Plant pathogenic fungi are a large and diverse assemblage of eukaryotes with substantial impacts on natural ecosystems and human endeavours. These taxa often have complex and poorly understood life cycles, lack observable, discriminatory morphological characters, and may not be amenable to in vitro culturing. As a result, species identification is frequently difficult. Molecular (DNA sequence) data have emerged as crucial information for the taxonomic identification of plant pathogenic fungi, with the nuclear ribosomal internal transcribed spacer (ITS) region being the most popular marker. However, international nucleotide sequence databases are accumulating numerous sequences of compromised or low-resolution taxonomic annotations and substandard technical quality, making their use in the molecular identification of plant pathogenic fungi problematic. Here we report on a concerted effort to identify high-quality reference sequences for various plant pathogenic fungi and to re-annotate incorrectly or insufficiently annotated public ITS sequences from these fungal lineages. A third objective was to enrich the sequences with geographical and ecological metadata. The results – a total of 31,954 changes – are incorporated in and made available through the UNITE database for molecular identification of fungi (http://unite.ut.ee), including standalone FASTA files of sequence data for local BLAST searches, use in the next-generation sequencing analysis platforms QIIME and mothur, and related applications. The present initiative is just a beginning to cover the wide spectrum of plant pathogenic fungi, and we invite all researchers with pertinent expertise to join the annotation effort.

The classification and phylogenetic position of Haplotaxidae, an enigmatic family of clitellate worms, have long been discussed. We estimate the phylogeny for Clitellata as a whole, using a taxon sample adhering to current definitions of Haplotaxidae and including species attributable to Haplotaxis, plus a representation of other clitellates. Sequences of five markers, 4087 bp in total, were analysed by Bayesian Inference. A well-supported tree shows that Haplotaxidae is polyphyletic, consisting of at least five separate clades scattered among other clitellate lineages. We redefine Haplotaxidae s.s. to only include Haplotaxis, and erect four new families: Pelodrilidae Martin et al.fam. nov. for Pelodrilus, Hologynus, and Delaya; Haplotaxoididae Martin et al.fam. nov. for Haplotaxoides Fend gen. nov., with Haplotaxoides decipiens Fend sp. nov. and Haplotaxoides tehama Fend sp. nov.; Limpluvidae Martin et al.fam. nov. for Limpluvia setoensis Fend & Torii gen. et sp. nov.; and Ohtakianidae Martin et al.fam. nov. for Ohtakiana kakidaensis Fend & Torii gen. et sp. nov. To adhere to standard classification systems, we assign all five families to separate orders, Pelodrilida ord. nov., Haplotaxoidida ord. nov., Othakianida ord. nov., Limpluvida ord. nov., and Haplotaxida. Haplotaxidae s.s. and Pelodrilidae seem to belong to an incompletely resolved group also including lumbriculids, leeches, and earthworms. Moreover, a close relationship between Limpluvia, Ohtakiana, and the lumbriculid-hirudinean clade (i.e. taxon Lumbriculata) is strongly supported, whereas Haplotaxoididae are placed more basally in the clitellate phylogeny than the other haplotaxids s.l., their exact position remaining unresolved. The analysed Holarctic Haplotaxis populations (representing up to 10 species) appear separated more by geography than by obvious morphological characters.

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.

Limnodrilus hoffmeisteri Claparède, 1862 is a common freshwater worm, often regarded as an indicator of organic pollution. The taxonomic status of this species is controversial due to great variation in morphological features. Numerous morphological forms of L. hoffmeisteri are recorded in the literature, especially from Europe and North America. Today, DNA-based species delimitation assumes that species boundaries can be more objectively and effectively estimated using genetic data rather than with morphological data alone. To investigate if L. hoffmeisteri is a single species, 295 worms identified as either L. hoffmeisteri or other similar (congeneric) morphospecies, using currently accepted morphological criteria, were collected from 82 locations in the northern hemisphere. The number of primary species hypotheses (PSHs) was first explored with cytochrome oxidase subunit I (COI), the proposed DNA barcode for animal species, and with data for all specimens. Both automatic barcoding gap discovery (ABGD) and the Bayesian general mixed Yule coalescent (bGMYC) model revealed the existence of ≥25 distinct PSHs (COI lineages) in our dataset. Then, smaller samples of individuals representing major COI lineages were used for exploration of a nuclear locus, the internal transcribed spacer (ITS) region. In the ITS gene tree (81 sequences), generated by BEAST, 16 well-supported terminal groups were found, but not all of these groups were congruent with the PSHs found in the COI tree. As results across these different analyses were inconsistent, we resorted to analyzing reciprocal monophyly between gene trees and used a minimum consensus of all evidence, suggesting that there are 13 separately evolving lineages (=13 species) within our sample. The smallest uncorrected COI p-distance between these species is 12.1%, and the largest intraspecific p-distance is 16.4%, illustrating the problem of species delimitation with a DNA-barcoding gap as the sole criterion. Ten of these species are morphologically identified as “L. hoffmeisteri,” the remaining three can be attributed to morphologically distinct congeneric species. An individual from the type locality in Switzerland was designated as a neotype of L. hoffmeisteri sensu stricto. This worm belongs to one of the ten species, and this lineage is widely distributed in Europe, Asia, and North America. The remaining nine species show a mixed distribution pattern; some appear to be endemic to a restricted area, others are Holarctic. Our results provide clues to the future revalidation of some of the nominal species today placed in synonymy with L. hoffmeisteri. A BEAST analysis, based on previously published and newly generated 16S data, suggested that this complex contains also other species than those studied by us. By integrating additional genetic data, it will be possible to identify these and additional specimens in future studies of Limnodrilus, and the neotype provides a baseline for further revisions of the taxonomy of the L. hoffmeisteri complex.