Limestone

High-resolution X-ray computed tomography (CT) scans were obtained for the cranium of UMZC T1041, which is the holotype of Mesochelys durlstonensis (Evans & Kemp, 1975 ). UMZC T1041 was found in the Early Cretaceous (Berriasian) Purbeck Limestone Group in Durlston Bay, United Kingdom (Evans & Kemp, 1975 ). Gaffney & Meylan (1988) referred the material to Pleurosternon bullockiiOwen, 1842 , albeit without justification. Milner (2004) compared the shell remains associated with the cranium of Mesochelys durlstonensis with those of Pleurosternon bullockii, and presented anatomical evidence for the synonymy suggested by Gaffney & Meylan (1988) . This has generally been accepted ever since (see review of Joyce & Anquetin (2019) (link)). Scans of UMZC T1041 were obtained by Roger Benson in 2017 at the Cambridge Biotomography Center, using a X-Tek H 225 µCT scanner (Nikon Metrology, Tring, UK). The cranium was scanned using a beam energy of 130 kV, a current of 250 µA, 500 ms exposure time, 1 frame per 1400 projections, and no filter, resulting in a voxel size of 0.03315 mm. The resulting CT-scans were segmented in the software Mimics (v. 16.0–19.0; http://biomedical.materialise.com/mimics), and 3D models were exported as .ply files. Figures of digital renderings were compiled using the software Blender v. 2.71 (www.blender.org/) . CT-slice data as well as the 3D models are deposited at MorphoSource (Evers, 2020 ).
We mostly compare UMZC T1041 to a selection of likely paracryptodires from the Late Jurassic to Cretaceous of North America and Europe based on description published in the literature, in particular Arundelemys dardeni (as described by Lipka et al., 2006 (link)), Compsemys victa (as described by Lyson & Joyce, 2011 ), Dorsetochelys typocardium (as described by Evans & Kemp, 1976 under the name Dorsetochelys delairi), Eubaena cephalica (as described by Rollot, Lyson & Joyce, 2018 (link)), Glyptops ornatus (as described by Gaffney, 1979a under the name Glyptops plicatulus), and Uluops uluops (as described by Carpenter & Bakker, 1990 ). For comparisons with Dorsetochelys typocardium, we used a 3D model of the holotype (DORCM G.00023) that was made available under a CC-BY-NC-SA license by the “GB3D Type Fossils” projects hosted by the British Geological Survey at http://www.3d-fossils.ac.uk. The comparisons with Compsemys victa (based on UCM 53971) and Uluops uluops (based on UCM 49223) were complemented by CT scans of these specimens. The original descriptions are cited for all previously described features, but the specimen numbers are cited when novel observations regarding these taxa are made based on these scans. These scans will be further discussed and made public in a forthcoming paper.

DNA of Begonia species is difficult to extract, especially for those collected from limestone substrates. Additionally, silica-gel dried leaves of Begonia tend to yield poor quality DNA. To circumvent these issues, total genomic DNA was extracted from fresh leaves of living collections using a modified CTAB protocol optimized for Begonia (Kopperud and Einset1995 (link)).
Two DNA sequence regions were used: the nuclear ribosomal DNA (nrDNA) internal transcribed spacer (ITS) region and the chloroplast DNA rpL16 intron. Previous analyses based on ITS (e.g., Tebbitt et al.2006 (link)) suggested its usefulness in resolving interspecific relationships while chloroplast sequences were more suitable for reconstructing the backbone structure of the Asian Begonia phylogeny (Thomas et al.2011a (link)). Although the rpL16 intron has thus far never been used in reconstructing the Begonia phylogeny, this region amplified and sequenced easily and shows adequate phylogenetic resolution in Asian Begonia. For each polymerase chain reaction (PCR), amplification was performed in a total volume of 25 μl, including 12.5 μl of Taq DNA Polymerase Master Mix Red (Ampliqon, Copenhagen, Denmark), 1 μl of each forward and reverse primer (10 μM), 2 μl of template DNA, and 8.5 μl of ddH₂O. For ITS, the primers 5P and 26S1Rev primers (Clement et al.2004 (link)) were used for both PCR amplification and sequencing. For rpL16 intron, three primers (rpL16-F: GCT ATG CTT AGT GTG TGA CTC G; rpL16-R: CGT CCY GCT TCT ATT TGT CTA G; Beg_rpL16: GTT TCA CAT TAT CTG GAT CG) were designed to optimize PCR amplification (rpL16-F and rpL16-R) and sequencing (Beg_rpL16 and rpL16-R) in Begonia. PCR reactions were carried out by a denaturation-step in 94°C for 5 min, 30 thermo-cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 90 s (60 s for ITS), and a final extension in 72°C for 5 min. PCR products were purified using QIAquick PCR purification Kit (Qiagen, Valencia, California, U.S.A.) and then sequenced using an ABI PRISM dye terminator cycle sequencer, model 3700 (Applied Biosystems, Foster City, California, U.S.A.).

All skin and prey samples were lipid-extracted, lyophilized, and homogenized by grinding them into a fine powder; as noted above the small set of subsamples that were analyzed to test the effects of lipid-extraction were not lipid-extracted (bulk tissue). Baleen plates were cleaned with a solution of 2:1 chloroform:methanol to remove surface contaminants. Sub-samples of keratin powder were collected with a Dremel rotatory drill fitted to a flexible engraving shaft at 1 cm intervals along the outer edge of each baleen, starting at the proximal section inserted in the gum (which represents the newest tissue) (Fig 2B). Baleen grows uniformly on the transverse perspective at a constant (but unknown) rate; thus our sampling strategy would yield equal time intervals between adjacent sub-samples [37 (link),42 –44 ,55 ,57 ,82 ]. Previous studies have confirmed the consistency of isotope values along the length of two adjacent baleen plates of a gray whale (Eschrichtius robusutu) [82 ] and two plates from opposing sides of the mouth of a bowhead whale (Balaena mysticetus) [43 ]. Consequently, we assumed that each baleen provides a consistent record of the past foraging history for each blue whale. Lastly, we compiled δ¹³C and δ¹⁵N data from the literature of blue whale prey from foraging zones in the northeast Pacific (S2 Table).
Approximately 0.5–0.6 mg of each tissue sample (dried skin, baleen, and prey) was weighed into a tin capsule. Carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope values were measured with a Costech 4010 elemental analyzer coupled to Thermo Scientific Delta V isotope ratio mass spectrometer at the Center for Stable Isotopes at the University of New Mexico (Albuquerque, NM). Isotope data are reported as delta δ values, δ¹³C or δ¹⁵N = 1000 [(Rsample / Rstandard)—1], where R = ¹³C/¹²C or ¹⁵N/¹⁴N ratio of sample and standard [83 ]. Values are in units of parts per thousand or per mil (‰) and the internationally accepted standards are atmospheric N₂ for δ¹⁵N and Vienna-Pee Dee Belemnite limestone (V-PDB) for δ¹³C [83 ]. Within-run analytical precision was estimated via analysis of two proteinaceous internal reference materials, which was ±0.2‰ for both δ¹³C and δ¹⁵N values. We also measured the weight percent carbon and nitrogen concentration of each sample and used the C/N ratio as a proxy of lipid content [84 (link)].

The study area of 1115.3
km² is located in Northwestern Turkey within the Çanakkale
province (Figure 1).
Kirazlı village is located about 40 km southeast of the city
center and around the Biga Peninsula, which is an active tectonic
region. Mountainous topography features are seen in the region. Kirazlı
Mountain is the most important hill in the region, 811 m above the
sea level and covered with forests, which provides the main means
of livelihood for the local people. In this peninsula, alternating
reddish-yellow-white-colored volcanic and sedimentary rock formations
are commonly seen.^{41 (link)} The former formations
are altered Neogene-age sedimentary covered with sand, silt, and clay,^{16 (link)} and both formations are covered by quaternary
alluvium, including sand and gravel grains. In the rock structures
of the region, lead (Pb)–zinc (Zn)–copper (Cu) and gold
(Au) metal deposits and industrial minerals such as clay (Al₂O₃·2SiO₂·2H₂O), coal,
and kaolinite (Al₂Si₂O₅(OH)₄) have been identified.^{42 (link)}In Çanakkale, Biga and some nearby towns
(Yenice, Can, and
Lapseki) are known for having a total of 204 metallic mineral deposits,
and the most important ones are Cu, Pb, Zn, antimony (Sb), and gold
(Au) reserves. Volcanic units at Kirazlı belong to the Miocene
age, which host alternating zones and precious metal mineralization
and contain feldspar, mafic minerals, and some quartz. The enrichment
of metals is Al + K in the argillic and Mg + Ca + Fe in the propylitic
alteration types. Moreover, two Au mineral deposit reserve places
are found—Kartal Dag and Maden Dag—and deposits of Fe
and Mn also have found been as small mass reserves. Environmental
changes (causing geogenic interaction between soil and water) affect
the enrichment and leaching of metals; for example, Ca, Mg, and Fe
were leached during argillic alteration, whereas strong Na leaching
is evident in all alteration types.^{43 (link)}The hydrogeology of the Kirazlı region generally comprises
volcanic units. Most of the springs in the study area are between
the silicified zone and the argillic zone. Several springs surface
from volcanic soils such as tuff and agglomerate in the Biga Peninsula.
These springs have flow rates between 0.01 and 3 L/s. In the region
Çanakkale and Koca streams discharge into the Atikhisar Reservoir,
which serves the water supply system of Çanakkale city.^{41 (link)} Generally, the main alluvial aquifers in the
region serve as the main water resources.^{41 (link)} As seen in Figure 1, the study area has three types of geological structures. J1, J2,
and J3 represent, respectively, high mineral soil, low mineral soil,
and alluvial soil. While J1 includes evaporite mineral sedimentary
rocks such as gypsum and carbonates with high solubility only in acidic
waters, travertine, caliche, limestone, marble, and calcschist formations,
J2 consists of aluminum silicate-containing soils, conglomerates,
sandstone, and silica-predominant formations.⁴⁴ X and Y in Figure 1 indicate the geologic coordinates, whereas W and S indicate water
and rock samples, respectively. The peninsula is in the Mediterranean
and Black Sea transition zone, affecting climate characteristics,
with summers being hot and dry and winters being cold and rainy. Maximum
precipitation is observed during the winter, whereas the least precipitation
is observed during summer.^{42 (link)}