This document summarizes an analysis of cytochrome c oxidase subunit 1 (cox1) DNA sequences from seven nematode genera to evaluate the effectiveness of DNA barcoding for nematode species identification and delimitation. The analysis found three patterns of the "barcoding gap" between intra- and interspecific genetic distances: 1) a clear gap, 2) a tendency towards a gap, and 3) no gap. It also estimated more putative species than labeled species for most genera, indicating potential cryptic diversity or misidentification in database sequences. While cox1 showed some ability to identify nematode species, incorrect database labels may limit its performance for this purpose.

1. Exploratory cox1 analyses
unveil operational biases and
blurry species boundaries
within Nematoda
Leonardo Tresoldi Gonçalves¹*
Filipe Michels Bianchi¹
Cláudia Calegaro-Marques¹
Maríndia Deprá¹
¹Universidade Federal do Rio Grande do Sul,
Porto Alegre, RS, Brazil
*tresoldigoncalves@gmail.comPicture credit:
Quickcrop

2. Nematodes are
traditionally identified
with morphology,
but this approach is
often complex and
innefficient.
But is DNA barcoding
effective to identify
nematodes?
Can cox1 aid in
nematode species
delimitation?
DNA barcoding uses a
standard mitochondrial
marker (cox1) to
improve organism
identification.
Detecting and
describing cryptic
nematode diversity
demands great effort.
Introduction

3. 2,928 cox1 sequences of seven
nematode genera were obtained:
Anguillicola
Caenorhabditis
Heterodera
Meloidogyne
Onchocerca
Strongyloides
Trichinella
Total of 111 species labels.
Methods
Pairwise comparisons (p-distance)
Sorted into inter- and intraspecific bins
Visualized in histograms
Putative species delimitation
Simple distance, default parameters
Identification success analysis
Scatter plot considering maximum
intraspecific distance and nearest-
neighbor distance
Alignment and quality control of sequences
Following Kvist (2016)
GenBank
Kvist (2016) - doi: 10.3109/19401736.2014.984166

4. Figure 1
Histograms with intraspecific distances (black) and interspecific distances (grey) for three genera,
illustrating the barcoding gap patterns detected. A. Caenorhabditis, with a consistent barcoding gap; B.
Anguillicola, with a barcoding gap flanked by atypical distances; C. Heterodera, without barcoding gap.
Results & Discussion
We found three barcoding gap patterns:
A. 'True' barcoding gap
A well-defined gap placed between the distribution
of intra- and interspecific distances. Detected in
Caenorhabditis and Trichinella.
B. Tendency to a barcoding gap
When atypical intra- and interspecific distances flank
the gap. Detected in Anguillicola and Onchocerca.
C. No barcoding gap
When intra- and interspecific distances consistently
overlap. Detected in Heterodera, Meloidogyne, and
Strongyloides.
Picture credit:
The Independent

5. Results & Discussion
Figure 2
Maximum intraspecific distance compared with nearest-neighbor
distance for 111 nematode species. One-third of the species (66.6%)
fall above the 1:1 line, which indicates identification success.
Cox1 showed efficiency for nematode identification to some
extent, but incorrect labeling of database sequences may hinder
its performance.
Table 1
Result summary, including number of species labels according GenBank, number of putative species
estimated by ABGD and statistics of the barcoding gap for each genus.
For most genera, ABGD delimits more putative species than the
number reported by GenBank. This indicates potential specimen
or sequence misidentification and/or cryptic diversity.