Winter 1999:
Regal Fritillaries in a Tailspin

a Story of East and West DNA and the Urgent Need for Conservation of a Flagship Species by Barry Williams

There is a subtle beauty to prairies. That prairies are not widely appreciated is not surprising given the amount of prairie one is likely to encounter while traveling through the Midwest. Those small parcels of pristine prairie that remain are often found along winding gravel roads away from tourist attractions, large towns, and interstates. This is unfortunate because a lack of knowledge contributes toward a lack of interest and thus to decreased public support for prairie preservation. Too few realize the pleasure that can come from visiting a prairie. There is an overwhelming sense of serenity that comes from standing amidst of a sea of grasses and prairie flowers as the wind gently rocks them back and forth in an almost hypnotic, wavelike motion.

A Regal Introduction
Of all the beautiful sights to be found on a prairie none are as m
agnificent as a Regal Fritillary. As its name implies, this species has a breathtaking beauty for those lucky enough to find it in flight. One of our largest butterflies, males can be seen even at a distance, patrolling the prairie for females. Flapping their wings as they move across the land, they stay just above the tops of the plants. Sometimes they soar with their wings parallel to the ground for short spurts; other times they will stop at clumps of dense vegetation where females may be hiding. When seen fleetingly, their orange ground color and black spots cause them to be confused with Monarchs. But, the underside, with silver spots on a black background, is a dead giveaway for this fritillary. Nothing else, not even those often difficult to identify relatives in the genus Speyeria, can be confused with it. People are usually pleasantly surprised when they see this species for the first time because there is a brilliant blue iridescence on the upperside hindwings that is rarely captured on film. Occasionally two individuals will encounter one another in flight, after which they will go upward several feet, circling one another in a swirling motion. If two males are involved, the dance stops there. But if a male finds a female they continue up and around at times racing down into the grass only to swirl up again. If the female is receptive, the pair will eventually land on a perch and the male will shutter his fully spread wings towards the female. If she is still receptive, they will eventually mate.

Regals can also be found nectaring on some of their favorite nectar sources like orange milkweed (Asciepias tuberosa), other milkweeds (Asciepias sp.), or thistle (Cirsium sp.). In some portions of their range females will aestivate in August, only to emerge from hiding late in the summer and walk along the ground laying eggs singly in the debris on the prairie floor. After roughly ten days the eggs will hatch and the tiny caterpillar, roughly 1 millimeter in length, will overwinter. In the spring the caterpillars will emerge to feed on their host plants, presumably North American violets (Viola sp.).

As large and beautiful as Regal Fritillaries are, relatively few people have had an opportunity to see one. One reason, as I have already mentioned, is that there are few prairies remaining. In much of the Midwest, more than 99% of the original prairie landscape has either been altered or destroyed. As the first plow became readily available to settlers in the mid-19th century, the fertile prairie soil was almost entirely transformed to row crop agriculture. This fact alone has doomed several species of prairie plants and animals. The Regal however, is in dire straights because, among the few remaining suitable patches of habitat, only a fraction of them still have Regals. This means that the Regal is in the unfortunate predicament of being a rare species in a rare habitat.

Decline of Regal Fritillaries
Just how rare are Regals? If you were to look at a range map in some butterfly identification guides it would indicate that Regals can be found anywhere from North Dakota and Colorado east to Virginia and Maine. Range maps are often constructed from historical observations and for most purposes that's fine. But we want to know whether a species is currently disappearing from some places which means that historical records are no longer useful. The problem is that up to date range maps are difficult to compile and it can be nearly impossible to prove that something doesn 't exist. For example, it is unlikely that "Bigfoot" is roaming the U.S. forests but no one has been able to actually prove that it does not. No matter how much we might search someone can always accuse us of not looking hard enough. The result is that showing a decrease in range size is extremely labor intensive. Luckily for us, locating Regal Fritillaries is an easier task than finding Bigfoot. First, we know that Regals can only be found in certain habitats, namely prairies and open areas with both the putative host plant and the nectar sources required for survival. Second, Regals are relatively large butterflies with males that spend most of their time looking for females. If one stands still in an open prairie, Regals will often fly up to you, almost as if they were curious. Both of these points mean that the butterfly is more likely to be found than our elusive friend Bigfoot and that makes the construction of a range map an easier task.

I set out to compile an up to date range map of the species by talking with all of the people I could who might have some information on where we could or could not find Regals. I am especially interested in finding populations of Regals rather than just single occurrences. A single sighting for a Regal is hard to verify and efforts to protect the species should be focused primarily on those populations which are large enough to be worth preserving. After talking with many active butterfly enthusiasts from around the country I was able to compile a map which has three major range components: 1) a region in the Great Plains (North Dakota south to Colorado and western Missouri) with several populations, 2) a region in the Midwest (Minnesota, Iowa, Wisconsin, Illinois, and Missouri) with relatively few, fragmented populations, 3) two isolated populations in the East (remnants of the former, much more extensive, range).

Many populations can still be found in the great plains. It has been my experience that much of the good prairie habitat I came across in this region still has Regals. As we move east of the Missouri river however, populations become harder to find. One of the main reasons populations are so difficult to find is that prairies themselves are so sparse. Even so, of the remaining prairies only a few still have Regals. It seems unlikely that we are overlooking the species in this region of the country. Several people are working on prairie butterflies in states like Iowa, Wisconsin, and Illinois (many have contributed to this magazine in the past) so if a large, mobile butterfly like the Regal was around we would expect that they would have found it by now. The disappearance of Regals has been so rapid that in many of the states where Regals are now feared extinct the species was never even listed as state threatened or endangered! For example, a survey compiled in 1973 by Roderick Irwin and John Downey at the Illinois Natural History Survey.

The situation becomes much worse once we move east of Illinois. No one recently has found a Regal Fritillary in any of the states east of the Illinois/Indiana border with the exceptions of Pennsylvania and Virginia, which harbor small, isolated colonies. The first colony is at Fort Indiantown Gap, Pennsylvania. This particular population is located on a military installation run by the United States Army and Pennsylvania national guard. Tank training and aerial bombing practices threaten to destroy valuable Regal habitat which has led to a hotly contested debate on the preservation of this population. Both the Nature Conservancy and NABA are working to protect it. NABA has been of particular importance by maintaining vocal opposition to proposed management plans for this population and by initiating litigation to protect it. The second population was a recent discovery in Virginia. Mark-recapture studies are currently underway but it appears that this is a relatively small population. Off-hand, this isn't a good sign. In most cases, Regals appear to be almost described three subspecies. So why is there no geographic variation in Regal Fritillaries? Paul Hammond, a Speyeria researcher from Oregon, points out that the Regal Fritillary is probably a highly mobile species that may be adapted to recolonizing large areas of prairie that have recently been burned. If the Regal can exchange several individuals among populations each year, then the continuous "mixing" of genetic material will prevent individuals in one region from diverging from those in another region. The result of all this mixing would a lack of geographic variation. If populations become separated from one another, as they have over most of the Regal's range, then mobility becomes an important issue. Once a population becomes isolated, then one bad year of weather or intense inbreeding can wipe out an entire population. A species which is not highly mobile cannot recolonize that area and the species' range starts to contract. So an important issue for the Regal centers around migratory ability.

Migratory ability is difficult to measure for several reasons. The first step is to mark all the individuals in several sites before they can move from one population to the next. The next step involves recapturing those same individuals to see if any of them have moved from one place to the next. If an unmarked individual arrives at a site it could be because it is a new immigrant, a newly emerged adult, or it was not captured on the first attempt. If an individual is captured that has moved between populations then one must establish that it and its offspring can survive in the new environment. All of these steps are required to show successful migratory ability in a species and are clearly labor intensive. But there is another way to get a similar sort of information. DNA.

Why DNA?
DNA is the one thing which is shared among almost all forms of life. It is the information that a single egg can use to construct a whole organism and is often referred to as the blueprint of life. But what does DNA have to do with migration and Regal Fritillaries? The answer lies with variation in the DNA. Some types of DNA, or genes, are so central to the function of the cell that they are unable to withstand the effects of mutation and therefore change at an extremely slow rate. Genes like this are used to examine relationships between organisms as distantly related as bacteria and humans. Conversely, some pieces of DNA are susceptible to mutation and can actually promote change. These pieces of DNA are so variable that they tend to be different among individuals, hence their use in DNA fingerprinting and human forensics.

As you might have already guessed, there are pieces of DNA that are ideal for looking at differences between populations as well. My graduate research has focused on using DNA to look at the relationships among populations of Regal Fritillaries. Despite the long-winded discussion above, the basic premise is rather simple. If all individuals from one population have the same types of DNA in the same proportion as those from another population, then the two populations are essentially the same and must be sharing several migrants. If the types of DNA and their respective proportions are very different among populations then they must not be sharing migrants or they would appear more similar. There are various mathematical techniques for deciphering this relationship in detail but the basic premise is the same. There are a couple of points that make this method rather attractive. First, because the information is genetic, only individuals moving between populations and successfully reproducing will be detected. Second, based on how quickly the DNA mutates you can get different measures of how long it has been since they last shared migrants. If the DNA mutates very quickly then differences among populations must be fairly recent. But differences at a more slowly mutating piece of DNA will reveal more ancient genetic exchange or lack thereof.

What Do DNA Studies Tell Us About Regal Fritillaries?
I have managed to examine individuals from 18 different populations, including the Pennsylvania population, from across the range of Regal Fritillaries. You might ask yourself how I managed to capture individuals from such sensitive locations. This is one of the biggest advantages of DNA. I am able to get all the DNA I need by simply removing a single leg and releasing the butterfly alive! While the removal of a leg certainly has some detrimental effects on the butterfly, the alternatives are either destruction of the specimens or no information.

The preliminary results of my analysis are still being prepared for publication and are therefore tentative until they can be reviewed by members of the scientific community, but I will provide a short summary here. There are very few differences among all populations in the great plains and Midwest. The Pennsylvania population, however, has several mutational differences that all Pennsylvania individuals share but no other population has. This particular piece of DNA mutates at a slow rate so that is not surprising that we don't find differences in the great plains and Midwest. Because we do see differences in the Pennsylvania population, it is likely that this populations has been separated from any of the other populations I examined for quite some time. The estimated rate of mutation for this region of DNA is roughly 2% per million years. If this rate holds for the Regal (there is variation in the estimated rate for this molecule) then it will have been on the order of 400,000 years since these populations last mixed.

There is some supportive evidence of such evolutionary separation in the literature. Despite my primary focus on prairies of the Midwest early in this paper, initial descriptions of Regal Fritillary in the eastern U.S. have described it as being associated with mesic (wet) habitats, such as marshes. Midwestern populations are most certainly associated with dry sand prairies. Differences in habitat type may be due to an underlying evolutionary pattern where butterflies from one region are adapted to specific ecological conditions unlike those in another region. If this is the case for Regals, than there are both genetic and ecological differences which make the eastern populations unique and worth protecting. Clearly, more research on host plant use and habitat requirements is needed.

The DNA data has particular importance in the ongoing litigation between the North American Butterfly Association and the Army base at Fort Indiantown Gap. It is possible that this analysis alone will lead to a subspecific standing for this population and therefore protection under the Endangered Species Act. I am working on more rapidly mutating regions of DNA to provide supporting evidence for this data and better resolution of the relationships among Midwestern populations. These data can then be used to look at the amount of inbreeding and estimate the number of breeding individuals in different populations.

Why don't they look different?
If the Pennsylvania population's DNA is so different from all the rest, why do they look the same? As it turns out, they don't. Based on advice from people like Harry Pavulaan, a lepidopterist in Maryland, I went to the American Museum of Natural History in New York to carefully examine morphological variation in the Regals. After measuring several physical features of the butterfly in over 350 specimens, I found that there are some differences after all. Species in the group Speyeria usually have differences which are based on color. The problem with color differences in butterflies is that they can exist because butterflies are raised under different conditions. This means that two populations may look different, but are actually the same. To avoid this problem I measured the size of spots on the underside of the wings. What I found is that not only is it quite variable, but there is a clear pattern to the variation. Butterflies from the plains states tend to have relatively larger spots and those from the east (remember these are museum collections and I was able to look at populations that are now extinct) are smaller or lack some spots altogether. It is not surprising that this variation has been overlooked in the past. It can be very difficult to tell species of Speyeria apart, especially in the western U.S. The Regal stands out as a unique species which is easily identified within the genus. It was therefore more likely to be overlooked while researches tried to sort out relationships among the confusing western species and subspecies of the genus Speyeria.

Future directions
While DNA can provide valuable information concerning the Regal Fritillary, much more needs to be learned. First, and most important, research is needed to provide information concerning host plant use by this species. While we know that Regal caterpillars can eat violets in the green house, it is not certain what they eat in the wild. Other species in the group have been documented eating violets in the wild and Regals often fly in prairies that have violets, but the direct link between the butterfly and its host plant has not been established. For example, one of the Illinois populations is found on a very small state nature preserve, less than one square mile in size, and surveys for Viola pedata and V pedatifida have turned up a total of three plants. Yet each year this site has hundreds of Regals. Some reports list the host plant as Viola pedata, others as Viola pedatifida and others as Viola sagittata. Which one is it? Is the host plant different in different places? Do the females prefer to lay eggs near different violets? Can they eat other plants? If they do prefer to lay eggs near and eat any violet then why don't they eat Viola soraria (otherwise known as Viola papilionacae). This violet is common along roads, sidewalks, gardens and is often found in disturbed prairies. We can raise Regals on this plant in the greenhouse so why doesn't it eat this plant in the wild?

This brings me to my final point concerning the Regal. How do you raise a Regal Fritillary? Speyeria have proven themselves a difficult group of butterflies to raise. Heroic efforts by people like Carol Boggs at Stanford University, Dave McCorkle in Oregon, Mark Roberts at Princeton University, and Dave Wagner at the University of Connecticut among others have led to a greater appreciation for what it takes to successfully raise a species like the Regal Fritillary. However, to my knowledge, no one has yet been able to successfully raise more than a single generation of Regals. Even those who can get adults in the first generation have trouble obtaining more than a few individuals. This number seems bleak given each female will lay hundreds of viable eggs.

The Regal Fritillary is a beautiful species in a beautiful setting. While we have gained much information over the last few years there is much more to be done. Hopefully articles like this one will lead to a better appreciation of this species. Maybe more people will stop on their way through the Midwest in hopes of spotting a glimpse of iridescent blue as the Regal glides across a prairie. Maybe people will start to demand that we do something to protect this rapidly disappearing "flagship species" of the prairie. One thing is certain, if we don't do something to protect it the rapid decline and extinction of the Regal Fritillary in the eastern U.S. will be a pattern repeated in the remainder of the species' range.

Copyright © 1999 by the North American Butterfly Association, Inc. All rights reserved.

19 March 2000
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