Dental calculus and isotopes provide direct evidence of fish and plant consumption in Mesolithic Mediterranean

Abstract

In this contribution we dismantle the perceived role of marine resources and plant foods in the subsistence economy of Holocene foragers of the Central Mediterranean using a combination of dental calculus and stable isotope analyses. The discovery of fish scales and flesh fragments, starch granules and other plant and animal micro-debris in the dental calculus of a Mesolithic forager dated to the end of the 8th millenium BC and buried in the Vlakno Cave on Dugi Otok Island in the Croatian Archipelago demonstrates that marine resources were regularly consumed by the individual together with a variety of plant foods. Since previous stable isotope data in the Eastern Adriatic and the Mediterranean region emphasises that terrestrial-based resources contributed mainly to Mesolithic diets in the Mediterranean Basin, our results provide an alternative view of the dietary habits of Mesolithic foragers in the Mediterranean region based on a combination of novel methodologies and data.

Full text

1
SCIEnTIfIC REPORtS | (2018) 8:8147 | DOI:10.1038/s41598-018-26045-9
www.nature.com/scientificreports
Dental calculus and isotopes
provide direct evidence of sh and
plant consumption in Mesolithic
Mediterranean
Emanuela Cristiani 1, Anita Radini 2, Dušan Borić3, Harry K. Robson 2, Isabella Caricola1,
Marialetizia Carra1, Giuseppina Mutri 1, Gregorio Oxilia 1, Andrea Zupancich 1,
Mario Šlaus4 & Dario Vujević5
In this contribution we dismantle the perceived role of marine resources and plant foods in the
subsistence economy of Holocene foragers of the Central Mediterranean using a combination of dental
calculus and stable isotope analyses. The discovery of sh scales and esh fragments, starch granules
and other plant and animal micro-debris in the dental calculus of a Mesolithic forager dated to the end
of the 8th millenium BC and buried in the Vlakno Cave on Dugi Otok Island in the Croatian Archipelago
demonstrates that marine resources were regularly consumed by the individual together with a variety
of plant foods. Since previous stable isotope data in the Eastern Adriatic and the Mediterranean region
emphasises that terrestrial-based resources contributed mainly to Mesolithic diets in the Mediterranean
Basin, our results provide an alternative view of the dietary habits of Mesolithic foragers in the
Mediterranean region based on a combination of novel methodologies and data.
The Central Mediterranean has yielded a unique funerary record whereby large and small islands in the
Tyrrhenian, Ionian and Adriatic Seas were selected as burial locations by Mesolithic foragers. Mesolithic burials
are known from Sicily, the Egadi Islands, Sardinia, Corsica as well as some islands of the Croatian archipelago1–9.
Despite the importance of marine localities for disposal of the dead, the dietary stable isotopic and zooarchaeo-
logical data for these Central Mediterranean foragers has emphasised that marine resources (and plant foods) had
a marginal role to their diets10–17. is pattern is in stark contrast to Mesolithic forgers inhabiting regions along
the Atlantic coastline18,19.
Recent methodological developments in the analysis of micro-fossils trapped in human dental calculus has
provided a new way for assessing neglected aspects of hunter-gatherer-sher subsistence along with non-dietary
information on human interaction with varied environments20,21. e potential of this method has mainly been
recognised for reconstructing the relative proportion of plant foods in human diets. It has emerged that the har-
vesting and processing of starchy resources, such as grasses, tubers or roots rich in carbohydrates, might not have
been a sole prerogative of agricultural societies22. Yet, it is more dicult to assess a direct correlation between
the presence of plant remains in calculus and estimations of the quantity of the plant foods consumed23 while
micro-debris of animal origin has rarely been recovered in ancient plaque. In addition, the recovery in dental
calculus of micro-particles of materials deliberately or accidentally ingested during the performance of various
activities has also proven the potential of the study of dental calculus to provide insights into aspects of individual
life-ways other than nutrition20,21,24.
In this contribution, we oer new insights into the complexity of Mesolithic diets in the Central Mediterranean
by presenting the results of our analysis of dental calculus remains from an individual buried at the site of Vlakno
Cave on Dugi Otok (Fig.1). e analysis of dental calculus presented here will be compared with the results
1Department of Oral and Maxillo Facial Sciences, “Sapienza” University of Rome, Via Caserta 6, 00161, Rome,
Italy. 2BioArCh, Department of Archaeology, University of York, York, YO10 5YW, United Kingdom. 3The Italian
Academy for Advanced Studies in America, Columbia University, 1161 Amsterdam Avenue, New York, NY, 10027,
USA. 4Anthropological Center, Croatian Academy of Sciences and Arts, 10000, Zagreb, Croatia. 5Department of
Archaeology, University of Zadar, Zadar, Croatia. Correspondence and requests for materials should be addressed to
E.C. (email: emanuela.cristiani@uniroma1.it)
Received: 13 February 2018
Accepted: 3 May 2018
Published: xx xx xxxx
OPEN
Content courtesy of Springer Nature, terms of use apply. Rights reserved

www.nature.com/scientificreports/
2
SCIEnTIfIC REPORtS | (2018) 8:8147 | DOI:10.1038/s41598-018-26045-9
obtained from stable isotope analysis in order to provide an alternative view of Holocene forager dietary and
non-dietary behaviour in the Mediterranean Basin as a whole.
The beginning of the Holocene marks a period of profound environmental changes in this part
of the Mediterranean, which deeply reshaped the geography of the Adriatic region and the nature of
human-environment interactions. To date, attempts to understand forager subsistence dynamics and dietary
preferences in this region have focused on traditional, protein-sensitive approaches, such as archaeozoological
and stable isotope analysis10–17,25–31.
Archaeological Background
e site of Vlakno cave is situated on the island of Dugi Otok along the Eastern Adriatic coast of present-day
Croatia (Fig.1). The cave has yielded evidence of Late Upper Palaeolithic (Epigravettian) to Mesolithic
hunter-gatherer-sher occupation5. Archaeological investigations at the site started in 200432 while systematic
excavations have been on-going since 2011. A 5 m-deep test trench did not reach the bedrock and covers a pos-
sibly continuous sequence from ca. 18,010–17,560 cal BC at 95% condence (Beta-302247: 16,330 ± 70 BP). Five
main cultural strata (Stratum 1 to 5) have been proposed covering the Epipalaeolithic and Mesolithic periods33
(Fig.2). e upper three strata were deposited during the Holocene. Mesolithic layers start from the surface of
Stratum 1 where Mesolithic deposits are mixed with intrusive Neolithic to Medieval artefacts5. Mesolithic Strata
2 and 3 roughly correspond with the Boreal and Preboreal chronozones. Based on the radiocarbon measure-
ments, Mesolithic occupation started ca. 9880–9370 cal BC at 95% condence (Beta-30276: 10,060 ± 50 BP)34.
e chipped stone tool assemblage shows a gradual transition towards a typical Mesolithic assemblage but with
strongly pronounced Epigravettian traditions. Similar subtle changes have also been identied in the ornamental
assemblage, the worked ints and subsistence strategies33.
Figure 1. Vlakno site location (a) and view of the entrance of the cave (b).
Figure 2. Stratigraphy of the site and chronology.
Content courtesy of Springer Nature, terms of use apply. Rights reserved

www.nature.com/scientificreports/
3
SCIEnTIfIC REPORtS | (2018) 8:8147 | DOI:10.1038/s41598-018-26045-9
e analysis of the faunal remains demonstrated a predominance of mammals (88.4% of the total vertebrate
assemblage) followed by sh and birds (11.6% of the total vertebrate assemblage)34. In the Early Mesolithic
(Stratum 3), the most common mammalian taxon was red deer (Cervus elaphus L.), followed by red fox (Vulpes
vulpes L.). In the chronologically later Stratum 2, the red fox is the predominant taxon followed by red deer.
Fish remains are present in the faunal assemblage from the beginning of the Mesolithic but there is a signicant
increase in Stratum 2 (increasing from 1.7% to 9.5% of the total assemblage per stratum)34. is dierence may
correspond with an environmental change, including sea level rise, with Dugi Otok becoming separated from the
mainland already before ca. 10,194–8351 cal BC at 95% condence (Z-3660: 9760 ± 280 BP)35. Consequently, the
local population of red deer may have become vulnerable and overexploited which in turn led to a diversication
in subsistence strategies. Fish bones have preliminarily been identied as Sparidae (sea-breams, porgies) and
Scombridae (tunas, mackerel). Taxonomically, there is a change in the bird assemblage between the two strata.
Wild pigeon (Columbidae) predominates in both, but its frequency decreases in the later deposits, coinciding
with an increase in taxa richness. Other taxa are represented by a few bones and include aquatic (cf. rail, duck),
steppe (great bustard, Otis tarda L.) and some other species (birds of prey, small passerines)34. ere was also
an abundance of mollusc shells. e most abundant are land snails Helix sp., showing a slight decrease in their
frequency towards the younger Mesolithic levels. e second most abundant group is made up of various taxa
of marine gastropods and bivalvia (Osilinus sp., Patella sp., Ostrea sp.) with a pattern of increased diversity in
Stratum 25.
In Mesolithic Stratum 2, a well-preserved skeleton of an adult male, aged between 30 and 40, was found in
an extended supine position (Fig.3). Paleopathological analyses undertaken according to established criteria
described in literature36,37 demonstrated mild osteophyte development on the distal right humerus and proximal
right ulna suggesting a mild form of degenerative osteoarthritis on the right elbow, as well as marginal osteophyte
development on the 2nd, 3rd, 4th and 5th lumbar vertebrae. Schmorl’s defects were also present on the 1st and 2nd
lumbar vertebrae. ese defects result from protrusions of the nucleus pulposus of the intervertebral disc through
the vertebral body endplate and into the vertebral body. During the excavation, because of the arid soil no traces
of a burial pit have been found, but given the age dierence between the radiocarbon measurements of Stratum 2
and the burial itself, it must have been dug into the earlier deposits.
e burial was directly AMS radiocarbon (14C) dated in two dierent labs: Beta-311088 measured a phalanx
that gave a radiocarbon age of 8420 ± 40 BP, which calibrates to ca. 7468–7185 cal BC at 95% condence; OxA-
34518 made on the right proximal ulna sha fragment yielded a radiocarbon age of 8490 ± 45 BP that calibrates to
Figure 3. Mesolithic burial from Vlakno cave: (a) View of the burial; (b) Close up of the skull; (c) Detail of the
teeth and dental calculus.
Content courtesy of Springer Nature, terms of use apply. Rights reserved

www.nature.com/scientificreports/
4
SCIEnTIfIC REPORtS | (2018) 8:8147 | DOI:10.1038/s41598-018-26045-9
ca. 7591–7496 cal BC at 95% condence. However, as discussed below, based on the stable isotope values obtained
on this individual, the radiocarbon ages are likely to have been aected by a marine reservoir eect. Since the 14C
results are probably several hundred years too old, the individual most likely falls somewhere towards the end
of the 8th millennium BC. At present, in the absence of a ‘perfect pair’ of datable material, which is contempora-
neous with the burial but unaected by a reservoir eect (e.g. the remains of terrestrial herbivores), it is not yet
possible to determine the correction factor with certainty. e global marine reservoir age (ΔR) is assumed to
be around 400 14C years. A more precise value for calibration purposes is 405 ± 22 14C years38 while more rened
estimates have recently been suggested for the Eastern Adriatic area depending on the type of sampled marine
organism39. However, three other Mesolithic radiocarbon measurements from Vlakno fall into the duration of the
Early Mesolithic and are at least a 1000 years older than the date obtained on the burial40. Presently, it is unclear
whether the burial was interred during the Late Mesolithic at an abandoned site with the remains of an earlier
Mesolithic occupation, or during the course of occupation in the Late Mesolithic.
Materials and Methods
Dental calculus analysis. Dental calculus or tartar is the mineralised biolm of dental plaque adhering to
the tooth enamel, composed primarily of calcium phosphate mineral salts deposited between and within rem-
nants of formerly viable micro-organisms41. e continuous production of saliva in the mouth ensures the pres-
ence of minerals – key for the formation of calculus42. As the deposition of mineralised plaque ceases at the death
of an individual with the production of saliva, the calcied plaque represents a sealed repository of unique human
biographic information related to the individual’s hygiene, dietary and non-dietary preferences and habits as well
as lifestyle20,22,43,44. To date, ancient dental calculus analysis has been applied to the study of humans and hom-
inins, even on several million year old fossils, providing insights into their diets and evidence of materials (e.g.
wood debris, charcoals and plant bres) ingested or inhaled while performing daily life activities or using teeth as
“third hand” in para-masticatory tasks20,21,24,45–47. Recently, it has been emphasised that non-dietary information
entrapped in dental calculus has only been partially explored and understood48.
Dental calculus was sampled from the well preserved and directly AMS radiocarbon (14C) dated skeleton
recovered in Stratum 2 (Fig.3). Mineralised plaque was removed using a metal dental pick. Gloves were worn at
all times to reduce contamination. Once removed, the calculus samples were stored in sterile Eppendorf tubes
and transported to the laboratory for analysis. Soil still adhering to the surface of the calculus was removed in the
laboratory under a microscope. A ne sterile acupuncture needle with 0.06 N HCl was used to gently scrape o
very small areas of dirt adhering to the surface. is procedure was essential due to the very small nature of the
fragments of calculus available for analysis. is process was conducted using a stereomicroscope at a magnica-
tion up to 100x. Once the surface was suciently clean, calculus samples were washed in ultrapure water (up to
three times) in order to remove any traces of sediment. is method allowed us to remove the dirt from the sur-
face, which minimally aected the calculus whilst ensuring that the surface was completely free of contaminants.
en, the calculus was degraded in a weak solution of 0.06 N HCl in order to extract the microfossils entombed
in the calculus matrix following established protocols44. e contaminated soil was examined as an additional
precaution. e dissolved calculus was mounted on slides using a solution of 50:50 glycerol and ultrapure water.
Examination of microfossils in tartar was carried out using Zeiss and Olympus compound polarized micro-
scopes (100x–630x) at the University of York and a Zeiss Imager2 polarized microscope (100x–600x) at Sapienza
University of Rome. Starches were identied on the basis of their 3D morphology, presence and shapes of features
(lamellae, hilum, bumps and depressions of their surface), characteristics of the extinction cross under polarized
light microscopy. e reference collections of modern plants native to the Balkans, the Mediterranean region and
Northern Europe housed at the Sapienza University of Rome and the University of York were used for compar-
ison. In addition, samples of modern plants were provided by the Botanical Garden “Jevremovac” of Belgrade,
the Genomics Research Centre (CREA) of Fiorenzuola d’Arda and the Agricultural Research Council (CRA) at
Sant’Angelo Lodigiano in Italy. e same reference collection has been successfully by the authors in both dental
calculus and stone tool analyses21,22,24,44,49,50. Previously published literature on local ora was also considered
during the analyses. It must be stressed that methodologies and criteria for the identication of archaeological
starch granules and microfossils entrapped in dental calculus were also based on the study of renowned literature
in the eld of modern and ancient starch research49,51.
Stable isotopes analysis. Carbon (δ13C) and nitrogen (δ15N) stable isotope analyses are routinely under-
taken to reconstruct the dietary habits of past populations. Human bone is a complex mixture of organic and
inorganic materials that is continually remodelled during a person’s lifetime, and therefore at death the chemistry
of the last 10–15 years of life is retained52. Since bone is sensitive to contamination and decomposition from the
burial environment, hydroxyapatite analyses were not performed. Carbon and nitrogen stable isotopes used in
palaeodietary studies are oen obtained from bone collagen and primarily relate to the protein content53. is
creates a bias towards the isotopic recognition of plant foodstus in comparison to other dietary resources54. δ13C
values primarily dierentiate between marine and terrestrial foodstus, and as such a higher δ13C value reects
the long-term consumption of marine derived protein. It is widely acknowledged that the marine and terrestrial
endpoints are −12 ± 1, and −21 ± 1‰ respectively19. It is also possible to distinguish between C3 and C4 photo-
synthetic plants on the basis of δ13C values. In comparison, δ15N is an indicator of the trophic level hierarchy and
can dierentiate between freshwater organisms and terrestrial mammals as well as plants. δ15N values in terres-
trial herbivores are generally between 4 and 7‰, while humans or carnivores higher up in the trophic level tend
to have δ15N values 3 to 4‰ higher than the animals they consume55.
Experimental analysis. e micro-debris found in the dental calculus was compared to a reference col-
lection composed of micro-remains extracted from modern plants native to the Mediterranean, North African
Content courtesy of Springer Nature, terms of use apply. Rights reserved

www.nature.com/scientificreports/
5
SCIEnTIfIC REPORtS | (2018) 8:8147 | DOI:10.1038/s41598-018-26045-9
regions and Northern Europe. Non-dietary items, which might have been used for cras or accidentally inhaled
as dust during cra and dietary related activities (e.g. wood, plant and animal bres, feathers, etc.), as suggested
elsewhere48, have also been considered as reference material. Agate mortars and distilled water were used to grind
fresh and dry botanical and non-botanical samples following an established experimental protocol49,50, which also
included the use of dierent grinding modalities in order to document distinctive states in the starch granules or
changes in size due to the preparation processes. Mortars were carefully cleaned between experiments with soap
and hot water for a prolonged period of time. Boiled, ground, and chewed seeds were also considered as exper-
imental reference items. e species relevant to the study are discussed in detail below (see results). Descriptive
and interpretative criteria have been chosen according to internationally recognised standards in the eld of
modern and ancient starch granule research56. Finally, due to the sh remains on site and contemporaneous sites
in the region30 an experimental reference collection of skin, esh and scales of Mediterranean marine taxa was
created. is collection included Atlantic mackerel (Scomber scombrus L.), gilthead sea bream (Sparus aurata L.),
white sea bream (Diplodus sargus L.) and European seabass (Dicentrarchus labrax L.) that had been processed by
a number of methods, including boiling or roasting. In addition, chewed remains were included as well as a range
of terrestrial animal tissues, including hair, skin and esh particles, bone, cartilage, etc. to aid the identication of
possible sh and mammalian tissues in tartar.
Results
Dental calculus. Plant residues. More than 30 starch granules were retrieved from the dental calculus.
ese were derived from two dierent morphological types, later assigned to the Triticeae (wheat and barley
tribe) potentially to the Poeae (oat tribe) tribes of the Poaceae Families (grasses).
Type I. Various small compound granules found in large sub-oval aggregates represent the rst and most
numerous typology of starch granules found in the Vlakno dental calculus (Fig.4a). e size and shape of these
lumps that were oen intact and oen still embedded in the calculus matrix, can be compared to those charac-
terising species of many genera of the Poeae Tribe (oats tribe) in our reference collection such as Avena sterilis
L., Avena barbata Link., Avena fatua L. and Phalaris minor Retz., although other tribes of grasses growing in the
Adriatic region of the Balkans, the Sesleriinae (e.g. Sesleria albicans Kit., Sesleria caerulea L., Sesleria Ard., S. ten-
uifolia Schrad., etc.), could not be excluded (Fig.5a–e).
Type II. e second type of starch granules is characterised by a “bimodal distribution”, which has been shown
to be a characteristic of most grasses of the Triticeae tribe (Fig.4b). ese granules were found intact or with
some damage but with characteristics still visible (including lamalleae), lodged close together in the amyloplast
and calculus matrix. In addition, some charcoal akes still adhered to them, possibly from smoke in the envi-
ronment or from roasted food. e bimodal distribution in the archaeological granules was characterised by the
co-presence of large and small granules, respectively known as Type A and B. In the Vlakno calculus, the large
starch granules (ca. 23μm in diameter) were oval to round in 2D and lenticular in 3D. ey had a central hilum,
deep ssures and no visible lamellae. eir morphology is not completely intact, which is very likely due to some
form of processing and/or cooking as well as chewing. e small granules (≤10 μm in diameter) were spherical in
shape with a central hilum. Based on our previous work, to which we demand for a detailed discussion49, we high-
light the following points. We exclude the possibility that the archaeological granules belonged to the Aegilops
genus as they are mostly spherical and oval and lacked lamellae, which, on the contrary, are visible through the
whole starch body of granules belonging to the six species of the Aegilops genus represented in the ora of region
(Aegilops cylindrica (Host.) Ces., Aegilops geniculata Roth., Aegilops lorentii Hochst., Aegilops neglecta Req. ex
Bertol., Aegilops triuncialis L., and Aegilops uniaristata Vis.)57. Indeed, the high number of Type B small round
granules in our sample as well as the absence of clear lamellae on the body of Type A granules suggest that the
identied archaeological starch granules may belong to one of the wild species of barley (Hordeum spp.) (e.g.
Hordeum bulbosus L., Hordeum marinum Huds., Hordeum murinum L., Hordeum secalinum Schreb.), which are
commonly found and identied in the ora of the region58 (Fig.5f–h).
A variety of plant remains not consisting of starch granules were also found. Although a portion was too
damaged to be diagnostic, some micro-remains were attributed to a non-dietary origin. In particular, two wood
fragments with characteristic conifer tracheid bres were identied (Fig.4c, cf. Supplementary Information), and
were very similar to those retrieved in a previous study24, demonstrating that this kind of evidence does preserve
in calculus. Bast bres still embedded in the calculus matrix were also retrieved (Fig.6a). When observed under
polarised light, these bres possessed characteristics such as a narrow lumen and a dislocation band, which are
known to be a characteristic of plants used for their brosity in cordage and textiles (e.g. nettle, ax and hemp)59.
On the basis of our reference collection, the region and chronological context we suggest that the bres could
belong to nettles (Urtica spp.). Two damaged grains of conifer pollen were also identied, supporting the identi-
cation of the wood debris as that of conifer (Fig.6b).
Animal micro-remains. Three fragments of fish scale(s) were found lodged in situ in the Vlakno calcu-
lus (Fig.4d,e). Although incomplete, the characteristic radii (Fig.4d) and circuli (Fig.4e) were clearly visible
under polarized light. ey were all whitish in colour. While dental calculus fragments can sometimes visually
appear similar to a sh scale visually (as they form by apposition of the bacterial concretions) this possibility was
excluded by injecting additional HCl to the slide where the remains were found. By doing so, it was possible to
further free a part of the scale and observe the dissolution of the calculus adhering to it. us, excluding the possi-
bility that the observed structure was a sh scale and not a fragment of calculus. Despite our reference collection,
we were unable to identify the scale to the family or species taxonomic levels due to a lack of diagnostic features.
at being said, structurally the remains were very similar to the Scombridae (tunas, mackerel) and gilthead sea
bream scales (Fig.7a–c) from our experiments. In addition, at least two fragments of animal micro-debris were
also identied and interpreted as sh muscle bands on the basis of comparison with our experimental reference
Content courtesy of Springer Nature, terms of use apply. Rights reserved

www.nature.com/scientificreports/
6
SCIEnTIfIC REPORtS | (2018) 8:8147 | DOI:10.1038/s41598-018-26045-9
collection. ey were white-yellowish in colour and composed of W-shaped parallel bres oen distorted but still
adhering to the calculus matrix. To our knowledge, this is the rst time that fragments of sh scale(s) and muscle
bres have been identied in dental calculus.
Among the animal micro-fragments, numerous barbule fragments were found in the archaeological calculus
and tentatively attributed to the Anatidae family based on their shape, features and distance of their nodes (Fig.5c
and Supplementary Information).
Other micro-remains. Two comparable diatoms were recovered entrapped in the calculus matrix (Fig.4f). e
morphological characteristics observed at transmitted light indicate that both remains belong to Cocconeis pla-
centula (Ehrenb.) a very widely-distributed diatom found in almost all freshwater or brackish water where the
pH is circum-neutral or alkaline. C. placentula (Ehrenb.) is very common in benthic habitats, where it attaches to
rocks, macrophytes and algae. It is a fast-growing, pioneer species that is able to colonise bare substrates quickly.
Although C. placentula (Ehrenb.) is found throughout the year, such the diatom is most abundant in rivers in the
summer where it can form 80–90% of all individuals present in an epilithic sample.
Stable isotope analysis. During the AMS radiocarbon (14C) dating, the δ13C and δ15N stable isotope val-
ues of −16.1 and 13.0‰ respectively were obtained by the Oxford Radiocarbon Accelerator Unit whilst a δ13C
Figure 4. Plant and animal micro-residues identied in Vlakno tartar: (a) Lumps of starch granules still
embedded in the calculus matrix; (b) Starch granules with bimodal distribution; (c) Conifer wood bre;
(d) Fragment of sh scale entrapped in calculus matrix; (e) Fragment of sh esh; (f) C. placentula diatom.
Content courtesy of Springer Nature, terms of use apply. Rights reserved

www.nature.com/scientificreports/
7
SCIEnTIfIC REPORtS | (2018) 8:8147 | DOI:10.1038/s41598-018-26045-9
isotope value of −16.6‰ was obtained by Beta Analytical. e δ13C values are presently some of the highest
obtained from human bone collagen dating to the Mesolithic in the Mediterranean basin14,15,17, and suggest a
signicant contribution of marine derived protein. Using a rudimentary linear model60 we have estimated that
the individual acquired protein of the order of 46.1% from marine organisms. Moreover, the δ15N is the highest
in the region14,15,17 and suggests that the foods consumed were rich in protein. In agreement with Mannino et al.15
marine organisms higher up in the trophic level hierarchy are likely to have been consumed although terrestrial
animals should not be ignored. In order to obtain a dietary baseline for the human interred at Vlakno, we also
analysed three dierent terrestrial mammal species, red deer, hare (Lepus sp.) and red fox. ese data are listed in
Figure 5. Modern starch granules used as reference: (a) Avena sterilis; (b) Avena fatua; (c) Sesleria cerulea;
(d) Phalaris minor; (e) Phalaris paradoxa; (f) Hordeum marinum; (g) Hordeum bulbosum; (h) Hordeum secalinum.
Figure 6. Non-dietary micro-debris identied in archaeological tartar: (a) Nettle bre; (b) Conifer pollen;
(c) Fragment of feather barbule.
Figure 7. Experimental sh tissues: (a) Scale fragment from a gilthead sea bream; (b) and (c) Fragments of
Atlantic mackerel esh.
Content courtesy of Springer Nature, terms of use apply. Rights reserved

www.nature.com/scientificreports/
8
SCIEnTIfIC REPORtS | (2018) 8:8147 | DOI:10.1038/s41598-018-26045-9
Table1 and presented in Fig.8. e δ13C and δ15N stable isotope values were within the range of values obtained
previously from Croatia27.
Discussion
In the last 15 years, a growing body of evidence has signicantly contributed to our understanding of dietary
habits in pre-agrarian societies of the Mediterranean basin. In light of this, the role of marine resources and plant
foods still remains unclear due to various methodological hindrances: a focus on protein-sensitive methods, the
poor preservation of plant and sh remains, and the lack of systematic recovery techniques for organic residues.
is ambiguity is particularly signicant for forager groups who lived in close proximity to the coast or on the
islands of the Central Mediterranean7,61–63.
At the beginning of the Holocene, a wide range of available vegetal resources became available due to the newly
established environmental conditions in the Eastern Adriatic region64–66. While the recovery of two dierent
group of plants from one individual is suggestive that carbohydrate-rich resources were  apart of the dietary hab-
its of the Mesolithic forager from Vlakno, understanding how  dierent plant foods contributed to theMesolithic
diet of theVlakno group is challenging as no direct correlation can still be drawn between the presence of plant
Sample Context Species Element δ13C VPDB δ15N AIR C:N atomic ratio
Beta-311088 Burial 1 Homo sapiens Phalanx −16.6 n.d. n.d.
OxA-34518 Ulna, dex. −16.1 13.0 3.2
1A
Sector East, sq. 15, context 14 Cervus elaphus Metatarsus
−20.5 5.2 3.3
1B −20.3 5.3 3.3
1 Mean −20.4 5.2 3.3
2A
Sector East, sq. I5, context 14 Cervus elaphus Phalanx I
−20.8 4.3 3.3
2B −20.7 4.2 3.3
2 Mean −20.8 4.3 3.3
3A
Sector East, sq. I5, context 14 Vulpes vulpes Radius
−20.6 6.6 3.6
3B −20.8 7.2 3.6
3 Mean −20.7 6.9 3.6
4A
Sector East, sq. I5, context 14 Vulpes vulpes Femur
−19.3 7.4 3.3
4B −19.4 7.5 3.3
4 Mean −19.3 7.5 3.3
5A
Sector East, sq. I5, context 14 Lepus s p. Tibia
−22.0 3.2 3.4
5B −21.8 3.2 3.4
5 Mean −21.9 3.2 3.4
Table 1. Stable isotope data obtained on the human bone collagen as well as those data obtained from the three
terrestrial species (that were measured in duplicate) from the Mesolithic levels of Vlakno Cave. Key: n.d., no data.
Figure 8. Stable isotope analysis. (A) δ13C and δ15N stable isotope data obtained on human and mammalian
bone collagen from Vlakno (squares) compared with data from Mesolithic human and faunal remains (circles)
throughout Croatia (data from)16,27; (B) δ13C and δ15N stable isotope data obtained on human bone collagen
from Vlakno (square) compared with data from 66 Mesolithic and/or Mesolithic-Neolithic humans (circles)
from Corsica (green), Croatia (red), Sicily (blue) and Spain (orange) (aer)10–17,25–27,61,76. Note that the plotted
data represents samples that were securely assigned to the Mesolithic, which also had a C:N atomic ratio
between 2.9–3.677.
Content courtesy of Springer Nature, terms of use apply. Rights reserved

www.nature.com/scientificreports/
9
SCIEnTIfIC REPORtS | (2018) 8:8147 | DOI:10.1038/s41598-018-26045-9
micro-remains in ancient calculus, the quantity of plant foods that entered the diet and the moment those were
consumed by the individual23. Yet, botanical microfossils recovered in the archaeologicaldental calculus seem to
strongly suggest dierent species of plants were selected and consumed by the individual and potentially local
foragers as a part of their diet and culinary tradition. Besides the botanical remains trapped in Vlakno calculus,
direct archaeological evidence for the consumption of plant foods in the Adriatic region during the Mesolithic
is restricted to the site of Vrbička Cave in Montenegro where fragments of hazelnut shells were recovered from
the Late Mesolithic layers aer extensive otation67. To the south, the remains of pears and a few peas since ca.
7300 cal BC and wild oats and barley aer ca. 7000 cal BC are known from the site of Franchti Cave in Greece68.
On the other side of the Adriatic, oats (Avena sp.) have been consumed since the Middle Upper Palaeolithic. At
the site of Apulia in Italy, starch granules and phytoliths have been identied on Gravettian ground stone tools
at the cave site of Paglicci69. During the Early Holocene data concerning the consumption of plant resources is
derived from Grotta di Pozzo in the Fucino Basin of Abruzzo where carpological remains of bearberry, cornelian
cherry and wild grapes were recovered70. Lastly, the possible consumption of plant foods is also available for the
Mesolithic foragers at Grotta dell’Uzzo in Sicily based on the stable isotope data13,14.
While animal micro-residues have rarely been found in the course of dental calculus microfossil analysis, the
analysis of the Vlakno individual yielded the exceptionally well-preserved fragments of sh esh and scales. is
was possible due to the anatomy of the sh scale, which may have provided an ideal substrate for the bacterial
to grow over and above it, as demonstrated by the need of adding further HCl to the slide to free the scale to
permit identication. Such evidence is unique and emphasises the potential of ancient tartar to capture other
lines of evidence than plant remains. While we cannot condently conrm when esh and scale(s) fragments
were entrapped in the tartar, the abundance of sh remains in the Mesolithic layers of Vlakno as well as the
stable isotope data demonstrate that marine resources were regularly consumed by the individual; alternatively,
de-scaling activities using teeth may have taken place. Tartar formation depends on dierent factors and can vary
from one individual to another. While it was not possible to identify the scale to the family or species taxonomic
levels, abundant Atlantic chub mackerel (Scomber colias Gmelin.), gilthead sea bream and cuttlesh remains have
been recovered in the Mesolithic layers of Vlakno cave34. e abundance of sh remains from the Mesolithic
layers at the site demonstrates that Vlakno cave may have been used seasonally for shing5. Not far from Vlakno
cave, the site of Vela Spila on the Island of Korčula yielded numerous sh remains30. e assemblage was dom-
inated by the cf.Atlantic chub mackerel (Scomber colias), and it was suggested that the specialised capture and
processing of this species took place during the Mesolithic and Neolithic periods. Although no comparison can
be made between the numerous sh remains recovered from Mesolithic sites in the westernmost part of the
Mediterranean71 or Northern Europe and the Atlantic72, at Vela Spila nearly half a ton of Atlantic chub mackerel
was processed during the Early Mesolithic30. Moreover, additional evidence relating to shing in the Central
Mediterranean derives from Sicily where at Grotta dell’Uzzo and Grotta d’Oriente the archaezoological remains
document an increase in the exploitation of sh throughout the Mesolithic and across the Mesolithic-Neolithic
transition73.
e stable isotope values obtained from the Vlakno individual corroborate the data obtained from the analy-
ses of the dental calculus and animal remains. As such these data demonstrate that protein-rich foods (probably
marine resources alongside C3 terrestrial animals) were consumed. Previous stable isotope analyses in the Eastern
Adriatic region suggests that terrestrial resources, particularly red deer and wild boar (Sus scrofa L.), were prefer-
entially consumed during the Mesolithic even at coastal localities15,27. ere are only a few examples that demon-
strate the consumption of marine derived protein, a long bone from El Collado (δ13C = –17.6‰, δ15N = 12.8‰),
and acranium from the site of Grotta dell’Uzzo (δ13C = –16.2‰, δ15N = 12.8‰)15,26. Whilst the current sample
size is based on one individual and possibly unrepresentative, it diers from the previously noted Mediterranean
Basin-wide trend of predominately terrestrial-based diets during the Mesolithic74,75, and which was recently also
emphasised for the Mesolithic of Sardinia, Sicily and the Egadi islands9,12,14.
Microfossils below 20μm in size, demonstrate the potential use of plant bres for non-dietary purposes, (e.g.
the masticatory apparatus). For instance, portions of wood fragments and xylem tissue are not edible parts of
plants, as noted previously24. Both bast bres and wood are potentially the remains resulting from non-dietary
activities, which may have included holding, soening or shredding plant bres for cordage, textile, basketry,
net-making and the use of tooth picks, wooden culinary tools, the exposure to smoke from replaces as well
as dust generated while plucking birds or other cras. e use of teeth as a “third hand” in para-masticatory
activities and oral hygiene may also be possible explanations for the presence of non-alimentary dust in the
tartar. Diatoms were also identied in the calculus matrix, which may have derived from a freshwater source in
the vicinity of the cave, which the individual had exploited. e recovery of these micro-residues in the calculus
matrix conrms the potential of tartar to act as an archaeological repository of environmental as well as occupa-
tional debris48.
Conclusions
is contribution details an osteobiography of an adult male individual from a lone Mesolithic burial found in
Vlakno Cave on the Island of Dugi Otok in the Croatian archipelago. As our data refer to a single individual
they are not expected to provide a comprehensive picture of dietary habits of the period and region. However,
our presentation of dietary information available for this individual based on the study of dental calculus along
with stable isotope data, and contextualised with the existing archaeological data, provides a signicant insight
into the lifeway’s of Adriatic and Mediterranean Holocene foragers. Methodologically, we have also shown the
potential of dental calculus to provide not only plant-related evidence but also to contribute to the reconstruc-
tion of non-dietary related practices of the everyday. By integrating two dierent strands of data—the analysis
of microfossils entombed in dental calculus and the stable isotope analysis of bone collagen—we were able to
document how carbohydrate-rich plants and marine resources contributed to Mesolithic food ways. While the
Content courtesy of Springer Nature, terms of use apply. Rights reserved

www.nature.com/scientificreports/
10
SCIEnTIfIC REPORtS | (2018) 8:8147 | DOI:10.1038/s41598-018-26045-9
data presented in this study remains limited, it potentially demonstrates that Mesolithic dietary strategies in the
Eastern Adriatic may have diered from areas in the central and western parts of the basin.
Statement. No experiments on live vertebrats have been performed for the study. All methods reported in
the paper were carried out in accordance with relevant guidelines and regulations.
References
1. Vigne, J.-D. & Desse-Berset, N. e exploitation of animal resources in the Mediterramean Islands during the Pre-Neolithic: the
example of Corsica. (Oxbow Boos, 1995).
2. Čeču, B. & adić, D. Vela spila: višeslojno pretpovijesno nalazište; Vela Luka-otok Korčula. (Centar za ulturu ‘Vela Lua’, 2005).
3. Martini, F. in La cultura del morire nelle società prei storiche e protostoriche italiane. Studio interdisciplinare dei dati e loro trattamento
informatico. (ed. Martini, F.) 3, 67–86 (Origines, Progetti, 2006).
4. adić, D. Vela spila: preliminarna analiza starijeneolitičih i mezolitičih naslaga iz sonde istražene 2004. godine. Opusc. Archaeol.
Rad. Arheol. zavoda 29, 323–348 (2005).
5. Vujević, D. & Bodružić, M. Mesolithic communities of Vlano cave. Diadora Glas. Arheol. muzeja u Zadru 26, 9–30 (2013).
6. omšo, D. Mezoliti u Hrvatsoj. Opusc. Archaeol. Rad. Arheol. zavoda 30, 55–92 (2008).
7. Mannino, M. & ichards, M. e role of aquatic resources in ‘Italian’ hunter-gatherer subsistence and diets. in Palaeolithic Italy.
Advanced studies on early human adaptation in the Apennine Peninsula (eds Borgia, V. & Cristiani, E.) (Sidestone Press).
8. Courtaud, P., Petersen, H. C., Zemour, A., Leandri, F. & Cesari, J. e Mesolithic burial of Campu Stefanu (Corsica, France). in
Mesolithic Burials-Rites, symbols and social organisation of early postglacial communities 719–731 (2016).
9. Floris, ., Melis, . T., Mussi, M., Palombo, M. . & Iacumin, P. La presenza umana nella Sardegna centro occidentale durante
l’Olocene antico: il sito di S’Omu e S’Oru, Arbus, VS. Atti della XLIV Riun. Sci. la Preist. e la protostoria della Sardegna Cagliari,
Barumini, Sassari 23-28 novembre 2009, vol. 2–3 Comun. vol. 4 Posters. 999–1004 (2012).
10. de Pablo, J. F.-L. et al. Late Mesolithic burials at Casa Corona (Villena, Spain): direct radiocarbon and palaeodietary evidence of the
last forager populations in Eastern Iberia. J. Archaeol. Sci. 40, 671–680 (2013).
11. Goude, G. et al. New Insights into Mesolithic Human Diet in the Mediterranean from Stable IsotopeAnalysis: e Sites of Campu
Stefanu and Torre d’Aquila, Corsica. Int. J. Osteoarchaeol. 27, 707–714 (2017).
12. Mannino, M. A. et al. Upper Palaeolithic hunter-gatherer subsistence in Mediterranean coastal environments: an isotopic study of
the diets of the earliest directly-dated humans from Sicily. J. Archaeol. Sci. 38, 3094–3100 (2011).
13. Mannino, M. A., omas, . D., Leng, M. J., Di Salvo, . & ichards, M. P. Stuc to the shore? Investigating prehistoric hunter-
gatherer subsistence, mobility and territoriality in a Mediterranean coastal landscape through isotope analyses on marine mollusc
shell carbonates and human bone collagen. Quat. Int. 244, 88–104 (2011).
14. Mannino, M. A. et al. Origin and diet of the prehistoric hunter-gatherers on the Mediterranean island of Favignana (Ègadi Islands,
Sicily). PLoS One 7, e49802 (2012).
15. Mannino, M. A. et al. Climate-driven environmental changes around 8,200 years ago favoured increases in cetacean strandings and
Mediterranean hunter-gatherers exploited them. Sci. Rep. 5, 16288 (2015).
16. Paine, C., O’Connell, T. & Miracle, P. T. Stable isotopic reconstruction of Early Mesolithic diet at Pupicina Cave. Mesolith. Horizons.
Oxford Oxbow Books 210–216 (2009).
17. Salazar-Garcia, D. C. et al. Isotope evidence for the use of marine resources in the Eastern Iberian Mesolithic. J. Archaeol. Sci. 42,
231–240 (2014).
18. Fischer, A. et al. Coast–inland mobility and diet in the Danish Mesolithic and Neolithic: evidence from stable isotope values of
humans and dogs. J. Archaeol. Sci. 34, 2125–2150 (2007).
19. ichards, M. P. & Hedges, . E. M. Stable isotope evidence for similarities in the types of marine foods used by Late Mesolithic
humans at sites along the Atlantic coast of Europe. J. Archaeol. Sci. 26, 717–722 (1999).
20. Hardy, . et al. Dental calculus reveals potential respiratory irritants and ingestion of essential plant-based nutrients at Lower
Palaeolithic Qesem Cave Israel. Quat. Int. 398, 129–135 (2016).
21. adini, A., Niita, E. & Shillito, L. M. Human dental calculus and a medieval urban environment. Everyday life Mediev. Eur. Environ.
artefactual approaches to D well. T. country. Belgium Brepols. https://doi.org/10.1073/pnas1018116108 (2016).
22. Bucley, S., Usai, D., Jaob, T., adini, A. & Hardy, . Dental calculus reveals unique insights into food items, cooing and plant
processing in prehistoric central Sudan. PLoS One 9, e100808 (2014).
23. Leonard, C., Vashro, L., O’Connell, J. F. & Henry, A. G. Plant microremains in dental calculus as a record of plant consumption: A
test with Twe forager-horticulturalists. J. Archaeol. Sci. Reports 2, 449–457 (2015).
24. adini, A. et al. Neanderthals, trees and dental calculus: new evidence from El Sidrón. Antiquity 90, 290–301 (2016).
25. Francalacci, P. Comparison of archaeological, trace element, and stable isotope data from two Italian coastal sites. Riv. di Antropol.
66, 239–250 (1988).
26. Garcia Guix, E., ichards, M. & Subir, M. E. Palaeodiets of humans and fauna at the Spanish Mesolithic site of El Collado. Cu rr.
Anthropol. 47, 549–556 (2006).
27. Lightfoot, E., Boneva, B., Miracle, P. T., Šlaus, M. & O’connell, T. C. Exploring the Mesolithic and Neolithic transition in Croatia
through isotopic investigations. Antiquity 85, 73–86 (2011).
28. Miracle, P. & Forenbaher, S. Prehistoric herders of northern Istria: the archaeology of Pupićina cave/Pretpovijesni stočari sjeverne
Istre: arheologija Pupićine peći. (2006).
29. Miracle, P., Galanidou, N. & Forenbaher, S. Pioneers in the hills: early Mesolithic foragers at Šebrn Abri (Istria, Croatia). Eur. J.
Archaeol. 3, 293–329 (2000).
30. ainsford, C. & O’Connor, T. & Miracle, P. Fishing in the Adriatic at the Mesolithic–Neolithic transition: Evidence from Vela Spila,
Croatia. Environ. Archaeol. 19, 311–320 (2014).
31. Via, E. & eodoropoulou, T. e-investigating sh consumption in Gree antiquity: results from δ 13 C and δ 15 N analysis from
sh bone collagen. J. Archaeol. Sci. 39, 1618–1627 (2012).
32. Brusić, Z. P. V. (Vlano Cave). Hrvat. Arheol. godišnjak 1, 197–199 (2005).
33. Cvitušić, B., adović, S. & Vujević, D. Changes in ornamental traditions and subsistence strategies during the Palaeolithic-
Mesolithic transition in Vlano cave. Quat. Int. (2017).
34. adović, S., Spy-Marquez, P. & Vujević, D. A tale of foxes and deer or how people changed their eating habitats during the Mesolithic
al Vlano cave (Croatia). in Holocene Foragers in Europe and Beyond (Papers Presented the Ninth International Conference on the
Mesolithic in Europe MESO 2015, Belgrade, Serbia) (eds Borić, D., Antonović, D., Mihailović, B. & Stefanović, S.) (Serbian
Archaeological Society, 2015).
35. Surić, M. Maša Surić, Promjene u oolišu tijeom mlađeg pleistocena i holocena – zapisi iz morem potopljenih siga istočnog Jadrana
(disertacija). (University of Zagreb, 2006).
36. Ortner, D. J. Identication of pathological conditions in human skeletal remains. (Academic Press, 2003).
37. Aufderheide, A. C., odriguez-Martin, C. & Langsjoen, O. e Cambridge encyclopedia of human paleopathology. 478, (Cambridge
University Press Cambridge, 1998).
Content courtesy of Springer Nature, terms of use apply. Rights reserved

www.nature.com/scientificreports/
11
SCIEnTIfIC REPORtS | (2018) 8:8147 | DOI:10.1038/s41598-018-26045-9
38. eimer, P. J. et al. IntCal13 and Marine13 radiocarbon age calibration curves 0–50,000 years cal BP. Radiocarbon 55, 1869–1887
(2013).
39. Faivre, S., Baran-Petricioli, T., Barešić, J. & Horvatinčić, N. New data on marine radiocarbon reservoir eect in the Eastern Adriatic
based on pre-bomb marine organisms from the intertidal zone and shallow sea. Radiocarbon 57, 527–538 (2015).
40. Vujević, D. & Bodružić, M. In Holocene Foragers in Europe and Beyond (Papers Presented the Ninth International Conference on the
Mesolithic in EuropeMESO 2015, Belgrade, Serbia)(eds Borić, D., Antonović, D., Mihailovic, D. & Stefanović, S.) (Belgrade: Serbian
Archaeological Society).
41. Lieverse, A. . Diet and the aetiology of dental calculus. Int. J. Osteoarchaeol. 9, 219–232 (1999).
42. Cowley, G. & MacPhee, T. Essentials of Periodontology and Periodontics (Blacwell, 1975).
43. Hardy, . et al. Starch granules, dental calculus and new perspectives on ancient diet. J. Archaeol. Sci. 36, 248–255 (2009).
44. Warinner, C. et al. Pathogens and host immunity in the ancient human oral cavity. Nat. Genet. 46, 336–344 (2014).
45. Blatt, S. H., edmond, B. G., Cassman, V. & Sciulli, P. W. Dirty teeth and ancient trade: evidence of cotton bres in human dental
calculus from Late Woodland, Ohio. Int. J. Osteoarchaeol. 21, 669–678 (2011).
46. Blondiaux, J. & Charlier, P. Palaeocytology in seletal remains: microscopic examination of putrefaction uid deposits and dental
calculus of seletal remains from French archaeological sites. Int. J. Osteoarchaeol. 18, 1–10 (2008).
47. Hardy, . et al. Neanderthal medics? Evidence for food, cooing, and medicinal plants entrapped in dental calculus.
Naturwissenschaen 99, 617–626 (2012).
48. adini, A., Niita, E., Bucley, S., Copeland, L. & Hardy, . Beyond food: e multiple pathways for inclusion of materials into
ancient dental calculus. Am. J. Phys. Anthropol. 162, 71–83 (2017).
49. Cristiani, E., adini, A., Edinborough, M. & Borić, D. Dental calculus reveals Mesolithic foragers in the Balans consumed
domesticated plant foods. Proc. Natl. Acad. Sci. 113, 10298–10303 (2016).
50. Lucarini, G., adini, A., Barton, H. & Barer, G. e exploitation of wild plants in Neolithic North Africa. Use-wear and residue
analysis on non-napped stone tools from the Haua Fteah cave, Cyrenaica, Libya. Quat. Int. 410, 77–92 (2016).
51. Madella, M., L ancelotti, C. & Garcia-Granero, J. J. Millet microremains:an alternative approach to understand cultivation and use of
critical crops in Prehistory. Archaeol. Anthropol. Sci. 8, 17–28 (2016).
52. ichards, M. P., Fuller, B. T., Sponheimer, M., obinson, T. & Aylie, L. Sulphur isotope measurements in archaeological samples:
some methodological considerations. Int J Osteoarchaeol 13, 37–45 (2003).
53. rueger, H. W. & Sullivan, C. H. In Stable Isotopes in Nutrition. ACS Symposium Series 258 (eds Turnlund, J. & Johnson, P.) 205–222
(ACS Publications, 1984).
54. Müldner, G. & ichards, M. P. Fast or feast: reconstructing diet in later medieval England by stable isotope analysis. J. Archaeol. Sci.
32, 39–48 (2005).
55. Schoeninger, M. J. & DeNiro, M. J. Carbon isotope ratios of apatite from fossil bone cannot be used to reconstruct diets of animals.
Nature 297, 577–578 (1982).
56. Torrance, . & Barton, H. Ancient Starch Research. (Le Coast Press, 2007).
57. Niolić, T. Flora Croatica Database. Department of Botany, Faculty of Science, University of Zagreb (2008).
58. Ljustjna, M. & Vitas, B. Zbornik istrazivackih radova Udruge studenata - Biologije - ‘BIUS’ u Parku prirode Telascica. (BIUS, 2002).
59. Bergord, C. & Holst, B. A procedure for identifying textile bast bres using microscopy: Flax, nettle/ramie, hemp and jute.
Ultramicroscopy 110, 1192–1197 (2010).
60. Arneborg, J. et al. Change of diet of the Greenland Viings determined from stable carbon isotope analysis and 14C dating of their
bones. Radiocarbon 41, 157–168 (1999).
61. Bocherens, H. Neanderthal dietary habits: review of the isotopic evidence. Evol. Hominin diets 241–250 (2009).
62. Jones, M. Moving north: archaeobotanical evidence for plant diet in Middle and Upper PaleolithicEurope. Evol. Hominin Diets
171–180 (2009).
63. ichards, M. P. 20. Stable isotope evidence for European Upper Paleolithic human diets. Hublin, J.-J., Richards, MP(Eds.), Evol.
Hominid Diets Integr. Approaches to Study Palaeolithic Subsist. Vertebr. Paleobiol. Paleoanthropology. Springer, Dordr. 251–257
(2009).
64. Andrič, M. et al. Late quaternary vegetation and hydrological change at Ljubljanso barje (Slovenia). Palaeogeogr. Palaeoclimatol.
Palaeoecol. 270, 150–165 (2008).
65. Favaretto, S., Asioli, A., Miola, A. & Piva, A. Preboreal climatic oscillations recorded by pollen and foraminifera in the southern
Adriatic Sea. Quat. Int. 190, 89–102 (2008).
66. ossignol-Stric, M. e Holocene climatic optimum and pollen records of sapropel 1 in the eastern Mediterranean, 9000–6000BP.
Quat. Sci. Rev. 18, 515–530 (1999).
67. Borić, D. et al. In Holocene Foragers in Europe and Beyond (Papers Presented the Ninth International Conference on the Mesolithic in
EuropeMESO 2015, Belgrade, Serbia(eds Borić, D., Antonović, D., Mihailovic, D. & Stefanović, S.) (Belgrade: Serbian Archaeological
Society).
68. Hansen, J. & enfrew, J. M. Palaeolithic–neolithic seed remains at Franchthi Cave, Greece. Nature 271, 349–352 (1978).
69. Lippi, M. M., Foggi, B., Aranguren, B., onchitelli, A. & evedin, A. Multistep food plant processing at Grotta Paglicci (Southern
Italy) around 32,600cal BP. Proc. Natl. Acad. Sci. 112, 12075–12080 (2015).
70. Lubell, D. et al. Exploitation of seasonal resources in the mountains of Abruzzo (central Italy): Epigravettian to Neolithic. L’ Eu ro p e
des derniers Chass. Epipaléolithique Mésolithique 5, 465–472 (1999).
71. Morales-Muñiz, A. & oselló-Izquierdo, E. Twenty thousand years of shing in the Strait. Hum. Impacts Anc. Mar. Ecosyst. A Glob.
Perspect. Ed. by Rick, TC Erlandson, JM, Univ. Calif. Press. Berkeley 243–277 (2008).
72. Engho, I. B. Regionality and biotope exploitation in Danish Ertebølle and adjoining periods. (Det ongelige Danse Vidensabernes
Selsab, 2011).
73. Tagliacozzo, A. Archeozoologia della Grotta dell’Uzzo, Sicilia: da un’economia di pesca ed allevamento. (Ist. poligraco e zecca dello
stato, 1993).
74. Schulting, . Sweet or salty? Isotopic evidence for the use of aquatic resources in Mesolithic Europe (2015).
75. Schulting, . & Borić, D. in e Neolithic of Europe: Papers in Honour of Alasdair Whittle (eds Bicle, P., Cummings, V., Hofmann,
D. & Pollard, J.) 82–105 (Oxbow Boos, 2017).
76. Pouydebat, E. Approche biogéochimique de l’alimentation humaine dans le site prénéolithique du Monte Leone (VIIIe millénaire av
J.-C.; Bonifacio Corse-du-Sud). Univ. Paris I , Paris (1997).
77. DeNiro, M. J. Postmortem preservation and alteration of in vivo bone collagen isotope ratios in relation to palaeodietary
reconstruction. Nature 317, 806–809 (1985).
Acknowledgements
We warmly thank Nicola Angeli (MUSE, Trento) for the characterisation of diatoms recovered in ancient tartar
and Dragana Filipović for productive conversations on wild Triticeae species in Croatia. E.C. is mostly grateful to
Renata Šostarić for she provided key information on the local ora and vegetation in Dalmatia. We acknowledge
funding received for this project through the European Research Council (ERC Starting Grant Project HIDDEN
FOODS, G.A. no. 639286 to E.C.). H.K.R. should like to thank Rick Schulting, Chris Meiklejohn and André Carlo
Content courtesy of Springer Nature, terms of use apply. Rights reserved

www.nature.com/scientificreports/
12
SCIEnTIfIC REPORtS | (2018) 8:8147 | DOI:10.1038/s41598-018-26045-9
Colonese for advice and the British Academy for funding. Research at Vlakno cave is funded through the project
of Croatian Science foundation (UIP 2014-09-1545, VLAKNO).
Author Contributions
E.C. conceived and planned the project. E.C., A.R. wrote the paper and performed analytical work and data
analysis on dental calculus. D.B. and H.K.R. performed the stable isotope analysis and interpreted the data; H.K.R.
compiled the Supplementary Information. E.C. and G.M. directed and executed the sampling of archaeological
materials, D.V. directed excavations, provided insights into the study region and the site. M.S. contributed on the
osteobiography of the Mesolithic individual. E.C. produced Figures 1–7. H.K.R. produced Figure 8. e satellite
imagery in Figure 1 was obtained from Natural Earth, http://www.naturalearthdata.com/ and the mapping
soware used was MapServer, http://mapserver.orgvia PHP MapScript, which was created by A.Z. All authors
read, revised and approved the nal version of the manuscript.
Additional Information
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-26045-9.
Competing Interests: e authors declare no competing interests.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional aliations.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre-
ative Commons license, and indicate if changes were made. e images or other third party material in this
article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the
material. If material is not included in the article’s Creative Commons license and your intended use is not per-
mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the
copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
© e Author(s) 2018
Content courtesy of Springer Nature, terms of use apply. Rights reserved

1.
2.
3.
4.
5.
6.
Terms and Conditions
Springer Nature journal content, brought to you courtesy of Springer Nature Customer Service Center GmbH (“Springer Nature”).
Springer Nature supports a reasonable amount of sharing of research papers by authors, subscribers and authorised users (“Users”), for small-
scale personal, non-commercial use provided that all copyright, trade and service marks and other proprietary notices are maintained. By
accessing, sharing, receiving or otherwise using the Springer Nature journal content you agree to these terms of use (“Terms”). For these
purposes, Springer Nature considers academic use (by researchers and students) to be non-commercial.
These Terms are supplementary and will apply in addition to any applicable website terms and conditions, a relevant site licence or a personal
subscription. These Terms will prevail over any conflict or ambiguity with regards to the relevant terms, a site licence or a personal subscription
(to the extent of the conflict or ambiguity only). For Creative Commons-licensed articles, the terms of the Creative Commons license used will
apply.
We collect and use personal data to provide access to the Springer Nature journal content. We may also use these personal data internally within
ResearchGate and Springer Nature and as agreed share it, in an anonymised way, for purposes of tracking, analysis and reporting. We will not
otherwise disclose your personal data outside the ResearchGate or the Springer Nature group of companies unless we have your permission as
detailed in the Privacy Policy.
While Users may use the Springer Nature journal content for small scale, personal non-commercial use, it is important to note that Users may
not:
use such content for the purpose of providing other users with access on a regular or large scale basis or as a means to circumvent access
control;
use such content where to do so would be considered a criminal or statutory offence in any jurisdiction, or gives rise to civil liability, or is
otherwise unlawful;
falsely or misleadingly imply or suggest endorsement, approval , sponsorship, or association unless explicitly agreed to by Springer Nature in
writing;
use bots or other automated methods to access the content or redirect messages
override any security feature or exclusionary protocol; or
share the content in order to create substitute for Springer Nature products or services or a systematic database of Springer Nature journal
content.
In line with the restriction against commercial use, Springer Nature does not permit the creation of a product or service that creates revenue,
royalties, rent or income from our content or its inclusion as part of a paid for service or for other commercial gain. Springer Nature journal
content cannot be used for inter-library loans and librarians may not upload Springer Nature journal content on a large scale into their, or any
other, institutional repository.
These terms of use are reviewed regularly and may be amended at any time. Springer Nature is not obligated to publish any information or
content on this website and may remove it or features or functionality at our sole discretion, at any time with or without notice. Springer Nature
may revoke this licence to you at any time and remove access to any copies of the Springer Nature journal content which have been saved.
To the fullest extent permitted by law, Springer Nature makes no warranties, representations or guarantees to Users, either express or implied
with respect to the Springer nature journal content and all parties disclaim and waive any implied warranties or warranties imposed by law,
including merchantability or fitness for any particular purpose.
Please note that these rights do not automatically extend to content, data or other material published by Springer Nature that may be licensed
from third parties.
If you would like to use or distribute our Springer Nature journal content to a wider audience or on a regular basis or in any other manner not
expressly permitted by these Terms, please contact Springer Nature at
onlineservice@springernature.com