Molecular phylogenetic analyses of mullets (Mugilidae, Mugiliformes) based on two mitochondrial genes

Molecular phylogenetic studies are very scarce for the Mugilidae family; the present analysis using DNA sequences of the mitochondrial 16S rRNA and cytochrome b genes is the first study involving Brazilian mugilids. The results corroborate the monophyly of Mugil and are elucidative for the taxonomy of Brazilian mugilids. Mugil curema is clearly divided into two genetically distinct taxa, M. curema type I being closer related to Mugil hospes and the true M. curema (type II) grouping significantly with M. incilis. The results also suggest that Mugil liza and Mugil platanus should be treated as a single species or even populations of Mugil cephalus.     

Molecular Phylogeny of Grey Mullets Based on Mitochondrial DNA Sequence Analysis: Evidence of a Differential Rate of Evolution at the Intrafamily Level

The phylogeny of the grey mullets is considered problematic both at the intra- and interfamily level. Such a difficulty arises from the highly homogeneous morphology displayed by this group of fish and, consequently, from the paucity of the key morphological characters suitable to address their phylogeny and evolution. In the present work, we have approached the phylogenetic and evolutionary relationships of seven species of Mugilidae, six of which from the Mediterranean Sea, on the basis of the DNA sequences of two mitochondrial genes (cytochromeband 12S rRNA). Despite the morphological homogeneity exhibited by the taxa considered, the two species of the genusMugil(M. cephalusandM. curema) showed a remarkable genetic divergence compared to all the other members of the family. The relative rate test revealed a significantly higher rate of evolution along theMugillineage.     

Molecular phylogeny of Mugilidae fishes revised

Systematics derived from morphological characters often does not correspond with the evolutionary processes underlying the divergence within a group of organisms. In the family Mugilidae (Teleostei) morphological similarities have resulted in inconsistencies between taxonomy and phylogeny among its species, and particularly for the genera Mugil, Liza and Chelon where both intrageneric and intergeneric phylogenetic clarifications are needed. To address these issues, the direct sequencing of the mitochondrial region that encodes Phenylalanine (69 bp), 12S rRNA (842 bp), cytochrome c oxidase subunit I (651 bp) and cytochrome b (702 bp) was carried out. The data reveal that Mugil platanus and Mugil liza represent a continuum of a single species, closely related to but distinct from Mugil cephalus which itself appears to comprise a grouping of multiple and closely related species. This species complex was genetically distinct from Mugil curema, which, based on three clearly diverged species identified in this study along the Atlantic coast of the Americas, requires extensive taxonomic revision throughout its world-wide distribution. Unlike the monophyly supported within Mugil, relationships within Liza are paraphyletic, and a taxonomic revision of the genera Liza, Chelon and Oedalechilus is needed.     

New insights into and limitations of the molecular phylogeny in the taxon-rich land snail genus Montenegrina (Mollusca: Gastropoda: Clausiliidae)

Rock-dwelling gastropods are usually patchily distributed in limestone habitats, presumably have low active and passive dispersal ability and often represent narrow-ranged endemic taxa. Their current taxonomy is predominantly shell morphology based, and it remains unknown whether the morphologically differentiated and geographically separated populations represent phylogenetic clades. In this study, we analysed the hyperdiverse, terrestrial door snail genus Montenegrina. Based on the current taxonomy defined by shell morphology, it contains 29 species and 106 subspecies distributed in the Balkan region. The constructed phylogenetic tree using three mitochondrial markers was used to test whether it agrees with the current taxonomy. In this comprehensive tree, about half of the species and subspecies are monophyletic. Some of the paraphylies could be reasonably resolved by taxonomic changes; that is, some subspecies should be reassigned or raised to species level. Other incongruencies probably arose due to introgression even between distant clades. The histone genes turned out to be unsuitable for elucidating the phylogeny of Montenegrina. In the species-delimitation tests, considerably more molecular operational taxonomic units were delimited than the number of presently described species. The present data indicate that (a) shell morphology-based taxonomy and taxon recognition can be problematic in such a large and morphologically highly variable genus; (b) the potential error due to incomplete sampling presents a problem in a genus as variable as Montenegrina; (c) multi-locus analyses should be conducted to arrive at a better basis for species delimitation; and (d) integrative approaches including genetic as well as morphological/anatomical data from a comprehensive geographic sample are necessary.     

A taxonomic revision and species delimitation of the genus Purpuraria Enderlein, 1929 (Orthoptera: Pamphagidae) using an integrative approach

Recent studies on the endemic Canarian genus Purpuraria have shown that the taxonomy of its only recognized species (P. erna) is probably erroneous. In this study, an integrative revision of the genus is performed, based on a large number of specimens and geographical sampling. As a result, (1) the diagnostic characters at the genus level are re‐described, (2) Purpuraria magna n. sp. based on morphological, morphometric and genetic data is described and (3) the taxonomic status of a formerly described subspecies is clarified. Intraspecific and interspecific morphometric differences have been found, indicating that the genus is undergoing a process of morphological diversification. Nevertheless, the possibility of interspecific mating between individuals of the two species is suggested, because no significant differences have been found between their respective calling songs. Genetic analyses using mitochondrial and nuclear DNA sequences suggest that P. erna and P. magna are recent species with evidences of secondary contact episodes in the past.     

Integrative insect taxonomy based on morphology,mitochondrial DNA,and hyperspectral reflectance profiling

Integrative taxonomy is considered a reliable taxonomic approach of closely related and cryptic species by integrating different sources of taxonomic data (genetic, ecological, and morphological characters). In order to infer the boundaries of seven species of the evacanthine leafhopper genus Bundera Distant, 1908 (Hemiptera: Cicadellidae), an integrated analysis based on morphology, mitochondrial DNA, and hyperspectral reflectance profiling (37 spectral bands from 411–870 nm) was conducted. Despite their morphological similarities, the genetic distances of the cytochrome c oxidase subunit I (COI) gene among the tested species are relatively large (5.8–17.3%). The species‐specific divergence of five morphologically similar species (Bundera pellucida and Bundera spp. 1–4) was revealed in mitochondrial DNA data and reflectance profiling. A key to identifying males is provided, and their morphological characters are described. Average reflectance profiles from the dorsal side of specimens were classified based on linear discriminant analysis. Cross‐validation of reflectance‐based classification revealed that the seven species could be distinguished with 91.3% classification accuracy. This study verified the feasibility of using hyperspectral imaging data in insect classification, and our work provides a good example of using integrative taxonomy in studies of closely related and cryptic species. © 2015 The Linnean Society of London     

Collembola,the biological species concept and the underestimation of global species richness

Despite its ancient origin, global distribution and abundance in nearly all habitats, the class Collembola is comprised of only 8000 described species and is estimated to number no more than 50 000. Many morphologically defined species have broad geographical ranges that span continents, and recent molecular work has revealed high genetic diversity within species. However, the evolutionary significance of this genetic diversity is unknown. In this study, we sample five morphological species of the globally distributed genus Lepidocyrtus from 14 Panamanian sampling sites to characterize genetic diversity and test morphospecies against the biological species concept. Mitochondrial and nuclear DNA sequence data were analysed and a total of 58 molecular lineages revealed. Deep lineage diversification was recovered, with 30 molecular lineages estimated to have established more than 10 million years ago, and the origin almost all contemporary lineages preceding the onset of the Pleistocene (~2 Mya). Thirty‐four lineages were sampled in sympatry revealing unambiguous cosegregation of mitochondrial and nuclear DNA sequence variation, consistent with biological species. Species richness within the class Collembola and the geographical structure of this diversity are substantially misrepresented components of terrestrial animal biodiversity. We speculate that global species richness of Collembola could be at least an order of magnitude greater than a previous estimate of 50 000 species.     

Mitochondrial genetic differentiation and morphological difference of Miniopterus fuliginosus and Miniopterus magnater in China and Vietnam

Because of its complicated systematics, the bent‐winged bat is one of the most frequently studied bat species groups. In China, two morphologically similar bent‐winged bat species, Miniopterus fuliginosus and Miniopterus magnater were identified, but their distribution range and genetic differentiation are largely unexplored. In this study, we applied DNA bar codes and two other mitochondrial DNA genes including morphological parameters to determine the phylogeny, genetic differentiation, spatial distribution, and morphological difference of the M. fuliginosus and M. magnater sampled from China and one site in Vietnam. Mitochondrial DNA gene genealogies revealed two monophyletic lineages throughout the Tropic of Cancer. According to DNA bar code divergences, one is M. fuliginosus corresponding to the Chinese mainland and the other is M. magnater corresponding to tropical regions including Hainan and Guangdong provinces of China and Vietnam. Their most recent common ancestor was dated to the early stage of the Quaternary glacial period (ca. 2.26 million years ago [Ma] on the basis of D‐loop data, and ca. 1.69–2.37 Ma according to ND2). A population expansion event was inferred for populations of M. fuliginosus at 0.14 Ma. The two species probably arose in separate Pleistocene refugia under different climate zones. They significantly differed in forearm length, maxillary third molar width, and greatest length of the skull.     

Integrative taxonomy reveals a new Gammarus species (Crustacea,Amphipoda) surviving in a previously unknown southeast European glacial refugium

The multidisciplinarity of integrative taxonomy is particularly useful for clarifying the systematics of speciose groups that are poorly differentiated morphologically, and this approach can also illuminate their evolutionary history and biogeography. Here, we utilize it to examine the systematics and taxonomy of a newly recognized amphipod species, Gammarus hamaticornis n. sp., which belongs to a highly diverse genus of endemic freshwater crustaceans that show very limited morphological differentiation. Since this species is endemic to northern Dobrogea, a region at the northwestern Black Sea coast devoid of permafrost during the Last Glacial Maximum, we hypothesized that it survived in situ during the Quaternary climatic oscillations. We first examined the phylogenetic position of Gammarus hamaticornis n. sp. within the genus and then compared its morphology, phylogeography, distribution, and climatic niche with that of its sister species. Results indicate that G. hamaticornis n. sp. is most closely related to its widely distributed northern neighbor, G. kischineffensis, and a remarkable agreement was observed among morphological, multilocus coalescent and climatic analyses which supported the distinctiveness of both taxa. These apparently diverged during the Pliocene from a common ancestor that likely colonized freshwaters from the adjacent brackish basins of the shrinking Paratethys. The results indicate that G. hamaticornis n. sp. has persisted throughout the Pleistocene in northern Dobrogea, a hitherto hypothesized refugium confirmed for the first time with molecular genetic data. Due to its narrow geographical range, rarity in the local communities and highly fluctuating nature of the streams it inhabits, this species should be in the focus of future conservation priorities.     

Evolution of Strigamia centipedes (Chilopoda): a first molecular assessment of phylogeny and divergence times

We present a first phylogenetic and temporal framework, with biogeographical insights, for the centipedes of the genus Strigamia, which are widespread predators in the forest soils of the Northern Hemisphere and comprise the evo‐devo model species Strigamia maritima. The phylogeny was estimated by different methods of maximum likelihood and Bayesian inference from sequences of two mitochondrial (16S, COI) and two nuclear (18S, 28S) genes, obtained from 16 species from all major areas of the global range of the genus and encompassing most of the overall morphological and ecological diversity. Divergence times were estimated after calibration upon the fossil record of centipedes. We found that major lineages of extant species of Strigamia separated most probably around 60 million years (Ma) ago. The two most diverse lineages diversified during the last 30 Ma and are today segregated geographically, one in Europe and another in Eastern Asia. This latter region hosts a hitherto underestimated richness and anatomical diversity of species, including three still unknown, yet morphologically well distinct species, which are here described as new: Strigamia inthanoni sp. n. from Thailand, Strigamia korsosi sp. n. from the Ryukyu Islands and Strigamia nana sp. n. from Taiwan. The northern European model species S. maritima is more strictly related to the Eastern Asian lineage, from which it most probably separated around 35 Ma ago before the major diversification of the latter.     

Phylogeography and phylogeny of the genus Acanthonyx (Decapoda,Epialtidae) in the north‐east Atlantic and Mediterranean

The genus Acanthonyx Latreille, 1828 (Majoidea, Epialtidae) contains 17 or 18 known species, depending on competing taxonomic views, that are widely distributed across the world. Morphologically, most species look superficially alike, and therefore, similar taxonomic concepts have been described under different names. Consequently, there is today a considerable list of synonyms, which further complicates the taxonomy of the genus Acanthonyx. In this study, we conducted a phylogeographical and phylogenetic analysis of populations of the genus Acanthonyx in the NE Atlantic, Mediterranean and Macaronesia using the mitochondrial cytochrome oxidase subunit I (COI) and the nuclear 28S rRNA loci. Our phylogenetic and phylogeographical results revealed that Acanthonyx lunulatus sensu lato is a complex of three distinct lineages: one corresponding to the previously described Acanthonyx brevifrons, another to A. lunulatus sensu stricto and a third to a yet undescribed group. Whereas our results confirms that A. brevifrons deserves the status of a species, as it can be easily distinguishable from A. lunulatus by a few morphological traits, we could not find any such traits suitable for the discrimination between A. lunulatus sensu stricto and the third lineage. Furthermore, the degree of COI divergence between this lineage and A. lunulatus is below average levels for Decapoda species. Yet, no shared haplotypes have been detected between them. The differences found in the nuclear gene (indels), together with the sympatric occurrence of the two forms, prompt for a more detailed analysis of this group. Overall, the results show that significant genetic differentiation between specimens with similar morphology occurs in the Epialtidae, thus reinforcing the importance of combining morphological and genetic tools to fully resolve the taxonomy of these decapods.     

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.     

Exploring taxonomic diversity and biogeography of the family Nemacheilinae (Cypriniformes)

Nemacheilidae, in the superfamily Cobitoidea, is comprised of many of morphologically similar fish species that occur in Eurasian water bodies. This large group shows inconsistencies between traditional morphological taxonomy and molecular phylogenetic data. We used mitochondrial genomes, recombinase‐activating gene proteins 1 (RAG1) and the mitochondrial cytochrome c oxidase I gene (COI) to study the phylogenetic relationships among Nemacheilidae species using Bayesian inference and maximum likelihood approaches. Phylogenetic analyses based on mitogenomes provided support for two clades (I and II). The mitogenomes, RAG1, and COI results indicated that several species and genera were not consistent with the traditional morphological subdivisions. The two clades inferred from mitogenomes showed clear geographical patterns. The Tibetan Plateau, Hengduan Mountains, and the Iran Plateau may act as a barrier dividing the clades. The estimated timing of clades separation (36.05 million years ago) coincides with the first uplift of the Tibetan Plateau. We conclude that the geological history of the Tibetan Plateau played a role in the diversification and distribution of the Nemacheilidae taxa. These results provided a phylogenetic framework for future studies of this complex group.     

Taxonomic revision of the Agaraceae with a description of Neoagarum gen. nov. and reinstatement of Thalassiophyllum

We confirmed the monophyly of the Agaraceae based on phylogenetic analyses of six mitochondrial and six chloroplast gene sequences from Agarum, Costaria, Dictyoneurum, and Thalassiophyllum species, as well as representative species from other laminarialean families. However, the genus Agarum was paraphyletic, comprising two independent clades, A. clathratum/A. turneri and A. fimbriatum/A. oharaense. The latter clade was genetically most closely related to Dictyoneurum spp., and morphologically, the species shared a flattened stipe bearing fimbriae (potential secondary haptera) in the mid‐ to upper portion. The phylogenetic position of Thalassiophyllum differed between the two datasets: in the chloroplast gene phylogeny, Thalassiophyllum was included in the A. clathratum/A. turneri clade, but in the mitochondrial gene phylogeny, it formed an independent clade at the base of the Agaraceae, the same position it took in the phylogeny when the data from both genomes were combined despite a larger number of bp being contributed by the chloroplast gene sequences. Considering the remarkable morphological differences between Thalassiophyllum and other Agaraceae, and the molecular support, we conclude that Thalassiophyllum should be reinstated as an independent genus. Dictyoneurum reticulatum was morphologically distinguishable from D. californicum due to its midrib, but because of their close genetic relationship, further investigations are needed to clarify species‐level taxonomy. In summary, we propose the establishment of a new genus Neoagarum to accommodate A. fimbriatum and A. oharanese and the reinstatement of the genus Thalassiophyllum.     

Genomic methods reveal independent demographic histories despite strong morphological conservatism in fish species

Human overexploitation of natural resources has placed conservation and management as one of the most pressing challenges in modern societies, especially in regards to highly vulnerable marine ecosystems. In this context, cryptic species are particularly challenging to conserve because they are hard to distinguish based on morphology alone, and thus it is often unclear how many species coexist in sympatry, what are their phylogenetic relationships and their demographic history. We answer these questions using morphologically similar species of the genus Mugil that are sympatric in the largest coastal Marine Protected Area in the Tropical Southwestern Atlantic marine province. Using a sub-representation of the genome, we show that individuals are assigned to five highly differentiated genetic clusters that are coincident with five mitochondrial lineages, but discordant with morphological information, supporting the existence of five species with conserved morphology in this region. A lack of admixed individuals is consistent with strong genetic isolation between sympatric species, but the most likely species tree suggests that in one case speciation has occurred in the presence of interspecific gene flow. Patterns of genetic diversity within species suggest that effective population sizes differ up to two-fold, probably reflecting differences in the magnitude of population expansions since species formation. Together, our results show that strong morphologic conservatism in marine environments can lead to species that are difficult to distinguish morphologically but that are characterized by an independent evolutionary history, and thus that deserve species-specific management strategies.Subject terms: Genetic variation, Phylogenetics, Speciation     

The genus Cyclops (Copepoda,Cyclopoida) in Europe

Although copepods of the genus Cyclops are among the most common and dominant plankton taxa of lakes in the northern temperate zone, their taxonomy is still unclear. We analysed an extensive array of Cyclops populations from Europe by means of molecular methods and evaluated morphological characters. Altogether, 68 populations of Cyclops species were sampled, assigned to morphospecies and sequenced for the 12S rRNA gene. Selected populations of each morphospecies were additionally sequenced for three mitochondrial (16S rRNA, cytochrome b, COI) and two nuclear genes (18S rRNA, ITS1) and analysed for micromorphological traits. Our analysis revealed fifteen lineages that can be regarded as separate species. Thirteen of these match currently accepted species, while the remaining two lineages were distinct from the other described species. Thus, their taxonomic status is open to further studies. Besides taxonomy, our study gives new insights into the ecology, distribution and phylogenetic relationships of these species. Finally, a set of morphological traits was selected to facilitate identification.     

Morphological and mitochondrial DNA variation revealed an undescribed lineage under the genus Sperata (Bagridae) in Bangladesh

Catfishes Sperata are popular, known for its taste and nutritional value, and are found naturally in wide variety of freshwaters in South Asia. The taxonomy of Sperata spp., sampled from Hakaluki Haor in Bangladesh, was re‐evaluated based on morphological variation and DNA barcoding. The collective variation in morphometric characters and mitochondrial DNA revealed an undescribed old and well‐separated lineage under the genus Sperata along with two previously known Sperata aor and Sperata seenghala in Bangladesh. Analyses of morphological traits suggested significant differentiation among Sperata species. The variation in mitochondrial DNA supported the distinct lineage and taxonomical discrimination. Sperata aor diverged earlier from the new lineage and Sperata seenghala with a divergence of 5.39 (CI: 3.91–7.19) Mya (PP > 90). Sperata seenghala and the new lineage shared a most recent common ancestry, which diverged from each other around 3.41 (CI: 2.24–4.62) Mya (PP > 90). Thus, the newly identified lineage could be a subspecies of S. seenghala or even a species under the genus Sperata. The information of the study will be useful for conservation, sustainable management and selective breeding of the putative species, including previously reported S. aor and S. seenghala in Bangladesh.     

Conflicting mitochondrial and nuclear paraphyly in small‐sized West African house bats (Vespertilionidae)

Vallo, P., Benda, P., Červený, J. & Koubek, P. (2012). Conflicting mitochondrial and nuclear paraphyly in small‐sized West African house bats (Vespertilionidae). —Zoologica Scripta, 42, 1–12. Hybridization between species may result in introgression of mitochondrial DNA from one species to another. Phylogenetic inference, therefore, may not recover true evolutionary relationships. In bats, there are only a few reported cases of introgressive hybridization. House bats are a genus with obscure phylogeny and taxonomy, caused mainly by morphological similarity. We undertook a detailed analysis of small‐sized West African house bats (Scotophilus), tentatively identified as S. nigritellus, to clarify relationships between two sympatric colour forms. These forms were recovered in paraphyletic position to each other in both mitochondrial and nuclear phylogenies, signifying that they are two distinct species. While the yellow‐bellied form could be assigned beyond doubt to S. nigritellus s. str., the white‐bellied form may be an as yet undescribed species. Moreover, the white‐bellied form clustered as a sister mitochondrial lineage to another species, Scotophilus leucogaster. These sister lineages differed by only 2.6–2.8% sequence divergence, which lies within the intraspecific range for this genus. Two nuclear markers, however, contradicted the sister relationship, showing them instead to be distantly related. The apparent conflict between the mitochondrial and nuclear signals suggests that past hybridization may have occurred between these morphologically distinct species.