Isoperla dicala Frison, 1942

Verdone, Chris J., Beaty, Steven R., Holland, Victor B. & Williams, Bronwyn W., 2023, Isoperla riverae, a new stonefly species from the southeast Nearctic, with notes on sympatric species including the larval description of Isoperla lenati Szczytko & Kondratieff, 2015 (Plecoptera: Perlodidae), Zootaxa 5270 (3), pp. 437-470 : 457-468

Isoperla dicala Frison, 1942

( Figs. 41–48 )

Sable Stripetail

http//lsid.speciesfile.org/urn:lsid: Plecoptera .speciesfile.org:TaxonName:468699

Isoperla dicala Frison 1942:321 . Holotype male (INHS), Free Soil , Great Sable River, Mason Co., Michigan, U.S.A.

Isoperla dicala: Hitchcock 1974:197 .

Isoperla dicala: Poulton & Stewart 1991:48 .

Isoperla dicala: Szczytko & Kondratieff 2015:86 .

Distribution. Canada —MB, NB, NS, ON, PQ. U.S.A. —AL*, AR, CT, GA, FL* IA, IN, KY, MA, MD, ME, MI, MN, MO, MS*, NC, NY, OH, PA, SC, TN, VA, WI, WV ( DeWalt et al. 2023). (records with * are considered dubious based on the present study)

Material examined. U.S.A. — Michigan: Presque Isle Co., Ocqueoc River, Ocqueoc Falls Hwy parking area, 45.39724, -84.01754, 11 June 2019, S. Beaty, A. Beaty, 1M, 2F ( NCDWR). Missouri: Shannon Co., Round Springs, Round Springs State Park, [37.28270, -91.40768], 22 June 1974, J. Morse, 1F ( NCDWR). Texas Co., Current River, Montauk State Park, [37.45059, -91.68704], 4 June 1976, B. Stark, J. Lackey, 5M, 9F ( INHS, Insect Collection 875889). North Carolina: Alleghany Co., Brush Creek, SR 1444, 36.49416, -81.00472, 3 April 2006, T. MacPherson, 2L ( NCDWR). Ashe Co., South Fork New River, US 221, 36.47380, -81.33694, 14 May 1990, D. Penrose, 1L, ( NCDWR). Buncombe Co., Flat Creek, SR 1741, 35.72833, -82.61528, 24 April 1986, D. Penrose, 5L, ( NCDWR); [Swannanoa River], Swannanoa, [35.59789, -82.39984], 19 June 1938, T.H. Frison, T.H. Frison Jr., 1M ( INHS, Insect Collection 875897). Burke Co., Linville River, NC 126, 35.79472, -81.89027, 10 April 1990, L. Eaton, D. Penrose, K. Lynch, 1L, ( NCDWR). Caldwell Co., Johns River, SR 1356 (Old Johns River Rd.), 35.93416, -81.69138, 6 April 2019, C. Verdone, S. Beaty, V. Holland, 1F ( NCDWR). Caldwell Co., Thorps Creek, Mortimer Campground, 35.99054, -81.76096, 13 April 2012, S. Beaty, V. Holland, E. Fleek, 1L, ( NCDWR); Wilson Creek, NC 90, 36.01381, -81.76700, 13 April 2012, S. Beaty, V. Holland, E. Fleek 1L ( NCDWR); Yadkin River, near SR 1516, 35.99166, -81.55805, 6 June 2006, M. Walters 1L, ( NCDWR); Yadkin River, off SR 1372, 36.06895, -81.58553, 23 May 2011, D. Black, S. Beaty, E. Fleek 1L ( NCDWR). Henderson Co., North Fork Mills River, SR 1343, 35.39425, -82.61503, 3 May 2011, T. Mormon, M. Walters, D. Black 1L, ( NCDWR); South Fork Mills River, SR 1340, 35.37569, -82.61440, 1 June 2009, T. MacPherson, M. Walters, E. Fleek 2L, ( NCDWR); same location, 35.37569, -82.61440, 3 May 2011, T. Mormon, M. Walters, D. Black, 4L, ( NCDWR); same location, 25 June 2002, T. Mormon, 1L, ( NCDWR). Macon Co., Cullasaja River, off Fox Ridge Rd, 35.17172, -83.36720, 28 May 2019, V. Holland, S. Beaty, C. Verdone, 1L ( NCDWR). Rockingham Co., Mayo River, Anglin Mill Rd., 36.53530, - 79.99070, 11 May 2018, C. Verdone, 2M, 1F ( NCDWR); same location, 22 April 2020, S. Beaty, V. Holland, C. Verdone, 1F, 1E ( NCDWR); same location, 2 May (no year given), D. Lenat, 3L ( NCDWR). Surry Co., Fisher River, near SR 1346, 36.41803, -80.71752, 4 May 2010, V. Holland, M. Walters, T. Mormon, 2L ( NCDWR). Swain Co., Nantahala River, Hwy 19, 35.33184, -83.60671, 27 May 1994, S.W. Szczytko, J.B. Sandberg, B. Stark, 1M ( INHS, Insect Collection 875908). Transylvania Co., French Broad River, SR 1129, 35.14861, -82.79972, 9 April 2019, C. Verdone, S. Beaty, V. Holland, E. Fleek, 10L ( NCDWR). Watauga Co., Cove Creek, off SR 1121, 36.23944, -81.82138, 15 May 2007, E. Fleek, T. MacPherson, S. Beaty 2L ( NCDWR). Yancey Co., Cane River, off SR 1114, 35.90437, -82.33250, 7 May 2008, D. Black, M. Walters, B. Crouch, 5L ( NCDWR); Cane River, US 19E, 35.91222, -82.34944, 14 May 2013, D. Lenat, 1L ( NCDWR). South Carolina: Oconee Co., Chattooga River, Hwy 28 at Georgia State Line, [34.91958, -83.16882], 24 May 1993, B. Kondratieff, R. Kirchner, 1M ( NCDWR). Pickens Co., South Saluda River, 4 mi N Pumpkintown, [35.06719, -82.61793], 22 June 1969, H. Douglass, 1F ( INHS, Insect Collection 875884). Tennessee: Blount Co., Abrams Creek, Abrams Creek Campground, Great Smoky Mountains National Park, 35.61030, -83.93270, 10 June 2001, B.D. Heinold, 2F ( INHS, Insect Collection 3491). Sevier Co., Little Pigeon River, Gatlinburg, [35.71417, -83.51028], 12 June 1935, H.H. Ross, 1F ( INHS, Plecoptera 2957 ); Gatlinburg, [35.71426, -83.51016], 11 June 1938, T.H. Frison, T.H. Frison Jr., 1F ( INHS, Insect Collection 875854); same location, 14 June 1940, [ T.H.] Frison, [C.O. Mohr, A.W. Hawkins] 2F, ( INHS, Plecoptera 2956 ); Sevierville, 35.86806, -83.56194, 11 June 1938 T.H. Frison, T.H. Frison Jr., 5F ( INHS, Insect Collection 875863); same data, 3F ( INHS, Insect Collection 875856); same data, 1M ( INHS, Insect Collection 875892). Virginia: Bland Co., Clear Fork, Hwy 61, 0.5 mi W of Clear Fork, 37.20992, -81.20296, 6 June 2016, C. Verdone, B. Kondratieff, 1F ( NCDWR); Wolf Creek, Grapefield Rd., Stephen Levitt Property, 37.14702, -81.26314, 4 June 2016, C. Verdone, B. Kondratieff, 3F ( NCDWR); same location, 6 June 2016, C. Verdone, B. Kondratieff, 2F ( NCDWR); s ame location, 8 June 2016, C. Verdone, B. Kondratieff, 2F ( NCDWR); same location, 23 May 2017, C. Verdone, 3F, 2F ( NCDWR); same location, 27 May 2017, C. Verdone, D. Fuller, 1F ( NCDWR); Wolf Creek, Grapefield Rd., Wolf Creek Picnic Area, 37.18026, -81.19496, 8 June 2016, C. Verdone, B. Kondratieff, 1F ( NCDWR); same location, 7 May 2017, C. Verdone, B. Kondratieff, S. Roble, 1M, 2F ( NCDWR); Wolf Creek, Hwy 61, War Memorial, 37.24324, -81.10800, 27 May 2016, C. Verdone, B. Kondratieff, 4F ( NCDWR); same location, 8 June 2016, C. Verdone, B. Kondratieff, 1F ( NCDWR). Botetourt Co., Craig Creek, Craig Creek Rec Area (2 mi SE of Oriskany), 37.61345, -79.95624, 9 June 2016, C. Verdone, B. Kondratieff, 1F ( NCDWR). Craig Co., Potts Creek, Rte 18, 37.59814, - 80.22697, 4 May 2019, C. Verdone, D. Fuller, 1M ( NCDWR). Floyd Co., Floyd, [36.91124, -80.32005], no date, no collector data, 3F ( VMNH). Franklin Co., Otter Creek, Philpott Reservoir, end of Rte 789, 2.4 mi SE of Endicott, [36.86837, -80.11660], 25 August 2000, A.C. Chazal, 2F ( VMNH); Pigg River, 0.3 km upstream of Rte 890 bridge SW Pennook, [36.94638, -79.64052], 26 June 2005, R.L. Hoffman, 1M, 2F ( VMNH); UT Nicholas Creek, VA 781, 2.5 mi W Ferrum, 36.91615, -80.06237, 6 July 2000, R.L. Hoffman, 1F ( VMNH). Giles Co., Sinking Creek, at VA 700 Bridge, 37.31149, -80.51610, 3 July 1992, R.L. Hoffman, 3F ( VMNH). Grayson Co., New River, Hwy 58 Boat Access, 36.61340, -81.04464, 29 May 2016, C. Verdone, B. Kondratieff, 1F ( NCDWR), same location, 11 May 2017, C. Verdone, B. Kondratieff, 1F ( NCDWR). Henry Co., South Mayo River, at VA 695, ca 5 mi SSW of Spencer, [36.55577, -80.02128], 6 June 1990, R.L. Hoffman, 2F ( VMNH); Toeclout Branch, at VA 637, 3 mi SE of Ridgeway, [36.54852, -79.84263], 12 May 1990, R.L. Hoffman, 1M, 2F ( VMNH). Patrick Co., Dan River, Rte 648, 36.62230, -80.44497, 11 May 2017, C. Verdone, B. Kondratieff, 2M ( NCDWR). Pittsylvania Co., Crooked Run, VA 841 6 mi SW Callands, [36.74825, -79.65121], 9 June 2000, R.L. Hoffman, 1F ( VMNH); Dan River, VA 860 bridge, ca 1 mi SE of Cascade, [36.54417, -79.66183], 31 May 1989, R.L. Hoffman, 1M ( VMNH); Sandy River, Rte 845 bridge 4.5 mi N Brosville, [36.67935, -79.59389], 7 June 1993, R.L. Hoffman, 1F ( VMNH). Prince William Co./ Fairfax Co., Bull Run, Manassas National Battle Field, [38.82435, -77.50375], 30 July 1997, C.S. Hobson, 1F ( VMNH). Pulaski Co., Little Reed Island Creek, Rte 607 at Boone Furnace, [36.92150, -80.76976], 10 June 1990, R.L. Hoffman, 1F ( VMNH). Russell Co., Clinch River, Rte 798 SE of Rte 645 nr Nash Ford, 36.96549, -82.07749, 24 May 2017, C. Verdone, 3M, 4F ( NCDWR). Scott Co., Clinch River, Rte 65, Dungannon Boat Access, 36.83108, -82.46252, 6 May 2017, C. Verdone, B. Kondratieff, S. Roble, 3M, 6F ( NCDWR). Smyth Co., North Fork Holston River, Carl Slate Memorial Boat Landing, 36.89335, -81.75539, 5 May 2017, C. Verdone, B. Kondratieff, S. Roble, 5M, 5F ( NCDWR); South Fork Holston River, jct. Riverside Rd./ Chestnut Ridge Rd., 36.76200, -81.63585, 8 May 2017, C. Verdone, B. Kondratieff, 8M, 3F ( NCDWR). Tazewell Co., spring pond on Smith Ridge, 1 mi N of Pounding Mill, [37.09095, -81.71416], 4 June 1999, Rodney Cordle, 1M, 2F ( VMNH). Washington Co., Wolf Creek, St. Rte 80 nr Conflu w/ North Fork Holston River, 36.82404, -81.92167, 4 May 2017, C. Verdone, B. Kondratieff, 1M ( NCDWR).

Adult male. Macropterous; forewing length 7.5–9.3 mm (n = 16), body length 6.9–8.9 (n = 16). Head pale yellow with brown bands connecting lateral ocelli to anterior ocellus. General body color pale yellow in life with few brown markings.

Dorsum of Head ( Fig. 16 ). Palpi pale yellow-brown. Labrum with stiff, short setae. Dorsum of head pale yellow with brown bands connecting median ocellus with lateral ocelli; interocellar area pale, open posteriorly; frons generally pale yellow. Antennal scape and pedicel yellow; flagellum brown with basal 7–8 segments pale, antennal length about ½ length of body with 32 segments.

Thorax. Pronotum with a wide, pale median stripe which extends to anterior margin ( Fig. 16 ); rugose area laterad of median stripe pale brown; pigment extends anteriorly to pronotal margin; rugose area with short stout brown setae interspersed; posterior edge of pronotum pale; lateral pronotal margins pale. Mesonota and metanota yellow with yellow-brown scutal humps; nota with sparse, short, fine pale setae. Wings hyaline with light brown veins ( Fig. 26 ). Mesobasisterna and metabasisterna pale yellow.

Legs. Anterior and posterior faces of femora pale yellow. Tibiae yellow with a thin transverse dark brown band near proximal ¼. Tarsi light brown, segment 3 darker apically. Leg vestiture dense with short dark setae giving legs a darker overall appearance; tibiae with widely spaced subdorsal and ventral rows of larger spines.

Abdomen. Terga pale yellow and clothed with short, fine golden setae. Sterna pale yellow. Sterna 6–9 with a posterior fringe of short, brush-like, brown setae. Sternum 8 with a deeply recessed, pale brown vesicle ( Fig. 17 ) 2.1–3.0X (n = 14) longer than wide when measured from apex to anterior sclerite / maximum subapical width; apex slightly expanded, evenly rounded, extending to base of segment 9. Paraprocts triangular, minimally sclerotized, pale yellow; apices narrow and acutely rounded; not recurved over tergum 10 ( Fig. 18 ). Cerci pale yellow; cercal length ½ as long as abdomen, with 12 segments, each segment with one long, light brown ventral seta near posterior margin, basal 4–6 segments with dorsal and ventral tufts of long stiff setae, setae becoming shorter and less numerous on each successive segment.

Aedeagus ( Figs. 19–25 ). Male appendage with a short, broad basal stalk, posteroventral lobe, posterior lobe, pair of small caudal lobes, conical apical lobe and paired hemispherical anterior lobes ( Figs. 19 , 22 ). Paired caudal lobes ( Figs. 19–22 ), small, often difficult to discern. Conical apical lobe ( Figs. 19 , 22 ) often depressed apically, flaplike, appearing triangular in dorsal and ventral aspects ( Figs. 20–21 ); apex broadly rounded. Anterior hemispherical lobes membranous ( Figs. 19–22 ).

Aedeagal armature ( Figs. 22–25 ). Basal stalk encircled with short stout spinulae ( Fig. 22 ); posteroventral lobe with sharp spinulae which transitions to dense hair-like spinulae dorsally ( Fig. 24 ). Mediolateral areas of aedeagus also covered in sharp spinulae ( Fig. 22 ). Posterior lobe with small mounds each adorned with a cluster of small sharp spinulae ( Fig. 25 ). Area dorsolateral to paired caudal lobes with linear brushes of hair-like spinulae and sensilla basiconica which transition to small, rounded knobs with apical filaments anteriorly ( Fig. 23 ).

Adult female ( Fig. 26 ). Macropterous; forewing length 8.1–10.1 mm (n = 11). Body length 7.9–10.3 (n = 11). General head pattern and body color similar to that of the male. Posterior margin of tergum 7 and posterolateral margins of terga 8–9 bearing brush-like setae.

Subgenital plate ( Fig. 27 ). Pale yellow, broadly triangular and produced posteriorly approximately ⅛–¼ the length of sternum 9; plate basally broad, about ¾ the width of segment 8 on most specimens, apex obtuse; plate with scattered short golden setae and a few longer setae interspersed.

Ovum ( Figs. 28–31 ). Length 268.7–277.4 μm (n = 2); width 200.3–202.2μm (n = 2). Color pale yellow. General shape oblong; cross section concave; chorion covered with subtle reticulate ridges; collar well-developed ( Figs. 28–29 ). Follicle cell impressions hexagonal or pentagonal, weakly developed and with clusters of globules at their center ( Figs. 30–31 ). Eclosion line weakly developed, micropyles positioned on eclosion line, spaced regularly around egg circumference ( Figs. 31 ). Collar height 28.7–34.3 μm, width 56.2–56.9 μm (n = 2); width either subequal or slightly narrowed from base to apex; densely covered in globules ( Figs. 28–29 ).

Mature larva ( Figs. 32–35 ). Pre-emergent larvae 6.7–9.2 mm (n = 17). Body with lightly contrasting body pattern.

Dorsum of head ( Fig. 33 ). Mottled brown with pale medial area anterior to median ocellus, a pair of large anterolateral pale spots, a pair of mediolateral pale spots, a small enclosed pale ocellar spot, and pale ocular areas. Anterior frontoclypeus with a dark bar that is obliterated medially by medial extension of pale median area. Pale medial area often shaped like a spinning top; sometimes with a short anteromedial extension and with small anterolateral projections, resembling a duck foot overall. Anterolateral pale spots variable and irregularly shaped, usually distinctly separated from pale medial area. Pale medial spot and anterolateral spot bordered by a dark M-line on posterior margins; pale ocellar spot small, usually round, fully enclosed and centered equidistant from ocelli; mediolateral pale spots subquadrate, extending anteromedially from epicranial suture but not reaching lateral ocelli; closed anterior to suture, confluent with pale ocular area. Pale ocular spots extend posteromedially towards posterior occipital margin; pale area divided by a thin angulate line of brown pigment and spicules. Epicranial stem with a pale oval spot covering posterior 2/3. Brown areas of occipital area with clusters of spicules. Clothing hairs on head lightly colored. Antennae yellow; apical segments slightly darker; scape and pedicel yellow to yellow-brown; flagellum with 30–39 antennomeres (n=13); each segment with an apical circlet of small spines.

Maxilla ( Figs. 36–37 ). Lacinia bidentate; receding evenly from base to subapical tooth; a thin, marginal seta ¾ subapical tooth length, proximal to subapical tooth; 5–6 (rarely 7) striated, stout marginal setae below subapical tooth, last few setae progressively smaller; 8–10 thin, unevenly-spaced setae along palm edge and curving dorsad; a submarginal row of 3–4 striated stout setae below apical tooth; ventral face of palm basally with 5–7 setae. Length of striated apical tooth of lacinia between 0.5–0.6X palm length and 0.6–0.7X the palm width. Subapical tooth 0.5–0.6X the length of the apical tooth. Galea 1.0–1.1X the lacinial palm length, with a ventral row of less than 30 setae and tipped with 2–4 apical spinous setae. Maxillary palp setose, 1.4–1.5X length of lacinia; segments 1–4 successively longer, segment 4 slightly longer than 5; segments 1 and 2 with transverse row of apical spinous setae; segment 3 sometimes with one apical spine, segment 5 tipped with 1–2 sensillae.

Mandibles ( Figs. 38–39 ). Right mandible ( Fig. 38 ) bicuspid, outer cusp with three teeth, ventral tooth largest and serrated basally, dorsal tooth smallest; a band of unorganized, long spinous setae on ventral surface from base of outer ventral tooth to base of mandible; inner cusp with three teeth, middle tooth longest and largest; a few sharply pointed acanthae from basal tooth on inner cusp to molar ridge ( Fig. 38 inset); molar ridge with a dense row of spine-like setae, setae longer and thinner towards base of mandible; dorsal surface of mandible with a band of unorganized setae from inner cusp towards base of mandible. Left mandible ( Fig. 39 ) similar to right except acanthae at base of inner cusp replaced by a dense brush of short spinous setae ( Fig. 39 inset).

Thoracic nota ( Figs 32–33 ). Pronotum ( Figs. 32–33 ) brown with contrasting pattern. Medial stripe yellow-brown, wider both anteriorly and posteriorly; medially interrupted on both anterior and posterior margins by brown triangular markings. Anterior pronotal flange with alternating stripes of pale and dark pigment; lateral edges pale, with sublateral brown stripes. Pronotal disk with an irregular pale crescent enclosed by brown markings. Posterior pronotal flange similar to anterior flange. Pronotum rimmed with short, closely set, stiff setae, intervening long setae mostly absent except at pronotal corners. Mesonota and metanota with irregular brown markings; wing pads yellow-brown with three sublateral longitudinal brown stripes ( Fig. 32 ). Lateral margins of nota fringed with a short, closely set row of stiff setae. All thoracic nota with sparse, short, pale to brown clothing hairs. Thoracic sterna pale, without distinctive markings; furcal pits and Y-stem slightly darker in most specimens. Clothing hairs on sternum pale, sparse.

Legs: Femora yellow to yellow-brown; ventral face mostly pale with a few stout, golden spines; anterior face yellow with an obscure submedial yellow-brown longitudinal band and regularly spaced stout golden setae; dorsally with a sparse fringe of silky setae. Tibiae pale yellow-brown with a broad ventral row of stout setae; anterior face with two longitudinal rows of stout setae separated by a row of long silky setae, setae twice as long as width of tibia. Tarsi pale yellow-brown with a ventral row of stiff setae and a sparse dorsal row of silky setae. Two moderately long claws darker apically, gradually curved.

Abdomen ( Figs. 32, 34–35 ). Terga ( Figs. 32, 34 ) with three dark brown longitudinal stripes bordered by pale lines, with intervening stripes wider and paler; in lateral aspect, anterior half of each segment with a pair of small, variably dark dots sometimes overlaid by a coalescing stripe on distal segments. Terga with numerous spicules with dark origins; posterior edges of abdominal segments bearing a transverse row of stout spines, longer spines interspersed among 2–3 shorter spines; Sterna ( Fig. 35 ) pale and with spicules with dark origins typically restricted to segments 7–10; spicules may be present on anterior segments, but rarely have dark origins; posterior edges of sterna bearing a transverse row of long clear spines; female larvae with row incomplete medially on tergum 8. Cerci yellow-brown with 18–22 segments (n = 13), each with an apical circlet of short spines; cerci with sparse dorsal fringe of silky setae on distal half.

Diagnosis. Isoperla riverae sp. n. is defined in the adult male stage by having a vesicle that is 2.1–3.0X as long as wide, dark bars connecting the anterior ocellus to the lateral ocelli, and an aedeagus that lacks an elongate posteromedian spine. Females are defined also by dark bars connecting the anterior ocellus to the lateral ocelli, in addition to having a broadly triangular subgenital plate that is produced posteriorly approximately 1/8–1/4 the length of sternum 9. Larvae are defined by the following combination of characters: the head pattern having a pale medial area anterior to median ocellus shaped like a spinning top or duck foot, a pair of large anterolateral pale spots, a small enclosed pale ocellar spot, and lateral ocelli that are separated from the mediolateral and ocular pale areas; three dark longitudinal abdominal stripes bordered by pale lines and sternal spicules with dark origins typically restricted to segments 7–10.

Genetic data results. We generated a total of 42 new COI sequences ( Table 1 ) for this study, including six for I. riverae sp. n., five for I. dicala , four for each Isoperla borisi Beaty, Holland & Lenat, 2017 , I. davisi , Isoperla evanescens Verdone & Kondratieff, 2016 , I. lenati , Isoperla poffi Szczytko & Kondratieff, 2015 , Isoperla zuelligi Szczytko & Kondratieff, 2015 , and Clioperla clio (Newman, 1839) (the outgroup taxon), and three for Isoperla decepta Frison, 1935 . Genetic distance within sampled I. riverae sp. n. was low (avg. p-dist = 0.67%; Table 2 ), and similar to that observed within each species clade (avg. p-dist 0.15%–1.27%; Table 2 ). Genetic distance was high between I. riverae sp. n. and I. dicala (avg. p-dist = 13.66%; Table 2 ) and among all included Isoperla species (avg. p-dist 11.40%–18.83%; Table 2 ). Maximum intraspecific distance for I. riverae sp. n. was 1.58%, and 1.27% for I. dicala . The minimum interspecific distance between the two taxa was 13.02%.

Maximum likelihood phylogenetic reconstruction placed I. riverae sp. n. as sister to I. dicala in a distinct moderately well-supported clade ( Table 2 , Fig. 40 ); average pairwise tree distance = 17.8% ( Table 2 ), bootstrap support, bs = 69%; Fig. 40 ). These two taxa are in turn weakly supported (bs = 57%; Fig. 40 ) as sister to I. poffi . All species clades were recovered with high nodal support (bs = 100% for; I. davisi , I. evanescens , I. poffi , I. riverae sp. n.; bs = 99% for I. borisi ; bs = 98% for I. decepta ; bs = 96% for I. lenati ; bs = 91% for I. dicala ; Fig. 40 ).

Taxonomic notes. Similar to other eastern Nearctic Isoperla described since 2015 ( I. evanescens , Isoperla dewalti Verdone & Kondratieff, 2017 ; and I. borisi ), I. riverae sp. n. had been lumped with previously described species; I. dicala in the adult stage ( Stark & Gaufin 1978; Stark & Harris 1986; Szczytko & Kondratieff 2015) and I. dicala and I. davisi in the larval stage (unpublished NCDEQ data). Although several vials containing the new species had been flagged by both Drs. Szczytko and Kondratieff, many specimens of the new species were identified as I. dicala ( Figs. 41–48 ) based on similarities of the vesicle ( Figs. 17 , 43 ) and subgenital plate ( Figs. 27 , 44 ) under the assumption that the head pattern was variable (B. Kondratieff personal comm.). Males of the new species and I. dicala both possess a long vesicle ( I. dicala : 1.7–2.9X L/W, n = 10; I. riverae sp. n.: 2.1–3.0X L/W, n = 14) and females have a broadly triangular subgenital plate. Adult specimens of I. dicala were examined from throughout its range and while a disjunctive population in Missouri had darker pigmentation around the anterior ocellus only, all other specimens completely lacked darkly colored bars ( Fig. 41 ). As such, the absence of dark bars connecting the anterior ocellus to the lateral ocelli consistently allows for the separation of I. dicala and the new species if the distinctive aedeagus ( Fig. 42 ) is not everted.

Larvae of the new species have been collected in North Carolina Biological Assessment Branch benthic samples dating back to 1985. However, much like the adult, the uniqueness of larval characteristics was considered to be intraspecific variation of either I. davisi or I. dicala . As a result of ongoing efforts to rear and associate Isoperla life stages, we found that the larva of I. lenati is superficially similar to the new species as well. A confounding factor that likely contributed to the past misidentification of I. riverae sp. n. is that it is sympatric with all the species mentioned above.

These four species have similar maculations, but with close examination of the head pattern, can be readily and confidently separated. The most reliable character state that distinguishes I. riverae sp. n. from the other three species is that the lateral ocelli are separated from the mediolateral and ocular pale areas. On both I. davisi ( Fig. 12 ) and I. lenati ( Fig. 2 ) the mediolateral pale spots are broadly confluent with the lateral ocelli. On I. dicala ( Fig. 46 ) this is less obvious because the pale ocular areas are somewhat obfuscated, but they are connected to the lateral ocelli via thin isthmi. When distinguishing the new species from I. dicala it is also helpful to look at spicules with dark origins on the abdomen ( Figs. 47–48 ). Most importantly, these spicules are present on all abdominal sterna on I. dicala ( Fig. 48 ), whereas on I. riverae sp. n. ( Fig. 35 ), they are only present on sterna 7–10. There are additional characters that distinguish the larvae of the new species from other congeners and will be detailed in a comprehensive review of North Carolina Isoperla (S. Beaty et al. in progress); however, the characters mentioned above are sufficiently diagnostic in most cases.

Type locality ( Fig. 49 ). Located in south-central North Carolina, Drowning Creek begins in, and flows through, the Sand Hills level IV Ecoregion (Griffith et al. 2002) and is the primary tributary of the Lumber River which is, in turn, a major tributary of the Little Pee Dee River . The collection site is 91 m ASL and has a drainage area of 214 km 2. Drowning Creek is typically clear and tannic and is complemented by substrates consisting of gravel, white sand, woody debris and coarse particulate organic matter. Water quality at the type locality has not been assessed using North Carolina Biotic Index ( NCBI) protocols ( Lenat 1993; NCDEQ 2015 ), but a long-term monitoring site approximately 4.5 km downstream has rated “Excellent” for 10 consecutive samples dating back to 1985. Although it has not been sampled in the spring during peak species richness, EPT richness often exceeds 30 taxa in the summer months.

Biological notes. Isoperla riverae sp. n. is widespread in the southeast Nearctic, occurring in four EPA level III Ecoregions (Blue Ridge, Piedmont, Southeastern Plains, Ridge & Valley) across eight states. The North Carolina Division of Water Resources Biological Assessment Branch has 90 verified larval records of I. riverae sp. n. dating back to 1985. Of these, 61 are from the Blue Ridge, 25 are from the Piedmont, and only four are from the Southeastern Plains. The new species is known to occur in small streams (stream width = 2 m; drainage area = 2.8 km 2) to large rivers (> 40 m; 1572 km 2). The North Carolina Biotic Index pollution tolerance value calculated for I. riverae sp. n. is 2.3, indicating relatively low pollution tolerance. Additionally, 84% of the streams assessed by NCDWR in which the new species was detected have rated either “Excellent” or “Good”.

Larvae can be found in riffle habitats and leaf packs in swift current. Adults typically emerge in mid-April through mid-May. Adult females have been collected as late as mid-July. Like most other Isoperla , a univoltineslow life cycle is presumed for this species.

Figures

FIGURE 40. Phylogenetic representation of the IQTREE-estimated maximum likelihood tree of cytochrome c oxidase I (COI). Bootstrap values of 50 or greater are shown at associated nodes; bootstrap values are not provided for short internodes within species clades. Tips are labeled with species name, sample number, and county and state. The scale bar represents estimated number of nucleotide substitutions per site.

FIGURE 49. Type locality: Drowning Creek, SR 1102, Moore/Richmond Co., North Carolina, 20 May 2018.

FIGURES 41–42. Isoperla dicala, adult male, Potts Creek, Craig Co., Virginia. 41. Head and pronotum, dorsal. 42. Terminalia, lateral.

FIGURES 43–44. Isoperla dicala, adults. 43. Male vesicle, ventral, South Fork Holston River, Smyth Co., Virginia. 44. Female subgenital plate, ventral, Clinch River, Tazewell Co., Virginia.

FIGURES 45–48. Isoperla dicala, larva. 45–46. Cullasaja River, Macon Co., North Carolina. 45. Habitus, dorsal. 46. Head and pronotum, dorsal. 47–48 French Broad River, Transylvania Co., North Carolina. 47. Abdomen, dorsal. 48. Abdomen, ventral.

FIGURES 16–18. Isoperla riverae sp. n., adult male. 16. Head and pronotum, dorsal, Drowning Creek, Moore/Richmond Co., North Carolina. 17–18. Grassy Fork, Franklin Co. Virginia. 17. Vesicle, ventral. 18. Paraprocts, caudal.

FIGURES 26–27. Isoperla riverae sp. n., adult female, Drowning Creek, Moore/Richmond Co., North Carolina. 26. Habitus, dorsal. 27. Subgenital plate, ventral.

FIGURES 19–21. Isoperla riverae sp. n., adult male terminalia, (A) posteroventral lobe, (B) posterior lobe, (C) pair of small caudal lobes, (D) conical apical lobe, (E) paired hemispherical anterior lobes, Mayo River, Rockingham Co., North Carolina. 19. Lateral, 20. Dorsal, 21. Ventral.

FIGURES 22–25. Isoperla riverae sp. n., adult male, aedeagus, (A) posteroventral lobe, (B) posterior lobe, (C) pair of small caudal lobes, (D) conical apical lobe, (E) paired hemispherical anterior lobes, Drowning Creek, Moore/Richmond Co., North Carolina. 22. Lateral. 23. Details of fig. 22 inset, hair-like spinulae, sensilla basiconica and small rounded knobs with apical filaments. 24. Sharp spinulae and hair-like spinulae of posteroventral lobe (A) 25. Small mounds with sharp spinulae on posterior lobe (B).

FIGURES 28–31. Isoperla riverae sp. n., ovum, Drowning Creek, Moore/Richmond Co., North Carolina. 28. Convex aspect. 29. Concave aspect. 30. Reticulation of egg chorion and globules. 31. Eclosion line and micropyles.

FIGURES 32–35. Isoperla riverae sp. n., larva, Naked Creek, Richmond Co., North Carolina. 32. Habitus, dorsal. 33. Head and pronotum, dorsal. 34. Abdomen, dorsal. 35. Abdomen, ventral.

FIGURES 36–39. Isoperla riverae sp. n., larva. 36–37. Naked Creek, Richmond Co., North Carolina. 36. Right lacinia, ventral. 37. Left lacinia, ventral. 38–39. Rocky Ford Branch, Richmond Co., North Carolina. 38. Right mandible, ventral; (inset) acanthae. 39. Left mandible, ventral; (inset) dense brush of short spinous setae.

FIGURES 11–14. Isoperla davisi, larva, Lumber River Hoke/Scotland Co., North Carolina. 11. Habitus, dorsal. 12. Head and pronotum, dorsal. 13. Abdomen, dorsal. 14. Abdomen, ventral.

FIGURES 1–4. Isoperla lenati, larva, Flat Creek, Hoke Co., North Carolina. 1. Habitus, dorsal. 2. Head and pronotum, dorsal, (a) pale medial area anterior to median ocellus, (b) anterolateral pale spots, (c) mediolateral pale spots, (d) enclosed pale ocellar spot, (e) pale ocular areas. 3. Abdomen, dorsal. 4. Abdomen, ventral.

Tables

TABLE 2. Average percent (%) pairwise tree distances, calculated from the COI-based ML tree using the Species Delimitation in Geneious Prime 2023.0.2, shown in white. Uncorrected average percent (%) pairwise genetic distances, calculated from the COI data using MEGA v11.0.8, shown in gray. Average percent (%) uncorrected intraspecific pairwise distances, along with sample size, are shown in green.

Species	Clioperla clio	Isoperla riverae n. sp.	Isoperla poffi	Isoperla borisi	Isoperla evanescens	Isoperla decepta	Isoperla dicala	Isoperla davisi	Isoperla zuelligi	Isoperla lenati
Clioperla clio	0.23; n=4	35.40	30.70	34.90	36.40	33.10	28.40	32.30	29.70	27.00
Isoperla riverae n. sp.	16.31	0.67; n=6	32.50	35.60	37.00	33.70	17.80	32.90	30.30	27.60
Isoperla poffi	15.00	16.16	0.87; n=4	32.10	33.50	30.20	25.50	29.40	26.80	24.10
Isoperla borisi	15.77	16.01	13.47	0.23; n= 4	29.90	26.70	28.50	25.90	23.20	20.50
Isoperla evanescens	16.19	16.16	13.61	11.40	0.15; n=4	14.40	29.90	27.80	28.00	25.30
Isoperla decepta	18.01	17.09	15.32	11.89	10.87	0.57; n=3	26.70	24.50	24.70	22.00
Isoperla dicala	17.63	13.66	18.83	18.60	18.33	16.42	0.63; n=5	25.90	23.20	20.50
Isoperla davisi	15.12	16.58	13.93	12.61	14.82	13.99	17.35	0.53; n=4	23.90	21.20
Isoperla zuelligi	14.63	14.96	13.54	13.34	13.53	14.22	16.33	12.99	0.86; n=4	12.50
Isoperla lenati	13.87	15.23	13.41	12.80	12.99	15.09	13.54	11.97	8.74	1.27; n=4