Trophic transfer of heavy metals through aquatic food web in the largest mangrove reserve of China.

Understanding the mechanism of trophic transfer of heavy metal through the aquatic food web is critical to ecological exposure risk assessments in mangrove ecosystems. Zhanjiang Mangrove National Nature Reserve (ZMNNR) is the largest and biologically richest mangrove reserve in China, but has been exposed to heavy metal pollutants caused by the progressive industrialization and urbanization. We collected a variety of aquatic consumers, and primary producers, as well as sediments from the ZMNNR and analyzed them for heavy metal (Cd, Cr, Cu, Ni, Pb and Zn) concentrations, and for both δ13C and δ15N values to establish the trophic levels. The trophic magnification factors (TMF) of Cd, Cu and Zn are 0.19 (p < 0.01), 0.07 (p < 0.01) and 0.33 (p < 0.05), respectively, indicating significant biodilution in a simplified food web composed of bivalves, crustaceans and fish. There are also potential tendencies of biodilution for Cr, Ni and Pb. Comparison of heavy metals in representative fish and shrimp in the ZMNNR with those in worldwide mangroves indicate a low risk level for aquatic consumers in our ecosystem. Quantitative source tracking is conducted based on principal component analysis and cluster analysis, which indicate that Cr, Ni and Pb are mainly originated from natural geological processes, Cu and Zn from shrimp farming and agriculture activities, and Cd from the deposition of aerosol released by regional metal smelting industry.

Atmospheric deposition as a direct source of particulate organic carbon in region coastal surface seawater: Evidence from stable carbon and nitrogen isotope analysis.

To assess the source characteristics of coastal aerosols and evaluate the contribution of atmospheric deposition to particulate organic matter in surface seawater, total suspended particulates (TSP) were collected at a shore-based site on the south coast of North Yellow Sea from December 2019 through November 2020. The samples were analyzed for total organic carbon (TOC) and nitrogen (TN) as well as stable carbon and nitrogen isotope (δ13C and δ15N). The results showed that the annual mean concentrations of TOC and TN were 5.36 ± 4.74 and 5.12 ± 6.52 µg m-3, respectively. δ13C fluctuated between -25.1 ‰ and -19.2 ‰ with an annual mean of -24.0 ± 1.0 ‰ and a significant seasonal variation (P < 0.05) characterizing by the enrichment in winter (-23.4 ± 0.6 ‰) compared to other seasons, which was probably related to the massive coal combustion. Besides, δ15N ranged from 7.9 ‰ to 21.1 ‰ with an annual mean of 12.5 ± 2.9 ‰ and a less pronounced seasonal pattern (P = 0.23). The Bayesian isotope-mixing model showed that, annually, the most important source of TSP was biogenic and biomass source (55.5 ± 10.8 %), followed by fossil fuel combustion (31.9 ± 9.0 %), while the marine contribution was less (12.6 ± 2.3 %). For TOC and TN, the dominated sources were fossil fuel combustion (47.7 ± 3.4 %) and biogenic and biomass source (57.3 ± 11.7 %), respectively. Furthermore, the model results indicated that the contribution of atmospheric deposition to suspended particulate matter in surface seawater was 18.0 ± 11.0 %, 17.1 ± 6.7 % and 10.2 ± 2.0 % in autumn, spring and summer, respectively. For particulate organic carbon in surface seawater, the contribution of atmospheric deposition was 35.2 ± 3.5 % in spring, highlighting the huge impact of atmospheric deposition on particulate carbon cycling in coastal waters.

[Distribution Characteristics, Source Analysis, and Pollution Evaluation of Organic Matter in Surface Sediments of Qingpu District, Yangtze River Delta Integration Demonstration Area].

The surface sediments were collected from 25 sampling points in the Qingpu District of the Yangtze River Delta integration demonstration area, and the total organic carbon (TOC), total nitrogen (TN), organic carbon isotope (δ13C), and nitrogen isotope (δ15N) content in the samples were analyzed. The distribution characteristics and potential sources of organic matter in the surface sediments were discussed in detail, and the organic pollution index was also evaluated. The results showed that the ω(TOC) in the surface sediments of 31 sampling points in Qingpu District was 0.21%-3.55%, with an average value of 1.18%; ω(TN) ranged from 0.02% to 0.23%, with an average value of 0.09%. The δ13C ranged from -28.04‰ to -10.80 ‰, with an average of -22.28‰, and the δ15N ranged from 2.28‰ to 11.19‰, with an average of 5.76‰. The TOC content was significantly correlated with the TN content. Source analysis showed that the organic matter in the surface sediments of the study area was mainly affected by soil organic matter, sewage organic matter, and terrestrial plants. The calculation results of the contribution rate based on IsoSource software showed that the relative contribution rate of soil organic matter was relatively high (0.3%-96.8%), and domestic sewage had a certain contribution to the organic matter source of most sampling points. In addition, the selected end-member substances had certain differences in the contribution of organic matter sources in samples of different land use types. The evaluation index of organic pollution in the surface sediments of the study area ranged from 0.006-0.745, with an average value of 0.163, indicating that the organic pollution of the study area was considered lightly polluted. Among them, the pollution of sampling points around the Jinze Reservoir was more serious than that in other areas, indicating that there was a potential risk of eutrophication in this area, which requires certain attention.

Effects of Temperature and Nitrogen Application on Carbon and Nitrogen Accumulation and Bacterial Community Composition in Apple Rhizosphere Soil.

Malus sieversii grows on the slopes of the Tianshan Mountains in Xinjiang where the difference in daily temperature is significant. In recent years, the rhizosphere soil health of Malus sieversii has been severely impacted by anthropogenic disturbance and pathogenic infestation. The soil nutrient content and soil microorganism diversity are the main components of soil health. Low temperature has negative effects on soil bacterial community structure by inhibiting the accumulation of carbon and nitrogen. However, the effects of temperature and nitrogen application on soil carbon and nitrogen accumulation and the bacterial community composition in the rhizosphere soil of Malus sieversii are unclear. We set two temperature levels, i.e., low temperature (L) and room temperature (R), combined with no nitrogen (N0) and nitrogen application (N1) to explore the response of plant carbon and nitrogen uptake, rhizosphere soil carbon and nitrogen accumulation and bacterial community composition to temperature and nitrogen fertilization. At the same temperature level, plant 13C abundance (P-Atom13C), plant 15N absolute abundance (P-Con15N), soil 15N abundance (S-Atom15N) and soil urease, protease and glutaminase activities were significantly higher under nitrogen application compared with the no-nitrogen application treatment. The bacterial community diversity and richness indices of the apple rhizosphere soil in the N1 treatment were higher than those in the N0 treatment. The relative abundances of Actinobacteria, Rhodopseudomonas, and Bradyrhizobium were higher in the LN1 treatment than in the LN0 treatment. Redundancy analysis (RDA) showed that plant 13C absolute abundance (P-Con13C) and plant 15N absolute abundance (P-Con15N) were the main factors affecting the soil bacterial community composition. In summary, Nitrogen application can alleviate the effects of low temperature stress on the soil bacterial community and is of benefit for the uptakes of carbon and nitrogen in Malus sieversii plants.

A guide for an anatomically sensitive dentine microsampling and age-alignment approach for human teeth isotopic sequences.

OBJECTIVES: Stable isotope analysis of sequential dentine samples is a potentially powerful method to reveal insights into early life-histories of individuals in the past. Dentine incremental growth structures are complex, however, and current approaches that apply horizontal sectioning of demineralized tooth halves or quarters risk combining multiple growth layers and may include unwanted cementum or secondary dentine. They also require destruction of large parts of a tooth. Here, we present a less destructive and relatively straightforward protocol that reduces damage, increases temporal resolution, and improves the accuracy of age-alignment between individuals. MATERIAL AND METHODS: We outline a protocol that includes the sampling of small (1 mm diameter) cylindrical plug transects from a thin section, along with an age-alignment scheme predicated on average growth rates for dentine areas. RESULTS AND DISCUSSION: The proposed protocol is readily applicable and more anatomically sensitive than horizontal slicing. Micro-samples are smaller (in both length and depth), hence minimizing temporal overlap and avoid directions that may contravene growth pattern. They completely avoid areas where secondary and tertiary dentine or cementum can be deposited. Age-alignment is improved by using growth ratios of anatomical tooth zones. CONCLUSION: This method minimizes destruction, enables finer temporal resolution and facilitates data comparison. It can be readily combined with fluorescence imaging-based or other pre-screening methods of dentine collagen preservation.

[Trophic level of main organisms in coastal water of Lyusi fishing ground based on stable isotope method]. / åŸºäºŽç¨³å®šåŒä½ç´ æ–¹æ³•çš„å•æ³—æ¸”åœºè¿‘å²¸æµ·åŸŸå¤å­£ä¸»è¦ç”Ÿç‰©è¥å »çº§.

We measured the ratio of δ13C and δ15N values to estimate the trophic level of main organisms based on a fisheries resource survey in coastal water of Lyusi fishing ground carried out in September 2018. The results showed that δ13C values of the main organisms ranged from -24.27‰ to -13.24‰, with an average of (-17.15±1.85)‰. The δ15N values ranged from 4.30‰ to 14.61‰, with an average of (11.21±1.90)‰. Results from cluster analysis demonstrated that the main organisms in the coastal water of Lyusi fishery ground could be divided into four trophic groups. The first group was consisted mainly by middle and small fishes, shrimps and other invertebrates. The second group included the small-sized fishes like Konosirus punctatus, Mugil cephalus, etc. and shrimps like Exopalaemon annandalei, Exopalamon carincauda, etc. The phytoplankton belonged to the third group. The fourth group was zooplankton. In the trophic spectrum, the trophic levels of fish, shrimps and crabs, shellfish were 3.2-4.7, 3.2-4.2 and 2.0-4.1, respectively. Most species belonged to the category of middle and high-level consumers. In this survey, the average trophic level of the same species in the coastal waters of Lyusi fishing ground was 0.6, which was higher than that in the East China Sea and Yellow Sea. There was niche overlap of most fish, shrimps and crabs. Those results indicated the growth environment and nutrient structure of most living organisms in this area were generally similar.

Tree water-use efficiency and growth dynamics in response to climatic and environmental changes in a temperate forest in Beijing, China.

Understanding the responses of local forests to the gradually rising atmospheric CO2 concentrations (ca) and changing environment is critical for appropriate management activities. This work used tree ring width measures (i.e. basal areal increment, BAI) and carbon (C) and nitrogen (N) signals to explore the intrinsic water use efficiency (iWUE) and tree growth dynamics of three major tree species (Pinus massoniana, P.tabuliformis and Larix gmelinii) in the Miyun Reservae Basin (MRB) of Beijing. The results indicate that ca was a primary contributor to tree growths, especially at the remote site where rising ca accounted for 92% and 74% of BAI changes for P. tabuliformis and L. gmelinii, respectively. N deposition was found to have a positive effect on BAI at this site. The controlling effect of ca on tree growth at the close-to-city site was smaller (52% and 44% of the contributions for P. tabuliformis and P. massoniana, respectively), while the negative influences of N deposition on BAI tends to be intensified. iWUE showed consistent increase during the entire growth period at all sites. Quadratic relationships between iWUE and BAI were observed, which indicated that the rising ca stimulated photosynthesis, contributing to the initial BAI and iWUE increase. However, the intensified water stress resulting from reduced precipitation and increased temperature led to a reduction in tree stomatal conductance causing the subsequent increase in iWUE but decrease in BAI. Of the site- and species-related responses of tree growth to ca, climatic and environmental changes in the MRB, the site-related variation dominated. The non-linear relationship between BAI and ca combined with the quadratic relationship between BAI and iWUE indicate a decreased ability of forests to capture atmospheric CO2 once the CO2 tipping point has passed.

The role of infant life histories in the construction of identities in death: An incremental isotope study of dietary and physiological status among children afforded differential burial.

OBJECTIVES: Isotope ratio analyses of dentine collagen were used to characterize short-term changes in physiological status (both dietary status and biological stress) across the life course of children afforded special funerary treatment. MATERIALS AND METHODS: Temporal sequences of δ15 N and δ13 C isotope profiles for incrementally forming dentine collagen were obtained from deciduous teeth of 86 children from four early-medieval English cemeteries. Thirty-one were interred in child-specific burial clusters, and the remainder alongside adults in other areas of the cemetery. Isotope profiles were categorized into four distinct patterns of dietary and health status between the final prenatal months and death. RESULTS: Isotope profiles from individuals from the burial clusters were significantly less likely to reflect weaning curves, suggesting distinctive breastfeeding and weaning experiences. This relationship was not simply a factor of differential age at death between cohorts. There was no association of burial location neither with stage of weaning at death, nor with isotopic evidence of physiological stress at the end of life. DISCUSSION: This study is the first to identify a relationship between the extent of breastfeeding and the provision of child-specific funerary rites. Limited breastfeeding may indicate the mother had died during or soon after birth, or that either mother or child was unable to feed due to illness. Children who were not breastfed will have experienced a significantly higher risk of malnutrition, undernutrition and infection. These sickly and perhaps motherless children received care to nourish them during early life, and were similarly provided with special treatment in death.

[Latitudinal trends in foliar δ13C and δ15N of Quercus variabilis and their influencing factors.] / æ “çš®æ Žå¶ç‰‡Î´13C和δ15Nçš„çº¬å‘è¶‹åŠ¿åŠå ¶å½±å“å› å­.

We aimed to reveal the latitudinal trends in foliar δ13C and δ15N of Quercus variabilis, a widely distributed species in East Asia, associated with two ontogenetic stages (juvenile and mature trees) along a North-South transect (26°-40° N). The results showed that mature trees had higher foliar δ13C and δ15N values than juveniles. Foliar δ13C and δ15N values of trees with both ontogenetic stages were nonlinearly increased and decreased with latitude, respectively. No interaction between ontogenetic stage and latitude for the changes of foliar δ15N and δ13C indicated that both ontogenetic stages across the transect consistently responded to latitudinal environmental variations. Results from the random forest models indicated that foliar δ15N of Q. variabilis was mainly affected by soil nutrient contents, e.g., soil organic matter, phosphorus, nitrogen, whereas dominated factors for foliar δ13C were related to moisture, such as relative humidity, precipitation of growing season.

Identifying the impacts of climate on the regional transport of haze pollution and inter-cities correspondence within the Yangtze River Delta.

Regional haze pollution has become an important environmental issue in the Yangtze River Delta (YRD) region. Regional transport and inter-influence of PM2.5 among cities occurs frequently as a result of the subtropical monsoon climate. Backward trajectory statistics indicated that a north wind prevailed from October to March, while a southeast wind predominated from May to September. The temporal relationships of carbon and nitrogen isotopes among cities were dependent on the prevailing wind direction. Regional PM2.5 pollution was confirmed in the YRD region by means of significant correlations and similar cyclical characteristics of PM2.5 among Lin'an, Ningbo, Nanjing and Shanghai. Granger causality tests of the time series of PM2.5 values indicate that the regional transport of haze pollutants is governed by prevailing wind direction, as the PM2.5 concentrations from upwind area cities generally influence that of the downwind cities. Furthermore, stronger correlation coefficients were identified according to monsoon pathways. To clarify the impacts of the monsoon climate, a vector autoregressive (VAR) model was introduced. Variance decomposition in the VAR model also indicated that the upwind area cities contributed significantly to PM2.5 in the downwind area cities. Finally, we attempted to predict daily PM2.5 concentrations in each city based on the VAR model using data from all cities and obtained fairly reasonable predictions. These indicate that statistical methods of the Granger causality test and VAR model have the potential to evaluate inter-influence and the relative contribution of PM2.5 among cities, and to predict PM2.5 concentrations as well.

Investigating economic specialization on the central Peruvian coast: A reconstruction of Late Intermediate Period Ychsma diet using stable isotopes.

OBJECTIVES: Hypothetical models of socioeconomic organization in pre-Columbian societies generated from the rich ethnohistoric record in the New World require testing against the archaeological and bioarchaeological record. Here, we test ethnohistorian Maria Rostworowski's horizontality model of socioeconomic specialization for the Central Andean coast by reconstructing dietary practices in the Late Intermediate Period (c. AD 900-1470) Ychsma polity to evaluate complexities in social behaviors prior to Inka imperial influence. MATERIALS AND METHODS: Stable carbon and nitrogen isotope analysis of archaeological human bone collagen and apatite (δ13 Ccol[VPDB], δ15 Ncol[AIR] , δ13 Cap[VPDB] ) and locally available foods is used to reconstruct the diets of individuals from Armatambo (n = 67), associated ethnohistorically with fishing, and Rinconada Alta (n = 46), associated ethnohistorically with agriculture. RESULTS: Overall, mean δ15 Ncol[AIR] is significantly greater at Armatambo, while mean δ13 Ccol[VPDB] and mean δ13 Cap[VPDB] are not significantly different between the two sites. Within large-scale trends, adult mean δ13 Cap[VPDB] is significantly greater at Armatambo. In addition, nearly one-third of Armatambo adults and adolescents show divergent δ15 Ncol[AIR] values. DISCUSSION: These results indicate greater reliance on marine resources at Armatambo versus Rinconada Alta, supporting the ethnohistoric model of socioeconomic specialization for the Central Andean coast. Deviations from large-scale dietary trends suggest complexities not accounted for by the ethnohistoric model, including intra-community subsistence specialization and/or variation in resource access.

Trace metal pollution and carbon and nitrogen isotope tracing through the Yongdingxin River estuary in Bohai Bay, Northern China.

A tide gate was built in 2010 to prevent seawater from moving upstream into the Yongdingxin River estuary in Bohai Bay, Northern China. We analysed the concentrations of Hg, Cd, Pb, TOC, TN, δ13C and δ15N and studied their variations in the surface layer and vertical profiles of sediment cores collected from the Yongdingxin River estuary. Contamination factors and geo-accumulation indices were calculated for each metal, which revealed high levels of contamination for Hg and Cd in the sediments, likely from anthropogenic sources. δ13C and δ15N were used as natural tracers to determine the sources of TOC and TN. The results revealed that sewage was the main source of TOC, while TN may have more than one source in the Yongdingxin River estuary. Sewage dominated trace metal pollution in the Yongdingxin River estuary. Our results provide a baseline for trace metal contamination in an estuary facing a large water project.

[Characteristics of Carbon and Nitrogen in the Downstream Columnar Sediment of Maozhou River, Shenzhen].

Based on the determinations of total nitrogen (TN), organic matter (OM), carbon-nitrogen ratio (C/N), δ15 N, and δ13 C on 12 sediment cores, the distribution characteristics and the sources of nitrogen and organic matter were analyzed in sediments from the lower reaches and main tributaries of the Maozhou River. The results showed that the average concentrations of total nitrogen (TN) and organic matter (OM) were 1815.37 mg·kg-1 and 22 401.68 mg·kg-1, respectively, in the Maozhou River sediments, which were higher than in TaihuLake and ChaohuLake. The levels of TN and OM in the study area were high and varied greatly with the increase of depth. The δ15 N content in the sediments of the MaozhouRiver downstream ranged from 2.20 ‰ to 32.78 ‰, while the δ13 C content ranged from -27.53 ‰ to -21.95 ‰. The average concentrations of δ15 N and δ13 C were 6.78 ‰ and -25.41 ‰. Moreover, the C/N ratio ranged from 0.49 to -18.23. The δ13 C varied slightly with the increase of depth, while δ15 N and C/N varied greatly. A source analysis in the study area indicated that C3 plants and chemical fertilizer were the main sources in the surface sediments (0-40 cm) downstream of the Maozhou River. Furthermore, algae were the main sources of organic matter in the deep sediments downstream of the Maozhou River and in tributary sediments of the Shajing River. Nitrogen in the surface sediments (0-40 cm) downstream of the Maozhou River mainly comes from inorganic fertilizer and soil organic nitrogen, while nitrogen in the Maozhou River deep sediments and Shajing River sediments mainly come from soil erosion and soil organic nitrogen.

Food Insecurity in Urban and Rural Areas in Central Brazil: Transition from Locally Produced Foods to Processed Items.

Aiming to investigate the effect of diet and food consumption with regard to health, environment, and economy in light of nutrition ecology, we studied the dimensions of nutrition and food security in urban and rural settings in the region of Chapada dos Veadeiros, Central Brazil. We tracked diet and food consumption through carbon and nitrogen stable isotope ratios in fingernails of these inhabitants together with food intake data as a proxy for their diet patterns. We estimated household food insecurity by using the Brazilian Food Insecurity Scale. Nutrition and food insecurity was observed in both urban and rural areas, but was accentuated in rural settings. The diet pattern had high δ(13)C values in fingernails and low δ(15)N. Both urban and rural areas have diets with low diversity and relying on low-quality processed food staples at the same time that nutrition and food insecurity is quite high in the region.

Stable isotopic variation in tropical forest plants for applications in primatology.

Stable isotope analysis is a promising tool for investigating primate ecology although nuanced ecological applications remain challenging, in part due to the complex nature of isotopic variability in plant-animal systems. The aim of this study is to investigate sources of carbon and nitrogen isotopic variation at the base of primate food webs that reflect aspects of primate ecology. The majority of primates inhabit tropical forest ecosystems, which are dominated by C3 vegetation. We used stable isotope ratios in plants from Kibale National Park, Uganda, a well-studied closed-canopy tropical forest, to investigate sources of isotopic variation among C3 plants related to canopy stratification, leaf age, and plant part. Unpredictably, our results demonstrate that vertical stratification within the canopy does not explain carbon or nitrogen isotopic variation in leaves. Leaf age can be a significant source of isotopic variation, although the direction and magnitude of this difference is not consistent across tree species. Some plant parts are clearly differentiated in carbon and nitrogen isotopic composition, particularly leaves compared to non-photosynthetic parts such as reproductive parts and woody stem parts. Overall, variation in the isotopic composition of floral communities, plant species, and plant parts demonstrates that stable isotope studies must include analysis of local plant species and parts consumed by the primates under study from within the study area. Am. J. Primatol. 78:1041-1054, 2016. © 2015 Wiley Periodicals, Inc.

Oral histories: a simple method of assigning chronological age to isotopic values from human dentine collagen.

BACKGROUND: Stable isotope ratios of carbon (δ(13)C) and nitrogen (δ(15)N) in bone and dentine collagen have been used for over 30 years to estimate palaeodiet, subsistence strategy, breastfeeding duration and migration within burial populations. Recent developments in dentine microsampling allow improved temporal resolution for dietary patterns. AIM: A simple method is proposed which could be applied to human teeth to estimate chronological age represented by dentine microsamples in the direction of tooth growth, allowing comparison of dietary patterns between individuals and populations. The method is tested using profiles from permanent and deciduous teeth of two individuals. SUBJECTS AND METHODS: Using a diagrammatic representation of dentine development by approximate age for each human tooth (based on the Queen Mary University of London Atlas), this study estimated the age represented by each dentine section. Two case studies are shown: comparison of M1 and M2 from a 19th century individual from London, England, and identification of an unknown tooth from an Iron Age female adult from Scotland. RESULTS AND CONCLUSIONS: The isotopic profiles demonstrate that variations in consecutively-forming teeth can be aligned using this method to extend the dietary history of an individual or identify an unknown tooth by matching the profiles.

Diet and gender in the Tiwanaku colonies: Stable isotope analysis of human bone collagen and apatite from Moquegua, Peru.

OBJECTIVES: Gender and other facets of social identity play important roles in the organization of complex societies. This study reconstructs dietary practices within the Middle Horizon (AD 500-1000) Tiwanaku colonies in southern Peru to increase our knowledge of gendered patterns of consumption within this early expansive state. METHODS: We use stable isotope analysis of 43 human bone samples representing 14 females, 20 males, 8 juveniles, and 1 indeterminate individual recovered from burial excavations at the sites of Rio Muerto and Omo in the Moquegua Valley. Data are contextualized by comparisons with previously published Tiwanaku isotope data from the period. RESULTS: Our results find mean values of δ(13) Capatite = -7.3 ± 1.6% (N = 36, 1SD), δ(13) Ccollagen = -12.3 ± 1.5% (N = 43, 1SD), and δ(15) Ncollagen = 8.4 ± 1.6% (N = 43, 1SD). Between the sexes, Mann-Whitney U tests demonstrate significant differences in δ(13) Ccollagen (U = 74, P = 0.021), but no differences in δ(13) Capatite (U = 58, P = 0.095) or δ(15) Ncollagen (U = 116, P = 0.755) values. CONCLUSIONS: These data indicate relatively high C4 plant consumption among the Tiwanaku colonies, and support paleobotanical and archaeological evidence that maize (Zea mays) was the staple crop. Dietary values are similar overall between the sexes, but significantly higher δ(13) Ccollagen values in males is consistent with a model of gendered norms of consumption similar to that of the later Inca (AD 1438-1533), where males consumed more maize than females, often in the form of beer (chicha). Results provide new insights on social dynamics within the Tiwanaku colonies and suggest the increased importance maize consumption for males during the Tiwanaku expansion.