The karyotype of the IUCN least concern red-backed toadlet Pseudophryne (P.) coriacea (Keferstein, 1868) from the New South Wales Central Coast is described following tissue culture of toe clipping macerates and conventional DAPI staining. The diploid number is 2n = 24. The karyotype is represented by six large and five small chromosomal pairs and one very small chromosomal pair. The very small chromosome 12 is 12% the size of chromosome 1. One of the large chromosomes is subtelocentric, two of the large chromosomes are submetacentric and the remaining chromosomes are metacentric. The putative nucleolus organiser region (NOR) is observed on chromosome 4. The diploid number and location of the putative NOR correlates to that of the previously published IUCN critically endangered P. corroboree (Moore 1953) and unpublished descriptions of the P. coriacea karyotype. This is the first described cell culture of a species from the genus Pseudophryne Fitzinger, 1843, first published analysis of the P. coriacea karyotype and the first published analysis of centromeric allocation of this genus. Globally there exists a large inventory of tissue samples in cryobanks that are not associated with known recovery mechanisms such as basic cell culture techniques. Detailed cytogenetic analyses of these cryobanked samples are therefore not possible. This work therefore enables: (i) a comparison of the P. coriacea karyotype with that of the critically endangered P. corroboree and (ii) a benchmark for repeat and future cytogenetic and genomic analyses of cryostored samples of this genus.

Cell culture, cryopreservation, karyotype, red-backed toadlet

Recently documented amphibian declines resulting from disease and habitat destruction have placed nearly one third of all amphibian species at risk of extinction (Silla and Byrne 2019). Examples of animals at the forefront of this decline are P. corroboree (the southern corroboree frog) and P. pengilleyi (Wells and Wellington 1985), both critically endangered species for which restorative husbandry programs are required and cryobanking proposed (Morgan et al. 2008; Kouba et al. 2013; McFadden et al. 2013; Clulow and Clulow 2016; Skerratt et al. 2016; Rojahn et al. 2018). To date, captive breeding programs have demonstrated some success towards the long-term reintroduction of these animals into the wild (McFadden et al. 2013; Silla et al. 2018). No examples of successful cell culture with or without cryobanking to provide a non-invasive technique for long term auxiliary and repeat genomic monitoring or assisted reproduction programs, however, have been reported for any representative of this genus.

The red-backed toadlet P. coriacea, an IUCN least concern listed species, is endemic to the east coast and ranges of Australia, north of Sydney to southern Queensland (White 1993; Donnellan et al. 2012). The genus Pseudophryne Fitzinger, 1843 comprises 14 known species (O’Brien et al. 2018) for which the karyotype of only P. corroboree has been published (Mahony and Robinson 1986). Reports from unpublished data (Morescalchi and Ingram 1974; Mahony and Robinson 1986) suggest a highly conserved 2n = 24 karyotype across nine species in this genus, including P. coriacea, with an NOR also highly conserved on chromosome set 4. Centromeric positions remain to be described for any species in this genus. Detailed karyotypic information available for interspecies comparisons within this genus and associated information for assistance in conservation management programs are therefore wanting (Mahony and Robinson 1986; Potter and Deakin 2018).

This report serves four aims: (1) to demonstrate successful cultivation, passaging and cryopreservation of cells from P. coriacea, (2) to formally describe their karyotype including centromeric positions and NOR locations, (3) to facilitate future genetic comparisons for conservation management programs of species within this genus, and (4) provide a tissue resource for future cytogenetic and genomic work that would not require harming living animals.

Toe macerates from an unsexed P. coriacea were placed in culture and individual cells were observed as attached single cells or within small expanding cell masses during the following two weeks (Figs 1, 2). Under high power inverted phase contrast microscopy, and at day 18 (D18), a mixed cell population comprising spindle-shaped and ovoid/polygonal morphology was observed. Rounded/semi-detached cells were presumed to be mitotic cells. At D18 and approximately 40% confluency, cells were trypsinised and cryopreserved in liquid nitrogen.

Figure 1.

Pseudophryne coriacea. Photographed by Michael Mahony at Wallingat State Forrest, New South Wales, Australia, 1982.

Figure 2.

Pseudophryne coriacea macerated and cultured toe clippings A–C primary culture prior to cryopreservation (P0) D–F passage 1 cells, post cryopreservation (P1) A, B, D, E low power C, F high power A, B P0 cells form an expanding cluster (c) adjacent to the reference debris (d) at days 13 (D13) and D18 C P0 cells are either ovoid/polygonal (o) or spindle-shaped (s); rounded cells (r) are also observed D, E post-cryopreservation, P1 cells form two expanding mass reference points (m2 and m3) to reach approximately 70% confluency by D13 F post-cryopreservation P1 cells are both ovoid/polygonal (o) and spindle-shaped (s); rounded cells (r) are also observed. Scale bars: 10 μM.

Following a 12 month period of cryopreservation, cells were thawed into two separate wells of a 24 well plate. Passage 1 (P1) cells post-thawing attached within 48 hours as both cell clumps and single cells, and formed colony outgrowths resulting in approximate 70% confluency by D13 (Fig. 1). Mixed spindle-shaped and ovoid/polygonal cell populations were observed, as well as more condensed rounded cells characterizing a presumed mitotic phenotype. Cells from one dish were processed for karyotyping, while cells from the second dish were passaged and subsequently processed for cryopreservation. A total of 200 000 passage two P. coriacea cells were cryopreserved in a seven week total culture period.

Of the first 27 metaphase P. coriacea chromosome spreads identified and counted, 26 displayed a 2N = 24 chromosomal count and one displayed a chromosomal count of 15, with the latter a probable artefact of the cell spreading technique (24_incidence = 96%; Fig. 3A). Six metaphase spreads were arranged in descending order of size to identify six large and five small chromosomal pairs and one very small chromosomal pair (Fig. 3B). A DAPI negative region was observed on the short arms of each chromosome pair number four (representing a presumptive NOR). Chromosomes 1, 2, 6, 7, 8, 9, 10, 11 and 12 are apparently metacentric, chromosomes 4 and 5 are apparently submetacentric and chromosome 3 is apparently subtelocentric (Table 1). Chromosome lengths were calculated relative to chromosome 1, not including measurements of the secondary restrictions on chromosome 4 (Table 1). The notably smaller chromosome 12 is 12% the size of chromosome 1.

Figure 3.

Pseudophryne coriacea karyotype A metaphase spread and B chromosomal pairs arranged in descending order relative to size and aligned by centromeric position. A 2N = 24 diploid chromosome number and the presence of a DAPI negative region on each of the short arms of chromosome 4 (arrows) are evident. Chromosomes 1 to 6 are larger, whereas chromosomes 7 to 11 are smaller in size, and chromosome 12 is smaller still.

While sperm cryobanking techniques have made significant advancements, methods for the cryopreservation of oocytes, embryos or amphibians suitable for conservation programs have not (Browne et al. 2019; Burger et al. 2022; Lampert et al. 2022). The cryostorage of karyotypically stable diploid nuclei amenable to recovery therefore represents a near term and important process for genomic and cytogenetic work and an additional resource for future conservation related assisted reproductive technologies (Kouba et al. 2013; Clulow and Clulow 2016; Zimkus et al. 2018).

The overall P. coriacea karyotype with 2n = 24 and the location of the presumptive NOR on chromosome 4 agrees with previously unpublished reports for this species (Morescalchi and Ingram 1978; Mahony and Robinson 1980). It is also consistent with that described for P. corroboree (Mahony and Robinson 1980) and the unpublished data (Morescalchi and Ingram 1978; Mahony and Robinson 1980) on a further seven species from this genus including P. pengilleyi. The description of the centromeric positions and relative lengths for any species of this genus is novel. This study serves as a prototype for future comparisons of centromeric descriptions and karyotypes of species from the genus Pseudophryne, thus aiding conservation management programs.

Richard Mollard has registered a company called Amphicell Pty Ltd (www.amphicell.com). Amphicell Pty Ltd received no funding for this work and privately provided the materials to execute the experimental procedures described in this study.

Tissues used in these studies were supplied from programs supported by Earthwatch Australia (Michael Mahony).

The authors have no funding to report.

Richard Mollard https://orcid.org/0000-0001-5820-7491