Volume 10 Number 4 1 May 2022

The Taxonomic Report

OF THE INTERNATIONAL LEPIDOPTERA SURVEY

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

Genomic DNA sequencing reveals two new North American

species of Staphylus (Hesperiidae: Pyrginae: Carcharodini)

Jing Zhang'’, Qian Cong'”, Jinhui Shen’, Leina Song’, and Nick V. Grishin?**

'Eugene McDermott Center For Human Growth & Development, Departments of “Biophysics and *Biochemistry, and

“Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX

75390-9050, USA; “Corresponding author: grishin@chop.swmed.edu

ABSTRACT. Two new skipper butterfly (Hesperiidae) species are described from the United States: Staphylus

floridus Grishin, sp. n. (type locality in Florida, Volusia Co.) and Staphylus ecos Grishin, sp. n. (type locality in Texas,

Brewster Co.). They are cryptic and hence escaped recognition. They differ from their sister species by the relative size and

morphology of genitalia and by genotype—including and beyond the COI barcode—thus suggesting genetic isolation that

argues for their species-level status. A lectotype is designated for Helias ascalaphus Staudinger, 1876. Staphylus opites

(Godman & Salvin, 1896), stat. rest. is a species-level taxon and not a synonym of Staphylus vincula (Plotz, 1886), while

Pholisora iguala Williams & Bell, 1940, syn. n. is a junior subjective synonym of S. vincula.

Additional key words: biodiversity, cryptic species, suture zones, genomics, phylogeny.

ZooBank registration: http://zoobank.org/CCFO18F4-01 88-45 1 A-BE75-006F64938595

INTRODUCTION

New methods bring new insights and reveal aspects not previously apparent. For centuries,

zoology relied on phenotype-based research and classification. New species were traditionally discovered

via phenotypic differences. Phenotypes are encoded by genotypes, and direct investigation of genotypes

by the analysis of DNA sequences may be a more direct and powerful approach to classifying nature.

Even short segments of DNA, such as COI barcodes, have been insightful in revealing species diversity

not readily evident from phenotypes (Hebert et al. 2004).

Genome-scale phylogenetic approaches show promise in revolutionizing our understanding of

butterfly evolution and refining their taxonomy (Toussaint et al. 2018; Allio et al. 2019; Li et al. 2019;

Zhang et al. 2019a; Zhang et al. 2019b; Toussaint et al. 2021; Robbins et al. 2022; Toussaint et al. 2022).

Accurate phylogenetic trees constructed from millions of base pairs give better confidence in the results

and frequently reveal inconsistencies with the current classification (Cong et al. 2019b; Zhang et al. 2021;

Toussaint et al. 2022; Zhang et al. 2022). While this approach is instrumental for higher classification and

taxonomy above species level, its application to species discovery and description has been limited

(Janzen et al. 2017; Cong et al. 2020). Even for COI barcodes, a major obstacle is the DNA analysis of

extant primary type specimens which is required to properly place discovered new species in the context

of previously named taxa (Pfeiler 2018).

Next-generation sequencing technology allows us to obtain whole genome shotgun sequences of

some of the oldest type specimens, thus resolving this impediment (Cong et al. 2021). Sequencing of

primary types is essential to confidently address nomenclatural questions. Imperative for cryptic species

complexes, it could also be helpful in seemingly less difficult cases. Oftentimes type localities were

incompletely documented or type specimens may have been mislabeled. Genomic comparison across a

Species’ range places type specimens among them and clarifies type localities (Cong et al. 2021).

Sequencing of primary types on a large scale (Zhang et al. 2022) offers unprecedented opportunities for

species-level taxonomy, both in correcting previous mistakes and new species discovery.

Our research group carries out large-scale butterfly genomic sequencing across taxonomic groups

and continents. As a result, specimens currently assigned to one species sometimes partition into distinct

and strongly supported clades in phylogenetic trees. We construct three maximum likelihood trees from

all protein coding genes encoded by: nuclear genome partitioned into (1) autosomes and (2) the Z

chromosome, and (2) mitochondrial genome. The Z chromosome holds a special place in the evolution of

Lepidoptera (Sahara et al. 2012; Mongue and Walters 2018; Cong et al. 2019a; Mongue et al. 2022) and

its phylogeny frequently agrees best with how species boundaries are defined based on previous work.

Well-supported clades observed in all three trees within "species" prompts a more detailed investigation.

Here, we analyze two such cases dealing with North American species of Staphylus Godman &

Salvin, 1896 (type species Helias ascalaphus Staudinger, 1876). Genomic trees that include primary type

specimens of the relevant names reveal a split into two clades in each of the two current species of

Staphylus. Following up on these results with morphological analyses, two new species are proposed.

MATERIALS AND METHODS

The specimens were inspected and photographed/sampled for DNA in the following collections:

American Museum of Natural History, New York, NY, USA (AMNH), Natural History Museum,

London, UK (BMNH), Carnegie Museum of Natural History, Pittsburgh, PA, USA (CMNH), Museum

fir Naturkunde, Berlin, Germany (MFNB), Texas A&M University Insect Collection, College Station,

TX, USA (TAMU), National Museum of Natural History, Smithsonian Institution, Washington, DC, USA

(USNM), Zoologische Staatssammlung Miinchen, Germany (ZSMC), and the personal collection of John

V. Calhoun, Palm Harbor, FL, USA. Historical documents, such as unpublished drawings, were inspected

in the Natural History Museum, London, UK and National Museum of Natural History, Smithsonian

Institution, Washington, DC, USA. Photographs of specimens and illustrations were taken with Nikon

D800 camera through 105 mm macro lens; dissected genitalia were photographed in glycerol with the

Nikon D200 camera without a lens and through a microscope at 4.5x magnification. Images were

assembled and edited in Photoshop CS5. Genitalia photographs were taken in several focus slices and

stacked in Photoshop to increase depth of field. Genomic DNA was extracted from a leg, libraries were

prepared, and sequenced as previously reported (Li et al. 2019). The details of phylogenetic tree

construction were given in Zhang et al. (2022). In brief, the maximum-likelihood tree was constructed

from protein-coding regions in the Z chromosome using IQ-tree v1.6.12 under GIR+GAMMA model

(Nguyen et al. 2015). To estimate the confidence of each node, we generated 100 replicates of 10,000

codons randomly sampled from the total set of codons in the Z chromosome and constructed maximum-

likelihood trees for each replicate. The support values of each node were summarized from these replicate

trees using sumtrees routine in dendropy package (Sukumaran and Holder 2010). The method to find

diagnostic DNA characters is described in Cong et al. (Cong et al. 2019b). Sequence datasets obtained in

this work are deposited in the NCBI database <https://www.ncbi.nlm.nih.gov/> as BioProject

PRJNA831579 (BioSample entries of the project contain the locality and collection data of the sequenced

Specimens shown in the trees), and COI barcodes have GenBank accessions ON351018—ON351022.

Exon sequences with diagnostic characters highlighted are also available from <https://osf.io/tygxc/>.

DNA characters are given as abbreviations, e.g., aly728.44.1:G672C means position 672 in exon | of

gene 44 from scaffold 728 of Cecropterus lyciades (formerly in Achalarus Scudder, 1872, thus aly)

reference genome (Shen et al. 2017) is C, changed from G in the ancestor. Similar notations are used for

the COI barcode characters, e.g., A78G means position 78 is G, changed from A in the ancestor, or

T264T(not C) means position 264 is T, unchanged from the ancestor, and not C as in sister taxon.

RESULTS

All three phylogenetic trees—based on nuclear genome: (1) autosomes and (2) Z chromosome, and (3)

mitochondrial genome—consistently reveal a well-supported split into two clades in each of the two

North American species of Staphylus: S. hayhurstii (W. H. Edwards, 1870) and S. ceos (W. H. Edwards,

1882). These two names have no known synonyms (Pelham 2008; Pelham 2022), and therefore one of the

clades in each species is likely to represent a new taxon. A combination of genomic and morphological

analyses suggests that these unnamed taxa are new species, which are herein described. In the first case,

nuclear genome diverged stronger than the mitogenome. In the second case, the opposite occurred.

Furthermore, genomic comparison of old primary type specimens placed among more recently collected

Specimens suggests a number of taxonomic changes that are detailed below.

Staphylus floridus Grishin, new species

http://zoobank.org/B7CE2D54-7EOF-4FBD-96B6-4A 96BCE02C6D

Definition and diagnosis. Genomic

analysis of specimens identified as

Staphylus hayhurstii (W.H. Edwards,

1870) (type locality USA: Missouri,

possibly in Pettis Co.) from across

its range, including the lectotype

(NVG-15096HO5), reveals their split

into two prominent clades with

100% statistical support in all three

trees (Fig. 1 red and blue clades).

The red clade consists of specimens

from Florida, and the blue clade

includes the rest, where the lectotype

of S. hayhurstii belongs. Genetic

differentiation between the two

clades is more pronounced in the

nuclear genome than in_ the

mitogenome: Fst/Gmin computed on

the Z chromosome are 0.49/0.02,

suggesting that the two clades are

two distinct species (Cong et al.

2019a); but mitogenomes of the two

species diverged less, e.g., their COI

barcodes differ by 1.1% (7 bp), a

value by itself insufficient to

substantiate species-level distinction.

Comparison of male and female

genitalia offers additional support for

the species-level status of the new

taxon, and we conclude that the red

clade (Fig. 1) represents a new

species. This species (Fig. 2)

superficially similar to its sister S.

hayhurstii, and we were not able to

find wing pattern characters that

(Figs. 1 part, 2, 3a, b, e)

Staphylus floridus| 18058E06|PT|M|USA:FL,Alachua Co.|1987

Staphylus floridus|20049A08|PT|F|USA:FL,Sumter Co.|1993

Staphylus floridus|20049A05|PT|M|USA:FL,Lee Co.|1983

Staphylus floridus|20049A06|PT|M|USA:FL,Volusia Co.|1994

Staphylus floridus|20049A04|PT|M|USA:FL,Volusia Co.|1987

Staphylus floridus]20049A03|PT|M|USA:FL,Volusia Co.|1993

a_sionuciear genome:

autosomes

Staphylus hayhurstii]15096A03|PLT|M|USA:MO|1869

Staphylus hayhurstii|18058E07|M|USA:TX,Comal Co.|1967

Staphylus hayhurstii|18058E08|M|USA:IL,Mason Co.|1985

Staphylus hayhurstii|20048F11|M|USA:MD,Montgomery Co.|1999

Staphylus hayhurstii|20048F12|F|USA:MD,Montgomery Co.|1999

Staphylus hayhurstii]4383|F]USA:TX,Dallas Co.|2015

Staphylus hayhurstii|4577|M|USA:TX,Dallas Co.|2015

Staphylus pag LE Ee TX, Delta Co.|2016

StpRente mararel 18058E10|M|Mexico: Veracruz|1979

Staphylus mazans|10068|M|USA:TX,Cameron Co.|2017

Staphylus mazans|18058E09|M|USA:TX,Comal Co.|1967

Staphylus ascalaphus|18011HO05|Panama|1983

0.004 Staphylus ascalaphus|18013G06|Costa Rica|2008

b nuclear genome:

Z chromosome

Staphylus floridus|20049A05|PT|M|USA:FL,Lee Co.|1983

Staphylus floridus|18058E06|PT|M|USA:FL,Alachua Co.|1987

Staphylus floridus|20049A06|PT|M|USA:FL,Volusia Co.|1994

Staphylus floridus|20049A04|PT|M|USA:FL,Volusia Co.|1987

Staphylus floridus|20049A08|PT|F|USA:FL,Sumter Co.|1993

“ Staphylus floridus|20049A03|PT|M|USA:FL, Volusia Co.|1993

Staphylus hayhurstii|15096A03|PLT|M|USA:MO|1869

}15096! |USA:M

Staphylus hayhurstii|20048F12|F|USA:MD,Montgomery Co.|1999

Staphylus hayhurstii|20048F11|M|USA:MD,Montgomery Co.|1999

, Staphylus hayhurstii|18058E07|M|USA:TX,Comal Co.|1967

Staphylus hayhurstii|7039|F|USA:TX,Delta Co.|2016

Staphylus hayhurstii|4383|F|USA:TX, Dallas Co.|2015

“ Staphylus hayhurstii|4577|M|USA:TX,Dallas Co.|2015

Staphylus hayhurstii|18058E08|M|USA: IL,Mason Co.|1985

Staphylus mazans|18058E10|M|Mexico:Veracruz| 1979

t Staphylus mazans|10068|M|USA:TX,Cameron Co.|2017

Staphylus mazans|18058E09|M|USA:TX,Comal Co.|1967

-Staphylus ascalaphus|15033F12|LT|M|Panama

oan pean mt 1 Staphylus ascalaphus|18011H0O5|Panama|1983

0.003 Staphylus ascalaphus|18013G06|Costa Rica|2008

Staphylus floridus|20049A05|PT|M|USA:FL,Lee Co.|1983

oStaphylus floridus|20049A06|PT|M|USA:FL,Volusia Co.|1994

aphylus floridus]20049A08|PT|F|USA:FL,Sumter Co. |1993

taphylus floridus| 18058E06|PT|M|USA:FL,Alachua Co.|1987

taphylus floridus|20049A03|PT|M|USA:FL,Volusia Co.|1993

aphylus floridus|20049A04|PT|M|USA:FL,Volusia Co. |1987

C_ mitochondrial genome

Staphylus hayhurstii|15096A03|PLT|M|USA:MO|1869

sgotaphylus hayhurstii|18058E07|M|USA:TX,Comal Co.|1967

Staphylus hayhurstii|7039|F|USA:TX, Delta Co.|2016

sgitaphylus hayhurstii|4383|F|USA:TX, Dallas Co.[2015

taphylus ea Tes crea NES TX, Dallas Co. RAR

lus ha stii| 15096 S|LTIMIL SA:MO,Pettis Co.|1869

Geprvice hayhurstii|18058E08|M|USA: Iv; Meron Co. /1985

taphylus hayhurstii|20048F11|M|USA:MD,Montgomery Co.|1999

taphylus hayhurstii|20048F12|F|USA:MD,Montgomery Co.|1999

Staphylus mazans|18058E09|M|USA:TX,Comal Co.|1967

Staphylus mazans|10068|M|USA:TX,Cameron Co.|2017

Staphylus mazans|18058E10|M|Mexico:Veracruz|1979

| 62 Staphylus ascalaphus|15033F12|LT|M|Panama —

0.007 5 Staphylus ascalaphus|18013G06|Costa Rica|2008

Staphylus ascalaphus|18011H05|Panama|1983

Fig. 1. Phylogenetic analysis of Staphylus floridus sp. n. and S. hayhurstii.

The trees are constructed from protein-coding regions in: a. autosomes, b. Z

chromosome, ¢. mitochondrial genome. Different species are shown in

different colors. Names of primary type specimens are highlighted in yellow.

Se ee ee a ee acm

Fig. 2. Staphylus floridus sp. n. holotype, male, NVG-18058E05 (a, b), USA: Florida, Volusia Co. and a female paratype

NVG-20049A08 (c, d), USA: Florida, Sumter Co., in dorsal (a, c) and ventral (b, d) views. Other data are in text.

b Fd ale,

Fig. 3. Genitalia of Staphylus floridus sp. n. paratypes (a, b. NVG-20049A03 and e. NVG-20049A07, data in text) and S.

hayhurstii from USA: Texas (e, d. NVG-12597, Dallas Co. and f. NVG-7042, Delta Co.), males (a—d) and females (e, f), in

left lateral (a, c), dorsal (b, d), and ventral (e, f) views. Whole genitalic capsule is shown for males and sterigma for females.

distinguish them. This is hardly surprising for Staphylus, a genus that includes dozens of species nearly

impossible to identify by facies even for more distant relatives, such as Staphylus mazans (Reakirt,

[1867]) (type locality in Mexico: Veracruz) (Fig. | green), which is sympatric with S. hayhurstii in central

Texas (e.g., in Comal Co., Fig. 1).

The new species differs from S. hayhurstii by less robust and relatively smaller genitalia, in both

males (Fig. 3a—d) and females (Fig. 3e, f): illustrated genitalia are of specimens with similar forewing

length. In male genitalia, tegumen longer and narrower, vs. more compact but broader tegumen in S.

hayhurstii; harpe narrower (in lateral view), straighter, and bent less from the body of valva at its junction

(best seen in dorsal view). In female genitalia, lamella postvaginalis less sculptured, typically with fewer

and shorter ridges; lamella antevaginalis with comparatively smaller tooth on each side and more

expanded middle section protruding caudad, with a central notch, vs. nearly crescent-shaped (tooth to

tooth) lamella with irregular middle margin in S. hayhurstii. These genitalic characters are rather subtle,

and due to individual variation the most confident identification can be achieved using DNA sequences.

Combinations of the following DNA characters are diagnostic: in COI barcode: C271T, T322A, C401T,

and T613C, and in the nuclear genome: aly1849.21.2:G3621A, aly1849.21.2:G3078A, aly1113.5.4:

T1282A, aly1849.21.2:A702G, aly1656.10.4:T414G. Each of the barcode characters taken individually

distinguishes the new species from S. hayhurstii in a sample of specimens we sequenced.

Barcode sequence of the holotype: Sample NVG-18058E05, GenBank ON351018, 658 base pairs:

AACTTTATATTTTATTTTTGGTATTTGAT gaccn TAGTAGGAACTTCTTTAAGTATTCTTATTCGTTCTGAATTAGGAACACCTGGATCTTTAATTGGAGATGATCAAATTTATAATACT

ATTGTAACAGCTCATGCTTTTATTATAATTTTTTTTATAGTTATACCTATTATAAT TGGAGGATTCGGAAATTGACTTGTTCCTCTTATAT TAGGAGCCCCTGATATAGCTTTTCCTCGAA

TAAATAATATAAGATTTTGATTATTACCTCCATCTTTAATACTTTTAATTT CAAGAAGAATTGTAGAAAATGGAGCAGGAACTGGAT GAACAGTT TACCCCCCCCTTTCAGCCAATATTGC

TCATCAAGGTTCTTCTGTAGATTTAGCTATTTTTTCCTTACATTTAGCAGGTATTTCTTCTATTTTAGGAGCAATTAATTTTATTACAACTATTATTAATATACGAATTAATAATTTATCA

TTTGATCAAATACCTTTATTTGTTTGAGCTGTTGGAATTACAGCATTACTTTTACTTTTATCTTTACCAGTATTAGCAGGTGCTATTACTATACTTTTAACAGATCGAAATCTTAATACAT

CATTCTTCGATCCTGCTGGTGGAGGAGATCCTATTTTATATCAACATCTATTC

Type material. Holotype: @ (Fig. 2a, b), deposited in the National Museum of Natural History,

Smithsonian Institution, Washington, DC, USA (USNM), bears the following three rectangular white

G. W. Rawson leg. ], [| DNA sample ID: | NVG-18058E05 | c/o Nick V. Grishin |], [ USNMENT | {QR

Code! | 01466715 |] and one red printed [HOLOTYPE < | Staphylus | floridus Grishin ]. Paratypes:

544 and 299, all from USA: Florida: 1d’ NVG-18058E06, Alachua Co., Alachua, leg. Scott W. Gross,

29-Aug-1987; others leg. John V. Calhoun: 34’ Volusia Co.: NVG-20049A04, New Smyrna Beach,

2495 N Dixie Fwy, 5-Mar-1987; NVG-20049A03, W side of USI, 1 mi N of Brevard Co. line, 7-Mar-

1993; and NVG-20049A06, Reed Grove Rd. N of Stacey Grove Rd., SW of Oak Hill, 20-Mar-1994; 14

NVG-20049A05 & 12 NVG-20049A07, Lee Co., W of IH75, off Carrell Rd. nr. Bonita Springs, 25-Mar-

1983; 12 NVG-20049A08, Sumter Co., E side SR301, N side of Shady Brook, 14-May-1993 (coll. J. V.

Calhoun, Palm Harbor, FL, USA).

Type locality. USA: Florida, Volusia County, New Smyrna Beach.

Distribution. Currently known only from USA: Florida with specimens sequenced from Alachua,

Sumter, Volusia, and Lee Counties. This species may be allopatric with S. hayhurstii, and geographically

separated from it by the lack of records in northern Florida and nearby parts of other states. This apparent

gap in the distribution of Staphylus would be interesting to investigate further.

Etymology. Being a reference to Florida, the state from which the type series is known, a Latin word

translated as flowery or blooming was chosen as the name for this not so garish species from Florida that

nevertheless flaunts rather elaborate pattern of browns, nearly as flamboyant as gothic styles. The name is

a masculine adjective.

Suggested English name. Florida Sootywing.

Lectotype designation for Helias ascalaphus Staudinger, 1876

Helias ascalaphus Staudinger, 1876 (type locality in Panama), the type species of Staphylus, was

described from a series of specimens (Staudinger 1876), and several syntypes are in the MFNB collection.

We sequenced a male syntype already bearing a red "Lectotypus" label (designation unpublished), and the

results agreed with the current application of this name (Fig. 1). To stabilize nomenclature, N.V.G. hereby

designates this sequenced syntype, in the Museum fiir Naturkunde, Berlin, Germany bearing the

Stdgr. |, | GEN.PREP., | MIELKE | 1996 ], [ {QR Code} http://coll.mfn-berlin.de/u/ | 908619 |, and

| DNA sample ID: | NVG-15033F12 | c/o Nick V. Grishin | as the lectotype of Helias ascalaphus

pisUeeer: 1876. The COI barcode sequence of the lectotype (GenBank ON351019) is:

TAAGTATTCTTATTCGTTCTGAATTAGGAACTCCTGGATCTTTAATTGGAGATGAT

TTGGAGGTTTTGGAAAT Tene) Gea Cane ma ties eae Se [GATAT

TAGGAACTTCTT

AACTTTATATTTTATCTTTGGTATTTGATCT oa TAG

T AGTTAT

[GTAACAGCTCATGCTTTTATTATAATTTTTTTTAT

TAAATAATAT

AAGAT ™mm GAT TATT

m7 q ny

TACCCTIATTICTT TTGGAAT

TGGAGGAGATCCTATTTTAT

momm TAATAC Mmmm

nmamm TACAT m™

TAGCAGGTATTTCTTCTATTT

TACAGCAT

ATCAACATTTATTC

TAGAATCGTAGAAAAT TGAACAGTTTATCCCCCCCTTT 1

TAGGAGCAATTAATTTTATTACAACTATTATCAATATACGAATTAATAATTTATCC

TAGCAGGTGCTATTACTATACTTTTAACAGATCGAAATCTTAAT

TATTATTACTTTTATCTTTACCAGTAT

Staphylus ecos Grishin, new species

http://zoobank. org/33 FF40E5-1E83-4E3E-8A0F-C85A81A0521A

Definition and diagnosis. Genomic

analysis of specimens identified as

Staphylus ceos (W. H. Edwards,

1882) (type locality in USA:

Arizona, Graham Co.) from across

its range, including the lectotype

(NVG-15096H06), reveals their split

into two prominent clades with

100% _ statistical support in the

mitochondrial genome tree (Fig. 4c

red and blue clades), substantiated

by a shallow but consistent split in

both nuclear genome trees (Fig, 4a,

b). The red clade consists of

Specimens from the eastern part of

the range and the blue clade includes

the rest, where the lectotype of S.

ceos belongs. Genetic differentiation

between the two clades is limited in

the nuclear genome, but is substantial

in the mitogenome. Fst/Gmin

computed on the Z chromosome are

0.08/0.15, well within the range of

one species (Cong et al. 2019a).

Mitogenomes of the two groups of

Specimens (red and blue) diverged

strongly, e.g., their COI barcodes

differ by 2.7% (18 bp), a value

typically sufficient to support

species-level distinction (Hebert et

al. 2003), but only in the presence of

phenotypic differences. Comparison

of male genitalia reveals the

differences that are typical of closely

(Figs. 4 part, 5, 6a, b)

Staphylus ecos|19125H12|PT|F|USA:TX,Val Verde Co.|1949

Staphylus ecos|19125HO08|PT|M|USA:TX,Bexar Co.|1966

Staphylus ecos|18058B05|PT|F|USA:TX,Bexar Co. |old

Staphylus ecos|19126A07|PT|F|USA:TX,Jeff Davis Co.|1969

Staphylus ecos|19126A06|PT|F|USA:TX,Brewster Co.|1971

Staphylus ecos|19126A01|PT|M|Mexico:Coahuila|1977

Staphylus ecos|19125H09|PT|M|USA:TX,Brewster Co.|1971

Staphylus ecos|19125H11|PT|M|USA:TX, Jeff pe Co.|1969

a ° Staphylus ecos|11137|PT|M|USA:TX,Jeff Davis Co.|2018

Staphylus ceos|10898|M|USA:AZ,Pinal Co.|2018

ews SSUES AZ, Pima Co. PAY

a__sinucilear genome:

autosomes

0.046

Staphyils ceos|9694]F|USA: AZ, Santa Cruz Co. 12017

Staphylus ceos|10988|M|USA: TX,El Paso Co.|2018

Staphylus ceos| 12119|F]USA:AZ,Pinal Co.|2019

Staphylus ceos|10941|M|USA:TX,El Paso Co.|2018

Staphylus opites|18011H0O7|Mexico:Oaxaca|1991

Staphylus opites| 18011H08|Mexico:Oaxaca|1988

Staphylus vincula (=iguala)|18059A11|M|Mexico:Oaxaca|1989

ae Staphylus cartagoa|18071A08|F|Costa Rica|2005

Staphylus ecos|19125H08|PT|M|USA:TX,Bexar Co.|1966

Staphylus ecos|19125H0O9|PT|M|USA:TX,Brewster Co.|1971

b nuclear genome:

Z chromosome

a Staphylus ecos|18058B05|PT|F|USA:TX, Bexar Co. |old

*Staphylus ecos|11137|PT|M|USA:TX,Jeff Davis Co.|2018

Staphylus ecos|19125H11|PT|M|USA: TX,Jeff Davis Co.|1969

Staphylus ecos]19126A06|PT| F|USA: TX, Brewster Co.|1971

Staphylus ecos|19126A07|PT|F|USA:TX, Jeff Davis Co.|1969

Staphylus ecos|19125H12|PT|F|USA:TX,Val Verde Co.|1949

Staphylus ecos|19126A01|PT|M|Mexico: aa eed

3 Staphylus ceos|10898|M|USA:AZ,Pinal Co.|20

: Staphylus ceos|10988|M|USA:TX, El Paso Co. rae

Staphylus ceos|10941|M|USA: TX,El Paso Co.|2018

Staphylus SRA REA a ae AZ, Pinal Co. [2019

1° 9H I j af = a

° Staphylus ceos|9694|F|USA: AZ, earita Cre Co, |2017

Staphylus ceos|8257|M|USA: AZ, Pima Co.|2017

[Sa Staphylus opites|18011H07|Mexico :Oaxaca [1991

Staphylus opites|18011H08|Mexico:Oaxaca|1988

Staphylus vincula (=iguala)|18059A11|M|Mexico:Oaxaca|1989

Staphylus cartagoa|18071A08|F|Costa Rica|2005

0.008 staphylus cartagoa|15097C04|HT|M|C : Rica

gg staphylus ecos|18058B05|PT|F|USA:TX, Bexar Co. |old

t ’ Staphylus ecos|19125HO8|PT|M|USA:TX, Bexar Co.|1966

eas ecos|19126A01|PT|M|Mexico: Coahuila | 1977

taphylus ecos|11137|PT|M|USA:TX,Jeff Davis Co.|2018

1@taphylus ecos|19126A06|PT|F|USA:TX,Brewster Co.|1971

,Staphylus ecos|19125H11|PT|M|USA:TX, eff Davis Co.|1969

Staphylus ecos|19125H09|PT|M|USA:TX, Brewster Co.|1971

‘Staphylus ecos|19125H12|PT|F|USA:TX Nal Verde Co.|1949

Staphylus ecos|19126A07|PT|F|USA:TX,Jeff Davis Co.|1969

Staphylus ceos|10898|M|USA:AZ, Pinal Co.|2018

7Staphylus ceos|12119|F|USA:AZ Pinal Co.|2019

-7Staphylus ceos|9694|F|USA:AZ, Santa Cruz Co.|2017

taphylus ceos|10988|M|USA: TX, El Paso Co.|2018

Staphylus ceos|8257|M|USA:AZ, Pima Co.|2017

°Staphylus ceos|10941|M|USA: uES El Paso Co. ars

A , 0 118 82

C_ mitochondrial genome

Staphyl aham

taphylus opites|18011H07|Mexico: Caracal 1991

taphylus opites| 18011H0O8|Mexico:Oaxaca|1988

*’Staphylus vincula (=iguala)|18059A11|M|Mexico:Oaxaca|1989

taphylus cartagoa|18071A08|F|Costa Rica|2005

0.02

Fig. 4. Phylogenetic analysis of Staphylus ecos sp. n. and S. ceos. The trees

are constructed from protein-coding regions in: a. autosomes, b. Z

chromosome, ¢. mitochondrial genome. Different species are shown in

different colors. Names of primary type specimens are highlighted in yellow.

| ICM

Fig. 5. Staphylus ecos sp. n. holotype, male, NVG-21113E02 (a, b) and a female paratype NVG-21113E03 (ce, d), from USA:

Texas, Brewster Co., Big Bend National Park, Chisos Mountains, in dorsal (a, c) and ventral (b, d) views. Other data are in text.

related but distinct species, and

therefore we conclude that the red

clade (Fig. 4) represents a new

species. This species (Fig. 5) is

superficially similar to its sister S. Yue

ceos, and we were not able to find tL x

meaningful wing pattern characters ra

to differentiate them. This is typical

for Staphylus, a genus that includes

a number of species with similar

appearance, even for more distant

relatives.

The new species differs from = ¢

S. ceos by more robust and relatively

larger male genitalia (Fig. 6):

illustrated genitalia are of specimens

with similar forewing _ length.

; Fig. 6. Male genitalia of Staphylus ecos sp. n. (a, b), paratype NVG-11137,

Ampulla of valva with nearly Ug. Texas, Jeff Davis Co. and S. ceos (ec, d), NVG-10941, USA: Texas, El

straight distal margin (in lateral — paso Co. in left lateral (a, c) and dorsal (b, d) views, whole genitalic capsule.

view), not concave as in typical S.

ceos; harpe longer relatively to its height (in lateral view), less curved dorsad, with longer dorsal ridge

that projects farther anteriad (towards vinculum) connecting to valva at several points that look like

"teeth" in lateral view, and with thicker short spines at the distal end, vs. S. ceos, which has a shorter,

more square-shaped harpe that is terminally more curved dorsad, the dorsal ridge that connects to valva

closer to the ampulla and well before the anterior portion of valva along ventral margin, the "teeth"

formed by connecting points of the ridge with valva that appear smaller, and distal spines that are more

minute. These genitalic characters are rather subtle, and due to individual variation, the most confident

identification can be achieved using DNA sequences. Combinations of the following DNA characters are

diagnostic: in COI barcode: T67C, T205C, T337A, T478C, and T514C, and in the nuclear genome:

aly1113.5.4:A1684G, aly1849.21.2:A8067G, aly1849.21.2:A8055G, aly1113.5.4:A1338C. Each of the

barcode characters taken individually distinguishes the new species from S. ceos in a sample of specimens

we sequenced.

Barcode sequence of a paratype: Sample NVG-19126A06, GenBank ON351020, 658 base pairs:

AACTTTATATTTTATTTTTGGTATTTGATCT oon TAGTAGGAACTTCTTTAAGTATTCTTATTCGCTCAGAATTAGGAACCCCAGGATCTTTAATTGGAGATGATCAAATTTATAATACT

ATTGTAACAGCTCATGCTTTTATTATAATTTTTTTTATAGTAATACCTATTATAATCGGAGGTTTTGGAAATTGATTAGTACCCCTAATATTAGGAGCCCCAGATATAGCTTTCCCTCGAA

TAAATAATATAAGTTTCTGATTATTACCCCCCTCTCTTATACTTTTAATTTCAAGTAGTATTGTAGAAAATGGAGCAGGTACAGGATGAACTGTATACCCCCCTCTTTCAGCTAATATTGC

TCATCAAGGATCATCTGTAGATTTAGCTATTTTTTCCCTTCATTTAGCCGGAATTTCTTCAATTTTAGGGGCAATTAATTTTATTACAACTATTATTAATATACGAATTAATAACTTATCA

TTTGATCAAATACCTTTATTTGTTTGAGCCGTAGGTATTACAGCATTACTTTTACTTTTATCTCTCCCAGTATTAGCTGGTGCTATTACTATACTTTTAACAGATCGAAATCTTAATACAT

CATTTTTTGACCCTGCAGGAGGTGGAGATCCTATTTTATACCAACATTTATTT

Type material. Holotype: @ (Fig. 5a, b), deposited in the National Museum of Natural History,

Smithsonian Institution, Washington, DC, USA (USNM), bears the following two rectangular white

printed labels: [ Green Gulch | Big Bend Nat. Park | Brewster Co. TX | 12-Jun-2004 USA | leg. Grishin N.

V. ], [| DNA sample ID: | NVG-21113E02 | c/o Nick V. Grishin ], and one red printed [ HOLOTYPE ¢ |

Staphylus | ecos Grishin ]. Paratypes: 84:4 and 59 2, most collected by Roy O. Kendall & C. A. Kendall

(ROK) and Nick V. Grishin (NVG), from USA: Texas: 14 NVG-19125H10, Comal Co., New Braunfels,

Panther Canyon, leg. W. W. McGuire, 6-May-1972; Bexar Co.: 14 NVG-19125H08, USH281 north at

Salado Creek, ROK, 4-Jun-1966; 12 NVG-18058B05, O. C. Poling, no date, estimated around 1900; 19°

NVG-19125H12, Val Verde Co., Del Rio, leg. Hugh Avery Freeman, 12-Jul-1949; Brewster Co., Big

Bend National Park: 1d NVG-19125H09, 22-Sep-1971 & 12 NVG-19126A06, 14-Sep-1971, K-Bar

Research Station, ROK; 12 NVG-21113E03, Chisos Basin Campground, NVG, 29-May-2004; Jeff Davis

o.: Io NVG-19125HI11, 10-Jul-1969 & 192 NVG-19126A07, 7-Jul-1969, Musquiz Canyon, SH118,

ROK; 14 NVG-11137, Jeff Davis Co., Fort Davis city limits, NVG, 18-May-2018; 2¢¢ NVG-21113E04

& 5, Jeff Davis Co., Davis Mts. State Park, nr. Indian Lodge, NVG, 25-Mar-2005; and 14 NVG-

19126A01, Mexico: Coahuila, Hwy 57 ca. 18 mi S of Monclova, ROK, 14-Sep-1977.

Type locality. USA: Texas, Brewster County, Big Bend National Park, Green Gulch, elevation 1960 m,

GPS 29.2769, —103.2836.

Distribution. Confirmed from USA: Texas, in the following counties: Jeff Davis, Brewster, Val Verde,

Bexar, and Comal; and from Mexico: Coahuila, ca. 18 mi S of Monclova. Specimens we sequenced from

El Paso County Texas were S. ceos (Fig. 4). The two sister species are currently allopatric, and the

boundary between them in the USA lies somewhere between the Davis Mountains and the Franklin

Mountains in Texas with no known intervening populations. Further studies of these species may be

productive in Chihuahua, Mexico, where they may closely approach one another.

Etymology. The species that ec[h|o[e]s ceos with distortion and the name is its anagram. For a simple

mnemonic, think of "e" for "east" for this more eastern species. The name is a noun in apposition.

Suggested English name. Texas Sootywing.

Staphylus opites (Godman & Salvin, 1896), reinstated status

Scantilla opites Godman & Salvin, 1896 (type locality in Guatemala) was placed as a junior subjective

synonym of Staphylus vincula (Plotz, 1886) (type locality in Panama) by Evans (1953). Genomic

sequencing of Tagiades vincula Plétz, 1886 syntype in ZSMC (NVG-18056G01, Fig. 7a—c) reveals that it

is in a different clade from the two specimens of S. opites from Mexico: Oaxaca (Fig. 4). This syntype, a

female (Fig. 7a—c), agrees with the original description of 7. vincula. It is close in appearance to the

Specimen from Costa Rica in BMNH that Godman considered to be similar to the original Pl6tz

illustration (Godman 1907), and is curated as a type in ZSMC. Therefore, we accept it as a true syntype,

and due to significant genetic differentiation between the two taxa, reinstate species-level status for

Staphylus opites (Godman & Salvin, 1896), stat. rest. The COI barcode sequence of the 7. vincula

syntype (GenBank ON351021) is:

AACTTTATATTTTATTTTTGGTATTTGATCCGGTATAGTAGGAACTTCTTTAAGTATTCTTATTCGTTCAGAACTAGGAACTCCTGGATCTTTAATTGGAGATGATCAAATTTACAATACT

ATTGTAACAGCTCATGCTTTTATTATAATTTTTTTTATAGTTATACCTATTATAAT TGGAGGATTTGGAAATTGATTAGTACCTTTAATAT TAGGAGCTCCTGATATAGCTTTCCCTCGAA

TAAATAATATAAGTTTTTGATTATTACCCCCTTCTCTTATACTTTTAATTTCAAGTAGTATTGTAGAAAATGGAGCAGGTACAGGAT GAACTGTT TACCCACCTCTTTCAGCCAATATTGC

TCATCAAGGAGCATCTGTAGATTTAGCTATTTTTTCCCTTCATTTAGCAGGAATTTCTTCAATT TTAGGAGCAATTAATTTTATTACAACTATTATTAATATACGAATTAATAATTTATCA

TTTGATCAAAT Wane Phage hae ee Tatas arte ee Guam oe We ee ee gee aes ge ee [GCTATTACTATACTTTTAACTGATCGAAATCTTAATACAT

CATTTTTTGATCCAGCAGGAGGAGGAGATCCAATTTTATATCAACATTTATT

Additionally, we found an illustration of 7. vincula (Fig. 7d) pinned in a Staphylus drawer in

USNM. This color illustration depicts the left dorsal surface and is rather similar to a Costa Rican

specimen from the Godman & Salvin collection in BMNH, which lacks the abdomen and is spread in a

similar manner. Furthermore, the illustration shows three subapical hyaline spots, as the BMNH

specimen, but the syntype has two, as stated in the original description. Hence, this illustration is possibly

of the BMNH specimen, rather than a copy of the original Pl6étz's drawing.

1 ZSM eo

Genitalprp.

_| | Samettea | Noh PIS

} — DNAsamole ID:

relate * - NVG-18056G01

Tag fades vineula , Pletz. - c/o Nick V. Grishin

iS9o0. Panama. d —_ a

Fig. 7. Staphylus vincula syntype, female, NVG-18056G01 in ZSMC: a. dorsal and b. ventral views, c. labels (lectotype

designation was not published and the specimen remains a syntype); d. an illustration pinned in a Staphylus drawer in USNM,

possibly of a specimen from Costa Rica in BMNH, which Godman (1907) considered to be similar to the original illustration

of Tagiades vincula by Plotz. Larger scale bar refers to the specimen and illustration and smaller scale bar refers to labels

(reduced compared to the specimen).

Pholisora iguala Williams & Bell, 1940 is a junior subjective synonym

of Staphylus vincula (Plotz, 1886)

As we have shown above, Staphylus opites (Godman & Salvin, 1896), stat. rest. (type locality in

Guatemala) and Staphylus vincula (Pl6tz, 1886) (type locality in Panama) are distinct, non-sister species

(Fig. 4). While S. vincula is sister to Staphylus cartagoa (Williams & Bell, 1940) (type locality in Costa

Rica), S. opites is sister to the clade consisting of Staphylus ceos (W. H. Edwards, 1882) (type locality in

USA: Arizona, Graham Co.) and Staphylus ecos sp. n. (type locality in USA: Texas, Brewster Co.).

Genomic sequencing of the holotype of Pholisora iguala Williams & Bell, 1940 (type locality in Mexico:

Guerrero) in the AMNH (NVG-18024G11), together with a more recently collected specimen identified

as S. iguala (NVG-18059A11), reveals that they are genetically close to the syntype of S. vincula, in both

nuclear and mitochondrial genomes. Moreover, their COI barcodes differ by only 0.3% (2 bp). Therefore,

we propose that Pholisora iguala Williams & Bell, 1940 syn. n. is a junior subjective synonym of

Staphylus vincula (Pl6tz, 1886). As a result of this change, current synonyms of P. iguala become junior

subjective synonyms of S. vincula. This genetic similarity with Mexican specimens casts certain doubt on

the Panamanian origin of the only known S. vincula syntype, and this question should be investigated

further by sequencing of additional specimens. Furthermore, the syntype bears two conflicting locality

labels: "Panama" and "S[outh] America" (Fig. 7d), but the original description gives Panama as the

locality, where it was supposedly collected by Ribbe. Ribbe is not mentioned on the labels of the syntype.

Genomic sequencing suggests that its South American (and maybe even Panamanian) origin is unlikely,

and therefore the specimen was possibly mislabeled. For these reasons, we refrain from designating it a

lectotype (it already bears a lectotype label, but this designation remains unpublished) waiting for further

research. However, this is the only credible syntype we located thus far, and we are using it to define the

identity of S. vincula. The COI barcode sequence of the P. iguala holotype (GenBank ON351022) is:

AACTTTATATTTTATTTTTGGTATTTGAT CCGGTATAG! TAGGAACTTCTTTAAGTATTCTTATTCGTTCAGAACTAGGAACTCCTGGATCTTTAATTGGAGATGATCAAATTTACAATACT

ATTGTAACAGCTCATGCTTTTATTATAATTTTTTTTATAGTTATACCTATTATAATTGGAGGATTTGGAAATTGATTAGTACCTTTAATATTAGGAGCTCCTGATATAGCTTTCCCTCGAA

TAAATAATATAAGTTTTTGATTATTACCTCCTTCTCTTATACTTTTAATTTCAAGTAGTATTGTAGAAAATGGAGCAGGTACAGGATGAACTGTTTACCCACCCCTTTCAGCCAATATTGC

TCATCAAGGAGCATCTGTAGATTTAGCTATTTTTTCCCTTCATTTAGCAGGAATTTCTTCAATTTTAGGAGCAATTAATTTTATTACAACTATTATTAATATACGAATTAATAATTTATCA

TTTGATCAAATACCTTTATTCGTTTGAGCTGTAGGAATTACAGCTTTACTTTTACTTTTATCTTTACCAGTATTAGCAGGTGCTATTACTATACTTTTAACTGATCGAAATCTTAATACAT

CATTTTTTGATCCAGCAGGAGGAGGAGATCCAATTTTATATCAACATTTATTT

DISCUSSION

Screening of butterfly populations across their distribution ranges by genomic sequencing to probe

genetic diversification is productive in revealing new taxa. Addition of primary type specimens to these

datasets puts this genetic approach on a solid nomenclatural footing. Traditionally done using only COI

barcodes, such screens have been insightful (Hebert et al. 2004; Burns and Janzen 2005; Burns et al.

2007; Burns et al. 2008), but total genomic coverage alleviates the problems with introgression and other

irregularities of COI barcodes. Here, we applied the genomic approach that led to the discovery of two

new, partly cryptic butterfly species in the United States. Genetic differences are reflected in genitalic

differences, although these are rather subtle and therefore difficult to evaluate without genetic

differentiation.

Geographically, the two allopatric species in each Staphylus pair are separated by well-

documented suture zones (Remington 1968; Rising 1983), which form known boundaries for other

butterfly taxa. For instance, the North Florida suture zone separates Erynnis somnus (Lintner, 1881) from

Erynnis brizo (Boisduval & Le Conte, [1837]) (Burns 2020), as well as Staphylus floridus sp. n. from S.

hayhurstii, among many subspecies-level taxa (Warren et al. 2016). The Central New Mexico suture zone

separates a number of species pairs, such as Megathymus violae D. Stallings & Turner, 1956 from

Megathymus ursus Poling, 1902 (Zhang et al. 2020), Poladryas minuta (W. H. Edwards, 1861) from

Poladryas arachne (W. H. Edwards, 1869), Microtia elada (Hewitson, 1868) from Microtia perse (W. H.

Edwards, 1882), and Junonia coenia Hiibner, [1822] from Junonia grisea Austin & J. Emmel, 1998

(Lalonde and Marcus 2019), among others, together with Staphylus ecos sp. n. from S. ceos. Notably, in

this case, the exact geographic position of the boundary between the two species in every pair varies

somewhat: it could be to the west (as for Megathymus) or to the east (as for Staphylus) of El Paso.

Textbooks can be written about species delimitation and its practice. For practical applications, we

reason that genetic differentiation is by itself sufficient to substantiate species-level status of a taxon

under certain conditions. This differentiation should be: 1) strongly supported by statistics, e.g., bootstrap-

like values near 100%; 2) consistent throughout the genomes, both nuclear and mitochondrial, e.g., Fig. 1

red and blue clades; and 3) of a magnitude documented for well-studied species (Cong et al. 2019a). This

is even more meaningful when coupled with differences in genitalia, although these differences would

necessarily be subtle for closely related species. The second case (Fig. 4 red and blue clades) provides

more food for thought. Although mitochondrial genome differentiation is prominent (Fig. 4c), and agrees

with that of closely related but distinct species (2.7% COI barcode difference), nuclear genomes did not

diverge much, and this divergence does not pass our conservative criteria for speciation. However,

nuclear genomic differences are consistent between autosomes and the Z chromosome (Fig. 4a, b) and

therefore are a reflection of distinct evolutionary paths of these clades. Due to the small divergence, we

considered proposing S. ecos sp. n. as a subspecies of S. ceos. However, this opinion changed after

genitalic comparison. The differences in male genitalia are consistent in magnitude with those used to

support new species even in the absence of DNA evidence. Further studies of S. ceos species complex in

Mexico are likely to enrich our understanding of speciation.

ACKNOWLEDGMENTS

We acknowledge Ping Chen and Ming Tang for excellent technical assistance. We are grateful to David

Grimaldi and Courtney Richenbacher (AMNH: American Museum of Natural History, New York, NY,

USA), Blanca Huertas, David Lees, and Geoff Martin (BMNH: Natural History Museum, London, UK),

Jim Fetzner, Bob Androw, Vanessa Verdecia, Cat Giles, and the late John Rawlins (CMNH: Carnegie

Museum of Natural History, Pittsburgh, PA, USA), Wolfram Mey, Viola Richter, and Theo Leger

(MFNB: Museum fiir Naturkunde, Berlin, Germany), Edward G. Riley, Karen Wright, and John Oswald

(TAMU: Texas A&M University Insect Collection, College Station, TX, USA), Robert K. Robbins, John

M. Burns, and Brian Harris (USNM: National Museum of Natural History, Smithsonian Institution,

Washington, DC, USA), and Axel Hausmann and Ulf Buchsbaum (ZSMC: Zoologische Staatssammlung

Miinchen, Germany) for granting access to the collections under their care and for stimulating

discussions; to Bernard Hermier for exchange of opinions, to John V. Calhoun for the loan of specimens,

suggestions, and critical review of the manuscript. We are indebted to Texas Parks and Wildlife

Department (Natural Resources Program Director David H. Riskind) for the research permit 08-02Rev, to

U. S. National Park Service for the research permits: Big Bend (Raymond Skiles) for BIBE-2004-SCI-

0011. We acknowledge the Texas Advanced Computing Center (TACC) at The University of Texas at

Austin for providing HPC resources. The study has been supported in part by grants from the National

Institutes of Health GM127390 and the Welch Foundation I-1505.

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