Description of two new species of freshwater leeches of the genus Helobdella (Hirudinea, Glossiphoniidae) from Mexico, with a redescription of Helobdella socimulcensis (Caballero, 1931)

141 Description of two new species of freshwater leeches of the genus Helobdella (Hirudinea, Glossiphoniidae) from Mexico, with a redescription of Helobdella socimulcensis (Caballero, 1931) Gerardo Torres-Carrera1,2 , Lucas Duarte-De Lima3, Alejandro Oceguera-Figueroa2 1 Posgrado en Ciencias Biológicas, Universidad Nacional Autónoma de México, Avenida Universidad 3000, Ciudad Universitaria, Coyoacán, Ciudad de México, C.P. 04510, Mexico 2 Laboratorio de Helmintología, Departamento de Zoología, Instituto de Biología, Universidad Nacional Autónoma de México, Ciudad Universitaria, Copilco, Coyoacán, A.P. 70-153, Ciudad de México, C.P. 14510, Mexico 3 State University of Maringá UEM, Graduate Program in Comparative Biology, Av. Colombo, 5790 - Zona 7, Maringá – PR, 87020-900 Maringá, Paraná, Brazil Corresponding author: Alejandro Oceguera-Figueroa (aoceguer[email protected]) Copyright: © Gerardo Torres-Carrera et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International – CC BY 4.0). Research Article Abstract Freshwater leeches of the genus Helobdella Blanchard, 1896 (Annelida: Clitellata) are widely distributed in the New World, with most of the species occurring in the neotropics and a single species native to Europe. Species of the genus are characterized by the presence of an eversible proboscis, dorsoventrally flattened body, presence of a single pair of eyespots, and a preference for feeding on the hemolymph of aquatic invertebrates. In this study, two new species of Helobdella are described based on specimens collected in Mexico, Helobdella papilloprocta sp. nov. and Helobdella gulloae sp. nov. These specimens were preliminarily identified as H. socimulcensis (Caballero, 1931), but detailed morphological and molecular analyses confirmed their status as separate species. In addition, a neotype for Helobdella socimulcensis is designated, and a redescription of the species is provided based on specimens from the type locality: Xochimilco, Mexico City, Mexico. Finally, H. austinensis in Nuevo León and H. europaea in Morelos, Mexico are reported for the first time, representing new records for the country. In total, ten species of Helobdella are known from Mexico. Key words: Freshwater leeches, Glossiphoniiformes, Mexico, new species, parental care Introduction The genus Helobdella Blanchard, 1896 (Annelida: Clitellata) includes proboscis-bearing freshwater leeches characterized by a dorsoventrally flattened body, gonopores separated by a single annulus, a single pair of cephalic eyespots, absence of esophageal organs (bacteriomes), and a feeding preference for the hemolymph of aquatic invertebrates (Govedich and Davies 1998; Siddall and Borda 2003). The genus Helobdella is the most speciose genus of Hirudinea with ~80 described species, with the majority of them distributed in the New World, particularly in the Neotropics (Christoffersen 2009; Magalhães et al. 2021). Helobdella stagnalis (Linnaeus, 1758), the type species of the genus, is widely Academic editor: Fredric Govedich Received: 17 June 2025 Accepted: 28 October 2025 Published: 26 November 2025 ZooBank: https://zoobank.org/ F999C799-D953-4609-B64EA0DCDAC6765C Citation: Torres-Carrera G, DuarteDe Lima L, Oceguera-Figueroa A (2025) Description of two new species of freshwater leeches of the genus Helobdella (Hirudinea, Glossiphoniidae) from Mexico, with a redescription of Helobdella socimulcensis (Caballero, 1931). ZooKeys 1261: 141–164. https://doi. org/10.3897/zookeys.1261.162279 ZooKeys 1261: 141–164 (2025) DOI: 10.3897/zookeys.1261.162279 142 ZooKeys 1261: 141–164 (2025), DOI: 10.3897/zookeys.1261.162279 Gerardo Torres-Carrera et al.: Two new species of freshwater leeches of the genus Helobdella distributed in Europe and is likely the only species to occur naturally outside the New World (Iwama and Arruda 2016; Saglam et al. 2018). In contrast, several species of Helobdella have been introduced to other continents, including Africa, Oceania, and Europe (Govedich and Davies 1998; Siddall and Budinoff 2005; Reyes-Prieto et al. 2014). Sawyer (1986) subdivided the genus Helobdella into two mayor groups or series: the “stagnalis” series, comprising species with a chitinous nuchal scute located on the anterior dorsal surface, and the “triserialis” series, which includes species lacking this chitinous scute and bearing multiple pigmented longitudinal stripes along the dorsal surface. Previous phylogenetic analyses supported the monophyly of both series, however, subsequent studies incorporating additional species, many of which were previously assigned to separate genera that now are synonymized with Helobdella, have complicated the diagnosis of both series (Siddall and Borda 2003). In general terms, the taxonomy of Helobdella species is based on the position and shape of eyespots; the presence, absence, and arrangement of dorsal papillae and pigmented stripes as well as in internal traits including the number and size of salivary glands, gastric caeca and testisacs (see Marchese et al. 2020). In Mexico, six species of Helobdella have been documented: H. atli Oceguera-Figueroa & León-Règagnon, 2005, H. elongata (Castle, 1900); H. adiastola Ringuelet, 1972; H. temiscoensis Salas-Montiel, Phillips, PérezPonce de León & Oceguera-Figueroa, 2014; H. socimulcensis (Caballero, 1931), and H. virginiae Oceguera-Figueroa, 2007 (Oceguera-Figueroa et al. 2010; Salas-Montiel et al. 2014). In addition to these, two potentially new species have been identified through mitochondrial DNA sequence analyses, one closely resembling H. socimulcensis and the other H. elongata (see Tessler et al. 2018). The “triserialis” series comprises at least ten species distributed across North and South America (Siddall and Borda 2003). Helobdella socimulcensis, originally described from Xochimilco Lake in Mexico City, is considered the only Mexican species of this complex. In addition to H. socimulcensis, two closely related forms from the Central Mexican plateau have been described: H. conchata Caballero, 1933 from Cuautla, Morelos and H. moorei Caballero, 1941, from León, Guanajuato. However, molecular and morphological analyses failed to differentiate specimens from these three nominal species collected at their respective type localities (Oceguera-Figueroa et al. 2010), thus supporting H. socimulcensis as the only valid species. During an ongoing project focused on the characterization of the Mexican diversity of leeches, specimens morphologically similar to H. socimulcensis were collected in Chiapas, and Veracruz, Mexico. Detailed morphological comparisons of these specimens revealed notable morphological differences with the specimens from Xochimilco, Mexico City, the type locality of the species. These findings, together with the evidence provided by DNA sequences, clearly indicate the presence of two previously undescribed species. Due the need for a thorough comparison with H. socimulcensis, a redescription of that species is also provided. Furthermore, since Caballero (1931) did not designate a holotype or paratypes and none of the original material for this species remains, we herein designate a neotype for Helobdella socimulcensis. 143 ZooKeys 1261: 141–164 (2025), DOI: 10.3897/zookeys.1261.162279 Gerardo Torres-Carrera et al.: Two new species of freshwater leeches of the genus Helobdella Materials and methods Collection of samples Specimens examined in this study were collected between February 2014 and November 2024 (see Table 1). Specimens were found in freshwater habitats, attached to submerged rocks, plant roots, and bivalve shells, and were collected by hand. Leeches were maintained in plastic containers filled with water from the original habitat and transported to the laboratory for further analysis. Table 1. Metadata associated with the specimens used in the present study. Numbers in bold denote newly generated sequences. CNHE = Colección Nacional de Helmintos. Taxon Locality CNHE catalog number GenBank accession number Reference GenSeq Nomenclature cox1ND1 ITS 28S rRNA Haementeria officinalis Michoacan, Mexico JN850906 JN850952 JN850928 JN850891 Oceguera-Figueroa 2012 Helobdella adiastola Ejido Rosendo salazar, Chiapas, Mexico 11446, 11465 MK354140 PX391404 PX380937 This study Genseq 4 Helobdella adiastola Laguna Escondida, Los Tuxtlas, Veracruz, Mexico 11054 MK354139 PX391403 PX380936 This study Genseq 4 Helobdella atli Totolcingo, Tlaxcala, Mexico HQ179851 PX391395 PX380935 PX375459 Oceguera-Figueroa et al. 2010; this study Genseq 3 Helobdella austinensis Shoal Creek, Austin, Texas, USA DQ995306 Bely and Weisblat 2006 Helobdella austinensis Austin, Texas, USA DQ995307 Bely and Weisblat 2006 Helobdella austinensis Shoal Creek, Austin, Texas, USA DQ995310 Bely and Weisblat 2006 Helobdella austinensis Austin, Texas, USA DQ995308 Bely and Weisblat 2006 Helobdella austinensis Shoal Creek, Austin, Texas, USA DQ995309 Bely and Weisblat 2006 Helobdella austinensis Austin, Texas, USA KC812736 Kutschera et al. 2013 Helobdella austinensis Rio San Juan, Nuevo Leon, Mexico PX366692 This study Genseq 4 PX366693 PX366694 PX366695 Helobdella austinensis Balneario las Lajas, Veracruz, Mexico 12857 PX366696 This study Genseq 4 Helobdella austinensis Balneario Florida, Gómez Farías, Tamaulipas 12858 PX366697 This study Genseq 4 Helobdella austinensis Sombreretillo, Nuevo Leon, Mexico 11672 PX366698 PX391402 PX380944 PX375460 This study Genseq 4 Helobdella elongata California, USA AF329045 AF329068 Siddall and Borda 2003 Helobdella eriensis Lake Erie, Toledo, Ohio, USA MN071313 Saglam et al. 2018 Helobdella europaea Berkeley, California, USA DQ995304 Bely and Weisblat 2006 Helobdella europaea Taipei, Taiwan FJ000349 Lai et al. 2009 Helobdella europaea Temixco Morelos, Mexico 11673 PX366685 PX391396 PX380938 PX375458 This study Genseq 4 Helobdella europaea Castellon, Valencia, Spain KC904242 Reyes-Prieto et al. 2014 Helobdella europaea Schobbach, Freiburg, Germany AY576008 Pfeiffer et al. 2004 Helobdella europaea New Zealand AY856049 Siddall and Budinoff 2005 Helobdella europaea Australia AF329052 Siddall and Borda 2003 Helobdella europaea South Africa AY856047 Siddall and Budinoff 2005 Helobdella farmeri California, USA OR075101 Kutschera 2023 Helobdella fusca Wild Goose Lake, Michigan, USA AF329038 AF329061 Siddall and Borda 2003 144 ZooKeys 1261: 141–164 (2025), DOI: 10.3897/zookeys.1261.162279 Gerardo Torres-Carrera et al.: Two new species of freshwater leeches of the genus Helobdella Taxon Locality CNHE catalog number GenBank accession number Reference GenSeq Nomenclature cox1ND1 ITS 28S rRNA Helobdella gulloae sp. nov. Laguna escondida, Los Tuxtlas, Veracruz, Mexico 11133 PX366688 PX391398 PX380939 PX375456 This study Genseq 2 Helobdella lineata Michigan, USA AF329039 AF329062 Siddall and Borda 2003 Helobdella modesta Connecticut, USA JF319993 JF326527 Richardson et al. 2017 Helobdella octatestisaca Taipei, Taiwan FJ000343 Lai et al. 2009 Helobdella papillata Virginia, USA AF329046 AF329069 Siddall and Borda 2003 Helobdella transversa Michigan, USA AF329044 AF329067 Siddall and Borda 2003 Helobdella papilloprocta sp. nov. Ejido Rosendo Salazar, Chiapas Mexico 11671, 11132 PX366689 PX391399 PX380941 This study Genseq 2 Helobdella papilloprocta sp. nov. Ejido Rosendo Salazar, Chiapas Mexico PX366690 PX391400 This study Genseq 2 Helobdella papilloprocta sp. nov. Ejido Rosendo Salazar, Chiapas Mexico PX366691 PX380940 PX375457 This study Genseq 2 Helobdella aff. robusta Sacramento, California, USA DQ995300 Bely and Weisblat 2006 Helobdella aff. robusta Sacramento, California, USA DQ995301 Bely and Weisblat 2006 Helobdella robusta Sacramento, California, USA AF178680 AF178680 Siddall and Borda 2003 Helobdella socimulcensis Xochilmilco, Mexico City, Mexico DQ995311 Bely and Weisblat 2006 Helobdella socimulcensis Xochilmilco, Mexico City, Mexico 11669, 11670 PX366686 PX391397 PX380942 PX375455 This study Genseq 3 Helobdella socimulcensis Guanajuato, Mexico 5561 HQ179870 Oceguera-Figueroa et al. (2010) Helobdella socimulcensis Cuautla, Morelos, Mexico HQ179872 Oceguera-Figueroa et al. (2010) Helobdella socimulcensis Queretaro, Mexico 5563 HQ179868 Oceguera-Figueroa et al. (2010) Helobdella socimulcensis Hidalgo, Mexico 5560 HQ179869 Oceguera-Figueroa et al. (2010) Helobdella cf. socimulcensis Jalisco, Mexico’’ 5562 HQ179866 Oceguera-Figueroa et al. (2010) Helobdella socimulcensis Camecuaro, Michoacan, Mexico PX366687 This study Helobdella cf. socimulcensis Emiliano Zapata, Chiapas, Mexico MG821615 Tessler et al. 2018 Helobdella temiscoensis Temixco, Morelos, Mexico HQ179861 Oceguera-Figueroa et al. (2010) Helobdella triserialis Bolivia AF329054 AF329077 Siddall and Borda 2003 Helobdella virginiae Laguna de Catemaco, Veracruz Mexico 12859 PX366699 PX391401 PX380943 PX375461 This study Genseq 3 Morphological procedures Leech specimens were narcotized by gradually adding 70% ethanol in a Petri dish filled with water until movement ceased. Subsequently, specimens were fixed in 96% ethanol and stored in 70% ethanol until further examination. Leeches intended for molecular analyses were fixed directly in 99% ethanol. To examine internal morphology, representative specimens were slightly flattened between two glass slides and posteriorly fixed and stained with a mixture of Meyer´s paracarmine and hematoxylin, cleared with methyl salicylate for 5–10 min, and mounted on glass slides using Canada balsam, following the protocols of Lamothe-Argumedo (1997). External and internal features were examined and illustrated using a Leica EZ4 stereomicroscope (Leica Microsystems, Schweiz) and an Olympus IX81 microscope (Olympus Optical CO, LTD, Tokyo, Japan), respectively. All measurements are provided in mm. Somite numbering follows Moore (1898). 145 ZooKeys 1261: 141–164 (2025), DOI: 10.3897/zookeys.1261.162279 Gerardo Torres-Carrera et al.: Two new species of freshwater leeches of the genus Helobdella Selected specimens were processed for scanning electron microscopy (SEM) following standard protocols. Samples were dehydrated through a graded ethanol series, critical-point dried with CO2, sputter-coated with a mixture of gold-palladium, and mounted on metal stubs using silver paste. Specimens were examined using a Hitachi Stereoscan Model SU1510 SEM at 10 kV at the Laboratorio Nacional de Biodiversidad (LANABIO), Instituto de Biología, Universidad Nacional Autónoma de México (IB-UNAM), Mexico. All specimens were deposited in Colección Nacional de Helmintos (CNHE), IB-UNAM. Molecular procedures Tissue samples from the caudal sucker of representative specimens were removed and used for genomic DNA extraction with the Invitrogen PureLink® Genomic DNA mini kit, following the manufacturer’s protocol. The following loci were amplified: mitochondrial cytochrome c oxidase subunit 1 (cox1), nicotinamide adenine dinucleotide dehydrogenase subunit I (nad1), and the nuclear internal transcribed spacers 1 and 2 (ITS1, ITS2), including the complete 5.8 rDNA, as well as a partial fragment of the large subunit of ribosomal RNA (28S). All the reactions were accomplished with the primers proposed by Folmer et al. (1994) for cox1, Light and Siddall (1999) for nad1, White et al. (1990) and Källersjö et al. (2005), for ITS and Whiting (2002) and Prendini et al. (2005) for 28S. Polymerase chain reactions (PCR) were performed in a total volume of 15 μl containing: 2 μl of DNA template, 0.2 μl of each primer, 3 μl of 5× reaction buffer, 0.1 μl of MyTaq DNA polymerase (Bioline Cat. BIO-21105) and 9.5 μl of DNase-free H2O. Amplification conditions followed protocols described by Oceguera-Figueroa et al. (2005) and de Carle et al. (2017). PCR products were visualized by electrophoresis on agarose gels. Successful amplifications were purified using CentriSep columns (Thermo Fisher Scientific) with 96-well filter plates with Sephadex G-50 (Cytiva). Sequencing reactions used 0.4 µl BigDye Terminator v. 3.1 (Applied Biosystems), 2 µl Buffer 5x, 4 µl ddH2O, 1 µl of primer at 10 µM, and 3 µl purified PCR product for a total volume of 10 µl. Samples were purified using Sephadex G-50 (Cytiva), then 25 µl 0.5 mM EDTA were added to each reaction. Sequencing reactions were performed using an ABI-PRISM® 3100 Genetic analyzer (Applied Biosystems) at LANABIO, IB-UNAM. DNA Contigs were assembled and edited using Geneious v. 5.1.7 (Biomatters Ltd. Auckland, New Zealand). All newly generated sequences were deposited in GenBank (Table 1). DNA sequence alignments and phylogenetic analyses DNA sequences of each locus were aligned independently in MAFFT (Katoh et al. 2019) using the default parameters. A concatenated dataset was assembled in MESQUITE v. 3.7 (Maddison and Maddison 2021). Haementeria officinalis de Filippi, 1849 was selected as outgroup, based on previous analyses (Light and Siddall 1999; Siddall and Borda 2003; Oceguera-Figueroa 2012; de Carle et al. 2017). The final matrix included 56 terminals and 3,211 aligned nucleotides. Phylogenetic analyses of the concatenated dataset were conducted under the maximum likelihood (ML) and Parsimony (P) optimality criteria. ML tree search was performed in IQ-TREE v. 2.0 (Nguyen et al. 2015). The appropriate model of 146 ZooKeys 1261: 141–164 (2025), DOI: 10.3897/zookeys.1261.162279 Gerardo Torres-Carrera et al.: Two new species of freshwater leeches of the genus Helobdella sequence evolution was selected using ModelFinder (Kalyaanamoorthy et al. 2017). Branch support was estimated with rapid bootstrap (Hoang et al. 2018) establishing automatic number of replicates. Each partition was specified in a separate file and called with -p function. The selected substitution models were: GTR+F+I+G4 for cox1, TN+F+I+G4 for nad1, TPM3+F+I for ITS and TPM3+F for 28S. Parsimony analyses were conducted in TNT (Goloboff et al. 2008) using New Technology search algorithms with 1,000 replicates. Branch support was calculated with 1,000 pseudoreplicates of bootstrap. Pairwise genetic distances of the cox1 dataset were calculated in MESQUITE v. 3.7 (Maddison and Maddison 2021) under the Kimura 2-parameter (K2P) model (Nei and Kumar 2000). Results Phylogeny and molecular analyses Maximum likelihood phylogenetic analysis resulted in a tree with a log-likelihood of -22729.420 (Fig. 1). Parsimony analysis recovered four most parsimonious trees of 2484 steps each. The strict consensus tree of the parsimony analysis and the ML tree were largely congruent. Both analyses recovered the monophyly of the “triserialis” series sensu Sawyer 1986 (bootstrap support value of 87 in ML), while the “stagnalis” series was recovered as a paraphyletic assemblage. Species included in the “triserialis” series are characterized by the absence of a chitinous nuchal scute, in most of the species, and also the presence of multiple pigmented dorsal longitudinal stripes and metamerically arranged papillae (see Table 2). Within the “triserialis” series, Helobdella fusca (Castle, 1900) from Michigan, USA was found as sister to the remaining species of the group. Two large clades were recovered: clade A includes H. papillata (Moore, 1906), H. transversa Sawyer, 1972, H. lineata (Verrill, 1874), and H. robusta Shankland, Bissen & Weisblat, 1992, from the USA; H. virginiae from Veracruz, Mexico; and multiple samples of H. austinensis Kutschera, Langguth, Kuo, Weisblat & Shankland, 2013 from Texas, USA and Nuevo León, México. Clade B includes H. triserialis (Blanchard, 1849) from Bolivia, H. cf. socimulcensis from Veracruz, Mexico, the invasive and worldwide-distributed H. europaea (Kutschera, 1985), H. socimulcensis from Mexico, and H. farmeri Kutschera, 2023 from California, USA. Finally, the two new species H. papilloprocta sp. nov. and H. gulloae sp. nov. are sister to each other, and together, sister to H. triserialis and H. cf. socimulcensis from Chiapas, Mexico, all together included in clade B. Taxonomy Order Hirudinida Siddall, Apakupakul, Burreson, Coates, Erséus, Gedlder Källersjö, & Trapido-Rosenthal, 2001 Suborder Glossiphoniiformes Tessler and de Carle, 2018 Family Glossiphoniidae Vaillant, 1890 Genus Helobdella Blanchard, 1896 Type species. Helobdella stagnalis (Linnaeus, 1758). 147 ZooKeys 1261: 141–164 (2025), DOI: 10.3897/zookeys.1261.162279 Gerardo Torres-Carrera et al.: Two new species of freshwater leeches of the genus Helobdella Figure 1. Maximum likelihood phylogenetic tree of species of Helobdella based on concatenated dataset of 3211 nucleotides. Values next to nodes indicate Maximum likelihood and Parsimony bootstrap values. New species described in this study are in bold. A and B indicate the two main species groups within the “triserialis” series. 148 ZooKeys 1261: 141–164 (2025), DOI: 10.3897/zookeys.1261.162279 Gerardo Torres-Carrera et al.: Two new species of freshwater leeches of the genus Helobdella Helobdella socimulcensis (Caballero, 1931) Figs 2–4 Helobdella moorei Caballero, 1933 – León, Guanajuato; Mexico. Helobdella conchata Caballero, 1941 – Cuautla, Morelos Mexico. Helobdella triserialis var. lineata Ringuelet, 1943 – Xochimilco, Mexico City (sensu Ringuelet 1981). Neotype designation. Mexico • A stained adult, mounted in a slide. 11.5 length and 4.5 maximum width at somite XIX; Xochimilco, Mexico City; 19°17'21"N, 99°06'31"W; Collected by G.T-C. CNHE 12299. Note. According to Article 75.3 of the ICZN, a neotype is designated due to the absence or loss of the original type material. Caballero (1931) did not designate any specimen as part of the type series, and no specimens attributable to the original description of Glossiphonia socimulcensis, 1931 have been found in the CNHE or any other scientific collection. Furthermore, the nomenclatural act presented here is justified by the recognition of five lineages with specimens that exhibit morphological similarities to H. socimulcensis. In the absence of type material, meaningful comparisons are hindered. The neotype is designated from material collected from the type locality (Lake Xochimilco) and after ensuring a morphological correspondence with the original description. Finally, DNA sequences were obtained from specimens collected in the present study; these sequences have been validated and comply with the recommendations outlined by Kvist et al. (2010). Material examined. Mexico • Neotype and 20 adult specimens in total: 13 stored in ethanol, six mounted on slides and one processed for SEM; same data as for Neotype; CNHE 12298. Mexico • 116 ethanol-preserved adult specimens; same data as for Neotype; collected by GT-C between April and November 2021; CNHE 13037. Mexico • six adult specimens preserved in ethanol and one slide of an immature specimen; same data as for Neotype; Collected by Salas-Montiel and AO-F; CNHE 8860. Mexico • One adult specimen; same data as for Neotype; collected by AO-F on February 2003; CNHE 4705 (Oceguera-Figueroa 2007. Mexico • two adult specimens; Lago de Tecocomulco, Hidalgo; 19°51'57"N; 98°23'51"W; collected by AO-F on 21 August 2004 CNHE 4704. Mexico • “Hellobdela conchata” Holotype Cuautla, Morelos CNHE 1852, and 2 adult paratypes; same data as for Holotype; CNHE 1853. Diagnosis. Small leeches, 4–16 length and 1.5–9 width (n = 115). Dorsal surface with ~34 brown stripes. Midline longitudinal paired stripes of solid color extend from Va2 to the anus, separated by a distance approximately equal to that between eyespots; the stripes join each other at a2 of each somite, creating a chain-like appearance. Three dorsal rows of longitudinal, dark browntipped papillae on a2: one median and two lateral rows. In some specimens, an additional pair of marginal rows more prominent in the posterior third of the body. Papillae smaller than the width of individual annuli. Eyespots punctiform and well separated. Full body with 68 annuli. Esophagus slightly globular, spanning six annuli. Crop with five pairs of digitiform caeca, the last pair forming post-caeca. Testisacs in five intersegmental pairs. Description. External morphology. Body lanceolate, 3–16 length and 1.5–9 maximum width at somite XIX (n = 115). Ground color pale yellow or cream; dorsal preocular zone unpigmented (Fig. 2A–C). Dorsal surface with ~34 stripes 149 ZooKeys 1261: 141–164 (2025), DOI: 10.3897/zookeys.1261.162279 Gerardo Torres-Carrera et al.: Two new species of freshwater leeches of the genus Helobdella of longitudinal brown stripes (visible in stained specimens). A pair of solid midline stripes, separated by a distance equivalent to the distance between eyespots, extends from Va2 to the anus at XXVII. The stripes join each other at a2 of each somite, creating a chain-like appearance. The remaining 32 stripes (16 pairs) are slender extend from Va2 or Va3 (annuli 6 or 7) and continue to the anus (Fig. 2A). Dorsum with three longitudinal rows of dark brown-tipped papillae located in a2 of each somite, one row median and two laterals. In some specimens, an additional pair of incomplete marginal rows may be present, papillae more prominent in the posterior third of the body, some papillae unpigmented (Fig. 2A). Median papillae row extends from X–XI to XXV–XXVI; lateral rows from XIII to XXVI (Fig. 2C). Papillae smaller than the width of an annulus. Dorsal white spots conspicuous, in three longitudinal rows metamerically arranged: one central and two laterals. White spots somewhat circular, approximately the size of an annulus and located in a2, beside lateral rows of papillae. Median row of white spots from VIII to XXVI; lateral rows from XII or XIII. Eyespots punctiform at IV a1/a2, well-separated. Anus dorsal, at XVII. Ventral surface whitish, without papillae. Mouth pore located at the anterior margin of Table 2. Key morphological characteristics of leeches in the Helobdella triserialis series sensu Sawyer (1986). Taxon Number of pairs of crop caeca Number of pairs of testisacs Number of dorsal brown stripes Segment on which papillae begin Papillae size with respect to width of annuli Arrangement of dorsal white spots Total number annuli Venter coloration Reference Helobdella austinensis Kutschera, Langguth, Kuo, Weisblat & Shankland, 2013 6 6 16–20 apparently X smaller irregular 70 grey-brown Kutschera et al. 2013 Helobdella brasiliensis (Weber, 1915) 6 6 absent IX larger apparently metameric 70 stripes and spots dark brown Ringuelet 1985 Helobdella chaviensis Iwama & Arruda, 2016 5 6 - apparently IX smaller metameric 68 -Iwama and Arruda 2016 Helobdella europaea (Kutschera, 1987) 5 5 14 Xlarger metameric 68 unpigmented to lightly pigmented Kutschera 1985; Govedich and Davies 1998; Kutschera 2004 Helobdella farmeri Kutschera, 2023 6 6 8–14 absent n/a orthogonal 70 uniformly light-brown Kutschera 2023 Helobdella fusca (Castle, 1900) 6 6 absent absent n/a irregular 70 present Castle 1900 Helobdella gulloae sp. nov. 5 4 37 X–XIII larger metameric 69 cream with brown stripes This study Helobdella lineata (Verrill, 1874) 5 6 12–14 irregularly scattered smaller metameric 70 unpigmented Sawyer 1972; Sawyer 2024 Helobdella papillata (Moore, 1906) 6 - - XIX–XX larger irregular - - Sawyer 1972; Siddall and Borda 2003 Helobdella papilloprocta sp. nov. 5 5 40 XII larger metameric 67 unpigmented This study Helobdella robusta Shankland, Bissen & Weisblat, 1992 5 - - - apparently smaller irregular - - Shankland et al. 1992 Helobdella socimulcensis (Caballero, 1931) 5 5 34 X–XI smaller metameric 68 unpigmented Caballero 1931 Helobdella transversa Sawyer, 1972 6 6 absent absent n/a metameric form transverse bands 73 unpigmented Sawyer 1972 Helobdella triserialis (Blanchard, 1849) 6 6 absent apparently X smaller metameric 70 unpigmented Ringuelet 1943 Helobdella virginiae Oceguera-Figueroa, 2007 6 6 30 XII smaller irregular 70 brown irregular spots Oceguera-Figueroa 2007 156 ZooKeys 1261: 141–164 (2025), DOI: 10.3897/zookeys.1261.162279 Gerardo Torres-Carrera et al.: Two new species of freshwater leeches of the genus Helobdella zone unpigmented (I + II to Va2) except for the anterior end of the dorsal midline stripes that extend to reach the eyespots (Fig. 8A). Dorsal surface with the characteristic brown longitudinal striping pattern of species of the “triserialis” series, with ~37 brown stripes visible in stained specimens. A pair of longitudinal midline stripes from IVa3 and fuse into a single, broad stripe from XVI to the anus; this band is interrupted by square-shaped white spots located at each a3/a1 from XXII to the posterior end. The remaining pairs of dorsal brown stripes extend from IVa3 to the anus. Three rows of dorsal papillae heavily pigmented on a2. Additional lateral may occur though these are typically incomplete. Papillae slightly exceed the width of individual annulus. Midline row of papillae extends from X, with conspicuous papillae evident from XII or XIII to XXVI (Fig. 8C). Lateral rows of papillae begin at XIV to XXV. White spots conspicuous, square to circular in shape, and smaller than the width of individual annulus; arranged in two paired rows on each side of the body, located on a2, flanking each papilla. White spots flanking central papillae begin at V or VI, and extend to XXVI, lateral rows of white spots extend from XII to XXVII. Eyespots semioval, at IVa1+a2, well-separated. Anus on XVII, typically surrounded by black pigment. Ventral surface ground cream-colored, with faint, thin longitudinal brown stripes (Fig. 8B). Oral sucker white; mouth located at the anterior margin. Gonopores separated by a single annulus (XII a2). Whole body with 69 annuli. Annulation: I and II fused; III and IV bi-annulate; V– XXIV tri-annulate; XXV bi-annulate, and XXVI, XXVII uni-annulate (Fig. 8C). Figure 8. Helobdella gulloae sp. nov. A. Dorsal view; B. Ventral view; C. SEM microphotograph showing somite numbering. Abbreviations: an = anus. Scale bar: 2 mm. 157 ZooKeys 1261: 141–164 (2025), DOI: 10.3897/zookeys.1261.162279 Gerardo Torres-Carrera et al.: Two new species of freshwater leeches of the genus Helobdella Internal morphology. Proboscis straight, not recurved, 1.52 length, extending from X to XIV. Salivary glands diffuse into parenchyma between XII and XV; ductules not forming bundle, insert independently into base of proboscis. Esophagus short, from at XVa1 to XV a2. Crop with five pairs of caeca; first four pairs digitiform and laterally directed; last pair with descending sinuous path or post-caeca, from XIX to XXII (Fig. 9A). Intestine with four pairs of digitiform caeca, the first three pairs directed anteriorly and last pair directed posteriorly. Testisacs in four intersegmental pairs, the first pair between XV/XVI, last pair XVIII/XIX. Ejaculatory ducts reaching the first pair of testisacs or XV/XVI (Fig. 9B). Ovisacs saccular, reaching XIX when it is filled (Fig. 9C). Reproductive information. No specimens were observed with spermatophores attached to the body wall, nor were any specimens found carrying eggs or young leeches on the ventral surface. Etymology. The species is named in honor of the Argentinian biologist Dr. Betina Sandra Gullo, in recognition of her significant contributions to the knowledge of South American leeches, in particular those within the genus Helobdella. Figure 9. Internal morphology of a stained paratype specimen of Helobdella gulloae sp. nov. A. Digestive tract; B. Male reproductive system; C. Male and female reproductive systems. Abbreviations: pr = proboscis, sg = salivary glands, cc = crop caeca, pc = post-caeca, int = intestine. 158 ZooKeys 1261: 141–164 (2025), DOI: 10.3897/zookeys.1261.162279 Gerardo Torres-Carrera et al.: Two new species of freshwater leeches of the genus Helobdella Remarks. The morphological characteristics of Helobdella gulloae sp. nov. are consistent with those diagnostic of the genus (Sawyer 1986; Siddall and Borda 2003); including their dorsoventrally flattened body, gonopores separated by a single annulus, one pair of cephalic eyespots and the absence of esophageal organs (bacteriomes). The presence of multiple, longitudinal dorsal stripes and metamerically arranged papilla clearly support its inclusion in the “triserialis” series sensu Sawyer 1986. Helobdella gulloae can be differentiated from other species of the “triserialis” series by difference of patterns of dorsal pigmentation and papillae distribution. Helobdella papillata has three rows of papillae restricted to the posterior third of dorsum (XIX or XX), while in H. gulloae sp. nov., dorsal rows of papillae begin at X. Additionally, H. papillata lacks dorsal pigmentation. In H. lineata, dorsal papillae are irregularly scattered, while in H. gulloae sp. nov. dorsal papillae are regularly arranged. Helobdella transversa lacks both longitudinal pigmented stripes and papillae (Sawyer 1972; Light and Siddall 1999), clearly contrasting with H. gulloae sp. nov. Helobdella lineata exhibits 12–14 longitudinal stripes on the dorsal surface, whereas H. gulloae sp. nov. display ~37. Helobdella fusca and Helobdella virginiae are characterized by irregularly arranged dorsal spots, clearly contrasting with the metameric pattern characteristic of H. gulloae sp. nov. (Table 2). Helobdella gulloae sp. nov. is morphologically and phylogenetically (see below) closely related to H. papilloprocta sp. nov. The presence of two prominent papillae adjacent to the anus in H. papilloprocta sp. nov. appears to be a consistent and reliable character to separate both species. Furthermore, H. gulloae sp. nov. seems to be the only species of the “triserialis” series with only four pairs of testisacs, contrary to five or six pairs, which are more common in this genus. The cox1 genetic distance, calculated under the K2P substitution model, between H. papilloprocta sp. nov. and H. gulloae sp. nov. is 3.33%. This value is comparable to the interspecific distance found between other sister species of Helobdella, such as H. austinensis and H. virginiae or H. socimulcensis and H. farmeri. The remaining pairwise genetic distances are presented in Table 3. Discussion Based on morphology, we describe two new species of Helobdella of the “triserialis” series. Both species differ in both dorsal coloration pattern and annulation. Also, their disparate geographic distribution allows us to consider both as independent species. Helobdella papilloprocta sp. nov. possesses a pair of papillae next to the anus. Additionally, H. gulloae sp. nov. differs from H. papilloprocta sp. nov. by possession of four pairs of testisacs instead of five (Table 2). Both species are sister to each other and have a cox1 genetic distance of 2.8%, a value that might be low, but they are clearly different in both. In addition to the two new species of the genus Helobdella described here, H. austinensis and H. europaea were recorded in Mexico for the first time. Helobdella austinensis was originally described from Austin, Texas, USA and here, we add three Mexican states to its geographic distribution: Nuevo León, Tamaulipas, and Veracruz. Helobdella europaea, on the other hand, is well known for its invasive ability, and here, we include Temixco, Morelos, Mexico, as a new locality for its distribution (Suppl. material 1). 159 ZooKeys 1261: 141–164 (2025), DOI: 10.3897/zookeys.1261.162279 Gerardo Torres-Carrera et al.: Two new species of freshwater leeches of the genus Helobdella Table 3. Cox1 genetic distances among Helobdella species under the Kimura 2-parameter model. New species are indicated in bold. Taxon 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 (1) Helobdella europaea 0.443 (2) Helobdella socimulcensis 3.949 0.330 (3) Helobdella triserialis 11.060 11.746 NA (4) Helobdella gulloae sp. nov. 11.000 11.584 8.522 NA (5) Helobdella papilloprocta sp. nov. 10.596 12.081 9.070 3.334 0.000 (6) Helobdella cf. socimulcensis Chiapas 13.669 14.987 10.807 11.824 11.843 NA (7) Helobdella fusca 16.904 17.555 16.058 17.193 16.800 16.414 NA (8) Helobdella austinensis 18.919 19.621 19.105 18.725 18.879 17.042 22.060 0.483 (9) Helobdella virginiae 17.094 17.568 16.950 17.579 17.704 17.009 21.409 3.789 NA (10) Helobdella lineata 18.305 18.225 18.497 18.594 18.270 16.664 20.985 11.771 12.078 NA (11) Helobdella papillata 17.414 18.047 17.254 17.748 17.143 15.874 18.742 9.745 10.305 6.853 NA (12) Helobdella transversa 17.331 18.366 18.089 18.620 17.634 16.693 20.302 10.286 10.305 8.334 4.753 NA (13) Helobdella cf. robusta 17.131 18.377 17.034 18.186 17.094 15.352 20.049 8.537 9.438 6.261 4.946 5.811 1.276 (14) Helobdella robusta 19.880 20.578 18.066 19.324 18.093 17.541 20.329 11.917 12.041 7.013 8.085 10.605 7.087 NA (15) Helobdella farmeri 4.851 2.884 12.397 10.726 11.579 14.156 16.557 19.765 17.830 19.063 18.618 18.591 18.350 20.284 NA (16) Helobdella cf. socimulcensis Jalisco 3.894 3.230 11.614 11.275 11.665 13.623 17.606 18.854 16.914 17.703 17.453 17.850 18.493 20.017 4.105 NA 160 ZooKeys 1261: 141–164 (2025), DOI: 10.3897/zookeys.1261.162279 Gerardo Torres-Carrera et al.: Two new species of freshwater leeches of the genus Helobdella With the description of two new species of Helobdella from Mexico and the two new country records provided here, a total of ten species of the genus are known in the country: H. elongata, H. atli, H. temiscoensis, H. socimulcensis, H. virginiae, H. octatestisaca, H. papillanis, H. gulloae, H. austinesis, and H. europaea. Conclusion Helobdella is the most speciose leech genus worldwide, with ~80 named species. The majority of this diversity occurs in the New World, especially in the Neotropics, where it is expected that new species remain to be described. In this study, we provide the morphological characterization of two sister species that can be distinguished based on a combination of both morphology and DNA sequences, thereby contributing to our understanding of leech diversity. Acknowledgements This study is part of the requirements for the Doctoral degree of GT-C at the Posgrado en Ciencias Biológicas, UNAM. We thank Secretaría de Ciencia, Humanidades, Tecnología e Innovación (SECIHTI) provided scholarship to GT-C (CVU 814372). Special thanks to the Willi Henning Society for subsidizing the program TNT and making it freely available. We thank Omar Lagunas-Calvo and Uriel Garduño-Montes de Oca for their assistance during fieldwork. We are also grateful to Luis García Prieto for incorporating the voucher specimens into the CNHE, IB-UNAM. Special thanks to members of LANABIO, UNAM: Laura Márquez Valdelamar, Nelly López Ortíz and A. Jiménez for their assistance in generating DNA sequences; Berenit Mendoza Garfias for preparing specimens for SEM and Camila Alcantara for support with multifocal microphotography. Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statement No ethical statement was reported. Use of AI No use of AI was reported. Funding This project was funded by the Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT-UNAM IN215722 and IN226525), granted to AO-F. Author contributions Conceptualization and visualization: G. T-C and A. O-F; Data Curation: G. T-C and L D-DL; Formal analysis: G. T-C; Funding Acquisition: A. 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The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.1261.162279.suppl1