Annelida ( Oligochaeta and Aphanoneura ) from the Natural Reserve of Isla Martín García ( upper Río de la Plata estuary , Argentina ) : biodiversity and response to environmental variables

The Island of Martín García – located in the Upper Río de la Plata, to the south of the Uruguay River – is an outcropping of the crystalline basement. Fourteen sampling sites were selected, five along the littoral section of the island and nine in inland ponds. Four major environmental variables were measured: water temperature, dissolved oxygen, electrical conductivity, and pH. A total of 34 species of Oligochaeta and Aphanoneura were found, 30 belonging to Naididae plus one species each of the Narapidae, Lumbricidae, Enchytraeidae, and Aeolosomatidae. The thirteen most frequent species were: A. leydigi (30%), N. bonettoi (13%), L. hoffmeisteri (11%), N. variabilis (10%), S. trivandrana (6.5%), A. pigueti (5.6%), D. sawayai (4.5%), D. digitata (3.5%), C. diastrophus (2.7%), A. costatus (2.5%), P. longiseta (2.0%), Enchytraeidae (1.5%), and A. p. paraguayensis (1.4%). UPGMA clustering of species based on their occurrence in different ecological conditions revealed two main species groups. Canonic-correspondence analysis (CCA) was conducted with the 15 most frequent and abundant species in the 9 sampling sites and the 4 environmental variables. Results from the CCA revealed that the order of fluctuation of the environmental variables during the sampling period was, from the greatest to the least: dissolved oxygen, conductivity, pH, and water temperature. Approximately 97.6% of the correlations between species and environmental variables were expressed on axis 1 of the ordination diagram. Species richness correlated with the four environmental variables in the following order, from the weakest to the strongest: water temperature, pH, electrical conductivity, and dissolved oxygen.


Introduction
The annelid class Oligochaeta -considered by many authors as a bioindicator of environmental conditionsis represented by a large number of genera and species present in continental waters within the benthic, pleuston, and periphyton communities (Hellawell, 1986;Rosenberg and Resh, 1993).Some genera exhibit a high degree of adaptation indicating a broad degree of plasticity with respect to environmental and evolutional challenges.A hundred species of Oligochaeta have been reported in Argentina, though most of the studies have been focused on the littoral fluvial zones and particularly on the Paraná-River basin, where 81 especies have been found (Marchese and Paggi, 2004).
Martín García Island is a Multiple-Use Reserve located in the upper Río de la Plata estuary and was declared a natural reserve in 1969 (Tratado del Río de la Plata y su Frente Marítimo).Because of the island's status, several investigations have been conducted on the flora and fauna of the island focusing on their biodiversity and ecology.Those studies mostly dealt with hydrophilic plants (Lahitte and Hurrell, 1996), vertebrates (Juárez, 1995;Lahitte et al., 1995;Lahitte and Hurrell, 1998), and aquatic and semiaquatic Insecta (Fernández and López Ruf, 1999).
The aim of the present investigation was to report the biodiversity of annelids of the classes Oligochaeta and Aphanoneura within the natural reserve and to determine the response of the most representative species to the range of environmental variables.

Material and Methods
The island study site Isla Martín García (Figure 1)located in the upper Río de la Plata estuary (34° 11'S 58° 15'W)-is an outcropping of crystalline Brazilian basement from the Precambrian Era covered by Quaternary-Era sediments (from both the Pleistocene and Holocene epochs (Ravizza, 1984)).The island's coasts are asymmetric in shape, with the western receiving constant contributions of suspended silty materials from the Paraná River.That silt deposits on the rocky bottom of the river and is progressively consolidated by the coastal vegetation.The eastern and southern coasts are rocky and owing to the strong action of southeastern winds may be occasionally covered by silty-sandy sediments.This coastal asymmetry functions in producing a distinctive plant distribution.Thus, along the northern coast (Station 5, Puerto Viejo) hydrophile plants -such as Typha latifolia L., Echinodorus grandiflorus (Chan and Schutdl.)Micheli, Panicum pernambucense (Spreng.)Mez ex Pilg.; Cyperus virens Michx., Pontederia rotundifolia L. f., Ludwigia elegans (Cambess.)Hara, Alternanthera philoxenoides (Mart.)Griseb, and Ranunculus flageliformis (Sm.) -predominate.In addition, submerged aquatic vegetation -such as Potamogeton gayii A. Benn, Egeria densa Planch, and Miryiophyllum aquaticum (Vell.)Verdc.-grow within beds of the reed Schoenoplectus californicus (C. A. Mey.) Soják in the permanently flooded areas (Lahitte and Hurrell, 1996).Station 4 (Playa Basural), located near the island's garbage dump, is an area with sandy-silty sediments.In Station 3 (Puerto Nuevo), sandy-silty sediments likewise predominate along with a scarce occurrence of reedbeds.
To the southeast of the island a small beach of light sand is present (Station 1), while further north lies a vast area of reedbeds (Station 2).
Inland, permanent and temporary ponds are present: some that have been formed in depressions in the former basalt quarries are located within several sectors surrounding the plateau (in the south-central portion of the island: stations 6, 7, 8 10, 11, 13); others -formed in depressions in the marginal forest near the coasts (stations 12 and 14) or in zones close to the inland's sandy plains -are small and temporary.These ponds contain floating vegetation -such as Lemna giba L., Spirodela intermedia W. Koch., Wolfia columbiana H. Karst., Wolffiella lingulata (Hegelm.)Hegelm, Hydrocotyle sp., Azolla sp., Salvinia sp., and Ludwigia sp.
Fourteen sites were surveyed from 1995 through 1997 -5 on the littoral zone of the island (stations 1-5) and 9 in ponds (stations 6-14) within the island (Figure 1) -during 8 seasonal collection trips.In the littoral zone of the island, samples were collected with an Ekman hand dredge (175 cm 2 ), whereas in the inner ponds the sampling was done with a net (frame size, 30 × 30 cm; mesh size, 150 mm).Samples were fixed in situ in 10% (v/v) formaldehyde.In the laboratory, the collected specimens were washed on a sieve (125 microns) and the Oligochaeta separated from the vegetation, stained with erythrosin B, and preserved in 70% (v/v) aqueous alcohol.This separation and the counting of the individuals were conducted under a stereomicroscope, but the species were identified under a conventional binocular microscope after Brinkhurst and Marchese (1992).The system and distribution proposed by Christoffersen (2007Christoffersen ( , 2011) ) was followed.
Four major environmental variables were measured at the study sites by means of portable digital sensors: water temperature (T °C), dissolved oxygen (mgl -1 ), electrical conductivity (mS cm -1 ), and pH.The relationships between the species and the environmental variables were examined through canonical-correspondence analysis (CCA), considering the fifteen most abundant and most frequently occurring species, 9 sampling stations, and 4 environmental variables (Ter Braak, 1986;Ter Braak and Verdonschot, 1995;Külköylüoğlu, 2003Külköylüoğlu, , 2005;;Külköylüoğlu and Dügel, 2004).The mean value for each environmental variable together with species-abundance data were used in the CCA.The associations among species were evaluated by clustering analysis according to the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) by means of the Jaccard Index (Crisci and López Armengol, 1983), with the species analyzed being the same as those used for the CCA.The Pearson correlation analysis was conducted and the statistical-significance values of the correlations determined by the Student t test.Statistical analyses were carried out through the use of the Multi-Variate Statistical Package (MVSP), version 3.1 (Kovach, 1998).
The UPGMA clustering analysis (Figure 2) revealed two main species groups.The first group comprised D. sawayai and A. p. paraguayensis (J=0.60).The second consisted of several subgroups, where the group Limnodrilus udekemianus Claparede, 1862 and S. trivandrana and the one composed of the Aelosomatidae, Enchitraeidae, L. hoffmeisteri, and A. leydigi became included in the maximum-similarity index.The third group in the maximum-similarity index is composed of A. pigueti and Pristina rosea (Piguet, 1906).
The CCA (Figure 3, Table 2) revealed that the degree of fluctuation of the environmental parameters during the sampling period was, in the following order, maximum to minimum: dissolved oxygen, conductivity, pH, and water temperature.Approximately 97.6% of the correlations between the species and these variables became expressed on axis 1 of the ordination diagram.
The correlation of species richness was weak with temperature (p <0.05 with 31% correlation), higher with pH (p <0.01, 54%), strong with electrical conductivity  3).N. variabilis was located in the portion nearest to the center of the ordination diagram, thus indicating a great plasticity with respect to environmental variables.This species was accordingly found along the coast and in the inland ponds (e. g., Station 6).Pristina a. aequiseta Bourne, 1891, P. longiseta, D. sawayai, and A. p. paraguayensis     exhibited medium adaptability and the closest relationship to conductivity.The prevalence of Narapa bonettoi and C. diastrophus correlated mainly with the pH (p <0.01, at a 54% and 65% correspondence, respectively); while the occurrence and abundance of the taxa Enchytraeidae and Aeolosomatidae, together with A. leydigi, A. pigueti, L. hoffmeisteri, P. rosea, S. trivandrana, and L. udekemianus -all being found along the coast -were mainly associated with levels of dissolved oxygen and temperatures near the mean values.

Discussion
Inland and littoral aquatic environments in the reserve Isla Martín García are rich in Oligochaeta and Aphanoneura (34 species).
Most of the studies on Oligochaeta from the littoral fluvial region in Argentina have been focused on the Paraná-River basin.Those studies provided information on the benthos, along with the fauna associated with macrophites, but the most frequently reported species belonged to the pleustonic community (Marchese and Paggi, 2004).Of the 25 species of Naidinae registered on the Isla Martín García, 9 (Table 1) were recorded only in the benthos, 7 in both the benthos and the pleuston, and 7 exclusively in the pleuston.With respect to the Tubificinae, Bothrioneurum americanum Beddard, 1894 was present in the pleustonic community, while the remaining 4 species were located in the benthic communities of the island's littoral.
Most of the species reported from Martín García Island had a general distribution throughout Argentina that also corresponded to that of the provinces included in the Paraná, Uruguay, and Río de la Plata river systems (Table 1). A. pigueti, A. costatus, A. furcatus, Eiseniella tetraedra Savigny, 1867, L. hoffmeisteri, L. udekemianus, P. longiseta andPristina osborni (Walton, 1906) were among the most widely distributed species in Argentina.As to other distributions, A. costatus, B. americanum, N. bonettoi, and D. sawayai are known to be neotropical species, while A. pigueti, Branchiura sowerbyi Beddard, 1892, L. hoffmeisteri, L. udekemianus, and N. variabilis are considered to be cosmopolitan.
The results from the clustering analysis revealed the presence of two main groups of species.The firstconsisting in D. sawayai, A. p. paraguayensis (J=0.60), and D. sawayai -had been recorded in the inland ponds and littoral environments of Martín García Island and also included A. lairdi as a shared species in the ponds (stations 6, 8, and 9).The second group contained several subgroups, one of which included the littoral species L. udekemianus and S. trivandrana that shared the maximum similarity index (J=1; stations 3-5) along with a second subgroup composed of littoral taxa, the Aelosomatidae and Enchitraeidae plus L. hoffmeisteri and A. leydigi (J=1).Finally, a third subgroup participating in the maximum similarity index comprised A. pigueti and P. rosea in addition to N. variabilis (J=0.8), with the latter species being also found inland.Similar associations among the species had been reported by Pavé and Marchese (2005) from urban rivers that flow into the middle Paraná River.
The results of the species responses to the environmental variables indicated that N. variabilis was the only species at the center of the ordination diagram, thus confirming the cosmopolitan nature of that species -namely, its ability to thrive in different habitats, including saline waters and mosses, and to tolerate high levels of organic pollution (Collado de La Peña, 1994).This species showed the greatest abundance at stations 4 and 5, with those sites -and especially number 5 -being associated with silty substrates and the presence of vegetation.N. variabilis was also registered at Station 6 (average conductivity = 1343.5mS cm -1 ).P. a. aequiseta, P. longiseta, D. sawayai, and A. p. paraguayensis, however, were the species most associated with medium values of conductivity.Pavé and Marchese (2005) had recorded medium to high values of conductivity (631-1501 mS cm -1 ) for those species in the urban rivers that flow into the middle Paraná River.The optimum pH values for N. bonettoi and C. diastrophus were similar to the optima for those same species previously reported in different sections of the Paraná River (Marchese, 1994;Pavé and Marchese, 2005;Behrend et al., 2009).
In his studies on the zoobenthos of the Arrecifes River (Buenos Aires province) and its main tributaries, Sampóns (1988) mentioned certain species of Oligochaeta -such as N. variabilis, P. aequiseta, L. hoffmeisteri, and A. piguetithat were also found on Martín García Island and considered here in the CCA.As to the response of these species to the environmental variables cited for the island, and in consideration of the values previously reported by Sampóns (1988), the populations of N. variabilis -and likewise of P. aequiseta -on Martín García Island developed under similar values of temperature and pH, and at medium to high levels of conductivity.
In contrast, the most frequent species on the island's littoral -e.g., A. leydigi, A. pigueti, L. hoffmeisteri, P. rosea, S. trivandrana, and L. udekemianus -were associated mainly with levels of dissolved oxygen and temperature near the mean values and, accordingly, are generally recognized as being cosmopolitan species of worldwide distribution.

Table 2 .
Main Results from the Canonical correspondence analysis (CCA).

Table 3 .
The numbers indicate strong correlations (* p< 0.05, ** p< 0.01) where n = 40 for all variables.The abbreviations are same as in