Land-bridge Islands Research Articles

Ecological correlates of mammal β‐diversity in Amazonian land‐bridge islands: from small‐ to large‐bodied species

AbstractAimMega hydroelectric dams have become one of the main drivers of biodiversity loss in the lowland tropics. In these reservoirs, vertebrate studies have focused on local (α) diversity measures, whereas between‐site (β) diversity remains poorly assessed despite its pivotal importance in understanding how species diversity is structured and maintained. Here, we unravel the patterns and ecological correlates of mammal β‐diversity, including both small (SM) and midsized to large mammal species (LM) across 23 islands and two continuous forest sites within a mega hydroelectric reservoir.LocationBalbina Hydroelectric Dam, Central Brazilian Amazonia.MethodsSmall mammals were sampled using live and pitfall traps (48,350 trap‐nights), and larger mammals using camera traps (8,160 trap‐nights). β‐diversity was examined for each group using multiplicative diversity decomposition of Hill numbers, which considers the importance of rare, common and dominant species, and tested to what extent those were related to a set of environmental characteristics measured at different spatial scales.Resultsβ‐diversity for both mammal groups was higher when considering species presence–absence. When considering species abundance, β‐diversity was significantly higher for SM than for LM assemblages. Habitat variables, such as differences in tree species richness and percentage of old‐growth trees, were strong correlates of β‐diversity for both SMs and LMs. Conversely, β‐diversity was weakly related to patch and landscape characteristics, except for LMs, for which β‐diversity was correlated with differences in island sizes.Main conclusionsThe lower β‐diversity of LMs between smaller islands suggests subtractive homogenization of this group. Although island size plays a major role in structuring mammal α‐diversity in several land‐bridge islands, local vegetation characteristics were additional key factors determining β‐diversity for both mammal groups. Maintaining the integrity of vegetation characteristics and preventing the formation of a large set of small islands within reservoirs should be considered in long‐term management plans in both existing and planned hydropower development in lowland tropical forests.

Understanding the long term consequences of fragmentation: lessons from the bats of Bijagós (Guinea-Bissau, West Africa)

The Bijagos archipelago consists of about 80 land-bridge islands located just off the coast of Guinea-Bissau (West Africa). The current faunas of such land-bridge archipelagoes reflect the isolation resulting from the fragmentation of the original coastal plains, caused by sea level rise about 11000 years ago. Therefore, they represent a unique opportunity to study long-term consequences of natural habitat fragmentation, analogous to the ongoing fragmentation processes caused by Man. We sampled and analyzed the bat assemblages of eight Bijagos islands, with up to 261 km 2 , and two nearby coastal regions to understand how insularity shaped island assemblages, and evaluate the potential impacts of fragmentation on African bat assemblages. Rarefaction using Chao2 estimator indicated that total species richness on Bijagos is only about 1/5 of that on the coast. Cluster and correspondence analyses demonstrated that bat faunas of different islands are quite similar, and not a random set of the mainland species assemblage. Moreover, trait analysis indicated that islands assemblages are composed by generalist and abundant species; presumably only generalist species can attain island population sizes viable in the long-term. The observed species depletion resulting from long-term isolation in Bijagos is much more accentuated than that observed in recent fragments, corroborating the view that such fragments are still losing species. Our results indicate that, despite the high mobility of bats, even relatively large fragments are unable to sustain viable populations of most species and that the impacts are worse in the case or rarer species. Most ongoing fragmentation resulting from habitat destruction involves matrices with a lower contrast than water, and this may lessen impacts. However, at least in the case of high contrast matrices, currently fragmented landscapes are likely to continue loosing species, and thus the true, long-term, impacts of fragmentation shall likely be worse than those so far reported.

Pathogen richness and abundance predict patterns of adaptive major histocompatibility complex variation in insular amphibians

The identification of the factors responsible for genetic variation and differentiation at adaptive loci can provide important insights into the evolutionary process and is crucial for the effective management of threatened species. We studied the impact of environmental viral richness and abundance on functional diversity and differentiation of the MHC class Ia locus in populations of the black-spotted pond frog (Pelophylax nigromaculatus), an IUCN-listed species, on 24 land-bridge islands of the Zhoushan Archipelago and three nearby mainland sites. We found a high proportion of private MHC alleles in mainland and insular populations, corresponding to 32 distinct functional supertypes, and strong positive selection on MHC antigen-binding sites in all populations. Viral pathogen diversity and abundance were reduced at island sites relative to the mainland, and islands housed distinctive viral communities. Standardized MHC diversity at island sites exceeded that found at neutral microsatellites, and the representation of key functional supertypes was positively correlated with the abundance of specific viruses in the environment (Frog virus 3 and Ambystoma tigrinum virus). Theseresults indicate that pathogen-driven diversifying selection can play an important role in maintaining functionally important MHC variation following island isolation, highlighting the importance of considering functionally important genetic variation and host-pathogen associations in conservation planning and management.

Non-random lizard extinctions in land-bridge Amazonian forest islands after 28 years of isolation

Major hydropower infrastructure has become a leading driver of biodiversity loss in the lowland tropics. Terrestrial species typically become stranded in post-isolation land-bridge islands within hydroelectric reservoirs. Understanding the resulting extinction dynamics of insular communities is critical to inform, if not to avert, the ongoing blitzkrieg of dam development. Here we assess the effects of forest patch and landscape metrics on diurnal lizard species richness and composition within the Balbina Hydroelectric Dam and surrounding areas in the Central Brazilian Amazon. This 28-yr-old dam created a reservoir of ~4438km2, comprising 3546 islands. We sampled 25 of these islands (0.83–1466ha) and five mainland continuous forest sites, one of which placed along stream banks. We further related morpho-ecological traits and the geographic distribution of lizard species to the spatial metrics of islands where they occurred. Using 100L-pitfall traps operated over 5447 trap-days, we recorded 1123 lizards from 17 taxa, two of which exclusively found along stream banks within continuous forest. Island area was the best predictor of species richness and composition. Small islands (≤2ha) harboured fewer than a third of all species typically observed in larger islands and continuous forest (≥8 species), and only islands ≥100ha retained nearly complete lizard faunas. Lizard assemblages inhabiting small, isolated islands consisted almost exclusively of an oversimplified set of widely distributed, large-bodied, habitat generalist, heliophile species associated with open areas and forest edges, and that feed on a wide spectrum of prey sizes. These wholesale changes in lizard community structure were characterized by severe losses in functional traits, and may profoundly affect ecosystem functioning.

Ethological and phenotypic divergence in insular fire salamanders: diurnal activity mediated by predation?

Phenotypic divergence can occur through both local adaptations and phenotypic plasticity in response to particular environmental pressures. Holocene land-bridge islands harbor recent isolated populations that offer an excellent natural framework to study the microevolutionary processes involved in rapid or ongoing phenotypic shifts. Two insular populations (Ons and San Martino) of Salamandra salamandra in NW Iberian Peninsula exemplify these phenotypic shifts with a rapid evolutionary transition in reproductive mode and genetic differentiation since their isolation ca. 8000 years ago. We evaluate parallel phenotypic changes in body size and behavior. In particular, we (1) collected observational fieldwork data during diurnal and nocturnal transects in both islands for over a decade (2004–2016); (2) investigated the climatic conditions associated to fire salamanders’ activity; (3) compared physical condition between insular and mainland populations; and (4) used plasticine models and camera trapping to identify potential predators that might cause the observed diurnal activity in San Martino Island. Our 13-year-long study shows that salamanders from San Martino show mainly diurnal activity and are significantly smaller than salamanders from Ons and nearby continental populations. Overall, we present an exceptional case of behavior and phenotypic differentiation of an insular population of fire salamanders and suggest that this unusual behavior in the small population of San Martino might be triggered by a response to predator pressure exerted by a dense population of Rattus rattus.

Ecological Correlates of Vulnerability to Fragmentation in Snakes on Inundated Subtropical Land-Bridge Islands

Ecological Correlates of Vulnerability to Fragmentation in Snakes on Inundated Subtropical Land-Bridge Islands

The small-island effect in amphibian assemblages on subtropical land-bridge islands of an inundated lake.

The small-island effect (SIE) has become more and more part of the theoretical framework of island biogeography and biodiversity research. However, previous methods for the detection of SIEs are often flawed in one way or another, including not accounting for model complexity, not comparing all relevant models, and not including islands with no species. Therefore, the existence and the prevalence of the SIE may be dubious. In this study, after controlling for all these methodological shortcomings, we tested for the existence of the SIE in amphibian assemblages on subtropical land-bridge islands created by the inundation of the Thousand Island Lake, China. We used the line transect method to determine the distribution of amphibian assemblages on 23 study islands during 3 breeding seasons from 2009 to 2011. To evaluate whether an SIE exists in amphibian assemblages, we compared the fit of a simple linearized power model with two most widely used breakpoint regression models. The information-theoretic multimodel inference approach based on Akaike’s information criterion identified the left-horizontal SIE model as the best single model. Thus, we found strong evidence for the existence of an SIE in our system. The upper limit of the SIE for amphibian assemblages was 39.95 ha. Below this threshold area, amphibian richness varied independently of island size. The SIE in amphibian assemblages may be due to episodic disturbances, stochastic events, and nutrient subsidies from the lake. Our results indicate that all the islands >39.95 ha should be protected for the effective conservation of amphibian assemblages in our system.

Dynamics of avian haemosporidian assemblages through millennial time scales inferred from insular biotas of the West Indies

Although introduced hemosporidian (malaria) parasites (Apicomplexa: Haemosporida) have hastened the extinction of endemic bird species in the Hawaiian Islands and perhaps elsewhere, little is known about the temporal dynamics of endemic malaria parasite populations. Haemosporidian parasites do not leave informative fossils, and records of population change are lacking beyond a few decades. Here, we take advantage of the isolation of West Indian land-bridge islands by rising postglacial sea levels to estimate rates of change in hemosporidian parasite assemblages over a millennial time frame. Several pairs of West Indian islands have been connected and separated by falling and rising sea levels associated with the advance and retreat of Pleistocene continental glaciers. We use island isolation following postglacial sea-level rise, ca. 2.5 ka, to characterize long-term change in insular assemblages of hemosporidian parasites. We find that assemblages on formerly connected islands are as differentiated as assemblages on islands that have never been connected, and both are more differentiated than local assemblages sampled up to two decades apart. Differentiation of parasite assemblages between formerly connected islands reflects variation in the prevalence of shared hemosporidian lineages, whereas differentiation between islands isolated by millions of years reflects replacement of hemosporidian lineages infecting similar assemblages of avian host species.

The effects of historical fragmentation on major histocompatibility complex class II β and microsatellite variation in the Aegean island reptile, Podarcis erhardii.

The major histocompatibility complex (MHC) plays a key role in disease resistance and is the most polymorphic gene region in vertebrates. Although habitat fragmentation is predicted to lead to a loss in MHC variation through drift, the impact of other evolutionary forces may counter this effect. Here we assess the impact of selection, drift, migration, and recombination on MHC class II and microsatellite variability in 14 island populations of the Aegean wall lizard Podarcis erhardii. Lizards were sampled from islands within the Cyclades (Greece) formed by rising sea levels as the last glacial maximum approximately 20,000 before present. Bathymetric data were used to determine the area and age of each island, allowing us to infer the corresponding magnitude and timing of genetic bottlenecks associated with island formation. Both MHC and microsatellite variation were positively associated with island area, supporting the hypothesis that drift governs neutral and adaptive variation in this system. However, MHC but not microsatellite variability declined significantly with island age. This discrepancy is likely due to the fact that microsatellites attain mutation‐drift equilibrium more rapidly than MHC. Although we detected signals of balancing selection, recombination and migration, the effects of these evolutionary processes appeared negligible relative to drift. This study demonstrates how land bridge islands can provide novel insights into the impact of historical fragmentation on genetic diversity as well as help disentangle the effects of different evolutionary forces on neutral and adaptive diversity.

Functional and phylogenetic structure of island bird communities.

Biodiversity change in anthropogenically transformed habitats is often nonrandom, yet the nature and importance of the different mechanisms shaping community structure are unclear. Here, we extend the classic Theory of Island Biogeography (TIB) to account for nonrandom processes by incorporating species traits and phylogenetic relationships into a study of faunal relaxation following habitat loss and fragmentation. Two possible mechanisms can create nonrandom community patterns on fragment islands. First, small and isolated islands might consist of similar or closely related species because they are environmentally homogeneous or select for certain shared traits, such as dispersal ability. Alternatively, communities on small islands might contain more dissimilar or distantly related species than on large islands because limited space and resource availability result in greater competitive exclusion among species with high niche overlap. Breeding birds were surveyed on 36 islands and two mainland sites annually from 2010 to 2014 in the Thousand Island Lake region, China. We assessed community structure of breeding birds on these subtropical land-bridge islands by integrating species' trait and evolutionary distances. We additionally analysed habitat heterogeneity and variance in size ratios to distinguish biotic and abiotic processes of community assembly. Results showed that functional-phylogenetic diversity increased with island area, and decreased with isolation. Bird communities on the mainland were more diverse and generally less clustered than island bird communities and not different than randomly assembled communities. Bird communities on islands tend to be functionally similar and phylogenetically clustered, especially on small and isolated islands. The nonrandom decline in species diversity and change in bird community structure with island area and isolation, along with the relatively homogeneous habitats on small islands, support the environmental filtering hypothesis. Our study demonstrates the importance of integrating multiple forms of diversity for understanding the effects of habitat loss and fragmentation, and further reveals that TIB could be extended to community measures by moving beyond assumptions of species equivalency in colonisation rates and extinction susceptibilities.

The role of ecological factors in determining phylogeographic and population genetic structure of two sympatric island skinks (Plestiodon kishinouyei and P. stimpsonii).

We conducted comparative phylogeographic and population genetic analyses of Plestiodon kishinouyei and P. stimpsonii, two sympatric skinks endemic to islands in the southern Ryukyus, to explore different factors that have influenced population structure. Previous phylogenetic studies using partial mitochondrial DNA indicate similar divergence times from their respective closest relatives, suggesting that differences in population structure are driven by intrinsic attributes of either species rather than the common set of extrinsic factors that both presumably have been exposed to throughout their history. In this study, analysis of mtDNA sequences and microsatellite polymorphism demonstrate contrasting patterns of phylogeography and population structure: P. kishinouyei exhibits a lower genetic variability and lower genetic differentiation among islands than P. stimpsonii, consistent with recent population expansion. However, historical demographic analyses indicate that the relatively high genetic uniformity in P. kishinouyei is not attributable to recent expansion. We detected significant isolation-by-distance patterns among P. kishinouyei populations on the land bridge islands, but not among P. stimpsonii populations occurring on those same islands. Our results suggest that P. kishinouyei populations have maintained gene flows across islands until recently, probably via ephemeral Quaternary land bridges. The lower genetic variability in P. kishinouyei may also indicate smaller effective population sizes on average than that of P. stimpsonii. We interpret these differences as a consequence of ecological divergence between the two species, primarily in trophic level and habitat preference.

Habitat fragmentation influences gene structure and gene differentiation among the Loxoblemmus aomoriensis populations in the Thousand Island Lake

Thousand Island Lake (TIL) is a fragmented landscape consisting of more than 1000 land-bridge islands isolated during reservoir formation. To evaluate the effects of fragmentation and island attributes on insect populations, we examined the genetic structure of Loxoblemmus aomoriensis, a species of cricket widely distributed in TIL, and compared genetic diversity between islands samples. Population genetic analyses was conducted based on mitochondrial DNA haplotype frequencies of 10 sample islands. By comparing three island attributes with population genetic diversity reveals that island area influenced population genetic diversity (r2 = 0.5094, p = 0.00204). Using Pairwise Fst values, we also found that long-distance isolation increased the genetic differentiation, while short-distance isolation can be offset by dispersal. These results indicate that fragmentation can impact populations on a genetic level.

Effects of habitat fragmentation on species diversity of ground dwelling beetles in the Thousand-Island Lake region, Zhejiang, China.

The ground dwelling beetles on 20 land-bridge islands and 8 mainland sites in the Thousand-Island Lake region were collected by pitfall trap methods for six times from July 2012 to April 2014 (in three seasons, spring, summer and autumn). The species abundance, composition, diversity and seasonal dynamics of ground dwelling beetles were analyzed. Besides, the relationship between island spatial attributes (e.g., island area and isolation) and the diversity of ground dwe-lling beetles were studied. There were 3370 litter-dwelling beetle individuals belonging to 26 families and 101 species.For species richness of ground dwelling beetles, there were significant diffe-rences between the large islands and mainland sites, the mean density on small islands was significantly higher than on mainland sites. The species turnover was higher among mainland sites than among islands. The species richness on islands was significantly associated with island area, and the density was significantly associated with the distance to mainland. The species richness of islands in summer was higher than that in spring and autumn. Shannon, Simpson and Pielou indices of islands and mainlands were all highest in summer and lowest in autumn.

Defaunation and biomass collapse of mammals in the largest Atlantic forest remnant

Large continuous rainforests are the main hope for sustaining the population of large-bodied vertebrates that cannot cope with fragmentation or unsustainable hunting. The Brazilian Atlantic forest is considered a biodiversity hotspot and although highly fragmented, it still contains large forest patches that may be important for the conservation of mammals that require large areas. Here, we estimated species richness, density and biomass of medium- and large-sized mammals along the largest remnant of the Atlantic rainforest, Brazil (the Serra do Mar bioregion), an estimated area of 8000 km2. We recorded 44 species based on 4090 km of diurnal line transects and camera traps, animal tracks and interviews in 11 continental regions and two large land-bridge islands. We found high levels of similarity in mammalian composition between pairs of sites in the continental forest sites (0.84–1), but much lower similarity between pairs from the continental forest sites and the two large land-bridge islands (0.29–0.74) indicating potential local extinctions or poor dispersal of continental mammals to these islands. In addition, we found that the density and biomass varied 16- and 70-fold among sites, respectively. Mammalian biomass declined by up to 98% in intensively hunted sites and was 53-fold lower than in other Neotropical non-fragmented forests. Although this large forest remnant is able to maintain a high diversity of medium- and large-bodied mammal species, their low density and biomass may affect the long-term persistence of these populations and the ecosystem services they provide.

Habitat Fragmentation Drives Plant Community Assembly Processes across Life Stages.

Habitat fragmentation is one of the principal causes of biodiversity loss and hence understanding its impacts on community assembly and disassembly is an important topic in ecology. We studied the relationships between fragmentation and community assembly processes in the land-bridge island system of Thousand Island Lake in East China. We focused on the changes in species diversity and phylogenetic diversity that occurred between life stages of woody plants growing on these islands. The observed diversities were compared with the expected diversities from random null models to characterize assembly processes. Regression tree analysis was used to illustrate the relationships between island attributes and community assembly processes. We found that different assembly processes predominate in the seedlings-to-saplings life-stage transition (SS) vs. the saplings-to-trees transition (ST). Island area was the main attribute driving the assembly process in SS. In ST, island isolation was more important. Within a fragmented landscape, the factors driving community assembly processes were found to differ between life stage transitions. Environmental filtering had a strong effect on the seedlings-to-saplings life-stage transition. Habitat isolation and dispersal limitation influenced all plant life stages, but had a weaker effect on communities than area. These findings add to our understanding of the processes driving community assembly and species coexistence in the context of pervasive and widespread habitat loss and fragmentation.

Extinction debt on reservoir land-bridge islands

Large dams cause extensive inundation of habitats, with remaining terrestrial habitat confined to highly fragmented archipelagos of land-bridge islands comprised of former hilltops. Isolation of biological communities on reservoir islands induces local extinctions and degradation of remnant communities. “Good practice” dam development guidelines propose using reservoir islands for species conservation, mitigating some of the detrimental impacts associated with flooding terrestrial habitats. The degree of species retention on islands in the long-term, and hence, whether they are effective for conservation is currently unknown. Here, we quantitatively review species' responses to isolation on reservoir islands. We specifically investigate island species richness in comparison with neighbouring continuous habitat, and relationships between island species richness and island area, isolation time, and distance to mainland and to other islands. Species' responses to isolation on reservoir islands have been investigated in only 15 of the >58,000 large-dam reservoirs (dam height >15m) operating globally. Research predominantly originates from wet tropical forest habitats and focuses on mammals, with species richness being the most widely-reported ecological metric. Terrestrial taxa are, overall, negatively impacted by isolation on reservoir islands. Reservoir island species richness declines with isolation time, and although the rate of loss is slower on larger islands, all islands exhibit depauperate species richness <100years after isolation, compared to continuous mainland habitats. Such a pattern of sustained and delayed species loss following large-scale habitat disturbance is indicative of an extinction debt existing for reservoir island species: this pattern is evident across all taxonomic groups and dams studied. Thus, reservoir islands cannot reliably be used for species conservation as part of impact mitigation measures, and should instead be included in area calculations for land impacted by dam creation. Environmental licensing assessments as a precondition for future dam development should explicitly consider the long-term fate of island communities when assessing biodiversity loss vs energy output.

Factors influencing tree diversity and compositional change across logged forests in the Solomon Islands

Tropical forests in the Solomon Islands have been heavily logged in the last century. However, little is known about forest recovery dynamics across this region. Extrapolating findings from logged forests in tropical mainlands or large continental landbridge islands to isolated archipelagos such as the Solomons is inappropriate because succession and diversification patterns and processes differ between the former and latter. We compared the taxonomic diversity and composition of trees between unlogged forest and sites that were logged 10, 30 and 50years previously to provide an indication of the potential dynamics of these forests following timber harvesting. The distance to logging roads and to unlogged forest influenced post-logging recovery, emphasising the importance of edge effects in previously logged forests. At least in the first 50years after logging, tree-community composition did not appear to converge toward that in unlogged forests over time. Although species assemblages in logged forests generally tend to shift from light demanding-pioneers to old-growth species over time, a long-lived pioneer Campnosperma brevipetiolata dominated the forest even 50years after logging. We suggest that recovery of the tree community in logged forests has been hindered by the persistence of C. brevipetiolata, and suggest that it could be thinned via careful silviculture techniques to enhance growth of mature-phase forest species. Removal of such persistent, long-lived pioneer trees could potentially help to accelerate recovery of heavily logged forests.

Selective extinction drives taxonomic and functional alpha and beta diversities in island bird assemblages.

Taxonomic diversity considers all species being equally different from each other and thus disregards species' different ecological functions. Exploring taxonomic and functional aspects of biodiversity simultaneously can better understand the processes of community assembly. We analysed taxonomic and functional alpha and beta diversities of breeding bird assemblages on land-bridge islands in the Thousand Island Lake, China. Given the high dispersal ability of most birds at this spatial scale (several kilometres), we predicted (i) selective extinction driving alpha and beta diversities after the creation of land-bridge islands of varying area and (ii) low taxonomic and functional beta diversities that were not correlated to spatial distance. Breeding birds were surveyed on 37 islands annually from 2007 to 2014. We decomposed beta diversity of breeding birds into spatial turnover and nestedness-resultant components, and related taxonomic and functional diversities to island area and isolation using power regression models (for alpha diversity) and multiple regression models on distance matrices (for beta diversity). We then ran simulations to assess the strength of the correlations between taxonomic and functional diversities. Results revealed that both taxonomic and functional alpha diversities increased with island area. The taxonomic nestedness-resultant and turnover components increased and decreased with difference in area, respectively, but functional counterparts did not. Isolation played a minor role in explaining alpha- and beta-diversity patterns. By partitioning beta diversity, we found low levels of overall taxonomic and functional beta diversities. The functional nestedness-resultant component dominated overall functional beta diversity, whereas taxonomic turnover was the dominant component for taxonomic beta diversity. The simulation showed that functional alpha and beta diversities were significantly correlated with taxonomic diversities, and the observed values of correlations were significantly different from null expectations of random extinction. Our assessment of island bird assemblages validated the predictions of no distance effects and low beta diversity due to pervasive dispersal events among islands and also suggested that selective extinction drives taxonomic and functional alpha and beta diversities. The contrasting turnover and nestedness-resultant components of taxonomic and functional beta diversities demonstrate the importance of considering the multifaceted nature of biodiversity when examining community assembly.

Effects of dispersal abilities on community dynamics of breeding birds on the land-bridge islands in the Thousand Island Lake, China

Effects of dispersal abilities on community dynamics of breeding birds on the land-bridge islands in the Thousand Island Lake, China

Characterization of transcriptome and development of novel EST-SSR makers based on next-generation sequencing technology in Neolitsea sericea (Lauraceae) endemic to East Asian land-bridge islands

Neolitsea sericea (Lauraceae), endemic to East Asian land-bridge islands, is an economically important tree species because of its characteristic aroma and timber uses. However, due to the lack of efficient molecular markers, the genetic diversity and historical demography of this endemic species is not clearly understood. In this study, we performed high-throughput transcriptome sequencing of N. sericea leaves using the Illumina HiSeq™ 2000 sequencing platform, and generated large transcript sequences for functional characterization and development of gene-associated SSR markers. A total of 68,624 unigenes (mean length 733 bp) were assembled from about 54.7 million reads, and 41,130 (59.94 %) unigenes of all the assembled unigenes showed similarity to public databases. From 68,624 unigenes, 13,213 expressed sequence tag–simple sequence repeats (EST-SSRs) were identified. Di-nucleotide SSRs were the most abundant motif (36.5 %), followed by mono- (32.3 %) and tri-nucleotide (26.5 %) repeats. From the 13,213 EST-SSRs, 1191 primer pairs were designed for marker mining. After selecting 131 of these pairs at random for further validation, 13 polymorphic pairs were identified as polymorphic SSR loci. These 13 EST-SSR markers showed intermediate levels of genetic diversity (e.g., N A = 7.15; mean H E = 0.51) when surveyed across six populations from East China (2), Taiwan (1), Korea (1), and the Ryukyus (1) and Honshu (1) of Japan. Both genetic distance and structure analyses identified two genetic clusters largely congruent with recent findings revealed by variations in chloroplast DNA sequences and genomic SSRs. The EST-SSR markers developed in our research will be an information resource for future studies on ecological, evolutionary, and conservation genomics in N. sericea.