Ancient DNA from the extinct New Zealand grayling (Prototroctes oxyrhynchus) reveals evidence for Miocene marine dispersal

Abstract

The evolutionary history of Southern Hemisphere graylings (Retropinnidae) in New Zealand (NZ), including their relationship to the Australian grayling, is poorly understood. The NZ grayling (Prototroctes oxyrhynchus) is the only known fish in NZ to have gone extinct since human arrival there. Despite its historical abundance, only 23 wet and dried, formalin-fixed specimens exist in museums. We used high-throughput DNA sequencing to generate mitogenomes from formalin-fixed P. oxyrhynchus specimens, and analysed these in a temporal phylogenetic framework of retropinnids and osmerids. We recovered a strong sister-relationship between NZ and Australian grayling (P. mareana), with a common ancestor ~13.8 Mya [95% highest posterior density (HPD): 6.1–23.2 Mya], after the height of Oligocene marine inundation in NZ. Our temporal phylogenetic analysis suggests a single marine dispersal between NZ and Australia, although the direction of dispersal is equivocal, followed by divergence into genetically and morphologically distinguishable species through isolation by distance. This study provides further insights into the possible extinction drivers of the NZ grayling, informs discussion regarding reintroduction of Prototroctes to NZ and highlights how advances in palaeogenetics can be used to test evolutionary hypotheses in fish, which, until relatively recently, have been comparatively neglected in ancient-DNA research.

Unlocking the genomes of formalin‐fixed freshwater fish specimens: An assessment of factors influencing DNA extraction quantity and quality

“…The degree of DNA fragmentation is an important factor influencing sequencing success. Typically, a higher proportion of DNA fragments above 300 bp increases the chances of amplifying targeted DNA and sequencing (Abdel-Latif and Osman 2017; Garg et al 2018;Scarsbrook et al 2023), although PCR fragment amplification and sequencing have been successfully performed on even shorter lengths (~200 bp; Shiozawa et al 1992;Hykin et al 2015). In the present study, muscle tissue yielded a significantly greater proportion of fragments over 300 bp in length for both Brown Trout and Slimy Sculpin specimens.…”

“…2022; Scarsbrook et al. 2023). A standard protocol was used for tissue washes that requires three sequential buffer washing steps following Hykin et al.…”

“…Preceding the heated alkali treatment with a series of glycine-tris-EDTA (GTE) buffer washes may more efficiently cleave cross-linkage complexes and bind excess formalin to improve extract quality (Splendiani et al 2017;Billerman and Walsh 2019;Rocha et al 2022;Scarsbrook et al 2023). A standard protocol was used for tissue washes that requires three sequential buffer washing steps following Hykin et al (2015).…”

“…However, since the amount of tissue sample can often be increased to achieve higher concentrations, the more pressing question for further examination becomes whether the purity and fragmentation levels are sufficient in real-world applications. Although contaminants can inhibit enzymatic reactions, including sequencing adapter ligation and PCR (Illumina 2023), past work has successfully sequenced samples despite contamination in DNA extractions from formalin-fixed maize (Abdel-Latif and Osman 2017), fish (Shiozawa et al 1992;Scarsbrook et al 2023), reptiles (Hykin et al 2015), and amphibians (Pyron et al 2022). If purity is the main consideration in a particular study, then our results suggest that caudal fin tissue may be superior to dorsal muscle.…”

“…These collections can be used to resolve taxonomic questions, monitor species that are relevant to public health and safety concerns, track the history and progress of biological invasions, and measure organismal responses to global change (Suarez and Tsutsui 2004;Winker 2004;Watson and Werb 2013;Holmes et al 2016). Additionally, the worldwide acceleration of extinction events, particularly in freshwater ecosystems (Jelks et al 2008;Burkhead 2012;Closs et al 2016), has further enhanced the value of phylogenetic and population genetic research using preserved specimens of rare or declining species (Burrell et al 2015;Splendiani et al 2017;Gaither and DiBattista 2022;Scarsbrook et al 2023). In combination with the monitoring of extant populations, the analysis-including genetic examinations-of specimens housed in natural history collections can play an important role in conservation status assessments and informing management actions (Shaffer et al 1998;Winker 2004;Sutherland et al 2009;Lister et al 2011;Watson and Werb 2013;Holmes et al 2016;Hahn et al 2021).…”