New method for the isotopic study of ancient conchiolin from archaeological shells of freshwater mussels (Unionoida)

doi:10.21203/rs.3.rs-2313604/v2

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Research Article

New method for the isotopic study of ancient conchiolin from archaeological shells of freshwater mussels (Unionoida)

https://doi.org/10.21203/rs.3.rs-2313604/v2

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published 18 Oct, 2024

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Freshwater mussels shells are common remains in archaeological sites of the Gumelnița culture (5^th millennium BC, Romania), and were part of the diet of its ancient inhabitants. The proteins of these shells are often preserved and can be used for paleodietary and paleoecosystem reconstructions by stable isotopes.

To obtain the empirical relationship between the isotopic composition of sell organic matrix and soft tissues, we analysed the body (foot and mantle), the muscle, the conchiolin of the shell and stomach contents of modern individuals of Unio tumidus from the Danube. In addition, modern shells were digested in 5M HCl and archaeological in 1M HCl to obtain the shell organic matrix or conchiolin.

We established a quality criterion for freshwater mussel shell conchiolin of 3.6 (± 0.3) for the C:N and obtained an offset of Δ¹⁵N_{conchiolin-defatted body}= +0.95‰, Δ¹³C_{conchiolin-defatted body} = +0.93‰ for the soft tissues, and an offset of Δ¹⁵N_{conchiolin-muscle}= +1.7‰, Δ¹³C_{conchiolin-muscle}= +0.3‰ for the muscle.

Freshwater mussels from Gumelnița showed that they came from different sources (rivers, lakes, ponds…) but which not necessarily have to come from long distances since all these different habitats were present in the vicinity of the site.

stable isotopes

freshwater mussels

Unio tumidus

conchiolin

Romania

Eneolithic

From the earliest times, molluscs have been an essential food source for fish, birds, mammals other invertebrates, and humans. Freshwater molluscs can live in a wide range of freshwater habitats. They belong to two main groups, Bivalves and Gastropods. Freshwater bivalves represent about 6% of the total freshwater mollusc in Europe (Cuttelod et al., 2011). They are divided into two orders: Unionoida (freshwater mussels or naiads) and Veneroida (clams and peaclams), and possess a shared suite of adaptations to life in freshwater, like larval brooding, direct development, and, in the case of freshwater mussels, obligate larval parasitism upon freshwater fishes (Araujo et al., 2009). Freshwater mussels are among the most threatened fauna, globally in decline (Strayer 2008), and particularly vulnerable to habitat loss and fragmentation, changes in flow regimes, pollution, climatic disturbances and introduction of invasive species (Strayer et al., 2004).

Seven species of naiads have been identified in Romania (Glöer & Sîrbu, 2005; Sîrbu et al., 2010): painter mussel (Unio pictorum Linnaeus, 1758), swollen river mussel (Unio tumidus Philipsson, 1788), thick-shelled river mussel (Unio crassus Lamarck, 1819), swan mussel (Anodonta cygnea Linnaeus, 1758), duck mussel (Anodonta anatine Linnaeus, 1758), Chinese pond mussel (Sinanodonta woodiana Lea, 1834) and depressed river mussel (Pseudanodonta complanata Rossmässler, 1835). Of this species, S. woodiana is an invasive species in Romania (Popa & Murariu, 2009). Following the European Red List of Non-marine Molluscs (Cuttelod et al., 2011) the most endangered species is U. crassus with vulnerable status, followed by A. cygnea and P. complanata which are near threatened. U. pictorum, U. tumidus and A. anatine are least concern.

1.1. Naiads ecology

Concerning the ecology, U. crassus, U. pictorum and U. tumidus live in rivers and lakes (Table 1), but U. crassus is a rheophilic water species that prefer moving waters (rivers, streams), but can be also found in lakes permanently supplied by rivers. U. pictorum and U. tumidus are slow-moving water species that prefer river branches and lakes. The genus Anodonta is stagnant water species that prefer standing water bodies (ponds, shallow waters with a frequently muddy substratum), but can also be found in tributary branches with low current. Anodonta is the sole genera that resist well at low oxygen concentrations. It appears especially when the waters are low or full of aquatic vegetation and the process of decomposition of organic material is high (Weber, 2005). All of these species are filter feeders animals, which feed on animal and vegetal particles, living or partially decomposed, which are present in suspension in these different water bodies. Usually, they fed on detritus and phytobenthic species (Makhutova et al., 2013), and for this reason, naiads qualify as a convenient isotopic baseline for comparisons of pelagic food webs (McKinney et al., 1999; Vuorio et al., 2007).

Table 1

Ecological requirements and distribution of the unionid molluscs studied in this work ( + = present and +/- = rare/absent)
	Unio crassus	Unio tumidus	Unio picturum	Anodonta cygnea
Ecological features
Sediment type	Sandy	Sandy-muddy	Sandy-muddy	Muddy
Water flow rate	High	Low/absent	Low/absent	Absent
Preferred location	Rivers bed	Rivers, channels, active ponds, floodplain	Rivers, channels, active ponds, floodplain	Lakes, ponds and channels
Distribution
Danube	+	+	+	+
Floodplain	+	+	+	+
Danube Delta	+/-	+/-	+/-	+/-
Rivers	+	+	+	+
Lakes and ponds	+/-	+	+	+

1.2. Archaeological data

During prehistory, molluscs were exploited opportunistically in relationship with the surrounding environment: as an essential source of food (Radu, 2011; Radu et al., 2016) and as raw material for producing tools or ornaments (Mărgărit et al., 2018, 2021).

The Gumelnița site (Călărași county) is located in the northern area of the Balkan region, in the Southern part of Romania. It is situated today on the left bank of the Danube River and about 2.7 km east of the Argeș River (Fig. 1) in the area where this river flows in the Danube (Lazăr et al., 2017, 2020). This position was strategic because it is situated in the floodplain of the Danube, and benefits from the freshwater resources which can be collected near the site. However, the contemporary landscape differs from prehistory, because the Danube has been channelled (Marin 2017). In support of this statement, our magnetometry survey led to the identification of a paleo-channel of the Argeș river, which flowed east of the tell, separating the settlement from the Danube terrace on which the cemetery used by this community is located.

In addition, the tell settlement is a multi-layered site, a kind of artificial mound, formed on the remnant of the terrace, and above the floodplain level and water river fluctuations. Gumelnița site is ¹⁴C dated (5035 − 4169 cal BCE, 95.4% probability) time spam attributed to Boian and Gumelnița cultures. (Lazăr et al., 2020).

A total of 636 mollusc shells/fragments were found during the excavations from 2017 to 2019 (Lazăr et al., 2017, 2020). Of the identified shells (Table 2), most of them (64.22%) belong to the three species of the Unio genus (U. pictorum, U tumidus, U. crassus), followed by Anodonta sp. (28.59%). The shells from molluscs that are not freshwater mussels only represent 7.03% of the remains. These data show the relevance of the naiads in the diet of the inhabitants of the Gumelnița tell settlement.

Table 2

Mollusc shells recovered during the excavations from 2017 to 2019 (Lazăr et al., 2017, 2020) at the Gumelnița site.
	Gumelnița		Boian
Specie	2017	2018–2019	2019	Total	%
Unio tumidus	35	91	6	132	21.09
Unio pictorum	21	20	11	52	8.31
Unio crassus	3	19		22	3.51
Unio sp.	34	144	18	196	31.31
Anodonta sp.	38	137	4	179	28.59
Pseudoanodonta sp.		1		1	0.16
Dreissena sp.	1			1	0.16
Viviparus sp.	4	30		34	5.43
Cepaea vindobonensis		4	5	9	1.44
Total Mollusca	136	446	44	626	100.00

1.3. Conchiolin

Mollusc shell has evolved to support and protect these soft-bodied animals. The shell is a biomaterial secreted by the mantle and composted by a mineral phase of calcium carbonate that accounts for 95–99% of the weight. The remaining 5% represents the organic matrix. It is organized in different superimposed calcium carbonate layers and an external organic layer, the periostracum (Saleuddin & Petit, 1983). Shell growth occurs in the distal border of the shell, which grows by adding subjacent layers, growing more in length than in thickness. The calcification occurs in the extrapallial space, which is delimitated by the growing shell, the periostracum and the calcifying mantle. The periostracum is secreted by the periostracal groove situated in the mantle, and also provides support for the calcium carbonate crystals. The calcifying matrix is secreted in the extrapallial space by specialized cells of the calcifying mantle. It is a complex mixture of proteins, glycoproteins, proteoglycans, polysaccharides, and chitin (Marin and Luquet 2004).

The shell organic matrix can be retrieved by dissolving the shell with a weak acid or with a calcium-chelating agent like EDTA (Cariolou & Morse, 1988; Carmichael et al., 2008; Simkiss, 1965). Then two fractions can be separated by centrifugation: a soluble fraction, and an insoluble fraction or conchiolin (Frémy, 1855). Conchiolin is a heterogeneous mixture of proteinaceous substances with three fractions: nacrin, nacrosclerotin and nacroin (Grégoire et al., 1955). Conchiolin showed a high content of glycine and alanine (30–60%) with a large proportion of hydrophobic residues (Gregoire, 1972).

The isotopic composition of carbon and nitrogen (δ¹³C and δ¹⁵N) in animal tissues reflects the isotopic composition of their diets and metabolic processes (DeNiro & Epstein, 1978). Therefore, conchiolin can be used as a proxy for the soft tissues in isotopic analyses (Delong & Thorp, 2009; O’Donnell et al., 2003; Watanabe et al., 2009). The values obtained in the conchiolin are similar to those of the animal's body (Watanabe et al., 2009), which is why this is promising data to take into account due to mollusc shells are common in archaeological sites, so it is further approximation to diets, but also ecosystems of the past. So far, only marine molluscs and not freshwater have been analysed to study the diets of past humans (Arnay-de-la-Rosa et al., 2009, 2010; Bownes et al., 2017; Schulting et al., 2022).

With this work we aim to propose a new method for studying archaeological freshwater mollusc shells (Unio and Anodonta), obtaining an offset that allows us to calculate the values of the animal's body and thus be able to apply these data to the study of past communities.

2.1. Modern molluscs

For this experiment, we analysed the stable isotopes of different tissues of modern Unio tumidus (n = 5) from the Danube river (at the mouth of the Mostiștea river) collected in August of 2022 to obtain the offset of the values of the animal body regarding the values of the shell conchiolin. To observe these offsets between the different parts of the animal, we analysed isotopically the body (mantle + foot), the adductor muscle, the conchiolin of the shell and also the stomach contents.

The height of the shells was measured using an electronic calliper (0.01 mm resolution) to compare later with the archaeological shells.

The first step was to freeze-drying the whole mollusc. After this, we separated the tissues. Next, the body was ground in a Retsch MM200 mill. From the resulting powder, we separated one part for direct analysis, and the other for lipid extraction, due to these animals have about 10–13% of lipids in their composition (Nichols & Garling, 2000). Lipids are depleted in δ¹³C compared to proteins and carbohydrates, and consequently, variations in the lipid content of organic tissues will influence the δ¹³C values (DeNiro & Epstein, 1978). The lipids were extracted using a solution of Methanol:Chloroform (2:1 v/v) (O’Connell & Hedges, 1999) in constant sonication for 1 h (two repetitions). After we eliminated the Methanol:Chloroform, the solution was washed in distilled water for 20 min (two repetitions). The resulting product was centrifuged and washed with water 3 times. Then the pellet was freeze-dried and analysed in an isotopic ratio mass spectrometer (IRMS). Adductor muscle was homogenized and analysed directly by IRMS.

The conchiolin was obtained by decalcification of the shells. First, the shells were cleaned to eliminate any remaining tissue. Next, they were placed in 100 ml beakers. Large shells were broken into fragments. They were immersed in 5M HCl (Arnay-de-la-Rosa et al., 2009) until the reaction stopped (CaCO₃ + 2HCl → CaCl₂ + H₂O + CO₂), then HCl was collected and placed in 15 ml centrifuge tubes. The samples were centrifuged to remove the supernatant and the resultant pellet was the conchiolin. This process was repeated until the shell’s carbonate was completely digested. It was then centrifuged to eliminate the HCl, washed with distilled water and centrifuged 3 times. Finally, the pellet was freeze-dried and analysed by IRMS.

2.2. Archaeological molluscs

The conchiolin was extracted from a total of 17 archaeological shells (3 U. crassus, 4 U. pictorum, 5 U. tumidus and 5 Anodonta sp.) from Gumelnița level and for comparison, two disarticulated modern shells from the Danube (at the mouth of the Mostiștea river) (1 U. tumidus, 1 Anodonta sp.) collected in August of 2020.

The conchiolin was also obtained by decalcification of the shells. First, the shells were pre-washed with distilled water to remove any remaining dirt from the soil. Then the shells were placed in 100 ml beakers. Large shells were broken into fragments. Due to diagenesis, conchiolin does not coat the entire shell, so it is easier to attack the inorganic part with acid than in modern shells. As 5M HCl can be aggressive, the shells were immersed in 1M (as a previous test, we tried 2.5M with GUM-77 to GUM-80). We waited until the reaction stopped (CaCO₃ + 2HCl → CaCl₂ + H₂O + CO₂), and then we collected the HCl, placed it in 15 ml centrifuge tubes and centrifuged it to remove the supernatant and keep the pellet, which will be the conchiolin. This process was repeated until the shell’s carbonate was completely digested. It was then centrifuged to eliminate the HCl, washed with distilled water and centrifuged 3 times. The pellet was freeze-dried and analysed by IRMS.

2.3. Stable isotopes analysis

Samples were pre-treated between the Stable Isotopes Laboratory of the ArchaeoScience Division of the University of Bucharest and the Molecular Palaeontology Laboratory of the University Institute of Geology (IUX) of the University of A Coruña (UDC).

The IRMS analyses were carried out in the Unit of Instrumental Techniques of Analysis (UTIA) of the UDC Research Support Services (SAI). For the stable isotope analysis, approximately 0.5 mg of lyophilised tissue/conchiolin was put into tin capsules and subjected to combustion The samples were combusted and analysed directly at the DeltaV Advantage (ThermoScientific) coupled with an interphase ConfloIV to an elemental analyser Flash IRMS EA IsoLink CNS (Thermo Scientific), with an analytical reproducibility greater than 0.2‰ for carbon and greater than 0.2‰ for nitrogen. Stable carbon and nitrogen isotope compositions were calibrated relative to VPDB and AIR. As secondary standards were employed: USGS 40 (-4.52‰), USGS41a (+ 47.55‰), IAEA-N-1 (+ 0.4‰), IAEA-N-2 (+ 20.3‰) and USGS-25 (-30.4‰) for δ¹⁵N, and USGS 40 (-26.39‰), USGS41a (+ 36.55‰) NBS 22 (-30.031‰), IAEA-CH-6 (-10.449‰) and USGS 24 (-16.049‰) for δ¹³C. To test the precision (standard deviation) acetanilide was employed as the internal standard, resulting in ± 0.15‰ (n = 10). Quantifications of each sample were done by duplicate when the sample was sufficient.

The results are presented under the delta (δ) notation, which reflects the proportion between both isotopes in the sample with the proportion in an international standard, in this case, the Vienna PeeDee Belemnite (VPDB) for carbon and atmospheric air (AIR) for nitrogen, as shown in the following equation: δX (‰) = (R_sample/R_standard) − 1*1000, where X is the heavier isotope and R is the ¹⁵N/¹⁴N or ¹³C/¹²C ratio.

It is not possible to compare directly the values of the δ¹³C of modern and archaeological shells due to the Suess effect (caused by the contribution of CO₂ depleted in δ¹³C due to the burning of fossil fuels) (Keeling, 1979). We applied a correction of the δ¹³C in modern shells, following García-Vázquez et al. (2022). The CO₂ δ¹³C value of the Neolithic is -6.5‰ (Vaiglova et al., 2020). The calibration curve of García-Vázquez et al. (2022) returns a result of -8,73 for the CO₂ of 2020 and − 8,82 for 2022, so we have to add + 2.2 to the 2020 shells and + 2.3 to the 2022 shells in the δ¹³C to compare the Neolithic shells with the modern ones.

Statistical calculations and plots were performed with PAST 4.05 (Hammer et al., 2001) and edited with Adobe Illustrator.

3.1. Modern molluscs

Results on modern U. tumidus are in Table 3, and their mean values, standard deviation, and maximum and minimum values are in Table 4. The size of modern U. tumidus is within the variation (Lazăr et al., 2017, 2020) of this species in the Gumelnița site, except for individual 3, which has a larger size. This could be due to the exploitation of these molluscs in the past, which prevented them from reaching larger sizes (Radu et al., 2016).

Table 3

Modern *U. tumidus* isotopic results.
Lab N	Ind.	Tissue	%N	δ ¹⁵N_AIR (‰)	%C	δ ¹³C_VPDV (‰)	C:N	Height (mm)
U-1-b	1	Body	4.6	7.1	27.9	-27.5	7.0	42.69
U-1-b-t		Body (defatted)	6.5	7.1	24.6	-26.4	4.4
U-1-m		Muscle	11.4	7.8	42.2	-27.2	4.3
U-1-c		Stomach contents	6.7	6.8	43.9	-28.2	7.7
U-1-s		Shell conchiolin	8.8	6.8	27.0	-27.1	3.6
U-2-b	2	Body	6.1	7.5	33.8	-27.6	6.5	35.63
U-2-b-t		Body (defatted)	8.8	7.4	34.4	-26.8	4.6
U-2-m		Muscle	11.8	7.8	42.2	-27.1	4.2
U-2-c		Stomach contents	7.8	7.1	44.9	-28.1	6.7
U-2-s		Shell conchiolin	13.0	6.1	40.6	-27.5	3.6
U-3-b	3	Body	3.6	7.8	24.8	-26.9	8.0	48.87
U-3-b-t		Body (defatted)	4.2	7.7	19.9	-26.4	5.5
U-3-m		Muscle	12.9	8.9	44.0	-26.8	4.0
U-3-c		Stomach contents	9.0	6.2	43.2	-27.7	5.6
U-3-s		Shell conchiolin	11.3	6.1	35.3	-27.5	3.6
U-4-b	4	Body	3.8	7.6	22.6	-27.2	6.9	38.76
U-4-b-t		Body (defatted)	4.4	7.6	17.7	-26.3	4.7
U-4-m		Muscle	11.7	8.7	42.4	-27.4	4.2
U-4-c		Stomach contents	6.4	7.5	40.7	-27.9	7.4
U-4-s		Shell conchiolin	12.1	7.1	37.2	-27.5	3.6
U-5-b	5	Body	5.4	7.0	34.8	-27.9	7.5	32.81
U-5-b-t		Body (defatted)	8.2	7.0	30.7	-27.2	4.4
U-5-m		Muscle	11.3	7.5	43.6	-27.4	4.5
U-5-c		Stomach contents	9.1	5.9	43.3	-28.3	5.5
U-5-s		Shell conchiolin	12.4	6.1	38.5	-28.1	3.6

Table 4

Media ± standard deviation (min value – max value) of modern *U. tumidus*.
Tissue	%N	δ ¹⁵N_AIR (‰)	%C	δ ¹³C_VPDV (‰)	C:N
Body	4.7 ± 0.94 (3.6–6.1)	7.4 ± 0.30 (7.0–7.8)	28.8 ± 4.83 (22.6–34.8)	-27.4 ± 0.35 (-27.9 – -26.9)	7.2 ± 0.53 (6.5–8.0)
Body (defatted)	6.4 ± 1.88 (4.2–8.8)	7.4 ± 0.27 (7.0–7.7)	25.5 ± 6.30 (17.7–34.4)	-26.6 ± 0.33 (-27.2 – -26.3)	4.7 ± 0.40 (4.4–5.5)
Muscle	11.8 ± 0.57 (11.3–12.9)	8.2 ± 0.54 (7.5–8.9)	42.9 ± 0.79 (42.2 –44.0)	-27.2 ± 0.21 (-27.4 – -26.8)	4.2 ± 0.17 (4.0–4.5)
Stomach contents	7.8 ± 1.12 (6.4–9.1)	6.7 ± 0.56 (5.9–7.5)	43.2 ± 1.39 (40.7–44.9)	-28.0 ± 0.21 (-28.3 – -27.7)	6.6 ± 0.89 (5.5–7.7)
Shell conchiolin	11.5 ± 1.45 (8.8–13.0)	6.4 ± 0.42 (6.1–7.1)	35.7 ± 4.69 (27.0 –40.6)	-27.5 ± 0.32 (-28.1 – -27.1)	3.6 ± 0.03

3.2. Archaeological molluscs

The results on the archaeological shells from Gumelnița and two modern disarticulated shells from the Danube (A-1-s and T-6-s) are shown in Table 5. GUM-77 to GUM-80 were decalcified with 2.5 HCl. Of these shells, we could only recover enough samples for 1 replicate. GUM-103 to GUM-116 were treated with a more gentle acid (HCl 1M). It was possible to do 2 replicates to all the samples except GUM-113, GUM-114 and GUM-115. On average, the %N is lower in the 2.5M treatment (3.4) than in the 1M (9.4), and the same happens with the %C (10.5 and 29.9, respectively). This gives us the idea that in addition to removing inorganic matter more quickly, it can also cause damage to the conchiolin, which in itself may not be very well preserved. Carmichael et al. (2008) observed that higher concentrations of acid reduced N recovery, and from our results, we can add that it has the same effect in C, and for extension the recovery of the organic matter.

For all archaeological samples, the mean of the %N is 8.1 (SD: 5.15; min-max: 1.2–15), for the %C is 25.6 (14,32; 4.9–45.2), for δ¹⁵N is 7.4 (0.73; 6.3–8.8), for δ¹³C is -26.2 (1.87; -29,9 – -22.1) and for C:N is 4.2 (1.53; 3.3–10.0).

Table 5

Raw data of archaeological naiads from Gumelnița (GUM) and disarticulated modern shells (A-1-s and U-6-s).
Lab N	Species	% N	δ¹⁵N_AIR (‰)	% C	δ¹³C_VPDV (‰)	C/N
GUM-80	Anodonta sp.	7.7	8.0	22.1	-26.6	3.3
GUM-112	Anodonta sp.	4.8	6.9	17.6	-26.6	4.3
GUM-113	Anodonta sp.	2.1	-	17.7	-28.3	10.0
GUM-114	Anodonta sp.	9.1	6.8	34.4	-26.4	4.4
GUM-115	Anodonta sp.	1.4	-	7.1	-25.7	5.8
GUM-116	Anodonta sp.	6.0	6.9	19.5	-25.2	3.8
A-1-s	Anodonta sp.	5.1	7.9	15.1	-25.4	3.5
GUM-78	U. crassus	3.1	8.8	9.6	-26.5	3.6
GUM-107	U. crassus	11.9	7.9	34.9	-26.7	3.4
GUM-108	U. crassus	14.9	6.9	44.7	-26.2	3.5
GUM-79	U. pictorum	1.2	6.7	5.2	-26.9	4.9
GUM-109	U. pictorum	14.2	6.3	42.1	-29.9	3.4
GUM-110	U. pictorum	12.4	8.3	36.7	-26.8	3.5
GUM-111	U. pictorum	13.7	7.6	40.3	-23.9	3.4
GUM-77	U. tumidus	1.5	8.2	4.9	-22.1	3.9
GUM-103	U. tumidus	2.2	6.4	7.1	-25.2	3.8
GUM-104	U. tumidus	11.8	7.1	34.9	-25.2	3.4
GUM-105	U. tumidus	12.5	7.9	36.6	-23.9	3.4
GUM-106	U. tumidus	15.0	7.4	45.2	-29.7	3.5
U-6-s	U. tumidus	2.0	6.1	6.1	-26.8	3.5

4.1. Modern molluscs

The mean of δ¹⁵N of the body tissues is 7.4‰, the same as the defatted body. However, there are differences in the δ¹³C of the body (-27.4‰) compared to the defatted body (-26.6‰). These differences support the suitability of lipid extraction, as lipids do not have nitrogen. Therefore the δ¹⁵N does not change, and fats have a more negative δ¹³C than proteins, which gives us confidence in the success of the extraction. The muscle, a proteinaceous tissue, has an enrichment of 0.8 in δ¹⁵N concerning the body tissues. This result is similar to the enrichment of ~ 1‰ of Elliptio sp. (McKinney et al., 1999), another unionid mussel.

If we represent the values of the modern individuals of U. tumidus in a bivariate graph of δ¹³C vs δ¹⁵N (Fig. 3), we can observe that not all the individuals are in the same position but there are trends between the different tissues like the defatted body has the less negative δ¹³C values, or that the conchiolin has the lowest δ¹⁵N values and the muscle the highest.

The enrichment of the body concerning the stomach contents is 0.7‰, which is inferior to the canonical trophic enrichment of 2–4‰ to the food source (Das et al., 2021). The stomach contents analysed have a lower δ¹⁵N than the body tissues (but not always than the shells). However, we are probably looking at a mixture of the consumed food together with the gastric fluids of the animal and possibly some of the animal's tissue. Therefore, we will disregard these results.

The mean of C:N of the body (7.2) is higher than the defatted body (4.7). Present-day Unionidae molluscs from a lake in Denmark show similar values: their defatted bodies are between 3.9 and 4.9, with a mean of 4.4 (Fischer et al., 2007).

The muscle shows a mean in the C:N of 4.2, similar to the defatted body. In the Manila clam (Ruditapes philippinarum), the C:N of the muscle is between 3.8 and 4.1 (Dang et al., 2009). Our values are similar and go from 4 to 4.5.

Not all mollusc species have the same amino acid proportion in the shell matrix protein and therefore do not have the same C:N ratio (Misarti et al., 2017). The mean C:N of the conchiolin of our samples (3.6) is similar to the 3.5 obtained in Ruditapes philippinarum (Watanabe et al., 2009). C:N is very stable in all 5 individuals compared with the other tissues analysed.

Demineralization is necessary before the isotopic measure of the conchiolin. Acidification can change the δ¹⁵N values (Schlacher & Connolly, 2014). The way to control this effect is using proxy controls of matched soft tissues because they are similar in composition to shell organic matrix but free of carbonate (Carmichael et al., 2008). So, for this, and assuming that the demineralization of the shell could have a systematic error, we propose two offsets. The first one for the transformation of conchiolin values in the defatted body: Δ¹⁵N_{conchiolin−defatted body} = + 0.95‰, and Δ¹³C_{conchiolin−defatted body} = + 0.93‰. And the second one, for the transformation of conchiolin in muscle values: Δ¹⁵N_{conchiolin−muscle} = + 1.7‰, Δ¹³C_{conchiolin−muscle} = + 0.3‰. This method can also have some errors since different tissues represent different times of the individual’s life. For example, muscle tissue turnover rates are on the order of months (Gorokhova & Hansson, 1999), while shell represents the lifetime of the individual.

Data have shown that bivalve shells could have enrichment, depletion or no difference in bulk isotope offset with soft tissues (O’Donnell et al., 2003; Vokhshohori et al., 2022; Watanabe et al., 2009; Whitney et al., 2019), so this could be species or genera specific. However, as other offsets were proposed (Mae et al., 2007; O’Donnell et al., 2003; Watanabe et al., 2009) for other species, we think that due to the differences between taxons and ecosystems, we have to propose one for U. tumidus and employ it in other Unioid freshwater mussels due to the lack of other studies.

4.2. Archaeological molluscs

As there are no criteria for the acceptance of isotopic values resulting from the analysis of conchiolin in archaeological or palaeontological shells, in particular freshwater mussels, we will rely on our results to propose possible criteria. Collagen is a protein preserved in bones often used in paleontological and archaeological stable isotope studies. The criteria for the preservation of this protein are based on C and N percentages (Ambrose, 1990; Bocherens et al., 2005), C:N (DeNiro, 1985) and extraction yield (Ambrose, 1990; Bocherens et al., 2005; van Klinken, 1999).

The first results that we have to discard are GUM-113 and GUM-115. Both shells did not yield a reliable δ¹⁵N value, so we automatically discard them. In addition, both individuals have the most distant C:N values (10 and 5.8 respectively) from the modern shells and also the rest of the archaeological samples.

The shells that were digested with HCl 2.5M show smaller percentages of C and N and only had enough samples for 1 replicate. Their % of C and N is smaller than most of the samples that were digested with HCl 1M, but the C:N is more similar to modern shells except for GUM-79. Most of the samples have a C:N that fluctuates between 3.3 and 3.9, that is 3.6 ± 0.3, almost the same amplitude that is used as criteria for archaeological/paleontological collagen (3.2 ± 0.4 or 2.9–3.6) (DeNiro, 1985). The shells with a higher C:N than these values are GUM-79, GUM-112, GUM-113, GUM-114 and GUM-115. Of these shells, GUM-79, GUM-113 and GUM-115 have small percentages of N (1.2, 2.1 and 1.4, respectively), but GUM-112 and GUM-114 present higher percentages of N (4.8 and 9.1), similar to other shells with C:N between 3.3 and 3.9.

Figure 4a and 4b show the %C and %N respective vs. C:N to see this effect graphically. Some of the specimens with a low % of C and/or N have highly variable C:N ratios, so for the acceptation or not, we should check how this proportion between the C and N is. For this, we represented these percentages in a bivariate plot (Fig. 4c). We observed that the data fit well to a regression line (R² = 0.9672). However, two values fall outside, which are GUM-113 and GUM-114, both specimens of Anodonta sp. GUM-113 results were already rejected, and GUM-114 has a C:N outside the range (4.4). The percentages of C and N of GUM-112 fit with the line, but we also did not accept it for its C:N value (4.3).

So, following these criteria, we did not accept the results from GUM-79, GUM-112, GUM-113, GUM-114, and GUM-115. All of these individuals belong to Anodonta sp. except GUM-79 which is a specimen of U. pictorum. It seems that the results for Anodonta sp. are not as good or at least as homogeneous as in the genera Unio, so further studies may be necessary to establish the values in modern specimens.

Modern disarticulated specimens (A-1-s and U-6-s) showed worse preservation values than complete individuals as they were subjected to diagenesis in the river, which may explain these less good data, although the C:N falls within the values established in this work.

4.3. Unioid molluscs and their ecology during the Neolithic

Once we disregarded the specimens with bad preservation, we represented the data of the accepted shells and their transformation in the defatted body tissue by adding + 0.95‰ in δ¹⁵N and + 0.93‰ in δ¹³C, in a bivariate plot (Fig. 5). To compare directly the archaeological shells with the modern ones, we applied the correction for the Suess effect described in section 2.3.

We observed that modern shells, all from the Danube, are concentrated. The archaeological shells have a wider distribution, which may indicate different origins in terms of different habitats, and not necessarily distant areas

The mean, standard deviation and minimum and maximum values of the shells of the species under study can be seen in Table 6. Of the different Unio species, U. crassus has a smaller standard deviation in δ¹³C than U. pictorum and U. tumidus. U. crassus is a species of waters with a high flow rate, while U. pictorum and U. tumidus prefer waters with less current or even without it (Table 1), which is reflected in the δ¹³C, since different watercourses may be more or less eutrophised, which will cause this variability to exist in the phytoplankton and be dragged into the rest of the trophic chain. Besides this effect, mussels that live near the river bank or lakeside may be influenced by terrestrial carbon input (Vuorio et al., 2007). This could be the case for those shells with less negative δ¹³C like GUM-77 or GUM-105. Modern shells are located in this area also, and they were collected from the river bank, which supports this hypothesis.

In the case of Anodonta, it is a species that is rare from rivers and prefers stagnant waters,. However, as we had to eliminate most of the values for this species, we only have two data, which makes it difficult to conclude, although the δ¹³C of the two samples is similar, so they could come from the same habitat.

Table 6

Mean ± standard deviation (min value – max value) of the raw data of the different species found in Gumelnița.
Species	n	%N	δ¹⁵N_AIR (‰)	%C	δ¹³C_VPDV (‰)	C:N
Anodonta sp.	2	6.8 ± 0.86 (6.0–7.7)	7.4 ± 0.57 (6.9–8.0)	20.8 ± 1.28 (19.5–22.1)	-25.9 ± 0.70 (-26.6 – -25.2)	3.6 ± 0.23 (3.3–3.8)
U. crassus	3	10.0 ± 4.99 (3.1–14.9)	7.9 ± 0.79 (6.9–8.8)	29.7 ± 14.77 (9.6–44.7)	-26.4 ± 0.22 (-26.7 – -26.2)	3.5 ± 0.06 (3.4–3.6)
U. pictorum	3	13.5 ± 0.77 (12.4–14.2)	7.4 ± 0.83 (6.3–8.3)	39.7 ± 2.24 (36.7–42.1)	-26.9 ± 2.46 (-29.9 – -23.9)	3.4 ± 0.01 (3.4–3.5)
U. tumidus	5	8.6 ± 5.64 (1.5–15.0)	7.4 ± 0.63 (6.4–8.2)	25.7 ± 16.51 (4.9–45.2)	-25.2 ± 2.51 (-29.7 – -22.1)	3.6 ± 0.20 (3.4 – 3.9)

The differences in the δ¹⁵N of these species are smaller than in the δ¹³C. All of these species have a media of 7.4‰ (corrected: 8.3‰), except U. crassus which is 7.9‰ (corrected: 8.8‰). Freshwater mussels are classified as primary consumers, and their trophic level can be compared with other primary consumers like fish. Interestingly, the media of δ¹⁵N in the collagen of Cyprinus carpio (omnivore fish) from Borduşani-Popină (Balasse et al., 2016) (an archaeological site from the same culture and chronology than Gumelnița that is also situated on the banks of the Danube), is 7.4‰ (± 0.78). Therefore, in the absence of fish remains, conchiolin could be used to predict the values of omnivorous fish, even better than the defatted body of the animal.

In the current paper, we established a quality criterion of 3.6 (± 0.3) for the C:N of the extracted conchiolin. However additional studies need to be done, mainly to check the differences or similarities between Unio and Anodonta. To transform the data obtained in archaeological shells of freshwater mussels into the animal body (defatted) values from the isotopic study of different tissues of present-day individuals of the species U. tumidus, we obtained an isotopic offset for the conchiolin-defatted body, of + 0.95 for δ¹⁵N and + 0.93 for δ¹³C, and conchiolin-muscle of + 1.7‰ for δ¹⁵N and + 0.3‰ for δ¹³C. Thus, this new method for the isotopic study of ancient conchiolin allows us to obtain an offset that is a helpful tool for reconstructing past diets and ecosystems with higher accuracy.

6.1. Acknowledgement

We like to thank Bogdan Manea and Mihaela Golea for helping to harvest the modern individuals of U. tumidus. To Aurora Grandal d’Anglade for access to the Molecular Palaeontology Lab of the University Institute of Geology (IUX) of the University of A Coruña (UDC). Last but not least, many thanks to our field team (Vasile Opris, Theodor Ignat, Madalina Dimache, Cristina Covataru, Valentin Parnic, Adelina Darie, Adrian Bălășescu, Gabriel Vasile, Adrian Șerbănescu, Cristian Roth, Carol Căpiță, and Gabriel Popescu) without which these archaeological discoveries would not have been possible.

6.2. Funding

This work was supported by a grant from the Ministry of Research, Innovation and Digitization, CNCS/CCCDI – UEFISCDI, project number PN-III-P4-ID- PCE-2020-2369, within PNCDI III. A. García-Vázquez was supported by a Research Fellowship for Visiting Professors within the Research Institute of the University of Bucharest (ICUB) under the project “Multi-isotopic approach to the life at the Gumelnița site” (515/10.01.2022).

6.3. Coflicts of interest

The authors have not disclosed any competing interests.

6.4. Ethics approval/declarations

No ethical issues related with the use of animals in the performed analyses were involved.

6.5. Consent to participate

Not applicable.

6.6. Consent for publication

Not applicable.

6.7. Availability of data and material

All data generated or analysed during this study are included in this published article.

6.8. Code availability

Not applicable.

6.9. Authors' contributions

AGV tested the methodology, prepared the samples for isotopic analysis and interpreted the isotopic data. VR identified the mollusc shells. AGV prepared the original draft with inputs from VR and CL. All authors read and approved the final manuscript.

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No competing interests reported.

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New method for the isotopic study of ancient conchiolin from archaeological shells of freshwater mussels (Unionoida)

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Abstract

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1. Introduction

1.1. Naiads ecology

1.3. Conchiolin

2. Material And Methods

2.1. Modern molluscs

2.2. Archaeological molluscs

2.3. Stable isotopes analysis

3. Results

3.1. Modern molluscs

3.2. Archaeological molluscs

4. Discussion

4.1. Modern molluscs

4.2. Archaeological molluscs

4.3. Unioid molluscs and their ecology during the Neolithic

5. Conclusions

Declarations

References

Additional Declarations

Status:

Journal Publication

Version 2