Molecular Systematics of Aphids and Their Primary Endosymbionts

Molecular Phylogenetics and Evolution Vol. 20, No. 3, September, pp. 437– 449, 2001 doi:10.1006/mpev.2001.0983, available online at http://www.idealibrary.com on

Molecular Systematics of Aphids and Their Primary Endosymbionts David Martinez-Torres, Celia Buades, Amparo Latorre, and Andres Moya Institut Cavanilles de Biodiversitat i Biologia Evolutiva, Universitat de Vale`ncia, Apartado de Correos 2085, 46071 Vale`ncia, Spain Received September 15, 2000; revised March 15, 2001

Aphids constitute a monophyletic group within the order Homoptera (i.e., superfamily Aphidoidea). The Aphidoidea originated in the Jurassic about 150 my ago from some aphidiform ancestor whose origin can be traced back to about 250 my ago. They exhibit a mutualistic association with intracellular bacteria (Buchnera sp.) related to Escherichia coli. Buchnera is usually considered the aphids’ primary endosymbiont. The association is obligate for both partners. The 16S rDNA-based phylogeny of Buchnera from four aphid families showed complete concordance with the morphology-based phylogeny of their aphid hosts, which pointed to a single original infection in a common ancestor of aphids some 100 –250 my ago followed by cospeciation of aphids and Buchnera. This study concentrated on the molecular phylogeny of both the aphids and their primary endosymbionts of five aphid families including for the first time representatives of the family Lachnidae. We discuss results based on two Buchnera genes (16S rDNA and the ␤ subunit of the F-ATPase complex) and on one host mitochondrial gene (the subunit 6 of the F-ATPase complex). Although our data do not allow definitive evolutionary relationships to be established among the different aphid families, some traditionally accepted groupings are put into question from both bacterial and insect data. In particular, the Lachnidae and the Aphididae, which from morphological data are considered recently evolved sister groups, do not seem to be as closely related as is usually accepted. Finally, we discuss our results in the light of the proposed parallel evolution of aphids and their endosymbionts. © 2001 Academic Press

INTRODUCTION Aphids are plant-sap-sucking insects comprising around 4000 species distributed mainly throughout the temperate regions of the globe (Dixon, 1990). They constitute a monophyletic group within the order Homoptera, which according to Heie (1980) has the rank of superfamily (i.e., superfamily Aphidoidea). The Aphidoidea and the Phylloxeroidea (aphids’ closest monophyletic group) probably diverged in the Jurassic

or earlier from some aphidiform ancestor whose origin can be traced back up to about 250 my ago (Heie, 1987). Based on morphological characters, Heie (1980, 1987) grouped aphids into 10 families and proposed a phylogenetic tree which is now generally accepted (Fig. 1). Aphids exhibit a mutualistic association with intracellular bacteria (Buchnera sp.) housed in special aphid cells designated bacteriocytes which aggregate to form a bilobed structure within the body cavity of aphids called mycetome (McLean and Houk, 1973; Douglas and Dixon, 1987; Munson et al., 1991a). The association is obligate for both partners: Buchnera cannot be cultured outside the aphid host and aphids treated with antibiotics grow slowly and are unable to reproduce (Douglas, 1998). Different kinds of evidence suggest a nutritional role for Buchnera that would provide their hosts with essential amino acids poorly represented in the plant phloem sap (Douglas, 1997). Analysis of Buchnera 16S rDNA sequences from different aphid species supported their belonging to a single monophyletic clade within the ␥3 subdivision of the class proteobacteria, having Escherichia coli and related members of the enterobacteriaceae as their closest known relatives (Unterman et al., 1989; Munson et al., 1991b; Moran et al., 1993). Moreover, the 16S rDNA-based phylogeny of Buchnera representatives from four aphid families showed complete concordance with the morphology-based phylogeny of their aphid hosts (Munson et al., 1991b; Moran et al., 1993; Moran and Baumann, 1994). These results pointed to a single original infection in a common ancestor of all modern Aphidoidea some 100 –250 my ago (see Fig. 1) followed by cospeciation of aphids and Buchnera (Moran et al., 1993). The parallel cladogenesis of Buchnera and their hosts seems to be the natural result of a long-term strict maternal transmission from mothers to daughters (Houk and Griffiths, 1980; Douglas, 1989; Baumann et al., 1997). Several other molecular-based phylogenetic reconstructions with different genes from Buchnera have also shown a congruence with the morphology-based phylogeny of aphids (Rouhbakhsh et al., 1996; Brynnel et al., 1998; Silva et al., 1998; Baumann et al., 1999; van Ham et al., 1999, 2000), although in most of them taxonomic sampling was limited and biased toward representatives of the family Aphididae.

437

1055-7903/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

438

MARTINEZ-TORRES ET AL.

extracted total DNA from single aphids following the method described by Martinez-Torres et al. (1992), omitting all the alkali-treatment-related steps. PCR Amplification

FIG. 1. Morphology-based phylogeny proposed by Heie (1987) for aphid families. The tree also includes the two families that according to Heie (1987) are aphids’ closest relatives (i.e., superfamily Phylloxeroidea). The five families analyzed in this report are underlined. The arrow indicates the original infection with Buchnera in an ancestor of modern aphids (Moran et al., 1993).

One report, however (van Ham et al., 1997), proposed a different topology for the phylogenetic relationships among Buchnera 16S rDNA sequences, therefore questioning either the traditionally accepted phylogeny of aphids or the perfect parallelism between the aphid and their primary endosymbiont phylogenies. In this report we address the question of the parallel evolution of aphids and their primary endosymbionts by comparing the phylogenies inferred from molecular data for both Buchnera and aphids representative of five different families within the Aphidoidea (see Fig. 1). MATERIALS AND METHODS Species and Genes Table 1 shows the classification of the species used in this study and the nomenclature used in the phylogenetic trees. Species representative of five different families within the Aphidoidea (i.e., families Pemphigidae, Thelaxidae, Drepanosiphidae, Lachnidae, and Aphididae) were selected. Sequences analyzed include two genes from Buchnera (most of the 16S rDNA gene and partial sequences from the atp-D gene encoding the ␤ subunit of the F-ATPase complex) and two genes from their hosts (the aphid homologous ␤ subunit and the mitochondrially encoded subunit 6 of the F-ATPase complex). Most of these sequences were obtained by us with a few exceptions (see Table 1 for details) DNA Preparation Isolation of total aphid DNA was the method of choice since it contains both host DNAs (chromosomic and mitochondrial DNAs) and endosymbiont DNA. We

Amplification of the Buchnera 16S rDNA was done as described (van Ham et al., 1997). The amplification of the central portion of the ␤ subunit of the F-ATPase complex from both Buchnera and their aphid hosts was done in a single PCR with a single pair of degenerate primers as described by Clark and Baumann (1993). Standard 1.4% agarose gel electrophoresis allowed the resolution of two bands of about 660 and 540 bp that corresponded to the host and the endosymbiont homologous subunits, respectively. For the amplification of the subunit 6 of the ATPase we designed two sets of nested degenerate primers based on conserved sequences present in the COX2 and COX3 genes among different insects obtained from the GenBank databases and from our own unpublished data with aphids. Two rounds of PCR allowed the amplification of a fragment of about 1.2 kb comprising the 3⬘ end of the COX 2 gene, two tRNAs, the subunit 8 of the ATPase, the complete subunit 6 of the ATPase, and a portion of the 5⬘ end of the COX 3 gene. The first PCR was done with the first set of primers on about 100 ng of total aphid DNA. The reaction was carried out with the GeneAmp 2400 system and cycling conditions consisted of 94°C for 3 min; 35 cycles of 94°C for 30 s, 45°C for 1.5 min, and 72°C for 1.5 min; and a final extension step of 7 min at 72°C. A second round of PCR was then done with the nested primers on a 1-␮l aliquot from the first PCR, keeping cycling conditions identical except for the increase to 47°C of the annealing temperature. In both PCRs we used Taq polymerase (Amersham–Pharmacia). Cloning and Sequencing All PCR products were purified by ammonium precipitation prior to subsequent steps. In some instances we proceeded to clone the amplified products into a T-tailed pBluescript II SK ⫹ plasmid (Stratagene) prepared according to Marchuk et al. (1992). Sequencing of cloned products was done on both strands with vector-based SK and KS primers (Stratagene) and insert internal primers when necessary. All the ␤ subunit fragments were sequenced in this way without the need of internal sequencing primers. 16SrDNA sequences were obtained either by direct sequencing with both PCR primers and three forward plus three reverse internal sequencing primers or through cloning when the presence of secondary endosymbiont-amplified products were detected. Finally, most of the subunit 6 of the ATPase fragments were sequenced directly with PCR primers plus two internal primers, although in a few instances we had to proceed through cloning steps. All primer sequences are available upon request. DNA

439

APHIDS AND THEIR PRIMARY ENDOSYMBIONTS

TABLE 1 Classification of Aphid Species Used (Heie, 1980) and Summary of the Sequences Analyzed in This Study Indicated by Their Accession Nos. Buchnera Family Pemphigidae

Subfamily Pemphiginae

Tribe

Species

Pemphigini

Pemphigus spyrothecae Pemphigus bursarius Pemphigus betae Pemphigus populi Eriosomatinae Eriosomatini Eriosoma lanuginosum Tetraneura caerulescens Tetraneura ulmi Fordinae Fordini Geoica utricularia Aploneura lentisci Forda formicaria Baizongia pistaciae Melaphidini Schlechtendalia chinensis Melaphis rhois Thelaxidae Thelaxes suberi Drepanosiphidae Drepanosiphinae Drepanosiphini Drepanosiphum oregonensis Phyllaphidinae Phyllaphidini Panaphis juglandis Hoplocallis pictus Chaitophorinae Chaitophorini Chaitophorus leucomelas Chaitophorus viminalis Lachnidae Lachninae Lachnini Lachnus roboris Tuberolachnus salignus Maculolachnus submacula Stomaphidini Stomaphis quercus Cinarinae Cinarini Cinara cupresi Schizolachnini Schizolachnus pineti Eulachnini Eulachnus sp. Aphididae Pterocommatinae Pterocommatini Pterocomma populeum Aphidinae Aphidini Rhopalosiphum padi Schizaphis graminum Macrosiphini Myzus persicae Acyrthosiphon pisum Phylloxeridae Phylloxera sp.1 (outgroup) Phylloxera sp.2

(Pemsp) (Pembu) (Pembe) (Pempo) (Erila) (Tetca) (Tetul) (Geout) (Aplole) (Forfo) (Baipi) (Schchi) (Melrho) (Thesu) (Dreor) (Panju) (Hoppi) (Chale) (Chavi) (Lacro) (Tubsa) (Macsu) (Stoque) (Cincu) (Schilac) (Eulsp) (Ptepo) (Rhopa) (Schgra) (Myzpe) (Acypi) (Physp1) (Physp2)

Aphids

16SrDNA ␤-subunit ␤-subunit

ATP6

AJ247133 AJ247132

AJ298660 AJ298661

M63254* AJ296750 AJ296748 AJ247134 AJ296749

AJ296760 AJ298665 AJ298666

AJ296751 AJ247135

AJ296752 Z19056* M63255* AJ296757 AJ247131 AJ296758 AJ247136 AJ296759 M63252* AJ296756 AJ247137 AJ296754 AJ247138 AJ296755 AJ296753

AJ296747 M63248* M63246* M63249* M27039*

AJ247126 AJ247127 Z15147** AJ247129

AJ298662 AJ298663 AJ298664

AJ296761 AJ298667 AJ298671 AJ296762 AJ298669 AJ298668 AJ298670 AJ296763 AJ298682 AJ298679 AJ298677 AJ298680 AJ298678 AJ298681 AJ296764 AJ298672 AJ296765 AJ298673 Z15145** AJ298676 AJ296766 AJ298674 AJ298675 AJ298683 AJ298684

Note. The name of the species is followed by the abbreviation used in the figures. * Sequences from Moran et al. (1993). ** Sequences from Clark and Bauman (1993).

sequencing was performed in a PE/ABI 373 automated sequencer with the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin–Elmer). Computer Analysis of DNA Sequences Chromatograms were analyzed and assembled with the Staden package (Staden et al., 1998). Multiple alignments were done with Clustal X (Thompson et al., 1997) with gap opening and gap extension penalties of 10.0 and 0.2, respectively. 16SrDNA alignments were improved by hand manipulation, trying to keep likely homologous secondary structures aligned by comparison to those proposed for the 16S rDNA of E. coli (Gutell, 1994) and Buchnera from the aphid Acyrtosiphon pisum (Unterman and Baumann, 1990). All alignments are available upon request. Amino acid sequences were obtained from coding

sequences with the Translate program [Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, WI] with the Drosophila mitochondrial genetic code for the ATPase 6 gene. Distance-based phylogenetic analysis was performed with the procedures implemented in MEGA (Kumar et al., 1993). For each gene we evaluated the likelihood of different tree topologies under different models of nucleotide substitution using PAML (Yang, 1998), and the model that best fitted our data was chosen. Parsimony analysis of nucleotide or amino acid sequences were carried out with PAUP 3.1 (Swofford, 1993) or PHYLIP 3.57c (Felsenstein, 1995), respectively. Statistical reliability of the inferred trees was determined by bootstrap analysis. Maximum-likelihood analysis was done with fastDNAml 3.3 (Olsen et al., 1994; Felsenstein, 1981).

440

MARTINEZ-TORRES ET AL.

TABLE 2 Summary of Some Sequence-Related Information for the Four Genes Analyzed in This Study

Gene Buchnera 16S Buchnera ATP␤ Aphid-ATP␤ Aphid-ATP6

No. of sequences a 21 12 8 23

Homogeneity test b

Phylogenetic signal c

Passed Passed Passed Passed

Yes Yes No Yes

Number of sites (amino acids)

Number of sites (nucleotides) Total d

1438 453 474 e 654

Variant

Informative

Total

Variant

Informative

400 198 98 376

267 140 57 297

— 151 158 218

— 44 12 121

— 20 2 100

a

Number of sequences without considering the outgroups (see Table 1 for details). A 5% level ␹ 2-test performed with the Puzzle 4.0 program (Strimmer and von Haeseler, 1996) to test whether sequences included in the analysis had homogeneous composition. c The g1 statistic of skewness for 10,000 random trees was compared to critical values (Hillis and Huelsenbeck, 1992). d Number of sites that remained after positions containing gaps were excluded. e Number of sites after positions corresponding to two small introns were excluded. b

Nucleotide Sequence Accession Numbers The nucleotide sequence data obtained in this report have been deposited in the GenBank/EMBL database under the accession numbers shown in Table 1. RESULTS Our results are presented in two main blocks. First we describe results of the phylogenetic analysis performed on the two Buchnera genes and then on the two aphid genes. For each gene, results obtained by different phylogenetic reconstruction approaches (distance-based, maximum-parsimony, and maximumlikelihood) are presented first followed by other complementary analyses (i.e., tree comparisons and relative-rates tests). Details on the sequences analyzed for the different genes are summarized in Table 2. Phylogenetic Analysis of Buchnera Sequences Buchnera 16S rDNA. Published sequences from E. coli, Enterobacter pyrinus, Salmonella typhi, and Klebsiella pneumoniae (Accession Nos. J01859, AJ010486, Z47544, and X87276, respectively), all members of the Enterobacteriaceae, were used as outgroups. Nucleotide composition among the 21 sequences from aphid bacteria was on average 49.3% G⫹C, significantly different from that of the free-living Enterobacteriaceae used as outgroups (average G⫹C, 54.5%) and confirming that amplified sequences do belong to the aphids primary endosymbionts rather than to the secondary endosymbionts described for some aphid species (Moran, 1996; Spaulding and von Dohlen, 1998). Distances were estimated with the Tamura and Nei (1993) (TN93) model of nucleotide substitution. Use of the neighbor-joining (NJ) method with the TN93 distances both with (alpha parameter 0.2) and without rate heterogeneity produced similar topologies (Fig. 2A). A bootstrap analysis involving 500 replicates cor-

roborated the monophyly of all endosymbiont sequences (100% of the replicates) and therefore confirmed that we had indeed analyzed Buchnera sequences and that no secondary endosymbiont sequences had been included in the analysis. However, bootstrap support for some major groupings within the Buchnera lineage was rather weak. Most notable is the basal position of Buchnera sequences from the Lachnidae. A parsimony analysis produced one most parsimonious (MP) tree with 1078 steps. Consistency index (C.I.) calculated with informative positions was 0.46. Up to 103 trees involving 1081 steps or less were found and their consensus (basically coincidental with the MP tree) is shown in Fig. 2B. As before, sequences from the Lachnidae occupied the basal position in the Buchnera lineage. Several heuristic searches were done with the fastDNAml program (Olsen et al., 1994; Felsenstein, 1981) with the jumble option and categorization of substitution rates into nine categories obtained with DNArates (G. J. Olsen, S. Pracht, and R. Overbeek, unpublished). One hundred trees were kept (K option⫽ 100) in each run and a consensus tree of the 50 trees nonsignificantly worse than the best tree was computed. Similar results were obtained for transition/transversion ratios of 1 and 2 (Fig. 2C). Again, Buchnera from the Lachnidae were placed basal to other Buchnera sequences in a rather high proportion of the trees (70%). Both the Kishino and Hasegawa (1989) and the Templeton (1983) tests did not find significant differences among a collection of alternative tree topologies including those reported above and (most important) the topology proposed by Heie (1987) for the aphids. However, it is worth mentioning that both tests agreed in placing the MP tree as the best tree and in determining that Heie’s topology was the worst. To investigate whether any of the Buchnera lineages

FIG. 2. Phylogenetic trees inferred from Buchnera 16S rDNA sequences from the aphid species indicated. (A) Neighbor-joining tree from use of the TN93 distances assuming among-site rate heterogeneity (alpha parameter of the gamma distribution set to 0.2). (B) Majority-rule consensus tree of the 103 trees whose length was 1081 or less (the MP tree was 1078). (C) Consensus of the 50 trees nonsignificantly worse than the ML tree. Values above branches are percentages of supporting trees. Values below branches correspond to bootstrap support in 500 replicates (only values higher than 50 are indicated). Abbreviations for species are as in Table 1. Those monophyletic groups that coincide with some monophyletic groupings of aphids in Heie’s classification are indicated (see Table 1). Aphid subfamilies: For, Fordinae; Phy, Phyllaphidinae; Eri, Eriosomatinae; Pem, Pemphiginae. Aphid families: PEM, Pemphigidae; DRE, Drepanosiphidae; THE, Thelaxidae; APH, Aphididae; LAC, Lachnidae.

APHIDS AND THEIR PRIMARY ENDOSYMBIONTS

441

442

MARTINEZ-TORRES ET AL.

TABLE 3 Monophyly of Taxa under the Three Genes Analyzed Taxon a

Buchnera 16S

Buchnera ATP␤

Aphid ATP6

Family Aphididae Family Lachnidae Family Pemphigidae Subfamily Eriosomatinae Subfamily Pemphiginae Subfamily Fordinae Family Drepanosiphidae Subfamily Phyllaphidinae Subfamily Chaitophorinae Subfamily Drepanosiphinae Family Thelaxidae

Yes Yes No Yes Yes Yes Yes Yes — ns —

Yes Yes? No — Yes — — — ns ns —

Yes Yes? No Yes Yes Yes? No Yes — — —

Note. Yes, monophyly is supported by high bootstrap values and by the different methods of tree reconstruction used; Yes?, either bootstrap support is low (less than 50%) or under some of the reconstruction methods used the taxon is not strictly monophyletic but species are sequentially added to the tree; No, the group has not been found to be monophyletic under the different analyses; —, a single species from that taxon has been analyzed; ns, no species were analyzed, a Taxon names according to Heie’s classification (see Table 1). Subfamily names are included only when monophyly of the family is not supported by some of the analysis.

had undergone accelerated evolution compared to other lineages, which might lead to artifactual attractions in phylogenetic reconstructions, we performed relative-rates tests (Robinson et al., 1998) on lineages shown to be monophyletic after the phylogenetic analysis (Table 3). Significantly higher rates of nucleotide substitution were found only in a few comparisons affecting almost exclusively Buchnera sequences from the Phyllaphidinae (Drepanosiphidae) and Thelaxidae (Table 4). Buchnera ␤ subunit of the F-ATPase complex. The E. coli sequence (Accession No. V00267) was used as outgroup. A marked nucleotide compositional bias toward a high A⫹T content compared with the homologous sequences from free-living bacteria relatives such as E. coli was evident. On average the A⫹T content for the different Buchnera sequences was 66%, whereas in E. coli the A⫹T content is 46% for the same region. This compositional bias is highest at the third codon positions (87% A⫹T in Buchnera vs 39% in E. coli). As a result, transitions were clearly saturated (especially at third codon positions) at divergence values higher than 10% (data not shown).

TABLE 4 Relative-Rate Tests Performed on 16S rRNA and ATP␤ Buchnera Sequences and on ATP6 Sequences with RRTree (Robinson et al., 1998) Outgroup

Lineage 1

Lineage 2

K1

K2

dK

sd-dK

Ratio

P

⫺0.018070 ⫺0.012540 ⫺0.019574 ⫺0.023087 ⫺0.018962 ⫺0.015410 ⫺0.018922 ⫺0.016985

0.007453 0.006134 0.006937 0.007962 0.008628 0.007211 0.007165 0.007770

⫺2.42437 ⫺2.04457 ⫺2.82178 ⫺2.89957 ⫺2.19767 ⫺2.13688 ⫺2.64095 ⫺2.18604

0.015 0.041 0.005 0.004 0.028 0.033 0.008 0.028

0.092030 0.041215 0.092030 0.054407 0.041215

⫺0.041976 0.030099 ⫺0.028864 0.037624 0.050815

0.014719 0.012333 0.014114 0.014653 0.014979

⫺2.85188 2.44054 ⫺2.04512 2.5676 3.39235

0.004 0.015 0.041 0.010 0.006

0.185137 0.18391 0.147005 0.147594 0.18391 0.18391

⫺0.035603 ⫺0.034375 0.038133 0.037544 ⫺0.036905 ⫺0.036316

0.015632 0.016974 0.015676 0.015176 0.017298 0.016565

⫺2.27753 ⫺2.02512 2.43247 2.47385 ⫺2.13346 ⫺2.19237

0.023 0.043 0.015 0.013 0.033 0.028

16S rRNA Buchnera sequences E. coli related (4)

Lachnidae (4) Aphididae (5) Aphididae (5) Aphididae (5) Pemphiginae (2) Fordinae (4) Fordinae (4) Chaitophorinae (1)

Phyllaphidinae (2) Eriosomatinae (2) Thelaxidae (1) Phyllaphidinae (2) Phyllaphidinae (2) Thelaxidae (1) Phyllaphidinae (2) Phyllaphidinae (2)

0.132874 0.127857 0.127857 0.127857 0.131982 0.132022 0.132022 0.133959

0.150944 0.140398 0.147431 0.150944 0.150944 0.147431 0.150944 0.150944

ATP␤ Buchnera sequences E. coli (1)

Pemphiginae (1) Eriosomatinae (1) Thelaxidae (1) Phyllaphidinae (1) Phyllaphidinae (1)

Phyllaphidinae (1) Aphididae (4) Phyllaphidinae (1) Lachnidae (2) Aphididae (4)

0.050054 0.071314 0.063166 0.092030 0.092030

ATP6 aphid sequences Phylloxera (2)

Pemphiginae (2) Pemphiginae (2) Fordinae (3) Fordinae (3) Chaitophorinae (1) Drepanosiphinae (1)

Fordinae (3) Phyllaphidinae (2) Chaitophorinae (1) Drepanosiphinae (1) Phyllaphidinae (2) Phyllaphidinae (2)

0.149535 0.149535 0.185137 0.185137 0.147005 0.147594

Note Only those comparisons in which significant differences were detected (P ⬍ 0.05) are shown. Number of species included in each lineage are indicated in parentheses. K, estimated distances between outgroup and lineages 1 and 2. For 16S rRNA sequences the Kimura two-parameter distance method was used. For coding sequences the number of nonsynonymous substitutions per nonsynonymous site was used. dK, K1-K2; sd-dK, standard error of dK; Ratio, standardized difference.

APHIDS AND THEIR PRIMARY ENDOSYMBIONTS

Due to the apparent saturation of transitions, transversion differences at the three codon positions were used to infer a phylogenetic tree by use of the neighborjoining method on both the Kimura two-parameter and the Tamura–Nei distances, obtaining identical topologies (Fig. 3A). Parsimony analysis was restricted to first and second codon positions plus transversion only changes at the third codon position, producing a single MP tree (Fig. 3B) needing 409 steps (C.I. calculated with informative positions was 0.44). Since for the analysis of deep branchings with coding sequences it is sometimes better to use amino acid sequences rather than nucleotide sequences (Adachi and Hasegawa, 1992), we performed a parsimony analysis on the inferred amino acid alignments. A total of 35 equally parsimonious trees needing 100 steps (C.I. with informative positions was 0.75) was found; the majority-rule consensus is shown in Fig. 3C. The Templeton test was performed to compare different tree topologies. With both nucleotide sequences and amino acid sequences, Heie’s topology was significantly worse than that of the MP trees (Buchnera from the Lachnidae was basal). Comparison of pairs of monophyletic Buchnera lineages (see Table 3) with the E. coli sequence as outgroup showed that the Phyllaphidinae (Drepanosiphidae) exhibited significantly higher rates than other lineages (see Table 4). Phylogenetic Analysis of Aphid Sequences

␤ subunit homologous sequences from aphids. Lack of phylogenetic signal was observed for this data set (see Table 2 for details). As a result, the different methods of phylogenetic reconstructions failed to give a coherent topology, not even supporting the well-established monophyly of the Aphididae, and therefore we will not discuss any phylogeny inferred from this aphid gene. Subunit 6 of the F-ATPase complex from aphids. Two species of the Phylloxeroidea superfamily were used as outgroups (see Tables 1 and 2 for sequence details). As expected for a mitochondrial gene, nucleotide composition was biased toward a high A⫹T content (82%), being extreme at the third codon positions (92%). As a result, transitions were clearly saturated for distances higher than 15%, especially at first and third codon positions, and consequently loss of linearity with the estimated distances was observed for synonymous replacements compared to nonsynonymous substitutions (data not shown). We estimated the number of nonsynonymous substitutions per nonsynonymous site by the method of Nei and Gojobori (1986) and used these distances to build a tree by the NJ method (Fig. 4A). Other NJ reconstructions (not shown) based on TN93 distances with first

443

and second codon positions, second positions only, or transversions at the three or at the first two positions, with or without consideration of rate heterogeneity among sites, produced similar topologies, with the major difference among them being the competition between some lachnid and some pemphigid representatives for occupation of a basal position in the tree. Two different distance measures were computed from the inferred amino acid sequences. Identical NJ topologies were obtained (Fig. 4B) from Poisson-corrected distances (i.e., with the assumption of homogeneity of rates among sites) and from the assumption that rate heterogeneity among sites followed a gamma distribution with alpha parameter set to 0.3, as obtained with the program PUZZLE 4.0 (Strimmer and von Haeseler, 1996), with the number of categories being fixed at eight. We performed an MP analysis based on the covarion model as described by Lopez et al. (1999). Briefly the method consists of replacement of the original amino acid data matrix by other matrixes in which those positions that appear to evolve very fast in a given lineage (need more than a fixed number of changes in a parsimony analysis restricted to that lineage) are encoded as missing (?) in all species belonging to that lineage (see details in Lopez et al., 1999). The aim of the method is to extract almost all of the ancient signal for deep nodes and the method takes into account that the evolutionary rate at a given position does not need to be the same in different lineages (as assumed with the usual weighting schemes). We built H0, H1, and H2 data matrixes. The H0 matrix was built by replacement of amino acids by question marks only in the lineages in which the number of steps needed by the most parsimonious tree obtained for that lineage was higher than zero (i.e., all the sequences in the lineage have the same amino acid at that position). In our case H0 had very little information left (only 2 informative sites remained). Matrix H1 (positions needing more than one step in a lineage were encoded by questions marks in that lineage) had only 41 informative positions and produced 50 equally parsimonious trees whose consensus was unable to resolve the relationships among families (species from the same family, including the Lachnidae and the Aphididae, clustered with species from other families). Finally, matrix H2 already had 81 informative positions and produced 21 equally parsimonious trees (C.I. calculated with informative positions was 0.586) whose consensus is shown in Fig. 4C. A Templeton test performed on the H2 amino acid matrix showed that when aphid species were arranged according to the phylogeny proposed by Heie or close to it, trees significantly worse than the MP tree were obtained. As with the other genes analyzed, relative-rates tests comparing sequences from monophyletic aphid

FIG. 3. Phylogenetic trees inferred from Buchnera subunit ␤ of the F-ATPase complex sequences from the aphid species indicated. (A) NJ tree obtained with both Kimura two-parameter and TN93 distances with transversions only at the three codon positions. (B) One of the three MP trees obtained with codon positions one and two and transversional changes at the third codon position. (C) Majority-rule consensus tree of the 35 equally parsimonious trees obtained from inferred amino acid sequences. Bootstrap values on 500 replicates are indicated below branches only when higher than 50%. In tree C values above branches correspond to the percentage of the 35 MP trees that support a given node if higher than 50%. The aphid families to which the different species belong are indicated: PEM, Pemphigidae; DRE, Drepanosiphidae; THE, Thelaxidae; APH, Aphididae; LAC, Lachnidae.

444 MARTINEZ-TORRES ET AL.

FIG. 4. Phylogenetic trees obtained for the subunit 6 of the F-ATPase complex from the aphid species indicated. (A) NJ tree obtained with distances based on nonsynonymous nucleotide positions. (B) NJ tree obtained with both Poisson-corrected and Gamma distances with inferred amino acid sequences. (C) 50% Majority-rule consensus tree of 21 equally parsimonious trees obtained with the H2 amino acid matrix (see text). Bootstrap values on 500 replicates are indicated only when higher than 50%. Family affiliations of aphid species are indicated on each tree: PEM, Pemphigidae; DRE, Drepanosiphidae; THE, Thelaxidae; APH, Aphididae; LAC, Lachnidae. Some of Heie’s (1980) subfamilies are indicated only on tree A (see Table 1). Aphid subfamilies: Aph, Aphidinae; Cin, Cinarinae; Eri, Eriosomatinae; For, Fordinae; Lac, Lachninae; Pem, Pemphiginae; Phy, Phyllaphidinae; Pte, Pterocommatinae.

APHIDS AND THEIR PRIMARY ENDOSYMBIONTS

445

446

MARTINEZ-TORRES ET AL.

lineages (see Tables 3 and 4) were performed. Significantly higher nucleotide substitution rates were found exclusively in Fordinae (Pemphigidae) and Phyllaphidinae (Drepanosiphidae) in some comparisons (see Table 4). DISCUSSION Phylogeny of Buchnera Genes Analysis of 16S rDNA and ATP␤ sequences of Buchnera from five aphid families in this study has confirmed the monophyly of the aphids’ primary endosymbionts as proposed by Munson et al. (1991b). This result agrees with the idea that all extant Buchnera are derived from a single species that successfully established in the ancestor of modern aphids (Munson et al., 1991b; Moran et al., 1993). Apart from the single representative of the Thelaxidae, some major groupings of Buchnera sequences are readily identifiable with some well-established aphid families (i.e., Lachnidae, Aphididae, and Drepanosiphidae). However, both genes failed to group all the representatives of the Pemphigidae into a single monophyletic clade (see Figs. 2 and 3). Since the family Pemphigidae is generally regarded the oldest extant aphid family (Heie, 1987), this points to a lack of resolving power of ancestral relationships of the studied genes, under the assumption that phylogenetic relationships among Buchnera sequences reflect relationships among their aphid hosts (Moran et al., 1993). However, some lower-level monophyletic groupings among Buchnera sequences have a correspondence with well-established lower-rank monophyletic aphid groupings, and relationships among Buchnera sequences within these lineages are fully concordant with their hosts’ generally accepted affiliations (see Table 1 and Figs. 2 and 3). ATP␤ sequences from the Aphididae revealed sistership relationships between Buchnera from the tribes Aphidini and Macrosiphini and between this clade and the pterocomatine representative as expected (Heie, 1987). 16S rDNA sequences from the two Buchnera representatives of the drepanosiphid subfamily Phyllaphidinae constitute a monophyletic clade clearly separated from the other drepanosiphid representative of the subfamily Chaitophorinae. Similarly, relationships among 16S rDNA Buchnera sequences from the four representatives of the aphid subfamily Fordinae (family Pemphigidae) do agree with the accepted subdivision of this aphid subfamily (i.e., tribes Fordini and Macrosiphini). The four Buchnera 16S rDNA sequences from the lachnid representatives (all members of the subfamily Lachninae) split into two well-supported lineages in an unexpected way with respect to Heie’s classification of aphids (see Table 1): the Buchnera sequence from Tuberolachnus salignus (a member of the tribe Lachnini according to

Heie) does not cluster with the other two Lachnini representatives (Lachnus roboris and Maculolachnus submacula) but instead groups with Stomaphis quercus (member of the tribe Stomaphidini). However, this topology agrees with Normark’s (2000) results who finds evidence for the existence of a new aphid tribe (Tuberolachnini) which would include T. salignus. With respect to relationships among Buchnera sequences from different aphid families there is a most notable result: all the reconstructions done with both genes coincide in placing all sequences from the Lachnidae as basal with respect to all the other Buchnera sequences and this has a relatively high support (bootstrap values around 60 –70%). This is a major point of disagreement with the accepted phylogeny of aphids since most authors coincide in the sistership relationship between the Aphididae and the Lachnidae, both of them usually referred to as the most recently evolved aphid lineage (Heie, 1987; Normark, 2000). Also, in all trees inferred from these two Buchnera genes, all representatives from the Drepanosiphidae (a single species for the ATP␤ gene) group with the single representative from the Thelaxidae, which coincides with accepted relationships between these two aphid families (see Fig. 1). Apart from these results, both genes failed to give a clear picture of the phylogenetic relationships among the primary endosymbionts of the different aphid families. This lack of resolving power of ancestral relationships of these two genes may be the consequence of the high evolutionary rates and bias toward an increase in A⫹T content attributed to Buchnera genes (Moran, 1996; Brynnel et al., 1998) leading to convergences that mask original relationships. Alternatively, or simultaneously, if extant aphid families diverged from their common ancestor fast enough during a very short period of time in their evolution (star radiation) it would be very difficult to reconstruct their phylogenetic relationships, and this would apply to both the insects (see below) and their associated bacteria genes. In this context, the observed tendency of the Fordinae (Pemphigidae) to group close to the Thelaxidae–Drepanosiphidae representatives (see Fig. 2) could be artifactual since accelerated rates of change have been shown for some of the Thelaxidae–Drepanosiphidae representatives (see Table 4). Phylogenetic Relationships among Aphids In all the above discussion we have been using the generally accepted morphology-based phylogeny of aphids (that of Heie, 1987) as a main frame for comparisons with the phylogenetic relationships inferred from molecular data for Buchnera species, given the lack of a comparable molecular phylogeny of aphids. In this report we have presented results of a wide survey aimed at elucidating phylogenetic relationships within the Aphidoidea by use of molecular data. Partial sequences from the aphid nuclear gene en-

APHIDS AND THEIR PRIMARY ENDOSYMBIONTS

coding the ATPase ␤ subunit completely failed to recover any congruent phylogeny due to the reduced number of informative positions found. Results obtained with the second aphid gene analyzed (the mitochondrially encoded subunit 6 of the F-ATPase complex) do not allow establishment of a definitive molecular phylogeny for the aphid families but deserve further discussion. Different weighting schemes aimed at keeping the ancient signal (Mindell and Thacker, 1996) yielded topologies congruent with that of the morphology-based phylogeny only for most recent splits. In particular, monophyly of the family Aphididae and relationships among the different tribes and subfamilies within this family are as expected (Heie, 1987). Similarly, most of the analysis also corroborated the monophyly of the lachnid representatives, although the position of Tuberolachnus salignus basal to all other Lachnidae would suggest its being placed in a new taxonomic unit other than the tribe Lachnini, as suggested by Normark (2000) (discussed above). Only lower-rank groupings within the remaining families are recovered in most of the analysis (see Table 1 and Fig. 4). Sequences from the three representatives of the Fordinae group into a single clade close to the two representatives of the Pemphiginae and both subfamilies are interspersed with the drepanosiphid representatives (see Figs. 4A and 4B) probably as a result of the faster evolutionary rates exhibited by some of these groups (see Table 4). The third Pemphigidae subfamily (subfamily Eriosomatinae) groups close to the other two subfamilies only when we applied a weighting scheme aimed at keeping only the ancient signal (see Fig. 4C). This was the only weighting scheme that, although in a paraphyletic fashion, recovered a topology that kept the integrity of all the aphid families including the Drepanosiphidae and the Pemphigidae, but the positions of the Lachnidae and Pemphigidae were far from those generally accepted (see Figs. 1 and 4). Most unexpectedly, the Lachnidae occupy the basal position, which coincides with results described for the endosymbiont sequences and is far from the accepted sistership relationships between the Lachnidae and the Aphididae. In this context it is important to mention that despite the general agreement on the modern origin of the Aphididae and the Lachnidae (Heie, 1987; Normark, 2000) some authors have in the past already proposed the Lachnidae as the most primitive group within the Aphidoidea (reviewed in Wojciechowski, 1992). Also interesting with respect to the above-discussed tendency of some pemphigid subfamilies to group close to the Thelaxidae and Drepanosiphidae is the proposed monophyletic clade Procnemidia, which included Pemphigidae and Thelaxidae (reviewed in Wojciechowski, 1992).

447

Implications for the Parallel Evolution of Aphids and Their Primary Endosymbionts Parallel evolution of aphids and their primary endosymbionts since the original infection in the ancestor of modern aphids some 100 –250 my ago has been proposed (Moran et al., 1993) in the light of the concordant phylogenies of Buchnera 16S rDNA and the morphology-based phylogeny of aphids (Heie, 1987) and interpreted as the logical result of strict vertical transmission of Buchnera from mothers to daughters (Houk and Griffiths, 1980; Douglas, 1989; Baumann et al., 1997). We have presented an attempt to test this proposal using molecular data to infer phylogenetic relationships both among aphids and among their primary endosymbionts, including for the first time representatives of the family Lachnidae, but failed to find a clear image of relationships among major groupings. However, we have presented some notable results in this context. Most important, the two bacterial genes analyzed in this study coincided in placing Buchnera sequences from the family Lachnidae as basal with respect to all other lineages, which, as already discussed, represents a major point of disagreement with the accepted phylogeny of their hosts. If we exclude the possibility of reconstruction artifacts, two possible explanations can account for this result. The first straightforward explanation would be that the so-claimed strict parallel evolution of aphids and their primary endosymbionts has not always been so strict and horizontal transfer would have to be invoked, since monophyly of all Buchnera sequences is not put into question by our results. One alternative to this explanation would involve revision of the phylogenetic relationships among aphid families. In this respect, and taking our results with caution, it is striking that in many of the phylogenetic reconstructions performed with an aphid gene, the lachnid representatives also occupy the basal position. Similarly, the tendency of some Buchnera sequences from the Pemphigidae to group close to bacteria from the Thelaxidae–Drepanosiphidae was also found in some of the ATP6 analysis. Therefore our results do not contradict the proposed parallel evolution of aphids and their primary endosymbionts but suggest that the phylogeny of the Aphidoidea should be revised. We cannot strongly propose an alternative solution to the phylogeny of the Aphidoidea but we believe that additional molecular approaches are needed to definitely establish such a phylogeny which might help to definitively establish the parallel evolution of aphids and their primary endosymbionts. ACKNOWLEDGMENTS This work was supported by the MICYT, project BFM2000-1383. We thank Dr. J. M. Michelena for providing and for identifying most

448

MARTINEZ-TORRES ET AL.

of the species analyzed in this work. We thank Dr. R. C. H. J. van Ham for kindly providing some of the total DNA from some of the species. We also thank Dr. M. Huille and Dr. A. Forneck for providing us with the two Phylloxera species. The facilities at SCSIE (Universitat de Vale`ncia) were used for sequencing, and the Servei de Bioinformatica (Universitat de Vale`ncia) provided computer support.

Kumar, S., Tamura, K., and Nei, M. (1993). MEGA: molecular evolutionary genetics analysis, version 1.01. The Pennsylvania State University.

REFERENCES

Marchuk, D., Drumm, M., Saulino, A., and Collins, F. S. (1992). Construction of T-vectors, a rapid general system for direct cloning of unmodified PCR products. Nucleic Acids. Res. 19: 1154.

Adachi, J., and Hasegawa, M. (1992). Amino acid substitution of proteins coded for in mitochondrial DNA during mammalian evolution. Jpn. J. Genet. 67: 187–197. Baumann, L., Baumann, P., Moran, N. A., Sandstrom, J., and Thao, M. L. (1999). Genetic characterization of plasmids containing genes encoding enzymes of leucine biosynthesis in endosymbionts (Buchnera) of aphids. J. Mol. Evol. 48: 77– 85. Baumann, P., Moran, N. A., and Baumann, L. (1997). The evolution and genetics of aphid endosymbionts. Bioscience 47: 12–20. Brynnel, E. U., Kurland, C. G., Moran, N. A., and Andersson, G. E. (1998). Evolutionary rates for tuf genes in endoymbionts of aphids. Mol. Biol. Evol. 15: 574 –582. Clark, M. A., and Baumann, P. (1993). Aspects of energy-yielding metabolism in the aphid, Schizaphis graminum, and its endosymbiont: Detection of gene fragments potentially coding for the ATP synthase beta-subunit and glyceraldehyde-3-phosphate dehydrogenase. Curr. Microbiol. 26: 233–237. Dixon, A. F. G. (1990). Ecological interactions of aphids and their host plants. In “Aphid–Plant Genotype Interactions” (R. K. Campbell and R. D. Eikenbary, Eds.), pp. 7–19. Elsevier, Amsterdam.

sequence data, and the branching order in Hominoidea. J. Mol. Evol. 29: 170 –179.

Lopez, P., Forterre, P., and Phylippe, H. (1999). The root of life in the light of the covarion model. J. Mol. Evol. 49: 496 –508.

Martinez-Torres, D., Moya, A., Latorre, A., and Fereres, A. (1992). Mitochondrial DNA variation in Rhopalosiphum padi (Homoptera: Aphididae) populations from four Spanish localities. Ann. Entomol. Soc. Am. 85: 241–246. McLean, D. L., and Houk, E. J. (1973). Phase contrast and electron microscopy of the mycetocytes and symbiotes of the pea aphid Acyrtosiphon pisum. J. Insect Physiol. 19: 625– 633. Mindell, D. P., and Thacker, C. E. (1996). Rates of molecular evolution: Phylogenetic issues and applications. Annu. Rev. Ecol. Syst. 27: 279 –303. Moran, N. A. (1996). Accelerated evolution and Muller’s rachet in endosymbiotic bacteria. Proc. Natl. Acad. Sci. USA 93: 2873–2878. Moran, N. A., and Baumann, P. (1994). Phylogenetics of cytoplasmically inherited microorganisms of arthropods. Trends Ecol. Evol. 9: 15–20. Moran, N. A., Munson, M. A., Baumann, P., and Ishikawa, H. (1993). A molecular clock in endosymbiotic bacteria is calibrated using the insect hosts. Proc. R. Soc. Lond. B 253: 167–171.

Douglas, A. E. (1989). Mycetocyte symbiosis in insects. Biol. Rev. 64: 409 – 434.

Munson, M. A., Baumann, P., and Kinsey, M. G. (1991a). Buchnera gen. nov. and Buchnera aphidicola sp. nov., a taxon consisting of the mycetocyte-associated, primary endosymbionts of aphids. Int. J. Syst. Bacteriol. 41: 566 –568.

Douglas, A. E. (1997). Parallels and contrasts between symbiotic bacteria and bacterial-derived organelles: Evidence from Buchnera, the bacterial symbiont of aphids. FEMS Microbiol. Ecol. 24: 1–9.

Munson, M. A., Baumann, P., Clark, M. A., Baumann, L., Moran, N. A., Voegtlin, D. J., and Campbell, B. C. (1991b). Evidence for the establishment of aphid– eubacterium endosymbiosis in an ancestor of four aphid families. J. Bacteriol. 173: 6321– 6324.

Douglas, A. E. (1998). Nutritional interactions in insect–microbial symbioses: Aphids and their symbiotic bacteria Buchnera. Annu. Rev. Entomol. 43: 17–37.

Nei, M., and Gojobori, T. (1986). Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Mol. Biol. Evol. 3: 418 – 426.

Douglas, A. E., and Dixon, A. F. G. (1987). The mycetocyte symbiosis of aphids: Variation with age and morph in virginoparae of Megoura viciae and Acyrtosiphon pisum. J. Insect Physiol. 33: 109 – 113.

Normark, B. B. (2000). Molecular systematics and evolution of the aphid family Lachnidae. Mol. Phylogenet. Evol. 14: 131–140.

Felsenstein, J. (1981). Evolutionary trees from DNA sequences: A maximum likelihood approach. J. Mol. Evol. 17: 368 –376. Felsenstein, J. (1995). Phylogeny Inference Package (PHYLIP). Version 3.5. University of Washington, Seattle. Gutell, R. R. (1994). Collection of small subunits (16S- and 16S-like) ribosomal RNA structures. Nucleic Acids Res. 22: 3502–3507. Heie, O. E. (1980). The Aphidoidea (Hemiptera) of Fennoscandia and Denmark. The families Mindaridae, HormAphididae, Thelaxidae, Annoecidae and Pemphigidae. Fauna Entomol. Scand. 9: 1–236. Heie, O. E. (1987). Paleontology and phylogeny. In “Aphids: Their Biology, Natural Enemies and Control.” “World Crop Pests, Vol. 2A” (A. K. Minks and P. Harrewijn, Eds.), pp. 367–391. Elsevier, Amsterdam. Hillis, D. M., and Huelsenbeck, J. P. (1992). Signal, noise, and reliability in molecular phylogenetic analysis. J. Hered. 83: 189 – 195. Houk, E. J., and Griffiths. (1980). Intracellular symbiotes of the Homoptera. Annu. Rev. Entomol. 25: 161–187. Kishino, H., and Hasegawa, M. (1989). Evaluation of the maximum likelihood estimate of the evolutionary tree topologies from DNA

Olsen, G. J., Matsuda, H., Hagstrom, R., and Overbeek, R. (1994). FastDNAml: A tool for construction of phylogenetic trees of DNA sequences using maximum likelihood. Comput. Appl. Biosci. 10: 41– 48. Robinson, M., Gouy, M., Gautier, C., and Mouchiroud, D. (1998). Sensitivity of the relative-rate test to taxonomic sampling. Mol. Biol. Evol. 15: 1091–1098. Rouhbakhsh, D., Lai, C. Y., von Dohlen, C. D., Clark, M. A., Baumann, L., Baumann, P., Moran, N. A., and Voegtlin, D. J. (1996). The tryptophan biosynthetic pathway of aphid endosymbionts (Buchnera): Genetics and evolution of plasmid-associated anthranilate synthase (trpEG) within the aphididae. J. Mol. Evol. 42: 414 – 421. Silva, F. J., van Ham, R. C. H. J., Sabater, B., and Latorre, A. (1998). Structure and evolution of the leucine plasmids carried by the endosymbiont (Buchnera aphidicola) from aphids of the family Aphididae. FEMS Microbiol. Lett. 168: 43– 49. Spaulding, A. W., and von Dohlen, C. (1998). Phylogenetic characterization and molecular evolution of bacterial endosymbionts in Psyllids (Hemiptera: Sternorrhyncha). Mol. Biol. Evol. 15: 1506 – 1513. Staden, R., Beal, K., and Bonfield, J. K. (1998). The Staden Package,

APHIDS AND THEIR PRIMARY ENDOSYMBIONTS Computer Methods in Molecular Biology, Eds. Stephen Misener and Steve Krawetz. Humana Press, Totowa, NJ. Strimmer, K., and von Haeseler, A. (1996). Quartet puzzling: A quartet maximum likelihood method for reconstructing tree topologies. Mol. Biol. Evol. 13: 964 –969. Swofford, D. L. (1993). PAUP: Phylogenetic Analysis Using Parsimony, version 3.1. Computer program distributed by the Illinois Natural History Survey, Champaign, IL. Tamura, K., and Nei, M. (1993). Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol. Biol. Evol. 10: 512–526. Templeton, A. R. (1983). Phylogenetic inference from restriction endonuclease cleavage site maps with particular reference to the evolution of humans and the apes. Evolution. 37: 221–244. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., and Higgins, D. G. (1997). The CLUSTAL_X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25: 4876 – 4882. Unterman, B. M., and Baumann, P. (1990). Partial characterization of the ribosomal RNA operons of the pea–aphid endosymbionts: Evolutionary and physiological implications. In “Aphid–Plant Ge-

449

notype Interactions” (R. K. Campbell and R. D. Eikenbary, Eds.), pp. 329 –359, Elsevier, Amsterdam. Unterman, B. M., Baumann, P., and McLean, D. L. (1989). Pea aphid symbiont relationships established by analysis of 16S rRNAs. J. Bacteriol. 171: 2970 –2974. van Ham, R. C. H. J., Gonzalez-Candelas, F., Silva, F. J., Moya, A., and Latorre, A. (2000). Post-symbiotic plasmid acquisition and evolution of the repA1-replicon in Buchnera aphidicola. Proc. Natl. Acad. Sci. USA 97: 10855–10860. van Ham, R. C. H. J., Martinez-Torres, D., Moya, A., and Latorre, A. (1999). Plasmid-encoded anthranilate synthase (TrpEG) in Buchnera aphidicola from aphids of the family Pemphigidae. Appl. Environ. Microbiol. 65: 117–125. van Ham, R. J., Moya, A., and Latorre, A. (1997). Putative evolutionary origin of plasmids carrying the genes involved in leucine biosynthesis in Buchnera aphidicola (endosymbiont of aphids). J. Bacteriol. 179: 4768 – 4777. Wojciechowski, W. (1992). “Studies on the Systematic System of Aphids (Homoptera, Aphidinea),” Uniw. Slaski, Katowice. Yang, Z. (1998). Phylogenetic Analysis Using Maximum Likelihood (PAML), Version 1.4. University College London.