DNA barcoding and species delimitation of the genus Oxynoemacheilus (Teleostei: Nemacheilidae) in Anatolia.

The DNA barcoding approach was used for the determination of evolutionary relationships and species delimitation of the genus Oxynoemacheilus (Teleostei: Nemacheilidae). The COI barcode region (615 bp amplicon) was used to barcode 444 individuals from 64 morphologically identified species in the genus Oxynoemacheilus and 189 haplotypes were identified. The average of the interspecific p distance (9.59%) was about 21-fold higher than the average intraspecific distance (0.44%). A general genetic threshold of 1.46% sequence divergence was defined for species delimitation. The multiple species delimitation methods (BCM, GMYC, bPTP and TCS) revealed a total of 62 molecular operational taxonomic units for 64 morphospecies with a new loach species from the BuyukMelen River. Neighbour-joining, maximum likelihood and Bayesian inference analyses indicated that all haplotypes were clustered into 62 clades, which corresponded to Oxynoemacheilus species, with strong bootstrap support (≥95%). Furthermore, all samples grouped in concurrence with the taxonomic status of the species except for species groups (O. germencicus-O. cinicus-O. mesudae and O. leontinae-O. namiri) that were showed intraspecific overlap in genetic diversity for COI-based barcodes. In conclusion, our analyses indicate that COI-based barcodes provide reliable species discrimination. Therefore, we currently recommend COI barcodes as the suitable barcode for genus Oxynoemacheilus.

Genetic structure and demographic history of endangered Alburnus tarichi (Güldenstädt, 1814) populations from Lake Van basin in Turkey inferred from mtDNA analyses.

Genetic diversity, genetic structure, and demographic history of the endemic and endangered cyprinid species Alburnus tarichi based on samples from 17 populations consisting of resident and potamodromous specimens from the Lake Van basin in eastern Turkey were analyzed using two mitochondrial DNA markers. A. tarichi populations in the Lake Van basin are genetically heterogeneous, as indicated by the high haplotype and low nucleotide diversity of 1233 bp of the 16S rRNA marker (44 haplotypes; 70 polymorphic sites, haploid diversity (Hd) = 0.9130, π = 0.0032) and 1140 bp of the cyt b marker (47 haplotypes; 82 polymorphic sites, Hd = 0.9339, π = 0.0057). Clades were separated by average sequence divergences of 1.94% (II vs. III), 1.80% (I vs. III), and 0.66% (I vs. II). Based on these clades, AMOVA analysis revealed that 80.76% of the total variation occurred among populations, 10.74% occurred within populations, and only 8.50% occurred between populations within groups for the concatenated 16S rRNA-cyt b sequences. Pairwise FST values varied from 0.0167 to 0.9705 for the concatenated 16S rRNA-cyt b dataset, emphasizing the high genetic variation among populations. The time since the endemic tarek populations split from their last common ancestor has been dated to 5.647 Ma (95% highest posterior density: 4.183-7.011 Ma) in the Messinian Stage. Recent population expansion for tarek populations has been determined by neutrality tests and mismatch distribution analyses. The results of this study provide valuable information on the genetic population structure, conservation, and management of this species.

Molecular systematics and phylogeography of the genus Alburnus Rafinesque, 1820 (Teleostei, Leuciscidae) in Turkey.

In this study, the phylogeny of Alburnus genus distributed in Turkish freshwaters was performed by analyzing mitochondrial cyt b gene (1141 bp) and COI gene (1551 bp) sequences from 1172 samples representing 112 populations of 24 species through their geographical distribution. According to our findings, 20 valid species are distributed in Turkey of which 18 have already been known. While six Alburnus species (A. battalgilae, A. istanbulensis, A. carinatus, A. schischkovi, A. nasreddini ve A. adanensis) have been synonomized, two new species (Alburnus sp.1 and Alburnus sp.2) from Dicle River and Çapraz Stream/Susurluk River have been identified. Extinct species such as A. akili and A. nicaeensis have not been observed in situ. Phylogenetic tree topologies and haplotype network of the 119 cyt b and 80 COI haplotypes detected in Alburnus species have indicated a consensus tree topology containing twenty lineages, each of corresponding to one species, and three Alburnus haplogroups corresponding to the geographical origins: Eastern Anatolia (I), Mediterranean (2) and Western & Northern Anatolia (3). The results indicate that the divergence between those haplogroups may have occurred during the Middle Miocene-Middle Pleistocene periods (from 14.9 to 5.29 million years).

A new species of trout from the northern drainages of Euphrates River, Turkey (Salmoniformes: Salmonidae).

Salmo fahrettini, a new species, is distributed in the northern tributaries of the Euphrates River. It differs from other Salmo species in adjacent waters by a combination of the following characters: a greyish body; one black spot behind the eye and on the cheek; three to six black spots on the opercle; numerous black spots on the back (missing on the predorsal area), flank and middle part of body, surrounded by a roundish white ring; red spots in the median part of the body, surrounded by a roundish white ring; short and narrow maxilla; increase in the number of black and red spots with an increase in size; adipose fin medium size, no or rarely one red spot at its posterior edge; 109-116 lateral line scales; 27-30 scale rows between dorsal-fin origin and lateral line; 20-23 scale rows between the lateral line and anal-fin origin; maxilla length 8.8-10.0% standard length in males, 8.8-9.6 in females.

Phylogeny and phylogeography of the genus Alburnoides (Teleostei, Cyprinidae) in Turkey based on mitochondrial DNA sequences.

Turkey has a rich freshwater biodiversity in terms of Cyprinid genus in respect to its geographical location. To elucidate the phylogeny of the Alburnoides genus, one of these genera, genetic data for the cytochrome b gene (1141 bp) was generated for 445 samples collected at 42 sampling sites across their geographical distribution. A total of 54 mitochondrial haplotypes identified were distrubuted among distinct twelve species that did not share haplotypes with each other. Pairwise sequence divergence among these species range from 1.37% (A. emineae and A. velioglui) and 10.99% (A. manyasensis and A. smyrnae). A new potential species in the River Dirgine that run into the Black Sea Basin was separated from the most closed known species with mean 6.3%. Network analysis and phylogenetic analysis indicated that all haplotypes were clustered into two major clades, which corresponded to twenty-three Alburnoides lineages, with moderate-high bootstrap supports and mutational steps, respectively. Application of a molecular clock to a Bayesian phylogeny indicates that Alburnoides diversified under the paleogeographic conditions such as tectonic uplift and faulting Miocene aged as well as climatic oscillation and sea-level fluctuations during late Miocene-middle Pleistocene. The genetic results of the present study indicated the inter-specific distance of cyt b gene sequences followed the ideal results for species identification and phylogeny of Turkish spirlins.

High cryptic diversity of bitterling fish in the southern West Palearctic.

South-east Europe, along with the adjacent region of south-west Asia, is an important biodiversity hotspot with high local endemism largely contributed by contemporary continental lineages that retreated to southern refugia during colder Quaternary periods. We investigated the genetic diversity of the European bitterling fish (Rhodeus amarus) species complex (Cyprinidae) across its range in the western Palearctic, but with a particular emphasis in the region of Balkan, Pontic and Caspian refugia. We genotyped 12 polymorphic microsatellite loci and a partial sequence of mitochondrial gene cytochrome b (CYTB) for a set of 1,038 individuals from 60 populations. We used mtDNA sequences to infer phylogenetic relationships and historical demography, and microsatellite markers to describe fine-scale genetic variability and structure. Our mtDNA analysis revealed six well-supported lineages, with limited local co-occurrence. Two lineages are distributed throughout central and western Europe (lineages "A" and "B"), with two zones of secondary contact. Another two lineages were restricted to the Ponto-Aegean region of Greece (lineages "C" and "D") and the final two lineages were restricted south of the Caucasus mountains (lineage "E" from the Black Sea watershed and lineage "F" from the Caspian watershed). A signal of recent expansion was revealed in the two widespread lineages and the Ponto-Aegean lineage "C". The geographic distribution of clusters detected by nuclear microsatellites corresponded well with mitochondrial lineages and demonstrated finely sub-structured populations. A profound population structure suggested a significant role of genetic drift in differentiation among lineages. Lineage divergence in the Ponto-Aegean and Caspian regions are substantial, supporting the validity of two described endemic species (Rhodeus meridionalis as lineage "D" and Rhodeus colchicus as lineage "E") and invite taxonomic evaluation of the other two southern lineages (Thracean "C" and Caspian "F").

Gobio baliki, a new gudgeon from Turkey (Teleostei: Cyprinidae).

Gobio baliki, new species, is described from the stream Büyük Melen in the northwestern Anatolian Black Sea basin. The new species is distinguished from other gudgeons in Anatolia by the breast being completely covered by scales, scales extending slightly behind the isthmus; 5-6 scales between the posterior extremity of the pelvic-fin base and the anus; the distance between the anus and the anal-fin origin 7-10% SL; the postorbital distance equal to or greater than the snout length; the depth of the anal-fin approximately equal to the length of the pelvic-fin; 8-9 midlateral black blotches, usually separated from each-other.

Three new species of Alburnoides from the southern Black Sea basin (Teleostei: Cyprinidae).

Three new Alburnoides species are described from the southern Black Sea basin. Alburnoides kurui, new species, from the Yesilirmak drainage, is distinguished from other species of Alburnoides in the southern Black Sea basin, the Marmara basin and the Anatolian Aegean basin by having a scaleless keel between the posterior pelvic fin base and the anus or with rarely 1 scale covering the anterior part of the keel; 50-59 total lateral-line scales; 13½-15½ branched anal-fin rays and 7-9 gill rakers. Alburnoides freyhofi, new species, from the Kizilirmak drainage, is distinguished by having a scaleless keel between the posterior pelvic fin base and the anus, 44-53 total lateral-line scales; 14½-16½ branched anal-fin rays and 5-7 gill rakers. Alburnoides kosswigi, new species, from the Sakarya drainage, is distinguished by having a scaleless keel between the posterior pelvic fin base and the anus or rarely with 1-3 scales covering the anterior part of the keel; 43-57 total lateral-line scales; 11½-13 (14)½ branched anal-fin rays and 5-7 gill rakers.

Alburnoides diclensis (Actinopterygii: Cyprinidae), a new species of cyprinid fish from the upper Tigris River, Turkey.

Alburnoides diclensis sp. n. is described from the Tigris River drainages (Persian Gulf basin) in southeastern Anatolia, Turkey. It is distinguished by a combination of the following characters (none unique to the species): a poorly developed ventral keel between pelvic and anal fins, completely scaled or rarely without one scale in front of anus; head short (length 24-27% SL), its upper profile markedly convex on level of nostril; mouth terminal, with marked chin; lips equal to or upper lip slightly longer than lower lip; snout with rounded tip, its length equal to or greater than eye diameter but smaller than interorbital distance; body moderately deep (depth at dorsal-fin origin 25-30% SL); anal-fin depth 1.2-1.4, mean 1.3 times in HL; 48-53 lateral-line scales, 12½-15½ branched anal-fin rays; 41-42 total vertebrae, comprising 20-21 abdominal and 20-21 caudal vertebrae.

Population structure and genetic analysis of narrow-clawed crayfish (Astacus leptodactylus) populations in Turkey.

The genetic differentiation among Turkish populations of the narrow-clawed crayfish was investigated using a partial sequence of cytochrome oxidase subunit I gene (585 bp) of 183 specimens from 17 different crayfish populations. Median joining network and all phylogenetic analyses disclosed a strong haplotype structure with three prominent clades diverged by a range between 20 and 50 mutations and substantial inter-group pairwise sequence divergence (5.19-6.95 %), suggesting the presence of three distinct clades within the Anatolian populations of Astacus leptodactylus. The divergence times among the three clades of Turkish A. leptodactylus are estimated to be 4.96-3.70 Mya using a molecular clock of 1.4 % sequence divergence per million years, pointing to a lower Pliocene separation. The high level of genetic variability (H d = 95.8 %, π = 4.17 %) and numerous private haplotypes suggest the presence of refugial populations in Anatolia unaffected by Pleistocene habitat restrictions. The pattern of genetic variation among Turkish A. leptodactylus populations, therefore, suggests that the unrevealed intraspecific genetic structure is independent of geographic tendency and congruent with the previously reported geographic distribution and number of subspecies (A. l. leptodactylus and A. l. salinus) of A. leptodactylus.

Use of rpoB sequences and rep-PCR for phylogenetic study of Anoxybacillus species.

This study was conducted to investigate the applicability of rpoB, which encodes the ß subunit of RNA polymerase, to be used as an alternative to 16S rRNA gene sequence similarity analysis in the thermophilic genus Anoxybacillus. Partial rpoB sequences were generated for the 14 type strains of Anoxybacillus species and 6 other strains of four Anoxybacillus species. The sequences and the phylogenetic tree of rpoB were compared with those obtained from 16S rRNA gene analysis. The rpoB gene was found to provide a better resolution for Anoxybacillus species, with lower interspecies sequence similarities. The rpoB sequence similarity analysis permitted a more accurate discrimination of the species within the Anoxybacillus genus than the more commonly used 16S rRNA gene. Furthermore, rapid and reproducible repetitive extragenic palindromic fingerprinting techniques (REP-, ERIC-, and BOX-PCR) were employed for the specimens of genus Anoxybacillus. Through comparison of the three methods, it was found that the BOX-PCR method generated more informative results than REP-PCR for the studied strains; BOX-PCR profiles were more distinct for the different strains, including a higher number of bands. Rapid and reproducible repetitive extragenic palindromic fingerprinting techniques (rep-PCR) constitute a suitable molecular approach for the validation and maintenance of taxonomy within the Anoxybacillus genus. The results of this study show that rpoB and rep-PCR provide rapid and reliable methods for molecular typing of Anoxybacillus species.