﻿Two new species of Aphis (Toxoptera) Koch (Hemiptera, Aphididae) from China

﻿Abstract Two new aphid species, Aphis (Toxoptera) fafuensis Cheng & Huang, sp. nov., feeding on Adinandramillettii (Pentaphylacaceae) from Fujian, China, and Aphis (Toxoptera) sennae Cheng & Huang, sp. nov., feeding on Sennabicapsularis (Fabaceae) from Yunnan, China, were described. Morphological characters and molecular data supported the taxonomic position of the new species within the subgenus Aphis (Toxoptera). A key for identifying species of apterous viviparous females in this subgenus is provided.


Introduction
The aphid genus Toxoptera was first proposed by Koch (1856), with T. aurantiae Koch, 1856 designated as the type species. However, the name was considered as a synonym and the type species was revised as T. aurantii Boyer de Fonscolombe, 1841 by Schouteden (1906). Baker (1920) defined Toxoptera as having alate with once-branched media of the forewing. Williams (1921) described another unique character of this genus, which is a stridulatory apparatus consisting of peg-like spines on the hind tibiae and ventro-lateral spinulose ridges on the posterior abdominal segments. After Börner (1930) erected the genus Schizaphis to include species characterized by the once-branched media of the forewing, Toxoptera had been distinguished from allied genera by the presence of the stridulatory apparatus. Kim and Lee (2008) investigated the phylogenetic relationships within the tribe Aphidini using several gene markers including tRNA/COII, 12S/16S and EF1-α, and their results showed that Toxoptera may be non-monophyletic. Based on COI sequences, Wang and Qiao (2009) showed that T. odinae was phylogenetically distinct from other Toxoptera species and should be reverted to Aphis (Aphis) odinae van der Goot, 1917(Blackman et al. 2011. Then a molecular phylogenetic study of Aphis species based on nuclear and mitochondrial genes confirmed that Toxoptera should be regarded as a subgenus of Aphis (Lagos et al. 2014).
Thus far, the subgenus Aphis (Toxoptera) has been represented by five species (Remaudière and Remaudière 1997;Favret 2023) Martin, 1991 and A. (T.) chaetosiphon Qiao, Wang & Zhang, 2008. However, it should be noted that A. (T.) celtis is considered a possible synonym of A. (T.) aurantii (Martin 1991;Blackman and Eastop 2023). The host plants for species of this subgenus are very diverse, including Theaceae, Rutaceae, Rubiaceae, and many other plant families. New host plants can provide novel ecological niches for herbivorous insects, contributing to different host preferences and genetic isolation. Therefore, host plant is key to the diversification of herbivorous insects and plays an important role in their speciation (Mitter et al. 1991;Futuyma and Agrawal 2009). Based on multiple gene fragments and the haplotype network analysis, Li et al. (2021) found that the population of A. (T.) aurantii feeding on Ficus showed great genetic difference from those feeding on other host plant groups, indicating that A. (T.) aurantii has been undergoing the evolution of host specialization on Ficus.
In recent years, while collecting aphid samples in southern China, we obtained some samples that may represent two undescribed species from Adinandra millettii (Hook. & Arn.) Benth. & Hook. f. ex Hance (Pentaphylacaceae) and Senna bicapsularis (L.) Roxb. (Fabaceae), respectively. By integrating morphological and molecular data, this paper describes the new species and confirms their taxonomic positions within Aphis (Toxoptera).

Field sampling
The specimens of A. (T.) fafuensis Cheng & Huang, sp. nov. were collected in Fujian, China on Adinandra millettii and the samples of A. (T.) sennae Cheng & Huang, sp. nov. were collected in Yunnan, China on Senna bicapsularis. The detailed collection information is provided in Suppl. material 1: table S1. All samples were preserved in 95% ethanol and kept at -80 °C for further morphological measurement and molecular experiments.

Morphological description
Six apterous viviparous females of A. (T.) fafuensis Cheng & Huang, sp. nov. and eight apterous viviparous females of A. (T.) sennae Cheng & Huang, sp. nov. were slide-mounted in Canada Balsam. Aphid terminology and the morphological measurements used in this paper followed Qiao et al. (2008) (Table 1). All specimens were examined and measurements and images were taken by using Nikon SMZ18 stereomicroscope. The measurements and the micrographs of mounted specimens were performed using a computer-connected Nikon set: Nikon Eclipse Ci-L upright microscope, 16MP digital camera with 0.55X adapter and imaging software NIS-Elements D ver. 4.60.00. The unit of measurement in this paper is millimeters (mm).
The following abbreviations have been used: BL, body length; BW, body width; URS, ultimate rostral segment; URS_BW, basal width of URS; WR, whole length of rostral; WA, whole length of antenna; Ant. I, Ant. II, Ant. III, Ant. IV, Ant. V, Ant. VIb, for antennal segments I, II, III, IV, V and the base of Ant. IV, respectively; Ant. III_WD, the widest diameter of Ant. III; PT, processus terminalis; PT_WD, the widest diameter of PT; HF, hind femur; HF_WD, the widest diameter To examine the possible morphological differences between the two newly discovered species and A. (T.) aurantii, a one-way analysis of variance (ANOVA) was conducted. Furthermore, to identify pairwise differences of the morphological characters of specimens, post hoc multiple comparisons were performed using the Least Significant Difference (LSD) test (Suppl. material 1: table S2). All statistical analyses were carried out using SPSS ver. 24 (IBM, Chicago, IL, USA).

Molecular analysis
The whole genomic DNA of each sample was extracted from the single individual preserved in 95% ethanol using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). The standard DNA barcode gene of aphids, cytochrome c oxidase subunit I (COI) was amplified with primer LepF (5'-ATTCAACCAATCATAAAGA-TATTGG-3') and LepR (5'-TAAACTTCTGGATGTCCAAAAAATCA-3') (Foottit et al. 2008). PCR amplifications were performed in a final volume of 25 µL reaction mixture containing 2 μL of template DNA, 0.5 μL of both forward and reverse primer (10 μM), 0.25 μL of Taq DNA polymerase (5 U/μL), 17.25 μL of double distilled H 2 O, 2.5 μL of 10× buffer and 2 μL of dNTP. PCR thermal regime was as follows: 5 min of initial denaturation at 95 °C, 35 cycles of 20 s at 94 °C, 30 s at 50 °C (the annealing temperature) and 2 min at 72 °C, and 10 min of final extension at 72 °C. The products of PCR were visualized by electrophoresis on a 1% agarose gel and then bidirectionally sequenced at Beijing Tsingke Biotech Co., Ltd. (Beijing, China).
The maximum-likelihood phylogenetic tree base on COI sequences includes thirty-six samples representing the two new species and three Aphis (Toxoptera) species including A.  Table 2). All sequences were as-sembled by ContigExpress (Vector NTI Suite 6.0, InforMax Inc.), and the reliability was checked by BLAST. Multiple alignment was conducted using MAFFT (Katoh and Standley 2013) based on the default setting. Maximum-likelihood phylogenies were inferred using PhyloSuite ver. 1.2.3 (Zhang et al. 2020) under the TIM2+I+F model for 5000 ultrafast bootstraps. The mean genetic distances among the seven Aphis species used for phylogenetic analysis were calculated using MEGA 7 (Kumar et al. 2016) under Kimura's two-parameter (K2P) model (Kimura 1980). Table 2. Voucher information and GenBank accession numbers of aphid samples used in molecular data analysis.

Species
Voucher number Host plant Location Accession Number  (Fig. 4A), dark brown in life, head is slightly lighter in color and the tibiae are markedly pale (Fig. 4B).
Thorax. Dorsal and ventral cuticle with polygon reticulations. Mesosternal furca with separated arms. Length of single arms 0.09-0.11 mm, 0.4-0.5 times as long as antennal segment III. Spiracles elliptical, spiracular plates dark brown. Prothorax with one pair of small marginal tubercles. Dorsal setae on thorax short and pointed, with small tuberculate bases. Legs normal. Distal part of femora, basal and distal part of tibiae dark brown, others brown. Hind femur 1.3-1.6 times as long as antennal segment III, hind tibia 0.5-0.6 times as long as body. Hind tibia with 7-8 peg-shaped spines, on basal two-thirds of inner side. Length of hairs on hind tibia 0.02-0.03 mm, 1.0-1.5 times as long as the widest diameter of antennal segment III. First tarsal chaetotaxy: 3, 3, 2. Second tarsal segments with transverse imbrications.
Thorax. Mesosternal furca with separated arms. Length of single arms 0.10-0.14 mm, 0.3-0.4 times as long as antennal segment III. Prothorax with one pair of small marginal tubercles. Dorsal hairs on thorax short and thin, with small tuberculate bases. Legs normal. Distal part of femora, basal and distal part of tibiae dark brown, others brown. Hind femur 1.4-1.5 times as long as antennal segment III. Hind tibia 0.5-0.6 times as long as body, with 8-10 peg-shaped spines, on basal two-thirds of inner side. Length of hairs on hind tibia 0.02-0.03 mm, 0.8-1.0 times as long as the widest diameter of antennal segment III. First tarsal chaetotaxy: 3, 3, 2. Second tarsal segments with transverse imbrications.
Etymology. The new species is named after the genus name of the host plant, Senna bicapsularis. The word 'sennae' is a noun, and does not change spelling based on gender.