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Abstract:
Background: Identification of species via DNA sequences is the basis for DNA taxonomy and
DNA barcoding. Currently there is a strong focus on using a mitochondrial marker for this
purpose, in particular a fragment from the cytochrome oxidase I gene (COI). While there is ample
evidence that this marker is indeed suitable across a broad taxonomic range to delineate species,
it has also become clear that a complementation by a nuclear marker system could be
advantageous. Ribosomal RNA genes could be suitable for this purpose, because of their global
occurrence and the possibility to design universal primers. However, it has so far been assumed
that these genes are too highly conserved to allow resolution at, or even beyond the species level.
On the other hand, it is known that ribosomal gene regions harbour also highly divergent parts.
We explore here the information content of two adjacent divergence regions of the large subunit
ribosomal gene, the D1-D2 region.
Results: Universal primers were designed to amplify the D1-D2 region from all metazoa. We show
that amplification products in the size between 800–1300 bp can be obtained across a broad range
of animal taxa, provided some optimizations of the PCR procedure are implemented. Although the
ribosomal genes occur in multiple copies in the genomes, we find generally very little intraindividual
polymorphism (<< 0.1% on average) indicating that concerted evolution is very effective
in most cases. Studies in two fish taxa (genus Cottus and genus Aphyosemion) show that the D1-D2
LSU sequence can resolve even very closely related species with the same fidelity as COI
sequences. In one case we can even show that a mitochondrial transfer must have occurred, since
the nuclear sequence confirms the taxonomic assignment, while the mitochondrial sequence would
have led to the wrong classification. We have further explored whether hybrids between species
can be detected with the nuclear sequence and we show for a test case of natural hybrids among
cyprinid fish species (Alburnus alburnus and Rutilus rutilus) that this is indeed possible.
Conclusion: The D1-D2 LSU region is a suitable marker region for applications in DNA based
species identification and should be considered to be routinely used as a marker complementing
broad scale studies based on mitochondrial markers.
