A split decision: molecular and biogeographical evidence support species-level status of Anguispira kochi and Anguispira occidentalis (Stylommatophora, Discidae)

241 A split decision: molecular and biogeographical evidence support species-level status of Anguispira kochi and Anguispira occidentalis (Stylommatophora, Discidae) Robert G. Forsyth1, Annegret Nicolai2,3 , Nathaniel F. Shoobs4, Reham F. Ali5,6 , Rodrigo B. Salvador7,8 1 Department of Natural History, Research and Collections Centre, New Brunswick Museum, Saint John, New Brunswick, Canada 2 Living Lab CLEF, Plélan-Le-Grand, France 3 Station Biologique de Paimpont, Ecobio, Université de Rennes, Rennes, France 4 Museum of Biological Diversity, Department of Evolution, Ecology and Organismal Biology, The Ohio State University, Columbus, USA 5 Department of Zoology and Agricultural Nematology, Faculty of Agriculture, Cairo University, Giza, Egypt 6 Faculty of Organic Agriculture, Heliopolis University for Sustainable Development, Cairo, Egypt 7 Zoology Unit, Finnish Museum of Natural History, University of Helsinki, Helsinki, Finland 8 Arctic Chronobiology and Physiology Research Group, Department of Arctic and Marine Biology, UiT – The Arctic University of Norway, Tromsø, Norway Corresponding author: Rodrigo B. Salvador (salvador[email protected]) Copyright: This is an open access article distributed under the terms of the CC0 Public Domain Dedication. Research Article Abstract Anguispira kochi (L. Pfeiffer, 1846), the banded tigersnail, is a North American member of the family Discidae. It is typically subdivided into two subspecies: A. kochi kochi (L. Pfeiffer, 1846) and A. kochi occidentalis (E. von Martens, 1882). Genetic data from throughout the distribution of A. kochi (sensu lato) was used to produce a phylogenetic analysis and, together with morphological and distributional data, assess the taxonomic status of its subspecies. Evidence for elevating the western subspecies, A. k. occidentalis, to the rank of species level is provided and the implications this change has for the conservation status (and future conservation plans) of both species is discussed. A lectotype is designated for A. occidentalis. The status of other infraspecific taxa of both A. kochi and A. occidentalis is discussed, as well as the potential validity of subgenus Zonodiscus Pilsbry, 1948. Finally, the new classification of Discus marmorensis H.B. Baker, 1932 as Anguispira marmorensis (H.B. Baker, 1932) comb. nov. is proposed. Key words: Banded tigersnail, conservation status, Discoidea, Eupulmonata, molecular phylogenetics, Zonodiscus Introduction The genus Anguispira Morse, 1864 (Eupulmonata, Discidae) is a group of rather large terrestrial snails typically inhabiting moist forested areas in both eastern and western North America (Rankin et al. 2021). It contains 13 species according to Turgeon et al. (1998) or 21 species and subspecies according to Schileyko (2002). The current classification of this group largely follows Pilsbry (1948), although Solem (1976) revised some species. Pilsbry (1948) recognised two subgenera, the nominate subgenus and a new subgenus, Zonodiscus Pilsbry, 1948, which he differentiated by characters of the shell and genitalia. Nearly all Academic editor: Eike Neubert Received: 4 September 2025 Accepted: 9 November 2025 Published: 2 December 2025 ZooBank: https://zoobank.org/ A9244B29-486E-4D9B-84A47A438F13A272 Citation: Forsyth RG, Nicolai A, Shoobs NF, Ali RF, Salvador RB (2025) A split decision: molecular and biogeographical evidence support species-level status of Anguispira kochi and Anguispira occidentalis (Stylommatophora, Discidae). ZooKeys 1261: 241–260. https://doi. org/10.3897/zookeys.1261.171098 ZooKeys 1261: 241–260 (2025) DOI: 10.3897/zookeys.1261.171098 242 ZooKeys 1261: 241–260 (2025), DOI: 10.3897/zookeys.1261.171098 Robert G. Forsyth et al.: Species-level status of Anguispira kochi and Anguispira occidentalis species of the nominate subgenus have an eastern and central North American distribution, with no representatives in the west and only one species in Canada: the type species, Anguispira alternata (Say, 1817). On the other hand, Anguispira (Zonodiscus) occurs in both western and eastern North America, but supposedly with a single species, A. kochi (L. Pfeiffer, 1846). Among at least all large terrestrial snail species, A. kochi is unusual in having remarkably disjunct eastern and western populations in North America; these are separated by a gap of more than 2,000 km. This disjunction is biologically real and not due to insufficient surveys or lack of knowledge (Pilsbry 1948; Hubricht 1985). Several purported infraspecific taxa, either as varieties or forms, of A. kochi have been described at various times (Walker 1906; Clapp 1916; Clench and Banks 1939; Pilsbry 1948) and have importance in the conservation assessment of A. kochi should they be evolutionarily significant. Three infraspecific taxa were described by Clapp (1916), including Pyramidula solitaria mynesites Clapp, 1916, P. s. strontiana Clapp, 1916, and P. s. roseo-apicata Clapp, 1916. Pilsbry (1948: 593) considered these as “minor strains” established as “pure races” and treated these taxa as a form (mynesites) and two subspecies (strontiana and roseoapicata) of A. kochi. Hubricht (1985) considered all Lake Erie infraspecific taxa to be synonyms of A. kochi, although he did not provide his reasons. Pilsbry (1948) recognised the western A. kochi occidentalis (E. von Martens, 1882) as a separate subspecies from the eastern stock, although the distinctions were not made entirely clear, and he wrote that some specimens from the east and west were nearly conchologically identical. The western group was first formally described by Martens (1882) as a variety of Patula solitaria (an earlier invalid name for A. kochi). Later, A. kochi eyerdami Clench & Banks, 1939, was described from Yakima County, Washington State by Clench and Banks (1939). It purportedly differs from A. k. occidentalis in having a smaller, darker, and flatter shell. Pilsbry (1948: 597) noted a broad range of colour, relative heights, and sizes that may be phenotypes found throughout the range of A. kochi occidentalis and gave eyerdami no special recognition (i.e. not a subspecies). However, most recently, Burke (2013) unconvincingly treated A. k. eyerdami as a separate subspecies. Thus, over the years, there have been plenty of new taxa proposed within A. kochi. In terrestrial malacology, the use of the subspecies category has often been applied rather loosely for commonplace variation that should not warrant formal recognition (e.g. Coan and Roth 1987). But on the other hand, cryptic and little-known taxa have often been first recognised as infraspecific, only later to be determined to represent species in their own right, either by morphological (e.g. Roth and Miller 1993) or molecular data (e.g. Weaver et al. 2006). Even so, Páll-Gergely et al. (2019) have shown that the ratio of subspecies described per species is positively correlated with morphological complexity, range size, and habitat type in multiple land-snail families, shedding light on some of the biological features that influence taxonomists’ decisions to describe and maintain subspecies rather than species. In 2017, the conservation status of Canadian populations of A. kochi was assessed by the Committee on the Status of Endangered Wildlife in Canada (COSEWIC 2017). Two of the three Lake Erie island taxa—strontiana from Middle Sister Island and roseoapicata from North Harbour, East Sister, and Middle islands—were deemed not to have any conservation value in their own right 243 ZooKeys 1261: 241–260 (2025), DOI: 10.3897/zookeys.1261.171098 Robert G. Forsyth et al.: Species-level status of Anguispira kochi and Anguispira occidentalis (COSEWIC 2017), but the western and eastern populations of A. kochi were treated as subspecies, following Pilsbry (1948), and assessed separately. The Western Banded Tigersnail, A. kochi occidentalis, was found to be Not at Risk, but the eastern banded tigersnail, A. kochi kochi, was assessed as Endangered (COSEWIC 2017). The Canadian population of this eastern subspecies is only present in small, isolated patches of habitat on Middle and Pelee Islands in Lake Erie. Subpopulations from other, smaller islands are probably now extirpated, caused by habitat destruction of over-abundant double-crested cormorants, Phalacrocorax auritus (Lesson, 1831), and by human activities (COSEWIC 2017). Anguispira kochi has not had a comparable nationwide assessment in the USA, although NatureServe (2025) provides national and subnational ranks for both the USA and Canada. In the USA, the eastern populations are considered Critically Imperilled (S1) in Michigan and West Virginia, Imperilled (S2) in Kentucky and Tennessee, Vulnerable (S3) in Pennsylvania, and Not Ranked in Illinois, Indiana, Missouri, and Ohio, although state-specific statuses may change following the 2025 update to U.S. State Wildlife Action Plans (Association of Fish & Wildlife Agencies 2025). The populations in Ontario are S1. Nowhere (when ranked) are the eastern populations Apparently Secure or Secure (S4 or S5). The western populations appear to be faring better and are ranked as Secure (S5) in Idaho and Montana, Vulnerable (S3) in Washington and British Columbia, and Not Ranked in Oregon. NatureServe’s national rank for A. kochi is Vulnerable (N3) for Canada and Secure (N5) for the USA, but this is misleading. In both countries, but especially in the USA, the most secure western populations (best S-ranks) appear to counteract the ranks of the less secure eastern populations, resulting in more secure national ranks than would be warranted if the western and eastern subspecies were actually separate species. Herein, we use genetic data of A. kochi from throughout its distribution and assess the taxonomic status of its populations and nominal subspecies. We provide evidence for elevating A. kochi kochi and A. kochi occidentalis to species level. Materials and methods The taxonomic status of the two subspecies was assessed through phylogenetic analyses (Bayesian inference) using molecular data from different genetic markers. To that end, we conducted two analyses (see below for parameters): (1) analysis with multiple markers, including the mitochondrial markers COI and 16S, alongside the consecutive nuclear markers 5.8S, ITS2, and 28S. This analysis relied heavily on sequence data of Discoidea generated by our team, part of which is new and part of which is already published (Salvador et al. 2020, 2023); to these, some other published sequences from other authors were added (Clutts 2008; Rankin et al. 2021), obtained from GenBank. See Table 1 for a full list. To that end, we assembled a representative set of Anguispira species, considering the sequences available for all markers (Table 1). This includes the type species Anguispira alternata, as well as the two problematic species pointed out in the phylogenetic study of Salvador et al. (2023): Anguispira nimapuna H.B. Baker, 1932 and Discus marmorensis H.B. Baker, 1932. Gonyodiscus rotundatus (O.F. Müller, 1774) was chosen as the outgroup, considering the previous phylogenies of Salvador et al. (2020, 2023); 244 ZooKeys 1261: 241–260 (2025), DOI: 10.3897/zookeys.1261.171098 Robert G. Forsyth et al.: Species-level status of Anguispira kochi and Anguispira occidentalis sequence data for this species were likewise obtained from GenBank (Dinapoli and Kluss mann-Kolb 2010). (2) Analysis using only the COI barcoding marker, due to the large availability of COI sequences of Anguispira spp. on GenBank (Suppl.material 1). This analysis included all sequences currently available for the taxa of interest (A. kochi kochi and A. kochi occidentalis), as well as the additional species and outgroup from the multi-marker analysis. Table 1. Species used for the multi-marker phylogenetic analysis, including GenBank accession numbers, collection localities, and data sources. Species COI 16S 5.8S+ITS2+28S Location Source Anguispira alabama (Clapp, 1920) –ON749857 ON749850 USA, AL, Jackson Salvador et al. 2023 Anguispira alternata (Say, 1817) MN792584 MN756711 MN782441 USA, IL, Sangamon Salvador et al. 2020 Anguispira alternata (Say, 1817) MN792583 MN756710 MN782440 Canada, ON Salvador et al. 2020 Anguispira cumberlandiana (I. Lea, 1840) MW543329 MW544210 – USA, TN, Battle Branch (N of Kimball) Clutts 2008; Rankin et al. 2021 Anguispira fergusoni (Bland, 1862) MW543321 MW544221 – USA, DE, Blackbird State Forest Clutts 2008; Rankin et al. 2021 Anguispira jessica Kutchka, 1938 MN792585 MN756712 MN782442 USA, NC, Macon Salvador et al. 2020 Anguispira nimapuna H.B. Baker, 1932 MN792588 MN756715 MN782445 USA, ID, Lowell Salvador et al. 2020 Anguispira nimapuna H.B. Baker, 1932 MW532808 MW525438 – USA, ID, Nez Perce National Forest Rankin et al. 2021 Anguispira picta (G. H. Clapp, 1920) MW543311 MW544248 – USA, TN, Buck Creek Cove Clutts 2008; Rankin et al. 2021 Anguispira strongyloides (L. Pfeiffer, 1855) MN792589 MN756716 MN782446 USA, AL, Stevenson / FL Salvador et al. 2020 Anguispira kochi occidentalis (E. von Martens, 1882) MW532916 MW533672 – USA, ID, Seven Devils Mountains Rankin et al. 2021 Anguispira kochi occidentalis (E. von Martens, 1882) MW532935 MW533691 – USA, MT, Mineral Co., McGee Creek Rankin et al. 2021 Anguispira kochi occidentalis (E. von Martens, 1882) MN792586 MN756713 MN782443 Canada, BC, Bear Creek Salvador et al. 2020 Anguispira kochi occidentalis (E. von Martens, 1882) ON751968 ON749858 ON749851 Canada, BC, Dodge Creek Salvador et al. 2023 Anguispira kochi kochi (L. Pfeiffer, 1846) MW543392 MW544259 – USA, OH, Ottawa Co., Green Island Rankin et al. 2021 Anguispira kochi kochi (L. Pfeiffer, 1846) PV365190 PV364846 – USA, OH, Delaware Co., Scioto River, Bellepoint This study (voucher OSUM -IZG-4959.91) Anguispira kochi kochi (L. Pfeiffer, 1846) PV365191 PV364847 – USA, OH, Erie Co., Kelleys Island This study (voucher OSUMIZG-15891.2) Anguispira kochi kochi (L. Pfeiffer, 1846) PV365192 PV364848 – USA, OH, Ottawa Co., Gibraltar Island This study (voucher OSUM -IZG-46225.1) Anguispira kochi kochi (L. Pfeiffer, 1846) MN792587 MN756714 MN782444 USA, IL, Brown Co. Salvador et al. 2020 Discus marmorensis H.B. Baker, 1932 MW543387 MW544270 ON749853 USA, ID, Slate Creek Road / Lucile Rankin et al. 2021; Salvador et al. 2023 Gonyodiscus rotundatus (O.F. Müller, 1774) FJ917285 FJ917265 FJ917240 Germany, Frankfurt am Main Dinapoli and KlussmannKolb 2010 245 ZooKeys 1261: 241–260 (2025), DOI: 10.3897/zookeys.1261.171098 Robert G. Forsyth et al.: Species-level status of Anguispira kochi and Anguispira occidentalis Abbreviations The following acronyms are used throughout the text for natural history collections: CM: Carnegie Museum of Natural History (Pittsburgh, PA, USA); DMNH: Delaware Museum of Nature and Science (Wilmington, DE, USA); MCZ: Museum of Comparative Zoology, Harvard University (Cambridge, MA, USA); NBM: New Brunswick Museum (Saint John, NB, Canada); NHMUK: Natural History Museum (London, UK); Lc: Mollusk Collection, Zoological Museum, Moscow Lomonosov State University, Moscow, Russia; LIEV: Mollusk Collection, Oberösterreichisches Landesmuseum (Linz, Austria); OSUM: Gastropod Collection, Division of Invertebrate Zoology, Museum of Biological Diversity, The Ohio State University (Columbus, OH, USA); USNM: Smithsonian National Museum of Natural History (Washington, DC, USA); ZMB: Museum für Naturkunde (Berlin, Germany). DNA extraction and amplification Given the poor representation of A. kochi kochi in GenBank, we sequenced additional specimens from this species, vouchers of which are housed in the OSUM collection (Table 1). A small tissue clip was obtained from the foot of each voucher specimen and DNA extraction followed the standard protocol of the QIAGEN DNEasy® Blood & Tissue Kit, adding one repetition of the final step to increase yield. For the COI marker, the invertebrate primers LCO/HCO of Folmer et al. (1994) were used, while for 16S, primers 16SarL/16SbrH were used (Simon et al. 1994). The protocol for PCR amplification consisted of an initial denaturation step of 3 min at 96 °C, followed by 35 cycles of denaturation at 95 °C (30 s), annealing at either 48 °C (COI) or 51 °C (16S) (1 min), and extension at 72 °C (2 min); final extension step of 5 min at 72 °C. The success of PCR was assessed via agarose-gel electrophoresis. PCR products were cleaned using ExoSAP-IT™ (Affymetrix Inc.) and samples were prepared and sent to Macrogen Europe (Amsterdam, The Netherlands) for Sanger sequencing. The sequences were de novo assembled and quality-checked (via Phred scores) in Geneious Prime (v. 2025, Biomatters Ltd) and then uploaded to GenBank (Table 1). Phylogenetic analyses Alignment of the sequences was conducted via the MUSCLE plugin in Geneious Prime (Edgar 2004). The resulting alignments for each marker were visually proofed for inconsistencies in Geneious. Alignments of the 16S and 5.8S+ITS2+28S markers were run through Gblocks (Talavera and Castresana 2007), using the least restrictive settings, to eliminate poorly aligned or data-deficient positions that could introduce noise into the analysis. The final alignments contained 644 bp (COI), 389 bp (16S), and 1279 bp (5.8S+ITS2+28S). All alignments were then concatenated for a single multi-marker Bayesian inference phylogenetic analysis. This was performed using MrBayes (v. 3.2.7, Ronquist et al. 2012) via the CIPRES Science Gateway (v. 3.3, Miller et al. 2015). Two concurrent analyses with four Markov chains each were run for 50 million generations, with the first 20% discarded as “burn-in”. The default priors were used, with nst = 6 (GTR), rates = invgamma, temperature parameter = 0.1; substitution model parameters were unlinked across the markers (COI, 16S, and 246 ZooKeys 1261: 241–260 (2025), DOI: 10.3897/zookeys.1261.171098 Robert G. Forsyth et al.: Species-level status of Anguispira kochi and Anguispira occidentalis 5.8S+ITS+28S). MCMC convergence was assessed by examining the standard deviation of split frequencies (<0.001), potential scale reduction factor (PSRF = 1.0), as well as trace plots (Ronquist et al. 2009). The COI-only analysis was done with the same settings, except for the number of generations (40 million). Compared to the multi-marker analysis, this analysis included 134 additional sequences of A. k. kochi and A. k. occidentalis (Suppl. material 1). Geographical distribution Data were downloaded from Global Biodiversity Information Facility (GBIF) and cleaned of obviously erroneous records. For records without geographical coordinates, Google Earth, Google Maps, or Bing Maps were used to find, where possible, approximate geopositions. When the coordinates were too generalised (e.g. centre of a state) or when locality data were too vague to determine coordinates, records were discarded. Additional data were added to the GBIF data from the following sources: OSUM database; RGF database of collections and observations; Ovaska et al. (2020). The resulting dataset included 971 unique data points (Suppl. material 2), which were mapped in QGIS v. 3.38.1. The national and subnational borders were downloaded as .shp files from Natural Earth v. 5.1.1. The map was later modified in Adobe Illustrator v. 29.5.1. Photography As images of shells were received from multiple institutions with different photography equipment, colours and white balances were corrected in Adobe Lightroom Classic CC v. 14.4 using calibration cards included in each raw image. Specimen photographs from NHMUK and CM, as well as Canadian specimens in Figure 4, did not include colour calibration cards, so white balance was manually adjusted. Figures of specimens were combined in Adobe Photoshop v. 26.11.0. Results The multi-marker phylogenetic analysis, i.e. containing both nuclear and mitochondrial markers, included a total of 2312 bp (1033 mitochondrial and 1279 nuclear) and 21 terminals. The resulting tree largely agrees with former studies (Salvador et al. 2020, 2023; Rankin et al. 2021). In it (Fig. 1), Anguispira is a well-supported clade (posterior probability PP = 1), with A. nimapuna being the sister taxon to a clade (low support, PP = 0.85) containing all other species. After the branching of Discus marmorensis, there is a strongly supported (PP = 0.98) clade containing two sister groups: the first (PP = 1) includes A. kochi kochi and A. kochi occidentalis; the second (PP = 1) includes the remaining Anguispira spp., including the type species A. alternata. Notably, A. kochi kochi and A. kochi occidentalis each form monophyletic clades with strong support (PP = 1). The genetic distance between these two purported subspecies clades is similar or larger than between most other Anguispira spp. pairs (Fig. 1). While specimens within each of the kochi and occidentalis clades have 97– 100% identity of their COI sequences, the two clades have only 81–82% identity 247 ZooKeys 1261: 241–260 (2025), DOI: 10.3897/zookeys.1261.171098 Robert G. Forsyth et al.: Species-level status of Anguispira kochi and Anguispira occidentalis between themselves. Level of identity between the other Anguispira species range roughly from 75% to 82%. The COI-only tree (Suppl. material 3) included a total of 645 bp and 154 terminals. Again, the monophyly of both A. kochi kochi (PP = 1) and A. kochi occidentalis (PP = 1) can be observed. However, this tree shows more geographically delineated clades, with A. kochi kochi as the sister of all other Anguispira spp., and A. kochi occidentalis nested well within the crown group, being the sister species to Discus marmorensis. The distribution map (Fig. 2) shows, as expected, two unambiguously isolated clusters, which agrees with Pilsbry’s (1948) understanding of the range. Anguispira kochi occidentalis is restricted to the northwestern United States and southeastern British Columbia, Canada. Anguispira kochi kochi is distributed along several states in the eastern portion of the USA, being mostly restricted to the eastern states of the Midwest but also occurring in a few other areas. Additionally, several nominal subspecies of A. kochi are known from the islands of western Lake Erie in Ohio, USA and Ontario, Canada. We also took the opportunity to reassess the problematic records of our species of interest coming from unexpected locations (i.e. Utah and Colorado). To that end, we tracked down the voucher specimens in museum collections and obtained photographs and further information from museum staff. Specimens from lot CM 102529 were mistakenly assigned to Utah, but are in fact from the Figure 1. Bayesian inference phylogenetic tree (50% majority-rule consensus) based on the concatenated nuclear and mitochondrial markers. The clades formed by Anguispira kochi and Anguispira occidentalis are marked in different colours. Posterior probabilities are shown on nodes; the scale bar represents substitutions per site. 248 ZooKeys 1261: 241–260 (2025), DOI: 10.3897/zookeys.1261.171098 Robert G. Forsyth et al.: Species-level status of Anguispira kochi and Anguispira occidentalis Bitterroot Mountains, northern Idaho (T. Pearce pers. comm. 2025). Specimens USNM 853254 and USNM 853255 from Colorado are not Anguispira and have been reidentified as Oreohelix cf. strigosa (A.A. Gould, 1846). Schileyko (2002: 1060, fig. 1384) figured the shell and reproductive anatomy of specimens he identified as A. kochi kochi (Lc-20459 and Lc-20442), from “Weeping Point, Zion Canyon, [Utah]”, likely from the same original lot as LIEV 2011/14/7095 (all are from the C. Frank-Fellner Collection). We were unable to examine Schileyko’s specimens but conclude that they are all likely Oreohelix cf. strigosa based on the location: “Weeping Point, Zion Canyon” is likely the same place as “Weeping Rock”, a famous landmark in Zion National Park where O. strigosa is known to occur (Gregg 1940). Discussion According to both the genetic data (both mitochondrial and nuclear) and the geographical distribution, we can consider Anguispira kochi and A. occidentalis as distinct species. Results from the phylogenetic analysis agree with previous studies (Salvador et al. 2020, 2023; Rankin et al. 2021). The genetic distance between A. kochi and A. occidentalis is similar or greater than that observed between most species pairs in the genus in the multi-marker phylogeny (Fig. 1). That is also seen in the pairwise identity values between clades. Furthermore, Figure 2. Map showing the geographical distribution of Anguispira kochi (red dots) and Anguispira occidentalis (blue dots). Occurrence data used to map the species are available in Suppl. material 2. 249 ZooKeys 1261: 241–260 (2025), DOI: 10.3897/zookeys.1261.171098 Robert G. Forsyth et al.: Species-level status of Anguispira kochi and Anguispira occidentalis the geographical ranges of A. kochi and A. occidentalis are entirely isolated, with a large gap (>2,00 km) between them (Fig. 2). When taken together, this evidence indicates that the split between A. kochi and A. occidentalis is an old one. The COI tree, with a large number of terminals, also argues in favour of this interpretation. This mitochondrial marker shows that there is a greater similarity between A. occidentalis and other Anguispira spp. than with A. kochi (Suppl. material 3). That could be expected in a tree consisting solely of COI, as it is observed that mitochondrial lineages of these slow-dispersing animals often form geographical clusters (e.g. Nekola et al. 2015, 2025b). Thus, such a pattern in mitochondrial markers is usually interpreted as evidence of mitochondrial introgression taking place between sympatric or parapatric congeners (e.g. Nekola et al. 2025a). Overall, the two species have shells of similar shape (Figs 3, 4), being discoid-conical to subglobose (varying in spire height and overall globosity of the shell), with 5–5½ whorls, a deeply incised suture, marked sinuous axial striation, a simple lip only slightly thickened in adults, and an open umbilicus. Shell colour varies from lighter to darker tones of brown, with two dark spiral bands (which can be externally invisible but still visible inside the aperture). Shell size averages 2.0–2.5 cm in width, with larger specimens reaching up to 3 cm (Clapp 1916; Forsyth 2004; Grimm et al. 2009; COSEWIC 2017). The animal’s head and tentacles are grey, and the foot is orange-red to brown. When disturbed, they produce a slightly orange mucus that fluoresces under UV light (Dourson and West Virginia DNR 2015; COSEWIC 2017). Typically, A. occidentalis has a darker shade of brown as base colour, the spiral bands are less conspicuous, and often the region between the spiral bands seems lighter. On the other hand, A. kochi typically has a lighter base colour that contrasts more with the dark spiral bands. This distinction in shell colour is the most straightforward manner to diagnose most specimens (besides geographical provenance, of course), but as Pilsbry (1948) noted, there is variation in both species, and some paler-shelled A. occidentalis can be very similar to A. kochi. In general, A. kochi appears to be the most variable in shell morphology, varying from coarsely striate to nearly smooth, and from bandless and straw-coloured to brown and banded (COSEWIC 2017). Anguispira species are detritivores, feeding on decaying plant material, fungi and lichens on logs or wood (Ali pers. obs.), but they can differ in their habitats. As expected from their distinct biogeographical provinces, A. kochi and A. occidentalis show differences in habitat use. Anguispira kochi has been recorded in ravines and upland forests, being a typical species of old forests and seldom found in second-growth areas (even in thick ones) (Hubricht 1985); they prefer a thick covering of leaf litter and limestone terrain (Pilsbry 1948; Dobbyn and Hoare 2009). Anguispira occidentalis is typically found in riparian habitats along rivers, streams, and creeks in mixed-wood or deciduous forests, frequently in the leaf-litter layer or under coarse woody debris (Forsyth 2004; Ovaska et al. 2020). Infraspecific taxa Anguispira kochi was described as Helix kochi L. Pfeiffer, 1846 from an unknown locality in the United States (Pfeiffer 1846). Its type locality was subsequently restricted to Cincinnati, Ohio, by Pilsbry (1948: 592), who also pinpointed the type specimen and, in doing so, designated the lectotype according to ICZN 256 ZooKeys 1261: 241–260 (2025), DOI: 10.3897/zookeys.1261.171098 Robert G. Forsyth et al.: Species-level status of Anguispira kochi and Anguispira occidentalis Author ORCIDs Robert G. Forsyth https://orcid.org/0000-0002-9637-0158 Annegret Nicolai https://orcid.org/0000-0001-7099-5603 Nathaniel F. Shoobs https://orcid.org/0000-0002-5272-6255 Reham F. Ali https://orcid.org/0000-0003-3368-8107 Rodrigo B. Salvador https://orcid.org/0000-0002-4238-2276 Data availability All data that support the findings of this study are available in the main text or Supplementary Information. 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Diversity & Distributions 12(6): 756–766. https://doi.org/10.1111/j.1472-4642.2006.00289.x Supplementary material 1 Sequences used for the COI-only phylogenetic analysis, with information on their GenBank accession numbers, the locality where the specimens were collected, and the source of the data Authors: Robert G. Forsyth, Annegret Nicolai, Nathaniel F. Shoobs, Reham F. Ali, Rodrigo B. Salvador Data type: xlsx Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). 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.1261.171098.suppl1 Supplementary material 2 Occurrence data used to produce the map of Anguispira kochi and A. occidentalis (Fig. 2) Authors: Robert G. Forsyth, Annegret Nicolai, Nathaniel F. Shoobs, Reham F. Ali, Rodrigo B. Salvador Data type: xlsx Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). 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.1261.171098.suppl2 260 ZooKeys 1261: 241–260 (2025), DOI: 10.3897/zookeys.1261.171098 Robert G. Forsyth et al.: Species-level status of Anguispira kochi and Anguispira occidentalis Supplementary material 3 Bayesian inference phylogenetic tree (50% majority-rule consensus) based on the COI barcoding marker Authors: Robert G. Forsyth, Annegret Nicolai, Nathaniel F. Shoobs, Reham F. Ali, Rodrigo B. Salvador Data type: pdf Explanation note: Posterior probabilities are shown on nodes; the scale bar represents substitutions per site. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). 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.1261.171098.suppl3