biological research 19

Journal of Biological Research-Thessaloniki 20: 00 – 00, 2013 J. Biol. Res.-Thessalon. is available online at http://www.jbr.gr Indexed in: WoS (Web of Science, ISI Thomson), SCOPUS, CAS (Chemical Abstracts Service) and DOAJ (Directory of Open Access Journals)

A molecular phylogenetic study of aphids (Hemiptera: Aphididae) based on mitochondrial DNA sequence analysis Vassilis PAPASOTIROPOULOS 1*, George TSIAMIS 2 , Charikleia PAPAIOANNOU 3 , Panagiotis IOANNIDIS 2,4 , Elena KLOSSA-KILIA 3 , Aristeidis P. PAPAPANAGIOTOU 1 , Kostas BOURTZIS 2,5 and George KILIAS 3 1

Faculty of Agricultural Technology, Department of Greenhouse Crops and Floriculture, Technological Educational Institute of Messolonghi, 30200 Nea Ktiria Messolonghi, Greece 2 Department of Environmental and Natural Resources Management, University of Western Greece, Agrinio, Greece 3 Department of Biology, University of Patras, Patras, Greece 4 Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD 21202, USA 5 Insect Pest Control Laboratory, Joint FAO/IAEA Programme of Nuclear Techniques in Food and Agriculture, Vienna, Austria Received: 1 November 2012

Accepted after revision: 12 March 2013

The phylogenetic relationships among aphids (Hemiptera: Aphididae), were studied using fragments of cytochrome c oxidase I (COI) and 12S rRNA genes. The majority of species, belonged to the subfamily Aphidinae, however members of the Chaitophorinae, Lachninae, Calaphidinae and Eriosomatinae subfamilies were also included. Genetic divergence ranged from 1.3% to 15.7% featuring Cinara tujafilina (Lachninae) as the most divergent species. Phylogenetic trees produced by Maximum Parsimony (MP) and Bayesian Inference analyses (BI) exhibited a similar topology, which to some extent, is in agreement with the systematic classification of aphids. Cinara tujafilina was the most basal group in the phylogram, indicating an early separation of Lachninae and a formation of another lineage distinct from the other aphids. The Aphidininae species formed the largest group in the phylogram; however, the clustering of Pterocomma pilosum with Cavariella aegopodii and Capitophorus elaeagni questions the monophyly of Macrosiphini and the systematic status of Pterocommatini. Within the Aphidini tribe, the Aphidina species appear to be monophyletic. On the contrary, the monophyly of Rhopalosiphum was not resolved. Aphis nerii and Toxoptera aurantii, were positioned as basal groups in the Aphidini-Aphidina cluster, while, within Aphis, several species with similar morphology were grouped together. Finally, the Macrosiphini were classified according to their genus with the exception of Myzus which appears to be paraphyletic. Our results corroborate the findings of previous studies although there are several issues which require further study. Key words: Aphids, mitochondrial DNA, monophyly, phylogeny, tribes. This open-access article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, printing, distributing, transmitting and reproduction in any medium, provided the original author and source are appropriately cited. Full text and supplementary material (if any) is available on www.jbr.gr

INTRODUCTION

world, although they are more common in temperate zones (Blackman & Eastop, 2000). Some of the aphid species are significant invasive pests, threatening the agricultural ecosystems worldwide (Capinera, 2002; Blackman & Eastop, 2006). Aphids not only feed on plants but also they are significant virus vectors, trans-

Aphids (Hemiptera: Aphididae) are small, soft-bodied phloem feeding insects, distributed throughout the * Corresponding author: tel.: +30 26310 58333, fax: +30 26310 58333, email: [email protected]

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Vassilis Papasotiropoulos et al. — A molecular phylogenetic study of aphids (Hemiptera: Aphididae)

mitting almost 30% of all plant virus species (Brault et al., 2010). Aphids’ main biological traits, such as polyphenism, host alternation and the ability to reproduce both asexually and sexually have made them an attractive model for evolutionary and ecological studies (Dixon, 1998; Nosil & Moers, 2005; Coeur d’Acier et al., 2007). Moreover, accurate taxonomy is also necessary for the detection of biological invasions and the efficient management of those pests (Lozier et al., 2008). More than 4000 valid species have been described so far within the family Aphididae. Speciation in aphids has been influenced by host plants, particularly by host shifts and thus regarded as a consequence to adaptation to new hosts (Peccoud et al., 2010). As it is common in many phytophagous insect groups, the major Tertiary diversification of aphids has probably been favored by the radiation of angiosperms (Heie, 1987; Peccoud et al., 2010). Fossil records and molecular clock calibration indicate that aphids common ancestor appeared nearly 84-99 million years ago (mya), concurrently with the diversification of woody angiosperms (von Dohlen & Moran, 2000). The largest taxon within the family is the subfamily Aphidinae. According to the traditional view, Aphidinae consists of three tribes: Aphidini with 750 species, Macrosiphini with approximately 2000 species and Pterocommatini with only 50 species (von Dohlen, 2009). Within Aphidini, two subtribes have been described: Aphidina (with Aphis as the major genus representative) and Rhopalosiphina. Macrosiphini is considered a unique tribe, although there have been several attempts in the past (Börner & Heinze, 1957; Shaposhnikov et al., 1998) to subdivide it. Historically, most researchers have recognized and concluded upon several subgroups within the family, but there are still strong arguments regarding their phylogenetic relationships and classification into higher taxa (von Dohlen, 2009). Aphid classification at various levels has been attempted using morphological characters, host-plants associations and life cycles (Heie, 1987; Blackman & Eastop, 1994; Shaposhnikov et al., 1998; Kim et al., 2010, 2011). However, morphological studies very often tend to be uncertain. Many aphids’ characters are difficult to interpret; in many cases, it is hard to define whether they are ancestral (plesiomorphes) or derived (apomorphies) (Heie, 2009). Moreover, development of convergent characters results finally to homoplasy (von Dohlen, 2009). Therefore, aphids’ taxonomies, especially at higher levels, have proven unsta-

ble and had to be revised (Blackman & Eastop, 2000; Margaritopoulos et al., 2006; Kim et al., 2010). Symbiosis has also played an important role in shaping aphid evolutionary history. Primary and secondary bacterial endosymbionts housed within specialized aphid host cells influence several aspects of aphid ecology, including host plant specialization and heat tolerance. These endosymbionts are also associated with adaptation and fitness under different environmental and ecological conditions (Douglas, 1997; Oliver et al., 2010). The first attempts to shed more light on the evolutionary and taxonomic relationships among aphids using mtDNA were reported by Stern et al. (1997) and Stern (1998). Since then, several mitochondrial and nuclear molecular markers have been employed in order to delineate the phylogenetic relationships at the subfamily, tribe, genus or species levels (e.g. von Dohlen & Moran, 2000; von Dohlen et al., 2006; Coeur d’Acier et al., 2007; Ortiz-Rivas & Martínez-Torres, 2010). Recently, Kim et al. (2011) presented evidence regarding timing and patterns of diversification and also host association and the biogeographic origin of aphids. Despite those efforts, there are still unresolved relationships at various taxonomic levels. Heie (1992), contrary to the common view that Aphidina and Rhopalosiphina are located within a monophyletic Aphidini group, suggested that Aphidini might be paraphyletic, placing Rhopalosiphina as sister group to Macrosiphini. Moreover, although there is strong support about the existence of a lineage containing the craccivora, fabae and spiraecola groups within Aphis (Coeur d’Acier et al., 2007), deeper relationships among this lineage and other species groups, subgenera and other distinct species still remain unresolved (von Dohlen, 2009). The grouping of Cavariella and Pterocomma demonstrated by many authors raises issues whether the Pterocommatines should be considered as an independent subfamily (Remaudière & Remaudière, 1997) or at least tribe within Aphidinae, or it should be nested into Macrosiphini according to von Dohlen et al. (2006). In addition, the subdivision of Macrosiphini into different tribes and the determination of their phylogenetic relationships still remain unresolved. Even at higher taxonomic levels, the taxonomic status and the origin of the main aphid subfamilies e.g. Lachninae and Eriosomatinae are still under investigation (Ortiz-Rivas et al., 2004; Ortiz-Rivas & Martínez-Torres, 2010).


Improvements in the resolution of the relationships among major aphid taxa would be extremely useful regarding their biology and their control as plant pests. In order to study the phylogenetic relationships within the family Aphididae, we undertook a broad survey of aphid species from several regions of Greece. To our knowledge, there has not been any study for aphid populations from Greece on such scale so far in the literature. We focused on the phylogenetic status of Aphidinae; for this reason, most of the species sampled are classified within the Aphidini and Macrosiphini tribes. On the other hand, in order to obtain a more global view of aphid phylogeny at higher levels, we included species belonging to the Chaitophorinae, Lachninae, Calaphidinae and Eriosomatinae subfamilies. Finally, since there are still unresolved issues regarding the position of Pterocommatini with respect to Aphidinae, we included in our analysis Pterocomma pilosum sequences deposited in GenBank.

MATERIALS AND METHODS Fifty four aphid species classified in the Aphidinae, Chaitophorinae, Lachninae, Calaphidinae and Eriosomatinae subfamilies were collected from different regions of Greece and included in our study (Table 1). Aphids were collected from infected parts (leaves, stems and shoot apices, inflorescences and seed pods) of annual and perennial plants during spring, summer and autumn of 2009 and 2010. Collection sites and dates are also shown in Table 1. The collected winged aphids were stored in vials filled with ethanol (95%) and kept at –20°C until species identification and DNA extraction. Aphid identification was based on the keys given by Jacky & Bouchery (1980), Taylor (1984), Stroyan (1984), combined with host identification (Blackman & Eastop, 1994, 2006). For the classification of aphids, we used the classification system of Tsitsipis et al. (2007).

DNA methods DNA was extracted from single individuals using the CTAB (hexadecyltrimethylammonium bromide) protocol (Doyle & Doyle, 1990) with minor modifications. For the amplification of a fragment of the cytochrome c oxidase I (COI) gene we designed the primers: APHF: 5ʹ-(CT)GG(AT)ATTTGATCAGG(AT)AT AAT(CT)GG-3ʹ and APHR: 5ʹ-C(AT)GC(AT)GGG TCAAA(AG)AATGA(AGT)GTAT-3ʹ based on a-

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phid COI sequences already deposited in GenBank. For 12S rRNA gene, we used the primers 12SCFR: 5ʹ-GAGAGTGACGGGCGATATGT-3ʹ and 12SCRR: 5ʹ-AAACCAGGATTAGATACCCTATTAT-3ʹ (Hanner & Fugate, 1997). PCR amplifications were carried out in 20 μl reactions containing 1 μl of DNA, 4 μl ×5 reaction buffer (Promega, Madison, WI, USA), 1.6 μl MgCl2 (25 mM), 0.1 μl deoxynucleotide triphosphate mixture (25 mM each), 0.5 μl of each primer (25 μM), 0.1 μl of Taq polymerase (1 U μl–1, Promega, Madison, WI, USA) and 12.2 μl dH2O. Amplifications were performed in a PTC-200 Thermal Cycler (MJ Research), using the following cycling conditions: 95°C for 5 min, followed by 34 cycles of 30 s at 94°C, 30 s at 53°C for 12S rRNA or 45 s at 55°C for COI, 1 min at 72°C and a final extension of 10 min at 72°C. Following PCR, the samples were PEG precipitated (Hartley & Bowen, 2003) and sequenced using the same primers. A dye terminator-labelled cycle sequencing reaction was conducted with the BigDye Terminator v3.1 Cycle Sequencing Kit (PE Applied Biosystems). Reaction products were analyzed using an ABI PRISM 310 (PE Applied Biosystems). All gene sequences generated in this study were assembled and manually edited with SeqManII by DNAStar. The sequences obtained in this study have been deposited in GenBank. Accession numbers for both COI and 12S rRNA genes are shown in Table 1. Pterocomma pilosum 12S rRNA, (GU457837.1) and COI (GU457803.1) sequences (Kim et al., 2011) obtained from GenBank were used in the analysis.

Sequence alignment Clustal W2 suite (Thomson et al., 1994) with the default parameters was used for the multiple sequence alignment of the COI fragment. COI sequences were fully translated with MEGA5 (Tamura et al., 2011), by applying the invertebrate mitochondrial genetic code. All 12S rRNA sequences were aligned using: 1) sequence-based methods in Clustal W2 suite and 2) Q-INS-i structural algorithm of the MAFFT v.7 multiple alignment program (Katoh & Standley, 2013), both with the default parameters. The gaps in the alignments were treated as missing data. As a constraint for the second alignment method, we used the structural alignment of Acyrthosiphon pisum and Schizaphis graminum 12S RNA sequences (GenBank accession number FJ411411.1 and AY531391.1, respectively) obtained by the R-Coffee software package

Aphidini-Aphidina

Aphidini -Rhopalosiphina

Macrosiphini-Anuraphidinae

Aphidinae

Aphidinae

Aphidinae

Macrosiphini-Dactynotinae

Tribe-Subtribe

Subfamily

Thessaloniki Thessaloniki Thessaloniki Thessaloniki Messolonghi Thessaloniki Thessaloniki Pieria Pieria Thessaloniki Thessaloniki Messolonghi Messolonghi Messolonghi

Zea mays Prunus domestica Triticum aestivum Triticum aestivum Cirsium arvense Prunus domestica Prunus armeniaca Malus sylvestris Pyrus communis Lactuca serriola Pisum sativum Solanum nigrum Petunia hybrida Rosa spp.

Rhopalosiphum maidis Fitch Rhopalosiphum nymphaeae L. Rhopalosiphum padi L. Schizaphis graminum Rondani Brachycaudus cardui L. Brachycaudus helichrysi Kaltenbach Brachycaudus persicae Passerini Dysaphis Pomaphis plantaginea Passerini Dysaphis Pomaphis pyri Boyer de Fonscolombe Acyrthosiphon Tija lactucae Passerini Acyrthosiphon pisum Harris Aulacorthum solani Kaltenbach Macrosiphum euphorbiae Thomas Macrosiphum rosae L.

Messolonghi Messolonghi Patra Messolonghi

Rubus spp. Malva neglecta Citrus aurantium Prunus persica

Aphis ruborum Börner Aphis umbrella Börner Toxoptera aurantii Boyer de Fonscolombe Hyalopterus pruni Geoffroy

Location

Aphis craccivora Koch Aphis cytisorum Hartig Aphis fabae Scopoli Aphis fabae solanella Theobald Aphis gossypii Glover Aphis hederae Kaltenbach Aphis nerii Boyer de Fonscolombe Aphis pomi De Geer Aphis punicae Passerini

Aphis craccae L.

Citrus aurantium

Host species Thessaloniki Messolonghi Vicia cracca Messolonghi Messolonghi Robinia pseudoacacia Thessaloniki Spartium junceum Thessaloniki Rumex crispus Pieria Solanum nigrum Messolonghi Cucumis sativus Messolonghi Hedera helix Thessaloniki Nerium oleander Thessaloniki Thessaloniki Malus sylvestris Thessaloniki Punica granatum Messolonghi

Aphis citricola Van der Goot

Genus Species

26/5/2009 16/5/2009 8/5/2009 4/6/2009 2/6/2010 15/4/2010

24/7/2009 27/5/2010 28/4/2010 27/4/2010 20/5/2010 21/4/2009 21/4/2009 26/5/2009

24/5/2009 17/4/2010 15/4/2009 10/5/2010

19/5/2009 24/4/2010 30/4/2009 24/4/2010 18/5/2009 13/5/2009 9/5/2009 25/4/2009 30/5/2009 26/5/2010 22/5/2009 20/5/2010 18/5/2010 19/4/2009

Date

JX966052 JX966037 JX966038 JX966039 JX966062 JX966063

JX966070 JX966071 JX966072 JX966075 JX966040 JX966041 JX966042 JX966051

JX966035 JX966036 JX966078 JX966055

JX966020 JX966021 JX966022 JX966023 JX966024 JX966025 JX966026 JX966027 JX966028 JX966029 JX966030, JX966031 JX966032 JX966033

JX965991 JX965976 JX965977 JX965978 JX966001 JX966002

JX965971 JX965972, JX965973 JX965974 JX965975 JX966017 JX965994, JX965995 JX966009 JX966010 JX966011 JX966014 JX965979 JX965980 JX965981 JX965990

JX965963 JX965964 JX965965 JX965966 JX965967 JX965968 JX965969

JX965961

JX965959

COI accession 12S accession number number

TABLE 1. Aphid species sequenced in this study, host plants and accession numbers for COI and 12S rRNA sequences deposited in GenBank

4 Vassilis Papasotiropoulos et al. — A molecular phylogenetic study of aphids (Hemiptera: Aphididae)

Castanea sativa Thessaloniki Populus nigra Thessaloniki Populus nigra var. kavaki Messolonghi

Pemphigini

Eriosomatinae

Myzocallis castanicola Baker Pemphigus bursarius L. Pemphigus spyrothecae Passerini

Chaitophorini Siphini Cinarini Panaphidini

Sonchus oleraceus Sonchus arvensis Prunus avium Prunus persica Prunus persica Cydonia oblonga Prunus insititia Daucus carota Populus nigra var. kavaki Hordeum murinum Thuja spp. Juglans regia

Hyperomyzus lactucae L. Hyperomyzus pallidus Hille Ris Lambers Myzus cerasi Fabricius Myzus persicae Sulzer Myzus varians Davidson Ovatus crataegarius Walker Phorodon humuli Schrank Semiaphis dauci Fabricius Chaitophorus leucomelas Koch Sipha Rungsia elegans Del Guercio Cinara tujafilina Del Guercio Callaphis juglandis Goetze

Capitophorus elaeagni del Guercio Cavariella aegopodii Scopoli Coloradoa rufomaculata Wilson Hayhurstia atriplicis Linnaeus

Macrosiphini-Myzinae

Thessaloniki Messolonghi Pieria Thessaloniki Arta Messolonghi Thessaloniki Messolonghi Messolonghi Thessaloniki Thessaloniki Thessaloniki

Thessaloniki Messolonghi Thessaloniki Thessaloniki

Thessaloniki Thessaloniki Thessaloniki Messolonghi Messolonghi Thessaloniki

Avena sterilis Triticum aestivum Hordeum murinum Cichorium intybus Sonchus oleraceus Brassica oleracea var. campestris Circium arvense Apium graveolens Chrysanthemus spp. Chenopodium album

Metopolophium dirhodum Walker Sitobion avenae Fabricius Sitobion fragariae Walker Uroleucon cichorii Koch Uroleucon sonchi Linnaeus Brevicoryne brassicae van der Goot

Location

Host species

Genus Species

Tribe-Subtribe

Chaitophorinae Chaitophorinae Lachninae Calaphidinae

Subfamily

TABLE 1. continued

JX966057 JX966058 JX966059 JX966060 JX966061 JX966066 JX966069 JX966076 JX966048 JX966077 JX966049 JX966044

JX966046 JX966047 JX966050 JX966053

JX966064 JX966073 JX966074 JX966079 JX966080 JX966043

JX965985 JX965986 JX965989 JX965992, JX965993 JX965996 JX965997 JX965998 JX965999 JX966000 JX966005 JX966008 JX966015 JX965987 JX966016 JX965988 JX965983, JX965984 JX966004 JX966006 JX966007

JX966003 JX966012 JX966013 JX966018 JX966019 JX965982

COI accession 12S accession number number

19/5/2009 JX966065 27/5/2010 JX966067 11/5/2009 JX966068

21/4/2010 20/4/2010 20/5/2009 5/5/2009 13/5/2010 24/4/2009 20/5/2009 5/5/2009 16/5/2009 17/5/2009 18/5/2009 20/5/2009

27/4/2010 21/4/2009 26/5/2010 21/5/2009

27/4/2010 27/4/2010 24/4/2009 30/5/2009 30/4/2009 30/10/2009

Date


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(Moretti et al., 2008). Both alignment methods showed little differences. The resulting secondary structure-based multiple alignment of the 12S rRNA sequences was applied to predict the consensus secondary structure with the RNAalifold server (Vienna RNA Servers, Gruber et al., 2008). The consensus structure was then used to separate stem and loop regions manually, so that they could be individually processed in the phylogenetic analysis.

Data and phylogenetic analyses Potential saturation in COI and 12S rRNA genes was individually assessed with transitions and transversions plotted against Kimura-2-Parameter (K2P) divergence using DAMBE 5.3.19 (Xia & Xie, 2001). In addition substitution saturation for both 12S rRNA and COI genes was evaluated using an entropy-based index, implemented also in DAMBE, which uses two simulated topologies (one perfectly symmetrical and one extremely asymmetrical) based on the supplied sequence data to calculate an Index of Substitution Saturation (ISS). High substitution saturation is indicated by an ISS greater than the Critical ISS (ISS.C) for both the perfectly symmetrical topology and an extremely asymmetrical topology (Xia et al., 2003; Xia & Lemey, 2009) determined with a two-tailed t-test. In order to examine whether the 12S rRNA and COI sequences have evolved with the same substitution pattern for all the species studied, we performed a disparity index test of pattern heterogeneity (Kumar et al., 2001), using MEGA5. The p-values were estimated using a Monte Carlo test (500 replicates) and all sites were taken into consideration. K2P pairwise genetic distance (Kimura, 1980) was estimated using MEGA5. An ILD test (Farris et al., 1995) using PAUP* v4.0b10 (Swofford, 2003) (100 replicates of heuristic searches) was conducted in order to examine whether the two data sets could be combined or not. Maximum Parsimony analysis was performed using PAUP* v4.0b10 and a 50% majority rule tree was generated (1000 bootstrap pseudoreplicates) (Felsenstein, 1985). A Bayesian Inference (BI) analysis was performed for all data sets with MrBayes v3.1.2 (Ronquist & Huelsenbeck, 2003), with clade support assessed by posterior probabilities. Two model setups were used: 1) a DNA model setup using the substitution model GTR+I+G and 2) a mixed DNA-RNA model setup using the substitution model GTR+I+G for the COI data set, the TIM2+I+G model for the stem regions

and finally the TIM3+G model for the loop regions of 12S rRNA data set. All models were selected according to the AIC criterion (Akaike, 1974) using jMODELTEST v0.1 (Posada, 2008). The Markov Chain Monte Carlo (MCMC) algorithm with the default parameters (4 chains and 2 runs) was applied for all data sets (number of generations=107) with both model setups. Conversion of the chains was established when the average standard deviation of split frequencies approached zero and the Potential Scale Reduction Factor was close to 1.0. Trees were sampled every 100 generations, with a 25% ‘burn-in’ from the total number of saved trees. For the construction of the phylogenetic trees, sequences of Adelges japonicus (FJ502415, AF275214) and Adelges laricis (FJ502446, AF275215), were used to root the trees. For the reconstruction of the phylogram we used Dendroscope v 2.7.4 software (Huson et al., 2007).

RESULTS & DISCUSSION Manual editing and trimming of the sequences resulted in a final segment size of 330 bp for 12S rRNA and 485 bp for COI. The COI gene fragment translates into 161 amino acids. Of the 485 bp, 297 sites were conserved, 188 were variable, whereas 161 were parsimony informative. For 12S rRNA, 226 out of the 330 sites were conserved, 104 were variable and 70 were parsimony informative. As a result the combined data set contained a total of 815 base pairs, with 523 conserved, 292 variable and 231 parsimony informative sites. Nucleotide composition for both sets of sequences showed an Adenine-Thymine bias. Average base composition was T: 40.5, C: 14.8, A: 34.9, G: 9.8 for COI and T: 38.0, C: 10.8, A: 45.5, G: 5.7 for 12S rRNA gene (Table 2). This is very similar to the nucleotide composition reported by Kim et al. (2011) at least for COI. The disparity index test for both data sets showed no significant sequence heterogeneity (p < 0.05) in almost all cases, despite the fact that the base frequencies were not all equal to 25%. Hence, both data sets are considered homogeneous and could be used for further analyses. The substitution saturation plots did not reveal saturation in any of the regions examined. Moreover according to Xia’s test substitution saturation was not significant for 12S, since ISS (0.103) was found to be lower than ISS.CSym (0.683) and ISS.CaSym (0.354) (p < 0.01). Likewise, COI did not exhibit evidence of saturation, when the three codon positions were taken in-


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TABLE 2. Qualitative analysis of the gene segments studied as well as for the combined dataset

Single individual datasets

Number of taxa Aligned sequence length (bp) Variable sites (%) Informative characters (%) Nucleotide composition (T:C:A:G) Pairwise sequence divergence Ti/Tv ratio

COI

12S

64 485 188 (38.8) 161 (33.2) 40.5:14.8:34.9:9.8 10.1 ± 2.5 1.79

64 330 104 (31.5) 70 (21.2) 38.0:10.8:45.5:5.7 5.2 ± 2.2 0.80

to consideration. The same results were also obtained when we tested the 1st and the 2nd codon position separately. However when the 3rd codon was taken into account, we obtained ISS values significantly lower than the ISS.C when the true topology is symmetrical, but almost equal or higher than the ISS.C, when the topology is assymetrical (Number of OTUs = 16 or 32). Nevertheless, while a degree of substitution saturation exists when the true topology is extremely asymmetrical and when the number of OTUs is greater than 16, such asymmetrical trees are probably not realistic for most cases (Xia & Lemey, 2009) and consequently all characters were retained for the phylogenetic analysis. The final result of the ILD test was p < 0.01, indicating according to Cunningham (1997), that we could proceed with a combined analysis of the two mtDNA data sets. Estimated genetic divergence was high between the two outgroups and the aphid species studied and also among the several tribes-subtribes (data not shown). The net divergence values for the combined data set ranged from 1.3% to 15.7%, with the Cinarini tribe being the most distant from all others. High genetic divergence was also observed within those tribes that comprised more than one species. COI exhibited the largest proportion of informative characters (33.2%) as well as the greatest pairwise sequence divergence (10.1%). Regarding the ratio of transitions to transversions (Ti/Tv), COI showed a moderate ratio (1.79), while 12S rRNA showed a predominance of Tv (0.80) (Table 2).

Phylogenetic analyses The phylogenetic reconstructions obtained by the MP and the BI analysis showed that clustering was overall in agreement with the currently accepted systematic classification of aphids at the subfamily and tribe/sub-

Combined dataset

64 815 292 (35.8) 231 (28.3) 39.5:13.2:39.1:8.1 8.2 ± 2.3 1.56

tribe level. Tree topologies were very similar, although statistical support was stronger in the BI analysis. Since the most highly resolved topologies were those obtained for the combined data set by the BI analysis, this analysis is discussed below. BI analysis with the DNA model setup identified several different groups (Fig. 1). The first one consists only of Cinara tujafilina (Lachninae); the second one of the species classified in the Calaphidinae subfamily, the third one of the Chaitophorinae and Eriosomatinae species and the fourth, which is the largest, includes all Aphidinae species plus Pterocomma pilosum. The Macrosiphini and the Aphidini species are separately clustered, while Pterocomma pilosum is positioned together with Capitophorus elaeagni and Cavariella aegopodii as the most basal group in this cluster. The separation of C. tujafilina was well supported (posterior probability, PP = 1.0). Positioning of Lachninae (although represented only by C. tujafilina) as the basal group in the phylogenetic tree is congruent with recent molecular studies considering this subfamily as a separate lineage within Aphididae far from Aphidinae (Ortiz-Rivas et al., 2004; Ortiz-Rivas & Martínez-Torres, 2010). These findings however, contradict the studies by Heie (1987) and Wojciechowski (1992) who proposed a close phylogenetic relationship between Aphidinae and Lachninae. Lachninae is one of the few subfamilies with conifer feeders retaining also an ancestral host relationship with gymnosperms (von Dohlen & Moran, 2000). As a consequence this issue is still open for further analysis using additional molecular markers and more subfamily representatives. There is a polytomy formed between the Calaphidinae and the Chaitophorinae-Eriosomatinae clades. This could be caused by the limited number of species studied and belong to the aforementioned subfa-

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FIG. 1. BI analysis tree obtained from the combined data set. Numbers next to nodes indicate Bayesian posterior probabilities (PP). The bar indicates the number of substitutions per site.


milies. Moreover, our data indicate (PP = 0.61) that members of the Chaitophorinae (Siphini and Chaitophorini tribes) and Eriosomatinae (Pemphigini tribe) subfamilies are grouped together. Ortiz-Rivas & Martínez-Torres (2010) suggested that the subfamilies Chaitophorinae and Calaphidinae belong to the same evolutionary lineage while the Eriosomatinae to a different one. However, their results differed, due to the phylogenetic reconstructions used and the gene segments (nuclear versus mitochondrial) studied. The group containing the Aphidinae and Pterocomma pilosum is the largest one in the phylogram. Within this group we have the strongly supported clustering (PP = 0.99) of Pterocomma pilosum with Cavariella aegopodii and Capitophorus elaeagni, while the rest of the Aphidinae were positioned in the same cluster (PP = 0.73) forming different subgroups. The existence of a Pterocomma and Cavariella (P-C) group has been reported by von Dohlen et al. (2006), OrtizRivas & Martínez-Torres (2010) and Kim et al. (2011). Moreover, Lee et al. (2010) reported the formation of a similar group with the addition of Capitophorus when they attempted to barcode aphids using COI. As pointed out by von Dohlen (2009) and Kim et al. (2011), this grouping is biologically meaningful because both Pterocomma and Cavariella share two common features: 1) host association with Salicaceae and 2) morphological characteristics of fundatrices identical to their offspring. Therefore, Cavariella should be transferred into Pterocommatinae and together within a larger clade of ‘liosomaphidines’. Recently, von Dohlen et al. (2006) reported that the P-C group was nested within Macrosiphini. On the other hand, Ortiz-Rivas & Martínez-Torres (2010) and Kim et al. (2011) suggested that the P-C group is the basal group to Aphidinae. Historically, Aphidinae are classified into three major tribes: Pterocommatini, Aphidini and Macrosiphini. However, according to Remaudière & Remaudière (1997), pterocommatines (due to their simple life cycle and also their morphological characteristics) can be classified to a special subfamily Pterocommatinae within the Aphididae and be regarded as sister group to all other aphidids (Heie, 2009; von Dohlen, 2009). Our proposed phylogeny is in agreement with the results of Ortiz-Rivas & Martínez-Torres (2010) and Kim et al. (2011) and also consistent with the view that the Pterocommatinae diverged early from the Aphidinae, and then the Aphidinae diverged into Aphidini and Macrosiphini (Kim et al., 2011). It is also

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consistent with phylogenies proposing that Pterocommatinae should be classified as an independent subfamily (Stroyan, 1984; Heie, 1986; Remaudière & Remaudière, 1997), although more recently Heie (2009) argued this hypothesis. The rest of the Aphidinae are clustered into different clusters/subgroups. The first cluster includes all the Macrosiphini species, while the subdivision of Aphidini results to a polytomy between the monophyletic Aphidina and the Rhopalosiphina subtribes. The monophyly of Aphidina observed in our study is congruent with the results of von Dohlen et al. (2006), Kim & Lee (2008) and Kim et al. (2011), based on molecular data. It is also in agreement with the previous morphological and ecological studies that have been conducted by Heie (1986) and Blackman & Eastop (1994, 2006). The relationship between Aphidina and Rhopalosiphina was not resolved, although the monophyly of Aphidini was proven when Pterocomma pilosum was not included in the analysis (dendrogram not shown). A sister relationship between Aphidina and Rhopalosiphina has been verified by von Dohlen & Teulon (2003), Kim & Lee (2008) and Kim et al. (2011) using molecular data. This relationship, although challenged before by Heie (1992) based on morphological and biological characters, is considered to be more valid according to Blackman & Eastop (2000). Within the Rhopalosiphina, a polytomy has been observed between Hyalopterus pruni and the rest of the Rhopalosiphina species. A similar situation has been reported by Lozier et al. (2008) when they studied the genus Hyalopterus. The monophyly of the genus Rhopalosiphum was not resolved in our study. An analogous situation has been reported by Kim & Lee (2008). Kim et al. (2011), who also did not resolve the monophyly of Rhopalosiphini, stated that the inconsistencies between the taxonomic and phylogenetic relationships for the genera of Rhopalosiphina are likely caused by faulty diagnoses and thus the generic division and classification within Rhopalosiphina needed to be revised. Aphis nerii and Toxoptera aurantii are grouped within, the robustly (PP = 0.99) supported, AphidiniAphidina cluster. Aphis nerii is morphologically distinct from the other congeneric species and has also different hosts. Thus, it is not believed to be part of a larger species complex (Blackman & Eastop, 2006; Lee & Kim, 2006). Regarding the rest of the Aphis species, some of the groupings observed here are congruent with previous studies (e.g. Coeur d’Acier et al., 2007; Kim et al., 2010). For example, the A. craccivo-

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ra/A. craccae/A. cytisorum group comprises of morphologically similar species, which have been identified as black backed aphids and classified in the craccivora group by Heie (1986). In addition, the black aphids A. fabae/A. fabae solanella/A. hederae which have several morphological and ecological characteristics in common are forming a lineage (Blackman & Eastop, 1984; Heie, 1986). Macrosiphini are also forming a big monophyletic cluster with strong nodal support (PP = 0.98). Within this group, all species (except Myzus sp.) are generally classified according to their genus. In addition some well supported subgroups were distinguished [e.g. most “dactynotines” (Macrosiphum, Acyrthosiphon, Sitobion) formed a clade]. However, we were not able to obtain a clear distinction among ‘myzines’ and ‘anuraphidines’. The subdivision of Macrosiphini has been rarely attempted because of existing convergent morphologies and/or cryptic morphological differences between species. This situation usually results in paraphyly or polyphyly, when these species are genetically studied (Coeur d’Acier et al., 2008). As already mentioned, Myzus species appeared to be paraphyletic although clustered with the rest of the other ‘myzines’. Valenzuela et al. (2007) and Footitt et al. (2008) reported similar results also using COI. Obviously, Myzus genus, as well as the rest of the Macrosiphini, need further molecular phylogenetic research combined also with extensive morphological data in order to fully resolve the complex relationships within this tribe. BI analysis with the mixed DNA-RNA model resulted to a similar clustering and identical groupings with the DNA model. All the groups previously recognized appear also in this phylogram. However, the basic difference between this phylogram and the one obtained from the DNA model is that polytomy appears between the groups, thus failing to provide enough resolution at this level.

Conclusion In this paper, we studied the phylogenetic relationships between members of the Aphididae family sampled from different regions of Greece. Our results confirm previous molecular studies and are consistent with some traditional views of aphids phylogeny derived from morphological and ecological studies. However, there are also some discrepancies and several issues, especially within the Aphidini and Macrosiphini tribes requiring further investigation. These

discrepancies could be attributed to several factors that are accepted to influence aphid phylogeny. For instance, aphid taxonomy is undermined by the difficulties in identifying or even defining an aphid species (Shaposhnikov, 1987; Lozier et al., 2008); even more, the use of species concept in aphid taxonomy is complicated by their reproductive biology. Aphids are characterized by cyclical parthenogenesis, but there are also anholocyclic populations without the sexual phase of the life cycle. For those aphids that are permanently parthenogenetic, formal species status could be given to a biologically recognizable anholocyclic group derived from a sexual ancestor (Miller & Foottit, 2009). Due to all these challenges the establishment of valid species boundaries and the determination of synonymies remain problematic (Eastop & Blackman, 2005). Polyphagy and morphological variation on different hosts may also play a role (Ilharco & Van Harten, 1987). Evidence suggests that phytophagous insects, such as aphids, acquire new plant hosts and adapt rapidly to new conditions (Raymond et al., 2001). As result increase of existing genetic diversity among aphid populations and even formation of sibling species has been expected. Genetic studies have shown that intraspecific variation is often much higher than the level of sequence divergence between species, indicating that entities considered as separate species actually represent a single genetic lineage (Eastop & Blackman, 2005; Coeur d’Acier et al., 2008). Symbiosis also seems to have played a major role in shaping aphid evolutionary history. Many aphids have established symbiotic relationships with several bacterial species, and many of their properties are shaped by the nature of the symbioses. Nearly all members of the Aphidoidea harbor obligate endosymbionts (Buchnera aphidicola), while occasionally secondary or facultative symbionts coexist with Buchnera potentially having beneficial effects on the aphid host (Russel et al., 2003; Oliver et al., 2010). These endosymbionts probably played a significant role in the ecology and evolution of aphids with respect to host adaptation and fitness (Brucker & Bordenstein, 2012). Among them, Wolbachia is well known for its ability to induce reproductive alterations, such as parthenogenesis, feminization, male-killing and, most commonly, cytoplasmic incompatibility in its hosts, thus promoting speciation (Saridaki & Bourtzis, 2010). Recently, Wolbachia was detected in several aphid species and two new supergroups were recognized (Augustinos et al., 2011). Although more effort is re-


quired in order to prove any contribution of Wolbachia to the evolutionary history of aphids, we cannot rule out the possibility of significant implications for the delineation of species and an impact in the determination of phylogenetic relationships. In conclusion, aphid phylogeny at several hierarchical levels poses a continuous challenge. Many relationships need to be further examined and there are also many occasions where morphology is ambiguous raising serious taxonomic issues. As pointed out by von Dohlen (2009), increased and more strategic sampling and additional molecular data emerging from the Pea Aphid Genome Project would help to deal with those issues. Under this point of view, although more populations within species and more species within the tribes-subtribes are required, our results corroborate some of the previously proposed relationships and add to the existing molecular and morphological data, improving the current understanding of aphids’ phylogeny.

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