Strengthening marine amphipod DNA barcode libraries for environmental monitoring
C. PrasannaKumar, G. Manikantan, J. Vijaylaxmi, B. Gunalan, S. Manokaran, S. R. Pugazhvendan
Abstract
Environmental DNA barcoding technology is gaining innovative applications. The effectiveness of current DNA barcode reference libraries in identifying amphipod barcodes and/or strengthening the existing library was tested. From 2500 amphipod individuals we barcoded 22 amphipod species belonging to 17 genera, 13 families among which 13 species were first time barcoded. More than 80 percent of the species were new distributional records. The minimum and maximum inter-specific pair-wise distance values was respectively 0.16 and 5.51 percent. Defining family specific species threshold values would be imperative, rather than expecting a universal barcode gap for amphipod species. The overall mean pair-wise distance, nucleotide diversity and Tajimas statistics were 3.59 percent, 0.27 and 2.62, respectively. There is a strong need to increase the number of amphipod species barcodes in the reference database. For better facilitation of environmental monitoring, the datasets could be exclusively accessed at BOLD through http://dx.doi.org/10.5883/DS-MAOI.
02 /Specification & Classification
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Claude Opus 5.5 · xhigh effort · 8 Nov 2026 · from the full text of version 4 (15 Oct 2020) · CC BY 4.0
COI barcodes for 22 amphipod species from Indian mangrove sediments, 13 barcoded for the first time, reveal gaps in GenBank and BOLD for environmental DNA monitoring.
Environmental DNA (eDNA) surveys need reference libraries, yet only 15 percent of animal species are barcoded and amphipod barcoding is rare in the Indian Ocean, where amphipod diversity is relatively rich. This work adds a vouchered, image-linked COI library for estuarine amphipods, a sediment bio-indicator group that morphology struggles to classify.
Key results
ShownThe authors built a COI library covering 22 estuarine amphipod species (17 genera, 13 families). For 13 of these species no barcode existed before, which shows that GenBank and BOLD cover this group poorly.
2869 individuals were sorted to 22 species (the abstract says 2500). Folmer primers amplified all 22. 13 species were barcoded for the first time (reported as 59% of sequences), and BOLD returned 'no match' for them. GenBank held no Isaeidae COI barcodes before this study. · Abstract; 3.1. Species composition and history of its…; 3.2. Sequence analysis and species…; 4. Conclusion
SuggestedDistances between species (K2P model) were small compared with published amphipod thresholds. The authors therefore argue for species cutoffs set per family rather than one universal barcode gap.
Minimum 0.16% (Isaea elmhirsti vs I. montagui), maximum 5.51% (Victoriopisa chilkensis vs Orchestia platensis), overall mean 3.59%. Canadian amphipods: 0.6% and 18.07%; deep-sea species: 13%. A 16% threshold and a proposed 26.8% Gammaridae value exceed every observed distance. · Abstract; 3.2. Sequence analysis and species…
SuggestedIn neighbour-joining trees, the authors report that study barcodes grouped with GenBank references of the same species, which supports tree-based identification, although first-time species had no references. Members of Gammaridae, Corophiidae and the genus Ampithoe did not cluster together.
22 study sequences clustered with 24 GenBank reference sequences of matching species. Over 90% of branches had bootstrap support above 75. The tree had 3 major clades, the largest holding 14 genera. Members of Gammaridae and Corophiidae were split, while Isaeidae formed one cluster. · 3.3. Tree based identification; Figure legends
SuggestedMost species were new records for the Vellar estuary. They include taxa previously known from distant regions or reported as invasive elsewhere, such as Elasmopus rapax, Melita nitida and Platorchestia platensis.
Only 4 of 22 species (Ampelisca scabripes, Grandidierella sp., Orchestia sp., Talorchestia sp.) had earlier Vellar estuary reports. More than 81 percent were new site records (the abstract says more than 80 percent), and 18 of 22 species were not recognized in earlier surveys. · Abstract; 3.1. Species composition and history of its…; 3.2. Sequence analysis and species…
ShownThe COI sequences had no stop codons. GC content was higher than reported for Arctic amphipods, and the share of conserved sites was higher than in Canadian amphipods.
Mean GC 39.25±0.93% vs 32.9% in Arctic amphipods. Nucleotide diversity 0.27; Tajima's statistic 2.62. 34.86% conserved and 58.2% parsimony-informative sites vs 23.5% and 69.25% in Canadian amphipods. · Abstract; 3.2. Sequence analysis and species…
Methods, data and reproducibility
Amphipods from mangrove sediment were sorted and identified by morphology. A ~658 bp fragment of the mitochondrial COI gene was amplified with Folmer primers and Sanger sequenced in both directions. Sequences were queried against BOLD and GenBank and analysed in MEGA X (K2P distances, diversity statistics, neighbour-joining trees).
- Mangrove sediment samples collected around Rhizopora annamalayana in the Vellar estuary, Southeast coast of India, in February 2012, sieved through 0.5mm mesh · n = 6 samples
- Amphipod specimens sorted and identified by morphology to 22 species, 17 genera and 13 families · amphipods (Malacostraca: Amphipoda) · n = 2869 individuals (abstract: 2500) · BOLD project DBMA
- COI barcode sequences (~658 bp) generated in this study, with specimen images and voucher metadata · amphipods (Amphipoda, 22 species) · n = 22 sequences · GenBank MT184213-MT184234; BOLD dataset 10.5883/DS-MAOI
- GenBank reference COI sequences retrieved by BLAST for tree-based identification · amphipods (Amphipoda) · n = 24 reference sequences
- Methods & tools
- DNeasy Blood & Tissue Kit (Qiagen), LCO1490/HCO2198 COI primers, Bidirectional Sanger sequencing (Macrogen), ChromasLite ver.2.1, BioEdit ver. 7.9, Clustal X ver. 2.0.6, MEGA X (K2P distances, NJ tree, nucleotide diversity, Tajima's test), BOLD identification engine, BLAST against GenBank, iTOL (Interactive Tree Of Life), Taxonomic keys via World Amphipoda Database, Nikon Eclipse E200 compound microscope
- Data & accessions
- http://dx.doi.org/10.5883/DS-MAOIhttp://www.boldsystems.org/
- Reagents & environment
- ChromasLite ver.2.1; BioEdit ver. 7.9; Clustal X ver. 2.0.6; MEGA version X (K2P model, all 3 codon positions); iTOL (version not stated).
- Reproducibility gaps
- No analysis code or scripts released. Cryopreserved genetic material is available only on request. Voucher specimens are held at Annamalai University's Marine Museum (Centre of Advanced Study in Marine Biology).
Limitations (6)
- Relying on one mitochondrial gene can bias identification at several processing steps and lacks an established barcode gap. COI-like sequences may also artificially inflate species-threshold estimates. · stated by the authors
- Several taxa (Ampelisca sp., Dexamine sp., Isaea sp., Leptocheirus sp., Pectenogammarus sp.) could not be resolved to species by either morphology or COI barcodes. · stated by the authors
- The library is a small local resource (n=22 species) with limited taxonomic reach. The authors call it only a small step toward a comprehensive amphipod barcode library. · stated by the authors
- The causes of the many new local occurrences were not studied and are left for future work. Examples are Othomaera othonis in shallow mangrove sediment and the possible invasiveness of Elasmopus rapax. · stated by the authors
- Each species is represented by a single sequence (22 sequences for 22 species), so variation within species and the barcode gap were not measured. The 0.16% distance between Isaea elmhirsti and I. montagui was never compared with within-species divergence. · noted in this summary
- The report is inconsistent in places: 2500 vs 2869 individuals, a list of 'four' unresolved taxa that names more than four, and a claim that all 22 sequences clustered with references although first-time species had none. Sampling was limited to 6 samples from February 2012. · noted in this summary
Who should read:Read this if you build or use COI reference libraries for eDNA monitoring of estuarine invertebrates, especially Indian Ocean amphipods, or are weighing barcode thresholds set per family.
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