USING QUANTITATIVE PCR TO DETERMINE THE DISTRIBUTION OF A SEMICRYPTIC BENTHIC DIATOM,NAVICULA PHYLLEPTA (BACILLARIOPHYCEAE)1

Evidence has accumulated during the last decade showing that many established diatom morpho‐species actually consist of several semicryptic or truly cryptic species. As these species are difficult or even impossible to differentiate by microscopic analysis, there is virtually no information on how they behave in natural environments. In this study, we developed a quantitative real‐time PCR (qPCR) assay using TaqMan probes^® targeted to the internal transcribed spacer 1 (ITS1) to assess the spatial distribution and seasonal dynamics of an important component of the microphytobenthos of intertidal sediments. Navicula phyllepta Kützing is a brackish‐marine morpho‐species with a cosmopolitan distribution. Axenic clones of this species were isolated from natural assemblages of benthic diatoms at different intertidal stations in the Westerschelde estuary (The Netherlands). At least two distinct semicryptic species of N. phyllepta were present, as shown by differences in the quantity of DNA per cell, the ITS1 sequences and the copy number of ITS per cell. DNA and chl a concentrations extracted from sediment surface samples were closely correlated, showing that the DNA used for subsequent analysis mostly belonged to the microalgal community. The results of real‐time qPCR from sites throughout the estuary and over several seasons agreed well with microscopic counts. Additionally, the seasonal pattern of the two forms of N. phyllepta showed an overlapping, but unique distribution along the estuary.     

PERIZONIUM AND INITIAL VALVE FORMATION IN THE DIATOM NAVICULA CUSPIDATA (BACILLARIOPHYCEAE)1

This paper describes the perizonium and initial valve formation in Navicula cuspidata Kütz., based on light microscope (LM) and scanning electron microscope (SEM) observations. The perizonium consists of concentric over-lapping bands, laid down sequentially at the tips of the expanding biconical auxospore during its elongation. The central perizonial band has fimbriate edges and is considerably more rigid than the more distal bands. During auxospore elongation and the band secretion, the chloroplasts continuously oscillate between the two ends of the cell; this oscillation ceases once the elongation is complete. The initial valves, formed within the perizonium, are molded into the basically biconical shape of the perizonium except for a central flattening of each valve face. In contrast to the raphes in gametangial and vegetative valves which are surrounded by a smooth axial area, the raphes in initial valves lie within a raised ridge running along the apical axis of the valve. The regular pattern of apically oriented ridges on the outer surface of vegetative valves is also lacking on initial valves. Comparison of pore–pore spacing within striae of gametangial valves, initial values and post-initial valves (first division and vegetative cells) reveals that the pore–pore distance within striae is conserved at all sexual stages. However, the distance between striae is considerably larger in initial valves than in gametangial and post-initial valves. Vegetative interstriae spacing as well as the planar morphology of the valve face is regained at the first division of the initial cell. This suggests that the spacing between striae is dependent on the sexual stage of the cell during valve formation (i.e. not directly dependent on the cell size) and can be altered independently of the pore–pore spacing.     

THE MORPHOLOGY AND INTEGRATION OF VALVES AND BANDS IN NAVICULA MUTICA VAR. MUTICA (BACILLARIOPHYCEAE)1

Navicula mutica (Kütz.) var. mutica was isolated from the air, cloned on agar, cultured in soil-water bottle, and studied with transmission and scanning electron micros-ropy. The frustules were lanceolate to ovoid with rounded apices, with the apical axis 8.5 ± 3.2 μ and the trans-apical and the transapical axis 3.6 ± 0.6 μm. Striae were composed of two or three puncta, and the mantle bore a single row of puncta aligned with the striae. The ends of the raphe turned away from an isolated punctual in the central area of the valve. The mantle puncta and one or two of the valve-face puncta in each stria opened into a series of transapical grooves in the interior of the valve, the grooves contributing to the appearance of striae in the light microscope. The interior of the mantle also possessed a pair of longitudinal grooves, discontinuous at the apices of the valves. An undulate advalvar margin of the valvocopula likely articulates along the interior longitudinal groove of the mantle. The projections of the undulate margin are perhaps positioned between the transapical grooves and along the longitudinal groove between the dentiform structures formed by the intersection of the double-grooved system. The girdle bands each had two (occasionally three) rows of pores. The pleurae margins were straight and not undulate.     

MOLECULAR DELINEATION OF SPECIES AND SPECIES RELATIONSHIPS IN THE RED ALGAL AGAROPHYTES GRACILARIOPSIS AND GRACILARIA (GRACILARIALES)1

Delineation of species in the economically important agarophyte genera Gracilaria and Gracilariopsis has proven extremely difficult using available morphological characteristics. In this study, we examine the usefulness of two transcribed spacers for molecular systematic studies of these genera. The polymerase chain reaction was used to amplify the internal transcribed spacers (ITSs) and the intervening 5.8S ribosomal DNA of the nuclear ribosomal repeat region. In addition, a plastid spacer region and flanking regions of coding genes were amplified from the RUBISCO operon. Both regions were sequenced for individuals and populations of Gracilariopsis lemaneiformis (Bory) Dawson, Acleto, et Foldvik to determine the usefulness of these spacers in delimiting populations. These studies reveal that there is as much variation among individuals of a population as there is between individuals of geographically separate populations. In addition, the ITS spacer regions were compared between different species of Gracilariopsis and Gracilaria. The nuclear ITS spacer region is conserved at a species level in both genera and provides phylogenetically informative characters that can be used to examine species interrelationships among relatively closely related taxa. However, because of the difficulties of aligning this entire region among species from the two genera, the ITS region is not useful for examining intergenera relationships. ITS interspecies sequence comparisons indicate that Gracilariopsis lemaneiformis from California is significantly different from G. lemaneiformis from China and that a species of Gracilariopsis from Peru is more closely related to G. lemaneiformis from North Carolina than it is to the other Gracilariopsis species examined. In addition, these studies indicate that Gracilaria chilensis Bird, McLachlan, et Oliveira from New Zealand and Gracilaria tenuistipitata Chang et Xia from southeast Asia are as closely related as are Gracilaria verrucosa (Hudson) Papenfuss, G. pacifica Abbott, and Gracilaria robusta Kylin. Phylogenetic analysis of aligned plastid spacer sequences from Gracilaria and Gracilariopsis taxa provide similar conclusions about species relationships.     

MONITORING RAPID VALVE FORMATION IN THE PENNATE DIATOM NAVICULA SALINARUM (BACILLARIOPHYCEAE)1

After each division of a diatom cell, a new siliceous hypovalve is formed inside the silica deposition vesicle (SDV). We present the sequence of this early formation of the new valve in the pennate marine diatom Navicula salinarum (Grunow) Hustedt, visualized by using the fluorescent probe 2‐(4‐pyridyl)‐5‐((4‐(2‐dimethylaminoethylamino‐carbamoyl)methoxy)phenyl)oxazole (PDMPO). Our observations confirm that two‐dimensional expansion of the growing valve is a rapid process of no more than 15 min; three‐dimensional completion of the valve appears to be slower, lasting most of the time valve formation takes. The results are relevant to studies of the timing of molecular processes involved in valve formation (i.e. the bio‐ and morphogenesis of the SDV) in relation to uptake and transport of silicic acid. Use of this probe helps us to identify specific developmental stages for further detail analysis of diatom basilica formation, which eventually could lead to obtaining enriched SDV fractions.     

GENETIC DIVERSITY AND INTROGRESSION IN TWO CULTIVATED SPECIES (PORPHYRA YEZOENSIS AND PORPHYRA TENERA) AND CLOSELY RELATED WILD SPECIES OF PORPHYRA (BANGIALES,RHODOPHYTA)1

We investigated the genetic variations of the samples that were tentatively identified as two cultivated Porphyra species (Porphyra yezoensis Ueda and Porphyra tenera Kjellm.) from various natural populations in Japan using molecular analyses of plastid and nuclear DNA. From PCR‐RFLP analyses using nuclear internal transcribed spacer (ITS) rDNA and plastid RUBISCO spacer regions and phylogenetic analyses using plastid rbcL and nuclear ITS‐1 rDNA sequences, our samples from natural populations of P. yezoensis and P. tenera showed remarkably higher genetic variations than found in strains that are currently used for cultivation. In addition, it is inferred that our samples contain four wild Porphyra species, and that three of the four species, containing Porphyra kinositae, are closely related to cultivated Porphyra species. Furthermore, our PCR‐RFLP and molecular phylogenetic analyses using both the nuclear and plastid DNA demonstrated the occurrence of plastid introgression from P. yezoensis to P. tenera and suggested the possibility of plastid introgression from cultivated P. yezoensis to wild P. yezoensis. These results imply the importance of collecting and establishing more strains of cultivated Porphyra species and related wild species from natural populations as genetic resources for further improvement of cultivated Porphyra strains.     

CALCIUM RELEASE FROM INTRACELLULAR STORES IS NECESSARY FOR THE PHOTOPHOBIC RESPONSE IN THE BENTHIC DIATOM NAVICULA PERMINUTA (BACILLARIOPHYCEAE)1

Complex photoreceptor pathways exist in algae to exploit light as a sensory stimulus. Previous studies have implicated calcium in blue‐light signaling in plants and algae. A photophobic response to high‐intensity blue light was characterized in the marine benthic diatom Navicula perminuta (Grunow) in van Heurck. Calcium modulators were used to determine the involvement of calcium in the signaling of this response, and the fluorescent calcium indicator Calcium Crimson was used to image changes in intracellular [Ca²⁺] during a response. A localized, transient elevation of Calcium Crimson fluorescence was seen at the cell tip at the time of cell reversal. Intracellular calcium release inhibitors produced a significant decrease in the population photophobic response. Treatments known to decrease influx of extracellular calcium had no effect on the population photophobic response but did cause a significant decrease in average cell speed. As the increase in intracellular [Ca²⁺] at the cell tip corresponded to the time of direction change rather than the onset of the light stimulus, it would appear that Ca²⁺ constitutes a component of the switching mechanism that leads to reversal of the locomotion machinery. Our current evidence suggests that the source of this Ca²⁺ is intracellular.     

GENETIC DIFFERENTIATION AMONG POPULATIONS OF THE PLANKTONIC MARINE DIATOM DITYLUM BRIGHTWELLII (BACILLARIOPHYCEAE)1

Population genetic structure was determined for the planktonic diatom Ditylum brightwellii (West) Grunow in two connected estuaries—Puget Sound and the Strait of Juan de Fuca (WA, USA). Three genetically distinct populations were detected that were characterized by different microsatellite allele distributions and unique alleles. Isolates from the two most genetically diverged populations displayed identical full‐length 18S rDNA sequences suggesting that either a single or two recently diverged species were sampled. The extent of genetic differentiation between populations was not correlated with distance between water samples or time between sampling. Instead, distinct populations were associated with different estuaries. In Puget Sound waters, one population was detected three times over the course of 28 months. Cells from this population were likely maintained inside Puget Sound over long periods through water recirculation within the Sound. In Strait of Juan de Fuca waters, two additional populations were detected. Maximum growth rates of Puget Sound isolates were significantly different from Strait of Juan de Fuca isolates, indicating that populations were composed of cells with different physiological capabilities. The genetic and physiological differentiation observed between populations from intermixing estuaries suggested that genetic exchange between populations was restricted through differential selection. Despite the potential for widespread dispersal in planktonic organisms, it appears that populations with distinct genetic and physiological characteristics can be maintained over long time periods through a combination of hydrology and differential selection.     

PSEUDO-NITZSCHIA PUNGENS AND P. MULTISENES (BACILLARIOPHYCEAE): NUCLEAR RIBOSOMAL DNAS AND SPECIES DIFFERENCES1

The region of the nuclear ribosomal DNA (rDNA) operon containing the small subunit (SSU), internal transcribed spacer 1 (ITS1), and a portion of the 5.8s rDNA gene was sequenced in one isolate each of Pseudo-nitzschia multiseries (Hasle) Hasle and Pseudo-nitzschia pungens (Grunow in Cleve & Möller) Hasle. The SSUs of these two species were highly similar, differing only in 14 point mutations and one insertion/deletion in 1774 bp. The ITS1 sequences were more variable, with 57 point mutations and three insertion/deletions in 257 bp. There were no differences in 44 bp of the 5.8S sequences. Restriction fragment patterns (RFPs) for the restriction endonucleases HaeIII, Hha1, and Rsa1 for 13 isolates of P. multiseries from the Atlantic, Pacific, and Gulf coasts of the United States and 16 isolates of P. pungens from the three coasts of the United States, in addition to Japan and China, were compared. There were differences between the RFPs of P. multiseries and P. pungens that corresponded to sites mapped by the DNA sequences, but no infraspecific variation in RFPs was observed for either species. The differences in RFPs correlate with morphological, immunological, and other rDNA differences and support the recognition of these taxa as separate species.     

New species Micractinium kostikovii (Chlorellaceae,Trebouxiophyceae) from Russia

Correctly identifying species of Chlorella-like microalgae is difficult because of their morphological simplicity and high phenotypic plasticity. The use of molecular tools has revolutionized research on algal diversity, enabling such advances as the discovery of numerous new taxa. This article presents the results of a study of strains that are newly isolated from a freshwater Lake Prudovikov (Samara region, Russian Federation). These strains had the typical Chlorella morphology, exhibiting spherical cells and a cup-shaped parietal chloroplast. The chloroplast contained a single pyrenoid enveloped by starch grains. However, 18S–ITS1–5.8S–ITS2 sequence analyses indicated that the studied strains are strongly allied with the well-supported genus Micractinium. Based on the results of a comparative analysis of the morphological, molecular and environmental characteristics of the studied strains (employing a polyphasic approach), we propose that they are new species of Micractinium: Micractinium kostikovii sp. nov.     

CRYPTIC DIVERSITY AND PHYLOGENETIC RELATIONSHIPS WITHIN THE MASTOCARPUS PAPILLATUS SPECIES COMPLEX (RHODOPHYTA,PHYLLOPHORACEAE)1

Mastocarpus papillatus (C. Agardh) Kütz. is a common intertidal red alga occurring along the west coast of North America from Baja California to Alaska. Sequencing of both the chloroplast‐encoded rbcL gene and the nuclear ribosomal internal transcribed spacer (ITS) regions of ～200 specimens from California to Alaska revealed that M. papillatus is actually a complex of at least five species. All five species have high bootstrap support in phylogenetic analyses of both genetic regions, and in the case of the ITS marker, the species also have distinctive patterns of indels. Three of the species are localized in the mid‐ to upper intertidal, whereas two of the species occur in the low intertidal. The species also have different geographic ranges that overlap in the Vancouver Island area of British Columbia. No distinctive, reliable morphological differences were observed among the species. Although a variety of names are available for species in the complex, it is not yet clear which name goes with which species. As part of the survey, I also sequenced other species of Mastocarpus in the northeast Pacific region, and I provide new distribution records for M. jardinii ( J. Agardh) J. A. West and for a nonpapillate and probably undescribed species of Mastocarpus.     

高产紫杉醇内生真菌J11的鉴定

从南方红豆杉(Taxus chinensis var.mairei)的幼茎中分离出一株产紫杉醇内生真菌J11.菌株J11的发酵提取物经高分辨质谱分析,证实J11菌株可产紫杉醇.提取该菌株的基因组DNA,扩增核糖体internal transcribed spacer(ITS)和28S核糖体large subunit rRNA gene(LSU)序列,经测序获得该菌的ITS序列和LSU序列.序列比对和检索结果表明,J11菌株为葡萄座腔菌(Botryosphaeria ssp.)属中的一个新菌株.形态学鉴定符合葡萄座腔菌属特征,高效液相色谱分析表明,J11菌株的紫杉醇含量约为615.1μg/L.本研究首次证实葡萄座腔菌J11是一株高产紫杉醇野生型菌株,具有潜在的应用前景.     

PHYLOGENY OF THE BATRACHOSPERMALES (RHODOPHYTA) INFERRED FROM rbcL AND 18S RIBOSOMAL DNA GENE SEQUENCES

The sequence data from the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase ( rbc L) gene and 18S ribosomal DNA (small subunit) of taxa in the freshwater rhodophyte order Batrachospermales were used to construct phylogenetic hypotheses. Taxa examined in this study represent four families, eight genera, and six sections of the genus Batrachospermum . In addition, Rhododraparnaldia oregonica Sheath, Whittick et Cole, was included in the analysis because it shares particular ultrastructural, reproductive, and morphological characteristics with members of the Batrachospermales and Acrochaetiales. The trees generated from each gene, as well as a combined data set, were largely congruent. Rhododraparnaldia consistently occurs on an early branch within the Acrochaetiales – Palmariales clade and does not appear to be a member of the Batrachospermales. In addition, Thorea violacea Bory de St. Vincent was not closely related to the other taxa of the Batrachospermales in all trees and hence the family Thoreaceae does not appear to be a natural grouping within this order. All other taxa analyzed, which are presently classified within this order, formed a monophyletic clade in most analyses. Psilosiphon scoparium Entwisle was not closely allied with the taxa of the Lemaneaceae, lending support to the newly proposed family Psilosiphonaceae. Sequence data from the remaining taxa of the Lemaneaceae support the concept of a derived monophyletic clade. The genus Batrachospermum appears to comprise many morphologically similar but distantly related taxa, which will need further investigation to resolve their taxonomic status. Tuomeya, Sirodotia and Nothocladus are retained at the generic level until further data are obtained.     

SYSTEMATIC STATUS AND PHYLOGENETIC RELATIONSHIPS OF THE FRESHWATER GENUS BALBIANIA (RHODOPHYTA)

The freshwater rhodophyte Balbiania investiens (Lenormand) Sirodot was collected as an epiphyte of Batrachospermum in small streams in England, Ireland, and Germany. Unialgal cultures and field collections of Balbiania were analyzed in terms of their morphology, ultrastructure, and the DNA sequences of the genes coding for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase ( rbc L) and 18S ribosomal RNA (rRNA), as well as the first internal transcribed spacer region (ITS1) of the rRNA genes. One of the major distinguishing features of this genus is the production of spermatangia on the tips of specialized, elongate cells. The spermatangia are cut off in a cluster together with similar nonspermatangial cells that are packed with starch granules. The only other rhodophyte genus with this feature is Rhododraparnaldia ; the two genera are differentiated from each other by the pattern of branching, substratum of attachment, and type of diploid phase. In the parsimony and distance trees inferred from the two gene sequences as well as in a combined parsimony tree, Balbiania and Rhododraparnaldia grouped together on a well-supported branch separate from other taxa of the Batrachospermales, Acrochaetiales, Nemaliales, and Palmariales. However, the two genera are positioned within a clade containing the last three orders. Based on these findings, it is clear that the genus Balbiania is a valid taxon and that it is phylogenetically associated with Rhododraparnaldia. A new order is proposed, the Balbianiales. The two specimens of Balbiania analyzed from England and Germany have identical sequences for all regions of DNA analyzed.     

GRACILARIA HUMMII SP. NOV. (GRACILARIALES,RHODOPHYTA), A NEW NAME FOR THE AGAROPHYTE “GRACILARIA CONFERVOIDES” HARVESTED IN NORTH CAROLINA DURING WORLD WAR II1

Gracilaria hummii Hommers. et Freshwater is proposed as a new name for the inshore cylindrical species found in North Carolina that was treated as Gracilaria confervoides (L.) Grev. during World War II, and more recently as G. verrucosa (Huds.) Papenf. Molecular evidence places G. hummii in the Gracilis‐group in Gracilaria together with G. gracilis (Stackh.) Steentoft, L. M. Irvine et Farnham, the name currently applied to specimens formerly identified as G. confervoides and G. verrucosa. G. hummii differs from G. gracilis in possessing shallower male conceptacles in which the spermatangial filament originates from a surface cortical cell rather than from a subcortical cell. The cystocarps are similar, except that the gonimoblasts of G. hummii are attached to the base of the pericarp by numerous, prominent thickened terminal tubular cells and because terminal tubular cells are absent above the midregion of the cystocarp or in the vicinity of the ostiole. The gonimoblasts are subtended by a bundle of longitudinally oriented, thick‐walled secondary filaments of a type that has not been described before in Gracilaria but that may be characteristic of some other species. G. hummii occupies a basal position in the Gracilis‐group and is distinct from all other cylindrical North Carolina Gracilaria species, according to the molecular and morphological evidence.     

CHARACTERIZATION OF OSTREOPSIS AND COOLIA (DINOPHYCEAE) ISOLATES IN THE WESTERN MEDITERRANEAN SEA BASED ON MORPHOLOGY,TOXICITY AND INTERNAL TRANSCRIBED SPACER 5.8S rDNA SEQUENCES1

Several isolates of epiphytic dinoflagellates belonging to the genera Ostreopsis Schmidt and Coolia Meunier from the western Mediterranean Sea were examined by LM and EM, toxicity assays, and internal transcribed spacer (ITS) regions of nuclear rDNA, and 5.8S rDNA were sequenced. Morphological comparisons based on the analyses of cell shape, size, thecal plates, and surface ornamentation revealed two distinct species in the western Mediterranean: O. cf. siamensis Schmidt from the Catalan, Andalusian, and Sicilian coasts and O. ovata Fukuyo from the Ligurian coast, southern Tyrrhenian Sea, and Balearic Islands. Both Ostreopsis species were toxic; however, no differences in toxicity were detected between the two Ostreopsis species. Coolia monotis Meunier was nontoxic. The morphological studies were supported by phylogenetic analyses; all western Mediterranean isolates of O. cf. siamensis showed ITS and 5.8S rDNA sequences identical to each other and so did those of O. ovata, whereas high genetic diversity was detected between the western Mediterranean and Asian isolates of O. ovata. The nucleotide sequence analyses of the C. monotis strains showed that all C. monotis isolates from Europe formed a homogeneous clade. Further, the genetic diversity was high between the European and Asian C. monotis isolates. In this study, genetic markers combined with morphology and toxicity analyses was useful in the taxonomic and phylogenetic studies of the Ostreopsidaceae in a temperate area.     

Phylogenetic relationships among Lactuca (Asteraceae) species and related genera based on ITS-1 DNA sequences

Internal transcribed spacer (ITS-1) sequences from 97 accessions representing 23 species of Lactuca and related genera were determined and used to evaluate species relationships of Lactuca sensu lato (s.l.). The ITS-1 phylogenies, calculated using PAUP and PHYLIP, correspond better to the classification of Feráková than to other classifications evaluated, although the inclusion of sect. Lactuca subsect. Cyanicae is not supported. Therefore, exclusion of subsect. Cyanicae from Lactuca sensu Feráková is proposed. The amended genus contains the entire gene pool (sensu Harlan and De Wet) of cultivated lettuce (Lactuca sativa). The position of the species in the amended classification corresponds to their position in the lettuce gene pool. In the ITS-1 phylogenies, a clade with L. sativa, L. serriola, L. dregeana, L. altaica, and L. aculeata represents the primary gene pool. L. virosa and L. saligna, branching off closest to this clade, encompass the secondary gene pool. L. virosa is possibly of hybrid origin. The primary and secondary gene pool species are classified in sect. Lactuca subsect. Lactuca. The species L. quercina, L. viminea, L. sibirica, and L. tatarica, branching off next, represent the tertiary gene pool. They are classified in Lactuca sect. Lactucopsis, sect. Phaenixopus, and sect. Mulgedium, respectively. L. perennis and L. tenerrima, classified in sect. Lactuca subsect. Cyanicae, form clades with species from related genera and are not part of the lettuce gene pool.     

COLEOFASCICULUS GEN. NOV. (CYANOBACTERIA): MORPHOLOGICAL AND MOLECULAR CRITERIA FOR REVISION OF THE GENUS MICROCOLEUS GOMONT1

Species currently classified within the cyanobacterial genus Microcoleus were determined to fall into two distinct clades in a 16S rDNA phylogeny, one containing taxa within the Oscillatoriaceae, the other containing taxa within the Phormidiaceae. The two lineages were confirmed in an analysis of the 16S–23S internal transcribed spacer (ITS) region sequences and secondary structures. The type species for Microcoleus is M. vaginatus Gomont, and this taxon belongs in the Oscillatoriaceae. Consequently, Microcoleus taxa in the Phormidiaceae must be placed in separate genera, and we propose the new genus Coleofasciculus to contain marine taxa currently placed in Microcoleus. The type species for Coleofasciculus is the well‐studied and widespread marine mat‐forming species Microcoleus chthonoplastes (Mert.) Zanardini ex Gomont. Other characters separating the two families include type of cell division and thylakoid structure.     

Cryptic diversity and species delimitation in the Xiphinema americanum‐group complex (Nematoda: Longidoridae) as inferred from morphometrics and molecular markers

The Xiphinema americanum‐group constitutes a complex of about 55 species of polyphagous plant‐ectoparasitic nematodes with a worldwide distribution. This group of plant‐parasitic nematodes is one of the most difficult dagger nematode species complexes for diagnosis because the morphology is very conservative and morphometric characters often overlap. We conducted nematode surveys in cultivated and wild olives in southern Spain from 2012 to 2014, from which we identified 16 nematode populations of the X. americanum‐group, five of which were tentatively identified as belonging to three new species and are described herein as X iphinema plesiopachtaicum sp. nov., X iphinema vallense sp. nov. , and X iphinema astaregiense sp. nov. , and 11 populations belonging to nine known species: Xiphinema brevisicum, Xiphinema duriense, Xiphinema incertum, Xiphinema luci, Xiphinema madeirense, Xiphinema opisthohysterum, Xiphinema pachtaicum, Xiphinema parapachydermum, and Xiphinema rivesi. A phenetic study based on multivariate factor analyses was developed to compare some of these related species by using morphometric features. In the factor analysis the first four factors accounted for 73.1% of the total variance of the selected characters, identifying body length, body length/maximum body width (a), body length/pharyngeal length (b), body length/tail length (c), and tail length/body width at anus (c′) ratios, distance from anterior end to vulva as percentage of body length (V), stylet length, oral aperture‐guiding ring distance, and lip region width as key morphometric characters to differentiate a restricted set of species within the X. pachtaicum‐subgroup that includes X. plesiopachtaicum sp. nov. and X. vallense sp. nov. Multivariate analysis of variance using these specific characters allowed to differentiate species in the X. pachtaicum complex or groups of them using morphometric characters (body length, a, b, c, c′, V, stylet length, lip region width, oral aperture‐guiding ring distance, female tail length, and hyaline region length). Phylogenetic analyses based on nuclear ribosomal DNA genes [D2‐D3 expansion segments of large ribosomal subunit 28S, and internal transcribed spacer 1 (ITS1)] and the protein‐coding mitochondrial gene, cytochrome c oxidase subunit 1 (coxI) were congruent, showing two main clades separating most of the species of X. americanum‐subgroup ‘sensu stricto’ from the X. pachtaicum‐subgroup. Agreement between phylogenetic trees and some morphological characters (viz. total stylet length, vulva position, and a ratio) were tested by reconstruction of their histories on rRNA‐based trees using parsimony and Bayesian approaches. Thus, integrative taxonomy, based on a combination of multivariate morphological and molecular analyses constitutes a new insight into the identification of X. americanum‐group species. © 2015 The Linnean Society of London