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Emendations and additions to “Paleomicrobiology: Revealing fecal microbiomes of ancient indigenous cultures”, the first indications of ancient fungiculture, and “PreCoprolite” a proposed new archaeological term

Posted by ErnestWilliams on 08 May 2015 at 15:21 GMT

Emendations and additions to “Paleomicrobiology: Revealing fecal microbiomes of ancient indigenous cultures”, the first indications of ancient fungiculture, and “PreCoprolite” a proposed new archaeological term

Ernest H. Williams, Jr.1, Lucy Bunley-Williams2, and Erilene X. Garcia-Roldan2
Department of Marine Sciences1, and Department of Biology2, PO Box 9000, University of Puerto Rico, Mayagüez, Puerto Rico 00680-9000

I (LBW) was very reluctant to make corrections on my own published paper, but since I did not see the manuscript before publication and EXGR only saw a preliminary draft, the Journal has an unusual (reversed?) post-publication review process, and some corrections are needed to improve the paper, I was so encouraged. We do not know if our emendations should be listed as “comments” and/or “corrections”? These revisions are presented to improve the quality and usefulness of the existing paper.

General: The term and adjective “fossil” should be removed or replaced throughout the paper. The term “fossil” has an arbitrary minimum age of 10,000 years before present. The archaeological coprolites in this paper are less than 10,000 years old, and therefore, are not fossils. The term “fossil” has been horrendously and shamelessly misused even in the scientific literature.
Some authors have suggested that archaeological coprolites are “partially fossilized” whatever that means. Actually, these are subfossils both because they have not been fossilized and because they are less than 10 thousand years old. Subfossils retain ample organic materials that have not been mineralized, fossils do not.

Title: Metazoan parasites cannot be included in “microbiomes” because they are not microbial. A more accurate title might be “Paleomicrobiology and Paleoparasitology: Revealing fecal microbiomes and metazoan parasites of ancient indigenous cultures”.

Authors’ Addresses: EGR was in the UPR Biology Department in Mayagüez, not San Juan. LBW’s “Mayaguez Campus” is in Mayagüez, not San Juan.

Introduction: Some mention of parasites or parasitology would be useful. Add: We examined archaeological coprolites for indications of parasites to compare the Huecoid and Saladoid cultures, compliment the microbiome portion of this study, and place Puerto Rico in the new world synthesis of prehistoric archaeological coprolite studies. EGR will publish a paper will more details of the parasites and their significance.

Materials and Methods
Sample Description, Lines 3-4: “A total of thirty-four coprolites were used in this study.” Actually only 15 coprolites were examined in this study. The microbiome analysis was conducted on 15 coprolites all from Vieques. Parasite analysis was conducted on all 34 available coprolites, but results from only 15 from Viequez appear to have been reported in the paper [1].

Lines 5-6: “Five of the Saladoid samples used were from Sorce, Vieques and the remaining from Tecla, Guayanilla.” Actually 7 of the Saladoid coprolites were from Vieques (Table 5 [1] lists 7).
Table 1: Why and how were 4 of the available Huecoid coprolites from Vieques not examined? Apparently, 8 of the 12 Huecoid and all 7 of the Saladoid coprolites from Vieques were chosen for microbiome analysis, but this was not explained [1].
Microscopic analysis for Parasite Eggs, Line 1: “A total of 34 coprolites were used to search for parasites in both cultures.” However, results were only shown for the examination of 15 coprolites in Results, Eukaryotic Parasites in Coprolites.

Results
Eukaryotic Parasites in Coprolites, Line 3: “There were twice as many infected Saladoid coprolites as there were Huecoid.” This statement is not correct. The parasite prevalencies in the Huecoid coprolites on Vieques was 41.6% and those of the Saladoid on Vieques 71.4%. To have twice as many the Saladoid infected coprolites, those from Guayanilla, Puerto Rico, (93.3%) would have to be combined (86.3%) with the Saladoid coprolites from Vieques. Using the Guayanilla data only once in the paper without explanation is very confusing. Not to mention without justification in combining different data sets.
Line 4: “Ascaris lumbricoides and Trichuris trichura were found in both cultures[,] but with greater number among the Saladoids.” The term “greater number” is confusing. We assume they mean “intensity.”
Table 5: This table apparently only compares coprolites examined from Vieques. However, this is never explained. None of their numbers agree with the original parasite data [2] in any way we can conceive. Their “percent positive” = prevalence. “Average number present” is confusing since it could be “mean intensity” (average per infected coprolite) or could be “mean abundance” (average per all examined coprolites). This table gives the impression that only 15 coprolites were examined for parasites, but all 34 were examined.

Discussion
Key observed differences in each culture's core microbiomes: Inferences on diet, Line 9: “amoebic parasites (Goussia spp.)” = coccidian pseudoparasites (Eimeria spp.) [3]. The report [1] does represent important new locality records for the group of Eimeria spp., which were formerly in Goussia, for Puerto Rico, the Caribbean, and the West Indies, however unnoted [1]. This is the first report of fish Eimeria in human coprolites. A number of mammal Eimeria have been found in human coprolites as pseudoparasites, e.g., [4].
The Eimeria spp., which were formerly in Goussia, are mostly parasites of freshwater fishes, but some species are found in marine fishes, frogs [5] (amphibian), geckos [6] (reptile), and insects and arachnids [6]. They are not known to infect mammals. A freshwater fish host was suggested in the paper. If we assume this was correct and consider the very limited freshwater fish fauna available in Vieques Island at the time [7], then this species of Eimeria occurred in a new host family. However, this could represent E. centropomi, (described in Goussia), which has only been reported from Florida (USA) [8], [9], but the reported host, Common Snook, Centropomis undecimalis, occurs in Puerto Rico [7]. We have examined specimens of all of the freshwater [7] and many of the marine species of fishes of Puerto Rico [10] extensively for parasites, bacteria, and fungi [11]; however, we seldom examined for coccidians unless lesions were present).
Line 11: Vibrio sp. is generally a marine, not a freshwater bacterium; therefore, it was probably from marine seafood rather than freshwater fishes.
Line 12: The Asian Lungworm, Paragonimus westermani, is the most widespread lungworm in humans. But it was originally found only in Asia, India, Phillipines, and New Guinea. A prehistoric occurrence in Puerto Rico would be unlikely. EXGR explained to the other authors that this identification had not been confirmed.
The eggs found in the present study could represent the Latin American Lungworm, P. mexicanus, which also infects humans in Central and South America. However, the egg morphologies of P. westermani and P. mexicanus differ. Therefore, this is probably The “North American” Lungworm, P. kellicotti, which has a similar egg morphology to P. westermani, infects humans, exists in Venezuela [12], and was probably brought to Puerto Rico from South America. This finding is important as the first locality record in Puerto Rico and further supporting the current suggestion that P. kellicotti occurs in South America [13].
The sensitivity of a fresh stool examination for Paragonimus eggs in an infected person is very low [14], [15]. The sensitivity in coprolites is likely much lower; therefore, the prevalence of P. kellicotti could have been higher than this study indicated.
Molecular characterizations of P. kellicotti from Missouri (USA) [16] and P. mexicanus from Guatemala and Equador [17] have been conducted. However, the molecular studies [1] did not examine for metazoan parasites. Our identification of P. kellicotti could be easily confirmed molecularly with eggs from the remaining samples.
Lines 15-17: “ … Paragonimus westermani … is commonly morphologicaly confused with [the Broad Fish Tapeworm,] Diphyobothrium latum.” This statement is very confusing. How can a mammal lung digenean be confused with a fish intestinal cestode? We can only assume this refers to their eggs. This could not have been D. latum because it only occurs in cool, temperate fishes, not tropical ones.
Lines 19-23: “Our results suggest that this culture may have helped introduce some of these maize strains to the Antilles during their migrations. Since they were detected in large proportions, Ascomycetes and Basidiomycetes also appear to have been important dietary elements of these cultures. However, it seems that the Huecoids had a preference for Basidiomycota fungi.” Ascomycetes are Sac Fungi and include morels, truffles, cup fungi and powdery mildews. Basidiomycetes are Club Fungi and include mushrooms, puff-balls, smuts, rusts and toadstools.
The “Basidiomycota fungi” Consumed by Huecoids could represent Corn Smut, Ustilago maydis, which was eaten by the ancient Aztecs as “Cuitlacoche” and is a delicacy in Mexico called “Huitlacoche” (Nahauti into Spanish). It has been eaten since precolombian times and has a flavor and texture similar to truffles.
However, this is a microfungal plant parasite, which does not produce sufficient material to constitute an “important dietary element.” Ustilago maydis can occur commonly, but never abundantly, and is seasonal. Macrofungi with large and visible fruiting bodies are the only ones sufficiently voluminous to be important in the diet.
Native Puerto Rican fungi are not sufficiently abundant and large to have been “important dietary elements” for Huecoids and Saladoids [18], [19]. This suggests hey must have raised fungi. This is not surprising for agricultural peoples, who cultured their own specific kinds of maiz [1]. Ancient fungiculture has long been suspected [18], but this is the first substantial indication of its occurrence.
We doubt they came to a strange land carrying kinds of maiz that were important to them, but without fungi that were also very important. They would not hunt for new, unknown, fungi to raise; they used some that they knew well, tasted good, was easy to culture, and produced a lot of material. Therefore, we suggest this indicates the first known importation of fungi from South America to the Caribbean.
The use of fungi in the ancient times is well documented; however, fungiculture has not been previously documented [18]. If fungiculture was conducted in Puerto Rico by the Huecoids and Saladoids, this indicates that the technology existed earlier in South America and was transferred.

Enteric Parasite infections, Lines 2-3: “… exposed to fecal material and thus parasite transmission.” Fecal exposure causes many diseases, but can only transmit the 4 species of nematodes (Giant Roundworm, Ascaris lumbricoides; Hookworm; Pinworm, Enterobius vermicularis; and Whipworm, Trichuris trichura) found in this study. The other 7 species of cestode, coccodia, digenean, and nematode parasites have more complex life cycles and 6 have intermediate hosts and any increases with them do not indicate fecal contamination. Only increases in the 4 nematode parasites would be an indication of fecal contamination.
Hookworms eggs cannot be identified morphologically to species and hookworms of European and Asian pre-Columbian origin have been found in South America [20].
Lines 9-10: “Dog … eat human feces … dogs being the vectors.” Dogs feeding on human feces cannot spread the reported parasites to humans. Even the 4 nematode species with direct life cycles require either a period in the soil (N=4) or are transmitted through direct contact among humans (E. vermicularis). They are not vectored directly from feces. Dogs only indirectly carry the Double-pored Dog Tapeworm, Dipylidium caninum (in their fleas). Humans must eat dog fleas infected with the intermediate stage of this parasite to become infected. Dogs were not vectors and did not cause the higher prevalence of Saladoid parasites.
Lines 13-16: “The presence of Dipylidium caninum supports this hypothesis, as does the high prevalence of most intestinal parasites found in the Saladoid coprolites.” The hypothesis referred to is unclear and the statement “parasites are passed from person to person with dogs being the vector” is incorrect.
Line 15: “zoonotic parasites” apparently refers only to D. caninum, but “Goussia spp.” [=Emeria] sp.] from freshwater fish (if one counts pseudoparasites) and Trichostrongylus sp. from herbivorous stock, also represent zoonosis.
Lines 16-18: “It is also intriguing that well-formed coprolites had such a high prevalence of enteric parasites, since many of these pathogens result in enteritis in present day populations.” Most all infections with the metazoan parasites listed in this study are asymptomatic in modern cases.
Lines 19-20: “… possible that multiple infections with parasites were common, and yet these infections failed to cause enteritis.” Metazoan parasite infections listed in this study do not cause enteritis. Rarely, infections with Dipylidium caninum can cause enteritis [21].

Differences in coprolite microbiota and the “Huecoid problem,” paragraph 1, last line: “Interaction with pets [= dogs]” only explains D. caninum, infection. None of the other listed parasites are even indirectly spread by dogs.


Reader (Dr. J. Allen Dove, Science Columnist) Comment 1: This comment is partially correct [22]. The term “fossil” should be removed throughout the paper and the term “coprolite” is incorrect. However, the term “coprolite” has sadly become an accepted misnomer in archaeology. We understand the reader’s frustration [23].
Author (Gary A. Toranzos) Reply 1: This comment is also partially true, if evasive. The term “coprolite” was useful in this paper, when not modified by “fossil” [24].
The term Coprolite is from the Greek Kopros (dung) and Lithikos (stone), which means “dung stone” (petrified fecal units), was coined by William Buckland in 1829 [25]. Paleontologists properly use the term for petrified, fossil, millions of year old, feces [26]. However, about 55 years ago archaeologists began using the same term for dried, intact, subfossil, hundred to thousands of years old, feces [24] and this stolen term has been widely used.
Paleontological coprolites and archaeological coprolites are preserved in completely different ways, are composed of different materials, are studied with different methods, and are of vastly different ages. No animal responsible for a paleontological coprolite has ever been responsible for an archaeological coprolite. Thus there is no similarity in their creation, composition, space, time, or field of study. They are completely different entities with the same name.
Science abhors homonyms. The same name should not be used for 2 very different things (rock vs. dried poop). Reinhard and Bryant [27] adequately describe the principal differences between these 2 entities, then strangely stated they are the same thing without discussion or justification. “Paleofeces” has been suggested as a replacement name for the “archaeological coprolite” [22]. However, this is a general term for any old fecal material, not necessarily whole preserved units containing useful, biological materials. The terms “coprolite” and “paleofeces” are used in the same archaeological papers demonstrating that they are distinct terms, e.g., [20]. Paleontologists, archaeologists, and other scientists use the term “coprolite” without definition or modification. Sometimes the kind of coprolite can be discerned from the context, sometimes not, and this is often hopeless in the general literature. A more specific term for the archaeological “coprolite” is required.
As paleontologists used the term “coporite” first (1829 vs. 1960) and often, they have priority. Since the term “coprolite” is already in use by archaeologists, we suggest a only a slight modification of this term to “PreCoprolite” as a proper term to replace the archaeological use of the term “coprolite.” This is from Prae (Latin) for “before.” This action would not only resolve the homonym, but would connect these forms as useful, logical units in a time line. PreCoprolites are definitely before coprolites in the timeline.
PreCoprolites are much more numerous than coprolites, although coprolites are common enough to have been mined for phosphorus [28]. Older precoprolites (50 thousand [29] and possibly 1 million years [26]) become partially mineralized [27], but can never become coprolites; therefore, precoprolites and coprolites are entirely different entities.
There are also different paleontological terms for coprolites (mineralized outside animals) and entrolites (mineralized inside animals). Surely the much more different paleontological coprolites and archaeological precoprolites deserve distinct names.

Coprolite = is a formal paleontological term for a hard fecal fossil unit, which was quickly buried, preserved by petrification, and is tens to ~485 million of years old. They sometimes contain fossilized biological remains or their chemical or mineral indicators. Only trace amounts of unmineralized biological material may rarely be present. Coprolites are usually marine and associated with fishes or reptiles. Terrestrial coprolites are quite rare.

PreCoprolite = is suggested as a formal archaeological term for a soft fecal subfossil unit preserved by drying and the Maillard Reaction, or sodium chloride, hundreds to thousands of years old, and containing biological materials useful for the study of diet, diseases, DNA, medications, microbes, parasites, phytoliths, spores, etc.
A few older possible precoprolites, 1 million years old, examined were partially mineralized and contained much less biological materials. PreCoprolites are usually found in archaeological investigations. PreCoprolites are strictly terrestrial since they largely disintegrate in water. They are usually associated with cool, dry regions or dry caves. However, they have recently been analyzed from a humid, tropical area [1].

References

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Media Coverage of the article [1] not listed by Plos One:

Annonymous (2014) Fossil feces tell the tale of ancient Caribbean migration. National Palentology, 14 September. http://typicalleaf5306.ji...
Anonymous (2014) Paleomicrobiology: Revealing Fecal Microbiomes of Ancient Indigenous Cultures. Anthropology Daily, 26 September http://anthropologydaily....
Fang J (2014) Fossilized Feces Differ Among Ancient Cultures. IFLSciences 25 September. http://www.iflscience.com...
Lowenberg D (2014) Fecal Matters: A Stepping Stool to Understanding Indigenous Cultures. EveryOne, 4 November, http://blogs.plos.org/eve...
Wilson N (2015) Retired Cal Poly professor uses fossilized feces to learn about ancient cultures. The Tribune, San Luis Obispo, California, 9 January. http://www.sanluisobispo....

No competing interests declared.