Research Article |
Corresponding author: Predrag Simonović ( pedja@bio.bg.ac.rs ) Academic editor: Jan Kotusz
© 2023 Ana Marić, Danica Srećković Batoćanin, Dubravka Škraba Jurlina, Miloš Brkušanin, Jelena Karanović, Tamara Kanjuh, Vera Nikolić, Danilo Mrdak, Predrag Simonović.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Marić A, Srećković Batoćanin D, Škraba Jurlina D, Brkušanin M, Karanović J, Kanjuh T, Nikolić V, Mrdak D, Simonović P (2023) A treatise about reliability in dating events of evolutionary history of brown trout Salmo cf. trutta (Actinopterygii) at Western Balkans: Impassable barriers, isolation of populations and assistance of geological timeframe. Acta Ichthyologica et Piscatoria 53: 1-18. https://doi.org/10.3897/aiep.53.97702
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A pool of data already existing about D-loop, i.e., the Control Region (CR) haplotypes of the mitochondrial DNA (mtDNA) of brown trout, Salmo trutta Linnaeus, 1758, tentative Adriatic trout Salmo farioides Karaman, 1938, and tentative Macedonian trout, Salmo macedonicus (Karaman, 1924), and their reconstructed phylogeography makes a good starting point for resolving their evolutionary history. That includes the dating of particular events in it. The events have hitherto been dated using the method of a molecular clock. Various calibrations were applied for the mutation rate, owing to the incongruence between the time of divergence that various authors notified and general knowledge about events in geological history and the periods in which they occurred in the Mediterranean region. Since geological history events were mandatory for setting the scene for the evolutionary history of brown trout, the incongruence between them has questioned the molecular clock calibration’s validity. From results about both the phylogeography and phylogenetic relations between native haplotypes (both partial and whole CR sequences) and the population genetics that characterized particular populations, we calculated the time of divergence between haplotypes in the regions of the western part of the Balkans: Iron Gate broader area in eastern Serbia, continental Montenegro and south-eastern Serbia. The distinct status of adjacent populations was verified by frequencies of microsatellites’ alleles and the STRUCTURE analysis that examined the significance of differences between them. In particular, we examined the populations that were clearly separated either by physical barriers, such as a waterfall in eastern Serbia (e.g., the upper and lower River Rečka supplemented by nearby rivers Vratna and Zamna), or by underground drops in Montenegro (e.g., upper and lower River Zeta, and rivers Nožica and lower River Mrtvica as isolated counterparts). We used the so far most common substitution rate of 1% in a million years’ (MY) period. The divergence times we obtained were compared to the events known for the region from available geological history data. There was a fairly good congruence between the dating obtained by the molecular clock method and that by geological history where the advanced, i.e., modern haplotypes, were concerned. In contrast, the congruence was worse for dating of divergence when more ancient haplotypes were in question, being much better if the mutational rate would be decreased to lower rates. That supported results both from the Rate Correlation Test about the independence of evolutionary rates in different lineages of brown trout, and from the Molecular Clock Test, which revealed that the evolutionary rate throughout the phylogenetic tree is not equal. That implies a difference in the speed of evolution in them, which was likely slower and faster, in the ancient, pre-Pleistocene haplotypes and the advanced, Pleistocene ones, respectively. The setting of the variable, or non-linear (i.e., logarithmic) speed of evolving seems helpful, since the early cladogenesis with the dominance of mutations was most likely combined afterwards with the acting of other evolutionary mechanisms, especially of genetic drift in populations that passed through the bottleneck episodes of the abrupt decrease in population size during the unfavourable periods of their evolutionary history.
brown trout, evolutionary history, geological history, tentative Adriatic trout, tentative Macedonian trout, molecular clock
Brown trout, Salmo trutta Linnaeus, 1758, is natively dispersed across a wide geographic area in the Northern Hemisphere and its overall variability is striking. Apart from the variability inherent in its geographic distribution, brown trout’s plasticity in life history traits in many local and often isolated populations resulted in particular ecological forms, i.e., the morphae that additionally complicated their taxonomy and nomenclature. Owing to that, by following the classification approach relying on the previously widely adopted typological species concept and employing predominantly external morphological features as taxonomic characters, more than 29 tentative taxa at both species and subspecies levels were assigned in time. Although new species concepts have been introduced since then, e.g., biological (
Brown trout have a wide geographical dispersal area, with many isolated populations. The explanation for their wide geographical distribution and intense biogeographical differentiation could be geological processes, primarily of orogenic nature, that led either to geographical separation of aquatic realms, or the birth of a new palaeogeographical area. For example, the ascent of the Alpine chain led to a partition of the Tethyan Ocean on two different biogeographical entities, the Mediterranean and the Paratethys Seas, yet around the Eocene/Oligocene boundary (
In contrast to evolutionary dating suggested by the fossil records of ancient brown trout, there is great variability in the dating by method of molecular clock that uses different average mutational rates. Using the molecular clock method relying on the average mutational rate of 1% at each 1 MY in the mitochondrial DNA (mtDNA),
Earlier reconstructions of phylogeny between brown trout haplotypes were accomplished on their partial CR sequences (
During the Pleistocene glaciations, the stream-dwelling brown trout in the River Danube’s drainage area passed through a very dynamic evolutionary history. Recent secondary connections between the River Danube’s tributaries during the Pleistocene (
The most common native haplotypes in Serbia are of DA haplogroup, with AT and AD haplogroups detected as well (
Observing the novel results obtained in Montenegro, it is necessary to emphasize that the whole area of Montenegro itself displays complex geological and tectonic evolution and significant differences in the distribution and abundance of water resources ranging from arid karst areas to areas rich in both surface and groundwater. The domination of carbonate rocks in geological composition of the mountains in Montenegro enabled the development of karst process and karst relief (
Water from the territory of Montenegro drains into two basins: the Adriatic Sea and the Black Sea, whose watersheds are separated by Triassic dolomites (
A broad karst massif, consisting of thick and permeable fluvioglacial sediments of the Würm glaciation stage, is situated between the River Morača Canyon and the lower River Zeta Plain, which considers tectonic depression formed at the Miocene/Pliocene boundary (
This study aims to clarify and support the evolutionary history of brown trout populations belonging to either the different haplogroups (e.g., in the Adriatic Sea basin), or different haplotypes of the same haplogroup (e.g., in the broader Iron Gate region of the River Danube drainage area) in the Western Balkans, including the results from use of microsatellites’ loci as molecular markers. The records that we have gathered about the adjacent brown trout populations which remained entirely isolated, and phylogenetic relations between them that we reconstructed, illustrate the dispersal of brown trout of particular haplogroups and haplotypes as evolutionary units in various time periods over the western part of the Balkans. That is related to the facts from the geological history known in the region and their timing, in order to check the estimation of the timing of the events dated using the molecular clock technique. Calibration of the molecular clock using calibration points from externally derived dates, such as biogeographical and geological events, can be used for interpolation of divergence times (i.e., the event to be estimated falls within the calibration points or within the calibration point and the tip of the branch), their extrapolation (i.e., the event to be estimated falls beyond the calibration points), or combination of both (Wilke at al. 2009;
Since a majority of haplotypes in both areas of interest was inferred as modern, i.e., derived (
The microsatellites’ analyses served to reveal the distinct population status of brown trout populations of the native haplogroups and haplotypes in streams in the Adriatic Sea basin in Montenegro and in the broader Iron Gate region, respectively.
In this study, three populations of brown trout from streams in Serbia and Montenegro with impassable barriers were analysed (Fig.
Regions in Serbia and Montenegro with the broader Iron Gate Gorge area (A), Adriatic Sea basin of Montenegro (B) and southwestern Serbia (C), where the populations of brown and tentative Adriatic and Macedonian trout were sampled (1, River Rečka; green quadrate denotes the Bledaria waterfall; 2, River Vratna; 3, River Zamna; 4, River Mrtvica; 5, River Nožica; 6, upper River Zeta; 7, lower River Zeta, red rectangle marks the subterranean drop; 8, River Studenačka; 9, River Vlasina; 10, River Džepska; 11, River Vrla; 12, River Božica). The dashed line denotes the peninsular divide between Adriatic, Black and Aegean Seas.
The River Nožica is situated also at the high karst plateau in southern Montenegro, in the drainage area of the River Morača, the main tributary of Lake Skadar in Montenegro. At the western rim of the plateau, it drops through the subterranean crevice, to emerge as the short River Mala that joins the River Morača. The River Rečka from eastern Serbia is situated at the Mt. Miroč in the broader Iron Gate Gorge area. It joins the River Danube under the name River Reka. In the headwaters’ section, it is formed by the confluence of the two forks, northern and western ones. The northern fork in its most upstream part flows through the high and narrow mountain valley built by impermeable Cretaceous clastic rocks that enable a surface stream. At its end, it drops down from the (about) 12 meters high Bledaria Waterfall that is impassable for fish from the downstream section, where tufa is precipitating even today. The western fork is free of barriers upstream, all along to the karstic spring in a deep forest. The drainage area is in the southernmost part of the so-called Dževrin Greda, formed along a fault of the same name, which extends from the Tekija in the North–South direction about 18 km. Evolving as the vertical one, the fault has brought Cretaceous and the Upper Jurassic rocks over the younger, Pliocene units. However, such movements came to an end and the fault started to act until recently as a strike-slip or lateral right fault (
Sampling of materials for phylogeographic and genetic researches was accomplished during the period from 2004 to 2015. Brown trout anal fin clips (approximately 16 mm2) were collected by electrofishing using AquaTech device IG200⁄1 (input 12 V per maximum 15A DC, output 500 V, and frequency 65 P s–1) in Serbia and Suzuki-Bosch (220V DC, Imax = 6A) in Montenegro and stored in 96% ethanol. All sampled fish from analysed streams were released alive immediately after the sampling.
Sequences of haplotypes from southeastern Serbia were already published in
Number of samples in analysed rivers in Serbia with recorded mtDNA CR haplotypes analysed using eight microsatellite loci.
Locality | Haplogroups with detected haplotypes | Micro-satellites | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
DA | AD | AT | ||||||||||||
Da1a | Da2 | Da23c | Da*Dž | Da*Vr | Da-Vl | Da-s6 | Adcs11 | Ad*Prz | Ad-M1 | Adcs1 | Ad*Bož | Atcs1 | ||
Džepska | 1 | |||||||||||||
Vrla | 2 | 1 | ||||||||||||
Vlasina | 2 | |||||||||||||
Studenička | 3 | |||||||||||||
Jerma | 4 | |||||||||||||
Božica | 7 | |||||||||||||
Upper Zeta | 27 | 1 | 2 | 32 | ||||||||||
Lower Zeta | 11 | 6 | 17 | |||||||||||
Nožica | 18 | 12 | ||||||||||||
Lower Mrtvica | 9 | 3 | 1 | 12 | ||||||||||
Rečka (N fork) | 11 | 11 | ||||||||||||
Rečka (W fork) | 2 | 1 | — | |||||||||||
Vratna | 10 | 7 | 3 | 10 | ||||||||||
Zamna | 6 | 6 | 6 | |||||||||||
Total | 74 | 1 | 14 | 1 | 2 | 2 | 3 | 20 | 9 | 1 | 5 | 7 | 5 | 100 |
Haplotypes’ sequences were aligned and edited using the Mega X (
The calculation of divergence times between haplotypes followed the calibration of
Eight microsatellite loci [SsoSL438 (
Alleles’ frequencies, number of alleles per locus, expected (Hexp), and observed (Hobs) heterozygosities, as well as Fst values representing a measure of differentiation between populations and gene flow (Nm) calculated (following
(1 – Fst)(4Fst)–1
were obtained using GENETIX 4.05 (
Population structure was analysed using the STRUCTURE 2.3.4 program (
Several mtDNA haplotypes were detected in analysed regions: seven of DA haplogroup, five of AD and one of AT haplogroup (Table
The ML tree reconstructed (Fig.
(A) Phyogenetic tree reconstructed from 13 CR mtDNA haplotypes’ sequences of brown trout using maximum likelihood (ML) method from three (DA, AD and AT) phylogenetic lineages in the western part of the Balkans (numbers at each clade denote bootstrap probabilities); (B) Timetree inferred by applying the RelTime method to the phylogenetic tree shown in a) and the Hasegawa–Kishino–Yano substitution model, with the branch lengths below each of them and time of divergence to the right of each node.
Estimation of the time of divergence between brown trout that have the ancestral haplotypes using the partial CR haplotypes’ sequences and under the λ1 = 1% and λ2 = 0.31% (Table
Time of divergence (tD) between brown trout of the native CR mtDNA haplotypes (in million years, MY) assessed using both only the first part of the CR to the poly T block (561 bp, above the diagonal) that was the only one available for particular haplotypes, and the maximal available common length (993 bp, below the diagonal) for haplotypes, whose complete CR sequences (as declared in the NCBI base) were available (dN, number of substitutions per site; λ1, substitution rate of 1%; λ2, substitution rate of 0.31%).
MY | Da1a | Da23c | Da2 | Da-Vl | Da-Dž | Da-s6 | Da-Vr | Ad*Prz | Adcs11 | Ad-M1 | Adcs1 | Ad*Bož | Atcs1 | |||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Da1a | dN | — | 1 | 1 | 2 | 4 | 1 | 5 | 9 | 9 | 11 | 9 | 9 | 9 | ||
λ1 | 0.178 | 0.178 | 0.357 | 0.713 | 0.178 | 0.891 | 1.604 | 1.604 | 1.961 | 1.604 | 1.604 | 1.604 | ||||
λ2 | 0.575 | 0.575 | 1.150 | 2.300 | 0.575 | 2.875 | 5.175 | 5.175 | 6.325 | 5.175 | 5.175 | 5.175 | ||||
Da23c | dN | 1 | — | 2 | 3 | 3 | 2 | 5 | 10 | 11 | 12 | 9 | 10 | 8 | ||
λ1 | 0.101 | 0.357 | 0.535 | 0.535 | 0.357 | 0.891 | 1.783 | 1.961 | 2.139 | 1.604 | 1.783 | 1.783 | ||||
λ2 | 0.325 | 1.150 | 1.725 | 1.725 | 1.150 | 2.875 | 5.750 | 6.325 | 6.900 | 5.175 | 5.750 | 5.750 | ||||
Da2 | dN | 1 | 3 | — | 1 | 4 | 2 | 5 | 10 | 11 | 12 | 9 | 8 | 10 | ||
λ1 | 0.101 | 0.302 | 0.178 | 0.713 | 0.357 | 0.891 | 1.783 | 1.961 | 2.139 | 1.604 | 1.426 | 1.604 | ||||
λ2 | 0.325 | 0.975 | 0.575 | 2.300 | 1.150 | 2.875 | 5.750 | 6.325 | 6.900 | 5.175 | 4.600 | 5.175 | ||||
Da-Vl | dN | — | 5 | 3 | 6 | 11 | 10 | 11 | 8 | 10 | 7 | |||||
λ1 | 0.891 | 0.535 | 1.070 | 1.961 | 1.783 | 1.961 | 1.426 | 1.783 | 1.248 | |||||||
λ2 | 2.875 | 1.725 | 3.450 | 6.325 | 5.750 | 6.325 | 4.600 | 5.750 | 4.025 | |||||||
Da-Dž | dN | — | 4 | 3 | 8 | 7 | 7 | 6 | 7 | 6 | ||||||
λ1 | 0.713 | 0.535 | 1.426 | 1.248 | 1.248 | 1.070 | 1.248 | 1.070 | ||||||||
λ2 | 2.300 | 1.725 | 4.600 | 4.025 | 4.025 | 3.450 | 4.025 | 3.450 | ||||||||
Da-s6 | dN | 1 | 3 | 3 | — | 4 | 9 | 8 | 8 | 8 | 8 | 6 | ||||
λ1 | 0.101 | 0.302 | 0.302 | 0.713 | 1.783 | 1.426 | 1.426 | 1.426 | 1.426 | 1.070 | ||||||
λ2 | 0.325 | 0.975 | 0.975 | 2.300 | 5.750 | 4.600 | 4.600 | 4.600 | 4.600 | 3.450 | ||||||
Da-Vr | dN | — | 7 | 7 | 8 | 6 | 6 | 6 | ||||||||
λ1 | 1.248 | 1.248 | 1.426 | 1.070 | 1.070 | 1.070 | ||||||||||
λ2 | 4.025 | 4.025 | 4.600 | 3.450 | 3.450 | 3.450 | ||||||||||
Ad*Prz | dN | 10 | 11 | 11 | 11 | — | 3 | 3 | 2 | 5 | 6 | |||||
λ1 | 1.007 | 1.108 | 1.108 | 1.108 | 0.535 | 0.535 | 1.070 | 0.891 | 1.070 | |||||||
λ2 | 3.249 | 3.573 | 3.573 | 3.573 | 1.725 | 1.725 | 3.450 | 2.875 | 3.450 | |||||||
Adcs11 | dN | 10 | 12 | 12 | 10 | 3 | — | 1 | 6 | 2 | 6 | |||||
λ1 | 1.007 | 1.208 | 1.208 | 1.007 | 0.302 | 0.178 | 1.070 | 0.357 | 1.070 | |||||||
λ2 | 3.249 | 3.898 | 3.898 | 3.249 | 0.975 | 0.575 | 3.450 | 1.150 | 3.450 | |||||||
Ad-M1 | dN | 14 | 17 | 15 | 14 | 4 | 2 | — | 2 | 3 | 7 | |||||
λ1 | 1.309 | 1.712 | 1.511 | 1.410 | 0.403 | 0.201 | 0.357 | 0.535 | 1.248 | |||||||
λ2 | 4.223 | 5.523 | 4.873 | 4.548 | 1.299 | 0.650 | 1.150 | 1.725 | 4.025 | |||||||
Adcs1 | dN | 9 | 9 | 9 | 2 | 1 | 3 | — | 1 | 5 | ||||||
λ1 | 0.906 | 0.906 | 0.906 | 0.906 | 0.201 | 0.101 | 0.302 | 0.178 | 0.891 | |||||||
λ2 | 2.924 | 2.924 | 2.924 | 2.924 | 0.650 | 0.325 | 0.975 | 0.575 | 2.875 | |||||||
Ad*Bož | dN | — | — | 4 | ||||||||||||
λ1 | 0.713 | |||||||||||||||
λ2 | — | 2.300 | ||||||||||||||
Atcs1 | dN | 10 | 12 | 11 | 13 | 7 | 7 | 7 | 6 | — | ||||||
λ1 | 1.007 | 1.208 | 1.108 | 1.309 | 0.705 | 0.705 | 0.705 | 0.604 | ||||||||
λ2 | 3.249 | 3.898 | 3.573 | 4.223 | 2.274 | 2.274 | 2.274 | 1.949 |
Calculation of divergence times by TimeTree of Mega XI software using the substitution rate λ = 0.31% (Fig.
As for the analysis of their microsatellite loci, in brown trout from the Adriatic Sea basin, the highest expected heterozygosity (Hexp) was detected in the lower Mrtvica (0.63) and the lowest one in the lower section of the River Rečka (0.20). The highest observed heterozygosity (Hobs) was in the lower River Zeta (0.64), as well, and the lowest (0.24) in the River Rečka. Generally, the majority of localities had slightly lower values for observed heterozygosity except the lower Zeta, Vratna and Rečka (Table
Expected (Hexp.) and observed (Hobs.) heterozygosities in analysed populations with their standard deviations and P values (with the P = 0.99 as a significance criterion), and the mean allele number (Ān).
NZ | UZ | LZ | LM | VR | ZM | RE | |
---|---|---|---|---|---|---|---|
H exp | 0.51 ± 0.23 | 0.53 ± 0.25 | 0.60 ± 0.32 | 0.63 ± 0.28 | 0.49 ± 0.29 | 0.37 ± 0.32 | 0.20 ± 0.26 |
H obs | 0.49 ± 0.29 | 0.45 ± 0.22 | 0.64 ± 0.36 | 0.54 ± 0.27 | 0.54 ± 0.32 | 0.30 ± 0.35 | 0.24 ± 0.34 |
P | 0.88 | 1 | 1 | 1 | 0.88 | 0.63 | 0.50 |
Ān | 4.25 | 8.25 | 5.57 | 8.00 | 4.13 | 2.63 | 1.63 |
Populations from upper and lower Zeta rivers had a greater number of alleles per locus than populations from rivers Nožica and Mrtvica (Table
Gene flow Nm (above the diagonal), and Fst values (below the diagonal) values between populations.
Nm | Nožica | Upper Zeta | Lower Zeta | Lower Mrtvica | Vratna | Zamna | Rečka |
---|---|---|---|---|---|---|---|
F st t | |||||||
Nožica | — | 0.83 | 0.52 | 0.58 | 0.79 | 0.65 | 0.28 |
Upper Zeta | 0.22612 | — | 0.43 | 0.46 | 1.13 | 1.02 | 0.4 |
Lower Zeta | 0.32416 | 0.36774 | — | 4 | 0.47 | 0.44 | 0.26 |
Lower Mrtvica | 0.29924 | 0.34928 | 0.05983 | — | 0.49 | 0.47 | 0.26 |
Vratna | 0.22336 | 0.17963 | 0.34985 | 0.33697 | — | 2 | 0.36 |
Zamna | 0.26494 | 0.19405 | 0.36175 | 0.34527 | 0.10744 | — | 0.21 |
Rečka | 0.45111 | 0.42333 | 0.48952 | 0.48348 | 0.42415 | 0.54356 | — |
Allelic richness in the population of brown trout from northern fork of the upper River Rečka above the Bledaria Waterfall was the lowest in Iron Gate region (1.75), as well as the observed heterozygosity (Hobs = 0.24), but still higher than expected heterozygosity (Hexp = 0.20). The presence of genetic bottleneck was not detected in this stream, but the deviation from L-shape allele frequency distribution under mode-shift test was present. This test does not show statistical significance, but the shape of the curve indicates deviation from Hardy–Weinberg equilibrium (HWE) in this river. In the previous studies (
Eight microsatellite loci with the number of alleles per locus detected in analysed streams, with the calculated allelic richness (Ar) and private allelic richness (Par) in each of them (in an absence of data for microsatellites for brown trout in the River Rečka western fork, data for rivers Vratna and Zamna closest to it were used).
Locus | Locality (haplogroup/haplotype) | ||||||
Upper Zeta | Lower Zeta | Nožica | Lower Mrtvica | Rečka (N fork) | Vratna | Zamna | |
(DA) | (AD) | (DA) | (AD) | (Da1) | (Da23c) | (Da23c) | |
Str73INRA | 5 | 3 | 2 | 3 | 1 | 1 | 1 |
Ssa85 | 4 | 2 | 4 | 2 | 1 | 2 | 1 |
SsaD71 | 8 | 13 | 6 | 7 | 3 | 3 | 4 |
Ssa410UOS | 11 | 10 | 7 | 5 | 2 | 7 | 3 |
SSsp2216 | 13 | 3 | 7 | 5 | 2 | 5 | 5 |
SsaD190 | 4 | 5 | 2 | 5 | 1 | 3 | 2 |
OMM1064 | 15 | 3 | 5 | 3 | 2 | 4 | 4 |
SsoSL438 | 8 | — | 2 | 3 | 2 | 3 | 2 |
Ar | 4.0 | 6.3 | 3.4 | 3.6 | 1.7 | 3.5 | 2.7 |
Par | 1.4 | 1.5 | 1.1 | 1.2 | 0.7 | 1.5 | 1.0 |
The STRUCTURE analysis included the novel tentative Adriatic trout samples from the upper and lower River Zeta and novel brown trout sample from the River Nožica, whose microsatellites were analysed here for the first time, and the tentative Adriatic trout samples from the lower River Mrtvica and brown trout samples from the rivers Zamna and Vratna in the Đerdap (Iron Gate) Gorge area we have already reported about (
The STRUCTURE analysis reveals the distinct population status (represented by distinct colour) of particular brown trout populations (NZ, River Nožica; UZ, upper River Zeta; REC, River Rečka with one genetic cluster; VRA + ZAM, rivers Vratna and Zamna; LM + LZ, lower River Mrtvica and lower River Zeta as one genetic cluster in putative Adriatic trout) . Y-axis presents the participation of alleles from all populations (expressed as a fraction of individual genome ranging from 0–1) in the genome of each individual in all populations, while bars in the x-axis represent individuals and colour-assigned obtained genetic clusters i.e., populations.
Comparison of dating that various authors gave for particular events in the evolutionary history of genus Salmo (
Another difficulty in calibrating the molecular clock lies in the weight, sensu
Earlier phylogeographic studies in eastern Serbia revealed the presence of few CR mtDNA haplotypes (
Indeed, the undertaken investigations on speleothem in the cave carved into the Barremian (Lower Cretaceous) reef limestones on the opposite, Romanian side of the Danube, showed that it was precipitated between ~75 ka and ~2 ka with at least two hiatuses (Constantin, unpublished
The dating of tectonic formation of the lower River Zeta valley and River Morača to Miocene–Pliocene boundary (
On the (1) high levels of diversity at the haplogroups’ level; (2) independent evolutionary histories and prominent endemism in tentative Adriatic and Macedonian, Salmo macedonicus (Karaman, 1924), trout at the Adriatic and Aegean Seas’ basins, respectively reported from results of molecular studies, as well as (3) ancestral character reconstructed in particular Balkans populations (
The contours of the whole Balkan area were finally shaped by geodynamic events during the Neoalpine history and the interaction of three geodynamic processes: Pannonian collapse, continuous epeirogeny upward movement and the Aegean collapse (
The course of the River Struma/Strymon is mainly controlled by the NNW–SSE trending Struma Lineament, a large tectonic structure that represents the tectonic boundary of the Serbo–Macedonian and the Rhodope massifs (
In the inferred phylogeny, the ancient Ad*Bož haplotype was a sister clade of all others in the AD haplogroup (
In conclusion, it seems that in addition to places of its occurrence, the dating of recent Salmo spp. evolutionary history remains vague. In addition to the phylogeography of brown trout, the reconstructed phylogenetic relations determining the ancestry and descendancy of particular evolutionary units (here referring to haplotypes) should be accounted in considering the dynamics of their evolving. The matters we again have posed here, e.g., (1) the speed of evolving in more recent, i.e., advanced and older, i.e., ancestral periods and evolutionary units (here referring to haplotypes), and (2) levels and the variability of parts of the D-loop, their usefulness in calibration of the molecular clock and their validation with the dating known from geological history, still need more research to get us closer to a more reliable estimation of dating of the divergence between brown trout lineages throughout their dispersal areas. While some dating of the more recent events of evolutionary history seems to correspond well to those of geological history and at local scale, others need more consideration in reaching that aim. It is certain that the ancient scene for evolving of ancestral brown trout haplotypes has been created from geological events either at a wider, continental geographical scale, e.g., gradual succession of the Parathetis Sea, or at the more regional scale, e.g., the formation of the river system in the Skadar Plane and during the Rhodopi Mountains uplifting at the Miocene–Pliocene boundary and mid-to-Late Miocene, respectively. The evolution and spread of the more recent haplotypes, on the other hand, has occurred on that scene in the circumstances of the Late Pliocene and Pleistocene cooling and glaciations, when glacial interconnections (e.g., at the Nikšić high karst plateau and that of the Širokar glacier bifurcating at the watershed divide between the River Vjeruša, i.e., the River Tara’s fork, and River Nožica) played a significant role in dispersal of more advanced haplotypes. It might be hypothesized that those two “acts” of brown trout evolution lasted differently, i.e., unrolled at a different speed and that the distinct molecular clock (i.e., substitution-mutational) rates should be established for them, or the polled, i.e., the logarithmic rate instead of the current uniform, linear one should be set. That period of advanced brown trout and related tentative trout taxa evolving during the Pleistocene glaciations most likely included the acting of evolutionary mechanisms other than mutations (e.g., genetic drift), when they likely used to decrease in population size and pass through the bottleneck episodes. However, the evolving of their CR, their phylogeography and phylogenetic relations give us an opportunity to trace their evolutionary history in the scene that geological history has set to them.
Work was granted by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Contracts No. 451-03-68/2022-14/200178, No. 451-03-68/2022-14/200007 and No. 451-03-68/2022-14/200126 for the University of Belgrade, Faculty of Biology, Institute for Biological Research “Siniša Stanković” and Faculty of the Mining and Geology, respectively).