Gekko gecko as a model organism for understanding aspects of laryngeal vocal evolution.

The ability to communicate through vocalization plays a key role in the survival of animals across all vertebrate groups. While avian reptiles have received much attention relating to their stunning sound repertoire, non-avian reptiles have been wrongfully assumed to have less elaborate vocalization types and little is known about the biomechanics of sound production and their underlying neural pathways. We investigated alarm calls of Gekko gecko using audio and cineradiographic recordings of their alarm calls. Acoustic analysis revealed three distinct call types: a sinusoidal call type (type 1), a train-like call type, characterized by distinct pulse trains (type 3), and an intermediary type, which showed both sinusoidal and pulse train components (type 2). Kinematic analysis of cineradiographic recordings showed that laryngeal movements differ significantly between respiratory and vocal behavior: during respiration, animals repeatedly moved their jaws to partially open their mouths, which was accompanied by small glottal movements. During vocalization, the glottis was pulled back, contrasting with what has previously been reported. In-vitro retrograde tracing of the nerve innervating the laryngeal constrictor and dilator muscles revealed round to fusiform motoneurons in the hindbrain-spinal cord transition ipsilateral to the labeled nerve. Taken together, our observations provide insight into the alarm calls generated by G. gecko, the biomechanics of this sound generation and the underlying organization of motoneurons involved in the generation of vocalizations. Our observations suggest that G. gecko may be an excellent non-avian reptile model organism for enhancing our understanding of the evolution of vertebrate vocalization.

Biomechanical behaviour of lizard osteoderms and skin under external loading.

Many species of lizards are partially enveloped by a dermal armour made of ossified units called osteoderms. Lizard osteoderms demonstrate considerable species-specific variation in morphology and histology. Although a physical/protective role (against predators, prey, conspecifics and impact loading during falls) is frequently advanced, empirical data on the biomechanics of lizard osteoderms are scarce, limiting our understanding of form-function relationships. Here, we report deformation recorded at the surface of temporal osteoderms during controlled external loading of preserved specimens of 11 lizard species (Tiliqua rugosa, Tiliqua scincoides, Corucia zebrata, Pseudopus apodus, Timon lepidus, Matobosaurus validus, Broadleysaurus major, Tribolonotus gracilis, Tribolonotus novaeguineae, Heloderma horridum and Heloderma suspectum). Based on the strain recorded in situ and from isolated osteoderms, the skin of the species investigated can be ranked along a marked stiffness gradient that mostly reflects the features of the osteoderms. Some species such as T. rugosa and the two Heloderma species had very stiff osteoderms and skin while others such as T. lepidus and P. apodus were at the other end of the spectrum. Histological sections of the osteoderms suggest that fused (versus compound) osteoderms with a thick layer of capping tissue are found in species with a stiff skin. In most cases, loading neighbouring osteoderms induced a large strain in the instrumented osteoderm, attesting that, in most species, lizard osteoderms are tightly interconnected. These data empirically confirm that the morphological diversity observed in lizard osteoderms is matched by variability in biomechanical properties.

Sound production in piranhas is associated with modifications of the spinal locomotor pattern.

In piranhas, sounds are produced through the vibration of the swim bladder wall caused by the contraction of bilateral sonic muscles. Because they are solely innervated by spinal nerves, these muscles likely evolved from the locomotor hypaxial musculature. The transition from a neuromuscular system initially shaped for slow movements (locomotion) to a system that requires a high contraction rate (sound production) was accompanied with major peripheral structural modifications, yet the associated neural adjustments remain to this date unclear. To close this gap, we investigated the activity of both the locomotor and the sonic musculature using electromyography. The comparison between the activation patterns of both systems highlighted modifications of the neural motor pathway: (1) a transition from a bilateral alternating pattern to a synchronous activation pattern, (2) a switch from a slow- to a high-frequency regime, and (3) an increase in the synchrony of motor neuron activation. Furthermore, our results demonstrate that sound features correspond to the activity of the sonic muscles, as both the variation patterns of periods and amplitudes of sounds highly correspond to those seen in the sonic muscle electromyograms (EMGsonic). Assuming that the premotor network for sound production in piranhas is of spinal origin, our results show that the neural circuit associated with spinal motor neurons transitioned from the slow alternating pattern originally used for locomotion to a much faster simultaneous activation pattern to generate vocal signals.

Sound production mechanism in triggerfish (Balistidae): a synapomorphy.

The ability to produce sounds for acoustic communication is known in different Balistidae species but the eventual synapomorphic aspect of the mechanism remains to be shown. In Rhinecanthus aculeatus, sounds result from alternate sweeping movements of the right and left pectoral fins, which push a system of three scutes against the swim bladder wall. In this study, we made a comparison between the sounds produced by this species and two additional ones (Balistapus undulatus and Rhinecanthus rectangulus) using hand-held specimens to provide a description of the sound mechanism. The results highlighted that the sound production mechanism is similar in the three species. According to recent phylogenetic data and shared morphological features, this mechanism could be common to the majority of Balistidae family members and all species could be capable of sound production using pectoral fins.

Environmental constraints drive the partitioning of the soundscape in fishes.

The underwater environment is more and more being depicted as particularly noisy, and the inventory of calling fishes is continuously increasing. However, it currently remains unknown how species share the soundscape and are able to communicate without misinterpreting the messages. Different mechanisms of interference avoidance have been documented in birds, mammals, and frogs, but little is known about interference avoidance in fishes. How fish thus partition the soundscape underwater remains unknown, as acoustic communication and its organization have never been studied at the level of fish communities. In this study, passive acoustic recordings were used to inventory sounds produced in a fish community (120 m depth) in an attempt to understand how different species partition the acoustic environment. We uncovered an important diversity of fish sounds, and 16 of the 37 different sounds recorded were sufficiently abundant to use in a quantitative analysis. We show that sonic activity allows a clear distinction between a diurnal and a nocturnal group of fishes. Moreover, frequencies of signals made during the day overlap, whereas there is a clear distinction between the different representatives of the nocturnal callers because of a lack of overlap in sound frequency. This first demonstration, to our knowledge, of interference avoidance in a fish community can be understood by the way sounds are used. In diurnal species, sounds are mostly used to support visual display, whereas nocturnal species are generally deprived of visual cues, resulting in acoustic constraints being more important.

Unusual sound production mechanism in the triggerfish Rhinecanthus aculeatus (Balistidae).

The ability to produce sound has been known for decades in Balistidae. Sounds of many species have been recorded and a variety of sound-producing mechanisms have been proposed, including teeth stridulation, collision of the buccal teeth and movements of the fins. The best-supported hypothesis involves movements of the pectoral fin against the lateral part of the swimbladder, called a drumming membrane. In this study, we describe for the first time the sounds made by the blackbar triggerfish Rhinecanthus aculeatus, which are like short drum rolls with an average duration of 85 ms, 193 Hz dominant frequency and 136 dB SPL level at 3 cm distance. The sounds are a series of pulses that result from alternate sweeping movements of the right and left pectoral fins, which push a system of three scutes that are forced against the swimbladder wall. Pulses from each fin occur in consecutive pairs. High-speed videos indicate that each pulse consists of two cycles. The first part of each cycle corresponds to the inward buckling of the scutes, whereas the second part of the cycle corresponds to an apparent passive recoil of the scutes and swimbladder wall. This novel sound production mechanism is probably found in many members of Balistidae because these peculiar scutes occur in other species in the family. Comparison of sound characteristics from fishes of different sizes shows that dominant frequency decreases with size in juveniles but not in adults.

A superfast muscle in the complex sonic apparatus of Ophidion rochei (Ophidiiformes): histological and physiological approaches.

In teleosts, superfast muscles are generally associated with the swimbladder wall, whose vibrations result in sound production. In Ophidion rochei, three pairs of muscles were named 'sonic' because their contractions affect swimbladder position: the dorsal sonic muscle (DSM), the intermediate sonic muscle (ISM), and the ventral sonic muscle (VSM). These muscles were investigated thanks to electron microscopy and electromyography in order to determine their function in sound production. Fibers of the VSM and DSM were much thinner than the fibers of the ISM and epaxial musculature. However, only VSM fibers had the typical ultrastructure of superfast muscles: low proportion of myofibrils, and high proportions of sarcoplasmic reticulum and mitochondria. In females, each sound onset was preceded by the onset of electrical activity in the VSM and the DSM (ISM was not tested). The electromyograms of the VSM were very similar to the waveforms of the sounds: means for the pulse period were 3.6±0.5 and 3.6±0.7 ms, respectively. This shows that the fast VSM (ca. 280 Hz) is responsible for the pulse period and fundamental frequency of female sounds. DSM electromyograms were generally characterized by one or two main peaks followed by periods of lower electrical activity, which suggests a sustained contraction over the course of the sound. The fiber morphology of the ISM and its antagonistic position relative to the DSM are not indicative of a muscle capable of superfast contractions. Overall, this study experimentally shows the complexity of the sound production mechanism in the nocturnal fish O. rochei.

Hearing capacities and otolith size in two ophidiiform species (Ophidion rochei and Carapus acus).

Numerous studies have highlighted the diversity of fish inner ear morphology. However, the function of the shape, size and orientation of the different structures remains poorly understood. The saccule (otolithic endorgan) is considered to be the principal hearing organ in fishes and it has been hypothesized that sagitta (saccular otolith) shape and size affect hearing capacities: large sagittae are thought to increase sensitivity. The sagittae of many ophidiids and carapids occupy a large volume inside the neurocranium. Hence they are a good structure with which to test the size hypothesis. The main aim of this study was to investigate hearing capacities and inner ear morphology in two ophidiiform species: Ophidion rochei and Carapus acus. We used a multidisciplinary approach that combines dissections, µCT-scan examinations and auditory evoked potential techniques. Carapus acus and O. rochei sagittae have similar maximal diameters; both species have larger otoliths than many non-ophidiiform species, especially compared with the intra-neurocranium volume. Both species are sensitive to sounds up to 2100 Hz. Relative to the skull, O. rochei has smaller sagittae than the carapid, but better hearing capacities from 300 to 900 Hz and similar sensitivities at 150 Hz and from 1200 to 2100 Hz. Results show that hearing capacities of a fish species cannot be predicted only based on sagitta size. Larger otoliths (in size relative to the skull) may have evolved mainly for performing vestibular functions in fishes, especially those species that need to execute precise and complex movements.

Sound production in Onuxodon fowleri (Carapidae) and its amplification by the host shell.

Onuxodon species are well known for living inside pearl oysters. As in other carapids, their anatomy highlights their ability to make sounds but sound production has never been documented in Onuxodon. This paper describes sound production in Onuxodon fowleri as well as the anatomy of the sound production apparatus. Single-pulsed sounds and multiple-pulsed sounds that sometimes last more than 3 s were recorded in the field and in captivity (Makemo Island, French Polynesia). These pulses are characterized by a broadband frequency spectrum from 100 to 1000 Hz. Onuxodon fowleri is mainly characterized by its ability to modulate the pulse period, meaning that this species can produce pulsed sounds and tonal-like sounds using the same mechanism. In addition, the sound can be remarkably amplified by the shell cavity (peak gain can exceed 10 dB for some frequencies). The sonic apparatus of O. fowleri is characterized by a rocker bone in front of the swimbladder, modified vertebrae and epineurals, and two pairs of sonic muscles, one of which (primary sonic muscle) inserts on the rocker bone. The latter structure, which is absent in other carapid genera, appears to be sexually dimorphic suggesting differences in sound production in males and females. Sound production in O. fowleri could be an example of adaptation where an animal exploits features of its environment to enhance communication.

Comparative analysis of osteoderms across the lizard body.

Osteoderms (ODs) are mineralized tissue embedded within the skin and are particularly common in reptiles. They are generally thought to form a protective layer between the soft tissues of the animal and potential external threats, although other functions have been proposed. The aim of this study was to characterize OD variation across the lizard body. Adults of three lizard species were chosen for this study. After whole body CT scanning of each lizard, single ODs were extracted from 10 different anatomical regions, CT scanned, and characterized using sectioning and nanoindentation. Morphological analysis and material characterization revealed considerable diversity in OD structure across the species investigated. The scincid Tiliqua gigas was the only studied species in which ODs had a similar external morphology across the head and body. Greater osteoderm diversity was found in the gerrhosaurid Broadleysaurus major and the scincid Tribolonotus novaeguineae. Dense capping tissue, like that reported for Heloderma, was found in only one of the three species examined, B. major. Osteoderm structure can be surprisingly complex and variable, both among related taxa, and across the body of individual animals. This raises many questions about OD function but also about the genetic and developmental factors controlling OD shape.

Sound production mechanism in Gobius paganellus (Gobiidae).

Gobiidae, the largest fish family (>1500 species), has species from at least 10 genera that produce sounds for communication. Studies focused on goby sound production mechanisms have suggested that sounds are produced by the forcible ejection of water through small apertures in the opercles (hydrodynamic mechanism). The present study was a multidisciplinary investigation (morphology, muscle histology, high-speed video, sound analysis and electromyography) of the sound emission mechanism in Gobius paganellus, which produces both pulsed and tonal calls. Two populations were used, from Brittany and Venice. In the French population, sounds were accompanied by a suite of coordinated movements of the buccal, branchial and opercular regions. This was not the case in the Venetian population, and thus the direct role of head movements in sound production was rejected. The hydrodynamic mechanism hypothesis was also rejected in G. paganellus on the basis of sound oscillogram shape and because sounds are still produced after the opercles and hyohyoid muscles are cut. The use of both electromyography and electron microscopy showed that the levator pectoralis muscle, which originates on the skull and inserts on the dorsal tip of the cleithrum, is involved in sound production. We propose that the contraction of this muscle and associated vibration of the large radials is used to make sounds. In addition, we propose that different sound types (pulsed sounds and tonal calls) could occur because of differences in fish size.

The role of cranial osteoderms on the mechanics of the skull in scincid lizards.

Osteoderms (ODs) are calcified organs formed directly within the skin of most major extant tetrapod lineages. Lizards possibly show the greatest diversity in ODs morphology and distribution. ODs are commonly hypothesized to function as a defensive armor. Here we tested the hypothesis that cranial osteoderms also contribute to the mechanics of the skull during biting. A series of in vivo experiments were carried out on three specimens of Tiliqua gigas. Animals were induced to bite a force plate while a single cranial OD was strain gauged. A finite element (FE) model of a related species, Tiliqua scincoides, was developed and used to estimate the level of strain across the same OD as instrumented in the in vivo experiments. FE results were compared to the in vivo data and the FE model was modified to test two hypothetical scenarios in which all ODs were (i) removed from, and (ii) fused to, the skull. In vivo data demonstrated that the ODs were carrying load during biting. The hypothetical FE models showed that when cranial ODs were fused to the skull, the overall strain across the skull arising from biting was reduced. Removing the ODs showed an opposite effect. In summary, our findings suggest that cranial ODs contribute to the mechanics of the skull, even when they are loosely attached.

Sexual dimorphism of sonic apparatus and extreme intersexual variation of sounds in Ophidion rochei (Ophidiidae): first evidence of a tight relationship between morphology and sound characteristics in Ophidiidae.

BACKGROUND: Many Ophidiidae are active in dark environments and display complex sonic apparatus morphologies. However, sound recordings are scarce and little is known about acoustic communication in this family. This paper focuses on Ophidion rochei which is known to display an important sexual dimorphism in swimbladder and anterior skeleton. The aims of this study were to compare the sound producing morphology, and the resulting sounds in juveniles, females and males of O. rochei. RESULTS: Males, females, and juveniles possessed different morphotypes. Females and juveniles contrasted with males because they possessed dramatic differences in morphology of their sonic muscles, swimbladder, supraoccipital crest, and first vertebrae and associated ribs. Further, they lacked the 'rocker bone' typically found in males. Sounds from each morphotype were highly divergent. Males generally produced non harmonic, multiple-pulsed sounds that lasted for several seconds (3.5 ± 1.3 s) with a pulse period of ca. 100 ms. Juvenile and female sounds were recorded for the first time in ophidiids. Female sounds were harmonic, had shorter pulse period (±3.7 ms), and never exceeded a few dozen milliseconds (18 ± 11 ms). Moreover, unlike male sounds, female sounds did not have alternating long and short pulse periods. Juvenile sounds were weaker but appear to be similar to female sounds. CONCLUSIONS: Although it is not possible to distinguish externally male from female in O. rochei, they show a sonic apparatus and sounds that are dramatically different. This difference is likely due to their nocturnal habits that may have favored the evolution of internal secondary sexual characters that help to distinguish males from females and that could facilitate mate choice by females. Moreover, the comparison of different morphotypes in this study shows that these morphological differences result from a peramorphosis that takes place during the development of the gonads.

Unravelling the structural variation of lizard osteoderms.

Vertebrate skin is a remarkable organ that supports and protects the body. It consists of two layers, the epidermis and the underlying dermis. In some tetrapods, the dermis includes mineralised organs known as osteoderms (OD). Lizards, with over 7,000 species, show the greatest diversity in OD morphology and distribution, yet we barely understand what drives this diversity. This multiscale analysis of five species of lizards, whose lineages diverged ∼100-150 million years ago, compared the micro- and macrostructure, material properties, and bending rigidity of their ODs, and examined the underlying bones of the skull roof and jaw (including teeth when possible). Unsurprisingly, OD shape, taken alone, impacts bending rigidity, with the ODs of Corucia zebrata being most flexible and those of Timon lepidus being most rigid. Macroscopic variation is also reflected in microstructural diversity, with differences in tissue composition and arrangement. However, the properties of the core bony tissues, in both ODs and cranial bones, were found to be similar across taxa, although the hard, capping tissue on the ODs of Heloderma and Pseudopus had material properties similar to those of tooth enamel. The results offer evidence on the functional adaptations of cranial ODs, but questions remain regarding the factors driving their diversity. STATEMENT OF SIGNIFICANCE: Understanding nature has always been a significant source of inspiration for various areas of the physical and biological sciences. Here we unravelled a novel biomineralization, i.e. calcified tissue, OD, forming within the skin of lizards which show significant diversity across the group. A range of techniques were used to provide an insight into these exceptionally diverse natural structures, in an integrated, whole system fashion. Our results offer some suggestions into the functional and biomechanical adaptations of OD and their hierarchical structure. This knowledge can provide a potential source of inspiration for biomimetic and bioinspired designs, applicable to the manufacturing of light-weight, damage-tolerant and multifunctional materials for areas such as tissue engineering.

A review of the osteoderms of lizards (Reptilia: Squamata).

Osteoderms are mineralised structures consisting mainly of calcium phosphate and collagen. They form directly within the skin, with or without physical contact with the skeleton. Osteoderms, in some form, may be primitive for tetrapods as a whole, and are found in representatives of most major living lineages including turtles, crocodilians, lizards, armadillos, and some frogs, as well as extinct taxa ranging from early tetrapods to dinosaurs. However, their distribution in time and space raises questions about their evolution and homology in individual groups. Among lizards and their relatives, osteoderms may be completely absent; present only on the head or dorsum; or present all over the body in one of several arrangements, including non-overlapping mineralised clusters, a continuous covering of overlapping plates, or as spicular mineralisations that thicken with age. This diversity makes lizards an excellent focal group in which to study osteoderm structure, function, development and evolution. In the past, the focus of researchers was primarily on the histological structure and/or the gross anatomy of individual osteoderms in a limited sample of taxa. Those studies demonstrated that lizard osteoderms are sometimes two-layered structures, with a vitreous, avascular layer just below the epidermis and a deeper internal layer with abundant collagen within the deep dermis. However, there is considerable variation on this model, in terms of the arrangement of collagen fibres, presence of extra tissues, and/or a cancellous bone core bordered by cortices. Moreover, there is a lack of consensus on the contribution, if any, of osteoblasts in osteoderm development, despite research describing patterns of resorption and replacement that would suggest both osteoclast and osteoblast involvement. Key to this is information on development, but our understanding of the genetic and skeletogenic processes involved in osteoderm development and patterning remains minimal. The most common proposition for the presence of osteoderms is that they provide a protective armour. However, the large morphological and distributional diversity in lizard osteoderms raises the possibility that they may have other roles such as biomechanical reinforcement in response to ecological or functional constraints. If lizard osteoderms are primarily for defence, whether against predators or conspecifics, then this 'bony armour' might be predicted to have different structural and/or mechanical properties compared to other hard tissues (generally intended for support and locomotion). The cellular and biomineralisation mechanisms by which osteoderms are formed could also be different from those of other hard tissues, as reflected in their material composition and nanostructure. Material properties, especially the combination of malleability and resistance to impact, are of interest to the biomimetics and bioinspired material communities in the development of protective clothing and body armour. Currently, the literature on osteoderms is patchy and is distributed across a wide range of journals. Herein we present a synthesis of current knowledge on lizard osteoderm evolution and distribution, micro- and macrostructure, development, and function, with a view to stimulating further work.

Morphological diversity of acoustic and electric communication systems of mochokid catfish.

Mochokid catfish offer a distinct opportunity to study a communication system transitioning to a new signaling channel because some produce sounds and others electric discharges. Both signals are generated using an elastic spring system (ESS), which includes a protractor muscle innervated by motoneurons within the protractor nucleus that also has a motoneuron afferent population. Synodontis grandiops and S. nigriventris produce sounds and electric discharges, respectively, and their ESSs show several morphological and physiological differences. The extent to which these differences explain different signal types remains unclear. Here, we compare ESS morphologies and behavioral phenotypes among five mochokids. S. grandiops and S. nigriventris were compared with Synodontis eupterus that is known to produce both signal types, and representative members of two sister genera, Microsynodontis cf. batesii and Mochokiella paynei, for which no data were available. We provide support for the hypothesis that peripheral and central components of the ESS are conserved among mochokids. We also show that the two nonsynodontids are only sonic, consistent with sound production being an ancestral character for mochokids. Even though the three sound producing-only species differ in some ESS characters, several are similar and likely associated with only sound production. We propose that the ability of S. eupterus to generate both electric discharges and sounds may depend on a protractor muscle intermediate in morphology between sound producing-only and electric discharge-only species, and two separate populations of protractor motoneurons. Our results further suggest that an electrogenic ESS in synodontids is an exaptation of a sound producing ESS.

Lizard osteoderms - Morphological characterisation, biomimetic design and manufacturing based on three species.

Osteoderms (OD) are mineralised dermal structures consisting mainly of calcium phosphate and collagen. The sheer diversity of OD morphologies and their distribution within the skin of lizards makes these reptiles an ideal group in which to study ODs. Nonetheless, our understanding of the structure, development, and function of lizard ODs remains limited. The specific aims of this study were: (1) to carry out a detailed morphological characterisation of ODs in three lizard species; (2) to design and manufacture biomimetic sheets of ODs corresponding to the OD arrangement in each species; and (3) to evaluate the impact resistance of the manufactured biomimetic sheets under a drop weight test. Skin samples of the anguimorphsH. suspectumandO. ventralis, and the skinkC. zebratawere obtained from frozen lab specimens. Following a series of imaging and image characterisations, 3D biomimetic models of the ODs were developed. 3D models were then printed using additive manufacturing techniques and subjected to drop weight impact tests. The results suggest that a 3D printed compound of overlapping ODs as observed inCoruciacan potentially offers a higher energy absorption by comparison with the overlapping ODs ofOphisaurusand the non-overlapping ODs ofHeloderma.Compound overlapping ODs need to be further tested and explored as a biomimetic concept to increase the shock absorption capabilities of devices and structures.

Neuroanatomical and neurophysiological mechanisms of acoustic and weakly electric signaling in synodontid catfish.

To what extent do modifications in the nervous system and peripheral effectors contribute to novel behaviors? Using a combination of morphometric analysis, neuroanatomical tract-tracing, and intracellular neuronal recording, we address this question in a sound-producing and a weakly electric species of synodontid catfish, Synodontis grandiops, and Synodontis nigriventris, respectively. The same peripheral mechanism, a bilateral pair of protractor muscles associated with vertebral processes (elastic spring mechanism), is involved in both signaling systems. Although there were dramatic species differences in several morphometric measures, electromyograms provided strong evidence that simultaneous activation of paired protractor muscles accounts for an individual sound and electric discharge pulse. While the general architecture of the neural network and the intrinsic properties of the motoneuron population driving each target was largely similar, differences could contribute to species-specific patterns in electromyograms and the associated pulse repetition rate of sounds and electric discharges. Together, the results suggest that adaptive changes in both peripheral and central characters underlie the transition from an ancestral sound to a derived electric discharge producing system, and thus the evolution of a novel communication channel among synodontid catfish. Similarities with characters in other sonic and weakly electric teleost fish provide a striking example of convergent evolution in functional adaptations underlying the evolution of the two signaling systems among distantly related taxa.

Limited possibilities for prezygotic barriers in the reproductive behaviour of sympatric Ophthalmotilapia species (Teleostei, Cichlidae).

Since prezygotic rather than postzygotic barriers are believed to maintain the diversity of closely related sympatric cichlids, differences in phenotypic traits and reproductive behaviours are likely involved in maintaining species boundaries. Here, we focused on the reproductive behaviour of three Ophthalmotilapia species with distributions that only overlap on a small stretch of the shore line of Lake Tanganyika. Repeated introgression of mitochondrial DNA between these species was previously reported, which suggested they can hybridise. Our aim is to test the hypothesis that reproductive behaviour acts as a prezygotic barrier that prevents frequent hybridisation in sympatric Ophthalmotilapia species. We performed a quantitative analysis of twelve reproductions (four for O. ventralis, six for O. nasuta, one for O. boops, and one between a female O. ventralis and a male O. nasuta). Although similar ethograms were obtained for these reproductions, the O. ventralis and O. boops males displayed a behaviour that was never performed by O. nasuta males. This behaviour was displayed during courtship and we called it 'invite'. In O. ventralis, we could show that it was associated with the emission of a single pulse sound. The comparison of O. nasuta and O. ventralis reproductive behaviours also revealed some quantitative differences: O. ventralis males showed the location of the bower more often to the female, whereas O. ventralis females followed the male more often. The similarity between the reproductive behaviours in O. ventralis and O. nasuta could explain the occurrence of the heterospecific spawning event recorded between an O. nasuta male and an O. ventralis female. Importantly, few eggs were laid and the maternal mouthbrooding that resulted from this heterospecific reproduction only lasted for two days, which suggested the abortion of egg development. Hence, in the absence of conspecifics, courtship and mating behaviours alone do not constitute perfect prezygotic barriers between these two species.