Microalgae community of the Huaytire wetland, an Andean high-altitude wetland in Peru Comunidade de microalgas do wetland Huaytire, uma área alagada de alta altitude nos Andes peruanos

Aim: The diversity and distribution of microalgae communities in a highaltitude (3,000 to 4,500 m a.s.l) Andean wetland, regionally known as bofedal, were examined to assess seasonal and spatial patterns. Methods: Samples were taken monthly from June to December, 2008 at 13 stations in the Huaytire wetland (16° 54’ S and 70° 20’ W), covering three areas (impacted by urban land use, impacted by camelid pasture, and non-impacted) and three climatologically induced periods (ice-covered, ice-melt and ice-free). Results: A total of 52 genera of algae were recorded. Diatoms were the predominant group in abundance and richness. We found a significantly higher abundance during the ice-melting period, when light exposure and runoff were intermediate, in comparison to the ice-covered (low light and flushing) and ice-free (high light and low runoff) periods. Microalgae abundance was significantly lower in the non-impacted area compared to the sites close to the urban area and to the camelid pastures. Alpha diversity ranged from 8 to 29 genera per sample. High genera exchange was observed throughout the wetland, showing a similar floristic composition (beta diversity = 4%). Conclusions: We found that diatoms were dominant and adapted to the extreme conditions of the Andean wetland, showing higher abundance during the ice-melt period and in the livestock area. Also, taxa richness was higher in the ice-melt period and in the most-impacted areas.

occur between 4,000 and 5,000 m a.s.l., and in the southern parts (27° S) between 2,000 and 3,000 m a.s.l.(Scott and Carbonell, 1986).These regions have a complex topography and geology, extreme climate conditions with large diel and seasonal variations in temperature and precipitation, very high insolation, and low atmospheric pressure (Jacobsen and Marin, 2008).The native wetland vegetation consists mainly of totorales, which are stands of a perennial reed (Cyperaceae), and bofedales, which are areas of heterogeneous, short and dense grasses, cross-crossed by shallow streamlets (Moreau et al., 2003).High Andean wetlands are sources of life and development for people who live and depend on these ecosystems.We evaluated the seasonal and spatial changes in abundance, richness and composition of the microalgae community in the Huaytire wetland, a high-altitude, low-latitude Andean wetland.As far as we know, this is the first microalgae ecological contribution in this type of extreme environment.

Material and Methods
Study Site -The study was carried out at the Huaytire wetland, which is located at 4,452 m a.s.l. in the tropical Andes (16° 54' S and 70° 20' W), near the Livicalani and Huaytire streams in Tacna, Peru.This wetland encompasses an area of 0.86 km 2 , and lies within the central Andean dry Puna (Baied and Wheeler, 1993;Otto et al., 2011) (Figure 1).The local climate, following the Köppen system

Introduction
The knowledge of high-altitude microalgae in the world is derived basically from European alpine lakes and streams (Rott and Pernegger, 1994;Tolotti, 2001).Wetlands in high-altitude temperate regions -peat bogs or Sphagnum bogs -represent one of the major vegetation types, in addition to forests and grasslands (Borics et al., 2003).Peat bogs are often dominated by diatoms, chlorophyceans and cyanobacteria; pH and electrical conductivity have been noted as the most important ecological variables influencing these communities (Kapetanović and Hafner, 2007).South American high-altitude aquatic systems have been little studied; most of these studies are taxonomic and have been carried out in lakes and streams.In these systems, diatoms are the main algal group (Donato-Rondón, 2001;Morales et al., 2007;Díaz-Quirós and Rivera-Rondón, 2004;Sylvestre et al., 2001;Tapia et al., 2003Tapia et al., , 2006)).In particular, diatoms from high altitude Andean wetlands, are reported mainly as floristic studies which include diatoms from systems located between 4,120 and 4,571 m a.s.l. in Argentina (Maidana and Seeligmann, 2006;Seeligmann et al., 2008;Maidana et al., 2011).
High-altitude wetlands in the Andes exist at the hydrological and altitudinal limits for plant life in the cold and arid high Andean grasslands of Peru, Bolivia, Chile and Argentina (Squeo et al., 2006).In the central Andes (16° S), these wetlands diversity (local genera richness in each sample); and iii) beta diversity (degree of genera turnover among the areas).To ensure that our samples were representative, we also used a rarefaction analysis between samples and number of genera.Intensities of runoff and light exposure were analyzed semi quantitatively, taking into account the ice cover and the seasonal period.
Differences in abundance and richness between areas or periods were determined using Kruskal-Wallis tests.If the differences were significant (p < 0.05), then a post hoc Dunn's test was used to identify differences.Statistical analyses were performed using Stat View © 5.01 and Sigma Plot v.11.

Results
Climatological, physical and chemical variables -During the entire year ( 2008), the total precipitation reached 192 mm and the mean temperature was 5 °C (Peru, 2008).The ice-covered and ice-melt periods occurred during the dry season, and were marked by lower temperature and precipitation (Table 1).During the ice-free period, the minimum and mean temperature increased, and the ice melted completely (Table 1).The landscape changed from brownish to greenish with the growing season, mainly Juncaceae.
The depth of the streamlets in this high Andean wetland reached ~0.30 m during the ice-covered and ice-melt periods, and increased to ~0.50 m at the beginning of the ice-free period.The median water temperature was higher during the ice-free period (8.7 °C) compared to the ice-covered (5.3 °C) and ice-melt periods (6.3 °C) (p < 0.001) (Table 2).
Low median dissolved oxygen values have been registered (31-38%), because of high altitude.Dissolved oxygen content during the ice-melt period was higher (p = 0.018) than in the icecovered and ice-free periods (Table 2).Most pH values (75%) were circumneutral (6.6-6.8), with no differences among periods (p < 0.558).Electrical conductivity followed the temperature pattern, with (1936) and updated by Kottek et al. (2006), is a tundra polar climate (ETk).Daily minimum temperature is generally below 0 °C and can reach below -10 °C during the winter (CONAMA, 2006).The streamlets are shallow (~0.5 m depth).The vegetation is mainly constituted by Juncaceae which are terrestrial or semi-aquatic plants, that occurs in sunny places.Genus Oxychloe, Patosia and Distichia grow at very high elevations, usually between 3,500-4,500 m a.s.l.(Balslev, 1996).Villages located around this type of wetland are sustained by raising camelids livestock (Morris and Panty, 1999).S a m p l i n g , s a m p l e a n a l y s i s a n d d a t a analysis -Samples were taken monthly, directly from the streams, for microalgae analysis (abundance and composition), from June to December 2008 at 13 sampling stations (91 total samples).Microalgae were fixed with formaldehyde at 4% final concentration.Electrical conductivity, pH, dissolved oxygen and temperature were measured in situ with a HANNA HI 9813 probe.According to the amount of ice-cover criteria, we recognized three periods: two during the dry season (period A, June to August, ice-covered; and period B, September to October, ice melt), and one in the early rainy season (period C, November to December, ice-free season).Also, three areas were selected according to the land use: area I (upper basin), which is subject to urban influence (impacted area), area II (intermediate, influenced by livestock); and area III (lower basin, the most preserved area, with minimum human and animal influences).
To estimate microalgae abundances, drops of a sample were transferred to a glass slide until they completed 1 mL (Vollenweider, 1974), and analyzed under an Olympus CX31 light microscope, at 400 x magnification.Microalgae were identified to the genus level and grouped in taxonomic classes according to Van den Hoek et al. (1997).Genus level was used because identification at the species level is uncertain in this relatively unknown system.Diversity was studied as i) gamma diversity (total pool of genera at all stations and dates); ii) alpha higher abundance was also found in the livestock area (508 ind.mL -1 ) than in the others (397 and 312 ind.mL -1 ) (Figure 2).The regional pool of genera (gamma diversity) contained a total of 52 genera (23 diatoms, 17 chlorophyceans, 12 cyanobacteria) (Table 3).
Microalgae community -In these extreme ecosystems, the predominant microalgae communities are mainly the metaphytic and benthic communities living in the shallow streamlets.During the study period, diatoms were the most abundant algal group, contributing 95% to the total abundance.Chlorophyceans and cyanobacteria contributed only 3 and 2% to the total, respectively.Significant differences were found in the total microalgae abundance among the three periods (p < 0.001) and areas (p = 0.006).Abundance was significantly higher during the ice-melt (566 ind.mL -1 ), in comparison to the ice-covered and ice-free seasons (324 and 411 ind.mL -1 , respectively) (Figure 2).Significantly  Hafner, 2007).Locally and temporally, diatoms can also constitute an important part of the algal communities in arctic and subarctic regions, because they are adapted to low temperature (Douglas and Smol, 1999).We found remarkable diatom dominance in the Huaytire wetland through the whole study period.Other studies also have reported diatom dominance, for example a small benthic diatom Fragilaria, in a high mountain lake in the northern Canadian Cordillera, where there are cold conditions with long periods of ice cover (Karst-Riddoch et al., 2005).Besides diatoms, chlorophyceans and cyanobacteria are numerous in subalpine peat bogs (Klemenčič et al., 2010); dinoflagellates and chrysophyceans are dominant in the plankton in the Italian Alps, but epilithic communities are dominated by diatoms (Tolotti, 2001).For Andean lakes, in particular, dominances of chlorophyceans and diatoms were reported for Chungará Lake (Chile), Tranca Grande and Titicaca lakes (Peru) (Dorador et al., 2003;Mariano-Astocondor, 2001;Dejoux and Iltis, 1991).
The gamma diversity in the Huaytire wetland is 52 genera.This number is derived through an beta diversity for the whole data set indicated a very similar composition at all 13 sampling stations (beta diversity = 4%).The rarefaction curve reached the asymptote at approximately 44 samples from a total of 91 samples (Figure 3).

Discussion
We found that the microalgae community of this Andean wetland located at tropical latitudes is dominated by diatoms both in richness and abundance showing higher abundance during the ice-melt period and in the area under livestock influence.The wetland, located in the dry Puna in the Andes, has little precipitation, perennial frost, intense solar radiation, and reduced atmospheric oxygen and carbon dioxide concentrations (Baied and Wheeler, 1993).We compared our data to other systems subject to extreme conditions, such as temperate high-altitude wetlands (peat bogs), high-altitude alpine lakes, and systems in polar or subpolar regions, because as far as we are aware, there is no available information on microalgae ecology from the central dry South-American Andean wetlands.
Diatoms were reported as the main algal group in subalpine peat bogs (Kapetanović and  Seeligmann et al., 2008), or even higher than in the Puna Nueva wetland (8 genera, Maidana and Seeligman, 2006).
Although we cannot directly compare our data to estimates of abundance and richness from other studies (because the lack of such information in the literature), aquatic high-altitude systems around the world show similar numbers of genera.For example, in high-altitude Swiss springs, 31 genera were recorded (Taxböck and Preisig, 2007); 49 genera were reported in high-altitude Andean Bolivian streams (Morales and Vis, 2007), and 17 genera in a high-altitude stream in the Alps (Rott et al., 2006).
In conclusion, the highest abundance and richness were found during the ice-melt season.Spatially, we found the highest abundance in the livestock area, and higher richness in the two more impacted areas.Diatoms were the predominant group throughout the study.This group seems to be well adapted to the extreme abiotic conditions (low

Figure 1 .
Figure 1.Map and location of the Huaytire wetland (Tacna-Peru) -(marked with a black dot).Area I = upper basin (impacted area); Area II = intermediate, influenced by livestock; Area III = lower basin, the most preserved area, with minimum human and animal influences.

Figure 3 .
Figure 3. Rarefaction curve and the 95% confidence interval for microalgae genera in the Huaytire wetland, Tacna Peru, in relation to the number of samples.

Table 1 .
Hydrological and climatological features through the seasonal periods in the Huaytire wetland, Tacna-Peru (June-December, 2008).The intensities of flushing and light exposure are semi quantitatively expressed by: X (low), XX (intermediate) and xxx (high).(T = temperature; Total P = precipitation).

Table 2 .
Median, minimum and maximum (between brackets)values of abiotic variables by season in the Huaytire wetland, Tacna-Peru (June-December, 2008).

Table 3 .
List of microalgae genus and relative abundance (%) in the Huaytire wetland,Tacna-Peru (June-December,  2008).effective sampling design; we reached 96% of the total expected flora in almost half of the samples.Diatoms comprised 42% of the total genera.The number of 23 diatom genera is similar to the number found in the Pululos wetland (22 genera, BORICS, G., TÓTHMÉRÉSZ, B., GRIGORSZKY, I., PADISÁK, J., VÁRBÍRÓ, G. and SZABO, S. 2003.Algal assemblage types of bog-lakes in Hungary and their relation to water chemistry, hydrological conditions and habitat diversity.Hydrobiologia,