Genomic Diversity Guides Conservation Strategies Among Rare Terrestrial Orchid Species When Taxonomy Remains Uncertain
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Annals of Botany Page 1 of 11 doi:10.1093/aob/mcx022, available online at https://academic.oup.com/aob Genomic diversity guides conservation strategies among rare terrestrial orchid species when taxonomy remains uncertain Collin W. Ahrens1,2,*, Megan A. Supple3, Nicola C. Aitken3, David J. Cantrill1, Justin O. Borevitz3 and Elizabeth A. James1 1Royal Botanic Gardens Victoria, Science Division, Melbourne, Victoria 3004, Australia, 2Hawkesbury Institute for the Environment, Western Sydney University, Locked Bag 1797, Penrith, NSW 2751, Australia and 3Australian National University, Research School of Biology, Centre of Excellence in Plant Energy Biology, Canberra, ACT 0200, Australia *For correspondence. E-mail [email protected] Received: 21 January 2017 Editorial decision: 30 January 2017 Accepted: 12 February 2017 Background and Aims Species are often used as the unit for conservation, but may not be suitable for species complexes where taxa are difficult to distinguish. Under such circumstances, it may be more appropriate to consider species groups or populations as evolutionarily significant units (ESUs). A population genomic approach was em- ployed to investigate the diversity within and among closely related species to create a more robust, lineage-specific conservation strategy for a nationally endangered terrestrial orchid and its relatives from south-eastern Australia. Methods Four putative species were sampled from a total of 16 populations in the Victorian Volcanic Plain (VVP) bioregion and one population of a sub-alpine outgroup in south-eastern Australia. Morphological measure- ments were taken in situ along with leaf material for genotyping by sequencing (GBS) and microsatellite analyses. Key Results Species could not be differentiated using morphological measurements. Microsatellite and GBS markers confirmed the outgroup as distinct, but only GBS markers provided resolution of population genetic struc- ture. The nationally endangered Diuris basaltica was indistinguishable from two related species (D. chryseopsis and D. behrii), while the state-protected D. gregaria showed genomic differentiation. Conclusions Genomic diversity identified among the four Diuris species suggests that conservation of this taxo- nomically complex group will be best served by considering them as one ESU rather than separately aligned with species as currently recognized. This approach will maximize evolutionary potential among all species during in- creased isolation and environmental change. The methods used here can be applied generally to conserve evolution- ary processes for groups where taxonomic uncertainty hinders the use of species as conservation units. Key words: Diuris, genotyping by sequencing (GBS), microsatellite markers, next-generation sequencing, Orchidaceae, population genomics. INTRODUCTION Peterson et al., 2012]. Having thousands of markers gives re- searchers and practitioners the power to uncover fine-scale Conservation of species relies on evidence-based, accurate and structure of a conservation unit regardless of current taxonomic meaningful data collected with consistency and scientific rigour classification. (Tear et al.,2005; Frankham et al., 2012). Conservation strate- Defining species boundaries can be a difficult task (Fazekas gies can be improved greatly with knowledge of genetic varia- et al.,2009; Hausdorf, 2011). Taxonomic uncertainty can lead tion identified through population genetic methodologies (e.g. to negative outcomes for conservation strategies (Frankham Xiao et al.,2006; Martins et al., 2015; Zhang et al.,2015; et al., 2012). Indeed, erroneous classifications can lead to po- Ahrens and James, 2016; Turchetto et al.,2016). Indeed, popu- tential financial, legal, biological and conservation repercus- lation genetics has recently been used as the basis for the devel- sions (Hey et al.,2003). Biological reality suggests that a opment of a conservation prioritization framework for rare species can be difficult to confirm when morphology is influ- plants (Ottewell et al.,2016). The utility of understanding the enced by phenotypic plasticity, hybridization or intraspecific genetic variation within a species can be crucial for prioritizing ploidy variability, among other characteristics (Fazekas et al., certain populations over others (Ahrens and James, 2015; 2009; Fitzpatrick et al., 2015). If maintaining evolutionary po- Dillon et al., 2015). Particularly with the advent of newer geno- tential is a part of the final conservation management goal, cate- mic technologies over the past 5 years, we are able to clarify gorization that is not constrained by species boundaries and and resolve patterns of population structure that were previ- includes genetic diversity metrics may lead to improved conser- ously unattainable. Reduced-representation molecular tech- vation outcomes. In such cases, next-generation sequencing niques are particularly useful in that they allow low-cost techniques have the potential to differentiate conservation units genome typing of hundreds or thousands of individuals [e.g. by identifying genetic lineages that are part of dynamic evolu- genotyping by sequencing (GBS), Elshire et al., 2011; double tionary processes (i.e. contemporary gene flow, mating dynam- digest restriction site-associated DNA sequencing (ddRADseq) ics and environmental resilience; Allendorf et al., 2010; VC The Author 2017. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: [email protected] Page 2 of 11 Ahrens et al.—Diuris population genomics Funk et al., 2012; Hoffmann et al.,2015). The use of evolution- GBS and microsatellite markers to test our hypothesis by ex- arily significant units (ESUs) provides an alternative categori- ploring genetic divergence and interspecific population struc- zation of biological entities (e.g. ecotypes, subspecies or ture at fine scales. The genetic results were compared with populations) and may be suitable as conservation units for man- morphological characteristics to guide the identification of ap- aging closely related species. ESUs represent distinct lineages propriate conservation targets, and we suggest new strategies and take into account genetic interactions among species that for conservation and recovery of this Diuris species complex. influence gene pools but are not constrained by species delimi- tations (Moritz, 1994). Classifying populations that have sub- stantial reproductive isolation is important for maximizing their MATERIALS AND METHODS potential for adapting to future environmental change (Funk Study species et al., 2012). The use of ESUs instead of species classifications has the power to improve conservation strategies by incorporat- Four co-occurring, closely related terrestrial orchid species on ing genetic diversity in addition to biological and morphologi- the Victorian Volcanic Plain (VVP; Fig. 1)wereusedtoexam- cal information. As currently constituted, existing international ine the relationship between current species delineations, mor- conservation policy tends to overlook genetic diversity as an phological differentiation and genetic divergence. The VVP important component of the species (Laikre, 2010). (Fig. 1) is a basalt-derived bioregion in south-eastern Australia Consider the family Orchidaceae, which exhibits highly di- that contains <1 % of its original vegetation due to fragmenta- verse floral morphology and is under pervasive threat from tion and urban development (Williams et al.,2015). Diuris global change (Swarts and Dixon, 2009). This floral diversity is basaltica is a nationally endangered terrestrial orchid that oc- the result of unprecedented genetic diversity that originated curs in only three known locations on the urban outskirts of the early in orchid evolution (Mondragon-Palomino and Theissen, greater Melbourne district. A fourth historical population is 2009). The breadth of floral variation within genera may create now extinct. At the time of sampling, two locations contained the illusion of greater species diversity than is actually present, only 2–5 individuals. The third site supposedly had hundreds of and has led to a history of taxonomic exaggeration (Pillon and individuals, but none flowered during the 2 years of the study. Chase, 2007). It has been found that taxonomically difficult However, an ex situ population, maintained at the Royal groups tend to have more endangered species within them Botanic Gardens Victoria glasshouses, contained several hun- (Pilgrim et al.,2004). Further, species delimitations in the dred individuals cultivated for conservation from plants sourced Orchidaceae remain tenuous in some groups (Flanagan et al., originally in the late 1990s and early 2000s from both the third 2006; Devey et al., 2007; Pillon and Chase, 2007; Bateman population and the fourth now-extinct population. Of the re- et al., 2010; Hedre´n et al.,2012), suggesting that using an ap- maining species, D. gregaria is considered endangered in the proach other than species boundaries may sometimes be war- state of Victoria and is mostly confined there, with some occur- ranted to guide conservation. Under circumstances where rences in South Australia (Fig. 1). It exhibits a clumping habit orchid species are hard to define, understanding patterns of ge- not found in the other species. Diuris chryseopsis and D. behrii netic diversity has been successful for developing conservation have large distributions that extend