PHYLOGENETIC RELATIONSHIPS OF PARANTHRENE (LEPIDOPTERA: SESIIDAE) USING MULTI- GENE ANALYSIS AND DETERMINATION OF MONOPHYLETIC RELATIONSHIPS ALONG COLOR FORM By William H Smith III A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Entomology- Master of Science 2024 ABSTRACT Paranthrene species are day flying moths that mimic wasps in both appearance and behavior. The genus has several species with diverse color patterns that are restricted geographically. Many of these color forms were originally described as species before becoming synonymized with each other due to similar behavior and morphology. To date, there have been no molecular multi-gene phylogenetic studies that investigate the monophyly of North American Paranthrene species boundaries. We reconstructed a phylogeny using partial DNA sequence from COI, EF-1alpha and wingless genes with 67 specimens representing all North American Paranthrene species, subspecies, and variants. Monophyly of any P. simulans color forms was not recovered in parsimony and Bayesian analyses. The results also suggest the synonymy of P. simulans, P. pellucida, and P. hilairemontis. Interestingly, the two-color forms of P. tabaniformis comprised two clades, one which was sister to a clade including P. dolli. Individuals of this clade also have a different flight period as P. tabaniformis which suggests the recognition of the clade as a species. Paranthrene robiniae presented as polyphyletic. Genitalic and genetic differences between P. robiniae populations suggest the recognition of two psuedocryptic species. ACKNOWLEDGEMENTS We would like to thank Mr. James Sogaard and Mr. Seth McCarthy for providing specimens for this research. We are also grateful to Clark County Wetland Park in Nevada for allowing us to collect on their land and assisting us in finding ideal habitat for trap placement. This research was funded in part by Mr. William Taft, The American Natural History Museum Theodore Roosevelt Grant, and the Michigan State University Scriber Fund. A special thank you is in order to Dr. Anthony Cognato, Dr. Henry Chung, and Dr. Jean Tsao for serving as my graduate committee and guiding me through this process iii TABLE OF CONTENTS INTRODUCTION ...............................................................................................................................1 CHAPTER 1: A NEW SPECIES OF PARANTHRENE (LEPIDOPTERA: SESIIDAE) FROM THE NORTHERN MIDWEST US ................................................................................................................5 CHAPTER 2: APOSEMATIC COLOR POLYMORPHISM IS A POOR INDICATOR OF SPECIES BOUNDARIES IN NORTH AMERICAN PARANTHRENE (LEPIDOPTERA: SESIIDAE) AS EVIDENCED BY A MULTI-GENE PHYLOGENY..................................................................................................... 19 CONCLUSION ................................................................................................................................. 51 BIBLIOGRAPHY............................................................................................................................... 52 iv INTRODUCTION Sesiidae, or clearwing moths, are a family of over 1400 species worldwide within the order Lepidoptera. Of those species, 135 of them reside in North America and include many pests of agriculture and forestry (Lair and Herbert, 2018). The genera Paranthrene and Synanthedon are of significant importance to nursery trees and timber production in the United States (McKern and Szalanski, 2007). Due to their diurnal nature and mimicry of Hymenoptera, their presence and distribution can be challenging to detect, making pheromone traps one of the better ways to monitor these moths (McKern and Szalanski, 2007). Pheromone traps provide an affordable way to monitor Sesiidae populations (McKern and Szalanski, 2007). The type of pheromone used in a trap can also help identify the species captured (Taft et al., 1991) although, there is some overlapping pheromone attraction among species (Taft et al., 1991). For example, both Paranthrene simulans and Paranthrene pellucida are attracted to the pheromone ZZA (3,13-octadecadien-1-o1 acetate) (Taft et al.,1991). This cross attraction, combined with the glue of sticky traps can often leave the moths damaged, making it difficult if not impossible to identify the specimen morphologically (McKern and Szalanski, 2007). To combat this identification problem researchers employ molecular identification, using the cytochrome oxidase I (COI) barcode region, to differentiate between species. (McKern and Szalanski, 2007). The majority of phylogenetic studies on Sesiidae have been COI based and there have been no multi-gene phylogenetic studies of Paranthrene. Several North American Paranthrene are known to have various color morphs that vary with their distribution (Eichlin and Duckworth, 1988). Some of these morphs have been recorded with differing behaviors from the rest of their species (Eichlin and Duckworth, 1988). A multi-gene analysis of the genus would shed light on the species boundaries of these morpho-types. 1 Biology and Life History Sesiidae life history Members of Sesiidae share many common traits; nearly all adults are diurnal mimics of wasps and bees, in both their appearance and their behavior (Eichlin and Duckworth, 1988). Clearwing moths can hover in the same patterns and speeds as their hymenopteran models (Taft et al, 1991, Skowron Volponi et al, 2018). Females oviposit single eggs on host plants, typically near natural or artificial openings, so to facilitate boring of newly hatched larvae into the plant (Eichlin and Duckworth, 1988). Most species have a narrow host plant range, often specializing on a single genus (Eichlin and Duckworth, 1988) or even a single species (Taft et al, 1991). Clearwing moth larvae bore into a variety of economically important plants, and are considered pests (Solomon, 1995). For example, The lesser peachtree borer, Synanthedon pictipes, attacks peach trees and Prunus species (Cottrell et al., 2008). There are also pests of hardwoods and ornamentals like the banded ash clearwing, Podosesia aureocincta (Solomon, 1995). The moth larvae continue to expand the galleries throughout the larval instars until they exit and pupate in the pupal chamber (Eichlin and Duckworth, 1988). This tunneling in plant tissue can lead to death of part or the whole of the plant. Even those species that typically restrict their damage to unmarketable wood, can exacerbate damage by providing opportunities for fungi and other insects to colonize the wood (Solomon and Morris, 1966). The boring of these subsequent insects into these trees increases the trees’ susceptibility to structural weakness and rot (Johnson and Lyon, 1988). Paranthrene life history Eight currently recognized Paranthrene species occur in the United States, Canada, and Mexico (Baja California) (Eichlin, 1992; Handfield and Handfield, 2021).). Larvae feed on oaks ( P. asilipennis, p. pellucida, P. simulans) and on willows and poplars (P. dollii, P. robiniae, P. tabaniformis) (Eichlin and Duckworth, 1988, Solomon, 1995). The remaining two species (P. fenestrata and P. hilairemontis) are believed to feed on oaks but documentation is lacking (Eichlin and Duckworth, 1988; Handfield and Handfield, 2021). Species typically have a 2-year 2 life cycle (Eichlin and Duckworth, 1988) but at least one species, P. dollii, has been observed with a shorter life cycle in select parts of its range (Solomon, 1995). Phylogeny of Paranthrene Phylogenetic studies of Sesiidae are few, with a majority of the work addressing the relationships among tribes and subfamilies (Cognato et al, 2023; Lait and Herbert, 2018; McKern et al, 2008). Current taxonomy places Paranthrene sister to Euhagena/Vitacea and together with Albuna they comprise Paranthrini which is supported by molecular data (Eichlin and Duckworth, 1988; McKern et al, 2008). A multi-gene phylogeny places Paranthrini sister to Synanthedonini as compared to a Paranthrini/Sesiini sister relationship in a previous COI based phylogeny (Lait and Herbert, 2018; Cognato et al., 2022). Phylogenies of North American sesiids only include P. simulans and P. tabaniformis (Cognato et al., 2023; Taft et al., 2016; McKern et al., 2008). There have been no molecular multi-gene phylogenetic studies conducted to determine the phylogenetic relationships of all North American Paranthrene species, nor has any study investigated species boundaries for species with vast ranges. Mitochrondrial DNA sequence (particularly the COI the barcode region) has been the primary data source for species identification and recognition for several sesiid species level studies (McKern et al., 2008; Taft et al., 2016; Taft and Cognato, 2017; Handfield and Handfield,2022). McKern and Szalanski (2007) report intraspecific mtDNA sequence variation among Arkansian Paranthrene simulans individuals and Cognato et al. (2023) show differences among populations from Minnesota and North Carolina. Handfield and Handfield, (2022) demonstrated monophyly of mt DNA haplotypes that were diagnostic for a population of P. simulans from Quebec and associated with a diurnal flight period asynchronous with other P. simulans. They described this group as a new species P. hilairemontis. Research Objectives Due to the lack of phylogenetic studies, the overall purpose of my research is to understand if there is a monophyletic relationship with color form in Paranthrene. I will attempt to accomplish this through two supporting objectives. The construction of a multi -gene phylogeny and the delineation of species boundaries along color forms. 3 Objective 1: Construct a phylogeny of North American Paranthrene Despite Neighbor Joining COI analysis of most North American Paranthrene by Handfield and Handfield (2022), the phylogeny is far from settled. Multi-gene analysis can help elucidate these phylogenetic relationships (Cognato et al., 2023). I intend to construct both parsimony and Bayesian phylogeny of North American Paranthrene utilizing both nuclear and mitochondrial genes. This will be the first multi-gene analysis of the genus and the only COI analysis of all North American species. Objective 2: Identify species boundaries of Paranthrene with diverse color morphologies Paranthrene simulans is a polymorphic species with differing biologies among the color forms and evidence of geographic mDNA variations (Cognato et al., 2023). This demonstrates a need for further investigations into species boundaries of the different forms. I will attempt to delineate the species boundaries for the three forms currently recognized. Several other species of Parathrene have multiple forms requiring a determination of species boundaries. Parathrene robiniae has three color morphologies within different localities of its range, P. tabaniformis has its typical and oslari forms along with an unnamed form investigated by this study and, P. fenestrata has two distinct color morphologies. I will conduct a multi-gene analysis of two of the P. robiniae color forms and all North American forms of P. tabaniformis and P. fenestrtata to identify molecular evidence of speciation. 4 CHAPTER 1: A NEW SPECIES OF PARANTHRENE (LEPIDOPTERA: SESIIDAE) FROM THE NORTHERN MIDWEST US The dusky clearwing, Paranthrene tabaniformis Rottenburg 1775 (Sesiidae: Lepidoptera) (Fig. 1) is holarctic species (Solomon 1995). Like all other Paranthrene species found in North America, P. tabaniformis is a diurnal wasp mimic (Eichlin and Duckworth 1988). Its North American range includes the continental United States, Alaska, and Canada, with the exception of California (Taft et al. 1991). The larval host plants are poplars and willowsand the species can also be a major pest of nursery plants (Solomon 1995). Adults emerge from their 2-year cycle in June and July (Taft et al. 1991). Several Paranthrene are known to have various color variants (Eichlin and Duckworth 1988). Paranthrene tabaniformis is no exception with three recognized color morphologies in North America (Eichlin 1989). This species is typically characterized by a blue-black head with a grey-black frons and white lateral margins, the thorax is blue-black with yellow on the anterior margins and beneath the wings, and the abdomen is blue-black with yellow bands on segments two, four, six and, seven (Eichlin 1989) (Fig. 1). The color forms “denotata” and “oslari” are largely similar with a few chromatic differences. Most notable is the form “oslari”, which is differentiated by a yellow band on all abdominal segments except segment one, with the bands on segments two and six wider than the rest (Eichlin 1989). In 2022, near Bath township, Clinton County, Michigan, a specimen originally identified as P. tabaniformis was collected with a unique color morphology and large transparent areas in the forewings which suggested it was a distinct species. This specimen was similar to one photographed by Mr. James Sogaard, in Minnesota in 2007. We investigate this color variant’s phylogenetic placement among Paranthrene species and morphology variation. We demonstrate that it is distinct from the typical P. tabaniformis color variant and we conclude that it is a new species. 5 Material and Methods Collection of specimens In July 2022 along Stoll and State Roads in Clinton County (Rose Lake State Game Area), Michigan the second author collected three male sesiid moths using Multi-Pher #1® pheromone canister traps (Distributions Solida Inc) baited with Western Poplar Borer pheromone lure, a blend of (ZZ 3,13 OH and EZ 3,13 OH (50:50) (Scentry Biologicals, Inc). Specimen preparation and imaging One specimen of P. tabaniformis and the new form was selected for genital comparison. Abdomens were removed at the 4th or 5th segment. The removed segments were placed in separate vials filled with water and two 116mg tablets of potassium hydroxide and allowed to sit on a hot plate set just below boiling for 2 hours. Softened abdominal segments were removed from the vials and teased apart under a dissecting microscope with fine-tipped forceps until genitalia were revealed. Genitalia were preserved in glycerin and placed in 5mm glass micro vials and pinned under the associated specimen. The genitalia were not stained. Specimens were photographed with a Visionary Digital Passport II system (Dun Inc., Palmyra, VA) using a Canon EOS 5D Mark II, 65.0-mm Canon Macro photo lens, two Dynalite (Union, NJ) MH2015 road flash heads, Dynalite RoadMax MP8 power pack and a Stack Shot (Cognisys, Inc, Traverse City, MI). Montage images were assembled using Zerene Stacker 1.04 and sized in Adobe Photoshop 2021 v. 22.5.1 (San Jose, CA). DNA sequence data and phylogenetic analyses DNA was extracted from a metathoracic leg from 25 frozen specimens representing P. tabaniformis and six other Paranthrene and outgroup species (Table 1) using a Qiagen DNeasy Blood and Tissue kit (Hilden, Germany) following the manufacturer’s protocol. The remaining bodies were vouchered in the A. J. Cook Arthropod Research Collection. The purified DNA was used in PCR for mitochondrial cytochrome oxidase I and wingless (Table 2). EXO -SAP-IT (USB Corp., Cleveland, OH, USA) was used to ready the PCR products for sequencing at the Michigan State University Research Technology Support Facility using Big-Dye Terminator v 1.1 (Applied Biosystems, Foster City, CA, USA) and an ABI 3730 Genetic Analyzer (Applied Biosystems). Sense and antisense strands were compiled using Sequencher (Ann Arbor, MI) to trim sequences of 6 primer sequences, align the sequences and to create consensus sequences. Final sequences were deposited in Genbank (Table 1) and assembled in a Nexus file for a total of 1060 nucleotides (639 from COI and 417 from Wingless) which included 205 parsimony-informative characters. Phylogenetic parsimony analysis of the aligned sequences consisted of a branch and bound search using default options in PAUP v4.0a (build 168; Swofford 2002). Gaps were treated as missing data. Bootstrap values were determined with 500 pseudo-replicates each conducted by heuristic search with simple stepwise addition. Percent pairwise DNA difference was calculated as p-distance in PAUP*. In addition, Bayesian analysis under a likelihood optimality criterion was used to assess phylogenetic relationships using this dataset. Using Mr. Bayes 3.2.6 (Ronquist et al. 2012) two simultaneous analyses were conducted in which both genes were partitioned by codon position and a model of general time reversal + gamma + proportion of invariable sites was applied to each partition (unlinked parameters). Four Metropolis-coupled Markov chain Monte Carlo searches (one cold, three heated) were analyzed for 5 million generations. Each analysis was sampled every 100th iteration and all parameters reached stability. Bayesian posterior probabilities of clades were based on 75,000 trees—the total of both runs after a 25% burn-in. Results The PAUP* analysis found six most parsimonious trees that were mostly resolved in the strict consensus of those trees (Fig. 2). Intraspecific relationships for two clades and one interclade relationship were unresolved (Fig. 2). All clades of species had 100 bootstrap values and values for internal nodes varied but were mostly >85 (Fig. 2). The topology of the Bayesian tree was generally similar to the parsimony tree but differed in the placement the “sogaardi” clade as sister to Vitacea and the remaining Parathrene species. Posterior probabilities were >0.9 for all species clades except “sogaardi” at 0.7. The average interspecific pairwise “p” distance among sister species within the genus ranged between 0.062-0.101 for COI and 0.008- 0.034 for Wingless. The average “p” distance between “sogaardi” and P. tabaniformis was 0.076 and 0.031 for COI and wingless, respectively, both of which are within interspecific ranges for other species within the genus. In comparison, intraspecific “p” distance amongst “soga ardi” 7 specimens was 0.005 and 0.003 while P. tabaniformis was 0.014 and 0.002. The genetic distance between “sogaardi” and P. tabaniformis is comparable to the interspecific distance between P. tabaniformis and P. asilipennis (Table 3). This genetic divergence, in addition to monophyly and morphological diagnostic characters, supports the recognition of a new species. 8 Taxonomy Paranthrene Hubner Paranthrene is characterized by the following: Males have an apical spine on the aedeagus (Eichlin & Duckworth 1988) Paranthrene sogaardi Taft & Smith, new species Diagnosis Paranthrene sogaardi n. sp. superficially resembles several other species of North American sesiidae, most notably Paranthrene pellucida and Synanthedon rileyana. Paranthrene sogaardi n. sp. differs from P. tabaniformis by its lack of scales in the discal cell of the forewing, a dark orange discal spot on the forewings, and uniform yellow bands along abdominal segments 2-7. Whereas the discal cell in the forewing of P. tabaniformis is covered in dark scales, without a discal spot, and has yellow bands only on abdominal segments 2, 4, 6 and 7. Concerning the male genitalia, the apical point of the valves of P. sogaardi n. sp. are more rounded than P. tabaniformis and the setae are denser at the base of the valves where setal density is relatively uniform along the valve margins in P. tabaniformis (Figs. 3 & 4). The distal end of the saccus in P. sogaardi n. sp. is flattened and squared off, while the saccus in P. tabaniformis ends in a point. Both aedeagi, both have a sclerotized, toothed ridge (Figs. 5 & 6). In contrast, this ridge is covered by a semi-translucent shingle like sclerite in P. tabaniformis, this feature is absent in P. sogaardi. The following characters distinguish P. sogaardi n. sp. from P. pellucida. The cell below the Cu vein to the anal margin in the forewing of P. sogaardi n. sp. is covered in orange-brown scales, whereas the same cell in P. pellucida is without scales and transparent. The yellow bands on P. pellucida’s abdomen are thicker distally from the thorax while all bands are roughly equal in thickness on P. sogaardi n. sp. Scaling on the wings also differs between P. sogaardi n. sp. and S. rileyana. Paranthrene sogaardi n. sp. has brown-black scales on the apical tips of the forewings and brown-black scales along the M vein in the hind wings. Synanthedon rileyana lacks scales in both the aforementioned areas. In addition, P. sogaardi n. sp. has yellow tarsi while S. rileyana has black tarsi. 9 Description Male (Fig. 7 & 8). Head: Vertex primarily flat black with fine light gray hair mixed near the base; Frons light translucent pearl with a blue sheen, white laterally (Fig. 9); occipital fringe straw yellow; labial palpus strongly roughened, pale yellow with brown-black scales mixed laterally; haustellum coiled, antenna orange with a narrow line of brown black dorsally; Thorax: Dorsum flat black with fine light gray hair-like setae mixed throughout, base of forewing surrounded by a yellow spot, mixed with pale white scales, a patch of yellow setae posterior to the cape (visible in lateral view (Fig. 8), yellow scales on the posterior and posterolateral margins of the metathorax. Legs with coxae flat black with bright yellow outer margins, femora dark brown- black; tibiae light orange-yellow with a small patch of brown-black setae medially covered by orange setae, tibial spurs, and tarsi light yellow-orange mixed with black setae. Forewing mostly hyaline with an outer brown-black margin, discal spot light orange with a light edging of brown scales around the margins; anal margin lined with orange and black scales; veins and fringe brown-black with pale yellow scales on outer margin between veins (often faded in older individuals). Hindwing hyaline with narrow margins; dark brown fringe transitions to pale yellow near the wing base. Abdomen: Brown black with yellow bands encircling segments two-seven (the band width appears variable across its range); anal tuft short with a mix of black and yellow scales. Male Genitalia: (Fig. 3) Valves rounded apically, with setal density thickest toward the base. Socci with dense light-colored setae. Saccus ending in a flat squared-off tip. Female. Unknown. Host Unknown but all Paranthrene moth species feed on trees and large shrubs (i.e., oak, poplar, aspen, and willow species). Distribution Known from Central Michigan and Minnesota. Additional images of specimens from western Quebec suggest that the range of P. sogaardi n.sp. may extend into Quebec (iNaturalist.com). Because of the scattered locations of both collected specimens and images we suspect their range may include parts of Wisconsin and Ontario.Types. Holotype: Male, 10 Michigan: Clinton Co., Bath Township, Rose Lake State Game Area, Lat/Long (42.7988, - 84.40185), July 2, 2022, Coll. William H. Taft, MSUC_ARC_320053, deposited in the Albert J. Cook Arthropod Research Collection, Michigan State University, East Lansing (MSUC). Michigan paratypes ( 2 males) and Minnesota paratypes ( 1 male) at MSUC and (3) in James Sogaard’s personal collection. Etymology The species is named after Mr. James Sogaard of Princeton, Minnesota who first captured and photographed a male specimen from the same area. Remarks In Michigan, the moth was collected in a wetland swale depression (Fig 10.) characterized by willow thickets and shrub wetlands surrounded by oak uplands. The common plant species of the type location were Quaking Aspen (Populus tremuloides), Northern Red Oak (Quercus rubra), Red Maple (Acer rubrum), Sand Bar Willow (Salix interior), Heart-leaved Willow (Salix eriocephala), Black Willow (Salix nigra), Elm (Ulmus sp.), Gray Dogwood (Cornus racemose), and a non-native plant - Common Buckthorn (Rhamnus cathartica). The herbaceous plants were Joe-Pye Weed (Eutrochium purpureum), Elderberry (Sambucus canadensis), Raspberry (Rubus sp.), Boneset (Eupatorium perfoliatum), and Cattail (Typha latifolia). The EPA ecoregion designation for the type location in Michigan is the Southern Michigan /North Indiana Drift Till Plains/ Lansing Loamy Plain with the mid-elevation forests and the foothills woodlands and shrub lands designation. In Minnesota, EPA ecoregion designation for the Princeton area is the Anoka Sand Plain and Mississippi Valley Outwash (White 2020). It is characterized by substantial agriculture, but much of the region is too wet and poorly drained to be cultivated, so is left as natural wetlands (Albert 1995). 11 Figures Figure 1. Paranthrene tabaniformis. 12 Figure 2. Strict consensus of six most parsimonious trees. Numbers above branches are bootstrap values and numbers below the branches are posterior probabilities. The arrow indicates the placement of P. sogaardi in Bayesian analysis. 13 Figure 3. Adult male Paranthrene sogaardi genitalia. Figure 4. Adult male Paranthrene tabaniformis genitalia. Figure 5. Adult male Paranthrene sogaardi aedeagus. 14 Figure 6. Adult male Paranthrene tabaniformis aedeagus. Figure 7. Paranthrene sogaardi dorsal view. Figure 8. Paranthrene sogaardi lateral view. 15 Figure 9. Paranthrene sogaardi frons. Figure 10. Michigan collection site. 16 Table 1. Species, specimens, localities and Genbank numbers. Tables Species C. laurelae V. admiranda V. admiranda P. simulans P. simulans P. fenestrata P. fenestrata P. fenestrata P. fenestrata P. asilipennis P. asilipennis P. tabaniformis P. tabaniformis P. tabaniformis P. tabaniformis P. tabaniformis P. robiniae P. robiniae P. dolli P. dolli P. sogaardi P. sogaardi P. sogaardi P. sogaardi P. sogaardi DNA voucher # BT 178 BT 204 BT 205 BT 129 BT 142 BT 179 BT 180 BT 181 BT 183 BT 175 BT196 BT 151 BT 152 BT 153 BT 210 BT 168 BT 148 BT 149 BT 160 BT 161 BT 154 BT 155 BT 211 BT 169 BT 170 Location Palmer Bridge, SC Medicine Park, OK Medicine Park, OK Jacksonville, NC Manistee County, MI Emory Pass, NM Emory Pass, NM Pinos Altos, NM Emory Pass, NM North Lima, OH Swanton, OH McClellanville, SC Bath Township, MI Bath Township, MI Princeton, MN Ravalli County, MT Grant County, NM Grant County, NM Clinton County, MI Clinton County, MI Bath Township, MI Bath Township, MI Princeton, MN Clinton County, MI Clinton County, MI Genbank # COI PP333557 PP333558 PP333559 PP333560 PP333561 PP333575 PP333576 PP333577 PP333578 PP333574 PP333579 PP333564 PP333565 PP333566 PP333580 PP333571 PP333562 PP333563 PP333569 PP333570 PP333567 PP333568 PP333581 PP333572 PP333573 Genbank # Wg PP338690 PP338691 PP338692 PP338693 PP338694 PP338706 PP338707 PP338708 PP338709 PP338705 PP338710 PP338697 PP338698 PP338699 PP338711 PP338702 PP338695 PP338696 NA NA PP338700 PP338701 PP338712 PP338703 PP338704 Table 2. PCR primers and cycling conditions. PCR Primers Gene Forward Primer Primer Sequence LCO1490 5’ GGT CAA CAA ATC ATA AAG ATA TTG G 3’ Reverse Primer HCO2198 LepWg1 5’ GAR TGY AAR TGY CAY GGY ATG TCT GG 3’ LepWg2 C O I W g Primer Sequence Annealing Reference 5’ TAA ACT TCA GGG TGA CCA AAA AAT CA 3’ 5’ ACT ICG CAR CAC CAR TGG AAT GTR CA 3’ 95℃ (15min); 5 cycles of 94℃ (30s) / 45℃ (90s) / 72℃ (90 s); 30 cycles of 94℃ (30s) / 50℃ (90s) / 72℃ (90 s); 72℃ (5min) 95℃ (15min); 36 cycles of 95℃ (30s) / 55℃ (45s) / 72℃ (60 s); 72℃ (10min) Folmer et al. (1994) Brower and DeSalle (1998) 17 Table 3. The intraspecific line is ordered as (Wg/COI). “P” distance values above the intraspecific line represent average interspecific Wg values and below the line are average interspecific COI values. Wg and COI P-Distance P. asilipennis P. dolli P. fenestrate P. robiniae P.simulans P. sogaardi P.tabaniformis C. laurelae V. admiranda P. asilipennis .002/.008 .085 .072 .084 .07 .073 .076 .149 .136 P. dolli 0 0/0 .104 .062 .095 .077 .08 .164 .149 P. fenestrata .008 0 .001/.002 .101 .071 .098 .101 .163 .139 P. robiniae .032 0 .028 .002/0 .092 .087 .09 .164 .149 P. simulans .013 0 .009 .034 .005/.006 .09 .081 .168 .134 P. sogaardi .036 0 .031 .023 .037 .003/.005 .076 .153 .133 P. tabaniformis .022 0 .018 .029 .028 .031 .002/.014 .155 .141 C. laurelae .204 0 .205 .202 .206 .201 .206 NA/NA .174 V. admiranda .075 0 .071 .066 .075 .065 .068 .213 .005/.005 18 CHAPTER 2: APOSEMATIC COLOR POLYMORPHISM IS A POOR INDICATOR OF SPECIES BOUNDARIES IN NORTH AMERICAN PARANTHRENE (LEPIDOPTERA: SESIIDAE) AS EVIDENCED BY A MULTI-GENE PHYLOGENY Color polymorphism is a phenomenon among animals that has been implicated as a factor in speciation (Huxley 1955, Gray and McKinnon 2006). Selection and genetic drift act upon the color variants and the balance with gene flow among variants determines the probability that color forms will become associated with a speciation event (Gray and McKinnon 2006). Specific isolating mechanisms that drive genetic divergence among color variants include, allopatric isolation, assortative mating, and frequency- dependent selection. While sexual selection and natural selection in the context of heterogenous environment play an important part for several animals, each mechanism (alone or combined) can contribute to either the maintenance of polymorphism within a species or drive speciation (Gray and McKinnon 2006). For example, many studies of aposematic coloration, or defensive warning colors, in the context of Mullerian and Batesian mimicry demonstrate the importance of predation and the frequency of color forms (Mallet and Barton 1989, Sherratt 2008, Chouteau et al 2016). However, both positive frequency dependent selection and male mate-choice act upon aposematic Heliconis polymorphic species (Lepidoptera) to drive speciation (Mallet and Barton 1989, Joron and Iwasa 2005, Kronforst et al. 2006). Discovering the role that color polymorphism plays in speciation requires observation and experimentation (Gray and McKinnon 2006). Most important, a phylogeny provides an evolutionary pattern of species and associated color variants which can direct experimentation (e.g., Brower 1996). Clearwing moths (Lepidoptera: Sesiidae) exhibit extensive aposematic color polymorphism within species (Eichlin and Duckworth 1988). All adults are diurnal mimics of wasps and bees, in both their appearance and their behavior (Eichlin and Duckworth, 1988). This exhaustive mimicry is demonstrated with some species mimicking their hymenopteran model’s flight patterns and acoustics (Skowron Volponi et al 2018, Skowron Volponi et al 2021). As larvae, these moths bore into a variety of economically important plants, and are considered pests (Solomon, 1995). Paranthrene typically spend two years in the larval stage and feed on oaks or willows and poplars (Eichlin and Duckworth, 1988, Solomon, 1995). Two or three color 19 forms which mimics hymenopterans occur among adult Paranthrene species (Eichlin and Duckworth, 1988). These forms appear to converge their color morphology to match various locally abundant hymenopterans (Fig.11) Currently, there are nine recognized Paranthrene species occurring in the United States, Canada, and Mexico (Baja California) (Eichlin, 1992; Handfield and Handfield, 2021; Smith et al, 2024). A few polymorphic species such as, P. simulans Grote 1881, P. fenestrata Barnes and Lindsey 1922, and P. robiniae Edwards 1880 have multiple color forms. The oak borer (Paranthrene simulans) has three unique color morphs, simulans, lugerii and, palmii forms (Fig. 12a-c). Its range includes the entire eastern United States and Canada, as far west as Texas and Minnesota (Solomon, 1995). The palmii form is found in the species’ southern range and the luggeri form occurs in the western portion (Eichlin and Duckworth, 1988). In the areas where simulans and palmii overlap intermediates between the forms have been observed (Eichlin and Duckworth, 1988). Host plants for all populations are various oak species and the larvae feed on different parts of the tree depending on their range (Solomon, 1995). In the south, they feed on larger more mature trees, while in the north they are more likely to feed on saplings and small branches (Johnson and Lyon, 1995). Prior to 1988, Paranthrene palmii was considered a separate species. Paranthrene palmii was synonymized with P. simulans due to its similar morphology, host plants, and pheromone attraction but considered a form (Eichlin and Duckworth, 1988). Likewise, luggeri was originally described in 1891 and later synonymized (Beutenmüller 1896). Despite the similarities, some behavioral and life history details have been noted between the forms. While all forms feed and have a preference for red oaks, palmii has been recorded on black oak, and never on white oaks, with the inverse observed for simulans (Solomon and Morris, 1966). Even their emergence timing is slightly different. Palmii emerges between April to June, and simulans emerges between June and July (Solomon, 1995). These differences, while not enough to convince Eichlin and Duckworth to recognize the palmii form as a distinct species, prompted the researchers to suggest the need for additional study of the species boundaries (Eichlin and Duckworth, 1988). McKern and Szalanski (2007) reported intraspecific mtDNA sequence variation among Arkansian P. simulans individuals and Cognato et al. (2023) show differences among populations from Minnesota and North Carolina. 20 Handfield and Handfield, (2021) demonstrated monophyly of mtDNA haplotypes that were diagnostic for a population of P. simulans from Quebec and associated with a diurnal flight period asynchronous with other P. simulans. They described this group as a new species, P. hilairemontis Handfield and Handfield 2021. Paranthrene fensetrata has two distinct sympatric color morphologies (Fig. 13a-b) with no evidence of hybrids. All specimens have been collected from high elevations in Arizona, New Mexico, Colorado, Utah, and the Mexican state of Hidalgo, otherwise, there is no life history available for this species (Eichlin and Duckworth, 1988). The western poplar clearwing (Paranthrene robiniae) is found along the Pacific Coast of the United States, the Rocky Mountains, and the provinces of British Colombia and Alberta (Canada) (Johnson and Lyon, 1988). Its preferred host plants, poplars and willows, occur throughout western North America in isolated populations separated by other habitats non- condusive to their growth, like deserts (Solomon, 1995, Cooke and Rood 2007), There are three recognized forms, robinae, perlucida, and palescens (Fig. 14a-b). The perlucida form occurs in Canada (British Columbia and Alberta), and the United States (the Pacific Northwest to Montana) (Engelhardt 1946). The palescens form occurs in deserts of southern California (Engelhardt 1946) and Nevada (this study). Phylogenetic studies of Sesiidae are few, with a majority of study addressing the relationships among tribes and subfamilies, and only P. simulans and P. tabaniformis have been included (Cognato et al, 2023; Taft and Cognato 2017, Taft et al. 2016 Lait and Herbert, 2018; McKern et al, 2008). There are no DNA-based phylogenies of North American Paranthrene species, nor has any study investigated species boundaries for these species including polymorphic individuals. In this study, we reconstructed a phylogeny using all known species of North American Paranthrene and examined the intraspecific relationships between known color forms. We hypothesize that monophyletic groups of different color forms associate with Paranthrene species which would suggest that polymorphism of aposematic coloration may have a role in Paranthrene speciation. 21 Materials and Methods Collection and identification of specimens All but two specimens were collected over a 5 year period using Multi-Pher #1® pheromone canister traps (Distributions Solida Inc) baited with various pheromone lures intended for different species (Table 4). Two P. dollii were collected in 1988. Most Paranthrene species identifications and color morphologies were confirmed using Eichlin and Duckworth 1988. The remaining species, having been described after that publication, were identified with Smith et al 2024 and Handfield and Handfield 2022 Specimen preparation and imaging A single representative of most color forms for 8 species was picked for genital comparisons. Paranthrene hilairemontis genitalia comparisons were done using images in Handfield and Handfield 2022. Access to the genitals was gained by removing the abdomen at the 4th or 5th segment. The extracted segments were placed in individual vials filled with water and two 116mg tablets of potassium hydroxide and allowed to sit on a hot plate set just below boiling for 2 hours. Softened abdominal segments were removed from the vials and teased apart under a dissecting microscope with fine-tipped forceps until genitalia were revealed. Unstained genitalia was prepared for photography by being placed on a microscope slide with glycerin and spread open with fine-tipped forceps then held in place temporarily with a slide cover. After the images were taken genitalia were preserved in glycerin and placed in 5mm glass micro vials and pinned under the associated specimen Specimens were photographed with a Visionary Digital Passport II system (Dun Inc., Palmyra, VA) using a Canon EOS 5D Mark II, 65.0-mm Canon Macro photo lens, two Dynalite (Union, NJ) MH2015 road flash heads, Dynalite RoadMax MP8 power pack and a Stack Shot (Cognisys, Inc, Traverse City, MI). Montage images were assembled using Zerene Stacker 1.04 and sized in Adobe Photoshop 2021 v. 22.5.1 (San Jose, CA). 22 DNA sequence data and phylogenetic analyses DNA was extracted from a metathoracic leg from 67 frozen specimens encompassing 9 species of Paranthrene and 5 color forms along with two outgroup species (Table 5) using a Qiagen DNeasy blood and tissue kit (Hilden, Germany) following the manufacturer’s protocol. The remaining bodies were vouchered in the A. J. Cook Arthropod Research Collection. The purified DNA was used in PCR for mitochondrial cytochrome oxidase I, elongation factor 1a, and wingless. All three genes have been used for previous phylogenetic studies of sesiids (Taft and Cognato 2017, Cognato et al 2023). EXO-SAP-IT (USB Corp., Cleveland, OH, USA) was used to ready the PCR products for sequencing at the Michigan State University Research Technology Support Facility using Big-Dye Terminator v 1.1 (Applied Biosystems, Foster City, CA, USA) and an ABI 3730 Genetic Analyzer (Applied Biosystems). Sense and antisense strands were compiled using Sequencher (Ann Arbor, MI) to trim sequences of primer sequences, align the sequences and to create consensus sequences. Final sequences were deposited in Genbank (Table 5) and assembled in a Nexus file for a total of 1421 nucleotides (639 from COI, 365 from ef1a, and 417 from Wingless) which included 254 parsimony-informative characters. Phylogenetic parsimony analysis of the aligned sequences consisted of a branch and bound search using default options in PAUP v4.0a (build 168; Swofford 2002). Gaps were treated as missing data. Bootstrap values were determined with 500 pseudo-replicates each conducted by heuristic search with simple stepwise addition. Percent pairwise DNA difference was calculated as p-distance in PAUP*. In addition, Bayesian analysis under a likelihood optimality criterion was used to assess phylogenetic relationships using this dataset. Using Mr. Bayes 3.2.6 (Ronquist et al. 2012) two simultaneous analyses were conducted in which each gene was partitioned by codon position and a model of general time reversal + gamma + proportion of invariable sites was applied to each partition (unlinked parameters). Four Metropolis-coupled Markov chain Monte Carlo searches (one cold, three heated) were analyzed for 5 million generations. Each analysis was sampled every 100th iteration and all parameters reached stability. Bayesian posterior probabilities of clades were based on 75,002 trees—the total of both runs after a 25% burn-in. 23 Results The PAUP* analysis found 600 most parsimonious trees that were mostly resolved in the strict consensus of those trees (Fig. 16). Intraspecific and interspecific relationships of all P. simulans forms, P. pellucida and P. hilairemontis were unresolved (Fig. 16) resulting in a single clade with a 100% bootstrap value. All other clades of species except P. robiniae and P. dollii had 100% bootstrap values and values for internal nodes varied (Fig. 16). The topology of the Bayesian tree was generally similar to the parsimony tree (Fig 17). Posterior probabilities (PP=1) were high for all species clades except P. pellucida, P. hilairemontis, P. robiniae and, P. dollii. Paranthrene pellucida and P. hilairemontis were intermixed with P. simulans. Within the P. simulans clade the placement of P. pellucida in both parsimony and Bayesian trees and P. hilairemomtis in the Bayesian tree rendered P. simulans as paraphyletic. Paranthrene simulans had an average intraspecific COI divergence of 1.56% and interspecific values of 1.17% and 1.27% compared to P. pellucida and P. hilairemontis respectively (Table 6). The divergence for nuclear genes between these species was <1% and <1.35% for Wg. These distances are similar to other intraspecific and interspecific distances of sister species within Paranthrene (Tables 9 and 12). Given the COI divergence was < 2%, as compared to the sister species, P. fenestrata (Table 6), the paraphyly of P. simulans, and a lack of genitalic differences among P. simulans, P. hilairemontis and P. pellucida, we synonymize the latter species with P. simulans. The luggeri form was the only monophyletic clade among P. simulans color forms and it rendered P. simulans paraphyletic. This clade is weakly supported in the parsimony (64% bootstrap value) and Bayesian trees (PP = 0.83) (Fig 16 and 17) and had a larger sequence divergence than any other color forms (Table 6). Neither the typical black or yellow forms of P. fenstrata were monophyletic in either tree. They resolved polyphyletic within the clade representing P. fenstrata (Fig. 16 and 17). There was no DNA divergence or morphological evidence to support the recognition of these forms as species (Table 6). Paranthrene robiniae and P. dollii resulted in a single weakly supported clade with specimens collected at different localities monophyletic with a 100% bootstrap value (Fig. 16). In the Bayesian tree, individual geographic clades of P. robiniae and P. dollii were less supported 24 with a posterior probability ranging from 0.68-1 (Fig. 17) but had similar placement to the parsimony tree. The placement of P. dollii rendered P. robiniae paraphyletic in both trees. The palescens form P. robiniae was polyphyletic, occurring in three separate clades (Fig. 16 and 17). The interspecific COI divergence among the three P. robiniae populations and P. dollii ranged between 6% and 9% (Table 7). The intraspecific COI divergence for each group was < 1%, except the Nevada population which was 7%. The great divergence observed in the Nevada population was caused by a single individual and removal of this single specimen decreased the average divergence to 1%. This specimen is an anomaly and requires investigation in future studies. External morphologies between the three populations showed striking similarities with little to negligible difference between the Nevada and New Mexico populations. The monophyletic Arizona and New Mexico populations had diagnostic differences of the male genitalia consistent with currently recognized species (Fig. 19 and 20). The two species are described below. 25 Taxonomy Paranthrene Hubner Males of Paranthrene have an apical spine on the aedeagus (Eichlin & Duckworth 1988) Paranthrene oasis Smith, Taft and, Cognato, new species Type material Holotype, male, United States: Arizona., Base and Meridian Wildlife Area, 280m ele., 33°22'37"N 112°18'24"W. 21vi2023. W, Smith Col (MSUC), second label, “DNA voucher BT195”. One paratype as previous except male genitalia dissected and with second label, “DNA voucher BT 194” (MSUC). Diagnosis Paranthrene oasis is most similar to the palescens form of P. robiniae. The two species are remarkably similar in gross appearance but can be differentiated by color pattern. In P. oasis the prothoracic collar and first abdominal segment are yellow in their entirety and in P. robiniae the collar is bicolored, brown-orange proximal to the head and yellow distally, and the first abdominal segment is pigmented orange to brown (Fig. 18c-d and 21). The tarsal spines of P. oasis are orange spines while spines of P. robiniae are black (Fig. 18a-b). The saccus of the male genitalia, of P. oasis is slender and terminates in a point, while the saccus of P. robiniae is spatulate. (Fig. 19 a-c). The aedeagus spine in P. oasis is pointed and thorn-like with few teeth, while the spine on P. robiniae is broad and ridge-like with many teeth (Fig 20). Description Male (Fig. 14c). Head: Vertex covered in long yellow to light orange scales; Frons light yellow; Labial palps roughened, yellow with burnt orange scales along ventral side; Haustellum coiled and longer than labial palps; Antenna burnt orange. Thorax: Prothorax covered in a solid collar of yellow scales (Fig. 18c). Scutum brown and burnt orange; Scutellum brown and burnt orange bordered posteriorly with light yellow; Tegulas brown and burnt orange anteriorly transitioning to yellow posteriorly; Pluerons with broad flat burnt orange and yellow scales; Metanotum yellow, lateral anterior margins with long dense burnt orange setae; Legs primarily yellow with some burnt orange ventrally on all segments; Coxae yellow and burnt orange; Prothoracic femur concaved posteriorly and line with burnt orange setae; Mid-tibia with a 26 single pair of ventrally apical spurs; Hind tibia with two pairs of yellow to burnt orange ventral spurs; All tarsi with ventral burnt orange spines (Fig. a). Forewing: Mostly burnt orange to brown scales. Orange scales brightest along the base and basal portion of the inner margin; A faint orange discal spot; R, M, and C veins with dark brown scales; Brown fringe along marginal edge. Hindwing: Hyaline with dark brown fringe. Small patch of basal orange scales; Dark brown scales along all veins except R-M and M veins closing the discal cell. Abdomen (Fig 21d): Predominantly yellow with a thin anterior dorsal band of burnt orange on segment two and thin dorsal posterior bands on segments five and six. Segment three yellow and burnt orange circumferentially with a dorsally posterior band of dark brown scales; Anal tufts short and golden yellow. Male Genitalia: Saccus is shaped in a narrow triangular prism and terminates in a point. The subscaphium squares off bluntly behind the transtilla. The spine on the aedeagus is pointed and thorn like (Fig. 20c). Female Unknown. Host Unknown Distribution Known only from two specimens collected in the Base and Meridian Wildlife Area south of Phoenix Arizona near Estrella Mountain. Etymology “Oasis” used as a noun in apposition. This species is named after the environment it was found, that is, in the narrow vegetated area along the Gila River in the deserts of Arizona. Remarks Paranthrene oasis was collected in a trap hanging from a Fremont cottonwood Poplus fremontii baited with Scentry Western Poplar Clearwing Moth Lures © from Great Lakes IPM © (Table 4). There was also an abundance of Gooding’s Willows Salix goodingii in the habitat. 27 Paranthrene gilaensis Smith, Taft, and Cognato, new species Type material Holotype, male, United States: New Mexico., Lake Roberts, Gila National Forest, 1846m ele., 33°01'44"N 108°09'04"W. 18vii2017. W, Taft Col.(MSUC), second label, “DNA voucher BT149”. Paratypes with same locality label. One with male genitalia dissected and second label “DNA voucher BT 148” and one with second label “DNA voucher BT 150” (2 MSUC). Diagnosis Paranthrene gilaensis most closely resembles P. robiniae. Diagnosis is difficult due to the color polymorphism in P. robiniae. Paranthrene gilaensis has darker forewings and the hindwing fringe is slightly darker and thicker. On the second abdominal segment of P. gilaensis there are three different colored bands, anterior band is black, followed by a thin red band, and a thicker yellow posterior band (Fig. 21c). In P. robiniae this middle red band is missing, although some red scales may occur but not enough to form a continuous band around the abdomen. On abdominal segment three, P. gilaensis is black dorsally and transitions laterally to red and ventrally to orange. In P. robiniae this band is black, or black and yellow for the entire circumference of the segment. Male genitalia provides unambiguous diagnostic an feature. The subscaphium in P. gilaensis is bifurcated basally while in P. robiniae ends in a single point. The presence of dark scales on the hindwing differentiate P. dollii from P. gilaensis in which the dark scales are absence. Description Male (Fig. 14d). Head: Vertex covered by long yellow to burnt orange scales; Frons covered in orange scales with occipital margins pale yellow, all covering a layer of smooth black scales underneath. These black scales might not be seen unless the upper layer of orange scales is rubbed off; Labial palps roughened, almost entirely light yellow and light orange except for the base which is burnt orange red and a scattering of long slender black scales; Antenna orange to orange-brown with black scales on the dorsal portion at the apical end. Thorax: Prothorax with a bicolored collar of burnt orange on the anterior margin and yellow on the posterior. The anterior orange scales cover a layer of smooth black scales similar to the frons; Scutum black with some long hair-like red scales: Scutellum black bordered posteriorly with 28 yellow, this yellow border may be speckled with occasional red scales; Tegulas black anteriorly with many long red hairlike scales, then yellow posteriorly giving the appearance of red and yellow “shoulders” (Fig 22b); Pluerons black; Legs, Coxae black with dorsal tufts of orange scales, Femurs orange on the anterior side and black on the posterior, Mid-tibia with a single pair of ventrally apical spurs; Hind tibia with two pairs of yellow to burnt orange ventral spurs, all Tibias orange and yellow, all Tarsi orange and yellow with black spines; Forewing: Densely covered with brown and dark orange scales, Ata the base of the wing dense black scales along the anterior margin and dense red scale posteriorly. Hindwing: Hayline with a thick dark brown fringe, bright orange scales along basal edge, all veins with brown scales except brown and burnt orange scales covering the R-M and M veins closing the discal cell. Abdomen: Predominantly yellow except for the first three segments, Segment one black, segment two with an anterior black band, then a thin red band followed by a posterior yellow band, abdominal segment three is black dorsally and transitions red laterally to yellow or orange ventrally, there is some variation with the lateral red scales forming a dorsal band between black bands, and the black scales forming a very thin circumferential anterior band; Male Genitalia: Saccus broadly spatulate and narrowed slightly at the base (Fig. 19d). The subscaphium long extending behind the transtilla and terminates in a bifurcated structure. Female Unknown. Host Unknown Distribution Known only from Lake Roberts New Mexico, within the Gila National Forest. Etymology This species is named after the type locality in the Gila National Forest. Remarks Paranthrene gilaensis was collected in traps baited with Western Poplar Borer (EZ 3,13 OH/ZZ 3,13 OH) 50:50 pheromone blend. 29 Paranthrene simulans Grote 1881 Trichilium simulans Grote, 1881, Bull. Brooklyn Ent. Soc., 3:78. Fatua palmii Hy. Edwards, 1887, Can. Ent., 19:145. Trochilium luggeri Hy. Edwards, 1891, Psyche, 6:108. Paranthrene pellucida Greenfield and Karandinos, 1979, Proc. Ent. Soc Washington, 81:499. New Synonymy Paranthrene hilairemontis Handfield and Handfield, 2021, Journ. Lep. Soc. 75(4):252- 258. New Synonymy Diagnosis Paranthrene simulans can be separated from other North American Paranthrene by a combination of hyaline hindwings, occipital fringe black dorsally. Redescription Male (Fig. 12). Head: Vertex black, except in palmii where it is black and yellow-orange; Occipital fringe black; Frons black with occipital margins white-yellow to orange-yellow; Labial palps white-yellow to orange-yellow, black at the base and various amounts of black ventrally; Antenna black with dark orange tips; Thorax: Prothorax with bicolored collar, anterior margin black and posterior band ranges from pale white-yellow, yellow to golden orange; Scutum and scutellum black with fringes ranging from pale white-yellow, yellow to golden orange: Tegulas black with a patch of “shoulder” scales ranging from pale white-yellow, yellow, to golden yellow; Legs, bicolored, black basally ending from the trochanter to the apical end of the tibia, second color ranges from pale white-yellow, yellow to golden orange-yellow; Forewings: except in the pellucida form, covered in brown, orange-brown, to black scales and hyaline in the tornus and outer margin, scaling much more dense in the palmii form, forewings of the pellucida form is hyaline with a brown discal spot and brown to black scales only along veins, brown to black fringe; Hindwings: Hyaline with brown to black scales along veins and brown to black fringe, base of inner margin either black, yellow or orange; Abdomen: Highly variable, Each segment may contain black scales ranging from small patches on the posterior margin (palmii), narrow bands (luggeri), to many whole segment black (hilairemontis), non-black portions of the abdomen range from pale white-yellow, yellow to golden orange-yellow; Male Genitalia: Valves 30 rounded, may have a slight point apically and many dense setae, Saccus spatulate, thickness of apical end may vary. Remarks Paranthrene simulans, P. pellucida, and P. hilairemontis are synonymized given the inclusion of the latter in a well-supported monophyletic group of widely distributed P. simulans individuals (Figs. 16 and 17) and the <2% interspecific DNA divergence of both mitochondrial and nuclear genes (Tables 6, 9, and 12). Genitalic morphology is undistinguishable between P. simulans, P. pellucida and P. hilairemontis and the extent of color polymorphism is consistent with the variability observed with other P. simulans color forms. 31 Discussion Traditional taxonomy studies synonymized many color forms (Engelhardt 1946, Eichlin and, Duckworth 1988). Several of these findings are upheld in this study such as the synonymies of the P. simulans and P. robiniae forms. Paranthrene pellucida and P. hilairemontis, along with palmii and luggeri, should be recognized as unique color forms of P. simulans, as well as, palescens as a form of P. robiniae. Conversely, this study and previous Sesiidae molecular phylogenetic studies have supported the recognition of color forms as species (Taft et al 2016, Smith et al 2024). However, of the nine color forms we examined only two are monophyletic, have comparable DNA sequence divergence, and associated with species diagnostic morphology. Thus, color pattern is not a predictable indicator for species boundaries. Maintenance of color polymorphism among Paranthrene species is likely a complexity of many factors including natural selection and genetic drift (Huxley 1955, Gray and McKinnon 2006). Color polymorphism of aposematic Lepidoptera can be maintained through selection against hybrids in narrow hybrid zones (Mattel and Barton 1989). A zone of overlap occurs for the simulans and palmii forms (Solomon, 1995, Eichlin and Duckworth 1988 and, Englehardt 1946), but this is not the case for the majority Paranthrene forms whose ranges are entirely sympatric. For example, the geographic ranges of the luggeri, pellucida, and hilairemontis forms are completely sympatric with the range of the simulans form (Handfield and Handfield 2021, Eichlin 1989, Eichlin and Duckworth 1988). The range of the P. robiniae palescens color form also completely overlaps with the robiniae color form (Englehardt 1946). Within these sympatric ranges, local abundance of particular models may provide selection at small geographic scales and/or for a limited time thus creating a patchwork of color forms without mating barriers (Mattel and Barton 1989). Little is known concerning the distribution of Paranthrene color forms and model hymnopteran species or predation intensity against poor mimics of local models. Alternatively, sexual selection may drive the diversity of color forms (refs). However, sesiids use pheromones to locate mates (Eichlin and Duckworth 1988). All color forms of P. simulans were collected with the same two semiochemical blends (Taft et al 1991). These include the semiochemicals used in the collection of the hilairimontis and pellucida forms 32 (Greenfield and Karandinos 1979, Taft et al 1991, Handfield and Handfield 2021). Also, all three P. robiniae populations and P. dollii were collected with the same semiochemical blend (Table 4). It is unlikely that color form and pheromone type are genetic linked. Thus, sexual selection likely does not contribute to maintenance of Paranthrene color forms although it is unknown if color pattern is used in mate recognition. We hypothesis genetic drift is an important factor in the fixation of color forms and it is coincidental with allopatric speciation. In this study and others, species were described or validated from mountainous sky islands and oasises (Taft and Cognato 2017, Cognato et al. 2023). The sky islands and deserts of southwestern United States are notable for their role in speciation due to geographic isolation (Masta 2000, Mitchell and Ober 2013, Edwards et al 2016). Given that much of sesiid diversity occurs in this region, it is likely that additional cryptic or pseudocryptic species exists among color polymorphic species given the apparent recent species radiation of several sesiid genera (Cognato et al. 2023). The abberant P. robiniae palescens form specimen (BT193) may represent a cryptic species where color form has not coincided with DNA divergence. Color pattern is a poor indicator of species boundaries in Paranthrene. A combination of monophyly, morphology, and DNA divergence reliably delimit Paranthrene species boundaries. These species may include populations of different color forms. Geographic barriers appear to play an important role in limiting gene flow, especially in the American Southwest. A more comprehensive survey of geographically separated populations of Paranthrene and of their hymenopteran models is needed to better understand the maintenance of color forms. 33 Figures Figure 11. Paranthrene spp (right)beside potential hymenoperan models (left). 34 Figure 12. a) Paranthrene simulans “simulans” b) Paranthrene simulans “luggeri” c) Paranthrene simulans “palmii” d) Paranthrene pellucida e) Paranthrene hilairemontis. 35 Figure 13. Paranthrene fenestrata a) yellow b) black. Figure 14. a)Paranthrene robiniae “robiniae” b) Paranthrene robiniae “palescens” c) Paranthrene oasis d) Paranthrene gilaensis e) Paranthrene dollii. 36 Figure 15. a) Paranthrene tabaniformis b). Paranthrene tabaniformis “oslari” c) Paranthrene sogaardi d) Paranthrene asilipennis. 37 Figure 16. Strict consensus of 600 most parsimonious trees. Numbers above branches are bootstrap values. 38 Figure 17. Bayesian analysis. Numbers above nodes are posterior probabilities based on 75,002 trees. 39 Figure 18. Paranthrene oasis and P. robiniae comparison a) Paranthrene oasis tarsal spines b) Paranthrene robiniae tarsal spines c) Paranthrene oasis prothoracic collar d) P. robiniae prothoracic collar. 40 Figure 19. Genitalia a) Paranthrene robiniae b) Paranthrene dollii c) Paranthrene oasis d) Paranthrene gilaensis. Figure 20. Adeagus spines a) Paranthrene robiniae b) Paranthrene dollii c) Paranthrene oasis d) Paranthrene gilaensis. 41 Figure 21. Abdominal color patterns a) Paranthrene robiniae “robiniae” b) Paranthrene robiniae “palescens” c) Paranthrene gilaensis d) Paranthrene oasis. Figure 22. Tegula “shoulder” scales a) Paranthrene robiniae b) Paranthrene gilaensis. 42 Tables Table 4. Pheromones used for species collection. Blend Retailer Target Species ZZ 3,13 OH/EZ 3,13 OH (50:50) Great Lakes IPM P. robiniae ZZ 3,13 A/EZ 2,13 A/Z 13 A (80: 15: 5 ) Alpha Scent EZ 3,13 A/ZZ 3,13 A (4:1) Alpha Scents ZZ 3,13 A/ZZ 2,13 OH (75:25) Great Lakes IPM P. dollii P. oasis P. gilaensis P. tabaniformis P. sogaardi P. fenestrata P. simulans P. pellucida P. simulans P. pellucida EZ 2,13 A Anglian Lepidopterist Supplies P. hilairemontis EZ2,13 A/Z 13 A (96:4) Alpha Scents P. asilipennis 43 Table 5. Species, specimens, localities and Genbank numbers. Species Color Form DNA voucher # Location Genbank # COI Genbank # Genbank # Ef1a V. admiranda V. admiranda BT 204 BT 205 Medicine Park, OK PP333558 Wg PP338691 PP661425 Medicine Park, OK PP333559 PP338692 PP661426 P. simulans Unkown BT103 Frankin, NC PP654332 PP661526 PP661427 P. simulans Unkown P. simulans P. simulans simulans simulans BT 177 BT 122 BT 141 North Lima, OH PP654361 PP661563 PP661471 Minneapolis, MN PP654333 PP661523 PP661428 Manistee County, MI PP654345 PP661536 PP661441 P. simulans simulans BT142 Manistee County, MI PP333561 PP338694 PP661442 P. simulans simulans P. simulans simulans P. simulans simulans P. simulans simulans luggeri luggeri luggeri luggeri luggeri luggeri palmii palmii palmii palmii palmii palmii palmii palmii palmii P. simulans P. simulans P. simulans P. simulans P. simulans P. simulans P. simulans P. simulans P. simulans P. simulans P. simulans P. simulans P. simulans P. simulans P. simulans P. pellucida P. pellucida P. pellucida P. pellucida P. Hilairemontis P. fenestrata P. fenestrata P. fenestrata P. fenestrata P. fenestrata P. fenestrata P. fenestrata P. gilaensis P. gilaensis typical typical typical yellow yellow yellow yellow typical typical BT 143 BT 144 BT 146 BT 147 BT 123 BT 134 BT 137 BT 138 BT 139 BT 145 BT 129 BT 130 BT 131 BT 132 BT 133 BT 135 BT 136 BT 172 BT 173 BT 156 BT 157 BT 158 BT 171 BT 174 BT 179 BT 181 BT 182 BT 180 BT 183 BT 184 BT 185 BT 148 BT 149 Manistee County, MI PP654346 PP661538 PP661443 Clinton County, MI PP654347 PP661539 PP661444 Clinton County, MI PP654349 PP661541 PP661446 Clinton County, MI PP654350 PP661542 PP661447 Minneapolis, MN PP654334 PP661524 PP661429 Princeton, MN PP654339 PP661530 PP661435 Princeton, MN PP654342 PP661533 PP661438 Princeton, MN PP654343 PP661534 PP661439 Fairfax, VA PP654344 PP661535 PP661440 Clinton County, MI PP654348 PP661540 PP661445 Jacksonville, NC PP333560 PP338693 PP661430 Godsden County, FL PP654335 PP661526 PP661431 Palatka, FL PP654336 PP661527 PP661432 Jacksonville, NC PP654337 PP661528 PP661433 Jacksonville, NC PP654338 PP661529 PP661434 Jacksonville, NC PP654340 PP661531 PP661436 Jacksonville, NC PP654341 PP661532 PP661437 Gainsville, FL Gainsville, FL PP654357 PP661558 PP661466 PP654358 PP661559 PP661467 Clinton County, MI PP654352 PP661551 PP661456 Gratiot County, Mi PP654353 PP661552 PP661457 Gratiot County, Mi PP654354 PP661553 PP661458 Clinton County, MI PP654356 PP661557 PP661465 Quebec, Canada PP654359 PP661560 PP661468 Emory Pass, NM PP333575 PP338706 PP661472 Pinos Altos, NM PP333577 PP338708 PP661474 Pinos Altos, NM PP654362 PP661567 PP661475 Emory Pass, NM PP333576 PP338707 PP661473 Emory Pass, NM PP333578 PP338709 PP661476 Pinos Altos, NM PP654363 PP661569 PP661477 Emory Pass, NM PP654364 PP661570 PP661478 Grant County, NM PP333562 PP661543 PP661448 Grant County, NM PP333563 PP661544 PP661449 44 Table 5 (cont’d) P. gilaensis P. robiniae P. robiniae typical typical typical BT 150 BT 190 BT 191 Grant County, NM PP654351 PP661545 PP661450 Henderson, NV PP654366 PP661572 PP661480 Henderson, NV PP654367 PP661573 PP661481 P. robiniae palescens BT 188 Henderson, NV PP654365 PP661571 PP661479 P. robiniae palescens BT 192 Henderson, NV PP654368 PP661574 PP661482 P. robiniae palescens BT 193 Henderson, NV PP654369 PP661575 PP661483 P. oasis P. oasis P. tabaniformis typical P. tabaniformis typical P. tabaniformis typical P. tabaniformis typical P. tabaniformis typical P. tabaniformis typical P. tabaniformis oslari P. dollii P. dollii P. dollii P. sogaardi P. sogaardi P. sogaardi P. sogaardi P. sogaardi P. asilipennis P. asilipennis P. asilipennis P. asilipennis BT 194 BT 195 BT 151 BT 152 BT 153 BT 168 BT 176 BT 210 BT 214 BT 159 BT 160 BT 161 BT 154 BT 155 BT 169 BT 170 BT 211 BT 175 BT 197 BT198 BT196 Pheonix, AZ Pheonix, AZ PP654370 PP661576 PP661484 PP654371 PP661577 PP661485 McClellanville, SC PP333564 PP338697 PP661451 Bath Township, MI PP333565 PP338698 PP661452 Bath Township, MI PP333566 PP338699 PP661453 Ravalli County, MT PP333571 PP338702 PP661462 North Lima, OH PP654360 PP661562 PP661470 Princeton, MN PP333580 PP338711 PP661489 Comanche County, OK PP654374 PP661583 PP661491 Clinton County, MI PP654355 Clinton County, MI PP333569 Clinton County, MI PP333570 NA NA NA PP661459 PP661460 PP661461 Bath Township, MI PP333567 PP338700 PP661454 Bath Township, MI PP333568 PP338701 PP661455 Clinton County, MI PP333572 PP338703 PP661463 Clinton County, MI PP333573 PP338704 PP661464 Princeton, MN PP333581 PP338712 PP661490 North Lima, OH PP333574 PP338705 PP661469 Swanton, OH Swanton, OH Swanton, OH PP654372 PP661579 PP661487 PP654373 PP661580 PP661488 PP333579 PP338710 PP661486 45 Table 6. Average percent COI "p" - distance between Paranthrene simulans, Paranthrene pellucida, Paranthrene hilairemontis and, Paranthrene fenestrata species and color forms. s i m u l a n s P . s i m u l a n s l u g g e r i P . s i m u l a n s p a m l i i P . s i m u l a n s P . h i l a i r e m o n t i s P . p e l l u c i d a " b l a c k " P . f e n e s t r a t a " y e l l o w " P . f e n e s t r a t a P. simulans simulans P. simulans luggerii P. simulans palmii P. hilairemontis 0.83% 0.65% 2.40% 2.03% 0.94% 0.55% 6.95% 2.63% 2.27% 2.66% 7.51% 6.76% 7.36% P. pellucida P. fenestrata "black" P. fenestrata "yellow" 1.15% 1.11% 0.90% 7.04% 6.84% NA 1.10% 6.36% 0.26% 7.17% 0.31% 6.26% 6.97% 0.23% 0.16% Table 7. Average percent COI "p" - distance between Paranthrene robiniae, Paranthrene dollii, Paranthrene oasis n. sp. and, Paranthrene gilaensis n.sp. species and color forms. P . o a s i s n . s p n . s p P . g i l a e n s i s t y p i c a l P . r o b i n i a e l p a e s c e n s P . r o b i n i a e P . d o l l i i P. oasis n.sp P. gilaensis n.sp P. robiniae typical 0.16% 7.43% 9.00% P. robiniae palescens 8.01% P. dollii 7.43% 0.00% 7.98% 7.25% 6.26% 0.63% 2.74% 8.29% 5.12% 7.88% 0.00% 46 Table 8. Average percent COI "p" - distance between Paranthrene species. P . d o l l i i P . a s i l l i p e n n i s P . f e n e s t r a t a P . h i l a i r e m o n t i s P . p e l l u c i d a P . o a s i s n . s p P . g i l a e n s i s n . s p P . r o b i n i a e P . s i m u l a n s P . s o g a a r d i P . t a b a n i f o r m i s V . a d m i r a n d a P. asillipennis 0.65% P. dollii 8.57% 0.00% P. fenestrata 7.28% 10.49% 0.22% P. hilairemontis P. pellucida 6.30% 8.76% 6.31% NA 7.00% 9.31% 7.06% 1.10% 0.26% P. oasis n.sp 7.39% 7.43% 8.11% 7.90% 8.45% 0.16% P. gilaensis n.sp P. robiniae 8.26% 6.26% 10.06% 9.08% 9.15% 7.43% 0.00% 8.21% 7.38% 10.20% 9.08% 9.43% 8.04% 7.62% 5.20% P. simulans 7.17% 9.41% 7.00% 1.27% 1.17% 8.55% 9.66% 9.55% 1.56% P. sogaardi 7.32% 7.67% 9.77% 8.29% 9.09% 7.56% 8.70% 8.40% 8.93% 0.47% P. tabaniformis V. admiranda 7.60% 8.03% 9.98% 7.38% 7.96% 7.49% 8.87% 8.65% 7.86% 7.46% 13.62% 14.87% 13.90% 13.22% 13.46% 12.83% 14.95% 14.28 % 13.51% 13.26 % 1.56 % 14.14 % 0.47% Table 9. Average percent EF1a "p" - distance between Paranthrene simulans, Paranthrene pellucida, Paranthrene hilairemontis and, Paranthrene fenestrata species and color forms. s i m u l a n s P . s i m u l a n s l u g g e r i P . s i m u l a n s p a m l i i P . s i m u l a n s P . h i l a i r e m o n t i s P . p e l l u c i d a " b l a c k " P . f e n e s t r a t a " y e l l o w " P . f e n e s t r a t a P. simulans simulans P. simulans luggerii P. simulans palmii P. hilairemontis 0.64% 0.48% 0.64% 0.45% 0.47% 0.34% 0.69% 0.50% 0.98% 0.35% 0.66% 0.56% 0.60% P. pellucida P. fenestrata "black" P. fenestrata "yellow" 0.36% 0.91% 0.26% 0.59% 0.53% N/A 0.70% 0.67% 0.57% 0.00% 0.39% 0.38% 0.29% 0.29% 0.29% 47 Table 10. Average percent EF1a "p" - distance between Paranthrene robiniae, Paranthrene dollii, Paranthrene oasis n. sp. and, Paranthrene gilaensis n.sp. species and color forms. P . o a s i s n . s p n . s p P . g i l a e n s i s t y p i c a l P . r o b i n i a e l p a e s c e n s P . r o b i n i a e P . d o l l i i P. oasis n.sp P. gilaensis n.sp P. robiniae typical 0.00% 0.29% 0.29% P. robiniae palescens 0.19% P. dollii 0.58% 0.19% 0.67% 0.49% 0.47% 0.00% 0.19% 0.86% 0.29% 0.78% 0.00% Table 11. Average percent EF1a "p" - distance between Paranthrene species. P . d o l l i i P . a s i l l i p e n n i s P . f e n e s t r a t a P . h i l a i r e m o n t i s P. asillipennis 0.15% P. dollii 2.07% 0.00% P. fenestrata 1.27% 2.64% 0.32% P. hilairemontis 1.35% 3.07% 0.62% NA P . p e l l u c i d a P . o a s i s n . s p P . g i l a e n s i s n . s p P . r o b i n i a e P . s i m u l a n s P . s o g a a r d i P . t a b a n i f o r m i s V . a d m i r a n d a P. pellucida 0.93% 2.26% 0.34% 0.70% 0.00% P. oasis n.sp 1.51% 0.58% 2.06% 1.99% 1.72% 0.00% P. gilaensis n.sp 1.83% 0.47% 2.35% 2.61% 2.12% 0.29% 0.19% P. robiniae 1.74% 0.81% 2.29% 2.33% 1.95% 0.23% 0.56% 0.22% P. simulans 1.28% 2.52% 0.60% 0.84% 0.32% 1.94% 2.34% 2.20% 0.48% P. sogaardi 2.90% 2.60% 3.18% 3.44% 2.83% 2.04% 2.40% 2.28% 3.17% 0.40% P. tabaniformis 2.06% 2.01% 2.55% 2.76% 2.29% 1.41% 1.80% 1.60% 2.40% 2.36% V. admiranda 5.95% 5.55% 5.69% 5.31% 5.53% 5.03% 5.17% 5.11% 5.62% 5.40% 0.42 % 4.48 % 3.44 % 48 Table 12. Average percent Wg "p" - distance between Paranthrene simulans, Paranthrene pellucida, Paranthrene hilairemontis and, Paranthrene fenestrata species and color forms. s i m u l a n s P . s i m u l a n s l u g g e r i P . s i m u l a n s p a m l i i P . s i m u l a n s P . h i l a i r e m o n t i s P . p e l l u c i d a " b l a c k " P . f e n e s t r a t a " y e l l o w " P . f e n e s t r a t a P. simulans simulans P. simulans luggeri P. simulans palmii P. hilairemontis 0.85% 0.43% 0.89% 0.00% 0.52% 0.74% 0.75% 0.97% 0.78% 1.04% 1.14% 0.77% 1.16% P. pellucida P. fenestrata "black" P. fenestrata "yellow" 0.70% 1.10% 0.95% 1.06% 1.08% NA 1.35% 0.94% 0.96% 0.28% 1.18% 0.16% 1.20% 0.10% 0.12% Table 13. Average percent Wg "p" - distance between Paranthrene robiniae, Paranthrene dollii, Paranthrene oasis n. sp. and, Paranthrene gilaensis n.sp. species and color forms. P . o a s i s n . s p n . s p P . g i l a e n s i s t y p i c a l P . r o b i n i a e l p a e s c e n s P . r o b i n i a e P . d o l l i i P. oasis n.sp P. gilaensis n.sp P. robiniae typical 0.00% 2.72% 2.62% P. robiniae palescens 2.62% P. dollii 0.00% 0.16% 0.08% 0.08% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 49 Table 14. Average percent Wg "p" - distance between Paranthrene species. P . d o l l i i P . a s i l l i p e n n i s P . f e n e s t r a t a P . h i l a i r e m o n t i s P . p e l l u c i d a P . o a s i s n . s p P . g i l a e n s i s n . s p P . r o b i n i a e P . s i m u l a n s P . s o g a a r d i P . t a b a n i f o r m i s V . a d m i r a n d a P. asillipennis P. dollii 0.36% 0.00% 0.00% P. fenestrata 0.83% 0.00% 0.11% P. hilairemonti s P. pellucida 1.23% 0.00% 0.95% NA 1.69% 0.00% 1.19% 1.35% 0.28% P. oasis n.sp 3.98% 0.00% 3.37% 0.00% 3.50% 2.82% 3.89% 3.93% 0.00% 3.64% 3.69% 2.72% 0.16% 3.24% 0.00% 2.79% 3.60% 3.68% 2.62% 0.08% 0.00% P. simulans 1.47% 0.00% 0.99% 0.95% 0.90% 3.76% 3.53% 3.51% 0.68% P. sogaardi 3.58% 0.00% 2.32% 0.00% 3.14% 1.85% 4.02% 2.47% 2.31% 2.23% 3.85% 0.29% 2.28% 2.70% 3.60% 2.89% 2.79% 2.51% 3.24% 7.51% 0.00% 7.08% 7.92% 7.34% 7.15% 6.58% 6.43% 7.71% 6.45% P. gilaensis n.sp P. robiniae P. tabaniformis V. admiranda 0.20 % 6.85 % 0.48 % 50 CONCLUSION Our phylogeny of North American Paranthrene does not demonstrate an association between color morphology and species boundaries. We hypothesize that other factors may be playing a role in speciation within the genus. Paranthrene demonstrates unique allopatric color polymorphism making them a potential model organism for the study of polymorphic color maintenance in Batesian mimicry. Future work This study was unable to evaluate all color morphologies for all North American species. More specifically the intraspecific relationships between all P. dolli and P. robiniae forms along with allopatric populations need further investigation. The phylogeny in Handfield and Handfield (2021) showed P. dolli rendering the perlucida and robiniae forms of P. robiniae as paraphyletic along with the single Robiniae anomaly recognized in chapter two demonstrate unresolved relationships within the species. In addition, observations of several P. robiniae specimens in the AJ Cook Arthropod Research Collection found unique, unnamed color morphologies of P. robiniae from the mountains of southern Arizona. A more comprehensive phylogeny of P. dolli forms and P. robiniae populations is still needed. Polymorphic color maintenance in Paranthrene remains under explored. Our phylogeny did not demonstrate monophyletic clades of allopatric color forms. Selective pressure for these morphologies still need investigation. We recommend a comprehensive survey of the abundance of potential hymenopteran models and their forms along corresponding Paranthrene ranges. Finally, phylogenetic analysis of Sesiidae centers on COI and a select few nuclear genes (Cognato et al, 2023; Lait and Herbert, 2018; McKern et al., 2008; Taft et al., 2016; Taft and Cognato, 2017; Handfield and Handfield,2021). These nuclear genes were relatively uninformative. In the multi-gene phylogenies of both chapters there was little support for some of the internal nodes. Exploration into other potentially informative genes is needed to help elucidate these interspecific relationships. 51 BIBLIOGRAPHY Albert, DA. 1995. 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