Phylogenetic relationships of Serpulidae (Annelida: Polychaeta) based on 18S rDNA sequence data, and implications for opercular evolution

Phylogenetic relationships of (19) serpulid taxa (including Spirorbinae) were reconstructed based on 18S rRNA gene sequence data. Maximum likelihood, Bayesian inference, and maximum parsimony methods were used in phylogenetic reconstruction. Regardless of the method used, monophyly of Serpulidae is confirmed and four monophyletic, well-supported major clades are recovered: the Spirorbinae and three groups hitherto referred to as the Protula-, Serpula-, and Pomatoceros-group. Contrary to the taxonomic literature and the hypothesis of opercular evolution, the Protula-clade contains non-operculate (Protula, Salmacina) and operculate taxa both with pinnulate and non-pinnulate peduncle (Filograna vs. Vermiliopsis), and most likely is the sister group to Spirorbinae. Operculate Serpulinae and poorly or non-operculate Filograninae are paraphyletic. It is likely that lack of opercula in some serpulid genera is not a plesiomorphic character state, but reflects a special adaptation.     

Phylogeny of Tunicata inferred from molecular and morphological characters

The phylogeny of the Tunicata was reconstructed using molecular and morphological characters. Mitochondrial cytochrome oxidase I (cox1) and 18S rDNA sequences were obtained for 14 and 4 tunicate species, respectively. 18S rDNA sequences were aligned with gene sequences obtained from GenBank of 57 tunicates, a cephalochordate, and a selachian craniate. Cox1 sequences were aligned with the sequence of two ascidians and a cephalochordate obtained from GenBank. Traditional, morphological, life history, and biochemical characters of larval and adult stages were compiled from the literature and analyzed cladistically. Separate and simultaneous parsimony analyses of molecular and morphological data were carried out. Aplousobranch ascidians from three different families were included in a molecular phylogenetic analysis for the first time. Analysis of the morphological, life history, and biochemical characters results in a highly unresolved tree. Aplousobranchiata form a strongly supported monophylum in the analysis of the 18S rDNA data, the morphological data, and the combined data set. Cionidae is not included in the Aplousobranchiata but nests within the Phlebobranchiata. Appendicularia (=Larvacea) nest within the 'Ascidiacea' as the sister taxon of Aplousobranchiata in the parsimony analysis of the 18S rDNA data and the combined analysis. A potential morphological synapomorphy of Aplousobranchiata plus Appendicularia is the horizontal orientation of the larval tail. In the 18S rDNA and the combined analysis, Thaliacea is included in the 'Ascidiacea' as the sister group to Phlebobranchiata. Pyrosomatida is found to be the sister taxon to the Salpidae in analyses of 18S rDNA and combined data, whereas the analysis of the morphological data recovers a sister group relationship between Doliolidae and Salpidae. Results of cox1 analyses are incongruent with both the 18S rDNA and the morphological phylogenies. Cox1 sequences may evolve too rapidly to resolve relationships of higher tunicate taxa. However, the cox1 data may be useful at lower taxonomic levels.     

Phylogeny of the arachnid order Opiliones (Arthropoda) inferred from a combined approach of complete 18S and partial 28S ribosomal DNA sequences and morphology

The phylogenetic relationships among the main evolutionary lines of the arachnid order Opiliones were investigated by means of molecular (complete 18S rDNA and the D3 region of the 28S rDNA genes) and morphological data sets. Equally and differentially weighted parsimony analyses of independent and combined data sets provide evidence for the monophyly of the Opiliones. In all the analyses, the internal relationships of the group coincide in the monophyly of the following main groups: Cyphophthalmi, Eupnoi Palpatores, Dyspnoi Palpatores, and Laniatores. The Cyphophthalmi are monophyletic and sister to a clade that includes all the remaining opilionid taxa (=Phalangida). Within the Phalangida the most supported hypothesis suggests that Palpatores are paraphyletic, as follows: (Eupnoi (Dyspnoi + Laniatores)), but the alternative hypothesis (Laniatores (Eupnoi + Dyspnoi)) is more parsimonious in some molecular data analyses. Relationships within the four main clades are also addressed. Evolution of some morphological characters is discussed, and plesiomorphic states of these characters are evaluated using molecular data outgroup polarization. Finally, Martens' hypothesis of opilionid evolution is assessed in relation to our results.     

Phylogeny of Hesionidae (Aciculata, Polychaeta), assessed from morphology, 18S rDNA, 28S rDNA, 16S rDNA and COI

We assess phylogenetic relationships within the polychaete family Hesionidae from morphological data combined with nucleotide data from 18S rDNA, 28S rDNA, 16S rDNA and COI. Parsimony and Bayesian analyses were performed on two data sets; the first was based on a more restricted set of terminals with both morphological and molecular data (17 ingroup terminals), while the second included additional taxa with morphological data only (25 ingroup terminals). The different sets of terminals yielded fully congruent results, as did the parsimony and the Bayesian analyses. Our results indicate high levels of homoplasy in traditionally used morphological characters in the group, and that Hesioninae, Gyptini and Gyptis are nonmonophyletic. Hesionini (mainly Hesione and Leocrates ), Psamathini (mainly Hesiospina , Micropodarke , Nereimyra , Psamathe and Syllidia ), Ophiodrominae (Gyptini and Ophiodromini) and Ophiodromini (mainly Heteropodarke , Ophiodromus and Podarkeopsis ) are monophyletic and agree with previous classifications, and Hesionini is probably the sister to all other hesionids. The placements of the small hesionids capricornia and Lizardia , the hydrothermal vent taxa Hesiodeira and Hesiolyra , and the newly described Hesiobranchia , remain uncertain.     

The interrelationships of all major groups of Platyhelminthes: phylogenetic evidence from morphology and molecules

We used a data matrix of 65 morphological characters from 25 ingroup and 6 outgroup taxa, and an alignment comprising complete 18S rDNA sequences from 82 species of parasitic and free-living Platyhelminthes and from 19 species of lower invertebrates to analyse phylogenetic relationships of various platyhelminth taxa. Of the 1358 unambiguously alignablc molecular positions, 995 were variable and 757 were phylogenctically informative (parsimony criterion); complete 18S rDNA sequences ranged in length from 1755 to 2873 bp. Main conclusions are: Ncodermata are monophylctic, and the Trematoda, Monogenca and Cestoda within them are monophylctic as well. The sister group of the Ncodermata is all the other Ncoophora; the Kalyptorhynchia, Typhloplanida, Dalyelliida and Tcmnocephalida form one clade, and the last three another. Monophyly of the Seriata is rejected, but Polycladida/ Macrostomida/Haplopharyngida are monophylctic, as arc the last two taxa. As a consequence, validity of the taxon Trepaxonemata is rejected. Further studies must show the correct position of the Acocla and Nemertodermatida. It is stressed that morphological and molecular data in some respects lead to contradictory results, for instance concerning the position of the Fecampiidac/ Urastoma/Ichthyophaga and the relative position of the Lccithoepitheliata. Denser sampling of taxa for molecular data, complementary sequences from independent genes, and inclusion of additional morphological data are necessary to resolve these contradictions.     

A Molecular Study of Euglenoid Phylogeny using Small Subunit rDNA

The euglenoids are an ancient and extremely diverse lineage of eukaryotic flagellates with unclear relationships among taxa. Synapomorphies for the euglenoids include a surface pellicle and a closed mitosis with a series of separate sub-spindles. The taxonomy currently in use is inconsistent with the available data and needs revision. Most euglenoid phylogenies are largely intuitive reconstructions based on a limited number of morphological characters. Therefore, we have added molecular characters from the Small Subunit (SSU) rDNA to generate an overall phylogenetic framework for the euglenoids. SSU rDNA sequences from photosynthetic, osmotrophic, and phagotrophic euglenoids were aligned based on secondary structure. Phylogenetic analysis using the conserved areas of the sequence was performed using parsimony, maximum likelihood, and distance methods. Trees derived using different criteria are in agreement. The euglenoids form a distinct monophyletic clade with phagotrophic members diverging prior to the phototrophic and osmotrophic members. Among photosynthetic members, the biflagellate form diverged prior to the uniflagellate form. Additionally, the genus Euglena appears to be paraphyletic, with osmotrophic taxa, such as Astasia and Khawkinea, diverging independently within the clade containing the photosynthetic genus Euglena.     

A phylogenetic hypothesis for Asilidae based on a total evidence analysis of morphological and DNA sequence data (Insecta: Diptera: Brachycera: Asiloidea)

An hypothesis of phylogenetic relationships of Asilidae and its constituent taxa is presented, combining morphological and DNA sequence data in a total evidence framework. It is based on 77 robber fly species, 11 Asiloidea outgroup species, 211 morphological characters of the adult fly, and approximately 7300 bp of nuclear DNA from five genes (18S and 28S rDNA, AATS, CAD, and EF-1α protein-encoding DNA). The equally weighted, simultaneous parsimony analysis under dynamic homology in POY resulted in a single most parsimonious cladogram with a cost of 27,582 (iterative pass optimization; 27,703 under regular direct optimization). Six of the 12 included subfamily taxa are recovered as monophyletic. Trigonomiminae, previously always considered as monophyletic based on morphology, is shown to be non-monophyletic. Two of the three Trigonomiminae genera, Holcocephala Jaennicke, 1867 and Rhipidocephala Hermann, 1926, group unexpectedly as the sister taxon to all other Asilidae. Laphriinae, previously seen in the latter position, is the sister group of the remaining Asilidae. Five other subfamily taxa, i.e. Brachyrhopalinae, Dasypogoninae, Stenopogoninae, Tillobromatinae, and Willistonininae, are also shown to be non-monophyletic. The phylogenetic relationships among the higher-level taxa are partly at odds with findings of a recently published morphological study based on more extensive taxon sampling. The total evidence hypothesis is considered as the most informative one, but the respective topologies from the total-evidence, morphology-only, and molecular-only analyses are compared and contrasted in order to discuss the signals from morphological versus molecular data, and to analyze whether the molecular data outcompete the fewer morphological characters. A clade Apioceridae+Mydidae is corroborated as the sister taxon to Asilidae.     

基于28S rDNA D2基因片段与形态特征的矛茧蜂亚科系统发育研究（膜翅目：茧蜂科）

本研究选取矛茧蜂亚科Doryctinae（昆虫纲Insecta：膜翅目Hymenoptera：茧蜂科Braconidae）的6族15属18种做内群,茧蜂科其它7亚科11属11种做外群,首次结合同源核糖体28S rDNA D2基因序列片段和100个形态学和解剖学特征对该亚科进行了系统发育学研究。利用“非圆口类"的小腹茧蜂亚科Microgastrinae为根,以PAUP*4.0和MrBayes 3.0B4软件分别应用最大简约法（MP）和贝叶斯法对矛茧蜂亚科的分子数据和分子数据与非分子数据的结合体进行了运算分析;并以PAUP*4.0对矛茧蜂亚科的28S rDNA D2基因序列片段的碱基组成与碱基替代情况进行了分析。结果表明：矛茧蜂亚科的28S rDNA D2基因序列片段的GC含量在39.33%～48.28%之间变动,而对于碱基替代情况来讲,矛茧蜂亚科各成员间序列变异位点上颠换（transversion）大于转换（transition）。不同的分析算法所产生的系统发育树都表明矛茧蜂亚科是一个界限分明的单系群;在矛茧蜂亚科内,除了吉丁茧蜂族Siragrini为单系群外,其他族（矛茧蜂族Doryctini和方头茧蜂族Hecabolini）都是并系群。对于矛茧蜂亚科内各属之间的相互亲缘关系,不同算法所得的系统发育树的拓扑结构不完全一致,表明矛茧蜂亚科内（属及族）的系统发育关系还有待于进一步研究。     

Phylogeny of benthic Phyllodocidae (Polychaeta) based on morphological and molecular data

A combined molecular (18S rDNA, 28S rDNA, 16S rDNA and COI) and morphological analysis of the benthic phyllodocids is presented for the first time. Nineteen phyllodocids and two outgroup taxa are assessed using parsimony, maximum likelihood and Bayesian analyses. We demonstrate high degrees in homoplasy in the traditionally used morphological phyllodocid characters, and show that all the three current subfamilies Phyllodocinae, Eteoninae and Notophyllinae are non-monophyletic. The genera Eulalia, Eumida, Protomystides, Pseudomystides, Pterocirrus and Sige form a well-supported group, as does Mystides and Nereiphylla. Another clade with strong support includes Eteone and Paranaitis, although with Eteone nested within a paraphyletic Paranaitis. The relationship between these two taxa indicate that the unusual arrangement of modified cirri on the first segments in Eteone is due to a fusion of segment 1 and 2 where the cirri of segment 1 have been reduced. Eulalia is non-monophyletic and should be split, minimally into two groups. Our results are ambiguous regarding the ancestral phyllodocid condition of absence-presence of median antenna or nuchal papilla and uniramous or biramous parapodia, but shows that the absence of cirri on segment 3 (previously an apomorphy, for e.g., Mystides, Pseudomystides and Hesionura) is maximally homoplastic.     

Combined molecular and morphological phylogeny of Eulophidae (Hymenoptera: Chalcidoidea), with focus on the subfamily Entedoninae

A new combined molecular and morphological phylogeny of the Eulophidae is presented with special reference to the subfamily Entedoninae. We examined 28S D2–D5 and CO1 gene regions with parsimony and partitioned Bayesian analyses, and examined the impact of a small set of historically recognized morphological characters on combined analyses. Eulophidae was strongly supported as monophyletic only after exclusion of the enigmatic genus Trisecodes. The subfamilies Eulophinae, Entiinae (=Euderinae) and Tetrastichinae were consistently supported as monophyletic, but Entedoninae was monophyletic only in combined analyses. Six contiguous bases in the 3e′ subregion of the 28S D2 rDNA contributed to placement of nominal subgenus of Closterocerus outside Entedoninae. In all cases, Euderomphalini was excluded from Entiinae, and we suggest that it be retained in Entedoninae. Opheliminae n. stat. is raised from tribe to subfamily status. Trisecodes is removed from Entedoninae but retained as incertae sedis in Eulophidae until its family placement can be determined new placement . The genera Neochrysocharis stat. rev. and Asecodes stat. rev. are removed from synonymy with Closterocerus because strong molecular differences corroborate their morphological differences. Closterocerus (Achrysocharis) germanicus is transferred to the genus Chrysonotomyia n. comb. based on molecular and morphological characters.     

Relationships among the major lineages of Dictyoptera: the effect of outgroup selection on dictyopteran tree topology

Abstract Dictyoptera, comprising Blattaria, Isoptera, and Mantodea, are diverse in appearance and life history, and are strongly supported as monophyletic. We downloaded COII, 16S, 18S, and 28S sequences of 39 dictyopteran species from GenBank. Ribosomal RNA sequences were aligned manually with reference to secondary structure. We included morphological data (maximum of 175 characters) for 12 of these taxa and for an additional 15 dictyopteran taxa (for which we had only morphological data). We had two datasets, a 59‐taxon dataset with five outgroup taxa, from Phasmatodea (2 taxa), Mantophasmatodea (1 taxon), Embioptera (1 taxon), and Grylloblattodea (1 taxon), and a 62‐taxon dataset with three additional outgroup taxa from Plecoptera (1 taxon), Dermaptera (1 taxon) and Orthoptera (1 taxon). We analysed the combined molecular?morphological dataset using the doublet and MK models in Mr Bayes , and using a parsimony heuristic search in paup . Within the monophyletic Mantodea, Mantoida is recovered as sister to the rest of Mantodea, followed by Chaeteessa; the monophyly of most of the more derived families as defined currently is not supported. We recovered novel phylogenetic hypotheses about the taxa within Blattodea (following Hennig, containing Isoptera). Unique to our study, one Bayesian analysis places Polyphagoidea as sister to all other Dictyoptera; other analyses and/or the addition of certain orthopteran sequences, however, place Polyphagoidea more deeply within Dictyoptera. Isoptera falls within the cockroaches, sister to the genus Cryptocercus. Separate parsimony analyses of independent gene fragments suggest that gene selection is an important factor in tree reconstruction. When we varied the ingroup taxa and/or outgroup taxa, the internal dictyopteran relationships differed in the position of several taxa of interest, including Cryptocercus, Polyphaga, Periplaneta and Supella. This provides further evidence that the choice of both outgroup and ingroup taxa greatly affects tree topology.     

Searching for natural lineages within the Cerylonid Series (Coleoptera: Cucujoidea)

Phylogenetic relationships within the diverse beetle superfamily Cucujoidea are poorly known. The Cerylonid Series (C.S.) is the largest of all proposed superfamilial cucujoid groups, comprising eight families and representing most of the known cucujoid species diversity. The monophyly of the C.S., however, has never been formally tested and the higher-level relationships among and within the constituent families remain equivocal. Here we present a phylogenetic study based on 18S and 28S rDNA for 16 outgroup taxa and 61 C.S. ingroup taxa, representing seven of the eight C.S. families and 20 of 39 subfamilies. We test the monophyly of the C.S., investigate the relationships among the C.S. families, and test the monophyly of the constituent families and subfamilies. Phylogenetic reconstruction of the combined data was achieved via standard static alignment parsimony analyses, Direct Optimization using parsimony, and partitioned Bayesian analysis. All three analyses support the paraphyly of Cucujoidea with respect to Tenebrionoidea and confirm the monophyly of the C.S. The C.S. families Bothrideridae, Cerylonidae, Discolomatidae, Coccinellidae and Corylophidae are supported as monophyletic in all analyses. Only the Bayesian analysis recovers a monophyletic Latridiidae. Endomychidae is recovered as polyphyletic in all analyses. Of the 14 subfamilies with multiple terminals in this study, 11 were supported as monophyletic. The corylophid subfamily Corylophinae and the coccinellid subfamilies Chilocorinae and Scymninae are recovered as paraphyletic. A sister grouping of Anamorphinae+Corylophidae is supported in all analyses. Other taxonomic implications are discussed in light of our results.     

基于28S rDNA D2基因片段与形态特征的优茧蜂亚科系统发育研究

本研究选取优茧蜂亚科Euphorinae(膜翅目Hymenoptera:茧蜂科Braconidae)的8族19属23种作为内群,茧蜂其它6个亚科的8属8种作外群,首次结合同源核糖体28S rDNA D2基因序列片段和41个形态学特征对该亚科进行了系统发育学研究。利用"圆口类"的内茧蜂亚科Rogadinae、茧蜂亚科Braconinae、矛茧蜂亚科Doryctinae的3个亚科为根,以PAUP*4.0和MrBayes3.0B4软件分别应用最大简约法(MP)和贝叶斯法对优茧蜂亚科的分子数据和分子数据与非分子数据的结合体进行了分析;并以PAUP*4.0对优茧蜂亚科的28S rDNA D2基因序列的片段的碱基组成与碱基替代情况进行了分析。结果表明:优茧蜂亚科的28S rDNA D2基因序列片段的GC%含量在40.00%~49.25%之间变动,而对于碱基替代情况来讲,优茧蜂亚科各个成员间序列变异位点上颠换(transversion)大于转换(transition);不同的分析和算法所产生的系统发育树都表明目前根据形态定义出的优茧蜂亚科Euphorinae不是一个单系群,而是一个与蚁茧蜂亚科Neoneurinae和高腹茧蜂亚科Cenocoelinae混杂在一起的并系群;在优茧蜂亚科内部,悬茧蜂族Meterorini和食甲茧蜂族Microctonini(排除猎户茧蜂属Orionis)为单系群,而宽鞘茧蜂族Centistini、大颚茧蜂族Cosmophorini、优茧蜂族Euphorini、瓢虫茧蜂族Dinocampini为并系群;悬茧蜂族Meterorini在优茧蜂亚科Euphorinae内位于基部位置的观点得到部分的支持,同时食甲茧蜂族Microctonini被判定为相对进化的类群。此外对于优茧蜂亚科内各属之间的相互亲缘关系,不同算法所得到的系统发育属的结果不完全一致,这表明优茧蜂亚科内(属及族)的系统发育关系还有待于进一步研究。     

A molecular phylogeny of annelids

We present parsimony analyses of annelids based on the largest taxon sample and most extensive molecular data set yet assembled, with two nuclear ribosomal genes (18S rDNA and the D1 region of 28S rDNA), one nuclear protein coding‐gene (Histone H3) and one mitochondrial ribosomal gene (16S rDNA) from 217 terminal taxa. Of these, 267 sequences are newly sequenced, and the remaining were obtained from GenBank. The included taxa are based on the criteria that the taxon must have 18S rDNA or at least two other loci. Our analyses show that 68% of annelid family ranked taxa represented by more than one taxon in our study are supported by a jackknife value > 50%. In spite of the size of our data set, the phylogenetic signal in the deepest part of the tree remains weak and the majority of the currently recognized major polychaete clades (except Amphinomida and Aphroditiformia) could not be recovered. Terbelliformia is monophyletic (with the exclusion of Pectinariidae, for which only 18S data were available), whereas members of taxa such as Phyllodocida, Cirratuliformia, Sabellida and Scolecida are scattered over the trees. Clitellata is monophyletic, although Dinophilidae should possibly be included, and Clitellata has a sister group within the polychaetes. One major problem is the current lack of knowledge on the closest relatives to annelids and the position of the annelid root. We suggest that the poor resolution in the basal parts of the trees presented here may be due to lack of signal connected to incomplete data sets both in terms of terminal and gene sampling, rapid radiation events and/or uneven evolutionary rates and long‐branch attraction. © The Willi Hennig Society 2006.     

Phylogeny of eriphioid crabs (Brachyura,Eriphioidea) inferred from molecular and morphological studies

The evolutionary relationships of the brachyuran crab superfamily Eriphioidea, commonly known as stone or rubble crabs, are examined. Analysis of three mitochondrial (12S, 16S and COI) and two nuclear loci (18S and Histone 3) was carried out for 51 taxa representing the Carpilioidea, Dairoidea, Eriphioidea, Goneplacoidea, Parthenopoidea, Pilumnoidea, Portunoidea, Pseudozioidea and Xanthoidea. Phylogenetic analyses of molecular data used three methods of inference that recovered similar topologies with minor differences. Maximum parsimony analysis of 20 morphological characters taken from first zoeas of 11 species yielded two equally parsimonious trees and generally supported the molecular analyses. None of the analyses recovered Eriphioidea as monophyletic, and each of the eriphioid families represented by two or more taxa was shown to be polyphyletic in both molecular and larval analyses. This study indicates that the present classification based on adult morphology is incongruent with phylogenetic relationships and that the diagnostic characters the result of convergence (particularly in feeding morphology) rather than shared ancestry.     

Analysis of the systematic relationships among ticks of the genera Rhipicephalus and Boophilus (Acari: Ixodidae) based on mitochondrial 12S ribosomal DNA gene sequences and morphological characters

A portion of mitochondrial 12S rDNA sequences (337-355 base pairs) and 63 morphological characters of 36 hard-tick species belonging to 7 genera were analyzed to determine the phylogenetic relationships among groups and species of Rhipicephalus and between the genera Rhipicephalus and Boophilus. Molecular and morphological data sets were first examined separately. The molecular data were analyzed by maximum parsimony (MP), maximum likelihood, and neighbor-joining distance methods; the morphological data were analyzed by MP After their level of congruence was evaluated by a partition homogeneity test, all characters were combined and analyzed by MP. The branches of the tree obtained by combining the data sets were better resolved than those of the trees inferred from the separate analyses. Boophilus is monophyletic and arose within Rhipicephalus. Boophilus species clustered with species of the Rhipicephalus evertsi group. Most of the clustering within Rhipicephalus was, however, consistent with previous classifications based on morphological data. Morphological characters were traced on the molecular reconstruction in order to identify characters diagnostic for monophyletic clades. Within the Rhipicephalus sanguineus complex, the sequences of specimens morphologically identified as Rhipicephalus turanicus were characterized by a high level of variability, indicating that R. turanicus-like morphology may cover a spectrum of distinct species.     

New molecular data for tardigrade phylogeny, with the erection of Paramacrobiotus gen. nov.

Up to few years ago, the phylogenies of tardigrade taxa have been investigated using morphological data, but relationships within and between many taxa are still unresolved. Our aim has been to verify those relationships adding molecular analysis to morphological analysis, using nearly complete 18S ribosomal DNA gene sequences (five new) of 19 species, as well as cytochrome oxidase subunit 1 (COI) mitochondrial DNA gene sequences (15 new) from 20 species, from a total of seven families. The 18S rDNA tree was calculated by minimum evolution, maximum parsimony (MP) and maximum likelihood (ML) analyses. DNA sequences coding for COI were translated to amino acid sequences and a tree was also calculated by neighbour-joining, MP and ML analyses. For both trees (18S rDNA and COI) posterior probabilities were calculated by MrBayes. Prominent findings are as follows: the molecular data on Echiniscidae (Heterotardigrada) are in line with the phylogenetic relationships identifiable by morphological analysis. Among Eutardigrada, orders Apochela and Parachela are confirmed as sister groups. Ramazzottius (Hypsibiidae) results more related to Macrobiotidae than to the genera here considered of Hypsibiidae. Macrobiotidae and Macrobiotus result not monophyletic and confirm morphological data on the presence of at least two large groups within Macrobiotus. Using 18S rDNA and COI mtDNA genes, a new phylogenetic line has been identified within Macrobiotus , corresponding to the ' richtersi-areolatus group'. Moreover, cryptic species have been identified within the Macrobiotus ' richtersi group' and within Richtersius . Some evolutionary lines of tardigrades are confirmed, but others suggest taxonomic revision. In particular, the new genus Paramacrobiotus gen. n. has been identified, corresponding to the phylogenetic line represented by the ' richtersi-areolatus group'.