Rare DNA Haplotypes Hint at Hidden Routes in the Blue Crab’s European Invasion

A four-year genetic monitoring study of the invasive blue crab Callinectes sapidus in the Guadalquivir Estuary, in the Gulf of Cadiz on Spain’s southwest coast, has uncovered two mitochondrial DNA haplotypes never before reported in the region, complicating what scientists thought was a fairly tidy story of how this species conquered European waters. The finding suggests that the crab’s invasion history in the eastern Atlantic may involve not only a well-documented westward spread from the Mediterranean, but also possible independent introductions from the American continent, the species’ native range.

The blue crab is native to the Atlantic coast of the Americas and was first recorded in Europe in the early 1900s, becoming established across the eastern Mediterranean during the 1950s. Because the species was initially found near ports and has a long-lived planktonic larval stage, ballast water discharge from ships has long been considered the principal route of introduction to Europe. There is also evidence, however, of deliberate introductions for aquaculture along the coasts of Turkey, where the crab has had the longest invasion history in the Mediterranean and where higher haplotype richness and genetic divergence have been recorded compared with other Mediterranean populations.

To reconstruct invasion routes, researchers rely on mitochondrial molecular markers, most commonly the COI barcoding gene, to compare the genetic makeup of introduced populations with that of candidate source populations. In its native range, the blue crab shows substantial genetic structure along the American Atlantic coast, with 174 known haplotypes grouped into three lineages arranged by latitude: one covering North America and the Gulf of Mexico, a second in the Caribbean, and a third in South America. European populations, by contrast, had previously shown a strong founder effect within the North American lineage, with only 12 haplotypes detected and two of them, CSWM1 and CSWM2, overwhelmingly dominant across the Atlantic and western Mediterranean. The other ten haplotypes were specific to Turkey, in the Aegean and Black Seas, and differ by two to ten mutations from the North American dominant haplotype and from CSWM1 and CSWM2.

That founder effect has been interpreted as the signature of a stepwise westward expansion from eastern Mediterranean source populations. Supporting this scenario, an earlier study detected an inversion in the relative frequencies of the two dominant haplotypes on either side of the Strait of Gibraltar, a pattern attributed to oceanographic barriers such as the Almeria-Oran front. In the Iberian Peninsula, the blue crab was first recorded in 1967 in the Tagus Estuary in Portugal, followed only by sporadic records, but established populations took hold from 2012 in the Ebro Delta, and from 2017 the species expanded into the Atlantic Ocean, reaching the Gulf of Cadiz.

Recent work has begun to complicate this picture. Elevated haplotype diversity has been reported along the Sicilian coast, with 28 haplotypes specific to that area, and along the Atlantic and Mediterranean coasts of Morocco, where 24 haplotypes were documented. While part of this diversity has been linked to functional adaptation and localised genetic diversification, the dominant haplotypes consistently show affinities with lineage 1, reinforcing North America as the principal source of introduction in Europe. Two Moroccan haplotypes were interpreted as evidence of independent introduction events from the North American lineage into the eastern Atlantic, which is geographically plausible given the region’s position between the native and invaded ranges. However, incomplete matching between newly detected and reference haplotypes has limited comparability among studies, leaving open the question of whether Iberian and North African Atlantic populations descend purely from Mediterranean colonisers or also carry variants from separate transatlantic introductions.

To address this, the new study characterised the genetic diversity of the blue crab population in the Guadalquivir Estuary, the first recorded population in the southwest Iberian Peninsula, over four years. The team obtained 100 mitochondrial COI sequences across six sampling events between 2021 and 2024: two events in 2021 with 15 and 12 individuals, two in 2022 with 35 and 11, one in 2023 with 6, and one in 2024 with 21. Sequences were compared against the GenBank and BOLD reference databases, and newly obtained sequences for the rarer haplotypes were deposited in GenBank under accession numbers PV382972 and PV382973. Haplotype richness and frequencies were estimated for the whole dataset and per sampling event, and a Pearson’s chi-squared test of independence, with p-values estimated by Monte Carlo simulation using 10,000 replicates because of the low expected frequencies of rare haplotypes, was used to test whether haplotype composition differed among sampling events.

The results confirmed the expected dominance of the two widespread haplotypes, with CSWM1 accounting for 66 percent and CSWM2 for 32 percent of the sequences across the whole study, and no statistically significant difference in haplotype composition among the six sampling events (chi-squared = 21.909; p = 0.1341). In most sampling events CSWM1 was the more common of the two, although in the second event, in 2021, its frequency of 42 percent was slightly below that of CSWM2 at 58 percent. This dominance pattern, together with the inversion of the two haplotypes’ relative frequencies compared with western Mediterranean populations, is consistent with the hypothesis that the Atlantic populations of the Gulf of Cadiz originated from Mediterranean ones, a westward expansion that has been associated with larval dispersal, adult swimming and human-mediated transport via maritime traffic.

The surprise lay in the remaining two percent. Two rare haplotypes, each detected at a total frequency below one percent across the study, appeared at specific times: a rare haplotype designated RH1 was found in the first sampling event in 2021 at a frequency of 7 percent within that event, and a second rare haplotype, matching haplotype H18, appeared in the fourth sampling event in 2022 at 9 percent within that event. RH1 matched 100 percent with 13 sequences from blue crab megalopae, the final larval stage, collected in the Gulf of Mexico, specifically from Grand Isle in Louisiana and Apalachicola in Florida. The second haplotype matched 100 percent with H18, a lineage 1 haplotype previously reported only from Brazil, from a specimen collected in the Estuário de São Vicente in São Paulo, although two other Brazilian haplotypes also belong to lineage 1 as exceptions to the latitudinal lineage arrangement.

The authors argue that the presence of these rare haplotypes from the beginning of the monitoring period, and their persistence at low frequencies, suggests they were introduced during the early stages of the invasion rather than representing recent arrivals. One possibility is that they travelled alongside the dominant haplotypes from the Mediterranean. A recent study in Tunisia detected CSWM1, CSWM2 and a third rare haplotype closely related to one of the rare haplotypes found in the Guadalquivir Estuary, sharing its phylogenetic origin and showing 99 percent similarity to sequences from Gulf of Mexico megalopae collected in Pensacola, Florida and Galveston, Texas. The small sample size in the Tunisian study precludes robust conclusions about frequencies, but the finding raises the prospect that such American-derived variants have been present but largely undetected in Mediterranean populations, given that genetic surveys of the blue crab across the Mediterranean have typically used relatively small sample sizes that may have missed low-frequency haplotypes.

At the same time, the study does not rule out alternative introduction pathways directly from the American continent. The detection of a Gulf of Mexico haplotype and a Brazilian haplotype, both belonging to lineage 1, raises the possibility that independent introductions from both North and South America contributed to the genetic diversity of populations in the southwestern Iberian Peninsula, echoing the independent introduction events recently proposed for the Atlantic Moroccan coast. The authors note that their results are consistent with previously observed patterns of genetic flow between the two basins, from west to east for an invasive fish species associated with the Azores current and from east to west for an invasive crab species associated with the weaker Alboran counter-current, but that their results do not provide direct evidence of these ocean-current-mediated patterns. Whether the Atlantic Iberian and North African populations result solely from stepwise Mediterranean expansion or also incorporate independently introduced variants remains an open question. What the four-year dataset does establish is that the Guadalquivir Estuary population is genetically stable over time, dominated by the two classic founder haplotypes, yet harbouring a small reservoir of genetic diversity that hints at a more tangled invasion history than the simple westward march from the Mediterranean once suggested.

Subject of Research: Genetic tracing of the introduction pathways of the invasive blue crab Callinectes sapidus in European Atlantic waters

Article Title: New insights into the potential introduction pathways of Callinectes sapidus Rathbun, 1896 ( Decapoda , Portunidae ) in European waters

Article References: Ortega Jimenez, E., González Ortegón, E., & Cuesta, J. A. (2026). New insights into the potential introduction pathways of Callinectes sapidus Rathbun, 1896 (Decapoda, Portunidae) in European waters. Biodiversity Data Journal, 14, Article e215033. https://doi.org/10.3897/bdj.14.e215033

Image Credits: AI Generated

DOI: 10.3897/BDJ.14.e215033

Keywords: blue crab, Callinectes sapidus, invasive species, mitochondrial DNA, COI haplotypes, Guadalquivir Estuary, Gulf of Cadiz, founder effect, ballast water, Mediterranean, introduction pathways, population genetics