New records and distribution extension of Hyphessobrycon itaparicensis Lima & Costa, 2001 (Characiformes: Characidae) in coastal drainages of Sergipe state, northeastern Brazil

The present study reports the first record of the small characid fish Hyphessobrycon itaparicensis Lima and Costa, 2001 in two coastal drainages of Sergipe State, Brazil. This species was collected in three sampling sites from Piaui and Sergipe river basins, both in the hydrographic ecoregion of Northeastern Mata Atlantica. Aspects of habitat, diet composition, phenotypic variation, molecular identification and distribution of H. itaparicensis are herein discussed.

during the day (SISBIO permit #20104-1).After capture, the specimens were euthanized with menthol, fixed in 10% formalin and stored in 70% ethanol.Some specimens were also fixed and stored in ethanol 95% for molecular analysis.Voucher specimens are deposited in the Universidade Federal de Sergipe (CIUFS) and Universidade Federal do Rio Grande do Norte (UFRN) fish collections (Appendix 1).Thirteen specimens of H. itaparicensis were dissected for sex determination and gut content analysis.
A molecular phylogeny analysis was performed to reinforce the morphological identification of the specimens from Sergipe and Piauí river basins.DNA was extracted with DNAeasy™ Tissue Kit (Qiagen) from six and four specimens of H. itaparicensis from Piauí and Sergipe river basin, respectively, and from four individuals of H. parvellus from Sergipe river basin.The mitochondrial cytochrome oxidase I (COI) was amplified and sequenced using the primers FISH-BCH2 and FISH-BCL (Tornabene et al. 2010).The PCRs were performed using an initial cycle at 95°C for 5 min, followed by 35 cycles at 94°C for 30 s, 50°C for 35 s, 72°C for 70 s, a final extension step at 72°C for 7 min, and then 20°C for 2 min.PCR products were purified using the QIAquick PCR Purification Kit (Qiagen).All sequencing reactions were performed using the big dye v3.1 (Applied Biosystems) and screened in ABI PRISM ® 3500 Genetic Analyzer (Applied Biosystems).Consensus sequences were aligned using Geneious 6.1 (www.geneious.com),including 10 sequences of Hyphessobrycon spp.and two of Hemigrammus marginatus Ellis, 1911 from GenBank (Appendix 1).These last species were used as outgroup.A maximum likelihood analysis was performed using Mega 5.1 (Tamura et al. 2011) under the HKY+Γ substitution model, defined by Akaike information Hyphessobrycon Durbin, 1908 is a species-rich characid genus comprising about 130 valid species, which occurs from Mexico to Argentina (Carvalho and Langeani 2013;Eschmeyer 2013).Currently, Hyphessobrycon is classified as Characidae incertae sedis genus due to its non-monophyletic nature (Lima et al. 2003;Carvalho and Bertaco 2006;Mirande 2010;Oliveira et al. 2011).
Hyphessobrycon itaparicensis is a poorly known species, with restrict occurrence on some small coastal basins (Baetantã, da Dona, Traíra, Patipe, Vermelho, Macacuá, and Sorojó drainages) on South of Itaparica island, until near Camamu municipality in the Bahia State, Northeastern Brazil (Burger et al. 2011).Burger et al. (2011) stated that this species also occurs in coastal basins from northern states, but its precise distribution range was not mentioned.This note extends the distribution of H. itaparicensis to Sergipe State, representing its northernmost known record, including notes on sexual dimorphism and diet.
The fishes were collected using trawl nets (5 mm mesh) criterion using the Modeltest 3.7 (Posada and Crandall 1998), and 1000 bootstrap replicates to verify the support for each node.Additionally, levels of genetic divergence (K2P distance) among H. itaparicensis from Sergipe State drainages were also calculated in Mega 5.1.
The new records of H. itaparicensis (Figure 1) were registered at tributaries of lower portion of Piauí and Sergipe river basins, Sergipe State, Northeastern Brazil (Figure 2).These streams are small and shallow with acidic blackwaters (pH 5.3-5.7).The streams grounds are mostly composed by mud and leaves in calm water; sand and gravel in lotic stretches (Figure 3).Both sampling sites are within the Atlantic Forest, a highly threatened Brazilian biome (Tabarelli et al. 2005) Individuals of H. itaparicensis were identified by presenting body depth from 35.4-40.7% of standard length (SL), 30-31 scales on longitudinal series, 27-30 anal-fin rays, 12-15 dentary teeth, two faint humeral spots, and a mid-lateral horizontal dark brownish stripe on the posterior half of body (Lima and Costa 2001).
While H. itaparicensis from the Sergipe river basin showed typical colour pattern in life, with body and fins predominately yellowish and maximum body size (43.0 mm SL), in Piauí river only small specimens were caught in some sampling surveys (14 and 16 March 2012).The largest specimen were 24.8 mm SL and presented a more intense coloration in live specimens, with body predominately metallic copper and red to orange fins, with inconspicuous humeral spots, and a conspicuous narrow longitudinal stripe on the lateral of the body from the vertical through the dorsal fin origin to the caudal peduncle.However, most of the meristic and morphometric data were in concordance suggesting that Hyphessobrycon specimens from both localities belongs to the same species.The only difference among H. itaparicensis from the type locality (Itaparica islands in Bahia State) and those from Sergipe State drainages are related to an increased number of maxillary teeth (3-5 vs. 6), but examinations including coastal intermediary populations showed a gradual variation from South to North (Angela Zanata, pers. comm.).
According to the molecular analysis, Hyphessobrycon from Piauí and Sergipe river basins comprises a monophyletic group with high bootstrap support.COI sequences (635 bp) presented only three polymorphic positions in which represents less than 0.5% in variation.According to the Barcode proposals COI variations less than 1% are consistently related to within-species variations in most cases of the Neotropical freshwater fishes (Pereira et al. 2013).Therefore, the present data are consistent with the occurrence of H. itaparicensis along both drainages.All four specimens from Sergipe river basin presented the same haplotype, also shared with one individual from the Piauí river basin.Hyphessobrycon itaparicensis from Piauí river basin also present two exclusive haplotypes, which suggests that the population could be genetically structured.Thus, the small molecular K2P distance (zero to 0.005)  and absence of reciprocal monophyly among basins corroborates the morphological identification and conspecific status of the H. itaparicensis from both drainages in Sergipe State (Figure 4).
Males of Hyphessobrycon itaparicensis are distinct from females by the presence of bony hooks on fins, length of pelvic fin, and shape of anal-fin distal profile (Figure 5).Reproductively mature males (19.4-24.0mm SL) present concave hooks at pelvic and anal fins, which are absent in females (20.9-22.0mm SL).The pelvic fin bears 4-7 hooks at the posterior half of first and second branched rays.The anal fin bears paired hooks (2-6) at the posterior half of the last unbranched ray and from the first to fifth branched rays, decreasing in number posteriorly.Hooks were also observed in other species of Hyphessobrycon: H. bifasciatus (Ellis 1911), H. scutulatus Lucena, 2003(Lucena 2003), H. hamatus Bertaco & Malabarba, 2007, H. socolofi (Weitzman, 1977), H. erythrostigma (Fowler, 1943) (Bertaco and Malabarba 2005), H. vinaceus (Bertaco et al. 2007)   Other dimorphic traits in H. itaparicensis are related to the length of pelvic fin and shape of anal-fin distal profile.On males the end of the pelvic fin surpasses the anal-fin origin, and the anal-fin distal profile is almost straight (vs.pelvic fin not reaching anal-fin origin and anal-fin profile with concave curvature from fifth to tenth branched ray on females) (Figure 5).The presence of a straight anal-fin margin on mature males was also observed in H. vinaceus (Bertaco et al. 2007), H. bifasciatus, H. heliacus Moreira, Landim & Costa, 2002, H. loweae Costa & Géry, 1994, and H. peugeoti Ingenito, Lima & Buckup, 2013(Ingenito et al. 2013).These variations in size and shape of fins may be associated to ornaments that can represent important role in reproductive processes (Nikolsky 1963).
Hyphessobrycon itaparicensis occurs in groups of 10-15 individuals in calm water and close to subsurface vegetation where they forage.Gut contents analysis (n=13) showed Arthropoda fragments (Hemiptera, Coleoptera, Diptera, Hymenoptera and Acari) and algae (Desmidiaceae).The presence of these food items seems common in Hyphessobrycon species due the alimentary strategies of pick up allochthonous items in surface and pasture in substratum (Barreto and Aranha 2006).
Apparently, H. itaparicensis was restricted to small streams with dark acidic water in the Atlantic Forest, with distribution range restricted to the coastal plain drainages from Bahia State (Burger et al. 2011).Nonetheless, the current distribution of H. itaparicensis is a remarkable evidence given it is inserted into the single freshwater ecoregion named Northeastern Mata Atlantica (Abell et al. 2008), which encompasses the coastal basins North of Paraíba do Sul river basin and South of the São Francisco river mouth.
These streams in Sergipe State are adjacent to two Conservation Units, Litoral Sul de Sergipe Environmental Protection Area (LSEPA) and Serra de Itabaiana National Park (SINP), but the area of occurrence of H. itaparicensis are subjected to the several anthropic impacts such as removal of native vegetation for planting of pasture and crops, elimination of wetlands, carciniculture, sand extraction and unplanned state development.
The differences in colour patterns found in H. itaparicensis from Piauí river basin could be related to the acid blackwaters of stream tributary of Fundo river.Other congeners also inhabit blackwater streams: H. griemi Hoedeman, 1954and H. flammeus Myers, 1924(Menezes et al. 2007).The latter is a small species (25.0 mm SL maximum size) found in the same biotope in the Atlantic Forest coastal basins of southeastern Brazil.This species figures in the Brazilian red list of threatened species as endangered due to destruction of streams by the disorderly occupation of lowland areas (Lima and Moreira 2008), the same kind of threats that H. itaparicensis is undergoing.
The presence of H. itaparicensis in other nearby coastal drainages is not dismissed, due to existence of other tributaries with similar environmental characteristics of the registered sites.Additional surveys in the coastal drainages are necessary to define the distribution of this species and a phylogeographic study is encouraged to determine the processes involved in its geographic distribution.help in algae and arthropods identification.CAPES for the master's degree scholarship granted to Waldir M. Berbel-Filho. Fapitec (#019.203.00909/2009-8) and CNPq (#483103/2010-1) for financial support.Rodrigo A.Torres is grateful to CNPq for the research fellowship provided (#Pq2 301208/2012-3).

Figure 2 .
Figure 2. Map of new records of Hyphessobrycon itaparicensis in coastal drainages of Sergipe State, Brazil.Star represents the type locality of H. itaparicensis in Itaparica island, Bahia State; square represents the new record in Piauí river basin; circle represents two new records in Sergipe river basin.

Figure 4 .
Figure 4. Maximum likelihood tree of Hyphessobrycon species based on sequences of the mitochondrial cytochrome oxidase I deposited in Genbank and from Sergipe coastal basins.Numbers on the branches indicate the bootstrap robustness of each node.