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OPEN Deep-sea hydrothermal vents

as natural egg-case incubators at the
Galapagos Rift
Received: 21 September 2017
Accepted: January 10, 2018
Published: xx xx xxxx
The discovery of deep-sea hydrothermal vents in 1977 challenged our views of ecosystem functioning
and yet, the research conducted at these extreme and logistically challenging environments still
continues to reveal unique biological processes. Here, we report for the frst time, a unique behavior
where the deep-sea skate, Bathyraja spinosissima, appears to be actively using the elevated temperature
of a hydrothermal vent environment to naturally “incubate” developing egg-cases. We hypothesize that
this behavior is directly targeted to accelerate embryo development time given that deep-sea skates
have some of the longest egg incubation times reported for the animal kingdom.
Similar egg incubating behavior, where eggs are incubated in volcanically heated nesting grounds, have
been recorded in Cretaceous sauropod dinosaurs and the rare avian megapode. To our knowledge, this is
the frst time incubating behavior using a volcanic source is recorded for the marine environment.

Despite being the largest biome, the deep sea remains the least explored ecosystem on earth1,2. In 1977, the dis
covers of the frst deep-sea ecosystems supported by hydrothermal vent fuid emissions at the Galapagos Rif,
challenged our views of ecosystem functioning and fueled new hypotheses about how life on earth could have
originated around these chemically reactive environments3–5 . Forty years later, we now know that hydrothermal
vent ecosystems exist in every ocean basin, supporting rich communities and unique biological processes
Initially considered isolated patches of life within a barren deep-sea foor, we are beginning to appreciate that these
ecosystems interact with the surrounding environment and infuence global geochemical cycles7,8 . Most
hydrothermal vent sites remain unexplored, and our understanding of the ecology of these ecosystems in most
parts of the world remains limited. Furthermore, some of these chemosynthesis based ecosystems are now under
threat from human activities and are targeted for exploitation of their mineral resources10,11. Here, we report for
the frst time a unique behavior where the Pacifc white skate Bathyraja spinosissima, one of the deepest living of
all known skate species12, uses active hydrothermal vent felds as a natural incubator for their external egg-
capsules. To the best of our knowledge and understanding, this is the frst time this incubating behavior at an active
hydrothermal vent feld has been recorded for a species within the marine environment.
Te Galapagos Platform in the eastern tropical Pacifc consists of 13 major volcanic islands and numerous
seamounts that straddle the equator13. To the north of the archipelago and parallel to the equator, the Galapagos
Spreading Center (GSC) extends for over 1000 km west to east, crossing the Galapagos Marine Reserve north of
Darwin Island (Fig. 1). Previous exploration of the area revealed the presence of active hydrothermal vents,

1 Charles Darwin Research Station, Av Charles Darwin s / n, Puerto Ayora, Santa Cruz, Galapagos Islands, Ecuador.
2Galapagos Marine Research and Exploration (GMaRE), joint CDF-ESPOL research program, Charles Darwin
Research Station, Santa Cruz, Galapagos Islands, Ecuador. Pristine
3 Seas, National Geographic Society, Washington, 4
5
DC, USA. Harvard Microrobotics Laboratory, Harvard University, Cambridge, MA, USA. Department of Ocean
Engineering, University of Rhode Island, Narragansett, RI, 02882, USA. 6Pacific Shark Research Center, Moss Landing
7
Marine Laboratories, 8272 Moss Landing Rd, Moss, CA, 95039, USA. Department of Ichthyology, California
Academy of Sciences, 55 Music Concourse Drive, San Francisco, CA, 94118, USA. 8 South African Institute for
Save9Our Seas Shark Research Center and Guy
Aquatic Biodiversity, Private Bag 1015, Grahamstown, 6140, South Africa.
Harvey Research Institute, Nova Southeastern University, 8000N Ocean Drive, Dania Beach, FL, 33004, USA.
10 Department of Biology, Pennsylvania State University, University Park, State College, PA, 16802, USA. 11 Ocean and
Earth Science, University of Southampton, Waterfront Campus, Southampton, SO14 3ZH, UK. 12 Marine Geoscience,
National Oceanography Center, European Way, Southampton, SO14 3ZH UK, UK. Correspondence and requests for
materials should be addressed to PS-d.-L. (email: [email protected])

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Figure 1. Bathymetric map of the Galapagos Marine Reserve (colored) with the location of the Galapagos
Spreading Center (green line) and the Iguana-Pinguinos hydrothermal vent site (red dot) located 45 km north of
Darwin Island. Gridded bathymetric data provided by the General Bathymetric Chart of the Oceans (GEBCO) 30 arc-
second grid (accessed via http://www.gebco.net/). Map created in ESRI ArcMap (version 10.3.1).

including the Iguanas-Pinguinos site that is located 45km north of Darwin Islands14. Tis particular hydrother mal
site was frst described in 2008 as being in a mature / waning phase with macrofauna dominated by crabs, bivalves,
anemones and shrimp14. These area is characterized by vigorous, active venting and dispersing clouds of 'black-
smoker' hydrothermal plumes.

Results
Remotely Operated Vehicle (ROV) Surveys. Our surveys using the Hercules ROV recorded a total of 157 egg-
cases in and around the Iguanas-Pinguinos vent feld (Fig. 2; Supplementary material 1). Egg-cases were
encountered right from the beginning of the dive when the ROV landed next to an active black smoker chimney
located at 1660–1670 m depth (Fig. 3a). Over the duration of the 24-hour dive, most of the egg-cases seen were
distributed within <150m of two active black smoke chimneys (Figs 2, 3a). The largest deposition of egg-cases was
on a basaltic ridge bathed in cloudy water venting from the nearby black smoker (Fig. 3b). Te colors of live egg-
cases ranged from golden to dark brown, suggesting that they were under diferent developmental stages (Fig. 3c –
f). Te majority of egg-cases visible lacked evidence of fouling suggesting they were recently deposited, however
there were ofen older spent egg-case remnants under these, indicating that this deposition site has been used for
many years (Fig. 3d). Some egg-cases were located within less than a meter from an active vent chimney (Fig. 3g).

Environmental data. A temperature probe and a CTD sensor (Conductivity, Temperature, Depth) recorded
temperatures during the duration of the dive. Te temperature probe was located ~ 10 cm above the bottom of the
ROV, while the CTD was located 1.5m above the bottom of the ROV. Mean ambient bottom water temperature was
2.76 ° C ± .0.01 SD and salinity was constant at 34.6 psu. Water temperature measured between 0.6–7.1 m above
the seafoor was generally higher closer to the active smokers, but difuse venting was also observed in some areas
of the vent feld along the survey track. Te highest number of egg-cases (58%) in both areas of the Iguanas-
Pinguinos vent feld were recorded within <20m of black smoker chimneys (Table 1). Anomalous tem peratures
exceeding 3.1 ° C were occasionally recorded as high as 2.78m above egg-cases in some places (Fig. 4).
Over 89% of the egg-cases were observed while the ROV measured temperature above the ambient bottom water
temperature of 2.76 ° C.

Species ID. We collected a total of 4 egg-cases using the Hercules ROV manipulator arm (Fig. 3f). During this
collection, the ROV altitude was <1m above the seafoor and the temperature probe measured 2.9 ° C. Egg-cases
were large, measuring 110mm in length (excluding horns), and the surface was rough and striated15. Te lateral
keels were narrow with 10% of the maximum egg-case width. Horns were fattened and tapered towards the tips
with tips becoming thin but not flamentous. Anterior horns where shorter and wider than posterior horns, the latter
being 2 times larger than the length of the anterior horns (Fig. 3f).
Based on visual examination, the egg-cases resembled those of Bathyraja spinosissima (Beebe and Tee-Van,
1941) a species previously reported associated with hydrothermal vents in the Eastern Pacifc and with a depth

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Figure 2. Higher resolution bathymetric map of the Iguanas-Pinguinos hydrothermal vent site at the Galapagos
Marine Reserve. Map displays Hercules ROV CTD temperature (grouped in 1 ° C increments) and position of
individual egg-cases (black stars). Gray shaded areas denote distance from an active black smoker chimney.
Contours generated from gridded bathymetric data provided by the General Bathymetric Chart of the Oceans
(GEBCO) 30 arc-second grid (accessed via http://www.gebco.net/). Map and associated shapefles created in
ESRI ArcMap (version 10.3.1).

range that extends beyond 2900 m15. Although we did not encounter any adult skate specimens on our survey of
the Iguanas-Pinguinos vent site, B. spinosissima were observed during a previous dive at the Tempus Fugit hydro
thermal vent site located about 750 km to the east, within the Galapagos Spreading Center (Fig. 3h).
In addition to the visual examination of egg-cases and ROV video footage, analysis of 603 basepairs of the 5 ÿ
mitochondrial COI region (GenBank Accession no. MF158863) resulted in a 100% identity match to a sequence in
the BOLD Systems database (GenBank Accession no. FJ164384). Although this specimen captured of Vancouver
Island (Canada) was reported as Bathyraja spinicauda, the reported range for B. spinicauda is restricted to the
North Atlantic and a taxonomic re-assessment of this specimen confrmed it to be B. spinosis sima15. Terefore, we
conclude that the egg-cases collected were B. spinosissima.

Discussion
Te presence of a B. spinosissima egg-case nursery (as described by16) in an active hydrothermal vent feld, where
even the temperatures several meters above the substrate were ofen higher than ambient water, implies that this
species is utilizing heat at the active Iguanas -Pinguinos vent site to incubate its egg-cases. We hypothesize that
this behavior is directly targeted to accelerate embryo development times. Deep-sea skates have some of the
longest egg incubation times reported for the animal kingdom, with species of the same genera, such as B.
parmifera in the Bering Sea, having incubation periods of 1290 days at water temperatures of 4.4 ° C17. Assuming
the ambi ent water temperature of about 2.76 ° C is relatively constant year-round, and a development rate similar
to B. parmifera, even a conservative correlation between temperature and embryonic development would suggest
an incubation time of over 1500 days. Tis direct relationship between temperature and development time has been
reported for several deep-sea organisms, including other oviparous Chondrichthyans17,18. While we recorded
temperature increases of <1 ° C above ambient in the water above where egg-cases were abundant, these are
con servative measurements considering that the temperatures reported here were collected at an average altitude
of 3.5m above the seafoor. These temperatures directly on the seafoor, where difuse venting and conductive
heating may occur, are likely to be considerably higher.
Previous evidence of egg incubation at hydrothermal sites exists in the fossil record, where a group of neosau
ropod dinosaurs used soil thermo-radiance and moisture of hydrothermal origin to incubate their unusually larger

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Figure 3. Hercules ROV imagery from the Iguanas-Pinguinos hydrothermal vent site at the Galapagos
Marine Reserve (excluding H). All images were taken between 1666-1649 m depth. Scale bar represents
10 cm. (a) High-temperature black-smoker chimney at Iguanas-Pinguinos East; (b) egg-cases observed
along ridge in close proximity to black-smoker; (c) clutch of egg-cases with dark brown coloration; (d)
bright-yellow egg case among associated vent fauna; (e) older egg-case with signs of fouling; (f) collection
of egg-case using the Hercules ROV robotic arm; (g) eggcase located within <1 m of active vent chimney
(temperature recorded by the probe was 4.52 ° C); (h) an adult Bathyraja spinosissima recorded on a previous
dive at the Tempus Fugit hydrothermal vent site located about 750 km to the east, but also within the
Galapagos Spreading Center. Footage and screenshots provided by Ocean Exploration Trust Inc (www.
oceanexplorationtrust.org/).

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Number of egg-case
observations
Distance from active
hydrothermal site West East Total

10m 3 4 7

20m 29 55 84

50m 2 14 16

100m 1 thirty 31

150m 0 10 10

Table 1. Number of egg-cases observed at diferent distances from the active Iguanas-Pinguinos hydrothermal sites.

Figure 4. Number of egg case observations recorded as a function of water temperature detected by the
Hercules ROV. Te measured average background water temperature of 2.76 ° C is indicated by the red vertical
line. ROV altitude, refecting the vertical position of the temperature probe, ranged 0.6–7.1m. Recorded temperatures
were consistently higher closer to the seafoor.

eggs during the Cretaceous19. In contemporary times, megapode birds, such as the Polynesian megapode
Megapodius pritchardii, burrow their nests in volcanically-heated soils on Niuafo'ou Island in Tonga20. Furthermore,
several additional species of reptiles and birds actively seek specifc soil temperatures to achieve optimal egg incubation.
In the marine environment, aggregation of the egg-brooding deep-sea fsh Pychrolutes phrictus and the cephalopod
Granelodone spp. Have been also recorded around “cold” seeps, where fows generate slight increases in tempera
ture of 0.1–0.2 ° C21. Despite these temperature anomalies, and the fact that Granelodone boreopacifca conducts
the longest-known egg-brooding period of any animal (with over 1500 days of incubation time) 18, egg occurrence
was not correlated with temperature. Tis suggests that there may be other reasons for egg deposition in this area. For
example, these cold seep sites could provide an additional food source that could infuence the location of egg deposition21.
In addition to decreasing egg incubation periods, this is the frst record of a hydrothermal vent habitat serving as
an egg-case nursery site, a discrete habitat with extremely high densities of egg cases when habit compared to sur
rounding similarats16. Among the elasmobranchs, skates are the only group known to be strictly oviparous, where
females produce large collagen egg cases containing a large yolk mass 22,23. Egg-case nursery sites for mem bers
of the genus Bathyraja have been previously identifed across most ocean basins and in diverse habitats like rocky
reefs, submarine canyons, seamounts or even methane cold seeps17,24,25. Hydrothermal vent sites may also have
other advantages as a juvenile nursery, although previous studies have revealed that juvenile skates of other species
of the genus Bathyraja leave egg-case nurseries afer hatching16.26.
One out of four species of Chondrichthyans are threatened with extinction, mainly as a result of over-fshing27.
Deep-water Chondrichthyans species are among the least productive given their long turnover times (ie slower growth,
later age at maturity and increased longevity) and, as a consequence, they may have higher extinction risk than other
oceanic and continental shelf species28,29. Terefore, understanding their reproductive processes and key habitat
requirements is vital to predict population stability and inform efective conservation strategies, especially under
conditions of global change17. In March 2016, the Ecuadorian government created a 40,000 km2 marine sanctuary
to protect unique underwater communities around Darwin and Wolf islands30,31. Tis fully non-extractive reserve also
protects adjacent seamounts, and it includes the Iguanas-Pinguinos vent site, thus protecting the frst known nursery
for deep-water predators associated with active hydrothermal vents. In 2015, the North Pacifc Fishery Management
Council designated eight deep-sea skate egg-nurseries in the eastern Bering Sea as habitat areas of particular
concern, and this represented the frst ofcial recognition of this habi tat type worldwide16. Further research should
focus on identifying and promoting the protection of additional Chondrichthyan deep-sea nurseries, given the
continuous expansion of fsheries towards the deep-sea and the intrinsic vulnerability of this group of species32–34.

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Methods
NA064 Galapagos cruise platforms. In June 2015 we conducted a 10-day collaborative research cruise (NA064)
aboard E / V Nautilus between the Ocean Exploration Trust, the Charles Darwin Foundation and the Galapagos
National Park Directorate to explore deep-sea environment of the Galapagos Marine Reserve. On June 30th
2015, we conducted dive H1439 to explore the active Iguanas-Pinguinos hydrothermal vent site (2 ° 6.322 ÿ
N, 91 ° 56.538 'W). All methods were carried out in accordance with relevant guidelines and regulations by the
Galapagos National Park Directorate under research permits PC-26-15 & PC-45-15. All experimental protocols
were reviewed and approved by a Galapagos National Park Directorate's committee that asses animal care in
research activities.

ROV surveys. Exploration of the seafoor was carried out using the two-body Remotely Operated Vehicle
(ROV) system Argus and Hercules, each rated for 4 km water depth. Video and still images of the sites were
acquired using Insite Pacific Zeus Plus HD color video cameras on both vehicles, each equipped with a 10 ×
mechanical zoom lens. CTD data was recorded using a calibrated Seabird FastCAT49 equipped with a circu
lating pump. Te temperature probe used was an Omega PT100 RTD sensor housed in a custom titanium
sheath (designed and fabricated by the Woods Hole Oceanographic Institution), which was calibrated against
the Seabird FastCAT49 unit. Tese two units reported temperatures within 0.04 ° C of each other. Seafoor
navigation was performed using a combination of sensors including a LinkQuest Tracklink 5000 USBL system,
RDI Workhorse Navigator 600 kHz DVL, IXSEA OCTANS 6000 fiber-optic gyrocompass, and a calibrated
Paroscientific DigiQuartz depth sensor. DVLNAV sofware was used to process these data35.

Sample collection and genetic analysis. Egg-case samples were collected using the ROV manipulator arm
and placed on sample boxes aboard Hercules ROV for recovery. Once aboard the ship, two egg-cases were
opened to sample for molecular analyzes and further examination. Both egg-cases were at a very early
develop ment stage, with no clear presence of the embryo.
DNA was extracted from egg-cases with the QIAGEN DNeasy Blood & Tissue Kit (QIAGEN Inc., Valencia,
CA). The 5 ÿ region of the mitochondrial COI gene was sequenced on an AB 3130 genetic analyzer via the primer
pair FF2d (5ÿ-TTCTCCACCAACCACAARGAYATYGG-3 ÿ) and FR1d (5ÿ-CACCTCAGGGTGTCCG
AARAAYCARAA-3 ÿ) following the protocol outlined in36. Sequences were assembled in geneious 7.1.737 and
subsequently compared to species level barcode data in the BOLD Systems database (www.boldsystems.org).

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Acknowledgements
We are thankful to the Ocean Exploration Trust as well as the pilots and crew aboard the E / V Nautilus during
cruise NA064 for their assistance in sample collection and exploration using the Hercules ROV. Thank you to the
NOAA Office of Exploration and Research for funding the E / V Nautilus Exploration Program (NA15OAR0110220).
Further acknowledgments and thanks go out to the Charles Darwin Foundation and the Galapagos National Park
Directorate for their collaboration and assistance in the exploration of the Galapagos Platform conducted under
research permit No. PC-45-15. We also gratefully recognize the Government of Ecuador via the Ecuadorian Navy
for permission to operate in their territorial waters. Tis research was supported by a grant from the Helmsley
Charitable Trust and Save Our Seas Foundation (genetics analysis). Tis is contribution number 2184 from the
Charles Darwin Research Station.

Author Contributions
Pelayo Salinas-de-León, Brennan Philips, David Ebert, Mahmood Shivji, Florencia Cerutti-Pereyra, Cassandra
Ruck, Chuck Fisher and Leigh Marsh performed the research and contributed to the paper.

Additional Information
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-20046-4.
Competing Interests: These authors declare that they have no competing interests.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and
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