Black flies (Diptera: Simuliidae) of Turkish Thrace, with a new record for Turkey

Abstract Background This paper includes 2742 specimens of 18 species of black flies (Diptera: Simuliidae) collected from 132 lotic sites in Turkish Thrace, the European part of Turkey, in the early summer of 2002 and 2003 and the spring of 2005 and 2006. New information All species are recorded from this region for the first time, and Metacnephia nigra (Rubtsov, 1940) is a new record for Turkey. Distributional and taxonomical remarks are given for each species.


Materials and methods
Study material was based on aquatic stages and reared adults from 132 rivers, streams and springs in the region (Fig. 1). The material included 2742 specimens (1168 larvae, 1369 pupae, 204 pharate adults and 47 reared adults) and was deposited in the LAB).
Larvae and pupae were collected into 80% ethanol, and reared flies with their pupal exuviae also were fixed in ethanol. For each species, the numbers of larvae, pupae and adults collected at a sampling site on a given date are recorded in the examined material below. Material was studied according to methods described by Bass (1998), with the aid of a stereomicroscope (Leica MZ 16).

Notes
We report this species for the first time from Turkey. The taxonomy of the genus Metacnephia requires revision. In Turkey, only two species, M. lyra and M. subalpina, of this genus have been known until now (Kazancı and Ertunç 2008). The pupal features of our specimens are similar to those of M. subalpina, reported from four localities in Anatolia, one in the northwestern and three in the eastern and southeastern parts of the country, by Crosskey and Zwick (2007). Metacnephia subalpina was described by Rubtsov (1956) as a variety of M. nigra, but Terteryan (1968) elevated it to species. According to Crosskey and Zwick (2007), the relationship of M. subalpina and M. nigra remains unsettled because of the problem of the true identity of M. nigra; the authors could not resolve the ambiguities. They noted that Yankovsky (1995) (pp. 4, 50) treated subalpina and nigra as synonyms before reverting [Yankovsky (2003)] to treating them as separate species. Crosskey and Zwick (2007) used the name subalpina for their material because it conformed in the shape of the ventral plate and pupal gill with Djafarov's illustrations of M. subalpina. Another species, Metacnephia uzunovi, described by Kovachev (1985) from Bulgaria, confuses the taxonomic situation by having similar structural characters to M. nigra. We have doubts about its validity. We recognize M. nigra as a species complex, following the recommendation of P.H. Adler (pers. comm.) who noted the similarity with Rubtsov's (1956, fig. 113) variety, saxicola, of M. nigra. Adler suggested that we use the name M. nigra for our material, with reference to Rubtsov's (1956) variety saxicola, as a conservative approach that will provide future workers with an idea of the morphology of our material until the correct name of the species can be determined. M. nigra is known from Azerbaijan (Nakhichevan), Romania, Russia (Caucasus) and Turkmenistan (Adler and Crosskey 2015).

Notes
Simulium erythrocephalum is the most abundant species of the subgenus Boophthora in the western Palearctic and is reported from the Far East of Asia (Adler and Crosskey 2015). Breeding sites of S. erythrocephalum are generally weedy, lowland streams and rivers, such as the Danube (Germany, Serbia), the Spree (Germany), the Warta (Poland), and the Morava (Czech Republich). In Central Europe, it occurs in various flowing-water systems such as lowland streams, outflows from ponds, irrigation constructions and large rivers (Werner and Kampen 2012). It can develop large populations and become a serious pest and nuisance to humans and farm animals (Ignjatović-Ćupına et al. 2006) In Anatolia, it has been recorded by several authors. Sirin and Sahin (2005) found it at two sites in the Sakarya river basin for the first time in Turkey. Kazancı and Ertunç (2008) Knoz (1965) and Bass (1998), the species can be identified by a pair of inconspicuous dorsolateral papillae on each segment of the larva, the pattern of the larval head capsule, the branching pattern of the six pupal gill filaments, and the shape of the pupal cocoon. The male genitalia of our specimens conform to the description by Rubtsov (1956).

Notes
Simulium velutinum occurs in most of Europe but is especially common in the Mediterranean subregion from the Canary Islands (type locality) to Israel and Jordan (Crosskey and Malicky 2001). According to Kazancı and Ertunç (2008), S. velutinum also inhabits southwestern Anatolia. Its existence in Turkish Thrace is, therefore, expected. Identification of this species is most reliably based on genitalia: the ventral plate of the male is smaller and more triangular and the spermatheca of the female is without the usual sclerotized nipple-like extension that is found in the other members of the S. aureum group.

Notes
Simulium cryophilum is a member of the S. vernum species-group, which includes more than one-fourth of the nominal species in the western Palearctic. According to Crosskey and Malicky (2001), there are difficulties in identification of the species of the S. vernum group. Another taxonomic difficulty is that S. cryophilum is a composite of at least two chromosomally distinct sibling species (Hunter 1987;Adler et al. 1999). We, therefore, refer to it in Turkey as the S. cryophilum complex. We found it in two streams in the southern part of our study region. The main structural features of our material were in the pupal stage: (1) (Bass 1998;Jedlićka et al. 2004); (4) thoracic microtubercles dense and rounded and thoracic trichomes simple (Bass 1998); and (5) cocoon fully covering pupa and with a short anteromedian horn. In our larvae, the postgenal cleft was clearly defined and pentagonal, conforming to the description by Bass (1998

Notes
This species occurs in the southwestern Palearctic from Iberia through central and southern Europe to the Transcaucasus and northern Middle East, and is present in northwestern Africa and most of the larger Mediterranean islands. It is common and widespread in Anatolia (Crosskey and Zwick 2007). Simulium bezzii, like Simulium kerisorum, also known from Turkey, has six pupal gill filaments, in contrast to most other species of the S. bezzii-group, which generally have eight filaments. It can be distinguished by large fenestrations posterior to the cocoon rim. Crosskey and Zwick (2007) stated that S. kerisorum is not a valid species and thought that the Turkish record of Kazancı and Clergue-Gazeau (1990) is questionable. The chromosomes of Simulium bezzii have been studied (mainly by Kachvoryan (1988), as Tetisimulium condici) but not those of S. kerisorum, and cytological comparison of the two nominal taxa is desirable (Crosskey and Zwick 2007

Notes
The type locality of Simulium bukovskii is in Crimea (Ukraine) and its distribution ranges from the Balkans to Armenia. It was recorded from Turkey by different authors (Crosskey and Zwick 2007). Furthermore, it is similar to S. degrangei Dorier and Grenier (1960), which is also common in most countries of southern Europe. Crosskey and Zwick (2007) emphasized the similarity of the two species and that synonymy should be considered. Notes Simulium (Simulium) noelleri is a well-known simuliid, with a general preference for outlets. It is distinguished by the branching arrangement of the pupal gills (3+3+2 or 3 +2+2+1). We encountered it at one locality, an outlet from a small pond. Our pupae have eight gill filaments arranged as 3+3+2, and the apotome pattern of the larval head capsule is like an ill-defined "H", as described by Bass (1998

Notes
This species is a member of the S. ornatum species-group, which presents some of the most difficult taxonomic problems among Palearctic simuliids, in part because of the descriptions of many nominal species. Until now, six species of this group (baracorne Smart, 1944, fontanum Terteryan, 1952, kiritshenkoi Rubtsov, 1940, caucasicum Rubtsov, 1940, ornatum Meigen, 1818, trifasciatum Curtis, 1839) have been recorded from Turkey by different authors, as well as ornatum s.l. Meigen reported from Thrace by Zwick (2007). Our material includes mainly aquatic stages, with only one reared adult, a male from Site 117. We identified this male as S. kiritshenkoi on the basis of the color of the antennae (orange-red) and legs (reddish yellow), as described by Crosskey (2002). The genitalia also conform to the description of Crosskey (2002): ventral plate beak-like process in profile narrow and slightly upturned, and dentate crest slightly overhanging the base of the beak-like process. We also have pupae and larvae from this locality and the taxonomic characters fit the description of S. kiritshenkoi by Rubtsov (1956), Terteryan (1968), Crosskey (2002) and Crosskey and Zwick (2007). The pupal characters are as follows: thoracic microtubercles dome-like and fairly sparse, cocoon slipper-shaped and with delicately to strongly perforated weave, gill filaments in four pairs on short common stems. The postgenal cleft of our larvae is comparatively small, subquadrate or slightly pentagonal and extended less than halfway toward the base of the hypostoma.  (2008) and Bernotiene and Stunzėnas (2009) showed, by analyzing mitochondrial cytochrome c oxidase 1 gene sequences, that S. galeratum is a distinct species. According to Bass (1998), Knoz (1965) and Jedlićka et al. (2004), the most distinctive pupal character between S. reptans and S. galeratum is the conformation and distribution of microtubercles on the cuticle of the head and thorax; in S. galeratum, the microtubercles are sparse and pointed, but dense in S. reptans, they are dense and rounded. In our study, the pupae have dense, rounded microtubercles. However, the head capsule pigmentation of our larvae conforms to that of S. reptans, as given by Bass (1998

Notes
The most diagnostic character of Simulium variegatum is the presence of two large thoracic bulges (patagia) near the pupal gill bases. We observed this feature in all specimens collected from 11 sites. This species is widely distributed in Turkey (Crosskey and Zwick 2007) and also common over most of Europe, western Asia and North Africa (Adler and Crosskey 2015).

Notes
This species was reported from the Yedi Göller region in Bolu Province as a new record for Turkey (Kazancı and Ertunç 2008), and then recorded from Eastern Marmara Region (Şirin et al. 2014). It also is known from adjacent Balkan countries such as Bulgaria and Romania. We encountered the species at four localities, and identified it by pupal gill characters, using the keys of Knoz (1965), Jedlićka et al. (2004) and Yankovsky (2003): the filaments are directed downward at the base and then curve anteriorly; they lie close together proximally.

Notes
According to Crosskey and Zwick (2007), Simulium auricoma is spread through southern Europe from the Iberian to the Aegean islands, such as Lesbos and Rhodes, and occurs in mountainous areas. They reported it from Turkey for the first time and emphasized that it shows wide variation in the length, taper and spread of the filaments. Crosskey and Malicky (2001) noted that gill filaments vary from short, stubby, and minimally tapered, to long and gradually tapered; in all forms, the lowermost filament tends to diverge downward from the others. The filaments of our pupae from the two sites are short, stubby, and minimally tapered. The cocoon and genitalia characters of pharate males conform to the description by Crosskey and Malicky (2001).

Notes
The pupae of Simulium equinum can be identified by the banana-like gill branches. Although S. equinum is found from central and northern Europe to northeastern Asia, it becomes progressively less common throughout the Mediterranean borderlands and islands (Crosskey and Zwick 2007). This species was reported from the Sakarya river system as a new record for Turkey (Sirin and Sahin 2005). Crosskey and Zwick (2007) also found it at a river near Kızılcahamam, Ankara. These authors note that S. equinum typically occurs with other species of the subgenus Wilhelmia. We record this species at nine sites, of which six were with S. pseudequinum and one with S. balcanicum.

Notes
Simulium pseudequinum is one of the most common black fly species in the Palearctic Region, from the Canary Islands to China. It is abundantly known from all Mediterranean countries except Egypt (Crosskey and Malicky 2001). In Anatolia, it has a wide distribution and has been reported by many authors [Zwick (1978) (as mediterraneum Puri, 1925), Kazancı and Clergue-Gazeau (1990), Sirin and Sahin (2005), Kazancı and Ertunç (2008), Gazyağcı and Aydenizöz (2011), Şirin et al. (2014)]. We encountered S. pseudequinum at 43 sites, the highest number for any species, and we consider it the most abundant black fly in the region. Crosskey and Malicky (2001) emphasized that in the western Mediterranean area it is reliably recognized in the pupa by the wrinkled bases of the middle group of gill tubes. This character, however, does not suffice in the eastern Mediterranean and Middle East, where S. pseudequinum can be confused with S. paraequinum, the two species having virtually identical gills. Simulium pseudequinum can be distinguished by the small spermatheca, resembling an unopened mushroom, and the characteristic shape of the male ventral plate, which is narrow apically with the inner margins resembling an isosceles triangle (Crosskey and Malicky 2001). In our specimens, the spermatheca of the females and the ventral plate of the males agreed with the description for S. pseudequinum given above.

Notes
Simulium paraequinum ranges from the Balkans to Pakistan; Anatolia lies centrally in the distribution. This species was recorded first from Anatolia by Kazancı and Clergue-Gazeau (1990) near the southwestern corner of Asia Minor. Crosskey and Zwick (2007) recorded it from five sites in the southern Taurus Mountains and northwestern Anatolia. It also occurs in Greece and Bulgaria, which are boundary countries of Turkish Thrace. This species can be distinguished from S. pseudequinum only by the genitalia of males and females. The genitalia of our pharate males and females collected from only one locality, Site 41, fit the figure and description of S. paraequinum given by Crosskey and Malicky (2001). We observed that the length of the gill tubes of our specimens of S. paraequinum were longer than all of those of S. pseudequinum.

Discussion
The simuliid species composition of Turkish Thrace is similar to that of Anatolia and the Balkan Countries, differing only by the presence of Metacnephia nigra in Turkish Thrace. The most two abundant species are respectively Simulium ( Wilhelmia) pseudequinum recorded at 43 sites and Simulium (Eusimulium) petricolum from 41 sites in the region. It is known that these two species are common in Meditaranean region and can be found in different types of running waters. On the other hand, Simulium (Simulium) noelleri and Simulium (Simulium) bukovskii are reported from only one site in the region depending on their special habitat preferences.
Our survey was based only on morphotaxonomic methods and did not reveal cryptic species in complexes such as the S. cryophilum complex. More comprehensive taxonomic surveys, including cytotaxonomical and molecular techniques, are required to obtain further information about the faunal structure of the family in the region.
Our results show that blood-feeding species, like Simulium erythrocephalum, S. reptans and S. bezzii, live also in the region. Therefore, monitoring programmes for pest populations of black flies in the region are needed to ensure the public health of citizens (e.g., about 13 million people in Istanbul) and protection of livestock, especially with regard to dam-construction projects, excessive pollution of freshwater systems caused by agricultural and industrial activities, and the effects of global warming.