A Bacterium Belonging to the Rickettsiaceae Family Inhabits the Cytoplasm of the Marine Ciliate Diophrys appendiculata (Ciliophora, Hypotrichia)

Bacteria of the family Rickettsiaceae (order Rickettsiales, α-Proteobacteria) are mainly known to be endosymbionts of arthropods with the capability to infect also vertebrate cells. Recently, they have also been found as leech endocytobionts. In the present paper, we report the first finding of a bacterium belonging to the family Rickettsiaceae in a natural population of a marine ciliate protozoan, namely Diophrys appendiculata, collected in the Baltic Sea. Bacteria were unambiguously identified through morphological characterization and the “full-cycle rRNA approach” (i.e., 16S rRNA gene characterization and use of specifically designed oligonucleotide probes for in situ detection). Symbionts are rod-shaped bacteria that grow freely in the cytoplasm of the host cell. They present two different morphotypes, similar in size, but different in cytoplasmic density. These are typical morphological features of members of the family Rickettsiaceae. 16S rRNA gene sequence showed that Diophrys symbionts share a high similarity value (>92%) with bacteria belonging to the genus Rickettsia. Phylogenetic analysis revealed that these new endosymbionts are clearly included in the clade of the family Rickettsiaceae, but they occupy an independent phylogenetic position with respect to members of the genus Rickettsia. This is the first report of a member of this family from a host protozoan and from a marine habitat. This result shows that this bacterial group is more diversified and widespread than supposed so far, and that its ecological relevance could until now have been underestimated. In light of these considerations, the two 16S rRNA oligonucleotide probes here presented, specific for members of the Rickettsiaceae, can represent useful tools for further researches on the presence and the spread of these microorganisms in the natural environment.     

Igniting taxonomic curiosity: The amazing story of Amazonocrinis with the description of a new genus Ahomia gen. nov. and novel species of Ahomia,Amazonocrinis, and Dendronalium from the biodiversity-rich northeast region of India

Five cyanobacterial strains exhibiting Nostoc-like morphology were sampled from the biodiversity hotspots of the northeast region of India and characterized using a polyphasic approach. Molecular and phylogenetic analysis using the 16S rRNA gene indicated that the strains belonged to the genera Amazonocrinis and Dendronalium. In the present investigation, the 16S rRNA gene phylogeny clearly demarcated two separate clades of Amazonocrinis. The strain MEG8-PS clustered along with Amazonocrinis nigriterrae CENA67, which is the type strain of the genus. The other three strains ASM11-PS, RAN-4C-PS, and NP-KLS-5A-PS clustered in a different clade that was phylogenetically distinct from the Amazonocrinis sensu stricto clade. Interestingly, while the 16S rRNA gene phylogeny exhibited two separate clusters, the 16S–23S ITS region analysis did not provide strong support for the phylogenetic observation. Subsequent analyses raised questions regarding the resolving power of the 16S–23S ITS region at the genera level and the associated complexities in cyanobacterial taxonomy. Through this study, we describe a novel genus Ahomia to accommodate the members clustering outside the Amazonocrinis sensu stricto clade. In addition, we describe five novel species, Ahomia kamrupensis, Ahomia purpurea, Ahomia soli, Amazonocrinis meghalayensis, and Dendronalium spirale, in accordance with the International Code of Nomenclature for algae, fungi, and plants (ICN). Apart from further enriching the genera Amazonocrinis and Dendronalium, the current study helps to resolve the taxonomic complexities revolving around the genus Amazonocrinis and aims to attract researchers to the continued exploration of the tropical and subtropical cyanobacteria for interesting taxa and lineages.     

16S rRNA GENE HETEROGENEITY IN THE FILAMENTOUS MARINE CYANOBACTERIAL GENUS LYNGBYA1

The SSU (16S) rRNA gene was used to investigate the phylogeny of the cyanobacterial genus Lyngbya as well as examined for its capacity to discriminate between different marine species of Lyngbya. We show that Lyngbya forms a polyphyletic genus composed of a marine lineage and a halophilic/brackish/freshwater lineage. In addition, we found morphological and genetic evidence that Lyngbya spp. often grow in association with other microorganisms, in particular smaller filamentous cyanobacteria such as Oscillatoria, and propose that these associated microorganisms have led to extensive phylogenetic confusion in identification of Lyngbya spp. At the species level, the phylogenetic diversity obtained from the comparison of 16S rRNA genes exceeded morphological diversity in Lyngbya. However, the expectation that this improved phylogeny would be useful to species and subspecies identification was eliminated by the fact that phylogenetic species did not correlate in any respect with the species obtained from current taxonomic systems. In addition, phylogenetic identification was adversely affected by the presence of multiple gene copies within individual Lyngbya colonies. Analysis of clonal Lyngbya cultures and multiple displacement amplified (MDA) single‐cell genomes revealed that Lyngbya genomes contain two 16S rRNA gene copies, and that these typically are of variable sequence. Furthermore, intragenomic and interspecies 16S rRNA gene heterogeneity was approximately of the same magnitude. Hence, the intragenomic heterogeneity of the 16S rRNA gene overestimates the microdiversity of different strains and does not accurately reflect speciation within cyanobacteria, including the genus Lyngbya.     

Genotypic and phenotypic diversity of cyanobacteria assigned to the genus <Emphasis Type="Italic">Phormidium</Emphasis> (Oscillatoriales) from different habitats and geographical sites

In this study, 30 strains of filamentous, non-heterocystous cyanobacteria from different habitats and different geographical regions assigned to diverse oscillatorian genera but here collectively referred to as members of the Phormidium group have been characterized using a polyphasic approach by comparing phenotypic and molecular characteristics. The phenotypic analysis dealt with cell and filament morphology, ultrastructure, phycoerythrin content, and complementary chromatic adaptation. The molecular phylogenetic analyses were based on sequences of the 16S rRNA gene and the adjacent intergenic transcribed spacer (ITS). The sequences were located on multiple branches of the inferred cyanobacterial 16S rRNA tree. For some, but not all, strains with identical 16S rDNA sequences, a higher level of discrimination was achieved by analyses of the less conserved ITS sequences. As shown for other cyanobacteria, no correlation was found between position of the strains in the phylogenetic tree and their geographic origin. Genetically similar strains originated from distant sites while other strains isolated from the same sampling site were in different phylogenetic clusters. Also the presence of phycoerythrin was not correlated with the strains’ position in the phylogenetic trees. In contrast, there was some correlation among inferred phylogenetic relationship, original environmental habitat, and morphology. Closely related strains came from similar ecosystems and shared the same morphological and ultrastructural features. Nevertheless, structural properties are insufficient in themselves for identification at the genus or species level since some phylogenetically distant members also showed similar morphological traits. Our results reconfirm that the Phormidium group is not phylogenetically coherent and requires revision.     

Application of <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">parE</Emphasis> sequence similarity analyses for differentiation of species within the genus <Emphasis Type="Italic">Geobacillus</Emphasis>

The primary structures of the genes encoding the β-subunits of a type II topoisomerase (gyrase, gyrB) and a type IV topoisomerase (parE) were determined for 15 strains of thermophilic bacteria of the genus Geobacillus. The obtained sequences were used for analysis of the phylogenetic similarity between members of this genus. Comparison of the phylogenetic trees of geobacilli constructed on the basis of the 16S rRNA, gyrB, and parE gene sequences demonstrated that the level of genetic distance between the sequences of the genes encoding the β-subunits of type II topoisomerases significantly exceeded the values obtained by comparative analysis of the 16S rRNA gene sequences of Geobacillus strains. It was shown that, unlike the 16S rRNA gene analysis, comparative analysis of the gyrB and parE gene sequences provided a more precise determination of the phylogenetic position of bacteria at the species level. The data obtained suggest the possibility of using the genes encoding the β-subunits of type II topoisomerases as phylogenetic markers for determination of the species structure of geobacilli.     

Evolutionary Relationships Among Old World Fruitbats (Megachiroptera: Pteropodidae) Based on 12S rRNA, tRNA Valine, and 16S rRNA Gene Sequences

Old World fruitbats were divided into the cynopterine, epomophorine, rousettine, eonycterine, and notopterine sections by Knud Andersen (1912). Among these, the eonycterine and notopterine sections together comprise the subfamily Macroglossinae, which includes forms with specializations for nectarivory. Single-copy DNA hybridization data argue against the monophyly of four of Andersen's sections and further suggest paraphyly or polyphyly of the Macroglossinae. DNA hybridization data provide support for an endemic African clade that includes Megaloglossus (an eonycterine), Epomophorus (an epomophorine), and Lissonycteris (a rousettine). Analyses of mitochondrial 12S rRNA-tRNA valine gene sequences corroborate the African clade but provide less resolution than hybridization data for most branches on the pteropodid tree. Here, we report 11 new 16S rRNA sequences and analyze a mitochondrial data set that includes 12S rRNA, tRNA valine, and 16S rRNA for 18 pteropodid genera. Parsimony, minimum evolution, and maximum likelihood were all employed in phylogenetic analyses. The addition of 16S rRNA sequences to the mitochondrial data set resulted in increased support for several clades, including Macroglossus + Syconycteris, Cynopterus + Thoopterus, Rousettus + the endemic African clade, and Eonycteris + Rousettus + the endemic African clade. Statistical tests suggest that another endemic African genus, Eidolon, is dissociated from the African clade and represents an independent invasion into Africa. We constructed a molecular phylogenetic framework that incorporated clades that were strongly supported by both single-copy DNA hybridization and 12S rRNA-tRNA valine-16S rRNA sequences. Using this framework as a backbone phylogenetic constraint, we then analyzed a morphological data matrix for 34 pteropodid genera with parsimony. Results of this analysis suggest that other epomophorines and Myonycteris (a cynopterine) are also part of the endemic African clade.     

Phylogenetic analysis of cultivation‐resistant terrestrial cyanobacteria with massive sheaths (Stigonema spp. and Petalonema alatum,Nostocales, Cyanobacteria) using single‐cell and filament sequencing of environmental samples

Molecular assessment of a large portion of traditional cyanobacterial taxa has been hindered by the failure to isolate and grow them in culture. In this study, we developed an optimized protocol for single cell/filament isolation and 16S rRNA gene sequencing of terrestrial cyanobacteria with large mucilaginous sheaths, and applied it to determine the phylogenetic position of typical members of the genera Petalonema and Stigonema. A methodology based on a glass‐capillary isolation technique and a semi‐nested PCR protocol enabled reliable sequencing of the 16S rRNA gene from all samples analyzed. Ten samples covering seven species of Stigonema from Europe, North and Central America, and Hawaii, and the type species of Petalonema from Slovakia were sequenced. Contrary to some previous studies, which proposed a relationship with heteropolar nostocalean cyanobacteria, Petalonema appeared to belong to the family Scytonemataceae. Analysis of Stigonema specimens recovered a unique coherent phylogenetic cluster, substantially broadening our knowledge of the molecular diversity within this genus. Neither the uni‐ to biseriate species nor the multiseriate species formed monophyletic subclusters within the genus. Typical multiseriate species of Stigonema clustered in a phylogenetic branch derived from uni‐ to biseriate S. ocellatum Thuret ex Bornet & Flahault in our analysis, suggesting that species with more complex thalli may have evolved from the more simple ones. We propose the technique tested in this study as a promising tool for a future revision of the molecular taxonomy in cyanobacteria.     

Phylogenetic analysis of Methanobrevibacter isolated from feces of humans and other animals

Comparative 16S rRNA gene sequence and genomic DNA reassociation analyses were used to assess the phylogenetic relationships of Methanobrevibacter fecal isolates. The 16S rRNA gene sequences of Methanobrevibacter smithii strain PS and the human fecal isolates B181 and ALI were essentially identical, and their genomic DNA reassociated at values greater than 94%. The analysis of 16S rRNA sequences of the horse, pig, cow, rat, and goose fecal isolates confirm that they are members of the genus Methanobrevibacter. They had a high degree of sequence similarity (97–98%) with the 16S rRNA gene of M. smithii, indicating that they share a common line of descent. The 16S rRNA genes of the horse and pig isolates had 99.3% sequence similarity. Sequence analysis of the 16S rRNA gene of the sheep fecal isolate showed that it formed a separate line of descent in the genus Methanobrevibacter. Genomic DNA reassociation studies indicate that the horse, pig, cow, and goose fecal isolates represent at least three new species. The horse and pig isolates were the only animal isolates that had > 70% genomic DNA reassociation and represent strains of a single species. The cow, goose, and sheep isolates had little or no genomic DNA reassociation with M. smithii or with each other. The relationship of the rat isolate to the other animal isolates was not determined. An evaluation of the relationship of 16S rRNA gene sequence similarity and genomic DNA reassociation of Methanobrevibacter and other methanogenic archaea indicated that genomic DNA reassociation studies are necessary to establish that two methanogenic organisms belong to the same species. Received: 17 November 1997 / Accepted: 16 January 1998     

Two new species of Phyllonema (Rivulariaceae,Cyanobacteria) with an emendation of the genus

Two untapered, heterocytous species were observed and collected from the intertidal and supratidal zones of the Mexican coastline of the Pacific Ocean near Oaxaca and from the Gulf of Mexico. These populations were highly similar in morphology to the freshwater taxon Petalonema incrustans in the Scytonemataceae. However, 16S rRNA sequence data and phylogenetic analysis indicated that they were sister taxa to the epiphyllic, Brazilian species Phyllonema aveceniicola in the Rivulariaceae, described from culture material. While genetic identity between the two new species was high, they differed significantly in morphology, 16S rRNA gene sequence identity, and sequence and structure of the 16S–23S ITS region. Their morphology differed markedly from the generitype of the previously monotypic Phyllonema, which has tapered, heteropolar, single‐false branched trichomes with very thin or absent sheath. The two new species, Phyllonema ansata and Phyllonema tangolundensis, described from both culture and environmental material, have untapered, isopolar, geminately false branched trichomes with thick, lamellated sheaths, differences so significant that the species would not be placed in Phyllonema without molecular corroboration. The morphological differences are so significant that a formal emendation of the genus is required. These taxa provide a challenge to algal taxonomy because the morphological differences are such that one would logically conclude that they represent different genera, but the phylogenetic evidence for including them all in the same genus is conclusive. This conclusion is counter to the current trend in algal taxonomy in which taxa with minor morphological differences have been repeatedly placed in separate genera based primarily upon DNA sequence evidence.     

Phylogeny of the American silverfish Cubacubaninae (Hexapoda: Zygentoma: Nicoletiidae): a combined approach using morphology and five molecular loci

Relationships within the subfamily Cubacubaninae, the dominant subfamily of Nicoletiidae in America, are appraised based on parsimony analysis of 20 morphological characters and sequence data from five loci (nuclear 18S and 28S rRNA, mitochondrial 16S rRNA, nuclear protein coding gene histone H3, and mitochondrial protein coding gene cytochrome c oxidase subunit I). The data, analyzed under direct optimization for a range of analytical parameter sets, indicated that species may show some biogeographical structure. It also indicated that the presence of articulated submedian appendages on urosternum IV is not a valid discriminating character in taxonomy. Species within the traditional genera Anelpistina, Cubacubana and Neonicoletia were found to belong to a group in which no clear morphological or molecular distinction was present. It is proposed that members of these three genera should be united within a single taxon. On the contrary, the genus Prosthecina is well supported by the data. © The Willi Hennig Society 2006.     

Ponticoccus gilvus gen. nov., sp. nov., a novel member of the family Propionibacteriaceae from seawater

A novel actinobacterium, designated strain MSW-19^T, was isolated from a seawater sample in Republic of Korea. Cells were aerobic, Gram-positive, non-endospore-forming, and non-motile cocci. Colonies were circular, convex, opaque, and vivid yellow in colour. A phylogenetic tree based on 16S rRNA gene sequences exhibited that the organism formed a distinct clade within the radius encompassing representatives of the family Propionibacteriaceae. The phylogenetic neighbors were the type strains of the genera Friedmanniella, Microlunatus, Micropruina, Propionicicella, and Propionicimonas. Levels of 16S rRNA gene sequence similarity between the isolate and members of the family were less than 95.3%. The cell wall peptidoglycan of the organism contained LL-diaminopimelic acid as the diagnostic diamino acid. The isolate contained MK-9(H₄) as the predominant menaquinone, ai-C_15:0 as the major fatty acid and polar lipids including phosphatidylglycerol, phosphatidylethanolamine, and an unknown phospholipid. The G+C content of the DNA was 69.6 mol%. On the basis of the phenotypic and phylogenetic data presented here, the isolate is considered to represent a novel genus and species in the family Propionibacteriaceae, for which the name Ponticoccus gilvus gen. nov., sp. nov. is proposed. The type strain is strain MSW-19^T (= KCTC 19476^T= DSM 21351^T).     

A widespread cyanobacterium supported by polyphasic approach: proposition of Koinonema pervagatum gen. & sp. nov. (Oscillatoriales)1

Several new genera originally classified as the genus Phormidium, a polyphyletic and taxonomically complex genus within the Oscillatoriales, were recently described. The simple morphology of Phormidium does not reflect its genetic diversity and the delimitation of a natural group is not possible with traditional classification systems based on morphology alone. Therefore, this study used morphological, ecological, and molecular approaches to evaluate four populations morphologically similar to Ammassolinea, Kamptonema, and Ancylothrix (simple, curved, and gradually attenuated at the ends trichome), found in subtropical and tropical Brazilian regions. 16S rRNA gene sequences grouped all the strains in a highly supported clade with other two European strains isolated from thermal springs surrounding areas. The 16S‐23S ITS secondary structure corroborated the phylogenetic analysis with all the strains having similar structures. Consequently, a genetically well‐defined and cryptic new genus, Koinonema gen. nov., is proposed containing the aquatic, mesophilic, and morphologically homogeneous new species, Koinonema pervagatum sp. nov.     

New records of two deep-sea eels collected from the Western Pacific Ocean based on COI and 16S rRNA genes

Background

Two deep-sea eels collected from the Western Pacific Ocean are described in this study. Based on their morphological characteristics, the two deep-sea eel specimens were assumed to belong to the cusk-eel family Ophidiidae and the cutthroat eel family Synaphobranchidae.

Methods and results

To accurately identify the species of the deep-sea eel specimens, we sequenced the mitochondrial genes (cytochrome c oxidase subunit I [COI] and 16S ribosomal RNA [16S rRNA]). Through molecular phylogenetic analysis based on mtDNA COI and 16S rRNA gene sequences, these species clustered with the genera Bassozetus and Synaphobranchus, suggesting that the deep-sea eel specimens collected are two species from the genera Bassozetus and Synaphobranchus in the Western Pacific Ocean, respectively.

Conclusions

This is the first study to report new records of the genera Bassozetus and Synaphobranchus from the Western Pacific Ocean based on COI and 16S rRNA genes

     

Morphological and molecular description of new species of squat lobster (Crustacea: Decapoda: Galatheidae) from the Solomon and Fiji Islands (South‐West Pacific)

The family Galatheidae is among the most diverse families of anomuran decapod crustaceans, and the South‐West Pacific is a biodiversity hot spot for these squat lobsters. Attempts to clarify the taxonomic and evolutionary relationships of the Galatheidae on the basis of morphological and molecular data have revealed the existence of several cryptic species, differentiated only by subtle morphological characters. Despite these efforts, however, relationships among genera are poorly understood, and the family is in need of a detailed systematic review. In this study, we assess material collected in different surveys conducted in the Solomon Islands, as well as comparative material from the Fiji Islands, by examining both the morphology of the specimens and two mitochondrial markers (cytochrome oxidase subunit I, COI, and 16S rRNA). These two sources of data revealed the existence of eight new species of squat lobster, four of which were ascribed to the genus Munida, two to the genus Paramunida, one to the genus Plesionida, and the last species was ascribed to the genus Agononida. These eight species are described along with phylogenetic relationships at the genus level. Our findings support the taxonomic status of the new species, yet the phylogenetic relationships are not yet fully resolved. Further molecular analysis of a larger data set of species, and more conserved genes, will help clarify the systematics of this group. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 156 , 465–493.     

Description of two new species of Nostoc (Nostocales,Cyanobacteria) from central Mexico,using morphological,ecological, and molecular attributes

The present study describes two new Nostoc species, N. montejanii and N. tlalocii, based on a polyphasic approach that combines morphological, ecological, and genetic characteristics. The five investigated populations, including those from newly collected material from central Mexico, were observed to possess morphological features characteristic of the Nostoc genus. Results showed that both new species are strictly associated with running water, and they show clear differences in their habitat preferences. The 16S rRNA gene sequences of the five strains displayed between 98% and 99% similarity to the genus Nostoc sensu stricto. The 16S rRNA gene phylogenetic analyses inferred using Bayesian inference, maximum likelihood, and parsimony methods, placed these five strains in two separate clades distinct from other Nostoc species. The secondary structures of the 16S–23S internal transcribed spacer rRNA region in the two new species showed >10.5% dissimilarities in the operons when compared with other Nostoc species. In addition, clear morphological differences were observed between the two Mexican species, including the color of the colonies (black in N. montejanii and green in N. tlalocii), the size of the cells (greater in N. montejanii), and the number of polyphosphate granules present in the cells (one in N. montejanii and up to four in N. tlalocii).     

Phylogenetic analysis of six species of pacific abalone (Haliotidae) based on DNA sequences of 16s rRNA and cytochrome c oxidase subunit I mitochondrial genes

Six species of abalones (Haliotidae) are found on the Korean coasts. Identification and characterization of these abalones are usually based on morphologic characters. In this research we compared the partial sequences of the mitochondrial 16S ribosomal RNA and cytochrome c oxidase subunit I genes to identify species using molecular data and to determine their phylogenetic relationships. Sequence alignments and phylogenetic analysis revealed that the 6 species fell into 2 distinct groups which were genetically distant from each other and exhibited little internal phylogenetic resolution. One group included Haliotis discus hannai, H. discus discus, H. madaka, and H. gigantea, while the other group contained H. diversicolor supertexta and H. diversicolor diversicolor. The 16S rRNA sequences were relatively more conserved than to the COI sequences, but both gene sequences provided sufficient phylogenetic information to distinguish among the 6 species of Pacific abalone, and thus could be valuable molecular characters for species identification.     

<Emphasis Type="Italic">Aliihoeflea aestuarii</Emphasis> gen. nov., sp. nov., a novel bacterium isolated from tidal flat sediment

A novel Gram-negative and rod-shaped bacterium, designated N8^T, was isolated from tidal flat sediment. Phylogenetic analysis based on 16S rRNA gene sequences showed that N8^T strain is associated with the family Phyllobacteriaceae: two uncultured clones (98.4 and 99.8% 16S rRNA gene sequence similarity) and the genus Mesorhizobium (≤97.0%). The novel strain formed a separate clade with uncultured clones in the phylogenetic tree based on 16S rRNA gene sequences. Cellular fatty acid profiles predominately comprised C_18:1 ω7c and C_19:0 cyclo ω8c. The major isoprenoid quinone is ubiquinone-10 and genomic DNA G+C content is 53.4 mol%. The polyphasic taxonomic study indicates that the novel strain N8^T represents a novel species of the new genus in the family Phyllobacteriaceae, named Aliihoeflea aestuarii. The type strain is N8^T (= KCTC 22052^T= JCM 15118^T= DSM 19536^T).     

Phylogeny of harvestmen family Gonyleptidae inferred from a multilocus approach (Arachnida: Opiliones)

Gonyleptidae is the second most diverse harvestmen family and the most studied in terms of morphology, behaviour, and ecology. Despite this, few phylogenetic studies have focused on gonyleptids, and those are based on a very limited number of taxa. We addressed this gap by constructing a phylogenetic hypothesis of the family using 101 taxa from all 16 gonyleptid subfamilies and four mitochondrial and nuclear loci (COI, 28S rRNA, 12S rRNA, and 16S rRNA). These were analysed under parsimony and likelihood optimality criteria (and using direct optimization for the former). Relationships among Gonyleptoidea and within each subfamily of Gonyleptidae were largely congruent between parsimony and maximum‐likelihood approaches. Taxonomic actions from our phylogeny include the following: Tricommatidae, new status, is restored as a family; Metasarcidae, new status, is recognized as a family and considered sister to the Cosmetidae; and Cranainae and Manaosbiinae are suggested as members of Gonyleptidae, restoring Roewer's concept of the family. Within Gonyleptidae, the “K92” group—composed of Sodreaninae, Caelopyginae, Hernandariinae, Progonyleptoidellinae, and Gonyleptinae—forms a clade, although the latter two subfamilies are not monophyletic. The genus Parampheres is here transferred to Caelopyginae, and “Multumbo” dimorphicus to Gonyleptinae. Gonyleptidae is characterized by the presence of a ventral process on the penis glans and a bifid apophysis on the male coxa IV. The long‐legged Mitobatinae can be considered monophyletic only if some short‐legged pachylines are included, or if we assume that elongate legs arose twice independently (in the true mitobatine genera and in Longiperna). Pachylinae, the most diverse gonyleptid subfamily, represents several distinct lineages. We further conclude that the traditional use of a small set of morphological characters in the systematics of Gonyleptidae is unable to explain the complex evolution of the family.