We explored the seed-associated bacterial endophytic microbiome in seeds of the endemic holoparasitic species Cistanche armena from a saline and arid habitat in Armenia. A combination of culture-dependent and molecular techniques was employed for identifying the seed endomicrobiome (culturable and unculturable). From surface-sterilized seeds, 10 phyla, comprising 256 endophytic bacterial genera, were identified. Of the culturable strains, we also investigated the plant growth-promoting (PGP) traits. Most of the isolates were spore forming, halotolerant and alkaliphile Bacillus spp., indicating that the endophytic bacteria of C. armena seeds own traits related to the natural habitat of their host plant. Our results confirm that Bacillus species are common and dominated endophytes from plants growing on saline and arid soils. Pantoea spp. and Stenotrophomonas spp. are more favourable PGP endophytes in seeds of C. armena. The PGP traits of these bacteria, such as production of indole, a precursor of auxin, ACC-deaminase and organic acids have the potential to improve the tolerance of their host plants against the abiotic stresses present in their natural habitat. To the best of our knowledge, this is the first report concerning bacterial seed endophytes of the C. armena.

The extensive diversity of the tribe Orobancheae, the most species-rich lineage of holoparasitic Orobanchaceae, is concentrated in the Caucasus and Mediterranean regions of the Old World. This extant diversity has inspired hypotheses that these regions are also centres of origin of its key lineages, however the ability to test hypotheses has been limited by a lack of sampling and phylogenetic information about the species, especially in the Caucasus region. First, we assessed the phylogenetic relationships of several poorly known, problematic, or newly described species and host-races of four genera of Orobancheae occurring in the Caucasus region–Cistanche, Phelypaea, Phelipanche and Orobanche–using nuclear ribosomal (ITS) and plastid (trnL–trnF) sequence data. Then we applied a probablistic dispersal-extinction-cladogenesis model of historical biogeography across a more inclusive clade of holoparasites, to explicitly test hypotheses of Orobancheae diversification and historical biogeography shifts. In sum, we sampled 548 sequences (including 196 newly generated) from 13 genera, 140 species, and 175 taxa across 44 countries. We find that the Western Asia (particularly the Caucasus) and the Mediterranean are the centre of origin for large clades of holoparasitic Orobancheae within the last 6 million years. In the Caucasus, the centres of diversity are composed both of long-branch taxa and shallow, recently diversified clades, while Orobancheae diversity in the Mediterranean appears to represent mainly recent diversification.

Obligate parasitic plant species from the Orobanchaceae family belong to the most critical genera of world flora. Due to their strongly reduced vegetative organ architecture, there arise many difficulties and mistakes in species identification, and extra characteristics of systematic importance are required. Seed micromorphology is a highly informative taxonomic criterion that helps to resolve ambiguities in plant taxonomy and evolution, and has proved to be a valuable complementary tool for Orobanchaceae species identification. In this study, seeds of 43 holoparasitic species from the Cistanche, Diphelypaea, Orobanche and Phelipanche genera collected in the Caucasus were subjected to micromorphological analysis. The Caucasus is one of the most important centres of world biodiversity, with many endangered and endemic parasitic plants. Twenty two quantitative/qualitative micromorphological seed parameters were analysed using stereo ZOOM, confocal scanning laser (CLSM) and scanning electron (SEM) microscopy. Three types of seed were established based on periclinal wall ornamentation: 1) clearly pitted sculpturing in all Cistanche, Diphelypaea, and most Orobanche seeds; 2) fibrillar and veined sculpturing in Phelipanche seeds; and 3) smooth, granular or rugged (very rarely visibly pitted) outer periclinal wall in O. coerulescens and O. colorata seeds. However, CLSM, used in present studies for Orobanchaceae seeds for the first time, revealed more details of wall lignification and ornamentation invisible in SEM images. The best micromorphological characteristics for species identification are the type of ornamentation of the periclinal wall, the width of anticlinal wall, the character of perforation, perforation length and width, the fibrillar character, and fibrillar and vein width. A comparison of the main characteristics distinguishing the seeds of Orobanchaceae is also presented and discussed.

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