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Molecular Ecology (2001)
10
, 661–671
© 2001 Blackwell Science Ltd
Blackwell Science, Ltd
Allochronic speciation, secondary contact, and reproductive
character displacement in periodical cicadas (Hemiptera:
Magicicada
spp.): genetic, morphological, and behavioural
evidence
JOHN R. COOLEY, CHRIS SIMON, DAVID C. MARSHALL, KAREN SLON and
CHRISTOPHER EHRHARDT
Department of Ecology and Evolutionary Biology, The University of Connecticut, Storrs, CT 06269, USA
Abstract
Periodical cicadas have proven useful in testing a variety of ecological and evolutionary
hypotheses because of their unusual life history, extraordinary abundance, and wide geograph-
ical range. Periodical cicadas provide the best examples of synchronous periodicity and
predator satiation in the animal kingdom, and are excellent illustrations of habitat partition-
ing (by the three morphologically distinct species groups), incipient species (the year classes
or broods), and cryptic species (a newly discovered 13-year species,
Magicicada neotredecim
).
They are particularly useful for exploring questions regarding speciation via temporal
isolation, or allochronic speciation. Recently, data were presented that provided strong
support for an instance of allochronic speciation by life-cycle switching. This speciation
event resulted in the formation of a new 13-year species from a 17-year species and led to
secondary contact between two formerly separated lineages, one represented by the new
13-year cicadas (and their 17-year ancestors), and the other represented by the pre-existing
13-year cicadas. Allozyme frequency data, mitochondrial DNA (mtDNA), and abdominal
colour were shown to be correlated genetic markers supporting the life-cycle switching/
allochronic speciation hypothesis. In addition, a striking pattern of reproductive character
displacement in male call pitch and female pitch preference between the two 13-year species
was discovered. In this paper we report a strong association between calling song pitch
and mtDNA haplotype for 101 individuals from a single locality within the
M. tredecim/
M. neotredecim
contact zone and a strong association between abdomen colour and mtDNA
haplotype. We conclude by reviewing proposed mechanisms for allochronic speciation and
reproductive character displacement.
Keywords
: allochronic speciation, hybridization,
Magicicada
, reproductive character displacement,
secondary contact, speciation
Received 4 June 2000; revision received 29 September 2000; accepted 29 September 2000
Introduction
Secondary contact between isolates can lead to complex
species interactions that challenge our understanding of
species and the speciation process (Harrison 1993; Noor
1999; Jiggins & Mallet 2000). Within the periodical cicadas
(
Magicicada
spp.) of eastern North America, the M. -decim
cognate species (described below) provide an example
of extrinsic isolation, divergence, and secondary contact,
with little evidence of hybridization. We present a review
of our research relating to speciation and secondary
contact in periodical cicadas and present new data on the
species-specificity of genetic, behavioural, and morphological
characters in the zone of contact between two sister
species. We conclude with some observations concerning
allochronic speciation in
Magicicada
.
Magicicada:
definitions and species
Three sympatric, morphologically distinct, synchronized
Magicicada
species with 17-year life cycles coemerge in the
Correspondence: John Cooley. Fax: (860) 486 6364; E-mail:
jcooley@sp.uconn.edu
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J. R. COOLEY
ET AL.
© 2001 Blackwell Science Ltd,
Molecular Ecology
, 10, 661–671
northern and plains states of the U.S. (
M. septendecim
,
M. cassini
, and
M. septendecula
); each corresponds to at least
one morphologically and behaviourally similar ‘cognate’
species inhabiting the midwestern and/or south-eastern
states (
M. tredecim
,
M. neotredecim
,
M. tredecassini
, and
M. tredecula
; Fig. 1). The cognate groups are abbreviated
M. -decim, M. -cassini, and M. -decula for convenience. On
the basis of shared morphology, behaviour, and genetic
markers (reviewed in Williams & Simon 1995; Simon
et al
.
2000; Marshall & Cooley 2000), each species appears most
closely related to a cognate with the alternative life cycle, a
pattern best explained by multiple allochronic speciation
events.
Brood and species formation
Superimposed on the species relationships of
Magicicada
is the complex biogeography of the ‘broods’, which are
largely parapatric populations of the same life cycle that
emerge in different years and are thus temporally isolated
(Marlatt 1907; Alexander & Moore 1962; Lloyd & Dybas
1966; Simon 1988). By convention, 17-year brood year-
classes are numbered sequentially from I to XVII, and 13-
year brood year-classes are numbered from XVIII to XXX,
although there are only 12 known 17-year broods and three
known 13-year broods (Simon 1988). With the exception of
Brood VII (at the extreme northern edge of
Magicicada
’s
range), each brood of a given life cycle contains species
belonging to all three species groups, although some
populations of each brood are lacking one or more species.
Brood formation appears to involve temporary life-cycle
anomalies that cause different populations to become
unsynchronized. Most temporal migrants are doomed,
because periodical cicadas reproduce successfully only
when densities are sufficient to ‘satiate’ local predators
(Marlatt 1907; Beamer 1931; Alexander & Moore 1962;
Dybas 1969; Karban 1982; Williams
et al
. 1993). Therefore,
the establishment of a new brood by temporal migrants
Fig. 1 Distribution of the seven periodical cicada (Magicicada) species, summarized from county-level maps in Simon (1988) and from 1993
to 1998 field surveys. The 17-year species are sympatric except in peripheral populations: M. cassini alone inhabits Oklahoma and Texas,
while only M. septendecim is found in some northern populations (Dybas & Lloyd 1974). Two 13-year species, M. tredecim and M. neotredecim,
overlap with each other only in the central U.S. The other 13-year species, M. tredecassini and M. tredecula, are sympatric with each other
and overlap with the former species in the south and the latter species in the north.
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663
© 2001 Blackwell Science Ltd,
Molecular Ecology
, 10, 661–671
must be a rare event, involving large numbers of cicadas.
Most of the modern
Magicicada
broods were probably
formed since the last glacial maximum, and their biogeo-
graphy has been hypothesized to reflect climatic processes
likely involved in their formation (Alexander & Moore
1962; Lloyd & Dybas 1966). Some evidence suggests that
temporary accelerations of one or four years may be most
important in brood formation (Lloyd & Dybas 1966; Lloyd
& White 1976).
While brood formation appears to result from temporary
life-cycle fluctuations, the existence of cognate species with
different life cycles indicates that permanent four-year life-
cycle alteration is one mechanism of species formation in
Magicicada
(Alexander & Moore 1962; Lloyd & Dybas 1966;
Lloyd & White 1976; Lloyd
et al
. 1983). Most
Magicicada
species of the same life-cycle type share nearly coincident
ranges, and most species likely predate the last glacial
cycle; thus biogeographic clues as to their origins have
been erased. One exception involves the 13-year -decim
species, one of which,
M. neotredecim
, appears to have ori-
ginated recently via allochronic speciation (Marshall &
Cooley 2000; Simon
et al
. 2000).
The discovery of
M. neotredecim
The discovery of
M. neotredecim
is the result of long-term
research. The first step was the discovery of two distinct
genetic lineages in the M. -decim species. The existence
of two different abdominal colouration morphs, both
ventrally orange, one lighter-coloured with few abdominal
markings and the other with dark transverse bands has
been recognized within the M. -decim species at least since
1962 (Alexander & Moore 1962; R. D. Alexander, personal
communication). Because the lighter coloured phenotype
is most common in 13-year M. -decim, individuals of this
type were deliberately chosen as the neotype and neoallo-
type for
M. tredecim
(R. D. Alexander, personal communica-
tion). Martin & Simon (1988, 1990) surveyed three genetic
markers for the M. -decim species from the largest brood
of 13-year cicadas (Brood XIX), which occupies parts of
the Midwest and South, and the largest brood of 17-year
cicadas (Brood X), which extends from the Midwest into
the north-eastern states. They discovered that 13-year
M. -decim cicadas from the northern part of Brood XIX
were indistinguishable in mitochondrial genotype, phospho-
glucomutase allozyme frequency, and mean abdominal
colour from 17-year Brood X
M. septendecim
. This shared
genetic lineage was termed ‘lineage A.’ In contrast, the south-
ern Brood XIX M. –decim differed from the lineage A cicadas
in abdomen colour, mitochondrial DNA (mtDNA)
haplotype, and phosphoglucomutase allozyme frequency.
This unique genetic lineage was termed ‘lineage B.’ Martin
& Simon (1988, 1990) suggested that the northern 13-year
cicadas were recently derived from 17-year cicadas by
life-cycle switching, perhaps by the deletion of a postulated
four-year dormancy period in the second nymphal instar
(White & Lloyd 1975). Biogeographic evidence suggested
that the northern 13-year cicadas were derived from
17-year ancestors rather than vice versa; otherwise rapid
and implausible range changes would be required to
explain the relative distributions of
M. septendecim
(wide-
spread) and northern 13-year cicadas (confined to an area
bounded on the west, north, and east by
M. septendecim
;
Fig. 1).
Secondary contact between the lineages
Martin and Simon’s discoveries were unexpected, and
their sampling scheme had not been designed to pinpoint
the genetic boundary within 13-year M. -decim or to locate
populations in which both genotypes existed. They post-
ulated that if these lineages existed in secondary contact,
random mating would reunite gene pools that had been
separated for more than a million years (Martin & Simon
1988, 1990; Williams & Simon 1995). Mating between
13- and 17-year cognate species had been experimentally
induced and no behavioural barriers to gene flow were
known (Lloyd & Dybas 1966; Alexander 1968). The next
opportunity to resample Brood XIX was in 1998.
Another large brood of 13-year cicadas, Brood XXIII,
inhabits the Mississippi Valley and has a range similar to
that of Brood XIX, though slightly smaller. Intensive sam-
pling within Brood XXIII revealed individuals of both the
A and B lineages (Simon
et al
. 2000). Simon
et al
. (2000) also
found that: (i) lineage A cicadas of Brood XXIII were indis-
tinguishable in abdominal colour, mitochondrial haplo-
type and geographical distribution from the 17-year Brood
X and northern 13-year Brood XIX lineage A individuals;
(ii) cicadas of both lineages coexist in a zone of overlap
within Brood XXIII; and (iii) genetic intermediates were
absent in the overlap zone, suggesting assortative mating
by cicadas of the two lineages. Intensive resampling of
Brood XIX in 1998 (Marshall & Cooley 2000; C. Simon
et al.
,
unpublished data) identified mixed populations in this
brood, within a narrow zone of overlap geographically
similar to that in Brood XXIII.
Newly discovered differences in mating behaviour
In 2000, 13-year mtDNA lineage A was named a new
species,
M. neotredecim
, on the basis of newly discovered
behavioural differences restricting gene flow between
lineage A (
M. septendecim/M. neotredecim
) and lineage B
(
M. tredecim
; Marshall & Cooley 2000). Within mixed
M. neotredecim/M. tredecim
choruses, male
M. neotredecim
produce calling songs with high dominant pitch (mean
1.71. kHz), while
M. tredecim
have calling songs with low
dominant pitch (mean 1.11 kHz; Marshall & Cooley 2000;
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664
J. R. COOLEY
ET AL.
© 2001 Blackwell Science Ltd,
Molecular Ecology
, 10, 661–671
Fig. 2). Courtship songs of the two species show similar
pitch differences. Female call pitch preferences, as
measured by female ‘wing flick’ signal responses to
male calls (Cooley 1999; J. R. Cooley & D. C. Marshall,
submitted), are species-specific (Marshall & Cooley 2000).
Females collected from mixed choruses of
M. neotredecim/
M. tredecim
fall into a bimodal distribution of pitch pre-
ference, males exhibit a bimodal distribution of call pitch,
and mixed choruses produce little sound at pitches inter-
mediate to those of the two species. These observations
provide further evidence that assortative mating is taking
place (Cooley 1999; Marshall & Cooley 2000). Analysis
of archived recordings (University of Michigan Museum
of Zoology) indicated that the same song pitch types
exist within Brood XXIII (D. C. Marshall & J. R. Cooley,
unpublished data). The importance of song pitch to the
Magicicada
mating system is underscored by recent dis-
coveries (Fonseca
et al
. 2000) that the neural architecture
of cicadas allows fine-scale pitch discrimination.
Reproductive character displacement
Geographic variation in male song pitch and female pitch
preference within
M. neotredecim
exhibits a striking pattern
of reproductive character displacement (Brown & Wilson
1956; Howard 1993; Fig. 3). Where
M. tredecim
overlaps
M. neotredecim
,
M. neotredecim
male calling songs and female
mating preferences are centred around 1.7 kHz, while
those of allopatric populations range between 1.3 and
1.5 kHz (Marshall & Cooley 2000). Extremely displaced
Fig. 2 Spectrogram (power spectrum vs. time) showing a two-banded, Magicicada tredecim/M. neotredecim chorus of male calls with one call
of each species standing out against the background chorus. Individual calls end with a downslur. Comparatively faint slurs of background
chorus males overlap and are not visible. Intervening time between calls has been removed.
Fig. 3 Geographic variation in dominant chorus pitch of
M
agicicada neotredecim, showing higher-pitch calls in and near the
region of overlap with M. tredecim. Lighter shaded circles indicate
higher-pitch calls. Shaded region is approximate M. tredecim
range. Reproduced with permission from Marshall & Cooley
(2000).
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665
© 2001 Blackwell Science Ltd,
Molecular Ecology
, 10, 661–671
M. neotredecim
song pitches are found exclusively within
and adjacent to the zone of contact. A weaker reciprocal
pattern in calling song pitch variation may exist in
M. tredecim
,
but this is based on less complete sampling (Marshall &
Cooley 2000). The pattern of character displacement in
M. neotredecim
song pitch lends strength to the argument
that
M. neotredecim
originated in the time since the last
glacial maximum, because the near-perfect correlation of
song displacement and sympatry and the biogeographic
relationships of
M. septendecim
,
M. neotredecim
and
M. tredecim
would likely not have persisted through range
changes during Pleistocene glacial cycles (Marshall &
Cooley 2000).
Derivation of
M. neotredecim
and its presence in
two broods
The presence of
M. neotredecim
or similar cicadas in both
major 13-year broods (XIX and XXIII; Simon
et al
. 2000)
could be explained in three ways (Fig. 4). In all schemes,
the ancestor is a periodical cicada with a 13-year life cycle
(node W, lineage B
′
), a hypothesis supported by the fact
that among- and within-population genetic variation is an
order of magnitude larger in present-day
M. tredecim
than
in
M. septendecim
. This ancestor gave rise to a 13-year proto-
Brood XIX + XXIII (node X, lineage B) that later split into
the current 13-year broods XIX and XXIII. The ancestor at
Fi
g.
4H
ypot
h
eses o
f
t
h
e
f
ormat
i
on o
f
13-
and 17-year M. -decim lineages. The spli
t
between mtDNA lineages A and B occurre
d
first (node W), followed by the Magicicad
a
septendecim/M. neotredecim split (node Y).
Numbered lines on tree branches mark th
e
addition or subtraction of four years to/
from the life cycle. The circled letters A an
d
B at the tips and nodes indicate lineag
e
type. (a) The evolution of M. neotredeci
m
involving one permanent life cycle chang
e
followed by the subdivision of a brood.
(b) The evolution of M. neotredecim in on
e
brood by a permanent life cycle chang
e
followed by its migration to another brood.
(c) The evolution of M. neotredecim involv
-
ing two separate permanent life cycle changes
.
See text for details.
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ET AL.
© 2001 Blackwell Science Ltd,
Molecular Ecology
, 10, 661–671
node W also gave rise via a permanent four year life-cycle
extension (event 1) to
M. septendecim
(node Y; lineage A).
Between nodes W and X, and W and Y, some amount of
evolution took place so that node X evolved into lineage
B and node Y evolved into lineage A. In scenario one
(Fig. 4a),
M. neotredecim
evolved from
M. septendecim
via
a single four year life-cycle reversion (event 2, forming
node Z lineage A) which caused it to join and gain pre-
dator protection from existing 13-year proto brood XIX +
XXIII. Later, temporary life-cycle anomalies caused this
combined lineage A + B proto-brood to split into the
current Broods XIX and XXIII with
M. neotredecim
already
in place in both. In this scheme, nodes X and Z are of the
same age. In scenario two (Fig. 4b), node X predates the
formation of
M. neotredecim
(event 2).
M. neotredecim
formed (event 2) from a single four year reversion event
from a 17-year ancestor and joined either Brood XIX or
XXIII. Then, via a temporary life-cycle anomaly, a subset
of
M. neotredecim
individuals became synchronized
with the other 13-year brood. Under this hypothesis,
node Z is more recent than node X. In scenario three
(Fig. 4c),
M. septendecim
(node Y) contributed separate
M. neotredecim
-like species to preexisting Broods XIX and
XXIII; this would have involved two independent 17- to
13-year life-cycle reversions (events 2 and 3). In scenario
three the order of origination of
M. neotredecim
in XIX
and XXIII is uncertain, and node X predates events 2
and 3.
The contact zone is not a hybrid zone
The pattern of reproductive character displacement
indicates that, subsequent to secondary contact, selection
has acted against heterospecific sexual interactions of
M. neotredecim
and
M. tredecim
. Instead of forming a stable
hybrid zone with at least limited introgression of genetic
material, these species have evidently evolved to reduce
opportunities for introgression. The mechanism driving
the species’ divergence could have taken different forms,
depending on current or past opportunities for gene flow.
Gene flow might have occurred in the past, if interspecific
mating occurred and hybrids were at least partially fertile;
however, current discussions of evolution following
secondary contact emphasize the difficulty of enhancing
assortative mating when significant gene flow occurs
between the species (e.g. Butlin 1987; Rice & Hostert 1993;
Noor 1999). Alternatively, the 13-year M. -decim species
may have been genetically isolated upon first contact by
incompatible sexual signals or hybrid sterility, in which
case the observed signal divergence would be explained
not by selection against hybrids
per se
, but by selection
against other forms of wasteful heterospecific sexual inter-
actions (e.g. signal interference or time wasted in ineffectual
courtships).
Present-day hybridization between
M. neotredecim
and
M. tredecim
may be rare or nonexistent within the contact
zone. Evidence from playback experiments suggests that
females in the overlap zone today rarely respond to the
calling songs of heterospecific males (Marshall & Cooley
2000). Additional information on the existence of present-
day gene flow in the overlap zone could be sought by
examining the relationship between song pitch and abdomen
colour within
M. neotredecim
or
M. tredecim
. If present-day
introgression is occurring, then lower song pitch should
tend to be associated with lighter abdomen colour within
the species
M. neotredecim
, and vice versa for individuals
of
M. tredecim
. If signal divergence of
M. neotredecim
and
M. tredecim
long ago eliminated opportunities for hybrid-
ization, then stabilizing selection within the species may
have removed introgressed alleles affecting the species-
specific song pitch. However, evidence of past hybrid-
ization could be observed in mtDNA haplotype or abdomen
colour variation, if, within the overlap zone, the alternative
abdomen colours and mtDNA haplotypes are ‘selectively
equivalent’ (cf. Hewitt 1993) in the genetic backgrounds
of both species. Under these conditions selection would
not tend to remove alleles affecting such traits unless they
were linked to song pitch.
Even if selection reinforcing reproductive character dif-
ferences later decreased levels of hybridization, historical
introgression would have eroded the correlations between:
(i) abdominal colour morphs; (ii) mtDNA haplotypes; and
(iii) calling-song pitch or pitch preference types. Previous
work established associations between: (i) abdomen colour
and mtDNA haplotype (Martin & Simon 1988, 1990); and
(ii) abdomen colour and male song pitch/female song
pitch preference (Marshall & Cooley 2000) but did not
examine correlations between song pitch and mitochon-
drial genotype. In this paper, we examine correlations among
mtDNA haplotype, calling song pitch, and abdominal
colour for individuals from a population within the zone
of overlap and find little evidence for introgression.
Materials and methods
During the 1998 emergence of 13-year Brood XIX, we col-
lected 150 males from a mixed
M. tredecim/M. neotredecim
chorus for analysis of song pitch, abdomen colour, and
mtDNA haplotype. The collection site was a privately
owned wood 0.25 miles south of County Road 62 on
County Road 51 at a powerline right-of-way, just outside
the north-west boundary of the Harold E. Alexander
Wildlife Management Area, Sharp Co., AR. We scored the
ventral abdomen colour of each individually recorded
male using a method similar to that of Martin & Simon
(1988), assigning each male a whole-number value from
1 (dark transverse marking present) to 4 (transverse
markings absent). An abdominal-sternite-colour chart can
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© 2001 Blackwell Science Ltd,
Molecular Ecology
, 10, 661–671
be viewed at http://www.eeb.uconn.edu/collections/
cicadacentral/magi/abcolor.htm. We recorded the calling
song of each male using a portable cassette recorder with a
microphone and parabola, or a videocassette recorder with
built-in microphone, and we analysed dominant call pitch
using Canary 1.2.4 (Cornell Bioacoustics Laboratory,
Ithaca, NY) (detailed methods in Marshall & Cooley 2000).
Specimens were then stored in 95% ethanol for later DNA
analysis.
For 101 males from the Sharp Co., AR, population,
whole-genome DNA was isolated from a small sample
of diced leg tissue from each cicada using a Qiagen
DnEasy™ tissue kit (Qiagen Corp.). An approximately
330 bp portion of domain III of the SSU (12s) rRNA gene
was amplified using primers SR-J-14233 and SR-N-14588
(Simon
et al
. 1994). These primers were chosen because
they flank a known 12s
Bgl
II restriction polymorphism
found to be a consistent discriminator between the A
and B mitochondrial lineages sampled over a broad geo-
graphical area (Martin & Simon 1990). Polymerase chain
reactions (PCRs) were carried out in 25
µ
L volumes con-
sisting of 1
µ
L template DNA, 1.25
µ
L each primer (10 m
m
),
2.5
µ
L of 10
×
PCR reaction buffer, 2.5
µ
L 8 m
m
DNTP’s,
0.125
µ
L Takara X-Taq™ (Takara Shuzo Co, LTD.),
Thermus
aquaticus
DNA polymerase (5 units/uL), and 16.375
µ
L
ddH
2
O. DNA was denatured initially at 94
°
C for 2 min,
then 30 cycles of amplification were carried out under the
following conditions: 92
°
C denturation for 45 s, 55
°
C anneal-
ing for 45 s, and 72
°
C extension for 75 s. Five microliters of
PCR product and negative control were electrophoresed
on a 1% agarose gel stained with ethidium bromide to
verify product size and purity. The PCR products were
cleaned with a Qiagen PCR purification kit (Qiagen Corp.),
then cycle sequenced using the ABI cycle sequencing Big
Dye™ kit (Perkin-Elmer Biosystems). Sequencing reac-
tions were carried out in 10
µ
L volumes using 2.5
µ
L tem-
plate DNA, 1
µ
L primer, 4.0
µ
L Big Dye (PE Biosystems)
mix, and 2.5
µ
L ddH
2
O, for 25 cycles (96 °C for 30 s, 50 °C
for 15 s, and 60 °C for 4 min). After cycle sequencing, the
DNA was cleaned using 0.5 g hydrated Sephadex™ (DNA
Grade, G-50 Fine; Pharmacia) in spin columns. All samples
were run for 9 h on an ABI Prism™ 377 automated sequencer.
All PCR fragments were sequenced in both directions,
and all sequences were aligned using Sequencher™ (Gene
Codes Corp.) after each chromatogram had been inspected.
The alignments were inspected by eye, but needed no
adjusting. For comparison and to aid in alignment, five
M. septendecim individuals from three broods were also
sequenced (see Collection localities below).
We divided our male sample into two groups, above and
below an intermediate pitch of 1.3 kHz, because song pitch
is bimodal and species-diagnostic (Marshall & Cooley
2000), with no intermediate phenotypes. We used Mann–
Whitney U-tests to evaluate any relationships between call
pitch and abdomen colour or genotype in the 101-male
sample. For the entire 150-male sample, statistical asso-
ciation of abdomen colour class and male song pitch
within M. neotredecim and within M. tredecim was measured
using a Kruskal–Wallis test. All statistical analyses were
conducted using systat Version 5.2.1 for the Macintosh
(Systat, Inc.).
Collection localities
M. septendecim. Brood III: Henderson County, IL 0.9 miles
east of County Road 164, 3 miles south of right angle bend
(9 June 1980, CS, JA); Mahaska County, IA, Lake Keoma
State Park (9 June 1997, CS, WS, JZ). Brood X: Owen
County, IN, Hoot Woods 5 miles off Highway 231 on Hoot
Road (21 May 1987, CS, AM). Brood XIII: Scott County, IA,
Scott County Park (16 June 1990, CS, WS); Peoria County,
IL, Jubilee College State Park (16 June 1990, CS, WS).
M. tredecim, M. neotredecim. Brood XIX: Sharp County, AR,
privately owned woods 0.25 miles South of County Road
62 on County Road 51 at a powerline right-of-way, just
outside the north-west boundary of the Harold E. Alexander
Wildlife Management Area (June 1998, JC, DM).
Results
Within the region sequenced, including the restriction site
polymorphism, there are 6 bp differences between the
two mtDNA lineages, all confined to unpaired regions
of 12s rRNA secondary structure (see Hickson et al. 1996).
In all individuals examined, these six differences do not
vary independently and are consistent within the lineage.
Calling song pitch and mtDNA haplotype were always
congruent; thus mtDNA haplotype was species-specific
(Table 1, Fig. 5). Abdominal colour was strongly congruent
with calling song and mtDNA haplotype, but was not
absolutely species-specific. Seven Magicicada neotredecim
Table 1 A Kruskal–Wallis test, with call pitch as dependent vari-
able, indicates overall relationship between pitch and abdomen
colouration in a mixed sample of 150 Magicicada neotredecim and
M
. tredecim (test statistic = 45.969, P < 0.001); the break between the
two species occurs within abdomen colour class 3
Abdomen colour Call
Class Count Pitch (mean ± SD) Rank-Sum
1 11 1.73 ± 0.09 1138.5
2 103 1.70 ± 0.08 8863.5
3 15 1.46 ± 0.33 880
4 21 1.16 ± 0.20 443
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668 J. R. COOLEY ET AL.
© 2001 Blackwell Science Ltd, Molecular Ecology, 10, 661–671
were classified in abdominal colour category 1, 64 in
category 2, four in category 3, and two in category 4. No
M. tredecim were classified in category 1, one in category 2, six
in category 3, and 17 in catgegory 4. Within each species,
we found no association between calling-song pitch and
abdomen colour (Table 2).
Discussion
Abdomen colour does not perfectly distinguish Magicicada
neotredecim and M. tredecim. However, the lack of cor-
relation between abdomen colour and calling-song pitch
within each species suggests that hybridization is not the
reason for this lack of species-specificity. The perfect cor-
respondence between mtDNA haplotype and call pitch
further supports the conclusion that gene exchange is not
occurring between these species. Even the strong pattern of
reproductive character displacement does not necessarily
imply gene flow, past or present, between M. neotredecim
and M. tredecim. Gene flow requires that hybrid offspring
reproduce successfully, but reproductive character dis-
placement can occur in response to heterospecific sexual
interactions other than mating or the production of hybrid
offspring. Time and effort wasted in interspecific court-
ship alone could lead to selection favouring elaboration
of differences between species’ sexual signals, especially
if the species already differed at first contact. The calling
song pitches of M. neotredecim and M. tredecim recorded
away from the zone of contact differ by approximately
300 Hz. If these allopatric populations accurately reflect
the precontact conditions for these species, then even
before any character displacement occurred the M. -decim
within 13-year mixed-species choruses would have had
some tendency to mate assortatively, although their pre-
ferences would likely not have been as exclusive as those
Fig. 5 Call pitch, abdomen colour and mtDNA haplotype in
M
agicicada neotredecim and M. tredecim at the Sharp County AR
site. Species were identified on the basis of call pitch, which is
b
imodal. Average abdomen colour differs significantly between
species (Mann–Whitney U = 62.65; P < 0.001). The distribution
of mtDNA haplotypes between the species is significantly
nonrandom (Fisher’s Exact Test P < 0.001); all M. neotredecim were
lineage A, and all M. tredecim were lineage B. Arrows indicate
mean values of call pitch and abdomen colour for each species.
Table 2 Kruskal–Wallis tests show no association between male
song pitch and abdomen colour class within (a) Magicicada
neotredecim (n = 125) or (b) M. tredecim (n = 26) from Sharp County,
AR, within the zone of overlap of the two species
(a) M. neotredecim
Abdomen Mean Pitch (kHz) Count Rank-Su
m
1 1.73 11 862.5
2 1.70 103 6296.5
3 1.71 9 567.5
4 1.72 2 148.5
Kruskal–Wallis test statistic = 2.470; P = 0.481
(b) M. tredecim
Abdomen Mean Pitch (kHz) Count Rank-Su
m
1 n/a n/a n/a
2 1.13 1 20.5
3 1.09 6 86.0
4 1.10 19 244.5
Kruskal–Wallis test statistic = 1.049; P = 0.592
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ALLOCHRONIC SPECIATION IN MAGICICADA 669
© 2001 Blackwell Science Ltd, Molecular Ecology, 10, 661–671
of present-day displaced populations (Marshall & Cooley
2000).
Our data, however, do not eliminate the possibility of
past gene flow. In another study, M. septendecim with espe-
cially light-coloured abdomens were found to characterize
Brood X populations near its boundary with Brood XIX
(A. Paradis & C. Simon, unpublished data). These unusual
M. septendecim could be evidence that: (i) some M. tredecim
abdomen-colour alleles are leaking across the M. tredecim/
M. septendecim species boundary; (ii) abdomen colour is
affected by environmental variation in a manner that
causes M. septendecim to converge on M. tredecim in the
southern part of its range; or (iii) other alleles leading to
light abdominal colour have appeared independently
in some populations of M. septendecim. The pattern of
abdomen-colour variation in Brood X supports the first
hypothesis; light-coloured M. septendecim are restricted
to populations adjacent to Brood XIX populations of
M. tredecim. The second hypothesis is diminished and the
third hypothesis is supported by the existence of a limited
population of surprisingly light-coloured M. neotredecim
in Brood XIX far from any populations of M. tredecim
(Allerton Park, Piatt County, IL; D. Marshall and J. Cooley,
unpublished data) and surprisingly light-coloured abdomens
in Brood XIV on Cape Cod, Massachusetts (A. Paradis &
C. Simon, unpublished data) supporting the interpretation
that abdomen colour, is in some cases, a homoplasious
character. Further evaluation of variation in abdominal
colour and other nuclear markers may reveal the extent
of gene flow between M. tredecim and M. neotredecim at
first contact.
Models of Magicicada speciation
Given that only seven Magicicada species are known, the
conditions promoting species formation and/or persistence
must be limited. Understanding speciation involves deter-
mining the mechanisms that isolate populations sufficiently
to allow their continuing evolutionary divergence. Two
mechanisms might foster allochronic isolation of Magicicada
populations: (i) genetic changes that influence life-cycle
length, thereby isolating mutant founders (Alexander &
Moore 1962; Lloyd & Dybas 1966; Marshall & Cooley 2000;
Simon et al. 2000); or (ii) environmental cueing of latent
phenotypic plasticity in life-cycle length, later made
more permanent by genetic changes selected under the
new environmental regime (Lloyd & Dybas 1966; Marshall
& Cooley 2000). These alternatives (mutation and plasticity)
are general and should apply to other forms of phenotypic-
ally mediated isolation, such as host-plant specificity (e.g.
Bush 1992). Speciation models involving only a few temporal
founders are implausible in Magicicada, because periodical
cicadas are dependent on high population densities for
predator satiation and successful reproduction (Marlatt
1907; Beamer 1931; Alexander & Moore 1962; Dybas 1969;
Karban 1982; Williams et al. 1993); this is perhaps a greater
problem for the mutation-based mechanism (i) above. How-
ever, Marshall & Cooley (2000) and Simon et al. (2000) note
that rare life-cycle mutants might escape predation and
establish an incipient species if they happen to coemerge
with an overlapping ‘nurse-brood’ of the same life-cycle
length. The ‘nurse-brood’ mechanism could also facilitate
survival of founders isolated by mechanism (ii); however,
environmental cueing of life-cycle plasticity has the potential
to isolate large numbers of individuals at once, given an
extrinsic cue of sufficient magnitude.
Species interactions at secondary contact
If secondary contact of populations leads to fitness losses
through hybrid sexual interactions, then selection favours
changes that reduce these losses. One outcome of second-
ary contact is the formation of a hybrid zone, in which
selection tends to reduce the impact of hybrid sexual inter-
actions by removing factors leading to hybrid unfitness.
For example, two subspecies of Chorthippus grasshoppers
that diverged in allopatric glacial refugia came into contact
and formed a hybrid zone along the crest of the Pyrenees
after deglaciation permitted reinvasion of territories
uninhabitable during the last glacial maximum (Cooper
& Hewitt 1993; Hewitt 1993). In Chorthippus, gene flow
and recombination resulting from incomplete hybrid
sterility appear to be overwhelming selection for assortative
mating (see Hewitt 1993; Butlin 1998), leading to a local
erosion of barriers to interbreeding and the formation of a
hybrid zone.
Another outcome of secondary contact is reproductive
character displacement, in which selection reduces the
possibility of hybrid sexual interactions. In Magicicada,
selection for assortative mating has apparently prevailed,
perhaps because high levels of hybrid sterility or slightly
incompatible mating signals already existed at the time
of contact. Magicicada and Chorthippus illustrate two
alternatives facing species achieving secondary contact.
The path taken will be determined by the degree to which
gene flow is possible and the degree to which members
of each species are sexually attractive to members of the
other at contact.
Concluding remarks
M. neotredecim inhabits midwestern habitat that will likely
be unsuitable for Magicicada during the next glacial
maximum (see Webb et al. 1993). What does the future hold
for this species? For all but the most mobile species, the
populations surviving in glacial refuges are most likely
founded from the edge of the earlier distribution located
farthest from the glacial boundary, while populations near
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670 J. R. COOLEY ET AL.
© 2001 Blackwell Science Ltd, Molecular Ecology, 10, 661–671
the glacial boundary simply go extinct (Hewitt 1996). Thus,
the most southern, song-displaced M. neotredecim have the
greatest likelihood of colonizing a refugium during the
next glacial cycle. If this scenario is realistic, then after
the next glacial retreat all undisplaced M. neotredecim will
have gone extinct, and the pattern of character displacement
linking M. neotredecim to an allochronic speciation event in
the M. septendecim lineage will have been erased. Repeated
glacial cycles may cause M. neotredecim to become even
more distinct by creating new contexts for signal evolution
through the elimination of intermediates or new instances
of secondary contact between species that have diverged
while in glacial refugia. Perhaps such a pattern of existence
in glacial refugia, allochronic speciation, evolution in
response to secondary contact, and extinction of inter-
mediates explains the existence of the distinct M. -decim,
M.-cassini, and M. -decula cognate groups.
Acknowledgements
We thank Richard D. Alexander, Steve Chiswell, Jerry Coyne,
Charles Henry, Thomas E. Moore, Daniel Otte, Mark Taper, and
Peter Turchin for thoughtful discussion of the research reviewed
in this paper. We are indebted to the Harold E. Alexander Wildlife
Management Area, Sharp County, AR, USA and to M. Downs,
Jr. for permission to work at the study sites. Carl McIntosh, Annie
Paradis, Walter Simon, and the U.S. Cooperative Extension Ser-
vice assisted with the field research. Funding was provided by the
Frank W. Ammermann Endowment of the UMMZ Insect Division
and by Japan Television Workshop Co., Ltd, and by NSF and
University of Connecticut grants and by NSF DEB 98-07113 and
DEB 99-74369 and University of Connecticut grants to CS.
GenBank accession numbers for domain III of the SSU (12s) rRNA
gene are: Magicicada neotredecim AF304453; M. tredecim AF304452;
M. septendecim are AF304454, AF304455, AF304456.
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