Broad geographic sampling and DNA barcoding do not support the presence of Helobdella stagnalis (Linnaeus, 1758) (Clitellata: Glossiphoniidae) in North America Author Iwama, Rafael Eiji Author Oceguera-Figueroa, Alejandro Author Carle, Danielle De Author Manglicmot, Claire Author Erséus, Christer Author Miles, Na’Ta’Ne Morning-Song Author Siddall, Mark E. Author Kvist, Sebastian text Zootaxa 2019 2019-09-16 4671 1 1 25 journal article 25461 10.11646/zootaxa.4671.1.1 f0b60062-33ae-4461-b3bf-39ec4b82cf26 1175-5326 3450074 76478201-D2BC-4FF3-B225-8828590BCA8D Helobdella stagnalis ( Linnaeus, 1758 ) ( Figures 1 , 2 ) (Synonyms modified from Harding 1930 ) Hirudo stagnalis Linnaeus, 1758 ; Pennant et al. 1768 ; Turton 1807 ; Dalyell 1853 . Glossophora stagnalis (Linnaeus) : Johnson 1817 . Glossiphonia stagnalis (Linnaeus) : Blanchard 1894 ; Scharff 1898 ; Evans 1905 . Erpobdella stagnalis (Linnaeus) : Templeton 1936 . Hirudo bioculata Bergman, 1757 ; Müller 1774 ; Müller 1776 ; Carena 1820 . Clepsine bioculata (Bergman) : Savigny 1809 ; Carena 1820 . Erpobdella bioculata (Bergman) : Lamarck 1838 . Glossophora bioculata (Bergman) : Johnson 1817 . Helluo bioculata (Bergman) : Oken 1815 . Helobdella bioculata (Bergman) : Bayer 1898 . ? Hirudo puligera : Daudin, 1800 . ? Hirudo stagnorum : Derheims, 1825 . ? Clepsine sowerbyi : Monquin-Tandon, 1846 . ? Glossiphonia circulans : Monquin-Tandon, 1846 . Neotype . SMNH Type-9177, fixed and stored in 95% ethanol, dissected, collected in October 2016 from north side of Lake Trehörningen ( 59 50.600’N ; 17 52.971’E ), Uppsala , Uppsala County , Uppland province , Sweden . Other material examined . One non-dissected specimen ( ROMIZ I11708 ) and one dissected specimen ( ROMIZ I11710 ) from same locality and date as the neotype . Diagnosis. One pair of circular or semicircular shaped eyespots; dorsal nuchal scute at 12/13, VIII a2/a3; no metameric pigmentation; papillae, sensillae, tubercles all absent; gonopores separated by one primary annulus, male gonopore at XII a1/a2, female gonopore at XII a2/a3; mid-body somites triannulate (a1+a2+a3) on both dorsum and venter; diffuse salivary glands; 5–6 pairs of digitiform crop caeca, last pair forming post caeca; 6 pairs of testisacs at interganglionic intervals. Body shape. Length 4.8–5 mm ; maximum width 1.7–1.8 mm ; body sublanceolate ( Fig. 1A, B ), dorsoventrally flattened; anterior sucker well developed, somewhat triangular with the mouth pore located on the anterior border; head somewhat triangular; posterior sucker circular, ventrally directed, not pedunculated, maximum width ± 1.4 mm . External morphology. Body yellowish, with scattered black chromatophores, no metameric pigmentation on either dorsum or venter; dorsal pair of paramedial lines; papillae and sensillae absent ( Fig. 1 A–C); one pair of eyespots on somite III ( Fig. 1E ), circular or semicircular in shape, well-separated from each other; dorsal nuchal scute at VIII a2/a3, oval ( Fig. 1F ); male gonopore at XII a1/a2, female gonopore at XII a2/a3, separated by one annulus ( Fig. 1E ); somites I–III: uniannulate; somites IV–V: biannulate; somites VI–XXIII: triannulate, (a1+a2+a3) on both dorsum and venter; somites XXIV–XXV: biannulate; somites XXVI–XXVII: uniannulate; one post anal annulus. Internal morphology. Pharynx eversible (proboscis in leech literature), J-shaped at base in fixed specimen, base at XIV; one pair of diffuse salivary glands extending from XII/XIII–XIV/XV ( Fig. 2 ); linear salivary ducts, no descendant or ascendant portion, connected to base of pharynx at XIII/XIV; esophagus short, limited to XIV; 5 pairs of simple, digitiform crop caeca ( Fig. 1D ), first pair at XV, last pair forming post caeca from XIX extending to XXIV; 4 pairs of simple intestinal caeca; male atrium located anterior to ganglia XII ( Fig. 1B ); atrial cornua laterally directed; ejaculatory ducts forming descendant and ascendant portions ( Fig. 2 ), descendant portion restricted to XII–XIV, ascendant portion from XIV to XI/XII; 6 pairs of testisacs; first pair at XIII/XIV ( Fig. 2 ), last pair at XVIII/XIX; simple ovisacs extending from XII to XV ( Fig. 1B ). FIGURE 1. Helobdella stagnalis ( Linnaeus, 1758 ) . Neotype (SMNH Type-9177) and non-type (ROMIZ I11710). A. Dorsal view (SMNH Type-9177). B. Dissection of the genital region (ROMIZ I11710). C. Ventral view (SMNH Type-9177). D. Dissection of median region of the body (ROMIZ I11710). E. Ventral surface of the genital region (SMNH Type-9177). F. Dorsal view of the nuchal region (SMNH Type-9177). Abbreviations: A, atrium; E, eyespots; FG, female gonopore; GC, gastric caeca; MG, male gonopore; O, ovisacs; P, pharynx; PC, post caeca; Sc, nuchal scute. FIGURE 2. Helobdella stagnalis ( Linnaeus, 1758 ) . Internal morphology based on a specimen from the type locality (ROMIZ I11710). Abbreviations: A, atrium; ED, ejaculatory ducts; G, ganglia; GC, gastric caeca; IC, intestinal caeca; O, ovisacs; P, pharynx; PC, post caeca; SD, salivary ducts; SG, salivary glands; T, testisacs. Remarks. Linnaeus did not assign type specimens for H. stagnalis , nor did he indicate the type locality. Kvist et al. (2010) , in their designation of a neotype for Hirudo medicinalis ( Linnaeus, 1758 ) , assumed that specimens were collected from Uppsala , Sweden , based on Linnaeus’s residency and intense research activity in the area ( Reid 2009 ). Following this assumption, we herein infer the type locality for H. stagnalis to be Uppsala , Sweden as required by article 75.3.6 of the ICZN (ICZN 1999). In his description of H. stagnalis, Linnaeus mentions only a few, non-diagnostic characters such as body shape and color. According to Linnaeus (1758) , this species is flat, blackish and the abdomen is greyish. Although subsequent records of H. stagnalis included information on size, pigmentation, and feeding behavior, the diagnostic character for the species was, for a long time, the presence of only two eyespots ( Dalyell 1853 ; Johnson 1817 b; Müller 1774 , 1776 ; Turton 1807 ). The chitinous scute was first described by Savigny (1809) , and was rarely incorporated into H. stagnalis redescriptions until the late 1800s ( Blanchard 1894 ; Monquin-Tandon 1846 ). The material that formed the basis for these redescriptions cannot be compared to our recently collected samples. Nonetheless, the neotype of H. stagnalis designated here possesses two eyespots and a chitinous scute, the two diagnostic features used since Blanchard (1894) and Monquin-Tandon (1846) , that differentiates this specimen from all other leech species recorded in Europe, as required by article 75.3.5 of the ICZN (ICZN 1999). In overall internal and external appearance, our specimens resemble those described by Saglam et al . (2018) as Helobdella stagnalis collected in Europe. However, our specimens differ minutely in the number of crop caeca and the proportional size of the ejaculatory ducts and ovisacs. Similar intraspecific variation is well documented in other species of the genus ( Shankland et al. 1992 ; Iwama & Arruda 2016 ). Another important difference is the J-shape formed by the base of the pharynx in the neotype specimen. This characteristic is not usually reported by other studies and might be due to differences in fixation methods. Several scute-bearing species of Helobdella can easily be confused with H. stagnalis s.str. . For example, Helobdella octatestisaca is only differentiated from H. stagnalis s.str. by the number of testisacs ( H. stagnalis = 6 pairs, H. octotestisaca = 4 pairs) ( Lai et al. 2009 ). Further, some morphological differences between Helobdella temiscoensis Salas-Montiel, Phillips, Pérez-Ponce de León & Oceguera-Figueroa, 2014 and specimens of H. stagnalis s.str. include the proportional size of the ejaculatory ducts, extending from XVII to XII in H. temiscoensis ( Salas-Montiel et al. 2014 ) ; note that Saglam et al. (2018) suggest that some specimens of H. stagnalis possess ejaculatory ducts of similar proportion to H. temiscoensis . Furthermore, both atrial cornuae are anteriorly directed in H. temiscoensis , whereas in H. stagnalis s.str. these structures are laterally directed. Also, the nuchal scute is triangular in H. temiscoensis and ovoid in H. stagnalis s.str. . By contrast, Helobdella atli Oceguera-Figueroa & Len-Rgagnon, 2005 ; Helobdella serendipitous Saglam, Kutschera, Sauders, Saidel, Balombini & Shain, 2018 ; and Helobdella californica Kutschera, 1988 are easily differentiated from H. stagnalis by their absence of post caeca ( Kutschera 1988 ; Oceguera-Figueroa & León-Règagnon 2005), contrary to the well developed post caeca of H. stagnalis s.str. . Also, Helobdella bowermani Moser, Fend, Richardson, Hammond, Lazo-Wasem, Govedich & Gullo, 2013 presents both external and internal differences when compared to H. stagnalis s.str. : for example, while no dorsal papillation is present in H. stagnalis s.str. , a single dorsal median longitudinal row of papillae is present in H. bowermani ( Moser et al. 2013 ) . The detailed morphological analysis of Helobdella modesta , performed by Saglam et al. (2018) , suggests that this species differs from H. stagnalis in the shape of the crop caeca: the crop caeca are digitiform in H. stagnalis s.str. , but exhibit an anteriorly directed tip in H. modesta . Saglam et al. (2018) also suggest that there is variation in the position of gonopores between scute-bearing species. In H. modesta , gonopores are located at XI a2/a3 and XI a3/XIIa1, and the same structures are present at XII a1/a2 and XII a2/a 3 in H. stagnalis s.str .. Moreover, the positioning of the male and female gonopores exhibited by H. modesta is also shared by Helobdella serendipitous Saglam, Kutschera, Saunders, Saidel, Balombini & Shain, 2018 , Helobdella eriensis Saglam, Kutschera, Saunders, Saidel, Balombini & Shain, 2018 and Helobdella echoensis Saglam, Kutschera, Saunders, Saidel, Balombini & Shain, 2018 (see Saglam et al. 2018 ). The 6th pair of gastric caeca in H. serendipitous does not form post caeca, as in H. stagnalis s.str .. The shape of the nuchal scute differs slightly between H. stagnalis s.str. , H. serendipitous , and H. echoensis : the nuchal scute is ovoid in H. stagnalis , is pentagonal in H. serendipitous , and forms two lobes in H. eriensis ( Saglam et al. 2018 ) .