Research Article |
Corresponding author: Ross Brookwell ( Rhipsalis@bigpond.com.au ) Academic editor: T Chassovnikarova
© 2021 Ross Brookwell, Kimberly Finlayson, Jason P. van de Merwe.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Brookwell R, Finlayson K, van de Merwe JP (2021) A comparative analysis of the karyotypes of three dolphins – Tursiops truncatus Montagu, 1821, Tursiops australis Charlton-Robb et al., 2011, and Grampus griseus Cuvier, 1812. Comparative Cytogenetics 15(1): 53-63. https://doi.org/10.3897/compcytogen.v15.i1.60398
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The aim of this study is to produce G-banded karyotypes of three dolphin species, Tursiops truncatus Montagu, 1821, Tursiops australis
Burrunan, chromosome, Common Bottlenose, G-band, Risso’s dolphin
The family Delphinidae contains 37 recognized species, excluding Tursiops australis
The three species of dolphin investigated here belong to the subfamily Delphininae, but it has been proposed that Grampus griseus Cuvier, 1812, should be attributed to the subfamily Globicephalinae, based on cytochrome b sequencing studies (
The tissue samples available for this study were from a male and female common bottlenose dolphin (T. truncatus), a male and a female Burrunan dolphin (T. australis), and a female Risso’s dolphin (G. griseus). Skin samples from T. truncatus and T. australis were obtained from captive individuals at SeaWorld, Queensland, Australia during routine vaccinations. The tissue was taken from the tail using a biopsy punch. One female T. truncatus (CB01) was wild caught in 1994 and is approximately 33 years old. The other (CB02) was a male wild caught in 1985 and is approximately 43 years old. Both individuals of T. australis were born in captivity; one male aged 40 was transferred to Sea World in 1990 from Marineland, South Australia (BD01), and the other was a female aged 10 born at Sea World (BD04). A lung sample from a stranded G. griseus was provided by Dolphin Marine Conservation Park, Coffs Harbour, New South Wales, Australia (RD01). All tissue samples were immediately placed in DMEM media with 10% fetal bovine serum, 1% penicillin/streptomycin (10,000 U/mL stock) and 1% amphotericin B (250 μg/mL stock) and kept at 4 °C until processing.
Samples were washed several times with DMEM media (as described above) and cut into 1–3 mm pieces in fresh media. Tissue pieces were transferred to 25 cm2 flasks, arranged evenly on the lower surface of the flask. The flasks were incubated in an inverted position at 37 °C, 5% CO2 for 24 hours. Five mL of media was introduced, and then the flasks were returned to the incubator in an upright orientation. When cells reached ~70% confluence, tissue pieces were detached and removed. Cells were cryopreserved in liquid nitrogen at a concentration of 1 × 106 cells/mL in DMEM media supplemented with 10% dimethyl sulfoxide, until ready to be used (
The Qiagen DNeasy Blood and Tissue kit was used to isolate DNA from ~2×106 cells, according to the manufacturer’s protocol for cultured cells. The resulting DNA was forwarded to the DNA Sequencing Facility at Griffith University, for confirmation of species. Around 660 bp of the mitochondrial COI gene was used for amplification by Platinum taq DNA polymerase (Invitrogen). The following primers were used – forward 5–3’ ATTCAACCAATCATAAAGATATTGG, reverse 5–3’ TAAACTTCTGGATGTCCAAAAAATCA (
A flask of cells for each dolphin at various passages (CB01: P7; CB02: P6; BD01: P6; BD04: P6; RD01: P6) was forwarded to the cytogenetics laboratory at Sullivan Nicolaides Pathology. Here, the cells were either incubated overnight at 37 °C prior to initiation of harvest, or sub-cultured into 25 cm2 flasks in Amniomax II medium (Gibco), then incubated at 37 °C until ready for harvest. The cells were harvested when approximately 80% confluent. This was initiated by adding colchicine (100 µg/mL,Sigma) for 2 hours, suspending the cells in the medium with trypsin (Trypsin/EDTA 1×, Sigma), and swelling the cells by treatment with hypotonic solution (0.075 M potassium chloride) at 37 °C for 10 minutes. A 10% prefix solution of 3% acetic acid was then added before methanol/glacial acetic acid (3:1) fixation. The resulting cell suspension was used to prepare slides by dropping via a glass pipette onto clean dry slides (
A Metafer slide scanner (Metasystems) was used to select cells for processing, and the Ikaros karyotyping system (Metasystems) was used to produce karyograms.
The template employed for chromosome grouping is consistent with that used by
Species identification confirmed both CB01 and CB02 to be T. truncatus with a 99.27% and 99.85% match of COI gene sequence, respectively. BD01 and BD04 were confirmed to be T. australis with a 99.71% COI gene sequence match for both individuals. RD01 was confirmed to be G. griseus with a 99.85% match of COI gene sequence.
The diploid number of all 3 species is 44. In all individuals studied, the karyotype consists of 2 large subtelocentric pairs (1–2), 9 submetacentric pairs (3–11), 6 smaller metacentric/submetacentric pairs (12–17), 4 acrocentric pairs (18–21), an X chromosome which closely resembles that observed in many mammalian species, and in the 2 males studied, a small Y chromosome. Five to 22 karyotypes per individual were prepared, depending on the availability of suitable metaphases, and these showed a consistent karyotype in each case. A representative karyogram from each of the five individuals studied is presented in Figs
G-banded karyotype of male T. truncatus (CB02). Note the size polymorphism in the distal short arm of chromosome 6.
G-banded karyotype of female T. truncatus (CB01). Note the size polymorphism in the short arm of chromosome 3 and the proximal long arm of chromosome 4.
There are a number of heterochromatic variants visible in these individuals. In the male T. truncatus there is a size polymorphism in the distal short arm of chromosome 6, the chromosome on the right has a larger G-negative band, and in the female, the short arm of chromosome 3 of the chromosome on the right has a larger pale band between the two dark bands, and the proximal long arm of chromosome 4 has a larger G-negative band just below the centromere. In the female T. australis, there are variant heterochromatic regions in the distal short arm of chromosome 2, where the chromosome on the right has a larger grey band distally, and the short and long arms of chromosome 4, where the chromosome on the right has a smaller pale band at the end of the short arm, and a smaller pale region just below the centromere. G. griseus has a variant on the proximal long arm of chromosome 18, the G-band negative region being larger in the chromosome on the right.
G-banded karyotype of female T. australis (BD04). Note the size polymorphism in the distal short arm of chromosome 2, and the short and long arms of chromosome 4.
G-banded karyotype of female G. griseus (RD01). Note the size polymorphism in the proximal long arm of chromosome 18.
Apart from the size polymorphisms attributable to heterochromatin variants, the results show that chromosome 1 in G. griseus has a significantly different morphology from the two Tursiops species. In the Tursiops karyograms, the short arm consists essentially of a proximal dark and distal light band, with a pale centromeric region, and a prominent dark band on the proximal long arm. In the G. griseus karyogram, the short arm has a darker distal region and a thin dark band in the proximal region, and it is also slightly longer. The centromeric region of G. griseus is not as distinctly pale, and there is no proximal dark band on the long arm. The remainder of the long arm is similar, but not completely identical. Overall, the chromosome is slightly shorter in G. griseus. Figure
A chromosome 1 from A T. truncatus B G. griseus C idiogram of chromosome 1 from T. truncatus to the left, G. griseus to the right D chromosome 2 from T. truncatus E T. australis, with arrows indicating the position of the centromere F idiogram of chromosome 2 from T. truncatus to the left, T. australis to the right, with arrows indicating possible breakage points of a pericentric inversion.
In both male and female karyograms of T. australis, the dark band on the proximal long arm of chromosome 2 in T. truncatus and G. griseus is present on the proximal short arm. Figure
The karyotypes of the three species of dolphin studied here are very similar, all having the same chromosome number (2n = 44) and gross morphology. It is only when studying the detail of the G-banding pattern that differences become apparent. This can be readily visualized by referring to Figure
As the number of individuals available is limited, reasons other than interspecific differences for the observed variation need to be considered. The presence of isolated populations can be a source of intraspecific variation, however in the karyotypes of the individual pairs studied, there was no heteromorphism that could not be assigned to heterochromatic size, relating the variant regions to the C-banded karyogram of Tursiops gilli Dall, 1873, now reclassified as T. truncatus, depicted in
Chromosome 1 appears very similar in T. truncatus and T. australis, and also, from the literature, in the delphinids S. clymene, and L. albirostris, and in the harbor porpoise P. phocoena (in the latter karyogram the short arm is smaller, lacking the prominent dark band, and the distal C-band positive region is lacking) (
The proximal dark band on chromosome 2 is on the long arm in S. clymene, L. albirostris, P. phocoena (
Hybrids between dolphin species occur rarely in the wild, more frequently in captive animals. In captivity, the most frequently observed hybrids result from crosses between T. truncatus and G. griseus (
The three species of dolphin species described here can be distinguished by their banding pattern, these differences being consistent in all cells within the individuals studied. The small number of individuals analysed makes it premature to draw firm conclusions, but it appears that these differences may potentially have use as an additional tool in determining the species of a particular animal where this is unclear, and in assessment of hybrids. Study of further individuals of these species, and of other dolphins, would enable karyotypic variation to be added to molecular and morphological differences in establishing the evolutionary relationships within this group. In the light of the study by