Evolutionary dynamics of Waxy and the origin of hexaploid Spartina species (Poaceae)

We investigated the evolutionary dynamics of duplicated copies of the granule-bound starch synthase I gene (GBSSI or Waxy) within polyploid Spartina species. Molecular cloning, sequencing, and phylogenetic analyses revealed incongruences between the expected species phylogeny and the inferred gene trees. Some genes within species were more divergent than expected from ploidy level alone, suggesting the existence of paralogous sets of Waxy loci in Spartina. Phylogenetic analyses indicate that this paralogy originated from a duplication that occurred prior to the divergence of Spartina from other Chloridoideae. Gene tree topologies revealed three divergent homoeologous sequences in the hexaploid S. alterniflora that are consistent with the proposal of an allopolyploid origin of the hexaploid clade. Waxy sequences differ in insertion–deletion events in introns, which may be used to diagnose gene copies. Both paralogous and homoeologous coding regions appear to evolving under selective constraints.     

Flow cytometry and GISH reveal mixed ploidy populations and Spartina nonaploids with genomes of S. alterniflora and S. maritima origin

Background

The genus Spartina exhibits extensive hybridization and includes classic examples of recent speciation by allopolyploidy. In the UK there are two hexaploid species, S. maritima and S. alterniflora, as well as the homoploid hybrid S. × townsendii (2n = 60) and a derived allododecaploid S. anglica (2n = 120, 122, 124); the latter two are considered to have originated in Hythe, southern England at the end of the 19th century.

Methods

Genomic in situ hybridization (GISH) and flow cytometry were used to characterize the genomic composition and distribution of these species and their ploidy levels at Eling Marchwood and Hythe, both near Southampton, southern England.

Key Results

GISH identified approx. 60 chromosomes each of S. maritima and S. alterniflora origin in S. anglica and 62 chromosomes from S. alterniflora and 30 chromosomes from S. maritima in a nonaploid individual from Eling Marchwood, UK. GISH and flow cytometry also revealed that most (94 %) individuals examined at Hythe were hexaploid (the remaining two individuals (6 %) were dodedcaploid; n = 34), whereas hexaploid (approx. 36 % of plants), nonaploid (approx. 27 %) and dodecaploid (approx. 36 %) individuals were found at Eling Marchwood (n = 22).

Conclusions

Nonaploid individuals indicate the potential for introgression between hexaploid and dodecaploid species, complicating the picture of polyploid-induced speciation within the genus. Despite the aggressive ecological habit of S. anglica, it has not out-competed S. × townsendii at Hythe (homoploid hybrids at a frequency of 94 %, n = 34), despite >100 years of coexistence. The success of GISH opens up the potential for future studies of polyploid-induced genome restructuring in this genus.     

Exploring the genome of the salt-marsh Spartina maritima (Poaceae, Chloridoideae) through BAC end sequence analysis

Spartina species play an important ecological role on salt marshes. Spartina maritima is an Old-World species distributed along the European and North-African Atlantic coasts. This hexaploid species (2n = 6x = 60, 2C = 3,700 Mb) hybridized with different Spartina species introduced from the American coasts, which resulted in the formation of new invasive hybrids and allopolyploids. Thus, S. maritima raises evolutionary and ecological interests. However, genomic information is dramatically lacking in this genus. In an effort to develop genomic resources, we analysed 40,641 high-quality bacterial artificial chromosome-end sequences (BESs), representing 26.7 Mb of the S. maritima genome. BESs were searched for sequence homology against known databases. A fraction of 16.91 % of the BESs represents known repeats including a majority of long terminal repeat (LTR) retrotransposons (13.67 %). Non-LTR retrotransposons represent 0.75 %, DNA transposons 0.99 %, whereas small RNA, simple repeats and low-complexity sequences account for 1.38 % of the analysed BESs. In addition, 4,285 simple sequence repeats were detected. Using the coding sequence database of Sorghum bicolor, 6,809 BESs found homology accounting for 17.1 % of all BESs. Comparative genomics with related genera reveals that the microsynteny is better conserved with S. bicolor compared to other sequenced Poaceae, where 37.6 % of the paired matching BESs are correctly orientated on the chromosomes. We did not observe large macrosyntenic rearrangements using the mapping strategy employed. However, some regions appeared to have experienced rearrangements when comparing Spartina to Sorghum and to Oryza. This work represents the first overview of S. maritima genome regarding the respective coding and repetitive components. The syntenic relationships with other grass genomes examined here help clarifying evolution in Poaceae, S. maritima being a part of the poorly-known Chloridoideae sub-family.     

Development and interspecific transferability of genic microsatellite markers in Spartina spp. with different genome size

Flow cytometry analysis showed variation of nuclear DNA content among different species of Spartina. Spartina alterniflora had the biggest genome (1763.9 Mbp) and S. cynosuroides had the smallest genome (756.35 Mbp), whereas the genomes of S. patens (969.36 Mbp) and S. spartinae (979.78 Mbp) were comparable. Mining simple sequence repeats (SSR) from 1227 expressed sequence tags (EST) generated from salt stressed S. alterniflora showed an abundance of di- and tri-nucleotide repeats. Of 100 ESSR (EST-derived SSR) loci with five or more repeats, 81 loci were successfully amplified in eight S. alterniflora genotypes and 15 (22.2%) ESSR markers were polymorphic. Eleven of the 15 polymorphic ESSRs showed amplification across six different species of Spartina while 100% cross transferability was observed with at least one species of Spartina. The average number of alleles per marker was 3.9 and 5.8 within S. alterniflora and among Spartina species, respectively. The ESSR markers discriminated different members within and between species of Spartina genus.     

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     

Effect of the Spartina alterniflora Root-Rhizome System on Salt Marsh Soil Denitrifying Bacteria

Nitrous oxide (N₂O) reductase activity was used as an index of the denitrification potential in salt marsh soils. In a short Spartina alterniflora marsh, the seasonal distribution of N₂O reductase activity indicated a causal relationship between S. alterniflora root-rhizome production and the denitrification potential of the soil system. The relationship was not discerned in samples from a tall S. alterniflora marsh. To further examine the in situ plant-denitrifier interaction in the short S. alterniflora marsh, plots with and without living S. alterniflora were established and analyzed for N₂O reductase activity 5 and 18 months later. In the plots without living Spartina there was a significant reduction in the soil denitrification potential after 18 months, indicating that in the SS marsh the denitrifiers are tightly coupled to the seasonal production of below-ground Spartina macroorganic matter. In plots with intact Spartina, the soil denitrification potential was not altered by NH₄NO₃ or glucose enrichment. However, in plots without living Spartina, there were significant changes in soil N₂O reductase activity, thus indicating that the plants can serve as a “buffer” against this form of pulse perturbation.     

Ecological control and integral utilization of Spartina alterniflora

Spartina alterniflora Loisel (S. alterniflora) is native to the Atlantic and Gulf Coasts of North America and was introduced to China in 1979. Over the years, S. alterniflora played an important role in protecting coastal banks, accelerating sediment deposition, improving local economies, and alleviating natural disasters. As an exotic species, S. alterniflora has a very strong capability of propagation, which has caused some negative effects, such as occupying ecological niches of some seashells and blocking harbors by fast sediment deposition. As a result, ecological control of S. alterniflora in some areas has drawn wide attention from biologists and ecologists in China and abroad. For this paper, we studied the technology of topography and hydrology modification to substitute S. alterniflora with P. asutralis and reported our results in a project applying this technology. Furthermore, we studied various ways to make use of biomass of S. alterniflora and found that such ways were a good alternative to ecological control of S. alterniflora.     

Spatial and temporal genetic structure in a hybrid cordgrass invasion

Invasive hybrids and their spread dynamics pose unique opportunities to study evolutionary processes. Invasive hybrids of native Spartina foliosa and introduced S. alterniflora have expanded throughout San Francisco Bay intertidal habitats within the past 35 years by deliberate plantation and seeds floating on the tide. Our goals were to assess spatial and temporal scales of genetic structure in Spartina hybrid populations within the context of colonization history. We genotyped adult and seedling Spartina using 17 microsatellite loci and mapped their locations in three populations. All sampled seedlings were hybrids. Bayesian ordination analysis distinguished hybrid populations from parent species, clearly separated the population that originated by plantation from populations that originated naturally by seed and aligned most seedlings within each population. Population genetic structure estimated by analysis of molecular variance was substantial (F_ST=0.21). Temporal genetic structure among age classes varied highly between populations. At one population, the divergence between adults and 2004 seedlings was low (F_ST=0.02) whereas at another population this divergence was high (F_ST=0.26). This latter result was consistent with local recruitment of self-fertilized seed produced by only a few parental plants. We found fine-scale spatial genetic structure at distances less than ∼200 m, further supporting local seed and/or pollen dispersal. We posit a few self-fertile plants dominating local recruitment created substantial spatial genetic structure despite initial long-distance, human dispersal of hybrid Spartina through San Francisco Bay. Fine-scale genetic structure may more strongly develop when local recruits are dominated by the offspring of a few self-fertile plants.     

Comparison of Nitrogen Fixation Activity in Tall and Short Spartina alterniflora Salt Marsh Soils

A comparison of the N₂ fixers in the tall Spartina alterniflora and short S. alterniflora marsh soils was investigated. Zero-order kinetics and first-order kinetics of acetylene reduction were used to describe the activity of the N₂ fixers in marsh soil slurries. It was found that the V_max values were approximately 10 times greater for the N₂ fixers in the tall Spartina than in the short Spartina marsh when raffinose was used as the energy source. In addition, the (K_s + S_n) values were approximately 4 to 15 times lower for the N₂ fixers in the tall Spartina than in short Spartina marsh. First-order kinetics of nitrogen fixation for several substrates indicate that the N₂ fixers in the tall Spartina marsh were two to seven times more active than those in the short Spartina marsh. Ammonium chloride (25 μg/ml) did not inhibit nitrogen fixation in the tall Spartina marsh, but there was a 50% inhibition in nitrogen fixation in the short Spartina marsh. On the other hand, sodium nitrate inhibited nitrogen fixation almost 100% at 25 μg/ml in both soil environments. Amino nitrogen (25 to 100 μg/ml) had little or no effect on nitrogen fixation. The results indicate that the N₂ fixers in the tall Spartina marsh were physiologically more responsive to nutrient addition than those in the short Spartina marsh. This difference in the two populations may be related to the difference in daily tidal influence in the respective areas and thus provide another explanation for the enhanced S. alterniflora production in the creek bank soil system.     

Spartina alterniflora invasions in the Yangtze River estuary,China: An overview of current status and ecosystem effects

The Yangtze River estuary is an important ecoregion. However, Spartina alterniflora, native to North America, was introduced to the estuary in the 1990s through both natural dispersal and humans and now it is a dominant species in the estuarine ecosystems, with its invasions leading to multiple consequences to the estuary. S. alterniflora had great competitive effects on native species, including Scirpus mariqueter and Phragmites australis, and could potentially exclude the natives locally. The presence of S. alterniflora had little influence on the total density of soil nematodes and macrobenthonic invertebrates, but significantly altered the structure of trophic functional groups of nematode and macrobenthonic invertebrate communities. The conversion of mudflats to Spartina meadows had significant effects on birds of Charadriidae and Scolopacidae, which might be attributable to the reduction of food resources and the physical alterations of habitats for shorebirds. S. alterniflora invasions increased the primary productivity of the invaded ecosystems, and altered carbon and nitrogen cycling processes. Our studies focused mainly on the effects of S. alterniflora invasions on the structure of native ecosystems; thus further studies are clearly needed to investigate how ecosystem functioning is affected by the modification of the structure of estuarine ecosystems by S. alterniflora invasions.     

Characterization of microsatellite loci in Spartina species (Poaceae)

The cordgrasses in the genus Spartina have become model organisms for studying biological invasions from both ecological and genetic perspectives. Here we characterize 11 disomic loci in Spartina alterniflora that show promise for population studies and for studying hybridization events between S. alterniflora and S. foliosa. Comparisons among invasive and native S. alterniflora populations showed that levels of allelic variation are lower in invasive populations. In addition, nearly all loci that amplified in S. foliosa populations and in a swarm of S. alterniflora×foliosa hybrids were polymorphic. We also found that several loci amplified successfully in other Spartina species.     

Impacts of an alien species (Spartina alterniflora) on the macrobenthos community of Jiangsu coastal inter-tidal ecosystem

Spartina alterniflora, a species vegetating on inter-tidal flats that was introduced from the eastern coast of United States, has become a hot topic, focusing on its invasion within local species in the coastal zone of China. Impacts of S. alterniflora on the inter-tidal macrobenthos community in the Jiangsu coastland are addressed by comparing the macrobenthos characteristics in a mudflat and in a four-year-old Spartina salt marsh that had earlier been a mudflat. During the period October 2002–July 2003, we studied the distribution pattern and diversity of macrobenthos, and discussed their correlation with environmental factors caused by Spartina vegetation. The results showed that a total of 43 macrobenthos species were found, mainly consisting of Mollusca, Crustacea, and Annelida. Ten macrobenthos species were found in the Spartina salt marsh, and 36 species were found in the mudflat. Life forms and functional groups of macrobenthos in the Spartina salt marsh were obviously distrinct from that of the mudflat. The study showed that macrobenthos diversity in the Spartina salt marsh decreased, and the community structure altered obviously, whereas the biomass showed no differences in different seasons. Statistical analysis demonstrated that seasonal change of macrobenthos diversity in the Spartina salt marsh negatively related to content of sediment organic matter, total N, bulk density, height and biomass of Spartina vegetation, and positively related to the density of Spartina. All these differences suggested the obvious effects of the Spartina vegetation on the Jiangsu inter-tidal benthic macroinvertebrate ecology. Furthermore, the investigation also showed that the niche of the native macrobenthos living in the mudflat has been transferred down, seaward, due to the invasion of Spartina in our study site.     

The positive and negative effects of exotic Spartina alterniflora in China

Spartina alterniflora is a perennial salt marsh grass native to the Atlantic and Gulf Coasts of North America. Recognized for its effects of diminishing strong tide even storms and accelerating sediment deposition, S. alterniflora was introduced into China in 1979. Now the artificial vegetation of Spartina has expanded to around 50 000 h m² along the east coast of China due to its strong capability of propagating by seeds and rhizome fragments. Although it was listed among 16 harmful exotic species in China 3 years ago, we need to evaluate its positive and negative effects in the coastal region of China objectively. This paper reviews some major positive effects of S. alterniflora in China, e.g. being a dominant primary producer, buffering against tides, accelerating accretion and reclamation, absorbing nutrients and digesting pollutants, as well as some major negative effects, e.g. occupying the niche of local species, altering the mudflat habitat, changing and even diminishing biodiversity, and damaging the aquiculture in the tidal land. In addition, its biomaterial uses and biological substitution as a means of ecological regulation is also discussed.     

The Root-Knot Nematode Producing Galls on Spartina alterniflora Belongs to the Genus Meloidogyne: Rejection of Hypsoperine and Spartonema spp.

Root-knot nematodes are a major group of plant-parasitic nematodes, but their sister group within the Tylenchida remains to be identified. To find the sister group and for any investigation of the evolutionary biology of the genus Meloidogyne, it would be useful to identify the most basal species within Meloidogyninae. Meloidogyne spartinae, a root-knot nematode parasitic on cordgrass (Spartina spp.), constitutes a potentially interesting early diverging (or at least highly divergent) root-knot nematode because it was originally described in a different genus, Hypsoperine (and later Spartonema), due to its unique anatomy and biology (although it was later put in synonymy by some, but not all, taxonomists). We have sequenced the whole 18S rDNA of this species and compared it to other sequences of this region that are available in GenBank for numerous Meloidogyne species. Phylogenetic analysis unambiguously locates the branch corresponding to M. spartinae as a lately diverging species, more closely related to M. maritima, M. duytsi or the M. ardenensis-hapla group. Thus, the distinction of a separate genus (Hypsoperine or Spartonema) for this species is not justified.     

Sulfur storage changed by exotic Spartina alterniflora in coastal saltmarshes of China

For the purpose of ecological engineering, Spartina alterniflora was introduced to China in 1979 and now covers about 112,000 ha of China's coastal lands. It was hypothesized that S. alterniflora could actively change the habitat environment, thus facilitating its competition over native species. In Yancheng Nature Reserve, sulfur storage of sediments and plant tissues was compared among marshes dominated by the exotic S. alterniflora and adjacent native Suaeda salsa and Phragmites australis and bare mudflat. Results showed that the S. alterniflora marsh contained the highest content of water-soluble, adsorbed, carbonate-occluded and total sulfur in the sediment. The sulfur levels were higher in the center than at the edges of the S. alterniflora marsh. Native marshes showed no significant difference in sediment sulfur levels. With greater biomass and higher tissue sulfur concentrations, plant sulfur storage of S. alterniflora vegetation was also larger than those of the native vegetations. Because higher concentrations of sulfur increase the competitive advantage of S. alterniflora over native halophytes, the results of the research showing that S. alterniflora increased marsh sulfur storage may shed light on the mechanism of expansion of monospecific vegetation in coastal China.