Rediscovery of Knipowitschia goerneri and its molecular relationships with other European northern Mediterranean Knipowitschia species (Teleostei: Gobiidae)

Abstract

The Corfu dwarf goby, Knipozvitschia goerneri, known only from the Korission Lagoon catchment on Kerkyra Island (Greece), was described in 1991 on the only existing material, consisting of five specimens. It has not been observed since 1983, and was thought to be possibly extinct. In 2014, we collected nine specimens of K. goerneri from Korission Lagoon. We found this species to be morphologically and genetically well distinguishable from all other Knipozvitschia species. Analyses of the mitochondrial gene cytochrome b placed it as a sister species of K. milleri, which is another goby from the northern Ionian Sea basin.

Full text

Ichthyol. Explor. Freshwaters, Vol. 26, No. 4
363
Ichthyol. Explor. Freshwaters, Vol. 26, No. 4, pp. 363-372, 3 figs., 2 tabs., March 2016
© 2016 by Verlag Dr. Friedrich Pfeil, München, Germany – ISSN 0936-9902
Rediscovery of Knipowitschia goerneri
and its molecular relationships with other European
northern Mediterranean Knipowitschia species
(Teleostei: Gobiidae)
Jasna Vukib*, Marcelo Kovacib**, Stamatis Zogaris*** and Radek Šanda****
The Corfu dwarf goby, Knipowitschia goerneri, known only from the Korission Lagoon catchment on Kerkyra Island
(Greece), was described in 1991 on the only existing material, consisting of five specimens. It has not been observed
since 1983, and was thought to be possibly extinct. In 2014, we collected nine specimens of K. goerneri from Koris-
sion Lagoon. We found this species to be morphologically and genetically well distinguishable from all other
Knipowitschia species. Analyses of the mitochondrial gene cytochrome b placed it as a sister species of K. milleri,
which is another goby from the northern Ionian Sea basin.
* Department of Ecology, Faculty of Science, Charles University in Prague, Vinicná 7, 128 44 Prague, Czech
Republic. E-mail: jvukic@seznam.cz (corresponding author)
** Prirodoslovni muzej Rijeka, Lorenzov prolaz 1, HR-51000 Rijeka, Croatia.
E-mail: marcelo@prirodoslovni.com
*** Hellenic Centre for Marine Research, Institute of Marine Biological Resources and Inland Waters, Anavissos,
Attiki 19013, Greece. E-mail: zogaris@gmail.com
**** Department of Zoology, National Museum, Václavské nám. 68, 115 79 Prague, Czech Republic.
E-mail: rsanda@seznam.cz
Introduction
A renewed interest in the European fish fauna has
resulted in the recent discovery or recognition of
many new species, especially from the Mediter-
ranean region (e. g., Freyhof et al., 2005; Kovacib,
2005; Kottelat, 2007; Miller & Šanda 2008; Buj et
al., 2010). This is an area with an exceptionally rich
freshwater ichtyofauna (Freyhof & Brooks, 2011),
and the number of species continues to increase
(e. g., Buj et al., 2014; Freyhof et al., 2014). Many
freshwater fish species in the region are endemic
to a single or a few neighbouring rivers, and are
under threat from human activities (Smith &
Darwall, 2006; Freyhof & Brooks, 2011).
Several fish species from the Mediterranean
area are currently considered extinct (Smith &
Darwall, 2006; Geiger et al., 2014). However, it is
possible that some of these have been overlooked
due to their cryptic behaviour, small size, absence
of real search effort, lack of interest, political or
social situation, or administrative obstruction.
One of the endemic species with an unclear
conservation, as well as taxonomic status, is the
Corfu dwarf goby, Knipowitschia goerneri Ahnelt,
1991. Known only from the Korission Lagoon

364
Vukib et al.: Rediscovery of Knipowitschia goerneri
catchment on Kerkyra Island (Corfu), Greece
(Ahnelt, 1991), this goby was described from the
only existing material (five specimens) collected
in 1968 and 1983, and taken from a freshwater
spring and the brackish coastal lagoon, respec-
tively. It was not observed in nature after 1983
(Kottelat & Freyhof, 2007), although at least one
ichthyological survey was conducted at various
localities on Kerkyra Island since this time (Kalo-
gianni et al., 2006), and K. goerneri was suggested
to be extinct (Economidis & Chrysopolitou, 2009).
In the IUCN red list, this goby is listed as data
deficient (Crivelli, 2006), as it is one of the least
studied freshwater fish species with practically
nothing known about its biology or ecology.
During an ichthyological survey on Kerkyra
Island in July 2014, several specimens of Knipo-
witschia were collected in Korission Lagoon
(Fig. 1). The aim of this work is to investigate
the taxonomic status of this population and to
provide information on its molecular relationships
with other Knipowitschia species known from the
European northern Mediterranean region.
Materials and methods
Counts and measurements follow Miller (1988),
as other recent descriptions of Knipowitschia spe-
cies also use the same methodology (Ahnelt 1995,
2011; Kovacib, 2005; Kovacib & Šanda, 2007; Miller,
2009). The length of the specimens is presented
as standard length, and the terminology of the
cephalic sensory systems follows Sanzo (1911)
and Miller (1972). All specimens were stained in
a 2 % solution of Cyanine Blue in distilled water
using a reversible staining method (Saruwatari et
al., 1997) to more clearly visualise scales and the
cephalic sensory systems.
Abbreviations: SL, standard length; COI,
cytochrome c oxidase subunit 1. Collection codes:
NMP, National Museum, Prague; NMW, Natur-
2
34
5
6
7
8
9
10 11
12
14
13
15
1
Korission
Lagoon
Southern Kerkyra (Corfu)20°
Albania
22° 24°
0 50 100 km
38°
40°
a
bc
A
e
g
e
a
n
S
e
a
P
i
n
d
o
s
M
o
u
n
t
a
i
n
s
I
o
n
i
a
n
S
e
a
Fig. 1. a, Location of Korission Lagoon, locality of Knipowitschia goerneri; b, sampling site at Korission Lagoon
(indicated by arrow); c, Localities from where populations of Knipowitschia have been recorded in Greek Ionian
Sea drainage (Economou et al., 2007; Barbieri et al., 2015): 1, Korission Lagoon on Kerkyra Island (K. goerneri);
2, Acheron River (K. milleri); 3-15, still unidentified populations: 3, Kalamas delta; 4, Louros River; 5, Arachthos
River; 6, Vlychos spring; 7, Voulkaria Lake; 8, Acheloos delta; 9, Trichonis Lake; 10, Evinos delta; 11, Mornos
delta; 12, Prokopos-Kotychi Lagoons; 13, Pinios River on Peloponnese; 14, Alfios delta; and 15, Zakynthos Island.

Ichthyol. Explor. Freshwaters, Vol. 26, No. 4
365
historisches Museum, Wien; PMR, Natural His-
tory Museum, Rijeka.
DNA extraction and PCR. DNA was extracted
from fin tissue preserved in 96 % ethanol using
JETQUICK Tissue DNA Spin Kit (GENOMED)
following the manufacturer’s instructions. Mito-
chondrial gene cytochrome b was analysed. The
amplification primers used were GluF and ThrR
(Machordom & Doadrio, 2001). PCR reactions,
amplification protocol, and PCR product purifica-
tion follow Šanda et al. (2008). Sequencing was
carried out by the Macrogen Service Centre (Seoul,
South Korea) using the amplification primers.
Molecular data analysis. Sequences were aligned
manually and revised in BioEdit (v.7.0.9). The
final alignment included 1119 base pairs of cy-
tochrome b. Pomatoschistus bathi was used as the
outgroup. From the Adriatic basin, K. panizzae
and K. montenegrina were included in the analyses
as the other two freshwater endemics from this
area (K. radovici and K. mrakovcici) are genetically
almost identical to K. panizzae (Geiger et al., 2014).
The best fitting model of nucleotide substitu-
tion was assigned using JModeltest 0.1.1 (Posada,
2008). Using Akaike information criterion (AIC),
the GTR+I model was selected. Two different ap-
proaches were used: Maximum Likelihood and
Bayesian inference. Maximum Likelihood phylo-
genetic analyses were performed using the BEST
approach implemented in PhyML, version 3.0.1,
which combines nearest neighbour interchanges
(NNI) and subtree pruning and regrafting (SPR)
algorithms to maximize tree likelihood (Guindon
& Gascuel, 2003). Five random starting trees were
used. Branch support was quantified by bootstrap
analysis with 1000 random replicates.
A Bayesian analysis was conducted using
MrBayes v. 3.1.2 (Huelsenbeck & Ronquist, 2001).
Two runs, each consisting of four Monte Carlo
Markov Chains, were run simultaneously for
1 000 000 generations with sampling trees every
100 generations. The first 20 % of trees were dis-
carded as burn-in and the remaining trees were
used to construct a 50 % majority-rule consensus
tree. The posterior probabilities were used to
indicate the branch supports in the final tree.
Genetic divergences between and within
species of Knipowitschia, based on cytochrome b
nucleotide sequences, were calculated in Mega
5.2 (Tamura et al., 2011) as uncorrected pairwise
genetic distance (p-distances).
Results
Knipowitschia goerneri
(Fig. 2)
Material examined. NMP P6V142862-142869, 1 male,
17.0 mm SL, 7 females, 13.1-20.6 mm SL; PMR VP3359,
1 female, 18.1 mm SL; Greece: Kerkyra Island: Korission
Lagoon, 39°27'02" N 19°54'20" E; R. Šanda & J. Vukib, 26
July 2014 (GenBank accession numbers: KT809439-
KT809446). – NMW 90253:1, 1, paratype, male, 17.8 mm
SL; Greece: Corfu (Kerkyra) Island: NW beach of Koris-
sion Lagoon; P. Keymar, Aug 1983.
Generic identification. The following combi-
nation of characters identify the specimens as
belonging to a species of the genus Knipowitschia:
1) suborbital row a of sensory papillae present;
2) pelvic disc with anterior transverse membrane;
3) cheek with several suborbital transverse rows of
sensory papillae; 4) anterior oculoscapular canal
ending in interorbital area, with paired pores λ,
or at doubled pore κ (K. goerneri), or head canals
absent; 5) no perianal organ; and 6) oculoscapu-
lar row tra of sensory papillae not reaching close
to suborbital row b.
Diagnosis. The genus Knipowitschia includes
16 species. There are no characters unique to
K. goerneri. Knipowitschia goerneri is easily dis-
tinguished from K. byblisia, K. ephesi, K. monte-
negrina, K. mrakovcici and K. thessala by having
the body squamation continuous from the axilla
to the caudal part along the lateral midline (vs.
body squamation not continuous). Knipowitschia
goerneri differs from K. milleri, K. ephesi and
K. montenegrina by the presence of at least some
head canals (vs. head canals absent). Knipo-
witschia goerneri is most clearly distinguished from
K. radovici, K. cameliae, K. caucasica, K. caunosi,
K. longecaudata and K. panizzae by the absence of
the preopercular head canal (vs. preopercular
head canal present). Knipowitschia goerneri differs
from K. iljini, K. cameliae, K. caucasica, K. caunosi,
K. longecaudata, K. panizzae and K. bergi by having
reduced squamation along the second dorsal fin,
i. e. dorsal naked along second dorsal fin up to
the base of the penultimate or of last articulated
ray (vs. squamation along the second dorsal fin
not reduced up to the base of the penultimate
or of last articulated ray). Knipowitschia goerneri
differs from K. mermere by having the base of the
last ray of the second dorsal fin before the vertical
through the base of the last anal-fin ray (vs. last

366
ray of second dorsal fin at vertical of the base of
the last anal-fin ray). Knipowitschia goerneri further
differs from K. mermere by having a wider head
(head width 17.9-22.3 % SL vs. 11.9-14.9; 58-69 %
HL vs. 38-48) and a shorter snout (snout length
16-21 % HL vs. 22-31).
Description. Body moderately elongate, cross
section circular anteriorly, compressed towards
caudal peduncle. Head large and moderately
depressed. Snout oblique, relatively longer in
smaller specimens, shorter in large specimens.
Eye large, dorsolateral, eye contour just slightly
above dorsal profile, i. e. above snout and predor-
sal area. Interorbital space distinctly smaller than
eye diameter and moderately narrow. Anterior
nostril short, tubular, erect, without process from
rim; posterior nostril pore-like, near orbit. Mouth
oblique, with lower jaw slightly projecting for-
ward to upper jaw, posterior angle of jaws below
pupil. Branchiostegal membrane attached along
entire lateral margin of isthmus. Body proportions
given in Table 1.
Fins. First dorsal VI; second dorsal I/7-8; anal
I/7-8; caudal 13 branched rays, 15-16 segmented;
pectoral 15-16; pelvic I/5 + 5/I. First dorsal fin
arising behind pectoral-fin base. First dorsal-fin
spines III-V when depressed extending to second
dorsal fin I in adult male, not reaching second
dorsal in female. Interdorsal space distinct. Second
dorsal fin commencing over or immediately be-
hind vertical of anus, with last ray before vertical
of last anal-fin ray. Anal fin commencing below
second to third articulated ray of second dorsal
fin. Caudal fin rounded. Pectoral fin extending
Table 1. Standard length and proportional measurements of Knipowitschia goerneri in this study and of type
material (Ahnelt, 1991). Morphometric ratios from Ahnelt (1991) were recalculated as percentages.
present research type material
range mean SD range
Standard length (mm) 13.1-20.6 17.8-21.8
In percent of standard length
Head length 30.0-33.8 31.7 1.3 30.0-34.2
Head width 19.1-22.3 20.3 1.1 17.9-20.0
Distance from snout to origin of first dorsal fin 39.2-42.2 41.2 1.0 39.1-43.9
Distance from snout to origin of second dorsal fin 56.1-61.7 59.1 2.0 54.6-61.3
Distance from snout to vertical of anus 55.2-59.7 57.1 1.4 55.2-61.3
Distance from snout to vertical of anal-fin origin 59.1-65.5 61.5 1.9 61.7-66.2
Distance from snout to vertical of pelvic-fin origin 31.2-35.9 32.9 1.5 30.3-34.5
Caudal-peduncle length 24.6-27.5 26.1 0.9 23.6-27.9
First dorsal-fin base 9.2-14.1 10.9 1.7 10.7-13.8
Second dorsal-fin base 13.0-16.0 14.9 1.1 16.1-18.3
Anal-fin base 13.5-17.1 15.2 1.2 16.1-17.9
Caudal-fin length 21.5-25.2 23.1 1.3 24.3-27.9
Pectoral-fin length 18.3-22.8 20.3 1.5 22.6-25.3
Pelvic-fin length 18.3-24.8 21.1 2.1 –
Body depth at pelvic-fin origin 21.0-23.8 22.7 1.1 20.7-23.9
Body depth at anal-fin origin 15.2-19.6 16.4 1.4 15.6-17.9
Body width at anal-fin origin 8.4-11.7 10.0 1.0 –
Caudal-peduncle depth 10.1-11.6 10.8 0.6 9.2-11.4
Distance from pelvic-fin origin to anus 22.5-30.4 25.9 2.4 –
In percent of head length
Snout length 17-21 19.2 1.3 16-20
Eye diameter 21-25 23.4 1.5 17-27
Postorbital length 49-61 54.9 3.7 53-61
Head width 58-69 63.9 3.6 59-61
Ratios
Caudal-peduncle depth in percent of its length 38-47 41.5 2.8 36-51
Interorbital width in percent of eye diameter 42-55 47.2 5.2 39-54
Pelvic-fin length in percent of distance from pelvic origin to anus 74-94 81.9 6.2 79-98
Vukib et al.: Rediscovery of Knipowitschia goerneri

Ichthyol. Explor. Freshwaters, Vol. 26, No. 4
367
back to below posterior end of first dorsal fin.
Pelvic disc rounded, nearly reaching anus in
male, shorter in female; anterior membrane in
midline 1
/
3 to 1
/
2 of pelvic spine length, free edge
of anterior membrane smooth. Lengths of fins and
fin bases in proportion to standard body length
given in Table 1.
Scales. Side of body and caudal peduncle covered
with ctenoid scales; predorsal and dorsal region,
back to base of penultimate or of last articulated
ray of second dorsal fin naked, breast and belly
naked; 30 to 33 in lateral series. Body squamation
continuous from axilla to caudal part along lateral
midline. Upper edge of scaled area extending from
behind upper part of pectoral axilla backwards
and up, reaching second dorsal fin at its posterior
end. Lower edge of scaled area extending from
behind pectoral-axilla bottom backwards and up
to vertical of anus and interdorsal space, and then
downwards to anal fin, variably reaching it. Nar-
rowest section of scaled area along lateral midline
between interdorsal space and anus.
Coloration. Preserved specimens. Body dark
brown above and whitish below; male darker
than female. Body, except most of underside in
female and belly in male, covered with numerous
melanophores, variably and irregularly mottled
with hardly recognizable patterns. Five pale
dorsal saddles: at nape opposite opercle, origin
of first dorsal fin, interdorsal space and origin
of second dorsal fin, posterior end of second
dorsal fin and posterior part of caudal peduncle.
In female several darker blotches recognizable
along lateral midline. Entire head pigmented in
male, in female underside mostly whitish and
poorly distinguished preorbital bars extending
to form a Y-shaped blotch on chin. First dorsal
fin in adult female with pigmentation anteriorly
at base, oblique transparent band in lower half
above pigmented area at base. Upper half of fin
with broad dark oblique band above transparent
band and transparent upper edge at fin-spine tips.
First dorsal fin in adult male entirely pigmented,
with more intensive black spot in mid-height of
fin between spine IV and V. Second dorsal fin
and caudal fin pigmented without recognizable
pattern. Pectoral fin with dark blotch on upper-ray
origin and rarely scattered melanophores on rest
of upper part of fin. Pelvic fin whitish in female,
pigmented and covered with melanophores in
male.
Lateral-line system. Cephalic canals present.
Anterior oculoscapular canal present, but much
reduced, divided into two or three parts: one
behind eye from pore κ (always paired) to pore α;
second more posteriorly from pore α' to pore ρ;
third short canal with pores λ and λ' between eyes
of variable occurrence in specimens. Posterior
oculoscapular and preopercular canals always
absent. Rows of sensory papillae: Preorbital
with three median rows and two rows lateral to
Fig. 2. Knipowitschia goerneri, NMP P6V142865, 17.0 mm SL, male, and NMP P6V142867, 18.9 mm SL, female;
Greece: Kerkyra Island: Korission Lagoon.

368
nostrils. Suborbital rows with infraorbital row
a extending forward to below anterior part or
anterior edge of pupil, posteriorly proliferated in
transverse row atp and a few shorter transverse
rows of 2-3 papillae. Longitudinal rows b not
extending anteriorly before vertical of posterior
border of orbit. Six or seven transverse c rows.
Longitudinal row d with continuous supralabial
and cheek parts. Three preopercular mandibular
rows: external row e and internal row i, both di-
vided, and mental row f. Oculoscapular rows tra,
z, x1, trp, x2, y present, with x1 divided by row trp
in anterior and posterior parts, additional papillae
present at position of missing preopercular canal
(below level of oculoscapular row z and slightly
in front of its vertical), and single papilla present
below posterior part of x1. Axillary rows as1, la1,
as2, la2 and as3 visible. Opercle with transverse
row ot, superior longitudinal row os and inferior
longitudinal row oi. Anterior dorsal row n trans-
versal, rows g and h longitudinal, rows o and m
absent. Interorbital longitudinal row p present.
Molecular relationships. Both Maximum Likeli-
hood and Bayesian inference analyses resulted in
an identical tree topology with a high statistical
support of branching (Fig. 3). Our results based on
cytochrome b nucleotide sequence analyses show
that K. goerneri is a sister species of K. milleri, the
species of Knipowitschia from the geographically
closest locality. The mean genetic divergence
between cytochrome b nucleotide sequences of
these two species is 3.2 % (Table 2), and they
form reciprocally monophyletic lineages (Fig. 3).
Discussion
Morphology. Our morphological examination
confirmed that the newly collected Knipowitschia
from Korission Lagoon is K. goerneri. This species
cannot be confused with any other known goby
species from the European coast of the Mediter-
ranean, including K. milleri (Ahnelt & Bianco,
1990), the only other Knipowitschia species from the
Fig. 3. Maximum Likelihood estimation of phylogenetic relationships of analysed Knipowitschia species based on
cytochrome b nucleotide sequences. Numbers on branches are posterior probabilities/bootstrap support of nodes.
0.05
NMP P6V141422
NMP P6V142869
NMP D1326
NMP P6V141242
NMP P6V141302
NMP P6V142644
NMP P6V142867
NMP P6V142868
NMP P6V142866
PMR VP1985
NMP P6V142863
NMP P6V88957
NMP P6V142642
NMP P6V141423
NMP CG089B
NMP P6V89874
NMP P6V141418
NMP D1325
NMP P6V142865
NMP P6V88956
PMR VP1905
NMP P6V142646
NMP P6V142864
NMP P6V142643
PMR VP3359
NMP P6V142645
NMP P6V141300
1/100
0.99/93
1/100
1/100
1/99
1/100
1/99
1/99
0.99/87
1/100
0.99/79
Knipowitschia
montenegrina
Knipowitschia
panizzae
Knipowitschia
thessala
Knipowitschia
milleri
Knipowitschia
goerneri
Knipowitschia
caucasica
Pomatoschistus bathi
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Ichthyol. Explor. Freshwaters, Vol. 26, No. 4
369
north-eastern part of the Ionian Sea basin. Unlike
K. goerneri, K. milleri completely lacks head canals.
The degree of reduction of both body squamation
and head canals in K. goerneri are unique among
all 16 named Knipowitschia species (Ahnelt, 1991,
1995, 2011; Miller, 2004a, 2009; Kovacib, 2005:
table 2; Kottelat & Freyhof, 2007; Kovacib & Šanda,
2007; Geiger et al., 2014) with the only exception
being K. mermere Ahnelt, 1995, a species from
Anatolia (Ahnelt, 1995) located so far away that
the presence of K. mermere in Korission Lagoon
is highly improbable. In addition, the values of
some morphometric characters of K. goerneri and
K. mermere do not overlap (head width, snout
length; see diagnosis), or overlap only slightly
(caudal-fin length 21.5-27.9 % SL, vs. 18.1-22.0;
body depth at pelvic-fin origin 20.7-23.9 % SL, vs.
19.2-21.6) (see Table 1 and Ahnelt, 1991, 1995).
Furthermore, K. mermere is a freshwater species,
which is related to K. thessala (Vinciguerra, 1921)
and is considerably divergent from K. milleri,
based on COI gene nucleotide sequence analysis
(Geiger et al., 2014), while K. goerneri is closely
related to K. milleri, based on cytochrome b gene
nucleotide sequence analyses (this study).
Comparison with the type material of K. goer-
neri showed that the morphology of the newly
collected specimens matches well with the original
species description (Ahnelt, 1991). Two exten-
sions of the original meristic character ranges
were observed in the new material of K. goerneri,
which is probably a result of the smaller number
of individuals used for the original description.
First, we observed both the second dorsal fin
and anal fin to have I/7-8, vs. I/8 for both fins
in the original description. Second, one specimen
exhibited 30 scales in the lateral series, vs. a range
of 32-33 in the original description. It was also
observed in the recent specimens that the pelvic
disc of a single male nearly reached the anus,
while in the original description the pelvic disc
of both males reached the anus.
The reduction of the cephalic lateral line canals
in the newly collected specimens of K. goerneri
matches with two types of reduction observed by
Ahnelt, 1991 (the types shown in his figures 3a,d
and 3b,e), both of which are well-represented in
the present sample. Some morphological details
missing in the original description were provided
in the present data (e. g. caudal-fin segmented-ray
count, exact limits of scaled area on body surface).
Ahnelt (1991) described the species based on only
five specimens, of which two were males and
three females. Measurements conducted on the
slightly larger sample size and better preserved,
freshly collected specimens, showed somewhat
higher morphological variability than previously
thought (Table 1; Ahnelt, 1991: table 1). However,
some morphometric characters (fin bases and fin
lengths) in the present study largely extend one
side of the range of values compared with the
original description. This is probably a result of
growth allometry, as five of the nine specimens
used in this study were small (13.1-15.7 mm SL)
compared to the comparatively larger specimens
used in the original description (17.8-21.8 mm
SL) (Ahnelt, 1991). The five new small specimens
had, proportionally to the standard length, shorter
dorsal- and anal-fin bases, as well as shorter cau-
dal- and pectoral-fin lengths compared with the
larger specimens in our sample and in the type
material.
Molecular relationships. Based on cytochrome b
nucleotide sequence analyses, K. goerneri is closely
related to K. milleri, another species from the
north-eastern part of the Ionian Sea basin (Fig. 3).
The cytochrome b haplotypes of both species
form unambiguous reciprocally monophyletic
lineages (Fig. 3), and together they form a well-
supported clade, which is in a sister position to the
cytochrome b haplotypes of K. thessala, a species
endemic to the Thessalian Pinios River drainage in
the north-western Aegean Sea basin (on the other
side of the Pindos Range, a major biogeographic
barrier). The genetic distance between K. goerneri
and K. milleri inferred from the cytochrome b gene
sequences is around 3 % (Table 2). The intraspe-
cific genetic variability of both species based on
cytochrome b is several times lower; in K. milleri
it does not exceed 0.9 %, whereas in K. goerneri it
is much lower, with a maximum of 0.2 %.
Several other populations of Knipowitschia,
whose taxonomic status is not clarified, are known
from the north-eastern Ionian Sea basin (from the
Kalamas River to the western Peloponnese Alfios
River; Economou et al., 2007, Barbieri et al., 2015;
Fig. 1). Only some of these have been genetically
studied thus far (Vanhove et al., 2012; Geiger et
al., 2014). These studies show that several Knipo-
witschia populations (from the Acheloos and
Evinos River basins and the Prokopos-Kotychi
drainage in north-western Peloponnese) are
closely related to K. milleri. Each study used dif-
ferent mitochondrial genetic markers (12S and
16S in Vanhove et al., 2012; COI in Geiger et al.,

370
2014; cytochrome b in our study), thus it is not
possible to compare the results directly due to
the different mutational rates in different genes
(Wan et al., 2004). As K. goerneri is also closely
related to K. milleri, it is necessary to compare
all populations of Knipowitschia from the north-
eastern Ionian Sea basin with these two species
in order to delimit their exact distribution range
and to clarify the taxonomy of the Knipowitschia
populations from this area. This should ideally be
done by combining the genetic and morphological
approaches, and could potentially result in the
discovery of additional species.
Ecology and distribution. Knipowitschia goerneri
was originally found in a freshwater spring, as
well as in the brackish lagoon of Korission (Ah-
nelt, 1991). We discovered that the species is still
present in the lagoon, though we sampled at two
sites and the species was only found at one. The
sampling point at the lagoon was very shallow
(maximum depth of 40 cm), with a muddy bottom
containing numerous shell remains and a dense
filamentous algal covering. From the locality
where it was found, we assume that K. goerneri
is a euryhaline and eurythermal species. It is
probable that the conditions in the lagoon vary
substantially during the course of the year. The
size and status of the population are unknown.
Further research of the Korission Lagoon catch-
ment and its surroundings on Kerkyra Island is
necessary in order to characterise the ecological
and biological requirements of K. goerneri, its habi-
tat preferences and its distribution. This species
may occur in other wetland sites on the island,
as well as on the mainland opposite Kerkyra in
Greece and in Albania.
Conservation and threats. Ahnelt (1991) de-
scribed K. goerneri based on specimens from a
freshwater spring south-west of Korission La-
goon (the type locality), as well as the north-west
beach of the lagoon. Since the work of Ahnelt
(1991), the lagoon has not been mentioned as the
locality for K. goerneri (Economidis, 1995; Miller,
2004b; Economidis & Chrysopolitou, 2009), and
there is no published evidence that it has been
investigated for its presence since 1983. The as-
sumption that this species is critically endangered
or possibly extinct (Miller, 2004b; Economidis &
Chrysopolitou, 2009) was based on the destruction
of Gardiki Spring, situated north of the lagoon
(Economidis, 1995), which was erroneously men-
tioned as the only locality of K. goerneri (Miller,
2004b; Economidis & Chrysopolitou, 2009), even
though the species has never been found there.
Although K. goerneri was listed in the Red Data
Book of Greece as critically endangered and pos-
sibly extinct (Economidis & Chrysopolitou, 2009),
no conservation actions have been proposed
for this species (Economidis & Chrysopolitou,
2009). Despite the Korission Lagoon catchment
being designated as a Natura 2000 site (European
Environment Agency, 2014), it is threatened by
development, pollution, fisheries, and over-
pumping groundwater.
Conclusions. The rediscovery of K. goerneri in
this study, as well as another freshwater fish spe-
cies, Telestes miloradi (Jelib & Jelib, 2015), shows
that even in Europe species can be overlooked
for decades. Hence, it is possible that some other
species currently thought to be extinct (Smith &
Darwal, 2006; Kottelat & Freyhof, 2007; Geiger et
al., 2014) could in fact still persist (e. g. K. cameliae,
Chondrostoma scodrense). Intensive field work is
Table 2. Mean uncorrected pairwise genetic distance (p-distance; %) between analysed species of Knipowitschia
based on cytochrome b nucleotide sequence analysis. Number of analysed specimens and range of intraspecific
genetic variability included next to species names in parentheses, and mean intraspecific genetic variability
included in bold font.
K. goerneri K. milleri K. thessala K. montengrina K. panizzae K. caucasica P. bathi
K. goerneri (8; 0-0.18) 0.045
K. milleri (5; 0-0.89) 3.2 0.55
K. thessala (4; 0-0.09) 7.8 7.6 0.06
K. montenegrina (3; 0.09-0.18) 8.2 8.8 8.6 0.12
K. panizzae (2; –) 8.5 8.9 8.1 3.9 1.43
K. caucasica (4; 0-1.43) 10.3 10.1 9.9 8.7 8.5 0.95
Pomatoschistus bathi (1; –) 16.4 17.1 16.8 16.3 16.2 15.2 –
Vukib et al.: Rediscovery of Knipowitschia goerneri

Ichthyol. Explor. Freshwaters, Vol. 26, No. 4
371
needed to better evaluate the possible presence
of such taxa in the Mediterranean region.
Comparative material. Knipowitschia milleri: PMR
VP3212, 1 female, 22.9 mm SL; PMR VP3213, 1 male,
20.2 mm SL; PMR VP3214, 1 juvenile, 17.5 mm SL;
Greece: Acheron River.
Material used in molecular genetic analysis: K. milleri:
NMP P6V142642-142646, 5; Greece: Acheron River,
39°14'34" N 20°28'40" E (GenBank accession numbers:
KT809434-KT809438).
K. thessala: NMP P6V141242, 1; Greece: Pinios drain-
age: Enipeas River, 39°33'43" N 22°4'45" E (GenBank
accession number: KT809433). – NMP P6V141418, 1;
NMP 141422-141423, 2, Greece: Pinios drainage: Lithe-
os River, 39°33'14" N 21°46'09" E (GenBank accession
numbers: KF214247, KT809431-KT809432).
K. montenegrina: NMP P6V89874, 1; NMP D1326-
D1325, 2; Montenegro: Moraca River, 42°18'37" N
19°11'56" E (GenBank accession numbers: KT809450-
KT809452).
K. panizzae: PMR VP1905, 1; Croatia: Privlaka,
44°16'37" N 15°07'53" E (GenBank accession number:
KT809453). – PMR VP1985, 1; Croatia: Cetina River,
43°26'21" N 16°45'02" E (GenBank accession number:
KT809454).
K. caucasica: NMP P6V88956-88957, 2; Bulgaria:
Pomorijsko Lake, 42°33'56" N 27°37'56" E (GenBank
accession number: KF214250, KT809449). – NMP
P6V141300, 1; NMP 141302, 1; Greece: Volvi Lake,
40°39'14" N 23°30'04" E (GenBank accession number:
KT809447-KT809448).
Pomatoschistus bathi: NMP CG089B, 1; Montenegro:
Becibi: Adriatic Sea, 42°16'26" N 18°53'16" E (GenBank
accession number: KT809430).
Acknowledgements
This work was supported by the Ministry of Culture of
the Czech Republic (DKRVO 2014/14, DKRVO 2015/15,
National Museum, 00023272) and by the institutional
resources of the Ministry of Education, Youth, and
Sports of the Czech Republic (JV). MK received support
from the SYNTHESYS Programme project CZ-TAF-3784
at the National Museum Prague, financed by European
Community Research Infrastructure Action under the
FP7 “Capacities” Programme. We thank Anja Palandacib
(NMW) for the loan of the type material of K. goerneri.
We acknowledge the assistance of the colleagues from
the Hellenic Centre for Marine Research and particu-
larly of Alcibiades N. Economou. Aris Vidalis provided
assistance with cartography. We thank the editor and
two anonymous referees for their valuable comments,
and T. Kohler for language correction.
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Received 26 February 2015
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Accepted 6 January 2016
Vukib et al.: Rediscovery of Knipowitschia goerneri