Shape, Microstructure, and Chemical Composition of Pearls from the Freshwater Clam Diplodon chilensis Native to South America

Simple Summary Natural pearls of Diplodon chilensis, a freshwater clam native to southern South America, are reported and characterized for the first time. The finding also constitutes the first record of pearls in a species of the genus Diplodon. The pearls have different shapes and sizes, and were found in both, male and female specimens. The microstructure and chemical composition of pearls is consistent with those reported in other bivalve species. Abstract The capability to produce pearls is widespread in the phylum Mollusca, including bivalves of the superfamily Unionoidea. Here, we identified and characterized natural pearls formed by Diplodon chilensis, a freshwater clam native to southern South America, using samples obtained from two lakes located in the Chilean Patagonia. Pearls were studied using light and scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), Fourier transform infrared spectroscopy (FTIR), and Raman spectroscopy. Naturally formed pearls were found in both male and female D. chilensis specimens. Pearls are produced in different shapes, including spherical, ellipsoidal, buttoned, and bumpy, ranging in size from 200 µm to 1.9 mm. The internal microstructure is composed of irregular polygonal tablets, about 0.40 to 0.55 μm in thickness. EDX analysis showed that pearls are composed of calcium carbonate. FTIR and Raman spectra recorded several peaks attributable to the aragonite in pearls of this species, as has been shown in other mollusks. In addition to these results, pearls of different colors are illustrated.


Introduction
Mollusks represent the second group with the largest number of species after the arthropods, reaching a diversity of between 85,000 and 120,000 species [1,2]. In addition to this high diversity, mollusks have a great variety of body plans [3], which has allowed them to colonize different environments across all climatic zones. Since ancient times, these animals have been used as food sources by humans [4,5], as demonstrated by the large number of archaeological records around the world [6,7].
Traditionally, many species of mollusks have been useful for human societies in religious ceremonies, folk medicine, building tools, the production of buttons, fertilizers, cattle feed, decoration, and ornamental purposes [8][9][10][11][12][13]. Moreover, shellfishing and cultured pearl production have been and continue to be important sources of income for the inhabitants of different countries, and in many parts of the world, some species have been included in commercial aquaculture [14,15].
Several species within the classes Bivalvia, Gastropoda, and Cephalopoda can produce pearls, mainly bivalves. The soft bodies of oysters, clams, mussels, and snails are covered by the mantle, a thin epithelial tissue that covers the body organs and secretes the molluscan shell, which is comprised of calcium carbonate and made up of two to five different layers

Materials and Methods
Adult D. chilensis individuals were obtained in January 2022 from a shellfisher in Playa Negra, Lake Caburgua (n = 100) and by a fisherman at the source of the Toltén River, Lake Villarrica, Southern Chile (n = 49) ( Figure 1). The clams from both lakes were assigned to D. chilensis since (i) our samples were obtained within the species range, (ii) it inhabits Lake Villarrica [32,33], (iii) the current nonexistence of Diplodon chilensis patagonicus (d'Orbigny, 1846) populations in Chile [34][35][36][37], and (iv) the absence of characters that differentiate the populations of both lakes. Shell length was measured (in mm) using a vernier caliper (precision 0.01 mm). The clams were dissected using a Motic SMZ-168 stereoscopic microscope and sexed by microscopic examination of gonad smears using a Leica light microscope. The pearls were isolated from the mantle tissue using surgical material, washed with distilled water, and observed using a Hitachi 3500 scanning electron microscope (SEM). SEM was coupled with Bruker model Quantax 100 energy dispersive X-ray spectroscopy (EDX) for the chemical determination of the samples through elemental mapping. Pearls from different individuals were wrapped in paper and then broken with the blunt part of a dissecting needle to observe the internal microstructure using SEM.
Representative pearls were analyzed with a 4 cm −1 resolution using a PerkinElmer Spectrum Two FTIR spectrophotometer. Pearls were also analyzed using the Ocean Onsight Raman spectroscopy model QEPRO-RAMAN-785-PLUS to detect the type of CaCO 3 polymorph present in the sample. The measurements were acquired with an excitation wavelength of 785 nm at 0.890 nW. The spectra were obtained with an 11 cm −1 optical resolution in a spectral range of 0-2000 cm −1 . Representative pearls were analyzed with a 4 cm −1 resolution using a PerkinElmer Spectrum Two FTIR spectrophotometer. Pearls were also analyzed using the Ocean Onsight Raman spectroscopy model QEPRO-RAMAN-785-PLUS to detect the type of CaCO3 polymorph present in the sample. The measurements were acquired with an excitation wavelength of 785 nm at 0.890 nW. The spectra were obtained with an 11 cm −1 optical resolution in a spectral range of 0-2000 cm −1 .

Results
Pearls of D. chilensis were found covered by the animal's mantle tissue ( Figure 2) and were isolated by making a fine cut on it. Pearls were found in both male and female clams and were of different shapes, including spherical, elliptical, buttoned, or with bumps ("baroque"). With the electron microscope, we observed the external appearance of the pearls, indicating that most of them were smooth ( Figure 3). However, some of them had small slits or holes as well as an irregular surface. Most pearls were white or grayish white in color, although a few silvers, light blue, pinkish, or brown were also found ( Figure 4). In some clams, there were small pearls in the adductor muscles. The size of the pearls varied between 200 µm and 1.9 mm (Table S1). In Lake Caburgua, we recorded 1% of clams with pearls, while in Lake Villarrica, we recorded 18.4%. In this lake, the smallest individual with a pearl had a length of 41.9 mm. The largest pearl (1.9 mm) was found in a 47.2 mm individual. Only one pearl (924.0 µm) was found in Lake Caburgua in a 65.6 mm individual.

Results
Pearls of D. chilensis were found covered by the animal's mantle tissue ( Figure 2) and were isolated by making a fine cut on it. Pearls were found in both male and female clams and were of different shapes, including spherical, elliptical, buttoned, or with bumps ("baroque"). With the electron microscope, we observed the external appearance of the pearls, indicating that most of them were smooth ( Figure 3). However, some of them had small slits or holes as well as an irregular surface. Most pearls were white or grayish white in color, although a few silvers, light blue, pinkish, or brown were also found ( Figure 4). In some clams, there were small pearls in the adductor muscles. The size of the pearls varied between 200 µm and 1.9 mm (Table S1). In Lake Caburgua, we recorded 1% of clams with pearls, while in Lake Villarrica, we recorded 18.4%. In this lake, the smallest individual with a pearl had a length of 41.9 mm. The largest pearl (1.9 mm) was found in a 47.2 mm individual. Only one pearl (924.0 µm) was found in Lake Caburgua in a 65.6 mm individual.      Scanning electron microscopy images indicate that the internal microstructure of D. chilensis pearls is composed of irregular polygonal tablets about 0.4 to 0.55 µm thick. EDX analysis revealed that they are composed principally of Ca, C, and O in stoichiometric amounts indicative of CaCO3 ( Figure 5, Tables 1, S2-S4). The FTIR spectrum showed signals located between 1410 and 1255 cm −1 , at 1065 cm -1 and between 2800 and 3000 cm −1 ( Figure 6). Raman spectra of pearls from different individuals present the main bands at 153, 206, 411, and 1085 cm −1 (Figure 6). Other bands of lesser intensity occurred at 701, 705, and 1340-1540 cm −1 .  Scanning electron microscopy images indicate that the internal microstructure of D. chilensis pearls is composed of irregular polygonal tablets about 0.4 to 0.55 µm thick. EDX analysis revealed that they are composed principally of Ca, C, and O in stoichiometric amounts indicative of CaCO 3 ( Figure 5, Tables 1 and S2-S4). The FTIR spectrum showed signals located between 1410 and 1255 cm −1 , at 1065 cm -1 and between 2800 and 3000 cm −1 ( Figure 6). Raman spectra of pearls from different individuals present the main bands at 153, 206, 411, and 1085 cm −1 (Figure 6). Other bands of lesser intensity occurred at 701, 705, and 1340-1540 cm −1 .

Discussion
In this study, the finding of natural pearls in two lacustric populations of the freshwater clam D. chilensis from southern Chile is reported for the first time, also constituting the first report made in the genus Diplodon. Diplodon chilensis can produce pearls in a

Discussion
In this study, the finding of natural pearls in two lacustric populations of the freshwater clam D. chilensis from southern Chile is reported for the first time, also constituting the first report made in the genus Diplodon. Diplodon chilensis can produce pearls in a

Discussion
In this study, the finding of natural pearls in two lacustric populations of the freshwater clam D. chilensis from southern Chile is reported for the first time, also constituting the first report made in the genus Diplodon. Diplodon chilensis can produce pearls in a variety of shapes, sizes, and colors. Pearl production in this species is not a rare phenomenon since many species of the superfamily Unionoidea Rafinesque, 1820, are potential producers of pearls [40]. However, in South America, only a few species have been described with this capability [23,41,42].
In a way, the finding of pearls in D. chilensis is quite surprising since this species has been relatively well studied in Chile and Argentina regarding its soft body [43][44][45][46][47][48][49], but until now the finding of gems had not been reported. In our lab, dissections of clams quickly revealed the presence of conspicuous pearls in the mantle tissue of adult individuals of this species. This could be indicative of differences between D. chilensis populations in pearl production, and, in fact, our results show that natural pearl formation is more common in Lake Villarrica than Lake Caburgua. It is unknown if these differences between populations in both water bodies have a genetic basis. It has been postulated that variations in certain life history traits between D. chilensis populations from lotic and lentic environments could be due to genetic differences [49], although a karyological study contradicted this hypothesis [50]. Consistent with the latter, it has been reported that the populations of this clam from different basins near the Pacific coast in southern Chile are not genetically structured [51], and that differences found in the growth rates can be associated with geographic and limnological parameters [32].
Our results suggest that the shell size does not seem to influence the ability to produce pearls because several individuals from Lake Villarrica in the 4.0 to 5.0 cm size class produced pearls, while twice or three times as many individuals from Lake Caburgua between 5.0 and 7.0 cm did not, except for the 65.6 mm specimen. According to Hohn and Costa [42], acidic waters prevent the proper formation of pearls in bivalves, but this would not be the case since the waters of lakes Caburgua and Villarrica have very similar pH [52], although some variation has been reported in this water body [53]. Rahman et al. [54] detected seven species of pearl-producing bivalves in waters off the coast of Bangladesh with a pH of 8.1 to 8.3.
The size of the pearls varies between mollusk species. The largest natural pearl found in the present study measured 1.9 mm. Pearls with similar sizes have been found in other bivalve species from South America with relatively similar shell lengths. Triplodon corrugatus (Lamarck, 1819) produces pearls from 2 to 3 mm, Castalia ambigua Lamarck, 1819, around 2 mm, and Prisodon obliquus Schumager, 1871, from 2.5 to 4 mm [23]. In Placuna placenta Linnaeus, 1758, from India, the diameter of pearls varies from 1.5 to 4 mm [54]. The cultured pearls in Mercenaria mercenaria (Linnaeus, 1758) and Pinctada margaritifera (Linnaeus, 1758) range from 9 to 13 mm, while in Venerupis aff. Decussata (Linnaeus, 1758), they generally do not exceed 6 mm [55,56]. In the production of cultured pearls, a typical 9-mm round non-beaded pearl takes about 4 years to form [42].
Peaks of the FTIR spectrum located between 1410 and 1255 cm −1 have been associated with the vibrations of the carbonyl group (C=O) while that at 1065 cm −1 with carbon-oxygen (C-O) confirming the presence of carbonated compounds (CO 3 =) in the samples [57,58]. Signals located between 2800 and 3000 cm −1 correspond to carbon-hydrogen (C-H) vibrations that can be attributed to the organic moiety present in the samples [59]. The main peak of pearls in D. chilensis obtained by Raman at 1085 cm -1 is the main vibration of the CO 3 = molecule in carbonates [60], which is also present in pearls of other mollusk species [22]. Bands at 1084-1087 cm −1 have been attributed to aragonite or calcite in different bivalve species [61][62][63][64][65][66]. However, distinguishing between the three carbonate polymorphs (aragonite, vaterite, and calcite) using only the most intense band v1 (symmetric stretching) located approximately at 1085 cm −1 is not enough to identify a particular phase [39]. This led some authors to focus on the v2, v3, and v4 vibrational modes of CO 3 = depicting peaks around 850-900, 1430-1600, and 680-750 cm −1 , respectively, to characterize some of the three phases [39,67]. Thus, the v4 band (a doublet) assigned to the out-of-plane vibrational modes of CO 3 = located at 701 and 705 cm −1 and peaks identified in bivalves at 153 and 206 cm −1 have been attributed to aragonite [22,39,63,[67][68][69][70][71], all of them detected and quantified in D. chilensis pearls ( Figure 5). No vaterite or calcite could be identified in this clam.
The causes of the variation in the color of pearls are still a subject of investigation [56,71,72]. Bands obtained near or between 1080, 1135, and 1530 cm −1 using Raman analysis, among other bands, suggest that the color of the pearls in several species of bivalves might be due to different chemical compounds, pigments, or optical effects of the sample [56,61,[71][72][73][74][75]. However, not all species that have pearls of different colors exhibit these bands [72,76]. On the other hand, there are also other factors involved in the color of pearls, including geographics, genetics, type of mollusk (species), harvest season, water characteristics, depth, quality, and quantity of food (plankton), and the thickness of the pearl [24,42,75,[77][78][79][80][81].
To improve the quality of cultured pearls, current trends show that the production of pearls has turned from the frequent improvement of traditional cultivation techniques to producing pearls through selective mollusk breeding [82]. In addition, several studies have detected genes related to the production of pearls in different species of the group, so this economic activity seems to have a very encouraging future [82][83][84]. In this context, the sequencing of the genomes and transcriptomes of some mollusk species [85][86][87][88][89][90] will be important for the identification of genes involved in shell and pearl biomineralization. To date, in D. chilensis, no progress has been made in this aspect of its biology.
Diplodon chilensis, an efficient filter feeder capable of depleting phytoplankton and bacteria from the water column [91][92][93], is one of the most abundant bivalves distributed in Patagonia [94][95][96]. However, this clam has been considered a threatened species in many Chilean water bodies since various localities in the central-southern parts of the country have experienced a decline in densities and even the disappearance of banks due to anthropic activities [32,50,96,97]. A drastic reduction in population density has also been reported in some lacustric towns in the Argentine Patagonia due to water pollution [98]. Despite these situations, the species has been classified as Least Concern (LC) by the IUNC Red List of Threatened Species as it has a widespread distribution throughout Chile and Argentina [99].
The culture of D. chilensis has not been implemented in Chile or Argentina. Although the species is not traded for food or any other purpose in the formal markets of Chile, it is edible and sold in informal urban and rural markets in the south of the country, considering the personal observation of the first author and reports by locals [100]. The use of the species as food is also evidenced by the presence of adult shells deposited in pre-Hispanic shell mounds in Patagonia [100][101][102]. The results reported in the present study may be the first step in boosting pearl production on culture farms in the country.

Conclusions
In this article, we provide a description of the main characteristics of the natural pearls produced by the freshwater clam Diplodon chilensis, considering shape, microstructure, and chemical composition. This is the first record of pearls produced in the genus Diplodon. Further studies are needed to investigate whether there are population differences in pearl production and culture feasibility for the species.