Albuna beutenmulleri Skinner (Lepidoptera: Sesiidae) is endemic to eastern and southern Utah, feeds on primrose (Oenothera pallida Lindl.), and is distinctly colored with opaque, orange-red wings. However, A. beutenmulleri was synonymized as a color variant of the widely distributed A. pyramidalis (Walker). Thus, given the criteria of monophyly, we tested the species status of A. beutenmulleri in the context of Albuna species. Phylogenetic analysis of mitochondrial DNA revealed that specimens of A. beutenmulleri and A. pyramidalis were reciprocally monophyletic. The nucleotide difference between species was ~8% which was similar to the distance among other Albuna species and exceeded the intraspecific difference by ten times. Given phylogenetic and life history evidence, we recognize A. beutenmulleri as a distinct species from A. pyramidalis and describe a yellow variant of A. beutenmulleri.
Walker (1856) described Albuna pyramidalis from a specimen collected along the Albany River near Hudson's Bay, Canada. Since then, specimens of the species have been collected within the endemic range of fireweed and willow-herb, (Chamerion angustifolium (L.) and C. latifolium (L.), (Onagraceae), respectively). Its current distribution includes Alaska, Canada, the Rocky Mountains, the northern tier of the United States and the Cascades and Sierra Nevada to the Pacific coast (Eichlin & Duckworth 1978, 1988). Color variants exist throughout its extensive range (Beutenmüller 1901)(Fig.1). Of these, beutenmulleri was the most distinct and was described as a new species from an individual collected in Stockton, Utah (Skinner 1903). However, it was synonymized with A. pyramidalis (Fig. 2 and 3) given that the color patterns showed degrees of intergradation throughout A. pyramidalis populations (Engelhardt 1946). Engelhardt (1946) recognized various color forms in addition to the typical form, which range from the nearly entirely black “coloradensis” Hy. Edwards to “rubescens” Hulst with orange-red wings, to “beutenmulleri” Skinner with opaque, orange-red wings. Eichlin and Duckworth (1988) investigated the genitalia among the various color forms of A. pyramidalis but they did not find differences that would support the recognition of different species and maintained these phenotypes as variants of A. pyramidalis. They maintained that A. pyramidalis was readily recognized by the broad oblique discal mark on the forewings despite distinct color variation throughout its range.
Fig. 1.
Current geographic distribution of Albuna pyramidalis (shaded area) and the Utah collection sites of Albuna beutenmulleri (black dots). Adapted from Eichlin and Duckworth, 1978.
However, along with the color differences, beutenmulleri exhibits differences in its geographic locality, phenology and host use. Specimens of the beutenmulleri color morph have only been collected or observed from localities in Utah between elevations of 1723–1905 meters. Adults emerge as early as April and no later then June 3 which is 2 weeks before the emergence of A. pyramidalis (Fig. 4, Table 1 and 2). The larvae of beutenmulleri are associated with various primrose species including (Oenothera pallida Lindl.) as compared to various fireweeds, which are used by the typical color morph (Engelhardt 1946). These observations suggest potential reproductive barriers and ecological differences between beutenmulleri and the typical color morph of A. pyramidalis.
Phylogenetic analysis using DNA sequence data offers a solution for delimiting species (Rubinoff & Powell 2004, Cognato & Sun 2007). Monophyly of suspect species with association of biological traits often provides evidence for the recognition of new species under the phylogenetic species concept (sensu Wheeler & Platnick 2000). Many cryptic Lepidoptera species, including sesiids, have been confirmed or discovered with the use of molecular phylogenies and morphology (e.g., Kallies 2002, Lumley & Sperling 2010, Dumas et al. 2015). Therefore, we reconstruct a phylogeny using mitochondrial cytochrome oxidase I DNA data for specimens of Albuna pyramidalis including the beutenmulleri and the typical color morph, A. fraxini, and species of Carmenta, Paranthrene, Synanthedon and Zenodoxus for outgroup comparison in order to test the monophyly of the beutenmulleri color morph.
Materials and Methods
Collection of specimens. In May of 2014, David Wikle collected two males of the beutenmulleri color morph along Lick Wash just off the Skutumpah Road (37.36428, -112.18711), elev. 1905 m, in Grand Staircase-Escalante National Monument in Kane County, Utah (Fig. 5). These adults were attracted to the lesser peachtree borer pheromone lure taped to his net. The habitat was at the interface between Ponderosa Pine above and Great Basin scrub below (Artemisia tridentata with abundant subshrubs and forbs). A year earlier in May 2013, Paul Opler caught a male and female of the beutenmulleri color morph at Cat Canyon (39.55083,-110.64098), elev. 1723 m, near Wellington, Carbon County, Utah. The female was net collected in steady flight and the male came to a pheromone strip taped to Opler's net rim. Here the habitat was open pinyon-juniper woodland at the base of a steep escarpment. The male was found amongst a patch of Oenothera pallida (Fig. 6) along a dirt track. The plants were all in full bloom in May 2013 but only a single plant was found in flower a year later apparently as a result of an ongoing drought (U.S. drought monitor, April 2014).
Table 1.
Albuna pyramidalis - North American records
Table 2.
Albuna beutenmulleri records
DNA sequence data and phylogenetic analyses. At Michigan State University, DNA was extracted from three hand-collected specimens initially killed with ethyl acetate and subsequently pinned and stored at 15–22° within C.P. Gillette Museum of Arthropod Diversity at Fort Collins (CUSU). Specimens were dissected by removing the meta-leg from the thorax. The leg was ground with a pestle in a 1.5 ml microfuge vial and DNA extractions were performed using a Qiagen DNeasy blood and tissue kit (Hilden, Germany) following the manufacturer's protocol. Specimens were vouchered in the A. J. Cook Arthropod Research Collection at Michigan State University. The purified DNA was used to amplify ~650 base pairs of the 5′ end of mitochondrial cytochrome oxidase I gene using PCR primers LCO1490 and HCO2198 and following the PCR protocol of Herbert et al. (2003). PCR products were purified with EXO-SAP-IT (USB Corp., Cleveland, OH, USA) and following the manufacturer protocols. Purified PCR products were sequenced in the Michigan State University Research Technology Support Facility using a Big-Dye Terminator v 1.1 (Applied Biosystems, Foster City, CA, USA) and visualized using an ABI 3730 Genetic Analyzer (Applied Biosystems). Sense and antisense strands were compiled using Sequencher (Ann Arbor, MI) to trim sequences of primer sequences, to examine for ambiguities and to create consensus sequences. Final sequences of 654 bp were deposited in Genbank (Table 3).
Table 3.
Specimens used for phylogenetic analysis and corresponding Genbank and BOLD identification numbers.
DNA sequences of the same locus were obtained from the BOLD data base (Ratnasingham & Hebert 2007) for 15 sesiid specimens representing Albuna pyramidalis (typical color morph), A. fraxini, Carmenta, Paranthrene, Synanthedon and Zenodoxus (Table 3). A phylogeny for these species and the specimens of the beutenmulleri color morph was reconstructed using PAUP* (Swofford 2003). Heuristic searches for the most parsimonious and most likely trees were conducted. Each search consisted of 500 stepwise random additions with tree bisection-reconnection. The model for the likelihood searches corresponded to HKY85+I+G. Bootstrap values for the parsimony tree were calculated by heuristic searches with simple sequence additions for 1,000 pseudoreplicates. Intraspecific and interspecific pairwise uncorrected “p” distances were computed in PAUP*.
Results
Inspection of the COI sequences produced for the three specimens of the beutenmulleri color morph revealed no evidence of nucleotide ambiguities or pseudogenes. As anticipated, the sesiid sequences aligned without the need of gaps. Subsequent phylogenetic analyses recovered mostly congruent trees. Four most parsimonious trees were reconstructed which were most resolved in a strict consensus (Fig. 7). The one most likely tree shared the topology of one of the four most parsimonious trees. All genera, Albuna species, and individuals of the beutenmulleri color morph were monophyletic. The likelihood tree suggests a sister relationship between the clades of the beutenmulleri color morphs and the typical color morphs however this relationship was unresolved among the four most parsimonious trees.
On average the intraspecific distance was 0.0097 for the beutenmulleri color morphs, which falls in the range (0–0.02446) of other Albuna species. The average interspecific distance among the beutenmulleri color morphs and the other Albuna species was 0.0868, which was greater than the interspecific distance observed between A. pyramidalis and A. fraxini (0.06575). The distance among species of the outgroup genera ranged between 0.07–0.17. Taken together, these values suggest that the amount of genetic divergence of the beutenmulleri color morphs is similar to the divergences observed for other Albuna species and sesiid genera.
Fig. 7.
Likelihood tree (-Ln likelihood = 3395.865) of Albuna species and outgroup genera which represents 1 of 4 parsimonious trees. Numbers are bootstrap values determined by parsimony analysis. Clades without bootstraps were unresolved in the strict consensus of the four most parsimonious trees.
The correspondence of monophyly, genetic divergence, and distinct biological characteristics strongly suggests that the beutenmulleri color morphs represent a lineage with a unique evolutionary trajectory. Given the phylogenetic species concept (Wheeler & Platnick 2000), there is much evidence to support the resurrection of A. beutenmulleri. For completion, we reprint the original description of A. beutenmulleri and describe a yellow morph of this species discovered during this investigation.
Albuna beutenmulleri Skinner, species resurrected
Black form female (Original type description, Skinner 1903) (Fig. 2): “Expanse 20 mm. Antennae, palpi, head, thorax, abdomen and legs, black. Segments of abdomen slightly differentiated by being somewhat bluish and shining. Under side of abdomen and thorax with blueblack shining metallic scales. Hind legs clothed with long black hairs. Wings bright red, edged narrowly with black. Fringes black. Fore wings with a translucent spot at outer third, divided by the red veins into four parts. There is also a similar spot at inner third in the centre of the wing, which is linear. These spots are covered with beautiful, very light greenish or bluish opalescent scales. The base of the wing is black and the black margin breaks and runs slightly into the wing from the inner margin. The hind wing has two spots of the same character “ one beyond the middle resting on the costa and divided into two parts, and a larger one near the base, divided into three parts by the veins. This spot extends the width of the wing. Base of wing black.
This species somewhat resembles Euhagena nebraskae H. Edw. but the red color is brighter and shining. E. nebraskae lacks the beautiful opalescent spots.”
Black Form Male (Fig. 8) Similar to the female description above except unlike the female the black form male has a black hind wing instead of red.
Specimens: Utah, Carbon Co. 5/12/2013. P.A. Opler. (CSUC-1 specimen)
Yellow Form, Male (Fig. 9): Head with vertex yellow; front pale yellow with white scales dorsally; occipital fringe pale yellow; labial palpus roughened, pale yellow with brown/black laterally; antennae bipectinate, dark orange dorsally and ventrally with upper half black; thorax black with yellow laterally near anterior margin and beneath wings, yellow band on collar, yellow scales on tegula; abdomen blue black dorsally with wide yellow banding on segments 4–7; anal tuft light orange, legs with coxa of fore leg mostly yellow mixed with a few black scales anteriorly; femora and upper third of tibia black; remainder of tibia covered with long yellow hair-like scales, tarsal spurs and tarsi yellow; Wings bright red with narrow black margins and black fringes. Forewing with a narrow elongated, translucent area before the distal mark, divided by red veins into 4 parts; behind the distal mark another translucent area, narrowly triangular, terminating before the wing base. Hindwing bright red on the outer two-thirds with two irregular translucent patches. Specimens: Utah, Kane Co. 5/19/2014. D. Wikle. (CSUC-1 specimen)
Female (Fig. 10): Similar to male except, Head with vertex yellow; front and occipital fringe pale yellow; labial palpus roughened, pale yellow; antennae orange; abdomen blue black dorsally with wide yellow banding on segments 2,4,6, and 7; anal tuft yellow.
Specimens: Utah, Carbon Co. 5/12/2013. P.A. Opler. (CSUC-1 specimen)
Discussion
Intraspecific color variability is a common phenomenon among sesiid species (e.g. Paranthrene fenestrata, Euhagena emphytiformis, Vitacea polistiformis, Synanthedon polygoni, Carmenta giliae) (Beutenmüller 1901, Engelhardt 1946, Eichlin & Duckworth 1988). These color variants often occur at the same location and respond to specific pheromones suggesting their unity as a species (Eichlin & Duckworth 1988). In the case of A. beutenmulleri, the apparent isolation by geography, flight period, and/or host plant has allowed for genetic divergence equivalent to divergence observed for other species. Although the red and black scaling and translucent areas of the fore and hind wing based diagnose A. beutenmulleri, color variation (black versus yellow) exists among individuals. The extent of this variation within the species is unknown because A. beutenmulleri is only known from a limited number of individuals from five locations in Utah. Further investigation, utilizing DNA data and detailed biological observations may yield additional cryptic sesiid species as observed with other Lepidoptera (e.g., Wilson et al. 2010).
Acknowledgments
The authors would like to thank J. D. Weintraub, Collection Manager, Department of Entomology Academy of Natural Sciences (ANSP), Philadelphia, PA. for providing the type photograph. For additional digital images and/or specimens, we thank David Wikle, Gary Anweiler, Todd Losee and the Chicago Botanic Garden/CLM internship program. For DNA COI data, we thank Franz Puhringer, project manager for Global Sesiidae initiative on the BOLD system.
