﻿Revision of the Chinese Pachynotus Kollar & L. Redtenbacher, 1844 (Coleoptera, Curculionidae), with descriptions of two new species

﻿Abstract The Chinese species of the highland weevil genus Pachynotus is revised, including a single known species, P.lampoglobus Chao & Y.-Q. Chen, 1980, and the descriptions of two new species, P.pilosussp. nov. and P.arcuatussp. nov. All Chinese Pachynotus species occur in Xizang (Tibet), China, and a key to these species is presented. Additionally, the COI sequences of two species, P.lampoglobus and P.pilosussp. nov., are provided, with details of the genetic distance.

Herein, we describe two new species and present a key to Chinese Pachynotus.Moreover, we provide the COI sequences of P. lampoglobus and P. pilosus and analyse the genetic distance of these two species based on COI sequences.

Materials and methods
All specimens, including types, examined for this study are collections of the Institute of Zoology, Chinese Academy of Sciences, Beijing, China (IZCAS), and the Natural History Museum, London, UK (NHMUK).The types of the new species are deposited in IZCAS.Label data are given as they are, verbatim, with pinyin romanisation and comments in square brackets if labels are in Chinese; labels are separated by double slashes and lines by slashes.
Specimens were dissected after soaking them in soapy water overnight for cleaning and softening, and the dissected parts were placed in a cold 10% KOH solution for 20 h to macerate the soft tissues.After dissection, all parts were photographed and stored in glycerine in microvials pinned beneath the specimen from which they were dissected.
The morphological terminology used in this study mainly follows Marshall (1916) and Aslam (1966).Measurements were made using an ocular micrometre as follows: standard length -dorsally from anterior margin of thorax to the apex of elytra along midline; pronotal length -dorsally from anterior margin to base along midline; pronotal width -dorsally across widest part; elytral length -dorsally along suture of elytra from base to apex; elytral width -dorsally across widest part; rostral length -dorsally in a straight line from apex to anterior margin of eye; rostral width -dorsally across base of rostrum.Measurements are made in millimetres.
All observations and dissections were performed using a Nikon SMZ1500 stereomicroscope.The habitus images were taken with a Canon-5D camera mounted on a Nikon SMZ1500 microscope.CombineZM and Helicon Focus software were used to combine the photos.Photoshop CC2019 was used to modify the photos if required.
DNA was extracted from all the specimens via DNeasy Blood & Tissue Kits (Qiagen, Germany).DNA was extracted from either 1, 3, or 6 legs or the whole body, depending on the size of specimen.Polymerase chain reaction (PCR) amplifications for COI sequences were conducted using the primers LCO1490 (GGTCAACAAATCATAAAGATATTGG) and HCO2198 (TAAACTTCAGGGTGAC-CAAAAAATCA).PCR reaction mixes (25 mL) contained 12.5 μL 2× Taq PCR MasterMix (Tiangen Biotech Co., Ltd, Beijing, China), 1 μL of forward and reverse primer each (Sangon Biotech Co. Ltd, Shanghai, China), 2 μL total undiluted DNA template, and 8.5 μL dd H 2 O. PCR profile as follows: 94 °C for 2 min, first cycle set (5 repeats): 94 °C for 40 s, 45 °C for 40 s and 72 °C for 60 s.Second cycle set (35 repeats): 94 °C for 40 s, 51 °C for 40 s and 72 °C for 60 s, followed by elongation at 75 °C for 5 min.PCR products were visualised through 1% agarose gel electrophoresis in TAE buffer.Successful PCR products were sent for sequencing in the Beijing Genomics Institute (BGI, Shenzhen, China).The raw data were assembled and edited via SeqMan v. 7.1.We failed to amplify the COI sequence of P. arcuatus.In order not to destroy the type specimen (only one specimen), we abandoned the idea of further amplification of the COI sequence.The intraspecific and interspecific K2P genetic distances of P. lampoglobus and P. pilosus were separately calculated using the MEGA v. 7. Description.Holotype, male.Measurements (in mm): standard length: 5.10; pronotal length: 1.70; pronotal width: 1.9; elytral length: 3.10; elytral width: 2.08; rostral length: 0.90; rostral width: 0.60.

Taxonomic treatment
Habitus and colour (Fig. 1A, B): body elongate-oval, small; integument dark reddish brown; antennae and legs reddish brown, with pale yellowish-brown scales; scales on dorsal and lateral surfaces of rostrum moderately dense, oval to elongate-oval; antennal scape and funicles without scales; pronotum with polygonal scales, moderately dense, not contiguous; scales on elytra polygonal, dense, not contiguous; scales on ventrites moderately dense, polygonal, elongate-oval; scales on legs dense; dorsal of tarsi surface without scales; body with erect to suberect and slightly longer setae; rostrum sparsely covered with suberect fine setae; antennal scape and desmomeres I-VII with long, fine, sparse setae; dorsal and lateral surfaces of pronotum with sparse, suberect setae; setae on interstriae long, erect to suberect, behind declivity slightly longer, erect, equal to 6 × diameter of one scale; setae on ventral surface moderately long, fine, erect.Head (Fig. 1C): moderately convex; dorsal surface smooth; punctures small, moderately dense; eyes flat, oval; forehead weakly convex, moderately elevated than base of rostrum in lateral view.
Rostrum (Fig. 1C): in dorsal view 1.50 × as long as wide, apex narrower than base; sides narrowed from base to middle, and then slightly broadened to apex; dorsal surface with a narrow and shallow median sulcus, reaching fore margin of forehead; epistome broad U-shaped, smooth, posterior angle of epistome >90°; mandibular scars oval; ventral margin of antennal scrobes visible at apical half in dorsal view; in lateral view, without triangular depression positioned laterally between eyes and antennal scrobes; prementum with two setae.
Pronotum: 0.89 × as long as wide; subquadrate in dorsal outline, strongly convex; sides strongly rounded, greatest width after midpoint, gradually narrowing towards both ends, fore margin shorter than posterior one; median sulcus fine, extremely shallow; dorsal surface smooth, punctures small, each puncture covered by a scale; postocular lobes absent, vibrissae fine, dense, yellow.
Scutellum: large and distinct, triangular, shiny, uncoated, reddish brown.Elytra: 1.49 × as long as wide, moderately convex, elongate-oval; base not raised as prominent flange, not bisinuate; sides subparallel before declivity, only slightly narrowed near the base; striae distinct, narrow, moderately deep, linear; punctures minute, spot like, moderately dense, space between punctures narrower than the diameter of punctures; interstriae wide, slightly flat, without tubercles, odd interstriae slightly raised than even ones.
Abdomen (Fig. 2A): sternite I depressed in middle, slightly convex at sides; suture between sternites I and II slightly curved forward in middle; sternite II slightly convex; sternite I longer than II, sternite II slightly shorter than III and IV combined; sternites III and IV equal in length; sternite V moderately convex, apex round, longer than sternites III and IV combined.
Legs: rather short; femora clavate; fore tibiae subcylindrical, slightly sinuate, slightly bent inwards at apical quarter, apex not projected inwards and outwards, with long and slender hairs, obviously different from other setae, somewhat sparse (Fig. 1D); inner margin of fore tibiae with several extremely small teeth; median and hind tibiae without teeth; corbels of hind tibiae closed.
Etymology.Pilosus, Latin adjective, meaning "hairy", in reference to the significantly longer hair on the male fore tibiae than on other parts.
Distribution.China (Xizang).Habitus and colour (Fig. 3C, D): body elongate-oval, small; integument dark reddish brown; antennae and legs reddish brown, with light-grey scales; scales on dorsal and lateral surfaces of rostrum moderately dense, oval to elongate-oval; antennal scape and funicles without scales; pronotum with polygonal scales, moderately dense, not contiguous; scales on elytra polygonal, dense, not contiguous; scales on ventrites moderately dense, polygonal to elongate-oval; scales on legs dense; dorsal of tarsi surface without scales; body with erect to suberect and slightly longer setae, sparser; rostrum sparsely covered with suberect fine setae; antennal scape and desmomeres I-VII with long, fine and sparse setae; dorsal and lateral surfaces of pronotum with sparse, suberect setae; setae on interstriae long, erect to suberect, behind declivity slightly longer, erect, equal to 6 × diameter of one scale; setae on ventral surface moderately long, fine, erect.

Pachynotus arcuatus
Head (Fig. 4B): moderately flat; dorsal surface smooth; punctures small and dense; eyes flat, oval; forehead weakly convex, moderately elevated than base of rostrum in lateral view.
Rostrum (Fig. 4B): in dorsal view, 1.35 × as long as wide, apex narrower than base; sides narrowed from base to middle, and then slightly broadened to apex; dorsal surface with a broad, deep, median sulcus, reaching vertex of head; posterior angle of epistome 90°, smooth; mandibular scars oval; ventral margin of antennal scrobes visible at apical half in dorsal view; in lateral view, without triangular depression positioned laterally between eyes and antennal scrobes; prementum with two setae.Antennae (Fig. 4F): scape slender, clavate, reaching posterior margin of eyes at rest, 0.98 × length of funicle; desmomere I 1.43 × length of II, both segments elongate clavate; desmomere III short, clavate, 0.51 × length of II; desmomere IV 0.93 × length of III, nearly equal width; desmomeres IV-VI moniliform, equal in length and width; desmomere VII moniliform, 1.08 × length, 1.03 × width of VI; club elongate-oval, apically sharp, three-segmented, uniformly pubescent, segment I 1.21 × length of II; segment II shorter than segment III.
Pronotum: 0.86 × as long as wide; subquadrate in dorsal outline, strongly convex; sides strongly rounded, greatest width after midpoint, gradually narrowing towards both ends, fore margin shorter than posterior one; median sulcus fine, extremely shallow; postocular lobes absent, vibrissae fine, dense, yellow.
Abdomen (Fig. 6A): sternite I depressed in middle, slightly convex at sides; suture between sternites I and II slightly bisinuate; sternite II slightly convex; sternite I longer than II, sternite II slightly shorter than III and IV combined; sternites III and IV equal in length; sternite V moderately convex, apical round, longer than sternites III and IV combined.
Legs: rather short; femora clavate; fore tibiae obviously bent inward at apical third, apex neither projected inwards nor outwards (Fig. 4D); inner margin of fore tibiae with several extremely small teeth; median-and hind tibiae without teeth; corbels of hind tibiae closed.

Male. Unknown.
Remarks.This new species resembles P. pilosus sp.nov.but differs by the following characters: scutellum U-shaped; rostrum with a broad and deep median sulcus, reaching head vertex; elytra base raised as prominent flange; fore tibiae obviously bent inward at apical third.
Etymology.Arcuatus, Latin participle, meaning "curved", in reference to the fore tibiae, which is bent inward at its apical third.

Pachynotus lampoglobus
Head: convex; dorsal surface smooth; punctures small and dense; eyes moderately convex, oval; forehead convex, moderately elevated than base of rostrum in lateral view.
Rostrum: in dorsal view, 1.10 × as long as wide; sides narrowed from base to apex; apex narrower than base; dorsal surface with a broad and deep median sulcus, reaching middle of forehead; posterior angle of epistome more than 90°, smooth; mandibular scars oval; ventral margin of antennal scrobes visible at apical two-thirds in dorsal view; in lateral view, without triangular depression positioned laterally between eyes and antennal scrobes; prementum with four setae.
Pronotum: 0.88 × as long as wide; subquadrate in dorsal outline, strongly convex; sides strongly rounded, greatest width at midpoint, gradually narrowing towards both ends, fore margin shorter than posterior one; median sulcus, fine, extremely shallow; dorsal surface of pronotum smooth, punctures small, each puncture covered by a scale; postocular lobes absent, vibrissae fine, dense, yellow.
Scutellum: large and distinct, U-shaped, shiny, uncoated, reddish brown.Elytra: 1.23 × as long as wide, moderately convex, elongate-oval; base not raised as prominent flange, not bisinuate; sides subparallel before declivity, only slightly narrowed near the base; striae distinct, narrow, moderately deep, linear; punctures minute, spot-like, moderately dense, spaces between punctures narrower than diameter of punctures; interstriae wide, flat, without tubercles, odd interstriae not more raised than even ones.
Abdomen: sternite I depressed in middle, slightly convex at sides; suture between sternite I and II slightly bisinuate; sternite II slightly convex; sternite I longer than II, sternite II slightly shorter than III and IV combined; sternites III and IV equal in length; sternite V moderately convex, apical round, shorter than sternites III and IV combined.
Legs: slightly short; femora clavate, densely with scales; fore tibiae bent inward at apical half, apex projected inwards and outwards; inner margin of fore tibiae with several moderately large teeth; median-and hind tibiae with moderately small teeth; corbels of hind tibiae closed.

Discussion
The three Chinese Pachynotus species occurring in Xizang are all endemic to China, and morphological variation and genetic differences among these species are conspicuous.In our study, the average interspecific genetic distance between P. lampoglobus and P. pilosus was 33 × that of the average intraspecific genetic distance.This confirm, by molecular means, the validity of the new species P. pilosus sp.nov.(Hebert et al. 2003(Hebert et al. , 2004)).In addition, the COI sequences of P. lampoglobus and P. pilosus are provided for the first time, which will help promote further DNA barcoding studies of this genus.
Pachynotus species in China are quite different in size from other Pachynotus species.P. globicollis (length 9.63 mm, width 2.89 mm), P. mayarami (length 9.55 mm, width 3.10mm), and P. kumaonensis (length 9.80 mm, width 3.25 mm) are larger than P. lampoglobus (average length 5.82 mm, average width 3.05 mm), P. pilosus (average length 5.12 mm, average width 2.21mm), and P. arcuatus (length 5.90 mm, average width 3.00 mm).It is worth noting that P. globicollis, P. mayarami, and P. kumaonensis are distributed in Uttarakhand, India; P. globicollis was found at an altitude of 1753-2286 m in Uttarakhand (Mahendiran and Ramamurthy 2013), and the other two species have no altitudinal information.P. lampoglobus, P. pilosus, and P. arcuatus are found in Xizang of China at altitudes of 3700 m, 4989 m, and 4685 m, respectively.This phenomenon of extreme altitude could be attributed to the advantages that diminutive bodies offer in the face of the relatively harsh conditions of high-altitude environments.In such conditions, these smaller weevils can thrive on minimal food resources and easily seek refuge by burrowing into rock crevices, thus evading predators and harsh climatic conditions.
As flightless weevils, the genus Pachynotus is inherently predisposed to geographical isolation, leading to the emergence of new species as observed in previous studies (Chen 1991;Ren 2008) and this study.Therefore, our findings suggest that there may be more Pachynotus species awaiting discovery, considering the geographic isolation and the unique ecological niches that Pachynotus species inhabit.

Figure 1 .
Figure 1.Habitus of Pachynotus pilosus sp.nov., holotype A dorsal view B lateral view C head and rostrum, dorsal view D left protibia, dorsal view E antenna, dorsal view.Scale bars: 1 mm (A, B, D); 0.5 mm (C, E).

Figure 5 .
Figure 5. Abdominal and genital structures of Pachynotus pilosus sp.nov.A ventrites, ventral view B sternite VIII and female genitalia, lateral view C sternum VIII, dorsal view D spermatheca, lateral view.Scale bars: 0.5 mm.

Figure 6 .
Figure 6.Abdominal and genital structures of Pachynotus arcuatus sp.nov.A ventrites, ventral view B sternite VIII and female genitalia, lateral view C sternum VIII, dorsal view D spermatheca, lateral view.Scale bars: 0.5 mm.