Next-generation phylogeography: a targeted approach for multilocus sequencing of non-model organisms

Abstract

The field of phylogeography has long since realized the need and utility of incorporating nuclear DNA (nDNA) sequences into analyses. However, the use of nDNA sequence data, at the population level, has been hindered by technical laboratory difficulty, sequencing costs, and problematic analytical methods dealing with genotypic sequence data, especially in non-model organisms. Here, we present a method utilizing the 454 GS-FLX Titanium pyrosequencing platform with the capacity to simultaneously sequence two species of sea star (Meridiastra calcar and Parvulastra exigua) at five different nDNA loci across 16 different populations of 20 individuals each per species. We compare results from 3 populations with traditional Sanger sequencing based methods, and demonstrate that this next-generation sequencing platform is more time and cost effective and more sensitive to rare variants than Sanger based sequencing. A crucial advantage is that the high coverage of clonally amplified sequences simplifies haplotype determination, even in highly polymorphic species. This targeted next-generation approach can greatly increase the use of nDNA sequence loci in phylogeographic and population genetic studies by mitigating many of the time, cost, and analytical issues associated with highly polymorphic, diploid sequence markers.

Figure 1. Graphical representation of the overall 454 experimental design and protocol.

Figure 2. Visual Representation of Library Quality.

A) Graph of the number of total reads by library, shaded by whether the reads were used or discarded. B) Histogram of reads by length for each library. Library A = New South Wales, Library B = Tasmania, and Library C = Southern Australia.

Figure 3. The average coverage per locus separated by species.

M. calcar in black and P. exigua in gray. Average coverage was significantly different between species (Two sided t-test, t = −6.96455, p<0.0001) and between loci within species (M. calcar oneway ANOVA, F = 12.6416, p<0.0001; P. exigua oneway ANOVA, F = 4.2989, p<0.0021). Bars represent standard errors.

Figure 4. Allelic networks for nDNA loci in Parvulastra exigua.

Networks on top were obtained via 454 sequencing and those on bottom from direct Sanger Sequencing. Loci from left to right are EFA1a, GPI, and TP. Samples from New South Wales are colored in blue, Tasmania in red, and Southern Australia in green. Numbers on network branches represent the number of mutational steps (including INDEL) between alleles.