DNA Reveals Hidden Amphipod Species That Look Exactly Alike

Deep in the cold waters of the North Atlantic and the Southern Ocean, tiny shrimp-like crustaceans are quietly defying the tools scientists use to tell species apart.

Liljeborgia is no obscure side branch of the crustacean tree. The family Liljeborgiidae contains 124 species, and Liljeborgia alone accounts for 79 of them, making it the richest genus in the family. Its members live everywhere from shallow coastal zones to abyssal depths exceeding 6,000 metres, with 12 species currently known from the North Atlantic, 25 from the Southern Ocean, 29 from the Pacific Ocean, and smaller numbers from the Arctic, Indian and South Atlantic Oceans. Yet the animals are notoriously hard to identify, distinguishable by only a very small number of characters, which is precisely why the researchers turned to genetics.

The COI alignment ultimately contained 155 sequences, 126 from the new study and the rest from NCBI GenBank and BOLD, each 585 base pairs long, while the 28S alignment included 35 sequences totalling 1,284 base pairs. Using the automatic species delimitation method ASAP, the best-scoring partition suggested a threshold distance of 14.2 percent and split the data into 17 putative Liljeborgia species, while a second partition at a 3.5 percent threshold suggested 24 species.

The two lineages are separated by COI distances of 7.4 to 9.1 percent, squarely within the range typically seen between amphipod species, and the nuclear 28S marker independently supported the split. Crucially, the two lineages occur around Iceland in a sympatric but apparently not syntopic distribution, meaning they share the same region but were never caught at the same station. Together, the genetic divergence and overlapping geography strongly support their status as separate, reproductively isolated species.

Here the study takes a twist worthy of a detective novel. Even detailed morphological analysis, including confocal laser scanning microscopy of mouthparts, antennae, gnathopods, pereopods, uropods and the telson, failed to reveal any obvious distinguishing traits between the two L. ossiani species. The researchers dissected one female from each lineage and documented the structures in high-resolution images, and while many characters varied within each species, differences between them were minimal to nonexistent. With 20 specimens of sp. 1 but only three of sp. 2, the authors caution that the variability of the rarer lineage might expand with larger samples, so they declined to formally describe sp. 2 as a new species for now.

The second case study delivered the opposite lesson. Liljeborgia charybdis and its forma caliginis had originally been described as distinct species based on two characters: the presence of a posterodorsal tooth on pleonite 3 in L. charybdis, and the presence of roughly five teeth on the dactylus of gnathopod 1 in forma caliginis, where L. charybdis has none. The new genetic data show that specimens of both morphotypes share identical COI and 28S sequences, and several individuals exhibit intermediate forms. The toothed individuals were all females measuring 4 to 10 millimetres, some apparently juvenile, while the toothless individuals were adults of 20 to 30 millimetres. The largest toothed individual had the smallest posterodorsal tooth and three dactylus teeth, an intermediate condition. The conclusion: with increasing age, the dorsoposterior robust seta of pleonite 3 is lost and the dactylus gains teeth, so the diagnostic characters change during ontogeny, and the two names refer to a single species.

This ontogenetic morphological change has real taxonomic consequences. The authors formally support the synonymy of L. caliginis with L. charybdis, a merger first proposed by d’Udekem d’Acoz in 2010, and they suggest a functional explanation: liljeborgiids are assumed to be micro-predators and/or detritus feeders, so the increasing number of robust setae on the first gnathopod’s dactylus may reflect changes in predation or feeding habits as the animal matures. For taxonomists, the message is sobering. Morphological stasis during speciation on one hand and dramatic change during an individual’s lifetime on the other make identification based on external anatomy alone genuinely treacherous in this genus.

The Southern Ocean told a different story. In Liljeborgia georgiana, the finer ASAP partition split the material into seven putative species, each recorded from only a single locality around the Weddell Sea or the South Atlantic, with each station yielding only one of the putative species, at depths from 226 to 969 metres. Pairwise COI distances between these putative species mostly matched or exceeded those between the two L. ossiani species, though one pair, sp. 5 and sp. 6, showed lower distances of about 4 percent. However, the nuclear 28S sequences of L. georgiana were nearly identical and failed to delimit any of the seven putative species, leaving it impossible to assess whether they are reproductively isolated. The authors conclude it may be a complex of several closely related cryptic species, a single genetically diverse species, or a mixture of both, and that further populations from across the species’ reported range, from South Georgia to the Ross Sea, will be needed to resolve the question.

Biogeographically, the two regions contrasted sharply. The North Atlantic species, L. brevicornis, L. charybdis and the two L. ossiani lineages, were found at several sites with relatively wide distributions around Iceland and extending toward Norway along the Greenland-Iceland-Faroer Ridge, with genetic diversity rather evenly spread and no clear association with geographic origin. L. charybdis and forma caliginis were collected north of Iceland and along the Norwegian coast at depths of 2,203 to 3,702 metres, often occurring syntopically at the same station. Depth is a known driver of differentiation in benthic amphipods and isopods and may have played a role here. In the Southern Ocean, by contrast, the strong geographic patterning of genetic diversity, with each putative L. georgiana species confined to one locality, contrasts starkly with the North Atlantic, and the authors speculate that fluctuating ice shields or pack ice accumulating in the Weddell Sea may act as barriers, while stressing that this explanation remains speculative.

The study also flags a practical hazard for biodiversity research: several sequences from public repositories, particularly those labelled L. fissicornis, were probably misidentified, grouping instead with L. charybdis or L. ossiani sp. 1, and such errors can confound studies of species distributions and diversity. None of the studied Liljeborgia species was recorded from more than one ocean, consistent with the limited dispersal expected of peracarids, whose females brood their young in a marsupium and lack a free-swimming larval stage. The authors also note intriguing hints of further cryptic diversity within L. brevicornis and L. charybdis, but too few specimens to draw firm conclusions. Overall, the work is a clear demonstration of why integrative taxonomy, combining mitochondrial and nuclear markers with morphology, remains the most reliable way to draw species boundaries in a genus where the animals themselves refuse to cooperate with the keys.

Subject of Research: Molecular and morphological diversity and distribution of the amphipod genus Liljeborgia in the North Atlantic and Southern Ocean

Article Title: New insights into the molecular and morphological diversity and distribution of Liljeborgia in the North Atlantic and Southern Ocean ( Amphipoda , Amphilochidea , Liljeborgiidae )

Article References: Schwentner, M., Timm, R., d’Udekem d’Acoz, C., & Lörz, A.-N. (2026). New insights into the molecular and morphological diversity and distribution of Liljeborgia in the North Atlantic and Southern Ocean (Amphipoda, Amphilochidea, Liljeborgiidae). Annals of the Natural History Museum Vienna, 127, 211–231. https://doi.org/10.3897/anhmw.186461

Image Credits: AI Generated

DOI: 10.3897/anhmw.186461

Keywords: amphipods, Liljeborgia, cryptic species, DNA barcoding, COI, deep sea, taxonomy, North Atlantic, Southern Ocean, integrative taxonomy, ontogeny, Iceland