Squalius species

Buj, Ivana, Marčić, Zoran, Čavlović, Kristina, Ćaleta, Marko, Tutman, Pero, Zanella, Davor, Duplić, Aljoša, Raguž, Lucija, Ivić, Lucija, Horvatić, Sven & Mustafić, Perica, 2020, Multilocus phylogenetic analysis helps to untangle the taxonomic puzzle of chubs (genus Squalius: Cypriniformes: Actinopteri) in the Adriatic basin of Croatia and Bosnia and Herzegovina, Zoological Journal of the Linnean Society 189 (3), pp. 953-974 : 964-966

Adriatic Squalius species, playing an important role in their recent genetic composition. Specimens of hybrid origin were found in the Konavočica, TrebiŠnjica, Krka, Zrmanja and BoljunŠćica Rivers, and in the Ravno karstic field. All samples from the Konavočica River (morphologically determined as S. svallize ), contain Cytb haplotypes corresponding to S. squalus (LJUT1– LJUT3 haplotypes), although both RAG1 alleles in all samples belong to S. svallize (Hrag4 haplotype) and both rhod alleles correspond to S. svallize (haplotypes Hrhod8, Hrhod9 and Hrhod10), implying a case of mitochondrial introgression of S. squalus DNA into the S. svallize population in the Konavočica stream. Several samples of hybrid origin are recognized in the TrebiŠnjica River. Samples SQTR3 and SQTR7 are outgroup Telestes morphologically determined as S. svallize and contain Cytb genescorrespondingto S.svallize (SVA1).However, sample SQTR7, in addition to both S. svallize RAG 1 alleles and one rhod allele, has a second rhod allele pertaining to S. squalus . For the SQTR3 sample, we were not able to amplify the RAG1 gene, although both its rhod alleles correspond to S. squalus . Three samples from the TrebiŠnjica River that are morphologically determined as S. squalus (samples SQTR2, SQTR8 and SQTR10) possess mtDNA corresponding to S. squalus , while S. sp. TrebiŠnjica (haploytpes LJUT1 and TRE2) have both rhod alleles and a RAG1 allele of the S. squalus type, while the second RAG1 allele corresponds to S. svallize (haplotypes Hrag4 and Hrag8), implying hybridization. Three samples from the BoljunŠćica River in Istria (SQBO1, SQBO6 and SQBO9) have Cytb haplotypes corresponding to S. illyricus (haplotype ILI1). Finally, one sample from the Zrmanja River (SQZR9) has S. illyricus mtDNA (haplotype ILI1), whereas one sample from the Krka River (SQKR2) contains S. zrmanjae mtDNA (haplotype ZRM2), implying mtDNA introgressions.

Based on several instances of incomplete lineage sorting and ancestral polymorphism retentions in both investigated nuclear genes, the Cytb dataset was employed for the evolutionary history reconstruction, and identification of fixed differences and shared polymorphisms. Timing of diversification events within the genus Squalius is shown in Fig. 8 . Separation of the two main phylogenetic groups occurred in the Early Miocene, explaining the high level of differentiation between species belonging to separate groups. The last common ancestor of S. lucumonis , S. microlepis , S. tenellus and three Dalmatian species ( S. illyricus , S. svallize and S. zrmanjae ) dates back to the Upper/ Middle Miocene. The ancestor of S. microlepis and S. tenellus separated from S. lucumonis in the Late Miocene, while the origination of the Dalmatian species seems to have occurred later, in the Pliocene and Pleistocene. Diversification within the ‘ S. cephalus group’ also started in the Middle Miocene. The lineage comprising the two distinct TrebiŠnjica haplotypes and S. prespensis separated from the main ‘ S. squalus group’ in the Pliocene, whereas the majority of diversification events within S. squalus likely occurred during the Pleistocene.

Fixed differences were found between each of the investigated taxon pairs, even between the genetically distinct unit of the TrebiŠnjica River and the remaining species ( Table 4). Moreover, within the Cytb gene, there are diagnostic sites for each Adriatic Squalius species ( Table 5) that differentiate certain species from the remaining ones and enable its reliable molecular identification. On the other hand, only several population pairs share polymorphisms (one shared polymorphism in each taxon pair; Table 4): S. illyricus / S. svallize , S. illyricus / S. microlepis , S. illyricus / S. squalus and S. illyricus /S. sp. TrebiŠnjica.

Of the six phylogenetic models compared using BFs, model E (identifying six Squalius species in the Adriatic basin and not recognizing S. janae nor the TrebiŠnjica lineage as separate species) is preferable over the remaining models ( Table 6). The second-best model, based on BFs, is model B, which recognizes haplotypes from the TrebiŠnjica as a distinct taxonomic unit, but in which S. janae is not a valid species. Model A corresponds with the current taxonomy and

RHOD

MORPHOLOGICAL PERTINENCE LOCALITY DETERMINATION OF HAPLOTYPES OF SPECIES PER SPECIES

Dragonja R. S. janae Unresolved ; possibly Krka R. S. illyricus retention of ancestral Neretva Prološko R blato. S. svallize polymorphism

S. microlepis

Dragonja R. S. janae Neretva R. S. svallize / S.illyricus / Ravno S. squalus S. zrmanjae Konavočica R. S. svallize incomplete lineage Krka R. S. illyricus sorting

Zrmanja R. S. zrmanjae

Dragonja R.

Pazinčica R. S. janae

Mirna R. S. squalus S. squalus

Krka R. S. svallize

Trebišnjica R.

S. illyricus

Ravno

S. microlepis

Prološko blato S. tenellus

Vrljika R.

S. microlepis S. illyricus

Ruda R. S. tenellus S. cephalus

Krka R. S. illyricus (with distinct Drava R. S. cephalus haplotypes,

but in unresolved polytomy)

1

change

recognizes S. janae as a separate species, but was found to be the least likely in describing phylogenetic patterns and was overpowered by all the remaining models.

Figures

Figure 8. Divergence time estimations of species and lineages within the genus Squalius, presented by mean values and 95% credibility range (in brackets) in million years ago. Numbers in square brackets are posterior probabilities (*posterior probability = 1).