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The genus Fluviphylax Whitley, 1965is comprized of five valid species (Fluviphylax pygmaeus Myers et Carvalho, 1955, Fluviphylax zonatus, Fluviphylax simplex, Fluviphylax obscurus Costa, 1996, and Fluviphylax palikur Costa et Le Bail, 1999), which are endemic to the Amazon region. These fishes are the smallest known South American vertebrates and among the smallest know vertebrates on Earth. All species but the type Fluviphylax pygmaeus have been described in late 1990’s, and much remains unknown about the biology, taxonomy and systematics of this group of fishes. The aims of the present study were to establish the diploid and haploid number of Fluviphylax zonatus and Fluviphylax simplex, and to find species-specific markers for the discrimination of taxa. The diploid number for both species was 48 chromosomes, with no sex chromosome heteromorphism. Fluviphylax zonatus exhibited the karyotypic formula 4m+8sm+22st+14a and FN=82, and Fluviphylax simplex exhibited 4m+16sm+18st+10a and FN=86. The determination of the total mean length of the chromosomes and their grouping into five size classes demonstrated different chromosome composition of the two species. This difference was further supported by the distribution of constitutive heterochromatin. The meiotic analysis revealed 24 bivalents in both species, but Fluviphylax zonatus exhibited chromosomes with late pairing of the telomeric portions in the pachytene. These data reveal that cytogenetic characterization is useful and important for the discrimination of these species. Our study further indicates that this method could be employed in the analysis of other species of small fishes that are difficult to distinguish using traditional morphological traits or are morphologically cryptic.
Chromosomes, heterochromatin, killifish, meiosis, mitosis
The Amazonian region has the most diverse freshwater fish fauna in the world, which, although only imperfectly known (
The cyprinodontiforms are a large and diverse group of teleostean fishes comprising the family Poeciliidae. The fishes of the family Poeciliidae are small and laterally compressed, widely distributed in American and African continent. The Poeciliidae include the subfamilies Poeciliinae, Aplocheilichthyinae and Procatopodinae, a group composed of the South-American Fluviphylax Whitley, 1965 and the African procatopodines (
The taxonomic history of the genus Fluviphylax is relatively recent. The first species was discovered and scientifically described by Myers and Carvalho in 1955. The genus Fluviphylax has paucity of systematic, taxonomic and genetic information, with our knowledge being almost entirely restricted to the information published in the original description. Therefore, cytogenetic studies significantly expand our knowledge base of this group, especially in the realm of understanding of chromosome evolution of Fluviphylax.
Cytogenetic studies have contributed significantly to the identification of fishes (
Specimens of Fluviphylax simplex were collected from Lua Beach (3°07'31.7"S, 60°10'38.9"W) near the confluence of the Negro and Solimões Rivers, Amazonas, Brazil. Specimens of Fluviphylax zonatus were collected from a small lake (3°00'19.2"S, 60°03'22.6"W) located near Manaus that gathers water from the Tarumã River, which is a tributary of the Negro River (Figs 1, 2). We analyzed 24 specimens of the Fluviphylax simplex and 37 of the Fluviphylax zonatus. The gender determination was made only for adults specimens of each species being 6 males, 8 females and 10 indeterminated for Fluviphylax simplex, and 7 males, 6 females and 24 interminated for Fluviphylax zonatus. Collections were performed under a license from the Brazilian Institute of the Environment and Renewable Natural Resources (IBAMA n. 11325-1/2007). Following the chromosome preparation, some specimens were fixed in 95% alcohol. Voucher specimens were deposited in the Fish Collection at the Instituto Nacional de Pesquisas da Amazônia (INPA) in Manaus, State of Amazonas, Brazil (number 25527), and in the Animal Genetics Tissue Collection of the Laboratory of Animal Evolution and Genetics of the Institute of Biological Sciences of the Universidade Federal do Amazonas (Brazil).
Due to the small size of the specimens (less than 20 mm in total length), the cell preparations were obtained through the maceration of the each individual in a cuvette containing 6 ml of hypotonic KCl solution with the aid of two pairs of tweezers. Eyes and intestines were removed prior to maceration. The cell suspension was infused with 0.3 ml of 0.0125% colchicine solution. This preparation was incubated for 40 minutes at 37°C. The subsequent fixation of cells was carried out following the method of
The chromosome preparations were analyzed under an optical microscope with an immersion objective. Selected cells were photographed with a Canon Power Shot A650 IS digital camera. The mounting of karyotypes was carried out with mitotic metaphase chromosomes, which were cut out and tentatively paired. The chromosomes were measured using the free ImageJ program and organized in decreasing order of size. Chromosome morphology was determined taking into account the position of the centromere, based on the method proposed by
Sampling locations circle and star indicate sampling points for Fluviphylax zonatus and Fluviphylax simplex, respectively
a Fluviphylax zonatus with 20.0 mm SL b Fluviphylax simplex with 18.4 mm SL.
In this study we analyzed 428 cells of the Fluviphylax zonatus, 16% corresponded to mitotic metaphase cells and the others were to meiotic cells, of which 46% leptotene/zygotene, 24% pachytene, 24 % diplotene/diakinesis/metaphase I and 6% in metaphase II. For Fluviphylax simplex wereobtained 384 cells corresponded to 36% mitotic cells in metaphase and the others were to meiosis cells, of which 12% leptotene/zygotene, 67% pachytene, 16 % diplotene/diakinesis/metaphase I and 5% in metaphasee II.
In the mitotic analysis, both species had a diploid number of 48 chromosomes, with symmetrical karyotypes and no sex chromosome heteromorphism. Fluviphylax zonatus karyotype consists of 2n=4m+8sm+22st+14a (Fig. 3a), and Fluviphylax simplex 2n=4m+16sm+18st+10a (Fig. 3f). Constitutive heterochromatin was detected in the pericentromeric region in the majority of chromosomes in the two species (Figs 3b, g). However, in Fluviphylax zonatus the constitutive heterochromatin occupied entire short arms of all chromosomes, with the exception of 1st and6thpairs. Also, in the 1st pair the constitutive heterochromatin was bitelomeric and in the 6th additional marks was found in long arms (Fig. 3b). In Fluviphylax simplex the heterochromatin blocks were less evident and in the 20th pair were found additional interstitial marks on the long arms (Fig. 3g). The mean total length of the chromosomes ranged from 1.47 to 3.06 μm in Fluviphylax zonatus and from 1.46 and 3.28 μm in Fluviphylax simplex (Table 1). The grouping of chromosomes into five size classes, also revealed the different length chromosome composition between the two species (Fig. 4). In Fluviphylax zonatus, there was a greater frequency of chromosomes in Class III, which encompasses pairs ranging in size from 2.21 to 2.57 µm, and heterogeneity in chromosomal frequencies among other classes. Moreover Fluviphylax simplex also had greater frequency of chromosomes in Class III, however, the distribution of chromosomal frequencies among other classes were homogeneous.
Gonadal cells of Fluviphylax zonatus and Fluviphylax simplex at interphase and prophase I had no heteropicnotic regions that indicated the presence of sex chromatin (Fig. 3c, h). The chromosomal behavior in some meiotic phases of both species was similar, but differences were detected. In pachytenic cells, both species had 2n=24 bivalents, but Fluviphylax zonatus showed chromosomes with late pairing in the telomeric portions (Fig. 3d), which did not occur in Fluviphylax simplex (Fig. 3i). The analysis of diplotene cells also revealed 2n=24 bivalents in both species, but Fluviphylax zonatus had 10 bivalents with a terminal chiasma and 14 with an interstitial chiasma (Fig. 3e), and Fluviphylax simplex had 12 bivalents with a terminal chiasma and 12 with an interstitial chiasma (Fig. 3j). For both species, metaphases I had 2n=24 bivalents and metaphases II had n=24 chromosomes (data not shown).
Average chromosome measurements (µm) and classifications in Fluviphylax zonatus and Fluviphylax simplex (Ch. Pair: Chromosome Pair; LA: Long arm; SA: Short arm; TL: Total length; AR: Arm ratio; CT: Chromosome type; m: metacentric; sm: submetacentric; st: subtelocentric; a: acrocentric). The LA, SA, TL are average values obtained from the measure of all karyotypes analyzed.
Fluviphylax zonatus | Fluviphylax simplex | |||||||||||
Ch. Pair | LA | SA | TL | AR | CT | Ch. Pair | LA | SA | TL | AR | CT | |
1 | 1.41 | 1.08 | 2.55 | 1.30 | M | 1 | 1.64 | 1.08 | 2.91 | 1.52 | M | |
2 | 0.89 | 0.56 | 1.68 | 1.58 | M | 2 | 1.01 | 0.63 | 1.63 | 1.61 | M | |
3 | 2.04 | 0.74 | 2.79 | 2.76 | SM | 3 | 2.14 | 0.82 | 3.13 | 2.61 | SM | |
4 | 1.55 | 0.53 | 2.40 | 2.92 | SM | 4 | 2.12 | 0.71 | 2.84 | 2.97 | SM | |
5 | 1.49 | 0.57 | 2.15 | 2.63 | SM | 5 | 1.79 | 0.79 | 2.54 | 2.26 | SM | |
6 | 1.22 | 0.51 | 1.75 | 2.40 | SM | 6 | 1.68 | 0.68 | 2, 33 | 2.46 | SM | |
7 | 2.33 | 0.64 | 3.06 | 3.65 | ST | 7 | 1.57 | 0.66 | 2.31 | 2.39 | SM | |
8 | 2.32 | 0.55 | 1.66 | 4.23 | ST | 8 | 1.30 | 0.53 | 1.93 | 2.43 | SM | |
9 | 1.95 | 0.54 | 2.57 | 3.58 | ST | 9 | 1.12 | 0.44 | 1.65 | 2.54 | SM | |
10 | 1.75 | 0.46 | 2.39 | 3.79 | ST | 10 | 0.72 | 0.31 | 1.46 | 2.33 | SM | |
11 | 1.62 | 0.40 | 2.22 | 4.04 | ST | 11 | 2.57 | 0.49 | 3.28 | 5.20 | ST | |
12 | 2.34 | 0.52 | 2.98 | 2.53 | ST | 12 | 2.66 | 0.49 | 3.21 | 5.38 | ST | |
13 | 2.32 | 0.38 | 2.72 | 6.05 | ST | 13 | 2.26 | 0.57 | 2.98 | 3.93 | ST | |
14 | 2.05 | 0.33 | 2.55 | 6.24 | ST | 14 | 2.29 | 0.60 | 2.92 | 3.80 | ST | |
15 | 2.15 | 0.62 | 2.77 | 3.47 | ST | 15 | 2.44 | 0.45 | 3.08 | 5.40 | ST | |
16 | 1.82 | 0.58 | 2.51 | 3.12 | ST | 16 | 1.99 | 0.61 | 2.72 | 3.27 | ST | |
17 | 1.80 | 0.47 | 2.37 | 3.85 | ST | 17 | 2.13 | 0.47 | 2.68 | 4.57 | ST | |
18 | 2.22 | 0.14 | 2.32 | 16.20 | A | 18 | 1.93 | 0.51 | 2.53 | 3.81 | ST | |
19 | 2.28 | 0.15 | 2.50 | 15.16 | A | 19 | 1.78 | 0.55 | 2.34 | 3.26 | ST | |
20 | 1.97 | 0.13 | 2.26 | 15.32 | A | 20 | 2.24 | 0.09 | 2.62 | 25.77 | A | |
21 | 2.04 | 0.21 | 2.25 | 9.82 | A | 21 | 2.00 | 0.18 | 2.35 | 11.21 | A | |
22 | 1.78 | 0.12 | 2.07 | 14.89 | A | 22 | 1.74 | 0.17 | 2.09 | 10.51 | A | |
23 | 1.79 | 0.09 | 2.11 | 20.34 | A | 23 | 2.06 | 0.27 | 2.37 | 7.58 | A | |
24 | 1.25 | 0.07 | 1.47 | 17.04 | A | 24 | 1.41 | 0.14 | 1.71 | 10.32 | A |
Data on Fluviphylax zonatus and Fluviphylax simplex: with conventional staining a, f C-banding b, g initial leptotene stage c, h pachytene stage revealing 2n=24II d, i arrow indicates late pairing in some telomeric regions d diplotene stage with 2n=24II, arrows indicate bivalents with interstitial chiasma; arrow indicates bivalents with terminal chiasma e, j
Analysis of chromosome size in Fluviphylax zonatus and Fluviphylax simplex; Y axis gives frequency of chromosomes with pair sizes in classes informed on X axis.
The Procatopodinae and their sister sub-family Poeciliinae belong to the family Poeciliidae within order Cyprinodontiformes (
Comparative analysis of chromosome size between Fluviphylax zonatus and Fluviphylax simplex revealed differences in the organization of the genome, that is reflected in difference of karyotype formulae, due occurrence of pericentric inversion rearrangements, which alter the karyotype formula without altering the diploid number.
Chromosomal rearrangements are considered an important mechanism of karyotypic differentiation in Aplocheiloidei and Cyprinodontiformes in general (
Although not commonly performed, meiotic analyses are an extreme powerful tool for chromosomal characterization (
Some species of Poeciliidae have visible sex chromosome, such as the ZW/ZZ sex determining system in Gambusia puncticulata (
Organisms with differentiated sex chromosomes generally display positive heteropycnotic corpuscles in the early stages of prophase I and differentiated meiotic behavior in these chromosomes (
As evident from our and other studies (
We would like to thanks INPA (Instituto Nacional de Pesquisas da Amazônia), IBAMA (Brazilian Institute of the Environment and Renewable Natural Resources) for field license. This work was supported by CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico).