The potential of distance-based thresholds and character-based DNA barcoding for defining problematic taxonomic entities by CO1 and ND1.

The mitochondrial CO1 gene (cytochrome c oxidase I) is a widely accepted metazoan barcode region. In insects, the mitochondrial NADH dehydrogenase subunit 1 (ND1) gene region has proved to be another suitable marker especially for the identification of lower level taxonomic entities such as populations and sister species. To evaluate the potential of distance-based thresholds and character-based DNA barcoding for the identification of problematic species-rich taxa, both markers, CO1 and ND1, were used as test parameters in odonates. We sequenced and compared gene fragments of CO1 and ND1 for 271 odonate individuals representing 51 species, 22 genera and eight families. Our data suggests that (i) the combination of the CO1 and ND1 fragment forms a better identifier than a single region alone; and (ii) the character-based approach provides higher resolution than the distance-based method in Odonata especially in closely related taxonomic entities.

The phylogeography of the Placozoa suggests a taxon-rich phylum in tropical and subtropical waters.

Placozoa has been a key phylum for understanding early metazoan evolution. Yet this phylum is officially monotypic and with respect to its general biology and ecology has remained widely unknown. Worldwide sampling and sequencing of the mitochondrial large ribosomal subunit (16S) reveals a cosmopolitan distribution in tropical and subtropical waters of genetically different clades. We sampled a total of 39 tropical and subtropical locations worldwide and found 23 positive sites for placozoans. The number of genetically characterized sites was thereby increased from 15 to 37. The new sampling identified the first genotypes from two new oceanographic regions, the Eastern Atlantic and the Indian Ocean. We found seven out of 11 previously known haplotypes as well as five new haplotypes. One haplotype resembles a new genetic clade, increasing the number of clades from six to seven. Some of these clades seem to be cosmopolitan whereas others appear to be endemic. The phylogeography also shows that different clades occupy different ecological niches and identifies several euryoecious haplotypes with a cosmopolitic distribution as well as some stenoecious haplotypes with an endemic distribution. Haplotypes of different clades differ substantially in their phylogeographic distribution according to latitude. The genetic data also suggest deep phylogenetic branching patterns between clades.

Character-based DNA barcoding allows discrimination of genera, species and populations in Odonata.

DNA barcoding has become a promising means for identifying organisms of all life stages. Currently, phenetic approaches and tree-building methods have been used to define species boundaries and discover 'cryptic species'. However, a universal threshold of genetic distance values to distinguish taxonomic groups cannot be determined. As an alternative, DNA barcoding approaches can be 'character based', whereby species are identified through the presence or absence of discrete nucleotide substitutions (character states) within a DNA sequence. We demonstrate the potential of character-based DNA barcodes by analysing 833 odonate specimens from 103 localities belonging to 64 species. A total of 54 species and 22 genera could be discriminated reliably through unique combinations of character states within only one mitochondrial gene region (NADH dehydrogenase 1). Character-based DNA barcodes were further successfully established at a population level discriminating seven population-specific entities out of a total of 19 populations belonging to three species. Thus, for the first time, DNA barcodes have been found to identify entities below the species level that may constitute separate conservation units or even species units. Our findings suggest that character-based DNA barcoding can be a rapid and reliable means for (i) the assignment of unknown specimens to a taxonomic group, (ii) the exploration of diagnosability of conservation units, and (iii) complementing taxonomic identification systems.

A low diversity of ANTP class homeobox genes in Placozoa.

Homeobox genes of the ANTP and PRD classes play important roles in body patterning of metazoans, and a large diversity of these genes have been described in bilaterian animals and cnidarians. Trichoplax adhaerens (Phylum Placozoa) is a small multicellular marine animal with one of the simplest body organizations of all metazoans, showing no symmetry and a small number of distinct cell types. Only two ANTP class genes have been described from Trichoplax: the Hox/ParaHox gene Trox-2 and a gene related to the Not family. Here we report an extensive screen for ANTP class genes in Trichoplax, leading to isolation of three additional ANTP class genes. These can be assigned to the Dlx, Mnx and Hmx gene families. Sequencing approximately 12-20 kb around each gene indicates that none are part of tight gene clusters, and in situ hybridization reveals that at least two have spatially restricted expression around the periphery of the animal. The low diversity of ANTP class genes isolated in Trichoplax can be reconciled with the low anatomical complexity of this animal, although the finding that these genes are assignable to recognized gene families is intriguing.

Current problems with the zootype and the early evolution of Hox genes.

"Hox cluster type" genes have sparked intriguing attempts to unite all metazoan animals by a shared pattern of expression and genomic organization of a specific set of regulatory genes. The basic idea, the zootype concept, claims the conservation of a specific set of "Hox cluster type genes" in all metazoan animals, i.e., in the basal diploblasts as well as in the derived triploblastic animals. Depending on the data used and the type of analysis performed, different opposing views have been taken on this idea. We review here the sum of data currently available in a total evidence analysis, which includes morphological and the most recent molecular data. This analysis highlights several problems with the idea of a simple "Hox cluster type" synapomorphy between the diploblastic and triploblastic animals and suggests that the "zootype differentiation" of the Hox cluster most likely is an invention of the triploblasts. The view presented is compatible with the idea that early Hox gene evolution started with a single proto-Hox (possibly a paraHox) gene. J. Exp. Zool. (Mol. Dev. Evol.) 291:169-174, 2001.

Isolation of Hox genes from the scyphozoan Cassiopeia xamachana: implications for the early evolution of Hox genes.

The isolation of Hox genes from two cnidarian groups, the Hydrozoa and Anthozoa, has sparked hypotheses on the early evolution of Hox genes and a conserved role for these genes for defining a main body axis in all metazoan animals. We have isolated the first five Hox genes, Scox-1 to Scox-5, from the third cnidarian class, the Scyphozoa. For all but one gene, we report full-length homeobox plus flanking sequences. Four of the five genes show close relationship to previously reported Cnox-1 genes from Hydrozoa and Anthozoa. One gene, Scox-2, is an unambiguous homologue of Cnox-2 genes known from Hydrozoa, Anthozoa, and also Placozoa. Based on sequence similarity and phylogenetic analyses of the homeobox and homeodomain sequences of known Hox genes from cnidarians, we suggest the presence of at least five distinct Hox gene families in this phylum, and conclude that the last common ancestor of the Recent cnidarian classes likely possessed a set of Hox genes representing three different families, the Cnox-1, Cnox-2, and Cnox-5 families. The data presented are consistent with the idea that multiple duplication events of genes have occurred within one family at the expense of conservation of the original set of genes, which represent the three ancestral Hox gene families.

Molecular resolution of marine turtle stock composition in fishery bycatch: a case study in the Mediterranean.

Based on an extensive sampling regime from both nesting populations and bycatch, frequency analyses of mitochondrial (mt) DNA control region haplotypes in the Mediterranean were used to assess the genetic structure and stock composition of the loggerhead sea turtle, Caretta caretta, in different marine fisheries. The analyses show the following. (i) In drifting longline fisheries working in Mediterranean pelagic habitats 53-55% of turtles caught originated from the Mediterranean stock; (ii) In bottom-trawl fisheries all turtle bycatch is derived from this regional stock; (iii) This regional stock contribution to fishery bycatch suggests that the population size of the Mediterranean loggerhead nesting population is significantly larger than previously thought. This is consistent with a recent holistic estimate based on the discovery of a large rookery in Libya. (iv) Present impact of fishery-related mortality on the Mediterranean nesting population is probably incompatible with its long-term conservation. Sea turtle conservation regulations are urgently needed for the Mediterranean fisheries. (v) The significant divergence of mtDNA haplotype frequencies of the Turkish loggerhead colonies define this nesting population as a particularly important management unit. Large immature and adult stages from this management unit seem to be harvested predominantly by Egyptian fisheries. (vi) Combined with other data, our findings suggest that all the nesting populations in the Mediterranean should be considered as management units sharing immature pelagic habitats throughout the Mediterranean (and possibly the eastern Atlantic), with distinct and more localized benthic feeding habitats in the eastern basin used by large immatures and adults. (vii) Between the strict oceanic pelagic and the benthic stages, immature turtles appear to live through an intermediate neritic stage, in which they switch between pelagic and benthic foods.

Homology of Hox genes and the zootype concept in early metazoan evolution.

The correct identification of homologous Hox genes within and between diplo- and triploblastic animals is of crucial importance for recent hypotheses on the anagenetic evolution of animal bauplans. While the homology discussion in general has reached new heights, we apply traditional homology criteria to assign homology to Hox genes from diploblastic animals. Comparison of the Trox-2 gene from the presumably most basal metazoan animal, the placozoan Trichoplax adhaerens, to other Hox genes suggests the presence of unambiguous homologs in Hydrozoa and Scyphozoa and the absence of any specific homolog in triploblasts. Furthermore, the comparisons provide support for the idea that Hox genes-at least in diploblastic animals-are composed of functional subunits (modules), which to some degree have undergone independent evolution. The findings are not readily compatible with the existence of the "zootype" in diploblastic animals.

Over one-half billion years of head conservation? Expression of an ems class gene in Hydractinia symbiolongicarpus (Cnidaria: Hydrozoa).

We report the isolation of an empty spiracles class homeodomain-containing gene, Cn-ems, from the hydrozoan Hydractinia symbiolongicarpus, the first gene of this class characterized in a lower metazoan. Cn-ems was found to be expressed in the head of gastrozooids, specifically in endodermal epithelial cells of the taeniolae of the hypostome. Cn-ems is not expressed in gonozooids, which lack taeniolae. Experimental conversion of the posterior region of the planula larva into head structures up-regulates expression of the gene. These findings establish that the association of ems-class genes with head structures preceded the evolution of bilateral symmetry.

Homeobox genes in the cnidarian Eleutheria dichotoma: evolutionary implications for the origin of Antennapedia-class (HOM/Hox) genes.

In order to test current hypotheses on the early evolution of Antp-class (HOM/Hox) genes in metazoan animals, we surveyed the diploblastic hydrozoan Eleutheria dichotoma for the presence of these genes. By means of PCR with different sets of degenerate primers, three new homeobox sequences were identified, in addition to two previously found fragments. Following RACE amplification of the 3' and 5' flanking regions, we classified all five genes, Cnox-1 to Cnox-5, as belonging to the Antp-class. Homeodomain identity to the most similar HOM/Hox genes from Drosophila and human ranges from 58 to 72%. Each of the Cnox genes contains diagnostic residues of more than one Antp-class family known from triploblastic animals. These data are at odds with a previously proposed ancestral HOM/Hox cluster composition, with respect to both the number and types of Antp-class homeobox genes found.

RAPD identification of microsatellites in Daphnia.

Simple sequence repeats (SSRs, or microsatellites) have been constantly gaining importance as single-locus DNA markers in population genetics and behavioural ecology. We tested a PCR-based strategy for finding microsatellite loci in anonymous genomes, which avoids genomic library construction and screening, and the need for larger amounts of DNA. In the first step, parts of a genome are randomly amplified with arbitrary 10mer primers using RAPD fingerprinting. Labelled SSR-oligonucleotides serve as probes to detect complementary sequences in RAPD products by means of Southern analyses. Subsequently, positive RAPD fragments of suitable size are cloned and sequenced. Using GA and GT probes, we applied this approach to waterfleas (Daphnia) and revealed 37 hybridization signals in 20 RAPD profiles. Thirteen positive RAPD fragments from three Daphnia species and two hybrid 'species' were cloned and sequenced. In all cases simple sequence repeats were detected. We characterized seven perfect repeat loci, which were found to be polymorphic within and between species.

The effects of nested primer binding sites on the reproducibility of PCR: mathematical modeling and computer simulation studies.

The polymerase chain reaction (PCR) has become an indispensable tool in modern biological research. Although the application of PCR is a standard routine, we widely lack a theoretical understanding of the dynamic processes involved, especially with respect to the amplification of nonreproducible and/or unexpected amplification products. For one potential source of uncertainty, the presence of nested primer binding sites within an amplifyable DNA locus, we consider a simple stochastic model for the dynamics of PCR amplification of competing products. For commonly used thermostable DNA polymerases lacking a 5'-3'-exonuclease activity, we predict the relative amplification frequencies of competing PCR products dependent on the primer binding probability, the number of PCR cycles, and the number of initial DNA template molecules. At low primer binding probabilities and low numbers of initial DNA template molecules and PCR cycles, we expect the amplification of two products. At high primer binding probabilities and/or high copy numbers of initial template molecules only one main amplification product is predicted at increasing cycle numbers. Furthermore, by means of computer simulation studies we quantify the stochastic variation for the amplification frequencies of competing products.

Arbitrarily amplified DNA in systematics and phylogenetics.

Different polymerase chain reaction (PCR) based techniques which use single primers of arbitrary sequence have become prominent and powerful tools for taxonomic and genealogical studies. While the use of arbitrarily amplified DNA (AAD) markers for the identification of lower taxonomic units (e.g. at the species level) has been proven to be more or less straightforward, their potential usefulness in phylogenetic studies has not been sufficiently addressed yet. The main advantages of AAD markers include that they require only very small amounts of DNA, no sequence information of the taxa under investigation and the relative ease of the generating high numbers of AAD markers in short time. On the other hand, the generation of AAD markers is susceptible to certain random and systematic errors, and for phylogenetic studies the taxonomic level at which informative markers can be generated is severely limited.

Developmental genes as a potential tool in population ecology of complex animal life cycles?

Many aquatic invertebrates possess complex life cycles, which include a switch from a vegetative to a sexual mode of reproduction. While sexual reproduction is usually linked to slow rates of propagation, vegetative reproduction leads to high rates of clonal propagation and a fast increase in population size. I have attempted to identify developmental genes which are differentially expressed between sexually and vegetatively reproducing individuals. quantitative assays of the expression of those diagnostic genes could be applied to field samples to gain information on the reproductive status of the sample. Here I outline a general strategy for identifying and testing the usefulness of homeobox genes as candidates for regulatory genes whose expression patterns are indicative of a sexual or vegetative developmental phase.

Natural hybridization in freshwater animals. Ecological implications and molecular approaches.

The number of cases where the phenomena of hybridization and gene introgression have been found in species interactions is steadily increasing, in both plant and animal taxa. During the last few years, many examples have been detected even in otherwise well-known freshwater animal taxa. We discuss the topic with respect to ecology and evolutionary processes and compare the main potentials and limitations of allozymes, mitochondrial DNA, and RAPD markers to address some important genetic issues of interspecific hybridization in natural populations of selected freshwater model systems.

The evolutionary ecology of Daphnia.

In order to generate genetic markers from both nuclear and mitochondrial DNA, we used three PCR-based techniques (RAPD, mtDNA-RFLPs, and sequencing of an amplified mtDNA fragment) to illuminate various aspects of the population genetics of large-lake Daphnia species. Estimations of genetic diversity at different taxonomic levels integrated with ecological data revealed insights into the genetic components of the evolutionary process of interspecific hybridization in these Daphnia species, which had previously been documented with allozyme markers. Our new molecular data suggest the occurrence of recent hybridization and backcrossing events, and allow the identification of the maternal species of hybrid clones.

Determination of paternity in dragonflies by Random Amplified Polymorphic DNA fingerprinting.

We used Random Amplified Polymorphic DNA (RAPD) fingerprinting to address issues of paternity in two odonate species. Amplification artifacts of RAPD markers were controlled by assessing paternity patterns relative to the banding patterns generated by quantitative mixtures of DNA from putative parents ('synthetic offspring'). In the aeshnid dragonfly Anax parthenope, for which the mating histories of both males and females were unknown, we found strong evidence for complete paternity success for the contact guarding male. In the highly polygamous libellulid dragonfly Orthetrum coerulescens, we detected and quantified mixed paternity in sequentially produced offspring clutches and demonstrated that fertilization success is correlated with the duration of copulation. Our results suggest that RAPD fingerprinting is suitable to address issues of paternity in systems which are genetically uncharacterized and produce large offspring clutches.

Class-level relationships in the phylum Cnidaria: evidence from mitochondrial genome structure.

The phylogenetic relationships of the Recent cnidarian classes remain one of the classic problems in invertebrate zoology. We survey the structure of the mitochondrial genome in representatives of the four extant cnidarian classes and in the phylum Ctenophora. We find that all anthozoan species tested possess mtDNA in the form of circular molecules, whereas all scyphozoan, cubozoan, and hydrozoan species tested display mtDNA in the form of linear molecules. Because ctenophore and all other known metazoan mtDNA is circular, the shared occurrence of linear mtDNA in three of the four cnidarian classes suggests a basal position for the Anthozoa within the phylum.

Applications of random amplified polymorphic DNA (RAPD) in molecular ecology.

Molecular genetic markers have been developed into powerful tools to analyse genetic relationships and genetic diversity. As an extension to the variety of existing techniques using polymorphic DNA markers, the Random Amplified Polymorphic DNA (RAPD) technique may be used in molecular ecology to determine taxonomic identity, assess kinship relationships, analyse mixed genome samples, and create specific probes. Main advantages of the RAPD technology include (i) suitability for work on anonymous genomes, (ii) applicability to problems where only limited quantities of DNA are available, (iii) efficiency and low expense.