NORTH-WESTERN JOURNAL OF ZOOLOGY 16 (2): 204-210 ©NWJZ, Oradea, Romania, 2020 Article No.: e201701 http://biozoojournals.ro/nwjz/index.html

Comparative analysis of the echolocation calls of the lesser horseshoe (Rhinolophus hipposideros) and the Mediterranean (Rhinolophus euryale) in the Carpathian Basin

Dorottya GYŐRÖSSY1,*, Krisztina GYŐRÖSSY2 and Péter ESTÓK2,3

1. University of Veterinary Medicine Budapest, Department of Ecology, 1077 Budapest, Rottenbiller u. 50., Hungary 2. Eszterházy Károly University, Department of Zoology, 3300 Eger, Eszterházy tér 1., Hungary 3. Bükk Mammalogical Society, 3300 Eger, Maklári u. 77/A, Hungary * Corresponding author, D. Győrössy, E-mail: [email protected]

Received: 01. December 2019 / Accepted: 29. April 2020 / Available online: 30. April 2020 / Printed: December 2020

Abstract. Although acoustic data collection is a widespread method to identify , it is not always adequate for precise species identification. The lesser horseshoe bat and the Mediterranean horseshoe bat have overlapping frequency intervals and the degree of this overlap has not been examined in the Carpathian Basin yet. Calls of the two species were recorded and analyzed in order to develop an acoustic identification key based on Hungarian sound database. Our results suggest that there is a considerable overlap between the frequency intervals (101.36–105.20 kHz) of both species, which questions the reliability of the previously used acoustic taxonomic keys. During our research we also examined the connections between sound parameters and the distinct traits of the individuals. We found that sex had a significant effect on the value of the mean frequency in the lesser horseshoe bat.

Key words: Rhinolophus hipposideros, Rhinolophus euryale, acoustic identification, frequency overlap, Doppler, bioacoustics.

Introduction body size and the height of the emitted frequencies of bats (Heller & Helversen 1989, Jacobs et al. 2007, Dietz et al. 2009) Bats have outstanding acoustic signals among . Re- our prediction was that larger horseshoe bats experience cording and analysing bat calls can provide valuable infor- lower frequencies. mation about these protected and vulnerable . Horseshoe bats (Chiroptera: Rhinolophidae) are mainly found in the tropical regions, although 15 species are present Material and Methods in the temperate zone as well. Out of the three species that Data collection occured in seven sampling areas of the Bükk Moun- occur in Hungary, the greater horsehoe bat (Rhinolophus fer- tains and the Aggtelek karst (Figure 1). We examined bats that were rumequinum, Schreber, 1774) uses a distinct frequency range captured in the close proximity of caves during the fall and summer that allows an easy acoustic identification (Heller & Helv- of 2013, 2016 and 2017. The nets were set up at the front of the caves ersen 1989, Siemers et al. 2005, Dietz 2009, Barataud 2015, before sunset. We identified the sex of the captured animals and Hackett et al. 2017). Our research focused particularly on the measured the forearm length as well as their body mass (Table 1). lesser horseshoe bat (Rhinolophus hipposideros, Bechstein, Then, we recorded their calls with the Pettersson D500X ultrasound

1800) and the Mediterranean horseshoe bat (Rhinolophus eu- bat detector. If the area and the structure of the forest allowed it, we set up a flight tent where the bats could and we could record their ryale, Blasius, 1853) because of the ambiguity of their acous- calls, otherwise the animals were hand-released close to the detector. th tic identification. In the second half of the 20 century, there Rhinolophus hipposideros is protected and Rhinolophus euryale is a has been a notable decrease in the European populations of strictly protected species by the law in Hungary. Due to this, the cap- these two species, some of which have become extinct. One ture and sample collection were carried out under license given by of the greatest problems is that many of their natural feeding the Hungarian authorities (permits No. OKTF-KP/3503-31/2016). ceased to exist (Stebbings 1988). As a result, data The aim of our research was to differentiate between these two collection in the field is highly important regarding conser- species based on the recordings of flying individuals. We also con- sidered the consequences of the Doppler effect which means that the vation of these species. The frequency intervals of the two height of vocal impulses changes. Thus we focused mainly on the species overlap (Dietz et al. 2009) and the degree of this analysis of calls of animals during their flight. However, we also ex- overlap has not been examined in the Carpathian Basin yet. amined the resting frequency of bats as supported in previous stud- Data from other geographical areas are not completely ade- ies (Neuweiler 1984, Jones et al. 1992, Siemers et al. 2005, Jiang et al. quate for our bat populations because their sound repertoire 2010, Puechmaille et al. 2014, Shahabi et al. 2019). The value of the presumably differs significantly in the various regions, as it body condition index (BCI) was also calculated, BCI = body has also been examined in the case of other bat species (Chen mass/forearm length, g/mm, according to Russo et al. (2001). et al. 2009, Jacobs et al. 2017). The aim of our research was to Based on the characteristics of their calls, horseshoe bats belong to the FM-CF-FM call type category (Schnitzler 1968, Siemers & develop an acoustic identification key based on Hungarian Schnitzler 2004). Their calls have a so-called first harmonic part sound database. We assumed that the threshold limit of the which is weaker than the second harmonic part that regularly con- overlap would be in a lower frequency than that of the for- tains the maximum energy of the sound (Neuweiler, 1984) (Figure 2). eign published data because we encountered such low calls We used Adobe Audition software for analysis. In the case of (below 102 kHz) during our previous field works that were flying frequencies on average 39 CF parts (from the second harmonic certainly from lesser horseshoe bats. We aimed at determin- part of the calls) and 19 FM parts (from the first harmonic part of the ing the acoustic traits and body parameters of these animals. calls) were measured from one recording. We measured the peak frequencies of the CF and FM parts. We aimed at selecting a period

As we know that there is a negative correlation between

Bioacoustic research on horseshoe bats 205

Figure 1. The locations of the sampling sites in Hungary: Aggtelek karst: 1, Bükk Mountains 2-7. The map was created using QGIS (QGIS Development Team, 2009, URL http://qgis.org).

Figure 2. Sonogram of the calls of Rhinolophus hipposideros, recorded in the Bükk Mountains.

of the emitted sound that surely contained the minimum and maxi- kHz) that constitutes only 0.04% of all measured CF parts of mum frequencies of the given individual, so that we could determine the flying frequencies of R. hipposideros (Table 1). We consid- the overlap. We averaged the values of CF and FM components re- ered this value statistically negligible. Our findings suggest sulting from the same recording thus we determined an average val- that there is a high frequency overlap between the two spe- ue of the frequency by individuals. During the analysis of the resting frequencies we averaged three impulses from one individual. cies that is from 101.36 kHz to 105.20 kHz (Figure 3). We used Welch’s Two Sample t-test to assign the difference be- Welch’s Two Sample t-test determined that the females tween the average value of the frequency between males and females of R. hipposideros (106.82 kHz (SD:1.17) n=25) had significant- used by both species. We applied general linear mixed model of R ly higher frequency (p<0.0001) than that of the males (104.00 3.3.1 statistical program with 5% significance level to evaluate the kHz (SD:1.38), n=34). In the case of R. euryale, no significant connections between body parameters and acoustic traits. The sam- difference was found between the frequencies of both sexes ple area served as random effect while fixed effects included two in- (p=0.54). The mean frequency of the females was 102.16 kHz teractions between species and sex as well as species and BCI. Con- (SD:0.91), n=24. Males had calls at 102.39 kHz (SD:0.69), sidering the main effect of males and females, contrast matrix was used. n=31 (Figure 4). According to the correlations between body parameters and the acoustic traits of bats, the females of both species were larger based on the body parameters. These Results calculations indicate that the females of R. hipposideros have higher BCI as well as higher mean frequency than males 141 bats were examined, including 61 individuals of R. hip- (Figure 5). There was no significant difference between posideros and 80 of R. euryale. Out of the FM-CF-FM calls, we males and females of R. euryale regarding the emitted mean measured 5433 CF and 1526 FM parts. frequency. We fitted general linear mixed model using the mean frequency as dependent variable. Our results show Flying frequency that sex had a significant effect (p<0.0001) on the value of the The frequency interval of R. euryale was 98.10–105.20 kHz, mean frequency regarding R. hipposideros (n=43) while R. eu- while that of R. hipposideros was 101.36–109.90 kHz. We also ryale (n=48) was not affected by sex (p=0.85). experienced one impulse lower than 101.36 kHz (100.48 FM components of the first harmonics of the calls of fly-

206 D. Győrössy et al.

Table 1. Descriptive statistics for R. hipposideros and R. euryale body mass, forearm length, BCI (body condition index), FF (flying frequency) and RF (resting frequency). CF_2 = CF parts of the calls from the second harmonic part, FM_1 = FM parts of the calls from the first harmonic part. No data = undetermined sex.

Species Variables Sex (sample size) Minimum Maximum Mean (SD) Rhinolophus hipposideros Body mass (g) male (27) 5.00 7.50 5.85 (0.57) female (16) 5.50 7.00 6.24 (0.47) Forearm length (mm) male (29) 37.31 40.63 38.81 (0.94) female (16) 37.80 40.74 39.60 (1.02) BCI (g/mm) male (27) 0.13 0.19 0.15 (0.01) female (16) 0.15 0.18 0.16 (0.01) FF: CF_2 (kHz) male (34) 100.48 107.61 104 (1.38) female (25) 103.74 107.23 106.82 (1.17) no data (2) 103.71 109.86 107.35 (2.11) FF: FM_1 (kHz) male (34) 41.6 48.04 44.98 (0.64) female (25) 43.35 48.04 45.48 (0.90) no data (1) 45.11 45.99 45.69 (0.62) RF: CF_2 (kHz) male (34) 103.41 109.57 105.49 (1.44) female (25) 104.88 109.86 108.60 (1.01) no data (2) 108,1 109.86 108.98 (1.24) Rhinolophus euryale Body mass (g) male (28) 10.00 15.00 12.29 (1.34) female (20) 10.00 15.50 12.71 (1.54) Forearm length (mm) male (30) 45.44 53.98 48.80 (1.35) female (20) 47.09 51.01 49.18 (1.17) BCI (g/mm) male (28) 0.21 0.31 0.25 (0.03) female (20) 0.21 0.31 0.26 (0.03) FF: CF_2 (kHz) male (31) 99.31 104.58 102.39 (0.69) female (24) 98.43 105.17 102.16 (0.91) no data (25) 98.14 104.29 101.11 (0.74) FF: FM_1 (kHz) male (8) 39.84 43.94 42.75 (1.42) female (12) 39.25 45.41 42.57 (1.09) no data (1) 40.13 40.13 40.13 (0.00) RF: CF_2 (kHz) male (31) 102.53 104.58 103.80 (0.50) female (24) 102.53 105.17 103.88 (0.66) no data (7) 102.83 104.58 103.83 (0.75)

6). Our findings are not adequately supported by this small amount of data. The disadvantage of this method is that we are often unable to see the first harmonic part of the calls on the records, so it is hard to measure the FM parts precisely (Figure 2). The value of the BCI (Body Condition Index) was also represented together with the mean frequency (Figure 7). We received similar results as our previous findings with CF parts. The females of R. hipposideros had larger body pa-

Figure 3. The overlap between the two species’ flying frequencies rameters as well as higher frequencies. Rhinolophus euryale based on the CF parts. did not display any significant variation in the emitted aver-

age frequencies of both sexes. We fitted general linear mixed ing specimens were also measured. We assumed that there is model to support the idea that the sex of the R. hipposideros a smaller overlap between the two species thus it would be exerts a significant (p=0.04) influence on the value of the av- easier to identify them. We determined the frequency inter- erage frequency. However, the sex of R. euryale had no effect val of R. hipposideros (41.60–48.00 kHz) and R. euryale (39.30– on this parameter (p=0.31). 45.70 kHz). Here, we also found an overlap that is around 41.60–45.70 kHz. During the analysis of the mean frequen- Resting frequency cies, R. hipposideros had calls at 45.20 kHz (SD: 0.79) and R. The CF parts of the resting frequencies that are emitted at euryale had calls at 42.52 kHz (SD: 1.29). During the analysis the same pitch in each vocal impulse were also analysed. of the sexes of the two species we found significant differ- Regarding R. hipposideros, we found that the minimum and ences (p=0.02) between males (44.98 kHz (SD:0.65); n=34) maximum value of the resting frequency of males are 103.41 and females (45.48 kHz (SD: 0.90); n=25) of the R. hippo- kHz and 109.57 kHz while that of the females are 104.88 kHz sideros. There was no significant difference (p=0.77) between and 109.86 kHz, respectively. Males (n=34) had calls at an the mean frequencies of the males (42.75 kHz (SD:1.42); n=8) average of 105.49 kHz (SD: 1.44) that is significantly and females (42.57 kHz (SD: 1.09); n=12) of R. euryale (Figure (p<0.0001) lower than the average frequency of females

Bioacoustic research on horseshoe bats 207

Figure 4. The distribution of the mean frequency of the CF parts regarding the flying and the resting frequencies in both species. Flying frequencies: R. hipposideros (female: mean=106.82 kHz, SD=1.17 kHz, n=25, male: mean=104 kHz, SD=1.38 kHz, n=34; p<0.0001) and R. euryale (female: mean=102.16 kHz, SD=0.91 kHz, n=24, male: mean=102.39 kHz, SD=0.69 kHz, n=31; p=0.54). Resting frequencies: R. hipposideros (female: mean=108.6 kHz, SD=1.01 kHz, n=23, male: mean=105.49 kHz, SD=1.44 kHz, n=34; p<0.0001) and R. euryale (female: mean=103.88 kHz, SD=0.66 kHz, n=24, male: mean=103.8 kHz, SD=0.5 kHz, n=31; p=0.64).

Figure 5. The value of the BCI (body condition index) represented along with the mean frequency of the CF parts of the flying individuals in both species: R. hipposideros (male: n=28, female: n=20) and R. euryale (male: n=28, female: n=20).

(n=23) that is 108.60 kHz (SD: 1.01) (Two Sample t-test). The resting frequency, while it does not influence (p=0.96) that minimum and maximum value of the resting frequency of R. value of R. euryale. euryale experienced 102.53 kHz and 104.58 kHz in males as well as 102.53 kHz and 105.17 kHz in females. T-test did not show significant difference (p=0.64) between males (103.80 Discussion kHz, SD: 0.50, n=31) and females (103.88 kHz, SD: 0.66, n=24) (Figure 4). Our findings revealed similar results to that Our study found a significant overlap in the frequency of of the flying frequency regarding the BCI values depending flying specimens of R. hipposideros and R. euryale (Figure 3). on the value of the resting frequency (Figure 8). The females This result modifies and questions the previously used of R. hipposideros had greater BCI values as well as higher acoustic identification methods that were based on pub- frequencies. There was no significant difference between lished data from other geographical areas (Dietz et al. 2009, males and females considering the values of the emitted rest- Barataud 2015). Measuring the CF parts we determined that ing frequencies of R. euryale. We fitted general linear mixed the minimum value of the overlap (101.36 kHz) is lower than model and our results demonstrate that the sex of R. hippo- in previous studies. Dietz et al. (2009) described the frequen- sideros influences significantly (p<0.0001) the value of the cy interval of R. hipposideros between 108–114 kHz and 104–

208 D. Győrössy et al.

Figure 6. The distribution of the mean frequency of the FM parts regarding the flying frequencies in both species: R. hipposideros (female: mean=45.48 kHz, SD=0.9 kHz, n=25, male: mean=44.98 kHz, SD=0.65 kHz, n=34; p=0.02) and R. euryale (female: mean=42.57 kHz, SD=1.09 kHz, n=12, male: mean=42.75 kHz, SD=1.42 kHz, n=8; p=0.77).

Figure 7. The value of the BCI (body condition index) represented along with the mean frequency of the FM parts of the flying individuals in both species: R. hipposideros (male: n=27, female: n=16) and R. euryale (male: n=7, female: n=9).

Figure 8. The value of the BCI (body condition index) represented along with the resting frequency of the CF parts in both species: R. hipposideros (male: n=26, female: n=14) and R. euryale (male: n=28, female: n=20).

109 kHz for R. euryale. It means that the overlap is between that the overlap is between 101.36–105.20 kHz. Following 108 and 109 kHz. However, according to Barataud (2015) the this, we advocate that during the acoustic identification of frequency interval of R. hipposideros is 102.5–113 kHz and the two species, frequencies lower than 101 kHz should indi- 100–106.4 kHz for R. euryale. Based on these previous studies cate R. euryale, while frequencies higher than 105.20 kHz are the overlap interval of 102.5–106.4 kHz were used during typical of R. hipposideros. Those calls that are between these acoustic identifications in the Carpathian Basin which may two values include both species, so they can be identified in have resulted in false faunistic data. Our findings suggest one category as R. hipposideros/R. euryale calls. Bioacoustic research on horseshoe bats 209

We have not encountered with such low frequencies of According to Barclay (1985) the height of the frequency R. hipposideros neither in the Carpathian Basin nor in foreign of the emitted calls of bats can affect the size-based selection published data. There is great geographical variation in the of prey. Bat calls with a higher frequency are more advanta- calls among bat species belonging to the family of Rhinolo- geous to sense smaller sized prey (Jones et al. 1992, Pye 1993, phidae and Hipposideridae that emit CF calls (Heller & Fenton 1999). In conclusion, we can say that female individ- Helversen 1989, Francis & Habersetzer 1998, Guillén et al. uals of R. hipposideros, due to their higher frequencies, can 2000, Chen et al. 2009, Jiang et al. 2010, Jacobs et al. 2017, prey more on smaller insects than males thus females are Mutumi et al. 2017, Xie et al. 2017, Najafi et al. 2018, Ahmim able to feed more efficiently due to their broader food spec- et al. 2019). Russo et al. (2007) examined the populations of trum in the energy-requiring breeding period. In contrast, R. hipposideros and R. euryale on the island of Sardinia and due to the similar frequencies of males and females of R. eu- peninsular Italy. They found that the Sardinian R. euryale ryale and their use of perch hunting (Goiti et al. 2003), we can emits significantly lower frequencies compared to that of the assume that according to Jones and Rayner (1989) both sexes Apennine bats. Furthermore, they found that in the case of of R. euryale capture larger prey during their hunt. Further- R. hipposideros females make calls that are higher by an aver- more, it is possible that hunting for those small insects, age with 4 kHz, while the males have calls that are higher by which the females of R. hipposideros can capture, is energeti- 3 kHz. This must be due to the presence of another species cally unfavorable for this larger body sized species. The dis- on the island of Sardinia: Rhinolophus mehelyi. This species advantage of the higher frequencies is that their echoes can has a frequency range that is between the frequency of the be absorbed and weakened more easily by the atmosphere other two species. This supports the idea that the shift in the thus animals must detect their prey from a shorter distance frequency is the result of some selection pressure in order to over a short period of time (Hartley 1989). avoid an overlap between the calls of the three species. Analysing the CF parts, the connections between the acoustic traits and body parameters indicate that the females Acknowledgement. We wish to thank Dr. Sándor András Boldogh, of R. hipposideros not only have larger body parameters but Kriszta Lilla Szabadi and Bernadett Sosovicska for helping in the they have a higher frequency. This is contradictory to our field. Special thanks to Dr. Zsolt Lang for his help with the statistical hypothesis that there is a negative correlation between body analysis. We are grateful to Dóra Teplánszki for creating the QGIS size and the height of the emitted frequencies of bats in the map. We would also like to thank Marcel Uhrin and one anonymous family of Rhinolophidae and Hipposideridae (Heller & reviewer for their valuable comments about the manuscript. Péter Helversen 1989, Dietz et al. 2009). Jones et al. (1992) also re- Estók was supported by the grant [EFOP-3.6.1-16-2016-00001] ceived similar results although they examined the resting ‘Complex improvement of research capacities and services at Eszterházy Károly University’. This research was supported through frequency of the animals, which is higher than their flying the New National Excellence Program of the Ministry of Human frequency. 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