Catching the rhizolith-makers: identification of calcified plant roots by DNA barcoding

Rhizoliths are organosedimentary structures resulting in the preservation of remains or traces of roots of higher plants in mineral matter. They are one of the most prominent features in palaeosols and may constitute the... [ view full abstract ]

Rhizoliths are organosedimentary structures resulting in the preservation of remains or traces of roots of higher plants in mineral matter. They are one of the most prominent features in palaeosols and may constitute the dominant fabrics in some forms of calcretes. Rhizoliths are most commonly made of calcium carbonate, typically precipitated through biological activity and biogeochemical processes in the root-soil system. Formation of rhizoliths can take place during a lifetime of roots, their senescence, or post-mortem, normally by microbial decay. A special type of rhizoliths are mm-sized intracellularly calcified roots (ICRs) which form entirely through plant-controlled calcite (CaCO3) biomineralisation of the cortex of living fine roots. Accumulation of CaCO3 in fine root cortical parenchyma, coupled with extrusion of protons to the rhizosphere, has been interpreted as an effective nutrient acquisition mechanism in plants, adapted to nutrient-poor calcareous soils, such as the present-day soils of the Mediterranean. Intracellular carbonate precipitation in fine roots has been reported from in vitro experiments on model plants, whereas no calcifying plants have been identified in their natural settings, although ICRs may locally, in the Mediterranean region, represent more than a half of the soil mass.
Plant roots growing in a mixed belowground community are difficult to identify at any taxonomic level, especially morphologically simple fine roots that acquire nutrients and take up water. Furthermore, it is virtually impossible to trace an individual plant root system from a single (calcified) fragment, excavated deep in a soil profile, to its coarse primary root and to the determinable aboveground part of a plant.
A DNA barcoding is a taxonomic identification tool, which uses a standard short genomic region, universally present in target biological material and has sufficient sequence variation to discriminate among taxa. We extracted DNA from very small amounts of plant tissue (mostly vascular cylinders) preserved in dehydrated fragments of ICRs collected in calcareous soils of the Alicante region, Spain. We used PCR amplification and tested sequencing using nuclear ITS and plastid (rbcL and matK) loci. Resulting sequences were edited and checked for similarity using BLAST tool and sequence database. Sequences (especially combination of rbcL and matK) have shown the best match with the data for genera Hedysarum, Onobrychis and Astragalus, all members of Leguminosae family. These molecular taxonomy results are in agreement with our preliminary survey of vegetation above the studied profiles, showing abundance of legume shrub species of the same genera.