Figures
Abstract
Color polymorphism among animal species can influence speciation. Factors such as natural and sexual selection, genetic drift and gene flow contribute to the maintenance of color polymorphism within the species or spur speciation. The evolutionary and ecological mechanisms for color polymorphism are taxon specific and only a few species have been studied. A phylogeny provides an evolutionary framework to understanding the association between color polymorphism and species. Paranthrene species are day flying aposematic moths that mimic wasps in both appearance and behavior. The genus has several polymorphic species and some color forms were originally described as species. Paranthrene presents the opportunity to test for an association between color polymorphism and species boundaries. We reconstructed a phylogeny using DNA sequence from COI, EF-1alpha, and Wingless genes from 67 specimens representing all North American Paranthrene species, nine color variants, and two outgroups. Parsimony and Bayesian analyses generally agreed in topology and support values. Paranthrene simulans (Grote, 1881) was polyphyletic, and monophyly of P. pellucida Greenfield and Karandinos, 1979 was not recovered. Paranthrene robiniae Hy. Edwards, 1880 was polyphyletic and genitalic and genetic differences among the three clades supported the recognition of two new pseudocryptic species, Paranthrene oasis Smith, Taft and, Cognato, new species and Paranthrene gilaensis Smith, Taft and, Cognato, new species. Paranthrene color variants did not overwhelming associate with species boundaries. Of the nine color forms we examined, only two were monophyletic, had DNA sequence divergence comparable to other species, and associated with species diagnostic morphology. It is likely that genetic drift and allopatric isolation explains the fixation of color variants with species. The mechanisms maintaining color polymorphisms within Paranthrene species will remain unknown until experimentation concerning the degree of aposematic protection in reference to wasp models is conducted.
Citation: Smith III WH, Taft WH, Cognato AI (2024) Aposematic color polymorphism is a poor indicator of species boundaries in North American Paranthrene (Lepidoptera: Sesiidae) as evidenced by a multi-gene phylogeny. PLoS ONE 19(11): e0312508. https://doi.org/10.1371/journal.pone.0312508
Editor: Bilal Rasool, Government College University Faisalabad, PAKISTAN
Received: May 1, 2024; Accepted: October 2, 2024; Published: November 27, 2024
This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Data Availability: All DNA sequences are available from the Genbank database (accession numbers are in Table 2).
Funding: WHS was funded by The American Natural History Museum Theodore Roosevelt Grant. This is a small travel grant (~$2000) and no grant number was given. WHS was also funded by The Scriber Scholar Award from the Department of Entomology, Michigan State University. The funders did not play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. More information about these funders can be found at the following links: (https://www.amnh.org/research/richard-gilder-graduate-school/academics/fellowship-and-grant-opportunities/research-grants-and-graduate-student-exchange-fellowships/roosevelt-memorial-fund) and (https://www.canr.msu.edu/ent/undergraduate/scholarships)
Competing interests: The authors have declared that no competing interests exist.
Introduction
Color polymorphism is a phenomenon among animals that has been implicated as a factor in speciation [1, 2]. Selection, genetic drift and countering gene flow determine the likelihood that color variants will associate with a speciation event [2]. 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 [2]. 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 [3–5]. However, both positive frequency dependent selection and male mate-choice act upon aposematic Heliconis (Lepidoptera) polymorphic species to drive speciation [3, 6, 7]. Discovering the role that color polymorphism plays in speciation requires observation and experimentation [2]. Most important, a phylogeny provides an evolutionary pattern of species and associated color variants which can direct experimentation [8].
Clearwing moths (Lepidoptera: Sesiidae) exhibit extensive aposematic color polymorphism within species [9]. All adults are diurnal mimics of wasps and bees, in both their appearance and their behavior [9]. This exhaustive mimicry is demonstrated with some species mimicking their hymenopteran model’s flight patterns and acoustics [10, 11]. As larvae, these moths bore into a variety of economically important plants, and are considered pests [12]. Paranthrene Hübner, 1819 typically spend two years in the larval stage and feed on oaks or willows and poplars [9, 12]. Currently, there are nine recognized Paranthrene species occurring in the United States, Canada, and Mexico (Baja California) along with approximately 20 species distributed in Eurasia and Africa [13–15]. Each Paranthrene species has two or three adult color forms, which mimic hymenopterans [9]. Likely, the color forms have converged to match various locally abundant hymenopterans (Fig 1).
a-c. Potential hymenopteran models and Paranthrene spp. a) Polistes sp. and Paranthrene robiniae “palescens”, b) Vespula sp. and Paranthrene simulans “palmii”, b) Pepsis sp. and Paranthrene fenestrata.
Several Paranthrene species have multiple color forms. The oak borer (Paranthrene simulans Grote, 1881) has three unique color morphs, simulans, luggeri, and palmii forms (Fig 2A–2C). Its range includes the entire eastern United States and Canada, as far west as east Texas and Minnesota [12]. The palmii form is found in the species’ southern range and the luggeri form occurs in the western portion [9]. In the areas where simulans and palmii overlap intermediates between the forms have been observed [9]. Host plants for all populations are various oak species and the larvae feed on different parts of the tree depending on their range [12]. 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 [16]. Prior to 1988, Paranthrene palmii Hy. Edwards, 1887 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 [9]. Likewise, luggeri was originally described in 1891 and later synonymized [17]. 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 [18]. Even their emergence is slightly different. Palmii emerges between April to June, and simulans emerges between June and July [12]. 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 [9]. Paranthrene pellucida, Greenfield and Karandinos, 1979, is a color-form of P. simulans that is currently recognized as a species given differential response to pheromones and flight period [19]. McKern and Szalanski [20] reported intraspecific mtDNA sequence variation among Arkansian P. simulans individuals and Cognato et al. [21] showed differences among populations from Minnesota and North Carolina. Handfield and Handfield [14] 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.
a-e. a) Paranthrene simulans “simulans” b) Paranthrene simulans “luggeri” c) Paranthrene simulans “palmii” d) Paranthrene pellucida e) Paranthrene hilairemontis.
Paranthrene fensetrata Barnes and Lindsey, 1922 has two distinct sympatric color morphologies (Fig 3A and 3B) 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 [9].
a-b. Paranthrene fenestrata a) yellow b) black.
The western poplar clearwing (Paranthrene robiniae Edwards, 1880) is found along the Pacific Coast of the United States, the Rocky Mountains, and the provinces of British Colombia and Alberta (Canada) [16]. Its preferred host plants, poplars and willows, occur throughout western North America in isolated populations separated by other habitats non-contusive to their growth, like deserts [12, 22], There are three recognized forms, robinae, perlucida, and palescens (Fig 4A and 4B). The perlucida form occurs in Canada (British Columbia and Alberta), and the United States (the Pacific Northwest to Montana) [23]. The palescens form occurs in deserts of southern California [23] and Nevada (this study).
a-e. a) Paranthrene robiniae “robiniae” b) Paranthrene robiniae “palescens” c) Paranthrene oasis d) Paranthrene gilaensis e) Paranthrene dollii.
The dusky clearwing (Paranthrene tabaniformis Rottemburg, 1775) (Fig 5A and 5B) is a Holarctic species [12]. Its North American range includes all of the continental United States, Alaska, and Canada, with the exception of California [24]. There are currently three recognized forms in North America, tabaniformis, denotata and oslari. Denotata is found along the Rocky Mountains from Colorado to Alaska, while oslari has been collected from Colorado and Oklahoma [23]. Recently, an unnamed color form of tabaniformis was collected and described as P. sogaardi Taft and Smith [15].
a-d. a) Paranthrene tabaniformis b). Paranthrene tabaniformis “oslari” c) Paranthrene sogaardi d) Paranthrene asilipennis.
The poplar/ cottonwood clearwing borer (Paranthrene dollii Neumoegen, 1894) (Fig 4E) is found in the eastern United States and to the Great Plains [12]. Larvae feed the trunks and branches of poplars and willows and individual larval galleries do not intersect [9, 12]. These infestations are associated with fungal attacks [25] and beetle larvae of Saperda and Cryptorhynchus [9]. Adult flight times and generation length is unclear but potentially P. dollii has a typical two-year life span in the northern part of its range [9, 12], while southern populations may have multiple generations in a single year [12]. Outside the typical dollii form there are two other recognized color forms. The castanea form is found in the southern states as far west as the gulf coast of Texas and the fasciventris form is found in the Mid-West states surrounding Lake Michigan [23].
Phylogenetic studies of Sesiidae are few, with a majority of studies addressing the relationships among tribes and subfamilies, and only P. simulans and P. tabaniformis have been included [21, 26–29]. 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. We found little association among color forms and species.
Materials and methods
Collection and identification of specimens
All but two specimens were collected over a 5-year period (year to year) using Multi-Pher #1® pheromone canister traps (Distributions Solida Inc) baited with various pheromone lures intended for different species (Table 1). Specimens were collected from public and private land where permits were not necessary. Paranthrene species and color forms were identified with several publications [9, 14, 15].
Specimen preparation and imaging
A single representative of most color forms for eight species was picked for genitalia comparisons. Paranthrene hilairemontis genitalia were compared to images in Handfield and Handfield [14]. The abdomen was removed at the 4th or 5th segment to access the genitals. The extracted segments were placed in individual vials filled with water and two 116 mg tablets of potassium hydroxide and allowed to sit on a hot plate set just below boiling for two hours. Softened abdominal segments were removed from the vials and teased apart under a dissecting microscope with fine-tipped forceps until genitalia were revealed. In preparation for photography, unstained genitalia were placed on a microscope slide with glycerin and spread open with fine-tipped forceps then held in place temporarily with a slide cover. Genitalia were preserved in 5 mm glass micro vials in glycerin 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).
DNA sequence data and phylogenetic analyses
DNA was extracted from a metathoracic leg from 67 frozen specimens encompassing nine Paranthrene species representing 9 of 13 color forms along with two outgroup species (Table 2) 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-1alpha, and wingless. 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 2) and assembled in a Nexus file for a total of 1421 nucleotides (639 from COI, 365 from EF-1alpha, 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; [30]). 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 [31] 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.
Species concept
We consider species as hypotheses of evolutionary lineages [32, 33]. In this study we use four criteria monophyly, differences of genitalic morphology, differences of mating behavior (pheromone blends and flight period) and DNA sequence divergence to test the hypothesis that Paranthrene color forms are species. Monophyly is a direct assessment of an of evolutionary lineage, differences of genitalic morphology and/or mating behavior suggest pre-mating barriers, and a 2–3% COI sequence divergence associates with Lepidopteran species boundaries [34, 35]. Concordance of a combination of these criteria validates species recognition.
Nomenclatural acts
The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix “http://zoobank.org/”. The LSID for this publication is: http://zoobank.org/urn:lsid:zoobank.org:pub:FAF78C13-980C-451E-A21C-7B30CD1B9773. The electronic edition of this work was published in a journal with an ISSN and has been archived and is available from the following digital repositories: LOCKSS.
Results
The PAUP* analysis found 600 most parsimonious trees that were mostly resolved in the strict consensus of those trees (Fig 6). Intraspecific and interspecific relationships of all P. simulans forms, P. pellucida and P. hilairemontis were unresolved (Fig 6) 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 6). The topology of the Bayesian tree was generally similar to the parsimony tree (Fig 7). 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.
Numbers above branches are bootstrap values > 80%. Color highlighted taxa are discussed in the text.
Posterior probabilities of 1 and those between 0.9–0.99 (black square) are shown. Color highlighted taxa are discussed in the text.
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 (Tables 3 and 4). The divergence for nuclear genes between these species was <1% and <1.35% for EF-1alpha and Wg, respectively (Tables 5 and 6). 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) (Figs 6 and 7) and had a larger COI sequence divergence than any other color form (Table 3).
N/A = not available.
Neither the typical black nor yellow forms of P. fenstrata were monophyletic in either tree. They resolved polyphyletic within the clade representing P. fenstrata (Figs 6 and 7). There was no DNA divergence or morphological evidence to support the recognition of these forms as species (Tables 3, 7 and 8).
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 6). In the Bayesian tree, individual geographic clades of P. robiniae and P. dollii were less supported with a posterior probability ranging from 0.68–1 (Fig 7) but had similar placement as in 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 (Figs 6 and 7). The interspecific COI divergence among the three P. robiniae populations and P. dollii ranged between 6% and 9%, EF-1alpha divergence ranged between .19% and .86%, and Wg divergence ranged between 0% and 2.72%, (Tables 5, 6 and 9–11). 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 (Figs 8 and 9). The two species are described below.
a-d. Genitalia a) Paranthrene robiniae b) Paranthrene dollii c) Paranthrene oasis d) Paranthrene gilaensis.
a-d. Adeagus spines a) Paranthrene robiniae b) Paranthrene dollii c) Paranthrene oasis d) Paranthrene gilaensis.
N/A = not available.
Paranthrene oasis Smith, Taft and, Cognato, new species
http://zoobank.org/urn:lsid:zoobank.org:act:668AE24C-B6C5-4315-A71D-32FD4CC99E4F.
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 (Figs 10C, 10D and 11). The tarsal spines of P. oasis are orange spines while spines of P. robiniae are black (Fig 10A and 10B). 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 8A–8C). 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 9).
a-d. 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.
a-d. Abdominal color patterns a) Paranthrene robiniae “robiniae” b) Paranthrene robiniae “palescens” c) Paranthrene gilaensis d) Paranthrene oasis.
Description
Male (Fig 4C). 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 10C). 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 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 10A). 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 11D): 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 9C).
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 1). There was also an abundance of Gooding’s Willows Salix goodingii in the habitat.
Paranthrene gilaensis Smith, Taft, and Cognato, new species
http://zoobank.org/urn:lsid:zoobank.org:act:3804C23C-E988-4DD7-A565-2F0D9587E90A.
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 has three colored bands, anterior band is black, followed by a thin red band, and a thicker yellow posterior band (Fig 11C). 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 dorsally black 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 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 4D). 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 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 12B); 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: Hyaline 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 8D). The subscaphium long extending behind the transtilla and terminates in a bifurcated structure.
a-b. Tegula “shoulder” scales a) Paranthrene robiniae b) Paranthrene gilaensis.
Discussion
Equating color form with sesiid species has been questioned in traditional taxonomic studies and often resulted in the synonymy species [9, 23]. The recognition of palescens as a color form of P. robiniae, as well as, palmii and luggeri, as a form of P. simulans is upheld in this study. The status of color-form is suggested for P. pellucida and P. hilairemontis given the lack of reciprocal monophyly with P. simulans, < 2% DNA sequence divergence for mitochondrial and nuclear genes (Tables 3–8), and morphologically similar male genitalia among these species. We, however, recognize these species as valid because of evidence for different mating behaviors among P. pellucida and P. hilairemontis and P. simulans [14, 19]. While rapid species radiation is suspected for Sesiidae [21], reciprocal monophyly and ultimately the validity of P. pellucida and P. hilairemontis will require testing with a better resolved and supported phylogeny based on genomic data. Conversely, this study and previous Sesiidae molecular phylogenetic studies have supported the recognition of color forms as species [15, 27]. However, of the nine color forms we examined three are monophyletic and only two have comparable DNA sequence divergence and associated with species diagnostic morphology (Figs 6 and 7). 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 [1, 2]. Color polymorphism of aposematic Lepidoptera can be maintained through selection against hybrids in narrow hybrid zones [3]. A zone of overlap occurs for the simulans and palmii forms [9, 12, 23], but this is not the case for the majority Paranthrene forms whose ranges are entirely sympatric. For example, the geographic ranges of the luggeri, P. pellucida, and P. hilairemontis forms are completely sympatric with the range of the simulans form [9, 14, 36]. The range of the P. robiniae palescens color form also completely overlaps with the robiniae color form [23]. Within these sympatric ranges, local abundance of particular models may provide selection at restricted geographic scales and/or for a limited time thus creating a patchwork of color forms without mating barriers [3]. Little is known concerning the distribution of Paranthrene color forms and model hymenopteran species or predation intensity against poor mimics of local models.
Alternatively, sexual selection may drive the diversity of color forms [2, 37]. However, sesiids use long-range pheromones to locate mates [9]. All color forms of P. simulans were collected with the same two semiochemical blends [24]. These include the semiochemicals used in the collection of the P. hilairimontis and P. pellucida forms [14, 19, 23]. Also, all three P. robiniae populations and P. dollii were collected with the same semiochemical blend (Table 1). It is unlikely that color form and pheromone type are genetically linked. Thus, sexual selection likely does not contribute to maintenance of Paranthrene color forms although it is unknown if color pattern is used in close-range mate recognition.
We hypothesize 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 oases [21, 24]. The sky islands and deserts of southwestern United States are notable for their role in speciation due to geographic isolation [38–40]. 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 [21]. The aberrant 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, mating behavior, 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.
Acknowledgments
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. Comments from Michael Greenfield, Andrzej Mazur, Silas Lendzele Sevidzem, and anonymous reviewers improved this publication.
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