Fishes from the Tusubres River basin , Pacific coast , Costa Rica : checklist , identification key and photographic album

A checklist of the fishes of the Tusubres River basin, Pacific coast of Costa Rica, compiled from field and museum surveys is herein presented. A total of 54 species, representing 47 genera and 27 families, were recorded. Peripheral species were dominant (64.8%), followed by secondary freshwater fishes (20.4%); primary freshwater fishes accounted only for 14.8% of the total fish diversity. Eleotridae (6 spp.), Gobiidae (6 spp.), Poeciliidae (5 spp.) and Characidae (4 spp.) were the most diverse. Two species (Caranx sexfasciatus, Carangidae; and Opisthonema libertate, Clupeidae) were new records for Costa Rican freshwaters, and two species (Gymnotus maculosus, Gymnotidae; and Lebiasina boruca, Lebiasinidae) was found to have expanded ranges. An identification key and a complete photographic album of all fish species recorded in the basin are presented. The results of this investigation provide a framework for future studies on biogeography, ecology and conservation on fishes from this area.


INTRODUCTION
The Tusubres River Basin (Figure 1), located on the Pacific slope of Costa Rica (09°47′-09°29′ N and 084°40′-084°18′ W), has a drainage area of 826 km 2 (Rojas 2011), which correspond to 1.6% of the total country area.Despite its small size, this basin has a relatively high diversity of fishes as a result of its environmental heterogeneity and geographic position (Alpírez 1985;Bussing 1998).
Biogeographically, the Tusubres River Basin is in cluded within the Chiriquí-Santa María area of endemism (sensu Matamoros et al. 2014), and represents the northern limit of this area.The area is characterized by the presence of several South American lineages that dispersed north into Middle America, representing, in most cases, the northern limit of their distributions (Matamoros et al. 2014).
Traditionally, several authors (e.g., Bussing 1987Bussing , 1998;;Angulo et al. 2013) have included the Tusubres River Basin within the Pirris River Basin.However, as noted by Rojas (2011), geographic, hydrological, climatic and ecological factors merit the recognition of the two basins as distinct units.
Despite its relatively high diversity and the risk of extirpation of some species due to human activities (mainly by deforestation, expansion of agricultural frontiers and intensive fishing; Bussing 1998;Rojas 2011), the fish fauna of this basin is relatively poorly known.Taking this in consideration, the aim of this study is to describe the composition and distribution of the ichthyofauna of the Tusubres River Basin as a framework for future studies of the biogeography, ecology and conservation of fishes from this area.A checklist, an identification key and a complete photographic album of the fish species are provided.

MATERIALS AND METHODS
A total of 19 sampling points were performed (Table 1; Figure 1).Fishes were collected using dip nets (30×40 cm  1. Angulo et al. | Fishes from the Tusubres River basin, Costa Rica frame and 1 mm net mesh size and 35×50 cm frame and 5 mm net mesh size), cast nets (1.2 m and 2.3 m radius and 15 mm net mesh size), gill nets (20×1.8m and 2 and 5 cm between knots), seine nets (6×2 m and 10×2 m and 5 mm between knots), and a Smith-Root LR-20B ® backpack electrofisher.Eleven sampling events were conducted: February and November 2011, February and November 2012, February, March, May, August and November 2013, and February and October 2014.Fishing gear was selected according to the environmental and hydrological conditions at each site.Collection and research permits (No-181-2010-SINAC, No 157-2012-SINAC and No 007-2013-SINAC) were issued by the Costa Rican Ministerio de Ambiente y Energía (MINAE) and the Sistema Nacional de Áreas de Conservación (SINAC).
The majority of the captured specimens were identified in the field following Bussing (1998) and Robertson and Allen (2008); these specimens were released alive.Some specimens of uncertain field identification were retained and were first preserved in 10% formalin and then transferred to 70% ethanol for laboratory identification.Prior to preservation, specimens captured alive were anaesthetized and euthanized with clove oil following Inoue et al. (2003).Voucher specimens (at least one) of all sampled species were retained, photographed (Figures 2-5) and deposited at the fish collection of the Museo de Zoología of the Universidad de Costa Rica (UCR) with catalog numbers provided in Table 3.In order to supplement the data obtained through this inventory, a thorough review of the material deposited at the fish collection of the UCR was also conducted (Table 2).
Ichthyofaunal data were recorded as presence-absence of individual fish species by sampled sites (Table 3).The family tolerance to salinity is listed (Table 3) according to the classification of Myers (1949).Taxonomic nomenclature follows Eschmeyer (2015).Measurements and counts follow Hubbs and Lagler (1958) and Bussing (1998).
Taking into consideration both sources of information (fish inventory and UCR material), the fish fauna of the Tusubres River Basin is comprised of a total of 54 species; representing 47 genera and 27 families (Table 3).Based on Bussing (1998), all listed species were native to the Tusubres River Basin.The number of species at a single locality ranged from 2 to 23 (mean=11.8).Highest species richness (12-23 species, mean=17.8) was recorded very close to the coast, between 9 and 14 m above sea level.Upstream, between 106 and 455 m, the number of species was relatively lower (8-17, mean=10.8),with localities 6, 11 and 12 (see Figure 1) having the low richness values (2-3 species, mean=2.33) Based on Myers's (1949) salinity tolerance classification of freshwater fishes, the majority of species were peripheral (35 species, 64.8%), followed by secondary freshwater fishes (11 species, 20.4%).Only four families (Characidae, Gymnotidae, Heptapteridae and Lebiasinidae) and 8 species (about 14.8% of the total fish diversity) of primary freshwater fishes were recorded (Table 3).

Key to species of fishes from the Tusubres River basin, Pacific coast, Costa Rica
The following key is based on our research and data available in the literature (Bussing 1987(Bussing , 1998;;Nelson 2006;Robertson and Allen 2008).Voucher specimens are listed in Table 3. *Pictures taken after fixation in 10% formalin and stored in 70% alcohol.Voucher specimens are listed in Table 3.  3.

DISCUSSION
The total diversity of fish species in the Tusubres River Basin (54 species) represents 21.6% of the total diversity of native inland water fishes in the country (250 species) (Angulo et al. 2013).Taking into consideration the relatively small area of the basin, this number can be considered relatively high, with about 6.5 species/100 km 2 vs. about 0.5 species/100 km 2 on average for the country.
On the basis of Angulo et al. (2013), this basin ranks seventh in the country in terms of total number of species; from a total of 19 basins, the Tusubres River Basin is surpassed only by the Tortuguero (115 species), the Térraba (88 species), the Parismina (83 species), the Matina (75 species), the Sixaola (62 species), and the Sarapiquí (60 species) river basins.It is notable that most of these basins (5) are located on the Atlantic versant and only the Térraba River Basin is located on the Pacific versant.In addition, the Tusubres River Basin includes about 27.9% of the total diversity of inland water fishes in the Pacific versant of the country (Alpírez 1985;Angulo et al. 2013).Its geographical position, at the transition between two ichthyofaunal areas of endemism (Matamoros et al. 2014), and its environmental heterogeneity (Rojas 2011) could explain the relatively high diversity of fish species in this basin.
In general terms, the diversity and composition of the fish assemblage within the basin increased from upstream to downstream sites.Downstream sites were dominated by peripheral species of the order Perciformes, including families such as Eleotridae, Gobiidae, Gerreidae and Centropomidae, whereas upstream sites were dominated by primary and secondary species of the families Characidae (Characiformes), Cichlidae (Perciformes) and Poeciliidae (Cyprinodontiformes).As noted by Bussing (1998), Espinoza (2007), and Rojas and Rodríguez (2008), these differences in the assemblage composition largely reflect local differences in environmental conditions among sampled sites.In general terms this pattern corresponds to that observed in neighboring basins, Térraba for example (Rojas and Rodríguez 2008), as well as in the rest of the country (Bussing 1998).The dominance of peripheral species, in proportion to the total number of species, also is in accordance with that reported in the literature for Costa Rican inland waters in general (Bussing 1998;Angulo et al. 2013).
Localities 6, 11 and 12 showed very low diversity values (2-3 species, mean=2.33).This can be explained by the presence of several natural and/or artificial barriers (e.g., waterfalls or small dams) that limit the movement upstream of several species and/or by difficulties during the sampling (e.g., inadequate climatic conditions and/or problems or limitations with the use of some specific sampling gears).These localities were included in this work because in some of them, despite of these limitations, some uncommon and/or interesting species were recorded (e.g., Pterobrycon myrnae Bussing 1974, andHyphessobrycon savagei Bussing 1967, Characidae; Gymnotus maculosus; and Cynodonichthys isthmensis (Garman 1895), Rivulidae).It is feasible that a more extensive and detailed sampling in these localities could result in higher values of fish diversity.
On the basis of Bussing (1987Bussing ( , 1998) ) and Angulo et al. (2013), two species (Caranx sexfasciatus, and Opisthonema libertate) are considered new records for Costa Rican inland waters.These peripheral species had already been recorded in coastal environments on the Pacific coast of the country (Bussing and López 2009); however, the present record is the first from inland waters.
Additionally, two primary freshwater species, Gymnotus maculosus and Lebiasina boruca, were found to have expanded distributional ranges.The previously known distribution of G. maculosus in the Pacific versant is between the San Nicolas River Basin (Chiapas, Mexico) and the Bebedero River Basin (Puntarenas, Costa Rica); the present record represents a southeastern range extension of about 85 km (straight-line distance).On the other hand, the previously known distribution of L. boruca, a species endemic to Costa Rica, is between the Térraba River Basin and the Coto River Basin (Puntarenas, Costa Rica); the present record represents a northwestern range extension of about 75 km (straightline distance).Probably both species are residents in this area and likely remained undetected due to their rarity (only a few specimens (2-4) were observed and captured during this inventory), habitat specificity (only found in small creeks, where the current is of low or moderate velocity with abundant riparian and submerged vegetation (Bussing 1998)), and lack of prior targeted sampling (Bussing 1998).
By providing up-to-date knowledge on their diversity and distribution patterns we hope that the results of this investigation provide a useful framework for future biogeographic work on fishes from this area.This work is also aimed at aiding biologists, non-governmental

Check
Figure 1.Map showing the sampled localities in the Tusubres River basin, Pacific coast, Costa Rica; associate data is provided inTable 1.
Angulo et al. | Fishes from the Tusubres River basin, Costa Rica organizations and governmental agencies in establishing conservation actions, and in the promoting of the sustainable use of the fishes in the basin.

Table 2 .
Sampled localities in the Tusubres River basin, Pacific coast, Costa Rica, with voucher specimens at the UCR collection (examined lots), prior to this inventory.

Table 3 .
Angulo et al. | Fishes from the Tusubres River basin, Costa Rica Fish fauna, by sampled localities, in the Tusubres River basin, Pacific coast, Costa Rica.After family names (in bold) the number of genera and species, separated by a comma, are indicated.UCR=Specimens with voucher at UCR, prior fieldwork.Tol.=Tolerance to salinity based on Meyers (1949); primary=Pri; secondary=Sec; and peripheral=Per.Fig.=Reference to Figure.Voucher specimens deposited at UCR; by each species catalog number is indicated.