Defining Boundaries for Ecosystem-Based Management: A Multispecies Case Study of Marine Connectivity across the Hawaiian Archipelago

Determining the geographic scale at which to apply ecosystem-based management (EBM) has proven to be an obstacle for many marine conservation programs. Generalizations based on geographic proximity, taxonomy, or life history characteristics provide little predictive power in determining overall patterns of connectivity, and therefore offer little in terms of delineating boundaries for marine spatial management areas. Here, we provide a case study of 27 taxonomically and ecologically diverse species (including reef fishes, marine mammals, gastropods, echinoderms, cnidarians, crustaceans, and an elasmobranch) that reveal four concordant barriers to dispersal within the Hawaiian Archipelago which are not detected in single-species exemplar studies. We contend that this multispecies approach to determine concordant patterns of connectivity is an objective and logical way in which to define the minimum number of management units and that EBM in the Hawaiian Archipelago requires at least five spatially managed regions

Next-Generation Phylogeography: A Targeted Approach for Multilocus Sequencing of Non-Model Organisms

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

Graphical representation of the overall 454 experimental design and protocol.

<p>Graphical representation of the overall 454 experimental design and protocol.</p

The average coverage per locus separated by species.

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

List of amplicon primers and development methods.

<p>Primer sequences used for 454 sequencing are found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034241#pone.0034241.s002" target="_blank">Table S1</a>. 1- Modified from universal primers in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034241#pone.0034241-Jarman1" target="_blank">[32]</a>, 2-Developed from ESTs from <i>Asterina pectinifera</i> (DB414458) and <i>Patiria miniata</i> (AY580177), 3-Developed from alignments of dog, frog, fish, and urchin GPI. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034241#pone.0034241-Keever1" target="_blank">[31]</a>. 4- Developed from an EST from the testis of <i>Patiria miniata</i> (EX452619.1), 5- Modified from universal primers in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034241#pone.0034241-Jarman1" target="_blank">[32]</a>.</p

Loci for which 454 sequencing detected additional variation in <i>Parvulastra exigua</i>.

<p>Loci for which 454 sequencing detected additional variation in <i>Parvulastra exigua</i>.</p

Genetic diversity and heterozygosity detected by both sequencing methods.

<p>Locus TP in <i>M. calcar</i> was only genotyped with sequence specific primers.</p

Costs of sequencing 3200 loci.

<p>Sequencing costs were estimated at $4.00 per Sanger read and five reads per cloned locus. Additionally, a$12.50 cost of cloning (based on $245 for 20 reactions of the Promega pGEM-T Easy Vector System) was added per sample. 12.5%, 25% and 50% are in reference to the proportion of individual loci that would need to be cloned to discern individual alleles.</p

Genetic diversity and heterozygosity of loci sequence only with the 454 method.

<p>Genetic diversity and heterozygosity of loci sequence only with the 454 method.</p