Scale morphology variability in cyprinid fishes and its significance in taxonomy using light and scanning electron microscopy: A case study of the genus Garra Hamilton, 1822 (Teleostei: Cyprinidae).

To evaluate scale morphological variability (shape, size, topological macro- and microstructures, ornamentation patterns) of cyprinid fishes, nine species of the genus Garra were selected and their scales from five different body regions were studied by using light and scanning electron microscopy. The scales of the examined species were thin with a central or antero-centrally positioned focus, with no cteni in the posterior part. In addition to these typical characteristics, some morphological variation was observed in the overall shape (irregular round, true oval, round-triangular, irregular pentagonal, hexagonal, irregular hexagonal, pentagonal, ovoid), and the focus shape. These variations were mainly dependent on the fish lengths and the flank region. Morphological analysis clustered the examined species into two distinct groups. Group I consisted of G. amirhosseini and G. gymnothorax, while Group II has three subgroups, which include G. persica and G. mondica (subgroup I), G. meymehensis, G. rossica, G. nudiventris, G. hormuzensis (subgroup II), and G. rufa (subgroup III). The grouping of the studied Garra species based on the current scale morphological characters and the molecular data was only consistent for G. rossica and G. nudiventris. In addition, in the phylogenetic tree, G. persica, G. mondica, G. amirhosseini, and G. hormuzensis formed a distinct clade. However, these species did not represent close relationships in the dendrogram obtained from the scale morphology. A possible explanation why the grouping of the studied Garra species based on their scale morphological characters does not match their phylogenetic relationships is that most of the scale morphological traits vary depending on the fish size and the location of the scales on the flank. Therefore, except for some traits, that is, central or antero-centrally positioned focus, having no cteni, a specific sectioned form so called "tetra-sectioned" type, the other examined variables are not useful enough to be used in the taxonomic study of the examined cyprinid fishes. Therefore, scale morphological characters should be used carefully for taxonomic purposes. RESEARCH HIGHLIGHTS: Shape, size, topological macro- and microstructures, and ornamentation patterns of nine species of the genus Garra were studied by using light and scanning electron microscopy. The scales were thin with a central or antero-centrally positioned focus, with no cteni in the posterior part. Morphological variation was observed in the overall shape (irregular round, true oval, round-triangular, irregular pentagonal, hexagonal, irregular hexagonal, pentagonal, and ovoid), and the focus shape. The grouping of Garra species based on the current scale morphological characters and the molecular data was only consistent for G. rossica and G. nudiventris.

An integrative insight into the diversity, distribution, and biogeography of the freshwater endemic clade of the Ponticola syrman group (Teleostei: Gobiidae) in the Caucasus biodiversity hotspot.

Freshwater habitats of the Caucasus biodiversity hotspot represent a center of endemism for the gobiid genus Ponticola Iljin, 1927. Hitherto, large-scale molecular studies, owing to restricted taxon and geographical sampling, have failed to give an elaborate picture of diversity and evolutionary history of these species. Here, to contribute to filling this gap, we assessed taxonomic diversity, phylogeography and evolutionary history for the south Caspian populations of Ponticola presently classified as P. iranicus and P. patimari, using an integrative taxonomic approach comprising an entire geographic range sampling, and analyses of mitochondrial DNA haplotypes, the head lateral line system, otolith shape, and meristic and morphometric variation. All freshwater samples of the P. syrman group belong to a monophyletic clade with two main subclades: a small subclade confined to the upper Sefidroud sub-basin including the type locality of P. iranicus and a large subclade with three geographically constrained haplogroups (Hg1, Hg2, and Hg3), comprising the rest of the distribution. Hg1 showed an eastern distribution including the type locality of P. patimari, while Hg2 and Hg3 are sister groups with central and western-central distributions, respectively. The freshwater clade diverged from P. syrman during the Tyurkyanian low stand (~150 m b.s.l. lasting ~0.1 Myr), while the divergence of P. iranicus and P. patimari and radiations within P. patimari took place during the Bakunian high stand (up to 50 m a.s.l. lasting ~378-480 kya). Species delimitation analyses indicated two distinct species, corresponding to each main subclade. Although the otolith shape and lateral line analyses did not reflect with phylogeographic pattern, PCA and DFA plots of meristic and morphometric data showed a clear separation of the two major subclades corresponding to P. iranicus and P. patimari, suggesting the presence of significant morphological variation meriting formal taxonomic recognition. Overall, our findings (i) reveal the presence of two freshwater endemic species in the P. syrman group, and pending further investigation, hypothesize the presence of a third cryptic species; (ii) revise and document a narrow distributional range and low diversity for P. iranicus, in contrast to a wider distributional range and high diversity for P. patimari; (iii) suggest that the climatic oscillations of the Pleistocene were associated with the cladogenesis within the P. syrman group; and (iv) allowed for the recognition of conservation units and proposition of management measures.

Comparative ultrastructure and ornamentation characteristics of scales in gobiid species (Teleostei: Gobiidae) using the scanning electron microscope.

To study scale based phylogenetic affinity, the ultrastructure and ornamentation characteristics of body key scales were studied for 12 gobiid species from the Iranian coast of the Persian Gulf including Qeshm and Hormuz Islands and the Makran coast of the Oman Sea using scanning electron microscopy (SEM) technique. The scales were removed from below the first dorsal fin, cleaned in potassium hydroxide solution 1%, and were prepared for the SEM imaging. The presence of both ctenoid and cycloid scales in the studied gobiids was revealed. The focus of ctenoid scales was positioned posteriorly, while the focus of cycloid scales was positioned in the postero-central part of the scale. In all the studied species, radii were located only on the anterior part of the scale, and the primary radii were dominant. Also, there were no granules in the inter circular space, but bifurcation was observed in some circuli. In most species, the teeth-like structures called lepidonts were present on the crest of circuli, which functionally help to firmly attach the scales to the epithelium. The dendrogram of the between-groups-linkage method sorted the gobiid species into the two main groups of five distinct clusters: (a) Cryptocentroides arabicus and Cryptocentrus cyanotaenia (the Cryptocentrus-lineage); (b) Bathygobius meggitti and Bathygobius cocosensis (the Glossogobius-lineage); (c) Coryogalops adamsoni and Coryogalops tessellatus (the Gobius-lineage); (d) Acentrogobius dayi, Istigobius ornatus, Favonigobius reichei, Aulopareia ocellata, and Silhouettea ghazalae (the Gobiopsis-lineage). It seems that the dendrogram topology obtained based on the macro-and microscopic structures of scales, reveals phylogenetic lineages of gobies that have already been proposed for these taxa. Hence, the results of this study are largely consistent with the previous molecular studies on the gobiid fishes and implied that besides other data, the analysis of scale shape and scale-surface microstructures could be served to study the diversification of gobiid species.

Assessment of genetic diversity of an endangered tooth-carp, Aphanius farsicus (Teleostei: Cyprinodontiformes: Cyprinodontidae) using microsatellite markers.

Genetic structure of an endemic tooth-carp fish, Aphanius farsicus from four different water bodies in the Maharlu Lake basin was investigated by applying five microsatellite markers. All of the five examined microsatellite loci showed polymor-phism pattern. A total of four alleles were detected at five microsatellite loci, with an average of 2.8 to 3.5 alleles per locus. Average values of observed and expected heterozygosity were 0.95±0.09 and 0.64±0.02 respectively. None of the tests of linkage disequilibrium were significant between each pair of loci and no deviation from Hardy-Weinberg equilibrium were detected to test for heterozygote deficiency within populations. The Nei's genetic distance values ranged between 0.03 - 0.13. Analysis of pairwise genetic differentiation between each pair of the populations revealed that fixation index (FST) values ranged from 0.013 to 0.039 and RST ranged from 0.005 to 0.065. High genetic diversity observed within the populations (99%) and low diversity (1%) among them indicating probably high level of gene flow among the studied populations of Fars tooth-carp at the present time or in the past. Regarding low genetic differentiation among the studied populations and results of population assignment test, two hypotheses are suggested and supporting evidence for each hypothesis are provided.

Molecular systematics and distribution review of the endemic cyprinid species, Persian chub, Acanthobrama persidis (Coad, 1981) in Southern Iran (Teleostei: Cyprinidae).

The Iranian Persian chub is an endemic species of the family Cyprinidae known only from few localities in drainages of Southern Iran. It was originally described in the genus Pseudophoxinus as (Pseudophoxinus persidis) and then Petroleuciscus (as Petroleuciscus persidis). In this study, we examined phylogenetic relationships of the Iranian Persian chub with other relatives in the family Cyprinidae based on the mitochondrial cytochrome b gene to estimate the phylogenetic (and taxonomic) position of the species. Our molecular phylogenies show that new fish sequences from the drainages in southern Iran are clustered with sequences of the genus Acanthobrama from GenBank while the sequences from two other genera (Pseudophoxinus and Petroleuciscus) are in distinct clade. Therefore, we conclude that the populations of Persian Chub in drainages of southern Iran (i.e., Kol, Kor, Maharlu and Persis) belong to the genus Acanthobrama and species Acanthobrama persidis. The predicted geographic distributions for the species showed a large area of suitable climate for A. persidis across south and west of Iran especially in the Kor River basin. Some other parts in the Persis and Tigris are also might have been suitable habitats for this cyprinid species showing possible dispersal route of Acanthobrama from Tigris to the Persis, Kor and Kol basins.