Using introns in phylogenetics and population genetics

Intron sequences have been used for phylogenetic resolution of relationships between closely related species for nearly twenty years (LESSA 1992; SLADE et al. 1993). It is in recent years, however, that they have begun to be used with any real frequency. Introns are amenable to be applied to most of the same things as mitochondrial DNA, though they will be less effective at reflecting recent population changes due to their larger effective population size (FRIESEN 2000). Despite accumulating changes more rapidly than coding DNA, when compared to the more commonly used mitochondrial DNA, diploid spliceosomal intron alleles have an effective population size four times larger and mutate at a rate one quarter the rate of animal mtDNA. MtDNA is thought to evolve 5-10 times faster than nuclear DNA, based on restriction digests of the mitochondrial genome and thermostability studies of single-copy nuclear DNA (BROWN et al. 1979). As a result mtDNA haplotypes coalesce (become monophyletic) more rapidly than introns and so track recent speciation events more effectively. As discussed above, however, there are uncertainties regarding complete dependence on mtDNA and so developing intron markers and understanding their evolution is highly desirable. Aside from recent discussion regarding mtDNA’s applicability for inferring population history, a gene tree obtained from mtDNA is still technically from only one locus and so may not be representative of the entire population history. By using more than one locus, we increase our ability to distinguish between the effects of selection and population demography, as the first will act locally and the second should present a common signature across many loci (HARE 2001).

'"! Intron-exon structure of a gene is generally conserved over wide evolutionary

expanses (PRYCHITKO and MOORE 1997). As a result, conserved exon sites flanking

introns offer ideal sites to place primers that may cross-amplify across a range of

species (FRIESEN 2000). These primers are commonly referred to as exon-primed,

intron-crossing primers (EPIC) (Fig. 1.6). This strategy was introduced over 15 years

ago (LESSA 1992; SLADE et al. 1993) but, unlike universal primers used in

mitochondrial DNA which amplify successfully across different animal species,

primers have yet to prove widely applicable (ZHANG and HEWITT 2003), and so a

certain amount of empirical testing is required before applying them. EPIC primers are generally placed such that there are stretches of exon sequence obtained large

enough to positively identify the amplification product (PRYCHITKO and MOORE

1997). There are different strategies one can take when amplifying intron markers. The first is to select previously used primers and markers and test them, preferentially choosing those which have been shown to work in your study species or which worked in a closely related species. Once a marker is successfully amplified, however, it is generally a good idea to create taxon-specific primers from the obtained sequence, as well as internal intron primers. The second approach is to utilize the abundant sequence data on GenBank (URL) and create EPIC primers from genomic and mRNA cDNA sequences, or generate genomic data for the species being studied and design primers from there.

Figure 1.6: Structure of the human beta-fibrinogen gene, including both coding (exon) and noncoding (intron) regions.

Expanded is intron 7 with flanking segments of exons 7 and 8, to illustrate the concept of the EPIC primer method. primers Intron 7 has the same position in chickens and humans, and is regularly used in avian phylogenetics. The 100 bp scale applies to exon regions only. Adapted from Prychitko & Moore (1997).

The use of introns in phylogenetics is more complex than that of mitochondrial DNA, due to its diploid nature and the frequency of length-variant heterozygotes and

""!

Intragenic recombination, largely absent from animal mtDNA, occurs throughout the nuclear genome. If the recombination rate approaches the substitution rate, haplotypes will have more than one ancestor and different segments within the haplotype will have independent histories (HARE 2001; ZHANG and HEWITT 2003). This can seriously affect phylogenetic reconstruction, biasing a gene tree to show a false signature of population expansion (SCHIERUP and HEIN 2000). Methods exist, however, to estimate recombination rates and identify recombinants in multiple sequence alignments (e.g., RDP3: (MARTIN et al. 2010)). Recombination can then be incorporated into evolutionary models during data analysis (ZHANG and HEWITT 2003). Aside from potentially functional regions within introns, the linkage of whole introns or nucleotide stretches to a functionally important gene, as can occur in regions of low recombination, will affect the evolution of the intron through hitchhiking (MAYNARD SMITH and HAIGH 1974). This should be taken into account when analyzing intron data. Insertion/deletion polymorphism (indels) can also complicate data analysis, and direct sequence reads may appear superimposed if the individual is a heterozygote. They make up a large part of intron polymorphism and can potentially contain phylogenetic information, though most phylogenetic methods do not use this information efficiently (ZHANG and HEWITT 2003). Heterozygosity and allele discrimination is another problem with using nuclear regions. At a given locus, heterozygotes will present two different alleles or haplotypes, which need to be determined in order to extract the most information for genetic analyses (ZHANG and HEWITT 2003). There are experimental and analytical approaches to address this issue. For example, cloning of PCR products is a universally applicable method to phase heterozygotes, however it is costly, laborious, requires the analysis of numerous clones in order to pick up poorly represented alleles. Artifacts can also be introduced due to recombination occurring upon transformation of bacterial cells (ZHANG and HEWITT 2003). Statistical approaches such as the one implemented in PHASE

(STEPHENS and DONNELLY 2003; STEPHENS et al. 2001) utilize the allelic information from homozygotes or heterozygotes differing only in one position to help resolve the phase of multi-site heterozygotes. Length-variant heterozygotes (the product of indel polymorphisms) can also be resolved analytically in some cases (DIXON 2010; DMITRIEV and RAKITOV 2008; FLOT 2007). These methods are not perfect, however, and incorrect phasing can affect downstream analyses (GARRICK et al. 2010).

'#! 1.5.4 Nuclear Introns for Penguins

A number of nuclear markers have been used previously in penguin phylogenetics.

Nuclear protein coding loci used for phylogenetics include RAG-1 (BAKER et al.

2006; ERICSON et al. 2006), and c-mos proto-oncogene (600bp exon fragment) (VAN

TUINEN et al. 2001). Nuclear introns used for phylogenetics of penguins include

370bp fragment of intron 11 of glyceraldehyde-3-phosphodehydrogenase (G3PDH)

(Adélie penguins) (VAN TUINEN et al. 2001), intron C of the gametologous avian

chromo-helicase-DNA-binding protein (CHD1Z/CHD1W) (Adélie penguins)

(SUNDSTRÖM et al. 2003), adenylate kinase intron 5 (AK1i5) (Spheniscus mendiculus)

(SHAPIRO and DUMBACHER 2001), ß-fibrinogen intron 7 (FGB7)(Eudyptula minor,

Spheniscus humboldti) (ERICSON et al. 2006; FAIN and HOUDE 2004), myoglobin

intron 2 (Spheniscus humboldti, Eudyptula minor) (ERICSON et al. 2006; HACKETT et

al. 2008), interferon regulatory factor 2 intron 2 (Eudyptula minor) (HACKETT et al.

2008), and ornithine decarboxylase intron 6-7 (Spheniscus humboldti) (ERICSON et al.

2006).

A large number of intron markers have now been developed and applied to avian phylogenetics. Of potential use for work in penguins are those developed or tested in closely related orders (e.g. Procellariformes, Ciconiiformes, Pelecaniiformes,

Gaviiformes (HACKETT et al. 2008)). For example, four intron markers isolated in

marbled murrelets (Brachyramphus marmoratus), which are coastal seabirds, may

cross-amplify in penguins (FRIESEN et al. 1997). Several studies have sought to

develop broadly applicable intron markers for avian phylogenetics from available

genomic sources that may potentially cross-amplify in penguins as well (BACKSTRÖM

et al. 2008; BORGE et al. 2005; KIMBALL et al. 2009; PRIMMER et al. 2002).