Molecular and morphological evidence for hybridization between endemic Canary Island Convolvulus

Morphological data and molecular data from the chloroplast trn H- psb A region and nuclear ribosomal ITS region are used to test the hypothesis that the problematic Canary Island endemic Convolvulus floridus var. virgatus is a hybrid between the endemic species C. floridus and C. scoparius . Analysis of mean leaf length and width of 58 individuals indicates that accessions referable to C. floridus var. virgatus are intermediate between the parental taxa in leaf dimensions. Direct sequencing of the ITS region of C. scoparius and C. floridus revealed two species-specific ribotypes distinguished by 10 base differences. Examination of ITS chromatograms for putative hybrids revealed polymorphisms at those sites that are diagnostic between species in all except one putative hybrid. Morphological intermediacy and ITS additivity therefore support the hybrid status of C. floridus var. virgatus . An analysis of intraindividual ITS variation confirmed the co-occurrence of both parental ribotypes in putative hybrids and demonstrated that some hybrid individuals contained chimaeric ITS types. It is proposed that chimaeric ITS types are the result of recombination following backcrossing. Sequencing of the trn H- psb A region revealed four haplotypes. Three were, for the most part, confined to C. floridus and putative hybrids although one C. scoparius accession was also found to share this haplotype. The remaining haploype was only found in C. scoparius and putative hybrid accessions. Patterns of haplotype distribution between parental and hybrid accessions suggest multiple, bidirectional hybridization events between C. floridus and C. scoparius . The nomenclature of the hybrid is discussed and the necessary new name C.  ×  despreauxii is proposed.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 154 , 187–204.     

Genetic diversity of introduced Manila clam Ruditapes philippinarum populations inferred by 16S rDNA

Manila clam Ruditapes philippinarum – synonym Venerupis philippinarum (Adams and Reeve, 1850) – is one of the most successful marine invaders, being introduced worldwide for aquaculture and fisheries. Genetic diversity and structure of its populations are largely unknown, especially in the invaded environments. Herein we present molecular genetic data on 12 introduced Manila clam populations in Italy, Spain, and Portugal from both Mediterranean (Adriatic) and Atlantic sampling sites. The phylogenetic information was investigated by the direct sequencing of 16S mitochondrial DNA. Results of mtDNA analyses showed the occurrence of 11 haplotypes for European introduced populations, as a consequence of multiple founder effects from different source populations. Rp hap1 was the most frequent, shared among all populations. The other 10 haplotypes were rare and distributed at local scale, in agreement with what was observed in other invasive bivalves. Biogeographic and phylogenetic analyses based on 16S rDNA of introduced Manila clam populations showed a complex scenario, dominated by multiple pools of individuals coming from different sources, as a consequence of multiple introductions.     

Molecular cloning and characterization of SOCS-2 from Manila clam Ruditapes philippinarum

Suppressor of cytokine signaling (SOCS) family members are key regulators of immunological homeostasis. In this study, we have discovered the SOCS-2 member from Manila clam Ruditapes philippinarum and further analyzed its immune responses against lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly I:C). Amino acid sequence of RpSOCS-2 consists of cytokine inducible SRC homology 2 (SH2) and SOCS box domains similar to vertebrate SOCS counterparts. It has the highest amino acid identity (41%) with Pacific oyster (Crassostrea gigas) SOCS-2 and showed close evolutional relationship with disk abalone (Haliotis discus discus) SOCS-2. Tissue specific expression results showed that RpSOCS-2 was constitutively expressed in all examined tissues with the highest level in gill tissue of un-challenged clams. RpSOCS-2 mRNA expression was up-regulated by LPS and poly I:C challenge in gills. Discovery of RpSOCS-2 homologue and expression analysis would support for understanding evolutional relationships and their role in innate immune responses in mollusks, respectively.     

Effects of shell morphological traits on the weight traits of Manila clam (Ruditapes philippinarum)

The shell length, height, and width, live body weight, and edible tissue weight of Manila clam of 1, 2, and 3 years of age were measured, and their correlation coefficients were calculated. The shell morphological traits were used as independent variables, and live body weight or edible tissue weigh used as a dependent variable for calculating the path coefficients, correlation index and determination coefficients. The results showed that the correlation coefficients between each shell morphological trait and the live body weight or edible tissue weight were all highly significant (P < 0. 01). The shell height at 1-year old clams was highly correlated with the live body weight and edible tissue weight. The shell width of 2- to 3-year-old clams was strongly associated with the live body weight, while the shell length was closely linked to the edible tissue weight. The results of coefficients of determination for the morphological traits against weight traits agreed well with the results of path analysis. The correlation indices for all morphological traits against weight traits were approximately the same as determination coefficients regardless of clam age. The correlation indices (R²) of morphological traits against the live body weight of clams of all ages and edible tissue weight of 1-year-old clams were larger than 0.85, but R² of morphological traits against the edible tissue weight of 2- and 3-year-old clams was smaller than 0.85, indicating that some other factors might be associated with the edible tissue weight of 2- and 3-year-old clams. Multiple regression equations were obtained to estimate shell length X₁ (cm), shell height X₂ (cm), shell width X₃ (cm) against live body weight Y (g), edible tissue weight Z (g): for 1-year-old clams: Y = ?4.317 + 0.18X₁ + 0.147X₂, (X₁ < 0.01, X₂ < 0.01), Z = ?1.011 + 0.095X₂, (X₂ < 0.01); for 2-year-old clams: Y = ?15.119 + 0.249X₁ + 0.176X₂ + 0.688X₃, (X₁ < 0.01, X₃ < 0.01), Z = ?4.248 + 0.198X₁, (X₁ < 0.05, X₃ < 0.01); and for 3-year-old clams: Y = ?25.013 + 0.415X₁ + 1.184X₃, (X₁ < 0.01, X₃ < 0.01), Z = ?7.082 + 0.119X₁ + 0.332X₃, (X₁ < 0.05, X₃ < 0.01).     

Genetic evidence for hybridization between the native Spartina maritima and the introduced Spartina alterniflora (Poaceae) in South-West France: Spartina × neyrautii re-examined

 Spartina alterniflora, a perennial grass native to the North American Atlantic coast, was introduced during the 19th century in western Europe (Southern England and western France) where it hybridized with the native Spartina maritima. In England, the sterile hybrid S. × townsendii gave rise by chromosome doubling to the highly fertile allopolyploid Spartina anglica, which has now invaded many salt marshes and estuaries in western Europe, and has been introduced in several continents. In South-West France, another sterile hybrid was discovered in 1892 in the Bidassoa Estuary, and named Spartina × neyrautii. According to their morphology, some authors suggested that S. × neyrautii and S. × townsendii result from reciprocal crosses. During the 20th century, the hybridization site was severely disturbed, and surviving of S. × neyrautii was questioned. In this paper, various Spartina populations are investigated in the Basque region (France and Spain), and compared to the hybrid taxa formed in England (S. × townsendii and S. anglica). The samples were analyzed using molecular fingerprinting (RAPD and ISSR) and Chloroplast DNA sequence (trnL-trnT spacer, trnL intron and trnL-trnF spacer). In the Bidassoa estuary, a hybrid isolated clone has been found, that displays additive species-specific nuclear markers of S. maritima and S. alterniflora, and that is subsequently considered as a surviving clone of S. × neyrautii. The molecular analyses indicate that S. × neyrautii and S. × townsendii share the same maternal (S. alterniflora), and paternal (S. maritima) parental species, but also that the two independent hybridization events have involved different parental (nuclear) genotypes in England and in South-West France. Received July 12, 2002; accepted October 4, 2002 Published online: March 20, 2003     

Comparison of competitive ability between native and introduced salmonids: evidence from pairwise contests

Brown trout, Salmo trutta, and rainbow trout, Oncorhynchus mykiss, have been introduced to freshwaters in Hokkaido, Japan. Today, it is recognized that these introduced salmonids have negative impacts on native salmonids such as white-spotted charr, Salvelinus leucomaenis, and masu salmon, O. masou. In particular, interspecific competition may be an important mechanism that could contribute to the exclusion for native salmonids. In this study, experimental pairwise contests were conducted to compare interference competitive ability between native and introduced salmonids. We demonstrated that brown trout were competitively superior to white-spotted charr and masu salmon whereas rainbow trout were superior to white-spotted charr. We suggest that introduced brown trout negatively impact both white-spotted charr and masu salmon, and introduced rainbow trout negatively impact white-spotted charr.     

Vibrio tapetis-like strain isolated from introduced Manila clams Ruditapes philippinarum showing symptoms of brown ring disease in Norway

The Manila clam Ruditapes philippinarum was introduced to Norway in 1987 and was produced in 2 hatcheries until 1991. Clam seed was planted at 6 sites. Two sites were on the Island of Tysnes, south of Bergen. Surviving adult Manila clams were recovered in 1995 and 1996. In the present study, Manila clams from the original seeding that displayed morphological signs of brown ring disease (BRD) were recovered in June 2003 (n=7) and in June 2004 (n=17). Samples from extrapallial fluid, tissues and haemolymph were inoculated on marine agar. Replicate subcultures on selective media were used to select potential Vibrio tapetis strains, and in total, 190 bacterial strains were isolated. One of these strains clustered within the V tapetis clade and was named NRP 45. DNA:DNA hybridisation with the type strain CECT4600 showed 52.7 and 57.3% DNA:DNA similarity. Hybridisation of NRP 45 and the V tapetis LP2 strain, isolated from corkwing wrasse Symphodus melops, produced 46.6 and 44.4% re-association. Partial gene segments encoding 16S rRNA, gyrase B protein (GyrB) and chaperonin 60 protein (Cpn60) were characterised and compared to CECT 4600. NRP 45 showed 5 differences in the 1416 nucleotides (nt) of the 16S rRNA encoding gene (99.6% similarity), while the GyrB encoding gene had 62 substitutions of 1181 nt compared (94.8% similarity) and the Cpn60 encoding gene had 22 substitutions out of 548 nt compared (96% similarity). This is the first finding of BRD and the first isolation of a V. tapetis-like bacterial strain from a bivalve in Norway.     

Molecular evidence for natural intergeneric hybridization between Liquidambar and Altingia

Since its establishment, a hybrid origin for Semiliquidambar has been proposed based on morphological intermediacy and sympatric distribution with Altingia and Liquidambar. This hypothesis, however, has lacked convincing molecular evidence. In this study, two nuclear genes, pin2 and cab4, and a chloroplast gene, matK, from Semiliquidambar cathayensis and its putative parental species Liquidambar and Altingia in Jianfengling, Hainan, and Heishiding and Nanling, Guangdong, China, were sequenced to test this hypothesis. Our results showed that L. formosana and L. acalycina were closely related and constituted an inseparable clade in the phylogenetic trees of both pin2 and cab4 genes. Phylogenetic analyses revealed two types of sequences for S. cathayensis, which were clustered with its putative parents, L. formosana–L. acalycina and A. obovata in Jianfengling, and with L. formosana–L. acalycina and A. chinensis in Heishiding and Nanling. The partial chloroplast matK gene sequences showed four nucleotide substitutions between L. formosana and A. obovata in Jianfengling; the sequences of the two individuals of S. cathayensis were identical with those of A. obovata. No diagnostic chloroplast markers including matK and three other chloroplast genes were found to distinguish L. formosana and A. chinensis in Heishiding and Nanling. Molecular data clearly demonstrated that S. cathayensis is of intergeneric hybrid origin between L. formosana–L. acalycina and A. obovata or A. chinensis and that A. obovata functions as the maternal parent in the hybridization event in Jianfengling, Hainan.     

Molecular data provide strong evidence of natural hybridization between native and introduced lineages of <Emphasis Type="Italic">Phragmites australis</Emphasis> in North America

Hybridization, both within and between taxa, can be an important evolutionary stimulus for bioinvasions. Novel intra-taxon hybridizations may arise either between formerly allopatric introduced lineages, or between native and introduced lineages. The latter can occur following a cryptic invasion of a non-native lineage, such as the nineteenth century introduction to North America of a European lineage of the common reed Phragmites australis. Previous studies found no evidence of natural hybridization between native and introduced lineages of P. australis, but produced some F₁ hybrids under experimental conditions when the seed parent was native and the pollen parent was introduced. In this study we used microsatellite data to compare genotypes of P. australis along a transect of approximately 2,000 km in eastern North America. Although hybridization appears uncommon, simulations and principle component analysis of genetic data provided strong evidence for natural hybridization at two sites adjacent to Lake Erie in which native and introduced lineages were sympatric. The seed parent was the native lineage in some cases, and the introduced lineage in other cases. There is now the potential for P. australis hybrids to become increasingly invasive, and managers should consider as a priority the removal of introduced stands from sites where they co-exist with native stands.     

Molecular and morphological evidence of hybridization between newts Triturus vulgaris and T. montandoni (Caudata: Salamandridae) in Slovakia

Natural hybridization and introgression is recorded between the newt species Triturus vulgaris and T. montandoni in Slovakia. To confirm a hybrid status of two putative hybrids, morphological and Randomly Amplified Polymorphic DNA (RAPD) markers specific for T. vulgaris and T. montandoni were used. The individuals found in Zbojská (Veporské vrchy Mts, Slovakia; analyzed morphologically and genetically) and Zubrohlava (Oravská kotlina basin, Slovakia; analyzed only morphologically) possessed markers of both species. Segregation of RAPD markers together with a model-based Bayesian analysis revealed that the specimen from Zbojská belonged to later generation hybrids.     

Molecular and morphological evidence of natural interspecific hybridization between the uncommon Eucalyptus aggregata and the widespread E. rubida and E. viminalis

Human activities can promote increased hybridization in the genus Eucalyptus with potentially detrimental consequences for the persistence of rare species. However, many hybrid combinations have not been investigated with combined use of genetic markers and morphology. We assessed the efficiency of the STRUCTURE program and morphological intermediacy for identifying hybrids between the uncommon tree, Eucalyptus aggregata, which putatively hybridizes with the common congeners, E. rubida and E. viminalis in south-eastern Australia. We sampled 1,005 seedlings across 27 populations, all seedlings were genotyped at 6 allozyme loci and scored for 22 stem and leaf characters. Both marker sets confirmed that E. aggregata is hybridizing with both E. rubida and E. viminalis. Allozymes revealed hybrids from E. aggregata trees in 88% of populations and hybrids comprised 7.3% of all seedlings. Both genetics and morphology indicated that ~50% were likely to be F₁ hybrids, and both simulations and morphological characteristics indicated that the remainder were mostly backcrosses. Morphological analysis correctly distinguished 71% of F₁ hybrids from parentals and was least accurate when dealing with potential backcrosses (50% success). Hence, techniques using genetic data (no prior information) and the assessment of appropriate admixture thresholds through simulations provided the most accurate estimates of hybrid frequency. In this study, potential introgression and the high frequency of hybrids in small populations (~30%), suggests that hybridization should be considered in the management and conservation of E. aggregata.     

Relationships between native small mammals and native and introduced large herbivores

Australia has a range of native and introduced large herbivores that could affect the abundance of small mammals through direct and indirect effects. Here we study the relationship between occurrence of the introduced rusa deer (Rusa timorensis) and the native swamp wallaby (Wallabia bicolor), and the abundance of four species of native small mammals in coastal heath vegetation with varying fire history. The abundance of two species, the brown antechinus (Antechinus stuartii) and bush rat (Rattus fuscipes), was related to occurrence of large herbivores and was dependent also on fire history. Abundance of swamp rats (R. lutreolus) and New Holland mice (Pseudomys novaehollandiae) was not related to the occurrence of any of the large herbivores, and did not depend on fire history. At sites burned within the last 9 years, captures of brown antechinus were negatively related to both deer and wallaby occurrence, and captures of bush rats were negatively related to deer occurrence. However, at sites that burned more than 15 years ago, captures of brown antechinus and bush rats were not related to large herbivore occurrence. Overall there was either no relationship, or a negative one, between small mammals and the large herbivores. This mensurative study has demonstrated relationships between deer and wallabies and small mammals, with fire as an additional important factor. From the results of the current study we put forward a series of hypotheses that need to be tested by future experiments.     

菲律宾蛤仔的精子发生和精子超微结构

用透射电镜研究了菲律宾蛤仔(Ruditapes philippinarum)精子结构和精子发生过程中细胞形态结构的变化及细胞器的演变规律。菲律宾蛤仔雄性生殖细胞的形态由椭圆形渐变为辣椒状,细胞核的形态由椭圆形逐渐拉长,渐变为锥形。染色质的凝集经历：小颗粒团块状一较大颗粒均匀状一粗颗粒均匀状的过程。线粒体在演化过程中数量先增多后逐渐减少,嵴数逐渐增多,电子密度和体积逐渐增大。高尔基体在初级精母细胞期已经发育,随后的各期中发育良好,分泌旺盛。精细胞Ⅱ期,高尔基体分泌的潴泡开始融合,形成前顶体囊。精细胞Ⅲ期,高尔基体的分泌物仍不断融合。精细胞分化的后期,前顶体囊逐渐发育形成顶体。菲律宾蛤仔成熟精子呈长辣椒状,为原生型,由头部、中段和尾部构成。头部的顶体为细长柱形,末端渐细,电子密度较小;细胞核为锥形;中段线粒体4个,尾部鞭毛为典型的“9 2”型结构。此外在成熟精子线粒体环横切面有一特殊“风车状”结构。     

庄河海域菲律宾蛤仔底播增殖区自身污染

采用生物沉积物捕集器和封闭式代谢瓶,周年现场研究了庄河海域菲律宾蛤仔的生物沉积速率、排氨率和排磷率.结果表明:菲律宾蛤仔的生物沉积速率、排氨率和排磷率均具有明显的季节变化.生物沉积速率为0.15～1.47 g·ind-1·d-1(年均0.61 g·ind-1·d-1);其排氨率及排磷率分别为0.02～0.40 mg·ind-1·d-1(年均0.17 mg·ind-1·d-1)和0.01～0.39 mg·ind-1·d-1(年均0.13 mg·ind-1·d-1).根据以上结果,估算庄河海域底播增殖菲律宾蛤仔每年产生的生物沉积物达到5.46×107t(干质量),折合有机物9.07×106t、有机碳1.00x106t和有机氮1.18xlO5t;而氨氮和磷酸盐分别为1.49x104t和1.15x104t.表明浅海高密度、规模化菲律宾蛤仔增养殖区自身污染严重,其对环境的影响不可忽视.     

Molecular and morphological evidence reveals hybridisation between two endemic cyprinid fishes

The geographic occurrence of moggel Labeo umbratus × mud fish Labeo capensis hybrids and the threat of hybridisation to the genetic integrity of L. umbratus were investigated. Genetic evidence from mtDNA cytb and nDNA s7 intron sequence data indicated probable interspecific hybridisation in two impoundments, Hardap Dam (Orange River Basin, Namibia) and Darlington Dam (Sundays River Basin, Eastern Cape, South Africa). Some putative hybrids were morphologically identifiable on account of their meristic and morphometric intermediacy to the parent species. Human activities appear to drive the observed hybridisation, either through dam construction and direct stocking of L. umbratus or via translocation of Labeo capensis and the previously isolated Orange River lineage of L. umbratus into the southern-flowing systems by inter-basin water transfers. It is recommended that further translocation of fish from these areas should be avoided.     

Purification and characterization of phenoloxidase from clam Ruditapes philippinarum

Using L-dihydroxyphenylalanine (L-DOPA) as a specific substrate, phenoloxidase (PO) from clam (Ruditapes philippinarum) was purified by Q Sepharose Fast Flow ion-exchange chromatography and Sephacryl S-100 gel-filtration, and characterized biochemically and enzymatically in this study. The molecular mass of PO in SDS-PAGE is about 76.9 kDa, and the prophenoloxidase (proPO) molecule, isolated as a monomeric protein, is 84.1 kDa. The PO molecule had a high oxidative activity, and the proPO molecule had almost no oxidative activity. The PO activity was optimal at pH 7.0 and temperature of 40 degrees C. The Km value of the PO for L-DOPA was 2.2 mmol l(-1). The PO was extremely sensitive to benzoic acid and sodium sulfite, very sensitive to citric acid, thio urea, 1-phenyl-2-thiourea and cysteine, but not sensitive to ascorbic acid. Combined with its specific enzyme activity on tyrosine and L-DOPA, it can be concluded that the Ruditapes PO is probably a kind of tyrosinase-type phenoloxidase. The PO activity was strongly inhibited by ethylenediaminetetraacetic acid (EDTA), diethyldithiocarbamate (DETC), Zn2+, Ca2+ and Cu2+, as well as by Mg2+. The results with EDTA, DETC, and some metal ions, combined with the perfect recovery effect of Cu2+ on DETC-inhibited PO activity, indicate that Ruditapes PO is most probably a copper-containing metalloenzyme.     

Community structure of insect herbivores on introduced and native Solidago plants in Japan

We compared community composition, density, and species richness of herbivorous insects on the introduced plant Solidago altissima L. (Asteraceae) and the related native species Solidago virgaurea L. in Japan. We found large differences in community composition on the two Solidago species. Five hemipteran sap feeders were found only on S. altissima. Two of them, the aphid Uroleucon nigrotuberculatum Olive (Hemiptera: Aphididae) and the scale insect Parasaissetia nigra Nietner (Hemiptera: Coccidae), were exotic species, accounting for 62% of the total individuals on S. altissima. These exotic sap feeders mostly determined the difference of community composition on the two plant species. In contrast, the herbivore community on S. virgaurea consisted predominately of five native insects: two lepidopteran leaf chewers and three dipteran leaf miners. Overall species richness did not differ between the plants because the increased species richness of sap feeders was offset by the decreased richness of leaf chewers and leaf miners on S. altissima. The overall density of herbivorous insects was higher on S. altissima than on S. virgaurea, because of the high density of the two exotic sap feeding species on S. altissima. We discuss the importance of analyzing community composition in terms of feeding guilds of insect herbivores for understanding how communities of insect herbivores are organized on introduced plants in novel habitats.     

Development of microsatellite markers for the Manila clam Ruditapes philippinarum

We isolated nine polymorphic microsatellites from the Manila clam Ruditapes philippinarum. These loci provide one class of highly variable genetic marker as the number of alleles ranged from six to 22, and the observed and expected heterozygosity ranged from 0.136 to 0.909 and from 0.553 to 0.954, respectively. We consider that these loci are potentially useful for detailing the genetic structure and gene flow among R. philippinarum populations.