Porcentaje del plan cumplido (PPC)

Geochemists traditionally pursue discovery based science, whereas ecologists often follow a more traditional and rigid hypothesis-based approach to their work. As a biogeochemist, I have followed both approaches and let my curiosity lead me to the most interesting science. Stable isotope methods and instrumen- tation “grew up” in earth science departments, but eventually ecologists and biologists have realised how powerful these methods are for figuring out major questions such as who is eating whom in a food web. Not only is the source of an animal’s diet important, but dietary resources and thus energy flow in an ecosystem is something that can be influenced by environmental parameters, specifically climate change over time. Answering questions about why organ- isms go extinct transcends the distinct disciplines of evolutionary biology and palaeontology. Stable isotope biogeochemistry has proven to be a strong factor in uniting and integrating these two fields of study.

In 1999, the first IsoEcol conference was held in Saskatoon, Saskatch- ewan, Canada, hosted by Len Wassenar and Keith Hobson. I was the invited keynote speaker. Everyone at the conference was measuring or was working with someone who could measure bulk isotopic compositions in plants and animals. For the first time while presenting isotope data like this to ecologists

and biologists, I didn’t get the usual question from the audience “Do you really

believe these magic numbers can tell us anything important?” However, only one or two other attendees were using compound specific isotope analysis (CSIA) methods, so our Geophysical Laboratory contingent, Mark Teece and Matt Fantle, really stood out from the crowd. The field of isotope ecology has grown so much that almost each issue of every ecological journal includes at least one stable isotope paper. While the eco-bio people have embraced the approach, it is the geochemists who have pushed the envelope by developing new techniques. Such innovations in techniques have all come from geochemists – in my field biogeochemists or organic geochemists. The compound specific isotope analysis field is an excellent example.

While our group at the Geophysical Laboratory forged ahead with CSIA using liquid chromatography methods, these were tedious, time-consuming

techniques, and really forced us to measure what Tom Hoering termed “five

well-chosen samples.” John Hayes and his students were pioneering continuous flow methods that worked directly with gas chromatography-IRMS (Matthews and Hayes, 1978), see Text Box 10.1.

Text Box 10.1 – Development of GC-C-IRMS

At this time, both of the major IRMS companies, Finnigan MAT and VG Instru- ments, were perfecting versions of an interface that allowed compounds in a stream of He to enter the IRMS source at pressures that could be handled by the source turbo pump. Computer software was also being drafted to handle ion counts from

distinct peaks rather than static measurements from dual inlet systems. Finnigan’s team included John Hayes (Indiana University), Martin Schoell (Chevron), Kate Freeman (Indiana University graduate student), Willi Brand (Finnigan), and Bob Dias (Chevron) (Freeman et al., 1989). They worked with software engineer Margaret Ricci (Penn State) to produce Finnigan’s first commercial model of the gas chromatograph-combustion system (GC-C-IRMS). At VG, Jeanette Jumeau, Steve Macko (University of Virginia), and Michael Engel (University of Oklahoma) developed a parallel instrument. Competition for customers was fierce!

I received NSF funding for one of these instruments in 1990, and Tom Hoering and I travelled to see both companies’ products. In the VG factory in Manchester, England, the PRISM IRMS system in their demo lab was not operational. We spent two days looking at the GC-C set up, but were unable to determine how it worked. We took a second trip to John Hayes’ lab where the Finnigan instrument was demonstrated by Kate Freeman, who has now been elected to the National Academy of Science. Kate started her earth science career as an undergraduate intern at the Geophysical Laboratory, washing glassware for me and working on some organic extractions for Tom, so we knew her well. The 252 IRMS was a real winner, and had the backing of people that we knew well, Freeman and Hayes, sealed the deal for us. Kate recalls, “I remember showing the instrument to Tom. It was an exciting moment for me (and a bit terrifying). Tom walked into the room and immediately disappeared. We were startled but quickly found him crouching behind the instrument to see the pumps and guts of the thing.“

The first system I received was relatively primitive compared to the GC- Isolink systems used today. There were four needle valves for controlling GC flow that needed to be fiddled with daily, if not before every sample. Combustion reactors were expensive, $1000 in 1992, and broke easily when they were installed. Jeff Silfer (Michael Engel’s Ph.D. student) joined the Geophysical Laboratory for a short time as a postdoc and helped get our system running. It worked slightly better than the old liquid chromatography methods, and we could measure hydrocarbons, fatty acids, and other molecules that liquid chromatography could not easily separate. Much of our work at that time was discovery-based and unpublished. Our questions were very simple: what is the

variation in the δ13_{C of certain compounds in a plant? How do the δ}13_{C values}

compare between species? Silfer brought his amino acid methods (Silfer et al.,

1991) to the Geophysical Laboratory and we were soon off and running on CSIA of amino acids, methods I still use today. Upwards of 40 % of presentations at the 2018 IsoEcol meeting included amino acid isotope measurements.

The impact and promise of CSIA in amino acids has been fully realised in marine science and animal ecology. A collaboration with colleagues at Univer- sity of Delaware on the growth of commercially important and tasty blue crabs (Callinectes sapidus) resulted in one of the first studies on CSIA-AA that tested

different diet sources for early growth stages in a lab setting (Fantle et al., 1999).

Our work using both bulk tissue and amino acid δ13C data showed that diet

quality was important for determining the isotope fractionation between diet and animal tissue, not simply the diet quantity or source. Using nitrogen as an additional tracer, it became clear that juvenile blue crabs not only lived in and

depended on Spartina marshes as a sanctuary while young, they also depended on marsh carbon and nitrogen for survival. CSIA-AA showed that detrital material is isotopically heterogeneous, cautioning those using bulk tissue isotope values for diet sourcing to think about diet quality. Kelton McMahon, a graduate student from Woods Hole Oceanographic Institution, started his CSIA “career” with me at the Geophysical Laboratory and investigated the idea of determining diet quantity, quality, and source even further with fish experiments. Kelton’s goal was to measure CSIA-AA in fish otoliths, the tiny ear bones that record a fish’s natal habitat in rings laid down annually over a fish’s

lifetime. After working out the procedures (McMahon et al., 2010), he went on

to use this approach coupled with δ13C fingerprints to study the importance of

mangrove habitat for reef fishes (McMahon et al., 2011).

Diane O’Brien, a postdoc from Stanford who came to the Geophysical Laboratory for long stretches of time, furthered CSIA-AA with a series of papers

on dietary resources for butterflies (O’Brien et al., 2002, 2003a,b, 2005). During

the caterpillar life stage of butterflies and moths, plants are their only source of nutrition for these insects. Once they hatch into butterflies and moths, the adults only consume sugary nectar that contains carbon, but no nitrogen. While the adults can make all of their non-essential amino acids from nectar, they must rely on their essential amino acids from the plants consumed during the cater- pillar life stage. Based on controlled feeding experiments, O’Brien confirmed that this was in fact the case, demonstrating that sugars were turned into non-essential amino acids, while essential amino acids were routed through the butterflies’ various life stages (Fig. 10.1).

One of my more unusual studies was on the periodical cicada which erupts in 13 or 17 year cycles. The larval stage of the cicada remains under- ground for 13-17 years, feeding primarily on the fluid from root xylem tissues of deciduous trees in the area where they are developing. When cicadas emerge from the ground, they then metamophose into adult flying insects, mate briefly, lay eggs, then perish. The hatched larvae fall to the ground after 6-8 weeks, then burrow into the soil where they exist for 13 or 17 years, arguably one of

the most amazing life cycles of any organism. The amino acid δ13_{C values and}

the concentration data showed that xylem tissue did not have all of the essential amino acids to support the larval stages. It is well known that bacteria inhabit the guts of these organisms, and we were able to show that a significant portion of the essential amino acids in the cicadas was supplied by this beneficial microbial symbiosis (Christensen and Fogel, 2011).

In the marine realm, Matt McCarthy, who was first a student from University of Washington, then a postdoc at the Geophysical Laboratory, and now Professor at UC Santa Cruz, worked with me to assess whether CSIA of amino acid isotope analyses might be useful in understanding the production and cycling of dissolved organic matter (DOM) in the ocean. DOM is composed of 40 % defined, characterised organic compounds, like carbohydrates, amino acids, cell membrane components, while the remaining 60 % is uncharacterised

organic matter. The relationship among bacteria, phytoplankton, zooplankton,

and DOM has intrigued scientists working in this field for many years (e.g.,

Hedges et al., 1997).

Figure 10.1 Carbon isotope compositions of essential amino acids in butterfly eggs come directly from plants that caterpillars consumed. Non-essential amino acid carbon is derived from a combination of original plant material and nectar. Modified from O’Brien et al. (2002).

In the 1990s, marine chemists were working out the methods for measuring DOM accurately as well as methods for concentrating this fraction from seawater. Ron Benner, at that time a Professor at UTMSI, my old alma mater, and John Hedges, University of Washington, were using ultra-filtration to separate DOM so that its complex chemistry could be studied with advanced instrumentation (Benner and Hedges, 1993; Hedges and Keil, 1995). One of

our first challenges was to measure the δ13_{C of D- and L-alanine. D-alanine is}

one of the principal components of bacterial cell walls and is found in DOM.

Our goal was to measure the δ13_{C of both isomers of alanine to compare a pure}

bacterial signal with a mixed isotope signal. Using material from sediment

traps, we measured the δ13_{C fingerprint of autotrophs (primarily cyanobacteria}

and diatoms), heterotrophic bacteria and microzooplankton, sinking particles,

and DOM from various depths. Patterns of δ13_{C in DOM more closely resem-}

bled autotrophs, whereas sinking particles were decomposed and resembled

bacterial fingerprints. D- and L-alanine δ13_{C from DOM were indistinguish-}

able suggesting that much of the DOM could be attributed to cyanobacteria. We concluded that the rapid cycling of POM in the surface ocean is decoupled

2004). Isotope fingerprinting of essential amino acids (Close, 2019) is currently the most popular use for carbon isotope data, but still remains an uncommon practice for most marine scientists.

CSIA of nitrogen in amino acids is another story and is primarily based on the work of McClelland and Montoya (2002) with marine zooplankton. They

proposed that in animals some amino acids have more positive δ15_{N values}

as they are metabolised, while other amino acids undergo minimal isotopic

fractionation as they move up the food chain and largely retain the δ15_{N values}

from primary producers at the base of the food chain. Those amino acids with more positive values are termed “trophic” amino acids, whereas those amino

acids with unchanging δ15N values are “source” amino acids. The concept has

spawned an entire new field within isotope ecology.

Our early work on human CSIA-AA δ15_{N in human bones compared}

just two specimens (Fogel et al., 1997) and never really took off in the archaeo-

logical community, probably because the methods are not routine. Ecologists, though, have embraced the trophic and source amino acid idea and heavily

exploited it (e.g., McMahon and McCarthy, 2016; Ohkouchi et al., 2017). That

said, the fundamentals behind the use of δ15_{N in amino acids for calculating}

trophic position are on uncertain ground. Inherent in all of the calculations,

is the beta (β) value, which is the difference between the δ15_{N of a trophic}

amino acid like glutamate (or proline) and that of a source amino acid like

phenylalanine. Based on a few original papers (e.g., McClelland and Montoya,

2002; McCarthy et al., 2007) this value (3.4 ‰ in marine systems) has been

assumed to be invariant. As more data are collected, we now know that there can be considerable variation in this measurement depending on the ecosystem.

Similarly, for terrestrial ecosystems, the β value has recently been shown to be

dependent on whether the tissue originates from a plant with a low amount

of lignin (e.g., aquatic plants) or a higher amount of lignin (e.g., tree leaves)

(Kendall et al., 2019) (Fig. 10.2).

An additional confounding factor is that with each step up the food chain, the nitrogen isotope fractionation between glutamate and phenylal- anine decreases because of direct routing of amino acids into animal tissue

(McMahon et al., 2015). Last, of the original proposed “source” amino acids,

only phenylalanine, tyrosine, and lysine remain as true source amino acids; the relatively simple (non-essential) glycine and serine are no longer considered to be reliable source amino acids. If recent work on the influence of gut microflora

pertains to most animal species, this amino acid δ15_{N-based method to estimate}

Figure 10.2 The difference between the δ15_{N of glutamate and phenylalanine or tyrosine} in plants is known as beta, a parameter used in determining trophic position using nitrogen isotopes in amino acids. Beta can be extremely variable in terrestrial systems, based on the amount of lignin in a plant’s tissues. Modified from Kendall et al. (2019).