Molecular confirmation of the North American leech Placobdella ornata (Verrill, 1872) (Hirudinida: Glossiphoniidae) in Europe

Specimens of the North American leech, Placobdella ornata (Verrill, 1872) were confirmed from the Donkmeer, a freshwater lake in the province of East Flanders, Belgium, by morphological and molecular analysis. Leech specimens from Belgium were morphologically consistent with the syntype series and description of P. ornata by Verrill (1872). Molecular comparison of the Belgian specimens to specimens of P. ornata from the type locality (New Haven, Connecticut, USA) using the cytochrome c oxidase subunit I (COI) gene revealed a similarity of 99.5%. Placobdella ornata from Belgium is the first documentation of a second species of the genus Placobdella in


Introduction
The Donkmeer (Figure 1), located in the municipality of Berlare in the East Flanders province in Belgium, is a shallow (max depth: 3.20m), 86 hectare, anthropogenically modified oxbow lake (paleopotamon) of the estuarine river Zeeschelde.The 'silted up' part of the old meander was used for peat extraction from the late 17 th century to the early 19 th century.After flooding of the peat pits, the extant lake was formed.The Donkmeer is a heavily utilized and disturbed lake with regular introductions of European eel Anguilla anguilla (Linnaeus, 1758), common roach Rutilus rutilus (Linnaeus, 1758), common bream Abramis brama (Linnaeus, 1758), ide Leuciscus idus (Linnaeus, 1758), northern pike Esox lucius (Linnaeus, 1758) and tench Tinca tinca (Linnaeus, 1758)

Collection of leeches
As part of a monitoring network to determine the biological quality of Vlaamse Milieumaatschappij/

DNA Analyses
Molecular analyses were conducted on one specimen of the newly collected material following the protocol of Richardson et al. (2010).Purified PCR products were sequenced using the HCO2198 primer and the LCO1490 primer for the Cytochrome c oxidase subunit I products by the W.M. Keck Foundation Biotechnology Resource Laboratory at Yale University.The DNA sequences were aligned using Clustal W version 2 (Larkin et al. 2007), checked manually using SeaView 4 (Gouy et al. 2010), analyzed using PAUP* 4.0b10 (Swofford 2002), deposited in GenBank (http://www.ncbi.nlm.nih.gov/genbank/), and compared to other leech DNA sequences contained within Genbank.Uncorrected p distance was calculated using PAUP*.

Results
Placobdella ornata was most abundant on plastic sheeting that was used to protect the shore against erosion.Live specimens of P. ornata ranged in length from 5 mm to 15 mm.Leech specimens from the Donkmeer were consistent with the syntype series and description of P. ornata by Verrill (1872) (Moser et al. 2012).Leech specimens from the Donkmeer had two coalesced eye spots; an unpigmented nuchal band, an unpigmented genital bar, and an anal patch; three rows of dorsal papillae; and two rows of five pre-anal papillae (Figures 2-4).
Molecular comparison of 603 nucleotides of CO-1 revealed a 99.5% similarity from a specimen of P. ornata from the Donkmeer Belgium (Gen-Bank KP176597) to five specimens of P. ornata collected from the type locality (West River, New Haven, Connecticut, USA) (GenBank JQ812128 -JQ812132) and a 99.2% to 99.5% similarity to four specimens of P. ornata collected from the type locality (Shivericks Pond, Falmouth, Barnstable County, Massachusetts, USA) of Placobdella phalera (junior synonym of P. ornata) (JQ812133-JQ812136).Placobdella ornata from the Donkmeer is identical to P. ornata from its type locality (West River, New Haven, Connecticut, USA) and P. ornata from Shivericks Pond, Falmouth, Barnstable County, Massachusetts, USA with the exception of 3 to 5 nucleotides.A difference of 14.4% (87 nucleotides) was found between a specimen of P. ornata from the Donkmeer, Belgium (GenBank KP176597) and the European congener Placobdella costata (Fr.Müller, 1846) (AY962461).

Discussion
This report of P. ornata is the first documentation of a second species of the genus Placobdella from Europe.A congener, Placobdella costata is widely distributed throughout Europe (Sawyer 1986;Bielecki et al. 2012).In addition to molecular differences, P. ornata also differs from P. costata morphologically.Placobdella ornata is distinguished from P. costata by its possession of five pair of pre-anal papillae, three rows of dorsal papillae, and unpigmented nuchal band, genital bar and anal patch, compared to three pair of pre-anal papillae, five rows of dorsal papillae, and an interrupted dorsal medial line in P. costata (see Table 1).No host of P. ornata has been documented.Although there is a large amount of literature reporting P. ornata as a turtle parasite in North America, the identifications of specimens are based on a series of taxonomic errors (Moser et al. 2012).Placobdella ornata, redescribed by Moser et al. (2012) based upon syntype series and contemporary specimens collected from the type locality, appears to be restricted to the northeastern region of the United States.
Specimens of P. ornata may have been brought into the Donkmeer with the introduction of its host.Moser et al. (2005) reported the eastern North American leech species Placobdella parasitica (Say, 1824) from California for the first time and surmised that the leech was introduced with its host.Introductions of alien blood-feeding leeches with their hosts have the risk of introducing blood parasites (e.g.trypanosomes, haemogregarines, etc.) which are transmitted by these leeches.
To date, P. ornata has a confirmed distribution in the US states Connecticut and Massachusetts (Moser et al. 2012), and now the Donkmeer in Belgium.Although Graf (1899) suggested that P. ornata is parasitic on the common musk turtle Sternothaerus odoratus Latreille, 1801, no host has been confirmed.
Knowledge on freshwater leeches is rather limited in Belgium compared to the well-studied neighboring countries Germany and The Netherlands.In both countries, the presence of at least four non-indigenous leech species has been confirmed: Helobdella europea Kutschera, 1987, Caspiobdella fadejewi Epshtein, 1961, Barbronia weberi (Blanchard, 1897) and Dina punctata Johansson, 1927(van Haaren et al. 2004).Placobdella costata is still to be found in Belgium but it is also present in other countries without freshwater turtles (Bielecki 2012;Elliott and Tullet 1982).Amphibians, mammals of which Castor fiber (Linnaeus, 1758) is the most suspected and birds are potential hosts of P. costata (Sawyer 1986;Grosser 1996).
In Belgium, there is a very recent record of the Asian leech Barbronia weberi (pers.comm.Nobby Thys) and in 2005, one specimen of the southeast European leech Hirudo verbana (Carena, 1820) was found in Edegem (province of Antwerp) (Vercauteren and Isate 2005).This medicinal leech was possibly released or escaped after having been used in a medicinal therapy; no further observations on this species are known.
Further investigation is needed to determine if P. ornata occurs in other localities in Europe.

Figure 1 .
Figure 1.The Donkmeer in landscape view.Photograph by Jan Soors.

Figure 2 .
Figure 2. Dorsal surface of living specimen of Placobdella ornata from the Donkmeer, Belgium.Note the characteristic white genital patch in the anterior third (see arrow).Photograph by Jan Soors.

Figure 4 .
Figure 4. Posterior end of Placobdella ornata from the Donkmeer, Belgium showing the papillar pattern of two rows of five pre-anal papillae (see arrows).Photograph by Jan Soors.

Figure 5 .
Figure 5. Dorsal surface of preserved specimen of Placobdella costata from Driebergen, The Netherlands.Scale bar equals 5 mm.Photograph by Ton van Haaren.