First record of the Black Bullhead Ameiurus melas (Pisces, Ictaluridae) in the Republic of Macedonia Прв податок за присуство на црното американско сомче Ameiurus melas (Pisces, Ictaluridae) во Република Македонија

The North American catfish – the black bullhead, Ameiurus melas (Rafinesque, 1820) is recorded for the first time in the Republic of Macedonia, in Pčinja River. Nine individuals of this ictalurid fish species were fished from the anglers in Pčinja River on September 2015 and another 8 samples on May 2016 were brought to the Institute of Biology, Faculty of Natural Science and Mathematics in Skopje. After a detailed observation of the external morphological features, osteology architecture, it was concluded that analysed individuals had all the distinguishing features of the species A. melas. The presence of different size class permitted the supposition that the black bullhead has established a selfsustaining population in this river.


Introduction
Invasive fish species constitute a major threat to biodiversity and ecosystem integrity and cause substantial economic damage (Pimentel et al. 2000;Copp et al. 2005).Environmental disturbance is known to greatly facilitate fish invasions.Habitat modification and fragmentation (e.g., water pollution, construction of dams and water diversions) threaten native fish faunas while favouring the invasion of more tolerant non-native species (Marchetti et al. 2004).It is the case for the brown bullhead Ameiurus nebulosus (Lesueur, 1819) and black bullhead Ameiurus melas (Rafinesque, 1820) which were introduced into many countries in Europe since the nineteenth century (Kottelat and Freyhof 2007).Both species have a negative impact on native populations of fish species (Rutkayová et al. 2013;Copp et al. 2016).
Concerning the Balkan Regions until now, A. nebulosus was recorded in Slovenia, Croatia, Bosnia and Herzegovina, Montenegro, Albania, Greece and Bulgaria and Macedonia, while A. melas was recorded in Slovenia, Cro-atia, Bosnia and Herzegovina and Serbia (Simonovic et al. 2013).Their impact in these countries (by FISK range) was calculated as a moderately high (A.melas) and high (A.nebulosus).
The reconstruction of the origin and paths of their distribution in Europe is very difficult due to the unclear taxonomic status of these species in the past, obstacles in the field identification and uncertainty of the literature data (Nowak et al. 2010).Moreover, the possibility of their hybridization (Scott and Crossman 1973) could also make additional problem in their identification.From the other side, osteological architecture, especially of the pectoral girdle and pectoral spine offer morphological clues for identification of this two species (Rutkayová et al. 2013).
Many years ago it was established that the brown bullhead, A. nebulosus and Ictalurus punctatus, were the only ictalurid species introduced into Macedonian waters (Naumovski 1995;Kostov et al. 2011).However, recent ichthyologic surveys brought to attention the introduction of another ictalurid species.In attempt to facilitate the identification of the newly introduced ictalurid species in Macedonia, this study presents a detail osteological analysis of Ameiurus melas from Pčinja River.

Material and Methods
In 2015, nine individuals of ictalurid fish were fished by the anglers in Pčinja River in September 2015 and another 8 specimens in May 2016 and all were brought to the laboratory for Comparative Anatomy in the Institute of Biology, Faculty of Natural Science and Mathematics in Skopje.
Pčinja River sources out from several streams on the western slopes of the Dukat Mountain which meet at the village of Radovnica (Serbia), and flows into the Vardar River in Taor Gorge, halfway between the cities of Skopje and Veles.The river's length is 135 km, of which 100 km are in Republic of Macedonia.Seventeen individuals of A. melas from Pčinja River were fished by the anglers and according to them individuals were fished in the parts of the river section named "Badar", characterised by a number of sand-extraction pits (Fig. 1).
Detailed morphometric analyses were performed according to Holčík (1998) (Fig. 2) as well as the meristic counts (Tab. 1 and 2).Seven specimens of A. melas were cleared and stained according to Taylor and Van Dyke's (1985) protocol for detailed of osteological analyses.A Camera Lucida attached to a Wild M3C microscope was used for examination of specimens and drawings (Fig 2;   1.

Results and discussion
In spite of the great morphological similarity in many features, according to Movchan et al. ( 2014) A. nebulosus significantly differs from A. melas by the number of gill rakers, serration of the first spine of the pectoral fin and coloration of the fin membrane.However, according to Kottelat and Freyhof (2007) the colour and anterior serrae on pectoral spine are not reliable characters and have led to much confusion regarding the identification of these species in Europe.
In the case of A. melas (Pčinja River) and A. nebulosus (Tikveš Reservoir), the results show that these selected morphological characters vary.The base of the anal fin in A. melas from Pčinja River (Fig. 3A) is lightly coloured and thicker compared to the fin of A. nebulosus from Tikveš Reservoir (Fig. 3B).However, the membrane between the rays of the anal and caudal fins is black in both species contrary to the foundlings of Movchan et al. (2014).
The first spine of the pectoral fin in A. melas is covered with a few sharp denticles only in the middle part (Fig. 4B) while in A. nebulosus the denticles extend along and almost reach the end of the spine (Fig. 4A).Anterior serration is absent in A. melas unlike the samples of A. nebuslosus from Tikveš Reservoir.But it should be stressed out that this anterior serration in A. nebulosus specimens was not observed in all samples, thus making this character questionable for identification purposes.
The number of gill rakers at the first ceratobranchial in A. melas vary from 15 to 19 (Fig. 5B), while in A. nebulosus usually this number is 15 (Fig. 6).However, in one of the samples of A. nebulosus the number of the gill rakers was 17.
The plastic characteristics analysed in this study are in accordance with the data published about other European populations of these two species.The osteological features still play an important role in the systematics and identification of ictalurid fishes (Paruch 1986;Lundberg 1982;Traynor et al. 2010;Rojo 2013, Rutkayová et al. 2013) and thus we made a complete osteological analysis of A. melas in addition to the standard morphometric measurements.Morphometric measurements and meristic counts including osteological ones are presented in Tab. 1 and 2.
Within the presented osteological results and in order to distinguish A. melas in Pčinja River from A. nebulosus we propose an identification key regarding their skeletal structural differences: It is well known that the number of vertebras and the

Ameiurus melas Ameiurus nebulosus
Possesses a secondary coracoid keel at the pectoral girdle (Fig. 8); Secondary coracoid keel is always absent in A. nebulosus Median notch in anterior margin of supraethmoid bone at least twice wide as deep (Fig. 7); The median notch in anterior margin of supraethmoid bone is as deep as wide; Entopterygoid bone in A. melas is much wider and higher, reaching the entire surface of the anterior margin from the metapterygoid bone (Fig. 9).
Entopterygoid (=ectopterygoid) bone (Rojo 2013), is much smaller, with height about one third of the height of anterior margin of the metapterygoid bone.number of branchiostegal rays are often used as taxonomic key characters (Kottelat and Frehof, 2007).Our results show that these characters offer useful information that can be considered for taxonomic discrimination between A. melas and A. nebulosus.Namely, in all examined specimens of A. melas from Pčinja River, the number of precaudal vertebra was 16, while caudal ones counted 25-26, corresponding with results for A. melas described by Lundberg (1982).In A. nebulosus described by Rojo (2013), the number of precaudal (abdominal) vertebra varies from 12 to 16, while the number of caudal ones from 22 to 29.In all examined specimens of A. me-     las from Pčinja River the number of the branchiostegal rays is 9 (Fig. 10), showing difference when compared with A. nebulosus described by Rojo (2013) where this number was 8 branchiostegal rays in 16 of 17 examined specimen, while 9 branchiostegal rays were detected in only one specimen.
The detailed analysis of the skeletal features of the ictalurid fish species from Pčinja River in comparison with samples from Tikveš Reservoir and the literature data, revealed the presence of A. melas in R. Macedonia.Osteological observations and results based on theme strongly support the importance of osteological markers in situation when external morphology is not conclusive.The presence of different size classes of A. melas in Pčinja River permits the supposition that the black bullhead has established a self-sustaining population in this river.The origin and introduction pathway of the black bullhead to the Pčinja River remains unclear, but knowing the fact that misidentification of A. melas occurred in many countries, it is reasonable to assume that the species might have been widely distributed throughout Macedonia for a long period.As many studies in Europe showed that distribution of A. melas in the water bodies is related to fading of A. nebulosus, it is necessary to undertake urgent surveys to ascertain the current distribution and the ecological impacts of this invasive species in Macedonian waters.The presence of this species in Macedonian waters should be considered in future management and conservation schemes.
Fig 4-10).The anatomical structures in the figures are shaded and labelled a consistent manner (dark, medium, and clear) to facilitate the real position.The nomenclature of skeletal elements follows Rojo (2013) and Rutkayová et al. (2013).Furthermore, already collected and stored samples of A. nebulosus from Tikveš Resirvoir were used for comparison.Dissection of some parts of the body (fins, branchial arches) from A. nebulosus, as well as technique of macer-ation with KOH solution of few skeletal structures was performed according toGreen (1952).In addition, literature data about osteological characters of A. nebulosus was used for comparison with A. melas from Pčinja River(Rojo 2013;Rutkayová et al. 2013

Figure 1 .
Figure 1.Location m ap show ing the first record of Ameiurus melas in R. Macedonia

First
record of the Black Bullhead Ameiurus melas (Pisces, Ictaluridae) in the Republic of Macedonia

Table 2 .
M eristic counts of Am eiurus m elas from P činja River and Am eiurus nebulosus from Tikveš