- A recent study in West Bengal used DNA barcoding and traditional methods of species identification to document fish diversity in the Purba Midnapore district.
- Ambiguities in species identities when determined either through DNA barcoding or traditional morphometry-based methods, highlight challenges in the taxonomic classification of these species.
- To avoid such ambiguities and accurately identify species, an integrative approach to taxonomy is important.
Waiting at the check-out as the cashier scans your weekly grocery haul is routine for many of us. Scanning an array of black and white lines to confirm whether it’s a packet of bread you’ve chosen, or a bag of chips, is done without missing a beat. But instead of scanning to identify a product in a grocery store, what if you could use such a scanning system to identify one of the 1.2 million described species on Earth?
Turns out, we may be able to do just that. DNA barcoding can use short genetic sequences from a standardised part of an organism’s genome to identify a species. It’s somewhat like a genetic fingerprint—unique to each species.
Researchers in West Bengal recently used DNA barcoding to document fish biodiversity in the coastal region of the Purba Midnapore district. The study, which also included traditional morphometry-based methods of species identification, revealed ambiguities in the current understanding of species classification in fish.
Results from a mixed approach
Traditionally, scientists use morphological classification, where body shape, size, structure, patterns, and other physical traits are documented, to identify and classify species. In this study, body length, body shape, fin structure, scale type, and colour patterns of the fishes were used to classify them into 19 species from 17 genera, 12 families, and 6 orders—revealing the rich biodiversity of the coastal areas near the Sunderbans.
However, the results of the DNA barcoding analysis brought up some questions. Although 19 distinct fish species were identified via DNA barcoding, only 70% of the samples identified as a particular species matched the morphological identification data available for that species. The species identities of 30% of the collected fish were ambiguous.
In addition to showing the correct species name identified through morphological traits, the DNA barcoding results also showed alternative species matches for some cases. For example, one fish morphologically identified as Salmostoma sardinella, was genetically very similar to two other species—Salmophasia phulo and Salmostoma sladoni—based on DNA barcoding with sample reference sequences. Another sample, initially identified as Apocryptus bato through morphological traits, showed a 99.6% genetic similarity with Scartelaos histophorus in the GenBank Core Nucleotide database. Similarly, a sample identified morphologically as Planiliza macrolepis showed very high genetic similarity with Paramugil parmatus.
“Morphological characteristics can be difficult to describe unless you are a good taxonomist. And using only molecular identification can result in ambiguities in species identification. So, combining both morphological and molecular techniques can help us be more accurate in identifying a species,” explains Barsha Sarkar, a research student at the University of Kalyani, West Bengal, and one of the authors of the study.
Limitations in traditional methods
Classification solely using physical traits has its limits. Some species, although genetically different, may look nearly identical to human eyes. These are known as cryptic species, a loose term used to describe “species that may be superficially similar but can be shown to differ in many characters after careful examination of their morphology,” explains Ishan Agarwal, an independent taxonomist from the Thackeray Wildlife Foundation. “In some cases, cryptic species may have different juvenile colouration but adults are indistinguishable; others may be very diverse in the pheromones they produce or in their calls,” he adds.
For example, mouse lemurs were once believed to be a single species and were dubbed with the scientific name Microcebus murinus. It was later revealed through DNA barcoding that the umbrella term ‘mouse lemur’ could refer to any one of 25 distinct species which are almost physically indistinguishable from each other. Agarwal comments that the strengths of DNA barcoding shines through in cases like this. “Things can look identical at first glance. But once you sequence their DNA, you find that they’re genetically actually quite divergent. And if you look closely at the morphology, you’ll find that there could be something that differs between them.”
Such cases highlight the value of a mixed approach to taxonomic classification—one that considers both morphology and molecular genetics.
Exploring reasons for genetic similarities
To further understand the evolutionary relationship between the different fish species in the coastal areas near the Sunderbans, the researchers also constructed a phylogenetic tree using a modelling software. Phylogenetics is the study of evolutionary relationships between different organisms, and a phylogenetic tree is a visual representation of how different species may have diverged from a common ancestor.
To construct the tree, the 20 DNA samples collected from the Sundarbans were compared with 101 similar reference samples collected from the National Centre for Biotechnology Information database. The findings revealed that in most cases, species from the same genus were grouped together in monophyletic groups, i.e., groups that include a common ancestor and all its descendants.
However, in some cases, species identified through physical characteristics from different genera were found in the same monophyletic group, suggesting that these fish species might be very closely related to each other. One case in particular stood out: the species Salmostoma sardinella and Salmophasia phulo, showed high genetic similarity despite being from two different genera.
However, existing literature indicates that Salmophasia is a junior synonym of Salmostoma — an earlier name for what is now recognised as the same genus. Ishan Agarwal, after reviewing the referenced literature, confirmed that Salmophasia is considered a junior synonym of Salmostoma under current taxonomic conventions.
In other cases where genetically similar species were identified, the authors provide several reasons. One is genetic polymorphism, or the occurrence of two or more variants in the DNA sequence of a species. Another reason could be incomplete lineage sorting, which occurs when species that share a common ancestor still carry overlapping genetic variants because there hasn’t been enough time for those differences to become distinct. The last reason could be hybridisation events, when two individuals from different species are able to breed.
“The sample size needs to be much larger. If a study with a larger sample size arrives at the same results, it can be said that the morphological taxonomy needs to be changed,” notes Sarkar, when asked to comment about the implications of these results on current taxonomic classifications. Beyond sample size, the authors state that more genetic markers are needed to confirm the results. They also point to potential data limitations with the genetic databases, which could affect the accuracy of classification.
Agarwal suggests that the observed genetic similarity might stem from errors in the existing reference DNA sequences: “It is more likely to be because of the reference DNA sequences being initially misidentified in the first place,” he says.
“GenBank IDs can sometimes be wrong. If you ever find something like that [referring to high genetic similarity], that’s the first thing you’ll try and rule out,” he explains, referring to barcoding work that may not have used morphological confirmations to establish species identity before submitting the sequences to genetics databases. However, Agarwal points out that the study did not explore this possibility further.
When asked whether these findings could impact current taxonomic classifications, he says, “If the possibility of incorrect reference DNA sequences is ruled out, and the suggested and identified species have overlapping distributions, then the classification may need to be re-evaluated; but this will have to be done with additional genetic markers to support the results.”
Is integrative taxonomy the way forward?
The future of taxonomy may ultimately lie in an integrative approach to species identification and classification known as integrative taxonomy. This approach makes use of various methods of identification such as physical traits, DNA analysis, behaviour, and even the types of environments species are adapted to, to arrive at an accurate taxonomic classification.
Despite having 1.2 million described species in the world, scientists estimate that more than 85% of species are yet to be identified and described. In a country as biodiverse as India, this gap highlights the need for better tools and strategies for species identification.
Agarwal, and Jahnavi Joshi, a senior scientist in the Centre of Cellular and Molecular Biology for the Council of Scientific and Industrial Research, suggest a national strategy for taxonomy and systematics research, in their joint paper on integrative taxonomy. Their proposed strategy includes 4 key areas: creating a national repository of voucher specimens, establishing a best-practices framework for taxonomic studies which is based on integrative taxonomy, improving education and training in taxonomy, and investing more into research and infrastructure.
This push for an integrative approach in taxonomy reflects broader global trends. Revisiting the case of the mouse lemurs, a recent study proposed reducing the officially recognised species count from 25 species to 19 species, after finding minimal differences in some of the species’ morphology, reproductive traits, climatic niches, and acoustic calls—reinforcing the need for multiple lines of evidence, along with DNA barcoding, for classification.
To improve consistency and reliability in DNA barcoding, the Ocean Biomolecular Observing Network program recently released a best-practices guide for creating and standardising DNA reference barcode sequences to genetics databases, particularly for marine species. The guide aims to support reliable monitoring of ocean biodiversity monitoring.
Sarkar, too, sees strong potential in DNA barcoding for documenting marine biodiversity along West Bengal’s coast. “I think molecular identification needs to be pursued vigorously,” she says. “It needs to be done in biodiversity-rich areas, in marketplaces—everywhere. This exercise of identifying species will benefit many sectors, especially future fisheries management.”
Read more: [Explainer] What is molecular ecology and how does it help in conservation?
Banner image: A river in East Midnapore, a coastal area where researchers collected fish specimens for DNA barcoding. Image by Biswarup Ganguly via Wikimedia Commons (CC BY-SA 4.0).
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