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Revealing Hidden Species Interactions Through the Building Blocks of Life
By: Kristen Hasenstab-Lehman, Ph.D., Conservation Geneticist and Lab Manager
One of the most foundational properties of living organisms on our planet is DNA. And the underlying molecular structure of DNA is the double-stranded helix, an image you’ve likely seen countless times, in various styles and iterations. The double helix is used as a motif in jewelry, art, and more, evoking its connection shared by nearly all living things. But for scientists, it is the link to understanding how species interact. We at Santa Barbara Botanic Garden are excited to be using advances in DNA science as a new tool in our conservation tool belt.
Let’s get into specifics for a moment. We know DNA, or deoxyribonucleic acid, codes the plans for cellular machinery necessary for life to function and replicate. Deoxyribonucleic acid is composed of repeating units of nucleotides often called the building blocks of life. Each nucleotide or building block (you can think of them like LEGO pieces) contains three parts: a phosphate group, a sugar group, and one of four types of nitrogen bases (A)denine, (T)hymine, (G)uanine, and (C)ytosine. The nitrogenous bases form the core of the nucleotide, with A only ever pairing with T, and G pairing with C. So, with these pairs plus phosphate and sugar as the backbone, you have one nucleotide block. The blocks then join together further to form two long, reverse-complement strands that we recognize as the double-stranded helix. Changes in the order of nucleotides over millions of years results in the dynamic and rich forms of biodiversity around us.
Leveraging Science To Protect Threatened Species
Each creature has a unique combination of A T G and C bases, and that unique sequence presents scientists with a novel way, known as DNA metabarcoding, to identify what animals have been eating. In the Garden’s Plant Genetics Lab, we’re using DNA metabarcoding to better characterize species interactions and food webs for taxa native to the Channel Islands, from foxes to snails. With that information, we can better protect these important food webs for their survival.
There is a rich body of literature describing the diet of imperiled animals, such as Channel Island foxes, based on the physical examination of their scat (or feces). In these studies, researchers collected scat and identified various prey items in the samples. However, this approach requires expertise in recognizing the small body parts of invertebrates, or soft-bodied animals, that form a large part of the diets of these charismatic omnivores. Soft-bodied animals and plants are particularly difficult to identify, as they do not preserve as well passing through the digestive tract of a fox. This is where DNA-based methods come in.
This cutting-edge metabarcoding technology improves our ability to examine predator-prey food webs in two ways. First, they can be applied to charismatic vertebrates like foxes to detect soft tissued components of their diet. Second, the approach works for any kind of animal, including invertebrates, with equal success in food item detection and precise identification.
The DNA Process at a Glance
To begin a diet study using DNA metabarcoding, scat of the focal species is collected either fresh or in the field. Back in the lab, we then grind up the scat and isolate any DNA present in the sample. We copy small, specific segments of DNA of the prey, often referred to as “barcodes,” that allow us to identify the DNA in the scat. We do this through a lab technique called Polymerase Chain Reaction (PCR) that can generate thousands of copies of these barcodes of prey items. Small identification tag molecules, which are also made out of A T G and C bases, are then added to each of these individual PCR samples. We then place all the identification-tagged PCR reactions into a single, small tube, which when full will appear as nondescript as water to the naked eye. However, once that clear liquid is put on a high throughput DNA sequencer, it is turned into billions of A T G and Cs.
Following an initial sorting of data by sample, we then compare those unknown sequences found in the scat samples to a large reference database of barcode sequences with known identities. Once we identify our unknown sample sequences using this reference database, a new understanding of the diet composition can emerge, one generated in a noninvasive way using nucleotides (those LEGO-like pieces that are the building blocks of life).
How Are We Using DNA To Advance Conservation?
At the Garden, we are applying these exciting DNA-based methods to better understand the diet of foxes on San Clemente and San Nicolas islands, as well as land snails across the archipelago. These studies are informing ongoing conservation efforts for these species.
San Clemente Island Fox
The largest land mammal and one of the top predators on San Clemente Island is the San Clemente Island fox (Urocyon littoralis ssp. clementae). Previous studies using manual examination of scat revealed a highly seasonal and diverse omnivorous diet for this island fox. Our metabarcoding results were consistent with finding some vertebrate prey items like the endemic San Clemente Island night lizard (Xantusia riversiana reticulata) and a variety of invertebrates. However, we also found foxes frequently consume a large number of moth taxa. (We suspect foxes are consuming these species while they are caterpillars and grubs.)
In addition, foxes are eating a surprising number of native plants, including a reliance on prickly-pear (Opuntia). These results give land managers evidence for better managing a robust fox population into the future.
San Nicholas Island Fox
For our study of the San Nicolas Island fox (U. littoralis ssp. dickeyi), scats were freshly collected by U.S. Navy staff during trapping season in the late fall and winter. In collaboration with former Garden Lab Technician Seth Kauppinen, our findings reflect those on San Clemente Island, and showed that invertebrates are the largest and seemingly most important component of the fox diet. Moths represented a novel and previously unknown portion of that diet on San Nicolas. Our detection of a high proportion of invasive garden snails (Cornu aspera) in fox diet was consistent with the physical examination of the scat. The top three vertebrates detected in fox diets included San Nicolas Island deer mouse (Peromyscus gambelii ssp. exterus), common side-blotched lizards (Uta standsburiana), and the introduced chukar (Alectoris chukar).
San Nicolas Island Snails
While our studies of island foxes represent important contributions to resource management on both San Nicolas and San Clemente, the exciting and novel application of metabarcoding also allows us to reveal the diet of animals for which physical scat examination was not possible before — enter island land snails. On San Nicolas Island, in collaboration with the U.S. Navy, we collected fresh scat from five native and two invasive species of snails. Excitingly, metabarcoding reveals that snails utilize both plants and fungi as food items. Plant species detected included giant coreopsis (Leptosyne gigantea), Menzies’ goldenbush (Isocoma menziesii),
California saltbush (Extriplex californica), pink sand verbena (Abronia umbellata), island morning glory (Calystegia macrostegia ssp. amplissima), and miner’s lettuce (Claytonia perfoliata).
Ambersnail (Cantinella) consume mosses growing in and around their preferred habitats, vernally wet ditches, and pools. While the plant results were interesting, our newly assembled fungal reference library demonstrated that a wide variety of fungi are consumed by all snail species — more than scientists ever suspected. The macrofungi consumed included a beautiful array of mushroom forms including deer mushrooms (Pluteus), oyster (Pleurotus), cups (Peziza), and hairy curtain crust (Stereum hirsutum). Our metabarcoding results also show that snails will consume lichens when they encounter them.
Partnering With the U.S. Navy To Improve Public Databases
The accuracy of DNA identification of diet items using the metabarcoding approach depends on a robust barcode reference library. Each sequence present in the reference library ought to be linked to museum specimens with expert taxonomic identifications. Though analyses are possible solely using DNA sequences available on publicly accessible databases, as we used on San Clemente Island, we know these may fail to detect important food items. Publicly available sequences from these databases may include misidentifications, and local species from the study area may be missing entirely.
The California Channel Islands are home to many endemic or native plants, animals, and fungi that occur nowhere else on Earth. Many have never been sequenced for barcode markers. In an effort to improve metabarcoding analyses for the Channel Islands, the Garden partnered with the U.S. Navy in 2019 to build a barcode reference library for San Nicolas Island. We sequenced all plants known to occur on the island. We also focused on adding local sequences for understudied groups such as lichenized fungi and macrofungi, bryophytes, and terrestrial invertebrates. Taxonomic experts — including Tucker Lichenologist and Curator of the Lichenarium Rikke Reese Næsborg, Ph.D.; Tucker Systematist and Curator of the Clifton Smith Herbarium Matt Guilliams, Ph.D.; Research Associate Christian Schwarz; biologist Adam Searcy; and former Conservation Technician Stephanie Calloway — collected, identified, and prepared museum specimens and tissue. The Garden’s Conservation Genetics Lab Technician Caitlin Hazelquist and former lab techs Isabel Rivera and Emily Thomas isolated DNA from each specimen and generated DNA barcodes for the reference library. As of today, our efforts added 545 species and 820 sequences to public databases that continue to serve as tools for conservation throughout California.
Contributing to Global Conservation Efforts
While the use of these sequences informed and improved our diet analyses for San Nicolas Island foxes, they are impactful for conservation beyond the Garden’s work. Metabarcoding is increasingly a tool used by land managers for biodiversity monitoring, and our reference library will be used by other scientists applying these methods to improve analyses beyond our own.
We have come far in our understanding of predator and prey relationships using the technology of metabarcoding, but there is much more to understand. Our conservation science is on the forefront of showing these connections using this code, and it will take a broad, diverse group of people using this information to ensure these delicate webs are preserved for future generations. It’s exciting and fascinating, and the Garden is determined to help lead the way!
