A new stonefly species (Plecoptera, Perlidae) from the Interior Highlands USA, with morphological and molecular comparison to other congeneric species

Abstract Thirty-one species of Nearctic Perlesta Banks, 1906 (Plecoptera: Perlidae) are recognized. A new species is described from western Arkansas and eastern Oklahoma, USA, Perlestasublobata South & DeWalt, sp. nov., from the adult male, adult female, and egg. Perlestasublobata males are differentiated from other congeners by a combination of a prominent ventral caecum and a distinct dorsal extension of the lateral sclerites of the aedeagus. A preliminary molecular phylogenetic hypothesis is proposed for Perlesta based on 17 congeners and three outgroup taxa using partial mitochondrial cytochrome c oxidase subunit I sequence data. Illustrations, stereomicroscope images, and scanning electron micrographs support the description and comparison to other Perlesta.


Introduction
described Perlesta Banks, 1906 as a Nearctic genus of small, brown, triocellate stoneflies with yellow costal wing margins, long cerci, and highly variable coloration of the head and wing membrane. For over a century, the name of the type species of the genus, Perlesta placida (Hagen, 1861), has been used for innumerable specimens that once critically reviewed, were revealed to encompass many cryptic species (Stark 1989, DeWalt et al. 2001. Stark (1989) revised the genus, removing several species from synonymy, describing seven new species, recognizing a total of 12 species, and providing the first useful key to the P. placida complex. Stark's revision prompted additional work, with eight new species described over the next 14 years (Poulton and Stewart 1991, Kirchner and Kondratieff 1997, Stark and Rhodes 1997, DeWalt et al. 1998, Kondratieff and Baumann 1999, Kondratieff and Kirchner 2002. Subsequently, a revised taxonomic key was necessary, in which was included 21 Nearctic species (Stark 2004). The genus has since expanded to 31 Nearctic species through the works of Kondratieff et al. (2006Kondratieff et al. ( , 2008, Kondratieff and Myers (2011), and Grubbs and DeWalt (2011. Two species are recognized from China (Murányi and Li 2016) and undetermined nymphs have been reported from Costa Rica (Gutiérrez-Fonseca and Springer 2011). Described species could easily surpass 40, given the amount of presumed new, undescribed Perlesta material currently present in North American collections (Grubbs and DeWalt 2018).
At least eight species of Perlesta co-occur in the United States Interior Highlands, a mountainous region defined by the United States Geological Survey as encompassing southern Missouri, western Arkansas, eastern Oklahoma, and extreme southeastern Kansas (Omernik 1987). This area of the central United States was extensively examined for stoneflies by Poulton and Stewart (1991). Surprisingly, a remarkably distinct and undescribed Perlesta species from the Interior Highlands was revealed through recent examination of undetermined Arkansas material donated to the Illinois Natural History Survey (INHS) Insect Collection by the late Kenneth W. Stewart ) and from eastern Oklahoma material borrowed from the K. C. Emerson Entomological Museum, Oklahoma State University (OKSU) at Stillwater. Using freshly collected and properly prepared specimens, we describe this new species, Perlesta sublobata sp. nov., and compare it to similar regional congeners. Moreover, we provide the first comparative molecular study of the genus by exploring partial mitochondrial cytochrome c oxidase subunit I (COI) DNA sequence data to examine monophyly of the new species, delimit congeners, and construct a preliminary phylogeny.
The holotype male and all paratypes are deposited in the Illinois Natural History Survey (INHS) Insect Collection. Other material is deposited in the INHS Insect Collection with the exception of nine vials borrowed and returned to the OKSU Insect Collection.

Collection and morphological analyses
Terminology of all stages follows Stark (1989). Fresh specimens of the new species (108 males and 40 females) were collected from six Arkansas stream systems, 13-19 June 2016 ( Fig. 1). Methods included sweep netting during the day and ultraviolet light trapping at night. Live male specimens were anesthetized in a dry ice CO 2 chamber and subsequently squeezed with forceps to evert the aedeagus, the source of the most informative morphological characteristics distinguishing species. All specimens were preserved in 95% EtOH. Select individuals of the fresh material and several related species were stack photographed and processed with a Zeiss AxioCam HRc Rev. 3 digital camera and Helicon Focus 6 software in the Sam W. Heads laboratory, INHS. The aedeagus and paraproct were sketched from the stereomicroscope images using Adobe Illustrator CC 2018. Scanning electron micrographs (SEM) of eggs and female terminalia were prepared at the Beckman Institute Microscopy Suite, University of Illinois by critical point drying, placing on an aluminum carbon disk, sputter coating with gold-palladium alloy, and imaged with a Thermo-Fisher FEI Quanta FEG 450 ESEM.

Molecular studies
Genomic DNA from 20 P. sublobata specimens, 17 congeners, and three outgroup taxa was extracted using the Qiagen DNeasy Kit, amplified for a fragment of the  Multiple specimens sharing the same haplotype are listed consecutively. All specimens collected from the USA except P. nelsoni Stark, 1989 (Canada). State or province is listed by standard postal abbreviation. Sequences obtained from GenBank are denoted with *. INHS = Illinois Natural History Survey Insect Collection record number. mitochondrial gene encoding for the COI subunit via polymerase chain reaction using either primers LCO1490 and HCO2198 (Folmer et al. 1994) or jgLCO1490 and jgHCO2198 (Geller et al. 2013), and sequenced with Sanger technology at the University of Illinois W. M. Keck Core Sequencing Facility. Thermocycling conditions consisted of one 94 °C for 5 min denaturation cycle, 40 cycles at 94 °C for 45 s, 50 or 53 °C for 1 min, 72 °C for 1.5 min, and one 72 °C for 5 min extension cycle. Amplification success was verified with gel electrophoresis. Forward and reverse sequences were aligned to create contigs, and all 63 aligned contigs were truncated to a uniform length of 606 nucleotides, visually edited with Sequencher 5.4, aligned in MUSCLE 3.8, and the sequences and supporting data deposited in GenBank (Table 1). Sequences were tested to determine the model of evolution in jModelTest2 (Darriba et al. 2012), and a gamma distribution with a proportion of invariable sites was used to model rate variation across sites (invgamma). Akaike Information Criterion (AIC) results indicated that the General Time Reversible nucleotide substitution model (GTR+I+G) was best for the Maximum Likelihood and Bayesian analyses. These models were applied in subsequent phylogenetic tree generation analyses. We generated a maximum likelihood tree using MEGA 7.0 (Kumar et al. 2016) and calculated pairwise genetic distances for both sequences generated for this study, as well as additional sequences accessioned from GenBank, using the Kimura 2-parameter model (K2P) (Kimura 1980), the de facto standard for measuring mitochondrial pairwise distances (Collins et al. 2012). A Bayesian analysis was performed for all haplotypes using MrBayes 3.2.6 (Huelsenbeck and Ronquist 2001) with a burn-in length of 500,000, subsampling frequency of 500, and a chain length of 5,100,000. Diagnosis. Males are distinguished by a combination of a prominent ventral caecum with a broad ventral setal patch and a distinct dorsal extension of the lateral sclerites of the aedeagus. Females possess a subgenital plate with a deep V-shaped notch and truncate lobes. Eggs have a smooth chorion and a well-developed, distally flanged collar.

Molecular analyses.
Perlesta sublobata formed a monophyletic group with strong support (ML bootstrap support = 97%, Bayesian posterior probability = 92%). The nearest neighbor species to P. sublobata was P. decipiens (Walsh, 1862) at 1.8% sequence divergence. Maximum intraspecific COI genetic distances were less than minimum interspecific distances within all tested Perlesta (Table 2). All intraspecific distances were less than the arbitrary threshold of 3.5%, suggesting that the new species was monophyletic without other cryptic species present within the new taxon (Hebert et al. 2003, Zhou et al. 2010. All haplotypes (total = 47) were confined to their respective genera and presumptive species in the ML and Bayesian analyses (Figs 7,8,respectively). The three tested species within the P. frisoni group, consisting of five Nearctic species that lack an aedeagal dorsal caecum, formed a monophyletic grouping. Four of the five "dark" species studied in Grubbs and DeWalt (2018) also formed a monophyletic grouping. The placement of P. adena Stark, 1989 outside this group may be spurious, indicating additional genes or populations are needed for further refinement. The relatively distant placement of P. golconda DeWalt & Stark, 1998 from P. sublobata is congruent with the species' distinctly different morphologies, apart from the male genitalic similarities.
Remarks. The shape and armature of the aedeagus are the most distinct morphological features of P. sublobata. Stark (1989) illustrated a lateral view of an undetermined species from Arkansas (P. sublobata), demonstrating spinule patterns and shape of the aedeagal telescoping sections: envelope, tube, and sac. He noted that lateral sclerites of the tube joined dorsally. This dorsal extension of the lateral sclerites was not illustrated or specified in the literature for any other Perlesta. Furthermore,  a ventral caecum is present in P. sublobata and only one other described congener, P. golconda. However, the ventral caecum of P. golconda is less prominent and without a distinct ventral patch of fine seta-like spines. Additionally, the dorsal caecum of P. sublobata is moderately developed, compared to the poorly developed dorsal caecum of P. golconda (Fig. 9). The known distribution of P. golconda, originally limited to Illinois (DeWalt and Stark 1998), has expanded to include Iowa, Indiana, Michigan, and Nebraska (DeWalt et al. 2019), as well as Missouri (Stark 2004) and Louisiana (INHS Insect Collection 564765). Arkansas is bordered by Missouri to the north and Louisiana to the south. A sympatric distribution with P. sublobata is expected due to this geographic adjacency and overlap of the Interior Highlands' habitat. Consequently, re-examination of some museum specimens may be required. The male and female habitus easily distinguish P. golconda from P. sublobata. The ocelli of P. golconda are usually connected by a moderately dark V-shaped pattern on a pale background (Fig. 10A, B), whereas P. sublobata has a dark subquadrate interocellar region. The pronotum of P. golconda is primarily pale with light tan rugosities on the lateral margins, whereas P. sublobata has a dark pronotum with a pale narrow median stripe. Additionally, P. golconda females are dis- tinguished by a very short egg collar (Grubbs and DeWalt 2008, their fig. 17) and rounded subgenital plate lobes (Fig. 11A, B).
Habitat. With the exception of one locality (OK, Washington Co., Caney River), all collection sites for P. sublobata are within or closely adjacent to the Interior Highlands, a region containing four contiguous U. S. Environmental Protection Agency (EPA) Level III Ecoregions: Ozark Highlands, Boston Mountains, Arkansas Valley, and Ouachita Mountains. Collection sites for P. sublobata within the Interior Highlands are partially canopied, hardwood forested, wadeable, low gradient streams (ca. 15-20 m wide) with substrata composed mostly of sand, gravel, and cobble. The type     locality is a low gradient run (ca. 25 m wide) of the Little Missouri River (Fig. 14), located 45 km downstream of Lake Greeson and 65 km upstream from its confluence with the Ouachita River in the extreme north EPA Level III Ecoregion 35 (South Central Plains). The substrate is primarily gravel and sand, with some large woody debris. Other stonefly species collected with the new species at the type locality included Acroneuria frisoni Stark & Brown, 1991, Acroneuria nr. ozarkensis Poulton & Stewart,  Stark & Lentz, 1988, N. robisoni Poulton & Stewart, 1986, P. decipiens, and Perlinella ephyre (Newman, 1839. Etymology. The specific epithet is derived from sub, Latin for under, and lobata, the feminine adjectival form of lobus, Latin for a rounded projection or protuberance (Brown 1956). The name references the ventral caecum of the aedeagus, a character shared by only one other described congener, P. golconda, though it is most prominent in P. sublobata.