Intercontinental karyotypic differentiation of Chironomus entis Shobanov, a Holarctic member of the C. plumosus group (Diptera, Chironomidae).

Analysis of banding sequences of polytene chromosomes in Palearctic (Russian) and Nearctic (North American) Chironomus entis shows strong karyotype divergence between populations on the two continents. Four out of seven chromosomal arms in the North American C. entis karyotype are characterized by sequences found only in the Nearctic. In total, 44 banding sequences are now known for this species across the Holarctic, including 22 exclusively Palearctic, 6 Holarctic, and 16 exclusively Nearctic sequences. The degree of cytogenetic differentiation between Palearctic and Nearctic C. entis populations is an order of magnitude greater than differentiation among populations within either continent, but is only one third as great as the cytogenetic distance between the sibling species C. entis and C. plumosus. C. entis is the only sibling species of C. plumosus uncovered during cytological identification of Chironomus species from more than 50 North American lakes, indicating that the plumosus sibling-species group is much smaller in the Nearctic than in the Palearctic, where a dozen sibling species are known. Cytogenetic distance values calculated between Nearctic and Palearctic representatives of both C. entis and its sibling species C. plumosus are similar, but result from different patterns of karyotype divergence. New World C. entis is distinguished from Old World populations by the 16 uniquely Nearctic sequences, four of which occur in the homozygous state. In contrast, North American C. plumosus has fewer uniquely Nearctic sequences, and only one that occurs as a homozygote. However, four chromosomal arms in C. plumosus that are polymorphic in the Palearctic show fixation, or near fixation, of Holarctic sequences in the Nearctic C. plumosus karyotype. Thus, both the fixation of Holarctic sequences, and the occurrence or fixation of distinctly Nearctic sequences, contribute significantly to karyotype divergence. Patterns of karyotype divergence in Palearctic and Nearctic populations of different Holarctic chironomid species are discussed relative to intercontinental cytogenetic differentiation in other dipterans.     

DNA barcode libraries provide insight into continental patterns of avian diversification

Background

The causes for the higher biodiversity in the Neotropics as compared to the Nearctic and the factors promoting species diversification in each region have been much debated. The refuge hypothesis posits that high tropical diversity reflects high speciation rates during the Pleistocene, but this conclusion has been challenged. The present study investigates this matter by examining continental patterns of avian diversification through the analysis of large-scale DNA barcode libraries.

Methodology and Principal Findings

Standardized COI datasets from the avifaunas of Argentina, the Nearctic, and the Palearctic were analyzed. Average genetic distances between closest congeners and sister species were higher in Argentina than in North America reflecting a much higher percentage of recently diverged species in the latter region. In the Palearctic genetic distances between closely related species appeared to be more similar to those of the southern Neotropics. Average intraspecific variation was similar in Argentina and North America, while the Palearctic fauna had a higher value due to a higher percentage of variable species. Geographic patterning of intraspecific structure was more complex in the southern Neotropics than in the Nearctic, while the Palearctic showed an intermediate level of complexity.

Conclusions and Significance

DNA barcodes can reveal continental patterns of diversification. Our analysis suggests that avian species are older in Argentina than in the Nearctic, supporting the idea that the greater diversity of the Neotropical avifauna is not caused by higher recent speciation rates. Species in the Palearctic also appear to be older than those in the Nearctic. These results, combined with the patterns of geographic structuring found in each region, suggest a major impact of Pleistocene glaciations in the Nearctic, a lesser effect in the Palearctic and a mild effect in the southern Neotropics.     

Molecular and morphological evidence for the Holarctic distribution of Urogonimus macrostomus (Rudolphi, 1803) Monticelli, 1888 (Digenea: Leucochloridiidae)

Species of Urogonimus Monticelli, 1888 (Leucochloridiidae Poche, 1907) are difficult to distinguish using adult morphology, and their taxonomy has been repeatedly subjected to revision. Some Nearctic species have been regarded as synonymous with the Palearctic type species Urogonimus macrostomus (Rudolphi, 1803) Monticelli, 1888. This implies that U. macrostomus is present in the Nearctic, but there is no additional evidence for this putative distribution. We collected trematodes morphologically indistinguishable from U. macrostomus from a house sparrow (Passer domesticus) in Edmonton, Alberta, Canada. Sequences 2958 bp in total length from the small and large subunits of ribosomal DNA from 2 specimens were 99.8-100% identical to those of U. macrostomus in the Ukraine and Japan. In light of the lack of morphological differences and small degree of genetic variation, we consider the specimens we collected to be conspecific with U. macrostomus in the Palearctic, and the Holarctic range of the species is thus supported. Sequences from a more rapidly evolving gene, cytochrome c oxidase 1, were obtained to aid future study of this and related species.     

Mind the gap! Integrating taxonomic approaches to assess ant diversity at the southern extreme of the Atlantic Forest

Understanding patterns of species diversity relies on accurate taxonomy which can only be achieved by long‐term natural history research and the use of complementary information to establish species boundaries among cryptic taxa. We used DNA barcoding to characterize the ant diversity of Iguazú National Park (INP), a protected area of the Upper Paraná Atlantic Forest ecoregion, located at the southernmost extent of this forest. We assessed ant diversity using both cytochrome c oxidase subunit 1 (COI) sequences and traditional morphological approaches, and compared the results of these two methods. We successfully obtained COI sequences for 312 specimens belonging to 124 species, providing a DNA barcode reference library for nearly 50% of the currently known ant fauna of INP. Our results support a clear barcode gap for all but two species, with a mean intraspecific divergence of 0.72%, and an average congeneric distance of 17.25%. Congruently, the library assembled here was useful for the discrimination of the ants of INP and allowed us to link unidentified males and queens to their worker castes. To detect overlooked diversity, we classified the DNA barcodes into Molecular Operational Taxonomic Units (MOTUs) using three different clustering algorithms, and compared their number and composition to that of reference species identified based on morphology. The MOTU count was always higher than that of reference species regardless of the method, suggesting that the diversity of ants at INP could be between 6% and 10% higher than currently recognized. Lastly, our survey contributed with 78 new barcode clusters to the global DNA barcode reference library, and added 36 new records of ant species for the INP, being 23 of them new citations for Argentina.     

The role of chromosomal polymorphism in divergence of populations and species of the genus Chironomus (Diptera)

The data on the structure and level of chromosomal polymorphism in natural populations of species of the genus Chironomus are summarized. A very high level of chromosomal polymorphism was noted for most species. Paracentric inversions prevailed among the chromosomal rearrangements found in natural populations. Changes in the set and frequency of inversion sequences are the most important factor of cytogenetic divergence of populations. Several cytogenetic types of populations were distinguished. The Palaearctic and Nearctic populations of Holarctic species diverged to a greater extent due to the formation of endemic Palearctic and Nearctic inversion sequences. The sequences common for both regions indicated a common ancestry of the populations. The cytogenetic distances between the Palearctic and Nearctic populations are greater by an order of magnitude than those between populations within each zoogeographic region. Divergence of species karyotypes was found to result from fixation of different inversion sequences in the course of evolution. The karyotypes of Palearctic and Nearctic species mainly differ by the presence of endemic Palearctic and Nearctic banding sequences. Several basic sequences common for some species allow the cytogenetic history of their origin to be revealed. A NJ phylogenetic tree was built for the genus Chironomus, demonstrating chromosomal evolution of its species.     

A revision of Benjaminia (Hymenoptera: Ichneumonidae, Campopleginae)

Abstract The campoplegine genus Benjuminia is herein revised, with ten species being described as new, in addition to the five previously known species. Phylogenetic relationships of Benjuminia to other genera are discussed, and a cladogram presented for the species. Two monophyletic species groups are recognized, one being confined to the Palearctic and the other to the Nearctic. Examination of larval morphology not only fails to reveal larval characters for distinguishing Benjuminia but leads to the conclusion that use of larval morphology to identify campoplegine species is a dubious practice.     

Cryptic diversity in ant-mimic Micaria spiders (Araneae,Gnaphosidae) and a tribute to early naturalists

Spiders of the genus Micaria are ground-living mimics of ants. Species delineation in these spiders is challenging, mainly because of exceptional high levels of intraspecific variation masking species boundaries. As implied by preliminary DNA barcode data from Central Europe, the Holarctic and very widely distributed glossy ant-spider M. pulicaria shows cryptic diversity. Here, we disentangle the hidden diversity by means of an integrative taxonomy approach, using mitochondrial DNA, morphometrics, traditional genitalic characters and ecology. Our data suggest the clear delineation of two distinct species, which supports the conception of 19th century taxonomists. These early naturalists distinguished M. pulicaria and a second closely related species based on morphology and natural history, which were synonymized in subsequent taxonomic studies. Therefore, we re-circumscribe M. pulicaria and revalidate the long forgotten M. micans. These two Micaria species co-occur sympatrically in vast areas of the western Palearctic, while the Nearctic region is populated by M. pulicaria alone. Male genitalic traits are more dissimilar in the area of sympatry than in allopatry, suggesting a decisive role of reproductive character displacement in species diversification. Our study emphasizes the value of the early taxonomic literature in integrative taxonomic studies, as it may contain crucial information on natural history that are not regularly recorded by modern taxonomists.     

Barcoding Neotropical birds: assessing the impact of nonmonophyly in a highly diverse group

In this study, we verified the power of DNA barcodes to discriminate Neotropical birds using Bayesian tree reconstructions of a total of 7404 COI sequences from 1521 species, including 55 Brazilian species with no previous barcode data. We found that 10.4% of species were nonmonophyletic, most likely due to inaccurate taxonomy, incomplete lineage sorting or hybridization. At least 0.5% of the sequences (2.5% of the sampled species) retrieved from GenBank were associated with database errors (poor‐quality sequences, NuMTs, misidentification or unnoticed hybridization). Paraphyletic species (5.8% of the total) can be related to rapid speciation events leading to nonreciprocal monophyly between recently diverged sister species, or to absence of synapomorphies in the small COI region analysed. We also performed two series of genetic distance calculations under the K2P model for intraspecific and interspecific comparisons: the first included all COI sequences, and the second included only monophyletic taxa observed in the Bayesian trees. As expected, the mean and median pairwise distances were smaller for intraspecific than for interspecific comparisons. However, there was no precise ‘barcode gap’, which was shown to be larger in the monophyletic taxon data set than for the data from all species, as expected. Our results indicated that although database errors may explain some of the difficulties in the species discrimination of Neotropical birds, distance‐based barcode assignment may also be compromised because of the high diversity of bird species and more complex speciation events in the Neotropics.     

DNA barcoding‐based species delimitation increases species count of Eois (Geometridae) moths in a well‐studied tropical mountain forest by up to 50%

Abstract The genus Eois comprises an important part of megadiverse assemblages of geometrid moths in mountain rainforests of southern Ecuador. In this study we report: (i) on the construction of a DNA barcode library of Eois for identification purposes; and (ii) the exploration of species diversity through species delimitation by pair‐wise distance thresholds. COI barcode sequences were generated from 408 individuals (at least 105 species) collected on a narrow geographic scale (～40 km²) in the Reserva Biológica San Francisco. Analyses of barcode sequence divergence showed that species delimitations based solely on external morphology result in broad overlap of intra‐ and interspecific distances. Species delimitation at a 2% pair‐wise distance threshold reveals a clear barcoding gap. Fifty‐two previously unrecognized species were identified, 31 of which could only be distinguished by an integrative taxonomy approach. Twelve additional putative species could only be recognized by threshold‐based delimitation. Most splits resulted in two or three newly perceived cryptic taxa. The present study increased the number of Eois species recorded from that small area of Andean mountain forest from 102 to 154 (morphology‐ plus integrative taxonomy‐based) or even 166 (sequence‐based), leaving the species accumulation curve still far from reaching an asymptote. Notably, in no case did two or more previously distinguished morphospecies have to be lumped. This barcode inventory can be used to match larvae to known adult samples without rearing, and will therefore be of vital help to extend the currently limited knowledge about food plant relationships and host specialization.     

Molecular phylogeny of the marmots (Rodentia: Sciuridae): tests of evolutionary and biogeographic hypotheses

There are 14 species of marmots distributed across the Holarctic, and despite extensive systematic study, their phylogenetic relationships remain largely unresolved. In particular, comprehensive studies have been lacking. A well-supported phylogeny is needed to place the numerous ecological and behavioral studies on marmots in an evolutionary context. To address this situation, we obtained complete cytochrome (cyt) b sequences for 13 of the species and a partial sequence for the 14th. We applied a statistical approach to both phylogeny estimation and hypothesis testing, using parsimony and maximum likelihood-based methods. We conducted statistical tests on a suite of previously proposed hypotheses of phylogenetic relationships and biogeographic histories. The cyt b data strongly support the monophyly of Marmota and a western montane clade in the Nearctic. Although some other scenarios cannot be rejected, the results are consistent with an initial diversification in North America, followed by an invasion and subsequent rapid diversification in the Palearctic. These analyses reject the two major competing hypotheses of M. broweri's phylogenetic relationships--namely, that it is the sister species to M. camtschatica of eastern Siberia, and that it is related closely to M. caligata of the Nearctic. The Alaskan distribution of M. broweri is best explained as a reinvasion from the Palearctic, but a Nearctic origin can not be rejected. Several other conventionally recognized species groups can also be rejected. Social evolution has been homoplastic, with large colonial systems evolving in two groups convergently. The cyt b data do not provide unambiguous resolution of several basal nodes in the Palearctic radiation, leaving some aspects of pelage and karyotypic evolution equivocal.     

DNA barcoding should accompany taxonomy - the case of Cerebratulus spp (Nemertea)

Many issues in DNA barcoding need to be solved before it can reach its goal to become a general database for species identification. While species delimitations are more or less well established in several taxa, there are still many groups where this is not the case. Without the proper taxonomic background/knowledge and corroboration with other kinds of data, the DNA barcoding approach may fail to identify species accurately. The classification and taxonomy of phylum Nemertea (nemerteans, ribbon worms) are traditionally based on morphology, but are not corroborated by an increasing amount of genetic data when it comes to classification either into species or into higher taxa. The taxonomy of the phylum needs to be improved before the full potential of DNA barcoding can be utilized to make sure that valid Linnean names accompany the barcode sequences. We illustrate the problematic situation in the phylum Nemertea by a case study from the genus Cerebratulus.     

Parallel evolution in ecological and reproductive traits to produce cryptic damselfly species across the holarctic

The damselfly genus Enallagma originated in the Nearctic, and two Nearctic lineages recently underwent radiations partly associated with multiple independent habitat shifts from lakes dominated by fish predators into lakes dominated by dragonfly predators. A previous molecular study of four Palearctic morphospecies and all representative Nearctic species identified the presence of two cryptic species sets, with each set having Palearctic and Nearctic representatives. However, the cryptic species within each set are not sibling species. Here, we present quantitative data on ecologically important larval morphologies and behaviors involved in predator avoidance and on adult male morphological structures involved in mate recognition to quantify the phenotypic relationships among these cryptic species sets. For the adult stage, our data indicate strong parallel evolution of the structures involved in specific mate recognition-the male cerci. For the larval stage, morphometric analyses show that the Palearctic species evolved a nearly identical morphology to the sibling-clade members in the Nearctic that live in waters where dragonflies are the top predators. This implicates the importance of dragonfly predation in the history of the Palearctic clade. Behavioral analyses suggest population differentiation in response to the actual predator environment in the Palearctic clade, consistent with the species differentiation seen in the Nearctic. Our results suggest parallel evolution of adult traits that influence specific mate choice and larval traits that influence ecological performance underlie the striking similarity of Enallagma species across continents. This concurrent parallel evolution in both stages of a complex life cycle, especially when both stages do not share the same selective environment, may be a very unusual mechanism generating cryptic species.     

DNA barcoding of economically important freshwater fish species from north‐central Nigeria uncovers cryptic diversity

This study examines the utility of morphology and DNA barcoding in species identification of freshwater fishes from north‐central Nigeria. We compared molecular data (mitochondrial cytochrome c oxidase subunit I (COI) sequences) of 136 de novo samples from 53 morphologically identified species alongside others in GenBank and BOLD databases. Using DNA sequence similarity‐based (≥97% cutoff) identification technique, 50 (94.30%) and 24 (45.30%) species were identified to species level using GenBank and BOLD databases, respectively. Furthermore, we identified cases of taxonomic problems in 26 (49.00%) morphologically identified species. There were also four (7.10%) cases of mismatch in DNA barcoding in which our query sequence in GenBank and BOLD showed a sequence match with different species names. Using DNA barcode reference data, we also identified four unknown fish samples collected from fishermen to species level. Our Neighbor‐joining (NJ) tree analysis recovers several intraspecific species clusters with strong bootstrap support (≥95%). Analysis uncovers two well‐supported lineages within Schilbe intermedius. The Bayesian phylogenetic analyses of Nigerian S. intermedius with others from GenBank recover four lineages. Evidence of genetic structuring is consistent with geographic regions of sub‐Saharan Africa. Thus, cryptic lineage diversity may illustrate species’ adaptive responses to local environmental conditions. Finally, our study underscores the importance of incorporating morphology and DNA barcoding in species identification. Although developing a complete DNA barcode reference library for Nigerian ichthyofauna will facilitate species identification and diversity studies, taxonomic revisions of DNA sequences submitted in databases alongside voucher specimens are necessary for a reliable taxonomic and diversity inventory.     

RECOGNIZING DINOFLAGELLATE SPECIES USING ITS rDNA SEQUENCES1

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

Single nucleotide polymorphism barcoding of cytochrome c oxidase I sequences for discriminating 17 species of Columbidae by decision tree algorithm

DNA barcodes are widely used in taxonomy, systematics, species identification, food safety, and forensic science. Most of the conventional DNA barcode sequences contain the whole information of a given barcoding gene. Most of the sequence information does not vary and is uninformative for a given group of taxa within a monophylum. We suggest here a method that reduces the amount of noninformative nucleotides in a given barcoding sequence of a major taxon, like the prokaryotes, or eukaryotic animals, plants, or fungi. The actual differences in genetic sequences, called single nucleotide polymorphism (SNP) genotyping, provide a tool for developing a rapid, reliable, and high‐throughput assay for the discrimination between known species. Here, we investigated SNPs as robust markers of genetic variation for identifying different pigeon species based on available cytochrome c oxidase I (COI) data. We propose here a decision tree‐based SNP barcoding (DTSB) algorithm where SNP patterns are selected from the DNA barcoding sequence of several evolutionarily related species in order to identify a single species with pigeons as an example. This approach can make use of any established barcoding system. We here firstly used as an example the mitochondrial gene COI information of 17 pigeon species (Columbidae, Aves) using DTSB after sequence trimming and alignment. SNPs were chosen which followed the rule of decision tree and species‐specific SNP barcodes. The shortest barcode of about 11 bp was then generated for discriminating 17 pigeon species using the DTSB method. This method provides a sequence alignment and tree decision approach to parsimoniously assign a unique and shortest SNP barcode for any known species of a chosen monophyletic taxon where a barcoding sequence is available.     

DNA barcoding of Pedicularis L.(Orobanchaceae): Evaluating four universal barcode loci in a large and hemiparasitic genus

One application ofDNA barcoding is species identification based on sequences of a short and standardized DNA region.In plants,various DNA regions,alone or in combination,have been proposed and investigated,but consensus on a universal plant barcode remains elusive.In this study,we tested the utility of four candidate barcoding regions (rbcL,matK,trnH-psbA,and internal transcribed spacer (ITS)) as DNA barcodes for discriminating species in a large and hemiparasitic genus Pedicularis (Orobanchaceae).Amplification and sequencing was successful using single primer pairs for rbcL,trnH-psbA,and ITS,whereas two primer pairs were required for matK.Patterns of sequence divergence commonly showed a “barcoding gap”,that is,a bimodal frequency distribution of pairwise distances representing genetic diversity within and between species,respectively Considering primer universality,ease of amplification and sequencing,and performance in discriminating species,we found the most effective single-region barcode for Pedicularis to be ITS,and the most effective two-region barcode to be rbcL +ITS.Both discriminated at least 78％ of the 88 species and correctly identified at least 89％ of the sequences in our sample,and were effective in placing unidentified samples in known species groups.Our results suggest that DNA barcoding has the potential to aid taxonomic research in Pedicularis,a species-rich cosmopolitan clade much in need of revision,as well as ecological studies in its center of diversity,the Hengduan Mountains region of China.     

Are subspecies useful in evolutionary and conservation biology?

The taxonomic rank of subspecies remains highly contentious, largely because traditional subspecies boundaries have sometimes been contradicted by molecular phylogenetic data. The most complete meta-analysis to date, for instance, found that only 3% of traditional avian subspecies represented distinct phylogenetic lineages. However, the global generality of this phenomenon remains unclear due to this previous study's narrow geographic focus on continental Nearctic and Palearctic subspecies. Here, we present a new global analysis of avian subspecies and show that 36% of avian subspecies are, in fact, phylogenetically distinct. Among biogeographic realms we find significant differences in the proportion of subspecies that are phylogenetically distinct, with Nearctic/Palearctic subspecies showing significantly reduced levels of differentiation. Additionally, there are differences between island and continental subspecies, with continental subspecies significantly less likely to be genetically distinct. These results indicate that the overall level of congruence between taxonomic subspecies and molecular phylogenetic data is greater than previously thought. We suggest that the widespread impression that avian subspecies are not real arises from a predominance of studies focusing on continental subspecies in North America and Eurasia, regions which show unusually low levels of genetic differentiation. The broader picture is that avian subspecies often provide an effective short-cut for estimating patterns of intraspecific genetic diversity, thereby providing a useful tool for the study of evolutionary divergence and conservation.     

Molecular Organization of the X Chromosome in Different Species of the Obscura Group of Drosophila

Nine single copy regions located on the X chromosome have been mapped by in situ hybridization in six species of the obscura group of Drosophila. Three Palearctic species, D. subobscura, D. madeirensis and D. guanche, and three Nearctic species, D. pseudoobscura, D. persimilis and D. miranda, have been studied. Eight of the regions include known genes from D. melanogaster (Pgd, zeste, white, cut, vermilion, RNA polymerase II 215, forked and suppressor of forked) and the ninth region (lambda DsubF6) has not yet been characterized. In all six species, as in D. melanogaster, all probes hybridize to a single site. Established chromosomal arm homologies of Muller's element A are only partly supported by present results since two of the probes (Pgd and zeste) hybridize at the proximal end of the XR chromosomal arm in the three Nearctic species. In addition to the centric fusion of Muller's A (= XL) and D (= XR) elements, the metacentric X chromosome of the Nearctic species requires a pericentric inversion to account for this result. Previously proposed homologies of particular chromosomal regions of the A (= X) chromosome in the three species of the D. subobscura cluster and of the XL chromosomal arm in the three species of the D. pseudoobscura cluster are discussed in light of the present results. Location of the studied markers has changed drastically not only since the divergence between the melanogaster and obscura groups but also since the Palearctic and Nearctic species of the obscura group diverged.     

Mitochondrial phylogeny of Locustella and related genera

We used maximum likelihood analysis of complete mitochondrial ND2 sequences (1041 bp) to clarify the taxonomy and relationships of various species and genera of grass and bush warblers. The tree revealed two clades of grass and bush warblers. One clade was comprised of all four western Palearctic Locustella and two species of Asian Bradypterus . The other clade included five eastern Palearctic Locustella (including the distinctive Sakhalin warbler Locustella amnicola ) and the marsh grassbird Megalurus pryeri . African Bradypterus and Australian little grassbird Megalurus gramineus were distantly related to their Asian congeners. Therefore, current taxonomy of these genera does not reflect their evolutionary history and needs revision. It is proposed that a phylogenetic analysis of morphology and ecological preferences would show that the current taxonomy of grass and bush warblers reflects species' habitat preferences and morphology related to locomotion and foraging in their habitats, rather than their shared ancestry. Distinct clades were found in grasshopper warbler Locustella naevia and Pallas's grasshopper warbler L. certhiola . Detailed phylogeographic studies are needed to elucidate the species status of the clades within these two species.