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Exploring the use of cytochrome oxidase c subunit 1 (COI) for DNA barcoding of free-living marine nematodes.

Derycke S, Vanaverbeke J, Rigaux A, Backeljau T, Moens T - PLoS ONE (2010)

Bottom Line: The taxon specific primers for the I3-M11 partition outperformed the universal M1-M6 primers in terms of amplification success (87.8% vs. 65.8%, respectively) and produced a higher number of bidirectional COI sequences (65.8% vs 39.0%, respectively).A threshold value of 5% K2P genetic divergence marked a clear DNA barcoding gap separating intra- and interspecific distances: 99.3% of all interspecific comparisons were >0.05, while 99.5% of all intraspecific comparisons were <0.05 K2P distance.The I3-M11 partition reliably identifies a wide range of marine nematodes, and our data show the need for a strict scrutiny of the obtained sequences, since contamination, nuclear pseudogenes and endosymbionts may confuse nematode species identification by COI sequences.

View Article: PubMed Central - PubMed

Affiliation: Marine Biology Research Group, Department of Biology, Ghent University, Ghent, Belgium. s.derycke@ugent.be

ABSTRACT

Background: The identification of free-living marine nematodes is difficult because of the paucity of easily scorable diagnostic morphological characters. Consequently, molecular identification tools could solve this problem. Unfortunately, hitherto most of these tools relied on 18S rDNA and 28S rDNA sequences, which often lack sufficient resolution at the species level. In contrast, only a few mitochondrial COI data are available for free-living marine nematodes. Therefore, we investigate the amplification and sequencing success of two partitions of the COI gene, the M1-M6 barcoding region and the I3-M11 partition.

Methodology: Both partitions were analysed in 41 nematode species from a wide phylogenetic range. The taxon specific primers for the I3-M11 partition outperformed the universal M1-M6 primers in terms of amplification success (87.8% vs. 65.8%, respectively) and produced a higher number of bidirectional COI sequences (65.8% vs 39.0%, respectively). A threshold value of 5% K2P genetic divergence marked a clear DNA barcoding gap separating intra- and interspecific distances: 99.3% of all interspecific comparisons were >0.05, while 99.5% of all intraspecific comparisons were <0.05 K2P distance.

Conclusion: The I3-M11 partition reliably identifies a wide range of marine nematodes, and our data show the need for a strict scrutiny of the obtained sequences, since contamination, nuclear pseudogenes and endosymbionts may confuse nematode species identification by COI sequences.

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Relative frequencies of K2P-genetic distances within species (black), between congeneric species (grey) and between species from different genera (white).
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pone-0013716-g005: Relative frequencies of K2P-genetic distances within species (black), between congeneric species (grey) and between species from different genera (white).

Mentions: Variability of I3-M11 was calculated using 27 sequences from the present study and 24 sequences from previous studies (Table S1). The I3-M11 partition was highly AT-rich (A: 27.1%, T: 42.8%, G: 18.6%, C: 11.5%). A very high variability was observed at the amino acid level with 95 out of 143 amino acids (66.4%) being variable (Table 2). Maximum pairwise K2P-distances within species varied between 0.005–0.121, while minimum K2P-distances between congeneric species ranged between 0.005–0.26 and minimum K2P-distances between species from different genera was 0.12. Although this suggests a strong overlap between intra- and interspecific genetic distances, the frequency distribution of the K2P-distances showed that 99.5% of all intraspecific comparisons showed genetic distances of less than 5%, while 99.3% of all interspecific comparisons were higher than 5% (Fig 5). The only species pair that was less than 5% different was Praeacanthonchus and Paracanthonchus.


Exploring the use of cytochrome oxidase c subunit 1 (COI) for DNA barcoding of free-living marine nematodes.

Derycke S, Vanaverbeke J, Rigaux A, Backeljau T, Moens T - PLoS ONE (2010)

Relative frequencies of K2P-genetic distances within species (black), between congeneric species (grey) and between species from different genera (white).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2965665&req=5

pone-0013716-g005: Relative frequencies of K2P-genetic distances within species (black), between congeneric species (grey) and between species from different genera (white).
Mentions: Variability of I3-M11 was calculated using 27 sequences from the present study and 24 sequences from previous studies (Table S1). The I3-M11 partition was highly AT-rich (A: 27.1%, T: 42.8%, G: 18.6%, C: 11.5%). A very high variability was observed at the amino acid level with 95 out of 143 amino acids (66.4%) being variable (Table 2). Maximum pairwise K2P-distances within species varied between 0.005–0.121, while minimum K2P-distances between congeneric species ranged between 0.005–0.26 and minimum K2P-distances between species from different genera was 0.12. Although this suggests a strong overlap between intra- and interspecific genetic distances, the frequency distribution of the K2P-distances showed that 99.5% of all intraspecific comparisons showed genetic distances of less than 5%, while 99.3% of all interspecific comparisons were higher than 5% (Fig 5). The only species pair that was less than 5% different was Praeacanthonchus and Paracanthonchus.

Bottom Line: The taxon specific primers for the I3-M11 partition outperformed the universal M1-M6 primers in terms of amplification success (87.8% vs. 65.8%, respectively) and produced a higher number of bidirectional COI sequences (65.8% vs 39.0%, respectively).A threshold value of 5% K2P genetic divergence marked a clear DNA barcoding gap separating intra- and interspecific distances: 99.3% of all interspecific comparisons were >0.05, while 99.5% of all intraspecific comparisons were <0.05 K2P distance.The I3-M11 partition reliably identifies a wide range of marine nematodes, and our data show the need for a strict scrutiny of the obtained sequences, since contamination, nuclear pseudogenes and endosymbionts may confuse nematode species identification by COI sequences.

View Article: PubMed Central - PubMed

Affiliation: Marine Biology Research Group, Department of Biology, Ghent University, Ghent, Belgium. s.derycke@ugent.be

ABSTRACT

Background: The identification of free-living marine nematodes is difficult because of the paucity of easily scorable diagnostic morphological characters. Consequently, molecular identification tools could solve this problem. Unfortunately, hitherto most of these tools relied on 18S rDNA and 28S rDNA sequences, which often lack sufficient resolution at the species level. In contrast, only a few mitochondrial COI data are available for free-living marine nematodes. Therefore, we investigate the amplification and sequencing success of two partitions of the COI gene, the M1-M6 barcoding region and the I3-M11 partition.

Methodology: Both partitions were analysed in 41 nematode species from a wide phylogenetic range. The taxon specific primers for the I3-M11 partition outperformed the universal M1-M6 primers in terms of amplification success (87.8% vs. 65.8%, respectively) and produced a higher number of bidirectional COI sequences (65.8% vs 39.0%, respectively). A threshold value of 5% K2P genetic divergence marked a clear DNA barcoding gap separating intra- and interspecific distances: 99.3% of all interspecific comparisons were >0.05, while 99.5% of all intraspecific comparisons were <0.05 K2P distance.

Conclusion: The I3-M11 partition reliably identifies a wide range of marine nematodes, and our data show the need for a strict scrutiny of the obtained sequences, since contamination, nuclear pseudogenes and endosymbionts may confuse nematode species identification by COI sequences.

Show MeSH