Cetaceans are recognized as key top-level controllers in marine ecosystems due to their roles in preserving the structure and function of marine communities (Heithaus et al., 2013; Ricci et al., 2021; Carlucci et al., 2021). The ecological health and population status of cetaceans can potentially be used as bio-indicators for the assessment of ecosystem health (Wells et al., 2004; Paudel et al., 2020). The implementation of effective strategies to conserve these species can spread downwards throughout the food chain and protect the entire ecosystem. Therefore, robust knowledge regarding population size, spatial-temporal habitat usage, and home ranges coupled with threat assessment are of critical importance not only for the species in question but also for the entire marine ecosystem (Panigada et al., 2017; Azzolin et al., 2018; Carlucci et al., 2021). The western and central Mediterranean Sea, including the northern and central Adriatic Sea have benefited from dedicated research effort on cetaceans since the 1980s (Notarbartolo di Sciara et al., 1993; Bearzi and Notarbartolo di Sciara, 1995; Bearzi et al., 1997; Miokovic et al., 1997; Bearzi et al., 1999; Pribanić et al., 2000; Bearzi et al., 2008; Bearzi et al., 2009). Conversely, the southern Adriatic region, and the eastern and southern Mediterranean Sea has limited baseline data, with dedicated research efforts starting in the early 2000s (Bearzi et al., 2009; Baş et al., 2017a; Akkaya et al., 2020).

Common bottlenose dolphins (Tursiops truncatus) are the most widely studied cetacean species, both visually and acoustically (Hill and Lackups, 2010), throughout the Mediterranean Sea due to their highly coastal distribution (Bearzi et al., 2008). According to the IUCN Red List, bottlenose dolphins have experienced a 30% decline in their population size since the 1940s in the Mediterranean Sea, while Adriatic populations declined by almost 50% in the latter half of the 20th century (Bearzi et al., 2004; Sackl et al., 2007; Bearzi et al., 2008; Bearzi et al., 2012). Due to the continuous decline in populations, this species was classified as “vulnerable” in the IUCN Red List in 2009 with its classification recently upgraded to “least concern” in 2021 (Bearzi et al., 2021). Basin-wide population estimates of bottlenose dolphins have historically relied on a handful of highly surveyed areas (e.g. Bearzi et al., 2008). Whilst an effort was made to address the disparities in survey effort between regions during the ACCOBAMS Survey Initiative (ACCOBAMS, 2021), smaller countries received only two or three transects and population estimates conducted during a single day of flying rendered their population sizes virtually unknown. Therefore, data-deficient regions (which may be at threat from uncontrolled and unregulated pressure) have the potential to considerably affect basin-wide estimates and year-round dedicated research at local scales remain integral to an accurate understanding of the status of the species in question (Awbery et al., 2022).

The majority of the effort in the Adriatic and Ionian Sea focuses on behavioural activity, individual identification, threat assessment and movement patterns (Bearzi et al., 1997; Fortuna et al., 2011; Baş et al., 2017a; Baş et al., 2017b; Awbery et al., 2019; Akkaya et al., 2021). Based on previous knowledge, bottlenose dolphins are generally known as a resident species with limited movement (Genov et al., 2009; Bearzi et al., 2016). Contrarily, a recent study documented an adult male bottlenose dolphin travelling between the Northern Adriatic Sea, passing through the Ionian and Tyrrhenian Sea, and finally reaching the Ligurian Sea with an estimated minimum distance of 1251 km across all three seas (Genov et al., 2022).

While traditional visual data collection can reveal critical information on species as highlighted in the example of an indivduals’ movement over a wider area as ever previously thought, it is limited by the duration that the species spends at the surface, and additionally, environmental conditions and daylight hours (Mellinger and Barlow, 2003; Mellinger et al., 2007). Considering that cetaceans spend the majority of their time under the surface and that sound emission is their primary source of information acquisition, acoustic monitoring as a research tool, increases our knowledge of underwater species and amplifies the potential of conservation strategies (Van Parijs et al., 2009; Davis et al., 2017). The marine soundscape has changed drastically since the beginning of the industrial revolution as humans have either deliberately added sound to the environment (e.g. when describing the bottom or sub-bottom of the seabed; Hildebrand, 2009; Estabrook et al., 2016) or as a by-product (e.g. the intensification of marine traffic; Duarte et al., 2021). As sound propagates further than light or chemicals in the marine environment, many marine animals have evolved to be sensitive to sound. Thus, the cumulative increase in anthropogenic sound may elicit many short and long-term effects, from behavioural and habitat alterations to area avoidance, to changes in population dynamics and even physical injuries and mortality (La Manna et al., 2013; Domit et al., 2016; Baş et al., 2017b; Fruet et al., 2017; Caruso et al., 2020).

Bottlenose dolphins are highly vocal animals with great plasticity and complexity in their vocal repertoire (Luís et al., 2021). The sounds emitted play a fundamental role in their social interactions, individual recognition, group coordination, foraging success, and recognition of an individual’s surroundings (Lind et al., 1996; Rendall et al., 1996; Janik and Slater, 1998; Azzolin et al., 2017; MacFarlane et al., 2017; La Manna et al., 2020). The acoustic repertoire of bottlenose dolphins includes echolocation clicks (broadband click trains), burst-pulsed sounds (closely spaced broadband click trains) and frequency modulated whistles (narrowband tonal sounds) (Caldwell et al., 1990; Janik, 2009; Herzing, 2014; La Manna et al., 2017; Luís et al., 2021; Pace et al., 2022). Echolocation clicks are known to be used primarily for navigation and foraging while whistles and pulsed sounds are considered to be used for individual recognition, social maintenance, group coordination, communication, as well as foraging activity (Au, 1993; Branstetter et al., 2012; Janik et al., 2012; MacFarlane et al., 2017). The vocal repertoires of dolphins are also known to vary significantly between populations at macro-and-micro geographic scales (Hawkins, 2010; Papale et al., 2013; La Manna et al., 2020). Within the Mediterranean Sea, acoustic studies on dolphins have mostly been assessed by categorisation of whistle type characteristics (Díaz López, 2011; La Manna et al., 2017; La Manna et al., 2020; Corrias et al., 2021; Terranova et al., 2021; La Manna et al., 2022; Terranova et al., 2022; Pace et al., 2022). These studies have mainly focused on the identification of species presence, geographical variation in vocalisation types, the impact of boat traffic, dolphin-fishery interactions, and whistle characteristics with bottlenose dolphins, being by far the most frequently studied species (Connor and Smolker, 1996; Boisseau, 2005; Díaz López and Shirai, 2010; La Manna et al., 2017).

This current study is the first attempt to understand the whistle characteristics of bottlenose dolphins in the Southern Adriatic Sea and provides additional information for the Northern Ionian Sea. This study assesses variation in their acoustic behaviour by examining the whistle characteristics of bottlenose dolphins and aims to evaluate the potential similarities or dissimilarities in regional repertoires between the two survey locations. The assessment of this baseline data aims to deepen our understanding of the vocal repertoire of bottlenose dolphins while reviewing the potential effect of geographical differences on their vocal behaviour.

The acoustic data was collected in two nearby regions: the Gulf of Taranto, Italy between 11.06.2018 and 20.06.2021 and Boka Bay, Montenegro between 01.04.2020 and 16.07.2022. The selected study locations are within a 400 km straight line distance from each other, and the presented data represents only part of a long-term research effort from each region.

The overall survey effort consisted of 346 days in the Taranto Gulf between 11.06.2018 and 20.06.2021 and 39 days in Boka Bay between 01.04.2020 and 16.07.2022. Bottlenose dolphin presence was recorded during 82 days in Taranto Gulf, of which the majority of survey days were conducted with visual surveys and an onboard hydrophone resulted in obtaining 10 acoustic recordings from bottlenose dolphins. The species was present for 21 days in Boka Bay, of which 14 days resulted in the acquisition of acoustic data. The acoustic data could only be collected when the vocalisations of the focal group were within detection range of the hydrophone. Regarding the total duration of recordings, Taranto Gulf had 01:44:38 hours of recording, where whistles were recorded during 01:04:16 hours. Whereas, Boka Bay had 08:08:05 hours of recording, of which whistles were present in 03:28:12 hours. Analysis of these audio files revealed 433 whistle detections in Taranto Gulf and 546 whistle detections in Boka Bay. Harmonics were present in 40% and 32% of tonal sounds in Taranto Gulf and Boka Bay, respectively. After evaluating the quality of each whistle and its background, a total of 423 whistles qualified as satisfactory for further analysis of which 238 whistles came from the Taranto Gulf and 185 whistles from Boka Bay.

Principal Component Analysis was performed on nine whistle variables. The first three principal components explained 87% of the variance with each component having eigenvalues greater than one ( Table 2 ). Therefore, the first three components were used for further analysis while the following six components were considered redundant and were not used in subsequent analysis. Principal Component 1 explained 51.3% of the variance in the dataset with the center frequency and high frequency having the highest loading scores, suggesting that they are the main drivers of variation in component 1. Principal component 2 formed 24.1% of the variance and the duration of the whistle and the number of inflection points had the highest loading scores in component 2. Lastly, component 3 comprised 11.5% of the variance with the highest loading score attained by the number of inflection points ( Table 2 ; Figure 5 ).

Whistle parameters did not show significant differences between Taranto Gulf and Boka Bay (F=0.11, df=1, p=0.94). Detected whistles fell in the range of 659 Hz and 26,122 Hz in Taranto Gulf, while they were between 850 Hz and 24,000 Hz in Boka Bay. The peak frequency had a mean of 10.121 ± 282 Hz in Taranto Gulf and 9,310 ± 256 Hz in Boka Bay. The average duration of the detected whistles was 570 and 500 milliseconds with a maximum whistle duration of 3,160 and 3,530 milliseconds for Taranto Gulf and Boka Bay respectively. The maximum number of inflection points was 17 (mean of two) in Taranto Gulf and 12 inflection points (mean of one) in Montenegro ( Table 3 ).

In addition, whistle variables showed significant variation between whistle types (F=24.84, df=5, p=0.001). The whistle variables of whistle type F showed significant variation from the other whistle types. Additionally, the whistle variables of whistle type C were also significantly different from the other whistle types, except whistle type B. Conversely, the whistle variables for whistle type E showed similar variation between each of the whistle types, except for whistle type C and F ( Table 4 ). Further, the whistles with highest frequencies were produced during whistle type F in both Taranto Gulf (median of 17,104 Hz) and Boka Bay (median of 17,832 Hz). The whistles with the highest peak frequencies were produced for whistle type D in both Taranto Gulf (median of 10,453 Hz) and in Boka Bay (median of 11,063 Hz). However, the whistle types with the highest central frequency showed variation between the study locations, with whistle types E and F possessing the highest medians (both of 10,986 Hz) in Taranto Gulf whilst whistle type D had the highest median (10,969 Hz) in Boka Bay. Finally, the duration of the whistle was longest for whistle type F in Taranto Gulf (median of 1,650 milliseconds) and Boka Bay (median of 1,160 milliseconds) ( Table 5 ).

When variation in whistle types between the study locations was assessed, there was a significant difference between Taranto Gulf and Boka Bay (x2 = 27.7, df=5, p=0.0004). Whistle type A was the most dominant whistle in each location and formed almost 40% of the overall produced whistles both for Taranto Gulf and Boka Bay. In contrast, the least detected whistles were type E for Taranto Gulf and type B and C whistles for Boka Bay ( Figure 6 ).

The results of this current study represent the first acoustic analysis of bottlenose dolphins in Montenegrin waters as well as providing additional information on the variation of whistle characteristics of two geographically separated populations residing in two neighbouring bodies of water: the Southern Adriatic and Northern Ionian Sea. The whistle characteristics of bottlenose dolphins reported here are consistent with those reported in the Mediterranean Sea (Rendell et al., 1999; Gannier et al., 2010; Díaz López, 2011; Papale et al., 2014a; Bolger et al., 2017; La Manna et al., 2019; Rako-Gospić et al., 2020; Papale et al., 2021) and elsewhere (Conner, 1982; Boisseau, 2005; May-Collado and Wartzok, 2008; Baron et al, 2008; Hawkins, 2010). The mean peak frequency was reported at 10kHz, with a frequency ranging between a mean low frequency of 7kHz and a mean maximum frequency of 14 kHz, with a mean duration of 500 ms in each of the locations. Although the frequency range, duration, and inflection points are similar to those reported for other populations, the mean peak frequency range was slightly higher in Croatian and other Italian studies with peak frequencies ranging from 13 to 15kHz (Díaz López, 2011; La Manna et al., 2013; La Manna et al., 2020). Previous studies have highlighted that bottlenose dolphins generally emit whistles ranging between 1 and 20 kHz, yet low frequency whistles reaching 200 Hz have also been detected (Richardson et al., 1995; Ding et al., 1995; Schultz et al., 1995; Lammers et al., 2003; Herzing, 2015). This study also recorded whistles below 1 kHz in each location, although these occurred rarely. On the other hand, the highest recorded fundamental whistles reached just over 25 kHz in each location with harmonics potentially extending beyond the 48 kHz window displayed in the analysed spectrograms, although these were discarded from the current analysis. Additionally, over 40% of whistles contained harmonics in Taranto Gulf while the ratio reached 30% in Boka Bay. Previously, it was noted that the occurrence of harmonics can be linked to behavioural activity and/or group composition (Henderson et al., 2011; La Manna et al., 2019). Henderson et al. (2011) reported that more complex whistles (harmonics) with increased number and duration were emitted when common dolphins (Delphinus sp.) were travelling, whilst whistles were less complex if they were engaged in milling activity. Therefore, it has been suggested that dolphins are likely to use these harmonics for directionality in order to maintain group cohesion and to convey changes in the orientation of the group (Miller, 2002; Rasmussen et al., 2006; Branstetter et al., 2013). It has been further suggested that increased underwater noise in the close proximity of boats may result in the production of harmonics, with the number of harmonic vocalisations having been observed to increase with calf presence and a larger group size (La Manna et al., 2019). However, it is important to consider that the production of harmonics may vary among species (Gannier et al., 2020).

It is important to understand the role of harmonics, specifically considering their high occurrence in each location during the current study. Although this study focuses on geographical differences of whistle variables between study locations, previous studies indicated that 50% of focal bottlenose dolphin groups in Montenegro were observed with the presence of subadults, with dolphins spending 70% of their time travelling, while milling and socialising behaviour only formed 10% or less of the total behavioural budget (Clarkson et al., 2020; Akkaya et al., 2021; Rudd et al., 2022). Above all, both of the locations have a significant amount of marine traffic; with approximately 4,130 vessels in the port of Taranto per year (Shipnext.com). Moreover, Boka Bay has faced a considerable annual increase in marine traffic between 2020-2021 and 2021-2022 respectively. A total of 579 vessels arrived in the Port of Kotor during the entirety of 2021, whilst 692 port calls were made within the first nine months of 2022 alone. It is critical to mention that both locations are utilised by large, environmentally disturbing marine vessels. Large merchant ships encompass approximately 57% of marine traffic in the port of Taranto (Marinetraffic.com), and an estimated 2,000 cruise ships were reported to visit the port of Kotor between 2013 and 2018 (Balkaninsight.com). It is plausible that the aforementioned behaviours, subadult presence, marine traffic presence, or the cumulative effects of these may explain the high occurrence of harmonics, but there is a clear need for future simultaneous visual and acoustic studies to understand the possible relationships influencing the production of harmonics by potentially extending this study to understand the potential role of behavioural activity as well as considering the importance of the direction of the hydrophone in relation to focal group the rate of frequencythat harnonics are recorded

When the whistle characteristics were investigated, the results of principal component analysis indicated that the centre frequency, high frequency, whistle duration, and the number of inflection points’ loading scores were the highest across principal components 1, 2 and 3. These acoustic features were also identified as the main contributors to whistle variation in the Mediterranean Sea by previous studies (Díaz López, 2011; Papale et al., 2014b; La Manna et al, 2020). Therefore, it is possible that these acoustic features play a critical role on the emitted whistles basin-wide, without a clear difference between groups or populations. Bottlenose dolphins are likely to adjust these features depending on the behavioural context and it is believed that these whistles carry information such as identification of an individual, group cohesion, stress level, the presence of food, or danger (Wells, 1991; Wang et al., 1995; Janik and Slater, 1998; Buckstaff, 2004; Esch et al., 2009a; Díaz López, 2011; Henderson et al., 2011). Previously, it has also been stated that an increased number of inflection points on a whistle could indicate the complexity of its contextuality (Steiner, 1981; Wang et al., 1995; Rendell et al., 1999; Azevedo et al., 2007; May-Collado and Wartzok, 2008; Amorim et al., 2016) and is directly linked to the duration of the whistle. It is important to take into account that multiloop whistles, which hold the highest inflection numbers, were responsible for only 11% of whistle types in Taranto Gulf and 17% of whistles in Boka Bay. Meanwhile, harmonics were responsible for almost half of the produced whistles of bottlenose dolphins in each location. The presence of harmonics vs multiloop whistles with the consideration of behavioural and environmental variation should be investigated to understand the role and differences of each acoustic feature.

The emitted whistle type did reveal considerable variation between Taranto Gulf and Boka Bay. While the dominant whistle type was the upsweep whistle (whistle type A) in each location comprising 40% of the whistles, the least emitted whistle was concave (whistle type E) in Taranto Gulf and flat (whistle type C) in Boka Bay. A neighbouring study revealed an absence of flat whistles in Croatia (Rako-Gospić et al., 2020), while the same whistle type was also identified as the least prominent whistle of bottlenose dolphins elsewhere in the Mediterranean Sea and its adjacent waters (Díaz López and Shirai, 2010; Bolger et al., 2017; Rako-Gospić et al., 2020). In the Mediterranean Sea, the dominant whistle type of bottlenose dolphins was reported to be either upsweep or multiloop whistles (Díaz López and Shirai, 2010; Gannier et al., 2010; Rako-Gospić et al., 2020). Adjacent Atlantic waters of the Mediterranean Sea also documented multiloop whistles as being the most produced whistle type (Bolger et al., 2017). Multiloop whistles were reported in moderate numbers at both locations with little difference between the two sites. Terranova et al. (2021) and Esch et al. (2009b) suggest dolphins often emit signature whistles in the form of a continuous connected multiloop whistle (an identical repeated unit in time without intervals) (Janik, 1999; Sayigh et al., 2007). Therefore, it may be likely that some of these multiloop whistles were representing signature whistles. Therefore, future studies should focus on the identification of signature whistles in the study locations. Previous studies suggested that produced whistle types are most likely to be linked with the behavioural activity that the dolphins are engaged in at the time, as well as the presence of marine traffic in the area (Quick and Janik, 2008; Díaz López and Shirai, 2010; Henderson et al., 2011; Jones et al., 2019; Rako-Gospić et al., 2020). Recently, Rako-Gospić et al. (2020) reported that whistle type does show significant variation during foraging activities, but also in the presence of trawlers or motorboats which also play a critical role in the dominant whistle type. Therefore, the number of anthropogenic activities that take place in the region should be considered in order to understand variation of whistle types under different behavioural activities and vice versa.

This current study represents the first attempt to understand whistle variability of bottlenose dolphins from the data deficient waters of the Southern Adriatic, while investigating potential differences in whistles produced in the relatively close region of the North Ionian Sea. Despite the small sample size and slightly variable survey methodology followed in each location, the acoustic features of whistles reported here are consistent with the results reported elsewhere in the Mediterranean Sea. Whistles are known to carry species-specific cues and it has been suggested that they carry information on social structure, behaviour, and the cohesion of the group (Miller, 2002; Lammers et al., 2003; Branstetter et al., 2013; Díaz López et al, 2017). Therefore, further research is needed to identify the role that behavioural, environmental, and anthropogenic states have on the acoustic features of whistles of bottlenose dolphins both in Taranto Gulf and Boka Bay.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by The Montenegrin Government.

AA, TA, PL, GC, MA, LI, OE, YA, PR, RCr, EP, and CF participated in the field surveys. Analysis was conducted by AA, TA, KM, and PL. AA and RCa supervised the manuscript. The manuscript was written by all the authors. All authors contributed to the article and approved the submitted version.