Volume 11 Number 5 December 10, 2023

The Taxonomic Report

OF THE INTERNATIONAL LEPIDOPTERA SURVEY

ISSN 2643-4776 (print) / ISSN 2643-4806 (online

Reassessment of Amblyscirtes hegon (Hesperiidae)

as a complex of four distinct species

revealed by genomic analysis

Harry Pavulaan!', Ricky Patterson’, and Nick V. Grishin?

'606 Hunton Place NE, Leesburg, Virginia, USA, 20176

7400 Winona Road, Vicksburg, Mississippi, USA, 39180

3Departments of Biophysics and Biochemistry, University of Texas Southwestern,

5323 Harry Hines Blvd., Dallas, Texas, USA, 75390-9050, grishin@chop.swmed.edu

ABSTRACT. After the discovery of a unique phenotype in the southern United States with a different

ventral ground color than nominotypical Amblyscirtes hegon (Scudder, 1863), which occurs in the northeastern United

States, genomic analysis revealed that A. hegon is a species complex. Phenotypic, genitalic, and genomic differences

of the complex are presented here. Four species are identified: A. hegon; A. nemoris (W. H. Edwards, 1864), stat.

rest.; A. matheri Patterson, Pavulaan & Grishin, sp. n. (TL: USA, Mississippi, Warren Co.); and A. ge/idus Grishin,

Patterson & Pavulaan, sp. n. (TL: USA, Michigan, Van Buren Co.).

Additional keywords: biodiversity, cryptic species, genitalia, skipper butterflies, pepper-and-salt skipper.

ZooBank registration: urn:|sid:zoobank.org:pub:466F6B4C-6BD9-43C5-9181-3CADE6C5F7EB

INTRODUCTION

One of the authors (Patterson) first noted a unique ventrally tan-brown colored

Amblyscirtes Scudder, 1872 in Warren County, Mississippi, in the early 1980’s. Despite ventral

coloration different from what was initially considered to be typical Amblyscirtes hegon (Scudder,

1863) in the same region, this unique color morph was identified by several knowledgeable

lepidopterists as A. hegon. One exception was Dr. Andrew Warren, Senior Collections Manager,

McGuire Center for Lepidoptera and Biodiversity, who felt there were differences from A. hegon

but, through analysis of the genitalia, could not identify differences that were clearly definitive.

He encouraged further work on this issue.

Early on in the present study, the undescribed ventrally tan-brown phenotype was further

documented from several locations in the Loess Bluff Hills region of Mississippi and the only

locality known for sympatry of the unique color morph with what was considered to be hegon is

the J. C. ‘Sonny’ Gilbert WMA (formerly the Sicily Island Hills Wildlife Management Area,

renamed in 2015), in Catahoula Parish, LA. This was discussed in The Butterflies of Louisiana

(Marks, 2018 [ref. pages 159-160]), which has the only published account and photo known to us

of the “A. hegon ventral (alternate coloring)” phenotype, having an underside colored differently

than the strongly grayish color of hegon. What we initially considered A. hegon and the new

species have been collected flying sympatrically and synchronously in the J. C. ‘Sonny’ Gilbert

WMA, with no evidence of integration. Marks (2018) explained that “one out of five” A. hegon

observed at Sicily Hills were of the tan-colored form. There are presently no other known localities

where they are sympatric.

After the initial phenotypic analysis, genomic analysis was performed (the Grishin lab) on

the nominotypical hegon including the neotype of its junior subjective synonym nemoris, as well

as specimens of the newly discovered tan-brown phenotype, and a broad distribution of samples

from across the eastern United States. Genomic analysis revealed a complex of four distinct

species within what has historically been recognized as A. hegon.

ORIGINAL DESCRIPTIONS OF PREVIOUSLY KNOWN TAXA

Hesperia hegon and H. samoset (Scudder, 1863)

The familiar “Pepper and Salt Skipper” was originally described as Hesperia hegon

(Scudder, 1863, sp. #77) (Fig. 1) from a female specimen. Scudder had also described Hesperia

samoset in the same paper (sp. #78) (Fig. 2) from a male specimen. The name /egon thus takes

precedence over samoset on the basis of line priority. Neither description was accompanied by

illustrations. Scudder (1889) first illustrated H. samoset on plate 10, in Vol. 3 of The Butterflies

of the Eastern United States and Canada with Special Reference to New England (Figs. 3, 4). In

that work, Scudder gave priority of the name samoset over hegon, likely due to samoset being the

male specimen (Fig. 3). The female is shown in Fig. 4, below. Scudder appears to have mistakenly

reversed the sexes on his plate. Specimen No. | of plate 10 (Fig. 4) is indicated as a female, yet

has the characteristic wing shape and thin abdomen of the male. Conversely, specimen No. 3 of

plate 10 (Fig. 3) is indicated as a male, yet has the characteristic wing shape and thicker abdomen

of the female. The hegon holotype is indicated in Fig. 5. A typical male from near the TL is

indicated in Fig. 6.

77. Hesperta Hecon nov. sp. Above and beneath uni-

form dull dark-brown, with faint white markings on both

surfaces of primaries situated as in H. Oneko; on under

surface of secondaries, a submarginal series of small indis-

tinct whitish spots, a small white spot in the centre, and

another between the costal and subcostal nervures, mid-

way between the base and the submarginal band. Ex-

panse of wings nearly 1 inch.

Ihave seen but a single specimen, a female taken by

myself at the White Mountains in the latter part of July.

Fig. 1. Original description of Hesperia hegon (Scudder, 1863).

78. Hesperta Samoser nov. sp. Above dark-brown with

a few ochraceous scales especially at base of primaries

and on disk of secondaries. Primaries with three small

yellowish spots one above the other on costal border a little

more than three-fourths the distance from the base ; below

these and a little further removed from the outer border

than they, between the uppermost branches of the median,

a small spot; in the space below, situated near the base,

another small spot or slender oblique line, and sometimes

another below it between median and submedian; a

double spot at the end of the cell.

Beneath dark-brown with profusely scattered pale-yel-

lowish scales most abundant toward the outer margin; a

very delicate purplish reflection, especially on secondaries.

Primaries with the markings of the upper surface repeated.

Secondaries with a narrow transverse pale yellowish band,

two-thirds the distance from the base, nearest the outer

margin at the lowest band of subcostal, where it is bent at

right angles, and whence towards the costal border it is

interrupted ; a small spot in centre and another between

costal and subcostal midway between the base and trans-

verse band ; fringe of both wings pale yellowish interrupt-

ed with dark-brown, most distinct upon primaries. Ex-

panse of wings fully Linch. Very rare; 1 have seen two

specimens from Mass. and N.H.

Fig. 2. Original description of Hesperia samoset (Scudder, 1863).

Interestingly the original descriptions of both hegon and samoset indicate a dorsal and

ventral ground color of “dark brown”. The specimen illustrated in Fig. 3 and imaged in Fig. 5,

reflect this description. This was confirmed by examining a series of hegon in the topotypical

region of northern New England, from specimens in the NMNH collection and specimens in the

collections of the present authors. Scudder (1889) gave a more detailed description of the adults,

again stating: “Wings above rich dark brown” and “Beneath of the same brown as above”, while

not mentioning any greyish or greenish scaling on the ventral hindwings which has been a popular

description of hegon’s ventral appearance. While the other described wing characters (spots,

fringes, etc.) can apply to most individuals of all populations comprising the hegon complex,

ventral ground color stands as the most definitive character, though there is considerable variation

with the grey “peppering”. Genomic analysis of specimens in the topotypical region of northern

New Hampshire establishes a baseline against which to compare additional populations from

across eastern North America.

Fig. 3. Hesperia samoset, specimen #3 indicated as male Fig. 4. Hesperia samoset, specimen #1 indicated as the

in Plate 10 (Scudder, 1889). female in Plate 10 (Scudder, 1889).

is Ce

Fig. 5. Holotype, 4. hegon. Images courtesy Museum of Comparative Zoology, Harvard University.

http://creativecommons.org/licences/by-nc-sa/3.0/legalcode

Fig. 6. Typical A. hegon near type locality. Male. June 18, 1966, Glencliff, Grafton Co., N.H., leg.

Charles G. Oliver. Images courtesy Museum of Comparative Zoology, Harvard University.

http://creativecommons.org/licences/by-nc-sa/3.0/legalcode

Hesperia nemoris (W. H. Edwards, 1864)

W. H. Edwards (1864) subsequently described Hesperia nemoris (Fig. 7):

HESPERIA NEMORIS, nov. sp.

Male. Fixpands one inch. Upper side glossy brown ; secondaries

covered with greenish hairs except a narrow space along the costal and

inner margins; primaries have three yellow dots in line, on the costa,

and two small spots on the disk; fringes long, whitish, dark brown at

tips of nervules on primaries only.

Under side greenish grey, except on inner margin of primaries,

which is brown; same spots as above on primaries, but enlarged; on

secondaries a sub-marginal band of small spots, not reaching the inner

margin; two minute spots on the costa and a third on the disk,

searcely paler than the ground color.

Taken at Portsmouth, Ohio, by Mr. John Bolton.

Fig. 7. Original description of H. nemoris (W. H. Edwards, 1864).

Fig. 8. Original illustrations of H. nemoris (W. H. Edwards, 1865).

In his description of nemoris, Edwards indicates a ventral ground color of “greenish grey”.

No life history notes were given. An illustration of nemoris was later published in Edwards (1865)

(Fig. 8). The difference when compared to the original illustrations of hegon and samoset indicates

a more complete ventral postmedian band of light spots in nemoris. However, the primary

difference is that nemoris appears to have a denser covering of light scales on the ventral hindwing,

giving specimens a grayer appearance (Fig. 9). As in the earlier descriptions of hegon and samoset,

the other described wing characters (spots, fringes, etc.) can apply to most individuals of the hegon

complex due to considerable variation. By comparison with the original descriptions of hegon and

samoset, the ventral ground color of nemoris, as originally described by Edwards, provides a

readily observable baseline character. Research with further access to larger series is suggested to

better differentiate the two phenotypes.

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Fig. 9. Neotype of A. nemoris. Ohio: Vinton Co., nr. Zaleski (leg. Harry K. Clench), 10 May 1970

Pyrgus argina Plétz, 1884

Pl6tz (1884) subsequently described Pyrgus Argina (Fig. 10):

| aun. Vil. nur mit den typischen weissen:

pan Lis oe Ques HO mit Seen

Mean, Querstrich, grauen

en im Bogen jinter dor Mitte. Unterseite grau mit

braunen Rippen: ee jen Pu

3 Kreis und einen in der Mitte. * ade

60. Argina Herr. Sch. i. L.

Fig. 10. Original description of P. argina Plotz (1 884). Plotz placed the descriptions beiae the names.

An approximate translation: “Upperside black-brown. Forewings only with the typical [for

Pyrgus| white spots: the one in the discal cell is split, the one in Cell 1 is divided and grey, in Cell

5 a horizontal streak. Hindwings with five gray dots in the arc past the middle. Underside grey

with brown veins: Forewings with the white spots as above, on the posterior half brown, hindwings

with eight white dots in the % circle and one in the middle.” The description is rather general and

could apply to any population of the hegon complex. TL is erroneously given as “Brisbane”.

Genomic analysis of a specimen collected in 1887, shortly after the description of P. argina and

resembling a drawing of P. argina (see p. 21) revealed it to be A. hegon by the Z chromosome.

AMBLYSCIRTES ‘HEGON’ IN LITERATURE

Amblyscirtes hegon has historically been recognized as a single species. The current

analysis views previously published life history accounts as broadly applying to a complex of four

newly-identified species under the guise of “hegon”. The following sampling of literature

treatments provides us with a general guide to the life histories of various populations, but cannot

be reliably attributed to any of the four species revealed in this study. The butterfly is generally

described as uncommon, though occasionally being found in numbers. A review of morphological

descriptions, as follows below, focuses on the ventral hindwing ground color, which is the most

revealing (though variable) character that we have found, to differentiate the species. All

populations of the hegon complex bear similar lightly colored markings set against a dark ground

color, especially the highly variable arc of light marks across the ventral hindwing, as well as

checkered wing margins. Interestingly, the ventral hindwing is often described as “greenish” or

“oreenish-gray”, based primarily on the effect of the peppering of pale scales set against the darker

ground color. This greenish look is more the result of human perception. The greenish appearance

apparently fades in collections (Forbes, 1960), but color analysis reveals a different ventral color

relationship, based on collected specimens of varying ages. Interestingly, not one of the published

images in the literature, nor in 900 images on inaturalist.org, butterfliesandmoths.org, or

butterfliesofamerica.com show the reported “greenish” appearance of the ventral hindwing; just

varying degrees of gray peppering on a variable brown background. The reference to “green” in

later works might simply be a repeat of earlier works subject to visual misinterpretation (Fernald,

1884; French, 1886; Scudder, 1889). [One of us (Pavulaan) previously worked at a printing

company, where a cost-saving “green” print was made by combining yellow with gray. This

combination might account for the apparent “green” look of the underside of the wings, where

pale, yellowish scales overlay a grayish ground color. |

The hegon complex is generally reported to be univoltine throughout its range, flying from

March (Gulf Coast region) to July (Canadian Maritimes), but there are curious reports of possible

second-generation individuals in August in Connecticut (O’Donnell, et al., 2007), Massachusetts

(Stichter, 2015), and North Carolina (Glassberg, 1999; LeGrand & Howard, 2023), and even into

September in Missouri (Heitzman & Heitzman, 1987). This requires future investigation and may

reveal either an extended univoltine flight of any of the four taxa in the hegon complex into August

in some places, or a partial second brood. Essentially, little is known of the hostplant choices of

the four species in the hegon complex. Several hosts have been identified for ‘A. hegon’, but most

published works simply appear to repeat earlier lists, including some which are certainly in error.

More fieldwork is needed here as well. The only images of the larvae we have found are those

shown in Allen (1997), Allen, et al. (2005), and Venable (2014).

William F. Kirby (1871) applied line priority, treating Hesperia hegon as a species over

H. samoset. No life history information was given.

Charles H. Fernald (1884) listed this butterfly as 4. samoset. The underside is described

as “lighter than above, and heavily overlaid with greenish scales.”

George H. French (1886) listed this butterfly as 4. samoset. The underside is described

as “lighter than the upper, overlaid with greenish scales...”

Samuel H. Scudder (1889), in his discussion of samoset at species rank, gave a rather

detailed description of the adults. He describes the ventral color as “uniformly and profusely

flecked with very pale greenish yellow scales, giving the wing a greenish gray appearance.” Life

history information concerning the broods, immature stages and the host was based on the

illustrations of John Abbot: “Nothing is known of the earlier stages but what may be gleaned from

Abbot.” However, Calhoun (2019 and pers. corr.) convincingly stated that Abbot’s drawings

depicted a well-marked Amblyscirtes alternata, as well as the immature stages and host of

alternata: “Scudder...misidentified the butterflies in the DBC and HLHO drawings as the species

now recognized as Amblyscirtes hegon.” Calhoun also stated: “While Abbot illustrated A.

alternata many times, I found no evidence that he encountered A. hegon.” Based on this

misidentification, Scudder erroneously believed that samoset has two broods in the southern states.

Scudder identified the host grass in Abbot’s illustration as Sorghastrum avenaceum

(Indiangrass), now known as Sorghastrum nutans per the USDA PLANTS Database

(https://plants.usda.gov/home/plantProfile?symbolI=-SONU2). [S. nutans should be stricken from

the record as a host of A. hegon, since Abbot’s image shows it associated with A. alternata. |

Scudder stated: “In the Boisduval MS, it is given as Sorghum secundum.”, now known as

Sorghastrum secundum (Lopsided Indiangrass). However, Abbot’s original drawing of A.

alternata depicts this grass accurately, matching the image of Sorghastrum secundum in the USDA

PLANTS Database (https://plants.usda.gov/home/plantProfile?symbol=SOSE5). = [Thus, 8S.

secundum should also be stricken from the record as a host of A. hegon. |

Charles J. Maynard (1891) listed this butterfly as A. samoset. He described the underside

as “brown, overwashed with whitish...”

William J. Holland (1898, 1931 [Revised Edition]) listed this butterfly as A. samoset. He

described the underside as “pale gray”.

William F. Fiske (1901) listed this butterfly as A. samoset.

Harrison G. Dyar (1902) listed this butterfly as A. samoset, with hegon, nemoris and

alternata listed as synonyms.

John H. Comstock & Anna B. Comstock (1912) listed this butterfly as 4. samoset. The

ventral surface of the wings is described as “being overlaid with greenish scales.”

James H. McDunnough (1938) listed this butterfly as 4. hegon, with samoset and nemoris

listed as synonyms.

Alexander B. Klots (1951) described the ventral hindwing of A. hegon as “heavily and

coarsely dusted with light, greenish gray on a dark background.” He erroneously followed Scudder

(1889) in description of the larvae and broods: “One brood in north, supposedly two southward”.

The hostplant is given as “grasses”.

Harrison M. Tietz (1952, 1972) correctly assigned hegon to species rank, and listed

samoset and nemoris as synonyms. Sorghum bicolor (as S. vulgare) (Grain Sorghum) 1s listed

under food plants.

Douglas C. Ferguson (1954) listed hegon as occurring in Nova Scotia, occurring from

May 31 to July 2, and being “scarce and local.”

William H. Evans (1955), citing Scudder (1889) as “first reviser”, treated samoset at

Species rank, and hegon as a synonym. Line priority of hegon over samoset in the original

descriptions (Scudder, 1863) shows this to be an erroneous treatment.

Bryant Mather & Katherine Mather (1958) list only two records for hegon, both from

Tishomingo County in extreme northeast Mississippi. These would most likely actually be

nemoris. The specimens of matheri were collected in southwest Mississippi after publication of

this work and it’s three supplements.

William T. M. Forbes (1960) provided a rather enlightening taxonomic account under A.

samoset: “The two names [referring to hegon and samoset| were published together, and Scudder

later chose samoset, as he then (and now) had a right to do; the use of hegon is based on a blind

following of “page priority”. Forbes described the underside: “Below evenly dusted with pale

yellow, giving a faint greenish effect when fresh (fading in a collection) ...”

Cyril F. dos Passos (1964) listed samoset at species rank, with hegon, nemoris and argina

as synonyms.

Lucien Harris, Jr. (1972) chose to refer to this as Amblyscirtes samoset. Flight dates in

Georgia are April through May, but one July record is listed.

Roderick R. Irwin & John C. Downey (1973) listed samoset at species rank.

Auburn E. Brower (1974) correctly assigned hegon to species rank, and listed samoset as

synonym.

Arthur M. Shapiro (1974) describes the flight of hegon as: “One brood, late vi-early vil,

northward; partially double-brooded in Finger Lakes, v1.3-vi.20 and vii.22.”

C. Don MacNeill in William H. Howe (1975) described the ventral hindwing of A. hegon

as being “heavily dusted with greenish gray scaling. The listed hostplants Sorghastrum nutans

(=avenaceum) and S. secundum are, no doubt, cited from Scudder and are in error.

Bryant Mather & Katherine Mather (1976) interestingly changed the Mississippi listing

of hegon to samoset. This would most likely actually be nemoris.

Lee D. Miller & F. Martin Brown (1981) listed hegon at species rank, with samoset,

nemoris and argina listed as synonyms.

Robert M. Pyle (1981) described the ventral side of hegon as “putty gray with understated

light spots; greenish cast over HW.”

Lee D. Miller & F. M. Brown in Ronald W. Hodges (1983) listed hegon at species rank,

with samoset, nemoris and argina listed as synonyms.

Paul A. Opler & George O. Krizek (1984) described the ventral hindwing as: “dusted

with light gray-green scales”. The habitat is described as “glades or at the edges of mixed or

coniferous forest as well as at the edges of bogs or boggy streams.” The authors correctly state

that “hegon is univoltine throughout its range, but rare, late-emerging adults are occasionally found

in late July [northern New York]”. The hostplants are given as Poa pratensis (Kentucky

Bluegrass), Sorghastrum nutans and Sorghastrum secundum |both likely in error as host of A.

alternata|, and Uniola latifolia (Indian Woodoats) [now recognized as Chasmanthium latifolium

(Indian Sea Oats)].

Bryant Mather & Katherine Mather (1985) interestingly changed the Mississippi listing

of samoset back to hegon. This would most likely actually be nemoris.

James A. Scott (1986) described the ventral hindwing as “greenish-gray.” The hostplants

are given as Poa pratensis, Sorghastrum nutans and S. secundum |both likely in error as host of A.

alternata|, and Uniola latifolia [pow recognized as Chasmanthium latifolium|.

Ernest M. Shull (1987) described the ventral hindwing of hegon as “heavily dusted with

greenish gray...”

J. Richard Heitzman & Joan E. Heitzman (1987) describe hegon as having “greenish-

gray scaling of the wings beneath.” They described the brood sequence in Missouri: “Most

specimens have been found in April and May, but there are June, July and September records,

indicating at least partial broods during the summer.”

Paul Klassen, A. Richard Westwood, William B. Preston & W. Brian McKillop (1989)

describe the underside of hegon as “dusted with pale gray... The veins on the underside of the

hindwings are highlighted with whitish-gray... Most specimens have a greenish-gray hue on the

underside of the wings.”

Paul A. Opler & Vichai Malikul (1992) described the ventral hindwing of hegon as “light

gray-green”. The flight period is described as “April-July, rarely early Aug. (1 brood), earliest in

the south.”

Jeffrey Glassberg (1993) described the underside as “yellowish tinged gray-brown

ground...”

Thomas J. Allen (1997) described the underside as “brownish gray”. He describes the

habitat preference as: “This skipper prefers wet areas and is found along streams, bogs, low-lying

wet meadows, and glades at the edges of mixed or coniferous forests.” He correctly stated that

hegon is univoltine throughout its range. The larvae is described as “pale green with 3 dark green

dorsal stripes and a pale lateral stripe” and the host is given as Glyceria striata (Fowl Mannagrass).

Ross A. Layberry, Peter W. Hall & J. Donald Lafontaine (1998) described A. hegon

with the ventral hindwing as “grey, with a slightly greenish tinge that is most noticeable in fresh

Specimens...”

Paul A. Opler & Amy B. Wright (1999) described the ventral hindwing of hegon as “light

gray-green”.

Jeffrey Glassberg (1999) described the underside as “olive-tinged gray-brown...” The

brood sequence is given as “1 brood + partial second north to Virginia and Missouri-mid April-

May, rare partial July-Aug.”

Mogens C. Nielsen (1999) described the underside of hegon as “grayish green.”

Jim P. Brock & Kenn Kaufman (2003) describe the ventral hindwing of hegon as

“frosted greenish gray”. The brood sequence is described as “late spring (mainly) to summer in

south (1-2 broods), early summer in north (1 brood).

Marc C. Minno, Jerry F. Butler & Donald W. Hall (2005) indicate a single hegon flight

in Florida, and suggest the host as “probably” Glyceria striata. The larva is illustrated.

Jane O’Donnell, Lawrence Gall & David Wagner (2007) describe the underside in

Connecticut specimens as “Wings below medium to dark brown with extensive gray frosting and

fainter spots.”

W. Mike Howell & Vitaly Charny (2010) repeat previous host lists, including Poa

pratensis and Chasmanthium latifolium. The listed hostplants Sorghastrum nutans (=avenaceum)

and S. secundum originate from Scudder and are certainly in error. The authors indicate a single

brood in Alabama, late March to late June.

John K. Bouseman, James G. Sternburg & James R. Wiker (2010) describe the venter

of hegon as “...the anterior half of the forewing is dusted with gray-green scales. Similar scales

extend over the entire hindwing.” The authors add Cinna arundinacea (Stout Wood Reed) to the

list of hosts.

Jim Patterson (2011) identified hegon as “A very small dark gray skipper with greenish

overtones on the ventral side.”

Peter W. Hall, Colin D. Jones, Antonia Guidotti & Brad Hubley (2014) described A.

hegon with the underside as: “Both wings flecked with greyish-green when fresh, creating the

“pepper and salt” effect. With age, the flecking wears away and the wings become more uniform

dark brown.”

Rita Venable (2014) noted, regarding the ventral color of hegon: “...they start out gray

and end up dark brown! Their gray scales just wear off with time.” This accurately describes the

difficulty observers will have, differentiating the four species described in this work based solely

on ventral ground color. The host listed for Tennessee 1s Chasmanthium latifolium (River Oats,

or Indian Woodoats), discussed extensively. Several hosts are repeated from previous authors,

one very likely in error: Sorghastrum sp. (Indiangrass), a host of A. alternata. The larva is

illustrated. A single brood is noted, flying in April through June.

Lori Spencer (2014) describes the ventral side of hegon as having “grayish green

shading”. Two broods are suggested: April-July.

Jeffrey Glassberg (2017) described the ventral side as “green-gray, sometimes

pink/purple-gray.” Interestingly he notes: “females usually tan [below]”. Here, he describes the

broods as “two broods, second is partial, Apr-May, July-Aug.”

James L. Monroe & David M. Wright (2017) described the ventral side with “extensive

gray overscaling...slight greenish sheen when fresh.” The habitat is described as “woodland

openings and edges, streamsides” and the host is given as Brachyelytrum erectum (Bearded

Shorthusk).

Craig Marks (2017) was first to publish a note of two ventral color forms: “I have found

two “forms” ... of the Pepper and Salt Roadside Skipper in LA.” Marks described the habitat as

“heavily wooded loess hills with deep ravines” and also noted “there was a great deal of cane

growing, both in the ravines and along the road.” Marks additionally noted: “These skippers were

extremely abundant, both on the road basking and taking nectar at wild garlic.” Lastly, it was

noted that one out of five seen that day were of the tan variety. The tan variant was illustrated for

the first time.

Craig Marks (2018) further noted the two ventral color forms: “At Sicily Island Hills

WMA, I would estimate that one out of five seen were tan colored dorsally [correctly: ventrally |

rather than the typical slate gray.” A single brood is reported for Louisiana, in March and April.

Phillip G. deMaynadier, John Klymko, Ronald G. Butler, W. Herbert Wilson, Jr.,

and John V. Calhoun (2023) described the ventral side of hegon in Maine and the Canadian

Maritime Provinces as: “brown with grayish-green scaling...becoming more dark brown with

wear.” They note one annual generation from late-May to mid-July with a flight peak in mid-June,

and that the host in that region is unknown.

METHODOLOGY AND GENERAL RESULTS

A combination of genomic and wing color analyses was performed on a series of specimens

across the historically recognized range of hegon. An additional sample of specimens was used to

determine differences in genitalia. Dr. Andrew Warren assisted, prior to this study, in extracting

the male genitalia of matheri and nemoris.

Genomic DNA extraction (from legs), sequencing, and computational analyses were

carried out using the Grishin lab published protocols (Li et al. 2019; Cong et al. 2021; Robbins et

al. 2022). In brief, a leg was detached from a specimen and non-destructively soaked in DNA

extraction solution. Genomic libraries were constructed from DNA and sequenced at 150 bp or

shorter. Every DNA sequence that made it into the libraries was sequenced, even very short ones

(30 bp), thus allowing us to sequence old specimens with DNA degraded to short fragments. The

resulting sequences were mapped on protein-coding genes of a reference genome of Lerema accius

(J. E. Smith, 1797) (1.e., a well-assembled genome of a close relative made from recently collected

Specimens), and phylogenetic trees constructed from such alignments: for a random sample of

positions from all nuclear genome genes in autosomes, those predicted to be in the Z chromosome

and those in the mitochondrial genome. The Z chromosome tree is typically best correlated with

speciation (Cong et al. 2019) and is shown in Fig. 11.

We used whole genome shotgun sequencing to learn about genetic differences between the

tan-brown and what we assumed at the time was “regular” A. hegon and sequenced several

specimens of each “form”. To our initial surprise, the two “forms” separated into two groups in

the Z chromosome. Genetic differentiation between them in the Z chromosome was strong and

suggestive of distinct species with Fy/Gnin values of 0.43/0.003 (Cong et al. 2019). Much intrigued

by this initial assessment, we broadened sequencing efforts, and got even more surprising results

(Fig. 11). What was considered a single species A. hegon, partitioned into four distinct and strongly

supported clades in the Z chromosome tree. The most genetically differentiated from other clades

is the tan-brown “hegon”. Three other clades are closer to each other, but they are overlapping in

distributions (Fig. 12), some quite significantly, over hundreds of miles and therefore are expected

to be sympatric. For instance, representatives of all three clades have been recorded from West

Virginia. Within each clade, specimens are close to each other regardless of the locality. This lack

of genetic differentiation within each clade suggests ongoing gene flow throughout the range of

the clade.

Conversely, the separation between the clades in the Z chromosome is prominent. Due to

the sympatry of the clades, individuals from different clades have the opportunity to meet and

exchange genes. If this exchange was frequent, it would have led to the equilibration of allele

frequencies (as we see within clades), and there would be no prominent clades in the tree as a

result. Therefore, a combination of this Z chromosome tree structure (distinct well-separated clades

and the lack of statistically supported clustering within clades, Fig. 11) with the sympatry of the

-s—«CTAAm@ibiyscirtes gelidus|21109C06|PT|MI,Van Buren Co.,Antwesp Twshp|1985

16 Amblyscirtes gelidus]21113G11|]RLP-22039|PT|MI,Van Buren Co.,Antwesp Twshp|1985

2 Amblyscirtes gelidus]22054A01|HT|M|MI,Van Buren Co.,Antwesp Twshp|1983

| ee Amblyscirtes gelidus|22054A07|PT|MI,Presque Isle Co.,Thompson Harbor SP|2014 :

| Amblyscirtes gelidus|22055F05|WVBAO0095|PT|WV,Wood Co.,Johnson Janes Park|2014 Cc

Amblyscirtes gelidus|22054C05|PT|MI,Van Buren Co.,Antwesp Twshp|1983 »

Amblyscirtes gelidus|21113G03|PT|WV,Randolph Co.,Spruce Knob Lake|2017 C.:

i 7D Amblyscirtes gelidus|22054B12|PT|MI,Van Buren Co.,Antwesp Twshp|1985

gelidus —— Amblyscirtes gelidus|22055F09|PT|WV,Pocahontas Co.,MNF Road 112

4 [28 Amblyscirtes gelidus|22054A08|PT|MI,Kalamazoo Co.,Portage, Gourdneck SP|2012

—|o Amblyscirtes gelidus|22054B01|PT|MI,Van Buren Co.,Antwesp Twshp|1985

Amblyscirtes gelidus|22055F11|PT|WV,Pocahontas Co., Thornwood|2013 =

Amblyscirtes gelidus|22054A11|PT|MI,Barry Co.,Yankee Springs Tnshp|1983 omen

o-——_———— Amblyscirtes gelidus|22054B06|PT|MI,Van Buren Co.,Antwesp Twshp|1985 oO

So)

iL Amblyscirtes gelidus|22055F08|WVBA09254|PT|WV,Pocahontas Co.,Buffalo Lake|2016

Amblyscirtes gelidus|22055F04|WVBA00595|PT|WV,Pocahontas Co.,Little River WMA|2013

Amblyscirtes gelidus|22054C06|RLP-7578|PT|WV,Pendelton Co,,Spruce Knob|2006

Amblyscirtes gelidus|22054C08|RLP-7581|PT|WV,Pendelton Co.,Spruce Knob|2006

Amblyscirtes gelidus|22054C07|RLP-7580|PT|WV,Pendelton Co.,Spruce Knob|2006

Amblyscirtes gelidus|22055F03|WVBAO00058|PT|WV,Randolph Co.,Great Bridge|2013

Amblyscirtes gelidus|22055F06|WVBA09712|PT|WV,Pocahontas Co.,Little River WMA|2016

Amblyscirtes hegon|19047E11|GA,Walker Co.|2018

a8 Amblyscirtes hegon|21089D12|RLP-8783|NC,Clay Co.,Buck Creek @ Hwy 164|2007

5 Amblyscirtes hegon|21109D01|GA,Union/Fannin Co.,Cooper Creek Rec. Area|1997

; Amblyscirtes hegon|21109C08|GA,Rabun Co.,Popcorn overlook, Hwy 76|2005

0 Amblyscirtes hegon|22031C11|PA,Westmorelnd Co.,Jones Mills|1988

Amblyscirtes hegon|21091A09|MS,Hinds Co.,Brownsville|1959

0 7a Amblyscirtes hegon|21109C11|AL,Madison Co.,Hale Mtn. area|2003

Amblyscirtes hegon|21113G10|RLP-19756|TN,Marion Co.,NE of Whitwell|2020

= Amblyscirtes hegon|22031C10|MO,Franklin Co.,Sullivan|1988

Amblyscirtes hegon|22054H02|RLP-22104|AR, Faulkner Co.,Wooly Hollow SP|2022

a Amblyscirtes hegon|21113H03|RLP-22101|AR, Faulkner Co.,Wooly Hollow SP|2022

TO Amblyscirtes hegon|22054H06|RLP-22124|AR, Faulkner Co.,Wooly Hollow SP|2022

8 Amblyscirtes hegon|22054F12|RLP-22099|AR, Faulkner Co.,Wooly Hollow SP|2022

Amblyscirtes hegon|22054H05|RLP-22122|AR, Faulkner Co.,Wooly Hollow SP|2022

Amblyscirtes hegon|21114A01|WI|1887

phates Amblyscirtes hegon|21109C12|AR,Logan Co.,Magazine Mt|2001

0 Amblyscirtes hegon|22055F07|WVBA00057|WV, Tucker Co.,Little Canaan WMA

Amblyscirtes hegon|22031C09|MD,Allegany Co.,Flintstone|1990

Amblyscirtes hegon|21109C05|ME,Penobscot Co.,Enfield|1976

aD Amblyscirtes hegon|21113G06|NH, Carroll Co.,Passaconaway|1984

Amblyscirtes hegon|21113E07|YPM ENT 589164|NH,Coos Co.,Second College Grant|1978

hegon rr Amblyscirtes hegon|21089D11|RLP-22020|Canada:Nova Scotia, Halifax|1977

14 Amblyscirtes hegon|21113G04|Canada:Nova Scotia,Hants Co.|2019

Amblyscirtes hegon|21113E06|YPM ENT 589163|NH,Coos Co.,Second College Grant|1978

Ts Amblyscirtes hegon|21113E08|/YPM ENT 849390|VT,Essex Co.,6 km NE Concord|1995

Amblyscirtes hegon|21113G07|NH,Carroll Co.,Passaconaway|1984

Amblyscirtes nemoris|21063F06|RLP-1244|MS, Tishomingo Co.,Woodall Mt.|1992

0 Amblyscirtes nemoris|22063H11|RLP-22360|MS,Lee Co.,Tombigbee SP|1977

Amblyscirtes nemoris|22063H10|RLP-22342|MS,Lee Co.,Tombigbee SP|1975

Amblyscirtes nemoris|15098A03|NT|OH,Vinton Co.,3 mi E of Zaleski|1970

Amblyscirtes nemoris|21109CO9|NC,Clay Co.,Buck Creek|2002

Amblyscirtes nemoris|21113G02|NC,Haywood Co.,Maggie Valley|2009

= 5 Amblyscirtes nemoris|21063F07|RLP-1248|MS, Tishomingo Co.,Woodall Mt.|1992

nemoris r Amblyscirtes nemoris|22055C10|FL,Gadsden Co.,nr. Chattahoochie|2021

7 Amblyscirtes nemoris|21063F10|RLP-3481|MS, Tishomingo Co.,Woodall Mt.|2000

Amblyscirtes nemoris|22055F10|WV,Richie Co.,North Bend SP|2014

F z Amblyscirtes nemoris|19047E12|LA,Catahoula Pa.,Gilbert WMA|2018

4 Amblyscirtes nemoris|22063HO5|LA,Catahoula Pa.,Gilbert WMA|2018

y Amblyscirtes nemoris|21063F11|RLP-0884|MS, Tishomingo Co.,5 mi W Belmont|1994

0 Amblyscirtes nemoris|22054H03|RLP-22105|AR, Faulkner Co.,Wooly Hollow Stake Prk|2022

100 Fi Amblyscirtes nemoris|21113G01|MO,St. Francois Co.,Nbonne Terre|1988

Amblyscirtes nemoris|22054H04|RLP-22106|AR, Faulkner Co.,Wooly Hollow SP|2022

= Amblyscirtes nemoris|21113HO2|RLP-22100|AR,Faulkner Co.,Wooly Hollow SP|2022

Amblyscirtes nemoris|22054H07|RLP-22077|LA,Catahoula Pa.,Gilbert WMA|2022

OS i= Amblyscirtes nemoris|19047F02|LA,Catahoula Pa.,Gilbert WMA|2018

i Amblyscirtes nemoris|22054H0O1|RLP-22103|AR, Faulkner Co.,Wooly Hollow SP|2022

5 Amblyscirtes nemoris|10674|TX,Smith Co., Tyler SP|1986

Amblyscirtes nemoris|22054H10|RLP-22091|LA,Catahoula Pa.,Gilbert WMA|2022

Amblyscirtes matheri|19047F01|PT|LA,Catahoula Pa.,Gilbert WMA|2018

ya Amblyscirtes matheri|22054H11|RLP-22093|PT|LA,Catahoula Pa.,Gilbert WMA|2022

: Amblyscirtes matheri|22054H12|RLP-22094|PT|LA,Catahoula Pa.,Gilbert WMA|2022

z Amblyscirtes matheri|21113F04|RLP-22079|PT|LA,Catahoula Pa.,Gilbert WMA|2022

Amblyscirtes matheri|21063F09|RLP-0123|HT|MS,Warren Co.,Vicksburg|1988

Aen Amblyscirtes matheri]21109C04|PT|AL, Jackson Co.,Hollytree|2014

4 Amblyscirtes matheri|21113E10|RLP-22090|PT|LA,Catahoula Pa.,Gilbert WMA|2022

Amblyscirtes matheri|22063H01|PT|LA,Catahoula Pa.,Gilbert WMA|2018

oe Amblyscirtes matheri|21063F02|RLP-0866|PT|MS,Warren Co.,Vicksburg|1994

; Amblyscirtes matheri|21063F04|RLP-0119|PT|MS,Warren Co.,Vicksburg|1988

Fi Amblyscirtes matheri|21063F05|RLP-5371|PT|MS, Yazoo Co.,3 mi E of Satartia|2004

0 Amblyscirtes matheri|21109C10|PT|AL, Jackson Co.,Hollytree|2020

; Amblyscirtes matheri|21113F05|RLP-22080|PT|LA,Catahoula Pa.,Gilbert WMA|2022

D Amblyscirtes matheri|22063H03|PT|LA,Catahoula Pa.,Gilbert WMA|2019

Amblyscirtes matheri|21063F03|PT|MS,Claiborne Co.,34 mi SW Clinton|1989

7 Amblyscirtes matheri|21113G09|RLP-22095|PT|LA,Catahoula Pa.,Gilbert WMA|2022

ic Amblyscirtes matheri|22054H08|RLP-22078|PT|LA,Catahoula Pa.,Gilbert WMA|2022

Amblyscirtes matheri|21063F08|RLP-5408|PT|MS, Yazoo Co.,3 mi E of Satartia|2004

Fr Amblyscirtes matheri|22063HO2|PT|LA,Catahoula Pa.,Gilbert WMA|2017

Pia Amblyscirtes matheri|21113F02|RLP-22081|PT|LA,Catahoula Pa.,Gilbert WMA|2022

Amblyscirtes matheri|21113G05|PT|SC, Fairfield Co.,Ridgeway|2007

1*i=5 Amblyscirtes matheri|21113F01|RLP-22088|PT|LA,Catahoula Pa.,Gilbert WMA|2022

: 0 Amblyscirtes matheri|22054H09|RLP-22089|PT|LA,Catahoula Pa.,Gilbert WMA|2022

matheri Amblyscirtes matheri|21113F03|RLP-22067|PT|LA,Catahoula Pa.,Gilbert WMA|2022

100 Amblyscirtes matheri|21109D02|PT|MS,Winston Co., Tombigbee NF|1999

Amblyscirtes matheri|21063F01|RLP-5369|PT|MS, Yazoo Co.,3 mi E of Satartia]2004

Amblyscirtes matheri|22063H04|PT|LA,Catahoula Pa.,Gilbert WMA|2018

0.003 Amblyscirtes matheri|21109C07|PT|SC,Laurens Co.,Garlington School Rd.|2012

98 2

Amblyscirtes hegon

tat. rest.

.nhemoris s

)

Fig. 11. The phylogenetic tree of the Amblyscirtes hegon complex inferred from protein-coding regions in the Z

chromosome. Different species are colored in different colors: A. gelidus (gray), A. hegon (blue), A nemoris (black),

and A. matheri (red). Primary type specimens are labeled in magenta. Values by nodes show statistical support values

(in %) for corresponding bipartitions.

clades suggests that the four clades represent four distinct species. Indeed, restricted (or nearly

absent) gene flow between the clades (meaning that they represent species) would explain the

prominence of these four clades. Further details of the genomic analysis of these specimens will

be presented elsewhere. This study focuses on the phenotypic analysis and description of the new

species. We only note that the mitochondrial DNA reveals a number of haplotypes and

introgression between these species but the most distinct tan-brown, which has a unique set of

haplotypes among sequenced specimens. The COI barcodes between the tan-brown species and

others differ by about 0.9%.

From the genomic analysis of the Z chromosome, we deduce that A. hegon is a complex of

four distinct species. Only one species reaches the extreme northeast of the range, where the type

locality of A. hegon was established. Therefore, we identify this species as A. hegon. Incidentally,

this is the most widespread species out of four (blue in Figs. 11, 12). We continue treating Pyrgus

argina Pl6tz, 1884 as conspecific with A. hegon (the blue clade) based on an old specimen in

MENB (sequenced as NVG-21114A01) resembling a drawing of P. argina (see p. 21). Genomic

sequencing of the neotype of Hesperia nemoris W. H. Edwards, 1864, places it in the black clade.

Therefore, the black clade is A. nemoris, stat. rest. The other two clades (red and gray) do not

have available names associated with them and represent new species that are described below.

In summary, genomic sequencing reveals that A. hegon is a complex of four species.

Combining this information with the type localities of available names and sequencing of primary

type specimens assigns names to two species and implies that two others are new.

Employing genomic analysis, specimens that were identified to species, and additional

specimens from several of these locations, were subjected to color analysis using the Color Grab!™

cellphone application (www.loomatix.com), version 3.9.2, to establish exacting RGB and HSB

color codes under “daylight” fluorescent lighting, in combination with the Colblindor™

application (www.color-blindness.com/color-name-hue/) to produce refined color swatches rather

than giving generalized color descriptions as is traditional with taxon descriptions. Six different

areas of the wings (Fig. 13) were measured for their red/green/blue (RGB) and

hue/saturation/brightness (HSB) color codes. Color codes of individual specimens were then

averaged to produce results for each species (Fig. 14). Color names in the description of each of

the four species reference the color names given in the Color Grab and Colblindor applications.

Of particular interest are the results of analyzing the ventral hindwings. While the human eye

perceives a “greenish” look on the ventral hindwing, particularly in nemoris, and as is often

described for “hegon’” in the literature, primarily due to the over-peppering of pale scales, both of

the color applications apparently view the base ground color and not the effect of peppering.

Additionally, wing measurements were made from the examined series. Measurements

were made of wingspan, forewing length, and the angle of the apical spot row from the leading

edge of the forewing (Fig. 15). These were then averaged and a range for each was determined

(Fig. 16).

Specimens subjected to genomic analysis were then mapped to determine their geographic

distribution (Fig. 12).

Amblyscirtes hegon @

Amblyscirtes nemoris @

Amblyscirtes matheri (n. sp.) @

Amblyscirtes gelidus (n. sp.) ©

1 — Dorsal ground color.

2 — Dorsal spots color.

3 — Ventral hindwing ground color.

4 — Ventral forewing apex color.

5 — Ventral forewing base color.

6 — Ventral spots color.

Fig. 13. Wing areas analyzed using color analysis.

Ventral Ventral HW Ventral Ventral FW Ventral Ventral FW Ventral | Ventral spots Dorsal | Dorsal ground Dorsal Dorsal spots

HW ground color FW apex apex color FW base base color spots color color ground color spots color color

ground | RGB color code color | RGB color code color | RGB color code RGB color code color | RGB colorcode RGB color code

color HSB color code HSB color code HSB color code HSB color code HSB color code HSB color code

121, 87, 90 87, 73, 54 62, 44, 33 204, 192, 159 59, 42. 30 208, 190, 154

355, 28, 47 34, 37, 34 22, 46, 24 43. 22, 80 24, 49, 23 40. 25. 81

112, 100, 86 91. 77, 61 64. 46, 30 204, 192, 160 69, 56, 37 | | 211, 195, 153

$4, 32:35 28, 53, 25 43, 21, 80 35, 46, 27 | 43,27, 82

32, 23, 43

A. hegon

A. nemoris

A. matheri 149, 124. 96 144. 116, 86 76, 50, 28 202. 184, 153 107, 77, 44 200, 179, 134

31, 35, 58 31, 40, 56 27, 63, 29 37, 24, 79 31, 58, 41 40, 32, 78

A. gelidus 144, 136, 123

105, 93, 79 79, 59, 52 207, 196, 167 84, 58. 48 215, 199, 159

32, 24, 41 15, 34, 30 43,19. 81 16, 42, 32 42. 26, 84

37, 14, 56

Fig. 14. Color analysis results (average colors) of four species in the study. Note ventral HW ground colors.

Wing measurements:

Red line — Wingspan.

Green line — Forewing Length.

Blue angle — Angle of subapical spots

from leading edge of forewing.

Fig. 15. Wing measurement areas.

Wingspan : FW length Angle of apical | Angle of apical

; Wingspan FW length

Species average average spots average | spots range

range (mm)

(mm) (degrees) (degrees)

| 23.4 | 20-26 10-13, || 78.0 | 65-90

22-25 11-14

24.1 23-25 11-13 || 75.3 | 6880

Fig. 16. Wing measurements.

Lastly, the genitalia of males of each of the four identified species was microscopically

examined for differences (Figs. 16 & 17).

bo mm

A. matheri sp. n

_ = a b

*/‘

A, nemoris stat. rest

A. gelidus sp. n.

g h

Fig. 16. Male genitalia of Amblyscirtes hegon complex from USA. a—b. A. matheri sp. n. paratype,

Mississippi, Warren Co., Vicksburg, 27-Mar-2000, R. Patterson leg., genitalia vial 401-02 Andrew

D. Warren. c-d. A. nemoris stat. rest., Mississippi, Tishomingo Co., Woodall Mt., 4-Apr-2000, R.

Patterson leg., DNA sample NVG-21063F10, genitalia vial #01-03 Andrew D. Warren. e-f. A.

gelidus sp. n., holotype, DNA sample NVG-22054A01, genitalia NVG230128-01. g—h. A. hegon,

Arkansas, Faulkner Co., Wooly Hollow State Park, 26-Apr-2022, R. Patterson leg., RLP#22101,

DNA sample NVG-21113H03, genitalia NVG230130-01. a. ¢. e. g. left lateral and b. d. f. h. dorsal

VIEWS.

A. matheri sp. n.

A. nemoris stat. rest.

Fig. 17. Male genitalia of Amblyscirtes hegon complex in posterior view. Green arrow points to

broad tooth on the inner surface of harpe, expanded differently in these species. a-b: A. matheri sp.

n. [a: ADW#01-02. b: NVG-21113F02, NVG230130-03], e-d: A. nemoris stat. rest. [¢: NVG-

21063F10. d: NVG-21113H02, NVG230130-02], e: A. gelidus sp. n., holotype [NVG-22054A01,

NVG230128-01], f: A. hegon [NVG-21113H03, NVG230130-01].

DESCRIPTIONS: THE AMBLYSCIRTES HEGON COMPLEX

Amblyscirtes hegon (Scudder, 1863)

syn: samoset (Scudder, 1863)

syn: argina (Pliétz, 1884)

DESCRIPTION AND PHENOTYPIC COMPARISON OF ADULTS

Color names are given per Color Grab™ and Colblindor™ applications used in the analysis

for sake of easy reference. Per Scudder (1863, 1889), this species is recognized primarily by the

dark brown color of the ventral hindwings, identified as “Buccaneer” (brown) in the present

analysis. The color swatches (Fig. 14) show hegon to have a very slight violet pigment, compared

to nemoris, matheri and gelidus. Some individuals vary to gray brown, with a perceived “peppery”

look, making differentiation from nemoris and gelidus problematic. The ventral forewing apex is

similarly colored to the hindwing, being slightly darker (“Brown Derby’). The last area of the

forewing that was analyzed was the darkest portion of the base of the forewing (“Bistre” brown).

There was no appreciable difference in color here, between the four species.

The dorsal brown ground color (“Bistre”’) differs little from either nemoris (“Mikado”) or

gelidus (“Very Dark Brown’’), but is considerably darker than matheri (“Horses Neck’) (Fig. 14).

Scudder (1889) gives detailed description of the dorsal and ventral spot pattern, which are

individually variable in extent across all four species in the hegon complex. The colors of the light

dorsal spots (“Double Spanish White”) and ventral spots (“Soft Amber”) differ very little from

either nemoris, matheri or gelidus (Fig. 14) and are essentially unreliable for differentiating the

species. In hegon the ventral hindwing spot pattern tends to be reduced in extent, frequently being

absent altogether. Specimens confirmed as hegon from northern New England, the Canadian

Maritimes and southern Appalachian Mountains have a high percentage of individuals with

unmarked ventral hindwings. Other features in the descriptions of hegon and samoset (Scudder,

1863, 1889) similarly apply to nemoris, matheri and gelidus and pose identification challenges to

observers.

Scudder (1889) provided measurements of the forewings in millimeters. Males (n=3)

ranged 11.6—12.2 mm and averaged 12.2 mm. The present analysis of forewing length measured

males (n=17) ranging 10.0-13.0 mm, and averaging 11.6 mm. Scudder measured female (n=3)

forewings, ranging 11.5-12.5 mm, and averaging 12.1 mm. The present analysis measured females

(n=7) ranging 12.0-13.0 mm, and averaging 12.4 mm. All sexes averaged together (Fig. 16) shows

hegon adults having forewing length ranging 10-13 mm, and averaging 11.9 mm. Wingspan of

males was measured at 20-25 mm (n=17), averaging 22.8 mm, and females (n=7) measured at 24-

26 mm, averaging 24.9 mm. All sexes averaged together (Fig. 16) show hegon adults having a

wingspan of 20-26 mm, averaging 23.4 mm; showing hegon to have the largest and smallest

individuals of the four species. Specimens from the northeastern portion of the species’ range

averaged slightly smaller, whereas specimens from the southern Appalachian Mountains averaged

slightly larger. The measurements show that hegon and nemoris have similar forewing length and

wingspan, whereas matheri and gelidus were both larger (Fig. 16). A larger sample would be

necessary to better define size differences between the two regions.

Of interest to us was the difference in the angle of the subapical spot row from the leading

edge of the forewing (Fig. 16), showing little difference between hegon and nemoris. Both matheri

and gelidus showed the alignment of the apical spots to have a sharper average angle. While the

measured angle varied greatly, hegon, nemoris and to a lesser degree, gelidus ranged closer to a

90° angle than matheri, which showed the apical spot row to sit at a sharper angle from the leading

edge of the forewing.

Differences in male genitalia between species are slight and difficult to assess due to

individual variation. Typically, a combination of two characters would distinguish the species.

First is the extent of development of the broad tooth on the inner surface of harpe, best seen in

posterior view (Fig. 17, note green arrow on panel b) and dorsal view (Fig. 16b, d, f, h). Second

is the shape of valva and harpe in lateral view (Fig. 16). In_A. hegon, the tooth is larger and more

robust (Fig. 17d), similar to 4. nemoris stat. rest., but different from both of the two new species,

in which the tooth is shallower and does not protrude much between the valvae. In 4. hegon, the

valva tends to broaden somewhat from the base to harpe, with its dorsal and ventral margins at an

angle (in lateral view). This broadening is not only due to expansion of the ampulla region on

costa, but also because harpe ventral margin is more convex near the base and somewhat expanded

ventrad (Fig. 16g). This valva shape is quite similar in A. gelidus, from which it can be

distinguished by a more robust tooth on the inner surface of valva.

IMMATURE STAGES

Scudder (1889) gave a rather detailed description of the caterpillar, chrysalis and host of

what he believed to be samoset, based on the illustrations of John Abbot. The host in Abbot’s

illustration is determined to be Sorghastrum nutans (=avenaceum) (Indiangrass). However,

Calhoun (2019 and pers. corr.) believes that Abbot’s drawings depicted the immature stages and

host of Amblyscirtes alternata.

SPECIMENS OF A. HEGON EXAMINED IN PRESENT STUDY:

Alabama: Madison Co., Hale Mtn. area (leg. Howard Grisham), 4 May 2003.

Arkansas: Faulkner Co., Wooly Hollow State Park (leg. Ricky Patterson), 26 April 2022 (2 ¢ RLP

#22101/NVG-21113H03, RLP #22099/NVG-22054F12, 1 9 RLP #22104/NVG-22054H02),

8 May 2022 (2 2).

Arkansas: Logan Co., Magazine Mountain (collection of Howard Grisham), 25 May 2001.

Georgia: Rabun Co., Hwy 76 @ Popcorn Overlook (leg. Ron Gatrelle), 25 May 2005.

Georgia: Union/Fannin Co., Cooper Creek Recreation Area (collection of Howard Grisham), 5 July

1997.

Georgia: Walker Co. (leg. Jeff Slotten), late April 2018.

Maine: Penobscot Co., Enfield (leg. L. Paul Grey), 5 June 1976 (collection of Howard Grisham).

Maryland: Allegany Co., Flintstone (Harry Pavulaan), 20 May 1990.

Mississippi: Hinds Co., Brownsville (leg. Bryant Mather), 24 March 1959 (1 3), NVG-21091A09.

Missouri: Franklin Co., Sullivan (leg. Harry Pavulaan), 28 May 1988.

New Hampshire: Carroll Co., Passaconaway (leg. Harry Pavulaan), 13 July 1984.

New Hampshire: Coos Co., Second College Grant (leg. Richard E. Grey), 11 June 1978 (2

specimens via PMNH collection).

North Carolina: Clay Co., Buck Creek @ Hwy. 164 (leg. Ricky Patterson), 17 May 2007 (2 3).

Nova Scotia (Canada): Halifax Regional Municipality, Fairview (leg. Chris T. Maier), 5 June 1995

(via PMNH collection).

Nova Scotia (Canada): Fairview, Halifax Regional Municipality (leg. Ken Neil), 26 June 1977 (1

Specimen).

Nova Scotia (Canada): Hants Co., Mt. Uniacke (leg. Derek Bridgehouse), 18 June 2019 (1

Specimen).

Pennsylvania: Westmoreland Co., Jones Mills (leg. Harry Pavulaan), 22 May 1988.

Tennessee: Marion Co., NE of Whitwell (leg. Ricky Patterson), 23 May 2020 (1 &).

Vermont: Essex Co., 6 km NE of Concord, (leg. Chris T. Maier) 5 June 1995 (via PMNH

collection).

West Virginia: Tucker Co., Little Canaan W.M.A. (leg. Susan Olcott), 31 May 2013 (3),

WVBA00057, NVG-22055F07.

“Wisconsin”, 1887, NVG-21114A01 in MFNB (Berlin, Germany) (Fig. 18b).

HABITAT AND DISTRIBUTION

The habitat is described as “open places in woods” (Scudder, 1889) but is primarily

associated with forest habitats, and mostly found along forest roads. Specimens confirmed as

hegon were taken along dirt roads through shale barren forest habitat near Flintstone, MD. At

Markham, VA., a single specimen was collected along a forest edge adjacent to a lake. Many

varied habitats are given for “hegon’, but extensive fieldwork and verification to species will be

required to determine the exact habitat requirements of the four species in this complex.

Amblyscirtes hegon flies in early to mid-spring (mid-March in Mississippi through mid-

June in Nova Scotia) which due to the wide distribution of this species will vary along with spring

from south to north following the distribution. We have confirmed specimens of Amblyscirtes

hegon from the northeast (Nova Scotia, New Hampshire, Pennsylvania, Maryland, Maine, and

Vermont), the Appalachian Mountains in southwest North Carolina, northern Georgia, northeast

Alabama, and southeast Tennessee, as well as the Ozark and Ouachita mountains of Arkansas and

Missouri, and near Brownsville in Hinds County, Mississippi. This last specimen is the odd one,

it is not from a mountain area, but a hardwood forested area in central Mississippi. Based on this,

it seems possible that Amblyscirtes hegon could appear in many places in the eastern and central

United States.

As with its sister species, the habitat of Amblyscirtes hegon is openings in or near shaded

wooded areas, often nectaring on flowers such as blackberry, clover, and other early spring

flowers. It also is often found at damp soil, or resting on bare areas on gravel roads and forested

foot trails. This species has been found flying sympatrically with Amblyscirtes nemoris in North

Carolina and Arkansas.

Amblyscirtes hegon seems to be a generally more northeastern species, but specimens have

been identified from as far west as Arkansas and Missouri, and as far southeast as northern

Georgia, and as far southwest as Hinds County, Mississippi.

Based on specimens examined by us, Mississippi and West Virginia are the only states that

have three of these species resident (Amblyscirtes hegon, A. matheri, and A. nemoris in Mississippi,

and A. hegon, A. matheri, and A. gelidus in West Virginia), but none were found flying

sympatrically with each other in these two states.

Analysis of Pyrgus (Syrichthus |sic]) argina Plétz, 1884

The name Pyrgus (Syrichthus [sic|) argina was published by Plétz (1884), who attributed

it to Herrich-Schaffer, from an unstated number of specimens with the locality given as “Brisbane.”

Pl6tz assigned this species to the genus Pyrgus Hiibner, [1819] but placed it near the end of his

identification key (the description was in the form of a key), next to a couple of species that are

currently not in Pyrgus. We regard that P. argina is differentiated from other species by the

following characters, as translated from Pl6étz (1884): “Black-brown upper side. Forewing only

with the typical white spots: that in the discal cell is split, that in cell 1 is divided and gray, in cell

5 a dash. Hindwing with 5 gray dots in the arch past the middle. Underside gray with brown

veins: FW with the white spots as above, brown on the posterior half, hindwings with 8 white dots

in the % [of a] circle and one [dot] in the middle.” By “typical white spots” for Pyrgus, Pl6étz

meant the postdiscal row and the discal forewing spot, not including the submarginal rows of spots

or dots characteristic of many Pyrgus and Burnsius Grishin, 2019 species.

In addition to the description, Plétz prepared drawings of many species included in his

keys, but the whereabouts of the original drawings remain unknown (Nakahara et al., 2022).

Godman studied these originals and recruited Horace Knight (and possibly other artists) to make

copies for the species he could not immediately recognize (Godman, 1907). A compilation of

these copies is in the library of the Natural History Museum, London (Zhang et al., 2022a; Zhang

et al., 2022b), and it contains P. argina under the number 903, reproduced here as Fig. 18a.

Inspecting these Godman’s copies of Plé6tz’s drawings, Evans (1949) concluded that P. argina was

not the Old World, but American species conspecific with A. hegon, which at that time was treated

as a junior subjective synonym of Hesperia samoset Scudder, 1864 (Evans, 1955). Evans’ opinion

has not been challenged since, and P. argina was included as a junior subjective synonym of A.

hegon in all subsequent literature (Evans, 1955; Mielke, 2005; Pelham, 2023).

With our discovery that A. hegon is a complex of four cryptic species, confidently

identifiable only by DNA, it became desirable to study the taxonomic identity of P. argina.

Because we were not aware of P. argina type specimens, we undertook a brief search for them in

the MFNB collection. We found an old specimen somewhat resembling the illustration of P.

argina, but nevertheless differing from it in several aspects, such as ventral hindwing postdiscal

row of pale spots being mostly connected into a band (instead of separated round spots as

illustrated in Fig. 18a) and the lack of a pale spot in the forewing cell Mi-M2. This specimen (Fig.

18b), a female unidentified in the MFNB collection that we placed in the 4. hegon complex by

visual inspection, came from the Méschler collection. Médschler was sending specimens to Pl6tz

for identification (Méschler, 1876), and Pl6tz used some of them in his drawings and descriptions.

However, this female was collected in 1887 according to its label, and therefore after the

description of P. argina, hence cannot be a syntype. The only locality information about this

female is “Wiscons.” There was also a second specimen, a male, with the same locality and

collection label, but identified as “samoset.” A leg of the female was sampled for genomic

sequencing (NVG-21114A01), and the Z chromosome tree implied that out of the four species, it

was conspecific with 4. hegon (Fig. 11) that is treated as a senior synonym of P. argina in nearly

all publications. Therefore, we concur with the previous assessment of P. argina being a junior

subjective synonym of 4A. hegon. We will conduct a more detailed search for P. argina syntypes

prior to proceeding with the neotype designation.

DNA sample ID:

NVG-—21114A01

c/o Nick V. Grishin

1cm

Coll. Méschl.

Fig. 18. Specimens and illustrations of Amblyscirtes hegon. a. Godman’s copy of an unpublished

Plotz’s illustration of Pyrgus (Syrichthus [sic]) argina Plotz, 1884, © of the Trustees of the Natural

History Museum London and are made available under Creative Commons License 4.0

(https://creativecommons.org/licenses/by/4.0/); b. an old specimen of A. hegon from Wisconsin and

its labels (above the ventral image) that resembles the illustration of P. argina [MFNB]. Dorsal (left

side of the panel letter) and ventral (right side of the panel letter) views are shown.

Amblyscirtes nemoris (W. H. Edwards, 1864) - reinstated status

DESCRIPTION AND PHENOTYPIC COMPARISON OF ADULTS

Genomic analysis of the holotype of Hesperia nemoris revealed that nemoris, long

considered a synonym of hegon, is, in fact, a species distinct from hegon.

Per W. H. Edwards (1864), this species is characterized by a “greenish gray” venter. Color

analysis shows no green background pigment, rather a brown color (“Pine Cone’’) that differs only

slightly from hegon, but differs appreciably from both the tan-brown venter of matheri and grayish

venter of gelidus (Fig. 14). The “greenish” appearance is certainly due to the visual effect of light

scales “peppered” over the brown ground color. Though there is considerable character overlap

with hegon, making identification to species challenging, nemoris generally has more of the light

ventral peppering of light scales (Fig. 9), whereas hegon is primarily brown-ventered with less of

the light peppering of nemoris. The ventral forewing apex is similarly colored to the hindwing,

being slightly darker (“Metallic Bronze”). The last area of the forewing that was analyzed was the

darkest portion of the base of the forewing (“Morocco Brown”). There was no appreciable

difference in color here, between the four species.

The dorsal brown ground color (“Bistre”’) differs little from either hegon (also “Bistre’”’) or

gelidus (“Very Dark Brown’), but is considerably darker than matheri (“Horses Neck”) (Fig. 13).

W. H. Edwards (1864) gives a detailed description of the dorsal and ventral spot pattern, which

are individually variable in extent across all four species in the hegon complex. The colors of the

light dorsal spots (“Tahuna Sands”) and ventral spots (“Soft Amber’) differ very little from either

hegon, matheri or gelidus (Fig. 14) and are essentially unreliable for differentiating the species.

In nemoris the ventral hindwing spot pattern tends to be well-developed, similar to both matheri

and gelidus, but dissimilar to hegon which has a tendency for a reduced or absent spot pattern.

Other features in the description of nemoris (W. H. Edwards, 1864) similarly apply to hegon,

matheri and gelidus and pose identification challenges to observers.

The present analysis measured the length of the forewing of the males (n=13), ranging

11.0-13.0 mm, and averaging 11.6 mm. The measurement of females (n=4) was consistently 12.0

mm, averaging 12.0 mm. All sexes averaged together (n=17) show nemoris adults having

forewings measuring 11.8 mm and ranging 11-13 mm (Fig. 16). W. H. Edwards (1864) provided

a single measurement for the male wingspan, expanding 1” (25.5 cm). In the present study,

wingspan of the males was measured at 22-24 mm (n=13), averaging 22.6 mm, with females (n=4)

measuring 23-24 mm and averaging 23.5 mm. All sexes averaged together (n=17) show nemoris

adults having a wingspan measuring 22.8 mm and ranging 22-24 mm (Fig. 16). The measurements

show that nemoris and hegon have similar forewing length and wingspan, whereas matheri and

gelidus were both larger (Fig. 16).

The difference in the angle of the subapical spot row from the leading edge of the forewing

(Figs. 15 & 16), shows little difference between nemoris and hegon. Both matheri and gelidus

showed the alignment of the apical spots to have a sharper average angle. While the measured

angle varied greatly, nemoris, hegon and to a lesser degree gelidus ranged closer to a 90° angle

than matheri, which showed the apical spot row to sit at a sharper angle from the leading edge of

the forewing.

In male genitalia of A. nemoris, the tooth on the inner surface of harpe is larger and more

robust (Figs. 17c, d, 16d), similar to 4. hegon (Figs. 17f, 16h), but different from both of the two

new species, in which the tooth is shallower and does not protrude much between the valvae.

Amblyscirtes nemoris differs from A. hegon by the valva with more or less parallel dorsal and

ventral margins in lateral view and the base of harpe along ventral margin is straighter and less

convex (or curved) (Fig. 16g).

SPECIMENS OF A. NEMORTIS EXAMINED IN PRESENT STUDY:

Neotype: Ohio: Vinton Co., 3 miles east of Zaleski (leg. Harry K. Clench), 10 May 1970 (Fig. 9).

Arkansas: Faulkner Co., Wooly Hollow State Park (leg. Ricky Patterson), 26 April 2022 (1 4 RLP

#22100/NVG-21113H02, 3 9 RLP #22103/NVG-22054H01, RLP #22105/NVG-22054H03,

RLP #22106/NVG-22054H04), 8 May 2022.

Louisiana: Catahoula Parish, J. C. ‘Sonny’ Gilbert WMA (leg. Ricky Patterson), 21 March 2022

(12), 25 March 2022 (1 ), (leg. Jeff Slotten), 23-Mar-2018 (2 9 NVG-19047E12 &

19047F02)

Mississippi: Tishomingo Co., 5 miles west of Belmont (leg. Ricky Patterson),16 April 1994 (2 2)

Mississippi: Tishomingo Co., Mt. Woodall (leg. Ricky Patterson), 4 April 1992 (2 @), 18 April 1993

(3g, 1 ), 16 April 1994 (1 3, 2 Y), 4 April 2000 (1 &, 1 9)

Missouri: St. Francois Co., Bonne Terre (leg. Harry Pavulaan), 24 April 1988

North Carolina: Clay Co., Buck Creek @ Hwy. 164 (leg. Ricky Patterson), 17 May 2007 (1

Specimen - genetic analysis NVG-21109C09 per tree).

North Carolina: Haywood Co., Maggie Valley (leg. Harry Pavulaan), 10 May 2009

Texas: Smith Co., Tyler State Park (leg. June and Floyd Preston), 15 March 1986 (via Texas A & M

collection)

HABITAT AND DISTRIBUTION

A. nemoris flies in early spring (mid-March through mid-May). Early and late dates are 21

March to 10 May. In Louisiana and Mississippi, it is found in late-March to late-April; in Arkansas

it flies from late-April to mid-May based on confirmed specimens.

The confirmed range of this species is from east Texas to Ohio (the Type Locality),

Louisiana, Arkansas and Mississippi to North Carolina. This species like the others is also found

in wooded areas, along gravel roads and small openings in wooded areas (hardwoods primarily),

nectaring on blackberry blooms, wild garlic, clover, and various spring flowers. In Louisiana at

the J. C. ‘Sonny’ Gilbert WMA this species flies sympatrically with Amblyscirtes matheri, and in

Arkansas at Woolly Hollow State Park and in North Carolina at Buck Creek/Hwy 164 in Macon

County, Amblyscirtes nemoris flies sympatrically with Amblyscirtes hegon. Amblyscirtes nemoris

seems to have a more east central United States distribution, but extremes range from east Texas,

thence east through Louisiana and Mississippi, and on to southwest North Carolina. It goes north

to Ohio (the Type Locality), the northern most record we have.

Amblyscirtes matheri Patterson, Pavulaan and Grishin - new species

ZooBank registration: urn:|sid:zoobank.org:act:7809EA3B-C188-428A-91 BE-9778BAE52327

DESCRIPTION AND PHENOTYPIC COMPARISON OF ADULTS

New species A. matheri differs from hegon, nemoris and gelidus primarily by the distinct

tan-brown (“Sorrell Brown”) ground color of the ventral hindwing surfaces. Wing marks differ

little between the four species.

Fig. 19. Holotype (<) of Amblyscirtes matheri. Warren Co., MS. 3 Apri

11988, RLP #0123/NVG-21063F09.

Size. Adults of A. matheri are generally of similar size to nominotypical hegon and

nemoris, though very slightly larger (Fig. 16). The length of the male forewings of the examined

matheri (n=28) series ranges 11-14 mm, averaging 12.4 mm. Male wingspan of A. matheri

[maximum wing spread] is measured at 22-25 mm, averaging 24 mm. Female A. matheri have

more rounded wings than males, with forewing length ranging 12-14 mm (n=31), averaging 12.4

mm. Female A. matheri have a wingspan ranging 23-28 mm, averaging 25.7 mm. All adults

analyzed in the study had a forewing length of 11-14 mm, averaging 12.4 mm (n=59) with a

wingspan of 22-25 mm, averaging 24 mm.

Dorsal ground color. The dorsal ground color of A. matheri males and females is a

uniform (“Horses Neck’) brown. Applying the Color Grab™ and Colblindor™ applications, males

and females of A. matheri averaged a red/green/blue (RGB) color code of 107, 77, 44, with a

hue/saturation/brightness (HSB) color code of 31, 58, 41 (Fig. 14). The dorsal ground color is a

lighter brown than in hegon, nemoris and gelidus.

Dorsal pattern and color of markings. The dorsal wing marking pattern of A. matheri is

similar to hegon, nemoris and gelidus. The markings are a light tan (“Yuma”) and do not differ

appreciably from hegon, nemoris and gelidus. The three apical spots near the FW apex of matheri

are at a sharper angle from the leading edge of the forewing than in hegon, nemoris and gelidus,

though there is considerable variation and overlap between the four species in this complex (Fig.

16).

Ventral ground colors. New species A. matheri differs from A. hegon, nemoris and

gelidus primarily by the distinct tan-brown (“Sorrell Brown”) ground color of the ventral hindwing

surfaces, as opposed to the ventral brown (“Buccaneer”) of hegon, the dark brown (“Pine Cone”’)

of nemoris, and the brown/gray (“Schooner”) of gelidus. Color analysis revealed an averaged

red/green/blue (RGB) color code of 149, 124, 96, with a hue/saturation/brightness (HSB) color

code of 31, 35, 58 (Fig. 14). In matheri the “peppering” of light scales on the ventral side of the

wings is absent. This peppering is present in nemoris and gelidus, and variably present in hegon,

and causes the perception of the “greenish” color of the ventral hindwings.

The apical area of the ventral forewings of matheri approximated the color of the ventral

hindwings closely, averaging red/green/blue (RGB) color code of 144, 116, 86, with a

hue/saturation/brightness (HSB) color code of 31, 40, 56 (Fig. 14). Additional color measurements

were made for the ventral postbasal area of the forewings, primarily within cell CuAz which ts the

darkest portion of the ventral side of the wings. In matheri, this area averaged a red/green/blue

RGB color code of 76, 50, 28 and a hue/saturation/brightness (HSB) color code of 27, 63, 29 (Fig.

14) and did not differ appreciably from hegon, nemoris and gelidus.

Ventral pattern and color of markings. Ventrally, the spot pattern of matheri is well-

developed and is similar to hegon, nemoris and gelidus, though the ventral pattern of hegon is

variable and frequently absent altogether. The markings are a light tan (“Yuma”) and do not differ

appreciably from hegon, nemoris and gelidus (Fig. 14).

Male genitalia. The tooth on the inner surface of harpe is shallower and does not protrude

as strongly between the valvae (Figs. 17a, b, 16b), in contrast to larger and more robust tooth in

both A. hegon (Figs. 17f, 16h) and 4. nemoris (Figs. 17c, d, 16d). Valva with somewhat expanded

ampulla that overlays the harpe, and a result, broadening somewhat from the base to harpe along

its costa, but not along the ventral margin (Fig. 16a), where harpe is not more expanded at the base

as in A. hegon (Fig. 16g) and A. gelidus (Fig. 16e). Harpe is usually straighter at the distal margin.

TYPES

Holotype:

USA: Mississippi: Warren Co., Vicksburg, (leg. Ricky Patterson), 3 April 1988 (Fig. 19), (@).

Deposited in the Carnegie Museum of Natural History, Pittsburgh, PA, USA.

Allotype:

Mississippi: Warren Co., Vicksburg, (leg. Ricky Patterson), 3 April 1988 (°).

Paratypes:

Alabama: Jackson Co., Hollytree (leg. Howard Grisham), 3 May 2014 (1 3), 28 April 2020 (1 3).

Louisiana: Catahoula Parish, J. C. ‘Sonny’ Gilbert WMA (leg. Craig Marks), 10 March 2012

(3 & - none of these are on genomic analysis chart). 1 4 NVG-19047F01 (leg. Jeff Slotten), 3

March 2018

Louisiana: Catahoula Parish, J. C. ‘Sonny’ Gilbert WMA (leg. Ricky Patterson), 21 March 2022

(4 4, including NVG-21113F02, 1 2), 25 March 2022 (5 ¢ 1 9), 7 March 2023 (8 ¢ 2 9).

Mississippi: Claiborne Co., Rocky Springs Campground 34 mi. SW of Clinton (leg. Drew

Hildebrandt and Maria Plonczynski), 7 April 1991 (1 4), 22 April 1989 (1 3).

Mississippi: Grenada Co., T12N, R3E, Section 7 SW (leg. Terry Schiefer), 9 April 1987

(14) (via MEM collection)

Mississippi: Holmes Co., Holmes County State Park (leg. Drew Hildebrandt and Maria

Plonczynski), 9 April 1988 (3 3)

Mississippi: Leflore Co., CR 518 north of Greenwood (leg. Leroy Koehn), 2-24 April, 1994 (4 2

Bae):

Mississippi: Warren Co., Vicksburg, (leg. Ricky Patterson), 3 April 1988 (7 ¢ 9 9), 4 April 1988

(1 419), 9 April 1988 (1 2), 24 March 1992 (6 d 1 Q), 28 March 1993 (4 4 3 &), 18

April 1993 (1 4), 9 May 1993 (1 4), 25 March 1994 (1 &), 2 April 1995 (1 3 2 &), 27

March 1998 (5 4), 27 March 2000 (2 4), 17 March 2002 (19°)

Mississippi: Warren Co., Vicksburg, (leg. Ricky Patterson), 27 March 1988 (1 3), 19/20 April

1993 (1 3 1 &) (via C. H. Grisham collection)

Mississippi: Winston Co., Tombigbee NF (leg. David Pollock and Terry Schiefer), 22-29 March 1999

(1 4) (via MEM collection)

Mississippi: Yazoo Co., 3 miles E of Satartia (leg. Ricky Patterson), 2 April 2004 (5 ¢ 13 9), 9 April

2004 (2 05 Q)

South Carolina: Fairfield Co., Ridgeway (leg. Harry Pavulaan), 6 May 2007 (1 3)

South Carolina: Laurens Co., Garlington School Road (leg. R. G. Simpson), 20 April 2012 (1 3)

In addition to the known type series, there appear to be recent photographic images of A.

matheri (as “A. hegon’’) on iNaturalist from Catahoula Parish, LA dated March 29, 2020; March

21, 2021; April 3 and 8, 2022.

Etymology: The species is named in honor of Bryant Mather, lifelong lepidopterist devoted to

the study of Mississippi Lepidoptera.

HABITAT AND DISTRIBUTION

Amblyscirtes matheri flies in early spring (mid-March through mid-April) in Mississippi

and Louisiana, early April in NE Alabama, and early April in South Carolina. Early/late dates are:

3 March (Louisiana) to 9 May (Mississippi).

The butterfly is found in openings in hardwood forest habitats in hilly areas, particularly in

the Loess Bluff Hills that border the Mississippi River alluvial plain. The loess deposits found

their origin in the Pleistocene ice age glaciers far to the north in Canada and the northern United

States. As the glaciers ground the bedrock into a fine flour-like deposit, the deposit was washed

down the Mississippi River and deposited onto the adjacent flood plains. The Loess deposits were

then blown by winds onto the bluffs on either side of the river. The loess bluffs trend north-south

throughout the state of Mississippi along the Mississippi River delta. This region is dominated by

deciduous hardwood and pine forest consisting primarily of various species of Oaks (Quercus sp.),

Loblolly Pine (Pinus taeda) and Shortleaf Pine (Pinus echinada), which comprise approximately

two-fifths of the Loess Bluff Hills in Mississippi. The remainder of the area is under cash crops

and pasture or hay farms.

Amblyscirtes matheri seems to be associated with this particular biogeographic region in

Mississippi and Louisiana. In northeast Alabama it has been confirmed in wooded, hilly areas that

are foothills of the Appalachians, where other more normally mountain species such as Evora laeta

have been found. A single South Carolina specimen was collected in the Sandhills region of the

eastern part of the state; a habitat in which Loblolly Pines predominate along with an understory

of Switch Cane (Arundinaria tecta). While presently recorded in seven counties in Mississippi, in

Louisiana the skipper has only been found in the J. C. “Sonny” Gilbert Wildlife Management Area

(formerly the Sicily Island Hills Wildlife Management Area until renamed in 2015), in northeast

Catahoula parish, which is in east central Louisiana. The complete range of A. matheri has yet to

be determined, but as stated above it is known to range from Louisiana to South Carolina. In

Mississippi and Louisiana, only one specimen has been found outside the Loess Bluffs region in

Mississippi/Louisiana. Whether this geographical tie to the Loess Bluffs is an artifact of the

collected specimens or due to a specialized habitat is not known. The specimens from northeast

Alabama are not in Loess Bluff hills, and this terrain is not found in South Carolina either. A

review of various collections, websites, and publications have not found any confirmed specimens

or photographs of adequate clarity to be determined as A. matheri outside of these four states. The

only specimens from the Mississippi River delta flatlands examined were collected just north of

Greenwood, very near the Loess Bluff Hills. It remains to be determined whether 4. matheri

ranges north through the Loess Bluffs region along the Mississippi River alluvial plain, potentially

as far as southern Illinois. It would be expected that gaps in distribution between Louisiana and

South Carolina will be filled as this species becomes known to collectors and watchers.

Adults can be found nectaring on flowers, especially white clover, wild garlic (as noted by

Craig Marks), fleabane, and occasionally at mud. They are not normally found in the middle of

fields or in thickly wooded areas, but along woodland edges and roadsides (especially gravel roads)

going through hardwood areas, similar to the habitat of A. hegon and A.nemoris. Craig Marks

(2018) describes the hostplants of related ‘Amblyscirtes hegon’ (actually nemoris) in Louisiana as:

“various types of grasses, including river oats, fowl manna-grass, and Indian grasses.” These

grasses should be investigated as potential hosts of A. matheri as well. One author (Patterson) has

obtained eggs from a female but was unable to get the emerged larva to feed on any of the grasses

offered.

Amblyscirtes gelidus Grishin, Patterson, Pavulaan - new species

ZooBank registration: urn:|sid:zoobank.org:act:24DEA 1 D8-3831-4D9F-9389-6FE3BAE642AA

DESCRIPTION AND PHENOTYPIC COMPARISON OF ADULTS

New species A. gelidus differs from hegon, nemoris and matheri primarily by the distinct

brownish-gray (“Schooner”) ventral hindwing. Freshly-emerged individuals display a “frosted”

appearance. Wing marks differ little between the four species.

Fig. 20. Holotype (<) of Amblyscirtes gelidus. Van Buren Co., MI. 5 June 1983, NVG-22054A01

Size. Adults of A. gelidus are generally of similar size to nominotypical hegon and

nemoris, though very slightly larger, and are approximately the same size as matheri (Fig. 16).

The length of the adult forewings of the physically examined series (n=6) ranges 11-13 mm,

averaging 12.3 mm. Unfortunately, since only a single female specimen was available for

examination, measurements are for all adults combined. The wingspan of ge/idus is measured at

22-25 mm, averaging 24.1 mm.

Dorsal ground color. The dorsal ground color of A. gelidus is a uniform (“Very Dark

Brown’) with a slight overlay of light scales in many individuals. Applying the Color Grab™ and

Colblindor™ applications, males and females of A. gelidus averaged a red/green/blue (RGB) color

code of 84, 58, 48, with a hue/saturation/brightness (HSB) color code of 16, 42, 32 (Fig. 14). The

dorsal ground color is similar to hegon, nemoris and matheri.

Dorsal pattern and color of markings. The dorsal wing marking pattern of A. gelidus is

similar to hegon, nemoris and matheri. The markings are a light tan (“Tahuna Sands”) and do not

differ appreciably from hegon and nemoris, but are lighter than matheri. The three apical spots

near the FW apex of ge/idus are at a slightly sharper angle from the leading edge of the forewing

than in hegon and nemoris but greater than matheri, though there is considerable variation and

overlap between the four species in this complex (Fig. 16).

Ventral ground colors. New species A. gelidus differs from A. hegon, nemoris and

matheri primarily by the distinct brownish-gray (“Schooner”) ventral hindwing which also

displays a peppering of light scales; as opposed to the ventral brown (“Buccaneer”) of hegon, the

dark brown (“Pine Cone”) of nemoris, and the tan-brown (“Sorrell Brown”) ground color of

matheri. Color analysis revealed an averaged red/green/blue (RGB) color code of 144, 136, 123,

with a hue/saturation/brightness (HSB) color code of 37, 14, 56 (Fig. 14). A. gelidus has the ventral

“peppering” of light scales as seen in nemoris and variably in hegon. This peppering causes the

perception of the “greenish” color of the ventral hindwings. Many individuals of this species

display a distinct overlay of light wing scales on the inner half of the dorsal forewing surface, and

elongated scales on the inner two-thirds of the dorsal hindwing that take on a more distinct

appearance of hair.

The apical area of the ventral forewings of ge/idus approximated the brownish-gray color

of the ventral hindwings, but is slightly darker, averaging red/green/blue (RGB) color code of 105,

93, 79, with a hue/saturation/brightness (HSB) color code of 32, 24, 41 (Fig. 14). Additional color

measurements were made for the ventral postbasal area of the forewings, primarily within cell

CuA2 which is the darkest portion of the ventral side of the wings. In ge/idus, this area averaged

a red/green/blue RGB color code of 79, 59, 52 and a hue/saturation/brightness (HSB) color code

of 15, 34, 30 (Fig. 14) and did not differ appreciably from hegon, nemoris and matheri.

Ventral pattern and color of markings. Ventrally, the spot pattern of ge/idus is variably

developed and is similar to hegon, nemoris and matheri, though the ventral pattern of hegon is

variable and frequently absent altogether. The markings are a light tan (“Soft Amber”) and do not

differ appreciably from hegon, nemoris and matheri (Fig. 14).

Male genitalia. The tooth on the inner surface of harpe is shallower and does not protrude

as strongly between the valvae (Figs. 17e, 16f), in contrast to larger and more robust tooth in both

A. hegon (Figs. 17f, 16h) and A. nemoris (Figs. 17c, d, 16d), but is somewhat more prominent

than in A. matheri (Figs. 17a, b, 16b). Valva tends to broaden from the base to harpe, with its

dorsal and ventral margins at an angle (in lateral view). This broadening is not only due to

expansion of the ampulla region on costa, but also because harpe ventral margin is more convex

near the base and somewhat expanded ventrad (Fig. 16e). This valva shape is quite similar in A.

hegon, from which it can be distinguished by less robust tooth on the inner surface of valva.

TYPES

Holotype:

USA: Michigan: Van Buren Co., Antwerp Township Section 14, (leg. W. A. Miller), 5 June 1983

(4). deposited in the Carnegie Museum of Natural History, Pittsburgh, PA, USA.

Allotype:

West Virginia: Pendleton Co., Spruce Knob, Monongahela National Forest (leg. Ricky

Patterson), 7 June 2006, (coll. of Ricky Patterson) ().

Paratypes:

Michigan: Barry Co., Yankee Springs Township, Section 31, (leg. W. A. Miller), 10 June 1983

(12).

Michigan: Kalamazoo Co., Portage, Gourdneck State Game Area, (leg. W. A. Miller), 17 May

2012 (240, 19).

Michigan: Presque Isle Co., Thompson Harbor State Park, (leg. W. A. Miller), 15 June 2014 (19).

Michigan: Van Buren Co., Antwerp Township Section 13, (leg. W. A. Miller), 17 May 1982 (12),

5 June 1983 (114, 49).

Michigan: Van Buren Co., Antwerp Township Section 14, (leg. W. A. Miller), 5 June 1983 (10).

Michigan: Van Buren Co., Antwerp Township Section 25, (leg. W. A. Miller), 29 May 1983 (13),

13 May 1985 (14, 19, 1 undet.), 14 May 1985 (54, 19), 22 May 1985 (10, 29).

West Virginia: Pendleton Co., Spruce Knob, Monongahela National Forest, (leg. Ricky

Patterson), 7 June 2006 (2).

West Virginia: Pocahontas Co., Lake Buffalo Recreation Area, Monongahela National Forest,

(leg. Susan Olcott), 8 June 2016 (14, dep. W.V. Butterfly Atlas Project).

West Virginia: Pocahontas Co., Little River WMA, Monongahela National Forest, (leg. Jane

Whitaker), 3 June 2013 (14%, dep. W.V. Butterfly Atlas Project).

West Virginia: Pocahontas Co., Little River WMA, Monongahela National Forest, (leg. Randall

Casto), 19 June 2016 (19, dep. W.V. Butterfly Atlas Project).

West Virginia: Pocahontas Co., Thornwood, Monongahela National Forest, (leg. Susan Olcott),

4 June 2013 (16, dep. W.V. Butterfly Atlas Project).

West Virginia: Randolph Co., Durbin, Cheat Bridge, (leg. Susan Olcott), 4 June 2013 (13, dep.

W.V. Butterfly Atlas Project).

West Virginia: Randolph Co., Spruce Knob Lake, Monongahela National Forest, (leg. Harry

Pavulaan), 30 May 2013 (1¢, dep. W.V. Butterfly Atlas Project), 2 June 2017 (10, H.

Pavulaan collection).

West Virginia: Wood Co., Parkersburg, Johnson T. Janes Park (leg. Susan Olcott), 5 June 2014

(13, dep. W.V. Butterfly Atlas Project).

Etymology: The species name reflects the frosted appearance of freshly emerged individuals.

HABITAT AND DISTRIBUTION

A. gelidus flies in late spring (mid-May through mid-June in Michigan, early June in West

Virginia). Early/late dates of confirmed specimens are: 13 May to 15 June in Michigan and 30

May to 19 June in West Virginia.

While we have had little experience with this species, it does not seem to be much different

in habitat or habits from A. hegon, A. matheri, or A. nemoris, though possibly more adapted to the

cold climates of Michigan and highlands of West Virginia. Several West Virginia specimens were

captured on and near Spruce Knob, the highest point in West Virginia. They were found on lightly

traveled gravel roads in Transition Zone hardwood forested areas. The area is known as the

Allegheny Plateau, on which the climate is considerably colder than surrounding lowlands, and

likely similar in climate and habitats to Michigan. Nothing is known about the precise habitat of

the evaluated Michigan specimens. Nielsen (1999) describes the habitat of A. hegon in Michigan

as “small, sunny forest openings, swamp edges and other partially shaded moist areas.” This is

similar to the habitat for the other species discussed in this work, and seems reasonable it would

apply to this new species.

The ones from the Spruce Knob area of West Virginia were resting at wet areas on a shaded

gravel road, and it can be assumed they nectar at flowers like the other Amblyscirtes discussed in

this work.

The confirmed range of this species consists of Michigan, primarily southwest Michigan,

plus one locality in NE Michigan, and in West Virginia, primarily the Spruce Knob area of West

Virginia. The photo of A. hegon in ‘Michigan Butterflies and Skippers’ (Nielsen, 1999) appears

to be this new species and states the distribution to be “throughout the Upper Peninsula and

scattered counties in the Lower Peninsula.” Since we have not examined any of these specimens

that he based these records on, we cannot confirm the distribution of ge/idus in Michigan based on

Nielsen (1999). We expect that additional states will be added to this disjunct distribution as more

Specimens are evaluated.

IMMATURE STAGES

Nielsen (1999) described Michigan ‘hegon’ larvae as “pale greenish white with dark green

dorsal and white lateral stripes; the head is dark brown with pale brown bands.” This might apply

to gelidus but needs to be confirmed. He also listed the host of Michigan ‘hegon’ as Kentucky

Bluegrass, Indian Grass, and possibly other grasses.

DESIDERATA

Based on the current study and historical literature, considerable fieldwork is required to:

(1) better determine reliable wing markings needed to differentiate 4. hegon, A. nemoris, and A.

gelidus (whereas A. matheri is more readily distinguished by its unique ventral coloration); (2)

confirm hostplants of each of the four species within the ‘hegon’ complex; and (3) determine the

complete range of all four species.

Genomic analysis found that a specimen from Tombigbee State Park, Lee County, MS

collected in June, 1979 was in the Amblyscirtes gelidus clade but appeared to potentially be a

different, albeit closely related, taxon. At this time this is the only specimen available, and before

any further conclusions can be made additional specimens need to be collected or located and

further analyses performed. As such, this potential new species or subspecies is not addressed here

but in a future study if deemed appropriate based on additional study.

CONCLUSIONS

Amblyscirtes hegon is a complex of four species: A. matheri sp. n., A. gelidus sp. n., A.

nemoris Stat. rest., and A. hegon. These species can be definitively identified using DNA of the Z

chromosome and display subtle differences in male genitalia. In facies, the species mostly differ

by their colors, A. matheri being the most distinct. To facilitate direct color comparison, four

specimens were photographed in a single frame instead of being combined in an image processing

matheri ee == gelidus

» nemoris

Fig. 21. Four species of Amblyscirtes hegon complex photographed together in one frame on 20 November 2023.

a, e: A. matheri sp. n. paratype [RLP#22081, DNA NVG-21113F02, genitalia NVG230130-03], b, f: A. gelidus sp.

n., holotype [DNA NVG-22054A01, genitalia NVG230128-01 ], c, g: A. nemoris stat. rest. [RLP#22100, DNA NVG-

21113H02, genitalia NVG230130-02], d, h: A. hegon [RLP#22101, DNA NVG-21113H03, genitalia NVG230130-

01] in dorsal (a-d) and ventral (e-h) views. Specimens of A. nemoris (RLP#22100) and A. hegon (RLP#22101) were

collected on 26 April 2022 in Wooly Hollow State Park, Faulkner Co., Arkansas.

software, and we illustrate the color differences discussed above (Fig. 21). Except for the older A.

gelidus holotype, others were collected in 2022 and have a similar degree of color fading.

ACKNOWLEDGEMENTS

Thanks go to David M. Wright for manuscript review; Dr. Andrew Warren (McGuire

Center for Lepidoptera and Biodiversity) for preparation and initial examination of genitalia;

Howard Grisham for access to specimens in his collection; Jeff R. Slotten for kindly sharing

information, specimens, and leg samples that were crucial for the studies of sympatric A. hegon

group species; and Craig Marks for additional paratype specimens. Thanks also to those who

provided specimens and access to their collections, including Jason Weintraub (ANSP: The

Academy of Natural Sciences of Drexel University, Philadelphia, PA, USA), Jim Fetzner, Bob

Androw, Vanessa Verdecia, Cat Giles, and the late John Rawlins (CMNH: Carnegie Museum of

Natural History, Pittsburgh, PA, USA), Richard L. Brown, Drew Hildebrandt, and personnel at the

Mississippi Entomological Museum (MEM: Mississippi Entomological Museum, Starkville, MS,

USA), Théo Léger, Wolfram Mey, and Viola Richter (MFNB: Museum fiir Naturkunde, Berlin,

Germany), Lawrence F. Gall (PMNH: Peabody Museum of Natural History, Yale University, New

Haven, CT, USA), Peter Michalik and Lara Lopardo (ZIMG: Zoological Institute and Museum

Greifswald, Germany), Axel Hausmann, Andreas Segerer, and UIf Buchsbaum (ZSMC:

Zoologische Staatssammlung Miinchen, Germany), and Maria Plonczynski. We acknowledge

Qian Cong, Jing Zhang, Jinhui Shen, Leina Song, Ping Chen, and Ming Tang for their help with

DNA studies, computer scripts, and excellent technical assistance without which the genomics part

of this project would have been impossible. Thanks also go to the Louisiana Department of

Wildlife and Fisheries for permits to collect on Wildlife Management Areas (permits #WL-

Research-2022-13, WDP-22-062, and WDP-23-004), to the Arkansas Department of Parks,

Heritage, and Tourism (Division of State Parks, permit # 098-2022 and 098-2023), and Texas

Parks and Wildlife Department (Natural Resources Program Director David H. Riskind, research

permit 08-02Rev) for permits to carry out research projects and collect specimens on State Park

lands. We acknowledge the Texas Advanced Computing Center (TACC) at The University of

Texas at Austin for providing HPC resources. This study was supported in part by the HHMI

Investigator funds and by grants from the National Institutes of Health GM127390 and the Welch

Foundation I-1505 (all to NVG).

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ADDITIONAL INFORMATION SOURCES

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