Cladistics

Cladistics 30 (2014) 26–66 10.1111/cla.12024

A total-evidence phylogenetic analysis of Hormaphidinae (Hemiptera: Aphididae), with comments on the evolution of galls

Jing Chena,b, Li-Yun Jianga and Ge-Xia Qiaoa,* aKey Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China; bCollege of Life Sciences, University of Chinese Academy of Sciences, No. 19 Yuquan Road, Shijingshan District, Beijing, 100049, China Accepted 20 February 2013

Abstract

A phylogenetic analysis of Hormaphidinae is presented based on a total-evidence approach. Four genes (two mitochondrial, COI and CytB, and two nuclear, EF-1a and LWO) are combined with 65 morphological and seven biological characters. Sixty- three hormaphidine species representing three tribes and 36 genera as well as nine outgroups are included. Parsimony and model-based approaches are used, and several support values and implied weighting schemes are explored to assess clade stabil- ity. The monophyly of Hormaphidinae and Nipponaphidini is supported, but Cerataphidini and Hormaphidini are not recov- ered as monophyletic. Based on the parsimony hypothesis from the total-evidence analysis, the phylogenetic relationships within Hormaphidinae are discussed. Cerataphidini is re-delimited to exclude Doraphis and Tsugaphis, and Hormaphidini is redefined to include Doraphis. Ceratocallis Qiao & Zhang is established as a junior synonym of Ceratoglyphina van der Goot, syn. nov. Lithoaphis quercisucta Qiao, Guo & Zhang is transferred to the genus Neohormaphis Noordam as Neohormaphis quercisucta (Qiao, Guo & Zhang) comb. nov. Galls have evolved independently within three tribes of Hormaphidinae. In Cerataphidini, pseudogalls are ancestral, both single-cavity and multiple-cavity galls have evolved once, and galls appear to have evolved towards greater complexity. Galling on secondary hosts has evolved twice in hormaphidines. © The Willi Hennig Society 2013.

Introduction and eastern North America (Heie, 1980; Ghosh, 1985, 1988; von Dohlen et al., 2002). The subfamily Hormaphidinae (Hemiptera: Aphidi- Hormaphidinae species have complex life cycles. dae) is a clade of extraordinary aphids characterized Many species are heteroecious, seasonally obligately by the possession of several intrinsically fascinating alternating between primary host plants where the sex- biological characteristics. It comprises more than 200 ual phase of the life cycle is completed and galls are species within 45 genera and three tribes worldwide. produced, and secondary host plants where only par- Many genera (17 of 45) are monotypic, whereas sev- thenogenetic generations occur (Ghosh, 1985, 1988; eral genera show great species diversity (e.g. Astegop- Moran, 1988, 1992). Also abundant within this sub- teryx Karsch contains 21 described species). The tribes family are non-alternating species, which are believed Cerataphidini and Nipponaphidini are restricted to to have descended from heteroecious ancestors by los- eastern and south-eastern Asia, whereas the Horma- ing one set of hosts (Moran, 1988, 1992; Dixon and phidini exhibits a widespread distribution in Europe as Kundu, 1994; Blackman and Eastop, 2000). Strong well as a disjunctive distribution between eastern Asia host specificity is well defined and represented in the Hormaphidinae, with different patterns of host associ- ation among tribes. The Cerataphidini is primarily associated with Styrax (Styracaceae), and the Horma- *Corresponding author: E-mail address: [email protected] phidini and Nipponaphidini occupy Hamamelis and

© The Willi Hennig Society 2013 J. Chen et al. / Cladistics 30 (2014) 26–66 27

Distylium (Hamamelidaceae), respectively, as their pri- might have evolved towards a better ability to mary hosts. The secondary host association is more manipulate their host plants, thus achieving higher relaxed, with Cerataphidini on Compositae, Grami- reproductive success by enlarging gall volume, chang- neae, Loranthaceae, Palmaceae, and Zingiberaceae; ing galling sites, or forming complicated gall struc- Hormaphidini on Betula (Betulaceae) and Picea (Pina- tures. So far, however, the evolution of galls in ceae); and Nipponaphidini on Fagaceae, Lauraceae, Hormaphidinae has not yet been thoroughly studied. and Moraceae. Based on the basic structure and mode of gall for- A great many hormaphidine aphids are known to mation, Fukatsu et al. (1994) suggested that multi- induce galls on their primary host plants (Chen and ple-cavity galls appear to be apomorphic and have Qiao, 2009, 2012a; Aoki and Kurosu, 2010). The mor- evolved only once in Cerataphidini. Stern (1995) phology and ontogeny of galls have been documented confirmed the single origin of multiple-cavity galls in in detail for many species (Kurosu and Aoki, 1990, Cerataphidini using the mitochondrial cytochrome 1991a,b, 1994, 1997, 2001, 2003, 2009; Aoki and oxidase subunits I (COI) and II (COII) genes. It Kurosu, 1992, 2010; Aoki et al., 1995, 2001, 2002; So- would be interesting to investigate the evolutionary rin, 1996, 2001; Qiao and Zhang, 2004; Kurosu et al., history of gall morphology within Homaphidinae 2006, 2008). Galls provide abundant nutrition (Price and to test the evolutionary trend that has been et al., 1986, 1987; Inbar et al., 2004; Koyama et al., revealed in Eriosomatinae. 2004; Zhang and Qiao, 2007a,b), a favourable micro- Hormaphidinae was once classified within Erioso- environment (Felt, 1940; Price et al., 1986, 1987; matinae or Thelaxinae in early studies (Mordvilko, Miller et al., 2009), and protection against natural ene- 1908, 1948; Borner,€ 1930; Borner€ and Heinze, 1957). It mies (Cornell, 1983; Price et al., 1986, 1987) to the was first regarded as a subfamily of Aphididae by inducer and its offspring. They can also mitigate clonal Baker (1920). Subsequently, Hille Ris Lambers (1964) mixing and maintain genetic integrity (Foster and considered Hormaphidinae a distinct clade closely Northcott, 1994; Stern and Foster, 1996). In Hor- related to Eriosomatinae and Thelaxinae, based on maphidinae, galls are quite diverse in terms of type, retaining three-faceted eyes in nymphs, which was site, shape, and structure (Ghosh, 1985, 1988; Chen approved by many authors (Zhang and Zhong, 1983; and Qiao, 2009, 2012a; Fig. 1). A few species form Ghosh, 1985, 1988; Blackman and Eastop, 1994, 2000; pseudogalls that appear as leaf rolls, leaf curls, or leaf Remaudiere and Remaudiere, 1997). Present knowl- blisters on the hosts (e.g. Aleurodaphis stewartiae Sorin edge of Hormaphidinae has resulted largely from the & Miyazaki, see Fig. 4k in Sorin and Miyazaki, 2004). great contributions of van der Goot (1917) and Noor- The majority produce variously shaped and structured dam (1991) on the Javan fauna; Takahashi (1931, true galls at different sites on the host plants (Fig. 1). 1935, 1936, 1939, 1941, 1957, 1958a,b,c,d, 1959a,b, Gall morphology is also highly specific to a particular 1962) and Takahashi and Sorin (1958) on the Chinese, species (Wool, 2004; Chen and Qiao, 2009, 2012a). Japanese, Sumatran, and Thai fauna; Sorin (1979, For instance, Astegopteryx spinocephala Kurosu, 1987, 1996, 1999, 2001, 2006) on the Japanese fauna; Buranapanichpan & Aoki forms banana-bunch-shaped Raychaudhuri et al. (1980) and Ghosh (1988) on the galls composed of several subgalls on twigs of Styrax Indian fauna; and Qiao and Zhang (1998, 1999, 2001, benzoides (see Fig. 1 in Kurosu et al., 2006); Hama- 2003, 2004) on the Chinese fauna. melistes miyabei (Matsumura) induces spherical galls The first high-level molecular phylogenetic analysis of with many spine-like hairs on twigs of Hamamelis the Aphididae including hormaphidines was based on japonica (see Fig. 1a in Aoki et al., 2001); and Neotho- mitochondrial ribosomal DNA (partial 12S and 16S) racaphis yanonis (Matsumura) produces spherical galls and included only three hormaphidine representatives, with a pointed bottom that protrude from both sides wherein the monophyly of Hormaphidinae was not of the leaves on Distylium chinense and D. racemosum recovered (von Dohlen and Moran, 2000). More recent (Fig. 1e). Therefore galls are commonly regarded as molecular studies conducted by Ortiz-Rivas et al. the extended phenotype of aphids (Dawkins, 1982; (2004) and Ortiz-Rivas and Martınez-Torres (2010) Stern, 1995; Stone and Schonrogge,€ 2003; Wool, included one and 12 hormaphidines, respectively. Both 2004). They are very helpful for species identifications, studies revealed a close relationship among Hormaphid- especially for species that are difficult to distinguish inae, Anoeciinae, Eriosomatinae, Mindarinae (not morphologically (e.g. Sorin, 2003), and also useful in included in the former study), and Thelaxinae. In the phylogenetic studies of aphids. latter phylogenetic analysis, the monophyly of Hor- Many studies have been conducted on the evolu- maphidinae was retrieved, albeit with low statistical tion of gall morphology in another galling aphid supports, based on the nuclear elongation factor-1a group, Eriosomatinae (Inbar et al., 2004; Zhang and (EF-1a) gene and the mitochondrial cytochrome oxidase Qiao, 2007a,b, 2008; Sano and Akimoto, 2011). It subunit II (COII) gene (excluding the third codon posi- has been suggested that the Eriosomatinae aphids tions). The monophyly of this subfamily was not estab- 28 J. Chen et al. / Cladistics 30 (2014) 26–66

(a) (b)

(c) (d)

(e) (f)

Fig. 1. Galls of Hormaphidinae. (a) A gall of Tuberaphis owadai Kurosu & Aoki on a twig of Styrax japonica (Yuanyang, Kunming, Yunnan, China; 10 June 2009): coral-like, single-cavity, with many small openings on the projections. (b) Leaf curls caused by Hamamelistes sp. on Betula sp. (Longsheng, Guilin, Guangxi, China; 19 May 2006). (c) Galls of Asiphonipponaphis vasigalla Chen, Sorin & Qiao on the leaf midribs of Distylium chinense (Jishou, Hunan, China; 15 April 2010): vase-shaped, single-cavity, opened on the bottom. (d) A gall of Metanipponaphis sp. on a petiole of Distylium chinense (Jishou, Hunan, China; 6 September 2009): global, single-cavity, closed. (e) Galls of Neothoracaphis yanonis (Matsumura) on the leaves of Distylium chinense (Jishou, Hunan, China; 21 May 2009): spherical with a pointed bottom, protruding from both sides of the leaves, single-cavity, closed. (f) A gall of Nipponaphis sp. on a twig of Distylium chinense (Jishou, Hunan, China; 5 September 2009): bottle-shaped, single-cavity, closed. lished robustly until recently. Huang et al. (2012) com- Previous phylogenetic studies within tribes have pleted a relatively detailed molecular phylogenetic study focused mostly on Cerataphidini and Hormaphidini. of hormaphidines, which tested the monophyly and Within the Cerataphidini, two monophyletic clades major relationships of Hormaphidinae using a broad were identified: one consisting of Astegopteryx, Cerato- taxonomic sample and a combined two-gene dataset. A glyphina, Ceratovacuna, Chaitoregma,andPseud- sister relationship between the tribes Hormaphidini and oregma; the other including Cerataphis and Tuberaphis Nipponaphidini was revealed, and the relationships of (Fukatsu et al., 1994; Stern, 1994, 1995, 1998; Stern constituent genera were discussed. However, only five et al., 1997; Huang et al., 2012). The genus Aleuroda- representatives of Nipponaphidini were included in that phis was once suggested to be a basal lineage to all study, leaving the generic relationships unresolved. remaining hormaphidines (Stern, 1994); however, Morphological cladistic analyses of Hormaphidinae Huang et al. (2012) confirmed its attribution to Cer- have been rare. Qiao (1996) estimated the phylogeny of ataphidini and proposed a sister relationship between hormaphidines based on limited morphological and bio- Aleurodaphis and Ktenopteryx. The phylogenetic posi- logical characters, suggesting a closer relationship tion of Glyphinaphis within Cerataphidini remains between Hormaphidini and Nipponaphidini. uncertain (Fukatsu et al., 1994; Stern, 1994, 1998; J. Chen et al. / Cladistics 30 (2014) 26–66 29

Huang et al., 2012), and the taxonomic positions and most genera in Nipponaphidini (20 of 29) were within this subfamily of several genera (Ceratocallis, sampled. An exemplar approach was implemented, Doraphis, and Tsugaphis) that have never been where one or several exemplar species were chosen for included in previous studies remain unclear (Aoki and each genus. All but three ingroup species were Kurosu, 2010). The phylogeny of Hormaphidini has represented by at least three genes (Table 1). The been tested on the basis of nuclear and mitochondrial combined dataset included 60 hormaphidine species genes (von Dohlen et al., 2002; Chen et al., 2011b). from the morphological dataset and 45 species from The monophyly of both Hamamelistes and Hormaphis the molecular dataset. Forty-two species were scored was well supported after transferring Hormaphis simili- for both morphology and molecules. betulae to Hamamelistes. However, the monotypic genus Protohormaphis has never been studied. Outgroup. On the basis of current phylogenetic Additionally, few studies have explored the phyloge- hypotheses for Aphididae (Heie, 1987; Wojciechowski, netic relationships of Nipponaphidini. 1992; Zhang et al., 1999; Ortiz-Rivas et al., 2004; The aims of the present study are to reconstruct reli- Ortiz-Rivas and Martınez-Torres, 2010), nine species able and detailed phylogenetic relationships within of four subfamilies (Anoeciinae, Eriosomatinae, Hormaphidinae and to trace the evolutionary scenar- Mindarinae, and Thelaxinae) were chosen to serve as ios of gall morphology. Compared with previous stud- outgroups. Six species of Eriosomatinae, the ies, a largely expanded dataset was used by (i) traditional sister group of Hormaphidinae (Heie, 1967; including four genes totaling more than 3 kb; (ii) add- Ghosh, 1985, 1988; Wojciechowski, 1992; Zhang et al., ing 72 morphological and biological characters; and 1999), were selected widely across three tribes. All but (iii) sampling widely across three constituent tribes in one outgroup species were represented by at least three Hormaphidinae. The resulting hypothesis will be useful genes (Table 1). The combined dataset included four for the phylogenetic classification of Hormaphidinae outgroup species from the morphological dataset and and will provide a comparative framework for the nine species from the molecular dataset. Four species understanding of character evolution. were scored for both morphology and molecules. All analyses were rooted with Kurisakia onigurumii (Shinji) which belongs to the subfamily Thelaxinae. Materials and methods Molecular data Taxon sampling Genes. Molecular analyses were based on the Sequenced taxa. The specimens used for molecular mitochondrial genes cytochrome oxidase subunit I work were preserved in 95% or 100% ethanol. Some (COI) and cytochrome b (CytB) and the nuclear genes published sequences were taken from von Dohlen elongation factor-1a (EF-1a) and long-wavelength et al. (2002), Foottit et al. (2008), Ortiz-Rivas et al. opsin gene (LWO). Mitochondrial genes were selected (2009), Ortiz-Rivas and Martınez-Torres (2010), Kim to provide resolution at lower taxonomic levels et al. (2011), Lee et al. (2011), and Huang et al. (generic and specific) (Coeur d’acier et al., 2007, 2008; (2012). A total of 54 species were represented by Kim and Lee, 2008; Zhang et al., 2011), whereas molecular data. Specimens from the same clonal nuclear genes were used to provide resolution deeper colony and preserved in 75% ethanol were kept for within the subfamily (Normark, 2000; Ortiz-Rivas slide voucher specimens and identification. Species et al., 2004; von Dohlen et al., 2006; Zhang and Qiao, identification was performed by G.X. Qiao based on 2008; Ortiz-Rivas and Martınez-Torres, 2010). exterior morphology of slide-mounted specimens, then verified by National Center for Biotechnology DNA extraction, amplification, and sequencing. Total Information (NCBI) Basic Local Alignment Search genomic DNA was extracted from a single aphid Tool (BLAST) searches of the cytochrome oxidase individual using a CTAB (hexadecyltrimethyl- subunit I (COI) sequences. All samples and voucher ammonium bromide) protocol modified from Doyle specimens were deposited in the National Zoological and Doyle (1987). All primers used in this study are Museum of China, Institute of Zoology, Chinese listed in Table 2. Typical polymerase chain reactions Academy of Sciences, Beijing, China (NZMCAS). were prepared in a 25 lL volume containing 10 9 Details of the sequenced taxa and voucher information EasyTaq DNA Polymerase Buffer (+Mg2+) are listed in Table 1. (TransGen Biotech, Beijing, China), 1.5 U EasyTaq DNA Polymerase (TransGen Biotech), 2.5 mM each Ingroup. Sixty-three species belonging to 36 genera dNTP (TransGen Biotech), 5 pmole each primer, and of Hormaphidinae were included in this study. All 1 lL DNA extract. The PCR thermal regime was as genera in Cerataphidini (13) and Hormaphidini (3) follows: 5 min initial denaturation at 95 °C followed 30

Table 1 Voucher information and GenBank accession numbers for the sequenced taxa

Voucher Species Host plant Collection locality number COI CytB EF-1a LWO

Hormaphidinae Ceretaphidini Aleurodaphis asteris Takahashi & Sorin Unknown Tibet, China 15371 JN032729* DQ493867 JX489721 Aleurodaphis blumeae van der Goot Unknown Guizhou, China 15597 JX489623 JX489655 JX489689 JX489722 Astegopteryx bambusae (Buckton) Fargesia semicoriacea Guangxi, China 14592 JX489624 JX489656 JX489690 JX489723 Astegopteryx rhapidis (van der Goot) Cocos nucifera Hainan, China Y8927 JX489625 JX489691 JX489724 Astegopteryx styracophila Karsch Zingiberaceae Hainan, China 26615 JX489626 JX489657 JX489692 JX489725 Cerataphis bambusifoliae Takahashi Chimonobambusa Fujian, China 14553 JN032723* DQ493861 JX489726 quadrangularis Cerataphis freycinetiae van der Goot Unknown Vietnam VT008 JX489627 JX489658 JX489693 JX489727 Ceratoglyphina bambusae van der Goot Bambusoideae Fujian, China 14466 JN032718* JX489659 DQ493856 JX489763 Ceratoglyphina phragmitidisucta Zhang Bambusoideae Fujian, China 14811 JX489628 JX489660 JX489694 JX489728 Ceratovacuna lanigera Zehntner Imperata cylindrica Guangxi, China 14655 JX282722 JX282621 JX282800 JX282866 Ceratovacuna panici (van der Goot) Indocalamus sp. Guangxi, China 14648 JX282702 JX282636 JX282816 JX282864 26–66 (2014) 30 Cladistics / al. et Chen J. Ceratovacuna silvestrii (Takahashi) Bambusoideae Guizhou, China 24537 JX282752 JX282647 JX282829 Chaitoregma tattakana (Takahashi) Bambusoideae Yunnan, China 24048 JX489629 JX489695 JX489729 Doraphis populi (Maskell) Populus sp. Hebei, China 16125 JX489630 JX489661 JX489696 JX489730 Glyphinaphis bambusae van der Goot Bambusoideae Zhejiang, China 17336 JX489631 JX489662 JX489697 JX489731 Ktenopteryx eosocallis Qiao & Zhang Styrax sp. Fujian, China 14438 JN032716* DQ493854 JX489764 Pseudoregma alexanderi (Takahashi) Bambusoideae Fujian, China 22022 JX489632 JX489663 JX489698 JX489732 Pseudoregma bambucicola (Takahashi) Bambusoideae Guangdong, China 22006 JN032731* JX489664 JX489699 JX489733 Pseudoregma koshunensis (Takahashi) Bambusoideae Guangxi, China 14602 JQ916430 JX489665 JX489700 JX489734 † Tuberaphis coreana Takahashi Unknown Japan CNC no. EU701940 HEM054817 Tuberaphis xinglongensis (Zhang) Cocos nucifera Hainan, China Y8928 JX489633 JX489666 JX489701 JX489735 Hormaphidinae Hormaphidini Hamamelistes similibetulae (Qiao Betula albosinensis Tibet, China 13549 JQ920920 JX489667 DQ493849 JX489736 & Zhang) Hamamelistes sp. Betula sp. Guangxi, China 18896 JX489634 JX489668 JX489702 JX489737 Hormaphis betulae (Mordvilko) Betula sp. Jilin, China 15214 JN032726* JX489669 DQ493864 JX489738 † ‡ Hormaphis cornu (Shimer) EU701682 AF454612 Hormaphidinae Nipponaphidini Dermaphis crematogastri (Takahashi) Fagaceae Guangxi, China 26041 JX489635 JX489703 JX489739 Euthoracaphis heterotricha Ghosh & Cinnamomum Yunnan, China Y8800 JX489636 JX489670 JX489704 JX489740 Raychaudhuri burmannii Euthoracaphis oligostricha Chen, Machilus Yunnan, China 15299 JN032727* DQ493865 JX489765 Fang & Qiao yunnanensis Metanipponaphis lithocarpicola Castanopsis Fujian, China 14539 JX489637 JX489671 JX489705 JX489741 (Takahashi) sclerophylla Metanipponaphis sp. Distylium chinense Hunan, China Y8914 JX489638 JX489706 JX489742 Neohormaphis wuyiensis Qiao & Jiang Quercus sp. Fujian, China 14525 JX489762 JX489672 DQ493858 JX489743 Neonipponaphis pustulosis Chen & Qiao Castanopsis eyrei Fujian, China 26868 JX489639 JX489673 JX489707 JX489744 Neothoracaphis elongata (Takahashi) Quercus sp. Yunnan, China 23871 JX489640 JX489674 JX489708 JX489745 Neothoracaphis quercicola (Takahashi) Quercus acutissima Yunnan, China 24087 JX489641 JX489709 JX489746 Neothoracaphis yanonis (Matsumura) Distylium chinense Hunan, China Y8970 JX489642 JX489675 JX489710 JX489747 Table 1 (Continued)

Voucher Species Host plant Collection locality number COI CytB EF-1a LWO § § § Nipponaphis coreana (Paik) Neolitsea sericea Korea 030513SH104 GU457804 GU457828 GU457844 ¶ ‡ Nipponaphis distyliicola Monzen GU978809 AF454614 Nipponaphis sp. 1 Machilus yunnanensis Yunnan, China Y8877 JX489643 JX489676 JX489711 JX489748

Nipponaphis sp. 2 Distylium chinense Hunan, China Y8917 JX489644 JX489677 JX489712 JX489749 26–66 (2014) 30 Cladistics / al. et Chen J. Parathoracaphis manipurensis Castanopsis sp. Yunnan, China 24888 JX489645 JX489678 JX489713 JX489750 (Pramanick, Samanta & Raychaudhuri) Parathoracaphis setigera (Takahashi) Fagaceae Yunnan, China 23861 JX489646 JX489679 JX489714 JX489751 Reticulaphis inflata Yeh & Hsu Ficus sp. Yunnan, China 24049 JX489647 JX489680 JX489715 JX489752 Reticulaphis sp. Ficus sp. Yunnan, China 24084 JX489648 JX489681 JX489716 JX489753 Schizoneuraphis gallarum Litsea sp. Guangxi, China 26139 JX489649 JX489682 JX489717 JX489754 van der Goot Thoracaphis quercifoliae Ghosh Quercus sp. Fujian, China 14526 JN032722* JX489683 DQ493851 JX489755 Anoeciinae Anoecia fulviabdominalis (Sasaki) Unknown Heilongjiang, China 17822 JX489650 JN847243* Eriosomatinae Epipemphigus yunnanensis (Chang) Populus yunnanensis Yunnan, China 18234 JX489651 JX489684 JX489758 JX489766 Eriosoma ulmi (Linnaeus) Ulmus sp. Hebei, China 14288 JX489652 JX489685 JX489718 JX489756 † †† Forda marginata Koch EU701668 FM163596** FM177108 Kaburagia rhusicola Takagi Rhus punjabensis Yunnan, China 18753 JX489761 JX489686 JX489759 var. sinica Prociphilus ligustrifoliae (Tseng & Tao) Ligustrum quihoui Yunnan, China 18235 JQ916897 JX489687 JX489719 JX489767 Tetraneura chinensis Mordvilko Ulmus pumila Beijing, China 16104 JX489653 JX489720 JX489757 Mindarinae Mindarus keteleerifoliae Zhang Keteleeria evelyniana Yunnan, China 18171 JX489654 JX489760 JX489768 Thelaxinae Kurisakia onigurumii (Shinji) Pterocarya sp. Guizhou, China 13303 JN847245* JX489688 DQ493825 JX489769 † ‡ § Reference sequences from previous studies: *Huang et al. (2012), Foottit et al. (2008), von Dohlen et al. (2002), Kim et al. (2011), ¶Lee et al. (2011), **Ortiz-Rivas et al. (2009), †† Ortiz-Rivas and Martınez-Torres (2010). 31 32 J. Chen et al. / Cladistics 30 (2014) 26–66

Table 2 Primers used in this study

Gene Primer Sequence References

COI LepF 5′-ATTCAACCAATCATAAAGATATTGG-3′ Foottit et al. (2008) LepR 5′-TAAACTTCTGGATGTCCAAAAAATCA-3′ CytB CP1 5′-GATGATGAAATTTTGGATC-3′ Harry et al. (1998) CP2 5′-CTAATGCAATAACTCCTCC-3′ CB2 5′-ATTACACCTCCTAATTTATTAGGAAT-3′ Jermiin and Crozier (1994) EF-1a EF3 5′-GAACGTGAACGTGGTATCAC-3′ von Dohlen et al. (2002) EF6 5′-TGACCAGGGTGGTTCAATAC-3′ EF2 5′-ATGTGAGCAGTGTGGCAATCCAA-3′ Palumbi (1996) LWO OPSETF1 5′-GGYRTYACNATTTTYTTCTTRGG-3′ Designed by B. Ortiz-Rivas OPSETR1 5′-GANCCCCADATYGTNAATAAYGG-3′ OPSETF2 5′-ATGTGYCCRCCRATGGTNTGGA-3′ OPSETR2 5′-GGWGTCCANGCCATRAACCA-3′ by 35 cycles of 95 °C for 30–60 s, 48–52 °C for Morphological and biological data 30–60 s, 72 °C for 1–1.5 min, and a 10 min final extension at 72 °C. The primer-specific annealing A total of 72 characters were scored for 64 taxa, temperatures of each primer set were 52 °C for COI, including 65 morphological characters and seven bio- 48 °C for CytB and LWO, and 50 °C for EF-1a. For logical characters (Table 3). The list of characters and the amplification of LWO genes of some samples, a their states is provided below. Sixty-seven discrete second nested PCR was necessary using primers characters were treated as non-additive. Five continu- OPSETF2 and OPSETR2 (Table 2) on 1 lL aliquot ous characters were coded as a range of one standard from the first PCR. Conditions were identical except error around the mean and treated as additive (Golob- for the increase of the annealing temperature to 50 °C. off et al., 2006). PCR products were purified using EasyPure Quick Gel Morphological characters were evaluated for apter- Extraction Kit (TransGen Biotech) and then ous and alate viviparous females. We followed Miya- sequenced directly. Sequencing reactions were zaki (1987) for the morphological terminology and performed using the corresponding PCR primers from Blackman and Eastop (1994, 2000) for the measure- both directions with BigDye Terminator v3.1 Cycle ment of body parts. For host-alternating species (Ano- Sequencing Kit (Applied Biosystems, Foster City, CA, eciinae, Eriosomatinae, and Hormaphidinae), the states USA) and run on an ABI 3730 automated sequencer of the morphological characters of apterae were evalu- (Applied Biosystems). In some cases, cloning of the ated for apterous exules, which were born on and colo- nuclear genes was necessary using pMD19-T Vector nized secondary host plants (Hille Ris Lambers, 1966). System (TaKaRa, Dalian, China) and Trans5a Either alate exules or sexuparae were used for the char- Chemically Competent Cell (TransGen Biotech) acter evaluation of alatae. They are both produced on following the manufacturer’s instructions. At least the secondary host and are very similar in morphology; three clones were sequenced. alate exules are hypothesized to have been derived from the alate sexuparae (Roberti, 1972; Akimoto, 1985; Sequence edition and alignment. Sequences were Aoki and Kurosu, 2010). Ceratocallis camellis Qiao & assembled by SeqMan II (DNAStar, Madison, WI, Zhang and Ktenopteryx eosocallis Qiao & Zhang were USA) and verified for protein coding frame-shifts scored based on their only known morphs, apterae using EditSeq (DNAStar). The positions of introns in from the putative primary host Camellia and primary EF-1a and LWO sequences were determined by host Styrax, respectively. Characters were scored from following the GT–AG rule and aligning sequences direct observation of the specimens under a Leica with the cDNA sequences from Hormaphis betulae DM2500 microscope and photographing with a Leica (GenBank accession no. AF454611) and Cerataphis sp. DFC490 digital camera system and Leica QWin Plus (GenBank accession no. AJ539465), respectively. software. The exemplar specimens examined in this Introns were removed before further analysis. All study are deposited in the National Zoological sequences have been deposited in GenBank (Table 1). Museum of China, Institute of Zoology, Chinese Acad- Multiple alignments were performed with ClustalX emy of Sciences, Beijing, China (NZMCAS). Character 1.83 (Thompson et al., 1997) and then verified evaluations of eight species were conducted on the manually. The final molecular dataset contained a basis of descriptions from the literature: Forda margin- total of 3006 characters, 997 of which were parsimony ata Koch (Sano and Akimoto, 2011), Hormaphis cornu informative characters (PICs). (Shimer) (von Dohlen and Stoetzel, 1991), Lithoaphis J. Chen et al. / Cladistics 30 (2014) 26–66 33

Table 3 Morphological data matrix

Characters Taxa 01234 Aleurodaphis asteris 0.25–0.27 0.32–0.34 0.54–0.68 3.12–3.34 1.78–1.86 Aleurodaphis blumeae 0.21–0.23 0.31–0.33 0.45–0.51 4.36–4.56 2.73–2.87 Astegopteryx bambusae 0.18–0.20 0.27–0.29 0.73–0.79 1.02–1.12 0.72–0.78 Astegopteryx rhapidis 0.27–0.29 0.25–0.27 0.87–0.99 1.13–1.23 0.57–0.59 Astegopteryx styracophila 0.21 0.23–0.30 1.11–1.12 0.82 0.58–0.60 Cerataphis bambusifoliae 0.20–0.21 0.36–0.38 0.64–0.68 0.91–0.99 0.61–0.65 Cerataphis freycinetiae 0.22 0.33–0.35 0.58–0.62 0.94–0.96 0.53–0.55 Ceratocallis camellis 0.20–0.21 0.38–0.42 0.34–0.36 0.65–0.71 0.46–0.48 Ceratoglyphina bambusae 0.17 0.61–0.65 0.40–0.44 1.22–1.28 0.51–0.53 Ceratoglyphina phragmitidisucta 0.16 0.35–0.37 0.44–0.46 1.04–1.14 0.43–0.49 Ceratovacuna lanigera 0.15 0.44–0.48 0.74–0.84 0.97–1.03 0.45–0.49 Ceratovacuna panici 0.15–0.17 0.38–0.42 0.82–0.90 1.00–1.08 0.43–0.45 Ceratovacuna silvestrii 0.15 0.35–0.37 0.33–0.36 1.13–1.29 0.63–0.69 Chaitoregma tattakana 0.16 0.40–0.42 1.08–1.18 1.14–1.28 0.54–0.58 Doraphis populi 0.02 ? ? ? ? Glyphinaphis bambusae 0.18 0.31–0.33 0.64–0.72 1.10–1.16 0.67–0.73 Ktenopteryx eosocallis 0.25–0.27 0.43–0.46 0.30–0.32 2.16–2.23 1.55–1.65 Pseudoregma alexanderi 0.16–0.17 0.36–0.43 0.50–0.58 1.37–1.49 0.50–0.53 Pseudoregma bambucicola 0.17 0.39–0.43 1.35–1.45 1.43–1.62 0.52–0.54 Pseudoregma koshunensis 0.15–0.17 0.23 0.39–0.45 1.40–1.42 0.53 Tsugaphis sorini 0.17–0.18 0.24–0.26 0.25 0.67 0.59 Tuberaphis coreana 0.25 0.24 0.83 1.29 0.75 Tuberaphis xinglongensis 0.25–0.26 0.38–0.42 0.42–0.82 1.15–1.23 0.64–0.68 Hamamelistes similibetulae 0.10 0.34–0.40 ? 1.42–1.48 1.84–1.96 Hamamelistes sp. 0.12 0.32–0.36 ? 1.41–1.51 1.16–1.42 Hormaphis betulae 0.04 0.30–0.32 ? 1.00–1.06 1.38–1.58 Hormaphis cornu ????? Protohormaphis piceae 0.45 0.38 ? 1.43 0.67 Allothoracaphis piyananensis 0.05 0.62–0.68 ? 1.94–1.99 1.59–1.83 Dermaphis crematogastri 0.07 0.13–0.19 ? 1.68–1.93 1.54 Euthoracaphis heterotricha 0.12 0.10–0.12 ? 1.75–2.09 1.40 Euthoracaphis oligostricha 0.12 0.08–0.10 ? 1.29–1.37 1.08–1.16 Indonipponaphis fulvicola 0.13–0.14 0.05 ? 2.00 1.14–1.44 Lithoaphis quercisucta 0.10 0.10–0.12 ? 0.78–0.84 0.80–0.84 Lithoaphis shiiae ? ? ? ? 2.00 Mesothoracaphis rappardi 0.08–0.10 0.33 ? ? 1.70–1.90 Metanipponaphis lithocarpicola 0.09–0.11 0.10–0.12 ? 1.16–1.22 0.87–0.95 Neohormaphis calva 0.11–0.16 ? ? ? 0.73–0.84 Neohormaphis wuyiensis 0.10–0.11 0.08–0.10 ? 1.09 0.73 Neonipponaphis shiiae 0.17 0.08–0.09 ? 1.09–1.19 1.42–1.50 Neonipponaphis pustulosis 0.16 0.08 ? 1.70 1.38–1.42 Neothoracaphis elongata 0.04 ? ? ? ? Neothoracaphis quercicola 0.03 ? ? 1.06–1.33 1.29–1.33 Neothoracaphis yanonis 0.04 ? ? 1.25–1.31 1.72–1.92 Nipponaphis distyliicola ? ? ? ? 1.70–1.80 Nipponaphis monzeni 0.16–0.17 0.06 ? 1.68–1.76 1.42–1.48 Nipponaphis sp. 1 0.17 0.05–0.08 ? 1.80–2.10 1.38–1.50 Paranipponaphis takaoensis ? ? ? ? 2.00–2.30 Parathoracaphis manipurensis 0.09 0.08–0.11 ? 0.88–1.10 0.78–0.84 Parathoracaphis setigera 0.04 0.27–0.31 ? 1.32–1.40 1.18–1.20 Parathoracaphisella indica 0.03 ? ? ? 0.70 Quadrartus agrifoliae 0.03 1.51–1.99 ? 1.81 3.17 Quadrartus yoshinomiyai 0.04 ? ? 2.20–2.90 ? Quernaphis tuberculata 0.06 1.00 ? 2.34–2.62 3.00 Reticulaphis inflata 0.07 0.25 ? 1.36–1.62 1.27–1.49 Reticulaphis sp. 0.08–0.09 0.28–0.30 ? 1.67 1.60 Schizoneuraphis gallarum 0.17–0.18 0.05 ? 1.67 1.11 Sinonipponaphis formosana 0.18 0.12–0.15 ? 1.46–1.64 1.38–1.50 Thoracaphis kashifoliae 0.05 0.44–0.52 ? 1.00–1.09 1.06–1.12 Thoracaphis quercifoliae 0.16–0.18 0.06 ? 1.30 0.79 Anoecia fulviabdominalis 0.43–0.46 0.35 0.86–0.92 2.50–2.88 0.95–1.05 Forda marginata ????? Mindarus keteleerifoliae 0.37–0.39 0.22–0.24 0.37–0.45 1.48–1.70 0.48–0.54 Kurisakia onigurumii 0.34–0.36 0.29–0.30 1.92–2.20 2.41–2.65 1.21–1.23 34 J. Chen et al. / Cladistics 30 (2014) 26–66

Table 3 (Continued)

First five characters are continuous, final 67 characters are discrete. Inapplicable characters and missing data are indicated by ‘–’ and ‘?’, respectively. Polymorphic states are represented as follows: a = [01]; b = [12]; c = [23]; d = [29]; e = [34]; f = [67]. J. Chen et al. / Cladistics 30 (2014) 26–66 35 shiiae Takahashi (Takahashi, 1959a), Neohormaphis tered on the marginal area of dorsum; (6) only present calva Noordam (Noordam, 1991), Nipponaphis distyliico- on abdominal tergite VIII. la Monzen (Takahashi, 1962; Ghosh and Raychaudhuri, 17 Development of wax gland plates: (0) devel- 1973b), Paranipponaphis takaoensis Takahashi oped, with well developed facets (Fig. 2c); (1) degener- (Takahashi, 1959a), Parathoracaphisella indica Prama- ate, without well developed facets (Fig. 2h). nick, Samanta & Raychaudhuri (Ghosh, 1988), and 18 Pustules or sculptures on dorsum: (0) absent; Quadrartus yoshinomiyai Monzen (Sorin, 2001). (1) present. 19 Shape of pustules or sculptures: (0) oval (Fig. 2j); (1) mosaic-like (Fig. 2d); (2) polygonal Continuous characters (Fig. 2i); (3) hemispherical; (4) conical. 20 Reticulations on dorsum: (0) absent; (1) present 0 Relative length of antennae to body length (Fig. 2h). (apterous viviparous females). 21 Dorsal setae: (0) fine; (1) stout (Fig. 3a); (2) 1 Relative length of processus terminalis to base of dagger-like (Fig. 3b). the last antennal segment (apterous viviparous females). 22 Submarginal setae on prosoma: (0) fine 2 Relative length of setae on antennal segment III (Fig. 3c); (1) stout (Fig. 3d); (2) dagger-like (Fig. 3e). to the widest diameter of the segment (apterous viviparous 23 Posteromesial setae at hind end of prosoma: (0) females). present; (1) absent. 3 Relative length of ultimate rostral segment to its 24 Submarginal setae on abdominal tergites II– basal width (apterous viviparous females). VII: (0) present; (1) absent. 4 Relative length of ultimate rostral segment to sec- 25 Submarginal setae on abdominal tergites II– ond hind tarsal segment (apterous viviparous females). VII: (0) fine; (1) stout; (2) dagger-like (Fig. 3f). 26 Posteromesial setae on abdominal tergite VII: (0) present; (1) absent. Discrete characters 27 Number of setae on abdominal tergite VIII: (0) two; (1) four; (2) 5–11; (3) more than 11. Apterous viviparous females 28 Frontal horns: (0) absent (Fig. 2a); (1) present (Fig. 2b). 5 Body shape: (0) elliptical (Fig. 2a); (1) round 29 Frontal horns: (0) pointed (Fig. 4a); (1) blunt (Fig. 2g); (2) ovate but asymmetrical (Fig. 2k). (Fig. 4b). 6 Aleyrodiform: (0) no (Fig. 2c); (1) yes (Fig. 2h). 30 Setae on frontal horns: (0) absent (Fig. 4a); (1) 7 Prosoma consisting of fused head, thorax, and present (Fig. 4b). abdominal segment I (Ghosh and Raychaudhuri, 31 Compound eyes: (0) three-faceted (Fig. 4c); (1) 1973a): (0) absent (Fig. 2c); (1) present (Fig. 2j). two-faceted (Fig. 4e); (2) multi-faceted. 8 Prosoma: (0) not subdivided (Fig. 2j); (1) subdi- 32 Compound eyes (if three-faceted): (0) three fac- vided (Fig. 2k). ets located close to each other (Fig. 4c); (1) one facet 9 Abdominal segments II–VII: (0) normal located a little apart from others (Fig. 4d). (Fig. 2c); (1) fused (Fig. 2l). 33 Number of antennal segments: (0) six; (1) five; 10 Abdominal segments II–VII (if fused): (0) not (2) four; (3) three; (4) two (Fig. 4g); (5) one (Fig. 4h). subdivided (Fig. 2l); (1) subdivided (Fig. 2k). 34 Setae on antennal segment III: (0) present; (1) 11 Prosoma and abdominal segments II–VII: (0) absent. completely separated (Fig. 2j–l); (1) indistinctly 35 Ultimate rostral segment: (0) blunt (Fig. 4i); (1) separated (Fig. 2i); (2) completely fused (Fig. 2d,g,h). pointed (Fig. 4j). 12 Head and thorax relative to abdomen: (0) 36 Fore and middle legs: (0) protruding behind shorter; (1) equal to or longer. body (Fig. 2a); (1) concealed under body (Fig. 2f). 13 Long marginal processes on dorsum: (0) absent; 37 Hind legs: (0) protruding behind body (Fig. 2l); (1) present (Fig. 2e). (1) concealed under body (Fig. 2h). 14 Crenulated submarginal area along the entire 38 Fore and middle tarsi: (0) well developed, two- margin of body: (0) absent; (1) present. segmented (Fig. 5a); (1) rudimentary, unsegmented; (2) 15 Wax gland plates on dorsum: (0) present; (1) absent (Fig. 5d). absent. 39 Hind tarsi: (0) well developed, two-segmented; 16 Arrangement of wax gland plates: (0) along the (1) rudimentary, faintly two-segmented (Fig. 5b); (2) entire margin of body (Fig. 2g); (1) two longitudinal rudimentary, unsegmented (Fig. 5c). rows on dorsum (Fig. 2b); (2) four longitudinal rows 40 Number of setae on the first tarsal segment of on dorsum; (3) six longitudinal rows on dorsum fore leg: (0) one; (1) two; (2) three; (3) four; (4) more (Fig. 2c); (4) scattered over dorsum (Fig. 2e); (5) scat- than four. 36 J. Chen et al. / Cladistics 30 (2014) 26–66

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

(j) (k) (l)

Fig. 2. Dorsal view of bodies of Hormaphidinae (apterous viviparous females): (a) Aleurodaphis blumeae van der Goot; (b) Ceratoglyphina phragmitidisucta Zhang; (c) Ceratovacuna silvestrii (Takahashi); (d) Doraphis populi (Maskell); (e) Ktenopteryx eosocallis Qiao & Zhang; (f) Tsu- gaphis sorini Takahashi; (g) Hormaphis betulae (Mordvilko); (h) Lithoaphis quercisucta Qiao, Guo & Zhang; (i) Metanipponaphis lithocarpicola (Takahashi); (j) Neonipponaphis shiiae Takahashi; (k) Quadrartus agrifoliae (Ferris); (l) Reticulaphis inflata Yeh & Hsu. Scale bars = 0.10 mm. J. Chen et al. / Cladistics 30 (2014) 26–66 37

(a) (b)

(c)

(d) (e)

(f)

Fig. 3. Setae of Hormaphidinae (apterous viviparous females). (a,b) Dorsal setae: (a) Glyphinaphis bambusae van der Goot; (b) Lithoaphis quer- cisucta Qiao, Guo & Zhang. (c–e) Submarginal setae on prosoma: (c) Quadrartus agrifoliae (Ferris); (d) Dermaphis crematogastri (Takahashi); (e) Lithoaphis quercisucta Qiao, Guo & Zhang. (f) Submarginal seta on abdominal tergites II–VII of Neohormaphis wuyiensis Qiao & Jiang. Scale bars = 0.10 mm.

41 Number of setae on the first tarsal segment of Alate viviparous females middle leg: (0) one; (1) two; (2) three; (3) four; (4) more than four. 52 Frontal horns: (0) absent; (1) present. 42 Number of setae on the first tarsal segment of 53 Frontal horns: (0) pointed; (1) blunt. hind leg: (0) one; (1) two; (2) three; (3) four; (4) more 54 Number of antennal segments: (0) six; (1) five than four. (Fig. 4f); (2) four; (3) three. 43 Dorsoapical setae on the second tarsal segment: 55 Number of secondary rhinaria on antennal seg- (0) pointed at apex (Fig. 5e); (1) capitate or flattened ment III: (0) 1–14; (1) 15–28; (2) more than 28. at apex (Fig. 5f). 56 Median vein of fore wings: (0) once-branched 44 Fore tarsal claws: (0) developed; (1) reduced; (Fig. 7a); (1) unbranched (Fig. 7b). (2) absent. 57 Two cubitus veins of fore wings: (0) fused at 45 Middle tarsal claws: (0) developed (Fig. 5a); (1) base (Fig. 7b); (1) close at base (Fig. 7a); (2) widely reduced; (2) absent (Fig. 5d). separated at base (Fig. 7c). 46 Hind tarsal claws: (0) developed; (1) reduced 58 Number of obliques of hind wings: (0) two (Fig. 5c); (2) absent (Fig. 5b). (Fig. 7a); (1) one (Fig. 7b). 47 Siphunculi: (0) present; (1) absent. 59 Number of abdominal spiracles: (0) seven 48 Siphunculi: (0) pore-like, on slightly raised pairs; (1) six pairs; (2) five pairs; (3) four pairs; (4) two cones (Fig. 6b); (1) pore-like, not raised (Fig. 6c). pairs. 49 Siphunculi setae: (0) absent (Fig. 6a); (1) pres- 60 Siphunculi: (0) present; (1) absent. ent (Fig. 6b). 61 Siphunculi: (0) pore-like, on slightly raised 50 Shape of cauda: (0) round at apex (Fig. 6d); (1) cones; (1) pore-like, not raised. knobbed (Fig. 6f). 62 Siphunculi setae: (0) absent; (1) present. 51 Shape of anal plate: (0) round at apex or broad 63 Shape of cauda: (0) round at apex; (1) knobbed; round (Fig. 6g); (1) bilobed (Fig. 6i). (2) tongue-shaped. 38 J. Chen et al. / Cladistics 30 (2014) 26–66

(a) (b)

(c) (d) (e)

(f) (j)

(g) (h) (i)

Fig. 4. Characters of head of Hormaphidinae (a–e, g–j, apterous viviparous females; f, alate viviparous female). (a,b) Frontal horns: (a) Cerata- phis freycinetiae van der Goot; (b) Astegopteryx bambusae (Buckton). (c–e) Compound eyes: (c) Cerataphis bambusifoliae Takahashi; (d) Reticula- phis inflata Yeh & Hsu; (e) Neothoracaphis elongata (Takahashi). (f–h) Antennae: (f) Pseudoregma bambucicola (Takahashi); (g) Parathoracaphis setigera (Takahashi); (h) Neothoracaphis yanonis (Matsumura). (i,j) Ultimate rostral segments: (i) Cerataphis bambusifoliae Takahashi; (j) Aleu- rodaphis blumeae van der Goot. Scale bars = 0.10 mm.

(a) Biology (c) 65 Primary host plants: (0) Styracaceae; (1) Hama- melis (Hamamelidaceae); (2) Distylium (Hamamelida- ceae); (3) Anacardiaceae; (4) Ulmaceae; (5) Theaceae; (6) others. (b) 66 Secondary host plants: (0) Compositae; (1) Gramineae; (2) Palmaceae; (3) Pinaceae; (4) Lorantha- (e) ceae; (5) Betulaceae; (6) Fagaceae; (7) Lauraceae; (8) Moraceae; (9) others. 67 Gall induction on primary host plants: (0) pres- (d) ent; (1) absent. 68 Type of galls on primary host plants: (0) pseu- dogalls; (1) true galls. (f) 69 Site of galls on primary host plants: (0) leaf blade (Fig. 1e); (1) leaf vein (Fig. 1c); (2) petiole (Fig. 1d); (3) twig (Fig. 1f). 70 Structure of galls on primary host plants: (0) Fig. 5. Characters of leg of Hormaphidinae (apterous viviparous single-cavity; (1) multiple-cavity. females). (a) Middle tarsus of Astegopteryx bambusae (Buckton). (b, 71 Colonies on secondary host plants: (0) free-liv- c) Hind tarsi: (b) Doraphis populi (Maskell); (c) Neothoracaphis elongata (Takahashi). (d) Middle leg of Doraphis populi (Maskell). ing; (1) gall-living; (2) root-living. (e,f) Hind tarsi, showing dorsoapical setae on the second tarsal seg- ments: (e) Tsugaphis sorini Takahashi; (f) Reticulaphis inflata Yeh & Phylogenetic analyses Hsu. Scale bars = 0.05 mm. Molecular dataset. Maximum parsimony (MP) 64 Shape of anal plate: (0) round at apex; (1) analyses were conducted for each gene, each genome bilobed. (mitochondrial: COI + CytB; nuclear: EF-1a + LWO; J. Chen et al. / Cladistics 30 (2014) 26–66 39

(a) (d) (g)

(b) (e) (h)

(c) (f) (i)

Fig. 6. Siphunculi, caudae, and anal plates of Hormaphidinae (apterous viviparous females). (a–c) Siphunculi: (a) Ceratovacuna silvestrii (Takah- ashi); (b) Astegopteryx rhapidis (van der Goot); (c) Lithoaphis quercisucta Qiao, Guo & Zhang. (d–f) Caudae: (d) Ceratocallis camellis Qiao & Zhang; (e) Ceratoglyphina bambusae van der Goot; (f) Cerataphis freycinetiae van der Goot. (g–i) Anal plates: (g) Ceratocallis camellis Qiao & Zhang; (h) Ceratoglyphina bambusae van der Goot; (i) Glyphinaphis bambusae van der Goot. Scale bars = 0.10 mm.

see Table 4), and the combined molecular dataset Bayesian information criterion (BIC) (Schwarz, 1978) (molecular, see Table 4) to discern their phylogenetic favoured TIM3+I+G for COI, TIM2+I+G for CytB signals. Datasets were analysed under equal weights and EF-1a, and HKY+I+G for LWO. Bayesian infer- using TNT v1.1 (Goloboff et al., 2003, 2008). New ence was carried out in MrBayes 3.1.2 (Huelsenbeck technology searches were applied consisting of 10 000 and Ronquist, 2001; Ronquist and Huelsenbeck, 2003) random addition sequence replicates, each employing under default priors, with each partition unlinked for default sectorial, ratchet, drift, and tree-fusing parameter estimations. Four Markov chains (three parameters. The best trees were then resubmitted for heated and one cold) were run, starting from a random tree bisection and reconnection (TBR) branch tree and proceeding for 1 000 000 Markov chain Monte swapping to check for additional most parsimonious Carlo (MCMC) generations, sampling the chains every trees. Clade supports were assessed based on Bremer 100 generations. Two concurrent runs were conducted support (BS) (Bremer, 1988, 1994) and bootstrapping to verify the results. A plot of sampled log-likelihood (Felsenstein, 1985). Bremer support values were scores against generation time was used to determine calculated from the suboptimal trees 1–10 steps longer the stationarity of the chains. The trees prior to sta- than the shortest trees, saving a maximum of 100 000 tionarity were discarded. For all runs, the first 2500 trees in each step. Standard bootstrap resampling was trees were discarded as burn-in samples. The remaining carried out with a traditional search, producing 1000 trees were used to compute a majority-rule consensus replicates with absolute frequencies reported. To tree with posterior probabilities (PP) (for the overcred- estimate the relative contribution of each gene ibility of Bayesian phylogenetics, see Suzuki et al., partition in the combined molecular analysis, 2002; Cummings et al., 2003; Simmons et al., 2004). partitioned Bremer support (PBS) (Baker and DeSalle, The maximum likelihood (ML) analysis was inferred 1997; Gatesy et al., 1999) was calculated in TNT using in RAxML v7.2.6 (Stamatakis, 2006; Stamatakis et al., a script written by Carlos Pena~ (“Pbsup.run”, 2008) with the GTRGAMMAI model for each gene available at http://nymphalidae.utu.fi/cpena/software. partition. All model parameters were estimated during html; Pena~ et al., 2006). the ML analysis. A rapid bootstrapping algorithm For the Bayesian analysis of the combined molecular with a random seed value of 12345 (command –fa–x dataset, the best-fit model of nucleotide substitution 12345) was applied with 1000 replicates (but see Sid- was selected for each gene using jModelTest 0.1.1 dall, 2010 for the overestimation problem of RAxML (Guindon and Gascuel, 2003; Posada, 2008). The bootstrap). 40 J. Chen et al. / Cladistics 30 (2014) 26–66

(a) (b)

(c)

Fig. 7. Wings of Hormaphidinae (alate viviparous females): (a) fore and hind wings of Aleurodaphis blumeae van der Goot; (b) fore and hind wings of Doraphis populi (Maskell); (c) fore wing of Quadrartus yoshinomiyai Monzen. Scale bars = 0.10 mm.

Table 4 Summary of parsimony analyses under equal weights

Number of Number Number of parsimony-informative Number MPT Dataset of taxa characters characters (% informative) of MPTs length CI RI

COI 54 658 234 (35.6) 12 1802 0.216 0.435 CytB 38 745 277 (37.2) 28 1595 0.344 0.435 EF-1a 53 864 233 (27.0) 6 1249 0.331 0.662 LWO 47 739 253 (34.2) 2 1004 0.441 0.701 Mitochondrial 54 1403 511 (36.4) 2 3433 0.273 0.427 Nuclear 53 1603 486 (30.3) 9 2290 0.374 0.670 Molecular 54 3006 997 (33.2) 2 5808 0.309 0.539 Morphological 64 72 64 (88.9) 2 327.700 0.400 0.740 Total-evidence 72 3078 1061 (34.5) 2 6174.100 0.312 0.552

MPT, most parsimonious tree; CI, consistency index; RI, retention index.

Morphological dataset. Maximum parsimony parsimony analysis was assessed by performing analysis was conducted in TNT with the same search analyses under implied weighting (Goloboff, 1993; options as described previously. Bremer support and Giribet, 2003). A range of values was used for the bootstrap values were used to determine the clade concavity constant K (1, 2, 3, 6, 8, 10, 12, 20, 35, 50, supports. Continuous morphological characters were 100). The stability of clades was displayed as “Navajo excluded from the Bayesian analysis. The Mk model rugs” in the strict consensus of the equal weights (Lewis, 2001) was implemented with equal state hypothesis. Discrete morphological characters were frequencies and a gamma-distributed rate variation optimized onto the strict consensus tree using across characters. Two independent runs of 3 000 000 WinClada ver. 1.00.08 (Nixon, 2002). For Bayesian generations were performed using four chains and analysis, a total of 11 000 000 generations were run, sampling every 100 generations. For each run, 7500 with sampling the chains every 500 generations. Burn- trees were discarded as burn-in samples. in was set at 5500 trees after MCMC convergence.

Total-evidence dataset. The total-evidence dataset comprised all taxa from both the molecular and Results morphological datasets (72 taxa, 3078 characters). The biological characters of eight species (Metanipponaphis Molecular data analyses sp., Nipponaphis coreana, Nipponaphis sp. 2, Epipemphigus yunnanensis, Eriosoma ulmi, Kaburagia The results of equally weighted parsimony analyses rhusicola, Prociphilus ligustrifoliae,andTetraneura of each gene, each genome, and the combined molecu- chinensis) were also included. Maximum parsimony lar dataset are summarized in Table 4 (see Table 5 for and Bayesian analyses were carried out as above. The clade monophyly; see strict consensus of each gene and stability of the result in equally weighted maximum each genome in Appendix 1: Figs A1–A6). Analysis of J. Chen et al. / Cladistics 30 (2014) 26–66 41 the combined molecular dataset (54 taxa, 3006 bp) with weak or moderate supports. Several clades consis- yielded two most parsimonious trees (MPTs) with a tent with those identified in the combined molecular length of 5808 steps (Table 4, Fig. 8). Support values hypothesis were revealed: Ktenopteryx + Aleurodaphis, for particular clades are shown in Table 5. Hormaphid- Parathoracaphis + Neohormaphis (excluding Lithoaphis inae, Hormaphidini, and Nipponaphidini were quercisucta), and Hamamelistes + Doraphis + Horma- retrieved as monophyletic, whereas Cerataphidini was phis. In Bayesian analysis (64 taxa, 67 characters; paraphyletic, with Doraphis being a sister group to Table 5, Fig. A10), the monophyly of Hormaphidinae Hormaphidini + Nipponaphidini. Within the clade of was supported. Three tribes were polyphyletic and their the remaining cerataphidines (i.e. without Doraphis), generic relationships were mostly unresolved. Tuberaphis coreana was positioned most basally. Kte- nopteryx and Aleurodaphis as well as Tuberaphis xing- Total-evidence analyses longensis and Cerataphis formed two sister groups. The latter pair was then sister to the remaining genera, with The maximum parsimony analysis of the total-evi- Glyphinaphis being a basal lineage. Most genera formed dence dataset (72 taxa, 3078 characters) under equal well supported clades, whereas Ceratovacuna was para- weights yielded two MPTs with a length of 6174.100 phyletic. Within the clade of Hormaphidini, the mono- steps (Tables 4 and 5). The strict consensus tree is used phyly of both Hormaphis and Hamamelistes was well as the working hypothesis for hormaphidines (Fig. 9). supported. The Nipponaphidini was split into two Autapomorphic or unambiguous synapomorphic mor- clades. One comprised Neothoracaphis, Dermaphis, Re- phological changes are represented in Fig. 10. ticulaphis, Parathoracaphis,andNeohormaphis,with Parathoracaphis paraphyletic; the other consisted of the Hormaphidinae (node 76). The monophyly of six remaining genera, with Metanipponaphis basally. Hormaphidinae was supported in all parameter sets, The maximum likelihood tree and the majority-rule although only with weak support. This result was in consensus tree inferred from Bayesian analysis were agreement with the analyses of single and combined essentially identical in topology, with the exception of nuclear genes as well as the morphological and the position of Glyphinaphis (Table 5, Fig. A7). They combined molecular datasets (Table 5, Figs 8, A3, A4, differed from the strict consensus of MPTs in the hor- A6, A7, A9, A10). Inner relationships within maphidine part of the phylogeny in the placements of Hormaphidinae were fully resolved. Two main clades Tuberaphis coreana, Doraphis populi, and Neonippona- were recovered, with node 75 sister to node 97. phis pustulosis, and the paraphyly of Nipponaphis. The four genes showed varied partitioned Bremer “Cerataphidini” (node 75). This clade included all support values across different clades in the combined cerataphidine representatives except for Doraphis and analysis (Fig. A8). COI supported 46 out of 47 nodes, Tsugaphis. Support value was low, but it was with positive values scattered from the deepest to the recovered in all parameter sets and retrieved by COI, shallowest nodes. CytB made relatively small contribu- EF-1a, and combined molecular dataset (Table 5, tions to the combined topology, supporting only nine Figs 8, A1, A3, A7). Ktenopteryx and Aleurodaphis nodes. The negative and zero values may be partially formed the most basal clade, followed by node 83 caused by missing sequences from a number of taxa. which included Tuberaphis + Cerataphis and the EF-1a contributed positively to 27 nodes, from the rel- remaining seven genera. Tuberaphis, Ceratoglyphina, atively deep to the shallowest nodes, especially for the and Ceratovacuna were paraphyletic. clade including Doraphis, Hormaphidini, and Nipp- onaphidini. Twenty-eight nodes including the topology Node 97. This node included the remaining nearer the root of the tree gained support from LWO, hormaphidine representatives, with a most basal group, whereas it made moderate contributions within the Protohormaphis, a sub-basal group, Tsugaphis,andasister major clades. group comprising Doraphis + Hormaphis + Hamamelistes and all nipponaphidine representatives. This clade was Morphological data analyses weakly supported, but was recovered under all parameter sets. Maximum parsimony analysis of the morphological dataset (64 taxa, 72 characters; Table 3) under equal Nipponaphidini (node 107). Nipponaphidini was weights resulted in two MPTs with a length of 327.700 recovered as monophyletic with weak support in six steps (Tables 4 and 5). The strict consensus tree parameter sets. Other weighting schemes clustered showed a pectinated pattern (Fig. A9). Hormaphidinae Quernaphis, Quadrartus, and sometimes also Lithoaphis was recovered as monophyletic, although weakly sup- shiiae with Doraphis, Hormaphis,andHamamelistes, ported. Three constituent tribes were not monophyletic. making Nipponaphidini non-monophyletic. The Some hormaphidine genera were retrieved as clades monophyletic Nipponaphidini was also retrieved by COI, 42

Table 5 Clade sensitivity to different datasets in the phylogenetic analyses performed in this study

COI CytB EF-1a LWO Mitochondrial Nuclear Molecular Morphological Total-evidence Node number Clade MP MP MP MP MP MP MP BI ML MP BI MP BI

76 Hormaphidinae n/a n/a 1/– 1/– n/a 1/53 1/– 1.00 85 2/58 0.99 2/– 0.95 75 “Cerataphidini” 2/– n/a 1/– n/a n/a n/a 1/– 1.00 90 n/a n/a 2/– n/a (excl. Doraphis and Tsugaphis) 74 Ktenopteryx + n/a n/a 1/– n/a n/a 5/72 9/78 1.00 99 + n/a 4/81 0.76 Aleurodaphis 87 Tuberaphis + n/a n/a 4/75 6/92 n/a 7/93 n/a n/a 50 n/a n/a 1/– n/a Cerataphis 82 Glyphinaphis + n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a 2/– 0.96 Ceratoglyphina + 26–66 (2014) 30 Cladistics / al. et Chen J. Ceratocallis + Astegopteryx + Chaitoregma + Ceratovacuna + Pseudoregma 89 Ceratoglyphina + n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a 2/67 0.96 Ceratocallis 92 Ceratovacuna + 2/– 4/– 2/65 n/a 4/60 n/a 7/67 0.73 + n/a n/a 4/54 n/a Pseudoregma 95 Doraphis + n/a n/a 6/58 5/71 n/a 4/85 3/71 1.00 96 + n/a 2/– 0.76 Hormaphis + Hamamelistes + Nipponaphidini 94 Doraphis + n/a n/a n/a 2/– n/a 1/– n/a 0.95 67 1/– 0.96 1/– n/a Hormaphis + Hamamelistes 107 Nipponaphidini 1/– n/a 4/73 6/86 n/a 11/98 16/98 1.00 99 n/a n/a 1/– n/a 122 Parathoracaphis + n/a n/a n/a n/a n/a n/a n/a n/a n/a ++2/– 1.00 Neohormaphis + Lithoaphis quercisucta 120 Neohormaphis + n/a n/a n/a n/a n/a n/a n/a n/a n/a 1/– n/a 1/– + Lithoaphis quercisucta

MP, maximum parsimony; BI, Bayesian inference; ML, maximum likelihood. Node number refers to that in the working phylogenetic hypothesis (result of equally weighted parsimony analysis of total-evidence dataset). Support values are shown for the recov- ered clades. A dash is shown if MP bootstrap value is below 50%. +, Clade recovered with BS = 0 and MP bootstrap < 50%, with ML bootstrap < 50%, or with posterior probabilities (PP) < 0.70. n/a, Clade not recovered or not enough information available for clade monophyly. J. Chen et al. / Cladistics 30 (2014) 26–66 43

Fig. 8. Strict consensus of the two most parsimonious trees resulting from the analysis of the combined molecular dataset under equally weighted parsimony. Numbers above each node indicate Bremer support (BS) and maximum parsimony (MP) bootstrap values. Numbers below each node indicate maximum likelihood (ML) bootstrap values and Bayesian posterior probabilities (PP) values. A dash is shown if bootstrap value is below 50%. 44 J. Chen et al. / Cladistics 30 (2014) 26–66

Fig. 9. Strict consensus of the two most parsimonious trees resulting from the analysis of the total-evidence dataset under equally weighted par- simony. Numbers above and below each node indicate Bremer support (BS) and bootstrap values (>50%), respectively. Navajo rugs indicate the reseult of stability analysis (black squares indicate clades recovered, white ones not). J. Chen et al. / Cladistics 30 (2014) 26–66 45

Fig. 10. Unambiguous morphological character optimization onto the strict consensus tree of the total-evidence parsimony analysis. Node num- bers are shown below each node. Numbers above and below circles on the branches indicate character numbers and states, respectively. White and black circles represent homoplasious and nonhomoplasious states, respectively. 46 J. Chen et al. / Cladistics 30 (2014) 26–66

EF-1a, LWO, combined nuclear genes, and combined ate support, and CytB showed limited signals and con- molecular dataset (Table 5, Figs 8, A1, A3, A4, A6, A7). flict at some nodes. COI and CytB have been Supports were weak for most clades. Thoracaphis considered very useful for resolving relationships at kashifoliae + Parathoracaphisella indica was positioned generic and specific levels given their high mutation most basally, followed by two lineages: node 105 and rates (Coeur d’acier et al., 2007, 2008; Kim and Lee, node 118. Several sister groups were recovered, namely 2008; Wang et al., 2011; Zhang et al., 2011). In this Quernaphis + Quadrartus, Dermaphis + Reticulaphis, study, COI contributed positively to both shallow and Lithoaphis shiiae + Neonipponaphis,andSchizoneuraphis + deep relationships when used in combination with the Euthoracaphis. Parathoracaphis and Neohormaphis were other three genes, whereas CytB primarily provided sig- paraphyletic. Lithoaphis, Nipponaphis,andThoracaphis nals for shallower relationships. EF-1a has been widely were polyphyletic. used in phylogenetic studies of aphids. It provides good In the total-evidence Bayesian analysis (72 taxa, 3073 resolution at generic and higher levels (Normark, 2000; characters; Table 5, Fig. A11), the relationships among von Dohlen et al., 2002, 2006; von Dohlen and Teulon, major clades were consistent with the parsimony 2003; Havill et al., 2007; Zhang and Qiao, 2007a,b, hypothesis, but it presented a decrease in resolution and 2008; Kim and Lee, 2008; Ortiz-Rivas et al., 2009; recovered different placements of several taxa. Ortiz-Rivas and Martınez-Torres, 2010). In our com- bined molecular analysis, EF-1a contributed positively to shallower relationships within the “cerataphidines” Discussion (excluding Doraphis), whereas it provided resolution from deep to shallow nodes within the clade including The preferred total-evidence hypothesis obtained Doraphis, Hormaphidini, and Nipponaphidini. LWO from the equally weighted parsimony analysis (Fig. 9) has been used in recent years to uncover deeper diver- supported the monophyly of Hormaphidinae, with gences in aphids (Ortiz-Rivas et al., 2004; Ortiz-Rivas high stability values but low support values. Cerata- and Martınez-Torres, 2010). In the present study, it phidini and Hormaphidini were polyphyletic. Nipp- provided support to the topology nearer the root of the onaphidini was retrieved as monophyletic, but the combined molecular tree, but moderate contributions support and stability values were not high, and its to the intratribal relationships. inner relationships were sensitive to weighting parame- ters. The monophyly of Hormaphidinae was recovered Implied relationships of Hormaphidinae by most datasets except for COI, CytB, and the com- bined mitochondrial genes (Table 5, Figs 8, 9, A3, A4, Monophyly of Hormaphidinae. The subfamily A6, A7, A9–A11), suggesting a poor resolving power Hormaphidinae was recovered as monophyletic, in of mitochondrial genes at high taxonomic levels. No agreement with Huang et al. (2012). Hormaphidinae is datasets supported the monophyly of Cerataphidini. considered as a distinct clade by many taxonomists from Both molecules and morphology placed Doraphis in a the morphological point of view (Hille Ris Lambers, cluster with Hamamelistes and Hormaphis (Table 5, 1964; Zhang and Zhong, 1983; Ghosh, 1985; Blackman Figs 9, A4, A6, A7, A9, A10), and in the preferred and Eastop, 1994, 2000; Remaudiere and Remaudiere, total-evidence hypothesis (Fig. 9), morphological parti- 1997). In the early molecular phylogenetic study of tion separated Tsugaphis from other cerataphidine rep- Aphididae (von Dohlen and Moran, 2000), it might resentatives. Hamamelistes and Hormaphis formed be sampling limitation that caused the failure to a robust monophyletic clade in most analyses (Figs 8, reconstruct a monophyletic Hormaphidinae. The 9, A4, A6, A7, A11). However, neither morphology following combination of unambiguous morphological alone nor combined molecular and morphological evi- synapomorphies supports the monophyly of dence clustered Protohormaphis with this clade, Hormaphidinae: wax gland plates distributed along the making Hormaphidini non-monophyletic (Figs 9, A9– entire margin of body or scattered over dorsum in A11). The Nipponaphidini was recovered as monophy- apterae (Fig. 2e,g); cauda knobbed, constricted at base in letic by molecular data (Table 5, Figs 8, A1, A3, A4, apterae and alatae (Fig. 6f; secondarily round at apex in A6, A7), whereas morphology alone placed Hamamel- Ceratocallis and Ceratoglyphina, Fig. 6d,e); two cubitus istes + Doraphis + Hormaphis distally within the nipp- veins of the fore wings fused at base in alatae (Fig. 7b; onaphidine clade (Fig. A9). When morphology was separated at base in Quadrartus yoshinomiyai,Fig.7c); combined with molecules, a monophyletic Nippona- and anal plate bilobed in apterae and alatae (Fig. 6i; phidini was retrieved once again (Table 5, Fig. 9). apterae with secondarily broad round anal plate in The PBS values of different genes in the combined Ceratocallis and Ceratoglyphina, Fig. 6g,h). Additional molecular analysis suggested a mixture of phylogenetic potential synapomorphies include apterae aleyrodiform signals, where COI lent positive support to the entire (Fig. 2a,d–l); body with wax gland plates (Fig. 2c; see combined topology, EF-1a and LWO provided moder- Chen and Qiao, 2012b for detailed information); eyes J. Chen et al. / Cladistics 30 (2014) 26–66 47 three-faceted (Fig. 4c; two-faceted eyes in Doraphis (Fig. 7b). Therefore the present study supports an populi, Neothoracaphis elongata, Neothoracaphis expanded circumscription of Hormaphidini to include quercicola,andNeothoracaphis yanonis, Fig. 4e); reduced Doraphis. antennal segments, up to five (Fig. 4f); annular secondary rhinaria in alatae (Fig. 4f); siphunculi ring- or Monophyly of Nipponaphidini. The monophyly of pore-like (Fig. 6a–c); wings flat in repose; rostrum Nipponaphidini was well corroborated by most datasets present in sexual morphs [absent in males of (Table 5, Figs 8, 9, A1, A3, A4, A6, A7). It is supported Astegopteryx spinocephala Kurosu, Buranapanichpan & by the unambiguous morphological synapomorphy of Aoki and Astegopteryx bambucifoliae (Takahashi) dorsoapical setae on the second tarsal segment capitate (Kurosu et al., 2006)]; and galling behaviour on primary or flattened at apex in apterae (Fig. 5f). Additional hosts (Fig. 1; Chen and Qiao, 2009, 2012a; Aoki and putative synapomorphies for Nipponaphidini include Kurosu, 2010). Hormaphidinae is also supported by 19 apterae aleyrodiform, body flattened dorsoventrally and molecular synapomorphies. strongly sclerotized (Fig. 2h–l); prosoma present, consisting of fused head, thorax, and abdominal segment Recircumscriptions of Cerataphidini and I(Fig.2i–l); abdominal segments II–VII fused Hormaphidini. All cerataphidine representatives except completely (Fig. 2h–l); head and thorax longer than for Doraphis populi and Tsugaphis sorini formed a abdomen; dorsum often bearing different shaped monophyletic lineage. The total-evidence parsimony analysis pustules or sculptures ornamentation (Fig. 2i–l); frontal placed Doraphis clustered with Hormaphis and Hamamelistes, horns absent; antennae short (Fig. 4g,h); antennal and Tsugaphis as sister to a clade including Doraphis, segments reduced (Fig. 4g,h); setae on antennal segment Hormaphis, Hamamelistes, and all nipponaphidines (Fig. 9). III absent if antennae 3- or 4-segmented; legs short Here, we recircumscribe the tribe Cerataphidini to exclude the (Fig. 2h–l); the induction of galls on primary hosts, genera Doraphis and Tsugaphis. It is supported by 21 Distylium; and mainly occupying Fagaceae and molecular synapomorphies. Putative synapomorphies for the Lauraceae as secondary hosts. Nipponaphidini genera newly delimited Cerataphidini include apterae without dorsal are mostly narrowly districted, monotypic or comprising pustulesorsculptures(Fig.2b,c,e;dorsumwithoval only a few species. Many nominal species have been sculptures in Aleurodaphis, Fig. 2a); wax gland plates well collected infrequently or only once. Nearly half the developed (Fig. 2a–c,e; Glyphinaphis with groups of minute nipponaphidine representatives in the present dataset wax pores over dorsum); frontal horns present (Fig. 2b,c; were represented only by morphological and biological absent in Aleurodaphis, Glyphinaphis, Ktenopteryx,and characters. Therefore the current proposed pattern of Tuberaphis, Fig. 2a,e); siphunculi present (Fig. 2a–c,e); alatae generic relationships should be considered cautiously. with six pairs of abdominal spiracles; and occupation of Additional information on molecules and life cycles, as Styracaceae as the primary hosts. well as the establishment of more combinations between Doraphis, Hormaphis, and Hamamelistes formed a the primary and secondary host generations, are greatly robust monophyletic clade (Table 5, Figs 9, A4, A6, needed for future study. A7, A9, A10), which is supported by 15 molecular syn- apomorphies and the following unambiguous morpho- Other taxonomic implications within logical synapomorphies: apterae body round (Fig. 2d, Hormaphidinae. Ktenopteryx and Aleurodaphis g); wax gland plates present (Fig. 2d,g); fore and mid- formed a basal lineage to all remaining cerataphidines dle tarsi absent (Fig. 5d); hind tarsi rudimentary, (hereafter referring to cerataphidines excluding Doraphis faintly two-segmented (Fig. 5b); fore, middle, and hind and Tsugaphis) (Table 5, Figs 9, A3, A7). This sister tarsal claws absent (Fig. 5b,d); and median vein of the group is supported by 18 molecular synapomorphies fore wings in alatae unbranched (Fig. 7b). In addition, and the following morphological synapomorphies: Doraphis has a similar life cycle to Hamamelistes in apterae aleyrodiform (Fig. 2a,e); abdominal segments that coccidiform first-instar nymphs hibernate on twigs II–VII fused completely (Fig. 2a,e); ultimate rostral of the host tree (Mordvilko, 1935; Aoki et al., 2001). segment pointed (Fig. 4j); and the first tarsal segments Both Populus (Salicaceae), which is the host of Dora- of the fore and middle legs with two setae. Other phis, and Betula (Betulaceae), which is the secondary synapomorphies include ultimate rostral segment long host of Hamamelistes and Hormaphis, are amentaceous (Fig. 4j); and the induction of leaf pseudogalls on plants. Furthermore, Doraphis populi shares with Ham- primary hosts (Qiao and Zhang, 2003; Sorin and amelistes similibetulae, Hormaphis betulae, and Horma- Miyazaki, 2004; Jiang and Qiao, 2011). Huang et al. phis cornu the aleyrodiform body; head, thorax, and (2012) suggested that Cerataphidini could be split into abdominal segments I–VII fused completely; a fringe two subtribes: one comprised Ktenopteryx and of crenulated wax gland plates; and the absence of sip- Aleurodaphis; the other included the remaining genera. hunculi in apterae (Fig. 2d,g). Both Doraphis and Hor- But they did not go so far as to actually name the maphis have a single oblique vein in the hind wings subtribes. In fact there seems to be inadequate 48 J. Chen et al. / Cladistics 30 (2014) 26–66 evidence for their suggestion. The absence of frontal monophyly of them as a whole. Neither Ceratovacu- horns is observed not only in Ktenopteryx and na nor Pseudoregma was retrieved as monophyletic. Aleurodaphis but also in Glyphinaphis and Tuberaphis. This finding indicates a possibility that they should They also share with other cerataphidines several be lumped into a single genus. biological characters, such as the primary association The morphological and total-evidence hypotheses with Styracaceae (Ktenopteryx on Styrax, Aleurodaphis positioned Lithoaphis quercisucta as sister to Neohor- sinojackiae Qiao & Jiang on Sinojackia xylocarpa) and maphis calva, making Neohormaphis paraphyletic the occupation of bamboos as the secondary hosts (Table 5, Figs 9, A9, A11). Lithoaphis quercisucta was [Aleurodaphis antennata Chakrabarti & Maity described on Quercus sp. in China (Yunnan) by Qiao (Chakrabarti and Maity, 1982)]. et al. (2005). We examined the type specimens of L. Tuberaphis and Cerataphis formed a monophyletic quercisucta carefully and found that this species group (Table 5, Figs 9, A3, A4, A6, A7), which is in shared with Neohormaphis species the morphological agreement with previous studies (Stern, 1994, 1995, characters of the prosoma completely fused with 1998; Huang et al., 2012). This clade is supported by abdominal segments II–VII (Fig. 2h); prosoma and four molecular synapomorphies and other potential abdominal tergites II–VII bearing dagger-like submar- synapomorphies, including the induction of single-cav- ginal setae (Fig. 3e,f); dorsum with five pairs of ity galls on twigs of Styrax (Fig. 1a) and the harbour- spinal setae; and the presence of siphunculi (Fig. 6c). ing of extracellular fungal symbionts instead of Therefore it is proposed that L. quercisucta should be prokaryotic intracellular symbionts Buchnera (Fukatsu transferred to the genus Neohormaphis as Neohorma- et al., 1994). The monophyly of Tuberaphis being not phis quercisucta comb. nov. Parathoracaphis clustered retrieved might be due to the limitation of the molecu- with Neohormaphis in most analyses (Figs 8, lar data of Tuberaphis coreana, for which only the 9, A2–A7, A9–A11). Together, they form a unique COI sequence was available. lineage within Nipponaphidini characterized by the In the total-evidence hypothesis, Ceratocallis camellis consolidated prosoma and abdominal segments II–VII clustered with Ceratoglyphina bambusae, making Cer- and dagger-like submarginal setae on the prosoma atoglyphina paraphyletic (Figs 9, A11). The monotypic and abdominal tergites II–VII (Fig. 3f). This clade is genus Ceratocallis was established by Qiao and Zhang also supported by 45 molecular synapomorphies. (1999) for C. camellis from China (Guizhou). Only apterous viviparae are known. After reviewing the type Evolution of gall morphology in Hormaphidinae specimens of C. camellis, we found that Ceratocallis was not distinctly different from Ceratoglyphina and Phylogenies can provide a useful framework for that they shared pointed frontal horns (Fig. 2b), round interpreting the evolutionary histories of organismal cauda (Fig. 6d,e), and broad round anal plate characters. To address the evolutionary questions con- (Fig. 6g,h) in apterae. Therefore Ceratocallis Qiao & cerning galls in Hormaphidinae, we reconstructed a Zhang is herein proposed as a junior synonym of Cer- phylogeny of this group. The total-evidence parsimony atoglyphina van der Goot, syn. nov. phylogenetic hypothesis for the Hormaphidinae was Ceratovacuna and Pseudoregma formed a mono- used. Gall morphology was depicted on the simplified phyletic clade, with Ceratovacuna paraphyletic and cladogram of the preferred phylogenetic hypothesis Pseudoregma monophyletic (Table 5, Figs 8, 9, A2, (Fig. 11) to trace its evolutionary scenarios within A3, A5, A7), which is in agreement with the previ- Hormaphidinae. ous study (Stern, 1998). This clade is supported by In Hormaphidinae, strong primary host specificity of seven molecular synapomorphies and the morpholog- galling species is represented by different tribes being ical synapomorphy of siphunculi bearing no setae in restricted and forming galls on different plant genera apterae (Fig. 6a). Additional putative synapomor- (indicated by arrows in Fig. 11). Phylogenetic con- phies include apterae dorsum with longitudinal rows straints on fundatrix are thought to have played an of well developed wax gland plates (see Fig. 4f,g,h,l important role in shaping this aphid–host plant associa- in Chen and Qiao, 2012b); the formation of banana- tion (Mordvilko, 1928; Shaposhnikov, 1985; Moran, bundle-shaped multiple-cavity galls on twigs of 1988, 1992). A fundatrix, the only morph that can Styrax; and the production of first-instar horned sol- induce a gall (with very few exceptions) (Wool, 2004, diers on the herbaceous secondary hosts (Fukatsu 2005), is highly specialized to the ancestral host and is et al., 1994; Stern, 1994, 1998; Aoki and Kurosu, less able to acquire new hosts compared with other mor- 2010). Ceratovacuna and Pseudoregma are very simi- phs over long evolutionary periods. The association of lar in morphology and relatively difficult to distin- galling hormaphidines with their specific primary hosts guish from one another. A multigene phylogenetic may be ancient. According to Huang et al. (2012), the study of these two genera with a much broader sam- Hormaphidini and Nipponaphidini are expected to have pling (Zhang et al., unpublished data) recovered the associated with Hamamelis and Distylium, respectively, J. Chen et al. / Cladistics 30 (2014) 26–66 49

Fig. 11. Simplified cladogram representing the preferred phylogenetic hypothesis of Hormaphidinae from the total-evidence parsimony analysis, with gall morphology depicted. near the Cretaceous–Tertiary boundary, accompanying the host plants. The evolutionary transition from leaf the origins of these two plant genera, and the Cerataphi- rolls to fully developed true galls has already been found dini is believed to have colonized Styrax during the Late in eriosomatine and pemphigine aphids (Akimoto, 1983; Cretaceous to Eocene. Given all the above, we hypothe- Zhang and Qiao, 2007b; Sano and Akimoto, 2011). In size that galls in the three tribes of Hormaphidinae galling sawflies and thrips, it has also been suggested that might have evolved along separate paths. leaf deformation (opened leaf folds or leaf rolls) is ances- Within the Cerataphidini, leaf pseudogalls appear to tral and appears to be an initial step preceding true gall- be ancestral, and single-cavity and multiple-cavity galls ing (Crespi and Worobey, 1998; Nyman et al., 1998, have each evolved once (Fig. 11). The basal lineage Kte- 2000). Relatively closed true galls are considered to have nopteryx and Aleurodaphis causes leaf deformation on more adaptive advantages to gallers. They serve as better 50 J. Chen et al. / Cladistics 30 (2014) 26–66 shelters than opened leaf pseudogalls, providing more species also form leaf pseudogalls or galls on their sec- protection to the inducer and its offspring from natural ondary host, Betula (Qiao and Zhang, 2004; Fig. 1b; enemies and adverse abiotic factors (e.g. rainwater, arid- also see Fig. 1b,d,f in Aoki et al., 2001). Compared ity, and high temperature) (Zhang and Qiao, 2007b; with Hormaphis, Hamamelistes is peculiar in having a Sano and Akimoto, 2011). biennial life cycle, a long period of which is spent on Single-cavity galls are the simplest and most com- Betula (Aoki et al., 2001). We suggest that the two-gall mon type of aphid galls, also found in Hormaphidini, system might be favourable for aphids to survive such Nipponaphidini, and many other groups of galling a long life cycle. aphids (Ghosh, 1985, 1988; Fukatsu et al., 1994; Aoki Up to the present, approximately 19 species and and Kurosu, 2010; Chen and Qiao, 2012a). In Cerata- one subspecies belonging to 13 genera in Nipponaphi- phidini, they appear to have a single origin and repre- dini are known to produce galls on their primary sent the ancestral state of true galls. Cerataphis species host, Distylium (Sorin, 2003; Chen et al., 2011a). generally form simple sac-like galls, whereas Cerata- These galls are single-cavity and mostly simple spheri- phis vandermeermohri (Hille Ris Lambers) and most cal or sac-like, but diverse in their galling sites galling Tuberaphis species induce branched galls con- (Fig. 11). In China, the galls of at least four nippona- sisting of a hollow tube that is ramified like a coral or phidine species occur at different sites on Distylium a bird nest (Fig. 1a). Galls of Tuberaphis takenouchii chinense, that is, Asiphonipponaphis vasigalla Chen, (Takahashi), which look like heads of broccoli, are Sorin & Qiao on the midrib (Fig. 1c), Neothoracaphis peculiar in having many solid projections on the inner yanonis (Matsumura) on the leaf blade (Fig. 1e), walls, largely enhancing the feeding area for aphids Metanipponaphis sp. on the petiole (Fig. 1d), and (see Fig. 1 in Aoki and Usuba, 1989). Multiple-cavity Nipponaphis sp. on the twig (Fig. 1f). Different aphid galls, which are composed of several subgalls, appear species coexist within the same host plant by coloniz- to have derived from single-cavity galls only once in ing different ecological niches. The galling site is clo- Cerataphidini, which would be consistent with previ- sely related to the sink strength of galls and thus ous studies (Fukatsu et al., 1994; Stern, 1995). Most greatly affects the reproductive success of fundatrices multiple-cavity galls are banana-bundle-shaped (see (Whitham, 1979, 1986; Wool, 2004, 2005). Intraspe- Figs 2a,c, 3a, 4a, 6a, 8d, 9a–d, 11a,b in Aoki and Ku- cific competition for galling sites between fundatrices rosu, 2010). Further modifications include short pro- has been reported in several aphid species (Aoki and jections on the inner walls of subgalls in Astegopteryx Makino, 1982; Whitham, 1986; Inbar, 1998), whereas nipae (van der Goot), A. pallida (van der Goot), A. interspecific competition seems weak (Inbar et al., spinocephala Kurosu, Buranapanichpan & Aoki, and 1995). In Fordini, considerable niche separation mini- A. styracophila Karsch (Aoki and Kurosu, 2010); mizes the interference competition among different developed projections on the inner walls in Cerato- galling aphid species and allows them to coexist glyphina styracicola (Takahashi) (see Figs 1, 2 in Aoki within the same host plant (Inbar and Wool, 1995). et al., 1977) and Pseudoregma sundanica (van der This may also be the case in Nipponaphidini. It has Goot) (see Fig. 1 in Kurosu and Aoki, 2001); and spi- been proposed that in fordine and pemphigine aphids rally twisted tubular subgalls in Ceratoglyphina roepkei and galling sawflies, the galling site might have (Hille Ris Lambers) (see Fig. 6c,d in Aoki and evolved gradually towards more central parts of the Kurosu, 2010). These complicated structures contrib- host plant, thus offering better control of the nutri- ute greatly to increasing the ratio of the inner surface tion flow and consequently achieving higher reproduc- area to volume, thus making the galls sustain more tive success (Nyman et al., 1998, 2000; Zhang and aphids per volume. For instance, the cauliflower-head- Qiao, 2007a,b). However, for the Nipponaphidini, the like gall of C. styracicola can harbour 100 000 aphids galls of many species are still unknown because they (Aoki, 1979). In addition, Tuberaphis macrosoleni have been collected only from the secondary hosts. (Noordam) and T. takenouchii cause leaf rolls and leaf Therefore the trend of shifts in galling sites in Nipp- folds on their secondary host, Loranthaceae (Noor- onaphidini is not clear based on current knowledge dam, 1991; Kurosu et al., 1994), suggesting an initial and needs to be investigated in the future. evolutionary step of galling on secondary hosts. In Hormaphidini, only Hamamelistes and Hormaphis are known to induce galls on their primary host, Conclusions Hamamelis (Fig. 11). Hamamelistes forms coral or spiny twig galls (see Fig. 1a,c,e in Aoki et al., 2001), We simultaneously analysed the available informa- and Hormaphis induces cone-shaped leaf galls (see tion (morphology, biology, and multiple genes) for Hor- Fig. 1 in Kurosu and Aoki, 1991c and Figs 1, 9 in von maphidinae to provide a reliable and detailed Dohlen and Stoetzel, 1991). Galling on secondary description of the phylogenetic relationships of this sub- hosts appears to have evolved once, as Hamamelistes family. The working phylogenetic hypothesis from the J. Chen et al. / Cladistics 30 (2014) 26–66 51 total-evidence parsimony analysis supports the mono- Acknowledgements phyly of Hormaphidinae, and the relationships of our taxonomic samples are fully resolved. Cerataphidini Great thanks are due to the associate editor and three and Hormaphidini are not recovered as monophyletic, anonymous referees for their constructive comments. whereas the monophyly of Nipponaphidini is sup- We are grateful to Prof. R.L. Blackman, Prof. V.F. Ea- ported, although with low support and stability values stop, and Prof. J. Martin for hosting G.X. Qiao at the and unstable inner relationships. The result of the total- Natural History Museum (London, UK) and for the evidence Bayesian analysis is consistent with the parsi- loan of specimens. Thanks to Prof. A.V. Stekolshchikov mony hypothesis with respect to major clades, but for hosting G.X. Qiao at the Zoological Institute of the exhibits lower resolution. Hormaphidinae monophyly is Russian Academy of Sciences (St. Petersburg, Russia). also recovered by nuclear genes, combined molecules, Thanks to Prof. M. Sorin for loaning and presenting and morphology alone, but not supported by single or specimens. Thanks to C.P. Liu and F.D. Yang for mak- combined mitochondrial genes. The analyses of mor- ing slides. Thanks to B. Chen and X.T. Li for providing phology alone using parsimony and Bayesian gall pictures and to H.J. Ge, C.X. Li, X.T. Li, C.Q. Lin, approaches reveal, respectively, a pectinated and a and X.N. Zhang for supplying samples for molecular mostly unresolved pattern of relationships within work. Thanks to Dr B. Ortiz-Rivas for providing primer Hormaphidinae. Our hypothesis for Hormaphidinae sequences for LWO and to L. Liu, Q.H. Liu, and R.L. relationships is based on all available information and Zhang for making unpublished sequences available. We provides a basis for future phylogenetic studies. More also thank Dr C. Pena~ for his help on the calculation of information on molecules and life cycles is needed for a partitioned Bremer support. This work was supported better understanding of the phylogenetic relationships by the National Science Fund for Distinguished Young within this subfamily, especially the Nipponaphidini. Scientists (No. 31025024), National Natural Sciences Foundation of China (No. 30830017), National Science Summary of taxonomic decisions Fund for Fostering Talents in Basic Research (No. J1210002), a grant from the Ministry of Science and Cerataphidini Baker recircumscription: Aleurodaphis Technology of the People’s Republic of China (MOST van der Goot, Astegopteryx Karsch, Cerataphis Lich- Grant No. 2011FY120200), and a grant from the Key tenstein, Ceratoglyphina van der Goot, Ceratovacuna Laboratory of the Zoological Systematics and Evolu- Zehntner, Chaitoregma Hille Ris Lambers & Basu, tion of the Chinese Academy of Sciences (No. Glyphinaphis van der Goot, Ktenopteryx Qiao & Zhang, O529YX5105). Pseudoregma Doncaster, Tuberaphis Takahashi. 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Fig. A1. Strict consensus of the 12 most parsimonious trees resulting from the analysis of COI under equally weighted parsimony. Numbers above and below each node indicate Bremer support (BS) and bootstrap values (>50%), respectively. J. Chen et al. / Cladistics 30 (2014) 26–66 57

Fig. A2. Strict consensus of the 28 most parsimonious trees resulting from the analysis of CytB under equally weighted parsimony. Numbers above and below each node indicate Bremer support (BS) and bootstrap values (>50%), respectively. 58 J. Chen et al. / Cladistics 30 (2014) 26–66

Fig. A3. Strict consensus of the six most parsimonious trees resulting from the analysis of EF-1a under equally weighted parsimony. Numbers above and below each node indicate Bremer support (BS) and bootstrap values (>50%), respectively. J. Chen et al. / Cladistics 30 (2014) 26–66 59

Fig. A4. Strict consensus of the two most parsimonious trees resulting from the analysis of LWO under equally weighted parsimony. Numbers above and below each node indicate Bremer support (BS) and bootstrap values (>50%), respectively. 60 J. Chen et al. / Cladistics 30 (2014) 26–66

Fig. A5. Strict consensus of the two most parsimonious trees resulting from the analysis of the combined mitochondrial genes (COI and CytB) under equally weighted parsimony. Numbers above and below each node indicate Bremer support (BS) and bootstrap values (>50%), respec- tively. J. Chen et al. / Cladistics 30 (2014) 26–66 61

Fig. A6. Strict consensus of the nine most parsimonious trees resulting from the analysis of the combined nuclear genes (EF-1a and LWO) under equally weighted parsimony. Numbers above and below each node indicate Bremer support (BS) and bootstrap values (>50%), respec- tively. 62 J. Chen et al. / Cladistics 30 (2014) 26–66

Fig. A7. Maximum likelihood tree resulting from the ML analysis of the combined molecular dataset. Numbers above and below each node indicate ML bootstrap values (>50%) and Bayesian posterior probabilities (PP) values (>0.70), respectively. J. Chen et al. / Cladistics 30 (2014) 26–66 63

Fig. A8. Strict consensus of the two most parsimonious trees resulting from the analysis of the combined molecular dataset under equally weighted parsimony. Numbers above each node indicate partitioned Bremer support (PBS) values for COI and CytB, numbers below each node indicate PBS values for EF-1a and LWO. 64 J. Chen et al. / Cladistics 30 (2014) 26–66

Fig. A9. Strict consensus of the two most parsimonious trees resulting from the analysis of morphological dataset under equally weighted parsi- mony. Numbers above and below each node indicate Bremer support (BS) and bootstrap values (>50%), respectively. J. Chen et al. / Cladistics 30 (2014) 26–66 65

Fig. A10. Bayesian tree resulting from the Bayesian analysis of morphological dataset. Numbers above each node indicate posterior probabilities (PP) values (>0.70). 66 J. Chen et al. / Cladistics 30 (2014) 26–66

Fig. A11. Bayesian tree resulting from the Bayesian analysis of the total-evidence dataset. Numbers above each node indicate posterior probabil- ities (PP) values (>0.70).