First documentation of the otoliths of the species of Gouania
(Teleostei: Gobiesocidae) in the Mediterranean Sea
Eleni A. Charmpila
| Maximilian Wagner
| Bettina Reichenbacher
Department of Earth and Environmental
Sciences, Palaeontology and Geobiology,
Institute of Biology, University of Graz, Graz,
Department of Biology, University of
Antwerp, Antwerp, Belgium
Universität München, Munich, Germany
Bettina Reichenbacher, Department for Earth
and Environmental Sciences, Palaeontology
and Geobiology, Ludwig-Maximilians-
Universität München, Richard-Wagner-Str. 10,
D-80333 München, Germany.
Austrian Academy of Science; Austrian
Research Association; University of Graz
Otolith morphology is a widely accepted tool for species identification in teleost fish,
but whether this holds true for very small species remains to be explored. Here,
the saccular otoliths of the cryptobenthic Mediterranean clingfish Gouania
(Gobiesocidae) are described for the first time. The new data, although preliminary,
indicate that otolith morphology and morphometry support the recognition of
the recently differentiated five species of Gouania in the Mediterranean Sea.
Furthermore, otoliths of phylogenetically closely related Gouania species resemble
each other more than do those of the more distantly related species.
clingfish, ecomorphotypes, Mediterranean, otolith morphology
The name “clingfish”collectively refers to small, cosmopolitan species of
the family Gobiesocidae found in intertidal (and freshwater) environ-
ments. The term itself derives from the fact that they attach themselves
to the substrate by means of a ventrally located adhesive disc
(Briggs, 1955; Conway et al., 2017, 2019). Their unusual lifestyle and
cryptobenthic, which in turn suggests that clingfish biodiversity has been
underestimated (Brandl et al., 2018; Wagner et al., 2019, 2020). Within
the Gobiesocidae, this applies in particular to the genus Gouania Risso
1810, which originally included only the species Gouania willdenowi.Nev-
ertheless, recent results from molecular and morphometric analyses
suggested that this endemic Mediterranean genus comprises four addi-
tional species (Wagner et al., 2019) and led to the taxonomic revision of
the genus (Wagner et al., 2020). Accordingly, (a) the species name
G. willdenowi Risso 1810 should be reserved for clingfish inhabiting the
western Mediterranean coasts, (b) two further species are present in the
Adriatic (Gouania pigra Nardo 1827 and Gouania adriatica Wagner
et al., 2020) and (c) two additional species occur in the eastern Mediter-
ranean (Gouania hofrichteri Wagner et al., 2020, and Gouania orientalis
Wagner et al., 2020). Notably, in both the latter regions, the two species
are congruent with two morphotypes –one slender bodied with a small
head and the other stout bodied with a larger head –which suggests
convergent evolution (Wagner et al., 2019).
Otoliths form three pairs of calcium carbonate structures in the
inner ear of teleosts (Popper et al., 2005). The saccular otoliths, usu-
ally the largest of the three pairs, are widely used for the identification
of species, as the morphology of most saccular otoliths has been
shown to be species specific (Nolf, 1985, 2013; Reichenbacher &
Reichard, 2014; Tuset et al., 2008). Hitherto, the otoliths of only a few
species of Gobiesocidae –fossil or extant –have been studied, mainly
from the genus Lepadogaster Goüan 1770 (Schwarzhans et al., 2017;
Smale et al., 1995; Tuset et al., 2008). Here, the saccular otoliths of
Gouania are described for the first time and compared between the
five species to examine the congruence between genetic data,
morphotypes and overall otolith morphology.
In total, 22 saccular otoliths were extracted from 12 specimens –
2 representatives of Lepadogaster lepadogaster Bonnaterre 1788 (from
St. Baska, Croatia, and Agni Beach, Corfu, Greece), 2 specimens of
Received: 19 March 2020 Accepted: 8 December 2020
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any
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© 2020 The Authors. Journal of Fish Biology published by John Wiley & Sons Ltd on behalf of Fisheries Society of the British Isles.
J Fish Biol. 2021;1–6. wileyonlinelibrary.com/journal/jfb 1
G. willdenowi from the western Mediterranean (Messina, Italy), 2 speci-
mens of each species from the Adriatic Sea (G. pigra and G. adriatica,
Glavotok, Otok Krk, Croatia) and 2 specimens of each species from the
eastern Mediterranean Sea (G. hofrichteri from Kapsali, Greece, and
G. orientalis from the Gulf of Corinth, Greece). See Supporting Information
for details on specimens and sites. The same set of specimens was used
in the molecular study published by Wagner et al. (2019, 2020).
Ethical statement: Fish collection and euthanasia were carried out
with the approval of the Ethics Committee of the University of Graz
(permit number: GZ. 39/54/63 ex 2019/20) and in accordance with
EU Directive 2010/63/EU, Annex IV, and the Austrian Animal Experi-
mentation Ordinance, §20.
Left and right saccular otoliths (termed “otoliths”in the following)
were extracted dorsally, and residual tissues were removed by immer-
sion in 1% KOH solution for 3 h. The otoliths were then rinsed in dis-
tilled water for 4 h; if necessary, the procedure was repeated, and the
otoliths were stored in distilled water overnight. Scanning electron
microscopy (SEM) images of all otoliths were obtained using a
HITACHI SU 5000 Schottky FE-SEM at the Department of Earth and
Environmental Sciences (Ludwig-Maximilians- Universität München,
Munich). Otoliths were mounted on aluminium pin stubs (12.5 mm in
diameter, 3.2 ×8 mm), to which adhesive tabs had already been
applied. A thin (20 nm) coating of gold was applied to the stubs (sput-
ter coating) in a high-vacuum coater. The pin stubs were then inserted
into the imaging system, and current (15 kV) was applied.
Morphological descriptions and otolith morphometry were based
on SEM images of all otoliths. The images were processed using
Adobe Photoshop. Figure 1A,B shows the otolith terminology and
morphometry used in this study. SEM images were oriented so that
the ventral margin of the otolith was essentially horizontal (Figure 1A,
B). For otolith morphometry, eight distances were measured from the
otolith images using Image J (Schneider et al., 2012): otolith length
(OL), otolith height (OH), sulcus length (SuL), sulcus height (SuH),
ostium length (OstL), cauda length (CaudL), rostrum length (RoL) and
rostrum height (RoH) (Figure 1B). All distances were measured to the
nearest 0.001 mm. In addition, the perimeter (P) and area (A) of each
otolith were determined (in mm and mm
, respectively). To measure
the lengths and heights of the otolith and sulcus, rectangles enclosing
the dorsal-most, ventral-most, anterior-most and posterior-most
points of the two structures were drawn (Figure 1B). The horizontal
and vertical edges of these rectangles were then taken to represent
the dimensions of interest. Ten otolith variables were calculated
according to Tuset et al. (2003), Reichenbacher et al. (2007) and Gierl
et al. (2018) (Figure 2; Table 1, Supporting Information). The outcome
of the morphometric measurements was then transformed into
descriptive statistics using Past (Hammer et al., 2001).
A summary of the otolith characters and morphometric results of
all studied otoliths is provided in Table 1. The general otolith outline
and sulcus traits are largely similar among the otoliths of G. willdenowi
and those of the four recently (re)described species from the Adriatic
and the eastern Mediterranean Sea (Figure 1C). The otolith outline is
oval to elliptical to slightly triangular; the anterior region is usually
blunt, and the posterior region is round. The sulcus acusticus has a
median to slightly supramedian position and is adjoined by well-
developed thickened (“swollen”) cristae that cover the entire (or almost
the entire) inner portion of the sulcus. The ostium extends to the anterior
margin (=heterosulcoid opening according to Tuset et al., 2008), and it is
separated from the cauda by a prominent structure, the collum. The
ostium is tubular in shape; the cauda is slightly shorter than the ostium
and round to oval in outline. The cauda is straight to slightly inclining and
ends far from the posterior margin. The rostrum is mostly short, round
and broad, whereas the antirostrum is usually absent or poorly defined.
The excisura is narrow and shallow. All otoliths are thick and robust and
exhibit a thicker posterior region when viewed from the ventral side (not
shown). Most of the otolith variables examined indicate overlapping
ranges among the five Gouania species, although there are some excep-
tions (see following text and Table 1).
The otoliths of L. lepadogaster are elliptical to trapezoid in outline
(Figure 1D). The rostrum is well developed. The main morphological
differences compared to the Gouania species are the (relatively) longer
sulcus (in % of OL, see Table 1), the reduced RoH (in % of OH, see
Table 1; Figure 2d) and the absence of “swollen”cristae on the inner
portion of the sulcus (see Figure 1D). In addition, the ranges of the
otolith variable “circularity”differ between Lepadogaster and all other
groups (Table 1; Figure 2a).
As the sample available for each species was small (and the body
sizes of specimens varied within and among groups), no conclusions
could be drawn with regard to within-species variability of the
otoliths such as sexual dimorphism (Teimori et al., 2020; Vaux
et al., 2019), ontogenetic variation (Vignon, 2012; Więcaszek
et al., 2020) or asymmetry between right and left otoliths (Lord
et al., 2012; Lychakov et al., 2008; Panfili et al., 2005). Nonetheless,
some preliminary remarks can be made based on the comparison
between otolith groups. It was observed that the otoliths of G. pigra
and those of G. willdenowi exhibit greater resemblance to each other
than to the otoliths from the other species with respect to circularity
(Figure 2a), RoL (% OL) and RoH (% OH) (Figure 2c,d) and also based
on overall comparison of their SEM images [Figure 1C(a–d)]. Only the
ratio of OstL to CaudL indicated non-overlapping ranges (Table 1).
This high similarity is compatible with their sister relationship
according to molecular data (Wagner et al., 2019; see Figure 2f). The
otoliths of the two stout morphotypes (G. adriatica and G. orientalis)
also exhibit close similarity with each other with regard to the
aforementioned otolith variables (circularity, RoL and height) and little
overlap with the other groups (Table 1; Figure 2a,c,d), which is again
consistent with their sister relation based on molecular data
(Figure 2f). In the case of the two slender morphotypes (G. pigra and
G. hofrichteri), the otolith variables circularity, rectangularity, RoL
(% OL) and RoH (% OH) display no overlap in range (Table 1;
Figure 2a–d). This implies possible differentiation between their oto-
lith morphologies, in spite of their similarity in body shape, and thus
supports the notion that G. pigra and G. hofrichteri are not closely
related and that their slender body shapes result from convergent
evolution (see Figure 2f). On the contrary, “rectangularity”was the
only variable that separated the otoliths of G. hofrichteri, the eastern
Mediterranean slender type, from almost all other otolith groups (the
2CHARMPILA ET AL.
FIGURE 1 Otolith morphology of the clingfish species studied here (left and right sagittal otoliths, inner face). (A) Left otolith of Gouania pigra
(GWK_03) with otolith nomenclature used in this study. (B) Left otolith of Gouania adriatica (GWK_05) with measurements according to
Reichenbacher et al. (2007) and Gierl et al. (2018). (C) Otoliths of Gouania species; (a, b) Gouania willdenowi, specimen numbers GWM_06,
GWM_05; (c, d) Gouania pigra (=slender ecomorphotype from the Adriatic), specimen numbers GWK_13, GWK_03; (e, f) Gouania adriatica (=stout
ecomorphotype from the Adriatic), specimen numbers GWK-01, GWK_05; (g, h) Gouania hofrichteri (=slender morphotype from the eastern
Mediterranean), specimen numbers KYT_22, KYT_23; (i, j) Gouania orientalis (=stout morphotype from the eastern Mediterranean), specimen
numbers GOK_38, GOK_37. (D) Otoliths of Lepadogaster lepadogaster; (k, l) specimen numbers LepKrk7, LGCorf_21. Abbreviations: CaudL, caudal
length; OH, otolith height; OL, otolith length; OstL, ostium length; RoH, rostrum height; RoL, rostrum length; SuH, sulcus height; SuL, sulcus
CHARMPILA ET AL.3
single exception being the stout eastern Mediterranean morphotype,
G. orientalis) (Table 1; Figure 2b). Based on the genetic analyses
reported by Wagner et al. (2019), a higher degree of divergence of
G. hofrichteri from all other groups would be expected.
The results of this study, although preliminary, are largely in
accordance with the genetic results in Wagner et al. (2019). It is
suggested that further exploration of otolith morphology from the
five Gouania species could provide additional support for species
FIGURE 2 (a–e) Summary of the results of otolith morphometry of the studied Gouania specimens, [n] indicates the number of otoliths that
could be used for the measurements; (f) phylogenetic tree modified from Wagner et al. (2019). stout and slender
4CHARMPILA ET AL.
TABLE 1 Description of otolith morphology based on specific otolith characters and ranges of the measured otolith variables among the five species of Gouania and Lepadogaster lepadogaster
Gouania willdenowi, n =3 Gouania pigra, n =4 Gouania adriatica, n =4 Gouania hofrichteri, n =3 Gouania orientalis, n =4 Lepadogaster lepadogaster,n=4
Shape outline Oval to elliptic Oval to elliptic Elliptic to triangular Elliptic squared Elliptic Elliptic to trapezoid
Sulcus position Median Median Median Median Median Median
Sulcus type Ostial Ostial Ostial Ostial Ostial Ostial
Sulcus opening Heterosulcoid Heterosulcoid Heterosulcoid Heterosulcoid Heterosulcoid Heterosulcoid
Ostium shape Tubular Tubular Tubular, slightly
Tubular Tubular Tubular
Cauda shape Round oval Round oval Round oval Round oval Round oval Round oval
Cauda position Ending far from the
Ending far from the
Ending far from the
Ending far from the
Ending far from the
Cauda curvature Straight Straight Straight Straight Straight Straight
Collum Solid bridge Solid bridge Solid bridge Solid bridge Solid bridge Solid bridge
Excisura Very narrow, shallow Very narrow, shallow Narrow, shallow Narrow, shallow notched Slightly deep
Narrow, shallow notched
Rostrum Very short, round, broad Very short, round, broad Long, round, broad Long, round, broad Short, round, broad Long, round to pointed
Antirostrum Absent Poorly defined Poorly defined Poorly defined or absent Short, round, broad Absent
Otolith margins Smooth Smooth Smooth Smooth Smooth Smooth
Posterior region Round Round Round to oblique Round Round Round to oblique
Cristae Well developed Well developed Well developed Well developed Well developed Less well developed
Otolith height (in % OL) 57.383–63.594 59.565–64.465 55.556–57.876 54.577–60.000 56.438–60.521 59.861–63.808
Rostrum height (in % OH) 52.058–55.797 32.439–59.500 53.831–60.412 62.739–67.647 52.330–68.024 40.515–48.519
Rostrum length (in % OL) 9.604–9.985 2.987–11.433 15.195–19.015 17.863–19.014 10.590–23.210 8.576–14.429
Sulcus height (in % OH) 35.024–35.351 34.750–42.265 37.585–42.944 33.226–35.987 31.541–37.740 35.308–39.578
Sulcus length (in % OL) 62.980–64.318 60.220–67.835 58.534–62.468 60.196–62.852 59.806–61.733 68.895–75.000
Ostium length (in % SuL) 55.854–56.490 45.550–54.831 53.620–56.531 49.837–58.523 50.903–57.505 49.798–58.857
Caudal length (in % SuL) 43.510–48.000 45.169–54.450 43.469–46.380 41.477–50.163 42.495–49.097 41.143–50.202
1.265–1.298 0.837–1.214 1.156–1.300 0.994–1.411 1.037–1.353 0.992–1.431
Circularity 15.703–16.391 15.632–16.423 16.742–17.746 17.106–17.205 16.774–19.039 16.499–16.737
Rectangularity 0.128–0.205 0.101–0.130 0.144–0.175 0.072–0.076 0.054–0.184 0.133–0.144
Note: Each species was represented by two specimens, of which both the right and left otoliths (sagitta) were extracted, except in the case of one specimen of G. willdenowi and G. hofrichteri (one otolith was
lost). Bold font indicates character differences between at least two species; circularity was calculated as (P
/A)(P, perimeter; A, area), and rectangularity was calculated as [A/(OL ×OH)]. n: number of otoliths;
OH: otolith height; OL: otolith length; SuL: sulcus length.
CHARMPILA ET AL.5
differentiation within Gouania. Finally, this study contributes to the
expansion of the hitherto-limited clingfish otolith record and offers
new insights into the otolith morphology of the group.
We thank Sandra Bracˇun (Morska Škola Pula, Croatia), Robert
Hofrichter (Mare Mundi, Austria), Samuel P. Iglesias (Muséum
National d'Histoire Naturelle, Paris, France), Enerit Sacdanaku
(University of Tirana, Albania) and Stamatis Zogaris (Hellenic Center
for Marine Research, Greece) for their help with sampling and/or
obtaining sampling permits; Paul Hardy (Düsseldorf, Germany) for crit-
ical reading of the manuscript; and two anonymous reviewers for their
constructive remarks. This work was supported in part by the Austrian
Research Association (ÖFG, to M.W.), the University of Graz (KUWI
stipend & Heinrich-Jörg Foundation, to M.W.) and the Austrian Acad-
emy of Science (ÖAW DOC Scholarship, to M.W.).
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Additional supporting information may be found online in the
Supporting Information section at the end of this article.
How to cite this article: Charmpila EA, Wagner M,
Reichenbacher B. First documentation of the otoliths of the
species of Gouania (Teleostei: Gobiesocidae) in the
Mediterranean Sea. J Fish Biol. 2021;1–6. https://doi.org/10.
6CHARMPILA ET AL.