Phylogenetic relationships and genetic diversity of the genus Squalius (Cypriniformes, Actinopteri) in Adriatic sea basin

Rod Squalius (Leuciscidae, Cypriniformes) živi u slatkovodnim vodotocima Europe i Azije, s velikom raznolikosti u mediteranskoj regiji. Cilj istraživanja bio je razriješiti taksonomske nedoumice klenova rijeka jadranskog slijeva u Hrvatskoj i BiH, te odrediti rasprostranjenosti pojedinih vrsta, povezanost populacija i razinu genske raznolikosti, te ugroženost pojedinih populacija u svrhu djelotvornije zaštite. Filogenetiĉka rekonstrukcija napravljena na mitohondrijskim i jezgrinim molekularnim biljezima (cyt b, rhod, rag1 i S7) pokazala je dvije nezavisne, genetiĉki udaljene evolucijske grupe: mediteransku (S. svallize, S. microlepis, S. tenellus, S. illyricus i S. zrmanjae) i S. cephalus grupu (S. squalus), zahvaljujući davnom vremenu divergencije. Na raznolikost navedenih vrsta najviše je utjecala alopatrijska specijacija, ĉemu je doprinijela i prirodna hibridizacija. Ovim istraživanjem nije potvrĊeno postojanje istarske vrste S. janae, te se predlaže da se taj naziv koristi kao mlaĊi sinonim vrste S. squalus. Manja zabilježena unutarvrsna genska raznolikost u dalmatinskoj regiji objašnjava se povećanom osjetljivosti klenova na antropogene utjecaje.Background: Genus Squalius (Leuciscidae, Cypriniformes) inhabits european and asian freshwater bodies, with particularly high diversity in Mediterranean area. The taxonomic status of many populations is still matter of debate. This paper constitute first attempt at deciphering mitochondrial and nuclear relationships of Adriatic Squalius species. Aim: The aim of this investigation was to resolve taxonomic uncertainties of the Adriatic chubs from Croatia and Bosnia and Herzegovina and to determine distribution of species, population's connection, status and genetic diversity for future conservation purposes. Also, to test biogeographical hypotheses that could have determined the current distribution of Adraitic Squalius species. Methods: In order to reveal phylogeographic and population genetic structure of the genus Squalius in the Adriatic watershed, samples from 19 localities in Croatia and Bosnia and Herezegovina were obtained. The isolation of DNA, gel electrophoresis, PCR and sequencing of DNA from frozen fin tissue. Selective neutrality of mutations and recombinations was tested by statistical tests D* and F*, Tajima test and recombination test. Haplotype variants of nuclear genes in heterozygous individuals were resolved using a Bayesian statistical method with 1000 iterations. Phylogenetic resconstruction was based on mitochondrial and nuclear genetic markers (cytochrome b, rhodopsin, recombination activating gene 1 and first intron of ribosomal protein S7). The complete mitochondrial cytochrome b (total of 1140 bp) from 166 sequences belonging to the Squalius species was obtain from Adriatic rivers, Europe and Asia. For the nuclear phylogenetic analysis, a subsets of 158 rhod sequences (782 pb), 136 rag1 sequences (1443 pb) and 47 S7 sequences (726 pb) was selected. Phylogenetic reconstruction was constructed using maximum parsimony (MP), maximum likelihood (ML) and Bayes (BAY) methods. For MP and ML analyses a heuristic search mode with 100 replicates and tree bisection-reconnection (TBR) branch swapping algorithm was used. Phylogenetic relationships among populations from Istria, Dalmatia and Herzegovina were investigated by a statistical parsimony and median joining methods. Divergence times estimation was conducted using the Bayesian MCMC coalescent method only on cyt b sequences (strict molecular clock model). Results: Neutrality tests suggested no evidence of selection, while recombination test showed no traces of recombination for all investigated genes. The results revealed high diversity of chubs in the investigated area. All genetic markers revealed the existence of two independent, genetically distinct evolutionary groups: The first one, Mediterranean group, comprising species S. svallize, S. microlepis, S. tenellus, S. illyricus and S. zrmanjae, whereas the second group corresponds with S. squalus. All Istrian samples clustered inside S. squalus lineage. Specimens of hybrid origin were found in Pazinĉica, Ljuta, Trebišnjica, Krka, Zrmanja as well as in the Ravno karstic field. Separation of two main phylogenetic groups occured in late oligocene-early miocene, while diversification of Adriatic Squalius species is of younger origin. Genetic distinctiveness of species belonging to different groups is very high, due to an old age of their divergence. Conclusions: The paraphyletic origin of Adriatic chubs imply multiple colonization events of chubs on Adriatic area. The main divergence force for Adriatic Squalius species was allopatric speciation together with the phenomenon of recorded ongoing and past natural hybridization events. This investigation did not confirm the existence of the S. janae species. It has been recommended that name of S. janae can be used as a junior synonim for S. squalus species. Surprisingly, no high genetic diversity was recorded for any of the investigated species which is alarming, and it should be taken into account while considering conservational plans. Smaller genetic diversity than expected is noticed in chubs from Dalmation area and explained by higher sensitivity on anthropogenic influences and pollution