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Genetic diversity and phylogenetic relationships of spined loaches (genus Cobitis) in Croatia based on mtDNA and allozyme analysis

Intensive morphological and genetic studies of specimens from different localities from Europe to Euroasia have revealed a high diversity among spined loaches (genus Cobitis) ; however the taxonomic positions and phylogenetic relationships of some of them remain uncertain. Croatian rivers belong to two watersheds: the Danube and Adriatic basins, separated by the Dinarid mountains. Both watersheds have their Cobitis complex population peculiarities. In order to estimate the phylogenetic position of Croatian populations of spined loaches within the European Cobitis phylogeny, as well as to revise their taxonomic status we sequenced 1140 bp of mitochondrial cytochrome b (cytb) gene for 29 individuals from 8 populations and a total of 828 bp of mitochondrial ATP synthase 8 and 6 (ATPase 8/6) genes for selected representatives of each population. Samples were collected from 8 rivers: Matica (specimens previously considered to be C. narentana), Neretva (C. narentana), Cetina (C. dalmatina), Zrmanja (loaches considered related or similar to Cobitis bilineata), Jadova (Cobitis sp.), Kupa (C. elongata and C. elongatoides), Sava (C. elongata and C. elongatoides) and Drava (C. elongatoides). The Neretva, Matica, Cetina, Zrmanja and Jadova Rivers belong to the Adriatic watershed, while the Kupa, Sava and Drava Rivers are part of the Danube watershed in Croatia. Samples from both analysed populations of C. elongatoides clustered together with other previously analysed members of this species and are characterised by a high haplotype but low nucleotide diversity. The remaining samples clustered within the “ Adriatic lineage” that is, as implicated in several earlier studies, composed of three main clusters. We named them “ Bilineata” , “ Elongata” and “ Ohridana-zanandreai” cluster. Croatian samples of C. elongata from two populations (from Kupa and Sava) belong to the “ Elongata” cluster ; this contains two sublineages with a maximal p-distance of 1.1% between them. Finally, five populations belong to the “ Bilineata” cluster that contains four mitochondrial lineages with p-distances between them ranging from 3.3– 5.7%, indicating an isolation time of 4.9 - 8.4 million years. The first lineage, “ C. bilineata” contains loaches from Zrmanja River and Italian samples of C. bilineata. The second lineage named “ Neretva-Cetina” includes samples belonging to the species C. dalmatina and C. narentana from the Cetina, and Neretva Rivers, respectively. These two species appear as two distinct monophyletic sublineages. However, the maximal p-distance of 1.6% observed between them is rather low and comparable to p-distances found within the C. bilineata species. The two remaining lineages “ Jadova” and “ Matica” represent new mitochondrial lineages. The p-distances between them (3.5– 3.6%) and three other species comprised within the “ Bilineata” cluster (3.3– 5.7%) are in the same range or even greater than those found between several well defined European Cobitis species. These results, based on the mtDNA analysis, suggest the need for a revision of the current systematics of the Croatian Cobitis populations.

Cobitis; Croatia; mtDNA; phylogeny; hybrids

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