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Achaeta (Enchytraeidae, Oligochaeta, Clitellata) is a genus of small, soil-dwelling annelids, peculiar by the absence of chaetae in all species. Here two new species of this genus are described from afforestation sites in southern Poland. Descriptions are based on the investigation of living and preserved material. The species are also characterized at the DNA level, using fragments of the COI and ITS genes, sequenced from the holotypes and paratypes. The worms are not longer than 4-6 mm, with about 30 to 35 segments, and with medium-large male and female reproductive organs. Achaeta florens sp. nov., discovered at a post-mining site, has four segmental pyriform glands, an oesophageal appendage restricted to segment V, a clitellum with distinct transverse rows and dorsal and ventral interruption, and spermathecae with blossom-like fields of vesicular gland cell apices around the ventrally located ectal pores. The species is without peculiar characters but the combination of characters is diagnostic. Its morphological distinction from A. nurmineni, described from the Indian subcontinent, is difficult due to the poor description of that species; A. nurmineni is therefore considered a species inquirenda. Achaeta gemmata sp. nov., discovered in high abundance at a post-fire site, is without pyriform glands but with conspicuous, gem-like lateral papillae in all preclitellar segments. The papillae are modified and enlarged epidermal gland cells that protrude into the body cavity. The species is without oesophageal appendage and has a dorsally closed clitellum and lateral spermathecal pores. It is superficially similar to Achaeta camerani, found at the same site, but can be distinguished based on sexual and non-sexual characters. A comparison of COI sequences showed 100% identity with a specimen from Sweden, erroneously identified as A. cf. brevivasa. Genetic distance between the two new species based on COI differences is 20%, distances to other species of Achaeta with publicly available COI sequences range from 16% to 26%.

The extremely difficult and challenging process is identifying pheretimoid species, genus Metaphire and Amynthas involving increased homoplasy in various morphological characteristics. The molecular identification, phylogenetic relationships, and evolutionary divergence time of earthworms belonging to the pheretimoid complex were investigated in this study using partial mitochondrial COI (cytochrome C oxidase subunit I) gene sequences ranging from 550-680 bp. Results revealed that 86 pheretimoid earthworms were morphologically different from a total of 342 mature worms. Moreover, 11 pheretimoid species were molecularly identified, including Metaphire posthuma (02), M. anomala (01), M. houlleti (02), M. californica (01), M. birmanica (02), Amynthas minimus (01), A. morrisi (01), and M. bununa (01). A phylogenetic tree was constructed with bootstrap values of 95%, which supported a monophyletic lineage of two well-supported clades formed by 12 partial COI sequences and 48 GenBank sequences using Hirudo medicinalis as an outgroup. The monophyly of these obtained genera indicated overall similarity at species level. Today, species like Amynthas, Metaphire and Pheretima have worm diversity in the form of pheretimoid earthworms, which dates to the Late Miocene (11.2-5.3 Mya) and the Pliocene (5.3-2.4 Mya). Compared to all relevant pheretimoid species, genetic p-distance values ranged from 0.0% to 0.57% (less than 1%). These low range values demonstrated that both genera Metaphire and Amynthas, supported the theory, which states that there are shared similarities among the species, despite different morphology. The current study is the first attempt in Pakistan to identify earthworms through DNA barcoding thus providing a genomic stamp. The work explored the significance of COI gene sequences to construct molecular tools that will be useful to overcome the different obstacles in morphologically similar earthworm identification and their phylogenetic study.

Background: Deep mitochondrial divergences were observed in Scandinavian populations of the terrestrial to semi-aquatic annelid Fridericia magna (Clitellata: Enchytraeidae). This raised the need for testing whether the taxon is a single species or a complex of cryptic species., Results: A total of 62 specimens from 38 localities were included in the study, 44 of which were used for species delimitation. First, the 44 specimens were divided into clusters using ABGD (Automatic Barcode Gap Discovery) on two datasets, consisting of sequences of the mitochondrial markers COI and 16S. For each dataset, the worms were divided into six not completely congruent clusters. When they were combined, a maximum of seven clusters, or species hypotheses, were obtained, and the seven clusters were used as input in downstream analyses. We tested these hypotheses by constructing haplowebs for two nuclear markers, H3 and ITS, and in both haplowebs the specimens appeared as a single species. Multi-locus species delimitation analyses performed with the Bayesian BPP program also mainly supported a single species. Furthermore, no apparent morphological differences were found between the clusters. Two of the clusters were partially separated from each other and the other clusters, but not strongly enough to consider them as separate species. All 62 specimens were used to visualise the Scandinavian distribution, of the species, and to compare with published COI data from other Fridericia species., Conclusion: We show that the morphospecies Fridericia magna is a single species, harbouring several distinct mitochondrial clusters. There is partial genetic separation between some of them, which may be interpreted as incipient speciation. The study shows the importance of rigorous species delimitation using several independent markers when deep mitochondrial divergences might give the false impression of cryptic speciation.

Limnodrilus species are annelid worms distributed worldwide in various freshwater sediments. The systematics of Limnodrilus has chiefly been based on morphology, but the genus has not been subject to any closer phylogenetic studies over the past two decades. To reconstruct the evolutionary history of Limnodrilus, and to assess the monophyly of this genus and its systematic position within the subfamily Tubificinae (Annelida: Clitellata: Naididae), 45 Limnodrilus specimens, representing 19 species, and 35 other naidid species (representing 24 genera) were sampled. The data consisted of sequences of three mitochondrial genes (COI, 12S and 16S rDNA) and four nuclear markers (18S and 28S rDNA, Histone 3, and ITS). The phylogeny was estimated, using Maximum Likelihood and Bayesian analyses of concatenated data of seven DNA loci, as well as a multi-locus coalescent-based approach. All analyses strongly suggest that Limnodrilus is monophyletic, but only if the morphospecies L. rubripenis is removed from it. Limnodrilus rubripenis and (at least) Baltidrilus, Lophochaeta and some species attributed to Varichaetadrilus comprise the sister group to the clade Limnodrilus sensu stricto, and the latter is further divided into three well-supported groups. One of them contains morphospecies characterized by short cuticular penis sheaths and enlarged chaetae in anterior segments (L. udekemianus, L. silvani and L. grandisetosus). The second is a small group of species with moderately long penis sheaths, i.e., L. sulphurensis and L. profundicola. The third, and largest group, includes not only the multitude of cryptic species in the L. hoffmeisteri complex, but also other, morphologically distinct, species nested within this complex. All studied species in this large group have long penis sheaths, which are exceptionally long in L. claparedianus, L. maumeensis, and a form morphologically intermediate between L. claparedianus and L. cervix. The identification and classification of these groups provide a framework for directed sampling in further phylogenetic studies, and for revisionary work on the L. hoffmeisteri complex and other unresolved Limnodrilus species., (Copyright © 2017 Elsevier Inc. All rights reserved.)

DNA-barcoding, using the mitochondrial marker COI, has been found successful for the identification of specimens in many animal groups, but may not be suited for species discovery and delimitation if used alone. In this study, we investigate whether two observed COI haplogroups in the earthworm Aporrectodea longa correspond to two cryptic species or if the variation is intraspecific. This is done by complementing COI with two nuclear markers, ITS2 and Histone 3. The variation is studied using distance methods, parsimony networks and Bayesian coalescent trees, and the statistical distinctness of the groups is tested on gene trees using the genealogical sorting index, Rosenberg's P AB and Rodrigo et al.'s P (RD) . We also applied multilocus species delimitation based on the multispecies coalescence model. The two haplogroups were found in COI, and all tests except P (RD) found them to be significantly distinct. However, in ITS2, the same groups were not recovered in any analyses or tests. H3 was invariable in A. longa, and was, therefore, included only in the multilocus analysis, which preferred a model treating A. longa as one species over a model splitting it into two. We also compared two measurements of size, body length, and no. of segments between the groups. No difference in body length was found, and although a significant difference in no. of segments was noted the haplogroup with the lower mean showed both the highest and the lowest value. When combined, these results led us to the conclusion that there is no support for the separation of A. longa into two cryptic species. This study again highlights the importance of complementing mitochondrial barcodes with more data when establishing species boundaries.

Background: Oligochaetes are valuable bioindicators of the quality of watercourse and lake sediments. The morphological identification of aquatic oligochaetes is difficult, prompting the development of new molecular oligochaete indices based on DNA barcoding and Next-generation sequencing of sorted specimens. In general, the samples for DNA barcoding are fixed in absolute ethanol. However, in the case of aquatic oligochaetes, this medium is not appropriate as it can induce a modification of specimen abundances and of the composition of communities. Therefore, we investigated the possibility to amplify and sequence aquatic oligochaetes fixed in formalin for a short time. We performed guanidine extraction and polymerase chain reaction (PCR) amplification/sequencing of the cytochrome c oxydase I (COI) gene on tissue fragments fixed in formalin for different periods of time (from 1 h to 1 week) and in ethanol., Results: The large majority of aquatic oligochaete specimens fixed in formalin for up to 1 week could be successfully amplified and all obtained sequences were of high quality. The amplification and sequencing success rate of formalin-fixed samples and ethanol-fixed samples was similar. These results suggest that formalin fixation of aquatic oligochaete tissues for a short time does not cause serious damages to DNA and inhibit PCR amplification., Conclusion: The possibility to fix aquatic oligochaetes with formalin before genetic analyses is very promising for diversity monitoring, for construction of a comprehensive DNA barcode library and for development of an index based on Next-generation sequencing analysis of samples composed of sorted specimens.

Eisenia fetida and Eisenia andrei are vermicomposting species that are used as model animals for testing chemical material toxicology. Eisenia spp. are grown commercially in various fields in Japan. However, these two species have not been classified because it is difficult to distinguish them morphologically; thus, all bred earthworms are called E. fetida. However, it has been proposed that these two species have different expression regulation mechanisms. Here, we classified a sample of earthworms purchased from several farms, confirming that both E. fetida and E. andrei are present in Japanese earthworm breeding programs. We also characterized two highly active endoglucanases (EfEG1 and EfEG2) from the E. fetida Waki strain, which contained strong fibrinolytic enzymes for improving human health. We confirmed that EfEG1 is 1371 bp long and belongs to GHF9. Thus, E. fetida Waki may have commercial application for biomass utilization and as a dietary health supplement.

Within Oligochaeta, Chaetogaster limnaei is unusual in exhibiting a parasitic relationship with freshwater pulmonate snails. Taxonomic confusion has been caused by differences in what have been considered 2 subspecies of this worm: Chaetogaster limnaei limnaei is an ectosymbiont and is present inside the mantle cavity of the snail, whereas Chaetogaster limnaei vaghini is parasitic and lives in the kidney of the snail. This study explored the distribution of these annelids in central New York and used mitochondrial DNA sequence data from the COI locus to examine the relationship, evolution, and species status of the ectosymbiotic and parasitic forms of C. limnaei. Snails ( Physa gyrina ) were collected from 6 streams and lakes in central New York, with additional specimens collected from a lake in Massachusetts for comparison. One hundred and forty snails were examined, and at least 1 form of Chaetogaster was present in 88 specimens, a prevalence of 62.9%. COI sequence data from New York and Massachusetts did not reveal separate ectosymbiotic and parasitic lineages. Instead, all parasitic forms were part of a mixed clade that included both ectosymbiotic and parasitic forms. This mixed clade was nested within clades of ectosymbiotic forms only, suggesting that a plastic lineage of C. limnaei, able to be both ectosymbionts and parasites, evolved from ectoparasitic ancestors.

Earthworms belonging to the family Lumbricidae are extremely abundant in terrestrial temperate regions. They affect soil properties and nutrient cycling, thus shaping plant community composition and aboveground food webs. Some lumbricids are also model organisms in ecology and toxicology. Despite the intense research efforts dedicated to lumbricids over the last 130years, the evolutionary relationships and taxonomic classification of these organisms are still subject to great debate. Resolution of their systematics is hampered by the structural simplicity of the earthworm body plan and the existence of cryptic species. We sampled 160 earthworm specimens belonging to 84 lumbricid species (28 genera) and 22 Lumbricoidea outgroups, sequenced two nuclear genes, four mitochondrial genes and seven mitochondrial tRNAs and examined 22 morphological characters. We then applied a combination of phylogenetic methods to generate the first robust genus-level phylogeny of the Lumbricidae. Our results show that the current Lumbricidae classification and the underlying hypotheses of character evolution must be revised. Our chronogram suggests that lumbricids emerged in the Lower Cretaceous in the holarctic region and that their diversification has been driven by tectonic processes (e.g. Laurasia split) and geographical isolation. Our chronogram and character reconstruction analysis reveal that spermathecae number does not follow a gradual pattern of reduction and that parthenogenesis arose from sexual relatives multiple times in the group; the same analysis also indicates that both epigeic and anecic earthworms evolved from endogeic ancestors. These findings emphasize the strong and multiple changes to which morphological and ecological characters are subjected, challenging the hypothesis of character stasis in Lumbricidae., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The importance of the Aporrectodea caliginosa species complex lies in the great abundance and wide distribution of the species which exist within it. For more than a century, chaos has surrounded this complex; morphological criteria has failed to solve the taxonomic status of these species. This present body of work aims to study the phylogeny of this complex by increasing the number of samples used in previous molecular works and by including morphologically-similar species that were never studied using molecular tools (A. giardi, Nicodrilus monticola, N. carochensis and N. tetramammalis). Two basal clades were obtained: one formed by A. caliginosa and A. tuberculata and the other by the rest of the species. This second clade was divided into two more: one with Eurosiberian and another with Mediterranean forms. A. caliginosa and A. longa were divided into two paraphyletic groups. Both A. giardi and A. nocturna showed characteristics consistent with monophyletic groups. Each of the two recovered lineages of A. trapezoides were phylogenetically related to different sexual species. While lineage I of A. trapezoides was monophyletic, lineage II resulted to be paraphyletic, as well as the three Nicodrilus 'species'. The diversification of the complex occurred during the Late Miocene-Early Pliocene (6.92-11.09 Mya). The parthenogenetic forms within the Mediterranean clade would have diversified before the ones in the Eurosiberian clade (3.13-4.64 Mya and 1.05-3.48 Mya, respectively), thus implying the existence not only of at least two different moments in which parthenogenesis arose within this complex of species, but also of two different and independent evolutionary lines. Neither the 4× rule nor the GMYC method for species delimitation were successful for distinguishing taxonomically-distinct species., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Traditional earthworm taxonomy is hindered due to their anatomical simplicity and the plasticity of the characteristics often used for diagnosing species. Making phylogenetic inferences based on these characters is more than difficult. In this study we use molecular tools to unravel the phylogeny of the clitellate family Hormogastridae. The family includes species of large to mid-sized earthworms distributed almost exclusively in the western Mediterranean region where they play an important ecological role. We analyzed individuals from 46 locations spanning the Iberian Peninsula to Corsica and Sardinia, representing the four described genera in the family and 20 species. Molecular markers include mitochondrial regions of the cytochrome c oxidase subunit I gene (COI), 16S rRNA and tRNAs for Leu, Ala, and Ser, two nuclear ribosomal genes (nearly complete 18S rRNA and a fragment of 28S rRNA) and two nuclear protein-encoding genes (histones H3 and H4). Analyses of the data using different approaches corroborates monophyly of Hormogastridae, but the genus Hormogaster is paraphyletic and Hormogaster pretiosa appears polyphyletic, stressing the need for taxonomic revisionary work in the family. The genus Vignysa could represent an early offshoot in the family, although the relationships with other genera are uncertain. The genus Hemigastrodrilus is related to the Hormogaster elisae complex and both are found in the Atlantic drainage of the Iberian Peninsula and France. From a biogeographic perspective Corsica and Sardinia include members of two separate hormogastrid lineages. The species located in Corsica and Northern Sardinia are related to Vignysa, whereas Hormogaster pretiosa pretiosa, from Southern Sardinia, is closely related to the Hormogaster species from the NE Iberian Peninsula. A molecular dating of the tree using the separation of the Sardinian microplate as a calibration point (at 33 MY) and assuming a model of vicariance indicates that the diversification of Hormogastridae may be ancient, ranging from 97 to 67 Ma., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We have constructed the first ever phylogeny for the New Zealand earthworm fauna (Megascolecinae and Acanthodrilinae) including representatives from other major continental regions. Bayesian and maximum likelihood phylogenetic trees were constructed from 427 base pairs from the mitochondrial large subunit (16S) rRNA gene and 661 base pairs from the nuclear large subunit (28S) rRNA gene. Within the Acanthodrilinae we were able to identify a number of well-supported clades that were restricted to continental landmasses. Estimates of nodal support for these major clades were generally high, but relationships among clades were poorly resolved. The phylogenetic analyses revealed several independent lineages in New Zealand, some of which had a comparable phylogenetic depth to monophyletic groups sampled from Madagascar, Africa, North America and Australia. These results are consistent with at least some of these clades having inhabited New Zealand since rifting from Gondwana in the Late Cretaceous. Within the New Zealand Acanthodrilinae, major clades tended to be restricted to specific regions of New Zealand, with the central North Island and Cook Strait representing major biogeographic boundaries. Our field surveys of New Zealand and subsequent identification has also revealed extensive cryptic taxonomic diversity with approximately 48 new species sampled in addition to the 199 species recognized by previous authors. Our results indicate that further survey and taxonomic work is required to establish a foundation for future biogeographic and ecological research on this vitally important component of the New Zealand biota., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: As currently defined, the genus Postandrilus Qui and Bouché, 1998, (Lumbricidae) includes six earthworm species, five occurring in Majorca (Baleares Islands, western Mediterranean) and another in Galicia (NW Spain). This disjunct and restricted distribution raises some interesting phylogeographic questions: (1) Is Postandrilus distribution the result of the separation of the Baleares-Kabylies (BK) microplate from the proto-Iberian Peninsula in the Late Oligocene (30-28 Mya)--vicariant hypothesis? (2) Did Postandrilus diversify in Spain and then colonize the Baleares during the Messinian salinity crisis (MSC) 5.96-5.33 Mya--dispersal hypothesis? (3) Is the distribution the result of a two-step process--vicariance with subsequent dispersal?, Methodology/principal Findings: To answer these questions and assess Postandrilus evolutionary relationships and systematics, we collected all of the six Postandrilus species (46 specimens - 16 locations) and used Aporrectodea morenoe and three Prosellodrilus and two Cataladrilus species as the outgroup. Regions of the nuclear 28S rDNA and mitochondrial 16S rDNA, 12S rDNA, ND1, COII and tRNA genes (4,666 bp) were sequenced and analyzed using maximum likelihood and Bayesian methods of phylogenetic and divergence time estimation. The resulting trees revealed six new Postandrilus species in Majorca that clustered with the other five species already described. This Majorcan clade was sister to an Iberian clade including A. morenoe (outgroup) and Postandrilus bertae. Our phylogeny and divergence time estimates indicated that the split between the Iberian and Majorcan Postandrilus clades took place 30.1 Mya, in concordance with the break of the BK microplate from the proto-Iberian Peninsula, and that the present Majorcan clade diversified 5.7 Mya, during the MSC., Conclusions: Postandrilus is highly diverse including multiple cryptic species in Majorca. The genus is not monophyletic and invalid as currently defined. Postandrilus is of vicariant origin and its radiation began in the Late Oligocene.

A multigene data set (12S, 16S, and COI mitochondrial DNA; 18S and 28S nuclear DNA) was analyzed by Bayesian inference to estimate the phylogeny of a sample of the clitellate family Enchytraeidae (86 species representing 14 nominal genera). Monophyly, as well as a basal dichotomy, of the family Enchytraeidae obtained maximum support, with one clade containing Hemienchytraeus and Achaeta, the other the remaining 12 genera analysed. The latter group is basally resolved in several well-supported clades. Lumbricillus and Grania are closely related. Bryodrilus, Oconnorella, Henlea and two species of Marionina (M. cf. riparia, and M. communis) form a well-supported clade. Cognettia is sister to Stercutus, and Cernosvitoviella sister to Mesenchytraeus, and the four together appear to be a monophyletic group. A large part of the taxonomically problematic Marionina appears to be a group not closely related to the type species (M. georgiana), and this group also includes Enchytronia. Further, this Marionina/Enchytronia group appears to be sister to a clade comprising the more or less littoral marine genera Stephensoniella and Enchytraeus. Hemifridericia, Buchholzia and Fridericia, the three genera characterized by two types of coelomocytes, also form a well-supported clade. The study corroborates most of the multi-species genera analysed (Cognettia, Cernosvitoviella, Mesenchytraeus, Oconnorella, Henlea, Enchytraeus, Grania, Buchholzia and Fridericia); only Lumbricillus and Marionina are non-monophyletic as currently defined., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Species delimitation of earthworms has been difficult to determine with certainty due to their structural simplicity. We sequenced fragments of COI, 16S, t-RNAs and 28S for 202 Hormogastridae individuals from the central Iberian Peninsula and three outgroup taxa. A morphological constancy was found but a high genetic diversity suggests the presence of five cryptic allopatric species. Results showed a pattern of isolation by distance and a positive but weak correlation between some soil properties (coarse sand and total loam content) and genetic distances, which indicates that these populations may have been shaped genetically but not morphologically, by the environment., (Copyright 2010 Elsevier Inc. All rights reserved.)

Opistocystidae Cernosvitov, 1936 is a largely Neotropical oligochaete taxon containing seven species. Its familial status has never been formally challenged, although possible close relationships with Naididae and Phreodrilidae have been noted. Mitochondrial 12S and 16S rDNA, and nuclear 18S rDNA, of a range of aquatic oligochaete taxa, including Trieminentia corderoi (Opistocystidae), were analysed by Bayesian inference. This showed that T. corderoi is a derived lineage within Naididae, closely related to Pristina and its monotypic subfamily Pristininae. Opistocystidae as a whole (with its three genera, Opistocysta, Trieminentia, and Crustipellis) is thus likely to be a group within Naididae.

The Aporrectodea caliginosa species complex includes the most abundant earthworms in grasslands and agricultural ecosystems of the Paleartic region. Historically this complex consisted of the following taxa: A. caliginosa s.s.Savigny, 1826, A. trapezoides Dugés (1828), A. tuberculata (Eisen, 1874), and A. nocturna Evans (1946). These four taxa are morphologically very similar and difficult to differentiate because of their morphological variability. Consequently, their taxonomic status and their phylogenetic relationships have been a matter of discussion for more than a century. To study these questions, we sequenced the COII (686 bp), 12S (362 bp), 16S (1200 bp), ND1 (917 bp), and tRNAs(Asn-Asp-Val-Leu-Ala-Ser-Leu) (402 bp) mitochondrial and 28S (809 bp) nuclear gene regions for 85 European earthworms from 27 different localities belonging to the A. caliginosa species complex and four outgroup taxa. DNA sequences were analyzed using maximum parsimony, maximum likelihood, and Bayesian approaches of phylogenetic inference. The resulting trees were combined with morphological, ecological, and genomic evidence to test species boundaries (i.e., integrative approach). Our molecular analyses showed that A. caliginosa s.s. and A. tuberculata form a sister clade to A. trapezoides, A. longa, and A. nocturna, which indicates that A. longa is part of the A. caliginosa species complex. We confirm the species status of all these taxa and identify two hitherto unrecognized Aporrectodea species in Corsica (France). Moreover our analyses also showed the presence of highly divergent lineages within A. caliginosa, A. trapezoides, and A. longa, suggesting the existence of cryptic diversity within these taxa.

The earthworms of the Metaphire formosae species group distributed in Taiwan are members of the Pheretima complex within the Megascolecidae. In this study, the systematics and phylogeography of this species group were investigated using DNA sequences of mitochondrial cytochrome c oxidase subunit I (COI), 16S ribosomal (r)RNA, and NADH dehydrogenase subunit 1 (ND1). The results indicated that the 13 taxa of the M. formosae species group form a clade, including a cryptic species discovered in this study. In addition, Metaphire hengchunensis (James, S.W., Shih, H.-T., Chang, H.-W., 2005. Seven new species of Amynthas (Clitellata: Megascolecidae) and new earthworm records from Taiwan. J. Nat. Hist. 39, 1007-1028) should be regarded as a subspecies of Metaphire paiwanna Tsai, C.-F., Shen, H.-P., Tsai, S.-C., 2000a. Native and exotic species of terrestrial earthworm (Oligochaeta) in Taiwan with reference to Northeast Asia. Zool. Stud. 39, 285-294, Tsai, C.-F., Tsai, S.-C., Liaw, G.-J., 2000b. Two new species of pontandric pheretimoid earthworms belonging to the genus Metaphire (Megascolecidae: Oligochaeta) from Taiwan. J. Nat. Hist. 34, 1731-1741, and Metaphire bununa glareosa Tsai et al. 2000 should be elevated to specific status. Phylogeographical inferences showed that allopatric speciation occurred in this species group during the rapid uplift of the main island of Taiwan between 5.0 and 2.5 million years ago. Our analysis exposes non-monophyly within each of the genera Amynthas and Metaphire, and more generally within the Pheretima complex. Further revisions of this speciose complex are urgently needed.

Phylogenetic relationships within the class Clitellata were examined using nucleotide sequences of nuclear 18S ribosomal RNA gene fragments. In the analysis, the already determined sequences for the individual species representing the class members Hirudinea, Acanthobdellida, Branchiobdellida, and Oligochaeta were included. Furthermore, newly determined sequences of the thirteen representatives of the family Lumbriculidae. including 12 Baikalian endemic species, were analyzed. The hypothesis on the close relatedness of these four groups of Clitellata was supported. Leeches, branchiobdellidans, and lumbriculides form three independent parallel branches of evolution. These results were consistent with the hypothesis on the role of the family Lumbriculidae as a connecting link, or the transition form between the parasitic and free-living groups of Clitellata. At the same time, these data refute the suggestion that Lumbriculidae could be the ancestral lineage of other Oligochaeta. Moreover, polymorphic group of Baikalian lumbriculides clustered independently from the other representatives of the family, pointing to the uniqueness of the Baikalian fauna of oligochaetes, which was formed within relatively closed system of this ancient lake.

A remarkable diversity of life history strategies, geographic distributions, and morphological characters provide a rich substrate for investigating the evolutionary relationships of arhynchobdellid leeches. The phylogenetic relationships, using parsimony analysis, of the order Arhynchobdellida were investigated using nuclear 18S and 28S rDNA, mitochondrial 12S rDNA, and cytochrome c oxidase subunit I sequence data, as well as 24 morphological characters. Thirty-nine arhynchobdellid species were selected to represent the seven currently recognized families. Sixteen rhynchobdellid leeches from the families Glossiphoniidae and Piscicolidae were included as outgroup taxa. Analysis of all available data resolved a single most-parsimonious tree. The cladogram conflicted with most of the traditional classification schemes of the Arhynchobdellida. Monophyly of the Erpobdelliformes and Hirudiniformes was supported, whereas the families Haemadipsidae, Haemopidae, and Hirudinidae, as well as the genera Hirudo or Aliolimnatis, were found not to be monophyletic. The results provide insight on the phylogenetic positions for the taxonomically problematic families Americobdellidae and Cylicobdellidae, the genera Semiscolex, Patagoniobdella, and Mesobdella, as well as genera traditionally classified under Hirudinidae. The evolution of dietary and habitat preferences is examined.

The phylogeny of the Tubificidae, and of most of its subfamilies and some of its genera, is revisited, on the basis of sequences of 18S ribosomal DNA in a selection of species. Forty-six new 18S sequences of Naididae (6), Tubificidae (37), Phreodrilidae (1), Lumbriculidae (1), and Enchytraeidae (1) are reported and aligned together with corresponding sequences of 21 previously studied taxa. The 18S gene of Insulodrilus bifidus provides the first molecular evidence that phreodrilids are closely related to tubificids, corroborating previous conclusions based on morphology. The data further support the monophyletic status of Tubificidae, provided that the "Naididae" is regarded a part of this family; "naidids" may not even constitute a monophyletic group. It is thus suggested that the family name Naididae is formally suppressed as a junior synonym of the Tubificidae. The 18S gene also resolves a number of relationships within the tubificids. Among the subfamilies, Tubificinae is supported, Rhyacodrilinae and Phallodrilinae are revealed as nonmonophyletic, and Limnodriloidinae remains unresolved. Most tubificid genera tested for monophyly are corroborated by the data, only one (Tubifex) is refuted, and two (Tubificoides and Limnodriloides) are unresolved from other taxa. It is concluded that it will be valuable to expand the taxonomic sampling for 18S rDNA in clitellates, and in annelids in general, as this is likely to improve the resolution at many levels. However, it will be equally important to combine the annelid 18S data with other gene sequences and nonmolecular characters, to estimate the phylogeny of these common and diverse worms with greater precision., ((C)2002 Elsevier Science (USA).)

Tubifex tubifex is a cosmopolitan freshwater oligochaete whose presence has been studied as a health indicator of the aquatic environment and as a host for several myxozoan parasites of fish. Unfortunately, current morphological criteria used to distinguish Tubifex spp. (Tubificidae) are inadequate. We therefore developed mitochondrial 16S ribosomal DNA markers to examine phylogenetic relationships among aquatic oligochaetes and to distinguish species of Tubifex that might serve as hosts for a particular myxozoan parasite, Myxobolus cerebralis. Our phylogenetic analyses of oligochaetes based on a 378-bp segment yielded one most parsimonious tree with three major groups that corresponded to the families Lumbricidae, Sparganophilidae, and Tubificidae. T. tubifex and T. ignotus formed a monophyletic assemblage, and a sister relationship between the genera Tubifex and Limnodrilus was strongly supported. A second analysis of the relationship within the genus Tubifex identified six genetically distinct lineages of T. tubifex from North America and Europe that were separated by genetic distances comparable to those found for "well-defined" species of Limnodrilus. Therefore, the existence of several morphologically indistinguishable, thus cryptic, species of Tubifex in North America and Europe is suggested., (Copyright 2001 Academic Press.)

It has been suggested that the bootstrap test of monophyly is too conservative, i.e. the test rejects the hypothesis of monophyly when it is true far too often. Here, a method called the iterated bootstrap is described which estimates by randomization the probabilities associated with rejecting the hypothesis of monophyly when it is true and accepting the hypothesis of monophyly when it is false. Using this method, the bootstrap test can be calibrated by taking account of the errors associated with the hypothesis test. This method is applied to the high-level phylogeny of the Platyhelminthes using 18S rRNA sequences. The analysis suggests that the data cannot unequivocally resolve the placement of the Platyhelminthes with respect to the Annelida and Insecta.