Reviving the sound of a 150-year-old insect: The bioacoustics of Prophalangopsis obscura (Ensifera: Hagloidea).

Determining the acoustic ecology of extinct or rare species is challenging due to the inability to record their acoustic signals or hearing thresholds. Katydids and their relatives (Orthoptera: Ensifera) offer a model for inferring acoustic ecology of extinct and rare species, due to allometric parameters of their sound production organs. Here, the bioacoustics of the orthopteran Prophalangopsis obscura are investigated. This species is one of only eight remaining members of an ancient family with over 90 extinct species that dominated the acoustic landscape of the Jurassic. The species is known from only a single confirmed specimen-the 150-year-old holotype material housed at the London Natural History Museum. Using Laser-Doppler Vibrometry, 3D surface scanning microscopy, and known scaling relationships, it is shown that P. obscura produces a pure-tone song at a frequency of ~4.7 kHz. This frequency range is distinct but comparable to the calls of Jurassic relatives, suggesting a limitation of early acoustic signals in insects to sonic frequencies (<20 kHz). The acoustic ecology and importance of this species in understanding ensiferan evolution, is discussed.

Introduction

Acoustic communication systems have long been a popular model for understanding ecological and evolutionary relationships within and between species. In insects, acoustic systems for both signal generation and recognition have evolved a substantial variety of forms, to facilitate a range of communication functions [1, 2], offering many routes to studying the evolution of acoustic communication. However, for extinct and rare insect species, we are often limited in our abilities to infer details of specific communication systems, as we are unable to record the sounds such species generate or measure their hearing capabilities.

In katydids (or bush-crickets; Orthoptera: Ensifera) and their allies, pure-tone and broadband sound production has evolved as a key mechanism for mate attraction and conspecific recognition [3-6]. These sounds are produced by tegminal stridulation–the process of moving a hardened scraper on one forewing, against a row of teeth (the file) on the other, producing vibrations on the wing which are then amplified by specialized wing cells (namely the harp and mirror) [4, 5] to radiate sound. This mechanism of sound production is evolutionarily conserved across a majority of the Ensifera, and its characteristics have been understood since the early 1900s [7, 8]. The retention of this mechanism across a diverse range of taxa, and the increasing ability of state-of-the-art imaging and acoustic technologies, is rapidly allowing researchers to re-visit once inaccessible specimens with novel methodologies to advance our understanding of Ensiferan acoustic communication.

Here we investigate the bioacoustics of Prophalangopsis obscura (Walker, 1869) (Ensifera: Prophalangopsidae), an insect belonging to an ancient katydid family of over 90 known species dominant during the Jurassic, with only eight extant members [9]. The genus Prophalangopsis, formerly Tarraga, has remained monotypic ever since the discovery of P. obscura, and thus received considerable interest in relation to the evolutionary history of the Ensifera [10-12]. The enigmatic nature of the type specimen has been compounded by no further male specimens being discovered in over 150 years, and only 2 potential female specimens ever found [13]. In addition, no works have explored the ecology of this species due to their uncertain geographic distribution [13, 14]. Therefore, a thorough study of their acoustic capabilities could improve our understanding of the communication systems and acoustic ecology of P. obscura and its long extinct relatives [15-17], and aid in future rediscovery of the species.

Using micro-scanning Laser-Doppler Vibrometry (LDV), we reconstruct the vibration patterns and resonances of the sound production organs (forewings) of the P. obscura type specimen. Furthermore, we investigate the morphology of the stridulatory apparatus and tegmina in detail to compliment LDV experiments and infer the likely carrier frequency (fc) of this species’ song over 150 years after specimen preservation. Employing existing validated models, and novel measurements from LDV, we obtain fc for the acoustic signal of P. obscura and use morphological data to calculate acoustic signal structure. Using knowledge of the wing biomechanics of other extant members of this ancient family, we reconstruct the calling song of P. obscura, and discuss the importance of this species in understanding the evolution of ensiferan acoustic communication.

Materials and methods

The holotype material

Prophalangopsis obscura (Walker 1869) is a large orthopteran (~10 cm; tegmina wingspan) represented by a single specimen housed at the London Natural History Museum, South Kensington, UK (specimen NHMUK 013806185). Collection details are scarce, with the location information listed only as ‘India’. The specimen was originally set in a resting position following collection, but sometime between 1898 and 1939, the specimen was re-mounted with both wings spread [10], a position which remains to this day (Fig 1). At an unknown time after 1939, the left foretibia was lost. The right foretibia, which contains the tympanic ear, remains intact (Fig 1C). Both forewings are present, with the stridulatory (sound producing) organs intact, however the left wing is torn along the apical axis (Fig 1A). In 2005, two female specimens identified as P. obscura were located in China, later published by Liu et al. [13]. While male specimens were not identified to confirm the identity of these specimens, they minimally belong to a close relative of P. obscura. No permits were required for the described study, which complied with all relevant regulations.

Fig 1

The holotype of Prophalangopsis obscura (collected in India, Walker 1869).

A, dorsal habitus; B, lateral habitus; C, tympanal organ.

Tegmina and stridulatory file anatomy

P. obscura possesses a stridulatory file on each forewing. The morphology of each file was imaged using an Alicona InfiniteFocus microscope (Bruker Alicona Imaging, Graz, Austria) at 20x objective magnification, resulting in one composite 3D-image of each file with a vertical and horizontal resolution of 0.7 and 7.8 μm, respectively. Using the built-in Alicona software, the length of the stridulatory file was measured, as well as the spacing between stridulatory teeth (inter-tooth distances), and length of each tooth. The inter-tooth distance was measured as the distance between the central tip (cusp) of adjacent teeth.

Forewing resonance and deflection pattern

The resonant frequency (f) and deflection patterns of the forewings was measured in the holotype of P. obscura using micro-scanning LDV (PSV-500, Polytec GmbH, Waldbronn, Germany), with approximately 1000 measuring points at a sampling frequency of 256 kHz. Acoustic signals for wing excitation were generated by the LDV internal data acquisition board (PCI-4451; National Instruments, Austin, TX, USA), and consisted of broadband periodic chirps ranging from 1 to 60 kHz at 60 dB SPL (re 20 μPa). The signal was amplified by a Pioneer A-400 amplifier (Pioneer, Kawasaki, Japan) and transmitted to a loudspeaker (Vifa, Avisoft Bioacoustics, Glienicke, Germany; flattened frequency response across the whole range) positioned 20 cm in front of the specimen. A reference signal to calculate the transfer function between the wing vibration and the stimulus was recorded using a 1/8” condenser microphone positioned horizontally at the wing plane between the wings (Model 4138-A015, Brüel & Kjaer, Nærum, Denmark). For further details of method, see [18].

Reconstruction of the sound

To reconstruct the sound of P. obscura, several characteristics of the acoustic signal are required. These are (1) the song carrier frequency (fc), (2) the decay of a single stridulatory tooth strike, (3) the number of oscillations produced during each stridulatory file strike (one full sound pulse), and (4) the timing between stridulatory file strikes. Previous investigations into the morphological parameters of katydid stridulatory apparatus have shown that the best predictors of the fc are the regions of mechanical displacement of the tegmina (the acoustically active wing cells), and the length of the stridulatory file [7]. In hagloids (Haglidae and Prophalangopsidae), which lack a specialised mirror area, it has been suggested that measurements of file length and LDV resonance, or the entire vibrating area, will be better predictors of fc [7, 8]. To predict fc, we used existing models [7] to compare the frequency derived from the file length, right tegmen vibrational area, left tegmen vibrational area, and resonance from vibrometry.

Following calculation of the mean fc predicted by these four techniques, an artificial impulse of a single tooth strike of P. obscura was produced at this frequency, including a decay caused by damping. Oscillations of the tegmina mirror cells usually exhibit a free decay of 3–4 ms in species communicating at the determined fc [3, 11, 19], thus a 4 ms exponential decay was used.

Members of the Prophalangopsidae have a high stridulatory tooth density and short functional file length, which permits the generation of uniquely pure-tone calls [15, 20, 21], and as pure-tone singing katydids display a 1: 1 relation between tooth strikes and the number of oscillations in the song pulse [7], we used fc, the number of functional stridulatory teeth, and the spacing of the teeth, to infer the pulse structure of the song of P. obscura. This was performed using a custom written Matlab code [15] which calculates the instantaneous period for each tooth impact based on the inter-tooth distance measurements. The resulting representative waveform of the acoustic signal of P. obscura was further analysed using the Signal Processing Toolbox in Matlab (R2021a, The MathWorks Inc., Natick, USA) with the following spectrogram parameters: FFT size 512, Hamming window, 50% overlap; frequency resolution: 512 Hz, temporal resolution: 0.15 ms.

Results

The anatomy of the tegmina (forewings) of P. obscura is similar to those observed in both extant and extinct relatives of the Prophalangopsidae. The left wing (LW) and right wing (RW) display stridulatory files that are similar enough to be considered functionally symmetrical. The pattern of tooth distribution is slightly gaussian (Fig 2), suggesting the file could be adapted for sound production during the opening or closing phase of the wings. File length and number of teeth of LW and RW file were 9.60 and 9.99 mm and 134 and 137 teeth, respectively. The inter-tooth distances, tooth lengths, and plectra are symmetrical, suggesting both might have been capable of producing sound pulses (S1 Table).

Fig 2

Stridulatory file anatomy and inter-tooth distances in Prophalangopsis obscura.

Orientation of both files is along the anal (left) to basal (right) axis.

Despite over 150 years of preservation, it was possible to obtain the deflection (vibratory) pattern of the forewings and f in P. obscura. An assessment of the regions of the wings theoretically involved in resonant sound production and the displacement of the wings in response to an acoustic stimulus (Fig 3A and 3D) confirmed that the mirror and pre-mirror are the most likely regions for sound production in this species, as with all extant members of this family [21]. Displacement was highest within the mirror area for both the LW (Fig 3B) and RW (Fig 3E). The normalised displacement amplitudes of the mirror area of the LW displayed a peak frequency at 6.3 kHz (Fig 3C). However, despite morphological symmetry of the wings, the RW displayed a peak of 4.8 kHz (Fig 3F).

Fig 3

Forewing resonance in Prophalangopsis obscura.

(A) Displacement map of the LW; (B) Deflection pattern of the white profile line in A; (C) Frequency spectrum of the left mirror; (D) Displacement map of the RW; (E) Deflection pattern of the profile line in D; (F) Frequency spectrum of the right mirror; (G) Angled view of the left forewing displacement pattern at 4.8 kHz.

Using fo, stridulatory file length, and vibrational areas of the tegmina resulting from LDV deflection measurements, fc was calculated (Table 1). Based on phylogenetically controlled linear models of several measurement parameters [7], we believe the fc to be ~ 4.7 ± 0.05 kHz (Table 1). The measurements of inter-tooth distances and fc allowed the calculation of a time vector of a single sound pulse of the species’ acoustic signal (For more details of the song reconstruction method, see [15]).

Table 1

Model measurement parameters for calculation of the likely carrier frequency (fc) of Prophalangopsis obscura.

Measurement parameter	Measurement (x)	Slope (m)	Intercept (c)	fc (kHz)
File length (mm)	9.6	-0.97	3.74	4.693
RW vibrating area (mm2)	45.31	-0.62	3.91	4.691
LW vibrating area (mm2)	39.05	-0.54	3.53	4.716
LDV resonance (kHz)				4.800
Average				4.725

For all estimates of fc: ln(fc) = m * ln(x) + c, where ln = natural logarithm.

The Matlab script for sound pulse reconstruction [15] revealed that the structure of a single call pulse (Fig 4) is similar to that of fossil relatives of the same family [15], but differs in frequency and duration (Fig 4). The duration of a single pulse was found to be 42 ms (Fig 4), which is very close to the predicted pulse duration from functional file length using an existing model (40.78 ms; Montealegre-Z et al. 2017). Surprisingly, a slight frequency modulation in each chirp of the call was observed (Fig 4). Extant species display similar modulations as a result of changing velocity over the course of each wing stroke. The first predictions of how crickets produced their sounds looked at tooth distribution to infer whether sounds are produced during opening or closing of the wings [22]. As the frequency of the sound is a function of tooth strikes per time period, any changes to wing velocity over the course of one wing stroke will cause frequency modulation in the sound. Looking at the almost gaussian distribution of seemingly functional teeth in P. obscura, we cannot confirm whether this species is able to stridulate during the opening or closing of the wings, or both. The final reconstruction of the sound (Fig 4; S1 Audio) therefore consists of a putative diplo-syllable containing two pulses with every other chirp artificially reversed, to leave this element of the reconstruction open for future interpretations.

Fig 4

Reconstruction and spectral analysis of a diplo-syllable containing two pulses of the sound of Prophalangopsis obscura.

Waveform of two chirps (top), with spectrogram below and frequency spectrum on the left marginal axis. The 2nd chirp is an artificial reversal of the 1st chirp, to demonstrate that frequency modulation (FM) will differ depending on whether sound is produced during the opening or closing wing stroke.

Discussion

Using LDV techniques, we were able to obtain the deflection pattern of the tegmina of P. obscura and use information on tegmina and stridulatory file anatomy to reconstruct the song of the 150-year-old preserved museum specimen. The anatomy of the tegmina and stridulatory file display similarities to both fossil and extant prophalangopsids [15, 16, 21], and this similarity is also represented in the frequency and structure of the song (Fig 4), although fc here is lower than that of related fossil species [15]. The resonant frequency (fo) of the tegmina provided by the LDV recordings matched the expected frequency from the models, and we were able to obtain the area of deflection, which was also used to calculate the potential calling song frequency (Table 1). Despite the matching frequency information provided by the right tegmen, the left tegmen did not predict a similar fc. We believe this discrepancy is due to a tear down the apical axis of the LW, given the similarities of the frequency predicted by the models to the resonance of the right tegmen (Table 1). In many singing ensiferans, the LW is found to be a better predictor of fc [7], however in the prophalangopsidae, it is known that the wings are functionally symmetrical [7], so we can be confident that the RW resonance is representative of the LW resonance. The retention of resonance in the RW may seem surprising, as insect cuticles become stiffer as they desiccate over time [23], and thus we may expect such stiffening to result in changes to resonance. However, in this case, we believe that the topology of the wings plays more of an important role in fo, and due to the size of the tegmina, the effect of drying is not so pronounced. The thickness and area of the tegmina dictate the resonant properties of the musical areas of the wing, and larger musical areas display less variation in frequency response with changes to thickness [24]. For example, in the gryllid Tarbinskiellus portentosus with a harp size of ~25 mm2, tegmina thickness would need to decrease by more than 30% before thickness would begin to greatly modify resonance [24]. Thus, for a large species like P. obscura which has a harp size of ~50 mm2, even a significant change in tegmina thickness from desiccation would be unlikely to result in large changes to resonance, explaining why the resonance is here maintained. Nonetheless, further studies into the effects of wing thickness and tissue desiccation on tegmina resonance across orthopterans would offer a rich dataset for future works to calculate taxon-specific frequency changes over time, increasing the information we can obtain from dry museum specimens.

Just like the other extant members of this family, and unlike modern katydids (Tettigoniidae), P. obscura is likely capable of using both wings for singing, with both tegmina possessing symmetrical stridulatory files, plectra, and acoustically functional areas [15, 16, 21]. The mirror region of the tegmina displayed the greatest deflection, and the pattern of deflection followed that of extant relatives [21]. However, as suggested by Zeuner [10], the tegmina are not as specialised for sound production as other closely related extant species such as the great Grig Cyphoderris monstrosa. The size and function of the wings is one of the key features of P. obscura that separates it from the other extant prophalangopsids and resembles the specimens of the fossil record [10, 15]. While all other extant prophalangopsids (e.g. Cyphoderris spp.) are flightless and use their wings exclusively for sound production and mate attraction/gifting [25], P. obscura has wings potentially large enough for short or sustained flight, resembling both the extinct prophalangopsids and many tettigoniids.

Reduced flight is a well-established evolutionary mechanism to reduce or avoid predation by aerial predators, and in particular, bats [26]. The other extant species in this family, all of whom have lost the ability to fly, exhibit novel anti-predator defences, namely ultrasonic sound production organs [27], which likely evolved to act as a deterrent to a new host of predators they now face after switching to a terrestrial lifestyle. Such anti-predator adaptations are not present in P. obscura, nor are any other morphological adaptations associated with predation by bats such as enlarged cuticular spines [26]. We may predict therefore that this species lives in a region with reduced selection pressure from ultrasonic aerial predators, allowing it to retain the Jurassic form even after the emergence of echolocating bats [28]. Similarly, low frequency calling songs such as that of P. obscura are indicative of reduced pressures from eavesdropping predators, as low frequency sounds travel larger distances and could give away the location of the signaller [29]. Tettigoniids regularly predated by bats benefit from the increased attenuation of ultrasonic conspecific signals by a reduced detection range by eavesdropping predators [26]. However, it should be noted that correlating call frequency to ecology in such a manner does not consider other factors which will be driving call frequency evolution [26, 29].

Unfortunately, further inferences on natural history remain challenging as the precise origins of the type specimen remain obscure. Previous literature on the specimen references a wide geographic area broadly synonymous with the extent of the former British India at the time of collection (e.g. Hindustan, E. Indies). The combined historical evidence suggests that the specimen was collected in northern India, although it is at present not possible to give a more precise location. If the female specimen described in Liu et al. [13] are confirmed to be P. obscura and not a closely related species, then the known range may be extended from northern India to include Tibet, a region certainly too cold to support an abundance of echolocating bats. Further collections from this area to confirm the association between males and females, and to investigate the local composition of potential predators, would be very valuable.

Following this song reconstruction, it may be plausible to deploy autonomous recording units (ARUs) into potential field sites and use signal detection algorithms to aid in the rediscovery of this species [30, 31]. We hope that in time, further specimens of Prophalangopsis obscura are located, to record the true song of this elusive species, and to validate the accuracy of the predictions presented here.

Morphological characters of the tegmina stridulatory files of P. obscura.

(DOCX)

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The reconstructed calling song of Prophalangopsis obscura.

(WAV)

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Visual reconstruction of Prophalangopsis obscura on a tree branch in a temperate montane habitat.

Illustrated by CW.

(JPG)

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24 Mar 2022

5 May 2022

Line 29: “…the bioacoustics of the orthopteran Prophalangopsis obscura are investigated.” Acoustics is treated as singular only when referring to the field of research in general.

Response: Modified as suggested.

Lines 35-36: The frequency range of extinct species is only reconstructed using similar models. Therefore, any comparison is confounded by the method itself.

Response: Here we state the frequency range is similar to Jurassic extinct relatives. This comparison is brought about by the fact that ‘modern’ ensiferans (i.e. Tettigoniidae) are often communicating at frequencies exceeding the ultrasonic range, whereas the Jurassic species and P. obscura are using lower frequency signals like their extant hagloid relatives. We appreciate the concern that the model may produce similarities in the signals it produces, however the model used is an improved version of the one with which we compare (Gu et al., 2012), following Montealegre et al., 2017, for which a reconstruction such as this has not yet been produced. For both models, the frequency produced is very sensitive to the geometry and anatomy of the tegmina, and in this instance, the forewings are just very similar in shape and size to known Jurassic fossils, and so the frequency produced by P. obscura (4.7 kHz) is close to, for example, the Jurassic species A. musicus (6.4 kHz; Gu et al., 2012). The anatomy of the stridulatory file is similar in length to the fossil example, which is the most accurate predictor of calling song frequency (Montealegre-Z et al., 2017), and explains why the frequencies are similar. However these two frequencies are still very distinct, and if the model were to be repeated on an ultrasonic katydid for example, it would produce a very different signal. Future works in development by the senior author confirm that the model can be used to obtain a wide variety of calling signals from fossil and extant data, which will help to reassure readers that the model does not produce intrinsic similarities which render the discussions inappropriate. We have re-worded the comparisons to existing species to highlight more generally that this species is limited to the sonic range, and added ideas in the discussion r.e. the discovery of further fossil species to validate the sensitivity and potential problems of the model.

Lines 72-74: The authors should mention the relatively recent discovery of possibly new specimens – even if only females; the relevant study is cited in the next sentence, but for a different reason altogether ([14] Liu et al. 2009).

Response: Clarified here that there have been no further male specimens found, and that there are 2 female specimens that have been identified as P. obscura in the mentioned study. Also expanded as suggested in the discussion.

Line 82: Bethoux (2012; ref. 13) in his Pl. 1CD in the appendix shows the results of exactly the same analysis – and the figure is basically identical to Fig. 2 in this manuscript. Therefore, it is not clear why you made this same effort a second time.

Response: We decided to assess the wing topology based on the advancements of the field since the study of Bethoux in 2012, and to permit student training/understanding of the anatomy. We agree that the anatomy does not differ from that of Bethoux 2012, and thus we have also moved the wing description figure (Figure 2) into the supplemental materials.

Line 83: The part after the semicolon is not a full sentence.

Response: Semicolon removed.

Line 96: The description of two females from China (caught 2005) by Liu et al. (2009) as Prophalangopsis obscura should be mentioned here. Even though one may claim that without a described male species identity is not demonstrated, these females in any case would belong to a very close relative.

Response: We have included in this section the suggested information. We agree the specimens identified by Liu et al, if not P. obscura, are certainly a close relative.

Lines 120-121: see comment to line 82: Bethoux did not just define conventions but shows an identical reconstruction.

Response: We have moved the reconstruction to the supplemental material as it does not add any novel information on tegminal anatomy. This sentence has been removed from the methodology.

Lines 158-159: One could shortly mention the relevant arguments for that for the reader’s ease.

Response: Relevant arguments briefly added here as suggested to provide context for the reader.

Paragraph 188 – 198: Measuring resonances of a completely dry wing is not what usually is done. The potential effects are not really discussed. While it obviously is not possible to make controls with P. obscura, one could rather easily make a kind of control with a long-winged bush cricket like Mecopoda elongata, which is reared in many labs. Also this species has low frequency components in its song and seeing whether and how the wing changes its resonances after drying would support (or contradict) arguments that the effect is rather minor. If you prefer a species with pure tone, then use a cricket like Gryllus or Teleogryllus. See your own discussion in lines 267-270.

Response: We agree that a model to account for the changes in resonances of fresh vs. dry wings would expand this study into further assessments of museum specimens, and this is something that we are experimentally investigating currently as part of a different study. However any changes to wing resonance as a result of drying will be very dependent on topology, thickness, and size of the wings (Godthi et al., 2022), and thus a species like Mecopoda would dry in a different way to P. obscura, as the mirror region in Mecopoda is much smaller, more specialised, and thinner compared to the rest of the wing, whereas P. obscura does not demonstrate the same level of specialisation of the forewings for sound production. The thickness and area of the tegmina dictate the resonant properties of the musical areas of the wing, and larger musical areas display less variation in frequency response with changes to thickness (Godthi et al., 2022). For example, in the gryllid Tarbinskiellus portentosus with a harp size of ~25 mm2, tegmina thickness would need to decrease by more than 30% before thickness would begin to greatly modify resonance (Godthi et al., 2022). Thus, for a large species like P. obscura which has a harp size of ~50 mm2, even a significant change in tegmina thickness from desiccation would be unlikely to result in large changes to resonance. At this scale, tegmina size and topology is the key driver of resonance.

We believe from the information above and the matching of the wing resonance with the frequency suggested by the other methods used in the paper that the resonance has been maintained. We have expanded upon this validation in the discussion, but highlighted that for application of this method into the diverse world of museum collections requires generation of thorough validated models such as those by Godthi et al (2022) to reconstruct resonances across species.

Lines 221-222: already detailed in Methods.

Response: Sentences removed so not to repeat methodology unnecessarily.

Lines 261-262: That the LW is damaged is quite unfortunate, since usually the LW gave a better prediction than the RW ([7] Montealegre-Z et al. 2017). One should at least mention this here, since peak frequencies of LW and RW differ clearly. That RW resonance fits better to the expectations alone is a risky argument.

Response: The LW is a better predictor of calling song frequency in many ensiferans, which is mostly a result of the specialisation of the wings, whereby the left wing becomes the sound radiator and the right wing the scraper for more efficient sound radiation, such as in the Tettigoniidae, and the wings are restricted to a ‘left wing over right wing’ arrangement. However in the Haglidae, the wings and their resonances are symmetrical, and these species can switch between singing with their tegmina left over right, or right over left. In other extant hagloids in the mentioned paper, it is shown that sound frequency is dependent on the size of the vibrating area, and file which is symmetrical across wings. Thus, if the LW of P. obscura was not damaged, it would very likely suggest the same frequency information, particularly given that the LW vibrating area also predicted a similar frequency (Table 1).

Line 280: If your sentence is true, it means that you question whether the species described by Liu et al. (2009) is even a prophalangopsid. Is that what you want to say? What are your arguments?

Response: This section has been reworded during addressing of the next comment. We clarify that the specimens of Liu et al are either P. obscura, or a close relative.

Lines 291-294: Solely relying on the “objective similarities between the external foretibial ear” without data is too speculative to suggest any functional similarities. Especially as it is not even clear which similarities the authors are referring to. Many bush cricket ears with open tympana look similar. The whole paragraph from line 284 on is rather general and speculative and should be shortened and merged with the following paragraph which mentions what is most desirable to get a better picture: find males of the females described from China.

Response: We have removed speculative comments of comparisons in foretibial ear anatomy and shortened the paragraph as suggested. We believe that our inferences of the acoustic ecology of P. obscura, in particular in relation to predation pressures by bats, are valid hypotheses to remain in the discussion.

Fig. 1: Consider using higher resolution photos.

Response: New higher resolution images used for this figure. Panels showing stridulatory file have been removed, as these are shown in figure 2.

Fig. 2: see above; identical to Bethoux (2012) Pl. 1CD; therefore, delimiting mirror, harp (and pre-mirror, if wished) in the wing in Fig. 4D (which could be shown larger in the foto) would be sufficient.

Response: Figure removed from main text and moved to supplementary materials. Areas of the wing added to figure 4 as suggested.

Fig. 2 could go to supplementary materials.

Figure removed from main text and moved to supplementary materials.

Reviewer #2: This interesting MS reconstruct the sound of a rare and possibly extinct ensiferan by anatomical and biophysical measurements and models of the sound producing structures, finding that the call frequency fundamental is around 4.5 kHz. I think this is a well-conducted experiment and only have a few minor suggestions.

Response: I suggest that you state the size of the animal somewhere in the text, although the reader can measure if from figure 1.

Specimen size briefly mentioned in the holotype material section of methods. As the specimen appears to have been eviscerated, which makes inferences of natural head to abdomen length challenging, we have reported wingspan as an indicator of size.

l. 146-148: I was a bit confused by this description: are you stating that the species here does not have a clearly defined mirror (although it is shown as a fairly well-defined area on fig 2) - btw you have not really defined what the mirror is at this stage in the MS.

Response: To clarify, we mean that although the region classed as a mirror based on venation is easy to identify, it is not as specialised for sound production as in the case of other ensiferan orthopterans. Clarity added to this section. Figure 2 has been moved to supplementary materials upon recommendation of reviewer 1.

Terminology for parts of the wing including mirror have been added to the introduction so the reader will be familiar with the term before this section.

l. 296-297: I think this sentence is unclear, suggest rephrasing as: 'allowing it to retain the Jurassic form even after the emergence of bats'

Response: Modified as suggested.

l. 302-304: The call frequency seems to be similar to the frequency of crickets, and it is also roughly the same size - are there similarities in lifestyles?

Response: The similarities in calling song frequency here definitely offers insights into predation pressures and ecology across the Ensifera. For example, crickets, as low frequency ground dwelling orthopterans, are under different predation pressures to katydids, which are often in vegetation and utilize much higher sound frequencies for communication. High frequency hearing and singing in katydids likely co-evolved (Song et al., 2020), maybe in response to the emergence of echolocating bats and the need to hear ultrasonic signals (Pulver et al., in review). Crickets on the other hand, not facing the predation pressure of bats due to their terrestrial lifestyle, have not faced such pressures to communicate at very high frequencies (although the Lebinthine crickets complicate the story). We may argue that this provides further evidence that P. obscura is not predated by bats, because as a large orthopteran clearly capable of flight, likely with a semi-arboreal lifestyle, we do not see adaptations for ultrasonic communication. However this argument is limited by a lack of information on hearing ranges in this species, and the presence of large, low frequency singing modern katydids such as Mecopoda, which are predated by bats. For the question you ask on similarities in the calling songs of crickets and P. obscura however, I believe this does not suggest similar lifestyles, just based on observations of the disparities in the lifestyles of similarly sized katydids and crickets.

Submitted filename: P_obscura_comments_to_reviewers.docx

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13 Jun 2022

Reviving the sound of a 150-year-old insect: the bioacoustics of Prophalangopsis obscura (Ensifera: Hagloidea)

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Academic Editor

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One author also had a very minor edit which you might want to incorporate.

Reviewers' comments:

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Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Thank you for addressing all our comments and concerns adequately and explaining all the subjects in detail!

Reviewer #2: I think the authors have responded adequately to the suggestions by me and the other referee.

Just one minor clarification:

Methods, l 103 states that: 'The right foretibia, which contains the tympanic ear, remains intact'

suggest to change to: 'The right foretibia, which contains the right tympanic ear, remains intact'

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Reviewer #1: No

Reviewer #2: No

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17 Jun 2022

PONE-D-22-01070R1

Reviving the sound of a 150-year-old insect: the bioacoustics of Prophalangopsis obscura (Ensifera: Hagloidea)

Dear Dr. Montealegre-Z:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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on behalf of

Dr. Vivek Nityananda

Academic Editor

PLOS ONE