A review of the genus Takecallis Mastumura in Korea with the description of a new species (Hemiptera, Aphididae)

Abstract The aphid genus, Takecallis Mastumura, 1917, was reviewed from Korea. Four species, T. alba Y. Lee, sp. n., T. arundicolens (Clarke), T. arundinariae (Essig), and T. taiwana (Takahashi), are recognized in Korea and morphological and molecular evidence are presented. Species descriptions and illustrations are given for the four species. A key to Korean species and the results of COI sequence analyses are also provided.


Introduction
The genus Takecallis was established by Matsumura (1917) based on the type species T. arundicolens. This is one of the small aphid genera of the tribe Panaphidini (Aphididae: Calaphidinae). In this genus, six species are known around the world (Remaudière andRemaudière 1997, Favret 2017). All known species have been described from Southeast Asian countries such as China (Qiao and Zhang 2004), India (Chakrabarti 1988), Japan (Higuchi 1968), Korea (Quednau and Lee 2001), and Taiwan (Quednau 2003).
In Korea, three species, Takecallis arundicolens, T. arundinariae, and T. taiwana, have so far been recorded in this genus (Quednau and Lee 2001). However, recent DNA barcoding revealed that there is an undescribed species in Korea (Lee et al. 2017). In this study, a large number of Takecallis samples were collected in Korea and examined together with museum specimens. We also conducted molecular analyses based on the partial mitochondrial cytochrome oxidase subunit I (COI) of fresh-ethanol preserved samples. Generally, COI barcoding provides a good enough resolution for species identification in aphids (Foottit et al. 2008, Lee et al. 2011.
A total of four species were recognized in Korea. Both morphological and molecular evidence strongly indicated that T. alba Y. Lee, sp. n. is a valid species. Here, together with a description of the new species, photographs of live aphids and illustrations are provided along with a key to species of the genus Takecallis in Korea. Pairwise distance analyses and a neighbor-joining tree based on the partial COI sequence are also provided.

Materials and methods
Aphid samples were collected in South Korea from 1999 to 2015. All samples were preserved in 90-95% ethanol for over one month, and then mounted in Canada balsam, following the methods of Blackman and Eastop (2000) and Martin (1983). Illustrations for each species were taken with a digital camera attached to the microscope (Leica 400B, Leica Microsystems, Germany) at a resolution of 600 dpi. Measurements for each specimen are taken from the digital images by using image analysis software, Active measure ver. 3.0.3 (Mitani Co. Ltd, Japan). All specimens are deposited in the National Academy of Agricultural Science (NAAS), Jeonju-si, Republic of Korea and the College of Agriculture and Life sciences, Seoul National University, Republic of Korea (CALS SNU).
Aphid samples were identified using keys to Takecallis species by Higuchi (1972) and Quednau (2003). For further confirmation, DNA barcoding results were also applied.
Abbreviations used for diagnosis, description, figures, and Table 1  Main morphological characters such as measurements (in mm), number of setae on antennal segments, number of rhinaria, and body part ratios of Korean Takecallis are given in Table 1. In total, 63 COI sequences of five Takecallis species were downloaded from GenBank (Suppl. material 1). All sequences were aligned using MEGA 7 (Kumar et al. 2016). Intra-and inter-specific distances were calculated by a pairwise distance method based on the Kimura-2-Parameter (K2P) model (Kimura 1980). A neighbor-joining analysis (NJ) based on the K2P model for the final data set of 658 bp was also constructed.

Type-species. Takecallis bambusae
Description. Alate viviparous female: Color in life. Head pale to yellow, compound eye red. ANT pale, marginal border of ANT I-II dusky, the top end of 1/3 of the segment, and distal joint of ANT III dark, distal joint of ANT IV -BASE dusky. Thorax and ABD TERG pale yellow to bright yellow. Legs pale, distal 2/5 of FEM with dark spot, tarsi dark. Wing veins dark, margins of wing veins with dark spots. SIPH pale. Cauda slightly dark. Entire body covered with white wax.
Distribution. This species has so far been collected from Gyeongsangnam-do, Gangwon-do, and Jeollanam-do of South Korea.

Host plants.
This species feeds on the underside of leaves of Pseudosasa sp., and Sasa spp. (Poaceae). The host plants were identified by the first author using Lee (2003).
Remarks. This species was first referred to as Takecallis sp. in Lee et al. 2017.
Distribution. This species is originally distributed in East Asian countries; Korea (Paik 1965), China (Qiao and Zhang 2004), Japan (Higuchi 1968), and eastern Russia (Blackman and Eastop 2017). It has been introduced into Europe (Pons andLumbierres 2004, Stroyan 1964), and USA (California) (Clarke 1903). However, the recent DNA barcoding result revealed that European populations are genetically different from Asian populations (Lee et al. 2017).

Remarks.
Among the examined specimens, it is described that four alate viviparous females were collected on Gramineae sp. However, this host plant is probably not a true host plant due to Takecallis species being recorded only on bamboo species, and is probably a misidentification. Fig. 5, Table 1 Therioaphis tectae Tissot, 1932.

Molecular analyses and discussion
The NJ tree of partial COI sequences suggested that 63 sequences are distinctly divided into six groups (Fig. 7). This result clearly represented each morpho-specific group except the T. arundicolens complex. The T. arundicolens complex was separated into two genetically distinct groups (Fig. 7). Genetic distances between the two T. arundicolens groups ranged from 7.16 % to 9.36 %. These intraspecific divergence values are much higher than the general species delimitation value of 2.5 % in the subfamily Calaphidinae (Lee et al. 2017). In the previous study, Lee et al. (2017) suggested that this species complex seems to include at least 2 distinct species. However, it is very difficult to determine which one is the original species because morphological differences between genetically distinct groups were only observed in alatoid nymphs (Lee et al. 2017). Therefore, to solve this issue explicitly, additional studies are needed. Except for the T. arundicolens complex, the rest of the four species showed 0 % of intraspecific genetic divergence (Table 2). Interspecific distances among the five species ranged from 5.71 % to 14.44 % (Table 2). T. sasae and T. taiwana showed the lowest interspecific distance level (Table 2). Overall mean genetic distance was 8.91 % for the 63 partial COI sequences of the five Takecallis species.
Molecular evidence strongly indicates the validity of T. alba sp. n. All of the individuals of T. alba sp. n. were grouped together and this group was clearly separated from other species groups with a high interspecific distance level that ranged from 9.36 % to 13.46 % (Table 2). Morphological characteristics of this species correspond   to molecular evidence. Although we could not test all Takecallis species from all over the world, this species also has morphological characteristics that distinguish it from all known species. Morphologically, T. alba sp. n. is most similar to T. affinis and T. assumenta. However, its number of accessory setae on URS and the arrangement of secondary rhinaria on ANT III are clearly distinct from the above two species.
In the present study, four Takecallis species were recognized from Korea. Our study demonstrated that the four species are clearly separated based on morphological and molecular evidence. However, the taxonomic status of genetically distinct groups within the T. arundicolens complex still needs to be resolved.