﻿Resurrection and redescription of Clepsinepallida Verrill, 1872 (Hirudinida, Glossiphoniidae) with a phylogeny of the genus Alboglossiphonia

﻿Abstract Alboglossiphoniapallida (Verrill, 1872) comb. nov. is resurrected and redescribed based on morphological and molecular data from specimens of the type locality (New Haven County, Connecticut, USA) that demonstrate it is distinct from North American Alboglossiphoniaheteroclita, European Alboglossiphoniaheteroclita, and Alboglossiphoniapapillosa. Alboglossiphoniapallida is characterized by having dark chromatophores on the dorsal surface arranged lateral to patrilaterally and medially as a thin line or interrupted thin line along with three pairs of eye spots (with the first pair closest together), six pairs of crop ceca, and a united gonopore. Additional sampling of specimens of the genus Alboglossiphonia is needed to understand its phylogeny especially as many species have not been collected since their description.

Hirudo heteroclita was originally described from Europe by Linnaeus (1761) and is characterized by the possession of six eye spots and a translucent body with black spots. Moquin-Tandon (1846) transferred H. heteroclita to the genus Glossiphonia Johnson, 1816. Carena (1820) and Blanchard (1894) stated that this species was very rare. Based upon pigmentation and the comparative distance between the first pair versus the second and third pair of eye spots, Lukin (1976) erected the subgenus Alboglossiphonia containing Glossiphonia (Alboglossiphonia) heteroclita. Klemm (1982) raised Alboglossiphonia to the genus rank, creating the combination Alboglossiphonia heteroclita as the type species.
In North America, Verrill (1872) described Clepsine pallida based on individuals from the West River of New Haven, Connecticut (Fig. 1). Clepsine pallida is characterized by the possession of six eyes and a pale body with scattered black specks and a median light line interrupted by a row of small black spots (Verrill 1872). Verrill (1874) updated the species concept by describing C. pallida Verrill, 1872 as Clepsine pallida var. a, and described Clepsine pallida var. b from New Haven, Connecticut and Colorado, respectively. As described by Verrill (1874), Clepsine pallida var. b is very similar to the North American Glossiphonia elegans (Verrill, 1872), a leech species resurrected by Siddall et al. (2005) and subsequently reaffirmed by Moser et al. (2012) and Mack and Kvist (2019).
In describing Glossiphonia concolor from Europe, Apáthy (1888) mentioned that the species was very similar to C. pallida in North America, but he did not indicate which of Verrill's varieties (var. a or var. b) was the most similar. In European studies, Blanchard (1894) considered G. concolor to be a simple variety of Glossiphonia complanata (Linnaeus, 1758), thus inferring similarity of C. pallida to G. complanata (including Glossiphonia elegans (Verrill, 1872) that was considered a synonym to G. complanata at that time). Castle (1900) synonymized C. pallida with Glossiphonia elegans (Verrill, 1872) while simultaneously recognizing G. heteroclita from the vicinity of Cambridge, Massachusetts. Clepsine pallida was subsequently ignored until Moore (1952) severed the association of C. pallida with G. complanata and determined C. pallida as a junior synonym of Glossiphonia heteroclita (Linnaeus, 1761) (Fig. 1). However, Soós (1969) caused further confusion by listing Clepsine pallida as a synonym of Glossiphonia complanata in his key and comprehensive list of all the species of the family Glossiphoniidae.
Hirudo swampina was described by Bosc (1802) as abundant in the swamps of "Carolina" and attached to turtles or frogs. As described, H. swampina has five eye

Hirudo heteroclita
Linnaeus, 1761 Alboglossiphonia heteroclita Klemm (1982) Clepsine pallida resurrected as  Moore (1952) synonymized by Moore (1952) synonymized by Castle (1900) resurrected by Siddall et al. (2005) reaffirmed by Moser et al. (2012) and Mack & Kvist (2019) as per this study resurrected by Dall (1982) as per this study as per Verrill, 1874 Clepsine pallida spots, a rough dorsum with green varied with brown, and the head, margins, and the posterior are spotted with white. The species description was updated by Moquin-Tandon (1846) and Diesing (1850). Although the species description indicated H. swampina possessed five eye spots, the redescriptions indicated that "five?" (Moquin-Tandon 1846) or six (Diesing 1850) eye spots were found, a character also present in several species of Alboglossiphonia and Glossiphonia. However, the rough dorsum, green/brown coloration, and attachment to turtles and frogs indicate that it is more similar to a species in the genus Placobdella, as species of Alboglossiphonia and Glossiphonia feed on invertebrates. Placobdella hollensis (Whitman, 1892) has up to five pairs of accessory "eyes" and has been found in North Carolina (Moser et al. 2017), and Sawyer (2021) (Bosc, 1802) from two localities in the coastal plain of South Carolina and deposited a neotype in the National Museum of Natural History (USNM 47122), Smithsonian Institution. Sawyer (1973) stated that G. swampina is distinct from the unpigmented, translucent G. heteroclita, because G. swampina has four to seven mid-dorsal pigment bars. However, such a pigmentation pattern also occurs in C. pallida (Fig. 2). Additional specimens of G. swampina were found in the coastal plain of North Carolina by Sawyer and Shelley (1976). Klemm (1976) suggested that G. swampina is a color variant of G. heteroclita, and after examining specimens from Quebec and Maryland, Klemm (1982) declared G. swampina a junior synonym of A. heteroclita.
In recent phylogenetic studies, Trontelj et al. (1999) used A. heteroclita from Italy and Apakupakul et al. (1999), Light and Siddall (1999), and Siddall et al. (2005) used A. heteroclita from Michigan, Jueg (2008) used A. heteroclita from Germany andMichigan, andBolotov et al. (2019) used A. heteroclita from Michigan and A. papillosa from Russia as a basis for molecular studies. However, A. heteroclita from Europe and A. heteroclita from North America have not been compared to A. papillosa nor to specimens of C. pallida in a molecular analysis.
The convoluted history of this assemblage is given in Fig. 1. In this study, we provide a molecular comparison of contemporary specimens that are morphologically consistent with the C. pallida of Verrill (1872) and C. pallida var. a of Verrill (1874) collected from the type locality of West River, Connecticut to specimens identified as A. heteroclita from Michigan, USA and from Germany, providing the basis for a redescription, resurrection, and molecular characterization of C. pallida.

Collection of leeches and morphological analysis
During the course of a survey of the leech fauna of south-central Connecticut, individuals matching the description of Clepsine pallida Verrill, 1872 were collected by hand from submerged substrate in the West River, New Haven, New Haven County, the type locality of C. pallida. Specifically, collections were made from the West River at Konolds Pond (41°20'52.1"N, 72°58'41.6"W) and Whalley Avenue Bridge (41°19'30.13"N, 72°57'26.76"W) south to the "Duck Pond" (41°18'51.30"N, 72°57'21.75"W) as illustrated on page 12 of Shumway and Hegel (1990) and Clark's Pond (41°24'47.9"N, 72°53'46.8"W) between May 2008 and September 2009, and later in September 2020 and July 2021. A collection was also made from Sturges Pond (41°11'50"N, 73°18'2"W), Larsen Sanctuary, Fairfield County Connecticut on 27 July 2021. Specimens were relaxed, examined, and fixed as described by Moser et al. (2006). Several specimens were pressed, stained with Semichon's acetocarmine and mounted in Canada Balsam for examination by light microscopy according to techniques outlined by Richardson (2006), as modified by Richardson and Barger (2006). Specimens were examined using an Olympus SZX16 dissecting microscope and were photographed with a Zeiss Stemi 2000-CS macroscope fitted with a Q-Capture 5.0 RTV Micropublisher camera. Images were acquired at different focal levels and the resulting stacks rendered with Helicon Focus 7 Pro to make an extended focus image. Post-processing was done using Adobe Photoshop CC 2015. Terminology for plane shapes follows Clopton (2004). Specimens were deposited in the Peabody Museum of Natural History (YPM), Yale University, New Haven, Connecticut, USA and the National Museum of Natural History (USNM), Smithsonian Institution, Washington, District of Columbia, USA.

DNA and phylogenetic analysis
Molecular analyses were conducted on newly collected material according to Richardson et al. (2010) as follows: DNA was isolated from the caudal suckers of five individual leeches (YPM IZ 058354, YPM IZ 062698, YPM IZ 109351-109353) with the DNeasy Blood & Tissue Kit from Qiagen (cat. no. 69504), following the protocol given for the purification of total DNA from animal tissues (spin-column). For the proteinase K treatment step, tissue samples were lysed overnight at 56 °C. DNA was eluted from the spin columns with 150 µl of buffer.
Polymerase chain reactions (PCR) were prepared using the Illustra PuRe Taq Ready-To-Go PCR beads from GE Health Care (cat. no. 27-9559-01). Primers were purchased from Invitrogen and were comprised of two primers each for cytochrome c oxidase subunit I (COI) as specified by Folmer et al. (1994) and Light and Siddall (1999). Specifically, the COI primers were LCO1490 (5'-GGTCAACAAATCATAAA-GATATTGG-3') and HCO2198 (5'-TAAACTTCAGGGTGACCAAAAAATCA-3'). Final volume of PCR reactions was 25 µl with 2 µl of leech genomic DNA added per reaction. DNA was amplified under the following PCR conditions: 94 °C for 5 min; 35 cycles of (94 °C for 30 s, 50 °C for 30 s, 72 °C for 45 s); 72 °C for 7 min. Following PCR, samples were cleaned up using a QIAquick PCR purification kit from Qiagen (cat. no. 28104).
Purified PCR products were sequenced using the HCO2198 and LCO1490 primers for the COI products by the W.M. Keck Foundation Biotechnology Resource Laboratory at Yale University. DNA sequences were edited and assembled using Geneious Prime (v. 2020.1.2, Biomatters Ltd.). Novel sequences were deposited in GenBank (Benson et al. 2018; Table 1). Comparable sequence data for seven recognized Alboglossiphonia species (24 sequences), sequences identified as Alboglossiphonia sp. (three individuals), Glossiphonia complanata (two individuals), and Glossiphonia elegans (two individuals) were downloaded from GenBank (Table 1). Additionally, five sequences identified as A. heteroclita were downloaded from BOLD (Ratnasingham and Hebert 2013; Table 1). The COI sequences were aligned using the MAFFT multiple sequence alignment plug-in for Geneious Prime (Katoh and Standley 2013) with default settings, checked by eye for gaps, and the sequences were translated to amino acids to assess sequence quality. Uncorrected pairwise sequence distances were calculated using Geneious Prime.
The best partitioning scheme was tested using ModelFinder within IQ-TREE (Kalyaanamoorthy et al. 2017) using the -m MF+MERGE command, as well as estimation of substitution models by codon position, resulting in the following models as best fit by partition by the Bayesian information criterion: first codon position = F81+F, second codon position = TN+F+I, and third codon position = K3Pu+F+G4. A maximum likelihood (ML) analysis was performed with IQ-TREE v. 1.6.12 (Nguyen et al. 2015), using the models suggested for each unlinked partition, the -spp command to allow each partition to have its own evolutionary rate, and 1,000 ultrafast bootstrap (UFBOOT2) approximations (Hoang et al. 2018). Glossiphonia complanata (AY047321 and HM246608) and Glossiphonia elegans (JQ073858 and JQ73860) served as outgroups. FigTree v. 1.4.4 (Rambaut 2018) was used to visualize trees that were then edited with Adobe Illustrator Creative Cloud (https://www. adobe.com).

Morphological analysis
Examination of the type series of Clepsine pallida (YPM IZ 00253) revealed a single specimen (Fig. 2). The more than 150-year-old holotype specimen is remarkably well preserved, but the pigmentation has faded and the eye spots are no longer discernible. The dorsal surface is smooth and there is a united gonopore. The original YPM Invertebrate Zoology Annelida ledger entry for YPM IZ 00253 indicates a single specimen of Clepsine pallida V. collected from West River, New Haven, Connecticut on 6 June 1871 and is labeled as type.
In further examination of the YPM Annelida Ledger, Clepsine pallida var. a and var. b of Verrill (1874) had not been assigned a catalog number. No specimens of Clepsine pallida var. a were found, and Verrill's (1874) account likely refers only to the holotype specimen, YPM IZ 00253. In Verrill (1874), Clepsine pallida var. b came from Colorado (US Geological and Geographic Survey of the Territories, i.e. Hayden's expedition) and again, Colorado (lake near Long's Peak, 9,000 feet elevation, Hayden's expedition, 1873). In the YPM uncataloged leech holdings, two lots were recently discovered that are likely the syntypes of Clepsine pallida var. b. One lot (now YPM IZ 106808) had an original label in Sidney Smith's handwriting that reads "Colorado Mts. & Plains, 1873" and another label in A.E. Verrill's handwriting as "Clepsine pallida var. b Colorado Mts. & Plains Haydens Exp. 1873." On the second label, pallida has been crossed out and "elegans" has been written in Verrill's handwriting, indicating an updated identification as Clepsine elegans. The single specimen (YPM IZ 106808) is in very good condition and morphologically consistent with Glossiphonia elegans (six eye spots; pair of dark paramedial lines; two pair of metameric white dots).
The second lot (now YPM IZ 106809) had a label in J. Percy Moore's handwriting as "Clepsine pallida Verrill, near Longs Peak, 9000 ft, Haydens Exp" with a reidentification as Glossiphonia complanata (Linnaeus) and a label written by former Yale Curator of Invertebrate Zoology Willard Hartman as "Glossiphonia complanata (Linn) Lake near Long's Peak, 9000 ft., Hayden's Expedition, 1873; Verrill's Ident: Clepsine pallida" -no Verrillera label was found. This information matches Verrill (1874) of Clepsine elegans var. b. YPM IZ 106809 containing three specimens of average condition which have likely dried out and subsequently been rehydrated without benefit of a restorative surfactant. Two of the specimens are morphologically consistent with Glossiphonia elegans (six eye spots and pair of paramedial dark lines). The third specimen is smaller and difficult to discern. Sawyer (1973) designated a neotype (USNM 47122) and an additional specimen (USNM 51436) of Glossiphonia swampina (Bosc, 1802) from French Quarter Creek, Clement's Ferry Road, Berkeley County, South Carolina at the National Museum of Natural History, Smithsonian Institution. The pigmentation has faded, but both specimens had small transverse bands (primarily in the medial region), six eye spots, and a united gonopore. In light of the findings in this study, G. swampina needs to be reassessed with molecular data. We conclude that Hirudo swampina, as described and illustrated by Bosc (1802)

Figs 2-5
Diagnosis. Dark chromatophores on the dorsal surface arranged lateral to patrilaterally and medially as a thin line or interrupted thin line along with three pair of eye spots (where the first pair are closest together), six pair of crop ceca, and a united gonopore.
External morphology. Body narrowly ovoid to narrowly pyriform. Rounded anterior region. Dorsum buff to translucent, smooth (without papillae), and with small, black chromatophores that form thin lines with scattered areas; thin, interrupted middorsal line with larger chromatophore patches (typically on sensory annuli); black chromatophores in a lateral pattern on the sensory annulus of the lateral to paralateral region (Figs 2, 3). Three pair of eye spots which are typically separate and arranged   linearly or with groupings of two and four eye spots in unpigmented cephalic area with the first pair of eye spots closest together (Figs 3, 4). Some individuals have five eye spots where the first pair is present and there are only three eye spots in the second and third pair. Caudal sucker of moderate size (half diameter of mid-body) without pigment or papillae. Ventrum without pigment or papillae and with united male and female gonopores (single opening) (Fig. 2).
Alimentary tract. Cylindrical, blunt-tipped protrusible proboscis (approximate length of 14 annuli), opening at the center of the oral sucker. Short esophagus and diffuse salivary glands that are distributed in the anterior third of the body (Fig. 5). Crop with six pair of ceca and last pair extend posteriad and diverticulated with four sections; four pair of simple, saccular intestinal ceca with hind gut saccate and rectum opening into anus, located one annulus anteriad of the caudal sucker (Fig. 5).
Reproductive anatomy. Male atrium opening into paired narrowly ovoid atrial cornua that extends laterally and narrows abruptly at junction with ejaculatory ducts and extends posteriad (Fig. 5). Six pair of testisacs between crop ceca. Pair of tubular ovisacs; length of ovisacs dependent on the reproductive state of the leech (Fig. 5). Male and female gonopores united.
The molecular dataset included COI sequences of 41 specimens (37 members of Alboglossiphonia and two specimens each of Glossiphonia complanata and Glossiphonia elegans that served as outgroups; Table 1) and a total of 631 aligned characters. The log-likelihood of the topology was −3174.987 and the topology is shown in Fig. 6.
Alboglossiphonia pallida was strongly supported by morphological and molecular evidence as a species within the genus Alboglossiphonia and distinct from North American A. heteroclita, European A. heteroclita, A. lata, A. weberi, A. iberica, and A. papillosa. Alboglossiphonia pallida is characterized by having dark chromatophores on the dorsal surface arranged lateral to patrilaterally and medially as a thin line or interrupted thin line along with three pair of eye spots (where the first pair are closest together, the defining characteristic of the genus Alboglossiphonia), six pair of crop ceca, and a united gonopore. The non-monophyly of A. heteroclita continues to pose a challenge. The A. heteroclita specimens from North America were 11.69-12.91% different from A. heteroclita from Europe, indicating that the North American specimens are not A. heteroclita and most likely represent an undescribed species. The A. heteroclita specimens from North America were 0.33-3.07% different from one another. These specimens form a strongly supported clade with A. pallida (bs = 100), although the North American A. heteroclita specimens as a group were 5.78-8.35% different from the A. pallida specimens. North American specimens assigned to A. heteroclita are typically characterized by a lack of pigmentation on translucent bodies (Sawyer 1972(Sawyer , 1973Klemm 1982;Moser et al. 2016). In North America, A. cf. heteroclita has been reported in the Great Lakes region and as far west as Nebraska in the USA and as far west as British Columbia in Canada (Klemm 1982;Moser 1991). Further collection is needed to elucidate the taxonomy and geographic distribution of the North American Alboglossiphonia specimens that were identified as A. heteroclita and its relationship with A. pallida.
In Europe, A. heteroclita had been a heterogenous concept and known more by the infraspecific varieties. These synonymies have since been elevated to the species rank with A. hyalina (O.F. Müller, 1774) having yellow chromatophores and no dark chromatophores and A. striata (Apáthy, 1888) with dark, transverse pigmentation (Lukin 1976;Nesemann and Neubert 1999;Jueg and Grosser per. comm.). As described and figured by Braun (1805), A. papillosa (Braun, 1805) has dark medial spots and some scattered dark chromatophores. This description is consistent with the description of A. heteroclita (Linnaeus, 1761) (Nesemann and Neubert 1999;Jueg and Grosser per. comm.). Alboglossiphonia heteroclita from Germany was 12.16-12.87% different than A. papillosa collected from Russia. As photographed by Klass et al. (2018), the specimen of A. papillosa has dark dorsal lines and is potentially a previously undescribed species.
Sequences of specimens from Asia, Australia, and Hawaii form a strongly supported clade, except for a single sequence from South Korea (MN503262). Sequences of A. lata form a clade with short internodes that were poorly supported for the most part. The clade predominantly consisted of sequences from South Korea, yet also included a single sequence from Primorsky Krai, Russia (MN295414) and the sequence of A. weberi from Hawaii, USA (AY962453). Alboglossiphonia lata is a widely distributed species that is considered invasive and spread via the aquatic plant trade. In particular, the specimen of A. weberi from Hawaii should be reexamined to determine if this might be an occurrence record of the invasive A. lata, which would be concerning for the Hawaiian island ecosystem. The sequences of Alboglossiphonia from Myanmar and Australia are supported as members of the genus and likely represent species distinct from one another and not otherwise represented in this analysis or publicly available databases (e.g., GenBank, BOLD), yet the specimens need to be examined to determine the species identification as there have been seven described species from Australasia and Oceania.
The sequence of Alboglossiphonia quadrata (AY962455) from Namibia has likely been assigned the incorrect name. Oosthuizen (1987) transferred the species name quadrata to the genus Hemiclepsis. This sequence is highly supported as a lineage within Alboglossiphonia and the specimen needs to be reexamined to determine if it belongs to one of the seven species of Alboglossiphonia described from Africa (Gouda 2010).
In this study, COI was largely successful at distinguishing congeners of Alboglossiphonia, but it had limited utility in resolving the relationships between species. Combining COI data with other loci, especially nuclear loci, is needed to determine relationships between glossiphonid species with confidence. The addition of sequences of more Alboglossiphonia species will improve our understanding of relationships within the genus. This study included all publicly available Alboglossiphonia sequences, although this represents only about one-third of the diversity of the genus.

Conclusion
Alboglossiphonia pallida (Verrill, 1872) is resurrected and redescribed based on morphological and molecular data that demonstrate it is distinct from the specimen assigned to A. heteroclita from Michigan and Wisconsin and A. heteroclita from Europe, as well as other species of Alboglossiphonia. Additional sampling of Alboglossiphonia is needed to understand its phylogeny especially as many species have not been collected since their original description. logistic insight. We are grateful to Dr Christian Albrecht, Justus-Liebig University, Giessen, Germany, and to Uwe Jueg, Ludwigslust, Germany, for providing the sequence data of Alboglossiphonia heteroclita from Germany and A. iberica. We thank Dr Milan Bull, Connecticut Audubon Society, for permission to collect at Sturges Pond, Larsen Sanctuary, Fairfield, Connecticut. We greatly appreciate Uwe Jueg and Clemens Grosser, Elstertrebnitz, Germany, for their insightful comments on the genus Alboglossiphonia. The State of Connecticut, Department of Environmental Protection provided permission to collect leeches. Funding for this project was provided in part by the Department of Biological Sciences and Schools of Arts and Sciences and Health Sciences at Quinnipiac University, Hamden, Connecticut.