An evaluation of LSU rDNA D1-D2 sequences for their use in species identification

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 intra-individual 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.

Figure 1

General organisation of eukaryotic ribosomal genes. All eukaryotes show a stereotypic arrangement, with often hundreds of tandemly repeated ribosomal transcription units. Each produces a large transcript which is processed into the small subunit, the 5.8S subunit and the large subunit. The external transcribed spacer (ETS) and the internal transcribed spacers (ITS1 and ITS2) do not become part of the mature rRNA, but their sequences and structures are required for the correct processing. The dot plot comparisons above the SSU and the LSU show the conservation profiles between a chordate and an arthropod sequence. In this presentation, the sequences of two species (mouse and Drosophila) are compared and a dot is placed in the diagram at each position where 10 consecutive nucleotides match. Conserved and divergent regions become thus directly apparent. It is evident that the SSU is more conserved, interrupted by a few less conserved regions, while the LSU show larger regions of divergence.

Figure 2

Primers used for amplification and sequencing of the D1-D2 LSU region. The primer positions in column 1 refer to the LSU sequence of Drosophila melanogaster (Acc. No. M21017). The sketch below indicates the approximate positions of the primers.

Figure 3

Two examples for ambiguous sequence positions in the forward and reverse sequencing direction of the same fragment (indicated by a red arrow). We interpret these ambiguities as polymorphic positions among the rDNA repeat units within an animal. Given that these ambiguities are very rare (see text), one can conclude that homogenization is usually very efficient.

Figure 4

A comparison between LSU and mtDNA sequences with respect to species level resolution for the genus Cottus. Twelve species of the genus Cottus were sequenced for both markers and trees were obtained via the neighbour joining algorithm in MEGA 3.1. For three species, multiple animals from given populations were sequenced. Both markers detect the same groupings, with the exception of the animals from the population "RotesW", where COI sequences generate a different assignment. Further analysis of this case has shown that this is due to mitochondrial transfer.

Figure 5

A comparison between LSU and mtDNA sequences with respect to species level resolution for the Aphyosemion calliurum species group with a complete taxon sampling. For species with larger distribution several populations were sampled. Both markers assign the samples to the same groups. See text for further details.

Figure 6

The assessment of hybrid status from heterozygosity of informative characters. The sequence traces were obtained from four F1 hybrid animals between Cottus perifretum and C. rhenanus. Four positions where fixed differences were known to occur are selected. Double peaks are evident in most, although not in all cases (see text).

Figure 7

Hybrid status analysis in a natural case. Samples were obtained from pure species of R. rutilus and A. alburnus, as well as from a suspected hybrid animal. The sequence traces show diagnostic positions for the two species and confirm double peaks at the respective positions for the apparent hybrid animal.