Molecular phylogenetics of Reig's short-tailed opossum (Monodelphis reigi) and its distributional range extension into Guyana

Reig's short-tailed opossum (Monodelphis reigi) was recently described morphologically, based on a single specimen from southeastern Venezuela. It was considered most similar to M. adusta, which is allopatrically distributed in the Andes and surrounding areas, but there has not been an explicit study of systematic relationships with other species of Monodelphis. We report the first occurrence from Guyana of this rare species that was previously known by only the holotype. In a molecular phylogenetic analysis of mitochondrial cytochrome b sequence variation, it groups within a well-supported monophyletic clade that includes M. adusta, M. handleyi, M. osgoodi, and M. peruviana. M. adusta was found to be the sister taxon to the rest of the adusta-complex. M. reigi is the basal taxon (sister species) of remaining adusta-complex forms. This corroborates earlier morphological studies suggesting close affinity of these taxa. As presently known, M. reigi is endemic to the highland regions (>1,000 m asl) of the Guiana Shield of northern South America, and is the only taxon within the M. adusta species complex that does not occur in the Andes or adjacent lowland regions. Based on previous molecular dating of Didelphidae, this suggests that there was a dispersal event from the Andes to the Guiana Highlands in the Miocene that gave rise to M. reigi.     

Petrosal and Bony Labyrinth Morphology Supports Paraphyly of Elephantulus Within Macroscelididae (Mammalia,Afrotheria)

Interest in the phylogeny of Macroscelididae (sengis or elephant shrews) has been prompted by molecular studies indicating that Elephantulus rozeti is best placed as the sister group of Petrodromus tetradactylus (this clade being in turn the sister taxon to Macroscelides proboscideus) than among other species of the genus Elephantulus. Until now, no discrete morphological characters have been proposed to support the grouping of E. rozeti, Petrodromus, and Macroscelides into this single so-called ‘Panelephantulus’ clade. Here, we employed μCT scanning in order to investigate the petrosal and bony labyrinth (bony capsule of the inner ear) morphology of most species of extant Macroscelididae. We performed a cladistic analysis on ear traits and found that despite some convergences (e.g., concerning the bony arterial canals in Macroscelides and Rhynchocyon) the middle and inner ear morphology furnishes significant support for the ‘Panelephantulus’ clade. In our analysis, this clade is unambigously supported by the presence of a fully ossified stapediofacial tube. Two additional characters (the presence of a bony septum at the mouth of the fenestra cochleae dividing the D3 sinus into two distinct cavities and the absence of an accessory lateral pneumatic fossa) could also support ‘Panelephantulus.’ These newly discovered morphological characters support the molecular phylogenies published and highlight the importance of coding hitherto difficult to sample morphologies within cladistic analyses using micro-CT techniques. Taxonomic implications are briefly discussed.     

Molecular Phylogeny of the Cliff Ferns (Woodsiaceae: Polypodiales) with a Proposed Infrageneric Classification

The cliff fern family Woodsiaceae has experienced frequent taxonomic changes at the familial and generic ranks since its establishment. The bulk of its species were placed in Woodsia, while Cheilanthopsis, Hymenocystis, Physematium, and Protowoodsia are segregates recognized by some authors. Phylogenetic relationships among the genera of Woodsiaceae remain unclear because of the extreme morphological diversity and inadequate taxon sampling in phylogenetic studies to date. In this study, we carry out comprehensive phylogenetic analyses of Woodsiaceae using molecular evidence from four chloroplast DNA markers (atpA, matK, rbcL and trnL–F) and covering over half the currently recognized species. Our results show three main clades in Woodsiaceae corresponding to Physematium (clade I), Cheilanthopsis–Protowoodsia (clade II) and Woodsia s.s. (clade III). In the interest of preserving monophyly and taxonomic stability, a broadly defined Woodsia including the other segregates is proposed, which is characterized by the distinctive indument and inferior indusia. Therefore, we present a new subgeneric classification of the redefined Woodsia based on phylogenetic and ancestral state reconstructions to better reflect the morphological variation, geographic distribution pattern, and evolutionary history of the genus. Our analyses of the cytological character evolution support multiple aneuploidy events that have resulted in the reduction of chromosome base number from 41 to 33, 37, 38, 39 and 40 during the evolutionary history of the cliff ferns.     

A New PCR-Based Method Shows That Blue Crabs (Callinectes sapidus (Rathbun)) Consume Winter Flounder (Pseudopleuronectes americanus (Walbaum))

Winter flounder (Pseudopleuronectes americanus) once supported robust commercial and recreational fisheries in the New York (USA) region, but since the 1990s populations have been in decline. Available data show that settlement of young-of-the-year winter flounder has not declined as sharply as adult abundance, suggesting that juveniles are experiencing higher mortality following settlement. The recent increase of blue crab (Callinectes sapidus) abundance in the New York region raises the possibility that new sources of predation may be contributing to juvenile winter flounder mortality. To investigate this possibility we developed and validated a method to specifically detect winter flounder mitochondrial control region DNA sequences in the gut contents of blue crabs. A survey of 55 crabs collected from Shinnecock Bay (along the south shore of Long Island, New York) in July, August, and September of 2011 showed that 12 of 42 blue crabs (28.6%) from which PCR-amplifiable DNA was recovered had consumed winter flounder in the wild, empirically supporting the trophic link between these species that has been widely speculated to exist. This technique overcomes difficulties with visual identification of the often unrecognizable gut contents of decapod crustaceans, and modifications of this approach offer valuable tools to more broadly address their feeding habits on a wide variety of species.     

Taxonomic status of Pseudopleuronectes yokohamae and P. schrenki (Pleuronectidae sensu Cooper and Chapleau, 1998)

In studying the variation of the taxa group Pseudopleuronectes yokohamae and P. schrenki from various areas in the Sea of Japan, significant differences in the combination of morphometric and meristic characters were not revealed. It was found that typical characters such as fin coloration from the blind side of the body, number of rays in the fins, number of scales in the lateral line, etc. that are used by most authors to distinguish P. yokohamae and P. shrenki strongly vary and are not adequate for assigning individuals to a certain taxon. A conclusion of the absence of differences and, hence, the homogeneity of the group studied is supported by genetic-biochemical studies, as well as by analysis of the lateral line system of these two groups of flatfish. Considering the results obtained and literature data on the morphology and ecology of flatfish taxa considered in the paper, it is justifiable to consider the P. yokohamae + P. schrenki group as one polymorphic species Pseudopleuronectes yokohamae Günther, 1877. The second taxon, P. shrenki, as a later species, should be considered a synonym of the species P. yokohamae.     

A preliminary phylogenetic analysis of the New World Helopini (Coleoptera,Tenebrionidae, Tenebrioninae) indicates the need for profound rearrangements of the classification

Helopini is a diverse tribe in the subfamily Tenebrioninae with a worldwide distribution. The New World helopine species have not been reviewed recently and several doubts emerge regarding their generic assignment as well as the naturalness of the tribe and subordinate taxa. To assess these questions, a preliminary cladistic analysis was conducted with emphasis on sampling the genera distributed in the New World, but including representatives from other regions. The parsimony analysis includes 30 ingroup species from America, Europe and Asia of the subtribes Helopina and Cylindrinotina, plus three outgroups, and 67 morphological characters. Construction of the matrix resulted in the discovery of morphological character states not previously reported for the tribe, particularly from the genitalia of New World species. A consensus of the 12 most parsimonious trees supports the monophyly of the tribe based on a unique combination of characters, including one synapomorphy. None of the subtribes or the genera of the New World represented by more than one species (Helops Fabricius, Nautes Pascoe and Tarpela Bates) were recovered as monophyletic. Helopina was recovered as paraphyletic in relation to Cylindrinotina. One Nearctic species of Helops and one Palearctic species of Tarpela (subtribe Helopina) were more closely related to species of Cylindrinotina. A relatively derived clade, mainly composed by Neotropical species, was found; it includes seven species of Tarpela, seven species of Nautes, and three species of Helops, two Nearctic and one Neotropical. Our results reveal the need to deeply re-evaluate the current classification of the tribe and subordinated taxa, but a broader taxon sampling and further character exploration is needed in order to fully recognize monophyletic groups at different taxonomic levels (from subtribes to genera).     

Phylogenetic Analyses of Meloidogyne Small Subunit rDNA

Phylogenies were inferred from nearly complete small subunit (SSU) 18S rDNA sequences of 12 species of Meloidogyne and 4 outgroup taxa (Globodera pallida, Nacobbus abberans, Subanguina radicicola, and Zygotylenchus guevarai). Alignments were generated manually from a secondary structure model, and computationally using ClustalX and Treealign. Trees were constructed using distance, parsimony, and likelihood algorithms in PAUP* 4.0b4a. Obtained tree topologies were stable across algorithms and alignments, supporting 3 clades: clade I = [M. incognita (M. javanica, M. arenaria)]; clade II = M. duytsi and M. maritima in an unresolved trichotomy with (M. hapla, M. microtyla); and clade III = (M. exigua (M. graminicola, M. chitwoodi)). Monophyly of [(clade I, clade II) clade III] was given maximal bootstrap support (mbs). M. artiellia was always a sister taxon to this joint clade, while M. ichinohei was consistently placed with mbs as a basal taxon within the genus. Affinities with the outgroup taxa remain unclear, although G. pallida and S. radicicola were never placed as closest relatives of Meloidogyne. Our results show that SSU sequence data are useful in addressing deeper phylogeny within Meloidogyne, and that both M. ichinohei and M. artiellia are credible outgroups for phylogenetic analysis of speciations among the major species.     

Cyatta abscondita: Taxonomy,Evolution, and Natural History of a New Fungus-Farming Ant Genus from Brazil

Cyatta abscondita, a new genus and species of fungus-farming ant from Brazil, is described based on morphological study of more than 20 workers, two dealate gynes, one male, and two larvae. Ecological field data are summarized, including natural history, nest architecture, and foraging behavior. Phylogenetic analyses of DNA sequence data from four nuclear genes indicate that Cyatta abscondita is the distant sister taxon of the genus Kalathomyrmex, and that together they comprise the sister group of the remaining neoattine ants, an informal clade that includes the conspicuous and well-known leaf-cutter ants. Morphologically, Cyatta abscondita shares very few obvious character states with Kalathomyrmex. It does, however, possess a number of striking morphological features unique within the fungus-farming tribe Attini. It also shares morphological character states with taxa that span the ancestral node of the Attini. The morphology, behavior, and other biological characters of Cyatta abscondita are potentially informative about plesiomorphic character states within the fungus-farming ants and about the early evolution of ant agriculture.     

Molecular phylogeny and morphology of <Emphasis Type="Italic">Ophiocordyceps unituberculata</Emphasis> sp. nov. (Ophiocordycipitaceae), a pathogen of caterpillars (Noctuidae,Lepidoptera) from Yunnan,China

Ophiocordyceps unituberculata, a new insect pathogenic fungus from southwestern China, is described using molecular phylogenetic and morphological data. This fungus differs from other Ophiocordyceps species by its enormously long monophialidic conidiogenous cells and a large periclinal protuberance often growing near the apex of conidiogenous cells, single conidia (lanceolate to fusiform) embedded in mucous sheath, and much larger conidial dimensions. Four-locus (nrSSU, nrLSU, tef-1α and rpb1) and ITS data phylogenetic analyses show that O. unituberculata belongs to the Hirsutella nodulosa clade within the genus Ophiocordyceps of Ophiocordycipitaceae and is a separate clade from other allied species. Molecular phylogeny and morphology both strongly support the distinctiveness of this taxon. The interspecific relationships in the H. nodulosa clade are discussed.     

Sweet or salty? The origin of freshwater gastrotrichs (Gastrotricha,Chaetonotida) revealed by molecular phylogenetic analysis

Chaetonotidae is the most diverse and widely distributed family of the order Chaetonotida (Gastrotricha) and includes both marine and freshwater species. Although the family is regarded as a sister taxon to the exclusively marine Xenotrichulidae, the type of environment, marine or freshwater, where Chaetonotidae originated is still not known. Here, we reconstructed the phylogeny of the family based on molecular sequence data and mapped both morphological and ecological characters to determine the ancestral environment of the first members of the family. Our results revealed that the freshwater genus Bifidochaetus is the earliest branching lineage in the paraphyletic Chaetonotidae (encompassing Dasydytidae and Neogosseidae). Moreover, we reconstructed Lepidochaetus-Cephalionotus clade as a monophyletic sister group to the remaining chaetonotids, which supports Kisielewski's morphological based hypothesis concerning undifferentiated type of body scales as a most primary character in Chaetonotidae. We also found that reversals to marine habitats occurred independently in different Chaetonotidae lineages, thus marine species in the genera Heterolepidoderma, Halichaetonotus, Aspidiophorus and subgenera Chaetonotus (Schizochaetonotus) or Chaetonotus (Marinochaetus) should be assumed as having secondarily invaded the marine environment. Character mapping revealed a series of synapomorphies that define the clade that includes Chaetonotidae (with Dasydytidae and Neogosseidae), the most important of which may be those linked to reproduction.     

Morphological variability and molecular characterisation of Dichelyne (Cucullanellus) pleuronectidis (Yamaguti, 1935) (Ascaridida: Cucullanidae) from the flatfish Pleuronichthys cornutus (Temminck & Schlegel) (Pleuronectiformes: Pleuronectidae) in the East China Sea

Cucullanid nematodes identified morphologically as Dichelyne (Cucullanellus) pleuronectidis (Yamaguti, 1935) were collected from the ridged-eye flounder Pleuronichthys cornutus (Temminck & Schlegel) (Pleuronectiformes: Pleuronectidae) in the East China Sea. Their examination using light microscopy and, for the first time, scanning electron microscopy, revealed several important, but previously unreported morphological features and the presence of remarkable morphological differences in the intestinal caecum and deirids among some individuals. Consequently, specimens of D. pleuronectidis were characterised using molecular methods by sequencing and analysing the internal transcribed spacer (ITS) of the ribosomal DNA to test whether the present material with broad range of morphological variability, represents a complex of sibling species or a single species. The results of molecular analyses proved that the differences in the intestinal ceacum and deirids should be considered as intraspecific variation and that the nematode material collected from P. cornutus in the East China Sea represented a single species, D. pleuronectidis. These new morphological and genetic data contributed to an accurate diagnosis of this hitherto insufficiently known nematode and also indicated that a more rigorous study based on morphological and genetic data with broader representation of the Cucullanidae is required to assess whether the traditionally used diagnostic character of absence or presence of intestinal ceacum is of generic importance in distinguishing Dichelyne and Cucullanus.     

Parahellenia,a new genus segregated from Hellenia (Costaceae) based on phylogenetic and morphological evidence

Previous studies recognized three major lineages of the family Costaceae: a South American clade, an Asian clade and a Costus clade. However, the genus Hellenia within the Asian clade has been shown to be non-monophyletic and its morphology has not been studied carefully. Therefore, the complete plastid genomes of Hellenia species were obtained and the monophyly of Hellenia was tested through four different datasets in this study. Plastid phylogenomic analyses of Costaceae revealed that Hellenia is strongly supported as paraphyletic. Two major clades are recovered, namely the Hellenia s.s. subclade and the Parahellenia subclade. Phylogenetic analyses based on an enlarged taxon sampling of the Asian clade using a two chloroplast markers dataset (trnK intron and trnL-F spacer) confirmed the paraphyly of Hellenia. Meanwhile, morphological analyses suggested that members of the Parahellenia subclade differ from the remaining Hellenia species in many characters including inflorescences, bracts, stigma, axillary buds, floral tubes and labellum. According to the present molecular and morphological evidence, the latter subclade is recognized as a new genus, Parahellenia. Two new species are described, four new combinations are made, and identification keys are also provided.     

The Basal Nodosaurid Ankylosaur Europelta carbonensis n. gen., n. sp. from the Lower Cretaceous (Lower Albian) Escucha Formation of Northeastern Spain

Nodosaurids are poorly known from the Lower Cretaceous of Europe. Two associated ankylosaur skeletons excavated from the lower Albian carbonaceous member of the Escucha Formation near Ariño in northeastern Teruel, Spain reveal nearly all the diagnostic recognized character that define nodosaurid ankylosaurs. These new specimens comprise a new genus and species of nodosaurid ankylosaur and represent the single most complete taxon of ankylosaur from the Cretaceous of Europe. These two specimens were examined and compared to all other known ankylosaurs. Comparisons of these specimens document that Europelta carbonensis n. gen., n. sp. is a nodosaur and is the sister taxon to the Late Cretaceous nodosaurids Anoplosaurus, Hungarosaurus, and Struthiosaurus, defining a monophyletic clade of European nodosaurids– the Struthiosaurinae.     

Phylogeny and character evolution of the Neotropical fern genus <Emphasis Type="Italic">Stigmatopteris</Emphasis> (Dryopteridaceae)

We did a phylogenetic analysis on 21 of the 23 species of Stigmatopteris, a neotropical montane genus of wet forest floors. The analysis was based on four plastid markers: rbcL, rps4-trnS, trnG-trnR, and trnL-trnF. Two of the most closely related families to the Dryopteridaceae (Didymochlaenaceae and Hypodematiaceae) and 12 dryopteroid genera were used as outgroups. Eight morphological characters were mapped on the resulting tree. Stigmatopteris was recovered as monophyletic and sister to a clade formed by polystichoid ferns such as Arachniodes, Dryopteris, and Polystichum. Synapomorphies that distinguish Stigmatopteris are 1-pinnate-pinnatifid laminae, visible punctate glands in the mesophyll, the presence of hydathodes, uniseriate cilia on the scale margins, and loss of indusia. Within the genus are two main clades. The first consists of four species characterized by pinnae long-decurrent in at least the distal third of the lamina. The second consists of the remaining 17 species in the analysis and has no known morphological synapomorphies. Nested within this second clade is a subclade of six species endemic to southeastern Brazil. Anastomosing veins, a character often used in keys to distinguish the species, evolved three times within the genus.     

The phylogenetic position of the Loimoidae Price, 1936 (Monogenoidea: Monocotylidea) based on analyses of partial rDNA sequences and morphological data

Phylogenetic analyses of partial sequences of 18S and 28S rDNA of some monogenoids, including monocotylids and a specimen of Loimosina sp. collected from a hammerhead shark off Brazil, indicated that the Loimoidae (as represented by the specimen of Loimosina sp.) represents an in-group taxon of the Monocotylidae. In all analyses, the Loimoidae fell within a major monocotylid clade including species of the Heterocotylinae, Decacotylinae, and Monocotylinae. The Loimoidae formed a terminal clade with two heterocotyline species, Troglocephalus rhinobatidis and Neoheterocotyle rhinobatis, for which it represented the sister taxon. The following morphological characters supported the clade comprising the Loimoidae, Heterocotylinae, Decacotylinae and Monocotylinae: single vagina present, presence of a narrow deep anchor root, and presence of a marginal haptoral membrane. The presence of cephalic pits was identified as a putative synapomorphy for the clade (Loimoidae (T. rhinobatidis, N. rhinobatis)). Although rDNA sequence data support the rejection of the Loimoidae and incorporating its species into the Monocotylidae, this action was not recommended pending a full phylogenetic analysis of morphological data.     

Cloning,tissue distribution of desert hedgehog (dhh) gene and expression profiling during different developmental stages of Pseudopleuronectes yokohamae

As a crucial member of the Hedgehog (Hh) protein family, desert hedgehog (dhh) plays a vital role in multiple developmental processes, cell differentiation and tissue homeostasis. However, it is unclear how it regulates development in fish. In this study, we cloned and characterized the dhh gene from Pseudopleuronectes yokohamae. The full-length cDNA of Pydhh comprises 3194 bp, with a 1386 bp open reading frame (ORF) that encodes a polypeptide of 461 amino acids with a typical HH-signal domain, Hint-N and Hint-C domains. Multiple sequence alignment revealed that the putative PyDHH protein sequence was highly conserved across species, especially in the typical domains. Phylogenetic analysis showed that the PyDHH clustered within the Pleuronectiformes. Real-time quantitative PCR showed that Pydhh was detected in fourteen different tissues in adult-female and adult-male marbled flounder, and nine different tissues in juvenile fish. During early embryonic development stages, the expression of Pydhh was revealed high levels at hatching stage of embryo development. Moreover, the relative expression of Pydhh was significantly higher in the juvenile liver than adults’, and higher in the female skin than the male skin. To further investigate its location, the in situ hybridization (ISH) assay was performed, the results showed that the hybridization signal was obviously expressed in the immune organs of Pseudopleuronectes yokohamae, with weak signal expression in the other tissues. Our results suggested that Pydhh is highly conserved among species and plays a vital role in embryonic development and formation of immune related organs.     

Phylogeny of Mesocyclops (Copepoda: Cyclopidae) inferred from morphological characters

Evolutionary relationships were investigated in the genus Mesocyclops, a pantropical freshwater cyclopid group. In the phylogenetic analyses that involved all 71 known species, and used 81 morphological characters (265 character states) mainly of the adult females, two different approaches were applied: global parsimony, and a new distance method based upon the recognition of sister‐groups on the basis of minimal distances iteratively corrected for unique character states (MICSEQ). In coding of the characters, half of which showed intraspecific variation, the ‘scaled’ method was employed, which assumes that any trait between its absence and fixed presence passes through a polymorphic stage. Impact of the reference points on topology of the trees generated by the parsimony method was tested in three ways where the outgroups comprised: (1) nine species representing six genera of two subfamilies; (2) three species from two genera supposedly not distant from Mesocyclops; and (3) one presumably close and one distant relative of Mesocyclops. The trees generated by the parsimony‐based and corrected distance methods agreed as to the monophyly of the following groups: reidae‐clade (M. reidae, M. chaci, M. yutsil); rarus‐clade (Mesocyclops annae, M. pseudoannae, M. splendidus, M. rarus, M. paludosus, M. darwini, M. dayakorum); annulatus‐clade (Mesocyclops intermedius, M. ellipticus, M. paranaensis, M. annulatus, M. tenuisaccus); meridianus‐clade (Mesocyclops meridionalis, M. varius, M. venezolanus, M. brasilianus, M. pseudomeridianus, M. meridianus); major‐clade (Mesocyclops major, M. pilosus, M. insulensis); dussarti‐clade (M. dussarti, M. dadayi, M. isabellae, M. thermocyclopoides); pubiventris‐clade (M. pubiventris, M. medialis, M. brooksi, M. notius). A majority of the analyses support a clade of the ‘true’Mesocyclops including all ingroup species except the reidae‐group, and point to monophyly of the Old World species lacking medial spine on P1 basipodite. There were, however, some components for which the two procedures, regardless of the outgroup choice and/or character set, suggested different relationships. Basal relationships of Mesocyclops[between M. edax (North and Central America), the Neotropical species (M. longisetus, M. araucanus, M. evadomingoi, meridianus‐ and annulatus‐clade), Old World group (P1 basipodite without medial spine) and the rarus‐clade (Old World; P1 basipodite with medial spine)] remained unresolved. © 2006 The Linnean Society of London, Zoological Journal of the Linnean Society, 2006, 147 , 1–70.