Phylogenetic Characterization and Molecular Evolution of Bacterial Endosymbionts in Psyllids (Hemiptera: Sternorrhyncha) Allen W. Spaulding and Carol D. von Dohlen Department of Biology, Utah State University Most sternorrhynchan insects harbor endosymbiotic bacteria in specialized cells (bacteriocytes) near the gut which provide essential nutrients for hosts. In lineages investigated so far with molecular methods (aphids, mealybugs, whiteflies), endosymbionts apparently have arisen from independent infections of common host ancestors and cospeciated with their hosts. Some endosymbionts also exhibit putatively negative genetic effects from their symbiotic association. In this study, the identity of endosymbionts in one major sternorrhynchan lineage, psyllids (Psylloidea), was investigated to determine their position in eubacterial phylogeny and their relationship to other sternorrhynchan endosymbionts. Small-subunit ribosomal RNA genes (16S rDNA) from bacteria in three psyllid species (families Psyllidae and Triozidae) were sequenced and incorporated into an alignment including other insect endosymbionts and free-living bacteria. In phylogenetic analysis, all sequences were placed within the g subdivision of the Proteobacteria. Three sequences, one from each psyllid species, formed a highly supported monophyletic group whose branching order matched the host phylogeny, and also exhibited accelerated rates of evolution and mutational bias toward A and T nucleotides. These attributes, characteristic of primary (P) bacteriocyte-dwelling endosymbionts, suggested that these sequences were from the putative psyllid P endosymbiont. Two other sequences were placed within the g-3 subgroup of Proteobacteria and were hypothesized to be secondary endosymbionts. The analysis also suggested a sister relationship between P endosymbionts of psyllids and whiteflies. Thus, a continuous mutualistic association between bacteria and insects may have existed since the common ancestor of psyllids and whiteflies. Calculations using a universal substitution rate in bacteria corrected for endosymbiont rate acceleration support the idea that this common ancestor was also the ancestor of all Sternorrhyncha. Compared with other P endosymbiont lineages, the genetic consequences of intracellular life for some psyllid endosymbionts have been exaggerated, indicating possible differences in population structures of bacteria and/or hosts.
Introduction Sternorrhynchans (Hemiptera: aphids, scales and mealybugs, whiteflies, and psyllids) and other insects that feed throughout their life cycles on nutritionally incomplete diets such as plant sap, blood, and wood often harbor mutualistic intracellular prokaryotes (Buchner 1965) that apparently provide nutrients essential for survival and reproduction of the hosts (Dadd 1985; Douglas 1998). These primary (P) endosymbionts are usually distinguished from other microorganisms (including secondary [S] endosymbionts) by their confinement to specialized host cells called mycetocytes (Buchner 1965; McLean and Houk 1973; Douglas 1989), or, more appropriately, bacteriocytes (Baumann et al. 1995). However, the original criterion used by Buchner (1965) defines the P endosymbiont as having the more ancient association with the host. In sternorrhynchans, bacteriocytes are organized into a large organ known as the mycetome or bacteriome, sometimes incorporating a cellular envelope or syncytial region that may contain the S endosymbionts (Buchner 1965; Baumann et al. 1995). Recent molecular phylogenetic analyses of 16S ribosomal DNA sequences have demonstrated that many insect endosymbionts belong to the Proteobacteria, mostly within the g subdivision (reviewed in Moran and Telang 1998). Within Sternorrhyncha, identities of enKey words: accelerated evolution, endosymbionts, Hemiptera, nucleotide compositional bias, psyllids, Sternorrhyncha. Address for correspondence and reprints: Carol D. von Dohlen, Department of Biology, Utah State University, UMC 5305, Logan, Utah 84322. E-mail:
[email protected]. Mol. Biol. Evol. 15(11):1506–1513. 1998 q 1998 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038
1506
dosymbionts in some host lineages are well characterized, while those of other lineages are essentially unknown (fig. 1). Aphid (Aphidoidea) P endosymbionts (Buchnera aphidicola) form a distinct lineage within the g-3 subgroup (Unterman, Baumann, and McLean 1989; Munson et al. 1991). Secondary endosymbionts from the bacteriome envelope cells of some aphids are members of the family Enterobacteriaceae within the g-3 subgroup (Unterman, Baumann, and McLean 1989; Chen and Purcell 1997). Whitefly (Aleyrodoidea) P endosymbionts were placed in the g subdivision but were unassignable to a specific subgroup; an S endosymbiont clustered within the family Enterobacteriaceae (Clark et al. 1992). In contrast to aphids and whiteflies, endosymbionts of mealybugs (Coccoidea, Pseudococcidae) were assigned to the b subdivision (Munson, Baumann, and Moran 1992), but the recent finding of a g subdivision symbiont in bacteriocytes of a different mealybug species (Kantheti, Jayarama, and Sharat Chandra 1996) suggests that the full diversity of pseudococcid endosymbionts is still to be determined. Coccoidea, as a whole, exhibit a rich diversity of symbiotic associations (Buchner 1965). The identity of endosymbionts in psyllids (Psylloidea) has not been investigated with molecular phylogenetic methods until this study. Knowledge of psyllid endosymbionts dates to early light microscopy studies (compiled in Buchner 1965) and later electron microscopy studies (Chang and Musgrave 1969; Waku and Endo 1987). Psyllids contain bilobed bacteriomes composed of round, uninucleate bacteriocytes, a multinucleate syncytial region, and a covering of envelope cells (Buchner 1965; Waku and Endo 1987). Bacteriomes usually house two symbiont types: irregular, rod-shaped
Bacterial Endosymbionts of Psyllids
FIG. 1.—Relationships of Sternorrhyncha and identities of their proteobacterial endosymbionts.
symbionts packed into bacteriocytes, and round or ovalshaped symbionts in the syncytium (Buchner 1965; Chang and Musgrave 1969; Waku and Endo 1987). As in aphids, both symbiont forms are enclosed by three structures: a plasma membrane and cell wall and an outer host-derived membrane (Chang and Musgrave 1969; Waku and Endo 1987). Both symbiont types are transmitted internally from maternal bacteriomes to developing eggs (Buchner 1965; Waku and Endo 1987). In at least two species (family Psyllidae), symbionts are absent from the syncytial region, possibly as a secondary loss (Buchner 1965). A secondary endosymbiont is located in cells of fatty tissue adjacent to the bacteriome in one of these species and is also transmitted to eggs (Buchner 1965). For several insect taxa, phylogenetic reconstructions have revealed a pattern of congruent host and P endosymbiont phylogenies, supporting the idea that extant associations arose from a single infection of the common host ancestor followed by vertical transmission of symbionts and parallel diversification of insect and bacterial lineages (Munson et al. 1991; Moran et al. 1993; Moran and Telang 1998). The ages of several associations are estimated to be 50–250 Myr old (Moran et al. 1993; Moran and Baumann 1994; Aksoy, Pourhosseini, and Chow 1995; Bandi et al. 1995; Schro¨der et al. 1996). Establishing the maximum age of continuous bacteriocyte endosymbioses becomes interesting in light of recent findings that endosymbiotic bacteria may suffer long-term, negative genetic consequences of the association that might ultimately affect host fitness (Moran 1996). Sternorrhyncha is a promising group in which to search for evidence of a more ancient, continuous mutualism. Because most extant sternorrhynchans feed on phloem sap, it is plausible to hypothesize that their common ancestor was phloem-feeding and had endosymbionts to supplement its diet. With a more complete knowledge of extant sternorrhynchan endosymbioses, specifically those of Psylloidea, it may be possible to characterize this ancestral relationship and determine whether a continuous bacteria–host association may have existed since the origin of the Sternorrhyncha, approximately 280 MYA (Szelegiewicz 1971; Hennig 1981; Wootton 1981). For example, although evidence so far suggests that there have been independent origins
1507
of the P endosymbionts in three of the four lineages (aphids, whiteflies, and mealybugs), if psyllid endosymbionts formed the sister group to endosymbionts of any other sternorrhynchan lineage, then that common association might represent the ancestral endosymbiosis. Alternatively, if psyllid endosymbionts make up an independent bacterial lineage, then the ancestral sternorrhynchan endosymbiosis (if it existed) may have been replaced in all lineages. The purpose of this study was thus to identify and characterize psyllid endosymbionts on the basis of 16S rDNA sequences and, through phylogenetic analysis, evaluate their position in the eubacterial tree and their relationships to other known endosymbionts. We also evaluate the extent to which psyllid endosymbionts show evidence of possibly detrimental genetic processes. Materials and Methods Molecular Methods and Sequence Alignment DNA was obtained from three species of psyllids. Pachypsylla venusta (Osten-Sacken) (Psyllidae) extractions were as described in von Dohlen and Moran (1995), and extraction was performed using the proteinsalting-out protocol of Sunnucks and Hale (1996) on bacteriomes dissected from individuals collected in April 1998 in Logan, Utah. Blastopsylla occidentalis Taylor (Psyllidae) was collected in March 1997 in Tucson, Ariz., and Trioza magnoliae (Ashmead) (Triozidae) was collected in March 1995 at Hilton Head Island, S.C. Both species were stored in 100% ethanol, and DNA was extracted from single individuals with the proteinsalting-out method of Sunnucks and Hale (1996). Voucher specimens were deposited in the Utah State University’s Insect Collection. Eubacterial 16S rDNA was amplified by polymerase chain reaction (PCR) with eubacteria-specific primers (from Munson et al. [1991], modified to omit the restriction sites). PCR products were cloned using the vector pCRt 2.1 (Invitrogen, Original TA Cloningt Kit, catalog number K-2000) or pCRt 2.1-TOPO (Invitrogen, catalog number 4500), and transformed into Escherichia coli (INVaF9) cells according to the instructions of the manufacturer. The presence of the 16S insert in E. coli cells, as indicated by blue/white colony selection, was confirmed by restriction digestion of reisolated plasmids with EcoRI or both EcoRI and HindIII. Plasmids were reisolated by the alkaline lysis method (Maniatis, Fritsch, and Sambrook 1982) or with Wizardt Plus SV Minipreps (Promega, catalog number A1330). After electrophoresis in 0.7% agarose, restriction digests were visualized by ethidium bromide staining and exposure to UV light. Samples for which insert fragment lengths summed to approximately 1,500 bp were used for sequencing. Unique clones were identified by sequences and/or electrophoresis patterns of EcoRI-HindIII double digests. Out of six positive clones for T. magnoliae, three clones were identical for each of two distinct sequences. Of six positive clones for B. occidentalis, five were identical for one sequence and one was a distinct
1508
Spaulding and von Dohlen
FIG. 2.—Maximum-likelihood phylogram, showing relative branch lengths, from a heuristic search (15 replicates). Bootstrap percentages (90 replicates) greater than 50% are shown above the branches. Hosts of psyllid endosymbionts are shown in bold, with clone numbers in parentheses. Hosts of other endosymbionts are aphids, Acyrthosiphon pisum (Ap), Schizaphis graminum (Sg), and Schlechtendalia chinensis (Sc); tsetse flies, Glossina morsitans (Gm), Glossina brevipalpis (Gb), and Glossina pallidipes (Gp); ants, Camponotus floridanus (Cf) and Camponotus rufipes (Cr); whiteflies, Siphoninus phillyreae (Sp) and Bemisia tabaci (Bt); the weevil, Sitophilus oryzae (So); and the bedbug, Cimex lectularius (Cl). GenBank accession numbers or RDP short id’s of taxa, from top to bottom, are: AF077605, AF077606, AF077604, Z11927, Z11925, D14555, M59157, Osp.japoni, X92549, X92552, AF077607, L37339, L37341, M63246, Z19056, AF077608, M99060, AF005235, J01695, X80681, U65654, M90801, X07652, Z11926, M27040, X74695, and M22508.
second sequence. Pachypsylla venusta yielded 10 identical positive clones: 1 from a whole individual, and 9 from the bacteriome preparation. Automated sequencing was performed at the Utah State University Biotechnology Center with an ABI 373 Stretch machine. Primers used for sequencing were the two amplification primers and two internal primers, 3411 (59-CCTACGGGAGGCAGCAGTGG-39) and 7661 (59-AAAGCGTGGGGAGCAAACAG-39). Another internal primer, 3981, was substituted for 3411 in sequencing the P. venusta clone (Rouhbaksh et al. 1994). With this combination of primers, approximately 50% of each sequence was derived from overlapping reads. Sequences were deposited in GenBank (see fig. 2 for accession numbers). Complete 16S sequences were submitted to the Ribosomal Database Project (RDP) (Maidak et al. 1997) small-subunit prokaryote database, using the SIMILARITYpRANK command, to obtain similarity scores for comparisons of our putative endosymbiont sequences to other known bacterial 16S sequences. In each case, the most similar sequences were from the g subdivision of Proteobacteria. A set of 43 taxa were selected for preliminary analyses from GenBank to represent the breadth of g subdivision Proteobacteria, including other insect endosymbionts. Several outgroup taxa were also
included, representing the a and b subdivisions of Proteobacteria, and a member of the low G1C gram-positive bacteria. A multiple-sequence alignment was prepared using the PILEUP program in the Wisconsin package (Genetics Computer Group 1994); length-variable and ambiguous regions of the alignment were removed manually. Phylogenetic Analyses Relative apparent synapomorphy analysis (RASA) (Lyons-Weiler, Hoelzer, and Tuesch 1996; Lyons-Weiler and Hoelzer 1997), and preliminary phylogenetic analyses indicated that there were extremely long branches leading to three of the new sequences. Because distance and parsimony methods are less likely to recover the correct tree under these conditions (Felsenstein 1978; Hillis, Huelsenbeck, and Swofford 1994), we chose maximum-likelihood (ML) analysis for the phylogeny estimate. Model selection for analyses using ML was carried out using the method of Sullivan, Markert, and Kilpatrick (1997), in which a series of likelihood ratio tests is performed for a set of various trees under several models. This allowed us to identify the simplest model that had a significantly better fit to the data, and thereby minimized the variance that could be introduced into
Bacterial Endosymbionts of Psyllids
distance estimates when parameters are added (Rzhetsky and Nei 1995). The test statistic, D 5 2((2ln L0) 2 (2ln L1)), is compared to the x2 distribution, where, in comparisons of nested models, 2ln L0 is the likelihood of the simpler model, and 2ln L1 is the likelihood of the model with added parameters. The number of degrees of freedom is equal to the difference in the number of parameters estimated by the models. Despite problems associated with the irregular nature of the tree parameter (Goldman 1993), the distribution of the likelihood ratio statistic appears to provide a good estimate of the x2 distribution of these kinds of tests (Yang, Goldman, and Friday 1995). The trees used in the tests were obtained by several methods: parsimony with transitions upweighted by a factor of 1.1, minimum evolution with LogDet corrected distances and Tamura-Nei (TN) corrected distances, and ML using the Hasegawa-KishinoYano (HKY) model with a transition/transversion ratio of 2.0 and base frequencies set to observed values. Likelihood scores (2ln L) were obtained for five test trees under four variations of five time-reversible models of nucleotide substitution (20 models in all). The five models were Jukes-Cantor, Kimura two-parameter, HKY, TN, and general time-reversible (see model descriptions in Swofford et al. 1996). The model variations involved four different assumptions of among-site rate heterogeneity: (1) substitution rates equal among all sites; (2) some sites invariable (I) and the rest with equal substitution rates; (3) variation in substitution rates among all sites gamma (G) distributed; and (4) some sites invariable, and variation in substitution rates among remaining sites gamma distributed (I1G). Where applicable, the discrete G model (Yang 1994) was used with mean rates from six rate categories. For each tree, and in each test, model parameters were estimated by ML. Additional tests that do not require phylogenetic trees (Rzhetsky and Nei 1995), from a computer program provided by A. Rzhetsky, were employed to confirm the results of some of the likelihood ratio tests. Successive model comparisons determined that, of the models tested, TN1I1G was most appropriate for our data set. A subset of 27 taxa was chosen for ML analysis using this model. In principle, the selected model and its estimated parameters were still valid for this subset (J. Felsenstein, personal communication). The b subdivision bacterium Alcaligenes faecalis was retained as the outgroup. The smaller data set was tested for presence of phylogenetic signal by two methods: comparison of an ML phylogeny estimate (similar to the one in fig. 2) with a star phylogeny using the Kishino-Hasegawa test, and a test for signal content (analytical option) with the program RASA 2.2 (Lyons-Weiler 1997). The above model, along with the ML estimates of its parameters, was used for a heuristic tree search using random addition of taxa followed by tree-bisection-andreconnection (TBR) branch swapping. Relative support of inferred relationships was obtained by bootstrap resampling. Phylogenetic estimations, likelihood ratio tests, and Kishino-Hasegawa tests were performed using the test versions 4.0d61 and 4.0d64 of PAUP*, written by David L. Swofford.
1509
Relative-Rates Test Relative-rates tests (Wu and Li 1985) were performed as in Moran (1996) to test whether putative psyllid endosymbiont 16S rDNA evolved faster than related free-living bacteria. Each test included a cloned sequence, a closely related free-living taxon, and a more distant free-living reference taxon that was also an outgroup. To maximize the power of the test, both freeliving relatives were chosen so that distances from the putative endosymbiont were minimized (Wu and Li 1985). The choice of free-living relatives was guided by the results of the phylogenetic analyses above. The tests we performed were not completely independent, because each included branches that were also included in another test. For each test, three-taxon alignments were performed with PILEUP of the Wisconsin package. Length-variable and ambiguous regions were removed manually before the test. Transition and transversion differences for each test were estimated using the TN1G distance correction of the program MEGA (Kumar, Tamura, and Nei 1993). Results Phylogenetic Analysis The 27-taxon data set was determined to have phylogenetic signal because an ML phylogeny estimate had a significantly better likelihood score (P , 0.0001) than the star phylogeny, and the RASA test for signal content (analytical option) returned a significant tRASA score (P , 0.001). Placement of all new sequences among g subdivision bacteria (fig. 2) was well supported by bootstrap resampling (88% and 93%). Blastopsylla occidentalis (9), P. venusta (6), and T. magnoliae (13) formed a monophyletic group (100% support) with a well-supported (91%) branching order that mirrored a morphological phylogeny of the psyllid hosts (White and Hodkinson 1985). The analysis suggested a sister group relationship between these sequences and the whitefly P endosymbiont, which was supported by 63% of bootstrap replicates. The extremely long branch leading to the B. occidentalis (9) 1 P. venusta (6) 1 T. magnoliae (13) clade reflected the high relative substitution rates and nucleotide bias found in these sequences (see below). Trioza magnoliae (14) and B. occidentalis (6) were placed among the enteric bacteria and relatives, within the g-3 subgroup (75% support). These sequences contained several signature oligonucleotides of the Enterobacteriaceae (Woese et al. 1985). Relative-Rates Tests and Mutational Bias In the relative-rates tests (Wu and Li 1985; Moran 1996), K01/K02 is the ratio that compares the number of substitutions along the branch between a recent common ancestor and the endosymbiont to the number of substitutions along the branch between that same ancestor and a free-living relative (table 1). The tests found significantly higher rates of substitution in the cloned 16S rDNAs than in their free-living relatives for all but B. occidentalis (6). Rates of evolution were highest for B.
1510
Spaulding and von Dohlen
Table 1 Relative-Rates Tests for Putative Psyllid Endosymbionts Compared with Related Free-Living Bacteria Taxon 1a
Taxon 2
Trioza magnoliae (13)
Oceanospirillum japonicum Pachypsylla venusta (6) Zymobacter palmae Blastopsylla occidentalis (9) Z. palmae T. magnoliae (14) P. vulgaris B. occidentalis (6) Salmonella typhimurium
Taxon 3
K12
K13
K23
K13 2 K23 6 SD
z
K01/K02
Proteus vulgaris
0.182
0.187
0.069
0.118 6 0.017
6.77*
4.71
Legionella pneumophila P. vulgaris Vibrio cholerae L. pneumophila
0.296
0.307
0.133
0.174 6 0.022
7.96*
3.88
0.319 0.152 0.077
0.331 0.176 0.187
0.148 0.094 0.183
0.183 6 0.022 0.082 6 0.014 0.004 6 0.014
8.24* 5.73* 0.27
3.72 3.32 1.10
NOTE.—Kij is the estimated number of substitutions per site between taxon i and taxon j. Taxon 1 is the endosymbiont, taxon 2 is a closely related free-living bacterium, taxon 0 is the most recent common ancestor to taxon 1 and taxon 2, and taxon 3 is a more distantly related reference bacterium. The estimated numbers of substitutions from the common ancestor to the endosymbiont and from the ancestor to the free-living relative, respectively, are K01 5 (K13 2 K23 1 K12)/2 and K02 5 K12 2 K01. For these one-tailed tests, H0: K01 # K02, H1: K01 . K02, and a 5 0.05. a The host of the putative endosymbiont is listed; the clone number is indicated in parentheses. * P , 0.001.
occidentalis (9), P. venusta (6), and T. magnoliae (13), ranging from 3.7- to 4.7-fold faster than free-living relatives. The substitution rate of T. magnoliae (14) was lower, but still 3.3-fold faster than that of the free-living relative. In comparisons of base frequencies of the new sequences with those of known insect endosymbionts and selected free-living taxa, the percentage of A1T for B. occidentalis (6) was similar to that found in free-living bacteria (;45%). A1T content for T. magnoliae (14) (57%) was higher than those for Buchnera and the tsetse fly P endosymbiont (both ;50%), and percentages of A1T for B. occidentalis (9), P. venusta (6), and T. magnoliae (13) averaged a striking 65%. This mutational bias was further evaluated in pairwise comparisons (Moran 1996) of new clones with a close free-living relative. Approximately 80% of all pairwise differences between B. occidentalis (9), P. venusta (6), T. magnoliae (13), and T. magnoliae (14) and a corresponding freeliving taxon were categorized as increased A1T for the new sequences (table 2). Differences for B. occidentalis (6) were more evenly distributed among categories. Several long stretches of A1T-rich insertions had to be removed from B. occidentalis (9), P. venusta (6), and T. magnoliae (13) sequences for the multiple alignment to ensure homology of all sites. If these stretches had been
included, the number of changes toward increased A1T in these taxa would have been even greater. Discussion The small but growing literature concerning the molecular genetics of insect endosymbionts suggests that several independent lineages of intracellular bacteria have followed similar paths of evolution (Moran and Telang 1998). First, the monophyletic statuses of symbionts within bacterial phylogeny, mostly coupled with branching patterns that are congruent with either host phylogeny or taxonomy, imply that such associations arose once in the ancestor of the host taxon, with subsequent cospeciation of host and endosymbiont lineages (Munson et al. 1991; Clark et al. 1992; Munson, Baumann, and Moran 1992; Moran and Baumann 1994; Aksoy, Pourhosseini, and Chow 1995; Bandi et al. 1995; Schro¨der et al. 1996). Second, once acquired and adapted to intracellular life, endosymbionts may be subject to detrimental genetic processes. Patterns observed for endosymbionts of aphids, whiteflies, mealybugs, tsetse flies, and Wolbachia show that rates of DNA substitutions have been accelerated in these lineages in comparison to free-living relatives (Moran 1996). Furthermore, some process has led to a mutational bias in sev-
Table 2 Numbers of Pairwise Differences in 16S rDNA Sequences of Putative Psyllid Endsymbionts and Related Free-Living Bacteria DIFFERENCES WITH ENDOSYMBIONT SHOWING:b PSYLLID ENDOSYMBIONTa Blastopsylla occidentalis (9) . . . . . . Pachypsylla venusta (6) . . . . . . . . . . Trioza magnoliae (13) . . . . . . . . . . . T. magnoliae (14) . . . . . . . . . . . . . . . B. occidentalis (6) . . . . . . . . . . . . . . a
RELATED BACTERIUM Zymobacter palmae Z. palmae Z. palmae Escherichia coli E. coli
More A1T
Less A1T
Same A1T
TOTAL DIFFERENCES
MORE A 1 T/TOTAL
183 190 176 109 20
17 21 17 14 18
21 20 25 11 13
221 231 218 134 51
0.83 0.82 0.81 0.81 0.39
The host of the psyllid endosymbiont is listed, with the clone number in parentheses. Nucleotide site differences between the endosymbiont and the free-living relative were placed in one of three classes: more A 1 T 5 the endosymbiont had an A or T, but the free-living relative had a G or C; less A 1 T 5 the endosymbiont had a G or C, but the free-living relative had an A or T; same A 1 T 5 differences between A and T, or G and C. b
Bacterial Endosymbionts of Psyllids
eral endosymbionts, favoring accumulation of A and T nucleotides (Moran 1996). The A1T bias in endosymbiont ribosomal genes lowers stability of rRNA secondary structure (Lambert and Moran 1998). In protein-coding genes of Buchnera, accelerated evolution and mutational bias have combined to elevate rates of amino acid replacements, which might affect enzyme function (Moran 1996). The speedup in endosymbiont lineages is interpreted to reflect accumulation of slightly deleterious mutations as a consequence of small, clonal populations and enforced population bottlenecks at each host generation. Endosymbionts presumably have survived the long-term effects of increased genetic drift due to compensatory processes in the association (Moran 1996). The 16S rDNA clones P. venusta (6), B. occidentalis (9), and T. magnoliae (13) exhibit the attributes documented above for insect P endosymbionts. They form a highly supported monophyletic group with a branching order that parallels host taxonomic designations and a morphologically based phylogeny (White and Hodkinson 1985). They collectively exhibit a highly accelerated substitution rate, averaging fourfold faster than those of related free-living taxa and more than twice the value found for other insect endosymbionts (Moran 1996). They are more A1T-rich and show even more pronounced A1T mutational bias than other insect endosymbionts. In addition, P. venusta (6) was identical to nine clones amplified from a bacteriome DNA preparation, indicating that it originated from the bacteriome. These combined results strongly suggest that the clones are derived from the bacteriocyte-dwelling psyllid P endosymbionts (Buchner 1965). This may be confirmed with in situ hybridization studies in future work. Results also suggest that the remaining two clones, B. occidentalis (6) and T. magnoliae (14), were amplified from the S endosymbionts noted by Buchner (1965). These two clones did not form a monophyletic group in the analysis. This is consistent with the idea that S endosymbionts represent younger associations (Buchner 1965) that have been formed independently in different host taxa after the radiation of major host lineages. These clones were placed with strong support in a clade of g-3 Proteobacteria with several other insect endosymbionts, including weevil and tsetse fly S endosymbionts. Trioza magnoliae (14) has a much higher mutational bias and substitution rate than does B. occidentalis (6). Because we expect mutational bias and rate acceleration within a given host lineage to reflect the age of the symbiosis, T. magnoliae (14) may indicate an older S endosymbiont association in Trioza. Alternatively, a possibility exists that T. magnoliae has harbored two long-term bacteriocyte endosymbionts of different lineages. For whiteflies, electron microscopy studies have detected two distinct bacterial forms in bacteriocytes (Costa et al. 1993, 1995), but the different morphs have not yet been linked to 16S sequences amplified from these species (Clark et al. 1992). Our results so far indicate that two species, T. magnoliae and B. occidentalis, harbor both P and S endosymbionts. In contrast, the molecular work and prelim-
1511
inary results from transmission electron microscopy (unpublished data) have detected only one form of bacterium in P. venusta. Pachypsylla venusta may be a case where S endosymbionts either are located in cells of fatty tissue next to the bacteriome or are absent (Buchner 1965). The phylogenetic position of psyllid P endosymbionts has implications for the history of endosymbiosis in Sternorrhyncha. Our analysis supported a sister-group relationship between whitefly and psyllid P endosymbionts. Thus, these endosymbionts may have shared a most recent common ancestor that infected the common ancestor of their hosts. The position (and age) of this host ancestor in sternorrhynchan phylogeny depends on the relationship between psyllid and whitefly lineages. Most morphologically based phylogenies recognize synapomorphies defining a sister group relationship between psyllids and whiteflies (Goodchild 1966; Szelegiewicz 1971; Hennig 1981; Carver, Gross, and Woodward 1991). Fossils purported to belong to the stem group of psyllids and whiteflies have been dated to 240 MYA (Hennig 1981). Recent molecular studies of sternorrhynchan phylogeny using 18S rDNA, however, could not resolve the position of whiteflies with confidence (Campbell et al. 1995; von Dohlen and Moran 1995). As in psyllid P endosymbionts, whitefly rRNA genes have accelerated substitution rates, and their long collective branch is problematic in phylogenetic reconstruction. The molecular data suggest that psyllids and whiteflies are not sister taxa, but have branched independently (von Dohlen and Moran 1995). If this is so, then the most recent common ancestor of psyllids and whiteflies is also the ancestor of all Sternorrhyncha. Under this scenario, our results would support the hypothesis that an endosymbiosis was established in the common sternorrhynchan ancestor and was maintained in psyllid and whitefly lineages but replaced by other Proteobacteria in aphid and coccid lineages. Fossils purported to be the common ancestor of Sternorrhyncha date to the lower Permian (;280 MYA) (Szelegiewicz 1971; Hennig 1981; Wootton 1981). A continuous bacterial endosymbiosis of this age would be older than any so far proposed for other insects. Comparisons of observed and predicted divergences between psyllid and whitefly P endosymbionts lend support to the latter hypothesis above. Given the phylogeny presented here, divergence between these endosymbionts departs from predicted values based on the estimated universal rate for free-living eubacteria (about 1%/50 MYA [Ochman and Wilson 1987]) in a manner expected by their rate acceleration. Using the previously mentioned competing dates for the common ancestor of psyllids and whiteflies, the universal rate predicts the divergence to be (240/50)(2) 5 9.6% if the hosts are sister taxa, and (280/50)(2) 5 11.2% in the case of independent branching of the hosts. The average rate acceleration for the psyllid P endosymbionts is 4.10 (table 1), and the average acceleration for whitefly P endosymbionts is 1.75 (Moran 1996). Using these to adjust the universal rate for each lineage, the estimated divergences become (9.6/2)(4.10 1 1.75) 5 28% and (11.2/
1512
Spaulding and von Dohlen
2)(4.10 1 1.75) 5 33%, respectively. The ML corrected (TN1I1G) divergence between the sequences is 35%. The proximity of this result to the latter value favors the hypothesis that psyllid and whitefly endosymbionts have been diverging for 280 Myr and thus date back to the origin of Sternorrhyncha. It is also consistent with the idea that hosts have branched independently from a common sternorrhynchan ancestor. The genetic consequences of long-term endosymbiosis appear to be considerably exaggerated in some psyllid endosymbionts. Because these processes are hypothesized to be related to population structure (Moran 1996), we speculate that these endosymbionts might have a different population structure than other insect endosymbionts; for example, they may experience tighter bottlenecks in each host generation or smaller host population sizes. Further research could reveal whether evolutionary rate increases and base compositional biases extend to other, protein-coding, genes and whether compensatory mechanisms exist. P endosymbionts of both aphids and tsetse flies produce exceptionally high amounts of the stress protein GroEL, a chaperonin (Ishikawa 1989; Aksoy 1995). Overproduction of GroEL is hypothesized to be a compensatory mechanism in which the chaperonin assists enzymes in maintaining functional conformation in the face of accumulating amino acid substitutions (Moran 1996). We predict that these rate-accelerated psyllid endosymbionts should exhibit similar, or higher, levels of GroEL expression. Psyllid endosymbionts further illustrate the idea that, once sequestered as intracellular mutualists, bacteria may experience long-term genetic effects that could ultimately reduce host fitness (Moran 1996; Lambert and Moran 1998). Perhaps the acquisition of S endosymbionts is a mechanism through which primary endosymbionts might ultimately be replaced (Moran and Baumann 1994), if S endosymbionts gradually take over the functions of P endosymbionts as they become less efficient due to mutational accumulation. Acknowledgments We thank N. Moran, P. Wolf, N. Youssef, and two anonymous reviewers for comments that improved the manuscript, and N. Moran for collecting Blastopsylla. J. Felsenstein and A. Rzhetsky kindly provided advice and/or computer programs for analysis. J. Hanks assisted with computer support. We are grateful to D. Swofford for making available the test version of PAUP*, without which our analyses would have been exceedingly difficult to implement. D. Hollis generously provided speedy identification of psyllid species. This project was funded by a New Faculty Research Grant from Utah State University and the Utah Agricultural Experiment Station; approved as journal paper 7044. LITERATURE CITED
AKSOY, S. 1995. Molecular analysis of the endosymbionts of tsetse flies: 16S rDNA locus and over-expression of a chaperonin. Insect Mol. Biol. 4:23–29.
AKSOY, S., A. A. POURHOSSEINI, and A. CHOW. 1995. Mycetome endosymbionts of tsetse flies constitute a distinct lineage related to Enterobacteriaceae. Insect Mol. Biol. 4:15– 22. BANDI, C., M. SIRONI, G. DAMIANI, L. MAGRASSI, C. A. NALEPA, U. LAUDANI, and L. SACCHI. 1995. The establishment of intracellular symbiosis in an ancestor of cockroaches and termites. Proc. R. Soc. Lond. B Biol. Sci. 259:293–299. BAUMANN, P., L. BAUMANN, C.-Y. LAI, D. ROUHBAKHSH, N. A. MORAN, and M. A. CLARK. 1995. Genetics, physiology, and evolutionary relationships of the genus Buchnera: intracellular symbionts of aphids. Annu. Rev. Microbiol. 49: 55–94. BUCHNER, P. 1965. Endosymbiosis of animals with plant microorganisms. John Wiley and Sons, New York. CAMPBELL, B. C., J. D. STEFFEN-CAMPBELL, J. T. SORSENSEN, and R. J. GILL. 1995. Paraphyly of Homoptera and Auchenorrhyncha inferred from 18S rDNA nucleotide sequences. Syst. Entomol. 20:175–194. CARVER, M., G. F. GROSS, and T. E. WOODWARD. 1991. Hemiptera (bugs, leafhoppers, cicadas, aphids, scale insects etc.). Pp. 429–509 in I. D. NAUMANN, P. B. CARNE, J. F. LAWRENCE, E. S. NIELSEN, J. P. SPRADBERY, R. W. TAYLOR, M. J. WHITTEN, and M. J. LITTLEJOHN, eds. The insects of Australia. A textbook for students and research workers. Melbourne University Press, Carlton, Australia. CHANG, K. P., and A. J. MUSGRAVE. 1969. Histochemistry and ultrastructure of the mycetome and its ‘symbiotes’ in the pear psylla, Psylla pyricola Foerster. Tissue Cell 1:597–606. CHEN, D. Q., and A. H. PURCELL. 1997. Occurrence and transmission of facultative endosymbionts in aphids. Curr. Microbiol. 34:220–225. CLARK, M. A., L. BAUMANN, M. A. MUNSON, P. BAUMANN, B. C. CAMPBELL, J. E. DUFFUS, L. S. OSBORNE, and A. N. MORAN. 1992. The eubacterial endosymbionts of whiteflies (Homoptera: Aleyrodoidea) constitute a lineage distinct from the endosymbionts of aphids and mealybugs. Curr. Microbiol. 25:119–123. COSTA, H. S., D. M. WESTCOT, D. E. ULLMAN, and M. W. JOHNSON. 1993. Ultrastructure of the endosymbionts of the whitefly, Bemisia tabaci and Trialeurodes vaporariorum. Protoplasma 176:106–115. COSTA, H. S., D. M. WESTCOT, D. E. ULLMAN, R. ROSELL, J. K. BROWN, and M. W. JOHNSON. 1995. Morphological variation in Bemisia endosymbionts. Protoplasma 189:194–202. DADD, R. H. 1985. Nutrition: organisms. Pp. 313–319 in G. A. KERKUTAND and L. I. GILBERT, eds. Comprehensive insect physiology, biochemistry, and pharmacology. Pergamon Press, Oxford. DOUGLAS, A. E. 1989. Mycetocyte symbiosis in insects. Biol. Rev. 64:409–434. . 1998. Nutritional interactions in insect-microbial symbioses: aphids and their symbiotic bacteria Buchnera. Annu. Rev. Entomol. 43:17–37. FELSENSTEIN, J. 1978. Cases in which parsimony or compatibility methods will be positively misleading. Syst. Zool. 27: 401–410. GENETICS COMPUTER GROUP. 1994. Program manual for the Wisconsin Package. Madison, Wis. GOLDMAN, N. 1993. Statistical tests of models of DNA substitution. J. Mol. Evol. 36:182–198. GOODCHILD, A. J. P. 1966. Evolution of the alimentary canal in the Hemiptera. Biol. Rev. 41:97–140. HENNIG, W. 1981. Insect phylogeny. Wiley, New York. HILLIS, D. M., J. P. HUELSENBECK, and D. L. SWOFFORD. 1994. Hobgoblin of phylogenetics? Nature 369:363–364.
Bacterial Endosymbionts of Psyllids
ISHIKAWA, H. 1989. Biochemical and molecular aspects of endosymbiosis in insects. Int. Rev. Cytol. 116:1–45. KANTHETI, P., K. S. JAYARAMA, and H. SHARAT CHANDRA. 1996. Developmental analysis of a female-specific 16S rRNA gene from mycetome-associated endosymbionts of a mealybug. Planococcus lilacinus. Insect Biochem. Mol. Biol. 26:997–1009. KUMAR, S., K. TAMURA, and M. NEI. 1993. MEGA: molecular evolutionary genetics analysis. Version 1.0. Pennsylvania State University, University Park. LAMBERT, J. D., and N. A. MORAN. 1998. Deleterious mutations destabilize ribosomal RNA in endosymbiotic bacteria. Proc. Natl. Acad. Sci. USA 95:4458–4462. LYONS-WEILER, J. 1997. RASA 2.1 software and manual for the Macintosh. http://loco.biology.unr.edu/archives/rasa/ rasa.html. LYONS-WEILER, J., and G. A. HOELZER. 1997. Escaping from the Felsenstein zone by detecting long branches in phylogenetic data. Mol. Phylogenet. Evol. 8:375–384. LYONS-WEILER, J., G. A. HOELZER, and R. J. TUESCH. 1996. Relative apparent synapomorphy analysis (I): the statistical measurement of phylogenetic signal. Mol. Biol. Evol. 13: 749–757. MCLEAN, D. L., and E. J. HOUK. 1973. Phase contrast and electron microscopy of the mycetocytes and symbiotes of the pea aphid, Acyrthosiphon pisum. J. Insect Physiol. 19: 625–633. MAIDAK, B. L., G. J. OLSEN, N. LARSEN, R. OVERBEEK, M. J. MCCAUGHEY, and C. R. WOESE. 1997. The RDP (ribosomal database project). Nucleic Acids Res. 25:109–111. MANIATIS, T., E. F. FRITSCH, and J. SAMBROOK. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Publications, Cold Spring Harbor, N.Y. MORAN, N. A. 1996. Accelerated evolution and Muller’s rachet in endosymbiotic bacteria. Proc. Natl. Acad. Sci. USA 93: 2873–2878. MORAN, N., and P. BAUMANN. 1994. Phylogenetics of cytoplasmically inherited microorganisms of arthropods. Trends Ecol. Evol. 9:15–20. MORAN, N. A., M. A. MUNSON, P. BAUMANN, and H. ISHIKAWA. 1993. A molecular clock in endosymbiotic bacteria is calibrated using the insect hosts. Proc. R. Soc. Lond. B Biol. Sci. 253: 167–171. MORAN, N. A., and A. TELANG. 1998. The evolution of bacteriocyte-associated endosymbionts in insects. BioScience 48:295–304. MUNSON, M. A., P. BAUMANN, M. A. CLARK, L. BAUMANN, N. A. MORAN, D. J. VOEGTLIN, and B. C. CAMPBELL. 1991. Evidence for the establishment of aphid-eubacterium endosymbiosis in an ancestor of four aphid families. J. Bacteriol. 173:6321–6324. MUNSON, M. A., P. BAUMANN, and N. A. MORAN. 1992. Phylogenetic relationships of the endosymbionts of mealybugs (Homoptera: Pseudococcidae) based on 16S rDNA sequences. Mol. Phylogenet. Evol. 1:26–30. OCHMAN, H., and A. C. WILSON. 1987. Evolution in bacteria: evidence for a universal substitution rate in cellular genomes. J. Mol. Evol. 26:74–86.
1513
ROUHBAKSH, D., N. A. MORAN, L. BAUMANN, D. J. VOEGTLIN, and P. BAUMANN. 1994. Detection of Buchnera, the primary prokaryotic endosymbiont of aphids, using the polymerase chain reaction. Insect Mol. Biol. 3:213–217. RZHETSKY, A., and M. NEI. 1995. Tests of applicability of several substitution models for DNA sequence data. Mol. Biol. Evol. 12:131–151. SCHRO¨DER, D., H. DEPPISCH, M. OBERMAYER, G. KROHNE, E. STACKEBRANDT, B. HOLLDOBLER, W. GOEBEL, and R. GROSS. 1996. Intracellular endosymbiotic bacteria of Camponotus species (carpenter ants): systematics, evolution and ultrastructural characterization. Mol. Microbiol. 21:479– 489. SULLIVAN, J., J. A. MARKERT, and C. W. KILPATRICK. 1997. Phylogeography and molecular systematics of the Peromyscus aztecus species group (Rodentia: Muridae) inferred using parsimony and likelihood. Syst. Biol. 46:426–440. SUNNUCKS, P., and D. F. HALE. 1996. Numerous transposed sequences of mitochondrial cytochrome oxidase I–II in aphids of the genus Sitobion (Hemiptera: Aphididae). Mol. Biol. Evol. 13:510–524. SWOFFORD, D. L., G. J. OLSEN, P. J. WADDELL, and D. M. HILLIS. 1996. Phylogenetic inference. Pp. 407–514 in D. M. HILLIS, C. MORITZ, and B. K. MABLE, eds. Molecular systematics. Sinnauer, Sunderland, Mass. SZELEGIEWICZ, H. 1971. Autapomorphous wing characters in the recent subgroups of Sternorrhyncha (Hemiptera) and their significance in the interpretation of the Paleozoic members of the group. Ann. Zool. 29:1–67. UNTERMAN, B. M., P. BAUMANN, and D. L. MCLEAN. 1989. Pea aphid symbiont relationships established by analysis of 16S rRNAs. J. Bacteriol. 171:2970–2974. VON DOHLEN, C. D., and N. A. MORAN. 1995. Molecular phylogeny of the Homoptera: a paraphyletic taxon. J. Mol. Evol. 41:211–223. WAKU, Y., and Y. ENDO. 1987. Ultrastructure and life cycle of the symbionts in a homopteran insect, Anomoneura mori Schwartz (Psyllidae). Appl. Entomol. Zool. 22:630–637. WHITE, I. M., and I. D. HODKINSON. 1985. Nymphal taxonomy and systematics of the Psylloidea (Homoptera). Bull. Br. Mus. Nat. Hist. 50:153–301. WOESE, C. R., W. G. WEISBURG, C. M. HAHN, B. J. PASTER, L. B. ZABLEN, B. J. LEWIS, T. J. MACKE, W. LUDWIG, and E. STACKEBRANDT. 1985. The phylogeny of purple bacteria: the gamma subdivision. Syst. Appl. Microbiol. 6:25–33. WOOTTON, R. J. 1981. Palaeozoic insects. Annu. Rev. Entomol. 26:319–344. WU, C.-I., and W.-H. LI. 1985. Evidence for higher rates of nucleotide substitution in rodents than in man. Proc. Natl. Acad. Sci. USA 82:1741–1745. YANG, Z. 1994. Maximum likelihood phylogenetic estimation from DNA sequences with variable rates over sites: approximate methods. J. Mol. Evol. 39:306–314. YANG, Z., N. GOLDMAN, and A. FRIDAY. 1995. Maximum likelihood trees from DNA sequences: a peculiar statistical estimation problem. Syst. Biol. 44:384–399.
HOWARD OCHMAN, reviewing editor Accepted July 20, 1998
