Morphology and genetics reveal the occurrence of Girardinus falcatus ( Eigenmann , 1903 ) ( Cyprinodontiformes , Poeciliidae ) in eastern Cuba

Girardinus is a Cuban genus of poeciliid fishes. It comprises 7 freshwater-restricted species most of them inhabiting specific ecosystems or regions within the island. Girardinus falcatus, the Gold-bellied Topminnow, is common in lowlands of western and central Cuba but thought not to be present in the East. Herein we provide the first wellsupported evidence of the occurrence of this species in eastern Cuba. Sixteen specimens were collected and identified primarily by body shape and coloration, and corroborated by gonopodial morphology and by sequences of the mitochondrial gene cytochrome b.


Introduction
Poeciliid fishes exhibit high endemism in the Caribbean islands (Rosen and Bailey 1963).There are 3 genera exclusively distributed in these islands, and 2 of them-Girardinus Poey, 1854 with 7 species and the monotypic Quintana Hubbs, 1934-are restricted to Cuba (Lara et al. 2010).The distribution of species of Girardinus is associated to specific habitats.For example, G. uninotatus Poey, 1860, G. creolus Garman, 1895, G. cubensis (Eigenmann, 1903), and some populations of G. microdactylus Rivas, 1944 inhabit streams and rivers in the mountain ranges of western Cuba, that is, in Pinar del Río province (Doadrio et al. 2009, Lara et al. 2010, Ponce de León and Rodríguez 2010).Girardinus denticulatus Garman, 1895 occurs in mountain rivers of central and eastern Cuba, whereas, G. metallicus Poey, 1854 and G. falcatus (Eigenmann, 1903) are mostly widespread in lowland freshwater ecosystems in the western and central regions.

NOTES ON GEOGRAPHIC DISTRIBUTION
in eastern Cuba and analyze the relationships among populations based on cytochrome b sequences.In addition, an updated list of localities for this species is provided.

Methods
A total of 29 rivers and streams were sampled during a survey to Holguín province (northeastern Cuba) in January 2016.Adult specimens of Girardinus falcatus were found in some of these water bodies.The specimens were collected using seines and hand nets (Perrow et al. 1996), anaesthetized with MS 222 (Tricaine Methanesulphonate) and preserved in 90% ethanol.Voucher specimens were deposited in independent lots according to the new localities in the collection of the Acuario Nacional de Cuba in La Habana, Cuba (ANC).
We followed the morphological and gonopodial features described by Rivas (1958) to distinguish the Girardinus species.We also used the photographic evidence of body and gonopodium morphology depicted in Ponce de León and Rodríguez (2010) for the identification of the male specimens collected in the field.To that purpose, and after the primary identification by body morphology, we captured images of the gonopodia using a stereoscopic microscope (40×) and a Nikon Coolpix digital camera.
The genetic identification of G. falcatus specimens from Holguín province was based on the mitochondrial gene cytochrome b (cytb).DNA was extracted, PCR amplified and sequenced following Lara et al. (2010).The primers used for amplification were Glufish (5ʹ-CCAATGACTTGAAGAACCACCGTTG; Meyer et al. 1990) and CB3 (5ʹ-GCCAAATAGGAARTATCATTC; Palumbi 1996).We compared the sequences of 1 individual from each new locality with those of G. falcatus included in Doadrio et al. (2009), and a sequence of G. uninotatus was used as outgroup (accession number: FJ178722.1).The new sequences were sent to GenBank database under the accession numbers KY606767-KY606770.Sequences were aligned using Clustal W (Thompson et al. 1994).The model of nucleotide substitution was selected using the Bayesian information criterion (BIC; Schwarz 1978) with jModelTest 2.1.7(Darriba et al. 2012).The Maximum Likelihood method based on the HKY model was used for the inference of G. falcatus phylogenetic relationships using MEGA 7.0.18(Kumar et al. 2016).The haplotype and nucleotide diversity (h and π, respectively) were estimated using DnaSP 5.10 (Librado and Rojas 2009).The genetic divergence (p-distance) between each pair of clades was also estimated using MEGA 7.0.18(Kumar et al. 2016).
A list of localities from which G. falcatus has been reported was made by reviewing previous records from 3 major ichthyology collections in Cuba: Museo Nacional de Historia Natural (MNHNCu), Instituto de Ecología y Sistemática (CZACC), and Museo de Historia Natural "Felipe Poey" (MFP).Additionally, the distribution information from published studies available online and from the Global Biodiversity Information Facility (GBIF) database (http://data.gbif.org,consulted on 4 July 2016) were used to update the list of known localities for G. falcatus.
The segments of the four rivers in which specimens  1.   Together with G. falcatus, we found specimens of Gambusia punctata Poey, 1854, Gambusia puncticulata Poey, 1854, andLimia vittata (Guichenot, 1853).In Mano river, we also found Girardinus denticulatus.All specimens found in Holguín province showed the unique morphological features that distinguish G. falcatus from its congeners: yellowish translucent body, golden belly, lateral scales with black edges, lower body edge with a black line, from the anal fin to the caudal peduncle, the origin of the dorsal fin above end of anal base (Rivas 1958), and males with a dark line along the gonopodium (Eigenmann 1903, Ponce de León and Rodríguez 2010) (Figs 4, 5).
Intra-specific variation in gonopodial structure from the reference individual from Ponce de León and Rodríguez (2010) and a male from Cacoyugüín river is shown in Figures 6 and 7, respectively.The following are some of the most relevant features of the gonopodium of the specimens collected in Holguín province.The gonopodia of the fish identified as G. falcatus showed similar coloration, and the proportion gonopodium length/body total length was also similar (0.4-0.5) among them.They also had the posterior branch of ray 5 with 6 or 7 serrae and its 2-4 terminal segments unarmed.These features are consistent with the gonopodial size and structure described by Rivas (1958), and used by Rodríguez et al. (1992) for G. falcatus identification.The reference specimen in Ponce de Leon and Rodríguez (2010) also presents the same gonopodium size, color, and the number of serrae in 5p.However, despite we found some unarmed segments distal to the serrae in 5p, the actual number of them is not clearly quantifiable in the photograph from Ponce de León and Rodríguez (2010).
The analysis of 804 base pairs of the cytb gene sequences from the 4 samples of Holguín and the 15 samples from Doadrio et al. (2009) showed 14 haplotypes and high haplotype (h = 0.965 ± 0.028) and nucleotide (π = 0.017 ± 0.003) diversities.The phylogenetic relationships are presented in Figure 8.We found 4 main clades which correspond to discrete geographical regions within the Cuban archipelago: 1-Western Pinar del Río, 2-Isla de la Juventud, 3-Pinar del Río/Artemisa/Matanzas/Western Central Cuba, and 4-Eastern Central Cuba/ Eastern Cuba.The genetic distance between each main clade ranged from d = 0.012 ± 0.003 to d = 0.038 ± 0.006.The lowest values were obtained between populations from Pinar del Río/Artemisa/Matanzas/Western Central Cuba and Eastern Central Cuba/Eastern Cuba, whereas the higher values were found between populations from Western Pinar del Río and Isla de la Juventud.

Discussion
Our results show that the distribution of G. falcatus reaches the northeastern region of Cuba.The westernmost locality among the 4 new ones, Mano river (number 71 in Fig. 1), is about 220 km southeast of Caonao river, Esmeralda (number 70 in Fig. 1), in the boundaries between Ciego de Ávila and Camagüey provinces, the most easterly location known for the species (Doadrio et al. 2009).We found a record referred to as G. falcatus (MCZ-Ich-33607) from Alto Songo, Santiago de Cuba province deposited in the fish collection of the Museum of Comparative Zoology, Harvard University.The voucher was collected by Carlos de La Torre in 1910.Unfortunately, no description is available and the specimens are damaged and not identifiable (K.Hartel, in litt., September 2016).Also, the geographic coordinate and the site name are unclear, which makes this record dubious.Moreover, despite the exact location is unknown, we conducted an extensive survey across eastern Cuba, including Granma, Santiago de Cuba and Guantánamo provinces, and we did not found G. falcatus in any of the 39 sampling localities; some of which are in the same area reported by Carlos de La Torre.All this increases the value of the present report as verifiable evidence for the occurrence of this species in eastern Cuba.All the sequences from Holguín cluster together in a node shared basally with the sample from Caonao river in Ciego de Ávila-Camagüey provinces boundary.This relationship is coherent with the geographic location of these sites.Regarding this, the tree topology provides new information about the geographic differentiation of G. falcatus.Although human-mediated translocations cannot be discarded (A.Fong and R. Teruel, pers.comm.), the divergence of Holguín sequences from Caonao river sequence (d = 0.075 ± 0.003) and the outgroup condition of the later, suggest that its distribution have followed a natural historical process.
The populations from Guanahacabibes region (western Pinar del Río) are highly differentiated compared to main western (d = 0.037 ± 0.007) and eastern (d = 0.038 ± 0.006) populations.These values are close to the cutoff criterion for species level divergence of Hebert et al. (2003).On the contrary, the clades composed by the rest of the western to central distributed populations (Pinar del Río/Artemisa/Matanzas/western Central Cuba, including Isla de la Juventud) and the eastern populations (eastern Central Cuba/Eastern Cuba) diverge only by d = 0.014 ± 0.003.According to our analysis of cytb sequences, G. falcatus seems to be composed by allopatric populations, as found in other Cuban freshwater fishes (Ponce de León et al. 2014, Hernández et al. 2016).Further multiloci analyses should be addressed to fully understand the genetic diversity and the relationships among G. falcatus populations.
The new localities reported in this work provide valuable information about the geographic distribution of the species and bring new insights on the biogeography of Girardinus.The geographic discontinuity of the new localities with respect to the previous eastern limit of the species distribution raises interesting questions concerning dispersion mechanisms and ancient distribution of the species.Future studies of population genetics on this species will help in answering these questions and will provide information about the evolutionary history of the genus in Cuba, in particular about possible patterns of dispersion of the populations and the events involved.

Figure 1 .
Figure 1.Map depicting the geographic distribution of Girardinus falcatus in the Cuban archipelago.Red dot: type locality; orange dots: known localities; green dots: new localities.Locality numbers as in Table1.
of G. falcatus were found were shallow and with perceptible water flow.The depth varied between 10 and 40 cm and the surrounding areas were composed by secondary forest with herbaceous riparian vegetation (Cyperacea) or secondary grassland and shrubby vegetation (Figs 2, 3).Aquatic vegetation was abundant and dominated by vascular plants (Hydrocharitaceae and Nelumbonaceae).

Table 1 .
Known localities of Girardinus falcatus in Cuba (geographic coordinates in decimal degrees; datum WGS84)."Voucher" refers to specimens deposited in museum collections and "Source" refers to publications where the locality was obtained from.AMNH: American Museum of Natural History, New York; ANC: Acuario Nacional de Cuba, La Habana; CAS: California Academy of Sciences, San Francisco; CZAAC: Instituto de Ecología y Sistemática, La Habana; MCZ: Museum of Comparative Zoology, Harvard University, Cambridge; MFP: Museo de Historia Natural "Felipe Poey", Universidad de La Habana, La Habana; MNHNCu: Museo Nacional de Historia Natural de Cuba, La Habana; UMMZ: University of Michigan Museum of Zoology, Ann Arbor; and USNM: National Museum of Natural History, Smithsonian Institution, Washington, DC.The asterisk represents specimens deposited in collection without a catalogue number assigned.