Phylogenetic information content of Copepoda ribosomal DNA repeat units: ITS1 and ITS2 impact

Abstract

The utility of various regions of the ribosomal repeat unit for phylogenetic analysis was examined in 16 species representing four families, nine genera, and two orders of the subclass Copepoda (Crustacea). Fragments approximately 2000 bp in length containing the ribosomal DNA (rDNA) 18S and 28S gene fragments, the 5.8S gene, and the internal transcribed spacer regions I and II (ITS1 and ITS2) were amplified and analyzed. The DAMBE (Data Analysis in Molecular Biology and Evolution) software was used to analyze the saturation of nucleotide substitutions; this test revealed the suitability of both the 28S gene fragment and the ITS1/ITS2 rDNA regions for the reconstruction of phylogenetic trees. Distance (minimum evolution) and probabilistic (maximum likelihood, Bayesian) analyses of the data revealed that the 28S rDNA and the ITS1 and ITS2 regions are informative markers for inferring phylogenetic relationships among families of copepods and within the Cyclopidae family and associated genera. Split-graph analysis of concatenated ITS1/ITS2 rDNA regions of cyclopoid copepods suggested that the Mesocyclops, Thermocyclops, and Macrocyclops genera share complex evolutionary relationships. This study revealed that the ITS1 and ITS2 regions potentially represent different phylogenetic signals.

Figure 1

Organization of eukaryotic tandemly repeated rDNA clusters. 18S, 5.8S, and 28S ribosomal RNA genes; ITS1 and ITS2 internal transcribed spacers; IGS intergenic spacer. Arrows indicate the locations of the DAMS18 and DAMS28 primers.

Figure 2

Phylogenetic relationships of Cyclopoida based on ~700 bp of the 28S rRNA gene. The consensus cladogram inferred from the 28S ribosomal DNA fragment sequence data of 16 Podoplea superorder species using maximum likelihood (ML) analysis under the HKY+G model and minimum evolution (ME) analysis. The numbers above branches indicate bootstrap percentages. The values are listed for ML/ME.

Figure 3

Phylogenetic relationships of Cyclopoida based on ~500 bp of concatenated ITS1/ITS2 rDNA sequences. The consensus cladogram inferred from the ITS1-ITS2 ribosomal DNA fragment sequence data of 10 species of the Cyclopidae family using maximum likelihood analysis under the HKY+G model and minimum evolution (ME) analysis. The numbers above branches indicate bootstrap percentages. The values are listed for ML/ME.

Figure 4

Reticulogram for concatenated ITS1/ITS2 sequences of 10 species of the Cyclopidae family. The red dashed line indicates the reticulation event connecting M. leuckarti to the Macrocyclops clade node. The number of internal vertices begins with n + 1, where n is the number of leaves. The order of internal vertices distribution corresponds to the increasing lengths of the 22 reticulogram edges.

Figure 5

Split networks for concatenated ITS1/ITS2 sequences of 10 species of the Cyclopidae family. Split network based on concatenated ITS1/ITS2 sequences; the split separating M. leuckarti, T. oithonoides, and T. crassus is indicated in bold red; the split separating M. leuckarti, M. distinctus, and M. albidus is indicated in bold blue; purple indicates the M. leuckarti reticulate relationship with Thermocyclops and Macrocyclops. The values on the branches indicate bootstrap percentages.

Figure 6

Phylogenetic relationships of Cyclopidae based on ITS1 and ITS2 sequences. The ITS1 consensus Clustal tree of ten Cyclopidae species constructed using (a) maximum likelihood and minimum evolution and (b) Bayesian inference. The ITS2 consensus Clustal tree of ten Cyclopidae species constructed using (c) maximum likelihood and minimum evolution and (d) Bayesian inference. The consensus Gblocks-treated MAFFT trees of eight Cyclopidae species constructed using maximum likelihood and minimum evolution: (e) ITS1, (f) ITS2. The numbers above the branches indicate bootstrap percentages and Bayesian posterior probabilities. The values are listed for ML/ME. Missing or weakly supported nodes (<50% or 0.5) are denoted by a ‘‘—”.