Variation in self-fertility,inbreeding depression and levels of inbreeding in four Cyclamen species

The ability to self in the absence of pollinators, i.e. reproductive assurance, and the detrimental consequences of inbreeding, i.e. inbreeding depression, are central factors influencing plant mating system evolution. The purpose of this study was to quantify whether self-fertility and inbreeding depression are related to levels of inbreeding in four Cyclamen species, namely C. balearicum (mean F_is = 0.930), C. creticum (mean F_is = 0.748), C. repandum (mean F_is = 0.658) and C. hederifolium (mean F_is = 0.329). C. balearicum showed a markedly greater capacity to autonomously self-fertilize than the three other species, which may have favoured inbreeding in this species. Levels of inbreeding depression were highest in C. creticum and C. hederifolium at the fruit maturation (δ = 0.18 and 0.20, respectively) and seed number (δ = 0.32 and 0.30, respectively) stages, and for C. repandum at the seed weight stage (δ = 0.23). Although C. balearicum showed inbreeding depression on seed germination (δ = 0.45), this may be an artefact of the generally low levels of seed germination in the experiment. Overall, we observed only limited evidence for the predicted negative relation between inbreeding coefficients and levels of inbreeding since C. creticum had high levels of inbreeding and inbreeding depression. Other factors may thus influence the relationship between inbreeding and inbreeding depression in these species.     

Floral trait variation in fourCyclamen (Primulaceae) species

The purpose of this study is to examine floral trait variation between fourCyclamen species that show variation in their ability to reproduce in the absence of pollinators and in levels of inbreeding. Pollen and ovule production, pollen/ovule ratio, pollen volume, petal length and width, diameter of the corolla mouth, pistil and stamen length, and stigma-anther separation varied significantly between the four study species. Flower, pollen and ovule production, pollen volume and corolla size were generally highest inC. hederifolium, the species with the lowest level of inbreeding (mean F_is = 0.329), intermediate in both species with relatively high levels of pollinator-mediated inbreeding,C. repandum (mean F_is = 0.658) andC. creticum (mean F_is = 0.748), and lowest in the highly inbredC. balearicum (mean F_is = 0.930). The two species with the most different inbreeding coefficients,C. hederifolium andC. balearicum, had lower pollen-ovule ratios and shorter longevities of stigma receptivity and pollen viability thanC. creticum andC. repandum. These patterns of variation in floral traits are discussed in relation to the relatedness, pollination ecology and levels of inbreeding of the four species.     

Genetic variation in Mediterranean Helichrysum italicum (Asteraceae; Gnaphalieae): do disjunct populations of subsp. microphyllum have a common origin?

The yellow‐flowered everlasting daisy Helichrysum italicum (Asteraceae, Gnaphalieae) is widely distributed in the Mediterranean basin, where it grows in continuous and widespread populations in diverse open habitats. Helichrysum italicum subsp. microphyllum has a disjunct distribution in the Balearic Islands (Majorca and Dragonera), Corsica, Sardinia, Crete and Cyprus. Numerous morphological intermediates between subsp. italicum and subsp. microphyllum are known from Corsica, where the two subspecies co‐occur. The aims of the study were to investigate if subsp. microphyllum has a common origin, constituting an independent gene pool from subsp. italicum, or if the morphological differences between subsp. microphyllum and subsp. italicum have arisen independently in different locations from a common wider gene pool. Our analyses of AFLP, cpDNA sequences and morphological characters show that there is geographic structure to the genetic variation within H. italicum, with eastern and western Mediterranean groups, which do not correspond with the division into subsp. microphyllum and subsp. italicum as currently circumscribed. Local selection on quantitative trait loci provides sufficient explanation for the morphological divergence observed and is consistent with genetic data. Within the western Mediterranean group of the species we found considerable polymorphism in chloroplast DNA sequences among and within some populations. Comparison with chloroplast DNA sequences from other Helichrysum species showed that some chloroplast haplotypes are shared across species.     

The xeric side of the Brazilian Atlantic Forest: The forces shaping phylogeographic structure of cacti

In order to investigate biogeographic influences on xeric biota in the Brazilian Atlantic Forest (BAF), a biodiversity hotspot, we used a monophyletic group including three cactus taxa as a model to perform a phylogeographic study: Cereus fernambucensis subsp. fernambucensis, C. fernambucensis subsp. sericifer, and C. insularis. These cacti are allopatric and grow in xeric habitats along BAF, including isolated granite and gneiss rock outcrops (Inselbergs), sand dune vegetation (Restinga forest), and the rocky shore of an oceanic archipelago (islands of Fernando de Noronha). The nucleotide information from nuclear gene phytochrome C and plastid intergenic spacer trnS‐trnG was used to perform different approaches and statistical analyses, comprising population structure, demographic changes, phylogenetic relationships, and biogeographic reconstruction in both spatial and temporal scales. We recovered four allopatric population groups with highly supported branches in the phylogenetic tree with divergence initiated in the middle Pleistocene: southern distribution of C. fernambucensis subsp. fernambucensis, northern distribution of C. fernambucensis subsp. fernambucensis together with C. insularis, southern distribution of C. fernambucensis subsp. sericifer, and northern distribution of C. fernambucensis subsp. sericifer. Further, the results suggest that genetic diversity of population groups was strongly shaped by an initial colonization event from south to north followed by fragmentation. The phylogenetic pattern found for C. insularis is plausible with peripatric speciation in the archipelago of Fernando de Noronha. To explain the phylogeographic patterns, the putative effects of both climatic and sea level changes as well as neotectonic activity during the Pleistocene are discussed.     

Cytogenetics of new Guizotia Cass. (Compositae) interspecific hybrids pertaining to genomic and phylogenetic affinities

 The following crosses were made using four recognized species/subspecies and a new population of Guizotia, referred to as Chelelu after the name of the locality in Ethiopia from which it was collected: G. scabra subsp. schimperi × Chelelu, Chelelu × G. scabra subsp. scabra, G. zavattarii × G. arborescens and Chelelu × G. zavattarii (all accessions with 2n=30). Plant morphology as well as mitotic and meiotic chromosome analysis confirmed the hybrid nature of the obtained progeny. At metaphase I of meiosis, the F₁ hybrid plants (2n=30) showed a mean of about 95%, 31%, 63% and 0.50% of the pollen mother cells with 15 bivalents, and a mean of about 14.95, 13.75, 14.40 and 7.86 bivalents per cell, respectively. The respective mean pollen stainability was about 67%, 19%, 31% and 2%. From the results it was concluded that Chelelu is more closely related to G. scabra subsp. schimperi than to G. scabra subsp. scabra but more to the latter than to G. zavattarii. Guizotia zavattarii and G. arborescens are closely related to each other. Based on the cytological observations made, the probable basic chromosome number for the genus is discussed. Received November 5, 1999 Accepted August 21, 2001     

Molecular phylogenetics of Hippophae L. (Elaeagnaceae) based on the internal transcribed spacer (ITS) sequences of nrDNA

 The genus Hippophae comprises 7 species and 8 subspecies according to the latest classification, and has shown enormous ecological, nutrient and medicinal values. Here we analyzed the phylogenetic relationships among 15 taxa of the genus by comparing sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA). ITS sequences in Hippophae varied in length from 651 bp to 666 bp. The aligned sequences were 690 bp in length and 269 (39.0%) were variable sites with 150 being parsimony-informative. The amount of polymorphism observed within a taxon was extremely low in most taxa except for two putative hybrid species. The aligned sequences were analyzed by maximum parsimony (MP) and neighbor-joining (NJ) methods. In the strict consensus trees of parsimony analysis, the monophyly of Hippophae was supported by 100% bootstrap value. H. tibetana was at the basal position of the genus, and the remaining taxa formed two clades with high bootstrap support. The first clade included subspecies of H.␣rhamnoides and the other one consisted of remaining species. Parsimony analysis also suggested that the species H. tibetana, H. neurocarpa and H.␣salicifolia were all distinct. Although the sequence divergence among subspecies of H. rhamnoides was also remarkably high, the molecular data supported the monophyly of H. rhamnoides when H. rhamnoides subsp. gyantsensis Rousi was excxluded. The NJ trees showed essentially the same topology. The taxonomical arrangement that divided the genus into two sections was not supported based on the ITS sequences. However, the hybrid origin of H. goniocarpa and H. litangensis proposed previously was supported by the present ITS data. Received January 7, 2002; accepted May 10, 2002 Published online: November 22, 2002 Addresses of the authors: Kun Sun, Xuelin Chen, Ruijun Ma, Qin Wang, Institute of Botany, Northwest Normal University, Lanzhou 730070, China. Changbao Li, Song Ge (e-mail: gesong@ns.ibcas.ac.cn or song_ge@hotmail.com), Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China.     

A bryophyte flora of Handa Island Sutherland

ABSTRACT

Background: Hybridization is a key driver of plant speciation, and understanding ongoing hybridization provides insights into the underlying process. We studied a hybrid complex in Mediterranean Cyclamen between common C. repandum and rare C. balearicum on Sardinia.

Aims: We analyzed variation in the frequency and morphology of different floral types to assess the degree of hybridization and analyze the ecological niche to better understand factors that influence the maintenance of hybrid populations.

Methods: We recorded the frequency of the different flower color-types in 45 populations and measured floral traits in 10 of them. We characterized the spatial extent and climatic niche of this hybrid complex. We analyzed floral type variation in four traits (flower size, style and anther length, herkogamy).

Results: Hybrid populations occupy a wide area on the NW coast of the island (~1000km2). Sites dominated by hybrid floral types are ecologically marginal for the common parent. The C. repandum-like type is present in all populations and shows a decrease in style and anther length and herkogamy in populations containing a greater proportion of hybrids.

Conclusions: This hybrid complex provides an ideal situation to examine reproductive isolation due to variation in the ecological niche of different floral types.     

Hares in Corsica: high prevalence of <Emphasis Type="Italic">Lepus corsicanus</Emphasis> and hybridization with introduced <Emphasis Type="Italic">L. europaeus</Emphasis> and <Emphasis Type="Italic">L. granatensis</Emphasis>

The Italian hare, Lepus corsicanus, was first described in Corsica more than 100 years ago, but the knowledge on the status of the species in this island remains scarce. Moreover, frequent introductions of thousands of individuals from other hare species, namely Lepus europaeus and Lepus granatensis, into Corsica are known to have occurred and an updated assessment of the prevalence of L. corsicanus in Corsica is therefore of utmost importance. Here, to estimate the relative prevalence of the hare species present in Corsica, we conducted a molecular analysis on 67 samples collected by hunters between 2002 and 2007 in 36 Corsican communes. Sequencing of portions of the nuclear gene transferrin and of the control region of the mitochondrial DNA allowed classifying most of the collected samples as belonging to L. corsicanus (70.1%). Of the sampled Corsican communes, 86.1% contained this species, while only in 11.1%, L. europaeus was present. Three of the analyzed specimens showed an inconsistent molecular assignment between markers suggesting a hybrid origin: L. corsicanus × L. europaeus, L. corsicanus × L. granatensis, and L. europaeus × L. granatensis. The first two cases of hybridization had never been described in nature, even in studies focusing on hares from Italy where L. corsicanus and L. europaeus are often sympatric. These results stress the real risk of corrosion of the native gene pool of L. corsicanus via hybridization with introduced species. We highlight the need of urgently rethinking the management plan of hare populations in Corsica.     

Interspecific molecular variation of Tunisian complex Lolium perenne L. and Festuca arundinacea Schreb. based on the internal transcribed spacer locus (ITS)

To establish phylogenetic relationships and estimate the intra and interspecific divergence, the amplification and the sequencing of the internal transcribed spacers of ribosomal DNA (ITS = ITS1 + 5.8S + ITS2) were analyzed in Tunisian complex Lolium–Festuca DNA. These spacer regions have evolved mainly by point mutations. Results revealed a high level of polymorphism within studied species. Significant similarities were observed between these two species and showed the existence of an important phylogenetic relationship. Besides, this molecular approach has revealed two new clusterings, with a homologous ITS gene namely: Bromus hordeaceus and Hordeum murinum subsp. This could be explained by the conservation of an ancestral ITS gene in some fescue plants. Thus, Tunisian tall fescue and perennial ryegrass may derive from Bromus hordeaceus and Hordeum murinum subsp. Considerable morphology and bioclimatic distribution similarities were discovered in ITS sequences within the same species. This study can be of great help to identify suitable accessions that could be used in local fescue and ryegrass improvement program.     

Distinct protoconchs recognised in three of the larger Mediterranean Cerithium species (Caenogastropoda: Cerithiidae)

The gastropod genus Cerithium includes several polymorphic species which are hardly distinguishable using a morphological approach based on teleoconch characters. Here we show that protoconch characters can be reliably used to identify the larger Mediterranean species (Cerithium alucastrum, C. repandum and C. vulgatum), and to assess their intraspecific variability. Based on a large amount of morphological data, we show that a multispiral, strongly sculptured protoconch (traditionally associated with C. vulgatum) is found in C. alucastrum. This species originated in the Pliocene. A multispiral, weakly sculptured protoconch, not observed previously, is reported for C. vulgatum. A paucispiral protoconch, distinct from those observed previously for C. lividulum and C. renovatum, is reported for C. repandum and can be used for recognising its wide intraspecific variation and understanding its geographical distribution. Cerithium repandum, previously doubtfully recorded outside its type locality, extends back to the mid-Pliocene and is today rather widely distributed in the Mediterranean, although absent from the Aegean Sea and the Alboran Sea. We describe the larval development of C. repandum and C. vulgatum for the first time, confirming their respective non-planktotrophic and planktotrophic development previously inferred from protoconch morphology. Also, we report a possible case of morphological convergence between C. repandum and C. vulgatum occurring in harbour sites, where we found a distinct (possibly Anthropocene) shell form not described previously from other Recent or fossil material.     

Mitochondrial and nuclear markers highlight the biodiversity of Kalotermes flavicollis (Fabricius, 1793) (Insecta, Isoptera, Kalotermitidae) in the Mediterranean area

The biodiversity of the European termite Kalotermes flavicollis is here studied through the analysis of mitochondrial (303 bp of control region and 912 bp of COI/tRNA(Leu)/COII) and nuclear (five microsatellite and 20 Inter-SINE loci) markers on 18 colonies collected in Southern France, Corsica, Sardinia, peninsular Italy, the Balkans and Greece. Different statistical analyses (Bayesian phylogenetic analysis,parsimony network, F-statistics, PCA) were performed. Mitochondrial sequences produced an unresolved polytomy including samples from peninsular Italy, Balkans and Greece, and three main clades: southern France, Corsica-Sardinia and Portoscuso(SW Sardinia). Nuclear markers confirm these data, further highlighting a more significant divergence at the regional scale. The results obtained for the peri-Tyrrhenian area agree with major paleogeographic and paleoclimatic events that shaped the biodiversity of the local fauna. K. flavicollis biodiversity and its phylogeographic pattern are also evaluated in the light of the data available for the other native European termite taxon (genus Reticulitermes), in order to produce a more complete scenario of the Mediterranean. In the area comprised between southern France and Italy, the degree of diversity is similar; however, in the eastern area, while K. flavicollis is differentiated only at the population level, the genus Reticulitermes comprises at least six entities of specific and/or subspecific level. This discrepancy may be explained by taking into account the different evolutionary histories of the two taxa.     

High outcrossing and random pollen dispersal in a planted stand of Acacia saligna subsp. saligna revealed by paternity analysis using microsatellites

The mating system, patterns of pollen mediated gene flow and levels of genetic contamination were investigated in a planted stand of Acacia saligna subsp. saligna via paternity analysis using microsatellite markers. High levels of outcrossing were detected within the stand (t _m = 0.98), and the average pollen dispersal distance was 37 m with the majority of progeny sired by paternal trees within a 50-m neighbourhood of the maternal tree. Genetic contamination from the natural background population of A. saligna subsp. lindleyi was detected in 14% of the progeny of A. saligna subsp. saligna and varied among maternal trees. Long distance inter-subspecific pollen dispersal was detected for distances of over 1,500 m. The results provide information for use in the breeding and domestication programme aimed at developing A. saligna as an agroforestry crop for the low rainfall areas of southern Australia.     

Distribution and ecological aspects of leptocephali collected 1979–1994 in North- and Central Atlantic. I. Congridae

This is the first report on the leptocephalus catches made during the last 15 years in North-and Central Atlantic during the course of 19 cruises of five different ships from Germany and one from Poland. This report comprises identification, geographical occurrence and abundance and in some cases depth preference, migratory routes and spawning area of Congridae in the North Atlantic. The largest part of this collection consists of 876Conger conger larvae. The likelihood that this species spawns in the Mediterranean is again confirmed. Decreasing density as well as increasing size from Gibraltar west-and north westward showed migration to be occurring in that direction. Age was determined by counting “daily rings” on the otoliths of up to 120-mm long larvae. It is suggested to be more than 300 days and is calculated to be 1 1/4 years for the largest larvae (TL=140–160 mm). Some 126 specimens of three otherConger species, i.e.C. oceanicus, C. triporiceps, C. esculentus, were identified in the collection. The major part originated from the western North Atlantic. The most numerous larvae, belonging to another genus of Congridae, wereAriosoma balearicum (n=265). They showed, also in consideration of other studies, quite a wide range in number of myomeres. Their systematic status is therefore uncertain, as is also the status of those known from the NW Indian Ocean and the NW Pacific. One larva, probably ofAriosoma selenops, of un-usually large size (TL=467 mm) and captured in the Iberian Basin is described and compared with specimens known from the literature. Two larvae of unknown identity were captured off NW Africa; they resembledA. balearicum but had too high a number of myomeres. The identity of most of theGnathophis larvae caught in the East Atlantic is uncertain. Leptocephali ofParaconger notialis in the East Atlantic, up to the area north of New Guinea, exhibited a higher number of myomeres than those known from the West Atlantic. Larvae ofXenomystax congroides (n-29), belonging to a population with a relatively high number of myomeres, were captured in the Sargasso Sea.     

Chromosome and breeding system evolution of the genus Mercurialis (Euphorbiaceae): implications of ITS molecular phylogeny

 The internal transcribed spacers (ITS1 and ITS2) of nuclear ribosomal DNA were amplified and sequenced from 19 samples representing all species of the genus Mercurialis and two outgroup species, Ricinus communis and Acalypha hispida. The length of ITS1 in the ingroups ranged from 223 to 246 bp and ITS2 from 210 to 218 bp. Sequence divergence between pairs of species ranged from 1.15% to 25.88% among the ingroup species in the combined data of ITS1 and ITS2. Heuristic phylogenetic analyses using Fitch parsimony on the combined data of ITS1 and ITS2 with gaps treated as missing generated 45 equally parsimonious trees. The strict consensus tree was principally concordant with morphological classification. Within the genus, the ITS sequences recognised two main infrageneric clades: the M. perennis complex including three Eurasian stoloniferous species (M.␣leiocarpa, M. ovata and M. perennis) and the western Mediterranean group including eight both annual and perennial species. Of the western Mediterranean clade, the annual and perennial species grouped respectively into two different groups, and the annual life form is revealed as a synapomorphic character derived from perennial, whereas in the Eurasian clade ITS phylogeny suggested M. leiocarpa as basal clade sister to M.␣perennis and M. ovata. ITS phylogeny failed to resolve the relationships among the different cytotypes of M. ovata and M. perennis. ITS phylogeny also suggested rapid karyotypic evolution for the genus. The karyotypic divergence among the perennial species of western Mediterranean region did not corroborate the nucleotide sequence divergence among the species. Optimisation of chromosome numbers onto the ITS phylogeny suggested x=8 to be the ancestral basic chromosome number of the genus. ITS phylogeny confirmed that the androdioecy of M. ambigua is derived from dioecy. The nucleotide heterozygosity and additivity in ITS sequences clearly confirm the interspecific hybridisation in the genus Mercurialis. Received December 22, 2001; accepted May 21, 2002?Published online: November 14, 2002 Address of the authors: Martin Kr?henbühl, Yong-Ming Yuan (correspondence) and Philippe Küpfer, Institut de Botanique, Laboratoire de botanique évolutive, Université de Neuchatel, Emile-Argand 11, CH-2007 Neuchatel, Suisse. (e-mail: yong-ming.yuan@unine.ch)     

Chromosome analysis of five Brazilian species of poison frogs (Anura: Dendrobatidae)

Dendrobatid frogs have undergone an extensive systematic reorganization based on recent molecular findings. The present work describes karyotypes of the Brazilian species Adelphobates castaneoticus, A. quinquevittatus, Ameerega picta, A. galactonotus and Dendrobates tinctorius which were compared to each other and with previously described related species. All karyotypes consisted of 2n = 18 chromosomes, except for A. picta which had 2n = 24. The karyotypes of the Adelphobates and D. tinctorius species were highly similar to each other and to the other 2n = 18 previously studied species, revealing conserved karyotypic characteristics in both genera. In recent phylogenetic studies, all Adelphobates species were grouped in a clade separated from the Dendrobates species. Thus, we hypothesized that their common karyotypic traits may have a distinct origin by chromosome rearrangements and mutations. In A. picta, with 2n = 24, chromosome features of pairs from 1 to 8 are shared with other previously karyotyped species within this genus. Hence, the A. picta data reinforced that the C-banding pattern and the NOR location are species-specific traits in the genus Ameerega. Moreover, the Ameerega monophyletism proposed by previous phylogenetic studies indicates that the karyotypic differences among species in this genus result from a long divergence time.     

Spatial genetic structure in Beta vulgaris subsp. maritima and Beta macrocarpa reveals the effect of contrasting mating system,influence of marine currents,and footprints of postglacial recolonization routes

Understanding the factors that contribute to population genetic divergence across a species' range is a long‐standing goal in evolutionary biology and ecological genetics. We examined the relative importance of historical and ecological features in shaping the present‐day spatial patterns of genetic structure in two related plant species, Beta vulgaris subsp. maritima and Beta macrocarpa. Using nuclear and mitochondrial markers, we surveyed 93 populations from Brittany (France) to Morocco – the southern limit of their species' range distribution. Whereas B. macrocarpa showed a genotypic structure and a high level of genetic differentiation indicative of selfing, the population genetic structure of B. vulgaris subsp. maritima was consistent with an outcrossing mating system. We further showed (1) a strong geographic clustering in coastal B. vulgaris subsp. maritima populations that highlighted the influence of marine currents in shaping different lineages and (2) a peculiar genetic structure of inland B. vulgaris subsp. maritima populations that could indicate the admixture of distinct evolutionary lineages and recent expansions associated with anthropogenic disturbances. Spatial patterns of nuclear diversity and differentiation also supported a stepwise recolonization of Europe from Atlantic‐Mediterranean refugia after the last glacial period, with leading‐edge expansions. However, cytoplasmic diversity was not impacted by postglacial recolonization: stochastic long‐distance seed dispersal mediated by major oceanic currents may mitigate the common patterns of reduced cytoplasmic diversity observed for edge populations. Overall, the patterns we documented here challenge the general view of reduced genetic diversity at the edge of a species' range distribution and provide clues for understanding how life‐history and major geographic features interact to shape the distribution of genetic diversity.     

A rapid PCR-based diagnostic test for the identification of subspecies of <Emphasis Type="Italic">Acacia saligna</Emphasis>

Subspecific taxa of species complexes can display cryptic morphological variation, and individuals and populations can often be difficult to identify with certainty. However, accurate population identification is required for comprehensive conservation and breeding strategies and for studies of invasiveness and gene flow. Using five informative microsatellite markers and a Bayesian statistical approach, we developed an efficient polymerase chain reaction-based diagnostic tool for the rapid identification of individuals and populations of the Acacia saligna species complex of Western Australia. We genotyped 189 individuals from 14 reference populations previously characterised based on morphology and used these data to investigate population structure in the species complex. High total genetic diversity (H _T = 0.729) and high population differentiation (θ = 0.355) indicated strong intraspecific structuring. With the provision of prior population information, the reference data set was optimally resolved into four clusters, each corresponding to one of the four main proposed subspecies, with very high membership values (Q > 97%). The reference data set was then used to assign individuals and test populations to one of the four subspecies. Assignment was unequivocal for all test individuals from two populations of subsp. lindleyi and for all but one individual of subsp. stolonifera. Individuals from populations of subsp. saligna and subsp. pruinescens showed a degree of genetic affinity for the two subspecies in their assignments, although the majority of individuals were correctly assigned to subspecies. The diagnostic tool will assist in characterising populations of A. saligna, especially naturalised and invasive populations of unknown origin.     

Production of γ-aminobutyric acid by <Emphasis Type="Italic">Streptococcus salivarius</Emphasis> subsp. <Emphasis Type="Italic">thermophilus</Emphasis> Y2 under submerged fermentation

Summary. γ-Aminobutyric acid (GABA), a major inhibitory neurotransmitter in the central nervous system, has several well-known physiological functions and has been applied to the production of many drugs and functional foods. The technology of GABA production via submerged fermentation by Streptococcus salivarius subsp. thermophilus Y2 was investigated in this paper. It indicated that the GABA production was related to the biochemical characteristics of glutamate decarboxylase (GAD) of S. salivarius subsp. thermophilus Y2. After 24 h of fermentation at 37 °C, which is the suitable culture conditions for GAD-production, then the culture condition were adjusted to the optimal temperature (40 °C) and pH (4.5) for the GAD reaction activity in biotransformation of cells and pyridoxal 5′-phosphate (0.02 mmol/l) were added to the broth at the 48 h, the GABA production was increased up to 1.76-fold, reaching 7984.75 ± 293.33 mg/l. The strain shows great potential use as a starter for GABA-containing yoghurt, cheese and other functional fermented food productions. Authors’ address: Zhao-Xin Lu, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, P.R. China     

Effect of citrate on growth of Lactococcus lactis subsp. lactis in milk

The effect of citrate on the growth of Lactococcus lactis subsp. lactis var. diacetylactis in milk has been investigated. Five strains of Lactococcus lactis subsp. lactis var. diacetylactis were compared to their citrate-negative variants, which lack the plasmid coding for citrate permease. In most cases, acidification kinetics and the final bacterial concentration of pure cultures of parental and variant strains did not differ significantly. Co-cultures of parental and variant strains, however, systematically tended towards the predominance of parental strains. Citrate metabolism is responsible for this change, since the predominance of citrate-positive strains was not observed in the absence of citrate. Continuous culture in milk enabled the difference in growth rates between the parental strain Lactococcus lactis subsp. lactis var. diacetylactis CDI1 and its citrate-negative variant to be quantified by following changes in the populations of the two co-cultured strains. At 26 °C, the growth rate of the parental strain was 7% higher than that of its citrate-negative variant. These results show that citrate metabolism slightly stimulates the growth of lactococci in milk. Received: 18 February 1997 / Received revision: 2 May 1997 / Accepted: 4 May 1997     

Effect of temperature and various nitrogen sources on L(+)-lactic acid production by Lactobacillus casei

 Two homofermentative strains, Lactobacillus casei NRRL B-441 and Lactobacillus casei subsp. rhamnosus NRRL B-445 were selected for further study from 17 lactic acid bacterial strains screened for lactic acid production. The effect of temperature on lactic acid production with the selected strains was investigated by adapting both strains to four different temperatures. The production of L(+)-lactic acid by both strains was most efficient at 37°C, although with L. casei the highest lactic acid concentration was obtained at 41°C. The maximal volumetric productivity with L. casei was 4.1 g l^-1 h^-1 and with L. casei subsp. rhamnosus 3.5 g l^-1 h^-1. The composition of the medium was studied in order to replace the costly yeast extract with less expensive sources of nitrogen and amino acids. From 11 different nitrogen sources investigated at 37°C, barley malt sprouts (88 g l^-1 lactic acid in 66 h) and grass extract (74 g l^-1 lactic acid in 73 h) were the best economic alternatives. The effect of different combinations of yeast extract, peptone and malt sprouts was further studied by using statistical experimental design, and an empirical second-order polynomial model was constructed on the basis of the results. With the right combination most of the yeast extract could be substituted by barley malt sprouts for efficient lactic acid production. A method for extraction of nutrients and growth factors from malt sprouts is also described. Received: 25 September 1995/Accepted: 24 October 1995