Anacroneuria flintorum Froehlich, 2002 : 93 Anacroneuria flintorum Froehlich 2002 (Plecoptera: Perlidae): Notes, distribution, and life stages association using molecular tools Almeida, Lucas Henrique De Gonçalves, Maísa De Carvalho Novaes, Marcos Carneiro Paresqui, Rayner Constantino Bispo, Pitágoras Da Conceição Zootaxa 2018 2018-01-12 4370 4 409 420 DBYG Froehlich, 2002 Froehlich 2002 [151,644,1206,1232] Insecta Perlidae Anacroneuria GBIF Animalia Plecoptera 2 411 Arthropoda species flintorum    Anacroneuria flintorum Froehlich, 2002: 93; Bispo & Froehlich, 2004: 99; De Ribeiro & Froehlich, 2007: 55; Froehlich, 2010: 56; Baldin et al., 2013: 394; Gonçalves et al., 2017: 146.    Materialexamined. BR, SP: Iporanga, Gruta da Tapagem,  15.xi.1991, 1 male; PEI, Rio do Carmo,  19.ii.1992, 1 male; PEI, Rio do Carmo,  16.ii.1993, 2 males; PEI, Rio Poços Altos,  10.xi.1993, CGF & HP col., 2 males; PETAR, Rio Betari, Bairro da Serra,  20.viii.1996, light trap, ASMcol., 1 male; PEI, Córrego Lajeado,  24.ii.1999, 2 males; PEI, Córrego Lajeado,  17.ix.1999, 1 male; PEI, Ribeirão Água Comprida,  27.x.1999, 3 males; PEI, Rio do Carmo,  28.x.1999, 1 male; PEI, Ribeirão Água Comprida,  23.xi.1999, 7 males; PEI, Ribeirão Água Comprida,  06.i.2000, 8 males; PEI, Rio Poços Altos,  01.ii.2000, 2 males; PEI, Rio do Carmo,  25.iii.2000, 1 male; PEI, Ribeirão Água Comprida, 24°17’38”S, 48°25’04”W, 30.x.2002, ASMcol., 1 male; PECB, Rio Taquaral,  09.ix.2006, 1 male; PEI, Córrego Roda D’Água,  13.xii.2014, light trap, PCB col., 1 female; PEI, Rio do Carmo, bridge, 24°18’15”S, 48°24’31”W,  14.xii.2014, light trap, PCB col., 1 maleand 1 female; PEI, Córrego do Mirante,  15.xii.2014, light trap, PCB col., 1 female; PEI, Rio do Carmo, bridge, 24°18’15”S, 48°24’31”W, 08.ii.2017, light trap, LHA et al. col., 1 male; PEI, Ribeirão Bocaina, bridge, 24°16’13”S, 48°27’09”W, 12.ii.2017, light trap, LHA et al. col., 1 male; PETAR Núcleo Santana, Riacho Furnas, 24°32’03”S, 48°42’02”W,  14.ii.2017, light trap, LHA et al. col., 2 males; PETAR Núcleo Santana, Rio Roncador, 24°32’00”S, 48°42’06”W,  15.ii.2017, light pan trap, LHA et al. col., 1 male; PECB, Braço do Rio Taquaral, 24°03’31”S, 47°59’41”W, 05.ii.2017, D-net, LHA et al. col., 1 nymph; PECB, Ribeirão de Pedras, 24°03’40”S, 47°59’51”W,  06.ii.2017, D-net, LHA et al. col., 1 nymph; PECJ, Córrego Campo do Meio 22°41’28”S, 45°28’26”W,  28.i.2009, light trap, 2 males. BR, ES: Caparaó, Rio Santa Marta,  18.ii.2016, FFST col., 3 females and 7 nymph; Caparaó, Pedra Menina, Base, 14-15.ii.2016, FFST col., 2 females; Caparaó, Pedra Menina, Base, 15-16.ii.2016, FFSTcol., 6 femalesand 1 male. BR, MG: Araponga, Serra do Brigadeiro, Valedas Luas,  14.i.2014, FFSTcol. 1 nymph. BR, SC: Chapadão, 28#12’09”S, 49#23’14”W,  15.ii.2013, light trap, L.C. Pinho, A.P. Amaral, M.C. Novaescol., 1 male; Orleans, Rio da Serra, 28°12’43”S, 49#27’28”W, 16.ii.2013, light trap, L.C. Pinho, A.P. Amaral, M.C. Novaes col., 1 male.   Description and association of nymph. Molecular association.We associated adults with nymphs of  A. flintorum(including teneral adults) from the states of Espírito Santoand São Paulo. The COI divergences ranged from 0–0.9% for specimens from the São PauloState and from 0–0.7% from Espírito SantoState. Intraspecific divergences ranged from 0–3.9%, probably due the geographic variation. For all species studied, interspecific divergences ranged from 12.4%, between  A. flintorum(PLE481a) and A.sp. 2 (PPI15) to 20.6% between  A. flintorum(PLE479a) and  A. debilis( Pictet, 1841)(PLE480b). A dendrogram and the matrix with pair divergence between specimens based on COI sequences are presented in Fig. 1and Table 2.   FIGURE 1.Neighbor-joining tree for cytochrome c oxidase subunit I (COI) sequences (433 bp) from  Anacroneuria flintorumFroehlichand related stoneflies from Espírito Santo and São Paulo States, Brazil, modeled by Kimura-2-parameter (K2P).  Description of nymph.General color dark brown in dorsal view and light yellow in ventral view. Head dark brown with light yellow areas near post-frontal suture, occiput and frontoclypeal region; M-line complete, with well-defined limits; eyes and ocelli black; ocelli with a translucent layer ( Fig. 2A); clypeus dark brown; labrum light yellow; maxillae light yellow with lacinia light brown and galea brown; mandible black with median region dark brown with f5 pointed teeth on distal margin, decreasing in size toward base, but teeth 3 and 4 similar in size ( Figs. 3A, 3B and 3C). Pronotum dark brown with thin median line and scattered rugosities light yellow ( Fig. 2A). Wing pad light with contour partially dark brown ( Fig. 2C). Leg brown, forefemur with a central pale line without setae and with lateral surface covered by sparse bristles, in dorsal view; forefemur anteriorly and posteriorly with higher density of thick bristles, and posteriorly with fringe of hairs; tibia anteriorly with sparse thick bristles, and posteriorly with a row of thick bristles and a well-developed band of hairs ( Figure 3D). Abdomen dark brown, almost black, with a drop-shaped light spot in the last segment ( Fig. 2B). Cerci brown with thick bristles; segments differ in shape and size from base to apex ( Fig. 3E). Thoracic gills ( Fig. 2D): ASC1, AT2, AT3 and PT3.  Measurements(n=4): head width 3-5 mm; head length 2-3 mm; antennae length 7-8 mm; pronotum width 3-3.5 mm; pronotum length 1.5- 2 mm; cercal length 9-12 mm; total length 10-15mm.   FIGURE 2.Nymph of  Anacroneuria flintorum: head and pronotum (A), abdominal segments and cercus (B), mesonotum and metanotum in dorsal view (C) and ventral view (D). Scale: 1 mm.  Adult morphological variation.Examination of specimens from different regions of Atlantic Forest (states of Espírito Santo, Minas Gerais, Santa Catarina, and São Paulo) indicated morphological variation. Forewing length of male adults of  A. flintorumrange from 10 to 15.2 mm(specimens studied herein and Bispo & Froehlich, 2004; Froehlich, 2010, and Baldin et al., 2013). The dorsal color pattern of head of adult of  A. flintorumcan be variable, even within the same population ( Bispo & Froehlich, 2004). In addition, it is possible to observe that the dark brown color of recently collected specimens can become ochraceous when preserved in 80% and 100% ethanol for an extended period of time ( Fig. 4). Therefore, it is difficult to identify the species by color pattern alone. For identification, it is essential to study the male penial armature, which in  A. flintorumis elongated, tapering progressively from base to the apex and has hooks well separated from distal vesicles ( Froehlich, 2002; Bispo & Froehlich, 2004). However, the keel of penial armature may diverge in specimens from the same population and in different populations. In dorsal view, the keel varies from rounded (male specimens from Rio de JaneiroState), pointed (male specimens from PECJ), to convergent or parallel lines (male specimens from PEI). The specimens collected farther north of the species distribution (e. g. ES, RJ and North of SP) have penial armature more robust than specimens from further south (e.g. South of SP and SC).   FIGURE 3.Nymph of  Anacroneuria flintorum: labium (A), right mandible (B), right maxilla (C), pronotum left leg, dorsal view (D); cerci in proximal, medial and distal view (E). Scales: 0.5 mm for A, B and C; 0.5 mm for D; 0.5 mm and 0.25 mm for E, respectively.   FIGURE 4.Males from ES (A), SC (B), PECJ (C) and Paranapiacaba mountains (D and E). Male collected in 2014 (D) and male collected in 1999 (E) not preserved in alcohol.  Teneral adult specimens.Teneral specimens of  A. flintorumstudied are generally brown in color with pale areas along the body; head and pronotum brown with a pale outline. In these specimens, the pronotum does not have lateral folds at the edges, wings are totally pale and the thoracic segments present remnants of gills ( Figure 5D, 5E and 5F). A similar pattern to  A. flintorumis also present in  A. debilis( Almeida & Duarte, 2017)suggesting that several species may be similar when teneral. Therefore, species described as having pale areas on the head and pronotum and milky wings (e.g.  A. petersi Froehlich, 2002) may have been based on teneral specimens, requiring caution for use in identifications.   Remarks.The nymph of  A. flintorumhas a different pattern than the other known  Anacroneurianymphs described from Brazil( Righi-Cavallaro & Lecci, 2010; Avelino-Capistrano et al., 2011a; Novaes et al., 2012; Righi-Cavallaro & Froehlich, 2013; Ribeiro & Gorayeb, 2014; Gonçalves et al., 2017). The color pattern of the head and the drop-shaped light spot in the last abdominal segment are apparently characteristic for this species. In addition to description of the nymph, we present new species records from the states of Minas Geraisand Santa Catarina.  Anacroneuria flintorumhas been collected from streams from protected areas of the Atlantic Forest ( Fig. 6). In general, these streams are pristine and have fast currents and rocky substrates, and undisturbed channels and banks. This suggests that  A. flintorummay be a sensitive taxon to environmental disturbances.