Ocean research is the key to a sustainable future

By Vivian Kuppermann Marco Antonio

Illustration by: Joana Ho

Did you know that the UN declared next decade (2021-2030) as the decade of ocean science?

The ocean covers 71% of the Earth's surface. It helps to regulate the climate and provides a number of essential and, in some cases, untouchable resources for man. The ocean is a source of food, raw materials, energy, and transportation, in addition to being used for recreation and leisure.

Currently, more than 40% of the global population lives in regions within 200 km of the sea. In addition, 12 out of 15 megalopolises are coastal.

However, rapid industrial development and population growth have deeply impacted the oceans. Climate change, unsustainable exploitation of natural resources, pollution, and habitat degradation threaten the productivity and health of our waters.

Storms, proliferation of toxic algae, and coastal erosion are just a few of the consequences of this, which are devastating to communities living in coastal regions. Throughout human evolution, we have devised strategies to increase our resilience to such sea damage. But for how long will that be enough?

In 2015, the southwest coast of Brazil recorded winds of 106 km/h – a light hurricane typically has speeds of about 115km/h – so it was almost there. From these winds, the area suffered much damage including fallen trees and billboards as well as the destruction of some buildings.

In 2017, a windstorm left 38,000 homes without electricity and knocked down more trees and commercial signs. In the Port of Santos (SP), the largest in Latin America, a man was trapped in a crane.

This all happened because the sea water was warmer than normal, generating areas of low pressure and creating instabilities that allowed for the development of these strong winds.

And that's not all. Let's think about food:

Research shows that more than 50% of the fish species consumed for food in the world are being exploited above the sustainable limit. According to a 2006 study, led by Boris Worm of the University of Halifax in Canada, fish and seafood stocks are expected to collapse by 2048 if nothing is done to contain the loss of marine biodiversity.

Brazilian sardines (Sardinella brasiliensis), for example, are widespread throughout Brazilian cuisine. It is an extremely important species for the Southern and Southeastern regions of Brazil. Rich in various nutrients, it has always been considered a low-cost and nutritious food.

However, due to overfishing, its price has been rising over the years. Sardine stocks have already collapsed twice, once in 1990 and again in 2000. In addition, sardines are a species that suffers directly from the influence of environmental variation. By 2016, the amount of fished sardines was once again reduced to frightening levels. Some experts even characterized the episode as yet another collapse of the species.

This shortage was caused by abnormal water warming, a process that is associated with both the El Niño phenomenon, which occurred that year, and global climate change. It is worth mentioning that the region is also under political instability, with constant government turnovers and a reduction in ocean investments, which does not help the scenario at all.

Now imagine if phenomena like El Niño became more frequent and more intense with climate change? How long will the species last?

We need to find new ways to use natural resources and use them conscientiously. However, according to estimates by the UNESCO Intergovernmental Oceanographic Commission (IOC), the average national expenditure on oceanographic surveys varies from 0.04 to 4% of the total invested in research and development. This tiny budget is too little to achieve high-quality studies that involve long-term processes. Oceanographic research is quite expensive, because it requires ships, on-board laboratories, equipment, qualified personnel, et cetera.

But there is still time to reverse this situation.

Scientists and activists have gradually organized a social movement that led the United Nations, at its General Assembly in December 2017, to declare the next decade as the Decade of Ocean Science for Sustainable Development.

The initiative aims to encourage further action for a more integrated and sustainable ocean observing system, in order to facilitate making new discoveries by monitoring the coast and deeper waters, thus broadening research to promote ocean conservation and natural resource management. Activities for this period will be the responsibility of UNESCO's Intergovernmental Oceanographic Commission (IOC).

The process was long and hard fought. The 2012 Rio + 20 final document named "The Future We Want" made extensive references to the ocean. In 2013, the Global Ocean Commission was created, and in 2016, released its report about ocean degradation and the need for more effective policies to help restore the health and productivity of these waters. The 2030 agenda for sustainable development, launched by the UN in 2016, also highlighted the oceans as protagonists for conservation actions.

This UN statement is a glimmer of hope for a more sustainable future, but it calls for greater engagement of researchers, politicians, government, and the general public. More research, incentives, and respect are essential if we seek to advance our knowledge about the waters around us; we must make better use of available resources to ensure their existence for future generations.

It is vital to find solutions that allow us to understand the changes that are taking place and to reverse the damage before it is too late.

The UN initiative aims to transform the way global society views and uses the ocean. As suggested by goal 14 of the Sustainable Development Goals (SDG), it will coordinate its actions to foster the conservation and sustainable use of the ocean, seas, and marine resources.

Before progress can be made, it is essential to understand the lack of knowledge that we still have when it comes to the blue immensity:

• There is no internationally accepted methodology for estimating the economic value of services provided by the ocean to the human race
• Science is not yet able to assess the cumulative impacts of climate change, marine pollution, or anthropogenic activities on ocean health
• Only 5% of the ocean floor has thus far been mapped
• Over 250 million km2 of the ocean floor is in complete darkness, yet it shelters possibly millions of still unknown species
• Only 3 people have ever explored the deepest point of the ocean

The next decade will be our time to support, demand, and celebrate new achievements for the health of our ocean, so that we can make the services and resources of the ocean available to future generations.

References

Global Ocean Commission. The Future of Our Ocean: Next steps and priorities Report. Available at http://www.some.ox.ac.uk/research/global-ocean-commission (Global Ocean Commission, 2016).

Ministry of the Environment. Management Plan for the sustainable use of Sardines-Verdadeira in Brazil. Source:Ibama:

http://www.ibama.gov.br/sophia/cnia/livros/planogestaosardinhaverdadeiradigital.pdf (2011).

UNESCO. United Nations Decade of Ocean Science for Sustainable Development (2021-2030) UNESCO press release. Available at: https://en.unesco.org/ocean-decade (2017).

United Nations General Assembly. The future we want. Rio+20 conference outcome document A/RES/66/288. Available at:

Big Bang to the Dawn of Life: A Brief History

By Amanda Bendia

English edit: Katyanne M. Shoemaker

Part I - Big Bang: the origin of atoms and explosion of stars

Fourteen billion years ago: from the singularity
 to the greatest explosion of all time, the Big Bang.
https://www.smithsonianmag.com/
smithsonian-institution/ what-astronomers-are-still-
discovering-about-big-bang-theory-180949794/

It is estimated that the number of species that inhabit the Earth currently exceeds 8.7 million.  Not included in this calculation are the bacteria and archaea, which are microscopic prokaryotes. These microscopic organisms are single celled and devoid of a nucleus and membrane-bound organelles. The number of species of these prokaryotic microorganisms, surprisingly, surpasses the estimated 8.7 million eukaryotic inhabitants of the planet  (eukaryotes have a more complex cellular structure with nuclei and membrane-bound organelles and encompass all animals, plants, fungi, protozoa, etc.). Such immense values make us reflect on how such incredible diversity may have arisen throughout the history of our planet and the Universe.

To begin to discuss this question, we need to go back 15 billion years ago, to a point where everything we now know was concentrated in one single point. Can you imagine this? All of the humans and all other organisms that have inhabited the Earth, all of the objects we have produce with our technology, all of the molecules that make up our planet, all of the atoms of the billions of stars that we have already detected in the Universe, all of the Cosmos, gathered in this singularity. And then, there was the biggest “explosion” of all time: the Big Bang.

The origin of our solar system:
the ingredients for the origin of life in a
 cloud of stellar dust.
http://www.abc.es/ciencia/20150115/abci
-otro-origen-sistema-solar-201501151033.html

The Universe expanded, cooled and darkened. The first atoms formed and their accumulation generated large clouds of cosmic dust that would give rise to the galaxies. Within the galaxies, the first generation of stars formed; within them, atoms fused, first of hydrogen, but then giving rise to heavier chemical elements. When the fuel was depleted, the stars exploded and released these elements, enriching the stellar gases.

A new generation of stars began recycling these elements, and even heavier atoms were formed. The accumulation of clouds filled with cosmic dust - the nebulae - gave rise to planetary systems, including our solar system. During the formation of planet Earth, approximately 4.5 billion years ago, organic molecules composed of carbon formed and created all of the ingredients essential for the development of life.

Water on Mars and the deep ocean

By Jana M. del Favero

English edit: Katyanne M. Shoemaker

   At the end of September of 2015, NASA scientists publically confirmed the existence of liquid water on Mars, the Red Planet (https://www.nasa.gov/press-release/nasa-confirms-evidence-that-liquid-water-flows-on-today-s-mars). I remember when this news was released and how it caused certain uproar over the possibility of finding life there.

Landscape of the mysterious Red Planet; from the movie The Martian (https://www.empireonline.com/movies/features/martian-trailer-breakdown/).

   We know that life depends on water: it is the largest constituent of every living being (e.g. the human body is composed, on average, of 60% water), it is necessary for photosynthesis, and it is indispensable for several other vital functions. However, the phrase just quoted neglects an important detail: life, AS WE KNOW IT, depends on water.

   This made me remember the following cartoon, about two giant tubeworms talking to each other:

http://www.beatricebiologist.com

   I had posted this cartoon on my personal Facebook page previously, but then I reflected: how many of my friends know what giant tubeworms are? Or what hydrothermal vents are?

   Tubeworms are marine invertebrates in the phylum Annelida (yes, the same as the earthworms) and the class Polychaeta (aquatic worms), but they are sessile, i.e. they live fixed on an underwater surface. Their body is rounded by a tube, which extends the length of the whole body. The one illustrated in the cartoon are of the species Riftia pachyptila, popularly known as the giant tubeworms. These worms can live several kilometers down in the ocean, and they can reach a length of 2.4 m with a diameter of 4 cm. (more information on: https://en.wikipedia.org/wiki/Giant_tube_worm)

Giant tubeworms (https://en.wikipedia.org/wiki/Giant_tube_worm#/media/File:Riftia_tube_worm_colony_Galapagos_2011.jpg)

   A hydrothermal vent is a fissure in a planet's surface from which geothermally heated fluid emerges. The water that penetrates the crust at deep depths reacts with the minerals present, undergoing physical and chemical changes along the way. Usually there is an “oasis” of life along the hydrothermal vents. This is due to chemosynthesis, a process in which microorganisms use chemical energy to produce organic matter from carbon dioxide. 

Hydrothermal vent (http://oceanexplorer.noaa.gov/explorations/05lostcity/background/chem/media/sully2.html)

   Prior to the discovery of hydrothermal vents in the 1970s, the scientific community assumed that all life in the ocean depended on photosynthetic production, mainly produced by phytoplankton. Since photosynthesis depends on sunlight, it was like saying that all of the life in the oceans depended solely on the sun! The hydrothermal vents and the abundance of organisms that live around them proved the opposite.

   And that's the point I wanted to get to in this post: WE KNOW AS LITTLE ABOUT THE OCEAN AS WE KNOW ABOUT SPACE!

   We have explored around 1% of the oceans, and they cover 80% of our planet.(http://noticias.terra.com.br/ciencia/pesquisa/cientista-brasileira-conhecemos-pouco-mais-de-1-dos-oceanos,58d9a38790aea310VgnCLD200000bbcceb0aRCRD.html)Most of the ocean is only about 3 km deep, but Mars is about 60 million miles away from Earth! I am not saying that scientific exploration of space is not important, but I wish that the amount of money invested in space studies and the media attention space discoveries receive would also be given to the oceans. We know so little still, and yet they are so much more present in our lives.

Two reasons to watch the documentary “Mission Blue”

By Jana M. del Favero and Catarina Marcolin

Translated by Lídia Paes Leme

Edited by Katyanne M. Shoemaker

In our first post in the Women's session “Old challenges for current women” we received a suggestion by Prof. Otto Muller P. Oliveira to post about the documentary “Mission Blue.” Indeed this documentary deserves a special mention in our blog because, aside from the excellent production, its content is simply inspiring.

The documentary “Mission Blue” was released in 2014 and tells the story of the incredible biologist Sylvia Alice Earle, explorer, author, mother, grandmother (amongst a thousand other possible titles) and her campaign to create a global coalition of marine protected areas, called “Hope Spots.”

When watching the movie, it is impossible not to fall in love with and be inspired by two “characters.” The first is the organization itself, also called Mission Blue (www.mission-blue.org), which was created in response to the prize Sylvia Earle earned in 2009 at “TED PRIZE WISH” (watch the talk here). In that talk, Dr. Earle encourages the use of all possible media (movies, expeditions, internet, new submarines) in a campaign to inspire public awareness and support for a worldwide network of marine protected areas. If these “Hope Spots” are wide enough, it could be possible to save and restore the planet's blue heart! Today, Mission Blue is a coalition of over 100 groups, from multinational corporations to groups of scientists, concerned with matters of ocean conservation. Mission Blue's website brings an interesting but scary statistic: only 2% of the World’s ocean is protected, hence the importance of this kind of effort.

Font: https://www.ted.com/participate/ted-prize/prize-winning-wishes/mission-blue

The second reason to fall in love with this film is the main character, Sylvia Earle, a woman that turned 80 in August 2015, who actively keeps studying, exploring, diving, and defending the ocean (learn more https://en.wikipedia.org/wiki/Sylvia_Earle). Sylvia completed high school at the age of 16, undergrad at 19 and her masters at 20. During her Doctorate, this rhythm slowed down, due to marriage and kids, but soon Sylvia returned to her frantic pace. In 1964, when her kids where only 2 and 4 she traveled for 6 weeks on an expedition in the Indian Ocean. According to Sylvia, she didn't know she'd be the only woman on board, for she was invited as the only botanist, not only woman. A reporter approached her in Mombassa, Kenya, from where the ship would depart, and Sylvia remembered being interested in talking about her work, but the reporter only wanted to know about what being on the ocean with so many men would be like. After all, the article was called “Sylvia sails away with 70 men, but she expects no problems.”

Despite everything appearing well, Sylvia implies in some interviews that her scientific expeditions may have lead to the end of her first marriage. This is a recurring difficulty faced in the scientific world; it is common to have campaigns where the scientists are away for weeks, sometimes months, without any communication with family. In 1966 Sylvia finished her Doctorate, and in 1968 she traveled 30m deep in the waters of the Bahamas in a submersible, 4 months pregnant with her 3rd child and in her second marriage.

In 1969 she signed up to participate in the project Tektite, where scientists lived weeks in a laboratory placed under the sea, at 15m depth. Despite her 1000+ hours of diving experience and her excellent written proposal, she was not allowed to live together with men underwater in Tektike I. The following year however, she was invited to lead the Tektite II project, with a women-only team. The success of this team was an important milestone for women in research, and it set a precedent for future aquatic and space expeditions to include women in their teams.

Picture: Bates Littlehales.
Font: http://images.nationalgeographic.com/wpf/media-live/photos/000/450/cache/sylvia-earle-habitat-window_45011_600x450.jpg

After her experience as a mermaid, Sylvia became a popular face in the media and her career took off (we'd say, all other qualities aside, she also has a lovely face). In 1979 Sylvia walked on the ocean floor at depths never before touched by any other human. This was done using what is called a JIM SUIT, and was used at a depth of almost 400m. This adventure resulted in the book “Exploring the Deep Frontier.”

Image: Dr. Sylvia Earle in Deep Rover Submarine. Font: http://ww2.kqed.org/quest/wp-content/uploads/sites/39/2012/05/Sylvia-Earle-in-a-Deep-Rover_horiz.jpg

In the 80's, together with the engineer Graham Hawkes, she started a company to create submersible vehicles, like Deep Rover. This partnership ultimately led to her third marriage, one where the offspring were the submarines created by them. One of her daughters currently works with her in her company.

When asked if she had problems reconciling family and career, Sylvia says yes, many, and that she tried to rearrange her life, having a laboratory and a library at home. For women that dream about following a scientific career, Sylvia advises “Try, you'll never know how it would be if you don't try.”

Font: http://mission-blue.org/wp-content/uploads/2013/01/IMG_1065.jpg

Other than the documentary itself, we recommend this short video: http://voices.nationalgeographic.org/2013/06/14/in-her-words-sylvia-earle-on-women-in-science/?source=newsbundlearticles

The extraordinary life of whale carcasses in the deep ocean

By Joan Manel Alfaro Lucas

Translated by: Lídia Paes Leme

Edited by: Katy Shoemaker

This story starts in 1987, when, during an oceanographic expedition lead by Dr. Craig Smith (University of Hawaii), the research robot Alvin found a whale carcass on the ocean floor in Santa Catalina Bay, California, 1240 meters deep (Smith et al., 1989). This discovery reinforced an idea that had been suggested before, that even though whale deaths are common in coastal zones, many die in spots far away from beaches and sink down to the depths of the ocean.

The deep ocean covers 63% of the planet's surface and is considered the biggest biome on Earth. It is unique and extreme due to its low temperatures, high pressure, and darkness (light doesn't penetrate more than 200 meters below surface, where the deep ocean starts). The absence of light makes organic matter production via photosynthesis impossible. Because of this, the deep ocean ecosystem is limited in food sources and depends almost exclusively on the sinking of organic matter produced in the surface waters. In the vast, cold, dark deserts of the deep ocean known as the abyssal plains, the few organisms that survive there filter water and sediments to take in the little organic matter that sinks down from the surface.

So now what about that Californian whale that Dr. Smith found? The carcass was completely missing meat, and other indicators suggested the whale carcass had been there for several years. However, the skeleton and the sediment around it were bursting with life! There were worms, snails, gastropods, dense mats of bacteria, and bivalves such as clams and mussels. The carcass was a real oasis of life in the deep desert of the bay. The scientists began to understand that, for an environment so poor in nutrients, the arrival of a whale carcass is an extraordinary event.

Whales are the largest animals that inhabit Earth. The blue whale can be 30 meters (~100 feet) long and weigh 120 kilotons and is the largest animal that has ever existed on our planet. To the desert depths of the ocean floor, their carcasses are the biggest source of organic matter that arrives from the surface. One carcass from a 40-kiloton whale is the equivalent of 2000 years worth of organic matter falling down at once!

Image 1 – Whale carcass on the deep ocean floor of Santa Catalina Bay, California, densely colonized by chemosynthetic bacterial mats. Photo by Craig R. Smith, University of Hawaii, USA.

Some of the organisms found for the first time on the carcass by Dr. Smith became much more interesting when identified. For example, some bivalve species found there are known to have symbiotic relationships with chemosynthetic bacteria. Those mussels feed on the matter produced by the bacteria, a process similar to what shallow water corals have with photosynthetic organisms. As it turns out, the dense bacterial mats found on the carcass were of that kind of bacteria.

Similar to vegetables in the terrestrial environment, these chemosythetic bacteria form the base of the food chain in the deep ocean. Chemosynthetic communities feed on organic compounds, some of which can be abundant on the sea floor. This is the case in hydrothermal vents, which form in parts of the floor where volcanic activity is elevated and hydrocarbons flow from underground reservoirs (post about hydrothermal vents here). The bivalve species associated with the whale carcass were discovered for the first time at cold hydrothermal vents! These similarities suggested that the whale carcass acts as a trampoline for the common habitants of different chemosynthetic communities to disperse, as they are usually separated by distances larger than can be reached by larval dispersion (Smith et al., 1989).

This discovery, other than being revolutionary for the ecology of chemosynthetic communities, led several groups of scientists to research more about these ecosystems. Rather than looking for a carcass on the vast ocean floor (a real needle in a haystack situation), scientists started to sink dead whale carcasses with weights. They were able to sink them in a determined spot where they could sample whenever needed. After these experiments, scientists began to understand that not only chemosynthetic communities developed in the carcasses, but also there were extremely diverse and abundant communities that explored the carcasses in amazing ways… for almost a century!

Image 2 – Hagfish feeding on a whale carcass during the ecological state of the mobile necrophagous organisms in Santa Catalina Bay, California, USA. Photo by Craig R Smith, University of Hawaii, USA

The whale carcasses develop mostly three ecological successive states, meaning three communities can be distinguished throughout time (Smith et al., 2015). The first stage starts with the arrival of the carcass in the bottom and includes the mobile necrophagous organisms. Hundreds of animals, like hagfish, drill the meat while sharks bite big chunks off. These communities, similar to vultures in a savanna, remove several dozen kilograms by day and can consume all the meat in up to two years, depending on the size of the carcass.

Image 3 – Crabs, snails, and anemones colonizing the skeleton during the enrichment and opportunist stage in a whale carcass in Monterey Canyon, California, USA. Photo by the Monterey Bay Aquarium Research Institute. USA.

The second stage involves the enrichment of opportunists and can also last up to two years. During this period, high densities of worms, crustaceans, and other invertebrates colonize the sediment around the skeleton that was exposed after the flesh was consumed. These invertebrates feed directly on the left over fat and meat left behind by the necrophagous organisms, as well as the bones, which are rich in protein and fat.

The last stage, the one Dr. Smith's whale was in when he found it, is the sulphophilic stage. Some microorganisms are able to penetrate the dense bone structure and access the big quantities of fat remaining in the interior of the bones. These organisms use the sulfur dissolved in water to digest the fat, creating inorganic compounds as secondary products. Similar process can also occur in the surrounding sediment, which was impacted by the organic matter of the carcass. This creates enough of a flux to develop a community based on chemosynthesis. This is the longest stage, lasting up to 80 years.

The discoveries around whale carcasses don't stop there. Since 1987, when Dr. Smith studied the first deep ocean carcass, 129 new species have been discovered, many of them only found in those communities. The most surprising one was discovered in 2002, when Osedax, a new kind of worm, was discovered in Monterey Canyon, California, at 2891 meters deep (Rousse et al., 2004). The species in this genre are sessile and don't have a mouth nor anus, nor any kind of digestive system, yet they feed on whale bones!

Osedax have a structure called a root, which helps to answer the multiple mysteries surrounding these organisms. This structure, with globular ramifications, fixes the organism to the bones and has pumps that acidify the bone matter. The “soup” produced in this process is sent up through the root into internal structures, where endosymbiotic bacteria are responsible for digestion. These worms are capable of completely decaying a whole juvenile skeleton (containing less calcified bone or fat then adults) in one decade. Impressive, no? Just wait…

All of these structures and endosymbionts only apply to female Osedax. The males are microscopic dwarves that live inside of the females, as simple sperm reservoirs. The Osedax larvae that are found on a skeleton develop as female, but if they find other females, they can get absorbed and develop as pedomorphic males, meaning they only develop sexually and not fully morphologically, retaining larval characteristics. Each female can absorb hundreds of males, which is believed to be a successful reproductive strategy.

Image 4 – Whale bone densely colonized by Osedax in Monterey Canyon, California, USA (left) and Osedax japonicus specimen with a yellow-colored root. Photos by Monterey Bay Aquarium Research Institute, USA, and Norio Miyamoto, from Japan Agency form Marine-Earth Science and Technology, respectively.

Organisms like Osedax show that whale carcasses are not only an oasis of life in the deep ocean, but also showcase uniquely evolved and specialized life forms. However, are the carcasses sustaining similar communities in all of the ocean basins? Or, like in hydrothermal vents, does each basin sustain communities with different evolutionary histories? This kind of question is still very hard to answer because practically all of the natural and placed carcasses have been studied in the Northern Pacific.

Only in 2010 was a natural carcass discovered on the seafloor near Antarctica, and, more recently in 2013, in the Southwest Atlantic off of the Brazilian coast. The latter is currently being studied by Brazilian and Japanese researchers, and is the topic of my Master's project at the University of São Paulo. This represents the first whale sink community to be studied in all of the deep Atlantic. The results of the research are beginning to emerge, reinforcing some previous hypotheses and explaining even more about the functioning of various ecological processes.

Many questions are still to be answered, and many more will be generated in the future. These extraordinary communities, not known 30 years ago, are a bottomless source of surprises!

References, links and videos:

Smith, C.R., Kukert, H., Wheatcroft, R.A, Jumars, P.A., Deming, J.W. (1989) Vent fauna on whale remais. Nature, 341. Pp 27-28.

Rouse, G.W., Goffredi, S.K., Vrijenhoek, R.C. (2004) Osedax: Bone-Eating Marine Worms with Dwarf Males. Science, 305.Pp 668-671.

Smith, C.R., Glover, A.G., Treude, T., Higgs, N.D., Amon, D.J. (2015) Whale-Fall Ecosystems: Recent Insights into Ecology, Paleoecology, and Evolution. Annual Review of Marine Science, 7. Pp 571-596.

About Joan Manel Alfaro Lucas:

A biologist from the Universitat Autonoma de Barcelona, Barcelona, I did a one year internship at the Federal University of Minas Gerais, which allowed me, among other things, to get to know Brazil and learn Portuguese. I'm passionate about the ecology of deep ocean communities, especially chemosynthetic ones. I did a Masters at the Oceanographic Institute of the University of São Paulo, where I had the opportunity to study the first whale carcass discovered in the deep Atlantic ocean. Other than that, I have experience in oceanographic cruises, sailed 2800 nautical miles across the southwest Atlantic, sampling, sorting and identifying benthic invertebrates, stable isotope analysis, and using the R language in ecological research.