Sample Set
The study's first prerequisite involved the establishment of a representative sample set. Initially, a total of more than 300 strains were identified as belonging to both H. lixii/T. harzianum by the ITS1 and 2 barcodes [11] and their diagnostic morphological and physiological characteristics [15, 16]. However, the sample was condensed to only 93 strains that best represented the genetic and geographic variations observed. The final sample set consisted of 10 strains collected from decaying wood as Hypocrea teleomorphs (Table 1), while the other strains isolated as Trichoderma anamophs were predominantly from various soil ecosystems. To determine the speciation history within the clade and establish genetic borders for morphological H. lixii and T. harzianum, representative strains of all neighbouring species (Harzianum-Catoptron Clade; [17, 18]) were included in the sample set.
. atrogelatinosa
CBS 237.63, tawa-
H. tawa
CBS 246.63, alni-
H. alni
CBS 120633, aggr-
T. aggressivum
CBS 433.95, epi-
H. epimyces
CBS 120534, pleuroti-
T. pleuroticola
Z. D. 56, pleurotu-
T. pleurotum
C. P. K. 2113, brunn-
H. brunneoviridis
CBS 120928, velu-
T. velutinum
DAOM 320014, stram-
H. straminea
G. J. S. 02-84, ceri-
T. cerinum
DAOM 230012, tom-
T. tomentosum
Z. D. 28, cato-
H. catoptron
G. J. S. 02-76, C. P. K. 410-
T
. sp. C. P. K. 410. Detection of the phylogenetic species was done by screening five potential markers in our study for their ability to differentiate within the population. In addition to the final markers analyzed, we tested an initial set consisting of exon fragments of RNA polymerase (rpb2
We analyzed the 93 strains of Heliothis parvus collected from 2014 to 2016 for their molecular genetic diversity, and found that there were four genes with significant variation between the three populations: calmodulin (
cal1
), translation elongation factor 1-alpha (
tef1
), GH18 chitinase gene (
chi18-5
) and a second gene. However, only three unlinked loci were sufficiently variable. The nucleotide characteristics of these genes are provided in Additional file 1. We analyzed them as a combined dataset as well as individually. As seen in Figures 1 and 2, all phylograms confirmed a monophyletic origin for selected 93 strains. Despite this monophyletic origin, the level of intraspecies genetic polymorphism within H. lixii/T. harzianum (as defined by Chaveri et al. [16]) was comparable to that of interspecific variation within the whole Harzianum-Catoptron Clade. We applied the genealogical concordance phylogenetic species recognition concept using the criteria of Dettman et al. [19], and found that our specimens belong to the genus Heliothis.
The identification of two distinct phylogenetic species in this study was based on the following criteria:
1. If a clade is supported by at least one gene tree and not contradicted in any of the others, then it can be considered an independent evolutionary lineage [phylogenetic species].
The first identified phylogenetic species consisted of a clade that contained the ex-type strain of T. harzianum (hz.07, = CBS 226.95), which was supported by high posterior probabilities (PP) in tef1, cal1, and in the combined phylograms, but not contradicted in the chi18-5 tree (Figures 1 and 2). This clade includes 15 strains isolated as Trichoderma anamphomes from North America, Europe, Siberia, and South Africa. Due to its presence of the ex-type strain, this clade is considered to represent T. harzianum sensu stricto, and will henceforth be referred to as "T. harzianum" throughout this manuscript.
The second identified phylogenetic species was supported with high significance by all three individual tree topologies. It comprises anamorphic strains of tropical origin, primarily African in origin, and is thus named T. sp.
The phylogenetic position of the ex-type strain of H. lixii (li13 = G.J.S. 97-96), the putative teleomorph of T. harzianum, occurs in a large clade (Lixii subclade, Figure 1). However, a comparison of individual gene trees (Figure 2) reveals that this separation is mainly caused by the chi18-5 topology, whereas it is contradicted in the tef1 and the cal1 trees. Nevertheless, a small subclade, which includes both teleomorphs and anamorphs from North America, is supported in the cal1 and tef1 trees and not contradicted in the chi18-5 tree.
We therefore consider this holomorphic subclade as a hypothetical species (H. sp. 'paralixii nom. prov.', Figure 1). The other strains in our sample are isolated as Hypocrea (Table S4).
Teleomorphs proved to be diverse and varied One group of teleomorphs, from Europe (li14), clustered in the vicinity of Hyphophilus sp. paralixii. Other European strains were found as terminal branches of Subclade III in the combined phylogram, but this relationship was not supported by individual loci. Two other teleomorph isolates, from Vietnam (X.12) and the USA (X.17), could not be attributed to any clade either. These three teleomorph isolates from Cameroon (li07, li08, and li09), however, formed a supported terminal clade in the tef1 and cal1 trees, which was not contradicted by chi18-5. As a result, we ranked them as another hypothetical species, Hsp. cameroonense nom. prov. (Figure 1).
The remaining teleomorph isolates had phylogenetic positions that were discordant with each other across trees derived from single loci. To accommodate these differences and maintain clarity in our discussion, we have adopted the term "pseudoharzianum matrix" for these strains and will refer to them as such throughout the paper (Figure 1). This designation emphasizes that their phylogenetic position is uncertain and may need to be revised based on further research.
In order to test the co-specificity of these 12 teleomorph strains with any of the "H. lixii" lineages identified by Chaverri et al, we also included them in our analysis (Table 1). The results showed that nine of these isolates clustered within the Lixii subclade or in Subclade III. Of the remaining three strains, one was found in Subclade IV, one formed a sister lineage to T. sp. 'afroharzianum' within Subclade IIa, and one had an unresolved position in the "pseudoharzianum matrix".
The detected phylogenetic species, hypothetical phylogenetic species, and newly introduced terms with and without taxonomic significance are listed in Table 2. We observed significant recombination between and within T. harzianum, T.sp. 'afroharzianum', and strains of the "pseudoharzianum matrix" (data not shown). These data support the idea that the Lixii group is likely to be a clade distinct from other Harzina groups, including T. sp. 'afroharzianum'.
We also tested whether the teleomorph strains were more closely related to H. lixii than those outside this group using 16 S rRNA gene sequences (Table 3). Our results showed that only six out of the twelve strains were closer to H. lixii than any other group. These six strains belonged to the following five lineages:
• Lineage I: G.J.S. 85-109
• Lineage IIa: G.J.S.97-114, G.J.S.107-119
• Lineage IIc: G.J.S.120-132, G.J.S.133-142, G.J.S.346-349, G.J.S.350-365
• Lineage IIIa: A.jp. 1057
• Lineage IIIb: A.jp. 1058
These results indicate that the teleomorph isolates were more closely related to H. lixii than any other group, including T. sp. 'afroharzianum'. Therefore, our data suggest that the Lixii group may represent a monophyletic group of teleomorph strains that includes both the T. sp. 'afroharzianum' strain and the previously known T. livii strain(s).
In the present study, we analyzed the karyotype of two species complex of the genus Harzianum (genus Harzianum in the taxonomic treatment below), which are closely related: H. lixii and T. harzianum. Specifically, we investigated their genetic relationship by means of sequence data analysis, specifically using three alternative computational methods for the detection of genetic recombination from DNA extracted from these fungi. We used the strains of T. harzianum as a control sample for clonal sterility, while seven representative strains of species from the Harzianum-Catoptron Clade, which are genetically most distant to both H. lixii and T. harzianum, were used as a control for interspecific sterility. The results of our study suggest that either the tested strains are virtually genetically identical and likely clonal (= clonal sterility) or that all teleomorph stains within the sample were completely reproductively isolated (= interspecific sterility).
The presence of teleomorph stains in the Lixii subclade of H. lixii, but not in other subclades, suggests a strong association between this subclade and T. harzianum, which is consistent with the hypothesis that these two species are closely related and possibly share similar mechanisms of sexual reproduction. However, the lack of teleomorph staining in the S.s. and T sp.'afroharzianum' subspecies suggests that these species may have evolved different reproductive strategies.
In conclusion, our findings provide evidence for the close relationship between H. lixii and T. harzianum, which may be further supported by future studies on their genetics and biochemistry. Furthermore, our computational approach has shown promise for detecting genetic recombination in complex species like Harzianum, opening up new avenues for studying evolutionary processes in this group of fungi.
In this paper, we present a new method for the analysis of gene trees. First, we use the partition homogeneity test (PHT) to examine the congruence between gene trees. This test produces artificial datasets by multiple (10 000) re-sampling and random swapping of observed datasets and subsequent construction of maximum-parsimony trees for every newly sampled 'gene' sequence. For clonally reproducing populations (= no sexual recombination), the sums of the lengths of the gene trees for the observed and re-sampled data should be similar. However, under recombination the sums of the tree lengths should be longer than that for the actual data because of introduction of homoplasy into unlinked sites. This test confirmed our analysis of topologies of single locus trees - the clades containing T. harzianum and T. sphaericus.
froharzianum is a genus of 18 species in the family Harziacinaceae. The genus is distributed throughout Africa and Asia. There are 350 described species, with another 27 described but pending review.
The Afroharzianum sp. 'afroharzianum' showed congruence of data suggesting their clonality (Figure 1). Other clades, which were incongruent in different trees, provided evidence for recombination. However, recombination was also not detected with strains occurring at unresolved positions on the combined tree (X.01 - X.16) including the ex-type strain of T. inhamatum (X.15 = CBS 273.78), and two strains isolated as teleomorphs (Figure 1). As none of these strains fulfilled the criteria of a phylogenetic species, we assume that they represent multiple relict lineages which are incompatible of recombination either due to the loss of sexuality or due to mating incompatibility evolved in a course of habitat isolation. The relatively long genetic distances between them support this view. The controls (= representatives of Harzianum-Catoptron Clade) produced essentially negative results (Figure 1, right inset).
In conclusion, it seems likely that the Afroharzianum sp.'afroharzianum' represents a monophyletic species, while other groups may be remnants of once-divergent lineages that were unable to undergo successful sexual recombinantation.
We performed a second type of analysis, the index of association (IA) test, on a subset of clone corrected data. In this test, individuals with identical alleles for each of the three loci were excluded to ensure that each haplotype was represented only once. The value obtained in this analysis indicated whether complete panmixia (sexual compatibility leading to recombination) was present; a value of 0 would indicate this and would be consistent with the null hypothesis (see [22], for details).
The Lixii subclade, and its Subclades III and IV did not yield such a value. This is despite the fact that no individual clades yielded a value greater than 1. This suggests that there is sexual compatibility between the strains within these groups.
In contrast, T. harzianum and T. sp. 'afrohazianum' both yielded values above 1, indicating that there is no evidence for recombination. As for Subclade V, which consisted mainly of two concatenated haplotypes, it was not analyzed further as it contained little genetic variation.
The IA values for strains at unresolved positions (X.01-X.16) also showed no evidence for recombination according to the results from PHT analysis.
Finally, we applied the Phi test to detect refined incompatibility based on pairwise homoplasy index (PHI, Ö). This method assumes the infinite sites model of evolution, where the detection of incompatibility for a pair of sites indicates recombination. The test was applied to the same subsamples based on phylogenetic species and clades from the combined tree, which confirmed previous results. No recombination was detected in T. harzianum and between representative strains of the Harzianum-Catoptron Clade (P = 0.1897, P = 0.2773, and P = 0.3406, respectively). However, all other subclades exhibited positive recombination signals (P < 0.05). An inset showing the corresponding network produced by Splitstree is provided on the right bottom of Figure 1.
Since the Phi-test is a very robust means which can detect recombination even in the presence of recurrent mutation, we decided to use this method to define the borders of recombining populations. To this end, we first set up a non-recombining sample consisting of the most terminal strains of a clade to be investigated, and the most distant strains from other phylogenetic species. Then we gradually added phylogenetically closer strains until evidence for recombination was detected (P
We analyzed all 90 meaningful combinations of species,clades, and subclades present in the phylograms (Figure 1). To determine the outer border of a recombining population, we started to add isolates that were more distant in phylogeny. This technique has the advantage that it also allows the analysis of small subclades which can not be analyzed alone due to insufficient data. For example, although the isolates of T. harzianum were proven to be clonal by all three methods used, they still revealed a recombination history with strains X.01-X.03 from Northern Africa which occupied an otherwise unresolved phylogenetic position in the tef1,
cal1
clades. We excluded the first strain indicating recombination to define the outer border of the recombining population. The positive results (recombination, P < 0.05) obtained are shown by shadowed areas on the main part of Figure 1, and provided interesting insights into the reproduction history of the clades found. In summary, the method described here allowed us to identify novel combinations of species/clades/subclades where recombination events occurred, thus enriching our knowledge of their genetic history and evolutionary relationships.
The agamospecies, Chi18-5, is composed of various tree species. Another recently discovered species, T. sp.'afroharzianum ', exhibits a history of recombination with five phylogenetically plesiotypic strains of different geographic origins (Figure 1). Not all strains of the Lixii subclade showed a recombination history in this test; however, two groups with positive recombination signals were detected. One group includes strains of H. sp.'paralixii' and two temperate strains from North America and Europe. The second group covers the latter two strains: H. sp.'cameroonense' and the ex-type strain of H. lixii.
limited recombination was also detected for strains from Subclade III occupying terminal positions. Three Austrian teleomorphs and two anamorphic strains isolated from Sardinian soils were recombining with each other but not with strains occupying basal branches of the subclade. Similarly, the four terminal strains from a Subclade IV (all from Africa) display a history of recombination. In addition, all strains of the panmictic subclade V showed evidence for recombination. This result corresponds to the fact that they occupy conflicting positions in the single gene trees but belong to the same subclade of ech18-5.
The phylogenetic analysis of Harzianum and its close relative Catoptron has revealed a complex evolutionary history for these two families, as well as an interesting role for sexual selection in the evolution of their reproductive strategies. The phylogeny of both groups is presented in Figure 1 (subclade X).
Figure 1: Phylogenetic analysis of Harzianum and its close relative Catoptron
The phylogenetic tree constructed by maximum parsimony showed that the most genetically polymorphic isolates of the 'pseudoharzianum matrix', which formed almost no phylogenetic structure, showed no evidence for recombination signal when confronted either with each other or with other subclades. This finding suggests that the members of this group are not closely related to each other and may have diverged from one another relatively recently.
However, when some of these isolates were analyzed together with representatives of the Harzianum-Catoptron Clade, a significant recombination signal was detected indicating traces of interspecific sexual compatibility. This is illustrated in Figure 1 by the open shadow area around the two groups. This finding provides additional evidence for the role of sexual selection in the evolution of Harzianum.
To estimate the degree of differentiation within the 'pseudoharzianum matrix', we applied methods for analysis of populations and computed the F ST values [25] for the main subclades of the combined phylogram. Qualitatively, an F ST value below 0.5 indicates a high degree of similarity between populations, while values above 0.5 indicate low genetic diversity.
In our case, the F ST value calculated for each subclade ranged from 0.2 to 0.7 (Table S1). This result suggests that the members of the 'pseudoharzianum matrix' are relatively homogeneous, but not so much that they can be considered a monophyletic group. In fact, several analyses suggested that they could be classified into at least three distinct lineages (Subclade A, Subclade B, and Subclade C) (Table S1). However, it should be noted that these results depend largely on the method used for the analysis and may change if alternative approaches are followed in future studies.
In conclusion, our study provides new insights into the evolutionary history of Harzianum and Catoptron. It also highlights the importance of considering sexual compatibility in the context of speciation events and highlights how complex evolutionary relationships can be in aquatic ecosystems.
The F ST value was a measure of differentiation (or fixation) between populations. Values close to 0 indicate low differentiation and close to 1 indicates great differentiation. When populations are composed of similar individuals, a value close to 1 indicates that the characters in one population differ significantly from those in another. On the other hand, when populations are composed of equally different individuals, a value close to 0 indicates that there is no significant difference between the characters in the two populations.
The F ST values between T. harzianum and other subclades were in the range of 0.67 - 0.83, suggesting that these species have essential genetic separation. The F ST value between Subclades III and IV of the 'pseudoharzianum matrix' was low (0.17), which may indicate that some genetic exchange still occurs between their strains.
Population parameters such as θ (for haploids, where N is the effective population size and m is the mutation rate per site and generation) and population growth rates were calculated using the LAMARC package. These parameters provide information on an individual's likelihood of surviving and reproducing, as well as their ability to transmit genetic information to the next generation.
Overall, these calculations provide valuable insights into the genetics and behavior of T. harzianum and its relationship with other subclades within the genus Harzianum. This information can be used for further research and conservation efforts aimed at protecting this species and its habitat.
. harzianum is a species complex that occurs in many habitats around the world, but its growth rate is relatively low compared to other subclades of this genus. This may indicate that it has a smaller effective population size and is less likely to expand into new areas (data not shown).
However, the recombining subclades of T. harzianum exhibit the highest growth rates among all the subclades in this genus. In contrast, T. harzianum has the lowest growth rate among all the subclades, with a value of 5.90 for population growth and a significantly smaller θ-value than the other subclades (5-20-fold smaller). This suggests that T. harzianum occupies a niche with limited potential for expansion, as other subclades are more likely to grow and expand into new areas.
