The Establishment of Intracellular Symbiosis in an Ancestor of Cockroaches and TermitesAuthor(s): Claudio Bandi, Massimo Sironi, Giuseppe Damiani, Lorenzo Magrassi, Christine A.Nalepa, Ugo Laudani, Luciano SacchiSource: Proceedings: Biological Sciences, Vol. 259, No. 1356 (Mar. 22, 1995), pp. 293-299Published by: The Royal SocietyStable URL: http://www.jstor.org/stable/50009Accessed: 09/10/2008 14:05

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The establishment of intracellular symbiosis in an ancestor of cockroaches and termites

CLAUDIO BANDI1, MASSIMO SIRONI1, GIUSEPPE DAMIANI2, LORENZO MAGRASSI3, CHRISTINE A. NALEPA4, UGO LAUDANI5 AND LUCIANO SACCHI6

Istituto di Patologia Generale Veterinaria, Universita di Milano, Via Celoria 10, 20133, Milano, Italy 2I.D.V.G.A.-C.N.R., Via Celoria 10, 20133 Milano, Italy 3Department of Biological Science, Florida State University, Tallahassee, Florida 32306-4075, U.S.A.

4Department of Entomology, North Carolina State University, Raleigh, North Carolina 27695-7613, U.S.A. 5 Istituto di Entomologia, Universita di Pavia, Viale Taramelli 24, 27100 Pavia, Italy 6 Dipartimento di Biologia Animale, Universita di Pavia, Piazza Botta 9, 27100 Pavia, Italy

SUMMARY

All cockroaches examined so far have been found to harbour a bacterial endosymbiont in specialized cells of the fat body, whereas Mastotermes darwiniensis is the only termite currently known to harbour an intracellular symbiont. The localization and mode of transmission of these bacteria are surprisingly similar, but so far no data have been published on their phylogenetic relationships. To address this issue, molecular sequence data were obtained from the genes encoding the small subunit ribosomal RNA of the M. darwiniensis endosymbiont, and compared with those obtained from endosymbionts of seven species of cockroaches. Molecular phylogenetic analysis unambiguously placed all these bacteria among the

flavobacteria-bacteroides, indicating that the endosymbiont of M. darwiniensis is the sister group to the cockroach endosymbionts examined. Additionally, nucleotide divergence between the endosymbionts appears to be congruent with the palaeontological data on the hosts's evolution. These results support previous claims that the original infection occurred in an ancestor common to cockroaches and termites. A loss of endosymbionts should subsequently have occurred in all termite lineages, except that which gave rise to M. darwiniensis.

1. INTRODUCTION

All cockroaches live in symbiosis with intracellular bacteria (Dasch et al. 1984). These uncultured endo-

symbionts inhabit specialized cells (mycetocytes or

bacteriocytes) of the cockroach fat body, are transo-

varially inherited and appear to be essential for the

growth and reproduction of their hosts (Douglas 1989; Sacchi & Grigolo 1989). The discovery of a similar

mycetocyte endosymbiont in the fat body of the Australian termite Mastotermes darwiniensis (Jucci 1932, 1952) led to the suggestion that the intracellular

symbiosis was established in a common ancestor of both cockroaches and termites (Grasse & Noirot 1959). Because it has been shown that M. darwiniensis alone harbours the mycetocyte bacteria, Buchner (1965) claimed that the symbiotic relation has disappeared over time in all termite lineages except that giving rise to M. darwiniensis. Further loss of symbiosis should have occurred in the praying mantids (Mantodea) (Buchner 1965), assuming that these are the sister group of cockroaches (for the relationships between cock-

roaches, termites and mantids, see Thorne & Carpenter (1992)). This scenario is controversial (Hennig 1981),

Proc. R. Soc. Lond. B (1995) 259, 293-299 Printed in Great Britain

but has been used by several authors as an example of the great age of intracellular symbiotic associations

(Grasse & Noirot 1959; Stanier et al. 1986; Douglas 1989). Data indicating that intracellular symbiosis in some insect groups has evolved independently on several occasions (O'Neill et al. 1993; Moran & Baumann 1994) highlights the possibility that myceto- cyte symbiosis in cockroaches and M. darwiniensis was

acquired independently. Data on the phylogenetic origin of insect endo-

symbionts can be obtained by analysis of the genes encoding for the small subunit ribosomal RNA (16S rDNA) (see references in Moran & Baumann 1994). Using this approach, the phyletic relationships of the

mycetocyte endosymbionts of cockroaches have re-

cently been investigated. Based on the rDNA sequence data, the cockroach endosymbionts have been assigned to the eubacterial taxon defined by the flavobacteria

(informally known as the 'flavobacter-bacteroides'

phylum; see Gherna & Woese 1992), and grouped into a coherent cluster corresponding to a mono-

phyletic unit (Bandi et al. 1994). This result supports the hypothesis that the cockroach hosts and their

endosymbionts have evolved from a common ancestral

? 1995 The Royal Society 293

294 C. Bandi and others Symbiosis in cockroaches and termites

symbiotic system. No evidence, however, has yet been obtained for the establishment of this symbiotic system in the common ancestor of cockroaches and termites.

Here, we compare 16S rDNA sequence data derived from the endosymbiont of the termite M. darwiniensis with previously reported sequences of cockroach

endosymbionts (Bandi et al. 1994). Two other cock- roach species were included in the analysis: the subsocial wood-eating cockroach Cryptocercus punctulatus (for its key role in the understanding of cockroach and termite evolution), and Blaberus craniifer.

2. MATERIALS AND METHODS

Samples of the visceral fat body (0.001-0.005 g) were obtained from wild-collected specimens of M. darwiniensis (Isoptera: Mastotermitidae; collected by L. R. Miller at Kapalga Research Station, Kakadu National Park, Northern Territory, Australia) and C. punctulatus (Blattaria: Crypto- cercidae; collected by C.A. Nalepa at Mountain Lake Biological Station, Giles County, Virginia, U.S.A.), and from laboratory specimens of B. craniifer (Blattaria: Blaberidae; strain held at the Dipartimento di Biologia Animale of the University of Pavia). A pooled sample of visceral fat bodies (obtained from five individual specimens) was used as the starting material for the amplification and sequencing of the endosymbiont rDNA from each host species. Individual fat body samples from two single specimens were also used for C. punctulatus and for M. darwiniensis. Crude DNA preparation, polymerase chain reaction (PCR) amplification, and direct sequencing of the amplified rDNAs were effected by using universal eubacterial primers following the procedure de- scribed by Bandi et al. (1994). PCR products obtained with the endosymbiont-specific primers BBf and BBr (see below) were also sequenced following the same procedure.

A PCR assay was performed to confirm that the sequences obtained were derived from the fat body endosymbionts. DNA preparations obtained from the fat body of specimens of all three species were assayed and DNA preparations from other body fragments (e.g. pharynx, leg, brain, eye, head) of the above specimens, from ovaries of Wolbachia-infected mosquitoes (Culex pipiens), and from bacterial strains repre- senting five species of eubacteria (Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Azospirillum lipoferum, Lactobacillus acidophilus) were included as negative controls. Four sets of primers were used in separate PCR reactions on the above DNA preparations: (i) BBf (5'-ATCACTAGTTGTTGG- ATT-3') and BBr (5'-TTAGAGATTAGCTTCTGA-3') (expected PCR product: an internal 460 base pair (b.p.) fragment of the 16S rDNA of the endosymbionts of cockroaches); (ii) 27fand 1495r (Bandi et al. 1994) (expected PCR product: almost the whole 16S rDNA of virtually all the eubacteria); (iii) 99f and 994r (O'Neill et al. 1993) (expected PCR product: an internal 950 b.p. fragment of the 16S rDNA of the insect-associated rickettsia Wolbachiapipientis) ; and (iv) 18Sa (5'-CCTGGTTGATCCTGCCAGT-3') and 18Sd (5'- AGTCTCGTTCGTTATCGGAAT-3') (expected PCR pro- duct: a 1400 b.p. fragment of the 5' end of the genes encoding for the small subunit ribosomal RNA of cockroaches and termites). Primer sets 27f-1495r, 99f- 994r, and 18Sa-18Sd were used to check for the presence and accessibility of the DNA in the negative controls. PCR conditions with the primer set 99f-994r were as described by O'Neill et al. (1993); PCR conditions for the other sets of primers were as described by Bandi et al. (1994).

The sequences obtained were checked for secondary struc- tural constraints and manually aligned with the previously

described 16S rDNA sequences of the endosymbionts of five cockroach species: Blattella germanica, Periplaneta americana, Periplaneta australasiae, Nauphoeta cinerea and Pycnoscelus suri- namensis (Bandi et al. 1994). They were also aligned with

groups of prealigned 16S rDNA sequences representing the main eubacterial lineages and the five subgroups of the flavobacter-bacteroides phylum (Woese 1987; Gherna & Woese 1992; Neefs et al. 1993).

Phylogenetic analysis was effected according to distance matrix (neighbour-joining, UPGMA and Fitch-Margoliash methods according to maximum likelihood, Jukes & Cantor and Kimura corrections) (see references in Van de Peer & De Wachter (1993)) and character state procedures (maximum likelihood and parsimony as implemented in PHYLIP 3.5 c) using TREECON 3.0 (Van de Peer & De Wachter 1993) and PHYLIP (Felsenstein 1989) on sequence alignments with and without the insertions/deletions (indels). The robustness of the results was evaluated by bootstrap resampling (see references in Van de Peer & De Wachter (1993)) and by constructing the trees after LogDet transformation (Lockhart et al. 1994) (bootstrap analysis was not performed with the maximum likelihood character state procedure). The per- centage nucleotide divergences reported below were esti- mated according to the model of Kimura by taking the indels into account. Relative rate comparisons of rDNA nucleotide

divergences were conducted by selecting outgroup reference taxa (see references in Sarich & Wilson 1967).

3. RESULTS

The approach used for the analysis of the endo-

symbiont 16S rDNA was PCR amplification with eubacterial primers followed by direct sequencing of PCR products. Using this approach, unambiguous and distinct sequences were obtained from the fat body samples of B. craniifer, C. punctulatus and M. darwiniensis, thus indicating that each PcR-examined prokaryotic population consisted predominantly or entirely of a

single and distinct bacterium. Moreover, no sequence variation was observed when two partial 16S rDNA

sequences (750 b.p., corresponding to the 3' 16S rDNA

half-region) were obtained independently from the fat

body samples of two single specimens of C. punctulatus and M. darwiniensis. However, the bacterial sequences obtained from the three host species showed a relatively high nucleotide divergence, ranging from 6.1 / (M. darwiniensis versus B. craniifer) to 4.5 % (C. punctulatus versus B. craniifer), with slight length variations (over a

sequence stretch corresponding to E. coli positions 51-1471 (Brosius et al. 1978), the bacterial sequences derived from B. craniifer, C. punctulatus, and M. darwiniensis had a length of 1416, 1417 and 1413 b.p., respectively). These sequences have been deposited in the EMBL Data Library (accession numbers: Z35664-

Z35666). Sequence alignments and the derived dis- tance matrices are available upon request from the authors.

The sequences obtained were aligned with those of various representatives of the main eubacterial lineages (Woese 1987; Neefs et al. 1993), and with the previously described sequences of the endosymbionts of five cockroach species (Bandi et al. 1994). According to McKittrick's classification (1964), these species should

represent the two phyletic lines of modern cockroaches

(i.e. the Blattoidea and Blaberoidea superfamilies).

Proc. R. Soc. Lond. B (1995)

Symbiosis in cockroaches and termites C. Bandi and others 295

M 1 2 3 4 5 6 7 8 9 10 11 12

kb

0.5- 0.4-

Figure 1. PCR assay to confirm that the sequences obtained derived from the endosymbionts of B. craniifer, C. punctulatus and M. darwiniensis. PCR products of the expected length (460 b.p.) were amplified from fat body samples obtained from individuals belonging to the three insect species (1-3). Other body fragments (4-6, leg; 7-9 head) from the same individuals and the ovaries of Wolbachia-infected mosquitoes (10-12) did not show any bands. The quality of the insect and Wolbachia DNAs was checked in separate PCR reactions using primers targeted for the 18S rDNA of eukaryotes and for the 16S rDNA of Wolbachia pipientis. M: Pharmacia 100 b.p. ladder.

After a sequence signature analysis (Woese 1987), the

target sites of two previously described primers (BBf and BBr), designed to be specific for the 16S rDNAs of the endosymbionts of cockroaches, were identified in all the new sequences, including that derived from the fat body of M. darwiniensis. These primers and three other primer sets were used in a PCR assay on the fat bodies of B. craniifer, C. punctulatus and M. darwiniensis, and on a collection of negative controls (see ?2): the main result of this assay is related as follows. Although the primers BBf and BBr produced amplifications of the expected length (460 b.p.) from the fat body of both cockroaches and M. darwiniensis, no amplifications were obtained with these primers from the negative controls (see figure 1). However, amplifications of the

expected length were obtained from each of the

negative controls with the appropriate primer set (see ?2). These results, as well as proving that closely related bacteria were located in the fat body of cockroaches and M. darwiniensis, ruled out the possi- bility that the DNA of contaminant microorganisms was amplified and sequenced. Moreover, the direct

sequencing of the 460 b.p. amplifications obtained with primers BBf and BBr from the fat body of B.

craniifer, C. punctulatus and M. darwiniensis generated distinct sequences in each case. These sequences overlap, at the expected positions, the sequences obtained from the same fat body samples with the universal eubacterial primers (see above).

Phylogenetic analyses confirmed the close relation-

ship of the endosymbionts of cockroaches and M. darwiniensis: 16S rDNA sequence data unambiguously placed all these bacteria into a coherent subgroup of the flavobacter-bacteroides phylum (figure 2a). This

endosymbiont subgroup is strongly supported by the

bootstrap analysis (1000 / after 100-1000 replicates

with both distance matrix and character state pro- cedures on data sets including the 62 flavobacteria- bacteroides reported in Neefs et al. (1993)). Because the

endosymbiont sequences show a slight bias in base

composition relative to the sequences of other flavo- bacteria-bacteroides (e.g. 46.6-47.5% G+C for the

endosymbionts versus 48.9-54.7% G+C for the flavobacteria-bacteroides included in figure 2a), trees were also constructed after using the LogDet trans- formation (Lockhart et al. 1994), a method designed to remove effects of base composition shifts. This method

gave the same grouping for the endosymbionts and thus further support for the node underlying the

endosymbiont clade. The relationships among the endosymbionts of

cockroaches and M. darwiniensis were found congruent with the host taxonomic relationship recognized by McKittrick (1964), with a minor difference for the

positioning of the endosymbiont of C. punctulatus. Figure 2 b is an example of a 16S rDNA-based tree illustrating the relationships amongst the endosymbionts; classifi- cation of the host species is shown on the right. The

endosymbiont of M. darwiniensis is found to be the sister

group of the cockroach endosymbionts; and among these the endosymbiont of C. punctulatus is found to be the earliest offshoot. In McKittrick's cockroach classifi-

cation, however, the Cryptocercidae are considered to be a deep branch of the superfamily Blattoidea. The

deep positioning of the endosymbionts of M. darwiniensis and C. punctulatus is supported by the bootstrap analysis, although the confidence values are not very high (69 % and 61 %, respectively). However, all distance matrix and character state procedures for phylogenetic re- construction tested resulted in trees showing identical

topologies, with bootstrap values (after 500-1000

replicates) for the positioning of the endosymbionts of M. darwiniensis and C. punctulatus ranging from about

60% (maximum parsimony) to about 90 % (UPGMA method; Jukes & Cantor correction).

If 16S rDNA evolves at an approximately constant

rate, then the number of substitutions observed between an outgroup taxon and different ingroup taxa should be similar. In relative rate comparisons, in some cases the tested endosymbiont sequences showed a similar divergence from the selected outgroup sequence (for example, all endosymbionts versus F. breve; 18.641-19.524%?; Blattidae endosymbionts versus B.

craniifer endosymbiont: 4.674-4.675 %; Blaberoidea

endosymbionts versus P. americana endosymbiont: 4.213-4.675%), but in other comparisons a slightly wider range of divergence was observed (for example, Blaberidae and Blattidae endosymbionts versus C.

punctulatus endosymbionts: 4.506-5.379 /; cockroach

endosymbionts versus M. darwiniensis endosymbiont: 4.762-6.164 ?). The sequences of the endosymbionts of P. surinamensis and B. germanica were mostly responsible for this range variation. Indeed, in most

comparisons these sequences showed the lowest nucleo- tide divergence from the selected outgroup, thus

providing an indication for a slightly lower rate of molecular evolution. Even after taking this variability into account, the rate of molecular evolution of the

endosymbionts still appears similar enough to allow for

Proc. R. Soc. Lond. B (1995)

296 C. Bandi and others Symbiosis in cockroaches and termites

(a) 0.05

1 A - Capnocytophaga ochracea

- - Capnocytophaga gingivalis - Cytophaga aquatilis .- Cytophaga lytica Flavobacterium breve - Weeksella zoohelcum

- Flavobacterium gleum - Flavobacterium balustinum

M.darwiniensis symb. C.punctulatus symb.

H N.cinerea symb. Cockroach _L-1~~ .. ' Isymbionts

L B.craniifer symb. Bacteroides distasonis

Bacteroides thetaiotaomicron I . Bacteroidesfragilis

-- Flexibacter elegans S- a. Saprospira grandis

Haliscomenobacter hydrossis ??-? Chlorobium vibrioforme

0.05 -H1 I 2 3

P.surinamensis symb. - N.cinerea symb.

B.craniifer symb.

B.germanica symb. I Blattellidae, "1-D1ila' iia

- P. americana symb. I Iatd

P.australasiae symb. lattidae a ._J-Blattoidea?-

C.punctulatus symb. I Cryptocercidae- .M.darwiniensis symb. Mastotermitidae Isoptera

Figure 2. Phylogenetic analysis of the endosymbiont of Mastotermes darwiniensis (Kimura's correction; neighbour- joining method). Numbers at nodes are the bootstrap confidence values obtained after 1000 replicates. The scale bars indicate the distances in substitutions per nucleotide: (a) representative 16S rDNA based tree illustrating the

positioning of the endosymbiont of M. darwiniensis (M. darwiniensis symb.) as shown after comparisons of its sequence with those of various representatives of the flavobacter-bacteroides phylum. The endosymbionts of three cockroach

species are included. The tree is based on an alignment of 1100 positions. Insertions/deletions (indels) have not been taken into account. Outgroup: Chlorobium vibrioforme; (b) representative 16S rDNA-based tree illustrating the

positioning of the endosymbiont of M. darwiniensis in relation to the endosymbionts of seven cockroach species. Classification of the host species is shown on the right (1, family; 2, superfamily; 3, suborder). The tree is based on an alignment of 1400 positions. Indels have been taken into account. Outgroup: Flavobacterium breve (not shown).

approximate comparisons between palaeontological dates and nucleotide divergences (see Discussion).

4. DISCUSSION

Phylogenetic analysis of 16S rDNA sequences reveals a close relationship between the endosymbiont of M. darwiniensis and those of modern cockroaches; all these bacteria make up a coherent subgroup within the flavobacter-bacteroides phylum, with no close relative

among the rDNA analysed flavobacteria-bacteroides. The use of the LogDet transformation results in the same grouping of endosymbionts. In addition, the 16S rDNAs show the endosymbiont of M. darwiniensis to be

the sister group of the cockroach endosymbionts examined. These results, although supporting the common origin of mycetocyte symbiosis in cockroaches and M. darwiniensis, do not exclude horizontal transfer of symbiotic bacteria to the ancestors of M. darwiniensis from cockroaches belonging to extinct lineages. This

possibility, however, appears quite unlikely if we consider the host-symbiont coadaptation required for

mycetocyte symbioses (Douglas 1989). Further evidence for the establishment of this

symbiotic association in an ancestor common to cockroaches and M. darwiniensis can be obtained by comparing the rDNA divergence of the endosymbionts with the divergence time of their hosts. According to

Proc. R. Soc. Lond. B (1995)

(b)

I

Symbiosis in cockroaches and termites C. Bandi and others 297

Handlirsch (1908), the termites diverged from cock- roaches during the Jurassic (210-135 Ma before pres- ent (BP)) ; palaeogeographic evidence suggests a slightly older split with the origin of the termites in the Triassic; 250-210 Ma BP (see references in Burnham 1978); and according to the classic textbook scenario the split between cockroaches and termites occurred during the late Palaeozoic (Buchner 1965) (although arguments for this ancient separation (Holmgren 1909, 1911) were questioned by Hennig (1981)). Indications for a post-Palaeozoic divergence of cockroaches and termites can also be derived from the DNA sequence data available for representatives of various insect orders showing cockroaches, termites and mantids as closely related compared with the insect lineages whose Palaeozoic split is unquestioned (De Salle et al. 1992; Liu & Beckenbach 1992). Assuming that the split between cockroaches and termites occurred sometime after the end of the Palaeozoic (250 Ma BP), and

certainly before the appearance of termites and modern cockroaches in the fossil record at the beginning of the Cretaceous (135 Ma BP) (Ross &Jarzembowsky 1993), the average divergence between the endosymbiont of M. darwiniensis and those of cockroaches (5.609%) indicates an approximate rate of 0.0056-0.01 substi- tutions per site every 50 Ma for the 16s rDNA of the endosymbionts. This rate is only slightly lower than that estimated for the prokaryotic 16S rDNA by Ochman & Wilson (1987) (0.01 every 50 Ma) and

overlaps the range estimated for the aphid proteo- bacterial endosymbionts (Moran et al. 1993) (0.0076- 0.0232 every 50 Ma). It also agrees with the rate estimated for the endosymbionts of cockroaches (0.0088 every 50 Ma) assuming that the split between the Blattoidea and Blaberoidea superfamilies occurred at the beginning of the Cretaceous period (Bandi et al.

1994). A more detailed comparison of the host and

endosymbiont divergence and investigation of the molecular evolutionary rate of the endosymbionts would require multiple and reliable host-based cali- bration points. In view of our uncertainty about the timetable of cockroach and termite evolution, we may limit ourselves to emphasizing the following results: (i) the cockroach hosts and their endosymbionts show a

congruent evolutionary history; (ii) the endosymbionts of M. darwiniensis are shown to be the sister group of the

endosymbionts of modern cockroaches; and (iii) the nucleotide divergence of the endosymbionts agrees with the available palaeontological data on cockroach and termite evolution. These results support the idea that the mycetocyte symbiosis was established in a common ancestor of cockroaches and termites, with a

subsequent loss of endosymbionts in termite lineages except that leading to M. darwiniensis. An interesting alternative would be to consider the termites as a

paraphyletic (or polyphyletic) group, and the as- sociation with the mycetocyte endosymbionts as a

synapomorphy for a phyletic line leading to modern cockroaches and M. darwiniensis (Vawter 1991). How-

ever, the hypothesis that M. darwiniensis is more closely related to cockroaches than to the rest of termites has not been substantiated (De Salle et al. 1992; Thorne &

Carpenter 1992; De Salle 1994). Our data do not of course exclude the possibility that the ancestors of cockroaches and M. darwiniensis independently es- tablished a mycetocyte symbiotic association with two closely related extracellular flavobacteria. This would, however, imply that: (i) of the hundreds of intracellular bacteria described so far (see references in Weiss & Dasch 1992; Douglas 1994), the only certain cases of intracellular flavobacteria (i.e. in cockroaches and M. darwiniensis) are of independent origin; (ii) the phyletic lines leading to these two independently established intracellular flavobacteria diverged at approximately the same time as the cockroach-termite divergence (for example, as a consequence of a previous extracellular symbiotic association); and (iii) the cockroach and M. darwiniensis flavobacteria independently acquired the same location and mode of transmission (Buchner 1965). In our opinion, the hypothesis of an independent acquisition of mycetocyte symbiosis in cockroaches and M. darwiniensis is therefore quite unlikely.

The evolutionary history of cockroaches and termites has been hotly debated since the beginning of this century (see references in Hennig .1981). A major issue in this debate is the appearance of termite eusociality relative to their wood diet (Thorne 1990, 1991; Nalepa 1991, 1994). M. darwiniensis and the wood-eating subsocial cockroaches belonging to the genus Cryptocercus are considered as keys for

understanding this issue (Cleveland et al. 1934; Wilson 1971; Hennig 1981). Indeed, only M. darwiniensis among termites and only Cryptocercus spp. among cockroaches show a double symbiosis (with the wood-

digesting protista of the gut and with the mycetocyte endosymbionts of the fat body), and both have been

regarded as survivors resembling the common ancestor of cockroaches and termites (Grasse & Noirot 1959). This classic view has been questioned by some authors

(Hennig 1981; Thorne 1991) and the phyletic lines

leading to Cryptocercus and Mastotermes have been derived from existing cockroach and termite families

(for differing positionings of the genus Cryptocercus see McKittrick 1964; Thorne & Carpenter 1992; Grand- colas 1994; for Mastotermes see Thorne & Carpenter 1992). To explain the shared attributes of Cryptocercus spp. and M. darwiniensis, a convergent evolution with horizontal transfer of the wood-digesting symbiotic protista has been proposed as an alternative hypothesis to heredity by ancestry with subsequent loss of

symbionts (Emerson 1935; Hennig 1981; Thorne 1990; Thorne 1991). Moreover, a third hypothesis claimed

Cryptocercus as the sister group of termites, and cockroaches as a paraphyletic assemblage (Hennig 1981). Our data places the endosymbiont of C.

punctulatus as the sister group of the endosymbionts of six unequivocal cockroach species: this result indirectly weakens the latter hypothesis. In addition, our endo-

symbiont rDNA data makes the hypotheses of hori- zontal transfer or independent acquisition for the

mycetocyte endosymbionts unlikely, and provides further evidence for the close phylogenetic relationship of cockroaches and termites. Indeed, the average rDNA nucleotide divergence observed between the

endosymbiont of the termite M. darwiniensis and those

Proc. R. Soc. Lond. B (1995)

298 C. Bandi and others Symbiosis in cockroaches and termites

of the examined cockroaches (5.609 %) is only slightly higher than that observed between the endosymbiont of C. punctulatus and those of the remaining cockroaches (4.907 0). Assuming that endosymbiont evolution matches host evolution, a relatively short time should have passed between the branching off of termites and the evolutionary radiation of modern cockroaches.

We thank L. L. Deitz, L. Gomulski, L. Ferretti, A. Minelli, M. Pearce and F. Scudo for helpful suggestions and com- ments on the manuscript. The authors are particularly grateful to L. H. Miller for providing the specimens of M. darwiniensis, to M. Nava and C. Violani for their logistic assistance and to M. Grandini for writing the software for LogDet transformation. M.S. is supported by a grant from the Fondazione Adriano Buzzati Traverso. L. M. is supported by a 'Borsa di Perfezionamento all'Estero' of the University of Pavia. Part of the research was carried out at the Dipartimento di Genetica e Microbiologia of the University of Pavia.

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Received 9 November 1994; accepted 16 December 1994

Proc. R. Soc. Lond. B (1995)