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Oviparity, viviparity or plasticity in reproductive mode of the olm Proteus anguinus: an epic misunderstanding caused by prey regurgitation?

In: Contributions to Zoology
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Hans Recknagel Department of Biology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000, Ljubljana, Slovenia, hans.recknagel@bf.uni-lj.si

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Ester Premate Department of Biology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000, Ljubljana, Slovenia, Ester.Premate@bf.uni-lj.si

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Valerija Zakšek Department of Biology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000, Ljubljana, Slovenia, Valerija.Zaksek@bf.uni-lj.si

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Gregor Aljančič Society for Cave Biology, Tular Cave Laboratory, Oldhamska cesta 8a, 4000, Kranj, Slovenia, gregor.aljancic@guest.arnes.si

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Rok Kostanjšek Department of Biology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000, Ljubljana, Slovenia, Rok.Kostanjsek@bf.uni-lj.si

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Peter Trontelj Department of Biology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000, Ljubljana, Slovenia, peter.trontelj@bf.uni-lj.si

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Abstract

Cave animals are biological models of fast evolutionary change induced by transition to extreme subterranean environments. But their concealed lifestyle makes it inherently difficult to study life-history changes. Therefore, currently very little is known on the reproduction of cave species, and even less is known on general patterns and potentially shared reproductive strategies. Theory predicts that the cave environment favours the production of a few well-developed offspring and live birth. For one of the most enigmatic cave animals, the olm (Proteus anguinus), it has been debated fiercely whether they reproduce by live birth (viviparity), egg-laying (oviparity) or facultatively. While successes in captive breeding after the 1950s report oviparity as the single parity mode, some historically older observations claimed viviparity. The controversial neo-Lamarckist Paul Kammerer even claimed to have induced changes in parity mode by altering environmental conditions. Here, we report on the feeding and regurgitation of fire salamander (Salamandra salamandra) larvae by olms. The salamander larvae showed clear teeth marks and other injuries on the head caused by the olm, yet one larva was still alive after regurgitation. We suggest that historical reports of olm viviparity could have been misled by regurgitated salamander larvae. Our data bring additional indications that at least some of Kammerer’s experiments were fraudulent.

Introduction

Reproduction in cave animals

Animals that colonize caves are faced with considerable changes in abiotic and biotic conditions compared to the ancestral life at the surface. Among these, the absence of light and low food availability probably represent the strongest selective pressures (Moldovan et al., 2018; Culver & Pipan, 2019). Evolutionary changes associated with cave colonization affect among other traits behaviour and life-history, for example through the loss of circadian rhythm, reduction in metabolism and aggressive behaviour, and a change in reproductive strategies (Poulson & White, 1969; Howarth & Moldovan, 2018). While currently not much is known on the reproductive biology of most cave animals, theory suggests that the low food availability favours an increase in egg and offspring size, a decrease of offspring number, iteroparity, and viviparity (Hüppop, 2000; Niemiller & Soares, 2014; Kováč, 2018; Fišer, 2019). Observational data under natural conditions are still extremely rare to absent; reproductive strategies have been inferred in a few cases from animals collected in the field (Fišer et al., 2013; Cieslak et al., 2014) and from cave animals that have been successfully bred in captivity (Poulson, 1963; Riesch et al., 2010).

Among amphibians, viviparity is a very rare strategy in general, and for urodeles only a few cases within the family of Salamandridae have been confirmed (Buckley, 2012; Blackburn, 2015). However, two species associated with caves, the cave-obligate olm (Proteus anguinus Laurenti, 1768) and Speleomantes sarrabusensis Lanza, Leo, Forti, Cimmaruta, Caputo & Nascetti, 2001, a salamander that is often found in caves, have been reported to be viviparous (Michahelles, 1831; Nusbaum, 1907; Kammerer, 1912; Lanza, 2006). Both cases are controversial, and more recent data suggest oviparity for both species as their natural parity mode (Lunghi et al., 2018; Blackburn, 2019).

Historical notes on olm reproduction

The reproduction of Proteus anguinus has been a long-standing mystery and debated vigorously in scientific literature (Aljančič, 2019). One of the most controversially discussed features – even today – is whether olms give birth to live young (viviparity), lay eggs (oviparity) or possess the ability to switch between the two parity modes (facultative oviparity/viviparity) (Aljančič & Aljančič, 1998; Blackburn, 2019; Nahm, 2021). The controversy partially stems from the lack of data on the biology of olms due to their subterranean lifestyle secluded from human sight and scientific observation. In addition, olms that have been found in the wild are often either not sexually active or not in a gravid state. In captivity, gravidity may occur less frequently than every seven years (Juberthie et al., 1996). To top it all, breeding olms in captivity has proven extremely difficult, with historically controversial results and even presently limited success. After its first description in 1768 by Laurenti, the olm’s reproduction remained a complete mystery for about 60 years, with failed attempts at discovering gravid females and/or breeding them (von Schreibers, 1801; Configliachi & Rusconi, 1819; Fitzinger, 1850; Kammerer, 1912).

The first report on olm reproduction in 1831 claimed viviparous reproduction, based on the observation of a local landowner on an olm caught from the vicinity of Stična on the morning of the 17th of June 1825 (Michahelles, 1831). This individual, described as unusually fat and about 25 cm in length was kept in a bottle with water and observed during the day by the landowner, his family and visitors. During the same day in the afternoon, the animal was reported to have given birth to two fully developed larvae of about 4 cm of length. The larvae were of a ‘dirty-yellow’ colouration, with the eyes free and clearly visible and of the size and shape of a poppy seed and still surrounded by the jelly capsule, which was subsequently removed by the mother after close inspection of the young. The landowner further reported that the mother frequently approached its young to inspect them in a nurturing way. This behaviour included the attempt to bring the two larvae together, touching and embracing them, and laying its head on them (Michahelles, 1831). Over the next night, another young was born, making it a total of three live-born offspring reported within 24 hours of the female’s capture.

Later reports on reproduction in olms refuted viviparity and claimed oviparity as mode of reproduction (Schulze, 1876; von Chauvin, 1882; Zeller, 1888). These studies were conducted in captivity, and it is notable that reproduction in the wild (or very shortly after capture) was not reported again. After the reports by Schulze, von Chauvin and Zeller the mystery seemed solved, and oviparity was accepted by researchers commenting on the topic (Brehm, 1878; Leunis, 1883). However, doubts reappeared after Nusbaum reported that one of his five captively held olms (under daylight conditions, no food provided) gave birth to a large, around 12.6 cm long young after being in captivity for 13 months (Nusbaum, 1907). The incident occurred during night, and the weak young died on the same day just a few hours later. He suspected that the olm’s embryo nourished itself from undeveloped eggs in the oviduct, similar to what had been described in Alpine salamanders (Salamandra atra). He further presumed that the unnatural, stressful conditions that the olm was kept in led to this unusual behaviour. At about the same time, Kammerer attempted to breed 40 olms that were held captive in an underground cistern at the Biologische Versuchsanstalt in Vienna since 1903. After observing an “apparently gravid” female, Kammerer isolated it from the group, and reported that it gave birth to two extremely large young (9.9 and 11.4 cm) two weeks later overnight (Kammerer, 1912). He did not identify any egg remains. In line with his experiments on observed plasticity in parity mode of fire salamanders (Salamandra salamandra) and common lizards (Zootoca vivipara) due to different temperature regimes, he claimed that olms naturally give birth to living young in caves (below 15°C), while oviparity is artificially induced by unnatural conditions experienced in captivity (above 15°C) (Kammerer, 1912). Indeed, it is now known that fire salamanders can give birth to larvae (larviparous) or fully developed offspring (pueriparous) (Velo-Antón et al., 2012; Rodríguez et al., 2017), and common lizards can be oviparous or viviparous (Mayer et al., 2000; Recknagel et al., 2018). However, parity mode is fixed within individuals, and variation does only occur at the level of separate and divergent evolutionary lineages. Interestingly, Wunderer (1910) noted that the differences in reproduction could also be attributed to the fragmentation of subterranean habitats and consequently structuring into distinct lineages. However, Kammerer stressed that his and Nusbaum’s material stem from animals caught in the Postojna-Planina Cave System, while the report from Michahelles (1831) is based on olms captured near Stična, representing alleged divergent lineages (Gorički & Trontelj, 2006).

After the discovery of Kammerer committing scientific fraud, some of his results were dismissed by the scientific community of his time, and studies from the 20th century unequivocally report oviparity as parity mode in captively bred olms (Vandel & Bouillon, 1959; Briegleb, 1962; Aljančič & Aljančič, 1998). In addition, the first washed-out eggs were found in the spring near Stična (Sket & Velkovrh, 1978), also suggesting oviparous reproduction in the natural habitat of olms. The mystery seemed solved, if not for the earlier reports of viviparity, and more recently expressed doubts on the alleged fraught by Kammerer; the discovery of epigenetic mechanisms explaining phenotypic plasticity have led some researchers to attempt to rehabilitate Kammerer and his results (Vargas et al., 2017; Nahm, 2021). Moreover, the recent discovery of the potential for facultative viviparity in a lizard (Laird et al., 2019) shows that biologically, some degree of plasticity in parity mode may not be an impossibility after all.

Diet and feeding habits of olms

While not much is known on reproduction in cave animals, their feeding habits are somewhat better studied (Moldovan et al., 2018; Culver & Pipan, 2019; Delić & Fišer, 2019). As other freshwater animals in caves, olms are generally limited to the food available to them in the subterranean aquifers. Direct observation of olm feeding habits in their natural environment are rare, except for an observation of feeding on invertebrates (Aljančič, 1961; Briegleb, 1962) and a single observation of an olm ingesting a minnow (Phoxinus sp.) (Bressi, 2004; Balázs & Lewarne, 2017). In captivity, olms seem to feed opportunistically, as seen in other salamanders (Delić & Fišer, 2019; Gorički et al., 2019). Olms usually slowly approach the prey, and then wait for the prey to move closer to their mouth (Briegleb, 1962). They have well-developed mechanosensory receptors along their body, and in high density on their snout (Istenič & Bulog, 1984; Bizjak Mali & Sket, 2019), presumably playing a crucial role in prey detection (Uiblein et al., 1992). These allow them to sense movements in the water, and eventually triggers their feeding response. In caves, their main potential prey constitutes of crustaceans such as Troglocaris and Niphargus, as well as snails that have been found in stomach contents (Aljančič, 1961; Briegleb, 1962; Delić & Fišer, 2019).

Caves are usually characterized by a generally low and periodic food supply. Faced with these conditions, most subterranean organisms are adapted to resist long periods of starvation and use their energy stores efficiently (Hervant et al., 2001). For olms, this includes using fat stores during starvation and saving muscle energy, as well as adjusting their behaviour, avoiding fast movements and using their long-range mechanosensory system to detect food (Hervant et al., 2001). Once food supply increases, olms can efficiently hunt and ingest their prey, quickly recovering and increasing their body mass (Hervant et al., 2001). One way to exploit a habitat rich in foods is accessing the surfacing of subterranean waters in karst springs. Here, the density and diversity of prey available to olms can be higher than in the subterranean environment (Gibert & Deharveng, 2002). It has been suggested that olms may visit springs for feeding on the multitude of small aquatic fauna, such as oligochaetes and tadpoles (Bressi et al., 1999). However, it is currently unknown how commonly olms access springs and ingest food items only available to them outside the subterranean environment.

Materials and methods

Sampling

Olm sampling at several sites across Slovenia is performed routinely as part of the development of a national monitoring scheme using genetic sampling (Zakšek et al., 2018). During these surveys, olms are captured by hand nets and transferred to a bucket with water sampled from the same location. After capture, their length (in a tray with a ruler, to the nearest 0.1 cm) and mass (with a spring scale, to the nearest 0.1 g) is recorded. Next, photographs and a swab of the skin mucus are routinely taken. Each individual is then released at the site where it was captured. Swabs are used later in the laboratory for dna extraction, and genetic identification of individuals and inference of population structure (Zakšek et al., 2018). Olms were caught under the permit no. 35601–37/2021–4, issued by the Slovenian Environment Agency.

Imaging

Images of the adult olm individual and the salamander larvae were taken using a Canon eos 90D camera with a Canon mp-e 65mm f/2.8 1-5x Macro Photo lens or a Canon ef 100mm f/2.8 Macro usm lens. In addition, both salamander larvae were prepared for scanning electron-microscopy (sem) as described previously (Bizjak-Mali et al., 2018) and images were taken by a jsm 7500F (Jeol, Japan) field emission scanning electron microscope.

Results

On the 8th of May 2021 an olm was captured at a temporary spring in the vicinity of the town of Stična in central Slovenia and kept in a bucket with water. At high water levels, the temporary spring is connected to a permanent stream, from which many invertebrates and amphibian larvae colonize it.

While measuring the captured olm (weight: 21.5 g; total length: 22.5 cm), it regurgitated two larvae of Salamandra salamandra (fig. 1). The larvae measured 3.1 cm (fig. 2A) and 3.2 cm (fig. 2B, C), with 0.184 g and 0.224 g of weight, respectively. The first larva was still alive and swimming (fig. 1B, C, D). It had minor abrasions on the dorsal surface of the head, but otherwise no external injuries inflicted by the predator were visible. Scanning electron microscopy revealed a set of parallel teeth marks (fig. 3A, B) corresponding to the olm’s jaw on the lateral and dorsal sides of the larva’s head. The second larva had substantial laceration above the left gills (fig. 2C) and was not moving. A combination of parallel teeth marks on the anterior edge of the laceration (fig. 3C) and above the eye on its right side (fig. 3D) were observed by sem, corresponding to the size and shape of the olm’s jaws.

Figure 1
Figure 1

Image of an olm (Proteus anguinus) regurgitating a fire salamander (Salamandra salamandra) larva. A) Image of an olm (22.5 cm of total length), moments before B) it started to regurgitate a salamander larva (3.1 cm of total length). C) shows a close-up of the salamander head and the larva fully regurgitated, with D) showing details of the still alive salamander larva.

Citation: Contributions to Zoology 91, 3 (2022) ; 10.1163/18759866-bja10029

Figure 2
Figure 2

Regurgitated salamander (Salamandra salamandra) larvae. The first image A) shows the larva (3.1 cm of total length) still alive after regurgitation with no obvious damage visible; B) shows the second larva (3.2 cm of total length), with heavier damage around the gills. A close-up of the respective damaged area of the C) neck and gills of the larva is shown.

Citation: Contributions to Zoology 91, 3 (2022) ; 10.1163/18759866-bja10029

Figure 3
Figure 3

Scanning electron micrographs of the olm’s teeth marks on regurgitated salamander (Salamandra salamandra) larvae. Heads of all larvae are facing towards the left. A) Teeth marks (arrowheads) on the dorsal side of the head corresponding to the position of the olm’s jaw and B) parallel teeth marks on the left side of the head above the gills (g) of the larvae shown in fig. 2A. C) Teeth marks on the anterior edge of laceration above the left gills (g) and D) above the right eye (e) of the head of the larva shown in fig. 2B and C. (Scale bars represents 500 µm).

Citation: Contributions to Zoology 91, 3 (2022) ; 10.1163/18759866-bja10029

The olm was released about 20 minutes after capture. External inspection revealed that its gut still contained one or more further food items roughly the size of a salamander larva, visible through the partly translucent body wall.

Two days later, on the 10th of May, a similar incident at the same location occurred, although regurgitation was not directly observed: Six olms were captured and transferred to a bucket with water. Minutes later, a free-swimming Salamandra salamandra larva was observed among the six captured olms.

Discussion

For the first time, here we recorded regurgitation of salamander larvae by olms. Previous research on the olm’s diet mainly reported ingestion of macroinvertebrates such as niphargids and cave shrimps, while a few other observations report that larger food items such as minnows (Aljančič, 1961; Balázs & Lewarne, 2017) and tadpoles (Bressi et al., 1999) may also be ingested. Importantly, these larger food items originate from outside the subterranean system and are only occasionally or seasonally available to olms. In springs, larger items are more readily available, and might constitute a regular seasonal food source. Certainly, our observation confirms previous reports that larger prey animals (more than 3 cm) can be captured by olms. The other important observation made here was the regurgitation of the captured food items. Food regurgitation has been previously observed in olms after capture (Freyer, 1850; Briegleb, 1963; Sket & Arntzen, 1994), indicating that this is a behaviour caused by stress.

By implication, the combination of salamander larvae feeding and later regurgitation may explain the first record of viviparous reproduction in olms (Michahelles, 1831). Admittedly, birthing is a logical explanation for the sudden appearance of aquatic salamander babies in a bottle with a single adult animal. The time of the historical observation, known as the Stratil protocol (Michahelles, 1831), coincides with the season of occurrence of Salamandra salamandra larvae. Mouth contact between the adult and the “babies” does take place (fig. 1). A century and a half later, eggs have been found at this location (Sket & Velkovrh, 1978), refuting the possibility that viviparity was a fixed trait of this lineage. The Stratil protocol contains further inconsistencies, including the observation of a conspicuously large cloaca of the alleged female and pigmented larvae. In olms, sex determination based on external characteristics is difficult, but a swollen cloacal region is mostly associated with males (Briegleb, 1962; Guillaume, 2001a, b), indicating that there is a fair chance that the observed adult animal was a male. Olm eggs had been reported as unpigmented already in earlier studies (Schulze, 1876; von Chauvin, 1882), while reports on the colouration of young larvae were more ambiguous, also due to the lighting conditions that they were exposed to (Zeller, 1888; Wiedersheim, 1890). More recent captive breeding in the dark revealed that larvae were very weakly pigmented (sparsely scattered melanized pigment cells), with a prominent whitish abdominal region from the yolk mass (Aljančič & Aljančič, 1998). Indeed, both eyes and pigmentation are visible in young olm larvae, although body proportions are clearly distinct (fig. 4).

Figure 4
Figure 4

Proteus anguinus larva (captive-bred from Tular Cave Laboratory, photograph by Gregor Aljančič) and Salamandra salamandra larva in comparison. Both larvae are about 3 cm in size. Note the difference in size and shape of the head and trunk length, but similarities in presence of eyes and pigmentation.

Citation: Contributions to Zoology 91, 3 (2022) ; 10.1163/18759866-bja10029

photograph of proteus anguinus larva by gregor aljančič and salamandra salamandra larva by james burgon

Hence, our observations of regurgitation of live salamander larvae provide the most plausible explanation of the historical event reported in the Stratil Protocol and interpreted as a case of viviparity in Proteus anguinus. A similar explanation for the sudden occurrence of “dead olm babies” alongside captured adult animals was offered already by Freyer in 1850, who attributed the regurgitated youngsters to cannibalism (Freyer, 1850).

The cases of Kammerer and Nusbaum are even more mysterious, as the new born reported are extremely large, corresponding to the length olms usually reach at the age of at least 2–3 years (Durand & Delay, 1981). There is currently no rational explanation for these supposed live births of large offspring other than accidents due to stressful rearing conditions (Nusbaum, 1907), data manipulation, or a yet unidentified mechanism. Distinct evolutionary lineages of olms exist (Gorički & Trontelj, 2006), and it is therefore conceivable that these differ in their parity mode, similar to fire salamanders or reproductively bimodal lizards (Smith et al., 2001; Rodríguez et al., 2017; Recknagel et al., 2018; Recknagel & Elmer, 2019). On the other hand, individuals available to historical breeding collections belong to the same two lineages that are reliably reported to lay eggs (Sket & Velkovrh, 1978; Aljančič, 2019; Blackburn, 2019). Unless parity mode is plastic within these two lineages, previous reports on olm viviparity are implausible. Long-term breeding experiments with captive olms in Moulis and the Tular Cave laboratory have not revealed a single case of viviparity (Aljančič, 2019). Therefore, we conclude that historical claims of viviparity are most probably the product of misinterpreted observation of regurgitated live fire salamander larvae or young olms. However, we cannot fully dismiss the possibility of viviparous reproduction in other evolutionary lineages of olms on which no reproductive data is available. These lineages are separated by millions of years of independent evolution (Trontelj et al., 2007). Given the rarity of viviparous reproduction across salamanders, it is unlikely that any of these may be viviparous.

The possibility of viviparous reproduction in olms was almost fully dismissed by researchers in the 20th century after repeated observations of egg-laying, if it were not for the re-interpretation of Kammerer’s results after the turn to the 21st century. The resurgence of phenotypic plasticity as an important factor for organismal survival and the identification of epigenetic modification as a driving molecular mechanism behind plasticity, a new enthusiasm for understanding Kammerer’s experiment and results have emerged (Vargas, 2009; Vargas et al., 2017; Sanchez et al., 2019; Nahm, 2021). After the discovery of data manipulation in some of Kammerer’s experiments during his lifetime, his research was dismissed by the scientific community of that time. Indeed, it is unequivocal that at least some of his experiments did not only lack data to prove his claims, but contained deliberate manipulations, such as the artificially swollen nuptial pads on midwife toads, the discovery of which eventually marked his scientific downfall (van Alphen & Arntzen, 2017).

editor: a. ivanović

Acknowledgements

We acknowledge the financial support from the Slovenian Research Agency to the Infrastructural Centre “Microscopy of Biological Samples” located in the Biotechnical faculty, University of Ljubljana, part of the mric ul network. We are further thankful for financial support from a Marie Skłodowska-Curie Actions (msca) individual global fellowship, project genevolcav (grant no. 897695) for hr. ep is supported by the Slovenian Research Agency through a PhD grant and by the University foundation of eng. Milan Lenarčič. Research on Proteus (vz, rk, pt) is supported by the Slovenian Research Agency through Research Project N1-0096 and Research Core Funding P1-0184. We would like to thank two anonymous reviewers for their constructive comments on improving this manuscript.

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