Bioacoustics of Scinax altae

Bioacoustics analysis The one-way ANOVA test for 13 acoustic variables of the three data sets showed significant differences for certain bioacoustic variables. When examining the multiple comparisons using the Tukey HSD tests, it was found that the characteristics of the calls of the populations of southeastern Costa Rica better fit the characteristics of the species Scinax altae , since no statistical differences were found in comparison with that species; while in contrast to Scinax staufferi , significant differences were found in several of the acoustic characteristics ( Table 2 ). Once the identity of the specimens from southeastern Costa Rica was ascertained, we added them to the Scinax altae data, leaving a total of 152 calls analyzed for this species. The summary of call measurements is presented in Table 3 . Using the note-centered approach, both advertisement calls share a single pulsed-type note, that is, each note is subdivided into acoustic units (pulses) separated from each other by modulations of amplitude less than 100% and duration less than 50 milliseconds (ms) ( Fig. 4 ). These pulsed notes do not present frequency modulations during the course of the call. The pulses that make up the notes are distinct, partly fused, without silent intervals. The majority of calls had an ascendant amplitude modulation from the first to the third pulse, until reaching a plateau, which was sustained throughout the call duration, where the first pulse always had a lower intensity ( Fig. 5 ). Acoustic measurements One-way ANOVA 2-1 Tukey HSD 3-1 3-2 Call (Note) duration 90% (s) F (2,6) = 0,218 p = 0,81 η2 = 0,068 0,848 0,811 0,999 Intercall interval F (2,6) = 0,514 p = 0,622 η2 = 0,146 0,959 0,835 0,610 Num. Call/ Call series F (2,6) = 11,07 p = 0,0097 η2 = 0,787 0,919 0,019 0,018 Call series duration F (2,6) = 13,85 p = 0,006 η2 = 0,822 0,803 0,010 0,012 Intercall series interval F (2,6) = 12,54 p = 0,019 η2 = 0,862 0,928 0,026 0,025 Bandwidth 90% (Hz) F (2,6) = 2,695 p = 0,146 η2 = 0,473 0,248 0,136 0,905 Frequency 5% (Hz) F (2,6) = 6,86 p = 0,028 η2 = 0,696 0,991 0,071 0,037 Frequency 95% (Hz) F (2,6) = 5,782 p = 0,040 η2 = 0,658 0,222 0,034 0,324 Dominant frequency (Hz) F (2,6) = 7,315 p = 0,025 η2 = 0,709 0,769 0,107 0,025 Peak Time (s) F (2,6) = 2,382 p = 0,173 η2 = 0,443 0,870 0,435 0,168 Pulse Duration 90% (ms) F (2,6) = 0,238 p = 0,796 η2 = 0,073 0,999 0,843 0,836 Num. of pulses/call-note F (2,6) = 1,737 p = 0,254 η2 = 0,367 0,313 0,956 0,317 Pulse repetition rate (Pulses /s) F (2,6) = 0,579 p = 0,589 η2 = 0,162 0,824 0,564 0,876 Acoustic measurements Scinax altae (n=152) Scinax staufferi (n=138) t-student Test Num. Notes/call 1 1 - Call duration 90% (s) 0,123 ± 0,007 (0,115- 0,133) 0,127±0,020 (0,101-0,150) t=-0,377 p= 0,718 Intercall interval (s) 0,487±0,078 (0,428-0,619) 0,425±0,101 (0,335-0,567) t=1,047 p= 0,330 Num. Call/ Call series 6,079±1,363 (2-8) 24,333±8,165 (17-35) t= -4,999 p= 0,002 Call series duration (s) 3,550±1,018 (2,217-4,736) 13,651±4,048 (8,763-17,739) t= -5,457 p= 0,0009 Intercall series interval (s) 3,358±0,642 (2,633-4,205) 6,140±0,429 (5,837-6,443) t= -5,490 p= 0,003 Frequency 5% (Hz) 1855,739±32,621 (1815,589-1899,229) 1622,684±127,448 (1528,846-1808,789) t= 3,993 p= 0,005 Frequency 95% (Hz) 4645,477±397,300 (4086,538-4968,297) 4118,188±189,966 (3937,500-4319,712) t= 2,418 p= 0,046 Bandwidth 90% (Hz) 2789,739±419,009 (2221,154-3136,017) 2495,504±2426,457 (2338,235-2790,865) t= 1,279 p= 0,242 Dominant frequency (Hz) 2424,093±431,496 (1981,055-2885,449) 3542,758±421,957 (2918,179-3837,696) t= -3,901 p= 0,006 Peak Time (s) 0,362±0,153 (0,219-0,540) 0,593±0,154 (0,362-0,680) t= -2,245 p= 0,060 Pulse Duration 90% (ms) 4,358± 0,624 (3,462- 5,100) 4,652±0,543 (3,890- 5,166) t= -0,742 p= 0,482 Num. of pulses/call-note 21,591±0,885 (20,727-22,800) 20,998±1,217 (19,444-22,353) t= 0,851 p= 0,423 Pulse repetition rate (Pulses /s) 131,734±5,336 (125,806-136,539 124,589±16,271 (105,676-145,200) t= 0,935 p= 0,381 In high FFT resolution, the spectrogram is automatically structured into frequency bands (similar to towers of coins) that should not be confused with harmonics. According to Jackson (1996) and Gerhardt (1998) , these frequency bands are caused by high rates of emission of acoustic structures (pulses), and thus reflect the pulse rate. Because each coherent entity of sound emission is separated by silent intervals longer than the duration of the units themselves, we do not consider them as a series of notes within a song, but as calls (consisting of one note) that are grouped together in call series with silent intervals between the series. Although the note-centered approach could consider the call series of songs as the coherent sound unit (call), Köhler et al . (2017) point out that it is important to compare homologous bioacoustic entities under a taxonomic perspective and apply the same name to them. In this way, comparative studies of advertisement calls of species in the genus Scinax ( Carvalho et al . 2015 , Bang & Giaretta 2016 , Ferrão et al . 2022 ) coincide in considering the advertisement calls consisting of a single note, grouped into call series ( Fig. 5 ). The advertisement calls of Scinax altae are structured in call series composed of 2-8 calls per series (= 6,079±1,363); their duration varied from 2,217 -4,736 s (=3,550±1,018), and they are separated by time intervals between 2,633 -4,205 s (=3,358±0,642). Call duration varied from 0,115 - 0,133 s (=0,123 ± 0,007), with intervals of 0,428 -0,619 s (=0,487±0,078). Calls had 20,7-22,8 pulses (=21,591±0,885), emitted at rates of 125,806-136,539 pulses per second (=131,734± 5,336). The pulse duration varied from 3,462 -5,100 ms (=4,358± 0,624). Regarding the spectral characteristics, dominant frequency varied from 1981,055-2885,449 Hz (=2424,093±431,496); minimum frequency at 5% of the energy (Freq 5%) varied between 1815,589- 1899,229 Hz (=1855,739±32,621) and maximum frequency at 95% (Freq 95%) between 4086,538-4968,297 Hz (=4645,477±397,300). The advertisement calls of Scinax staufferi are structured in call series composed of 17 -35 calls per series (( =24,333±8,165); their duration varied from 8,763 - 17,739 s (=13,651±4,048), and were separated by time intervals between 5,837 -6,443 s (=6,140±0,429). Call duration varied from 0,101 -0,150 s (=0,127±0,020), with intervals of 0,335 -0,567 s (=0,425±0,101). Calls had 19,444 -22,353 pulses (=20,998±1,217), emitted at rates of 105,676-145,200 pulses per second (=124,589±16,271). The pulse duration varied from 3,890 - 5,166 ms (=4,652±0,543). Regarding the spectral characteristics, dominant frequency varied from 2918,179-3837,696 Hz (=3542,758±421,957); minimum frequency at 5% of the energy (Freq 5%) varied between 1528,846-1808,789 Hz (=1622,684±127,448) and maximum frequency at 95% (Freq 95%) between 3937,500-4319,712 Hz (=4118,188±189,966). Despite the similarity, the acoustic and statistical analyses (t-student test) show that there are significant differences between the advertisement calls of these species for some structural variables (Number of notes per call); temporal variables (Call series duration, Intercall series interval); and spectral variables (Frequency 5%, Frequency 95%, Dominant frequency). The variability of the acoustic parameters can be visualized in box-and-whisker plots ( Fig. 6 ). The most striking difference is in Dominant frequency, where S, altae shows 2424,093±431,496 Hz while S. staufferi is much higher (3542,758±421,957 Hz). Likewise, it was found that the way in which their calls are organized is different. Series of short calls (6,079±1,363 calls per series) characterize S. altae , while in S. staufferi calls are organized in series of greater numbers (24,333±8,165 calls per series), showing the importance of examining and integrating these types of structural characteristics of advertisement calls, in addition to the traditional temporal and spectral characteristics. Previous call analyses of both species ( León 1969 , Duellman 1970 ) failed to provide modern statistical data. Observations on natural history The Buenos Aires population is in a rapidly reducing lagoon in the middle of the city. Many species called on the night of April 30, 2018 , after heavy but short rainfall; other species calling included Dendropsophus microcephalus (Cope, 1886) , D. ebraccatus (Cope, 1874) , Scinax elaeochrous , Leptodactylus poecilochilus (Cope, 1862) . Scinax altae call randomly every several minutes. The call is striking but difficult to locate among so many other loud calling species. In La Florida , only two males were located by MC, and only once each time, when the photos and recordings were made. Both individuals were seen around an artificial pond made to attract local amphibians, such as Agalychnis callidryas , A. spurrelli , Scinax boulengeri, Engystomops pustulosus, and Leptodactylus savagei , among others. It appears that the species is a new arrival to the area, as there is not an apparent established population, but, rather, a few single males calling and probably dispersing.