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Research Article
Helobdella blinni sp. n. (Hirudinida, Glossiphoniidae) a new species inhabiting Montezuma Well, Arizona, USA
expand article infoRebecca K. Beresic-Perrins‡, Fredric R. Govedich§, Kelsey Banister‡, Bonnie A. Bain§, Devin Rose‡, Stephen M. Shuster‡
‡ Northern Arizona University, Flagstaff, United States of America
§ Southern Utah University, Cedar City, United States of America

Corresponding author: Fredric R. Govedich ( govedich@suu.edu )

Academic editor: Samuel James
© 2017 Rebecca K. Beresic-Perrins, Fredric R. Govedich, Kelsey Banister, Bonnie A. Bain, Devin Rose, Stephen M. Shuster.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Beresic-Perrins RK, Govedich FR, Banister K, Bain BA, Rose D, Shuster SM (2017) Helobdella blinni sp. n. (Hirudinida, Glossiphoniidae) a new species inhabiting Montezuma Well, Arizona, USA. ZooKeys 661: 137-155. https://doi.org/10.3897/zookeys.661.9728
ZooBank: urn:lsid:zoobank.org:pub:963C2520-3C29-419B-A5D1-0518E9713DD5
Open Access

Abstract

A new leech species Helobdella blinni sp. n., is described from Montezuma Well, an isolated travertine spring mound located in central Arizona, USA. In its native habitat, H. blinni had been previously identified as Helobdella stagnalis (Linnaeus, 1758), which was later reclassified to Helobdella modesta (Verrill, 1872). Similar to the European H. stagnalis and North American H. modesta, H. blinni has six pairs of testisacs, five pairs of smooth crop caecae, one lobed pair of posteriorly-directed crop caecae, one pair of eyes, a nuchal scute, and diffuse salivary glands. However, the pigmentation of this new species ranges from light to dark brown, unlike H. modesta which tends to be light grey in color. Also, H. modesta produces a clutch of 12-–35 pink eggs, whereas H. blinni produces smaller clutches of white eggs (7–14, 0.5 ± 0.15 mm, N = 7) and consequently broods fewer young (1–14, 7 ± 3.3 mm, N = 97). Helobdella blinni are also able to breed year-round due to the constant warm water conditions in Montezuma Well. Their breeding season is not restricted by seasonal temperatures. These species are morphologically similar, however, comparing the COI mtDNA sequences of H. blinni with sequences from nearby populations of H. modesta and other Helobdella species from GenBank indicate that H. blinni is genetically distinct from these other Helobdella populations.

Keywords

Leech, Glossiphoniidae , Helobdella blinni sp. n., new species, Montezuma Well

Introduction

Montezuma Well is a collapsed travertine spring mound located 72 km south of Flagstaff in the Verde Valley of Northern Arizona (34.6491°N,111.7522°W (DD)) (Fig. 1A). The age of Montezuma Well is estimated to be ~11,000 years (Wagner and Blinn 1987). This location is thermally constant year-round (19–24˚C) and is continuously replenished by two vents located at the well bottom. Montezuma Well is 0.76 ha in area and approximately 20 m deep. Most of the shoreline drops off immediately into open water, except at the northeast corner where water drains through a shallow region called the “swallet” and empties into Wet Beaver Creek which is located east of Montezuma Well (Fig. 1A–B). The water within Montezuma Well has unique water chemistry, containing high levels of arsenic (>100μg/L) and dissolved CO2 (>300mg/L) (Cole and Barry 1973).

Four leech species are known to inhabit Montezuma Well, including an endemic pelagic predator (Govedich et al. 1998), the erpobdellid Motobdella montezuma (Davies et al. 1985), and three other glossiphoniid species currently identified as Helobdella papillata (Moore, 1952), H. elongata (Castle, 1900), and a species currently thought to be H. stagnalis (Linnaeus, 1758), all of which inhabit the swallet (Fig. 1B). These Montezuma Well leech populations are thought to have been isolated from other leech populations for as long as 11,000 years (Wagner and Blinn 1987).

In support of this hypothesis, Beresic-Perrins’s (2010) description of brood size, parental behavior, and life history of the Montezuma Well population of H. stagnalis suggests that this leech is distinct from other known populations of H. stagnalis, a species originally described from Europe and which had until very recently been considered to be a widespread cosmopolitan leech species, inhabiting both Europe and North America. Siddall et al. (2005) addressed this problem by resurrecting the original species description for the North American leech, Helobdella modesta (Verrill, 1872) which had long been considered to be a synonym of the European H. stagnalis (Moore 1898). The molecular analysis by Moser et al. (2011) provided confirmation for the resurrection of H. modesta by Siddall et al. (2005). Even though the two species are morphologically indistinguishable (Verrill 1872, Moore 1898, Moore 1952), they differ genetically. Henceforth, we will refer to the North American H. stagnalis as H. modesta.

Here, we compare key traits, both morphological and molecular, among members of the Montezuma Well Helobdella sp. population, several other nearby populations of H. modesta, and several other Helobdella species. Our molecular analysis includes the cytochrome c oxidase subunit I (COI) mitochondrial gene region to test the hypothesis that the Montezuma Well population of H. modesta is a distinct species and warrants a new species description. This region is known to be sufficiently variable to reveal interspecific differences and unlikely to suggest differences due to elevated mutation rates (Apakupakul et al. 1999).

Figure 1. 

Location of Helobdella blinni sp. n. A The northeast side of Montezuma Well; and B The swallet where the leeches were collected.

Materials and methods

Sampling

A total of 34 individuals of Helobdella sp. inhabiting Montezuma Well were collected from the underside of rocks in the swallet: five specimens were collected in June 2011 for molecular analysis and 29 were collected in June 2012 to assess morphological characteristics. For the molecular analysis, the leeches were preserved in 95% ethanol and others, for museum collections, were fixed with buffered formalin overnight and preserved in 70% ethanol. Additionally, a total of 10 specimens of H. c. f. modesta from Rio de Flag ponds near the Rio de Flag Waste Water Facility outflow in Flagstaff, Arizona (35.18418°N, 111.63294°W (DD)) and Oak Creek, AZ near the Cave Springs campground (34.9961°N, 111.7394°W (DD)) were collected for molecular analyses. These specimens were also fixed in 95% ethanol.

Morphological examination

We documented number of eyes and their placement, color pattern, presence of papillae, number of and structure of gastric caecae, body size, presence of nuchal scute, gonopore placement, egg size and number, and number of offspring using a Nikon binocular dissecting microscope. We then deposited the examined materials in the Invertebrate Zoology collection at the Smithsonian Institution, National Museum of Natural History (USNM).

Molecular analysis

Whole DNA was extracted from the caudal suckers of the individual leeches using a Qiagen DNeasy Blood & Tissue Kit (Cat. No. 69504), with each sample incubated overnight in a water bath set at 54°C. Using Siddall and Borda’s (2002) PCR method, the mitochondrial gene region, cytochrome c oxidase subunit I (COI) was amplified. The primers were LCO1490 5’-GGTCAACAAATCATAAAGATATTGG-3’ and HCO2198 5’-TAAACTTCAGGGTGACCAAAAAATCA-3’ (Folmer et al. 1994). The PCR product was purified through the use of the QIAquick PCR Purification Protocol (Cat. No. 28104), checked for PCR product using gel electrophoresis, and sequenced with an ABI Prism 3730 sequencer (Applied Biosystems). We imported the seven “cleanest” sequences and 71 comparative sequences (Table 1) from previous studies (Siddall and Burreson 1998, Siddall and Borda 2002, Siddall and Budinoff 2005, Siddall et al. 2005, Williams and Burreson 2005, Bely and Weisblat 2006, Williams and Burreson 2006, Lai et al. 2009, Oceguera-Figueroa et al. 2010, Kutschera 2011, Moser et al. 2013) from GenBank (http://www.ncbi.nlm.nih.gov/genbank/) into MEGA7.0.18 (Kumar et al. 2016). We aligned the sequences automatically using MUSCLE (Edgar 2004) and then corrected the alignments by hand. We partitioned the data and performed the substitution model test by codon in Partitionfinder (Lanfear et al. 2012). The best substitution model test was General Time Reversal (GTR) +gamma which we used in our maximum-likelihood (ML) analysis (Lanave et al. 1984, Tavare 1986, Rodriguez et al. 1990). For ML analysis, we used RAxML v. 8 (Stamatakis 2014) and included 1,000 nonparametric bootstrap replicates. We used MrBayes for Bayesian inference analysis with ten million generations with a 25% burn-in and our support was assessed based on clade posterior probabilities (Ronquist and Huelsenbeck 2003). These analyses were conducted through CIPRES (Miller et al. 2010). We used PAUP* 4.0 (Swofford 2003) to construct parsimony phylogenies with 100 random additions. We performed the parsimony analysis twice, treating the deletions in the sequences as a 5th state and then as missing data. We performed an uncorrected p-distance analysis to examine nucleotide differences between sequences with 1,000 replicates in MEGA7.0.18 (Kumar et al. 2016).

Table 1.
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Helobdella and outgroup taxa used for our molecular analysis.

Taxon Locality Reference
Cystobranchus salmositicus Outgroup Williams and Burreson 2006
Ozobranchus margoi Outgroup Siddall and Burreson 1998
Gonimosobdella klemmi Outgroup Williams and Burreson 2005
Myzobdella lugubris Outgroup Siddall and Burreson 1998
Helobdella atli French Guiana Oceguera-Figueroa et al. 2010
Helobdella atli Uruguay Oceguera-Figueroa et al. 2010
Helobdella atli Mexico Oceguera-Figueroa et al. 2010
Helobdella blinni sp. n. Montezuma Well, AZ, USA This study
Helobdella blinni sp. n. Montezuma Well, AZ, USA This study
Helobdella blinni sp. n. Montezuma Well, AZ, USA This study
Helobdella bolivianita Bolivia Siddall and Borda 2002
Helobdella bowermani Oregon, USA Moser et al. 2013
Helobdella bowermani Oregon, USA Moser et al. 2013
Helobdella bowermani Oregon, USA Moser et al. 2013
Helobdella californica California, USA Kutschera 2011
Helobdella “elongata” Mexico Oceguera-Figueroa et al. 2010
Helobdella europaea Taiwan Lai et al. 2009
Helobdella europaea Taiwan Lai et al. 2009
Helobdella europaea Taiwan Lai et al. 2009
Helobdella europaea Taiwan Lai et al. 2009
Helobdella europaea South Africa Siddall and Budinoff 2005
Helobdella lineata Michigan, USA Siddall and Borda 2002
Helobdella fusca Michigan, USA Siddall and Borda 2002
Helobdella melananus Taiwan Lai et al. 2009
Helobdella melananus Taiwan Lai et al. 2009
Helobdella melananus Taiwan Lai et al. 2009
Helobdella michaelseni Chile Siddall and Borda 2002
Helobdella modesta Columbus, Ohio, USA Siddall and Borda 2002
Helobdella modesta Washington, USA Oceguera-Figueroa et al. 2010
Helobdella modesta Washington, USA Oceguera-Figueroa et al. 2010
Helobdella c. f. modesta Rio de Flag, Flagstaff, AZ, USA This study
Helobdella c. f. modesta Rio de Flag, Flagstaff, AZ, USA This study
Helobdella c. f. modesta Oak Creek, AZ, USA This study
Helobdella c. f. modesta Oak Creek, AZ, USA This study
Helobdella nununununojensis Bolivia Siddall and Borda 2002
Helobdella nununununojensis Bolivia Siddall and Borda 2002
Helobdella octatestisaca Taiwan Lai et al. 2009
Helobdella octatestisaca Taiwan Lai et al. 2009
Helobdella octatestisaca Taiwan Lai et al. 2009
Helobdella octatestisaca Taiwan Lai et al. 2009
Helobdella octatestisaca Taiwan Lai et al. 2009
Helobdella octatestisaca Taiwan Lai et al. 2009
Helobdella octatestisaca Taiwan Lai et al. 2009
Helobdella octatestisaca South Africa Oceguera-Figueroa et al. 2010
Helobdella octatestisaca Mexico Oceguera-Figueroa et al. 2010
Helobdella octatestisaca Mexico Oceguera-Figueroa et al. 2010
Helobdella octatestisaca Mexico Oceguera-Figueroa et al. 2010
Helobdella octatestisaca Mexico Oceguera-Figueroa et al. 2010
Helobdella octatestisaca Mexico Oceguera-Figueroa et al. 2010
Helobdella papillata Michigan, USA Siddall and Borda 2002
Helobdella papillata Virginia, USA Siddall and Borda 2002
Helobdella papillornata Australia Siddall and Borda 2002
Helobdella paranensis Uruguay Siddall and Borda 2002
Helobdella pichipanan Bolivia Siddall et al. 2005
Helobdella “robusta” TXAU1 Texas, USA Bely and Weisblat 2006
Helobdella “robusta” California, USA Bely and Weisblat 2006
Helobdella “robusta” CASA 1 California, USA Bely and Weisblat 2006
Helobdella “robusta” NYTA New York, USA Bely and Weisblat 2006
Helobdella simplex Argentina Moser et al. 2006
Helobdella simplex Argentina Moser et al. 2006
Helobdella simplex Argentina Moser et al. 2006
Helobdella socimulcensis Mexico Oceguera-Figueroa et al. 2010
Helobdella socimulcensis Mexico Oceguera-Figueroa et al. 2010
Helobdella socimulcensis Mexico Oceguera-Figueroa et al. 2010
Helobdella socimulcensis Mexico Oceguera-Figueroa et al. 2010
Helobdella socimulcensis Mexico Oceguera-Figueroa et al. 2010
Helobdella socimulcensis Mexico Oceguera-Figueroa et al. 2010
Helobdella socimulcensis Mexico Oceguera-Figueroa et al. 2010
Helobdella sp. Xochimilco Mexico Oceguera-Figueroa et al. 2010
Helobdella sorojchi Bolivia Siddall and Borda 2002
Helobdella sorojchi Bolivia Siddall and Borda 2002
Helobdella stagnalis United Kingdom Siddall and Borda 2002
Helobdella “stagnalis” Mexico Oceguera-Figueroa et al. 2010
Helobdella “stagnalis” Mexico Oceguera-Figueroa et al. 2010
Helobdella transversa Michigan, USA Siddall and Borda 2002
Helobdella triserialis Bolivia Siddall and Borda 2002
Helobdella triserialis California, USA Bely and Weisblat 2006
Helobdella virginiae Mexico Oceguera-Figueroa et al. 2010

Results

Family Glossiphoniidae Vaillant, 1890

Genus Helobdella Blanchard, 1896

Helobdella blinni sp. n.

http://zoobank.org/B1B3D234-BC3F-4126-BF25-52DA00BA7EB9
Figs 2, 3, 4

Type materials

Holotype.USNM 1186106 (Table 2).

Table 2.
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Holotype and paratype collection data and voucher numbers.

Family Species Collection data Voucher #
Glossiphoniidae Helobdella blinni sp. n. USA: AZ: Yavapai Co., Montezuma Well 34.6491°N, 111.7522°W (DD), 10.VI.2010, aquatic system, under rocks, RK Beresic-Perrins, Holotype (USNM) 1186106
Glossiphoniidae Helobdella blinni sp. n. (14 specimens) USA: AZ Yavapai Co., Montezuma Well 34.6491°N, 111.7522°W (DD), 10.VI.2010, aquatic system, under rocks, RK Beresic-Perrins, Paratypes (USNM) 1186107
1186108
1186109
1186110
1186111
1186112
1186113
1186114
1186115
1186116
1186117
1186118
1186119
1186120

Additional materials

Paratypes. (14 specimens) (USNM 1186107, 1186108, 1186109, 1186110, 1186111, 1186112, 1186113, 1186114, 1186115, 1186116, 1186117, 1186118, 1186119, 1186120) (Table 2)

Type locality

USA, Arizona: Yavapai County, Montezuma Well (34.6491°N, 111.7522°W (DD)), aquatic system, under rocks, 10 June 2012, R.K. Beresic-Perrins.

Etymology

We have named this new species, Helobdella blinni in honor of Dr. Dean W. Blinn for his dedication to natural history research at Montezuma Well. For over 20 years at Northern Arizona University, Dr. Blinn studied a wide range of organisms and their interactions at Montezuma Well including predator-prey interactions between Motobdella montezuma and the endemic amphipod, Hyalella montezuma Cole & Watkins, 1977.

Description

External morphology. Length of specimens 11 to 22 mm (mean + SE 16.6 + 3.2 N=24) and width 3 to 8 mm (5.7 + 1.1 N=28) (Table 3, Figs 2, 3). Individual color ranges from translucent with brown spots to dark brown (Fig. 4). No dorsal papillae; one pair of eyes located at somite II (0.07 + 0.02 mm diameter, N = 11), distance between eyes 0.1 to 0.03 mm apart (N = 13). A scallop-shaped nuchal scute is present on the dorsal side, length 0.293 to 0.432 mm (0.335 + 0.05 N=9) and width 0.27 to 0.386 mm (0.32 + 0.04 N=9). One annulus separates the female and male gonopores. The caudal sucker diameter averages 1.6 + 0.3 mm (N = 27). The eggs (diameter 0.5 + 0.15 mm, N = 28) are laid on the ventral side of the parent in soft-walled transparent cocoons (7–11 eggs per cocoon, N = 7). The mouth is located subterminally in the oral sucker (Figs 2, 3).

Internal morphology. Average oral sucker diameter is 0.7 + 0.19 mm (N = 15), proboscis length is 3.5 + 1.1 mm (N = 17) (Table 3). Diffuse salivary glands are located near the anterior of the first pair of crop caecae. There are five pairs of smooth crop caecae and one lobed pair of posteriorly directed post caecae. Six pairs of compact testisacs are located in between each of the crop caecae. The intestine contains four pairs of caecae, with the first two pairs anteriorly directed and the other two pairs posteriorly directed. The intestine leads into an unraised anus located two annuli from the caudal sucker (Figure 3).

Development and growth. This species breeds year-round with peaks in spring and fall. Our specimens had an average of 7 to 11 white eggs (diameter 0.5 + 0.15 mm, N = 7) fixed to their ventral surface. Laboratory collections (2007–10) of H. blinni documented the eggs hatching 1 to 2 weeks after ovipositing (Beresic-Perrins 2010). Once hatched, the young attach to the ventral surface of the parent, allowing the parent to hunt for food and feed the young, occasionally feeding along with them. Prey consists of oligochaetes and other invertebrates. The average number of young per adult is 7 + 3.3 (N = 97) ranging from 1 to 14 offspring. The young remain attached to the parent for an additional four to five weeks after hatching. Once the juveniles leave the parent, they tend to aggregate together on rocks (Beresic-Perrins 2010).

Figure 2. 

Internal and external morphology of Helobdella blinni sp. n. A dorsal view of the eyes and extended proboscis B crop and post caecae C testisacs D ventral view of internal eggs which have not been oviposited yet E ventral view of white eggs that have been oviposited F ventral view of attached and detached offspring.

Figure 3. 

Diagram of the external and internal morphology of Helobdella blinni sp. n. (drawn by Rebecca Beresic-Perrins and Fredric Govedich).

Table 3.
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Morphological measurements of Helobdella blinni sp. n.

Trait Ave SE Min Max N
body length relaxed (mm) 16.6 3.18 11.3 22.5 24
body width relaxed (mm) 5.7 1.15 3.1 8.0 28
caudal diameter (mm) 1.7 0.3 1.0 2.3 27
egg diameter (mm) 0.5 0.15 0.2 0.7 28
gonopore separation (mm) 0.1 0.08 0.1 0.3 13
nuchal scute length (mm) 0.335 0.05 0.284 0.432 9
nuchal scute width (mm) 0.32 0.04 0.27 0.386 9
proboscis length (mm) 3.5 1.10 2.0 6.2 17
oral sucker diameter (mm) 0.7 0.19 0.4 1.0 15
progeny length (mm) 3.6 1.68 1.6 6.6 18
progeny width (mm) 1.5 0.8 0.7 2.9 18
# eggs 10.0 2.73 7.0 16.0 7
# progeny 7.2 3.35 1.0 14.0 97
eye diameter (mm) 0.1 0.02 0.0 0.1 11
eye distance (mm) 0.1 0.04 0.0 0.2 13
Figure 4. 

Typical pigmentation of Helobdella blinni sp. n.

Molecular analysis

A Bayesian inference phylogenetic tree of the COI sequence data is presented in Figure 5. We include the posterior probabilities and maximum-likelihood branch supports >50. The Arizona populations of Helobdella c. f. modesta formed a sister clade to Helobdella blinni sp. n., supported by both the Bayesian and parsimony analyses. The results of the uncorrected p-distance analysis revealed a difference of 13.3% (233 nucleotides included) between the two groups (Table 6). The two groups form a larger clade with H. modesta (Ohio), H. stagnalis (UK), and H. modesta (Washington) which is supported by both Bayesian inference and maximum-likelihood. H. blinni differed from H. modesta (Ohio) by 13.7%, H. stagnalis (UK) by 16.3%, and H. modesta (Washington) by 16.3% (Table 6).

When we aligned all 78 sequences, there were four, ten-codon deletions within all of the Arizona sequences and H. atli (Oceguera-Figueroa and León-Regagnon 2005, Oceguera-Figueroa et al. 2010). When we performed the parsimony analysis, we included deletions as a 5th state in our first analysis and in our second, we treated the deletions as missing data. In the resulting 5th state tree, the two Arizona species remained sister taxa (100% support), but included in the clade was H. atli (100% and 58% support). The missing data tree placed H. blinni ancestral to H. modesta (Washington), H. modesta (Ohio), H. stagnalis (UK), and H. c. f. modesta with 100% branch support (Fig. 5).

Figure 5. 

Bayesian Inference phylogenetic tree with 25% burn-in and support was assessed based on clade posterior probabilities tree. We included COI sequences from 31 species of Helobdella (family Glossiphoniidae). The Arizona populations are from Oak Creek (OC), Rio de Flag (RDF), and Montezuma Well (MW). Our outgroup included Cystobranchus salmositicus (Meyer, 1946), Gonimosobdella klemmi (Williams & Burreson, 2005), Myzobdella lugubris (Leidy, 1851), and Ozobranchus margoi (Davies, 1978). The shaded branches are the Arizona sample sequences. Branch labels include the Bayesian / ML probability. The blue nodes are supported by Bayesian Inference, Maximum-Likelihood, and parsimony analyses. The yellow nodes are supported by Bayesian Inference and Maximum-Likelihood analyses. The green nodes are supported by Bayesian Inference and parsimony analyses. The red nodes are supported by Bayesian Inference analysis only.

Table 4.
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Morphological comparison of Helobdella species.

Traits H. blinni sp. n. H. modesta H. californica H. papillornata H. temiscoensis H. atli H. bowermani H. octatestisaca
(current paper) (Kutschera 1988: Sawyer 1986) (Kutschera 1988; 2011) (Govedich and Davies 1998) (Salas-Montiel et al. 2014) (Oceguera-Figueroa and León-Regagnon 2005) (Moser et al. 2013) (Lai et al. 2009)
crop caecae 5 pairs, smooth 5 pairs, smooth 6 pairs, lobed 5–6 pairs, lobed 4 pairs 6 pairs 5 pairs, smooth 5 pairs
post caecae 1 pair 1 pair none none 1 pair none 1 pair 1 pair
eyes 1 pair 1 pair 1 pair 1 pair) 1 pair 1 pair 1 pair 1 pair
distance between eyes 0.1 mm ? ? 0.06 mm ? ? ? ?
nuchal scute yes yes yes no yes yes yes yes
pairs of testisacs 6 pairs 6 pairs 6 pairs 5 pairs 6 pairs 6 pairs 6 pairs 4 pair
salivary glands diffuse diffuse ? diffuse diffuse ? diffuse diffuse
proboscis length 3.5mm ? 0.7mm 2mm ? ? ? ?
color transparent with spots to dark brown transparent to light grey dark grey transparent with stripes and papillae pale brown, blackish - on posterior and mid-body white-yellowish pale yellow/buff, papillae present brown, pale, gray, and pink
body length 11–22 mm 8–12 mm 10–18 mm 15–40 mm 7.9–13.6 mm 7.5 mm 5.2–9.7 mm 9–14 mm
feeding small invertebrates small invertebrates small invertebrates small invertebrates ? ? ? small invertebrates
brooding period 6–7 weeks 6–7 weeks 3–4 weeks 4–6 weeks ? ? ? ?
egg color white pink pink pink ? ? ? ?
egg diameter 0.5 mm ? 0.5 mm 0.2 mm ? ? ? ?
# eggs 7–14 12–35 8–56 20–50 ? ? ? ?
Table 5.
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Differences in brooding season and size between H. blinni sp. n., H. stagnalis, and H. c.f. tp:taxon-name-part taxon-name-part-type="species" full-name="modesta">modesta.

Location Brooding Season Average # of offspring Author
H. blinni sp. n.
Montezuma Well, AZ		 Year-round 1–14 Beresic-Perrins (2010)
H. modesta
Utah Lake, UT		 Late spring through summer 12.6–17.4 Tillman and Barnes (1973)
H. modesta
Lake Washington, WA		 Spring and Summer 14.5 Thut (1969)
H. modesta
Marion Lake, BC, CA		 Spring and Summer 17.2–19.7 Davies and Reynoldson (1976)
H. modesta
Newsome Pond, AB, CA		 Late spring through summer 21.3
H. modesta
Cambridge, MA		 Spring 31 Castle (1900)
H. modesta
Michigan		 Late spring through summer 35.3 Sawyer (1972)
H. stagnalis
Iceland		 Late spring through summer No data Bruun (1938)
H. stagnalis
River Ely, South Wales		 Late spring through summer No data Murphy and Learner (1982)
H. stagnalis
Whiteknights Lake, UK		 Late spring through summer 13–17 Mann (1957)
H. stagnalis
Eglwys Nunydd, UK		 Late spring through summer 14 Learner and Potter (1974)
H. stagnalis
Denmark		 Late spring through summer 20 Bennike (1943)
Table 6.
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Uncorrected p-distance pairwise analysis.

Species Distance - H. blinni Distance - H. c. f. modesta
H. atli 14.1–15% 16.7%
H. bolivianita 18.5% 19.3%
H. bowermani 15.9% 15.5%
H. blinni 0.0% 13.3%
H. californica 16.7% 17.6%
H. c. f. modesta 13.3% 0.0%
H. elongata 18.0% 19.3%
H. europaea 15.5% 16.3%
H. fusca 19.3% 20.6%
H. lineata 16.3% 14.6%
H. melananus 16.3% 17.2%
H. michaelseni 23.2% 20.6%
H. modesta OH 13.7% 8.6%
H. modesta WA 16.3% 14.6%
H. nununununojensis 17.5–19% 17.5–19.7%
H. octatestisaca 19.7% 16.3%
H. papillata 17.6% 15.8–16.3%
H. papillornata 15.9% 16.7%
H. paranensis 16.3% 13.7%
H. pichipanan 17.2% 19.3%
H. robusta 17.6% 14.6%
H. aff robusta CASA 18.9% 17.2%
H. aff robusta NYTA 17.6% 15.9%
H. aff robusta TXAU1 17.2% 18.0%
H. simplex 16.3% 13.7–14.2%
H. socimulcensis 15.9% 16.7–17.2%
H. sorojchi 18–18.5% 17.6%
H. sp. Xochimilco 15.5% 17.2%
H. stagnalis 20.6% 17.2%
H. stagnalis UK 16.3% 11.6%
H. transversa 16.3% 15.0%
H. triserialis 15.9% 16.7–18.5%
H. virginiae 16.3% 16.7%
Outgroup1 C. salmositicus 24.9% 23.2%
Outgroup2 G. klemmi 21.5% 21.5%
Outgroup3 M. lugubris 18.0% 19.3%
Outgroup4 O. margoi 23.2% 20.2%

Discussion

Helobdella blinni sp. n. has morphological and life-history traits similar to other Helobdella species, including possession of a nuchal scute, diffuse salivary glands, six pairs of testisacs, and extended parental care for the young (6–7 weeks; Tables 4, 5). Helobdella blinni, H. bowermani (Moser et al. 2013), H. octatestisaca (Lai et al. 2009), and H. c. f. modesta each have five pairs of smooth crop caecae as opposed to six pairs of lobed crop caecae in H. californica (Kutschera 2011) and H. papillornata (Govedich and Davies 1998). Helobdella blinni and H. temiscoensis (Salas-Montiel et al. 2014) share pigmentation characteristics, but they differ internally. Helobdella temiscoensis has only four pairs of crop caecae and one descending post caecae as opposed to five pairs and one descending post caecae in H. blinni. Helobdella modesta, H. californica, H. atli, H. bowermani, and H. octatestisaca do not resemble the pigmentation of H. blinni, running the spectrum from grey to pink. Additionally, they have a descending pair of post caecae, whereas H. atli, H. californica, and H. papillornata do not. Helobdella blinni, H. c. f. modesta, H. californica, H. temiscoensis, H. atli, and H. bowermani possess six pairs of testisacs, whereas H. papillornata has five pairs and H. octatestisaca has four pairs. Helobdella blinni also has a larger proboscis than the other Helobdella species (mean + SE, H. blinni 3.5 mm + 1.1, N=17, H. californica mean = 0.7 mm, H. papillornata mean= 2 mm). Furthermore, breeding periods also differ between H. blinni and the other Helobdella species (Tables 4, 5).

Helobdella blinni, unlike the other Helobdella species discussed here, breeds year-round, living in the thermally stable environment of Montezuma Well, with constant (19–24˚C) year-round temperatures (Table 5). Monthly samples have individuals carrying cocoons every month of the year, with peak seasons in the spring and fall, a situation quite different than that for other Helobdella species, which have seasonally-constrained reproductive cycles, with egg-laying and brooding beginning in the spring and ending in the fall every year (Table 5). In addition to breeding year-round, H. blinni produces smaller broods (7–14 young) when compared to H. modesta and H. stagnalis (12–35 young) (Tables 4, 5) and has white eggs, unlike the characteristically pink eggs of H. modesta, H. californica, and H. papillornata (Table 4). The external pigmentation of H. blinni also tends to be dark brown, whereas most other Helobdella species are grey/brown in color (Fig. 4). Helobdella blinni are slightly longer (body length 11–22 mm, 16.6 + 3.2, N=24) than H. c. f. modesta (8–12 mm) and H. californica (10–18 mm), but slightly shorter in length than H. papillornata (15–40 mm) (Table 4).

The results from our molecular analysis show H. blinni to be genetically distinct from other Helobdella species, both from the same region (Rio de Flag and Oak Creek, Arizona populations) and from Europe (UK sample). The sequences yielded a 13.3%–17.4% genetic difference between H. blinni and both the Arizona H. c. f. modesta, H. modesta (Ohio and Washington), and the United Kingdom H. stagnalis populations (Table 6). The three Arizona populations belong to their own separate clade, but are closely related to H. stagnalis (UK) and the H. modesta. Helobdella atli, H. bowermani, and H. californica are located on separate branch tips, but they comprise what Oceguera-Figueroa et al. (2010) designated as the “stagnalis” series (Fig. 5).

Based on morphological, life-history, and molecular differences, we propose the Helobdella sp. leeches found at Montezuma Well should be considered a new species, likely the result of allopatric isolation. This concept supports our hypothesis that the leech species inhabiting Montezuma Well may have become isolated from other populations as far back as 11,000 years ago (Wagner and Blinn 1987). Helobdella blinni sp. n. can be considered a distinct species found in Montezuma Well and may also turn out to be endemic to the area. Further sampling and analyses are needed in order to verify endemism.

Although currently classified as Helobdella c. f. modesta, the Arizona populations from the Rio de Flag and Oak Creek may be an additional undescribed species based on our molecular analysis. Our next step is to investigate these populations more closely, comparing them to other local populations, including White Horse Lake and J.D. Dam Lake, AZ which also contain H. modesta.

Acknowledgements

We would like to thank the National Park Service rangers and volunteers at the Montezuma Castle National Monument, especially Rex Vanderford, who assisted with collections and permits. Funding for the molecular and morphological analyses was provided by the National Science Foundation’s Integrative Graduate Education and Research Traineeship. William Moser, Smithsonian Institution, National Museum of Natural History, Department of Invertebrate Zoology, provided valuable assistance in specimen management. Dr. Tina Ayers and Tamara Max (Northern Arizona University, Department of Biological Sciences) provided valuable assistance and insight with molecular data analyses. All the members past and present of Dr. Stephen Shuster’s laboratory were indispensable with all aspects of data collection for this manuscript.

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