Sensory evolution of hearing in tettigoniids with differing communication systems

Sensory evolution of hearing in tettigoniids with differingcommunication systems

J. STRAUß* , A. W. LEHMANN† & G. U. C. LEHMANN‡*Institute for Animal Physiology, AG Integrative Sensory Physiology, Justus-Liebig-Universit€at Gießen, Gießen, Germany

†Friedensallee, Stahnsdorf, Germany

‡Department of Biology, Behavioral Physiology, Humboldt University Berlin, Berlin, Germany

Keywords:

animal communication;

insect hearing;

regressive evolution;

sensory ecology;

sensory evolution;

tympanal organ.

Abstract

In Tettigoniidae (Orthoptera: Ensifera), hearing organs are essential in mate

detection. Male tettigoniids usually produce calling songs by tegminal stridu-

lation, whereas females approach the males phonotactically. This unidirec-

tional communication system is the most common one among tettigoniids.

In several tettigoniid lineages, females have evolved acoustic replies to the

male calling song which constitutes a bidirectional communication system.

The genus Poecilimon (Tettigoniidae: Phaneropterinae) is of special interest

because the ancestral state of bidirectional communication, with calling

males and responding females, has been reversed repeatedly to unidirec-

tional communication. Acoustic communication is mediated by hearing

organs that are adapted to the conspecific signals. Therefore, we analyse the

auditory system in the Tettigoniidae genus Poecilimon for functional adapta-

tions in three characteristics: (i) dimension of sound-receiving structures

(tympanum and acoustic spiracle), (ii) number of auditory sensilla and (iii)

hearing sensitivity. Profound differences in the auditory system correlate

with uni- or bidirectional communication. Among the sound-receiving

structures, the tympana scale with body size, whereas the acoustic spiracle,

the major sound input structure, was drastically reduced in unidirectional

communicating species. In the unidirectional P. ampliatus group, auditory

sensilla are severely reduced in numbers, but not in the unidirectional

P. propinquus group. Within the P. ampliatus group, the number of auditory

sensilla is further reduced in P. intermedius which lost acoustic signalling due

to parthenogenesis. The auditory sensitivity correlated with the size of the

acoustic spiracle, as hearing sensitivity was better with larger spiracles, espe-

cially in the ultrasonic range. Our results show a significant reduction in

auditory structures, shaped by the differing sex roles during mate detection.

Introduction

Understanding of animals’ behaviour and decision-mak-

ing requires insights into how they detect and perceive

their environment and the many signals around them

(Dusenbery, 2001). Contact with their environment is

made by specialized peripheral sensory receptors (Smith,

2009; Ryan & Cummings, 2013), which act as an inter-

face between the outside and the central nervous system

(Dangles et al., 2009). In general, the mechanistic base of

sensory systems is broadly studied, revealing the fascinat-

ing diversity of sensory systems (Barth, 2002; Greenfield,

2002; Smith, 2009). Sensory ecology provides the frame-

work for analysing receptor systems in an environmental

context (Dusenbery, 1992; Barth & Schmid, 2001; Phelps,

2007; Stevens, 2013). However, we still need an integra-

tion of ecological and evolutionary concepts into sensory

ecology studies by comparing sensory traits among spe-

cies (Chittka & Briscoe, 2001; Ryan & Cummings, 2013).

Central for selection acting on any expressed trait is the

Correspondence: Johannes Strauß, Institute for Animal Physiology, AG

Integrative Sensory Physiology, Justus-Liebig-Universit€at Gießen,

IFZ –Heinrich-Buff-Ring 26, 35392 Gießen, Germany.

Tel.: +49 641 99 35253; fax: +49 641 99 35279;

e-mail: [email protected]

200ª 2 01 3 THE AUTHORS . J . E VOL . B I OL . 2 7 ( 2 0 1 4 ) 2 0 0 – 21 3

JOURNAL OF EVOLUT IONARY B IOLOGY ª 2013 EUROPEAN SOC I E TY FOR EVOLUT IONARY B IO LOGY

doi: 10.1111/jeb.12294

https://www.researchgate.net/publication/24239343_Variability_in_Sensory_Ecology_Expanding_the_Bridge_Between_Physiology_and_Evolutionary_Biology?el=1_x_8&enrichId=rgreq-95c86339-979b-489c-a7f5-1638fa24df9f&enrichSource=Y292ZXJQYWdlOzI2MzY2MDc2NztBUzoxMTU5NTg1MDkyMTU3NDdAMTQwNDY1ODA3MjU1Mw==

https://www.researchgate.net/publication/260427107_Perceptual_Biases_and_Mate_Choice?el=1_x_8&enrichId=rgreq-95c86339-979b-489c-a7f5-1638fa24df9f&enrichSource=Y292ZXJQYWdlOzI2MzY2MDc2NztBUzoxMTU5NTg1MDkyMTU3NDdAMTQwNDY1ODA3MjU1Mw==

evolutionary perspective that individuals balance their

costs with the benefits realized by that trait. In regard to

sensory systems, we expect animals to adapt their costs of

developing and maintaining expensive neural tissue with

the needs for environmental perception and signal detec-

tion (Phelps, 2007; Chittka & Niven, 2009).

Animal signalling is shaped by sexual and natural

selection with respect to signals, signalling behaviour

and communication systems (Gerhardt & Huber, 2002;

Greenfield, 2002; Bailey, 2003; Bradbury & Vehren-

camp, 2011). Among insects, intraspecific acoustic sig-

nalling over long distances has evolved repeatedly

(Bailey, 1991; Yager, 1999; Stumpner & von Helversen,

2001; Greenfield, 2002; Robinson & Hall, 2002).

Orthopteran insects (grasshoppers, crickets and tettigon-

iids) have served as model organisms for studying both

acoustic signalling behaviour as well as the sensory and

neuronal systems mediating signal detection (Bailey,

1991; Gwynne, 2001; Gerhardt & Huber, 2002; Green-

field, 2002; Robinson & Hall, 2002). However, adaptive

signal characteristics are better understood than the ori-

gins of communication systems and the (co-)evolution

of the receiver’s sensory organs (Gerhardt & Huber,

2002; Greenfield, 2002). The growing accumulation of

phylogenetic data in combination with comparative

functional analysis provides the possibilities to track

adaptive changes in sensory systems along evolutionary

routes (Heller, 2006; Strauß & Lakes-Harlan, 2014).

Male tettigoniids (Orthoptera: Ensifera: Tettigoniidae)

produce calling songs by tegminal stridulation, whereas

females usually perform the phonotactic approach to

the singer (Robinson, 1990; Gwynne, 2001; Greenfield,

2002; Robinson & Hall, 2002). This ancestral state of

unidirectional communication in tettigoniids has been

modified into bidirectional communication systems in

independent lineages (Robinson, 1990; Gwynne, 1995;

Spooner, 1995; Greenfield, 2002; Robinson & Hall,

2002; Bailey, 2003). Here, females produce an acoustic

response upon perceiving the male calling song, estab-

lishing acoustic duets with their partners. Such duetting

has evolved independently in different Tettigoniidae

subfamilies: the Phaneropterinae, the Bradyporinae and

probably the Pseudophyllinae (Nickle & Carlysle, 1975;

Robinson & Hall, 2002). The change to bidirectional

communication has several evolutionary consequences:

it shifts the phonotactic role between sexes (Spooner,

1995), and thus also predation risk during the process

of mate localization, from females towards males (Hel-

ler, 1992). Hearing sensitivity may become tuned to the

song frequencies of the opposite sex (Heller et al.,

1997a). As differences in signal amplitude exist

between male and female signals, the ears may show

sex-specific hearing sensitivity (Stumpner & Heller,

1992; Heller et al., 1997a). Within the bidirectional

communicating Phaneropterinae, several species have

secondarily lost the acoustic female response, returning

to unidirectional acoustic communication. Notably,

all known species with reversal back to acoustic

unidirectionality are restricted to the European genus

Poecilimon and occur in species of three distinct groups

(Heller, 1984, 1990; Stumpner & Heller, 1992; Choba-

nov & Heller, 2010). For Poecilimon, the ancestrally bidi-

rectional communication is clearly supported by

comparative studies including out-group species (Heller,

1990) as well as a recent molecular phylogeny of the

genus (Ullrich et al., 2010). We use the genus Poecilimon

Fischer, 1853 to analyse the consequences of different

communication systems for adaptive features of the

auditory system. Species were selected to include repre-

sentatives of bidirectional species from different

lineages, based on current phylogenetic hypotheses

(Ullrich et al., 2010). Species with an abolished female

response were chosen from the eight species of the

P. propinquus group (Lehmann, 1998) and the P. ampli-

atus group, which includes five different species (Heller

& Lehmann, 2004). The latter species group contains

the only obligate parthenogenetic Poecilimon species,

P. intermedius (Fieber, 1853) (Lehmann et al., 2011). As

no stridulating males exist, in this parthenogenetic spe-

cies, females might be under relaxed selection for hear-

ing (Lehmann et al., 2007). The bidirectional Isophya

modestior is included to represent a closely related out-

group genus (Ullrich et al., 2010).

The functional anatomy of tettigoniid ears shows

common features across different species (Bailey, 1990;

R€ossler et al., 2006). Sounds are perceived by tympanal

organs in the foreleg’s proximal tibia with membranes

of thinned cuticle located on both the anterior and pos-

terior tibia. The main sound input to this sensory organ

comes through an auditory trachea: sound enters the

auditory system mainly via the acoustic spiracle in the

thorax rather than the tympana in the legs (Lewis,

1974; Nocke, 1975; Michelsen et al., 1994). Accordingly,

the size of the spiracle opening correlates positively

with hearing sensitivity, especially in the ultrasonic

ranges (Stumpner & Heller, 1992; Bailey, 1998). The

relative size of the spiracle also varies between species,

occasionally to the extent of evolutionary regression to

rather small diameters (Bailey & R€omer, 1991; Lakes-

Harlan et al., 1991; Lehmann et al., 2007). Tympana are

backed by air-filled cavities, connected to the auditory

sensilla which lay in a row, called crista acustica. Com-

parative anatomical studies show no direct correlation

between the length of this auditory organ and the num-

ber of auditory sensilla, not even in related species.

Therefore, major evolutionary changes in the crista acus-

tica can be identified more clearly by the number of

auditory sensilla than by differences in length (Schum-

acher, 1979; Lakes & Schikorski, 1990).

We investigate the adaptation of the auditory organs

in Poecilimon in a phylogenetic framework on different

structural levels: (i) the morphology and size of sound-

receiving structures, (ii) the neuroanatomy of auditory

sensilla and (iii) the hearing sensitivity.

ª 2 01 3 THE AUTHORS . J . E VOL . B I OL . 2 7 ( 2 0 1 4 ) 2 0 0 – 21 3

JOURNAL OF EVOLUT IONARY B IO LOGY ª 20 1 3 EUROPEAN SOC I E TY FOR EVOLUT IONARY B IO LOGY

Evolution of tettigoniid communication 201

https://www.researchgate.net/publication/230246967_Do_large_bushcrickets_have_more_sensitive_ears_Natural_variation_in_hearing_thresholds_within_populations_of_the_bushcricket_Requena_verticalis_Listroscelidinae_Tettigoniidae?el=1_x_8&enrichId=rgreq-95c86339-979b-489c-a7f5-1638fa24df9f&enrichSource=Y292ZXJQYWdlOzI2MzY2MDc2NztBUzoxMTU5NTg1MDkyMTU3NDdAMTQwNDY1ODA3MjU1Mw==

https://www.researchgate.net/publication/226904727_Risk_shift_between_males_and_females_in_the_pair-forming_behavior_of_bushcrickets?el=1_x_8&enrichId=rgreq-95c86339-979b-489c-a7f5-1638fa24df9f&enrichSource=Y292ZXJQYWdlOzI2MzY2MDc2NztBUzoxMTU5NTg1MDkyMTU3NDdAMTQwNDY1ODA3MjU1Mw==

In bidirectional communicating species, hearing is

essential for mating in both sexes. Hence, our hypothe-

sis is that selection favours a high sensitivity in combi-

nation with large sound-receiving structures and

presumably also a relatively high number of auditory

sensilla in the crista acustica, forming elaborate hearing

organs. The spiracle size should be under especially

strong selection pressure to be enlarged. Enhancement

in size would not only allow for better hearing, but also

favour a better detection of high frequencies which is

especially important for directionality in hearing

(Stumpner & Heller, 1992). Female Poecilimon respond

with signals reduced in both duration and intensity

compared with the male song (Heller & von Helversen,

1986; Heller et al., 1997b). Consequently, males should

be selected for higher sensitivity and to evolve larger

spiracles than their conspecific females.

After loss of female acoustic signalling, male ears in

unidirectional communicating species are no longer

under selection for detecting the female response. This

can be hypothesized to lead to decreased sensitivity in

hearing, in combination with relatively smaller spiracles

and lower sensitivities compared with bidirectional spe-

cies. In particular, we expect the reversal to unidirec-

tional communication to be accompanied by a

reduction in auditory sensilla number. As male signal-

lers have still to be detected by females, we ask

whether females have more sensitive hearing or show

similar reductions in hearing as males.

Materials and methods

Animals

Fifteen species of the genus Poecilimon were selected for

morphometric analysis of auditory structures (see Sup-

plementary Table 1). The neuroanatomy and hearing

sensitivity was investigated in a subset of five selected

Poecilimon species (P. ampliatus Brunner von Watt-

enwyl, 1878, P. chopardi Ramme, 1933, P. elegans Brun-

ner von Wattenwyl, 1878, P. gracilis (Fieber, 1853) and

P. ornatus (Schmidt, 1850)) (see Supplementary

Table 1). Isophya modestior Brunner von Wattenwyl,

1882 was used as out-group for all analyses, based

upon its position basal to the genus Poecilimon (Ullrich

et al., 2010; Grzywacz et al., in press). The communica-

tion mode of each species was extracted from previous

studies (Heller, 1990; Heller & von Helversen, 1993;

Lehmann & Lehmann, 2000, 2006, 2008; Lehmann

et al., 2001, 2007). The unidirectional species belong to

the so-called P. propinquus group and the P. ampliatus

group, respectively. Animals were collected at several

locations in Greece, Slovenia and the Czech Republic at

different time points (see Supplementary Table 1).

Specimens for anatomical measurements had been

collected between 1994 and 1996 and were stored

in ethanol. For neuroanatomical and physiological

experiments, we collected the individuals in May/June

2011 and June 2012.

Animals used for neuroanatomical and physiological

experiments were maintained at room temperature

under a 12:12 light-dark regime at the Institute for

Animal Physiology, Justus-Liebig-Universit€at, Gießen.

They were kept individually in 200-mL plastic boxes

(Drosophila rearing boxes, Greiner Bio-one GmbH, Fric-

kenhausen, Germany; www.GreinerBioone.com) and

fed with a mixture of Taraxacum leaves and flowers ad

libitum.

The physiological experiments and morphological

measurements carried out in this study comply with

the principles of animal care of the Justus-Liebig-Uni-

versit€at Gießen and with the current law of the Federal

Republic of Germany.

Morphometry

For data sampling, animals were individually marked

and subsequently used for morphometrical analysis.

The same individuals were also used in experiments on

sensory physiology and neuroanatomy. Dimensions of

the auditory tympana and spiracles as well as hind

femur length were determined on a Leica binocular

microscope with an ocular micrometre (Wild, Heerb-

rugg, Switzerland, accuracy � 0.01 mm). For hind

femur length, different numbers of specimens were

measured (Table S1). We chose hind femur length as a

measure of body size, because all species in the genus

Poecilimon have similar life histories as bush climbers

and scrub dwellers. Therefore, hind femur length can

reliably be used (Lehmann, 1998). All species in this

study have open tympana without a covering, and

therefore, the maximum extension of the tympanum

was readily accessible. Both the anterior and the poster-

ior tympana were measured in the dorso-ventral and

proximo-distal dimension. For the acoustic spiracle, the

dorso-ventral spiracle diameter was measured because

it is the maximal extension of the spiracle opening (see

also Stumpner & Heller, 1992) and is given as the spira-

cle’s ‘major axis’.

Sensory physiology

The auditory threshold of the animals was determined

in a Faraday cage covered with sound-absorbing mate-

rial. For these electrophysiological experiments, animals

were briefly cold-anaesthetized and fixed on a metal

holder with the ventral side up using a 1:1 mixture of

bee wax and colophony (both from Carl Roth, Kar-

lsruhe, Germany). The forelegs were attached to metal

wires in a position resembling the normal standing

position of the animal as closely as possible. The pro-

thoracic ganglion was exposed by opening up the ster-

nal cuticle, and the coxa, trochanter and proximal

femur were opened ventrally.



202 J. STRAUß ET AL.

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Acoustic stimuli were delivered from a broad-band

loudspeaker (Dynaudio, Skanderborg, Denmark DF

21/2) placed in the recording cage at a distance of

38 cm from the animal. Stimuli were computer-gener-

ated and computer-amplified (Lang et al., 1993). Stimu-

lus intensities were calibrated with a sound level meter

(Br€uel and Kjær Type 2203, Br€uel and Kjær, Copenha-

gen, Denmark) and a microphone (Br€uel and Kjær

Type 4135). During the experiments, frequencies rang-

ing from 3 to 40 kHz were presented to the animals.

For each frequency, these signals increased over-

defined intensities ranging from 30 to 80 dB SPL. Steps

between intensities were of 5 dB SPL for stimuli

between 30 to 60 dB SPL and of 10 dB SPL for stimuli

between 60 to 80 dB SPL. For each sound intensity,

five stimuli of 100 ms duration were presented.

Summed action potentials were recorded from the

nerve with a silver wire hook electrode close to the

entrance to the ganglion. A reference electrode was

inserted in the hemolymph close to the recording site.

The obtained signal was amplified 1000-fold by a pream-

plifier (ISO-80; World Precision Instruments, Sarasota,

FL, USA) and displayed on an oscilloscope and transferred

to earphones. The auditory threshold was determined as

the lowest stimulus intensity at which a neuronal excita-

tion was detectable for at least three out of five stimuli of

that specific intensity level. During recordings, the

sequence of frequencies tested was chosen at random.

Neuroanatomy of the auditory sensilla in thetympanal hearing organ

Auditory sensilla in the foreleg tympanal organ were

stained by retrograde filling of the tympanal nerve

(N5B1) using a 5% cobalt chloride solution dissolved in

distilled water (cobalt chloride from Merck, Darmstadt,

Germany) as a tracer (Pitman et al., 1973). For dissection

of the tympanal nerve, the forelegs were cut off and

fixed with needles in glass dishes covered with silicone

under locust saline (Clements & May, 1974) (pH = 7.4).

The tympanal nerve was dissected from the ventral side

of the femur up to the femur-tibia joint. It was cut with

iridectomy scissors and placed in a glass capillary

containing the tracer. Preparations were incubated for

48 hours at 4 °C. The cobalt was precipitated with

an aqueous solution of 1% ammonium sulphide

(Fluka, Buchs, Switzerland) during incubation for

10–15 minutes. Given sufficient incubation time, the

tracing method allows staining of the entire set of scolo-

pidial sensilla in the complex tibial organ, including the

auditory sensilla. The legs were then fixed in chilled 4%

paraform aldehyde (Sigma Chemicals, St. Louis, MO,

USA) in phosphate buffer (0.04 M Na2HPO4, 0.00574 M

NaH2PO4 9 2 H2O; pH = 7.4) for 60–120 minutes. The

preparations were briefly rinsed in phosphate buffer,

dehydrated in a graded ethanol series (Carl Roth,

Karlsruhe, Germany) and cleared in methyl salicylate

(Fluka). After clearing, the individual tibiae were cut

open from the dorsal side to allow a view onto the crista

acustica for documentation and counting of auditory

sensilla. The complex tibial organ was examined under

an Olympus BH-2 microscope and photographed with a

Leica DCF-320 digital camera (2088 9 1055 pixels) con-

nected to the microscope. Photographs were adjusted for

contrast and brightness and assembled to panels using

CorelDraw 11 (Corel, Ottawa, Canada).

Statistical analysis

Statistical analysis was carried out with Prism 4 soft-

ware (Graph Pad, San Diego, CA, USA) or R (by Thor-

sten Dickhaus).

Results

Tympana size

Sound can reach the tympanic membrane of tettigoni-

ids via two pathways: (i) directly from the outside or

(ii) from the interior entering the acoustic trachea via

the acoustic spiracle in the thorax (Fig. 1).

All species in this study have open tympana without

cuticular covering, and the anterior tympana were lar-

ger and more slender than the posterior tympana (on

average 7% longer and 3% broader) (Table 1). Thus,

the anterior and posterior tympana differ in shape; the

greatest width in the dorso-ventral axis was 44% of the

length in the proximo-distal axis on the anterior and

47% on the posterior side. This general pattern holds

for all species with the exception of P. elegans: here,

tympana from both sides are nearly equal in length,

being relatively wider than in the other species (50%

width to length ratio on the anterior and 54% on the

posterior side). Because the relationship is very similar

between all four measurements taken from the tym-

pana (R2 = 0.84–0.88), we restrict the remaining analy-

ses to the anterior tympanal length.

The anterior tympana differ profoundly in the abso-

lute dimension of proximo-distal length between spe-

cies, the largest having twice the length (females of

P. ornatus) of the smallest in P. ebneri (Ramme, 1933)

males. This size difference scales with body size, mea-

sured as hind femur length (Table S2; ANCOVA: t-test:

covariate hind femur length: T156 = 2.56, P = 0.011).

Adjusted for hind femur length, the factor species

exhibited a highly significant influence on tympanum

size (ANCOVA: F17,156 = 61.03, P < 0.001). However, a

correlation of the tympana size to the communication

system of the respective species is not evident: similar

tympana sizes occur in bidirectional communicating

species both within the genus Poecilimon and the out-

group species I. modestior, as well as unidirectional com-

municating species (P. propinquus and P. ampliatus

group) (Fig. 2). In the majority of species, females have




Table

1Morphometric

analysisofPoecilimon

,givingthemeanmeasurementdata.Forcomparison,meanvaluesare

calculatedindependentlyforfemale

andmale

individuals.Species

are

groupedinto

therespectivespeciesgroups.

Species

Females

Males

nPronotum

Hindfemur

Spira

cle

Tym

panum

anterio

r

Tym

panum

posterio

r

Sensillae

numbers

nPronotum

Hindfemur

Spira

cle

Tym

panum

anterio

r

Tym

panum

posterio

r

Sensillae

numbers

Outgroup

Isophya

modestior

12

5.05�

0.27

19.96�

0.64

0.68�

0.09

0.94�

0.06

0.90�

0.07

33.83�

1.72(6)

10

4.86�

0.11

20.00�

0.51

0.97�

0.10

0.95�

0.08

0.91�

0.04

33.67�

1.63(6)

Poecilimonornatusgroup

ornatus

78.79�

0.32

23.54�

1.25

1.39�

0.07

1.31�

0.05

1.24�

0.05

37.86�

1.07(7)

88.19�

0.29

21.58�

0.42

1.62�

0.14

1.27�

0.05

1.20�

0.07

38.33�

1.03(6)

hoelzeli

27.05�

0.07

20.85�

0.07

1.53�

0.04

1.25�

0.00

1.13�

0.04

47.38�

0.62

20.70�

0.55

1.38�

0.05

1.10�

0.09

1.06�

0.06

gracilis

65.05�

0.35

17.43�

0.67

1.16�

0.10

0.96�

0.06

0.89�

0.06

34.00�

1.41(4)

44.98�

0.91

16.20�

2.16

1.59�

0.11

0.99�

0.14

1.01�

0.08

Poecilimonjonicusgroup

jonicus

44.58�

0.23

15.25�

0.76

0.89�

0.05

0.73�

0.05

0.70�

0.00

44.23�

0.25

14.25�

0.31

0.95�

0.04

0.75�

0.06

0.74�

0.05

laevissim

us

56.30�

0.10

18.73�

0.64

1.40�

0.13

0.85�

0.13

45.30�

0.20

17.92�

0.49

1.30�

0.09

0.85�

0.09

Poecilimonthoracicusgroup

macedonicus

24.90�

0.28

17.10�

0.57

0.83�

0.04

0.75�

0.07

0.75�

0.07

54.56�

0.27

16.80�

1.10

1.22�

0.12

0.74�

0.08

0.67�

0.04

elegans

84.61�

0.27

16.18�

1.01

0.63�

0.03

0.71�

0.04

0.70�

0.06

31.80�

0.45(5)

64.25�

0.11

14.85�

0.67

0.85�

0.10

0.69�

0.05

0.69�

0.03

31.83�

0.98(6)

Poecilimonpropinquusgroup

chopardi

54.90�

0.14

16.06�

0.30

0.33�

0.04

0.78�

0.04

0.75�

0.07

29.67�

0.58(3)

54.92�

0.18

15.82�

0.45

0.28�

0.03

0.75�

0.05

0.69�

0.05

mariannae

54.92�

0.13

17.80�

0.22

0.29�

0.04

0.80�

0.10

0.73�

0.07

54.92�

0.08

16.36�

0.59

0.29�

0.02

0.76�

0.04

0.70�

0.04

zimmeri

55.46�

0.44

17.06�

0.63

0.39�

0.04

0.90�

0.07

0.92�

0.06

55.16�

0.13

16.24�

0.43

0.43�

0.33

0.94�

0.05

0.87�

0.04

veluchianus

10

5.50�

0.70

17.40�

0.10

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Allmeasurements

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forsensillaecounts

givenin

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larger tympana in absolute size than their males. This

difference in tympana size is also an effect of the larger

body size in females; when measurements of tympana

size were adjusted for hind femur length, sex showed no

significant influence (t-test: T156 = �0.789, P = 0.43).

Acoustic spiracle size

The second entrance to the acoustic system in tettigoni-

ids is via the acoustic spiracle, opening at the posterior

border of the first thorax segment (Figs 1 and 3).

In contrast to most morphological traits, spiracle size

in our cross-species comparison showed no allometric

correlation with hind femur length (ANCOVA covariate

hind femur length: t-test: T172 = 1.24.20, P = 0.21).

After adjusting for body size, the factors species and sex

had a highly significant influence on spiracle size (ANCO-

VA: F31,172 = 318.20, P < 0.001), with sizes 11 times

longer in major axis for the species with the largest spi-

racles (P. ornatus) in comparison with the one with the

smallest (P. intermedius).

Spiracle size was correlated with the communication

system of the respective species and showed clustering

into discrete groups (Fig. 4): the largest spiracles are

consistently found in the bidirectional species, both

within the genus Poecilimon and in the out-group spe-

cies I. modestior. In reversed unidirectional communicat-

ing species, spiracle sizes were distinctively smaller in

both species groups (P. propinquus group and P. amplia-

tus group). Corrected for body size, the spiracle in uni-

directional species is significantly reduced by 27 to 37%

in the P. propinquus group, and 48–51% in the P. ampli-

atus group, compared with species with a bidirectional

communication system (Table S3).

Males have absolutely and relatively larger spiracles

than their conspecific females. After adjustment for

hind femur length, sexual dimorphism in spiracle size

differs between species (ANCOVA interaction species 9

sex: t-test: T172 = 9.45, P < 0.001). Setting the bidirec-

tional singing I. modestior as out-group, the sexual size

dimorphism in favour of relatively larger male spiracles

is similar or even greater (P. gracilis by 16%) than in

I. modestior (Table S3). This male biased larger spiracle

size is not found in the unidirectional species of the

P. propinquus group and the P. ampliatus group.

0.5

0.8

1.1

1.4

11 13 15 17 19 21 23 25

Tym

pana

- an

terio

r, m

ajor

axi

s [m

m]

Hind femur [mm]

Fig. 2 Anterior tympana (= proximo-distal) length scales with

body size, measured as hind femur length, presented as species

means � SD for females (solid symbols) and males (open

symbols). Scaling was independent of the communication system.

Triangles: bidirectional out-group species Isophya modestior,

diamonds: bidirectional Poecilimon species, squares: unidirectional

species of the P. propinquus group, circles: unidirectional species of

the P. ampliatus group.

(a) (b)

Fig. 1 The auditory system in a Poecilimon bushcricket. (a) The main sound input is via the acoustic spiracle (as) located at the thorax,

partly covered in some species by the lateral pronotum. From here, sound travels through the acoustic trachea (at) to the inside of the

tympana (ty), located below the knee of the front leg. (b) In most Phaneropterinae including Poecilimon, the tympana are uncovered and

located on the anterior (aty) and posterior (pty) side of the front leg. Several sensory neurons (sn) form the crista acustica between the

tympana and locate on the acoustic trachea (at).




Relationship of spiracle and tympanal sizes

Based on the two independent analyses for tympanum

and spiracle size, it became evident that these two

structures are not size matched. The tympana scale

with body size (Fig. 2), whereas the spiracles differ

between uni- and bidirectional acoustically communi-

cating species and between sexes (Fig. 4). A clear rela-

tionship between both structures is not suggested, but

spiracles are similar in size to the tympanum in the

out-group species Isophya modestior or even greater than

the tympana in most bidirectional communicating

Poecilimon species. In species with unidirectional com-

munication, the spiracles are much smaller than the

tympana, both in the P. propinquus group and especially

in the P. ampliatus group (Fig. 5).

Neuroanatomy of the auditory organ

In all tettigoniid species investigated, the hearing organ

is located below the femur-tibia-joint of the front leg

and conforms to the general structure of the Tettigonii-

dae ear: the auditory sensilla of the crista acustica are

part of the complex tibial organ, which also contains

the subgenual and intermediate organ (Fig. 6). All

three sensory organs are well developed and similar in

anatomy between species (Fig. 6). The auditory sensilla

of the crista acustica are located between the anterior

and posterior tympanum, with the cell body to the

anterior side, and dendrites pointing to the posterior

side of the leg and then dorsally.

(a) (b)

(c) (d)

Fig. 3 Acoustic spiracles in the

bidirectional out-group species

I. modestior female (a) and male (b) and

the bidirectional Poecilimon ornatus

female (c) and male (d). Measurement

of the spiracle major axis is indicated in

(a); the animal’s head (anterior) is to

the right. All scales: 500 lm.

0

0.3

0.6

0.9

1.2

1.5

1.8

2.1

11 13 15 17 19 21 23 25

Spira

cle

- maj

or a

xis

[mm

]

Hind femur [mm]

Fig. 4 Size of the acoustic spiracle in relation to body size, both

presented as species means � SD for females (solid symbols) and

males (open symbols). Spiracles in the bidirectional species

(triangles: out-group species Isophya modestior, diamonds:

bidirectional Poecilimon species) are consistently larger than in

unidirectional species of the P. propinquus group (squares) and the

P. ampliatus group (circles).




Clear differences between species exist in the num-

bers of sensilla in the crista acustica, ranging from a min-

imum of 17 sensilla on average in P. intermedius females

to a maximum of 38 sensilla in both sexes of P. ornatus

(ANCOVA: F8,51 = 332.90, P < 0.001; Table S4). This large

interspecies difference is not attributable to differences

in body size (ANCOVA covariate hind femur length: t-test:

T51 = �1.29, P = 0.20). Furthermore, the number of

sensilla is identical between sexes (t-test: T51 = �0.36,

P = 0.72). The sensilla number is highest in the three

bidirectional communicating Poecilimon species and the

bidirectional out-group species I. modestior, with aver-

ages ranging from 32 to 38 cells (Fig. 7). Restricting the

analysis to the bidirectional Poecilimon species reveals a

clear correlation between the number of auditory sen-

silla and body size (y = 0.76x + 20.03, R² = 0.96). In

the unidirectional species P. chopardi, belonging to the

P. propinquus group, the number is slightly lower with

29 auditory sensilla. A drastic reduction is found in the

unidirectional species P. ampliatus, with sensilla num-

bers lowered on average to 21 cells. In the parthenoge-

netically reproducing P. intermedius, this number is

further lowered to 16–18 cells. This shift between

the sister species P. ampliatus and P. intermedius is even

larger than what can be explained by body size

differences.

Auditory sensitivity

Auditory thresholds are similar for conspecific females

and males over the entire frequency range, with the

minor exception that males of I. modestior are more sen-

sitive than females at mid-range frequencies between

12 and 20 kHz (pairwise t-test: T8 = 2.248–2.55,P = 0.024–0.043) (Fig. 8a). Although the sexes within a

species were similar, auditory sensitivity differed clearly

between species (Fig. 8). Representatives of those spe-

cies with large spiracles (P. ornatus, I. modestior) have

the highest sensitivity (Fig. 8a, b). Whereas these two

species show the expected correlation of spiracle size

and sensitivity, the tuning curve of P. elegans clearly

deviates from this correlation. This species has moder-

0

0.3

0.6

0.9

1.2

1.5

1.8

0.5 0.8 1.1 1.4

Spira

cle

- maj

or a

xis

[mm

]

Tympana - major axis [mm]

Fig. 5 Spiracle size in correlation with the length of the

tympanum, both presented as species means � SD for females

(solid symbols) and males (open symbols). The line represents

equal lengths for both hearing structures. Out-group species

Isophya modestior (triangles), bidirectional communicating Poecilimon

species (diamonds), unidirectional species of the P. propinquus

group (squares) and the P. ampliatus group (circles).

(a) (b) (c) (d) (e)

Fig. 6 Neuroanatomy of the auditory organ in the front tibia below the knee. CA = crista acustica, SGO = subgenual organ,

IO = intermediate organ, at = anterior tympanum, pt = posterior tympanum. All scales: 100 lm.




ately large spiracles which are only slightly smaller than

in I. modestior. However, hearing is largely reduced in

the low frequencies between 3 and 20 kHz, more so

than in any other species. Furthermore, sensitivity does

not decrease but in fact increases in the ultrasonic

range (Fig. 8c). In P. ampliatus, a representative of a

unidirectional communicating species with small acous-

tic spiracles, hearing thresholds are rather high, mainly

above 50 dB SPL (Fig. 8d).

Discussion

The two acoustic signalling systems studied here

resulted in functionally relevant auditory differences

between species and between sexes within species. By

far the greatest differences exist between bidirectional

and unidirectional communicating species regardless of

song characters. These differences are found for sound

input structures (spiracle size), neuroanatomy (num-

bers of auditory sensilla) and hearing sensitivity (hear-

ing thresholds).

Tympana are important structures for sound percep-

tion in Tettigoniidae (Bangert et al., 1998; Hummel

et al., 2011), and neither the acoustic communication

system nor sex greatly affects their size. In our analysis,

tympana show an allometric relationship, scaling line-

arly with the hind femur length, a marker of overall

body size. However, tympanal dimensions might be

limited by front leg size. Therefore, we can assume that

overall body size might be the evolutionary target, if

tympanal structures come under positive selection. The

second source of sound input in Tettigoniids, the acous-

tic spiracle, leads the sound waves through an acoustic

trachea to the inner side of the tympanum (Michelsen

et al., 1994). Above a critical frequency, the acoustic

trachea and the acoustic vesicle in the thorax amplify

the sound with a gain of 10–20 dB (Heinrich et al.,

1993; Michelsen et al., 1994). Poecilimon species have in

general relatively large acoustic spiracles (Heller, 1984)

compared with other Tettigoniid species (Bailey, 1993),

but size variation was extensive between species stud-

ied here (see Fig. 4). Male songs of the species studied

are rather broad-banded but usually have the main fre-

quency components between 15 and 40 kHz (Heller,

1988).

In bidirectional communicating species, hearing is

essential in both sexes for mate finding. Hence, selec-

tion might favour a high sensitivity in combination

with large sound-receiving structures. The spiracle

should be under especially strong selection pressure to

be enlarged, not only allowing for better hearing

(Nocke, 1975) but also favouring a better detection of

high frequencies (R€omer & Bailey, 1998) which is espe-

cially important for directionality in hearing (Stumpner

& Heller, 1992). Male songs of the species studied are

rather broad-banded and have their main frequency

components between 15 and 40 kHz (Heller, 1988).

Female Poecilimon respond with signals of reduced dura-

tion and intensity compared with the male song

(Stumpner & Heller, 1992; Heller & von Helversen,

1993; Heller et al., 1997b; von Helversen et al., 2001).

Consequently, males should evolve larger spiracles than

their conspecific females. This prediction is fully met, as

bidirectional species have consistently large spiracles,

which are again larger in males than in conspecific

females.

In unidirectional communicating species, the selec-

tion for sensitivity in hearing may be weakened for

males compared to the bidirectional species. After the

loss of female acoustic signalling, male sensitivity and

related morphological structures of the ear are no

longer under selection for detection of the female

response. In line with the hypothesis, we found that

unidirectional species have significantly reduced small

spiracles. However, due to unknown reasons, the

extent of vestigialization differs between species groups:

Within the P. propinquus group, spiracles are reduced in

mean by one-third, whereas members of the P. amplia-

tus group have even smaller spiracles reduced to half

the size of bidirectional species (Fig. 4). For males, this

reduction can be seen as a consequence of relaxed

selection after the loss of the female response, which

requires less sensitive hearing (Stumpner & Heller,

1992). In contrast to the expectations, the small spira-

cles are not restricted to the signalling males but are of

the same size in females. This implies that communica-

tion distances might be restricted, resulting in less

requirements for high auditory sensitivity in both sexes

(Stumpner & Heller, 1992), as previously shown for

Fig. 7 Number of auditory sensilla in the crista acustica in relation

to hind femur length, both presented as species means � SD.

Mean numbers of auditory sensilla are not different between the

sexes; therefore, only data for females are shown, making the

comparison to the parthenogenetic P. intermedius more obvious.

Regression line calculated for bidirectional Poecilimon species. Data

for P. ampliatus and P. intermedius females from Lehmann et al.

(2007).




P. ampliatus (Lehmann et al., 2007). Nonetheless, the

evolutionary sequence of events remains unclear: was

there first a return from bi- to unidirectional communi-

cation leading to reduced spiracles, or, was there an ini-

tial reduction in spiracle size enforcing the loss of

female responses? If spiracles became initially smaller

due to unknown reasons, they may have become inad-

equate for detection of faint female replies, which

would no longer have been effective and therefore

could lead to the loss of the female response songs.

Currently available functional and phylogenetic data

cannot resolve these two scenarios. However, a very

plausible explanation comes from results on the bidirec-

tional P. affinis (Frivaldsky, 1867): experimentally

muted females had reduced mating frequency under

low population densities, but got equal number of mat-

ings in a high-density situation (von Helversen et al.,

2012). Females obviously balance the predation risk

during mate approach (Heller, 1992) against the bene-

fits of receiving the nutritious spermatophore during

mating (Lehmann, 2012). A further evolutionary trig-

ger for reducing spiracle size in high densities might be

noise level reduction for both sexes. Such a scenario is

proposed for the recently discovered P. jablanicensis

Chobanov et Heller, 2010. This species is placed within

a phylogenetic branch of bidirectional species, closely

related to P. gracilis. It occurs in high densities and has

reduced female wings that are no longer able to pro-

duce sound (Chobanov & Heller, 2010).

From a functional perspective, it is astonishing how

flexible spiracle dimensions are in comparison to tymp-

anal dimensions within the genus Poecilimon. As a con-

sequence, no functional correlation was found between

acoustic spiracle and tympanal size. It seems that selec-

tion on tympana size takes routes quite different from

selection on spiracle size. Despite the huge impact of

(a) (b)

(c) (d)

Fig. 8 Auditory thresholds for females (open symbols) and males (solid symbols) (n = 5–8 individuals for the different sexes of the

respective species). (a) The bidirectional out-group species I. modestior, (b) the bidirectional P. ornatus (large body size), (c) the bidirectional

P. elegans (small body size) and (d) the unidirectional P. ampliatus. Threshold differences between sexes were only significant for three

hearing frequencies in I. modestior, indicated by asterisk at the abscissa (t-test: T8 = 2.248–2.55, P = 0.024–0.043). Data for P. ampliatus

females from Lehmann et al. (2007).




intraspecific communication, hearing and therefore spi-

racle size might also be influenced by other factors,

especially predator avoidance (Gerhardt & Huber,

2002). This might limit the reduction of hearing espe-

cially at sonic frequencies, as, for example, silent Podis-

mini grasshoppers have reduced but full functional ears

(Lehmann et al., 2010). Such hearing might allow the

detection of approaching predators, like recognizing the

wing beat of hovering birds (Fournier et al., 2013).

In all species studied here, the neuroanatomical orga-

nization of the crista acustica is similar to those known

from other tettigoniids (Schwabe, 1906; Schumacher,

1979; Lakes & Schikorski, 1990; Strauß et al., 2012)

and related Ensifera taxa (Strauß & Lakes-Harlan, 2008,

2009). Among tettigoniids, there are typically 25–50sensory neurons in the crista acustica, with few known

exceptions having less than 20 sensilla (Schumacher,

1979; Lakes & Schikorski, 1990). As a rule, closely

related species also have highly similar numbers of

sensilla, as shown by intrageneric comparisons

(Schumacher & Houtermans, 1975; Lakes & Schikorski,

1990; Kalmring et al., 1992; Lehmann et al., 2007;

Kowalski & Lakes-Harlan, 2013). In contrast, the range

of sensilla numbers within the genus Poecilimon is far

greater than within other genera studied. During evolu-

tion, a reduction of sensory cells may occur in a sense

organ in a process of sensory specialization (Dangles

et al., 2009). Remarkably, in P. chopardi as a member of

the unidirectional P. propinquus group, the number of

auditory sensilla is – compared with a body-size-

adjusted expectation based on the bidirectional species

– only marginally reduced from the minimum 32 sens-

illae present in P. elegans to 29 neurons. This highlights

the special situation within the P. ampliatus group:

based on phylogenetic interference, sensilla numbers

are secondarily reduced in species of the P. ampliatus

group to 21 sensilla (P. ampliatus) resp. 17 (P. intermedi-

us females) (Lehmann et al., 2007). In contrast, neuro-

sensory elements of tettigoniid ears are rather

conservative in changes over evolutionary time. Only

three other tettigoniid species with such a low sensilla

number of 20 or less are known. In the oak-living

Meconema thalassinum (De Geer, 1773), acoustic com-

munication by tegminal stridulation was replaced by

hindleg substrate drumming (Schumacher, 1979). In

the related Meconema meridionalis (Costa 1860), also a

low number of 15 sensilla are documented (Schumach-

er, 1979). The third species, Phasmodes ranatriformis

(Westwood 1843), also lost acoustic communication and

the auditory tympana as well (Lakes-Harlan et al.,

1991). For these hearing organs, the ancestral neuron

number is unknown, due to a lack of a robust phyloge-

netic hypothesis and comparative neuroanatomical stud-

ies. Therefore, the drastic decrease of sensilla number in

the P. ampliatus group is the first case of a secondary ear

reduction among tettigoniids that is inferred from and

supported by phylogeny. This finding raises the question

why about one-third of auditory sensilla apparently

have been lost in both species. It has been shown that

auditory sensilla in the crista acustica are individually

tuned to a specific frequency of highest sensitivity

(Oldfield, 1982, 1988; Stumpner, 1996; St€olting &

Stumpner, 1998; Udayashankar et al., 2012), although

the sensitivity for specific frequencies may overlap

between sensilla. If auditory input is detectable at a high

intensity, for example, due to high population densities

resulting in short interindividual distances, less sensitiv-

ity is needed to provide adequately strong input to the

central nervous system. In crickets, the sensory input

from low- and high-frequency neurons is modulated by

their synaptic efficacy (Pollack, 1994; Pollack & Faulkes,

1998). It can be speculated that the elimination of sensil-

lae in the P. ampliatus group represents an alternative

way to reduce the sensory input. Apart from decreasing

overall hearing sensitivity, elimination of sensilla might

result in decreased discrimination of sound frequencies

or sound amplitude. However, the further reduction

in P. intermedius compared with its sister species

P. ampliatus is even greater than what can be expected

by allometric scaling with body size in the bidirectional

species. Due to parthenogenetic reproduction (Lehmann

et al., 2011), P. intermedius is no longer subject to stabi-

lizing selection for auditory sensitivity of male calling

songs (Lehmann et al., 2007). Therefore, the reduction

in P. intermedius is the result of the overall reduction in

the P. ampliatus lineage that was further enhanced by

vestigialization of hearing due to the loss of sexual

communication.

For tettigoniids, larger acoustic spiracles are linked to

higher auditory sensitivity especially at higher frequen-

cies as repeatedly shown by blocking experiments

(Lewis, 1974; Nocke, 1975) and comparative studies

(Stumpner & Heller, 1992; Bailey, 1998). For the

unidirectional species P. ampliatus and P. intermedius

(Lehmann et al., 2007) which have very small spiracles,

hearing is restricted, as less sound input reaches the

sensory organ. Our results broadly support the correla-

tion of spiracle size and hearing threshold. Comparing

sexes, bidirectional communicating males have larger

spiracles than females. As expected, this is associated

with better male hearing in the bidirectional I. modestior

above 7 kHz (Fig. 8a). Such an increased higher male

hearing sensitivity has been documented in two South

Asian duetting Phaneropterinae species for frequencies

represented in the female song (Heller et al., 1997a). In

the other bidirectional species, hearing is only slightly

better in male than in female P. elegans or is even

highly similar between the sexes in P. ornatus despite

the 20% larger spiracles in males. This may be due to

differences in the sound propagating auditory trachea

or the auditory vesicles within the prothorax.

Projecting data from the auditory system onto the

molecular phylogeny of Poecilimon (Ullrich et al., 2010)

allows determining the direction of changes during




auditory evolution in this genus. The numbers of audi-

tory sensilla in representative present-day species sug-

gest that a set of around 30 auditory sensilla reflects

the ancestral situation. The reversal to unidirectional

communication is a loss of the female response (Heller,

1990) which is correlated with a drastically reduced size

of the openings for sound input (spiracles) and reduced

hearing, especially in the ultrasonic range. In conclu-

sion, a consistent correlation between audition and the

acoustic communication mode is obvious. These differ-

ences as well as intraspecific differences between sexes

indicate a co-adaptation between audition and the

acoustic communication systems.

Acknowledgments

We are indebted to Reinhard Lakes-Harlan for making

the neurobiological study in his laboratory possible as

well as insightful discussions and comments on the

manuscript. We thank Florin Rutschmann, Z€urich, for

his efforts in collecting fresh P. ebneri and P. chopardi

specimens. Thorsten Dickhaus supported the statistical

analysis. We thank Klaus-Gerhard Heller, Michael Rei-

chert, Mike Ritchie and three anonymous referees for

comments which largely improved the manuscript. The

last author received funding from the German Society

of general and applied Entomology (DGaaE).

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Supporting information

Additional Supporting Information may be found in the

online version of this article:

Table S1 List of individuals studied from the genus

Poecilimon and the out-group genus Isophya.

Table S2 Full model of the ANCOVA for the anterior

tympanum size.

Table S3 Full model of the ANCOVA for spiracle size.

Table S4 Full model of the ANCOVA for the number of

sensilla in the crista acustica.

Data deposited at Dryad: doi:10.5061/dryad.49jg0

Received 1 August 2013; revised 31 October 2013; accepted 1

November 2013




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Sensory evolution of hearing in tettigoniids with differing communication systems