Evolutionary trend of phylogenetic diversity of nitrogen fixation genes in the gut community of wood-feeding termites

Nitrogen fixation by gut microorganisms is one of the crucial aspects of symbiosis in wood-feeding termites since these termites thrive on a nitrogen-poor diet. In order to understand the evolution of this symbiosis, we analysed the nitrogenase structural gene nifH in the gut microbial communities. In conjunction with the published sequences, we compared approximately 320 putatively functional NifH protein sequences obtained from a total of 19 termite samples that represent all the major branches of their currently proposed phylogeny, and from one species of the cockroach Cryptocercus that shares a common ancestor with termites. Using multivariate techniques for clustering and ordination, a phylogeny of NifH protein sequences was created and plotted variously with host termite families, genera, and species. Close concordance was observed between NifH communities and the host termites at genus level, but family level relationships were not always congruent with accepted termite clade structure. Host groups examined included basal families (Mastotermitidae, Termopsidae, Kalotermitidae, as well as Cryptocercus), the most derived lower termite family Rhinotermitidae, and subfamilies representing the advanced and highly diverse apical family Termitidae (Macrotermitinae, Termitinae, and Nasutitermitinae). This selection encompassed the major nesting and feeding styles recognized in termites, and it was evident that NifH phylogenetic divergence, as well as the occurrence of alternative nitrogenase-type NifH, was to some extent dependent on host lifestyle as well as phylogenetic position.     

Wood-feeding cockroaches as models for termite evolution (Insecta: Dictyoptera): Cryptocercus vs. Parasphaeria boleiriana

Isoptera are highly specialized cockroaches and are one of the few eusocial insect lineages. Cryptocercus cockroaches have appeared to many as ideal models for inference on the early evolution of termites, due to their possible phylogenetic relationship and several shared key attributes in life history. Recently, Pellens, Grandcolas, and colleagues have proposed the blaberid cockroach Parasphaeria boleiriana to be an alternative model for the early evolution in termites. We compare the usefulness of Cryptocercus and P. boleiriana as models for termite evolution. Cryptocercus and lower Isoptera (1) can both feed on comparatively recalcitrant wood, (2) have an obligate, rich and unique hypermastigid and oxymonadid fauna in the hindgut, (3) transfer these flagellates to the next generation by anal trophallaxis, (4) have social systems that involve long-lasting biparental care, and, finally, (5) are strongly suggested to be sister groups, so that the key attributes (1)-(4) appear to be homologous between the two taxa. On the other hand, P. boleiriana (1) feeds on soft, ephemeral wood sources, (2) shows no trace of the oxymonadid and hypermastigid hindgut fauna unique to Cryptocercus and lower Isoptera, nor does it have any other demonstrated obligate relationship with hindgut flagellates, (3) is likely to lack anal trophallaxis, (4) has only a short period of uniparental brood care, and (5) is phylogenetically remote from the Cryptocercus+Isoptera clade. These facts would argue against any reasonable usage of P. boleiriana as a model for the early evolution of Isoptera or even of the clade Cryptocercus+Isoptera. Cryptocercus thus remains an appropriate model-taxon-by-homology for early termite evolution. As compared to P. boleiriana, some other Blaberidae (such as the Panesthiinae Salganea) appear more useful as model-taxa-by-homoplasy for the early evolution of the Cryptocercus+Isoptera clade, as their brooding behavior is more elaborate than in P. boleiriana.     

Phylogenetic relationship among cockroach families inferred from mitochondrial12S rRNA gene sequence

A number of phylogenies exist for cockroaches that differ in the postulated relationships among families and genera. The relationship of the wood-feeding genus, Cryptocercus, to other cockroach families and to termites, has generated considerable debate. Grandcolas (1994), based on morphological analysis, synonymized the family Cryptocercidae with Polyphagidae and placed the genus Cryptocercus in the subfamily Polyphaginae. To determine if an independent set of characters supports the placement of Cryptocercus in Polyphaginae, a phylogenetic analysis of relationships among representative genera of the five cockroach families was undertaken. DNA sequence of a -430 base pair portion of the mitochondrial small ribosomal subunit gene from representatives of Blattidae, Blattellidae, Blaberidae and Cryptocercus, previously published by Kambhampati (1995) and Kambhampati et al. (1996), and the homologous sequence from representatives of Polyphagidae were used in the analysis. A total of twenty cockroach taxa and three termite genera were included in the study. Because a recent study showed that Cryptocercus punctulatus consists of a species complex, DNA sequence from four individuals collected in different parts of the U.S.A. was included in the study. The trees estimated from parsimony and neighbour-joining analyses indicated that Cryptocercus is a monophyletic clade which is most closely related to members of Blattidae. Polyphagidae is indicated as a sister group to the Blattidae + Cryptocercus complex, suggesting that Polyphagidae may belong to the superfamily Blattoidea rather than to Blaberoidea as proposed by McKittrick (1964). Blaberidae and Blattellidae were sister groups as previously proposed. Based on the present analysis, I propose that the genus Cryptocercus be retained in the family Cryptocercidae. Cockroaches     

Cospeciation of termite gut flagellates and their bacterial endosymbionts: Trichonympha species and 'Candidatus Endomicrobium trichonymphae'

Symbiotic flagellates play a major role in the digestion of lignocellulose in the hindgut of lower termites. Many termite gut flagellates harbour a distinct lineage of bacterial endosymbionts, so-called Endomicrobia, which belong to the candidate phylum Termite Group 1. Using an rRNA-based approach, we investigated the phylogeny of Trichonympha , the predominant flagellates in a wide range of termite species, and of their Endomicrobia symbionts. We found that Trichonympha species constitute three well-supported clusters in the Parabasalia tree. Endomicrobia were detected only in the apical lineage (Cluster I), which comprises flagellates present in the termite families Termopsidae and Rhinotermitidae, but apparently absent in the basal lineages (Clusters II and III) consisting of flagellates from other termite families and from the wood-feeding cockroach, Cryptocercus punctulatus . The endosymbionts of Cluster I form a monophyletic group distinct from many other lineages of Endomicrobia and seem to have cospeciated with their flagellate host. The distribution pattern of the symbiotic pairs among different termite species indicates that cospeciation of flagellates and endosymbionts is not simply the result of a spatial separation of the flagellate lineages in different termite species, but that Endomicrobia are inherited among Trichonympha species by vertical transmission. We suggest extending the previously proposed candidatus name ' Endomicrobium trichonymphae ' to all Endomicrobia symbionts of Trichonympha species, and estimate that the acquisition by an ancestor of Trichonympha Cluster I must have occurred about 40–70 million years ago, long after the flagellates entered the termites.     

Termites and trees: a review of recent advances in termite phylogenetics

Summary: Modern termite phylogenetics is critically reviewed, with an emphasis on tree topologies as phylogenetic hypotheses. Studies have especially concentrated on (1) the position of Isoptera among the Dictyoptera and (2) the family group relationships within the Isoptera. The first of these problems is still controversial; although the weight of evidence now suggests that termites are nested within the cockroaches, thus making "Blattaria" as presently constituted paraphyletic. The exact position of termites within the cockroaches is uncertain, although Cryptocercus is the most plausible sister group.¶Family groups relationships are rather better resolved. Mastotermitidae is now generally accepted to be the most basal termite group. Termopsidae, Hodotermitidae and Kalotermitidae are all basal to (Termitidae + Serritermitidae + Rhinotermitidae), although their relative positions within that part of the tree are disputed. Most recent studies support a sister group relationship for Serritermitidae and (Termitidae + Rhinotermitidae). However, no study has yet unambiguously found the Rhinotermitidae monophyletic. The Termitidae are well established as monophyletic and as the most apical termite family. However, within the Termitidae the monophyly of none of the subfamilies is well established, making subfamily level analyses unreliable.¶A number of problem areas are identified: (1) poor taxon sampling is a universal problem, (2) higher taxonomic groupings are often assumed to be monophyletic a priori without adequate support, (3) datasets are collected from different taxa and character systems without consideration of the overall international effort.     

A case for ancestral transfer of symbionts between cockroaches and termites

Living species of the cockroach family Cryptocercidae have intestinal symbionts that are congeneric with some of the gut protozoa found in Isoptera. Presence of such closely related symbionts in cryptocercids and in termites has been frequently interpreted as a uniquely derived homologous character shared between the two xylophagous groups. This may not be the most parsimonious interpretation. Cryptocercus nymphs placed into Zootermopsis (dampwood termite) colonies were killed and eaten by the termites. Termites placed into a Cryptocercus nest box were also fully consumed. Modern Cryptocercus punctulatus and Zootermopsis are often found in the same decaying logs in the Pacific Northwest of the U.S.A., and it is likely that their ancestors also cohabited in at least a portion of their ranges. By occasionally killing and consuming an intruder from the other group, gut protozoa could have been acquired and exchanged between termites and Cryptocercus or their ancestors, under natural conditions and before the life histories of the protozoa became specialized within the host orders. Implications for assessing the phylogeny of the two dictyopteroid groups are also discussed.     

The evolutionary transition from subsocial to eusocial behaviour in Dictyoptera: phylogenetic evidence for modification of the "shift-in-dependent-care" hypothesis with a new subsocial cockroach

Cockroaches have always been used to understand the first steps of social evolution in termites because they are close relatives with less complex and integrated social behaviour. Termites are all eusocial and ingroup comparative analysis would be useless to infer the origin of their social behaviour. The cockroach genus Cryptocercus was used as a so-called "prototermite" model because it shows key-attributes similar to the termites (except Termitidae): wood-feeding, intestinal flagellates and subsocial behaviour. In spite of these comparisons between this subsocial cockroach and eusocial termites, the early and remote origin of eusocial behaviour in termites is not well understood yet and the study of other relevant "prototermite" models is however needed. A molecular phylogenetic analysis was carried out to validate a new "prototermite" model, Parasphaeria boleiriana which shows a peculiar combination of these key-attributes. It shows that these attributes of Parasphaeria boleiriana have an independent origin from those of other wood-eating cockroaches and termites. The case of P. boleiriana suggests that a short brood care was selected for with life on an ephemeral wood resource, even with the need for transmission of flagellates. These new phylogenetic insights modify evolutionary hypotheses, contradicting the assumption made with Cryptocercus model that a long brood care is necessary for cooperation between broods in the "shift-in-dependent-care" hypothesis. An ephemeral wood resource is suggested to prompt generation overlap and the evolution of cooperation, even if brood care is shortened.     

Inheritance and diversification of symbiotic trichonymphid flagellates from a common ancestor of termites and the cockroach Cryptocercus

Cryptocercus cockroaches and lower termites harbour obligate, diverse and unique symbiotic cellulolytic flagellates in their hindgut that are considered critical in the development of social behaviour in their hosts. However, there has been controversy concerning the origin of these symbiotic flagellates. Here, molecular sequences encoding small subunit rRNA and glyceraldehyde-3-phosphate dehydrogenase were identified in the symbiotic flagellates of the order Trichonymphida (phylum Parabasalia) in the gut of Cryptocercus punctulatus and compared phylogenetically to the corresponding species in termites. In each of the monophyletic lineages that represent family-level groups in Trichonymphida, the symbionts of Cryptocercus were robustly sister to those of termites. Together with the recent evidence for the sister-group relationship of the host insects, this first comprehensive study comparing symbiont molecular phylogeny strongly suggests that a set of symbiotic flagellates representative of extant diversity was already established in an ancestor common to Cryptocercus and termites, was vertically transmitted to their offspring, and subsequently became diversified to distinct levels, depending on both the host and the symbiont lineages.     

Molecular phylogeny of Asian termites (Isoptera) of the families Termitidae and Rhinotermitidae based on mitochondrial COII sequences

The families Termitidae and Rhinotermitidae are the most evolved and diverse groups of the social insects, termites (Order Isoptera), showing elaborated morphology and complex behavior. Molecular phylogeny of termites with the emphasis on these families was examined by Bayesian and maximum-likelihood analyses based on DNA sequence of mitochondrial cytochrome oxidase II (COII) gene of 31 genera sampled in Asia (mainly Thailand and Japan) along with those reported previously. Termitidae was monophyletic and originated from within polyphyletic Rhinotermitidae. Among the four subfamilies of Termitidae, Macrotermitinae was monophyletic suggesting a single common origin of fungus-growing habit characteristic for this subfamily, and was placed in the basal position in the family. A group consisting of other subfamilies Termitinae and Nasutitermitinae, though some important groups were still untouched, was the most apical but neither Termitinae nor Nasutitermitinae formed a monophyletic lineage. It was implied that, as defense systems of the soldier castes, the appearance of snapping mandibles has occurred at a single event, but the development of nasus for chemical secretion has probably not. Our tree provides some evidence concerning contradictions in the previously proposed phylogeny of termites.     

The effects of fossil placement and calibration on divergence times and rates: An example from the termites (Insecta: Isoptera)

Among insects, eusocial behavior occurs in termites, ants, some bees and wasps. Isoptera and Hymenoptera convergently share social behavior, and for both taxa its evolution remains poorly understood. While dating analyses provide researchers with the opportunity to date the origin of eusociality, fossil calibration methodology may mislead subsequent ecological interpretations. Using a comprehensive termite dataset, we explored the effect of fossil placement and calibration methodology. A combined molecular and morphological dataset for 42 extant termite lineages was used, and a second dataset including these 42 taxa, plus an additional 39 fossil lineages for which we had only morphological data. MrBayes doublet-model analyses recovered similar topologies, with one minor exception (Stolotermitidae is sister to the Hodotermitidae, s.s., in the 42-taxon analysis but is in a polytomy with Hodotermitidae and (Kalotermitidae + Neoisoptera) in the 81-taxon analysis). Analyses using the r8s program on these topologies were run with either minimum/maximum constraints (analysis a = 42-taxon and analysis c = 81-taxon analyses) or with the fossil taxon ages fixed (ages fixed to be the geological age of the deposit from which they came, analysis b = 81-taxon analysis). Confidence intervals were determined for the resulting ultrametric trees, and for most major clades there was significant overlap between dates recovered for analyses A and C (with exceptions, such as the nodes Neoisoptera, and Euisoptera). With the exception of isopteran and eusiopteran node ages, however, none of the major clade ages overlapped when analysis B is compared with either analysis A or C. Future studies on Dictyoptera should note that the age of Kalotermitidae was underestimated in absence of kalotermitid fossils with fixed ages.     

Ancestral transfer of symbionts between cockroaches and termites: an alternative hypothesis.

Closely related cellulolytic protozoa reside in the hindguts of extant woodroaches (Cryptocercidae) and termites (Isoptera). The evolutionary origin of these symbiotic relationships in the two lineages is uncertain. Transfer of protozoa between ancestors of modern Cryptocercus and termites remains a valid alternative theory to the established hypothesis of symbiont inheritance from a common ancestor. Nalepa's (Proc. R. Soc. Lond. B 246, 185 (1991] concerns regarding the protozoan transfer hypothesis focus on the biology of modern species, and neglect to consider the evolutionary framework of an ancestral dynamic postulated to occur among Palaeozoic insects. Legitimacy of the symbiont transfer theory removes the constraint of interpreting presence of cellulolytic protozoa as a synapomorphy between Cryptocercidae and Isoptera, with potential impact on objective resolution of dictyopteran phylogeny.     

Armed reproductives: Evolution of the frontal gland in imagoes of Termitidae

The frontal gland of termites is a structure without any equivalent among other animals. Although this gland is well known in soldiers, it received almost no attention in other castes. Recently, we described it in imagoes of Rhinotermitidae and Serritermitidae. In order to provide a complete picture of the evolution of this gland in termite imagoes, we studied it in additional 34 species of Termitidae, representing 7 of the 8 subfamilies. The frontal gland of these species is formed by class 1 secretory cells only, and occurs in two basic shapes: epithelial with reservoir in Foraminitermitinae and Macrotermitinae, and epithelial without reservoir in all other subfamilies. The size variability of the gland is high, not only among Termitidae subfamilies, but also within subfamilies. Our data suggest that the ancestral form of the frontal gland is epithelial with reservoir, as found in Rhinotermitidae, Serritermitidae, and basal Termitidae. The reduction of the reservoir occurred at least two times and the gland was lost two times independently: in Protermes sp. and in Microtermes toumodiensis (both Macrotermitinae).     

The fine structure of colleterial glands in two cockroaches and three termites, including a detailed study of Cryptocercus punctulatus (Blattaria, Cryptocercidae) and Mastotermes darwiniensis (Isoptera, Mastotermitidae)

The colleterial glands of insects are organs associated with the female genital apparatus. In cockroaches, these glands produce secretions that cover two parallel rows of eggs during oviposition, and in oviparous species, these secretions become the tanned, sculpted, rigid outer casing of the ootheca. The goal of this study was to compare the gross anatomy of the colleterial glands and the ultrastructure of their component tubules in the phylogenetically significant genera Cryptocercus (Blattaria) and Mastotermes (Isoptera). Recent studies indicate that cockroaches in the genus Cryptocercus are the sister group of termites, and Mastotermes is the only termite known to produce a cockroach-like ootheca. One additional oviparous cockroach, Therea, and two additional termites, Zootermopsis and Pseudacanthotermes, were also examined. As in other cockroaches, the colleterial glands of Cryptocercus and Therea are asymmetrical, with a well developed bipartite left gland and a smaller right gland. In the termites Mastotermes, Zootermopsis, and Pseudacanthotermes, the colleterial glands are composed of a well-developed, paired, anterior gland and a small posterior gland; histological staining and cytological evidence suggest that these are homologues of the left and the right colleterial glands of cockroaches, respectively. At the ultrastructural level, colleterial gland tubules are made of cells belonging to a modified class 1 type cell in the cockroaches, in Mastotermes, and in Zootermopsis; the latter lays its eggs singly, without a surrounding ootheca-like structure. In the advanced termite Pseudacanthotermes, the tubules are made of secretory units belonging to the class 3 cell type. This study demonstrates that the cytological characteristics of colleterial glands in basal termites are similar to those of cockroaches, whether the termite secretes an oothecal casing that covers two parallel rows of eggs, as in Mastotermes, or lays its eggs singly, as in Zootermopsis. The function of colleterial glands in non-mastotermitid termites is unknown.     

Morphological phylogenetics of termites (Isoptera)

Isoptera (termites) are an ecologically important order, with both a high abundance and biomass in tropical ecosystems. However, there have been few phylogenetic hypotheses for termites, and we present here the first comprehensive cladistic analysis for the group. We analysed relationships between all seven termite families, including representatives of all known feeding group, plus a number of systematically critical taxa. Termite species richness is biased towards the higher termites (Termitidae), and our taxon sampling reflects this. Our analysis was based essentially on morphological characters (96 workers, 93 soldiers) plus seven biological characters. The cladistic analysis gave four equally parsimonious trees, representing two islands of topologies. The strict consensus tree is fully resolved for the higher termites, but less so for the lower termites. Overall there is low statistical support for the suggested topology, and this can be explained by the high incongruence between the data sets (worker, soldier and biological). This study highlights the particular problems of coding morphological characters in social insects with multiple castes. Without the input of additional data sets, e.g. alates, biological, behavioural and molecular, it will not be possible to obtain a well-supported termite phylogeny.     

Symbiotic nitrogen fixation in the New Zealand dampwood termite (

This study investigates symbiotic microorganisms in the New Zealand dampwood termite Stolotermes ruficeps using culture-independent techniques to describe the diversity of nitrogen-fixing organisms within this termite. Phylogenetic analysis of a portion of the nifH gene (encoding dinitrogenase reductase) revealed 19 phylotypes (>98% sequence identity) with 77?86% similarity to published nucleotide sequences from uncultured microorganisms described from termite guts. The majority of sequences obtained in this study were most closely related to sequences obtained from basal families Kalotermitidae, Termopsidae and the closely related wood-feeding cockroach species Cryptocercus. This adds to the growing amount of evidence suggesting that the composition of nifH sequences is characteristic of a termite family. This study also identifies wood-dwelling termites as a potentially important source of nitrogen input into temperate forests, something previously neglected and warranting further investigation.     

The Cockroach Origin of the Termite Gut Microbiota: Patterns in Bacterial Community Structure Reflect Major Evolutionary Events

Termites digest wood and other lignocellulosic substrates with the help of their intestinal microbiota. While the functions of the symbionts in the digestive process are slowly emerging, the origin of the bacteria colonizing the hindgut bioreactor is entirely unknown. Recently, our group discovered numerous representatives of bacterial lineages specific to termite guts in a closely related omnivorous cockroach, but it remains unclear whether they derive from the microbiota of a common ancestor or were independently selected by the gut environment. Here, we studied the bacterial gut microbiota in 34 species of termites and cockroaches using pyrotag analysis of the 16S rRNA genes. Although the community structures differed greatly between the major host groups, with dramatic changes in the relative abundances of particular bacterial taxa, we found that the majority of sequence reads belonged to bacterial lineages that were shared among most host species. When mapped onto the host tree, the changes in community structure coincided with major events in termite evolution, such as acquisition and loss of cellulolytic protists and the ensuing dietary diversification. UniFrac analysis of the core microbiota of termites and cockroaches and construction of phylogenetic tree of individual genus level lineages revealed a general host signal, whereas the branching order often did not match the detailed phylogeny of the host. It remains unclear whether the lineages in question have been associated with the ancestral cockroach since the early Cretaceous (cospeciation) or are diet-specific lineages that were independently acquired from the environment (host selection).     

Diet is the primary determinant of bacterial community structure in the guts of higher termites

The gut microbiota of termites plays critical roles in the symbiotic digestion of lignocellulose. While phylogenetically ‘lower termites’ are characterized by a unique association with cellulolytic flagellates, higher termites (family Termitidae) harbour exclusively prokaryotic communities in their dilated hindguts. Unlike the more primitive termite families, which primarily feed on wood, they have adapted to a variety of lignocellulosic food sources in different stages of humification, ranging from sound wood to soil organic matter. In this study, we comparatively analysed representatives of different taxonomic lineages and feeding groups of higher termites to identify the major drivers of bacterial community structure in the termite gut, using amplicon libraries of 16S rRNA genes from 18 species of higher termites. In all analyses, the wood‐feeding species were clearly separated from humus and soil feeders, irrespective of their taxonomic affiliation, offering compelling evidence that diet is the primary determinant of bacterial community structure. Within each diet group, however, gut communities of termites from the same subfamily were more similar than those of distantly related species. A highly resolved classification using a curated reference database revealed only few genus‐level taxa whose distribution patterns indicated specificity for certain host lineages, limiting any possible cospeciation between the gut microbiota and host to short evolutionary timescales. Rather, the observed patterns in the host‐specific distribution of the bacterial lineages in termite guts are best explained by diet‐related differences in the availability of microhabitats and functional niches.     

The phylogeny of leaf beetles (Chrysomelidae) inferred from mitochondrial genomes

The high-level classification of Chrysomelidae (leaf beetles) currently recognizes 12 or 13 well-established subfamilies, but the phylogenetic relationships among them remain ambiguous. Full mitochondrial genomes were newly generated for 27 taxa and combined with existing GenBank data to provide a dataset of 108 mitochondrial genomes covering all subfamilies. Phylogenetic analysis under maximum likelihood and Bayesian inference recovered the monophyly of all subfamilies, except that Timarcha was split from Chrysomelinae in some analyses. Three previously recognized major clades of Chrysomelidae were broadly supported: the ‘chrysomeline’ clade consisting of (Chrysomelinae (Galerucinae + Alticinae)); the ‘sagrine’ clade with internal relationships of ((Bruchinae + Sagrinae) + (Criocerinae + Donaciinae)), and the ‘eumolpine’ clade comprising (Spilopyrinae (Cassidinae (Eumolpinae (Cryptocephalinae + Lamprosomatinae)))). Relationships among these clades differed between data treatments and phylogenetic algorithms, and were complicated by two additional deep lineages, Timarcha and Synetinae. Various topological tests favoured the PhyloBayes software as the preferred inference method, resulting in the arrangement of (chrysomelines (eumolpines + sagrines)), with Timarcha placed as sister to the chrysomeline clade and Synetinae as a deep lineage splitting near the base. Whereas mitogenomes provide a solid framework for the phylogeny of Chrysomelidae, the basal relationships do not agree with the topology of existing molecular studies and remain one of the most difficult problems of Chrysomelidae phylogenetics.