Molecular diagnosis of the arowanas Osteoglossum ferreirai Kanazawa , 1966 and O . bicirrhossum ( Cuvier , 1829 ) from the Orinoco and Amazon River basins

The arowanas, fishes of Gondwanan origin, are represented in South America by the genus Osteoglossum. All species were initially reported as being exclusive to the Amazon region, with O. ferreirai restricted to the Negro River basin and O. bicirrhosum to the Amazon and Essequibo Rivers basin. Starting in the mid 1970’s it was reported that O. ferreirai also occurs in the Orinoco River basin. In all regions the arowanas assumed significant socio-economic importance due to their popularity in the international ornamental fish trade, leading to over-exploitation of both species in some areas. The Orinoco populations are particularly heavily exploited, and thus conservation and management measures are needed. Both depend on the clarification of taxonomic status, and phylogenetic distinctness of the Orinoco populations. With the goal of molecularly characterizing the two species of Osteoglossum, and comparing populations of Osteoglossum from the Orinoco and Amazon basins, we characterized individuals sampled from eight localities, one in the Orinoco River basin and seven in the Amazon River basin. We sampled 39 individuals, obtaining 1004 base pairs, of which 79 were synapomorphies. Genetic distance between the two species calculated using the HKY + G model of molecular evolution was 8.94%. Intraspecific distances ranged from 0.42% in O. bicirrhosum to 0.10% in O. ferreirai. The genetic characterization confirmed the taxonomic status of O. ferreirai in the Orinoco basin, and suggested that its distribution in the Orinoco basin is unlikely to be the result of vicariance or natural dispersal, but rather an anthropic introduction.


Introduction
The osteoglosids, known as the bony tongue fishes, are ancient creatures whose diversification is associated with the breakup of Gondwana (Carroll, 1988;Kumazawa & Nishida, 2000).Currently, ten species are known with distribution in the following regions: three in South America, one in Africa, four in Asia and two in Australia (Nelson, 2006).In South America, osteoglossids are represented by Arapaima gigas of the family Arapaimidae, and Osteoglossum bicirrhosum and Osteoglossum ferreirai of the family Osteoglossidae, distributed in the Amazonas, Essequibo and Orinoco basins (Reis, et al., 2003;Watkins, et al., 2004;Maldonado-Ocampo, et al., 2008).The existence of arowanas in South America was reported first by the Portuguese naturalist Alexandre Rodriguez Ferreira during expeditions in the Brazilian Amazon between the years 1783 to 1792.Based on specimens he collected and his illustrations of the white arowana, Cuvier erected the genus and described the species O. bicirrhosum.It was Wallace during his 1845 expedition to the Negro River (Brazil) who found and illustrated for the first time the black arowana, however, this individual was lost during his return trip to England.The black arowana was described in 1966 by Kanazawa as Osteoglossum ferreirai, and its distribution was restricted to The Negro River basin, with rest of the Amazon and Essequibo basin being occupied by O. bicirrhossum.Based on an ichthyological survey performed by Cala (1973), the range of O. ferreirai was expanded to the lower Tomo River in the Colombian Llanos.By the turn of the millennium, O. ferreirai was being reported in the Bita, Tomo and Tuparro Vichada Rivers by Mojica (1999), Lasso et al. (2005) and Maldonado-Ocampo et al. (2006;2008).
The arowanas from the Bita River appear to differ in their reproductive biology from those of the Negro River.A study of Gutiérrez et al. (2009) showed that the Bita River population had lower fecundity, producing 38 to 127 oocites per female and maturing at 62 cm TL, while in the Negro River populations females produced an average of 136 oocites, and matured sexually at 70 cm TL (Rabello-Neto, 1999;2002).These differences were viewed by the authors as indication of population or even species level differences.
The two South American species of arowanas began to be exported in large numbers for the aquarium trade in the 1970's when they started to replace the Asian arowana Scleropages formosus, also known as dragon fish, after S. formosus was listed as endangered by CITES (Yue, et al., 2003).Starting in the 1970's, over-exploitation of South American arowanas turned these species threatened with extinction in Colombia (Alvarez-León, 2002a;2002b).In 2005, Traffic and WWF reunited prominent ichthyologists in the "International Workshop of ornamental fishes from northern South America".They discussed the high vulnerability of both Osteoglossum species, and proposed to include them in Appendix II of CITES.They considered as first action the necessity to clarify the taxonomic status of O. ferreirai in the Orinoco basin, its divergence from the Negro basin populations, and an assessment of genetic variability of O. bicirrhossum in its area of distribution.
All tissue samples were preserved in absolute alcohol and processed in the Laboratório de Evolução e Genética Animal (LEGAL) at the Universidade Federal do Amazonas (UFAM), Manaus, Brazil.DNA extraction was performed with the CTAB method (Doyle & Doyle, 1987), and quantified by means of electrophoresis in a 0.8% agarose gel against a known quantity of a molecular size standard (Fermentas).
The PCR reactions were tested on 1% agarose gel.Sequencing reactions were performed using the BigDye Terminator Kit V.3.1.according to the manufacturer's guidelines and the product was analyzed on an ABI 3130xl automated sequencer.The sequences obtained were viewed and edited by using the program BioEdit (Hall, 1999).The dataset was aligned in Crustal W (implemented within Bioedit) and then adjusted manually (Thompson, et al., 1996).The composition of nucleotide bases, identification of variable sites, stop codons, transversions/transitions and genetic distances was estimated in the program MEGA5 (Tamura, et al., 2011).The number and frequency of haplotypes was determined in the program DnaSP v.5 (Librado & Rozas, 2009).
The program Modeltest 3.7 was used to test for the best molecular evolutionary model fitting the dataset (Posada & Crandall, 1998).Phylogenetic reconstruction was performed using the maximum likelihood method (ML) implemented in the program Treefinder (Jobb, et al., 2004) and support for relationships was assessed via 1000 bootstrap pseudoreplications.We used Scleropages formosus and Scleropages leichardti as outgroups (Genbank No. DQ023143, FJ890319).
Phylogenetic species were identified using the Population Aggregation Analysis algorithm, that involves a search for fixed differences between local populations, followed by successive rounds of aggregation of populations and previously aggregated population groups that are not distinct from each other (Davis & Nixon, 1992), and complementary analytical procedures described by Cook et al. (2010).

Results
The final aligned sequences had a length of 1004 bp and 79 of which were diagnostic for the two species.The 79 synapomorphies were divided between 73 transitions and 6 transversions.The base pair composition of the data set was A = 27.2%,T = 29.1%,C = 30.2%,G = 13.4%.In total eleven haplotypes were identified for both species, seven in O. bicirrhosum and four in O. ferreirai.In both species a single haplotype was the most frequent (online Supplement 1-3).Haplotypes are available under the GenBank accession numbers JQ436740 to JQ436750.
The maximum likelihood topology using the HKY+G model of molecular evolution (Posada & Crandall, 1998) showed the formation of two evolutionary lineages representing the two species.The clade representing O. bicirrhosum split into two sub-clades.The first comprised the majority of individuals from the Putumayo (Puerto Leguizamo), Jurua (Eirunepé) and Solimões (Leticia) Rivers and the second was formed by majority of individuals in the Madeira (Humaitá), Solimões (Tefé) and Amazon (Santarém) Rivers.The clade representing O. ferreirai indicated the existence of a single evolutionary lineage consisting of individuals from the Bita (Orinoco basin) and from the Negro (Amazon basin) Rivers (Fig 2).Genetic divergence between the two species of Osteoglossum averaged at 8.94%, while intraspecific divergence in O. bicirrhosum was 0.42% and O. ferreirai was 0.10% (Tables 1-2).

Discussion
Phylogenetic and Population Aggregation Analysis clearly separate and diagnose the two species of Osteoglossum, and place the Colombian populations within the species O. ferreirai.This diagnosis confirms that of Cala (1973).There were no unique haplotypes in the Orinoco basin samples of O. ferreirai, indicating a very recent divergence of the populations of the Orinoco and Negro River basins.We suggest that the lack of divergence of the Orinoco basin samples is most likely due to a recent introduction from the present in these areas (Maldonado-Ocampo, et al., 2006;Maldonado-Ocampo, et al., 2008;Winemiller et al., 2008;Lasso, et al., 2009;Miller-Hurtado, et al., 2009;Winemiller & Willis, 2011).Additionally, based on fauna surveys (Cala, 1973;Mojica, 1999;Lasso et al., 2005;Maldonado-Ocampo et al., 2006;2008) the expansion in the distribution of O. ferreirai in Colombia appears to have proceeded from north to south, i.e. towards the Negro River basin.Therefore, we consider it highly unlikely that the occupation of the Orinoco basin would proceed via geographically intermediate areas between the Orinoco and Negro basins.Natural long distance dispersal and colonization also appears an unlikely mechanism.Although we could not find any reference or report that would suggest that O. ferreirai in the Orinoco basin was or could have been introduced, we consider an introduction the most likely explanation.The area of origin of O. ferreirai from the Orinoco basin is unclear, however, and will require a detailed characterization of O. ferreirai from the Negro basin.
The possibility and what we consider to be the most likely scenario that O. ferreirai has been introduced in the Orinoco basin, constitutes a management and conservation dilemma.The Orinoco O. ferreirai have genetic diversity comparable to that of the fishes found in the Negro River basin, and appear to have expanded their geographic distribution.Thus they appear likely to increase in density and expand geographically if an ecological opportunity presents itself.On one hand, O. ferreirai makes an important contribution to the local economy, and is a highly desirable export species in the aquarium trade.In the Orinoco basin it appears to be over-exploited (Alvarez-León, 2002b;CCI, 2009), hence implementing a management and/or conservation program may result in not only its recovery, but also its expansion to other areas where it is currently not found.Being a top of the pyramid predator, and predating not only aquatic but also terrestrial vertebrates, could have a major impact on population ecology of the areas where densities O. ferreirai will increase.On the other hand, conservation programs normally try to eliminate introduced species.However, it seems unlikely that a program to eradicate O. ferreirai would succeed due to the area already colonized, and the remoteness of these areas.A third option is to do nothing.Under this scenario, the global aquarium market will largely determine if the species increases in density and colonizes new areas, remains at status quo, or its densities will decrease and possibly will even experience local extinctions.Ultimately, however, conservation and management decisions will be mandated by governmental agencies and will likely tend to balance socioeconomic needs with environmental protection.

Fig. 2 .
Fig. 2. Maximum likelihood phylogenetic hypothesis of relationships of Osteoglossum individuals representing the species O. bicirrhossum and O. ferreirai.
Negro to the Orinoco river basin.Based on ichthyofaunal surveys of areas that can potentially serve as biological corridors and links between the two basins such as the Casiquiare channel and the floodplain of the Inirida and Atabapo Rivers, neither O. ferreirai nor O. bicirrhossum is O. bicirrhossum O. ferreirai Negro O

Table 1 .
Table of K2P distances

Table 2 .
(Kimura, 1980)specific and intragroup K2P distances(Kimura, 1980)observed in the two species of Osteoglossum and the Negro and Orinoco River populations of O. ferreirai.