Forest Focus Episode 43: Wandering after Wolves - Transcript

Jamie Hinrichs [00:00:12] Welcome to episode 43 of Forest Focus, brought to you by the USDA Forest Service Pacific Southwest Region. We're continuing in our explorations of threatened or endangered wildlife species, and this time it's all about gray wolves.

Tom Rickman [00:00:28] I've been on the Eagle Lake Ranger District since 1985. I never thought we would have one of the most iconic species recolonize public lands in this area. As a biologist, I think it's very cool that they're here. It's an exciting time in terms of biodiversity, in terms of restoring some of the species that had been extirpated.

[Acoustic music with harmonica plays underneath following narration]

Jamie Hinrichs [00:00:49] The gray wolf, Canis lupus, became extirpated or locally extinct in California in the 1920s during a nationwide eradication effort. During this time, wildlife management gave greater focus to conserving birds and hoofed animals like deer, and the nation did not yet have the Endangered Species Act, which today gives explicit attention to conserving species showing signs of significant decline.

But the gray wolf was once a native species in California that ranged widely, both here and throughout much of the United States, and as Forest Service wildlife biologist Tom Rickman just expressed, in recent years, gray wolves have been returning to California. Their slow comeback is a natural progression of population growth happening in other western states, including Idaho, Washington and Oregon.

Wildlife specialists with the USDA Forest Service and the California Department of Fish and Wildlife are working together to monitor and conserve this recovering species by looking for signs of their whereabouts with boots on the ground, satellite collars and DNA analysis. In this episode, we'll hit the road in two national forests and make a stop at a forensic lab.

We begin in the Lassen National Forest, where we meet our first guide.

[Music ends]

Tom Rickman [00:02:05] My name is Tom Rickman. I'm the wildlife biologist on the Eagle Lake Ranger District of the Lassen National Forest.

Jamie Hinrichs [00:02:10] Tom gives us a brief introduction to the gray wolf.

Tom Rickman [00:02:14] Gray wolves are large canids or large members of the dog family, generally 80 pounds or larger. They prey primarily on native ungulates like deer and elk. They live in packs. Generally, there's a breeding pair, and then the puppies of that year, along with a few yearlings. Yearlings tend to disperse their second winter, so maybe 7 to 10 animals in a pack at any given time. Counting puppies.

Jamie Hinrichs [00:02:39] It is common for yearlings, the year-old offspring, to leave their birth pack in order to find new territories, join another pack or form their own pack. The forming of new packs and search for new terrain has allowed wolves to return to California.

Tom Rickman [00:02:53] The returning because of increasing wolf populations in Washington and Oregon. That is just a natural progression. As population increases, they keep going into new areas looking for additional habitat.

Jamie Hinrichs [00:03:04] Wolves move through lands of all ownerships in search of this habitat. So, while they may travel through or temporarily den within national forests, they may do the same in other kinds of federal land, along with state, Tribal or private land. But for now, we'll focus on the history of wolves coming and going in parts of the Lassen National Forest.

Tom Rickman [00:03:24] In this period where wolves have been returning to California, the first known wolf to have ventured on to the Lassen was OR7, the radio collared wolf from Oregon that came in 2011, and it traveled on to and through the Lassen National Forest. And then it established a pack in southern Oregon. And then it wasn't until either late 2015 or early 2016 where we had the pair that eventually became the Lassen Pack showed up on the forest. They've had at least one litter every year since 2017.

And there's a newspaper article from August of 1883 about wolves being active in our district. You know, it's pretty cool to be able to see an article from the 1880s that talked about wolves in a location where 140 years later almost, because of the Lassen Pack, wolves had returned.

Jamie Hinrichs [00:04:16] As of 2023, the Lassen Pack is one of seven confirmed packs in California. Most are in the northern part of the state, but the most recently confirmed pack is in Tulare County, within the southern Sierra Nevada mountains. How do we know about this? There are several methods that wildlife biologists use to confirm the establishment of a new pack, and to monitor the whereabouts of known packs. Tom gives us a taste of two, starting with motion-detecting camera stations.

[Beeping, indicating a car door is open. Car door shuts.]

Tom Rickman [00:04:46] Today we're out checking some trail cameras, exchanging SD cards, and then, when we have time back at the office, we'll check the photos to see if any wolves were in front of these cameras.

[steps walking on grass and gravel]

Tom Rickman So, in this area I have about 12 cameras out on trails or in drainages that might form trailways for the wolves. If you happen to get a photograph of a wolf, they could be anywhere within maybe a 20-mile radius of that camera. Or it could just be a dispersing wolf that may never come back. So, it takes a lot of effort and a lot of time to go from a single trail photo on a trail cam to finding out if it is a pack and where their center of activity is.

Jamie Hinrichs [00:05:35] There is another aspect of Tom's efforts to learn where the wolves might be within the national forest, and what areas they are using as travel routes, and this informs where he might set up cameras in the future.

[Steps walking on a dirt road]

Tom Rickman [00:05:56] We're traveling roads, looking for wolf tracks to see if we can get an idea of where the wolves are traveling to. We know they're traveling past the cameras we have, but we don't quite know where they're going to or where their activity center is. So, we're doing a wider look in the area to get an idea of where their activity center is.

Jamie Hinrichs [00:06:17] By "activity center", Tom means the place the wolves are using temporarily as a denning site, and then what are called rendezvous sites, locations where pups are reared when they are old enough to eat solid food. We'll learn more about both on our next forest stop. But first, back to tracking.

Tom Rickman [00:06:34] Since wolves often travel on roads and trails, my process is to slowly drive roads and watch the margins of the roads for wolf tracks, or to get out and walk around ponds where there's mud to look for prints. I typically drive by waters or areas with easy travel that might be a good connection corridor, not only for ease of travel, but also for the presence of water or prey like deer or elk.

Jamie Hinrichs [00:07:01] In other words, this type of survey requires that we think like a wolf. And if a track is found, what happens next?

Tom Rickman [00:07:09] If it's on Forest Service lands, I'll put out a trail camera to see if the animal is routinely traveling in that area. I'll use that track as a hub to go out and search other areas. Then I'll share that information with the state biologists so they can have another data point to help their search.

Jamie Hinrichs [00:07:27] Through implementation of the Endangered Species Act, the U.S. Fish and Wildlife Service has a responsibility to conserve threatened and endangered species. As part of this work, the U.S. Fish and Wildlife Service coordinates with many partners, including state and federal agencies, Tribes, local governments, and interested stakeholders to monitor and recover federally-listed and at-risk species.

Depending on the state and the species, state wildlife agencies may lead the coordination of conservation and management activities. In California, a state where the gray wolf is endangered under both the California and Federal Endangered Species Act, the California Department of Fish and Wildlife coordinates monitoring of and responses to wolf activity on the ground. Forest Service wildlife biologists work closely with state wildlife biologist to assist with conservation and monitoring efforts.

Tom Rickman [00:08:19] The state has a huge land area to work in and a very elusive, wide-ranging animal. My role is to try to help document the wolves, cutting down the area they have to go survey. So, whenever I get a trail cam photo or if I see a track, I'll send documentation of that to the California Department of Fish and Wildlife, to their biologist.

Kent Laudon [00:08:39] The Forest Service has been helpful with giving us information on wolf sightings, tracks...

Jamie Hinrichs [00:08:45] This is Kent Laudon, leading wolf biologist for the California Department of Fish and Wildlife. Kent's been working with wolves since 1997, when he worked with the Nez Perce Tribe in their efforts to collar and monitor wolves in central Idaho. But today, he's speaking with us on the Klamath National Forest, where the Whaleback Pack has been documented. As Tom just shared with us back on the Lassen National Forest, the California Department of Fish and Wildlife is the lead agency in monitoring wolves in California, so they are tasked with getting as many details as possible about wolf packs and their whereabouts.

Kent Laudon [00:09:19] We're being very thorough through a very large area to ultimately get any detections that we can. Any time that we have a detection of two or more wolves in an area, we will start to survey that entire area looking for sign, often tracks and scat. And because wolves love to follow roads and trails, we survey roads and trails a lot by driving, mountain biking, hiking, and sometimes trail running. And then we look for patterns that can hopefully narrow us down to a specific spot.

Jamie Hinrichs [00:09:54] This detective work is made easier when the state biologists are able to fit wolves with satellite collars, and then safely release them back to the wild. By communicating with satellites, the collars take a reading of the collared wolf's exact location at preset times during the night and day. And those data points are logged on a digital map.

Kent Laudon [00:10:13] Most of our collars in California are programed for three nighttime activity points, so 11:00 at night, 3:00 in the morning and 7:00 in the morning. These are times where wolves are likely to be active. Then we have another point, that's a midday point, that is essentially where they're probably sleeping or bedded or loafing.

Jamie Hinrichs [00:10:35] So while the collar is not gathering location information constantly, which would drain its batteries too quickly, Kent and his staff can see where the wolves are at set hours, which allows them to start to detect a pattern in their movements.

This can be seen on the digital map on his phone, which shows an array of dots and lines. Each dot marks for a collared wolf was located at a specific date and time, and the lines connect the chronological order of the dots. This helps the biologists see the sequence of wolf movements through the landscape during a single day and over multiple weeks. Some dots on the map look like a hub of a wheel, with lines radiating outward like spokes, that indicates the wolves are going out from and returning to the same spot. This suggests a possible den or rendezvous site.

Kent Laudon [00:11:22] Home sites are the pup-rearing sites, which include the den site and rendezvous sites. So starts with the den, of course. Often, it's an excavation in the ground, maybe six feet in, with a chamber in the back where you can picture a female wolf nursing. And at some point, maybe June early June, they will move the pups to a new location. And we call that a rendezvous site. And there will be a series of rendezvous sites through the summer pup-rearing season until pups are big enough to start traveling with the pack.

Jamie Hinrichs [00:11:59] While examining the map with the collar GPS locations on his phone, Kent describes how patterns begin to emerge, giving the biologists insight on the probable location of the rendezvous site.

Kent Laudon [00:12:10] You'll start to have a new spot where the wolves are coming and going. Here's 7:00 in the morning, could be heading in. There's your 2:00 in the afternoon, 2:00 in the afternoon, 2:00 in the afternoon. Guess where the pups are.

Jamie Hinrichs [00:12:28] The wolf collars also have a VHS or very high frequency transmitter. This emits a pulsed radio signal that allows the wolf biologist with a radio receiver to get a sense of how close a wolf is to their present location when they are in the field.

Kent Laudon [00:12:43] The other part of the collars, the old VHF, it puts out a signal. We'll hear the classic beeps and I'm just connecting the omni antenna. It's an omnidirectional, so I can't tell direction with it. All I can tell is signal strength as your position relates to the wolf on the landscape relative to topography. So, if you're losing your line of sight, the signal's going to start disappearing. 

[Radio static starts, then evenly-spaced beeps]

Kent Laudon So that's OR85. OR85 is a breeding male of the Whaleback Pack. And he is from northeast Oregon. He came into California in November of 2020. By I think, Thanksgiving, we had identified that he is running with another wolf, and it was a female. 2021, they had seven pups. 2022, they had eight pups. This year it's a minimum of five. It's a really strong signal. He's really close.

[Beeping fades. Steps walk on dirt road]

Jamie Hinrichs [00:14:04] In case the pack is close by. Kent tries to be quiet during his surveys, which often requires whispering with those who are with him. Using the information the caller provided, Kent looks for signs of the pups on a dirt road here in the Klamath National Forest. In this case, sign takes the shape of tracks and scat, and there is evidence of both here ... Especially the latter.

Kent Laudon [00:14:28] {whispering} Okay, so that could be a pup poop.

Jamie Hinrichs [00:14:30] The pup scats contain the DNA of individual wolves. The samples he collects here will go to a lab for analysis. More on that later.

Here on the road, Kent uses what he calls the "chopstick method" to collect the samples we found on this road. In short, he takes a nearby stick, snaps it tow, and uses the fragments to pick up the scat and place it in a paper lunch sack.

[paper crinkles as something drops inside a paper bag]

Kent Laudon [00:14:56] {whispering} Sometimes pups mess around the culverts. I'm going to look in there.

Jamie Hinrichs [00:15:00] Kent makes his way over to the culvert.

[Feet walk on dirt and then on branches]

Jamie Hinrichs a small metal tunnel that carries water from a rain event under a road. With no recent rain in this area, the culvert is likely dry, so he bends his head down, but straightens up immediately and jogs

[steps jog softly]

Jamie Hinrichs over to the other side of the road, where the other end of the culvert is located. Here we see...

Kent Laudon [00:15:25] {whispering} Three ... four ...

Jamie Hinrichs [00:15:25] Five pups total, quietly exiting the culvert opening on the other side of the road, one at a time. They are nearly silent in their movements as they head away into the trees at a brisk but not urgent stride.

[faint snap of twigs]

Jamie Hinrichs The only sound of their passing is a few quiet snaps of brittle twigs under paw. Although wolf biologists use the methods we've just learned about - motion detecting camera stations, satellite collars and radio signals - to help them know where wolves might be, going from collar signal to pup viewing isn't uncommon experience, as is seeing them this close.

Kent Laudon [00:16:00] {whispering} It's your lucky day.

Jamie Hinrichs [00:16:02] Indeed it is. It is also important to note here that wolves are wild animals. Why they generally fear and avoid people, so rarely pose a direct threat to human safety, us non-experts should never look for, approach, feed, or otherwise interact with the wolf. We are only out looking for signs today because we are with a professional wolf expert.

Additionally, Kent's intention was to visit the road to look for signs of the wolves without the wolves detecting the visit. This is to avoid disturbing the wolves, which may cause them to find a new secure spot for themselves and the pups, one without human intrusion. It came as a surprise to find so much pup scat on the road, which indicated the pups were much closer to the road rather than the 300 yards upslope, in the cover of trees, as the GPS collar data indicated. It came as an even greater surprise that they were in the culvert.

So, Kent does not linger long here. After setting up a camera near the culvert and one on the road in the hopes of getting photos of the pups we just saw. We leave the area.

Kent Laudon [00:17:06] {whispering} We should just get out of here.

[steps walking on dirt road for a few seconds, then fades]

Jamie Hinrichs [00:17:08] But we're not done with our pursuit of wolves. Several weeks later, we follow the scat we collected to the California Department of Fish and Wildlife forensic lab in Sacramento to better understand why it was so important that we gathered it.

[music interlude: upbeat, electronic, 80s vibe]

[doorbell]

Unidentified [00:17:36] Morning.

Erin Meredith [00:17:40] We'll start in the warehouse. A lot of our boxes right over in that area are actually scat samples that we have analyzed this year. And you can smell the eau de scat, a little bit.

Jamie Hinrichs [00:17:53] This is our first forensic guide.

Erin Meredith [00:17:55] I'm Erin Meredith, I'm a senior wildlife forensic specialist with the California Department of Fish and Wildlife. And we fall under the law enforcement division. I've been around here for 24 years.

Right now, we're in the warehouse space where we sample a lot of our cases and investigations because we have a large metal table that allows us to wash down any of the excess debris. We predominantly do genetic analysis here. And so, when we're sampling in this room, that's what we are sampling for.

Since the inception, we've been working with predominantly two packs in California: the Whaleback Pack, which you visited and then the Lassen Pack. And so, we've been monitoring those breeders and their pups for several years now. But this year, one that hit the news fairly recently, was the group down in Sequoia National Forest. And so, some of the scats we have over there were the original samples that came from Sequoia to let us know that we did actually have wolves down in that area.

Kent is out in the field sampling during late spring early summer, trying to size differentiate the scat a little bit so that we know this is a pup versus it's one of the adults, and then try and target those smaller ones so that we can get every pup, their genetic profile, into the database.

Kent said to let you know, the camera that was on the culvert, where you saw the pups...

Jillian Adkins [00:19:33] You got to see the pups?

Jamie Hinrichs [00:19:34] The second voice is Jillian Adkins, also a senior wildlife forensic specialist with the California Department of Fish and Wildlife. She works closely with Erin on wolf forensics and other wildlife investigations.

Erin Meredith [00:19:46] Kent said a couple of times what he thinks was the female would walk by, and the one that you guys set on the road did get the pups for about another week. So those scats that you collected with him, the pup scats, detected six different individuals.

Jillian Adkins [00:20:04] We saw five of those pups with our eyes, but the scat we collected had genetic information for six individuals. And since our visit, some trail cam footage found evidence of even more pups.

Erin Meredith [00:20:16] So we thought we had them all. And someone else said they had two more on trail cam video. He went back to that same site and collected a few more scat. That's what these are. We're going to hope that we catch the other two.

Jamie Hinrichs [00:20:31] By "catch", Erin means identify genetically.

Erin Meredith [00:20:35] And so the scats that you collected with Kent and what he just collected now are going to be really important.

Jamie Hinrichs [00:20:41] Many of us have seen scat as something to avoid stepping on while out on a national forest. But scat is not merely a waste product. It also contains a wealth of genetic information that helps specialists monitor and conserve wolves.

Erin Meredith [00:20:55] For the wolves, we are actually using genetics to identify each of the individuals of the different packs that are present in California. We can use the genetics to tell their sex, their individual identity, and also their coat color. We've been doing that predominantly through scat samples that are collected in the field. And then we keep a database of their DNA profiles. So even if an animal isn't collared with a GPS collar, we can track them genetically through time. So, we'll know the year they were born ... if they've dispersed and we find their scat somewhere else, we know they've moved to a different location ... if they have produced offspring, we can use their genetic profile to trace that. So, it's very helpful in knowing what individuals are out there on the landscape.

Jamie Hinrichs [00:21:48] As we're about to learn. There is a lot of patient work and precise technique that goes into extracting the DNA we seek from the scat samples.

Erin Meredith [00:21:56] Scat might contain the DNA of what the animal has eaten. And what we're trying to really get is a host's DNA from the scat. Well, what's the best way to do that? Well, you target the tapered end. Now we are pros in tapered ends, I would say. {laughs}

Jamie Hinrichs [00:22:16] The exterior surface of the scat, especially the tapered or pointed end, are the portions most likely to have supplies of DNA-carrying epithelial or skin cells, which cover internal and external surfaces of the body. These cells, which are shed from the intestinal tract, attach to the scat during their digestion process. So, Erin and Jillian start the DNA extraction process by cutting off the tapered end of the scat, or shaving off a bit of the exterior surface here in the warehouse.

[Paper rips. Paper bags crinkles as bag is opened. Scat shaken from bag, clunks onto metal table]

Jillian Adkins [00:22:57] So each sample has a six-by-six piece of weigh paper, one razor blade, and one wooden applicator stick that will help us handle the scat. And we also have a tube rack that I'm placing our bead tubes into, which we'll label with the corresponding scat identifiers.

Erin Meredith [00:23:23] In scat, you have bacterial components, but you still have those epithelial cells that are on that external surface that's depositing the host DNA there. That's what we're really looking for. The extraction that we do has a lot of special components in it to eliminate the inhibitors. We really just take, razor blade and cut right into the end.

[soft slicing, fibrous materials rips quietly]

Erin Meredith maybe shave a little bit off of the side trying to get that external surface more specifically. And it goes into a special tube that has beads in it that kind of beat it up and get the DNA to release from the fecal matter. Okay. The lab. Here we go.

[Quick beep. Door opens and closes. Loud hum of warehouse fades to quiet of lab]

Jillian Adkins [00:24:19] So this is called our pre-PCR lab.

Jamie Hinrichs [00:24:22] PCR stands for polymerase chain reaction.

Jillian Adkins [00:24:25] Where we're going to be isolating and extracting all of our DNA.

Erin Meredith [00:24:29] We started out with a certain kit. It's all done by hand. And we're very fortunate here. We've got a couple of different DNA-extraction robots and having really good success so far. So, we're still kind of in our testing phase where we've been doing things in duplicate the old way and the new way, and making sure that everything is connecting up the way we would like it. And so far, the new way with the robot is winning. So, we're very happy about that, because if we do a batch of 24 scats, that's going to take maybe 3 to 4 hours with a lot of hands-on time, and with the robot, there's a lot more walking away and leaving it for the robot to do. So, that's really slick, and hopefully it's going to free up some time for us.

Jamie Hinrichs [00:25:18] Before the robot can begin its work, some human, hands-on preparations are still required. It involves a series of steps, sometimes repeated. Let's lay out the mental checklist before we see those steps play out:

[music with ticking and snaps starts]

Jamie Hinrichs

At the start, liquid substances, called reagents are added to the tubes with the scat shavings.

Those tubes are put on to a vortex, which shakes, allowing the beads within the tubes to break down the fibrous scat material and release the cells that contain the host DNA.

Those tubes then go into a centrifuge, which spins the samples, thereby separating the liquid and remaining solid components. The remaining fibrous scat material sinks to the bottom of the tubes.

The liquid in the tubes, which has the cells containing the host DNA, is then transferred into new tubes that have additional reagents in them.

Those tubes are then heated in an incubator to encourage the breakdown of undesirable substances from the scat, and the cells themselves, which release the DNA into the liquid within the tubes.

The samples get another reagent added, one final shake and spin, and then the liquid is transferred again into yet another new, but final, set of tubes ready for the robot.

[music ends abruptly with guitar riff, then moment of silence]

Jamie Hinrichs Got all that? Don't worry, we will not be asked to recite those steps for memory. The only thing we need to remember at the end is there are a lot of steps and precise techniques involved. Now let's just enjoy the lab journey as we see those steps play out.

Erin Meredith [00:26:46] We start with those bead tubes and add the buffer to take out some of those inhibitors and release the DNA from the scat. Jillian's going to show you what that looks like.

Jillian Adkins [00:27:02] This is one of our pipetters.

Jamie Hinrichs [00:27:04] A pipette might be described as a thicker and less sharp syringe, which can extract or add liquid and precise measurements, usually amounts of less than one millimeter.

Jillian Adkins [00:27:13] We're adding one of the special components that Aaron Erin has been talking about.

[soft squirt of liquid]

Erin Meredith [00:27:20] And we put it on the vortex.

[unscrews metal top]

Erin Meredith which is going to shake it.

[snaps tubes into place, soft shaking of beads begins]

Erin Meredith So the next step, we're going to centrifuge at very high speed to get all of the remaining solids and the beads to go down to the bottom of the tube. And then that will leave that liquid, what they call the lysate that hopefully is going to have our host DNA in it, which will be able to transfer off.

[whirr of spinning builds speed, then fades]

Jamie Hinrichs [00:28:04] At the end of the spin cycle, the lysate - the fluid component that contains DNA carrying cells which have been released from the scat with the shaking and spinning - is extracted and transferred into new tubes. The liquid is sort of a scat-cell smoothie, if you will.

Erin Meredith [00:28:19] You can still see quite a bit of solid, but you can also see the discolored fluid at the top. That's what we're going to be transferring off.

Jillian Adkins [00:28:28] We're trying to avoid remaining solid. So, I'm holding the tube at an angle to attempt to just get the lysate.

[soft squirt of fluid]

Jamie Hinrichs [00:28:39] Once the lysate is added to the new tubes, the tubes hit the DNA sauna to further remove the undesirable components and release the DNA. It's a bit like sweating it out for humans.

Erin Meredith [00:28:50] So now it's going to go and just incubate for 15 minutes at 56 Celsius.

[faint whirr, and then a timer beeps]

Erin Meredith The idea of this solution is it's going to pull some of those impurities out and keep those to the bottom, so that you can draw off the DNA that you want. And you'll see once I vortex it.

[liquid sloshes with buzzing of vibration]

Erin Meredith So now you can see they're all a little lighter, a little thicker looking. So now they get to ride the Gravitron again.

[spin builds, then fades]

Erin Meredith Okay, so now we're on our last step before we get to loaded up the robot. So same thing. We're just going to try to avoid that solid clump that's at the bottom. So, if I can just angle the tube and take that supernatant off the top.

Jamie Hinrichs [00:29:44] Now we meet the robot.

Erin Meredith [00:29:46] So that's a DNA-extraction robot. It's a big rectangular box that has a moving robotic arm that can pick things up, transfer the fluids around. And I think it's smarter than I am.

Jamie Hinrichs [00:30:01] To extract the DNA. The robot will add the lysate, the cell smoothie we learned about earlier, which is now in a more purified form to a set of tubes with magnetic beads.

Erin Meredith [00:30:11] So DNA has a charge, and you can use that to essentially bind the DNA to the magnetic beads. While they're holding the DNA, magnetic rods hold the beads to them. The rods shake around, wash away all the impurities, and then the final step is going to release the DNA from the beads and just leave you with a solution of your DNA, only. Let me open up the sample drawer.

[draw squeaks]

Erin Meredith So it recognizes that there's 14 samples there. Now it's where I'm telling it what protocol to run.

[mechanic groans begin and continue, interspersed with pauses and various forms of mechanic chirps and hums]

Erin Meredith Now it's picking up the magnetic beads. That's actually it mixing them. It's probably about an hour to go from you load your sample to you have your DNA.

Jamie Hinrichs [00:31:08] We'll let the robot continue to do its work as we learn about what comes next in the process. In short, Erin and Jillian take the extracted DNA and make many copies of it, a process called amplification.

Erin Meredith [00:31:21] We just take that extract straight from the robot, and we put it through a process called PCR - polymerase chain reaction. We have a number of very specific markers that have been tested over time. These markers can differentiate individuals from one another. It'll give us the color of the individual and we can tell if the individuals male or female. We're going to set up a reaction that essentially is going to use those markers to copy very specific regions of the genome.

Jamie Hinrichs [00:31:51] To better grasp what we're learning here. We need a quick dip into molecular biology. If we stick together, we can come out on the other side with knowledge that can up our competitive edge at future trivia nights. So, let's go:

[mechanic groans and hums fade as soft music begins. Music has a soft series of beats, suggests being in outer space or underwater]

Jamie Hinrichs First, a genome contains the genetic information of an organism, and this genetic information is made up of DNA.

DNA can be thought of as written instructions that live inside the cells of living things. More specifically, DNA resides in chromosomes: discrete, thread like structures at the center of cells.

These written instructions, called DNA, use the language of four chemical building blocks, also known as bases. Just as the arrangement of words become written instructions, the arrangement of these four bases form a sequence of genetic instructions. These genetic instructions determine the species and character traits of an organism, and ultimately make each individual unique.

A specific location on a chromosome with a known DNA sequence is called a genetic marker.

To conceptualize how these genetic components come together, we can think of the genome as an encyclopedia. Each chromosome is a volume of the encyclopedia, and each marker is a specific page or even a single sentence on the page.

In the case of wolves, the markers can be used to determine the species, sex, coat color, family line, and individual identification. Taken together, the combination of specific markers forms a DNA profile.

To further wrap our heads around this, let's think of a DNA profile as a chemical fingerprint. At a distance, one human fingerprint tends to look much like another, but a close examination reveals distinct patterns of whorls and lines that are unique to an individual.

A close examination of a DNA profile can do the same, but since DNA is invisible to the human eye, seeing a DNA profile requires another multi-step process accomplished here in the laboratory.

This is how it works. A chemical reaction performed with a DNA copying machine, targets, tags and copies the precise regions of the genome where the DNA markers reside. During the process, colorful dyes are added to each copy of every marker. Then, computer software translates the chemical language of each marker into a digital genetic portrait that human eyes can read and analyze.

[music stops]

Erin and Jillian are masters of this process here in the lab. Erin said earlier she thought the robot might be smarter than her. But after this brief sip of molecular biology, I think we respectfully, but firmly, disagree.

Erin Meredith [00:34:36] So this is the post PCR side of the lab.

[background whirring of machines]

Erin Meredith These instruments here at both thermal cyclers, through different heating and cooling steps, cause the copying of the DNA. And while it's copying, each one of those markers has a florescent tag, associated with it, and that's how we're going to visualize it. Each cycle, a particular region of DNA, it's going to double. We do a total of 36 cycles for wolves. So, two to the 36 is how many copies you would end up with.

Jamie Hinrichs [00:35:09] That's 68 billion copies, billion with a "B", for anyone trying to do the mental math.

Erin Meredith [00:35:15] Once it's done copying on this machine, we take it over to this genetic analyzer. And really, it's just detecting the fluorescence that we added in the PCR cycle. It has these really fine little capillary tubes that are going to suck up the sample. And those fragments are going to get detected by the laser, transmitted to the computer, and some really high capability software translates your DNA profile.

[Background softens, followed by quiet steps, then quick keyboard typing]

Jamie Hinrichs [00:35:48] Erin shows us some profiles back in our office. They look a bit like a line graph with peaks on some sections in various colors.

Erin Meredith [00:35:55] So this is kind of what it looks like after it's gone through the software, and the software's cleaned it up. We've got results on every marker for sure. Here comes some blue peaks from the markers that we have labeled with the blue dye. Here are some red peaks from the markers that are labeled with the red dye.

Jamie Hinrichs [00:36:12] Over the past seven years, the team has analyzed more than 1500 wolf scats, detecting the genetic profiles of over 90 different wolves who've at least pass through California, leaving a "deposit".

There are many important things we've learned from this dive into wildlife forensics. For one, when we next encounter a story about wolves in California and there is a mention of "DNA analysis", we now know that there are countless hours of recent work and years of research silently embedded in that two-word phrase.

Erin Meredith [00:36:44] I really like the wolves because you feel an individual connection with each one of them. We don't see them in the field. It's not that way. But it's more like, "Oh, look! There's so-and-so's profile again." You know, "he's still around." Or, "Oh, that's a new one coming in here." Being able to know the counts and where this individual is going and, and follow it genetically ... there's just a little bit of a personal feel to it.

Jamie Hinrichs [00:37:11] Back in the field, the lab results help state and Forest Service wildlife specialists to better understand changes and movement of wolf populations. Photographs, tracks and scats are working together to enhance collaborative efforts to conserve this elusive keystone species.

Jamie Hinrichs [00:37:30] Thanks for listening. For those who are curious to learn more, go over to the show description page and click on the links to find more episodes. Search for Forest Focus podcast wherever you get your podcasts and if you have questions or suggestions for the show, email us at SM.FS.R5FFpodcast@usda.gov.

Until next time, enjoy your public lands. Please remember to recreate responsibly. Know before you go and learn what you can do to prevent wildfires. Take care.

This podcast is sponsored by the U.S. Department of Agriculture, Forest Service, and the USDA is an equal opportunity provider, employer, and lender.