Reproductive ethology and association with sounds
We identified nine male behavioural categories within three distinct
reproductive phases in O. croaticus : Chase, Lead, Approach and
Circling (courtship phase), Nest display, Frontal display, Nest rubbing,
Pre-mating (pre-spawning phase) and Spawning (spawning phase). The
category Pre-mating included all behaviours that occurred after the
female entered the male nest and prior to belly-up position
(oviposition ). Spawning was not observed, but we observed the
female’s repeated upside-down or belly-up position (an indication ofoviposition ), during which she probably laid eggs on the nest
ceiling. Briefly, the behaviours Chase, Lead, Approach and Circling were
performed by the male outside the nest (recognised as the “courtship”
phase), while Nest display, Frontal display, Nest rubbing, Pre-mating
and Spawning were performed within the nest (“pre-spawning” and
“spawning” phases). In some cases, Nest display and Frontal display
were performed by the male occupying the nest with or without a female
inside. However, Nest rubbing, Pre-mating and Spawning were always
performed by the male when the female was inside the nest. The
frequency, duration and overall percentage of these categories were
scored during ethological analysis. The first dataset included seven
video recordings (210 min) where at least one sound occurred per
recording. Overall, we observed 410 behavioural acts (102.5 per male).
The behavioural categories Nest display (29.3%), Pre-mating (22.7%)
and Approach (19.1%) were most frequently observed, while Circling
(1.2%), Chase (1.0%) and Lead (0.7%) were rarely recorded
(Figure 5a-d ). Concerning the association of sound production
with behavioural acts, of the 410 behavioural acts, 99 acts (24.1%)
were accompanied by sound production. The behaviours Pre-mating (303
sounds), Nest display (27 sounds), Frontal display (20 sounds), Nest
rubbing (16 sounds) and Spawning (5 sounds) were coincided with sounds,
while Approach, Circling and Lead never co-occurred with sound
production (Figure 5a ). During four Chase acts, only one sound
was recorded. Only one spawning act (Spawning) was observed in this
study, during which five sounds were produced. Unfortunately, spawning
sounds were not used in the comparative purposes due to their limited
occurrence. The chi-square (χ2) test of independence
indicated that behavioural categories Nest display and Pre-mating were
significantly associated with sound production
(χ 2 = 138.3; d.f. = 5; P <
0.05; residual score: 1.5 and 41.5, respectively), while other
categories failed to support this hypothesis (Figure 5a ).
To compare male behaviour when soniferous or mute, a second dataset of
eight video recordings was considered (190.5 min) of the same four males
but in which no sound production was documented. In these recordings, we
have observed 324 male behavioural acts (averaging 81.0 per male), of
which Approach (38.3%), Lead (31.8%) and Nest display (20.1%) were
the most frequent categories (Figure 5b ). Contrary, Chase,
Circling, Pre-mating or Spawning were not documented within these
recording sessions. In general, there is an obvious dissimilarity
between the frequency of the behavioural categories that were or were
not accompanied by sounds. Specifically, Pre-mating, one of the two
behaviours significantly associated with sound production, decreased
from an average of 22.7% in the trials with sounds to 0% in the trials
without sound. In addition, Nest display, Nest rubbing and Frontal
display acts produced during sound emission decreased from 29.3%,
13.9% and 12%, to 20.1%, 3.7% and 6.2% in frequency in experiments
without the sounds, respectively. On the other hand, Approach and Lead
were more frequent during the mute behaviours (31% and 38%,
respectively) than during sound production (19% and 0.7%,
respectively) (Figure 5b ). Overall, the behavioural rate
decreased from 55.8% to 44.1% when males produced sounds in comparison
to when they were mute (soniferous vs. mute males: means 2.79vs. 1.55), though the differences were not significant
(Wilcoxon signed-rank test, P > 0.05)
(Figure 6a ). Importantly, the number of times the females
entered the male nest differed significantly between the two datasets
(means 3.71 vs 0.71), as female nest entrance was more frequent
when males produced sound than when they were mute (Wilcoxonsigned-rank test, P < 0.05) (Figure 6b ).
When producing sounds, the occurrence frequency between the courtship
(Chase, Lead, Approach and Circling) and pre-spawning (Nest display,
Frontal display, Nest rubbing, Pre-mating) phases of reproduction did
not differ between soniferous males (Wilcoxon signed-rank test,P > 0.05), though their duration did
(Wilcoxon signed-rank test, P < 0.05).
Generally, males exhibited courtship-related behaviours less frequently
and for a shorter period compared to pre-spawning behaviours (x̄ =
22.5 vs. 79.7 for frequency and x̄ = 173.4 vs.2393.3 for duration).