A new cryptic Sympistis from eastern North America revealed by novel larval phenotype and host plant association (Lepidoptera, Noctuidae, Oncocnemidinae)

Abstract A Triosteum-feeding species of Sympistis is described from eastern North America: Sympistis forbesi sp. n. Identity of the new species is most reliably determined from larval morphology and host plant association—both adult scaling and genitalic characters overlap with those of Sympisitis chionanthi, a Chionanthus and Fraxinus feeder.


Introduction
Sympistis Hübner is the second largest genus of North American macrolepidopterans, with 176 recognized species (Troubridge 2008, Lafontaine andSchmidt 2010) and many others awaiting formal description. Sympistis forbesi sp. n. was first mentioned as a Triosteum-feeding variant of Adita chionanthi (J. E. Smith) by Rummel (1921) Two additional genitalic preparations were left in glycerin. Six slide mounted genitalic preparations made by John G. Franclemont, identified as Triosteum-feeding strains of S. chionanthi, were borrowed and examined from CUIC. Thirteen S. chionanthi genitalic preparations (from New York, Connecticut, Manitoba, Ontario, and Saskatchewan) were examined. COI sequences were generated by the Barcodes of Life Project. Sequences for two S. forbesi specimens (Barcodes of Life Project Numbers CNCLEP 81921 and CNCLEP 81922) and six S. chionanthi from Ontario and Quebec (Barcodes of Life Project Numbers CNCLEP 81919, CNCNoctuoidea 7959, DH007094, DH009854, 2005-ONT-1897, 2005-ONT-1928 have been deposited at GenBank.  Etymology. We name the species after William T. Forbes, North America's premier lepidopterist over a 40-year period from 1920 to 1960. Forbes' understanding of the species and higher-level taxonomy of eastern Macrolepidoptera was extraordinary, with the vast majority of his taxonomic decisions standing the test of time (and additional data). His four-volume treatise on the Lepidoptera of New York and Neighboring States remains the definitive work on eastern moths, especially for most Microlepidoptera.
Diagnosis. Adult. Sympistis forbesi averages slightly smaller than S. chionanthi. The scales over the thorax are smaller, more densely packed. In most individuals there are fewer white scales on the thorax and forewing: e.g., the costal margin, wing base, and orbicular and reniform spots have fewer white scales than most individuals of S. chionanthi. Additionally, the anal dash is often more J-shaped and the fringe is only faintly checkered, lacking the pure white scales seen in many S. chionanthi. In the hindwing, there is a more distinctive terminal line and the apex tends to have more black scales extending onto the fringe. The rami of male antennae through the basal half of the antenna average 0.50-0.65 mm in S. forbesi, and 0.55-0.70 mm in S. chionanthi. Larva. The larva provides unambiguous morphological characters that allow recognition of this new species. The last instar is mostly green with a reddish dorsum (red coloration is added through mid to late instars); there are no black or brown markings as in S. chionanthi (in particular, the black subdorsal stripe characteristic of S. chionanthi is absent from all instars of S. forbesi). Body smaller, more elongate and modestly tapered at both ends, especially relative to the robust habitus of S. chionanthi. Head width of ultimate instar of S. forbesi 2.5-2.8 mm; head width of S. chionanthi 3.0-3.2 mm. Spiracular height consistently smaller in S. forbesi compared to S. chionanthi-mean spiracular heights of A1-A6 are 0.30 and 0.36 mm, respectively. Mean crochet number of S. forbesi on A3-A6 and A10 are 17, 19, 20, 21, and 20; mean crochet number of S. chionanthi on A3-A6 and A10 are 27, 28, 32, 32, and 33.
Description of adult. Male. Forewing length: 14.5-16 mm (n=23, reared from wild larvae). Ground color warm gray. Head. Antenna biramous; rami approximately 0.50-0.65 mm through basal half of antenna. Forward-facing tuft of scales just above faint black line between eyes. Thorax. Gray, medial prothoracic tuft, edged with black, preceded by conspicuous transverse black line. Black edging of tuft continues laterad to wing base. Tegula steely gray, indistinct thick band of dark scales at back. Legs with mix of dark and light scales. Tarsi dark brown or black. Forewing. Thin, smoothly curved basal and antemedial lines. Thickened antemedial line tapering to inner margin. Orbicular spot gray centrally and pale gray peripherally, thinly edged with black. Medial line ill defined; field proximal to reniform spot with numerous dark scales, forming two dark fascia along costa above orbicular and reniform spots. Black line or open triangle in position of claviform spot. Postmedial line running parallel to medial line, connecting to base of reniform and looping around toward margin, finally connecting to dark fascia along costa. Anal dash usually crisp, occasionally absent, subtended by sharp or diffuse black spot basad, forming J-shape. Subterminal line forming black fascia at costa, but otherwise pale gray, weakly developed to nearly obsolescent. Fringe weakly checkered, without white scaling. Hindwing. Pearly white with thin, crisp terminal line except at apex where diffuse field of black scales extends through fringe. Postmedial line obsolescent in males. Abdomen. Mixture of light and dark scales and hairs. Whitish scales along posterior margin of pregenital abdominal terga. Male genitalia. Valves elongate, nearly parallel sided with flat-topped projection from apex; bulbous clasper with claw-like apex that curves mesad; corona of fine setae of variable lengths. Juxta poorly differentiated. Uncas curved, gradually tapering, apex drawn into fine, curved spine. Saccus V-shaped, drawn into point anteriorad. Aedeagus cylindrical, variously sclerotized with vesica bearing approximately one dozen spines on elbowbend and numerous longer, narrower spines over bulbous subapical region; terminus armed with single stout spine nearly 1 mm in length (as large as uncus).
Description of pupa (Figs 28, 29). 16-19 mm long, 4.4-5.0 mm wide. Orange brown to deep chestnut brown, mostly smooth except for deeply pitted anterior portion of abdominal segments A4-A7. Primary setae extremely short, difficult to locate. Labial palpus visible, subequal to visible portion of profemur. Foreleg with cuminate apex, ending in abrupt spine. Proboscis extending just beyond antenna and midleg, nearly reaching end of wing. Labrum roughly shovel shaped with truncated apex. Eyepiece and frons ornamented with dense micro-ridging. Spiracular scars elongate, five times longer than wide. Cremaster ending in pair of minute thorn-like spines; cremaster deeply wrinkled and heavily sclerotized at base.  (Figs 31, 32). Ground color sea to mint green with pink to red dorsum and pale longitudinal striping along sides of trunk; A8 modestly humped. Reddish dorsum composed of pink to pale red middorsal stripe flanked by darker red addorsal stripes; dorsal pinacula white. Mostly broken white pinstripe zigzags through D1 pinacula. Red dorsal area bounded by pale (green to white) subdorsal pinstripe. Two supraspiracular pinstripes edged below with darker green. Lateral stripe, greenish white, roughly equal to height of spiracles, extending along lower end of spiracles. Prolegs on A3 and A4 about half size of those on A5 and A6. Head pale to dark brown above, often with pink to reddish flush; labrum greenish white, shallowly rugose frons, gena with three whitish lines that anastamose about stemmata.

Description of preserved final instar
Distribution. Locally common in Midwest, especially prairies. Most commonly found in Iowa, Illinois, and Minnesota. Believed to be extirpated from eastern portion of range in New York and New Jersey. Given that the genus Triosteum occurs from southern Canada to Texas and eastward, it is probable that the range of the new species is more extensive than circumscribed here.
Biology. So far as known larvae are specialists on members of the genus Triosteum, also known as horse-gentian or feverwort, of the family Caprifoliaceae. Nearly all our larval collections are from T. perfoliatum (feverwort). We found a few larvae of what appeared to be the same species on T. aurantiacum in Iowa. In the laboratory, larvae from T. perfoliatum readily accepted and matured on T. aurantiacum. In two separate instances, larvae were successfully reared to pupation on Fraxinus as well, a widely used host plant of S. chionanthi. S. forbesi larvae grew more slowly on Fraxinus, and maintained their typical green and pink coloration (MJH unpublished data, M. Keene personal communication). S. forbesi is univoltine with a single generation that emerges, flies, and mates in late summer, mostly in early September. Females presumably lay eggs on or near the stems of Triosteum. Above-ground tissues of the host die and senesce over the winter. MJH has found first instars on unopened leaves that were just pushing forth from the ground in early March (in Iowa). Larvae complete their development by mid-June. Early instars feed exclusively on new leaves, principally of the apical meristem, before the leaves have had a chance to open and expand to full size. Where the moth is common and when collections are made through the first half the season, partially opened leaf fascicles often yield larvae that were not seen at the time of collection. Last instars also consume new leaves, but are content to feed on fully expanded leaves and flowers (Figs 34-36). All instars are cryptic in both color and habit. The late instars rest along a shoot head down, often near flowers, where their coloration is well matched to that of the stem and reddish-pink Triosteum petals and sepals. Densities can be high with more than a dozen larvae on a single shoot; on several occasions we noted cases where the larvae of S. forbesi severely damaged the apical portions of their host plant. Prepupae form a slight cocoon below ground; the summer months are passed as a pupa.
Barcoding. In a neighbor-joining tree based on J. D. Lafontaine's unpublished barcodes for 137 North American oncocnemidine noctuids (representing 687 individuals), S. chionanthi (n=7; CT, Quebec, Ontario, Alberta) and the new species (n=2; both Iowa) grouped together in a "cluster" separate from other North American Sympistis, and each taxon was reciprocally "monophyletic," although the two groups differed by less than 1% from one another. In a second analysis, focused on "Aditagroup" Sympistis that included 13 individuals from across North America, again the two Triosteum feeders grouped in their own cluster.
Discussion. S. chionanthi was described by J. E. Smith in Abbot and Smith 1797 based on a painting of the adult, caterpillar, pupa, and the host Chionanthus virginica Linn. (fringetree) (family Oleaceae) by Abbot. As is the case for all of taxa drawn by Abbot, there is no type specimen for S. chionanthi. The common name "Grey O Moth" was given for the "O" shaped orbicular spot on each forewing. Abbot's rendering of the larva depicts a robust caterpillar that is pale brown laterally, shaded with darker brown dorsally, and bears a thick black subdorsal stripe, a slight stripe behind the head and a moderately humped A8 segment. His illustration is undoubtedly a match for the current-day S. chionanthi caterpillars from Fraxinus (Fig 33). Despite not having any type material for S. chionanthi, we are confident in our assessment that the original species description agrees with the current understanding of S. chionanthi and that S. forbesi represents a distinct species.
S. chionanthi was described as being very rare in Georgia (Abbot and Smith 1797). There are no recent reports of S. chionanthi living in Georgia (J. Adams pers. comm.), and even in North Carolina it is an extremely rare mountain taxon (Bo Sullivan pers. comm.).
Although the adults of S. forbesi and S. chionanthi are difficult (and sometimes impossible) to distinguish even upon dissection, their larvae are distinct in size, coloration, habitus, and life history. Presumably these coloration and morphological differences reflect, at least in part, the structural differences in their preferred hosts. Triosteum is an herbaceous perennial that dies back to the ground each winter; Fraxinus and Chionanthus are trees. The brown, bark-like coloration of late instar S. chionanthi is suggestive that larvae rest off of foliage by day and perhaps even near the ground along the trunk or off the host in leaf litter. We know of no brown noctuoid larvae that rest on foliage by day, and many, like Catocala Schrank, Melipotis Hübner, and Zale Hübner, may wander far from the foliage when not feeding. The coloration of last instar S. forbesi (Figs 32,33) is reflective of its preferred resting site: the green stems of feverwort. Likewise, it is our guess that the less robust body, smaller prolegs, and reduced crochet hook number of S. forbesi reflect the fact that larvae rest adjacent to suitable foliage. By contrast, the caterpillars of S. chionanthi on a mature ash or fringetree may well have to traverse meters in search of suitable food each night. Surprisingly, no differences in mandible morphology of the two sister taxa were noted.
Despite the differences between the hosts of S. forbesi and S. chionanthi, the plants share secondary metabolites which may elucidate how the ancestral host plant switch was able to occur. Triosteum are members of the Caprifoliaceae, whereas the hosts of S. chionanthi (Chionanthus and Fraxinus) (Troubridge 2008, Robinson et al. 2012) are both Oleaceae. Both families are known to contain iridoid glycosides (Bowers 1991, Seigler 1998, Jensen et al. 2002, Lee et al. 2010. While larval hosts are known for only a small fraction of North American Sympistis (Troubridge 2008), at least among the known hosts, plants with iridoid glycosides figure prominently (Wagner et al. 2011). In western North America, Penstemon, in particular, well known to have iridoids (Stermitz et al. 1988, Krull andStermitz 1998), supports numerous Sympistis species (DLW unpublished data).
Prior to Troubridge's (2008) oncocnemidine revision, S. chionanthi was classified in the monobasic genus, Adita Grote, 1874. Troubridge synonymized the genus into Sympistis and regarded chionanthi to be a highly derived species within Sympistis related to the S. dentata species group. In addition to the species that we describe here, there may be additional cryptic species in collections sorted as "Adita chionanthi." George Godfrey and Tim McCabe have beaten caterpillars of an "Adita" group species from Symphoricarpos Duhamel in the Upper Midwest. Images taken by Godfrey of these larvae closely approach those of S. chionanthi, but differ in having more gray in the ground color and some brick red over the dorsum. Unfortunately, neither McCabe nor Godfrey reared adults or preserved larvae. John Franclemont collected a large series of "chionanthi" in Montana and reared an ex ova cohort on Fraxinus. Based on the number of pinned specimens, larval photographs, and genitalic dissections it seems likely that Franclemont believed the Montana populations might represent a new species. Adults average larger and brighter than material from eastern North America. Three individuals of "chionanthi" from nearby Alberta, also included in the barcoding dataset, clustered separately from the eastern individuals. Given the above, we caution that our figured male and female genitalic preparations (Figs 15-17) for S. chionanthi are from central Canada; without larval or genetic data, we cannot with certainty know that these are nominate S. chionanthi. We were unable to find consistent differences in male or female genitalia in S. chionanthi (representing five states and provinces), or between S. chionanthi and S. forbesi. If our findings about the differences between S. chionanthi and S. forbesi are indicative for other members of the species group, or Sympistis more widely, larvae, life history data, and molecular data will be needed to tease apart the biological species in this complex.
Part of our interest in the new species derives from our desire to document instances where rates of phenotypic evolution in Lepidoptera differ markedly among life stages. For example, in Acronicta Oschenehimer and some notodontid genera (e.g., Datana Walker and Schizura Doubleday) larval phenotypes differ substantially among related species that are otherwise difficult to determine using external and genitalic features of the adults. Adults of Acronicta hastulifera (J. E. Smith) and A. dactylina Grote are sometimes impossible to separate by eye or dissection, but each has a distinctive larva that readily distinguishes the second to final instars of both species (Wagner et al. 2011;Schmidt and Anweiler unpublished data). Conversely, plusiine caterpillars are remarkably undifferentiated relative to their adults, and often require microscopic examination even to make generic and tribal assignments (Crumb 1956, Lafontaine andPoole 1991). The adults of Sympistis forbesi and S. chionanthi are mixed in collections that we have examined (under the latter name). By contrast, their larvae, are immediately distinct, with the coloration of each approximating that of the stem-color of their primary hosts: feverwort (Triosteum) for S. forbesi and ash (Chionanthus and Fraxinus) for S. chionanthi. With careful morphological analysis, it may be possible to quantify these differing rates of phenotypic evolution within and between species using newly developed phylogenetic techniques (e.g., Adams 2013).