Assessing herbarium material with novel molecular techniques reveals a wealth of new data from old treasure troves

Assessing herbarium material with novel molecular techniques reveals a wealth of new data from old treasure troves

Assessing herbarium material with novel molecular techniques reveals a wealth of new data from old treasure troves

Using large-scale genome skimming to build a resilient resource for the future
Hamersley Range, Pilbara, Western Australia PHOTO CREDIT: Stephen van Leeuwen

Herbaria are valuable sources of extensive curated plant material that are important reference specimens for plant identification. These plant materials are now also accessible to genetic studies because of advances in high-throughput, next-generation sequencing (NGS) methods.

In our study, we conducted a large-scale applied assessment of one such NGS approach – genome skimming – and its ability to recover plastid and ribosomal genome sequences from a broad taxonomic sampling of herbarium material for the Western Australian flora. We sequenced 672 samples covering 21 families, 142 genera, and 530 named and proposed named species, and explored the impact of sample age, DNA concentration and quality, read depth and fragment length on plastid assembly error.

We demonstrate that herbaria are a valuable source of plant material for building a comprehensive DNA sequence database which serves various applications from modernizing plant surveys to improving the resolution of plant phylogenies.

Gastrolobium grandiflorum, Pilbara, Western Australia PHOTO CREDIT: Stephen van Leeuwen

Genome skimming1 was effective at producing genomic information at large scale. Substantial sequence information on the chloroplast genome was obtained from 96.1% of samples, and complete or near-complete sequences of the nuclear ribosomal RNA gene repeat were obtained from 93.3% of samples.

Eucalyptus kingsmillii, Pilbara, Western Australia PHOTO CREDIT: Stephen van Leeuwen
Grevillea wickhamii, Pilbara, Western Australia PHOTO CREDIT: Stephen van Leeuwen

We extracted sequences for plastid markers rbcL and matK – the core DNA barcode regions – from 96.4% and 93.3% of samples, respectively. Read quality and DNA fragment length had significant effects on sequencing outcomes and error correction of reads proved essential. Assembly problems were specific to certain taxa with low GC and high repeat content (e.g. Goodenia, Scaevola, Cyperus, Bulbostylis, Fimbristylis), suggesting the influence of biological rather than technical factors. The structure of related genomes was needed to guide the assembly of repeats that exceeded the read length. DNA-based matching proved highly effective and showed that the efficacy for species identification declined in the following order: total chloroplast DNA >> ribosomal DNA > matK >> rbcL.

Ptilotus rotundifolius, Pilbara, Western Australia PHOTO CREDIT: Stephen van Leeuwen

Our success is important as it demonstrates that herbaria can be used as a source of plant material for building a comprehensive DNA sequence database. These data form the basis of development of a molecular identification system for the Western Australian flora. This will enable identification of specimens throughout the year (e.g., non-flowering times) and for hard-to-identify species (e.g., those with constrained or reduced morphological characters) or for specimens where only fragments of non-diagnostic material are available. The availability of this technology will modernize plant surveys by reducing constraints on survey effort through moderating sampling timing restrictions and seasonal effects, as well as enabling rapid verifiable identification. It will also have practical applications in a wide range of ecological contexts using eDNA metabarcoding, such as gut and scat analysis of animals to determine dietary preferences of threatened species and livestock, and checking the integrity of seed collections for seed banking and use in land restoration/revegetation programs. Other potential uses of extensive plastid sequence data, beyond species identification, include improving the resolution of plant phylogenies and studies on the evolution of plastid genome function, including understanding adaptive changes.

References:

1. Straub S, Parks M, Weitemier K, Fishbein M, Cronn R, Liston A (2012) Navigating the tip of the genomic iceberg: Next-generation sequencing for plant systematics. American Journal of Botany 99(2), 349-364. https://dx.doi.org/10.3732/ajb.1100335

For full details, please refer to the publication in BMC Plant Methods.

Written by

Paul Nevill

Paul Nevill

Curtin University, School of Molecular and Life Sciences, ARC Centre for Mine Site Restoration

& Trace and Environmental DNA (TrEnD) Lab, Perth, Western Australia

February 4, 2020
https://doi.org/10.21083/ibol.v10i1.5934 

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Discovering Fiji’s native bees: hidden secrets in a biodiversity hotspot

Discovering Fiji’s native bees: hidden secrets in a biodiversity hotspot

Discovering Fiji’s native bees: hidden secrets in a biodiversity hotspot

Researchers provide new insights into biodiversity using DNA barcoding in Fiji's topographically complex archipelago.

Homalictus hadrander, one of the four described species previously known from Fiji.
PHOTO CREDIT: James Dorey

Fiji’s entomological diversity has historically been considered depauperate. Recent widespread DNA barcoding efforts, however, from the South Australian Museum, Flinders University, and University of South Australia, along with a flurry of undergraduate, honours, and PhD students, have helped to uncover some of the hidden secrets of biodiversity within this topographically complex archipelago.

Since 2010, funding from the Australian & Pacific Science Foundation and Australian Commonwealth New Colombo Plan, along with support from students, has enabled fieldwork focused on collecting bees, wasps, and butterflies across all the major Fijian islands. Trekking up the tallest mountains, four-wheel driving across challenging terrain, and following the meandering rivers of inland Fiji has revealed that initial estimations of Fiji’s entomological fauna have been severely underestimated.

DNA barcoding over 1,000 bee specimens has increased species richness estimates from 4 species (known since 1979) up to 26 endemic species in the genus Homalictus. Interestingly, 60% of these new species are only found above 800 m elevation which comprise a mere 2% of land area of Fiji, and they are often restricted to single mountain tops (Figure 1). From extensive DNA barcoding, mitochondrial haplotype diversity was used to explore the level of intraspecific gene flow in the widespread species of the genus (Figure 2).

Figure 1: (a) The number of species (species richness) plotted against land area available at each elevational gradient. (b) Map of Fiji showing the land area available. Colours correspond to those used in (a).

CREATED BY: Cale Matthews

These results also indicate that gene flow is being restricted within highland localities of the most widespread Homalictus species. Dispersal from a species home range does not appear to be occurring in Fiji, which may be presenting a contemporary model of speciation that is predominantly influenced by past climatic fluctuations. There is an estimated crown age of 400 ka for the initial Fijian Homalictus colonisation, which would result in the genus being present for several glacial cycles. During glacial maxima, cooler climates would be ubiquitous throughout Fiji, however during glacial minima and interglacial periods there is a distinction between cool highland and warm lowland climate. DNA barcoding results indicate that the largest diversification of this genus is concordant with the most recent glacial minima, as species that were freely dispersing during glacial maxima are forced to retreat into highland refugia. Combined with the inferred haplotype networks, these results indicate that restriction due to low thermal tolerance of lowland climate is driving the extraordinary highland species richness in Fiji.

 

Figure 2: (a) Haplotype network of all sequenced Homalictus fijiensis (N=358) coloured by sampling locality. Hash marks represent nucleotide changes between each haplotype. Shared haplotypes represented by circles with multiple colours. Circle outline representing highland or lowland sampling. (b) Sampling map of H. fijiensis coloured by geographic sampling locality.

CREATED BY: Cale Matthews

Further to the work on bees, we have also started barcoding Fiji’s butterfly fauna, along with the first-ever species of Gasteruption, a parasitoid wasp genus, found in Fiji. The species, Gasteruption tomanivi (Published in Zootaxa by PhD student Ben Parslow), was found at the peak of Fiji’s highest mountain. These discoveries have highlighted how little is known about the entomofauna of Fiji and how the use of DNA barcoding has helped to uncover Fiji’s hidden secrets of biodiversity.

 

Written by

Cale Matthews

Cale Matthews

School of Biological Sciences, Flinders University, Adelaide, Australia

James Dorey

James Dorey

School of Biological Sciences, Flinders University, Adelaide, Australia

Scott Groom

Scott Groom

School of Agriculture, University of Adelaide, Australia

Olivia Davies

School of Biological Sciences, Flinders University, Adelaide, Australia

Elisha Freedman

Elisha Freedman

School of Biological Sciences, Flinders University, Adelaide, Australia

Justin Holder

School of Biological Sciences, Flinders University, Adelaide, Australia

Ben Parslow

School of Biological Sciences, Flinders University, Adelaide, Australia

Michael Schwarz

School of Biological Sciences, Flinders University, Adelaide, Australia

Mark Stevens

Mark Stevens

School of Biological Sciences, Flinders University, Adelaide, Australia

April 7, 2019
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https://doi.org/10.21083/ibol.v9i1.5482

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