Redalyc.Molecular Evolution of Aphids and Their Primary (Buchnera SP

Interciencia ISSN: 0378-1844 [email protected] Asociación Interciencia Venezuela

Sabater, Beatriz; Van Ham, Roeland C. H. J.; Martínez, David; Silva, Francisco; Latorre, Amparo; Moya, Andrés Molecular evolution of aphids and their primary (buchnera SP.) and secondary endosymbionts: implications for the role of symbiosis in insect evolution Interciencia, vol. 26, núm. 10, octubre, 2001, pp. 508-512 Asociación Interciencia Caracas, Venezuela

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How to cite Complete issue Scientific Information System More information about this article Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Journal's homepage in redalyc.org Non-profit academic project, developed under the open access initiative MOLECULAR EVOLUTION OF APHIDS AND THEIR PRIMARY (BUCHNERA SP.) AND SECONDARY ENDOSYMBIONTS: IMPLICATIONS FOR THE ROLE OF SYMBIOSIS IN INSECT EVOLUTION BEATRIZ SABATER, ROELAND C.H.J. VAN HAM, DAVID MARTÍNEZ-TORRES, FRANCISCO SILVA, AMPARO LATORRE and ANDRÉS MOYA

he term symbiosis (from One special case of sym- With respect to the role the Greek simbios or liv- biosis is endosymbiosis, where the prokary- played by symbiosis in evolution, there are ing together) was first ote is literally sequestered within an eu- two prevailing views. The first acknowl- introduced by Anton de Bary in 1879. karyotic cell. The paradigm of endosym- edges that symbiosis is an important com- This author explicitly included parasitism biosis is the origin of the eukaryotic cell, ponent in the biology of many organisms, as a type of symbiosis and excluded in which different bacteria successively but yet does not consider it to be a major short-term associations. Some authors do entered a protoeukaryotic cell giving rise evolutionary force per se, because many not accept Bary’s definition and hold that to mitochondria first, and chloroplasts mutualistic associations have limited short-term interactions should be consid- later (Margulis, 1970; Fisher, 1989; Gray, chances of promoting significant evolu- ered true symbiosis and parasitic associa- 1989). Insects are particularly receptive to tionary innovation (Maynard Smith, 1989). tions must be rejected as such. Most re- endosymbiotic processes. It has been esti- Margulis (1991) holds a completely oppo- searchers consider, in a more restrictive mated than more than 10% of the extant site view and broadly argues against the meaning, that there is symbiosis only insect species harbor endosymbionts at Neodarwinian theory as an incomplete the- when both partners benefit from the different stages of accommodation to their ory of evolution. Her argument is based assocation. Douglas (1994) narrows the host. Some of them are recent and casual, on the scarcity of cases of observed spe- term even further and confines symbiosis but others are well established associations ciation and she maintains that the major to associations in which at least one part- that are as relevant to the insect and its taxonomic groups (i.e. phyla, kingdoms) ner bestows the other with some sort of physiology as true organelles are for an have evolved through engagements in novel metabolic capability. In most of the eukaryotic cell (Buchner, 1965). symbiotic associations. According to cases one member of the association is Empirical research on Charlesworth (1991) Margulis’ view is an eukaryote. Contrary to most prokary- symbiosis, specially at the molecular level, rather radical. Although it is possible to otes, eukaryotes have rather limited meta- is a growing and promising field. The im- admit evolutionary accommodations after a bolic capabilities and hence, symbiosis pact of symbiosis on active speciation symbiotic event between two species, has provided and continues to provide an processes, on the genomic transformations there is not an a priori reason to discard evolutionary strategy through which eu- of both partners, and on the evolutionary compatibility between this fact and the karyotes have access to a wider range of success of many taxonomic lineages is be- presence of natural selection as a driving metabolic resources. ginning to be unveiled. force promoting the evolution of the sym-

KEYWORDS / Aphids / Buchnera sp. / Secondary Endosymbionts / Phylogenetic Analysis / Genome Reduction / Recibido: 26/03/2001: Aceptado: 20/09/2001

Beatriz Sabater Muñoz. Bachelor in Biology. University of Valencia, Spain. e-mail: [email protected]. Roeland C.H.J. van Ham. Ph.D. in Biology, University of Utrecht, The Netherlands. Postdoctoral researcher, Centro de Astrobiología (INTA-CSIC), Spain. e-mail: [email protected] David Martínez Torres. Ph.D. in Biology. Assistant Professor, University of Valencia, Spain. e-mail: [email protected] Francisco Silva Moreno. Ph.D. in Biology. Associate Professor, University of Valencia, Spain. e-mail: [email protected] Amparo Latorre Castillo. Ph.D. in Biology. Associate Professor, University of Valencia, Spain. e-mail: [email protected] Andrés Moya Simarro. Ph.D. in Biology, Ph.D. in Philosophy and Full Professor, University of Valencia, Spain. Address: Instituto Cavanilles de Biodiversidad y Biología Evolutiva, Departamento de Genética, Universidad de Va- lencia, Spain, Apartado de Correos 2085, 46071, Valencia, Spain. e-mail: andré[email protected]

508 0378-1844/01/10/508-05 $ 3.00/0 OCT 2001, VOL. 26 Nº 10 biotic association. Moreover, the relevance TABLE I of symbiotic events determining the ap- SUMMARY OF THE MAJOR METABOLIC CAPABILITIES ACQUIRED BY THE pearance of major evolutionary lineages or HOST AS A BY PRODUCT OF THE SYMBIOTIC ASSOCIATION** more precisely, the role played by symbiosis in traits that define higher taxa, should Metabolic novelty Symbiont Host be evaluated. Photosynthesis Algae and Cyanobacteria Several protists, invertebrates, lichenized fungi Endosymbiosis: A Classification Chimiosynthesis Gamma-proteobacteria Several invertebrates

In most, if not all, endo- N2 fixation Rhizobium, Frankia Several plants symbiotic associations one of the two part- and cyanobacteria ners is eukaryotic (Table I). Although they Methanogenesis Methanogenic bacteria Some protists display great diversity and morphological Celulases Bacteria, ciliated protists Herbivore vertebrates complexity, eukaryotes show limited meta- and phycomicetes fungi and termites bolic capabilities, are rarely anaerobic, Luminiscence Vibrio and Photobacterium Teleosts, cephalopds, lack photosynthesis (except for plants and some protists), cannot fix nitrogen, and Synthesis of Bacteria Some insects, protists some groups have lost the ability to syn- essential nutrients thesize essential amino-acids and coen- **Modified from Douglas, 1994. zymes necessary for their basic metabo- lism. Some eukaryotes solve these limita- “telescopic generations” is often observed, Comparative Phylogenetics tions by means of a symbiotic association in which embryogenesis starts in yet un- of Buchnera sp. and Aphids with another eukaryotic or prokaryotic or- born daughters, resulting in the enclosure ganism. The examples shown in Table I of up to three generations developing Analysis of Buchnera should be considered as recent endosymbi- within an adult individual. These high re- spp. 16S rDNA sequences from different otic events compared with respiration and productive rates make of aphids an impor- aphid species supported their monophyly photosynthesis carried out by eukaryotic tant agricultural pest, promoting in their within the γ3 subdivision of the class cells, where mitochondria and chloroplasts hosts the loss of nutrients, sheet ruffling, proteobacteria, having Escherichia coli and are endosymbionts derived from ancestral galls, and other forms of structural dam- related members of the enterobacteriaceae free living proteo- and cyanobacteria, re- age. In addition, they are vectors of im- as their closest known relatives (Moran et spectively. portant plant viral diseases. al., 1993). In addition, the 16S rDNA The phloem which aphids based phylogeny of representatives of Aphids and their Endosymbionts feed upon is rich in sugars but poor in ni- Buchnera from four aphid families showed trogen compounds. In particular, the diet is complete concordance with the morphol- Within insects, the Ho- deficient in essential amino acids, vitamins ogy-based phylogeny of their aphid hosts moptera are a highly specious group, with and several lipids that are also essential (Moran and Baumann, 1994). These re- more than 30000 extant species (Remau- (Dixon, 1975; Houk and Griffiths, 1980; sults pointed to a single original infection dière and Remaudière, 1997). The most Raven, 1983; Sasaki et al., 1991; Douglas, in a common ancestor of all modern Aphi- relevant superfamilies within this order are 1993). doidea ocurred aproximately 100-250 MY Phylloseroidea, Aphidoidea, Psylloidea, Species of the genus followed by co-speciation of aphids and Aleurodoidea, and Coccoidea. Buchnera are the primary endosymbionts Buchnera (Moran et al., 1993). With more than 4400 ex- of aphid species and they are required for Several molecular phylo- tant species, the superfamily Aphidoidea is its hosts’ normal development and fecun- genetic studies using different genes from one of the most diversified and widely dity. These bacteria are located in spe- Buchnera have also shown a congruence distributed groups of Homoptera (Black- cialized, polyploid cells, called bacterio- with the morphology-based phylogeny of man and Eastop, 1994). Representatives cytes that form an organ (bacteryome) lo- aphids (Brynnel et al., 1998; Baumann et occur from Ecuador to the Polar Circle, calized in the hemocoel above the diges- al., 1999; van Ham et al., 1999, 2000). with the Temperate Zones showing the tive tube. They are maternally inherited However, in most of these phylogenetic highest diversity. Their evolutionary suc- by means of infection of the embryos studies taxonomic sampling was limited to cess might be attributed to their ability to during the blastoderm stage (Hinde, representatives of the family Aphididae. suck phloem from plants or from the ac- 1971). The symbiosis is an example of Indeed, van Ham et al. (1997) proposed a quisition of bacterial endosymbionts. Both obligatory mutualism, in the sense that topology for the phylogenetic relationship facts seem to be related because the phlo- the aphid cannot survive without Buch- among 16S rDNA sequences of Buchnera em diet, poor in essential amino acids is nera and Buchnera cannot survive outside from some aphid families which might compensated by essential nutrients sup- the aphid. For example, aposymbiotic question either the traditionally accepted plied by endosymbionts (Buchner, 1965; aphids (aphids treated with antibiotics) phylogeny of aphids or the perfect paral- Douglas, 1989; Fukatsu et al., 1994). have a slow growth rate and have no off- lelism between aphids and their primary Aphid populations are enormous in size, spring, and Buchnera spp. cannot be cul- endosymbiont phylogenies (see Figure 1). with densities reaching up to 5x105 aphids/ tured in artificial media (revised in Bau- In fact, since most of the molecular phylo- m2. This reproductive potential is due pri- man et al., 2000). Several experiments genetic reconstructions done with Buch- marily to their exceptional mode of repro- have led to the conclusion that Buchnera nera were in good agreement with the ac- duction. Beside the sometimes complex al- species are the main source of essential cepted phylogeny of their host (Heie, ternation of sexual and non-sexual phases, amino acids for aphids, and responsible 1980; 1987), the latter was never ques- many species of aphids pass through a in part of the high growth rate of their tioned before the report by van Ham et al. cycle of viviparous, parthenogenetic repro- hosts (Prosser et al., 1992; Sasaki et al., (1997). Moran et al. (1995) compared the duction. In this cycle, the phenomenon of 1991; Liadouze et al., 1995). evolutionary rates in bacteria and their

OCT 2001, VOL. 26 Nº 10 509 host, but the study was restricted to three aphid families. More recently, von Dohlen and Moran (2001) have used mitocondrial ribosomal DNA sequences to reconstruct the phylogeny of aphids. They found, in general, good correlation with morphological data at the level of tribes. Finally, Martínez-Torres et al. (2001) have carried out a large study using genes from either Buchnera and aphid species representative from five aphid families, including Lach- nidae. Although the results of Martínez- Torres et al. (2001) do not define completely evolutionary relationships among aphid families, some traditionally accepted groupings are questioned from both bacterial and insect data. In particular the Lach- nidae and the Aphididae, which on the ba- Figure 1. Phylogenetic trees based on Buchnera 16S rDNA gene (van Ham et al., 1997) sis of morphological data are considered and morphological traits and geological data records from aphids (Heie, 1980; 1987). to be sister groups, do not seem to be as closely related as expected. plasmid was described (Bracho et al., Tetraneura caerulescen (pLeu-BTc) en- Molecular changes experienced 1995) in Buchnera from Rhopalosiphum codes only one repA copy plus ORF1, by primary (Buchnera sp.) padi (pLeu-BRp) a member of the probably representing the ancestral replicon, and secondary endosymbionts Aphidinae subfamily. It contains the genes related to the IncFII plasmids in which the leuABCD, for the biosynthesis of leucine, other genes were relocated (van Ham et al. Buchnera is present in all two copies of repA and ORF1, a putative 1997) (see Figure 2). aphid species studied to date, with the ex- integral membrane protein. The same gene The complete genome of ception of some species from the Hor- content and in the same order was found in Buchnera from Acyrthosiphon pisum has maphididae family (Baumann et al., 1995). other species from the same subfamily shown the presence of a set of biosyn- Molecular and genetic studies have shown (Bauman et al., 1999). However, a different thetic genes that are absent in intracellular that Buchnera overexpresses the essential gene arrangement has been found in pLeu- pathogenic bacteria, suggesting that most amino acids tryptophan and leucine by BPp, the plasmid of Pterocomma popu- of these genes may be involved in the pro- means of gene amplification into plasmids leum, a species belonging to Pterocomma- vision of small molecules to the host (Lai et al., 1994; Bracho et al., 1995; tini, the most divergent tribe of the Aphid- (Shigenobu et al., 2000). This has been Bauman et al., 1997; 1999; van Ham et al., idae family (Silva et al., 1998). The plas- demonstrated at least in the case of the vi- 1997; 1999; 2000; Silva et al., 1998). The characteristic and evolutionary history of Family Localization Gene order Species these plasmids are complex as not all the families of aphids carry plasmids and not all plasmids have the same gene content and/or gene order. In the case of tryptophan, the genes trpEG, the two first genes of the pathway, are carried in several copies, arranged as tandem repeats on a low copy-number plasmid from species of the family Aphididae. The remaining genes of the pathway are chromosomal (Lai et al., 1994). The amplification of the same genes has been shown by van Ham et al. (1999), but in a different replicon in Pem- phigus spyrothecae, a member of the Pemphiginae, the most divergent family of aphids (Heie, 1980), whereas in Schendalia chinensis, a Pemphigidae belonging to a different tribe, trpEG was shown to be Figure 2. Genetic organization of the leucine and tryptophan gene cluster in Buchnera from five aphid families. E. coli is used as reference of bacterial operon. Localization: chromosomally encoded, though unlinked chromosomal (c), plasmidic (p). from the remaining genes from the pathway (Figure 1). In the case of leucine mid of Buchnera of Thelaxes suberi (pLeu- tamin riboflavin (Nakabachi and Ishikawa, plasmids, only one single replicon, named BTs), a member of a different family 1997; 1999). repA1 has been found, but the gene content (Thelaxidae) differed from the previous by Wernegreen and Moran and/or gene order is different in different the presence of a small heat shock gene (1999) have carried out a large study of lineages of the family Aphididae, indicating (ibp) and in the order of the leu and repA the effects of selection and random ge- a great plasticity of the Leu plasmids dur- genes (van Ham et al., 1997). Finally, the netic drift on the evolution of several loci ing Buchnera evolution. The first leucine plasmid of Buchnera of the pemphigid of Buchnera, as compared to free-living

510 OCT 2001, VOL. 26 Nº 10 bacteria. The major conclusion of their symbiotic bacteria, belonging to the five study, which also supports previous find- subdivisions of the Proteobacteria. Com- ings (Moran, 1996), is that random drift pared to the homologous genes of the last affects the potential effect of selection in group, both genes belonging to Buchnera the evolution of Buchnera in a crucial behave in a similar way, showing a higher way. Due to its maternal inheritance, the A+T content, forming a monophyletic effective population size of Buchnera is group, a loss in codon bias, especially in much lower than the effective population third base position, an evolutionary accel- size of free-living bacteria. This has at eration and an increase in the number of least three important evolutionary conse- non-synonymous substitutions. Some of quences at the molecular level: a negative the putative secondary endosymbionts effect on the process of optimization of show a high A+T content, an intermediate, codon usage, an increase in the number of unresolved phylogenetic position between Figure 3. Number of orthologous genes that non-synonymous substitutions, and an ac- Buchnera and the other γ-Proteobacteria, a have been found in ten regions of the Buch- celeration of the evolutionary rate. Accord- loss in codon bias, and a significant evolu- nera sp APS genome (see text for more de- ing to Li (1987), the coefficient of selec- tionary acceleration in the case of the tails) when compared with E. coli and V. tion needed to drive a population from a three-atpD genes. These results lead us to cholerae genomes. less to a more efficient codon usage (i.e., conclude that some of them are true endo- translational selection) should be larger symbionts at different stages of symbiotic than 2/Ne. Hence, those populations having accommodation to the host (Moya et al., Discussion and Perspectives great effective size (Ne) will experience unpublished results). the effects of translational selection with Although it is apparent weaker coefficients of selection than those Genome Reduction of Buchnera sp. that symbiosis has played a major role in others with less effective population size. the adaptation and evolution of insects, Besides Buchnera as pri- The comparative analysis theoretical problems remain as to how the mary simbiont, a number of aphids are of Buchnera sp. APS genome (Shigenobu accommodation process of endosymbionts known to contain other intracellular bac- et al., 2000), with respect to those from E. has taken place. Primary endosymbionts are teria denoted as secondary symbionts coli and Vibrio cholerae, the two closest maternally inherited; they have small effec- (Buchner, 1965; Fukatsu and Ishikawa, free living bacteria whose genomes have tive population sizes and the continuous 1993; 1998; Sandström et al., 2001). The been sequenced, reveal that the endosym- bottlenecking they experience drives the in- location, presence and pattern of inherit- bionts not only have experienced large ge- crement of the average number of deleteri- ance of the secondary endosymbionts in nome reductions, but also large genome ous mutations they carry. The effects of this aphids have been less studied and most reorganizations (Silva et al., 2001). This is process on endosymbiotic genomes, known of the current information has been ob- inferred when considering that sinteny is as Muller’s ratchet, have been demonstrated tained from those of A. pisum and only preserved in a number of short length in Buchnera (Moran, 1996), but also in sec- Macrosiphum rosae (Unterman et al., fragments, being the average number of ondary endosymbionts of aphids as well 1989; Chen and Purcell, 1997; Fukatsu et genes per fragment larger when comparing (Moya et al., unpublished results). Such al., 1998; 2000;). In situ hybridization Buchnera versus E. coli than when com- process should finish with the extinction of and electron microscopy revealed that, at paring with V. cholerae, as expected if we the corresponding bacterial lineages and, least in A. pisum, secondary endosym- consider that E. coli is closer phylogeneti- concomitantly, with the insects harboring bionts are located in a different type of cally to Buchnera than to V. cholerae. In them. However, Buchnera and other pri- bacteriocytes adjacent to those formed by order to determine if the genome reduc- mary endosymbionts of insects have sur- Buchnera (Fukatsu et al., 2000). Avail- tion involved large genome fragments, we vived for over 100 MY. From that we can able data indicate that secondary endo- divided the circular E. coli chromosome in hypothesize that some bacterial lineages symbionts have been acquired several ten sections, being the first center on the have evolved one or more compensatory times during aphid evolution and, con- origin and the sixth on the replication ter- mechanisms to solve the problem of accu- trary to the origin of Buchnera, they do minus, and screened the Buchnera chro- mulation of deleterious mutations. Genome not represent a monophyletic clade mosome for orthologous protein coding reduction or active presence of proteins (Fukatsu and Ishikawa, 1993; Moran and genes. The number of genes of each sec- able to compensate defective gene functions Telang, 1998). Recently, three types of tion (Figure 3) showed that the actual have been advanced by Wernegreen and bacteria belonging to the Enterobacteri- Buchnera genes come from every section Moran (1999). Demonstrating the active aceae have been identified in several spe- of the E. coli chromosome, except the re- presence of these and other factors is of cies from the Macrosiphini tribe, named gion around the terminus. A more detailed particular relevance in the future research U-, R-, and T- type (Sandström et al., examination shows that there is only 4 in endosymbiont evolution. 2001). The phylogenetic distribution of orthologous genes in the E. coli region be- An intriguing aspect of secondary endosymbionts indicates occur- tween ter site C and A. This large absence symbiont evolution in insects is the poten- rence of repeated horizontal transmis- could be due to a large deletion of a re- tial interplay between old-primary and re- sions. gion higher than 300kb in the ancestor of cent-secondary endosymbionts, which also A+T content, phyloge- Buchnera. However, the fact that a similar deserves research. As already mentioned netic relationships, codon usage, evolution- circumstance occurred in the comparison the presence of secondary endosymbionts ary rates, and ratio of synonymous versus with V. cholerae chromosome (Figure 3), indicates that new symbiotic accommoda- non-synonymous substitutions have been and that the region around ter sites is not tion processes are taking place in insect studied in partial sequences of the atpD well-conserved between the E. coli strains evolution. The point then is in what respect and aroQ/pheA genes of primary (Buch- K-12 and O157:H7 (Perna et al., 2001) secondary endosymbionts are replacing and/ nera) and putative secondary endosym- points to this region being very unstable or complementing metabolic functions of bionts of aphids and a set of selected non- and subjected to lateral transfer events. the primary endoysmbionts. In the case of

OCT 2001, VOL. 26 Nº 10 511 Buchnera, for instance, it is of great rel- Fukatsu T, Ishikawa H (1993) Occurrence of bacteria is calibrated using the insect hosts evance to clarify if the fate of Buchnera is chaperonin 60 and chaperonin 10 in primary Proc. R. Soc. Lond. B.253: 167-171. and secondary bacterial symbionts of aphids: Moran NA, von Dohlen CD, Baumann P (1995) to become replaced by a new generation of implications for the evolution of an endosym- Faster evolutionary rate in endosymbiotic bac- symbionts. Although it is too preliminary, biotic system in aphids. J. Mol. Evol. 36: 568- teria than in cospeciating insect hosts. J. Mol. we have evidence (unpublished results) that 577. 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Insect structure of their galls: Implications for host- Physiol. 45: 1-6. eventually, go to extinction. symbiont coevolution and evolution of sterile Perna NT and 27 authors (2001) Genome sequence soldier castes. Zool. Sci. 11: 613-623. of enterohaemorrhagic Escherichia coli O157: ACKNOWLEDGEMENTS Fukatsu T, Watanabe K, Sekiguchi Y (1998) Spe- H7. Nature 409: 529-533. cific detection of intracellular symbiotic bacte- Prosser WA, Simpson SJ, Douglas A (1992) How The authors thank the ria os aphids by oligonucleotide-probed in situ an aphid (Acyrtosiphum pisum) symbiosis re- hybridization. Appl. Entomol. Zool. 33: 461- sponds to variation in dietary nitrogen. J. In- Servicio Central de Soporte a la Investi- 472. sect. 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