Did the earliest Vertebrates have four eyes?

Vertebrates have complex, camera-type eyes which have been a source of interest to evolutionary biologists since the nineteenth century, when this seemed an unusually complex system, which it was difficult to imagine arising through a series of gradual steps. Modern evolutionary biologists are less concerned by this, recognising that even a very simple eye is better than no-eye-at-all, and that therefore a complex eye could arise step-wise from the simplest cluster of light-sensitive cells, but beyond this have been able to give no real explanation of what the eyes of our earliest Chordate ancestors looked like. 

Camera eyes comprise a comprise of a spherical lens, a retina, an iris, and a set of muscles exterior to the main eye structure, which can be used to alter the shape of the lens, enabling it to focus an image on the hemispherical retina, which are detected by the optic nerve, and transmitted to the brain.

Almost all modern Vertebrates have two lateral camera eyes, although some groups have lost these, and, curiously, some Lizards have a third such eye on the top or back of their heads, which is derived from the pineal complex of the brain.

Eyes in Vertebrate fossils are often identified by the preservation of the pigmentation from the retinal epithelium, which is rich in melanin, as dark stains, and/or by impressions left by the hard lens. The oldest purportative fossil Vertebrate eyes are seen in Metasprigginna walcotti, a probable Chordate from the Burgess Shale of Canada, dated to about 505 million years before the present. In these fossils a hemispherical shape has been interpreted as the retina, and an associated circular area as the lens. The earliest known example of melanostomes (the cells which contain the pigment melanin) being preserved in the eye of a Vertebrate is the Devonian Jawless Fish Euphanerops longaevus, from the Escuminac Formation of Canada, which has lateral eyes with abundant such cells, inferring the presence of a retina.

No non-Vertebrate Chordates possess a camera eye. The Lancets, or Amphioxi, have four clusters of photoreceptor cells, but are not thought to be able to produce an image (unsurprising since they also lack a brain). Salps, which are planktonic Tunicates, have a multiple stage life cycle, with an colonial adult phase which reproduces sexually, and a solitary adult phase which reproduces asexually. The larval form of the colonial Salp has three pigment cup eyes, while the larval form of the solitary stage has a single eye. During the embryonic development of Vertebrates, the paired eyes arise from a section of the anterior neural plate which also gives rise to the pineal organ, leading some biologists to speculate that these three organs are analogous to the three eyes of the Salp larvae. 

In a paper published in the journal Nature on 21 January 2026, Xiangtong Lei of the Center for Vertebrate Evolutionary Biology and Institute of Palaeontology at Yunnan University, Sihang Zhang, also of the Center for Vertebrate Evolutionary Biology, and of the State Key Laboratory for Vegetation Structure, Functions and Construction at Yunnan University, Peiyun Cong, also of the Center for Vertebrate Evolutionary Biology, and State Key Laboratory for Vegetation Structure, Functions and Construction at Yunnan University, as well as the Oxford University Museum of Natural History, Jakob Vinther of the Palaeobiology Research Group and School of Biological Sciences at the University of Bristol, Sarah Gabbott of the Centre for Palaeobiology & Biosphere Evolution at the University of Leicester, Fan Wei again of the Center for Vertebrate Evolutionary Biology, and State Key Laboratory for Vegetation Structure, Functions and Construction at Yunnan University, and Xing Xu, once again of the Center for Vertebrate Evolutionary Biology and Institute of Palaeontology at Yunnan University, and of the Institute of Vertebrate Paleontology and Paleoanthropology of the Chinese Academy of Sciences, identify organs which they believe are eyes in two species of Myllokunmingids (Early Chordate Animals which may be ancestral Vertebrates) from the approximately 518-million-year-old Chengjiang Biota of Yunnan Province.

Lei et al. consider Myllokunmingids such as Haikouichthys ercaicunensis and Myllokunmingia fengjiaoa to be the earliest known Vertebrates. For their study they examined six specimens of Haikouichthys ercaicunensis and four slabs which each contained multiple specimens of an as yet unnamed new Myllokunmingid. In both species they found that the head region typically has four black spots, two larger spots being placed laterally on the head, and two smaller spots facing forward. Previous studies have identified the larger of these spots as eyes, while the forward-pointing spots have been identified as nasal sacs. 

General morphology of the lateral eyes and pineal complex with their preserved melanosomes in two species of Myllokunmingidae from the Chengjiang biota. (a)-(b) Haikouichthys ercaicunensis (YNGIP-90281) with its enlarged eye region (b). (c) Carbon (red) and iron (green) element mapping of the same region in (b), arrows denote the position of figured melanosomes in (g) and (h). (d) General morphology of the unnamed Myllokunmingid  (YNGIP 90291-b,). (e) Enlarged eye region of the unnamed Myllokunmingid (YNGIP 90292-a), illustrating lateral eyes (circles in dotted line) and pineal/parapineal organs (arrows). (f) Carbon (red) and iron (green) element mapping of the same region in (e), arrows denote the position of figured melanosomes in (i), (j). (g)-(h) Melanosomes in the eyes (g) and pineal complex (h) of Haikouichthys ercaicunensis. (i)-(j) Melanosomes in the eyes (i) and pineal complex (j) of the unnamed Myllokunmingid. Scale bars are 2 mm (a); 1 mm (d); 200 μm (b), (c), (e), and (f); 500 mm (g)-(j). Lei et al. (2026).

Energy dispersive X-ray, Raman spectroscopy, and X-ray photoelectron spectroscopic analysis of the lateral eyes and the forward facing spots are enriched in organic carbon. Examination under a scanning electron microscope revealed that these organic patches are made up of oblong or cylindrical microbodies, which measure 200-1200 nm in length, and 200-900 nm in width. Most of these microbodies appear deformed or fused together, and they are associated with pyrite minerals and a clay matrix.

In the lateral eyes of Haikouichthys ercaicunensis these microbodies are consistently oval in shape, ranging from 250 to 900 nm in length and from 200 to 800 nm in width. Element mapping suggests that these objects are carbonaceous structures with a small central hole. In the unnamed Myllokunmingid, there are two morphotypes of microstructures present, the first similar to those seen in Haikouichthys ercaicunensis, and the second being cylindrical in shape and between 400 and 1200 nm in length and between 200 nm and 550 nm in width. These structures also have a central hole. Transverse sections of the melanosomes of some living Vertebrates have also shown such a central hole.

Lei et al. next investigated the molecular composition of the microstructures using Time-of-Flight Secondary Ion Mass Spectrometry. This revealed that in both species the microstructures contained the pigments eumelanin and phaeomelanin, both of which are found in living Vertebrates, confirming that these structures are in fact melanosomes. 

The melanosomes in the lateral eyes of Haikouichthys ercaicunensis appear to be largely distributed on the horizontal axis, while those of the unnamed Myllokunmingid are spread along a diagonal axis, with the two types of melanosomes present having different distributions and pigment contents; the cylindrical cells have a higher eumelanin content (which would have made them browner in colour) while the ovoid cells have a higher phaeomelanin content (which would have made them oranger in colour). 

In living Vertebrates, melanosomes are found in the iris, choroid and retinal pigment epithelium, but layers of ovoid and cylindrical melanosomes are found only in the retinal pigment epithelium. The observed structures in the eyes of the unnamed Myllokunmingid are consistent with a retinal pigment epithelium with a similar structure. However, in the six specimens of Haikouichthys ercaicunensis examined only ovoid melanosomes could be observed. However, rather than interpreting this as a more primitive state, Lei et al. note that in the Lamprey Mayomyzon pieckoensis and the Cartilaginous Fish Bandringa rayi from the Carboniferous Mazon Creek Fauna of Illinois, a preponderance of ovoid melanosomes have also been observed in eye structures, and that relatively few living Vertebrates have have been investigated to determine what forms of melanosomes are present in their retinas.

In both Chengjiang Myllokunmingids, the central spots are smaller than the lateral spots, about 160-240 µm in diameter in Haikouichthys, and about 90-120 µm in diameter in the unnamed Myllokunmingid. These were also found to be carbonaceous in composition, and to contain microbodies which appeared to be melanosomes; in each species these were consistent with the bodies found in the larger lateral eyes, with only oval melanosomes in Haikouichthys and both cylindrical and oval forms in the unnamed Myllokunmingid. Based upon this, Lei et al. conclude that these medial organs are also preserved retinas.

Carbonaceous preservation of Myllokunmingids eyes and median dark s pots (a-h). (a)-(b) Haikouichthys ercaicunensis (YNGIP-90285) showing lateral eyes (grey) and pineal eyes (green) with lens (blue). (c) Carbon element map of Haikouichthys ercaicunensis (YNGIP-90285) head. (d)-(e) Haikouichthys ercaicunensis (YNGIP-90296) showing lateral eyes (grey) and pineal eyes (green) with lens (blue). (f) Carbon element map of Haikouichthys ercaicunensis  (YNGIP-90296), arrows indicating left pineal eye. (g)-(i) Eyes of Haikouichthys ercaicunensis showing lens (arrows). (g) YNGIP-90283. (h) YNGIP-90284. (i)  YNGIP-90289. (j), (m) lens in Elonichthys peltigerus (ROM56794). (k), (n) Lens in Platysomus circularis (PF7333). (l), (o) Lens in Bandringa rayi (ROM56789). Scale bars are 200 μm (a)-(f); 50 μm (g)-(i); and 500 mm (m)-(o). Lei et al. (2026).

As well as melanosomes within their retinas, both species show preserved lenses, which are ovoid in structure, and about one fifth of the size of the associated retinas. These structures are preserved as impressions with some relief, suggesting that they represent an original structure which was somewhat decay resistant. This placement, size, and composition is consistent with the interpretation of these structures as eye lenses, which are harder tissue than other components of the eyes, and have been found in other Vertebrate fossils, including the Middle Cambrian vertebrate Metaspriginna walcotti.

The similarity of the lateral eyes of the two Myllokunmingid species from the Chengjiang Fauna to those found in later Vertebrate fossils is taken by Lei et al. to indicate that camera eyes had appeared by the Early Cambrian. The combination of a large retinal pigment epithelium and smaller lens is consistent with a fluid-filled retinal sphere with an iris opening within which the lens is suspended, as seen in living Vertebrates. Such eyes would almost certainly have been capable of image formation, although the quality of such images is impossible to know. 

The median, forward-facing spots on Myllokunmingids have previously been interpreted as nasal sacs, or possibly pineal organs. The former explanation seems unlikely, as nasal sacs otherwise appear to have been quite a late development, not found in many later stem Vertebrates, and probably first evolving in Galeaspids (probable stem Gnathostomes) between 435 and 370 million years ago. Lei et al. report the discovery of melanosome-bearing tissues and lenses in these spots, which are again inconsistent with an interpretation as nasal sacs. They instead interpret them as paired pineal organs functioning as a second pair of camera eyes.

Lei et al. also note that the Middle Cambrian stem Vertebrate Metaspriginna walcotti also has a pair of dark spots between the lateral eyes, preserved as carbonaceous films, and that these also appear to have associated spherical objects, which may also have been lenses, suggesting that this species may also have had a second pair of median eyes.

In Lampreys, the pineal organ is photosensitive, helping the Animal to respond to changes in light levels within the environment. In Mammals, the pineal organ is entirely internal, but it is associated with aligning the neuroendocrine system with the day/night cycle. In Lizards, the pineal organ is also associated with the neuroendocrine system, but in some species retains a photoreceptive capacity. It has therefore previously been suggested that the pineal organ may have developed from some sort of precursor eye, something that has entered popular culture as the 'third-eye' theory. Lei et al. suggest that the pineal organ may have begun as a pair of photosensitive organs acting as additional camera eyes. 

The presence of complex visual systems in the earliest Vertebrates suggests that this sense was of key importance to the success of the group from very early in its history. Both the photoreceptive cells of Vertebrates and the cells of the retinal ganglion arise from nurosensory cell precursors also present in Tunicates. A theoretical model has previously been developed in which the camera eye developed via two rounds of whole-genome duplication, the first allowing for a divergence between the photoreceptor cells and the optical ganglion cells, the second between the pineal complex and the lateral eyes. The apparent presence of a second pair of camera eyes associated with the pineal complex in Early Cambrian Myllokunmingids may represent a transitional stage, in which the genes associated with the development of the eyes have been duplicated, but only just started to evolve towards the modern pineal complex.

Evolutionary scheme of visual system in early Vertebrates. (a) Thalia (Tunicata). (b) Haikouichthys. (c) Euphanerops. (d) Generalised Lamprey. (e) Sacabambaspis. (f) Shuyu. (g) Aphyocharax. Coloured regions show positions of key sensory organs: blue, eyes; red, pineal. Light grey lines represent body outlines. Coloured bars represent the suggested acquisition of key characters. Abbreviations: br, brain; p, pineal; pp, parapineal; TG, total group. Cyclostome represents the Petromyzontidae and Myxinoidea total groups and Gilpichthys, which was recovered in a polytomy with those two groups. Cyclostome and Gnathostome total groups in this topology recovered in a polytomy with Metaspriggina and (Haikouichthys + Myllokunmingia). Lei et al. (2026).

Euphanerops longaevus, an anaspid-like fossil from the Devonian Escuminac Formation of Canada, which has been suggested as a stem-Agnathan (jawless Fish) also has paired median dark patches which have been shown to be carbonaceous films with structures identical to the melanosomes of its lateral eyes. Living Lampreys have a pineal eye and a smaller parapineal eye, both of which have functioning retinas (but not lenses) and are used to detect changes in light conditions. The stem Gnathostome (jawed Fish) Sacabambaspis has two pineal openings, which Lei et al. suggest are analageous to the pineal and parapineal eyes of Lampreys. Later stem Gnathostomes, such as the Galeaspids, only have a single such opening, suggesting a progressive loss of this system. Crown Gnathostomes have lost this opening completely, but some have a preserved pineal window, with an area of thin, semitransparent skull overlaying a pigmented area associated with the pineal complex. Thus an image-forming pineal complex was slowly replaced with a light sensitive organ regulating the production of the hormone melatonin, which regulates sleep patterns. Most crown Vertebrates possess both pineal and parapineal organs, sugesing that this complex was originally paired.

During the Cambrian Explosion, early Animals went through a phase of remarkable morphological innovation, with each new development changing the ecological environment in which all Animals lived, particularly as predation became more common. It has been suggested that higher levels of ultraviolet radiation in shallow waters during the Cambrian may have made the rapid evolution of vision more important, although it is likely that the evolution of predator-prey relationships would have been sufficient to drive this. The appearance of large (for the Cambrian) predators such as Radiodonts, gilled Lobopods, and stem Chaetognaths, all of which developed complex visual systems, would have made it important for smaller, non-predatory Animals such as Myllokunmingids to develop equivalent systems to evade predation and survive. 

See also...

Neobolus wulongqingensis: A Cambrian Brachiopod with encrusting kleptoparasites.

Parasitism is an enduring symbiotic relationship in which the parasite is nutritionally dependent upon the host for at least part of its life cycle, increasing its own fitness in the process and directly impinging upon the biological fitness of the host. Parasite–host interactions form a significant proportion of the biotic interactions in extant global ecosystems, influencing many characteristics of species networks including behavior, population structure, and ecological function. The antagonistic relationship between parasites and hosts has also been proposed as the primary mechanism leading to the evolution and maintenance of sexual reproduction, due to the negative frequency-dependent selection associated with parasitism. Despite its obvious importance, the origins and early evolution of Metazoan parasitism remains enigmatic. Molecular phylogenies predict the emergence of parasitic clades in the Cambrian and putative instances of shell damage, shell scarring and occasional bioclaustration from the early Cambrian represent circumstantial evidence that hint at possible parasitism, but the rarity of well-preserved specimens precludes decisive identification of parasite–host interactions in the earliest Phanerozoic. Possible examples of epibiontism, commensal infestation, and hitchhiking are also known from the early Cambrian, but none of these constitute definitive instances of parasitism with a clear negative biological effect on the host. This absence of clear evidence for parasitism in the earliest animal communities may, in part, be due to a lack of cross-sectional quantitative analyses on Cambrian material of the type that have been demonstrated as necessary to identify and discriminate instances of animal parasitism in deep time.

In a paper published in the journal Nature Communications on 2 June 2020, Zhifei Zhang and Luke Strotz of the State Key Laboratory of Continental Dynamics, Shaanxi Key Laboratory of Early Life & Environments and Department of Geology at Northwest University, Timothy Topper, also of the State Key Laboratory of Continental Dynamics, Shaanxi Key Laboratory of Early Life & Environments and Department of Geology at Northwest University, and of the Department of Palaeobiology at the Swedish Museum of Natural History, Feiyang Chen, again of the State Key Laboratory of Continental Dynamics, Shaanxi Key Laboratory of Early Life & Environments and Department of Geology at Northwest University, and of the Department of Biological Sciences at Macquarie University, Yanlong Chen and Yue Liang, again of the State Key Laboratory of Continental Dynamics, Shaanxi Key Laboratory of Early Life & Environments and Department of Geology at Northwest University, Zhiliang Zhang, also of the State Key Laboratory of Continental Dynamics, Shaanxi Key Laboratory of Early Life & Environments and Department of Geology at Northwest University, of the Department of Biological Sciences at Macquarie University, Christian Skovsted, also of the State Key Laboratory of Continental Dynamics, Shaanxi Key Laboratory of Early Life & Environments and Department of Geology at Northwest University, and Department of Palaeobiology at the Swedish Museum of Natural History, and Glenn Brock, once again of the State Key Laboratory of Continental Dynamics, Shaanxi Key Laboratory of Early Life & Environments and Department of Geology at Northwest University, of the Department of Biological Sciences at Macquarie University, describe a new species of Lingulid Brachiopod from the early Cambrian Guanshan Konservat-Lagerstätte, and the encrusting kleptoparasites which cover many specimens of this species.

The early Cambrian (Stage 4) Guanshan Konservat-Lagerstätte occurs mostly in the lower 40m of the Wulongqing Formation, which crops out over a geographically wide area in eastern Yunnan, located in southern China. The Guanshan Biota is unusual in being proportionately dominated by Brachiopods, and so, is strongly differentiated from other Cambrian Konservat-Lagertätten such as the Chengjiang, Sirius Passet, Emu Bay Shale and the Burgess Shale, which are Euarthropod-dominated assemblages. 

 

Locality map and stratigraphic position of the Guanshan Konservat-Lagerstätte in Yunnan Province. (a) Locality map for the Guanshan Konservat-Lagerstätte in Yunnan Province. All specimens of Neobolus wulongqingensis included in Zhang et al.'s study were sampled from the Gaoloufang section (Locality 3). (b) Cambrian stratigraphic scheme, both international and for China, showing the relative position of the Wulongqing Formation. Zhang et al. (2020).

The new species is placed in the genus Neobolus, and given the specific name wulongqingensis, meaning 'from Wulongqing', in reference to the Wulongqing Formation, from which the fossils were extracted. It is an Organophosphatic Linguliform Brachiopod with an adult shell subcircular, no visible pits or pustules on surface, peripherally ornamented with distinct growth lines; metamorphic shell, average of 2396 μm in width and 1907 μm in length (based upon 13 measured specimens); ventral pseudointerarea orthocline to apsacline with wide and triangular pedicle groove; ventral propareas vestigial or indistinguishable; dorsal pseudointerarea forming narrow, crescent-shaped rim; ventral visceral field short, slightly thickened and not extending beyond midvalve; dorsal interior with long median septum extending to or beyond ⅓ valve length. Short spirolophe lophophore present.

 

The Brachiopod Neobolus wulongqingensis, with associated obligate, encrusting kleptoparasitic tubes. (a) ELI GB-N-0301, densely aggregated valves of Neobolus wulongqingensis forming distinctive shell beds with their associated kleptoparasites. Scale bar 4 mm. (b), (c) Specimens of Neobolus wulongqingensis with varying numbers of encrusting kleptoparastic tubes; (b) ELI GB-N-0650, (c) ELI GB-N-0648-5. (d) ELI GB-N-0008, Neobolus wulongqingensis with baculate mantle canals preserved. (e) ELI GB-N-0261-18, Neobolus wulongqingensis with encrusting kleptoparastic tubes. (f) ELI GB-N-0255-6, internal view of a pair of conjoined valves with kleptoparasitic tubes encrusted to both valves. (g) ELI GB-N-0869-2-1. Neobolus wulongqingensis with encrusting kleptoparasitic tubes and Trilobite cranidium (exuviae) lacking attached tubes. Scale bars 2 mm, unless otherwise stated. Zhang et al. (2020).

Neobolus wulongqingensis is the most numerically abundant taxon in the Wulongqing Formation, with many thousands of specimens forming dense concentrations of monotypic, mostly conjoined shells, clustered closely on bedding plane surfaces. Remarkably, many of the Brachiopod shells are encrusted with elongate, tapering biomineralised tubes. Symbiotic relationships such as this are seldom directly observed in the fossil record because taphonomic biases generally impede the preservation of direct interaction between organisms. The high-fidelity preservation and great abundance of specimens in the Wulongqing Formation provides a rare opportunity to investigate this unique interaction between a Brachiopod host and their associated encrusting tube-dwelling organisms.

 

Additional examples of Neobolus wulongqingensis from the Guanshan Biota encrusted with varying numbers of obligate, kleptoparasitic tube-dwelling organisms. (a) ELI GB-N- 263A5-1. (b) ELI GB-N-254-1-1, Neobolus wulongqingensis with visceral region preserved (c) ELI GB-N-253-19- 1-D2, Neobolus wulongqingensis with tube outlines visible from the interior of the Brachiopod shell (d) ELI GB-N-290-8-1. (e) ELI GB-N-263A-2-3, Neobolus wulongqingensis with the outline of the kleptoparasitic tubes visible from the interior of the Brachiopod shell (f) ELI GB-N-0263B-3. (g) ELI GB-N-0253. (h) 284-3-1. i, ELI GB-N-0255-8. Zhang et al. (2020).

Zhang et al. assess differences in biomass between Brachiopod individuals of the species Neobolus wulongqingensis encrusted with tubes and those individuals lacking tubes, with biomass representing a proxy for the biological fitness of an individual. This analyses suggests that the tube-dwelling organisms reduced the biological fitness of the host and, when considered in combination with observations of the preferred growth orientation of the encrusting tubes, these results suggest the interaction between the tube-dwelling organisms and their host Brachiopod represents kleptoparasitism. This instance in a Cambrian epibenthic marine community likely represents the oldest known parasite–host relationship in the fossil record and reveals that parasite–host interactions emerged in conjunction with the rise of the earliest Animal communities during the Cambrian radiation.

 

Exceptionally preserved examples of Neobolus wulongqingensis from the Guanshan biota with chaetae preserved together with morphological details of encrusting biomineralised tubes. (a), (b) exceptionally preserved specimens of Neobolus wulongqingensis with chaetae preserved: (a) ELI GB-N 632-1. (b) 648-6-1-GLF. (c) ELI GB-N 561-2-1, close up of kleptoparsitic tube extending into the chaetal fringe of a Neobolus wulongqingensis individual. (d), (e) ELI GB-N 258-1-1. d, twisted dorsal and ventral valves with encrusting kleptoparasitic tubes. (e) enlarged view of boxed area in (d). (f) 258 -1-2 –GLF, Close up of biomineralised tubes showing clear surface annulations. (g), (h) 250 -1 -1-2- GLF. Detail and micro X-ray fluorescence elemental mapping of biomineralised tubes detached from Brachiopod valve. (g) Light photo. (h) Micro X-ray fluorescence elemental mapping of potassium. (i) Micro X-ray fluorescence elemental mapping of silicon. (j) Micro X-ray fluorescence elemental mapping of iron. (k) Close up of the three biomineralised tubes. (l) Close up of kleptoparasitic tubes still attached to Neobolus wulongqingensis. Zhang et al. (2020).

The preservation of marginal chaetae, mantle canals, visceral areas, and, rarely, the lophophore in the Brachiopods indicates rapid burial and minimal transport by episodic obrution deposits. Despite this, the soft body of the tube-dwelling Animal is not well-preserved, and its biological affinities are not self-evident. The greyish-white tubes, normally flattened by post depositional compaction, are immediately apparent. The tubes, some with preserved accretionary growth increments, encrust the exterior of both dorsal and ventral valves of Neobolus wulongqingensis with the open apertures exclusively oriented toward the anterior commissure of host Brachiopods, indicating an intimate, life-long, in-vivo association. The tubes exclusively encrust the exterior of the host shell, which occasionally shows signs of minor damage or disruption of shell growth lines, but there is no evidence of boring into the interior of the brachiopod by the tube-dwelling organism. The tubes are not found attached to any other hosts or substrates, such as the Trilobite or Palaeoscolecid exuviae that occasionally occur in the shell beds. Consequently, Zhang et al. interpret this interaction as representing an obligate relationship, as there is no evidence to suggest that the tube-dwelling organisms can adopt a free-living lifestyle in the absence of their Brachiopod host.

 

The basibiont Neobolid (Lingulata) Brachiopod Neobolus wulongqingensis from the Guanshan biota (Cambrian Stage 4) of eastern Yunnan. (a) Holotype, ELI GB-N-0377-1, a composite mould with dorsal and ventral valves strongly compressed. Note the fringe of chaetae and proximal pedicle. (b) ELI GB-N-0297-4, a 3- dimensional ventral valve with a preserved elongated pedicle attached to an exoskeleton of a Trilobite. (c) ELI GB-N-0625, compressed dorsal and ventral valves, showing the dorsal and ventral chaetae cross to form a fine sieve or mesh. (d), (e) ELI GB-N-0385: (d) View of dense anterior marginal chaetae; (e) Magnified view of boxed area in Fig. (d). (f), (h) ELI GB-N-SJJ-1308, shell interior, showing paired spiral lophophore: (f) light photograph; (g) enlarged view of boxed area in (f); (h) Micro X-ray fluorescence elemental mapping of aluminium, slilicon and iron shows paired spiral lophophore in high contrast. Zhang et al. (2020).

Bayesian estimation analysis (a widely used technique for estimating the probability density function of random variables with unknown parameters) demonstrates that a credible difference in biomass exists between Brachiopods with encrusting tubes (205 specimens) compared to those without (224 specimens). There is no overlap in the 95% highest density interval of the posterior distribution for the means of the two groups and the highest density interval for effect size does not overlap with zero. Mean biomass for individuals with encrusting tubes is thus credibly lower than for those without tubes. A null hypothesis significance testing approach also identified a significant difference between encrusted and non-encrusted individuals with a small effect size. Zhang et al. therefore contend that individual Brachiopods encrusted with tubes have reduced fitness when compared with their non-encrusted counterparts. On the basis of the difference in the values for mean biomass between the two groupings, encrustation results in a 26.08% reduction in overall fitness across the entire measured cohort.

 

Results of analyses demonstrating encrusted tubes were parasitic. (a) Posterior distribution of mean biomass derived from Bayesian estimation for Brachiopods without attached tubes (μ1; left) versus those brachiopods with encrusted tubes (μ2; right). Highest density interval denotes highest density interval and represents credible values for mean biomass for each grouping. (b) Posterior distribution of effect size for μ1 versus μ2 derived from Bayesian estimation. Highest density interval exceeds 0, indicating that a credible difference exists between the mean values for Brachiopods with encrusted tubes versus those Brachiopods without tubes. (c) Plot of Attachment Distance versus Biomass. Attachment distance from the posterior margin of Neobolus wulongqingensis represents a proxy for the duration of the symbiotic relationship between an individual Brachiopod and its associated encrusted tubes. Correlation between these two variables therefore indicates that those Brachiopods with enduring symbiotic relationships are reduced in biomass in comparison to those where time of attachment has been short. Zhang et al. (2020).

Although Zhang et al.'s analyses indicate that Brachiopods with encrusting tubes are reduced in biomass compared to those without, there is no clear relationship between the biomass of host individuals and increasing numbers of encrusted tubes per individual. In some symbiotic relationships, the impact on the host is amplified depending on the number of parasites present, but this relationship can be highly variable. For Zhang et al.'s dataset, the biomass of the Brachiopod host decreases when a single tube is encrusted to the shell surface, but no further decline is associated with an increasing number of tubes. Both proxies for increasing total parasite load also show no correlation with biomass. This suggests the tube-dwelling organism did not directly inhibit the feeding capability of the host, as larger numbers of parasites do not result in decreased fitness. However, a significant relationship exists between the attachment point of the encrusting tubes and the biomass of the brachiopod host, indicating encrustation earlier in ontogeny results in greater reduced biomass relative to hosts that have been infected at later ontogenetic stages, regardless of the number of symbionts present. In living Brachiopods, smaller individuals generally display an increased growth rate compared to larger individuals. It would therefore be expected that the impact on fitness would be greater for host individuals that are settled by parasites during earlier ontogenetic stages. The increase in median attachment distance for larger numbers of symbionts and the larger size of specimens with greater than four tubes establishes that higher infection rates can only occur when Brachiopod hosts have already managed to grow to larger adult sizes and there is sufficient Brachiopod shell surface area to accommodate a larger number of encrusting tubes.This also indicates that the tube-dwelling organisms do not preferentially encrust smaller Brachiopod individuals, as Brachiopods are clearly encrusted in large numbers later in their ontogeny, when they have reached larger sizes.

 

Supplementary plots exploring relationship between Brachiopod biomass and characteristics of attached tubes. (a) Box and whisker plot of number of attached tubes versus Brachiopod biomass. Whilst 1-3 attached tubes results in lower median biomass compared to individuals without tubes, individuals with 4+ tubes are indistinguishable from those with no attached tubes. (b) Plot of total tube width versus biomass. Total tube width for each individual is calculated as the sum total width of all tubes present on the relevant individual. (c) Plot of total tube area versus biomass. Total tube area for each individual is calculated as the sum total area of the shell surface covered by the attached tubes for the relevant individual. (d) Box and whisker plot of number of attached tubes versus attachment distance. Increasing the number of tubes per individual results in an increase in attachment distance. With attachment distance representing a proxy for time of attachment, this result suggests that large numbers of parasites are present for shorter durations and are only possible on larger, older shells. Number of biologically independent specimens used for each plot: (a) 429; (b) 408; (c) 383; (d) 167. Zhang et al. (2020).

Zhang et al.'s analyses demonstrate that the tube-dwelling organism directly impinges upon the biological fitness of the host, supporting the assertion that the encrusting tube-dwelling organisms are parasitic, rather than being either mutualistic or commensal with the Brachiopod host. A reduction in host biomass or growth rate has been directly attributed to the presence of a parasite in a variety of extant symbiotic relationships. Parasites typically increase the energetic requirements of infected organisms, as the host must generate sufficient energy to not only maintain its own requirements but also the needs of the parasite. This commonly leads to hosts with decreased biomass when compared with uninfected individuals. This result represents the first definitive and statistically supported instance of parasitism from the Cambrian and indicates that parasite–host systems were well established by Cambrian Stage 4, suggesting this type of interaction probably emerged even earlier during the main pulse of the Cambrian radiation.

Variations in biomass between individuals and assemblages of the same species have also been previously attributed to regional variation in environmental stressors. All specimens of Neobolus wulongqingensis included in this analysis occur in dense aggregations (estimated 60 000 individuals per m²) from the same geographic locality and stratigraphic package with similar sedimentological features subject to similar environmental and depositional conditions. Consequently, the reduced biomass of tube-encrusted Neobolus wulongqingensis individuals cannot be attributed to environmental factors and a parasitic affect is the most strongly supported probable cause.

 

Aggregations of Neobolus wulongqingensis with associated attached obligate kletoparasitic tube-dwelling organisms, showing the density of individuals per unit area. Each square equals 1 cm² and each black dot equals one individual Brachiopod. (a) ELI GB-N-N-0300. (b) ELI GB-N-N-0301. Zhang et al. (2020).

In all instances, the apertures of tubes are orientated toward the Brachiopod commissure, spanning an arc (plan view) of about 150°. No tubes have been observed orientated toward the hinge line of the Brachiopod. Tubes consistently grow beyond the commissural margin of Neobolus wulongqingensis into, and slightly above but rarely beyond, the Brachiopod chaetal fringe. Critically, the dominant growth direction of the tubes aligns tightly along a vector between 40° and 70° either side of the median plane of symmetry of the Brachiopod; this alignment is most pronounced in shells with a single encrusting tube but the same orientation pattern occurs in shells with all numbers of tubes, strongly supporting a preferential growth direction in the tubes toward the antero-lateral margin of the Brachiopod shell.

 

Evidence demonstrating the associated encrusted tube-dwelling organisms were kleptoparasitic. (a) Shell interior of specimen ELI GB-N-0595A of Neobolus wulongqingensis from Wuding showing the presence of a paired spirolophe lophophore (as indicated by white arrows). Scale bar is 1 mm. (b) Micro X-ray fluorescence elemental mapping of iron for ELI GB-N-SJJ-0595A provides a high contrast image of the spirolophe lophophore (as indicated by white arrows). (c), (d) Rose diagrams of attached tube orientation for: (c) All measured individuals of Neobolus wulongqingensis (146 specimens) and; (d) Neobolus wulongqingensis individuals with only one attached tube (31 specimens). Each division represents a 10° interval. Intervals coloured in orange are those that correspond to the inhalant laminar currents generated by Neobolus wulongqingensis. For all numbers of attached tubes, orientations that align with inhalant laminar currents are preferred but for individuals with only one attached tube, where the symbiont has all available orientations still available, orientations that align with the inhalant laminar currents are strongly preferred. Zhang et al. (2020).

Five specimens of Neobolus wulongqingensis from Wuding Quarry preserve a partial spirolophe lophophore. A spirolophe lophophore produces two separate inhalant laminar feeding currents at the antero-lateral edge of the shell margin that match the preferred orientation and growth position of the encrusting tubes on shells of Neobolus wulongqingensis. The preferred orientation of growth demonstrates that the tube-dwelling organisms were not purely utilising the Brachiopod as a hard substrate on which to construct their tubes. This data when combined with the demonstrated empirical cost to the host in the form of reduced biomass, strongly supports kleptoparasitic behavior. Kleptoparasitism is a form of competition, where food that is either already in the possession of the host or which the host has expended energy on obtaining and capture is imminent, is stolen by the parasite. In Zhang et al.'s scenario, this involves the tube-dwelling organisms acting as intercept feeders, stealing a portion of the Brachiopod feeding stream before it reached the chaetal fringe. Erika Iyengar recognised six distinct morphological, behavioral and physiological criteria that characterise living sedentary/sessile kleptoparasitic interactions in a 2002 study of such relationships. At least five of these criteria can be directly applied to the relationship between the encrusting tube-dwelling organism and Neobolus wulongqingensis further reinforcing a kleptoparasitic relationship.

 

Additional rose diagrams of attached tube orientation for all values of attached tubes. Each division represents a 10º interval. Intervals coloured in orange are those that correspond to the inhalant laminar currents generated by Neobolus wulongqingensis. The radii of each sector is equal to the square root of the relative frequencies of observations for each group. (a) Individuals with 2 attached tubes. (b) Individuals with 3 attached tubes. (c) Individuals with 4 attached tubes. (d) Individuals with 5 attached tubes. (e) Individuals with 6 attached tubes. (f) Individuals with 7+ attached tubes. Zhang et al. (2020).

Kleptoparasitism is rarely identified in the fossil record, and no instances of kleptoparasitism, as far as Zhang et al. are aware, have been proposed for Cambrian communities. Detailed empirical investigations of the energetic and nutritional cost of kleptoparasitism to the host, even for extant systems, are few. For this reason, it is currently difficult to assess if the reduction in host fitness (about 26%) Zhang et al. detect for Neobolus wulongqingensis is typical of sessile kleptoparasitic relationships. Brachiopods are particularly vulnerable to exploitation by kleptoparasites, since active filter feeding represents the greatest energy expenditure in the life of Brachiopods, and the time lag between collection and ingestion of nutritionally beneficial particles also provides potential for other organisms to exploit this resource. Combined with the fact that the biotic interaction  Zhang et al. document is interpreted as obligate for the parasite, this suggests that the effect observed is likely greater than would be the case in facultative kleptoparasitic associations. Intriguingly, obligate kleptoparasitism is exceedingly rare in modern marine systems, which might suggest that this novel ecological relationship is always rare in benthic communities or has been secondarily lost some time during the Phanerozoic.

 

Artist’s reconstruction of the Wulongqing Formation benthic community, showing the dense aggregations of monotypic Neobolus wulongqingensis forming benthic ‘meadows’ on the soft sediment with their associated obligate encrusting kleptoparasitic tube-dwelling organisms. Rebecca Gelernter/Near Bird Studios in Zhang et al. (2020).

Verification of this kleptoparasitic relationship reveals that the heritage of parasite–host interactions can be traced back more than half a billion years to the rise of Bilaterian Animal communities during the Cambrian and further establishes the importance of the early Cambrian as a primary source of ecological novelty. Antagonistic biotic interactions have also been proposed as the drivers of widespread evolutionary phenomena such as the maintenance of sexual reproduction and genetic polymorphism at disease loci. Both of these phenomena are known drivers of biodiversity increase, suggesting that the already established presence of parasitic relationships in Cambrian communities potentially had a fundamental role in the upsurge in evolutionary innovation associated with the Cambrian Radiation.

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