Evolution of 13- and 17-Year Periodical Cicadas
(Homoptera: Cicadidae: Magicicada)
CHRIS SIMON
A great observer who hath lived long in New England did upon occasion
relate to a friend of his in London where he lately was, that some few years
since there was such a swarm of a certain sort of insects in that English
colony, that for the space of 200 miles they poyson 'd and destroyed all the
trees of that country. There being found innumerable little holes in the
ground, out of which those insects broke forth in the form of maggots,
which turned into flyes that had a kind of taile or sting, which they struck
into the tree, and thereby envenomed and killed it.
H. Oldenburg (1666)
P
RIODICALCICADAS have been of interest to biologists
since they were first described in the scientific literature
more than 300 years ago (Oldenburg 1666). Their appeal has
to do with their unique prime-numbered life cycle and their rare
but recklessly theatrical appearances by the millions-large, noisy,
black bugs with bright red eyes and orange legs and wing veins.
Today, periodical cicadas are of interest to biologists because
they offer a unique system with which to address current problems
in evolutionary biology. Specifically, how is genetic isolation reflected in evolutionary divergence at the phenotypic and genotypic
levels? How do ecologically and evolutionarily similar species differ
in their response to genetic isolation? How is genetic divergence affected by inter- and intraspecific gene flow? How useful are various
kinds of data for reconstructing evolutionary histories? My laboratory is using periodical cicadas to study these questions by examining DNAand allozyme data. This article reviews our work to date
and points out how these data can be used in a complementary
fashion. The article concludes with a brief description of DNAmethodology, a general discussion of the usefulness of DNAdata for evolutionary srudies of insects, and examples of currem research efforts in this area.
Periodical cicadas are an ideal organism for study because they
can be collected in large numbers from a wide geographic range of
locations. Their distribution has been mapped since the early
18005, and except for shrinkage of local populations, they can still
be found in the same locations (Fig. 1). Males and females are
abundant and equally easy to collect.
Periodical cicadas are divided into year classes, known as
broods, which are potential incipient species, isolated in time because the adults never meet. These broods have various patterns of
distribution that suggest interesting ecological and evolutionary
comparisons: Some occupy only a few hundred square miles; others occur in as many as 15states. Some are entirely southern, some
entirely northern. Some range from the southern to the northern
states and encompass a wide range of forest ecosystems. Some
broods overlap spatially, some do not. Finally,these interesting sets
of comparisons can be made not for just one but for three morphologically distinct species, each of which exhibits both a 13-year and
a 17-year life cycle.
0ll1.i·H~'4'HH!OIb.i·OI"6S0!.OO!O (l~ 19HHEnlllmolo!(ical Society of America
Life History
Periodical cicadas of the genus Magicicada are commonly known
as 13- or 17-year locusts due to their habit of emerging in plague
proportions every 13 or 17 years. Dybas and Davis (1962) counted
1,500,000 per acre in a particularly dense flood plain population in
Illinois. That translates into more than a ton and a half of cicadas
per acre.
Periodical cicadas are native to the United States east of the Great
Plains. The 17-year cicadas are found in the nonhern, eastern, and
western edges of the distribution, and the 13-year cicadas predominate in the southern states and in the Mississippi River Valley However, 13-year cicadas can be found as far north as central Indiana,
and 17-year cicadas are seen as far south as the Red River Valley of
Texas (Fig. 1). Although the 17-year cicada has been known since
1666 in the literature (and by native American Indians before that
time), the 13-year life cycle was not discovered until the mid-1800s
(Walsh & Riley 1868).
Although periodical cicadas are not the longest lived insects, they
do exhibit the longest time to maturity (egg to adult). All but four
to six weeks of their lives are spent as subterranean nymphs. Metamorphosis to the adult stage occurs immediately after the emergence of the fifth instar. Mating begins during the first and second
weeks of the emergence (Fig. 2A) and is followed immediately by
egg laying (Fig. 2B). Eggs are laid and develop in slits in tree
branches and hatch in six to eight weeks. Detailed discussions of
periodical cicada life histories can be found in Marlatt (1907) and
Snodgrass (1921).
Periodical cicadas can be serious pests of orchard trees. Nymphal
feeding has been shown to affect tree growth (Karban 1980, 1982a),
and growers are advised not to plant young trees in years immediately preceding large emergences. Egg punctures can kill young
trees ,ind damage mature trees. Wounds can cause breakage, give
ingress to disease, and harbor scale, wooly aphids, and other pests.
CHRIS SIMON is affiliated with the Departments of General Science and Zoo
ology and the Hawaiian Euolutionary Biology Program, Unil'ersity of Hawaii, Honolulu, Hawaii 96822.
163
Fig. 1. Extant broods of periodical cicadas (Decim, Cassini, and Decula combined). Broods I - X, XIII, and XN 07-year) and Broods
XIX, XXII, and XXIII 03-year). Maps are updated from Marlatt (907) with small black dots representing smaller populations and large
black dots representing larger populations. Black dots with white centers represent populations that have become extinct or for which
there are unclear records. Updating information came from: numerous publications since Marlatt; personal observations of Broods I X, XlV, XIX, XXII, and XXlII 0974 - 1988); Cooperative Extension records; and USDAAnimal and Plant Health Inspection Service files.
164
BULLETIN OF THE ESA
Notable differences from Marlatt include Brood N localities added in southern Oklahoma and Brood XIX in Alabama (G. S., unpublished data), Brood X in Missouri (Haseman 1915), and Brood XIX in Indiana (Deay 1952). Kritsky (1988a,b) gives fine scale distribution data for all broods in Indiana and for Brood X in Ohio. Persons with locality information inconsistent with these maps are urged
to contact tbe autbor. Tom Moore (personal communication) is compiling distribution records from field observations and from museum specimens for each of the tbree morphologically distinct species.
WINTER
19HH
165
Fig. 2. A. Mating M. septendecim. B. M. septendecim laying eggs in tree hranches.
The peculiar periodicity exhibited by Magicicada is thought to
be maintained by strong stabilizing selection. Individuals that
emerge in "off' years appear to be quickly eliminated by birds and
other predators (Marlatt 1907,Alexander & Moore 1962, Karban
1982b). Their bright colors, large size, loud song, and unwillingness to fly are all characteristics that have lead to their description
as predator foolhardy (Lloyd & Dybas 1966). Karban (198Ia, 1982b,
1984) showed thar earlier speculation about the adaptive value of
predator satiation is supported by empirical evidence: The individual's risk of capture decreases as cicada density increases, and reproductive success increases with adult density. Several mathematical models have been devised to describe ways in which the long
life cycle and periodicity could have evolved (Hoppensteadt &
Keller 1976, Bulmer 1977,May 1979). Lloyd & Dybas (1966 a,b) developed a descriptive model.
Broods
In the spring of 1987,the eastern United States experienced the
emergence of Brood X, the largest brood year class of the 17-year
cicadas. There was no 17-year cicada brood in the spring of 1988,
however, because Brood XI, which was at one time located in the
Connecticut River Valley, has not been seen since the turn of the
century (Lloyd & White 1976). Nevertheless 1988 has not been without periodical cicadas, Brood XXII of the 13-year cicadas appeared
in May in southwestern Mississippi and pans of Louisiana. This
emergence will be followed in 1989 by that of Brood XXIII of the
13-year cicadas throughout the Mississippi drainage from Louisiana
to central Indiana.
Broods of periodical cicadas are loosely geographically contiguous, sequentially numbered year classes. Brood membership is
determined solely by the year of adult emergence. Seventeen-year
cicada broods were assigned the numerals I-XVII and 13-year
broods XVIII-XXX by Charles Marian (1898), who studied their
distributions. He chose 1893 arbitrarily as the year to begin numbering; such that Broods I and XVIII emerged in that year, II and
XIX emerged in the subsequent year, and so on. Because of the difference in the life cycles, he noted, 17-year Brood I and 13-year
Brood XVIII would nOt emerge simultaneously for another 221
years.
166
Not all broods of 13- and 17-year cicadas that have been numbered are known to exist. Some (Broods XI and XXII) are known to
have become extinct. Others (Broods XII, xv, XVI,XVII,XVIII, XX,
and XXIV-XXX) have never been documented or are represented
by a few scattered reports that may involve stragglers (those that
emerge in off years) from well-established broods or that might be
by-products of hybridization between year classes. There are three
well-documented, extant broods of 13-year cicadas and a dozen
broods of 17-year cicadas (Fig. 1).
The Three Species
There are three morphologically distinct species of 17-year cicada (Magicicada septendecim (L.), M. cassini Fisher, and M. septendecula Alexander and Moore). They differ in coloration, size,
song, mating behavior, and habitat preference, although the ranges
of many of these characters overlap considerably and differences
have not been quantified (Alexander & Moore 1962; Dybas & Lloyd
1962, 1974;Lloyd & White 1976; Dunning et al. 1979). Furthermore,
microhabitat separation is by no means strict, and it breaks down in
disturbed situations. The same three distinct species occur in the
l3-year cicada but have been named separately (M. tredecim Walsh
and Riley,M. tredecassini Alexander and Moore, and M. tredecula
Alexander and Moore) solely on the basis of the life cycle difference
(Alexander & Moore 1962).
The three morphologically distinct forms are abbreviated here to
Decim, Cassini, and Decula. Most broods contain all three forms,
with Decim predominating in the northern extremes, Cassini predominating in the Mississippi River Valley and in the extreme
southwest of the range, and Decula never predominating but reaching its highest densities in the south (Dybas & Lloyd 1974).
J. White (1973) succeeded in crossing the larger Decim and
smaller Cassini to produce first instars of intermediate size. She induced cross mating experimentally by crowding Decim and Cassini adults together in small cages placed over twigs suitable for
oviposition. After the eggs hatched, she dissected the twigs and
counted empty egg cases and aborted eggs. Decim females suffered a 15% reduction in fertility when mated with Cassini males;
Cassini fertility was 27% lower with Decim females.
BULLETIN OF THE ESA
Life Cycles
Lloyd and Dybas (1966) succeeded in cross mating 13- and 17year forms. They transported M. septendecim and M. cassini from
Iowa (Brood UI) and mated them with M. tredecim and M. tredecessini, respectively, from southern Illinois (Brood XXIII). This was
done in large cages, where females could easily reject unwanted
males. There was not the slightest indication of a behavioral barrier
to cross mating, nor would one be expected, because the songs of
13- and 17-yearsiblings are indistinguishable. The hybrid eggs later
hatched into first instal's that appeared normal in every respect.
No consistent differences in any characteristics have been found
between 13- and 17-year sibling pairs. In the first published description of the 13-year cicada, Walsh and Riley (1868) wrote,
"There are absolutely no perceptible differences between the 17year and B-year broods, other than in the time of maturing." Riley
later (1869) wrote: "Mr. Walsh informs me that Charles Darwin, Asa
Gray and Dr. Hooker all agree in the belief that the 17-year and 13year forms ought not to be ranked as distinct species, unless other
differences besides the period of development could be discoven.~d."
Alexander & Moore (1962, ,2 and frontispiece) noted a possible
color difference in the abdominal sternites of 13- and 17-year Decim siblings-solid
orange versus orange and black with orange
stripes, respectively. This distinction holds in general, although I
have found some variability in this character: 13-year Decim populations have predominantly orange abdominal color patterns, and
17-year Dccim populations have color patterns that are predominantly black with various degrees of orange striping.
Lloyd & White (1976) found no differences in oviposition preference between members of the 13- and 17-year pairs. However, I
have found differences in wing morphology and allozymes to be no
greater between the 13- and 17-year pairs than among some broods
of 13- or 17-year cicadas (Simon 1979a,b, 1983; Archie et al. 1985).
Current research in my laboratory examining mitochondrial DNA
(mtDNA) restriction site polymorphism has turned up substantial
differences between 13- and 17-year siblings (A. Martin & c. Simon
1988).
Gene Flow Among Broods or Species
Periodical cicadas have been cited as classic examples of sibling
species, the 13- and 17-year pairs, and of allochronically isolated incipient species, the broods within life cycles (White 1978). Neither
of these generalizations is strictly true.
13- and 17-Year Life Cycles. Individual 13- and 17-year cicada
broods emerge together once every 221 years, but few 13- and 17year broods overlap. Although the maps in Fig. 1 indicate that some
13- and 17-year cicadas occur in the same counties, I have found
only three localities (two in southeast Iowa 'Ind one in southeast
Oklahoma) where 13- and 17-year cicadas sing, mate, and oviposit
on the same trees. The ranges of all broods are shrinking, and the
overlap may have been greater in the past. It also is possible that
overlap was less in the past and that the cases I have found are the
result of invasion of 17-year territory by 13-year cicadas (Lloyd et a!.
1983). Examination of mtDNA genotypes has told us something
about the prevalence in nature of hybridization between life cycle
forms (A. Martin & c. Simon unpublished data).
\X'INTER 1988
Broods Within a Life Cycle. By definition, broods of periodical
cicadas within a given life cycle never emerge in the same year, but
isolation of broods within a life cycle is by no means perfect. For
example, stragglers are commonly sighted one year before, one
year after, and four years before particularly dense emergences of
17-year cicadas. The appearance of stragglers is not surprising because contrary to what one might expect in a species where the
vast majority of adults are so highly synchronized in emergence,
nymphs grow at varying rates. White & Lloyd (197,) examined 13and 17-year cicadas after nine years of growth and invariably found
three instal'S coexisting (Table 1). They also found that 17-year cicada nymphs grow more slowly than 13-year cicada nymphs and
that the slmving of growth takes place during the second instal'.
What is remarkable is that there are not more stragglers, given the
enormous numbers of cicadas and the variation in their developmental rates.
Imperfect isolation leads to the possibility of gene flow,but most
broods do not overlap geographically. Broods separated by one
year in time have never been found to overlap geographically (although they may come within less than a mile of one another).
Broods separated by four years or more can overlap geographically.
These areas of overlap are more common than overlap between Band 17-year populations; hut they are still infrequent. We have identified several areas of overlap of 17-year broods. Allozymic data
cannot be used to distinguish overlapping broods because they are
not significantly different in gene frequency (Simon & Lloyd 1982).
We are searching for mtDNA markers that could provide evidence
for gene flow.
Morphologically Distinct Species. The morphologically distinct
species have been found to hybridize infrequently in nature. The
distinctive mating calls and aSSOciatedbehavior appear to be effective barriers to mating (Dunning et al. 1979). Dybas & Lloyd (1962)
found 7 of 725 mating pairs collected in the field to be interspecific-ali M. septendecim females mating with M. cassini males. In
14 years of collecting individuals from the entire range of each
brood, I have found only two interspecific matings, both of them M.
tredecim males mating with M. tredecassini females.
In all of these cases, interspecific mating pairs were found in extremely dense populations. Occasionally, individuals are found in
the field that appear to be hybrids between two morphologically
distinct species, but it is difficult to be sure because the obvious
Table 1. Instar distributions in three collections of 9·year old periodical
cicada nymphs.
Broods(Year)Studied
III (1963)
Lifecycle
Collectiondate
17yrs.
August
1972
Iowa
State
Meanmonthly
temperature
9.0°C
Meanannual
rainfall
81cm
Instal':
Second
27
Third
106
Fourth
457
Fifth
0
Source:WhiteandLloyd(1975).
XXIlI (1963)
IV (1964)
13yrs.
October
1972
illinois
17yrs.
October
1973
Oklahoma
13.9°C
lS.6°C
112cm
105cm
0
2
39
334
0
12
306
68
167
morphological differences among the species (color and size) are
not great and appear at times to integrade (Lloyd & Dybas 1966, C.
S., unpublished data). Restriction site mapping of mtDNA has allowed us to verify hybrids between Decim and Cassini that appeared morphologically intermediate when they were collected
(C.S. & A.M., unpublished data). This line of investigation continues.
Evolutionary Hypotheses
Alexander & Moore (1962) note that groups of 17-yearbroods are
related both geographically and temporally, such that broods that
appear in successive years border one another. The clearest pattern
of this kind is displayed by four eastern broods: Brood VII is located to the north of Brood VIII, which lies to the north of Brood
IX, which is surrounded by Brood X (Fig. 1). They also note that in
every pair of related broods, the brood to the north appears one
year earlier than its relative. They suggest that climatic conditions
in the past (for example, a freeze causing death of young spring
leaves followed by a second leafing out) could cause cicadas to
count an extra year and emerge one year early to the north. These
researchers do not offer further discussion of the relationships
among the broods but focus on the evolution of the life cycles and
species. They propose that an ancestral species was separated into
three divergent populations. Subsequently, these three species became largely sympatric, with a later division into 13- and 17-year life
cycles concurrently in all three. Brood formation followed the onetime 13-17-year split.
Lloyd & Dybas (1966) agree that the most logically consistent theory begins with the origin of the three morphologically distinct
species followed by brood and life cycle formation. But they hypothesize that rather than one major 13-17-year split, life cycle
changes could have evolved more than once. They suggest that 13year broods could have evolved repeatedly from one 17-year brood
or independently from different 17-year broods. An important part
of their theory is that it allows for at least partial brood formation in
17-year cicadas prior to the formation of 13-year cicadas.
Lloyd & Dybas (1966) published a detailed scheme for the evolution of the 17-year broods (Fig. 3). They discuss the one-year differences in adjacent broods recognized by Alexander & Moore (1962)
and agree that the differences probably are the result of climatic
factors. Furthermore, they note that the largest broods of 17-year cicadas overlap widely (i.e., they occur in many of the same counties)
and are separated by four years. They suggest that these major
broods could have been derived from one another by a four-year
shortening of the life cycle, which they suggest could be the result
of the temporary deletion of a postulated supernumerary sixth instar.
White & Lloyd's later (1975) study of nymphal growth demonstrates that rather than possessing an extra instar, the 17-year
nymphs grow more slowly during the first four years of life than 13year cicadas do. Lloyd & White (1976) postulated that this four-year
inhibition in growth-which
they call a four-year dormancy period-might be broken by the stimulus of early nymphal crowding,
leading part of the population to emerge four years ahead of schedule. There is evidence that more periodical cicada nymphs can be
supported per unit area of forest if they are subdivided into two
groups four years apart (Simon et al. 1981).
168
Lloyd & Dybas's (1966 a,b) four-year acceleration hypothesis was
unexpectedly supported three years after publication by a massive
emergence in suburban Chicago of hundreds of thousands of periodical cicadas, four years ahead of schedule. Only one brood of periodical cicadas (Brood XIII) had ever been recorded from that
area, so these cicadas could have had no other origin. The remainder of the brood emerged on schedule, in 1973, in enormous abundance (many millions) with a few appearing during the intervening
years. The many thousands of cicadas that emerged in 1969 were
not enough to satiate predators, and apparently they left few descendants; no egg nests could be found. A similar four-year acceleration was again observed in that area in 1986.
Since that first published report of a four-year acceleration, I have
witnessed several others. In 1975, hundreds of cicadas emerged in
the same suburban yards where hundreds of thousands later
emerged on schedule in 1979 (Brood II). I found a similar situation
for Brood VI (1983) and Brood X (1987) in suburban Washington,
and there is documented evidence of a four-year acceleration of
Brood X in Cincinnati in 1983 (M.L.,personal communication). Recently, Gene Kritsky (in press a,b) has gathered extensive historical
records of 4-year accelerations in 20% of the counties of Indiana
and 30% of the counties of Ohio. Four-year accelerations are probably responsible for the peculiar pattern of cicada broods on Long
Island (Simon & Lloyd 1982). Our studies of mtDNAhave provided
genetic evidence for at least one massive 4-yr acceleration (Martin
& Simon 1988).
Testing the Theories
To test the proposed theories of cicada evolution, I examined 20
enzyme loci for two broods of 17-year cicadas (XIII and XIV) and
three broods of 13-year cicadas (XIX, XXII, and XXIII) (Simon
1979a,b). Three broods were sampled extensively for geographic
variation in allele frequency. I found no alleles that were unique to
any brood or species. The morphologically distinct species generally differed in having a different second most common allele. All
broods were homogeneous in gene frequency within their ranges.
Only Decim were significantly different in allele frequency among
broods; Cassini showed little among-brood differentiation, and Decula displayed little or no allozyme variability. Therefore, phylogenetic comparisons based on allozymes could be made for the Decim species only.
Six loci varied and were used in the phylogenetic analysis. Rogers's (1972) genetic distance indicated that 13- and 17-year siblings
are no more different from one another in allozyme frequency at
these loci than are the local popultions of most other species of animals that have been examined. There were no clines in gene frequency, and no systematic variation was found with climate or habitat. Populations found in the sandy pine-oak forests of Long Island
and Cape Cod were no more likely to be similar to one another
than they were likely to be similar to populations collected from
the diverse moist montane forests of the Great Smoky Mountains.
For phylogenetic analysis, allele frequencies were used as characters. These data were analyzed using a distance Wagner procedure (Farris 1985), the available method at that time. More properly,
they should have been analyzed using a non distance (character
state) minimum-length tree method, such as that proposed by Rogers (1984,1986) or Swofford & Berlocher (1987). These methods inBULLETIN OF THE ESA
Four-Year
Accelerations ~
::xri[ ~X
~JZI
~
One-Year
Accelerations
~
XIII
~
:m
~
XI
~
IX ~
~
mn:
~
mr
~
~
:sz:
~
TIL
~
m
IT
~
I
~
:xw
~
:xJZI
~
x:sz:
Fig. 3. The hypothesis of Lloyd and Dybas (1966), showing proposed relationships among the broods of 17-year cicadas. Reprinted with permission from Lloyd and Dybas (1966b).
corporate restrictions on ancestral character states and thereby satisfy earlier objections to using frequency data for phylogenetic
analysis (Penny 1982, Farris 1985). Nevertheless, these data are free
enough from homoplasy that the same solution is reached regardless of the method; even an unweighted pair group method using
averages (llPGMA) phenetic analysis produced an identical branching diagram (Simon 1979b). The Wagner tree was rooted using
Cassini and Decula as outgroups.
The phylogenetic
tree based on allozyme data agrees with pre-
dictions made by Lloyd & Dybas (I966a,b) in that it hypothesized
brood formation in the 17-year cicadas prior to formation of 13-year
broods; Brood XIVwas most ancestral; and it could be easily interpreted in terms of the four-year acceleration model (Fig. 4). The
three 13-year broods that were studied form a monophyletic group
according to the allozyme data. Because no other 13-year broods
are extant it is impossible to say, based on these data, whether 13year cicada broods are all more derived than 17-year broods are or
whether the 13-year cicadas arose independently several times.
Studies of the mitochondrial genomes of 13- and 17-year cicadas
will provide information that will allow us to determine whether
either group or both are monophyletic.
Spatial Patterning
A1lozymic studies of six additional broods of 17-year cicadas (1Vl) provided the following results: The eastern broods I, II, V,and
Vl are very similar to one another in allozyme frequency. The two
western broods (III and IV), which are separated from other 17year broods by the 13-year broods, are similar to one another but
different from the 13-year broods and from the other 17-year
broods. Unlike the broods I had examined previously (XXII, XXIII,
and XIV), local populations within the western broods exhibited
some variability in allozyme frequency.
Characters that respond to local environmental factors are not
appropriate for phylogenetic analysis; the resulting dendrogram
would unite groups from similar environmental regimes rather
than groups with common evolutionary histories. For this reason, it
is important to check spatial patterning in allozyme data that are
used for phylogenetic analysis from a representative portion of the
WINTER
1988
geographic range of the species under investigation. If allozyme
frequencies are influenced by environment rather than by phylogenetic history, spatial patterning should be apparent. Spatial autocorrelation analysis was used to make such a check for Broods I-VI,
XIII, and XIV of the 17-year cicadas and for Broods XXII and XXIII
of the 13-year cicadas (Archie et al. 1985) for a total of 69 localities.
Spatial autocorrelation analysis tests whether the observed value
of a variable at one locality is significantly correlated with values of
the same variable at neighboring localities. The results of our analyses, based on the three most variable loci, show that there are
three major areas of gene frequency similarity: the eastern United
States (I7-year Broods I, II, V,and VI), the western United States (17year Broods III and IV), and the central United States (13-year
Broods XXII and XXIII).
Within each of these regions there is no significant spatial patterning within or between broods. In biological terms, broods appear to be homogeneous breeding units. Differentiation among
populations within broods and among broods within regions exists
but is less pronounced than the differentiation among geographic
regions, and it forms no spatial pattern. For the particular loci examined, allozyme frequency differences appear to reflect the phylogenetic history of the broods rather than their environmental surroundings.
These results also suggest something interesting about the relationship between the 17-yearM. septendecim and the 13-year M. tredecim. The allozyme data do not neatly separate these two taxa,
rather, they reveal three groups: one 13-year group and two 17-year
groups
(eastern
and western).
For one enzyme (phosphoglucomu-
tase), the western 17-yearcicadas are more similar to the 13-year cicadas than they are to the eastern 17-yeargroup. For another (esterase), the eastern 17-year cicadas are more similar to the 13-year
cicadas than they are to the western I7-year group. In the third enzyme examined (alpha glycerol phosphate dehydrogenase), the
western 17-year cicadas are intermediate in gene frequency between the eastern 17-year cicadas and the 13-year cicadas. Given
that all authorities agree that the three morphologically distinct
species of Magicicada evolved in parallel, these data suggest that
there should be either three species, or nine, but not six.
To date, four additional17-year broods (VII-X) and one additional 13-year brood (XIX) have been examined electrophoretically.
Results of the analyses of these data will be forthcoming. There is
some indication that gene flow has occurred among broods and
species in a few boundary populations. This hypothesis will be
tested further with nucleotide sequencing of mtDNA.
mtDNA As a Systematic Tool
Because allozyme frequencies for broods within the three geographic regions were discovered to be relatively undifferentiated, it
was apparent that more sensitive data (i.e., those with a greater
amount of differentiation per unit time) were needed to continue
phylogenetic studies. Restriction site mapping of mtDNA provides
such data (Avise 1986). mtDNA has a high average mutation rate
(Wilson et al. 1985). This observed rapid rate of evolution is what
gives these data the potential for use in species and population
level studies.
Mitochondrial DNA is a circular molecule. It is small (15,000 to
20,000 base pairs) (Borst & Grivelll981), well studied, maternally
169
E
DECIM
:x:::nl:17
13- a 17- yr
CASSINI
(LUMPED)
13- a 17- yr
DECULA
(LUMPED)
Fig. 4. Proposed sequence of splitting events leading to the current distributions of the broods studied, explained in terms of Lloyd and
Dybas's (1966b) four-year acceleration hypothesis. Shading indicates one-year accelerations, cross-hatching indicates four-year jumps:
(A) A temporary one-year acceleration divides the ancestral range in half (B) A four-year acceleration separates future Broods X/X,
XXI/, and XXI/I from Brood XII/. Thisprocess occurs repeatedly to produce 13-year cicadas. (C) A mosaic of 13-year cicadas centering
around the Blufflands of the Mississippi River becomes four years out of synchrony, separating Brood XX/IJ from Brood X/X. (D) A oneyear temporary acceleration produces Brood XXI/ from Brood XX/IJ. (E) Phylogenetic hypothesis produced by Wagner parsimony analysis of allozyme data. Reprinted with permission from Simon (1979a).
inherited (Giles et al. 1980, Lansman et al. 1983), and easy to extract
and clone. By examining mtDNA, one can look for mutational
changes at the nucleotide level. Populations and species can be
characterized and a phylogenetic hypothesis can be constructed
based on the most likely sequence of mutational events. Rather than
sequencing the entire mitochondrial genome, a random subset of
DNA sequences can be examined, using an indirect technique
known as restriction site mapping.
Restriction sites are palindromic sequences of nucleotide pairs
(usually four to six) recognized by specific enzymes-restriction
endonucleases-which
cut double-stranded DNAat a specific place
within the recognition site to produce fragments of different
lengths. If one population gains a restriction site or loses a restriction site found in another population, its mtDNA will be cut into a
greater number of smaller pieces or into fewer, larger pieces, respectively (Fig. 5). Four-base recognizing enzymes, by random
chance, find more cutting sites than six-base enzymes and so provide greater resolution (Kreitman & Agaude 1986).
Different sized pieces of DNA can be visualized electrophoretically on agarose gels, and the resulting bands of DNAcan be made
visible either by staining them with ethidium bromide or by making them radioactive and exposing them to X-ray film. Radioactive
labeling is much more sensitive than ethidium bromide staining,
and it allows the detection of nanogram amounts of mtDNA. Bands
are made radioactive by attaching a radiolabel before electrophoresis (end-labeling) or by hybridizing radioactive probe DNAto bands
170
after electrophoresis (e.g., Southern transfer [Southern 1975] followed by probing). Southern transfers can also be visualized using
probes labeled with nonradioactive avidin:biotinylated enzyme systems (e.g., from Vector Labs).
The Southern transfer-probing technique uses DNA consisting
of mtDNA contaminated with nuclear DNA (approximately 100
times as much nuclear DNAas mtDNA). Both kinds of DNAare present in the electrophoretic gel. mtDNA is made visible by first transferring the gel to a special nitrocellulose or nylon filter to make it
easier to handle (Southern 1975), then making the sample DNAsingle stranded by denaturing it with sodium hydroxide, and finally
hybridizing it to a single-stranded labeled probe. The probe is
made by purifying mtDNA from a pooled sample of the species under investigation or a closely related species, inserting labeled nucleotides in place of some of the existing nudeotides, and making
that DNA single stranded usually by heat denaturation. The probe
DNA hybridizes to the sample DNA and makes the bands visible.
These techniques are described in detail by Lansman et al. (1981).
End-labeling is more sensitive than transfer-probing methods in
that it can detect smaller pieces of DNA.But, for end-labeling, sample mtDNA must be highly purified prior to electrophoresis. This
requires larger sample volumes than probing does, and in the past
made studies of variation among individuals more difficult for small
organisms. For this reason, larger organisms, such as vertebrates,
were studied first by molecular population biologists in preference
to smaller organisms, such as insects. With the improvement of exBULLETIN
OF THE ESA
8g1 II
Restriction
17-year
quencing machines become commonplace-in
5 or 10 years-restriction mapping techniques will become obsolete.
For studies in which it is necessary to examine variation within
and among populations for a large number of individuals, the level
of effort currently required for sequencing can be avoided when
restriction site mapping provides sufficient information to differentiate taxa and to construct phylogenetic trees. However, there is a
tradeoff. Although restriction mapping is less expensive and faster
than sequencing, restriction site data are more difficult to interpret
(DeBry & Slade 1985).
Digest
13-year
14400
9800
Rate Determines Application
5500
Rates of evolution (mutation and fixation of nucleotides) vary
among taxa (Britten 1986). Rates also vary for different classes of
DNA.Certain functional genes are highly conserved, whereas others evolve more rapidly (Minghetti et al. 1985,Martin & Meyerowitz
1986, Sharp & Li 1987). Noncoding DNAregions (introns, spacer regions, pseudogenes) and redundant (silent) codon positions evolve
faster than other positions, presumably because they lack functional constraints (Brown et al. 1982,Li et al. 1984,Li 1986, 1987).
Unlike nuclear DNA,mtDNA has few noncoding regions (Borst &
GriveIl1981). Nevertheless, data from mammals reveal that mtDNA
has a higher average rate of evolution than nuclear DNA(Brown et
al. 1982, Wilson et al. 1985). This more rapid rate of change has
been postulated to be due to higher mutation rates (Miyata et al.
1982), or a higher probability of fixation of mildly deleterious mu-
4600
9800
Fig. 5. Bglll
restriction
enzyme
digest of the mitochondrial
ge-
nomes of 13- and 17-year periodical cicadas. The recognition sequence for Bglll is Ai GATCT (up-arrow indicates cleavage site).
Whenel'er this enzyme encounters these six bases in the mtDNA
molecule, a cut is made. 17-year cicadas have three Bgl II sequences and 13-year cicadas have two. The circular mtDNA molecule of these species is cut into three and two pieces, respectively, by
this enzyme.
traction techniques, we can now purify mtDNA from single cicadas
and single Hawaiian Drosophila. A new cell-free cloning technique,
the polymerase chain reaction (PCR) (Saiki 1985, Mullis et al. 1986,
Wr~schnik et al. 1987,Marx 1988) allows rapid selection, purification, and amplification of subsections of a DNAmolecule from minute amounts of tissue so that the size of an organism is no longer a
consideration.
Once the restriction sites are identified, they can be mapped
onto the circular mtDNAmolecule and this information can be used
to construct a phylogenetic estimate using parsimony methods
(such as those found in PAUp,D. Swofford, Illinois Natural History
Survey; or PHYLIp,J. Felsenstein, Dept. Genetics, U. Washington).
1empleton (1987), Nei & Tajima (1987), and DeBry & Slade (1985)
have discussed specific methods for analyzing mtDNAdata.
Sequencing of the mitochondrial genome, at one time prohibitively expensive and time consuming, is becoming feasible. Advances in sequencing technology inciude improvement of polymerase enzymes and the use of PCR, first, to select and amplify a
segment of DNAand then to separate complementary strands prior
to sequencing. Using PCRand commercially available kits, a person
working in a laboratory equipped to sequence mtDNA could sequence about two kilobases of DNAper week. When automated seWINTER
1988
tations or, more likely, tQ some combination
of these factors. Hy-
potheses put forward to explain the higher mutation rate of mtDNA
involve greater exposure to damage, slower or nonexistant repair, a
system that is more prone to errors of replication, a higher rate of
turnover, and relaxed functional constraints (Brown et al. 1982,
Cann et al. 1982, Cann and Wilson 1983, Brown 1985, Cann et al.
1987). In nonmammalian species, average evolution rates for these
two types of DNA may be more equal (Brown 1985, Powell et al.
1986,Vawter & Brown 1986, DeSalle & Hunt 1987), however, few of
the estimated rates are corrected for intraspecific divergence. Furthermore, the rates of evolution being discussed are averages. Like
nuclear DNA,mtDNA has conserved (Carr et al. 1987,Huysmans &
DeWachter 1986) and hypervariable (Cann et al. 1987) regions.
The divergence rate of a particular segment of DNA will determine the systematic level at which it is appropriate for phylogenetic
study. Rapidly evolving DNA is useful for studies of recently diverged taxa, but it may be too different to be useful in differentiating genera or species belonging to particularly ancient genera. If
taxon divergence time is long, many sites are likely to become saturated (that is, they change more than once) and homologies will
be difficult to determine (Brown et al. 1982,DeSalle et al. 1987).
Population biologists have made use of the ribosomal region of
nuclear DNA (rONA) for higher level systematic studies. rONA
genes in the nucleus and mitochondrion contain conserved sequence blocks (separated by more variable regions; Huysmans &
DeWachter 1986, DeSalle et al. 1987) such that probes from human
and mouse will cross react with samples of cicada and Drosophila.
A preliminary examination of the Magicicada nuclear ribosomal
region suggests that populations within periodical cicada broods,
and broods within species, are not different in their rONA restriction site patterns, but that the morphologically distinct species are
171
Table 2. Examples of current
alphabetically
by institution
unpublished
applications of DNA technology to questions
(collaborators,
in parentheses,
may be at other institutions
LaboralOry/lnvestigator( s)
Insect taxa
Harrison/R. Harrison (D. Weisman)
R. Harrison (I ODell)
R. Harrison
in insect systematics,
).
(R. Carde)
Honeycutt/W Wheeler
LewontinID. Rand
Techniques"
Cornell University
Gryllus spp., field crickets
Lymantria dispar (L.),
gypsy moth
Ostrinia nubilalis Hubner,
European corn borer
Harvard University
Paleoptera, Neoptera
Gryllus spp., Drosophila
melanogaster
LouiSiana State University
Spodopterafrugiperda U. E. Smith),
fall armyworm
Lepidopteran
superfamilies
Holometabolous
orders
Pashley/D. Pashley
]. Martin
B. McPheron
Zimmeri). Spatafora
Bush/S. Williams,].
Cockburn/A.
listed
Broad questions
mtDNARSM
Phylogenetics,
mtDNARSM
Population
mtDNA RSM
Strain markers
rDNA NS
Phylogenetics
Population structure;
sequence evolution;
mtDNANS&
RFLP
subgroup
ecology, and evolution,
species
markers
markers
transI11ission
genetics
mtDNA&
rONA RSM
Strain markers
rRNANS
Phylogenetics
rRNA NS
Colony markers
rmDNAHY/
rDNA RSM
Evolutionary rate;
phylogenetics; race markers
mtDNA&
rDNA RSM
DNARFLP
Species identification
Strain identification
Coptotermes formosanus
Feder
Cockburn,
Hall/G. Hall
MaruniaktJ. Maruniak
Danyluck)
S. Mitchell
(G.
Shiraki, Formosan
subterranean
termite
Michigan State University
Tephritid genera,
especially Rhagoletes
University of florida
Anopheles
quadramaculatus (Say)
Apis mellifera (L.), honey bee
lrichoplusia ni (Hubner),
cabbage
looper;
Spodoptera frugiperda,
fall armyworm
]. Maruniak
Simon/C. Simon, A. Martin, C.
McIntosh
C. Simon (I Shelly,]. Strazanac)
PalumbilB.
Kessing
introduced fire ants
University of Hawaii, Manoa
Magicicada spp.,
periodical cicadas
Banza spp., endemic
katydids
Lycosid spiders
different. Thirty three percent nuclear DNA restriction sites examined were different in at least two species (compared with 87% of
mtDNA restriction sites) (C.S., unpublished data).
In an older animal genus, Hillis & Davis (1986) found that 32 species of Rana (leopard frog), which last shared a common ancestor
approximately 50 million years ago, showed extensive variation in
nuclear rONA and that this information could be used to construct
a phylogeny estimate. Hillis & Davis (1986) point out that nuclear
rONAcontains slowly evolving regions (the 185, 5.85, and 285 rRNA
genes) and more rapidly evolving ones (the transcribed and nontranscribed spacers). (Mitochondrial rRNA contains no spacer regions.)
Applications to Evolutionary Studies of Insects
As DNA-based systematic techniques become more refined, more
laboratories are adopting them for studies of insect systematics and
evolution. Much of this work is in progress and not yet published. I
have provided examples in Table 2.
172
mtDNARFLP
Solenopsis spp.,
mtDNARFLP
mtDNARSM
&NS
DNA &
mtDNA
RSM
Verifying cell lines
Marker to identify source
population
Phylogenetics; population
structure; species markers
Population markers;
phylogenetics
Mitochondrial DNAand nuclear DNAalready have proved useful
in phylogenetic studies of Hawaiian Drosophila (reviewed by DeSalle & Hunt 1987). mtDNA markers have been useful for delineating hybrid zones in closely related cricket species (Harrison et a!.
1987). Many other insects are being studied from a phylogenetic or
population genetics viewpoint (Table 2).
Applications of mtDNA research to economically important insect groups should provide information vital to pest control and
population management. Researchers at Cornell University and the
N.E. Forest Experiment Station (Hamden, Conn.) are exploring the
application of high-resolution techniques (Kreitman & Aquade
1986) to mtDNA to distinguish local populations of gypsy moth, Lymantria dispar L. Similarl)\ it may soon be possible to identify the
two pheromone strains of European corn borer, Ostrinia nubilalis
(Hubner) and to answer questions concerning the presence and extent of interbreeding. mtDNA markers are being used for similar
purposes in studies of the European and Africanized honey bees
Apis mellifera L., the fall armyworm, Spodoptera frugiperda
E.
Smith), the imported fire ant, Solenopsis spp., and Anopheles spp.
a.
BULLETIN OF THE ESA
Thble 2. Continued
Laboratorytl nvestigator( s)
Insect taxa
University
Iluntl). Hunt, R. Thomas
TechniqueS"
of Hawaii, Manoa (cont"d)
Hawaiian drosophilids,
species groups/genera
University
Scotti). Scott (E Howarth,
E Stone)
crickets
lbnpleton/A.
A. lcmpleton,
S. Lawler
1empleton
OrthopteroicilBla
orders
H. Hollacher, L. Park,
DeSalle/R. DeSalle, (K. Kaneshiro,
). Archie)
R. DeSalle (A. 1empleton)
R. DeS:i1le
Washington University,
Polisles spp., paper wasps
7Hmerotropis spp.,
grasshoppers
Drosopbila repleta group
Yale University
Hawaiian Drosophila,
modified-mouthparts
group
Hawaiian Drosophila,
planatibia subgroup
Hawaiian Drosophila,
picturewing subgroups
Hawaiian
mtDNARSM
Phylogenetics
rDNANS
Phylogenetics
mtDNARSM
Phylogenetics
mtDNA&
rDNARSM
mtDNA&
rDNARSM
Population structure;
phylogetics
Population structure;
ecological genetics
species
markers
Md.
51. Louis
Phylogenetics
mtDNARSM
&NS
mtDNARSM
Phylogenetics;
structure
population
mtDNARSM
Phylogenetics
mtDNANS
Phylogenetics
mtDNARSM
Strain identification
Drosophila, all
major groups
Anopbeles gambiae Giles,
Powell/DeSalle
mosquito
Anopheles gambiae
N. Bcsansky
complex
Powell/). Powell
A. Caccone,
Phylogenetics;
noid
USDA Beltsville,
Apis mellifera, honey bee
Heliothine moths
(S. Davis)
mtDNARSM
of Michigan
Apis mellifera, honey bee
S. Sheppard
Phylogenetics
of Hawaii (Hila)
University
Huenel/W
DNA NS
(ADH region)
Caconemobius spp.,
endemic
Brown/D. Smith
L. Vawter
Broad questions
K. Goddard
DNAHY
Drosophila
pseudoobscura group
D. obscura group
(Europe
and America)
mtDNARSM
Population
structure
DNA HY
Phylogenetics
"DNA, nuclear DNA;mtDNA, mitochondrial DNA;rRNA,nuclear ribosomal RNA;RFLP,restriction fragment length polymorphism; RSM,restriction site mapping; HI';
hybridization; NS, nucleotide sequencing; markers are genotypes that can be used for taxon identification and tracing gene noW.
mosquitO (Table 2). A researcher at Louisiana State University is using nuclear DNAmarkers to differentiate colonies of the introduced
Formosan subterranean termite, Coptotermes Jormosanus (Shiraki), to determine the number of introductions and to follow migration among populations.
Progress also is being made in studies of higher level insect systematics. Several laboratories are exploring the use of ribosomal
RNA(rRNA) and rONA sequencing to create a phylogeny of insect
orders. rRNAsequencing has already proven useful in higher plant
systematics (E. Zimmer, personal communication). A Harvard University researcher is sequencing the 18S rONA region to examine
the systematics of the Paleoptera and to determine the relationship
of the orders of Holometabola to the other neopterous orders (Table 2).
Hawaii
Hawaii has a fascinating insect fauna with many possibilities for
application of molecular systematic techniques (Simon et al. 1984;
WINTER 1988
Simon 1987). Researchers from the University of Hawaii at Hilo and
the B. P.Bishop Museum in Honolulu are collaborating in the study
of endemic cave crickets of the genus Caconemobius. Phylogenetic
studies of Hawaiian drosophilids are continuing with restriction
site mapping of the mitochondrial genome and sequencing of the
alcohol dehydrogenase region of the nuclear genome, and pan of
the large ribosomal subunit of the mitochondrial genome for representatives of all species groups and genera. Other researchers at
the University of Hawaii, Manoa are studying gene flow among endemic Iycosid cave- and surface-dwelling spiders, and systematics of
endemic katydids (Banza). And, as discussed above, the periodical
cicada project continues.
We are using Magicicada as a model system to develop techniques that will be used to study harder-to-collect native Hawaiian
organisms. We are making mitochondrial maps for the six Magicicada species, and we are surveying variation among the broods
and species using restriction enzymes and direct sequencing. I
have frozen and pickled samples of 12 broods and plan to collect
the remaining three. In collaboration with Svante Paabo and Allan
173
Wilson, I have extracted
and sequenced
DNA from dried and
ethanol preserved Magicicada
specimens
using the polymerase
chain reaction and the Sequinase enzyme system (United States
Biochemical Company, Cleveland). This new technology greatly expands our source of material and increases the value of museum
specimens.
mtDNA data have already proven extremely valuable for studies
of hybridization among the 13- and 17-year siblings (A. M. & c. 5.,
unpublished
data) and will be useful for estimating
gene flow
among broods with life cycles. In addition, by examining mutational changes at the DNA level, we hope [0 be able [0 characterize
genetic differences within and among species, life cycles, broods,
and local population of Magicicada
and to use these data to construct a phylogenetic history of the group. Our data also will speak
to the rate evolution of various mtDNA genes in insects.
•
Acknowledgment
Because the work reviewed in this paper spans a to-year period, there is
insufficient space to acknowledge all the individuals and organizations who
contributed (many of them have been acknowledged in earlier papers), but
1 would like to single out my collaborators and colleagues James Archie,
Richard Karban, Monte Lloyd, and JOAnnWhite for their many contributions.
1 thank Tom Moore for suggesting that 1 work on periodical cicads and for
sharing field notes. Support from R. K. Koehn was invaluable during the
early stages of this work. The cooperative extension agencies in all of the
MSTAT-C
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174
states in which 1 worked provided considerable assistance in locating populations in the field. This work has been supported by NSF grams BSR8509164, BSR-8411083,DEB-8107038,and DEB-7810710;grants from Sigma Xi
and the Theodore Roosevelt Memorial Fund; and NSFgrant BMS20522 to R.
K. Koehn. 1thankJohn Anderson,James Archie, Rebecca Cann, Milton Huettel, Andrew Martin, and Richard Karban for reviewing all or part of the manuscript.
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AVAILABLE FALL 1988
Nymphs
if North
American Sconif/y Genera (Plecoptera)
Thomas Say Foundation Series, Volume 12
by Kenneth W Stcwart and Bill P. Stark
illustrated by Jean Stanger
The definitive sequel to P. W. Claassen's 1931 book Plecoptera Nymphs
of America (North of Mexico), this volume is the result of a decadc of
comparative study by the authors.
Contents:
Introductory chapters on classification, phylogeny, biogeography,
ecology of nymphs, and behavior of Plecoptera.
Review of all major systematic and ecological literature on nymphs
through 1987.
244 illustrations including 99 full page nymphal habitus and 99 sets of
characters for the type or other representative species of all current
genera.
New family and generic keys; diagnoses of nymphs of known
congener species.
Referencing of all previous nymph descriptions and illustrations.
Complete species list and distribution tOr North America.
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reference work for serious study of North American Plecoptera and for
stream ecological studies.
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