The role of oceanic currents in the dispersal and connectivity of the mangrove Rhizophora mangle on the Southwest Atlantic region.

Dispersal is a crucial mechanism to living beings, allowing them to reach new resources such that populations and species can occupy new environments. However, directly observing the dispersal mechanisms of widespread species can be costly or even impractical, which is the case for mangrove trees. The influence of ocean currents on mangrove dispersal is increasingly evident; however, few studies mechanistically relate the patterns of population distribution with the dispersal by oceanic currents under an integrated framework. Here, we evaluate the role of oceanic currents on connectivity of Rhizophora mangle along the Southwest Atlantic. We inferred population genetic structure and migration rates, simulated the displacement of propagules and tested our hypotheses with Mantel tests and redundancy analysis. We observed populations structured in two major groups, north and south, which is corroborated by other studies with Rhizophora and other coastal plants. Inferred recent migration rates do not indicate ongoing gene flow between sites. Conversely, long-term migration rates were low across groups and contrasting dispersal patterns within each one, which is consistent with long-distance dispersal events. Our hypothesis tests suggest that both isolation by distance and isolation by oceanography (derived from the oceanic currents) can explain the neutral genetic variation of R. mangle in the region. Our findings expand current knowledge of mangrove connectivity and highlight how the association of molecular methods with oceanographic simulations improve the interpretation of the dispersal process. This integrative approach is a cost- and time-efficient strategy to include dispersal and connectivity data into marine protected areas planning and management.

Incidence of Coffee Leaf Rust in Vietnam, Possible Original Sources and Subsequent Pathways of Migration.

This research focused on the incidence and population genetics of coffee leaf rust (CLR) fungus, Hemileia vastatrix, to estimate the possible original source(s) and subsequent migration pathways of wind-borne and human-aided spores in three main coffee production regions (Northwest, Central Highlands, and Southeast) in Vietnam. In southern Vietnam (Central Highlands and Southeast), Coffea canephora covers the majority area, while Catimor lines of C. arabica accounts for 95% of the coffee plantations in northwestern Vietnam. Field surveys conducted at eighty-five plantations, show coffee leaf samples infected by the rust fungus across forty-one plantations. Catimor varieties exhibited high levels of susceptibility with severe rust symptoms, while robusta varieties had varying degrees of susceptibility. We analyzed 863-869 base pairs of internal transcribed spacer (ITS) region from 83 samples (41 sequences from Vietnam, 2 from Thailand, and the remaining 40 from American countries); and fifty-two haplotypes consisting of 123 polymorphic sites were detected. Although the analysis of molecular variance (AMOVA) indicates significant genetic differentiation in the H. vastatrix populations in Vietnam, there was no clear genetic structure with respect to the three geographic areas surveyed. Based on the haplotype network, NeighborNet analysis, and geographical distribution patterns of the haplotypes, five haplotypes were identified as early established, from which most other haplotypes in Vietnam were derived. The early established haplotypes were found in the highest frequency in Northwest Vietnam. This finding corresponds to the earliest record of CLR in Vietnam. The phylogenetic network analysis also illustrated that H. vastatrix had expanded from the northwest to southern Vietnam. Pairwise genetic distance analysis and the geophylogenetic tree also suggests that CLR was first established in the Northwest. In addition, some scattered individuals on the principal coordinate analysis (PCoA) diagram and several separated haplotypes in the phylogenetic networks indicated that other branches of CLR in Vietnam were initiated in the Central Highlands. Hemileia vastatrix from these branches have been spreading in southern Vietnam.

Possible northern persistence of Siebold's beech, Fagus crenata, at its northernmost distribution limit on an island in Japan Sea: Okushiri Island, Hokkaido.

Siebold's beech, Fagus crenata, is widely distributed across the Japanese Archipelago and islands in Japan Sea. Similar to the northern limit of the geographical distribution of F. crenata on the mainland of Hokkaido, the northern limit of the distribution of F. crenata on islands in the Japan Sea is observed on Okushiri Island (ca 42°N). To understand the genetic relationships of F. crenata on Okushiri Island, we examined chloroplast (cp) DNA haplotypes and 11 nuclear microsatellite (SSR) loci among 1,838 individuals from 44 populations from Okushiri Island, mainland Hokkaido, and the northern part of the Tohoku region on Honshu Island. We identified 2 cpDNA haplotypes, which represent not only populations on the Japan Sea coast but also those on the Pacific coast and this suggested the Okushiri Island populations might not be formed by single colonization. Genetic diversity of the Okushiri Island populations of nuclear SSR was not lower than the mainland and the STRUCTURE analysis revealed the Okushiri Island individuals were admixed between Hokkaido and Tohoku clusters. Approximate Bayesian computation inferred that divergence between Tohoku and Hokkaido, and admixture between two populations which generated Okushiri populations occurred before the last glacial maximum (LGM), that is, 7,890 (95% hyper probability density (HPD): 3,420 - 9,910) and 3,870 (95% HPD: 431- 8,540) generations ago, respectively. These inferences were well supported by a geological history which suggested an isolation of Okushiri Island from Hokkaido started prior to the Middle Pleistocene. We discuss the possible persistence of F. crenata during the last glacial maximum on northern islands in the Japan Sea such as Okushiri Island.

Mating system evolution and genetic structure of diploid sexual populations of Cyrtomium falcatum in Japan.

Evolution of mating systems has become one of the most important research areas in evolutionary biology. Cyrtomium falcatum is a homosporous fern species native to eastern Asia. Two subspecies belonging to a sexual diploid race of C. falcatum are recognized: subsp. littorale and subsp. australe. Subspecies littorale shows intermediate selfing rates, while subsp. australe is an obligate outcrosser. We aimed to evaluate the process of mating system evolution and divergence for the two subspecies using restriction site associated DNA sequencing (RAD-seq). The results showed that subsp. littorale had lower genetic diversity and stronger genetic drift than subsp. australe. Fluctuations in the effective population size over time were evaluated by extended Bayesian skyline plot and Stairway plot analyses, both of which revealed a severe population bottleneck about 20,000 years ago in subsp. littorale. This bottleneck and the subsequent range expansion after the LGM appear to have played an important role in the divergence of the two subspecies and the evolution of selfing in subsp. littorale. These results shed new light on the relationship between mating system evolution and past demographic change in fern species.

Isolation and characterization of microsatellites from a cicada, Yezoterpnosia nigricosta (Hemiptera: Cicadidae), distributed in subarctic and cool temperate forests.

The cicada Yezoterpnosia nigricosta (Hemiptera: Cicadidae) is distributed in subarctic and cool temperate forests in Japan, China and the Russian Far East. Due to its limited and isolated distribution at higher altitudes in cool temperate forests on the main island of Japan, especially in the central and southern areas, this species is listed as a threatened or near-threatened species on the Red List of 12 prefectures in Japan. Moreover, there are concerns about the impacts of climate change on the species' distribution and population demography. In this study, seventeen microsatellite markers were developed for Y. nigricosta, and marker suitability was evaluated using 32 individuals from two populations in Nagano prefecture (central Japan) and Hokkaido, a northern island of Japan. The number of alleles, expected heterozygosity and fixation index at each locus were 1-15 (mean = 4.294), 0.000-0.914 (mean = 0.519) and -0.225-0.456 (mean = 0.108), respectively. Furthermore, there was moderate genetic differentiation between the two populations (FST = 0.111, F'ST = 0.237). These markers will be useful to evaluate the genetic structure and to infer population demographic history of Y. nigricosta populations, which can contribute to population genetics studies of this species.

Efficient PCR Amplification Protocol of Nuclear Microsatellites for Exuviae-Derived DNA of Cicada, Yezoterpnosia nigricosta.

Although insect exuviae-based genetics is challenging, it can be a valuable method for obtaining reliable DNA resources by non-invasive sampling. This approach is especially effective when the target species is endangered/endemic or when sampling the adult is difficult. One example is cicadas, which during molt leave their exoskeletons on tree trunks, making them easily collectable. While cicada exuviae-derived DNA has previously been employed for mitochondrial DNA sequencing, this study aimed to develop a reliable method for the PCR amplification of nuclear microsatellite loci from cicada exuviae derived DNA for application in molecular ecology, conservation and population genetics. Five different PCR amplification protocols were performed, and the fragment patterns compared with those obtained using DNA extracted from adult individuals. Moreover, the relationship between the freshness of the exuviae and genotyping success was evaluated. TaKaRa LA Taq provided the best performance in the PCR amplification of DNA isolated from cicada exuviae and the electropherogram showed a clear fragment pattern that was equivalent to that obtained from the DNA extracted from the adult individual. This result suggests that cicada exuviae-derived DNA can be amplified by PCR and that multiple independent loci of nuclear DNA microsatellite markers can be easily genotyped. This study demonstrates that fresh cicada exuviae provide high quality DNA, which can be used for microsatellite genotyping. The methods developed in this study are applicable not only for cicada but other insect species for which exuviae are available. Thus, this study can make a significant contribution to insect sciences.

Development of microsatellite markers for the endangered sleeper Eleotris oxycephala (Perciformes: Eleotridae).

The amphidromous sleeper Eleotris oxycephala (Perciformes: Eleotridae) is mainly distributed along the Kuroshio Current in East Asia, and this current is thought to be the main driver of the species' dispersal. Due to anthropogenic environmental changes in rivers, E. oxycephala is ranked as a threatened or near-threatened species in the red lists of 12 prefectures in Japan. Moreover, there is concern that the species' dispersal pattern could be changed due to fluctuations in the Kuroshio Current caused by global warming. In this study, 40 microsatellite markers were developed for E. oxycephala, and their suitability was tested on 43 individuals from two populations of E. oxycephala from Kanagawa and Miyazaki Prefectures. The number of alleles, expected heterozygosity and fixation index at each locus were 2-10 (mean = 5.350), 0.034-0.860 (mean = 0.650) and -0.261-0.448 (mean = 0.065), respectively. Furthermore, there was a lack of genetic difference between the two populations (FST = 0.008, F'ST = 0.024), indicating widespread gene flow via the Kuroshio Current. These markers will be useful to evaluate the genetic structure and infer population demographic history of E. oxycephala populations, which may assist in the conservation of this species.

The presence of a cryptic barrier in the West Pacific Ocean suggests the effect of glacial climate changes on a widespread sea-dispersed plant, Vigna marina (Fabaceae).

Ocean currents are an important driver of evolution for sea-dispersed plants, enabling them to maintain reciprocal gene flow via sea-dispersed diaspores and obtain wide distribution ranges. Although geographic barriers are known to be the primary factors shaping present genetic structure of sea-dispersed plants, cryptic barriers which form clear genetic structure within oceanic regions are poorly understood. To test the presence of a cryptic barrier, we conducted a phylogeographic study together with past demographic inference for a widespread sea-dispersed plant, Vigna marina, using 308 individuals collected from the entire Indo-West Pacific (IWP) region. Chloroplast DNA variation showed strong genetic structure that separated populations into three groups: North Pacific (NP), South Pacific (SP) and Indian Ocean (IN) (F'CT among groups = 0.954-1.000). According to the Approximate Bayesian computation inference, splitting time between NP and SP was approximately 20,200 years (95%HPD, 4,530-95,400) before present. Moreover, a signal of recent population expansion was detected in the NP group. This study clearly showed the presence of a cryptic barrier in the West Pacific region of the distributional range of V. marina. The locations of the cryptic barrier observed in V. marina corresponded to the genetic breaks found in other plants, suggesting the presence of a common cryptic barrier for sea-dispersed plants. Demographic inference suggested that genetic structure related to this cryptic barrier has been present since the last glacial maximum and may reflect patterns of past population expansion from refugia.

Phylogeography of lethal male fighting in a social spider mite.

When males fight for access to females, such conflict rarely escalates into lethal fight because the risks and costs involved, that is, severe injury or death, are too high. The social spider mite, Stigmaeopsis miscanthi, does exhibit lethal male fights, and this male-male aggressiveness varies among populations. To understand the evolution of lethal fighting, we investigated aggressiveness in 42 populations and phylogenetic relationships in 47 populations along the Japanese archipelago. By analysis of the male weapon morph, a proxy for aggressiveness, we confirmed the existence of a mildly aggressive (ML) form, besides the low aggression (LW) and high aggression (HG) forms reported earlier. To evaluate demographic history of these three forms, we employed the approximate Bayesian computation approach using mtCOI sequences and taking into consideration the postlast glacial expansion history of the host plant, Miscanthus sinensis. As results, hierarchical split models are more likely to explain the observed genetic pattern than admixture models, and the ML form in the subtropical region was considered the ancestral group. The inferred demographic history was consistent with the one reconstructed for the host plant in a previous study. The LW form was split from the ML form during the last glacial period (20,000-40,000 years BP), and subsequently, the HG form was split from the ML form at the end of or after the last glacial period (5,494-10,988 years BP). The results also suggest that the mite invaded Japan more than once, resulting in the present parapatric distribution of LW and HG forms in eastern Japan.

Multispecies genetic structure and hybridization in the Betula genus across Eurasia.

Boreal and cool temperate forests are the major land cover of northern Eurasia, and information about continental-scale genetic structure and past demographic history of forest species is important from an evolutionary perspective and has conservation implications. However, although many population genetic studies of forest tree species have been conducted in Europe or Eastern Asia, continental-scale genetic structure and past demographic history remain poorly known. Here, we focus on the birch genus Betula, which is commonly distributed in boreal and cool temperate forests, and examine 129 populations of two tetraploid and four diploid species collected from Iceland to Japan. All individuals were genotyped at seven to 18 nuclear simple sequence repeats (nSSRs). Pairwise FST' among the six species ranged from 0.285 to 0.903, and genetic differentiation among them was clear. structure analysis suggested that Betula pubescens is an allotetraploid and one of the parental species was Betula pendula. In both species pairs of B. pendula and B. plathyphylla, and B. pubescens and B. ermanii, genetic diversity was highest in central Siberia. A hybrid zone was detected around Lake Baikal for eastern and western species pairs regardless of ploidy level. Approximate Bayesian computation suggested that the divergence of B. pendula and B. platyphylla occurred around the beginning of the last ice age (36 300 years BP, 95% CI: 15 330-92 700) and hybridization between them was inferred to have occurred after the last glacial maximum (1614 years BP, 95% CI: 561-4710), with B. pendula providing a higher contribution to hybrids.

The Relationship between Mating System and Genetic Diversity in Diploid Sexual Populations of Cyrtomium falcatum in Japan.

The impact of variation in mating system on genetic diversity is a well-debated topic in evolutionary biology. The diploid sexual race of Cyrtomium falcatum (Japanese holly fern) shows mating system variation, i.e., it displays two different types of sexual expression (gametangia formation) in gametophytes: mixed (M) type and separate (S) type. We examined whether there is variation in the selfing rate among populations of this species, and evaluated the relationship between mating system, genetic diversity and effective population size using microsatellites. In this study, we developed eight new microsatellite markers and evaluated genetic diversity and structure of seven populations (four M-type and three S-type). Past effective population sizes (Ne) were inferred using Approximate Bayesian computation (ABC). The values of fixation index (FIS), allelic richness (AR) and gene diversity (h) differed significantly between the M-type (FIS: 0.626, AR: 1.999, h: 0.152) and the S-type (FIS: 0.208, AR: 2.718, h: 0.367) populations (when admixed individuals were removed from two populations). Although evidence of past bottleneck events was detected in all populations by ABC, the current Ne of the M-type populations was about a third of that of the S-type populations. These results suggest that the M-type populations have experienced more frequent bottlenecks, which could be related to their higher colonization ability via gametophytic selfing. Although high population differentiation among populations was detected (FST = 0.581, F'ST = 0.739), there was no clear genetic differentiation between the M- and S-types. Instead, significant isolation by distance was detected among all populations. These results suggest that mating system variation in this species is generated by the selection for single spore colonization during local extinction and recolonization events and there is no genetic structure due to mating system.

Development and characterization of 27 microsatellite markers for the mangrove fern, Acrostichum aureum (Pteridaceae).

PREMISE OF THE STUDY: Twenty-seven nuclear microsatellite markers were developed for the mangrove fern, Acrostichum aureum (Pteridaceae), to investigate the genetic structure and demographic history of the only pantropical mangrove plant. METHODS AND RESULTS: Fifty-six A. aureum individuals from three populations were sampled and genotyped to characterize the 27 loci. The number of alleles and expected heterozygosity ranged from one to 15 and 0.000 to 0.893, respectively. Across the 26 polymorphic loci, the Malaysian population showed much higher levels of polymorphism compared to the other two populations in Guam and Brazil. Cross-amplification tests in the other two species from the genus determined that seven and six loci were amplifiable in A. danaeifolium and A. speciosum, respectively. CONCLUSIONS: The 26 polymorphic microsatellite markers will be useful for future studies investigating the genetic structure and demographic history of of A. aureum, which has the widest distributional range of all mangrove plants.

The extent and meaning of hybridization and introgression between Siberian spruce (Picea obovata) and Norway spruce (Picea abies): cryptic refugia as stepping stones to the west?

Boreal species were repeatedly exposed to ice ages and went through cycles of contraction and expansion while sister species alternated periods of contact and isolation. The resulting genetic structure is consequently complex, and demographic inferences are intrinsically challenging. The range of Norway spruce (Picea abies) and Siberian spruce (Picea obovata) covers most of northern Eurasia; yet their geographical limits and histories remain poorly understood. To delineate the hybrid zone between the two species and reconstruct their joint demographic history, we analysed variation at nuclear SSR and mitochondrial DNA in 102 and 88 populations, respectively. The dynamics of the hybrid zone was analysed with approximate Bayesian computation (ABC) followed by posterior predictive structure plot reconstruction and the presence of barriers across the range tested with estimated effective migration surfaces. To estimate the divergence time between the two species, nuclear sequences from two well-separated populations of each species were analysed with ABC. Two main barriers divide the range of the two species: one corresponds to the hybrid zone between them, and the other separates the southern and northern domains of Norway spruce. The hybrid zone is centred on the Urals, but the genetic impact of Siberian spruce extends further west. The joint distribution of mitochondrial and nuclear variation indicates an introgression of mitochondrial DNA from Norway spruce into Siberian spruce. Overall, our data reveal a demographic history where the two species interacted frequently and where migrants originating from the Urals and the West Siberian Plain recolonized northern Russia and Scandinavia using scattered refugial populations of Norway spruce as stepping stones towards the west.

Halfway encounters: meeting points of colonization routes among the southern beeches Nothofagus pumilio and N. antarctica.

The Patagonian region is characterized by a complex biogeographic history, with evidence of deep phylogeographic breaks shared among species. Of particular interest to conservation is the nature of colonization and settlement patterns after the last glacial period, including the detection of secondary contact between different lineages and/or hybridization among related species around phylogeographic breaks. Here we studied population demography and past hybridization of two widespread tree species endemic to South America, Nothofagus pumilio and N. antarctica. Using 8 nuclear microsatellites we genotyped 41 populations of both species. Genetic variation and structure across the geographic region were evaluated within and among species and the past demographic history of hybridization between the two species was inferred using Approximate Bayesian Computation (ABC). Northern and southern lineages were identified in each species, and Bayesian clustering revealed their convergence at mid latitudes (42°S). Spatial genetic structure (SGS) also indicated the existence of a genetic discontinuity at these latitudes, which is in agreement with previous data from maternal DNA markers. Several populations around 42-44°S presented high levels of genetic diversity with a decrease toward southern populations. Even though the species are clearly differentiated (G'ST=0.335), admixed gene pools were observed in both species. Two independent runs of ABC suggested that inter species admixture-like patterns occurred within the timescale of the Last Glacial Maximum (around 20,000 BP). We also provide evidences of recent and bi-directional hybridization/introgression between the two Nothofagus species and describe features of the populations demography in the past. The settlement of a secondary contact zone in Nothofagus species around 42-44°S coincides with the phylogeographic breaks and hotspots of genetic diversity found in other plant and animal species in Patagonia, highlighting its importance as reservoir of diversity. The characterization of the population history of native species can contribute substantially to long-term conservation and management policies.

Clinal variation at phenology-related genes in spruce: parallel evolution in FTL2 and Gigantea?

Parallel clines in different species, or in different geographical regions of the same species, are an important source of information on the genetic basis of local adaptation. We recently detected latitudinal clines in SNPs frequencies and gene expression of candidate genes for growth cessation in Scandinavian populations of Norway spruce (Picea abies). Here we test whether the same clines are also present in Siberian spruce (P. obovata), a close relative of Norway spruce with a different Quaternary history. We sequenced nine candidate genes and 27 control loci and genotyped 14 SSR loci in six populations of P. obovata located along the Yenisei river from latitude 56°N to latitude 67°N. In contrast to Scandinavian Norway spruce that both departs from the standard neutral model (SNM) and shows a clear population structure, Siberian spruce populations along the Yenisei do not depart from the SNM and are genetically unstructured. Nonetheless, as in Norway spruce, growth cessation is significantly clinal. Polymorphisms in photoperiodic (FTL2) and circadian clock (Gigantea, GI, PRR3) genes also show significant clinal variation and/or evidence of local selection. In GI, one of the variants is the same as in Norway spruce. Finally, a strong cline in gene expression is observed for FTL2, but not for GI. These results, together with recent physiological studies, confirm the key role played by FTL2 and circadian clock genes in the control of growth cessation in spruce species and suggest the presence of parallel adaptation in these two species.

Genetic structure and hierarchical population divergence history of Acer mono var. mono in South and Northeast China.

Knowledge of the genetic structure and evolutionary history of tree species across their ranges is essential for the development of effective conservation and forest management strategies. Acer mono var. mono, an economically and ecologically important maple species, is extensively distributed in Northeast China (NE), whereas it has a scattered and patchy distribution in South China (SC). In this study, the genetic structure and demographic history of 56 natural populations of A. mono var. mono were evaluated using seven nuclear microsatellite markers. Neighbor-joining tree and STRUCTURE analysis clearly separated populations into NE and SC groups with two admixed-like populations. Allelic richness significantly decreased with increasing latitude within the NE group while both allelic richness and expected heterozygosity showed significant positive correlation with latitude within the SC group. Especially in the NE region, previous studies in Quercus mongolica and Fraxinus mandshurica have also detected reductions in genetic diversity with increases in latitude, suggesting this pattern may be common for tree species in this region, probably due to expansion from single refugium following the last glacial maximum (LGM). Approximate Bayesian Computation-based analysis revealed two major features of hierarchical population divergence in the species' evolutionary history. Recent divergence between the NE group and the admixed-like group corresponded to the LGM period and ancient divergence of SC groups took place during mid-late Pleistocene period. The level of genetic differentiation was moderate (FST  = 0.073; G'ST  = 0.278) among all populations, but significantly higher in the SC group than the NE group, mirroring the species' more scattered distribution in SC. Conservation measures for this species are proposed, taking into account the genetic structure and past demographic history identified in this study.

Genetic structure and demographic history of the endangered tree species Dysoxylum malabaricum (Meliaceae) in Western Ghats, India: implications for conservation in a biodiversity hotspot.

The impact of fragmentation by human activities on genetic diversity of forest trees is an important concern in forest conservation, especially in tropical forests. Dysoxylum malabaricum (white cedar) is an economically important tree species, endemic to the Western Ghats, India, one of the world's eight most important biodiversity hotspots. As D. malabaricum is under pressure of disturbance and fragmentation together with overharvesting, conservation efforts are required in this species. In this study, range-wide genetic structure of twelve D. malabaricum populations was evaluated to assess the impact of human activities on genetic diversity and infer the species' evolutionary history, using both nuclear and chloroplast (cp) DNA simple sequence repeats (SSR). As genetic diversity and population structure did not differ among seedling, juvenile and adult age classes, reproductive success among the old-growth trees and long distance seed dispersal by hornbills were suggested to contribute to maintain genetic diversity. The fixation index (F IS) was significantly correlated with latitude, with a higher level of inbreeding in the northern populations, possibly reflecting a more severe ecosystem disturbance in those populations. Both nuclear and cpSSRs revealed northern and southern genetic groups with some discordance of their distributions; however, they did not correlate with any of the two geographic gaps known as genetic barriers to animals. Approximate Bayesian computation-based inference from nuclear SSRs suggested that population divergence occurred before the last glacial maximum. Finally we discussed the implications of these results, in particular the presence of a clear pattern of historical genetic subdivision, on conservation policies.

Clone identification in Japanese flowering cherry (Prunus subgenus Cerasus) cultivars using nuclear SSR markers.

Numerous cultivars of Japanese flowering cherry (Prunus subgenus Cerasus) are recognized, but in many cases they are difficult to distinguish morphologically. Therefore, we evaluated the clonal status of 215 designated cultivars using 17 SSR markers. More than half the cultivars were morphologically distinct and had unique genotypes. However, 22 cultivars were found to consist of multiple clones, which probably originate from the chance seedlings, suggesting that their unique characteristics have not been maintained through propagation by grafting alone. We also identified 23 groups consisting of two or more cultivars with identical genotypes. Most members of these groups were putatively synonymously related and morphologically identical. However, some of them were probably derived from bud sport mutants and had distinct morphologies. SSR marker analysis provided useful insights into the clonal status of the examined Japanese flowering cherry cultivars and proved to be a useful tool for cultivar characterization.