First DNA barcoding based record of Echinogammarus trichiatus ( Martynov , 1932 ) ( Crustacea , Gammaridae ) in Belarus

A new alien Ponto-Caspian amphipod species Echinogammarus trichiatus (Martynov, 1932) was identified in the Belarusian part of the Dnieper River using DNA barcoding. This species expands the national checklist of Ponto-Caspian amphipods to nine species. We found individuals of both sexes, including ovigerous females and juveniles. The structure of E. trichiatus population and low abundance suggest that it is still being established. We conclude that further expansion of E. trichiatus into Belarusian waters is possible.


Introduction
Rapid and accurate species identification is critical for detecting alien species as well as for control and eradication programs.DNA barcoding as proposed by Hebert et al. (2003) is now widely used for species identification.A standard short nucleotide sequence of a 658 base-pair region in the mitochondrial cytochrome c oxidase 1 gene (COI) has been used for identifycation of crustaceans since the early 2000s (Lefébure et al. 2006;Costa et al. 2007).This method can be more accurate and informative especially when attempting to identify immature specimens or when morphological features are not evident or damaged.
The spread of alien species of Ponto-Caspian origin to the Belarusian territory was caused by construction of several dam reservoirs on the Dnieper River in Ukraine and by intentional introduction of amphipods as food source for fish (Mordukhai-Boltovskoi 1964;Karatayev et al. 2008;Semenchenko et al. 2009).There are three interbasin canals connecting the Dnieper River with several rivers from the Baltic Sea basin, therefore Belarus is a transit zone through which Ponto-Caspian species spread to the Baltic region (Bij de Vaate et al. 2002;Karatayev et al. 2008;Jazdzewski et al. 2002).The Dnieper River with its large tributary, Pripyat River, is a substantial part of the Central European invasion corridor (Bij de Vaate et al. 2002) and it plays an important role in the movement of alien aquatic species to Central Europe.According to several recent articles (Semenchenko et al. 2009;Semenchenko et al. 2013;Semenchenko et al. 2016), eight alien species of amphipods are established in the water bodies of Belarus.

Material and methods
Samples were collected in May and September 2016 by hand net (ISO 7828) and hand-picked from submerged logs and other natural and artificial substrates floating or submerged in the water.Crustaceans were immediately fixed in cold 96% ethanol and stored on ice for return to the laboratory.Additional samples from a previous collection (August 2010) were also used.The keys of Cărăuşu et al. (1955), Morduchai-Boltovskoy (1969) and Dobson (2013) were used for species identification.Specimens were stored it the fridge until subsampling for DNA extraction.All details regarding taxonomy and vouchers can be found on the Barcode of Life Datasystem (BOLD) website (Ratnasingham and Hebert 2007) in the "Echinogammarus from Belarus" dataset (DS-BELCRUST) section.The samples collected in 2010 were processed in 2015 during the Global Taxonomy Initiative Training Course, whereas the sequencing of samples collected in 2016 was obtained in 2017 as a part of inventory of alien species list using DNA barcoding.
DNA barcodes were compared with other COI sequences of Echinogammarus species available in BOLD (Table 1) and the most probable to be found in Belarus.A phylogenetic tree was constructed in MEGA 6 (Tamura et al. 2013) using the Neighbor-Joining method (Saitou and Nei 1987) based on the p-distance (Nei and Kumar 2000) with a bootstrap test performed on 10000 replicates (Felsenstein 1985).New sequence of Gammarus lacustris Sars, 1863 was used as an outgroup.Mean Kimura 2-parameter (K2P) genetic distance between E. trichiatus sequences and standard error (SE) were also calculated in Mega 6 (Kimura 1980).

Results
The only location, where E. trichiatus is established in Belarus (Figure 1), is in the Dnieper River near Nizhnie Zhary vill.(51º17′40.5″N;30º34′21.1″E).The sampling site is characterized by a sandy bottom with numerous empty shells of Lymnaeidae species and submerged beams (Figure 2).Chemical parameters were as following: pH 8.5, dissolved oxygen 8.5 mg O 2 /l, conductivity 173 S.
The analysis of our own data and existing literature revealed that this species was accidentally unnoticed and omitted from the checklist of nonnative benthic macroinvertebrates in the Dnieper River basin published by Semenchenko et al. (2016).
One sequence (BOLD sample ID SPCB-ET004) belonged to E. ischnus due to misidentification.This sequence clustered with 100 % value within one clade with BOLD obtained sequences of E. ischnus from Ukraine, Belarus, Romania, Netherlands, Canada, and Russia (Table 1; Figure 3).The mean K2P distance between 46 compared sequences of E. ischnus was 0.0164 (± 0.0053 SE).The mean genetic distance between specimens of each species was below 0.03 upper threshold for the intraspecific genetic distance defined by Hebert et al. (2003) and Costa et al. (2009).The mean interspecific genetic distance between the two species was 23.54 %.

Discussion
The native distribution region of E. trichiatus includes the Black Sea, the Azov Sea, their deltas as well as the coastal lakes of Bulgaria and Romania   ( Cărăuşu et al. 1955;Mordukhaj-Boltovskoj 1969).
The first record of this species outside the native area was made by Weinzierl et al. (1997) in the upper part of the Danube River in Germany.Subsequently, E. trichiatus has spread in German waterways and navigation canals due to human-mediated transport (Tittizer et al. 2000;Podraza et al. 2001;Bernerth and Stein 2003;Eggers 2005;Hirt and Schödel 2005;Müller and Eggers 2006).This species has also been found in Slovakia and Hungary (Borza 2009), France (Labat et al. 2011), Belgium (Boets et al. 2012), the Netherlands (Duijts 2011;Tempelman 2011), Poland (Rachalewski et al. 2013), and recently in Turkey (Rewicz et al. 2016).The easternmost record came from the lowest flow of the Caucasian river Khosta (Dedju 1967), which is the locus typicus of this species (Martynov 1932).
In Belarus, few specimens of E. trichiatus were sampled for the first time in 2008 by Vezhnovetz V. (unpublished data) and identified as a subspecies of Chaetogammarus ischnus (Stebbing, 1899).Due to this misidentification, E. trichiatus was not included into the first national checklist published in 2009 (Semenchenko et al. 2009).Afterwards, four specimens of E. trichiatus were found at the same location in 2010 and their identification was checked by DNA barcoding in 2015 (results presented in this study).Data on alien amphipods distribution, including Ch. ischnus with two subspecies Ch. ischnus behningi Martynov, 1919 and Ch.ischnus major (= E. trichiatus) Сǎrǎusu, 1943, was published recently (Makarenko and Vezhnovetz 2014).The material for the abovementioned study was sampled in 2011-2013.At the beginning of 2015, another article was published by Makarenko (2015), where E. trichiatus was mentioned as a separate species and morphological data for this species were provided.This taxonomic confusion has been discussed in depth by Rachalewski et al. (2013).
It is known that E. trichiatus was introduced in the Kiev reservoir early in 1998 (Grigorovich et al. 2002) and it was mentioned for the Kremenchuk and Dniprodzerzhynsk reservoirs (Rachalewski et al. 2013) in Ukraine.As described above, this species was found in Belarus only in one location situated very close to the Kiev reservoir.We can conclude that dispersal of E. trichiatus into the Belarusian part of Dnieper River is the result of natural spread upstream.However, it is not clear why this species was found only at one site since 2010.
Individuals of both sexes, including ovigerous females and juveniles were found in the River Dnieper.It should be noted that only few specimens were caught by hand net while most of them were found on submerged logs near a place where boats are usually floated and lifted by fishermen.Taking into consideration its high viability outside the water bodies for six days between mussel layers (Boets et al. 2012), E. trichiatus could be unintentionally spread by overland transportation of fishing boats and nets.The structure of its population and low abundance suggests that this species is still establishing and further expansion of E. trichiatus into Belarusian waters is possible.
In conclusion, the presence of Echinogammarus trichiatus was confirmed by DNA barcoding in the Belarusian part of the Dnieper River at least since 2010.Echinogammarus trichiatus expands the national checklist of Ponto-Caspian amphipods to 9 species.All the Ponto-Caspian species known from Belarus occur in this locality of the Dnieper River making it a regional hotspot for alien amphipods.

Figure 1 .
Figure 1.Map of Belarus with indication of the sampling location of E. trichiatus records.

Table 1 .
Cytochrome oxidase I (COI) sequences of Echinogammarus ischnus and Echinogammarus trichiatus used in the study.Samples from Belarus are in bold.Localities with approximate coordinates are marked by asterisk (*).