Chloroplast microsatellite markers for the mangrove tree species Bruguiera gymnorrhiza, Kandelia candel, and Rhizophora stylosa, and cross-amplification in other mangrove species

Chloroplast microsatellite (cpSSR) markers were developed for three ecologically and economically important tree species in the mangrove family, Rhizophoraceae: Bruguiera gymnorrhiza, Kandelia candel, and Rhizophora stylosa. Noncoding regions of chloroplast DNA (cpDNA) from each species were separately amplified using universal chloroplast primers. Six, two, and three polymorphic cpSSR loci in B. gymnorrhiza, K. candel, and R. stylosa, respectively, were developed from amplified noncoding cpDNA regions. Characterization of 216, 156, and 253 individuals of B. gymnorrhiza, K. candel, and R. stylosa, respectively, collected from different natural mangrove populations (B. gymnorrhiza, 9; K. candel, 7; R. stylosa, 9) on Iriomote Island in Japan showed that these loci provide cpSSR markers with polymorphisms ranging from two to four alleles per locus and gene diversity between 0.027 and 0.480. These cpSSR markers will be useful for analyzing the maternal lineage distributions and population genetic structures of the three species. Several of these markers may also be useful in similar studies of other mangrove species.     

Growth and physiological responses of two mangrove species (Bruguiera gymnorrhiza and Kandelia candel) to waterlogging

Effects of duration of waterlogging on growth and physiological responses of two mangrove species, Bruguiera gymnorrhiza and Kandelia candel, were investigated. The relative growth rate of B. gymnorrhiza decreased significantly with waterlogged time, with the highest value found for drained plants and the lowest in plants under 12 weeks waterlogging. On the contrary, no significant difference was found between waterlogged and drained K. candel plants. The shoot to root biomass ratio of K. candel increased when subjected to 8 or 12 weeks waterlogging but little change was recorded in B. gymnorrhiza, indicating a shift in biomass allocation from roots to shoots in K. candel under prolonged waterlogging but not in B. gymnorrhiza. These different growth responses between the two mangrove species supported the hypothesis that K. candel is more tolerant to waterlogging than B. gymnorrhiza. Under 12 weeks waterlogged treatment, root oxidase activity significantly decreased in B. gymnorrhiza but increased in K. candel. Chlorophyll contents of K. candel increased more rapidly in response to waterlogging than B. gymnorrhiza. Activities of both peroxidase and superoxide dismutase increased significantly in leaves of K. candel when the waterlogging period was longer than 8 weeks, while only the peroxidase activity of B. gymnorrhiza showed a significant increase, indicating that K. candel had stronger resistance to the oxidant damage resulting from waterlogging. These physiological indicators further supported the hypothesis that K. candel is more tolerant to waterlogging than B. gymnorrhiza.     

Evaluation of genetic introgression from domesticated pigs into the Ryukyu wild boar population on Iriomote Island in Japan

We evaluated genetic introgression from domesticated pigs into the Ryukyu wild boar (RWB) population on Iriomote Island based on their genetic structure and diversity. We used a combination of mitochondrial DNA D‐loop region (596 bp) polymorphisms and 23 microsatellite markers. RWBs (n = 130) were collected from 18 locations on Iriomote Island and compared with 66 reference samples of European and Asian domestic pigs. We identified six distinct haplotypes, involving 22 single nucleotide polymorphisms (including one insertion) in the RWB population. The phylogenetic tree had two branches: the RWB group and domestic lineage. Fourteen of 130 RWBs (10.8%) belonged to the European domestic lineage, including 11 RWBs from the Panari Islands, northwest of Iriomote Main Island (IMI). The heterozygosity values, total number of alleles, number of effective alleles and polymorphism information content of the RWB groups were lower than those of the European domestic groups. The RWB population on IMI had a lower heterozygous deficiency index (F_IS = 0.059) than did the other populations, which indicates that this population was more inbred. There was a large genetic distance (F_ST = 0.560) between RWBs on IMI and the Meishan populations. Structure analysis using the 23 microsatellite markers revealed that 16 RWBs had an admixture pattern between RWB and domesticated pig breeds. These results suggest that gene flow may have occurred from domestic pigs to RWBs and demonstrate that there was low genetic variation in the IMI population.     

Structure and productivity along a tree height gradient in a Kandelia obovata mangrove forest in the Manko Wetland, Okinawa Island, Japan

Tree height (H) of Kandelia obovata trees decreased sharply from 5 m at the forest interior behind the terrestrial forest to 1.5 m at the forest edge near the river bank according to an increase in the yearly waterlogged period along a belt transect. The decreasing tree stature was attributed to a decrease in the asymptote of H in the D _0.1 (stem diameter at H/10)-H relationship toward the edge. The K. obovata trees were well classified into interior and edge types using a discriminant function based on the habitat-specific D _0.1–H relationships. Allometric equations, as a function of D _0.1² H, differed significantly between the interior and edge types in the estimation of the phytomasses of stems and leaves, and the leaf area per tree. On the other hand, common allometric equations were successfully established in the estimation of respective phytomasses of aboveground parts and branches. Biomass and leaf area index decreased toward the forest edge. The biomass allocation to stems decreased toward the edge, whereas those to branches and leaves increased. A dramatic change in stem diameter increment resulted in differences in the D _0.1–H relationship along the tree height gradient. Relative growth rate of biomass and light-saturated net photosynthesis, which paralleled net assimilation rate from the interior to the edge, showed their maximum peaks in the middle of the belt transect. This indicates that there exists an optimal environmental condition for growth of K. obovata trees. Leaf nitrogen content tended to increase to the edge with increasing waterlogged period.     

Vegetative and reproductive phenology of the mangrove Bruguiera gymnorrhiza (L.) Lam. on Okinawa Island, Japan

Vegetative and reproductive phenology of the subtropical mangrove Bruguiera gymnorrhiza was investigated at Manko Wetland, Okinawa Island, Japan. Phenology was assessed using litterfall data over 4 years. Leaf and stipule litterfall occurred throughout the year, with distinct seasonal patterns. Kendall’s coefficient of concordance, W, revealed that the monthly changes in leaf and stipule litterfall were strongly and significantly concordant among years. Leaf litterfall was linked to monthly day length and maximum wind speed, and stipule litterfall was linked to monthly mean air temperature, monthly rainfall, and maximum wind speed. Branch litterfall showed no clear monthly pattern, but tended to vary with monthly maximum wind speed. Mean leaf longevity was 19 months. Mean total litterfall was 10.1 Mg ha^?1 year^?1, with the largest component being vegetative organs (58 %). Flower and propagule litterfall were highest in autumn and summer, respectively, and lowest in winter. The W values revealed that, of the reproductive organs, only flower litterfall had a significant monthly trend. Flower litterfall was correlated with monthly mean air temperature and relative humidity. The average development periods from flower buds to flowers and from flowers to mature propagules were approximately 1 and 8 months, respectively. It took 9 months to produce mature propagules from flower buds. Except for branches, all vegetative and reproductive components of litterfall had clear annual cycles.     

Reproductive biology and population genetic structure of Kandelia candel (Rhizophoraceae), a viviparous mangrove species

The pollination biology, mating system, and population genetic structure of Kandelia candel were investigated. Field observations on its pollination and reproductive biology suggested that this species is pollinator dependent for fruit set, and bee activities can lead to substantial geitonogamous selfing. Quantitative analysis of the mating system parameters was performed using progeny arrays assayed for allozyme markers. Multilocus outcrossing rates (t(m)) were estimated to be 0.697 ± 0.091 and 0.797 ± 0.062 in two populations. In comparison to other plant species with mixed-mating system, the level of allozyme variation was very low in the 13 populations sampled along the coastlines of Hong Kong. At the species level, the proportion of polymorphic loci was 20%, number of alleles per locus was 1.2, and heterozygosity was 0.0362. The total gene diversity was primarily distributed within populations (H(S )= 0.0339), and the coefficient of genetic differentiation among populations was low (G(ST )= 0.064). This pattern of population genetic structure suggests that gene flow, primarily in the form of water-dispersed seedlings in viviparous mangrove species, is not as limited as previously thought. However, microgeographic pattern in allele frequency at the marker loci could still be detected between the western and eastern coastal populations.     

A new species of Gnathia (Isopoda: Cymothoida: Gnathiidae) from Okinawajima Island, Ryukyu Archipelago, southwestern Japan

Gnathia limicola sp. nov. is described from Okinawajima Island, Ryukyu Archipelago, southwestern Japan. Burrows of this species were found in a small intertidal creek bank on a muddy tidal flat near mangrove trees. Adult males differ from those of other Gnathia species in the following features: (1) fine setae cover peduncle articles 1 and 2 of antenna 1, peduncle articles 1-3 of antenna 2, and the erisma of the mandibles; (2) the ventral frontal border of the cephalon is medianly notched, and the lateral parts extend beyond the dorsal frontal border; and (3) the penes are fused into a thin rectangular blade directed posteriorly. Adult females and praniza larvae were also distinguished morphologically from other Gnathia species. Based on field and laboratory observations, the mating behavior of this species appears similar to that of Paragnathia formica (Hesse, 1862), which inhabits salt marshes in Europe and North Africa.     

Mating system,pollen and propagule dispersal,and spatial genetic structure in a high-density population of the mangrove tree Kandelia candel

Mangrove tree species form ecologically and economically important forests along the tropical and subtropical coastlines of the world. Although low intrapopulation genetic diversity and high interpopulation genetic differentiation have been detected in most mangrove tree species, no direct investigation of pollen and propagule dispersal through paternity and/or parentage analysis and spatial genetic structure within populations has been conducted. We surveyed the mating system, pollen and propagule dispersal, and spatial genetic structure in a natural population of Kandelia candel, one of the typical viviparous mangrove tree species, using nuclear and chloroplast microsatellite markers. High diversity and outcrossing rates were observed. Paternity and parentage analysis and modelling estimations revealed the presence of an extremely short-distance component of pollen and propagule dispersal (pollen: 15.2 ± 14.9 m (SD) by paternity analysis and 34.4 m by modelling; propagule: 9.4 ± 13.8 m (SD) by parentage analysis, and 18.6 m by modelling). Genetic structure was significant at short distances, and a clumped distribution of chloroplast microsatellite genotypes was seen in K. candel adults. We conclude that the K. candel population was initiated by limited propagule founders from outside by long-distance dispersal followed by limited propagule dispersal from the founders, resulting in a half-sib family structure.     

Descriptions of two azooxanthellate Palythoa species (Subclass Hexacorallia,Order Zoantharia) from the Ryukyu Archipelago,southern Japan

Two new species of zoantharians (Hexacorallia, Zoantharia, Sphenopidae), Palythoa mizigama sp. n. and Palythoa umbrosa sp. n., are described from the Ryukyu Archipelago, southern Japan. Unlike almost all other known Palythoa spp., both species are azooxanthellate and inhabit low-light environments such as floors or sides of caves, crevasses, or hollows of shallow coral reefs. The two species were initially considered to be the same species from their similar habitat environments and highly similar morphological features. However, phylogenetic analyses of nuclear internal transcribed spacer (ITS) ribosomal DNA, mitochondrial 16S ribosomal DNA, and cytochrome oxidase subunit I (COI) sequences revealed that these two species have a genetically distant relationship within the genus Palythoa. Morphological characteristics, including polyp size, tentacle number, external/internal coloration, and types and sizes of cnidae were examined in this study. As a result, only tentacle coloration was found to be useful for the morphological distinction between the two species. Palythoa mizigama possesses white tentacles with black horizontal stripes while Palythoa umbrosa possesses white tentacles without any stripe patterns. Considering their distant phylogenetic relationship, it can be assumed that their unique yet similar morphological and ecological characteristics developed independently in each species as an example of parallel evolution.     

A new genus and species of Renaudarctidae (Heterotardigrada: Arthrotardigrada) from Ryukyu Archipelago,Japan

A new armoured marine tardigrade, Nodarctus hallucis gen. et sp. nov., is described from specimens collected from sandy beaches of Haterumajima, Ishigakijima and Okinawajima Islands, Ryukyu Archipelago, Japan. It is a member of the rare family Renaudarctidae, which is characterized by a stocky body with dorsal and ventral plates and stumpy legs with digits, claws and accessory filaments. However, the collected animals have prominent characters that differ from the two known species of the family: subdivided dorsal segmental plates I–III lacking lateral processes, an acute caudal spike, indented sensory plates, short spikes and peculiar papillae on legs I–III, basal spikes of legs IV and only two digits on legs IV. These differences justify the erection of a new genus and species for the animal. Morphological affinities with other marine taxa, the function of the leg papillae and the distribution pattern of the new species are discussed.http://zoobank.org/urn:lsid:zoobank.org:pub:041878AA-B437-45BF-80E3-32591D21D965     

中国红树林北缘区秋茄树种群空间分布格局和结构特征分析

对位于中国红树林北缘区福鼎市的秋茄树〔Kandelia candel(Linn.)Druce〕天然林和人工林的高度结构和径级结构进行了分析,并以"空间替代时间"的方法研究了秋茄树天然林和人工林的空间分布格局以及天然林的种群结构特征。结果表明:不同滩位秋茄树天然林和人工林的高度结构及径级结构明显不一致。天然林中株高0.0~0.5和1.5~2.0 m的个体占多数;而人工林以株高1.5~2.0 m的个体为主,无株高0.0~0.5 m的幼苗,株高超过2.0 m的植株也极少,表明天然林的高度结构均匀而人工林的高度结构极不合理。根据基径(D)可将秋茄树种群分为13个径级,其中天然林不同径级个体数量随径级增大依次减少,而人工林中径级Ⅲ(6 cm≤D<10 cm)的植株数量最多,且没有径级Ⅸ(30 cm≤D<34 cm)以上的个体,表明天然林的径级结构良好。从分布格局看,天然林幼苗群呈现聚集分布,而其小树群、大树群和老树群均为随机分布;人工林无幼苗群和老树群,小树群呈均匀分布,大树群呈聚集分布。种群静态生命表分析结果表明:秋茄树天然林种群的期间死亡率和消失率在龄级1(D<2 cm)和龄级7(22 cm≤D<26 cm)达到最大,在其他龄级均较小且处于稳定状态;其个体生存率单调下降、累计死亡率单调上升,仅在龄级1时个体生存率高于累计死亡率,从龄级2(2 cm≤D<6 cm)开始均表现为累计死亡率高于生存率;其存活曲线属于典型的Deevey-Ⅲ型,表明秋茄树天然林种群中幼苗丰富但期间死亡率较高,成树的期间死亡率相对较低且个体数量稳定。     

Two new species of Asellota (Crustacea,Isopoda) from coral reefs on Iriomote Island,Okinawa, Japan

Pleurocope iriomotensis sp. n. and Prethura tuberculata sp. n. are described from Iriomote Island, Ryukyu Archipelago, southern Japan. These are the first records of Pleurocope from the Pacific and of Prethura from the Asian Pacific coast. Pleurocope iriomotensis differs from its congeners in having lateral spine-like processes on pereonite 4 and coxal plates of pereonite 7. Prethura tuberculata can be distinguished from its single congener in having a lateral short projection of protopod of pleopod 2.     

Small-scale population genetic structure of the sand bubbler crab Scopimera ryukyuensis in the Ryukyu Islands,Japan

Molecular Biology Reports - Generally, the gene flow of marine organisms is well maintained, but some local populations of coastal species are genetically differentiated even on a small scale...     

A new genus and two new species of Peltogastridae (Crustacea: Cirripedia: Rhizocephala) parasitizing hermit crabs from Okinawa Island (Ryukyu Archipelago, Japan), and their DNA-barcodes

A new genus and two new species of Peltogastridae, Peltogaster postica sp. nov. and Ommatogaster nana gen. et sp. nov., are described from Okinawa Island, Ryukyu Islands, southwestern Japan. The two new rhizocephalans were found to be parasitic on the estuarine hermit crabs, Pagurus minutus Hess, 1865 and Diogenes leptocerus Forest, 1956, respectively. Peltogaster postica sp. nov. is allied to P. curvata Kossmann, 1874, P. paguri Rathke, 1842 , and P. reticulata Shiino, 1943 , but is distinguished by its relative length and internal and external structures of the mature externa. Ommatogaster gen. nov. is established for the present new species O. nana based on the morphologies of the visceral mass of the externa and the presence of a nauplius eye in the larvae. Partial COI sequences were obtained from the two new species and one known species, Dipterosaccus indicus Van Kampen and Boschma, 1925, to test the possible usefulness of the sequences as tags for species identification.     

Reproductive biology and genetic population structure of two alien Lolium species inhabiting the sandy coasts of Japan

Understanding the characteristics of alien species is a prerequisite for any biological study or anti-invasion management strategy. Lolium rigidum (Poaceae) is an alien species that has become naturalized on the sandy coasts of Japan; however, it exhibits extensive morphological variation, leading to speculation that several taxa of Lolium have become naturalized. Here, we compared the morphology, reproductive biology and genetic structure of this Lolium species by growing individuals from different locations in the same environment to clarify whether the observed morphological variation is genetically based or is caused by intraspecific variation as a result of environmental differences. Principle component analyses of 11 morphological traits separated the study species into two types. Bagging experiments showed that one type exhibited an outcrossing life history, whereas the other type selfed exclusively. Nuclear DNA microsatellite analyses supported this distinction between these morphological types, with no intermediate individuals being found. The outcrossing type exhibited high genetic diversity, whereas the selfing type exhibited almost no polymorphism, reflecting the differences in their breeding systems. Moreover, both types differed from the economically important outcrossing species Lolium multiflorum and Lolium perenne. These two types of Lolium are expected to have different introduction histories and invasive potential on the coasts of Japan. In conclusion, it is important to distinguish between these types through additional biological studies to design effective control measures.     

Two new species of diplosoma (Ascidiacea: Didemnidae) bearing prokaryotic algae prochloron from Okinawajima (Ryukyu Archipelago, Japan)

Two new species of didemnid ascidians, Diplosoma ooru sp. nov. and Diplosoma simileguwa sp. nov., are described from coral reefs on Okinawajima (Ryukyu Archipelago, Japan). These two species form green colonies, having a symbiotic association with a prokaryotic alga Prochloron sp. The former species was found at the reef edges in the subtidal zone and the latter was found in a shallow reef lagoon. In these species, the colonies are thinner and the zooids are smaller than those of any other Prochloron-bearing Diplosoma species so far described. Moreover, each of the present new species has a unique combination of stigmatic numbers: 5 stigmata in the first and third rows, 6 in the second row, and 4 in the fourth in D. ooru; 4 stigmata in the first and third rows, 5 in the second row, and 3 in the fourth in D. simileguwa. In both of the new species, the retractor muscle emerges from the underside of the thorax. Larval morphology of D. ooru is also described.     

Population genetic structure of three tree species in the mangrove genus Ceriops (Rhizophoraceae) from the Indo West Pacific

Ceriops is a viviparous mangrove with widespread species Ceriops decandra and C. tagal, and an endemic species C. australis. Genetic diversity of the three species was screened in 30 populations collected from 23 locations in the Indo West Pacific (IWP) using Inter-simple sequence repeats (ISSR) and sequences of partial nuclear gene (G3pdh) and chloroplast DNA (trnV-trnM). At the species level, the total gene diversity (Ht) revealed by ISSRs was 0.270, 0.118, and 0.089 in C. decandra, C. tagal, and C. australis, respectively. A total of six haplotypes of G3pdh and five haplotypes of trnV-trnM were recognized among the three species. Only C. decandra was detected containing more than one haplotype from each sequence data set (four G3pdh haplotypes and three trnV-trnM haplotypes). At the population level, genetic diversity of Ceriops was relatively low inferred from ISSRs (He = 0.028, 0.023, and 0.053 in C. decandra, C. tagal, and C. australis, respectively). No haplotype diversity within population was detected from any of the three species. Cluster analysis based on ISSRs identified three major geographical groups in correspond to the East Indian Ocean (EIO), South China Sea (SCS), and North Australia (NA) in both C. decandra and C. tagal. The cladogram from DNA sequences also detected the same three geographical groups in C. decandra. Analysis of molecular variance (AMOVA) revealed that most of the total variation was accounted for by differentiation between the three major geographical regions of both C. decandra and C. tagal. The significant genetic structure may result from the geological events in these regions during the recent Pleistocene glaciations. This study also provided insights into the phylogenetics of Ceriops. Yelin Huang and Fengxiao Tan contributed equally to this work.     

Spatial structure and genetic diversity of two tropical tree species with contrasting breeding systems and different ploidy levels

Analyses of the spatial distribution pattern, spatial genetic structure and of genetic diversity were carried out in two tropical tree species with contrasting breeding systems and different ploidy levels using a 50-ha demographic plot in a lowland dipterocarp forest in Peninsular Malaysia. Shorea leprosula is a diploid and predominantly outcrossed species, whereas S. ovalis ssp. sericea is an autotetraploid species with apomictic mode of reproduction. Genetic diversity parameters estimated for S. leprosula using microsatellite were consistently higher than using allozyme. In comparisons with S. leprosula and other tropical tree species, S. ovalis ssp. sericea also displayed relatively high levels of genetic diversity. This might be explained by the lower pressure of genetic drift due to tetrasomic inheritance, and for autotetraploids each locus can accommodate up to four different alleles and this allows maintenance of more alleles at individual loci. The observed high levels of genetic diversity in S. ovalis ssp. sericea can also be due to a random retention of more heterogeneous individuals in the past, and the apomictic mode of reproduction might be an evolutionary strategy, which allows the species to maintain high levels of genetic diversity. The spatial distribution pattern analyses of both species showed significant levels of aggregation at small and medium but random distribution at the big diameter-class. The decrease in magnitude of spatial aggregation from small- to large-diameter classes might be due to compensatory mortality during recruitment and survival under competitive thinning process. Spatial genetic structure analyses for both species revealed significant spatial genetic structure for short distances in all the three diameter-classes. The magnitude of spatial genetic structure in both species was observed to be decreasing from smaller- to larger-diameter classes. The high spatial genetic structuring observed in S. ovalis ssp. sericea at the small-diameter class is due primarily to limited seed dispersal and apomictic mode of reproduction. The similar observation in S. leprosula, however, can be explained by limited seed and pollen dispersal, which supports further the fact that the species is pollinated by weak fliers, mainly of Thrips and Megalurothrips in the lowland dipterocarp forest.     

A new genus and species of hatschekiid copepod (Siphonostomatoida) from groupers (Actinopterygii: Serranidae) collected off the Ryukyu Archipelago, Japan

A new genus and species of copepod, Mihbaicola sakamakii n. g., n. sp., belonging to the siphonostomatoid family Hatschekiidae, is described based on the females collected from inside the tissue of the branchiostegal membrane in three species of the groupers, Epinephelus fasciatus (Forsskål) (type-host), E. merra Bloch and Cephalopholis leopardus (Lacépède), collected off Okinawa-jima Island and Iriomote-jima Island, Ryukyu Archipelago, North Pacific Ocean. The new genus can be distinguished from other hatschekiid genera by a combination of the following characters in the female: the head is composed of the cephalosome and the pedigerous somite; the cephalothorax is expanded into a pair of posteroventral lobes carrying leg 1; legs 1 and 2 are biramous and composed of the protopod and both rami are 2-segmented; leg 3 is absent; and leg 4 is represented by a rounded lobe with a chitinous pointed apical process.     

Mating system and population genetic structure of an androdioecious tree, Fraxinus lanuginosa Koidz. (Oleaceae) in northern Japan

Models for the maintenance of androdioecy have suggested that selfing of hermaphrodites decreases the frequency of males in a population (the "male frequency"). To test this hypothesis, we used electrophoretic allozyme methods to study the mating system and population genetics of an androdioecious tree, Fraxinus lanuginosa, which exhibits large variations in male frequency among subpopulations in central Hokkaido (northern Japan). We estimated the outcrossing rates by using seeds assayed at three polymorphic loci, and found that the multilocus outcrossing rate (t(m)) increased with increasing male frequency (q) (t(m) = 0.69 to 0.99, q = 0.11 to 0.59). Fixation indices (F(j)) estimated from these t(m) values ranged from 0.006 to 0.185, and were significantly greater than zero in plots with a low male frequency (q or = 0.47). However, fixation indices at the late life stage (F(a)) were not significantly different from zero at five loci (F(a) = 0.056 to 0.101, q = 0.11 to 0.61), and the F(a) values were lower than the F(j) values in several of the plots where both values were measured. These results indicate that inbreeding depression substantially decreases the proportion of selfed progeny in partially self-fertilising subpopulations. A theoretical model for the maintenance of androdioecy showed expected male frequencies significantly lower than the observed values in plots with high male frequency (q = 0.59 to 0.61), although the differences between the expected and observed values of male frequencies were not significant in plots with intermediate or low male-frequencies (q = 0.11 to 0.47). In this paper, the factors are discussed that affect variations in the male frequency among subpopulations of F. lanuginosa based on these findings.