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Combining niche modeling & next- generation sequencing of DNA from museum specimens John McCormack Director/Curator, Moore Laboratory of Zoology Assistant Professor, Biology Department Occidental College

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Misperception of natural history collections as antiquated and Victorian

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Goal of this talk: show how older museum collections can leverage todays technology Dr. Edwards pointed out last week how useful museum specimens are for exploring phylogeography and species limits My goal is to show how research specimens provide links to todays technologies and data sources Which allow a holistic appraisal of an organisms phenotype, genotype, and ecological niche

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We have incredible new technologies and data sources Oxford Nanopore MinIon 40 kB reads DNA sequencing Remote-sensing satellites

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Every vouchered specimen provides a unique opportunity to link phenotype, genotype, and the ecological niche in the pursuit of outstanding questions in evolutionary biology Mission Statement

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The Phenotype: observable characteristics Genes + Environment http://www.nytimes.com/2013/02/05/science/pigeons-a-darwin-favorite-carry-new-clues-to-evolution.html http://learning.blogs.nytimes.com/2013/02/06/darwins-pigeons-learning-about-evolution-from-bird-traits/

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Phenotypes have always been studied, but we are discovering new ways to unlock the data 3D CT scanning Multivariate statistics

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Phylophenomics?

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The Genotype: DNA from museum specimens Frozen tissue collection at the American Museum of Natural History Fresh frozen tissue Ancient DNA Moore Laboratory of Zoology

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Traditional DNA sequencing with ancient DNA is laborious time Three times the work for the same fragment! design internal primers three PCRs, sequencing rxns High quality genomic DNA Somewhat degraded DNA Badly degraded DNA Repeat this for each locus (Scott showed why we prefer to have many loci)

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New methods and technologies are especially well-suited to ancient DNA Sequence capture, target enrichment, in-solution hybridization Species1 ACTGA Species2 TGCAT Species3 CCCTC 24 hours

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All fragments can be pooled and sequenced on a next-generation sequencing machine 12-15 million reads per lane But what are these mysterious probe sequences?

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The difference in throughput is perhaps over 1,000,000x Traditional Sanger sequencing Sequence capture with next-generation sequencing

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The Niche: Ecology from Space Georeferencing in real time by GPS retrospectively w/ Google Earth, field notes, etc.

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Remote-sensing satellites Weather Stations Temperature Rain Seasonality Greenness Leaf Area Index Tree Cover Canopy Height The Niche: Ecology from Space Museum specimens allow us to tap into these data through the record of an individual of a species occurring at a certain place and time on Earth

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The Niche: Ecology from Space NASA and the Jet Propulsion Lab are releasing new environmental data layers for every square kilometer of Earth all the time Canopy Height

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Testing for ecological differences among species Extract the environmental data from all the occurrence points (where it does live) As well as from many random points from the range of the species to represent the suite of habitats that it could live in Reduce the variation to a couple axes and visualize Niche model Localities (dots) + enviro. layers + analysis (Maxent) = model of where a species lives

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species occurs Where species could occur (but doesnt for whatever reason) Arteaga et al. 2011 Evolution McCormack et al. 2010 Evolution

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Testing for ecological differences among species Visualizing species and their backgrounds together Are niches different just because of where they live? Or are they more similar/different than you might guess based on where they live? McCormack et al. 2010 Evolution Remember: the primary data are drawn from museum specimens (and other sources of georeference data)

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Moore Laboratory of Zoology Founded in 1950 by Robert T. Moore, private bird collector Over 60,000 skins (highest bird to student ratio worldwide) Most specimens pre-date knowledge about DNA = virtually no tissue collection Largest Mexican bird collection in the world (larger than both big collection in Mexico City) Extinct Imperial Woodpeckers

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Is use of museum collections really declining? MLZ georeference data first made available

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Our research at the Moore Lab Every vouchered specimen provides a unique opportunity to link phenotype, genotype, and the ecological niche in the pursuit of outstanding questions in evolutionary biology Mission Statement

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Mexico is topographically complex What is the role of mountains in species diversification? Describing the basic units of biodiversity Our research focus

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Madhvi Venkatraman Undergraduate Unicolored Jays Aphelocoma unicolor

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Whitney Tsai Laboratory Technician Wood-quail Dendrortyx

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Fiona Gowen Masters Student Western Scrub-Jay Aphelocoma californica

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Field Work Collections Work Molecular Work

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Why is it important to make a one-to-one link between phenotype and genetics? Mexican Jay Aphelocoma wollweberi Transvolcanic Jay Aphelocoma ultramarina In mitochondrial DNA, the two species are divergent with no evidence for gene flow McCormack et al. Molecular Ecology 2008

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Why is it important to make a one-to-one link between phenotype and genetics? Mexican Jay Aphelocoma wollweberi Transvolcanic Jay Aphelocoma ultramarina In nuclear DNA, a cline of Transvolcanic markers to the north suggested ancient gene flow Is ancient gene flow detectable in the appearance of the individuals carrying the markers? McCormack & Venkatraman submitted ms.

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Why is it important to make a one-to-one link between phenotype and genetics? Mexican Jay Aphelocoma wollweberi Transvolcanic Jay Aphelocoma ultramarina McCormack & Venkatraman submitted ms.

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Why is it important to make a one-to-one link between phenotype and genetics? Mexican Jay Aphelocoma wollweberi Transvolcanic Jay Aphelocoma ultramarina We could conclude that ancient gene flow had left no detectable trace in the mtDNA or appearance of Mexican Jays! But only because we could make the one-to-one link between genotype and phenotype. McCormack & Venkatraman submitted ms.

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Why is it important to make a one-to-one link between phenotype and genetics? Mexican Jay Aphelocoma wollweberi Transvolcanic Jay Aphelocoma ultramarina Also, the fact that Transvolcanic Jays were recognized as different species was due to research linking phenotypes and genotypes of museum specimens McCormack et al. Molecular Ecology 2008 Genotype

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Why is it important to make a one-to-one link between phenotype and genetics? Mexican Jay Aphelocoma wollweberi Transvolcanic Jay Aphelocoma ultramarina Also, the fact that Transvolcanic Jays were recognized as different species was due to research linking phenotypes and genotypes of museum specimens MJs TJs UJs McCormack et al. Molecular Ecology 2008 Phenotype

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Spearfishing vs. trawling for DNA Traditional Sanger sequencing Sequence capture with next-generation sequencing

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New methods and technologies are especially well-suited to ancient DNA Sequence capture, target enrichment, in-solution hybridization What are these mystery probes designed from? Somewhat conserved so they work on a broad swath of species Not so conserved that there is no variation to build phylogenies with

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Ultraconserved elements (UCEs) as universal markers for sequence capture phylogenomics and ancient DNA UCEs = stretches of DNA that are remarkably conserved across highly divergent species

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Brant Faircloth UCLA UCEs found in mammals and birds Found over 5,000 UCE regions shared between birds and lizards.

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Faircloth et al. 2012 Syst Biol And all amniotes!

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Core UCE is conserved (anchor) & variation found in the flanks Frequency variant bases Distance from core UCE Faircloth et al. 2012 Syst Biol

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All fragments can be pooled and sequenced on a next-generation sequencing machine Illumina HiSeq or MiSeq

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For very rapid divergences, randomness in gene histories needs to be accounted for (Edwards Lecture)

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Applications of UCEs Evolutionary origin of turtles

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Sunbittern + tropicbirds? shorebird + hoatzin??? McCormack et al. 2013 PLoS One Bird phylogeny from 1,500 loci

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UCE sequence capture of ancient DNA from museum skins Would revolutionize older museum collections that pre-date knowledge about DNA (like the Moore Lab)

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Unicolored Jays Aphelocoma unicolor Tissue Indiv1: 1147 UCE loci Indiv2: 1347 Recent Toe Pad - 1990 Indiv1: 273 Indiv2: 323 Indiv3: 199 Indiv4: 563 Tissue Indiv1: 528 Indiv2: 612 Old Toe Pad - 1940 as high as 151

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Summary Exciting new technologies are available to mine genetic and ecological information from museum specimens Sequence capture using conserved probe sets are promising for ancient DNA from study skins Ecological data from satellites allow for construction and comparison of species niches These advances promise to make older natural history collections increasingly relevant to modern research