Ribosomal DNA gene and phylogenetic relationships of Diplura and lower Hexapods

HennigdividedInsectas.lat.(=Hexapoda)intotwowelldefinedtaxonomicgroups:Entog-nathaandEctognatha[1].ThemostdistinctivecharacterofEntognatha(includingProtura,Col-lembolaandDiplura)liesintheenclosedmouthpartscondition,whereasthepresenceofexposedmouthpartsisthemainfeatureofEctognatha(includingMicrocoryphia,Zygentomaandtheptery-goteinsects).ControversiesaboutthephylogeneticrelationshipsofhightaxaEntognathahavekeptgrowinginrecentyears,withthemonophylyofDipluraandthephylogeneticpositionsofDiplur…     

MicroRNA evolution provides new evidence for a close relationship of Diplura to Insecta

The phylogenetic interrelationships among four hexapod lineages (Protura, Collembola, Diplura and Insecta) are pivotal to understanding the origin of insects and the early diversification of Hexapoda, but they have been difficult to clarify based on the available data. In this study, we identified 91 conserved microRNA (miRNA) families from 36 panarthropod taxa, including seven newly sequenced non-insect hexapods. We found major clade differentiation accompanied by the origin of novel miRNA families, and most miRNA clusters are conserved with a high degree of microsynteny. Importantly, we were able to identify two miRNA families unique to Hexapoda, and four miRNA families and a miRNA cluster that exist exclusively in Diplura and Insecta, suggesting a close relationship between Diplura and Insecta as well as the monophyly of Hexapoda. Combined with a phylogenetic analysis based on the presence/absence matrix of miRNA families, our study demonstrates the effectiveness of miRNA in resolving deep phylogenetic problems.     

Testing for misleading effects in the phylogenetic reconstruction of ancient lineages of hexapods: influence of character dependence and character choice in analyses of 28S rRNA sequences

The present analyses employ the almost complete sequence of the 28S rRNA gene to investigate phylogenetic relationships among Pancrustacea, placing special emphasis on the position of basal hexapod lineages. This study utilizes a greater number of characters and taxa of Protura, Collembola and Diplura than previous analyses to focus on conflicts in the reconstruction of the early steps in hexapod evolution. Phylogenetic trees are mainly based on Bayesian approaches, but likewise include analyses with Maximum Likelihood and Maximum Parsimony. Different analyses, including the application of a mixed DNA/RNA substitution model, were performed to narrow possible misleading effects of non-stationarity of nucleotide frequencies, saturation and character independence down to a minimum. This is the first time that a mixed DNA/RNA model is applied to analyse 28S rRNA sequences of basal hexapods. All methods yielded strong support for the monophyly of Collembola, Diplura, Dicondylia and Insecta s.str. , as well as for a cluster composed of Diplura and Protura ('Nonoculata-hypothesis'). However, the last cluster may be an artifact caused by a shared GC bias of the 28S sequences between these orders, in combination with a long branch effect. The instability of the position of the 'Nonoculata' within Pancrustacea further bears out the misleading effect of non-stationarity of nucleotide frequencies. Protura and Diplura either form the sister-group to Collembola (Entognatha) or cluster with branchiopod crustaceans. Overall, the phylogenetic signal of the complete sequences of the 28S rRNA gene favours monophyly of Hexapoda over paraphyly. However, further corroboration from independent data is needed to rule out the competing hypothesis of mutually paraphyletic Crustacea and Hexapoda.     

Is Ellipura monophyletic? A combined analysis of basal hexapod relationships with emphasis on the origin of insects

Hexapoda includes 33 commonly recognized orders, most of them insects. Ongoing controversy concerns the grouping of Protura and Collembola as a taxon Ellipura, the monophyly of Diplura, a single or multiple origins of entognathy, and the monophyly or paraphyly of the silverfish (Lepidotrichidae and Zygentoma s.s.) with respect to other dicondylous insects. Here we analyze relationships among basal hexapod orders via a cladistic analysis of sequence data for five molecular markers and 189 morphological characters in a simultaneous analysis framework using myriapod and crustacean outgroups. Using a sensitivity analysis approach and testing for stability, the most congruent parameters resolve Tricholepidion as sister group to the remaining Dicondylia, whereas most suboptimal parameter sets group Tricholepidion with Zygentoma. Stable hypotheses include the monophyly of Diplura, and a sister group relationship between Diplura and Protura, contradicting the Ellipura hypothesis. Hexapod monophyly is contradicted by an alliance between Collembola, Crustacea and Ectognatha (i.e., exclusive of Diplura and Protura) in molecular and combined analyses.     

Brain organization in Collembola (springtails)

Arthropoda is comprised of four major taxa: Hexapoda, Crustacea, Myriapoda and Chelicerata. Although this classification is widely accepted, there is still some debate about the internal relationships of these groups. In particular, the phylogenetic position of Collembola remains enigmatic. Some molecular studies place Collembola into a close relationship to Protura and Diplura within the monophyletic Hexapoda, but this placement is not universally accepted, as Collembola is also regarded as either the sister group to Branchiopoda (a crustacean taxon) or to Pancrustacea (crustaceans + hexapods). To contribute to the current debate on the phylogenetic position of Collembola, we examined the brains in three collembolan species: Folsomia candida, Protaphorura armata and Tetrodontophora bielanensis, using antennal backfills, series of semi-thin sections, and immunostaining technique with several antisera, in conjunction with confocal laser scanning microscopy and three-dimensional reconstructions. We identified several neuroanatomical structures in the collembolan brain, including a fan-shaped central body showing a columnar organization, a protocerebral bridge, one pair of antennal lobes with 20-30 spheroidal glomeruli each, and a structure, which we interpret as a simply organized mushroom body. The results of our neuroanatomical study are consistent with the phylogenetic position of Collembola within the Hexapoda and do not contradict the hypothesis of a close relationship of Collembola, Protura and Diplura.     

Molecular phylogeny of Collembola inferred from ribosomal RNA genes

The classification of taxa within Collembola (Springtails, Hexapoda) has been controversial. In this study, we combined complete 18S rRNA gene with partial 28S rRNA gene (D7-D10) sequences to investigate the phylogeny of Collembola. About 2500 aligned sites of thirty species representing 29 genera from14 families of Collembola were analyzed, including one species of Neelipleona from which no sequence has been reported previously.The phylogenetic trees were obtained by different methods (maximum parsimony, maximum likelihood, and Bayesian analysis). Our results supported the monophyly of two of the four taxonomic groups of Collembola summarized by Deharveng [Deharveng, L., 2004. Recent advances in Collembola systematics. Pedobiologia 48, 415–433.], namely of Poduromorpha and of Symphypleona. Within Poduromorpha, Neanuridae was monophyletic with high support, but Hypogastruridae was not. Entomobryomorpha was paraphyletic, as the Tomoceroidea (Tomoceridae and Oncopoduridae) was found to be apart from the other entomobryomorphs. In the latter Isotomoidea and Entomobryoidea joined into a group with moderate support. Within Symphypleona, the phylogenetic relationship [(Sminthuridae + Bourletiellidae) + Sminthurididae] was consistent with traditional morphological studies. Neelipleona grouped with Symphypleona in all trees, with moderate support in the ML and Bayesian analyses.     

The phylogenetic positions of three Basal-hexapod groups (protura, diplura, and collembola) based on ribosomal RNA gene sequences

This study combined complete 18S with partial 28S ribosomal RNA gene sequences ( approximately 2,000 nt in total) to investigate the relations of basal hexapods. Ten species of Protura, 12 of Diplura, and 10 of Collembola (representing all subgroups of these three clades) were sequenced, along with 5 true insects and 8 other arthropods, which served as out-groups. Trees were constructed with maximum parsimony, maximum likelihood, Bayesian analysis, and minimum-evolution analysis of LogDet-transformed distances. All methods yielded strong support for a clade of Protura plus Diplura, here named Nonoculata, and for monophyly of the Diplura. Parametric-bootstrapping analysis showed our data to be inconsistent with previous hypotheses (P < 0.01) that joined Protura with Collembola (Ellipura), that said Diplura are sister to true insects or are diphyletic, and that said Collembola are not hexapods. That is, our data are consistent with hexapod monophyly and Collembola grouped weakly with "Protura + Diplura" under most analytical conditions. As a caveat to the above conclusions, the sequences showed nonstationarity of nucleotide frequencies across taxa, so the CG-rich sequences of the diplurans and proturans may have grouped together artifactually; however, the fact that the LogDet method supported this group lessens this possibility. Within the basal hexapod groups, where nucleotide frequencies were stationary, traditional taxonomic subgroups generally were recovered: i.e., within Protura, the Eosentomata and Acerentomata (but Sinentomata was not monophyletic); within Collembola, the Arthropleona, Poduromorpha, and Entomobryomorpha (but Symphypleona was polyphyletic); and in Diplura, the most complete data set (> 2,100 nt) showed monophyly of Campodeoidea and of Japygoidea, and most methods united Projapygoidea with Japygoidea.     

有关节肢动物分类的几个问题

本文简要讨论了近年来有关节肢动物特别是昆虫高级分类研究中争论较大的几个关键问题,包括节肢动物的分类基础、“单肢亚门Ｕｎｉｒａｍｉａ”的单系性、六足总纲的单系性及昆虫纲（狭义）Ｉｎｓｅｃｔａ ｓ．ｓｔｒ．的范围等,以期引起我国动物学者的注意与重视。     

低等六足动物系统学研究进展

低等六足动物包括原尾纲、弹尾纲和双尾纲三个类群,是探讨六足动物起源和进化问题的关键类群,近十年来成为节肢动物系统进化研究中的焦点之一。低等六足动物的系统发育地位以及它们之间的关系一直是备受争论的问题。通过介绍三类低等六足动物最新的分类系统,从经典分类学和系统发育两个方面对低等六足动物近十年来的研究进展进行了综述。迄今,对于三类低等六足动物都建立了比较完备的分类体系,原尾纲划分为3目10科,弹尾纲划分为4目30科,双尾纲划分为2亚目3总科10科。系统发育研究中,大多数的系统发育分析结果不支持传统的缺尾类假说,缺尾纲应摒弃不用。分子数据分析的结果普遍支持原尾纲与双尾纲近缘,但仍需要进一步探讨。线粒体基因组、比较胚胎学和比较精子学的研究结果表明,原尾纲可能经历了长期的趋异进化历史。最近的比较精子学研究支持了双尾纲的单系性。总之,三类低等六足动物系统学研究均取得了长足的发展,但仍然存在诸如研究人员匮乏和研究水平不均衡等问题。系统发育研究中,分子系统学研究成为关注的焦点,而基于核基因和线粒体基因的数据分别建立的系统发育假说存在分歧,亟需开发更优的数据分析方法。此外,需加强低等六足动物比较形态学、比较胚胎学、发育生物学等方面的研究,以便将来进行全证据的系统发育研究。     

The phylogeny of the social Anelosimus spiders (Araneae: Theridiidae) inferred from six molecular loci and morphology

We use fragments of three nuclear genes (Histone 3, 18SrDNA, and 28SrDNA) and three mitochondrial genes (16SrDNA, ND1, and COI) totalling approximately 4.5kb, in addition to morphological data, to estimate the phylogenetic relationships among Anelosimus spiders, well known for their sociality. The analysis includes 67 individuals representing 23 of the 53 currently recognized Anelosimus species and all species groups previously recognized by morphological evidence. We analyse the data using Bayesian, maximum likelihood, and parsimony methods, considering the genes individually as well as combined (mitochondrial, nuclear, and both combined) in addition to a 'total evidence' analysis including morphology. Most of the data partitions are congruent in agreeing on several fundamental aspects of the phylogeny, and the combined molecular data yield a tree broadly similar to an existing morphological hypothesis. We argue that such congruence among data partitions is an important indicator of support that may go undetected by standard robustness estimators. Our results strongly support Anelosimus monophyly, and the monophyly of the recently revised American 'eximius lineage', although slightly altered by excluding A. pacificus. There was consistent support for the scattering of American Anelosimus species in three clades suggesting intercontinental dispersal. Several recently described species are reconstructed as monophyletic, supporting taxonomic decisions based on morphology and behaviour in this taxonomically difficult group. Corroborating previous results from morphology, the molecular data suggest that social species are scattered across the genus and thus that sociality has evolved multiple times, a significant finding for exploring the causes and consequences of social evolution in this group of organisms.     

The problem with "the Paleoptera Problem:" sense and sensitivity

While the monophyly of winged insects (Pterygota) is well supported, phylogenetic relationships among the most basal extant pterygote lineages are problematic. Ephemeroptera (mayflies) and Odonata (dragonflies) represent the two most basal extant lineages of winged insects, and determining their relationship with regard to Neoptera (remaining winged insects) is a critical step toward understanding insect diversification. A recent molecular analysis concluded that Paleoptera (Odonata Ephemeroptera) is monophyletic. However, we demonstrate that this result is supported only under a narrow range of alignment parameters. We have further tested the monophyly of Paleoptera using additional sequence data from 18SrDNA, 28S rDNA, and Histone 3 for a broader selection of taxa and a wider range of analytical methodologies. Our results suggest that the current suite of molecular data ambiguously resolve the three basal winged insect lineages and do not provide independent confirmation of Odonata + Neoptera as supported via morphological data.     

Molecular phylogeny and divergence time of the water penny genus Eubrianax (Coleoptera: Psephenidae) in Japan

The phylogenetic relationships among the Japanese members of the genus Eubrianax (Coleoptera: Psephenidae) were examined using the mitochondrial cytochrome oxidase subunit I (COI) gene and nuclear 28S rRNA gene sequences. Based on the molecular phylogeny as well as morphological features, the species status of Eubrianax brunneicornis Nakane, 1952 was proposed. The phylogenetic analyses recovered monophyly of the previously proposed pellucidus species group with four Japanese species, whereas a single Japanese species of the granicollis group was included in the lineage of the ramicornis group with five Japanese species. The divergence times of the species were estimated by dating the phylogenetic tree against the fossil record and a molecular clock based on the COI gene. The divergence of the Japanese species was inferred to have occurred during the Pliocene epoch.     

From a comb to a tree: phylogenetic relationships of the comb-footed spiders (Araneae, Theridiidae) inferred from nuclear and mitochondrial genes

The family Theridiidae is one of the most diverse assemblages of spiders, from both a morphological and ecological point of view. The family includes some of the very few cases of sociality reported in spiders, in addition to bizarre foraging behaviors such as kleptoparasitism and araneophagy, and highly diverse web architecture. Theridiids are one of the seven largest families in the Araneae, with about 2200 species described. However, this species diversity is currently grouped in half the number of genera described for other spider families of similar species richness. Recent cladistic analyses of morphological data have provided an undeniable advance in identifying the closest relatives of the theridiids as well as establishing the family's monophyly. Nevertheless, the comb-footed spiders remain an assemblage of poorly defined genera, among which hypothesized relationships have yet to be examined thoroughly. Providing a robust cladistic structure for the Theridiidae is an essential step towards the clarification of the taxonomy of the group and the interpretation of the evolution of the diverse traits found in the family. Here we present results of a molecular phylogenetic analysis of a broad taxonomic sample of the family (40 taxa in 33 of the 79 currently recognized genera) and representatives of nine additional araneoid families, using approximately 2.5kb corresponding to fragments of three nuclear genes (Histone 3, 18SrDNA, and 28SrDNA) and two mitochondrial genes (16SrDNA and CoI). Several methods for incorporating indel information into the phylogenetic analysis are explored, and partition support for the different clades and sensitivity of the results to different assumptions of the analysis are examined as well. Our results marginally support theridiid monophyly, although the phylogenetic structure of the outgroup is unstable and largely contradicts current phylogenetic hypotheses based on morphological data. Several groups of theridiids receive strong support in most of the analyses: latrodectines, argyrodines, hadrotarsines, a revised version of spintharines and two clades including all theridiids without trace of a colulus and those without colular setae. However, the interrelationships of these clades are sensitive to data perturbations and changes in the analysis assumptions.     

Molecular phylogeny of the family Tephritidae (Insecta: Diptera): New insight from combined analysis of the mitochondrial 12S, 16S,and COII genes

The phylogeny of the family Tephritidae (Diptera: Tephritidae) was reconstructed from mitochondrial 12S, 16S, and COII gene fragments using 87 species, including 79 tephritid and 8 outgroup species. Minimum evolution and Bayesian trees suggested the following phylogenetic relationships: (1) A sister group relationship between Ortalotrypeta and Tachinisca, and their basal phylogenetic position within Tephritidae; (2) a sister group relationship between the tribe Acanthonevrini and Phytalmiini; (3) monophyly of Plioreocepta, Taomyia and an undescribed new genus, and their sister group relationship with the subfamily Tephritinae; (4) a possible sister group relationship of Cephalophysa and Adramini; and (5) reconfirmation of monophyly for Trypetini, Carpomyini, Tephritinae, and Dacinae. The combination of 12S, 16S, and COII data enabled resolution of phylogenetic relationships among the higher taxa of Tephritidae.     

Phylogenetic analysis of mitochondrial protein coding genes confirms the reciprocal paraphyly of Hexapoda and Crustacea

Background

The phylogeny of Arthropoda is still a matter of harsh debate among systematists, and significant disagreement exists between morphological and molecular studies. In particular, while the taxon joining hexapods and crustaceans (the Pancrustacea) is now widely accepted among zoologists, the relationships among its basal lineages, and particularly the supposed reciprocal paraphyly of Crustacea and Hexapoda, continues to represent a challenge. Several genes, as well as different molecular markers, have been used to tackle this problem in molecular phylogenetic studies, with the mitochondrial DNA being one of the molecules of choice. In this study, we have assembled the largest data set available so far for Pancrustacea, consisting of 100 complete (or almost complete) sequences of mitochondrial genomes. After removal of unalignable sequence regions and highly rearranged genomes, we used nucleotide and inferred amino acid sequences of the 13 protein coding genes to reconstruct the phylogenetic relationships among major lineages of Pancrustacea. The analysis was performed with Bayesian inference, and for the amino acid sequences a new, Pancrustacea-specific, matrix of amino acid replacement was developed and used in this study.

Results

Two largely congruent trees were obtained from the analysis of nucleotide and amino acid datasets. In particular, the best tree obtained based on the new matrix of amino acid replacement (MtPan) was preferred over those obtained using previously available matrices (MtArt and MtRev) because of its higher likelihood score. The most remarkable result is the reciprocal paraphyly of Hexapoda and Crustacea, with some lineages of crustaceans (namely the Malacostraca, Cephalocarida and, possibly, the Branchiopoda) being more closely related to the Insecta s.s. (Ectognatha) than two orders of basal hexapods, Collembola and Diplura. Our results confirm that the mitochondrial genome, unlike analyses based on morphological data or nuclear genes, consistently supports the non monophyly of Hexapoda.

Conclusion

The finding of the reciprocal paraphyly of Hexapoda and Crustacea suggests an evolutionary scenario in which the acquisition of the hexapod condition may have occurred several times independently in lineages descending from different crustacean-like ancestors, possibly as a consequence of the process of terrestrialization. If this hypothesis was confirmed, we should therefore re-think our interpretation of the evolution of the Arthropoda, where terrestrialization may have led to the acquisition of similar anatomical features by convergence. At the same time, the disagreement between reconstructions based on morphological, nuclear and mitochondrial data sets seems to remain, despite the use of larger data sets and more powerful analytical methods.

     

Morphology and molecular phylogeny of Prasiola sp. (Prasiolales, Chlorophyta) from Myanmar

Morphological and molecular phylogenetic studies were carried out on a freshwater green alga from Myanmar. Most specimens exhibited a gross morphology similar to Enteromorpha, however, their thalli were basically monostromatic and chloroplasts were axile and stellate. In addition, the phylogenetic analysis inferred from the 18S rRNA gene strongly supported a monophyly with Prasiola japonica and a more distant relationship with other chlorophytan taxa, including Enteromorpha. From our results, it has been shown that this alga belongs to Prasiola and has a close phylogenetic relationship with P. japonica.     

Phylogeny of the mud worm genus Marenzelleria (Polychaeta, Spionidae) inferred from mitochondrial DNA sequences

The taxonomy of the cryptic morphospecies of the mud worm genus Marenzelleria is particularly difficult and the phylogenetic relationship within the genus is unknown. Herein we reconstructed the phylogeny of all five species of this genus using sequence data of three mitochondrial genes (16SrDNA, cytochrome b, cytochrome oxidase subunit I) from 104 specimens out of 26 populations. For the three invasive species of the genus, Marenzelleria neglecta, M. viridis and M. arctia , individuals from native populations as well as from recently invaded populations were included. Nuclear 18S rDNA sequences were used to evaluate the appropriate outgroup taxon among several spionid polychaete species. The results supported the monophyly of Marenzelleria , and Malacoceros fuliginosus was found to be a suitable outgroup for the analysis of the mitochondrial gene segments. All phylogenetic reconstructions revealed a basal position of M. arctia and M. wireni , which have primarily Arctic distribution, with M. arctia obtaining the most basal position. Together with the present-day distribution of the species, this indicates an origin of the genus in the Arctic region. The relationship of the species M. neglecta , M. viridis and M. bastropi could not be resolved sufficiently due to genealogical discordance that might reflect relatively young cladogenetic events.     

Phylogeny and biogeography of the genus Cephalenchus (Tylenchomorpha,Nematoda)

The phylogenetic position of Cephalenchus is enigmatic with respect to other tylench nematodes. In this study, Cephalenchus populations representing 11 nominal species were sampled worldwide for molecular and morphological characterization. Species identification was based on light microscopy (LM) and scanning electron microscopy (SEM). Molecular analyses were based on the genes (i.e. 18S, 28S, 5.8S) and internal transcribed spacers (ITS‐1 and ITS‐2) of the ribosomal RNA (rRNA). Phylogenetic analyses (i.e. full and reduced alignments) of either concatenated or single genes always supported the monophyly of Cephalenchus. A sister relationship between Cephalenchus and Eutylenchus excretorius was recovered by most analyses, although branch support varies depending on the dataset used. The position of Cephalenchus + E. excretorius within Tylenchomorpha nevertheless remains ambiguous, thus highlighting the importance of sampling additional genes as well as taxa. Placement of Cephalenchus + E. excretorius as sister of Tylenchinae or Boleodorinae could not be rejected on the basis of 18S and 28S rRNA genes. Within Cephalenchus, amphidial opening morphology shows congruence with molecular‐based phylogenetic relationships, whereas the number of lines in the lateral field is likely to be a convergent trait. Morphometric analyses clearly distinguished short tail from medium–long tail species, and SEM observations seem to suggest a relation between tail length and amphidial opening. In addition, molecular phylogenies support the non‐monophyly of Cephalenchus cephalodiscus, Cephalenchus cylindricus, Cephalenchus daisuce and Cephalenchus leptus. The known extent of Cephalenchus diversity is increased with the inclusion of two new species, and the biogeography of the genus is discussed.     

基于18S rRNA基因序列的直翅目主要类群系统发育关系研究

基于78种直翅目昆虫的18S rRNA基因全序列构建了直翅目各主要类群间的系统发育关系。本研究的结果支持直翅目的单系性,但不支持蝗亚目和螽亚目各自的单系性;直翅目下除蜢总科和蝗总科外各总科的划分多数与Otte系统相一致;蜢总科的单系性得不到支持;蝗总科的剑角蝗科、斑腿蝗科、斑翅蝗科、网翅蝗科和槌角蝗科5科均不是单系群,各物种间的遗传距离差异不大,应合并为一科,即蝗科;本研究支持将Otte系统中蚱总科和螽蟖总科下各亚科级阶元提升为科级阶元;18S rRNA基因全序列可以作为划分科级阶元的工具,当位于同一分支上互成姐妹群的类群间的遗传距离超过1%时,这几个类群属于不同的科;但由于其在进化上的保守性,18S rRNA基因只能用于纲目等高级阶元间关系的研究,而由其获得的总科以下阶元间的关系并不可靠。     

The ant subfamily Pseudomyrmecinae (Hymenoptera: Formicidae): phylogeny and evolution of big-eyed arboreal ants

Abstract. The ant subfamily Pseudomyrmecinae comprises three genera of hyperoptic, arboreal ants, widely distributed in tropical and subtropical regions: Pseudomyrmex (∼200 species, New World), Myrcidris (two species, South America) and Tetraponera (∼100 species, Palaeotropics). The phylogenetic relationships among these ants were investigated using DNA sequence data (∼5.2 kb from 18S rDNA, 28S rDNA, wingless, abdominal-A, and long-wavelength rhodopsin genes) and 144 morphological characters, both separately and in combination. Data were gathered from a representative set of forty-nine pseudomyrmecine species, plus eighteen species from various outgroups. There was substantial agreement among the results obtained from different datasets, and from different methods of phylogenetic inference (parsimony, Bayesian inference). The monophyly of the following groups is strongly supported (100% bootstrap support and 1.00 posterior probability in the molecular dataset): Pseudomyrmecinae, Pseudomyrmex, and Pseudomyrmex + Myrcidris. The status of the genus Tetraponera is less clear: the DNA sequence data indicate that the genus is paraphyletic, but morphological features and a unique insertion in the 28S gene support the monophyly of this taxon. Seven of nine Pseudomyrmex species groups, established previously on the basis of morphology alone, are strongly upheld, but monophyly is rejected for the P. pallens group and the P. viduus group. In the latter case, molecular evidence indicates the existence of two independent clades, associated with the ant-plants Triplaris and Tachigali, respectively, whose convergent morphological features had caused them to be placed erroneously in the same species group. The present results confirm an earlier assertion that obligate associations with domatia-bearing plants have arisen at least twelve times in the subfamily. Molecular and morphological data support the hypothesis of a sister-group relationship between Pseudomyrmecinae and Myrmeciinae (84% parsimony bootstrap, combined dataset), which implies a Cretaceous origin of the stem-group pseudomyrmecines in the southern hemisphere. Pseudomyrmecines appear to have arisen in the Palaeotropics and later dispersed from Africa to South America, where they experienced a pronounced burst of diversification.