Molecular Investigations of Food-Borne Cladosporium and Fusarium Species from Nigeria

    

    

    Figure 6: Gene genealogies for the tef1α locusin multiple Cladosporium
species. The respective outgroup species (H1) used for inferred gene tree
A was C. salinae and for inferred gene tree B was C. cladosporioides. No
ITS ARGs could be inferred because too few haplotypes resulted when the
sequences were collapsed. These coalescent-based trees show mutations
as dots along branches and time is scaled to The Most Recent Common
Ancestor (TMRCA) of 1.0 for each locus. The haplotype designations for each
locus are shown in Table 3.
    
    

A fairly recent bifurcation in the second lineage appeared to segregate C. tenuissimum into multiple haplotype branches. One of those branches showed the speciation events associated with C. cladosporioides and C. oxysporum. The most recent bifurcation for this group resulted in the segregation of the Nigerian isolates (H2 and H4).The gene tree without C. salinae (Figure 6B) offered more resolution for the group of fungi sharing the second lineage in Figure 6A. We observed bifurcations resulting in haplotypes H1-H6. Haplotypes H7-H9 were not resolved and appeared to undergo clonal amplification since their divergence from H2-H5. No gene tree could be inferred for the ITS region due to having only two haplotypes.

Discussion

It has been shown, through these investigations, that two of the Nigerian Fusarium isolates were consistently identified using different genomic loci. This would indicate that ITS and tef1α are adequate species delimiters for this particular genus. The exception for this group of isolates was SRRC1606, identified as F. equiseti based on ITS sequence, but also as F. incarnatum based on its tef1αsequence. Both species are part of the Fusarium Incarnatum-Equiseti Species Complex (FIESC) [30] and share a most recent common ancestor. In SRRC1606 we see evidence of recombination whereby the ancestry of the ITS region originated with an F. equiseti strain and the tef1α locus originated with an F. incarnatum strain. Reports of F. equiseti and F. incarnatum as specifically pathogenic to yams are lacking although both species have been reported as pathogens of potatoes [31,32]. Some of the mycotoxins associated with F. equiseti are nivalenol, A and B trichothecenes and zearalenone [31,33], while F. incarnatum has been reported to produce beauvericin and zearalenone [32]. Isolate SRRC1633 was sampled from bread and identified as F. incarnatum based on ITS and tef1α regions. Although reports of F. incarnatum as a cereal grain pathogen are rare, they do exist [34]. Isolate SRRC1616 was consistently identified as F. oxysporum based on both genomic regions examined. This isolate/species was sampled from cooked rice which correlated with its reported pathogenicity of cereal grains, and beauvericin and moniliformin are among the mycotoxins that may be produced by F. Oxysporum [32,35]. ITS and tef1α did not offer consistency for species delimitation for sampled Cladosporium species from Nigeria. We determined the respective identifications of isolates SRRC1616 and SRRC1634 as C. Cladosporioides (ITS) and C. Tenuissimum (tef1α). Taxonomically, C. Cladosporioides shares no synonymy with C. tenuissimum, which supported that these are distinct species. However, these two species are part of the same (C. cladosporioides) complex [36] and share a most recent common ancestor. Interestingly, no evidence of recombination was observed in the histories of these isolates. This likely relates to the necessity of removing indels and infinite sites violations in order to proceed with our haplotype associations and comparative inferences. SRRC1616 was sampled from cooked rice and SRRC1634 was sampled from groundnut (peanut). Our findings supported previous reports of a host-pathogen relationship between these two fungal species and cereal grains such as rice, but only C. cladosporioides has been reported on groundnut [37,38]. There have been no reported mycotoxins produced by Cladosporium species [39]. The exact reason for two conserved loci offering different taxonomy is uncertain, but may be the results of ancient inter specific recombination, or horizontal gene transfer, of the loci examined from one species to another that has maintained in isolates like SRRC1606, SRRC1616 and SRRC1634.

From a phylogenetic perspective, the Fusaria exhibited an expected segregation for the loci examined. Cladal associations could be observed, and overall the species/complex distinctions were maintained. For example, F. Chlamydosporum Represents Its Own Species Complex (FCSC) but is considered a closely-related lineage to the FIESC complex that includes the F. incarnatum and F. equiseti species [30]. The most divergent lineage of species examined was F. oxysporum which represents its own species complex (FOSC). Inferring phylogenetic association for the Cladosporia; we were unable to infer a phylogeny for the ITS region because 99% of the sequences obtained were identical. The examined species are encompassed within the C. cladosporioides species complex. The observation that not enough haplotype diversity existed for the Cladosporium ITS region suggests that these are indeed closely-related species whose diversity will likely exist in other loci. For example, we observed greater sequence diversity for the tef1α locus. As expected for closelyrelated species, two C. Cladosporioides sequences were observed sharing a clade with C. tenuissimum while a third C. cladosporioides sequence was inferred as its own lineage. The high degree of diversity among C. tenuissimum sequences could relate to geography or host specificity, but could also relate to misidentifications which can occur for closely-related species. Future studies should involve an increase in the numbers of sampled isolates for more refined inferences of intra-species population dynamics.

Contemporary recombination is an occurrence that is more likely within panmictic populations, but with conserved loci it is still possible to infer ancient recombination events, despite geographic isolation, among coevolved species [39,40]. We were able to infer recombination within the histories of multiple Fusarium species based on two different genomic regions that are considered “conserved” [41]. This finding would support that (1) historical recombination likely contributed to species diversity among these fungi [42,43], and (2) true species delimitation for these fungi would require genomescale comparisons and a more holistic approach to identification [44]. Recombination among Cladosporium species was only observed for the tef1α locus, but the richness of species diversity may require examining other loci and additional species. Based on our inferences of recombination, the different species within each genus, are either exhibiting ancient genetic configurations that have been maintained in certain species, or are exchanging genetic material on a recent time scale as part of their convolution. This is because they share a common ancestor,

Coalescent analysis removes the influence of recombination from species evolution by simply counting mutations and applying them to a time scale based on the assumption of a neutral mutation rate [29]. Basically, the greater the number of mutations along a sequence, the longer that sequence has been in existence. The finding that ITS sequences from three F. Oxysporum isolates segregated as a distinct lineage, with no divergence, suggests it is an ancient organism that long ago diverged from the common ancestor of the other examined Fusarium species. Despite being species from closelyrelated complexes, F. chlamydosporum, F. equiseti and F. incarnatum exhibited recent divergence of their respective ITS regions with no further resolution. More evident patterns of divergence/speciation were observed for the tef1α locus. For example, in the F. oxysporum lineage isolate SRRC1630 was derived from a recent divergence event. The divergence of FCSC and FIESC occurred millions of years ago, and not too long after their divergence a speciation event segregated F. equiseti and F. incarnatum. The tef1α locus for the Cladosporia also suggested an ancient divergence for which the C. salinae lineage is shown as much older than the other species examined. Resolution of the divergence of the remaining Cladosporium species is moderate, but what can be inferred are recent divergence/speciation events for species that share a complex. As well, the Nigerian isolates appeared to be the most recently derived.

In conclusion, the sampling of Fusarium species on foodstuffs in Nigeria is import when one considers the potential for the identified species to produce mycotoxins. We did not perform any mycotoxin assays for our sampled isolates in this study. The identification of a fungus on a particular host for which it is not considered a pathogen may indicate the presence of new species that happen to share genomic sequence with a known species/pathogen. Our findings support the need for a more holistic approach to species identification; particularly, when those fungi are contaminating foods and feeds and even cosmetics [45].

Author Contribution

GG Moore and SO Fapohunda conceived and designed the experiment. Fapohunda did the isolation of the fungi from the food samples while Moore performed molecular analyses. Both contributed to the writing of the paper.

Acknowledgements

The authors would like to thank Mrs. Shannon B. Beltz for her assistance in the morphological identification of the Nigerian isolates, and all reviewers who helped to improve this body of work.

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