Mitochondrial diversity in Gonionemus (Trachylina:Hydrozoa) and its implications for understanding the origins of clinging jellyfish in the Northwest Atlantic Ocean

Sample collection

Atlantic and Pacific medusae were collected by snorkeling or by net from boats or floating docks, or from colleagues (Table 1). We refer to locations in the Northwest Atlantic as NWA, Northeast Atlantic as NEA, Northwest Pacific as NWP, and Northeast Pacific as NEP. Medusae were either used live or preserved in ethanol for later DNA processing.

Molecular methods

For the mitochondrial DNA assessment, genomic DNA was extracted using DNEasy Blood & Tissue kits (Qiagen) following the manufacturer’s instructions. An approximately 600 base pair part of the mitochondrial COI gene was amplified using universal primers (Folmer et al., 1994) under the following conditions: 95° 3 min, 35 cycles of 95° for 30 s, 48° for 30 s, 72° for 1 min, and a final cycle of 72° for 5 min. PCR products were visualized on a 1.2% agarose gel stained with GelRed (Biotium) and purified using Qiaquick PCR purification kits (Qiagen). Products were quantified with a Nanodrop 2000 spectrophotometer (Nanodrop Technologies, Wilmington, Delaware, USA) and sequenced in both directions by MWG Eurofins Operon. Chromatograms were assembled and DNA sequences were analyzed using the Geneious R9 software platform (Kearse et al., 2012). Sequences were aligned using ClustalW (Larkin et al., 2007). The alignment was verified by eye, and the ends were trimmed so that all sequences were the same length. In order to facilitate direct comparisons with other published studies (e.g., Ortman et al., 2010; Zheng et al., 2014), mean Kimura 2-parameter (K2P) distances between locations were calculated with MEGA 7 (Kumar, Stecher & Tamura, 2016) and a neighbor-joining haplotype tree based on K2P distances was constructed using the PAUP* 4 (Swofford, 2003) plugin in Geneious. We confirmed that the K2P haplotype tree was consistent with a haplotype tree based on the best-fit model (GTR+I, as determined in ModelTest using the Akaike Information Criterion in Geneious; Posada & Crandall, 1998). DNA summary statistics were calculated with DnaSP 5.10.01 (Librado & Rozas, 2009).

Population delineation and taxonomy

Our results refine, rather than resolve, the Gonionemus zoogeographic puzzle. Pairwise distances between the NEP/NEA Gonionemus and the NWP/NWA Gonionemus are typical for interspecific, rather than intraspecific, comparisons (Ortman et al., 2010; Zheng et al., 2014) and thus suggest the possibility that the two groups may represent different species. Cnidarian lineages differ substantially in their mitochondrial substitution rates (Shearer et al., 2002; Govindarajan, Halanych & Cunningham, 2005; Wares, 2014). And, based on a relative rate test on COI in the closely related Maeotias marginata, Limnomedusae may exhibit relatively slow rates (Shearer et al., 2002). Thus, the difference observed in NEP/NEA and NWP/NWA forms could reflect especially deep divergences. It would be possible to calculate the substitution rate between the NEP/NEA and NWP/NWA forms if their divergence date could be reasonably estimated (e.g., from the opening of an Arctic passage, Wares & Cunningham, 2001; Govindarajan, Halanych & Cunningham, 2005); however, there is too much uncertainty at this point in the origin and dispersal history of Gonionemus as described below, to do this with confidence.

Species-level genetic divergence is corroborated by our incidental morphological observations, which are consistent with those from Russell (1953). A future taxonomic re-assessment that incorporates morphology and multiple genetic markers could suggest that the NWP/NWA form should be reclassified as G. murbachii Mayer 1901. Because G. murbachii was first described in the NWA, this line of reasoning would suggest that Gonionemus could actually be native to the NWA. However, the situation is complex as our findings also refute previous taxonomic thought and do not necessarily dismiss an invasion event. We also cannot rule out the possibility that none of the contemporary NWA lineages correspond to G. murbachii. For clarity, we will refer to the contemporary NWA/NWP form as “Gonionemus sp.” for the remainder of our discussion.

Naumov (1960) considered G. vertens and G. murbachii to be morphologically distinguishable subspecies. He described the Pacific form (including both NWP and NEP forms) as G. vertens vertens, and the Atlantic form as G. vertens murbachii. Naumov (1960) also noted that only the G. vertens vertens form is toxic to humans (although only in some Russian locations). Thus, while Naumov (1960) recognized two forms, as our results suggest, his delineation of those two forms differs substantially from ours. Naumov (1960) assumed each form was found in a different ocean, but our results show that both the vertens and Gonionemus sp. forms have disjunct distributions—with the vertens form in the NEP and NEA and the Gonionemus sp. form in the NWP and NWA. This is suggestive of anthropogenic dispersal in both forms. But again, the situation is complex, and possible explanations include both natural and anthropogenic causes. We now evaluate 4 possible invasion history scenarios in light of these results.

NWA invasion scenarios

Scenario 3—“Lineage admixture”

Here we also consider the NWA/NWP and NEP/NEA forms to be different species as in Scenario 2. The NWA and NWP forms are also different from each other, although they represent different lineages of a single species (G. murbachii or Gonionemus sp.) rather than different species. In this scenario we assume that the NWA contains at least 2 ostensibly native cryptic lineages (possibly segregated geographically, southern and northern New England; Fig. 3) that are distinct from each other and from the assumed native NWP lineages. There are also multiple NWP lineages (and likely more than what we observed with our limited sampling), only one of which is highly toxic (possibly represented by Haplotype IV, although mitochondrial haplotype should not be construed as equivalent to toxicity). The NWA experienced a single invasion (likely in the late 1980s) of the toxic NWP lineage, which is now interbreeding with the native NWA lineages. This hybridization may be facilitating NWA blooms and regional range expansion.

While a naturally disjunct distribution for G. murbachii/sp. seems unlikely, it could be explained by a trans-Arctic migration. Around 3 $\frac{1}{2}$ million years ago, a well-documented sea passage opened up connecting the north Pacific and the north Atlantic oceans (Vermeij, 1991). This passage enabled a large-scale migration of organisms, primarily in the direction of the North Pacific to the North Atlantic. Migrating seaweeds and seagrasses would likely have carried associated fauna such as Gonionemus. More recent (e.g., in the Pleistocene; Palumbi & Kessing, 1991) or earlier (e.g., Olsen et al., 2004) trans-Arctic migrations are also potentially possible. The presence of unique haplotypes separated by several substitutions in both the NWA and NWP suggests independently diverging populations consistent with this explanation. As described above, the mitochondrial substitution rate might be particularly slow in Limnomedusae so that differences represent deeper divergences consistent with an earlier migration. It is also possible that multiple trans-Arctic migrations have occurred—for example, the first, resulting in the NEP/NEA and NWP/NWA split, and a second, resulting in the different NWP and NWA lineages.

An alternative variant of the “Lineage Admixture” scenario is that G. murbachii/sp. is native to the NWP, where it went unnoticed until the 20th century, and was introduced multiple times into the NWA, beginning in the late 19th century. This variant seems less likely, given the number of steps between the two unique NWA haplotypes to each other and to the NWP haplotypes. However additional sampling is necessary to definitively rule this out, and importantly, this variant still involves lineage admixture which has evolutionary and ecological implications as described below.

Evolutionary processes influencing NWA Gonionemus

Multiple evolutionary processes can influence colonizing populations, including introduced populations. It is often assumed that colonizing populations harbor only a subset of the genetic diversity found in parent populations. As genetic diversity is thought to promote population persistence, it follows that the low diversity in founding populations would make it difficult for them to become established. The fact that many colonizing populations do become established despite their assumed low diversity is called the “genetic paradox”. Multiple inputs followed by lineage admixture may be a mechanism to overcome the “genetic paradox” (Kolbe et al., 2004; Dlugosch & Parker, 2008). In some cases, incoming populations may come from different source areas, and subsequent interbreeding could generate novel genetic combinations that also may help the nascent population become established (Kolbe et al., 2004).

Hybridization (or population admixture) may be an important mechanism leading to evolutionary change in NWA Gonionemus populations. This process could be occurring either between multiple anthropogenic inoculation events (e.g., Scenario 1) or between long-diverged intraspecific lineages (e.g., Scenarios 3 and 4). The resulting genetic changes could be promoting the prominent blooms and apparent rapid range expansion that has been observed since the 1990s. After the 1930s eelgrass dieoff, NWA Gonionemus was known primarily from a single pond on the island of Martha’s Vineyard, Massachusetts, and very occasional reports from the Gulf of Maine (Govindarajan & Carman, 2016). These populations, which were apparently marginal for decades, may have been re-invigorated when mixed with recent NWP individuals, which provided the genetic material to enhance the jellyfish’s fitness. Because our dataset is limited to a single mitochondrial marker, we cannot evaluate here if introgression or hybridization has occurred.

In contrast to the genetic paradox paradigm, the assumption that genetic diversity is low in colonizing populations may not hold true for many invading populations (e.g., Darling et al., 2008), and may be sufficient to allow for adaptive changes driven by natural selection (Koskinen, Haugen & Primmer, 2002; Wares, Hughes & Grosberg, 2005). Adaptive evolution could be responsible for differences in phenotype between invading and source populations in some species. Similarly, it is possible that contemporary NWA populations may be experiencing rapid adaptive evolution. However, detection of adaptation can be obscured by phenotypic plasticity (Tepolt, 2015; Krueger-Hadfield et al., 2016). Thus, future studies that use nuclear markers capable of finer-scale resolution and detection of hybridization, coupled with phenotypic characterization in Pacific and NWA regions, are necessary to assess both the lineage admixture and adaptive evolution hypotheses in Gonionemus.

The toxicity phenotype

The toxicity phenotype stands out with special importance due to the concerns about envenomations, and it may also play a role in the NWA range expansion. Gonionemus medusae are predators that consume a variety of zooplankton prey (Arai & Brinckmann-Voss, 1980), and so increased toxicity could improve prey capture or deter predators and lead to population growth. The current NWA toxicity phenotype appears more aggressive than in pre-1990 populations, as evidenced by the history of stings to humans (Govindarajan & Carman, 2016). This observation could be due to lineage admixture between contemporary and historical NWA populations, recent adaptation in contemporary NWA populations, and/or plasticity due to environmental conditions.

The contemporary NWA toxicity phenotype could be subtly different than the NWP toxicity phenotype. In the NWP, especially off of Vladivostok, Gonionemus stings are a well-known hazard and there have periodically been mass-stinging events. In an extreme example, on just one day in June, 1966, over 1,000 people were stung by Gonionemus off of the Amur Bay recreational area (Mikulich& Naumov, 1974). Stings seem to be more common in the Vladivostok region in hot, dry years, suggesting environmental factors play a role. Also, cold winters associated with ice cover may be unfavorable for Gonionemus as a result of scour on the eelgrass beds and the pulse of fresh water when the ice melts (Mikulich& Naumov, 1974).

Govindarajan & Carman (2016) described a number of Gonionemus sting symptoms reported by victims including severe pain, respiratory difficulty, and temporary paralysis. It should be noted that these symptoms were based on the victims’ personal accounts of the stings and not medical reports. Russian accounts describe additional symptoms including blindness and hallucinations (Michaleff, 1974; Yatskov, 1974) that have not been reported in the NWA as far as we are aware. However, because sting symptoms may be described differently by individuals, and individuals likely vary in their physiological sting responses, a toxicity assay that is objective and quantitative is necessary to accurately compare jellyfish toxicities. Given the apparent increase in Gonionemus bloom frequency and NWA range expansion, this is an area where more research is urgently needed.

The biogeography of Gonionemus has perplexed scientists for decades. New sightings in the NWA and recent reports of extremely painful stings speak to an urgent need for a better understanding of its origins, which can come from an approach that integrates its taxonomy, genetics, life cycle dynamics, and historical records (e.g., Pringle et al., 2009). While our study resolves some questions, it leads to many more new ones. We found that NEP G. vertens is distinct from the form found in the NWA and NWP. Gonionemus in the NWA and NWP may be G. murbachii, although a comprehensive taxonomic assessment is required to confirm the classification of this form. Our results show that both the NWA and NWP contain independent Gonionemus lineages, possibly as a result of trans-Arctic migrations. We also resurrect the hypothesis that Gonionemus could be native to the NWA, in contrast with the long held assumption that it was introduced by anthropogenic means. One lineage, which we term Haplotype IV, is shared with the well—known highly toxic populations in the coastal Vladivostok-area in the Sea of Japan. However, because the distribution of Haplotype IV does not correspond with the geographic distribution of NWA sting reports and mitochondrial lineage is not equivalent to toxicity, we cannot assume that this form was introduced recently. While it seems less likely, it is also possible that an invasion occurred in the reverse direction—from the NWA to the NWP. More research is needed to resolve these competing scenarios.