Crocus heuffelianus-A New Species for the Bulgarian Flora from Series Verni (Iridaceae).

In the Pirin Mountains, at an elevation of around 1000 m, three populations of a new species of Bulgarian flora from the genus Crocus, series Verni, were discovered. The species was compared to the morphologically related C. veluchensis, and presented with diagnostic morphological and anatomical features. Despite the high degree of morphological similarity, the molecular analysis, which included sequences from all related species (C. cvijicii, C. dalmaticus, C. jablanicensis, C. rujanensis, C. sieberi subsp. atticus, and C. veluchensis), distinguished the Pirin Mountains' populations, and revealed the closest relationship to C. heuffelianus. Despite the C. heuffelianus/C. verni complex's uncertain taxonomic status, our findings on the local population, based on morphometric, anatomical, molecular, and geographic analyses, indicate its belonging to the putative allotetraploid C. heuffelianus of south-eastern Europe and the Balkans, and an expansion of its range to the southeast. Given the taxonomic uncertainty and unclear phylogenetic relationships of the taxa in the Crocus vernus complex, we considered it appropriate to accept our taxon as Crocus heuffelianus. So far, only C. tommasinianus Herb. has been found in Bulgarian flora from the Crocus series Verni, but in terms of altitude and morphological features, the species from our collection is close to the Balkan endemic C. veluchensis, which belongs to the C. sieberi aggregate. Morphologically, it differs by the dark, heart-shaped spots on the tip of the tepals, and the presence of one bract. A detailed comparative anatomical analysis between the three species of crocuses from the series Verni in Bulgaria shows discrete differences: the width of the white stripe and lacunar area are good distinguishing features, as are the number of conducting vessels.

Identification of Bulgarian Sourdough Microbiota by Metagenomic Approach Using Three Commercially Available DNA Extraction Protocols.

Research background: Sourdough is a spontaneously formed, complex microbial ecosystem of various lactic acid bacteria (LAB) and yeast which, by producing specific metabolites, determines the quality of the baked products. In order to design and control the sourdough with preferred nutritional characteristics, it is crucial that the LAB diversity of the product of interest be elucidated. Experimental approach: Using the opportunities of next-generation sequencing (NGS) of the V1-V3 hypervariable gene region of 16S rRNA, we studied the microbial ecosystem of a whole grain sourdough made of Triticum monococcum, originating from Southwestern Bulgaria. Since the DNA extraction method is considered crucial for the accuracy of the sequencing results, as it can introduce significant differences in the examined microbiota, we used three different commercial kits for DNA isolation and analyzed their impact on the observed bacterial diversity. Results and conclusions: All three DNA extraction kits provided bacterial DNA which passed quality control and was successfully sequenced on Illumina MiSeq platform. The results received from the different DNA protocols showed variations in the microbial profiles. Alpha diversity indices (ACE, Chao1, Shannon, and Simpson) were also different among the three groups of results. Nevertheless, a strong dominance of phylum Firmicutes, class Bacilli, order Lactobacillales, represented mostly by family Lactobacillaceae, genus Lactobacillus (relative abundance of 63.11-82.28%) and family Leuconostocaceae, genus Weissella (relative abundance of 3.67-36.31%) was observed. Lactiplantibacillus plantarum and Levilactobacillus brevis with relative abundance of 16.15-31.24% and 6.21-16.29% respectively, were the two dominant species identified in all three DNA isolates. Novelty and scientific contribution: The presented results give insight into the taxonomic composition of bacterial community of a specific Bulgarian sourdough. Having in mind that the sourdough is a difficult matrix for DNA isolation on the one hand, and that there is no standardized DNA extraction protocol for this matrix on the other hand, this pilot study aims to give a small contribution to the future establishment and validation of such a protocol, which will allow accurate assessment of the specific microbiota of sourdough samples.

Crocus speciosus (Iridaceae)-A New Species for the Bulgarian Flora.

This is the first report on the autumn-flowering species Crocus speciosus, belonging to C. ser. Speciosi from the Bulgarian flora. The species was found in Southeastern Bulgaria, in the area between Ahtopol and Rezovo. Re-examining the Bulgarian collections, the earliest specimen was collected in 1975, was probably overlooked, and most likely determined as C. pulchellus. The nearest known localities of the species are on the territory of Türkiye. In this study, we compared C. pulchellus and C. ibrahimii using DNA sequence data from the nuclear ITS1/2 region and morphological features. Our study showed a close relationship between the specimens from Bulgaria and the recently deposited data of C. speciosus and their separation from the closely related C. pulchellus. Together with the previously cited white anthers as a key feature for determination, the molecular data confirmed a clear distinction between the samples with white anthers in the two species. The morphological data of our taxon overlapped with the description of C. ibrahimii. The molecular data strongly supported the affiliation of C. speciosus s.l., but did not support the recognition of C. ibrahimii as a separate species and it should be referred to as a subspecies of C. speciosus subsp. ibrahimii Ruksans.

Crocus pallidus (Iridaceae)-A Neglected Species for the Bulgarian Flora and Critical Taxon in the Balkans.

For a long time, the Balkan endemic species Crocus pallidus has been unconfirmed and neglected for the flora of Bulgaria. It has remained an uncertain species from the Balkans, often listed as a synonym of C. weldenii. The morphological resemblance to the albinistic forms of C. chrysanthus has led to incorrect identification in the past, resulting in uncertainty regarding the distribution of this species in Bulgaria. In this regard, a detailed morphological and anatomical study of Bulgarian natural populations was carried out. A phylogenetic comparison in the ITS region of two Bulgarian populations of C. pallidus with other related species was conducted, revealing the distinction of C. pallidus populations from the closely related C. weldenii. The recently described new endemic species from Turkey, C. thracicus, shows very similar morphological and anatomical characteristics to C. pallidus and logically continues the distribution range in Eastern Thrace, along the Black Sea coast. We have a basis for suggesting that it should be treated as a synonym of C. pallidus.