Oribatid mites are a very diverse taxon (Hansen 2000) of minute animals that live in the soil matrix or litter. Most of them have long life cycles and low reproductive rates, which makes them unsuitable for lab-cultures. Direct observation of oribatid mites therefore is almost impossible except for a few fast reproducing species that can be cultured easily.
Taxonomic and ecological studies on soil organisms are always invasive since individuals need to be extracted from their natural habitat and determination to species level in oribatid mites are time-consuming due to their high local diversity in most habitats. Although oribatid mites are ubiquitous and abundant in soil habitats all over the world and easily extracted, working with mites is not trivial and, beside morphology and distribution patterns, we do not know much about them.
Molecularbiology, however, provides an excellent toolkit to study oribatid mites. The heredity information in biological macromolecules enables time-travels to an organisms evolutionary history. All genetic information descends from a common ancestor but over evolutionary time replication errors generated variation that again have changed in frequency due to selection or chance events in populations. Different types of molecules provide different genetic information and are suited to investigate past temporal horizons (Avise 2004, Fig. 1.2). Therefore, studies with molecular markers provide tools to resolve (i) evolutionary divergence ranging from recent to distant (phylogeny), (ii) genetic identity from non-identity (sexual from asexual origin) and (iii) genetic parentage (population genetics), comprising questions of relatedness and population structure.
1.4.1 ribosomal RNA
The eukaryotic 18S rRNA locus in particular has become standard in molecular analyses of metazoan relationships (Giribet 2002). Ribosomal RNA comprises one part of the ribosome complex and is therefore essentially involved in bio-protein-synthesis. Its nucleotide sequence does not code for a certain protein but its three-dimensional structure, forming single-stranded loops and double-single-stranded stems, is functional. Due to its importance in
fundamental cell processes its evolution is under strong selective pressure, the accumulation of mutations and substitutions of nucleotides are rare and evolution therefore is slow, i.e.
about 1% sequence divergence per 50 million years (Fig. 1.3). According to the neutral theory of molecular evolution (Kimura 1968), variability in genes is mainly due to neutral substitutions that are unaffected by selection. Neutral substitutions are stochastic events that occur constantly over long periods of time. The genetic distance of a particular gene therefore depends on its mutation rate and correlates with time since separation of two species (molecular clock theory, Zuckerkandl and Pauling 1965). If genetic distances in a phylogenetic tree can be correlated with palaeontological data, like fossil dates or dated colonisation events of islands, the age of each node in the phylogenetic tree can be inferred. The molecular clock is a powerful tool for reconstructing the evolutionary past of life on earth in a temporal context but is also subject to controversial debates. Large discrepancies between molecular
Fig. 1.2 Examples of the phylogenetic resolution of different genetic markers. The resolution of genetic markers determines if the genetic common ancestor is a distant associate (a), a distant cousin of the same species (b) or a close relative in the same population (c). For phylogenies (a), conserved genes with low mutation and substitution rates and without intraspecific variation, like ribosomal RNA loci, are used. For medium resolution (b) mitochondrial DNA, especially the COI gene, have become standard in relationship analyses, due to its accelerated mutation and substitution rate compared to nuclear genes and intraspecific variation. Deep splits cannot be inferred with these markers. Genealogies are equivalent to phylogenies on population levels and usually generated with several fast evolving, non-coding regions of the genome, e.g.
variable number of tandem repeats (VNTR) or single nucleotide polymorphisms (SNP) loci, and are suitable for pedigree reconstructions.
clock estimates and the fossil record are hard to explain and both methods, molecular and paleontological, are susceptible to errors. The rapid diversification of metazoa in Cambrian times hampers reliable resolutions of ancient splits and heterogeneous substitution rates between species or over time are inherent problems of molecular time estimates (Bromham et al. 1998, Aris-Brosou and Yang 2003, Rokas et al. 2005). The fossil record, however, is famous for its incompleteness and can never infer the origin of a species, but only reflects a period after its origin. Further, inaccurate dating of geological strata and misconceptions of phylogenetic relationships between fossils and extant taxa are inherent problems of palaeontology (Donoghue and Benton 2007). However, the discrepancies between molecular and paleontological dates should be viewed as uncertainties in todays scientific knowledge and should encourage close cooperation between both disciplines.
1.4.2 mitochondrial DNA Cytochrome Oxidase I
Molecular clocks can be applied to any gene, but caution has to be taken on the time horizon of resolution. Mitochondrial DNA for example evolves faster, on average 10-15 times, than nuclear DNA due to its independent and more frequent replication cycles of mitochondria and the simpler genetic code. The mitochondrial code is more degenerated than the nuclear code; every third codon position is synonymous (Baker 2000). That means substitutions of the third codon-position never generate aminoacid exchanges, resulting in a less constraint evolution. On average, mitochondrial DNA will be saturated in about 10-20 million years, that means after a linear, clock-like rise in sequence divergence, the number of substitutions over time declines (Fig. 1.3) due to constraints of protein function and depletion of variable sites; protein sequences can provide phylogenetic resolution of up to > 200 million years. Mitochondrial genes therefore are useful genetic markers for phylogenies that resolve more frequent divergences than 18S rRNA and their intraspecific variation and maternal inheritance makes them ideal genetic markers for studies of gene flow, population variability, historical biogeography and intraspecific phylogeography.
1.4.3 Microsatellites
An even more recent time horizon can be investigated using simple sequence loci with variable numbers of tandem repeats (VNTR). The eukaryotic genome is interspersed at high frequency with tandemly repeated copies, usually 10-50 times, of short sequence motifs of 2-5 basepairs (e.g. (GT)n, (GCG)n, (GCAG)n). These loci, called microsatellites, usually lie in non-coding regions and are therefore selectively neutral. Trinucleotid microsatellite motifs with
variable length within coding-regions in humans have been associated with Huntingtons disease and spinobulbar muscular atrophy but are assumed to be exceptional. Di- and tetranucleotid repeat arrays are generally considered as non-coding and neutral DNA (Goldstein and Schlötterer 1999).
Mutations of microsatellite loci are expressed as addition or loss of one repeat-unit, much less frequently of several repeat units (Goldstein and Schlötterer 1999). Mutation rates are among the highest reported with rates estimated at 10-2 to 10-5 per haploid genome per generation (Baker 2000). High mutation rates are proposed to arise by replication errors due to polymerase slippage. Misalignments of associating strands during DNA replication can cause loop formation on either the newly generated strand, which will be one repeat unit longer than the template strand (Fig. 1.4a), or the loop forms at the template strand and the nascent strand will be one repeat unit shorter than its template after replication (Fig. 1.4b).
Microsatellites are ideal markers to detect microevolutionary processes, like the degree of population subdivision, genetic variation within and among populations, reconstruction of gene flow in populations, pedigree analyses and to infer breeding structures in populations.
However, microsatellite motifs are under constraint of maximal length and in combination
Fig. 1.3 Rate of sequence divergence and time frames of resolution for three molecular markers.
Percent sequence divergence against divergence time in million years ago for neutral nuclear (microsatellites) and non-neutral mitochondrial (COI) and nuclear (18S rRNA) genes. Microsatellites (VNTRs) resolve recent divergences of a few hundred years and are rapidly saturated. Mitochondrial DNA evolves with about 2% sequence divergence per million years. Beyond 15-20 million years, mtDNA sequence divergence begins to plateau, probably due to limited numbers of substitutions at the variable sites (sequence saturation) and inferences about divergence times become problematic. The 18S ribosomal locus evolves at a rate of 1% per 50 million years and resolves divergence events deep in time, but inferences of divergences younger than 50 million years are difficult. Graphic adapted from Avise (2004).
with their high mutation rates, the convergence of allele sizes that is inpendent of a common ancestry (homoplasy) is common among populations. Microsatellites therefore fail to reflect separation times past some threshold and their application to higher taxonomic groupings is problematic (Baker 2000). Trees constructed with highly variable markers like microsatellites are therefore called genealogies, describing the evolutionary relationships of alleles in contrast to phylogenies, which describe evolutionary relationships of species and higher taxonomic groups.
Microsatellite analyses constitute of PCR based lengths polymorphism analyses and primers - once established - are highly species specific and applicable in any standard molecular laboratory. They are assumed to be randomly distributed throughout the genome and therefore physically independent (not linked) and they are multi-allelic loci with high levels of variation; both are important qualities for powerful statistical analyses. Further, the co-dominant pattern of inheritance provides additional information for pedigree construction by characterising each allele as either hetero- or homozygotic.