Two ancient bacterial endosymbionts have coevolved with the planthoppers (Insecta: Hemiptera: Fulgoroidea)

Background

Pseudoscorpions are chelicerates and have historically been viewed as being most closely related to solifuges, harvestmen, and scorpions. No mitochondrial genomes of pseudoscorpions have been published, but the mitochondrial genomes of some lineages of Chelicerata possess unusual features, including short rRNA genes and tRNA genes that lack sequence to encode arms of the canonical cloverleaf-shaped tRNA. Additionally, some chelicerates possess an atypical guanine-thymine nucleotide bias on the major coding strand of their mitochondrial genomes.

Results

We sequenced the mitochondrial genomes of two divergent taxa from the chelicerate order Pseudoscorpiones. We find that these genomes possess unusually short tRNA genes that do not encode cloverleaf-shaped tRNA structures. Indeed, in one genome, all 22 tRNA genes lack sequence to encode canonical cloverleaf structures. We also find that the large ribosomal RNA genes are substantially shorter than those of most arthropods. We inferred secondary structures of the LSU rRNAs from both pseudoscorpions, and find that they have lost multiple helices. Based on comparisons with the crystal structure of the bacterial ribosome, two of these helices were likely contact points with tRNA T-arms or D-arms as they pass through the ribosome during protein synthesis. The mitochondrial gene arrangements of both pseudoscorpions differ from the ancestral chelicerate gene arrangement. One genome is rearranged with respect to the location of protein-coding genes, the small rRNA gene, and at least 8 tRNA genes. The other genome contains 6 tRNA genes in novel locations. Most chelicerates with rearranged mitochondrial genes show a genome-wide reversal of the CA nucleotide bias typical for arthropods on their major coding strand, and instead possess a GT bias. Yet despite their extensive rearrangement, these pseudoscorpion mitochondrial genomes possess a CA bias on the major coding strand. Phylogenetic analyses of all 13 mitochondrial protein-coding gene sequences consistently yield trees that place pseudoscorpions as sister to acariform mites.

Conclusion

The well-supported phylogenetic placement of pseudoscorpions as sister to Acariformes differs from some previous analyses based on morphology. However, these two lineages share multiple molecular evolutionary traits, including substantial mitochondrial genome rearrangements, extensive nucleotide substitution, and loss of helices in their inferred tRNA and rRNA structures.     

Seasonal aspects of the life cycle of solifuges (Arachnida, Solifugae) as compared with pseudoscorpions (Arachnida, Pseudoscorpiones)

Solifuges (order Solifugae) and pseudoscorpions (order Pseudoscorpiones) united into the superorder Haplocnemata (Shultz, 2007) are nevertheless characterized by essential differences both in morphological and biological characters. Analysis of available information on the biology and life cycles of these arachnids revealed a clear difference between the daily rhythms of activity: their presence in solifuges and their absence in pseudoscorpions. However, this concerning the seasonal adaptations in the two orders is not simple since they demonstrate not only differences but also a lot of similarities. All the studied solifuges are characterized by the seasonally timed stenochronous (heterodynamic) type of development which is characteristic of species with uni-, bi-, and semi-voltine development (i.e., to life cycles completed within a year, half a year, and several years), as well as to species combining different forms of voltinism. This type of development is not only prevalent in solifuges (as in pseudoscorpions and other arachnids) but appears to be the only one, since no cases of eurychronous (homodynamic) development have been found in solifuges; whereas pseudoscorpions and other arachnids develop both steno- and eurychronously. The initial ontogenetic stages remain in underground shelters (brood burrows in solifuges and brood chambers in pseudoscorpions). The first nymphal stages (I instar nymphs in solifuges, protonymphs in pseudoscorpions) are embryonized; active life outside the brood burrows starts with II instar nymphs in solifuges and with deutonymphs in pseudoscorpions.     

Reinterpretation of Dracochela deprehendor (Arachnida: Pseudoscorpiones) as a stem‐group pseudoscorpion

Abstract: The middle Devonian (Givetian–Eifelian) pseudoscorpion Dracochela deprehendor Schawaller, Shear and Bonamo is redescribed from the type material and an additional palpal fragment. Dracochela differs from extant pseudoscorpions in having numerous spinules on the leg tarsi, the femur at least as long as the patella on the posterior legs, the stem of the arolia thick, most blades of the serrulae only weakly fused and in lacking a spinneret on the chelicera. The blades of the cheliceral rallum are shown to have been arranged in two rows, as in most Heterosphyronida. The cheliceral serrulae are compared with analogous structures in other arachnids (Notostigmata, Opiliones, Palpigradi, Schizomida and Scorpiones), and it is concluded that the panctenal state (all lamellae attached to finger) is plesiomorphic relative to the hemictenal state (apical lamellae raised), which has evolved independently in Heterosphyronida and Neobisioidea. The trichobothriotaxy of the chela of Dracochela is shown to be similar to that of the extant family Pseudotyrannochthoniidae. The growth of the chelal fingers followed the same pattern as that seen in modern pseudoscorpions, with most of the increase in length occurring at the base of the fingers. The family Dracochelidae Schawaller, Shear and Bonamo is treated as a plesion and assigned to the stem‐group of Pseudoscorpiones. The ordinal name Chelonethi Thorell is restricted to crown‐group pseudoscorpions, and the superordinal name Pseudoscorpiones Latreille is adopted for the total‐group (i.e. stem‐group plus Chelonethi).     

The first cytogenetic characterization of atemnids: pseudoscorpions with the highest chromosome numbers (arachnida: pseudoscorpiones)

The karyotypes of pseudoscorpions of the family Atemnidae (Arachnida: Pseudoscorpiones) were studied for the first time. Karyotype data for 7 species have been obtained. The diploid chromosome numbers of most species considerably exceed the numbers reported in pseudoscorpions so far, with males ranging between 65 and 143. In spite of this, the sex chromosome system of atemnids is characterized by the same features that are found in the majority of other pseudoscorpions with an X0 system; the X chromosome is metacentric and is the largest chromosome or one of the largest chromosomes of the karyotype. Male meiotic cells of Atemnus politus contain 1 or 2 autosome multivalents; most specimens had 2 multivalents. The multivalents were composed of 4, 6, 8 or 10 chromosomes. Multivalent number and structure was consistent within each of the studied individuals. The same number of chromosomes in all of the males examined suggests that multivalents are generated by reciprocal translocations. The high diversity of multivalents suggests considerable range of translocation heterozygosity in the studied population.     

中国伪蝎名录（蛛形纲：伪蝎目）

本文根据我国伪蝎目已有种类记述,归纳并列出10科34属73种（包括亚种）伪蝎的中文名、拉丁学名、引证及其地理分布,以供今后研究参考。     

Phylogenetic position of Protoopalina intestinalis based on SSU rRNA gene sequence

A robust recognition of phylogenetic affinities of Opalinidae-the peculiar multinucleated intestine commensals of frogs-is hindered by the absence of reliable molecular data. Up to now all attempts to sequence opalinid genes failed, as the obtained sequences labeled as Protoopalina intestinalis, Cepedea virguloidea, and Opalina ranarum in GenBank apparently originate from a zygomycete contamination. In this paper, we present the first molecular data for the family Opalinidae-SSU rRNA gene of P. intestinalis. Our phylogenetic analyses undoubtedly show opalinids as a sister group to Proteromonas within the Stramenopila clade, confirming the monophyly of Patterson's order Slopalinida. The enigmatic genus Blastocystis is resolved with high statistical support as a sister group to Slopalinida. The information contained in the SSU rRNA gene proved insufficient to uncover broader affinities of this group to other groups of Stramenopila. Nevertheless, our analyses clearly demonstrate that Cavalier-Smith's phylum Bigyra, which comprises Oomycetes and their relatives together with Slopalinida and Blastocystis, is not monophyletic.     

Morphology of locomotor appendages in Arachnida: evolutionary trends and phylogenetic implications

The morphological diversity of locomotor appendages in Arachnida is surveyed lo reconstruct phylogenetic relationships and discover evolutionary trends in form and function. The appendicular skeleton and musculature of representatives from the ten living arachnid orders ate described, and a system of homology is proposed. Character polarities are established through comparison with an outgroup. Limulus polyphemus Xiphosura). Cladistic analysis suggests that Arachnida is monophyletic and that absence of extensor muscles is a primitive condition. Extensors are primitively absent in Araneae. Amblypygi, Uropygi, Palpigradi, Ricinulei and Acari. Most similarities in the appendages of these orders are symplesiomorphic so that phylogenetic relationships among the 'extensorless' groups cannot be resolved solely on the basis of appendicular characters. Extensor muscles appear to have evolved once, and their presence is considered a synapomorphic feature of Opiliones, Scorpiones, Pseudoscorpiones and Solifugae. Solifugae lack extensors, but a parsimonious interpretation of other characters indicates that this is a secondary, derived condition. The phylogenetic relationships among these four orders are clarified by modifications of the patellotibial joint. Cladistic analysis indicates that Opiliones may be the sister group of the other three orders and that Scorpiones is the sister group of Pseudoscorpiones and Solifugae. Conclusions concerning phylogenetic relationships and evolutionary morphology presented here differ substantially from those of earlier studies on the locomotor appendages of Arachnida.     

Morphology of locomotor appendages in Arachnida: evolutionary trends and phylogenetic implications

The morphological diversity of locomotor appendages in Arachnida is surveyed lo reconstruct phylogenetic relationships and discover evolutionary trends in form and function. The appendicular skeleton and musculature of representatives from the ten living arachnid orders ate described, and a system of homology is proposed. Character polarities are established through comparison with an outgroup. Limulus polyphemus Xiphosura). Cladistic analysis suggests that Arachnida is monophyletic and that absence of extensor muscles is a primitive condition. Extensors are primitively absent in Araneae. Amblypygi, Uropygi, Palpigradi, Ricinulei and Acari. Most similarities in the appendages of these orders are symplesiomorphic so that phylogenetic relationships among the ‘extensorless’ groups cannot be resolved solely on the basis of appendicular characters. Extensor muscles appear to have evolved once, and their presence is considered a synapomorphic feature of Opiliones, Scorpiones, Pseudoscorpiones and Solifugae. Solifugae lack extensors, but a parsimonious interpretation of other characters indicates that this is a secondary, derived condition. The phylogenetic relationships among these four orders are clarified by modifications of the patellotibial joint. Cladistic analysis indicates that Opiliones may be the sister group of the other three orders and that Scorpiones is the sister group of Pseudoscorpiones and Solifugae. Conclusions concerning phylogenetic relationships and evolutionary morphology presented here differ substantially from those of earlier studies on the locomotor appendages of Arachnida.     

The road to the Janiroidea: Comparative morphology and evolution of the asellote isopod crustaceans

This paper presents a new phylogenetic estimate of isopod crustaceans of the suborder Asellota with the aim of clarifying the evolution of the superfamily Janiroidea, a large and diverse group inhabiting all aqueous habitats. The phylogenetic analysis is based on a morphological evaluation of characters used in past classifications, as well as several new characters. The evolutionary polarity of the characters was determined by outgroup analysis. The characters employed were from the pleopods, the copulatory organs, the first walking legs, and the cephalon. The resulting character data set was analyzed with numerical phylogenetic computer programs to find one most parsimonious clado-gram, which is translated into a classification using the sequencing convention. The new phylogenetic estimate is significantly more parsimonious than previous trees from the literature, and several of its monophyletic groups have robust confidence limits. The superfamily Stenetrioidea belongs to the clade including the Janiroidea, not with the Aselloidea as previously suggested. The sister group of the Janiroidea is the family Pseudojaniridae, which is elevated to superfamily rank. The clade including the families Gnathostenetroididae and Protojaniridae is not the sister group of the Janiroidea, and is derived earlier in janiroidean evolution than the Stenetrioidea. Within the Janiroidea, the family Janiridae is not the most primitive taxon as previously believed. The clade including the families Munnidae and Pleurocopidae contains the earliest derived janiroideans. The data also indicate that the unusual sexual morphology of the Janiroidea did not appear suddenly but developed as a series of independent steps within the Asellota.     

Phylogenetic relationships among the Malagasy lemuriforms (Primates: Strepsirrhini) as indicated by mitochondrial sequence data from the 12S rRNA gene

Numerous phylogenetic hypotheses have been advanced for the Malagasy lemuriform radiation, drawing on data from morphology, physiology, behaviour and molecular genetics. Almost all possible relationships have been proposed and most nodes have been contested. We present a phylogenetic analysis, using several analytical methods, of a partial sequence from the 12s rRNA mitochondrial gene. This gene codes for the small ribosomal subunit, and functional constraints require that the secondary structure of the molecule is strongly conserved, which in inturn exerts constraints on the primary sequence structure. Although previous studies have suggested a very wide range of phylogenetic applicability for this molecule, our results indicate that it is most useful in strepsirrhine primates for estimating relationships among genera within families and among relatively recently diverged families (mean sequence divergence about 11%). Relationships among families separated by larger genetic distances (>12% divergence; e.g. Cheirogaleidae, Daubentoniidae, Megaladapidae) are difficult to resolve consistently. Our data show strong support for an Indridae-Lemuridae sister group and for monophyly of the Lemuridae with Varecia as the sister to all other lemurids. They also support, albeit less strongly, sister group relationships between Lemur and Hapalemur within the Lemuridae and between PmpLthecus and Avahi in the Indridae.     

Phylogeny of mysticete whales based on mitochondrial and nuclear data

Mysticetes or baleen whales are comprised of four groups: Eschrichtiidae, Neobalaenidae, Balaenidae, and Balaenopteridae. Various phylogenetic hypotheses among these four groups have been proposed. Previous studies have not satisfactorily determined relationships among the four groups with a high degree of confidence. The objective of this study is to determine the relationships among the mysticete whales. Mitochondrial and nuclear DNA were sequenced for phylogenetic analysis. Most species relationships determined using these data were well resolved and congruent. Balaenidae is the most basal group and Neobalaenidae is the second most basal and sister group to the balaenopterid-eschrichtiid clade. In this phylogenetic study, the resolution of Eschrichtiidae with two main lineages of Balaenopteridae was problematic. Some data partitions placed this group within the balaenopterids, and other partitions placed it as a sister taxon to the balaenopterids. An additive likelihood approach was used to determine the most optimal trees. Although it was not found in the combined phylogenetic analyses, the "best" tree found under the additive likelihood approach was one with a monophyletic Balaenopteridae.     

Using RAD seq for reconstructing phylogenies of highly diverged taxa: A test using the tribe Scandiceae (Apiaceae)

The angiosperm Apiaceae tribe Scandiceae includes four major clades—subtribes Daucinae, Ferulinae, Torilidinae, and Scandicinae—that originated ca. 20 Mya. Although all four subtribes are highly supported in molecular analyses, and morphological data indicate a sister relationship between Daucinae and Torilidinae, their branching order has not been resolved using standard Sanger multilocus data. Therefore, in this study, we test the utility of genomic RAD seq data in resolving deep phylogenetic relationships (up to 20 Mya) in Apiaceae subfamily Apioideae, with special emphasis on tribe Scandiceae using 12 representative species. We used two bioinformatic pipelines, pyRAD and RADIS (based on STACKS), to assemble RAD seq data and we tested the influence of various combinations of parameters on the robustness of the inferred tree topologies. Although different data processing approaches produced alignments with various amounts of missing data, they converged to two well‐supported topologies, irrespective of the phylogenetic method applied. Highly supported trees showed Scandicinae as sister to all other clades and indicated that Daucinae and Torilidinae are sister groups, thus confirming the relationship inferred from morphology. We conclude that the RAD seq method can be successfully used to resolve deep relationships formed 20 Mya within Apiaceae. We provide recommendations for parameter settings in RADIS and pyRAD for the analysis of taxa that have accumulated considerable genomic divergence.     

The impact of parsimony weighting schemes on inferred relationships among toucans and neotropical barbets (Aves: piciformes)

The development of new schemes for weighting DNA sequence data for phylogenetic analysis continues to outpace the development of consensus on the most appropriate weights. The present study is an exploration of the similarities and differences between results from 22 character weighting schemes when applied to a study of barbet and toucan (traditional avian families Capitonidae and Ramphastidae) phylogenetic relationships. The dataset comprises cytochrome b sequences for representatives of all toucan and Neotropical barbet genera, as well as for several genera of Paleotropical barbets. The 22 weighting schemes produced conflicting patterns of relationship among taxa, often with conflicting patterns each receiving strong bootstrap support. Use of multiple weighting schemes helped to identify the source within the dataset (codon position, transitions, transversions) of the various putative phylogenetic signals. Importantly, some phylogenetic hypotheses were consistently supported despite the wide range of weights employed. The use of phylogenetic frameworks to summarize the results of these multiple analyses proved very informative. Relationships among barbets and toucans inferred from these data support the paraphyly of the traditional Capitonidae. Additionally, these data support paraphyly of Neotropical barbets, but rather than indicating a relationship between Semnornis and toucans, as previously suggested by morphological data, most analyses indicate a basal position of Semnornis within the Neotropical radiation. The cytochrome b data also allow inference of relationships among toucans. Supported hypotheses include Ramphastos as the sister to all other toucans, a close relationship of Baillonius and Pteroglossus with these two genera as the sister group to an (Andigena, Selenidera) clade, and the latter four genera as a sister group to Aulacorhynchus.     

Adaptations for matrotrophy in the female reproductive system in the pseudoscorpion Chelifer cancroides (Chelicerata: Pseudoscorpiones,Cheliferidae)

Pseudoscorpiones (pseudoscorpions, false scorpions) is an order of small terrestrial chelicerates. While most chelicerates are lecithotrophic, that is, embryos develop due to nutrients (mostly yolk) deposited in the oocyte cytoplasm, pseudoscorpions are matrotrophic, that is, embryos are nourished by the female. Pseudoscorpion oocytes contain only a small amount of yolk. The embryos develop within a brood sac carried on the abdominal site of the female and absorb nutrients by a pumping organ. It is believed that in pseudoscorpions nutrients for developing embryos are produced in the ovary during a postovulatory (secretory) phase of the ovarian cycle. The goal of our study was to analyze the structure of the female reproductive system during the secretory phase in the pseudoscorpion Chelifer cancroides, a representative of the family Cheliferidae, considered to be one of the most advanced pseudoscorpion taxa. We use diverse microscopic techniques to document that the nutritive fluid is produced not only in the ovaries but also by the epithelial cells in the oviducts. The secretory active epithelial cells are hypertrophic and polyploid and release their content by fragmentation of apical parts. Our observations also indicate that fertilization occurs in the oviducts. Moreover, in contrast to previous findings, we show that secretion of the nutritive material starts when the fertilized oocytes reach the brood sac and thus precedes formation of the pumping organ. Summing up, we show that C. cancroides exhibits traits of advanced adaptations for matrotrophy due to coordinated secretion of the nutritive fluid by the ovarian and oviductal epithelial cells, which substantially increases the efficiency of nutritive fluid formation. Since the secretion of nutrients starts before formation of the pumping organ, we suggest that the embryos are able to absorb the nutritive fluid also in the early embryonic stages.     

The position of Cetacea within mammalia: phylogenetic analysis of morphological data from extinct and extant taxa

Knowledge of the phylogenetic position of the order Cetacea (whales, dolphins, and porpoises) within Mammalia is of central importance to evolutionary biologists studying the transformations of biological form and function that accompanied the shift from fully terrestrial to fully aquatic life in this clade. Phylogenies based on molecular data and those based on morphological data both place cetaceans among ungulates but are incongruent in other respects. Morphologists argue that cetaceans are most closely related to mesonychians, an extinct group of terrestrial ungulates. They have disagreed, however, as to whether Perissodactyla (odd-toed ungulates) or Artiodactyla (even-toed ungulates) is the extant clade most closely related to Cetacea, and have long maintained that each of these orders is monophyletic. The great majority of molecule-based phylogenies show, by contrast, not only that artiodactyls are the closest extant relatives of Cetacea, but also that Artiodactyla is paraphyletic unless cetaceans are nested within it, often as the sister group of hippopotamids. We tested morphological evidence for several hypotheses concerning the sister taxon relationships of Cetacea in a maximum parsimony analysis of 123 morphological characters from 10 extant and 30 extinct taxa. We advocate treating certain multistate characters as ordered because such a procedure incorporates information about hierarchical morphological transformation. In all most-parsimonious trees, whether multistate characters are ordered or unordered, Artiodactyla is the extant sister taxon of Cetacea. With certain multistate characters ordered, the extinct clade Mesonychia (Mesonychidae + Hapalodectidae) is the sister taxon of Cetacea, and Artiodactyla is monophyletic. When all fossils are removed from the analysis, Artiodactyla is paraphyletic with Cetacea nested inside, indicating that inclusion of mesonychians and other extinct stem taxa in a phylogenetic analysis of the ungulate clade is integral to the recovery of artiodactyl monophyly. Phylogenies derived from molecular data alone may risk recovering inconsistent branches because of an inability to sample extinct clades, which by a conservative estimate, amount to 89% of the ingroup. Addition of data from recently described astragali attributed to cetaceans does not overturn artiodactyl monophyly.     

Molecular phylogeny of parasitic zygomycota (Dimargaritales, zoopagales) based on nuclear small subunit ribosomal DNA sequences

We analyzed sequence data of the 18S rDNA gene from representatives of nine mycoparasitic or zooparasitic genera to infer the phylogenetic relationships of these fungi within the Zygomycota. Phylogenetic analyses identified a novel monophyletic clade consisting of the Zoopagales, Kickxellales, Spiromyces, and Harpellales. Analyses also identified a monophyletic mycoparasitic-zooparasitic Zoopagales clade in which Syncephalis, Thamnocephalis, and Rhopalomyces form a sister group to a Piptocephalis-Kuzuhaea clade. Although monophyly of the mycoparasitic Dimargaritales received strong bootstrap and decay index support, phylogenetic relationships of this order could not be resolved because of the unusually high rate of base substitutions within the 18S rDNA gene. Overall, the 18S gene tree topology is weak, as reflected by low bootstrap and decay index support for virtually all internal nodes uniting ordinal and superordinal taxa. Nevertheless, the 18S rDNA phylogeny is mostly consistent with traditional phenotypic-based classification schemes of the Fungi.     

A molecular approach to arthrotardigrade phylogeny (Heterotardigrada,Tardigrada)

The marine order Arthrotardigrada (class Heterotardigrada, phylum Tardigrada) is known for its conspicuously high morphological diversity and has been traditionally recognized as the most ancestral group within the phylum. Despite its potential importance in understanding the evolution of the phylum, the phylogenetic relationships of Arthrotardigrada have not been clarified. This study conducted molecular phylogenetic analyses of the order encompassing all families except Neoarctidae using nuclear 18S and 28S rRNA fragments. Data from two rare families, Coronarctidae and Renaudarctidae, were included for the first time. The analyses confirmed the monophyly of Heterotardigrada and inferred Coronarctidae as the sister group to all other heterotardigrade taxa. Furthermore, the results support a monophyletic Renaudarctidae + Stygarctidae clade, which has been previously suggested on morphology. Our data indicated that two subfamilies currently placed in Halechiniscidae are only distantly related to this family. We propose that these taxa are each elevated to family level (Styraconyxidae (new rank) and Tanarctidae (new rank)). The morphology of tardigrades is discussed in the context of the inferred phylogeny.     

Phylogeny of the millipede order Spirobolida (Arthropoda: Diplopoda: Helminthomorpha)

This study examines relationships within the millipede order Spirobolida using an exemplar approach, sampling within families to maximize geographical and morphological diversity; due to lack of available material, Allopocockiidae and Hoffmanobolidae were not included in analyses. The focus of this study was to test monophyly of the order, the suborders, and the families of Spirobolida and to propose interfamilial relationships using morphological and molecular data in a total‐evidence approach. Both maximum‐parsimony analyses and Bayesian inference were employed to analyse two datasets consisting of combined morphological and molecular data, one aligned using progressive alignment methods and the second aligned by secondary structure models. Rhinocricidae was recovered sister to all remaining spirobolidan millipedes and is elevated to suborder status as suborder Rhinocricidea. Trigoniulidea was recovered as monophyletic as was Spirobolidea excluding Rhinocricidae; Spirobolidea is redefined to reflect this change. All previously recognized families were recovered, with the exception of Spirobolidae; in all instances, this family was paraphyletic or part of a polytomy that lacked sufficient resolution to assess its monophyly. The results reaffirm much of the existing taxonomic foundation within Spirobolida. This study provides the first phylogenetic test of higher‐level relationships within Spirobolida and will serve as a foundation for future work in this group at finer levels. © The Willi Hennig Society 2010.     

Phylogenetic relationships of African microhylid frogs inferred from DNA sequences of mitochondrial 12S and 16S rRNA genes

The phylogenetic relationships of microhylid frogs are poorly understood. The first molecular phylogeny for continental African microhylids is presented, including representatives of all subfamilies, six of the eight genera, and the enigmatic hemisotid Hemisus. Mitochondrial 12S and 16S rRNA sequence data were analysed using parsimony, likelihood and Bayesian methods. Analyses of the data are consistent with the monophyly of all sampled subfamilies and genera. Hemisus does not nest within either brevicipitines or non-brevicipitines. It is possibly the sister group to brevicipitines, in which case brevicipitines might not be microhylids. Phrynomantis and Hoplophryne potentially group with non-African, non-brevicipitine microhylids, in agreement with recent morphological and molecular data. Within brevicipitines, Breviceps is recovered as the sister group to a clade of Callulina+Spelaeophryne+Probreviceps. The relationships among the genera within this latter clade are unclear, being sensitive to the method of analysis. Optimal trees suggest the Probreviceps macrodactylus subspecies complex might be paraphyletic with respect to P. uluguruensis, corroborating preliminary morphological studies indicating that P. m. rungwensis may be a distinct species. P. m. loveridgei may be paraphyletic with respect to P. m. macrodactylus, though this is not strongly supported. Some biogeographic hypotheses are examined in light of these findings.