Authentication of five <Emphasis Type="Italic">Barilius</Emphasis> species from Indian waters using DNA barcoding

Authentic identification of fish species is essential for conserving them as a valuable genetic resource in our environment. DNA barcoding of living beings has become an important and ultimate tool for establishing their molecular identity. Among cyprinids, Barilius is an important genus having nearly 23 species in Indian region whose morphological identification is often difficult due to minute differences in their features. Five species collected from Indian waters and primarily identified as Opsarius bakeri (syn. Barilius bakeri), B. gatensis, B. vagra, B. bendelisis and B. ngawa were authenticated by their DNA barcoding based on mitochondrial COI gene sequences. Five individuals of each species were taken for barcode preparation by COI gene sequencing which yielded one barcode for B. ngawa, two barcodes each for O. bakeri, B. gatensis, B. bendelisis and three barcodes for B. vagra. The order of inter and intra-specific variation was estimated to know a preliminary status of variation prevailing in these cold stream fish species significant for evolution and conservation of these valued species of our ichthyofauna. Average variation within genera was found to be 13.6% with intra-specific variation ranging from 0.0% (B. ngawa) to 0.6% (B. gatensis). These distance data are in the same order found by various researchers globally using COI barcode sequences in different fish species. Phylogenetic relatedness among Barilius species and some other cyprinids validate their status of individual species as established by conventional taxonomy.     

DNA barcoding of freshwater zooplankton in Lake Kasumigaura,Japan

Although DNA barcoding is a promising tool for the identification of organisms, it requires the development of a specific reference sequence library for sample application. In the present study we developed a Lake Kasumigaura, Japan, zooplankton DNA barcode library to increase the sensitivity of future zooplankton monitoring for detecting lake ecosystem condition changes. Specifically, the mitochondrial cytochrome c oxidase subunit I (mtCOI) haplotype, i.e., the primary DNA barcode, was examined for each zooplankton taxon. In crustaceans, 37 mtCOI haplotypes were obtained from 99 individuals, representing four and 15 morpho-species of Copepoda and Cladocera, respectively. Comparing these sequences with those in GenBank shows that the lake harbors putative non-indigenous species, such as Daphnia ambigua. In rotifers, 132 mtCOI haplotypes were obtained from 302 individuals, representing 11 genera and one unclassified taxon. The automatic barcode gap discovery (ABGD) algorithm separated these haplotypes into 43 species. Brachionus cf. calyciflorus was divided into five ABGD species, and different ABGD species tended to occur in different seasons. Seasonal ABGD-species succession was also observed within Polyarthra spp. and Synchaeta spp. These seasonal successions were not detected by inspections of external morphology alone. Accepting up to 7% sequence divergence within the same species, mtCOI reference sequences were available in GenBank for three, 13, and 17 species in Copepoda, Cladocera, and Rotifera, respectively. The present results, therefore, reveal the serious shortage of mtCOI reference sequences for rotifers, and underscore the urgency of developing rotifer mtCOI barcode libraries on a global scale.     

A report on identification of sequence polymorphism in barcode region of six commercially important <Emphasis Type="Italic">Cymbopogon</Emphasis> species

Cymbopogon

is an important member of grass family Poaceae, cultivated for essential oils which have greater medicinal and industrial value. Taxonomic identification of Cymbopogon species is determined mainly by morphological markers, odour of essential oils and concentration of bioactive compounds present in the oil matrices which are highly influenced by environment. Authenticated molecular marker based taxonomical identification is also lacking in the genus; hence effort was made to evaluate potential DNA barcode loci in six commercially important Cymbopogon species for their individual discrimination and authentication at the species level. Four widely used DNA barcoding regions viz., ITS 1 & ITS 2 spacers, matK, psbA-trnH and rbcL were taken for the study. Gene sequences of the same or related genera of the concerned loci were mined from NCBI domain and primers were designed and validated for barcode loci amplification. Out of the four loci studied, sequences from matK and ITS spacer loci revealed 0.46% and 5.64% nucleotide sequence diversity, respectively whereas the other two loci i.e., psbA-trnH and rbcL showed 100% sequence homology. The newly developed primers can be used for barcode loci amplification in the genus Cymbopogon. The identified Single Nucleotide Polymorphisms from the studied sequences may be used as barcodes for the six Cymbopogon species. The information generated can also be utilized for barcode development of the genus by including more number of Cymbopgon species in future.

     

Gutsy genetics: identification of digested piscine prey items in the stomach contents of sympatric native and introduced warmwater catfishes via DNA barcoding

A major focus of ecology is understanding trophic relationships and energy flows in natural systems, associated food web dynamics and changes in food webs due to introduced species. Predator-prey interactions are often assessed by examining stomach contents. However, partially digested remains may be difficult to accurately identify by traditional visual analysis. Here we evaluate the effectiveness of DNA barcoding to identify digested piscine prey remains in invasive Blue Catfish Ictalurus furcatus, non-native, but established Channel Catfish Ictalurus punctatus and native White Catfish Ameiurus catus from Chesapeake Bay, USA. Stomach contents were examined and piscine prey items were scored as lightly digested, moderately digested or severely digested. A 652 base pair region of the cytochrome c oxidase subunit I (COI-5P) mitochondrial DNA gene was sequenced for each prey item. Edited barcode sequences were compared to locally-caught and validated reference sequences in BOLD (Barcode of Life Database). A large majority of prey items were sufficiently digested to limit morphological identification (9.4 % to species and an additional 12.1 % to family). However, overall barcoding success was high (90.3 %) with little difference among the digestion classifications. Combining morphological and genetic identifications, we classified 91.6 % of fish prey items to species. Twenty-three fish species were identified, including species undergoing active restoration efforts (e.g., Alosa spp.) and commercially important species, e.g., Striped Bass Morone saxatilis, White Perch Morone americana, American Eel Anguilla rostrata and Menhaden Brevoortia tyrannus. We found DNA barcoding highly successful at identifying all but the most heavily degraded prey items and to be an efficient and effective method for obtaining diet information to strengthen the resolution of trophic analyses including diet comparisons among sympatric native and non-native predators.     

DNA Barcoding for Identification of Sugarcane Borers in China

Sugarcane borers are economically damaging insects with species that vary in distribution patterns both geographically and temporally, and vary based on ecological niche. Currently, identification of sugarcane borers is mostly based on morphological characters. However, morphological identification requires taxonomic expertise. An alternative method to identify sugarcane borers is the use of molecular data. DNA barcoding based on partial cytochrome c oxidase subunit 1 (COI) sequences has proven to be a useful tool for rapid and accurate species determination in many insect taxa. This study was conducted to test the effectiveness of DNA barcodes to discriminate among sugarcane borer species in China. Partial sequences of the COI gene (709 bp) were obtained from six species collected from different geographic areas. Results showed that the pairwise intraspecies genetic distance was < 0.02, whereas the interspecies genetic distance ranged from 0.117 to 0.182. Results from a neighbor-joining tree showed that the six sugarcane borer species were certainly separated. These results suggested that the partial COI sequences had high barcoding resolution in discriminating among sugarcane borer species. Our study emphasized the use of DNA barcodes for identification of the analyzed sugarcane borer species and represents an important step for building a comprehensive barcode library for sugarcane borers in China.     

Uncovered Diversity of a Predominantly Andean Butterfly Clade in the Brazilian Atlantic Forest: a Revision of the Genus <Emphasis Type="Italic">Praepedaliodes</Emphasis> Forster (Lepidoptera: Nymphalidae,Satyrinae, Satyrini)

The genus Praepedaliodes Forster, 1964, the only representative of the mega-diverse mostly Andean Pedaliodes complex lineage in the Brazilian Atlantic Forest, is revised. Prior to this study, four species were known, P. phanias (Hewitson, 1862), P. granulata (Butler, 1868), P. amussis (Thieme, 1905) and P. exul (Thieme, 1905). Here, a further six are described, all from SE Brazil, expanding to 10 the number of species in this genus. Lectotypes are designated for P. phanias, P. granulata and P. amussis. The genus is most diverse in the Serra da Mantiqueira (São Paulo, Rio de Janeiro, Minas Gerais) and in the Serra Geral (Paraná, Santa Catarina) with seven species occurring in both ranges. Praepedaliodes phanias is the most widespread species and the only one found in the western part of the Atlantic Forest; only this species and P. duartei Dias, Dolibaina & Pyrcz n. sp. occurring to near sea level. Other species, P. zaccae Dolibaina, Dias & Pyrcz n. sp., P. francinii Freitas & Pyrcz n. sp., P. sequeirae Pyrcz, Dias & Dolbaina n. sp., P. landryi Pyrcz & Freitas n. sp. and P. pawlaki Pyrcz & Boyer n. sp. are strictly montane and the highest species richness is reached at 1400–1800 m. One species, P. sequeirae n. sp., is a narrow endemic found only at timberline in the Agulhas Negras massif above 2300 m. Immature stages are described for two species, P. phanias and P. landryi n. sp. Molecular data (barcode region of cytochrome oxidase, subunit I) and adult morphology, including male and female genitalia, support the genus as monophyletic, belonging to a predominantly Andean clade of the Pedaliodes Butler, 1867 complex. Morphological evidences, in particular female genitalia comparative analysis, indicate the genera Physcopedaliodes Forster, 1964 and Panyapedaliodes Forster, 1964 as possibly the closest relatives to Praepedaliodes. Molecular data are inconclusive in this respect.     

Inferring the potential of plastid DNA-based identification of derived ferns: a case study on the <Emphasis Type="Italic">Asplenium trichomanes</Emphasis> aggregate in Europe

The utility of three plastid DNA regions to identify fern species was explored with focus on the European representatives of the Asplenium trichomanes aggregate. The sampling included representatives of the three diploid and the four tetraploid taxa recognized in the European flora plus Macaronesia. Besides European samples, the compiled data set comprised specimens of a putative Hawaiian endemic and one species occurring in Southeast Asia. By combining the sequences of three non-coding plastid regions, 13 haplotypes were recovered of which four were found in more than one taxon. Evidences for four distinct diploid lineages were found that correspond to Asplenium anceps, A. inexpectans, A. trichomanes s.s., and A. tripteropus. The four tetraploids occurring in Europe shared haplotypes with A. inexpectans. Thus, DNA barcoding can successfully identify the diploids, but fail to separate the tetraploids from their diploid ancestors. As a consequence, barcoding analyses of ferns need to take into account the differences of ploidy level measured by evidence independent from the DNA barcode. Evidence for uneven accumulation of intra-species DNA variation was recovered by comparing all species. Furthermore, the study provided evidence that the current taxonomy of these ferns requires to be revised. The two European diploids form well-separated clades and need to be recognized as A. inexpectans and A. trichomanes s.s. To keep name consistency for all European tetraploids, a new name Asplenium jessenii is introduced to replace A. trichomanes subsp. hastatum.     

Identification of <Emphasis Type="Italic">Colletotrichum</Emphasis> spp. associated with fruit rot of <Emphasis Type="Italic">Capsicum annuum</Emphasis> in North Western Himalayan region of India using fungal DNA barcode markers

The DNA barcode approach was used to identify and establish association of Colletotrichum species complex with fruit rot disease of chili (Capsicum annuum L.) in North-Western Himalayan region of India. Twenty isolates of five morphologically identified Colletotrichum species collected from commercial chili growing areas were identified using deoxyribonucleic acid (DNA) barcode marker genes, 5.8S ribosomal ribonucleic acid flanking internal transcribed spacers 1 & 2 and β-tubulin gene. Morpho-cultural identification requires expertise to delineate C. gloeosporioides, C. boninense and C. acutatum complexes from each other, as these species possess minute variation in spore shape and size. Ribosomal DNA and β-tubulin sequence analysis along with species-specific marker amplification established the association of seven Colletorichum spp. viz., C. truncatum (syn. Colletotrichum capsici), C. coccodes, C. karstii, C. kahawae, C. nymphaeae, C. fructicola and C. gloeosporioides complex with fruit rot of chili. Phylogenetic analysis of 35 Colletotrichum sequences including authentic type sequences validated the identified sequences with strong bootstrap support. This approach delineated morphologically identified species with great ease into more reliable genotype based speciation of various Colletorichum complexes. The DNA barcode markers have direct implications for plant pathologists in relation to diagnostics in fields and for the purpose of quarantine and disease management.     

<Emphasis Type="Italic">Paulogramma hydarnis</Emphasis> (n. comb.) (Nymphalidae: Biblidinae): Distribution,Systematic Position,and Conservation Status of a Rare and Endangered Butterfly

The nymphalid Paulogramma hydarnis (Godart) (n. comb., previously in the genus Callicore) is an endangered butterfly present in a few montane sites in the Atlantic Forest in the Southeastern Brazil. The precise systematic position of P. hydarnis was previously unknown. Based on molecular data, we find that it is sister to Paulogramma pygas (Godart) (n. comb., also previously in Callicore), a common and widespread species in the Neotropics. In addition, we find that Callicore is not monophyletic and that “Callicore” hydarnis (along with other species) is more related to the genus Paulogramma, and should thus be placed in that genus. The genus Paulogramma is now composed by the following species: Paulogramma pyracmon (Godart), Paulogramma eunomia (Hewitson) n. comb., Paulogramma hydarnis (Godart) n. comb., Paulogramma hystaspes (Fabricius) n. comb., Paulogramma pygas (Godart) n. comb., and Paulogramma tolima (Hewitson, 1852) n. comb. Museum specimens and field data report P. hydarnis in four sites in Southeastern Brazil. Recently, P. hydarnis was recorded for the first time at Parque Nacional do Caparaó, states of Espírito Santo and Minas Gerais, expanding its distribution about 200 km northward of the previously known limit. Although regularly recorded in some sites, most records are historic, before the 1960s, and the current conservation situation of this species is delicate, deserving attention.     

From species to individuals: combining barcoding and microsatellite analyses from non-invasive samples in plant ecology studies

By means of DNA barcoding and microsatellite analyses, we studied the species and individuals of legitimate seed dispersers of the Mediterranean shrub Pistacia lentiscus, a keystone species that represents the main source of food in winter for frugivorous birds. We collected dropping of birds containing seeds, and after DNA extraction we amplified and sequenced a fragment of the mitochondrial COI gene. Through BLASTN queries of the sequenced fragments against registered sequences in the GenBank database we identified the bird species that are currently dispersing P. lentiscus seeds. Further, through the amplification of specific nuclear microsatellite loci we calculated standard genetic diversity parameters of the population of birds from the genus Sylvia (the blackcap and Sardinian warbler), the most important dispersers of P. lentiscus. Five bird species were identified as seed dispersers through their barcode match. Further, we found that S. melanocephala displayed slightly lower levels of genetic diversity than S. atricapilla. In this study we show how the genetic analyses of environmental faecal samples can be a useful and convenient tool for the study of plant-frugivore interactions through the ascertainment of the identity of the species involved and through the analyses of genetic variability of their populations.     

Extensive cryptic diversity in the cosmopolitan sludge worm <Emphasis Type="Italic">Limnodrilus hoffmeisteri</Emphasis> (Clitellata,Naididae)

Limnodrilus hoffmeisteri Claparède, 1862 is a common freshwater worm, often regarded as an indicator of organic pollution. The taxonomic status of this species is controversial due to great variation in morphological features. Numerous morphological forms of L. hoffmeisteri are recorded in the literature, especially from Europe and North America. Today, DNA-based species delimitation assumes that species boundaries can be more objectively and effectively estimated using genetic data rather than with morphological data alone. To investigate if L. hoffmeisteri is a single species, 295 worms identified as either L. hoffmeisteri or other similar (congeneric) morphospecies, using currently accepted morphological criteria, were collected from 82 locations in the northern hemisphere. The number of primary species hypotheses (PSHs) was first explored with cytochrome oxidase subunit I (COI), the proposed DNA barcode for animal species, and with data for all specimens. Both automatic barcoding gap discovery (ABGD) and the Bayesian general mixed Yule coalescent (bGMYC) model revealed the existence of ≥25 distinct PSHs (COI lineages) in our dataset. Then, smaller samples of individuals representing major COI lineages were used for exploration of a nuclear locus, the internal transcribed spacer (ITS) region. In the ITS gene tree (81 sequences), generated by BEAST, 16 well-supported terminal groups were found, but not all of these groups were congruent with the PSHs found in the COI tree. As results across these different analyses were inconsistent, we resorted to analyzing reciprocal monophyly between gene trees and used a minimum consensus of all evidence, suggesting that there are 13 separately evolving lineages (=13 species) within our sample. The smallest uncorrected COI p-distance between these species is 12.1%, and the largest intraspecific p-distance is 16.4%, illustrating the problem of species delimitation with a DNA-barcoding gap as the sole criterion. Ten of these species are morphologically identified as “L. hoffmeisteri,” the remaining three can be attributed to morphologically distinct congeneric species. An individual from the type locality in Switzerland was designated as a neotype of L. hoffmeisteri sensu stricto. This worm belongs to one of the ten species, and this lineage is widely distributed in Europe, Asia, and North America. The remaining nine species show a mixed distribution pattern; some appear to be endemic to a restricted area, others are Holarctic. Our results provide clues to the future revalidation of some of the nominal species today placed in synonymy with L. hoffmeisteri. A BEAST analysis, based on previously published and newly generated 16S data, suggested that this complex contains also other species than those studied by us. By integrating additional genetic data, it will be possible to identify these and additional specimens in future studies of Limnodrilus, and the neotype provides a baseline for further revisions of the taxonomy of the L. hoffmeisteri complex.     

Tracking the diversity of the flatworm genus <Emphasis Type="Italic">Imbira</Emphasis> (Platyhelminthes) in the Atlantic Forest

The genus Imbira Carbayo et al., 2013 encompasses two species, Imbira guaiana (Leal-Zanchet & Carbayo, 2001) and Imbira marcusi Carbayo et al., 2013, which occur in south Brazil, in areas originally covered by the Atlantic Forest. In the present study, we examine the genetic diversity within the genus, investigate the occurrence of molecular autapomorphies for its species and describe a new species for the genus based on an integrative approach. The Bayesian and maximum likelihood analyses based on DNA barcoding recovered the monophyly of the genus Imbira, but indicate that specimens representing I. marcusi correspond to five distinct lineages. These analyses, as well as sequence divergence data, revealed that the new species herein described is closely related to I. guaiana and that the specific status of specimens of I. marcusi available in GenBank should be reviewed. In addition, sequence analysis revealed 32 molecular autapomorphies for all independent evolutionary units within the genus. The new species described herein seems to be endemic to its type locality, a private area without legal protection.     

DNA barcoding of fishes in Irtysh River China

DNA barcoding was a molecular diagnostic method that provided rapid and accurate species identification. The 650 bp-length cytochrome c oxidase subunit I (COI) gene of 33 species in Irtysh River China was sequenced and analyzed in this study. The average intra-species, -genus, -family, and -order of Kimura two parameter (K2P) distances were 0.003, 0.060, 0.163 and 0.240, respectively. The genetic distance between genus Barbatula and Cobitis was the largest whereas that between genus Hypophthalmichthys and Aristichthys was the smallest. The neighbour-joining tree constructed by all 44 haplotypes was divided into two major clusters: Cypriniformes fishes and other fishes. A cryptic species of Barbatula barbatula was detected according to 2% genetic threshold.     

A new species of the genus <Emphasis Type="Italic">Morellia</Emphasis> Robineau-Desvoidy (Diptera: Muscidae) from Yunnan,China, with analysis of available DNA barcoding sequences

A new species of the genus Morellia Robineau-Desvoidy, 1830, Morellia (Morellia) trifurcata sp. n., collected from Yunnan, China is described. Four DNA sequences of the partial mitochondrial cytochrome c oxidase subunit I (mtCOI) gene of this new species are provided. In order to evaluate the availability of DNA barcoding for identifying Morellia species, 38 currently available, non-identical COI sequences of 16 Morellia species are involved in a molecular analysis using the neighbor-joining (NJ) method. The intra- and interspecific p-distances are summarized.     

Efficiency of DNA barcoding for phylogenetic analysis and species identification in flying fish (Exocoetidae)

The article reports DNA barcoding (sequencing of the cox 1 mitochondrial gene fragment) of five South Atlantic flying fish species belonging to family Exocoetidae together with the results of the comparative analysis of cox 1 variability in the Exocoetidae and its closely related family Hemiramphidae. It has been demonstrated that DNA barcoding can be used as an extra tool for species identification and phylogenetic analysis in flying fish, since species identification accuracy using the cox 1 gene sequence proved to be 88%, or 78% when intraspecies variability level is taken into account. We have confirmed monophyletic origin of certain species, genera, and subfamilies of flying fish except for the genus Cheilopogon, which was represented on the phylogenetic tree by three paraphyletic clades. One of them shows close relationship to the genus Cypselurus, while another encompasses Hirundichthys genus species. The Exocoetidae is characterized by much lower overall genetic divergence level compared to the Hemiramphidae (average intraspecies differences: 10.2 ± 0.4% vs. 18.1 ± 0.8%; average intrageneric differences: 13.3 ± 1.1% vs. 21.3 ± 1.8%), which may indicate that the former group is relatively young in terms of evolution. No intraspecies differentiation was observed for Exocoetus obtusirostrus across a significant geographic distance (>3000 km). Phylogenetic reconstructions based on the variability of conservative (cox 1 mtDNA) and more variable regions of mitochondrial and nuclear genomes and on the adaptive morphological traits associated with gliding flight development were shown to coincide to a large extent.     

DNA barcoding from the hooked squids (Cephalopods: Onychoteuthidae) of the Sargasso Sea

Squids of the family Onychoteuthidae are ecologically important in pelagic food webs and have been reported from every ocean except the Arctic. Although they are abundant and caught frequently as bycatch in fisheries, the biogeography of many species remains poorly understood. Species identification within the Atlantic Ocean is usually restricted to two species: Onychoteuthis banksii and Onykia carriboea. Here, we report the occurrence of four species of the family Onychoteuthidae (Onychoteuthis cf. banksii, Onykia carriboea, Walvisteuthis jeremiahi, and Onychoteuthis sp. AL 2) from the Sargasso Sea in the western Atlantic, identified using DNA barcoding (cytochrome c oxidase subunit I) and morphology. Our results have doubled the known onychoteuthid biodiversity in the Sargasso Sea, which has implications for the ecology of this oceanic region.     

Diversification of <Emphasis Type="Italic">Caiophora</Emphasis> (Loasaceae subfam. Loasoideae) during the uplift of the Central Andes

Andean orogeny and the ecological changes that followed promoted diversification in plant and animal lineages since the Early Miocene. The angiosperm genus Caiophora (Loasaceae, subfam. Loasoideae) comprises around 50 species that are endemic to South America. These are distributed from southern Ecuador to Central Chile and Argentina. Bee pollination and distribution at low-intermediate elevations probably represent the ancestral condition in the lineage that includes Caiophora and its allied genera. The majority of Caiophora species grow at high elevations in the Andes, where some depend on vertebrate pollination. Previous studies did not resolve phylogenetic relationships within Caiophora, which precluded the dating of the origin and divergence of this group. We used markers of one nuclear (ITS) and one plastid region (trnS ^GCU -trnG ^UUC ) to solve phylogenetic relationships among 19 Caiophora species (including different accessions). We also included 10 species of the allied genera Blumenbachia and Loasa. Aosa rostrata and Xylopodia klaprothioides were used as outgroups. Phylogenetic reconstruction strongly supports the monophyly of Caiophora, and although several clades within this genus are poorly supported, our study yielded a better infra-generic resolution than previous studies. The origin of Caiophora is dated to the Early-Middle Miocene and can be related to the uplift of the Cordilleras Frontal and Principal and to Pacific marine transgressions. According to our estimations, Caiophora began to diversify during the Middle-Late Miocene and this unfolding proceeded eastwards during the Pliocene and the Pleistocene, in parallel to the uplift of different Andean mountain ranges.     

A new efficient method for analyzing fungi species using correlations between nucleotides

Background

In recent years, DNA barcoding has become an important tool for biologists to identify species and understand their natural biodiversity. The complexity of barcode data makes it difficult to analyze quickly and effectively. Manual classification of this data cannot keep up to the rate of increase of available data.

Results

In this study, we propose a new method for DNA barcode classification based on the distribution of nucleotides within the sequence. By adding the covariance of nucleotides to the original natural vector, this augmented 18-dimensional natural vector makes good use of the available information in the DNA sequence. The accurate classification results we obtained demonstrate that this new 18-dimensional natural vector method, together with the random forest classifier algorthm, can serve as a computationally efficient identification tool for DNA barcodes. We performed phylogenetic analysis on the genus Megacollybia to validate our method. We also studied how effective our method was in determining the genetic distance within and between species in our barcoding dataset.

Conclusions

The classification performs well on the fungi barcode dataset with high and robust accuracy. The reasonable phylogenetic trees we obtained further validate our methods. This method is alignment-free and does not depend on any model assumption, and it will become a powerful tool for classification and evolutionary analysis.

     

Molecular phylogeny of the genus <Emphasis Type="Italic">Bolusiella</Emphasis> (Orchidaceae,Angraecinae)

Recent molecular studies have suggested the monophyly of Bolusiella, a small orchid genus comprising five species and one subspecies from Continental Africa, but sampling has been limited. Using the species delimitation presented in the recent taxonomic revision of the genus, this study aimed to confirm the monophyly of Bolusiella and assess the interspecific relationships using a comprehensive sampling and various analytical methods. DNA sequences of one nuclear spacer region (ITS-1) and five plastid regions (matK, rps16, trnL–trnF, trnC–petN, and ycf1) from 20 specimens representing all five species of the genus were analyzed using static homology, dynamic homology, and Bayesian methods. The monophyly of both the genus Bolusiella and each of its five species was confirmed, corroborating the previously published taxonomic revision. The use of dynamic homology methods was not conclusive for this particular group. The results of the total evidence analysis (combining all six sequence regions) using the dynamic homology approach yielded a slightly different hypothesis regarding interspecific relationships (namely the exchange of B. talbotii and Bolusiella iridifolia as the earliest diverging lineage), probably because the nodes in question are supported by a small subset of conflicting characters, compared to the hypotheses resulting from the static homology and Bayesian methods, which are congruent with the results of previous studies.