4%) in a population of 125 B. bassiana isolates [25]. The number of introns found in the 57 isolates was in agreement with the 199 introns detected in 125 B. bassiana isolates by Wang et al. [25]; the 44 introns detected in 26 M. anisopliae isolates by Márquez et al. [31], and the 69 introns found in 28 representative

members of the genus Cordyceps by Nikoh and Fukatsu [26]. However, only four intron insertion patterns were present in our B. bassiana collection while greater variability was found in other studies: 13, 7 and 9 insertion patterns within 125 B. bassiana [25], 26 M. anisopliae [31] and 47 B. brongniartii Thiazovivin ic50 [23] isolates, respectively. The MP tree based on intron sequences shows that they were distributed in four large groups, with bootstrap values of 100%, corresponding to four insertion positions (Figure 1). As could be expected [25, 28], the introns inserted at the same site always belonged to the same subgroup: IC1 at positions 2 and 4, and IE at position 1. Although the ARRY-438162 chemical structure origin and transmission mechanisms of group I introns have generated controversy [26], this distribution of sequences is in agreement with previously reported observations [25] and means that introns inserted at the same position have a monophyletic origin and are transmitted vertically. In subsequent events intron speciation

and diversification take place as occurs at position 4, where B. bassiana introns are separated from Metarhizium and Cordyceps introns, and two B. bassiana IC1 sequence sizes were located in two different sub-clades, supported by high bootstrap values. Rehner and Buckley’s study [8] based on EF1-α and ITS phylogenies has revealed that i) six clades can be resolved within Beauveria (A-F) and, excepting those corresponding to B. bassiana (A and C), they are closely

to species previously described on the basis of their morphology, and ii) B. bassiana s.s. (A) was determined almost entirely from nucleotide variation at EF1-α. Further phylogenetic studies carried out with nuclear and/or mitochondrial DNA regions of B. bassiana from all continents have served to resolve BCKDHB lineage diversity within this species [7, 12, 18, 21]. Since phylogenetic species by continent and in the order of their discovery have been designated previously [7], we followed this nomenclature to refer the new phylogenetic subgroups identified among the Spanish B. bassiana s.s. isolates as Eu-7, Eu-8 and Eu-9. The results obtained from MP analyses (Figure 2), using a 1.1 kb fragment of the EF1-α gene from 56 isolates from our collection, confirmed that 53 isolates were B. bassiana s.s. (A), and three isolates grouped in three different phylogenetic subgroups within B. cf. bassiana (C). As in a previous study [7], the collection of Spanish isolates of B. bassiana s.s. was separated in five phylogenetic subgroups.