global climate change (GCC) on ecosystems and species is flourishing, a fundamental component of biodiversity – molecular variation – has not yet received its due attention in such studies. Here we present a methodological framework for projecting the loss of intraspecific genetic diversity due to GCC.MethodsThe framework consists of multiple steps that combines 1) hierarchical genetic clustering methods to define comparable units of inference, 2) species accumulation curves (SAC) to infer sampling completeness, and 3) species distribution modelling (SDM) to project the genetic diversity loss under GCC. We suggest procedures for existing data sets as well as specifically designed studies. We illustrate the approach with two worked examples from a land snail (Trochulus villosus) and a caddisfly (Smicridea (S.) mucronata).ResultsSampling completeness was diagnosed on the third coarsest haplotype clade level for T. villosus and the second coarsest for S. mucronata. For both species, a substantial species range loss was projected under the chosen climate scenario. However, despite substantial differences in data set quality concerning spatial sampling and sampling depth, no loss of haplotype clades due to GCC was predicted for either species.ConclusionsThe suggested approach presents a feasible method to tap the rich resources of existing phylogeographic data sets and guide the design and analysis of studies explicitly designed to estimate the impact of GCC on a currently still neglected level of biodiversity.