Karyotypic description and comparison of Litoria (L.) paraewingi (Watson et al., 1971), L.ewingii (Duméril et Bibron, 1841) and L.jervisiensis (Duméril et Bibron, 1841) (Amphibia, Anura)

Abstract

The karyotype of Litoria (L.) paraewingi (Watson et al., 1971) (Big River State Forest, Victoria) is described here for the first time. It is prepared following tissue culture of toe clipping macerates, cryopreservation, reculture and conventional 4',6-diamidino-2-phenylindole (DAPI) staining. The L.paraewingi karyotype is then compared to similarly processed IUCN (International Union for the Conservation of Nature) least concern members L.ewingii (Duméril et Bibron, 1841) (southern Victoria) and L.jervisiensis (Duméril et Bibron, 1841) (Myall Lakes National Park, New South Wales), all members of the same L.ewingii complex/group. The L.paraewingi diploid number is 2n = 26, the same as for the other two species. Litoriaparaewingi chromosomes 1, 2, 6 and 7 are submetacentric, chromosomes 3 and 5 are subtelocentric and the remainder are metacentric. No secondary constriction or putative nucleolus organiser region (NOR) was readily identifiable following conventional DAPI staining in any scored L.paraewingi metaphase spread. Conversely, a putative NOR was readily identifiable on the long arm of chromosome 1 in all examined metaphase spreads for the other two species. The karyotypes of L.ewingii and L.jervisiensis here further differ from L.paraewingi with chromosome 1 being metacentric and chromosomes 8 and 10 being submetacentric for both former species. The L.jervisiensis karyotype differs from those of L.ewingii and L.paraewingi by DAPI staining with: (i) apparent relative length inversion of subtelocentric chromosome 3 and metacentric chromosome 4 and (ii) chromosome 6 being metacentric rather than submetacentric. All three species have a highly conserved chromosome morphology with respect to chromosomes 2, 5, 7, 9, 11, 12 and 13. The greatest gross morphological difference karyotypically is observed between L.paraewingi and L.jervisiensis. These karyotype data support the previous phylogenetic separation of these three species based upon genetic compatibility and behavioural, biochemical and molecular genetic analyses.

Keywords: Cell culture; Plains brown tree frog; cryopreservation; karyotype.

Figure 1.

L.ewingii; photographed by Matthew West at Merri Creek, Australia, 2020. L.paraewingi; photographed by Stephen Mahony at Wangaratta, Victoria, Australia, 2017. L.jervisiensis; photographed by Stephen Mahony at Mungo Brush Park Myall Lakes National Park, New South Wales, Australia, 2021.

Figure 2.

Representative metaphrase spreads of cryopreserved, thawed and cultured cells AL.ewingiiBL.paraewingiCL.jervisiensis. Arrows indicate DAPI negative regions, or presumptive NORs. No DAPI negative regions were apparent in the L.paraewingi metaphase spreads. As per Table 1, underlined numbers represent those morphologies differing to L.ewingii and italicised (in red) numbers represent L.jervisiensis morphologies differing to those of L.paraewingi.

Figure 3.

Metaphrase spreads of cryopreserved, thawed and cultured cells from L.ewingii. A–C three individual metaphase spreads. Arrows indicate DAPI negative regions, or presumptive NORs.

Figure 4.

Metaphrase spreads of cryopreserved, thawed and cultured cells from L.paraewingi. A–C three individual metaphase spreads. No DAPI negative regions, or presumptive NORs, were apparent.

Figure 5.

Metaphrase spreads of cryopreserved, thawed and cultured cells from L.jervisiensis. A–C three individual metaphase spreads. Arrows indicate DAPI negative regions, or presumptive NORs.