Persea boninensis (Lauraceae) is an endemic tree species distributed throughout the Bonin Islands. It grows in a wide range of environments from dry to mesic forests, and has multiple flowering peaks that may correspond to different habitats on Chichijima Island of the Bonin Islands. We predicted that P. boninensis is differentiated into two groups with different habitats on these islands. We examined and compared the flowering phenology, morphology, and genetics of populations of species growing in dry and mesic forests. We also performed preliminary artificial crossing experiments. Based on our results, P. boninensis on the Chichijima Islands can be clearly divided into two genetic groups with different habitats and flowering times. Although the flowering time difference could act as an effective pre‐zygotic isolation mechanism between the two groups, there was still a 1‐month overlap in flowering time. Furthermore, our artificial crossing experiments between the two groups resulted in plants that set seeds. Therefore, there was no evidence of reproductive isolation after fertilization. Differences in flowering time as well as in growth habitat will have to occur to maintain genetic differentiation between the two groups of P. boninensis.

Eight microsatellite loci were identified and characterized for the endangered Machilus pseudokobu (Lauraceae), an endemic tree species of the Bonin Islands. The observed number of alleles at each locus ranged from 1 to 20 with an average of 6.2, and the expected heterozygosity ranged from 0.00 to 0.83 with an average of 0.47. All eight loci were screened in cross-amplification tests for two other endemic Machilus species that also inhabit the Bonin Islands. All loci were successfully amplified in these species.Keywords The Bonin (Ogasawara) Islands Á Cross-species amplification Á Genetic differentiation Á Lauraceae Á Machilus Á MicrosatelliteThe Bonin (Ogasawara) Islands comprise *20 oceanic islands located in the northwest Pacific Ocean. The flora of the Bonin Islands is characterized by a high degree of endemism and several examples of adaptive radiation (e.g., Crepidiastrum, Pittosporum, Symplocos; Ito 1998). Plants of the genus Machilus (Lauraceae) in the Bonin Islands are considered to represent an example of adaptive radiation (Ito et al. 1990). Three endemic species of Machilus are distributed in the Bonin Islands. Machilus pseudokobu. Koidz. occurs in the highly mesic forests in the central region of Chichijima Island, and is listed as endangered in the Japanese Red Data Book (Environment Agency of Japan 2007). It is difficult to distinguish M. pseudokobu. Koidz. from the other two Machilus species, M. kobu Maxim. and M. boninensis Koidz. based only on morphology, although their habitat preferences seem to be differentiated (Toyoda 2003). Thus, this species might be at an early stage of speciation by adaptive radiation. In this work, we developed eight microsatellite markers for the endangered M. pseudokobu in order to estimate its genetic diversity and to assess genetic differentiation among the Machilus species endemic to the Bonin Islands.Fresh leaves of M. pseudokobu were collected from Chichijima Island and genomic DNA was extracted using a DNeasy plant miniprep kit (Qiagen). Microsatellite loci were isolated using an improved technique for isolating codominant compound microsatellite markers (Lian et al. 2006). Briefly, DNA was digested with the blunt-end restriction enzymes SspI and BsrBI. The restriction fragments were then ligated with a specific blunt adaptor (consisting of the 48-mer: 5 0 -GTAATACGACTCACTATAGGGCACGCGT GGTCGACGGCCCGGGCTGGT-3 0 ; and an 8-mer with the 3 0 -end capped with an amino residue: 5 0 -ACCAGCCC-NH 2 -3 0 ) using a DNA ligation kit (Takara). The ligated fragments were treated with ddGTP using AmpliTaq Gold (Applied Biosystems) to block polymerase-catalyzed extension of the 8-mer adaptor strand. Fragments were amplified from the SspI and BsrBI DNA libraries using one of the compound SSR primers, (AC) 6 (AG) 5 or (AC) 6 (TC) 5 , and an adaptor primer (5 0 -CTATAGGGCACGCGTGGT-3 0 ). The amplified fragments were cloned using the Qiagen PCR Cloning plus kit (Qiagen) in accordance with the manufacturer's instructions. Insert-positive clones were amplified using forward and rev...

In ferns, intra-gametophytic selfing occurs as a mode of reproduction where two gametes from the same gametophyte form a completely homozygous sporophyte. Intra-gametophytic selfing is considered to be prevented by lethal or deleterious recessive genes in several diploid species. In order to investigate the modes and tempo of selection acting different developmental stages, doubled haploids obtained from intra-gametophytic selfing within isolated gametophytes of a putative F1 hybrid between Osmunda japonica and O. lancea were analyzed with EST_derived molecular markers, and the distribution pattern of transmission ratio distortion (TRD) along linkage map was clarified. As the results, the markers with skewness were clustered in two linkage groups. For the two highly distorted regions, gametophytes and F2 population were also examined. The markers skewed towards O. japonica on a linkage group (LG_2) showed skewness also in gametophytes, and the TRD was generated in the process of spore formation or growth of gametophytes. Also, selection appeared to be operating in the gametophytic stage. The markers on other linkage group (LG_11) showed highest skewness towards O. lancea in doubled haploids, and it was suggested that the segregation of LG_11 were influenced by zygotic lethality or genotypic evaluation and that some deleterious recessive genes exist in LG_11 and reduce the viability of homozygotes with O. japonica alleles. It is very likely that a region of LG_11were responsible for the low frequencies of intra-gametophytic selfing in O. japonica.

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