Pleotrichophorus blackmani Barjadze, Halbert & Kanturski, 2022

Barjadze, Shalva, Halbert, Susan E., Moore, Matthew R. & Kanturski, Mariusz, 2022, Morphology, molecular phylogenetics, and DNA barcoding revealed a new unusual species of the aphid genus Pleotrichophorus from the USA (Insecta, Hemiptera Aphididae), Zootaxa 5183 (1), pp. 390-422 : 397-416

Pleotrichophorus blackmani Barjadze, Halbert & Kanturski , sp. n.

( Figs 1–13 ; Table 2 )

Material examined: HOLOTYPE. Apterous viviparous female, UNITED STATES OF AMERICA, Florida, St. Lucie Co., Port St. Lucie, 06.i.2003, on Euthamia graminifolia (= E. caroliniana as it is written on the original label), Kenneth L. Hibbard leg., (FSCA# E2003-166), BM-2003-16a, BMNH.

PARATYPES. The following specimens have the same data as the HOLOTYPE (FSCA# E2003-166): Alate viviparous female, BM-2003-16b, BMNH; oviparous female, BM-2003-16c, BMNH; apterous viviparous female, 03/01/01/1- US, DZUS; alate viviparous female 03/01/01/2- US, DZUS; apterous viviparous female, FSCA N E2003- 166(10), FSCA; apterous viviparous female, FSCA N E2003-166 (18), FSCA; apterous viviparous female, FSCA N E2003-166(19), FSCA; apterous viviparous female, FSCA N E2003-166(22), FSCA; apterous viviparous female, FSCA N E2003-166(22), FSCA; apterous viviparous female, FSCA N E2003-166(23), FSCA; apterous viviparous female, FSCA N E2003-166(24), FSCA; apterous viviparous female, FSCA N E2003-166(25), FSCA; apterous viviparous female, N TA–T–00046, IZISU; apterous viviparous female, FSCA N E2003-166(27), FSCA; apterous viviparous female, N TA–T–00047, IZISU; apterous viviparous female, FSCA N E2003-166(29), FSCA; alate viviparous female, FSCA N E2003-166(4), FSCA. Paratypes with other collection data include: apterous viviparous female, USA, Florida, St. Lucie Co., Port St. Lucie, 16.i.2003, on Euthamia graminifolia, Kenneth L. Hibbard , leg., FSCA N E2003-257, N TA–T–00048, IZISU; apterous viviparous female, 16.I.2003, FSCA N E2003-257(2), FSCA; apterous viviparous female, 16.i.2003, FSCA N E2003-257(3), FSCA; apterous viviparous female, FSCA N E2003-257(4), FSCA; apterous viviparous female, FSCA N E2003-257(5), FSCA; apterous viviparous female, 16.i.2003, FSCA N E2003-257(6), FSCA; apterous viviparous female, FSCA N E2003-257(7), FSCA; apterous viviparous female, FSCA N E2003-257(8), FSCA; apterous viviparous female, N TA–T–00049, IZISU; apterous viviparous female, FSCA N E2003-257(11), FSCA; apterous viviparous female, FSCA N E2003-257(12), FSCA; apterous viviparous female, FSCA N E2003-257(13), FSCA; apterous viviparous female, FSCA N E2003-257(14), FSCA; apterous viviparous female, FSCA N E2003-257(15), FSCA; apterous viviparous female, FSCA N E2003- 257(16), FSCA; apterous viviparous female, FSCA N E2003-257(17), FSCA; apterous viviparous female, FSCA N E2003-257(19), FSCA; apterous viviparous female, N TA–T–00050, IZISU; alate viviparous female, USA: Florida, St. Lucie Co., Port St. Lucie, 16.i.2003, on Euthamia graminifolia, Kenneth L. Hibbard leg., FSCA N E2003- 258(1), FSCA; alate viviparous female, FSCA N E2003-258(2), FSCA; alate viviparous female, N TA–T–00051, IZISU, other data as in the holotype; alate viviparous female, USA, Florida, Collier Co., Immokalee, SW FL R&E Center, 21-28.vi.2012, suction trap, Scott Croxton leg., FSCA N 2012 5221, TA–T–00052, IZISU; alate viviparous female, USA, Florida, Polk Co., Winter Haven, DPI Citrus Arboretum, 05-12.XII.2013, tall suction trap, Peggy Sieburth leg., FSCA N E2013-9129, N TA–T–00053, IZISU; alate viviparous female, USA, Florida, Collier Co., Immokalee, SW Florida R&E Center, 06-13.xii.2013, short suction trap, Scott Croxton, leg., FSCA N E2013- 15 FSCA; 5 apterous viviparous females, USA, Florida, Alachua Co., Gainesville, Morningside Nature Center, 15.x.2020, on Euthamia graminifolia, Catherine White & Susan Halbert , leg., FSCA N E2020-4081, FSCA. 1 alate viviparous female on SEM stub, FSCA N E2020-4081 as DZUS-SEM-USA-1; Molecular vouchers (slide mounted in balsam): 3 apterous viviparous females (extractions 1, 2, 4), 1 nymph (extraction 3), and 1 partial specimen (extraction 5), all FSCA N E2020-4081, data as above.

Description. Apterous viviparous female (n=31) ( Figs 1–3 ; Table 2 )

Colour in life: body in general light green with yellowish-green head, light green thorax and green abdomen. Antennal flagellum brown with pale basal part of ANT III. Legs with pale femora and brown tibiae with paler central sections. Tarsi brown. SIPH and cauda light green ( Fig. 1a, b ). Pigmentation on slide: head yellow, slightly sclerotized; ANT brown with ANT I, ANT II and basal part of ANT III yellow. Femora yellow; tibiae dark with paler middle sections, tarsi dark; abdomen pale. SIPH and cauda yellow ( Fig. 2a ).

Morphological characters: HW 0.15–0.19 × ANT. Head with short and very short, rigid setae with blunt or slightly capitate tips, 0.009 –0.026 mm long. ANT tubercles each with 0–1 seta on internal angles ( Fig. 3a ). ANT 1.45–1.83 × BL. ANT III with 1–5 small and rounded secondary rhinaria with poorly-developed sclerotized rims ( Fig. 3b ), ANT IV shorter than ANT V. PT 5.99–7.63 × BASE. Other antennal ratios: VI:III 1.86–3.30, V:III 0.73– 0.87, IV:III 0.69–0.82, PT:III 1.60–2.01, PT:IV 2.07–2.66, PT:V 2.10–2.56. ANT chaetotaxy: ANT have very short, thick, rigid setae with blunt apices ( Fig. 3c ). ANT III setae 0.009 –0.013 mm long, LS ANT III 0.29–0.37 × BD III. ANT I with 6–9, ANT II with 2–5, ANT III with 20–30, ANT IV with 8–18, ANT V with 7–12 setae. ANT VI with 2–4 basal, 4 apical and 3–7 setae along the PT. Rostrum reaching middle coxae. URS 0.15–0.19 × ANT III, 0.07– 0.10 × ANT VI, 0.08–0.11 × PT, 0.55–0.71 × BASE and 0.73–0.88 × HT II with 3–5 fine and pointed accessory setae. Mesosternal furca fused, wide, without stem. III FEMORA have very short, stiff, rigid and blunt setae, 0.007 – 0.016 mm long. Setae on III TIBIAE short to medium-long, blunt in the proximal part and pointed in the distal part, 0.010 –0.026 mm long. HT I with 3:3:3 setae, HT II 0.19–0.23 × ANT III, 0.09–0.11 × ANT VI, 0.10–0.13 × PT and 0.70–0.84 × BASE. Abdomen membranous, without sclerotization and sometimes with 0–4 marginal tubercles. Setae on ABD dorsum are very few, very short, rigid and blunt or slightly capitate, 0.004 –0.008 mm long.ABD VIII with 5–7 moderately capitate setae, without sclerotised bar. SIPH tubular, tapering without subapical reticulation and without flange ( Fig. 3d, e ). SIPH 1.54–2.25 × cauda, 0.18–0.26 × BL and 0.61–0.80 × ANT III. Genital plate with 2–3 anterior, 0–4 median and 4–8 posterior setae. Cauda triangular, 1.33–1.88 × its width at base and 0.11–0.16 × BL with 4–5 long and pointed setae ( Fig. 3f ).

Alate viviparous female (n=10) ( Figs 1–3 ; Table 2 )

Colour in life: body green with yellowish-green thorax, dark brown antennae and tibiae of legs. Wings hyaline with brown venation. SIPH and cauda green ( Fig. 1c, d ). Pigmentation on slide: head pale to yellow, slightly sclerotized; ANT yellow with darker ANT III and sometimes basal part of ANT IV. Femora pale to yellow; tibiae brown with paler middle sections, tarsi brown;. Abdomen pale; SIPH and cauda pale to yellow ( Fig. 2b ).

Morphological characters: HW 0.13–0.17 × ANT. Head with short and very short, rigid setae with blunt or slightly capitate setae, 0.008 –0.017 mm long. ANT tubercles each with 2–3 setae on internal angles. ANT 1.65–2.10 × BL. ANT III with 13–19 small and rounded secondary rhinaria with well-developed sclerotized rims ( Fig. 3g ), ANT IV with 5–10 secondary rhinaria, ANT V with 0–3 secondary rhinaria. PT 6.82–8.01 × BASE. Other antennal ratios: VI:III 2.04–2.50, V:III 0.80–0.91, IV:III 0.90–1.03, PT:III 1.79–2.21, PT:IV 2.20–2.56, PT:V 2.18–2.62.ANT chaetotaxy: ANT have very short, thick, rigid setae with blunt apices. ANT III setae 0.008 –0.014 mm long, LS ANT III 0.27–0.38 × BD III. ANT I with 5–9, ANT II with 4–5, ANT III with 17–26, ANT IV with 9–16, ANT V with 9–13 setae. ANT VI with 2–4 basal, 4 apical and 4–10 setae along the PT. Rostrum reaching mesosternum. URS ( Fig 3h ) 0.14–0.18 × ANT III, 0.06–0.08 × ANT VI, 0.07–0.09 × PT, 0.49–0.68 × BASE and 0.74–0.91 × HT II with 4 fine and pointed accessory setae. Forewing with typical venation, media twice-branched ( Fig. 3i ). III FEMORA have short, stiff, rigid and blunt setae, 0.011 –0.015 mm long. Setae on III TIBIAE short to medium-long, blunt in the proximal part and pointed in the distal part, 0.021 –0.028 mm long. HT I with 3:3:3 setae, HT II 0.19–0.22 × ANT III, 0.08–0.09 × ANT VI, 0.10–0.11 × PT and 0.66–0.84 × BASE. Abdomen membranous, without sclerotization and sometimes with 3–4 marginal tubercles. Setae on ABD very few, very short, rigid and blunt, 0.006 –0.011 mm long. ABD VIII with 5–8 setae, without sclerotised bar. SIPH tubular, slightly tapering, without subapical reticulation and without flange. SIPH 1.51–1.75 × cauda, 0.15–0.23 × BL and 0.52–0.63 × ANT III. Genital plate with 2–3 anterior, 1–4 median and 5–8 posterior setae. Cauda triangular, 1.38–1.94 × its width at base and 0.10–0.14 × BL with 4–5 long and pointed setae.

Oviparous female (n=1) ( Fig. 4 ; Table 2 )

Colour in life: unknown. Pigmentation on slide: head slightly sclerotized, yellow; ANT brown with ANT I, ANT II and basal part of ANT III yellow. Femora yellow; tibiae brown with paler middle sections, tarsi brown; abdomen pale; SIPH and cauda pale-yellow ( Fig. 4a ). Morphological characters: HW 0.17 × ANT. Head with short and very short, rigid setae with blunt or slightly capitate setae, 0.007 –0.017 mm long. ANT tubercles each with 0–1 seta on internal angles. ANT 1.32–1.33 × BL. ANT III with 4–7 small and rounded secondary rhinaria with poorlydeveloped sclerotized rims, ANT IV shorter than ANT V. PT 6.40–6.60 × BASE. Other antennal ratios: VI:III 1.81– 1.90, V:III 0.80–0.81, IV:III 0.73–0.75, PT:III 1.57–1.65, PT:IV 2.13–2.20, PT:V 1.95–2.20. ANT chaetotaxy: ANT have very short, thick, rigid setae with blunt apices. ANT III setae 0.009 –0.010 mm long, LS ANT III 0.23–0.25 × BD III. ANT I with 3, ANT II with 4, ANT III with 20–21, ANT IV with 10–13, ANT V with 8–9 setae. ANT VI with 3–4 basal, 4 apical and 5–7 setae along the PT. Rostrum reaching middle coxae. URS 0.14–0.15 × ANT III, 0.07– 0.08 × ANT VI, 0.09 × PT, 0.60 × BASE and 0.75–0.78 × HT II with 4 fine and pointed accessory setae. Mesosternal furca fused, wide, without stem. III FEMORA have very short, stiff, rigid and blunt setae, 0.007 –0.010 mm long. Setae on III TIBIAE short to medium-long, blunt in the proximal part and pointed in the distal part, 0.008 –0.040 mm long. III TIBIAE very slightly swollen in the proximal part with 25–31 rounded or ∞-shaped pseudosensoria, which are sometimes fused into larger and irregular ones ( Fig. 4 b, c ). Pseudosensoria distributed only in the distal part of tibiae. HT I with 3:3:3 setae, HT II 0.19 × ANT III, 0.10 × ANT VI, 0.11–0.12 × PT and 0.76–0.80 × BASE. Abdomen membranous, without sclerotization. Setae on ABD very few, very short, rigid and blunt, 0.005 –0.007 mm long. ABD VIII with 4 setae, without sclerotised bar. SIPH tubular, tapering without subapical reticulation and without flange. SIPH 1.95–2.00 × cauda, 0.19–0.20 × BL and 0.70–0.73 × ANT III. Genital plate with 3 anterior, 5 median and 15 posterior setae. Cauda triangular, 1.83 × its width at base and 0.10 × BL with 4 long and pointed setae.

Biology: Colonies of the new species live in the inflorescence of Euthamia graminifolia (L.) Nutt. ( Asteraceae ). Probably this species is monoecious holocyclic on Euthamia . Despite the fact that males of this taxon have not been detected yet, we do not exclude that it lives anholocyclically in warm areas such as in Florida, USA. A parasitoid wasp, Aphelinus asychis Walker , was reared from one of the original collections (FSCA N E2003-257).

Distribution: The new species is known in four localities (Port St. Lucie, Immokalee SW FL R&E Center, Winter Haven DPI Citrus Arboretum, and Gainesville Morningside Nature Center) from Florida, USA. A COI sequence (BOLD GMOAG1830-15) of a single unidentified Pleotrichophorus from Ontario, Canada revealed a 99.3% match with COI sequences of P. blackmani sp. n. These COI data suggest that P. blackmani sp. n. may occur in eastern Canada, but the veracity of this barcode match needs to be evaluated with additional sequences and examination of specimens.

Taxonomic comments

The generic placement of our new species was not obvious. It does not have widely capitate setae or the acute URS present in many species. We compared P. blackmani sp. n. to other species already accepted in Pleotrichophorus , with the conclusion that the new species falls within the morphological range of variation for the genus. Our final decision about generic placement was made based on morphology, supplemented by molecular data. These latter data will be discussed in a later section.

Six species of Pleotrichophorus have short accessory setae on URS, shorter than the primary setae on the segment: P. blackmani sp. n., P. lagacei , P. magnautensus ( Knowlton & Smith, 1936) , P. pycnorhysus ( Knowlton & Smith, 1936) , P. stroudi ( Knowlton, 1948) and P. utensis ( Pack & Knowlton, 1929) ( Hille Ris Lambers 1969; Corpuz-Raros & Cook 1974; our observation). Of these species— P. lagacei and P. stroudi have styleto-shaped or acute URS, while P. blackmani sp. n., P. magnautensus , P. pycnorhysus and P. utensis have parallel-sided and bluntly pointed URS ( Hille Ris Lambers 1969; Corpuz-Raros & Cook 1974). The SIPH is distinctly clavate in P. lagacei , while other above-mentioned species have cylindrical or sometimes slightly thickened SIPH. Pleotrichophorus blackmani sp. n. is related morphologically to P. pycnorhysus by the shape of the URS, SIPH, SIPH L/CAUDA L ratio and presence of short accessory setae on the URS but can be differentiated easily by the shape of dorsal setae on the body, which are slightly capitate in the new species and cone- or funnel-shaped in P. pycnorhysus (CorpuzRaros & Cook 1974; our observation). It should be mentioned that the last new Pleotrichophorus species from the Nearctic realm was described 48 years ago ( Favret 2022).

Six species of aphids— Aphis gossypii Glover , Illinoia goldamaryae (Knowlton) , Pemphigus bursarius (Linnaeus) , Prociphilus erigeronensis (Thomas) , Uroleucon lanceolatum (Patch) and U. olivei Moran —are reported from Euthamia spp. (Asteraceae) in North America (Blackman & Eastop 2022; Jensen personal communication). Aphis gossypii is a polyphagous and widely distributed species. Similarly, Pemphigus bursarius and Prociphilus erigeronensis are distributed widely on the roots of many North American Asteraceae . Uroleucon lanceolatum is found on Euthamia graminifolia and Solidago spp. in Canada and USA, while U. olivei is on Aster spp. , Erigeron annuus (L.) Pers. and Euthamia graminifolia in Canada and USA (Blackman & Eastop 2022). Illinoia goldamaryae is known from various Asteraceae throughout North America. There is a recent Florida record (E2014-2360) from Erigeron .

Key to apterous viviparous females of Euthamia- feeding aphids (partially based on keys given in Robinson 1985; Blackman & Eastop 2022)

1 ANT PT/BASE less than 1. Eyes with 3 facets. SIPH absent. Dorsal wax glands present............................. 2

- ANT PT/BASE more than 1. Eyes multifaceted. SIPH present, tubular. No discrete wax glands....................... 3

2 URS shorter than last antennal segment, and without any accessory setae. Setae at apices of tibiae and first tarsal segments short and thick. Abdomen without posterior projection........................................... Pemphigus bursarius

- URS longer than last antennal segment and bearing 4–8 accessory hairs. Setae at apices of tibiae and first tarsal segments long and fine. Anus borne on posterior projection of the abdomen............................. Prociphilus erigeronensis

3 SIPH without polygonal reticulation...................................................................... 4

- SIPH with polygonal polygonal reticulation................................................................ 5

4 ANT PT/BASE 6.0–7.6. SIPH pale, thin, cylindrical, and flangeless................. Pleotrichophorus blackmani sp. n.

- ANT PT/BASE 2.0–3.5. SIPH dark, robust with distinct flange..................................... Aphis gossypii

5 SIPH pale or only with dusky/dark apices, slightly swollen near the tip and with polygonal reticulation on subapical 0.04–0.08 of its total length...................................................................... Illinoia goldamaryae

- SIPH dark at least on distal part, with polygonal reticulation on distal 0.25–0.40 of length............................ 6

6 Length of SIPH less than 0.65mm. Cauda with 12–18 setae................................ Uroleucon lanceolatum

- Length of SIPH more than 0.65mm. Cauda with 21–36 setae..................................... Uroleucon olivei

Notes on SEM morphology and sensilla of Pleotrichophorus blackmani sp. n. and type species of the genus— P. glandulosus

General morphology and body surface. Scanning electron microscopy of viviparous females of P. blackmani sp. n. and the type species P. glandulosus revealed differences in body chaetotaxy and flangeless siphunculi ( Figs 5 , 14 ). The body surface of apterous viviparous females of P. blackmani sp. n. is covered by a thin but visible wax layer and is characterized by short and very short trichoid sensilla with capitate apices. In lateral view, the sockets of the sensilla are well-developed, mostly trapezoid or sometimes slightly rounded ( Fig. 6 ). The dorsal surface of the abdomen is covered by very few very short and inconspicuous trichoid sensilla. The cuticle is visibly wrinkled, especially on the abdomen, and the rather irregular sculpture of the cuticle is visible even when covered by wax, which has a granulose structure ( Fig. 7 ). In alate viviparous females of the new species the trichoid sensilla are also very short and inconspicuous. The head is characterized by rounded and well-developed compound eyes and ocelli ( Fig. 12a, b, c ). In contrast to the apterous females, the cuticle of the body especially on abdomen of the alate is smoother, and the wax layer is much thinner ( Fig. 12d, e ). Wings are covered by sparse scale-like elements, which become much more numerous and denser in the marginal areas and pterostigma ( Fig. 12g ). High magnification of the wings shows that the surface is covered by a thin layer of granulose wax secretions ( Fig. 12h ). Hind wings bear two hamuli ( Fig. 12i ). Siphunculi and cauda are very similar to those in apterous viviparae ( Fig. 12 j-l).

Apterous viviparous females of P. glandulosus have long and thick trichoid sensilla on the body surface, and siphunculi have a well-developed flange. Trichoid sensilla covering the dorsal body surface are long and thick. Their sockets are more evident than in P. blackmani sp. n. and are trapezoid or with bulbous bases in lateral view. Each sensillum, especially those on the abdomen, seem to arise from slightly tuberculate bases ( Fig. 15 a-e). The apices of the sensilla are wide-capitate, sometimes with more or less evident indentation ( Fig. 15f, g ). The dorsal body surface, especially on the abdomen, is densely wrinkled, and the sculpture is irregular with partially fused polygons reminiscent of a maze ( Fig. 15h, i ). The body of apterous viviparous females is not covered by wax.

Antennal sensilla. Antennae of P. blackmani sp. n. bear a majority of the sensilla known in aphids, besides two types of trichoid sensilla. The pedicel has type I trichoid sensilla ( Fig. 8b, c ), one campaniform sensillum on the dorsal side ( Fig. 8a ), and one rhinariolum ( Fig. 8b, c ). The Type I trichoid sensilla are short and thick with blunt or slightly rounded apices that may be slightly capitate ( Fig. 8d ). The opening of the rhinariolum is rather inconspicuous and small, but the tip of the coeloconic sensillum is visible ( Fig. 8e, f ). The campaniform sensillum is rounded with an evident pore near the margin of the inner disc ( Fig. 8g ). Type I trichoid sensilla of the remaining antennomeres are very similar to those on the pedicel, but their sockets are more bulbous, especially at the base, and the apices are evidently capitate ( Fig. 8h, i ). Type II trichoid sensilla, which have been found on the PT, are thick, rigid, and tapering with trapezoid sockets. Their apices are rather blunt, granulose and wrinkled ( Fig. 8 j-l). Antennal segment III of the apterous viviparous females bears small multiporous placoid sensilla near the basal part ( Fig. 9a ), besides type I trichoid sensilla. The placoid sensilla are small, rounded, situated in cavities, and surrounded by poorly developed sclerotized rims ( Fig. 9b ). There is what appears to be waxy secretion, especially in the marginal part of the sensilla near the sclerotized rims, but the overall surface of the plates is clear, covered by numerous pores ( Fig. 9c, d ). Antennal segment V bears a large multiporous placoid sensillum near the tip ( Fig. 9 e-g). The sensillum plate is rounded, flat and surrounded by a sclerotized rim with numerous short and long projections ( Fig. 9h, i ). The basal part of ANT VI is characterized by a group of tightly adjoined sensilla distally ( Fig. 9j ). The large multiporous placoid sensillum is covered by waxy secretion and adheres to two small multiporous placoid sensilla behind which three sunken coeloconic sensilla are visible. All sensilla are surrounded by projections ( Fig. 9 k-o). Alate viviparous females in general are characterized by trichoid and small placoid sensilla on ANT III and IV, quite similar to the apterous morphs ( Fig. 13 ). Small placoid sensilla are characterized by better developed sclerotized rims, and the whole surface is covered by what appears to be waxy secretion with granulose structure ( Fig. 13 g-i). Large multiporous placoid sensilla on ANT V and VI are very similar to those of the apterous viviparous females, except that the sunken coeloconic sensilla are divided into two groups: one sensillum in the upper part between the large multiporous placoid sensillum and the upper small multiporous placoid sensillum, and three remaining in the lower part near the second small placoid sensillum ( Fig. 13 l ).

In the case of P. glandulosus , type I trichoid sensilla are similar to those of the new species, with capitate apices, but the small multiporous placoid sensilla are smaller, deeper, and surrounded with better developed sclerotic rims ( Fig. 16 a-e). The morphology of sensilla on ANT V and VI is very similar to that in P. blackmani sp. n., and the arrangement of sensilla on ANT VI also is similar to that of alate viviparous female P. blackmani sp. n., with two groups of sunken coeloconic sensilla ( Fig. 16 f-h). The type II trichoid sensilla of PT of P. glandulosus are thick, tapering, and have blunt apices ( Fig. 16i ).

Mouthparts and legs sensilla. The URS of P. blackmani sp. n. is isosceles triangle-shaped with well-visible trichoid sensilla and very short basiconic sensilla ( Fig. 10 a-c). Trichoid sensilla along the rostrum and URS are long, rather fine, pointed and of similar length ( Fig. 10d ), while type II basiconic sensilla on the proximal part of the URS are stiff, and the almost pointed apices are slightly blunt in higher magnification ( Fig. 10e ). Sockets of the sensilla are well-developed, and in the case of trichoid sensilla they are more often trapezoid than bulbous based as in type II basiconic sensilla ( Fig. 10e, f ). There are about six pairs of type III basiconic sensilla on the very tip of the URS. They have tapering with slightly rounded apices and clearly visible molting pores ( Fig. 10 g-i). In P. glandulosus the URS is also isosceles triangle-shaped but evidently narrow with a narrow distal part and two lengths of trichoid sensilla ( Fig. 17a, b ). Type III basiconic sensilla are slightly longer and are represented by seven pairs ( Fig. 17c ). Trichoid sensilla on the legs of both species are rather similar. They are very short (especially on the femora), thick, and have capitate apices. In many cases molting pores can be observed at the basal part of sensilla. On the tibiae the sensilla become longer and finer and the apices are less capitate. The ventral distal part of the tibia bears long, thick and pointed trichoid sensilla. One campaniform sensillum can be found on the proximal lateral side of HT II ( Figs 11 , 17 d-h). The parempodia (empodial setae) of P. blackmani sp. n. seems to be shorter than in P. glandulosus ( Figs 11n, o , 17i ).

COI and gnd Barcodes

Gnd barcodes were 14.3% divergent between P. ambrosiae and P. blackmani sp. n. These gnd data appear to the first such DNA barcodes for Pleotrichophorus . Buchnera gnd sequencing for supporting aphid identification is still under development and evaluation (e.g., see Zhu et al. 2017; Théry et al. 2018). Taxon sampling for this symbiont gene target is still poor across Aphidoidea, making interpretation of Pleotrichophorus gnd divergences unclear. GenBank BLASTn searches reveal 84%–85% similarity to gnd sequences from Acyrthosiphon , Artemisaphis , Brevicoryne , Macrosiphum , Microlophium , and Sitobion @ species. COI barcodes had a much lower K2P distance of 5.1% between P. ambrosiae and P. blackmani sp. n. BOLD queries of P. ambrosiae COI barcodes revealed high 97%–99.8% matches to other Pleotrichophorus sequences. The 99% matches suggest P. ambrosiae inclusion in BIN BOLD:ACV0112, along with two specimens from California and Saskatchewan. BOLD queries of P. blackmani sp. n. COI barcodes revealed a 99.3% match to a single unidentified Pleotrichophorus sequence from Ontario. The 99% match suggests P. blackmani sp. n. inclusion in BIN BOLD: ACZ4273 (sequence record GMOAG1830-15). These close COI barcode matches will require examination of specimens to confirm.

It was important to determine whether the new species was similar enough to the type species of the genus, P. glandulosus , to warrant belonging to Pleotrichophorus . The COI barcode alignment was 673 bp long and included 618 Pleotrichophorus and Capitophorus terminals. Neighbor-joining analysis included 534 bp positions and yielded an optimal tree with the sum of branch lengths equaling 0.92958335 ( Fig. 18 ). Pleotrichophorus barcodes formed a clade with high support ( Fig. 18 ). Pleotrichophorus blackmani sp. n. formed a terminal cluster of barcodes along with sequence GMOAG1830-15 from eastern Canada ( Fig. 18 ). Sequences from specimens identified as P. glandulosus , P. sp., and P. pseudoglandulosus collected from France, Russian Far East, and eastern Canada form a terminal cluster among the other sampled Pleotrichophorus ( Fig. 18 , cluster in purple). Specimens identified as P. glandulosus sensu Choi et al. (2018) from Korea form a terminal cluster along with samples identified as Capitophorus sp. from central China and California ( Fig. 18 , cluster in orange). This cluster is nested among the other Capitophorus barcodes. The photograph of the alate specimen of P. glandulosus sensu Choi et al. has a dorsal patch, suggesting it may belong in the genus Capitophorus .

Phylogenetic Analyses

Pleotrichophorus and Capitophorus have been regarded as closely related taxa in various morphological literature (Corpus-Raros & Cook 1974). The molecular data from Choi et al. (2018) and this study suggest that it might not be the case.

Maximum-likelihood analyses recovered a topology largely consistent with that of Choi et al. (2018). Aphidini was sister to Macrosiphini sensu lato with strong support in all analyses ( Fig. 19 ). Pterocomma -group species formed a strongly supported clade (except for parsimony) sister to the remaining macrosiphines (with weak support) ( Fig. 19 ). The recovered Pterocomma-group clade included species in the genera Cavariella Del Guercio, 1911 , Pterocomma Buckton, 1879 , Plocamaphis Oestlund, 1923 , Liosomaphis Walker, 1868 , Vesiculaphis Del Guercio, 1911 , Capitophorus van der Goot, 1913 , and a species misidentified as Pleotrichophorus glandulosus . Like Choi et al. (2018), relationships among the macrosiphines were generally weakly supported except at the genus-level. For example, strongly supported clades united Megoura (6 species), Hyperomyzus (2 species), most of Uroleucon (8 species; internal nodes not shown), Sitobion , and New World Pleotrichophorus (2 species) ( Fig. 19 ). New World Pleotrichophorus species were sister to Megoura in all analyses without strong support ( Fig. 19 ).

Our ability to determine the phylogenetic position of New World Pleotrichophorus species within the Macrosiphini sensu stricto was facilitated by the dataset of Choi et al. (2018). Each of our molecular analyses clearly placed P. blackmani sp. n. within the current concept of Pleotrichophorus . Our COI barcoding of P. blackmani sp. n., P. ambrosiae , and Capitophorus hippophaes helped to confirm our morphological concepts of these genera. Based on the available phylogenetic and barcoding evidence, it appears that the Choi et al. (2018) specimens of P. glandulosus might be a species of Capitophorus that occurs in Korea, China, and has been sampled at least once in California. However, the identity of P. glandulosus , the type species of Pleotrichophorus , in molecular datasets may require confirmation. COI barcodes identified as P. glandulosus and P. pseudoglandulosus from France, Russian Far East, and eastern Canada cluster together in our analyses. The French specimens were identified and sequenced at INRA. These French specimens of P. glandulosus were identified in 1999 (and re-identified in 2020) using the dichotomous keys of Heie (1994) and Blackman & Eastop (2022) (Armelle Coeur d'acier pers. comm., December 2020). Future research should seek to confirm the identity of P. glandulosus in the Old and New World, establish synonymies, and tease out relationships in available data.

Figures

FIGURE 1. Pleotrichophorus blackmani sp. n. on Euthamia graminifolia in life: (a, b) apterous viviparous females; (c, d) alate viviparous females. Photographs by Lyle J. Buss, University of Florida.

FIGURE 2. Habitus of slide-mounted specimens of Pleotrichophorus blackmani sp. n.: (a) apterous viviparous female; (b) alate viviparous female.

FIGURE 3. Detailed morphological characters of Pleotrichophorus blackmani sp. n. Apterous viviparous female: (a) head setae; (b) secondary rhinaria on ANT III; (c) ANT III chaetotaxy; (d) posterior part of abdomen; (e) SIPH; (f) cauda; alate viviparous female: (g) secondary rhinaria on ANT III; (h) URS; (i) fore wing.

FIGURE 4. Pleotrichophorus blackmani sp. n. slide-mounted specimen of oviparous female: (a) habitus; (b) arrangement of pseudosensoria on hind tibiae; (c) shape of pseudosensoria.

FIGURE 5. Scanning electron microscopy (SEM) of apterous viviparous female of Pleotrichophorus blackmani sp. n.: (a) dorsal habitus; (b) head and pronotum; (c) posterior part of abdomen dorsally; (d) surface of SIPH; (e) distal tip of SIPH; (f) lateral side of the end of abdomen; (g) cauda, H—head, Pr—pronotum, MS—mesonotum, Mt—metanotum, I-VIII-abd. tergites I-VIII, s—siphunculi, c—cauda, ap—anal plate, gp—genital plate.

FIGURE 6. SEM of Pleotrichophorus blackmani sp. n. apterous viviparous female, head surface: (a) general view; (b) general structure of the head surface and compound eye; (c) head trichoid sensilla; (d) structure of the sensillum on frons; (e, f, g) structure of the surface of cuticle and trichoid sensilla; (h) ultrastructure od the trichoid sensillum socket and basal part with molting pore (arrow); (i) ultrastructure of the trichoid sensillum apex.

FIGURE 7. SEM of Pleotrichophorus blackmani sp. n. apterous viviparous female, thorax and abdomen surface: (a) thorax surface with trichoid sensillum (arrow); (b) abdomen general surface structure with trichoid sensilla (arrows); (c) abdomen surface with wax layer; (d) ultrastructure of the wax secretion; (e) structure of the surface of cuticle without wax (f) ultrastructure of the surface without wax.

FIGURE 8. SEM of Pleotrichophorus blackmani sp. n. apterous viviparous female. antennal sensilla: (a) dorsal side of the pedicel with campaniform sensillum (star); (b, c) ventral side of the pedicel with rhinariolum (arrow); (d) structure of trichoid sensillum on the pedicel; (e, f) ultrastructure of the rhinariola; (g) ultrastructure campaniform sensillum; (h) type I trichoid sensilla on ANT III; (i) ultrastructure of type I trichoid sensillum; (j-l) structure and ultrastructure of type II trichoid sensilla on PT.

FIGURE 9. SEM of Pleotrichophorus blackmani sp. n. apterous viviparous female. antennal sensilla: (a) small multiporous placoid sensilla (red) and type I trichoid sensilla (blue) on ANT III; (b, c) structure of small multiporous placoid sensilla with secretion; (d) ultrastructure of the sensillum membrane; (e-g) large multiporous placoid sensillum (yellow) and type I trichoid sensilla (blue) on ANT V general view; (h-i) ultrastructure of the large multiporous placoid sensillum; (j) ANT VI BASE with sensilla on the distal end; (k) large multiporous placoid sensillum (yellow); (l) small multiporous placoid sensilla (green) and sunken coeloconic sensilla (pink); (m) ultrastructure of the secretion on sensilla surface; (n) ultrastructure of large multiporous placoid sensillum; (o) ultrastructure of sunken coeloconic sensillum.

FIGURE 10. SEM of Pleotrichophorus blackmani sp. n. apterous viviparous female. mouthparts sensilla: (a) ventral view of URS with well-visible trichoid sensilla (arrows); (b) lateral view of URS with well-visible trichoid sensilla (arrow); (c) distal end of URS with long trichoid sensilla and very short type III basiconic sensilla (asterisk); (d) structure of type I trichoid sensillum; (e) structure of type II basiconic sensillum; (f) ultrastructure of the socket of trichoid sensillum; (g, h) ventral and lateral view of the apical tip of URS with six pairs of type III basiconic sensilla; (i) ultrastructure of the basiconic sensillum with molting pore (star) on the base.

FIGURE 11. SEM of Pleotrichophorus blackmani sp. n. apterous viviparous female. sensilla on legs: (a) III FEMUR with very short trichoid sensilla; (b) structure of femoral trichoid sensillum; (c) proximal part of III TIBIA with trichoid sensilla; (d) structure of trichoid sensillum with visible molting pore (star); (e) ultrastructure of the molting pore; (f) trichoid sensilla on the middle part of III TIBIA; (g) structure of trichoid sensillum from the middle part; (h) trichoid sensilla on the proximal part of III TIBIA; (i, j) structure of the sensilla from the distal part; (k) hind tarsus with visible campaniform sensillum (arrow); (l) HT I with three ventral setae; (m) structure of the campaniform sensillum; (n) distal end of HT II with short empodial seta; (o) structure of an empodial seta.

FIGURE 12. SEM of general characters of the alate viviparous female of Pleotrichophorus blackmani sp. n.: (a) head and pronotum; (b) compound eye and ocellus; (c) frontal ocellus and trichoid sensilla; (d) cuticle surface; (e) ultrastructure of the cuticle surface; (f) fore wing; (g) distal end of pterostigma and radial sector; (h) ultrastructure of wing surface and the scale-like element; (i) hind wing with hamuli enlarged; (j) abdomen; (k) SIPH; (l) cauda, s-siphunculi, c-cauda.

FIGURE 13. SEM of Pleotrichophorus blackmani sp. n. alate viviparous female. head and antennal sensilla: (a) frontal ocellus and trichoid sensilla; (b, c) structure of trichoid sensilla of different lengths; (d) small multiporous placoid sensilla (red) and type I trichoid sensilla (blue) on ANT III; (e) structure of type I trichoid sensillum; (f) structure of the small multiporous placoid sensilla; (g-i) ultrastructure of small multiporous placoid sensillum and the secretion; (j) large multiporous placoid sensillum (yellow) on ANT V; (k) ultrastructure of the large multiporous placoid sensillum; (l) large multiporous placoid sensillum (yellow), small multiporous placoid sensilla (green) and sunken coeloconic sensilla (pink) on ANT VI BASE; (m) ultrastructure of the small multiporous placoid sensillum; (n, o) ultrastructure of sunken coeloconic sensilla.

FIGURE 14. SEM of apterous viviparous female of Pleotrichophorus glandulosus (Kaltenbach, 1846): (a) dorsal habitus; (b) head and pronotum; (c) end of dorsal side of abdomen; (d) surface of SIPH; (e) distal tip of SIPH; (f) lateral side of the end of abdomen; (g) cauda, H-head, Pr-pronotum, MS-mesonotum, Mt-metanotum, I-VIII-abd. tergites I-VIII, s-siphunculi, c-cauda, ap-anal plate, gp-genital plate.

FIGURE 15. SEM of Pleotrichophorus glandulosus (Kaltenbach, 1846): head and body surface: (a) head and pronotum; (b) head trichoid sensilla; (c) trichoid sensilla of abdomen; (d) structure of the sensillum; (e) ultrastructure of the trichoid sensillum socket; (f, g) ultrastructure of the apices of trichoid sensilla; (h) abdominal cuticle surface; (i) ultrastructure of the cuticle surface.

FIGURE 16. SEM of Pleotrichophorus glandulosus (Kaltenbach, 1846): apterous viviparous female. antennal sensilla: (a) small multiporous placoid sensilla (red) and type I trichoid sensilla (blue) on ANT III; (b) structure of the type I trichoid sensillum; (c, d, e) structure of small multiporous placoid sensilla; (f) large multiporous placoid sensillum (yellow) on ANT V general view; (g) ultrastructure of the large multiporous placoid sensillum; (h) large multiporous placoid sensillum (yellow), small multiporous placoid sensilla (green) and sunken coeloconic sensilla (pink) on ANT VI BASE; (i) type II trichoid sensilla on the PT.

FIGURE 17. SEM of Pleotrichophorus glandulosus (Kaltenbach, 1846): apterous viviparous female. mouthparts and legs sensilla: (a, b) URS with trichoid sensilla of two lengths (arrows) and very short type III basiconic sensilla (asterisk); (c) seven pairs of type III basiconic sensilla on the very apical end of URS; (d, e) trichoid sensilla on III FEMUR; (f) trichoid sensilla on the proximal part of III TIBIA; (g) trichoid sensilla on the distal part of III TIBIA; (h) HT I with three ventral setae setae and basal part of HT II with campaniform on the dorsal side (arrow); (i) distal end of HT II with long empodial setae (arrow).

FIGURE 18. K2P neighbor-joining tree of Capitophorus and Pleotrichophorus COI barcodes with auto-collapsed clades with 0.005> average branch length. Collapsed terminal clusters are indicated by red, green, blue, or orange circles. Bootstrap values higher than 80% are indicated on branches with gray circles. Capitophorus species are indicated by blue branches. Pleotrichophorus glandulosus sensu Choi et al. 2018 and Capitophorus sp. from South Korea, China, and California are indicated in orange. Pleotrichophorus species are indicated by red branches. Pleotrichophorus blackmani sp. n., is indicated in green. COI barcodes identified as Pleotrichophorus glandulosus, P. pseudoglandulosus, and P. sp. from France, Russian Far East, and Eastern Canada are indicated in purple.

FIGURE 19. Maximum-likelihood bootstrap consensus tree of Macrosiphini taxa. Pleotrichophorus samples new to this study are highlighted by the peach-colored box. Capitophorus species are highlighted by the blue box. The misidentified specimen of Pleotrichophorus glandulosus specimen reported by Choi et al. (2018) was recovered in a clade along with members of the genus Capitophorus. Support values from left to right are ML ultrafast bootstrap (>95; blue dot), standard non-parametric ML bootstrap (>75; red dot), and MP ultrafast bootstrap (>95; green dot). Nodes without labeled support values indicate that the support value was not significant for that analysis or was not recovered by that analysis.

Tables

TABLE 2. Measurements (in mm) of Pleotrichophorus blackmani sp.n. Explanations about abbreviations used in the table are given in the Material and Methods section. Values in parentheses are the measurements of the holotype.

Character	Apterous viviparous female (n=31)	Alate viviparous female (n=10)	Oviparous female (n=1)
BL	1.48–2.10 (1.70)	1.41–1.77	2.2
HW	0.42–0.48 (0.46)	0.39–0.45	0.5
ANT	2.28–3.05 (2.64)	2.55–3.15	2.92–2.93
ANT III	0.45–0.59 (0.55)	0.70–0.84	0.60–0.61
ANT IV	0.34–0.48 (0.38)	0.40–0.50	0.45
ANT V	0.35–0.50 (0.41)	0.41–0.48	0.49
ANT VI	0.91–1.29 (1.04)	1.03–1.38	1.11–1.14
BASE	0.13–0.15 (0.14)	0.12–0.17	0.15
PT	0.78–1.14 (0.90)	0.90–1.22	0.96–0.99
URS	0.08–0.10 (0.97)	0.08–0.09	0.09
FEMORA III	0.49–0.73 (0.61)	0.51–0.67	0.70
TIBIAE III	0.89–1.28 (1.05)	0.95–1.21	1.25–1.27
HT II	0.10–0.12 (0.11)	0.09–0.15	0.11–0.12
SIPH	0.31–0.46 (0.39)	0.26–0.34	0.43–0.44
CAUDA	0.18–0.25 (0.21)	0.16–0.21	0.22