AMPHIDINIUM REVISITED. II. RESOLVING SPECIES BOUNDARIES IN THE AMPHIDINIUM OPERCULATUM SPECIES COMPLEX (DINOPHYCEAE), INCLUDING THE DESCRIPTIONS OF AMPHIDINIUM TRULLA SP. NOV. AND AMPHIDINIUM GIBBOSUM. COMB. NOV.1

Amphidinium operculatum Claparède et Lachmann, the type species of the dinoflagellate genus Amphidinium, has long had an uncertain identity. It has been considered to be either difficult to distinguish from other similar species or a morphologically variable species itself. This has led to the hypothesis that A. operculatum represents a “species complex.” Recently, the problem of distinguishing A. operculatum from similar species has become particularly acute, because several morphologically similar species have been found to produce bioactive compounds of potential interest to the pharmaceutical industry. In this study, we cultured and examined existing cultures of several species of Amphidinium, most of which have been previously identified as A. operculatum or as species considered by some to be synonyms or varieties of A. operculatum. Thirty strains were examined using comparative LM, SEM, and partial large subunit (LSU) rDNA sequence data. Through morphological and molecular phylogenetic analyses, six distinct species were identified, including Amphidinium trulla sp. nov. and Amphidinium gibbosum comb. nov. Amphidinium operculatum was redescribed based on four cultures. Genetic variability within the examined Amphidinium species varied greatly. There was little difference among strains in partial LSU rDNA for most species, but strains of A. carterae and A. massartii Biencheler differed by as much as 4%. In both A. carterae and A. massartii, three distinct genotypes based on partial LSU rDNA were found, but no morphological differences among strains could be observed using LM or SEM. In the case of A. carterae, no biogeographically related molecular differences were found.     

中国阿果蝇属五新种和二新记录种（双翅目：果蝇科）

记述采自中国大陆的阿果蝇属5新种和2新记录种:尖叶阿果蝇Amiota acutifoliolata sp.nov.,缺茎阿果蝇A.apenis sp.nov.,小叶阿果蝇A.minufoliolata sp.nov.,曲叶阿果蝇A.undulata sp.nov.,爪叶阿果蝇A.ungulfoliolata sp.nov.,须刺阿果蝇A.balaenodentata Takada,Beppu&Toda,1979和钩茎阿果蝇A.orchidea Okada,1968;这7种阿果蝇不能指派于本属已建立的7个种团中的任何一个。     

Isoenzyme and Total Protein Analysis by Agarose Isoelectric Focusing, and Taxonomy of the Genus Acanthamoeba

Thirty axenically grown reference strains belonging to 15 different Acanthamoeba spp. were investigated for isoenzyme patterns by agarose isoelectric focusing in the pH range 3–10. Zymograms of acid phosphatase, leucine amino peptidase, malate dehydrogenase, propionyl esterase, glucose phosphate isomerase, phosphoglucomutase, and alcohol dehydrogenase were compared. The same strains were also analyzed for protein patterns separated by agarose isoelectric focusing in a pH gradient of 5–8. The results suggested changes in taxonomy within morphology group II of Pussard & Pons. Acanthamoeba paradivionensis becomes a synonym of A. divionensis. Although this species seems to be related to A. rhysodes, it could not be concluded that the species names are synonyms since the type strain of A. rhysodes was not available for comparison. In the subgroup A. polyphaga–A. quina–A. lugdunensis, A. lugdunensis becomes the species name for pathogenic strains of this subroup, A. quina for the nonpathogenic strains, while A. polyphaga is the species name for an atypical strain. Two strains of A. castellanii showed different zymograms from strain Neff of this species, but related protein patterns. In group III, A. pustulosa is found to be a synonym of A. palestinensis, while one strain of A. lenticulata is also found to belong to the A. palestinensis species. All other species names in both morphology groups could be retained as valuable, on the basis of the techniques used. Group I was not investigated, as axenic cultures could not be obtained.     

The impact of an invading alien grass (Agropyron cristatum) on species turnover in native prairie

Abstract. Alien invasions typically reduce species richness of habitats, but few studies have examined their effects on species turnover, the difference in species composition between localities. Agropyron cristatum (L.) Gaertn. (crested wheat grass) has been planted on 6–10 million ha of North American prairie, and is invading native prairie. We studied the invasion of A. cristatum into native prairie by measuring species composition along a gradient from maximum to minimum A. cristatum abundance. As A. cristatum increased, the abundance of most common native species decreased, but one appeared to be unaffected (Bouteloua gracilis (H.B.K.) Lag.), and another (Poa sandbergii Vasey) increased. The effect of A. cristatum on species turnover was investigated by examining species–area curves for areas from 0.5 m² to 8.0 m². Species diversity was reduced by 35% at high A. cristatum abundances at all areas. A. cristatum reduced the intercept of the species–area curve, but not the slope, suggesting that A. cristatum affected species turnover proportionally in all areas and habitats. This unusual result may indicate a homogeneous environment where species are distributed randomly. A. cristatum produced almost twice as many seeds as all native grasses combined. The number of seeds collected of native grasses and A. cristatum was highly correlated with the number of seed heads immediately nearby, but not with transect position. This suggests most seeds were dispersed over distances less than 5 m. In sum, the invasion of native prairie by A. cristatum might be related to high rates of seed production, and has the effect of decreasing species turnover by reducing the intercept of the species–area curve.     

The trimorphic flea-beetle, <Emphasis Type="Italic">Alagoasa extrema</Emphasis>, not suitable for biocontrol of <Emphasis Type="Italic">Lantana camara</Emphasis> in Africa

The biology and host range of the flea-beetle, Alagoasa extrema (Harold) (Coleoptera: Chrysomelidae: Galerucinae: Alticini), was studied under quarantine laboratory conditions to evaluate the insect’s suitability for release as a biological control agent for the noxious weed, Lantana camara L. (Verbenaceae), in South Africa. Identification of this species proved noteworthy. The adults of A. extrema display three freely interbreeding and very distinct colour morphs, which can be confused with no less than 11 species of Alagoasa found in Mexico and the southwestern U.S. Initially, specimens were identified as two species of the genus Alagoasa Bechyné, i.e. A. quadrilineata (Harold) and A. extrema. Thirteen additional Alagoasa species and Kushelina petaurista (F). that can be confused with A. extremabased on external morphology of adults or larvae are discussed. Favourable biological characteristics included long-lived adults, several overlapping generations per year, and high adult and larval feeding rates. Observations from the insect’s native Mexican range and studies in South Africa suggest that A. extrema would probably be more suited to subtropical, rather than temperate areas in Africa. Host-specificity studies showed A. extrema to be an oligophagous species, capable of feeding and developing on several non-target species, especially two indigenous, African Lippia species (Verbenaceae). The host suitability of these indigenous species was only marginally lower than that of L. camara, and the potential risk to them was deemed to be too high to warrant release. It was therefore recommended that A. extrema be rejected as biocontrol agent for lantana in Africa.     

Phenetic investigation of non-nodulating African species ofAcacia (Leguminosae) using morphological and molecular markers

Morphological and RAPD markers were used to assess the relationships among nodulating and non-nodulating species of AfricanAcacia. Non-nodulating species of AfricanAcacia are only found within subg.Aculeiferum sect.Monacanthea. African species of sect.Monacanthea examined were found to form a group distinct from the other African species examined on a morphological and molecular basis. All lack the ability to nodulate, suggesting that non-nodulation may be used as a taxonomic tool. The species of sect.Aculeiferum were separated by RAPD and morphological analysis into two groups depending on whether they were armed with prickles in pairs and/or prickles in threes, or solitary. A third group of species was identified within sect.Acacia: further subdivision of this group was achieved into subsectt.Pluriseriae andUniseriae. The position ofA. albida relative to other AfricanAcacia species was found to be distinct but not totally independent of the genus. The partitioning and distribution of the genetic variability within the genus is further elucidated by the RAPD analysis of populations ofAcacia species. A population analysis ofA. polyacantha demonstrated geographical and site-specific variation.     

Karyotypic analysis of Cajanus,Atylosia, and Rhynchosia species

Summary Somatic chromosomes of two cultivais of Cajanus cajan, eight species of Atylosia (A. albicans, A. cajanifolia, A. lineata, A. platycarpa, A. scarabaeoides, A. serica, A. trinervia and A. volubilis), and of Rhynchosia rothii were analysed. All species had 2n=22. Eight of the 10 species studied had two pairs of satchromosomes while A. scarabaeoides and A. sericea had only one sat-chromosome pair. Based on relative chromosome length (L%), arm ratio (pa-value) and presence or absence of secondary constriction, a karyotype formula for each species was formulated. Based on these parameters the chromosome pairs could also be assigned to groups ranging from 8 to 10 in different species. Except for the asymmetrical karyotype of A. albicans, the other species had rather moderately symmetrical karyotypes.     

Comparison of genetic variability in remnant and wide-spread rainforest understorey species of Austromyrtus (Myrtaceae)

This study investigated the genetic variability, stand structure, and reproductive activity of four species of Austromyrtus which showed differing levels of rarity and patterns of distribution. The focus of the study was A. gonoclada which is extremely rare due to extensive urban expansion in Brisbane, Australia. The total number of known individuals in the species is only 27. Because nothing was known about the genetic structure of Austromyrtus comparison with other species was made to provide a context for studies on A. gonoclada. Population structure and levels of reproductive activity were not correlated to species rarity or distribution. A. gonoclada showed maximum genetic variability in comparison to the other species of Austromyrtus, despite the small population size. Three of six variable loci were in Hardy-Weinberg equilibrium in A. gonoclada populations, in contrast to populations of the other species which had few heterozygous genotypes. The higher variability of the remaining individuals is suggested to be a legacy of the relatively short time since they were part of a larger population. There were very few seedlings of A. gonoclada, in contrast to the other species of Austromyrtus and the seeds produced fell from the tree while still green and were unable to be germinated. A. gonoclada is under continuing threat from development and has low reproductive success. Active programmes for recovery of this species will be necessary as its continued survival is dependent on population increase. Management of the genetic resources of the species will enable survival with long-term evolutionary potential.     

Indigenous and Ammophila arenaria-dominated dune vegetation on the South African Cape coast

Abstract. Communities formed by the potentially invasive European Ammophila arenaria (marram grass) are compared with those dominated by indigenous dune plant species in coastal dune systems. Sampling of communities was carried out along the Cape coast for species richness, species diversity, importance values and species associations. The influence of soil and other environmental factors on vegetation were also compared. While species richness values in A. arenaria communities appear similar to those of indigenous dune plant communities, diversity indices are significantly lower. However, on the basis of importance values of individual species, A. arenaria does not show extreme dominance to the exclusion of other species, as it does on the North American Pacific coast, where it has also been introduced. Because of its growth in dense tufts, A. arenaria is accompanied mostly by small chamaephytes and therophytes, while indigenous stands support more phanerophytes. Moreover, A. arenaria forms weaker species associations than dominant indigenous dune plant species. The alien status of A. arenaria in South Africa is confirmed by applying classification and ordination analyses which failed to differentiate A. arenaria communities according to their geographical origin as achieved with indigenous communities. This may be attributed to the lack of vigorous indigenous plants in A. arenaria communities, which accounts for the low variety in species composition of A. arenaria communities along the coastline. With regard to environmental factors, A. arenaria communities were observed to be less sensitive to extrinsic factors, such as climate, than indigenous dune plant communities. Results confirm that A. arenaria is an alien plant species in South Africa, but do not imply its invasiveness in the present or near future.     

Comparative study of the phylogenetic structure in six Apodemus species (Mammalia,Rodentia) inferred from ISSR-PCR data

The utility of the Inter Simple Sequence Repeat-Polymerase Chain Reaction (ISSR-PCR) was explored in order to determine genetic variation in six species of the genus Apodemus (A. flavicollis, A. sylvaticus, A. uralensis, A. agrarius, A. mystacinus and A. epimelas) at the individual level, population level, in separate geographic samples and in the species as a whole. Six optimized primers produced highly reproducible and polymorphic DNA markers with 98.3% polymorphic bands on a total sample of 91 individuals from 32 localities in Europe and Asia. Moreover, each primer allowed for an exact diagnosis of each of the six Apodemus species and thus provides a simple and reliable tool for the hitherto problematic discrimination of species from the subgenus Sylvaemus. Genetic distances between species ranged from 0.079, among the closely related A. flavicollis and A. sylvaticus, to 0.203 between A. mystacinus and A. agrarius. A. flavicollis, A. uralensis and A. sylvaticus display a strong population substructure. The range of genetic distances among geographic samples within last two species reaches the values obtained for closely related species. ISSR markers proved to be a simple and reliable tool for species diagnosis, as well as for estimating genetic diversity below the species level and for closely related species, but they showed questionable reliability for larger genetic distances.     

Phylogenomic,morphological, and niche differentiation analyses unveil species delimitation and evolutionary history of endangered maples in Acer series Campestria (Sapindaceae)

Accurate species delimitation is crucial for biodiversity conservation. The Acer series Campestria comprises four species, A. campestre L., A. miyabei Maxim., A. miaotaiense P. C. Tsoong, and A. yangjuechi Fang & P. L. Chiu. To clarify controversies over the taxonomic status of the latter three endangered species, we undertook phylogenomic, morphological, and niche differentiation analyses in series Campestria. Our coalescent species tree of 544 and 77 single-copy nuclear genes supported series Campestria as monophyletic, with A. yangjuechi having the closest relationship with A. miaotaiense. However, in the plastome-derived tree based on 64 protein coding sequences, the four species did not cluster together, and each of them grouped with some other sympatric Acer species. Given this nuclear-cytoplasmic conflict, we hypothesize that A. yangjuechi have been subject to nuclear gene introgression and plastid (pt) capture involving another sympatric maple, that is, A. amplum Rehder. Principal component analysis and machine learning based on morphological data could not separate A. yangjuechi and A. miaotaiense, but they both could be clearly distinguished from A. miyabei. Moreover, the niche overlap tests of the two more widespread species, A. miyabei and A. miaotaiense, showed they clearly occupy distinct niches. Overall, we conclude that A. miyabei and A. miaotaiense are distinct species, while A. yangjuechi (endemic to Mt. Tianmu/East China) should be treated as a subspecies of A. miaotaiense. Our study points out that multiple lines of phylogenomic, morphological, and ecological evidence prove highly useful in species delimitation. Additionally, our results should help to inform conservation measures for endangered species of the genus Acer/series Campestria in East Asia.     

Are psyllids affiliated with the threatened plants Acacia ausfeldii,A. dangarensis and A. gordonii at risk of co‐extinction?

Secondary extinctions of dependent affiliate species may exceed the numbers of species directly extirpated with habitat loss. We investigated the host specificity, and hence the co‐extinction risk, of multiple psyllid species (Hemiptera: Psylloidea) on the threatened plants Acacia ausfeldii, A. dangarensis and A. gordonii by sampling from these and other sympatric common plants across multiple sites, years and plant phenologies. We found one specialist psyllid species (Psyllidae: Acizzia sp.) on each of the plants A. ausfeldii and A. gordonii but no specialists on A. dangarensis. The A. gordonii specialist only occurred when its host was in flower. Specialist psyllids considered at risk of extinction represented 10% of all psyllid species observed on the three threatened acacias. Potential risks to co‐endangered affiliates need to be considered when implementing threatened plant recovery actions. Conservation of plant populations and plant species diversity is also necessary to maintain high insect diversity.     

Reproductive isolation is mediated by pollen incompatibility in sympatric populations of two Arnebia species

To explore uncertain aspects of the processes that maintain species boundaries, we evaluated contributions of pre‐ and postpollination reproductive isolation mechanisms in sympatric populations of Arnebia guttata and A. szechenyi. For this, we investigated their phylogenetic relationships, traits, microenvironments, pollinator visits, action of natural selection on floral traits, and the outcome of hand pollination between the two species. Phylogenetic analysis indicates that A. szechenyi is a derived species that could be closely related to A. guttata, and both could be diploid species. Arnebia guttata flowers have larger parts than A. szechenyi flowers, but smaller nectar guides. Soil supporting A. szechenyi had higher water contents than soil supporting neighboring populations of A. guttata (in accordance with their geographical distributions). The pollinators shared by the two species preferred A. szechenyi flowers, but interspecific visitations were frequent. We found evidence of conflicting selection pressures on floral tube length, flower diameter and nectar guide size mediated via male fitness, and on flower diameter and floral tube diameter via female fitness. Hand‐pollination experiments indicate complete pollen incompatibility between the two species. Our results suggest that postpollination prezygotic mechanisms are largely responsible for reproductive isolation of sympatric populations of the two Arnebia species.     

Taxonomic implications of genome size and pollen colour and vitality for species of Agapanthus L’Héritier (Agapanthaceae)

Nuclear DNA content (2C) and pollen vitality and colour are used as new criteria to investigate all species of the genus Agapanthus LHéritier. The species have the same chromosome number (2n=2x=30), with exception of four triploid plants found. The nuclear DNA content of the diploids, as measured by flow cytometry with propidium iodide, is demonstrated to range from 22.1–31.6 pg. This implies that the largest genome contains roughly 10¹⁰ more base pairs than the smallest. The species could be divided in two groups based on pollen colour and DNA content: a group with lilac pollen and a DNA content between 22.3 and 24.1 pg containing the species A. campanulatus Leighton, A. caulescens Sprenger and the rarer A. coddii Leighton, and a group with yellow/brownish pollen and a DNA content from 25.2–31.6 pg containing the species A. praecox Willd., A. inapertus Beauv. and A. africanus (L.) Hoffmanns. Four other taxa, recognized by Leighton (1965) are treated as follows: A. comptonii Leighton, has a nuclear DNA content similar to A. praecox and is considered to be a synonym of A. praecox subsp. minimus Leighton. A. walshii L. Bol., has with 31.6 pg the same high amount of DNA as A. africanus from the same area and is therefore renamed as a subspecies (A. africanus subsp. walshii (Leighton) Zonn. & Duncan comb. nov.). The nuclear DNA amounts of A. dyeri Leighton, including the geographically isolated plants from Mozambique, are shown to be identical to A. inapertus. Therefore A. dyeri is considered synonymous with A. inapertus subsp. intermedius Leighton. A. nutans Leighton is identical in DNA content to A. caulescens and is considered to be synonymous with that species. Concluding there are six species: A. campanulatus Leighton, A. caulescens Sprenger, A. coddii Leighton, A. praecox Willd., A. inapertus Beauv. and A. africanus (L.) Hoffmanns. Nuclear DNA content as measured by flow cytometry and pollen colour are shown to be relevant traits to throw light on the relationships between Agapanthus species.     

Integrative taxonomy reveals too extensive lumping and a new species in the moss genus Amphidium (Bryophyta)

An integrative taxonomic approach, including molecular phylogenetic reconstructions based on plastid rps4-trnF and nuclear ITS sequences, statistical analysis of morphological-anatomical characters, and classical taxonomy, indicates that the reduction of 13 Amphidium species to three in a recent morphological revision represents a case of too extensive lumping. Instead, six Amphidium species can be distinguished based on molecular and morphological data, the widespread Amphidium lapponicum, A. mougeotii, and A. tortuosum, as well as the Macaronesian endemic A. curvipes, the North American endemic A. californicum, and a newly discovered species from Central Asia (southern Siberia and northern Mongolia), A. asiaticum sp. nov. Diagnostic morphological characters for all six species are discussed. The present data confirm that species diversity of Amphidium is highest in the Holarctic, where all six species occur.     

Phenotypic and genotypic identification and phylogenetic characterisation of <Emphasis Type="Italic">Taphrina</Emphasis> fungi on alder

All Taphrina species are dimorphic with a mycelium stage biotrophic on vascular plants and a saprophytic yeast stage. European species of Taphrina on Alnus species (Betulaceae) were identified using morphological, physiological and molecular characteristics, the latter including determination of PCR fingerprints and of nucleotide sequences from selected nuclear ribosomal DNA regions. PCR fingerprinting gives a good overview of species identification, as do nucleotide sequences, which in addition, help to clarify phylogenetic relationships. Taphrina alni is a homogeneous species that exhibited more than 50% similarity in PCR fingerprinting with three different primers. Morphologically, it produces tongue-like outgrowths from female catkins of Alnus incana. Taphrina robinsoniana from A. rugosa and A. serrulata in North America is phylogenetically closely related to T. alni, but the two species could be separated by their PCR fingerprints, partial sequences of 26S rDNA (D1/D2) and ITS1/ITS2 sequences. T. epiphylla and T. sadebeckii are two phylogenetically closely related species. T. epiphylla causes witches brooms in crowns of A. incana. In addition, T. epiphylla forms slightly yellow white-grey leaf spots in midsummer on A. incana. Yellow white-grey leaf spots up to 10 mm on A. glutinosa are characteristic for T. sadebeckii. Both species can be separated well by PCR fingerprinting. Different from T. epiphylla, T. sadebeckii is genotypically more heterogeneous. Only two out of three different primers showed similarity values above 50% in different European strains of T. sadebeckii. Although genetic variability was not detected in complete sequences of the 18S ribosomal DNA of T. sadebeckii, ITS1/ITS2 sequences appeared to be more heterogeneous, too. Taphrina tosquinetii is a genotypically homogeneous species causing leaf curl on Alnus glutinosa. It was not possible to distinguish the yeast phases from different Taphrina species on Alnus using morphological and physiological characteristics only. Dedicated to Prof. Dr. Hanns Kreisel on the occasion of his 70^th birthday     

Protospirura siamensis n. sp. (Nematoda: Spiruridae) from rodents in Thailand

A large number (828) of rodents belonging to nine genera (Bandicota, Berylmys, Chiropodomys, Hapalomys, Leopoldamys, Maxomys, Mus, Niviventer and Rattus) were trapped in four Thai provinces between 2008 and 2010. A new species of Protospirura (Nematoda: Spiruridae) was identified and described. Protospirura siamensis n. sp. was found in 10 rodent species from the four Thai provinces surveyed. The new species can be distinguished from known species of the genus by the arrangement of the papillae on the male and the morphology and length of the spicules. This is the first species of Protospirura to be described from South-East Asia.     

The comparison of seed protein patterns within the genusArachis by polyacrylamide gel electrophoresis

The seed protein patterns of 12Arachis species were compared by polyacrylamide gel electrophoresis (PAGE), similarities between patterns were measured by the Jaccard index. Results obtained confirm the close relationships established between members of the genus on morphological grounds and support the more recent classification schemes.A. villosa andA. correntina could well be regarded as distinct species on grounds of protein differences whileA. macedoi andA. villosulicarpa (although members of the same section, Extranervosae) show considerable differentiation of their protein patterns. Surprisingly, the formA. ×batizogaea showed less similarity in protein pattern to those of its parental species than might have been expected. The principle value of seed protein pattern data appears to be in distinguishing species within sections.