Goats, bookworms, a monk’s kiss: Biologists reveal the hidden history of ancient gospels

OXFORD, U.K.—Behind locked doors in one of the oldest libraries in Europe, two dozen scholars mill around a conference table where rare medieval manuscripts perch on lecterns, illuminated by natural light streaming in from floor-to-ceiling windows. Most scholars simply look at these precious books while librarians turn the pages for them. But evolutionary biologist Blair Hedges, wearing gray rubber gloves, approaches one book with a mini–cotton swab. He gently dabs the circumference of a hole in the original white leather binding of a rare 12th century copy of the Gospel of Luke. Then, he inserts a tiny gum brush—the kind teenagers use to clean their braces—into another hole to swab its edges. His goal? "To collect bookworm excrement for ancient DNA analysis," says Hedges, who works at Temple University in Philadelphia, Pennsylvania.

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As Hedges magnifies the holes with a lens on his iPhone, book conservator Andrew Honey of the University of Oxford notices that the holes extend all the way back to the oak boards beneath the binding. Honey suggests that furniture beetles laid eggs in the oak before the bookmaker bound the wood in leather. The larvae lurked there for years before developing into adults that exited through the leather. That means it's likely that "the holes were made by beetles 900 years ago … the oldest example of wormholes I've ever seen," says Hedges, who uses DNA and the size of the holes to assess the type of beetle and so help identify where books were made; the DNA will also help him trace the evolution of the bookworms themselves.

This intimate exploration of wormholes was an unlikely scene at Oxford's historic Bodleian Libraries, which prohibit invasive sampling of books they own. Books are such privileged objects here that scholars are forbidden to bring in pens, purses, sharp objects, or drinks. But scientists have recently figured out how to sample books for ancient DNA and proteins without damaging them. Such studies are revealing the organisms that interacted with ancient books, from the animals whose skins are preserved as parchment to the bookworms and people who once lingered over the pages. Researchers can even isolate the microbes spewed on manuscripts when people kissed, coughed, or sneezed on them.

"Medieval manuscripts represent a relatively untapped store of biological information," says biochemist Matthew Collins, whose team has studied this Gospel of Luke over the past 5 years. Parchment alone is a "rich palimpsest of molecular data," notes Collins, who has a joint appointment at the University of York in the United Kingdom and the University of Copenhagen.

In May, he shared new ways of analyzing ancient books at an unusual symposium at the Bodleian, which brought together biologists, librarians, medievalists, and even a modern scribe. They explored how biological clues can reveal hidden aspects of medieval life, from husbandry and economics to disease. As this new field of research cracks open, researchers hope to expose "the whole bustling medieval world of monks, scribes, readers, poets, country gentlemen" who touched books over the centuries, says Timothy Stinson, a medieval poetry scholar at North Carolina State University in Raleigh.

At a Sotheby's auction in 2009, the Gospel of Luke was sold to book historian William Zachs. Its contents, decoration, and distinctive "prickly" style script suggest that the book was produced around 1120 C.E. by scribes at St. Augustine's Abbey in Canterbury, U.K., says Bruce Barker-Benfield, curator of manuscripts at the Bodleian. The manuscript contains both the Gospel of Luke text and commentary on it, called a gloss, written in the margins. It is one of the few books to survive from that time with its original cover, and is in remarkable condition, Honey says.

Zachs, an honorary fellow at the University of Edinburgh, wanted to know what kind of animal hide was used to make the white leather cover of his book, and in 2012 agreed to open the book to investigation by Collins's lab. The manuscript became the centerpiece of the May symposium, which organizers describe as a model for "a 360-degree study of any book."

Collins already had developed a method to identify ancient species by differences in the amino acid sequences of collagen and other proteins preserved in fossils. His team soon realized that the proteins in parchment offered a new line of evidence about the animals whose skin was used and about the economics of bookmaking in medieval Europe. "With books, you have year after year of pristine information about the medieval world of animal husbandry," says Stinson, who collaborates with Collins.

But no librarian would let them snip a bit of ancient parchment for collagen testing. "It's even harder to sample a rare book than human fossils or teeth," Collins says.

So Collins's postdoctoral fellow in York, Sarah Fiddyment, developed a nondestructive method to extract ancient proteins from parchment. Librarians often "dry clean" a rare manuscript by rubbing it lightly with a polyvinyl chloride eraser, which pulls tiny fibers off the page in curled debris that's usually swept away. Fiddyment discovered that the eraser shavings captured proteins from the parchment, which she could isolate and analyze with a mass spectrometer.

She had just developed this method when Zachs reached out to Collins, and the researchers applied it to the Gospel of Luke. They found that the book's cover was made of the skin of roe deer, a species common in the United Kingdom. But the strap was made from a larger deer species, either native red deer or fallow deer introduced from continental Europe, possibly by the Normans after their invasion in 1066. Fiddyment speculates that the book may have captured a transitional moment when native roe deer were declining and landowners and monasteries stocked parks with bigger deer.

The Normans established scriptoriums in many monasteries in the 11th century, and their appetite for animal skins must have had a "huge impact" on the animals raised, notes Naomi Sykes, a zooarchaeologist at the University of Nottingham in the United Kingdom. But few historical records preserve clues to this shift.

As for the stuff of the manuscript itself, the Sotheby's catalog described it as vellum, or fine parchment usually made from young animals, "probably calfskin." But Jiří Vnouček, a conservator of parchment and paper at the Royal Library of Copenhagen and a graduate student of Collins's at York, noticed while studying the book that some pages were of lower quality. They had dark hair follicles and pores, as well as scars from parasites—a far cry from the uniform white parchment typical of calfskin. "This was some strange kind of cow," says Vnouček, who suggested systematically sampling all the pages with the eraser method.

The researchers did so, and found to their surprise that those unusual pages were goat, thought to be rare and used only in less wealthy areas, Collins says. This suggests that even established scriptoriums sometimes ran out of desirable skins from calves and lambs. Perhaps the monks were letting sheep live to adulthood in order to get more wool, which was the backbone of the medieval economy, Collins says, or perhaps herds were simply too small.

Further analysis showed an unusual pattern: The book was comprised of skins from an estimated 8.5 calves, 10.5 sheep, and half a goat. When the team showed a graphic (below) of the complicated organization of the manuscript at the meeting, the scholars were startled. "This interleaving of calf and sheep is unexpected," says Peter Stallybrass, a historian at the University of Pennsylvania and co-organizer of the symposium. "This shows what these new kinds of scientific approaches can do."

Mining the molecules of an ancient book Researchers applied new methods to probe the history of a single 12th century book, the Gospel of Luke , treating the parchment as a biological sample. They uncovered a pattern of interleaved calfskin and sheepskin, with goatskin used as a last resort. version="1.0" encoding="utf-8"? 1.4 mm 2.3 mm Front flyleaf Back flyleaf 3 1 10 11 2 18 19 26 27 34 35 42 43 50 51 58 59 66 67 74 75 82 83 90 91 98 99 106 107 114 115 122 123 130 131 138 139 146 147 155 154 156 * + Change in commentary scribe Change in main scribe Leather binding Strap I VII VIII IX II III IV V VI X XI XII XIII XVI XV XVII XVIII XIX XIV Calf Sheep Goat Roe deer Fallow deer or red deer Hair side Flesh side Each quire is sewn into the cord Estimated number of skins used Binding The Gospel of Luke is made of 19 quires, each consisting of four skins laid with flesh sides facing each other and folded together to make 16 pages. Ancient proteins Collagen samples collected by rubbing pages with an eraser can reveal the kinds of animals used to make parchment. Wormholes Beetle larvae tunnel in wooden covers and emerge as adults, leaving exit holes that can be used to identify their species. Ancient DNA DNA left on books by bacteria, beetles, humans, and other organisms can be sequenced to learn about their identity. Pigments and dyes Researchers can shine non- destructive lasers on ink to identify the source of components, such as blue lapis lazuli from Afghanistan. Cutting-edge methods provide information beyond textual content and style. * * + Oak board Oak board Sheep 10.5 Calf 8.5 Goat 0.5 Making a manuscript How scientists “read” ancient books Credits: (Graphic) K. Sutliff/ Science ; (Data) Sarah Fiddyment, Jiří Vnouček/University of York Bioarch

The use of goatskin on pages 109–112 of the 156-page manuscript came right before a new scribe with a distinct script took over the main text. That change, along with spelling errors and omitted words in the commentary flanking the main text, suggests a crisis. "A major trauma happened in the history of this book," Barker-Benfield says. "I suspect the idiot scribe [who made mistakes] was sacked."

During the May viewing of the Gospel of Luke, the researchers took a break to examine other medieval manuscripts from the Bodleian's collections. Almost immediately, they spotted pages in another manuscript that resembled those now identified as goat in Zachs's book. Visual inspection suggested that this manuscript, from the Christ Church scriptorium in Canterbury and dating to about 1125 C.E. to 1150 C.E., was also made of interleaved calf, sheep, and goat. "We are finding out more about how the calves and sheep were resourced, right down to the animals being killed to order, as the scribe works out how he will have to switch from sheep to calf," Stallybrass says.

Parchment eraser shavings also yield DNA that can trace specific breeds and their use over time. For example, in a 2014 study of DNA from two pieces of parchment from the 1600s and 1700s, Collins's team showed that a big shift occurred in the breed of sheep raised in the midlands of the United Kingdom, from a scrappier, black-faced, highland variety to a meatier, lowland breed.

At the symposium, Matthew Teasdale, a postdoc in Dan Bradley's lab at Trinity College in Dublin, reported on the biology of another valuable text: the York Gospels, thought to have been written around 990 C.E. DNA from this book's eraser shavings showed that, aside from some sheep, its pages were mostly calfskin—mainly from female calves, which was unexpected because cows were usually allowed to grow up to bear offspring. Historic records report that a cattle disease struck the area from 986–988 C.E., so perhaps many sick and stillborn calves were used for parchment, says zooarchaeologist Annelise Binois-Roman of the Université Paris 1 Panthéon-Sorbonne.

The York Gospels also offer a rare record of the people of the book: Almost 20% of the DNA Teasdale extracted from its eraser shavings came from humans or microbes shed by humans, he announced at the symposium. This is the only surviving Gospel book to contain the oaths taken by U.K. clergymen between the 14th and 16th centuries, and it's still used in ceremonies today. Pages containing oaths were read, kissed, and handled the most, and these pages were particularly rich in microbial DNA from humans, Teasdale reported.

For example, researchers identified DNA from bacteria known to live in human skin and noses, including an abundance of two genera—Propionibacterium, which causes acne, and Staphylococcus, which includes strains that cause staph. Thus the "crud" that mars the surface of many books and documents is a well-preserved bioarchive of bacteria that infected people who handled the books, Stallybrass says.

Of course, how much of that DNA is contamination by recent handlers of a manuscript is tough to tell. Researchers are seeking creative solutions to find out. Many medieval manuscripts contain pages with darkened or discolored areas and smudged fingerprints, signs of being regularly touched or kissed long ago. If Teasdale could sample such smudges in devotional prayer books used heavily by one person, he predicts that "the main user's original DNA could be retrievable." For example, an image of Christ on the cross in the Missal of the Haarlem Linen Weavers Guild (circa 1400 C.E.) was apparently kissed repeatedly by a Dutch priest, who may have left secretions from his lips and nose on the feet of Christ and on the cross. Eraser shaving DNA might reveal that priest's hair and eye color, ailments, and ancestry.

By sampling roughly dated parchment documents, researchers could also trace changes in the ethnic identity of people who made and used books over time, and perhaps identify some of their diseases. Bradley is seeking samples from books that have identifiable contrasts between early and later users, such as books that have been moved from one continent to another.

With funding from the European Union and book owners, Collins is now traveling the world, from the J. Paul Getty Museum in Los Angeles, California, to the Royal Library in Copenhagen, gathering eraser debris from all manner of illustrious books. Other scholars are sending samples and entering the field as well: Researchers studying the Dead Sea Scrolls in Israel are reportedly getting ancient DNA from those parchments. And Stinson has tried to extract DNA from a medieval poem. "We want to build a parchment DNA library," he says.

Collins is seeking advice on what to do with any DNA his team finds and sequences. What hypotheses should they address? "We're fishing for proteins and DNA and catching a lot of stuff," Collins says. "But we scientists come up with questions humanities scholars think are dumb," such as the cause of a death already described in mortuary records. And scientists and humanities scholars have different approaches: Given the concerns about DNA contamination, scientists prefer to touch books only with gloves. But among humanities scholars, the tradition is to use bare hands to ensure that people handle the pages gently; those wearing gloves are thought be rougher.

Some scholars at the Bodleian meeting had lofty ideas—would it be possible to get DNA of famous people such as Isaac Newton, who left behind many diaries and documents? Others were more interested in the bookworms. Hedges announced that the wormholes he measured in the Gospel of Luke were 1.3 millimeters in diameter, suggesting that they were made by Anobium punctatum, a northern European beetle. That would confirm that the book was made in the United Kingdom or northern Europe, rather than in southern Europe. The DNA of bookworms "can provide clues as to when and where objects such as books originated and were transported," Hedges says.

Some medievalists are enthused about the idea that biologists might be able to aid their studies, filling in the blanks left by written records. "I look at handwriting and dialect analysis to figure out a manuscript's age—ridiculous!" laments Stinson, given the Herculean effort required to do so. Now, he says, "I could go ask a biologist."