Amphibians in Eurasian otter Lutra lutra diet: osteological identification unveils hidden prey richness and male-biased predation on anurans

Introduction

Predator–prey interactions play a major role in shaping community composition and dynamics at local scales, and can shape species richness at the global scale in combination with environmental gradients and interspecific competition (Sandom et al. 2013, Terborgh 2015).

Although terrestrial mammalian predators are, in general, highly specialised, only a minority of species are obligate specialists, i.e. rely on a single food or a handful of food resources; most usually integrate preferred food items with a number of locally and seasonally varying ‘secondary resources’ (Pineda-Munoz & Alroy 2014).

A positive effect of prey diversity on predator population stability was first hypothesised by MacArthur (1955), who envisaged that diversity increases the reliability of the resource pool on which each predator depends. Further mechanisms that enhance predator stability when several prey species are available have been revised by Petchey (2000) and, briefly, include increased total prey biomass, lowered temporal variation in food availability, and increased opportunities to combine complementary foods to achieve a balanced nutrient intake. Diversity of the prey community may also lower resource competition, as food partitioning increases with increasing prey diversity (Sánchez-Hernández et al. 2017).

Assessing the nutritional requirements of a predator and its position along the specialist–generalist continuum is essential for reconstructing food webs and understanding the predator's function in the ecosystem (Remonti et al. 2016). Unfortunately, the investigation in detail of the diversity of species preyed on by elusive mammalian carnivores is often hindered by the difficulty of identifying prey remains to the species level through the analysis of the undigested remains found in either faeces or, to a lesser extent, stomachs (Britton et al. 2006). Low levels of prey identification can lead to unpredictable biases in the estimate of major food-niche and community parameters of mammalian carnivores (Greene & Jaksic´ 1983).

The Eurasian otter Lutra lutra is a top predator of freshwater habitats (Chanin 2003, Prenda et al. 2006, Miranda et al. 2008, Ruiz-Olmo & Jimenez 2009, Clavero et al. 2010, Almeida et al. 2012). During the 20^th century, this mustelid declined dramatically throughout Europe, mainly due to water pollution, hunting and poaching, increased road traffic, and habitat loss (Prigioni et al. 2007). In Italy, the otter is currently recovering (Balestrieri et al. 2016), but it is still classified as Endangered by the International Union for Conservation of Nature (IUCN; Panzacchi et al. 2010).

The semi-aquatic lifestyle of otters is metabolically costly compared to that of other mammals of similar size (Pfeiffer & Culik 1998, Kruuk 2006). Because of this, otters are ravening predators, but they have the most restricted trophic niche in the mustelid family (Jedrzejewska et al. 2001, McDonald 2002). The bulk of the diet of otters is formed by fish, which can constitute up to 95% of their prey (Ruiz-Olmo & Palazón 1997). Nonetheless, in most areas otters also rely on alternative prey, mainly amphibians, crabs, and crayfish (Mason & Macdonald 1986, Ruiz-Olmo & Palazón 1997, Jedrzejewska et al. 2001, Clavero et al. 2003, Prigioni et al. 2006, Remonti et al. 2008, 2009, Smiroldo et al. 2009), and, occasionally, birds (Mason & Macdonald 1986, de la Hey 2008). Habitat type and stability (Jedrzejewska et al. 2001, Smiroldo et al. 2009, Lanszki et al. 2016) and, ultimately, fish availability (Remonti et al. 2008) are the main factors determining the diversity of otter diet.

Amphibian remains are frequently recorded in otter spraints (faeces; e.g. Weber 1990, Brzezinski et al. 1993, Parry et al. 2011), and in several localities amphibians form a major portion of otter diet, particularly during late winter, when the availability of fish prey is reduced (Carss et al. 1990), and in spring, when anurans aggregate in high densities to spawn (Clavero et al. 2005). In Poland (Brzezinski et al. 1993, Jedrzejewska et al. 2001, Pagacz & Witczuk 2010) and Belarus (Sidorovich et al. 1998), frogs have been reported to outweigh fish in otter diet.

However, unlike fish remains, amphibian remains are rarely assigned to species level in dietary studies, and therefore information on the diversity of this important alternative resource is generally scarce (but see Clavero et al. 2005 and Parry et al. 2015). Although several previous reviews have analysed range-scale variability in otter diet, they were mainly focused on fish prey and the effects of variation in its use or availability on the consumption of secondary food resources (Jedrzejewska et al. 2001, Clavero et al. 2003, Balestrieri et al. 2013, Krawczyk et al. 2016, Lanszki et al. 2016). Following on from Ruiz-Olmo's (1995) review on otter predation of reptiles, we reviewed the available literature with the aim of outlining the level of detail currently applied to the class Amphibia in otter dietary studies, and the overall pattern of use of this resource by otters throughout their European range.

Italy has the highest amphibian diversity in Europe (43 species, of which 15 are endemic; Sindaco et al. 2006, Sillero et al. 2014), and amphibians are the main alternative food resource for otters in freshwater ecosystems in Italy (Prigioni et al. 2006, Remonti et al. 2008, Smiroldo et al. 2009). The consumption of amphibians by otters is usually higher in Italy than the average for the Mediterranean region (Remonti et al. 2009). Thus, southern Italy may represent an area of choice for investigating amphibian prey diversity in otter diet. For these reasons, we applied a comparative osteological approach usually used for palaeontological and zooarchaeological studies, with the aim of identifying species and, whenever possible, gender and age of the amphibians preyed on by the Eurasian otters of the Sila Piccola Massif (Calabria region, southern Italy).

Methods

Case study: osteological identification of amphibians

Spraints were collected in a ca. 2040 km² large area of southern Italy (the Sila Piccola Massif, Calabria region), as part of the long-term monitoring of a recently rediscovered otter population (see Gariano & Balestrieri 2018). Otters occurred on six rivers at a total of 12 sampling stations, and 357 spraints were collected, of which 91 in summer 2014 and 2015, and 266 in 2016–2017 (summer: 34; autumn: 30; winter: 85; spring: 117).

Undigested remains were sorted according to standard methodologies (e.g. Smiroldo et al. 2009), and the bones of amphibians were identified using a comparative osteological approach. Taxonomic identification was based on available keys (e.g. Haller-Probst & Schleich 1994, Ratnikov & Litvinchuk 2007, 2009, for caudates; Bailon 1999, for anurans; Figs 1 and 2) and reference collections (see Appendix S1 for a brief description of the most significant diagnostic features).

Bailon's diagnostic keys (1999) are based on anurans found in France, and do not allow discrimination between Rana italica and Rana dalmatina, both currently occurring on the Sila Piccola Massif. Consequently, most brown frog remains were attributed to Rana dalmatina/italica. Nonetheless, based on our observations on available comparative material, the frontoparietals of Rana italica (Fig. 2a–c) show a wide and deep groove on the posterolateral part of their dorsal surface which does not occur in Rana dalmatina (Fig. 2d–e). This feature allowed us to distinguish the two species whenever we found well-preserved frontoparietals.

When identification to species level from only osteological criteria was not possible (e.g. for Lissotriton spp. and Pelophylax spp.), a biogeographical rationale was applied, assigning the remains to the only species currently reported for the study area. Whenever possible, the age (juveniles vs. adults) and sex of each specimen were assessed. Age identification was mainly based on the size and degree of ossification of the bones. The sex of adult (large) Ranidae and Bufonidae was assessed on the basis of the sexually dimorphic mesial crest of the humerus (more robust in males than in females; Bailon 1999).

Diagnostic bones were photographed using a Leica M205 microscope equipped with the Leica application suite V 4.10. This apparatus allows multiple pictures of a single specimen to be taken at different focus planes (‘Z-stacks’). The different pictures are then merged by the image acquisition software to create a new, virtual picture showing the specimen entirely in focus.

Chi-squared tests were used to test for seasonal variation in the raw frequencies of amphibians in otter diet; Kruskal–Wallis tests were used to test for seasonal variation in the mean number of prey individuals per spraint.

Results

Amphibians in otter diet

Sixty-four studies, published as 48 articles between 1969 and 2017, met our criteria (Table 1). They were carried out in 20 European countries, from Sweden in the north to Greece in the south and from Portugal in the west to Lithuania in the east (Fig. 3). The %RF of amphibians in otter diet ranged between 0% in north-east Spain (Melero et al. 2008) to 43% in Poland (Pagacz & Witczuk 2010), averaging 12% ± 3.2 (mean %RF ± standard error; Fig. 4). In most studies, amphibians accounted for <15% of otter diet (Fig. 5). No latitudinal trend could be outlined, but predation of amphibians by otters increased with longitude (Spearman's rho = 0.47, P < 0.001), and from the Atlantic and Mediterranean biogeographical regions (mean %RF = 8.9% and 11%) to the Alpine region (mean %RF = 23%); the Pannonian, Continental, and Boreal regions showed intermediate values (%RF = 12–14%).

Table 1. Amphibians in the diet of the Eurasian otter Lutra lutra in the 64 studies included in this review. Location (country), bio-regions, study period, latitude and longitude, number of otter spraints (faeces) that were analysed (N), classification level used for prey fragments, and percent relative frequency (%RF) of amphibians in otter diet for each of the 64 studies in Europe. Biogeographical regions: 6-BOR = Boreal, 9-MED = Mediterranean, 4-ATL = Atlantic, 7-CON = Continental, 1-ALP = Alpine, 11-PAN = Pannonian. Locations of study codes are shown in Fig. 3, and codes are used in Table 2

Study code	Country	Bio-region	Period	Latitude	Longitude	N	Classification level	%RF	Article reference
1	Lithuania	6-BOR	2006-07	55°17′N	23°55′E	1593	Amphibians	3.4	Baltru−naite˙ (2009)
2	Portugal	9-MED	1991-92	37°38′N	08°39′W	2883	Frogs-toads	9.6	Beja (1996)
3	England	4-ATL	1998-99	50°36′N	03°41′W	161	Amphibians	4	Bonesi et al. (2004)
4	Romania	7-CON	2012-14	45°43′N	27°00′E	233	Species	20.2	Bouros and Murariu (2017)
5	England	4-ATL	2014-15	51°03′N	01°48′E	140	Amphibians	10	Britton et al. (2017)
6	Poland	7-CON	1 year	54°00′N	23°00′E	1202	Amphibians	11.5	Brzezinski et al. (2006)
7		7-CON	1 year	50°20′N	22°40′E	513	Amphibians	12.4
8		7-CON	1 year	53°00′N	23°00′E	1374	Amphibians	12.9
9		1-ALP	1 year	49°20′N	22°40′E	950	Amphibians	27.2
10	Spain	9-MED	2009-10	39°11′N	04°15′W	731	One species	2.17	Bueno-Enciso et al. (2014)
11	Spain	9-MED	1974-79	42°45′N	03°30′W	367	One species	0.5	Callejo and Delibes (1987)
12	Scotland	4-ATL	1987-88	57°01′N	03°15′W	324	Amphibians	2.9	Carss et al. (1990)
13	England	4-ATL	1972-73	50°16′N	03°39′W	389	Amphibians	0.5	Chanin (1981)
14		4-ATL		50°36′N	03°41′W	253	Amphibians	0.6
15	Spain	9-MED	2001-02	36°00′N	05°36′W	1682	Amphibians	6.5	Clavero et al. (2004)
16	Spain	9-MED	1981-82	37°00′N	06°26′W	307	Amphibians	10.7	Delibes et al. (2000)
17	Sweden	6-BOR	1966-68	58°00′N	15°15′E	350	Amphibians	1.6	Erlinge (1969)
18	Portugal	9-MED	2003-04	38°02′N	8°22′W	1680	Amphibians	2.03	Freitas et al. (2007)
19	Italy	9-MED	2001	40°16′N	15°22′E	564	Species	33.73	Fusillo et al. (2003)
20	Germany	7-CON	1994-96	51°20′N	14°58′E	1099	Amphibians	2.5	Geidezis (2002)
21	Bulgaria	1-ALP	2005-06	42°40′N	25°32′E	1155	Species	9.4	Georgiev (2006)
22	Poland	1-ALP	1987-89	49°21′N	22°04′E	379	Species	14.5	Harna (1993)
23	Poland	7-CON	1988-96	52°41′N	23°53′E	396	Species	11.61	Jedrzejewska et al. (2001)
24	Greece	9-MED	2010-12	40°36′N	21°33′E	414	Amphibians	29	Karamanlidis et al. (2013)
25	Hungary	11-PAN	1989	47°17′N	19°13′E	270	Amphibians	0.82	Kemenes and Nechay (1990)
26	Poland	7-CON	1997-98	51°03′N	23°00′E	478	Amphibians	10.7	Kloskowski et al. (2013)
27	Austria	7-CON	1992-93	49°00′N	15°00′E	175	Amphibians	3.1	Knollseisen (1995)
28	Czech Rep.	7-CON	2003-04	50°52′N	14°14′E	349	Amphibians	2.9	Kortan et al. (2010)
29	Ireland	4-ATL	1984-86	53°33′N	07°21′W	2349	Amphibians	15	Kyne et al. (1989)
30	Hungary	11-PAN	1992	46°24′N	17°59′E	873	Amphibians	9.9	Lanszki and Körmendi (1996)
31	Hungary	11-PAN	1996-98	46°14′N	17°29′E	801	Amphibians	4.3	Lanszki and Molnar (2003)
32		7-CON	1999-01	46°18′N	16°52′E	116	Anura	18.3
33		11-PAN	2001-02	46°44′N	17°45′E	234	Anura	34.3
34	France	4-ATL	1982-87	46°16′N	00°43′W	165	Amphibians	9.85	Libois and Rosoux (1991)
35	France	7-CON	1991	45°06′N	02°40′E	704	Amphibians	9.66	Libois (1997)
36	Spain	9-MED	1979	38°22′N	03°49′W	2145	One species	10	López-Nieves and Hernando (1984)
37	Portugal	9-MED	2003-05	40°17′N	06°57′W	206	Species	11.56	Marques et al. (2007)
38	Spain	9-MED	2002-05	41°49′N	01°53′E	108	-	0	Melero et al. (2008)
39	England	4-ATL	2004-05	51°22′N	02°52′W	358	Amphibians	1.96	Miranda et al. (2008)
40	Spain	9-MED	1997	40°00′N	07°00′W	426	Species	6.2	Morales et al. (2004)
41	Ireland	4-ATL	1996	52°12′N	08°06′W	287	Anura	17.1	Ottino and Giller (2004)
42	Poland	1-ALP	2008-09	49°08′N	22°40′E	284	Amphibians	43	Pagacz and Witczuk (2010)
43	Czech Rep.	7-CON	2000-02	49°38′N	18°43′E	400	Anura	15.4	Polednik et al. (2004)
44		7-CON		49°38′N	18°43′E	136	Anura	17.3
45		7-CON		49°35′N	18°45′E	358	Anura	30
46	Italy	9-MED	1987-88	42°40′N	11°35′E	122	Rana spp.	7.6	Prigioni et al. (1991)
47		9-MED		40°35′N	16°25′E	461	Rana spp.	9.1
48		9-MED		42°40′N	11°35′E	148	Rana spp.	10.2
49		9-MED		40°10′N	16°10′E	490	Rana spp.	12
50	Italy	9-MED	1996-97	40°30′N	15°30′E	193	Amphibians	7.2	Prigioni et al. (2006)
51		9-MED	2001-03	40°16′N	16°00′E	555	Amphibians	17.5
52	Italy	9-MED	2001	40°00′N	16°30′E	1323	Amphibians	23.9	Remonti et al. (2008)
53	Spain	9-MED	1984-96	42°00′N	00°00′E	610	Amphibians	1.67	Ruiz-Olmo and Palazón (1997)
54		9-MED		42°00′N	00°00′E	755	Amphibians	4.8
55		9-MED		42°00′N	00°00′E	1432	Amphibians	6.16
56		9-MED		42°00′N	00°00′E	596	Amphibians	9.32
57	Portugal	9-MED	1996-97	40°21′N	08°09′W	1328	Species	3.8	Sales-Luís et al. (2007)
58	Belarus	6-BOR	1988-95	56°00′N	32°00′E	641	Amphibians	35.6	Sidorovich et al. (1998)
59	Italy	9-MED	2006	40°16′N	16°00′E	838	Amphibians	35	Smiroldo et al. (2009)
60	Finland	6-BOR	1988-93	62°15′N	24°25′E	1506	Amphibians	16.5	Sulkava (1996)
61	Denmark	4-ATL	1990-91	56°20′N	09°06′E	587	Amphibians	2.3	Taastrom and Jacobsen (1999)
62		4-ATL		56°20′N	09°06′E	391	Amphibians	19.26
63	Scotland	4-ATL	1987	57°08′N	02°05′W	919	Amphibians	23.5	Weber (1990)
64	Italy	9-MED	2014-17	39°20′N	16°40′E	357	Amphibians	21.6	Smiroldo et al. (in press)

Information on seasonal variation in otter diet was available for 34 studies. Otters preyed on amphibians mostly in winter (mean %RF = 53%) and/or spring (68%), but in some studies peaks in their frequency of use were reported for summer and autumn (mean %RF = 15% and 21%, respectively; χ² = 23.5, 3 d.f., P < 0.001).

Only a minority of studies (17%) classified amphibian prey at the species level: the number of species recorded ranged between 1 and 5 (mean = 3.0). Considering all available studies consulted for this review and our own results (see below), 28 amphibian species have been reported to be preyed on by otters (Table 2), comprising eight Salamandridae (Order Urodela) and three Discoglossidae, two Pelodytidae, three Bufonidae, three Hylidae, and nine Ranidae (all Order Anura). Four of the 28 species (Lissotriton boscai, Discoglossus galganoi, Pelodytes ibericus, and Rana iberica) are endemic to the Iberian Peninsula, and three (Lissotriton italicus, Hyla intermedia, and Rana italica) are endemic to Italy (Sindaco et al. 2006, Speybroeck et al. 2016).

Table 2. Amphibian species preyed on by Eurasian otters Lutra lutra as assessed by reviewing the available literature, including data collected for this study

Family	Species	Country	Study code (see Table 1), references
Salamandridae	Triturus marmoratus	Spain, Portugal	40, 57
	Triturus cristatus	Denmark, England		Britton et al. (2006), Bringsøe and Nørgaard (2018)
	Lissotriton vulgaris	Hungary		Lanszki et al. (2015)
	Lissotriton italicus	Italy	This study
	Lissotriton helveticus	Wales		Parry et al. (2015), Poledník et al. (2007)
	Lissotriton (Triturus) boscai	Portugal	57
	Pleurodeles waltl	Spain		Cogaˇlniceanu et al. (2010)
	Salamandra salamandra	Portugal, Spain	37, 40
Discoglossidae	Discoglossus galganoi	Spain		Romero and Guitián (2017)
	Bombina bombina	Hungary		Lanszki et al. (2015)
	Bombina variegata	Bulgaria, Poland	21, 22
Pelodytidae	Pelodytes ibericus	Spain		Clavero et al. (2005)
	Pelodytes cultripes	Spain		Clavero et al. (2005), Cogaˇlniceanu et al. (2010)
Bufonidae	Bufo bufo	Belarus, Bulgaria, England, Finland, France, Hungary, Italy, Poland, Portugal, Scotland, Spain	21, 23, 57,this study	Beja (1996), Lodè (1996), Sulkava (1996), Sidorovich and Pikulik (1997), Lanszki et al. (2001), McCafferty (2005), Ayres and García (2007), Parry et al. (2011)
	Bufotes viridis	Bulgaria	21
	Bufotes balearicus	Italy	This study
Hylidae	Hyla intermedia	Italy	This study
	Hyla meridionalis	Spain		Clavero et al. (2005)
	Hyla arborea	Romania, Hungary	4	Lanszki et al. (2009)
Ranidae	Pelophylax lessonae	England		Forman et al. (2012)
	Pelophylax (Rana) perezi	Portugal, Spain	10, 11, 40, 57
	Pelophylax (Rana) esculenta	Hungary, Italy, Poland, Romania	4, 19, 23	Lanszki et al. (2015)
	Pelophylax (Rana) ridibunda	Bulgaria, Hungary, Romania, Spain	4, 21, 36	Lanszki et al. (2001)
	Pelophylax bergeri/ hispanicus	Italy	This study
	Rana temporaria	England, Italy, Poland, Romania, Scotland	4, 19, 22, 23	Forman et al. (2012), McCafferty (2005)
	Rana dalmatina	Bulgaria, France, Hungary, Italy, Romania	4, 21, this study	Lodè (1996), Lanszki et al. (2001)
	Rana iberica	Portugal, Spain	40, 57
	Rana italica	Italy	This study

Discussion

Eurasian otters have been recorded to prey on 28 amphibian species, corresponding to 35% of all species available throughout the European range of the otter (Speybroeck et al. 2016). These numbers, which are certainly underestimates, highlight the opportunistic feeding behaviour of the otter, which, although shaped by evolution as a fish-specialist predator, is able to shift to a large variety of semi-aquatic species when fish availability is low (Clavero et al. 2004, Remonti et al. 2008, Smiroldo et al. 2009).

With eight species, Order Urodela consistently contributed to the diversity of otter diet. In the 1980s, Chanin (1985) suggested that newts are either not taken by otters, or they are missed through standard spraint analysis. In the last decade, newt remains have been recovered in the stomachs of otter carcasses in both south-west England (Triturus cristatus; Britton et al. 2006) and Hungary (Lissotriton vulgaris; Lanszki et al. 2015), and in otter spraints in Wales (Lissotriton helveticus; Parry et al. 2015) and in the Czech-Moravian Highlands (Poledník et al. 2007). The negligible percentage of spraint-based studies reporting newts may be due to the difficulty in identifying amphibian remains (Parry et al. 2015).

Further evidence has been obtained by direct observations of otters preying on Triturus cristatus in North Jutland (Bringsøe & Nørgaard 2018) and on Iberian ribbed newts Pleurodeles waltl in southern Spain (Cogaˇlniceanu et al. 2010). In southern Spain, otters ate only the internal organs of ribbed newts, a behaviour that may lead to the underestimation of their contribution to the mustelid's diet (Cogaˇlniceanu et al. 2010).

Many newts produce tetrodotoxin, a powerful neurotoxin and anti-predatory skin secretion, at various concentrations depending on the species (Hanifin 2010), which may explain why otters do not eat newts whole. Otherwise, otters may avoid the ingestion of the poison by skinning the newt, a behaviour that has been described in otters eating toads in Portugal (Beja 1996), Spain (Lizana & Pérez Mellado 1990), Belarus (Sidorovich & Pikulik 1997), Finland (Sulkava 1996), and Wales (Slater 2002). As reported for toads (Lizana & Pérez Mellado 1990), the large amount of time spent handling newts may explain otters’ general preference for frogs, but recent observations (Cogaˇlniceanu et al. 2010) suggest that local availability may also play a major role.

Previous reviews focused on the geographical variability in otter diet. Mason and Macdonald (1986) reported that in southern Europe otters generally eat more amphibians and reptiles than in the north, and Adrián and Delibes (1987) suggested that the frequency of occurrence of amphibians, reptiles, and insects in otter spraints decreases as latitude increases. In contrast, Jedrzejewska et al. (2001) did not find any latitudinal pattern in otter diet in Eurasia, while Clavero et al. (2003) did not include coastal otters in their analyses and found that otters inhabiting Mediterranean localities had more diverse diets than those in northern regions. Both Jedrzejewska et al. (2001) and Clavero et al. (2003) pointed out that habitat features (especially water availability) are important factors influencing the diet of otters. More recently, Balestrieri et al. (2013) found no relationship between otter dietary breadth and latitude, suggesting that habitat-related variations in fish assemblage richness and stability play a major role in shaping otter diet. Accordingly, otters prey on fish more frequently in standing waters surrounded by riparian vegetation than in flowing waters in open habitats (Krawczyk et al. 2016), and fish consumption decreases with stream elevation (Remonti et al. 2009).

In agreement with previous studies (Jedrzejewska et al. 2001, Balestrieri et al. 2013), we could not highlight any latitudinal trend, but the frequency of amphibians in otter diet increased with longitude: the highest %RFs were recorded in the eastern Alpine region (Poland and Bulgaria) and in Boreal regions (Finland, Belarus; see also Lanszki et al. 2016). As suggested by Kruuk (2006), this trend may result from the availability of large numbers of frogs and toads in those regions. Despite this general trend, we recorded a wide variation in amphibian contribution to otter diet (e.g. in southern Italy, %RF ranged between 7.2% and 34%), suggesting that it is shaped by the local availability of prey species, mainly fish (Remonti et al. 2008, Smiroldo et al. in press).

While Anura may be most abundant in north-eastern Europe, the richness of amphibians communities is the highest in Mediterranean Europe (Sillero et al. 2014), as confirmed by the relatively large number of endemic species preyed on by otters in the Iberian and Italian peninsulas. Therefore, to unveil the actual diversity of otter prey in south-western Europe, a detailed analysis of bone remains should be undertaken.

This task is time-consuming and, despite the availability of several useful keys, requires a certain degree of laboratory experience. For this reason, and because research has often been focused on fish prey, only a few of the selected studies reported sufficient information on the diversity of amphibian prey in otter diet. Osteological identification of amphibian remains allowed us to document otter predation on seven out of the 11 species recorded in our study area (64%; Sindaco et al. 2006). Three – Italian newt Lissotriton italicus, Italian tree frog Hyla intermedia, and Italian stream frog Rana italica – are endemic to the Italian peninsula and are listed as Least Concern in the Italian Red List of threatened species (Rondinini et al. 2013). The discrimination of Rana italica from Rana dalmatina was allowed by a newly described diagnostic feature, based on the morphology of frontoparietals, which, unfortunately, were seldom preserved in a condition good enough to allow species identification. The mean number of individual amphibians per spraint (2.2) was higher than that reported in the only previous study in which an attempt was made to assess numbers of otter prey (1.6; Clavero et al. 2005). As recorded throughout Europe, frogs formed the bulk of the amphibian fraction of the diet of the otter.

According to published data, the frequency of otter predation on amphibians is usually the highest in late winter and spring, when amphibians aggregate in large numbers to spawn. However, in our study area, amphibian remains occurred in otter spraints throughout the year, with the exception of autumn, when otters mainly preyed on cyprinid fish (Smiroldo et al. in press). As frogs are often preyed on during spawning, it is hard to discriminate whether seasonal diet shifts depend on their availability or on shortages of fish (Kruuk 2006); nonetheless, in Mediterranean habitats, predation on amphibians is generally inversely related to fish biomass (Remonti et al. 2009, Novais et al. 2010, Krawczyk et al. 2016, but see Ayres & García 2011 for an opposing opinion). Most brown frogs recovered in otter spraints in summer probably belonged to the widespread species Rana italica, which is more strictly associated with freshwater habitats throughout the year than Rana dalmatina (Romano et al. 2012), and which, in summer, is mainly crepuscular and nocturnal (Sindaco et al. 2006). Accordingly, in our study area, Rana italica has been reported to be far more widespread than Rana dalmatina (the two species were found at 41% and 8.0% of 36 sampling stations, respectively; Montillo 2017).

The analysis of bone remains allowed us to document male-biased predation on anurans. Sex-biased mortality induced by predation is a general phenomenon, and males are often the sex paying the heavier toll (Christe et al. 2006). Otters, as predators, are not an exception to this general rule: male Atlantic salmon Salmo salar are more likely to be killed by otters than females (Carss et al. 1990). As with fish, male-biased predation on anurans is likely to result from the breeding behaviour of the prey. First, the number of male anurans at breeding sites greatly exceeds that of females (Merrell 1968, Calef 1973), as females leave the pond soon after laying their eggs (Hartel et al. 2007), while males stay close to breeding sites for longer in order to mate with more females. Moreover, during the breeding season advertisement calls are mainly produced by males, exposing them to a greater risk of being detected by predators. Although we do not know to what extent mustelids rely on auditory cues to find their prey, European polecats Mustela putorius take large numbers of male agile frogs during their spawning season (Lodé 1996, Lodé et al. 2004). Otters preyed on juvenile amphibians only in spring and summer, when their availability is highest, but in very low numbers, suggesting that otters prefer the more profitable adult individuals.

Conclusions

Our results show that, although Eurasian otters prey mainly on fish, amphibians represent an important secondary food resource. The contribution of amphibians to the richness of the otter's prey community is higher than commonly perceived, and osteological analyses allowed us to investigate to a deeper extent the feeding behaviour of this top predator of freshwater habitats. Throughout its wide range, the otter can rely on amphibians wherever fish availability is insufficient, and thus this resource may play a major role in the ongoing recovery of otter populations, especially in parts of the otter's range where freshwater habitats have been deeply altered by human activities (Smiroldo et al. in press).

The dramatic decline affecting amphibian populations at the global scale (Wake & Vredenburg 2008, Blaustein et al. 2011) might threaten the viability of otters living in freshwater systems that have been depleted of their fish communities. In turn, expanding otter populations may affect the survival of small populations of endemic amphibian species, as otters are able to kill several individual amphibians in a very short period of time (Cogălniceanu et al. 2010, Parry et al. 2015). Further studies are needed to understand the potential role played by otters in amphibian conservation, and a detailed knowledge of predator–prey interactions is a first step in that direction.