Description of a new Oriental stonefly species, Phanoperla constanspina (Plecoptera: Perlidae) from Mindanao, Philippines and association of life stages using DNA barcoding Dela Cruz, Ian Niel B. Nuñeza, Olga M. Lin, Chung-Ping Zootaxa 2016 4193 1 102 116 8N8PS [151,459,843,869] Insecta Perlidae Phanoperla Animalia Plecoptera 2 104 Arthropoda species constanspina sp. nov.     Materialexamined: Philippine Islands, Mindanao, Mt. Malindang, Layawan River. Holotypemale, 185 masl, N 08°25.418’ E 123°41.806’,  3 October 2013(A-G23.m). Paratypes 5 males: 1 male, 336 masl, N 08°23.690’ E 123°40.056’, 1 October, 2013 (A-E23.m); 4 males, 161 masl, N 08°25.971’ E 123°42.492’, 4 October 2013(A- H7.m, A-H11.m, A-H12.m, A-H16.m). Nymphal paratypes 3 males: 185 masl, N 08°25.418’ E 123°41.806’, 3 October, 2013 (N-G49.m); 161 masl, N 08°25.971’ E 123°42.492’, 4 October, 2013 (N-H27.m); 73 masl, N 08°27.290’ E 123°44.697’, 6 October, 2013 (N-J26.m). Nymphal paratypes: 2 males, 3 females, 956 masl, N 08°19.417’ E 123°38.017’, 16 April 2014(N-B30.f); 1 male at 264 masl, N 08°24.452’ E 123°40.887’, 2 October 2013(N-F20.f). The Holotype is deposited in the collection of the Natural Science Museum, MSU-IIT,  Philippinesand paratypesin the Insect Collection, National Museum of Natural Science, Taichung,  Taiwan( NMNS).  Allspecimens were collected and prepared by INDC.  Adult habitus. General color brown, sometimes appearing dark brown when membranous dark wings fold flat at rest. Head wider than pronotum, bearing a distinctive stretched M-shaped pattern, appearing to be paler to surrounding area ( Figs. 1A and 1B). Frons with dark brown pigment along occipital line and ridge ( Fig. 1A). Compound eyes bulging, relatively large. Ocelli of 0.76 ± 0.1 mmin diameter in males and 1.0 ± 0.0 mm diameter in females, with lateral inner dark rings. Median occipital line conspicuous and well-marked. Median suture forms a bare depression with raised margins. Antennae and palpi pale brown basally, distally dark. Pronotum rectangular with rounded corners, dark lines occur on margins and median suture, rugosities conspicuous on pronotum. Smaller hairs cover pronotum and most of the head. Wings brown and slightly infuscated with dark venation ( Figs. 2A and 2B). Legs dark brown from the proximal half of femora towards tibia and tarsus ( Fig. 3). Small thorn-like hairs present particularly on femoral margins.   Male. Body length 9.12 ± 0.51 mm, forewing 9.35 ± 0.47 mm, hindwing 8.25 ± 0.2 mm. Width of head at pronotum 1.62 ± 0.13/1.5 ± 0.1 mm( Fig. 1A). Femur 1.91 ± 0.03 mm, tibia 2.28 ± 0.06 mm, tarsus 0.62 ± 0.03 mm( Fig. 3). Brushes of long setae on abdominal sternites S4-S8, usually strongest on S5-S7 ( Fig. 4). Tergites simple (T7 and T8), T9 with a median depression but slightly raised at sides with short conical-shaped setae ( Fig. 5). Anterior process of the hemitergite unbranched and of variable shape ( Fig. 5). Penial capsule short, 0.76 ± 0.1 mm, but tubular at base, faintly sclerotized for muscle attachment ( Fig. 6). Everted sac almost equal to penial tube in length and armed with small jet-black posterior spines, fewer and becoming shortest towards the base ( Fig. 6). Apex of the sac cylindrical and black covered with short and fine black hair-like spines of uniform length on anterior surrounding structure ( Fig. 6).  Female. Body length 12.6 mm, forewing 12.46 mm, hindwing 12.1 mm. Head/pronotum width 2.23/ 2.09 mm( Fig. 2A). Femur 2.06 mm, tibia 2.69 mm, tarsus 0.85 mm. Subgenital plate undeveloped, displaying a simple posterior margin on S8.  Egg. General outline droplet-shaped, somewhat elongated, and very similar to  P. batac( Fig. 8A). Total length ca. 522.98 ± 0.06 µm and equatorial width ca. 198.6 ± 0.12 µm, with long and slender collar with a length of 137.75 ± 0.23 µm. Rugose processes are observed on the collar extension while the apex is flanged with a cork-like rim ( Fig. 8C). Chorionic surface even and simple but with few, fine punctations ( Fig. 8B) around the subapical ring of the anterior end ( Fig. 8D).   FIGURES 1.  Phanoperla constanspina  sp. nov.head and pronotum. (1A) Male adult, indicating occipital line. (1B) Female adult. Scale = 1.00 mm.   FIGURE 2.  Phanoperla constanspina  sp. nov.male wing. (2A) Right forewing, showing the forking of Cu2 from Cu1. (2B) Right hindwing. Scale = 1.00 mm.   FIGURES 3–4.  Phanoperla constanspina  sp. nov.male. (3) Right foreleg. (4) Abdominal sternite showing hair brushes on each segment (S5–S8). Penial capsule visible on S9. Scale = 1.00 mm.   FIGURE 5.  Phanoperla constanspina  sp. nov.male dorsal terminalia showing the hemitergal (arrow) and paired clumps of short setae located on medial tergum 9. Scale = 1.00 mm.   FIGURE 6.  Phanoperla constanspina  sp. nov.penial tube showing the everted penial sac at the base. Scale = 0.2mm.  Nymphal habitus. Females are considerably larger than males. General pigment pattern variable in mature individuals, ranging from light to dark brown and sometimes black with varying hues and pigment intensity on a few structures such as tips of wingpad and tergal margins ( Figs. 11A and 11B). Heads always with darker frons and paler M-line pattern, which is evident on the adults. Well-marked horizontal suture separating labrum from head, making a protruding labrum slightly noticeable. Pronotal disc usually wider than head, occipital ridge positioned more forward from posterior margin of head base ( Figs. 11A and 11B). Pronotum oval-shaped. Wingpads medium and simple. Abdomen slender relative to body size with obvious stripes separating terga from each other ( Figs. 11A and 11B). Antennal flagellum long and setaceous, cercus medium-sized and bearing short, stiff hairs. Basal attachment of cerci is a little plump extending wide from the base of T10 while the apical tip becomes thin. Mandible ovate, base plumper than the rest of the structure ( Fig. 9). Teeth uneven in length and size, slightly bent towards the inner side, brushes of bristled hair present laterally and adjacent to the shortest tooth on the apex. Leg completely light brown with numerous fine and long lateral tibial and femoral hairs ( Fig. 10). Thorn-like hairs sparsely distributed on the proximal half of the femur and sometimes along tibial margins, but none on the tarsus.  Male nymph.Body length 10.52 ± 0.58 mm, abdominal length 4.75 ± 0.32 mm( Fig. 11A and 11B). Width of head/pronotal disc 2.12 ± 0.12/2.22 ± 0.16 mm( Fig. 11A and 11B). Ocelli 1.06 ± 0.28 mmdiameters apart. Mandible 0.74 ± 0.03 mm( Fig. 9). Mesothoracic and metathoracic width 2.53 ± 0.14 mmand 2.43 ± 0.36 mm, respectively. Femur 1.67 ± 0.19 mm, tibia 1.59 ± 0.13 mm, tarsus 0.8 ± 0.07 mm( Fig. 10). Cercus 3.53 ± 0.35 mm.  Female nymph. Body length 14 ± 0.98 mm, abdominal length 6.54 ± 0.46 mm. Width of head against pronotal disc 2.73 ± 0.25/2.94 ± 0.17 mm, ocelli 1.5 ± 0.23 mmdiameters apart, mandible 1.06 ± 0.07 mm. Mesothoracic and metathoracic width 3.31 ± 0.26 and 3.19 ± 0.28 mm, respectively. Femur 2.14 ± 0.35 mm, tibia 2.02 ± 0.29 mm, tarsus 1.02 ± 0.15 mm. Cercus 4.29 ± 0.48 mm.  Habitat. Adults of  P. constanspinawere collected from Layawan River (alt. 73–1,218 masl) of Mt. Malindang, northern Mindanao, Philippines. This fast flowing river has an average width of approximately 10 m, depth of 80 cmand is canopied with forest trees of mainly dipterocarps and palm species, as well as shrubs near the riverbank. Nymphs of  P. constanspinaare often sympatric with  Neoperlaspecies occurring under small to medium-sized cobbles, or can be found in leaf pack litter accumulated on rocks in fast currents.   Etymology. The species name refers to the constant array of fine, spine-like hairs of uniform length covering the apical tip of penial sac.  Association of sexes and life stages. The pairwise distances of CO1barcode sequences between males and presumptive female of  P. constanspinarevealed 100% similarity (no sequence divergence), suggesting that they belong to the same species. Between adults and presumptive nymphs, the sequence divergence is 0%, showing both life stages belong to the same species (see Table 2).   Diagnosis. The new species is a member of the  P. anomalaspecies complex and morphologically most similar to  P. bakeri, known from the Philippinesand Borneo. Similarities between  P. constanspinaand  P. bakeriare in the structure of the penial capsule with a short, plump and unbranched penial tube. However, the everted sac of  P. constanspinais distinguished by a blackened and rounded apical tip covered by small, spine-like hairs surrounding and covering the apex, where the jet-black hooks are positioned posteriorly forming a triangular shape, but with fewer hooks towards the base.  Phanoperla constanspinahas a droplet-shaped egg with long, slender collar, which 1325789622 2013-10-03 Philippines Material 8.423634 Mt. Malindang 1 123.69677 Mindanao 2 104 1 holotype 1325789623 Philippines Taichung 2 104 1 Taichung paratype 1325789620 [1189,1386,1238,1263] NMNS Taiwan Taiwan 2 104 1 paratype 1325789621 INDC Taiwan All 2 104 1 paratype