Svante Martinsson

Improving ITS sequence data for identification of plant pathogenic fungi

SummaryPlant 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.

Integrative taxonomy of the freshwater worm Rhyacodrilus falciformis s.l. (Clitellata: Naididae), with the description of a new species

The genetic and morphological variation within Rhyacodrilus falciformis Bretscher, 1901 (Clitellata: Naididae) in Europe was explored using an integrative approach, with three unlinked genetic markers [the mitochondrial cytochrome c oxidase subunit I (COI), the nuclear histone 3 (H3) and internal transcribed spacer region (ITS)] combined with morphology, to investigate whether this taxon constitutes a single or several species. Using Automatic Barcode Gap Discovery on the COI data set, the specimens were divided into seven clusters, used as hypothetical species that were further tested with the other data sources. Single‐gene trees were estimated for all three markers, using coalescence analysis and they were in many parts incongruent with each other. Only one of the clusters was supported by all trees; it was also morphologically differentiated from the other clusters by the shape of its modified penial chaetae. This group consists of two specimens from the Crotot Cave in south‐eastern France, and morphologically they fit a previously described but invalid variety, ‘pigueti’, which is here described as a new species, Rhyacodrilus pigueti Achurra & Martinsson sp. n. The study highlights the fact that a single data source (e.g. COI barcodes) seldom provides a sufficient basis for taxonomic decisions such as species delimitation.

Revision of Cognettia (Clitellata, Enchytraeidae): re-establishment of Chamaedrilus and description of cryptic species in the sphagnetorum complex

The oligochaete worm, Cognettia sphagnetorum (Vejdovský, 1878), is widely used as a model organism in soil biology, and therefore it is important that its taxonomy is firmly established. A previous study based on both mitochondrial and nuclear genetic markers showed that this taxon is an assemblage of at least four species that do not form a monophyletic group. Also the validity of the genus Cognettia Nielsen and Christensen, 1959 is subject to debate, since the existence of two putative senior synonyms, Euenchytraeus Bretscher, 1906 and Chamaedrilus Friend, 1913 has been pointed out. Herein we revise the generic assignment of the species currently placed in Cognettia: two species, C. clarae Bauer, 1993 and C. piperi Christensen and Dózsa-Farkas, 1999, are transferred to Euenchytraeus, together with its type Eu. bisetosus Bretscher, 1906, whereas the remaining species, including Cognettia sphagnetorum, are being transferred to Chamaedrilus. Five species within the Chamaedrilus sphagnetorum complex are revised: the type species of Chamaedrilus, Ch. chlorophilus Friend, 1913, as well as the type species of Cognettia, Ch. sphagnetorum s. str. are redescribed, and a neotype is designated for the latter; and the cryptic species Ch. pseudosphagnetorum sp. nov. and Ch. chalupskyi sp. nov. are described as new to science and discussed against a paratype of Ch. valeriae (Dumnicka, 2010) comb. nov. DNA-barcodes are provided for all the named species in the complex except Ch. valeriae. A key to the species in the complex is given and the value of different somatic characters for separating and identifying species of Chamaedrilus is discussed. No morphological feature seems to distinguish Ch. sphagnetorum from Ch. pseudosphagnetorum. Thus, for a reliable identification of these species, molecular methods, e.g. DNA barcoding, are recommended. http://zoobank.org/urn:lsid:zoobank.org:pub:F840CD92-F784-429E-B4BF-3E61F6632A8D

Barcoding gap, but no support for cryptic speciation in the earthworm Aporrectodea longa (Clitellata: Lumbricidae)

Abstract 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 PAB 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.

Towards a molecular phylogeny of the fungus gnat genus Boletina (Diptera: Mycetophilidae)

Martinsson, S., Kjærandsen, J. & Sundberg, P. (2011). Towards a molecular phylogeny of the fungus gnat genus Boletina (Diptera: Mycetophilidae). —Zoologica Scripta, 40, 272–281.

Extensive cryptic diversity in the cosmopolitan sludge worm Limnodrilus hoffmeisteri (Clitellata, Naididae)

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.

Phylogeny and species delimitation of North European Lumbricillus (Clitellata, Enchytraeidae)

The enchytraeid genus Lumbricillus comprises about 80 described species of clitellate worms, which are up to a few centimetres long, and they mostly inhabit the littoral zone of non‐tropical marine and brackish waters world‐wide. The phylogeny of this genus is poorly studied, but previous work has suggested that Lumbricillus is a non‐monophyletic group. In this study, species boundaries and the phylogeny of this genus is re‐assessed using more than 300 DNA‐barcoded specimens (using COI mtDNA), part of which was also sequenced for two additional mitochondrial and four nuclear molecular markers. Statistical and coalescent based applications were used for the delimitation of a total of 24 species, of which 20 were identified as belonging to 17 described morphospecies; one morphospecies was found to be a complex of four delimited species, and another four delimited species could not be matched with any described species. Furthermore, gene trees, concatenation and multispecies coalescent based species trees were estimated using Bayesian inference. The estimated phylogenies confirm a non‐monophyletic Lumbricillus as L. semifuscus is clearly excluded from the genus. Furthermore, the placement of a monophyletic clade consisting of L. arenarius, L. dubius, and an unidentified species varies between analyses; they are either found as the sister‐group to the genus Grania or as sister‐group to the remaining Lumbricillus, where the latter relationship is supported by the multispecies coalescent, which we consider as the most reliable method.

On the identity of Chamaedrilus glandulosus (Michaelsen, 1888) (Clitellata, Enchytraeidae), with the description of a new species

Abstract The taxonomy of Chamaedrilus glandulosus (Michaelsen, 1888) s. l., most commonly known previously as Cognettia glandulosa, is revised. A recent molecular systematic study has shown that this taxon harbours two cryptic, but genetically well separated lineages, each warranting species status. In this study these two lineages are scrutinized morphologically, on the basis of Michaelsen’s type material as well as newly collected specimens from Central and Northern Europe. Chamaedrilus glandulosus s. s. is redescribed and Chamaedrilus varisetosus sp. n. is recognized as new to science. The two species are morphologically very similar, differing mainly in size, but seem to prefer different habitats, with Chamaedrilus glandulosus being a larger aquatic species, and Chamaedrilus varisetosus being smaller and mainly found in moist to wet soil.

DNA-barcoding of invasive European earthworms (Clitellata: Lumbricidae) in south-western Australia

Using DNA-barcoding, we studied the diversity of invasive European earthworms in the south-western corner of Australia. We found six Molecular Operational Taxonomic Units belonging to five morphospecies: Aporrectodea caliginosa, A. trapezoides, Dendrobaena cf. attemsi, Eiseniella tetraedra and Octolasion cyaneum. These were variously collected from indigenous forests and/or alienated land. Two cryptic lineages were found within A. trapezoides, and high intraspecific genetic variation was also found within E. tetraedra—variation that had previously been documented in Europe. Our study demonstrates the usefulness of DNA-barcoding for the identification of earthworms, including cryptic species. Correct identification and high taxonomic resolution is crucial for the monitoring of cryptic diversity, detecting new introductions and monitoring spread of established exotic earthworms.

Placing the forgotten: on the positions of Euenchytraeus and Chamaedrilus in an updated enchytraeid phylogeny (Clitellata : Enchytraeidae)

Abstract. The phylogeny of Enchytraeidae was re-estimated to establish the relationships of the now resurrected Chamaedrilus and Euenchytraeus and to confirm their status as separate taxa. The former Cognettia (Enchytraeidae) was recently revised and split into its two senior synonyms, Chamaedrilus and Euenchytraeus, with the majority of the species transferred to Chamaedrilus. Euenchytraeus was re-established for three species sharing a unique anatomical trait, but has never before been represented in any phylogenetic study. We included representatives from 21 (of 33) valid enchytraeid genera and used three mitochondrial and four nuclear genes. The dataset (4164 base pairs) was analysed using multi-species coalescent (MSC) and maximum likelihood (ML) methods. Chamaedrilus (represented by eight species) and Euenchytraeus (represented by Eu. clarae) were found in a clade together with the monotypic Stercutus. Chamaedrilus was found to be monophyletic with maximum support in both analyses. The ML tree supported Euenchytraeus and Chamaedrilus as sister groups, whereas the MSC tree placed Euenchytraeus together with Stercutus, both with low support. A Bayes factor test weakly supported Euenchytraeus and Chamaedrilus as sister groups over Euenchytraeus + Stercutus. Possible morphological synapomorphies for these genera are discussed, and we conclude that Chamaedrilus and Euenchytraeus are closely related, but their status as separate genera is justified.