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Zoological Journal of the Linnean Society, 2014, 172, 831–845. With 6 figures
Identification of gobies (Teleostei: Perciformes:
Gobiidae) from the North and Baltic Seas combining
morphological analysis and DNA barcoding
THOMAS KNEBELSBERGER1* and RALF THIEL2
1
Senckenberg am Meer, German Centre for Marine Biodiversity Research (DZMB), 26382
Wilhelmshaven, Germany
2
University of Hamburg, Biocenter Grindel and Zoological Museum, 20146 Hamburg, Germany
Received 17 April 2014; revised 2 July 2014; accepted for publication 14 July 2014
Gobies are difficult to identify, as they are very similar in appearance. Here, we identified (sub)adult specimens
of 12 goby species from the North Sea and the Baltic Sea by carefully analysing meristic characters, coloration
patterns, papillae row patterns and morphometric measurements. The results of the morphological identifications
were congruent with those obtained with the analysis of COI DNA barcodes; sequences from morphological conspecific
specimens were clustered together in clades with bootstrap values ≥ 99%. Mean intra- and interspecific distance
(uncorrected p) was 0.37 and 18.97%, respectively. A gap between the maximum intraspecific distance and the
distance to the nearest neighbour was apparent in every species and ranged from 2.35 to 16.11%. The Barcode
Index Number (BIN) analysis performed on the Barcode of Life Data Systems (BOLD) web platform, assigned the
DNA barcodes to 12 separate clusters corresponding to sequence- and morphology-based identification. In 25% of
the investigated species, the BIN clusters showed taxonomic discordances, as they contained sequences assigned
to more than one species. This result demonstrates the importance of accurate morphological species identification at the beginning of the barcoding pipeline.
© 2014 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 172, 831–845.
doi: 10.1111/zoj.12189
ADDITIONAL KEYWORDS: COI – meristic characters – mitochondrial DNA – morphology – Pomatoschistus
– species identification.
INTRODUCTION
The teleost family Gobiidae (Perciformes) includes more
than 1700 valid species belonging to more than 200
genera (Eschmeyer, 2014). Somewhat earlier, Nelson
(2006) reported up to 1950 species belonging to 210
genera. These values indicate that the family Gobiidae
is a very species-rich taxon comprising more than 5%
of all recent fish species. Gobiid fishes exhibit a high
capacity to adapt and diversify and show remarkable
morphological and ecological variability, which is reflected by the large number of species (Zander, 2011)
*Corresponding author.
E-mail: [email protected]
by gobies are globally distributed and occur in marine,
freshwater and brackish habitats. However, the NorthEastern Atlantic region is dominated by marine gobiid
species by gobies are rare in western and northern European freshwaters (Freyhof, 2011).
A reliable checklist of the North-Eastern Atlantic
gobiid species fauna is currently missing. However,
Kovačić & Patzner (2011) counted 47 gobiid species
for the North-Eastern Atlantic, and Carneiro et al.
(2014) listed 33 species of Gobiidae for Portuguese
waters in a recently updated checklist of marine fishes.
According to the information provided by Miller (1986),
about 14 species of Gobiidae occur in the North Sea
and its transitional area to the Baltic Sea. This number
can be considered a benchmark of the actual species
number. By contrast, the fauna of the Baltic Sea is
© 2014 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 172, 831–845
831
832
T. KNEBELSBERGER AND R. THIEL
well described and comprises at least 15 gobiid species
(see HELCOM, 2012).
In northern European shelf waters, including the
North and Baltic Seas, five species belonging to the
genus Pomatoschistus can be found: the sand goby
P. minutus (Pallas, 1770); Lozanos goby P. lozanoi (de
Buen, 1923); the Norway goby P. norvegicus (Collett,
1903); the common goby P. microps (Krøyer, 1838); and
the painted goby P. pictus (Malm, 1965). These species
are known to be among the most abundant demersal
fishes in the coastal waters of the continental shore,
the Wadden Sea and estuaries (Hamerlynck, 1990). Only
P. norvegicus appears in offshore habitats as well. Species
of the genus Pomatoschistus are difficult to distinguish as they are very similar in appearance and widely
use similar North Atlantic habitats (Hamerlynck, 1990).
In particular, the correct species identification of
P. minutus, P. lozanoi and P. norvegicus, the so-called
‘Pomatoschistus minutus complex’ (Webb, 1980), is
known to be problematic. Pomatoschistus lozanoi is in
fact morphologically intermediate between the other
two species, and is suspected to interbreed with
P. minutus and P. norvegicus in their natural habitat
(Webb, 1980). The identification of gobies is traditionally based on external morphology and osteology. Particular features such as the cuteaneous papillae patterns,
the head canal system and the sensory pores on the
head provide the most reliable key to species identification (Miller, 1986; Van Tassel, Tornabene & Taylor,
2011). Due to miniaturization and reduction of morphological features of these mostly small sized species
(generally below 10 cm in length), the search for accessible diagnostic characters has generally been unsuccessful. However, additional characters such as
pigmentation patterns or detailed morphometric measurements may help in the distinction among closely
related species, as demonstrated by Hamerlynck (1990)
and Apostolou et al. (2011) for species of the genus
Pomatoschistus.
The morphological features used to discriminate (sub)
adult specimens might be useless for the identification of (post)larval stages, damaged individuals or
remains of animals found in gut contents and biopsies (Larmuseau et al., 2008). In particular, a number
of sympatric gobiids share morphological characters
during their early larval stages, thus inhibiting their
accurate identification. In other cases, important
external distinguishing features (e.g. the sensory papillae or pigmentation) might be damaged and invisible due to the capture procedure or the digestion
process.
A number of goby species, especially of the genus
Pomatoschistus, occur in high abundance and play an
important ecological role in North Atlantic coastal
waters, and are often used as model species in ecological, behavioural and genetic studies (Leitão et al.,
2006; Rodrigues et al., 2006; Jones et al., 2001;
Lindström, St. Mary & Pampoulie, 2006; Gysels et al.,
2004a, b; Pampoulie et al., 2004). Additionally, gobies
are known as successful invasive species. The round
goby Neogobius melanostomus (Pallas, 1811) is one of
the most wide-ranging invasive gobiid fish species on
earth, with substantial introduced populations in several
European rivers systems, the Baltic Sea and the
Laurentian Great Lakes (Kornis, Mercado-Silva &
Vander Zanden, 2012). Recently, new populations of
N. melanostomus were found in the North Sea region,
e.g. in the Weser river and Elbe estuary (Brunken et al.,
2012; Hempel & Thiel, 2013). The naked goby
Gobiosoma bosc (Lacepède, 1800), a western Atlantic
gobiid species, has been recently reported for the first
time in the Weser estuary (Thiel, Scholle & Schulze,
2012). The record of both adult male and female individuals could be a sign of possible future establishment in German waters.
Due to the ecological importance of gobies as model
organisms, their accurate identification is essential for
many research fields. Fast and reliable molecular
methods, such as PCR-RFLPs (Larmuseau et al., 2008),
have been used as morphological identification of gobiid
species is very difficult in many cases. Thus far, Gobiidae
DNA sequences have been used to analyse phylogenetic
relationships among taxa (Thacker & Roje, 2011;
Agorreta et al., 2013; Tornabene, Chen & Pezold, 2013)
or to investigate phylogeographical patterns at the population level (Gysels et al., 2004a, b). Furthermore, DNA
sequences were used for species identification based
on divergence of a defined part of the mitochondrial
cytochrome c oxidase subunit I (COI) gene (Hyung-Bae,
Seung-Ho & Ho, 2012; Viswambharan et al., 2013). This
technique, known as DNA barcoding (Hebert et al., 2003),
has already proven its utility as a molecular tool for
species identification throughout all major fish taxa,
including species of marine as well as freshwater ecosystems from different geographical regions (Ward et al.,
2005, 2008a; Hubert et al., 2008; Steinke et al., 2009a;
April et al., 2011; Mabragaña et al., 2011; Cerutti-Pereyra
et al., 2012; Costa et al., 2012; Zhang & Hanner, 2012;
Keskin & Atar, 2013; McCusker et al., 2013; Geiger
et al., 2014; Knebelsberger et al., 2014). Moreover, DNA
barcodes provide the opportunity to identify different
life stages (e.g. larvae) to species level (Pegg et al., 2006;
Victor et al., 2009; Hubert et al., 2010), whereas morphological features might be barely available. In marine
fishes, species discrimination rates based on DNA barcodes are usually high. In several studies, more than
98% of the investigated species were clearly delimited
(e.g. Ward et al., 2005; Steinke et al., 2009a; Steinke,
Zemlak & Hebert, 2009b; Costa et al., 2012; Zhang &
Hanner, 2012; Knebelsberger et al., 2014), as genetic
distances within species were considerably lower than
interspecific distances. In some cases, high intraspecific
© 2014 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 172, 831–845
IDENTIFICATION OF GOBIES
distances may suggest the existence of cryptic diversity (Ward et al., 2008b; Zemlak et al., 2009; Hubert
et al., 2012; Puckridge et al., 2013). Nevertheless, the
successful application of DNA barcodes for species identification is strongly dependent on the quality of the
barcode reference library. Therefore, an accurate morphological identification of the analysed material is an
essential prerequisite at the beginning of the barcoding
pipeline. The comparison of newly generated barcodes
with published data may help to detect misidentifications, taxonomic uncertainties or real cases of haplotype
sharing among species. In this context, the Barcode of
Life Data Systems (BOLD, Ratnasingham & Hebert,
2007) provides a useful resource. The BOLD platform
also provides an automated barcode sequence clustering system, where a Barcode Index Number (BIN) is
assigned to each record (Ratnasingham & Hebert, 2013).
In an ideal case, all sequences that were assigned to
the same species have identical BIN numbers.
According to the Fish Barcode of Life website
(FISH-BOL, 2012), barcodes for approximately onethird (more than 10,000) of all known fish species are
currently available. Nevertheless, the permanent growth
of the database is essential, as the taxonomic reliability of the data will increase, if independent observers
produce congruent results.
In this paper we present a detailed and accurate
species identification matrix based on the integration
833
of morphological characters and molecular DNA barcodes. The analysis includes 12 common gobiid species
from the North and Baltic Seas and all five members
of the genus Pomatoschistus known from this region.
For comparison with published barcodes, the whole
dataset was uploaded to BOLD.
MATERIAL AND METHODS
SAMPLING
In total, 73 specimens representing 12 gobiid fish species
were sampled from the North Sea, the Baltic Sea, the
Kiel canal, and the estuary regions of the rivers Weser
and Trave (Table S1, Fig. 1). Main sampling was performed during several cruises conducted by the Thünen
Institute of Sea Fisheries (Hamburg, Germany) and
the research vessel of the Senckenberg Institute
(Wilhelmshaven, Germany). A smaller part of the material was collected during several other research fisheries using bottom trawls, stow nets, fyke nets, beach
seines and hand nets. A few additional individuals were
obtained from two fishermen. Most specimens were fixed
in 96% ethanol immediately after catch. All specimens of N. melanostomus and some of the specimens
of P. minutus and P. lozanoi were deep frozen (−20 °C)
initially and later fixed in 96% ethanol. All specimens of G. bosc were fixed in 70% ethanol upon capture
60°
58°
56°
54°
-2°
0°
Scale: 1:15000000
2°
4°
6°
8°
10°
12°
-2°
0°
2°
4°
6°
8°
Pomatoschistus lozanoi
Pomatoschistus microps
Pomatoschistus minutus
Pomatoschistus norvegicus
Pomatoschistus pictus
10°
12°
14°
Aphia minuta
Buenia jeffreysii
Crystallogobius linearis
Gobius niger
Gobiusculus flavescens
Gobiosoma bosc
Neogobius melanostomus
Figure 1. Sampling sites of gobiid species of the genus Pomatoschistus (left) and species belonging to other genera (right).
© 2014 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 172, 831–845
834
T. KNEBELSBERGER AND R. THIEL
and later transferred to 96% ethanol. Muscle or fin
tissue samples were taken from fixed specimens, preserved in 96% ethanol and stored at the tissue collection of the Senckenberg’s German Center for Marine
Biodiversity Research (DZMB, Wilhelmshaven,
Germany) for subsequent molecular analyses. Voucher
specimens were catalogued and morphologically investigated at the Zoological Museum of the University of Hamburg (ZMH, Hamburg, Germany). The
material was then stored for future reference in
the ZMH fish collection. All metadata (including ZMH
registration and DZMB sample numbers, see Table S1)
related to the voucher specimens were uploaded
to the Barcode of Life Data System (http://www
.boldsystems.org; Ratnasingham & Hebert, 2007) in the
public project folder ‘Barcoding Gobiidae from the North
Sea and the Baltic Sea’ (BGNBS).
MORPHOLOGICAL
IDENTIFICATION
Meristic parameters, coloration, papillae row characters and body dimensions were estimated from the lefthand side of the specimens. Basic characters (Table 1)
of all specimens were counted and then fish were identified according to characters given by Miller (1986),
Thiel et al. (2012) and Hempel & Thiel (2013). The
number of vertebrae, dorsal-fin spines and rays, as well
as anal-fin rays of all specimens were counted from
radiographs (Fig. 2) using an X-ray imaging system
(Faxitron LX-60).
Table 1. Data for morphological characters used for species identification (except genus Pomatoschistus; n.e., character
not estimated)
Species
Am
Bj
Cl
Gb
Gn
Gf
Nm
Number of specimens analysed
Meristic characters
Lower jaw teeth in single row (JTSR)
First dorsal-fin spines (D1S)
Second dorsal-fin rays (D2R)
Anal-fin rays (AR)
Pectoral-fin rays (PR)
Body scaled (BS)
Total vertebrae (Ve)
Coloration
Large dark blotches laterally or on lateral
midline (DBLM)
Large black spot at base of caudal fin (BSCF)
Dark blotch in upper anterior corner of each
dorsal fin (DBCD)
D1 with dark spot in posterior part (D1DS)
Papillae rows
Sub-orbital papillae row a present (SPRP)
Second transverse row of sensory papillae
descending below level of row d (TR2D)
10
4
6
4
4
6
7
Yes
5
11–12
13–14
17–18
Yes
26–28
No
6
8–9
7–8
18
Yes
30
Yes
2–3
18–20
20–21
30–31
No
29–31
No
7–8
12–13
9–10
18
No
27–28
No
6
12–13
11
17–19
Yes
28
No
7
9–10
9–10
17
Yes
32
No
6
13–16
11–13
18
Yes
31–34
No
Yes
No
No
Yes
No
Yes
No
No
No
No
No
No
No
No
No
Yes
Yes
No
No
No
No
No
No
No
No
No
Yes
n.e.
No
Yes
No
n.e.
No
No
No
No
No
Yes
No
No
No
Am, Aphia minuta; Bf, Buenia jeffreysii; Cl, Crystallogobius linearis; Gb, Gobiosoma bosc; Gn, Gobius niger; Gf, Gobiusculus
flavescens; Nm, Neogobius melanostomus.
Figure 2. Radiograph of the sand goby Pomatoschistus minutus (Pallas, 1770) indicating the meristic characters evaluated from X-ray pictures. Depicted specimen: ZMH 200017, total length 85.4 mm.
© 2014 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 172, 831–845
IDENTIFICATION OF GOBIES
For the specimens of Pomatoschistus with problematic identifications, we investigated additional characters and morphometric measurements (Table 2)
according to the method described by Hubbs & Lagler
(1958). Measurements were taken with a Vernier caliper
with a precision of 0.1 mm. The occurrence of papillae rows was evaluated according to Akahito (1986)
and Miller (1986).
DNA
ISOLATION,
PCR AMPLIFICATION
AND SEQUENCING
Total genomic DNA was extracted at the DZMB using
the Qiagen DNeasy Blood and Tissue Kit for single
columns as described by Knebelsberger & Stöger (2012).
A 652-bp fragment of the COI gene was amplified for
54 samples using the primers FishF1 and FishR1 (Ward
et al., 2005). Failed PCR amplifications were repeated using a cocktail of two forward and two reverse
primers (C_FishF1t1-C_FishR1t1) tagged with M13 tails
at 5′ ends (Ivanova et al., 2007). Each PCR reaction
mixture contained 1 μL DNA template, 2.25 μL of 10×
reaction buffer (including MgCl2), 0.5 μL dNTPs (2 mM
each), 0.25 μL of each primer (10 pmol μL−1), 0.25 μL
Taq polymerase (5 U μL−1; Qiagen) and molecular grade
water for a total volume of 25 μL. Thermal cycling was
performed with an initial denaturation for 2 min at
94 °C, followed by 35 cycles of 30 s at 94 °C, 30 s at
the annealing temperature of 54 °C, 60 s at 72 °C with
a final extension of 10 min at 72 °C. PCR products were
checked by a 1% agarose gel. Amplicons were purified by incubating 10 μL of PCR products with 0.5 μL
of exonuclease I (20 U μL−1) and 2 μL alkaline phosphatase (1 U μL−1) for 15 min at 37 °C followed by 20 min
at 75 °C. Purified amplicons were sequenced by
Macrogen Europe (Amsterdam, Netherlands). DNA
aliquots of 19 specimens were analysed at the Canadian Centre for DNA Barcoding (BIO, Guelph, Canada)
following a protocol which has been described elsewhere (Steinke et al., 2009a).
SEQUENCE ALIGNMENT AND
DATA ANALYSES
Forward and reverse sequences were assembled
and edited using Geneious (version 5.4.5, http://
www.geneious.com). Consensus sequences were submitted to GenBank (accession numbers KM077806–
KM077878) and are also available in the corresponding
BOLD project folder ‘BGNBS’ together with the raw
sequence chromatograms. Variance in sequence length,
base composition, number of invariable sites and the
presence of stop codons was analysed in Geneious. The
sequences were aligned using MUSCLE (Edgar, 2004)
with default settings as implemented in MEGA version
5.02 (Tamura et al., 2011). Uncorrected p and Kimura
two-parameter (K2P) distances (Kimura, 1980) were
835
calculated in MEGA. Uncorrected p distances have been
shown to perform better for species identification than
model-corrected ones (Collins et al., 2012); nevertheless, K2P distances were suggested by Hebert et al. (2003)
as standard for barcoding analyses and enable direct
comparison with other studies. Neighbour-joining (NJ,
Saitou & Nei, 1987) topology was built in MEGA using
the ‘pairwise deletion’ option for the treatment of gaps
and missing data, to retain all sites initially, excluding
them as necessary. Node support for the NJ topology
was evaluated by a non-parametric bootstrap analysis
(Felsenstein, 1985) with 10 000 replicates. Genetic distances were calculated on species, genus and family
levels. The barcoding gap was calculated for each species
comparing the maximum intraspecific genetic distance with the minimum distance to the nearest neighbour. BINs were calculated for the whole dataset using
the ‘BIN Discordance Report’ sequence analysis tool
available on BOLD (Ratnasingham & Hebert, 2013).
RESULTS
MORPHOLOGICAL
IDENTIFICATION AND
SPECIES COMPARISON
Meristic and morphometric characters of the 12 examined species of Gobiidae are given in Tables 1 and
2. The number of individuals per species ranged from
three to ten. The genus Pomatoschistus was represented by five species and all other seven genera (Aphia,
Buenia, Crystallogobius, Gobiosoma, Gobiusculus,
Gobius, Neogobius) by one species each.
Seven of the 12 species could be identified using meristic characters and coloration (e.g. Miller, 1986), whereas
the identification of the other five Pomatoschistus species
was possible by evaluating additional meristic characters, occurrence of several papillae rows and
morphometric measurements.
In the following section, the morphological identification procedure and discrimination criteria are
presented for each of the species analysed in this study.
The species Aphia minuta (Risso, 1810) and
Crystallogobius linearis (von Düben, 1845) are unique
in having the lower jaw teeth arranged in a single row
(Tables 1 and 2). However, C. linearis differs from
A. minuta by its lower number of first dorsal-fin spines,
a higher number of total vertebrae, higher numbers
of second dorsal-fin, anal-fin and pectoral-fin rays and
the lack of scales on the body (Table 1).
Buenia jeffreysii (Günther, 1876) was distinguished
from all the other species by its lowest number of analfin rays in combination with a relatively low number
of dorsal-fin rays (Tables 1 and 2).
The identification of Gobiusculus flavescens (Fabricius,
1779) is based on the occurrence of a large black
spot at the base of the caudal fin. Additionally, the
© 2014 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 172, 831–845
No
5–6
8–9
9
18–19
No
Yes
40–47
No
No
30–31
No
No
No
No
No
Yes
No
23.8–41.7
16.0–22.9 (± 2.2)
25.6–29.0 (± 1.1)
10.1–15.8 (± 1.9)
12.2–18.2 (± 1.9)
5.8–7.6 (± 0.7)
5.8–8.0 (± 0.8)
1.5–2.9 (± 0.5)
7.1–8.2 (± 0.5)
22.8–25.6 (± 1.1)
25.2–27.4 (± 0.8)
29.4–33.2 (± 1.3)
29.5–39.9 (± 4.0)
55.4–58.6 (± 1.2)
12.9–17.6 (± 1.5)
17.0–27.6 (± 3.3)
21.3–26.1 (± 1.7)
0.5–1.2 (± 0.2)
25.7–30.3 (± 1.6)
2.8–4.9 (± 0.9)
No
6
9–10
10–11
18
Yes
Yes
59–62
Yes
Yes
32
No
No
No
No
Yes/no
Yes
Yes
46.1–47.7
15.5–18.4 (± 1.6)
27.0–32.1 (± 2.6)
13.0–18.4 (± 3.1)
13.3–18.0 (± 2.3)
6.8–8.0 (± 0.7)
7.2–8.1 (± 0.5)
2.1–3.3 (± 0.7)
6.3–7.2 (± 0.5)
21.0–23.3 (± 1.1)
28.2–31.0 (± 1.4)
28.7–36.2 (± 3.9)
35.7–36.7 (± 0.5)
56.0–56.9 (± 0.5)
16.8–17.6 (± 0.4)
20.3–21.9 (± 0.8)
20.8–25.6 (± 2.7)
0.5–0.6 (± 0.1)
22.8–28.1 (± 2.7)
2.1–2.4 (± 0.1)
14.9–19.6 (± 1.5)
26.2–29.0 (± 0.8)
13.3–16.5 (± 1.0)
15.2–19.1 (± 1.1)
6.0–8.4 (± 0.9)
5.3–8.1 (± 0.8)
1.4–2.5 (± 0.3)
6.5–8.8 (± 0.7)
19.4–24.9 (± 1.6)
26.4–29.3 (± 0.9)
26.6–33.7 (± 2.0)
31.6–36.2 (± 1.7)
54.2–61.2 (± 1.9)
11.8–17.8 (± 1.8)
18.8–24.5 (± 1.7)
19.2–26.1 (± 2.0)
0.3–0.6 (± 0.1)
23.8–29.2 (± 1.4)
1.6–2.2 (± 0.2)
53.8–72.8
Yes
No
No
No
No
No
Yes
No
6
10–11
10–11
19–20
Yes
Yes
58–66
Yes
Yes
32–33
10
Pm
12.4–17.4 (± 2.0)
20.0–29.6 (± 3.0)
12.1–14.8 (± 1.0)
11.1–14.1 (± 1.1)
6.0–6.9 (± 0.4)
6.5–8.9 (± 0.8)
1.6–2.1 (± 0.2)
5.4–6.9 (± 0.5)
24.2–28.7 (± 1.6)
25.9–30.0 (± 1.4)
28.8–35.3 (± 2.0)
34.1–38.7 (± 1.6)
52.6–59.1 (± 1.9)
13.5–17.9 (± 1.3)
17.9–23.3 (± 1.7)
18.1–22.6 (± 1.5)
0.6–1.1 (± 0.2)
25.7–28.2 (± 0.8)
3.0–3.3 (± 0.2)
38.5–44.3
Yes
Yes
No
No
No
No
No
No
6
8–10
9–10
16–17
Yes
Yes
55–60
Yes
No
32
8
Pn
Pl, Pomatoschistus lozanoi; Pmic, Pomatoschistus microps; Pm, Pomatoschistus minutus; Pn, Pomatoschistus norvegicus; Pp, Pomatoschistus pictus.
7
3
Number of specimens analysed
Meristic characters
Lower jaw teeth in single row (JTSR)
First dorsal-fin spines (D1S)
Second dorsal-fin rays (D2R)
Anal-fin rays (AR)
Pectoral-fin rays (PR)
Pelvic disc anterior membrane with villose rear edge (PAME)
Body scaled (BS)
Lateral line scales (LL)
Predorsal area scaled (PAS)
Breast scaled (BRS)
Total vertebrae (Ve)
Coloration
Large dark blotches laterally or on lateral midline (DBLM)
Large black spot at base of caudal fin (BSCF)
D1&D2 with 1–2 rows of large dark spots (DRDS)
Dark blotch in upper anterior corner of each dorsal fin (DBCD)
D1 with dark spot in posterior part (D1DS)
Papillae rows
Sub-orbital papillae row a present (SPRP)
Second transverse row of sensory papillae descending below level
of row d (TR2D)
Morphometric measurements
Standard length (SL, mm)
%SL
Body depth (BD)
Head length (HL)
Head depth (HD)
Head width (HW)
Snout length (SnL)
Orbit diameter (OD)
Interorbital width (IW)
Caudal peduncle depth (CPD)
Caudal peduncle length (CPL)
Prepectoral length (PPL)
Prepelvic length (PVL)
Predorsal length (PD1L)
Preanal length (PAL)
First dorsal-fin hight (D1H)
Pectoral-fin length (PL)
Pelvic-fin length (VL)
Orbit diameter/head length (OD/HL)
Pelvic-fin insertion to anal-fin origin (VI-AO)
Pelvic-fin insertion to anal-fin origin/pelvic-fin length ((VI-AO)/VL)
Pmic
Pl
Species
13.3–18.8 (± 2.3)
25.9–31.1 (± 2.5)
15.4–16.0 (± 0.2)
15.7–18.5 (± 1.2)
5.5–8.3 (± 1.2)
7.5–8.4 (± 0.4)
2.3–3.3 (± 0.5)
7.0–8.6 (± 0.7)
22.7–26.0 (± 1.6)
26.7–32.7 (± 2.6)
32.2–35.8 (± 1.8)
33.1–38.1 (± 2.2)
57.6–59.9 (± 1.0)
15.7–16.1 (± 0.2)
23.3–30.7 (± 3.1)
23.5–26.5 (± 1.3)
0.7–0.9 (± 0.1)
23.5–32.0 (± 3.6)
2.8–3.5 (± 0.3)
34.4–36.1
Yes
No
Yes
No
Yes
No
No
No
6
8–9
9
18
No
Yes
35–40
No
No
29–30
4
Pp
Table 2. Data for morphological characters used for identification of Pomatoschistus species (n.e., character not estimated; morphometric measurements given
with their ranges as proportion of SL; standard deviations in parentheses)
836
T. KNEBELSBERGER AND R. THIEL
© 2014 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 172, 831–845
IDENTIFICATION OF GOBIES
specimens of G. flavescens and Gobiosoma bosc
(Lacepède, 1800) were characterized by the highest
number of first dorsal-fin spines among the analysed
species (Tables 1 and 2). Gobiosoma bosc (Thiel et al.,
2012) was easily distinguished from G. flavescens based
on its lack of body scales (Thiel et al., 2012). Furthermore, G. bosc has more second dorsal-fin rays and fewer
total vertebrae than G. flavescens (Table 1).
Neogobius melanostomus (Pallas, 1811) is identified by a typical large black spot in the posterior part
of the first dorsal fin (Hempel & Thiel, 2013) in combination with dark lateral blotches and a relatively high
number of total vertebrae (Table 1). Gobius niger
(Linnaeus, 1758) has also dark lateral blotches similar
to those of N. melanostomus, but it differs from the
latter species in having a significantly lower number
of total vertebrae, dark blotches in the upper anterior corner of each dorsal fin and the absence of a dark
spot in the posterior part of the first dorsal fin (Table 1).
The five species of Pomatoschistus were distinguished from all the other species by a combined occurrence of the following characters: teeth of lower jaw
in more than one series; body always scaled; more than
eight anal-fin rays; fewer than seven first dorsal-fin
spines; and presence of sub-orbital papillae row a.
Pomatoschistus pictus (Malm, 1865) and P. microps
(Krøyer, 1838) were distinguished from the other three
Pomatoschistus species based on their significantly lower
numbers of lateral line scales and total vertebrae, the
lack of a villose rear edge on the pelvic disc anterior
membrane and the lack of scales in the predorsal area
(Table 2).
Pomatoschistus pictus differed from P. microps in the
presence of one or two rows of large dark spots on the
first and second dorsal fins.
The three remaining Pomatoschistus species,
P. minutus (Pallas, 1770), P. lozanoi (de Buen, 1923)
and P. norvegicus (Collett, 1903), are referred to as the
‘P. minutus complex’ owing to their high morphological similarity.
Pomatoschistus minutus differed from the other two
species based on its second transverse row of sensory
papillae not descending below the level of row d
(Table 2). Pomatoschistus lozanoi was distinguished from
P. norvegicus by the following characters: the higher
number of pectoral fin rays; the scaled breast; the lower
length of the caudal peduncle; and the lower ratio of
pelvic-fin insertion to anal-fin origin/pelvic-fin length
(Table 2).
DNA
BARCODING
–
SPECIES DELINEATION
The mitochondrial COI barcoding region was obtained from 73 individuals of 12 gobiid species (Table
S1). The COI sequences did not show any stop codons,
and no insertions or deletions were found after se-
837
quence alignment. Sequence length ranged from 546
to 652 bp (mean and standard deviation: 648.9 ± 12.9
bp). The average base composition was 21.8% adenine
(A), 29.7% cytosine (C), 19.6% guanine (G) and 28.8%
thymine (T) (GC content 49.3%). The total number of
ambiguous base calls (Ns) was six. A number of 396
sites (60.7%) were identical throughout the entire 652bp-long sequence alignment.
The results of the morphological species identification were confirmed by DNA barcoding. In the NJ analysis of the COI genetic uncorrected p-distances,
haplotypes from the same morphological species were
grouped in non-overlapping clusters (Fig. 3). No single
haplotype was shared between species. Bootstrap values
for species clusters were 99% in one case (P. norvegicus)
and 100% in all the others. In the NJ phenogram all
species of the genus Pomatoschistus were monophyletic
with a bootstrap support of 99% (Fig. 3). Surprisingly, Gobiusculus flavescens appeared within this clade.
The species discrimination success within a barcoding
dataset depends on the presence of a gap between the
maximum intraspecific distance and the distance to
the nearest neighbouring species. This ‘barcoding gap’
was found in the comparison between all intraspecific
and interspecific distance values (Fig. 4). For every
species the maximum intraspecific distance was smaller
than the minimum distance to the nearest neighbouring species (Table 3). This was visualized by plotting
the maximum intraspecific distance against the distance to the nearest neighbour (Fig. 5). The barcoding
gaps ranged from 2.35 to 19.21%. The lowest values
were found within the clade including the genus
Pomatoschistus and Gobiusculus flavescens (2.35–
11.90%). Even the apparently very closely related species
P. norvegicus and P. lozanoi exhibited barcoding gaps
to one another of 2.35 and 2.38%, respectively.
Intraspecific distance values ranged from 0.0 to 3.37%
(mean and standard deviation: 0.37 ± 0.55%), and most
of the values were below 1% (Table 3). Two species
showed maximum intraspecific values between 1 and
2%, namely P. norvegicus (1.41%) and P. lozanoi (1.38%).
Sequences of C. linearis showed a maximum intraspecific
distance of 3.37% (1.75 ± 0.91%), even though a nearestneighbour distance of 19.48% guaranteed its unambiguous identification. In the NJ phenogram, C. linearis
split into two well-supported clusters (Fig. 3) with bootstrap values > 90%. This indicates a remarkable degree
of cryptic diversity within a population as the specimens analysed here were taken from the same locality. Interspecific distances ranged from 3.76 to 24.85%
(18.97 ± 4.25%) (Table 4). In this study, only the genus
Pomatoschistus was represented by more than one
species. The values among Pomatoschistus species
ranged between 3.76 and 15.05% (11.62 ± 2.61%).
Genetic distances between congeners were on average
31-fold higher than those within species. At the family
© 2014 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 172, 831–845
838
T. KNEBELSBERGER AND R. THIEL
Figure 3. Tree based on the neighbour-joining (NJ) analysis of pairwise genetic p-distances. Numbers next to branches
indicate bootstrap values > 50 for 10 000 replicates. Numbers in bold represent bootstrap support for species clades. Scale:
2% p-distance.
© 2014 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 172, 831–845
IDENTIFICATION OF GOBIES
839
Figure 4. Frequency distribution of 217 intraspecific and 2411 interspecific pairwise genetic p-distance values. Enlarged area displays the ‘barcoding gap’ (grey) between intra- and interspecific distances.
Table 3. Minimum and maximum intraspecific genetic p-distances for each species including mean and standard deviation
Mean
No. of
intraspecific
Species specimens distance
SD
Minimum
Maximum
Nearest
Distance to
intraspecific intraspecific neighbouring nearest
distance
distance
species
neighbour
Am
Bj
Cl
Gb
Gf
Gn
Nm
Pl
Pmic
Pm
Pn
Pp
0.00
0.31
0.15
0.00
0.00
0.00
0.00
1.28 (1.29)
0.00
0.00
0.15
0.00
10
4
6
4
6
4
7
3
7
10
8
4
0.26
0.62
1.75 (1.78)
0.18
0.00
0.00
0.00
1.31 (1.33)
0.38
0.23
0.54
0.23
± 0.19
± 0.24 (± 0.25)
± 0.91 (± 0.94)
± 0.12
± 0.00
± 0.00
± 0.00
± 0.06
± 0.35
± 0.21
± 0.48 (± 0.49)
± 0.16
0.61 (0.62)
0.94
3.37 (3.47)
0.31
0.00
0.00
0.00
1.38 (1.40)
0.92 (0.93)
0.61 (0.62)
1.41 (1.42)
0.46
Barcoding
gap
Cl, Nm (Nm) 19.48 (22.90) 18.87 (22.28)
Gf
16.10 (18.28) 15.16 (17.34)
Am
19.48 (23.17) 16.11 (19.70)
Am
19.52 (23.07) 19.21 (22.75)
Pp
10.58 (11.66) 10.58 (11.66)
Pmic
18.41 (20.89) 18.41 (20.89)
Gn
18.87 (22.26) 18.87 (22.26)
Pn
3.76 (3.88)
2.38 (2.40)
Pn
12.82 (14.29) 11.90 (13.36)
Pn
8.43 (9.06)
7.82 (8.44)
Pl
3.76 (3.88)
2.35 (2.46)
Gf
10.58 (11.66) 10.12 (11.22)
The barcoding gap indicates the difference between the maximum intraspecific and the minimum interspecific genetic
distance (nearest neighbour). K2P genetic distances additionally reported in parentheses, when differing from p-distances.
For abbreviations see Tables 1 and 2.
level distances ranged from 10.58 to 24.85%
(20.40 ± 2.77%). The overlapping of distances on the
family and genus levels was caused by G. flavescens
sequences (see arrows in Fig. 6). This species oc-
curred within the Pomatoschistus cluster within the
NJ tree (Fig. 3), suggesting it to be closely related with
this genus. K2P values were equal to uncorrected
p-distances for most of the intraspecific comparisons
© 2014 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 172, 831–845
840
T. KNEBELSBERGER AND R. THIEL
Table 4. Summary of intraspecific, interspecific, intrageneric (only for Pomatoschistus) and interfamilial pairwise genetic
p-distances; K2P genetic distances are additionally given in parentheses when differing from p-distances
Comparisons
No. of
comparisons
Minimum
distance
Maximum
distance
Mean distance
SD
Intraspecies
Interspecific
Within genus
Within family
217
2411
393
2018
0.00
3.76 (3.88)
3.76 (3.88)
10.58 (11.66)
3.37 (3.47)
24.85 (31.00)
15.04 (17.02)
24.85 (31.00)
0.37 (0.38)
18.97 (22.41)
11.62 (12.87)
20.40 (24.27)
± 0.55 (± 0.56)
± 4.25 (± 5.55)
± 2.61 (± 3.07)
± 2.77 (± 3.72)
Figure 5. Maximum intraspecific genetic p-distance versus
minimum distance to the nearest neighbour for each species.
The latter value (y) is always higher than the former (x).
(Table 3). On higher taxonomic levels K2P distances
were always higher than the uncorrected ones (Table 4).
This is also true for all of the distances to the nearest
neighbours and all barcoding gaps (Table 3).
BIN
REPORT
The BIN analysis assigned the 73 COI sequences to
12 separate clusters (Table 5 and Table S1) corresponding to sequence- and morphology-based identification. Six BINs representing the species A. minuta,
C. linearis, G. bosc, G. flavescens, N. melanostomus and
P. minutus show taxonomic concordance; in each BIN
sequences clustered together with already published
sequences assigned to the same species. The BIN clusters of B. jeffreysii, P. norvegicus and P. pictus only contained sequences from the present study.
The BINs of P. lozanoi, G. niger and P. microps showed
discordances at different taxonomic levels. The se-
Figure 6. Boxplot of pairwise genetic p-distances on different taxonomic levels. A, within-species variation; B and
C, variation on the genus (B) (comprising only the genus
Pomatoschistus) and family (C) level. D, summary of all
interspecific distance values. The box comprises the 25–
75th percentile of the data, the vertical line the median,
the ‘whiskers’ the 5th to 95th percentile while dots depict
outliers. The grey bar indicates the ‘barcoding gap’ between
intra- and interspecific distances. Between arrows: values
between Gobiusculus flavescens and species of the genus
Pomatoschistus.
quences of P. lozanoi clustered together with five sequences assigned to the species Gaidropsarus biscayensis
belonging to the order Gadiformes. These data were
all provided by the same source and probably represent a case of misidentification or contamination. The
G. niger BIN contained 46 records, of which 41 were
identified as G. niger by various researchers. The remaining five records were assigned to gobiid species
of the genus Pomatoschistus, as for instance P. microps,
P. pictus and P. minutus. The BIN including P. microps
exhibited only conflicts on the species level. The seven
barcodes generated in the present study clustered
together with ten sequences identified as P. lozanoi or
P. minutus.
© 2014 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 172, 831–845
IDENTIFICATION OF GOBIES
841
Table 5. Analysis of Barcode Index Numbers (BINs) performed with the ‘BIN Discordance Report’ tool available on BOLD
Species
Species with discordant BINs:
Pomatoschistus lozanoi
Gobius niger
Pomatoschistus microps
Species with concordant BINs:
Aphia minuta
Buenia jeffreysii
Crystallogobius linearis
Gobiosoma Bosc
Gobiusculus flavescens
Neogobius melanostomus
Pomatoschistus minutus
Pomatoschistus norvegicus
Pomatoschistus pictus
Highest rank
of conflict
BIN
BIN total
members
Order
Genus
Species
BOLD:AAE3251
BOLD:AAC0219
BOLD:AAD1389
8
46
17
–
–
–
–
–
–
–
–
–
BOLD:ABU8824
BOLD:AAU0731
BOLD:AAU2778
BOLD:ABX8247
BOLD:ACG5215
BOLD:AAC0218
BOLD:AAU0729
BOLD:AAU0730
BOLD:ACL0958
16
4
10
9
10
77
14
8
4
DISCUSSION
SPECIES
IDENTIFICATION AND DELINEATION
Our study provides robust species-level identifications of North Sea and Baltic Sea gobiid species by
applying a detailed analysis of a wide range of morphological characteristics, including meristic characters such as dentition of lower jaws, number of first
dorsal-fin spines, number of second dorsal-fin, analfin and pectoral-fin rays, occurrence of scales on the
body, number of total vertebrae as well as several coloration and papillae row patterns. For the successful
identification of Pomatoschistus species, we evaluated additional characters such as morphology of pelvic
disc anterior membrane rear edge, number of lateral
line scales, occurrence of scales at breast and predorsal
area as well as a number of different morphometric
measurements.
Few meristic characters were sufficient to identify
A. minuta, C. linearis and B. jeffreysii, while further
meristic characters and/or coloration patterns were necessary for the identification of G. flavescens, G. bosc,
N. melanostomus and G. niger. Two of the five
Pomatoschistus species (P. pictus, P. microps) were discriminated after analysing the papillae row pattern,
and in particular the presence/absence of the suborbital papillae row a and its combination with other
meristic features. The three species of the P. minutus
complex were the most difficult to distinguish (Webb,
1980). We identified P. minutus, P. norvegicus and
P. lozanoi considering the extension of the second transverse papillae row in combination with several
meristic features and additional morphometric measurements. In this context, the caudal peduncle length
and ratio of pelvic-fin insertion to anal-fin origin/pelvic-
fin length were considered here for the first time as
diagnostic characters for the distinction among species
of the P. minutus complex. However, further investigations are recommended to test the reliability of these
new distinguishing features.
Our morphological identifications were confirmed by
analyses of the DNA barcodes. Sequences derived from
morphologically conspecific specimens clustered together with very high bootstrap support. Barcoding gaps
were apparent for all species, allowing an undoubted
assignment of specimens to species clusters. Even the
five closely related species of the genus Pomatoschistus
were clearly delimitated by COI sequences.
It is standard procedure in DNA barcoding studies
to use K2P distances as the species clustering method,
but it has been shown elsewhere that these values may
fit poorly at the species level (Collins et al., 2012). We
calculated both uncorrected p and K2P distances and
found no qualitative difference in barcoding success
between the two metrics. All barcoding gaps in our study
were more pronounced using K2P values. This demonstrates that K2P still represents a useful model for
analysing barcoding data. In addition, the use of K2P
enables direct comparisons with other studies. Within
the P. minutus species complex, P. lozanoi and
P. norvegicus appear as closest relatives separated by
a minimum genetic K2P distance of 3.88%. Intraspecific
K2P distances of these two species were 1.33 and 0.54%,
respectively. For COI barcodes, intraspecific K2P distances are generally below 2% (Hebert et al., 2003;
Hebert, Ratnasingham & deWaard, 2003) which has
also been observed in fish (Ward, 2012; Knebelsberger
et al., 2014) and has been confirmed for barcoding of
gobiid fishes (Viswambharan et al., 2013). Low
intraspecific distances were also demonstrated for
© 2014 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 172, 831–845
842
T. KNEBELSBERGER AND R. THIEL
extremely distant fish populations (Ward et al., 2008b).
Therefore, we would not expect considerably higher
intraspecific distances for P. lozanoi and P. norvegicus,
even if a more extended sampling within the species
distribution range was considered.
Pomatoschistus norvegicus had a minimum p-distance
of 8.43% from its nearest neighbour P. minutus, allowing a clear separation among P. minutus, P. lozanoi
and P. norvegicus. However, P. lozanoi is considered to
be morphologically intermediate between the other two
species and is suspected to interbreed with P. minutus
and P. norvegicus in the wild (Webb, 1980). In this study,
we estimated intermediate numbers of second dorsalfin and pectoral-fin rays as well as of lateral line scales
for P. lozanoi (Table 2). Pomatoschistus norvegicus
appears in offshore habitats, and it is unlikely to occur
together with the other species (Hamerlynck, 1990).
However, successful instances of hybridization in the
wild cannot be excluded, as at least P. minutus and
P. lozanoi occur in the same habitats in the NorthEast Atlantic (Wallis & Beardmore, 1980). The remaining two Pomatoschistus species analysed in this
study, P. microps and P. pictus, exhibit K2P distances
to their nearest neighbour above 11%. For all the other
species in our dataset, K2P distances to the nearest
neighbour were higher than 16%, probably because no
further congeneric species were analysed. The species
of Pomatoschistus were grouped together in a clade
with G. flavescens, and its genetic distance to the
Pomatoschistus species suggests a congeneric status
(Fig. 6). However, a closer relationship between
G. flavescens and Pomatoschistus could not be confirmed by morphological characteristics.
EXTERNAL
CONGRUENCE OF
DNA
BARCODE DATA
The BIN analysis demonstrated that our data were
congruent with published sequences for most of the
species. In some cases, DNA barcodes assigned to the
same species were included in more than one BIN, probably because of specimen misidentifications. Besides
that, incongruence may be caused by contamination,
synonymy and syntax errors (Tautz et al., 2003;
Radulovici, Archambault & Dufresne, 2010; Ward, 2012).
Species of the genus Pomatoschistus are well known
for being taxonomically difficult to identify (Webb, 1980).
For instance, several sequences assigned to P. minutus
and P. lozanoi were found within the P. microps BIN.
Our results, however, demonstrated that DNA barcoding
discriminates all five Pomatoschistus species by assigning the COI sequences to five different BIN
clusters. The BIN analysis clearly demonstrated the
importance of accurate morphological species identification at the beginning of the barcoding pipeline
to minimize misidentifications causing discordant
results.
CONCLUSION
Here we present a DNA barcode library for the reliable identification of about two-thirds of the expected
gobiid fauna of the North Sea and Baltic Sea, including all species of the genus Pomatoschistus known from
this region. The molecular analysis was based on a detailed analysis of morphological diagnostic characters. The integration of molecular and morphological
data for species identification was essential to provide
a robust and accurate database, which is now available for the end-user. The library represents a contribution towards the effort of barcoding the fish fauna
of the North-East Atlantic. All sequences as well as
metadata were submitted to the BOLD (Ratnasingham
& Hebert, 2007) database and are available as a public
project.
ACKNOWLEDGEMENTS
We thank the Thünen Institute of Sea Fisheries for
supporting our sampling and Mattias Hempel, Dr
Joachim Horstkotte, Hermann Neumann, Jörg Scholle,
Inken Rottgart, Dr Andrés Velasco and the fishermen Mr Brauer and Mr Ihnken for collecting specimens. Many thanks go to Irina Eidus for preparing
the X-rays and to Renate Thiel for her help in managing the morphological data. We thank the Biodiversity Institute of Ontario (BIO) for providing the
sequencing service for part of the data. The molecular part of the project (T.K.) was funded by the Federal
Ministry of Education and Research (Grant no.
03F0499A) and the Land Niedersachsen.
REFERENCES
Agorreta A, San Mauro D, Schliewen U, Van Tassell JL,
Kovačić M, Zardoya R, Rüber L. 2013. Molecular
phylogenetics of Gobioidei and phylogenetic placement of
European gobies. Molecular Phylogenetics and Evolution 69:
619–633.
Akahito P. 1986. Some morphological characters considered
to be important in gobiid phylogeny. In: Uyeno T, Arai R,
Taniuchi T, Matsuura K, eds. Indo-Pacific fish biology. Proceedings of the second international conference on IndoPacific Fishes. Tokyo: The Ichthyological Society of Japan,
629–639.
Apostolou A, Ivanova P, Velkov B, Vassilev M, Dobrev
D, Dobrovolov I. 2011. Pomatoschistus marmoratus (RISSO
1810), is it really a ‘new’ species for Bulgarian ichthyofauna?
Acta Zoologica Bulgarica 63: 289–294.
April J, Mayden RL, Hanner RH, Bernatchez L. 2011.
Genetic calibration of species diversity among North America’s freshwater fishes. Proceedings of the National Academy
of Sciences of the United States of America 108: 10602–
10607.
© 2014 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 172, 831–845
IDENTIFICATION OF GOBIES
Brunken H, Castro JF, Hein M, Verwold A, Winkler M.
2012. First records of round goby Neogobius melanostomus
(Pallas, 1814) in the river Weser. Lauterbornia 75:
31–37.
Carneiro M, Martins R, Landi M, Costa FO. 2014. Updated
checklist of marine fishes (Chordata: Craniata) from Portugal and the proposed extension of the Portuguese continental shelf. European Journal of Taxonomy 73: 1–73.
Cerutti-Pereyra F, Meekan MG, Wei N-WV, O’Shea O,
Bradshaw CJA, Austin CM. 2012. Identification of rays
through DNA barcoding: an application for ecologists. PLoS
ONE 7: e36479.
Collins RA, Boykin LM, Cruickshank RH, Armstrong KF.
2012. Barcoding’s next top model: an evaluation of nucleotide
substitution models for specimen identification. Methods in
Ecology and Evolution 3: 457–465.
Costa FO, Landi M, Martins R, Costa MH, Costa ME,
Carneiro M, Alves MJ, Steinke D, Carvalho GR. 2012.
A ranking system for reference libraries of DNA barcodes:
application to marine fish species from Portugal. PLoS ONE
7: e35858.
Edgar RC. 2004. MUSCLE: multiple sequence alignment with
high accuracy and high throughput. Nucleic Acids Research 32: 1792–1797.
Eschmeyer WN, ed. 2014. Catalog of fishes. San Francisco:
California Academy of Sciences. Available at: http://
research.calacademy.org/research/ichthyology/catalog/
fishcatmain.asp). Electronic version accessed 6 February 2014.
Felsenstein J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783–791.
FISH-BOL. 2012. The fish Barcode of life initiative.
Retrieved 21 January 2014. Available at: http://www.fishbol.org/
index.php
Freyhof J. 2011. Diversity and distribution of freshwater
gobies from the Mediterranean, the Black and Caspian Seas.
In: Patzner RA, Van Tassell JL, Kovačić M, Kapoor BG, eds.
The biology of gobies. New York: Science Publishers, 3–22.
Geiger MF, Herder F, Monaghan MT, Almada V, Barbieri
R, Bariche M, Berrebi P, Bohlen J, Casal-Lopez M,
Delmastro GB, Denys GP, Dettai A, Doadrio I,
Kalogianni E, Kärst H, Kottelat M, Kovačić M, Laporte
M, Lorenzoni M, Marčić Z, Ozuluğ M, Perdices A, Perea
S, Persat H, Porcelotti S, Puzzi C, Robalo J, Sanda R,
Schneider M, Slechtová V, Stoumboudi M, Walter S,
Freyhof J. 2014. Spatial heterogeneity in the Mediterranean biodiversity hotspot affects barcoding accuracy of
its freshwater fishes. Molecular Ecology Resources. Online
in advance of print. doi: 10.1111/1755-0998.12257.
Gysels E, Hellemans B, Pampoulie C, Volckaert FAM.
2004b. Phylogeography of the common goby, Pomatoschistus
microps, with particular emphasis on the colonization of the
Mediterranean and the North Sea. Molecular Ecology 13: 403–
417.
Gysels E, Hellemans B, Patarnello T, Volckaert FAM.
2004a. Current and historic gene flow of the sand goby
Pomatoschistus minutus on the European Continental Shelf
and in the Mediterranean Sea. Biological Journal of the
Linnean Society 83: 561–576.
843
Hamerlynck O. 1990. The identification of Pomatoschistus
minutus (Pallas) and Pomatoschistus lozanoi (de Buen) (Pisces,
Gobiidae). Journal of Fish Biology 37: 723–728.
Hebert PDN, Cywinska A, Ball SL, deWaard JR. 2003. Biological identifications through DNA barcodes. Proceedings of
the Royal Society of London Series B 270: 313–321.
Hebert PDN, Ratnasingham S, deWaard JR. 2003. Barcoding
animal life: cytochrome c oxidase subunit 1 divergences among
closely related species. Proceedings of the Royal Society of
London Series B 270: 96–99.
HELCOM. 2012. Checklist of the Baltic Sea Macro-species. Baltic
Sea Environment Proceedings No. 130.
Hempel M, Thiel R. 2013. First records of the round goby
Neogobius melanostomus (Pallas, 1814) in the Elbe River,
Germany. BioInvasions Records 2: 291–295.
Hubbs CL, Lagler KF. 1958. Fishes of the Great Lakes Region,
revised edition. Michigan: University of Michigan Press.
Hubert N, Delrieu-Trottin E, Irisson J-O, Meyer C, Planes
S. 2010. Identifying coral reef fish larvae through DNA
barcoding: a test case with the families Acanthuridae and
Holocentridae. Molecular Phylogenetics and Evolution 55: 1195–
1203.
Hubert N, Hanner R, Holm E, Mandrak NE, Taylor E,
Burridge M, Watkinson D, Dumont P, Curry A, Bentzen
P, Zhang J, April J, Bernatchez L. 2008. Identifying Canadian freshwater fishes through DNA barcodes. PLoS ONE
3: e2490.
Hubert N, Meyer CP, Bruggemann HJ, Guérin F, Komeno
RJL, Espiau B, Causse R, Williams JT, Planes S. 2012.
Cryptic diversity in Indo-Pacific coral reef fishes revealed by
DNA-barcoding provides new support to the centre-ofoverlap hypothesis. PLoS ONE 7: e28987.
Hyung-Bae J, Seung-Ho C, Ho YS. 2012. Exploring the utility
of partial cytochrome c oxidase subunit 1 for DNA barcoding
of gobies. Animal Systematics, Evolution and Diversity 28:
269–278.
Ivanova NV, Zemlak TS, Hanner RH, Hebert PDN. 2007.
Universal primer cocktails for fish DNA barcoding. Molecular Ecology Notes 7: 544–548.
Jones AG, Walker D, Kvarnemo C, Lindström K, Avise
JC. 2001. How cuckoldry can decrease the opportunity for
sexual selection: data and theory from a genetic parentage
analysis of the sand goby, Pomatoschistus minutus. Proceedings of the National Academy of Sciences of the United States
of America 98: 9151–9156.
Keskin E, Atar HH. 2013. DNA barcoding commercially important fish species of Turkey. Molecular Ecology Resources
13: 788–797.
Kimura M. 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies
of nucleotide sequences. Journal of Molecular Evolution 16:
111–120.
Knebelsberger T, Landi M, Neumann H, Kloppmann M,
Sell AF, Campbell PD, Laakmann S, Raupach MJ,
Carvalho GR, Costa FO. 2014. A reliable DNA barcode
reference library for the identification of the North
European shelf fish fauna. Molecular Ecology Resources
14: 1060–1071.
© 2014 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 172, 831–845
844
T. KNEBELSBERGER AND R. THIEL
Knebelsberger T, Stöger I. 2012. DNA extraction, preservation, and amplification. In: Kress WJ, Erickson DL, eds.
DNA barcodes: methods and protocols. Berlin: Springer
Science+Business Media, LLC 2012, Methods in Molecular
Biology, vol. 858. 311–338.
Kornis MS, Mercado-Silva N, Vander Zanden MJ. 2012.
Twenty years of invasion: a review of round goby Neogobius
melanostomus biology, spread and ecological implications.
Journal of Fish Biology 80: 235–285.
Kovačić M, Patzner RA. 2011. North-Eastern Atlantic and
Mediterranean gobies. In: Patzner RA, Van Tassell JL, Kovačić
M, Kapoor BG, eds. The biology of gobies. New York: Science
Publishers, 3–22.
Larmuseau MHD, Guelinckx J, Hellemans B, Van Houdt
JKJ, Volckaert FAM. 2008. Fast PCR-RFLP method facilitates identification of Pomatoschistus species from the North
Atlantic. Journal of Applied Ichthyology 24: 342–344.
Leitão R, Martinho F, Neto JM, Cabral H, Marques JC,
Pardal MA. 2006. Feeding ecology, population structure and
distribution of Pomatoschistus microps (Krøyer, 1838) and
Pomatoschistus minutus (Pallas, 1770) in a temperate estuary,
Portugal. Estuarine, Coastal and Shelf Science 66: 231–239.
Lindström K, St. Mary CM, Pampoulie C. 2006. Sexual selection for male parental care in the sand goby, Pomatoschistus
minutus. Behavioral Ecology and Sociobiology 60: 46–51.
Mabragaña E, Díaz de Astarloa JM, Hanner R, Zhang J,
González Castro M. 2011. DNA barcoding identifies Argentine fishes from marine and brackish waters. PLoS ONE
6: e28655.
McCusker MR, Denti D, Van Guelpen L, Kenchington E,
Bentzen P. 2013. Barcoding Atlantic Canada’s commonly
encountered marine fishes. Molecular Ecology Resources 13:
177–188.
Miller PJ. 1986. Gobiidae. In: Whitehead PJI, Bauchot M-L,
Hureau JC, Nielson J, Tortononese E, eds. Fishes of the
North-eastern Atlantic and the Mediterranean. Paris: UNESCO,
1019–1085.
Nelson J. 2006. Fishes of the world, 4th edn. New York: John
Wiley and Sons, Inc.
Pampoulie C, Gysels ES, Maes GE, Hellemans B, Leentjes
V, Jones AG, Volckaert FAM. 2004. Evidence for finescale genetic structure and estuarine colonization in a
potential high gene flow marine goby (Pomatoschistus minutus).
Heredity 92: 434–445.
Pegg GG, Sinclair B, Briskey L, Aspden WJ. 2006. MtDNA
barcode identification of fish larvae in the southern Great
Barrier Reef, Australia. Scientia Marina 70S2: 7–12.
Puckridge M, Andreakis N, Appleyard S, Ward RD.
2013. Cryptic diversity in flathead fishes (Scorpaeniformes:
Platycephalidae) across the Indo-West Pacific uncovered
by DNA barcoding. Molecular Ecology Resources 13:
32–42.
Radulovici AE, Archambault P, Dufresne F. 2010. DNA
barcodes for marine biodiversity: moving fast forward?
Diversity 2: 450–472.
Ratnasingham S, Hebert PDN. 2007. BOLD: the
barcode of life data system. Molecular Ecology Notes 7: 355–
364.
Ratnasingham S, Hebert PDN. 2013. A DNA-based registry for all animal species: the barcode index number (BIN)
system. PLoS ONE 8: e66213.
Rodrigues P, Reis-Henriques MA, Campos J, Santos MM.
2006. Urogenital papilla feminization in male Pomatoschistus
minutus from two estuaries in northwestern Iberian
Peninsula. Marine Environmental Research 62:
258–262.
Saitou N, Nei M. 1987. The neighbor-joining method: a new
method for reconstructing phylogenetic trees. Molecular Biology
and Evolution 4: 406–425.
Steinke D, Zemlak TS, Boutillier J, Hebert PDN. 2009a.
DNA barcoding of Pacific Canada’s fishes. Marine Biology 156:
2641–2647.
Steinke D, Zemlak TS, Hebert PDN. 2009b. Barcoding nemo:
DNA-based identifications for the ornamental fish trade. PLoS
ONE 4: e6300.
Tamura K, Peterson D, Peterson N, Stecher G, Nei M,
Kumar S. 2011. MEGA5: molecular evolutionary genetics
analysis using maximum likelihood, evolutionary distance,
and maximum parsimony methods. Molecular Biology and
Evolution 28: 2731–2739.
Tautz D, Arctander P, Minelli A, Thomas RH, Vogler AP.
2003. A plea for DNA taxonomy. Trends in Ecology and
Evolution 18: 70–74.
Thacker CE, Roje DM. 2011. Phylogeny of Gobiidae and identification of gobiid lineages. Systematics and Biodiversity 9:
329–347.
Thiel R, Scholle J, Schulze S. 2012. First record of the naked
goby Gobiosoma bosc (Lacepède, 1800) in European waters.
BioInvasions Records 1: 295–298.
Tornabene L, Chen Y, Pezold F. 2013. Gobies are deeply
divided: phylogenetic evidence from nuclear DNA (Teleostei:
Gobioidei: Gobiidae). Systematics and Biodiversity 11: 345–
361.
Van Tassel JL, Tornabene L, Taylor MS. 2011. A history
of gobioid morphological systematics. In: Patzner RA, Van
Tassell JL, Kovačić M, Kapoor BG, eds. The biology of gobies.
New York: Science Publishers, 3–22.
Victor BC, Hanner R, Shivji M, Hyde J, Caldow C. 2009.
Identification of the larval and juvenile stages of the Cubera
Snapper, Lutjanus cyanopterus, using DNA barcoding. Zootaxa
2215: 24–36.
Viswambharan D, Pavan-Kumar A, Singh DP,
Jaiswar AK, Chakraborty SK, Nair JR, Lakra WS.
2013. DNA barcoding of gobiid fishes (Perciformes,
Gobioidei). Mitochondrial DNA. doi:10.3109/19401736.2013
.834438.
Wallis GP, Beardmore JA. 1980. Genetic evidence
for naturally occurring fertile hybrids between two
goby species, Pomatoschistus minutus and P. lozanoi
Pisces, Gobiidae. Marine Ecology Progress Series 34: 309–
315.
Ward RD. 2012. FISH-BOL, a case study for DNA barcodes.
In: Kress WJ, Erickson DL, eds. DNA barcodes: methods
and protocols. Berlin: Springer Science+Business Media,
LLC 2012, Methods in Molecular Biology vol. 858, 423–
439.
© 2014 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014, 172, 831–845
IDENTIFICATION OF GOBIES
Ward RD, Costa FO, Holmes BH, Steinke D. 2008b. DNA
barcoding of shared fish species from the North Atlantic and
Australasia: minimal divergence for most taxa, but Zeus
faberand Lepidopus caudatus each probably constitute two
species. Aquatic Biology 3: 71–78.
Ward RD, Holmes BH, William TW, Last PR. 2008a.
DNA barcoding Australasian chondrichthyans: results and
potential uses in conservation. Marine and Freshwater Research 59: 57–71.
Ward RD, Zemlak TS, Innes BH, Last PR, Hebert PDN.
2005. DNA barcoding Australia’s fish species. Philosophical
Transactions of the Royal Society of London Series B 360:
1847–1857.
845
Webb CJ. 1980. Systematics of the Pomatoschistus minutus
complex (Teleostei: Gobiidae). Philosophical Transactions of
the Royal Society of London Series B 291: 201–241.
Zander CD. 2011. Morphological adaption to special environments of gobies. In: Patzner RA, Van Tassell JL, Kovačić M,
Kapoor BG, eds. The biology of gobies. New York: Science
Publishers, 345–366.
Zemlak TS, Ward RD, Connell AD, Holmes BH, Hebert
PDN. 2009. DNA barcoding reveals overlooked marine fishes.
Molecular Ecology Resources 9: 237–242.
Zhang J, Hanner R. 2012. Molecular approach to the
identification of fish in the South China Sea. PLoS ONE 7:
e30621.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online version of this article at the publisher’s web-site:
Table S1. Supplementary metadata.
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