See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/347491916 First DNA-based Records of New Alien Freshwater Species in the Republic of Georgia Article in Acta Zoologica Bulgarica · December 2020 CITATIONS READS 0 65 6 authors, including: Bella Japoshvili Tatsiana Lipinskaya Ilia State University National Academy of Sciences of Belarus 53 PUBLICATIONS 339 CITATIONS 47 PUBLICATIONS 92 CITATIONS SEE PROFILE SEE PROFILE Helen Gajduchenko Sinchuk Aleh National Academy of Sciences of Belarus Belarusian State University 20 PUBLICATIONS 24 CITATIONS 12 PUBLICATIONS 0 CITATIONS SEE PROFILE SEE PROFILE Some of the authors of this publication are also working on these related projects: Research and Restoration of the Essential Filters of the Sea (REEFS) View project Current karyologic and meristic description of Carassius auratus s. lato populations from Belarus View project All content following this page was uploaded by Levan Mumladze on 19 December 2020. The user has requested enhancement of the downloaded file. ACTA ZOOLOGICA BULGARICA Acta Zool. Bulg. 72 (4), December 2020: 545-551 Published online 18 December 2020 Special Issue: http://www.acta-zoologica-bulgarica.eu/00SIO_1_13 ESENIAS and DIAS Scientific Reports 4 Research Article First DNA-based Records of New Alien Freshwater Species in the Republic of Georgia Bella Japoshvili1, Tatsiana Lipinskaya2, Helen Gajduchenko2, Aleh Sinchuk3, Ani Bikashvili1& Levan Mumladze1* 1 Institute of Zoology, Ilia State University, Tbilisi, Georgia; E-mail: [email protected] 2 Scientific and Practical Center for Bioresources, National Academy of Sciences of Belarus, Minsk, Belarus 3 Biology Department, Belarussian State University, Minsk, Belarus Abstract: The aim of the present study was to check the identity of a newly recorded freshwater crab and a goby species using DNA barcoding. The specimens were collected from the fresh waters of the western and eastern areas of Georgia. DNA barcodes (CO1) were obtained from samples, which turned out to belong to Rhithropanopeus harrisii (Decapoda: Panopeidae) and Rhinogobius lindbergi (Gobiiformes: Oxudercidae), detected in Georgia for the first time. Our results demonstrated the usefulness of the molecular methods for the identification of new species introductions that can play an important role in the early detection and the timely decision making. The results encourage the increase of taxon sampling and DNA barcode libraries development in order to effectively deal with the biodiversity inventory and monitoring. Key words: Rhinogobius lindbergi, Rhithropanopeus harrisii, Georgia, new records, freshwater. and the bivalve Mytilopsis leucophaeata (Conrad, Introduction 1831) have been reported from the Georgian inland The Ponto-Caspian region is one of the main sources waters during the last two decades (Vinarski & of the worst invasive species worldwide (Bij de Palatov 2018, Mumladze et al. 2019a). Such an Vaate et al. 2002). On the other hand, the region is unusual low number of fresh/ brackish water aliens presumably a recipient of many alien species, some in Georgia is most probably due to the scarcity of of which can have significant effect on the structure observation/ research activities rather than the rare and functioning of the local ecosystems (Gozlan invasion events. et al. 2010, Zhulidov et al. 2018). However, due In the recent years, the DNA barcoding has to the ethno-cultural heterogeneity and constant emerged as a tool for the rapid evaluation of species political oscillations in the region, the biodiversity identification along with the traditional taxonomic research, documentation and communication is expert-based approach. Since the seminal paper of apparently insufficient for the timely identification Paul Hebert (Hebert et al. 2003), the DNA barcoding of invasive species and the establishment of effective gained increasing importance in the species monitoring programmes (Mumladze et al. 2020). identification and biodiversity exploration, having For instance, only two alien invertebrate species, many different applications. The DNA barcoding the gastropod Ferrissia californica (Rowell, 1863) has already been recognised as an effective aid in *Corresponding author 545 Japoshvili B., T. Lipinskaya, H. Gajduchenko, A. Sinchuk, A. Bikashvili & L. Mumladze the early detection of the alien species even without Stream) located in the eastern Georgia (N41.560567, having the respective taxonomic expertise (Kvist et E45.993493, altitude of 445 m a.s.l.). The collected al. 2018). The only requirement for using the DNA goby specimens were very small (max total length barcoding in the detection of alien species is the of 54 mm) and it was impossible to match any of the availability of relevant DNA barcode libraries that species known for the region according to Kottelat can be used to evaluate the sequence identity. To this & Freyhof (2007) and Ninua et al. (2013). end, the publicly available DNA data in the large The crab and goby samples were submitted to databases such as BOLD systems (Ratnasingham the Scientific and Practical Center for Bioresources & Hebert 2007) and GenBank (Agarwala et al. of the National Academy of Sciences of Belarus for 2018) is already sufficient to effectively diagnose DNA barcoding. most of the widespread non-native species for the For the barcoding purposes, DNA was extracted regions with limited barcode coverage (Weigand et from one or two pereiopods of the crabs and fin-clip al. 2019). muscles of the fish, using a spin column based DNA The aim of the present study was to provide Preparation Kit (Jena BioscienceTM). The 658-bp evidence of two new alien freshwater species: the barcode region of the mitochondrial cytochrome crustacean Rhithropanopeus harrisii (Gould, 1841) c oxidase subunit 1 (COI) gene (Hebert et al. and the fish Rhinogobius lindbergi Berg, 1933, 2003) was amplified using Lepidoptera and Folmer for the Republic of Georgia based on the DNA primer cocktail (C_LepFolF / C_LepFolR, 1:1) barcoding approach (i.e. using traditional and the (Hernández-Triana et al. 2014) for the crabs, and reverse taxonomic approach sensu Markmann & fish primers (L6468/H7696) (Thacker 2003) for the Tautz 2005). gobies. The polymerase chain reaction was performed in the solution totalling 25 μl: 200 μM of each dNTP, Materials and Methods 0.5 μM of each primer, 1.5 mMol of MgCl2, Taq Buffer “A” (65 mMTris-HCl, 16.6 mM (NH4)2SO4, The data reported in this article are based on the 0.02% Tween 20 at a pH of 8.8) 0.5 units and with by-catch material collected during 2016–2019 for a Taq polymerase, approximately 100 ng DNA matrix project studying the freshwater molluscs of Georgia (2 μl). The thermocycling regime used for the crabs (Mumladze et al. 2019b). was the following: initial denaturation at 94°C for 1 Within the fieldwork campaigns, the samples min, 5 cycles of 94°C for 40 s, 45°C for 40 s and 72°C of benthic macroinvertebrates were collected using for 1 min, followed by 35 cycles of 94°C for 40 s, a kick-net and hand-collecting. The samples were 51°C for 40 s and 72°C for 1 min, and a final step of fixed in 96% alcohol and sorted afterwards. The 72°C for 5 min. The thermocycling regime used for large macroinvertebrates (such as crabs and some the gobies was the following: initial denaturation at species of molluscs) and accidentally collected fish 94°C for 3 min; 35 cycles of 94°C for 30 s, 53°C for were preserved separately. One of the investigated 30 s, and 72°C for 60 s; with a final extension at 72°C locality (N42.116841, E41.702946; at the sea level) for 2 min. Bidirectional sequencing was done using is in the westernmost coast of Lake Paliastomi a BrilliantDyeTM Terminator v3.1 Cycle Sequencing near the ranger station (Fig. 1). The water in Lake Kit (Life Technologies, 2018). The sequences Paliastomi is brackish and its salinity (ranging from obtained thereafter were submitted to the Barcode 1 to 10‰ or more) is increasing towards the sea due of Life Data System (BOLD) under the project to the regular water exchange. Crabs were collected INSGE. The sequence analysis tools available in the in this locality for the first time in September 2016 BOLD systems were used to identify the specimens and then in July 2018 and August 2019. In all by sequences, and calculate the sequence divergence sampling cases only one single species, apparently statistics (based on uncorrected p distance) different from the native Potamon species, was (Srivathsan & Meier 2012). We also downloaded recorded. The second locality, where similar crabs the available sequences of the most closely related were collected, was the left tributary of the Kulevi species (sequences within the same Barcode Index River (N42.258500, E41.644500, altitude of 2 m Number – BIN) and the other congenerics in order a.s.l.), northwards from Lake Paliastomi. For the to construct phylogenetic trees. The sequences from preliminary identification of the crab specimens, we Projecto-Garcia et al. (2010) and Simakova et al. used Jensen (2010) and Zaitsev & Öztürk (2001). (2017) were used for the crabs, while the sequences Small goby fish (family Oxudercidae, from Xie et al. (2015), Yamasaki et al. (2015) and subfamily Gobionellinae) were collected on 28 Chang et al. (2017) were used for the fish. The April 2017 in an intermittent brook (the Ozaani sequence alignments and neighbour-joining trees 546 First DNA-based Records of New Alien Freshwater Species in the Republic of Georgia Fig. 1. Map of Georgia with sampling localities and habitats: 1 – left tributary of the Kulevi River (R. harrisii); 2 – Lake Paliastomi (Rhithropanopeus harrisii); and 3 – the Ozaani Stream (Rhinogobius lindbergi). were constructed and illustrated by MEGA X specimens (belonging to BIN ACB4145) most software using default parameters (Saitou & Nei closely related (from 99.43% to 99.81% similarity 1987, Kumar et al. 2018). based on uncorrected p-distance) to our specimens (BOLD System ID: NEOCR018-18; NEOCR030-19, NEOCR03-19).