Iria Fernandez-Silva

ezRAD: a simplified method for genomic genotyping in non-model organisms

Here, we introduce ezRAD, a novel strategy for restriction site–associated DNA (RAD) that requires little technical expertise or investment in laboratory equipment, and demonstrate its utility for ten non-model organisms across a wide taxonomic range. ezRAD differs from other RAD methods primarily through its use of standard Illumina TruSeq library preparation kits, which makes it possible for any laboratory to send out to a commercial genomic core facility for library preparation and next-generation sequencing with virtually no additional investment beyond the cost of the service itself. This simplification opens RADseq to any lab with the ability to extract DNA and perform a restriction digest. ezRAD also differs from others in its flexibility to use any restriction enzyme (or combination of enzymes) that cuts frequently enough to generate fragments of the desired size range, without requiring the purchase of separate adapters for each enzyme or a sonication step, which can further decrease the cost involved in choosing optimal enzymes for particular species and research questions. We apply this method across a wide taxonomic diversity of non-model organisms to demonstrate the utility and flexibility of our approach. The simplicity of ezRAD makes it particularly useful for the discovery of single nucleotide polymorphisms and targeted amplicon sequencing in natural populations of non-model organisms that have been historically understudied because of lack of genomic information.

Sympatric Speciation in the Post “Modern Synthesis” Era of Evolutionary Biology

Sympatric speciation is among the most controversial and challenging concepts in evolution. There are a multitude of definitions of speciation alone, and when combined with the biogeographic concept of sympatric range overlap, consensus on what sympatric speciation is, whether it happens, and its importance, is even more difficult to achieve. Providing the basis upon which to define and judge sympatric speciation, the Modern Evolutionary Synthesis (Huxley in Evolution: the modern synthesis. MIT Press, Cambridge, 1942) led to the conclusion that sympatric speciation is an inconsequential process in the generation of species diversity. In the post Modern Synthesis era of evolutionary biology, the PCR revolution and associated accumulation of DNA sequence data from natural populations has led to a resurgence of interest in sympatric speciation, and more importantly, the role of natural selection in lineage diversification. Much effort is currently being devoted to elucidating the processes by which the constituents of an initially panmictic population can become reproductively isolated and evolve some level of reproductive incompatibility without the complete cessation of gene flow due to geographic barriers. The evolution of reproductive isolation solely due to natural selection is perhaps the most controversial manner by which sympatric speciation occurs, and it is that which we focus upon in this review. Mathematical model simulations indicate that even strict definitions of sympatric speciation are possible to satisfy, empirical data consistent with sympatric divergence are accumulating, but irrefutable evidence of sympatric speciation in natural populations remains elusive. Genomic investigations are advancing our ability to identify genetic patterns caused by natural selection, thereby advancing our understanding of the power of natural selection relative to gene flow. Overall, sympatric lineage divergence, especially at the sub-species level, may have led to a substantial portion of biodiversity.

Microsatellites for next-generation ecologists: a post-sequencing bioinformatics pipeline.

Microsatellites are the markers of choice for a variety of population genetic studies. The recent advent of next-generation pyrosequencing has drastically accelerated microsatellite locus discovery by providing a greater amount of DNA sequencing reads at lower costs compared to other techniques. However, laboratory testing of PCR primers targeting potential microsatellite markers remains time consuming and costly. Here we show how to reduce this workload by screening microsatellite loci via bioinformatic analyses prior to primer design. Our method emphasizes the importance of sequence quality, and we avoid loci associated with repetitive elements by screening with repetitive sequence databases available for a growing number of taxa. Testing with the Yellowstripe Goatfish Mulloidichthys flavolineatus and the marine planktonic copepod Pleuromamma xiphias we show higher success rate of primers selected by our pipeline in comparison to previous in silico microsatellite detection methodologies. Following the same pipeline, we discover and select microsatellite loci in nine additional species including fishes, sea stars, copepods and octopuses.

Multilocus evidence for globally distributed cryptic species and distinct populations across ocean gyres in a mesopelagic copepod

Zooplanktonic taxa have a greater number of distinct populations and species than might be predicted based on their large population sizes and open‐ocean habitat, which lacks obvious physical barriers to dispersal and gene flow. To gain insight into the evolutionary mechanisms driving genetic diversification in zooplankton, we developed eight microsatellite markers to examine the population structure of an abundant, globally distributed mesopelagic copepod, Haloptilus longicornis, at 18 sample sites across the Atlantic and Pacific Oceans (n = 761). When comparing our microsatellite results with those of a prior study that used a mtDNA marker (mtCOII, n = 1059, 43 sample sites), we unexpectedly found evidence for the presence of a cryptic species pair. These species were globally distributed and apparently sympatric, and were separated by relatively weak genetic divergence (reciprocally monophyletic mtCOII lineages 1.6% divergent; microsatellite FST ranging from 0.28 to 0.88 across loci, P < 0.00001). Using both mtDNA and microsatellite data for the most common of the two species (n = 669 for microsatellites, n = 572 for mtDNA), we also found evidence for allopatric barriers to gene flow within species, with distinct populations separated by continental landmasses and equatorial waters in both the Atlantic and Pacific Ocean basins. Our study shows that oceanic barriers to gene flow can act as a mechanism promoting allopatric diversification in holoplanktonic taxa, despite the high potential dispersal abilities and pelagic habitat for these species.

Phylogeny of deepwater snappers (Genus Etelis) reveals a cryptic species pair in the Indo-Pacific and Pleistocene invasion of the Atlantic

Evolutionary genetic patterns in shallow coastal fishes are documented with dozens of studies, but corresponding surveys of deepwater fishes (>200m) are scarce. Here we investigate the evolutionary history of deepwater snappers (genus Etelis), comprised of three recognized Indo-Pacific species and one Atlantic congener, by constructing a phylogeny of the genus with two mtDNA loci and two nuclear introns. Further, we apply range-wide Indo-Pacific sampling to test for the presence and distribution of a putative cryptic species pair within E. carbunculus using morphological analyses and mtDNA cytochrome b sequences from 14 locations across the species range (N=1696). These analyses indicate that E. carbunculus is comprised of two distinct, non-interbreeding lineages separated by deep divergence (d=0.081 in cytochrome b). Although these species are morphologically similar, we identified qualitative differences in coloration of the upper-caudal fin tip and the shape of the opercular spine, as well as significant differences in adult body length, body depth, and head length. These two species have overlapping Indo-Pacific distributions, but one species is more widespread across the Indo-Pacific, whereas the other species is documented in the Indian Ocean and Western Central Pacific. The dated Etelis phylogeny places the cryptic species divergence in the Pliocene, indicating that the biogeographic barrier between the Indian and Pacific Oceans played a role in speciation. Based on historic taxonomy and nomenclature, the species more widespread in the Pacific Ocean is E. carbunculus, and the other species is previously undescribed (referred to here as E. sp.). The Atlantic congener E. oculatus has only recently (∼0.5Ma) diverged from E. coruscans in the Indo-Pacific, indicating colonization via southern Africa. The pattern of divergence at the Indo-Pacific barrier, and Pleistocene colonization from the Indian Ocean into the Atlantic, is concordant with patterns observed in shallow coastal fishes, indicating similar drivers of evolutionary processes.

Global divergence of the human follicle mite Demodex folliculorum: Persistent associations between host ancestry and mite lineages

Significance Mites live in human hair follicles and have been implicated in medically important skin disorders, but we know surprisingly little about these residents of our skin. By analyzing the variation segregating among 241 mite sequences isolated from 70 human hosts, we showed that hosts with different regional ancestries harbor distinct lineages of mites and that these associations can persist despite generations spent in a new geographic region. These results suggest that some mite populations are better able to survive and reproduce on hosts from certain geographic regions. Improving our understanding of human follicle mites promises to shed light on human evolution and to provide important contextual information for their role in human health. Microscopic mites of the genus Demodex live within the hair follicles of mammals and are ubiquitous symbionts of humans, but little molecular work has been done to understand their genetic diversity or transmission. Here we sampled mite DNA from 70 human hosts of diverse geographic ancestries and analyzed 241 sequences from the mitochondrial genome of the species Demodex folliculorum. Phylogenetic analyses recovered multiple deep lineages including a globally distributed lineage common among hosts of European ancestry and three lineages that primarily include hosts of Asian, African, and Latin American ancestry. To a great extent, the ancestral geography of hosts predicted the lineages of mites found on them; 27% of the total molecular variance segregated according to the regional ancestries of hosts. We found that D. folliculorum populations are stable on an individual over the course of years and that some Asian and African American hosts maintain specific mite lineages over the course of years or generations outside their geographic region of birth or ancestry. D. folliculorum haplotypes were much more likely to be shared within families and between spouses than between unrelated individuals, indicating that transmission requires close contact. Dating analyses indicated that D. folliculorum origins may predate modern humans. Overall, D. folliculorum evolution reflects ancient human population divergences, is consistent with an out-of-Africa dispersal hypothesis, and presents an excellent model system for further understanding the history of human movement.

Coral reef grazer-benthos dynamics complicated by invasive algae in a small marine reserve

Blooms of alien invasive marine algae have become common, greatly altering the health and stability of nearshore marine ecosystems. Concurrently, herbivorous fishes have been severely overfished in many locations worldwide, contributing to increases in macroalgal cover. We used a multi-pronged, interdisciplinary approach to test if higher biomass of herbivorous fishes inside a no-take marine reserve makes this area more resistant to invasive algal overgrowth. Over a two year time period, we (1) compared fish biomass and algal cover between two fished and one unfished patch reef in Hawai’i, (2) used acoustic telemetry to determine fidelity of herbivorous fishes to the unfished reef, and (3) used metabarcoding and next-generation sequencing to determine diet composition of herbivorous fishes. Herbivore fish biomass was significantly higher in the marine reserve compared to adjacent fished reefs, whereas invasive algal cover differed by species. Herbivorous fish movements were largely confined to the unfished patch reef where they were captured. Diet analysis indicated that the consumption of invasive algae varied among fish species, with a high prevalence of comparatively rare native algal species. Together these findings demonstrate that the contribution of herbivores to coral reef resilience, via resistance to invasive algae invasion, is complex and species-specific.

Optimizing Selection of Microsatellite Loci from 454 Pyrosequencing via Post-sequencing Bioinformatic Analyses

The comparatively low cost of massive parallel sequencing technology, also known as next-generation sequencing (NGS), has transformed the isolation of microsatellite loci. The most common NGS approach consists of obtaining large amounts of sequence data from genomic DNA or enriched microsatellite libraries, which is then mined for the discovery of microsatellite repeats using bioinformatics analyses. Here, we describe a bioinformatics approach to isolate microsatellite loci, starting from the raw sequence data through a subset of microsatellite primer pairs. The primary difference to previously published approaches includes analyses to select the most accurate sequence data and to eliminate repetitive elements prior to the design of primers. These analyses aim to minimize the testing of primer pairs by identifying the most promising microsatellite loci.

Molecular Phylogeny Demonstrates the Need for Taxonomic Reconsideration of Species Diversity of the Hydrocoral Genus Millepora (Cnidaria: Hydrozoa) in the Pacific

Millepora (Cnidaria: Hydrozoa: Milleporidae) spp. are distributed throughout shallow subtropical and tropical marine environments in the Indo-Pacific and Caribbean-Atlantic, and have traditionally been identified using pore characteristics and colony form. Until now, representatives of Millepora spp. on the island of Okinawa-jima, Japan, have been divided into five species; three branching species (Millepora intricata, M. tenera, M. dichotoma), one species with plate-like morphology (M. platyphylla), and one encrusting species (M. exaesa). There have been only a few reports from the Indo-Pacific that have studied the genetic diversity within Millepora spp., although phylogenetic analyses in the Caribbean-Atlantic have proven useful in delimiting closely-related species, while demonstrating that morphologically-based identification systems may have problems. In the present study, we sought to clarify taxonomic confusion of Millepora spp. in the Pacific by using sequence data of the ribosomal internal transcribed spacer (ITS-rDNA) of specimens from Okinawa, Japan and other localities (Johnston Atoll, Great Barrier Reef). Four separate clades were recovered from the ITS-rDNA analyses. Although we examined specimens of all three branching Millepora spp. previously reported from Okinawa-jima Island, in our phylogenetic analyses they were concentrated within a single clade, with only three specimens in other clades. Encrusting Millepora specimens were found within all clades, although it should be noted all species initially start as encrusting forms, and plate-like specimens were found within three clades. Our data also point to the existence of a previously unknown lineage within Millepora characterized by its ability to overgrow live scleractinian corals.

Whole-genome assembly of the coral reef Pearlscale Pygmy Angelfish ( Centropyge vrolikii)

The diversity of DNA sequencing methods and algorithms for genome assemblies presents scientists with a bewildering array of choices. Here, we construct and compare eight candidate assemblies combining overlapping shotgun read data, mate-pair and Chicago libraries and four different genome assemblers to produce a high-quality draft genome of the iconic coral reef Pearlscale Pygmy Angelfish, Centropyge vrolikii (family Pomacanthidae). The best candidate assembly combined all four data types and had a scaffold N50 127.5 times higher than the candidate assembly obtained from shotgun data only. Our best candidate assembly had a scaffold N50 of 8.97 Mb, contig N50 of 189,827, and 97.4% complete for BUSCO v2 (Actinopterygii set) and 95.6% complete for CEGMA matches. These contiguity and accuracy scores are higher than those of any other fish assembly released to date that did not apply linkage map information, including those based on more expensive long-read sequencing data. Our analysis of how different data types improve assembly quality will help others choose the most appropriate de novo genome sequencing strategy based on resources and target applications. Furthermore, the draft genome of the Pearlscale Pygmy angelfish will play an important role in future studies of coral reef fish evolution, diversity and conservation.