A new species of medicinal leech, Macrobdella mimicus n. sp., is described from specimens collected in Maryland; this is the first description of a North American macrobdellid since 1975. Superficially, the new species resembles the well-known Macrobdella decora, as both species possess 4 accessory pores arranged symmetrically on the ventral surface, yet the new species is distinguished from M. decora in possessing 4–4½ annuli (rather than 3½) between the gonopores and 4 annuli (rather than 5 annuli) between the female gonopore and the first pair of accessory pores. Phylogenetic analyses, based on 2 mitochondrial and 2 nuclear loci for a set of closely related taxa, confirms the placement of the new species within the family Macrobdellidae and places it as the sister taxon to M. decora and M. diplotertia.
The leech genus Macrobdella Verrill, 1872 includes 4 species of jawed blood-feeding leeches exclusively restricted to freshwater ecosystems (Sawyer, 1986; Moser et al., 2016): Macrobdella decora (Say 1824), Macrobdella sestertia Whitman 1886, Macrobdella ditetra Moore 1953, and Macrobdella diplotertia Meyer 1975. Members of Macrobdella are known as the New World medicinal leeches as a reflection of their blood-feeding behavior, using vertebrates, including humans, as hosts. Although Macrobdella species have not been widely used for bloodletting (Munro et al., 1991, 1992), decorsin, an inhibitor of platelet aggregation, was characterized from the saliva of M. decora (Seymour et al., 1990). Recently, analysis of the salivary transcriptome of the species revealed the expression of a suite of polypeptides related to anticoagulation that facilitate blood feeding (Min et al., 2010; Kvist et al., 2013). Members of Macrobdella are easily recognizable by their olive-green dorsum with black flecks and red-orange metameric spots (in some species), and conspicuous external accessory glands on the ventral surface posterior to the female gonopore (Sawyer, 1972, 1986; Klemm, 1982).
Previous phylogenetic analyses that have included members of Macrobdella found strong support for the monophyletic status of the genus and recovered M. ditetra as sister to a clade formed by M. decora and M. diplotertia (Phillips and Siddall, 2005, 2009), albeit with shallow sampling of the species and corresponding geographic distribution. Macrobdella sestertia has yet to be included in phylogenetic analyses based on molecular data because of the lack of fresh tissue with viable DNA (Phillips et al., 2016).
Members of Macrobdella are geographically distributed in North America east of the Rocky Mountains (Klemm, 1982; Sawyer, 1986). Of the 4 species, M. decora exhibits the broadest distribution, having been reported from temperate freshwater habitats throughout the northern United States, and southern Canada east of the Rocky Mountains (Klemm, 1982), except for a single disjunct population in Nuevo León, Mexico (Caballero y Caballero, 1952). Macrobdella sestertia is only known from New Hampshire, Maine, Massachusetts, and South Carolina (Smith, 1977; Smith and Hanlon, 1997; Phillips et al., 2016; Poly, 2018), M. diplotertia occurs throughout Arkansas, Kansas, and Missouri (Connior and Trauth, 2010), and M. ditetra inhabits the Atlantic Coastal Plain ecoregion of the southeastern United States from eastern Texas to Florida and northwards as far as North Carolina (Meyer, 1975; Phillips and Siddall, 2005).
During a reassessment of species delimitation among Macrobdella species, specimens of a previously unrecognized species of Macrobdella were found. Herein, we present the description of the new species based on morphological data from stereomicroscopy, dissection, and scanning electron microscopy and investigate its phylogenetic placement based on analyses of DNA sequence data.
MATERIALS AND METHODS
Six leech specimens were collected in freshwater wetlands in eastern Maryland (038°23′40.3″N, 77°14′23.4″W) in 2014 and 2015. Collectors waded into the water and periodically examined their legs for attached leeches to collect; investigators also used dip nets. Leeches were transported to the laboratory in plastic containers with water from the habitat and relaxed by the gradual addition of 70% ethanol; all specimens were fixed in 95% ethanol. Additionally, individuals of the new species were also collected in Georgia and South Carolina in 2016 and North Carolina in 2017 (Table I). Examination of external and internal morphology was accomplished with Zeiss® Discovery V8 (Carl Zeiss Microscopy, LLC, Thornwood, New York) and Leica® M125 stereomicroscopes (Leica Microsystems, Inc., Buffalo Grove, Illinois). Photographs were taken with a Leica® IC80 HD camera (Leica). Drawings were made by the superposition of vector art over images placed in Adobe Illustrator® CS6 and Adobe Photoshop® CS6 (Adobe, Inc., San Jose, California). Somites are indicated by Roman numerals (e.g., prostomium is I) and annuli in each somite by alphanumeric designations following Castle (1900) and Moore (1900). The holotype and 3 paratypes were deposited at the National Museum of Natural History (NMNH), Smithsonian Institution in Washington, DC, and 2 paratypes were deposited in the Colección Nacional de Helmintos (CNHE) at Universidad Nacional Autónoma de México, México. Additional specimen lots from natural history museum collections were examined and the identifications redetermined to be the new species: 3 specimen lots at the NMNH, including a single specimen lot from the U.S. National Parasite Collection (USNPC); 7 specimen lots at the North Carolina Museum of Natural Sciences (NCSM) in Raleigh, North Carolina; and 4 specimen lots from the Virginia Museum of Natural History (VMNH) in Martinsville, Virginia (Table I).
Scanning electron microscopy (SEM) was used to examine the jaw morphology of 2 individuals of the new species (USNM 1532253 and USNM 1532306). The anterior end of 2 leeches of the new species were removed and dissected to expose the jaws and the entire piece was then prepared for SEM. Each sample was placed in a solution of 4% glutaraldehyde and distilled water for 15 days, followed by a rinse in deionized water for 10 min. The samples were transferred to 1% osmium tetroxide solution overnight, and then rinsed 3 times in deionized water for 30 min, and twice for 15 min. The samples were dehydrated in a graded ethanol series (15 min at each stage), consisting of 15% ethanol, 25% ethanol, 50% ethanol, and left overnight in 75% ethanol, and for 20 min each in 85% ethanol, 95% ethanol, and twice for 20 min in 100% ethanol. The samples were transferred to hexamethyldisilazane (HMDS; Ted Pella, Inc., Redding, California) for 15 min and then allowed to air dry in a fume hood. Each sample was mounted on an aluminum stub with double-sided carbon tabs (Ted Pella, Inc.) and then sputter-coated with 30 nm of gold palladium and examined with a Philips XL-30 ESEM scanning electron microscope (ThermoFisher Scientific, Waltham, Massachusetts). The number of teeth (denticles) per jaw was assessed for both samples by counting teeth craters that were evenly spaced along the ridge of the jaw; where mucus obscured the craters, an estimate of the number of teeth was extrapolated from the number of visible craters of approximately the same distance.
Total genomic DNA was extracted from tissue of the caudal sucker of 11 individuals; this tissue was selected in order to avoid contamination of host DNA from the gastric or intestinal regions. DNeasy® blood and tissue kit (Qiagen, Inc., Valencia, California) was used for tissue lysis and DNA purification. Partial sequences of the mitochondrial cytochrome c oxidase subunit I (COI) and nicotinamide adenine dinucleotide dehydrogenase subunit I (ND1), as well as the nuclear ribosomal DNA of the small (18S rDNA) and large subunits (28S rDNA) of the new species were obtained with the use of the primers detailed in Phillips et al. (2010) for COI, 18S rDNA, and 28S rDNA, and from de Carle et al. (2017) for ND1, in order to investigate the phylogenetic position of the new species. Additionally, three specimens of M. decora from the Royal Ontario Museum in Toronto, Ontario, Canada (ROMIZ I10058, ROMIZ I10059, ROMIZ I10060) were sequenced and included in the present study. Polymerase chain reaction (PCR) was performed in 10-μl reactions with 1 μl of genomic DNA and final concentrations of 3 μmol of each primer, 500 μM dNTPS, 3mM MgCl, 0.25 mg/μl of BSA (bovine serum albumin), and 0.05 U/μl of BiolaseTM DNA polymerase (Bioline, Inc., Memphis, Tennessee) with buffers provided by the manufacturer. PCR reactions were performed in an Applied Biosystems 2720 Thermal Cycler (Applied Biosystems, Foster City, California) under the following thermal profiles: for COI, 95 C (5 min), followed by 35 cycles of 95 C (30 sec), 49 C (30 sec), 72 C (30 sec), and final extension at 72 C (7 min); for 18S rDNA and 28S rDNA, 95 C (5 min), followed by 35 cycles of 95 C (30 sec), 52 C (30 sec), 72 C (30 sec), and final extension at 72 C (7 min). ExoSAP-IT® (Affymetrix/ Thermofisher Scientific) was used to purify the PCR products, and cycle sequencing was performed with BigDye® Terminator v3.1 (Applied Biosystems). Cycle-sequenced products were purified using SephadexTM G-50 Fine (GE Healthcare, Chicago, Illinois), and DNA sequencing was performed on an ABI 3730 at the Laboratories of Analytical Biology (LAB) at the National Museum of Natural History, Smithsonian Institution. Contig assembly and sequence editing was performed using Geneious version 10.2.3 (http://www.geneious.com; Kearse et al., 2012). Comparative DNA sequence data for species of the genus Macrobdella, representatives of other related genera, and the outgroup, Limnatis paluda (Tennent, 1859), were downloaded from GenBank (Table II; Phillips and Siddall 2005, 2009; Oceguera-Figueroa et al., 2012). Sequence alignment of each partition was accomplished with the use of the online version of MAFFT ver. 7 (Katoh and Standley, 2013) applying default settings and the final concatenated dataset was created by Mesquite ver. 3.04 (Maddison and Maddison, 2015).
Phylogenetic analyses employed both maximum-likelihood (ML) and parsimony approaches. A total of 11 individuals of the new species, 6 from the type locality, were included and the data set was augmented by 17 terminals belonging to both Macrobdellidae and Praobdellidae; for this purpose, data for all loci were downloaded from GenBank (see Table II); all trees were rooted with L. paluda in accordance with the results recovered by Phillips and Siddall (2009). For the ML analysis, PartitionFinder ver. 1.1.1 (Lanfear et al., 2012) was used both to optimize the partitioning scheme and to estimate the best-fitting model of nucleotide evolution, with the use of the “greedy” search algorithm and the Akaike Information Criterion (AIC). All loci were separately assessed and codon positions for protein coding sequences (COI and ND1) were also proposed as partitions. Subsequently, a ML analysis was carried out using RAxML ver. 8 (Stamatakis, 2014) on the CIPRES online platform (Miller et al., 2010). Tree searches consisted of 1,000 iterations with 25 initial GAMMA rate categories and final optimization with the use of 4 GAMMA shape categories. Support values were estimated through 1,000 pseudoreplicates of the rapid bootstrap algorithm. The parsimony analysis was conducted in TNT ver. 1.5 (Goloboff et al., 2008), employing a New Technology search for 1,000 iterations with 5 rounds of ratcheting and tree fusing and finally, 3 rounds of sectorial searches after the initial Wagner tree builds. Support values were estimated from 1,000 pseudoreplicates using standard bootstrapping, applying the same settings as mentioned above.
Uncorrected p-distances for the COI locus within the entire data set were calculated in PAUP* ver. 4.0b10 (Swofford, 2002) applying the following settings: function of minimal evolution, ignoring gaps for affected pairwise comparisons (although no gaps were present in the resulting alignments) and equal rates for variable sites.
Sawyer and Pass, 1972, partim—Sawyer, 1973, partim
Diagnosis (based on 6 specimens [4 dissections])
Macrobdellid in general aspect. Somite XXIV quinquannulate. Four complete annuli between gonopores. Two rows of 2 accessory pores each in furrows, first pair situated 4 complete annuli posterior to female gonopore. Fifty-six to fifty-nine denticles per lateral jaw.
Body up to 86 mm length (n = 6; Fig. 1A, B) and 15 mm maximum width. Somites I–III uniannulate; somites IV, V biannulate; somite VI, VII triannulate; somite VIII quadrannulate; somites IX–XXIV quinquannulate (complete), somite XXV triannulate, somite XXVI biannulate. Annuli of somites VI a3, VII a1, a3, and VIII a1, slightly subdivided, longer than adjacent annuli. Dorsum olive green, median field dark green; 1 pair lateral slender broken dark green lines extending from somite V to somite XXVII; median longitudinal line of 22 reddish orange spots, 1 pair submarginal longitudinal lines of black flecks, on a2 of somites V–XXVII. Ventral surface reddish to orange in life, pale yellow when preserved, occasional black flecks. Clitellum inconspicuous, from X b5 to XII a2. Five pairs of eyespots arranged in parabolic arc on somites II, III, IV a1, V a1, VI a2; 1 annulus separating annuli with third and fourth pairs of eyespots and 2 annuli separating annuli with fourth and fifth pairs of eyespots. One pair of ventral nephridiopores on posterior margin of each b2 from somites VIII–XXIV, 17 pairs total. Male gonopore on somite XII b1. Female gonopore on somite XIII b1, occasionally in furrow of somite XIII b1/b2. Four complete annuli between gonopores. Two rows of 2 accessory pores; first pair in furrow of somite XIII/XIV, second pair in somite XIV in furrow of annuli b2/a2. Four complete annuli between female gonopore and first pair of accessory pores (Fig. 1C, D). Anus located on the dorsal surface at XXVII a3.
Three well-developed jaws, one dorsal and two ventrolateral; monostichodont (Fig. 2). Each jaw armed with 56–59 denticles. Pharynx short. Crop from somite VIII to somite XIX. Crop in somites VIII and IX with single pair of lateral caeca. Somites X–XII with 2 pairs of equal caeca, XIII and XIV with anterior pair shorter (posterior pair slightly bilobed), XV–XVII 2 pairs equal size, XVIII anterior pair larger and posteriorly directed, posterior pair reduced. Somite XIX with pair of large postcaeca or diverticula extending to XXIV. Intestine simple, acaecate tube from somite XX to XXVII.
Testisacs 10 pairs at interganglionic intervals, from somites XIII/XIV to XXII/XXIII, first pair embedded in accessory gland. Sperm ducts forming anterio-lateral loop, in somites XI–XII. Epididymes compact, tightly coiled, mostly in somite XI (Fig. 3). Ejaculatory ducts widening slightly proximally. Male atrium compact, covered by pigmented tissue, cross-hatched muscle fibers near base. Ovisacs simple, spheroidal. Oviducts insert directly into anterior of vagina. Accessory gland posterior to female gonopore, in somites XIII–XIV; size highly variable, largest observed deeply lobed, overtaking female gonopore, extending from posterior of somite XII to somite XIV, smallest observed extending from posterior of somite XIII to anterior of somite XVI.
Holotype National Museum of Natural History, Smithsonian Institution USNM 1532255: fixed and preserved in 95% ethanol, dissected. Length 79 mm, maximum width 11 mm. Paratypes: USNM 1532253 and USNM 1532254, 1 specimen each specimen lot; Colección Nacional de Helmintos, Mexico CNHE 10262 and CNHE 1123, 1 specimen each.
Additional material examined
USNM 1532261: 1 individual from extension of Thorne Gut Marsh, Nanjemoy, Charles Co., Maryland (038°23′40.3″N, 77°14′23.4″W); USNM 1532304: 6 individuals from Walnut Creek wetlands, Chicopee Woods/Elachee Nature Preserve, Hall Co., Georgia (34°14′45.0″N, 83°48′40.3″W); USNM 1532305: 7 individuals from Nancy Town Lake, Chattahoochee National Forest, Habersham Co., Georgia (34°30′04.4″N, 83°28′55.7″W); USNM 1532306: 25 individuals from Lake Wattacoo, Ashmore Heritage Preserve, Greenville Co., South Carolina (35°05′11.8″N, 82°34′45.5″W); USNM 1532307: 6 individuals from Bunched Arrowhead Heritage Preserve, Greenville Co., South Carolina (34°59′33.0″N, 82°24′28.1″W); USNM 1532309: 10 individuals from Clemson Experimental Forest, Pickens Co., South Carolina (34°44′53.6″N, 82°51′29.7″W); USNM 1532302: 20 individuals from Stone Mountain State Park, Alleghany Co., North Carolina (36°23′09.8″N, 81°01′44.5″W); USNM 1532303: 19 individuals from Stone Mountain State Park, Alleghany Co., North Carolina (36°23′07.9″N, 81°01′41″W); specimens of NCSM and VMNH (Table I).
USA, Maryland, Charles County, Nanjemoy, extension of Thorne Gut Marsh, swamp by the bridge on Riverside Road (Highway 224) (038°23′40.3″N, 77°14′23.4″W) on 19 July 2015.
Known from eastern North America, ranging from northern Georgia, through North and South Carolina, Virginia, and Maryland to Long Island, New York.
The specific name mimicus (“imitator” or “actor” in Greek) refers to the same number and arrangement of accessory pores in the new species and M. decora.
Macrobdella mimicus n. sp. belongs in the genus Macrobdella because of its possession of a single row of more than 50 teeth per lateral jaw, ventral accessory pores symmetrically arranged posterior to the female gonopore, and a male median region. This suite of characters is exclusive to the genus and clearly different from the other members of Macrobdellidae sensu Phillips et al. 2010 (i.e., Philobdella and Oxyptychus). The morphology of M. mimicus n. sp. most closely resembles that of M. decora, yet M. mimicus n. sp. differs from M. decora in its possession of 4–4½ annuli between the gonopores, rather than 3½ annuli, 4–4½ annuli between the female gonopore and the first pair of accessory pores, rather than 5 annuli, 56–59 teeth per jaw rather than approximately 65 teeth, and the quinquannulate nature of somite XXIV, rather than quadrannulate. Historically, the number and arrangement of accessory pores and the number of annuli between gonopores has been used to distinguish congeners of Macrobdella (Klemm, 1982; Sawyer, 1986; Phillips et al., 2016). Macrobdella sestertia has 24 accessory pores arranged in 4 transverse rows of 6 pores each and gonopores separated by 2½ annuli. Macrobdella ditetra exhibits 8 accessory pores arranged in 2 transverse rows of 4 pores each and gonopores separated by 2 annuli. Macrobdella diplotertia presents 3 transverse rows of 2 pores each for a total of 6 accessory pores and gonopores separated by 4½–5 annuli. Therefore, the new species is readily distinguished from M. sestertia, M. ditetra, and M. diplotertia based on the number and arrangement of accessory pores.
Uncorrected p-distances for mitochondrial COI gene fragments (28 sequences) are presented in Table III. Among Macrobdella species, pairwise distances of COI sequences were: 6.23–6.55% (avg. 6.42%) between M. mimicus (11 sequences) and M. decora (3 sequences), 8.36–8.78% (avg. 8.62%) between M. mimicus and M. diplotertia (1 sequence), and 8.32–9.47% (avg. 8.94%) between M. mimicus and M. ditetra (3 sequences). Variation within Macrobdella species range from 0 to 0.91% (avg. 0.5%). Pairwise distances of COI sequences comparing M. mimicus to the Philobdella species were 11% between M. mimicus (3 sequences) and Philobdella floridana, and 12% between M. mimicus (3 sequences) and Philobdella gracilis.
The combined COI, ND1, 18S rDNA, and 28S rDNA data set contained 28 terminals with 3,903 aligned characters. PartitionFinder suggested 3 partitions for the data set: (1) 18S rDNA, 28S rDNA, first codon position of COI, (2) second codon position of COI and second codon position of ND1, (3) third codon position of COI, first codon position of ND1, and third codon position of ND1. In addition, PartitionFinder suggested that the GTR+I+Γ model be used for partition 1 and the GTR+ Γ model for partitions 2 and 3. Maximum-likelihood analysis of the combined data set produced a topology (Fig. 4) with a log-likelihood of −11611.345009. Parsimony analysis of the combined data set recovered 12 most parsimonious trees with 1,233 steps (Consistency Index = 0.73; Retention Index = 0.73) and both optimality criteria recovered identical topologies with the exception of a polytomy within the M. mimicus clade retrieved by parsimony and the relative placement of the Philobdella clade. In both trees, specimens of M. mimicus formed a well-supported clade (likelihood bootstrap support [LBS] = 98, parsimony bootstrap support [PBS] = 100), which placed as the sister group to M. decora (monophyletic with LBS = 100, PBS = 100). Both methods recovered a strongly supported monophyletic group of North American members of Macrobdellidae, including species of Macrobdella and Philobdella (LBS and PBS = 99); however, they strongly differ in the placement of the monophyletic Philobdella (LBS = 100, PBS = 99). The maximum-likelihood analysis recovered Philobdella as the sister group to M. ditetra whereas parsimony analyses placed Philobdella as the sister group to M. diplotertia, M. decora, and M. mimicus. It is important to note that both methods recovered Macrobdella as paraphyletic; however, this result is poorly supported (LBS = 51).
Macrobdella mimicus represents the fifth species in the genus of medicinal leeches from North America. Morphological and molecular data confirm its inclusion within the family Macrobdellidae and within Macrobdella, morphologically most closely resembling M. decora.
Specimens of M. mimicus form a well-supported monophyletic clade (LBS = 98, PBS = 100) well within Macrobdella, as the sister group to M. decora and M. diplotertia, regardless of optimality criterion. Macrobdella was recovered as paraphyletic by virtue of the inclusion of the monophyletic clade of Philobdella species. Parsimony analyses placed Philobdella as sister to M. diplotertia, M. decora, and M. mimicus, and ML analyses placed Philobdella sister to M. ditetra. Philobdella species have been closely allied with Macrobdella in prior taxonomic schemes based on jaw morphology and presence of denticles as well as by the possession of accessory pores (Richardson, 1969; Sawyer, 1986). Philobdella floridana (Verrill, 1874) was described as a subgenus of Macrobdella based on the deep copulatory pit and accessory pores surrounding the female gonopore, although Verrill (1874) suggested that Philobdella could warrant being elevated to genus. Moore (1901) elevated Philobdella to the generic level when he described Philobdella gracilis (Moore, 1901); P. gracilis was the last species to be added to the genus. Previous analyses based on COI have placed Philobdella within Macrobdella, whereas ribosomal DNA loci (18S rDNA, 28S rDNA, and 12S rDNA) and combined analyses of nuclear and mitochondrial loci placed Philobdella as the sister group to Macrobdella (Phillips and Siddall, 2005, 2009; Phillips et al., 2010; Oceguera-Figueroa et al., 2012). Importantly, these analyses lacked specimens of M. sestertia, the rarely collected New England medicinal leech, a species that to date has yet to be included in a molecular phylogeny (Smith, 1977; Smith and Hanlon, 1997; Phillips et al., 2016). Deeper gene and population-level sampling of Philobdella species and deeper taxon sampling of Macrobdella species, especially M. diplotertia and M. sestertia, is needed to evaluate whether or not Philobdella is a distinct genus within Macrobdellidae.
Morphological data support the results of the molecular phylogeny and effectively separate M. mimicus from congeners. The new species resembles M. decora in possessing four accessory pores arranged in 2 rows in 2 columns. Specifically, the male gonopore, the accessory pores, and the nephridiopores are located in the same position in the new species and in M. decora; the difference between these species is that the female gonopore in the new species is a single annulus posterior to the position of the female gonopore in M. decora.
Species delimitation within Macrobdella has centered around the number and arrangement of the accessory pores, though this is the first instance of congeners having the same arrangement of accessory pores. The new species has not been recognized based on the traditional character set used to delimit Macrobdella species, which is likely the reason why this species has been overlooked until now. Macrobdella mimicus is well-represented in scientific collections with significant holdings of eastern U.S. fauna, albeit labeled as M. decora, and identifying this species is straightforward with distinctive external morphological characters. In this study, scientific collections were invaluable for comparing morphology of historical specimens to molecular voucher specimens and determining the extent of the geographic distribution of this new species.
Macrobdella mimicus is distributed in eastern United States, ranging from northern Georgia through North and South Carolina, Virginia, and Maryland to Long Island, New York. The geographic distribution is nestled between that of M. ditetra in the Atlantic Coastal Plain and M. decora to the west and north. So far, there has not been overlap of the distribution of M. mimicus based on verified specimens and the distributions of its congeners. Localities of verified specimens of M. mimicus fall within 3 ecoregions as defined by the Commission for Environmental Conservation (Wiken et al., 2011): predominantly Piedmont, but also Blue Ridge and Atlantic Coastal Pine Barrens (Fig. 5). Macrobdella decora displays a much broader geographic distribution, including much of northern North America, than its congeners. Macrobdella ditetra is considered to be distributed along the coastal plain from Virginia through eastern Texas and up the Mississippi River drainage as far north as Arkansas (Phillips et al., 2016), whereas M. diplotertia has been found only in the Mississippi River drainage of Arkansas, Kansas, and Missouri (Moser et al., 2006; Trauth and Neal, 2004). It is possible that additional species diversity is present within the currently recognized Macrobdella species, especially M. decora.
Prior to this study, the last description of a medicinal leech species from North America was published more than 40 yr ago (Meyer, 1975) suggesting that additional species diversity within this group could lie within concepts of named species. The leech fauna of the southeastern United States has been less frequently sampled than other regions of North America, such as the midwest and northeast (Moore, 1901; Sawyer, 1968, 1972; Klemm, 1972, 1977, 1982); further sampling of this region has great potential to reveal additional diversity in other leech genera. The additional specimens examined from the collections of the NMNH, the North Carolina Museum of Natural Sciences, and the Virginia Museum of Natural History spanned 63 yr of collecting from 1937 to 2000 and expands the distribution of M. mimicus to include northwestern Georgia and Long Island, New York as well as points in between including North Carolina and Virginia. This study demonstrates the power of scientific collections when combined with DNA sequence data from voucher specimens for identifying and characterizing overlooked diversity.
Thank you to Mary Bunch (SC DNR), Ed Cory (NC DNR), Nelson González-Süllow (U.S. Forest Service), and Scott Smith (MD DNR) for assistance with collecting permits, access to field sites, and general knowledge about the landscape. We thank Alan Cressler (USGS), Bill Phillips, Don Stacey, Nallely Ruiz Torres, Luca Varone, Kelly Varone, and Joe Varone for their assistance in the field. Special thanks to Andrea Jiménez Marín, Laura Márquez, Ofelia Delgado, and Herman Wirshing for their participation in generating DNA sequences, Susana Gómez for her assistance with the light microscopy images, Freya Goetz for SEM assistance and image improvement, and Dan Cole for generating maps. For access to collections and assistance with collections management we thank William E. Moser and the Invertebrate Zoology collections management staff of the NMNH, Bronwyn Williams of the North Carolina Museum of Natural Sciences, Kal Ivanov of the Virginia Museum of Natural History, and Luis García Prieto of the CNHE. Thank you to the Willi Hennig Society for providing TNT as freely available software to the scientific community. This project was partially funded by the project PAPIIT (IN210318) and CONACYT (220408) to AO-F. SK thanks NSERC (RGPIN-2016-06125), the Royal Ontario Museum (Schad Conservation Grant) and the Olle Engkvist Byggmästare foundation for funding.
Version of Record, first published online with fixed content and layout, in compliance with ICZN Arts. 22.214.171.124, 8.5, and 21.8.2 as amended, 2012.