Bridging Archaeology and Marine Ecology: Coral Archives of Hellenistic Coastal Change

1. Introduction

Stony corals are calcifying cnidarians that are responsible for the formation of large, shallow-water, coastal reef ecosystems [1,2] that house ~25% of described marine fishes and invertebrates [3,4]. They have adapted to thrive across relatively large gradients of environmental/abiotic factors including temperature, turbidity, flow, light and nutrient availability [5]. At the species level, stony corals exhibit abundant examples of phenotypic plasticity, or visible responses to environmental factors despite shared genotypes. Plasticity of coral growth location, morphology, physiology, and gene expression has been documented in response to light [6,7,8,9], water flow [10,11], nutrient availability [12], and seawater chemistry [13], and aims to maintain energy acquisition across environmental gradients. As such, stony corals have persisted, as a taxonomic order (Scleractinia), for more than 240 million years, enduring multiple catastrophes in Earth's history [14], and leaving them as records of not only fairly static environmental growth conditions over the long term (e.g., presence/absence, macromorphology, and bulk skeleton analysis) but also of decadal, seasonal, and impending terminal ecosystem changes. For instance, it has been well-documented that the trace element and stable isotope composition of scleractinian coral skeletons records environmental signals from the surrounding seawater in species-specific ways review by [15], allowing paleoceanographic reconstructions of seawater temperature, cation concentrations, and pH [16,17] Recently Gothmann et al. [18] extended the use of fossil corals as geochemical archives into the Mesozoic. In addition, the symbiotic relationship between scleractinian corals and photosynthetic dinoflagellates can be detected from the isotopic composition of organic matter embedded within the coral skeleton (SOM), which reflects photosymbiotic activity (and hence light conditions) [19,20,21]. This isotopic signatures also provide insights into the relative contributions of auto- versus heterotrophy to both fixed carbon and nitrogen nutritional sources [19,20,22,23,24]. Multiple lines of evidence suggest that the nitrogen isotopic composition (δ¹⁵N) of corals' SOM and other calcifying organisms can remain stable over geological timescales [19,22,25,26,27,28]. As such, coral-bound δ¹⁵N serves as a robust proxy for assessing photosymbiotic status in fossil corals [19,22], with well-preserved aragonitic skeletons retaining these isotopic signatures for over 200 million years following the death of the holobiont (the coral host and its associated symbiotic community)[e.g., [22]].

Cladocora caespitosa (Linnaeus, 1767; family Cladocoridae) is a colonial symbiotic stony coral, endemic to the Mediterranean Sea [29]. It is considered a remnant reef-building species and a vital evolutionary link between tropical coral reefs and the ancient reef ecosystems that existed in the Mediterranean prior to the end of the Messinian period [30]. Although large reef structures formed by this coral are frequently observed in the fossil record [30], they have become exceptionally rare in the modern Mediterranean [31,32]. Due to its longevity and distinctive calcification patterns, C. caespitosa is increasingly studied as a natural archive for reconstructing past environmental and climatic conditions in the Mediterranean region [33,34].

The Mediterranean of the Hellenistic period (232- 63 BCE) was heavily populated along the coasts [35], with a sea level and sea surface temperatures probably lower and cooler than today, respectively [36]. Recent studies have revealed significant changes in sea surface temperatures (SSTs) in the eastern Mediterranean over the past millennia, recording a warming trend in the region, with the 20th century being the warmest period in the last millennium [36,37]. The Hellenistic period was unstable in the human history of the Syria-Palestine region, withhe “Syrian wars”, the longest sequence of wars in the ancient world [38], spanning from 301 BCE or earlier to 103-101 BCE. Modern Akko (also called Acre and known at the time as Ptolemais-Ake; Figure 1) became a Greek polis at the end of the fourth century BCE, and was an important Egyptian military port and city during the “Syrian Wars” [39]. The city became active in regional and maritime trade, and it fairly quickly became one of the most important and prosperous cities in the region. During the Roman period, the harbor was the most important gateway to Europe and Rome and was favored by the Roman troops as the regular landing place for over two centuries [40]. Surveys and reconstructions by the British Mandate Department of Antiquities (1920s - 1940s) revealed and then confirmed the importance of greater Akko to past geopolitical conflicts in the region [41]. Later archaeological surveys and excavations of Akko then began in the mid-1960s [42], revealing a variety of underwater breakwaters, foundations, and shipwrecks. Underwater excavations of coastal Akko in the 2010s under the auspices of the Israeli Antiquities Authority established six archaeological strata from the Hellenistic period (i.e., sub-fossil) to the British Mandate [40,43]. During these underwater excavations eight square (2x2m.) pits were dug among them only three were dug up to the bedrock, in which several well-preserved corals of genus Cladocora were identified as colonizers of the ancient bedrock [40,43]. The colonies were and radiocarbon dated to 335-94 BCE [44]. Based on their size, corallite length and known modern growth rates of Cladocora [45,46], they were likely decades old at the time of burial. The relatively large colonies and attachment to the bedrock at a depths that have remained shallow (~10 m) over the past two kiloanna; led the authors to suggest that the site was minimally turbid - allowing sufficient sunlight for coral growth - prior to 94 BCE, with subsequent burial following an acute sedimentation event leading to excellent macro- and microscopic skeletal feature preservation [40,43]. These Cladocora corals can thus have the potential to reveal the underwater environment and human uses of the Akko harbor from ~2300 years ago.

In this study, we apply multiple analyses to several shallow sub-fossil Cladocora sp. colonies excavated from portions of the Akko port dated to the Hellenistic period as well as to live Cladocora caespitosa from 10 and 30 m depth in Haifa Bay (~10 km north and south, respectively, of Akko Bay; Figure 1). Both micromorphology and DNA sequencing (modern and sub-fossil) support taxonomic identification. Colony growth form (macromorphology) and SOM amino acid stable isotope analysis from both modern and sub-fossil specimens suggests that they experienced roughly similar water energy environments and trophic position, although the sub-fossil Cladocora appears to have experienced higher light levels, at shallow depths, and exposure to anthropogenic terrestrial waste (i.e., sewage). Further, the very high degree of colony preservation suggests a rapid burial event. By comparing various measurements from genetically similar corals separated by 2,000 years, this study offers a more integrated and comprehensive reconstruction of the eastern Mediterranean’s historical physical and social environments than analyses conducted in isolation.

2. Materials and Methods

2.1. Collection

Modern Specimens: Small fragment (approximately 2 cm each) from five live colonies identified as Cladocora caespitosa were collected via SCUBA from the Israeli Mediterranean Sea in January 2021 at 30 m depth in Haifa Bay, Israel, from the Leonid ship wreck (32.86975N 34.95405E) (Figure 1). One fragment was immediately frozen in dry nitrogen for genetic analysis, and the rest were transported on ice and stored in -20 °C until fragmentation for ex situ analyses. Additionally, five newly discovered colonies at a depth of 10 meters on Naharia, ranging in size from 3 to 10 cm in diameter, were examined in situ for photophysiology.

Sub-Fossil Specimens: Five Sub-fossil Cladocora sp. colonies were collected via SCUBA diving during the excavation of the Hellenistic-early Roman Harbour of Akko in May-June 2012 and 2014 at 2.5 m depth. Excavation pits 1, 2, and 7 are located at (32.92094N 35.07138E). All excavations were carried out under the auspices of the Israeli Antiquities Authority under permits A6557, A6829, and A7120. Colonies were documented in situ, detached carefully from the bedrock and put together with seawater in a closed plastic box. Boxes were tagged with the following data: permit no., dive and date, pit no. depth; samples of sediments from the surrounding area were taken separately. The coral boxes were transferred to the lab and stored at 4^oC until further analysis [43]. The sub-fossil skeletal fragments used for scanning electron microscopy and computed tomography analyses as well as some comparable modern specimens of Cladocora are housed at the Institute of Paleobiology, Polish Academy of Sciences (abbreviated ZPAL).

Both modern and sub-fossil corals were collected for subsequent physiological, skeletal, and Compound specific stable isotope analysis of amino acids (CSIA-AA).

2.2. Photosynthetic Parameters Assessment

In situ physiological assays were conducted 20 September 2023 at 10 and 30 m on five Cladocora colonies each at 10 m (Naharia, Israel) and 30 m (Leonid shipwreck, Haifa Bay, Israel) depth using a Diving-Fluorescence Induction and Relaxation (FIRe) fluorometer [47], following methods previously described [48]. In brief, to account for intra-colony differences in light exposure, five readings were collected per colony and averaged to reflect the photophysiology of the entire colony in the analysis. As Diving-FIRe is based on a kinetic approach, it offers instantaneous measurements of ETR_max without dark adaptation of the sample, therefore allowing in-situ measurements during the daytime. Photophysiology data were determined to display normal distribution and homogenous variance between depths so that parametric statistical tests were applied.

2.3. Skeletal Morphology

2.3.1. Scanning electron Microscopy

Modern coral fragments were airbrushed and then submerged in 3% sodium hypochlorite for one hour to remove any remaining tissue, rinsed in freshwater, and dried at 60^oC. Sub-fossil coral fragments were sonicated in deionized water three times for five minutes each to remove debris and loosely cemented sand and then dried at 60^oC. Skeletal micro-morphology was visualised using a Thermo Fisher (Philips) XL20 scanning electron microscope (SEM) at the Institute of Paleobiology, Polish Academy of Sciences. All samples were sputter coated with platinum and photographed. SEM was also used to visualize the micro-structural features of polished sections that were lightly etched in Mutvei’s solution following Schöne et al., [49].

2.3.2. Computed Tomography Scanning

3D visualization of the internal structure of the coralla was made with a Zeiss XRadia MicroXCT-200 system at the Institute of Paleobiology, Polish Academy of Sciences. Scans were performed using the following parameters: voltage: 60 kV, power: 10 W, exposure time: 6 s, pixel size: 19.47 μm, 1201 projections. Three-dimensional images were obtained by processing with the AVIZO7.1 Fire Edition software.

2.4. DNA Extraction

2.4.1. Modern Corals

Five sub-samples of modern C. caespitosa genomic DNA were extracted using the Wizard Genomic DNA Purification Kit (Promega, USA). PCR amplifications were performed using Kodaq 2X PCR MasterMix (ABM, Richmond, BC Canada) [50]. To verify coral and photosymbiont taxonomy, we queried the highly conserved cytochrome oxidase subunit 1 (COI) using the following primers—FOL-LDEG (forward) 5′-TCWACHAAY CAT AAR GAY ATWGG-3′ and FOL-HDEG (reverse) 5′-TCWACHAAY CAT AAR GAY ATWGG-3′ (modified from [51]). In addition, the internal transcribed spacer (ITS2) region of Symbiodiniaceae rDNA was amplified using Symbiodiniaceae-specific

primers CS1F (forward) 5′-ACA CTG ACG ACA TGG TTC TAC ATG TGA ATT GCA GAA CTC CGT G-3′ and CS2R (reverse) 5′-TAC GGT AGC AGA GAC TTG GTC TTA CTT ATA TGC TTA AAT TCR GCGG-3′ taken from [52], respectively (Table S1). The host COI region was sequenced by the Sanger sequencing method using the ABI 3730xl DNA Analyser while Symbiodiniaceae ITS2 was sequenced on the Illumina Miseq using a v2-500 cycle kit to generate 250x 2, paired-end reads. COI sequences from the five samples were aligned using ClustalW to create a consensus sequence, which was BLASTed in NCBI’s GeneBank for species identification. Symbiodiniaceae ITS2 data were demultiplexed by the Illumina software, and the paired forward and reverse fastq.gz files were submitted to SymPortal [53] to assess the diversity and relative abundance of symbiont species within each sample.

2.4.2. Sub-Fossil Corals

Excavated sub-fossil Hellenistic Cladocora sp. was always handled independently of either the modern C. caespitosa or any other modern corals. Specimens were first examined by light microscopy; no large exogenous biological material was observed while clays and some recrystallization was apparent as dense, shiny regions similar to secondary calcite (SI Figure S1). Specimens were sonicated for 2.5 hours in deionized water with frequent water changes (every 10-15 minutes) to remove debris and loosely cemented sand. Cleaned fragments were dried at 40^oC. After cleaning, sub-fossil coral fragments were only handled in UCLA’s dedicated ancient DNA(aDNA) lab under established ‘clean’ conditions [54,55]. Standard contamination prevention protocols employed included: UV irradiation of the entire lab space before and after each use; only unopened consumables packages were brought into the aDNA lab and boxes were wiped with bleach and UV irradiated before opening and use; PPE always included disposable smocks, hairnets, foot covers, surgical masks, and double-gloving with nitrile gloves; and no modern specimens are ever allowed into the aDNA lab.

aDNA was extracted from eight fragments of cleaned Cladocora sp. using a Qiagen PowerSoil kit, per manufacturer’s instructions, after four fragments were manually broken to fit into the extraction tubes and four fragments were ground in a mortar and pestle. Each fragment was extracted independently and DNA was eluted in Qiagen solution C6 for four fragments and sterile water for four fragments. Extracted DNA was stored at -80^oC.

To compare extracted aDNA quality from Cladocora to modern coral skeletons that have seen similar depositional conditions (dead >10 years; stored in coastal sandy sediments near to the surf zone), a colony of Pocillopora verrucosa from West Ambae, Vanuatu in co-author JLD’s personal collection was cleaned, extracted, and stored as above, with the only difference being that all steps were performed in labs outside of the aDNA lab, using completely different PPE, and on different days from when sub-fossil Cladocora sp. specimens were handled.

Prior to sample shipment for library preparation and sequencing, aliquots of all extracted DNA were analyzed by Qubit and TapeStation to assess DNA quantity and quality (SI Table S4). Cladocora sp. aDNA was pooled according to grinding and elution buffer, yielding four fossil coral DNA samples in total which were shipped on ice. P. verrucosa extracted DNA was not sent for sequencing.

Pooled aDNA was prepared into libraries via the “Spotlight” single stranded library preparation method Santa Cruz Reaction (SCR) optimized specifically for ancient (i.e., degraded) DNA [56], using Illumina P5 and P7 adapters, followed by Qubit and fragment analyzer assessment, and then sequenced on a NextSeq v2.5 - 150 cycles, mid-output - at the UC Santa Cruz Paleogenomics Laboratory. Forward and reverse paired-end reads were imported together to Geneious Prime software for assembly and alignment. Paired reads were trimmed of adapters using the BBDuck plugin with a Kmer value of 27 and a quality score of 20 (99% likelihood of correct base calling). Paired and merged reads from all four pooled aDNA libraries were mapped to mitochondrial genomes from fellow faviids Cyphastrea, Platygyra, and Dipsastraea downloaded from NCBI. Because of the low return rate of reads mapped to faviid mitogenomes, all merged reads from the most promising pooled library (SCS284) were also extracted for further analysis. Mapped reads and all SCS284 merged reads were then separately BLASTed against the NCBI core nucleotide database and best BLAST hit taxonomy was assigned using the taxonomizr package in RStudio.

2.4.3. Species Relationships

Modern C. caespitosa CO1 DNA sequences were BLASTed in NCBI and then aligned with all isolates from the same study as the best BLAST hit [57]. These were then aligned with other scleractinian CO1 sequences from the same portion of the gene, as well as a Nematostella vectensis (class Anthozoa, order Actiniaria) CO1 sequence as an outgroup. An ancient Cladocora CO1 DNA sequence pulled from the full aDNA dataset was aligned with other scleractinian CO1 sequences from the same portion of the gene - further downstream from the location of the modern C. caespitosa sequence, as well as the Nematostella vectensis CO1 gene as for the modern alignment. All alignments were performed in T-Coffee [58]. Best model fit was determined using default settings in the ModelFinder tool of IQTree [59] and then trees were predicted in IQTree using 1000 bootstraps; best models based on BIC scores were HKY +F +G4 and K2P +G4 for modern-only upstream and ancient DNA-included downstream CO1 sequences, respectively.

2.5. Compound Specific Stable Isotope Analysis of Amino Acids (CSIA-AA):

2.5.1. Sample Preparation

Tissue from the live coral nubbins collected from Haifa Bay was removed from the skeleton using an airbrush with ultra-pure water, homogenized with an electric homogenizer, and centrifuged three times at 500 xg for 10 min at 4°C to separate host tissue from symbionts. Symbiont pellets were further washed with ultra-pure water after each triplicate centrifugation. Both fractions were freeze-dried before subsequent analysis. Sub-fossil and modern (after tissue removal) coral skeletal fragments were incubated in 3% bleach overnight, then washed in ultra-pure water and sonicated to remove all debris before decalcification for analysis of skeletal organic matter. Approximately 1.5 g of cleaned skeleton from each colony was hydrolyzed in 0.9 M HCl at room temperature until complete decalcification, concentrated via 15 ml Amicon centrifugal filtration units (30kd), and freeze-dried.

2.5.2. CSIA-AA

Amino acid nitrogen and carbon isotopic composition of tissue fractions (modern corals) and skeleton organic matter were determined by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). The acid-hydrolyzed host, symbiont, and skeleton product samples were first derivatized using the EZ:faast kit [24]. Briefly, ~4 mg of the hydrolyzed samples were derivatized with the EZ:faast kit with a slight modification of replacing reagent 6 with dichloromethane as the solvent. Amino acids were separated on a Zebron ZB-50 column (30 m, 0.25 mm, and 0.25 µm) on a Thermo Scientific Trace 1300 Gas Chromatograph using helium as the carrier gas at a constant flow of 1.5 ml/min. For carbon analysis, 1.5 µl was injected in split mode (1:15) at 250°C, while 2 µl was injected in splitless mode at 250°C for nitrogen analysis. The separated amino acids were split on the MicroChannel device into two direction flows: Thermo Scientific ISQ quadruples for amino acid identification and Thermo Scientific Delta-V advantage for C and N isotope analysis. In order to determine the isotopic ratio of carbon and nitrogen, the separated amino acids were combusted in a Thermo scientific GC isolink II at 1000°C for CO2 and N2. Before the sample was run into the Delta-V for N2 analysis, it passed through a cold trap with liquid nitrogen to freeze all other gases. Duplicates for carbon and triplicates for nitrogen were injected from each sample. Stable isotope ratios were expressed in standard δ notation, with the standard for carbon being Vienna PeeDee Belemnite (VPDB) and for nitrogen atmospheric N₂ (air). To account for carbon atoms incorporated during the derivatization process, we followed the correction factor of Docherty et al. [60] for each amino acid. Since the samples are from different time periods all modern samples were applied with Seuss correction to compensate for the changes in modern CO₂ emissions [61]. The trophic position (TP_Glu-Phe) was calculated using glutamic acid and phenylalanine with the predefined equation of Chikaraishi et al. [62] and constants from Martinez et al. [24].

3. Results

In 2012-2014, during three season of underwater excavation at Akko Port, within the larger Haifa Bay area (Figure 1; SI Figure 1) eight metal molds were placed on the harbor sea floor at a distance of 20 to 40 meters from the city wall in order to locate the edge of the wharf . During the excavation, stones up to 1.8 m in diameter were moved away to expose the bedrock in three square pits to a depth of 2.2-2.6 m, while only wharf pavement stones were exposed for cleaning in the remaining squares. Coral colonies and fragments were found in situ in the three fully exposed pits. Across pits 1, 2, and 7, a large preserved coral colony 20x20 cm remained affixed to the bedrock, one smaller colony remained connected to the bedrock, and some pottery shards covered with preserved corals were found, respectively [43]. ¹⁴C tests of several coral samples dated them to the mid second century to early 1st century BCE during the Hellenistic - Early Roman Period [43]. The preservation of organic fragments, most likely remnants of the polyp (Figure 2A), along with the aragonitic mineralogy and the preservation of the finest skeletal structural elements, such as the nano-granulate structure in rapid accretion deposits (RADs) (Figure 2B,C), confirms that the corals were rapidly buried alive and tightly sealed by a protective layer of sediment.

3.1. Morphology:

The almost identical skeletal structure, from the colony level to the micrometer scale, between the sub-fossil coral and the modern Cladocora caespitosa suggests that the sub-fossil coral represents the same taxon (Figure 2). For both specimens, small clumped colonies are composed of tubular, compact, upward facing corallites, each several mm in diameter, with their own distinct corallite wall (i.e., phaceloid) (Figure 2E, H). Microtomographic reconstructions of modern and sub-fossil corallites, along with their virtual sections (Figure 2 F-G, I-J, respectively), reveal identical internal organization in both specimens. Interestingly, some calices of sub-fossil Cladocora show clear constrictions known as rejuvenescence (arrows in Figure 2 I,J). SEM micrographs of the distal view of calices show well-developed septal granulations on the septal faces in both Recent (Figure 2K,L) and sub-fossil (Figure 2O,P) specimens. Etched transverse sections of septa and wall show an identical microstructural organization in Recent (Figure 2M,N) and sub-fossil (Figure 2Q,R) skeletons. The mid-septal zone consists of closely spaced rapid accretion deposits (RADs), which are also visible as part of the wall (the so-called trabeculotheca). Regular, fine-scale banding of thickening deposits (Figure 2N,R) is typical of symbiotic corals [22,63]. The sub-fossil Cladocora’s septa hexamerally arranged in approximately four cycles are well-preserved, with excellent preservation of RADs and lower inner edges of S1 septa merging with the columella (Figure 2A, C,D), as is observed for modern C. caespitosa. To support the visual taxonomic identification, we extracted DNA from both the recent intact Cladocora tissue from the Leonid shipwreck and the sub-fossil Akko Cladocora’s preserved skeletons with remaining tissue. In addition, we compared these exceptionally-preserved sub-fossil coral remains to living Cladocora corals immediately north (Naharia; 10 m specimen for in situ FIRe analyses) and south (Leonid shipwreck; 30 m specimen for in situ FIRe, external DNA sequencing and CSIA-AA analyses) to better understand the underwater environment of Akko Port during the Hellenistic-Roman period.

3.2. DNA Results:

Analyses of cytochrome c oxidase subunit 1 (CO1; Table S1) gene sequences of the modern coral host collected at Haifa Bay from 30 m depth approximately 12 km south of Akko Bay showed 100% match to vouchers for eastern Mediterranean Cladocora caespitosa mitochondrial partial COI gene via NCBI nucleotide Blast (accession numbers: MW032500.1-MW032523.1) (Table S2; [57]), confirming the taxonomic assignment based on macro-morphology. The dominant ITS2 (Table S1) symbiont type was identified as Breviolum psygmophilum (synonym of Symbiodinuium psygmophiulm, clade B2; Table S3).

Ancient DNA extracted from the best quality pooled sub-fossil Cladocora sp. sample (SCS284; Figure 3A) was dominated by bacterial sequences, with cnidarians, and more specifically scleractinians, making up 0.3% (non-redundant) and 0.06% (total) BLAST taxonomic assignments of 706,328 merged reads. Of those merged reads, 50-90 mapped to one of the three Faviid mitogenomes used for comparison (Table S4); 10 of these reads mapped to Cyphastraea had best BLAST hits to anthozoan nicotinamide adenine dinucleotide (NADH) subunits and cytochrome oxidase 1 (CO1) (Figure 3C). One read mapped to Oculina sp. CO1 (accession no. OQ625380) clusters with the modern Haifa Bay Cladocora caespitosa (Figure 3D).

3.3. Photophysiology

Photophysiological measurements of modern C. caespitosa growing in Haifa Bay at 10 and 30 m (Naharia and Leonid shipwreck, respectively; Figure 1, SI Figure S2), and therefore different light intensities, show that the connectivity parameter, p, a measure of photoprotection (energy transference between photosystems II and I) and the functional absorption of light, σ_PSII (a measure of efficiency) did not differ between the two depths (p > 0.05; Kruskal-Wallis; SI Figure S3A, B). However, total photosynthetic efficiency (F_v/F_m) was significantly lower in 10 m corals and maximum photosynthetic yield (P_max) was significantly lower in 30 m corals (p = 6.182e-08, 1.909e-06; Kruskal-Wallis; respectively Figure 4A, B).

3.4. CSIA-AA

Carbon CSIA-AA analysis of four essential AA (valine, leucine, isoleucine, and phenylalanine) indicates that there is no significant difference between the carbon isotopic signature of the host, symbiont, and skeleton of the contemporary Cladocora (PERMANOVA monte-carlo p=0.53, Figure 4G) indicating resource sharing between the coral host and its symbionts. The sub-fossil coral skeleton δ¹³C of 3 out of 4 of the essential AA is at least 2‰ higher than all the contemporary compartments (Figure 4 C-F).

Similarly to carbon CSIA-AA analysis, the nitrogen CSIA-AA shows no significant difference between the host, symbiont, and skeleton of the contemporary Cladocora (PERMANOVA monte-carlo p=0.66, Fig-5). However, the sub-fossil coral skeleton δ¹⁵N values of individual amino acids are higher than the contemporary ones by an average of 2-13‰ (Fig-5A-I) except for threonine and isoleucine, which fall within their range. Interestingly, the highest difference (>13‰) is observed in source AA phenylalanine (Figure 5G) which is not affected by trophic discrimination in the food web. There is also no significant difference in the trophic position (TP_Glu-Phe) between the host, symbionts, and skeleton of the contemporary coral, and the sub-fossil coral TP_Glu-Phe skeleton is similar to the contemporary one (Figure 5K).

4. Discussion

The Mediterranean Sea is recognized as a long term hotspot of biodiversity, shaped by geological events, climatic fluctuations, and its limited connection to the Atlantic Ocean [64,65]. The region harbors a diverse array of temperate and subtropical species, along with a significant number of endemics [66]. Extensive coral reef systems thrived in the Mediterranean Sea during warmer geological periods, as part of the ancient Tethys more than 200 million years ago [67,68]; however, a major shift in salinity at the end of the Miocene (e.g. six million years ago), termed the Messinian salinity crisis [69] resulted in the near-complete loss of zooxanthellate corals and the disappearance of established shallow-water coral reefs [70]. The sudden opening of Gibraltar filled the Mediterranean again 5.5 million years ago, introducing new scleractinian species to the region [71]. In the present day, shallow rocky seabeds are predominantly covered by frondose algae, while long-lived, filter-feeding organisms prevail in light-deprived benthic communities [72]. The scleractinian coral diversity of the Mediterranean today is relatively low, with 37 representatives [73] compared with over 700 species in the Caribbean and Indo-Pacific, [5].

Since the Messinian crisis, the colonial coral Cladocora caespitosa (Linnaeus, 1767; family Cladocoridae) is the only remnant of ancient Mediterranean reefs, resembling tropical reef-builders in its ability to form reef-like structures named “reef banks” [31,74]. Ancient dead banks of C. caespitosa (dated from around 2500–3000 yr ago) are known from the coasts of Tunisia and Corsica (central Mediterranean) [75]. While it was abundant during the warmer Pleistocene, still forming reef-like structures [46], modern distribution of this taxon has declined dramatically in the eastern Mediterranean while retaining a fairly abundant presence in the central-western Mediterranean (mainly found near the Tunisian coast, Aegean Sea, Spain, France and Croatia [5,29,31,76,77,78], from shallow waters to depths of about 50m [32,79], on both hard and soft bottoms [80]. C. caespitosa forms small-sized globose to hemispherical colonies ranging from a few hundred polyps to larger banks [76,81], typically of compact arrangements of separated polyps that retain relatively short corallite walls [5], as observed in both the modern and sub-fossil corals examined here (Figure 2). However, C. caespitosa colonies can grow as extended and diffuse corallites under extremely low flow conditions ([82]; SI Figure S4). The frequency for smaller colonies may result from high mortality rates or fragmentation due to climate changes [83]. It is a slow-growing coral (2.55 ± 0.79 mm year–1 [32]), and is differentiated from other Cladocora taxa both by its upward-only facing polyps and its limited geographic distribution [5,84]. Hence, macro and micro-morphology, as well as DNA sequencing (SI Table S2D) supports identification of the modern Cladocora examined here as C. caespitosa. C. caespitosa is abundant in the Mediterranean between the surface and 50 m [46,85], the sub-fossil Cladocora likely grew at <5 m depth [43], and shallower depths are typically sites of higher flow and elevated temperature. The compact clustering of the sub-fossil Cladocora colonies’ polyps suggests that they grew in a higher flow environment (Figure 2D,G versus SI Figure S4; [82]). Additionally, the presence of calicular constrictions known as rejuvenescence suggests that this colony survived a past stressor event (Figure 2 I, J) [86]. The ancient specimen’s excellent preservation is evidenced in the nano-granular structure of the septa (Figure 2), also observed in rapid accretion deposits of modern coral skeletons collected live [1]. Both macro- and microstructure of the sub-fossil Cladocora adhere to descriptions for modern C. caespitosa. The intact microstructure and appearance of tissue, combined with evidence of rapid encasement in sediment, support its use in molecular analyses.

Ancient DNA (aDNA) sequencing, along with macro- and micromorphology (Figure 2), supports the sub-fossil Cladocora specimen’s identification and highlights the increasing role that aDNA analysis can play in historical ecological understanding [87], particularly when species preservation is sufficient. For invertebrates, this can be made particularly difficult by the lack of tissue occlusion in the mineral; however, it is not without precedent as aDNA has been sequenced from ornamental mollusk shells and allowed taxonomic identification [88,89]. It is not surprising that the majority of the sequences from even the best quality aDNA extraction in the present study are off-target (Figure 3A, B). In other aDNA studies, as little as <10% of the mapped reads have been aligned to target nuclear and mitochondrial genomes [90,91], including in the original validation of the library preparation method used here [56], with much of the remaining reads mapping to microbial DNA even when steps are taken to remove microbial DNA accepting sacrificial loss of target DNA as well [92].

Of the two species of Cladocora found in the Mediterranean, C. caespitosa is the only one with documented photosymbiotic ability [80]. Its plasticity in its ability to shift between heterotrophy and autotrophy for fixed carbon acquisition is often related to strong seasonal variability in light availability [93,94], water motion as waves and currents, type of substrata, and seafloor morphology [29,31,32]. Highly variable physiology and morphology, and unresolved genetic relationships of the Favidae family, have left genus Cladocora - and many other taxa in the family - grouped loosely together and considered ‘problematic’ in terms of classification [95]. However, recent study supports a close phylogenetic relationship between Oculina patagonica and Cladocora caespitosa, suggesting their placement within the same family, and potentially even the same genus [96]. This variability and unstable phylogenetic classification, combined with its relative scarcity in the eastern Mediterranean, has precluded its previous utility in reconstructing past shallow water ecology in the Levant. However, the regular, fine-scale banding of thickening deposits, typical of symbiotic corals [22,63] observed here both in the modern and sub-fossil coral (Figure 2) suggest that the sub-fossil coral is indeed C. caespitosa.

The coral diet appears to have remained remarkably consistent over the past 2000 years, with a high trophic position (TP) indicating a significant reliance on heterotrophic feeding (here ~2.5 compared with TP ~1 for autotrophy [97]. However, the skeletal δ13C values of sub-fossil Cladocora are notably heavier (more positive) than those of their modern counterparts, likely due to differences in light environment [24]. The decoupling between high Fv/Fm and low Pmax observed at the deeper site (Figure 4A-B) suggests that while the photosynthetic apparatus remains functionally efficient, light or downstream metabolic limitations may constrain the overall carbon fixation capacity [98,99,100,101,102,103]. Under low-light conditions, isotopic discrimination against ¹³C by Rubisco remains high, favoring the assimilation of ¹²C and resulting in lighter δ¹³C values in the coral skeleton. However, the observation of heavier δ¹³C in the sub-fossil samples (Figure 4C-D) indicates that the corals were not experiencing light limitation at the time of growth. Instead, the enriched ¹³C signature suggests that photosynthetic rates were sufficiently high to reduce isotopic discrimination, consistent with an environment characterized by ample light availability. This interpretation is supported by the paleoenvironmental context, as sub-fossil Cladocora colonies likely grew in shallower and less turbid waters, where higher light availability facilitated faster photosynthesis and heavier δ¹³C signatures[100,101,102,103]. Furthermore, the stable δ¹³C values we observe between the two time points ~2000 years apart indicate that the balance between autotrophic and heterotrophic nutrition in the sub-fossil Cladocora has remained fairly constant, despite environmental and human-induced changes.

In contrast, significant shifts are evident in nitrogen isotope signals, with nitrogen being heavier in sub-fossil specimens. This difference can be attributed to two interacting factors: (1) historical documents reveal that during the Middle Ages (Crusader period), the city's sewage was discharged directly into the harbor, creating a nutrient-rich environment that is typically turbid; in general, Hellenistic and Roman cities had impressive sanitary systems that collected the sewage and discharged it away from population centers [104,105,106]. The presence of heavier nitrogen isotope in the sub-fossil coral indicates that sewage was discharged into the port of Akko as early as the Hellenistic period, making anthropogenic land-based nitrogen the primary source for corals then, similar to observations near modern cities experiencing recent rapid growth [107]. (2) The widespread adoption of synthetic fertilizers after 1913, produced using the Haber–Bosch process, which converts atmospheric nitrogen into ammonia, introduced nitrogen with isotopic values close to 0‰. This has contributed to the lowering of nitrogen isotope values observed in modern corals [23,108].

The δ¹⁵N distribution in organic matter preserved within coral skeletons has proven to be a valuable tool for reconstructing historical changes in nitrogen sources. For example, examination of skeletal δ15N in modern Indonesian poritid corals grown between 1970 and 2003 revealed that corals from fertilizer-affected reefs experienced a steady decline in bulk δ¹⁵N values, from ＋10.7‰ to ＋3.5‰, reflecting increased use of chemical fertilizers with low δ¹⁵N values and a reduction in organic manure application in upstream agricultural fields [109]. In contrast, corals and algae from sewage-affected reefs maintained consistently high bulk δ¹⁵N values (>8‰), underscoring the isotopic difference between chemical fertilizers and organic nitrogen sources such as sewage [109]. While changes in bulk δ¹⁵N in coral can be attributed to changes in their heterotrophic diet, CSIA-AA demonstrates that the coral maintained its trophic position. Furthermore, using the source amino acid phenylalanine, which remains unchanged between TP, we show that the modern corals exhibit a relatively low δ¹⁵N value averaging 0.3‰, aligning with the isotopic signature of the Haber–Bosch process. Conversely, sub-fossil corals, unaffected by synthetic fertilizers, show much heavier δ¹⁵N-phenylalanine values, approximately 13‰, resembling those found in sewage-affected environments.

The remarkably high degree of preservation of the sub-fossil C. caespitosa - intact microstructures and lack of secondary infillings [110] and the fact that the corals were found below the Hellenistic quay was likely due to a rapid burial process [43]. This burial was possibly triggered by a sudden singular sedimentation event caused by anthropogenic activity, such as the foundation of the harbor, that could cause changes in sediment movement or other changes inside the harbor, as supported by archaeological evidence from the region [43]. Additionally, the discovery of multiple Cladocora specimens in all the pits that reached the bedrock during the excavation in Akko suggests a historically larger population. In contrast, no Cladocora colonies were documented by the surveys of the Morris Kahn Marine Research Station Long Term Ecological Research program (MKMRS LTER; established in 2014—https://med-lter.haifa.ac il/index.php/en/data-base) conducted along the Israeli coasts which indicate a limited distribution with only two known sites harboring a few colonies each.

The discrepancy between historical and current Cladocora population, along with the rapid increase in temperature in the eastern Mediterranean [36] suggests that the population reduction may be related to climate change, pollution, and habitat destruction [111]. Persistence of the species in the eastern Mediterranean today therefore suggests that the population was able to adapt, migrate locally, or repopulate from sites further west [50], or some combination of these. The species may continue to use these mechanisms today in response to the effects of Anthropocene climate change. Minimal reef accretion observed in the eastern Mediterranean over the past several thousand years precludes coring for a fine-resolution temporal study of ecosystem shifts as performed in tropical locations [e.g., [112]]; however, the present study provides important timepoints and geographic locations of comparison in an anthropogenically dynamic environment [113]. Further, the present and similar recent studies comparing between data sets from modern organisms and ancient environments, utilizing private collections and newly collected specimens as well as emerging sources in museum-held collections and government- and NGO-directed cultural excavations may also serve as lessons for furthering our understanding of how other coral taxa will meet climate change impacts [e.g., [107,114,115].

5. Conclusions

Through the interdisciplinary comparison of a 2000 year old coral skeleton with modern taxonomic brethren, we propose a framework of the ecological structure of Akko port ~2000 years ago. At that time, shallow water Cladocora caespitosa, a zooxanthellate stony coral, settled on appropriately hard substrates at a relatively shallow depth with sufficient water flow to promote the formation of compact colonies. Our results suggest that these corals thrived in a high-flow, nutrient-rich, and turbid environment; however, despite the turbidity, light availability was adequate to sustain efficient photosynthesis, likely due to the colony’s position in water shallow enough to allow sufficient light penetration. The colonies possibly survived one or more stressor events. These corals were mixotrophic, like their modern counterparts, and occupied a similar trophic position, with port water clear enough to support photosymbiosis supplemented by phyto- and zooplankton communities, possibly supported by nutrient-rich effluent from the city of Akko similar to anthropogenic coastal nutrient loading observed today in urbanizing coastal systems [108,116,117]. A sudden catastrophic event entombed the colony studied here, leaving it akin to a hard drive containing much and varied data about the past ecology of the port. Because C. caespitosa colonies are rarely found in the eastern Mediterranean today under similar conditions, the present comparison shows how temperate stony corals, and the structures they provide, respond (adapt, migrate, or die out) to local physical conditions. Similar ecological reconstructions in other locations will also require very well-preserved specimens at both the microscopic (e.g., mineralogical for geochemical analyses) and macroscopic scales as well as modern local analogs for holistic comparison.