Geographic parthenogenesis and the common tea‐tree stick insect of New Zealand

Worldwide, parthenogenetic reproduction has evolved many times in the stick insects (Phasmatidae). Many parthenogenetic stick insects show the distribution pattern known as geographic parthenogenesis, in that they occupy habitats that are at higher altitude or latitude compared with their sexual relatives. Although it is often assumed that, in the short term, parthenogenetic populations will have a reproductive advantage over sexual populations; this is not necessarily the case. We present data on the distribution and evolutionary relationships of sexual and asexual populations of the New Zealand stick insect, Clitarchus hookeri. Males are common in the northern half of the species’ range but rare or absent elsewhere, and we found that most C. hookeri from putative‐parthenogenetic populations share a common ancestor. Female stick insects from bisexual populations of Clitarchus hookeri are capable of parthenogenetic reproduction, but those insects from putative‐parthenogenetic populations produced few offspring via sexual reproduction when males were available. We found similar fertility (hatching success) in mated and virgin females. Mated females produce equal numbers of male and female offspring, with most hatching about 9–16 weeks after laying. In contrast, most eggs from unmated females took longer to hatch (21–23 weeks), and most offspring were female. It appears that all C. hookeri females are capable of parthenogenetic reproduction, and thus could benefit from the numerical advantage this yields. Nevertheless, our phylogeographic evidence shows that the majority of all‐female populations over a wide geographic area originate from a single loss of sexual reproduction.     

Phylogeography and ecological niche modelling of the New Zealand stick insect Clitarchus hookeri (White) support survival in multiple coastal refugia

Aim Increasing our understanding of the effects of the Last Glacial Maximum (LGM) and determining the location of refugia requires studies on widely distributed species with dense sampling of populations. We have reconstructed the biogeographic history of Clitarchus hookeri (White), a widespread species of New Zealand stick insect that exhibits geographic parthenogenesis, using phylogeographic analysis and ecological niche modelling. Location New Zealand. Methods We used DNA sequence data from the mitochondrial cytochrome c oxidase subunit I gene to reconstruct phylogenetic relationships among haplotypes from C. hookeri and two undescribed Clitarchus species. We also used distribution data from our own field surveys and museum records to reconstruct the geographic distribution of C. hookeri during the present and the LGM, using ecological niche modelling. Results The ecological niche models showed that the geographic distribution of C. hookeri has expanded dramatically since the LGM. Our model predicted large areas of suitable LGM habitat in upper North Island, and small patches along the east coast of South Island. The phylogeographic analysis shows that populations in the northern half of North Island contain much higher levels of genetic variation than those from southern North Island and South Island, and is congruent with the ecological niche model. The distribution of bisexual populations is also non-random, with males completely absent from South Island and very rare in southern North Island. Main conclusions During the LGM C. hookeri was most likely restricted to several refugia in upper North Island and one or more smaller refugia along the east coast of South Island. The unisexual populations predominate in post-glacial landscapes and are clearly favoured in the recolonization of such areas. Our study exemplifies the utility of integrating ecological niche modelling and phylogeographic analysis.     

Growth form evolution and hybridization in Senecio (Asteraceae) from the high equatorial Andes

Changes in growth forms frequently accompany plant adaptive radiations, including páramo–a high‐elevation treeless habitat type of the northern Andes. We tested whether diverse group of Senecio inhabiting montane forests and páramo represented such growth form changes. We also investigated the role of Andean geography and environment in structuring genetic variation of this group. We sampled 108 populations and 28 species of Senecio (focusing on species from former genera Lasiocephalus and Culcitium) and analyzed their genetic relationships and patterns of intraspecific variation using DNA fingerprinting (AFLPs) and nuclear DNA sequences (ITS). We partitioned genetic variation into environmental and geographical components. ITS‐based phylogeny supported monophyly of a Lasiocephalus‐Culcitium clade. A grade of herbaceous alpine Senecio species subtended the Lasiocephalus‐Culcitium clade suggesting a change from the herbaceous to the woody growth form. Both ITS sequences and the AFLPs separated a group composed of the majority of páramo subshrubs from other group(s) comprising both forest and páramo species of various growth forms. These morphologically variable group(s) further split into clades encompassing both the páramo subshrubs and forest lianas, indicating independent switches among the growth forms and habitats. The finest AFLP genetic structure corresponded to morphologically delimited species except in two independent cases in which patterns of genetic variation instead reflected geography. Several morphologically variable species were genetically admixed, which suggests possible hybrid origins. Latitude and longitude accounted for 5%–8% of genetic variation in each of three AFLP groups, while the proportion of variation attributed to environment varied between 8% and 31% among them. A change from the herbaceous to the woody growth form is suggested for species of high‐elevation Andean Senecio. Independent switches between habitats and growth forms likely occurred within the group. Hybridization likely played an important role in species diversification.     

Loss and gain of sexual reproduction in the same stick insect

The outcome of competition between different reproductive strategies within a single species can be used to infer selective advantage of the winning strategy. Where multiple populations have independently lost or gained sexual reproduction it is possible to investigate whether the advantage is contingent on local conditions. In the New Zealand stick insect Clitarchus hookeri, three populations are distinguished by recent change in reproductive strategy and we determine their likely origins. One parthenogenetic population has established in the United Kingdom and we provide evidence that sexual reproduction has been lost in this population. We identify the sexual population from which the parthenogenetic population was derived, but show that the UK females have a post‐mating barrier to fertilisation. We also demonstrate that two sexual populations have recently arisen in New Zealand within the natural range of the mtDNA lineage that otherwise characterizes parthenogenesis in this species. We infer independent origins of males at these two locations using microsatellite genotypes. In one population, a mixture of local and nonlocal alleles suggested males were the result of invasion. Males in another population were most probably the result of loss of an X chromosome that produced a male phenotype in situ. Two successful switches in reproductive strategy suggest local competitive advantage for outcrossing over parthenogenetic reproduction. Clitarchus hookeri provides remarkable evidence of repeated and rapid changes in reproductive strategy, with competitive outcomes dependent on local conditions.     

Molecular and morphological characterization of a poorly known marine ciliate, Myoschiston duplicatum precht 1935: implications for phylogenetic relationships between three morphologically similar genera -- Zoothamnium, Myoschiston, and Zoothamnopsis (Ciliophora, Peritrichia, Zoothamniidae)

We studied the morphology and molecular phylogeny of Myoschiston duplicatum, a peritrich ciliate that has been recorded as an epibiont of crustaceans, but which we also identified on marine algae from Korea. The important morphological characteristics revealed by silver staining of Myoschiston species have not been described because they are rarely collected. Using morphological methods, we redescribed the type species of the genus, Myoschiston duplicatum, and provided an improved diagnosis of Myoschiston. In addition, the coding regions for nuclear small subunit (SSU) rRNA and internal transcribed spacer 1‐5.8S‐internal transcribed spacer 2 sequences were sequenced. Phylogenetic analyses that included available SSU rDNA sequences of peritrichs from GenBank strongly supported a position of M. duplicatum within the family Zoothamniidae. In addition, phylogenetic analyses were performed with single datasets (ITS1‐5.8S‐ITS2) and combined datasets (SSU rDNA + ITS1‐5.8S‐ITS2) to explore further the phylogenetic relationship in the family Zoothamniidae between the three morphologically similar genera—Zoothamnium, Myoschiston, and Zoothamnopsis.     

Genetic distinctness of parthenogenetic forms of European Polydrusus weevils of the subgenus Scythodrusus

Abstract Among eight species of Polydrusus weevils which belong to subgenus Scythodrusus, at least two possess parthenogenetic forms: P. (S.) inustus and P. (S.) pilifer. Both of these species consist of dioecious populations in the Caspian area and of parthenogenetic populations in Eastern Europe (P. (S.) inustus), the Caucasus region (both species) and Middle Asia (P. (S.) pilifer). The origin of parthenogenesis in this subgenus is unresolved; however some data suggest that the parthenogenetic forms are of hybrid ancestry. The genetic distinctness of parthenogenetic Scythodrusus was assessed on the basis of COII, ITS2, EF1‐α and Wolbachiawsp, 16S ribosomal DNA, ftsZ and hcpA sequence comparisons. Both taxa turned out to be monophyletic for all markers, which is an evidence against hybridization of their dioecious ancestors. On the other hand, a high frequency of heterozygous P. (S.) inustus females suggests an origin resulting from hybridization between genetically distinct dioecious representatives of this species. Very similar strains of Wolbachia supergroup A were found in both species, indicating that they have been either inherited from a common ancestor or were transmitted between parthenogenetic Scythodrusus weevils and probably spread randomly across their ranges.     

A REASSESSMENT OF SPECIES BOUNDARIES IN CYSTOSEIRA AND HALIDRYS (PHAEOPHYCEAE,FUCALES) ALONG THE NORTH AMERICAN WEST COAST1

Application of phylogenetic species recognition to morphologically recognized species in the genera Cystoseira Agardh and Halidrys Lyngbye on North American west coasts revealed little genetic variation despite a remarkable degree of morphological variation currently used to recognize and delineate species. Whereas morphological characteristics allow recognition of two genera, four morphological species and three informal forms, maximum genetic variation among them was similar to that characteristic of the intraspecific level in European congeners and other Fucales. Among morphological species and forms, nucleotide variation in a combined 26S (large subunit (LSU)) and internal transcribed spacer (ITS) ribosomal DNA analysis was below 3% while it was 1% or less for the RUBISCO spacer of the chloroplast DNA. Comparison of the LSU data to available data for European congeners showed that the genera Cystoseira and Halidrys are not monophyletic and that the previously recognized Cystoseiraceae should be included within the family Sargassaceae. These observations suggest that the current taxonomy for the Sargassaceae fails to reflect evolutionary history because Atlantic and Pacific Cystoseira and Halidrys appear to have arrived at similar morphologies independently. Our results indicate a comparatively recent establishment on the west coast of North America of a sargassacean progenitor whose descendant taxa have experienced limited genetic divergence and are characterized by a high capacity for phenotypic variation despite their overall genetic similarity.     

Molecular phylogeny of fig wasp pollinators (Agaonidae, Hymenoptera) of Ficus section Galoglychia

The obligate mutualism between fig trees and their fig wasp pollinators, together with the general tendency for each host species to be pollinated by one fig wasp species, led to the hypothesis that these two lineages have cospeciated. The pollinators of African figs of section Galoglychia form a diverse group of genera whose species seem to be less constrained to a specific host than other pollinating fig wasp genera. Various authors have suggested remarkably different phylogenetic relationships between the seven genera associated with section Galoglychia. These uncertainties concerning the classification make it difficult to understand the historical patterns of association between these wasps and their hosts. The phylogenetic tree for the pollinators was reconstructed with 28S, COI and ITS2 DNA sequence data and compared with morphological classification of the hosts. Pollinator genera were monophyletic in all analyses. However, the relative position of some genera remains unresolved. Investigation of host−fig association suggests that there have been frequent host jumps between host subsections. This indicates that cospeciation between fig trees and fig wasps is not as stringent as previously assumed. In addition, pollinators of the genus Alfonsiella associated with three host figs (Ficus craterostoma, F. stuhlmannii and F. petersii) are morphologically very similar in South Africa. We investigated the possibility that these pollinators form a complex of species with host‐based genetic differentiation. Molecular analyses supported the distinction of the pollinator of F. craterostoma as a good species, but the pollinators of F. stuhlmannii and F. petersii clustered within the same clade, suggesting that these two host species share a single pollinator, Alfonsiella binghami. Based on both molecular data and morphological re‐evaluation, a new Alfonsiella species is described, Alfonsiella pipithiensis sp. nov., which is the pollinator of F. craterostoma in southern Africa. A key to both females and males of all described species of Alfonsiella is provided.     

Dispersal flight and colony development in the fungus-growing termites <Emphasis Type="Italic">Pseudacanthotermes spiniger</Emphasis> and <Emphasis Type="Italic">P. militaris</Emphasis>

Pseudacanthotermes spiniger and P. militaris are two African fungus-growing termites (Termitidae, Macrotermitinae) which may become pests in disturbed agrosystems where they often live in sympatry. To study their development and their reproductive strategies, colonies of both species were reared in the laboratory for 20 and 17 years, respectively, after their foundation from reproductive pairs. The first steps of development were in great part similar in both species, although P. spiniger favoured the defence during the juvenile period, while P. militaris tended to favour a rapid development. While P. spiniger colonies did not produce alates until year 7 of colony life, P. militaris colonies were able to produce a fertile progeny 4 years after their foundation. In contrast, major soldiers were more rapidly differentiated in the incipient colonies of P. spiniger. Dispersal flights occurred every year for 10 years in P. spiniger. In P. militaris, dispersal flights did not occur regularly although alates appeared yearly. The annual number of alates produced by P. spiniger increased with the colony age to reach a maximum of 25,000 individuals and global production of alates was estimated at ca. 150,000 individuals in the life of a colony. The longevity of P. spiniger and P. militaris colonies was around 20 years. These species were shown to be reproductively isolated by multiple pre-mating mechanisms. While chronological differences in dispersal flights contribute to reproductive isolation of the two species, the non-viability of experimental hybrid colonies also indicates the involvement of post-mating mechanisms of isolation.     

USE OF RIBOSOMAL DNA INTERNAL TRANSCRIBED SPACERS FOR PHYLOGENETIC STUDIES IN DIATOMS1

Sequence variation of ribosomal DNA internal transcribed spacers (ITS) among populations, species, and genera of the diatom genus Stephanodiscus was investigated. ITS 1 and ITS 2, including the 5.8S gene, were sequenced from geographically distant and nearby populations of S. niagarae Ehrenberg. In addition, repeats from S. hantzschii Grunow and Cyclotella meneghiniana Kützing were sequenced to determine the taxonomic range over which the ITS region could be used for diatom systematics. The morphologically distinct S. yellowstonensis Theriot & Stoermer, thought to have evolved from S. niagarae in Yellowstone Lake between 12,000 and 8000 years ago, also was sequenced to assess its relationship to nearby S. niagarae populations. The organization and relative sizes of ITS 1 and ITS 2 in Stephanodiscus species were similar to those reported for other eukaryotes. In general, ITS 2 was slightly larger and more variable than ITS 1. Cladistic analysis of ITS sequences did not resolve relationships of nearby S. niagarae and S. yellowstonensis populations. However, central North American S. niagarae populations were in a clade supported by two nucleotide changes. For Cyclotella, much of the ITS region was not alignable with that for Stephanodiscus species; therefore, generic-level comparison within the Thalassiosiraceae may not be possible. The variation (95–96% similarity) between S. hantzschii and other Stephanodiscus species suggests that interspecific relationships could be assessed with ITS sequences. Although S. yellowstonensis is morphologically distinct from S. niagarae, no autapomorphic nucleotide sites were identified. Two S. niagarae populations (Heart and Lewis Lakes), however, did possess autapomorphic ITS sites.     

Are you my mother? Phylogenetic analysis reveals orphan hybrid stick insect genus is part of a monophyletic New Zealand clade

The hybrid stick insect genus Acanthoxyla Uvarov 1944 is unusual for an obligate parthenogen, in the extreme morphological diversity it exhibits that has led to eight species being recognised. The New Zealand sexual species Clitarchus hookeri [White, A. 1846. The zoology of the Voyage of H.M.S. Erebus and Terror. In: 1 Insects of New Zealand. E.W. Janson, London.] is the putative parental species in the hybridization that gave rise to the hybrid lineage Acanthoxyla. In an effort to identify the maternal ancestor of Acanthoxyla we sequenced nuclear 28S rDNA and/or mtDNA COI & COII of all nine endemic New Zealand stick insect genera, representing 17 of the 22 described species. We also sequenced 28S from eight non-New Zealand stick insects to supplement published 28S sequence data that provided a taxonomically and geographically broad sampling of the phasmids. We applied a novel search algorithm (SeqSSi=Sequence Similarity Sieve) to assist in selection of outgroup taxa for phylogenetic analysis prior to alignment. Phylogenetic reconstructions resolved an exclusively New Zealand clade to which the maternal lineage of Acanthoxyla belonged, but did not support existing higher level taxonomy of stick insects. We did not find a sexual maternal species for Acanthoxyla but phylogenetic relationships indicate that this species lived in New Zealand and could be classified among the New Zealand Phasmatinae. Among the available taxa, the nearest evolutionary neighbours to the New Zealand phasmid fauna as a whole were predominantly from the New Zealand region (Fiji, Australia, New Guinea, New Caledonia and South America). As it appears to be an orphan, it is interesting to speculate that a combination of parthenogenetic reproduction and/or hybrid vigour in Acanthoxyla may have contributed to the extinction of its mother.     

Morphological and molecular characterization of <Emphasis Type="Italic">Mariannaea aquaticola</Emphasis> sp. nov. collected from freshwater habitats

Mariannaea aquaticola sp. nov. is described, illustrated, and compared with closely related taxa. Phylogenetic analysis of rDNA ITS and 28S sequences show that Mariannaea aquaticola has affinity with Nectriaceae, which is in agreement with other Mariannaea species. Other morphologically similar genera such as Verticillium and Clonostachys are shown to be phylogenetically distant to Mariannaea. The generic concept of Mariannaea is discussed, and the affinity to Nectriaceous teleomorphs is suggested as additional generic character. A key to Mariannaea species is provided.     

Phylogenetic species delimitation in ectomycorrhizal fungi and implications for barcoding: the case of the Tricholoma scalpturatum complex (Basidiomycota)

Population studies have revealed that the fungal ectomycorrhizal morphospecies Tricholoma scalpturatum consists of at least two genetically distinct groups that occur sympatrically in several geographical areas. This discovery prompted us to examine species boundaries and relationships between members formerly assigned to T. scalpturatum and allied taxa using phylogenetic analyses. Sequence data were obtained from three nuclear DNA regions [internal transcribed spacer (ITS), gpd and tef], from 101 carpophores collected over a large geographical range in Western Europe, and some reference sequences from public databases. The ITS was also tested for its applicability as DNA barcode for species delimitation. Four highly supported phylogenetic clades were detected. The two previously detected genetic groups of T. scalpturatum were assigned to the phylospecies Tricholoma argyraceum and T. scalpturatum. The two remaining clades were referred to as Tricholoma cingulatum and Tricholoma inocybeoides. Unexpectedly, T. cingulatum showed an accelerated rate of evolution that we attributed to narrow host specialization. This study also reveals recombinant ITS sequences in T. inocybeoides, suggesting a hybrid origin. The ITS was a useful tool for the determination of species boundaries: the mean value of intraspecific genetic distances in the entire ITS region (including 5.8S rDNA) was <0.2%, whereas interspecific divergence estimates ranged from 1.78% to 4.22%. Apart from giving insights into the evolution of the T. scalpturatum complex, this study contributes to the establishment of a library of taxonomically verified voucher specimens, an a posteriori correlation between phenotype and genotype, and DNA barcoding of ectomycorrhizal fungi.     

Characterization of geographically isolated accessions in five Alstroemeria L. species (Chile) using FISH of tandemly repeated DNA sequences and RAPD analysis

In fifteen geographically isolated populations of five species of Alstroemeria L. (A. aurea, A. hookeri, A. ligtu, A. pelegrina and A. presliana) collected in Chile, karyotypes and variation of RAPD markers were investigated. Tandemly repeated DNA sequences - 5S and 18/25S rDNA genes and the sequence A001-1 (De Jeu et al. 1997) were used to characterize karyotypes by fluorescence in situ hybridization (FISH). Ten somatic metaphases per population were used for measurement of chromosome length. Differences in RAPD marker bands were used for characterization of populations, creating a similarity index. FISH with all three DNA probes shows a high degree of polymorphism between and sometimes also within accessions of A. aurea, A. hookeri and A. ligtu. The number of chromosome pairs showing 5S rDNA signals is more different for the investigated species A. aurea, A. hookeri, A. ligtu, A. pelegrina and A. presliana with 5, 7, 5, 3 and 7, respectively, than the number of 18/25S rDNA signals in this succession with 7, 7, 6, 5 and 7 chromosome pairs, showing a high evolutionary dynamics within the genus. Furthermore, among the four populations of A. hookeri, accession 4181 was different in arm length of chromosome 3. RAPD markers (index of similarity) also showed a greater genetic distance of accession 4181 from the other three accessions of A. hookeri. The possible evolutionary mechanisms providing these polymorphisms were discussed.     

Resolving the unresolved tribe: a molecular phylogenetic framework for the Merremieae (Convolvulaceae)

Tribe Merremieae, as currently circumscribed, comprise c. 120 species classified in seven genera, the largest of which (Merremia) is morphologically heterogeneous. Previous studies, with limited sampling, have suggested that neither Merremieae nor Merremia are monophyletic. In the present study, the monophyly of Merremia and its allied genera was re‐assessed, sampling 57 species of Merremieae for the plastid matK, trnL–trnF and rps16 regions and the nuclear internal transcribed spacer (ITS) region. All genera of Merremieae and all major morphotypes in Merremia were represented. Phylogenetic analyses resolve Merremieae in a clade with Ipomoeae, Convolvuleae and Daustinia montana. Merremia is confirmed as polyphyletic and a number of well‐supported and morphologically distinct clades in Merremieae are recognized which accommodate most of the species in the tribe. These provide a framework for a generic revision of the assemblage. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015.     

Genetic variation and evolutionary origins of parthenogenetic Artemia (Crustacea: Anostraca) with different ploidies

Using two nuclear (ITS1 and Na⁺/K⁺ ATPase) and three mitochondrial (COI, 16S and 12S) markers, we determined the genetic variation and evolutionary relationship of parthenogenetic and bisexual Artemia. Our analyses revealed that mitochondrial genes had higher genetic variation than nuclear genes and that the 16S showed more variety than the other mitochondrial genes in parthenogenetic populations. Triploid parthenogens showed lower genetic variation than diploid ones, whereas the tetra‐ and pentaploids had greater genetic distance than diploid parthenogens. No shared haplotype was found between individuals of parthenogenetic populations and Asian bisexual species with the exception of Na⁺/K⁺ ATPase (Artemia tibetiana). Only mitochondrial markers can demonstrate phylogenetic relationships, and showed that the parthenogenetic Artemia is a polyphyletic group in which the diploid lineages share a common ancestor with Artemia urmiana while tetraploids are closely related to Artemia sinica. The triploid and pentaploid linages are likely to be directly derived from diploid and tetraploid parthenogens, respectively. Subsequently, west Asia is origin for di‐/triploids, and tetra‐/pentaploids rose from East Asia.     

Species distinctions among closely related strains of Eustigmatophyceae (Stramenopiles) emphasizing ITS2 sequence-structure data: Eustigmatos and Vischeria

ABSTRACT

There is an increasing interest in the Eustigmatophyceae, a class of stramenopile microalgae, because they offer a variety of high-value health-beneficial compounds, e.g. polyunsaturated fatty acids (PUFAs), while concomitantly producing high biomass. Clarification of the taxonomy of these organisms at the species level is important in order to achieve reproducible results and constant yields of valuable compounds in their exploitation. Here the distinction of the, so far exclusively, morphologically defined species of the genera Eustigmatos and Vischeria was tested. Distinctions inferred from almost full 18S and ITS2 rRNA as well as plastid-encoded rbcL gene sequences were evaluated following a morphological investigation. The ITS2 secondary-structure-based phylogenies separated independent lineages (species) with long internal branches. This recommends ITS2 as a promising marker for a DNA metabarcoding approach (culture-independent biodiversity assessment). In contrast, the 18S V4 region which is commonly used in metabarcoding was almost invariant, whereas the almost full length sequences distinguished eight groups/types of strains. Monophyly of the species was supported by shared ITS2 secondary structure features, making them distinct from other eustigmatophyte lineages in concordance with phylogenetic analyses. No groups of strains were congruently supported by all three markers. Consequently, the previous distinction of two genera on the basis of morphology cannot be retained and the species should be accommodated in a single genus, Vischeria. Taxonomic changes among the species with the definition of epitypes, on the basis of cryopreserved strains, are recommended. Two findings point to a more complex evolutionary history of the species. The rbcL and nuclear markers resulted in disparate groupings of strains. In three species divergent intragenomic ITS2 paralogues were revealed. Therefore, a still broader taxon sampling, in conjunction with a deep sequencing approach, is needed for a more comprehensive understanding of the complex evolution of eustigmatophyte species.     

RECOGNIZING DINOFLAGELLATE SPECIES USING ITS rDNA SEQUENCES1

Dinoflagellate taxonomy is based primarily on morphology and morphometric data that can be difficult to obtain. In contrast, molecular data can be rapidly and cost‐effectively acquired, which has led to a rapid accumulation of sequence data in GenBank. Currently there are no systematic criteria for utilizing taxonomically unassigned sequence data to identify putative species that could in turn serve as a basis for testable hypotheses concerning the taxonomy, diversity, distribution, and toxicity of these organisms. The goal of this research was to evaluate whether simple, uncorrected genetic distances (p) calculated using ITS1/5.8S/ITS2 (ITS region) rDNA sequences could be used to develop criteria for recognizing putative species before formal morphological evaluation and classification. The current analysis used sequences from 81 dinoflagellate species belonging to 14 genera. For this diverse assemblage of dinoflagellate species, the within‐species genetic distances between ITS region copies (p=0.000–0.021 substitutions per site) were consistently less than those observed between species (p=0.042–0.580). Our results indicate that a between‐species uncorrected genetic distance of p≥0.04 could be used to delineate most free‐living dinoflagellate species. Recently evolved species, however, may have ITS p values <0.04 and would require more extensive morphological and genetic analyses to resolve. For most species, the sequence of the dominant ITS region allele has the potential to serve as a unique species‐specific “DNA barcode” that could be used for the rapid identification of dinoflagellates in field and laboratory studies.     

PHYLOGENY AND GENETIC VARIANCE IN TERRESTRIAL MICROCOLEUS (CYANOPHYCEAE) SPECIES BASED ON SEQUENCE ANALYSIS OF THE 16S rRNA GENE AND ASSOCIATED 16S–23S ITS REGION1

Thirty‐one strains of Microcoleus were isolated from desert soils in the United States. Although all these taxa fit the broad definition of Microcoleus vaginatus (Vaucher) Gomont in common usage by soil algal researchers, sequence data for the 16S rRNA gene and 16S–23S internal transcribed spacer (ITS) region indicated that more than one species was represented. Combined sequence and morphological data revealed the presence of two morphologically similar taxa, M. vaginatus and Microcoleus steenstrupii Boye‐Petersen. The rRNA operons of these taxa were sufficiently dissimilar that we suspect the two taxa belong in separate genera. The M. vaginatus clade was most similar to published sequences from Trichodesmium and Arthrospira. When 16S sequences from the isolates we identified as M. steenstrupii were compared with published sequences, our strains grouped with M. chthonoplastes (Mertens) Zanardini ex Gomont and may have closest relatives among several genera in the Phormidiaceae. Organization within the 16S–23S ITS regions was variable between the two taxa. Microcoleus vaginatus had either two tRNA genes (tRNA^Ile and tRNA^Ala) or a fragment of the tRNA^Ile gene in its ITS regions, whereas M. steenstrupii had rRNA operons with either the tRNA^Ile gene or no tRNA genes in its ITS regions. Microcoleus vaginatus showed no subspecific variation within the combined morphological and molecular characterizations, with 16S similarities ranging from 97.1% to 99.9%. Microcoleus steenstrupii showed considerable genetic variability, with 16S similarities ranging from 91.5% to 99.4%. In phylogenetic analyses, we found that this variability was not congruent with geography, and we suspect that our M. steenstrupii strains represent several cryptic species.     

Morphology of the early zoeal stages of Macropipus tuberculatus (Roux, 1830) (Crustacea, Brachyura, Portunidae)

The first three zoeal stages of the portunid crab Macropipus tuberculatus (Roux, 1830) are described and illustrated from laboratory-reared material obtained from one ovigerous crab captured in the western Mediterranean Sea. Zoeae of M.tuberculatus can be distinguished from other known polybiinid species from the northeastern Atlantic and Mediterranean waters by a combination of meristic and morphometric features, especially by the number of setae in the outer lobe of the maxilla endopod. Larval morphology presents additional evidence supporting the split of the genus Macropipus into the genera Liocarcinus and Macropipus sensu stricto based on adult characters.