Combining morphology and genetics in resolving taxonomy--a systematic revision of spined loaches (Genus Cobitis; Cypriniformes, Actinopterygii) in the Adriatic watershed

Abstract

Taxonomic investigation of spined loaches from Dalmatia and Herzegovina was conducted on specimens from 14 localities. The results of the detailed morphological investigations were combined with genetic data (based on one mitochondrial and two nuclear genes) in order to resolve the taxonomic status of each Cobitis population. Among the investigated features of external morphology, the appearance of spots on the caudal fin base turned out to have the greatest diagnostic value. Furthermore, the number of branched fin rays enabled the discrimination of several species. No morphometric character alone could ensure determination of any Cobitis species. Nevertheless, groups of populations that are more similar in their body shapes correspond to mitochondrial phylogenetic lineages. Based on molecular genetic markers, Dalmatian and Herzegovinian spined loaches form independent lineages inside the Adriatic phylogenetic group. Mitochondrial DNA phylogenetic reconstruction revealed six monophyletic lineages, corresponding to six species distributed in the investigated area. The population distributed in Mostarsko blato karstic field in Bosnia and Herzegovina is described as a new species based on a unique combination of morphological characters: a single triangular Canestrini scale; usually 51/2 branched anal fin rays, 61/2 branched dorsal fin rays, 14 branched caudal fin rays; no spots in the surface pigmentation layer on the caudal fin base; scales on the body very small.

Figure 1. Map of investigated area with sampling localities marked, as well as distribution of Cobitis species based on literature.

Legend: A = C. jadovaensis; B = C. bilineata; C = C. dalmatina; D = C. illyrica; E = C. narentana; 1 = Jadova; 2 = Zrmanja (HE Velebit accumulation); 3 = Cetina (Blato); 4 = Neretva in Metković; 5 = Mislina; 6 = Norin; 7 = Modro oko; 8 = Baćinska Lakes; 9 = Matica; 10 = Prološko blato; 11 = Trebišnjica; 12 = Hutovo blato; 13 = Mostarsko blato; 14 = Krenica; A = Austria; H = Hungary; SLO = Slovenia; HR = Croatia; I = Italy; BIH = Bosnia and Herzegovina; RO = Romania; SRB = Serbia; MK = The former Yugoslav Republic of Macedonia.

Figure 2. Plot of scores for factors 1 and 2 based on size-independent measures for females (A) and males (B) from all investigated populations.

Legend: Z = Zrmanja (C. bilineata); C = Cetina (C. dalmatina); Me = Neretva in Metković (C. narentana); Mi = Mislina (C. narentana); N = Norin (C. narentana); Mo = Modro oko (C. narentana); H = Hutovo blato (C. narentana); T = Trebišnjica (C. narentana); M = Matica (C. illyrica); P = Prološko blato (C. illyrica); K = Krenica (C. illyrica); MB = Mostarsko blato (C. herzegoviniensis Buj & Šanda, sp. nov.).

Figure 3. ML phylogram of cyt b haplotypes showing position of and phylogenetic relationships among Dalmatian and Herzegovinian spined loaches.

Numbers at nodes represent ML BS, MP BS and BPP values. The species delimitation (each mtDNA sublineage = separate species) is based on the results of this investigation, and is not completely concordant with literature data.

Figure 4. 95% parsimony network of cyt b haplotypes of spined loaches from Matica R., Baćinska Lakes, Prološko blato, Krenica and Mostarsko blato.

The size of ovals corresponds to haplotype frequency. Small circles are missing (unobserved) haplotypes.

Figure 5. ML phylogram of RAG1 haplotypes.

Numbers at nodes represent ML BS, MP BS and BPP values. Species delimitation is based on the results of this investigation, and is not completely concordant with literature data.

Figure 6. ML phylogram of S7 first intron haplotypes.

Numbers at nodes represent ML BS, MP BS and BPP values. The species delimitation is based on the results of this investigation, and is not completely concordant with literature data.

Figure 7. Median-joining network of S7 first intron haplotypes.

Black circles represent median vectors. The number of mutational steps is displayed by the branches, when there were two or more mutations. Haplotype clusters are marked with roman numbers, corresponding to the numbers used to mark the mtDNA sublineages in Figure 3. The species designation is based on the results of this investigation.

Figure 8. Median-joining network of RAG1 haplotypes.

Black circles represent median vectors. The number of mutational steps is displayed by the branches, when there were two or more mutations. Haplotypes from the investigated area are grouped into two clusters: cluster A corresponds to the “Dalmatian mtDNA lineage” and cluster B to the “Imotski mtDNA lineage”.

Figure 9. Pictures of the caudal parts of specimens from investigated Cobitis species.

Legend: A = C. jadovaensis; B = C. bilineata; C = C. dalmatina; D = C. narentana; E = C. illyrica; F = C. herzegoviniensis Buj & Šanda, sp. nov. Characteristic spots on the base of the caudal fin can be seen.

Figure 10. Cobitis herzegoviniensis Buj & Šanda, sp. nov., a newly described species from Mostarsko blato; holotype.

Figure 11. Photograph of a live specimen of C. herzegoviniensis Buj & Šanda, sp. nov.

Figure 12. Photographs of representative individuals for each species.

Legend: A = C. jadovaensis; B = C. bilineata; C = C. dalmatina; D = C. narentana; E = C. illyrica; F = C. herzegoviniensis, paratype PMR VP2950.