## 2 Materials and Methods  
Trout samples included in this study were collected from several tributaries of Nestos River, during different periods of 2007-2014 (Table 1, Figure 1). All sampling sites are within the limits of the protected area of the Rodopi Mountain Range National Park, according to the Joint Ministerial Decision of the Greek State 40379/01-10-2009. Fish were caught by electrofishing and a small piece of the fin tissue was obtained from each specimen for the DNA extraction, such that after the release of the fish back into the water, their swimming ability was not affected, and their survival was secured. DNA extraction was performed using the protocol of [PERSON] et al. [13] and samples were genotyped using a combination of PCR-RFLP and sequencing analysis in different mitochondrial DNA segments.  
Restriction Fragment Length Polymorphisms (RFLPs) were examined on a mtDNA segment, embedding NADH dehydrogenase subunits 5 and 6 genes (ND5 and ND6 genes). The primers (ND5/6-A, ND5/6-B) and the PCR conditions were applied as described by [PERSON] et al. [14]. Amplified segments (ND5/6), approximately 2500 bp in length, were digested with the restriction endonucleases _All_1 and _Hae_III and restriction profiles were observed in 2.5% agarose gels. Digestion of the ND5/6 region with those two enzymes in the Nestos trout samples, produces two different genotypes that distinguish the indigenous from the non-indigenous translocated fish [12].  
For the sequence analysis, the entire control region (CR) was amplified with the primer pair HN20-LN20 [15] in 18 individuals comprising at least 2 individuals from each sampling site. PCR products were purified using the NucleoSpin PCR Clean-up Kit (Macherey-Nagel, Duren, Germany) and sequencing was performed bidirectionally with both HN20 and LN20 in an ABI Prism 3730 XL automatic capillary sequencer and applying the BigDye Terminator v3.1 kit (Applied Biosystems(r), Foster City, CA, USA). Sequences were edited and aligned with the software MEGA version 5.05 [16]. Finally, in order to visualize the relationships among different haplotypes a maximum likelihood dendrogram was constructed using MEGA v. 5.05.  
\begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Code**} & \multicolumn{2}{c}{**Sampling Location in the**} & \multirow{2}{*}{**Number of Samples**} & \multirow{2}{*}{**Sampling Time**} \\  & & **Greek Territory** & \\ \hline MOU & Mousda stream & 9 & July 2008 \\ VATH1.1 & Vathyrema (Kalikarpo) & 5 & May 2007 \\ VATH1.2 & Vathyrema (Kalikarpo) & 3 & September 2008 \\ VATH1.3 & Vathyrema (Kalikarpo) & 5 & June 2012 \\ VATH1.4 & Vathyrema (Kalikarpo) & 3 & March 2014 \\ VATH2 & Vathyrema (Elatia forest) & 7 & July 2013 \\ DIAV & Diavolorema & 6 & June 2012 \\ ARK1 & Arkoudorema-Drama1 & 11 & June 2012 \\ ARK2 & Arkoudorema-Drama2 & 6 & June 2014 \\ FAR & Farasino & 11 & June 2012 \\ Total & 66 & & \\ \hline \hline \end{tabular}
\end{table}
Table 1: Sample collection information.  
Figure 1: Nestos tributaries (southern Balkans) where the trout samples were collected. Sampling stations are indicated as a black dot [1. Vathyrema (Elatia forest), 2. Vathyrema (Kalikarpo), 3. Mousda stream, 4. Diavolorema, 5. Farasino, 6. Arkoudorema-Drama1, 7. Arkoudorema-Drama2].