MATERIAL AND METHODS

Field and laboratory activities. Sampling was carried out in an unnamed floodplain lake (31°40'36.7"S; 60°32'2.2"W) (near Paraná city) on August - November 2011, during the low water level season. This lake is characterized by two permanent connections with the main channel of the Paraná River and a secondary channel (Mini stream). This double connection implies a bidirectional exchange of benthic invertebrates between this lake and both rivers (Mesa et al., 2012). As a result, this is a representative floodplain lake of the Middle Paraná River. The area of this lake is 0.28 km² ha and its maximum depth is 8 m. For this study, we selected some of the most common taxa in the floodplain of the Middle Paraná River (summarized in ^{Marchese et al., 2002}; ^{Ezcurra de Drago et al., 2007}; ^{Zilli & Montalto, 2012}) that were present in the lake during the studied period. Thus, the invertebrates selected to perform the diet analysis were adults of Dero vagus Leidy, 1880, Nais communis (Piguet, 1906), Pristina leidyi Smith, 1896 and Aulodrilus pigueti Kowalewski, 1914 (Oligochaeta), late instars larvae of Parachironomus Lenz, 1921 and Monopelopia Fittkau, 1962 (Diptera, Chironomidae), late stages nymphs of Campsurus violaceus Needham & Murphy, 1924, Americabaetis Klunge, 1992 and Caenis Stephens, 1835 (Ephemeroptera), Sympetrum Newman, 1833 (Odonata: Libellulidae), Cyrnellus Banks, 1913 (Trichoptera, Polycentropodidae) and adults of Pomacea canaliculata Lamarck, 1828 (Mollusca, Gastropoda, Ampularidae). Specimens were deposited in Benthos collections of the Instituto Nacional de Limnología (Santa Fé, Argentina).

Benthic invertebrates (except P. canaliculata) were collected with Ekman grab of 225 cm² and were fixed in 10% formaldehyde in the field. Chironomids, oligochaetes and ephemeropterans were sampled in clay silt patches. Cyrnellus sp. and Sympetrum sp. were collected in patches of leaf litter. In laboratory, organisms were handpicked under stereoscopic microscope (4X) and preserved in 70% alcohol. Pomacea canaliculata (Gastropoda) individuals were handpicked in field and frozen at -18°C, and then the anterior gut was dissected for diet analysis. The consumers were identified to genus or species level using taxonomic keys (^{Brinkhurst & Marchese, 1991}; ^{Domínguez & Fernández, 2009}; ^{Trivinho-Strixino, 2011}). Gut content of Pomacea canaliculata, Sympetrum sp. and C. violaceus were extracted and the proportional contribution of food items was quantified under the microscope (100X). Oligochaeta individuals were cleared with lactophenol, while chironomids, Caenis sp., Americabaetis sp. and Cyrnellus sp. were cleared with Hoyer, then squashed and analyzed directly from slides. We analyzed the whole gut content of all taxa, under the microscope (100X). The sets of slides were prepared from larvae collected from each sample (10-30 individuals). The proportion of each food item in the guts was estimated from the relative area of the particles in each slide (magnification at 100 X) using a microscopic ocular grid. Five categories of food resource were recognized: detritus (Det), vegetal tissues (Veg), filamentous algae (Algae), Bacillarophycea (Bacil), Chironomidae (Chir), Ostracoda (Ostr), Copepoda (Cop), Diptera (Dipt), Coleoptera (Col), Acari (Acar), Hyalella (Hyal), Cladocera (Clad) and not identifiable animal tissues (Anim). The importance of each item was measured according to the Index of Relative Importance (%IRI) (^{Cortés, 1998}).

Data analysis. Additional diet information of other common taxa in the area such as Hyalella curvispina Shoemaker, 1942 (Amphipoda), the chironomids Procladius Skuse, 1889, Ablabesmyia Johannsen, 1905, Pelomus Reiss, 1989, Cladopelma Kiefer, 1921, Chironomus Meigen, 1803, Endotribelos Grodhaus, 1987 Phaenopsectra Kieffer, 1921 and Polypedilum Kieffer, 1912 were used from previous studies by ^{Saigo et al. (2009}) and Gallizzi et al. (2012).

We analyzed the patterns of niche overlap in order to verify if analyzed taxa were structured into guilds. In that case, some taxa would present high niche overlap (those belonging to the same guild) while others would show lower overlap (those of different guilds). Therefore, the observed variance in niche overlap would be greater than expected by stochastic processes (^{Inger & Colwell, 1977}; ^{Winemiller & Pianka, 1990}). For that propose, we tested the niche overlap variance, using Pianka's index, against null models using the software EcoSim 7.0 (^{Gotelli & Entsminger, 2005}).

After confirming the presence of guilds, we performed a cluster analysis using Morisita similarity index, considering a similarity threshold of 0.6 (^{Wallace & Ramsey, 1983}) in order to define groups. Moreover, we applied a non metric multidimensional scaling (NMDS) using the same similarity index. Likewise, we compared niche overlap within and among guilds with simulated null models with EcoSim 7.0.

Randomization algorithms. Null models provide a quantitative basis to assess observed niche overlapping among species. To generate random matrices two decisions must be taken. First, niche breadth can be retained or relaxed. If niche breadth is retained the trophic specialization of taxa is preserved in simulated matrices. Second, zero states can be retained or reshuffled. If zeros are retained, resources that are not consumed by certain consumer are not consumed in simulated matrices either. Therefore, there might be four different scenarios, R1-R4. Randomization algorithm R1 relaxes niche breadth and reshuffles zeroes and tends to simulate "too null" matrixes. On the other hand, matrixes simulated with the algorithm R4, which retains both niche breadth and zeroes tend to be too conservative and subject to error type II. Therefore, recommended randomization algorithms are R2, which relaxes niche breadth and retains zeroes, and R3 which retains niche breadth and reshuffles zeroes (^{Gotelli & Entsminger, 2005}). In the present study, as we aimed at including observed niche breadth in the analysis, we used R2 as randomization algorithm.

This classification was then, compared to that performed in the Holarctic Region (^{Cummins & Klug, 1974}; ^{Merritt & Cummins, 1996}).

RESULTS

Diet analysis. Detritus was the most important feeding item for most of analyzed taxa. Except from Monopelopia sp., Sympetrum sp. and Pomacea canaliculata all the taxa presented detritus as their dominant food item. In some cases, indeed, its importance reached almost 100% of the relative importance (Americabaetis sp., Caenis sp., Campsurus violaceus and Nais communis) (Fig. 1).

Sympetrum sp. was the taxon for which animal tissues (Hyalella, Coleoptera, Diptera, Acari, Oligochaeta, Ostracoda, Cladocera and Copepoda) were the most important food item. On the other hand, P. canaliculata presented a great dominance of vegetal tissues in its diet, although detritus, algae and even animal tissues were registered as ingested items for this species. The chironomid Monopelopia sp, in turn presented a wide trophic spectrum including algae (Bacillariophyceae and filamentous algae), detritus, vegetal and animal tissues (Fig. 1).

Fig. 1 Relative importance (IRI) of food items for analyzed taxa of dominant benthic invertebrates in a floodplain lake of Paraná River, Argentina (parenthesis indicate sample size). 

These data were combined with those of ^{Saigo et al. (2009}) and ^{Galizzi et al. (2012}) to analyze patterns in niche overlap and trophic similarity. Niche overlap variance turned out to be greater than expected by chance (p<0.05) indicating that some taxa presented higher levels of niche overlap than others (Appendix 1). This result indicated that analyzed taxa were structured into guilds.

Cluster analysis using Morisita similarity index with a threshold of 0.6 yielded 5 groups (Fig. 2): one group included A. pigueti, P. leidyi, D. vagus, N. communis, Parachironomus sp., Polypedilum (Tripodura), Chironomus gr. decorus, Cladopelma sp., Phaenopsectra sp., Endotribelos sp., Pelomus sp., Crynellus sp., Caenis sp., Americabaetis sp., C. violaceus and H. curvispina. This group is characterized by a strong dominance of detritus as feeding item, thus it will be hereafter referred to as gatherer collectors.

Fig. 2 Cluster plot depicting trophic similarity (Morisita index) among species of dominant benthic invertebrates in a floodplain lake of Paraná River, Argentina. Dotted line depicts the threshold similarity of 0.6. 

Coelotanypus sp., Procladius sp., and Ablabesmyia (Karelia) built up a different group characterized by animal tissues (predators). Although Sympetrum was placed in a different group, its diet is also characterized by animal tissues (see discussion). The diet of P. canaliculata was characterized by vegetal tissues and therefore this species will be hereafter referred as herbivorous (see discussion). Monopelopia sp. in turn, presented a wide trophic spectrum with even proportions of detritus algae and animal tissues, and then we considered this taxon as omnivorous. This classification was supported as well by the NMDS that yielded a consistent multidimensional ordination with a stress value of 0.02 (Fig. 3).

Fig 3 Non Metric Multidimensional scaling plot. Circles depicts taxa classified as gatherer collectors (Aulodrilus pigueti, Pristina leidyi, Dero vagus, Nais communis, Pelomus sp., Cladopelma sp., Endotribelos sp., Polypedilum sp., Chironomus sp., Parachironomus sp., Phaenopsectra sp., Americabaetis sp., Baetis sp., Campsurus violaceus, Hyalella curvispina, Crynellus sp.) [Triangles: Tanypodinae (Coelotanypus sp., Procladius sp. and Ablabesmyia (Karelia); inverted triangle: Sympetrum sp.; square: Monopelopia sp.; cross: Pomacea canaliculata]. 

We performed an intra group overlap analysis only for those FFG that contained more than one species (gatherer collectors and predators). This analysis pointed out that niche overlap within gatherer collectors was higher than expected by chance (p=0.0001). Likewise, niche overlap within predators was not significantly different than null simulations (the index obtained between Coelotanypus and Procladius was 0.90 and Ablabesmyia (Karelia), 0.99; while between Procladius and Ablabesmyia (Karelia) it was 0.84). On the other hand, niche overlap among groups was lower than expected by chance (p=0.017).

Nearly one third of analyzed taxa (7 of 22) were classified in a different functional feeding group than in the Holarctic region (Tab. I).

Tab. I Comparison between functional classification of taxa in the Holartic region (according to Merrit & Cummins, 1996; Cummins et al., 2005) and the studied lake in the Neotropical region. Shaded rows refer to taxa which, in this study, were classified in a different functional feeding group than in the Holartic region. 

DISCUSSION

The present study provides a functional classification for some abundant benthic invertebrate taxa in a floodplain lake of the Middle Paraná River. Taxa such as H. curvispina, Phaenopsectra sp., Endotribelos sp., Parachironomus sp. and Polypedilum sp. were, in this study, classified as gatherer collectors although in the Holarctic region they are allocated in different FFGs. Indeed, most of analyzed taxa were classified as gatherer collectors with detritus as the most important feeding item. The great importance of the detritus energy pathway in the floodplain of the Middle Paraná River has been evidenced in previous studies (^{Ezcurra de Drago et al., 2007}; ^{Marchese et al., 2014}). Primary production of macrophytes in this system is very high, for example, the biomass fluctuated between 0.30 and 8.67 t dry weight ha^-1 (^{Sabattini & Lallana, 2007}). This production and the high bacterial activity result in a great amount of fine detritus, which is food source for gatherer collectors. This would explain that certain taxa which in temperate regions were classified as shredders, in this study they (or taxonomically related taxa) were classified as gatherer collectors (i.e. Polypedilum sp., Endotribelos sp., H. curvispina).

However, some studies have suggested the concept of "snail shredder" for environments in which hard substrata is scarce and snails scrape on plants, accelerating their breakdown (^{Mulholland et al., 1985}; ^{Ramirez & Gutierrez-Fonseca, 2014}). While our gut content analysis revealed that P. canaliculata feed on vegetal tissues, we allocated this species in the guild "herbivorous". Moreover, in the context of a general scarcity of shredders in Neotropical streams and their replacement by large omnivores in floodplain rivers (^{Wantzen & Wagner, 2006}), the great importance of vegetal tissues in the diet of P. canaliculata, suggests a role of this species in plant breakdown.

Likewise, comparative morphological studies have suggested that Tanypodinae were predators (^{Gouin, 1959}; ^{Bryce & Hobart, 1972}). Although Ablabesmyia (Karelia), Coelotanypus sp. and Procladius sp. were confirmed to be predators by this study, Monopelopia sp. appears to be an exception. Indeed, this genus is classified as a predator in temperate regions but in this study we classified it as omnivorous. Some diet analysis concluded that, under certain conditions, species of Chironomidae may utilize a wider range of trophic resources (^{Baker & McLachlan, 1979}; ^{Motta & Uieda, 2004}; ^{Galizzi et al., 2012}; ^{Saito & Fonseca-Gessner, 2014}; ^{Butakka et al. 2014}). Our results pointed out that, because of its wide trophic spectrum, Monopelopia sp. may play different roles by predating, grazing algae or consuming detritus. Further efforts should be devoted in order to know the trophic selectivity and plasticity of this taxon.

Sympetrum sp. was identified as a predator, and Hyalella sp. was its most important prey followed by copepods (Cyclopoda) and Cladocera. Although vegetal tissues and detritus were found in the diet of this genus, probably these items were in the stomach of digested prey organisms as suggested by ^{Walker (2009)}. However, some copepods and cladocerans can inhabit benthos and littoral plankton and their presence in the diet of Sympetrum sp. could suggest a role of this genus linking benthic and pelagic/planktonic food webs, as previous studies have reported predation of zooplankton by Odonata (^{Timms & Moss, 1984}; ^{Corbet, 1999}; ^{Burks et al., 2001}; ^{Gonzales Sagrario et al., 2009}). Although animal tissues are the main feeding item for this taxon as well as for Tanypodinae (Ablabesmyia, Procladius and Coelotanypus), in the cluster analysis and the NMDS they were placed in different groups. We consider that the bigger size of the dragonfly larvae allows the ingestion of relatively big preys such as Hyalella sp. which are inaccessible for Tanypodinae larvae. In this context, Tanypodinae and Sympetrum may belong to different guilds (sensu^{Ramirez & Gutierrez-Fonseca, 2014}) as they rely on different preys but, while they predate on other consumers, their FFG is Predators.

The present study provides a functional classification of common taxa in a floodplain lake of the Middle Paraná River based on gut content analysis. Nearly one third of analyzed taxa were classified in a different FFG than in the Holarctic region. This result indicates that classifications performed in the Holarctic region should be used with care in the Neotropical region, even in subtropical systems. Further efforts should be devoted to classify other common macroinvertebrate taxa of this system. As well, quantitative analysis should be performed to measure the relative importance of each FFG since even single species guilds, like herbivores, could be highly abundant and therefore crucial for system dynamics.