Cells from a total of ten different Australian native frog species have been cultured, passaged, cryopreserved and validated by karyotyping following freeze-thaw cycles. A further four species have been successfully cultured, however, final validation for cryobank submission has not yet been completed. Below current work on these 14 individual species representing nine individual genera is briefly described (see Donnellan et al., 2025 for classifications). Four of these frog species are IUCN critically endangered, one is endangered, one is vulnerable and the remainder are of least concern. Furthermore, two of the species described below are represented by both frog and tadpole derived cells.

  1. Drymomantis fallax (eastern dwarf tree frog, eastern sedgefrog; formerly Litoria fallax; two frogs).
  2. Dryopsophus spenceri (Spencer’s river tree frog, spotted tree frog; formerly Litoria spenceri; one tadpole and five frogs),
  3. Limnodynastes tasmaniensis (spotted marsh frog, spotted grass frog; one frog),
  4. Nictimystes infrafrenatus (white-lipped tree frog; formerly Litoria infrafrenata; one tadpole and two frogs),
  5. Phyloria frosti (Baw Baw frog; one frog)
  6. Pseudophryne coriacea (red-backed toadlet; one frog),
  7. Pseudophryne corroboree (corroboree frog; one frog),
  8. Ranoidea aurea (golden and green bell frog; formerly Litoria aurea, one frog)
  9. Ranoidea raniformis (growling grass frog; formerly Litoria raniformis; one frog) – in preparation for publication, coming soon,
  10. Rawlinsonia ewingii (southern brown tree frog, whistling tree frog; formerly Litoria ewingii; two frogs),
  11. Rawlinsonia jervisiensis (Jervis Bay tree frog; formerly Litoria jervisiensis; two frogs),
  12. Rawlinsonia paraewingi (plain’s brown tree frog; formerly Litoria paraewingi; two frogs, no remaining samples),
  13. Rawlinsonia verreauxii alpina (alpine tree frog; formerly Litoria verreauxii alpina; two frogs) – in preparation for publication, coming soon, and
  14. Rhyaconastes lesueuri (Lesueur’ tree frog; formerly Litoria lesueurii; two frogs, no remaining samples).
Cells from Drymomantis fallax have been successfully grown, passaged, freeze – thawed and karyotyped. The correct karyotype of 2n = 26 is described (one frog, 86% of first 42 counted karyomaps). A nucleolus organiser region was allocated to chromosome pair 11 (red arrow) as previously described (Schmid, 2018, Cytogenet Genome Res 2018;155:55–221) (Frog image source).

Both frog and tadpole cells from Dryopsophus spenceri have been successfully grown, passaged, freeze – thawed and karyotyped. The correct karyotype of 2n = 26 is described (one tadpole, 94% of the first 33 counted karyomaps; five frogs, 71% of 24, 84% of 31, 100% of 16, 82% of 11 and 75% of 12 first counted karyomaps, respectively). These data are described in a peer review publication (Mollard et al. 2018). Of interest, frogs were collected from three different locations and found to vary in the context of their nucleolus organiser regions. Furthermore, crosses between specific frogs from different locations resulted in tadpoles with a mixed nucleolus organisation region phenotype. These data point to the possible existence of distinct Dryopsophus spenceri varieties. Here, the tadpole nucleolus organiser regions can be seen on chromosomal pairs 8 and 11, with chromosomal pair 11 displaying the hybrid phenotype (red arrows). Here, the frog nucleolus organiser regions can be seen on each chromosome of chromosomal pairs 8 and 11 (red arrows) (Frog photo source, tadpole image source).
Cells from Limnodynastes tasmnaniensis have been successfully grown and karyoptyped following freezing. The correct karyotype of 2n = 24 is described (one frog, 78% of first 27 counted karyomaps), with a nucleolus organiser region present on chromosome pair 9 as previously described (Moreschalchi and Ingram 1978). An additional potential nucleolus organiser region has also been described on the long arms of chromosomes 10, yet this was not apparent with the techniques used here (Frog image source).
Both frog and tadpole cells from Nictimystes infrafrenatus have been successfully grown, passaged, freeze – thawed and karyotyped. The correct karyotype of 2n = 24 is described (one tadpole, 91% of first 33 counted karyomaps, two frogs, 89% of 36 and 92% of 24 first counted karyomaps, respectively). These data are described in two peer reviewed publications (Mollard et al., 2018a, Mollard et al., 2018b). (Frog and tadpole photos provided by R. Mollard).
Cells from Philoria frosti were successfully grown in culture and cryopreserved at passage 0. Cells were unfortunately lost in a shared liquid nitrogen facility. This work will be repeated, however, these data demonstrate that cells from this species can be successfully grown in culture (Frog image source)
Cells from Pseudophryne coriacea have been successfully grown, passaged, freeze – thawed and karyotyped. The correct karyotype of 2n = 24 (one frog, 96% of 27 karyomaps) is described with a nucleolus organiser region observed on chromosome pair 4 (red arrow). This is the first description of the karyotype of Pseudophryne coriacea and these data are described in a peer reviewed publication. (Mollard et al., 2023) (Frog image source).
Cells from Pseudophryne corroboree have been successfully grown, passaged, karyotyped and frozen. Cells have not yet been thawed from the liquid nitrogen facility. These data demonstrate that cells from this species can be successfully grown in culture. The correct karyotype of 2n = 24 is described (one frog, 100% of five counted karyomaps) with a nucleolus organiser region allocated to chromosome pair 4 as previously described (see Mahony and Robinson, 1986, Genetica 68, 119-127). (Frog image source).
Cells from Ranoidea aurea were successfully grown in culture and cryopreserved at passage 0. Cells are yet to be thawed from liquid nitrogen for karyotyping. This work is ongoing and these data demonstrate that cells from this species can be successfully grown in culture (Frog image source)
Cells from Ranoidea raniformis have been successfully grown, passaged, freeze – thawed and karyotyped. The correct karyotype of 2n = 26 has been determined. (Frog image source)

Cells from Rawlinsonia ewingii have been successfully grown, passaged, freeze – thawed and karyotyped. The correct karyotype of 2n = 26 is described (one frog, 70% of the first 23 counted karyomaps). A nucleolus organiser region was apparent on chromosome pair 1. These data have been the subject of a peer reviewed publication (Mollard et al., 2024) (Frog image source).
Cells from Rawlinsonia jervisiensis have been successfully grown, passaged, freeze – thawed and karyotyped. The correct karyotype of 2n = 26 is described (one frog, 94% of the first 71 karyomaps counted). A nucleolus organiser region was apparent on chromosome pair 1. These data have been the subject of a peer reviewed publication (Mollard et al., 2018) (Frog image source).
Cells from Rawlinsonia paraewingi have been successfully grown, passaged, freeze – thawed and karyotyped. The correct karyotype of 2n = 26 is described (87% of the first 15 karyomaps counted). No nucleolus organiser region was apparent using the techniques here. Samples have unfortunately been lost from a shared LN2 facility and this work will be repeated. This is the first demonstration of the Rawlinsonia paraewingi karyotype and has been the subject of a peer reviewed publication (Mollard et al., 2025) (Frog image source)
Cells from Rawlinsonia verreauxii alpina have been successfully grown, passaged, freeze – thawed and karyotyped. The correct karyotype of 2n = 26 has been determined. Note the image here is of Ralwinsonia verreauxii verreauxii and not Ralwinsonia verreauxii alpina, an image of which is not currently available (Frog image source).
Cells from Rhyaconastes lesueuri were successfully grown in culture and cryopreserved at passage 1. Cells were unfortunately lost in a shared liquid nitrogen facility. This work will be repeated, however, these data demonstrate that cells from this species can be successfully grown in culture (Frog image source)

Ethics

All tissues were collected in compliance with relevant State governmental and ethical licensing requirements, The Code of Ethics of the World Medical Association of The Declaration of Helsinki and the EU Directive 2010/63/EU for animal experiments.

Dr Richard Mollard: provision of tissue from Rawlinsonia ewingii (Victorian Department of Environment, Land, Water & Planning Permit number 10008085; University of Melbourne Animal Ethics Committee Notification of Scavenged Animal Tissue).

Prof Michael Mahony: provision of tissue from Drymomantis fallax, Limnodynastes tasmaniensis, Pseudophryne coriacea, Ranoidea aurea and Rawlinsonia jervisiensis (New South Wales National Parks Scientific Licence SL00190).

Gerry Marantelli: provision of tissue from Dryopsophus spenseri, Philoria frosti, Pseudophryne corroboree, Ranoidea raniformis and Rawlinsonia verreauxii alpina (Victoria Wildlife Research Permit No. 10007968).

Deborah Pergolotti: provision of tissue from Nyctimystes infrafrenatus (S12(F) Nature Conservation (Administration) Regulation 2006, Scientific Purposes Permit number WISP15208614).

Dr Matt West: provision of tissue from Dryopsophus spenseri, Rawlinsonia paraewingi and Rhyaconastes lesueuri (Victoria Wildlife Research Permit No. 10009587).

 Abbreviations

D = day; HP = high power; LP = low power; P = passage