Phylogenomics and diversification in subtribe Malinae

Hybridisation, polyploidy (the possession of more than two chromosome sets) and apomixis (asexual reproduction via seeds) constitute an evolutionary syndrome considered key for organismic diversification in several major plant families including the sunflower family, buttercup family, or rose family. We will study the consequences of this syndrome for organismic diversification in the subtribe Malinae (i.e. apple, pear and their about 25 related genera, all of which distributed in the Northern Hemisphere) and genus Crataegus (hawthorns including medicinally important species) as models. We address two main aims: (1) We expect that unintended sequencing of duplicated genes (leading to false placement of affected individuals in phylogenetic reconstructions), incomplete lineage sorting (the stochastic sorting of ancestral molecular variants into phylogenetic lineages), and reticulate events (i.e. hybridisation) all played a significant role in the evolution of the Malinae and Crataegus. We aim to identify the relative role of these processes and causes and to solve existing phylogenetic conflicts in the placement of genera and species. (2) We will study the origin of species diversity in Eurasian hawthorns. We are particularly interested to understand the role of ancient sexual species (so-called orthospecies) in the origin of derived, often asexual species (so-called nothospecies) and to clarify their validity as species of their own right. Traditional phylogenetic studies were hampered by low numbers of available and affordable markers, lack of knowledge of genomic location of markers, and in Malinae, by low sequence divergence and resolution of markers. Our study promises to overcome these limits in basing phylogenetic reconstructions on a dense sample of orthologs (markers which have a single and identical origin) covering the whole genome. We will use High-throughput sequencing to identify non-linear relationships using phylogenetic network techniques and build species phylogenies from mutually congruent gene trees. High-throughput sequencing revolutionized the methodological repertoire of DNA-molecular techniques available to biologists and provides access to unchallenged high levels of resolution and accuracy of data and analyses. Although extensive work has been attributed to taxonomic characterization, applications of modern approaches to study the systematics of Eurasian Crataegus are virtually missing. Here we make the theoretically and analytically important step from traditional morphology-based taxonomy to an integrative evolutionary approach for species delimitation based aside of classical morphology on molecular phylogenetics and cytological data (flow cytometry by which we estimate number of chromosome sets and reproductive mode of individuals).

There are several large plant families, including the rose family (Rosaceae), which has long and very effectively made use of nature's toolbox, employing the full repertoire of tools for speciation and the evolution of their species. These tools (processes) include, among others, hybridization, polyploidization (formation of multiple chromosome-sets), and asexual reproduction through seeds (apomixis). Although these processes can be advantageous for plants, they often lead to "inconsistencies" in phylogenetic history. Nevertheless, scientists do not shy away from delving into the complex and tangled past to, among other things, draw clear lines between species and reconstruct the phylogeny, which shows how,when,and where the "explosions of species diversity" (radiation) occurred, where a small group of ancestral species in a short time diversified into a large number of descendant species. One such project was carried out by a team of scientists from the Austrian-Research-Centre-for-Forests (BFW) in Vienna, Charles-University in Prague, and the Czech-Academy-of-Sciences in Prhonice. The aim was to clarify the phylogeny and complexity of species diversity formation in the subtribe Malinae of the rose family (which also includes apple, pear, and hawthorn). A special focus was placed on the genus Crataegus (hawthorn). First, the team adapted the methodological and analytical repertoire. They moved away from purely traditional, morphology-based analyses and the limited number of available molecular markers with unknown positions in the genome. Instead, they adopted an integrative approach. This was mainly based on molecular phylogenies using high-throughput sequencing like Hyb-Seq and newly developed bioinformatic tools allowing the phylogenetic reconstruction based on markers covering the entire genome with high density, as well as cytological data. The latter refers to the so-called flow-cytometry,which was used to determine the number of chromosome sets (ploidy level) and the reproductive mode (sexual or asexual) of the studied individuals. The research revealed that the radiation of the Malinae plant group is linked to climatic and geographical changes. There were even several significant speciation explosions (radiations). The first radiation occurred shortly after a genome duplication in the early Eocene (56-48 million years ago) in the so- called Madrean Region (now a region of the southwestern-United-States and northwestern-Mexico),when the Northern Hemisphere experienced a climatic cooling. The second radiation occurred in the middle Eocene (48-38 million years ago),when the plants spread to East-Asia. Delving into the phylogeny of over 100 hawthorn species confirmed some trends,such as the division of species into four main lineages (subgenera),but also brought surprises in the form of newly discovered relationships,new groupings of species, and deviations from traditional taxonomy. Additionally, it was possible to genetically define three Central European hawthorn species and understand how they interbreed and mix, which is the basis for their correct identification in practice.