Biochemical and Structural Diversification of C4 Photosynthesis in Tribe Zoysieae (Poaceae).

C4 photosynthesis has evolved independently multiple times in grass lineages with nine anatomical and three biochemical subtypes. Chloridoideae represents one of the separate events and contains species of two biochemical subtypes, NAD-ME and PEP-CK. Assessment of C4 photosynthesis diversification is limited by species sampling. In this study, the biochemical subtypes together with anatomical leaf traits were analyzed in 19 species to reveal the evolutionary scenario for diversification of C4 photosynthesis in tribe Zoysieae (Chloridoideae). The effect of habitat on anatomical and biochemical diversification was also evaluated. The results for the 19 species studied indicate that 11 species have only NAD-ME as a decarboxylating enzyme, while eight species belong to the PEP-CK subtype. Leaf anatomy corresponds to the biochemical subtype. Analysis of Zoysieae phylogeny indicates multiple switches between PEP-CK and NAD-ME photosynthetic subtypes, with PEP-CK most likely as the ancestral subtype, and with multiple independent PEP-CK decarboxylase losses and its secondary acquisition. A strong correlation was detected between C4 biochemical subtypes studied and habitat annual precipitation wherein NAD-ME species are confined to drier habitats, while PEP-CK species prefer humid areas. Structural adaptations to arid climate include increases in leaf thickness and interveinal distance. Our analysis suggests that multiple loss of PEP-CK decarboxylase could have been driven by climate aridization followed by continued adaptive changes in leaf anatomy.

Intraspecific divergence of diploid grass Aegilopscomosa is associated with structural chromosome changes.

Aegilopscomosa Smith in Sibthorp et Smith, 1806 is diploid grass with MM genome constitution occurring mainly in Greece. Two morphologically distinct subspecies - Ae.c.comosa Chennaveeraiah, 1960 and Ae.c.heldreichii (Holzmann ex Boissier) Eig, 1929 are discriminated within Ae.comosa, however, genetic and karyotypic bases of their divergence are not fully understood. We used Fluorescence in situ hybridization (FISH) with repetitive DNA probes and electrophoretic analysis of gliadins to characterize the genome and karyotype of Ae.comosa to assess the level of their genetic diversity and uncover mechanisms leading to radiation of subspecies. We show that two subspecies differ in size and morphology of chromosomes 3M and 6M, which can be due to reciprocal translocation. Subspecies also differ in the amount and distribution of microsatellite and satellite DNA sequences, the number and position of minor NORs, especially on 3M and 6M, and gliadin spectra mainly in the a-zone. Frequent occurrence of hybrids can be caused by open pollination, which, along with genetic heterogeneity of accessions and, probably, the lack of geographic or genetic barrier between the subspecies, may contribute to extremely broad intraspecific variation of GAAn and gliadin patterns in Ae.comosa, which are usually not observed in endemic plant species.

Biodiversity of rolB/C-like Natural Transgene in the Genus Vaccinium L. and Its Application for Phylogenetic Studies.

A variety of plant species found in nature contain agrobacterial T-DNAs in their genomes which they transmit in a series of sexual generations. Such T-DNAs are called cellular T-DNAs (cT-DNAs). cT-DNAs have been discovered in dozens of plant genera, and are suggested to be used in phylogenetic studies, since they are well-defined and unrelated to other plant sequences. Their integration into a particular chromosomal site indicates a founder event and a clear start of a new clade. cT-DNA inserts do not disseminate in the genome after insertion. They can be large and old enough to generate a range of variants, thereby allowing the construction of detailed trees. Unusual cT-DNAs (containing the rolB/C-like gene) were found in our previous study in the genome data of two Vaccinium L. species. Here, we present a deeper study of these sequences in Vaccinium L. Molecular-genetic and bioinformatics methods were applied for sequencing, assembly, and analysis of the rolB/C-like gene. The rolB/C-like gene was discovered in 26 new Vaccinium species and Agapetes serpens (Wight) Sleumer. Most samples were found to contain full-size genes. It allowed us to develop approaches for the phasing of cT-DNA alleles and reconstruct a Vaccinium phylogenetic relationship. Intra- and interspecific polymorphism found in cT-DNA makes it possible to use it for phylogenetic and phylogeographic studies of the Vaccinium genus.

Diversity of Arbuscular Mycorrhizal Fungi in Distinct Ecosystems of the North Caucasus, a Temperate Biodiversity Hotspot.

BACKGROUND: Investigations that are focused on arbuscular mycorrhizal fungus (AMF) biodiversity is still limited. The analysis of the AMF taxa in the North Caucasus, a temperate biodiversity hotspot, used to be limited to the genus level. This study aimed to define the AMF biodiversity at the species level in the North Caucasus biotopes. METHODS: The molecular genetic identification of fungi was carried out with ITS1 and ITS2 regions as barcodes via sequencing using Illumina MiSeq, the analysis of phylogenetic trees for individual genera, and searches for operational taxonomic units (OTUs) with identification at the species level. Sequences from MaarjAM and NCBI GenBank were used as references. RESULTS: We analyzed >10 million reads in soil samples for three biotopes to estimate fungal biodiversity. Briefly, 50 AMF species belonging to 20 genera were registered. The total number of the AM fungus OTUs for the "Subalpine Meadow" biotope was 171/131, that for "Forest" was 117/60, and that for "River Valley" was 296/221 based on ITS1/ITS2 data. The total number of the AM fungus species (except for virtual taxa) for the "Subalpine Meadow" biotope was 24/19, that for "Forest" was 22/13, and that for "River Valley" was 28/24 based on ITS1/ITS2 data. Greater AMF diversity, as well as number of OTUs and species, in comparison with that of forest biotopes, characterized valley biotopes (disturbed ecosystems; grasslands). The correlation coefficient between "Percentage of annual plants" and "Glomeromycota total reads" r = 0.76 and 0.81 for ITS1 and ITS2, respectively, and the correlation coefficient between "Percentage of annual plants" and "OTUs number (for total species)" was r = 0.67 and 0.77 for ITS1 and ITS2, respectively. CONCLUSION: High AMF biodiversity for the river valley can be associated with a higher percentage of annual plants in these biotopes and the active development of restorative successional processes.

Hybridization and diversity of aquatic macrophyte Sparganium L. (Typhaceae) as revealed by high-throughput nrDNA sequencing.

Sparganium is an emergent aquatic macrophyte widely spread in temperate and subtropical zones. Taxa of this genus feature high phenotypic plasticity and can produce interspecific hybrids. By means of high-throughput sequencing of the internal transcribed spacer (ITS1) of 35S rDNA, the status of 15 Eurasian Sparganium species and subspecies was clarified and the role of hybridization events in the recent evolution of the genus was investigated. It has been shown that a number of species such as S. angustifolium, S. fallax and S. subglobosum have homogenized rDNA represented by one major ribotype. The rDNA of other taxa is represented by two or more major ribotypes. Species with high rDNA heterogeneity are apparently of hybrid origin. Based on the differences in rDNA patterns, intraspecific diversity was identified in S. probatovae and S. emersum. Thus, we have concluded that Sparganium has extensive interspecific hybridization at the subgenus level, and there may also be occasional hybridization between species from different subgenera.

Conservation of an Agrobacterium cT-DNA insert in Camellia section Thea reveals the ancient origin of tea plants from a genetically modified ancestor.

Introduction: Many higher plants contain cellular T-DNA (cT-DNA) sequences from Agrobacterium and have been called "natural genetically modified organisms" (nGMOs). Among these natural transformants, the tea plant Camellia sinensis var. sinensis cv. Shuchazao contains a single 5.5 kb T-DNA fragment (CaTA) with three inactive T-DNA genes, with a 1 kb inverted repeat at the ends. Camellia plants are allogamous, so that each individual may contain two different CaTA alleles. Methods: 142 Camellia accessions, belonging to 10 of 11 species of the section Thea, were investigated for the presence of CaTA alleles. Results discussion: All accessions were found to contain the CaTA insert, showing that section Thea derives from a single transformed ancestor. Allele phasing showed that 82 accessions each contained two different CaTA alleles, 60 others had a unique allele. A phylogenetic tree of these 225 alleles showed two separate groups, A and B, further divided into subgroups. Indel distribution corresponded in most cases with these groups. The alleles of the different Camellia species were distributed over groups A and B, and different species showed very similar CaTA alleles. This indicates that the species boundaries for section Thea may not be precise and require revision. The nucleotide divergence of the indirect CaTA repeats indicates that the cT-DNA insertion took place about 15 Mio years ago, before the emergence of section Thea. The CaTA structure of a C. fangchengensis accession has an exceptional structure. We present a working model for the origin and evolution of nGMO plants derived from allogamous transformants.