Biodiversity Data Journal :
Research Article
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Corresponding author: Levan Mumladze (levan.mumladze@iliauni.edu.ge)
Academic editor: Rupert Collins
Received: 21 Aug 2020 | Accepted: 24 Sep 2020 | Published: 23 Oct 2020
© 2020 Giorgi Epitashvili, Matthias Geiger, Jonas Astrin, Fabian Herder, Bella Japoshvili, Levan Mumladze
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:
Epitashvili G, Geiger M, Astrin JJ, Herder F, Japoshvili B, Mumladze L (2020) Towards retrieving the Promethean treasure: a first molecular assessment of the freshwater fish diversity of Georgia. Biodiversity Data Journal 8: e57862. https://doi.org/10.3897/BDJ.8.e57862
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In this study, we provide a first estimation of the molecular diversity of the freshwater fishes of Georgia. In addition to field collections, we integrated DNA barcode data obtained from recent works and public databases (BOLD and NCBI GenBank). Currently, the DNA barcode reference library for freshwater fishes of Georgia comprises 352 DNA barcodes for 50 species, 36 genera and 15 families (52% of total Georgian freshwater fish diversity), from which 162 DNA barcodes belonging to 41 species were newly generated as part of this study. A total of 22 species are reported from the Caspian Sea basin and 31 from the Black Sea basin. Amongst the studied taxa, seven species were found with large interspecific divergences (> 2%) while 11 species were found to share DNA barcodes within our dataset. In the course of the study, we found the first evidence of the existence of Gymnocephalus cernua (Linnaeus, 1758) and also confirm the second occurrence of invasive Rhinogobius lindbergi (Berg, 1933) in Georgia. Based on the evaluation of currently-available barcode data for Georgian fishes, we highlighted major gaps and research needs to further progress DNA-based biodiversity studies in Georgia. Though this study lays a solid base for DNA, based biodiversity assessment and monitoring approaches, further efforts within the recently started CaBOL (Caucasus Barcode Of Life) project are needed to obtain reference data for the species still lacking DNA barcodes.
DNA barcoding, COI, molecular assessment, Caucasus
Around 15000 freshwater fish species are known worldwide (
Broadly, the Georgian ichthyofauna can be divided into the eastern Black Sea and Caspian Sea basins. According to
Since the late eighteenth century, industrial and economic developments have led to severe environmental changes in the whole Caucasus region (
Along with traditional faunistic assessments, molecular genetic tools (such as DNA barcoding) have emerged as an important aid to deal with uncertainties related to taxonomy, species boundaries or cryptic diversity and have helped to enable innovative and efficient ways of biomonitoring (
The important step for DNA barcoding to be useful in biodiversity study/monitoring, is to develop a DNA barcode library for a particular taxa or area. The successful completion of this step, however, requires the integration of traditional taxonomic expert knowledge and DNA technology. While traditional taxonomic expertise (not only in ichthyology), based on academic training, has been largerly neglected in Georgia, as well as in many other countries — part of the phenomenon known as the ‘taxonomic impediment’ (e.g.
In July 2018 and July 2019, concerted collecting activities (BioBlitzes) were organised by the Ilia State University - ISU (Georgia) and the Zoologisches Forschungsmuseum Alexander Koenig - ZFMK (Germany) in the Kintrishi areas in Western Georgia (N41.76 E42.02) and in the Kazbegi region in Northern Georgia (N42.65 E44.64), respectively (
Genomic DNA was extracted from sub-samples using a BioSprint96 magnetic bead extractor (Qiagen, Hilden, Germany). PCRs, targeting the standard DNA barcode region COI, were carried out in 20 μl reaction volumes including 2 μl undiluted DNA template, 0.8 μl of each primer (10 pmol/μl; LCO1490-JJ: 5´-CHACWAAYCATAAAGATATYGG-3´ and HCO2198-JJ: 5´ AWACTTCVGGRTGVCCAAARAATCA-3´, (
Data processing and sequence assembly was done with the software Geneious Pro v.7 (
In addition to the newly-generated DNA barcodes, we included all BOLD-deposited DNA barcodes that originated from Georgia. Sequences from the BOLD database were included in our dataset if the specimen metadata explicitly stated the origin of the sample and provided geo-referenced data (Suppl. material
DNA barcodes of 352 individuals representing 50 species, 36 genera and 15 families (52% of Georgian freshwater fish diversity) are currently available for Georgian fishes. From these, 162 COI sequences were newly generated for this study, through the GGBC (Georgian-German Biodiversity Center) initiative, 153 were contributed through the FREDIE (Freshwater Diversity Distribution for Europe) project (https://www.fredie.eu/), 19 sequences stem from the "Russian Freshwater Fishes" project on BOLD and 18 DNA barcodes were mined from GenBank through BOLD. In the final dataset of all 352 barcodes, the length of the COI sequences was, on average, 648 base pairs (minimum 465 and maximum 658) including no stop codons, insertions or deletions. A total of 82 positions out of 658 (13%) were variable, from which 60 positions (9%) were diagnostically informative. On average, nucleotide base frequency (A-24.47%, C-27.67%, G-18.56%, T-29.29%) and GC content (46.24%) were well within the range known for fishes (see, for example,
Summary table of K2P genetic distances within the different taxonomic levels derived from 349 specimens analysed. The list of studied species is provided in Suppl. material
Label |
N |
Taxa |
Comparisons |
Min Dist (%) |
Mean Dist (%) |
Max Dist (%) |
S.E. |
Within Species |
349 |
50 |
1835 |
0.00 |
0.33 |
2.46 |
0.01 |
Within Genera |
253 |
13 |
1268 |
0.00 |
4.1 |
9.54 |
0.00 |
Within Families |
295 |
5 |
21804 |
4.00 |
16.1 |
27.98 |
0.00 |
Amongst the 50 barcoded species, 26 were represented by more than four sequences in the dataset allowing intraspecific distance estimates (Fig.
Due to small interspecific genetic distances (i.e. distance to nearest neighbour), several species were not predicted to be separate taxa by barcode gap analyses. For instance, nearest-neighbour distances for species belonging to Salmo, Barbus, Capoeta, Chondrostoma, Gobio and Squalius, showed < 2% divergence, indicating a possible need for re-evaluating species limits. As an example, all three specimens from Natanebi and Supsa rivers (Western Georgia, Black Sea Basin), which were identified as Gobio artvinicus (sequence IDs in BOLD: EUFWF3080-18; EUFWF3079-18; EUFWF4984-19) according to
Seven species (Chondrostoma cyri, Rutilus lacustris, Alburnus derjugini, Phoxinus colchicus, Oxynoemacheilus sp., Neogobius fluviatilis and Proterorhinus nasalis) (Fig.
Loaches of the genus Oxynoemacheilus from the Black Sea basin have recently been studied including the description of a new species – O. cemali – from the Coruh river drainage (
Maximum Likelihood estimation of the phylogenetic relationships of Oxynoemacheilus loaches, based on the mitochondrial COI barcode region (Kimura 2-parameter model, discrete Gamma distribution for rate differences with three categories + G parameter = 0.0610). Nucleotide positions with less than 95% site coverage were eliminated, resulting in 637 analysed positions. Numbers near nodes indicate bootstrap support values from 1000 pseudoreplicates. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis includes 45 nucleotide sequences taken from
DNA barcoding confirmed for the first time an occurrence of Gymnocephalus cernua (Linnaeus, 1758) and contributed the second record of alien Rhinogobius lindbergi (Berg, 1933) in Georgia. The former species has never been considered to occur in the country (
Georgia, as part of the Caucasus and Irano-Anatolian biodiversity hotspots, is distinguished by its unique biodiversity and rich freshwater resources, which have been strongly impacted by anthropogenic pressure throughout the 20th century until the present. During the Soviet time, large-scale industrial projects presumably had a strong influence on the Georgian biodiversity and especially on the freshwater fauna. An example of this is the construction of the Mingachevir Dam in Azerbaijan which acted as an insurmountable obstacle for anadromous fishes such as sturgeons, Caspian lampreys and salmon (
We would like to thank Laura von der Mark, Jana Thormann and Katharina Kurzrock for their help with field and laboratory work. This study has been funded through the German Federal Ministry of Education and Research (BMBF), see below. Reviewers and the editor contributed significantly to improve the readability of the manuscript. The two central field expeditions were supported by Deutsche Gesellschaft für Internationale Zusammenarbeit GIZ Georgia under the Integrated Biodiversity Management South Caucasus Initiative, funded by the German Federal Ministry for Economic Cooperation and Development (contract number 2018: 83291308, contract number 2019: 83322223). Collecting permits were kindly provided by the Agency of Protected Areas of Georgia and the Ministry of Environment Protection and Agriculture of Georgia. This study also benefited from the German Barcode of Life initiative (BMBF FKZ 01LI1101 and 01LI1501), by the DAAD and Rustaveli Foundation and the SAW Leibniz project FREDIE (www.fredie.eu).
GGBC (Georgian-German Biodiversity Centre), BMBF: 01DK17048
CaBOL (Caucasus Barcode of Life), BMBF: 01DK20014A
MFG, GE, BJ and LM developed conception and design of the study, collected material and analysed data. FH, JA and LM supported the research logistics. GE, LM and MFG drafted manuscript. All authors participated in revising the article and contributed with intellectual content. All authors gave final approval of the submitted version.
The authors have declared no conflicts of interest.
Details on barcoded specimens from Georgia currently available in BOLD. Abbreviations used in the table: NA - information is not available; ISU - Ilia State University; FJF - Fischsammlung Jorg Freyhof; PC - Prior Collection i.e. before 2018-2019 bioblitz; RAS - Russian Academy of Sciences, Zoological Institute; ZFMK - Zoologisches Forschungsmuseum Alexander Koenig. Samples in bold face are mined from GenBank with BOLD ID same as GenBank accession number.
Taxonomic structure and BOLD summary data of barcoded Georgian fishes with mean and maximum intraspecific distances and nearest-neighbour statistics.