Ichthyofaunal diversity in the upper Jejuí River watershed in eastern Paraguay

ABSTRACT The upper Jejuí River is a major tributary of the Paraguay River in eastern Paraguay and is the principal drainage for the Mbaracayú Forest Biosphere Reserve. Despite the international significance of the biosphere reserve and detailed documentation of its terrestrial fauna and flora, the fish fauna in its streams remains relatively unknown, with 48 species previously recorded. We sampled 35 sites within the biosphere reserve over five years (2007–2011) to assess the richness of its ichthyofauna. These surveys yielded a new total of 105 known species for the biosphere reserve, including two newly-recorded orders, Gymnotiformes and Cyprinodontiformes, and 14 newly-recorded families. Fish community composition resembled that of previously-reported fish communities from the Paraguay River drainage, with characiforms and siluriforms comprising the majority of the species (57.1% and 31.4%, respectively). Post hoc analyses showed significantly greater ichthyofaunal diversity at sites within the Mbaracayú Forest Nature Reserve, a core protected forest area within the biosphere reserve, compared to areas outside the nature reserve, suggesting a negative impact from deforestation and land conversion on fish assemblages in these headwater streams. Broad regional deforestation places greater emphasis on important management decisions that will protect current biodiversity.


Introduction
The diversity of fishes in the Neotropical biogeographic region, with greater than 6,000 described species, surpasses that of other biogeographic regions [1]. Currently, this diversity is threatened by numerous anthropogenic activities [2] that impact rivers and streams both directly (e.g. dams and channelization) and indirectly (e.g. agriculture and urbanization) [3]. Moreover, it is argued that freshwater fishes are inadequately protected throughout the Neotropical region [4,5]. Freshwater ecosystems often receive ancillary conservation benefit only from the creation of terrestrial-focused protected areas [6,7]. Establishment of protected areas has become an increasing priority for many government and non-government entities. One such entity is the Mbaracayú Forest Biosphere Reserve (hereafter biosphere reserve) which lies within the Upper Paraná Atlantic Forest in eastern Paraguay. The biosphere reserve occurs entirely within the Jejuí River watershed, streams of which are left margin tributaries of the Paraguay River, and features the Mbaracayú Forest Nature Reserve (hereafter nature reserve) as its core protected area.
The Upper Paraná Atlantic Forest (locally referred to as the Bosque Atlántico del Alto Paraná (hereafter BAAPA) [8]) is part of the Paraguay-Paraná Complex aquatic ecoregion [9] and the Paraguay Freshwater Ecoregion [10]. It is estimated that, as recently as 1940, the BAAPA totaled almost 9 million hectares [11]. In the 1990ʹs and early 2000ʹs this ecoregion experienced some of the highest deforestation rates in the world [12]. As a result, by 2000, only about 20% of the original Atlantic Forest in Paraguay remained [13], which declined to less than 10% by 2016 [14]. Canindeyú Department, where the biosphere reserve is located, experienced a 32.8% forest loss between 1999 and 2016 [14]. A 5-km buffer zone area surrounding the biosphere reserve experienced a 39% loss of forest cover between 1989 and 2000 [13].
Paraguay's deforestation rate remains one of the highest in Latin America [15][16][17]. Most habitat loss comes from conversion of forest to agricultural land for soybean production, cattle grazing, and subsistence farming [18,19]. BAAPA has been rapidly and extensively fragmented due to changing land use patterns, infrastructure development in the form of roads and dams, and unsustainable exploitation of timber for construction, furniture, and firewood [8]. In addition to fragmenting the terrestrial landscape, these activities can negatively impact stream hydraulics, water quantity, water quality (via soil erosion and agrochemical pollution), and canopy cover (which raises water temperature and decreases leaf litter and woody debris) [20,21]. Thus, the biosphere reserve is an important remnant of BAAPA that helps protect both terrestrial and aquatic environments.
Despite a reasonably well-documented terrestrial vertebrate fauna for the biosphere reserve [22] and BAAPA [23][24][25][26], there exists wide variation among estimates of aquatic vertebrate diversity, and more intensive sampling of headwaters in the Paraguay River Basin has been recommended [27]. Neotropical fish assemblages are often speciose and are typically dominated by characiforms and siluriforms [26,[28][29][30][31][32][33]. Estimates as high as 450 fish species have been proposed for Paraguay based on its geographic position and resulting variety of intersecting habitats [34].
Diversity of fishes in the Paraguay Freshwater Ecoregion is strongly influenced by the unique evolutionary history experienced by Neotropical fishes that included periodic interbasin exchanges between the Amazon and La Plata, the latter of which includes the Paraguay River and its tributaries [28]. Additionally, given that habitat diversity promotes species diversity [35][36][37][38], the numerous aquatic habitats found within the Paraguay Freshwater Ecoregion is a strong predictor of high fish diversity. Modest estimates for the Paraguay Freshwater Ecoregion, which includes all of eastern Paraguay but does not include the western Chaco region, suggest 333 species, of which approximately 35% are considered endemic [39,40]. Numerous reports of Paraguay fish diversity emphasize the taxonomic dominance of Characiformes and Siluriformes [41][42][43][44][45][46][47].
As of 2005, the list of fishes known for the nature reserve included 48 species with 19 identified to genus only [22]. This list was consistent with results from Paraguay fish surveys by Chernoff et al. (Paraguay River basin only) [42] and Ramlow (a country-wide inventory) [44] in that 56% of species presented in the nature reserve management plan were characiforms and 29% were siluriforms [22]. Despite somewhat thorough survey treatments for mammals, birds, and plants, there existed only sporadic surveys of aquatic organisms in waterbodies near the reserve headquarters [22]. The lack of specific identifications (some fish records for the nature reserve identified only genus) coupled with the wide range of aquatic environments within the nature reserve and broader biosphere reserve suggested that species richness in the area could be higher. Therefore, we aimed to document fish diversity in the headwaters of the upper Jejuí River in the biosphere reserve by conducting an ichthyofaunal survey. Subsequently, we used post-hoc tests to assess patterns that appeared to emerge from our survey results.

Study area
The Mbaracayú Forest Nature Reserve was recognized in 1991 by Paraguay Law No. 112/91 as a joint ecosystem management effort between the Fundación Moisés Bertoni (FMB; a Paraguayan conservation nongovernment organization) and The Nature Conservancy [22]. Initially 57,715 hectares of Atlantic Forest habitat, over the next 5 years additional and mostly Cerrado (savannah) habitat was added to create what is now a 64,405-hectare nature preserve, one of the largest protected areas in Paraguay [22]. The terrestrial composition of the reserve is a habitat mosaic of 19 natural communities [22]. The Mbaracayú Forest Biosphere Reserve was included in the world network of biosphere reserves in 2000 by the United Nations Educational, Scientific and Cultural Organization (UNESCO) through the Man and the Biosphere Programme (MAB) when an additional 280,000 hectares of buffer zone was included around the nature reserve to serve as a multiple use area and to help protect the core forested region [22]. A hallmark of the biosphere reserve, and of Paraguay in general, is the large number of streams and rivers that traverse this land-locked country.
The upper Jejuí River watershed is the primary drainage of the biosphere reserve [9] with two major branches, the Jejuí mi and Jejuí guasu. The Jejuí mi occurs largely within the protected core forest area (nature reserve) and the Jejuí guasu occurs largely outside the core area (biosphere reserve) ( Figure 1).

Fish sampling
We sampled 35 locations within the biosphere reserve over a 5-year period (2007-2011); 20 were within the nature reserve and 15 were outside that core forested area (Table 1 and Figure 1). Over the five years, we gathered 77 samples. We  (Table 1). Three sites (rio Jejuí mi at the nature reserve headquarters, arroyo Guasu immediately west of the nature reserve headquarters, and arroyo Guyra Keyha at the eastern limit of the nature reserve) were sampled each year. Two sites were sampled during each of the first four years, eight were sampled three times between 2007 and 2011, eight were sampled twice, and 14 were sampled during one year only. Landowner permission was obtained prior to sampling on privately-owned land. Geographic location data were obtained using Garmin handheld GPS units (Garmin GPSMAP 60 cs). Geographic coordinates for all survey sites (Table 1) Table 1); the Jejuí mi (northern branch) and Jejuí guasu (southern branch) rivers are indicated by thicker blue lines, the border of the biosphere reserve is indicated by the red line, and the forest reserve is indicated by the black line.
The streams we surveyed presented a range of characteristics (e.g. depth, width, stream velocity, canopy cover; Figure 2). At each site, a point of entry into the river or stream formed the center of the sampling area. All sites were sampled for approximately 50 m upstream and 50 meters downstream from the Table 1.. Sampling sites in Mbaracayú Forest Biosphere Reserve, Canindeyú, Paraguay. Listings include site code (which corresponds to locations identified on Figure 1), site name, location coordinates (latitude, longitude), watershed within which the site resides, and years a sample was collected. Sites are identified by the year in which they were first sampled: sites beginning with 1 were first sampled in 2007, 2 in 2008, 3 in 2009, 4 in 2010, and 5 in 2011.

Site Code
Site Name  Latitude  Longitude  Watershed  2007  2008  2009  2010  2011  1A Rio Jejuí mi at Jejuí mi North central stream -Aguara Ñu 24°9ʹ13.0"S 55°15ʹ53.0"W Jejuí mi X  Fishes were identified to species, when possible, through use of multiple taxonomic keys and references  and compared to a comprehensive catalog of fish species for Paraguay [47]. Taxonomy herein follows the Eschmeyer's Catalog of Fishes electronic database of the California Academy of Sciences [72].
All collected fishes were assigned a functional role which combined their principal adult diet with their primary foraging stratum in the water column based on their reported foraging ecology and based on prior assessments [45,59,73,74]. Fish assemblages often are categorized into functional groups, which can be defined by ecomorphology, a combination of physical features and habitat characteristics [75][76][77][78]. A functional group consists of several species that perform a similar ecological role while not necessarily sharing an immediate taxonomic relationship. Thus, functional groups are defined by a shared niche rather than a shared taxonomy [79]. Principal adult diets were categorized as omnivore (feeds on a wide variety of plant and animal materials), invertivore (feeds primarily on aquatic and/or terrestrial invertebrate animals), piscivore (feeds primarily on other fishes), algivore (feeds primarily on single-or multi-celled algae), or detritivore (feeds primarily by ingesting organic detritus and extracting nutrients from the material itself and any organisms residing within) [52,73]. Foraging strata within the water column water were defined as surface (forages primarily at the air /water interface), mesopelagic (forages primarily in the mid-section of the water column), benthopelagic (forages primarily at, or within several centimeters of, the substrate), and benthic (forages primarily on or below the substrate surface) [74]. Functional diversity is defined in our study as the number of functional groups, formed from a combination of primary diet and primary water foraging stratum.

Statistical analyses
Upon examination of the data following survey completion, patterns appeared to emerge that led us to employ post hoc, one-tailed t-tests to test a posteriori hypotheses that fish species richness, family-level diversity, functional diversity, and total captures from sites inside the nature reserve were higher than from sites outside the nature reserve. Because the sampling focus shifted from fishes to freshwater decapod crustaceans during 2010 and the sampling in 2011 did not include sites outside the nature reserve, we excluded 2010 and 2011 data from these analyses. In accordance with the recommendation of Dayton [80], we set alpha = 0.10 to decrease the probability of Type II error, concurring that failure to detect patterns that do exist can result in dire conservation consequences. While survey methods were consistent within each year, we elected to test each year separately due to changes in survey methods between years.

Survey summaries
During the 5 years of sampling the upper Jejuí River watershed in the biosphere reserve, we collected 7,110 individuals and identified 97 fish species, belonging to seven orders, 26 families, and 66 genera (Supplemental Information). Two orders, Gymnotiformes and Cyprinidontiformes, were new records for the biosphere reserve. Of the 26 families, 14 were new records for reserve, including five within Characiformes (Acestrorhynchidae, Anostomidae, Iguanodectidae, Lebiasinidae, Parodontidae) and three within Siluriformes (Auchenipteridae, Cetopsidae, Pseudopimelodidae). The remaining six new families reside within the two newly-documented orders, Gymnotiformes (Gymnotidae, Hypopomidae, Rhamphichthyidae, Sternopygidae) and Cyprinidontiformes (Cynolebiidae, Poeciliidae). Our surveys yielded 65 species not previously identified for the biosphere reserve (Supplemental Information). Fishes were categorized into one of 14 functional groups (Supplemental Information).
Of the 48 species previously recorded for the reserve [22], we detected 37 in our surveys. Of the remaining 11 species previously recorded, three have been discounted (Astyanax bimaculatus, Astyanax brevirhinus and Hemigrammus belottii) because they are known only from the Amazon River basin [72]. The remaining eight species were not detected in our surveys and are so noted in the Supplemental Information. When added to the 97 species detected by our surveys, a minimum fish species richness of 105 is obtained for the biosphere reserve. Characiformes represented 57.1% of the species (60) while Siluriformes represented 31.4% (33).

Richness and diversity
Species richness, family-level diversity, and functional diversity were higher inside the nature reserve than outside the nature reserve (but still within the biosphere reserve) for the three years tested, while no significant differences occurred in the total number of individuals caught inside compared to outside during each survey year ( Table 2).
In 2007, the number of fish species at sites within the nature reserve varied from 3 to 23 while the number outside the nature reserve ranged from 2 to 14, and the number of individuals captured varied from 22 to 240 inside to 9 to 166 outside the nature reserve. Richness inside the reserve was 82.3% greater than that found outside the reserve (one-tailed t = 2.14, df = 15, p = 0.028; Figure 3(a)). Family-level diversity inside the reserve was 40.6% higher than outside the reserve (one-tailed t = 1.82, df = 15, p = 0.047; Figure 3 (b)) while functional diversity was 60.9% greater inside the reserve compared to outside (one-tailed t = 3.51, df = 15, p = 0.002; Figure 3(c)). While not statistically significant, a greater number of individuals was collected from inside the reserve than outside in 2007 (one-tailed t = 1.36, df = 15, p = 0.168; Figure 3(d)).
In 2008, the number of fish species at sites inside nature reserve varied from 3 to 25 while the number outside the nature reserve ranges from 3 to 21, and the number of individuals captured varied from 17 to 319 inside and 7 to 324 outside the nature reserve. Richness inside the nature reserve was 74.4% greater than outside the reserve (one-tailed t = 1.36, df = 15, p = 0.084; Figure 3(a)). Family-level diversity was 37.2% higher inside the nature reserve than outside (onetailed t = 1.486, df = 15, p = 0.083; Figure 3(b)) while functional diversity was 28.6% greater inside the reserve than outside (one-tailed t = 1.41, df = 15, p = 0.093; Figure 3(c)). In 2008, more individuals were collected from outside the reserve, though no significant difference was detected (one-tailed t = 1.34, df = 15, p = 0.478; Figure 3(d)).
In 2009, the number of fish species at sites inside the nature reserve varied from 11 to 23 while the number outside the nature reserve ranged from 4 to 23, and the number of individuals captured varied from 13 to 431 outside and 50 to 184 inside the nature reserve. Richness was again significantly higher (47.5%) at sites inside the reserve compared to sites outside the reserve (one-tailed t = 1.85, df = 15, p = 0.044; Figure 3(a)). Family-level diversity of fishes inside the reserve was 69.9% more than outside (one-tailed t = 2.63, df = 15, p = 0.012; Figure 3(b)) while functional diversity was 44.1% higher inside the reserve than outside (one-tailed t = 3.40, df = 15, p = 0.003; Figure 3(c)). Though not statistically significant, there were more fish collected outside the reserve than inside (one-tailed t = 0.60, df = 15, p = 0.280; Figure 3D).

Discussion
The 105 species identified within the biosphere reserve represent 31.5% of the estimated 333 species for the Paraguay Freshwater Ecoregion [39,40]. The Paraguay Freshwater Ecoregion consists only of the immediate ecoregion associated with the left margin Paraguay River watershed and does not consider the fish species associated with the Chaco region of western Paraguay [9] that are included in country-wide diversity estimates. This substantial proportion of the ecoregion's fish fauna that occurs within the biosphere reserve highlights the importance of management in the reserve that maintains aquatic habitat quality to support healthy fish assemblages.
Our results yielded an increase in the number of fish species known to inhabit the Mbaracayú Forest Biosphere Reserve by 65 species including two new orders and 14 new families. This increase can likely be attributed to the increase in sampling effort, including sampling more of the varied aquatic habitats found there. Moreover, while results from each year's surveys (both number of species captured and species composition) were similar to those in other studies from southern South America [31][32][33]81], the cumulative number of species detected was increased by our repeated effort over 5 years.
Twenty-five common species were detected during each and every year of the survey. Composition of this group was similar to the overall fish community composition, with Characiformes representing 52.0% (13) and Siluriformes representing 36% (9). This group of commonly encountered species in the streams of the biosphere reserve reflects the general community composition of broader fish community structure patterns throughout the neotropics [39].
Not previously documented for the biosphere reserve, gymnotiforms were detected every year during our surveys. That gymnotiforms had not previously been recorded is surprising given the number of species (15) in Paraguay [47], their regular occurrence in our surveys, both temporally and spatially, and their prominence in fish assemblages and widespread distributions throughout the Neotropics [39,82]. Detection of cyprinodontiforms did not occur until 2009, the third year of our survey, and corresponded with new survey locations, including two in Cerrado (savannah) habitat (sites 3B and 3C in Table 1 and Figure 1).
The composition of fishes detected during our survey was similar to that of other fish assemblages compiled by Chernoff et al. [42] for the upper Paraguay River and by Ramlow [44] for all of Paraguay. While the collections reported by Ramlow [44] originated from throughout Paraguay, the majority occurred in the vicinity of the Paraguay River and its immediate left margin tributaries. Therefore, it appears that the ichthyofaunal diversity of Paraguay's Freshwater Ecoregion is well represented in the biosphere reserve, particularly in the relatively pristine forested core protected area of the nature reserve. Thus, the fish assemblage of the nature reserve may represent a model for similar headwater regions of the Paraguayan Atlantic Forest Ecoregion prior to expansive deforestation.
Significantly fewer fish species were collected at sites outside the core nature reserve, illustrating the negative impact of deforestation and land conversion on fish assemblages in these headwater streams, and demonstrating the benefit of protected areas for conservation of biodiversity. Deforestation is known to negatively impact diversity in many Neotropical aquatic systems [35,83]; homogenization of the physical environment around streams in the neotropics leads to reduced overall species diversity despite equal, or even greater, abundance of fish [84]. We found no statistical differences in abundance of total fish captures inside the nature reserve versus outside for all three years tested, which adds support to our conclusion that results for the three diversity measures presented (species richness, family-level diversity, functional diversity) reflect real difference between years. That functional diversity was greater inside the nature reserve than outside during each year could be attributed to the decreased heterogeneity of the aquatic system with landscape changes that have occurred outside the core protected area, a situation documented in other regions of the neotropics, where deforestation negatively impacts fish communities [35,[84][85][86].
Terrestrial habitat modification is a global threat to aquatic biodiversity [87] and this is especially problematic for freshwater fish diversity in the Neotropical region [3]. Conversion of natural habitat in Paraguay into human-altered landscapes has been extensive and has accelerated in recent decades [17]. Forest cover within Paraguay's Atlantic Forest Ecoregion was reduced from 73.4% to 24.9% between the 1970ʹs and 2000ʹs, and this reduction occurred throughout the region irrespective of proximity to protected areas [13,17]. This rapid loss in forest cover is causing major declines in biodiversity within Paraguay, highlighting the importance of conserving natural areas [88]. Considering that the fish fauna of the Mbaracayú Forest Biosphere Reserve includes approximately onethird of the regional fish diversity, management of the reserve is extremely important for maintaining what is likely an excellent representation of fish assemblages in headwaters of the Paraguay Freshwater Ecoregion. As landscape change continues due to increasing agricultural and urbanization demands, the value of the biosphere reserve for conserving fish diversity is likely to increase. Managers of the biosphere reserve should consider the impact of actions not only on the terrestrial environment but also on the aquatic environment.