Morpho‐anatomical and molecular characterization of the mycorrhizas of European Polygala species

Abstract

The mycorrhizas of 12 species of Polygala (Polygalaceae), including herbs, subshrubs and one shrub, collected from Germany, Mallorca (Spain) and Malta, were investigated by morpho-anatomical and molecular methods. Aseptate hyphae, arbuscules and vesicles indicate an arbuscular mycorrhiza in all species examined. Hyphal spread in Polygala is predominantly, but not exclusively, intracellular and comprises three characteristic stages of colonization: (i) intracellular, linear hyphal growth in a cascading manner after penetration towards the penultimate parenchyma layer (layer 2), (ii) initially linear hyphal growth in the cells of layer 2 from where hyphal branches repeatedly penetrate the anatomically distinct innermost parenchyma layer (layer 1), forming arbuscule-like structures therein which are subject to degeneration, (iii) more branches from the linear hyphae in layer 2 develop, but coil and make contact to the layer outside layer 2 (layer 3) in which arbuscule-like structures similar to those in layer 1 form and degenerate. This general colonization pattern differs in details between the species, and critical comparisons, in particular between the woody P. myrtifolia, the herbaceous Polygala spp. and the mycoheterotrophic Epirixanthes spp. (Polygalaceae) suggest an evolutionary shift of mycorrhizal features within the family towards an optimization of plant benefit through the fungus. Based on the molecular marker 18S rDNA mycorrhizal fungi detected in roots of Polygala spp. are largely restricted to five clades of Glomeraceae 1 (Glomus Group A). This result rejects the hypothesis of a strict symbiotic specificity in Polygalaceae but may stimulate a discussion on functionally compatible groups of fungi.

Evolution of mycoheterotrophy in Polygalaceae: The case of Epirixanthes

“…All plants of Salomonia were fl owering and additionally bore mature fruits. Additional fungal sequence data represented virtual taxa (i.e., fungal phylogroups based on 97% sequence similarity and bootstrap support) from the Maarj AM database ( Öpik et al, 2010 ), as well as mycorrhizal symbionts of Polygala from Rath et al (2013) .…”

“…From each plant of the two species a single root fragment (1-3 cm) was used for molecular identifi cation of the mycorrhizal symbionts. The PCR procedure for the 18S rDNA region of the fungal symbionts basically followed the DNA extraction protocol described in Rath et al (2013) , although here we used dried root material instead of CTAB fi xed material. Amplifi cation of fungal DNA was done using a nested PCR approach.…”

“…In only two cases ( S. longiciliata i2 and S. cantoniensis i1), initial sequencing of the PCR product with primer AM1 resulted in high sequence quality, which allowed immediate use for the alignment. The remaining PCR products of S. longiciliata i1 and i2 and S. cantoniensis i2 were used for cloning and subsequent sequencing (see Rath et al [2013] for detailed information on DNA processing).…”

“…We use this framework to compare the fungal specifi city between Epirixanthes and its close relatives in Salomonia and Polygala by measuring the phylogenetic diversity (PD; Faith, 1992 ) of the associated AMF. The results were compared with published insights on AMF of Polygalaceae ( Imhof, 2007 ;Rath et al, 2013 ) as well as to recent studies of mycoheterotrophic specifi city ( Hynson and Bruns, 2009 ;Merckx et al, 2009Merckx et al, , 2012Roy et al, 2009 ).…”

“…Imhof (2007) suggested a degree of adaptation to these fungi and reported similarities in AMF features with unrelated mycoheterotrophic plants and with published drawings of mycorrhizal symbionts of Polygala amara ( Marcuse, 1902 ). Within Polygalaceae, the chlorophyllous genus Polygala was found to grow in symbiosis with several clades of AMF of the family Glomeraceae, based on DNA sequence data ( Rath et al, 2013 ). Moreover, a single fungal sequence from Acaulosporaceae was extracted from P. myrtifolia.…”