Phylogeny and species delimitation of North European Lumbricillus (Clitellata,Enchytraeidae)

The enchytraeid genus Lumbricillus comprises about 80 described species of clitellate worms, which are up to a few centimetres long, and they mostly inhabit the littoral zone of non‐tropical marine and brackish waters world‐wide. The phylogeny of this genus is poorly studied, but previous work has suggested that Lumbricillus is a non‐monophyletic group. In this study, species boundaries and the phylogeny of this genus is re‐assessed using more than 300 DNA‐barcoded specimens (using COI mtDNA), part of which was also sequenced for two additional mitochondrial and four nuclear molecular markers. Statistical and coalescent based applications were used for the delimitation of a total of 24 species, of which 20 were identified as belonging to 17 described morphospecies; one morphospecies was found to be a complex of four delimited species, and another four delimited species could not be matched with any described species. Furthermore, gene trees, concatenation and multispecies coalescent based species trees were estimated using Bayesian inference. The estimated phylogenies confirm a non‐monophyletic Lumbricillus as L. semifuscus is clearly excluded from the genus. Furthermore, the placement of a monophyletic clade consisting of L. arenarius, L. dubius, and an unidentified species varies between analyses; they are either found as the sister‐group to the genus Grania or as sister‐group to the remaining Lumbricillus, where the latter relationship is supported by the multispecies coalescent, which we consider as the most reliable method.     

A new type of 3‐D peripheral ultrastructure in Glaucocystis (Glaucocystales,Glaucophyta) as revealed by ultra‐high voltage electron microscopy

The coccoid glaucophyte genus Glaucocystis is characterized by having a thick cell wall, which has to date prohibited examination of the native ultrastructural features of the protoplast periphery. Recently, however, the three‐dimensional (3‐D) ultrastructure of the protoplast periphery was revealed in two divergent Glaucocystis species, with the world's most powerful ultra‐high voltage electron microscope (UHVEM). The two species exhibit morphological diversity in terms of their 3‐D ultrastructural features. However, these two types do not seem to encompass actual ultrastructural diversity in the genetically diverse genus Glaucocystis. Here, we report a new type of peripheral 3‐D ultrastructure resolved in “G. incrassata” SAG 229‐2 cells by 3‐D modeling based on UHVEM tomography using high‐pressure freezing and freeze‐substitution fixation. The plasma membrane and underlying flattened vesicles in “G. incrassata” SAG 229‐2 exhibited grooves at intervals of 200–600 nm, and the flattened vesicles often overlapped one another at the protoplast periphery. This 3‐D ultrastructure differs from those of the two types previously reported in other species of Glaucocystis. The possibility of classification of Glaucocystis species based on the 3‐D ultrastructure of the protoplast periphery is discussed.     

Evolution,biogeography and systematics of the western Palaearctic Zamenis ratsnakes

The phylogenetic relationships between western Palaearctic Zamenis and Rhinechis ratsnakes have been troubled, with recent estimates based on the supermatrix approach questioning their monophyly and providing contradictory results. In this study, we generated a comprehensive molecular data set for Zamenis and closely related ratsnakes to assess their phylogenetic and systematic relationships and infer their spatial and temporal modes of diversification. We obtained a fully resolved and well‐supported phylogeny, which is consistent across markers, taxon‐sets and phylogenetic methods. The close phylogenetic relationship between Rhinechis and Zamenis is well‐established. However, the early branching pattern within this clade, and the position of R. scalaris and Z. hohenackeri, remains poorly supported. The Persian ratsnake Z. persicus is sister to the Mediterranean species Z. situla, Z. longissimus and Z. lineatus, of which Z. situla is sister to a clade containing the latter two species. These results are consistent with a recent phylogenomic study on ratsnakes based on hundreds of loci. Whereas, topological tests based on our data and evidence from such phylogenomic study strongly rejected previous phylogenetic estimates based on the supermatrix approach and demonstrate that these “mega‐phylogenies”, with hundreds of taxa and high levels of missing data, have recovered inconsistent relationships with spurious nodal support. Biogeographical and molecular dating analyses suggest an origin of the ancestor of Rhinechis and Zamenis in the Aegean region with early cladogenesis during the Late Miocene associated with the Aegean arch formation and support a scenario of east‐to‐west diversification. Finally, while we have little morphological and phylogenetic evidence for the distinctiveness between Rhinechis and Zamenis, a classification of them in a single genus, and the designation of Zamenis scalaris (Schinz, 1822), reflects better their evolutionary relationships.     

Diaporthe amygdali,a species complex or a complex species?

Delimitation of species boundaries within the fungal genus Diaporthe has been challenging, but the analyses of combined multilocus DNA sequences has become an important tool to infer phylogenetic relationships and to circumscribe species. However, analyses of congruence between individual gene genealogies and the application of the genealogical concordance principle have been somehow overlooked. We noted that a group of species including D. amygdali, D. garethjonesii, D. sterilis, D. kadsurae, D. ternstroemia, D. ovoicicola, D. fusicola, D. chongqingensis and D. mediterranea, commonly known as D. amygdali complex, occupy a monophyletic clade in Diaporthe phylogenies but the limits of all species within the complex are not entirely clear. To assess the boundaries of species within this complex we employed the Genealogical Concordance Phylogenetic Species Recognition principle (GCPSR) and coalescence-based models: General Mixed Yule-Coalescent (GMYC) and Poisson Tree Processes (PTP). The incongruence detected between individual gene phylogenies, as well as the results of coalescent methods do not support the recognition of lineages within the complex as distinct species. Moreover, results support the absence of reproductive isolation and barriers to gene flow in this complex, thus providing further evidence that the D. amygdali species complex constitutes a single species. This study highlights the relevance of the application of the GCPSR principle, showing that concatenation analysis of multilocus DNA sequences, although being a powerful tool, might lead to an erroneous definition of species limits. Additionally, it further shows that coalescent methods are useful tools to assist in a more robust delimitation of species boundaries in the genus Diaporthe.     

Description of a new species and phylogenetic analysis of the subtribe Cynopoecilina,including continuous characters without discretization (Cyprinodontiformes: Rivulidae)

A new species of the subtribe Cynopoecilina is described from the rio Gravataí basin, laguna dos Patos system, southern Brazil. The relationships of the new species among taxa of the subtribe Cynopoecilina is discussed based on two analyses: one using 71 discrete characters and other with the addition of six continuous characters analyzed without discretization. The addition of the continuous characters resulted in the first fully resolved phylogenies for Cynopoecilus and Leptolebias species, not obtained in the analysis including only discrete characters. The new species is assigned to Cynopoecilus as sister group to the remaining species of the genus. A new diagnosis is proposed for Cynopoecilus to accommodate the new species. The resulting phylogeny indicates that the occupation of the grasslands of the Pampa biome by the species of Cynopoecilus occurred along the evolution of the genus and that this event was significant for the diversification of the genus. © 2014 The Linnean Society of London     

Molecular phylogeny of the New World gecko genus Homonota (Squamata: Phyllodactylidae)

The genus Homonota was described by Gray (1845) and currently includes 10 species: Homonota andicola, H. borellii, H. darwinii, H. fasciata, H. rupicola, H. taragui, H. underwoodi, H. uruguayensis, H. williamsii & H. whitii and one subspecies of H. darwinii (H. darwinii macrocephala). It is distributed from 15° latitude south in southern Brazil, through much of Bolivia, Paraguay, Uruguay and Argentina to 54° south in Patagonia and across multiple different habitats. Several morphological taxonomic studies on a subset of these species have been published, but no molecular phylogenetic hypotheses are available for the genus. The objective of this study is to present a molecular phylogenetic hypothesis for all the described species in the genus. We sequenced two mitochondrial genes (cyt‐b & 12S: 1745 bp), seven nuclear protein coding (RBMX, DMLX, NKTR, PLRL, SINCAIP, MXRA5, ACA4: 5804 bp) and two anonymous nuclear loci (30Hb, 19Hb: 1306 bp) and implemented traditional concatenated analyses (MP, ML, BI) as well as species‐tree (*beast ) approaches. All methods recovered almost the same topology. We recovered the genus Homonota as monophyletic with strong statistical support. Within Homonota, there are three strongly supported clades (whitii, borellii and fasciata), which differ from those previously proposed based on scale shape, osteology, myology and quantitative characters. Detailed morphological analyses based on this highly resolved and well‐supported phylogeny will provide a framework for understanding morphological evolution and historical biogeography of this phenotypically conservative genus. We hypothesize that extensive marine transgressions during Middle and Late Miocene most probably isolated the ancestors of the three main clades in eastern Uruguay (borellii group), north‐western Argentina‐southern Bolivia (fasciata group), and central‐western Argentina (whitii group). Phylogeographic and morphological/morphometric analyses coupled with paleo‐niche modelling are needed to better understand its biogeographical history.     

Phylogeny and species diversity of the genus Herichthys (Teleostei: Cichlidae)

We provide a review of the systematics of Herichthys by evaluating the usefulness of several mitochondrial and nuclear genetic markers together with morphological data. The nDNA next‐generation sequencing ddRAD analysis together with the mtDNA cytochrome b gene provided well‐resolved and well‐supported phylogenies of Herichthys. On the other hand, the nDNA S7 introns have limited resolution and support and the COI barcoding analysis completely failed to recover all but one species of Herichthys as monophyletic. The COI barcoding as currently implemented is thus insufficient to distinguish clearly distinct species in the genus Herichthys that are supported by other molecular markers and by morphological characters. Based on our results, Herichthys is composed of 11 species and includes two main clades (the H. labridens and H. cyanoguttatus species groups). Herichthys bartoni is in many respects the most plesiomorphic species in the genus and has a conflicting phylogenetic position between mtDNA and nDNA markers, where the robust nDNA ddRAD data place it as a rather distant basal member of the H. labridens species group. The mtDNA of H. bartoni is on the other hand only slightly divergent from the sympatric and syntopic H. labridens, and the species thus probably have hybridized in the relatively recent past. The sympatric and syntopic Herichthys steindachneri and H. pame are supported as sister species. The Herichthys cyanoguttatus species group shows two well‐separated basal species (the northernmost H. minckleyi and the southernmost H. deppii) followed by the closely related and centrally distributed species H. cyanoguttatus, H. tepehua, H. carpintis, and H. tamasopoensis whose relationships differ between analyses and show likely hybridizations between themselves and the two basal species as suggested by conflicts between DNA analyses. Several instances of introgressions/hybridizations have also been found between the two main clades of Herichthys.     

Frogs at the summits: phylogeny of the Andean frogs of the genus Telmatobius (Anura,Telmatobiidae) based on phenotypic characters

A phylogenetic hypothesis for the frogs of the genus Telmatobius that includes a comprehensive sample of the morphological and geographical variation is lacking. Obtaining such a hypothesis constitutes the main focus of this contribution. A phylogenetic matrix was generated based on 97 phenotypic characters and 56 terminals. A parsimony analysis of this matrix was performed with TNT. Telmatobius is found to be monophyletic and well supported by 11 synapomorphies. Although the consensus tree shows several polytomies, four main groups have been recovered. The well‐supported T. verrucosus Group includes forest and sub‐paramo species from Bolivia and Peru, and is the sister group of the remaining species. The T. bolivianus Group includes forest and inter‐Andean valley species from Argentina and Bolivia but it is poorly supported. Two supported high‐altitude groups have been recovered, the T. macrostomus Group from the Central Andes of Peru, and the T. marmoratus Group from the Altiplano‐Puna Plateau of Argentina, Bolivia, Peru and Chile and its adjacent Pacific and Northern slopes. The synapomorphies proposed for Telmatobius are discussed as well as the evolution of some of these synapomorphies and other characters within the genus.     

BIODIVERSITY OF CORALLINE ALGAE IN THE NORTHEASTERN ATLANTIC INCLUDING CORALLINA CAESPITOSA SP. NOV. (CORALLINOIDEAE,RHODOPHYTA)1

The Corallinoideae (Corallinaceae) is represented in the northeastern Atlantic by Corallina officinalis L.; Corallina elongata J. Ellis et Sol.; Haliptilon squamatum (L.) H. W. Johans., L. M. Irvine et A. M. Webster; and Jania rubens (L.) J. V. Lamour. The delimitation of these geniculate coralline red algae is based primarily on morphological characters. Molecular analysis based on cox1 and 18S rRNA gene phylogenies supported the division of the Corallinoideae into the tribes Janieae and Corallineae. Within the Janieae, a sequence difference of 46–48 bp (8.6%–8.9%) between specimens of H. squamatum and J. rubens in the cox1 phylogeny leads us to conclude that they are congeneric. J. rubens var. rubens and J. rubens var. corniculata (L.) Yendo clustered together in both phylogenies, suggesting that for those genes, there was no genetic basis for the morphological variation. Within the Corallineae, it appears that in some regions, the name C. elongata has been misapplied. C. officinalis samples formed two clusters that differed by 45–54 bp (8.4%–10.0%), indicating species‐level divergence, and morphological differences were sufficient to define two species. One of these clusters was consistent with the morphology of the type specimen of C. officinalis (LINN 1293.9). The other species cluster is therefore described here as Corallina caespitosa sp. nov. This study has demonstrated that there is a clear need for a revision of the genus Corallina to determine the extent of “pseudocryptic” diversity in this group of red algae.     

Phylogenetic and mixed Yule‐coalescent analyses reveal cryptic lineages within two South American marine snails of the genus Crepipatella (Gastropoda: Calyptraeidae)

The presence of sibling species within the marine gastropod genus Crepipatella has complicated the taxonomy of members of the group. Since the establishment of the genus, 15 species have been described, but recent studies have indicated that there are only five valid species, two of which inhabit the coasts of Chile, namely C. dilatata and C. fecunda. The two species are morphologically indistinguishable as adults, but can be differentiated on the basis of their encapsulated developmental stages. The primary aim of this study was to reconstruct phylogeny within the genus, and to establish species limits of C. dilatata and C. fecunda, using mitochondrial DNA data. To this end, we used maximum parsimony, maximum likelihood, and Bayesian inference to reconstruct phylogenies using 589 bp of the cytochrome oxidase I (COI) gene. The mtDNA phylogenies were then used as input in a general mixed Yule‐coalescent (GMYC) analysis to estimate species boundaries. In addition, quarter likelihood mapping was used to test a posteriori the confidence of inner branch patterns in the phylogenetic tree. Both DNA tree‐based and GMYC methods provide support for five isolated lineages within this species complex. Our data also suggest that Late Pleistocene and Holocene fragmentation and subsequent range expansion events may have shaped contemporary genetic patterns of Crepipatella in South America.     

Out of South America: phylogeny of non‐biting midges in the genus Labrundinia suggests multiple dispersal events to Central and North America

Non‐biting midges of the genus Labrundinia (Chironomidae: Tanypodinae) are minute dipterans with immature stages living in a variety of unpolluted water bodies, from small streams and ponds to lakes and bays. Extensively recorded in ecological studies, the genus comprises 39 species, all except one described from areas outside the Palearctic region. Internal structure among Labrundinia species was postulated by S. S. Roback, who recognized four species groups based on morphological characters of immature stages. We examined phylogenetic relationships among known Labrundinia species using partial DNA sequences of the nuclear protein‐coding gene CAD and morphological characters. Both analyses with Bayesian inference and parsimony methods recovered the monophyly of Labrundinia, strongly supported by five morphological synapomorphies. Internal relationships within the genus partly supported Roback's species groups with the addition of later described species. Biogeographical inferences were obtained by applying Bayesian binary MCMC (BBM) analysis and favoured a scenario where Labrundinia had its initial diversification in the Neotropical region and that current presence in the Nearctic region and southern South America is due to subsequent dispersal.     

Miocene diversification of an open‐habitat predatorial passerine radiation,the shrikes (Aves: Passeriformes: Laniidae)

Diversification of avifaunas associated with savannah and steppes appears to correlate with open habitats becoming available, starting in the Miocene. Few comparative analyses exist for families for which all species are predominantly adapted to these habitats. One such group is Laniidae (Passeriformes), which are small‐ to medium‐sized predatory passerines known for their distinctive behaviour of impaling prey. We used multispecies coalescent‐based and concatenation methods to provide the first complete species‐level phylogeny for this group, as well as an estimate of the timing of diversification. Our analyses indicate that Laniidae as currently delimited is not monophyletic, as the genus Eurocephalus is not closely related to the remaining species. The two species currently assigned to the monotypic genera Urolestes and Corvinella are part of the same clade as the Lanius species, and we propose that they are included in the genus Lanius, making Laniidae monogeneric. The initial diversification of the clade is inferred to have occurred very rapidly, starting about 7.2–9.1 million years ago, timing depending on calibration method, but in either case coinciding with the expansion of C4 grasses. An African origin is inferred in the biogeographic analysis. In the redefined Laniidae, cooperative breeding is inferred to be restricted to a single clade, characterized by gregarious behaviour and rallying. Migratory behaviour evolved multiple times within the family.     

Comprehensive phylogeny of the laughingthrushes and allies (Aves,Leiothrichidae) and a proposal for a revised taxonomy

DNA phylogenies have gradually shed light on the phylogenetic relationships of the large babbler group. We focus in this study on the family Leiothrichidae (laughingthrushes and “song babblers”), which represents the largest clade of babblers in terms of species diversity. Our phylogeny includes all genera and 82% of the recognized species, using mitochondrial and nuclear loci. The sister group to Leiothrichidae is composed of the Pellorneidae (“jungle babblers”) plus the genus Alcippe. Within Leiothrichidae, four strongly supported primary clades (A–D) are recovered. Clade A includes Grammatoptila, Laniellus and Cutia. Clade B includes a large group of laughingthrushes, all of them classified in Trochalopteron. In Clade C, the two laughingthrushes endemic to southern India, T. fairbanki and T. cachinnans, which have recently been proposed to be placed in the newly erected genus Montecincla, form a sister clade to the group comprising the “song babblers” (Lioptila, Leiothrix, Heterophasia, Minla, Liocichla, Actinodura, Chrysominla, Siva, and Sibia). Clade D includes the African babblers (Turdoides, Phyllanthus, Kupeornis), Asian relatives (Argya, Acanthoptila, Chatarrhaea) and all remaining laughingthrushes (Garrulax). The time estimates suggest that the early diversification of the Leiothrichidae occurred in the mid‐Miocene, a period that corresponds to the diversification of many passerine groups in Asia. A revised taxonomic classification of the family is proposed in the light of these results.     

Accounting for molecular stochasticity in systematic revisions: Species limits and phylogeny of Paroaria

Different frameworks have been proposed for using molecular data in systematic revisions, but there is ongoing debate on their applicability, merits and shortcomings. In this paper we examine the fit between morphological and molecular data in the systematic revision of Paroaria, a group of conspicuous songbirds endemic to South America. We delimited species based on examination of >600 specimens, and developed distance-gap, and distance- and character-based coalescent simulations to test species limits with molecular data. The morphological and molecular data collected were then analyzed using parsimony, maximum likelihood, and Bayesian phylogenetics. The simulations were better at evaluating the new species limits than using genetic distances. Species diversity within Paroaria had been underestimated by 60%, and the revised genus comprises eight species. Phylogenetic analyses consistently recovered a congruent topology for the most recently derived species in the genus, but the most basal divergences were not resolved with these data. The systematic and phylogenetic hypotheses developed here are relevant to both setting conservation priorities and understanding the biogeography of South America.     

Phylogeny and biogeography of the dung beetle genus Phanaeus (Coleoptera: Scarabaeidae)

Abstract The dung beetle genus Phanaeus as currently recognized by Edmonds (1994) consists of 51 species placed in 13 species groups and two subgenera. Here, I examine the phylogeny and biogeography of this genus by analysing the mitochondrial cytochrome oxidase subunit I (530 bp), nuclear large subunit ribosomal RNA (28S, D2 region), and 67 morphological characters for 28 species of Phanaeus. Both maximum parsimony and Bayesian analyses from the combined data yielded well‐resolved trees, although low bootstrap and posterior probability support were found for basal nodes. The phylogenetic hypotheses presented here suggest that the subgenera Phanaeus s.str. and Notiophanaeus should each be elevated to the status of full genus. With the exception of the eucraniine outgroups, the paleano species group of the genus Phanaeus is recovered as sister to all other taxa, including the outgroups Oxysternon, Sulcophanaeus and Coprophanaeus. High bootstrap values and posterior probabilities supported the species groups endymion, tridens and vindex. Biogeographical analyses suggest an ancestral distribution for Phanaeus in the Andes in South America, although numerous dispersal events evidently have produced a complicated biogeographical history.     

The ITS region of groundwater amphipods: length,secondary structure and phylogenetic information content in Crangonyctoids and Niphargids

The phylogenetic analysis of groundwater amphipods is challenging due to the lack of suitable morphological characters. However, molecular phylogenies based on the 18S and 28S nuclear genes of two Crangonyctoidea species endemic to Iceland, Crymostygius thingvallensis and Crangonyx islandicus, support the taxonomy of these species on the basis of morphological characters. Molecular analyses suggest that the genus Crangonyx is paraphyletic, with the species that is found in Eurasia being highly divergent genetically from the species present in North America and Iceland. Studies of the phylogenetic relationships within the genus Niphargus also warrant further work. The nuclear ITS2 region has recently been proposed as a barcoding marker for plants and animals. In addition, ITS2 has been used to build phylogenies at high taxonomic levels by including its secondary structure. In this study, we want to evaluate the applicability of the ITS region for this group of species and describe its characteristics. The taxonomy of C. thingvallensis, as well as the paraphyly of the genus Crangonyx, is supported herein by phylogenies based on the ITS2 variation. The secondary structure and the length of the ITS2 sequences of the Crangonyctoidea and the Niphargidae species studied are highly variable and are characterized by duplications. The ITS2 sequence of Niphargus plateaui is the longest metazoan sequence deposited in the ITS2 database so far. Although saturation was observed in the nucleotide variation of this marker, the addition of the secondary structure information for the reconstruction of the phylogeny did not add support to the phylogenetic trees. The ITS1 region, which is known to be more variable than ITS2 and bears a large duplication within C. islandicus, was found to be less useful for phylogenetic reconstruction.     

Historical biogeography and diversification within the Neotropical parrot genus Pionopsitta (Aves: Psittacidae)

Aim We investigate spatial and temporal patterns of diversification within the Neotropical avifauna using the phylogenetic history of parrots traditionally belonging to the genus Pionopsitta Bonaparte 1854. This genus has long been of interest for those studying Neotropical biogeography and diversity, as it encompasses species that occur in most Neotropical forest areas of endemism. Location The Neotropical lowland forests in South and Central America. Methods Phylogenetic relationships were investigated for all species of the genus Pionopsitta and five other short‐tailed parrot genera using complete sequences of the mitochondrial genes cyt b and ND2 as well as 26 plumage characters. The resulting phylogeny was used to test the monophyly of the genus, investigate species limits, and as a framework for reconstructing their historical biogeography and patterns of diversification. Results We found that the genus Pionopsitta, as previously defined, is not monophyletic and thus the Chocó, Central American and Amazonian species will now have to be placed in the genus Gypopsitta. The molecular and morphological phylogenies are largely congruent, but disagree on the position of one of the Amazon basin taxa. Using molecular sequence data, we estimate that species within Gypopsitta diversified between 8.7 and 0.6 Ma, with the main divergences occurring between 3.3 and 6.4 Ma. These temporal results are compared to other taxa showing similar vicariance patterns. Main conclusions The results suggest that diversification in Gypopsitta was influenced mainly by geotectonic events, marine transgressions and river dynamics, whereas Quaternary glacial cycles of forest change seem to have played a minor role in the origination of the currently recognized species.     

Rapid diversification associated with ecological specialization in Neotropical Adelpha butterflies

Rapid diversification is often associated with morphological or ecological adaptations that allow organisms to radiate into novel niches. Neotropical Adelpha butterflies, which comprise over 200 species and subspecies, are characterized by extraordinary breadth in host plant use and wing colour patterns compared to their closest relatives. To examine the relationship between phenotypic and species diversification, we reconstructed the phylogenetic history of Adelpha and its temperate sister genus Limenitis using genomewide restriction‐site‐associated DNA (RAD) sequencing. Despite a declining fraction of shared markers with increasing evolutionary distance, the RAD‐Seq data consistently generated well‐supported trees using a variety of phylogenetic methods. These well‐resolved phylogenies allow the identification of an ecologically important relationship with a toxic host plant family, as well as the confirmation of widespread, convergent wing pattern mimicry throughout the genus. Taken together, our results support the hypothesis that evolutionary innovations in both larvae and adults have permitted the colonization of novel host plants and fuelled adaptive diversification within this large butterfly radiation.