Abstract
For more than two decades, mitochondrial DNA sequences and simple sequence repeats (SSRs, or microsatellite loci) have served as gold standards in population genetics. More recently, next generation sequencing (NGS) has enabled researchers to address biological questions that can benefit from hundreds or even thousands of nuclear single-nucleotide polymorphisms (SNPs) generated by restriction-site associated DNA sequencing (RAD-seq). Here we compare the performance of SSR and RAD-seq SNP methods to characterize clonal patterns in a self-fertilizing and highly inbred killifish, Kryptolebias marmoratus (mangrove rivulus) in Florida. RAD-seq analyses conducted on 18 inbred lineages of mangrove rivulus obtained from western Florida and a distant location in eastern Florida unveiled 481 polymorphic RAD loci of which 129 were homozygous within individuals and 352 loci were heterozygous in at least one individual. An initial UPGMA phenogram was constructed, based on 32 microsatellite loci, and used as a benchmark for comparisons with SNP-based phenograms, using a number of different criteria for SNP selection. A phenogram produced by the homozygous SNPs was in excellent agreement with the one generated from 32 microsatellite loci. However, heterozygous SNP data and RAD loci with more than one polymorphic site contributed more noise than usable signal and were unable to resolve clades consistently. This is likely due to errors in identifying homologous loci in the absence of a reference genome. In summary, although the RAD data were powerful in distinguishing the clonal lineages identified by SSR analyses, they also carried considerable phylogenetic noise. Our results suggest that RAD-seq methods should be used with caution for inferring fine population structure, and that stringent quality controls are necessary to reduce false phylogenetic signals.
Highlights
Historical advancements in empirical population genetics often have been tightly connected with the development of new molecular methodologies to uncover genetic variation
DELINEATION OF CLONAL LINEAGES BY MICROSATELLITES Our multilocus (32 loci) SSR analyses of 18 mangrove rivulus fish samples uncovered two isogenic lineages in western Florida: Clone 1 consisting of specimens ERIN12, −14, −15, −18, FDS02, −04, −05, −07, −08, −09, −10, and −11; and Clone 2 consisting of specimens EPP01, −02, −04, and −07 (Figure 1A)
All fish were completely homozygous and the two lineages were distinct from each other at 16 microsatellite loci. These two isogenic lineages were spatially well-separated from a third mangrove rivulus clonal lineage: Clone 3, consisting of specimens PC12 and -13 that were sampled over 600 kilometers away from a location in eastern Florida (Figure 1A)
Summary
Historical advancements in empirical population genetics often have been tightly connected with the development of new molecular methodologies to uncover genetic variation. For years SNP analyses were restricted to “model” organisms for which sequences of full genomes were available (Hinds et al, 2005; Clark et al, 2007), but recent generation sequencing technologies enabled population genomics studies of non-model organisms, including characterization of phylogeography and phylogenetics (Brito and Edwards, 2009; McCormack et al, 2013). One of the most popular NGS methods to date is restriction-site associated DNA sequencing (RAD-seq) (Baird et al, 2008; Davey et al, 2011), which in recent years has figured prominently in phylogeographic and population-genetics studies (Emerson et al, 2010; Reitzel et al, 2013; Chu et al, 2014). The methodology is considered a reproducible way to generate multitudes of nuclear markers, with individual SNPs detected by short NGS reads nearby or between restriction sites scattered throughout the nuclear genome (Peterson et al, 2012)
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