Three new species of the genus Neuquenaphis (Hemiptera, Aphididae, Spicaphidinae) from southernmost South America Author Nafría, Juan Manuel Nieto Author Von Dohlen, Carol D. Author Brown, Paul A. Author Durante, M. Pilar Mier Author Ortego, Jaime Author López Ciruelos, Sara I. Author Licht, Megan text Zootaxa 2019 2019-05-01 4590 5 525 545 journal article 26940 10.11646/zootaxa.4590.5.2 d8837133-c1e6-4024-b530-3de27907aa51 1175-5326 2656391 685DF8B3-F249-40CB-AA50-EF931C287FB8 Neuquenaphis blackmani Nieto Nafría & Brown , sp. n. 2003. Blackman, R.L., Brown, P.A., Ramírez, C.C., Niemeyer, H.M.: Neuquenaphis “ sp. A”. 2018. Blackman, R.L. and Eastop V.F.: Neuquenaphis sp. A of Blackman et al . (2003) . Types. Holotype : apterous viviparous female (number 1 of measurement series), CHILE , Los Ríos region , Valdivia province , Valdivia [approx. 39°50’S , 74°18’W ], 5 December 2000 , on Nothofagus dombeyi , P. A. Brown & C. C. Ramírez leg . ; Karyotype number RLB4587 ; Natural History Museum London collection . Paratypes : 13 apterous viviparous females [apt.], 4 alate viviparous females [al.] and 1 alatoid nymph [nym.], Natural History Museum London and University of León collections. CHILE , Los Ríos region , same data as holotype , 11 apt . and 1 nym .; CHILE , Los Lagos region , Chiloe province , Ancud at Senda Darwin [approx. 41°53’S , 73°39’W , 20 m ], 7 December 2000 , on Nothofagus nitida , 1 nym., P. A. Brown & C. C. Ramírez leg. ; Karyotype number RLB4583 . CHILE , Los Ríos region , Valdivia province , Niebla [ 39°52'S , 73°24'W , 25 m ], 3 February 2016 , on Nothofagus dombeyi , 2 apt. and 4 al., Mier Durante , Nieto Nafría & Ortego leg . Etymology. The specific name of the new species, blackmani , is devoted to Roger L. Blackman (scientific associate of the Natural History Museum, London), who studied the karyotype of the species and recognised the specimens as being of a new species. FIGURE 1. Neuquenaphis blackmani sp. n. A , apterous viviparous female. B , alate viviparous female. Description. Apterous viviparous females ( Figures 1A , 2 A–H). From 14 specimens . When alive pale green. Body 2.15–2.95 mm . Other measurements are in Table 2 . Mounted specimens very pale in general, with cephalic dorsum in part, antennae, clypeus, mandibular and marginal lames, penultimate and ultimate segment of rostrum, legs and finger-shaped processes (whole or mostly) pale brown, other segment of rostrum, siphunculi, genital and anal plates and cauda very pale brown. Spiracular and intersegmental sclerites inconspicuous. No wax pores observed. Finger-shaped processes are ornamented with rings of wrinkles on its proximal part and scarce and small spinules on middle and distal part, with tenuously imbricated margin, and ending in sharp spines, which are much shorter than the apical setae, which are pointed, more or less lancet-shaped or tapering. Head with four fingershaped processes, two anterior with two setae each, and two posterior, and two pairs of seated setae, one pair behind the base of anterior processes, sometimes on small elevations or in several cases sited on the basal part of the process, and the other pair lateral to posterior processes, minute and pointed. Frons sinuate, with conspicuous lateral and medial tubercles, and a tenuous epicraneal line. Ventral face of head smooth in general with a pair of lateral setae, delicate and pointed, and with a medial oval-shaped bulge, rough and with two pairs of setae, thick and pointed. Antennae longer than body and progressively pigmented to apex. Antennal segment I with scarce and feeble striae and 10–12 setae, thick and pointed. Antennal flagellum progressively imbricate from almost smooth segment III proximal part to apex. Inner margin of antennal segment III with 8–12 setae, pointed and thinner than those of segment I. Base of antennal segment VI with (1)2(3) setae, fine and pointed. Primary sensoria small and ciliate; satellite sensoria also ciliate and distal relative to primary. Rostrum short, never reaching beyond middle coxae. Ultimate rostral segment with 3–4 accessory setae, long and delicate. Prothorax with four spinal fingershaped processes and always with a broad marginal bulge, rugose and carrying one seta on its anterior part, and sometimes with a submarginal wart-shaped process, with 1–3 setae, plus a marginal small wart-shaped process or tubercle. Mesothorax and metathorax with spinal, pleural, submarginal and marginal finger-shaped processes on each. Legs slender. Femora smooth; femoral setae scarce, short and pointed. Tibiae with scarce and little marked striae and setae longer and more abundant than those in femora, mainly on its distal third, and also pointed. Tarsi imbricated. First tarsal segments usually with two dorsal setae (sometimes lacking or lost) and (5)7 ventral setae, which are long and delicate. Empodial setae spatulate. Abdominal segments 1 to 4 with spinal, pleural, submarginal and marginal finger-shaped processes on each; abdominal segments 5 and 6 only with three pairs each, lacking submarginal ones. Siphunculus with slight and much spaced striae on proximal part, 2 (sometimes one of them incomplete) to 4 rows of cells at the apex, and a broad flange. Abdominal segment 7 with two pairs of fingershaped processes; segment 8 with one pair of finger shaped processes and 1–3 marginal setae in all, pointed and relatively thick. Knob of cauda elongated, with 27–38(42) setae, long and delicate. FIGURE 2. Neuquenaphis blackmani sp. n. A–H , apterous viviparous female. A , antero-cephalic finger-shaped process. B , prothoracic submarginal wart-shaped process. C , prothoracic marginal wart-shaped process. D, prothoracic posterolateral seta. E , finger-shaped process on abdominal segment 8. F , proximal part of antennal segment VI. G , siphunculus. H , cauda. I–V , alate viviparous female. I , anterior cephalic tubercle. J , K , spinal rod-shaped process on abdominal segments 2 and 3, respectively. L , M , marginal and submarginal, respectively, rod-shaped process on abdominal segment 4. O , P , spinal and marginal, respectively, conical-shaped tubercle on abdominal segment 7. Q , R , abdominal seated setae. S , rod-shaped process on abdominal segments 8. T , middle portion of antennal segment III, with secondary sensoria. U , siphunculus. V , cauda. NOTE: The scales vary according to pieces drawn; see the range of measurements in Table 2. Alatoid nymphs. From 1 specimen . Body 2.35 mm . Very similar to apterous viviparous females, with: (1) frons less sinuous; (2) three or four secondary sensoria on proximal part of antennal segment III, transverse-oval and ciliate, (3) front femora more robust than middle and hind femora; (4) first tarsal segments without dorsal setae; and (5) siphunculi tapering. Measurements in Table 2 . Alate viviparous females ( Figures 1B , 2 I–V). From 4 specimens , measured. When alive pale green with head and thorax brown. Body 2.22–2.55 mm . Other measurements are in Table 2 . Mounted specimens with head, thorax and appendages brown, abdomen very pale, with anal plate and cauda dusky, and siphunculi dark brown. Four pairs of abdominal intersegmental sclerites, small and pale; spiracular sclerites also pale. Tegument smooth in general. No wax pores observed. Head with two anterior conical-shaped tubercles, blunt and smooth, sometimes absent, with two setae, thin, rigid and pointed. Frons gently wavy. Dark brown to black ring around each ocellus; the ring around the anterior ocellus contacts the dark epicraneal line. Seated dorso-cephalic setae similar in shape to those on cephalic processes. Ventro-cephalic setae delicate and pointed. Antennae much longer than body. Antennal segment I with 9–11 setae, shorter than dorso-cephalic ones, delicate and pointed. Antennal flagellum progressively imbricate from smooth proximal part of segment III to apex. Antennal segment III with 20–26 setae on its inner margin, pointed and thinner than those of segment I, and with 1–4 secondary sensoria, placed on the end of second fifth of its length, oval-transverse and with thick margin. Base of antennal segment VI with two setae, fine and pointed. Primary and satellite sensoria ciliate. Rostrum short, never reaching beyond middle coxae. Ultimate rostral segment also short and with 3–4 accessory setae, long and delicate. Prothorax with setae long, thin, rigid and pointed; two spinal setae sometimes placed on conical-shaped tubercles or elevations. Mesothorax sometimes with 2 tubercles similar in shape and size to prothoracic ones. Front legs with dark femorotibial articulation and different in shape to other legs: femur rather more strongly developed than other femora and with a conspicuous subapical tooth, and tibia widened at base. Middle and hind legs slender. Cuticular ornamentation and setae of legs similar to those in apterous viviparous females, except for the presence of 2–4 tooth-like setae (or coarse spines) at the end of each tibia. Abdomen with 8 spinal pairs of processes, rod-shaped on segments 1 to 3 and 8, conical-shaped tubercles on segments 4 to 7, and 3–4 marginal pairs and 1–3 submarginal pairs of processes, conical-shaped tubercles of varied heights, blunt and smooth, or cuticular elevations, all of them with thin, rigid, pointed and long apical seta. Dorsal abdominal setae when they are not on processes, including pleural ones, sometimes placed on very small elevations. Siphunculi as usual in alate females of Neuquenaphis , with striae on the proximal part, 11–15 rows of cells in the distal reticulate part and slightly flanged. Abdominal segment 8 with 2–3 marginal setae in all. Caudal knob elongated and relatively pointed, with 20 – 22 setae. Karyotype. From Blackman et al. (2003) , female number 2n = 12. Nucleotide sequences. Neighbor-joining dendrograms generated from the two mitochondrial loci placed this species closest to N. aurata sp. n. ( Figure 7 ). Genetic distances between N. blackmani sp. n. and N. aurata sp. n. at the two loci are low (1.1–1.8%; Table 3 ) but within the range for other well-accepted species ( Foottit et al ., 2008 ; Nieto Nafría et al ., 2016 ; Nieto Nafría et al. , 2019 ). N. blackmani sp. n. is genetically well differentiated from the remaining Neuquenaphis species sampled, with distances ranging from 7.1–18.7% across the two loci ( Table 3 ). Biology. Nothofagus dombeyi (Mirb.) Oerst. and N. nitida (Phil.) Krasser (Nothofagaceae) are the host-plant species of Neuquenaphis blackmani sp. n. Oviparous females and males have not been collected, but we predict that the species is holocyclic, because other species of Neuquenaphis are known to be holocyclic, althoug these two species of Nothofagus are evergreen trees. Distribution. Neuquenaphis blackmani sp. n. has been recorded in localities of the provinces of Valdivia ( Los Ríos ) and Chiloe ( Los Lagos ), although it is possible that it has a wider distribution, following the distribution of its host plants. TABLE 2. Metric features of apterous viviparous females [apt. viv. fem.], alate viviparous females [al. viv. fem.] and alatoid nymphs [alat. nym.] of Neuquenaphis blackmani sp. n. , N. aurata sp. n. and N. ramirezi sp. n. Processes and their setae in italics.

N. blackmani	N. blackmani	N. blackmani	N. aurata	N. aurata	N. ramirezi	N. ramirezi
apt. viv. fem.	alat. nym.	al. viv. fem	apt. viv. fem.	al. viv. fem	apt. viv. fem.	alat. nym.
Antenna [mm]	2.62–3.50	3.25	4.30–4.88	2.99–4.05	4.10	1.90–2.45	1.60–1.90
Antenna / body [times]	1.19–1.40	1.38	1.78–1.93	1.10–1.51	1.94–1.95	0.94–1.30	0.85–1.31
Antennal segment III [mm]	0.81–0.96	0.69	1.22–1.32	0.74–1.10	1.03–1.04	0.43–0.63	0.31–0.37
Antennal segment IV [mm]	0.58–0.72	0.61	0.89–1.03	0.53–0.89	0.78–0.82	0.32–0.49	0.26–0.30
Antennal segment V [mm]	0.51–0.64	0.64	0.78–0.91	0.49–0.77	0.81–0.82	0.34–0.46	0.29–0.35
Antennal segment VI – base [mm]	0.14–0.20	0.17	0.17–0.20	0.16–0.23	0.18–0.19	0.11–0.18	0.11–0.15
Antennal segment VI – processus terminalis [mm]	0.75–0.83	0.92	0.92–1.10	0.68–0.90	1.02–1.04	0.50–0.78	0.49–0.58
Antennal segment VI – processus terminalis / – base [times]	3.8–5.4	5.4	4.8–5.8	3.1–4.8	5.5–5.6	3.3–6.0–	3.5–4.7
Antennal segment VI – processus terminalis / Antennal segment III [times]	0.8–0.9	1.3	0.7–0.8	0.7–0.9	1.00	1. 0–1. 6	1.4–1.74
Setae on inner margin of antennal segment III [μm]	10–15	12–13	12–13	10–15	18	8–15	7–10
Setae on inner margin of antennal segment III [times D ]	0.2–0.4	0.3	0.3	0.2–0.3	0.4	0.3–0.5	0.2–0.3
Cephalic anterior process [mm]	0.14–0.19	0.14	0.02–0.03	0.12–0.20	0.04	0.09–0.20	0.15–0.20
Cephalic anterior process / Antennal segment I [times]	0.84–1.14	0.94	0.14–0.24	0.8–1.2	0.3	1.0–1.9	1.4–2.3
Setae on cephalic anterior process [µm]	10–15	?	25	9–18	25	5–20	?

…continued on the next page TABLE 2 . (continued)

N. blackmani	N. blackmani	N. blackmani	N. aurata	N. aurata	N. ramirezi	N. ramirezi
apt. viv. fem.	alat. nym.	al. viv. fem	apt. viv. fem.	al. viv. fem	apt. viv. fem.	alat. nym.
cephalic posterior process [mm]	0.13–0.18	0.13	////	0.11–0.19	T////	0.10–0.21	0.16–0.18
Setae on cephalic posterior process [µm]	7–13	?	////	5–13	////	3–15	3–5
Setae behind cephalic anterior process [μm]	15–25	20	25	17–28	30	13–35	8–15
Setae cephalic postero-lateral [μm]	10–12	10	20–25	11–19	10	5–8	5
Setae on ventral oval bulge [μm]	20–33	37	25–38	17–37	25	15–37(43)	20–33
Setae cephalic ventro-lateral [μm]	27–50	47	32–43	25–50	28	(15)20–40	17–30
Ultimate rostral segment [mm]	0.09–0.10	0.08	0.08	0.09–0.10	0.08	0.09–0.12	0.09–0.10
Ultimate rostral segment / its basal width [times]	1.1–1.4	1. 5	1.0–1.1	0.9–1.3	1.1	1.5–1.9	~1.8.
Ultimate rostral segment / second segment hind tarsus [times]	0.5–0.6	0.5	0.6	0.5–0.6	0.5	0.7–0.9	0.7
Prothoracic spinal processes [mm]	0.14–0.17	0.14	0.02–0.03	0.10–0.31	///	0.10–0.30	0.15–0.16
Setae on prothoracic spinal processes [µm]	9–11	11	20–25	8–15	///	3–13	2–10
Setae on prothoracic marginal processes [µm]	10–15	25	22–35	8–23	///	5–13	4–5
Setae on hind tibia, middle third, outside [μm]	20–38	25	17–25	15–38	18	10–25	18–27
Setae on hind tibia, middle third, outside / diameter of tibia at insertion point [times]	0.4–0.8	0.6	0.5–0.7	0.3–0.8	0.5	0.4–0.8	0.6–0.8
Second segment hind tarsus [mm]	0.16–0.17	0.15	0.13–0.14	0.15–0.18	0.16	0.12–0.16	0.13–0.14

…continued on the next page TABLE 2 . (continued)

N. blackmani	N. blackmani	N. blackmani	N. aurata	N. aurata	N. ramirezi	N. ramirezi
apt. viv. fem.	alat. nym.	al. viv. fem	apt. viv. fem.	al. viv. fem	apt. viv. fem.	alat. nym.
Abdominal segments 1–3 spinal processes [mm]	0.14–0.20	0.20	0.05–0.09	0.12–0.21	0.10	0.08–0.27	0.15–0.19
Setae on abdominal segments 1–3 spinal processes [µm]	7–10	11	25–38	10–18	28	2–8	2–4
Abdominal segments 1–4 marginal processes [mm]	0.14–0.20	0.20	0.02–0.04	0.12–0.21	0.03	0.08–0.27	0.15–0.19
Setae on abdominal segments 1–4 marginal processes [µm]	7–10	11	22–35	10–18	33	2–8	2–4
Abdominal segments 1–4 submarginal processes [mm]	0.11–0.16	0.06	0.02–0.04	0.10–0.23	0.05	(0.01)0.07– 0.13	0.04–0.08
Setae on abdominal segments 1–4 submarginal processes [µm]	7–15	12–13	20–30	8–18	30	5–15	2–7
Siphunculus [mm]	0.17–0.22	0.13	0.15–0.18	0.15–0.23	0.15	0.10–0.19	~ 0.13
Siphunculus / body [times]	0.06–0.08	0.05	0.06–0.08	0.05–0.08	0.07	0.05–0.09	~ 0.06
Abdominal segment 8 process [mm]	0.16–0.22	0.14	0.03–0.08	0.17–0.27	0.11	0.10–0.22	0.15–0.28
Setae on abdominal segment 8 process [µm]	13–20	15	35–40	15–18	35	5–20	2–5
Lateral setae on abdominal segment 8 [μm]	25–40	32	25–45	25–50	37–38	10–35	7–8
Caudal knob [mm]	0.15–0.22	///	0.12–0.13	0.17–0.27	0.11	0.10–0.14	///
Caudal knob / its maximal width [times]	1.2–1.9	///	1. 7–1.7	1.2–2.0	1.6	1.3–2.3	///

TABLE 3 . Genetic distances between Neuquenaphis samples sequenced in this study. Kimura 2-parameter distances for the COI barcode locus are shown above the diagonal and Tamura-Nei distances for the tRNA-COII locus are shown below the diagonal. We were unable to obtain tRNA-COII sequences for CHI-393, CHI-261, and CHI-463. NOTE: In the heading row numbers of samples to which the analysed specimens belong, starting with CHI, and in parentheses accession GenBank numbers are presented. In the first column only respective sample numbers are included. Sample data: CHI-255, Guardería Grey (Magallanes, Última Esperanza), Nothofagus betuloides , 5-February-2016; CHI-262, Pehoé island (Magallanes, Última Esperanza), Nothofagus pumilio , 5-February-2016; CHI-244, Las Vacas (Magallanes, Magallanes), Nothofagus pumilio , 4-February-2016; CHI-261, Pehoé island (Última Esperanza), Nothofagus betuloides , 5-February-2016; CHI-292, Fuerte Bulnes (Magallanes, Magallanes), Nothofagus betuloides , 7-February-2016; CHI-215, Niebla (Los Ríos, Valdivia), Nothofagus dombeyi , 3-February-2016; CHI-393, Petrohué (Los Lagos, Llanquihue), Nothofagus dombeyi , 13-February-2016; CHI-213, Villarrica volcano slopes (La Araucanía, Cautín), Nothofagus dombeyi , 2-February-2016; CHI-385, Cochamó (Los Lagos, Llanquihue), Nothofagus dombeyi , 13-February-2016.

N. similis CHI-255 ( MK226131 )	N. similis CHI-262 ( MK226130 )	N. edwardsi CHI-244 ( MK226137 )	N. ramirezi sp. n. CHI-261 ( MK226135 )	N. aurata sp. n. CHI-292 ( MK226132 )	N. blackmani sp. n. CHI-215 ( MK226133 )	N. palliceps CHI-393 ( MK226138 )	N. palliceps CHI-213 ( MK226139 )	N. bulbicauda CHI-385 ( MK226135 )	N. michelbacheri CHI-463 ( MK226134 )
N. similis – CHI-255	0	4.3%	10.2%	10.8%	9.8%	8.2%	8.4%	18.5%	15.2%
N. similis – CHI-262	0	4.3%	10.2%	10.8%	9.8%	8.2%	8.4%	18.5%	15.2%
N. edwardsi – CHI-244	3.20%	3.20%	12.3%	12.9%	11.9%	10.3%	10.4%	20.5%	17.3%
N. ramirezi sp. n. – CHI-261	/	/	/	13.8%	12.8%	11.2%	11.4%	21.5%	18.2%
N. aurata sp. n. – CHI-292	11.50%	11.50%	11.3%	/	1.8%	8.0%	8.2%	19.7%	16.4%
N. blackmani sp. n. – CHI-215	11.20%	11.20%	11.0%	/	1.1%	7.1%	7.2%	18.7%	15.5%
N. palliceps – CHI-393	/	/	/	/	/	/	0.3%	17.1%	13.9%
N. palliceps – CHI-213	8.30%	8.30%	8.2%	/	7.7%	7.4%	/	17.3%	14.0%
N. bulbicauda – CHI-385	12.00%	12.00%	11.9%	/	13.8%	13.5%	/	10.6%	17.9%

Taxonomic discussion, diagnostic characteristics. Neuquenaphis blackmani sp. n. must be included in the nominotypical subgenus. Neuquenaphis essigi , N. edwardsi , N. schlingeri and N. blackmani sp. n. have the same diploid chromosome number, 12, but their idiograms are different ( Blackman et al. , 2003 ). Apterous and alate viviparous females of N. essigi have 2 pairs of cephalic processes and have no marginal setae on abdominal segment 8 (for this reason this species is included in the subgenus Spicaphis ) and antennae are shorter than body. Apterae of N. edwardsi also have antennae shorter than body, have no submarginal abdominal processes and apical setae on processes are different in shape (blunt or with thickened apex) and size (short) than those in the new species; alatae of N. edwardsi have antennae somewhat shorter (at most 1.5 times body), few abdominal processes and pigmented marginal sclerites. Both apterae and alata of N. schlingeri have no abdominal spinal processes. The pattern of abdominal processes of the apterous viviparous females of N. blackmani sp. n. and N. valdiviana (whose presence is limited to Valdivia province [ Los Ríos ]) is similar to each other. Apterous females of N. valdiviana have antennae shorter and setae on processes thickened at apex and shorter than those of N. blackmani ; alate females of N. valdiviana have many dorso-abdominal pigmented sclerites and antennae also relatively short. The diploid chromosome number of N. valdiviana is 6. Apterous viviparous females of N. palliceps also have long antennae, short ultimate rostral segment and poorly pigmented abdomen; specimens of summer and autumn generations have six longitudinal series of finger-shaped processes, but apterae of early generations, including fundatrices, also have submarginal processes on presiphuncular abdominal segments; they can be distinguished from those of N. blackmani by having processes that are not so spinulated and apical setae that are blunt, and they have no prothoracic marginal bulge. Alate viviparous females of N. palliceps have fewer protruding processes than N. blackmani and their antennal segment VI processus terminalis is relatively longer, 5.5–8.8 in N. palliceps (from Quednau, 2010 and our data) versus 4.7–5.8 in N. blackmani . Diploid chromosome number is an additional difference: 6 in N. palliceps and 12 in N. blackmani . Apterous viviparous females of N. staryi (diploid chromosome number = 14) and N. michelbacheri (unknown karyotype) are not known; they could resemble those of the new species; in the absence of them we can use the characteristics of their alatoid nymphs, which are known. Apical setae on processes of both species are different to those of N. blackmani sp. n. : short and very thick at the apex in N. michelbacheri and shorter than the apical spines in N. staryi . Alatae of both species can be distinguished from those of N. blackmani by the ultimate rostral segment (longer in them than in N. blackmani ); in addition, alatae of N. michelbacheri have antennae only slightly longer than body and carry more secondary sensoria on antennal segment III, and alatae of N. staryi have no abdominal presiphuncular marginal processes. The identification of apterous and alate viviparous females of N. blackmani sp. n. that can be collected in the future can be done using the dichotomous key included in the taxonomic discussion of Neuquenaphis ramirezi sp. n.