Phylogenomics of the lepidopteran endoparasitoid wasp subfamily Rogadinae (Hymenoptera: Braconidae) and related subfamilies

Rogadinae are a cosmopolitan, species‐rich braconid wasp subfamily whose species are endoparasitoids that attack larvae of a number of lepidopteran families. Members of this subfamily are characterized by pupating within the mummified host larval skin. The subfamily contains six tribes whose relationships have only been partially clarified: Aleiodini, Betylobraconini, Clinocentrini, Rogadini, Stiropiini and Yeliconini. The limits and composition of the closely related subfamilies to the Rogadinae, Hormiinae and Lysiterminae, also remain unclear. Here, we generated ultraconserved element data to reconstruct an almost fully resolved phylogeny for the members of Rogadinae and related subfamilies. Based on our best estimate of phylogeny, we confirm the monophyly of Rogadinae including Betylobraconini, synonymize Xenolobus Fahringer and Bequartia Cameron within the species‐rich genus Aleiodes Wesmael ( syn.n. ) based on DNA, and synonymize Promesocentrus van Achterberg with Pilichremylus Belokobylskij ( syn.n. ) based on morphology. We also consistently recovered Hormiinae and Lysiterminae as not reciprocally monophyletic, and thus propose to unite their members under Hormiinae. The ancestral host preference for Rogadinae was probably attacking concealed lepidopteran larvae, with the occurrence of at least two main subsequent transitions to attack both concealed and exposed hosts, one within Rogadini and a second within Aleiodini. We highlight the importance of natural history collections as a source for conducting genomic‐based studies using techniques that allow to obtain a substantial amount of data from considerably old preserved insect specimens.     

A new, putatively primitive Cretaceous fossil braconid subfamily from New Jersey amber (Hymenoptera, Braconidae)

Protorhyssalus goldmani gen. n., sp. n., in a new subfamily of braconid wasps, the Protorhyssalinae, is described from Late Cretaceous amber fossils from New Jersey, USA. The Protorhyssalinae appears to be cyclostome and shows a similar set of plesiomorphic characters to the extant Rhyssalinae. However, it possesses hindwing vein 2-CU, a feature only found among the cyclostome braconids in the rare and putatively primitive Chilean subfamily Apozyginae.     

基于28S rRNA D2序列的内茧蜂亚科的分子系统发育

首次利用同源28S rRNA D2基因序列对内茧蜂亚科Rogadinae （昆虫纲Insecta：膜翅目Hymenoptera：茧蜂科Braconidae）进行了分子系统学研究。本研究从95%～100%乙醇浸渍保存的标本中提取基因组DNA并扩增了10种内群种类和5种外群种类的28S rDNA D2片段并测序（GenBank序列号AY167645-AY167659）,利用BLAST搜索相关的同源序列, 采用了GenBank中13个种类的28S rRNA D2同源序列,然后据此进行分子分析。利用3个外群（共8个种类）和3种建树方法 (距离邻近法distance based neighbor joining, NJ; 最大俭约法maximum parsimony, MP; 和最大似然法maximum likelihood, ML)分析了内茧蜂亚科内的分子系统发育关系。结果表明,由分子数据产生的不同的分子系统树均显示内茧蜂亚科是一个单系群。内茧蜂亚科内依据形态和生物学特征的分群（族和亚族）及其系统发育关系得到部分支持。NJ、MP和ML分析结果均表明内茧蜂族Rogadini不是一个单系,而是一个并系,其余3族则得到不同程度的支持。内茧蜂族可分成2个分支：“脊茧蜂属Aleiodes+弓脉茧蜂属Arcaleiodes”和“沟内茧蜂属Canalirogas+锥齿茧蜂属Conspinaria+刺茧蜂属Spinaria+内茧蜂属Rogas”,二者不是姐妹群。脊茧蜂属Aleiodes和弓脉茧蜂属Arcaleiodes始终是姐妹群。脊茧蜂属Aleiodes是一个单系,并可分成2个姐妹分支,这与依据形态和生物学特征的亚属分群相一致。弓脉茧蜂属Arcaleiodes Chen et He,1991是一个独立的属。分支“沟内茧蜂属Canalirogas+锥齿茧蜂属Conspinaria+刺茧蜂属Spinaria+内茧蜂属Rogas”的单系性仅得到部分分子数据的支持;因形态特异（腹部成甲壳状）而列为亚族级的刺茧蜂属Spinaria,分子分析没有证实这一点。横纹茧蜂族Clinocentrini是个单系,并在内茧蜂亚科的系统发育中处于基部（原始）的位置。我们研究结果还表明,阔跗茧蜂属Yelicones和潜蛾茧蜂属Stiropius相对应的阔跗茧蜂族Yeliconini和潜蛾茧蜂族Stiropiini为2个独立的分支, 与形态和生物学的结果一致,但它们在内茧蜂亚科的系统发育的位置不明,有待今后进一步研究。     

Phylogeny of the genera of the parasitic wasps subfamily Doryctinae (Hymenoptera: Braconidae) based on morphological evidence

The evolutionary relationships among most (143 genera) of the currently recognized genera of the braconid wasp subfamily Doryctinae were investigated using maximum parsimony analysis, employing 100 characters from external morphology and four additional, less well‐studied character systems (male genitalia, ovipositor structure, venom apparatus and larval cephalic structure). We investigated the ‘performance’ of characters from external morphology and the other character systems and the effects of abundant missing entries by comparing the data decisiveness, retention and consistency indices of four different character partitions. The results indicate that the performances of the different partitions are not related to the proportions of missing entries, but instead are negatively correlated to their proportion of informative characters, suggesting that the morphological information in this group is subject to high levels of homoplasy. The external morphological partition is significantly incongruent with respect to a data set comprising the other character systems based on the ILD test. Analyses supported neither the monophyly of the large tribes Doryctini and Hecabolini, nor the monophyly of the Spathiini and Heterospilini. Relationships obtained from successive approximation weighting analysis for the complete data differ considerably from the currently accepted tribal and subtribal classifications. The only exceptions were the Ypsistocerini and the Ecphylini, whose recognized members were recovered in single clades. A close relationship between the Binaerini and Holcobraconini, and also Monarea, is consistently supported by venom apparatus and ovipositor structure characters but is not indicated by external morphological data. Low bootstrap values obtained for most of the recovered clades in all analyses do not allow us to propose a meaningful reclassification for the group at this time. A complete list of the recognized genera and their synonymies is given. © 2004 The Linnean Society of London, Zoological Journal of the Linnean Society, 2004, 142 , 369–404.     

Host range of braconid species (Hymenoptera: Braconidae) that attack Asphondyliini (Diptera: Cecidomyiidae) in Japan

We reared six idiobiont braconids, Bracon asphondyliae, B. sunosei, B. tamabae, Simplicibracon curticaudis, Testudobracon longicaudis and T. pleuralis from 22 identified species and 11 unidentified segregates of Asphondyliini (Diptera: Cecidomyiidae) in Japan. A total of 22 cecidomyiid species and segregates were newly recorded as hosts of the braconids. Analysis of cytochrome oxidase subunit I (COI) did not show any evidence of host races among the braconids. Bracon sunosei, which was synonymized with B. asphondyliae, is restored to a valid species. The host range of the braconid species seemed to be related to the lineage of host genera within Asphondyliini.     

Host location by the fruit fly parasitoid Diachasmimorpha krausii: role of fruit fly species,life stage and host plant

• 1 Diachasmimorpha krausii is a braconid parasitoid of larval tephritid fruit flies, which feed cryptically within host fruit. At the ovipositor probing stage, the wasp cannot discriminate between hosts that are physiologically suitable or unsuitable for offspring development and must use other cues to locate suitable hosts.
• 2 To identify the cues used by the parasitoid to find suitable hosts, we offered, to free flying wasps, different combinations of three fruit fly species (Bactrocera tryoni, Bactrocera cacuminata, Bactrocera cucumis), different life stages of those flies (adults and larvae) and different host plants (Solanum lycopersicon, Solanum mauritianum, Cucurbita pepo). In the laboratory, the wasp will readily oviposit into larvae of all three flies but successfully develops only in B. tryoni. Bactrocera tryoni commonly infests S. lycopersicon (tomato), rarely S. mauritianum (wild tobacco) but never C. pepo (zucchini). The latter two plant species are common hosts for B. cacuminata and B. cucumis, respectively.
• 3 The parasitoid showed little or no response to uninfested plants of any of the test species. The presence of adult B. tryoni, however, increased parasitoid residency time on uninfested tomato.
• 4 When the three fruit types were all infested with larvae, parasitoid response was strongest to tomato, regardless of whether the larvae were physiologically suitable or unsuitable for offspring development. By contrast, zucchini was rarely visited by the wasp, even when infested with B. tryoni larvae.
• 5 Wild tobacco was infrequently visited when infested with B. cacuminata larvae but was more frequently visited, with greater parasitoid residency time and probing, when adult flies (either B. cacuminata or B. tryoni) were also present.
• 6 We conclude that herbivore‐induced, nonspecific host fruit wound volatiles were the major cue used by foraging D. krausii. Although positive orientation to infested host plants is well known from previous studies on opiine braconids, the failure of the wasp to orientate to some plants even when infested with physiologically suitable larvae, and the secondary role played by adult fruit flies in wasp host searching, are newly‐identified mechanisms that may aid parasitoid host location in environments where both physiologically suitable and unsuitable hosts occur.

     

Molecular phylogenetics of Braconidae (Hymenoptera: Ichneumonoidea), based on multiple nuclear genes,and implications for classification

This study examined subfamilial relationships within Braconidae, using 4 kb of sequence data for 139 taxa. Genetic sampling included previously used markers for phylogenetic studies of Braconidae (28S and 18S rDNA) as well as new nuclear protein‐coding genes (CAD and ACC). Maximum likelihood and Bayesian inference of the concatenated dataset recovered a robust phylogeny, particularly for early divergences within the family. This study focused primarily on non‐cyclostome subfamilies, but the monophyly of the cyclostome complex was strongly supported. There was evidence supporting an independent clade, termed the aphidioid complex, as sister to the cyclostome complex of subfamilies. Maxfischeria was removed from Helconinae and placed within its own subfamily within the aphidioid complex. Most relationships within the cyclostome complex were poorly supported, probably because of lower taxonomic sampling within this group. Similar to other studies, there was strong support for the alysioid subcomplex containing Gnamptodontinae, Alysiinae, Opiinae and Exothecinae. Cenocoeliinae was recovered as sister to all other subfamilies within the euphoroid complex. Planitorus and Mannokeraia, previously placed in Betylobraconinae and Masoninae, respectively, were moved to the Euphorinae, and may share a close affiliation with Neoneurinae. Neoneurinae and Ecnomiinae were placed as tribes within Euphorinae. A sister relationship between the microgastroid and sigalphoid complexes was also recovered. The helconoid complex included a well‐supported lineage that is parasitic on lepidopteran larvae (macrocentroid subcomplex). Helconini was raised to subfamily status, and was recovered as sister to the macrocentroid subcomplex. Blacinae was demoted to tribal status and placed within the newly circumscribed subfamily Brachistinae, which also contains the tribes Diospilini, Brulleiini and Brachistini, all formerly in Helconinae.     

Venom gland and reservoir morphology in the Doryctinae and related braconid wasps (Insecta, Hymenoptera, Braconidae)

Detailed venom reservoir and venom gland intima morphology has been investigated in reprsentatives of 84 genera and 10 subfamilies of Braconidae with particular reference to the Doryctinac, basal cyclostomes and related groups including the Opiinae, Alysiinae and Rogadinae sens. lat . Several new phylogenetically significant characters are described and illustrated. Extcnsivc secretory ductules on the primary venom duct is suggested as a synapomorphy for the subfamilies Braconinac, Doryctinae, Opiinae, Alysiinae and Gnamptodontinae. The presence of two separate venom gland insertions and their position beyond the spiral sculpture of the reservoir are suggested as synapomorphies for the subfamilies Doryctinae and Ypsistocerinac. An anterior, weakly sclerotized, probably glandular. expansion of the reservoir characterizes the Histeromerinae and Rhyssalinae and also the enigmatic genera Thoracoplites Fischer and Doryctomorpha Ashmead which are hcre transferred to the Rhyssalinae. Within the Doryctinac, venom reservoir morphology suggests three generic groups, one comprising the genera of the Odontohraconini and possibly also the genera Acanthodoryctes , Binareu and Monarea , all of which have an essentially undivided reservoir with extremely fine and uniform spiral sculpture. the second comprising the genera Doryctophasmus , Euscelinus, Gildoria, Halycea and Schlettereriella , which share horn-like processes at the base of the venom duct insertions, and a third including Acrophasmus. Dendro- soter, Heterospilus, Megaloproctus, Rutheia, Paraspathius, Schlettereriella. Spathius and Syngaster based on the presence of two markedly different densities of annulation of the reservoir. The degree to which external and other characters support these groups is discusscd.     

Phylogenetic relationships of Combretoideae (Combretaceae) inferred from plastid,nuclear gene and spacer sequences

Phylogenetic relationships of the subfamily Combretoideae (Combretaceae) were studied based on DNA sequences of nuclear ribosomal internal transcribed spacer (ITS) regions, the plastid rbcL gene and the intergenic spacer between the psaA and ycf3 genes (PY-IGS), including 16 species of eight genera within two traditional tribes of Combretoideae, and two species of the subfamily Strephonematoideae of Combretaceae as outgroups. Phylogenetic trees based on the three data sets (ITS, rbcL, and PY-IGS) were generated by using maximum parsimony (MP) and maximum likelihood (ML) analyses. Partition-homogeneity tests indicated that the three data sets and the combined data set are homogeneous. In the combined phylogenetic trees, all ingroup taxa are divided into two main clades, which correspond to the two tribes Laguncularieae and Combreteae. In the Laguncularieae clade, two mangrove genera, Lumnitzera and Laguncularia, are shown to be sister taxa. In the tribe Combreteae, two major clades can be classified: one includes three genera Quisqualis, Combretum and Calycopteris, within which the monophyly of the tribe Combreteae sensu Engler and Diels including Quisqualis and Combretum is strongly supported, and this monophyly is then sister to the monotypic genus Calycopteris; another major clade includes three genera Anogeissus, Terminalia and Conocarpus. There is no support for the monophyly of Terminalia as it forms a polytomy with Anogeissus. This clade is sister to Conocarpus. Electronic Publication     

Systematics of the cyclostome subfamilies of braconid parasitic wasps (Hymenoptera: Ichneumonoidea): a simultaneous molecular and morphological Bayesian approach

Phylogenetic relationships among 95 genera collectively representing 17 of the 18 currently recognized cyclostome braconid wasp subfamilies were investigated based on DNA sequence fragments of the mitochondrial COI and the nuclear 28S rDNA genes, in addition to morphological data. The treatment of sequence length variation of the 28S partition was explored by either excluding ambiguously aligned regions and indel information (28SN) or recoding them (28SA) using the 'fragment-level' alignment method with a modified coding approach. Bayesian MCMC analyses were performed for the separate and combined data sets and a maximum parsimony analysis was also carried out for the simultaneous molecular and morphological data sets. There was a significant incongruence between the two genes and between 28S and morphology, but not for morphology and COI. Different analyses with the 28SA data matrix resulted in topologies that were generally similar to the ones from the 28SN matrix; however, the former topologies recovered a higher number of significantly supported clades and had a higher mean Bayesian posterior probability, thus supporting the inclusion of information from ambiguously aligned regions and indel events in phylogenetic analyses where possible. Based on the significantly supported clades obtained from the simultaneous molecular and morphological analyses, we propose that a total of 17 subfamilies should be recognized within the cyclostome group. The subfamilial placements of several problematic cyclostome genera were also established.     

Recovery of species diversity and composition of braconid parasitic wasps after reforestation of degraded grasslands in lowland East Kalimantan

Human-induced disturbances and wildfires can transform areas of tropical rainforest into Imperata-dominated grassland, but it may be possible that recovery of biodiversity is facilitated by reforestation with fast-growing trees. We compared the assemblages of braconid wasps as parasitoids of taxonomically diverse groups of insects among Imperata grasslands, young and mature plantations of Acacia mangium, young secondary forests after wildfires, and old secondary forests in the lowland of East Kalimantan. The abundance and species richness of braconids, which had declined in Imperata grasslands, somewhat increased in Acacia plantations, and also the species composition of braconids in Acacia plantations was transitional between Imperata grasslands and old secondary forests. Parasitoids of detritivores and wood borers increased markedly after plantation, while those of herbivores showed a distinct turnover of species all over the range from grasslands to old secondary forests. The plantation of A. mangium had most likely facilitated the recovery of the diversity of host forest and their parasitoids, but the recovery was just at the rudimentary stage even in mature plantations. Monitoring of parasitic wasps would be useful to test the continuous recovery of forest biodiversity in plantation stands.     

Venom induces alarm behaviour in the social wasp Polybioides raphigastra (Hymenoptera: Vespidae): an investigation of alarm behaviour, venom volatiles and sting autotomy

Chemicals from the venom gland elicited alarm behaviour and attack in the Asian polistine wasp Polybioides raphigastra. When presented with crushed venom glands workers of this wasp respond with a mass stinging attack. Gas chromatography–mass spectrometry analyses show that the major volatiles in the venom gland are alkanes, monounsaturated alkenes and 2-alcohols. Several pyrazines, a spiroacetal and aromatics were also identified as trace compounds. The anatomy and morphology of the sting apparatus are reported, and we describe sting autotomy in this wasp. This is the first such report for the Ropalidiinae. The structures responsible for autotomy are likely to be large barbs present on the sting lancets, and a conspicuous tooth present on the medial side of the left lancet. Sting autotomy in P. raphigastra probably plays an important role in the localization of sites of attack by wasps defending the nest.     

Prevalence and evolutionary history of endosymbiont Wolbachia (Rickettsiales: Anaplasmataceae) in parasitoids (Hymenoptera: Braconidae) associated with Bactrocera fruit flies (Diptera: Tephritidae) infesting carambola

Wolbachia endosymbiont is a maternally inherited bacteria that infects a wide range of hosts, including parasitoids and their respective hosts. In this study, a total of 171 individuals of braconid endoparasitoids, consisting of Fopius arisanus, F. vandenboschi, Diachasmimorpha longicaudata, Psyttalia sp.1, Psyttalia sp.2, P. fletcheri and P. incisi, and their host tephritid fruit flies of Bactrocera dorsalis, B. papayae and B. carambolae infesting carambola were screened molecularly by the Wolbachia surface protein (wsp) gene. Interestingly, 21 (24.14%) wsp gene sequences were successfully isolated from 87 braconid samples tested, showing a low infection rate of Wolbachia. However, despite the close ecological contact between parasitoids and their hosts, none of the tephritid individuals were infected by Wolbachia. A comparison of wsp and host mitochondrial cytochrome c oxidase subunit I (COI) sequences found that braconids did not cluster in connection with Wolbachia infection, suggesting that selective sweep has not yet occurred because Wolbachia may have recently infected the braconid populations in Peninsular Malaysia (≈0.1 MYA). Despite of relatively recent infections of Wolbachia, the history of Wolbachia infection into F. arisanus populations of Peninsular Malaysia is complex, involving at least two independent occasions of infection and two secondary losses.     

Relationships among the cyclostome braconid (Hymenoptera: Braconidae) subfamilies inferred from a mitochondrial tRNA gene rearrangement

The arrangement of mitochondrial tRNA genes for lysine (K) and aspartate (D) from the junction of the cytochrome oxidase II and ATPase 8 genes was determined in a range of hymenopteran taxa. This indicated that the ancestral arrangement for the order is 'KD', as found in the Diptera (represented by Drosophila and Anopheles) and basal Orthoptera. Most Hymenoptera that evolved after the appearance of parasitism also have the 'KD' arrangement, including noncyclostome braconids. However, most cyclostome braconids have either a 'DK' or a 'DHK' arrangement (where 'H' refers to the tRNA gene for Histidine). In both cases, the aspartate tRNA gene is encoded on the mitochondrial N-strand, rather than the J-strand as is usually the case. This rearrangement identified a monophyletic group not previously recognized, consisting of Rogadinae + Braconinae + Gnamptodontinae + Histeromerinae + Rhyssalinae + Betylobraconinae + Opiinae + Alysiinae. Only one cyclostome subfamily (Doryctinae) retained the 'KD' arrangement, suggesting this to be the most basal of the cyclostome subfamilies, consistent with ectoparasitism being plesiomorphic for the cyclostomes. However, the Aphidiinae also retained the 'KD' arrangement, leaving unresolved the issue of whether they should be included within the cyclostomes.     

腰带长体茧蜂毒液器官和卵巢的形态学及其超微结构

应用超薄切片和电镜技术,观察内寄生蜂腰带长体茧蜂Macrocentrus cingulum Brischke毒液器官和卵巢的形态结构。腰带长体茧蜂毒液器官由1个毒囊和2条毒腺组成,毒腺接于毒囊的顶端。毒腺由单层分泌细胞、退化的外胚层细胞和环腔的内膜构成,分泌细胞主要由1个明显的细胞核和1个较大囊状细胞器构成,囊状细胞器的功能是分泌毒液。毒囊由肌肉鞘和扁平细胞层构成,但没有分泌细胞。腰带长体茧蜂卵巢1对,每个卵巢由10条左右卵巢小管组成,与侧输卵管相接处略微膨大形成卵巢萼区。2条侧输卵管在产卵管基部会合形成1条总输卵管与产卵管相接。毒液器官通过毒囊的毒液导管附着在总输卵管上。对寄生蜂毒液器官的生物学、细胞学及在分类进化上的意义进行研究。     

A new species of Syntretus Foerster (Hymenoptera: Braconidae: Euphorinae), a parasitoid of the stingless bee Trigona carbonaria Smith (Hymenoptera: Apidae: Meliponinae)

A new species of euphorine braconid, Syntretus trigonaphagus sp. nov., is described and illustrated. This is the first Australian Syntretus species to be described and the first record of braconids parasitising Trigona Jurine stingless bees. Egg-laying by adults and emergence of larvae from the host Trigona carbonaria is detailed. A 12S ribosomal rRNA gene fragment was sequenced to confirm the association of larvae and adults of the wasp parasitoid.     

Phylogenetic study of the Characinae (Teleostei: Characiformes: Characidae)

The Characinae is a subunit of the Characidae of special significance in including Charax, the type genus of the family and the order Characiformes. Twelve genera and 79 species have been traditionally assigned to the Characinae, but the subfamily still lacks a phylogenetic diagnosis. Herein, a data matrix including 150 morphological characters and 64 taxa (35 species representing all genera of the Characinae and 29 included in other lineages within the Characiformes) was submitted to two cladistic analyses that differ in the inclusion/exclusion of Priocharax due to the difficulty of coding most of the character states in the miniature species of this genus. Both analyses resulted in a non‐monophyletic Characinae and this subfamily is herein restricted to only seven of the original 12 genera forming the clade (Phenacogaster((Charax Roeboides)(Acanthocharax(Cynopotamus(Acestrocephalus Galeocharax))))), which is supported by ten non‐ambiguous synapomorphies and is more closely related to other genera of the Characidae than those traditionally placed in the subfamily. A second clade includes the members of the tribe Heterocharacini (Lonchogenys(Heterocharax Hoplocharax)) as the sister‐group of Gnathocharax, supported by seven non‐ambiguous synapomorphies. This clade is more closely related to a taxon formed by Roestes and Gilbertolus based on seven non‐ambiguous synapomorphies. Results do not corroborate a close relationship between Roestes–Gilbertolus and the Cynodontinae. Inclusion of the genus Priocharax suggests that it is related more closely to the Heterocharacini, but the profound modifications in its anatomy possibly related to ontogenetic truncations obscure a better understanding of its relationships. A new classification of the Characinae and the Heterocharacinae is proposed. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 165 , 809–915.     

Molecular phylogeny of Pottiaceae (Musci) based on chloroplast rps4 sequence data

Comparative sequencing of the chloroplast rps4 gene was used to reconstruct the phylogenetic relationships within the family Pottiaceae (Musci). The results confirm that Ephemerum spinulosum, Splachnobryum obtusum, Goniomitrium acuminatum and Cinclidotus fontinaloides are clearly positioned within the Pottiaceae and that Hypodontium dregei is not a member. At subfamily level, the data support the subfamily Pottioideae as being a monophyletic clade. The Trichostomoideae are probably paraphyletic. Neither the subfamily Chionolomideae, represented in this study by Pseudosymblepharis schimperiana, nor the subfamily Erythrophyllopsoideae, represented by both known species, Erythrophyllastrum andinum and Erythrophyllopsis fuscula, are supported by the sequence data. The Timmielloideae should be excluded from the Pottiaceae. The Merceyoideae, represented in this study by Scopelophila cataractae, might form a sister clade to all other Pottiaceae, but their position is not fully resolved. At the genus level, Barbula is clearly polyphyletic since Barbula bolleana and Barbula indica appear in a clade clearly separated from Barbula unguiculata. Pottiopsis caespitosa and Leptobarbula berica are placed within the Trichostomoideae. Likewise, the genera Gymnostomum and Anoectangium are excluded from the Pottioideae and placed within the Trichostomoideae. Leptophascum leptophyllum is closely related with Syntrichia; Aloina is not closely related to Tortula or Crossidium. Evidence of a clade within the Pottioideae, formed of Leptodontium and Triquetrella, is provided.