Genetic and anatomic relationships among three morphotypes of the echinoid Echinocrepis rostrata

Abstract. Three morphotypes—purple, tan, and white—of an irregular echinoid of the genus Echinocrepis are commonly observed in the deep North Pacific Ocean. Mitochondrial (mt) 16S DNA and cytochrome c oxidase subunit I (COI) DNA sequences were amplified from gonadal tissue samples from specimens of each morphotype. These portions of the mitochondrial genome are commonly used in species barcoding, and their similarities were compared and placed in 95% connection limit parsimony networks. All three morphotypes have similar 16S mtDNA and COI mtDNA sequences, and are thus likely representatives of the same species, Echinocrepis rostrata. Specimens of the white morphotype were smaller than the specimens of the other two morphotypes, had less gonad tissue, and had a different body shape, suggesting that it represents the juvenile form of E. rostrata. Resolving the three morphotypes into one species simplifies the identification of E. rostrata from photographs and leads to a greater understanding of the life history and reproductive cycle of a species vital to deep-sea bioturbation and carbon sequestration.     

The 5S DNA units of bread wheat (Triticum aestivum)

A collection of 44 cloned 5S DNA units fromTriticum aestivum cv. Chinese Spring were grouped into 12 sequence-types based on sequence similarity and the respective consensus sequences were then produced. The relationship between these 12 consensus sequences (T. aestivum S 1-S 8 andT. aestivum L 1-L 4), together with two clones sequenced byGerlach andDyer, and the 5S DNA consensus sequences from diploidTriticum spp. were then determined by numerical methods. Both phenetic and cladistic analyses were carried out. The following wheat 5S DNA sequences were found to group with respective sequences from diploidTriticum spp.:T. aestivum S 4, S 6 withT. tauschii S;T. aestivum S 3 withT. monococcum S andT. monococcum S-Rus 7;T. aestivum L 1 andT. aestivum L-G&D withT. speltoides L;T. aestivum L 2, L 3 withT. tauschii L;T. aestivum L 4 withT. monococcum L andT. monococcum L-Rus 12. The analyses suggested that 5 out of the 65S Dna loci present in wheat were identified at the sequence level. The locus that could not be identified in this analysis was the5S Dna-B 1 locus. A group ofT. aestivum sequences (T. aestivum S 1, S 7, S 8, S-G&D) were found to be distinct from the other 5S DNA sequences in the data base. The existence of the distinct group of 5S DNA sequences suggests that there is a gap in our current understanding of wheat evolution with respect to the5S Dna loci.     

Subgenome analysis of two southern hemisphere allotriploid species in Sphagnum (Sphagnaceae)

Sphagnum ×australe s.l. and S. ×falcatulum s.l. are both cryptic species complexes of gametophytically allodiploid and allotriploid cytotypes, with the allodiploid cytotype being one parent of the respective allotriploid cytotype. Phylogenetic analyses of S. ×australe s.l. and S. ×falcatulum s.l. were undertaken using sequences from two plastid loci and cloned sequences from three nuclear loci. Subgenomes from three subgenera were detected in allotriploid S. ×australe. The haploid parent of allotriploid S. ×australe was S. fimbriatum (subg. Acutifolia). One of the two subgenomes contributed by allodiploid S. australe was associated with S. strictum (subg. Rigida); the parental species associated with the other subgenome could not be identified, either at the species level or in terms of subgenus association. It may represent an early diverging lineage of subg. Sphagnum or a previously undetected subgenus. Allotriploid S. ×falcatulum has subgenomes from two subgenera. This study confirms prior findings that S. cuspidatum (subg. Cuspidata) was the haploid parent of allotriploid S. ×falcatulum. One of the two subgenomes contributed by allodiploid S. ×falcatulum was associated with subg. Cuspidata (species unidentified) and the second was associated with subg. Subsecunda (species unidentified). Recurrent allopolyploidy appears to have been associated with the complex evolutionary processes (inter-subgeneric hybridization, interploidal hybridization, double allopolyploidy) which resulted in each of these two allotriploids. The highest level of divergence detected among the subgenomes in each of these Sphagnum allotriploids is comparable to the average genetic divergence reported for angiosperm allopolyploids.     

Systematics of Cladophora spp. (Chlorophyta) from North Carolina,USA, based upon morphology and DNA sequence data with a description of Cladophora subtilissima sp. nov.

Identification of Cladophora species is challenging due to conservation of gross morphology, few discrete autapomorphies, and environmental influences on morphology. Twelve species of marine Cladophora were reported from North Carolina waters. Cladophora specimens were collected from inshore and offshore marine waters for DNA sequence and morphological analyses. The nuclear‐encoded rRNA internal transcribed spacer regions (ITS) were sequenced for 105 specimens and used in molecular assisted identification. The ITS1 and ITS2 region was highly variable, and sequences were sorted into ITS Sets of Alignable Sequences (SASs). Sequencing of short hyper‐variable ITS1 sections from Cladophora type specimens was used to positively identify species represented by SASs when the types were made available. Secondary structures for the ITS1 locus were also predicted for each specimen and compared to predicted structures from Cladophora sequences available in GenBank. Nine ITS SASs were identified and representative specimens chosen for phylogenetic analyses of 18S and 28S rRNA gene sequences to reveal relationships with other Cladophora species. Phylogenetic analyses indicated that marine Cladophorales were polyphyletic and separated into two clades, the Cladophora clade and the “Siphonocladales” clade. Morphological analyses were performed to assess the consistency of character states within species, and complement the DNA sequence analyses. These analyses revealed intra‐ and interspecific character state variation, and that combined molecular and morphological analyses were required for the identification of species. One new report, Cladophora dotyana, and one new species Cladophora subtilissima sp. nov., were revealed, and increased the biodiversity of North Carolina marine Cladophora to 14 species.     

Rhodotorula lamellibrachii sp. nov., a new yeast species from a tubeworm collected at the deep-sea floor in Sagami Bay and its phylogenetic analysis

A new species of the genus Rhodotorula was isolated from a tubeworm (Lamellibrachia sp.) collected at a depth of 1156 m in Sagami Bay, Japan. Strain SY-89 had physiological properties quite similar to R. aurantiaca. Two phylogenetic trees, one based on internal transcribed spacer (ITS) regions and 5.8S rDNA sequences and the other based on the D1/D2 region of the large subunit (26S) rDNA sequences, united strain SY-89 to the type strain of Sakaguchia dacryoides through a considerable evolutionary distance. Strain SY-89 was differentiated from S. dacryoides by the G+C content of the nuclear DNA and differences in the ability to utilize specific carbon and nitrogen compounds. The low complementarity of strain SY-89 DNA to that of the type strain of S. dacryoides confirmed that this strain was genetically unrelated to previously known species. The tubeworm isolates are described as R. lamellibrachii sp. nov. The type strain of R. lamellibrachii is strain SY-89 (= JCM 10907). R. lamellibrachii formed a cluster with Erythrobasidium hasegawianum, R. lactosa, S. dacryoides and Sporobolomyces elongatus on the ITS and 5.8S rDNA phylogenetic tree. These five species shared a signature sequence in 26S rDNA, although this relationship was not supported by phylogeny based on the D1/D2 region of 26S rDNA.     

Organellar DNA restriction fragment length polymorphism (RFLP) and nuclear random amplified polymorphic DNA (RAPD) analyses of morphotypes of Gracilaria (Gracilariales,Rhodophyta) from Chile

The extreme phenotypic variability recognized among the species of Gracilaria has highlighted the need for the application of refined methods to help solve taxa identifications. In Chile, there still exists uncertainty about the exact number of Gracilaria species. Our investigations are centered on DNA analyses of morphotypes collected from different geographical locations, namely Lenga and Isla Santa María, Region VIII (36°00 S to 38°00 S), and Maullín, Region X (39°30 S to 43°40 S). These two regions of Chile are considered as areas of confluence of G. chilensis, G. verrucosa, and a species of Gracilariopsis. In this study four morphotypes, from a natural bed located in Maullín, were analyzed for RFLP of plastid DNA and the results compared with data of four morphotypes from a bed in Lenga. The DNA banding patterns from each enzyme digest were identical irrespective of morphotypes and/or locations. In an attempt to unravel the nature of the morphological differences found among Lenga and Maullín morphotypes, RAPD analyses of nuclear DNA were also performed; however, no polymorphism has been found yet. Therefore, the data of this study, as well as concurrent data from preliminary interfertility tests, suggest that all morphotypes belong to a single taxon, Gracilaria chilensis.Departamento de Botánica, Facultad de Ciencias Naturales y Oceanográficas     

Phenotypic variability in the Caribbean Orange Icing sponge Mycale laevis (Demospongiae: Poecilosclerida)

Sponge species may present several morphotypes, but sponges that are morphologically similar can be separate species. We investigated morphological variation in Mycale laevis, a common Caribbean reef sponge. Four morphotypes of M. laevis have been observed (1) orange, semi-cryptic, (2) orange, massive, (3) white, semi-cryptic, and (4) white, massive. Samples of M. laevis were collected from Key Largo, Florida, the Bahamas Islands, and Bocas del Toro, Panama. Fragments of the 18S and 28S rRNA ribosomal genes were sequenced and subjected to phylogentic analyses together with sequences obtained for 11 other Mycale species and additional sequences retrieved from GenBank. Phylogenetic analyses confirmed that the genus Mycale is monophyletic within the Order Poecilosclerida, although the subgenus Aegogropila is polyphyletic and the subgenus Mycale is paraphyletic. All 4 morphotypes formed a monophyletic group within Mycale, and no genetic differences were observed among them. Spicule lengths did not differ among the 4 morphotypes, but the dominant megasclere in samples collected from Florida and the Bahamas was the strongyle, while those from Panama had subtylostyles. Our data suggest that the 4 morphotypes constitute a single species, but further studies would be necessary to determine whether skeletal variability is due to phentotypic or genotypic plasticity.     

Genetic diversity and phylogeography of Corynosoma australe Johnston, 1937 (Acanthocephala: Polymorphidae), an endoparasite of otariids from the Americas in the northern and southern hemispheres.

Adult specimens of Corynosoma australe Johnston, 1937 were recorded from the intestines of California sea lions, Zalophus californianus (Lesson), from Baja California Peninsula, Mexico, whereas larval forms were collected from two fish species on the Argentinian coast. Adult specimens of C. australe were morphologically characterized by having a cylindrical proboscis with 18–20 rows of 12–14 hooks per row and a cylindrical trunk expanded anteriorly into a disk with tiny, triangular spines spreading almost to three quarters of the hind-trunk in males and to the posterior body end in females. The aim of this study was to explore the genetic diversity and systematic position of C. australe distributed in the Americas. Newly generated sequences of the mitochondrial cytochrome c oxidase subunit 1 (cox 1) gene were compared with sequences available from GenBank.Phylogenetic analyses performed with the cox 1 dataset using maximum likelihood and Bayesian inference showed that the 11 new sequences of C. australe recovered from the California sea lion in northern Mexico plus the six sequences from Argentinian seashores formed a clade with other sequences of specimens previously identified as C. australe. The intraspecific genetic divergence among the isolates was very low, ranging from 1 to 1.7%, and in combination with the phylogenetic trees confirmed that the isolates belonged to the same species. The cox 1 haplotype network inferred with 27 sequences revealed 18 haplotypes divided into two clusters clearly separated from each other by 5 substitutions. The first cluster corresponded to specimens from the Northern Hemisphere (United States of America and Mexico), and the second corresponded to specimens from the Southern Hemisphere (Argentina and Brazil). The current evidence suggests that C. australe has an amphitemperate distribution and is associated mainly with otariids with secondary and independent colonization events to other mammals and the Magellanic penguin in the Southern Hemisphere.     

Molecular phylogenetic and morphological analyses of Oidium heveae, a powdery mildew of rubber tree

Powdery mildew of rubber tree caused by Oidium heveae is an important disease of rubber plantations worldwide. Identification and classification of this fungus is still uncertain because there is no authoritative report of its morphology and no record of its teleomorphic stage. In this study, we compared five specimens of the rubber powdery mildew fungus collected in Malaysia, Thailand, and Brazil based on morphological and molecular characteristics. Morphological results showed that the fungus on rubber tree belongs to Oidium subgen. Pseudoidium. Nucleotide sequence analysis of the ribosomal DNA internal transcribed spacer (ITS) region and the large subunit rRNA gene (28S rDNA) were conducted to determine the relationships of the rubber powdery mildew fungus and to link this anamorphic fungus with its allied teleomorph. The results showed that the rDNA sequences of the two specimens from Malaysia were identical to a specimen from Thailand, whereas they differed by three bases from the two Brazilian isolates: one nucleotide position in the ITS2 and two positions in the 28S sequences. The ITS sequences of the two Brazilian isolates were identical to sequences of Erysiphe sp. on Quercus phillyraeoides collected in Japan, although the 28S sequences differed at one base from sequences of this fungus. Phylogenetic trees of both rDNA regions constructed by the distance and parsimony methods showed that the rubber powdery mildew fungus grouped with Erysiphe sp. on Q. phillyraeoides with 100% bootstrap support. Comparisons of the anamorph of two isolates of Erysiphe sp. from Q. phillyraeoides with the rubber mildew did not reveal any obvious differences between the two powdery mildew taxa, which suggests that O. heveae may be an anamorph of Erysiphe sp. on Q. phillyraeoides. Cross-inoculation tests are required to substantiate this conclusion.     

Pycnogonids of the Eastern Weddell Sea (Antarctica), with remarks on their bathymetric distribution

The current work presents new data on the pycnogonids collected during the ANTXXI/2 cruise on board of “Polarstern” R/V during December 2003 and January 2004 in the Eastern Weddell Sea (Antarctica). Twenty-eight samples were taken, with different trawls, from depths between 120 and 1,866 m. In total, 251 specimens of pycnogonids, belonging to 31 species, were collected. Five species were observed to increase their depth range while six were found for the first time in the Weddell Sea, exhibiting an expansion in their geographical distribution, and confirming the general trend toward the circumpolarity of this group (23 of 31 species were circumpolar). Pallenopsis kupei is new for Antarctic waters. The most abundant species were Colossendeis megalonyx and Nymphon australe. Current data were completed with the samples collected from the same region during Polarstern cruise ANTXIII/3 (EASIZ I) in February–March 1996. Bathymetric patterns of distribution were analyzed for the total of 1,564 specimens (82 species, 14 genera). The results showed a difference in the composition between the continental shelf (from 100 to 900 m depth) and the slope (below 900 m), where the genus Nymphon dominated. Depth seems to be an influential factor in the structure of pycnogonid assemblages.     

The occurrence of two genotypes of the planktonic foraminifer Globigerinoides ruber (white) and paleo-environmental implications

Planktonic foraminifera provide a record of the upper ocean environment in the chemical and isotopic composition of individual shells. Globigerinoides ruber is a common tropical–subtropical planktonic foraminifer, and this species is used extensively for reconstruction of the paleo-environment. The different stable isotopic compositions of two morphotypes, G. ruber sensu stricto (s.s.) and G. ruber sensu lato (s.l.), first identified in sediments, suggested that G. ruber s.s. was dwelling in the upper 30 m of the water column and G. ruber s.l. at greater depths. Plankton tows and sediment trap experiments provided additional evidence distinguishing the two morphotypes and their habitats and invited the question as to whether the two morphotypes could be distinguished genetically. In this study, using phylogenetic analysis of nuclear partial small subunit ribosomal DNA (SSU rDNA) gene sequences representing 12 new and 16 known sequences, we identified four genotypes within G. ruber white variation; one of which is a sister group of Globigerinoides conglobatus, whereas the three others were sister groups of the G. ruber pink variation. Moreover, these two major groups corresponded to morphological differences described as G. ruber s.l. and s.s., respectively. This genetic evidence corroborates differences between the two morphotypes in the isotope record, and it will contribute to a more precise reconstruction of the thermal structure of the water column.     

Non-invasive methods for identifying oocysts of Sarcocystis spp. from definitive hosts

Because the excreted sporocysts and/or oocysts of various species of Sarcocystis may not be discriminated morphologically, we sought to validate a diagnostic technique based on variation in the 18S rDNA sequence. Oocysts and/or sporocysts from three taxa of Sarcocystis were collected from human, feline, and canine definitive hosts that had fed upon meats infected with the muscle cysts of Sarcocystis hominis, Sarcocystis fusiformis and a species of Sarcocystis from water buffalo that could not be distinguished from Sarcocystis cruzi. Using a new collection method employing filter paper, these excreted oocysts and sporocysts were subjected to DNA extraction, as were the corresponding muscle cysts. Methods employing PCR–RFLP and DNA sequencing of a partial 18S rDNA gene (ssrRNA) sequence were then used to successfully distinguish among the three taxa. The same, unique restriction digestion pattern characterizes the tissue cysts and oocysts and/or sporocysts of each parasite taxon. The technique makes possible amplification and identification of species specific gene sequences based on DNA extracted from as few as 7 excreted sporocysts (the equivalent of 3 and 1/2 oocysts) from freshly prepared material, or as few as 50 sporocysts from feces samples that had been stored in potassium dichromate (K₂Cr₂O₇) for as long as 6 years. This represents the first report using molecular diagnostic procedures to diagnose oocysts of Sarcocystis in faecal samples, describing a valuable new tool for studying the epidemiology of various Sarcocystis species.     

Molecular phylogeny of Armillaria from the Patagonian Andes

A number of species in the plant pathogen genus Armillaria are known from South America where they cause root rot disease on a wide variety of hosts. Knowledge pertaining to phylogenetic relationships of these species with those of other Armillaria species is almost non-existent. In addition, very few cultures representing these species are available, making DNA-based phylogenetic analyses impossible. The aim of this study was to characterise a collection of Armillaria isolates from the Patagonian Andes using DNA sequences and to determine their phylogenetic relationships with other Armillaria species. DNA sequences were obtained from the internal transcribed regions (ITS1, 5.8S and ITS4) and ribosomal large subunit (LSU) gene and used in phylogenetic analyses. Phylogenetic trees generated from the sequences separated the Armillaria isolates into four lineages. Lineages I and II represented A. novae-zelandiae and A. luteobubalina, respectively. Isolates belonging to A. novae-zelandiae from Malaysia, New Zealand, Australia and South America showed considerable intra-clade sub-structure. Lineages III and IV are probably distinct species and are most closely related to A. hinnulea and an unnamed species isolated from New Zealand and Kenya. This is the first comprehensive study of the phylogenetic relationships of Armillaria species from Patagonia and it provides a foundation for future research in this region.     

The karyotype and 5S rRNA genes from Spanish individuals of the bat species <Emphasis Type="Italic">Rhinolophus</Emphasis><Emphasis Type="Italic">hipposideros</Emphasis> (Rhinolophidae; Chiroptera)

The karyotype of individuals of the species Rhinolophus hipposideros from Spain present a chromosome number of 2n = 54 (NFa = 62). The described karyotype for these specimens is very similar to another previously described in individual from Bulgaria. However, the presence of one additional pair of autosomal acrocentric chromosomes in the Bulgarian karyotype and the differences in X chromosome morphology indicated that we have described a new karyotype variant in this species. In addition, we have analyzed several clones of 1.4 and 1 kb of a PstI repeated DNA sequence from the genome of R. hipposideros. The repeated sequence included a region with high identity with the 5S rDNA genes and flanking regions, with no homology with GenBank sequences. Search for polymerase III regulatory elements demonstrated the presence of type I promoter elements (A-box, Intermediate Element and C-box) in the 5S rDNA region. In addition, upstream regulatory elements, as a D-box and Sp1 binding sequences, were present in flanking regions. All data indicated that the cloned repeated sequences are the functional rDNA genes from this species. Finally, FISH demonstrated the presence of rDNA in nine chromosome pairs, which is surprising as most mammals have only one carrier chromosome pair.     

Characterization of freshwater benthic biofilm‐forming Hydrocoryne (Cyanobacteria) isolates from Antarctica

The aims of this work were to study cyanobacterial isolates resembling the genus Hydrocoryne using a combination of morphology and phylogeny of 16S rRNA and nifH sequences and to investigate genes involved in cyanotoxin and protease inhibitor production. Four new cyanobacterial strains, isolated from biofilm samples collected from King George Island, Antarctica, were studied. In terms of morphology, these new strains share traits similar to true Anabaena morphotypes (benthic ones), whereas phylogenetic analysis of their 16S rRNA gene sequences grouped them with the sequence of the type species Hydrocoryne spongiosa (H. Schwabe ex Bornet and Flahault 1886–1888), but not with sequences of the type species from the genus Anabaena. This cluster is the sister group of Anabaena morphotypes isolated only from the Gulf of Finland. In addition, this cluster is related to two other clusters formed by sequences of Anabaena isolated from different sites. Partial nifH genes were sequenced from two strains and the phylogenetic tree revealed that the Antarctic nifH sequences clustered with sequences from Anabaena. Furthermore, two strains were tested, using PCR with specific primers, for the presence of genes involved in cyanotoxins (microcystin and saxitoxin) and protease inhibitor (aeruginosin, and cyanopeptolin). Only cyanopeptolin was amplified using PCR. These four Hydrocoryne strains are the first to be isolated and sequenced from Antarctica, which improves our knowledge on this poorly defined cyanobacterial genus.     

The Miocene Architectonicidae (Gastropoda) of Chile

Three new species of Architectonicidae Heliacinae,Heliacus (Torinista) chonos, Heliacus (Grandeliacus) antu andSolatisonax bieleri, are described. These include the first reports ofGrandeliacus andSolatisonax from Chile.Solarium australe Philippi, 1887 is an earlier name forHeliacus (Torinista) bahamondei Frassinetti & Covacevich, 1981, but is a junior homonym ofS. australe Philippi, 1849. Type material and new specimens of the five previously described architectonicid species from Chile are described and figured for comparison. Together with other gastropod taxa from the same deposits, Architectonicidae provide evidence for tropical to subtropical water temperatures in central Chile during the early Miocene.     

Genetic divergence between and within two color morphotypes of <Emphasis Type="Italic">Parapercis sexfasciata</Emphasis> (Perciformes: Pinguipedidae) from Tosa Bay,southern Japan

The anterior half of the mitochondrial DNA control region (mtCR) sequence (ca. 400 base pairs) was compared between two color morphotypes (A, B) of Parapercis sexfasciata from Tosa Bay, southern Japan, using 16 and 21 specimens, respectively. Intramorphotypic mtCR divergences were only 0.0–0.5% and 1.0–2.5% for morphotypes A and B, respectively. In contrast, intermorphotypic mtCR divergence was much greater, 12.7–14.0%. Furthermore, phylogenetic analysis using a neighbor-joining algorithm, with P. multifasciata as an outgroup, showed that each morphotype was reciprocally monophyletic. These results and the distinct coloration and overlapping distribution indicate that the two color morphotypes of P. sexfasciata represent two distinct species. Mismatch distribution analysis suggested that both morphotypes had undergone population expansion; however, estimates of initial population sizes and mutational timescales suggested that morphotype B comprises historically larger and older populations than morphotype A.     

Raphidiopsis mediterranea Skuja represents non-heterocytous life-cycle stages of Cylindrospermopsis raciborskii (Woloszynska) Seenayya et Subba Raju in Lake Kastoria (Greece), its type locality: Evidence by morphological and phylogenetic analysis

The taxonomical relationship of Cylindrospermopsis raciborskii and Raphidiopsis mediterranea was studied by morphological and 16S rRNA gene diversity analyses of natural populations from Lake Kastoria, Greece. Samples were obtained during a bloom (23,830 trichomes mL⁻¹) in August 2003. A high diversity of apical cell, trichome, heterocyte and akinete morphology, trichome fragmentation and reproduction was observed. Trichomes were grouped into three dominant morphotypes: the typical and the non-heterocytous morphotype of C. raciborskii and the typical morphotype of R. mediterranea. A morphometric comparison of the dominant morphotypes showed significant differences in mean values of cell and trichome sizes despite the high overlap in the range of the respective size values. Additionally, two new morphotypes representing developmental stages of the species are described while a new mode of reproduction involving a structurally distinct reproductive cell is described for the first time in planktic Nostocales. A putative life-cycle, common for C. raciborskii and R. mediterranea is proposed revealing that trichome reproduction of R. mediterranea gives rise both to R. mediterranea and C. raciborskii non-heterocytous morphotypes. The phylogenetic analysis of partial 16S rRNA gene (ca. 920 bp) of the co-existing Cylindrospermopsis and Raphidiopsis morphotypes revealed only one phylotype which showed 99.54% similarity to R. mediterranea HB2 (China) and 99.19% similarity to C. raciborskii form 1 (Australia). We propose that all morphotypes comprised stages of the life cycle of C. raciborkii whereas R. mediterranea from Lake Kastoria (its type locality) represents non-heterocytous stages of Cylindrospermopsis complex life cycle.     

DNA Barcodes indicate members of the Anopheles fluviatilis (Diptera: Culicidae) species complex to be conspecific in India

Anopheles fluviatilis, a major vector of malaria in India has been described as a complex of three sibling species members, named as S, T and U, based on variations in chromosomal inversions. Also, ribosomal DNA markers (repetitive Internal Transcribed Spacer 2 (ITS2) and 28S D3 region) were described to differentiate these three sibling species members. However, controversies prevail on the genetic isolation status of these cryptic species. Hence, we evaluated this taxonomic incongruence employing DNA barcoding, the well established methodology for species identification, using 60 An. fluviatilis sensu lato specimens, collected from two malaria endemic eastern states of India. These specimens were also subjected to sibling species characterization by ITS2 and D3 DNA markers. The former marker identified 31 specimens among these as An. fluviatilis S and 21 as An. fluviatilis T. Eight specimens amplified DNA fragments specific for both S and T. The D3 marker characterized 39 specimens belonging to species S and 21 to species T. Neither marker identified species U. Neighbor Joining analysis of mitochondrial cytochrome c oxidase gene 1 sequences (the DNA barcode) categorized all the 60 specimens into a single operational taxonomic unit, their Kimura 2 parameter (K2P) genetic variability being only 0.8%. The genetic differentiation (F_ST) and gene flow (N_m) estimates were 0.00799 and 62.07, respectively, indicating these two ‘species’ (S & T) as genetically con‐specific intermixing populations with negligible genetic differentiation. Earlier investigations have refuted the existence of species U. Also, this study demonstrated that An. fluviatilis and the closely related An. minimus could be taxonomically differentiated by the DNA Barcode approach (K2P = 5.0%).