Taxonomy, the science of categorizing organisms, has historically relied on observable characteristics. The scientific practice first began in the 18th century, and used observations on the physical traits of organisms to lay the discipline’s foundation. This traditional practice is called morphology.
In recent years, molecular biology tools have been applied to taxonomy. These techniques have revealed errors in morphology-based assumptions. Most modern taxonomic studies now combine morphologic and molecular approaches. With this in mind, it’s important for taxonomists to understand the molecular biology tools available across the spectrum of taxonomic research.
In this article, we will explain why taxonomic researchers have added molecular biology to their toolkit. Then, we discuss six different molecular biology techniques and instances where they’ve been used in recent taxonomic studies.
Morphology Alone Isn’t the Answer
Even without subjective differences between researchers and a scarcity of experts, morphological taxonomy encounters obstacles. Many species that appear similar often have complex genetic differences. These distinctions aren’t always easy to identify via observable characteristics. Morphology also has limited use for partial specimens or specimens at early life stages.
However, at the genetic level, tools can detect phenomena like:
Convergent evolution, when two unrelated species develop similar characteristics independently. An example of this is echolocation, which evolved separately in bats and whales. Similar observable traits can suggest an evolutionary relationship when there is none.
Cryptic species, which are genetically distinct from another species, but cannot be distinguished via morphological traits. African elephants made waves when researchers conducted genetic analysis, finding that savanna and forest elephants are actually distinct species. Until this project, many researchers argued that these animals should be classified as one species.
Hybridization and intergrading species, caused by existing species mixing and potentially interbreeding. In flowering plant species Packera crocata and Packera dimorphophylla, for example, plants share and mix many of the same characteristics, making morphological characterization difficult. It was only with next-generation sequencing that researchers were able to create a species tree (Fig. 1) identifying subspecies and refining the relationship between P. crocata and P. dimorphophylla.
Phenotypic plasticity, a phenomenon where the same genome can create multiple phenotypes based on environmental factors. These organisms may appear to be distinct species, but genetic analysis reveals them to be the same. Mosquito populations, for example, have significant phenotypic differences based on environmental factors. These delayed phenotypic variations are critical to predicting the severity of vector-borne disease outbreaks like dengue fever.
Dormant, degraded, or incomplete specimens. Morphology depends on having an observable specimen. Molecular biology tools, however, can obtain valuable genetic information, even when a whole specimen is unavailable. This can include samples extracted from feces, parts of whole organisms, decomposed specimens, and more.
Ancient DNA (aDNA) techniques can even help us understand the taxonomic context of significantly aged specimens and fossils. That was the case in this study on central European pre-Roman equid samples, where aDNA analysis combined with morphological analysis of bone specimens were used to refine historic taxonomic classification of these species.
Phylogeny, the study of evolutionary relationships between animals, is about more than outer appearance. This understanding has led to a major shift in how taxonomists work. A majority of taxonomic studies published today use molecular biology tools in conjunction with morphological analysis. This intersection of old school and new school techniques is called integrative taxonomy.
Fig. 1 : Two phylogenetic trees generated using molecular data for Packera species. The first tree used nrITS sequencing data with Bootstrap support. The second used NGS data with ASTRAL for estimations.
Tools Used in Integrative Taxonomy
Integrative taxonomy was initially controversial among taxonomic researchers, many of whom believed molecular technologies were not a sufficient replacement for traditional techniques. The current state of taxonomy combines morphological and biological analysis. Often, these techniques are used to tackle the difficult species relationships or conditions detailed above.
Some of these molecular analysis tools include:
1. DNA barcoding
With this technique, a small genetic region is compared to a reference for the purpose of identifying a species. A region of the COI gene was proposed as a basis for identifying all animal species. COI can be amplified from a wide variety of species. It often varies just enough between species to provide a distinction. As a mitochondrial gene, it provides insights into maternal inheritance. In many cases, the COI gene is used to better distinguish between species or to create reference datasets, such as this project on marine zooplankton.
Sanger sequencing is often the technology of choice for DNA barcoding, since the technology can provide the necessary accurate, small-scale sequence. In higher throughput or larger scale projects, some DNA barcoding is done with next-generation sequencing technologies. In this study on pet food mislabeling, for example, NGS-based barcoding was used to identify the species present in a wide survey of commercial dog foods. In several cases, this technique successfully identified species present in the food that were not listed on the label.
In plants, the COI gene is not a valid target for DNA barcoding. This region does not vary significantly between plant species. In many cases, plant identification requires the sequencing of more than one genetic region (a technique referred to as multilocus sequence typing or multilocus sequence analysis).
In this study on Indonesian medicinal plants, for example, researchers compared four barcoded regions of 61 plant species. This study generated 212 barcode regions, and identified new DNA regions that could be used for future plant studies.
2. Whole genome sequencing
In contrast to DNA barcoding that only analyzes a single gene or a group of genes, WGS covers every gene in the organism. In fungi particularly, taxonomy is challenging because of species’ complex lifecycles and ability to create multiple phenotypes in different circumstances.
A 2025 study on fungal species analyzed 18 Colletotrichum isolates, including species known to have a symbiotic relationship with ferns. WGS identified evolutionary links between species more effectively than multilocus sequence typing. This full-picture view reduced species misidentification and improved our understanding of the Colletotrichum genus.
3. Mitochondrial DNA sequencing
Mitochondrial DNA sequencing is also a common method used in taxonomy. This takes advantage of mitochondrial DNA’s maternal inheritance, rapid mutation, and lack of recombination, giving researchers a new lens through which to analyze evolutionary relationships.
In 2025, Psomagen customers used mitochondrial genome sequencing to characterize a group of small mesocarnivore species. This included olingo, ringtail, coati, and raccoon species. Six species’ mitochondrial genomes were assembled and used to create a probable phylogenetic tree. This categorization grouped species much differently than traditional morphological taxonomic analysis.
In plants, researchers sometimes compile the chloroplast organelle’s genome for similar reasons. Recent research on a genus of temperate trees and shrubs, for example, compiled Karpatiosorbus bristoliensis’s plastome and mitogenome. Analysis solidified the plant’s relationship to a genus of rose plants.
4. Transcriptomics
RNA sequencing provides insights into expressed and unexpressed genes, which can offer valuable evolutionary insights in taxonomic studies. RNA data can also be informative for hybrid species studies, when DNA may be very similar, but regulated differently in the transcriptome. Transcriptomics was used in this study on new generations of sea buckthorn hybrids, confirming that H. goniocarpa is a hybrid and not a distinct species.
Animals and plants not commonly used as model organisms often have limited genomic and transcriptomic data available. Crustaceans, for example, are underrepresented in publicly available datasets despite their diversity and universality. A 2023 research project provided transcriptomic data for 189 crustaceans, 30 of which were never before published. This CrusTome mRNA transcriptome dataset is searchable for similarities between species, and has already been applied to phylogenetic analyses.
5. Metagenomics
Metagenomics is used in taxonomy for identifying and categorizing microorganisms. Shotgun metagenomics is a valuable option when there are appropriate reference datasets available, like this analysis of soil invertebrates.
However, full metagenome assembly can be necessary for identifying unresearched microbes without applicable reference datasets. Infant and child gut microbiome studies, for example, are hampered by reference datasets typically sourced from adult samples. Assembling a microbiome dataset from infant to 7-year-old child microbiome samples profiled thousands of unique microbial species. Lower abundance of 54 species was then associated with preterm infant microbiomes, and were associated with childhood allergies.
6. Epigenetics
Epigenetics can be used to analyze genetic expression changes not caused by changes to the underlying DNA sequence. Epigenetic techniques have been used with success in cancer taxonomy, like in this study that conducted epigenetic testing on 100 primary prostate carcinomas. Results indicated three subtypes of prostate cancer tightly regulated by the Androgen Receptor transcription factor.
As the field of taxonomy evolves, molecular biology tools have become essential for uncovering relationships that morphology alone cannot resolve. Whether it's distinguishing cryptic species, analyzing hybrid lineages, or identifying organisms from degraded or environmental samples, molecular techniques provide precision, scalability, and insight that were once out of reach.
While morphology remains a valuable piece of the taxonomic puzzle, especially for field identification and historical continuity, it is the integration of genomic, transcriptomic, and metagenomic data that now drives the most accurate and robust species classification. Together, these tools are not just reshaping taxonomy—they are redefining how we understand biodiversity.
