Girardia tigrina ( Girard, 1850 )

Kanana, Yuliia & Riutort, Marta, 2019, First record of freshwater planarian Girardia igrina (Platyhelminthes, Tricladida, Continenticola) in Eastern Europe, Zootaxa 4624 (4), pp. 597-600 : 597-599

Girardia tigrina ( Girard, 1850) have been recorded in thirteen countries so far. It is commonly accepted that this species is native to North America, where it is widely distributed, forming a polytypic group ( Kenk 1974, 1989; Sluys et al. 1995; Vila-Farré et al. 2011). In the 1920s, it was introduced in Europe ( Meinken 1925) and after that it started to expand southwards and eastwards during the next decades reaching southern France, Spain (including the Balearic Islands) and Italy in the late 1960s to early 1970s ( Baguñà et al. 1980; Gourbault 1969). There are also records from Australia and Japan ( Kawakatsu et al. 1993; Sluys et al. 1995; Tamura et al. 1985).

In this paper, we present the first record of this species in Eastern Europe. We identified morphologically freshwater planarians found in the Desna River ( Ukraine) as Girardia tigrina . The identity of these planarians was subsequently confirmed by molecular analysis of Cytochrome Oxidase I gene (COI) sequences.

Specimens were found in Desna River (vicinity of Pushkari village, Novhorod-Siverskyi district , Chernihiv region, Ukraine, 52°11’08.3”N 33°18’20.8”E, June 2015, coll. A.V. Martynov), with sand and silt at the bottom. Specimens were collected by hand. They were preserved in 96% ethanol and are deposited in the collections of the Laboratory of the Department of Genetics, Microbiology and Statistics, University of Barcelona, Barcelona, Spain, under registration numbers MR0472-01 to MR0472-11 . Photographs of fixed animals were taken for identification using camera Leica M165 C. Histological anatomical investigations were not undertaken at this time.

A small piece of the body was taken from the ethanol-fixed individuals for molecular analysis. Genomic DNA was extracted using Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA). We amplified a fragment of approximately 600 bp from COI gene by polymerase chain reaction (PCR) using the primers BarS (GTTATGCCTGTAAT- GATTG) ( Álvarez-Presas et al. 2011) and PlatRGi (CATCCTGAGGTTTATATWTTGATT). The PCR reaction was performed in 25 μl, containing 1 μl of DNA, 1× PCR buffer, 2.5 mM MgCl 2, 0.1 mM of each dNTP, 0.5 μM of each primer, and 0.15 units of Taq DNA polymerase (Qiagen). The amplification protocol was: 2′ at 95°C, followed by 30 cycles of 94°C for 50′′, 44°C for 30′′, 72°C for 50′′, with a final extension at 72°C for 5′. Amplification products were purified using a vacuum manifold (Multi-Well Plate Vacuum Manifold, Pall Corporation, MI, USA) and sequenced in both directions on a DNA Analyzer 96-capillary sequencer at Macrogen Inc. (Amsterdam, The Netherlands). A contig was obtained using Genious v. 10 (https://www.geneious.com), the resulting sequence had been deposited in GenBank under accession number MN 092348 . The sequence obtained was compared to the GenBank database content using nBLAST. The best hits, with similarities between 89-90% and e-values <7 x e -159, were to three Girardia species ( G. sinensis KP 091892 ; G. tigrina KM 200930 and G. dorotocephala KM 200929 ). We then downloaded the COI sequences from these three Girardia species from GenBank plus that of G. schubarti ( DQ666041 ) and obtained an alignment including as well sequences of two Girardia sister genera, two Dugesia ( D. sigmoides KY498849 and D. gonocephala FJ646985 ) and four Schmidtea ( KM821047 , JF837062 S. mediterranea-1 and 2 respecively; MG457275 S. lugubris ; MG457277 S. nova). Finally, we included COI sequences of two Planarioidea ( Polycelis coronata KY057186 and Crenobia alpina KY569153 ) as outgroups. We inferred a phylogenetic tree by maximum likelihood using RAxML v 7.2.6 ( Stamatakis 2006).

The phylogenetic tree ( Fig. 1 ) shows that the sequence obtained in the present work forms a highly supported clade with G. tigrina , G. dorotocephala and G. sinensis , that together constitute the sister group of G. schubarti . The new sequence hence clearly belongs to the genus Girardia , it does not show a closest relationship to any of the three species, probably due to the variability of the COI gene. Having only one sequence per species does not allow to have all the variability of the gene within each species represented and, on the other hand, the three species are quite similar for their COI (compare the short branches separating G. sinensis and G. tigrina with the ones separating two individuals of S. mediterranea ). Although G. sinensis was described from China ( Chen et al. 2015), its close relationship to G. tigrina and G. dorotocephala in the phylogenetic tree most probably indicates that it is also a case of a recent introduction in that country coming from North America. Sequences from more individuals of all these species will be needed to molecularly identify the sequence we obtained from the Ukrainian specimen, and also to clarify the relationships among the three species and even their species status. From their external appearance nonetheless, the specimens ( Fig. 2 ) collected in the studied locality fall within type I (sensu Hyman 1939) and class A of Girardia tigrina according to Ribas et al. (1989). These are fissiparous specimens, externally spotted (numerous small black spots intermingled with bigger white spots) on a brownish background and with a pharynx clearly pigmented. The previous reports on distributions of European populations show that the type I-class A populations are the commonest type found. These fissiparous populations are mixoploids, presenting diploid (2n = 16) and triploid (3n = 24) cells, while the sexuals are always diploids ( Ribas et al. 1989). After studying the populations of the western Mediterranean, Ribas et al. (1989) suggested that several introductions of different stocks, races, or subspecies, living within the area of origin, have occurred since populations in Europe differ from the commonly described fissiparous population both in pigmentation patterns and reproductive biology. Girardia tigrina has also been recorded from Brazil, Marcus (1946) considered the species to be indigenous in Brazil rather than descended from North America. Kawakatsu and Ponce de León (1990) did not exclude the possibility that the Brazilian populations of the species were introduced from North America. Finally, Sluys et al. (2005) pointed slight anatomical differences between North and South American specimens of G. tigrina and suggested that it might well indicate that the North and South American forms concerned sibling species. Though, they refrained from coining a new species name for the South American animals.

In conclusion, we report the first case of a Girardia species, G. tigrina, in Ukraine and Eastern Europe. Moreover, our phylogenetic results showing the tight relationship between G. tigrina, G. dorotocephala and G. sinensis, together with the existing literature on these species, point to the need of a thorough revision for the whole genus. This situation derives from a poor sampling and systematic study of Girardia in its area of origin, America. As well, whether single, or multiple and independent introductions of Girardia have occurred in Europe is still an open question. These riddles can only be solved by a comprehensive comparison of distantly located European populations to the original lineages from North America. Therefore, more sampling efforts and an in-depth systematic study should be conducted to clarify all these issues.

Figures

FIGURE 1. Maximum-Likelihood tree of COI gene, the scale indicates the branch length in substitutions per site. Numbers at nodes indicate bootstrap supports.

FIGURE 2. A preserved specimen of Girardia tigrina (Girard, 1850) collected from Desna River near Pushkari village (Ukraine). A. Ventral view. B. Anterior part in dorsal view, posterior part in ventral view. C. Dorsal view. ph—pharynx, m—mouth, e—eye.