ZooKeys 111: 11–18, doi: 10.3897/zookeys.111.1284
Taxonomic position of Hormaphis similibetulae Qiao & Zhang, 2004 (Hemiptera, Aphididae): molecular and biological evidences
Jing Chen1,2, Li-Yun Jiang1, Ge-Xia Qiao1
1 Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing 100101, P.R.China
2 Graduate University of Chinese Academy of Sciences, No. 19 Yuquan Road, Shijingshan District, Beijing 100049, P.R.China

Corresponding author: Ge-Xia Qiao (qiaogx@ioz.ac.cn).

Academic editor: Mike Wilson

received 29 March 2011 | accepted 19 May 2011 | Published 22 June 2011

(C) 2011 Jing Chen. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

For reference, use of the paginated PDF or printed version of this article is recommended.


The taxonomic position of Hormaphis similibetulae Qiao & Zhang, 2004 has been reexamined. The phylogenetic position of Hormaphis similibetulae was inferred by maximum parsimony, maximum likelihood and Bayesian analyses on the basis of partial nuclear elongation factor-1α and mitochondrial tRNA leucine/cytochrome oxidase II sequences. The results showed that this species fell into the clade of Hamamelistes species, occupying a basal position, and was clearly distinct from other Hormaphis species. A closer relationship between Hormaphis similibetulae and Hamamelistes species was also revealed by life cycle analysis. Therefore, we conclude that Hormaphis similibetulae should be transferred to the genus Hamamelistes as Hamamelistes similibetulae (Qiao & Zhang), comb. n.


Hormaphidinae, Hamamelistes similibetulae, molecular evidence, biological evidence, new combination, China


The aphid tribe Hormaphidini in subfamily Hormaphidinae (Hemiptera: Aphididae) consists of three genera, Hamamelistes, Hormaphis and Protohormaphis (Remaudière and Remaudière 1997). Hamamelistes and Hormaphis are disjunctively distributed in Eurasia and North America (Guo and Qiao 2005), where they are primarily associated with Hamamelis and secondarily associated with Betula. The taxonomy of these two genera was once in a mess at both the genus and species levels. They are easily confused with one another, and species of the same genus are difficult to distinguish morphologically. This confusion in the taxonomy was due partly to a limitation of diagnostic characteristics and partly to the fact that no combination had been established between different aphid forms on primary and secondary hosts. Distinction of Hamamelistes and Hormaphis is based mainly upon alatae, galls and life cycles. However, it is not easy to collect all morphs, and the observation of life cycles takes a long time. Molecular studies have shed light on these issues. Based on the mitochondrial cytochrome oxidase II (COII) gene, Aoki et al. (2001) clarified the Japanese Hamamelistes species, established the combination between generations on primary and secondary hosts, and elucidated their life cycles. von Dohlen et al. (2002) estimated the phylogeny of Hormaphidini using partial nuclear elongation factor-1α (EF-1α) and mitochondrial tRNA leucine/cytochrome oxidase II (COII) sequences, the monophyly of both Hamamelistes and Hormaphis was retrieved with strong support.

Qiao and Zhang (2004) described Hormaphis similibetulae based on specimens of apterous viviparous females collected from small conical galls on leaves of Betula albosinensis in China (Tibet); the specimens were closely related to Hormaphis betulae (Mordvilko) but differed from the latter in body color and living habits. In this study, the taxonomic position of Hormaphis similibetulae was reassessed on the basis of nuclear EF-1α and mitochondrial tRNA/COII sequences. A discussion of life cycles was also included.

Materials and methods

The samples used in this study and the corresponding collection information are listed in Table 1. Eight species of Hormaphidini, covering all the species of Hamamelistes and Hormaphis were used as ingroups. Three species of Nipponaphidini were chosen as outgroups because Nipponaphidini is considered the sister group of Hormaphidini based on biological and phylogenetic data (Ghosh 1985, von Dohlen and Moran 2000, Ortiz-Rivas and Martínez-Torres 2010). Voucher specimens were preserved in 75% ethanol and deposited in the National Zoological Museum of China, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.

Total genomic DNA was extracted from single aphids preserved in 95% or 100% ethanol using a CTAB protocol modified from Doyle and Doyle (1987). Partial leucine tRNA and the cytochrome oxidase II (COII) gene was amplified with primers 2993+ (Stern 1994) and A3772 (Normark 1996). Sequencing reactions were performed using the corresponding PCR primers from both directions with BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and run on an ABI 3730 automated sequencer (Applied Biosystems). Sequences were assembled by Seqman II (DNAStar, Inc., Madison, WI, USA) and verified for protein coding frame-shifts to avoid pseudogenes (Zhang and Hewitt 1996) using Editseq (DNAStar, Inc.). All sequences were deposited in GenBank under the accession numbers JF730745–JF730749. All EF-1α sequences used in this study were downloaded directly from GenBank (for accession numbers see Table 1), and only exons were used for further analysis. Multiple alignments were done with ClustalX 1.83 (Thompson et al. 1997) and then verified manually. Nucleotide composition and pairwise distances based on Kimura’s two-parameter model (K2P) (Kimura 1980) of the aligned sequences were calculated using MEGA 4.0 (Tamura et al. 2007).

Phylogenetic reconstructions were conducted by maximum parsimony (MP), maximum likelihood (ML) and Bayesian analyses for each single gene and a combined dataset. The partition homogeneity test (Farris et al. 1994) based on 100 replicates of a heuristic search algorithm was performed with PAUP*4.0b10 (Swofford 2002) to examine the incongruence between EF-1α and mtDNA. Unweighted MP and ML analyses were carried out using PAUP*. For ML analysis, the best-fit model of nucleotide substitution was selected for each dataset according to the Akaike information criterion (AIC) in Modeltest 3.7 (Posada and Crandall 1998). Heuristic searches were performed with 1000 (MP) or 100 (ML) random-addition sequences and tree-bisection-reconnection (TBR) branch swapping. Bootstrap (BS) analyses were used to assess the relative robustness of branches of the MP (1000 replicates) and the ML (100 replicates) trees (Felsenstein 1985). Bayesian analysis was conducted using MrBayes 3.1.2 (Ronquist and Huelsenbeck 2003) based on the model selected by Modeltest 3.7. In the combined analysis, the mitochondrial and nuclear data were partitioned, and a heterogeneous model was used for each gene partition. The parameters of the model were treated as unknown variables with uniform prior probabilities and were estimated during the analysis. Four Markov chains (three heated and one cold) were run, starting from a random tree and proceeding for one million Markov chain Monte Carlo generations, sampling the chains every 100 generations. Two concurrent runs were conducted to verify the results. The first 2500 trees were discarded as burn-in samples, the remaining trees were used to compute a majority-rule consensus tree with posterior probabilities (PP).

Table 1.

Collection information and GenBank accession numbers for aphid samples used in this study.

Species Host Locality Date Voucher EF-1× tRNA/COII
Hamamelistes betulinus (Horvath) Betula davurica Japan: Yamanashi, Masutomi 17 Jul. 1998 98081 AF454599* AF328782*
Hamamelis japonica Japan: Aomori, Temmabayashi 7 Aug. 1998 98132 AF454596* AF328775*
Betula platyphylla Japan: Tokyo, Okutamako 20 May 1999 99121 AF454597* AF328780*
Betula platyphylla Japan: Hokkaido, Sapporo 15 Jun. 1999 99187 AF454598* AF328781*
Hamamelistes kagamii (Monzen) Hamamelis japonica Japan: Yamanashi, Masutomi 17 Jul. 1998 98084 AF454600* AF328772*
Betula grossa Japan: Yamanashi, Sanjonoyu 20 May 1999 99118 AF454601* AF328779*
Hamamelis japonica Japan: Saitama, Shomaru Pass 8 Jul. 1999 99209 AF454603* AF328773*
Hamamelis japonica Japan: Saitama, Shomaru Pass 8 Jul. 1999 99220 AF454602* AF328774*
Hamamelistes miyabeiHamamelistes miyabei (Matsumura) Hamamelis japonica Japan: Yamanashi, Masutomi 17 Jul. 1998 98086 AF454595* AF328771*
Betula maximowicziana Japan: Hokkaido, Sapporo 5 Sep. 1998 98151 AF454593* AF328776*
Betula maximowicziana Japan: Gumma, Mt. Akagi 25 May 1999 99146 AF454594* AF328777*
Betula maximowicziana Japan: Hokkaido, Sapporo 15 Jun. 1999 99182 AF454592* AF328778*
Hamamelistes spinosus Shimer Hamamelis japonica USA: Washington, DC May 1993 93-23 AF454606* AF328783*
Betula nigra USA: UT, Logan 28 May 1999 99-54 AF454607* AF454619*
Betula nigra USA: WI, Madison 28 Jun. 1999 99-57 AF454608* None
Hormaphis betulae (Mordvilko) Betula platyphylla Japan: Yamanashi, Masutomi 17 Jul. 1998 98078 AF454609* None
Hamamelis japonica Japan: Saitama, Shomaru Pass 21 May 1999 99130 AF454610* AF454622*
Betula platyphylla Japan: Tokyo, Kazahari Pass 26 Jul. 1999 99224 AF454611* AF454623*
Betula sp. China: Jilin, Ji’an 13 Aug. 2004 15214 DQ493864* JF730745
Hormaphis cornu (Shimer) Hamamelis virginiana USA: Georgia, Athens 8 Jun. 1994 94-93 AF454612* AF454621*
Hormaphis hamamelidis (Fitch) Hamamelis virginiana USA: Connecticut, Danielson 1 Aug. 1998 98-05 AF454613* AF454620*
Hormaphis similibetulae Qiao & Zhang Betula albosinensis China: Tibet, Gongbo’gyamda 5 Jul. 2002 13549 DQ493849* JF730746
Betula albosinensis China: Tibet, Linzhi 6 Aug. 2003 15318 DQ493866* JF730747
Neohormaphis wuyiensis Qiao & Jiang Quercus sp. China: Fujian, Mt. Wuyi 18 Jul. 2003 14525 DQ493858* JF730748
Nipponaphis distyliicola Monzen Quercus glauca Japan: Shinkiba, Tokyo 16 Apr. 1999 99008 AF454614* AF454626*
Thoracaphis quercifoliae Ghosh Quercus sp. China: Fujian, Mt. Wuyi 18 Jul. 2003 14526_2 DQ493851* JF730749

* Sequences from GenBank.

Results and discussion

The final alignments of EF-1α (excluding three introns) and tRNA/COII sequences consisted of 826 and 761 sites, with 131 and 165 parsimony-informative sites, respectively. A single 1- to 2-base-long indel was found in the tRNA. The genetic distance between two distinct samples of Hormaphis similibetulae was 0 for EF-1α and 0.001 for tRNA/COII. The distances of both genes between Hormaphis similibetulae and Hamamelistes species were much smaller than those between Hormaphis similibetulae and the other Hormaphis species (EF-1α: average of 0.040 and range of 0.038–0.042 to Hamamelistes, average of 0.082 and range of 0.078–0.092 to Hormaphis; tRNA/COII: average of 0.080 and range of 0.071–0.085 to Hamamelistes, average of 0.106 and range of 0.102–0.112 to Hormaphis).

For phylogenetic analyses, the partition homogeneity test found no significant conflict between EF-1α and mtDNA (P=0.05), indicating that information from both genes could be combined. Combined analysis resulted in similar topology to that obtained in single gene analyses and with higher support for most nodes, so only the combined dataset results were presented. MP analysis yielded eight most parsimonious trees with a length of 611 steps (CI=0.705401, RI=0.845626). ML analysis produced one ML tree based on the optimal model GTR+G selected by AIC in Modeltest 3.7. The 50% majority-rule consensus tree inferred from Bayesian analysis is shown in Fig. 1 and resulted in a topology essentially identical to that obtained in ML analysis, but was different from the strict consensus of MP trees in the position of Hormaphis similibetulae. All ingroup taxa constituted a monophyletic group with respect to these outgroups and formed two clades. Clade I (100% MP BS, 100% ML BS, 1.00 PP) was comprised of Hormaphis betulae, Hormaphis cornu, and Hormaphis hamamelidis. Clade II (99% MP BS, 99% ML BS, 1.00 PP) consisted of all the Hamamelistes species and Hormaphis similibetulae. Within clade II, two distinct samples of Hormaphis similibetulae clustered together (100% MP BS, 100% ML BS, 1.00 PP) and were placed as the outermost branch in ML and Bayesian analyses, just as the results based on EF-1α. However, MP analysis revealed the same topology as the mitochondrial analysis: Hormaphis similibetulae and Hamamelistes spinosus were sister groups, although the support value was low (53% BS), and together formed the basal lineage within clade II.

Figure 1.

Phylogenetic tree reconstructed from the combined dataset of EF-1α and tRNA/COII sequences. The Bayesian topology and branch lengths are shown. Values above the branches are MP and ML bootstrap percentages, respectively, and Bayesian posterior probabilities are shown below the branches. The broken line indicates inconsistent branch.

The results of genetic distances and phylogenetic analyses strongly suggested that Hormaphis similibetulae was more closely related to Hamamelistes than to Hormaphis. Hormaphis similibetulae was distinguished by its unique biology, forming galls on leaves of Betula. Because of the high morphological similarity with Hormaphis betulae (Mordvilko), it was placed under the genus Hormaphis (Qiao and Zhang 2004). However, the distinction of apterae of Hamamelistes and Hormaphis from the secondary host Betula is very difficult: both of them are aleyrodiform, dorsoventrally compressed, have body segments fused, short antennae with only 2–4 segments, fore and middle legs without tarsi, and hind legs with rudimentary unsegmented tarsi and lack claws. These reductions appear to be related to the organisms’ sedentary habits on Betula and represent the adaptive convergences selected by their temperate habitat. Although species of both genera migrate between Hamamelis and Betula, their life cycles are quite different and have proven extremely valuable in distinction (Pergande 1901, von Dohlen and Gill 1989, Aoki and Kurosu 1991, von Dohlen and Stoetzel 1991, Aoki et al. 2001). Firstly, Hamamelistes have two-year life cycles due to a long gall phase, while Hormaphis complete their life cycles within one year. Secondly, on Hamamelis, Hamamelistes induce spiny or coral-like galls on leaf or flower buds, whereas Hormaphis cause conical galls on the leaves. Lastly, Hamamelistes inhabit cockscomb-like or blister-like galls on leaves of Betula, but Hormaphis live freely on the leaves, not causing any deformation. In China, there is only one species of Hamamelis, Hamamelis mollis, distributed in Sichuan, Hubei, Anhui, Zhejiang, Jiangxi, Hunan and Guangxi Provinces (Zhang and Lu 1995). According to the absence of primary host at high elevations in the Tibetan Plateau, Qiao and Zhang (2004) inferred that Hormaphis similibetulae was autoecious on Betula albosinensis. We agree with their inference, as Hamamelis betulinus and Hormaphis betulae were also observed living all year round parthenogenetically on Betula in Europe due to lack of primary host (Heie 1980). Although the life cycle of Hormaphis similibetulae requires further research, it appears to be more similar to that of Hamamelistes than to that of Hormaphis.


The phylogenetic position of Hormaphis similibetulae was inferred by MP, ML and Bayesian analyses on the basis of nuclear EF-1α and mitochondrial tRNA/COII sequences. In all phylogenetic analyses, Hormaphis similibetulae clustered firmly with Hamamelistes and was placed as a basal lineage, clearly differed from other Hormaphis species. Life cycle similarities also indicated that Hormaphis similibetulae was more closely related to Hamamelistes species. We therefore conclude that Hormaphis similibetulae should be transferred to the genus Hamamelistes as Hamamelistes similibetulae (Qiao & Zhang), comb. n.


Thanks are due to X. L. Huang, G. X. Qiao and N. Qiao for their collections. The work was supported by the National Natural Sciences Foundation of China (Nos. 30830017, 31061160186), National Science Funds for Distinguished Young Scientists (No. 31025024), National Science Fund for Fostering Talents in Basic Research (No. J0930004), and a grant (No. O529YX5105) from the Key Laboratory of the Zoological Systematics and Evolution of the Chinese Academy of Sciences.

Aoki S, Kurosu U (1991) Host alternation of Hormaphis betulae (Homoptera, Aphidoidea). Japanese Journal of Entomology 59:164.
Aoki S, von Dohlen CD, Kurosu U (2001) Revision of the Japanese species of the aphid genus Hamamelistes (Hemiptera, Aphididae, Hormaphidinae) based on the mitochondrial DNA sequence data. Entomological Science 4:59-67.
Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemistry Bulletin 9:11-15.
Farris JS, Källersjö M, Kluge AG, Bult C (1994) Testing significance of incongruence. Cladistics 10: 315–319. doi: 10.1111/j.1096-0031.1994.tb00181.x
Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783–791. doi: 10.2307/2408678
Ghosh AK (1985) Hormaphidinae: distribution, phylogeny and systematics. In: Szelegiewicz H (Ed) Evolution and Biosystematics of Aphids. Proceedings of the International Aphidological Symposium at Jablonna, 1981. Polska Akademia Nauk, Warsaw, 303–336.
Guo K, Qiao GX (2005) Study of geographical distributional pattern of the subfamily Hormaphidinae (Homoptera, Hormaphididae). Acta Zootaxonomica Sinica 30:252-256.
Heie OE (1980) The Aphidoidea (Hemiptera) of Fennoscandia and Denmark. I. General Part. The Families Mindaridae, Hormaphididae, Thelaxidae, Anoeciidae, and Pemphigidae. Scandinavian Science Press Ltd., Klampenborg, 236 pp.
Kimura M (1980) A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution 16: 111–120. doi: 10.1007/BF01731581
Normark BB (1996) Phylogeny and evolution of parthenogenetic weevils of the Aramigus tessellatus species complex (Coleoptera: Curculionidae: Naupactini): Evidence from mitochondrial DNA sequences. Evolution 50: 734–745. http://www.jstor.org/stable/pdfplus/2410846.pdf [accessed 21.III.2011] doi: 10.2307/2410846
Ortiz-Rivas B, Martínez-Torres D (2010) Combination of molecular data support the existence of three main lineages in the phylogeny of aphids (Hemiptera: Aphididae) and the basal position of the subfamily Lachninae. Molecular Phylogenetics and Evolution 55: 305–317. doi: 10.1016/j.ympev.2009.12.005
Pergande T (1901) The life history of two species of plant-lice inhabiting both the witch-hazel and birch. United States Department of Agriculture, Division of Entomology, Technical Series 9:1-44.
Posada D, Crandall KA (1998) MODELTEST: testing the model of DNA substitution. Bioinformatics 14: 817–818. doi: 10.1093/bioinformatics/14.9.817
Qiao GX, Zhang GX (2004) Record of Hormaphis Osten-Sacken (Aphididae: Hormaphidinae) from China, with description of a new species. Oriental Insects 38:277-282.
Remaudière G, Remaudière M (1997) Catalogue of the World’s Aphididae. Institut National de la Recherche Agronomique, Paris, 473 pp.
Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574. doi: 10.1093/bioinformatics/btg180.
Stern DL (1994) A phylogenetic analysis of soldier evolution in the aphid family Hormaphididae. Proceedings of the Royal Society of London Series B: Biological Sciences 256: 203–209. http://www.jstor.org/stable/pdfplus/50356.pdf [accessed 21.III.2011] doi: 10.1098/rspb.1994.0071
Swofford DL (2002) PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4.0b10. Sinauer Associates, Sunderland, Massachusetts.
Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24: 1596–1599. doi: 10.1093/molbev/msm092
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25: 4876–4882. doi: 10.1093/nar/25.24.4876
von Dohlen CD, Gill DE (1989) Geographic variation and evolution in the life cycle of the witch-hazel leaf gall aphid, Hormaphis hamamelidis. Oecologia 78: 165–175. doi: 10.1007/BF00377152
von Dohlen CD, Kurosu U, Aoki S (2002) Phylogenetics and evolution of the eastern Asian–eastern North American disjunct aphid tribe, Hormaphidini (Hemiptera: Aphididae). Molecular Phylogenetics and Evolution 23: 257–267. doi: 10.1016/S1055-7903(02)00025-8
von Dohlen CD, Moran NA (2000) Molecular data support a rapid radiation of aphids in the Cretaceous and multiple origins of host alternation. Biological Journal of the Linnean Society 71: 689–717. doi: 10.1006/bijl.2000.0470
von Dohlen CD, Stoetzel MB (1991) Separation and redescription of Hormaphis hamamelidis (Fitch 1851) and Hormaphis cornu (Shimer 1867) (Homoptera: Aphididae) on witch-hazel in the eastern United States. Proceedings of the Entomological Society of Washington 93: 533–548. http://biostor.org/cache/pdf/30/f8/1c/30f81c9b6406e63f5df75ad2c1d9ff87.pdf [accessed 21.III.2011]
Zhang DX, Hewitt GM (1996) Nuclear integrations: challenges for mitochondrial DNA markers. Trends in Ecology & Evolution 11: 247–251. doi: 10.1016/0169-5347(96)10031-8
Zhang ZY, Lu AM (1995) Hamamelidaceae: geographic distribution, fossil history and origin. Acta Phytotaxonomica Sinica 33:313-339.