The identification of citrus aphid species and their morphometric characteristics was conducted based on the samples collected from the citrus orchards in Zhejiang Province. A total of five citrus aphid species, all belonging to two subgenera (Aphis and Toxoptera) within the genus Aphis, were identified, including A. (Aphis) craccivora Koch (2.37%), A. (A.) gossypii Glover (46.99%), A. (A.) spiraecola Patch (44.30%), A. (Toxoptera) aurantii (Boyer de Fonscolombe) (2.41%), and A. (T.) citricidus (Kirkaldy) (3.93%) (Fig. 2). However, A. (A.) gossypii and A. (A.) spiraecola were the most dominant species in this region. The identification and characteristics of citrus aphid species were based on the references provided by Zhang and Zhong (1983), Blackman and Eastop (2006), and Yu and Wang (2019).

The occurrence rate of the citrus aphid species in Zhejiang Province, China

Citrus aphid species and their characteristics were referenced from Zhang and Zhong (1983), Blackman and Eastop (2006) and Yu and Wang (2019):

Aphis (Aphis) craccivora Koch, 1854

Common name: cowpea aphid, which colonize various plants, particularly leguminous crops.

Description: BL about 1.35 ~ 1.60 mm (n = 12). Apterous viviparous morphs black, paint black or thick purple, shiny. Antennae segment 1 and 2, and the end dark brown, pale in the remaining segments. The end femur, tibia, and tarsus dark brown. Antennae (ANT) tubercles weakly developed, ANT PT/BASE about 1.91 ~ 3.11. R IV + V mostly 0.82 ~ 1.33 × HT II. Dorsal abdomen with an extensive black shield and a solid black patch. SIPH black, more than 3.00 × their basal width, and 1.60 ~ 2.05 × cauda. Cauda black, as black as SIPH, with distal part tapering, more than 1.50 × longer than its basal width (Fig. 3, Table S1).

Aphis (Aphis) craccivora. a Photo of microscope slide; b colony on citrus; c antenna; d siphunculi and cauda

Aphis (Aphis) gossypii Glover, 1877

Common name: cotton aphid, melon aphid, cotton-melon aphid. This polyphagous pest exhibits wide host range.

Description: BL about 1.31 ~ 1.49 mm (n = 15). Apterous viviparous morphs with varying body colors, commonly dark blackish green, grass green (larger species usually in spring and autumn), or pale whitish yellow (small species usually in hot summer). Antennae segment 1, 2, 6 and the distal 2/5 of segment 5 black. The end 1/5 of tibia and tarsus dark brown. ANT tubercles weakly developed, not projecting beyond middle part of frons in dorsal view, usually 6-segmented, sometimes 5-segmented in small individuals, and ANT PT/BASE about 2.45 ~ 3.29. R IV + V 0.09 ~ 0.11 mm, and less than 1.50 × HT II. Dorsal abdomen unpigmented, without any clearly defined dark markings. SIPH uniformly dark, which has no constriction, and clearly darker than cauda, more than 0.10 × BL, and tapering from base to flange, with no trace of swelling. Cauda pale to dusky, without any constriction (Fig. 4, Table S2).

Aphis (Aphis) gossypii. a Photo of microscope slide; b colony on citrus; c antenna; d siphunculi and cauda

Aphis (Aphis) spiraecola Patch, 1914

Common name: green citrus aphid, spiraea aphid. A cosmopolitan pest with wide range of hosts, and recognized as the major pest of citrus crops.

Description: BL about 1.41 ~ 1.62 mm (n = 14). Apterous viviparous morphs bright greenish yellow to apple green. Antennae segment 1 and 2, the 1/3 basal part of segment 6, and the end of the femur, the tibia, and the tarsus dark brown, pale with the rest. ANT tubercles weakly developed. ANT PT/BASE less than 3.0, and ANT III 0.70 ~ 0.93 × SIPH. R IV + V 1.00 ~ 1.22 × HT II. Dorsal abdomen without any dark markings. SIPH dark. Cauda black like SIPH, usually has an evident constriction between basal and distal part (Fig. 5, Table S3).

Aphis (Aphis) spiraecola. a Photo of microscope slide; b colony on citrus; c antenna; d siphunculi and cauda

Remarks: multiple studies have historical ambiguity about this aphid species as “Aphis citricola” van der Goot, 1912 (the details are in the discussion part).

Aphis (Toxoptera) aurantii (Boyer de Fonscolombe, 1841)

Common name: black citrus aphid. Polyphagous.

Description: BL about 1.42 ~ 1.69 mm (n = 11). Apterous viviparous morphs black to dark brown. Antennae segment 1, 2, the end of segment 3, 4, 5, and the 1/2 basal part of segment 6 black, pale with the rest. The femur of middle-leg and hindleg, the base and the distal part of the tibia black, pale with the rest. ANT tubercles weakly developed, PT/BASE 4.10 ~ 4.89. R IV + V 1.33 ~ 1.86 × HT II. SIPH and cauda entirely dark. SIPH 1.00 ~ 1.53 × cauda (Fig. 6, Table S4).

Aphis (Toxoptera) aurantii. a Photo of microscope slide, ventral view; b colony on citrus; c antenna; d siphunculi and cauda

Aphis (Toxoptera) citricidus (Kirkaldy, 1907)

Common name: brown citrus aphid. An oligophagous aphid mainly on Rutaceae plants, and which is a principal virus vector of citrus.

Description: BL about 1.96 ~ 2.24 mm (n = 12), and usually more than 2.0 mm. Apterous viviparous morphs black to dark brown, shiny. Antennae segment 1, 2, the end of segment 4, the end 1/2 of segment 5, and segment 6 black, pale with the rest. The 1/2 basal part of profemur, and the 2/3 middle part of each tibia pale, black with the rest. ANT tubercles weakly developed, PT/BASE 4.55 ~ 6.22. Thoracic segments often partly sclerotised. R IV + V 1.17 ~ 1.60 × HT II. SIPH and cauda both dark, SIPH 1.10 ~ 1.65 × cauda (Fig. 7, Table S5).

Aphis (Toxoptera) citricidus. a Photo of microscope slide, ventral view; b adult; c antenna; d siphunculi and cauda

Remarks: the aphid species Aphis (Toxoptera) citricida is an unjustified emendation of Dr. Stoetzel (Nafría et al. 2005).

The COI fragments of 21 citrus aphid individuals were sequenced yielding a range of 671 to 704 bp. Following the trimming for alignment, 661 bp were used for the phylogenetic and cluster analysis. The results of transitions and transversions showed that the sequences did not reach saturation.

The Neighbour-Joining analysis (Fig. 8) distinctly revealed five independent clades with strong support of Aphis (Aphis) craccivora (100, gray clade), A. (A.) gossypii (99, reddish orange clade), A. (A.) spiraecola (99, green clade), Aphis (Toxoptera) aurantii (99, blue clade), and A. (T.) citricidus (100, claybank clade). These molecular analyses support our morphological results, about the occurrence of five citrus aphid species in Zhejiang province.

The Neighbour-Joining phylogenetic tree of citrus aphid samples based on the COI sequences; black triangles represent the samples collected in this study (Table S6)

The intraspecific genetic distances of the Aphis (Aphis) craccivora and = A. (A.) gossypii clade were 0.000, the A. (A.) spiraecola clade ranged from 0.000 to 0.002, the Aphis (Toxoptera) aurantii clade ranged from 0.000 to 0.003, and the A. (T.) citricidus clade ranged from 0.000 to 0.033. The interspecific distance of the Aphis (Aphis) craccivora clade was 0.819 to 0.821, the A. (A.) gossypii clade ranged from 0.783 to 0.809, the A. (A.) spiraecola clade ranged from 0.813 to 0.833, the Aphis (Toxoptera) aurantii clade ranged from 0.805 to 0.816, and the A. (T.) citricidus clade ranged from 0.803 to 0.845, compared with Myzus persicae and Diuraphis noxia, respectively. The genetic divergences within and between each clade of the aphids are provided in Supplementary Table S8 to S12, respectively.