In this study 85 specimens representing two species of genus Ammodytes and two species of genus Hyperoplus were sampled from the North and the Baltic Seas (Suppl. material 1 and 2, Figure 1). For the molecular analysis 70 samples were collected from the North Sea during several cruises conducted by the Thünen Institute of Sea Fisheries (Hamburg, Germany) and the research vessel of the Senckenberg Institute (Wilhelmshaven, Germany). Tissue samples were taken from each of the 70 specimens and preserved in 96% ethanol for molecular analysis at Senckenberg’s German Center for Marine Biodiversity Research (DZMB, Wilhelmshaven, Germany). Specimens were preserved in 70% ethanol. The remaining 15 individuals belonging to the species A. tobianus were used for morphological analyses only and collected from the Baltic Sea during three different cruises conducted by the German Oceangraphic Museum (Stralsund, Germany). Immediately after catch, specimens were preserved in 4% formaldehyde solution. All 85 voucher specimens were databased and morphologically investigated at the Zoological Museum of the Center of Natural History of the University of Hamburg (ZMH, Hamburg, Germany). Finally, the material was stored for future reference in the ZMH fish collection. All COI sequences and related metadata belonging to the 70 voucher specimens from the North Sea are available on the Barcode of Life Data System (www.barcodinglife.org; Ratnasingham and Hebert 2007). DNA barcodes of eight specimens of H. immaculatus and 22 specimens of A. marinus were obtained from the BOLD project “Barcoding North Sea Fish I” (BNSFI) (Knebelsberger et al. 2014). Newly generated barcodes belonging to five specimens of A. marinus, six of A. tobianus, and 29 of H. lanceolatus were uploaded to the BOLD project “Barcoding North Sea Sand eels” (BNSSE). In addition to COI, nuclear Rhodopsin DNA sequences were generated from all 70 North Sea specimens (Suppl. material 1). For comparison, published Rhodopsin data was downloaded from GenBank (A. tobianus: AY141306; H. lanceolatus: EU492010 and EU492011).

Figure 1. 

Sampling sites of sand lance species of the genera Ammodytes and Hyperoplus.

Meristic parameters (Table 1) were analysed at the left-hand side of the specimens and supplemented with right-side counts when the left side was damaged. Counts of dorsal, ventral and principal caudal-fin rays as well as of vertebrae were taken from radiographs (Figure 2) made by an X-ray imaging system (Faxitron LX-60). The first caudal vertebra was defined as the first centrum with a long haemal spine, and the centrum fused to the hypural plate was counted as the last vertebra. Counts of dorsal-fin rays were made using the method of Nizinski et al. (1990). Counting dorsal-fin rays began with the first visible ray and excluded the one or two anterior rayless pterygiophores. However, these counts included the last two rays that were each supported by a pterygiophore. Counts of anal-fin rays included all rays visible from the outside. Gill rakers were counted on the lower and upper arch separately. Gill rakers of the lower arch included the raker at the junction between upper and lower parts of the arch. Dermal plicae included those anterior and posterior to the lateral-line pores.

Figure 2. 

Radiograph of Common sand eel Ammodytes tobianus (Linnaeus, 1758) indicating the meristic characters evaluated from X-ray pictures. Depicted specimen: ZMH 26098-3, standard length 128.1 mm.

Morphometric measurements (Table 2) were taken by vernier calipers to one tenth of a millimetre. Measurements were done following Hubbs and Lagler´s (1958) method, with the following changes: standard length (SL) was measured from the front of the upper lip in the median plane to the midbase of the caudal fin (end of hypural plate). The front of the upper lip was used as the anterior point of all other horizontal measurements. Head length (HL) was measured from the front of the upper lip to the posterior end of the opercular membrane. Body depth was measured twice, as the depth at the beginning of the base of the dorsal fin (BDD) and as the depth at the beginning of the base of the anal fin (BDA). Body width was measured as the maximum width at the beginning of the base of the dorsal fin (BWD). Orbit diameter (OD) is the maximum fleshy diameter. Interorbital width (IW) is the least fleshy width. Caudal-peduncle depth (CPD) is the smallest depth, and caudal peduncle length (CPL) the horizontal distance between verticals at the rear base of the anal fin and the caudal-fin base. Height of dorsal-fin rays (DFH) and anal-fin rays (AFH) were measured from their tips to the body contour. Caudal-fin length (CFL) was taken horizontally from the caudal-fin base to a vertical point at the tip of the longest ray. The values obtained were standardised by multiplying them by 100 and dividing them by the standard length.

All morphological data were statistically processed, involving ranges, means, and standard deviations. Morphological data of all specimens that had a complete suite of meristic and morphometric character data were used to conduct two multiple discriminant function analyses (DFA) to determine if the four species of sand lances could be differentiated based on meristic and/or morphometric parameters using XLSTAT (version 2013.0.04, Addinsoft), a statistical analysis add-in for Microsoft Excel®. DFA was used to demonstrate the degree of separation in multivariate space defined by the main patterns of morphological variation among species which is described via the discriminant functions. It also shows which character contributes more to the differentiation. The standardised discriminant function coefficients represent the contributions of every variable to the discriminatory power of the function. Hence, the larger the standardised coefficient, the larger the weight of the variable in the function. Both discriminant analyses were conducted for 76 individuals (22 A. marinus, 20 A. tobianus, 8 H. immaculatus, 26 H. lanceolatus). Morphological variables without any variation (e.g. principal caudal-fin rays (CR)), variables, where other variables are included (e.g. total vertebrae (TV)) and qualitative variables (e.g. premaxillae clearly protrusible (PCP)) were excluded from the DFA procedures. The first DFA was performed for the following eight quantitative meristic characters: dermal plicae, dorsal-fin rays, anal-fin rays, pectoral-fin rays, upper arch gill rakers, lower arch gill rakers, precaudal vertebrae, and caudal vertebrae. The second DFA was conducted for the following 19 morphometric parameters: body depth at dorsal-fin origin, body depth at anal-fin origin, body width at dorsal-fin origin, head length, snout length, orbit diameter, interorbital width, upper jaw length, caudal peduncle depth, caudal peduncle length, prepectoral length, predorsal length, preanal length, pectoral-fin length, dorsal-fin base length, anal-fin base length, caudal-fin length, dorsal-fin height, and anal-fin height.

Genomic DNA was extracted at the DZMB using the Qiagen DNeasy Blood and Tissue Kit for single columns as described by Knebelsberger and Stöger (2012). A 652-bp fragment of the mitochondrial (mt)COI gene was amplified for 38 samples using a M13 tailed primer cocktail (C_FishF1t1-C_FishR1t1) including FishF2_t1 (5’-TGTAAAACGACGGCCAGTCGACTAATCATAAAGATATCGGCAC), FishR2_t1 (5’-CAGGAAACAGCTATGACACTTCAGGGTGACCGAAGAATCAGAA), VF2_t1 (5’-TGTAAAACGACGGCCAGTCAACCAACCACAAAGACATTGGCAC) and FR1d_t1 (5’-CAGGAAACAGCTATGACACCTCAGGGTGTCCGAARAAYCARAA) (Ivanova et al. 2007). As a second marker, a 464 bp fragment of the nuclear (nc) Rhodpsin gene was amplified for all 70 samples using the forward primer Rh545 (5’-GCAAGCCCATCAGCAACTTCCG) and the reverse primer Rh1039r (5’-TGCTTGTTCATGCAGATGTAGA) developed by Chen et al. (2003). Each PCR reaction mixture contained 1 µl DNA template, 2.25 µl of 10X reaction buffer (including MgCl₂), 0.5 µl dNTPs (2mM each), 0.5 µl of each primer (10 pmol/µl), 0.5 µl Taq polymerase (5 U/µl; Qiagen) and molecular grade water for a total volume of 25µl. 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 50°C, 60 s at 72°C with a final extension of 10 min at 72°C. All 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) and 2 µl of Alkaline Phosphatase (1 U/µl) for 15 min at 37°C followed by 20 min at 75°C. Purified amplicons were sequenced by Macrogen Europe (Amsterdam, Netherlands).

Forward and reverse sequences of COI and Rhodpsin were assembled and edited using Geneious (version 7.1.9. http://www.geneious.com). Consensus sequences were submitted to GenBank (for accession numbers see Suppl. material 1). Variance in sequence length, base composition, number of invariable sites and the presence of stop codons were analysed using Geneious. The nc and mt sequences were aligned independently using MUSCLE (Edgar 2004) with default settings as implemented in MEGA version 6.06 (Tamura et al. 2013). Primer sequences were cut from the alignment. Rhodopsin gene sequence alignment was checked by eye for species specific diagnostic characters. For COI, Kimura-2-parameter (K2P) distances were calculated in MEGA, as K2P is used as standard model for barcoding analyses and enables direct comparison with other studies. Neighbour-Joining (NJ) topology (Saitou and Nei 1987) was built in MEGA using the “pairwise deletion” option for the treatment of gaps and missing data, in order 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. In order to quantify the distinctness between species at the barcode locus, genetic distances were used to calculate the difference between the maximum intraspecific genetic distance and the minimum distance to the nearest neighbor (barcode gap). For the calculation of genetic distances at genus and family level, we used BOLDs “Distance Summary” tool by choosing K2P distance model and MUSCLE (Edgar 2004) alignment algorithm.

On BOLD, DNA barcodes were automatically assigned to operational taxonomic units (OTUs), generated through Refined Single Linkage (RESL) analyses (Ratnasingham and Hebert 2013). Finally, a unique alphanumeric code is assigned to each of the OTUs, constituting the so called barcode index number (BIN). It has been shown that BINs are highly congruent with existing species assignments (Ratnasingham and Hebert 2013). Here, the ‘BIN Discordance Report’ analysis tool was applied to analyse our dataset together with public sequences on BOLD, and to get hints on cryptic diversity (species) or to identify cases of haplotype sharing between species.

Furthermore, BOLD’s “Diagnostic Characters” sequence analysis tool was applied to the COI dataset choosing MUSCLE (Edgar 2004) alignment algorithm. Sequences were grouped by species names in order to categorise consensus bases by their diagnostic potential.

Meristic characters and morphometric measurements of the four examined species of sand lances are given in Table 1. The number of individuals per analysed character ranged from 24 to 27 for A. marinus, from 20 to 21 for A. tobianus, from 28 to 29 for H. lanceolatus, and was eight individuals for H. immaculatus (Table 1).

The data of the present study confirmed that the two genera of Ammodytes and Hyperoplus can be distinguished by qualitative meristic characters, i.e. by having a clear protrusible premaxillae (PCP) and no vomerine teeth (VTP) (Ammodytes) or no clear protrusible premaxillae and two vomerine teeth (Hyperoplus) (Table 1). Hyperoplus can also be separated from Ammodytes by its significantly higher number of dermal plicae (DP). It is also possible to distinguish Hyperoplus from Ammodytes by its obviously lower pectoral-fin length (PFL), and to a somewhat lesser significance, also by its greater mean snout length (SNL), since no sexual dimorphism has been reported for the last two characters in both genera.

Hyperoplus lanceolatus can be separated from H. immaculatus by the presence of a conspicuous dark spot on either side of snout (DSSS) which is lacking in H. immaculatus (Table 1). Furthermore, H. lanceolatus differs from H. immaculatus by its lower numbers of total and lower arch gill rakers (GR, LR), total and caudal vertebrae (TV, CV), as well as dorsal and anal-fin rays (DR, AR).

Ammodytes tobianus can be distinguished from A. marinus by having belly scales that are organised in tight chevrons (BSTC) and having scales present over the musculature at the base of the caudal fin (SBCF) and in the midline anterior to dorsal fin (SADF), whereas these characters are not present in A. marinus (Table 1). Ammodytes tobianus differs from A. marinus also by its lower numbers of dermal plicae (DP), dorsal-fin rays (DR), and precaudal and total vertebrae numbers (PV, TV).

DFA based on meristic characters provided three significant functions (Box-Test with χ² = 790.916 and p<0.0001; Wilks´ lambda = 0.0003 and p<0.0001). These three functions explain 100% of the total variation in the data. The first two functions explain 91,755% of the total variation in the data (Table 2), which is sufficient for the further detailed analysis. The third discriminant function explains 8.245% of total variation.

Individual specimens are projected onto the first two discriminant functions in Figure 3. Because all four species were clearly separated in the discriminant space defined by the first two functions, the third function was not used. The first discriminant function explains 71.438% of total variation (Table 2). It mainly separates A. tobianus and H. immaculatus and to a lesser extent the species pairs of A. tobianus and H. lanceolatus, A. marinus and H. immaculatus, A. marinus and H. lanceolatus as well as H. lanceolatus and H. immaculatus (Figure 3). Ammodytes tobianus and A. marinus cannot be so clearly separated by the first discriminant function.

Figure 3. 

Plot of all analysed Ammodytes and Hyperoplus specimens onto the first and second discriminant functions based on a set of eight meristic characters. Circles include 95% of specimens in each species.

From the standardised coefficients (Table 2), the two characters that have the greatest influence on the first discriminant function (characters most discriminatory) are the dermal plicae (DP) and lower arch gill rakers (LR) (Table 2). In general H. immaculatus and H. lanceolatus have a much higher number of dermal plicae than A. tobianus and A. marinus (Table 1). The numbers of DP of both species of Hyperoplus are overlapping, whereas the Ammodytes species have different numbers of DP. The number of lower arch gill rakers (LR) is higher in H. immaculatus in comparison with the other three species, which have overlapping numbers of LR.

The second discriminant function accounts for 20.317% of total variation. Ammodytes tobianus and A. marinus are clearly and the species pairs of A. tobianus and H. immaculatus, A. marinus and H. lanceolatus as well as H. immaculatus and H. lanceolatus are to a lesser extent discriminated by this function. Ammodytes tobianus and H. lanceolatus and A. marinus and H. immaculatus cannot be clearly separated by the second discriminant function. The contrasts between the numbers of dorsal-fin rays (DR) and the numbers of precaudal vertebrae (PV) of the species are mainly responsible for this discrimination. DR is lowest in A. tobianus and highest in H. immaculatus (Table 1). PV is lowest in A. tobianus and highest in A. marinus and H. immaculatus.

Three significant DFA functions were estimated based on morphometric measurements (Box-Test with χ² = 944.979 and p < 0.0001; Wilks´ lambda = 0.003 and p < 0.0001). Together these functions explain 100% of the total variation in the data. The first two functions explain 93.144% of the total variation in the data (Table 3), which is sufficient for the further detailed analysis. The third discriminant function explains 6.856% of total variation.

Figure 4 presents the individual specimens projected onto the first two discriminant functions. Because all four species were clearly separated in the discriminant space defined by the first two functions, the third function was not used. The first discriminant function explains 78.576% of total variation (Table 3). It mainly separates A. tobianus and H. lanceolatus and to a lesser extent the species pairs of A. tobianus and H. immaculatus, A. marinus and H. lanceolatus as well as A. marinus and H. immaculatus (Figure 4). The species pairs of A. marinus and A. tobianus as well as of H. immaculatus and H. lanceolatus cannot be separated by the first discriminant function.

Figure 4. 

Plot of all analysed Ammodytes and Hyperoplus specimens onto the first and second discriminant functions based on a set of 19 morphometric characters. Circles include 95% of specimens in each species.

The two measurement characters that have the greatest weight on the first discriminant function are pectoral-fin length (PFL) and the snout length (SNL) (Table 3). Both species of the genus Ammodytes have a greater PFL than both species of the genus Hyperoplus (Table 1). In contrast, both Hyperoplus species have a greater SNL than both Ammodytes species. PFL and SNL are relatively similar for the species of the same genera.

The second discriminant function accounts for 14.568% of total variation. Especially the species within the genera Ammodytes and Hyperoplus, namely A. tobianus and A. marinus as well as H. immaculatus and H. lanceolatus are separated by this function (Figure 4).

Upper jaw length (UJL) and caudal peduncle depth (CPD) are the two measurements, for which no sexual dimorphism is known, and that have the greatest weight on the second discriminant function (Table 3).

Mitochondrial DNA barcodes were obtained for 70 specimens belonging to four species of the family Ammodytidae investigated in this study (Suppl. material 1). The DNA sequences did not show any ambiguous base calls (Ns) or stop codons, and no insertions or deletions were found within the sequence alignment. Sequence length ranged from 619 to 652 bp (mean and standard deviation: 650.5 ± 5.7 bp). The average base composition was 22.8% adenine (A), 29.7% cytosine (C), 18.3% guanine (G) and 29.3% thymine (T); GC content was 48%. The sequence alignment showed 588 identical sites.

The NJ analysis of the K2P distances revealed well supported monophyletic clusters for the species A. marinus and H. immaculatus with bootstrap values of 97 and 100, respectively (Figure 5). In contrast, A. tobianus and H. lanceolatus sequences were grouped together in one monophyletic cluster with a bootstrap support of 100. Within this cluster the sequences of A. tobianus were grouped together without bootstrap support indicating that there is no sharing of haplotypes between these two species. The analysis of the K2P genetic distances revealed an overlap between intraspecific (range: 0.0-0.77%; mean and standard deviation: 0.22 ± 0.17%) and interspecific distances (0.15-7.27%; 4.73 ± 1.7%). The overlap was caused by the two species A. tobianus and H. lanceolatus: in A. tobianus, the minimum distance to the nearest neighbour species was even lower than the maximum intraspecific distance, whereas both values were equal in H. lanceolatus (Table 4). In contrast, the species A. marinus and H. immaculatus exhibited barcode gaps of 2.73% and 3.34% respectively, which indicates an undoubtedly separation from the other species. At genus and family level, the genetic distances between species of the same genus varied between 4.46-7.09% and the distances between species belonging to different ranged from 0.15-7.27%.

Figure 5. 

NJ dendrogram based on K2P pairwise genetic distances. Values at nodes indicate the result of the bootstrap test (10.000 pseudo replicates). Only values ≥ 50 are shown. For Ammodytes tobianus (grey box) and Hyperoplus lanceolatus all analysed individuals are shown. In case of Ammodytes marinus and Hyperoplus immaculatus the number of specimens is given in brackets.

The BIN discordance report tool on BOLD assigned three different BIN numbers to the 70 COI haplotypes. The BINBOLD:ACF3320 was found to be “concordant” and exclusively comprised 32 specimens of the species Ammodytes marinus, of which five individuals were not provided by this study. The “discordant” BINBOLD:AAC5676 comprised 57 specimens, 14 identified as Ammodytes tobianus and 43 as Hyperoplus lanceolatus. From the former species eight specimens and from the latter 14 specimens were not provided by our study but also support the findings of this study. The third BINBOLD:AAJ2299 was also specified as discordant and comprised ten specimens, eight (in our study) identified as Hyperoplus immaculatus and two identified as Ammodytes marinus. The two A. marinus entries may represent cases of misidentification as 32 A. marinus individuals were grouped together in BINBOLD:ACF3320.

The analysis revealed four diagnostic characters for the species A. marinus and 16 for H. immaculatus (results not shown). The two species A. tobianus and H. lanceolatus did not show any diagnostic characters on species level. Consequently, only two of the four investigated species can be identified using diagnostic characters on the basis of COI barcode sequences.

The nc Rhodopsin sequence alignment showed a length of 464 bp after primer trimming. The complete fragment could be amplified and sequenced for all 70 specimens used for the mt DNA barcode analysis. The number of variable sites was very low and the alignment could be easily evaluated by eye. One diagnostic character was found for each of the species A. marinus (Table 5; site 460: C instead of A), H. lanceolatus (site 82: T instead of C), and H. immaculatus (site 433: A instead of G). Ammodytes tobianus showed no species specific mutation but could be characterised by a combination of all tree variable sites (Table 5, underlined bases). The Rhodopsin sequences from GenBank were compared with our sequences; the two H. lanceolatus sequences (GenBank accessions: EU492010, EU492011) showed concordant results. In the case of the A. tobianus sequence (GenBank accession: AY141306) no data was available for site 460 but the two other sites were in agreement with our results.

The primary objective of this study was to contribute to robust genera- and species-level identifications, combining morphological and molecular methods, of four closely related species of sand lances of the genera Ammodytes and Hyperoplus occurring in the northeast Atlantic Ocean and adjacent waters.

The detailed morphological analyses confirmed findings described by other authors (e.g. Duncker and Mohr 1939, Reay 1986): the genus Ammodytes can be distinguished by two morphological characters from the genus Hyperoplus. Ammodytes has clear protrusible premaxillae and no vomerine teeth. In contrast, Hyperoplus has no clear protrusible premaxillae and a pair of vomerine teeth. It should be noted here that Kayser (1961) found out that Hyperoplus has no real vomerine teeth, but anterior hooked ends of the prevomer instead.

Subsequently, Ida et al. (1994) pointed out that the tip of the prevomer in Ammodytes is straight, not protruded from the roof of the mouth, whereas in Hyperoplus the tip of the prevomer curved downwards, protruding from the roof of the mouth. According to Wiecaszek et al. (2007), the genus Ammodytes also has a longer lower jaw when compared to the length of pectoral-fin, while this relationship is reversed in Hyperoplus.

This study adds three more characters helpful in distinguishing between both genera of sand lances based on the four species considered. Firstly, the number of dermal plicae is significantly higher in Hyperoplus compared to Ammodytes. Secondly, Hyperoplus has a lower pectoral-fin length in relation to standard length (SL) than Ammodytes. Goltberg (1910) also reported a lower value of pectoral-fin length expressed as a proportion of head length for H. lanceolatus than for A. tobianus. Thirdly, Hyperoplus has a larger mean snout length in % SL than Ammodytes. However, the last mentioned character is less recommended for practical taxonomical assignments, since its ranges overlap between the genera to a relatively large extent. Therefore, a combination of the following four characters remains, which seems to be useful to distinguish between the genera Hyperoplus and Ammodytes: protrusibility of premaxillae, presence of the hooked ends of prevomer, number of dermal plicae, and pectoral-fin length in % SL.

As indicated by the results of discriminant function analysis, morphometric measurements seem not to be characters of the first choice to distinguish the two species of each of the two genera, since they could not be discriminated by the first discriminant function.

According to the results presented here, six meristic characters (the number of lower arch gill rakers, the total number of gill rakers, the number of caudal vertebrae, the number of total vertebrae, and the number of dorsal-fin and anal-fin rays) are more useful than morphometric measurements to distinguish between H. immaculatus and H. lanceolatus. The use of these additional characters would support and refine the current methods to separate H. lanceolatus from H. immaculatus. Searching only for the occurrence of a conspicuous dark spot on either side of snout below anterior nostril could be unsuccessful in the case of preserved specimens.

In the case of A. tobianus and A. marinus, these results support the information on useful distinguishing characters between both species reported for instance by Reay (1986): A. tobianus differs from A. marinus by its belly scales that are organised in tight chevrons, scales which are present over musculature at base of caudal fin, as well as by lower numbers of dermal plicae, dorsal-fin rays and vertebrae. It should be mentioned that our analyses included also A. tobianus from the Baltic Sea, for which no meristic or morphometric data had been published, except for the number of vertebrae and pectoral-fin length (Wiecaszek et al. 2007).

The successful discrimination of the two sand lance species A. marinus and H. immaculatus by DNA barcoding was already demonstrated by Knebelsberger et al. (2014) and could be confirmed by the present study. An additional three specimens of A. marinus were added to the dataset and the NJ analysis revealed well-supported monophyletic species clusters for A. marinus and H. immaculatus, indicating an unambiguous separation of these two species. Successful species discrimination can also be demonstrated by the presence of gaps between intra- and interspecific genetic distances (Meyer and Paulay 2005), which were in case of A. marinus and H. immaculatus 2.73 and 3.34 respectively. The BIN analysis performed on BOLD revealed two separate species BINs: one concordant BIN exclusively contained sequences which were taxonomically annotated as A. marinus, and a second discordant BIN contained all specimens of H. lanceolatus and two further entries referring to as A. marinus. These two individuals were provided by other sources and may represent cases of misidentification, as all other A. marinus entries appeared in the concordant BIN.

Surprisingly, the two species A. tobianus and H. lanceolatus belonging to different genera cannot be clearly separated on the basis of genetic distances, as the lowest distance (K2P) between these two species was only 0.15% and within species variation was found to be 0.15 and 0.62% respectively. In the NJ dendrogram both species appeared together in a well supported clade and were also found within the same BIN cluster when analysed together with data on BOLD. However, A. tobianus and H. lanceolatus do not show haplotype sharing, as A. tobianus sequences appeared together in a separate cluster. The two species may therefore be separated by applying tree-based approaches like GMYC or model-based ones like ABGD.

In contrast to the barcoding results, both genera of Ammodytes and Hyperoplus can undoubtedly be separated by morphological character traits as discussed above. DNA barcoding failure between closely related congeneric species is usually more common than between species belonging to different genera (e.g. McCusker et al. 2013, Knebelsberger et al. 2015). For congeneric species of the genus Ammodytes inconsistencies between morphological data and DNA barcodes have already been demonstrated. For instance A. americanus DeKay, 1842 and A. dubius Reinhardt, 1837 from the northwest Atlantic Ocean could not be separated by DNA barcoding, possibly caused by inadequate taxonomy (McCusker et al. 2013), which may also concern the two species A. personatus Girard, 1856 and A. hexapterus Pallas, 1814 from the north Pacific (Turanov and Kartavtsev 2014).

In the present work, inadequate taxonomy, erroneous species designation or identification error can be excluded as possible explanation for DNA barcoding failure in unambiguously separating A. tobianus from H. lanceolatus. In addition, true biological phenomena such as the occurrence of hybridisation or incomplete lineage sorting seem to be unlikely, as no interspecific haplotype sharing was found. In cases where mitochondrial COI sequences fail to distinguish between species, the application of nuclear DNA markers may be tested alternatively. In fish, the nuclear Rhodopsin gene has already been proposed as supplementary marker in order to identify species (Sevilla et al. 2007). However, most studies demonstrated reduced species discrimination success using nuclear Rhodopsin sequences compared to COI barcodes (Hanner et al. 2011, Collins et al. 2012, Behrens-Chapuis et al. 2015). In our study, the analysis of a short nuclear Rhodpsin gene fragment revealed diagnostic nucleotides for the species Ammodytes marinus, H. lanceolatus and H. immaculatus. The species Ammodytes tobianus can be characterised by the lack of species specific mutations compared to the other three species. Consequently, all four species of sand lances can be identified using the diagnostic character approach in combination with nuclear Rhodopsin sequences. In contrast to that, COI provided diagnostic characters only for the two species A. marinus and H. immaculatus. Ammodytes tobianus and H. lanceolatus cannot be characterised by this approach.

Our study clearly demonstrated that nuclear Rhodopsin constitutes a preferable alternative marker to discriminate successfully between the four investigated species of sand lances.

Finally, it should be pointed out that the present results are not meant to provide a phylogenetic reconstruction with regard to the genera Ammodytes and Hyperoplus, since the latter requires a more detailed study of more species of both genera, as well as other members of the group. However, accurate identification of these sand lance species is the basis to assess the status of their stocks and to implement appropriate measures of fisheries management or conservation, and as such, the aim of successfully identifying the NE Atlantic species has been accomplished.