In the present study, we have identified different B. bassiana genotypes and phylogenetic subgroups in a collection of 57 isolates of this fungus, based on intron insertion patterns and EF1-α phylogenies, respectively.
The variability in group I introns from rDNA genes has been used as a molecular tool for the identification of polymorphisms in entomopathogenic fungi [23, 30, 31]. Our study of B. bassiana LSU rDNA identified 99 introns among the 57 isolates analyzed. Four specific sites of intron insertion have been described previously in Beauveria species [23, 25], but in our collection introns were only detected at positions 1, 2 or 4.
Particularly, our study shows that 100% of B. bassiana s.s. isolates had an intron inserted at position 4. This position was also preferential for intron insertion (84.4%) in a population of 125 B. bassiana isolates . 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. ; the 44 introns detected in 26 M. anisopliae isolates by Márquez et al. , and the 69 introns found in 28 representative members of the genus Cordyceps by Nikoh and Fukatsu . 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 , 26 M. anisopliae  and 47 B. brongniartii  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 origin and transmission mechanisms of group I introns have generated controversy , this distribution of sequences is in agreement with previously reported observations  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  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 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 , 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 , the collection of Spanish isolates of B. bassiana s.s. was separated in five phylogenetic subgroups. However, only isolate Bb38, sampled from insects, was grouped with one (Eu-3) of the five phylogenetic species proposed by those authors working with a Danish collection of B. bassiana s.s. , including insect isolates only. Interestingly, three phylogenetic subgroups (Eu-7, Eu-8 and Eu-9) were only formed by isolates from Spanish and Portuguese isolates. However, most of the isolates in our collection (39 out of 56) were grouped with isolates from Romania and the USA in the world-wide phylogenetic subgroup Wd-2, which includes isolates from Europe, Africa and North America .
When the different intron insertion patterns were mapped on the B. bassiana EF1-α phylogeny (Figure 2), the existence of a same intron genotype in a given phylogenetic subgroup could be indicative of its clonal origin as it is the case of Eu-7 and Eu-8. Previous studies have shown that Eu-3, where Bb38 is located, is a clonal group . Isolate Bb51 was the only member of Eu-9 and the separated phylogenetic grouping of this isolate is supported by a characteristic intron insertion pattern and the production of statistically significant smaller conidia than those from any other intron genotype (data not shown). The two different intron genotypes observed among the isolates from the complex phylogenetic subgroup Wd-2, may indicate that homologous recombination is involved in the IE intron loss at position 1. Previous studies have shown frequent intron losses of group I introns in the nuclear rDNAs of Cordyceps . Recently, a low frequency of sexual reproduction was observed in Eu-1 ; this could also be the case of Wd-2 where the absence of an IE intron at position 1 was only observed in 6 out of 39 isolates of this phylogenetic subgroup.
The genetic diversity of Spanish B. bassiana s.s. isolates was compared in relation to their hosts and geographical provenance and according to the latter view , no general correlation can be observed between the molecular variability among isolates and host and/or geographical origin. Although most of the isolates in our study were collected from soil, 8 out of 9 isolates from insects were grouped together in the subgroup Wd-2 although they derived from different insect orders. Phylogenetic subgroups only indicated a tenuous dependence upon geographic origin (i.e., Bb2-5 located in Eu-7 or Bb23-26 and Bb29-31 located in Wd-2). A recent phylogeographic report  has provided evidence that the genetic distance of Brazilian B. bassiana isolates correlates with geographical distance, suggesting that according to Rehner's study  allopatry plays an important role in the phylogenetic diversification of B. bassiana. The authors of another recent study  concluded that multiple phylogenetic species of B. bassiana s.s. co-exist in sympatry within the limited natural habitat of a bordering hedgerow. We observed that isolates sampled in close locations were placed in different phylogenetic subgroups (i.e., Bb35 and Bb36, from Cádiz, belong to Eu-7 and Wd-2, respectively; and Bb38, and Bb39-40 and Bb42, from Badajoz, group within Eu-3 and Wd-2, respectively, Bb39-40 and Bb42 having different intron genotypes). According to Meyling's study , the high phylogenetic diversity of the Spanish isolates of B. bassiana s.s. could be explained by the untilled habitats where most of them were sampled (i.e., olive, oak, pine, meadow or scrubland).
Previous studies have suggested that the saprophytic phase of entomopathogenic fungi exerts evolutionary pressure on the genotype and that adaptation to a habitat type is associated with their environmental preferences . Recent studies have also pointed out the importance of climatic conditions in the prevalence and distribution of B. bassiana genotypes . Our study was carried out on 51 isolates from subtropical Mediterranean climate locations that were distributed within the phylogenetic subgroups Eu-3, Eu-7, Eu-8, Wd-2 and clade C; 4 isolates were from continental climate sites and grouped in Eu-7, Wd-2 and clade C; and 2 isolates came from a humid oceanic climate zone, being located in Eu-9 and clade C. Interestingly, the only B. bassiana s.s. from a humid oceanic climate was the singular isolate Bb51. The fact that isolates from Mediterranean or continental climates overlapped in different phylogenetic subgroups, could be due to lower differences among the abiotic conditions existing in Spain, a country covering far smaller geographical surface and with much less variability than that considered in other Canadian, Brazilian or world-wide studies where phylogenetic species showed a better correlation with climate characteristics , biogeographic distribution  and habitat . In a thermal growth study  it was described that B. bassiana genetic groups from three different habitats in Canada were associated with temperature preferences. When we explored the thermal preferences within a set of Spanish B. bassiana s.s. isolates belonging to the two main intron genotypes (A1B2B3A4 and B1B2B3A4) and four phylogenetic EF1-α subgroups (data not shown), a correlation between intron genotypes and the mean optimal and maximum temperatures for growth was observed, both growth temperatures being significantly lower in the B1B2B3A4 genotype with respect to A1B2B3A4. However, no correlation was observed between thermal preferences and the climatic origin of the Spanish B. bassiana isolates.