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.
|Abstract (english)|| |
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.