Mark E. Siddall

The unholy trinity: taxonomy, species delimitation and DNA barcoding

Recent excitement over the development of an initiative to generate DNA sequences for all named species on the planet has in our opinion generated two major areas of contention as to how this ‘DNA barcoding’ initiative should proceed. It is critical that these two issues are clarified and resolved, before the use of DNA as a tool for taxonomy and species delimitation can be universalized. The first issue concerns how DNA data are to be used in the context of this initiative; this is the DNA barcode reader problem (or barcoder problem). Currently, many of the published studies under this initiative have used tree building methods and more precisely distance approaches to the construction of the trees that are used to place certain DNA sequences into a taxonomic context. The second problem involves the reaction of the taxonomic community to the directives of the ‘DNA barcoding’ initiative. This issue is extremely important in that the classical taxonomic approach and the DNA approach will need to be reconciled in order for the ‘DNA barcoding’ initiative to proceed with any kind of community acceptance. In fact, we feel that DNA barcoding is a misnomer. Our preference is for the title of the London meetings—Barcoding Life. In this paper we discuss these two concerns generated around the DNA barcoding initiative and attempt to present a phylogenetic systematic framework for an improved barcoder as well as a taxonomic framework for interweaving classical taxonomy with the goals of ‘DNA barcoding’.

Recent advances in our knowledge of the Myxozoa

Abstract In the last few years two factors have helped to significantly advance our understanding of the Myxozoa. First, the phenomenal increase in fin fish aquaculture in the 1990s has lead to the increased importance of these parasites; in turn this has lead to intensified research efforts, which have increased knowledge of the development, diagnosis, and pathogenesis of myxozoans. The hallmark discovery in the 1980s that the life cycle of Myxobolus cerebralis requires development of an actinosporean stage in the oligochaete, Tubifex tubifex, led to the elucidation of the life cycles of several other myxozoans. Also, the life cycle and taxonomy of the enigmatic PKX myxozoan has been resolved: it is the alternate stage of the unusual myxozoan, Tetracapsula bryosalmonae, from bryozoans. The 18S rDNA gene of many species has been sequenced, and here we add 22 new sequences to the data set. Phylogenetic analyses using all these sequences indicate that:1) the Myxozoa are closely related to Cnidaria (also supported by morphological data); 2) marine taxa at the genus level branch separately from genera that usually infect freshwater fishes; 3) taxa cluster more by development and tissue location than by spore morphology; 4) the tetracapsulids branched off early in myxozoan evolution, perhaps reflected by their having bryozoan, rather than annelid hosts; 5) the morphology of actinosporeans offers little information for determining their myxosporean counterparts (assuming that they exist); and 6) the marine actinosporeans from Australia appear to form a clade within the platysporinid myxosporeans. Ribosomal DNA sequences have also enabled development of diagnostic tests for myxozoans. PCR and in situ hybridisation tests based on rDNA sequences have been developed for Myxobolus cerebralis, Ceratomyxa shasta, Kudoa spp., and Tetracapsula bryosalmonae (PKX). Lectin-based and antibody tests have also been developed for certain myxozoans, such as PKX and C. shasta. We also review important diseases caused by myxozoans, which are emerging or re-emerging. Epizootics of whirling disease in wild rainbow trout (Oncorhynchus mykiss) have recently been reported throughout the Rocky Mountain states of the USA. With a dramatic increase in aquaculture of fishes using marine netpens, several marine myxozoans have been recognized or elevated in status as pathological agents. Kudoa thyrsites infections have caused severe post-harvest myoliquefaction in pen-reared Atlantic salmon (Salmo salar), and Ceratomyxa spp., Sphaerospora spp., and Myxidium leei cause disease in pen-reared sea bass (Dicentrarchus labrax) and sea bream species (family Sparidae) in Mediterranean countries.

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.

Success of Parsimony in the Four-Taxon Case: Long-Branch Repulsion by Likelihood in the Farris Zone

The accuracy of phylogenetic methods is reinvestigated for the four‐taxon case with a two‐edge rate and a three‐edge rate. Unlike previous studies involving computer simulations, the two‐edge rate relates to branches that are sister taxa in the model tree. As with previous studies, certain methods are found to behave inaccurately in a portion of the parameter space where the two‐edge rate is proportionally large. This phenomenon, to which parsimony is immune, is termed “long‐branch repulsion” and the region of poor performance is called the Farris Zone. Maximum likelihood methods are shown to be particularly prone to failure when closely related taxa have long branches. Long‐branch repulsion is demonstrated with an empirical case involving Strepsiptera and Diptera.

Diverse molecular data demonstrate that commercially available medicinal leeches are not Hirudo medicinalis

The European medicinal leech is one of vanishingly few animal species with direct application in modern medicine. In addition to the therapeutic potential held by many protease inhibitors purified from leech saliva, and notwithstanding the historical association with quackery, Hirudo medicinalis has been approved by the United States Food and Drug Administration as a prescription medical device. Accurate annotation of bioactive compounds relies on precise species determination. Interpretations of developmental and neurophysiological characteristics also presuppose uniformity within a model species used in laboratory settings. Here, we show, with mitochondrial sequences and nuclear microsatellites, that there are at least three species of European medicinal leech, and that leeches marketed as H. medicinalis are actually Hirudo verbana. Beyond the obvious need for reconsideration of decades of biomedical research on this widely used model organism, these findings impact regulatory statutes and raise concerns for the conservation status of European medicinal leeches.

Biases in Maximum Likelihood and Parsimony: A Simulation Approach to a 10-Taxon Case

Biases present in maximum likelihood and parsimony are investigated through a simulation study in a 10‐taxon case in which several long branches coexist with short branches in the modeled topology. The performance of these methods is explored while increasing the length of the long branches with different amounts of data. Also, simulations with different taxonomic sampling schemes are examined through this study. The presence of a strong bias in parsimony is corroborated: the well‐known long‐branch attraction. Likelihood performance is found to be sensitive to the mere presence extreme of branch length disparity, retrieving topologies compatible with long‐branch attraction and long‐branch repulsion, irrespective of the correctness of the model used.

Phylogenetic analysis of Perkinsus based on actin gene sequences.

Perkinsus species presently are classified within the phylum Apicomplexa. This placement, however, is controversial. Based upon morphological observations and phylogenetic analyses of the small subunit ribosomal RNA gene, it has been suggested that Perkinsus may be more closely related to dinoflagellates. To reevaluate the phylogenetic position of Perkinsus, we obtained nucleotide sequence data for actin genes from Perkinsus marinus and 2 dinoflagellates, Prorocentrum minimum and Amphidinium carterae. Results indicated that there are 2 closely related actin genes in the genome of P. marinus. Phylogenetic comparisons of these actin gene fragments of P. marinus to available actin gene sequences for several ciliates and apicomplexans and to the 2 actin gene sequences from dinoflagellates obtained in this study supported a closer affinity of P. marinus to dinoflagellates than to apicomplexans.

PHYLOGENY OF THE LEECH FAMILY GLOSSIPHONIIDAE BASED ON MITOCHONDRIAL GENE SEQUENCES AND MORPHOLOGICAL DATA

The phylogenetic relationships of the Glossiphoniidae (Rhynchobdellida) were investigated using morphological characters and the mitochondrial genes cytochrome c oxidase subunit I and nicotinamide adenine dinucleotide dehydrogenase subunit 1. Thirty-five taxa representing 10 of the 23 currently recognized glossiphoniid genera were sampled, including more than 70% of known North American species, as well as others from Europe, South America, Africa, and a species endemic to Lake Baikal. Outgroup taxa included species from the Piscicolidae and Ozobranchidae. Cladistic analysis resulted in 1 most-parsimonious tree. Subfamily distinctions, i.e., Haementeriinae, Theromyzinae, and Glossiphoniinae, that have been based on eye morphology and reproductive biology are not corroborated. Results also provide insights into several problematic genus-level classifications. For example, relationships of Placobdella and Haementeria are clarified and elimination of Desserobdella may be necessary. Bloodfeeding from vertebrates is seen to be a primitive characteristic that has been lost twice within the clade. The hypothesis that the biannulate leech, Oligobdella biannulata, represents an important transitional form is re-evaluated in a phylogenetic context.

Molecular phylogeny of the Haplosporidia based on two independent gene sequences.

The phylogenetic position of the Haplosporidia has confounded taxonomists for more than a century because of the unique morphology of these parasites. We collected DNA sequence data for small subunit (SSU) ribosomal RNA and actin genes from haplosporidians and other protists for conducting molecular phylogenetic analyses to help elucidate relationships of taxa within the group, as well as placement of this group among Eukaryota. Analyses were conducted using DNA sequence data from more than 100 eukaryotic taxa with various combinations of data sets including nucleotide sequence data for each gene separately and combined, as well as SSU ribosomal DNA data combined with translated actin amino acids. In almost all analyses, the Haplosporidia was sister to the Cercozoa with moderate bootstrap and jackknife support. Analysis with actin amino acid sequences alone grouped haplosporidians with the foraminiferans and cercozoans. The haplosporidians Minchinia and Urosporidium were found to be monophyletic, whereas Haplosporidium was paraphyletic. “Microcell” parasites, Bonamia spp. and Mikrocytos roughleyi, were sister to Minchinia, the most derived genus, with Haplosporidium falling between the “microcells” and the more basal Urosporidium. Two recently discovered parasites, one from abalone in New Zealand and another from spot prawns in British Columbia, fell at the base of the Haplosporidia with very strong support, indicating a taxonomic affinity to this group.

Poly-paraphyly of Hirudinidae: many lineages of medicinal leeches

BackgroundMedicinal leeches became infamous for their utility in bloodletting popularized in the 19th century, and have seen a recent resurgence in post-operative treatments for flap and replantation surgeries, and in terms of characterization of salivary anticoagulants. Notorious throughout the world, the quintessential leech family Hirudinidae has been taken for granted to be monophyletic, as has the non-bloodfeeding family Haemopidae.ResultsThis study is the first to evaluate molecular evidence from hirudinid and haemopid leeches in a manner that encompasses the global scope of their taxonomic distributions. We evaluated the presumed monophyly of the Hirudinidae and assessed previous well-accepted classification schemes. The Hirudinidae were found not to be monophyletic, falling instead into two distinct and unrelated clades. Members of the non-bloodfeeding family Haemopidae were scattered throughout the tree and among traditional hirudinid genera. A combination of nuclear 18S rDNA and 28S rDNA with mitochondrial 12S rDNA and cytochrome c oxidase I were analyzed with Parsimony and with Bayesian methods.ConclusionThe family Hirudinidae must be refined to include only the clade containing Hirudo medicinalis (European medicinal leech) and related leeches irrespective of bloodfeeding behavior. A second clade containing Macrobdella decora (North American medicinal leech) and its relatives may yet be recognized in Semiscolecidae in order to avoid paraphyly. The African distribution of species from each of the divergent hirudinid clades suggests that a deep divergence took place in the history of the medicinal leeches hundreds of millions of years ago.