Characterization and analysis of simple sequence repeat (SSR) loci in seashore paspalum (Paspalum vaginatum Swartz)

A size-fractionated TaqI genomic library of seashore paspalum (Paspalum vaginatum Swartz) was screened for the presence of (GA) _n and (CA) _n simple sequence repeats (SSRs). A total of 54 clones with a positive signal were detected among 13,000 clones screened. Forty-seven clones having repeats of n 3 were identified, of which 85% were perfect, 13% were imperfect and 2% were compound repeat sequences. Five of ten primer pairs synthesized to amplify selected loci resulted in a product in the expected size range and were subsequently used to examine SSR polymorphisms among 46 ecotypes of P. vaginatum. The number of alleles resolved on agarose or polyacrylamide gels were similar and ranged from 6 to 16 with an average of 14 per locus. Phenetic analysis of SSR polymorphisms revealed genetic relationships among the P. vaginatum ecotypes that were in general agreement with relationships determined previously by RAPD analysis of the same plant materials. Further screening of the genomic library did not identify (AT) _n , trimeric or tetrameric repeats. Hybridization of an (ATT)₈ oligonucleotide probe to genomic DNA isolated from I. batatas, E. coli, Citrullis lanatus and P. vaginatum suggested that the P. vaginatum genome contained significantly fewer ATT repeats than either the I. batatas or C. lanatus genome.     

Genetic diversity and relationships of sweetpotato and its wild relatives in Ipomoea series Batatas (Convolvulaceae) as revealed by inter-simple sequence repeat (ISSR) and restriction analysis of chloroplast DNA

Genetic diversity and relationships of 40 accessions of Ipomoea, representing ten species of series Batatas, were examined using ISSR markers and restriction-site variation in four non-coding regions of chloroplast DNA. A total of 2071 ISSR fragments were generated with 15 primers in these accessions and, on average, 52 bands per accession were amplified. Most of the primers contained dinucleotide repeats. The ISSR fragments were highly polymorphic (62.2%) among the 40 accessions studied. Restriction analysis of chloroplast (cp) DNA revealed 47 informative restriction-site and length mutations. Phylogenetic analyses of ISSR and cpDNA datasets generally revealed similar relationships at the interspecific level, but the high polymorphism of ISSRs resulted in a better separation of intraspecific accessions. However, the combined ISSR and cpDNA dataset appeared to be appropriate in resolving both intra- and interspecific relationships. Of the species examined, I. trifida was found to be the most closely related to cultivated sweetpotato, the hexaploid I. batatas, while I. ramosissima and I. umbraticola were the most distantly related to I. batatas within the series. Ipomoea triloba, hitherto considered to be one of the ancestors of sweetpotato, was only distantly related to sweetpotato based on ISSR similarity index. Received: 4 January 1999 / Accepted: 27 September 1999     

GENETIC RELATIONSHIPS AND PATTERNS OF ALLOZYMIC DIVERGENCE IN THE IPOMOPSIS AGGREGATA COMPLEX AND RELATED SPECIES (POLEMONIACEAE)

The Ipomopsis aggregata complex consists of diploid, outcrossing, perennial herbs. The group is highly variable morphologically and is treated as three species: I. aggregata, I. tenuituba, and I. arizonica. Geographic races of I. aggregata and I. tenuituba are recognized as subspecies. Enzyme electrophoresis was used to examine genetic relationships among populations and taxa in the Ipomopsis aggregata complex and some related species. Genetic data for 23 allozyme loci from 60 populations were also used to determine how genetic variation is distributed geographically. Populations in the southwestern United States were more variable than those in the northwest: the center of genetic diversity corresponded to the center of species diversity. Allozymic data provided no evidence of loss of genetic variability associated with recent and rapid divergence. Genetic relationships based on Nei's genetic identity did not correspond to taxonomic relationships. For example, populations of both I. arizonica and I. tenuituba clustered within I. aggregata. Despite relatively high levels of genetic diversity among populations, diversity among taxa was low. Results indicated that floral divergence and concomitant speciation have occurred recently in the Ipomopsis aggregata complex. Allozymic patterns also reflected convergent evolution for floral morphology and possible introgression. Despite morphological differences among species, insufficient evolutionary time has elapsed for allelic fixation at neutral or near-neutral allozyme loci.     

Chloroplast DNA variations in sect. Cyclachaena of Iva (Asteraceae)

Much argument has been occasioned by questions having to do with generic concepts in the tribe Heliantheae, subtribe Ambrosiinae, especially involving the genus Iva. Some workers recognize five genera (e.g., Leuciva, Chorisiva, Oxytenia, Cyclachaena, and Iva s. str.), while others recognize only a single genus Iva s. 1. Although chemical, pollen ultrastructure, and other morphological data have challenged the latter concept, relationships among the segregate taxa are still unclear. The current study used cladistic analyses of chloroplast DNA variation to investigate evolutionary relationships within Iva s. l., especially within the controversial section Cyclachaena. Nuclear rDNA was also utilized to test for hybrid origin of selected taxa. The results support the dismemberment of Iva s. l. and recognition of Iva s. str., largely because Iva s. l. is shown to be paraphyletic. Most members of the section Cyclachaena were found to have relatively close interspecific relationships. Nevertheless, cpDNA data strongly support two lineages within Cyclachaena. One lineage, including I. xanthifolia, I. acerosa, I. nevadensis, and I. dealbata, has a close relationship with the genus Euphrosyne. The second lineage includes only one species, I. ambrosiaefolia, which is closely related to Dicoria. The origin of I. ambrosiaefolia was shown to be monophyletic, not involving hybridization.     

Restriction enzyme analysis of the nuclear 45s ribosomal DNA of six cultivated Alliums (Alliaceae)

Estimates of the phylogenetic relationships among cultivated and wildAllium species would benefit from identification of objective molecular characters. Restriction fragment length polymorphisms in the nuclear 45s ribosomal DNA (rDNA) were identified among two of five accessions of each of six cultivated Alliums. Restriction enzyme sites forBamHI,DraI,EcoRI,EcoRV,SacI, andXbaI were mapped. Different lengths of the rDNA repeat unit among the cultivated Alliums were due to sizes of the intergenic spacer. Nineteen polymorphic restriction enzyme sites were discovered and used to estimate phylogenetic relationships. Cladistic analyses based on Wagner parsimony were completed without an outgroup and resulted in two equally most parsimonious trees of 22 steps. A combined analysis of differences at RE sites in the ribosomal (19 characters) and chloroplast (15 characters) DNA generated a single most parsimonious tree of 39 steps. Single trichotomies were observed at 40 and 41 steps. Strict consensus of the three trees of 41 or fewer steps consisted of a lineage forA. tuberosum, a second forA. ampeloprasum andA. sativum, and a third forA. cepa, A. fistulosum, andA. schoenoprasum. Estimates of phylogenetic relationships based on variability at restriction enzyme sites in the rDNA and chloroplast DNA agree with the classification scheme ofTraub. Because of the predominance of autapomorphies, restriction enzyme analysis of the nuclear 45s rDNA is of limited use in estimating phylogenies amongAllium sections. However it is useful in the establishment of interspecific hybridity.     

DArT for high-throughput genotyping of Cassava (Manihot esculenta) and its wild relatives

Understanding the distribution of genetic diversity within and among individuals, populations, species and gene pools is crucial for the efficient management of germplasm collections. Molecular markers are playing an increasing role in germplasm characterization, yet their broad application is limited by the availability of markers, the costs and the low throughput of existing technologies. This is particularly true for crops of resource-poor farmers such as cassava, Manihot esculenta. Here we report on the development of Diversity Arrays Technology (DArT) for cassava. DArT uses microarrays to detect DNA polymorphism at several hundred genomic loci in a single assay without relying on DNA sequence information. We tested three complexity reduction methods and selected the two that generated genomic representations with the largest frequency of polymorphic clones (PstI/TaqI: 14.6%, PstI/BstNI: 17.2%) to produce large genotyping arrays. Nearly 1,000 candidate polymorphic clones were detected on the two arrays. The performance of the PstI/TaqI array was validated by typing a group of 38 accessions, 24 of them in duplicate. The average call rate was 98.1%, and the scoring reproducibility was 99.8%. DArT markers displayed fairly high polymorphism information content (PIC) values and revealed genetic relationships among the samples consistent with the information available on these samples. Our study suggests that DArT offers advantages over current technologies in terms of cost and speed of marker discovery and analysis. It can therefore be used to genotype large germplasm collections.Electronic Supplementary Material Supplementary material is available for this article at     

Morphological and molecular description of new species of squat lobster (Crustacea: Decapoda: Galatheidae) from the Solomon and Fiji Islands (South‐West Pacific)

The family Galatheidae is among the most diverse families of anomuran decapod crustaceans, and the South‐West Pacific is a biodiversity hot spot for these squat lobsters. Attempts to clarify the taxonomic and evolutionary relationships of the Galatheidae on the basis of morphological and molecular data have revealed the existence of several cryptic species, differentiated only by subtle morphological characters. Despite these efforts, however, relationships among genera are poorly understood, and the family is in need of a detailed systematic review. In this study, we assess material collected in different surveys conducted in the Solomon Islands, as well as comparative material from the Fiji Islands, by examining both the morphology of the specimens and two mitochondrial markers (cytochrome oxidase subunit I, COI, and 16S rRNA). These two sources of data revealed the existence of eight new species of squat lobster, four of which were ascribed to the genus Munida, two to the genus Paramunida, one to the genus Plesionida, and the last species was ascribed to the genus Agononida. These eight species are described along with phylogenetic relationships at the genus level. Our findings support the taxonomic status of the new species, yet the phylogenetic relationships are not yet fully resolved. Further molecular analysis of a larger data set of species, and more conserved genes, will help clarify the systematics of this group. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 156 , 465–493.     

Effects of contextual and social variables on contact call production in free-ranging ringtailed lemurs (Lemur catta)

Ringtailed lemurs (Lemur catta)often emit a characteristic vocalization when resting as a troop. This “meow call” is believed to serve an intragroup contact function. I investigated the effects of behavioral context and proximity on call production and acoustic features of the vocalization in a free-ranging troop in the Berenty Reserve, Madagascar. When group members were dispersed, the meow call was given frequently, and the acoustic features of the call probably increased the locatability of the emitter. I evaluated social relationships among the troop members from grooming interactions and spacing data. I also studied the call exchange network among the troop members, the results of which indicate that calls are exchanged between the two subgroups of females, which they connect.     

Towards a phylogeny of Cyclops (Copepoda): (in)congruences among morphology,molecules and zoogeography

The phylogeny of Cyclops (~30 spp.), a predominantly Palearctic cold‐adapted genus, was reconstructed based on morphological and molecular characters. The morphological analysis used extensive taxon sampling from the entire Holarctic range of the genus and included 53 morphological characters. Polymorphic traits were coded by the “unordered,” “unscaled” and “scaled” methods; maximum parsimony criterion was applied in tree building. Molecular phylogenetic reconstructions utilized partial nuclear 18S and 28S ribosomal genes, mitochondrial cytochrome oxidase I and complete internal transcribed spacer regions I and II, albeit with limited taxon sampling. Bayesian inference and maximum likelihood were used in these tree reconstructions. The molecular characters were used both in combination with morphology and as an independent test of the basal relationships inferred from morphology. Monophyly of the genus received strong support in both the morphological and molecular phylogenies; the basal relationships remain unresolved. The morphology‐based phylogenies, along with the geographic distribution patterns and ecological traits, supported monophyly of the ankyrae?ladakanus clade, scutifer‐clade (C. scutifer, C. jashnovi, C. columbianus), kolensis‐clade (C. kolensis, C. kikuchii, C. vicinus, C. furcifer, C. insignis, C. alaskaensis), abyssorum‐clade (C. abyssorum s. str., C. abyssorum larianus, C. ricae, C. sevani) and divergens‐clade (South Carpathian “Cyclops sp. Y,” C. mauritaniae, C. divergens, C. bohater, C. lacustris). Relationships among European and North American populations of C. scutifer and C. columbianus based on partial sequences of the 12S mitochondrial gene show C. scutifer to be paraphyletic, suggesting two independent invasions into North America via the Bering Land Bridge from Siberia to Alaska.     

Phylogenetic relationships of Andean-Ecuadorian populations of Anastrepha fraterculus (Wiedemann 1830) (Diptera: Tephritidae) inferred from COI and COII gene sequences

Phylogenetic relationships among Andean-Ecuadorian and other Neotropical populations of Anastrepha fraterculus and related species have been studied using two regions of mtDNA : 405 base pairs within Cytochrome Oxidase I ( COI) and 224 base pairs within Cytochrome Oxidase II (COII). Phylogenetic relationships were inferred using Maximun Parsimony (MP) method and haplotype networks. Andean-Ecuadorian populations of A. fraterculus are monomorphic at the COI locus and fall within a clade of South-American lowland populations of A. fraterculus. They appear to be unrelated with populations of northern Andes of Colombia and Venezuela also assigned to A. fraterculus, meaning that this species, as currently circumscribed, is not monophyletic and is composed of different biological entities that are little differentiated morphologically. At the COII locus, Andean-Ecuadorian populations of A. fraterculus show a major haplotype with a few variants, and form a clade with the lowland populations of southern Brazil an Argentina, but are clearly differentiated from them. Andean-Ecuadorian populations of Anastrepha fraterculus appear to be homogeneous with respect to their mitochondrial genome and thus their identity as members of a single gene pool is confirmed by these results.     

A phylogenetic analysis of the Illiciaceae based on sequences of internal transcribed spacers (ITS) of nuclear ribosomal DNA

Sequences of the internal transcribed spacers (ITS) of nuclear ribosomal DNA were determined for 15 species ofIllicium (Illiciaceae) to examine phylogenetic relationships. The ITS trees show a major dichotomy between the two North American species (I. floridanum andI. parviflorum) and the remaining east Asian species. This suggests that the existing division between two sections (sect.Illicium and sect.Cymbostemon) ofIllicium based on tepal characters in unnatural. The ITS phylogeny shows congruence with palynology: of the species examined, the three species (I. angustisepalum, I. anisatum andI. fargesii) from sect.Illicium that possess trizonocolpate pollen consistently form a clade, although nesting within a clade consisting of the species of sect.Cymbostemon, which generally have trisyncolpate pollen. The low ITS sequence divergence and the close relationship among east Asian species suggest a recent diversification of this group of species or an unusual slowdown of sequence mutations.     

Species limits and hybridization zones in Icterus cayanensis–chrysocephalus group (Aves: Icteridae)

One of the best examples of differentiation and hybridization among South American passerine birds is exhibited by Icterus cayanensis (Epaulet Oriole) and Icterus chrysocephalus (Moriche Oriole). Icterus chrysocephalus is a monotypic species restricted to northern South America. Icterus cayanensis is a polytypc species that ranges from Suriname and French Guyana to northern Argentina. Five subspecies are recognized to I. cayanensis. Hybrid zones are known between I. cayanensis and I. chrysocephalus as well as between subspecies of I. cayanenis, even though character variation has never been adequately assessed and mapped. Although molecular data support the hypothesis that I. cayanensis and I. chrysocephalus form a monophyletic group, they do not support the species limits currently recognized within this group. We analysed the geographic variation of plumage characters along the range of this group to map the geographic variation of individual plumage characters and identify the populations that have uniform phenotypic character expression and therefore represent genuine phylogenetic species. We also used molecular data to investigate the phylogenetic relationships among these species. Geographic variation of plumage characters, habitat preferences and molecular data identified four species within I. cayanensis–chrysocephalus clade: an Amazonian species group, formed by I. cayanensis and I. chrysocephalus and a Southern species group composed of I. pyrrhopterus and I. tibialis. The Amazonian species are separated by a relatively narrow hybrid zone along the Amazon valley, whereas the Southern species are separated by a hybrid zone that is larger than the ranges of the two species individually. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95 , 583–597.     

Molecular phylogeny and divergence time of the water penny genus Eubrianax (Coleoptera: Psephenidae) in Japan

The phylogenetic relationships among the Japanese members of the genus Eubrianax (Coleoptera: Psephenidae) were examined using the mitochondrial cytochrome oxidase subunit I (COI) gene and nuclear 28S rRNA gene sequences. Based on the molecular phylogeny as well as morphological features, the species status of Eubrianax brunneicornis Nakane, 1952 was proposed. The phylogenetic analyses recovered monophyly of the previously proposed pellucidus species group with four Japanese species, whereas a single Japanese species of the granicollis group was included in the lineage of the ramicornis group with five Japanese species. The divergence times of the species were estimated by dating the phylogenetic tree against the fossil record and a molecular clock based on the COI gene. The divergence of the Japanese species was inferred to have occurred during the Pliocene epoch.     

Isolation and characterization of polymorphic microsatellites in Iris ensata (Iridaceae)

• Premise of the study: Microsatellite primers were developed in Iris ensata (Iridaceae) to provide polymorphic markers for further studies into population genetics. • Methods and Results: Thirteen polymorphic microsatellite loci were isolated from I. ensata. These loci were successfully amplified in two natural populations of I. ensata from eastern China (Longwangshan, Zhejiang Province) and northeastern China (Jinchuan, Jilin Province). There was no significant linkage disequilibrium found for any pair of loci. These loci contained between two and 12 alleles per locus across all 48 individuals of I. ensata. The number of alleles per locus varied from two to 10 at the population level and the observed and expected heterozygosities ranged from 0.167 to 0.958 and from 0.284 to 0.853, respectively. • Conclusions: These loci showed high levels of polymorphism and could be used to study the population genetic structure, genetic relationships, and phylogeography of I. ensata.     

Phenotypic plasticity and character differentiation in a subdivided population ofIris pumila (Iridaceae)

Variation patterns in phenotypic plasticity and broad sense heritability of 26 characters were examined within and among closely adjacent habitats of the bearded iris,Iris pumila. It was found thatI. pumila has considerable differentiation for phenotypic plasticity and genetic variation over short distances. An analysis of relationships between character differentiation and phenotypic plasticity suggests that they could have evolved independently. Possible mechanisms for maintaining local differentiation of the observed plastic and genetic variation are also discussed.     

Phylogeny and reclassification of species groups in Aquarius Schellenberg, Limnoporus Stål and Gerris Fabricius (Insecta: Hemiptera-Heteroptera, Gerridae)

Abstract. We investigated phylogenetic relationships of water striders (Hemiptera‐Heteroptera: Gerridae) from the three principal Holarctic genera, Aquarius Schellenberg, Limnoporus Stål and Gerris Fabricius with parsimony analyses of sixty‐six morphological characters and DNA sequences from mitochondrial (cytochrome c oxidase subunit I + II; large mitochondrial ribosomal subunit) and nuclear (elongation factor 1‐alpha) genes. The taxon sampling included all species of Aquarius and Limnoporus, and a dense, near complete, sample of Gerris species with representatives from all subgenera and species groups, and Gigantometra gigas (China) was selected as an outgroup species. A simultaneous analysis of all data sets gave eight equally parsimonious trees, and a strict consensus tree left only a few relationships within Gerris unresolved. While Limnoporus and Gerris each were resolved as monophyletic entities, Aquarius was found to be polyphyletic, because the Nearctic Aquarius remigis‐group, comprising A. remigis (Say), A. amplus (Drake and Harris), A. nyctalis (Drake and Hottes) and A. remigoides Gallant and Fairbairn, was placed as sister group to Gerris, while the Andean Aquarius chilensis (Berg) was sister group to all three genera. Remaining species of Aquarius comprised a sister group to the Gerris + the A. remigis‐group clade. Based on our phylogenetic reconstruction we discuss relationships within and among the three genera, reassess and diagnose species groups, and discuss zoogeographical relationships among all taxa.     

The molecular phylogeny of the sea star Echinaster (Asteroidea: Echinasteridae) provides insights for genus taxonomy

In this study, we used sequences of two mitochondrial genes, cytochrome c oxidase I (COI) and 16S rRNA, and one nuclear gene, 28S rRNA, to test the monophyly of the sea star genus Echinaster, and understand the phylogenetic relationships among species and subgenera within this genus. Phylogenetic analyses based on Bayesian inference and maximum likelihood methods revealed three clades with high values of genetic divergence among them (K2P distances for COI over 23%). One of the clades grouped all Echinaster (Othilia) species, and the other two clades included Echinaster (non‐Othilia) species and Henricia species, respectively. Although the relationships among Henricia, Othilia, and Echinaster could not be completely clarified, the Othilia clade was a well‐supported group with shared diagnostic morphological characters. Moreover, the approximately unbiased test applied to the phylogenetic reconstruction rejected the hypothesis of the genus Echinaster as a monophyletic group. According to these results, we suggest the revalidation of Othilia as a genus instead of a subgenus within Echinaster. Our study clarifies important points about the phylogenetic relationships among species of Echinaster. Other important systematic questions about the taxonomic classification of Echinaster and Henricia still remain open, but this molecular study provides bases for future research on the topic.     

Molecular identification and phylogenetic relationships of seven Indian Sciaenids (Pisces: Perciformes,Sciaenidae) based on 16S rRNA and cytochrome c oxidase subunit I mitochondrial genes

The partial sequences of 16S rRNA and cytochrome c oxidase subunit I (COI) mitochondrial genes were analyzed for species identification and phylogenetic relationships among the commercially important Indian sciaenids (Otolithes cuvieri, Otolithes ruber, Johnius dussumieri, Johnius elongatus, Johnieops vogleri, Otolithoides biauritus and Protonibea diacanthus). Sequence analysis of both genes revealed that the seven species fell into three distinct groups, which were genetically distant from each other and exhibited identical phylogenetic resolution. Partial sequences of both the genes provided sufficient phylogenetic information to distinguish the seven sciaenids indicating the usefulness of mtDNA-based approach in species identification.     

Taxonomy and biogeography of Diapensia (Diapensiaceae) based on chloroplast genome data

Diapensia L. is the second largest genus of Diapensiaceae. The taxonomic treatment within Diapensia and relationships within Diapensiaceae have been disputed. Chloroplast genome sequence data have proved to be useful for plant phylogenetic analyses and species delimitation. In this study, we de novo sequenced and assembled 22 chloroplast genomes of 15 species of Diapensiaceae, including all recognized species of Diapensia with multiple samples. A super‐matrix containing a total of 107 genes and 18 taxa was constructed for phylogenetic analyses to resolve phylogenetic relationships among genera of the family and within Diapensia. The resulting phylogenetic tree showed the following strongly supported relationships: (Galax, (Pyxidanthera, (Berneuxia, ((Schizocodon, Diapensia), and Shortia s.s.)))). The dated phylogeny and reconstructed lineage‐through‐time plot for the family indicated rapid diversification in the Neogene and an acceleration of diversification rate after c. 8 Ma. Biogeographic analysis suggested that Diapensia originated in the Northeast Asian mountains approximately 6.06 Ma, followed by northward dispersal to the Arctic and southwestward dispersal to the Himalaya–Hengduan Mountains. Phylogenetic relationships within Diapensia were well resolved. Based on the phylogenetic results, we proposed to reinstate the species status of Diapensia bulleyana Forrest ex Diels, and raised D. purpurea f. albida to the species rank (D. albida [W. E. Evans] J. F. Ye comb. & stat. nov.). The distribution ranges of all species delineated based on the phylogenetic results were revised accordingly based on specimen occurrences. Our study adds new examples for the power of plastid genome data for resolving phylogenetic relationships and clarifying taxonomic disputes among closely related species.     

Patterns of relationships inTrichostomoideae (Pottiaceae, Musci)

Ribosomal DNA sequences were employed to infer relationships among EuropeanPottiaceae. Intragenic spacer I was sequenced for sixTrichostomoideae (Eucladium verticillatum, Pleurochaete squarrosa, Tortella flavovirens, T. nitida, T. tortuosa, Trichostomum brachydontium) and seven taxa from the other European subfamilies included inPottiaceae (Bryoerythrophyllum recurvirostrum, Didymodon vinealis, Microbryum starkeanum, Tortula muralis, Syntrichia ruralis, Weissia controversa andTimmiella cf.barbuloides). Cladistic analysis of sequence data shows close relationships betweenPleurochaete, Tortella andWeissia. Tortella appears to be paraphyletic, as it includesPleurochaete, Weissia andTrichostomum. Weissia, therefore, seems to be better placed inTrichostomoideae than, as traditionally done, inPottioideae. NeitherMerceyoideae norPottioideae appear to be monophyletic sensu stricto, at least within the limits of the taxa in analysis. Within the latter subfamily,Syntrichia is clearly separated fromTortula. These results would suggest that taxonomy inPottiaceae does not depict the pattern of descent, and therefore is in need of revision.