#ZooKeys ZooKeys 1228: 69-97 (2025) DOI: 10.3897/zookeys.1228.142202 Research Article A cryptic new species of tiger swallowtail (Lepidoptera, Papilionidae) from eastern North America Charles J. DeRoller’, Xi Wang?2, Julian R. Dupuis’, B. Christian Schmidt*© Kingston, Ontario, Canada Fe wo NY PO Box 374, Victor, New York, 14564, USA Department of Entomology, University of Kentucky, Lexington, KY, 40546, USA Canadian National Collection of Insects, Arachnids, and Nematodes, Biodiversity Program, Agriculture and Agri-Food Canada, Ottawa, ON, Canada Corresponding author: B. Christian Schmidt (christian.schmidt@agr.gc.ca) OPEN Qaccess Academic editor: Shinichi Nakahara Received: 19 November 2024 Accepted: 14 January 2025 Published: 14 February 2025 ZooBank: https://zoobank.org/ FOAEECC8-82AD-48E3-8FBE- SCA6F883420F Citation: DeRoller CJ, Wang X, Dupuis JR, Schmidt BC (2025) A cryptic new species of tiger swallowtail (Lepidoptera, Papilionidae) from eastern North America. ZooKeys 1228: 69-97. https://doi.org/10.3897/ zookeys.1228.142202 Copyright: © This is an open access article distributed under the terms of the CCO Public Domain Dedication. Abstract In the eastern Great Lakes region of North America, two tiger swallowtail species have pre- viously been recognized, Papilio glaucus Linnaeus, 1758 and Papilio canadensis Rothschild & Jordan, 1906. A third entity, the Midsummer Tiger Swallowtail, has been treated as a P. glaucus x canadensis hybrid, and exhibits a mosaic of both intermediate and unique mor- phological and biological traits. Here we demonstrate that rather than being a localized, his- torically recent hybrid phenomenon, the Midsummer Tiger Swallowtail maintains its mor- phological and physiological distinctness over a large geographic region in the absence of one or both putative parental species, and was first documented in the literature nearly 150 years ago. Papilio solstitius sp. nov. is physiologically unique in delaying post-diapause development, which results in allochronic isolation between the spring flights of P. glau- cus and P. canadensis, and the late summer flight of P. glaucus. Similarly, the geographic range of Papilio solstitius spans the region between the northern terminus of P. glaucus and southern limits of P. canadensis, remaining distinct in areas of sympatry. Defining the taxonomic identity of this unique evolutionary lineage provides an important baseline for further inquiry into what has served as an exemplary species group in evolutionary study. Key words: cryptic species, hybrid, Papilio glaucus, Papilionidae, Pterourus, speciation Introduction The North American Papilio glaucus species group (Lepidoptera: Papilionidae) is amodel study system in insect evolutionary biology. The recognition and de- limitation of P. glaucus L., 1758 and P. canadensis as a classic sibling species pair (Hagen et al. 1991; Sperling 1993) led to three decades of study in specia- tion, host plant adaptation, hybridization, and molecular evolution (e.g., Ryan et al. 2017 and references therein). More recently, the discovery of a third species, P. appalachiensis Pavulaan & Wright, 2002, has provided unprecedented insight into speciation via hybridization (Scriber and Ording 2005; Kunte et al. 2011; Cong et al. 2015; Vernygora et al. 2022). Papilio appalachiensis is now recog- nized as a homoploid hybrid species with origins from P. glaucus x P. canaden- sis crosses some 0.4 million years ago (Cong et al. 2015; Kunte et al. 2011). 69 Charles J. DeRoller et al.: A new species of tiger swallowtail The Papilio glaucus group previously comprised nine species (Kunte et al. 2011; Pavulaan 2024), and the five eastern North American species discussed herein are termed the glaucus complex. All are very similar in external appear- ance, and prior to 1991, were included within the concept of a single species, P glaucus. Subsequently, three species were recognized: The Eastern Tiger Swal- lowtail (P. glaucus) which occurs across most of eastern USA and as far north as southwestern Ontario, and south of the Adirondack and Catskill Mountains in New York; the more northern Canadian Tiger Swallowtail (P. canadensis) that oc- curs across the boreal region from Newfoundland to Alaska, and as far south as southern Ontario and the northern Appalachians; and the Appalachian Mountains endemic P. appalachiensis, found from Pennsylvania to Georgia (Pavulaan and Wright 2002). The recently described New England Swallowtail, Papilio bjorkae Pavulaan, 2024, may be conspecific with P. canadensis or P. glaucus; as detailed below in “Comparative morphology of the Papilio glaucus-complex,’ incomplete knowledge of P. bjorkae’s morphology, range, biology, and taxonomic status cur- rently precludes full comparison to the remainder of the P. glaucus group. The Papilio glaucus group is part of a larger, predominantly New World clade of swallowtails of the subgenus Pterourus Scopoli, sometimes recognized as a distinct genus (e.g., Pelham and Pohl 2023). The broader concept of the genus Papilio L. is used herein, in agreement with the results and reasoning presented by Condamine et al. (2023). Each of the glaucus-complex species show adaptation to different thermal niches that can be broadly characterized as warm (P. glaucus), intermediate (P. appalachiensis), and cool (P. canadensis) climatic regions; all have broad larval host plant diets, and are not restricted by the distributions thereof. At coarse geographic scales, species distributions appear parapatric, but at finer spatial scales, multiple taxa can overlap (Fig. 1). The transition or contact zone between P. glaucus and P. canadensis has received considerable study. West of Lake Michigan, introgression and hybridization have been well-documented through morphometric and molecular studies (Luebke et al. 1988; Ryan et al. 2016, 2017, 2018). Here, a narrow hybrid zone (50-100 km wide) is maintained by strong selective pressure for adaptation to either warm or cool thermal re- gimes, with a rapid geographic shift from P. glaucus to P. canadensis across a threshold thermocline (Fig. 1; Scriber 2010; Ryan et al. 2016, 2017, 2018). In the topographically and climatically complex region of eastern Ontario and adjacent New York, the relationship between P. canadensis and P glaucus is less straightforward. Unlike the central Great Lakes region to the west, the ranges of P canadensis and P glaucus are more poorly defined as a result of confusing phenotypes and phenologies, making identification difficult. In northern New York, Vermont and eastern Ontario, univoltine tiger swallowtails with a July flight period have variously been called P. glaucus (Shapiro 1974; Layberry et al. 1998; Hall et al. 2014), “false second generation” (Hagen and Lederhouse 1985), “late flight P canadensis” (Scriber and Ording 2005; Kunte et al. 2011), “hybrid types” (Scriber 1990), “late flight” (Scriber 2010), “late flight hybrids” (Wang 2018), “delayed ‘late flight’ hybrid swarm” (Scriber 2010), “a stable hybrid” (Zhang et al. 2013), “intermediate individuals” (Vernygora et al. 2022), “midsummer tiger swallowtail” (Schmidt 2020), and “divergent ecomorphs” (Vernygora et al. 2022). Hagen and Lederhouse proved that this taxon is not the second annual gener- ation of any spring-flying swallowtails, instead representing a single-brooded ZooKeys 1228: 69-97 (2025), DOI: 10.3897/zookeys.1228.142202 70 Charles J. DeRoller et al.: A new species of tiger swallowtail ZAHA LL Lge LE MT tL Figure 1. Geographic ranges of the Papilio glaucus-complex in eastern North Ameri- ca. Papilio glaucus (diagonal lines), P. canadensis (horizontal lines), P. appalachiensis (blue), and P. solstitius sp. nov. (red). In the central Great Lakes region, a sharp transition or hybrid zone occurs between P glaucus to the south and P canadensis to the north, indicated by the orange dashed line. In the northern Appalachian region this transition zone is much larger as a result of topography-induced climatic variation, with elevational rather than latitudinal separation. Considerable uncertainty exists in the northern range limit of P glaucus in NY (see “Habitat and distribution” section). Distribution data based on Luebke et al. (1988); Stump et al. (2003); Pavulaan and Wright (2002); Mcnaughton et al. (2020) and specimens verified in this study (Suppl. material 1). taxon physiologically distinct from P glaucus and P. canadensis (Hagen and Le- derhouse 1984; Scriber and Ording 2005). This taxon is now referred to by the common name Midsummer Tiger Swallowtail (MST; Schmidt 2020). Here, we present evidence that MST is not the result of historically recent hybridization between P glaucus and P. canadensis as suggested by Kunte et al. (2011); literature and specimen records of MST date back 150 and 50 years, respectively. MST was also previously thought to be geographically localized to areas of P glaucus - P canadensis overlap, but this is also not the case. MST exhibits a large geographic range that includes regions where one or even both putative parent species are absent (Fig. 1). Lastly, the unique late-season flight acts as an allochronic reproductive barrier between MST and other tiger swallowtails. Based on combined molecular, phenological, morphological, and natural history data, the Midsummer Tiger Swallowtail is described as a new species, Papilio solstitius sp. nov. Methods and materials Field studies and specimen collections were carried out from 1999 to 2023 in Pennsylvania, Virginia, Kentucky, and the Finger Lakes region of New York (CJD); and from 2008 to 2023 in eastern Ontario (XW, BCS). Host plant suit- ZooKeys 1228: 69-97 (2025), DOI: 10.3897/zookeys.1228.142202 71 Charles J. DeRoller et al.: A new species of tiger swallowtail ability, larval development, and adult emergence were studied based on ex ova and ex larva rearings from 2008 to 2011 in Hamilton, Ontario, and from 2015 to 2022 in Kingston, Ontario (XW). All larvae were reared indoors at a constant 23 °C under outdoor ambient light conditions. Larvae were provided with cut- tings of the host they were found on, either green ash (Fraxinus pennsylvanicus Marshall) or black cherry (Prunus serotina Ehrhart), held in small vials of water. Pupae that were entering diapause rather than direct development did not ex- hibit melanization of the eyes (visible by transillumination) after 2-3 weeks and were placed in cold storage, either in a conventional refrigerator or unheated garage. After removal from cold storage, they were again kept at a constant 23 °C and time to eclosion recorded. Adult genitalia were prepared following the protocol detailed in Schmidt (2018) and imaged using a Leica DFC 450 camera mounted on a Leica M205C stereo microscope. Where confident identification was possible, distribution and phenology data were augmented with records from iNaturalist (inaturalist.org), eButter- fly (e-butterfly.org), and the Ontario Butterfly Atlas (Macnaughton et al. 2020). Manual calipers precise to the nearest 0.1 mm were used for wing measure- ments. Occurrence maps were created with SimpleMappr (https://www.sim- plemappr.net). Voucher specimens examined in this study (Suppl. material 1) are found in the following collections: CNC Canadian National Collection of Insects, Arachnids and Nematodes, Ottawa, CAN CMNH_ Carnegie Museum of Natural History, Pittsburgh, PA, USA CJDC Charles J. DeRoller Collection XWC Xi Wang Collection Molecular datasets Publicly available sequences and previous DNA barcoding efforts in the P. glau- cus group have focused on both the 5’ region of the mitochondrial cytochrome oxidase subunit | (COI) gene (the standard barcode region (Hebert et al. 2003) using primers LCO1490 and HCO2198 (Folmer et al. 1994)) and the 3’ region of COI (primers Jerry and Pat, as used in Kunte et al. 2011; Vernygora et al. 2022). Unfortunately, few, if any, specimens have been sequenced for both re- gions, so we are limited to considering these regions separately and hereafter refer to them as COI5 and COl3, respectively. Fourteen MST specimens were sent to a private COI5 barcoding service; Sanger sequencing was performed by Azenta Life Sciences (Chelmsford, Massachusetts, United States), and consensus sequences were constructed using de novo assembly in Geneious Prime v. 2024.0 (uploaded to BOLD with accessions provided in the associated figure). Additionally, COI5 barcodes were generated for two P. appalachiensis (UASM400650 and UASM400651, also sequenced by Vernygora et al. 2022), to ensure representation of that species in the COI5 dataset (NCBI GenBank ac- cessions: PQ578215.1 and PQ578216.1). Sequencing and analysis were con- ducted as in Vernygora et al. (2022). COI5 and COI3 sequences were retrieved from GenBank (September 2024) for all species in the glaucus-complex and aligned to a complete P glaucus mitogenome (NC_027252.1). Outgroup taxa ZooKeys 1228: 69-97 (2025), DOI: 10.3897/zookeys.1228.142202 72 Charles J. DeRoller et al.: A new species of tiger swallowtail were also selected as in Vernygora et al. (2022). Unique and pertinent COI5 se- quences in the BOLD database (i.e., those of P glaucus and P. canadensis from NE USA and SE Canada) were added to this dataset, and we used AliView v1.28 (Larsson 2014) to align sequences either manually or using default settings with MUSCLE (Edgar 2004). We used IQ-Tree v. 2.3.5 (Nguyen et al. 2015) to conduct maximum likelihood tree searches using the best model identified by Bayesian Information Criterion with ModelFinder (Kalyaanamoorthy et al. 2017). One thousand replicates of ultra-fast bootstrap (ufBS, Hoang et al. 2018) and the Shimodaira-Hasegawa approximate likelihood ratio test (SH-aLRT, Guindon et al. 2010) were used to assess nodal support. The genomic phylogeny using 3,733 single nucleotide polymorphisms (SNPs) from Vernygora et al. (2022) was also considered (we focused on the majority rule consensus tree generat- ed from MrBayes (Ronquist et al. 2012), although see Vernygora et al. (2022) for more details on their thorough analysis), and we reevaluated the morpholo- gy of those specimens noted as “intermediates” in their analyses. All trees were visualized with FigTree v. 1.4.4 (Rambaut and Drummond 2010). Results Taxonomic names currently in synonymy under P. glaucus and P. canadensis were reviewed and revised by Pavulaan and Wright (2002). Our review of these synonymies confirms that all taxon names are correctly attributed to their re- spective species, and do not apply to the Midsummer Tiger Swallowtail. As such, a new name is proposed here. Papilio solstitius sp. nov. https://zoobank.org/A9B99C5C-E8EC-4AA 1-A6E6-B09E610E3389 Figs 3a, 4, 5, 6a, 7a, 8a, 9c-d, 10a, 11 Type locality. Canada, Ontario, Ottawa-Carleton District, Long Swamp, Old Al- monte Rd., 45.249°N, 76.079°W. Type material. Holotype (Fig. 4a) * male. Ontario, Ottawa-Carleton Dist., Old Al- monte Rd. at Long Swamp, 45.249°N, 76.079°W, 3.Jul.2020, B.C. Schmidt, CNC voucher # CNCLEP00342771 [CNC]. Allotype (Fig. 4b) - female. Ontario, Fron- tenac Co., Vanalstine Lake, 44.858°N, 76.847°W, 5.Jul.2021, B. C. Schmidt, ob- served ovipositing on Prunus serotina [CNC]. Paratypes + 53 in CNC, 9 in XWC, 8 in CJDC; complete data and specimen deposition are given in Suppl. material 1. Etymology. The epithet solstitius is derived from solstitium, the Latin term for solstice. The species’ unique midsummer flight period commences near the summer solstice. Differential diagnosis. Papilio solstitius is closely related to P. glaucus, P. canadensis and P. appalachiensis, but differs from all in a suite of charac- ters (Table 1). The most significant differences are apparent in developmen- tal biology and phenology. Papilio solstitius is unique in its long post-diapause emergence delay, with adult eclosion beginning in late June to early July, com- pared to May for all other species (Fig. 2). Unlike the facultatively multivoltine P. glaucus, P. solstitius is obligately univoltine (like P. canadensis and P. ap- palachiensis). In the northern part of its range, P. solstitius overlaps with P. ZooKeys 1228: 69-97 (2025), DOI: 10.3897/zookeys.1228.142202 73 Charles J. DeRoller et al.: A new species of tiger swallowtail Table 1. Comparison of morphological traits among species of the Papilio glaucus-complex. FW = forewing; DFW = dor- sal forewing; VFW = ventral forewing; HW = hindwing; DHW = dorsal hindwing; VHW = ventral hindwing. Forewing length is geographically variable in P canadensis and P glaucus, and values are based on Ontario specimens. Trait Head - setation of frons Average FW length (range): male Average FW length (range): female FW shape - distal margin DFW - frequency of medial band black scales extending beyond Cu2 (male) VFW margin: submargin- al band VFW margin: inner (prox- imal) border HW shape HW tails HW margin HW anal cell black band width (male) DHW (female) submar- ginal orange lunule in cell Sc+R1 DHW female blue scaling VHW marginal lunules VHW marginal lunule ScR1 of female VHW submarginal black band: inner border of 3 interspaces between Sc to M2 VHW anal margin se- tation Abdomen shape Abdomen subdorsal yellow stripe Male valve scales Larva: 1* instar posterior white patch Larva: 1* instar anterior white patch P. solstitius sp. nov. intermediate, compact 51 mm (42-57 mm) 53 mm (48-56 mm) usually straight to slight- ly concave; concave frequency 40-50% 10-15% broadly coalescent lunules, usually with scalloped inner border moderate amount of yellow dusting over black inner half elongate spatulate less scalloped 40-50% Smaller than remaining lunules, sometimes a mere dot sparse lunules rectangular to slightly crescentic length less than that of other lunules, often much more so slightly scalloped sparse setation narrow, attenuated anteriorly broad, bright yellow, lateral black line well defined but narrow solid yellow scales; clasper same shade abdomen usually present; rarely absent or faint usually present; rarely absent or faint P. canadensis long and diffuse 46 mm (41-50 mm) 48 mm (47-50 mm) usually straight to slightly convex; concave frequency 25-30% 55-70% continuous band with straight inner and outer border; varying to coales- cent rounded-rectangular elements, but lunules never well-separated by black extensive yellow dusting over black inner half broad / rounded aspatulate to slightly spat- ulate less scalloped 55-90% Smaller than remaining lunules, sometimes a mere dot none to minimal lunules more rectangular length less than that of other lunules, often much more so more linear than scalloped long, dense setation shorter, broad anteriorly narrower, less vivid yellow; sublateral black line wide yellow with sparse black scales, clasper often appear- ing darker than abdomen always present and well-de- veloped always present and well-de- veloped * based on images and information in Pavulaan (2024a). P. glaucus short and compact spring: 50 mm (43-52 mm); summer: 54 mm (45-58 mm) spring: 53 mm (50- 55 mm); summer: 57 (49-64 mm) usually concave; concave frequency >80% < 15% discrete lunules distinctly separated by black line along veins; varying to coalesced lunules with scalloped inner and outer margin extensive yellow dusting over black inner half elongate spatulate scalloped 10-40% (summer); 20-50% (spring) Much larger than remain- ing lunules sparse to extensive crescentic lunules conspicuously larger/ deeper than other lunules scalloped sparse to very sparse setation narrow, attenuated an- teriorly broad, bright yellow, sub- lateral black line faint or partially absent solid yellow scales; clasp- er same shade abdomen absent absent P appalachiensis intermediate, compact 50-62 mm 50-65 mm usually straight < 20% broadly coalescent lunules, usually with scalloped inner border extensive yellow dust- ing over black inner half more triangular than glaucus aspatulate to slightly spatulate less scalloped average ~50% Slightly larger than remaining lunules sparse lunules more rectan- gular similar in size to other lunules slightly scalloped sparse setation moderately attenuated anteriorly broad, bright yellow, lateral black line well defined but narrow solid yellow scales; clasper same shade abdomen absent or faint (tan) absent P. bjorkae* short and compact male and female combined: 49.2 mm (43-55 mm) (unknown) concave (unknown) continuous band, some- times with coalescent lunules anteriorly extensive yellow dust- ing over black inner half more angular than glaucus slightly to well-spat- ulate less scalloped 40-50% (based on 2 illustrated specimens) Slightly larger than remaining lunules sparse lunules rectangular to slightly crescentic conspicuously larger/ deeper than other lunules scalloped (not given) (not given) (not given) (not given) unknown unknown canadensis, and in the south with P. glaucus; it is not known to overlap with P appalachiensis (Fig. 1). Identification difficulties are therefore largely limited to confusion with either P canadensis or P. glaucus. In combination with location and date, the comparative morphological characters summarized in Table 1 ZooKeys 1228: 69-97 (2025), DOI: 10.3897/zookeys.1228.142202 74 Charles J. DeRoller et al.: A new species of tiger swallowtail 100 40 20 joe tc) oy. LU E Mar M Mar L Mar E-Apr M-Apr L-Apr E-May M-May L-May E-Jun M-Jun L-Jun E-Jul M-Jul L-Jul E-Aug M-Aug L-Aug M-Sep L-Sep E Oct M Oct L Oct E Nov Figure 2. Phenology of Papilio glaucus group species from three regions, based on combined observations for all spe- cies and grouped by 10-day intervals a eastern Ontario (Hastings Co., Frontenac Co.; Mcnaughton et al. 2020), with P canadensis peaking in late May — early June and P solstitius in early to mid-July b finger Lakes region, New York (Naturalist), with a late May — early June peak of spring P glaucus and a July — August peak of P. solstitius and summer P glaucus; note later seasonal persistence and late-shifted peak resulting from summer P. glaucus, which is absent in eastern Ontario (2a) c Greater Toronto region, with a spring peak comprised of P canadensis and spring brood P glaucus, and a July peak of P. solstitius; summer brood (August) P. glaucus are very rare. imm Figure 3. Dorsal view of head (antennae removed for clarity) comparing profile of frontal setae ina P. solstitius b P canaden- sis c P glaucus and d P. appalachiensis. Scale bar: 1 mm. ZooKeys 1228: 69-97 (2025), DOI: 10.3897/zookeys.1228.142202 75 Charles J. DeRoller et al.: A new species of tiger swallowtail Figure 4. a dorsum of Papilio solstitius, male, holotype, ventrum on right. Long Swamp, Old Almonte Rd., Ottawa, Ontario, CAN. CNC voucher # CNCLEP00342771 b dorsum of Papilio solstitius, female allotype, ventrum on right. Vanalstine Lake, Frontenac Co., Ontario, CAN; ovipositing on Prunus serotina. Scale bar:10 mm. and discussed in the “Comparative Morphology” section below will serve to identify most specimens. Description of adult. Head (Fig. 3) and thorax: setation of frons of mod- erate length, intermediate between P. canadensis and P. glaucus; dorsum of ZooKeys 1228: 69-97 (2025), DOI: 10.3897/zookeys.1228.142202 76 Charles J. DeRoller et al.: A new species of tiger swallowtail Submarginal R 3 band Rg Anal band Cuz Cu, Figure 5. Papilio solstitius, ventrum, wing vein and pattern terminology. head and thorax with limited sublateral yellow scaling; ventral thorax vestiture pale lemon yellow, legs black. Forewing (Figs 4, 5, 6): Male forewing length 50.7 mm (46.7-55.0 mm; n = 17), female 53.4 mm (47.7-57.0 mm; n = 8); dorsal ground color of male mustard yellow (Ridgway 1912), of female light orange yellow (Ridgway 1912), like that of P. glaucus but slightly richer in tone than P. canadensis; female mimetic dark phase absent; all pattern elements flat black; antemedial band an elongate wedge variable in thickness and edge, on average attenuating more strongly between Cu and anal margin than in P. canadensis; medial band an irregular rectangular bar across discal cell, vari- ably extending as far as vein Cu, or slightly beyond (in P. canadensis the medial band is more extensive, more frequently extending past Cu, and sometimes to 2A); subapical black bar well-defined in cell R,-R,, diminishing across R,-M,, more strongly so than in P. canadensis; costa and subapical bar with diffuse yellow streaking, generally more so than in P. canadensis; females with wider, more diffuse transverse black bands than males; marginal band solid black with 6-8 yellow rounded-ovoid submarginal spots in interspaces; pattern elements repeated on ventral forewing, but ground color paler yellow, and black elements of distal half of wing with a flush of yellow scales; submarginal band variable but comprised of essentially D-shaped yellow spots usually separated by black lines along veins; yellow spots wider and more confluent than in P. glaucus, but more discrete and irregular than the essentially continuous, even-bordered band of P. canadensis. Hindwing: (Figs 4, 5, 7): Like P. glaucus, the scalloping of the hindwing outer margin is more pronounced than in P. canadensis, as a result of the disc margins oriented closer to the perpendicular of the long axis of the hindwing; the tail and Cu, angle are slightly more lunate/lobate than in P. canadensis; ground color identical to that of forewing; inner margin bordered ZooKeys 1228: 69-97 (2025), DOI: 10.3897/zookeys.1228.142202 77 Charles J. DeRoller et al.: A new species of tiger swallowtail C Figure 6. Comparison of variation in ventral forewing submarginal band in a P solstitius b P canadensis and c P glaucus. in black across 35-50% of cell 2A-Cu,; narrow, straight medial line attenuating towards juncture with anal band near Cu,,; end of discal cell veins black-scaled; black marginal band extending along distal quarter of wing, with diffuse yellow dusting from vein M, to anal angle; yellow submarginal lunules in the four cell spaces between Rs and Cu7; lunules of cell ScR,-Rs and Cu,-Cu, (i.e., the upper- most and lowermost lunules) reduced or absent, orange or orange and yellow when present; anal angle with orange crescent capped proximally with blue, black bordered crescent; males with diffuse blue crescent in cell Cu,-Cu,, often faint, rarely traces of blue crescent in adjacent cell Cu,-M,; females with more extensive blue scaling, often with diffuse crescents extending to costal edge of submarginal band; ventral hindwing paler than dorsum, and with dusting of yellow scales across marginal band, and with more prevalent orange scaling in submarginal lunules and basad of marginal band in cells M,-2A; yellow setae ZooKeys 1228: 69-97 (2025), DOI: 10.3897/zookeys.1228.142202 78 Charles J. DeRoller et al.: A new species of tiger swallowtail Figure 7. Comparison of variation in ventral hindwing anal band ina P solstitius and b P canadensis and ¢ P glaucus. along anal band shorter and sparser than in P. canadensis. Abdomen: dorsum black, pale yellow laterally and ventrally with black sublateral line; vestiture of mixed yellow and black fine, setae; scales of male clasper entirely yellow (Fig. 8); clasper of male valve with two dorsal tines (Fig. 9). Description of larva. First instar (Fig. 10) with well-developed white medial saddle, comprised of predominantly white dorsal pigmentation of segments A3-A4; three additional, variably developed white bands, one each comprised of T1 and T3, and a posterior band formed by A8; Anterior and posterior bands rarely absent (entirely brown pigmentation); mature larva (Fig. 11) indistin- guishable from that of P glaucus and P. canadensis. Comparative morphology of the Papilio glaucus-complex Adult morphology of all eastern North American species in the glaucus-com- plex can be deceivingly similar, and any single morphological character should not be relied upon for identification. Most similar to P. solstitius are P. glau- cus, P canadensis and potentially P. bjorkae, another new species in the glaucus-complex proposed in 2024 (Pavulaan 2024). Given its recency, the taxonomic status of P bjorkae has not yet been scrutinized by the scientific community, but it is necessary to do so here. For the reasons detailed below the recognition and diagnosis of P. bjorkae is currently problematic, although based on the spring flight period and comparison of the figures in the original description (Pavulaan 2024), it is certain the name does not apply to MST. The justification for treating P. bjorkae as a distinct species hinges on rec- ognition of three distinct, partially sympatric, spring-flying taxa, recognized by adult phenotypes (P. glaucus, P. “near canadensis,’ P. bjorkae) which correlate with slightly different flight periods (Pavulaan 2024). No diagnostic differences in immature stages, biology, larval hosts, or molecular markers of P. bjorkae have been documented to date (Pavulaan 2024), nor is there evidence in previ- ous research that might hint at the existence of such (e.g., Ording et al. 2010; ZooKeys 1228: 69-97 (2025), DOI: 10.3897/zookeys.1228.142202 79 Charles J. DeRoller et al.: A new species of tiger swallowtail Figure 8. Comparison of scale coloration of male valve in a P solstitius and b P canadensis. Clasper color in P. glaucus (not shown) is identical to P. solstitius Figure 9. Inner surface of male right valve of a P solstitius b PR glaucus c, dP canadensis c and d show variation in dorsal clasper tines from the same individual (image of left valve (d) is flipped for ease of comparison). Kunte et al. 2011). Using seasonal adult abundance peaks combined across the glaucus-complex, flight phenologies for taxa present within the range of P. bjor- kae are attributed to spring (P. glaucus, P. canadensis, and P. bjorkae), summer ZooKeys 1228: 69-97 (2025), DOI: 10.3897/zookeys.1228.142202 80 Charles J. DeRoller et al.: A new species of tiger swallowtail (midsummer swallowtail), and late summer (Second-generation P. glaucus) (Pavulaan 2024: figs 3-5). During spring (May through June), P. bjorkae flies in “late spring,’ versus “early spring” for P. glaucus and P. canadensis. However, only a single spring abundance peak is evident and attributed to P. bjorkae, whereas neither P. glaucus nor P. canadensis peaks are distinguishable due to the relative scarcity of observations for these species (Pavulaan 2024: 7, figs 3, 4). No additional data are provided to define late- versus early spring, leaving it unclear to what extent the phenology of P. bjorkae differs. Life history data that could corroborate such a difference are currently lacking. The differential diagnosis of P. bjorkae is based largely on differences in wing pattern and shape, especially of the female (Table 1). Males are described as intermediate between P. glaucus and P. canadensis; comparative differences are given compared to P. appalachiensis and P. canadensis, but not P. glaucus (Pavulaan 2024: 16). Without an indication of sample size and a full description of male and female morphology, it is currently difficult to gauge intra- versus in- terspecific variation. Lastly, P bjorkae is stated to be larger than spring P. glau- cus and P. canadensis, but conflicting information on p. 9 states that P. glaucus is the largest species in the study region. No size measurements specific to male or female are given for P. bjorkae (including the holotype), nor is it possi- ble to infer size of specimens from figures since scale bars are not given; size as a diagnostic trait for P. bjorkae therefore remains undefined. The adult phenotype of P. bjorkae is very similar to that of P. canadensis and P. glaucus, so attributing phenotypic variation to three different putative taxa requires careful assessment. A potential additional source of phenotyp- ic variation which remains unstudied stems from seasonal polymorphism in P. glaucus. Contrary to the assumption that P. glaucus is obligately bivoltine at the northern range edge (Pavulaan 2024), Ryan et al. (2016) demonstrate that it can be uni- or bivoltine depending on thermal constraints. In other words, temperature and day length experienced during the larval stage of P. glaucus dictate whether or not pupae develop directly into second generation adults, or enter winter diapause to emerge the following spring (Ryan et al. 2016). Since adult phenotype of P. glaucus is influenced by different tempera- ture-photoperiod profiles (different spring and summer forms are well-known in P. glaucus, e.g., Pavulaan and Wright 2002), populations that comprise uni- and bivoltine cohorts would be expected to exhibit bimodal spring phe- notypes (i.e., those developed from previous year’s spring versus summer adults). If proven, phenotypic variation driven by facultative voltinism in P } ~ — i 7. ? ¥% A. ¥ i “S mr As a ‘- i ars n= ak: gh ees ZooKeys 1228: 69-97 (2025), DOI: 10.3897/zookeys.1228.142202 8] Charles J. DeRoller et al.: A new species of tiger swallowtail Figure 11. Mature larva of P solstitius on hop-tree (Ptelea trifoliata), Ottawa, Ontario, CAN (H. Goulet, photograph). glaucus could account for the perception of phenotypes that are unaccount- ed for with existing taxonomy. It is evident that the descriptive and diagnostic information defining P. bjor- kae is currently incomplete and partially contradictory, and corroborating evi- dence for its distinctness as a species, outside of adult morphology, is lacking. This renders the recognition of P. bjorkae as a valid species tenuous at best. To spur further inquiry and study, we nevertheless include the known comparative phenotypic traits in Table 1. Despite the overall similarity of P solstitius to P glaucus, we have found that it is possible to confidently identify the vast majority of individuals when multi- ple diagnostic traits are assessed. Papilio solstitius is most similar to the north- ernmost populations of spring generation P. glaucus, and some specimens are not distinguishable based on wing pattern alone. Papilio solstitius differs from P. glaucus in smaller overall size, greater tendency for the ventral forewing sub- marginal band to be band-like (broken into rounded crescents interrupted by black veins in typical P. glaucus); less scalloped outer border of the ventral hindwing submarginal band, and the absence of dark phase females (present in both P glaucus and P. appalachiensis). The forewing outer margin is less frequently concave than in P. glaucus. Variation in these wing pattern traits often overlap with those of P. glaucus, and specimen identification requires consideration of seasonal timing and location. In P. solstitius, the tuft of setae projecting from the frons is much more prominent than in summer generation P glaucus, where it is greatly reduced (Fig. 4); spring generation P glaucus have similar setation to that of P. solstitius. The spring generation of P glaucus can have some P. canadensis-like traits (Scriber 1990) that make it more difficult to differentiate from P. solstitius based on adult morphology alone. However, throughout much of the range of P. solstitius, there is no overlap with the more southern P. glaucus. Male genitalic structure is generally regarded as being homogenous among the glaucus-complex (Brower 1959; Hagen et al. 1991), ZooKeys 1228: 69-97 (2025), DOI: 10.3897/zookeys.1228.142202 99 Charles J. DeRoller et al.: A new species of tiger swallowtail but our limited sample suggests that there may be quantitative differences in the number of dorsal tines on the clasper, with P. canadensis and P. solstitius ranging from one to two spines and P. glaucus from one to three (Fig. 9). Compared to sympatric P canadensis populations, P. solstitius can usually be separated with confidence. It is larger with less extensive black markings, most consistently so in the narrower black border of the hindwing anal margin (Fig. 7; Table 1). The narrower margin also results in the large black V (formed by the medial line bridging to the distal part of the anal margin) appearing more U-shaped, versus more sharply V-shaped in canadensis (Fig. 7). The ground color is a slightly richer yellow tone. The body vestiture and color differ signifi- cantly between the two: the setation of P. solstitius is more sparse and short- er, particularly evident on the frons (Fig. 3), the dorsal thorax, and along vein 2A through the black anal margin band of the ventral hindwing (Fig. 7). The head and dorsal thorax are brighter yellow, as is the abdomen. The abdominal subdorsal yellow band is also wider, the male clasper is solid yellow, not inter- spersed with grey-black scales as in P canadensis (Fig. 8). Best observed on the underside of the hindwings, the anal margin black band relative to the width of the entire cell containing the band is approximate- ly 10-40% wide in P glaucus and 55-90% wide in P canadensis (Scriber and Ording 2005). The band width averages greater in females than males, but the relative difference between species persists. In P. solstitius, this width ranges between approximately 30-55%. Also, on the underside of the hindwings, the lateral interface separating the basal yellow from the black submarginal region is typically somewhat straight in P canadensis (though a common exception being in cell Rs-M, where the line can be bowed inward), noticeably scalloped in P glaucus, with P. solstitius demonstrating intermediacy. The hindwing un- derside submarginal lunules tend toward those of P canadensis in being more rectangular than crescentic. Comparison of the larval morphology indicates that the color pattern of the first instar is diagnostic for P. glaucus and P. canadensis (Hagen et al. 1991; Scriber 1998). Papilio solstitius differs from P glaucus and P. canadensis in the white dorsal banding pattern (Fig. 10). The prominent white medial saddle, comprised mostly of segments A3-A4, is present in all species. In P canaden- sis, there are three additional, smaller white bands: two anterior bands formed by white pigmentation on T1 and T3, and a posterior band formed by A8. This banding pattern, with additional anterior-posterior (AP) bands, is consistent in P canadensis. In P. glaucus, only the A3-A4 medial saddle is present, and AP bands are absent, the pigmentation on T1, T3, and A8 being dark brown. Papilio solstitius shows intermediacy and variability in the development of the AP bands. Typically, the AP bands are not as prominently white as in P canadensis, but not completely brown as in P. glaucus. Development of the AP patterns varies and can be absent (glaucus-like) or highly developed (canaden- sis-like), although such variants are rare (< 10% of individuals reared). However, canadensis-like larvae never express the same intensity of white pigmentation as that species, although dark variants are essentially undistinguishable from P. glaucus. Examples of glaucus-like first instars are limited to one field-collec- tion event on a single ash sapling (Kingston, 22.Jul.2023), where five of nine larvae were glaucus-like. Clearly, further study of larval variation is needed. ZooKeys 1228: 69-97 (2025), DOI: 10.3897/zookeys.1228.142202 93 Charles J. DeRoller et al.: A new species of tiger swallowtail Larval host plants In the southern range parts, Papilio solstitius seems to prefer ovipositing on tulip tree (Liriodendron tulipifera L.) and hoptree (Ptelea trifoliata L.), like P glau- cus. Larvae can occur regularly on hoptree where it is planted as an ornamental shrub outside of the natural range (Fig. 12). North of the native ranges of both of these plants (approximately north and east of the region of Toronto, Ontar- io), P solstitius feeds on Fraxinus pennsylvanica and Prunus serotina, based on wild-collected ova and larvae and observation of oviposition (Fig. 4b). Lar- vae demonstrate high survival rates on tulip tree, unlike P canadensis, and also demonstrate survival on quaking aspen (Populus tremuloides Michaux), unlike P glaucus, but at a rate lower than that of P canadensis (Mercader et al. 2009). Diapause and phenology Papilio solstitius exhibits delayed post-diapause pupal development, producing a single summer flight. In Ontario, the flight period commences in late June to early July, peaking in the first half of July (Fig. 2a). Studies on the effect of tem- perature on pupal development show a similar phenology in New York (Ording et al. 2010). Rearing field-collected ova and larvae from the Kingston region of Ontario further confirm that P solstitius is univoltine with obligate diapause like P canadensis, differing from P glaucus which is facultatively multivoltine (Scriber 2013). Notably, some lab reared pupae overwintered twice, not eclos- ing until the second year. Pupae removed from cold storage to a constant temperature of ~23 °C eclosed after 30.4 +/- 5.5 days (male and female combined), or an average of 699 degree-days (DD). Papilio canadensis pupae emerged 19.4 +/- 4.2 days (p < 0.0001), or 446 DD, under the same conditions. In eastern Ontario, accumu- lated degree-days (above a minimum threshold of 6 °C) for these values corre- Figure 12. Distribution of examined specimens of P solstitius (voucher data in Suppl. material 1). ZooKeys 1228: 69-97 (2025), DOI: 10.3897/zookeys.1228.142202 84 Charles J. DeRoller et al.: A new species of tiger swallowtail spond to the second week of June (446 DD) and the first week of July (699 DD) (Schmidt and Layberry 2016), precisely when peak emergences of P. canadensis and P. solstitius are occurring (Fig. 2a). Difference in post-diapause pupal emer- gence therefore perfectly accounts for the staggered emergence peaks between P canadensis and P solstitius in eastern Ontario. Male and female P solstitius differ in the length of post-diapause development delay. On average, males re- quired approximately 26.6 +/- 3.2 days to eclose compared with 34.2 +/- 4.2 days for females (p = 0.02; n = 10; two-tailed T-test). In the wild, this would be expected to translate to a difference in peak flight times between the sexes of approximately 15 days, which matches well with field observations (Fig. 2). Bivoltine P glaucus populations occur primarily to the south of the range of P. solstitius. However, P. glaucus is facultatively univoltine or bivoltine at the northern range periphery, contrary to the initial hypothesis that it is unable to switch to univoltinism and limited to regions where it can undergo two annual generations (Hagen et al. 1991). In Ohio and Michigan populations, pupae are induced to enter winter diapause when 4"-5" instars experience photoperiods of less than 14 hours (Ryan et al. 2017). Facultative uni- vs. bivoltinism is also demonstrated by our rearing results from the Hamilton, Ontario region, which is north of the bivoltine thermal threshold (Scriber 2013). Lab-reared larvae of spring P glaucus on L. tulipifera developed directly into a second genera- tion of adults, despite the rarity of naturally occurring second-flight P glaucus here. Univoltine P glaucus populations probably occur more widely than pre- viously recognized and have added to the complexity of defining the taxa in- volved in the glaucus-complex. Indeed, this could explain the perception of two spring-flying phenotypes (Pavulaan 2024) in regions where both uni- and bivol- tine P. glaucus occur: offspring developing from either spring-flight (univoltine) or summer-flight (bivoltine) parents experience differing temperature-photope- riod profiles as larvae (known to influence adult phenotype), but both cohorts emerge the following spring. In southern Ontario and the Finger Lakes region of New York, the presence of both spring and summer P. glaucus likely accounts for a longer spring abundance peak and a more protracted late summer abun- dance peak (Fig. 2a, c; see also Schmidt 2020: fig. 7). Habitat and distribution Since Papilio solstitius, like its congeners, uses a range of unrelated host plants, it has a similarly broad habitat tolerance for a range of forest, forest edge and woodland habitats. Although habitats of P. solstitius and P canadensis overlap widely, the former reaches its highest abundance in or near mesic or moist woodlands, particularly ash-dominated swamps, where ash is common. Con- versely, P canadensis is most common in drier upland habitats where trembling aspen is common. The core range of Papilio solstitius includes eastern and southcentral Ontar- io, northern and central New York and adjacent Vermont, New Hampshire, and Pennsylvania (Fig. 12), encompassing a minimum land area of approximately 174 000 km? (by comparison, the range extent of P. appalachiensis is ~ 140,000 km?). In New York, P. solstitius inhabits most of the state except the southeast and greater New York City area. In Canada, P solstitius extends from the Mon- tréal, Québec region west to the Bruce Peninsula of Ontario, south to the Niag- ZooKeys 1228: 69-97 (2025), DOI: 10.3897/zookeys.1228.142202 95 Charles J. DeRoller et al.: A new species of tiger swallowtail ara region (Fig. 12; Wang 2018; Schmidt 2020; Macnaughton et al. 2020). The western limit appears to be the eastern shores of Lake Huron; we have not seen any verifiable specimens west of there. The glaucus-complex has received con- siderable study in the lower peninsula of Michigan and in Wisconsin, and there is no evidence of delayed flight (July) swallowtails there (Luebke et al. 1988; Stump et al. 2003). The northern range limit of P. solstitius is easily defined since adult morphol- ogy and phenology differ considerably from P canadensis. Furthermore, the range limit is climatically constrained since P solstitius larval development is shifted about a month later than P. canadensis, and development must be com- pleted before autumnal leaf abscission and frost. The current northern limit is the southern edge of the Algonquin Dome, the lower Ottawa River valley, and the southern edge of the Gatineau/Laurentide escarpment as far east as the Montréal region. Papilio solstitius has undergone a northward range expansion of several hun- dred kilometers since the 1970s (Schmidt 2020), as has P. glaucus elsewhere (Scriber et al. 2014). In 2022, P. solstitius was recorded for the first time near Montebello, Québec. Continuous monitoring at this location since 1994 indi- cates that P solstitius was not present prior to 2022 (P. Legault, pers. comm). Based on the climatic zones given in Scriber et al. (2014), the distribution of P. solstitius approximates the 1300-1400 degree-day (°C) climatic envelope. For context, the northern limit of bivoltine P. glaucus is ~1444 DD. The southern (warm) limit of P canadensis appears to be slightly north of this, and is possibly limited by pupal mortality due to prolonged high summer temperatures (Kukal et al. 1991). The northern range limit of Papilio solstitius is likely determined by minimum thermal requirements, given the late seasonal phenology of a July flight period that dictates a shorter window for larval development before au- tumnal host plant senescence. The southern range limits of P. sol/stitius are currently difficult to define owing to overlap and confusion with single- and double-brooded P. glaucus, and the uncertainty in the northern range limit of P. glaucus. Swallowtails that are mor- phologically consistent with P. solstitius and eclosing in the first half of July, when P glaucus is between flights, extend south to approximately 41, 42°N to the eastern seaboard (Fig. 12). In Pennsylvania, the southern extent of P. sol- stitius coincides approximately with the northern limit of RP glaucus contain- ing dark morph females (Scriber 1996), extending from Erie to just north of Pittsburgh and east to New York City. It may also extend to the Atlantic coast through Connecticut and Rhode Island based on the phenology information in Pavulaan (2024), but this warrants further study. The occurrence of P. canadensis at the southern range edge, near that of P. solstitius, may be more limited than depicted in some range maps (e.g., Pavulaan and Wright 2002; Cech and Tudor 2005; Monroe and Wright 2017). Our examination of putative P canadensis photos from New York and Penn- sylvania indicate that most are spring flight P glaucus; CJD has been unable to verify the presence of typical P. canadensis in New York state south of the Adirondacks. It is possible and indeed expected that P. canadensis is undergo- ing a northward range contraction with warming climates (Scriber 2013), but this remains unexamined. In the Finger Lakes region of New York, members of the glaucus-complex can be observed continuously from mid-May to early Sep- ZooKeys 1228: 69-97 (2025), DOI: 10.3897/zookeys.1228.142202 86 Charles J. DeRoller et al.: A new species of tiger swallowtail tember (Fig. 2b). In this region, a pale canadensis-like phenotype emerges first, followed by a tiger swallowtail in late May which has historically been referred to as “spring form” P. glaucus, and then finally P. solstitius in late June to July, and possibly a partial second flight of P. glaucus in August (although not all taxa are sympatric everywhere). Phylogenetic analyses Both regions of COI recover the same general relationships between members of the P. glaucus group, including P. multicaudata Kirby, 1884, P eurymedon Lu- cas, 1852, P rutu/lus Lucas, 1852, and the glaucus-complex clade of P glaucus, P canadensis, and hybrid taxa (P. appalachiensis, P. solstitius, etc.) (Figs 13, 14). Within the latter, PR glaucus and P. canadensis almost form reciprocally mono- phyletic clades in both COI5 and COI3, but in each gene, a handful of specimens fall in the opposing clade (marked with asterisks in Figs 13, 14), and P. appala- chiensis falls throughout the P. glaucus clades in both genes. Papilio solstitius clusters within the P canadensis clade, as does a handful of P glaucus. Notably, there are few nodes with strong branch support within this clade of P. glau- cus/P. canadensis/P. appalachiensis/P. solstitius, indicating close genetic sim- ilarity between all of these entities in their mitochondrial genomes. Excluding specimens with missing data in the 5’ or 3’ ends of their sequences, pairwise sequence identity for haplotypes in this P glaucus/P. canadensis clade were > 98% for COI3 and > 97.5% for COIS. We re-evaluated identification of specimens sequenced in Vernygora et al. (2022) and conclude that specimens noted as “intermediate” therein are mostly P. glaucus, but one is P. solstitius (samples annotated with asterisks in Fig. 15). In their SNP-based phylogeny (remade in Fig. 15), these specimens form a paraphyletic grade between typical (and more geographically distant) P canadensis and P glaucus; Papilio appalachiensis also falls out in this grade, and as with COl, only a handful of nodes within this broad clade were strongly supported and many of these specimens appeared admixed in Vernygora et al.'s population genetics-oriented analyses. Discussion Comparison of physiological and morphological traits of the P glaucus-com- plex taxa in the eastern Great Lakes — northern Appalachian region reveals that the midsummer tiger swallowtail, Papilio solstitius sp. nov., is a distinct, locally common species rather than occasional F1 hybrid individuals between P. glau- cus X canadensis. It is geographically widespread over thousands of square kilometers outside of established hybrid zones and is allochronically isolated from its sibling species. Nevertheless, the evolutionary origin of P. solstitius through hybridization between P. glaucus and P. canadensis is likely, as is con- tinued hybridization between the three. How has a large, conspicuous swallowtail butterfly gone unrecognized in a well-studied region of North America for so long? In hindsight, an earlier study of two univoltine tiger swallowtail populations near Ithaca, NY established the existence of a taxon that was clearly not attributable to either P glaucus or P canadensis, although both entities were referred to as P glaucus (Hagen and ZooKeys 1228: 69-97 (2025), DOI: 10.3897/zookeys.1228.142202 87 Charles J. DeRoller et al.: A new species of tiger swallowtail glaucus_BOLD_KYBU_230719-1 glaucus_BOLD_NYON_A_20230626_2 glaucus_EF126470.1 glaucus_BOLD_MECD382-06 glaucus_EF126454.1 glaucus_EF126450.1 glaucus_EF126473.1 glaucus_EF126446.1 glaucus_EF126447.1 glaucus_EF126474.1 glaucus_KM548369.1 glaucus_EF126463.1 glaucus_EF126462.1 glaucus_EU141368.1 glaucus_EF126469.1 glaucus_EF126453.1 glaucus_EF126456.1 glaucus_EF126466.1 glaucus_GU090088.1 glaucus_EF126455.1 canadensis_EF126440.1* glaucus_GU090087.1 glaucus_EF126468.1 glaucus_EF126448.1 glaucus_EF126471.1 glaucus_AF044013.1 glaucus_EF126465.1 glaucus_KF491982.1 glaucus_EF126467.1 glaucus_BOLD_BBLOC645-11 appalachiensis_PQ578216.1 laucus_EF126460.1 appalachiensis_PQ578215.1 glaucus_EF126461.1 glaucus_EF126464.1 glaucus_EF126452.1 glaucus_EF126459.1 glaucus_NC027252.1 glaucus_EF126449.1 laucus_EF126451.1 glaucus_EF126472.1 glaucus_EF126458.1 glaucus_garcia_KP174096.1 laucus_garcia_KP174097.1 glaucus_EF126457.1 canadensis_MG465384.1 canadensis_KM547277.1 canadensis_MG464258.1 canadensis_KM544174.1 canadensis_KM545095.1 canadensis_KM540752.1 canadensis_KT129609.1 canadensis_EF126443.1 canadensis_EF126444.1 canadensis_KT145966.1 canadensis_EF126434.1 canadensis_EF126438.1 canadensis_EF126433.1 canadensis_EF126436.1 canadensis_KU875739.1 canadensis_EF126445.1 canadensis_KM545469.1 canadensis_KT146981.1 canadensis_KT135240.1 canadensis_KM549449.1 canadensis_AF044014.1 canadensis_EF126439.1 canadensis_EF126435.1 canadensis_EF126442.1 canadensis_KM554515.1 canadensis_EF126441.1 canadensis_KM545398.1 canadensis_KT130133.1 canadensis_KM542389.1 canadensis_FJ808888.1 canadensis_KM540526.1 canadensis_FJ808894.1 canadensis_KM550886.1 canadensis_KU875740.1 canadensis_KM541646.1 canadensis_EF126437.1 e canadensis_FJ808889.1 canadensis_FJ808886. 1 canadensis_FJ808892. 1 canadensis_FJ808890. 1 canadensis_FJ808893. 1 canadensis_FJ808887. 1 canadensis_FJ808891.1 MST_BOLD_NYON_GAN_140823_3 MST_BOLD_CNCBF603-14 MST_BOLD_NYTO_GP_20230705_6 canadensis_KT133738.1 MSTtype_BOLD_NYON_A_230713_3 canadensis_BOLD_LCHQ915-08 MST_BOLD_NYTO_GP_20230705_1 MST_BOLD_NYON_A_230713_4 canadensis_BOLD_RDBBC015-05 canadensis_BOLD_BBLPA286-10 canadensis_BOLD_RDBBC196-05 MST_BOLD_CNCBF604-14 laucus_BOLD_CNCBF605-14* ST_BOLD_XAJ969-06 canadensis_EF126432.1 canadensis_BOLD_RDHP191-05 MST_BOLD_NYON_GAN_140823_2 MST_BOLD_XAE574-04 lee ie ae rutulus_EF126476,1 rutulus_JN275688. 1 rutulus_JN275686.1 rutulus_HQ561170.1 rutulus_HQ561168.1 rutulus_HQ561169.1 rutulus_JN275687.1 eurymedon_MW807709.1 rutulus_HQ561235.1 rutulus_AY954560.1 rutulus_KT144703.1 é rutulus_HM428654.1 rutulus_HM428655.1 rutulus_AF044015.1 ; eurymedon_HQ561236.1 multicaudata_pusilllus_KP174092.1 o multicaudata_AF044016.1 multicaudata_EF126475.1 C) multicaudata_multicaudata_KP174094.1 rr regaled PSPS soa 0.02 avg. changes/site multicaudata_multicaudata_| . g g multicaudata_pusilllus_KP174091 .1 birchalli_AY457596.1 scamander_AF044020.1 troilus_AF044017.1 Figure 13. Maximum likelihood tree for COI5. Specimens are labeled with a species epithet determination and NCBI or BOLD accession numbers. Papilio solstitius samples indicated in blue as “MST.” Specimens with asterisks indicate those that fell outside of their typical respective clade. Grey circles indicate strong node support (> 0.95 ufBS and > 0.8 SH-aL- RT). All outgroup branch lengths have been edited for space. ZooKeys 1228: 69-97 (2025), DOI: 10.3897/zookeys.1228.142202 88 Charles J. DeRoller et al.: A new species of tiger swallowtail canadensis_AA378_JF764396.1 canadensis _| wv oMatBes FJ808889.1 glaucus_WV. canadensis_SD_OM243 $08.1 canadensis haplotype1_FJ808886.1 canadensis_haplotype3 See canadensis_Pc03_EF126434.1 canadensis_SD_OM243994.1 canadensis MA59_JF764412.1 canadensis_SD_OM243992.1 canadensis_AA413_JF764403.1 MST_NY_OM244004.1 canadensis_SD_OM243996.1 glaucus_|IL_OM243974.1* canadensis_WI|_OM244006.1 canadensis_Pc08_EF126439.1 canadensis_SD 244003. canadensis_Pc07_EF126438.1 canadensis_AK_OM244002.1 canadensis_AB_OM243982.1 canadensis_ND OM243977-1 glaucus_NY_OM243959.1* canadensis _Pc11 ert 26442.1 MST_NY_OM244007. canadensis AK OM244000.1 canadensis_PcT2 Fa ence canadensis_AB 43983.1 canadensis _MA61 Hereadt4 canadensis_AB_OM243988.1 canadensis_SD_ -OMB43900 canadensis_SD_OM243! canadensis_AA391 5764309.1 canadensis_W1|_OM244005.1 canadensis_MA60_JF764413.1 canadensis_Pc4078 ri ara canadensis_SD_OM243997.1 canadensis_| OE Net sas 1 canadensis__AF0440 canadensis _MA58 we 764411. 1 canadensis PcanadensisSp_EF126445.1 canadensis_A M2. canadensis_AA392_JF764400.1 canadensis_Pc05_EF126436.1 canadensis_haplotype9_FJ808894.1 canadensis_SD_OM243993.1 canadensis_MA57 Jereadiat canadensis_SD_OM243991. canadensis_MB _ OM243980.1 glaucus_WV_OM243962. canadensis_AK OMo004 1 glaucus_NY_OM243964. canadensis AB _OM243987.1 glaucus_AA028_JF764418.1 glaucus AA544 Jee! 553 SE reda4o sf re ABO02_. JE 764448. Qlaucus_AA554_JF764441, ia glaucus_PgFB43_EF126451 appalachiensis ARSO0 JPredo0s.1 glaucus_AA560_JF764447. glaucus_AA549_JF7! 6447 appalachiensis_AA318_JF764388.1 a ape ras laucus_PgFB66_EF126452.1 3 palachiensis AA299_JF/764376.1 glaucus_AB010_JF764455. glaucus_PgPY13_EF126470.1 glaucus_AA366_JF764426.1 glaucus_ABO09 _JF764454.1 glaucus_NY_OM243967.1 glaucus_ABO17_JF/64461.1 PgMBT1 EF 126468.1 glaucus_AA540_JF76443i appalachiensis_AA348 ereago4. glaucus_AB008_JF764453 glaucus_ABOO7_, SE 7e44ze. | appalachiensis ANS, JF764382.1 glaucus_AB003_JF764449.1 glaucus_ABO06_., SE ve4asi, 1 glaucus_PgFB42_EF126450.1 glaucus 7252.1 appalachiensis_AA306_JF764381.1 glaucus_AB012_JF764457. appalachiensis_AA312_JF764385.1 glaucus_AA537_JF764428.1 al palachiensis Boges J JPreagee,4 glaucus_PgGB 7_EF 126466 glaucus_Pg' GB 130047 EF 1264601 al palachiensis AAS15_JF764387.1 glaucus_AA558_JF764445.1 glaucus_AA552__ Eee glaucus_AA543_J Picante 1 glaucus_MD_OM243' ol glaucus_MD_ OMeaso7 a palachisnais MASON ‘i 764378.1 elleerione aplo' 243986.1 e6 Pi08801. 1 canadensis_Pc10_EF126441.1 canadensis_AA380_JF764398.1 aucus PoOBO3 EF126474.1 canals Aaags aineaate ag ao glaucus_ABO04_J appalachiensis ASO T764380.1 glaucus_AA365_JF764425.1 appalachiensis_, WASo6- E764390. 1 appalachiensis_AA335_JF764393.1 glaucus_PgMB12_EF126469.1 glaucus_PgGB13197 ET sessed appalachiensis_AA333_JF764392.1 glaucus_MD_OM2 P5073 1 appalachiensis_AA329 "1764391. 1 glaucus_AA548_JF764436.1 glaucus_PgFY14_EF126459.1 canadensis_haj aplotype> Fs Fea canadensis _ MB canadensis A BC_OM2 yee 8.1 canadensis_| haplotype 7. Fe FJ808802.1 canadensis_S canadensis_WI GMie43988 t canadensis_ha eplotypee tse FJ606687.1 canadensis_B canadensis _Pc06. EF 1204971 canadensis_AA379_JF764397.1 canadensis_AA418_JF764404.1 QO) canadensis_ MAGt -JF764415.1 canadensis. MA56_JF764409.1 yea aerate canadensis POO) KF136435 1 glaucus-PGBOT-EFIZe47é.1 JaUCUS. cenecones DA pot emer glaucusAA027—JF76441 7.1 rutulus_CA _OM244020.1 rutulus_ID OMe aes 1 enriea PgFB32 ER 26449.1 glaucus_AB016_JF764460.1 aaa AB022 —IF764466 4 eurymedon_WA_OM244031.1 eurymedon_WA_O eurymedon_CA_OM244034.1 siaucs Fo ate EF126447.1 glaucus_AB013 srresas o. glaucus_AA325_JF76442. glaucus_PgPY15 ERI 2e4/1, 1 glaucus_ OM243969.1* Canadensis _PcO9 EF126440.1* glaucus_PgFY04_E . glaucus_AA556_JF764. appalachiensis_VA_OM243957.1 glaucus_AA545_JF764433.1 glaucus_| Wess. Sep glaucus_AA555_JF76444: glaucus_AA319 -IF764423 | glaucus_AA056 roe. glaucus_ABO21_JF764465.1 glaucus_NY_OM243966.1 glaucus_CT_OM243968.1 rutulus_| ws, OM244018.1 rutulus_CA_OM244021.1 rutulus CA _( OM244019.1 rutulus_OR_OM244010 rutulus_BC_| -GMo4aao1 2:4 eurymedon_OR_OM244054.1 eurymedon_CA. eee rutulus_ID_OM244025.1 eurymedon_CA_OM244039.1 eurymedon_OR_OM244036.1 eurymedon_CA aS oe ae rutulus_CA_OM244022.1 eurymecon CA OM244048,1 eurymedon_CA_OM244040.1 eurymedon_CA_OM244035.1 rutulus_MT_OM244026.1 eurymedon CA OM244050.1 rutulus_OR_OM2441 Oa 0.02 avg. changes/site muitos TX "0OM244057.1 multicaudata_WA_ OM244028-1 multicaudata_NV_OM244060.1 © multicaudata_MT_OM244062.1 multicaudata_OR_OM244058.1 scamander__AF044020.1 © birchalli__AY457596.1 garamus__| ONi243954, 1 troilus__OM243952.1 Figure 14. Maximum likelihood tree for COI3. Specimens are labeled with a species epithet determination, state/prov- ince, or additional unique identifier, and NCBI accession numbers. Papilio solstitius samples indicated in blue as “MST.” Specimens with asterisks indicate those that fell outside of their typical respective clade. Grey circles indicate strong node support (> 0.95 ufBS and > 0.8 SH-aLRT). All outgroup branch lengths have been edited for space and dotted grey line is a visual link between independently shown parts of the tree. ZooKeys 1228: 69-97 (2025), DOI: 10.3897/zookeys.1228.142202 89 Charles J. DeRoller et al.: A new species of tiger swallowtail wee glaucus_AR_FS333 glaucus_WV_JRDE068 glaucus_MD_JRDE066 glaucus_WV_JRDE067 glaucus_CT_JRDE074 glaucus_MD_FS039 glaucus_MD_FS069 ° glaucus_MD_JRDE065 glaucus_MD_FS346 glaucus_FL_FS269 glaucus_VA_FS347 + glaucus_IL_JRDEO76 MST_NY_FS336* glaucus_NY_JRDE071* glaucus_NY_JRDE0O70* glaucus_NY_FS0028* appalachiensis_VA_12284 appalachiensis_VA_12285 glaucus_NY_JRDE062* glaucus_NY_JRDEO69* glaucus_NY_JRDE072* canadensis_WI_FS349 canadensis_WI_FS117 glaucus_NY_JRDE061* canadensis_AK_FS306 canadensis_AK_JRDE018 canadensis_AK_FS268 canadensis_AK_FS324 canadensis_SD_FS246 canadensis_WI_FS348 e canadensis_AB_JRDE0O11 canadensis_MB_JRDE013 canadensis_MB_JRDE014 canadensis_AB_JRDE0O16 canadensis_BC_JRDE012 canadensis_AB_FS241 canadensis_MB_JRDE015 canadensis_SD_FS244 canadensis_ND_JRDE010 canadensis_SD_FS249 canadensis_SD_FS250 canadensis_SD_FS253 canadensis_SD_FS247 canadensis_SD_FS243 canadensis_SD_FS331 e) canadensis_AB_JRDE017 canadensis_SD_FS252 canadensis_SD_FS245 canadensis_SD_FS251 canadensis _SD_FS248 canadensis_ND_JRDE0O9 canadensis_AB_FS304 canadensis_BC_JRDE029 eurymedon_CA_JRDE046 eurymedon_CA_JRDE0O57 eurymedon_CA_JRDE052 eurymedon_OR_JRDE044 eurymedon_CA_JRDE055 eurymedon_CA_JRDEO56 eurymedon_WA_JRDE040 eurymedon_CA_JRDE041 eurymedon_OR_JRDE0O45 eurymedon_WA_JRDE038 eurymedon_CA_JRDE050 eurymedon_CA_JRDE047 eurymedon_CA_JRDE042 eurymedon_BC_JRDE037 eurymedon_WA_FS309 O eurymedon_OR_FS418 = eurymedon_WA_FS169 eurymedon_OR_JRDE043 eurymedon_WA_JRDE0O39 eurymedon_CA_JRDE049 eurymedon_CA_JRDEO58 eurymedon_CA_JRDE054 eurymedon_CA_JRDE048 eurymedon_CA_FS314 Y eurymedon_CA_JRDEO51 eurymedon_CA_JRDE053 rutulus_AZ_JRDEOOS rutulus_AZ_JRDE032 rutulus_AZ_JRDEO30O Of rutulus_AZ JRDEO31 e rutulus_AZ_JRDEOO6 O rutulus_MT_JRDE027 rutulus_NV_JRDE034 rutulus_ID_JRDE024 rutulus_CA_JRDE021 rutulus_CA_JRDE023 rutulus_ID_JRDE025 rutulus_CA_JRDE002 rutulus_CA_JRDE019 rutulus_WA_JRDE004 rutulus_CA_JRDE020 rutulus_CA_JRDE022 rutulus_OR_JRDEOO3 rutulus_OR_FS272 rutulus_I|D_JRDE026 rutulus_OR_FS305 rutulus_BC_JRDE001 multicaudata_NV_JRDEOO7 multicaudata_TX_JRDEO63 multicaudata_TX_JRDE064 multicaudata_MT_JRDE028 O multicaudata_WA_JRDE008 —— ' multicaudata_WA_FS414 0.06 avg. changes/site multicaudata_OR_FS041 birchalli_FS070 garamus_FS341 scamander_FS408 troilus_FS029 Figure 15. Majority rule consensus tree generated from 3,733 SNPs from Vernygora et al. (2022). Specimens are labeled with a species epithet determination, state/province, and unique identifier, and specimens with asterisks indicate those called “intermediates” in the original publication, including one we identify now as P solstitius (“MST” in blue). Grey circles indicate strong node support (> 0.9 posterior probability support). All outgroup branch lengths have been edited for space. ZooKeys 1228: 69-97 (2025), DOI: 10.3897/zookeys.1228.142202 90 Charles J. DeRoller et al.: A new species of tiger swallowtail Lederhouse 1985). Papilio canadensis was subsequently recognized as a dis- tinct species (Hagen et al. 1991), but the late-flight tiger swallowtails remained a taxonomic enigma and were attributed to a hybrid zone phenomenon (Scriber 1990; Ording et al. 2010; Kunte et al. 2011). Papilio solstitius was documented as early as the 1970s in upstate New York (Hagen and Lederhouse 1985) and eastern Ontario (CNC specimens). However, the earliest literature reference to P. solstitius that we could find dates to the mid-1800s from southcentral On- tario. Saunders (1874) noted: “[The tiger swallowtail] appears first on the wing from the middle to the latter end of May, but becomes much more plentiful in July. Whether these July insects are a second brood, or whether the bulk of the chrysalids which have wintered do not mature until about this time we are unable to determine.” As it were, it was not until 1984 that it was proven that the July swallowtails are in fact not a second generation (Hagen and Lederhouse 1985). At the time located near London, Ontario, Saunders’ observations are now easily explained by what would have been either May-flying P. canadensis or P. glaucus (likely the latter based on current ranges), and P. solstitius with its unique July flight time. Although P solstitius exhibits a mosaic of characters of both P. glaucus and P. canadensis (Table 1) which might suggest that it is a hybrid, it differs from artificial hybrids in several significant ways (Table 2). Based on the novel de- tection of late-emerging populations in western Vermont, Ording et al. (2010) suggested Papilio solstitius to be of very recent hybrid origin mediated by cli- matic amelioration. The historic documentation and large geographic range, much of it beyond the contact zone between P glaucus and P. canadensis, counter this hypothesis. Notably, the delayed pupal emergence with a single summer flight differs from lab hybrids which emerge in the spring (Ording et al. 2010). Our data for post-diapause pupal development of Ontario P. sol- stitius are comparable to values given by Ording et al. (2010) from Vermont Table 2. Comparison of genetic and ecological traits among species of the Papilio glaucus-complex. Sourced from Kunte et al. (2011), Scriber and Ording (2005), and this paper. The recently described P. bjorkae is excluded because most traits remain undefined or unknown (see Introduction). Trait Thermal habitat Pupal diapause Voltinism Larval survival: aspen Larval survival: tuliptree Body size Female polymorphism Pupal emergence Flight season mtDNA Z: Kettin Z: TH Z: Tpi Z: Period Z: PAH LDH allozyme LDH20 “hybrizyme” PGD allozyme P glaucus P. appalachiensis P. solstitius sp. nov. P. canadensis F7 lab hybrid cool na obligatory obligatory obligatory Z-linked univoltine univoltine univoltine photoperiod (Z) o = | age small intermediate mimetic “‘non-mimetic W-linked | early | early heterozygous (Z) early +late. : early - early ; n.a. —— glaucus-like canadensis-like canadensis-like | maternal — glaucus canadensis canadensis | heterozygous | heterozygous (Z) — glaucus canadensis | canadensis canadensis | heterozygous (Z) —— glaucus canadensis | canadensis. | ‘canadensis heterozygous (Z) — glaucus canadensis canadensis canadensis heterozygous (Z) -— glaucus canadensis canadensis canadensis heterozygous (Z) 100 80 / 40 80 / 40 80 / 40 | heterozygous (Z) ‘ na. 100/50 (40-50%) 125/80/150 heterozygous (Z) ZooKeys 1228: 69-97 (2025), DOI: 10.3897/zookeys.1228.142202 91 Charles J. DeRoller et al.: A new species of tiger swallowtail populations: under controlled laboratory conditions, post-diapause pupae of Papilio solstitius and P canadensis emerged after an average of 828 DD and 450 DD, respectively (Ording et al. 2010), versus our results for Ontario popu- lations of both species at 690 DD and 437 DD. In eastern Ontario, the average peak flight period of Papilio solstitius is 11-20 July, compared to 1-10 June for P canadensis (Fig. 2a). Papilio solstitius is distinct from artificial F1 hybrids and both parental species in this regard, which emerge in the spring (Ording et al. 2010). Importantly, this difference results in temporal reproductive iso- lation between P. solstitius and P canadensis/glaucus. Within the P glaucus group, delayed pupal emergence is unique to P. solstitius, and understanding the adaptive significance of this may provide key insights into its evolutionary history. Possibly it is a mechanism to escape pupal mortality due to summer heat, to which P canadensis is susceptible (Kukal et al. 1991). Hybridization between P. glaucus and P canadensis has been well-docu- mented using molecular and morphological evidence, and only some purported hybrid populations can be attributed to Papilio solstitius. The most extensive- ly studied hybrid zone between P glaucus and P canadensis is a narrow geo- graphic zone across Michigan's lower peninsula and into Wisconsin (Luebke et al. 1988; Hagen et al. 1991). Here, the hybrid zone is dictated by a narrow band of the thermal landscape that limits the occurrence of P canadensis to the north and P glaucus to the south. There is no evidence that Papilio solstitius occurs this far west. To the east, the biogeography of the P glaucus group is more difficult to untangle, influenced by the complex topography of the north- ern Appalachians, Frontenac Arch, Alleghany Plateau, and Adirondack Mtns with the added complexity of Great Lakes weather effects. Unlike the region west of Lake Michigan, large gaps occur between the ranges of P glaucus and P canadensis here, but there is undoubtedly ongoing gene flow between P sol- stitius and its sibling species and is fertile ground for future molecular study. Some of the initial genetic work on the glaucus-complex included samples of P solstitius and indicated different allele frequencies of alpha-galactosamin- idase compared to P. canadensis (Hagen and Lederhouse 1985). Papilio sol- stitius also possesses a unique allozyme, LDH-20, not present in other P. glau- cus group species (Scriber and Ording 2005). The presence of molecular traits unique to P solstitius not known from either putative parent species cannot easily be explained by ongoing hybridization. Considering recent genetic data together, it is clear that the standard barcoding gene, COl, is unable to confidently separate P solstitius from P canadensis. The handful of specimens falling outside their respective clades for COI may be indicative of geographic variation that has been historical- ly unsampled/unsequenced, or more varied hybrid interactions between P glaucus and P. canadensis. Ignoring these specimens that fall outside of their respective clades, P. solstitius clearly has more P. canadensis maternal influence, but its nuclear genome is less clear as our sampling is more lim- ited and shows a paraphyletic grade in phylogenetic analyses (Fig. 15) and varied signals of admixture in the results of Vernygora et al. (2022). More comprehensive population genomic sampling will be required to tease apart the genetic situation of P. solstitius, P appalachiensis (Cong et al. 2015), and the other hybrids/entities documented in this species group (Ryan et al. ZooKeys 1228: 69-97 (2025), DOI: 10.3897/zookeys.1228.142202 92 Charles J. DeRoller et al.: A new species of tiger swallowtail 2016, 2017, 2018; Pavulaan 2024). Although Scriber and Ording (2005) and Kunte et al. (2011) potentially addressed P solstitius with other putative hy- brids within the P glaucus group, modern genomic methods should be used to properly characterize population-wide genetic variation throughout this broad geographic region and other hybrid entities within the P glaucus group (Ryan et al. 2016). Current evidence is consistent with the possibility that P so/stitius has a recombinant evolutionary origin similar to that of P appalachiensis. How- ever, most questions regarding the evolutionary origin of this taxon, and its role within the speciation of the P. glaucus-complex, remain to be answered. It is our hope that recognizing and defining the taxonomic identity of this unique evolutionary lineage provides a staging point in the fertile grounds for future research. Acknowledgements Whitney Carleton of New York State Office of Parks, Recreation and Preserva- tion permitted study by CJD within NY state parks. Michael Galban, Historic Site Manager of Ganondagan State Historic Site Seneca Art & Culture Center, approved study on Haudenosaunee traditional lands. Dr. Mark Scriber provided insights resulting from his vast studies. Henri Goulet and Pierre Legault kind- ly provided new records and photographs of P solstitius, and Christi Jaeger provided technical and field assistance. We also thank Rick Cavasin, Ross Layberry, Peter Hall, Ricky Patterson, and Alan McNaughton for providing feed- back, discussion, additional data, and specimens that significantly aided the re- search presented here. Dr. Felix Sperling and an anonymous reviewer provided suggestions that improved the manuscript. Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statement No ethical statement was reported. Funding No funding was reported. Author contributions All authors have contributed equally. Author ORCIDs B. Christian Schmidt © https://orcid.org/0000-0003-41 60-7629 Data availability All of the data that support the findings of this study are available in the main text or Supplementary Information. ZooKeys 1228: 69-97 (2025), DOI: 10.3897/zookeys.1228.142202 93 Charles J. DeRoller et al.: A new species of tiger swallowtail References Brower LP (1959) Speciation in butterflies of the Papilio glaucus group. |. 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The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.1228.142202.suppl1 ZooKeys 1228: 69-97 (2025), DOI: 10.3897/zookeys.1228.142202 97