Introduction
Mountain viscachas are large caviomorph rodents that inhabit arid regions of western and southern South America, from the highlands of Ecuador through the Andes of Peru and Bolivia to the coastal mountains of Chile and Patagonian steppe of Argentina. This rodent is found in rocky outcrops and is highly gregarious, living in colonies that may range widely in size. The presence of viscachas is readily detected by distinctive fecal pellets that can be found throughout the colony, on top of rocks or in sheltered crannies (Spotorno and Patton, Reference Spotorno, Patton, Patton, Pardiñas and D'Elía2015).
In the last years, several studies have been carried out in different areas of Patagonia involving micromammals fossil remains. The available data indicate that these communities had minor changes during the last 10 000 years. This holocene stability, despite the occurrence of climatic pulses, contrasts with the current structure of the assemblages, which shows a remarkable loss of diversity and even local or regional extinctions (e.g. Pardiñas et al., Reference Pardiñas, Moreira, García-Esponda and De Santis2000, Reference Pardiñas, Teta, D'Elia and Lessa2011, Reference Pardiñas, Udrizar Sauthier and Teta2012; Udrizar Sauthier, Reference Udrizar Sauthier2009; Pardiñas and Teta, Reference Pardiñas and Teta2013; Teta et al., Reference Teta, Formoso, Tammone, de Tommaso, Fernández, Torres and Pardiñas2014). It has been suggested that these recent changes have been unleashed by anthropogenic activities, such as overgrazing, deforestation or the implantation of agroecosystems (Teta et al., Reference Teta, Formoso, Tammone, de Tommaso, Fernández, Torres and Pardiñas2014 and references cited therein).
Paleoparasitology is the study of parasites found in the archaeological or paleontological material. In a broad sense, paleoparasitologists are interested in the evolution of parasite–host–environment relationships, as well as in the origin and the evolution of infectious diseases within a paleoepidemiological perspective (Araújo et al., Reference Araújo, Jansen, Bouchet, Reinhard and Ferreira2003). Beltrame et al. (Reference Beltrame, Sardella, Fugassa and Barberena2012, Reference Beltrame, Fugassa, Udrizar Sauthier and Sardella2014, Reference Beltrame, De Porras, Barberena, Llano and Sardella2016) performed paleoparasitological studies on mountain viscacha coprolites from Patagonia, where nematode and cestode eggs were reported. The species found were also recorded in studies with modern mountain viscachas (Hugot and Sutton, Reference Hugot and Sutton1989a; Denegri et al., Reference Denegri, Dopchiz, Elissondo and Beveridge2003; Tantaleán et al., Reference Tantaleán, Quispe and Angulo2013). The environmental changes occurred in the last 100 years in Patagonia reflected in diversity changes of micromammals, could also be reflected in the evolution of parasite–mountain viscacha–environment relationships throughout time.
The aim of the present study was to examine the parasite remains from mountain viscacha fecal pellets from the site ‘Cueva Peligro’, Patagonia, through the last 1200 years and to discuss the paleoparasitological findings in temporal and paleoecological contexts. This is the first paleoparasitological examination of pellets of mountain viscachas throughout time, which include samples from modern, recent and ancient times. This study is part of an ongoing project in the area, where the impact of anthropic activities on micromammal community is under study.
Material and Methods
The paleontological site ‘Cueva Peligro’ (CP; 43°40′18″ S, 66°24′52″ W) is located near the southern margin of the Chubut River, about 6 km downstream of the Villa Dique Florentino Ameghino, Chubut Province, Argentina (Fig. 1). The site is a cave emplaced on a rocky front of approximately 35 m of height (Marifil Formation, Jurassic), with a single entrance (4.57 m wide) and a single long gallery (ca.30 m). The general dip of the floor of the cave is towards the outside, with some sectors that can reach 45° of inclination and others that approach 0°. From a phytogeographic point of view, CP is located in the Monte Phytogeographic Province (León et al., Reference León, Bran, Collantes, Paruelo and Soriano1998). A 1 × 1 m2 square grid was defined and the sequence was excavated by artificial layers of 3 cm thick until reaching the basement rock at a depth of 0.42 m. The extracted sedimentary material was sieved through a 5 mm mesh.
Geographic location of the paleontological site ‘Cueva Peligro’, Chubut Province, Patagonia Argentina.
Feces samples from three layers were processed at the Laboratorio de Tritio y Radiocarbono (LATYR), Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata (LP), Argentina, to obtain radiocarbon dates. Calibration for the Southern Hemisphere was made through SHCal13 curve (Hogg et al., Reference Hogg, Hua, Blackwell, Niu, Buck, Guilderson, Haeton, Palmer, Reimer, Reimer, Turney and Zimmerman2013) using OxCal 4.2 software (Bronk Ramsey, Reference Bronk Ramsey2009) and weighted average was used as age estimator (Telford et al., Reference Telford, Heegaard and Birks2004; Table 1). All the sequence was dated from the Late Holocene, including the historic period and actual times, approximately 1200 years throughout time. The radiocarbon dates of these layers range between 1220 ± 70 14C years BP to modern years (Table 1).
Depth, time period, radiocarbon dates, calibrated age, other fecal pellets present and parasite egg found from all studied layers from ‘Cueva Peligro’
The area has been under a progressive transformation of natural environments, mainly due to extensive sheep farming. The beginning of this activity corresponds to the 1880–1885 AD period. Based on the number of sheep feces found in each level, three time periods were defined: modern (1950–2015 AD, high sheep feces content), recent (1885–1950 AD, medium sheep feces content and ancient (previous to 1885 AD, low/null sheep feces content). Taking into account that: low/null: 0–15 sheep feces units 1.200 cm−3 of sediment, medium: 15–80 sheep feces units 1.200 cm−3 of sediment, high: >80 sheep feces units 1.200 cm−3 of sediment.
Six fecal pellets assigned to mountain viscachas from each layer were examined for parasites. A total of 84 samples were observed. Samples were inventoried and processed individually. Each sample was fully processed by rehydration in a 0.5% water solution of tris-sodium phosphate in a glass tube for at least 3 days, followed by homogenization, processed by spontaneous sedimentation (Lutz, Reference Lutz1919) and preserved in 70% ethanol. Twenty slides were prepared from each sample, along with the addition of one drop of glycerin to each slide and were examined using light microscopy. Eggs of parasites were measured and photographed at 400 × magnification.
Differences in parasite assemblages composition among modern, recent and ancient pellets were tested using permutational multivariate analyses of variance (PERMANOVA, Anderson, Reference Anderson2001). This is a non-parametric technique that uses label permutation to estimate the distribution of the test statistics under the hypothesis that within-group distances are not significantly different from between-group distances. The Sørensen similarity index was used as the distance measure for occurrence data. The Sørensen index of similarity was calculated on the basis of presence/absence between samples, according to the formula: S = 2C/(A + B), where S = index of similarity, A = number of species in one sample, B = number of species in the other sample and C = number of species common to both samples. A test of homogeneity of dispersions (PERMDISP, Anderson, Reference Anderson2006) was done in order to test differences in dispersions among groups because PERMANOVA is sensitive to differences in dispersions. A one-way PERMANOVA was performed using ‘Period’ as a factor (with three levels: modern, recent and ancient). Type III error and unrestricted permutation of raw data were used for unbalanced designs. Besides, a non-metric multidimensional scaling (NMDS, Clarke and Warwick, Reference Clarke and Warwick2001) was used to ordinate samples in an ordination space. NMDS was used to avoid assumptions of linearity. Multivariate analyses were performed using the software PERMANOVA + for PRIMER (Plymouth Routines In Marine Ecological Research) (v. 5.2) (Anderson et al., Reference Anderson, Gorley and Clarke2008).
Results
Fecal pellets were dark brown and cylindrical, with smooth surfaces and a flat end and the other point (Fig. 2). Average measurements of feces (N = 84) were 13.77 ± 2.38 mm long by 4.97 ± 0.49 mm wide. All layers examined contained parasite eggs (Table 1). Thirty-eight pellets were positive for parasites. Eggs of the parasite found were assigned to the nematodes Heteroxynema (Cavioxyura) viscaciae Sutton and Hugot, 1989, Helminthoxys effilatus Schuurmans-Stekhoven, 1951 (Oxyurida: Oxyuridae), Trichuris sp. Roederer, 1761 (Trichinellida: Trichuridae) and one anoplocephalid (Cestoda: Anoplocephalidae).
Fecal pellets studied from the paleontological site ‘Cueva Peligro’.
Eggs of H. viscaciae (Fig. 3a), with single thick walls and with a rounded pole and the other sharp, without operculum, were found in five pellets from modern, recent and ancient times. Average egg measurements (N = 31) were 135.5–142.5 (135.2 ± 7.18) μm long by 57.5–70.0 (64.75 ± 3.59) μm wide.
Egg found from ‘Cueva Peligro’ (A) Heteroxynema viscaciae (Nematoda: Oxyuridae), (B) Helminthoxys effilatus (Nematoda: Oxyuridae), (C) Trichuris sp. (Nematoda: Trichuridae), (D) Anoplocephalid (Cyclophyllidea: Anoplocephalidae). Bar: 20 µm.
Twelve pellets contained eggs of nematode attributed to H. effilatus. The samples belong to recent and ancient times. This nematode was not found from modern samples. The eggs were oblong, brown, with the striated and thick wall. One rounded pole and the other sharp, with a subterminal and notorious operculum (Fig. 3b), and some of them were embryonated. Average egg measurements (N = 29) were as follows: 110.0–122.5 (119.57 ± 4.77) μm long by 62.5–75.0 (67.74 ± 3.64) μm wide.
Eggs of Trichuris sp. (Fig. 3c) were observed in one pellet from modern times. These eggs were lemon-shaped, with a smooth surface and polar plugs. Measurements of eggs (N = 4) were 70.0–75.0 (71.63 ± 2.88) μm long by 37.5–42.5 (39.95 ± 2.5) μm wide.
Twenty-one samples were positive for anoplocephalids (Fig. 3d). Samples belong to modern, recent and ancient period. The eggs presented square or circular shape, with a piriform apparatus. The size ranges (means) of the eggs that were measured (N = 81) were 62.5–82.5 (69.53 ± 6.75) μm long by 62.5–80.5 (56.7 ± 1.5) μm wide.
Multivariate analysis performed on parasite occurrences in coprolites showed no significant differences in dispersions between groups (PERMDISP, P = 0.156), but significant differences between periods (PERMANOVA, P ⩽ 0.01, Table 2). The Pair-wise result showed a significant difference between modern and ancient periods (P ⩽ 0.01, Table 1). NMDS plot evidenced these differences with a stress value of 0.01 (Fig. 4). This difference between modern and ancient periods could be explained by the presence of Trichuris sp. and the absence of H. effilatus in actual coprolites in comparison with ancient samples.
NMDS plot of presence-absence of parasites in mountain viscacia fecal pellets from ‘Cueva Peligro’.
PERMANOVA and pair-wise PERMANOVA results based on Sorensen similarity coefficient between parasite occurrences in fecal pellets from ‘Cueva Peligro’ of different periods
Discussion
This is the first paleoparasitological study of mountain viscacha fecal pellets that includes a temporal period through the last 1.200 years to modern times. Previously, Beltrame et al. (Reference Beltrame, Sardella, Fugassa and Barberena2012) studied mountain viscacha coprolites collected from Cueva Huenul 1, northern Neuquén (Patagonia, Argentina), an archaeological site that provides stratified sequences of archaeological and palaeontological remains, dated from Late Pleistocene/Early Holocene Transition to Late Holocene period (13 844 ± 75–1416 ± 37 years BP). The feces were positive for Viscachataenia quadrata and Monoecocestus sp. (Cestoda: Anoplocephalidae), and for H. viscaciae. Furthermore, coprolites from fossil rodent middens (dated among 9240 ± 130 and 2540 ± 80 14C years B.P.) attributed to mountain viscachas were studied. Parasite eggs assigned to H. viscaciae, Helminthoxys sp., Trichuris sp. and one unidentified nematode (Beltrame et al., Reference Beltrame, De Porras, Barberena, Llano and Sardella2016) were found. Mountain viscacha coprolites (dated at 2210 ± 70 BP to present) from the paleontological site ‘Los Altares Profile’, Chubut Province were also studied and the samples were positive for eggs of H. viscaciae and for two morphotypes of anoplocephalids (Beltrame et al., Reference Beltrame, Fugassa, Udrizar Sauthier and Sardella2014).
In the present study, the parasitic species H. viscaciae and an anoplocephalid, were registered in modern, recent and ancient pellets. However, H. effilatus was just observed from recent and ancient samples. Trichuris sp. was only recorded in one sample from the modern period.
Species of oxyurid nematodes are monoxenic parasites that live in the posterior third of the digestive tract of various vertebrates and arthropods. Eggs are dispersed to the environment, where with favorable conditions of humidity and temperature can become a continual source of eggs for oral infection (Anderson, Reference Anderson2000). Oxyuroidea from vertebrates can be grouped into three families: Pharyngodonidae, Oxyuridae and Heteroxynematidae (Petter and Quentin, Reference Petter, Quentin, Anderson, Chabaud and Willmott2009). Heteroxynematidae includes nematodes that evolved in sciuromorph, caviomorph and miomorph mammals. Heteroxynema viscaciae (Heteroxynematidae) is a parasite found in the caecum and large intestine from L. viscacia from Chubut Province, Argentina, first described by Hugot and Sutton, Reference Hugot and Sutton1989a. It was also observed from mountain viscachas and wild viscachas (Lagostomus maximus) from other areas from Argentina and Perú (Foster et al., Reference Foster, Branch, Marchiote, Kinsella, Villarreal and Forrester2002; Ferreira et al., Reference Ferreira, Uhart, Romano, Beldoménico, Samartino, Paolicchi, Lauricella, Jorge, Schettino, Guida and Martín2007; Tantaleán et al., Reference Tantaleán, Quispe and Angulo2013).
Helminthoxys sp. (Oxyuridae: Syphaciinae) is a parasite of neotropical caviomorph rodents. The genus Helminthoxys comprises seven species: Helminthoxys caudatus (syn. Helminthoxys pujoli), Helminthoxys effilatus (syn. Helminthoxys velizi), Helminthoxys freitasi, Helminthoxys tiflophila, Helminthoxys quentini, Helminthoxys urichi, Helminthoxys gigantea and Helminthoxys abrocomae (Hugot and Sutton, Reference Hugot and Sutton1989b). Several studies have demonstrated the high host-specificity among Oxyuridae (Hugot, Reference Hugot and Page2003). In particular, these studies showed that the distribution of Syphaciinae belonging to the same family of rodent hosts has a close parallel phylogeny with them. Helminthoxys effilatus is a parasite of the mountain viscacha (Hugot and Sutton, Reference Hugot and Sutton1989b). Eggs of Helminthoxys spp. are similar. Nevertheless, due to the specificity of this genus, we can hypothesize that the eggs found in the studied samples belong to the species H. effilatus. This would be the first time that H. effilatus is reported from ancient times. This oxiurid was also registered in actual samples of mountain viscacha from Argentina and Perú (Hugot and Sutton, Reference Hugot and Sutton1989b; Tantaleán et al., Reference Tantaleán, Quispe and Angulo2013). Nevertheless, in the present study, it was not found in samples from modern times. Due to the favourable conditions of humidity and temperature that these eggs need to continue the biological cycle, the environmental changes along the last period could be responsible for the absence/presence of this species in the samples under study.
Trichuris spp. include intestinal parasites of the caecum and colon of mammals, mainly humans, primates, pigs, ovines, goats, cervids, rodents and canids, with eggs that mature in the soil. They hatch in the small intestine of the definitive hosts and the larvae migrate to the large intestine, where they penetrate the intestinal mucosa and develop through four molts before reaching the adult stage (Anderson, Reference Anderson2000). To date, a total of 24 species of Trichuris have been described from 10 families of American rodents (Robles and Navone, Reference Robles and Navone2014). Trichuris spp. in South American rodents from ancient material were cited (Beltrame et al., Reference Beltrame, Fugassa, Udrizar Sauthier and Sardella2014). Moreover, there is a previous record of two Trichuris species in coprolites of L. viscacia from fossil rodent middens of Cueva Huenul 1 archaeological site, northwest Patagonia, dated at 2540 ± 80, 5730 ± 70 and 8438 ± 37 years B.P. (Beltrame et al., Reference Beltrame, De Porras, Barberena, Llano and Sardella2016). Species of Trichuris have been infrequently recorded in high latitudes. This infrequent recording is probably related to the behavior of host species, characteristics of the environments where eggs are deposited, and/or lower degree of sampling effort in the area with respect to other areas of the country (Robles and Navone, Reference Robles and Navone2014).
The anoplocephaline cestodes (Cyclophyllidea: Anoplocephalidae) represent a diverse group of parasites infecting both terrestrial mammals (placentals and marsupials) and birds. Based on the number of genera present in these hosts, the most important radiation of anoplocephalines has been in rodents and lagomorphs (Beveridge, Reference Beveridge, Khalil, Jones and Bray1994; Wickström et al., Reference Wickström, Haukisalmi, Varis, Hantula and Henttonen2005). The intermediate hosts of these cestodes are oribatid mites, which are ingested by their herbivorous definitive hosts (Beveridge, Reference Beveridge, Khalil, Jones and Bray1994). Anoplocephalids are parasites of zoonotic importance for animals and humans (Denegri et al., Reference Denegri, Wilbert, Pérez-Serrano and Rodriguez-Caabeiro1998). As previously mentioned, V. quadrata and Monoecocestus spp. were found in modern and ancient samples. In the present study, only one anolocephalid morphotype egg was found with similar characteristics to those found from Los Altares and Cueva Huenul (Beltrame et al., Reference Beltrame, Fugassa, Barberena, Udrizar Sauthier and Sardella2013).
The difference between modern, recent and ancient periods could be explained by the presence of Trichuris sp. and the absence of H. effilatus in actual coprolites in comparison with recent and ancient samples. The fact that two of the recorded species (i.e. H. viscaciae and an anoplocephaline species) along the span of time studied had remained constant, may be due to a different capacity of adapting to a changing environment or/and that the conditions were not different enough to affect them. The record of trichurid eggs in just one sample in the modern period strengthens the previous discussion that this species is infrequently recorded in high latitudes and very rare in the ancient material. Nonetheless, the pattern presented by H. effilatus where the high abundances recorded in the ancient period declined in the recent period, to finally be absent in modern times, is remarkable. This can be a consequence of a higher sensibility of this species to environmental changes. Indeed, oxyurid species are geohelminthes which undergo a period of development in the soil before being ingested. In a greater or lower degree, all these geohelminthes suffer the influence of the alterations of the soil characteristics and climate of the region (Chieffi, Reference Chieffi2015). However, evidence showed that eggs of different species vary their resistance to extreme temperatures (Bunday and Cooper, Reference Bunday and Cooper1989).
Concluding remarks
This is the first study of mountain viscacha parasites along the time. The presence of H. viscaciae and anoplocephalids in fecal pellets from mountain viscachas from CP have not changed significantly during the last 1200 years, although environmental conditions fluctuated throughout this period, indicating the stability of these associations over time. Helminthoxys effilatus was reported for the first time in ancient samples and was absent in modern fecal pellets studied. New studies are now needed and could contribute with the study of mountain viscacha–parasite–environment relationships throughout time.
Acknowledgements
The site CP was detected by Analía Andrade and Daniel Udrizar Sauthier. Fieldwork was done with Pablo Teta, Julio Torres, Ulyses Pardiñas and Erica Cuellar assistance. We thank Claudio Iglesias, Piedra Grande S.A. and the Secretaría de Cultura de la Provincia del Chubut for permits to work in the area. Lurdes López helped in samples observation. Roberta Callicó kindly reviewed the English version of this manuscript. Two anonymous referees made suggestions. To the mentioned persons and institutions, our deep gratitude.
Financial support
This research was funded by CONICET and ANPCyT (PICT 2008-0547 to Ulyses Pardiñas and PIP 2015–2018 1122015 to Beltrame M. Ornela).
Conflicts of interest
None.
Ethical standards
Not applicable.
