Molecular cytogenetic analysis of the Appenine endemic cyprinid fish Squalius lucumonis and three other Italian leuciscines using chromosome banding and FISH with rDNA probes

Karyotype and other chromosomal characteristics of the Appenine endemic cyprinid fish, Toscana stream chub Squalius lucumonis, were analysed using conventional banding and FISH with 45S and 5S rDNA probes. The diploid chromosome number (2n = 50) and karyotype characteristics including pericentromeric heterochromatic blocks and GC-rich CMA₃-positive sites corresponding to both positive Ag-NORs and 45S rDNA loci on the short arms of a single medium-sized submetacentric chromosome pair were consistent with those found in most European leuciscine cyprinids. On other hand, 5S rDNA FISH in the Toscana stream chub and three other Italian leuciscines, S. squalus, Rutilus rubilio and Telestes muticellus, revealed a species-specific hybridization pattern, i.e. signals on four (S. lucumonis), three (S. squalus and R. rubilio) and two (T. muticellus) chromosome pairs. Whereas all the species shared the 5S rDNA loci on the largest subtelocentric chromosome pair, a “leuciscine” cytotaxonomic marker, S. lucumonis showed both classes of rDNA loci tandem aligned on the short arms of chromosome pair No. 12. The present findings suggest that the observed high variability of 5S rDNA loci provides a powerful tool for investigation of karyotype differentiation in karyologically conservative leuciscine fishes.     

Genetic structure of three Sorbus latifolia (Lam.) Pers. taxa endemic to northern Bavaria

The Franconian Alb (Bavaria, Germany) is rich in endemic Sorbus taxa, considered as apomictic microspecies and derived by hybridization between Sorbus aria aggregate and Sorbus torminalis (Sorbus latifolia aggregate). Molecular studies using the AFLP technique, neighbour joining, Bayesian clustering, principal coordinate analysis (PCo) and voucher studies were used to investigate genetic structure and origin of adult plants and cultivated offspring of three endemic S. latifolia taxa, namely Sorbus cordigastensis, Sorbus franconica and Sorbus adeana and probable parental species from the S. aria agg. and S. torminalis. The S. latifolia taxa, adults and progeny, showed low genetic variability and a more or less clonal structure, confirming an apomictic mode of reproduction. The investigated S. latifolia taxa were remarkably different among each other, confirming their status as microspecies. The AFLP data confirmed the hybrid origin of the S. latifolia taxa, they were 1.3–1.5 times more closely related to S. aria agg. than to S. torminalis. The S. aria agg. showed a complicate genetic structure and fell into four main groups, two intermediate groups besides Sorbus pannonica and Sorbus aria sensu stricto (S. aria s.str.). Some progeny of S. pannonica was more variable than expected and clustered partly with other groups indicating gene flow within S. aria agg. Different subgroups of the S. aria aggregate may be parental for the S. latifolia taxa, contributing to the remarkable genetic distances between them.     

Evolution of the Portulaca oleracea L. aggregate in Egypt on molecular and phenotypic levels revealed by morphology,inter-simple sequence repeat (ISSR) and 18S rDNA gene sequence markers

Molecular markers including inter simple sequence repeats (ISSR) and 18S rDNA gene sequence markers were combined with a detailed morphological analysis to seek characters that discriminate four taxa of Portulaca oleracea s.l. These taxa were identified as the microspecies Portulaca nitida, Portulaca oleracea, Portulaca rausii and Portulaca granulatostellulata. Morphological characters did not provide a clear distinction among the four taxa of P. oleracea s.l. It was found that mixed populations of the taxa occur in several locations and morphological similarities between the microspecies were detected. ISSR analysis indicates that gene flow among populations of different taxa was high and most of the genetic variation (61.9%) occurs within population. These results are inconsistent with the general characteristics of P. oleracea as a self- or 5% cross-pollinated species. A close relationship between P. oleracea, P. rausii and P. granulatostellulata was supported by ISSR and 18S rDNA gene sequence. The ISSR and 18s data support the specific status of P. nitida as a recently evolved taxon. We conclude that P. oleracea exists as a polymorphic species and is not divisible into microspecies based on seed surface as the primary morphological trait, and inter simple sequence repeats (ISSR) and 18S rDNA gene sequence markers. We suggest that the taxa do not merit specific rank as morphological characters and absence of a breeding barrier fail to separate the different populations when they become sympatric.     

An evaluation of the species boundaries of two putative taxonomic entities of Euterpe (Arecaceae) based on reproductive and morphological features

Past taxonomic treatments have classified Euterpe espiritosantensis as a synonym of E. edulis. However, both continue to be identified as two distinct species and are enumerated in the threatened species list. The goal of this study was to compare the reproductive biology of sympatric populations of these two morphs so as to identify morphological characters and mechanisms of reproductive isolation that could help to clarify species boundaries. Individuals of E. edulis and E. espiritosantensis show differences in size and duration of the peduncular bract, and differ in regard to inflorescence and immature fruit coloration. With an overlapping flowering period, but with distinctly different flowering peaks, the two taxa share the main visitors, viz. different Apidae and some Vespidae. Fruit and viable seeds are produced after self-, cross- and inter-specific cross-pollination in both morphs. Seed germination is significantly higher in E. edulis than in E. espiritosantensis. Differences in morphological and reproductive features suggest that these sympatric populations of two Euterpe morphs are to a reasonable degree reproductively isolated, which supports the recognition of E. espiritosantensis as a distinct species from E. edulis. The potential for hybridization does not reject the hypothesis of species distinctness, but points to a potential case of sympatric speciation that merits further investigations. Given that natural populations of Euterpe are nowadays fragmented and reduced in area of occurrence, retaining the high conservation status for E. espiritosantensis will help to safeguard this taxonomic entity under considerable threat.     

Genetic connectivity between sympatric populations of closely related char species,Dolly Varden <Emphasis Type="Italic">Salvelinus malma</Emphasis> and white char <Emphasis Type="Italic">Salvelinus albus</Emphasis>

The closely related chars Salvelinus malma and Salvelinus albus, which sympatrically inhabit the Kamchatka River basin and Kronotsky Lake (Kamchatka), attract the attention of the researchers because of their debated origin and taxonomic status. Previous studies of sympatric populations of these chars revealed small but statistically significant genetic differences between these species at a number of molecular markers, suggesting the presence of the genetic exchange and hybridization. In this study, based on genotypic characterization of nine microsatellite loci, a considerable level of historical and contemporary genetic migration between sympatric populations of these chars was demonstrated. At the individual level, a high degree of hybridization was observed, mainly among the Dolly Varden individuals from the studied populations. The obtained evidences on the genetic connectivity between sympatric S. malma and S. albus do not support the separate species status of S. albus.     

Molecular differentiation of Chenopodium album complex and some related species using ISSR profiles and ITS sequences

The present study was undertaken to understand the genetic differentiation and relationships in various components of C. album complex, C. giganteum and some related species using inter simple sequence repeats (ISSR) profiles and internal transcribed spacer (ITS) sequences. The relationships based on UPGMA dendrograms have shown the heterogenous nature of C. album complex. The 2x taxa while showing close relation among themselves are sharply segregated from 4x and 6x taxa belonging to C. album and C. giganteum. Among the three cytotypes from North Indian plains the 4x shows greater similarity to 6x than to 2x which is corroborated by the karyotypic studies. Furthermore, the 6x C. album and C. giganteum accessions of American and European origin are clearly segregated from those of Indian origin which may show their separate origin. Other related species show relationships according to their taxonomic position. The present study based on ISSR profiles and ITS sequences has therefore been very useful in explaining the relationships between various components of C. album complex and related species. However, more work needs to be done using different CpDNA loci to define correct species boundary of the taxa under C. album complex from Himalayas and North Indian Plains.     

Taxonomy and species delimitation in Cryptosporidium

Amphibians, reptiles, birds and mammals serve as hosts for 19 species of Cryptosporidium. All 19 species have been confirmed by morphological, biological, and molecular data. Fish serve as hosts for three additional species, all of which lack supporting molecular data. In addition to the named species, gene sequence data from more than 40 isolates from various vertebrate hosts are reported in the scientific literature or are listed in GenBank. These isolates lack taxonomic status and are referred to as genotypes based on the host of origin. Undoubtedly, some will eventually be recognized as species. For them to receive taxonomic status sufficient morphological, biological, and molecular data are required and names must comply with the rules of the International Code for Zoological Nomenclature (ICZN). Because the ICZN rules may be interpreted differently by persons proposing names, original names might be improperly assigned, original literature might be overlooked, or new scientific methods might be applicable to determining taxonomic status, the names of species and higher taxa are not immutable. The rapidly evolving taxonomic status of Cryptosporidium sp. reflects these considerations.     

An insight into molecular taxonomy of bufonids,microhylids, and dicroglossid frogs: First genetic records from Pakistan

The current study was focused on documentation of amphibian assemblage in North Punjab and Islamabad Capital Territory, Pakistan, by using mitochondrial gene sequences of 16S rRNA. Our study entailed 37% of the known amphibian species of the country. We provided a phylogenetic analysis based on 74 newly generated mitochondrial 16S rRNAs from nine species of genus Microlyla, Duttaphrynus, Allopaa, Nanorana, Sphaerotheca, Minervarya, Hoplobatrachus, and Euphlyctis. We employed the maximum‐likelihood inference and Bayesian analysis to assess the taxonomic status of the samples obtained from Pakistan, with respect to other congeneric species from surrounding regions. Our findings confirmed the taxonomic status of South Asian anuran species Duttaphrynus stomaticus, Duttaphrynus melanostictus, Microhyla nilphamariensis, Allopaa hazarensis, Nanorana vicina, Sphaerotheca maskeyi (synonym: S. pashchima), Minervarya pierrei, Hoplobatrachus tigerinus, and Euphlyctis kalasgramensis in Pakistan. We have reported new country records of genus Minervarya ( M. pierrei). Minervarya pierrei was previously misidentified as Fejervarya limnocharis, due to dearth of genetic information. We provided the first genetic records of our endemic species N. vicina. The results revealed the taxonomic placement of N. vicina with respect to its congeners and validated the taxonomic status of N. vicina from its type locality (Murree) for the first time. The findings of the present study also indicated the paraphyletic relationship of A.‐ hazarensis with Nanorana species. So, based on our phylogenetic inferences, morphological characters, and habitat preferences, validity of generic status of A. hazarensis is undecided. As our data were not enough to resolve this issue, we suggest sequencing of additional mitochondrial and nuclear genes in the future studies to get a better resolution. We recommend carrying out extensive surveys throughout the country for proper scientific documentation of amphibians of Pakistan. Many new species, some of them might be endemic to Pakistan, are expected to be discovered, and taxonomic status of other species would be resolved.     

Norfolk Island Robins are a distinct endangered species: ancient DNA unlocks surprising relationships and phenotypic discordance within the Australo-Pacific Robins

Uncertain taxonomy hinders the effective prioritization of taxa for conservation. This problem is acute for understudied island populations in the southwest Pacific Ocean, which are increasingly threatened by habitat loss, predation and climate change. Here, we offer the first test of taxonomic limits and phylogenetic affinities of the iconic Pacific Robin radiation (Petroica multicolor) in order to prioritize the conservation of its nominotypical subspecies, the endangered Norfolk Island Robin (P. m. multicolor). We integrate phylogenetic analyses of ancient DNA and quantitative measures of plumage and morphometric variation to show that the Norfolk Island Robin should be recognized as a distinct species. Phenotypic and genetic datasets contradict the longstanding treatment of Pacific Robins (including Norfolk Island Robins) and Scarlet Robins (P. boodang) as a single species. Instead, we show that Norfolk Island Robins are deeply divergent from Scarlet Robins and have more genetic similarity to Red-capped Robins (P. goodenovii) than to other Pacific Robins. This finding is unrepresentative of the current taxonomic and conservation status of the Norfolk Island Robin, which we propose should be recognised as an endemic endangered species. Our study clearly shows that in the absence of contemporary tissues, ancient DNA approaches using historical museum specimens can address taxonomic questions that morphological traits are unable to resolve. Further, it highlights the need for similar studies of other threatened Norfolk fauna with uncertain taxonomic status in order to ensure appropriate conservation prioritization.     

Molecular and chemical characters to evaluate species status of two cuckoo bumblebees: Bombus barbutellus and Bombus maxillosus (Hymenoptera,Apidae, Bombini)

Many methods, based on morphological, molecular or chemical characters, have been used to address the question of species taxonomic status. Integrative taxonomy aims to define stronger supported taxonomic hypotheses by considering complementary datasets from different characters. By following an integrative approach, the present study includes molecular, chemical and morphological criteria to establish the taxonomic status of two rare and doubtful cuckoo bumblebee taxa: Bombus (Psithyrus) barbutellus and Bombus (Psithyrus) maxillosus. These two sympatric taxa are discriminated by few morphological criteria (mainly wing darkness and hair length). We used these morphological character diagnoses to establish an a priori status of our samples (23 specimens). We developed a combined molecular dataset from one nuclear gene, elongation factor 1α (EF‐1α), and one mitochondrial gene, cytochrome c oxidase subunit I (COI), spanning 1623 bp, and a chemical dataset of sexual marking pheromones (73 compounds). The molecular data were subjected to maximum‐likelihood and Bayesian phylogenetic inference under partitioned model and maximum parsimony. The chemical data were analysed by clustering and the two‐group k‐means method to test divergences between the two species. The resulting phylogenetic trees show no consistent divergence between the two taxa. Moreover, we found no divergence in the sexual marking pheromones in the clustering and two‐group k‐means analyses. These converging results support the conspecificity of both taxa. Nonetheless, our determinations using the traditional morphological criteria separated our samples into two taxa. We conclude that the morphological criteria seem to relate to intraspecific variations: B. maxillosus is regarded as a syn.n. of B. barbutellus.     

Gnaphalium teydeum and Gnaphalium luteo-album: two taxa of the Canary Islands with different genetic histories

Gnaphalium teydeum and G. luteo-album (Asteraceae) are two closely congeneric taxa native to the Canary Islands. While G. luteo-album is widespread in the Macaronesian Region, G. teydeum is endemic to the island of Tenerife and considered endangered by IUCN. Using the RAPD technique this study investigated the level and apportionment of genetic diversity of these taxa, trying to solve a taxonomic dispute related to G. teydeum. Based on the 102 DNA fragments generated by 11 primers, a high level of genetic differentiation was found between the taxa (F _ST  = 0.366), with G. luteo-album showing levels of genetic variability (P = 100%; H = 0.246) higher than those found in G. teydeum (P = 75.5%; H = 0.173). UPGMA dendrogram and Bayesian cluster analysis clearly separated populations from both the species. Overall, results show that although morphological differentiation between G. teydeum and G. luteo-album is not strong, they show marked molecular divergence, supporting the current taxonomic status.     

Integrative analyses unveil speciation linked to host plant shift in Spialia butterflies

Discovering cryptic species in well‐studied areas and taxonomic groups can have profound implications in understanding eco‐evolutionary processes and in nature conservation because such groups often involve research models and act as flagship taxa for nature management. In this study, we use an array of techniques to study the butterflies in the Spialia sertorius species group (Lepidoptera, Hesperiidae). The integration of genetic, chemical, cytogenetic, morphological, ecological and microbiological data indicates that the sertorius species complex includes at least five species that differentiated during the last three million years. As a result, we propose the restitution of the species status for two taxa often treated as subspecies, Spialia ali (Oberthür, 1881) stat. rest. and Spialia therapne (Rambur, 1832) stat. rest., and describe a new cryptic species Spialia rosae Hernández‐Roldán, Dapporto, Dinc?, Vicente & Vila sp. nov. Spialia sertorius (Hoffmannsegg, 1804) and S. rosae are sympatric and synmorphic, but show constant differences in mitochondrial DNA, chemical profiles and ecology, suggesting that S. rosae represents a case of ecological speciation involving larval host plant and altitudinal shift, and apparently associated with Wolbachia infection. This study exemplifies how a multidisciplinary approach can reveal elusive cases of hidden diversity.     

Taxonomy of the Solidago virgaurea Group (Asteraceae) in Poland,with Special Reference to Variability along an Altitudinal Gradient

The morphological differentiation and taxonomic treatment of lowland and high-mountain morphotypes within the Solidago virgaurea group are controversial. To clarify the taxonomic status of these taxa, we conducted a morphometric analysis of 1,746 individuals from 80 localities along an altitudinal gradient from the lowlands of northern Poland to the Carpathians and Sudetes of southern Poland. Multivariate morphometric analyses, cluster analyses and principal component analyses, were used to examine the morphological differentiation within the S. virgaurea group in Poland. Canonical discriminant analysis was applied to determine the morphological characters that best discriminate among the taxa. The stability of the high-mountain Solidago minuta morphotype was tested in an experimental field established in lowland Poland; individuals transplanted from various mountain sites were cultivated at this site, and the morphotypes remained stable in terms of their floral and vegetative characters. Multivariate analyses revealed two morphologically distinct taxa in the S. virgaurea group, which correspond to lowland S. virgaurea s. str. and high-mountain S. minuta as recognised in some European floras. The most important morphological characters for distinguishing the taxa are the number of tubular florets per capitulum, inner involucral bract width and involucre height. Vegetative and inflorescence characters appear to have less taxonomic value because they changed continuously with altitude. A key for identifying S. virgaurea and S. minuta in Poland is presented.     

Genotypic and phenotypic evaluation of Rutilus spp. from Skadar, Ohrid and Prespa Lakes supports revision of endemic as well as taxonomic status of several taxa

One mtDNA gene (cytochrome b), one nuclear DNA fragment, five microsatellites and a suite of morphological characters were evaluated in samples of Rutilus spp. from Skadar, Ohrid and Prespa Lakes. Both genetic and phenotypic data supported two sympatric taxa in Lake Skadar, whereby Prespa and Ohrid Lakes revealed only a single taxon each. One of the taxa from Lake Skadar was similar to samples from Lake Prespa, whereas the second taxon was the most divergent in the data set. The estimated time to the most recent common ancestor of these two sympatric taxa in Lake Skadar was between 125 000 and 500 000 years. The data did not support existing taxonomic schemes for Rutilus in these lakes. This study poses the following working hypothesis: (1) Rutilus prespensis lives both in Lake Prespa and Lake Skadar and therefore is not endemic to Lake Prespa, (2) Rutilus ohridanus lives in Lake Ohrid only and therefore could be considered an endemic if its species status is retained and (3) a third recently described taxon (Rutilus albus) sympatric to R. prespensis lives in Lake Skadar.     

Phylogenetic and conservation status of the endangered terrestrial orchid Nervilia nipponica (Orchidaceae) in Korea

The putative Japanese endemic Nervilia nipponica was discovered recently on Jeju Island in Korea. However, Japanese populations of N. nipponica have since been shown to comprise two polyphyletic taxa. To confirm the taxonomic status of the Korean Nervilia populations, we compared ITS1, matK and trnL intron sequence data with that from the two cryptic Japanese taxa. Our results confirmed all Korean populations as belonging to the true N. nipponica s.s, which must shed its endemic status. Korean accessions showed no distinct genotypic patterning, with samples from all populations and representing two distinct colour forms forming a single monophyletic clade. The extremely low level of genetic diversity detected within N. nipponica on Jeju Island underscores the urgent need for measures to secure the species from local extirpation. We conclude that N. nipponica is a priority for conservation and recommend that systematic controls and sustained efforts are required to ensure its protection.     

Systematic studies in sedum section ternata (Crassulaceae)

An investigation of flavonoid compounds, pollen grains, and seed coats of the four taxa of the genusSedum sectionTernata, S. nevii, S. glaucophyllum, S. pulchellum, andS. ternatum, was undertaken to resolve the taxonomic status ofS. glaucophyllum. Flavonoid compounds were tentatively identified utilizing UV spectroscopy and shift reagents. Both flavones and fla vonols, as apigenin, luteolin, quercetin, and kaempferol aglycones and glycosides, were detected. Pollen grains and seed coats were viewed and photographed with the aid of a scanning electron microscope. Pollen grains of all four taxa were uniformly tricolporate; interspecific variation was negligible. Seed coats of all four taxa differed significantly with respect to ornamentation. Phytochemical and morphological data both support the recognition ofS. glaucophyllum as a species distinct from S.nevii.