diversity

Article Increased Genetic Diversity via Gene Flow Provides Hope for Acacia whibleyana, an Endangered Wattle Facing Extinction

Colette Blyth 1,2,* , Matthew J. Christmas 3,*, Doug C. Bickerton 4, Renate Faast 1, Jasmin G. Packer 1,5, Andrew J. Lowe 1 and Martin F. Breed 2,6

1 School of Biological Sciences, University of Adelaide, North Terrace, SA 5005, Australia; [email protected] (R.F.); [email protected] (J.G.P.); [email protected] (A.J.L.) 2 Group on Earth Observations Biodiversity Observation Network (GEO BON) Genetic Composition Working Group, 04103 Leipzig, Germany; martin.breed@flinders.edu.au 3 Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala 753 41, Sweden 4 Northern Territory Herbarium, Palmerston, NT 0831, Australia; [email protected] 5 Environment Institute, University of Adelaide, North Terrace, SA 5005, Australia 6 College of Science and Engineering, Flinders University, Bedford Park, SA 5042, Australia * Correspondence: [email protected] (C.B.); [email protected] (M.J.C.)



Received: 29 June 2020; Accepted: 24 July 2020; Published: 30 July 2020


Abstract: In this paper we apply a conservation genomics approach to make evidence-based management recommendations for Acacia whibleyana, an endangered shrub endemic to Eyre Peninsula, South Australia. We used population genomic analysis to assess genetic connectivity, diversity, and historical inbreeding across all known stands of the species sampling remnant stands, revegetated stands of unknown origin, and a post-fire seedling cohort. Our results indicate a degree of historical connectivity across the landscape, but habitat loss and/or pollinator community disruption are potential causes of strong genetic structure across the remnant stands. Remnant stands had low genetic diversity and showed evidence of historical inbreeding, but only low levels of intra-stand relatedness indicating that risks of contemporary inbreeding are low. Analysis of a post-fire first generation cohort of seedlings showed they likely resulted from intra-stand matings, resulting in reduced genetic diversity compared to the parents. However, admixed seedlings in this cohort showed an increase in heterozygosity relative to likely sources and the non-admixed seedlings of the same stand. Assisted inter-stand gene flow may prove an effective management strategy to boost heterozygosity and corresponding increases in adapting capacity in this endangered species.

Keywords: conservation genetics; endangered species; genetic diversity

1. Introduction The likelihood that a species is able to adapt to both short-term environmental variability and long-term change is positively correlated with its level of genetic diversity [1]. Small and/or fragmented populations often suffer from restricted gene flow and small effective population sizes, which increase the effects of genetic drift. In small populations, the chances of inbreeding are high, resulting in further reductions in genetic diversity. Genetic drift and inbreeding are therefore potential drivers of the extinction vortex and reduce the adaptive potential of small populations [1–6]. The maintenance and, where possible, enhancement of genetic diversity is therefore a critical aim of conservation genetic strategies and can be achieved through increasing connectivity and gene flow between subpopulations [7].

Diversity 2020, 12, 299; doi:10.3390/d12080299 www.mdpi.com/journal/diversity Diversity 2020, 12, 299 2 of 18

Connecting isolated populations via the translocation of individuals involves an element of risk [7]. Admixture may in fact lead to reduced population fitness due to outbreeding depression, where the introduction of non-local genotypes can disrupt local adaptation and/or lead to the breakdown of adaptive gene complexes [8]. In the case of rare and threatened species, however, the risks of outbreeding depression are often outweighed by the consequences of inbreeding and low adaptive potential if no action is taken [7,9,10]. Indeed, the primary risks for threatened species, especially those that occur in small and fragmented populations, are the deleterious inbreeding and genetic drift effects [9,10]. In such situations, the introduction of non-local genotypes may instead increase a population’s chances of survival due to a genetic rescue effect [11–13]. The development of high throughput sequencing technologies over the last decade has facilitated research into conservation genetics in plant species, with an aim to inform conservation practice on the appropriate management of genomic diversity for improved conservation outcomes [14–16]. Such studies can provide insights into the genetic diversity within plant populations, and the historical gene flow between populations, as well as identifying where genetic breaks may occur [16]. This information can be used to guide when and how to enhance the genetic diversity of an endangered species while minimising the risks associated with moving genetic material across large genetic breaks in a species range, such as outbreeding depression [16]. As a result of such studies, there have been increasing calls for a paradigm shift in conservation management thinking—away from the separate management of isolated populations and towards a strategy that seeks to increase connectivity and diversity across the range of a species in the hope it will lead to genetic rescue [13,17]. First described in the 1990s, the endangered Acacia whibleyana (family Fabaceae) is restricted to the Tumby Bay area of the Eyre Peninsula, South Australia. There have been significant investments to aid its conservation, and it is part of the Australian Government’s 30 by 2020 Threatened Species Strategy [18]. Land clearing has limited its suitable habitat, and ongoing low seed set and lack of natural recruitment, as well as competition from weeds, are some of its key threatening processes [19–21]. One of the priority conservation actions for A. whibleyana is to establish new translocated populations [19–21]. Conservation management decisions should ideally be underpinned by a sound understanding of its genetic structure. However, to date, there has been no published research on the levels of genetic variation and structure within this species. Here, we address this knowledge gap by using high throughput sequencing to construct a high-resolution picture of the population genetic structure and diversity in A. whibleyana. We generated a genome-wide single nucleotide polymorphism (SNP) dataset for the last remaining stands of A. whibleyana, including all remnant and revegetated stands, plus a cohort of seedlings that recruited post-fire. We used this population genomic information to provide practical conservation management recommendations for the conservation of this species.

2. Materials and Methods

2.1. Study Species and Sampling Acacia whibleyana is a narrow-ranged endemic shrub that is restricted to five subpopulations near Tumby Bay, Eyre Peninsula, South Australia (Figure1). The species is not known to hybridise [21]. Diversity 2020, 12, 299 3 of 18 Diversity 2020, 12, x FOR PEER REVIEW 3 of 20

Figure 1. Map of collection stands. The Southerns and Marshalls Rd locations had collections of more thanthan oneone statusstatus (i.e.,(i.e., aa combinationcombination ofof remnant,remnant, revegetatedrevegetated andand/or/or seedlingseedling plants).plants). The figurefigure waswas generated using Quantum Geographic InformationInformation System (Quantum GIS Development Team, 2016, Open sourcesource geospatial geospatial foundation foundation project, project, http: http://qgis.osgeo.org//qgis.osgeo.org). Map). Map layer layer was downloaded was downloaded from https:from //https://www.divawww.diva-gis.org-gis.org/Data/Data. .

Acacias have have an an important important nitrogen nitrogen fixation fixation and and nutrient nutrient cycling cycling role in role many in manyecosystems, ecosystems, which iswhich of particular is of particular ecological ecological importance importance in the predominantly in the predominantly arid, nutrient arid, poor nutrient soils of Australia poor soils [22 of]. ItAustralia produces [22]. flowers It produces between Augustflowers and betw Octobereen August [23], with and fruits October maturing [23], with from fruits December maturing to January. from ItDecember has four to January. six seeds It per has pod, four and to six seed seeds dispersal per pod, is believedand seed todispersal be assisted is believed by ants to [23 be]. assisted Like other by wattleants [23]. species, Like the other seeds wattle of A. species, whibleyana the have seeds a ofhard A. seed-coat whibleyana dormancy have a hard mechanism seed-coat that dormancy requires disturbancemechanism that to scarify requires the disturbance seed and trigger to scarify germination. the seed and trigger germination. A species census census in in April April 2019 2019 recorded recorded ~1800 ~1800 individuals individuals across across all known all known subpopulations. subpopulations. The Themaximum maximum geographic geographic distance distance between between subpopulations subpopulations was was 38 km. 38 km. The The total total area area of o ofccupancy occupancy of ofAcaciaAcacia whibleyana whibleyana waswas 14.2 14.2 ha (0.142 ha (0.142 km2) km based2) based on site on-validated site-validated data of data each of remnant each remnant stand. stand.Many Manyplants plants were restricted were restricted to remnant to remnant vegetation vegetation on roadside on roadside verges, with verges, some with found some on foundprivately on- privately-ownedowned land which land is grazed which by is livestock grazed by [21 livestock] (Table A1 [21 in] (TableAppendix A1 Ain). AppendixOne of theA existing). One of stands the existing(Marshalls stands Rd) (Marshalls was established Rd) was in established 2004 through in 2004 two through separate two translocations, separate translocations, with plants with from plants the fromQuarry the stand. Quarry The stand. oldest The Marshalls oldest Marshalls Rd stand Rd was stand experimentally was experimentally burned in burned October in October2018 to explore 2018 to explorethe effect the of e fffireect on of recruitment. fire on recruitment. Initially,Initially, wewe collectedcollected A.A. whibleyanawhibleyana samplessamples inin latelate 2017.2017. SamplingSampling was rangewide and included 5 remnantremnant standsstands andand aa revegetatedrevegetated stand.stand. On average, we aimed to collect 20 individualsindividuals per standstand wherewhere possiblepossible toto allowallow forfor extractionextraction andand genotypinggenotyping failures,failures, butbut thisthis variedvaried dependingdepending onon censuscensus sizesize and and accessibility accessibility (Table (Table A1 A1)). We. W targetede targeted young young leaves leaves to maximise to maximise DNA quality,DNA qualit and oncey, and picked, once leavespicked, were leaves placed were in placed gauze in bags gauze and bags desiccated and desiccated on silica on gel si untillica gel DNA until extraction. DNA extraction. InIn MayMay 2018,2018, aa prescribedprescribed burnburn ofof thethe revegetatedrevegetated MarshallsMarshalls RdRd standstand waswas donedone toto encourageencourage natural regeneration. regeneration. An An additional additional 46 46 seedlings seedlings were were collected in October 2018 post-burnpost-burn for for genotyping. Seedling leaves were placed directly into DNA extraction plates for processing of thethe freshfresh leafleaf material.material. In February 2019, an additional sampling trip took place, at which time samples were collected from both a newly-discovered stand of A. whibleyana within the Salt Lake subpopulation and from a known stand previously not sampled. Samples were also collected from a site on the western edge of the Quarry subpopulation referred to as Southerns.

Diversity 2020, 12, 299 4 of 18

In February 2019, an additional sampling trip took place, at which time samples were collected from both a newly-discovered stand of A. whibleyana within the Salt Lake subpopulation and from a known stand previously not sampled. Samples were also collected from a site on the western edge of the Quarry subpopulation referred to as Southerns.

2.2. DNA Extraction, Sequencing and Filtering DNA extraction, library preparation, and sequencing were done at the Australian Genome Research Facility. We used the Machery-Nagel Nucleospin Plant II Kit for DNA extraction. Double digest restriction-associated DNA sequencing (ddRADseq; [24] was used to generate reduced representations of the genome for SNP calling across all samples. Full details of the method can be found in Peterson, Weber [24]. Briefly, ddRAD library preparation protocol involved the following steps: (1) DNA digestion with two restriction enzymes (Pstl and MseI); (2) ligation of barcoded adaptors, specific to each sample, to restriction site overhangs; (3) a wide size selection of pooled digested-ligated fragments 280–342bp using Blue Pippin (Sage Science, Beverly, MA, USA) (Wide); (4) amplification of library via polymerase chain reaction (PCR) for 11 cycles using indexed primers. The libraries were assessed by gel electrophoresis (Agilent D1000 ScreenTape Assay), quantified by quantitative polymerase chain reaction (qPCR; KAPA Library Quantification Kits for Illumina) and then sequenced with 150 bp single reads on the NextSeq 500 system using NextSeq 500 high Output Kit v2 (150 cycles) reagents. Following sequencing, reads were processed with the STACKS pipeline [25,26] at Australian Genome Research Facility(AGRF; Melbourne, Australia). Briefly, reads were deconvoluted by inline barcodes, checked for read quality, and for the presence of a restriction site, creating per-sample FASTQ files. These FASTQ files were then trimmed to the size of the shortest read minus 2 bp to compensate for differences in read length due to any variation in barcode length. After trimming, stacks of similar reads were created for each sample individually; these read stacks are also known as tags. Tags which appear across all samples were collated (catalogue tags), and genotypes were then allocated to the common polymorphic sites. The collated SNPs across all individuals were then filtered with the following settings: minimum number of reads required at a stack to call a homozygous genotype = 5; minor allele frequency, below which a stack is called a homozygote = 0.05; minimum minor allele frequency to call a heterozygote = 0.1 (above 0.05 but below 0.1, a stack is called ‘unknown’). Computed genotypes were then exported as a single .vcf file for further filtering and analysis. We used VCFtools [27] to remove SNPs that had a minimum depth of coverage of <10 reads per individual or a minor allele frequency of <0.1. VCFtools was also used to assess missing data, removing SNPs if they had >25% missing data across all individuals and removing individuals if they had >25% missing SNP calls. The –indep-pairwise command in PLINK [28] was then used to identify SNPs in linkage disequilibrium, whereby one SNP in a pair of linked SNPs (r2 > 0.8) was pruned from the data set. Inbreeding coefficient (F) values were estimated using the G-stats calculator in GENODIVE v. 3.2 [29] for each SNP across all samples, and SNPs with significantly negative F removed using VCFtools (significance assessed using permutation tests with 10,000 permutations and a significance threshold of p < 0.05). Negative F indicates greater than expected heterozygosity under Hardy-Weinberg equilibrium, which may be indicative of paralogous reads being stacked together in the STACKS pipeline [30].

2.3. SNP Calling We generated 467,346,192 sequences across the 237 A. whibleyana samples in our study. A total of 281,779 SNPs were called after read alignment. After filtering on depth of coverage, minimum minor allele frequency, percentage of missing data, linkage disequilibrium, 16,776 SNPs across 228 individuals remained in our dataset (see Table 3 for final sample sizes). Computed genotypes were then exported as a single .vcf file for further filtering and analysis (Supplementary file S1). The final, filtered SNP set was then used to investigate population genetic structure, diversity, and inbreeding. Diversity 2020, 12, 299 5 of 18

2.4. Genetic Structure Analysis

We estimated pairwise genetic differentiation (FST) between all 11 stands (including the remnant, revegetated, translocated, and the seedling cohort) in GENODIVE v3.2 [29]. We also used two different genetic clustering analyses to identify the most likely number of genetic clusters (K) in our dataset. Firstly, we used the non-model based Discriminant Analysis of Principal Components (DAPC) using the R package adegenet [31]. DAPC is a multivariate approach that is not based on population genetic models, but rather seeks discriminating functions between groups of individuals while minimising variation within clusters. Genetic data were first transformed into uncorrelated components using principal component analysis (PCA). The number of genetic clusters was then defined using k-means, a clustering algorithm that looks for the value of K that maximises the variation between groups. The Bayesian Information Criterion (BIC) was calculated for K = 1 to 20, and the K value with the lowest BIC was selected as the optimal number of clusters. A discriminant analysis was then performed on the first 80 principal components using the function dapc, implemented in R, in order to efficiently describe the genetic clusters and assign samples to each cluster. The model-based genetic clustering algorithm ADMIXTURE [32] was also used to estimate the most likely number of clusters (K) in our dataset. ADMIXTURE includes a cross-validation procedure for identifying the value of K for which the model has best predictive accuracy (see ADMIXTURE manual for full details of model and cross validation procedure). ADMIXTURE was also run for K values 1 to 10 and the most likely value of K was assessed by comparing the cross-validation errors (cv errors) between runs, with the lowest cv error indicating greatest support. We repeated each ADMIXTURE analysis ten times and used CLUMPAK to combine the results and construct bar plots of individual assignment to clusters for the most supported values of K using DISTRUCT [33,34]. GENODIVE v3.2 [29] was used to run a nested analysis of molecular variance (AMOVA) to explore genetic variation within individuals, among individuals nested within collection stand, and among collection stands.

2.5. Genetic Diversity and Inbreeding Analysis

We used GENODIVE to estimate the inbreeding coefficient (F), and observed (HO) and expected (HE) heterozygosity. Wealso ran these analyses on only Marshalls Rd and the Marshalls Rd seedlings, comparing admixed and non-admixed individuals (as identified from the DAPC and ADMIXTURE analyses) in the seedling cohort. In order to compare levels of heterozygosity in the Marshalls Rd seedlings that were identified as admixed and non-admixed (see results), we calculated individual heterozygosity using the -het flag in VCFTools. These values were then imported into R for plotting using the ggplot2 package [35]. To further explore historical inbreeding and future risks of inbreeding, we estimated pairwise kinship coefficients in GENODIVE. Kinship varies between 0 and 1, where 0 indicates no genetic evidence of recent co-ancestry and 1 indicates samples are genetically identical (i.e., clones).

2.6. Redundancy Analysis All stands were located in close proximity to each other (the maximum distance between any two stands was 38 km). We ran a redundancy analysis (RDA) on the allele frequencies of all SNPs in order to assess how much of the genetic variation among our populations can be explained by spatial factors (i.e., resulting from isolation by distance) using the VEGAN analytical package v2.5-6 in R (https://cran.r-project.org/web/packages/vegan/). First, spatial coordinates for each sampling stand were centred and converted into third degree polynomials. An RDA of allele frequencies~all spatial polynomials was then performed using the ‘rda’ function. Analysis of variance (ANOVA) was used to assess the significance of the RDA using the ‘anova.cca’ function. Value of BIC versus number of clusters Diversity10 2020, 12, 299 6 of 18 10 1520 10

3. Results1510 Value of BIC BIC versusValue number of BIC of clusters 10versus numberValue of ofclusters BIC 10 versus numberValue of of BIC clusters versus number of clusters 3.1. Genetic1500 1010 10 Structure Analysis Collection Stand

10 1520 10 10 10 1520

1520 Collection Stand 10 10 10 Collection Stand 1520 10 5 1490 10 10 10 Foothills Both the1510 DAPC and ADMIXTURE analyses indicated significant genetic structure in our data

BIC Foothills 1510 5 1510 5 5 10 15 20 Foothills 1510 BIC BIC 10 Marshalls Rd (Figure2). TheBIC BICNumbervalues of10 clusters10 from DAPC10 revealed similar levels of support for K = 3, 4, or 5. 1500 10Value of BIC 10 Marshalls RdCollectionMarshalls Stand Rd 1500 versus number of clusters

1500 Collection Stand 1500 Value of BIC CollectionCollection Stand StandMarshalls Rd seedlings Cross validation error values from the ADMIXTURE10 analysis showed greatestCollectionCollection Stand support Stand Collection for K-valuesStand of 10 10versus5 number10 of clusters CollectionCollection Stand StandMarshallsCollection Rd seedlingsStandMarshallsFoothills Rd seedlings 1490 10 10 Value of BIC 10 10 CollectionFoothills StandCollectionCollection Stand Stand 5 1490 1010 10 Foothills Collection StandFoothills 1490 5 10 10 5 versus1490 number of clusters 10 1010 FoothillsFoothillsFoothillsMoonlight Bay 5–7.(a) However,55 5 beyond5 K1010=10 5 the15 ADMIXTURE20 (b) results appeared noisy and clustering closelyFoothillsCollection matched Stand

1520 Foothills Marshalls Rd 5 5 5 10 15 20 10 Foothills FoothillsCollectionMarshalls StandMoonlight Rd Bay 10 5 5 105 105 10 15 15 20 20 FoothillsMarshallsMoonlight Rd Foothills Bay 5 Number5 5of clusters 10 Marshalls Rd Foothills Collection Stand 10 ValueNumber of BIC of clusters 10 Marshalls Rd 1520 10 Marshalls Rd Collection Stand Number10 Value Numberof clusters of of BICclusters 105 10 MarshallsMarshalls MtRdCollection RdLiverpoolMarshalls StandMarshallsFoothills FoothillsRd Rd seedlings individual source population5 versusValue number10 ofValue BIC of of clusters BIC for K values below six. CollectionCollectionMarshalls Stand RdStandMarshalls Rd seedlings 10 10 10versus10 numberValue ofof BICclusters MarshallsMarshallsMarshalls Rd Rd RdMarshalls seedlingsMarshalls Rd RdMt Liverpool versus versusnumber number of Valueclusters of ofclusters BIC Marshalls MtRd LiverpoolseedlingsCollection Stand 1520 10 Collection StandMarshalls Rd 1510 versusValue number Valueof BIC of of clusters BIC 10 10 2) MarshallsMarshallsCollection Rd seedlingsRd Stand seedlingsFoothillsMoonlight BayFoothills 10 10 5 105 versus number of clusters 1010 MarshallsCollection Rd StandMarshallsFoothills seedlingsMarshallsCollection Rd seedlings StandRd seedlings BIC Valueversus of BIC10versus5 number number of clusters of clusters 10 10 Marshalls QuarryCollectionRd seedlingsFoothills Stand Marshalls Rd 10 10 Value105 of10 BIC5 105 10 MarshallsMarshalls Rd seedlingsRdFoothillsMarshalls seedlingsMarshallsFoothillsMtMarshallsFoothillsCollection Rd Liverpool seedlings RdQuarryMoonlight Rd seedlingsseedlings Stand Bay 0 10 1510 Value of BIC 105 Foothills (a) 1010 10 versusversusValue number ofnumber BIC of clusters of clusters (b) 5 MoonlightMoonlight FoothillsBay Bay (a) 10 0versus number of clusters (b) 5 CollectionFoothillsCollection StandFoothills Stand

1520 Moonlight Bay BIC 10 5 Quarry Marshalls RdMarshalls Rd 1520 5 CollectionMarshalls StandMoonlight Rd Bay 10 10(a) versus number of clusters (b)1010 MoonlightCollectionCollectionMarshalls BayMarshalls StandMoonlight Rd Rd StandMarshalls Bay Rd 0 (a) 10 10 1520 1010 (b) MoonlightMoonlightCollection Bay BayCollection StandMoonlightMarshallsCollectionCollectionQuarryMoonlight RdBay Stand StandStand Bay 1520 10 MarshallsCollection Rd Marshalls Stand Rd 1510 10 10 10 0 Moonlight BayCollection StandMoonlight BayMarshalls Rd seedlings 1500 10 SaltMarshalls Lake Rd remnantCollectionMarshallsMt Rd Liverpool Stand 1520 10 1520 Collection StandFoothills 1010 Value5 of10 BIC 10510 MtMoonlight MtLiverpool LiverpoolMarshallsFoothills BayMarshallsFoothillsCollection Rd seedlings Rd seedlings StandSalt Lake remnant BIC 1520 5 FoothillsFoothills 10 5105 105 Mt LiverpoolCollectionMarshallsFoothills RdMtMarshalls seedlingsStand LiverpoolCollectionMarshallsFoothillsMtMarshalls LiverpoolRd Standseedlings Rd Rd seedlings 10 10 1520 10 FoothillsMt LiverpoolSalt Lake remnant (A) 10 versus number of clusters105 (B) 510 Mt LiverpoolCollectionMarshallsFoothills StandMtMarshalls Liverpool Rd seedlings Rd seedlingsMarshalls Rd seedlings 1500 Value of BIC 5 Mt MtLiverpool LiverpoolSaltCollection Lake StandFoothills remnantMarshalls Rd seedlings

1520 Marshalls Rd

1520 Marshalls Rd

1510 Moonlight BayFoothillsMarshalls Rd seedlings 10 1510 105 10 2)2) Mt LiverpoolCollection StandMoonlightMt Bay FoothillsLiverpool 1520 5 5 10 Marshalls Rd 1510 2) CollectionCollectionMoonlightCollection BayStandFoothills Stand StandMoonlightMoonlight Bay Bay BIC versus number of clusters5 10 Collection Stand Quarry Quarry BIC Mt LiverpoolCollectionCollection Stand StandSaltMarshallsFoothills LakeMarshalls FoothillsrevegetationRd Rd 1520 Quarry MarshallsMarshallsCollection MarshallsRd Rd Stand SaltRd Lake revegetation 1510 5 QuarrySaltMoonlight LakeMoonlight Bay FoothillsrevegCollection Bay Stand BIC 05 100 105 2) Quarry MtMarshallsFoothillsCollection LiverpoolMarshallsFoothillsCollectionMarshallsQuarry Rd seedlingsMoonlight Rd Standseedlings Rd Stand Bay 10 0 1510 5 105 Mt LiverpoolQuarry 0 1510 FoothillsCollectionCollectionMoonlight Stand Stand MoonlightBay Bay 1500 5 MarshallsQuarry Rd seedlings BIC

1490 10 Mt Liverpool 0 1510 0 0 Quarry FoothillsFoothillsMarshalls RdMoonlight Bay BIC 0 5 Quarry FoothillsFoothillsMarshallsMoonlightMt Liverpool Rd Bay BIC 5 QuarryCollectionFoothillsMarshallsCollectionCollection Rd MarshallsseedlingsStandSalt StandLake Stand remnant Rd PC2 (3.3% explained var.) PC2 (3.3% explained 5 Quarry

BIC 10 MarshallsMt Liverpool Rd MarshallsMarshalls RdRd seedlings 5 0 0 1510 5 Quarry CollectionSaltMoonlightMarshalls StandLake BayMarshalls Rd revegSebs Rd Patch 10 0 0 105 Quarry CollectionMarshallsCollectionQuarry StandQuarryMoonlight Rd Stand BayFoothills 1510 0 CollectionMoonlight StandFoothills BayMarshalls Rd seedlingsMt Liverpool 0 1500 Foothills Quarry Mt LiverpoolCollectionMt Liverpool Stand 1510 BIC 5 Salt Lake remnantCollectionMarshalls RdMarshalls Stand SaltRd seedlings Lake remnant PC2 (3.3% explained var.) PC2 (3.3% explained 0 Quarry Marshalls Rd 1500 Foothills 1510 Value of BIC MarshallsSalt Lake Rd remnant seedlings PC2 (3.3% explained var) PC2 (3.3% explained 0 Mt LiverpoolMarshallsMarshalls RdRd seedlings 1490 1500 Salt Lake remnantCollectionMtMarshalls LiverpoolFoothills Rd StandSebs Patch 5 Salt LakeSebs PatchremnantFoothillsCollectionMarshallsMarshalls Rd SaltRd Stand Lake remnant BIC Value of BIC 1520 10 Value of BIC CollectionMoonlightFoothillsSaltFoothills Stand Lake Bay remnantFoothills 0 5 10 5 15 20 Mt Liverpool SebsMarshallsQuarry PatchRd seedlingsQuarry 5 BIC 5 Marshalls Rd Collection StandCollectionFoothillsMt Liverpool Stand Salt Lake revegetation BIC Value5 ofversus BIC number of clusters 10 Salt LakeMarshalls remnant Rd seedlingsSouthernsMoonlightFoothillsMarshalls BayRdremnant seedlings 5 1510 Marshalls Rd seedlingsMarshallsMarshalls Rd Rd seedlings seedlings 10 500 5 CollectionCollection StandMt Liverpool Stand Moonlight Bay 1500 CollectionCollection StandCollection FoothillsMtStand Liverpool SaltFoothillsStand LakeMt remnant LiverpoolMarshalls Rd versus number of clusters Salt Lake remnant Salt Lake reveg 1500 versusValue number of BIC of clusters 55 SaltSalt Lake Lake remnant MarshallsremnantCollectionMarshallsMarshalls RdSaltFoothills seedlings RdMarshalls Lake RdStand remnant seedlings MtRd seedlingsLiverpool Value of BIC Southerns reveg

1500 Salt Lake remnant Quarry 1500 Value of BIC Salt Lake remnant

PC2 (3.3% explained var) PC2 (3.3% explained 5 Value of BIC BIC MarshallsMarshalls Rd seedlings versus number of clusters MarshallsSalt Lake remnant Rd seedlings

1520 MoonlightMarshallsMoonlight Bay Rd seedlings Bay Number of versusclusters number of clusters 0 Southerns remnant CollectionCollectionCollection Stand Stand SebsCollectionStandMarshallsQuarryFoothillsSaltFoothillsMt Liverpool QuarryLake PatchRd Stand remnant 1490 versus number of clusters CollectionSalt Lake StandMoonlightSaltMarshalls Foothillsrevegetation MoonlightLakeMarshallsSalt Bay revegetationRd Lake Bayremnant Rd seedlings 5 5 versusversus number10 5 number of clusters of clusters15 20 var.) PC2 (3.3% explained 5 05 CollectionCollectionCollectionSalt LakeFoothills Stand StandrevegMarshallsQuarryMoonlightStandFoothillsCollectionQuarryMoonlightQuarry RdBay Quarry Bay StandMarshallsSebs PatchQuarry Rd 10 1500 10 Value 0of BIC 0505 SaltCollectionSaltSalt Lake Lake CollectionLake revegSaltCollectionCollection Moonlight remnantLake revegStand StandSaltMarshallsCollectionFoothillsMarshallsSaltMarshalls Foothillsrevegetation Lake BayStand LakeSaltMarshalls FoothillsrevegetationRd RdStandSouthernsMtMarshalls Moonlightseedlings LakerevegetationRd Liverpool Stand SouthernsMoonlightFoothillsSaltrevegetationRd Marshalls RdBayrevegetation seedlings Lake revegetation BayRdremnant seedlings Marshalls Rd seedlings 1500 0 5 05 5 10 10 1490 5 0 −5 MoonlightMoonlight BayMoonlight Bay BayMarshalls Rd 1500 Foothills MarshallsMoonlight RdSalt Lake Bay remnant 1500 1490 Collection StandMt LiverpoolMoonlight Bay 1490 versus number of clusters CollectionCollection StandSalt LakeQuarryMarshalls Stand remnantMoonlightQuarry RdBay 5 var.) PC2 (3.3% explained FoothillsCollectionSaltFoothills Lake SouthernsrevegCollectionMtSebsMarshallsMarshalls Liverpool StandSaltMarshallsFoothills Patch Lake RdRd Stand seedlingsSaltMarshalls FoothillsrevegetationRd Moonlight remnant Lake revegetationRd Bay Value of BIC 0510 Quarry 3) CollectionCollectionSalt Stand LakeCollection StandSaltFoothills revegLakeCollection StandMt remnantLiverpool Stand 10 10 Number of clusters var.) PC2 (3.3% explained SaltCollection Lake revegSebsMarshallsMarshallsCollection PatchStandSalt RdRd Lake seedlings Stand remnant Quarry 5 05 var.) PC2 (3.3% explained 5 FoothillsMarshallsMarshallsMt Liverpool RdRd seedlingsSalt Lake revegetation Foothills Salt Lake reveg 105 10 10 Quarry Collection Stand FoothillsCollectionMt LiverpoolSebsMoonlightSalt Patch Lake Bay StandMarshallsSoutherns remnant Rd remnant seedlings 10 var.) PC2 (3.3% explained 10 Quarry Foothills QuarryMt Liverpool

1500 5 1510 10 Collection Stand Mt Liverpool PC2 (3.3% explained var) PC2 (3.3% explained 5 10 15 20 MarshallsMarshalls RdRd seedlings

1490 Foothills Sebs Patch 5 10 1490 0 5 CollectionMt Liverpool StandMoonlight Bay Sebs PatchFoothills Salt LakeMtMoonlight Liverpool remnant Bay PC2 (3.3% explained var.) PC2 (3.3% explained Quarry Foothills Marshalls Rd seedlings 1520 0 Foothills CollectionMtFoothills Liverpool Stand PC2 (3.3% explained var) PC2 (3.3% explained 10 MarshallsMarshalls RdRd seedlings versus number of clusters Mt Liverpool 1490 SebsFoothills PatchQuarry SebsSalt Patch Lake remnant 5 var.) PC2 (3.3% explained 10 15 20 Marshalls Rd Sebs PatchFoothills Salt LakeMt Liverpool revegetation PC2 (3.3% explained var) PC2 (3.3% explained 5 CollectionCollectionFoothills Stand StandMarshallsMarshalls RdRd seedlings 1490 Value of BIC 1520 MoonlightMarshallsSaltMarshalls Lake BayRd seedlings remnantSebsSalt PatchRd LakeSalt revegetation Lake remnant PC2 (3.3% explained var.) PC2 (3.3% explained 105 var.) PC2 (3.3% explained 10 FoothillsFoothillsSouthernsSouthernsFoothills remnant remnant 5 Mt Liverpool 5 10 5 10 15 20 50 CollectionSebsSebs PatchSalt FoothillsStand Patch LakeQuarry revegSebsMarshallsMarshallsMoonlight Patch Bay RdRd seedlings Marshalls Rd Marshalls Rd 1520 BIC 5 Salt Lake revegetationFoothills Sebs Patch 10 FoothillsSalt LakeFoothills reveg Mt LiverpoolMarshalls Rd 10 5 10Value5 of BIC 15 20 1010 Quarry Mt LiverpoolCollectionSebs Stand PatchSouthernsMoonlightMarshallsFoothillsQuarry BayRdremnant seedlings Moonlight Bay 5 Marshalls Rd 0 Collection Stand Moonlight Bay Mt Liverpool 10 105 10 5 5 Southerns reveg MarshallsMarshallsFoothillsSalt Rd LakeRd revegetation Southerns revegetation Value of BIC SebsSalt Patch Lake reveg QuarryMoonlightMarshallsMt BayRd Liverpool seedlings 5 var.) PC2 (3.3% explained 10 SouthernsMt Liverpool remnant Foothills

PC2 (3.3% explained var) PC2 (3.3% explained 5 10 0 CollectionMarshalls Stand Rd Mt LiverpoolMoonlight Bay versus number of clusters 5 Salt Lake remnant CollectionSebs Stand Patch

1510 Southerns reveg MarshallsSalt Rd Lake remnantMarshalls Rd seedlings Mt Liverpool PC2 (3.3% explained var.) PC2 (3.3% explained 1520 QuarrySalt Lake reveg MtSoutherns Liverpool revegetation

Bayesian information criterion criterion information Bayesian 0 Moonlight Bay Marshalls Rd Southerns remnant

PC2 (3.3% explained var) PC2 (3.3% explained Number of clusters MarshallsMarshalls RdRd seedlings PC2 (3.3% explained var) PC2 (3.3% explained 1490 MarshallsFoothills Rd SebsSaltQuarry Patch Lake remnant SebsSalt Lake remnant Patch versusNumber number of clusters of clusters Southerns reveg Collection Stand 1490 Sebs PatchFoothills 1520 MarshallsSalt LakeQuarry remnant Rd seedlings PC2 (3.3% explained var.) PC2 (3.3% explained Moonlight Bay

1520 Quarry 5 10 15 20 0 Southerns remnant FoothillsMt Liverpool PC2 (3.3% explained var) PC2 (3.3% explained 10 MarshallsFoothillsFoothillsSebs Rd Patch −5 Mt Liverpool Salt Lake remnant Marshalls Rd 1520 5 5 versus number10Number of of clusters clusters 15 20 FoothillsSebsSebs Patch PatchQuarryMarshalls Rd seedlings 1490 −50 MtMoonlight LiverpoolSalt Bay LakeSalt Lake reveg revegetation Marshalls Rd 1520 SouthernsSaltSoutherns LakeSalt remnant FoothillsLake revegetation revegetation reveg 5 var.) PC2 (3.3% explained FoothillsQuarry Salt Lake remnant 5 10 15 20 0 Southerns remnant Value5 10 of BIC var.) PC2 (3.3% explained 10 FoothillsSebsMarshalls PatchMoonlightMarshallsSouthernsMoonlight MarshallsRd seedlings BayRd seedlings BayRdremnant seedlings Marshalls Rd seedlings MarshallsSoutherns Rd remnant BIC 5 5 Southerns reveg FoothillsSebs Patch Number of clusters 0 Collection StandMtMoonlight LiverpoolSalt Bay LakeMoonlight Bay revegetationSebs Patch 105 1490 50 5 SouthernsMarshallsSaltFoothills revegetation Lake Rd remnant 5 10 10 15 20 var.) PC2 (3.3% explained −5 MarshallsSalt Lake reveg SouthernsMoonlight MarshallsRdMarshalls seedlingsQuarryMt Liverpool BayRd remnantRd seedlings 10 Value of BIC Marshalls Rd seedlings Marshalls Rd Sebs Patch 5 5 0 FoothillsMarshallsSaltSalt LakeMtMarshalls MoonlightLake remnant Liverpool Rd remnantSalt RdBay Lake revegetation 5 Southerns reveg SouthernsMarshalls remnant RdSouthernsMarshallsFoothills revegetation Rd 10 −5 Southerns remnant Marshalls RdSalt LakeMarshallsQuarry remnantMoonlight MarshallsRd seedlings BayRd seedlings

PC2 (3.3% explained var) PC2 (3.3% explained Southerns remnant Marshalls Rd seedlings Marshalls Rd −5 5 versus number0 Value of of clusters BIC 3) MarshallsSalt Lake remnant Rd QuarrySoutherns remnant 10 1520 5 Number of clusters Southerns remnant Collection Stand Number ofValue clusters of BIC −50 Southerns3) remnant FoothillsSebsMarshallsFoothills PatchMoonlightSouthernsSalt RdLakeSalt revegetation remnant LakeMarshalls remnantBaySaltMtSalt Liverpool LakeLake Salt Rd revegetationremnant Lake revegetation 1490 10 Number of clusters MarshallsQuarryMarshallsMt LiverpoolSalt Lake RdQuarry seedlings remnant

PC2 (3.3% explained var.) PC2 (3.3% explained Foothills 5 5 1510 versus5 number5 of10 clusters010 15 15 20 20 FoothillsFoothillsSouthernsMarshallsSouthernsSaltMoonlight Lake MarshallsSebsremnant revegetationRd remnant BayPatch seedlingsQuarry Rd revegQuarry RdQuarryMarshallsSoutherns Mtseedlings Liverpool Rd remnant seedlings Value of BIC 5 Marshalls Rd seedlings Quarry Marshalls RdQuarryMt Liverpool seedlingsSouthernsMtMoonlight LiverpoolSalt Bayrevegetation Lake revegetation 1510 5 versus10 numberNumber ofof clusters clusters15 20 0 3) FoothillsMarshallsQuarry Rd seedlings 1500 105 10 0 MoonlightMarshalls Bay SouthernsMoonlight Rd seedlings Bayremnant 0 Marshalls Rd seedlings Salt Lake revegetation Quarry FoothillsMarshallsMarshalls Rd Saltseedlings Rd LakeSalt revegetation Lake remnant 10 5 var.) PC2 (3.3% explained versus50 number of clusters 0 Foothills SouthernsMarshalls remnantQuarryMarshallsSouthernsMt Liverpool RdMarshalls Rd remnantseedlings seedlingsSoutherns RdMoonlight remnant Bay BIC −5 Sebs Patch 1510 5 10 15 20 MarshallsMarshallsSalt Rd LakeSaltMarshalls revegetation Lake remnant Rd 5 10 0 var.) PC2 (3.3% explained 0 Foothills MarshallsSoutherns Rd remnantMoonlightSaltSouthernsMtMoonlight Lake Liverpool Bay remnant Bayrevegetation Quarry Marshalls Rd BIC Sebs Patch Marshalls RdSebs seedlings Patch −5 versus number of clusters var.) PC2 (3.3% explained 0 SouthernsSebs remnant PatchMtMarshallsMoonlight Liverpool RdBay

1510 Marshalls Rd seedlings 10 Mt Liverpool Marshalls Rd Southerns revegetation

0.36 Moonlight Bay Sebs Patch Southerns revegetation Salt Lake revegetation PC2 (3.3% explained var.) PC2 (3.3% explained Southerns revegetation Value of BIC Marshalls Rd MarshallsMoonlightSalt BayRd LakeSalt revegetation Lake remnant PC2 (3.3% explained var.) PC2 (3.3% explained Number ofNumber clusters of clusters 3) Foothills SouthernsCollectionSebsSaltQuarry Patch Lakeremnant Stand remnantQuarry 10 var.) PC2 (3.3% explained

1510 Marshalls Rd seedlings

1520 Moonlight Bay Mt Liverpool Salt Lake revegetationQuarryFoothills

BIC Marshalls Rd seedlingsSebs Patch Foothills Southerns revegetationMoonlight Bay Number− of5 clusters Marshalls Rd CollectionSaltMoonlightQuarryMt LakeLiverpoolSoutherns remnantBay StandMarshalls remnantSebsSalt Lake Patch Rd revegetation 1520 MarshallsCollection RdMarshalls Stand seedlings MtRdMoonlight seedlings Liverpool Bay 10 5 −50 MarshallsSoutherns SaltRd Lakereveg reveg QuarryMt LiverpoolSouthernsMarshalls Rdrevegetation 1510 Marshalls Rd seedlingsSalt LakeSalt Lake remnant remnant 5 10 15 20 Sebs Patch Mt LiverpoolMoonlight Bay 1500 PC2 (3.3% explained var.) PC2 (3.3% explained 0−5 var.) PC2 (3.3% explained −05 BIC 5 1520 Southerns reveg Marshalls SebsRdMoonlight Patchseedlings Bay −50 Quarry Salt Lake reveg Sebs Patch

1510 Southerns reveg Marshalls Rd seedlings 0 Quarry Mt Liverpool Marshalls Rd seedlingsSalt Lake remnant PC2 (3.3% explained var.) PC2 (3.3% explained MarshallsSoutherns Rd revegetationseedlings 10 Valueversus of numberBIC of clusters Marshalls1) Rd Moonlight Bay BIC Number of clustersValue of BIC Southerns reveg Salt Lake remnant SebsSalt QuarryPatch Lake remnant 10 var.) PC2 (3.3% explained Marshalls MarshallsRd CollectionSalt Rd Lake StandSalt revegetation LakeSoutherns remnant revegetation PC2 (3.3% explained var.) PC2 (3.3% explained Marshalls Rd seedlings Southerns remnant Foothills Foothills Moonlight Bay 1510 10 Southerns remnantMt Liverpool Number− of5 clustersValue of BIC MarshallsSalt Lake remnant Rd Sebs Patch −5 1520 Marshalls Rd seedlings BIC 0 Sebs Patch −5 SouthernsSouthernsSalt Lakereveg reveg remnantMarshalls Rd Southerns remnant MoonlightCollection MtBay Liverpool Stand Salt LakeSoutherns remnant revegetation −5 var.) PC2 (3.3% explained Mt LiverpoolSaltMtMoonlight Lake Liverpool revegetation MoonlightBay Bay BIC Marshalls Rd Southerns revegetation 10 Southerns reveg SaltSalt LakeLake revegetationremnant −5 Sebs Patch SouthernsSebs remnant Patch Quarry PC2 (3.3% explained var.) PC2 (3.3% explained MoonlightCollection BayFoothills Stand Salt Lake remnant versus number of clusters SouthernsCollectionMarshallsSebs PatchMt SaltrevegQuarry Liverpool LakeRdSebsMoonlightSalt remnantMarshallsSoutherns seedlingsPatch Stand LakeCollection revegetation Rd remnantBay seedlings Stand 10 1510 Salt Lake revegetationMarshalls Rd seedlings versus number ofValue clusters of BIC Mt Liverpool Salt Lake remnant QuarryMt Liverpool 5 Marshalls Rd seedlings MarshallsCollection Rd Southerns Stand revegetation PC2 (3.3% explained var.) PC2 (3.3% explained FoothillsMoonlightSoutherns Bay remnant Number ofversus clusters number of clusters Marshalls Rd Mt Liverpool MarshallsQuarry RdSaltQuarryMarshalls seedlingsLake remnantSaltMarshalls Rd Lake seedlingsMt remnantRd Liverpool 1500 Southerns reveg Southerns remnant BIC 0 Marshalls Rd CollectionQuarryMt StandLiverpoolSouthernsSebs Patch remnant 5 Marshalls Rd seedlings MarshallsMarshalls Rd seedlings SouthernsRd Moonlightseedlings remnant Bay −5 10 MtSoutherns LiverpoolMoonlight remnant BayMarshalls Rd seedlings 1500 −50 −5 Moonlight Bay 1520 0 MarshallsCollection Rd Stand seedlingsSalt Lake revegetation 0 Moonlight Bay Moonlight Bay 10 0 −5 MarshallsSoutherns Rd seedlingsSouthernsSalt Lakereveg revegSaltMtSaltMoonlight Liverpool Lake LakeremnantSalt revegetationremnantSalt LakeBay Lake revegetation remnant PC2 (3.3% explained var.) PC2 (3.3% explained 10 var.) PC2 (3.3% explained Salt Lake reveg

−BIC 5 versus number0 of clusters Southerns reveg MarshallsFoothillsQuarrySebs Rd PatchMarshallsSoutherns seedlings Rd remnant seedlings −5 0.36 MoonlightSoutherns BaySoutherns reveg revegetationSaltQuarryMt Lake Liverpool revegetationSebs Patch 10 Value of BIC −10 5 Marshalls Rd seedlings Southerns reveg

PC2 (3.3% explained var.) PC2 (3.3% explained Salt Lake remnant 0.35 1520 MarshallsMoonlight RdQuarryFoothills Bay

0.36 Moonlight Bay 1500 Collection StandMtSouthernsSebsSalt LiverpoolLake Patch revegetation revegetationSalt Lake revegetation 0 Southerns remnant PC2 (3.3% explained var.) PC2 (3.3% explained Marshalls Rd seedlingsSoutherns remnant 1490 Marshalls Rd Moonlight Bay

1520 Moonlight Bay 10 5 QuarryFoothillsSaltSalt Lake Lake remnant remnant −5 1500 −5 Collection StandSebsSalt Lake PatchMt revegetation Liverpool 0 0 Quarry CollectionMarshallsSoutherns Rd Stand remnant 5 Southerns remnant MoonlightCollectionSalt LakeBay remnant Stand 1500 1520 Value of BIC −05 1) 4) Marshalls Rd FoothillsseedlingsMt LiverpoolSalt Lake revegetation

10 var.) PC2 (3.3% explained SaltSalt LakeLake revegetationremnant −5 Quarry Marshalls Rd seedlingsSoutherns revegetation Value of BIC Foothills Foothills Southerns remnant 1510 versus number of clusters 1) MarshallsQuarry SebsRdMarshalls seedlings PatchSaltMarshalls Lake Rd revegetationRd seedlingsseedlings 0.36 10 var.) PC2 (3.3% explained SouthernsMoonlightSoutherns revegBaySoutherns revegetationQuarryMt revegetationSalt LiverpoolSouthernsSoutherns Lake revegetationremnantrevegetation Marshalls Rd seedlings Moonlight Bay Foothills PC2 (3.3% explained var.) PC2 (3.3% explained 5 Southerns remnant 1510 Mt Liverpool PC2 (3.3% explained var.) PC2 (3.3% explained Southerns revegetation 5 1520 MoonlightQuarryFoothillsSaltSebsMoonlight BayLake PatchrevegetationMoonlight Bay Bay 10 1500 0 −5 CollectionMt Liverpool StandSebsSaltQuarry Patch LakeSebsCollection remnant Patch Stand 5 CollectionMarshallsSebs Patch Moonlight Stand RdCollection seedlings BayMarshallsSouthernsMoonlightSouthernsMoonlight Rd remnant Stand Bayrevegetation BayMt Liverpool 1510 10 versus number of clusters Salt Lake remnant 1500 −05 −5 Marshalls Rd seedlings Collection Stand SaltQuarry Lake remnant PC2 (3.3% explained var.) PC2 (3.3% explained Mt Liverpool Salt Lake remnant Sebs Patch CollectionSaltMarshalls LakeMt remnantRd Liverpool Stand Salt Lake revegetation 0 −5 Quarry CollectionMoonlight Stand Bay BIC versusValue numberof BIC of clusters Marshalls Rd CollectionMarshalls Stand RdQuarrySouthernsSebs seedlings Patch remnant PC2 (3.3% explained var.) PC2 (3.3% explained 1) Mt Liverpool SaltMarshallsMarshalls Lake remnantRd Rd seedlings Foothills 1490 Mt Liverpool −105 var.) PC2 (3.3% explained −50 −15 −10 −5 0 Moonlight5 Bay BIC Marshalls Rd SouthernsSebsSalt LakePatchSoutherns remnantrevegetationMoonlight Bay revegetationQuarry Foothills 1510 Marshalls Rd seedlingsSebs Patch

0.36 Moonlight Bay PC2 (3.3% explained var.) PC2 (3.3% explained −50 Moonlight Bay SouthernsMt Liverpool revegetationSalt Lake revegetation 10 Salt SaltLake reveg Lake revegetation Moonlight Bay

PC2 (3.3% explained var.) PC2 (3.3% explained Foothills

1520 5 Mt MoonlightLiverpoolQuarryFoothills BaySebs Patch

PC2 (3.3% explained var.) PC2 (3.3% explained Southerns revegetation Salt Lake reveg Moonlight Bay 10 −10 Southerns reveg Collection StandFoothillsSebsSalt Lake Patch revegetation BIC 0 0.35 −5 Quarry 1500 Mt Liverpool MarshallsSebs SaltRd Patch Lake remnant 5 Sebs Patch Collection Stand −10 Southerns reveg Salt Lake remnant

1500 Mt Liverpool 1490 −5 CollectionMoonlight Stand Bay 0.35 versus number of clusters Salt Lake remnant 5 5 0 10 15 20 var.) PC2 (3.3% explained 0 CollectionMt LiverpoolMt Liverpool Stand PC2 (3.3% explained var.) PC2 (3.3% explained Quarry Mt Liverpool SaltMarshalls LakeMt remnantRd LiverpoolMoonlight BaySoutherns remnant 1490 Collection Stand Sebs Patch 5 Southerns remnant Salt Lake remnant

BIC Marshalls Rd seedlings Value of BIC Marshalls Rd SouthernsSebs Patch remnant 1520 1) 4) FoothillsMt Liverpool Southerns revegetation 10 10 var.) PC2 (3.3% explained Collection Stand MarshallsSoutherns Rd revegetation seedlings 0 Southerns remnant Moonlight Bay Mt Liverpool 1520 −5 4) Salt Lake revegetation Foothills Foothills

1510 Marshalls Rd seedlingsMoonlight Bay 0.34 Moonlight Bay SouthernsSaltSoutherns Lake revegetation revegetationremnantSebsSoutherns Patch remnant Quarry PC2 (3.3% explained var.) PC2 (3.3% explained Southerns remnant 5 var.) PC2 (3.3% explained PC1 (5.9% explained var.) 10 10 −5 MoonlightMt LiverpoolBay QuarrySouthernsSaltMarshallsSouthernsMoonlightSebsCollection SaltLake Lake Patch revegSoutherns revegetationRd revegetation remnantBay seedlingsMoonlightSouthernsSebs Stand remnant Patch Bayrevegetation 5 −10 var.) PC2 (3.3% explained −5 Moonlight BayMt Liverpool 1510 −10 Southerns reveg QuarrySaltQuarry Lake remnant Marshalls Rd seedlings SebsCollection Patch Stand 10 Sebs Patch 0.35 CollectionSaltSalt LakeLake revegetationremnantStand −5 CollectionFoothillsMtCollection MoonlightLiverpool StandMt Bay LiverpoolStand

1510 Quarry versus number of clusters Marshalls Rd seedlings Collection Stand PC2 (3.3% explained var.) PC2 (3.3% explained 1490 SaltSalt LakeLake revegetationremnant Mt Liverpool Salt Lake remnantMarshallsFoothills Rd 1490 −15 −10 −5 0 5 1520 −5 QuarryMt LiverpoolFoothillsSaltMarshalls LakeMt remnantRd Liverpool BIC Marshalls Rd SouthernsSebsFoothillsQuarry Patch remnant Southerns revegetation 1490 CollectionMt Liverpool Stand 0 −15 −10 −5 Southerns remnant 0 5 Marshalls Rd seedlings 1520 −50 4) MoonlightFoothillsFoothills Bay SaltQuarry Lake revegetation PC2 (3.3% explained var.) PC2 (3.3% explained 5 5 10 15 20 Mt Liverpool Moonlight Bay CV error Mt Liverpool 0 FoothillsSebs Patch QuarryFoothills 0 BIC Foothills

Number of var.) PC2 (3.3% explained clusters 5 1500 5 SaltMarshalls Lake revegetationRd seedlings 10 Mt LiverpoolFoothills Salt Lake revegSouthernsSoutherns revegetationremnant SebsSalt Lake Patch revegetation

BIC Moonlight Bay Mt Liverpool 0 0 var.) PC2 (3.3% explained

1500 5 10 15 20 Southerns remnant 5 5 var.) PC2 (3.3% explained MoonlightSoutherns BaySebsMt Liverpool Patch remnant −10 Southerns reveg Mt Liverpool SebsMoonlight Patch Bay Southerns remnant 5 Marshalls Rd SebsCollection Patch StandMarshalls Rd 10 5 0 10 15 20 var.) PC2 (3.3% explained Southerns revegetation 0.35 105 5 Mt LiverpoolSebsMoonlight Patch BaySouthernsMt Liverpool remnantSalt Lake remnant 1510 Salt Lake remnant 1510 5 −5 Marshalls Rd seedlings SouthernsMt Liverpool revegetationQuarry 1490 0.34 Southerns remnantSaltSalt LakeLake revegetationremnant 10 Salt Lake remnant 0 −5 Mt Liverpool SouthernsMoonlight revegetation Bay PC1 (5.9%Quarry explained var.) QuarryMt Liverpool 0.34 CollectionQuarry StandSoutherns remnantFoothills 1490 −5−15 −10 Quarry−5 Southerns remnant 0 5 Salt Lake reveg 1520 0−10−15 −10 PC1 (5.9%4) −explained5 var.) 0 QuarryFoothillsQuarry5 Mt LiverpoolQuarryQuarry −10−5 FoothillsCollectionCollection StandMarshalls Stand Rd Southerns reveg MarshallsQuarryFoothills MarshallsRd SaltMarshalls Lake revegetationRd Rdseedlings 5 Foothills 1520 −5 CollectionMoonlightFoothillsFoothills Stand Bay SouthernsSouthernsSouthernsSoutherns revegetationremnant revegetation remnantSoutherns revegetation PC2 (3.3% explained var.) PC2 (3.3% explained BIC 1500 50 Marshalls Rd 1520 0 1500 FoothillsQuarry MoonlightSebsCollectionSoutherns Patch Bay revegetation Stand Salt Lake remnant BIC 10 5 var.) PC2 (3.3% explained 10 15 20 Mt Liverpool

CV error Mt Liverpool Sebs Patch 5 0 0 5 Number of clusters0 10 15 20 var.) PC2 (3.3% explained QuarryFoothillsMt Liverpool 5 1510 Number of clusters PC2 (3.3% explained var.) PC2 (3.3% explained Southerns remnant 0 5 10 15 20 Marshalls Rd seedlings Salt Lake revegetation CV error Mt Liverpool MoonlightSebs Bay Patch 5 Sebs Patch SebsQuarryFoothills PatchSaltSalt LakeLake revegetationremnant 0 Southerns remnant 10 5 Number of clusters −5 Mt Liverpool Quarry SouthernsSebsSalt Lake Patch revegetation revegetation Marshalls Rd SouthernsMt Liverpool remnant

0.33 Marshalls Rd 5 Mt Liverpool Mt LiverpoolFoothills 1490 −15 −10 −5 0 5 0.34 SouthernsSalt Lake remnant remnant Marshalls Rd SouthernsMt Liverpool remnant 1510 −55 Salt Lake remnantQuarry MarshallsQuarry Rd 0 PC1 (5.9%Quarry explained var.) QuarryMt LiverpoolSouthernsSaltMoonlight revegetation BayLake remnant 0 1510 −5 −5 Salt Lake remnantSalt Lake remnantSouthernsQuarry revegetation 0 −5 FoothillsQuarrySouthernsMoonlight revegetation Bay 5 −10 QuarrySalt Lake reveg 1490 0 FoothillsQuarrySalt LakeSalt remnantLake remnant 0 BIC −15 −10 −5 0 Collection5Foothills MarshallsQuarry StandSalt LakeRd reveg 1500 PC1 (5.9% explained var.) Collection Stand 0 −50 −15 −10 −5 0 Southerns reveg Collection5 MtMarshallsMarshalls LiverpoolMarshalls Stand RdRd Rd 1520 CollectionFoothills StandSoutherns revegetation

0 var.) PC2 (3.3% explained 5 10 15 20 Marshalls Rd seedlings Southerns reveg Marshalls Rd seedlings 5 1500 −10 Marshalls Rd Southerns revegetation PC2 (3.3% explained var.) PC2 (3.3% explained −5 −5 Quarry FoothillsMarshallsSouthernsSoutherns Rd revegetation remnant Salt Lake remnant SebsMoonlight Patch Bay 50 BIC SaltMarshalls Lake remnantRd 1500 Number of clusters Quarry Southerns revegetation 5 var.) PC2 (3.3% explained 10 15 20 Salt Lake remnant 10 BIC CV error 0 Mt Liverpool Sebs Patch QuarryFoothills SouthernsSebs Patch remnant 5 0 NumberNumber of clusters of clusters Southerns remnant Mt Liverpool 0.34 0 Quarry SouthernsSebs Saltremnant Lake Patch revegetation 5 0.33 PC1 (5.9%Quarry explainedSoutherns remnant var.) Mt Liverpool Marshalls Rd seedlings

0.33 Quarry 0 5 −5 −10−5 Marshalls Rd SaltSalt LakeQuarry Lake remnantSouthernsQuarry remnantSouthernsMt Liverpool revegetationMarshallsSalt remnant Lake SaltremnantRd Lake revegetation 0 1510 −5 SouthernsQuarryQuarry revegetation 1490 −5 Salt Lake remnant Foothills Salt Lake remnant 0 Collection StandSalt LakeSoutherns remnantMoonlight revegetation Bay 1490 PC1 (5.9% explained var.) MarshallsSalt LakeSaltSalt remnantRdLake Lake remnant reveg 1520 −100 CollectionFoothills Stand PC1 (5.9% explained var.) Salt Lake reveg 1510 −10 Marshalls Rd seedlings MarshallsQuarry Rd seedlingsSoutherns revegetation 0 var.) PC2 (3.3% explained −50 CollectionFoothillsMarshalls Stand Rd 0 −010−15 Marshalls−10 Rd seedlings −5 0 5 Marshalls Rd seedlingsSaltMarshalls Lake remnantRd PC2 (3.3% explained var.) PC2 (3.3% explained CollectionFoothillsMarshallsMarshalls RdStand Rd −5 Southerns reveg 5 var.) PC2 (3.3% explained 10 15 20 SaltMarshallsSouthernsSebs Lake PatchSalt remnantRd remnantLake remnant

CV error Mt LiverpoolMarshalls Rd 5 0 −10 Number of clusters −5 QuarrySebs Patch SaltSouthernsSebs QuarryFoothillsLake PatchSoutherns remnantremnant revegetation 5 1500 QuarryQuarry MarshallsMarshallsSebsSalt RdSalt Lake Patch Rd Lakeseedlings seedlingsrevegetation revegetation Mt Liverpool Collection Stand Salt Lake remnant BIC 5 Number of clusters MarshallsFoothillsMt MarshallsLiverpoolSalt Lake Rd Rd revegetation seedlings seedlings Marshalls Rd SouthernsMarshalls remnant Rd 0 5 Quarry Salt Lake revegetation Southerns remnant Salt Lake remnantSalt Lake remnant 1510 −5 −15 −10 −5 0 Salt5 LakeSalt revegLakeQuarry remnant 0.33 0 −10 Moonlight Bay 1510 Southerns revegetation

BIC 5 10 15 20 Salt Lake reveg 1500 0 −10 0 1510 5 0 −5 FoothillsSaltQuarry LakeSaltSalt Lakeremnant Lake revegSouthernsQuarry remnant revegetation 0.32 −10 0 −15 −10 −5 0 Collection5 MarshallsSaltSalt Lake Lake StandSalt remnantLakeRdremnant remnant

1490 Marshalls Rd seedlings −10 Southerns reveg CollectionMarshallsMarshallsMarshalls SaltStand RdRd SaltLake Rdseedlings seedlingsLakeSalt remnantrevegetationLake remnant validation error validation −5 PC1 (5.9% explained var.) CollectionFoothills StandSalt Lake revegetationSoutherns revegetation 0 Quarry Marshalls Rd seedlings 1500 CollectionFoothills Stand −10 −15 Marshalls−10 Rd seedlings Quarry−5 0 5 SaltQuarry LakeMarshalls remnantSalt Rd Lake seedlings reveg PC2 (3.3% explained var.) PC2 (3.3% explained FoothillsMarshalls Rd Salt Lake remnant BIC −5 −15 −10 −5 0 5 Salt Lake remnantSebs Patch BIC 5 10 15 20 SaltMarshalls Lake remnantRd - 1500 Number of clusters BIC 1490 0 5 10 15 20

1500 Salt Lake reveg 5 Foothills SouthernsSebs Patch remnant −15 −10 −5Southerns remnant 0 5 0.32 SaltMarshalls Lake revegetationRd seedlings 5 Foothills Mt Liverpool Quarry 0.32 5 0.33 Marshalls SouthernsRdSoutherns seedlings revegetationremnant Marshalls Rd FoothillsMoonlightMarshallsSalt BayRdSalt Lake Lakeseedlings revegetation revegetation PC2 (3.3% explained var.) PC2 (3.3% explained 0 var.) PC2 (3.3% explained −5 PC1 (5.9% explained var.) SaltSaltCollection Lake Lake reveg remnantSouthernsQuarry Stand revegetation Moonlight Bay Sebs PatchSalt Lake reveg Sebs Patch Sebs Patch 1490 1490 −15 −10 −5 0 5 SaltSaltSalt LakeLake Lake revegSalt remnantLake remnant SebsMoonlight Bay Patch 1490 SaltSalt Lake Lake reveg SaltMarshallsremnant Lake revegetationRd seedlings −10 −15 −10 PC1 (5.9%−5 explained var.)0 Mt Liverpool 5 1510 SaltMarshalls Lake revegetationRd seedlings

PC2 (3.3% explained var) PC2 (3.3% explained Southerns remnant 0 Mt Liverpool Southerns revegetation Number of clusters 0 Marshalls Rd CollectionMoonlightMarshalls Stand BayRdSalt Lakeseedlings revegetationSalt Lake reveg 5 Marshalls Rd seedlings Marshalls RdSoutherns Southernsseedlings revegetationremnant −10 Marshalls Rd Foothills

PC2 (3.3% explained var.) PC2 (3.3% explained Moonlight Bay PC2 (3.3% explained var.) PC2 (3.3% explained PC2 (3.3% explained var.) PC2 (3.3% explained −5 PC1 (5.9% explained var.) CollectionFoothillsMarshallsFoothillsMarshalls Stand RdSalt LakeseedlingsRdSalt revegetation Lake remnant PC2 (3.3% explained var.) PC2 (3.3% explained PC2 (3.3% explained var.) PC2 (3.3% explained Collection StandSalt SebsLake Patch MoonlightremnantSaltMarshalls Lake Bay remnantRd PC1 (5.9% explained var.) Southerns reveg Moonlight Bay −10 −10 MarshallsSebs PatchRdSebs Moonlightseedlings BaySouthernsSebs Patch Patch remnant −5 SaltSaltCollection Lake LakeMarshalls remnant remnant Stand RdSaltSebsMoonlight seedlingsLake Bay Patchrevegetation PC2 (3.3% explained var) PC2 (3.3% explained Quarry 5 Number of clusters FoothillsSaltSebs Lake reveg Patch Quarry −10 5 var) PC2 (3.3% explained 5 Number of clusters Salt Lake reveg Marshalls Rd seedlings −15 −10 −5 0 5 Salt Lake remnantSalt Lake remnant Salt Lake revegetation −15 −10 −5 PC1 (5.9%0 explainedSoutherns reveg var.) 5 −5 −10 Southerns reveg

BIC 5 10 15 20 1500 −5 −10 Sebs Patch Salt Lake reveg 5−10 Southerns remnant Foothills Sebs Patch −0.32 10 −15 −10 −5 0 5 SaltSalt Lake remnant LakeMoonlight reveg Bay −10 Salt Lake remnant 0 1510 −15 −10 −5 PC1 (5.9%(5.9%0 explainedexplained var.)var.) 5 Salt LakeMoonlight reveg Bay Southerns remnant PC2 (3.3% explained var.) PC2 (3.3% explained Salt LakeMarshalls reveg Rd Southerns remnant MoonlightSaltFoothills BayLakeSalt remnantLake remnant Cross −10 MoonlightSaltCollectionSalt Lake Bay Lake reveg reveg StandSebs Patch MoonlightSebs Patch Bay PC2 (3.3% explained var.) PC2 (3.3% explained MoonlightFoothillsMarshallsMarshalls Bay Rd RdSalt Lakeseedlings revegetation

PC2 (3.3% explained var.) PC2 (3.3% explained CollectionMarshalls RdStand 1490 5 10 15 20 −15 −10 −5 0 CollectionCollectionSalt5Foothills LakeMarshallsFoothills reveg StandSaltStandSebs SebsLake Rd Patch Patch seedlingsrevegSaltMarshallsSoutherns Lake revegetationRd remnant seedlings 1500 Quarry Mt Liverpool PC2 (3.3% explained var) PC2 (3.3% explained 5 Salt Lake reveg SouthernsSoutherns revegetationremnant 5 10 15 20 −15 Marshalls Rd −10 −5 0 5 Salt MoonlightLake remnantSoutherns BaySalt Lakeremnant revegetation

1500 Marshalls Rd seedlings 5 10 15 20 Marshalls Rd seedlings PC2 (3.3% explained var) PC2 (3.3% explained FoothillsSoutherns remnant PC2 (3.3% explained var.) PC2 (3.3% explained 1500 PC2 (3.3% explained var.) PC2 (3.3% explained

BIC 5 10 15 20 PC2 (3.3% explained var) PC2 (3.3% explained 5 PC2 (3.3% explained var) PC2 (3.3% explained 1500 PC1 (5.9% explained var.) Collection Stand

5 var.) PC2 (3.3% explained Moonlight Bay Marshalls Rd seedlings Foothills Sebs Patch 5 var) PC2 (3.3% explained Moonlight Bay PC2 (3.3% explained var.) PC2 (3.3% explained 0.32

2 var) PC2 (3.3% explained 4 6 8 10 Sebs Patch 2 4 var.) PC2 (3.3% explained 6 8 10 −10 Salt Lake remnantSebs Patch SebsMoonlight Patch Bay

PC2 (3.3% explained var) PC2 (3.3% explained 2 4 6 8 10 −10 5 −−1010 Number of clusters −−−101015−15 −10−10 −−55 −5 00 0 55 SebsSebsSaltSebs5 Patch Patch Lake Patch Saltreveg LakeSebs revegSebsMoonlightSouthernsSebsMoonlightSoutherns Patch Patch Bay revegetationBay revegetationSoutherns revegetation Southerns reveg Sebs Patch −5 −10 Sebs Patch Mt Liverpool Mt Liverpool

1490 Mt Liverpool 1490 1490 SebsSalt Patch Lake reveg −10 1490 −15 −10 −5 0 5 MtMarshallsMoonlightMarshallsMtMoonlight Liverpool Liverpool Rd BayMtMarshallsMoonlightRdBay seedlings LiverpoolseedlingsSouthernsSouthernsSaltMarshalls RdBay Lake remnant remnantseedlings revegetationRdSoutherns seedlings remnant −15 −10 −5 PC1 (5.9%0 explained var.) Mt Liverpool 5CollectionMarshallsSalt LakeStand remnant RdMarshallsSouthernsSoutherns Rd revegetationremnant PC1Marshalls Rd(5.9% explained var.) CollectionFoothillsFoothillsFoothillsMoonlightMarshallsCollection Stand BayRdSalt Lakeseedlings Stand revegetation PC2 (3.3% explained var.) PC2 (3.3% explained PC1 (5.9% explained var.) PC2 (3.3% explained var.) PC2 (3.3% explained Salt Lake reveg −5 PC1 (5.9% explainedSoutherns remnant var.) SaltCollectionSalt Lake LakeMoonlight Saltreveg reveg Stand FoothillsLakeSoutherns SebsBay reveg PatchremnantMoonlight Bay −5 −−10Number10Number−5 of of clusters clusters Salt MoonlightLake remnantSoutherns Bay remnantSebsMoonlight BaySoutherns Patch remnant −10Number of clusters var.) PC2 (3.3% explained FoothillsMarshallsSoutherns Rd revegetation PC2 (3.3% explained var) PC2 (3.3% explained 5 5 5 Number of clusters Collection SouthernsStandSalt Lake reveg revegetation

PC2 (3.3% explained var) PC2 (3.3% explained Quarry Sebs Patch 5 var) PC2 (3.3% explained −15 −10KK −5 0 PC1 (5.9%Quarry 5 explainedexplained var.)var.) SaltSouthernsSoutherns Lake reveg remnant remnant Southerns revegetation Southerns reveg PC2 (3.3% explained var) PC2 (3.3% explained −15 5 −1010 15 −520 −10 0 5 Quarry Southerns remnant

1500 K −5 PC2 (3.3% explained var) PC2 (3.3% explained 5 PC1 (5.9% explained var.) Southerns remnant Foothills Sebs Patch −15 −10 −5 0 5 Foothills −10 −−55 −15 Marshalls−10 Rd seedlings −5 0 5 Sebs Patch PC2 (3.3% explained var) PC2 (3.3% explained −15 −10 −5 0 5 Sebs Patch PC2 (3.3% explained var) PC2 (3.3% explained Salt Lake remnant PC2 (3.3% explained var) PC2 (3.3% explained PC2 (3.3% explained var.) PC2 (3.3% explained −15 −10 −5 0 5 MtMt Liverpool Liverpool 2 4 6 8 10 −5−−1010−15 −10 PC1 (5.9%−5 explained var.) 0 MtMarshallsMtMarshallsFoothills Liverpool Liverpool5 Rd Rd seedlings seedlingsSebs Patch PC2 (3.3% explained var) PC2 (3.3% explained Salt Lake reveg −10 −10−15 −10 −5 Southerns remnant 0 5 MarshallsMarshallsMoonlightSebs PatchRd MtRd BayLiverpoolSebsMoonlight Patch Bay CollectionSebsSebs Patch Patch Stand Southerns revegetation −10 CollectionSebsSebs PatchFoothillsFoothills PatchMoonlight StandSebsMt Patch BayLiverpool PC2 (3.3% explained var.) PC2 (3.3% explained FoothillsMarshallsMarshalls Rd Rd seedlings Mt Liverpool Mt Liverpool Mt Liverpool Collection Stand 1490 Salt MtLake Liverpool revegSebs Patch 5 10 15 20 Marshalls Rd MarshallsMoonlight RdBay seedlingsSoutherns remnant 55 5 5 10 10 15 15 20 20 MarshallsCollectionSebsSebs PatchFoothillsSouthernsSoutherns Patch RdSouthernsSouthernsMt SouthernsMtrevegetation revegetation LiverpoolStand Liverpool remnant remnant remnant

1500 Collection Stand PC2 (3.3% explained var) PC2 (3.3% explained PC1 (5.9% explained var.) Foothills Moonlight Bay 5 Moonlight Bay Mt Liverpool SaltSouthernsSoutherns Lake reveg remnant remnant −−1515 −−1010 −−55 0 Marshalls Rd seedlings 55 QuarryQuarry PC2 (3.3% explained var) PC2 (3.3% explained 5 10 15 20 Marshalls Rd PC1 (5.9% explained var.) SaltSalt Lake Lake reveg reveg 5 2 4 var.) PC2 (3.3% explained −10Number−5 6 of clusters 8 10 −−55 PC1 (5.9% explained var.) SouthernsSoutherns remnant remnantSouthernsSouthernsSoutherns remnantSoutherns revegetation revegetationSouthernsMt revegetation Liverpool remnant PC2 (3.3% explained var) PC2 (3.3% explained PC2 (3.3% explained var) PC2 (3.3% explained Southerns reveg Sebs Patch Moonlight Bay −105 −10−−1010−15 −10 −5 Southerns0 reveg 5 QuarryQuarry SouthernsSebs revegetation Patch 1490 1490 MoonlightMtMoonlightMt Liverpool Liverpool Bay Bay Moonlight Bay −15 var) PC2 (3.3% explained −10 K 00 −5 0 PC1 (5.9%Quarry explained5 var.) MarshallsMarshallsSouthernsSebs Rd SouthernsRd Patch seedlings seedlings remnantSoutherns remnantQuarry revegetation −15 −10 −5 0 5 SaltSalt Lake Lake remnant remnant −−1515 −−1010 −15 −5 −10 PC1−−55 00 (5.9% explained00 var.)55 55 MarshallsMarshalls Rd Rd SouthernsMtSalt LakeLiverpool reveg remnantSoutherns revegetation 1490 PC1PC1 (5.9%(5.9% explained−5 var) SebsSebsFoothillsFoothills Patch PatchMtMarshallsMoonlight Liverpool RdBay seedlingsSoutherns remnant −5 −15 −10 −5 SouthernsSoutherns remnant remnant 0 Foothills 5 MarshallsSebs PatchRd PC2 (3.3% explained var) PC2 (3.3% explained PC2 (3.3% explained var) PC2 (3.3% explained −−55 −10 Southerns reveg Collection StandQuarry 1490 0505 0 NumberNumber of of clusters clusters −10 PC1 (5.9% explained var.) SaltSouthernsSoutherns FoothillsMarshallsMtLake Liverpool reveg MarshallsMoonlightMtreveg reveg RdLiverpool seedlings RdBay seedlings −−1510−5 −10 −15 −5 −10 −5 0 0 5 5 MarshallsSaltSoutherns Lake remnant remnant Rd 5 Number of clustersPC1 (5.9% explained var) CollectionSebsSebs PatchFoothills PatchSebs FoothillsStand PatchSoutherns revegetation Southerns remnant SouthernsSoutherns remnant remnant PC2 (3.3% explained var) PC2 (3.3% explained −5 K Marshalls Rd seedlings PC1 (5.9%Quarry explained var.) Mt Liverpool QuarryQuarrySoutherns remnant 05 5−15 5 Number− 10of clusters10 15 −5 20 Marshalls0 Rd PC1 (5.9%5 explainedexplained var.)var.) MarshallsQuarryMoonlightMarshallsQuarryMoonlight Rd BayRdBay seedlings seedlingsSouthernsSouthernsMt revegetation Liverpool remnant −15 −10 −−55 00 5Foothills5Southerns remnant −5 −15 −10 −5 0 Southerns5 SouthernsSebs remnantSoutherns Patch remnantMoonlight revegBay PC2 (3.3% explained var) PC2 (3.3% explained −−15−55 −10 PC1PC1− (5.9%(5.9%5 explained var)0 5 FoothillsMarshallsFoothillsMarshallsMt Liverpool Rd Rd SouthernsQuarrySouthernsQuarryQuarry revegetation revegetation 00 5 10 15 20 −10−5 PC1 (5.9% explained var.) MarshallsMt LiverpoolSoutherns SaltRd Lake reveg seedlings remnantSoutherns revegetation PC2 (3.3% explained var) PC2 (3.3% explained 0 5 10 15 20 SouthernsSalt Lake remnant remnant 0 Southerns reveg Salt Lake remnant −10 Sebs PatchQuarry Marshalls Rd seedlings CollectionSebs Patch Stand 55 1490 0 00 PC1 (5.9% explained var.) SouthernsSouthernsFoothillsMarshallsMoonlightMt Liverpool Quarryreveg reveg RdBay seedlings −−10−1015 −10 −15 −5 −10 PC1−5 (5.9%0 explained0 var.)Mt Liverpool 5 5 MarshallsSouthernsSouthernsSalt Lake remnantMarshallsMoonlight revegRd reveg RdBay seedlings 5 10−−55 PC115 (5.9%20 explained−15 Marshalls var) Rd −10 −5 0 SebsFoothills5 PatchSouthernsSoutherns remnantSouthernsMt revegetation Liverpool remnant −5 5 −5 Southerns remnant SaltMoonlightMtQuarrySaltMtQuarry Liverpool LiverpoolLake Lake remnant BayremnantFoothillsMarshalls Rd SouthernsQuarry revegetation 0 Number of clustersPC1 (5.9% explained var) PC1 (5.9% explainedexplained var.)var.) MoonlightSoutherns Bay remnantQuarryMoonlight Bay 5−15 −10 −5 0 Quarry 5 Quarry Marshalls Rd seedlings 0 −−55 5 10 15 20 −5 PC1 (5.9% explained var.) SouthernsSouthernsMarshallsMarshallsSoutherns Southernsremnant remnant Rd RdSalt Lake seedlings reveg remnantSoutherns Saltrevegetation Lake remnant 5 var) PC2 (3.3% explained −10 PC1 (5.9% explained var.) Southerns reveg Foothills Quarry 00−1490 −55 0 Foothills MarshallsMoonlightMt LiverpoolSouthernsSoutherns RdBay seedlings remnant reveg 0 NumberNumber of of clusters clusters Marshalls Rd seedlings (c) −−105 −15 PC1 (5.9%−10 explained−15 var)−5 −10 PC1 −(5.9%5 0 explained var.)0 5 5 SebsMarshallsFoothillsMarshallsQuarryMoonlight PatchSalt Lake remnant RdRdBaySoutherns seedlings reveg −05(c) −5 −−1010 −15 −10 −5 Southerns remnant 0 5SebsSebsSoutherns Patch Patch MarshallsSouthernsSoutherns SaltMtQuarryremnant revegRdLiverpool revegLake remnant Mt Liverpool SaltFoothills Lake remnantMoonlightSouthernsQuarry Bay revegetation 5 Number of clusters PC1 (5.9% explained var) PC1 (5.9% explained Mtvar.) Liverpool Salt Lake remnant Quarry Marshalls Rd SaltMt Liverpool Lake remnant 0 5 10 15 20 Quarry Marshalls Rd Quarry SouthernsSouthernsMoonlightMoonlightSaltMtSoutherns Liverpool LakeSoutherns reveg reveg Bay remnantMarshallsBay revegSaltSalt remnant Lake SaltLake Rd Lakeremnant remnant seedlings remnant Marshalls Rd seedlingsMoonlight Bay −5 5 PC1 (5.9% explained var.) MarshallsMarshalls Rd Rd seedlingsSaltSaltMoonlight BayLake Lake revegetation revegetation SouthernsSouthernsSalt Lake reveg reveg remnant 0 Salt Lake reveg −5 Marshalls Rd seedlings FoothillsQuarry 0 −5 Foothills −10 Foothills Southerns reveg −5 Southerns reveg Quarry Number of −clusters5 −−1515 −−1010 −15 −5 −10 PC1 (5.9%−5 00 explained var.)Southerns0 reveg 55 5 SouthernsMarshallsMoonlightSaltMtQuarry Liverpool Lake remnant RdBayremnant seedlings −5 SaltMarshallsMoonlight Lake remnant Rd Bay (c) −−55 PC1 (5.9% explained var) PC1 (5.9% explained var.) FoothillsSalt Lake remnant SouthernsQuarry revegetation −10 Quarry Southerns remnant MarshallsSebs Patch Rd seedlings −05 5 10 15 20 Southerns remnant SouthernsSaltMtQuarrySaltQuarry LiverpoolMarshalls Lake Lake reveg remnantrevegremnant RdSouthernsSalt Lake reveg remnant Mt Liverpool Mt LiverpoolSalt Lake remnant 5 PC1 (5.9% explained var.) SebsSebs Patch Patch Moonlight Bay 0 −5 Quarry Marshalls Rd FoothillsSouthernsMarshallsMoonlightSoutherns reveg RdMoonlightSalt MtBay seedlingsrevegLiverpool Lake remnantBay Salt Lake remnant −−10Number05 of clusters Mt Liverpool SouthernsMarshallsSouthernsSoutherns reveg Rd seedlings reveg reveg 0 Mt Liverpool Marshalls Rd seedlingsMoonlight Bay Marshalls Rd seedlings Marshalls Rd 0 Marshalls Rd Salt Lake revegetation (c) Marshalls Rd seedlings Salt Lake remnant Marshalls Rd Sebs PatchMtQuarry Liverpool −10 −−1515 −−1010 −5 PC1 (5.9%0 0explained var.) 55 MoonlightSoutherns Bay revegSalt Lake reveg −5 −10 Quarry Foothills Mt Liverpool MarshallsSalt Lake Rdremnant seedlings Quarry −10 Marshalls Rd Southerns reveg SouthernsMarshallsSalt Lake remnant Rdremnant −−5−55 K=3 −15 −10 −5 0 Salt5 MoonlightMt Lake Liverpool reveg BayMoonlightSaltMoonlightSalt Bay Lake Bay SaltLake Lakerevegetation revegetation remnant K=3 var.) PC2 (3.3% explained SouthernsSaltSalt Lake Lake reveg reveg

PC2 (3.3% explained var.) PC2 (3.3% explained PC1 (5.9% explained var.) Foothills Quarry −5 Marshalls Rd seedlingsMoonlight Bay PC1 (5.9% explained var.) MtSalt SaltLiverpool LakeLake reveg revegSalt Lake remnant −5 0 MtQuarry MarshallsSaltLiverpool Lake reveg RdSebsSebsSalt Patch Patch Lake revegetation Number of clusters MarshallsMoonlight RdBay Saltseedlings Lake reveg −5 Foothills Southerns remnant MarshallsMoonlight SaltRdBay seedlings Lake reveg (c) Southerns reveg Southerns reveg MtQuarrySalt Liverpool Lake remnant 00 −10−15 −10 −5 Southerns reveg 0 Sebs Patch5 Sebs Patch SouthernsMoonlight reveg Bay Mt Liverpool Salt Lake remnant Quarry Salt Lake remnant −5 var) PC2 (3.3% explained −10 SouthernsMarshalls reveg Rd seedlings Marshalls Rd Salt Lake remnant Quarry Quarry Salt Lake remnant Mt Liverpool PC2 (3.3% explained var) PC2 (3.3% explained −10 Moonlight Bay Salt Lake remnant −−1010 Mt Liverpool Southerns remnant Salt Lake remnantSouthernsSaltQuarry Lake reveg revegremnant 0 −15 −10 −5 0 Foothills 5 SebsMarshallsMt Patch Liverpool Rd seedlingsMoonlight Bay Sebs Patch Foothills Salt Lake remnantreveg Marshalls Rd −15SouthernsPC1 remnant (5.9%−Marshalls10 Rd seedlingsexplained−5 var.) 0 5 QuarrySebs Patch Salt Lake revegetation −5 −15 −10 SouthernsPC1 remnant (5.9%−5 explained var.)Foothills 0 SouthernsMtMoonlightQuarrySalt 5LiverpoolMoonlightMarshalls Lake BayremnantrevegSalt LakeRdBay reveg seedlings Southerns reveg Mt Liverpool −15 −10 −5 Mt Liverpool 0 5 Sebs Patch Moonlight Bay 0 −10 Sebs Patch −5 Marshalls Rd Marshalls Rd seedlings Mt Liverpool MoonlightSalt Bay Lake revegetation 0 Salt Lake remnant −5 K=3 var.) PC2 (3.3% explained Marshalls Rd Mt Liverpool Salt Lake reveg 0 var.) PC2 (3.3% explained −15 −10 −5 0 5

PC2 (3.3% explained var.) PC2 (3.3% explained Marshalls Rd PC2 (3.3% explained var) PC2 (3.3% explained Salt Lake reveg PC1 (5.9% explained var.)Southerns remnant QuarrySebs Patch

PC2 (3.3% explained var) PC2 (3.3% explained Salt Lake revegSalt LakeMoonlight reveg Bay PC2 (3.3% explained var) PC2 (3.3% explained −10 MtQuarrySalt Liverpool LakeMt remnant LiverpoolSebs Patch Sebs Patch (C)−5 var) PC2 (3.3% explained Moonlight BayMoonlightSalt Bay Lake revegetation K=3 PC1 (5.9% explained var.) Sebs Patch Salt LakeSalt LakeMoonlight reveg revegSoutherns Bay remnant PC2 (3.3% explained var.) PC2 (3.3% explained SebsMoonlight Patch Bay −15 −10 PC1 (5.9%−5 explained var.)0 5 SaltMarshalls Lake reveg Rd seedlings Marshalls Rd seedlings PC1 (5.9% explained var.) SouthernsSebsMarshallsSalt Patch Lake reveg Rdreveg Southernsseedlings remnant Marshalls Rd seedlings −15 −10 Southerns reveg −5 0Southerns reveg 5 QuarrySaltSebsMtQuarry Lake PatchSaltLiverpool Lake remnant reveg Sebs Patch Mt Liverpool Southerns reveg MtQuarrySalt LiverpoolMoonlight Lake remnant Bay 0 0 MoonlightSaltMarshalls Lake remnant Bay Rd seedlings Marshalls Rd Marshalls Rd seedlings Mt Liverpool Southerns reveg MoonlightSalt Lake remnant Bay Quarry Southerns remnant PC2 (3.3% explained var) PC2 (3.3% explained −10 −10−15−15 −10 −5−5 0 0 5 5 0 0 −10−15 −10 −10−10 −5 0 Southerns remnant 5 Salt Lake remnant 0 Quarry Moonlight Bay PC2 (3.3% explained var) PC2 (3.3% explained −5 var) PC2 (3.3% explained −10 Salt Lake revegetation K=3 Salt Lake remnant FoothillsSalt Lake reveg PC2 (3.3% explained var.) PC2 (3.3% explained PC2 (3.3% explained var) PC2 (3.3% explained −10−15SouthernsPC1 remnant (5.9%− 10explainedPC1 (5.9% var.) −explained5 var.) 0 SaltQuarry5 LakeQuarrySebsSalt reveg Lake Patch remnantreveg PC1 (5.9% explained var.) Mt SaltLiverpool Lake reveg Foothills Southerns remnant Sebs Patch −15Southerns remnant Marshalls−10 Rd seedlings PC1 (5.9%−5 explained var.)0 5 SaltSebsSouthernsMoonlightQuarrySebsSalt Lake Patch Lake PatchremnantrevegMt remnant BayremnantrevegLiverpool PC1 (5.9%Marshalls Rd seedlings explained var.) QuarrySaltSebsMarshalls Lake Patch remnantMoonlightreveg Rd seedlings Bay Southerns reveg MtQuarry LiverpoolMtMoonlight LiverpoolSebs Patch Bay 0 MtSebs Patch Liverpool 0 Mt Liverpool Southerns remnant PC2 (3.3% explained var.) PC2 (3.3% explained Marshalls Rd Quarry Mt Liverpool PC2 (3.3% explained var) PC2 (3.3% explained 0 −10 Southerns remnant

PC2 (3.3% explained var.) PC2 (3.3% explained Marshalls Rd Salt Lake remnant PC2 (3.3% explained var) PC2 (3.3% explained 0 −10 Southerns revegetationSebs Patch PC2 (3.3% explained var) PC2 (3.3% explained PC2 (3.3% explained var) PC2 (3.3% explained 0 Sebs PatchMoonlight Bay Foothills PC2 (3.3% explained var) PC2 (3.3% explained −15 −10 −5 0 5 SebsSalt Lake Patch remnantrevegSouthernsMoonlight Bay revegetation SouthernsPC1PC1 remnant (5.9% (5.9% explained explainedPC1 var.) (5.9% var.) explained var.) SouthernsQuarry remnant Salt Lake reveg PC1 (5.9% explained var.) Southerns SebsremnantSalt Lake reveg Patch Southerns remnant Quarry −15 PC1 (5.9%− 10explained var.)−5 0 Salt Lake5 reveg Southerns remnant Marshalls Rd Marshallsseedlings Rd seedlings −15 −10 −5 0 5 SaltSouthernsMtSebs Lake Liverpool PatchremnantSebs remnant Patch Southerns reveg Sebs Patch Southerns reveg SouthernsMoonlightSalt remnant Bay Lake remnant Quarry SaltSalt Lake Lake Quarryreveg remnant −5 −15 −10 −5 Quarry 0 5 MtQuarrySaltSebs Patch LiverpoolQuarry Lake remnant Quarry QuarryMt LiverpoolMt Liverpool 0 −5 −15 −10 −5 0 Mt Liverpool5 Mt MoonlightLiverpoolSalt Lake remnant MoonlightSalt Bay Lake remnant Bay PC2 (3.3% explained var.) PC2 (3.3% explained −10 Marshalls Rd Foothills Foothills Southerns remnant PC2 (3.3% explained var) PC2 (3.3% explained −10−15 −10 −5 0Foothills Southerns remnant Foothills5 Sebs Patch

PC2 (3.3% explained var) PC2 (3.3% explained −10 0 0 var) PC2 (3.3% explained 0 −10−15 −10 −5 0 5 Moonlight Bay

PC2 (3.3% explained var) PC2 (3.3% explained PC1 (5.9% explained var.) Salt Lake reveg 0 Sebs Patch PC2 (3.3% explained var) PC2 (3.3% explained Sebs Patch Southerns remnant PC2 (3.3% explained var) PC2 (3.3% explained −15 −10 −5 0 5 Southerns reveg Marshalls Rd seedlings Sebs PatchSebsSouthernsSebs Patch Patch remnant PC1 (5.9% explained var.)SouthernsMt reveg Liverpool SaltQuarrySalt Lake Lake reveg remnant Marshalls Rd Sebs Patch SouthernsSebs Patch revegremnant PC1 (5.9% explained Mtvar.) Liverpool SaltSaltSoutherns LakeLake remnant reveg remnant Marshalls Rd seedlings QuarryMtSaltSebs Liverpool Lake Patch remnantreveg Marshalls Rd Sebs Patch MoonlightSalt Lake remnant Southerns BaySoutherns remnant revegetation Marshalls Rd seedlings Salt Lake SaltSebsremnant LakeMoonlight Patch Bay remnantreveg Mt Liverpool QuarryQuarry Southerns remnant Southerns revegetation Mt LiverpoolSalt Lake reveg 0 Marshalls− Rd10−15 −10 −5 Mt Liverpool 0 5 QuarryMoonlight Bay Southerns reveg SaltMt Lake reveg Liverpool −50 Marshalls Rd Southerns remnant

PC2 (3.3% explained var) PC2 (3.3% explained PC1 (5.9% explained var.) PC2 (3.3% explained var) PC2 (3.3% explained −5 Southerns reveg 0 −5 PC1 (5.9% explained var.) Salt Lake remnant Moonlight Bay 0 var) PC2 (3.3% explained PC1 (5.9% explained var.) Southerns reveg SouthernsSoutherns revegetation remnant −−105 Southerns remnant SouthernsSebsSouthernsSalt Lake remnantPatch reveg remnantMoonlight Bay 0 Marshalls Rd seedlings SaltSouthernsSaltSebsSalt Lake reveg Lake Patch reveg reveg remnantreveg −10 PC1 (5.9% explained var.)Southerns remnant Southerns reveg QuarrySaltSaltSebsQuarrySouthernsSalt Lake Lake Patch reveg remnant reveg remnant Southerns revegetation −10 −15 −10Quarry PC1 (5.9%−5 Southerns explained reveg 0 var.) 5 QuarrySaltMtSaltSoutherns Lake Liverpool Lake Southernsremnant remnantreveg remnant remnant K=4 −15 −10 −5 Southerns reveg Quarry0 5 Quarry Southerns remnant −10−5 K=4 −5 −15 −Quarry10 −5 Marshalls Rd seedlings 0 5 Mt LiverpoolSaltSoutherns Lake remnantreveg remnant 0 var) PC2 (3.3% explained −50 Marshalls Rd seedlings Salt Lake remnant −5 −10 Quarry Foothills Foothills Quarry Southerns revegetation PC2 (3.3% explained var) PC2 (3.3% explained 0 −5 −15 −10 −5 0 Salt Lake remnant 5 Mt Liverpool Southerns remnant Southerns remnantreveg PC2 (3.3% explained var) PC2 (3.3% explained 0 PC1 (5.9% explained var.) SebsSoutherns Patch remnant Quarry Foothills SaltSouthernsFoothillsSoutherns Lake reveg remnantreveg remnant −10 Southerns remnant SaltSebsSaltSebsSalt PatchSouthernsSebs Lake Lake Patch Lake Patchremnant reveg remnantreveg revegremnant Quarry Mt Liverpool QuarrySalt Lake reveg PC2 (3.3% explained var) PC2 (3.3% explained 0 −10 −5 SaltMt Lake Liverpool remnant −10 −15 −10 Marshalls Rd−5 0 Sebs Patch 5 SaltQuarry Lake remnant −10−5 Sebs Patch Southerns revegetation −10 Foothills Foothills SouthernsMoonlight reveg Bay Mt Liverpool PC2 (3.3% explained var) PC2 (3.3% explained −10 PC1 (5.9% explained var.) SebsSoutherns Patch remnant −15 −10 −5 0 5 Mt Liverpool −5 SaltSalt Lake revegLake reveg

PC2 (3.3% explained var) PC2 (3.3% explained 0 Marshalls Rd Marshalls Rd Sebs Patch

PC2 (3.3% explained var) PC2 (3.3% explained PC1 (5.9% explained var.) −5 −Marshalls15 Rd seedlings −10 −5 0 Southerns reveg 5 Salt Lake remnant 0 PC1 (5.9% explained var.) SebsSouthernsSebs Patch Patch Quarryremnant Southerns revegetation PC2 (3.3% explained var) PC2 (3.3% explained Marshalls Rd seedlings SouthernsSebs Patch revegremnant Moonlight Bay Mt Liverpool Salt Lake reveg −5 Mt Liverpool SaltSebsSouthernsSalt Lake SaltPatch Lake remnant remnant remnant reveg Moonlight Bay Marshalls Rd Sebs Patch SaltSoutherns Lake remnant reveg Salt Lake reveg −15Marshalls Rd −10−10 −5 0 5 Southerns remnant SaltQuarrySaltSouthernsSebs Lake Lake Patch remnantreveg reveg revegSoutherns revegetation −5 Southerns reveg Salt Lake reveg Moonlight Bay 0 −1015 −10 −5 0 Mt Liverpool 5 PC1 (5.9% explained var.) Salt LakeSalt Lakeremnant reveg PC2 (3.3% explained var) PC2 (3.3% explained Quarry −10 Marshalls− Rd5 −15 −10 −5 Southerns reveg 0 5 Salt Lake remnant Moonlight Bay Southerns reveg −−−105010 var) PC2 (3.3% explained K=4 PC1 (5.9% explained var.) Southerns Southernsreveg reveg PC2 (3.3% explained var) PC2 (3.3% explained −10 Southerns remnant Southerns remnant −5 Marshalls Rd seedlings Southerns reveg PC2 (3.3% explained var) PC2 (3.3% explained Sebs Patch −5 −5 Sebs SaltPatch Lake remnant SaltSalt Lake revegMoonlight reveg Bay 0.36 −10 PC2 (3.3% explained var) PC2 (3.3% explained 0 PC1 (5.9% explained var.) Salt Lake remnantSaltSouthernsSoutherns Lake reveg remnantreveg reveg −10 Southerns remnant SouthernsSebs PatchQuarrySalt reveg Lake remnant SebsSalt SaltPatch Lake reveg reveg 0.36 −10 −15 −15−10 −10−5 SouthernsPC1 remnant 0(5.9%−5 explainedSoutherns reveg var.)5 0 5SaltSaltSebsSoutherns Lake Lake Patch reveg remnant remnantreveg −10K=4 −5−15 −15 −10−15−10 −−510−5 0−5 0 Southerns reveg 50 5 5 QuarrySalt Lake reveg −5 −5 K=4−15 −10 −5 0 Marshalls Rd seedlings 5 Salt Lake remnant Marshalls Rd seedlings PC2 (3.3% explained var) PC2 (3.3% explained 0 −5 var) PC2 (3.3% explained Salt Lake remnant −−−10510 PC1 (5.9%Quarry explained var) Southerns reveg PC2 (3.3% explained var) PC2 (3.3% explained 0 Salt Lake remnantSoutherns remnant −−105 var) PC2 (3.3% explained −10 PC1 (5.9%Quarry explainedSoutherns remnantvar) SaltSebsSoutherns Lake Patch revegSebsSoutherns remnantreveg Patch remnantreveg

PC2 (3.3% explained var) PC2 (3.3% explained PC1 (5.9% explained var.) Southerns remnantreveg −15 −10 Southerns remnant−5 0Foothills 5 SebsSebs Patch PatchSalt Lake reveg Marshalls Rd seedlings SaltSaltSoutherns Lake Lake Saltreveg revegremnant remnant Lake remnant −10 −10 −5 −−1515 −−1010 −−55 0 Foothills55 SebsSalt Lake Patch remnantreveg −5−10 −5− var) PC2 (3.3% explained 10−−−15515 −−1010 PC1−5− (5.9%5 explained0 var)0 5 5 Southerns reveg Sebs Patch

PC2 (3.3% explained var) PC2 (3.3% explained Southerns remnant

PC2 (3.3% explained var) PC2 (3.3% explained PC1 (5.9% explainedPC1 (5.9% var) explained var.) PC2 (3.3% explained var) PC2 (3.3% explained −−1015 −−1015 −15 PC1−5− (5.9%10−10 explainedPC10 var) (5.9%−−55 explained5 0 var.)0 5 Sebs5Southerns Patch reveg Marshalls Rd seedlings PC1 (5.9% explained var) Mt Liverpool Sebs Patch Southerns remnant MarshallsMarshalls Rd Rd seedlings SebsSoutherns Patch remnant PC2 (3.3% explained var) PC2 (3.3% explained Sebs PatchSoutherns remnant −10 Mt Liverpool Sebs Patch −5 Marshalls Rd SaltSoutherns LakeSalt remnantreveg remnantreveg Lake reveg Sebs PatchSalt MoonlightLake Bay remnant 0.36 −−155 −10 −5 PC10 (5.9% explained var.)5 Salt Lake reveg −15 − var) PC2 (3.3% explained 10−−1515 −10 −−1010 −−55−5 0 0 5 5 5 SouthernsSalt Lake reveg reveg Moonlight Bay

PC2 (3.3% explained var) PC2 (3.3% explained PC1 (5.9% explained var) Southerns remnant −10 −15 −10 −5 0 5 SebsSalt LakePatch reveg −5 −15 −10 −5 0 Southerns reveg 5 Southerns reveg PC2 (3.3% explained var) PC2 (3.3% explained 0.35 −10 K=5 PC1 (5.9% explained var) SebsSouthernsSoutherns Patch remnant remnantreveg PC2 (3.3% explained var) PC2 (3.3% explained PC1 (5.9% explained var) Southerns reveg PC2 (3.3% explained var) PC2 (3.3% explained

0.35 −10 Salt Lake remnant Sebs Patch 0.36 −10 −5 K=5 PC1PC1 (5.9% (5.9% explainedQuarry explainedPC1 var) var)(5.9% explained var.) SaltSoutherns LakeSouthernsSebs reveg remnantreveg Patch remnantreveg 0.36 −5 −−155−15 −10−10 −5 −5 PC1Southerns remnant 0(5.9%0 explained5 var.)5 Salt Lake remnant SouthernsSaltSebs Lake Patch Saltreveg reveg Lake reveg −15−15−5 −15 −10−10 −−1010 −5 −5−5 Southerns remnant0 00 Foothills55 5 SouthernsSoutherns remnant revegremnant −5 −10 var) PC2 (3.3% explained −10 PC1PC1 (5.9% (5.9% explainedQuarry explained var) var) Southerns remnantreveg PC2 (3.3% explained var) PC2 (3.3% explained −15 −5 −10 −5 0 5 SebsSoutherns Patch reveg PC2 (3.3% explained var) PC2 (3.3% explained PC1 (5.9% explained var) PC1 (5.9% explained var) Sebs Patch Salt Lake reveg −10 MarshallsPC1 Rd seedlings (5.9%PC1 (5.9%Quarry explained explained var) var) SouthernsSebs PatchSoutherns remnant remnantreveg −5 Foothills Marshalls Rd seedlings Mt Liverpool Southerns SaltSebsremnantreveg Lake Patch reveg −5 −15 Marshalls Rd −10 −5 0 5 −5 Foothills Southerns remnantreveg −5 var) PC2 (3.3% explained −−1515 −10−10 PC1−5 (5.9%−5 explained0 var) 0 5 5 Sebs Patch Southerns reveg Moonlight Bay

PC2 (3.3% explained var) PC2 (3.3% explained −5 PC1 (5.9% explained var) Southerns remnant −15 −10 −5 0 5 SebsSalt LakePatch reveg −10 Mt Liverpool Southerns reveg PC2 (3.3% explained var) PC2 (3.3% explained −5 PC1 (5.9% explained var) Southerns remnant −15 −10Marshalls Rd −5 0 5 SouthernsSebs Patch Southernsreveg reveg −15PC1 (5.9%− explained10 var)−5 Southerns reveg 0 5 −10 Sebs Patch 0.35 −5 K=5 PC1PC1 (5.9% (5.9% explained explained var) t var) SouthernsSebs Patch remnant −5 −15 −10 −5 Mt Liverpool0 5 Salt Lake remnantSoutherns reveg Moonlight Bay 0.34 PC2 (3.3% explained var) PC2 (3.3% explained −15 −10 Marshalls Rd −10 −5 Quarry 0 t 5 SouthernsSalt Lake reveg reveg −5 Southerns remnant . 0.34 −5 PC1 (5.9% explained var) Southerns remnant −10−5 Quarry Southerns reveg SebsSoutherns Patch reveg 0.35 K=5 PC1 (5.9% explained var) Foothills SouthernsSoutherns .reveg remnant Moonlight Bay PC2 (3.3% explained var) PC2 (3.3% explained −15 −10 −10 −5 s 0 5 Salt Lake reveg −5 −10 PC1PC1 (5.9% (5.9% explainedQuarry explained var)PC1 (5.9% var) explained var.)Foothills Salt Lake remnantSouthernsSoutherns reveg revegremnant −5 −10 −15 s −10 Southerns remnant−5 Southerns reveg 0 5 Sebs Patch CV error −5 Sebs Patch 0.35 K=5−10 MarshallsPC1 Rd seedlings (5.9% explainedQuarry var)PC1 (5.9% explained var.)Foothills Southerns revegremnant −10 −15 −10 −5 Mt Liverpool 0 5 CV error −5 −5 Marshalls− Rd10 Foothills Sebs Patch Salt Lake Salt LakeSoutherns remnant remnantreveg PC1 (5.9% explained var) Mt Liverpool Salt Lake −5 Marshalls Rd SouthernsSoutherns remnant Saltemnant Lake Salteveg Lake Moonlight Bay −10 −15 −10Marshalls Rd seedlings −5 Southerns0 Southerns 5 r emnant Salt Lake revegr Southerns revegremnant −5 Moonlight Bay −10 −15 −10−10 −5 remnan0 Southernseveg. reveg 5 Sebs Patch r Southernseveg reveg −10 −PC115 (5.9% explained−10 var)PC1remnan (5.9%−5 explainedSouthernsr var.)0 Sebs Patch 5 SouthernsSouthernsSalt Lake revegr remnant reveg −5 −15 Marshalls Rd −10 −5 PC1 0(5.9%t Southernsexplainedeveg. reveg Mt Liverpool var.)5 Salt Lake remnant −5 −15 −10 Marshalls Rd −5 Southerns remnant0 r Mt Liverpool5 Salt Lake remnant 0.34 Marshalls Rd seedlings 0.33 −10 Marshalls −Rd10 Quarry Moonlight Bay −5 −15 −10−15 Marshalls Rd −−510 Southerns remnant0−5 50 5 SouthernsSoutherns .reveg remnantreveg 0.33 seedlingPC1 (5.9% explained var) Moonlight Bay s t Foothills Southerns reveg

0.34 seedlingPC1 (5.9% explained var) −10 −10 Marshalls Rd seedlings −5 −−105 −15 −10−15 −−510Quarry PC1 (5.9%0−5 explained var.)50 5 . CV error −5−10 −15 −10 −10 Marshalls Rd seedlings −5 0 5 Sebs Patch PC1 (5.9% explained var) FoothillsFoothills −15 s PC1− (5.9%10 explained var)−5 Mt Liverpool 0 5 SouthernsSoutherns reveg reveg −10 −10 Marshalls Rd PC1 (5.9%Quarry explainedPC1 var)(5.9%t explained var.) Salt Lake Salt Lake −10 −15 −10 Southerns−5 0 5 emnant Moonlight Bay 0.34 CV error −10 −5 Sebs Patch eveg −5 −10 −15 −10 −5 Quarry 0 5 Foothills r Salt Lake reveg −10 PC1 (5.9% explained var) SouthernsMt Liverpool r . Marshalls Rd remnan Southernseveg. reveg Sebs Patch Salt Lake PC1 (5.9%Quarry explainedPC1Southerns var) (5.9% explained var.) Foothills emnant Salt SouthernsLake reveg −10 −−1510 −15 −10−10 s −5−5 00 r Mt Liverpool55 Salt Lake remnant eveg Moonlight Bay Marshalls Rd Quarry Southerns Foothills r Salt Lake revegr 0.32 −10 −−1015−15 −10−10 Marshalls Rd −5−5Quarry PC1Southernsremnan remnant 0(5.9%0 explained55 var.) Sebs Patch 0.33 −10 −15 −10 −5 Southernseveg. reveg 0 5 CV error −5 PC1 (5.9% explained var.) Sebs Patch Moonlight Bay Foothills

0.32 Foothills −10 −15 −10 seedlingPC1 (5.9%−5 explained var)0 r Mt LiverpoolMt Liverpool5 Sebs Patch Salt Lake remnant −15 Marshalls Rd Marshalls Rd −10 Marshalls Rd −5 Southerns remnant0 Mt Liverpool 5 Salt Lake Salt Lake Marshalls Rd Sebs Patch 0.33 −10−−1015−15 −10−10Marshalls Rd seedlings PC1− (5.9%−55 explainedSoutherns var)0 55 emnant Moonlight Bay Moonlight Bay −10 −15 −10Marshalls Rd −5 0 Mt Liverpool 5 r Moonlight Bayeveg −15 −10 seedlingPC1PC1PC1 (5.9% (5.9% (5.9%−5 explained explained explained var) var)0 Southerns 5 Salt Lake reveg Salt Lake revegr PC1 (5.9% explainedremnan var) Sebs Patch Moonlight Bay −10 SouthernsSoutherns revegeveg. reveg −10 −15Figure−15 2. (a) The −Bayesian10−10Marshalls Rd Information seedlings − 5Criterion−5 (BIC)PC1 values 00(5.9% for each explained number 5of5 var.)clusters, with the lowest BICSalt Lake indicatingreveg support for 3 or −10−10 PC1 (5.9% explained var) Southerns revegr Mt Liverpool Salt Lake remnant Salt Lake remnant FigureMarshalls 2. Rd(a) The Bayesian InformationPC1 (5.9% Criterion explained (BIC) valuesSoutherns var) remnant for each number of clusters, with the lowest BIC indicating support for 3 or −15 −15 −10 −Marshalls10 Rd PC1PC1− 5(5.9% (5.9%−5 explained explainedSoutherns var) remnantvar)00 5 5 Salt Lake remnant

0.33 PC1 (5.9% explained var) −10 Southerns remnant Moonlight Bay −−1010 −−10154− clusters15 plus the− ADMIXTURE10−10seedlingPC1 CV PC1(5.9% error− (5.9%−55 results explainedQuarry explained -The cv var)errorsvar)0 are plotted for5 K=15 -10, with the lowest cv error indicating greatest Marshalls Rd seedlings Foothills

0.32 PC1PC1 (5.9% (5.9% explained explained var)var) 2 4 6 8 −10 10 −104 clusters−15 plus theMarshalls ADMIXTURE− Rd10 seedlings CV error−5 results -The cv errors0 are plotted for5 K=1-10, with the lowest cv error indicating greatest −15 −15 −10 −10 Marshalls Rd seedlings −5−5 Quarry 00 5 5 2 4 6 8 10 −10 −10 −15 −10 PC1 (5.9%−5 explained var)0 5 Sebs Patch −10 −15 −10 −5 0 5 Foothills

0.32 support at K=7 (b) PrincipalPC1 Component (5.9% Analysis explained showing var) population genetic structure across all samples. Colours represent stands and PC1 (5.9% explained var) Mt Liverpool −10 −15 Marshalls Rd −10 PC1PC1 (5.9% (5.9%−5 explained explained var)0 5 −10 support at K=7 (b) Principal Component Analysis showing population genetic structure acrossSebs Patch all samples. Colours representMoonlight Baystands and −10 −15 −10 −5 Quarry 0 5 K −−1015−15 −10−10 PC1− (5.9%5−5 explained var)00 Mt Liverpool 55 Salt Lake reveg −10 Marshalls Rd Southerns reveg K Figureellipses− 152. (a) identify The Bayesian the genetic−10 Information PC1clustersPC1PC1 (5.9% (1 (5.9%Criterion- 4)(5.9%− 5identified explained explainedQuarry explained (BIC) in valuesthe var) DAPCvar)var)0 for analysiseach number for K=4.5 ofFoothills (c) clusters,Bar plots with represent the lowest individual BIC indicating genetic cluster support for 3 or PC1 (5.9% explained var) Salt Lake remnant Moonlight Bay ellipses identify the genetic clusters (1-4) identifiedQuarry in the DAPC analysis for K=4. (c) Bar plots represent individualSalt Lake reveg genetic cluster −10 −15 −15 −15 −10 −10−10 PC1−5− 5(5.9%−5 explainedSoutherns remnantvar)000 5 Foothills5 Sebs Patch −10 Southerns reveg −10Figure−15 2. (a) The Bayesian−10 Information Criterion−5 (BIC) values0 for each numberMt Liverpool of5 clusters,Foothills with the lowest BIC indicating support for 3 or Marshalls Rd 0.32 Figureassignment 2.−15(A) The from Bayesian ADMIXTURE−10 PC1 InformationresultsPC1 (5.9% (5.9%from−5 K=3explained explained to Criterion K=5 (*note var) var)0 - beyond (BIC) K=5 values the5 ADMIXTURE forSebs each Patch resultsSalt number Lake remnant appeared ofnoisy clusters, but see Fig. with A1 for the PC1 (5.9% explained var) Mt Liverpool Moonlight Bay 4 clusters plus the ADMIXTURE CV error results -TheSoutherns cv errorsremnant are plotted for K=1-10, with the lowest cv error indicating greatest −15assignment−15 fromMarshalls −ADMIXTURE Rd10−10Marshalls Rd seedlings resultsPC1− 5from(5.9%−5 K=3 explained to K=5 (*note var)00 - beyond K=5 the5 ADMIXTURE Sebsresults Patch appearedSalt Lake reveg noisy but see Fig. A1 for 2 4 6 8 −10 −15−10 −10 −5 0 Southerns reveg 5 Moonlight Bay −10 −15 −10 PC1− (5.9%5 explained var)0 Mt Liverpool5 Salt Lake reveg 4 clustersMarshalls plus Rd the ADMIXTURE CVPC1 error (5.9% results explained -The cv errorsvar) are plotted for K=1-10, with theSalt Lake lowest remnant cv error indicating greatest full output). Marshalls Rd seedlingsPC1PC1 (5.9% (5.9% explained explainedSoutherns var) remnant Southerns reveg 2 4 6 8 10 −10support−15 at K=7 (b) Principal−10 ComponentPC1− 5Analysis(5.9% explained showing var) 0population genetic5 structure acrossSalt allLake remnant samples. Colours represent standsMoonlight Bay and −15 −15full output).−10 −10 −5 −5 Southerns remnant00 5 5 Salt Lake reveg −10 −10 PC1PC1 (5.9% (5.9% explained explained var) var) Southerns reveg Figurelowest 2. (a) BIC The indicatingBayesian Information supportMarshalls Rd seedlings Criterion for 3 or (BIC) 4 clusters values plusfor each the number ADMIXTURE of clusters, cross-validation with the lowest BIC error indicating (cv error) support for 3 or K support at K=7 (b) Principal ComponentPC1PC1 (5.9% (5.9% Analysis explainedQuarry explained showing var) population genetic structure across all samples.Salt Lake remnant Colours represent stands and −10ellipses−−1515 identify the −genetic−1010 Marshalls clusters Rd seedlings (1-4)−−5 5identified in Southernsthe DAPCremnant00 analysis for K=4.55 Foothills (c) Bar plots represent individual genetic cluster K −10 −15 −10 PC1− 5(5.9%Quarry explained var)0 5 PC1 (5.9% explained var) Sebs Patch ellipses identify the genetic clustersPC1 (1(5.9%-4) identified explained in the DAPCvar) analysis for K=4.Foothills (c) Bar plots represent individual genetic cluster 4 clusters− 15plus the ADMIXTURE−10 CV error −results5 -The cv errors0 are plottedMt Liverpool 5for K=1-10, with the lowest cv error indicating greatest assignment−15 fromMarshalls ADMIXTURE Rd Marshalls−10 Rd seedlings results from−5 K=3 to K=5 (*note0 - beyond K=5 the5 ADMIXTURE results appeared noisy but see Fig. A1 for 2 4 6 8 10 −10 −15results. The cv−10 errors are plottedPC1− 5(5.9% for explainedQuarry K = 1–10, var)0 with the lowest5 cvSebs error Patch indicating greatestMoonlight Bay support PC1 (5.9% explained var) Mt Liverpool Salt Lake reveg assignment−15 fromMarshalls ADMIXTURE Rd −10 PC1 results (5.9% from−5 K=3explainedQuarry to K=5 (*note var)0 - beyondSoutherns reveg K=5 the5 Foothills ADMIXTURE results appeared noisy but see Fig. A1 for Salt Lake remnant Moonlight Bay supportfull at K=7output). (b) Principal ComponentPC1 Analysis (5.9% explained showing var) population geneticFoothills structureSebs Patch across all samples.Salt Lake reveg Colours represent stands and Southerns remnant PC1 (5.9% explained var) Southerns reveg Mt Liverpool −10 −15 −10Marshalls Rd −5 0 5 Sebs Patch Salt Lake remnant Moonlight Bay K full output). Marshalls Rd PC1 (5.9%Quarry explainedSoutherns remnantvar) Mt Liverpool Salt Lake reveg Marshalls Rd seedlingsPC1 (5.9% explained var) Southerns reveg at K = 7. (B) Principal Component Analysis showing population genetic structure acrossMoonlight Bay all samples. ellipses identify the genetic clusters (1-4) identified in the DAPC analysis for K=4.Foothills (c) Bar plots represent individual genetic cluster −15 −10 −5 0 5 Salt Lake remnant Salt Lake reveg Southerns remnant Southerns reveg Marshalls Rd seedlings PC1 (5.9% explained var) SebsSalt Patch Lake remnant Southerns remnant Marshalls Rd Mt Liverpool assignment−15 from ADMIXTURE−10 resultsMarshalls Rd seedlings from−5 K=3 to K=5 (*note0 - beyond K=5 the5 ADMIXTURE results appeared noisy but see Fig. A1 for Moonlight Bay Colours represent standsMarshallsPC1 Rd andseedlings (5.9% solid explainedQuarry line ellipses var) identify the genetic clusters (1–4) identifiedSalt Lake reveg in the Southerns reveg Foothills full output). Quarry Salt Lake remnant Southerns remnant Sebs Patch PC1 (5.9% explainedQuarry var) Foothills Marshalls Rd Mt Liverpool Foothills Quarry Sebs Patch Moonlight Bay Discriminant AnalysisMarshalls Rd seedlings of Principal Components (DAPC)Mt Liverpool analysis for K = 4. CircleSalt Lakewith reveg the dashed line Marshalls Rd Southerns reveg Foothills Sebs Patch Mt Liverpool Marshalls Rd Salt Lake remnant Moonlight Bay Southerns remnant Sebs Patch Salt Lake reveg Moonlight Bay Marshalls Rd Southerns reveg Mt Liverpool Salt Lake reveg Southerns reveg Salt Lake remnant Moonlight Bay Southerns remnant Salt Lake remnant Salt Lake reveg highlights the seedlingsMarshalls Rdthat seedlings grouped withSoutherns a remnantdifferentSoutherns reveg genetic cluster (cluster 4) to the Marshalls Rd Quarry Salt Lake remnant Marshalls Rd seedlings Southerns remnant Marshalls Rd seedlings Foothills Marshalls Rd seedlings Quarry Sebs Patch Mt Liverpool mature plansMarshalls Rd and non-admixed seedlings (cluster 1). (C) Bar plotsFoothills represent individual genetic cluster Quarry Moonlight Bay Quarry Sebs Patch Salt Lake reveg Foothills Marshalls Rd Southerns revegMt Liverpool Foothills Quarry Salt Lake remnant SebsSebs Patch Patch Moonlight Bay Southerns remnant Mt Liverpool Foothills Salt Lake reveg assignment fromMarshalls RdMarshalls ADMIXTURE Rd results from K = 3Southerns to reveg KMt Liverpool= 5 (* note-beyond K = 5 the ADMIXTURE Sebs Patch Salt Lake remnant Moonlight BayMoonlight Bay Southerns remnant Mt Liverpool Salt LakeSalt reveg Lake reveg Marshalls Rd SouthernsSoutherns reveg Marshalls Rd seedlings Moonlight Bay Salt LakeSalt remnantLake remnant Salt Lake reveg SouthernsSoutherns remnant remnant Marshalls Rd seedlings Southerns reveg Salt Lake remnant Southerns remnant results appeared noisy butMarshalls Rd seedlings see Figure A1 for full output). Marshalls Rd seedlings

Marshalls Rd seedlings Quarry Quarry Foothills Foothills Sebs Patch Quarry Mt Liverpool Sebs Patch Marshalls Rd Foothills Marshalls Rd Mt Liverpool Moonlight Bay Sebs Patch Salt Lake revegMoonlight Bay Southerns reveg Salt Lake reveg Marshalls Rd Mt Liverpool Southerns reveg Salt Lake remnant Salt Lake remnant Moonlight Bay SouthernsSoutherns remnant remnant Salt Lake reveg Southerns reveg Salt Lake remnant Southerns remnant Marshalls Rd Marshallsseedlings Rd seedlings

Marshalls Rd seedlings

Quarry Foothills

Sebs Patch Marshalls Rd Mt Liverpool Moonlight Bay Salt Lake reveg Southerns reveg Salt Lake remnant Southerns remnant

Marshalls Rd seedlings Diversity 2020, 12, 299 7 of 18

At K = 3, individuals were clustered into groups that aligned with geography: a northern cluster containing samples from Quarry and all but the one above-mentioned Marshalls Rd individual (Cluster 1), and a second cluster containing all samples from Southerns (Cluster 2), and a southern cluster, containing all individuals collected from stands in the south (Salt Lake reveg. and remnant, Sebs Patch, Moonlight Bay, Foothills Rd, and Mt Liverpool, and one individual from Marshalls Rd; Cluster 3). In the ADMIXTURE analysis, ten of the Marshalls Rd seedlings showed approximately 50/50 admixture between Clusters 1 and 3 and, in the DAPC analysis, clustered with Cluster 3. For K = 4, substructure was revealed within the revegetated and remnant Salt Lake stands, with 17 individuals having almost 100% assignment to this 4th cluster (Cluster 4), with the rest displaying admixture between clusters 1 and 4. Individuals from the nearby Sebs Patch stand displayed a low level of admixture with Cluster 4 (average assignment 16%). The Marshalls Rd individual that showed 100% assignment to Cluster 3 when K = 3 had 100% assignment to Cluster 4, and the admixed seedlings showed 50% assignment to Cluster 4. Further substructure was revealed for K = 5, in Foothills Rd and Mt Liverpool, with the Foothills Rd and Mt Liverpool stands forming a distinct genetic cluster (Cluster 5) which distinguished them from Moonlight Bay plants. The majority of the Marshalls Rd seedlings clustered with the Quarry and Marshalls Rd samples in all genetic structure analyses. However, of the 46 seedlings genotyped, 10 of the samples showed ca. 50/50 ancestry with Marshalls Rd and Salt Lake in the ADMIXTURE analysis, suggesting that they are likely to be offspring from a cross between a Marshalls Rd individual and a Salt Lake individual. These seedlings were also located halfway between the Marshalls Rd samples and Salt Lake samples in the PCA, and clustered with some Salt Lake revegetation and remnant samples in the DAPC (Figure1). The genetic proximity of clusters generally reflected geographic proximity. This was supported by results of pairwise genetic differentiation (FST) between stands (Table1) and the RDA. Pairwise F ST between stands was reasonably high (a maximum of 0.193, Table1). However, there was high differentiation between the two geographically close stands Foothills and Mt Liverpool (pairwise FST = 0.185, Table1). These stands also displayed comparatively high genetic di fferentiation from all other stands (Mt Liverpool pairwise FST = 0.100 to 0.185 and Foothills pairwise FST = 0.129 to 0.193, Table1). The redundancy analyses of allele frequencies ~ space revealed that the spatial variables explained 83% of the variation in allele frequencies among stands (ANOVA, F = 6.43, p = 0.017), showing spatial variation to be a strong explanatory variable of the observed patterns of genetic differentiation. Results showed all historical revegetated stands clustered with their nearby remnant stands. Marshalls Rd (revegetated) and Quarry (remnant) were genetically very similar (Pairwise FST = 0.017, Table1) and clustered together in PCA, DAPC, and ADMIXTURE analyses, most likely reflecting the use of the Quarry stand as the source population for the Marshalls Rd stand. The samples taken from Salt Lake revegetation were genetically similar to samples taken from the remnant individuals at Salt Lake (Pairwise FST = 0.000; Table1.) and also clustered together in the PCA, DAPC, and ADMIXTURE analyses. Similarly, the samples collected from the Southerns revegetation stand were genetically similar to those from the Southerns remnant stand (Pairwise FST = 0.013; Table1.), again corroborated by the PCA, DAPC and ADMIXTURE analyses. Analysis of molecular variance revealed that the majority of genetic variation is found among all individuals (73.6%). Significant levels of genetic variation are also found among individuals within a collection stand (15.7%, p < 0.001), as well as between collection stands (10.7%, p < 0.001, Table2), as highlighted by the genetic structure analysis. Diversity 2020, 12, 299 8 of 18

Table 1. Pairwise genetic differentiation (measured by FST) between stands (values vary from 0-1 and values of 0 indicate no evidence of differentiation).

Marshalls Marshalls Rd Southerns Southerns Mt Salt Lake Salt Lake Moonlight Collection Stand Quarry Foothills Sebs Patch Rd Seedlings Remnant reveg. Liverpool remnant reveg. Bay Marshalls Rd 0 Marshalls Rd seedlings 0.008 0 − Quarry 0.017 0.032 0 Southerns remnant 0.090 0.101 0.087 0 Southerns reveg. 0.101 0.114 0.101 0.013 0 Mt Liverpool 0.156 0.158 0.153 0.122 0.145 0 Foothills 0.192 0.193 0.189 0.147 0.176 0.185 0 Sebs Patch 0.109 0.117 0.111 0.083 0.090 0.104 0.134 0 Salt Lake remnant 0.088 0.099 0.097 0.073 0.077 0.100 0.129 0.037 0 Salt Lake reveg. 0.115 0.123 0.126 0.097 0.104 0.122 0.148 0.061 0.000 0 Moonlight Bay 0.136 0.145 0.137 0.111 0.116 0.135 0.161 0.084 0.075 0.097 0 Diversity 2020, 12, 299 9 of 18

Table 2. Nested analysis of molecular variance (AMOVA) including standard deviations (SD; obtained through jackknifing over loci) and 95% confidence intervals (c.i. 95%; obtained through bootstrapping over loci) of F statistics.

Source of Variation Nested in %var F-Value SD c.i.95% Within Individual - 73.6 0.263 0.002 0.004 ± Among Individual Collection stand 15.7 0.175 0.002 0.004 ± Among Population 10.7 0.107 0.001 0.002 ±

3.2. Genetic Diversity and Inbreeding

Expected heterozygosity varied significantly but by a small degree between stands (HE = 0.117 to 0.141, Table3). Observed heterozygosity was consistently lower and less variable than expected heterozygosity (HO = 0.102 to 0.118, Table3), which is reflected in the positive inbreeding coefficients (F) found in all collection stands (F ranged from 0.110 to 0.226; Table3), indicating the likely presence of historical inbreeding. Mt Liverpool and Foothills had significantly lower inbreeding coefficients than other stands (F = 0.11, 0.13 respectively, Table3). Moonlight Bay, Sebs Patch, and Salt Lake remnant stands had the highest inbreeding coefficients (F = 0.226 to 0.211, Table3). The confidence intervals from the kinship analysis spanned zero for every stand, suggesting that relatedness, and therefore contemporary inbreeding, was low within stands (Table3). We found significant differences in HE,HO and F between the mature Marshalls Rd plants and both the non-admixed and admixed seedlings (Table3, Figure A2). The non-admixed seedlings displayed lower heterozygosity and higher F compared to the mature plants (Table3, Figure A2). In contrast, the admixed seedlings had higher heterozygosity compared to both the non-admixed seedlings and mature plants (Table3, Figure A2). Further, we found the inbreeding coe fficient was significantly lower in the admixed seedlings compared to the non-admixed seedlings (F = 0.036 and 0.181 respectively, Table3, Figure A2). Diversity 2020, 12, 299 10 of 18

Table 3. Stand status, sample size after filtering (n), census population size, location, observed (HO) and expected (HE) heterozygosity (both vary from 0 to 1, with 0 indicating no diversity), inbreeding coefficient (F) (varies from 0 to 1 and values of 0 indicate no evidence of inbreeding), and kinship (varies between 0 and 1, where 0 indicates no recent co-ancestry and 1 indicates samples are genetically identical). Includes a focused analysis for both the admixed and non-admixed Marshalls Rd seedlings.

Collection Stand Status n HO HE F Kinship All stands Marshalls Rd Translocation 12 0.114 (0.111 to 0.116) b,c 0.137 (0.135 to 0.140) c,d 0.173 (0.163 to 0.182) b 0.008 (0.018 to 0.027) a,b − − Marshalls Rd seedlings (all) Seedlings 44 0.107 (0.105 to 0.109) a,b 0.131 (0.128 to 0.133) b 0.183 (0.176 to 0.189) b 0.001 (0.003 to 0.002) a − Quarry Remnant 15 0.111 (0.109 to 0.114) b 0.136 (0.134 to 0.139) c,d 0.183 (0.174 to 0.192) b 0.004 (0.011 to 0.003) a − Southerns remnant Remnant 39 0.118 (0.116 to 0.121) c 0.145 (0.142 to 0.147) d 0.183 (0.177 to 0.189) b 0.000 (0.003 to 0.003) a − Southerns reveg. Revegetation 5 0.118 (0.115 to 0.121) c 0.140 (0.137 to 0.143) c,d 0.157 (0.143 to 0.170) b 0.006 (0.043 to 0.031) a,b − Mt Liverpool Remnant 14 0.109 (0.107 to 0.112) b 0.123 (0.120 to 0.126) a 0.110 (0.100 to 0.121) a 0.009 (0.026 to 0.007) a − Foothills Remnant 13 0.102 (0.099 to 0.105) a 0.117 (0.114 to 0.120) a 0.130 (0.119 to 0.141) a 0.008 (0.023 to 0.006) a − Sebs Patch Remnant 24 0.109 (0.107 to 0.111) b 0.138 (0.136 to 0.141) c,d 0.211 (0.204 to 0.218) c 0.001 (0.006 to 0.003) a − Salt Lake remnant Remnant 14 0.110 (0.108 to 0.112) b 0.141 (0.139 to 0.144) d 0.221 (0.212 to 0.229) c 0.011 ( 0.008 to 0.014) b − − − Salt Lake reveg. Revegetation 25 0.117 (0.115 to 0.120) b,c 0.138 (0.136 to 0.141) c,d 0.151 (0.144 to 0.159) b 0.001 (0.009 to 0.006) a − Moonlight Bay Remnant 23 0.104 (0.102 to 0.106) a 0.135 (0.132 to 0.137) b,c 0.226 (0.219 to 0.234) c 0.004 (0.000 to 0.008) a,b Marshalls Rd focus − − Marshalls Rd adults Translocation 12 0.114 (0.111 to 0.116) b 0.137 (0.135 to 0.140) b 0.173 (0.163 to 0.182) b 0.008 (0.018 to 0.027) a − − Marshalls Rd seedlings (non-admixed only) Seedlings 34 0.103 (0.101 to 0.106) a 0.126 (0.124 to 0.129) a 0.181 (0.173 to 0.188) b 0.008 (0.010 to 0.002) a − Marshalls Rd seedlings (admixed only) Seedlings 10 0.120 (0.117 to 0.123) c 0.125 (0.122 to 0.127) a 0.036 (0.023 to 0.049) a 0.117 (0.122 to 0.005) a − 95% confidence intervals in parentheses and homogeneous subgroups indicated by a, b, c, and d. Diversity 2020, 12, 299 11 of 18

4. Discussion Our analysis of genomic diversity and differentiation among the remaining stands of the endangered Acacia whibleyana has revealed many of the hallmarks of a species trapped in an extinction vortex. There was strong genetic structure among stands, indicating that gene flow between stands is likely restricted and genetic drift has driven divergence among stands. Positive inbreeding coefficients across all stands suggest the presence of historical inbreeding, however analysis of kinship shows the relatedness of remaining plants to be low within stands, suggesting the risks of contemporary inbreeding are low and potentially that this species has mechanisms for inbreeding avoidance. We found that seedlings resulting from admixture events between the distinct genetic clusters had higher genetic diversity compared to progeny of single-parent stands. Together, these results indicate that while the species presents low risks of contemporary inbreeding, the establishment of mixed genetic cluster stands may be required to boost the genetic diversity of subsequent generations and provide an escape route from the extinction vortex this species is facing.

4.1. Genetic Structure and Sources of Genetic Variation The level of genetic structure found among A. whibleyena stands was high, given the small geographic range of the species (<38 km), and spatial location was a strong explanatory variable of the genetic variation in the species. The fact that the remaining A. whibleyana plants were distributed across small subpopulations suggests that genetic drift has played a part in shaping the genetic differentiation of the stands, as has been found for other endangered plant species, including the comparable Acacia pinguifolia [36]. Acacias are mostly pollinated by generalists, with bees their most common pollinators [37]. Concerns have been raised over the generalised threat of pollinator limitation to genetic diversity and inbreeding [38], with supporting evidence from other endangered Acacias [37]. Pollinator limitation could, therefore, be part of the explanation for the high genetic structure and low levels of genetic diversity found in A. whibleyana and may represent a major threat to the long-term survival of the species. Another possible contributing factor is that the seed is predominantly dispersed by ants and therefore only over short distances [39]. This, coupled with the lack of alternative suitable habitats for seed to disperse to, may also limit gene flow, however pollen dispersal is thought to have a far greater effect on genetic structure [40]. Interestingly, we found strong genetic structure among stands that were closely located. The Southerns site was found to be significantly differentiated from the Marshalls Rd and Quarry sites despite its proximity to them (<1 km). Further, the highest level of genetic differentiation was between two of the most geographically proximate stands—Foothills and Mt Liverpool. Despite the high FST between these stands reflecting large differences in allele frequencies, they cluster together and separately from all other sites at K = 5 in the ADMIXTURE analysis, suggesting that they share variation not found elsewhere. The small population size of these stands is likely to have led to a pronounced effect of genetic drift increasing relative divergence between them.

4.2. Opportunistic Test of Admixture on Genetic Diversity We present evidence that seedlings at Marshalls Rd likely resulted from crosses between individuals from distinct genetic clusters. The likely source of this admixture is a single Marshalls Rd adult that clusters with Salt Lake individuals. This individual is likely to have originated from a translocation event, such as from seed from the Salt Lake site that was included in the original Quarry/Marshalls Rd translocation project. Plants were propagated for revegetation projects at both sites in the same nursery during this time, so we believe this genetic transfer is likely (Geraldine Turner, pers. Comm, May 2020.). While we cannot rule out the presence of other Salt Lake-like individuals at the Marshalls Rd site, as we did not genotype all mature plants and seedlings, the presence of at least one Salt Lake-like individual is strongly indicative of an occurrence of within-stand mating resulting in the admixed seedlings, rather than gene flow between the geographically distant Marshalls Rd and Salt Lake stands. Diversity 2020, 12, 299 12 of 18

The admixed seedlings provided us with an unexpected opportunity to explore the effects of inter-population crossing on heterozygosity and inbreeding. When comparing the admixed to the non-admixed seedlings, our results show a striking reduction in the inbreeding coefficient and increase in observed heterozygosity in the admixed seedlings (admixed vs. non-admixed seedlings: F = 0.181 vs. 0.036; Ho = 0.103 vs. 0.120; He = 0.126 vs. 0.125). These large differences suggest that including individuals from a non-local population could bolster the genetic diversity of a population by encouraging matings between genetically dissimilar individuals. We also saw reduced observed heterozygosity in the non-admixed seedlings compared to mature Marshalls Rd plants, indicating that the species is experiencing a reduction in genetic diversity across generations, likely due to increased inbreeding, pushing the species further down the extinction vortex. Alternatively, these results could indicate that the seedlings included in our study were more inbred than would normally make it to maturity, and that insufficient time has passed for inbreeding depression to manifest. Unfortunately, all of the seedlings genotyped in this study died, and so the fitness effects of higher or lower genetic diversity in the admixed and non-admixed seedlings respectively is unknown. Whilst it is promising to see that the admixed progeny display increased levels of heterozygosity, whether assisted gene flow among genetic clusters would prove an effective conservation strategy for increasing fitness in future generations is unclear. We see this as the next research goal for this species, with controlled crosses and genotype-fitness associations required to test these ideas.

4.3. Genetic Diversity and Inbreeding Levels of heterozygosity were similar across all stands and observed heterozygosity was consistently lower than expected heterozygosity in all stands, which is indicative of historical inbreeding. It is difficult to assess whether or not genetic diversity is low in this species in the absence of a reference species, ideally a more widespread Acacia species. To our knowledge, no other studies using genome-wide SNP markers have been carried out on Acacia species. However, studies on other predominantly outcrossing tree species that have utilised SNP markers report heterozygosity values of between 0.1 and 0.4 [41–45], suggesting that A. whibleyana is at the lower end of this range. Additionally, the increase in heterozygosity that we observe in the admixed seedlings demonstrate that the genetic diversity we observe is not evenly distributed among the stands and can potentially be boosted in future generations via admixture. However, the fitness consequences of this boost in genetic diversity requires further exploration. Australian Acacia species are generally preferential outcrossers [46–48], and therefore are likely to display mechanisms of inbreeding avoidance. In a study of Acacia myrtifolia [46], it was found that as relatedness between individuals increased, the number of set seed pods decreased. Low seed set has been observed in some stands of A. whibleyana [49], however our kinship analysis revealed relatedness among individuals in stands to be low. We also show there to be substantial genetic variation within individuals, meaning that the risks of contemporary inbreeding are low. There has also been suggestion that some endangered Acacia are intrinsically rare or can at least adapt to having lowered genetic diversity and small population size by increasing the levels of selfing or, in extreme cases, reproduce vegetatively [50,51]. It is unclear if A. whibleyana could respond in this manner.

4.4. Management Recommendations The results we present here demonstrate that the remaining stands of A. whibleyana exhibit low inter-stand gene flow with evidence that genetic drift, and potentially inbreeding, will draw the species further down the extinction vortex in subsequent generations. Our findings are supported by demographic observations of A. whibleyana, where low seed set, a lack of recruitment, and poor seedling survival have been identified as threats to the its survival [21]. Similar patterns were also observed for A. pinguifolia, which is another threatened species endemic to a similar region to A. whibleyana. A. pinguifolia presented high levels of historical inbreeding, strong fine-scale genetic structure, and limited Diversity 2020, 12, 299 13 of 18 inter-stand gene flow [36]. Crossing between populations was recommended as a strategy to help reduce the risk of extinction in this species [36]. The boost to genetic diversity that we observed in the seedlings resulting from natural crossings between two of the genetic clusters provides hope for the species and a potential escape from the extinction vortex. These chance crossings demonstrate that genetic diversity can substantially increase within a single generation. However, since a significant proportion of the diversity found between the remaining stands is an ongoing concern, we suggest that a long-term genetic rescue trial should be established. This trial should focus on simultaneously determining the effects and trade-offs of inbreeding vs. outbreeding depression, heterosis as a result from inter-stand crosses, and pollination deficiency. This trial would require experimentally crossing a range of selfed, intra-stand, and inter-stand individuals, spanning the extent of genetic distance observed, and monitoring plants into adulthood. This genetic rescue trial could be efficiently combined with an assessment of pollination deficiency; this pollination/pollinator effect is important to determine as it may be contributing to the poor demographic rates observed [21,38].

5. Concluding Remarks All too often, it is easy to lose optimism when managing an endangered species faced with extinction. Here, through an unexpected natural experiment, we have been given a glimmer of hope and guidance for the management of Acacia whibleyana. Through the analysis of the seedlings, we have evidence that mixing between genetically different stands can increase genetic diversity, at least for a period of time in the first generation. Further, our results suggest that without action, we may experience a decrease in genetic diversity with each generation. We hope that a cautious approach to genetic recue will help lessen the extinction risk faced by Acacia whibleyana by increasing its levels of genetic diversity and hopefully its adaptability in the face of environmental change.

Supplementary Materials: The following are available online at http://www.mdpi.com/1424-2818/12/8/299/s1, Supplementary file S1—The variant call format (vcf) file is available as supplementary (Acacia_whibleyana_paper.vcf). Reads and mapping files will be archived at the NCBI SRA (accession number TBA). Author Contributions: Conceptualization, C.B., M.F.B., A.J.L. and D.C.B.; Data curation, C.B. and M.F.B.; Formal analysis, C.B., M.J.C. and M.F.B.; Funding acquisition, M.F.B., A.J.L. and C.B.; Methodology, C.B., M.F.B., M.J.C., A.J.L., D.C.B., J.G.P. and R.F.; Project administration, C.B., M.F.B., M.J.C., A.J.L, D.C.B., J.G.P. and R.F.; Resources, M.F.B.; Supervision, M.F.B., A.J.L. and M.J.C.; Visualization, C.B., M.F.B., and M.J.C.; Writing—Original draft, C.B.; Writing—Review & editing, C.B., M.F.B., M.J.C., A.J.L., D.C.B., J.G.P., R.F. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Commonwealth Government’s Threatened Species Recovery Fund, Eyre Peninsula Natural Resources Management Board (EPNRMB), and the Australian Research Council (grant numbers DE150100542 and DP150103414). This project was supported by The Holsworth Wildlife Research Endowment & The Ecological Society of Australia. Acknowledgments: The authors wish to thank Geraldine Turner and Seb Drewer from the Eyre Peninsula Natural Resources Management Board (EPNRMB) for providing their assistance and expertise throughout this study, and everyone else who assisted us with sample collection. Thanks also go to Sam Turner-Blyth for help early on in the writing process. Finally, a special thanks to Jeremy Austin for his assistance behind the scenes. Conflicts of Interest: The authors declare no conflict of interest. Diversity 2020, 12, 299 14 of 18

Appendix A

Table A1. Summary of collection stands, habitat type, census counts, sample size after filtering (n), and land use history of Acacia whibleyana.

Collection Stand Habitat Type Census Count n Land Use History Road reserve. Control burn in 2018 resulted in A. Marshalls Road Roadside vegetation. 42 reveg. 12 whibleyana plants emerging post fire. 100+ seedlings 44 Post-disturbance regeneration in disused quarry, Council-owned land. Quarried up to 1950’s, stockpiling Quarry 189 remnant 15 surrounded by remnant eucalypt woodland gravel then woodchips (to 1993). 59 remnant 39 Southerns Remnant eucalypt woodland. Private land. 40ha fenced to exclude stock in May 2012. 8 reveg. 5 Private land subdivided for rural living & stock removed Mount Liverpool Remnant eucalypt woodland. 50 remnant 14 2005. Road reserve, burnt in 2005 bushfires. A. whibleyana plants Foothills Road Roadside vegetation. 17 remnant 13 emerged post fire. Private land under different land management regimes Remnant chenopod scrub along salt-scalded Sebs Patch 107 remnant 24 (grazed by sheep intermittently, a small portion fenced & drainage line and salt lake. revegetated) 137 remnant 14 Council-owned land of road intersection, fenced in 1995 to Salt Lake Remnant chenopod scrub adjacent to salt lake. 26 reveg. 25 exclude vehicles and rabbits. Private land. Approx. one third of population fenced Moonlight Bay Remnant mallee woodland on salt lake lunettes. 557 remnant 23 from stock. Remainder accessible to sheep on intermittent basis. Area around salt lake burnt in 2005 bushfires. Note. Adapted from Faast, R.; Blyth, C.; Breed, M.F.; Packer, J.G. Acacia Whibleyana: Part 1—Literature Synthesis to Inform Conservation of a Threatend Acacia; Eyre Peninsula Natural Resources Management Board, 2019, Port Lincoln, South Australia, Australia. Quarry Foothills

SebsPatch Marshalls RdMarshalls LiverpoolMt Moonlight BayMoonlight Salt Lakereveg Salt Southernsreveg Salt Lake remnantLake Salt Southernsremnant

Marshalls Rdseedlings Marshalls Diversity 2020 12 Diversity 2020, , 1,2 299, x FOR PEER REVIEW 7 of15 20 of 18 Quarry Foothills

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Marshalls Rdseedlings Marshalls

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.

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SebsPatch Marshalls RdMarshalls LiverpoolMt Moonlight BayMoonlight Salt Lakereveg Salt Southernsreveg Salt Lakeremnant Salt Southernsremnant Quarry Foothills SebsPatch Rdseedlings Marshalls K=3 MarshallsRd MtLiverpool MoonlightBay SaltLake reveg Southernsreveg SaltLake remnant Southernsremnant Quarry MarshallsRd seedlings Foothills

SebsPatch Marshalls RdMarshalls LiverpoolMt Moonlight BayMoonlight Quarry revegLake Salt Foothills Southernsreveg SebsPatch remnantLake Salt MarshallsRd MtLiverpool Southernsremnant MoonlightBay SaltLake reveg K=4 Southernsreveg SaltLake remnant Rdseedlings Marshalls Southernsremnant

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5 MarshallsRd K=5 MoonlightBay BayMoonlight 3 SaltLake reveg Lakereveg Salt . Southernsreveg Southernsreveg 0 SaltLake remnant Lakeremnant Salt Southernsremnant Southernsremnant

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0 SebsPatch MarshallsRd MtLiverpool K=7 MoonlightBay SaltLake reveg Southernsreveg SaltLake remnant Southernsremnant 2 4 6 8 10 MarshallsRd seedlings

K Quarry Foothills SebsPatch MarshallsRd MtLiverpool K=8 MoonlightBay SaltLake reveg Southernsreveg SaltLake remnant Southernsremnant

MarshallsRd seedlings

Quarry Foothills SebsPatch K=9 MarshallsRd MtLiverpool MoonlightBay SaltLake reveg Southernsreveg SaltLake remnant Southernsremnant

MarshallsRd seedlings

Quarry Foothills SebsPatch MarshallsRd MtLiverpool K=10 MoonlightBay SaltLake reveg Southernsreveg SaltLake remnant Southernsremnant

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s l e y d d y s ls h e r n o il c k k a R r r n o t t R Quarry t r h a a B s a e p t a L n l s h n e r Foothills L . t l ll u t h o P t a t h a s a t . e o SebsPatch l n l g a Q u n MtLiverpool v s a g MarshallsRd h g o u g i F a e li s h n b S m v MoonlightBay r s i S m o e L e e SaltLake reveg S n r l e Southernsreveg v t r e a d r S S SaltLake remnant r o a Southernsremnant e o M e r M M e M MarshallsRd seedlings s

(b)

FigureFigure A1. A1.(a ()a) ADMIXTURE ADMIXTURE errorerror resultsresults - - the the cv cv errors errors are are plotted plotted for for K K= =1–1–10,10, with with the the lowest lowest cv cv errorerror indicating indicating greatest greatest support support at at KK == 7. ( (bb)) Bar Bar plots plots represent represent individual individual genetic genetic cluster cluster assignment assignment fromfrom ADMIXTURE ADMIXTURE results results from from KK == 2 to K = 1010..

Diversity 2020, 12, 299 16 of 18 Diversity 2020, 12, x FOR PEER REVIEW 8 of 20

Figure A2. Boxplots of the Marshalls Rd mature plants, admixed seedlings, and non-admixed seedlings. Figure A2. Boxplots of the Marshalls Rd mature plants, admixed seedlings, and non-admixed (A) proportions of single nucleotide polymorphisms (SNPs) that are heterozygous, (B) inbreeding seedlings. (A) proportions of single nucleotide polymorphisms (SNPs) that are heterozygous, (B) coefficient (F), and (C) the kinship values. Homogeneous subgroups are indicated by a, b, and c. inbreeding coefficient (F), and (C) the kinship values. Homogeneous subgroups are indicated by a, b, Note the greater proportion of heterozygous SNPs in admixed seedlings, demonstrating that admixture and c. Note the greater proportion of heterozygous SNPs in admixed seedlings, demonstrating that between genetic clusters may lead to greater genetic diversity compared to non-admixed seedlings and admixture between genetic clusters may lead to greater genetic diversity compared to non-admixed the parent populations. seedlings and the parent populations. References References 1. Reed, D.H.; Frankham, R. Correlation between fitness and genetic diversity. Conserv. Biol. 2003, 17, 230–237. 1. [Reed,CrossRef D.H.;] Frankham, R. Correlation between fitness and genetic diversity. Conserv. Biol. 2003, 17, 230– 2. Nicotra,237, doi:10.1046/j.1523 A.B.; Beever, E.A.;-1739.2003.01236.x. Robertson, A.L.; Hofmann, G.E.; O’Leary, J. Assessing the components of adaptive 2. capacityNicotra, to A.B.; improve Beever, conservation E.A.; Robertson, and management A.L.; Hofmann, efforts G.E.; under O’Leary, global J.change. AssessingConserv. the components Biol. 2015, 29 of, 1268–1278.adaptive capacity [CrossRef to][ improvePubMed conservation] and management efforts under global change. Conserv. Biol. 3. Leimu,2015, 29 R.;, 1268 Mutikainen,–1278, doi:10.1111/cobi.12522. P.; Koricheva, J.; Fischer, M. How general are positive relationships between plant 3. populationLeimu, R.; size,Mutikainen, fitness and P.; Koricheva, genetic variation? J.; Fischer,J. Ecol. M. How2006 ,general94, 942–952. are positive [CrossRef relationships] between plant 4. Aguilar,population R.; Quesada, size, fitness M.; Ashworth, and genetic L.; Herrer variation?ías-Diego, J. Ecol. Y.; Lobo,2006, J. Genetic94, 942 consequences–952, doi:10.1111/j.1365 of habitat- fragmentation2745.2006.01150.x. in plant populations: Susceptible signals in plant traits and methodological approaches. 4. Mol.Aguilar, Ecol. R.;2008 Quesada,, 17, 5177–5188. M.; Ashworth, [CrossRef L.;] Herrerías-Diego, Y.; Lobo, J. Genetic consequences of habitat 5. Keller,fragmentation L. Inbreeding in plant eff ectspopulations: in wild populations. Susceptible signalsTrends Ecol.in plant Evol. traits2002 and, 17 methodological, 230–241. [CrossRef approaches.] Mol. 6. Spielman,Ecol. 2008,D.; 17, Brook,5177–5188, B.W.; doi:10.1111/j.1365 Frankham, R. Most-294x.2008.03971.x. species are not driven to extinction before genetic factors 5. impactKeller, them.L. InbreedingProc. Natl. eff Acad.ects in Sci. wild USA populations.2004, 101, 15261–15264. Trends Ecol. Evol. [CrossRef 2002], 17, 230–241, doi:10.1016/s0169- 7. Weeks,5347(02)02489 A.; Sgr-ò8., C.M.; Young, A.G.; Frankham, R.; Mitchell, N.J.; Miller, K.A.; Byrne, M.; Coates, D.J.; 6. Eldridge,Spielman, M.D.B.; D.; Brook, Sunnucks, B.W.; Frankham, P.; et al. R. Assessing Most species the benefitsare not driven and risks to extinction of translocations before genetic in changing factors environments:impact them. Proc. A genetic Natl. Acad. perspective. Sci. USAEvol. 2004 Appl., 101,2011 15261, 4–,15264, 709–725. doi:10.1073/pnas.0403809101. [CrossRef] 8.7. Barmentlo,Weeks, A.; H.; Sgrò, Meirmans, C.M.; Young, P.G.; Luijten, A.G.; S.H.; Frankham, Triest, L.; R.; Oostermeijer, Mitchell, N.J.; G. OutbreedingMiller, K.A.; depression Byrne, M.; and Coates, breeding D.J.; systemEldridge, evolution M.D.B.; in Sunnucks, small, remnant P.; et populations al. Assessing of the Primula benefits vulgaris: and risks Consequences of translocations for genetic in changing rescue. Conserv.environm Genet.ents: 2017A genetic, 19, 545–554. perspective. [CrossRef Evol. ]Appl. 2011, 4, 709–725, doi:10.1111/j.1752-4571.2011.00192.x. 9.8. Frankham,Barmentlo, R.; H.; Ballou, Meirmans, J.D.; Eldridge, P.G.; Luijten, M.D.B.; S.H.; Lacy, Triest, R.C.; Ralls, L.; Oostermeijer, K.; Dudash, M.R.; G. Outbreeding Fenster, C.B. depression Predicting theand probabilitybreeding system of outbreeding evolution depression. in small, remnantConserv. populations Biol. 2011, 25 o,f 465–475.Primula [vulgaris:CrossRef ]Consequences for genetic rescue. Conserv. Genet. 2017, 19, 545–554, doi:10.1007/s10592-017-1031-x.

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