Drawing on my work in evolutionary ecology, wilderness ethics, and machine learning, I reflected on the tension between our desire to intervene and our limited ability to forecast long-term ecological outcomes. Using examples like the Chernobyl exclusion zone—where many species are thriving in the absence of people despite nuclear contamination—I argued that ecological recovery is often less about precision intervention and more about restraint. We discussed how machine learning can help us simulate alternative futures and understand potential tradeoffs, but that ultimately, the most powerful conservation tool may be humility. More wilderness, not more control, might be the best way to meet the uncertainties ahead.

Listen to the episode here or wherever you get your podcasts.

I am deep in the Alaskan Arctic,  300 miles from the nearest road system, attempting to conduct the kind of science that usually requires a specialized laboratory. We rowed 30 miles of meandering flatwater today, bringing our total to 200 river miles in 12 days since we landed at a lonely gravel bar on the headwaters of Ambler River in Gates of the Arctic National Park.

Mosquitoes spangle the tent canopy arching over me. Backlit by summer solstice sun, the silhouettes of the insects make an inverted night sky of shifting constellations. The sun never sets on the banks of the Kobuk River this time of year. It hangs high above the horizon even now at 11 p.m., transforming my tent into a solar oven as I, ironically, work to uncover the secrets of a frog that can turn into ice.

… Read the rest of the article here.

Take me to the photos!

Step 1: Come up with a hair-brained scheme.

My labmate Yara and I had been dreaming up the idea studying wood frog genomes from across the species’ range since she started her PhD. Wood frogs have the largest range of any North American amphibian. They also happen to be the only North American amphibian that can survive North of the Arctic circle.

Dr. Julie Lee-Yaw had done a similar study back in 2008. She embarked on a road trip from Quebec all the way up to Alaska to collect wood frog tissue. So, out first step was to ask Dr. Lee-Yaw if she would collaborate and share her samples.

Those samples gave us a solid backbone across the wood frog range, but we were missing population in expansive regions north and west of the road systems. We worked with the Peabody Museum to search for tissue samples that were already housed in natural history collections around the world. We filled a few gaps, but huge portions of the range were still missing.

We knew that there must be samples out there sitting in freezers and labrooms that were not catalogued in museum databases. So, our next step was to begin sleuthing. We looked up author lists from papers and cold-called leads. I even reached out to friends on Facebook (…which actually turned out to be a big success. The aunt of a friend from undergrad happens to do herpetology research in Galena, Alaska and was able to collect fresh samples for us this year!). This effort greatly expanded our sample coverage with new connections (and friends) from Inuvik and Norman Wells in the Northwest Territories, Churchill on the Hudson Bay, and the Stikine River Delta in Southeast Alaska.

But as the points accumulated on the map, we noticed some glaring holes in our coverage. Most importantly, we had no samples from Northwestern Alaska. Populations in this region are the most distant from the ancestral origin of all wood frogs in the southern Great Lakes. If we wanted a truly “range-wide” representation of wood frog samples, we needed tissue from that blank spot on the map!

Step 2: Convince your advisor and funders it’s a good idea.

This might be the hardest step. In our case, Yara and I were lucky that our advisor, Dave, was immediately supportive of the project. After we made the case for the importance of these samples, funders came around to the idea as well.

Step 3: Make a plan …then remake it …then make a new plan yet again.

Once we knew where we required samples from, we needed to figure out how to get there. Alaska in general is remote, but northwestern Alaska is REALLY remote. The road system doesn’t stretch farther than the middle of the state. All of the communities–mainly small villages–are only accessible by plane, and most of them only have runways for tiny prop planes. Travelling out from the villages into the bush is another layer of difficulty. Most people here either travel by boat on the river or by snowmachine during the winter. Traveling on land, over the soggy and brush-choked permafrost, is brutal and most locals only do it when necessary, if at all.

Prior to academia, I made a career of organizing expeditions to the most remote places in the rugged southeastern archipelago of Alaska. Despite my background, the logistic in the Arctic were even inscrutable to me. Fortunately, I had a couple of friends, Nick Jans and Seth Kantner, who know the area well. In fact, Seth grew up in a cabin out on the Kobuk. (Seth and Nick are both talented authors. I suggest checking out Ordinary Wolves by Seth and The Last Light Breaking by Nick). With their help, I was able to piece together the skeleton of a trip.

After many logistic iterations, Yara and I decided to follow in the footsteps of local hunters who, for generations, have used the rivers as conduits into the heart of the wilderness. Our plan was to travel down one of the major arterial rivers and hike inland to search for frog as we went.

Our original itinerary was to raft the 100 mile section of the Kobuk River from just north of Ambler village to the village of Kiana. But at the last minute (literally), our plans changed. As we were loading up the plane, the pilot told us that he couldn’t fly into our planned starting point. Instead, he suggested that we fly into a gravel bar 30 miles up river in Gate of the Arctic. Those “30 miles” turn out to be AIR MILES. Following the river, it ended up adding over 60 miles to our trip.

We packed two inflatable oar rafts, almost 150 pounds of food, and another 300 pounds of camping, rescue, and science gear, into the balloon-wheeled plane. For the next two weeks, we rowed down the swift Ambler River from the headwaters to the confluence of the Kobuk. Then, we rowed down the massively wide and meandering Kobuk River, eventually extending our trip by an additional 30 miles, by-passing Kiana, and continuing to Noorvik, the last village on the river.

Step 4: Recruit a crew.

Despite being the worlds first and only Saudi Arabian Arctic Ecologist with limited camping experience, I knew Yara would be a stellar field partner. But I never like traveling in brown bear country with fewer than four people. Plus, expedition research involves too many daily chores for the two of us to manage alone. So, we recruited a team.

Sam Jordan is a dry land ecologist, but he had been willing to help me with my dissertation fieldwork in wetlands before, so I knew he would be willing to defect for a good adventure. Sam is also an exceptional whitewater paddler and all-around outdoor guru. Plus, he’s just a great guy (when he leaves his banjo at home). He and I spend two weeks floating the Grand Canyon in the dead of winter and there are few people I would want along on a remote river trip.

Kaylyn Messer and I guided sea kayak expeditions in Southeast Alaska back in our youth. I am a bit particular about how I manage my camp system (read: “extremely picky and fastidious to a fault”) on big trips. Kaylyn is one of the few people as scrupulous as me, but she’s also a super amenable Midwesterner at heart. I knew she’d be a huge help out in the field.

We fell into an effective rhythm on the trip.  Each morning we woke, made breakfast, broke camp, packed the boats, and launched early in the day. While one person on each boat rowed, the other person checked the maps for frog surveying spots, fished, or photographed. We stopped along the way to bushwhack back into wetlands we’d identified from satellite images. We typically arrived at camp late. Yara and I would set up one tent to process the specimens from the day while Same and Kay made camp and cooked dinner. One of the hidden disadvantages of 24-hour Arctic sunlight is that it is easy to overwork. Most nights we only managed to get sampled finished, dinner cleaned up, and camp bearproofed with enough time to crawl into tents with just eight hours till beginning again the next day.

Step 5: Do the science.

Doing science in the field is difficult. Tedious dissections seem impossible while baking in the omnipresent sun and being alternately hounded by hundreds of mosquitoes or blasted by windblown sand. Trading lab coats for rain jackets and benchtops for sleeping pads covered in trashbags compounds the trouble. Not to mention, keeping tissues safe and cool. Organization and adaptability go a long way.

On remote, self-supported trips, it is inevitable that equipment fails or is lost. On one of the first days, we discovered that our formalin jar was leaking—and formalin is not something you want sloshing around! We cleaned the boats and found a creative solution to replace the offending container: a 750ml Jack Daniel’s bottle!

Planning ahead and engineering backup plans also helps. One of our main struggles was figuring out how to preserve specimens and get them home. It is illegal to ship alcohol by mail and you can’t fly with the high-proof alcohol needed for genetic samples. You can ship formalin, but it is difficult to fly with. To make matters worse, we were flying in and out of “dry” or “damp” villages where alcohol is strictly regulated or forbidden. Also, we happened to be flying out on a Sunday, making it impossible to mail samples home. The solution we arrived at was to ship RNAlater and formaldehyde to our hotel room ahead of time. Tissue would remain stable in RNAlater for a couple of weeks and we could make formalin to fix the specimens. After fixing, we cycled the specimens through water to leach out the formalin. This made it possible for me to fly with all of the tissue tubes and damp specimens in my carry on. Other than a few concerned looks from the TSA folks, all of the samples made it back without issue!

Step 6: Enjoy the adventure.

Despite the hard work, there was a lot to appreciate about the Arctic. We witnessed major changes in ecology as we travelled from the steep headwater streams in the mountains to the gigantic Kobuk. Every day was an entirely new scene.

Here’s an illustrated thread on our collecting trip to the Alaskan Arctic for the @Yale @yalepeabody this past summer. 1/22 pic.twitter.com/Jh3VCqVEgx

— A. Z. Andis Arietta (@azandisarietta) August 24, 2021

Step 7: Forget the hardships

Looking back, it is really easy to forget the sweltering heat, swarms of mosquitoes, inescapable sun, and freak lightning storms. And, it’s probably better to forget those anyway!

Arctic CircleSam illustrates that we are, in fact, above the Arctic Circle.

GearGear (minus food and rafts) ready for weigh-in before loading the plane.

Upper Ambler RiverOur drop-off point on the braided upper reach of the Ambler River in Gate of the Arctic National Park.

Gates of the ArcticGetting dropped off on a gravel bar in Gates of the Arctic National Park.

The first dayUnloading the plane on the first day of the trip.

Camp, night 1Campsite along the Kobuk River.

Rigging the raftsPacking up the two-person rafts on the first day.

Kaylyn Messer at the helm.In the upper rivers, near the headwaters, the current was pleasantly swift.

Upper riverSam and Yara rowing down the Kobuk River.

Early morning rowingRowing the swiftwater on the headwaters of the Ambler River in Gates of the Arctic National Park.

Searching for frogs

First frogs!Yara and I after finding the first frogs of the trip!

Science in the fieldYara preparing to do the sciencing with a garbage back as a makeshift lab bench.

Dinner. night 6Dinner and a view on the banks of the Kobuk River.

Midnight sunTrying to avoid getting a sunburn while sleeping in the Arctic.

Midnight sunThe sun baking our tent at 1130pm.

Rainy rowingSam and Yara floating downstream.

Arctic frogYara proudly presents her first wood frog collection.

Kobuk viewsSmoke and clouds mute the midnight sun.

Sam JordanSam Jordan in his happy place.

Chocolate con culicidae.With hundreds of bugs circling your head at all times, it is impossible not to end up with a few in every meal.

CutbanksMassive cutbanks made much of the shore inaccessible for sampling.

StormA freak lightning storm.

SunsetBeautiful light from the midnight sun.

Field dissection Taking tissue samples for sequencing.

TadpolesTadpoles from "Seth's Pond".

Prepping collectionsYara and I taking tissue samples and prepping specimens.

Arctic Wood FrogBeautiful arctic wood frog.

Fixing specimensHere, I am fixing specimens with formalin to preserve them for the Peabody Museum.

Bushwhacking The view while bushwhacking in to search for frogs.

BugsA random sample of friendly mosquitoes.

BearGrizzly bear prints on the beach.

FishingSam with his first of many greyling.

The Kobuk River valleyThe Kobuk Valley watershed is just an endless expanse of wetlands.

The end of the tripFlying back to Kotzebue in a packed plane at the end of the trip.

Heading homeCatching a ride out to the airstrip with our truckload of gear on the last day of the trip.

The study:

One of the most consistent findings arising from 20 years of study in our lab is that wood frogs seem to adapt to life in cold, dark ponds. In general, cold-blooded animals like reptiles and amphibians are not suited for the cold and function much better in warmer conditions. So, wood frogs that live in colder ponds should have a harder time competing against their neighbors in warmer ponds.

In response, cold-pond wood frogs seem to have developed adaptations that level the playing field. In separate experiments, we’ve found that wood frog tadpoles in cold-ponds tend to seek out warmer water (like in sunflecks) and have lower tolerance to extremely warm temperatures. Most importantly, they can mature faster as eggs and larvae.

But I’ve always struggled with a lingering question: if cold pond frogs have adapted these beneficial adaptation to compete with warm-pond frogs, what is keeping those genes out of the warm-ponds? Shouldn’t cold-pond genes in a warm pond mean double the benefits? One would expect the extrinsic environmental influence and the intrinsically elevated growth rates to produce super tadpoles that metamorphose and leave the ponds long before all the others.

Kaija, who was an undergrad in the lab at the time, decide to tackle that question for her senior thesis.

We hypothesized that there might be a cost to developing too quickly. Studies in fish suggested that the trade-off could be between development and performance. The idea is that, like building Ikea furniture, if you build the tissue of a tadpole too quickly, the price is loss of performance.

So we collected eggs from 10 frog ponds that spanned the gradient from sunny and warm to dark and cold. We split clutches across two incubators that we set to bracket the warmest and coldest of the ponds.

Then we played parents to 400 tadpoles, feeding and changing water in 400 jars two to three times a week.

We reared the tadpoles to an appropriate age (Gosner stage 35ish). Those in the warm incubator developed about 68% faster than those in the cold incubator. In addition to our lab-reared tadpoles, we also captured tadpoles from the same ponds in the wild as a comparison. Development rates in the lab perfectly bounded those in the wild.

Once they reached an appropriate size, we put them to the test. We simulated a predator attack by a dragon fly naiad by poking them in the tale. Dragonfly naiads are fast, fierce, tadpole-eating machines and a tadpole’s fast-twitch flight response is a good indicator of their chance of evading their insect hunters. It’s a measure of performance that directly relates to a tadpole’s fitness.

Above the test arenas, we positioned highspeed cameras to capture the tadpoles’ burst responses. We recorded 1245 trials, to be exact—way more than we ever wanted to track by hand. Fortunately, Kaija is a wiz at coding; and with a bit of help, she was able to write a Matlab script that could identify the centroid of a tadpole and record its position 60 times per second.

We measured the tadpoles’ speed during the first half second of their burst response and looked for an association with their developmental rates. One complicating factor is that a tadpole’s fin and body shape can influence burst speeds. So, a weak tadpole with a giant fin might have a similar burst speed to a super fit tadpole with a small fin. To account for this, we took photos of each tadpole and ran a separate analysis mapping their morphometry and included body shape into our models.

As we had hypothesized, tadpoles reared at warmer temperatures show much slower burst speed than their genetic half-sibling reared in the cold incubator. We even saw a similar, but weaker effect for the tadpoles that were allowed to develop in their natal ponds. It seems that developing too fast reduces performance.

Thus, it certainly seems that the counter-gradient pattern we see of faster development in cold-pond populations, but not in warm-pond populations, is at least partially driven by the trade-off between development rate and performance.

In fact, it may even be the case that we’ve been viewing the pattern backwards all along. Perhaps instead we should consider if warm-pond populations have developed adaptively slower development rates to avoid the performance cost. This especially makes sense given the range of wood frogs. Our populations are at the warm, southern end of the range. Maybe this tradeoff is also a factor constraining wood frogs to the cold north of the continent?

If warm weather and faster development are a real liability for wood frogs, it is only going to get worse in the future. We know from another recent study that our ponds have been warming quickly, especially during the late spring and early summer months. But climate change is also causing snow to fall later in the winter forcing frogs to breed later. The net result is that wood frogs may be forced to develop fast intrinsic developmental rates in response to a contracting developmental window, while at the same time, extrinsic forces drive development even faster. That’s a double whammy in the trade-off with performance. And might lead to too many “Ikea furniture mistakes” at the cellular level.

As a separate part of this study, we also measured metabolic rates in out tadpoles in hopes of understanding the relationship between developmental rates, performance, and cellular respiration. I’m in the process of analyzing those data, so stay tuned for more!

Our chapter tells the story of green frogs and the feral condition of their life with suburban human neighbors. We especially highlight the way that the human built-environment of lawns, pavement, sewers, and septic systems is infused into the biology of green frogs (a topic that Max and Dave have studied in depth). As a counter example, I told the story of the wood frog, a species that has escaped a feral fate by clinging to the remnants of wild space away from humans (a topic I study in depth).

I rarely get to write about wood frogs outside of academic articles, so it was a pleasure to contribute to this piece. I think it is some of my best natural history writing. I’ve excerpted my section below (or, read the chapter):

“To better understand why our housing patterns influence frogs, it is worth taking a frog’s-eye-view of suburbanization. Most frogs exhibit distinct life-stages. Like humans, frogs begin development as shell-less and fragile eggs, but while human embryos float within the protection of a womb, frog embryos are buoyed among the vegetation and flotsam of ponds. The embryos have an umbilical relationship to the water that surrounds them. Nutrients and oxygen easily pass through the transparent jelly and are consumed through delicately branching gills. Any contaminants in the water also suffuse the embryos.
Even before their eyes or mouths have formed, the developed embryos hatch as free-swimming larvae not much larger than a grain of rice. Hatchlings are vulnerable. Thus, frogs hedge their bets by producing hundreds of eggs per clutch, hoping that at least a few will win the lottery of life. Some species, like wood frogs, additionally safeguard their offspring by choosing impermanent pools that are devoid of fish as relatively safe nurseries.

Those hatchlings that survive develop into recognizable tadpoles with bulbous bodies and slender tails. A pond’s version of cows, tadpoles graze along the bottom with scraper-like teeth. They consume algae and detritus along with any solid matter that washes into the pond basin. A long digestive tract allows the tadpoles to incorporate nutrients into a growing body. Where ponds neighbor septic systems, this means that human waste makes up a prodigious portion of a tadpole’s body.

The transition from a tadpole to a frog is a remarkable change. It makes the squeaking voice and acne of human puberty seem like a blessing. Every system in the tadpole’s body transforms. The tail gives way to bony limbs. The narrow, disc-shaped mouth morphs into a wide, insect-capturing, gape. The goggle eyes, so fine-tuned to underwater vision, mutate into something much like our own. Even the long and coiled digestive tract shortens and distends. At the end of this metamorphosis, the aquatic vegetarian leaves the water’s edge and becomes a terrestrial carnivore.

Green frogs are parochial and prefer a pond-side life. For a short time as juveniles they might range far and occupy any standing water from lakes and ponds to swimming pools and tire ruts. Upon adulthood though, they settle along freshwater shores where they patiently wait for dragonflies and other insects to approach within range of a lunging gulp. Since green frogs inhabit permanent ponds, they can breed throughout the summer, and without the threat of the pond drying out from beneath them, their tadpoles can be leisurely in development. When snow falls and the pond freezes, both adults and overwintering tadpoles take refuge deep in the insulating layer of pond muck. Because a green frog’s life is so reliant on a pond, they can survive in just about any permanent water with at least a narrow perimeter of vegetation. As long as a homeowner neglects the tufts of grass along the bank, green frogs are more than happy to remain neighbors.

Unlike green frogs, wood frogs become sylvan nomads after metamorphosis. As their home ponds dry up in the summer and fall, they wander the forest floor hunting among the leaves, only briefly returning to recently filled pools in early spring to breed. During the winter months, wood frogs burrow just under the blanket of leaves dropped in fall. This enables them to be the first out of hibernation as the forest thaws in spring. For these reasons, wood frogs rely on leaf-littered landscapes. Manicured lawns where leaves are regularly raked and bagged make inhospitable places for them. Where the balance of forest gives way to lawns, wood frogs disappear…”

Overall, this was a really fun project to work on that gave me a chance to switch up my writing style. It was also a lot of fun to be able to collaborate with my partner, Bayla, who painted the featured image for the chapter.

The online version of the book is a little counter-intuitive to navigate (I think this was intentionally designed as a rhetorical device), but if you can figure it out, it is worth checking out some of the other cool stories of our feral world!

Update (Nov. 2020): And, I’m especially thrilled that our piece will be the cover article for the journal, featuring a pair of breeding wood frogs from our population! My hands nearly froze trying to get this underwater shot, so I’m glad it was worth the effort!

Over the past 20+ years, our lab has been monitoring over 50 populations of wood frogs at Yale Myers Forest. Each year in early spring, we listen for the duck-like clucks of the male frogs which means that they have emerged from under the snow and moved into the breeding ponds. Shortly afterward, we head out into the freezing ponds to count the egg masses as a way to monitor population density over time.

In this study, we looked at how the oviposition date (the day on which frogs deposited eggs) has changed over time. As climates warm, we usually expect for the timing life-history events (like oviposition, emergence from hibernation, flowering time, etc.) called ‘phenology’ to advance in the year as winters get shorter. That’s just what most species do. And the trend of advancing phenology is strongest for amphibians.

Given that annual temperatures at our field site have increased by almost 0.6 C in the past two decades, we expected frogs to breed and lay eggs earlier. If our frogs were like other amphibians, we might expect oviposition to come around 6 days earlier.

Surprisingly, we found the opposite. Our frogs seem to be breeding 3 days LATER.

To figure out what might be going on with our frogs, we decided to look more closely at climate across the season, not just annual averages. It turns out that most of the increase in annual temperatures are felt later in the summer, but relatively less when frogs are breeding. Snowpack, on the other hand, is actually accumulating later and lasting longer. In the figure (Fig. 3 from the paper) below, you can see these trends. On the left are the comparisons between temperature, precipitation, and snowpack between 1980 and 2018. On the right, we plot only the difference in trends over time. At the top-right, we plot the oviposition dates to show how seasonal changes in climate line up with frog breeding.

We also looked at how the timing of oviposition correlated with climate across the season. We found that breeding occurs later when there is more snow at the beginning of the breeding window. Also colder temperatures just before breeding correlate with delayed oviposition (which makes sense if colder temps mean more snow and less melting).

So, we think that frogs may be kind of stuck. Persistent snowpack might be keeping them from breeding earlier. But at the same time, warmer summer temperatures might be drying up their ponds faster. If so, this could be a big problem for tadpoles that need to maximize their time for development. The figure below shows that frogs tend to breed earlier when winter and early spring air temperature are high. As we’d expect, more snowpack correlates with later breeding. High precipitation during the spring delays breeding (probably because it is falling as snow).

Twenty years is a long time to be collecting ecological data, but it is a pretty short window into the evolutionary history of wood frogs. And, we don’t know how long snow and temperatures may have been working against these frogs. So, as a final piece of our analysis, we used a machine learning technique called a random forest to predict oviposition dates backwards in time an additional 20 years. It doesn’t seem like much has changed over the past half-century or so. In one way, that could be good news in that at least things don’t seem to be getting any worse.

The big question is, how will frogs cope with these climate changes? If tadpoles are faced with an ever-shrinking window of time to develop into frogs, will they be able to keep up? Or, will they lose the race and end up as tadpole-shaped raisins in our ponds?

I won’t give away any spoilers, but I’m looking at our long-term larval datasets to ask that question next.

Here’s my talk from the conference, “Evolution of Intrinsic Rates: Can adaptation counteract environmental change?“:

I’m excited to announce that my paper “A new, noninvasive method for batch marking amphibians across developmental stages” is now published at Herpetological Conservation and Biology.

This project originally grew out of frustration that no methods existed to be able to track amphibian larvae through metamorphosis into adulthood. This is a key bit of information needed in understanding amphibian ecology and asking questions like “How many tadpoles survive to adulthood?” “What percentage of frogs return to the same pond?” “Do tadpoles tend to school with kin or strangers?” etc. And ultimately this information is integral to estimating dispersal kernels and defining microgeographic variation.

Originally, I was looking into using radioactive isotopes to mark tadpoles, but just as my digging made that method seem intractable, I came upon a paper (Mohler 2003) that used a calcein fluorochrome solution to mark salmon.

Calcein binds to calcifying tissues like bones and scales. Although it had been tested in fish and bivalves, it had never been trialed on amphibians or any terrestrial applications. My study demonstrates that this technique is extremely promising for herps and solves a major limitation of marking amphibians. Below are the pertinent figures from the paper and supplemental materials, and also a couple extras.

Video of a wood frog tadpol approximately 24 hours after administration of calcein label.

This technique allows for both short and long term labeling. Short-term marking is detectable throughout the entire integument for 3-4 days and is visible in internal structures for up to 20 day in tadpoles marked within 28 days of metamorphosis. Labels are most useful for long-term marking (over 146 days) across metamorphosis when applied within 10 days of metamorphosis with 99% detection rate. If marked within 16 days of metamorphosis, the detection rate falls to 90% and sharply declines if tadpoles are marked earlier in development.

Check out the paper for more info. And also check out my poster on the project.

Not GFP!

A lot of folks ask me if this is technique is similar to the GFP (green fluorescent protein) that Shimomura, Chalfie, and Tsien discovered in the 60s (Tsien 1998). The answer is, no. GFP is a gene that can be introduced to animal genomes to induce production of a growing protein originally derived from jellyfish genomes. GFP is a genetic technique and so must introduced in germline or other stem cells. In contrast, calcein is a molecule that binds chemically to calcium. This means that calcein can be administered to any tissue for immediate fluorescence without interacting with the genome.

Thanks

This project required keeping lots of tiny frogs in the lab for almost 8 months, which turned out to be a major cleaning and feeding project a couple times a week. I couldn’t have done this work without loads of help from our lab manager and undergrad researcher–they deserve glowing medals for their service.

I especially would like to thank my brother Wes for spending part of his vacation playing scientist and helping me set up the experiment.

References:

Mohler, J. W. (2003). Producing fluorescent marks on Atlantic Salmon fin rays and scales with calcein via osmotic induction. N. Am. J. Fish. Manage. 23, 1108–1113.

Tsien, R. Y. (1998). The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544.

I had a ton of fun presenting a poster detailing my soon-to-be published study exploring the use of calcein fluorescence marking in amphibians at this year’s Joint Meeting of Ichthyologists and Herpetologists in Rochester, New York. This method solves a lot of problem areas we run into in trying to mark and re-identify amphibians across time and is one of the only methods for reliably marking larval anurans across developmental stages for identification as adults.

Keep an eye out for the paper in the next couple of week in Herpetological Conservation and Biology.  Check out my blog post and download the paper!

Here is a link to a PDF version of the poster if you’d like a closer look.

If you are interested in this method, please feel free to contact me: a.andis@yale.edu.

Here is the product page for SE-MARK brand calcein.

Much thanks to everyone who came to talk with me about the poster!

UPDATE: I was thrilled to win the Victor Hutchinson Poster Award from SSAR! This is quite an honor for my first real conference presentation and goes to show that there are at least a few benefits of taking a long time off and gaining some ancillary skills in graphic design before heading back to graduate school. I also have to make a huge shout out to Better Posters blog where I found tons of tips, tricks, good advice in turning academic content into a visual story.

As part of my dissertation research, I spent the spring monitoring the development of wood frog eggs. Some of the eggs I brought to the lab, removed from the jelly coat and reared in incubators. I left the rest of the eggs in the ponds and checked on them daily until they hatched. Although not part of my research, I thought it would be interesting to take photos comparing the wild eggs and algae symbionts to the lab-reared eggs without. The relationship between this alga species and only a handful of amphibians is one of the most unique stories in biology. To the best of my ability, I’ve chronicled the Green Egg symbiosis story below.

Green Eggs

Folks have been fascinated by the coincidence of algae and amphibian embryos for a long time. One of the first notes was published in 1888. Orr was studying amphibian skeletal and nervous system development. He needed embryos and had collected egg masses of a few local species. He was surprised to see that the salamander eggs he procured seemed “to present a remarkable case of symbiosis.” He noticed that there were algae cells situated inside of the egg membrane.

This is surprising because that membrane, called the vitelline membrane, is impermeable to everything but ions and small molecules, which certainly excludes entire algae cells. So the only other option is that the algae entered the embryo before the vitelline membrane had formed. But this would be no less surprising because, like basically all animals, the vitelline membrane is formed in the ovaries, and in amphibians, extra layers of jelly are added in the oviducts, long before the egg is exposed to pond water.

In the figure above (from Gilbert 1942) depicting the layers surrounding a salamander egg, it is clear to see that there are many layers acting as barriers between the pond water and the egg.

Which came first–the algae or the egg?

So, Orr was left with the plaguing question: Which came first—the algae or the egg? Perplexed, he wrote, “I have not discovered how the Algae enter the membrane, nor what physiological effect they have on the respiration of the embryo, but it seems probable that in the latter respect they may have an important influence.”

It turns out that Orr was right, algae do have an important influence on the embryos, but it was not demonstrated until the 1940s.  The phenomenon continued to perplex scientists for decades. By the 1940s, algae had been reported in the eggs of multiple salamander species and also wood frogs across the continent from California to New England and Virginia. Gilbert (1942) was the first to explore how the algae came to enter the egg and also studied the importance of the algae-egg relationship.

As to the entrance of the algae into the egg, he first assumed that the algae were present in the ovitract of the female salamander. He washed the oviducts of salamanders and tried to culture the solution, but no algae developed. If algae were not present in the ovitract, then they enter from the pond after the eggs are deposited. To confirm, Gilbert took a recently inseminated female and allowed her to lay eggs in an aquarium with pond water then removed her and allowed her to lay another clutch in an aquarium with algae-free tap water. Algal cells were present in the eggs laid in pond water within hours, but no algae grew within the tap water eggs after a few days. At the end of this experiment, Gilbert put the eggs from the tap water aquarium in a natural pond. Within a few days, algae had penetrated the jelly membrane.

By closely studying the eggs, he found that the algae transformed as they penetrated the egg. When free-swimming in the pond, the algal cells are long and oval with flagella used for locomotion. As the algae penetrate the egg layers, they grow in size, become spherical in shape, and lose their flagella. It is these large, non-mobile cell-types that rest on the inner membrane and create the “verdant blanket” (as Gilbert describes it; 1942, p. 220).

To demonstrate symbiosis, he compared hatching rates in egg masses kept in light and dark to prevent photosynthesis, finding that light and algae increased hatching success. Although algalogistis (yep, that’s a thing) hadn’t officially recognized the alga species, folks who worked with amphibian eggs called it Oophila amblystoma (“Oo” and “phila” come from the Greek words “egg” and “love,” and “amblystoma” is the genus name of the salamander species the algae cohabitates with. So, the name just means “an algae that likes salamander eggs”).

Just a few years ago, researchers took a closer look at the Oophila algae. They wondered if this was really one species or a clade of algal species, and if it the latter, did different algal species associate with specific amphibian species? To answer those questions, they sequenced the genome of algae from four species across the continent.

It turned out that the Oophila are closely related, close enough to be considered a single clade or species, but within the group there are subclades that tend to associate with each host. However, the researchers also found evidence that different subclades of the algae occasionally defect and switch to other host species.

Friends or just roommates?

Since Gilbert’s era, we’ve learned a lot about the economy of the biological transactions between the algae and the embryo. Using microelectrodes inserted within the egg membrane, Bachmann et al. (1986) measured oxygen concentration within the egg in contrast to the outside water in both light and dark. In the light, the algae worked overtime, producing so much oxygen through photosynthesis that the oxygen production exceeded that needed by the embryo and the environment inside of the egg became super-oxygenated, even when the surrounding water was anoxic (oxygen-poor).

This extra photosynthetic boost is more than just a little helpful. Most amphibian embryos get oxygen by diffusion across the egg membrane from surrounding water or air. Rinder and Friet (1994) wondered if diffusion of oxygen from the surrounding water alone could support amphibian species that had evolved with algal symbionts. They measured oxygen gradients across the cell membrane and injected dye in egg masses to see how much water would be able to flow past the eggs. In wood frogs, they found that the egg mass was loose enough and contained enough channels that water diffusion alone could support respiration. However, spotted salamander egg masses are dense and water does not penetrate to the interior eggs. In their case, the innermost eggs would suffocate without their personal algal oxygen factories. (Note: Hutchinson and Hammen (1958) inferred this relationship many decades before through experimentation rather than directly measuring O² within the eggs.)

This plant-animal relationship is a multifaceted exchange. The reciprocity extends past simple O² and CO² cycling. The algae also benefit from the nitrogen waste produced by the embryo, which limits the ammonia buildup in the egg which is toxic to embryos (Goff and Stein 1978).

Friends or more-than-just-friends?

This symbiotic relationship is certainly interesting, but not altogether surprising. After all, lots of animals host symbiotic microbes, even humans house a panoply in our guts and all over our skin (Gilbert et al. 2018). These types of ectosymbionts, commensal organisms that live in and on other organims’ bodies but outside of the tissue are common.

But the green egg story gets even more fascinating. In 2011, Kerney et al. used a technique that binds only to algae DNA and causes it to glow under fluorescent lights (fluorescent in situ hybridization (FISH)). In doing so, they could see algae cells inside(!) of the salamander tissue and cells. This makes this salamander-algae relationship the only example of endosymbiosis in a vertebrate ever seen! Previously, this type of animal-plant union was thought to only be possible in simpler organisms like coral and mollusks.

Friends or frenemies?

So, not only do these free-swimming algae manage to invade the egg membrane, but they even make it all the way inside of the embryonic cells. To some extent, this makes sense for the algae. Why would you want to live out in the pond water with variable temperatures and predators when you could live inside a nice cozy egg with abundant CO2 and nutrients? But once inside the egg, why would algae invade inside an animal cell where there is less access to light for photosynthesis?

A team of researchers (Burns et al. 2017), including Kerney, wanted to figure out the riddle. They isolated and sequenced the genes expressed by salamander cells with and without algae cells from inside and outside of the animal cell. Inside the animal cells, algae seemed to be under more stress, to the point that they switch from photosynthesis to fermentative energy production, something that would normally only happen in highly unfavorable environments. On the other hand, salamander cells with algae were unfazed and even seemed to suppress their immune response which would ordinarily attack foreign cells, perhaps to keep the algae within. For algae, then, there appears to be a risk-benefit balance that drives them inside of the egg where conditions are favorable, but risk being captured inside of the embryonic animal cells.

Better than Seuss

After over a century of investigation, the story of the Green Eggs has gotten more and more interesting with finer and finer resolution. The saga goes to show how the naturalist’s intrigue can inspire a cascade of illuminating research.

Understanding this complex relationship can also help us understand threats to amphibian populations. Herbicides can kill the symbiotic algae and result in lower hatching success and reduced developmental rates. Olivier and Moon (2009) tested the effect of the herbicide atrazine on spotted salamander egg masses. They found that even low concentrations of the chemical killed the algae. In addition to the direct effects of the chemical, the loss of the symbiont had negative repercussions.

References:

Bachmann, M. D., Carlton, R. G., Burkholder, J. M., and Wetzel, R. G. (1986). Symbiosis between salamander eggs and green algae: microelectrode measurements inside eggs demonstrate effect of photosynthesis on oxygen concentration. Can. J. Zool. 64, 1586–1588.

Burns, J. A., Zhang, H., Hill, E., Kim, E., and Kerney, R. (2017). Transcriptome analysis illuminates the nature of the intracellular interaction in a vertebrate-algal symbiosis. Elife 6. doi:10.7554/eLife.22054.

Gilbert, J. A., Blaser, M. J., Caporaso, J. G., Jansson, J. K., Lynch, S. V., and Knight, R. (2018). Current understanding of the human microbiome. Nat. Med. 24, 392–400.

Gilbert, P. W. (1942). Observations on the Eggs of Ambystoma Maculatum with Especial Reference to the Green Algae Found Within the Egg Envelopes. Ecology 23, 215–227.

Goff, L. J., and Stein, J. R. (1978). Ammonia: basis for algal symbiosis in salamander egg masses. Life Sci. 22, 1463–1468.

Hutchison, V. H., and Hammen, C. S. (1958). Oxygen utilization in the symbiosis of embryos of the salamander, Ambystoma maculatum and the alga, Oophila amblystomatis. Biol. Bull. 115, 483–489.

Kim, E., Lin, Y., Kerney, R., Blumenberg, L., and Bishop, C. (2014). Phylogenetic Analysis of Algal Symbionts Associated with Four North American Amphibian Egg Masses. PLoS One 9, e108915.

Olivier, H. M., and Moon, B. R. (2010). The effects of atrazine on spotted salamander embryos and their symbiotic alga. Ecotoxicology 19, 654–661.

Orr, H. (1888). Memoirs: Note on the Development of Amphibians, chiefly concerning the Central Nervous System; with Additional Observations on the Hypophysis, Mouth, and the Appendages and Skeleton of the Head. J. Cell Sci. s2-29, 295–324.

Pinder, A., and Friet, S. (1994). Oxygen transport in egg masses of the amphibians Rana sylvatica and Ambystoma maculatum: convection, diffusion and oxygen production by algae. J. Exp. Biol. 197, 17–30.