Here we provide evidence of intragenomic copy number variation of the nrDNA operon in the Rhizoplaca melanophthalma species group, ranging from nine to 43 copies. Our estimates for members of the Rhizoplaca melanophthalma species group were in line with recent genome-based estimates for symbiotrophic fungi . Given the rather limited intragenomic variation among copies (Additional file 3), it appears that specimen identification using the standard fungal DNA barcoding marker, the ITS , is not biased by intragenomic variation in the nrDNA region for the Rhizoplaca melanophthalma species group . While copy number variation in fungi has recently been investigated and discussed in light of our current understanding , below we highlight the implications of our findings as they relate to the utility of nrDNA for specimen identification, patterns of intron evolution, and the development of complete nrDNA reference libraries.
We used short reads from metagenomes of lichenized fungi to infer limited intragenomic variation in members of the R. melanophthalma species complex. Potentially variable sites among nrDNA operon copies did not coincide with diagnostic, fixed nucleotide position characters separating species in the Rhizoplaca melanophthalma species group (Additional file 3), providing additional evidence that distinct clades in the nrDNA topologies are not merely a reflection of variable nrDNA copies. Similarly, consistent clades are recovered from alignments of different regions of nrDNA, e.g., 28S, intergenic spacer region, and intronic regions (Fig. 3) and distinct clades in the ITS are not merely idiosyncratic in the ITS region alone.
For members of the R. melanophthalma species complex, we note that the distinct clades recovered from phylogenetic analyses of nrDNA do not correspond to phylogenies reconstructed from genome-scale data, specifically for members of the R. porteri group—R. occulta, R. polymorpha, and R. porteri [31, 32]. Rhizoplaca occulta, R. polymorpha, and R. porteri are recovered as divergent lineages in analyses of nrDNA, none of which are each another’s sister clade (Fig. 3), while genome-scale data consistently recover these three taxa as closely related and intermixed in a well-supported clade [31, 32]. This is in contrast to R. haydenii, R. melanophthalma, R. novomexicana, R. parilis, and R. shushanii that are recovered as distinct in phylogenies inferred from both nrDNA alignments and phylogenomic data matrices.
The origin of highly divergent nrDNA clades among closely related, or even conspecific, lineages remains enigmatic. The three previously undetected nrDNA clades recovered here within the R. melanophthalma complex, ‘nrDNA clade I’, ‘nrDNA clade II’, and ‘nrDNA clade III’ (Fig. 3), may represent previously unsampled species-level lineages, other members of the R. porteri clade with divergent nrDNA, or some other unexplained phenomenon. The role of hybridization/introgression has recently been proposed as an important mechanism in the diversification of the Rhizoplaca melanophthalma species group (Keuler et al. in review).. It is possible that divergent nrDNA clades may represent evidence of reticulate evolution with extinct or as-of-yet unsampled species or simply artifacts of hybridization/introgression. However, additional broader sampling will be required to infer the relationship of these newly found divergent nrDNA lineages. Similarly, the number and diversity of lichen associated symbionts has recently been shown to be far more complex than initially thought . These studies suggest that the dynamics of symbiotic genetics may be synergistic with intricate interactions that combine to support these unique and complicated symbiotic systems. However, with the use of additional high-throughput sequencing methods, including single molecule sequencing and/or long-read sequencing technologies it will be possible to more directly characterize intragenomic variation and copy number of nrDNA [1, 34, 35].
Here, intragenomic variation was observed to be low in the Rhizoplaca melanophthalma species group. Concerted evolution is considered to be responsible for the maintenance of sequence similarity among copies of rDNA repeats in the operon potentially leading to low intragenomic variation [36, 37]. Concerted evolution is driven by unequal crossing over and gene conversion and although these two processes both lead to the homogenization of rDNA repeat units their underlying mechanisms differ from each other [37, 38]. Gene conversion is caused by replacement of DNA sequence by another in a unidirectional manner during homologous recombination. Unequal crossing over, on the other hand, involves the misalignment of homologous chromosomes in meiosis or sister chromatids in mitosis followed by nonreciprocal transfer of DNA sequence from one chromosomal location to another resulting in a deletion in one of the chromosomes and a duplication in the other. While both these mechanisms lead to homogenization of rDNA repeat units, unequal crossing over also has the potential to affect the copy number of the repeat units [36, 39].
The results of this study revealed striking patterns of multiple intron gains and losses in the R. melanophthalma group. Eleven of the 23 introns found in the 18S and 28S nrDNA genes are present in all Rhizoplaca specimens (i1, i5, i9, i10, i11 in the 18S gene and i3, i5, i7 to i10 in the 28S gene), but the pattern of all other introns is highly variable indicating multiple gains and losses in the evolution of Rhizoplaca. When comparing the intron pattern with the phylogenetic tree, only the occurrence of three introns can be most parsimoniously explained by a single event, i. e. the loss i7 from the 18S gene in some R. porteri, the loss of i13 from the 18S gene in all R. porteri, and a gain of i4 in the 28S gene after the split of R. novomexicana and R. melanophthalma from the remaining Rhizoplaca. All other introns would require at least require two (i2, i3, i4, i6, i12 in 18S and i1, i2, i6 in 28S) or four losses (i8 in 18S) to reassemble the intron pattern. This intron pattern confirms that ntDNA introns are highly mobile and the dynamic variability of the nrDNA region .