When Did the Chicken Cross the Road: Archaeological and molecular evidence for ancient chickens in Central Asia

The origins and dispersal of the chicken across the ancient world remains one of the most enigmatic questions regarding Eurasian domesticated animals1,2. The lack of agreement regarding the timing and center of origin is due, in large part, to issues with morphological identications, a lack of direct dating, and poor preservation of thin bird bones. Historical sources attest to the prominence of chickens in southern Europe and southwest Asia by the last centuries BC3. Likewise, art historical depictions of chickens and anthropomorphic rooster-human chimeras are reoccurring motifs in Central Asian prehistoric and historic traditions4-6. However, when this ritually and economically signicant bird spread along the trans-Eurasian exchange routes has remained a mystery. Here we show that chickens were widely raised by people at villages across southern Central Asia from the third century BC through medieval periods for their eggs and likely also meat. In this study, we present archaeological and molecular evidence for the cultivation of chickens for egg production from 12 different Central Asian archaeological sites spanning a millennium and a half. These eggshells were recovered in high abundance at all of these sites, suggesting that chickens were widely raised by people at villages across southern Central Asia from the third century BC through medieval periods and that they were an important part of the overall diet. Contrary to views that ancient peoples of Central Asia were primarily herding sheep, goat, and cattle, these data show that chicken was also important in the subsistence economy and that it was widely spread along the ancient Silk Road. LC-MS/MS a Q-Exactive HF spectrometer coupled an ACQUITY UPLC M-Class system AG). composition the two channels 0.1% formic channel A and 0.1% formic acid, 99.9% ACN B. each 4 μl of peptides were loaded on a commercial MZ Symmetry C18 Trap Column (100 5 μm, μm × 20 mm, Waters) followed by nanoEase MZ C18 HSS T3 Column (100 Å, 1.8 μm, μm 250 mm, Waters). The peptides were eluted at a ow rate of 300 nL/min by a gradient from 5 to 40% B 120 min and 98% B in 5 min. The column was cleaned after each run with 98% solvent B for 5 min and holding 98% B for 8 min prior to re-establishing loading condition. The mass spectrometers were operated in data-dependent mode performing HCD (higher-energy collision dissociation) fragmentation on the most intense signals per cycle. Full-scan MS spectra (300–1500 m/z) were acquired at a resolution of 120,000 at 200 m/z after accumulation to a target value (AGC) of 3,000,000, while HCD spectra were acquired a resolution of 30,000 a normalized collision energy injection time: AGC singly-charged ions and


Introduction
Debate over the origin(s) and spread of domesticated chickens (Gallus gallus spp. domesticus) has intensi ed in recent years with the introduction of genetic and molecular methods, reigniting old controversies over the enigmatic bird. For a summary of the domestication debates, as they currently stand, see Supplementary Discussion 1. Specialists agree that domestication traits evolved in the South Asian red jungle fowl (possibly involving hybrids of subspecies) somewhere across its expansive range from Thailand to India. Scholars have also argued for widely diverging dates and routes of spread, and some of this confusion comes from unclear identi cations of birds in ancient art historical depictions 3 and overlap in morphological features of chicken bones with those of certain wild avian species.
Additionally, brittle hollow bones and eggshells are far less likely to be preserved, recovered, and identi ed than those of other animals 7 . Further blurring the narrative of the early stages of chicken spread is the fact that pheasants (Phasianidae) and ducks (Anatidae) in China and geese (Alopochen aegyptiaca) in Egypt have, at times in the ancient past, been cultivated or heavily hunted. Biomolecular techniques, such as peptide mass ngerprinting and ancient DNA are uniquely poised to overcome these taphonomic and morphological obstacles to clarify one of the greatest remaining mysteries in the domestication and spread of Old World animals. In this manuscript, we illustrate the success of combining morphological analysis and peptide mass ngerprinting by identifying ancient chicken eggshell fragments from twelve archaeological sites in Central Asia spanning ca. 300 BC to AD 1220. The lack of eggshells from any older Central Asian archaeological sites hints to a rapid rise of egg-laying and chicken rearing across Hellenistic and Zoroastrian West Asia, an economic tradition that appears to have remained prominent through the medieval period in this part of the world.
In a large-scale reassessment of the zooarchaeological material from the southern Levant, Perry-Gal et al. 1 argued that chickens occupied a symbolic and social domain prior to the Hellenistic period (fourth to second centuries BC), after which point their bones sharply rise in ubiquity and abundance in archaeological assemblages. One often-propagated claim is that the chicken served an entertainment role in cock ghts before it took on its signi cant economic status, and some scholars have hypothesized that breeding sports birds may have segued into their role as food 8,9 . Historians have also suggested that the birds were a sacri cial animal for divination or an elite commodity for their plumage and as live caged displays prior to their rapid dispersal across West Asia 10 . They maintained a ritual role into the Roman period as attested from Classical texts and archaeological contexts. For example, at a second century AD shrine to Mithras in Germany an assemblage of more than 7,500 chicken remains was recovered 11 (the linkage between the chicken and a deity associated with Persian origins is unlikely to be a coincidence). Cicero mentioned their power of divination in De Divinatione 12 . The imagery of cock ghting also continued into the Greco-Roman period and accounts for many of the earliest chicken images in the Mediterranean, such as on coins from the Temple of Artemis at Ephesus, estimated to date to 625 -600 BC 9 .
The sudden cultural shift in the role of the chicken to an important food source across the ancient world may have been tied into the development of more proli c egg-laying phenotypes. In this article, we present new evidence for a prominence of chicken egg production in Central Asia starting in the last centuries BC and continuing into the medieval period. The archaeological data that we present consist of fragments (1.5-4.0mm) of eggshells recovered from sediments in archaeological contexts at the core of the ancient Silk Road trade routes. The data come from Bash Tepa, an Hellenistic forti ed site on the edge of the Bukhara Oasis (ca. 3 rd century BC -1 st century AD), several occupation layers at Paykend dated from the Hellenistic (ca. 3 rd -2 nd centuries BC) to medieval period (10 th -12 th centuries AD), the medieval cities of Ka r Kala (7 th -9 th centuries AD), Bukhara (9 th -11 th centuries AD), Afrasiab (10 th -12 th centuries AD), ancient Panjikent (citadel and main town, 5 th -8 th centuries AD), Kok-Tosh or pre-Mongol Panjakent (9 th -12 th centuries AD), Sanjar-Shah settlement (5 th -9 th centuries AD) and the highelevation urban site of Tashbulak (10 th -12 th centuries AD). We propose two hypotheses: 1) the rapid and widespread dispersal of chickens across the ancient world during the last centuries BC coincided with greater egg producing variants; and 2) poultry farming and egg production were an important and regular part of the economy in villages and urban sites across Central Asia from the Hellenistic through at least the Qarakhanid periods.

Rapid Dissemination across the Ancient World
Zooarchaeologists claim to have identi ed domesticated chickens at a handful of southwest Asian sites from contexts dating to the late second and early rst millennia BC 2,13 (see Supplementary Discussion 2).
However, these nds consist of one or two bones in assemblages of tens of thousands of animal bones. The earliest sites with identi able chicken bones that appear in abundance are Tel Kedesh (last centuries BC 2 ) and Maresha (4 th -2 nd centuries BC 1 ), both in Israel ( Figure 1). Shortly thereafter, the bird is reported from archaeological sites in Japan and Korea -the Yayoi period (ca. 300BC -AD300 13 ) and in England by the rst centuries AD 14 . One argument suggests that the species rapidly disseminated with the Macedonian expansion, another suggests that the expansion of the Persian Empire by Cyrus in 539 BC opened the long-distance exchange networks that allowed the chicken to cross the world 7 . Much of the contention over the earliest spread of chickens has focused on the reliability of pre-Hellenistic reports of the bird. For example, there have been many claims of pre-Ptolemaic chickens in Egypt, but the earliest indisputable evidence comes from the last centuries BC 15 . Fascinatingly, the chicken appears to have spread via coastal routes to Central Africa prior to its dispersal in Egypt 16 . There are also a handful of early reports of chicken bones in Europe, pre-dating the Roman expansions. Historians have suggested that the chicken spread through the Mediterranean with Phoenician traders during the mid-rst millennium BC, rising to prominence in the Greco-Roman period, when specialized poultry farms developed for the rst time 17,18 . Chicken bones are a common feature at nearly all Roman sites, including ritual centers, villages, and farmsteads ( Figure 1), and the Classical importance of chickens is well attested in textual sources.

Archaeological Eggshells
Eggshells were recovered from all archaeological sites from central and southern Central Asia dating to later than the third century BC that our team has examined thus far. Additionally, shell fragments remain absent from any earlier occupation sites (bearing in mind that absence of evidence is not necessarily evidence for absence). The shell fragments were recovered from anthropogenic sediments using a 1.4mm geological sieve as part of the heavy fraction recovery portion of archaeobotanical studies. A summary of the recovered remains and the density of shell fragments in the sediments are presented in Table 1. The data used in this study come from 12 archaeological sites, broadly speaking, 11 of which are urban or village sites and have been associated with agropastoral communities. One additional site, LVD-HA-K7 (in the Bukhara Oasis), is a burial mound and was linked to a different population of people than those living in the urban sites. Interestingly, palaeoproteomics analysis of the eggshells from the burial mound suggest that they were not from chickens; all other tested eggshells were identi ed as chicken. Two archaeological sites date to the Hellenistic period, Bash Tepa and Paykend 1; both of which had eggshells in their sediments in relatively high densities, which were recovered from more than 50 percent of the sampled contexts. In Samanid and Qarakhanid period sediments, we recovered eggshells at even higher densities, being prominent in samples from Ka r Kala, Bukhara, Afrasiab, Panjakent 2, and Paykend 2 and 3. Collectively, these data suggest continual and regular deposition of eggshells across all sites and throughout the sediment accumulation period.

Peptide Mass Fingerprinting
Peptide mass ngerprints were obtained for eggshell fragments from four archaeological sites, Afrasiab (n=5), Paykend (n=5), Bash Tepa (n=5), Tashbulak (n=1), and burial mound LVD-HA-K7 (n=2) (see Extended Data Table 2 for a detailed overview of the sample numbers, context information and identi cation). The majority of the samples (16 out of 18) were identi ed as chicken ( Figure 4). The identi cation to G. gallus was con rmed with LC-MS/MS data to rule out a possible origin to a genetically closely related genus in the Galliformes family, such as pheasant (see Supplementary Table 1 and Extended Data Table 3). All of the chicken samples were from residential contexts. The only two samples not identi ed as chicken were from a non-residential context, burial mound LVD-HA-K7. One sample was identi ed as Anseriformes, based on earlier published markers 19 . The other has a low-quality spectrum more consistent with Anseriformes than chicken, but there were too few peaks present for con dent taxonomic identi cation. Within Anseriformes, it is not possible to identify either sample to a more precise taxonomic level, since the majority of the MALDI peptide markers published are identical in all studied species from this order 20 .

Discussion
Evidence for Non-Seasonal Egg Laying We argue that the data in this manuscript provide strong evidence, not only for the rearing of chickens for eggs, but also for a prominence in egg production across a signi cant portion of the year. We present evidence for ancient chicken eggshells from 12 different archaeological settings, spanning a period of a millennium and a half. More informatively, eggshells were recovered at high ubiquity from these sites, meaning they were located in most of the stratigraphic layers and archaeological contexts that we examined. It is well-established by archaeobotanists that artefacts recovered in many different archaeological contexts are more likely to represent frequent-occurrence events, as opposed to seasonal or semi-annual events [21][22][23][24] . In archaeobotany, this reasoning is often applied to grain chaff, suggesting that high ubiquity of remains likely represents a situation where a household processes grain daily and stores the crop in its chaff, as opposed to one seasonal grain threshing event and the storage of grain in a cleaned state. The same reasoning applies to the eggshells in these assemblages -their abundance and ubiquity, not only illustrate their importance in the dietary economy, but also their regular use as a daily or common food over much of the season. The wild reproductive cycle of a chicken progenitor consists of one brood of eggs a year, with a clutch of less than 6 eggs 7 . We suggest that, our identi cation of eggshells is evidence that these chickens had lost their habits of reproduction from the wild and were producing eggs at a regular rate for a signi cant part of the year.
While few archaeological projects have speci cally sought out eggshell fragments in anthropogenic sediments, they are occasionally reported. However, evidence from before the rst century BC for chicken eggs is completely lacking. Eggshell fragments were reported from Roman Mons Claudianus in Egypt, and Van Neer et al. 25 argued that the prominence of medullary bones at the Roman site of Berenike is indicative of an egg-focused chicken industry. Evidence for egg production in Roman Britain comes from tablets found at Hadrian's Wall, noting receipt of, among other items one or two hundred eggs (chickens are among the other listed items 26 ). More informatively, eggshells have been identi ed at 38 Roman period sites in Britain 27 ; using a combination of SEM and peptide mass ngerprinting identi cation techniques, the eggshells from the amphitheater at Chester, Cheshire (AD 70-80), were identi ed as chicken 26 . Further evidence for egg production in the Roman period comes from Diodorus of Siculus, who mentioned a way to incubate chicken eggs. Columella 18 (Book VIII: II:3-8) discussed specialized chicken farmers and suggested that certain kinds of chickens were better for cock ghting and others for egg laying. Some of the breeds he mentions appear to have originated on islands, and may have already diverged through insularity over the previous few centuries. He speci cally references a form of poultry from Adria, near modern-day Venice; the Adrian chicken is also noted by Pliny the Elder 9 . Aristotle's 28 famous chicken embryo evo-devo studies may further attest to the prominence of eggs in ancient Greece.
Eggs are mentioned in Apicius' Cook Book 29 (Book 6, chapter 248:2-3). Pliny claimed that the best birds could lay daily, but most historians agree that this is a signi cant exaggeration, as were many of Pliny's claims 30 . Additionally, Varro 31 (Book III, 481) discusses how to care for hens when they are laying and further illustrated the prominence of chicken egg production by the early Roman period.
As a nal line of evidence for a deeper legacy of egg-laying chickens in West Asia, historical landraces or ecotypes attest to a long legacy (stretching back at least for several centuries) of egg-laying chickens. Genetic studies suggest that there has been long-term isolation and maintenance of insular breeds in remote areas of Iran and Turkey 32,33 . This deep continuity of egg-laying breeds may suggest that the traits of many reproductive cycles within a season evolved before the dispersal of chickens into Europe. That said, the rapid and ongoing extinction of ancient landraces in southwest Asia is evidence that recently developed (within the last century) breeds are far more productive than the ancient ones 32 . In fact, modern commercial poultry production has largely erased the genetic legacy of ancient chicken landraces or ecotypes globally, further complicating studies of the origins and dispersal of this enigmatic animal 34 .

The Rise in Symbolic and Economic Prominence of the Chicken in Central Asia
Specialization in chicken farming and the focus on breeds and large-scale egg laying, as described by Columella, did not continue in Europe after the Roman period and was not resurrected until the early nineteenth century 9 . In medieval Europe the chicken took on the role of a barn-yard scavenger and may have been of some economic importance to members of lower socioeconomic status; during the early nineteenth century, the prominence of chickens in Europe again rose and specialized poultry farms developed. The situation in Central and southwest Asia appear to be different -we report the presence of chicken eggs in high ubiquity, density, and abundance at all sites that we studied for this paper through Hong's (AD 592), also from Xinjiang, the tails are clearly "cock-like" 39 ). Two rooster priests also ank a re on An Jia's tympanum above the doorway to the funerary chamber 39 . Ancient Chinese depictions of funerary birds, identi ed as phoenixes or vermilion birds, invariably have clear features that link them in style to pheasants, whereas Central Asian funerary birds have up-turned tails and leg spurs like roosters. The clear representations of leg spurs on most of the rooster priest chimeras, veri es that they are Galliformes. In Aramaic document C-1 (line 13) from ancient Bactria, dated to the month of Kislev of the 1 st year of Artaxerxes V (the usurper Bessus, November -December 330 BC), among the supplies provided to Bessus as he passed from Bactra (modern Balkh) eastward to Varnu, were 30 chickens (in addition to ve geese, 33 lambs, 133 sheep, one donkey, four bovines, one calf, one horse, as well as oil, wine, our, etc 40 ).
The earliest report for chickens in Central Asia currently comes from bones recovered at the site of Kyzyl Tepa, dating between the sixth and fourth centuries BC in the Syrkhandarya region of Uzbekistan 41 . Identi cations of the Kyzyl Tepa zooarchaeological remains were conducted separately by Pam Crabtree and Norbert Benecke, both of whom identi ed chicken bones in the assemblage. However, they represent the lowest abundance of any domesticated food animal (NISP = 12 out of 2,900) and are not even presented in the summary table for domesticated animals in the study. Notably, wild goose bones are more abundant in the assemblage than chickens 41 . Other evidence for early chickens comes from a coin produced around 300 BC depicting the Satrap of Bactria, Sophytes, which is an Indian name -possibly suggesting links to the south (Figure 2c). The coin was minted in a Greek style, depicting a rooster on one side. Faunal studies from the 2012 excavations at the site of Ulug-Depe in Turkmenistan resulted in the identi cation of chicken remains from the last phase of occupation at the site (Pre-Achaemenid and Achaemenid (Yaz II) 1100-329 BC 42 ). While these remains have not yet been directly dated, it is informative to point out that they are absent from all earlier layers at the site. Wooden slab documents recovered from a military guard tower or postal station near the Silk Road town of Dunhuang, dating to 62 BC mention the import and export of chickens 43 . Also discovered in Xinjiang, faunal remains from Yuansha Gucheng have been reported to contain chicken bones dating to the third or fourth centuries AD 44 .
Chicken bones have also been reported at the medieval Islamic capital of Shahr-e Gholgholah, located at 2,600masl in the Bâmiyân Valley of Afghanistan 45 . Two roosters were also depicted anking a re alter in Temple B in Surkh-Kotal, Afghanistan, dating to the second century AD 39 . Two half-bird creatures were also said to have once anked an image of Mithras on the two Buddhas of Bamiyan 46 . Further south, at the Iranian site of Dasht Qal'eh, chicken bones have been reported from layers dating to the fth or sixth centuries AD, but other large (non-chicken) bird bones were also recovered, identi ed as pheasants, waterfowl, and raptors 47 . Lerner 48 compiled Sasanian (7 th -8 th centuries AD) stamp seals with rooster depictions, which she argues had apotropaic functions. One of the key cookbooks that has survived from the Golden Age of Islam, which was originally compiled in Syria for the Ayyubid rulers, is the Kitab al-Wuslah ila l-Habib Wasf al-Tayyibat wal-Tib (Scents and Flavors the Banqueter Favors). This book contains 635 recipes and medicinal concepts, such as presenting ways to balance the humors, and idea popular from Europe to East Asia by this time 49 . Eggs merit a special section in this book, which contains thirty-eight types of egg-based dishes. Chickens and eggs remain prominent in Central Asian imagery through the medieval period, one example is provided in Figure 2, with a red clay egg on display at the Arc Museum in Bukhara, dated to sometime between the 10 th to 12 th centuries AD.
Linguistic evidence also suggests recent links between the bird, as it spread across West Asia. The common Iranian word for "chicken" is *kr ka-(Avestan kahrka-, Middle Persian kark, Ossetic kark, Wakhi kэrk, etc), which is explained as phonosemantic formation meaning "cackling one", the similar Indo-European formation sometimes means chicken too (Tocharian kranko, Greek kérkos, old Slavic kurъ for rooster), and sometimes other birds (Old Indian kr kāra for partridge 50 ). The word for rooster such as Persian xurōs is derived from another root meaning to cry. In many modern languages the hen retaines the meaning of "bird" in general, e.g. Tajik murγ or Uzbek paranda. The word for "chicken" (Sogdian cwz'kk, Persian jujeh, etc) is again onomatopoetic.

Conclusions
Chickens and chicken eggs have been an important aspect of Central Asian culture and economy for more than two millennia. Beyond a source of food, archaeological evidence has rmly established that the chicken, speci cally the rooster, has long been a symbol of virility in Inner Asia, and, to the Zoroastrian faith, the rooster represents a spirit that calls at dawn to praise the triumph of light over dark in the eternal struggle between night and day. This domesticated bird's importance at the heart of the Silk Road may have facilitated its rapid dispersal across two and a half continents by roughly two millennia ago. In this manuscript, we present evidence for regular consumption of chicken eggs at many of the largest urban centers of medieval Central Asia, including Afrasiab, Paykend, Panjikent, and Bukhara. We also discuss new nds from the ancient mountain village of Tashbulak and the Hellenistic forti ed sites of Paykend and Bash Tepa. The earliest clear evidence for non-seasonal egg laying dates to the Hellenistic period, at which time the more productive birds were already present across Central Asia and the eastern Mediterranean. By the Roman period, specialized poultry farms existed in southern Europe and northern Africa. However, the role of the chicken in Medieval Europe switched to that of a farm-yard scavenger; whereas across Central and southwest Asia, during the Golden Age of Islam, chicken egg production appears to have remained important and continued on a large scale in cities and villages. Chickens express an impress reaction norm of developmental plasticity and are therefore easy to spread into different climates and environmental zones. The eggshell fragments from these sites at the center of the ancient trans-Eurasian trade routes illustrate: 1) that chicken eggs were a regular part of the diet starting in the last centuries BC; and 2) the chicken and the egg remained important in urban centers across Central Asia until at least the Qarakhanid period. Given the di culties in morphologically identifying chicken bones, the highly fragmentary state they are usually preserved in, and their paucity in early contexts, peptide mass ngerprinting is well suited to clarify the domestication and dispersal narrative for the most enigmatic of the Old World domesticated animals (see Supplementary Discussions 1

Sampling and Morphological Identi cation
For most of the sites discussed in this text, systematic collection of sediment samples for archaeobotanical oatation was conducted. The heavy fraction of the samples was wet screened though a 1.4mm geological sieve. The eggshells were collected from the wet screened sediments and recorded as to how many fragments per liter of sediment were recovered. For the site of Ka r Kala, eggshell fragments were handpicked by excavators during excavation, and further sediment samples have been collected but remains un oated at present. The samples were t into contexts using both radiocarbon dating and stratigraphic sequencing. All of the case studies in this paper are part of larger excavations and ongoing projects. The identi cation of eggshells was further conducted using a scanning electron microscope, which allowed us to verify that the breathing pores on the eggs matched those of modern chicken eggs in morphology and size. A selection of the shell fragments was then sent to the ZooMS facilities at the Max Planck Institute for the Science of Human History for further identi cation certainty.

Peptide Mass Fingerprinting
Eggshell fragments were analyzed alongside a blank based upon previously published methods 19,51 . Eggshell fragments of approximately 20 mg were cleaned with 400 µl 0.5 M EDTA, washed three times with 400 µl ultra-pure water and left to dry overnight. Fragments were then crushed into a powder and incubated in 200 µl NaOCL (12% w/v) for 5 days. The supernatant was discarded and the samples were rinsed ve times with 200 µl ultra-pure water, then they were resuspended in methanol (100%) and left to dry overnight. Samples were demineralized in 500 µl 0.6 M hydrochloric acid (HCl) for 10 min after which 500 µl of the supernatant was transferred to a 10 kDa ultra lter (Sartorius, Vivaspin ® ) and centrifuged until completely passed through the lter. 500 µl of 50 mM ammonium bicarbonate (AmBic) was then added to the ultra lter and the samples were centrifuged a second time. The ltrates were resuspended in 200 µl AmBic. Half of the supernatant was transferred to a second tube, which was stored as a back-up. 11 µl of 100 mM CAA (2-chloracetamide)/100 mM TCEP (tris [2-carboxyethyl]phospine) solution was added to the remaining sample supernatant, and then they were digested with 1 µl 0.4 µg/µl of trypsin solution (Pierce™ Trypsin Protease, Thermo Scienti c) for 18h at 37°C. Following enzymatic digestion, peptides were puri ed and concentrated using 100 µl C18 resin ZipTips (Pierce™ C18 Tips, Thermo Scienti c) with conditioning and eluting solutions composed of 50% acetonitrile (v/v) and 0.1% TFA and a lower hydrophobicity wash buffer of 0.1% TFA. Peptides were eluted in 50 µl conditioning solution.
The samples were spotted in triplicate onto an MTP AnchorChip 384-target plate, together with matrix solution (10 mg α-cyano-4-hydroxycinnamic in 7 mL 85% acetronitrile [ACN]/0.1% tri uoracetic acid In order to con rm taxonomic identi cation, one eggshell sample with a high-quality MALDI spectra, identi ed as chicken (sample CP565, Afrasiab) was selected for further analysis using liquid chromatography with tandem mass spectrometry (LC-MS/MS) to obtain peptide sequence data. 20 µl of the sample extract was dried down and sent for LC-MS/MS analysis at the Functional Genomics Center Zurich. LC-MS/MS was conducted using a Q-Exactive HF mass spectrometer (Thermo Scienti c) coupled with an ACQUITY UPLC M-Class system (Waters AG). Solvent composition at the two channels was 0.1% formic acid for channel A and 0.1% formic acid, 99.9% ACN for channel B. Column temperature was 50°C . For each sample, 4 μl of peptides were loaded on a commercial MZ Symmetry C18 Trap Column (100 Å, 5 μm, 180 μm × 20 mm, Waters) followed by nanoEase MZ C18 HSS T3 Column (100 Å, 1.8 μm, 75 μm × 250 mm, Waters). The peptides were eluted at a ow rate of 300 nL/min by a gradient from 5 to 40% B in 120 min and 98% B in 5 min. The column was cleaned after each run with 98% solvent B for 5 min and holding 98% B for 8 min prior to re-establishing loading condition. The mass spectrometers were operated in data-dependent mode performing HCD (higher-energy collision dissociation) fragmentation on the 12 most intense signals per cycle. Full-scan MS spectra (300-1500 m/z) were acquired at a resolution of 120,000 at 200 m/z after accumulation to a target value (AGC) of 3,000,000, while HCD spectra were acquired at a resolution of 30,000 using a normalized collision energy of 28 (maximum injection time: 50 ms; AGC 10,000 ions). Unassigned singly-charged ions and ions were excluded. Precursor masses previously selected for MS/MS measurement were excluded from further selection for 30 s, and the exclusion window was set at 10 ppm. The samples were acquired using internal lock mass calibration on m/z 371.1012 and 445.1200.
The masses of all observed MALDI m/z peaks were compared to the list of peptides found in Byonic to identify corresponding peptide sequences (protein FDR 1%, LogProb ≥ 1, unique peptides ≥ 2, and at least 2 peptides with a PEP2D score lower than 0.01). These sequences were aligned with published markers, checked for uniqueness using NCBI Blast and mapped back onto the corresponding protein sequences using Geneious Prime 2020.1.

Methods references 51
Van der Sluis, L. G. et al. Combining histology, stable isotope analysis and ZooMS collagen ngerprinting to investigate the taphonomic history and dietary behaviour of extinct giant tortoises from Key data points in the dispersal of the chicken, information for this map is provided in Extended Data Table 1 and Supplementary Discussion 3. Given the quantity of evidence for chickens in Roman and medieval Europe and West Asia, this map is not comprehensive, but rather provides an idea of the rapid rate of adoption of chicken rearing.