The numbers of deep-sea fish species and their genetic diversities are poorly understood because of taxonomic confusion and the lack of robust diagnostic features. However, DNA barcoding using mitochondrial DNA sequences may offer an effective approach to identifying cryptic species and characterizing their genetic diversities. To validate the genetic differentiation identified by DNA mitochondrial barcoding, it is necessary to show that these reflect variations present in nuclear genomic markers. Here, we performed DNA barcoding using cytochrome c oxidase subunit I (COI) sequences and also carried out multiplexed intersimple sequence repeat genotyping by sequencing (MIG-seq) for mesopelagic and demersal fish species from the continental shelf and upper slope of the northwestern Pacific Ocean. We obtained the COI sequences of 115 species from 48 families; the species were identified using the Barcode of Life Data System. Phylogenetic analyses using COI sequences showed high levels of intraspecific genetic differentiation (Kimura 2-parameter distances >2%) in 20 of 115 species, suggesting many cryptic species or intraspecific genetic differentiation previously unknown in these species. We performed phylogenetic and population genetic analyses using multiple single-nucleotide polymorphism loci obtained by MIG-seq of 3 species that showed high levels of intraspecific genetic differentiation in COI sequences. The nuclear markers confirmed the genetic differentiation in all 3 species identified by the COI sequences. The high concordance between these different genetic markers indicates the effectiveness of DNA barcoding for identifying cryptic deep-sea species and characterizing genetic differentiation in these species.

In forest ecosystems, understanding the relationship between the vertical distribution of fine roots and residual soil nitrogen is essential for clarifying the diversity-productivity-water purification relationship.Vertical distributions of fine-root biomass (FRB) and concentrations of nitrate-nitrogen (NO 3 -N) in soil water were investigated in a conifer plantation with three thinning intensities (Control, Weak and Intensive), in which hardwood abundance and diversity were low, moderate and high, respectively.Intensive thinning led to the lowest NO 3 -N concentration in soil water at all depths (0-100 cm) and highest FRB at shallow depths (0-50 cm). The NO 3 -N concentration at a given depth was negatively correlated with total FRB from the surface to the depth at which NO 3 -N concentration was measured, especially at shallow depths, indicating that more abundant fine roots led to lower levels of downward NO 3 -N leaching. FRB contributed positively to nitrogen content of hardwood leaves.These findings demonstrate that a hardwood mixture in conifer plantations resulted in sufficient uptake of NO 3 -N from soil by well developed fine-root systems, and translocation to canopy foliage. This study suggests that productivity and water purification can be achieved through a hardwood mixture in conifer plantations.

SAYA TAMURA, TOMOKO FUKUDA, ELENA A. PIMENOVA, EKATERINA A. PETRUNENKO, PAVEL V. KRESTOV, SVETLANA N. BONDARCHUK, OLGA A. CHERNYAGINA, YOSHIHISA SUYAMA, YOSHIHIRO TSUNAMOTO, AYUMU MATSUO, HAYATO TSUBOI, HIDEKI TAKAHASHI, KEN SATO, YOKO NISHIKAWA, TAKASHI SHIMAMURA, HIROKO FUJITA & KOH NAKAMURA An alpine plant Saxifraga yuparensis is endemic to a scree consisting of greenschist of Mt. Yubari in Hokkaido, Japan and it has been proposed as an immediate hybrid derived from two species of the same section Bronchiales based on morphological intermediacy: namely S. nishidae, a diploid species endemic to a nearby cliff composed of greenschist and tetraploid S. rebunshirensis comparatively broadly distributed in Japan and Russian Far East. Saxifraga yuparensis is red-listed and it is crucial for conservation planning to clarify whether this is an immediate hybrid and lacks a unique gene pool. The immediate-hybrid hypothesis was tested by molecular and cytological data. In nuclear ribosomal and chloroplast DNA trees based on maximum parsimony and Bayesian criteria, S. yuparensis and S. rebunshirensis formed a clade with several other congeners while S. nishidae formed another distinct clade. Genome-wide SNP data clearly separated these three species in principal coordinate space, placing S. yuparensis not in-between of S. rebunshirensis and S. nishidae. Chromosome observation indicated that S. yuparensis is tetraploid, not triploid directly derived from diploid-tetraploid crossing. Additionally, observation of herbarium specimens revealed that leaf apex shape of S. yuparensis fell within the variation of S. rebunshirensis. These results indicate that S. yuparensis is not an immediate hybrid of S. rebunshirensis and S. nishidae but a distinct lineage and an extremely narrow endemic species, that deserves for intensive conservation.

， 5 (2020) Salt Tolerance Seedlings Derived from Putative Hybrid Pines (Pinus × densi-thunbergii) : Possibility of Candidate Materials for Coastal Forest Restoration. J Jpn For Soc 102: 101-107 Japanese black pine (Pinus thunbergii) is tolerant against salty wind but susceptible to pinewood nematodes, whereas Japanese red pine (Pinus densiflora) is more resistant against pinewood nematodes than P. thunbergii but weaker against salty wind. To produce materials with high resistance against pinewood nematodes and high tolerance against salty wind, we focused on seedlings derived from putative hybrid clones (Pinus × densi-thunbergii) . In this study, we investigated the nuclear DNA genome composition of two putative hybrid clones. Using a PCR-RFLP method for analyzing chloroplast DNA, we subsequently identified the paternal species of progenies of two putative hybrids. Branches and potted seedlings from the progenies of P. densiflora, P. thunbergii, and the hybrids were immersed in seawater. We harvested the needles from them and measured the potential maximal PS Ⅱ quantum yield (F v /F m ) . We also dried some needle samples and used inductively coupled plasma mass spectrometry (ICP-MS) to determine Na concentrations. Nuclear DNA analysis of the putative hybrid clones revealed that they were F1 hybrids (P. × densi-thunbergii) . Chloroplast DNA analysis revealed that progenies of the hybrid clones were sired by P. densiflora. Those progenies were significantly more salt tolerant than P. densiflora. Moreover, the Na concentration of needles of hybrid clones was lower than those of P. densiflora. Our results suggest that the progenies of hybrids are potential candidates for plantation in coastal pine forests, due to high resistance against pinewood-nematode and high tolerance against salt water.