Lydia L. Smith

Local phylogenetic divergence and global evolutionary convergence of skull function in reef fishes of the family Labridae

The Labridae is one of the most structurally and functionally diversified fish families on coral and rocky reefs around the world, providing a compelling system for examination of evolutionary patterns of functional change. Labrid fishes have evolved a diverse array of skull forms for feeding on prey ranging from molluscs, crustaceans, plankton, detritus, algae, coral and other fishes. The species richness and diversity of feeding ecology in the Labridae make this group a marine analogue to the cichlid fishes. Despite the importance of labrids to coastal reef ecology, we lack evolutionary analysis of feeding biomechanics among labrids. Here, we combine a molecular phylogeny of the Labridae with the biomechanics of skull function to reveal a broad pattern of repeated convergence in labrid feeding systems. Mechanically fast jaw systems have evolved independently at least 14 times from ancestors with forceful jaws. A repeated phylogenetic pattern of functional divergence in local regions of the labrid tree produces an emergent family-wide pattern of global convergence in jaw function. Divergence of close relatives, convergence among higher clades and several unusual ‘breakthroughs’ in skull function characterize the evolution of functional complexity in one of the most diverse groups of reef fishes.

Exploring the radiation of a diverse reef fish family: Phylogenetics of the damselfishes (Pomacentridae), with new classifications based on molecular analyses of all genera

The damselfishes (Perciformes, Pomacentridae) are an important family of marine reef fishes that occupy a range of ecological positions in the worlds oceans. In order to determine the evolutionary pattern of their radiation, we used multiple methods to examine molecular data from 104 species representing all extant genera. The analysis of 4291 DNA nucleotides (1281bp were parsimony informative) from three nuclear genes (rag-1, rag-2 and bmp-4) and three mitochondrial genes (12s, 16s and nd3), produced well-resolved phylogenies with strong evidence for a monophyletic Pomacentridae, and support for five major damselfish clades. We found that the monotypic subfamily Lepidozyginae evolved early in the pomacentrid radiation. The placement of the genus Altrichthys indicates that brood care has evolved at least twice among the damselfishes. The subfamilies Chrominae and Pomacentrinae, and the genera Abudefduf, Chromis, Chrysiptera, Plectroglyphidodon, and Stegastes, were always found to be polyphyletic, and monophyly was rejected for the genus Amphiprion by almost every analysis. All phylogenetic studies of the Pomacentridae have indicated that their taxonomy is in need of revision at multiple levels. We provide a new classification scheme wherein each subfamily is now monophyletic, and this reorganization is consistent with all previous molecular studies of the damselfishes. The Chrominae are restricted to the genera Chromis and Dascyllus; the Pomacentrinae now represent a lineage of 16 genera that constitute a major, and relatively recent, radiation of coral reef fishes throughout the Indo-West Pacific; we erect the new subfamlies Abudefdufinae and Stegastinae; we relegate the anemonefishes (the Amphiprioninae sensu Allen) to the tribe Amphiprionini within the Pomacentrinae, and synonomize the genus Azurina with Chromis.

Sequence capture using PCR‐generated probes: a cost‐effective method of targeted high‐throughput sequencing for nonmodel organisms

Recent advances in high‐throughput sequencing library preparation and subgenomic enrichment methods have opened new avenues for population genetics and phylogenetics of nonmodel organisms. To multiplex large numbers of indexed samples while sequencing predominantly orthologous, targeted regions of the genome, we propose modifications to an existing, in‐solution capture that utilizes PCR products as target probes to enrich library pools for the genomic subset of interest. The sequence capture using PCR‐generated probes (SCPP) protocol requires no specialized equipment, is highly flexible and significantly reduces experimental costs for projects where a modest scale of genetic data is optimal (25–100 genomic loci). Our alterations enable application of this method across a wider phylogenetic range of taxa and result in higher capture efficiencies and coverage at each locus. Efficient and consistent capture over multiple SCPP experiments and at various phylogenetic distances is demonstrated, extending the utility of this method to both phylogeographic and phylogenomic studies.

Phylogenetic relationships and the evolution of regulatory gene sequences in the parrotfishes

Regulatory genes control the expression of other genes and are key components of developmental processes such as segmentation and embryonic construction of the skull in vertebrates. Here we examine the variability and evolution of three vertebrate regulatory genes, addressing issues of their utility for phylogenetics and comparing the rates of genetic change seen in regulatory loci to the rates seen in other genes in the parrotfishes. The parrotfishes are a diverse group of colorful fishes from coral reefs and seagrasses worldwide and have been placed phylogenetically within the family Labridae. We tested phylogenetic hypotheses among the parrotfishes, with a focus on the genera Chlorurus and Scarus, by analyzing eight gene fragments for 42 parrotfishes and eight outgroup species. We sequenced mitochondrial 12s rRNA (967 bp), 16s rRNA (577 bp), and cytochrome b (477 bp). From the nuclear genome, we sequenced part of the protein-coding genes rag2 (715 bp), tmo4c4 (485 bp), and the developmental regulatory genes otx1 (672 bp), bmp4 (488bp), and dlx2 (522 bp). Bayesian, likelihood, and parsimony analyses of the resulting 4903 bp of DNA sequence produced similar topologies that confirm the monophyly of the scarines and provide a phylogeny at the species level for portions of the genera Scarus and Chlorurus. Four major clades of Scarus were recovered, with three distributed in the Indo-Pacific and one containing Caribbean/Atlantic taxa. Molecular rates suggest a Miocene origin of the parrotfishes (22 mya) and a recent divergence of species within Scarus and Chlorurus, within the past 5 million years. Developmentally important genes made a significant contribution to phylogenetic structure, and rates of genetic evolution were high in bmp4, similar to other coding nuclear genes, but low in otx1 and the dlx2 exons. Synonymous and non-synonymous substitution patterns in developmental regulatory genes support the hypothesis of stabilizing selection during the history of these genes, with several phylogenetic regions of accelerated non-synonymous change detected in the phylogeny.

An evaluation of transcriptome‐based exon capture for frog phylogenomics across multiple scales of divergence (Class: Amphibia, Order: Anura)

Custom sequence capture experiments are becoming an efficient approach for gathering large sets of orthologous markers in nonmodel organisms. Transcriptome‐based exon capture utilizes transcript sequences to design capture probes, typically using a reference genome to identify intron–exon boundaries to exclude shorter exons (<200 bp). Here, we test directly using transcript sequences for probe design, which are often composed of multiple exons of varying lengths. Using 1260 orthologous transcripts, we conducted sequence captures across multiple phylogenetic scales for frogs, including outgroups ~100 Myr divergent from the ingroup. We recovered a large phylogenomic data set consisting of sequence alignments for 1047 of the 1260 transcriptome‐based loci (~561 000 bp) and a large quantity of highly variable regions flanking the exons in transcripts (~70 000 bp), the latter improving substantially by only including ingroup species (~797 000 bp). We recovered both shorter (<100 bp) and longer exons (>200 bp), with no major reduction in coverage towards the ends of exons. We observed significant differences in the performance of blocking oligos for target enrichment and nontarget depletion during captures, and differences in PCR duplication rates resulting from the number of individuals pooled for capture reactions. We explicitly tested the effects of phylogenetic distance on capture sensitivity, specificity, and missing data, and provide a baseline estimate of expectations for these metrics based on a priori knowledge of nuclear pairwise differences among samples. We provide recommendations for transcriptome‐based exon capture design based on our results, cost estimates and offer multiple pipelines for data assembly and analysis.

Squeezing water from a stone: high-throughput sequencing from a 145-year old holotype resolves (barely) a cryptic species problem in flying lizards

We used Massively Parallel High-Throughput Sequencing to obtain genetic data from a 145-year old holotype specimen of the flying lizard, Draco cristatellus. Obtaining genetic data from this holotype was necessary to resolve an otherwise intractable taxonomic problem involving the status of this species relative to closely related sympatric Draco species that cannot otherwise be distinguished from one another on the basis of museum specimens. Initial analyses suggested that the DNA present in the holotype sample was so degraded as to be unusable for sequencing. However, we used a specialized extraction procedure developed for highly degraded ancient DNA samples and MiSeq shotgun sequencing to obtain just enough low-coverage mitochondrial DNA (721 base pairs) to conclusively resolve the species status of the holotype as well as a second known specimen of this species. The holotype was prepared before the advent of formalin-fixation and therefore was most likely originally fixed with ethanol and never exposed to formalin. Whereas conventional wisdom suggests that formalin-fixed samples should be the most challenging for DNA sequencing, we propose that evaporation during long-term alcohol storage and consequent water-exposure may subject older ethanol-fixed museum specimens to hydrolytic damage. If so, this may pose an even greater challenge for sequencing efforts involving historical samples.

Constructing a database of terrestrial radiocarbon measurements

Terrestrial Radiocarbon Database Workshop; Berkeley, California, 20–22 July 2011 Soils play a large role in the global carbon (C) cycle, but soil C stocks and dynamics remain highly uncertain. Radiocarbon (14C) observations provide critical information on the rates of exchange of soil C with the atmosphere and hydrosphere and how those rates vary with edaphic (soil-related) factors and over a range of time scales. For example, the degree to which radio decay has affected 14C demonstrates the importance of short-range-order minerals for stabilizing organic C on millennial time scales in some soils. Time series that track the infiltration of “bomb” 14C help identify the components of soil C that cycle on decadal to centennial time scales.

Cryptic genetic diversity, population structure, and gene flow in the Mojave rattlesnake (Crotalus scutulatus)

The Mojave rattlesnake (Crotalus scutulatus) inhabits deserts and arid grasslands of the western United States and Mexico. Despite considerable interest in its highly toxic venom and the recognition of two subspecies, no molecular studies have characterized range-wide genetic diversity and population structure or tested species limits within C. scutulatus. We used mitochondrial DNA and thousands of nuclear loci from double-digest restriction site associated DNA sequencing to infer population genetic structure throughout the range of C. scutulatus, and to evaluate divergence times and gene flow between populations. We find strong support for several divergent mitochondrial and nuclear clades of C. scutulatus, including splits coincident with two major phylogeographic barriers: the Continental Divide and the elevational increase associated with the Central Mexican Plateau. We apply Bayesian clustering, phylogenetic inference, and coalescent-based species delimitation to our nuclear genetic data to test hypotheses of population structure. We also performed demographic analyses to test hypotheses relating to population divergence and gene flow. Collectively, our results support the existence of four distinct lineages within C. scutulatus, and genetically defined populations do not correspond with currently recognized subspecies ranges. Finally, we use approximate Bayesian computation to test hypotheses of divergence among multiple rattlesnake species groups distributed across the Continental Divide, and find evidence for co-divergence at this boundary during the mid-Pleistocene.

Rapid divergence of mussel populations despite incomplete barriers to dispersal

Striking genetic structure among marine populations at small spatial scales is becoming evident with extensive molecular studies. Such observations suggest isolation at small scales may play an important role in forming patterns of genetic diversity within species. Isolation‐by‐distance, isolation‐by‐environment and historical priority effects are umbrella terms for a suite of processes that underlie genetic structure, but their relative importance at different spatial and temporal scales remains elusive. Here, we use marine lakes in Indonesia to assess genetic structure and assess the relative roles of the processes in shaping genetic differentiation in populations of a bivalve mussel (Brachidontes sp.). Marine lakes are landlocked waterbodies of similar age (6,000–10,000 years), but with heterogeneous environments and varying degrees of connection to the sea. Using a population genomic approach (double‐digest restriction‐site‐associated DNA sequencing), we show strong genetic structuring across populations (range FST: 0.07–0.24) and find limited gene flow through admixture plots. At large spatial scales (>1,400 km), a clear isolation‐by‐distance pattern was detected. At smaller spatial scales (<200 km), this pattern is maintained, but accompanied by an association of genetic divergence with degree of connection. We hypothesize that (incomplete) dispersal barriers can cause initial isolation, allowing priority effects to give the numerical advantage necessary to initiate strong genetic structure. Priority effects may be strengthened by local adaptation, which the data may corroborate by showing a high correlation between mussel genotypes and temperature. Our study indicates an often‐neglected role of (evolution‐mediated) priority effects in shaping population divergence.