Chromosomal evidence for a putative cryptic species in the Gymnotus carapo species-complex (Gymnotiformes, Gymnotidae)
© Milhomem et al; licensee BioMed Central Ltd. 2008
Received: 31 May 2008
Accepted: 25 November 2008
Published: 25 November 2008
In this study we examined the karyotypes of morphologically indistinguishable populations of the electric knifefish Gymnotus carapo sensu stricto from the Eastern Amazon of Brazil. These were identified unambiguously on the basis of external morphology, meristics, and pigmentation.
Specimens from one of five localities exhibited a karyotype previously not documented for Gymnotus species in the Amazon basin: 2n = 40 (34M/SM+6ST/A). Samples from the other four localities exhibited a different karyotype: 2n = 42 (30M/SM+12ST/A), which we had previously described. Specimens from all five localities presented constitutive heterochromatin in the centromeric region of almost all chromosomes, including in the distal and interstitial regions. Staining with 4'6-Diamidino-2-phenylindole revealed C-positive banding. In both karyotypes the Nucleolar Organizer Region (NOR) was located on the short arm of pair 20, and Chromomycin A3 stained the NORs. Fluorescent in situ hybridization with telomeric probes showed an Interstitial Telomeric Sequence (ITS) in the proximal short arm of a metacentric pair in the 2n = 40 karyotype.
The difference between the two karyotypes on the diploid number and chromosome morphology can be explained by rearrangements of the fusion-fission type and also by pericentric inversions. The presence of ITS in a metacentric pair of the 2n = 40 karyotype suggests that the difference in the diploid number of the karyotypes results from a fusion. The consistent 2n = 42 karyotype at four localities suggests an interbreeding population. However, because fusion-fission and pericentric inversions of this nature typically result in reproductive isolation, we speculate that the form with the 2n = 40 karyotype is a different species to that of the 2n = 42 form. Nonetheless, we did not observe evident differences in external morphology, meristics and pigmentation between the two forms, which suggest that they represent cryptic sympatric species in the G. carapo species complex. We speculate that the chromosomal speciation occurred recently, allowing insufficient time for the fixation of other differences following post-zygotic isolation.
Gymnotus (Gymnotiformes, Gymnotidae) is the most diverse known Neotropical electric knife fish genus. It currently holds 33 valid described species and many additional undescribed species are known from museum collections [1–4]. Gymnotus has the ability to generate a pulsed electrostatic field from a specialized electric organ and detect electrostatic fields with electroreceptors. These Electric Organ Discharges (EODs) permit electrolocation, the detection of objects within the electrostatic field, and also electrocommunication (review in ; ).
The diploid number of chromosomes in Gymnotus has been documented to vary from 2n = 39–40 (with sex chromosomes of the type X1X2Y) to 2n = 54, exhibiting variation in the karyotype formula, the quantity of heterochromatin, and the position of the Nucleolar Organization Region (NOR) [6–9].
The species Gymnotus carapo (L.) sensu stricto, as currently defined , occurs over large areas of northern South America: in the Amazon and Orinoco basins, the coastal drainages of the Guyanas, and some coastal basins of Northeastern Brazil. The Linnaean syntypes of G. carapo were collected in the first half of the 18th Century near Paramaribo, Surinam .
A complex of additional morphologically similar or cryptic species that closely resemble G. carapo sensu stricto are known to extend as far south as Northern Argentina (Albert, Crampton, pers. obs). Likewise, some populations within the currently defined range of G. carapo sensu stricto may also prove to be distinct species within this species-complex. This G. carapo species-complex is not to be confused with the G. carapo species-group, which currently comprises 18 well-defined species in which there are two (vs. one) laterosensory pores in the dorsoposterior portion of the preopercle (and which includes G. carapo sensu stricto). G. carapo senso stricto can be distinguished from all other members of the G. carapo species-group by a combination of characters that are listed in the description of the species . It has a distinct color pattern comprising 16–27 dark obliquely oriented pigment bands or pand-pairs, with irregular wavy margins, often broken into spots above the lateral line. This distinct pigmentation pattern is shared only with G. arapaima and G. diamantinensis, from which G. carapo senso stricto can be distinguished on the basis of morphological and meristic characters.
At least four new species from the G. carapo species-group are currently being described from southern Brazil, Uruguay and Argentina (Crampton, Albert, Cognato and Richer-de-Forges, in review). However, many taxonomic uncertainties will be resolved only by using a combination of traditional taxonomy (based on morphology), molecular systematic techniques, cytogenetic analysis, and an examination of the diversity of EODs.
Cytogenetic variation has probably played an important role in the diversification of species in the G. carapo species-complex. Previous studies have indicated that forms identified as G. carapo exhibit different karyotypes (e.g. 2n = 48 in Amazonas, 2n = 42 in Pará, 2n = 54 and 2n = 52 from Southern Brazil) (reviewed in ). Whether this diversity is the manifestation of cryptic species diversity in a G. carapo species-complex or, alternatively, intraspecific cytogenetic diversity is currently unknown. Nonetheless, this question is of great relevance to our understanding of diversification in the genus. In this contribution, which forms the second in a series on the evolutionary cytogenetics of Gymnotus, we describe a novel karyotype in a population of Gymnotus carapo from the Eastern Amazon. We speculate that this may have evolved from chromosomal rearrangement of the karyotype of a more common, sympatrically co-occurring form.
Sample localities in the State of Pará, Brazil
MPEG 13332, 15099
Ponta de Pedra
01° 20' 25.4" S, 048° 58' 06.2" W
São Miguel do Guamá
01° 32' 09.5" S, 047° 36' 18.7" W
MPEG 13331, 15098
Lago Segredo and Lago Açaiteua
01° 07' 30" S, 047° 07' 30" W
01° 16' 34.8" S, 048° 20' 17.0" W
MPEG 13329, 15100
01° 31' 34.2" S, 052° 33' 37.9" W
Morphometric data for adult specimens of two distinct karyotypic forms belonging to the Gymnotus carapo species complex from the Eastern Amazon (EA) (G. carapo 2n = 42 and G. carapo 2n = 40).
G. carapo 2n = 42
G. carapo 2n = 40
G. carapo EA
Meristic data for adult specimens of two distinct karyotypic forms belonging to the Gymnotus carapo species complex from the Eastern Amazon (EA) (G. carapo 2n = 42 and G. carapo 2n = 40).
G. carapo 2n = 42
G. carapo 2n = 40
G. carapo EA
Metaphase chromosomal preparations were undertaken following the methods described by . In brief: specimens were injected with a 0.025% colchicine solution in the proportion 0.5 ml/100 g body weight. After dissection, the kidney cells were suspended in 0.075 M KCl and incubated at 37°C for 30 min. The hypotonically treated cell suspension was then centrifuged and the pellet was re-suspended in fixative (3:1 methanol: acetic acid solution) and centrifuged twice. The final pellet was suspended in fresh fixative and dropped onto warmed slides. The slides were analyzed after conventional Giemsa staining, C-banding , silver staining of NORs , CMA3 staining , DAPI staining , and Fluorescente in situ Hybridization (FISH) with telomeric probes (All Telomere Probes, Oncor). Chromosomes were morphologically classified according to . Fish specimens for which cytogenetic analysis was conducted were vouchered at the Museu Paraense Emilio Goeldi (MPEG) and the lot numbers are listed in Table 1.
The examined specimens measured between 150 and 350 mm total length and weighed from 10 to 160 g. All had fully developed gonads and were easily sexed.
The C-banding in the 2n = 42 karyotype shows that the constitutive heterochromatin (CH) exhibits a centromeric location in all the chromosomes and interstitial regions on the short arms of chromosome 6 (Figure 3b). The C-banding in the 2n = 40 karyotype shows that the CH exhibits a centromeric location on almost all the chromosomes (with the exception of pairs 13, 14 and 17) and interstitial regions on the short arms of chromosome 9 (Figure 4b). One of the NOR-bearing chromosomes also possesses a block on the distal region of the short arm.
The karyotype with the diploid number of 2n = 42 (30M/SM+12ST/A) found in G. carapo from localities 1–4 (Table 1) is the same as that previously characterized from Santa Cruz do Arari, Ilha do Marajó . An almost identical karyotype, 2n = 42 (32M/SM+10ST/A), was also described  from G. carapo collected near Belém, Pará. The difference in morphology of one chromosomal pair may be attributed to a pericentric inversion. Alternatively, the other authors  may have interpreted the morphological classification of the pair differently, perhaps due to different degrees of condensation of the metaphase chromosomes. In either case, the data suggest that there is one common form of G. carapo with a homogenous 2n = 42 karyotype in the Eastern Amazon region of Belém and Marajó Island.
The 2n = 40 karyotype (28M/SM+12ST/A) from Almeirim (locality 5, Table 1) has not previously been documented from Gymnotus of the Amazon basin. Nonetheless, this diploid number has previously been documented for other species from more southerly regions of South America, but with variation in the karyotypic formula. G. sylvius (38M/SM+2ST/A ; 30M/SM+10ST/A, [20, 21]; 36M/SM+4ST/A [22, 23]; Gymnotus sp (14M/SM+26ST/A [7, 8]) and G. pantanal (14M/SM+26ST/A ; 14M/SM+26ST/A in females and 15M/SM+24ST/A in males, with an multiple sex chromosome system X1X1X2 X2/X1X2Y ). The disparate phylogenetic positions of G. sylvius, G. pantanal and G. carapo senso stricto hypothesized by  indicates that the diploid number of 2n = 40 is homoplastic.
The location of NOR in both the 2n = 42 and 2n = 40 karyotypes were detected in the short arm of a unique pair of chromosomes, resembling the condition for karyotypes previously described in other species of Gymnotus [6, 8, 9, 19, 22, 25, 26].
The banding with fluorescent stains CMA3 and DAPI corroborates results previously obtained in other species of Neotropical freshwater fishes, where CMA3 preferentially stains the NOR, and where DAPI exhibits a banding model similar to that of C-banding .
The occurrence and apparent abundance of the same 2n = 42 form of G. carapo at multiple localities around the mouth of the Amazon: Belém , Santa Cruz do Arari  and localities 1–4 (Table 1, Figure 1), and the absence of substantial morphological variation between or within these samples suggest that they together constitute an interbreeding population of a single species. This same situation has been reported by  for four species of Gymnotus occurring in southeast Brazilian drainages: G. "carapo" (in fact an undescribed species that is not G. carapo) from twelve localities; G. inaequilabiatus (in fact also an undescribed species that is not G. inaequilabiatus) from two localities, G. sylvius from eight localities, and G. pantherinus from four localities. The authors of this study also observed that individuals of the same species always possessed the same karyotype in different localities, for example, 2n = 54, 52M/SM+2ST/A in G. "carapo".
The difference in the diploid number (2n = 40 and 2n = 42) reported here can be explained by a fusion-fission rearrangement. The presence of an ITS in a metacentric pair in the 2n = 40 karyotype (Figure 5b) supports the fusion hypothesis, where two ST/A pairs from the 2n = 42 karyotype fused originating a M/SM pair in the 2n = 40 karyotype. The morphologic variation in the karyotypic formula (2n = 40: 34M/SM+6ST/A e 2n = 42: 30M/SM+12ST/A) can be explained also by pericentric inversions. According to , multiple pericentric inversions are an important post-zygotic reproduction isolation mechanism. The absence of morphologic differentiation suggests that the putative chromosomal speciation event occurred recently and that there was consequently insufficient time for the fixation of phenotypic differences. These considerations lead us to speculate that the Almeirim sample may be a cryptic species that is isolated by post-zygotic reproductive barriers from an extant sister taxon (the 2n = 42 form), and from which it derived by chromosomal rearrangement. This hypothesis is amenable to empirical test with molecular phylogenetic data and by examination of EOD signal variation.
Localities and diploid number for karyotpes of Gymnotus species in Brazil (see also Figure 1).
Localities and 2n
Miracatu-SP , Botucatu-SP , Jundiaí-SP , Rio Claro-SP , Americana-SP , Salto Grande-SP , Primeiro de Maio-SP , Mococa-SP , São Simão-SP , Santa Maria da Serra-SP , Jacareí-SP  (2n = 54); Pirassununga-SP  (2n = 54 e 2n = 81); Brotas-SP , (2n = 52); Humaitá-AM  (2n = 48); Belém-PA , Benfica-PA1, São Miguel do Guamá-PA1, Capanema-PA1, Ponta de Pedras-PA1, Santa Cruz do Arari-PA , (2n = 42); Almeirim-PA1 (2n = 40).
Miracatu-SP , São Simão-SP [19, 20], Americana-SP , Represa de Capivara-SP , Sta. Maria da Serra-SP , Corumbataí-SP , Jacareí-SP , Paraibuna-SP  (2n = 40), Guaíra-PR , Alfenas-MG , (2n = 40).
Sta. Cruz do Arari-PA , (2n = 54).
We expect that the wide geographic range, diversity of species, and cytogenetic diversity of the Gymnotus carapo species-complex will provide a good testing ground for exploring the role of chromosomal evolution in speciation. A holistic approach involving cytogenetics, phylogenetic systematics, population genetics, and the study of electric communication signals (putative pre-zygotic reproductive isolating barriers, see [29–31]) may allow us to trace individual speciation events (including those separating extant sister species) to documented chromosomal rearrangement events, such as the one we hypothesize here.
The difference in the diploid number and FN between a 2n = 42 form of Gymnotus from the Eastern Amazon, and a morphologically identical form with 2n = 40 can be explained by a fusion/fission and pericentric inversions rearrangements. We speculate that these two forms are cryptic species, isolated from each other by post-zygotic reproductive isolating barriers.
We acknowledge the Museu Paraense Emilio Goeldi (MPEG) for vouchering specimens. We thank Geoffrey Shetka for assistance with the morphological measurements (funded by NSF Research Experience for Undergraduates internship, DEB-0724280 to W. Crampton). Funds were supported by CNPq, CAPES, UFPA. W. Crampton received funding from National Science Foundation grant DEB-0614334 (Evolution of Species and Signal Diversity in the Neotropical Electric Fish Gymnotus). Collecting was authorized by IBAMA (Instituto Brasileiro do Meio Ambiente) permit 020/2005 (IBAMA Registration: 207419).
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