Friday 21 June 2024

The deep history of the Hagfish.

The deep oceans serve as refugia for many groups of Animals which have effectively vanished in the shallow seas, including Coelacanths, Vampire Squid, Crinoids and Brittlestars, the living graptolite genus Rhabdopleura and other colonial Hemichordates, and several lineages of deep-sea Isopods, all of which seem to have diverged from their closest shallow-marine relatives more than 200 million years ago. Although generally thought of as evolutionary relicts, most of these groups appear to have undergone significant evolutionary diversification since entering the deep seas.

The Vertebrates underwent their first major evolutionary radiation in the oceans between the Ordovician and the Devonian, or between about 480 and 360 million years ago. Most living deep-sea Vertebrates, however, belong to a few relatively young groups, stemming from diversification events less than 100 million years ago. 

Although the first Vertebrates were jawless, by the End of the Devonian these had largely been eclipsed by jawed taxa, and today only two groups of Jawless Vertebrates survive, the Hagfish, Myxiniformes, and the Lampreys, Petromyzontiformes. The relationship between these groups, as well as the timing of divergence events between them, and within each group, remains unclear, though comparative genomic analysis has now confirmed that the two groups can be regarded as sister taxa (a variety of other relationships had been proposed, including a sister relationship between the Lampreys and Jawed Vertebrates, with the Hagfish being more distantly related to the two).

Hagfish form a significant proportion of the total biomass of Vertebrates on the deep ocean floor, with most species found on the continental slopes and ocean floors, between 200 m and 3 km beneath the surface, where they form an important part of the benthic ecosystem. Some species are found on the shallower ocean shelves, but these are rare. 

Hagfish have an Eel-like body with poorly developed eyes, a loose, scale-less skin, a minimal skeleton comprising a cartilaginous skull and rudimentary vertebrae, several auxiliary hearts, a mouth surrounded by barbels (short tentacles), a single nostril, and a single semicircular canal. The tongue of the Hagfish comprises a cartilaginous plate with two pairs of horny teeth, used to seize food and draw it into the mouth. 

In a paper published in the journal BMC Ecology & Evolution on 13 June 2024, Chase Doran Brownstein of the Department of Ecology and Evolutionary Biology at Yale University, and Thomas Near, also of the Department of Ecology and Evolutionary Biology at Yale University, and of the Yale Peabody Museum, present a time-calibrated phylogenetic tree for the Myxiniformes using data from fossils as well as a genetic dataset which includes 60% of living species of Hagfish.

Brownstein and Near were able to obtain sequences for the mitochondrial COI and 16S ribosomal DNA genes for 44 species of Hagfish from the GenBank database. This sample included two species of Rubicundus, two species of Neomyxine, 14 species of Myxine, and 26 species of Eptatretus, with an additional three potential species of Eptatretus from India, Japan, and Korea. the problematic ‘Notomyxine’ (= Myxine) tridentiger and several species   previously classified in ‘Quadratus’ and ‘Paramyxine'. 

This represents more than 50% of all known extant Hagfish species, although it does not include the problematic genus Nemamyxine, known only from two preserved specimens collected in the mid-twentieth century, with no genetic material available. This makes it impossible to place the genus Nemamyxine within a phylogenetic tree based upon genetic analysis, although Brownstein and Near note that it is thought to have close affinities to the genus Rubicundus, but also that there are problems with the validity of the genus. Both Nemamyxine and Rubicundus are defined as having an extremely slender body and an anteriorly placed ventral finfold that originates anterior of the ventral gill apertures, but this is also seen in many members of the genera Myxine and Eptatretus, as well as the Late Cretaceous fossil Hagfish, Tethymyxine tapirostrumNemamyxine is also defined as having a slender body depth and high slime pore counts, but these are also widespread in elongated Hagfish.

The first of two known specimens of Nemamyxine elongata, one of two described species in the genus Nemamyxine, which was found dead in a net in the Kaituna River on the Bay of Plenty on North Island, New Zealand, in 1958, and thought to have been a fishery discard. A second specimen was later recovered by a trawler from  the Canterbury Bight on the east coast of South Island, from a depth of between 132 and 140 m. Museum of New Zealand Te Papa Tongarewa.

Brownstein and Near constructed phylogenies using both maximum likelihood and Bayesian methods, and the online Clustal Omega tool at the European Molecular Biology Laboratory - European Bioinformatics Institute online resource portal to aid in 16S alignments. For outgroups they used the jawed Ornate Birchir, Polypterus ornatipinnis, West African Lungfish, Protopterus annectens, and Australian Ghostshark, Callorhincus milii, and the Lampreys Geotria australis, Petromyzon marinus, and Lampetra fluviatilis.

A molecular clock methodology with fossils was used to calibrate the divergence of clades. This is challenging for Hagfish, as the fossil record for the group is extremely limited, and most fossils assigned to the group are poorly preserved and/or of dubious placement. The putative stem-hagfish Myxinikela siroka from the Late Carboniferous Francis Creek Shale of Illinois was included in the study, as was the Late Cretaceous crown group Hagfish Tethymyxine tapirostrum from the Hâdjula Lagerstätte of Lebanon, as were a number of fossil Lampreys (phylogenetically the closest group to the Hagfish).

The putative stem-Hagfish Myxinikela siroka from the Late Carboniferous Francis Creek Shale of Illinois. Miyashita (2020).

Data on the habitat preference of Hagfish species was collected from the FishBase database, with two identified environments, continental shelf (less than 200 m) and continental slope (more than 200 m). These were used to make a probability-based estimation of the ancestral state of Hagfish groups using the R package, with the possibility that species might be flexible in their choice of habitat taken into account using the fitpolyMk function.

Brownstein and Near consistently recovered the three major lineages of Hagfish (the Rubicundinae, Eptatretinae and Myxininae) as  valid and distinct taxa. The genus Neomyxine was recovered as the sister taxon the genus Myxine within the family Myxininae,  rather than being the sister group to all other extant Hagfish, as have been found by some previous studies. The Family Rubicundinae was recovered as the outgroup to other extant Hagfish, something which has been found by some previous studies. The study also suggests that the genera Quadratus and Paramyxine should be included within the genus Eptatretus, and the species Notomyxine tridentiger should be included within the genus Myxine.

Hagfish phylogeny and tempo of diversification. Tip-dated Bayesian maximum clade credibility phylogeny of jawless fishes from two independent runs in BEAST 2.6.6 showing the interrelationships of the major lineages of hagfishes. Bars indicate 95% highest posterior density intervals for divergence times at nodes. Outgroups not shown. Grey bars are at nodes supported by posterior values of 0.90 or more, clear bars are at nodes supported by posterior values of 0.89 or less. Gray columns indicate mass extinction events. Dagger (†) indicates extinct species known from the fossil record. Pie charts indicate ancestral state reconstructions of habitat for each node, where purple represents the probability of a slope component (either slope or shelf-slope) at each node and salmon indicates the probability of continental shelf habitat being ancestral. Inset includes the transition matrix from the polymorphic character ancestral reconstruction analysis (note that purple here is exclusively slope, as opposed to purple denoting slope/shelf-slope at nodes in the phylogeny). Photograph of Eptatretus stoutii is courtesy Douglas FudgeBrownstein & Near (2024).

The results of the study suggest that the three major Hagfish groups diverged from one-another during the Palaeozoic. This did not change when the Carboniferous Myxinikela siroka was included in the matrix, suggesting that the use of this taxon as a calibration point is valid. Brownstein and Near note that they excluded the Mazon Creek 'Hagfish' Gilpichthys greenei from the study, as the affinities of this abundant fossil are now considered highly doubtful. Other phylogenetic studies have included this species, recovering it as either a stem Hagfish, or a Jawless Fish of uncertain affinities. Brownstein and Near suggest that these fossils may be difficult to interpret phylogenetically as most had decayed somewhat before preservation.

The putative Hagfish Gilpichthys greenei from the Mazon Creek fossil beds. Earth Science Club of Northern Illinois.

Brownstein and Near consistently found that the crown Hagfish (a crown Hagfish is any species, living or fossil, which is descended from the last common ancestor of all living Hagfish) arose in the Early Permian, and the split between the Eptatretinae and Myxininae occurred in the Early Triassic. Both events are substantially older than previous studies have suggested, with the diversification of major Hagfish clades until now assumed to have happened in the Middle-to-Late Cretaceous. Brownstein and Near note that the use of mitochondrial DNA has been linked to the overestimation of the age of some groups of Ray-finned Fish, but cannot see how this would lead to the discrepancy between their study and earlier studies of Hagfish which also used mitochondrial DNA. Instead they suggest that the variance is due to the increased number of living species in their study, combined with a stricter approach to the inclusion of fossil species, with less certain species such as Gilpichthys greenei excluded. 

This revised timeline removes a 120 million year gap between the separation of the Hagfish and their closest relatives (the Lampreys), as well as showing that the group have survived three major extinction events, including the End Permian, which wiped out 81% of marine species. This makes the crown group Hagfish one of the oldest known Vertebrate crown groups, and far older than most other marine Vertebrate groups. 

The reconstruction of the Ancestral habits of the Hagfish suggests that the oldest members of the group occupied the continental slopes (more than 200 m beneath the surface) during the Late Palaeozoic. This is despite all known fossil Hagfish coming from coastal slope or estuarine environments. All the major Hagfish groups apparently first appeared on the continental slopes, or at least as organisms with flexible requirements able to inhabit both the continental slopes and shelves.

Hagfish and Lampreys have been the sole surviving jawless Vertebrates since the Triassic Extinction. This makes them important to our understanding of the earliest Vertebrates, although probably atypical of these. 

Brownstein and Near's study suggests that the crown group Hagfish emerged during the Permian, with the three major extant groups having appeared by the end of the Early Triassic, 20-30 million years after the oldest putative Hagfish fossils. It is likely that the stem group Hagfish appeared during a significant radiation event after the extinction of the jawless Ostracoderms at the end of the Devonian. 

Hagfish have a simple bodyplan, which has remained essentially unchanged for a very long time, notably so compared to other ancient Vertebrate groups such as the Teleosts, Chondrichthyans, and Lissamphibians. This highly specialised anatomy appears to have developed before the End of the Permian.

This deep diversification is different to the situation seen in Lampreys, where the extant groups all appear to have derived from a series of regional diversification events within the past 100 million years. Hagfish species appear to have diverged from their closest relatives an average of 31.6 million years ago, compared to 1-2 million years for most Lampreys. The most ancient division for a single species is that for Eptatretus cheni, which appears to have diverged from other members of the genus Eptatretus in the Jurassic. This is a similar timing for the division between the living Neoselachian Sharks and Rays, the Tuatara, Sphenodon punctatus, and the Squamates, or the Salamanderfish, Lepidogalaxias salamandroides, and all other Teleosts.

Eptatretus cheni, not notably different to other members of the genus Eptatretus, but separated from them since the Jurassic. Fish Database of Taiwan/FishBase.

Hagfish taxonomy is a challenging field, due to the conservative morphology of these organisms, and the inaccessible environments in which they live. The widespread genus Rubicundus is the only genus in the family Rubicundinae, and forms the sister group to all other Hagfish, but was not recognised as a distinct genus until 2013. Brownstein and Near's study implies that this genus split from its closest living relatives in the Permian. 

A Pink Hagfish, Rubicundus eos. The genus Rubicundus appears to have diverged from all other extant Hagfish in the Permian. Museum of New Zealand Te Papa Tongarewa.

Brownstein and Near's study also highlights that deep marine habitats have been utilised by Hagfish since the origin of the group in the Permian. Lineages of Myxine and Eptatretus found in shallower continental shelf environments appear to have diversified into these shallower waters relatively recently, with fossil Hagfish from shallow marine environments probably the result of similar diversification events. This makes the Hagfish the Vertebrates the group with the longest history in deep marine environments, with a continuous habitation of these environments long predating the arrival of the ancestors of any extanct Chondrichthyan or Teleost found in the deep seas. 

This inhabiting of deep-sea environments may explain how the group has persisted so long with relatively little apparent evolutionary innovation. Although the group has not  occupied deep marine environments for as long, the oldest surviving Chondrichthyan lineages, such as the Goblin Sharks, Frilled and Sevengill Sharks, Chimeras, and Ratfish, all inhabit deep environments. Thus thee deep sea. environment appears to be a refugia for Vertebrate groups able to live there, offering a degree of protection against  extinction events which heavily impact the shallow seas.

Nevertheless, Hagfish appear to have undergone significant diversification within deep sea environments, with many distinct lineages arising over the time they have dwelt there.

Most Vertebrate groups found in the deep seas have colonised these environments within the last 100 million years. In contrast, many Invertebrate groups have long deep marine lineages. This has led to the view that the deep seas can act as a refugia for groups that can live there during mass extinction events that affect the shallow seas.  Until now, no Vertebrate group has been seen as truly endemic to this refugium, but Brownstein and Near's study suggests that the deep seas are the principle habitat for Hagfish, with modern and fossil shallow-water species being the result of repeated colonisations from deeper marine environments.

See also...