Conservation Genetics

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Understanding the exchange of individuals between wildlife populations, particularly those with naturally fragmented habitats, is important for the effective management of these species. This is of particular consequence when the species is of conservation concern, and isolated populations may be lost due to pressures from predation or competition, or catastrophic events such as wildfire. Here we demonstrate the use kinship and population structure analysis to show potential recent movement between colonies in metapopulations of yellow-footed rock-wallaby ( Petrogale xanthopus Gray 1854) at two sites in the Grey Range of Queensland, and at four sites in the Gawler Ranges of South Australia. These colonies are also compared to a single colony from the Flinders Ranges, a connected landscape of rock-wallaby habitat. Using reduced representation next-generation sequencing, we acquired and filtered a set of ~ 17,000 single-nucleotide polymorphisms to examine population genetic variation, structure and relationships within populations, and also identify putative migrants. Initial STRUCTURE analysis re-confirmed each population should be considered separately. Tests of population genetic variation identify several colonies appearing to be experiencing genetic erosion, also with low calculated effective population sizes (N e = 4.5–36.6). Pairwise comparisons of individual relatedness (relatedness coeffiecients; r ) implied several contemporary movement events between colonies within both the Gawler and Grey Ranges ( r > 0.125), which was then affirmed with tests for putative first generation migrants. These results are of particular note in South Australia, where threat abatement (management of key predators and competitors) may facilitate dispersion. Additionally, in Queensland, colonies are separated by anthropogenic barriers: predator exclusion fencing designed to exclude dingoes ( Canis familiaris ) from grazing land, which may hinder dispersal. This work highlights the usefulness of population genetics to inform management outcomes in wildlife, in this case, highlighting the need for threatened species management at the landscape level.
The spatiotemporal genetic variation at early plant life stages may substantially affect the natural recolonization of human-altered areas, which is crucial to understand plant and habitat conservation. In animal-dispersed plants, dispersers’ behavior may critically drive the distribution of genetic variation. Here, we examine how genetic rarity is spatially and temporally structured in seedlings of a keystone pioneer palm (Chamaerops humilis) and how the variation of genetic rarity could ultimately affect plant recruitment. We intensively monitored the seed rain mediated by two medium-sized carnivores during two consecutive seasons in a Mediterranean human-altered area. We genotyped 143 out of 309 detected seedlings using 12 microsatellite markers. We found that seedlings emerging from carnivore-dispersed seeds showed moderate to high levels of genetic diversity and no evidence of inbreeding. We found inflated kinship among seedlings that emerged from seeds within a single carnivore fecal sample, but a dilution of such FSGS at larger spatial scales (e.g. latrine). Seedlings showed a significant genetic sub-structure and the sibling relationships varied depending on the spatial scale. Rare genotypes arrived slightly later throughout the dispersal season and tended to be spatially isolated. However, genetic rarity was not a significant predictor by itself which indicates that, at least, its influence on seedling survival was smaller than other spatiotemporal factors. Our results suggest strong C. humilis resilience to genetic bottlenecks due to human disturbances. We highlight the study of plant-animal interactions from a genetic perspective since it provides crucial information for plant conservation and the recovery of genetic plant resilience.
Map illustrating the likely historical distribution of clouded apollo in Sweden (dashed lines), as well as the sampling regions of the three remaining natural populations.
Principal component analysis (PCA) illustrating the genetic variation across clouded apollo butterflies in Sweden. The analysis was based on SNPs in 4D-sites. The X-axis (PCA1) explains 7.1% and the Y-axis (PCA2) 4.7% of the total variation.
A dendrogram illustrating the sequence divergence at autosomal 4D-sites for the 38 clouded apollo individuals included in the analysis.
A haplotype network based on 931 bp of the mitochondrial CO1-gene. A subset of individuals from a previous study (Gratton et al. 2008) was used to get information about genetic relationships between individuals in different populations in Sweden and other populations in the distribution range in Europe. Roslagen L and Roslagen B correspond to the two sampling sites Lötaholmen and Brudskäret in the region, respectively.
Summary of the counts of fixed, private and shared alleles for all variable 4D-sites in each of the six clouded apollo population comparisons.
The clouded apollo ( Parnassius mnemosyne ) used to have a wide distribution in Fennoscandia. Recent population declines have, however, led to regional extinctions and in Sweden it is currently one of the most endangered butterflies, confined to three geographically separated metapopulations: Blekinge, Roslagen and Västernorrland. Especially the Blekinge population has declined dramatically and few imagines have been observed during recent census efforts (< 10 in some localities). The clouded apollo is subject to a species action plan which includes both habitat restorations and captive breeding to produce individuals for release and reintroductions. Here, we apply whole-genome resequencing of clouded apollo individuals collected in the three natural populations and the captive population in Sweden and apply population genomic approaches to get a better understanding of the genetic structure and levels of genetic diversity in the species. We find that the clouded apollo populations in the different geographic regions have similar, but comparatively low levels of genetic diversity and we find evidence for significant genetic differentiation between the northernmost population and the populations in southern Sweden. Additional analysis, including previously available mitochondrial data, unveil that a bi-directional re-colonization of Fennoscandia after the latest glacial maximum most likely is the explanation for the considerable differentiation between some Swedish populations. Finally, we find evidence for population sub-structure in one of the Swedish populations. The results provide insights into the genetic consequences of population size declines and fragmentation in general and provide important information for direct conservation actions for the clouded apollo in Sweden in particular.
Population genetic structuring within subpopulations of the Kuranda Treefrog (Litoria myola). A Map showing the sampled range of the species, with occurrence records as white dots and sampling localities as coloured dots. Sampling localities abbreviations are provided in the legend. OF: Oak Forest; DC: Dismal Creek; QC: Queens Creek; NB: North Barron; CD: Cadagi Drive; OC: Owen Creek; WC: Warril Creek; NM: North Myola; BA: Barron Alambi; FA: Fairyland; JC: Jumrum Creek. NS represents sites where L. myola was detected but not sampled. The insets show the location of the study area within Queensland (QLD), Australia, and a photo of an adult male by Conrad Hoskin. The red line represents the Kennedy Highway. B, D, F. NETVIEW networks for the three time periods: (B) T2001–2016, (D) T2001–2004 and (F) T2007–2009. k-NN values are 25, 25 and 15 respectively. Individuals are coloured based on sampling locality. C, E, G. Structure plots for the three time periods: (C) T2001–2016, (E) T2001–2004 and (G) T2007–2009, for K values of 2, 2 and 3 respectively. Vertical black lines separate sampling localities, with the coloured dots underneath matching the localities in panel A
Effective population size (Ne) and census population size (N) for the whole species and the Eastern genetic population cluster (essentially, Jumrum Creek). Ne estimates from this study are provided for all three time periods investigated (T2001–2016, T2001–2004 and T2007–2009). The average value from independent runs is reported, and individual estimates are displayed as black points. Point estimates of N were available only for time periods T2001–2004 and T2007–2009 (Hoskin 2007; Hoskin et al. 2018). Arrows highlight the detected decline between T2001–2004 and T2007–2009
Visual summary of management recommendations for Litoria myola. All known records for the species are depicted as circles, with coloured circles representing breeding sites sampled for this study. Colours of sampled breeding sites match Fig. 1A. Green symbols are Jumrum Creek, yellow is the Fairyland site, and the remaining coloured sites form the Western genetic cluster. The red line shows the Kennedy Highway and blue lines represent streams within the region. The thicker blue line is the Barron River, the main waterbody in the region
The Kuranda Treefrog occurs in tropical north-east Australia and is listed as Critically Endangered due to its small distribution and population size, with observed declines due to drought and human-associated impacts to habitat. Field surveys identified marked population declines in the mid-2000s, culminating in very low abundance at most sites in 2005 and 2006, followed by limited recovery. Here, samples from before (2001–2004) and after (2007–2009) this decline were analysed using 7132 neutral genome-wide SNPs to assess genetic connectivity among breeding sites, genetic erosion, and effective population size. We found a high level of genetic connectivity among breeding sites, but also structuring between the population at the eastern end of the distribution (Jumrum Creek) versus all other sites. Despite finding no detectable sign of genetic erosion between the two times periods, we observed a marked decrease in effective population size (Ne), from 1720 individuals pre-decline to 818 post-decline. This mirrors the decline detected in the field census data, but the magnitude of the decline suggested by the genetic data is greater. We conclude that the current effective population size for the Kuranda Treefrog remains around 800 adults, split equally between Jumrum Creek and all other sites combined. The Jumrum Creek habitat requires formal protection. Connectivity among all other sites must be maintained and improved through continued replanting of rainforest, and it is imperative that impacts to stream flow and water quality are carefully managed to maintain or increase population sizes and prevent genetic erosion.
Genetic diversity among and within populations of all species is necessary for people and nature to survive and thrive in a changing world. Over the past three years, commitments for conserving genetic diversity have become more ambitious and specific under the Convention on Biological Diversity’s (CBD) draft post-2020 global biodiversity framework (GBF). This Perspective article comments on how goals and targets of the GBF have evolved, the improvements that are still needed, lessons learned from this process, and connections between goals and targets and the actions and reporting that will be needed to maintain, protect, manage and monitor genetic diversity. It is possible and necessary that the GBF strives to maintain genetic diversity within and among populations of all species, to restore genetic connectivity, and to develop national genetic conservation strategies, and to report on these using proposed, feasible indicators.
Genetic diversity is critical to a population’s ability to overcome gradual environment change. Large-bodied wildlife existing in regions with relatively high human population density are vulnerable to isolation-induced genetic drift, population bottlenecks, and loss of genetic diversity. Moose ( Alces americanus americanus ) in eastern North America have a complex history of drastic population changes. Current and potential threats to moose populations in this region could be exacerbated by loss of genetic diversity and connectivity among subpopulations. Existing genetic diversity, gene flow, and population clustering and fragmentation of eastern North American moose are not well quantified, while physical and anthropogenic barriers to population connectivity already exist. Here, single nucleotide polymorphism (SNP) genotyping of 507 moose spanning five northeastern U.S. states and one southeastern Canadian province indicated low diversity, with a high proportion of the genomes sharing identity-by-state, with no consistent evidence of non-random mating. Gene flow estimates indicated bidirectionality between all pairs of sampled areas, with magnitudes reflecting clustering and differentiation patterns. A Discriminant Analysis of Principal Components analysis indicated that these genotypic data were best described with four clusters and indicated connectivity across the Saint Lawrence River and Seaway, a potential physical barrier to gene flow. Tests for genetic differentiation indicated restricted gene flow between populations across the Saint Lawrence River and Seaway, and between many sampled areas facing expanding human activity. These results document current genetic variation and connectivity of moose populations in eastern North America, highlight potential challenges to current population connectivity, and identify areas for future research and conservation.
Distribution of the three lineages of the Erebia manto species complex across Europe. Each dot depicts an individual occurrence based on data from (accessed 16th of March 2022 with additional data from Lelo 2000; Burnaz 2008; Sanz et al. 2017; Jakšic 2019). A few doubtful occurences, as well as a few occurrences in the French Alps south of Grenoble, which could not be attributed unambiguously to one subspecies, have been omitted. Colors depict the different recognized E. manto subspecies (see Cupedo & Doorenweerd 2020). The inset depicts the Alps with numbers highlighting the populations sampled (see Supplementary Information 1 for details). The attribution to some occurrences to the different subspecies have been made following Cupedo (1997), Schmitt et al. (2014) and Cupedo & Doorenweerd (2020). For simplicity, manto and mantoides are merged and treated as "manto" given the uncertainties regarding the type locality of manto (see Cupedo & Doorenweerd 2020). Map sources: European Comission GISCO:; EU-DEM
Phylogram artificially rooted on the node that separates E. eriphyle from the E. manto complex comprising manto, bubastis and vogesiaca. Numbers depict bootstrap support for nodes with > 90% support. Colors indicate taxa. Numbers at the terminal branches indicate sampling location as given in Fig. 1. For each E. manto lineage, a picture of the male genital valve is given
of the individual based clustering analysis and their spatial relationship. A Individual-based assignments using Admixture for samples belonging to the E. manto complex. The case for four genetic clusters (K = 4) is shown as a bar diagram. B Map of all genotyped E. manto complex individuals (map modified from Bing Maps C Principal component (PC) scores for all genotyped E. manto complex individuals. D–E Relationship between longitude and the PC1 scores for PC analyses done seperatenly on manto and bubastis individuals only. For B-E pie charts depict the Admixture assignment for each individual as shown in A
of the degree of genetic differentiation (FST) and recent migration (m) as estimated by BA3-SNPs. Black arrows indicate the direction of migration with the respective estimate of m ± 1 SD. For E. manto the FST between un-admixed individuals of the two inferred genomic clusters is given. All FST values are significant (p < 0.001)
Distribution of Erebia manto (filled circles) and E. bubastis (open circles) across Switzerland. Colours indicate different major substrate types (map source: Lithological-petrographic map of Switzerland; Federal Office of Topography swisstopo, Georesources Switzerland Group)
A problem to implement conservation strategies is that in many cases recognized taxa are in fact complexes of several cryptic species. Failure to properly delineate species may lead to misplaced priorities or to inadequate conservation measures. One such species complex is the yellow-spotted ringlet Erebia manto, which comprises several phenotypically distinct lineages, whose degree of genomic isolation has so far not been assessed. Some of these lineages are geographically restricted and thus possibly represent distinct units with conservation priority. Using several thousand nuclear genomic markers, we evaluated to which degree the bubastis lineage from the Alps and the vogesiaca lineage from the Vosges, are genetically isolated from the widespread manto lineage. Our results suggest that both lineages are genetically as strongly differentiated from manto as other taxonomically well separated sibling species in this genus from each other, supporting a delineation of bubastis and vogesiaca as independent species. Given the restricted and isolated range of vogesiaca as well as the disjunct distribution of bubastis, our findings have significant implication for future conservation efforts on these formerly cryptic species and highlight the need to investigate the genomic identity within species complexes.
Map of Lake Saimaa showing the locations of seal samples used in this study
Distributions of a number of runs of homozygosity (RoH), b number of long RoH, c percentage of RoH and d median RoH length in kilobases per sample group. The IDs are: SAI = Saimaa, BAL = Baltic reference. The boxplots represent the first quartile (box base), the median (horizontal black line at the box center), and the third quartile (box top) in the sample. The box whiskers extend to 1.5*interquartile range. Round dots are considered outliers with respect to the rest of the data points in the dataset
Distributions of a number of runs of homozygosity (RoH), b number of long RoH, c percentage of RoH and d median RoH length in kilobases, per group within Lake Saimaa. The group IDs are: MHV Main Haukivesi, PV Pihlajavesi, SS Southern Saimaa, KV Kolovesi, NS Northern Saimaa
Examining inbreeding depression in Saimaa ringed seals between stillborns and pups aged under 12 months. Distributions of a number of RoH per individual across subpopulations, b distributions of the number of long RoH, c percentages of individuals with RoH exceeding 2–5 megabase-pairs (Mbp) and d distributions of the number of long RoH. In subplots b-d, the boxplots represent the first quartile (box base), the median (horizontal black line at the box center), and the third quartile (box top) in the sample. The box whiskers extend to 1.5*interquartile range. The black dots are data points, and black triangles mark the To-20/A1702 seal pup that was found in poor condition and died in captivity
Overlaps of the 731,209 genomic positions with single-nucleotide variance between the Lake Saimaa groups. The group IDs are: MHV Main Haukivesi (n = 10), PV Pihlajavesi (n = 20), KV Kolovesi (n = 3), SS Southern Saimaa (n = 6), NS Northern Saimaa (n = 2)
Fragmentation of isolated populations increases the risk of inbreeding and loss of genetic diversity. The endemic Saimaa ringed seal ( Pusa hispida saimensis ) is one of the most endangered pinnipeds in the world with a population of only ~ 400 individuals. The current genetic diversity of this subspecies, isolated in Lake Saimaa in Finland for ca. 1000 generations, is alarmingly low. We performed whole-genome sequencing on Saimaa ringed seals (N = 30) and analyzed the level of homozygosity and genetic composition across the individual genomes. Our results show that the Saimaa ringed seal population has a high number of runs of homozygosity (RoH) compared with the neighboring Baltic ringed seal ( Pusa hispida botnica ) reference population ( p < 0.001). There is also a tendency for stillborn seal pups to have more pronounced RoH. Since the population is divided into semi-isolated subpopulations within the Lake Saimaa exposing the population to deleterious genomic effects, our results support augmented gene flow as a genetic conservation action. Based on our results suggesting inbreeding depression in the population, we recommend Pihlajavesi as a potential source and Southern Saimaa as a potential recipient subpopulation for translocating individuals. The Saimaa ringed seal is a recognized subspecies and therefore translocations should be considered only within the lake to avoid an unpredictable risk of disease, the introduction of deleterious alleles, and severe ecological issues for the population.
Captive propagation is widely used for the conservation of imperiled populations. There have been concerns about the genetic effects of such propagation, but few studies have measured this directly at a genomic level. Here, we use moderate-coverage (10X) genome sequences from 80 individuals to evaluate the genomic distribution of variation of several paired groups of Chinook salmon (Oncorhynchus tshawytscha). These include (1) captive- and natural-origin fish separated by at least one generation, (2) fish within the same generation having high fitness in captivity compared to those with high fitness in the wild, and (3) fish listed as different Evolutionarily Significant Units (ESUs) under the US Endangered Species Act. The distribution of variation between high-fitness captive and high-fitness natural fish was nearly identical to that expected from random sampling, indicating that differential selection in the two environments did not create large allele frequency differences within a single generation. In contrast, the samples from distinct ESUs were clearly more divergent than expected by chance, including a peak of divergence near the GREB1L gene on chromosome 28, a gene previously associated with variation in time of return to fresh water. Comparison of hatchery- and natural-origin fish within a population fell between these extremes, but the maximum value of FST was similar to the maximum between ESUs, including a peak of divergence on chromosome 8 near the slc7a2 and pdgfrl genes. These results suggest that efforts at limiting genetic divergence between captive and natural fish in these populations have successfully kept the average divergence low across the genome, but at a small portion of their genomes, hatchery and natural salmon were as distinct as individuals from different ESUs.
Sample locations, flower, fruit and seed of M. odoratissima
Analysis of the population structure of the 70 studied M. odoratissima accessions using ADMIXTURE. a Admixture graph showing the individual cluster values corresponding to each K value. The different samples are shown on the x-axis. The y-axis quantifies the membership probability of samples belonging to different groups. Colors in each row represent structural components. b Admixture estimation of the number of groups for K values ranging from 1 to 10. c population structure based on K = 3
A PCoA plots of M. odoratissima based on the analysis of 180,650 SNPs; B DAPCplots of M. odoratissima based on the analysis of 180,650 SNPs; C Mantel test based on five wild populations of M. odoratissima; D Gene flow from TreeMix analysis. Arrows correspond to migration events, and the darkness of the arrow color indicates the weight of the migration edge
Phylogenetic tree generated from 70 M. odoratissima samples and based on the analysis of 180,650 SNPs
Demographic history of M. odoratissima inferred by Stairway Plot2. The 95% confidence interval of the estimated effective population size is shown by light color. A Cluster 1; B Cluster 2; C Cluster 3.
Magnolia odoratissima is a highly threatened species, with small distribution and scattered populations due to habitat fragmentation and human activity. In this study, the genetic diversity and population structure of the five remaining natural populations and two cultivated populations of M. odoratissima were analyzed using single nucleotide polymorphisms (SNPs) derived from specific-locus amplified fragment sequencing. A total of 180,650 SNPs were identified in seventy M. odoratissima individuals. The Nei’s and Shannon-Wiener diversity index across all M. odoratissima population were 0.35 and 0.51, respectively, while the observed heterozygosity (Ho) and expected heterozygosity (He) were 0.27 and 0.34, respectively. Our results suggest that M. odoratissima has relatively high genetic diversity at the genomic level. The FST and AMOVA indicated that high genetic differentiation exists among populations, and a phylogenetic neighbor-joining tree, Bayesian model–based clustering and discriminant function analysis of principal component all divided the M. odoratissima individuals into three distinct clusters. The Treemix analysis showed that there was low gene flow among the natural populations. Demographic history inferences indicated show that three clusters of M. odoratissima experienced at least three bottlenecks and resulted in a decrease of effective population size. Our results suggest that three distinct evolutionary significant units should be set up to conserve this critically endangered species.
Karyotypes of female (left) and male (right) D. melanogaster, illustrating the XX and XY sex chromosomes (bottom), the metacentric chromosomes 2 and 3 and the dot fourth chromosomes (Morgan 1916, Fig. 52:
Method used to obtain homozygous chromosomes 2 in D. melanogaster and to test for recessive lethal chromosomes (after Sved 1971). Cy is a balancer chromosome (e.g. SM1 or SM5: Kaufman 2017) with inversions to prevent recombination, and that is marked by the dominant Curly wing (Cy) mutation, which is lethal when homozygous. Note that a single Cy/ + 1 male is selected in the F1 generation, and this + 1 chromosome is made homozygous. If the + 1 chromosome contains a recessive lethal, there will be no wild-type progeny in the F3 generation, while if it contains a recessive sterility allele, interbreeding of wild-type individuals will fail
Estimates of susceptibility to inbreeding depression for total fitness are needed for predicting the cost of inbreeding and for use in population viability analyses, but no such valid estimates are available for any wild invertebrate population. I estimated the number of lethals equivalents for total fitness in recently wild-caught populations of Drosophila melanogaster using published data on the total fitness of homozygosity versus heterozygosity for each of the major chromosomes (the X, second, and third) under competitive conditions. As there are no data for the fitness effects of homozygosity for the small fourth chromosome which represents 1.0% of the euchromatic genome, this was accounted for by attributing the homozygosity for the three large chromosome to an inbreeding coefficient of 0.99 when computing lethal equivalents for total fitness. Total genomic homozygosity is predicted to be essentially lethal in D. melanogaster. The corresponding haploid lethal equivalents estimate for total fitness was 5.04. The lethal equivalent value lies within the range for vertebrates but tends to be higher than for most outbreeding plants which are often purged as they exhibit up to 20% selfing (by definition). As D. melanogaster has its genome sequenced and annotated and has lethal equivalent estimates for total fitness for individual chromosomes as well as its total genome, it provides an excellent opportunity for evaluating genomic estimates of mutation load.
a Distribution ranges of Eurasian lynx evolutionary lineages according to Kitchener et al. (2017). Sampling within wild populations is indicated by stars (1 Scandinavian, 2 Harz, 3 Carpathian, 4 Baltic, 5 Kirov, 6 Irkutsk, 7 Sacha, 8 Primorsky Krai). b Locations of breeding facilities included in this study, their full names are given in Table S1. Distribution ranges of particular European lynx populations according to IUCN Red List Mapping 2012–2016 (including corrections LCIE et al. 2020) are displayed as a background, their pertinence to the Carpathian and the Northern lineage is indicated in a legend by green and blue type colour, respectively
Microsatellite-based genetic separation of wild and captive lynxes from 42 zoos and wildlife parks using factorial correspondence analysis (FCA) (N = 210). Approximate borders between recognized lineages are illustrated by lines
Microsatellite-based genetic sub-structuring of captive and wild Eurasian lynx using Bayesian clustering in software STRUCTURE for K = 2, 5 and 6. Each column corresponds to one animal, the colour of each column corresponds to the probability of assignment to a certain cluster
The assignment of captive lynx to evolutionary lineage according to their putative origin stated by breeding facilities (the agreement is indicated by green, non-agreement or missing previous data are indicated by blue colour). The circles are displayed proportionally to the number of assigned individuals, which is given in or near the circle
The main aim of ex situ programmes in conservation is to provide a suitable source of individuals for future reintroductions or reinforcement of existing populations. A fundamental prerequisite is creating and maintaining healthy and sustainable captive populations that show high levels of phenotypic and genetic similarity to their wild counterparts. The Eurasian lynx (Lynx lynx) is a model of a locally extinct species that has been subject to long-term captive breeding and of past and ongoing reintroduction efforts. To test for genetic suitability of ex situ population, a comparative genetic evaluation including in situ populations was undertaken. The assignment analysis of 97 captive lynx from 45 European zoos, wildlife parks and private breeds was performed using 124 lynx from different wild Eurasian populations belonging to three evolutionary lineages: the Carpathian, the Northern, and the Siberian lynx. The results showed a high proportion of Siberian lynx (51%) in the European captive lynx population. Remaining captive animals were assigned to either the Carpathian (28%), or the Northern lynx lineage (13%). Admixture between lineages was rather low (8%). Notably, no or very low difference in genetic diversity was detected between the wild and captive lynx populations. Our results support the potential of the captive population to provide genetically suitable individuals for genetic rescue programmes. The transfer of genes between isolated populations, including those in captivity, should become an important management tool to preserve genetic variability and prevent inbreeding depression in native and reintroduced populations of this iconic predator.
Mainland Australian Alps, showing the contiguous Kosciuszko Plateau and fragmented southern plateaus, and current elevational range of C. praealtus (white, > 1,250 m). Shading corresponds to a 1 s digital elevation model (Gallant et al. 2011), where gray represents potential historic upland connectivity ~ 20 ka. Letters on Kosciuszko Plateau refer to nominal site groupings, where N northern, E eastern, LN lower-northern, C central, LC lower-central, S southern. Sampled southern plateaus were the Bogong High Plains, Mt Hotham/Loch area, Lankey/Omeo Plains and Wellington Plain (see Fig. 4 for specific sample locations and codes). Black represents lowlands (< 700 m). Dotted lines show jurisdiction boundaries. Stars depict capital cities. Hatched areas represent ocean. Inset shows the location of the study area within Australia
Discriminant analysis of principal components (DAPC) among a 259 C. praealtus individuals from 58 sites across both regions; b 88 individuals from 34 sites on Kosciuszko Plateau; and c 171 individuals from 24 sites on the southern plateaus. Analyses are based on a 32,739 SNPs and b 13,905 SNPs for Kosciuszko Plateau; and c 10,202 SNPs for the southern plateaus (minor allele count = 3). Clear genetic structure is evident between regions and among sites
Bayesian genetic clustering of C. praealtus individuals from Kosciuszko Plateau and the southern plateaus, using fastSTRUCTURE. Analyses are based on a 32,739 SNPs and b 13,905 SNPs for Kosciuszko Plateau; and c 10,202 SNPs for the southern plateaus (minor allele count = 3). When analysis included Kosciuszko Plateau and the southern plateaus (a) each region comprised two distinct genetic clusters with minimal sharing of genetic signal between regions. Admixture is evident among Kosciuszko Plateau populations. Analysis of Kosciuszko Plateau samples (b) provided greater resolution of genetic structure and fine-scale admixture. A key difference was noted within the southern plateaus; when all individuals were included, signal from Kosciuszko Plateau is seen in the individual from Wellington Plain (WP) (a). However, this signal is lost when only the southern plateaus individuals were analyzed (c)
from the three genetic clustering algorithms used to identify populations of C. praealtus from a Kosciuszko Plateau and b the southern plateaus. Analyses are based on a 13,905 SNPs for Kosciuszko Plateau; and b 10,202 SNPs for the southern plateaus (minor allele count = 3). Diamonds on each map show site locations, and are coloured as per DAPC results. Pie charts reflect the results from fastSTRUCTURE, and show the degree of admixture among populations (squared brackets on map edges group the sites that share 100% pie charts). Dashed lines on each map show geo-referenced genetic clusters identified via ‘geneland’ (raw ‘geneland’ outputs are presented in Online Resource Fig. 6)
Sky island species face climate-driven and anthropogenic habitat loss and population fragmentation, and are therefore vulnerable to genetic erosion. We conducted a genetic study of the cryptic and threatened alpine she-oak skink (Cyclodomorphus praealtus) throughout its range, across two regions of the mainland Australian Alps; an extensive high elevation plateau in the north (‘Kosciuszko Plateau’) and several smaller plateaus in the south (‘southern plateaus’). We investigated whether extensive potential habitat across Kosciuszko Plateau supported larger, connected populations with better genetic health than more fragmented southern plateaus. Our analyses of genome-wide markers confirmed effective isolation of the two regions. We identified three populations from the southern plateaus, largely aligning with discrete landforms, and four populations on Kosciuszko Plateau. Only one individual, from the southern-most population, showed evidence of admixture between the two regions. Across its range, C. praealtus populations had low genetic diversity and small effective population sizes. In contrast to our expectations, Kosciuszko Plateau populations were smaller, with greater genetic differentiation and a higher degree of inbreeding than the southern populations. We detected admixture between populations on Kosciuszko Plateau, while the southern plateaus had limited admixture. We found no evidence of local adaptation, suggesting plateaus represent interglacial refugia. Our results suggest that C. praealtus has little capacity to withstand further disturbance or rapid environmental changes. Maintaining or restoring habitat quality in occupied and suitable connecting habitats across the species’ range is paramount. ‘Genetic rescue’ should be investigated as an option to mitigate the effects of isolation and improve population resilience.
Median-joining network for 153 cheetah individuals based on the discriminatory 3-bp deletion and 15 SNPs of the Cheetah Subspecies-specific Amplicons (CSAs). Pie-chart diameters represent sample sizes supporting a specific haplotype. Colors represent subspecies assignment based on sample origin. We highlighted subspecies haplogroups, and mutations between haplotypes are indicated by dashes
Phylogeographic distribution and population structure based on mitochondrial DNA. a) Map showing expected historic (light colors) (Marker 2019) and current distribution (dark colors) (Durant et al. 2017) of the five cheetah subspecies (Acinonyx jubatus ssp.). b and c) Scatterplots showing the coordinates for the individuals (retained principal components of the DAPC) plotted on the principal axis of the DAPC (discriminant functions) using the 15 SNPs and 3-bp deletion of the CSAs from 153 individuals suggesting 5 clusters based on (b) haplogroup-based subspecies assignment or (c) geographical origin-based subspecies assignment. The cumulated variance of the PCA and the discriminative analysis (DA) eigenvalues, are shown as in-figure barplots respectively. d and e) Results of the DAPC membership probability analysis based on (d) geographical origin-based subspecies assignment and (e) haplogroup-based subspecies assignment. Each vertical line represents a single individual. Colored segments indicate the individual’s estimated proportion of membership to the haplogroups
Electrophoresis results of the A. j. soemmeringii ARMS PCR in 26 cheetahs. 189 bp control amplicon and 110 bp A. j. soemmeringii-specific amplicon. Cheetah subspecies were assignment based on geographic origin. LAD = GeneRuler Ultra Low Range DNA Ladder. Individual 190 originates from southern Ethiopia, the border of A. j. soemmeringii and A. j. raineyi. It was tentatively assigned to A. j. soemmeringii, but probably belongs to A. j. raineyi
Unlabelled: There are only about 7,100 adolescent and adult cheetahs (Acinonyx jubatus) remaining in the wild. With the majority occurring outside protected areas, their numbers are rapidly declining. Evidence-based conservation measures are essential for the survival of this species. Genetic data is routinely used to inform conservation strategies, e.g., by establishing conservation units (CU). A commonly used marker in conservation genetics is mitochondrial DNA (mtDNA). Here, we investigated the cheetah's phylogeography using a large-scale mtDNA data set to refine subspecies distributions and better assign individuals to CUs. Our dataset mostly consisted of historic samples to cover the cheetah's whole range as the species has been extinct in most of its former distribution. While our genetic data largely agree with geography-based subspecies assignments, several geographic regions show conflicting mtDNA signals. Our analyses support previous findings that evolutionary forces such as incomplete lineage sorting or mitochondrial capture likely confound the mitochondrial phylogeography of this species, especially in East and, to some extent, in Northeast Africa. We caution that subspecies assignments solely based on mtDNA should be treated carefully and argue for an additional standardized nuclear single nucleotide polymorphism (SNP) marker set for subspecies identification and monitoring. However, the detection of the A. j. soemmeringii specific haplogroup by a newly designed Amplification-Refractory Mutation System (ARMS) can already provide support for conservation measures. Supplementary information: The online version contains supplementary material available at 10.1007/s10592-022-01483-1.
Geographical locations of the sampled populations of Trifolium alpestre in Estonia, Poland, and Czechia. Gray area marks continuous distribution of this species according to Hultén and Fries 1986
(a) Principal Coordinates Analysis (PCoA) based on Jost’s differentiation among 16 populations of T. alpestre. PCoA1-, PCoA2- and PCoA3-axis explain 40.5% and 17.1% and 11.0% of the total variation, respectively (b) The STRUCTURE plots show the membership probabilities of each individual in two (upper graph) and four (lower graph) genetic clusters
Relationship between pairwise genetic distances [FST/(1-FST] and log geographic distances (km) of all sampled populations of T.alpestre. Comparisons within regions are represented by filled triangles (Estonia), empty squares (Poland), filled squares (Czechia); empty circles denote all other comparisons. Lines indicate significant correlations (continuous line – all populations; R² = 0.354; dotted line: Czech populations; R² = 0.278)
Plant size (estimated as the number of leaves × stem length), seed number in a flower head and weight of 100 seeds in marginal (Estonia) and central (Czechia) populations of T. alpestre (± SE). Letters above bars indicate differences between regions according to GLM-s
The maintenance of genetic variation is crucial at the margins of a species’ distribution range where plants grow in a suboptimal environment and often put less effort into sexual recruitment. The main focus of this study was on exploring the variation in genetic patterns and plant fitness of the long-lived clonal legume Trifolium alpestre in marginal populations in comparison to the distribution centre with the purpose to plan adequate conservation actions for this species. We used highly variable microsatellite loci to explore genetic patterns in 16 populations of varied size in Trifolium alpestre at the different parts of its range (marginal/central populations in Estonia, Poland and Czechia) of this species. We also studied overall genetic structure and population divergence, and historical and contemporary gene flow within each region. To estimate the potential for sexual reproduction at the marginal and central area, we measured the amount and weight of seeds produced in Estonian and Czech populations. Our study revealed high HE and AR in all studied populations that were unconnected with population size, and the occurrence of unique alleles both in central as well as in marginal northernmost (Estonian) populations. Overall genetic structure reflected the geographical location of populations. Very weak population structure together with high historical migration at the distribution margin imply a past, more continuous occurrence of T. alpestre in the northernmost region. Recent bottlenecks, lowered seed production and lighter seeds in marginal populations point to the local suboptimal conditions and indicate the need to pay more attention to management to prevent loss of genotypes and maintain diverse populations in this region.
(a) Map showing the sampling area (red box) within the Kruger National Park (green polygon); (b) Kruger National Park’s location and total extent within South Africa; (c) photograph of a black rhinoceros taken during the 2019 census (photo credit: SANParks, C. Dreyer)
Median-joining haplotype network among 296 mtDNA sequences from three D. b. minor populations. The circle sizes are proportionate to the numbers of individuals representing each haplotype. Colours represent black rhinoceros populations. Hatch marks represent the number of mutation steps between haplotypes. The label above or below the circles represent the haplotype number (H1 – H7). n = number of samples. Numbers in brackets indicate numbers of individuals from the colour-coded population within that haplotype
Current mtDNA control region haplotype distribution in the Zambezi River, Zimbabwe (ZIM), KwaZulu-Natal (KZN) and Kruger National Park (KNP) source populations. n = sample size. H1–H7 = haplotype number
The proportion of Zambezi Valley (ZIM) and KwaZulu-Natal (KZN) animals in the (a) founder, and (b) current Kruger populations
Globally, wildlife populations are becoming increasingly small and isolated. Both processes contribute to an elevated risk of extinction, notably due to genetic factors related to inbreeding depression and a loss of adaptive potential. Wildlife translocation is a valuable conservation tool to reintroduce species to previously occupied areas, or augment existing populations with genetically divergent animals, thereby improving the viability of endangered populations. However, understanding the genetic implications of mixing gene pools is key to avoid the risk of outbreeding depression, and to maximise translocation effectiveness. In this study we used mitochondrial and microsatellite DNA collected from 110 black rhinoceroses (Diceros bicornis minor) in Kruger National Park, South Africa, to determine levels of genetic diversity, inbreeding and relatedness. We compared this diversity with the two source populations (KwaZulu-Natal, South Africa and Zambezi River, Zimbabwe) using data from previously published studies, and assessed changes in the relative contribution of source lineages since their reintroduction in the 1970s. Our results show that Kruger’s black rhinoceroses are genetically more diverse than those from KwaZulu-Natal, with levels closer to those from the Zambezi Valley. Furthermore, our findings indicate a relative increase in the Zimbabwean lineage since reintroduction, suggesting a possible selective advantage. From a conservation perspective, our results demonstrate the benefits of mixing multiple source populations to restore gene flow, improve genetic diversity and thereby help protect small, isolated populations from extinction.
Maps of Ceratina sampling location and land use classes for our study. A Map of Canada showing the sampling point location (red dots) in Caledon, Ontario. Land use classification from B historic (1972) and C contemporary (2018) time points. The bee pictured is a C. calcarata female. Photo credit: Sandra Rehan; Map of Canada taken from
Climatic variables collected from Ceratina sample points. A Maximum, mean, and minimum temperatures expressed in Celsius degrees for the historic and contemporary time-bins. On the left is a historic bin ranging from 1958 to 1988. On the right is a contemporary bin ranging from 1989 to 2019. B Total annual precipitation expressed in cm for the same two historical and contemporary time bins
Principal component analysis (PCA) results from C. calcarata and C. dupla samples. Individuals from museums are colored as red dots and individuals recently collected are colored as blue dots. PLINK v.2.0 was used to generate PCA and ggplot2 and tidyverse packages were used to create these plots
ADMIXTURE results from C. calcarata and C. dupla samples show the genetic ancestry proportions for each individual from K = 1 to K = 4. Both are best supported as one breeding population (K = 1; Fig. S2)
Historic and contemporary data can shed light on a species’ conservation status and work together to address two main goals in conservation biology: (1) identifying species under extinction risk and (2) the forces shaping this process. Museomics is the study of historical DNA acquired from museum specimens that allows researchers to answer myriad questions across many taxa. Museomics is an effective way to understand how populations have been affected by human and climate factors from a historic perspective. Here, our goal is to investigate changes in wild populations of two small carpenter bee species (Ceratina calcarata and C. dupla) across a 50-year time span. We sampled museum specimens and recent collections to determine their genetic diversity, population structure, effective population size, signatures of selection, and local adaptation. Both species displayed reduced genetic diversity and effective population size through time. We identified signatures of adaptation in both species across human-altered land use and climate change scenarios. We found signatures of selection in genes related to biochemical defense, insecticide, and thermal tolerance, which are consistent with the observed increase in agricultural land use development and rising temperatures over the past 50 years. Our findings suggest that these species are facing population inbreeding, possibly attributable to human land-use change and agrochemicals in their environment. Overall, this study highlights the use of museomics to understand species declines, threats to populations, and targets for remediation.
Yearly changes in the number of individuals in five ex situ captive sub-populations derived from wild Acheilognathus longipinnis individuals in the Kiso River, as well as the pattern of individual exchanges among captive sub-populations. Solid frames include the number of breeding individuals; dashed frames include the number of individuals carried over from the previous year, including hatched individuals and perennial individuals. Arrows and the associated numbers indicate the sub-population from which they originated and the number of individuals delivered, respectively. unk, unknown data. Population abbreviations are shown in Table 1
Annual fluctuation of genetic diversity and genetic estimates for ex situ captive and wild Acheilognathus longipinnis populations. A expected heterozygosities (HE); B observed heterozygosities (HO); C allelic richness (AR); D inbreeding coefficient (F); E effective population size (Ne); and F relatedness coefficients (r), respectively
Two-dimensional plot of microsatellite allele composition based on axis one and two of ex situ captive and wild Acheilognathus longipinnis populations in the Kiso River for cohort levels assessed via factorial correspondence analysis
Comparison of genetic diversity fluctuations between observed values (solid line) and estimated values (dotted line) with population viability analysis (see text). The gray straight line shows the initial value (relative value one). Generation 1 corresponds to the 2014 cohort
EX situ conservation management is an effective method that conserves endangered species that are on the decline owing to anthropogenic alteration of natural habitats. This entails the management of a captive population while maintaining its genetic variability and preventing its adaptation to the captive environment. However, implementation of such efforts is largely limited to experimental animals and zoo-managed animals with pedigree information. In this study, ex situ management of endangered Itasenpara bitterling (Acheilognathus longipinnis) was conducted, while practicing recommended conservation procedures, for the purpose of conserving this species. In this 11 year long study, we conducted multi-locus microsatellite DNA analyses to evaluate the genetic dynamics of an ex situ captive population of A. longipinnis, as well as the wild A. longipinnis population of the Kiso River. Genetic diversity generally varied between yearly cohorts in each of the captive sub-populations, and some showed a stable increasing trend with generations. When all sub-populations were considered as one population, genetic diversity was maintained at a high value, while effective population size generally reached target values, thereby preventing inbreeding. These results were achieved by maintaining multiple captive sub-populations and exchanging individuals between them. Simultaneously, the introduction of additional individuals from the wild population produced genetic variability in the captive population. These fluctuating patterns of genetic diversity in the captive A. longipinnis population were desirable compared to previously predicted values. Consequently, these findings show that the current ex situ conservation program is suitable for maintaining the genetic composition of the captive population of A. longipinnis.
Map of the study area in the Province of Viterbo, Central Italy. Landscape is reclassified according to the features utilized in the IBR analysis. RA represent the only railway intersecting the study area. Population codes are shown in Table 1
Strip chart of pairwise FST distances between sampled populations. Lines connect the same pairwise comparison between the wood mouse and the bank vole
Population assignment test performed with STRUCTURE. Bar plots represent the genetic composition of single individuals (thin vertical columns) from K = 2 to K = 4. A wood mouse; b bank vole. Maps of the study area with the genetic composition of each population for K = 3 in the wood mouse (left) and the bank vole (right)
Individual-based EEMS analysis of effective migration rates (m) for the wood mouse (left) and the bank vole (right). The effective migration rate is represented on a log10 scale. Areas showing negative values (orange) represent possible barriers to gene-flow while zones with positive values (blue) correspond to places of increased gene-flow, both with respect to the Isolation-by-Distance background (white). Migration surfaces are averages of 3 runs each with 50, 100, 200, 300, and 400 demes. The scale is different in the two panels, indicating that the increase (blue colors) or decrease (red colors) of the rates relative to the average rate are more pronounced in the wood mouse compared to the bank vole
Goodness of fit for models of landscape resistance. Panels show the coefficient of determination (R²) for models analyzing genetic differentiation (panel a–c: wood mouse; panel b–d: bank vole) in relation to resistance (a, b) and permeability (c, d) distance matrices plotted against resistance values for different landscape features. Circles with black outline showed significant P-values
The negative impact of habitat fragmentation due to human activities may be different in different species that co-exist in the same area, with consequences on the development of environmental protection plans. Here we aim at understanding the effects produced by different natural and anthropic landscape features on gene flow patterns in two sympatric species with different specializations, one generalist and one specialist, sampled in the same locations. We collected and genotyped 194 wood mice (generalist species) and 199 bank voles (specialist species) from 15 woodlands in a fragmented landscape characterized by different potential barriers to dispersal. Genetic variation and structure were analyzed in the two species, respectively. Effective migration surfaces, isolation-by-resistance (IBR) analysis, and regression with randomization were used to investigate isolation-by-distance (IBD) and the relative importance of land cover elements on gene flow. We observed similar patterns of heterozygosity and IBD for both species, but the bank vole showed higher genetic differences among geographic areas. The IBR analysis suggests that (i) connectivity is reduced in both species by urban areas but more strongly in the specialist bank vole; (ii) cultivated areas act as dispersal corridors in both species; (iii) woodlands appear to be an important factor in increasing connectivity in the bank vole, and less so in the wood mouse. The difference in dispersal abilities between a generalist and specialist species was reflected in the difference in genetic structure, despite extensive habitat changes due to human activities. The negative effects of fragmentation due to the process of urbanization were, at least partially, mitigated by another human product, i.e., cultivated terrains subdivided by hedgerows, and this was true for both species.
Distribution of primary and guest box author host institutions across continents for the three editions of this book. Note, North America is defined here as the United States of America and Canada, as Mexico was not represented. Australasia includes only Australia and New Zealand, as no other countries in Melanesia were represented
Conservation genetics is a relatively new discipline, and yet has rapidly evolved in the last decade with massive advances in sequencing technologies. Here, we review the newest edition of an influential textbook in the field, “Conservation and the Genomics of Populations”, which seeks to bridge the transition from population genetics to genomics and its application to conservation management. This textbook—complete with 24 chapters (one completely new), 25 guest boxes, and two new authors over the previous edition—navigates the rich and sometimes complex history of conservation and population genetics, while also providing a comprehensive catalog of how genomics broadens our understanding of diversity in a changing world. Despite some sections requiring an advanced understanding of population genetic theory, we foresee this text being used as a reference for conservation geneticists and for teaching upper level undergraduate or graduate students. While we anticipate the field of conservation genetics will continue to rapidly advance with new technologies, this textbook provides a strong foundation of population genetics, while also celebrating the new horizon of genomics for conservation management.
Map showing the different sampling locations. The populations were grouped into East (Green), North (Red) and South (Blue) clusters and are colour coded as per the labels of the haplotype network. Inset: Male adult blackbuck
Haplotype network of all samples showing unique haplotypes for all locations
Neighbour joining tree built using pairwise genetic distance between all samples, from 7 microsatellite loci. The highlighted region shows the clusters where samples from Bhetanai (East) are present. The sample labels are colour coded according to the North (red), South (blue) and East (green) clusters
a Graph showing the DeltaK values for each K assigned in STRUCTURE. b Bar plot of STRUCTURE analyses arranged according the three clusters (K = 3)
Historical demographic scenarios tested using DIYABC. The green, red and blue regions correspond to the East (Pop1), North (Pop2) and South (Pop3) respectively. Results indicated Scenario 3 as the most likely scenario (See supplementary figure S3)
Genetic diversity of organisms is an indicator of their long-term survival and can potentially be shaped by the extent of geneflow between populations. Geographical features and anthropogenic interferences can both obstruct and also facilitate animal movement, directly or indirectly. Such patterns have not been extensively studied across grasslands in the Indian subcontinent which is a mosaic of both natural and man-made topography. This study looks at genetic variation in an endemic ungulate, the Antilope cervicapra or blackbuck, throughout its distribution range. Using mitochondrial and nuclear (microsatellite) information, we find that different markers shed light on different aspects of their evolutionary history. Absence of robust geographical clustering in mitochondrial DNA indicate recent isolation in these populations, while lack of shared haplotypes between sampling locations suggests female philopatry. Nuclear data shows the presence of three genetic clusters in this species, pertaining to the Northern, Southern and Eastern regions of India. Our study also shows that an ancestral stock separated into two groups that gave rise to the North and South clusters and the East population was derived from the South at a later time period. Both microsatellite and mitochondrial data indicate that the population from the Eastern part of India is genetically distinct and the species as a whole shows signatures of having undergone recent genetic expansion. In spite of immense losses in grassland habitats across India, blackbucks seem to have well-adapted to human altered landscapes and their numbers are beginning to show an upward trend.
Analysis of genetic structure in natural populations of Castanea dentata including reference samples of known identity. (a) STRUCTURE analysis with K = 4 genetic groups for all natural populations including all Castanea reference samples. (b) STRUCTURE analysis with K = 3 genetic groups for all natural populations and C. dentata reference samples, excluding all suspected misidentified and hybrid trees. Each vertical line represents an individual sample and colour depicts their membership coefficients to each genetic group
Geographic locations of the 13 American chestnut populations and proportional representation of the K = 3 genetic clusters, identified in the STRUCTURE analysis. The dashed line represents the N-S transect to investigate predictions of the central-marginal hypothesis
Relationship between pairwise genetic distance (linearized FST) and geographic distance (km) for (a) all Castanea dentata population pairs, and from population pairs in (b) Canada, and (c) U.S.
Between-class correspondence analysis (bCA) of the genetic composition of Castanea dentata populations. The populations served as the factor for variance maximization. Plots a and b demonstrate the dispersion of individual samples across the first two axes of the bCA, 43.3% and 17.1% of the variation on bCA1 and bCA2, respectively, accounting for a total of 60.4% of the variation. Plot b includes the loadings of each locus on plot a. Dots represent individual samples surrounded by 67% inertia ellipses for each population. Numbers within ellipses indicate the following populations in Canada (red) and the U.S. (blue): (1) CA-1, (2) CA-2, (3) CA-3, (4) CA-4, (5) CA-5, (6) CA-6, (7) CA-7, (8) US-1, (9) US-2, (10) US-3, (11) US-4, (12) US-5, (13) US-6
The relationship between two measures of genetic diversity within populations and their distance from the central population (US-4). (a) expected heterozygosity (He), (b) average number of alleles per locus (Na)
Knowledge of the magnitude and geographic patterns of genetic diversity is instrumental for recovery of endangered tree species whose persistence is limited by genetic variation. One such species is American chestnut (Castanea dentata), which has experienced a dramatic reduction in population size in North America in association with the spread of the parasitic fungus, Cryphonectria parasitica, causing chestnut blight. To examine the impact of the bottleneck and role of genetic diversity on population dynamics and recovery, we conducted a population genetic assessment of native American chestnut populations in the understudied northern range in Canada and along a transect towards the center of the U.S. range. Leaf tissue from 13 natural populations in Canada (N = 7) and northern U.S. (N = 6) were genetically characterized using 16 microsatellite loci and compared to a sample of reference Castanea species. Genetic diversity and population structure were assessed within and among populations to determine population connectivity and the presence of admixture with other Castanea spp. Populations throughout the range displayed high genetic diversity and significant inbreeding, with no significant difference in diversity between those at the center and edge of the range. We found evidence of infrequent interspecific hybridization in some Canadian populations but no relationship between admixture and tree health, assessed in a previous demographic survey. Unexpectedly, Canadian populations clustered separately from U.S. populations. American chestnut appears to have retained substantial genetic diversity following the population bottleneck, which is at odds with the limited incidence of blight resistance/tolerance in extant populations.
Narrowly endemic species are particularly vulnerable to catastrophic events. Compared to widespread species, they may also be less capable of adapting to shifts in environmental pressures as a result of specialisation on a narrow range of local condition and limited ability to disperse. However, life-history traits, such as preferential outcrossing and high fecundity can maintain genetic diversity and evolutionary potential, and boost species resilience. The endangered Grevillea bedggoodiana (Enfield Grevillea) is an understorey shrub restricted to an area of ca. 150 km 2 in southeastern Australia with a legacy of large-scale anthropogenic disturbance. Prior to this study little was known about its biology and population structure. Here, its breeding system was assessed through a controlled pollination experiment at one of its central populations, and eight populations were sampled for genetic analysis with microsatellite markers. The species was found to be preferentially out-crossing, with no evidence of pollination limitation. In most populations, allelic richness, observed heterozygosity and gene diversity were high (Ar: 3.8-6.3; H o : 0.45-0.65, H e : 0.60 − 0.75). However, the inbreeding coefficients were significant in at least four populations, ranging from F i-0.061 to 0.259 despite high outcrossing rates. Estimated reproductive rates varied among sampled populations but were independent of gene diversity and inbreeding. Despite its small geographic range, the species' populations showed moderate differentiation (AMOVA: F ST = 0.123), which was largely attributable to isolation by distance. We interpret these results as suggesting that G. bedggoodiana is reproductively healthy and has maintained high levels of genetic diversity despite recent disturbance.
a Localities sampled for the two morphological forms of C. pristinus. Circles identify localities for the Caney Fork Form and diamonds denote those for the Sequatchie Form. Symbols filled with color denote localities where C. pristinus was encountered and colors corresponds to those used to depict phylogeographic relationships in (b, c). White-filled symbols denote localities sampled where C. pristinus was not detected. The white star is where the Caney Fork River becomes > 4th order. The Cumberland Plateau ecoregion is shaded gray. The thick black line represents the Western Escarpment of the Cumberland Plateau. Numbers denote site ID and correspond to those in Table 1: 1—Caney Fork, 2—Pokepatch Creek, 3—West Fork, 4—Meadow Creek, 5—Little Laurel Creek, 6—Puncheoncamp Creek, 7—Spring Creek, 8—West Fork Little Cane Creek, 9—Camp Creek, 10—Long Fork, 11—Flatrock Branch. b The two haplotype networks recovered from mitochondrial COI sequences of C. pristinus. Circles represent haplotypes and the size of each circle is proportional to the number of individuals with a given haplotype; connecting lines represent mutations among haplotypes. Colors correspond to localities depicted in Fig. 1a and open circles represent unsampled haplotypes. c Maximum-likelihood 50% majority rule consensus tree from the unique mitochondrial COI haplotypes recovered for C. pristinus. Bootstrap support values are found at each node and average pairwise sequence divergence estimates between clades or taxa are in parentheses
Population structure and EEMS results based on 19 microsatellite loci examined for the Caney Fork form of C. pristinus. a EEMS overlay indicating areas of higher and lower than expected migration rates for the Caney Fork Form of C. pristinus. Blue areas indicate areas of higher-than-expected migration rates and orange areas indicate areas of lower-than-expected migration rates. Colors in pie charts indicate proportion of individuals from each locality assigned to a distinct population cluster identified from the DAPC analysis; colors correspond across a, b, and c. Triangle colors indicate cluster assignment from the STRUCTURE analysis. Numbers represent site ID: 1—Caney Fork, 2—Pokepatch Creek, 3—West Fork, 4—Meadow Creek, 5—Little Laurel Creek, 6—Puncheoncamp Creek, 7—Spring Creek, 8—West Fork Little Cane Creek as used in Fig. 1 and Table 1. b DAPC results showing first two discriminate functions. Colors represent the six distinct population clusters recovered from STRUCTURE analysis and shapes indicate the original locality assignment for each individual. c STRUCTURE analysis results using loc priors to assess population structure. Black boxes represent sites and colored bars within each box represent population of ancestry for each individual. Numbers denote site ID and letters denote population cluster assignment
Isolation by distance analysis for the eight sites of the Caney Fork Form of C. pristinus showing the relationship between geographic (log-transformed river and Euclidean kilometers) and genetic distances (FST and Jost’s D) based on genotypes of 19 microsatellite loci. For corresponding pairwise values refer to Table 4. Comparisons using log-transformed river distances are in black and those using log-transformed Euclidean distances are in gray (River-FST: R² = 009; p = 0.13, Jost’s D: R² = 0.14; p = 0.08; Euclidean-FST: R² = 0.16; p = 0.05, Jost’s D: R² = 0.16; p = 0.05). Jost’s D values are indicated with circles and FST values are indicated with diamonds
Assessments of genetic diversity for imperiled species can provide a baseline for determining the relative impacts of contemporary anthropogenic threats (e.g., habitat fragmentation) on population connectivity and identify historical factors contributing to population structure. We conducted a population genetics and phylogeographic assessment of the imperiled Pristine Crayfish (Cambarus pristinus) sampled throughout its range encompassing two morphologically distinct forms. Pristine Crayfish exhibit a disjunct distribution throughout lower order tributaries suggesting they are headwater-adapted species. The two morphologically distinct forms of the Pristine Crayfish are found in the upper Caney Fork (nominal Caney Fork form) and the Big Brush Creek and Cane Creek systems (Sequatchie form). We used 19 microsatellite loci and the cytochrome oxidase subunit I (COI) gene to assess population connectivity and genetic diversity of the Pristine Crayfish. Haplotypes recovered from the COI gene revealed that historic connectivity was maintained within each form of the Pristine Crayfish. However, the divergence between forms was higher (2.3%) than within forms (< 2.0%), suggesting each form is on an independent evolutionary trajectory, supporting recognition of the Sequatchie form as a distinct taxon. Microsatellite analyses for the Caney Fork form recovered a high degree of population isolation and support for six genetically isolated population. In addition, genetic diversity metrics per population and for the Caney Fork form were low suggesting that the Caney Fork form is at an increased risk of extinction under anthropogenic disturbances. We suggest that each form receive continued listing protection and conservation resources and that the Sequatchie form be treated as a unique taxon.
(a) Known L. europaeus introduction sites (brown circles) in Ireland (showing county boundaries for orientation) as reported by Barrett-Hamilton (1898) though all died out except those at Baronscourt Estate, west Tyrone. Subsequently, sightings (tan 10km squares) were reported in north Donegal (Fairley 2001; Sheppard 2004) and Mid-Ulster spanning south-east Derry and east Tyrone (Caravaggi et al. 2016). (b) Infographic showing 53 specimens from Mid-Ulster identified using species-specific mitochondrial (bottom lane) and nuclear (top lane) DNA as L.t. hibernicus (green) or L. europaeus (brown) or mixed sequences (hatched green/brown) and species ID (top). (c) Distribution of genetically identified samples relative to the non-native range of L. europaeus as surveyed during 2012-13 (Caravaggi et al. 2016) showing named urban areas (grey) for orientation. Pie charts show the percentage of specimens at each location which were native (green), non-native (brown) or hybrids (hatched green/brown with bold boundary) with charts scaled to represent sample sizes (1 < n < 15). Hare thumbnails show L.t hibernicus (left ©Mike Brown), L. europaeus (right ©Mark Hamblin) and a europaeus x timidus hybrid during winter (middle ©Mark Hamblin)
Introduced non-native species can threaten native species through interspecific hybridisation and genetic introgression. We assessed the prevalence of hybridisation and introgression between introduced European brown hare, Lepus europaeus , and the endemic Irish hare, L. timidus hibernicus . Roadkill hares ( n = 56) were sequenced for a 379bp section of the mitochondrial DNA D-loop and a 474bp segment of the nuclear transferrin ( Tf ) gene. A species-specific indel in the transferrin gene was present in L.t. hibernicus and absent in L. europaeus . Excluding three hares from which molecular data could not be recovered, 28 hares (53%) were native L.t. hibernicus , 7 (13%) were non-native L. europaeus and 18 (34%) were hybrids; of which 5 (28%) were first generation (F1) involving bidirectional crosses with mismatched nuclear and mtDNA (3 ♂ europaeus x ♀ hibernicus and 2 ♂ hibernicus x ♀ europaeus ). Mixed nuclear transferrin sequences suggested 13 (72%) of hybrids were at least 2nd generation (F2) with 9 (69%) possessing L.t. hibernicus and 4 (31%) L. europaeus mtDNA (the latter indicative of hybrid backcrossing with the non-native). The prevalence of hybridisation at similar mountain-brown hare contact zones throughout Europe is notably lower (4–16%) and typically unidirectional (♂ europaeus x ♀ timidus ). A high prevalence of bidirectional hybridisation and introgression (in association with projected climate change) may favour the introduced species over the native. Genetic surveillance and population monitoring are needed to further explore the potential conservation implications of European brown hare in Ireland.
Identifying units for appropriate management and conservation of rare species is an important and challenging process, and population genetics can inform this decision making. Using Phlox hirsuta, a rare species restricted to serpentine soils in Northern California and with a geographic range of less than 15 km, we examined genetic variation within and among populations, using tunable Genotyping-by-Sequencing (tGBS) to generate single nucleotide polymorphisms (SNPs) as well as 11 microsatellite loci, to identify population structure, patterns of migration and selection, and units for conservation. Multiple methods recognized three geographically structured population clusters. The species has undergone a recent genetic bottleneck, and the increase in population size may be influenced by the changing climate. Patterns of gene flow are greater from south to north than in the opposite direction. Some of the genes under selection are putatively involved in adaptation to edaphic conditions, and genes under selection differ among the populations. Four population units were identified as suitable for conservation purposes based on various partitions of the SNPs.
Caribou sampled in western Canada for genomic analyses (n = 190). Black numbered circles indicate sampled populations (also referred to as “herds”, as they might not be genetically or ecologically distinct). Circle size is proportional to sample size by population (mean = 8.63, SD = 7.56, range 1–29). Grey-scale polygons show the distribution of currently recognized subspecies and Designatable Units (DUs). The Barren-ground subspecies (R. t. groenlandicus) is currently recognized as its own DU while the Woodland caribou subspecies (R. t. caribou) includes the Northern Mountain, Central Mountain, and Boreal DUs
Population structure of caribou in western Canada. Bar plots from Structure (a) and Admixture (b) analyses, respectively, indicate proportions of caribou individuals belonging to either a Northern Caribou (blue color) or Southern Caribou cluster (red color). Currently recognized subspecies and Designatable Unit (DU) assignments of individuals are indicated: BG refers to the Barren-ground subspecies; NM, CM, and Boreal refer to the Northern Mountain, Central Mountain, and Boreal DUs, respectively, within the Woodland subspecies. For NM individuals, we also indicate their location in either Yukon (NM-YT, farther North), or British Columbia (NM-BC). Map from Tess3 analyses c showing geographic prediction of Southern or Northern Caribou clusters. Boundaries between the two clusters can be predicted with less confidence where the color is paler. Most likely number of clusters (K) obtained with d Structure (higher values best), e Admixture, or f Tess3 (lower values best)
Principal Component Analysis (a PCA) and neighbor-joining tree (b) of caribou in western Canada. Dots in the PCA and branches of the neighbor-joining tree were calculated examining SNP data and represent caribou individuals, while colors represent caribou groups. PC 3 and 4 explained 2.89% and 2.23% of variance, respectively. Caribou groups include Barren-ground (BG), Northern Mountain—Yukon (NM-YT), Northern Mountain-BC (Northern Mountain British Columbia), Central Mountain (CM), and Boreal
Caribou individuals’ assignment into K = 2 clusters when using X-linked SNPs. a Caribou assignment obtained analyzing X-linked SNPs. Each bar represents an individual and colors represent genetic clusters (blue and red indicate Northern and Southern Caribou clusters, respectively). b Cross-validation plot of Admixture analyses conducted for X-linked SNPs and three sets of random autosomal SNPs. Random SNPs were chosen in equal number to those located on the X-chromosome (n = 781 out of 28,260, see Methods). Admixture analyses conducted for these random sets of SNPs did not support any K
Signatures of divergent selection detected between caribou groups in western Canada using the BayeScan program. Outlier SNPs detected between Northern and Southern Caribou clusters (i.e. clusters detected at K = 2 with Structure program) (a), and outliers detected between currently recognized Barren-ground and Woodland subspecies (b). The horizontal axis indicates the log10 of the q value (the false discovery rate (FDR) analog to the p-value) and the vertical axis is the mean genetic differentiation (FST). Each point represents a SNP and significant outliers are visible right of the grey vertical line. Outliers located on the X-chromosome are marked with an “x”
Within-species, biodiversity can be organized in units, ranging from subspecies to evolutionarily significant units (ESUs), populations and social groups. To define ESUs, researchers often focus on the concordant distribution of traits that exhibit likely adaptive significance, including genetic and ecological variation. Caribou is a Species at Risk in Canada, and are conserved at the level of both subspecies and designatable units (DUs), which are conceptually similar to ESUs. However, the use of genomics has been suggested to provide better delineation of units that are based upon variation of genes—not just neutral genetic markers. Here, we analyzed single nucleotide polymorphisms (SNPs) for 190 caribou belonging to two recognized subspecies and four DUs found throughout western Canada. We confirmed two major genetic clusters, which we refer to as the Northern Caribou and Southern Caribou, characterized by divergence at numerous SNPs and genes with known functions in other mammals. Notably, the distribution of these two clusters did not fully overlap with currently recognized subspecies. A discrepancy with current classification was detected for Mountain DUs, which were thought to belong to the Woodland subspecies, but with significant northern-type ecological traits described in the literature, indicating more work is needed to refine our understanding of this transitional zone. We also detected genetic signals of male-biased dispersal, which may be natural or affected by habitat fragmentation effects on females. This work illustrates the value of genomics in rethinking subspecies and conservation unit designations and better conserve biodiversity within terrestrial species at risk.
Map of study sites on the Cataract River and Cataract Dam, New South Wales, Australia. Study area is highlighted in red on inset map. MDB Murray-Darling Basin; HNB Hawkesbury-Nepean Basin. Site 1: Below Cataract Dam; Site 2: Jordans Pass; Site 3: Broughtons Pass Weir
PCoA analysis of genotypes for Cataract Dam (N = 56) and Cataract River (N = 51) using a 370 loci, and b 2777 loci, respectively. Pie charts on scatterplot indicate assignment to HNB nuclear lineage (black) and MDB nuclear lineage (white) by STRUCTURE. Cataract River fish assigned to the MDB mitolineage have a blue outline (N = 20) and fish assigned to the HNB mitolineage have a red outline (N = 31). Cataract Dam fish have a black outline as their mitolineage was not assigned. MDB Murray-Darling Basin; HNB Hawkesbury-Nepean Basin
Results of STRUCTURE analysis, built from a 370 loci most useful at distinguishing between Cataract Dam (N = 56) and Cataract River (N = 51) populations and b 2777 loci (Cataract Dam N = 56 and Cataract River N = 51). Membership of each individual to River (black, QR) cluster is indicated by histogram bars. River individuals identified as being part of the MDB mitolineage are highlighted in blue (N = 14), fish with HNB mitolineage are highlighted in red (N = 26). Fish assigned as F1 × Cataract Dam, F1 × Cataract River and F2 in NEWHYBRIDS are shown in detail in inset graphs (QR-values and mitolineage (MDB = blue; HNB = red; N = 10). MDB Murray-Darling Basin; HNB Hawkesbury-Nepean Basin; Dam Cataract Dam; River Cataract River
Restoring levels of genetic diversity in small and declining populations is increasingly being considered in biodiversity conservation. Evidence-based genetic management requires assessment of risks and benefits of crossing populations. Because risks are challenging to assess experimentally, e.g. through multi-generational crosses, decision-support approaches utilize proxy risk factors such as time since separation of lineages. However, the paucity of empirical datasets on fitness consequences of longer separation times tends to favour crossing lineages with conservatively short separations, restricting wildlife managers’ options. Here, we assessed the genetic outcomes of interbreeding in the wild between lineages of a threatened Australian freshwater fish (Macquarie perch) separated by an estimated 119,000–385,000 years of evolution in distinct environments. Fish belonging to the Murray-Darling Basin (MDB) lineage escaped from Cataract Dam—into which they were translocated in ~ 1915—into the Cataract River, where they interbred with the local Hawkesbury-Nepean Basin (HNB) lineage. Analyses of reduced-representation genomic data revealed no evidence of genetic incompatibilities during interbreeding of the two lineages in the Cataract River: assignment to genotypic clusters indicated a spectrum of hybrid types including second generation hybrids and backcrosses to both parental lineages. Thus, no adverse effects were detected from genetic mixing of populations separated by > 100,000 years. We are not advocating purposely crossing the two lineages for management purposes under present cost–benefit considerations, because there are currently sufficient intra-lineage source populations to beneficially mix. Instead, this study presents a useful calibration point: two morphologically different lineages evolved in different habitats for 119,000–385,000 years can successfully interbreed.
Map of the 31 bushmeat trading places surveyed in Côte d'Ivoire, with main roads and protected areas. 1-Abobo; 2-Abidjan; 3-Agbaou; 4-Agboville; 5-Akabréboua; 6-Alépé; 7-Bouaflé; 8-Boundiali; 9-Dabou; 10-Dagbégo; 11-Daloa; 12-Dassioko; 13-Diapé; 14-Dimbokro; 15-Dinaoudi; 16-Duékoué; 17-Fresco; 18-Grand_Lahou; 19-Grogoua; 20-Issia; 21-Korhogo; 22-Kouto; 23-Lélédou; 24-Man; 25-Odienné; 26-Tiébissou; 27-Touba; 28-Toumodi; 29-Yopougon; 30-Zéban; 31-Zuénoula. GPS coordinates are given in Appendix 1
Rarefaction curve of the expected number of species found in the bushmeat trading places of Côte d’Ivoire. The maximum x-axis value (32) corresponds to the number of places sampled in our study
Distribution of intra- and inter-species pairwise genetic distances among the bushmeat species from Côte d’Ivoire for the four genes used in this study. A Cyt b; B COX1; C 12S; D 16S. Intra- and inter-species distances are given as white and black bars, respectively. x-axis represents K2P distance values; y-axis shows the number of pairwise comparisons
Accuracy of field-based taxonomic identifications among mammalian orders
Surveying and quantifying the bushmeat crisis in Africa requires up-front, reliable species-level identification. We conducted a comprehensive survey of 31 trading places where bushmeat are sold in Côte d’Ivoire (West Africa) and two seizures from Europe, using a multi-gene DNA-typing approach and a dedicated species-assignment pipeline (DNAbushmeat). We identified 47 wild and five domestic species-level taxa from 348 collected carcasses, including mammals (15 Cetartiodactyla, 10 Rodentia, seven Carnivora, seven Primates, two Pholidota, two Lagomorpha, one Hyracoidea, one Chiroptera), reptiles (two Squamata), birds (one Bucerotiformes, one Galliformes, one Otidiformes) and fish (one Perciformes). Our DNA-based approach allowed the detection of two separate lineages of red-flanked duikers (Cephalophus rufilatus), a yet unreferenced cane rat (but possibly Thryonomys gregorianus) and two cryptic species of Gambian rat (Cricetomys). We also observed important levels of intraspecific diversity in several mammals and squamates, suggesting additional cryptic diversity within bushmeat species from Côte d’Ivoire. More than half of the bushmeat carcasses were inaccurately identified, with European customs peaking at 100% inaccuracy. Our study also explored the use of diversity indices among bushmeat markets to identify ‘hotspot’ market places where biodiversity would be the most impacted. Overall, 12 protected species (including pangolins, crocodiles, primates and antelopes) were impacted by the bushmeat trade in Côte d’Ivoire, indicating weak law enforcement related to game protection. We suggest that the recognition of the bushmeat sector by the state and its DNA-based surveillance is necessary to reach a sustainable management of the bushmeat trade in Côte d’Ivoire.
Study area and genetic structure. A Sampling localities. Labels correspond to population IDs (Pop1, 2, …, 9) in Table 1. The broad-area map shows the location of the Shiretoko Peninsula and routes of the Tsushima Current, including its branches. B The values of posterior probability of the data (LnP(D)) from 20 runs for each value of K (1–9) and Evanno’s delta K in the STRUCTURE analysis. C Population structure estimated in STRUCTURE. Barplots display the proportion of the membership coefficient in the inferred clusters at K = 2 and 4 for all individuals, and the numbers indicate the population IDs
Comparison of genetic diversity between west and east subpopulations. A, B Boxplots showing differences in the level of allelic richness (A) and expected heterozygosity (B) between the western and eastern populations. C, D Approximate Bayesian skyline plots showing the demographic history of C. hangiongensis for west (C) and east (D) subpopulations. The solid line shows the median estimates of historical effective population size (Ne), and the dashed lines show the 95% highest posterior density estimates of the Ne
Despite the global crisis facing migratory benthic fishes, conservation genetic knowledge of these species remains scarce. In this study, we conducted a population genetic analysis using seven microsatellite loci to obtain basic information for determining conservation units and priorities of Cottus hangiongensis in Shiretoko, a mountainous peninsula where sculpin habitats are thought to be in decline throughout the region. The genetic structure was clearly divided between west and east coastal populations, and there was little recent migration between them. The western populations, which are closer to the center of the species’ range, had significantly higher genetic diversity than the eastern populations. However, a bottleneck analysis and the inference of demographic history using approximate Bayesian computation showed that only the west group had experienced a significant recent bottleneck, probably due to recent habitat losses. These results suggest that the western and eastern populations should be different conservation units and that the western populations should be prioritized for conservation despite their high genetic diversity. This study contributes to the conservation genetics of diadromous sculpin and reiterates the importance of analyzing not only the current levels but also temporal changes in assessing genetic diversity.
Rafflesia species (Rafflesiaceae) are among the flagship plants of South-East Asian countries in which they occur. Three species of Rafflesia, i.e. Rafflesia patma, R. rochussenii, and R. zollingeriana, are known from Java, Indonesia. All three species are threatened with extinction due to human activities that cause habitat loss and fragmentation. Conservation efforts such as determining conservation units for prioritization of those species have been difficult due to the lack of data on their population genetics. Availability of genetic information is important to develop appropriate conservation measures. Our study evaluates genetic diversity and structure of the three Rafflesia species using a total of 166 samples across the island. We used single nucleotide polymorphism (SNP) markers obtained via MIG-seq. The three species of Rafflesia in Java bear much lower genetic diversity compared to what was previously shown for R. speciosa and R. lagascae on Borneo, the Philippines and the Malayan Peninsula. Low genetic diversity within the Javanese Rafflesia species, particularly in R. patma and R. zollingeriana, is attributed to bottleneck events and population expansion in the past. We also provide evidence of clonality and existence of different genotypes within Tetrastigma host plants in two species of Rafflesia. Scattered and fragmented populations as reconstructed in the genetic structure analyses are important to be considered in designing appropriate conservation strategies. Furthermore, we demonstrate how the establishment of Rafflesia ex-situ collections can conserve genetic diversity that may no longer be present in nature and could be used in future reintroduction programs.
Map of locations of spiny daisy populations in mid-north South Australia, showing Melrose, Telowie, Thornlea, Yangya, Rusty Cab and Hart
Principal component analysis of genetic distance between individuals. Samples are coloured by sampling location
Likelihood of ancestry to each population for each individual based on the Bayesian clustering model of K = 6. Individuals are represented by vertical lines, ordered by geographic population along the x-axes. Proportion of ancestry to each population is illustrated by colour on the y-axis
Heatmap showing the distribution of absolute fixed differences between populations represented as a dissimilarity matrix. Darker colours represent a greater number of fixed differences between the two corresponding populations
Observed Heterozygosity (Ho: black) and Expected Heterozygosity (He: white) within each population (N = 15)
Understanding population structure and genetic diversity is important for designing effective conservation strategies. As a critically endangered shrub, the six remaining extant populations of spiny daisy ( Acanthocladium dockeri ) are restricted to country roadsides in the mid-north of South Australia, where the species faces many ongoing abiotic and biotic threats to survival. Currently the spiny daisy is managed by selecting individuals from the extant populations and translocating them to establish insurance populations. However, there is little information available on the genetic differentiation between populations and diversity within source populations, which are essential components of planning translocations. To help fill this knowledge gap, we analysed population structure within and among all six of its known wild populations using 7,742 SNPs generated by a genotyping-by-sequencing approach. Results indicated that each population was strongly differentiated, had low levels of genetic diversity, and there was no evidence of inter-population gene flow. Individuals within each population were generally closely related, however, the Melrose population consisted entirely of clones. Our results suggest genetic rescue should be applied to wild spiny daisy populations to increase genetic diversity that will subsequently lead to greater intra-population fitness and adaptability. As a starting point, we suggest focussing on improving seed viability via inter-population crosses such as through hand pollination experiments to experimentally assess their sexual compatibility with the hope of increasing spiny daisy sexual reproduction and long-term reproductive fitness.
Map showing mtDNA control region haplotype frequencies per sampling site for ridley sea turtles (Lepidochelys spp.). Red circled lines depict non-rookery populations (i.e., foraging grounds, bycatch, or ghost-net). Haplotypes characteristic of Atlantic olive ridley populations are shown in blue shades, Indian in aqua shades, Indian-West Pacific in red/pink shades, and East Pacific in orange shades, while Kemp’s ridley haplotypes are shown in green. Rare haplotypes (found in less than three individuals) are not included on the map. Smaller circles indicate sampling locations with only one sample. Coordinates for all populations are approximate. Literature sources and haplotype frequencies are given in Tables S1 and S2. Putative geographical barriers are shown by dotted bars
Median-joining network showing the relationships among Kemp’s and olive ridley mtDNA 653 bp control region haplotypes. Circles are proportional to the number of individuals. Black circles indicate missing haplotypes, and two or more mutational steps are shown as numbers. Colors represent sampled regional rookeries and non-rookeries as described in Fig. 1. Loggerhead sea turtles (the outgroup) are shown in gray. IWP = Indian-West Pacific rookeries, IWP-GP = Indian-West Pacific Ghost net and Pelagic, IN = Indian nesting and foraging, ATL = Atlantic, ATL-CA = Atlantic feeding and Capture at-sea, EP = East-Pacific rookeries, EP-FP = East Pacific Foraging and pelagic/stranded
Bayesian tree with divergence times of olive ridley mtDNA sequences (ATL, IWP, EP, IN) and Kemp’s ridleys. Abbreviations and colors as in Fig. 2. Terminal clades were represented by triangles in which deep points correspond to diversification times. Loggerhead sea turtles were used as the outgroup (not shown). Gray bars represent 95% High Posterior Density estimates for divergence times. The asterisk denotes the only major clade subdivision with low Posterior Probability values (PP = 0.02)
(A) STRUCTURE barplot of individual admixture proportions from olive ridley microsatellite genotypes for K = 2 and K = 3 without prior population information. (B) STRUCTURE barplot using oceanic region of origin as prior information. Arrows show individuals with Q-values from 0.2 to 0.8 (admixed, grey arrows) or recent migrants (Q > 0.8, black arrows). (C) DAPC barplot with membership probabilities, similar to the STRUCTURE barplots, with K = 12 and the 12 main sampling sites (in the same order as in A-B) as prior information. (D) DAPC scatterplot with K = 3 and the 12 main sampling sites as prior information. (E) DAPC scatterplot with K = 3 without prior population information. Codes used below the horizontal axis represent sample collection location. Only individuals with a maximum of one missing locus are shown. SL = Sri Lanka; MA = Malaysia; AU-nwCY = Australia north-western Cape York; AU-NT = Australia Northern Territory; GB = Guinea-Bissau; SU = Surinam; FG = French Guiana; BR = Brazil; BC = Baja California; ME = Mexico; CR = Costa Rica and EP-FEED = East Pacific (Mexican waters) foraging grounds
Globally distributed marine taxa are well suited for investigations of biogeographic impacts on genetic diversity, connectivity, and population demography. The sea turtle genus Lepidochelys includes the wide-ranging and abundant olive ridley (L. olivacea), and the geographically restricted and 'Critically Endangered' Kemp's ridley (L. kempii). To investigate their historical biogeography, we analyzed a large dataset of mitochondrial DNA (mtDNA) sequences from olive (n = 943) and Kemp's (n = 287) ridleys, and genotyped 15 nuclear microsatellite loci in a global sample of olive ridleys (n = 285). We found that the ridley species split ~ 7.5 million years ago, before the Panama Isthmus closure. The most ancient mitochondrial olive ridley lineage, located in the Indian Ocean, was dated to ~ 2.2 Mya. Both mitochondrial and nuclear markers revealed significant structure for olive ridleys between Atlantic (ATL), East Pacific (EP), and Indo-West Pacific (IWP) areas. However, the divergence of mtDNA clades was very recent (< 1 Mya) with low within- clade diversity, supporting a recurrent extinction-recolonization model for these ocean regions. All data showed that ATL and IWP groups were more closely related than those in the EP, with mtDNA data supporting recent recolonization of the ATL from the IWP. Individual olive ridley dispersal between the ATL, EP, and IN/IWP could be interpreted as more male- than female-biased, and genetic diversity was lowest in the Atlantic Ocean. All populations showed signs of recent expansion, and estimated time frames were concordant with their recent colonization history. Investigating species abundance and distribution changes over time is central to evolutionary biology, and this study provides a historical biogeographic context for marine vertebrate conservation and management. Supplementary information: The online version contains supplementary material available at 10.1007/s10592-022-01465-3.
Inter- and intra-specific genetic differentiation in the Fregata genus, calculated from a 550-bp fragment of the cytochrome b gene: A inferred phylogenetic relationships among species, and correspondence with the different geographical groups defined from hierarchical analysis of molecular variance (AMOVA; Table 3); B spatial patterns of genetic groups; and Principal Coordinates Analysis (PCoA) using pairwise differences among genetic groups based on C corrected p-distance and D average Tamura and Nei (1993) distance (values in Table S2). Colors correspond to morphological species and numbers to genetic groups (Table 2). In the map, black dots represent sampling sites and polygons the known species distribution (source: IUCN): F. magnificens (stippled red), F. aquila (orange vertical striped), F. ariel (purple diagonal striped), F. minor (continuous green) and F. andrewsi (brown horizontal striped)
Haplotype network and distribution of Fregata magnificens. Known species distribution range is shown (shade; source: IUCN) and details of sampling sites are described in in Table 1. In the network, circle size is proportional to the frequency of the respective haplotype and dashes in between haplotypes represent single mutation events. The genetic groups identified by AMOVA are labelled. Arrows highlight two detected migration events, including a vagrant F. magnificens sampled in the Ascension Island (see Table S1). Species illustration from (Accessed September 2018)
Haplotype network and distribution of AFregata aquila and F. andrewsi, BF. ariel and CF. minor. Known species distribution range is shown (shade; source: IUCN) and details of sampling sites are described in Table 1. In the network, circle size is proportional to the frequency of the respective haplotype and dashes in between haplotypes represent single mutation events. The genetic groups identified by AMOVA are labelled. Species illustrations from (Accessed September 2018)
In the present context of the ramping up of the global biodiversity crisis, improving our understanding on the genetic and biogeographic patterns of ill-known taxa is central to conservation planning. This is especially relevant for geographically isolated populations that suffer from little or no gene flow and an increased extinction risk. The few studies available on the molecular phylogeny and phylogeographic diversity of Frigatebirds have only focused on populations of three species, Fregata magnificens, F. andrewsi from Christmas Islands and F. minor in the Galapagos. To improve our knowledge on these tropical seabirds, our study aimed at assessing worldwide phylogeographic patterns and relationships among all five extant species of the genus Fregata. To accomplish it, we sampled museum specimens corresponding to 18 frigatebird populations spatially distributed by Brazil, Mexico, Ascension Islands, Cabo Verde and the Indo-Pacific region, and fresh samples from Cabo Verde, and amplified them for a mtDNA cytochrome b fragment. We complemented our dataset with previously available data representing a total of 36 populations in this study. Similar to the well-known endemic populations of the Galapagos and Christmas Island, the isolated ultraperipheral populations in the Atlantic were shown to be genetically divergent from their main populations for the three widespread species, F. magnificens, F. ariel and F. minor. We provide the first genetic data for F. ariel, whilst building upon the existing knowledge of the genetic patterns of F. magnificens, F. aquila and F. minor. Furthermore, our molecular data comes in support of most but not all the morphologically recognized frigatebird subspecies. This study provides important genetic insights into the evolutionary history of the genus Fregata and acts as a baseline for future molecular work and conservation efforts.
The folded site frequency spectrum (SFS) of European and American eel with the sample size of European eel downsized to n = 30, corresponding to the sample size of American eel. The proportion rather than number of SNPs in each frequency class is shown. The full SFS of both species can be seen in Supplementary Material Fig. S1
Demographic histories of Atlantic eels inferred by Stairway Plot 2, assuming generation length of 13 years for European eel (Anguilla anguilla) and 10.7 for American eel (A. rostrata). The solid line and dotted lines indicate the median estimate and the 2.5 (upper) and 97.5 (lower) percentile estimation of the effective population size, respectively. The gray shadows from left to right indicate five historical events: the last glacial maximum (LGM), the Brunhes-Matuyama magnetic reversal, secondary contact between the two species and time of speciation inferred using δaδi (Nikolic et al. 2020), and the closure of the Panama Gateway. Inferred demographic histories assuming the same generation length for both species are shown in Supplementary Material Fig. S2
European (Anguilla anguilla) and American eel (A. rostrata) are panmictic catadromous fish species, which have experienced recent drastic population declines amounting to just a few percent recruitment relative to levels prior to 1980. However, little is known about the extent of recent population declines relative to historical fluctuations. We analyzed demographic histories of the species using a method for reconstructing skyline plots based on site frequency spectra, in this case derived from restriction site-associated DNA (RAD) markers. The results showed very high effective population sizes ranging in the millions for most of the time span covered. Both species experienced ancient declines coinciding with the time of speciation (ca. 170,000 generations ago) and at a later stage where secondary contact occurred (ca. 90,000 generations ago). Whereas the demographic histories of the species were similar most of the time, they followed widely different trajectories from ca. 70,000 to 40,000 generations ago. However, for the past ca. 30,000 generations both species have shown demographic stability, even across glacial and interglacial periods. We discussed the possible environmental factors, including ocean current changes and geomagnetism reversal that could have affected demographic history and further suggest that southward displacement of spawning regions and continental distribution could explain the apparent stability even during glaciations. The recent declines appear unprecedented against a backdrop of long-term demographic stability, underpinning concerns that low density of spawners in the huge spawning region could lead to detrimental Allee Effects.
Sampling sites of Neocaridina shrimps in Lake Biwa and the surrounding rivers, central Japan. See Table 1 for details of sampling sites
Comparison of five diagnostic characters in Neocaridina shrimps between genetic clusters. Left column: measurement points for the five characters; a proportion of rostrum length to carapace length (RL/CL), b number of dorsal rostrum teeth (RT), c curvature of propodus of 3rd pereopod (TP), d aspect ratio of the endopod of the 1st pleopod (AE), e excavation of carpus of 1st pereopod (FP). Middle column: trait values of the five characters mapped on PCA using SNPs. Right column: box plots or stacked bar plots of the trait values using only individuals with ancestry proportions of ≥ 95% for any genetic clusters in the ADMIXTURE analysis (see Fig. 3). The black dots in the box plots represent the measured values for each individual, the white circles represent the mean values for each cluster, and the arrows and lines represent the measurements for the male specimen collected in 1915 (Zoological Collection of Kyoto University). The numbers below the bar plots are sample sizes. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001. Illustrations of the characters were modified from Kakui and Komai (2022) with the permission of the authors
Results of SNP-based and mtDNA analyses for Neocaridina spp. in and around Lake Biwa. Identification of each cluster/clade is based on this study. a Ancestry bar plots of ADMIXTURE analysis based on 183 SNPs showing K = 2 and 3. The optimal number of clusters estimated via cross-validation error comparison was three. The color of the plot above the bar plot represents the mtDNA clade (see c). b Principal component analysis results showing PC1 and PC2 plots based on 183 SNPs. The pie chart of the plots represents the ancestry proportion estimated from the ADMIXTURE analysis in K = 3 (SNP cluster 1, 2, 3). c Phylogenetic relationships of the mtDNA haplotypes estimated by the maximum likelihood method using IQ-TREE2 based on the 596 bp COI region. The tip label represents the haplotype name and the number in parentheses represents the number of individuals. The numbers in each node represent the support values or posterior probability estimated by SH-like approximate likelihood ratio test 1000 times, approximate Bayes test, and the ultrafast bootstrap 1000 times
Invasive species prevention involves avoiding two aspects: introduction and secondary spread. The latter is essential in places that can become hubs for spreading invasive species. Lake Biwa, the largest lake in Japan, is an important area for biodiversity and fisheries. However, several invasive fish and crustaceans were established in the lake last century. One of the conservation problems in the Lake Biwa region is the unresolved suspicion that the native freshwater shrimp Neocaridina denticulata have been replaced with alien Neocaridina species. To verify whether exotic species have replaced the native Neocaridina population in this region, we estimated the population structure of Neocaridina spp., collected from 19 sites in and around Lake Biwa, based on genome-wide SNPs and mitochondrial DNA. The three detected genetic clusters were characterized by quantitative analysis of multiple morphological traits. Two clusters were identified as non-native N. davidi and the other as native N. denticulata. However, species discrimination based solely on morphological analysis was difficult, highlighting the importance of genetic analysis. We rediscovered the native populations in the region for the first time in a century; however, in 11 sites, the invasive species were dominant. These findings suggest that the native populations are in a critical situation. Furthermore, fishery resource stocking throughout Japan from Lake Biwa can cause the secondary spread of the invasive shrimps from the lake, acting as a hub, to other parts of the country.
The tiger habitats within the Indian part of Terai-Arc landscape (TAL), encompassing both protected (National Parks, Tiger Reserves, Conservation Reserves and Wildlife Sanctuaries) as well as non-protected areas (Forest Divisions and Social Forestry Divisions). The top plot a shows the entire landscape with marked ‘Tiger Habitat Blocks’ (THBs) and identified corridors (Johnsingh et al. 2004). The bottom plot b presents the ‘Tiger Genetic Blocks’ (TGBs) along with the genetic structure results from program TESS (Chen et al. 2007). These TGBs roughly correspond to the western, central and eastern parts of the landscape
Assessment of tiger source-recipient dynamics within each TGB in the Indian TAL. The direction and magnitude of gene flow has been presented by different color allows among the protected and non-protected areas. The top plot (a), middle plot (b) and the bottom plot (c) show the gene flow patterns in TGB I, II and III, respectively
Results of the CIRCUITSCAPE analyses to identify the corridor conductances across TAL. Both ‘least-cost pathways (LCPs)’ as well as the critical corridors (pinchpoints) are shown here in a. The critical corridor areas to maintain contiguous landscape and require urgent management attention are highlighted in b, c and d. Refer Table 4 for details of these critical areas
India led the global tiger conservation initiatives and has doubled its wild tiger population to 2967 (2603–3346) since 2006. As the extant habitats are shrinking continuously, the persistence of these growing populations can only be ensured through focused landscape-scale conservation planning across all the existing tiger landscapes of Indian. We used intensive field-sampling, genetic analyses and GIS modelling to investigate tiger population structure, source-recipient dynamics and functionality of the existing corridors across the Indian part of Terai-Arc landscape (TAL). Using a 13 microsatellite marker panel we identified 219 individual tigers across Indian TAL. Further genetic analyses revealed three weakly, but significantly differentiated tiger subpopulations, termed as ‘Tiger Genetic Blocks (TGBs)’. Genetic migrant detection and gene flow analyses distinguished seven source and 10 recipient areas within this landscape. Circuitscape analyses ascertained total 19 (10 high, three medium and six low conductance) corridors across this landscape, of which 10 require immediate conservation attention. Overall, the tiger populations residing in the western, central and eastern TAL still maintain functional connectivity through these corridors. We suggest urgent management plan involving habitat recovery and protection of ~ 2700 sq. km. identified area to establish landscape connectivity. Further, mitigation measures associated with ongoing linear infrastructure developments and transboundary coordination with Nepal will ensure habitat and genetic connectivity and long-term sustainability of tigers in this globally important landscape.
(a) Map of the study area showing sites (black dots) where cutthroat trout were sampled in Arapaho and Roosevelt National Forests and Rocky Mountain National Park in northern Colorado, USA. Streams are shown as light blue lines, and the Grand Ditch is indicated by the dark blue line. The Continental Divide, a major biogeographic barrier, is indicated by the dashed black line, and the greenback cutthroat trout reclamation area is shaded in green. Direction of water flow is indicated by the light blue arrow. Due to low sample size, multiple stream reaches were pooled for sites on Grand Ditch and Baker Gulch. (b) Photo of the Grand Ditch, an interbasin water transfer, near the Continental Divide. The majority of the Grand Ditch is a low gradient, open water channel bound by an earthen levee. Photo provided by Audrey Harris
Matrix of pairwise FST comparisons of cutthroat trout for all pairs of sites. Values range from 0.009 (purple) to 0.538 (yellow). BG = Baker Gulch; GD = Grand Ditch; LPPA = La Poudre Pass Creek Above Long Draw Reservoir; NE = Neota Creek; LPPB = La Poudre Pass Creek Below Long Draw Reservoir
(a) STRUCTURE analysis (K = 2 and K = 3) of 225 cutthroat trout genotyped at 38 single nucleotide polymorphisms (SNPs) from Baker Gulch (BG), Grand Ditch (GD), La Poudre Pass Creek Above Long Draw Reservoir (LPPA), Neota Creek (NE), and La Poudre Pass Creek Below Long Draw Reservoir (LPPB). Each individual is represented as a vertical bar whose colors correspond to the probability of assignment to different clusters. Sites and individuals within Grand Ditch are ordered from west to east, and location of the Continental Divide is indicated by the dashed black line. (b) Map of the study area showing mean cluster assignment probabilities for each site under the K = 2 scenario. The Continental Divide, a major biogeographic barrier, is indicated by the dashed black line, and the greenback cutthroat trout reclamation area is shaded in green. Direction of water flow is indicated by the light blue arrow
Discriminant analysis of principal components (DAPC) of 225 cutthroat trout genotyped at 38 single nucleotide polymorphisms (SNPs) from Baker Gulch (BG), Grand Ditch (GD), La Poudre Pass Creek Above Long Draw Reservoir (LPPA), Neota Creek (NE), and La Poudre Pass Creek Below Long Draw Reservoir (LPPB). Each individual is represented by a point plotted along discriminant function 1 (x-axis; eigenvalue = 638.1) and discriminant function 2 (y-axis; eigenvalue = 304.8). Colors of individual points and inertia ellipses correspond to sampling sites
DivMigrate analysis of 225 cutthroat trout genotyped at 38 single nucleotide polymorphisms (SNPs) from Baker Gulch (BG), Grand Ditch (GD), La Poudre Pass Creek Above Long Draw Reservoir (LPPA), Neota Creek (NE), and La Poudre Pass Creek Below Long Draw Reservoir (LPPB). Sites are shown as circles, and arrows indicate the direction and magnitude of relative migration rates. (a) Relative migration network estimated using GST and (b) significantly asymmetric relative migration rates estimated using GST and 10,000 bootstrap replicates
Interbasin water transfers are becoming an increasingly common tool to satisfy municipal and agricultural water demand, but their impacts on movement and gene flow of aquatic organisms are poorly understood. The Grand Ditch is an interbasin water transfer that diverts water from tributaries of the upper Colorado River on the west side of the Continental Divide to the upper Cache la Poudre River on the east side of the Continental Divide. We used single nucleotide polymorphisms to characterize population genetic structure in cutthroat trout (Oncorhynchus clarkii) and determine if fish utilize the Grand Ditch as a movement corridor. Samples were collected from two sites on the west side and three sites on the east side of the Continental Divide. We identified two or three genetic clusters, and relative migration rates and spatial distributions of admixed individuals indicated that the Grand Ditch facilitated bidirectional fish movement across the Continental Divide, a major biogeographic barrier. Previous studies have demonstrated ecological impacts of interbasin water transfers, but our study is one of the first to use genetics to understand how interbasin water transfers affect connectivity between previously isolated watersheds. We also discuss implications on native trout management and balancing water demand and biodiversity conservation.
Summed number of times, a particular cluster solution (K) was supported in the eight STRUCTURE analyses of the middle spotted woodpecker and great spotted woodpecker. Noloc and loc refer to analyses without and with a priori location information used by STRUCTURE, respectively. Explanations of the settings of the eight analyses per species are given in Table S4 and detailed results per species for each of the 31 parameters used to infer K are found in Tables S6 and S7. Note that for the great spotted woodpecker, noloc_1 and noloc_2 yielded exactly the same results, as indicated by their identical line and marker
Q-membership plots for STRUCTURE analyses using prior information on sampling locations. Each vertical bar corresponds to one individual. K = number of clusters. Plots were generated with CLUMPAK based on loc_1 analysis output (see Table S4; results for loc_2-loc_4 look virtually the same). Q = membership coefficients. For abbreviations of population names see Table 1
Genetic structure of seven local populations of middle spotted woodpeckers (based on 50 PCs) and great spotted woodpeckers (45 PCs) according to discriminant analysis of principal component (DAPC). For abbreviations of population names see Table 1
Correlations between genetic distance [FST/(1-FST)] and geographic distance (ln kilometers) in middle spotted woodpeckers and great spotted woodpeckers. Upper panels include all population pairs, lower panels only Swiss population pairs, with the excluded German-Swiss pairs shown as red points for information
Species are often arranged along a continuum from “specialists” to “generalists”. Specialists typically use fewer resources, occur in more patchily distributed habitats and have overall smaller population sizes than generalists. Accordingly, the specialist-generalist variation hypothesis (SGVH) proposes that populations of habitat specialists have lower genetic diversity and are genetically more differentiated due to reduced gene flow compared to populations of generalists. Here, expectations of the SGVH were tested by examining genetic diversity, spatial genetic structure and contemporary gene flow in two sympatric woodpecker species differing in habitat specialization. Compared to the generalist great spotted woodpecker (Dendrocopos major), lower genetic diversity was found in the specialist middle spotted woodpecker (Dendrocoptes medius). Evidence for recent bottlenecks was revealed in some populations of the middle spotted woodpecker, but in none of the great spotted woodpecker. Substantial spatial genetic structure and a significant correlation between genetic and geographic distances were found in the middle spotted woodpecker, but only weak spatial genetic structure and no significant correlation between genetic and geographic distances in the great spotted woodpecker. Finally, estimated levels of contemporary gene flow did not differ between the two species. Results are consistent with all but one expectations of the SGVH. This study adds to the relatively few investigations addressing the SGVH in terrestrial vertebrates.
A) Bayesian phylogeny based on Ampithoe species COI haplotypes. Small grey circles indicate nodes with 70% maximum likelihood bootstrap support and black circles indicate 95% posterior probability from Bayesian search. Clade A (orange), B (green) and C (blue) were defined by Pilgrim and Darling 2010. Numbers after each haplotype indicate the number of repeated haplotypes sequenced (B) Admixture proportions based on three putative clusters using SNP genotype likelihoods (k = 3). Each bar represents one individual and is separated into colored segments that represent that individual’s likely proportion of membership to a cluster. Black lines separate individuals of different populations. Populations are labeled below the figure with their regional affiliations above (Arg = Argentina)
Mitochondrial COI vs. nuclear SNPs. Pie charts based on mtDNA represent clade frequencies (Orange = Clade A, Green = Clade B, Blue = Clade C) for each population; pie charts based on nuclear SNP data represent the average admixture proportions based on three putative clusters (k = 3) for each population
(A) Principal component analysis (PC1 against PC2) of SNPs. Individuals are colored by their mitochondrial clade assignment (Orange = Clade A, Green = Clade B, Blue = Clade C). Circles of a single individual represent outliers from the Eastern USA and a square of a single individual represents an outlier from San Francisco Bay (SFB). (B) Genotypes of individuals for a subset of 335 loci that are ‘fixed’ between US Atlantic populations and Pacific (US and Japan) populations. Red blocks represent homozygotes for Pacific alleles, yellow blocks represents homozygotes for Atlantic alleles, and orange blocks represent heterozygotes. The first plot to the right indicates the COI allele assignment for each individual and the second plot indicates the proportion of each individual’s genome that is of Atlantic origin. Note the strongest cytonuclear mismatch are those genotypes in SFB that have predominantly Atlantic nuclear background (red) coupled with Pacific mitochondrial clades (COI-B)
Principal component analysis based on a K = 3 for individuals (A) within the native eastern US, (B) within the East Atlantic, (C) within the native western US and (D) within the native North Pacific. Colors represent the following populations-for (A & B) red-Allan Harbor, black-Sandwich Marina, blue-Charleston Harbor, purple-Chesapeake Bay; orange-Playa Bonita Beach, Argentina; for (C & D) red-Elkhorn Slough, black-Tomales Bay, purple-Willapa Bay; orange-Mangoku-ura, green-Moune Bay, blue-Matsushima Bay
A summary of our current understanding of cryptic native, introduced and hybrid lineages of Ampithoe valida from this study, Pilgrim and Darling (2010) and Faasse (2015)
Biological invasions can pose a severe threat to coastal ecosystems, but are difficult to track due to inaccurate species identifications and cryptic diversity. Here, we clarified the cryptic diversity and introduction history of the marine amphipod Ampithoe valida by sequencing a mtDNA locus from 683 individuals and genotyping 10,295 single-nucleotide polymorphisms (SNPs) for 349 individuals from Japan, North America and Argentina. The species complex consists of three cryptic lineages: two native Pacific and one native Atlantic mitochondrial lineage. It is likely that the complex originated in the North Pacific and dispersed to the north Atlantic via a trans-arctic exchange approximately 3 MYA. Non-native A. valida in Argentina have both Atlantic mitochondrial and nuclear genotypes, strongly indicating an introduction from eastern North America. In two eastern Pacific estuaries, San Francisco Bay and Humboldt Bay, California, genetic data indicate human-mediated hybridization of Atlantic and Pacific sources, and possible adaptive introgression of mitochondrial loci, nuclear loci, or both. The San Francisco Bay hybrid population periodically undergoes population outbreaks and profoundly damages eelgrass Zostera marina thalli via direct consumption, and these ecological impacts have not been documented elsewhere. We speculate that novel combinations of Atlantic and Pacific lineages could play a role in A. valida’s unique ecology in San Francisco Bay. Our results reinforce the notion that we can over-estimate the number of non-native invasions when there is cryptic native structure. Moreover, inference of demographic and evolutionary history from mitochondrial loci may be misleading without simultaneous survey of the nuclear genome.
The adoption of measures to protect the viability of threatened populations should be supported by empirical data identifying appropriate conservation units and management strategies. The global population of the majorera limpet, P. candei candei d’Orbigny, 1840, is restricted to the Macaronesian islands in the NE Atlantic, including near-to-extinct and healthy populations in Fuerteventura and Selvagens, respectively. The taxonomic position, genetic diversity and intra- and interspecifc relationships of these populations are unclear, which is hindering the implementation of a recovery plan for the overexploited majorera limpet on Fuerteventura. In this study, ddRAD-based genome scanning was used to overcome the limitations of mitochondrial DNA-based analysis. As a result, P. candei candei was genetically diferentiated from the closely related P. candei crenata for the first time. Moreover, genetic diferentiation was detected between P. candei candei samples from Selvagens and Fuerteventura, indicating that translocations from the healthy Selvagens source population are inadvisable. In conclusion, the majorera limpet requires population-specifc management focused on the preservation of exceptional genetic diversity with which to face future environmental challenges.
Sampling locations of all samples in this study. The left map shows sampling locations of all the samples by watershed, including wild, hatchery rainbow trout, “Other Rainbow Trout”, and Kern River golden trout and rainbow trout. Inset area is the Upper McCloud River (UMCR) watershed. The red box shows sampling locations within the Upper McCloud River watershed, the area above the Middle Falls. Highlighted river in purple is the mainstem McCloud River. Golden Trout Creek (GTCR), South Fork Kern River (SFKR), Kern River (KRNR), Eagle Lake (EGLK), North Fork American River (NFAR), Lower Stanislaus River (LSTN), Lower Yuba River (LYBA), Coleman Hatchery (COLE), Eagle Lake Hatchery (EGLH), Hot Creek Strain (HTCS), Mt. Shasta Hatchery (MTSH), Pit Strain Hatchery (PITS), Warner Valley (WARV), Goose Lake (GOSL), Surprise Valley (SPRV), North Fork Pit River (NFPT), South Fork Pit River (SFPT), Upper Pit River (UPIT), Lower Pit River (LPIT), Yuba North Fork (YUBA), Upper McCloud River (UMCR): Swamp Creek (SWPC), Edson Creek (EDSN), Sheepheaven Creek (SHPN), Dry Creek (DRYC), Moosehead Creek (MOHD), Bull Creek (BLLC), Cow Creek (COWC), Trout Creek (TRTC), Shady Gulch Creek (SHGU), Raccoon Creek (RCCN), McCloud River (MCLD), McKay Creek (MCKY), Blue Heron Creek (BLHN), Tate Creek (TATE)
Population Structure of all samples. Top plot: all samples PCA, color represents watershed. Three main groups are distinguishable: Golden Trout Complex (GTCX), Rainbow Trout Group (RBTG), and McCloud River Redband Trout (MRRT). PC1(8.7% variance explained) / PC2(7.65% variance explained). Bottom plot: all samples admixture plots at K = 2 (top admixture plot) and K = 3 (bottom admixture plot). Blue represents MRRT ancestry group which is different from GTCX (green) and RBTG (red). Golden Trout Creek (GTCR), South Fork Kern River (SFKR), Kern River (KRNR), Eagle Lake (EGLK), North Fork American River (NFAR), Lower Stanislaus River (LSTN), Lower Yuba River (LYBA), Coleman Hatchery (COLE), Eagle Lake Hatchery (EGLH), Hot Creek Strain (HTCS), Mt. Shasta Hatchery (MTSH), Pit Strain Hatchery (PITS), Warner Valley (WARV), Goose Lake (GOSL), Surprise Valley (SPRV), North Fork Pit River (NFPT), South Fork Pit River (SFPT), Upper Pit River (UPIT), Lower Pit River (LPIT), Yuba North Fork (YUBA), Upper McCloud River (UMCR)
PCA and admixture analyses of MRRT with a group of a potential source of introgression – a small subset of RBTG. Top plot: PCA of MRRT with the RBTG small subset, color represents populations. The RBTG small subset includes: wild rainbow trout from Eagle lake (EGLK) and North Fork American River (NFAR), Steelhead from Lower Yuba River (LYBA) and Stanislaus River (LSTN), hatchery strains from Mt. Shasta (MTSH), Coleman (COLE), Eagle Lake (EGLH), Hot Creek (HTCS), and three from the “Other Rainbow Trout” group: Lincoln (LCLN) and Lost (LOST) from Lower Pit River watershed and Nelson (NLSN) creeks from Yuba watershed, PC1(11.6% variance explained) / PC2(3.44%, variance explained). Bottom plot: admixture plot of MRRT and the RBTG subset cluster. Five pure populations are identified within the MRRT population: Swamp creek (SWPC), Edson Creek (EDSN), Sheepheaven Creek (SHPN), Dry Creek (DRYC), and Moosehead Creek (MOHD)
Population structure within MRRT group. MRRT group admixture plot at K = 3 and K = 4. Four major genetic groups are distinguishable: red represents Swamp and Sheepheaven creeks genetic group, blue represents Edson and Dry creeks genetic group, purple represent Bull creek genetic group, and green represents rainbow trout genetic group. The population’s order is based on increasing in the percentage of rainbow trout ancestry.
Swamp Creek (SWPC), Edson Creek (EDSN), Sheepheaven Creek (SHPN), Dry Creek (DRYC), Moosehead Creek (MOHD), Bull Creek (BLLC), Cow Creek (COWC), Trout Creek (TRTC), Shady Gulch Creek (SHGU), Raccoon Creek (RCCN), McCloud River (MCLD), McKay Creek (MCKY), Blue Heron Creek (BLHN), Tate Creek (TATE)
The McCloud River Redband Trout (MRRT; Oncorhynchus mykiss stonei ) is a unique subspecies of rainbow trout that inhabits the isolated Upper McCloud River of Northern California. A major threat to MRRT is introgressive hybridization with non-native rainbow trout from historical stocking and contemporary unauthorized introductions. To help address this concern, we collected RAD-sequencing data on 308 total individuals from MRRT and other California O. mykiss populations and examined population structure using Principal Component and admixture analyses. Our results are consistent with previous studies; we found that populations of MRRT in Sheepheaven, Swamp, Edson, and Moosehead creeks are nonintrogressed. Additionally, we saw no evidence of introgression in Dry Creek, and suggest further investigation to determine if it can be considered a core MRRT conservation population. Sheepheaven Creek was previously thought to be the sole historical lineage of MRRT, but our analysis identified three: Sheepheaven, Edson, and Dry creeks, all of which should be preserved. Finally, we discovered diagnostic and polymorphic SNP markers for monitoring introgression and genetic diversity in MRRT. Collectively, our results provide a valuable resource for the conservation and management of MRRT.
R. raviventris trapping locations within the USFWS Suisun Bay Area Recovery Unit (white outline). Red circles indicate trapping locations where ≥ 10 mice were caught; yellow circles indicate where ≤ 3 R. raviventris were caught. We did not successfully trap any R. raviventris at Bay Point (indicated by a white circle). The five marsh management units (marsh complexes) are named
Population tree of R. raviventris sampling locations across the marshes of Suisun Bay, CA. Based on locations with > 10 individuals. Distance is Nei’s DA. The value at the node of Point Edith and McNabney is bootstrap support based on 200 replicates. Support at all other nodes was < 50%, consistent with a little population substructure across the Northern Marshes
Population structure of R. raviventris across the Suisun Bay, California, based on K = 4 as estimated in the program Structure. Representing all 538 salt marsh harvest mice genotyped at 16 microsatellite loci. Sampling sites are arranged in order from west to east, first considering the Northern Marshes, then Ryer Island, and finally the southern marshes along the Contra Costa Shoreline. Marsh complexes are indicated at the bottom
Cumulative current map modelling connectivity among 20 R. raviventris trapping locations within USFWS Suisun Bay Area Recovery Unit, CA. The current map was generated in the program Circuitscape based on the best supported model that contained Elevation (resistance weight = 500) and Water (200). Darker blue indicates areas with higher resistance that may restrict gene flow. Areas shaded in light blue to green have higher current density and may facilitate gene flow (higher connectivity). Narrower areas of conductance tend to have higher density current, while broader areas tend to have more diffuse current
Preserving the genetic diversity of endangered species is fundamental to their conservation and requires an understanding of genetic structure. In turn, identification of landscape features that impede gene flow can facilitate management to mitigate such obstacles and help with identifying isolated populations. We conducted a landscape genetic study of the endangered salt marsh harvest mouse ( Reithrodontomys raviventris ), a species endemic to the coastal marshes of the San Francisco Estuary of California. We collected and genotyped > 500 samples from across the marshes of Suisun Bay which contain the largest remaining tracts of habitat for the species. Cluster analyses and a population tree identified three geographically discrete populations. Next, we conducted landscape genetic analyses at two scales (the entire study area and across the Northern Marshes) where we tested 65 univariate models of landscape features and used the best supported to test multivariable analyses. Our analysis of the entire study area indicated that open water and elevation (> 2 m) constrained gene flow. Analysis of the Northern Marshes, where low elevation marsh habitat is more continuous, indicated that geographic distance was the only significant predictor of genetic distance at this scale. The identification of a large, connected population across Northern Marshes achieves a number of recovery targets for this stronghold of the species. The identification of landscape features that act as barriers to dispersal enables the identification of isolated and vulnerable populations more broadly across the species range, thus aiding conservation prioritization.
Few studies have evaluated the genetic status of medicinal plants exposed to commercial harvesting. Here, we examine the genetic variability of Pilocarpus microphyllus, an endemic and threatened medicinal plant species from the eastern Amazon, across its largest remaining wild population. Popularly known as jaborandi, species of Pilocarpus genus are the unique known natural source of pilocarpine, an alkaloid used to treat glaucoma and xerostomia. However, Populations of P. microphyllus has experienced a severe decline in the last decades. Using RAD sequencing, we identified a total of 5,266 neutral and independent SNPs in 277 individuals collected from the Carajás National Forest (CNF). We quantified genetic diversity and gene flow patterns and estimated the minimum number of individuals necessary to establish a germplasm bank. Our results revealed high genetic diversity and four spatially distinct clusters of P. microphyllus with substantial admixture among them. Geographic distance and temperature dissimilarity were the factors that best explained the relatedness patterns among individuals. Additionally, our findings indicate that at least 40 matrices sampled randomly from each population would be required to conserve genetic diversity in the long term. In short, P. microphyllus showed high levels of genetic diversity and an effective population size (NE) sufficient to reduce the likelihood of extinction due to inbreeding depression. Our results indicate that diversity has been maintained despite the continuous harvesting of raw leaf material in the area over recent decades. Finally, the results provide information essential for the design of a germplasm bank to protect the endangered medicinal plant species.
A Mitochondrial haplotype network of the cytochrome B gene for all 61 Gyrinophilus salamanders found within General Davis Cave (both G. porphyriticus and G. subterraneus) and two nearby populations of G. porphyriticus (Harts Run and Buckeye Creek Cave, ~ 17 and 28 km away). Colors are based on the genetic identification from the nuclear data. B Map of sampling localities included in the study. Range of G. porphyriticus indicated in blue, and range of G. subterraneus indicated in purple based on shapefiles downloaded from Sciencebase
Results of the nuclear genetic analyses of Gyrinophilus subterraneus and G. porphyriticus samples. A Principal component analysis of all 81 samples. Axis length is proportional to variation explained. B Admixture analyses at K = 2 for the 52 individuals found inside General Davis Cave. Every bar represents the assignment probability for a given individual to each species and samples are ordered by population. C Hybrid index analyses of all 52 samples collected within General Davis Cave. The x-axis is a representation of the proportion of the genome corresponding to each of the parental species. The y-axis represents the hybrid index (derived from estimates of heterozygosity) and ranges from 0 (no signal or very ancient hybridization) to 1 (a first generation hybrid between parental species). D Genetic identification of all samples collected within General Davis Cave with known distance along the cave stream transect (N = 35). The three F1 hybrids with known sampling locations are centered at the start of the cave stream transect. Samples without exact collecting location information were not included
Results of the demographic modelling based on the Site Frequency Spectrum of nuclear SNPs of the 20,20 downprojection. A Scheme depicting the best ranked demographic model (asym_mig) with the estimated effective population sizes (Nµ) and gene flow parameters indicated. B Graphic representation of the best ranked model and the empirical data of the site frequency spectrum with residuals between model and data. C Distribution of Pearsons chi-squared scores of the empirical data in the blue line, compared to 300 SFS simulations
A 3D surface models of the skulls of adult, metamorphosed Gyrinophilus subterraneus (USNM 525271), F1 hybrid (USNM 525272), and G. porphyriticus (USNM 525273) presented in rostral (top panel), dorsal (middle panel), and ventral (bottom panel) views. The premaxilla (pm) is indicated to highlight the variable condition between the species. The F1 hybrid illustrates the condition typical of G. porphyriticus.B Size-corrected eye diameter for larval specimens split by species. C Size-corrected eye diameter for transformed specimens split by species. The F1 hybrid (USNM 525272) was field identified as G. porphyriticus, but presented an intermediate eye diameter (see arrow). D Snout-vent length for specimens, split by both species and life-stage demonstrating that compared to G. porphyriticus, larval G. subterraneus grow to larger sizes before transforming
Population trend in AGyrinophilus subterraneus and CG. porphyriticus population density over time in General Davis Cave. The blue line is the fitted mean, the observed data are the open circles, and the 95% CI is in grey. Panels (B) and (D) are histograms of the mean stochastic population growth rate (r) for G. subterraneus (panel B) and G. porphyriticus (panel D) in General Davis Cave for surveys conducted between 1973 and 2018. Red line is the stochastic growth rate of a stable population
Due to their limited geographic distributions and specialized ecologies, cave species are often highly endemic and can be especially vulnerable to habitat degradation within and surrounding the cave systems they inhabit. We investigated the evolutionary history of the West Virginia Spring Salamander (Gyrinophilus subterraneus), estimated the population trend from historic and current survey data, and assessed the current potential for water quality threats to the cave habitat. Our genomic data (mtDNA sequence and ddRADseq-derived SNPs) reveal two, distinct evolutionary lineages within General Davis Cave corresponding to G. subterraneus and its widely distributed sister species, Gyrinophilus porphyriticus, that are also differentiable based on morphological traits. Genomic models of evolutionary history strongly support asymmetric and continuous gene flow between the two lineages, and hybrid classification analyses identify only parental and first generation cross (F1) progeny. Collectively, these results point to a rare case of sympatric speciation occurring within the cave, leading to strong support for continuing to recognize G. subterraneus as a distinct and unique species. Due to its specialized habitat requirements, the complete distribution of G. subterraneus is unresolved, but using survey data in its type locality (and currently the only known occupied site), we find that the population within General Davis Cave has possibly declined over the last 45 years. Finally, our measures of cave and surface stream water quality did not reveal evidence of water quality impairment and provide important baselines for future monitoring. In addition, our unexpected finding of a hybrid zone and partial reproductive isolation between G. subterraneus and G. porphyriticus warrants further attention to better understand the evolutionary and conservation implications of occasional hybridization between the species.
Canadian Grass Pickerel (Esox americanus vermiculatus) in A lateral view from Walpole Island, ON, on Lake St. Clair, and B dorsal view from Twenty-Mile Creek near Hamilton, ON. Photo A by FAM, photo B by George Coker
Genomic population structure of Grass Pickerel (Esox americanus vermiculatus) in Canada described by principal coordinates (A, B) and STRUCTURE analyses (C, D) of 66 individuals and 740–950 SNPs per individual. Given the considerable genomic distinctiveness of samples from the Georgian Bay-Severn River (A), and the lack of apparent admixture from this cluster to others (STRUCTURE results not shown), these samples were excluded from subsequent PCoA (B) and STRUCTURE analyses (D). In map C, mean pairwise Hudson Fst values are given on arrows between clusters. See Table 2 for Fst estimate confidence intervals. STRUCTURE results presented as barplot (D) in which each column is an individual and each color is a distinct genomic cluster, with percentage of an individual’s genome assignable to a cluster shown by color proportion within columns. Analysis of Evanno’s delta K yielded K = 3 and K = 6 as optimal subdivisions of genomic diversity (see Fig. S1). Percent variation explained by each PCoA axis (A, B) given on axis. Icon colors in PCoA scatterplot (A, B) derived from STRUCTURE analysis. Map C derived from > 14,200 fish sampling events dating from 2002 to 2017 in the Fisheries and Oceans Canada (DFO) Biodiversity Science database
Genomic population structure of Grass Pickerel (Esox americanus vermiculatus) in the Niagara Peninsula described by principal coordinates (A) and STRUCTURE (B, C) analyses of 26 individuals and over 1,690 SNPs per individual. Mean pairwise Hudson Fst values given on arrows between PCoA clusters (A). See Table 3 for Fst estimate confidence intervals. Percent variation explained by each PCoA axis (A) given on axis. Icon colors in A derived entirely from STRUCTURE results. STRUCTURE results presented as barplot (C) in which each column is an individual and each color is a distinct genomic cluster, with percentage of an individual’s genome assignable to a cluster shown by color proportion within columns. Analysis of Evanno’s delta K yielded K = 4 as an optimal number of subdivisions of genomic diversity. Sample sites Hahn Marsh and Long Point Bay (denoted with asterisk) are outside map boundaries (B) but are shown in Fig. 2C. Map B derived from fish sampling events dating from 2002 to 2017 in the Fisheries and Oceans Canada (DFO) Biodiversity Science database. Small, open circles represent sampling events that did not yield Grass Pickerel
Locality of origin for 66 tissues of Grass Pickerel (Esox americanus vermiculatus) examined in this study
Eighty nine (42%) of Canada’s 215 freshwater fish species have been assessed as at risk by the Committee on the Status of Endangered Wildlife in Canada. This study examines genomic population structure of the at-risk Grass Pickerel (Esox americanus vermiculatus), a small (≤ 33 cm) predatory fish that in Canada has a range spanning approximately 114,000 km² of southern Ontario. Within this range it occupies approximately ten sites that are mostly shallow, weedy, and slow-flowing. Its populations and habitat are declining. This study defines population clusters and quantifies genomic diversity within and between populations based on > 5500 loci and > 950 SNPs from genomes of 66 individuals representing the subspecies’ entire Canadian range. Ordination and STRUCTURE analyses revealed four major geographic/genomic clusters centered in the Georgian Bay-Severn River, southeastern shore of Lake Huron, Niagara Peninsula, and upper St. Lawrence River. Major clusters were distinguished by relatively high Hudson Fst values (0.205–0.480), with Georgian Bay-Severn River being consistently most distinct. The Niagara Peninsula major cluster contained an additional three discernable sub-clusters differentiated by Fst values as great or greater than major clusters, despite spanning only ca. 200 km². Genomically distinct Niagara sub-clusters occurred in Abino Drain, Big Forks Creek, and Tea Creek. Samples from sites between both major and minor clusters exhibited admixture from adjacent clusters. Despite current management of Grass Pickerel under a single designatable unit throughout its Canadian range, we map considerable geographic population structure that should help guide the designation of additional conservation units.
Distribution of P. cinereus (black) and distribution of P. nettingi (green). The green area represents the five-county region in West Virginia in which P. nettingi is found. All sampling was performed within the known distribution of P. nettingi. Specific sampling sites are shown in panel at right. (Color figure online)
MtDNA haplotype networks and geographic distribution of (a) P. nettingi and (b) P. cinereus haplotypes. Each circle represents a unique haplotype, and the size of each circle is proportional to the number of individuals
Geographic distribution of mtDNA haplotypes for (a) P. nettingi (n = 6) and (b) P. cinereus (n = 14) across the sampled area in west Virginia; each color represents a unique haplotype. Geographic distribution of STRUCTURE population clusters for (c) P. nettingi (n = 5) and (d) P. cinereus (n = 4). Each color represents a unique genetic cluster; substructure is represented by dashed or dotted patterns. * indicates the locality (Snowshoe) where individuals were not unambiguously assigned to a cluster. (Color figure online)
Relationship between geographic (km) and genetic (FST) distance between localities for P. nettingi (solid circles) and P. cinereus (open circles). The correlation between distance matrices was only significant for P. nettingi (dashed line: r² = 0.25, P < 0.05)
Proportion of kinship observed for each species that is more than expected levels due to chance in each of four kinship categories
Comparative population genetic studies of closely related taxa provide a powerful framework for evaluating if and to what degree a species of conservation concern has been negatively impacted by factors such as habitat fragmentation, decreased population connectivity, inbreeding and genetic drift. In this study, we take advantage of a paired sampling strategy to compare the population genetics of the geographically restricted, federally threatened Cheat Mountain salamander (Plethodon nettingi) to those of its partially sympatric, but much more widely distributed congener, the red-backed salamander (P. cinereus), where the two species overlap in the Appalachian mountains of West Virginia. Mitochondrial DNA haplotype and nucleotide diversity were lower in P. nettingi, as were a variety of metrics of nuclear genetic diversity estimated from microsatellite data. Population differentiation and structuring were greater in P. nettingi, suggesting reduced gene flow following fragmentation. Significant inbreeding and evidence of recent population bottlenecks were also seen in P. nettingi and estimated population sizes were smaller. Estimates of contemporary gene flow, as measured through kinship, also showed more restricted gene flow in P. nettingi. Overall, our comparative study provides strong evidence that the small and highly fragmented nature of its geographic distribution has resulted in a suite of negative genetic consequences for the federally threatened Cheat Mountain salamander. Management efforts aimed at enhancing the genetic health and long-term viability of this species should focus on increasing population connectivity through establishment of forest habitat corridors where possible and exploring the potential merits of translocations.
Briefly considered extinct in the wild, the future of the Wyoming toad (Anaxyrus baxteri) continues to rely on captive breeding to supplement the wild population. Given its small natural geographic range and history of rapid population decline at least partly due to fungal disease, investigation of the diversity of key receptor families involved in the host immune response represents an important conservation need. Population decline may have reduced immunogenetic diversity sufficiently to increase the vulnerability of the species to infectious diseases. Here we use comparative transcriptomics to examine the diversity of toll-like receptors and major histocompatibility complex (MHC) sequences across three individual Wyoming toads. We find reduced diversity at MHC genes compared to bufonid species with a similar history of bottleneck events. Our data provide a foundation for future studies that seek to evaluate the genetic diversity of Wyoming toads, identify biomarkers for infectious disease outcomes, and guide breeding strategies to increase genomic variability and wild release successes.
Cerrado is one of the largest biomes in Brazil and has undergone constant changes in its landscape over the years. Modifications driven by the expansion of agriculture and uncontrolled deforestation endanger the permanence of several species in the territory. Ecological imbalances demand the application of methodologies that seek the conservation of species, however, conflicts between theoretical expectations and empirical observations have suggested the need for studies that carefully assess the impacts of fragmentation on different species. In this study, we investigated the impact of changes in the seed and pollen dispersal of an isolated population of Caryocar brasiliense Camb. through in silico analyzes, in order to evaluate their impact on genetic variability of populations. We simulated different scenarios with changes in seed dispersal and pollination patterns in an isolated population. Populations with well preserved pollinators had lower rates of inbreeding and maintained greater genetic diversity over time, even in the absence of the seed disperser. In contrast, in scenarios with reduction of pollen dispersal distance or pollination efficiency, populations had an increase in inbreeding and in extinction probability, and reduction in genetic diversity over time. Small areas of conservation of C. brasiliense can harbor viable populations only if pollination service is maintained, so conservation strategies must consider both tree and pollinator protection.
Top-cited authors
Martin F Breed
  • Flinders University
Andrew John Lowe
  • University of Adelaide
Kym Ottewell
  • Department of Parks and Wildlife
Michael G Gardner
  • Flinders University
Michael Graham Stead