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F I G U R E 3 in The future of fish-based ecological assessment of European rivers: from traditional EU Water Framework Directive compliant methods to eDNA metabarcoding-based approaches

F I G U R E 3 Comparison of trait-based metrics expressed in relative number of individuals computed from eDNA () and traditional electro-fishing (TEF;) samples in the five river stretches (RS), A, B, C, D and E. Trait categories: BEN, benthic; EUR, eurytopic; INS, insectivorous; OMN, omnivorous; PHY, phytophilic; POT, potamodromous; RHE, rheophilic; TOL, tolerant; PEL pelagic. Significance of the differences between eDNA and TEF metrics are shown: ns, not significant (P> 0.05)

F I G U R E 4 in The future of fish-based ecological assessment of European rivers: from traditional EU Water Framework Directive compliant methods to eDNA metabarcoding-based approaches

F I G U R E 4 Boxplots (, median value;, interquartile range;, full range;, outliers) showing the variability in the eDNAadapted fish index (six metrics) computed at three sites (Brangues, Rhins and Usses) where 10 eDNA water samples were collected once. At each site, the eDNA-based six-metric fish index was computed for each of the 45 possible pairs of samples

F I G U R E 2 in The future of fish-based ecological assessment of European rivers: from traditional EU Water Framework Directive compliant methods to eDNA metabarcoding-based approaches

F I G U R E 2 Comparison of trait-based metrics expressed in number of species computed from eDNA () and traditional electrofishing (TEF;) samples in the five river stretches (RS), A, B, C, D and E. Trait categories: BEN, benthic; EUR, eurytopic; INS, insectivorous; OMN, omnivorous; PHY, phytophilic; POT, potamodromous; RHE, rheophilic; TOL, tolerant; PEL pelagic. Significance of the differences between eDNA and TEF metrics are shown: P <0.05; P <0.01; ns, not significant (P> 0.05)

F I G U R E 7 in Unravelling the taxonomy of an interstitial fish radiation: Three new species of Gouania (Teleostei: Gobiesocidae) from the Mediterranean Sea and redescriptions of G. willdenowi and G. pigra

F I G U R E 7 Gouania hofrichteri sp. nov., PMR VP4595, holotype, male, 30.35+3.67 mm, Souda Beach, Plakias, Crete, Greece. Lateral view of specimen preserved in 4% formaldehyde (top). Lateral, dorsal and ventral view, alive (below). Photographs by M. Wagner and M. Kovacˇic

F I G U R E 1 in The future of fish-based ecological assessment of European rivers: from traditional EU Water Framework Directive compliant methods to eDNA metabarcoding-based approaches

F I G U R E 1 Sampling locations along river stretches (RS) A to E () of the main channel of the Rhône River, France, using both traditional electro-fishing (TEF) and eDNA., Sites sampled every 2 months (September 2015– August 2016);, sites where ten eDNA water samples (filtration capsules) were collected once;, sites located on the tributaries or the Rhône River itself sampled once for eDNA. The 10 metric fish index and the adapted six-metric fish index were computed at all sites with a black filled symbol within natural water bodies

F I G U R E 9 in Unravelling the taxonomy of an interstitial fish radiation: Three new species of Gouania (Teleostei: Gobiesocidae) from the Mediterranean Sea and redescriptions of G. willdenowi and G. pigra

F I G U R E 9 Gouania willdenowi (Risso 1810), PMR VP4574, neotype, male, 46.11+6.56 mm, Cagnes-sur-mer, Nice, France. Lateral view of specimen preserved in 4% formaldehyde (top). Lateral, dorsal and ventral view, alive (below). Photographs by M. Wagner and M. Kovacˇic

F I G U R E 1 0 in Unravelling the taxonomy of an interstitial fish radiation: Three new species of Gouania (Teleostei: Gobiesocidae) from the Mediterranean Sea and redescriptions of G. willdenowi and G. pigra

F I G U R E 1 0 Results of DNA-barcoding analyses. (a) Net between-group mean distances (mean and S.E.). The phylogeny shown in this figure is based on Wagner et al. (2019). (b) DNA-barcoding gap represented by boxplots showing maximum intraspecific and minimum interspecific divergences in %

F I G U R E 6 in Unravelling the taxonomy of an interstitial fish radiation: Three new species of Gouania (Teleostei: Gobiesocidae) from the Mediterranean Sea and redescriptions of G. willdenowi and G. pigra

F I G U R E 6 Gouania orientalis sp. nov., PMR VP4585, holotype, male, 32.8+4.93 mm, Plakias, Crete, Greece. Lateral view of specimen preserved in 4% formaldehyde (top). Lateral, dorsal and ventral view, alive (below). Photographs by M. Wagner and M. Kovacˇic

F I G U R E 8 in Unravelling the taxonomy of an interstitial fish radiation: Three new species of Gouania (Teleostei: Gobiesocidae) from the Mediterranean Sea and redescriptions of G. willdenowi and G. pigra

F I G U R E 8 Gouania pigra (Nardo, 1827), PMR VP3529, neotype, female, 43.12+5.03 mm, Glavotok, Krk, Croatia. Lateral view of specimen preserved in 4% formaldehyde (top). Lateral, dorsal and ventral view, alive (below). Photographs by M. Wagner and M. Kovacˇic

F I G U R E 5 in Unravelling the taxonomy of an interstitial fish radiation: Three new species of Gouania (Teleostei: Gobiesocidae) from the Mediterranean Sea and redescriptions of G. willdenowi and G. pigra

F I G U R E 5 Head osteology of Gouania species. (a) G. adriatica sp. nov. (PMR VP4618 – Holotype), (b) G. orientalis sp. nov. (PMR VP4585 – Holotype), (c) G. hofrichteri sp. nov. (ZSM-PIS- 047656 – Paratype), (d) G. pigra (Nardo 1827) (PMR VP3531 – Other material) and (e) G. willdenowi (Risso 1810) (ZSM-PIS-0476654 – Other material). Red, ceratobranchial 5; orange, premaxillary bone; blue, maxillary bone; green, nasal bone

F I G U R E 3 in Unravelling the taxonomy of an interstitial fish radiation: Three new species of Gouania (Teleostei: Gobiesocidae) from the Mediterranean Sea and redescriptions of G. willdenowi and G. pigra

F I G U R E 3 Gouania adriatica sp. nov., PMR VP4618, holotype, male, 41.41+6.77 mm, Stoja, Pula. Lateral view of specimen preserved in 4% formaldehyde (top). Lateral, dorsal and ventral view, alive (below). Photographs by M. Wagner and M. Kovacˇic

F I G U R E 4 in Unravelling the taxonomy of an interstitial fish radiation: Three new species of Gouania (Teleostei: Gobiesocidae) from the Mediterranean Sea and redescriptions of G. willdenowi and G. pigra

F I G U R E 4 Sucking discs and disc-papillae of Gouania species. (a) G. adriatica sp. nov. (PMR VP4618 – Holotype), (b) G. orientalis sp. nov. (PMR VP4585 – Holotype), (c) G. hofrichteri sp. nov. (PMR VP4595 – Holotype), (d) G. pigra (Nardo 1827) (ZSM-PIS-047649 – Other material) and (e) G. willdenowi (Risso 1810) (PMR VP4574 – Neotype). (f) Males of Gouania can have seemingly perfused prominent finger-like extensions on sucking disc edge in region A.A, B and C correspond to disc regions. Photographs by M. Wagner

F I G U R E 1 in Unravelling the taxonomy of an interstitial fish radiation: Three new species of Gouania (Teleostei: Gobiesocidae) from the Mediterranean Sea and redescriptions of G. willdenowi and G. pigra

F I G U R E 1 Geographical distribution ranges and ecology. (a) Distribution ranges of single Gouania species. The data shown are based on genetic, morphological and field observations as well as on historical findings mentioned by Hofrichter (1995) (all his records combined the species in G. willdenowi described here). Type and location of water currents are based on data provided by El-Geziry and Bryden (2010). Numbers 1–23 indicate sampling sites (compare with Supporting Information Table S1). (b) Female Gouania pigra (Nardo, 1827) in the interstitial of pebbles (photo taken in aquarium); see Supporting Information Video S1 for behaviour. (c) Trstenik (Pelješac, Croatia) – a site where G. pigra, G. adriatica sp. nov. and G. hofrichteri sp. nov. were found in sympatry. Photographs by M. Wagner

FIGURE 3 in An overview of fish bioacoustics and the impacts of anthropogenic sounds on fishes

FIGURE 3 Schematic drawing of the ear of Gadus morhua (anterior is to the left): (a) top view of the body showing the location of the ears in the cranial cavity as well as the proximity of the rostral end of the swim bladder to the ear; (b) lateral and (c) top view of the same ear. Each ear is set at an angle relative to the midline of the fish., The otolith organs,, the semicircular canals (enlarged areas are the ampullae regions that contain the sensory cells);, the dense calcarious otolith lying in close proximity to the sensory epithelium (). Also see Figure 4. Fig. © 2018 Anthony D. Hawkins, all rights reserved

FIGURE 5 in An overview of fish bioacoustics and the impacts of anthropogenic sounds on fishes

FIGURE 5 The sensory epithelia of the end organs of the inner ear have numerous mechanoreceptive sensory hair cells. The apical ends of these cells, directed into the lumen of the epithelia, have ciliary bundles (inserts in the figure) consisting of a single kinocilium (longest of the cilia) and graded stereocilia. Bending of the ciliary bundle during sound stimulation results in neurotransmitter release to stimulate the 8th cranial nerve. The sensory cells on the otolith maculae are organized into orientation groups, with all of the cells in each group having their kinocilia in the same general direction. In this typical saccular epithelium (anterior to the left, dorsal to the top), the cilia on the rostral end are oriented rostrally or caudally, while the cells on the caudal end are oriented dorsally and ventrally., The approximate dividing lines between orientation groups)

FIGURE 2 in An overview of fish bioacoustics and the impacts of anthropogenic sounds on fishes

FIGURE 2 Masking in the Gadus morhua and Salmo salar by ambient noise. The thresholds were determined using a pure tone signal at a frequency of 160 Hz. The ambient noise (natural sea noise, augmented by white noise from a loudspeaker) is expressed as the spectrum level at that same frequency (dB re 1 μPa/Hz). Closed symbols, thresholds to natural levels of ambient noise; open symbols, thresholds to anthropogenic noise. n.b., The thresholds in S. salar were only influenced by high noise levels, above the natural ambient levels of noise (data from Hawkins, 1993). Fig. © 2018 Anthony D. Hawkins, all rights reserved

FIGURE 4 A in An overview of fish bioacoustics and the impacts of anthropogenic sounds on fishes

FIGURE 4 A frontal view of the head of Gadus morhua showing a section of the saccule (). The saccular chamber is filled with perilymph and contains the otolith (), which lies close to the sensory hair cells of the epithelium (macula). The hair cells are innervated by the eighth cranial nerve. Fig. © 2018 Anthony D. Hawkins, all rights reserved

FIGURE 1 in An overview of fish bioacoustics and the impacts of anthropogenic sounds on fishes

FIGURE 1 Fish hearing sensitivity (thresholds) obtained under open sea, free-field, conditions in response to pure tone stimuli at different frequencies. The lower the thresholds (y-axis), the more sensitive the fish is to a sound. Thus, Clupea harengus has best hearing of all of these species over a wider range of frequencies. Note that the thresholds in Gadus morhua and C. harengus obtained under quiet conditions may be below natural ambient noise levels, especially at their most sensitive frequencies. In the presence of higher levels of noise, the thresholds would be raised, a phenomenon referred to as masking. Gadus morhua and C. harengus are sensitive to both sound pressure and particle motion, whereas Limanda limanda and Salmo salar are only sensitive to particle motion. The reference level for the particle velocity is based on the level that exists in a free sound field for the given sound pressure level. n.b., For the particle velocity levels in this figure to match the sound pressure levels in a free sound field it is necessary to calculate an appropriate particle velocity reference level. If the standard reference levels are used, then the curves will not match one another and so they are not included here to keep the figure relatively simple. Fig. © 2018 Anthony D. Hawkins, all rights reserved

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F I G U R E 5 in Length-weight relationships of 55 mesopelagic fishes from the eastern tropical North Atlantic: Across- and within-species variation (body shape, growth stanza, condition factor)

F I G U R E 5 Log–log plot of the relative condition factor (Krel) vs. standard length (cm) calculated from length–weight relationships (LWRs) of the species (a) Argyropelecus affinis, (b) Argyropelecus sladeni, (c) Ceratoscopelus warmingii, (d) Diaphus dumerilii, (e) Electrona risso, (f) Lampanyctus nobilis, (g) Lepidophanes guentheri, (h) Notoscopelus resplendens and (i) Scopelogadus mizolepis (Table 3). Geographic regions are indicated by linetype, symbol and colour (EQ–C, dotted line, dark-blue square; EQ–N, two-dashed line, turquoise triangle; LO–E, solid line, red circle; LO–W, dashed line, violet diamond). If present, vertical dashed grey line indicates breakpoint in the LWR estimated by segmented regression analysis (cf. Table 2)