Two centuries later, on the Caribbean island of Hispaniola, a Florida Museum of Natural History researcher was attracted to a group of insects he calls “Darwin’s butterflies,” because of their similarly high degree of diversity derived from a common ancestor. But it wasn’t until 20 years after beginning his research on the genus Calisto as a University of Florida Ph.D. student that Andrei Sourakov found the missing link for understanding how the group should be classified.

Calisto confusa is named for its tendency to be confused with other butterfly species. Florida Museum lepidopterist Andrei Sourakov used DNA bar coding in a recent study to distinguish C. debarriera as a separate species, rather than a subspecies of C. confusa. © Photo by Andrei Sourakov

“DNA bar coding was the perfect tool to look at this genus because a lot of these species were separated based on only wing patterns, and it’s difficult to prove whether these differences correspond to species, or just represent variation,” said Sourakov, Florida Museum Lepidoptera collection coordinator. “DNA actually allows us to evaluate if and when the gene exchange occurred.”

In a study published Aug. 24, 2011, in Comparative Cytogenetics, Sourakov revised the genus Calisto to include 34 species and 17 subspecies, nine fewer species than previously established, providing better organization for the 54 known taxa. His results are based on his knowledge of morphology, ecology and genetics of these butterflies, and they accompany a strong warning not to abandon other methods of taxonomic classification.

“In general, a lot of people look at DNA sequencing as a substitute for taxonomy and it really doesn’t work this way,” Sourakov said. “One should really know the group they work on to understand what they are looking at – there might be errors in sequencing or in specimen identification, genetic introgressions and other unforeseen circumstances, and if a person isn’t familiar with the group, it can result in extremely erroneous conclusions. DNA is another dataset, like larval or adult morphology, not a substitute.”

The genus Calistoboasts the greatest diversity of butterflies native to the Caribbean, from individuals living in the lowland deserts to their immediate relatives in the mountains. The second-largest Caribbean Island, Hispaniola, formed when two land masses collided more than 50 million years ago, then separated from the archipelago about 25 million years ago. Previous theories suggested these geological occurrences explained the high diversity within the genus and its presence on Cuba, Jamaica, Puerto Rico and the

Calisto hysius has similar characteristics to Calisto batesi, but DNA bar coding confirmed each as a separate species. © Photo by Andrei Sourakov

Bahamas, but calculations from the study establish a younger evolutionary history than researchers previously expected.

“This study falsifies the hypothesis that ancient geological events of continental movement were main factors of diversification in this group,” said Vladimir Lukhtanov, leading research scientist at the Zoological Institute in St. Petersburg and professor at St. Petersburg State University in Russia. “Rather, Darwinian adaptive evolution triggered the rapid species formation in Calisto. I think this work is a great step to understanding the phenomenon of accelerated species evolution on oceanic islands.”

Hispaniola is humid and tropical, but its unique geography includes flooded grasslands, pine forests, savannas and five major mountain ranges. Before researching the island’s fauna, Sourakov studied the host plants of the butterflies to better understand their correlation with the evolution of the genus and how different niches were occupied.

While the different Calisto species look similar – small and brown with eyespots on the undersurface – the caterpillars develop to feed on diverse plants, from bamboos and canes to desert bunch grasses and their roots.

“Some of them have names like confusa, enigma, obscura for a reason – it’s because people have a very hard time identifying them,” Sourakov said. “They are described as a single genus based mostly on distribution, and studying their molecules can allow us to see the deeper differences in these butterflies.”

One species, Calisto pulchella, has had economic importance as a wide-ranging pest of sugarcane, and the DNA bar coding showed greater diversification than expected, supporting a previous separation into an additional species, Calisto darlingtoni.

“I knew that this butterfly fed on sugarcane, but I searched and searched and could never find the caterpillar,” Sourakov said. “Then finally it occurred to me to pull off the leaf, and there it was – feeding practically inside the stem! These butterflies are really adapted to their specific host plants, something I’d never have guessed by simply studying dead specimens in the collections.”

On Hispaniola, there are about 200 species of butterflies in more than 100 genera, and Calisto represents over 20 percent of the total, a phenomenon that is “quite remarkable,” Sourakov said.

“Names only exist in the minds of people, but in order to talk to each other, we have to have names and these names have to have meaning, and taxonomic revisions allow for that,” Sourakov said. “But, this paper is a little more to me than just a revision because it has so many implications for the evolutionary history of Calisto, and for understanding butterfly evolution in general, especially on tropical islands.”

The study was co-authored by Evgeny Zakharov of the Biodiversity Institute of Ontario at the University of Guelph.

Calisto obscura is part of the 20 percent of about 200 butterfly species found on Hispaniola that belong in the same genus. Sourakov’s recent study improved organization of the genus. © Photo by Andrei Sourakov

Calisto pulchella, pictured here in the Florida Museum collections, has economic importance as a wide-ranging pest of sugar cane on the island of Hispaniola. Sourakov described this caterpillar and immature stages of many other species of the genus for the first time in 1996. His recent research shows Calisto pulchella may actually be two separate species that were once found in different habitats, but now converge on the same introduced host plant – the sugar cane. © Photo by Andrei Sourakov

A study appearing online this week in the Proceedings of the National Academy of Sciences unravels 100 million years of evolution through an extensive analysis of plant genomes. It targets one of the major moments in plant evolution, when the ancestors of most of the world’s flowering plants split into two major groups.

Together the two groups make up nearly 70 percent of all flowering plants and are part of a larger clade known as Pentapetalae, which means five petals. Understanding how these plants are related is a large undertaking that could help ecologists better understand which species are more vulnerable to environmental factors such as climate change.

Shortly after the two groups split apart, they simultaneously embarked upon a rapid burst of new species that lasted 5 million years. This study shows how those species are related and sheds further light on the emergence of flowering plants, an evolutionary phenomenon described by Charles Darwin as an abominable mystery.

“This paper and others show flowering plants as layer after layer of bursts of evolution,” said Doug Soltis, study co-author and UF distinguished professor of biology. “Now it’s falling together into two big groups.”

Pentapetalae has enormous diversity and contains nearly all flowering plants. Its two major groups, superrosids and superasterids, split apart between 111 million and 98 million years ago and now account for more than 200,000 species. The superrosids include such familiar plants as hibiscus, oaks, cotton and roses. The superasterids include mint, azaleas, dogwoods and sunflowers.

Earlier studies were limited by technology and involved only four or five genes. Those studies hinted at the results found in the new study but lacked statistical support, said study co-author Pam Soltis, distinguished professor and Florida Museum of Natural History curator of molecular systematics and evolutionary genetics.

The new study at UF’s Florida Museum of Natural History analyzed 86 complete plastid genome sequences from a wide range of plant species. Plastids are the plant cell component responsible for photosynthesis.

Previous genetic analyses of Pentapetalae failed to untangle the relationships among living species, suggesting that the plants diverged rapidly over 5 million years. Researchers selected genomes to sequence based on their best guess of genetic relationships from the previous sequencing work.

Genome sequencing is more time-consuming for plants than animals because plastid DNA is about 10 times larger than the mitochondrial DNA used in studying animal genomes. But continual improvements in DNA sequencing technology are now allowing researchers to analyze those larger amounts of data more quickly.

The study provides an important framework for further investigating evolutionary relationships by providing a much clearer picture of the deep divergence that led to the split within flowering plants, which then led to speciation in the two separate branches.

Eventually, researchers hope to match these evolutionary bursts with geological and climatic events in the earth’s history. “I think we’re starting to get to a point with a dated tree where we could start looking at what was happening at some of those time frames,” Pam Soltis said.

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Source: Pam Soltis, 352-273-1964, psoltis@flmnh.ufl.edu
Doug Soltis, 352-273-1963, dsoltis@ufl.edu
Writer: Bill Kanapaux, bkanapaux@flmnh.ufl.edu
Media contact: Paul Ramey, 352-273-2054, pramey@flmnh.ufl.edu

GAINESVILLE, Fla. — The Florida Museum of Natural History recently added specimen number 20,000 to its Genetic Resources Repository, a nitrogen-cooled freezer with a temperature of minus 300 degrees Fahrenheit.

The freezer at Dickinson Hall stores tissue samples and DNA preparations from physical specimens in the museum at extremely low temperatures to preserve the integrity of the samples for future research. During a brief ceremony marking the occasion Dec. 2, the temperature gauge read minus 308 degrees Fahrenheit.

“The Florida Museum has the world’s largest comprehensive collection of DNA and tissue samples,” said Pam Soltis, Florida Museum curator of molecular systematics and evolutionary genetics. “It’s unique because it includes specimens of invertebrate, vertebrate and plant species.”

The Florida Museum received a National Science Foundation grant to install the freezer in 2006. About two-thirds of the specimens have been added in the last 18 months.

“It’s really exciting to have hit this point so quickly,” Soltis said. “Too often molecular analyses lose track of the actual organisms. By providing DNA and tissue samples that are linked to our own specimens, we can encourage a more comprehensive analysis of biodiversity.”

The repository receives specimens from the museum’s mammal, herpetology, bird, invertebrate zoology, ichthyology and Lepidoptera collections, as well as its herbarium and molecular lab. Invertebrates make up 54 percent of the samples, the result of a large grant from the National Science Foundation to curator Gustav Paulay to prepare 25,000 DNA samples as part of an international barcoding project. Other museums and institutions have also contributed samples of their specimens to the repository.

The Florida Museum has processed 850 transactions relating to requests for DNA and tissue subsamples stored in the repository from researchers within and outside the University of Florida.

Sample requests are batched together to limit the number of times the freezer is open, said Lorena Endara, a graduate student and research assistant in charge of maintaining the system and processing requests.

The cryogenic freezer runs on a vapor system that does not require samples be immersed in liquid nitrogen, Endara said. Instead, 5 to 8 inches of liquid at the base of the unit keep the samples frozen as the nitrogen evaporates. The liquid nitrogen must be recharged every 10 to 13 days.

Providing researchers subsamples from the repository prevents the original specimens from being repeatedly sampled and potentially damaged. And keeping all the museum’s samples in a cryogenic freezer prevents the samples’ DNA from degrading and ensures that the samples remain viable for generations to come.

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Source: Pam Soltis, 352-273-1964, psoltis@flmnh.ufl.edu
Writer: Bill Kanapaux
Media contact: Paul Ramey, 352-273-2054, pramey@flmnh.ufl.edu

GAINESVILLE, Fla. — Florida Museum of Natural History researchers are collecting marine invertebrates on the French Polynesian island of Moorea as part of a massive effort to inventory the DNA sequence of every living species there.

The genetic information collected by scientists from the Florida Museum is part of a whole-system approach that will be used to study ecological processes in depth across the entire island. Moorea’s coral reefs in particular are considered crucial indicators of how natural systems respond to climate change.

“Nobody has ever sequenced a single place to this level,” said Gustav Paulay, the project’s team leader for marine invertebrates and the Florida Museum’s curator of marine malacology. “And nobody has ever investigated coral reef biodiversity this thoroughly in one place.”

The three-year Moorea Biocode Project is now in its second year. Several Florida Museum researchers are in the field using scuba gear, snorkels and wading nets to collect specimens for the first-of-its-kind project.

The Florida Museum scientists are one of seven teams collecting specimens on everything from terrestrial vertebrates to algae. Marine invertebrates make up about 50 percent of the species on the island, which is about 37 miles in circumference and 11 miles from Tahiti.

Based at the UC Berkeley Richard B. Gump South Pacific Research Station on Moorea, the UF team collects specimens up to three times a day. The catch includes crabs, shrimp, plankton, mollusks and worms.

Back at the research station lab, the larger specimens are grouped by appearance. The researchers select individual specimens from each grouping to photograph and take tissue samples. The samples are sent to an on-site DNA extractor for immediate preparation, and the DNA is shipped to the Smithsonian Institution for sequencing.

“We can answer all these things in ecology and evolution and resource management if we have a dictionary to the DNA,” Paulay said. “We’re building that dictionary.”

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Attempting to collect all species of marine invertebrates is a daunting task. To meet the challenge, Paulay’s team divides the works into three sizes: macro (anything longer than four-tenths of an inch), meso (smaller but visible) and micro (less than 1 mm in length).

Paulay hopes to effectively cover the macro fauna, but even that is impossible, he said. Rare things live in strange places. Other things migrate into the area every couple of years. Meso-organisms are even more difficult. Paulay’s team uses a number of extractive methods, such as shaking them out of sand and rock, to get as many unique specimens as possible.

Microfauna are the most challenging of all. The coral reefs are full of them, like microscopic plankton in sea water and minute worms in sand and rock. Paulay’s team is exploring methods of community DNA extraction. This approach involves running a simultaneous DNA analysis on all organisms found in a sample such as sand. Cluster patterns in the DNA sequences will help indicate individual species. Modern technology can sequence millions of genes at once, making the technique possible.

Larger invertebrates are easier to find, but they still provide plenty of surprises.

“You’re always expecting to see things you haven’t seen before,” Paulay said. “Just about every day there’s some really weird thing coming in.”

Paulay estimates more than 5 percent of the macrofauna his team collects are new genera and species. The team recently found a new species of crab in deep water that looks like a transition species between a hermit crab and a free-moving crab. It wears a clam shell instead of a snail shell, and its main body has a crab-like triangular shape.

“I knew enough about hermit crabs to know this was pretty special, “Paulay said. “So I e-mailed a picture of it to a specialist and got an answer back the next day.”

Having such a vast store of genetic information for Moorea is also bound to attract a lot of additional research, Paulay said. The island’s coral reefs are being studied under a long-term ecological research project funded by the National Science Foundation. And DNA information from the Moorea project is being uploaded to a global sequencing effort known as the Barcode of Life, which hopes to collect a DNA sequence for every living thing on the planet.

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Source: Gustav Paulay, 352-273-1828 or 352-273-1829, paulay@flmnh.ufl.edu
Writer: Bill Kanapaux, bkanapaux@flmnh.ufl.edu
Media contact: Paul Ramey, 352-273-2054, pramey@flmnh.ufl.edu

This burst of speciation over a 5-million-year span was one of three major radiations of flowering plants, known as angiosperms. The study focuses on diversification in the rosid clade, a group with a common ancestor that now accounts for one-third of the worlds flowering plants. The forests that resulted provided the habitat that supported later evolutionary diversifications for amphibians, ants, placental mammals and ferns.

“Shortly after the angiosperm-dominated forests diversified, we see this amazing diversification in other lineages, so they basically set the habitat for all kinds of new things to arise,” said Pamela Soltis, study co-author and curator of molecular systematics and evolutionary genetics at UFs Florida Museum of Natural History. “Associated with some of the subsequent radiations is even the diversification of the primates.”

The study appearing online in next weeks Proceedings of the National Academy of Sciences is the first to show the evolutionary relationships of these plants and provide evidence for their rapid emergence and diversification.

Because the diversification happened so quickly, at least in evolutionary terms, molecular methods were needed to sort out the branches of the rosid clades phylogenetic tree, a sort of family tree based on genetic relationships. Only after sequencing many thousands of DNA base pairs are genetic researchers able to tease apart the branches and better understand how plant species evolved.

“Often, when scientists discuss the rapid radiation of flowering plants, they talk as if there had been one massive burst of early diversification,” said Doug Soltis, co-author and chair of UFs botany department. “I think one thing that becomes very clear from our phylogenetic trees when you look at them closely is that its not just one big explosion of species within the flowering plants. There’s a series of explosions.”

The rosid clades diversification is one of at least three bursts in the early evolution of flowering plants. More than 300,000 species of angiosperms exist, classified into an estimated 15,000 genera and more than 400 families. Understanding how these plants are related is a large undertaking that could help ecologists better understand which species are more vulnerable to environmental factors such as climate change.

“We really need to know on a finer scale how these species are related and on different parts of the planet how members of the clade are related,” Doug Soltis said. “That’s where the action is going to be in terms of how this clade responds to climate change. How members of this large clade respond is really going to determine the fate of most of the organisms on the planet.”

The study’s authors sequenced 25,000 base pairs of DNA and sampled a broad range of 104 species from the rosid clade. Using a phylogenetic tree to date the diversification of lineages requires the use of a molecular clock, which calibrates the degree of change that has occurred over time.

“You can assume that over time DNA sequences accumulate change, and things that are more similar to each other in general would have diverged from each other more recently than things that are more different,” Pam Soltis said.

But different genes have different rates of evolution, as do different clades. To compensate, the study used algorithms that accommodate the different rates. Rosid fossils selected by co-author Steven Manchester, the museum’s curator of paleobotany, were used to help calibrate that clock by setting minimum ages for member species.

The study’s first author is Hengchang Wang, who worked at the Florida Museum as a post-doctoral fellow but is now with The Chinese Academy of Science. Other authors include former post-doctoral fellows Michael J. Moore from Oberlin College and Charles D. Bell from the University of New Orleans. UF botany graduate students Samuel F. Brockington and Maribeth Latvis, former UF undergraduate Roolse Alexandre, and Charles C. Davis of Harvard University also contributed to the study.

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Source: Pamela Soltis, 352-273-1964, psoltis@flmnh.ufl.edu
Writer: Bill Kanapaux, bkanapaux@flmnh.ufl.edu 
Media Contact: Paul Ramey, 352-273-2054, pramey@ufl.edu

GAINESVILLE, Fla. — The Florida Museum of Natural History received $186,000 from the Alfred P. Sloan Foundation Tuesday to identify and prepare 25,000 marine specimens as part of a new international DNA barcoding project.

Florida Museum invertebrate zoology researchers will analyze specimens from about 5,000 species in the museum’s collections for barcoding, or genetic sequencing. Florida Museum Malacology Curator Gustav Paulay expects the project to eventually yield public, online databases for species identification that also will create evolutionary tree diagrams with the click of a button.

“The point of this is to make the taxonomic information as available as possible,” said Paulay, a world-renowned coral reef expert and co-principal investigator on the two-year project known as the Marine Barcode of Life. “The funds will help us sort through our batch samples – some of which are identified only to the family level – and make proper IDs to the genus and species level. Then we’ll select representative tissue samples for DNA sequencing.”

The Florida Museum’s role is part of a larger $997,000 project linking its collections with those at the Paris Museum and Australia’s Queensland Museum. Together, these institutions will photograph and prepare 85,000 specimens from 20,300 species. Dirk Steinke of the Biodiversity Institute of Ontario at the University of Guelph is leading the project, which begins in January.

“As a tool, DNA barcoding is currently gaining a lot of momentum in the marine sciences,” Paulay said. “The limiting factor for us is getting access to good materials because museums don’t always have specimens appropriate for genetic testing. But this project links the three institutions worldwide that are most suited for this work.”

Writer: DeLene Beeland
Source: Gustav Paulay, (352) 273-1948, paulay@flmnh.ufl.edu

]]> http://www.flmnh.ufl.edu/pressroom/2007/12/17/fla-museum-receives-186000-as-part-of-international-dna-barcoding-project/feed/ 0 http://www.flmnh.ufl.edu/pressroom/2005/11/28/study-first-to-quantify-validity-of-dna-i-d-tool-using-marine-snails/ http://www.flmnh.ufl.edu/pressroom/2005/11/28/study-first-to-quantify-validity-of-dna-i-d-tool-using-marine-snails/#comments Mon, 28 Nov 2005 16:37:46 +0000 Gerber,Logan R http://slurm.flmnh.ufl.edu/blogs/pressroom/?p=1601 GAINESVILLE, Fla. — A trendy holiday gift within a decade may be a hand-held device that instantly identifies any species from a snippet of animal tissue, says a Florida Museum of Natural History researcher.

That may be possible thanks to scientific advances that include the first test quantifying the effectiveness of a DNA identification tool among brightly colored shells. With an error rate as low as 4 percent, two UF scientists have been able to identify cowries collected from around the world by analyzing tissue samples from the marine organisms and comparing them to a comprehensive catalog of species they compiled.

The findings are published in the December issue of PLOS Biology.

“DNA barcoding — the ability to take a remnant of animal tissue or blood and compare it with a known data base — has attracted widespread attention with its promise as a valuable aid in species identification and discovery,” said Christopher Meyer, a UF biologist and one of the researchers. “However, few comprehensive datasets are available to test its performance. This is the first study to actually put realistic numbers on it.”

Because species around the world are disappearing faster than biologists can identify them, the need for a quick and accurate method of classifying life has never been more pressing, Meyer said. With millions of animal species on Earth, DNA barcoding can be a helpful identification tool for ecologists who may not necessarily be taxonomy experts, he said.

“This new technology is seen as kind of a fancy, cool tool that will revitalize museums, which will house the reference collections, and generate ‘gee whiz’ appreciation from the general public as well,” he said.

Much of the analysis was done at the Florida Museum of Natural History at UF — where Meyer and his co-author Gustav Paulay are curators — because of its world-renowned collection of cowries. After 10 years of collecting and sequencing cowries from around the world, Meyer and Paulay assembled a database from 218 species. The public has long been fascinated by the shiny, colorful shells, ardently collecting them for centuries, Meyer said.

“The question is what happens as you move away from cowries or birds into nematodes or sea spiders and other creatures that people don’t know much about,” he said. “That’s where the problem in identifying different species is greatest, where the bulk of the diversity of life is, including large numbers of undescribed forms.”

In those cases where the data is incomplete because the collection of known species is small, scientists currently rely on threshold values to identify the likelihood of a particular specimen being a brand new species vs. being distantly related to an existing one, he said.

Using their database of these well-known animals, the accuracy of thresholds was examined supposing that their identity was unspecified. In these cases, the researchers determined that thresholds would yield a 17 percent error rate.

Besides its benefits to ecology, DNA barcoding has some forensic applications, Meyer said. One applied use already being employed is identifying the bird species responsible when a carcass damages an airplane engine, he said. “Engines are built to withstand strikes by birds up to a certain size, but not a large crane or goose,” he said. “Thus, it’s helpful to know which brand of shredded tweet went through the combine.”

And because the technology also can identify eggs or other different life stages it could be used to help stop the spread of invasive species, Meyer said. “A border guard may come across some eggs or larvae in an orange shipment and wonder if they are from a dangerous fruit fly or something else to be concerned about,” he said.

For that matter, this ability to detect species in earlier stages of development could benefit ecologists in their work as well, Meyer said.

“Scientists studying butterflies are able to link caterpillars to adults in the field without having to rear them in the lab anymore to see them pupate and grow up,” he said. “They can just sequence the caterpillar and link it to the adult butterfly.”

Probably the most common application, for scientists and consumers alike, would be the ability to instantly analyze the DNA of a plant or animal with a hand-held device, Meyer said. “It’s very Star Treky if you can imagine McCoy having this kind of hand-held device, something like his tricorder,” he said. “Is that really a cod fillet you’re buying at the fishmonger?”

Although the availability of such a device might be 10 or 15 years off, it could allow scientists to have a small lab within the rain forest, collecting biodiversity data and being instantly linked via satellite to the encyclopedia of life.

Media Contact: Paul Ramey, (352) 846-2000, pramey@ufl.edu
Writer: Cathy Keen, ckeen@ufl.edu, (352) 392-0186
Source: Christopher Meyer, cmeyer@flmnh.ufl.edu, (510) 684-9060