A perfect duet? The acoustic behaviour of Anaulacomera almadaenis sp. nov., a species with an unusual chromosome complement, discovered in the footsteps of the explorers Spix and Martius in Brazil

Abstract

To find a mate, males and females of phaneropterine bush-crickets engage in duets. The male starts a duet and a female responds if she is prepared to mate. This pattern is exactly followed in the acoustic communication of a species of the genus Anacaulomera from the State of Bahia in Brazil which we newly describe in this manuscript. However, the male also acoustically defends the duet. As soon as it hears a female response, it adds an additional component to its own signal to make the localisation of the female difficult for rivals. At the same time the male reduces the interval between its songs for a faster exchange of information about the location of the partner, and reduces the intensity of the song that it can be heard only from nearby. Besides the nearly perfect anti-eavesdropping modifications of the duet the new species is remarkable concerning its karyotype. The meiotic behaviour of chromosomes in the male may indicate the origin of a new type of sex determination mechanism.

Full text

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SPIXIANA 43 1 105-118 München, Oktober 2020 ISSN 0341-8391
A perfect duet?
The acoustic behaviour of Anaulacomera almadaenis sp. nov.,
a species with an unusual chromosome complement,
discovered in the footsteps of the explorers Spix and Martius
in Brazil
(Orthoptera, Tettigonioidea, Phaneropterinae)
Klaus-Gerhard Heller, Marianne Volleth, Varvara Vedenina,
Anna Maryañska-Nadachowska & Elybieta Warchałowska-Šliwa
Heller, K.-G., Volleth, M., Vedenina, V., Maryañska-Nadachowska, A. &
Warchałowska-Šliwa, E. 2020. A perfect duet? The acoustic behaviour of Anaula-
comera almadaenis sp. nov., a species with an unusual chromosome complement,
discovered in the footsteps of the explorers Spix and Martius in Brazil (Orthoptera,
Tettigonioidea, Phaneropterinae). Spixiana 43 (1): 105-118.
To find a mate, males and females of phaneropterine bush-crickets engage in
duets. The male starts a duet and a female responds if she is prepared to mate. This
pattern is exactly followed in the acoustic communication of a species of the genus
Anacaulomera from the State of Bahia in Brazil which we newly describe in this
manuscript. However, the male also acoustically defends the duet. As soon as it
hears a female response, it adds an additional component to its own signal to make
the localisation of the female difficult for rivals. At the same time the male reduces
the interval between its songs for a faster exchange of information about the loca-
tion of the partner, and reduces the intensity of the song that it can be heard only
from nearby. Besides the nearly perfect anti-eavesdropping modifications of the
duet the new species is remarkable concerning its karyotype. The meiotic behaviour
of chromosomes in the male may indicate the origin of a new type of sex determi-
nation mechanism.
Klaus-Gerhard Heller (corresponding author) & Marianne Volleth, Grillenstieg
18, 39120 Magdeburg, Germany; e-mail: heller.volleth@t-online.de
Varvara Vedenina, Institute for Information Transmission Problems, Russian
Academy of Sciences, Moscow, Russia
Anna Maryañska-Nadachowska & Elybieta Warchałowska-Šliwa, Institute of
Systematics and Evolution of Animals, Polish Academy of Sciences, Kraków, Po-
land
Introduction
In 1891, at the time of the last review of the subfamily
Phaneropterinae (Brunner von Wattenwyl 1891), the
genus Anaulacomera Stål, 1873 was the largest genus
of this subfamily containing 40 species, distributed
in Central and South America. Now (Cigliano et al.
2018, abbreviated OSFO) it comprises 114 species,
but despite this increase it has less species than
the East Asian genus Elimaea (164 spp. + 7 sspp.) or
the Western Paleartic genus Poecilimon (136 + 31),
which had only 21 and 30 species respectively in
1891. Comparison of the species numbers and their
increase suggest that Anaulacomera is very species-

106
rich but poorly studied genus with probably many
undescribed species. Poecilimon, for example, had
attracted the attention of orthopterologists already
long time ago with the first review in 1933 (Ramme
1933) and even its subgroups are now subject of
revisions (e. g., Chobanov & Heller 2010). There are
also many studies on its distribution as well as on
bioacoustics, phylogeny and genome organization
(e. g., Heller 1984, Ullrich et al. 2010, Ünal 2010,
Grzywacz et al. 2014). Using these information,
identification of previously unknown forms becomes
much faster, allowing an understanding of the dis-
tributional patterns of species and species groups
as well as phylogenetic studies. For Anaulacomera,
however, a first key to species groups was published
only in 2015 (Cadena-Castañeda 2015). Before that
the various species were nearly unidentifiable and
studies with comments like “Anaulacomera sp. 1-8”
(Braun 2002, 2008) had to be published. Up to now
neither the male song nor the female response nor
any acoustical or ecological factors permitting the
coexistence of Anaulacomera species are known for
any described species of the genus. In Macaulay
Library there are 30 recordings of Anaulacomera from
Trinidad & Tobago made by Walker in 1966 (Walker
1966), but all remained unidentified.
In Anaulacomera as well as in Poecilimon the struc-
ture of the male genitalia presents the most important
morphological characteristics for identification,
showing a much higher diversity in Anaulacomera
than in Poecilimon. Otherwise the genera differ
strongly. Poecilimon is micropterous and flightless
with many local forms, while all Anaulacomera have
well developed wings and are good flyers. However,
nothing can be said about the exact ranges of the
species because of the difficulties with identification.
We came in contact with Anaulacomera during a
short excursion after the 12th International Congress
of Orthopterology, held in October/November
2016 in Ilheus (Bahia, Brazil), when we could stay
some days on the nearby Fazenda Almada. To our
surprise we discovered later that nearly exactly 200
years earlier (December 1818) the famous naturalists
and explorers of Brazil, Johann Baptist von Spix and
Carl Friedrich Philipp von Martius, had visited the
same place (Martius 1828, p. 681; see also https://
de.wikipedia.org/wiki/Johann_Baptist_von_Spix)
and met the German settlers who had founded the
fazenda. During our stay we found several phanero-
pterine bush-crickets, among them one adult male
and three nymphs which successfully moulted to
adults. It turned out that they all belonged to one
and the same species of the genus Anaulacomera. They
are morphologically very characteristic and different
from all known species, so we describe them here
as new species and as starting point for the study
of Anaulacomera bioacoustics.
As typical for phaneropterine bush-crickets, fe-
males respond acoustically to the male song (Heller et
al. 2015). Using this system the male needs to signal
only with relatively large intervals, waiting to hear
a response. If successful, he should reduce interval
length for a fast phonotactic approach. In addition he
should make the female response difficult to locate
for eavesdropping rivals or conceal it completely.
Our species seems to use several of such tactics, not
observed or at least not described and quantitatively
measured in any other bush-cricket species before.
Material and methods
The animals were held in plastic containers, differing
in size depending on the size of the animals, to avoid
dehydration in the laboratory and fed with Taraxacum
officinale, replaced daily.
Measurements. Total body length, lateral aspect, refers
to the mid-line length of the insect from fastigium verti-
cis to tip of abdomen including the subgenital plate. In
females, the ovipositor is not included in the measure-
ment of the body length. Measurements of the oviposi-
tor are taken laterally in straight line from tip to base not
regarding the curvature. To obtain the mass data, living
animals and spermatophores were weighed to the near-
est 2 mg (balance Tanita Professional Mini 1210-100).
Acoustics. The male calling song was recorded in
the laboratory using a digital bat detector (Pettersson
D1000X) with a sampling rate of 100 kHz. Duets were
recorded in stereo using a Sony ECM-121 microphone
(frequency response relatively flat up to 30 kHz ac-
cording to own tests) and an Uher M645 audio micro-
phone connected to a personal computer through an
external soundcard (Transit USB, “M-Audio”; 44.1 kHz
or 64 kHz sampling rate). In these experiments male
and female were placed separately into two plastic
tubes (Drosophila tube 28.5 × 95 mm, Biosigma, Cona
(VE), Italy) standing side by side, with one microphone
placed inside or on top of each vial. Both microphones
typically picked up male and female sounds, but with
different amplitudes. Duration of echemes and echeme
intervals and amplitude data were measured using
Canary (http://www.birds.cornell.edu/brp/). Other
song measurements and spectrograms were obtained
using Amadeus II and Amadeus Pro (Martin Hairer;
http://www.hairersoft.com). For frequency analysis
additionally the program ZeroCrossing v5 (kprestwi@
holycross.edu) was used. Here the singers were caged
in gauze cages with microphone fixed at a distance of
about 80 cm. Oscillograms of the songs were prepared
using Turbolab (Bressner Technology, Germany). All
recordings were made at temperatures between 20 and
25 °C. Data are presented as mean ± standard deviation.

107
Acoustical terminology. Tettigonioids produce their
songs by repeated opening and closing movements of
their tegmina. The sound resulting during one cycle
of movements is called a syllable, often separable in
opening and closing hemisyllable (Ragge & Reynolds
1998). Syllable duration: time period measured from
the beginning of the pulse to its end (or first impulse
to the last); syllable period: time period measured from
the beginning of a syllable to the beginning of the next
(reciprocal value: syllable repetition rate); echeme: first-
order assemblage of syllables; pulse: undivided train of
sound waves increasing in amplitude at the beginning
and containing many similarly sized wave maxima and
minima (cricket-like song structure; example see Fig. 7);
impulse: a simple, undivided, transient train of sound
waves (here: the damped sound impulse arising as the
effect of one tooth of the stridulatory file); latency time:
interval between beginning of male (female) song to
beginning of female (male) response.
Chromosomal analysis. Two males (CH8308, CH8309)
and two females (CH8310, CH8311) were used for chro-
mosomal analyses. Preparations were obtained from
testes, ovarioles, and hepatic caeca incubated in hypo-
tonic solution (0.9 % sodium citrate), fixed in ethanol :
acetic acid (3 : 1), and squashed in 45 % acetic acid.
C-banding was carried out using the version of Sumner
(1972), and the silver staining method (AgNO3) for lo-
calization of the nucleolus organizer regions (NORs)
was performed as previously reported (Warchałowska-
Šliwa & Maryañska-Nadachowska 1992). In order to
identify GC- and AT-rich regions, the prepared samples
were stained with CMA3 and DAPI, respectively
(Schwei zer 1976). Chromosomes were studied with a
Nikon Eclipse 400 microscope with a CCD DS-U1 cam-
era and NIS-Elements BR2, and the imagines were op-
timized using Adobe Photoshop. For each individual at
least three oogonial/spermatogonial mitotic metaphase
and 25 meiotic divisions in male were examined.
Results
The animals we found were identified as members of
the genus Anaulacomera Stål, 1873 using the keys in
Brunner von Wattenwyl (1891) and Cadena-Castañe-
da (2015). Within the genus, they are possibly similar
to the species of the group alfaroi according to the
size of the cerci as described by Cadena-Castañeda
(2012, 2015). However, the cerci are even shorter
than in this group where they are said to be slightly
longer than the subgenital plate. In shape they differ
distinctly from all figured species. Therefore, we
describe them as a new species.
Anaulacomera almadaensis Heller sp. nov.
urn:lsid:Orthoptera.speciesfile.org:TaxonName:503430
Material examined and depository. Holotype M, allo-
type W and 2 paratypes, 1 M, 1 W. All pinned, original
labels “BRAZIL: Bahia, Ilheus, Uruçuca, Fazenda Al-
mada, 95 m, 29 x - 1 xi 2016, leg. K.-G. Heller, M. Volleth,
Varja Vedenina”. “Holotype Anaulacomera almadaen-
sis” [red handwritten label]. Holo- (CH8308) and allo-
type (CH8310) in Zoologische Staatssammlung Mün-
chen (ZSM), Germany, Paratypes (CH8309, CH8311) in
Collection Heller. One hind leg of CH8308,10-11 sepa-
rate in pure ethanol in Collection Heller.
Sound files are deposited at OSFO and bio.acusti.ca.
Measurements (in mm; holotype bold). Males.
Body length: 23-24; pronotum length: 5.5-5.9; pro-
notum height: 4.0; hind femur: 20-21; hind tibiae:
23-24; tegmina: 34; length of hind wings: 37-38.5;
tegmina width: 8.5-9. Females. Body length: 22-23;
pronotum length: 5.6; pronotum height: 3.8-4; hind
femur: 21; hind tibiae: 24; tegmina: 32-33.5; length
of hind wings: 36-38; tegmina width: 7.5-8.5; ovi-
positor 11-12.
Locality and time. The type locality is situated at
Fazenda Almada at about 14.6563° S, 39.1884° W,
26 m a.s.l., as read from Google maps using http://
www.mapcoordinates.net/en. For details of the area
see http://www.fazenda-almada.com/en/willkom-
men/. One male was caught as adult (30th October)
and sang in the night after the capture, the other
moulted to adult more than three weeks later, on
24th November. The two females became adults on
18th and 23th November.
Description
Habitus and colour. Predominantly green, medium-
sized bush-crickets (Fig. 1), large for the genus
(compared to the data from Brunner von Wattenwyl
1878, 1891). Male stridulatory area in both tegmina
brown. Female tegmina only with with-brown spot
at articulation. In living specimens of both sexes
with a series of about six white spots distributed
along the radius.
Male. Head with round eyes, fastigium verticis
expanded at tip (see Figs 2b, 3a), half as wide as
scapus of antenna, not in contact with fastigium
frontis (Fig. 3a); fronto-genal carinae very indistinct;
antennal sockets prominent; antennae about as
long as tegmina. Pronotum without lateral carinae,
slightly longer than high, covered with very fine
hairs; prozona hardly separable from metazona,
anterior margin straight, posterior margin rounded,
with evident lateral excisions where wings are in-
serted. Prothoracic spiracle narrow, long, reaching

108
to about two third of pronotal height. Ventral edge
of paranota rounded. Tegmina wider than prono-
tal length. The stridulatory area of the left tegmen
brown and green (Fig. 1), with distinct and weakly
elevated stridulatory vein (Fig. 2a), of right tegmen
with several irregular veins, without glossy mirror
(Fig. 2b). The stridulatory vein on the underside of
the left tegmen with 57-63 (n = 2) relatively evenly
spaced teeth, only at the distal end a little bit denser
(Fig. 5a). Hind wings longer than tegmina. Meso- and
metasterna with two lobes each, rounded.
Fore coxae armed, fore femora ventrally with
a few small spines, fore tibiae ventrally with five
spines each on inner and outer edge, rounded, dorsal
side rounded, without dorsal spurs. Tympana open
on inner and on outer side. Mid femora below with
several, mid tibiae with about ten spinules. Hind
femora armed ventrally with a few spines distally,
genicular lobes without spines. Hind tibiae armed
ventrally and dorsally, furrowed on all sides. Hind
tibiae longer than femora.
Abdomen. Subgenital plate long, strongly
curved upwards, tapering into a weakly incised
expanded caudal part with drawn out tips, but with-
out styli (Fig. 4e-f); very large, vertically oriented
supraanal plate, which prevents the view on a pos-
sibly existing sclerotized titillator. Cerci (Fig. 4a-d)
short and stumpy, with a thick basal part carrying
an upper and lower terminal branch; upper branch
ending in two inward-curved tips, the outer with one
sharp spine, the inner with a broad ending; lower
part outwardly-curved with two spines, the lower
much larger than the upper; connection between
both parts concave, opening directed outwards.
Cerci densely covered with long hairs.
Fig. 1. Anaulacomera almadaensis sp. nov., habitus: a-b, male; c-d, female; a, c, alive; b, (mirror image); d, dried.
Scale 5 mm.
Fig. 2. Anaulacomera almadaensis sp. nov., dorsal view of head, pronotum and base of tegmina: a, male; b, female.
Note the single stridulatory vein in the right female tegmen (arrow). Scale 1 mm.

109
Female. As male, except stridulatory organs and
genitalia. Proximal quarter of right tegmen (dorsal
area) with one distinct stridulatory vein carrying
about 15 teeth (Fig. 5b). Ovipositor long, evenly
curved (Fig. 1d). Subgenital plate short and broad,
lateral sclerites complicated corresponding to the
male cercus shape (Fig. 4g).
Fig. 3. Anaulacomera almadaensis sp. nov., head and pronotum: a, head in frontal view; b, lateral view of pronotum.
Scale 1 mm.
Fig. 4. Anaulacomera almadaensis sp. nov., cerci and sub genital plate: a-d, male cerci; a, latero-posterior view; b, view
from above; c, view from below; d, view from behind; scale 1 mm (b-d); e-f, male subgenital plate; e, dorsal view
with cerci (left one near natural resting position, right one opened widely); f, lateral view; g, female subgenital plate
and lateral sclerites, lateral view; scale 5 mm (e-g).

110
Eggs. Mature eggs were taken from the females
after death and preserved dried. They show the flat,
ovoid shape, typical for phaneropterines (length
4.70 ± 0.07 mm, width 1.68±0.11 mm, n = 4 (2 per
female)).
Diagnosis. A. almadaensis differs from the other spe-
cies of the genus by its short, stumpy cerci ending in
an upper and lower branch (Fig. 4a-d). Tips of the
cerci are directed inwards while in resting position,
but even when opened shorter than subgenital plate.
Derivatio nominis. The name refers to the locality, the
fazenda and the river Almada, therefore almada-ensis,
adjective.
Mating behaviour. One male (body mass 876 mg
and 986 mg) copulated with both females (body mass
994 mg, 650 mg) at an interval of 8 days (10.12.2016
and 18.12.2016). They mated at night with a mating
duration of ca. 17 minutes (no data for other mating)
and transferred spermatophores of 172 and 217 mg
(mean of male loss and female gain), thus about 20 %
of the male body mass. The spermatophylax was
correspondingly large, the ampulla orange.
Acoustics. The calling song of an isolated male
consisted of echemes, repeated in intervals of
around 15 s (M1, 30.10.2016, T = 25 °C; 16.8 ± 5.4 s;
26.11.2016, T = 20 °C; 11.1 ± 2.2 s; M2, 3.12.2016,
T = 23 °C; 17.0 ± 6.2 s; n = 10 each). Each echeme
contained 5-7 syllables, produced with a rate of ca.
30 Hz (syllable period 30.7 ± 0.8 ms; 36.0 ± 0.8 ms;
33.3 ± 0.7 ms; as above; Fig. 6a). The syllables were
very short, less than 5 ms (3.6 ± 0.5 ms; T = ca. 22 °C,
n = 12), typically consisting of one cricket-like pulse
(Fig. 7a). The duty cycle can thus be estimated as
(6 × 3.6 ms) × 4/60 000 ms = 0.1 %.
The females ready to mate reacted to the
male song by emitting their own acoustic signals.
Fig. 5. Stridulatory files in male and female Anaulacom-
era almadaensis sp. nov.: a, male stridulatory file on
lower side of left tegmen; b, female stridulatory file on
upper side of right tegmen.
They always answered after the last syllable of
an echeme with a latency of ca. 100 ms (Fig. 6b-c;
W1: 103 ± 13 ms, W2; 102 ± 13 ms, range 74-118 ms;
T = 22-23 °C, n = 12 each). In one female the response
often consisted of one impulse, in the other two or
three impulses were observed with an interval of up
to 10 ms between the first and the last.
After having heard a female response, the
acoustic behaviour of the male changed (Fig. 9).
Immediately after the response – within the next
50-150 ms (102 ± 20 ms, range 44-127 ms, n = 25), it
produced (an) additional sound(s). This fast reaction
happened even after quite long times without female
response. After at least 93, 53 and 52 minutes (the
three longest available intervals) without female
response the male immediately modified its song.
It added one to four additional syllables (Fig. 6c).
After one interval of usual duration (or even longer
than the preceding; Fig. 9 b,d) the male produced
echemes at a faster rate than before the response. In
addition, it modified again the pattern of the echeme
and its amplitude. These changes were most obvious
when the male had heard one or several responses
before (as in Fig. 9b). Often the next echemes had
low amplitude, the syllables consisted of several
(im)pulses, contained clearly visible opening and
closing hemisyllables and, at the time when a fe-
male response was to expect, the male produced a
series of impulses (Fig. 6d). In situations when the
female responded more frequently her response
arrived always before these impulses (Fig. 6e). This
elongation of the echeme disappeared at first if the
female did not continue to respond, but the other
modifications could be relatively long lasting (longer
than 1 min). If the female responded regularly, oc-
casionally combinations of both echeme modifica-
tions (adding an impulse series and syllables) were
observed. Concerning loudness, however, it cannot
be excluded that the reduction of sound amplitude
was at least partly an effect of a changed position
of the male relative to the microphone (see Discus-
sion). All these song modifications were observed
only in the song of the male caught as adult; in the
only 2 h-recording of the younger male at the age
of 10 days it behaved as if was deaf – no reaction to
female responses at all.
The carrier spectrum of male calling song is
relatively narrow-banded with its maximum at
11-12 kHz (Fig. 8). The syllables showed a slight
frequency modulation, often upwards in the first
syllables. The last syllables of an echeme were mostly
flat, but sometimes also downward modulated
(rarely also seen in the first syllable) (see Fig. 7b
for examples). The spectrum of the soft song of the
male, however, is more broad-banded, similar to the
spectrum of the female responses. The peak value

111
of these broad-banded songs is similar to that of the
male calling songs (Fig. 8).
Chromosomes. The analysis of mitotic and meiotic
chromosomes of Anaulacomera almadaensis revealed a
diploid chromosome number 2n = 29, X0 and 2n = 30,
XX in males and females, respectively (Fig. 10a-b).
All autosomes were acrocentric, except of the first
pair, and can be divided into two groups: nine
large and medium (1-9) and five small ones (10-
14), both gradually decreasing in size, which com-
plicates their identification. The sex chromosome
(X) was metacentric and the largest element in the
karyotype, about twice as long as the first pair of
Fig. 6. Time-amplitude pattern of songs of Anaulacomera almadaensis sp. nov.: a, calling song of isolated male; b, male
song and female response (rarely observed); c, male song, female response and male reaction (nearly always ob-
served); d, soft song of the male after he heard a female response; e, soft song of the male with female response.
Red colour indicates female sounds visible in the male sound track.

112
autosomes. A heteromorphic long pair (1st), clearly
subacrocentric/subtelomeric was found both in
females (Fig. 10a) and males (Fig. 10b-c). During
spermatogonial metaphase and diplotene/diakinesis
a secondary constriction, located interstitially in one
arm on the X, was observed (Fig. 10b-c, marked by
arrowheads).
Heterochromatic blocks revealed by C-banding
were located in the paracentromeric regions of all
chromosomes. Thin C-positive regions were gener-
ally negative for both DAPI and CMA3, whilst a
C-band in the heteromorphic 5th pair was DAPI-
negative and CMA3-positive. The heterochromatic
region located interstitially near the distal ends of
the 3rd pair (in males in heteromorphic conditions)
was C/DAPI/CMA3-positive (Fig. 10b-e, marked
by asterisks).
The biarmed first pair demonstrating heterochro-
matic characters is called “megameric” (m). In sper-
matogonial, oogonial and somatic (cells from hepatic
caeca of females) metaphases, a heteromorphism of
both C-bands and fluorochrome bands was observed
in these chromosomes in terms of the size/strength
of bands on homologs. Short arms, differing in size
between homologs, were euchromatic, whereas
heterochromatin in long arms differs concerning
C/DAPI/CMA3-bands. In one of the homologues
of this pair two heterochromatic blocks of unequal
size were separated by a short euchromatic region
(Fig. 10c-e).
The analysis of meiotic cells in males demon-
strates the behaviour of this bivalent which creates
a pseudo-trivalent with the X chromosome (marked
as m+X). This heterozygous (unequal) bivalent
shows strong heterochromatic characteristics (high
condensation), from prophase to metaphase II,
whereas the X was weakly positive heteropycnotic.
In zygotene-pachytene, both unequal bivalents
and the X chromosome probably exhibited partial
synapsis only at their terminal parts and small in-
terstitial part in one arm of the metacentric X. Thus,
a large region remained unpaired forming a loop.
Alternatively, they may be connected only in a short
part, which would suggest that these chromosomes
are not homologous (Fig. 10 f). Generally, from early
diplotene to early diakinesis, the trivalent showed
end-to-end association (probably without chiasma)
(Fig. 10g,n,o). Part of the megameric bivalent is
probably connected in the secondary constriction
of the X (Fig. 10b,c,g). In the late diakinesis and
metaphase I, the heteromorphic pair and the X
chromosomes were arranged separately (Fig. 10 i,p).
In anaphase I, unequal chromosomes underwent an
equational segregation pattern, whereas the first
meiotic division was reductional for the other auto-
somes (Fig. 10 h). As a result, in metaphase II 14 or 15
(with X) chromosomes and rarely 15 or 16 (with X)
in both cells with an extra element (e) were observed
(Fig. 10j-k). This small euchromatic additional ele-
ment (e) was often present at different meiotic stages
(Fig. 10b, g-i,k, p). At diplotene, it was often seen
near that region of the X which was in association
with the unequal bivalent (Fig. 10g). Additionally, in
some analysed spermatogonial metaphases (6 of 15)
30 or 31 chromosomes (with one or two additional
elements) were observed (e. g. Fig. 10 b-e). On the
other hand, in oogonial and somatic metaphases of
females (from hepatic caeca) these element/s were
not found. However, such cells were not analysed
in males. In anaphase II the megameric “m” chro-
mosomes divided reductionally and segregated to
opposite poles (Fig. 10l).
Two active nucleolus organizer regions (NORs)
located distally on the long arms of the unequal m
bivalent were heterozygous in sizes, and always
located near the X chromosome (Fig. 10 m-o). NORs
coincided with large GC-rich blocks that were in-
tensively stained by CMA3 and positive and thick
Fig. 7. Anaulacomera almadaensis sp. nov., details of male song: a, time-amplitude pattern of a syllable of the male
song; b, cycle-by-cycle frequency of three syllable of one echeme.

113
step further and modifies its song in several ways
only after heard a female response. One of the four types of modification it uses, i. e. to increase the duty
Fig. 9. Examples for changes in male calling behaviour after he had heard a female response: reduction of echeme
intervals (blue squares) and sound amplitude (red triangles), increase in duration of echemes (green dots): a, reac-
tion to first female response after 40 min of singing; b, reaction after having heard a female response shortly be-
fore; c-d, reaction with two female responses. Blue: interval to following echeme (note different scales in a-d); green:
echeme duration; red: relative sound pressure amplitude (linear scale); *, 2nd female response.
Fig. 8. Power spectra of sounds of Anaulacomera alma-
daensis sp. nov.: a, male calling song (blue: two different
males) and example of soft song (lila); b, female response
(red: two different females).
C-bands. Additionally, in some cells small silver
dots were observed in the early meiotic prophase on
the X, probably a “secondary NOR” associated with
the intercalary secondary constriction (not shown).
Discussion
Concerning bioacoustics, the subfamily Phanero-
pterinae is certainly the most diverse group among
ensiferan Orthoptera. This relates to the fact that in
most species the females respond acoustically to the
male song and establish a duet with the male (for a
review, see Heller et al. 2015). Elements of the male
song must indicate the female when to respond,
and, perhaps even more important, the male should
defend the duet against silent, eavesdropping rivals.
For this purpose, the males often add self-made fe-
male imitations to the song, making the localization
of a responding female difficult. Most prominent
examples are e. g. Ducetia antipoda Heller & Rentz,
2017, where the male song ends in a long series
of well-separated impulses (Heller et al. 2017), or
Gonatoxia helleri, where the male produces special
signals which imitate the female signals in carrier
frequency (Heller & Hemp 2017). In all these spe-
cies the female imitations are part of the male song and are emitted independently of the presence of
females. Anaulacomera almadaensis, however, goes one

114
Fig. 10. Examples of C-banded (a, b, c, f-l), fluorochrome stained heterochromatin (d, e) as well as silver stained
(m-p) chromosomes of A. almadaensis sp. nov.: a, oogonial metaphase and somatic metaphase (in the inset) of a
female with 30 chromosomes; b, karyogram arranged from a spermatogonial metaphase (in the inset) of a male with
29 chromosomes plus two “e” elements; b, c, heteromorphic heterochromatic/megameric long chromosomes (prob-
ably 1st pair marked in b) are indicated with “m” and show clearly short euchromatic arms; arrowheads indicate a
secondary constriction in one of arms of X. Thin paracentromeric C-bands (c) are negative for DAPI (blue) and CMA3
(green) bands (d, e, respectively); an asterisk marks the heteromorphic paracentromeric region in 5th pair which is
DAPI-negative and CMA3-positive and the heteromorphic 3rd pair with interstitially located C/DAPI/CMA3-positive
region; heterochromatin in long arms of the megameric chromosome differs concerning C/DAPI/CMA3-bands (ar-
rows); f-l, the behaviour of the heteromorphic bivalent which forms a pseudo-trivalent with X chromosomes (marked
such m+X) in meiosis: the X chromosome shows weakly positive heteropycnosis, m bivalent is strongly heterochro-
matic, both from pachytene to metaphase II. In pachytene (f) and diplotene/diakinesis (g, m-o) chromosomes of the
m+X complex form a loop or are connected to each other only in the short part. . . .

115
cycle, is well known also from other duetting species.
After a female response, the male typically increases
its duty cycle by reducing the intervals between its
songs. In fact, this variability is considered as the
most important advantage of bidirectional com-
munication in Orthoptera (e. g., Heller & Helversen
1993, Helversen et al. 2012). Hartley et al. (1974:
p. 387) gave the first figure demonstrating this ef-
fect in a bradyporine duetting species. It should be
typical for duetting species, but only few examples
are known. The second type of song modification
concerns loudness. Reducing song amplitude may
be similarly common, but data are even rarer. Also
the data presented here have problems. In the four
examples presented in Figure 9, there is always a
clear drop in intensity with the first echeme(s) after
the response. However, we cannot exclude the pos-
sibility that the male moved rapidly into another
position less favourable for the microphone. Since
we did not detect any opposite example in all the
recordings, we assume that males really reduced the
intensity. In theory, the male should use the lowest
intensity to elicit a response making it most incon-
spicuous to predators and rivals. The continuous
increase in amplitude after a single female response
(Fig. 9b) fits nicely to this hypothesis.
The most unexpected song modifications were
the two types of changes in the amplitude pattern.
The male always started by adding one or several
additional syllables after the female response. These
sounds had a latency very similar to that of the
female response itself. This is possibly the shortest
time for an acousto-motoric reflex (sensu Robinson
et al. 1986) in this species. However, it may be too
slow to disturb an eavesdropping rival completely.
So the other type of modification, an added impulse
series, is certainly much more effective. But what is
the function of the added syllable? We must assume
that it has a special negative effect on rivals – and it is
very rarely missing, even after long times without re-
sponse. Such context-specific changes have not been
described in detail for any phaneropterine species,
but are mentioned in Spooner’s (1968) large descrip-
. . . In late diakinesis/metaphase I (h) heteromorphic
megameric bivalent is not connected with X and during
anaphase it undergoes an equational segregation pattern
(i); as result, in metaphase II there are 14 and 15 chro-
mosomes (j) with heteromorphic autosome megameric
pair, whereas both chromosomes segregate to opposite
poles (l). Some elements (e) are seen in different sper-
matogonial (the lower inset in b, c-e) and meiosis stage
(g-i, k, p). The arrowheads indicate active NORs located
in unequal (m) bivalent. X-, sex chromosome. Scale bar
10 µm.
/
tive study for two or three species. In Microcentrum
rhombifolium he refers mainly to the unpublished
dissertations of Alexander (1956) and especially of
Grove (1959). The latter interpreted “an irregular
shuffling sound” – with a possibly similar timing as
the impulse series in A. almadaensis – already as “to
confuse the location of the female” (Spooner 1968).
Males of Scudderia cuneata and S. furcata seem to
produce ticks when responsive females are nearby,
and subside this ticking after a few minutes when the
females are removed (Spooner 1968). This behaviour
has similarities with adding the impulse series in
A. almadaensis, which is also done only for a short
time. The increased width of the spectrum of these
male signals compared to the typical calling song
may be a consequence of the low intensity, but makes
the sounds also more similar to the female responses.
A. almadaensis shows all types of song modifica-
tion presently known to speed up the localisation
process and to conceal the duet at the same time from
predators and rivals. Therefore we call it a perfect
duettist. Single components of this behaviour are
certainly widespread among phaneropterines, but
even for the most common types (increase of duty
cycle and reducing sound amplitude) the data are
very limited. This is true, e. g. for increase in duty
cycle described only for Leptophyes punctatissima on
the basis of daily activity measurements (Hartley
& Robinson 1976) and for a bradyporine species on
hourly rates (Hartley 1993). The combined occur-
rence of the whole set of modifications may be rarer
and not so easy to observe. If a female is older or
more eager to mate she will respond continuously
to many or even all male songs and the effects of a
single answer will be difficult to recognize. It should
be noted that nothing is known about modifications
of the female signal which also may occur.
As mentioned above, Anaulacomera is a huge
genus but with very few data on acoustics and none
from any identified species. From the figures in Braun
(2002) and the recordings by Walker (1966) the pres-
ence of short syllables is typical for many species, but
not for all (e. g., Anaulacomera sp. 3 in Braun 2002).
Their spectrum is often narrow-banded (Braun 2002;
the recordings of Walker, now available as mp3-
audio, cannot be evaluated in this respect). Brunner
von Wattenwyl (1878, 1891) had placed Anaulacomera
together with the East African/Malagasy Parapyr-
rhicia in his group Anaulacomerae (including also
the South American Grammadera and Abrodiaeta),
but currently it is placed together with some South
American genera in the subtribe Anaulacomerina
of Phaneropterini, while Parapyrrhicia is separate.
Surprisingly, however, the biogeographically very
unusual assemblage of Brunner von Wattenwyl is
supported by bioacoustics. In both genera the male

116
calling songs consist of short, resonant syllables and
also the female stridulatory organs are built from
few and very conspicuous toothed veins at least in
A. almadaensis (see Hemp et al. 2017 and above). This
female file structure has not been studied systemati-
cally in phaneropterines, but the presence of many
female stridulatory veins is quite common (e. g.,
Heller et al. 1997, Hemp et al. 2016), while some
Anaulacomera females from French Guiana have the
same type of veins as seen in A. almadaensis (own
unpublished observations).
Up to now, the chromosome numbers and
morphology of only five species of the subtribe
Anaulacomerina were described. Anaulacomera horti
Piza, 1975, A. linguata Piza, 1952, A. chelata Brunner
von Wattenwyl, 1878, A. sp. 1 and Grammadera clara
Brunner von Wattenwyl, 1878 (described under the
synonym Anaulacomera dimidiata Piza, 1952) from
the State of São Paulo have 30 acrocentric autosomes
and a meta-/submetacentric X chromosome in males
(2n = 31, FN = 32) with X0/XX sex mechanism (Fer-
reira 1977, Ferreira & Mesa 2007). In Anaulacomera
brasiliae Piza, 1977 (from Goiás) the chromosome
number is reduced to 2n = 23, FN = 28 (X0) as a result
of two independent Robertsonian translocations
between autosomes (two metacentric pairs), a peri-
centric inversion forming a biarmed X chromosome
and some tandem fusions/centric fusions followed
by pericentric inversions (Ferreira & Mesa 2007).
The chromosome characteristics including male
and female karyotypes, meiosis and sex chromosome
of A. almadaensis show an advanced karyotype evolu-
tion. In this species, similar to the above-mentioned
species of Anaulacomera, (1) a biarmed X chromo-
some (submeta-/metacentric) arises as result of a
pericentric inversion; (2) the ancestral chromosome
number is reduced to 2n = 29 (FN = 32) probably as
a result of one Robertsonian translocation (as ob-
served in A. brasiliae) between long and small pair
of chromosomes creating a biarmed heteromorphic,
megameric long pair. Such a long chromosome pair
considered as megameric was described in other
phaneropterines, for example in the genus Isophya
(e. g., Warchałowska-Šliwa & Bugrov 1998).
In A. almadaensis the heteromorphic biarmed
pair of chromosomes with distally located active
NOR/GC-rich heterochromatin and unequal both
heterochromatic and euchromatic arms may have
arisen by paracentromeric inversion or pericentric
inversion. Both of these aberrations lead to the lack
of complete homology in the heteromorphic pair.
The behaviour of the trivalent (pseudo-trivalent)
observed during meiosis could suggest that some
other kind of translocation occurred between the
megameric pair of autosomes and the X chromosome.
However, the origin and behaviour of this complex
is difficult to interpret. The result of these changes
is a reduction in chromosome number (2n = 29/30)
and occurrence of additional elements which are
often connected with the trivalent in meiosis. Thus,
different chromosome numbers in meiotic and mi-
totic stages in the male can be explained by presence
of the supernumerary elements (centric/acentric)
which are not homologous with autosomes and
could be a result of some rearrangements of the
karyotype. However, the question remains, why
there are no additional elements in the mitosis in
females (meiosis was not observed). Possibly this
could be explained by the fact that only a small
number of good divisions was observed. Among
phaneropterine species a pericentric inversion plays
an important role in the appearance of biarmed X
chromosomes which does not lead to a change in the
number of chromosomes (e. g., Warchałowska-Šliwa
1998, Warchałowska-Šliwa et al. 2008, Hemp et al.
2013). Re-structuring the karyotype of Anaulacomera
almadaenis sp. nov. implies a few more (individual)
rearrangements. However, it cannot be excluded
that the karyotypic changes consist in the simultane-
ous occurrence of pericentric inversions in the sex
chromosome (formation of biarmed chromosome)
and translocation between the megameric pair and
the X. On the other hand, based on our observation
of the structure and meiotic behaviour of the trivalent
(m+X) in the meiosis, it cannot be excluded that these
events may indicate the origin of the first stage of a
neo-sex system (multiple neo-X1X2Y/X1X1X2X2) start-
ing from the X0/XX system. This hypothesis, based
on the location of active NOR/s on both autosomal
pairs and the X (forming a trivalent), may indicate
that the NOR-bearing chromosomes take part in
karyotype changes. Cytogenetic analysis of some
other tettigoniids, e. g., Odontura (Phaneropterinae)
or Spalacomimus (Hetrodinae) also revealed one or
two NORs located only on the sex chromosomes
of species with a neo-sex determination system:
neo-X (neo-XY) or both neo-X1 and neo-Y (with
neo-X1X2Y) (Warchałowska-Šliwa et al. 2011, 2015).
An autosome-sex chromosome translocation often
lead to a change in the type of sex determination
when it causes polymorphism of autosomes (e. g.,
in grasshoppers, Castillo et al. 2010). However, the
heteromorphic pair was observed in oogonial and
somatic metaphase of females A. almadaenis (present
paper), which may contradict the assumption of an
initial state of the differentiation process of a neo-sex
chromosome system.

117
Author’s contributions
K-GH, MV and VV collected the specimens; K-GH re-
corded and analysed the songs; EW-S and AM-N ana-
lysed the chromosomes; K-GH led the writing.
Acknowledgements
At first we are grateful to Juliana Mauthe de Cerqueira
Lima and her team for the support during our stay at
Fazenda Alamada. Our thanks go also to Juliana Cham-
orro-Rengifo, to an anonymous referee and especially
to Holger Braun for many useful comments.
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