The molecular phylogeny of the sea star Echinaster (Asteroidea: Echinasteridae) provides insights for genus taxonomy

In this study, we used sequences of two mitochondrial genes, cytochrome c oxidase I (COI) and 16S rRNA, and one nuclear gene, 28S rRNA, to test the monophyly of the sea star genus Echinaster, and understand the phylogenetic relationships among species and subgenera within this genus. Phylogenetic analyses based on Bayesian inference and maximum likelihood methods revealed three clades with high values of genetic divergence among them (K2P distances for COI over 23%). One of the clades grouped all Echinaster (Othilia) species, and the other two clades included Echinaster (non‐Othilia) species and Henricia species, respectively. Although the relationships among Henricia, Othilia, and Echinaster could not be completely clarified, the Othilia clade was a well‐supported group with shared diagnostic morphological characters. Moreover, the approximately unbiased test applied to the phylogenetic reconstruction rejected the hypothesis of the genus Echinaster as a monophyletic group. According to these results, we suggest the revalidation of Othilia as a genus instead of a subgenus within Echinaster. Our study clarifies important points about the phylogenetic relationships among species of Echinaster. Other important systematic questions about the taxonomic classification of Echinaster and Henricia still remain open, but this molecular study provides bases for future research on the topic.     

Morphology,phylogeny and taxonomy of the genus Sectonema (Nematoda,Aporcelaimidae)

This contribution provides new insights in the phylogeny and taxonomy of the nematode genus Sectonema with an integrative approach. A brief historical outline of the matter is presented. Then, the morphological pattern of the genus is revised and illustrated, the nature of its stomatal protruding structure, either a reduced odontostyle or a mural tooth, being its most relevant diagnostic feature. The existence of cilia‐ or seta‐like structures in the perioral area and/or at the anterior part of cheilostom of some species is evidenced by SEM observations for the first time. Available molecular data (D2–D3 LSU‐rRNA gene) are analysed, including two new sequences of S. septentrionale from Spain. The monophyly of the genus is confirmed, and two species groups with geographical projection are tentatively identified. A close relationship with Metaporcelaimus is also demonstrated as both taxa constitute a highly supported clade. A likely polyphyly of the family Aporcelaimidae is once more demonstrated. Finally, an updated taxonomy of the genus is proposed, including revised diagnosis, list of species, identification key and a compendium of their main morphometrics.     

Molecular phylogeny, taxonomy and evolution of the land snail genus Iberus (Pulmonata: Helicidae)

Partial DNA sequences of two mitochondrial genes [cytochrome oxidase subunit I (COI) and 16S rRNA] from 59 specimens of Iberus were used to test the validity of the described morphospecies of this genus, and examine genetic divergences within and between main phylogenetic groups. Both gene fragments showed phylogenetic concordance. The COI gene was found to be faster evolving than the 16S gene and was fully protein-coding with no insertions or deletions. 16S rRNA was more informative than COI for resolving basal nodes. Both individual and combined analyses of the two gene fragments revealed five main phylogroups. These five groups are genetically unique lineages that are allopatrically distributed and considered to have full species status. Further subdivisions were also considered. Shell morphology was suitable for delimiting species boundaries, but several incongruences between morphology and mtDNA phylogeny were observed. These incongruences were considered consequence of hybridization between Iberus cobosi and Iberus marmoratus , and the result of shell shape polymorphism in Iberus rositai . According to spatial patterns of sequence divergence, life habits and shell morphology may be concluded that the keeled-flat shelled snails independently originated several times within Iberus and they could represent cases of similar shell adaptation to a karstic arid environment.     

Morphology and molecular phylogeny of the marine diatom genus Nagumoea (Bacillariophyceae) from Japan

The canal-bearing diatom genus Nagumoea, described based on only morphological evidence, was tentatively assigned to the order Bacillariales, although its phylogenetic position remained unclear. Because three isolates of Nagumoea (SK002, SK024 and SK053) were successfully established from Japanese coasts, we performed their morphological observations and molecular phylogenetic analyses to discuss the phylogeny and taxonomic position of this genus. Strains SK002 and SK024 were identified as Nagumoea africana, whereas SK053 conformed with Nagumoea serrata. There was high interspecific divergence between N. africana and N. serrata in the rbcL sequences (8.03–8.17%), indicating their distinctness. Furthermore, intraspecific variations were detected within N. africana (2.35%) in the rbcL, implying its cryptic diversity. The maximum likelihood and Bayesian phylogenetic trees inferred from the plastid rbcL, psbC and nuclear 18S rDNA genes recovered Nagumoea as monophyletic with strong statistical support and embedded within an unresolved, poorly supported lineage containing Achnanthes, Craspedostauros, Staurotropis and Undatella in the canal-bearing order Bacillariales (= the family Bacillariaceae). Although the constrained tree based on the monophyly of Nagumoea and the other canal-bearing clade (Surirellales and Rhopalodiales) was statistically rejected by the topology tests, the phylogenetic position of Nagumoea with other Bacillarialean members remains equivocal. The possession of two plastids positioned fore and aft, observed in the present study, and lack of keel, typical of the Bacillariales, indicate the possibility of Nagumoea being part of the ingroup of the Bacillariales or its closely related outgroup.     

The genus Morinda (Morindeae, Rubioideae, Rubiaceae) in New Caledonia: taxonomy and phylogeny

The genus Morinda (Morindeae, Rubioideae. Rubiaceae) is closely allied to Coelospermum, Gynochthodes , and Pogonolobus , but differs from these genera in inflorescence and pollen morphology. In this revision of Morinda in New Caledonia fourteen species are recognized, one of which is subdivided into four varieties. Eleven species are endemic to the area. Two new species, M. mumtana and M. truneata , and one new variety, M. myrtifolia var. vrevifolia , are described. Two recombinations, M. myrtifolia var. choriophylla and M. myrtifolia var. velutina , are made. All taxa are illustrated with line drawings and their distributions mapped, and a key to the species is presented. Phylogenetic analyses including nineteen species are presented.     

Integrative taxonomy uncovers hidden species diversity in the rheophilic genus Potamometra (Hemiptera: Gerridae)

The genus Potamometra Bianchi, 1896 represents big rheophilic semi-aquatic bugs that typically inhabit middle-altitude mountainous streams. Here, we integrated molecular and morphological data for delimiting species boundaries and understanding the evolutionary history of the genus Potamometra. Twenty-seven complete mitochondrial genomes of Potamometra were sequenced, with samples representing most of the known geographically distributed locations around the Sichuan Basin. The results of different species delimitation methods (ABGD, bPTP, GMYC and BPP) based on the monolocus or multilocus data strongly supported the existence of two cryptic new species (Potamometra anderseni Zheng, Ye & Bu, sp. nov. and Potamometra zhengi Zheng, Ye & Bu, sp. nov.) although more entities were found in the tree-based delimitation methods. The two new species were successfully validated using morphological characters within a detailed taxonomic framework. Phylogenetic analyses supported the reciprocal monophyly of the seven highly node-supported clades, which were matched with the five known species and two new taxa. A novel gene arrangement pattern that two trnF (trnF1 and trnF2) genes separated by an intergenic spacer (IGS) were found in all the species except the sister group of Potamometra berezowskii Bianchi, 1896 and Potamometra linnavuorii Chen, Nieser & Bu, 2016. This gene rearrangement event could be explained by the tandem duplication and random loss (TDRL) model. Our study emphasized that the combination of molecular sequence data, morphological characters and mitochondrial structural information could improve the accuracy of species delimitation.     

A molecular phylogeny for the frog genus Limnodynastes (Anura: myobatrachidae)

Members of the genus Limnodynastes are a prominent and widespread feature of the Australian frog fauna. Yet despite their potential to be informative about biogeographic history and mechanisms of speciation, the relationships among these taxa are not well known. We investigated phylogenetic relationships within the genus Limnodynastes via sequencing of mitochondrial (mt)DNA from current members of the genus Limnodynastes and the monotypic genus Megistolotis. a 450-bp fragment of the 16S rRNA gene and a 370-bp fragment of the protein-coding gene ND4 were used to infer a molecular phylogeny. We revise traditional species groupings and now recognize four species groups within Limnodynastes: the L. ornatus group (L. ornatus and L. spenceri), the L. peronii group (L. peronii, L. tasmaniensis, L. fletcheri, the L. depressus), the L. salmini group (L. salmini, L. convexiusculus, and L. lignarius), and the L. dorsalis group (L. dorsalis, L. terraereginae, L. dumerilii and L. interioris). The L. ornatus species group forms a highly distinctive clade that is a sister group to the other Limnodynastes groups. Pending broader phylogenetic studies it could be removed from the genus Limnodynastes. Our results concur with previous suggestions that Megistolotis lignarius is nested within Limnodynastes, and we therefore reclassify this species as Limnodynastes lignarius. Furthermore, specimens identified as L. depressus form a mtDNA lineage distinct from other species in the genus, confirming the validity of the species. Specimens of species from the L. dorsalis group (L. dorsalis, L. dumerilii, L. interioris, and L. terraereginae) are closely related such that L. dumerilii is paraphyletic with two other species. Finally, our study provides broad support for previous phylogenies based on microcomplement fixation.     

New Macroderoides (Digenea: Macroderoididae) from Florida gar, with molecular phylogeny of the genus

Macroderoides minutus n. sp. is described based on specimens collected from the Florida gar (Lepisosteus platyrhincus) from Orange Lake, Alachua County, Florida. The new species is the ninth recognized species of Macroderoides and is morphologically closest to Macroderoides flavus and Macroderoides typicus. It differs from both species in having a smaller body, ventral sucker larger than the oral sucker, substantially lower body length to width ratio, fewer and larger eggs, and other characters. A comparison of sequences of nuclear rDNA (partial 18S, complete ITS region, and partial 28S) of the new species, with sequences of 5 other Macroderoides, convincingly supports the status of M. minutus as a new species. Molecular phylogenetic analysis, including all North American Macroderoides species except for Macroderoides parvus, has demonstrated the presence of 2 distinct clades, 1 including Macroderoides spinifer + (Macroderoides minutus n. sp. + Macroderoides texanus) and the other including Macroderoides trilobatus + (M. typicus + M. flavus). The tree topology suggests that the similarity in body proportions among M. minutus n. sp., M. typicus + M. flavus is not a result of close phylogenetic relationship. It has also revealed for the first time the phylogenetic affinities of M. flavus, which is the only North American member of Macroderoides that switched from holostean fishes to teleosts.     

Integrative taxonomy of the stalk-eyed bug genus Chauliops (Heteroptera: Malcidae: Chauliopinae) reveals orogeny-driven speciation

Economically significant bean pests of the genus Chauliops are species rich in the areas surrounding the Qinghai–Tibet Plateau and provide an excellent system for speciation studies. Here, an integrative taxonomic approach, employing morphological analyses, population genetic methods, and multiple molecular species delimitation methods, was used to clarify the taxonomy of Chauliops in East and Southeast Asia. Four new species (Chauliops parahorizontalis Li & Bu, sp. nov., Chauliops albida Li & Bu, sp. nov., Chauliops bicoloripes Li & Bu, sp. nov., and Chauliops paraconica Li & Bu, sp. nov.) were described, which increases the number of Chauliops species in this area from six to 10; a key for Chauliops species is also provided. Phylogenetic analysis and divergence time estimation revealed that Chauliops was divided into four clades: Clade A (Chauliops bisontula + [Chauliops horizontalis + C. parahorizontalis sp. nov.]), Clade B (C. albida sp. nov. and C. bicoloripes sp. nov.), Clade C (Chauliops quaternaria and Chauliops zhengi), and Clade D (Chauliops fallax + [Chauliops conica + C. paraconica sp. nov.]). Two species diversification events of Chauliops estimated to have occurred 7–1 million years ago (Ma) and 25–13 Ma were detected. These speciation events were consistent with the two historical uplift events of the Qinghai–Tibet Plateau, suggesting that orogeny might have provided opportunities for the diversification of Chauliops species on the southeastern margin of the Qinghai–Tibet Plateau. Our findings show that population genetic analyses can be used to delimit related species and that orogeny is a key driver of species diversification on the southeastern margin of the Qinghai–Tibet Plateau.     

A molecular phylogeny of the neotropical butterfly genus Anartia (Lepidoptera: Nymphalidae)

While Anartia butterflies have served as model organisms for research on the genetics of speciation, no phylogeny has been published to describe interspecific relationships. Here, we present a molecular phylogenetic analysis of Anartia species relationships, using both mitochondrial and nuclear genes. Analyses of both data sets confirm earlier predictions of sister species pairings based primarily on genital morphology. Yet both the mitochondrial and nuclear gene phylogenies demonstrate that Anartia jatrophae is not sister to all other Anartia species, but rather that it is sister to the Anartia fatima-Anartia amathea lineage. Traditional biogeographic explanations for speciation across the genus relied on A. jatrophae being sister to its congeners. These explanations invoked allopatric divergence of sister species pairs and multiple sympatric speciation events to explain why A. jatrophae flies alongside all its congeners. The molecular phylogenies are more consistent with lineage divergence due to vicariance, and range expansion of A. jatrophae to explain its sympatry with congeners. Further interpretations of the tree topologies also suggest how morphological evolution and eco-geographic adaptation may have set species range boundaries.     

A molecular phylogeny of the genus Ichthyocotylurus (Digenea, Strigeidae)

Three nucleotide data sets, two nuclear (ribosomal internal transcribed spacer regions 1 and 2, ITS1 and ITS2) and one mitochondrial (cytochrome c oxidase subunit 1, CO1), were analysed using distance matrix and maximum likelihood methods to determine the inter-relationships amongst the four species attributed to the genus Ichthyocotylurus Odening, 1969. Sequence data obtained from all gene loci investigated supported the position of Ichthyocotylurus variegatus as a species discrete from Ichthyocotylurus platycephalus. Phylogenetic analyses yielded congruent trees, with I. variegatus isolates comprising a common clade to which I. platycephalus constitutes a sister taxon. Ichthyocotylurus erraticus and Ichthyocotylurus pileatus were found to demonstrate a similarly close inter-specific relationship. The greatest intra-generic divergence occurred in the CO1 region (16% variability), with resultant disparities in three to eight encoded amino acids. PCR amplification yielded multiple ITS1 products for all Ichthyocotylurus spp. Analyses of equivalent-sized amplicons showed 5.4% intra-generic variation and several point mutations between I. variegatus isolates from different geographical localities and from different piscine hosts. The ITS2 locus was extremely conserved, with less than 1% variation between species. No intra-specific variation was recorded for any CO1 or ITS2 sequences.     

3D morphology of pharyngeal dentition of the genus Capoeta (Cyprinidae): Implications for taxonomy and phylogeny

Capoeta is a herbivorous cyprinid fish genus, widely distributed in water bodies of Western Asia. Recent species show a distinct biogeographic pattern with endemic distribution in large fluvial drainage basins. As other cyprinids, the species of this genus are characterized by the presence of the pharyngeal bone with pharyngeal teeth. Despite this, the detailed morphology of the pharyngeal teeth, its interspecific and topologic variations, and the importance for taxonomy and phylogeny of the genus Capoeta are still not established. For the first time, a detailed comprehensive study of the pharyngeal dentition of 10 Capoeta species has been provided. The morphologic study of the pharyngeal dentition bases on the 3D microtomography and follows the purpose to evaluate the potential taxonomic and phylogenetic signals of these elements, as well as to study interspecific and topologic variations of the pharyngeal teeth. In this study, we propose a new methodology to categorize the studied pharyngeal teeth in 18 shape classes. The results of this study show that the detailed 3D morphology of the pharyngeal teeth is a useful tool for the identification of isolated teeth at the generic and/or specific level and that in certain cases, the tooth position in the teeth rows can be identified. Additionally, the preliminary analysis shows that the morphology of the pharyngeal teeth provides a potential phylogenetic signal. Both these patterns are very important for the taxonomy of cyprinid fishes and especially can be applied to fossil records.     

Molecular phylogeny of the genus Gobio Cuvier, 1816 (Teleostei: Cyprinidae) and its contribution to taxonomy

The phylogenetic relationships among gudgeons that represent most nominal taxa within Gobio gobio sensu lato were examined by mitochondrial and nuclear genome sequencing. The molecular analyses confirmed the separate generic status of Gobio as a monophyletic group and revealed 15 Eurasian lineages divided into two main clades, the Northern European and the Ponto-Caspian. The validity of eleven nominal taxa as distinct species was confirmed, gudgeons from the Volga River basin were described as a new species G. volgensis, and three revealed phylogenetic lineages were submitted for a comprehensive revision as "species-in-waiting". The species G. gobio showed a wide range extending from the British Isles to the Black Sea coast and overlapped the areas of several other species. Four pure lineages were detected in the middle Danube River basin. The Crimean Peninsula was found to be a region with the occurrence of individuals of hybrid origin. This region will require special investigation to define species participating in hybridization events, and to establish further steps for the conservation of endemic native gudgeon species. A simple diagnostic method, based on different lengths of the PCR products, called "S7indel diagnostics" is presented for further taxonomic investigations in the genus Gobio.     

Integrative taxonomy of European parasitic flatworms of the genus Metorchis Looss, 1899 (Trematoda: Opisthorchiidae)

Metorchis spp. are flukes (Platyhelminthes: Digenea) that infect vertebrates, including humans, dogs, cats, poultry and wild game, with cyprinid freshwater fish serving as typical second intermediate hosts. In their definitive hosts, the Metorchis spp. are difficult to identify to species. We provide and analyze sequences of two nuclear (18S rDNA and ITS2) and two mitochondrial (CO1 and ND1) DNA loci of four morphologically identified European species of the Metorchis, namely Metorchis albidus, Metorchis bilis, Metorchis crassiusculus and Metorchis xanthosomus, and of another opisthorchiid, Euamphimerus pancreaticus. DNA analysis suggests that the Metorchis specimens identified morphologically as M. albidus (from Lutra lutra), M. bilis (from Phalacrocorax carbo) and M. crassiusculus (from Aquila heliaca and Buteo rufinus) represent a single species. Thus, M. albidus (Braun, 1893) Loos, 1899 and M. crassiusculus (Rudolphi, 1809) Looss, 1899 are recognized as junior subjective synonyms of M. bilis (Braun, 1790) Odening, 1962. We also provide comparative measurements of the Central European Metorchis spp., and address their tissue specificity and prevalence based on the examination of extensive bird cohort from 1962 to 2015. M. bilis and M. xanthosomus can be morphologically diagnosed by measuring the extent of genitalia relative to body length and by the size ratio of their suckers. They also differ in their core definitive hosts, with ducks (Anas, Aythya) and coots (Fulica) hosting M. xanthosomus, and cormorants (Phalacrocorax), the birds of prey (Buteo, Aquila, etc.), piscivorous mammals (Lutra, Vulpes, Ursus, etc.) and humans hosting M. bilis. Previous reports on the Metorchis spp. contain numerous suspected misidentifications.     

Hagfish phylogeny and taxonomy,with description of the new genus Rubicundus (Craniata,Myxinidae)

A recent phylogenetic analysis of the Myxinidae based on the 16S rRNA gene resulted in synonymization of Paramyxine with Eptatretus. This created homonymy of Paramyxine fernholmi with Eptatretus fernholmi and Paramyxine wisneri with Eptatretus wisneri. In order to resolve this nomenclatural dilemma, we made a more extensive phylogenetic assessment of the Myxinidae and examined the nomenclature of the family. We used 75 sequences (37 of which new for this study) of a 561 bp fragment of the 16S rRNA gene, representing 33 species, and 72 sequences (37 of which new for this study) of a 687 bp fragment of the cytochrome c oxidase subunit I (COI) gene, representing 23 species, to reconstruct the phylogeny of Myxinidae. The monophyly of the subfamily Myxininae, traditionally characterized by having a single pair of external gill openings, was rejected (0.50 Bayesian posterior probability) by the 16S analysis, but supported by the COI and combined COI+16S analyses (0.99 and 0.81 Bpp, respectively). The monophyly of the subfamily Eptatretinae, characterized by having several pairs of external gill openings, was not supported by the 16S analysis and rejected by the COI and combined COI+16S analysis due to the placement of Eptatretus lopheliae as the earliest branch of Myxinidae (0.71 and 0.57 Bpp, respectively). Eptatretus lopheliae and Eptatretus rubicundus formed a monophyletic group and were allocated to a new genus, Rubicundus, characterized by the presence of an elongated tubular nostril and reddish coloration. A new monotypic subfamily, Rubicundinae, was proposed for Rubicundus. The synonymy of the genera Paramyxine and Quadratus with Eptatretus was confirmed. E. fernholmi is renamed Eptatretus luzonicus. Eptatretus wisneri was renamed Eptatretus bobwisneri. Petromyzon cirrhatus Forster, 1801, Homea banksii Fleming, 1822, and Bdellostoma forsteri Müller, 1836 are synonyms, but no type specimens are known to exist. Petromyzon cirrhatus was designated as type species of Eptatretus, conserving present usage. Gastrobranchus dombeyi Shaw, 1804 has priority over other names for Chilean myxinids. Bdellostoma stoutii was designated as type species of Polistotrema Gill. The validity of the Western Atlantic Myxine limosa as distinct from the Eastern Atlantic Myxine glutinosa was confirmed.     

The molecular phylogeny of Alpinia (Zingiberaceae): a complex and polyphyletic genus of gingers

Alpinia is the largest, most widespread, and most taxonomically complex genus in the Zingiberaceae with 230 species occurring throughout tropical and subtropical Asia. Species of Alpinia often predominate in the understory of forests, while others are important ornamentals and medicinals. Investigations of the evolutionary relationships of a subset of species of Alpinia using DNA sequence-based methods specifically test the monophyly of the genus and the validity of the previous classifications. Seventy-two species of Alpinia, 27 non-Alpinia species in the subfamily Alpinioideae, eight species in the subfamily Zingiberoideae, one species in the subfamily Tamijioideae, and three species in the outgroup genus Siphonochilus (Siphonochiloideae) were sequenced for the plastid matK region and the nuclear internal transcribed spacer (ITS) loci. Parsimony analyses of both individual and combined data sets identified six polyphyletic clades containing species of Alpinia distributed across the tribe Alpinieae. These results were supported by a Bayesian analysis of the combined data set. Except in a few specific cases, these monophyletic groupings of species do not correspond with either Schumann's (1904) or Smith's (1990) classification of the genus. Here we build on previous molecular analyses of the Alpinioideae and propose the next steps necessary to recognize new generic boundaries in the Alpinieae.