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.     

DNA barcodes from century‐old type specimens using next‐generation sequencing

Type specimens have high scientific importance because they provide the only certain connection between the application of a Linnean name and a physical specimen. Many other individuals may have been identified as a particular species, but their linkage to the taxon concept is inferential. Because type specimens are often more than a century old and have experienced conditions unfavourable for DNA preservation, success in sequence recovery has been uncertain. This study addresses this challenge by employing next‐generation sequencing (NGS) to recover sequences for the barcode region of the cytochrome c oxidase 1 gene from small amounts of template DNA. DNA quality was first screened in more than 1800 century‐old type specimens of Lepidoptera by attempting to recover 164‐bp and 94‐bp reads via Sanger sequencing. This analysis permitted the assignment of each specimen to one of three DNA quality categories – high (164‐bp sequence), medium (94‐bp sequence) or low (no sequence). Ten specimens from each category were subsequently analysed via a PCR‐based NGS protocol requiring very little template DNA. It recovered sequence information from all specimens with average read lengths ranging from 458 bp to 610 bp for the three DNA categories. By sequencing ten specimens in each NGS run, costs were similar to Sanger analysis. Future increases in the number of specimens processed in each run promise substantial reductions in cost, making it possible to anticipate a future where barcode sequences are available from most type specimens.     

Historical biogeography of the hyperdiverse hidden snout weevils (Coleoptera,Curculionidae, Cryptorhynchinae)

The first dated phylogeny of the weevil subfamily Cryptorhynchinae is presented within a framework of Curculionoidea. The inferred pattern and timing of weevil family relationships are generally congruent with previous studies, but our data are the first to suggest a highly supported sister-group relationship between Attelabidae and Belidae. Our biogeographical inferences suggest that Cryptorhynchinae s.s. originated in the Late Cretaceous (c. 86 Ma) in South America. Within the ‘Acalles group’ and the ‘Cryptorhynchus group’, several independent dispersal events to the Western Palaearctic via the Nearctic occurred in the Late Cretaceous and Early Paleogene. A second southern route via Antarctica may have facilitated the colonization of Australia in the Late Cretaceous (c. 82 Ma), where a diverse Indo-Australian clade probably emerged c. 73 Ma. In the Early Eocene (c. 50–55 Ma), several clades independently dispersed from Australia to proto-New Guinea, i.e. the tribe Arachnopodini s.l., the ‘Rhynchodes group’ and the genus Trigonopterus. New Zealand was first colonized in the Late Palaeocene (c. 60 Ma). Divergence time estimations and biogeographical reconstructions indicate that the colonization of New Guinea is older than expected from current geological reconstructions of the region.     

Phylogeny and taxonomy of the Prenolepis genus‐group of ants (Hymenoptera: Formicidae)

Abstract. We investigated the phylogeny and taxonomy of the Prenolepis genus‐group, a clade of ants we define within the subfamily Formicinae comprising the genera Euprenolepis, Nylanderia, gen. rev. , Paraparatrechina, gen. rev. & stat. nov. , Paratrechina, Prenolepis and Pseudolasius. We inferred a phylogeny of the Prenolepis genus‐group using DNA sequence data from five genes (CAD, EF1αF1, EF1αF2, wingless and COI) sampled from 50 taxa. Based on the results of this phylogeny the taxonomy of the Prenolepis genus‐group was re‐examined. Paratrechina (broad sense) species segregated into three distinct, robust clades. Paratrechina longicornis represents a distinct lineage, a result consistent with morphological evidence; because this is the type species for the genus, Paratrechina is redefined as a monotypic genus. Two formerly synonymized subgenera, Nylanderia and Paraparatrechina, are raised to generic status in order to provide names for the other two clades. The majority of taxa formerly placed in Paratrechina, 133 species and subspecies, are transferred to Nylanderia, and 28 species and subspecies are transferred to Paraparatrechina. In addition, two species are transferred from Pseudolasius to Paraparatrechina and one species of Pseudolasius is transferred to Nylanderia. A morphological diagnosis for the worker caste of all six genera is provided, with a discussion of the morphological characters used to define each genus. Two genera, Prenolepis and Pseudolasius, were not recovered as monophyletic by the molecular data, and the implications of this result are discussed. A worker‐based key to the genera of the Prenolepis genus‐group is provided.     

Phylogenetics of Eulophiinae (Orchidaceae: Epidendroideae): evolutionary patterns and implications for generic delimitation

Eulophiinae comprise c. 270 species divided into nine genera, with the species‐rich terrestrial genus Eulophia representing 60% of this diversity. Remarkable ecological and morphological variation, and an absence of clear diagnostic characters have led to uncertain generic delimitation in the subtribe. Using a combination of new and previously published DNA sequences, we created a dataset representing 122 taxa and all genera of Eulophiinae and inferred a complete generic‐level phylogeny for the subtribe for the first time. Our sampling focused on analysing Afro‐Madagascan taxa and therefore included representatives of the four mostly epiphytic Madagascan endemic genera, the near Madagascan endemic Oeceoclades and additional sampling of the predominantly African genera Eulophia and Orthochilus. In total, 104 new accessions were collected for this study in Zambia and Madagascar (88 of which represented 36 Eulophia spp. and 12 Oeceoclades spp.). Independent plastid and nuclear phylogenetic trees were inferred using Bayesian and maximum‐likelihood algorithms, which recovered strong support for a monophyletic Eulophiinae, the first‐branching position of the mostly epiphytic Madagascan endemic genera, and increased support for recognition of the terrestrial genera Oeceoclades and Orthochilus. Eulophia, the largest genus in the group, was recovered as polyphyletic, but with implications for its classification and that of Geodorum, that was nested in the main Eulophia clade. Although relationships among several genera were resolved with some confidence, the positions of the South African endemic genus Acrolophia and the epiphytic Madagascan endemic Paralophia require further work. Taxon sampling of Asian Eulophia is a priority for future work on the systematics of this group. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179 , 43–56.     

Evolutionary history of Gymnocarpos (Caryophyllaceae) in the arid regions from North Africa to Central Asia

Gymnocarpos has only about ten species distributed in the arid regions of Asia and Africa, but it exhibits a geographical disjunction between eastern Central Asia and western North Africa and Minor Asia. We sampled eight species of the genus and sequenced two chloroplast regions (rps16 and psbB–psbH), and the nuclear rDNA (ITS) to study the phylogeny and biogeography. The results of the phylogenetic analyses corroborated that Gymnocarpos is monophyletic, in the phylogenetic tree two well supported clades are recognized: clade 1 includes Gymnocarpos sclerocephalus and G. decandrus, mainly the North African group, whereas clade 2 comprises the remaining species, mainly in the Southern Arabian Peninsula. Molecular dating analysis revealed that the divergence age of Gymnocarpos was c. 31.33 Mya near the Eocene and Oligocene transition boundary, the initial diversification within Gymnocarpos dated to c. 6.69 Mya in the late Miocene, and the intraspecific diversification mostly occurred during the Quaternary climate oscillations. Ancestral area reconstruction suggested that the Southern Arabian Peninsula was the ancestral area for Gymnocarpos. Our conclusions revealed that the aridification since mid‐late Miocene significantly affected the diversification of the genus in these areas.     

Phylogenetics of Escallonia (Escalloniaceae) based on plastid DNA sequence data

Escallonia (Escalloniaceae) is a New World genus of c. 39 species distributed mainly in the South American highlands. Plastid DNA sequence data from the intergenic spacers trnS‐trnG and 3′ trnV‐ndhC and the ndhF gene for 32 species were used to examine the relationships among species and related genera and to analyse the relationship between phylogeny and the geographical distribution of the species. Maximum parsimony and Bayesian inference were employed to analyse the data. The sister relationship of Escallonia to Forgesia and Valdivia was corroborated. We recovered five strongly supported clades that are geographically structured, suggesting that the evolutionary history of the genus may be linked to historical processes, including the uplift of mountainous systems in South America. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173 , 442–451.     

Phylogenetic systematics of Colotis and associated genera (Lepidoptera: Pieridae): evolutionary and taxonomic implications

We investigated the genetic diversity and phylogenetic placement of the butterflies in the genus Colotis and eight related pierid genera using sequence information from two mitochondrial and two nuclear genes. To establish the status of species, we initially barcoded 632 specimens representative of all genera and most species and subspecies in those genera. A subset was then selected for phylogenetic analysis where additional gene regions were sequenced: 16S rRNA (523 bp), EF‐1α (1126 bp) and wg (404 bp). DNA barcode results were largely congruent with the traditional classification of species in the Colotis group, but deep splits or lack of genetic divergence in some cases supported either species‐level differentiation or synonymy. Despite using information from four genes, the deeper nodes in our phylogeny were not strongly supported, and monophyly of the ‘Colotis group’ and the genera Colotis and Eronia could not be established. To preserve the monophyly of Colotis, we revive the genus Teracolus for three outlying species previously in Colotis (i.e. Colotis eris, Colotis subfasciatus and Colotis agoye), as well as the genus Afrodryas for Eronia leda. The position of Calopieris is unresolved although it appears to be well outside the molecular variation in Colotis (s.l.). A dispersal/vicariance analysis suggested that major diversification in Colotis (s.str.) occurred in Africa with subsequent dispersal to India and Madagascar.     

Evolution of the male genitalia in the genus Limnebius Leach, 1815 (Coleoptera,Hydraenidae)

We investigated the evolution of the male genitalia of species of Limnebius (Coleoptera, Hydraenidae). This genus is very homogeneous externally but characterized by highly diverse male genitalia, which are in some cases extraordinarily complex. We reconstructed a molecular phylogeny for 70 of the c. 150 known species of the genus with six fragments of mitochondrial and nuclear genes. We found two main lineages of Miocene origin, largely corresponding to the synonymized subgenera Bilimneus and Limnebius, which are here re‐erected. Within the Holarctic Limnebius s.s. we found four well‐supported lineages, although with poorly supported relationships between them: the L. piceus, L. gracilipes, L. parvulus and L. nitidus species groups. We describe the aedeagus and its different appendages in detail based on the study of 116 species, including serial histological sections to reconstruct the internal structure of seven of them. Using Bayesian methods we reconstructed the ancestral Limnebius as having small males (c. 1.2 ± 0.5 mm), with a small aedeagus (c. 0.4 ± 0.3 mm) with a free left paramere, probably an externally fused right paramere, and a possible additional appendage. The species of Bilimneus experienced a reduction in size and a simplification of their genitalia, without free parameres and with a very simple, homogenous structure. Within Limnebius s.s. several independent increases in male body and genital size took place, with a strong correlation as measured with independent contrasts. There was, however, no overall correlation between genital size and number of appendages, even though smaller genitalia tend to be less complex.     

Molecular phylogeny of the Korean Rivellia species (Diptera: Tephritoidea: Platystomatidae) based on 16S rDNA sequences: Testing morphological hypotheses using molecular data

The phylogeny of the genus Rivellia Robineau‐Desvoidy was inferred from mitochondrial 16S ribosomal (r)DNA gene sequences of 13 Korean Rivellia species and six species representing other platystomatid genera and the family Tephritidae. We compared the inferred molecular phylogeny with the previously published morphological cladogram. As a result, the following phylogenetic relationships were recognized: (i) monophyly of the genus Rivellia; (ii) monophyly of the R. syngenesiae species group; (iii) R. depicta and R. apicalis (which were not previously placed in any species group) were recognized as a sister group of the R. syngenesiae species group; and (iv) monophyly of the R. basilaris species group was recognized to a limited extent. These results, even though geographically limited, provide a new insight into the phylogeny of the genus Rivellia. They clearly show the utility of 16S rDNA for phylogenetic analysis of the genus Rivellia. Additional study involving samples from different geographical areas will be needed to gain a better understanding of the adaptive radiation of this species‐rich genus.     

Old views and new insights: taxonomic revision of the Bukovina blind mole rat,Spalax graecus (Rodentia: Spalacinae)

As a result of their rather uniform external appearance and gross cranial morphology, the systematics of blind mole rats has been hotly debated over the last century; however, the separation of the large‐bodied and small‐bodied blind mole rats at the genus level (Spalax and Nannospalax, respectively), suggested earlier on morphological grounds, is strongly supported by recent molecular biological evidence. The species of Spalax have so far been distinguished from each other by cranial traits only, especially the outline of sutures of the cranium, and the shape and relative size of the nasal and parietal bones. Based on mitochondrial DNA sequences (with the widest taxonomic and geographic coverage so far) and detailed anatomical comparisons of museum specimens, we herewith provide a revision of the taxonomic and phylogenetic status of the westernmost representative of the genus, Spalax graecus s.l. We clarify that antiquus and istricus – presently regarded as synonyms of graecus – are well‐defined species, and they together form a separate clade within Spalax. The robustness of our conclusions is supported by the combined evidence of morphology, multilocus phylogeny, species distribution, and taxon history (species congruence with past tectonic and climate events). © 2013 The Linnean Society of London     

Unexpected distribution patterns of Carduiceps feather lice (Phthiraptera: Ischnocera: Philopteridae) on sandpipers (Aves: Charadriiformes: Scolopacidae)

The louse genus Carduiceps Clay & Meinertzhagen, 1939 is widely distributed on sandpipers and stints (Calidrinae). The current taxonomy includes three species on the Calidrinae (Carduiceps meinertzhageni, Carduiceps scalaris, Carduiceps zonarius) and four species on noncalidrine hosts. We estimated a phylogeny of four of the seven species of Carduiceps (the three mentioned above and Carduiceps fulvofasciatus) from 13 of the 29 hosts based on three mitochondrial loci, and evaluated the relative importance of flyway differentiation (same host species has different lice along different flyways) and flyway homogenization (different host species have the same lice along the same flyway). We found no evidence for either process. Instead, the present, morphology‐based, taxonomy of the genus corresponds exactly to the gene‐based phylogeny, with all four included species monophyletic. Carduiceps zonarius is found both to inhabit a wider range of hosts than wing lice of the genus Lunaceps occurring on the same group of birds, and to occur on Calidris sandpipers of all sizes, both of which are unexpected for a body louse. The previously proposed family Esthiopteridae is found to be monophyletic with good support. The concatenated dataset suggests that the pigeon louse genus Columbicola may be closely related to the auk and diver louse genus Craspedonirmus. These two genera share some morphological characters with Carduiceps, but no support was obtained for grouping these three genera together. Based on mitochondrial data alone, the relationships among genera within this proposed family cannot be properly assessed, but some previously suggested relationships within this proposed family are confirmed.     

The biogeography and phylogeny of schizothoracine fishes (Schizopygopsis) in the Qinghai‐Tibetan Plateau

Freshwater fish belonging to the genus Schizopygopsis are widespread in drainages throughout the Qinghai‐Tibetan Plateau and, thus, a model group with which to investigate how paleo‐drainage changes linked to historical uplifting within the Qinghai‐Tibetan Plateau influence speciation. To date, the phylogenetic and taxonomic relationships within Schizopygopsis remain controversial. In this study, we constructed a comprehensive molecular phylogeny of Schizopygopsis based on six mitochondrial gene sequences. We compared the taxonomic relationships revealed by this phylogeny with those obtained from morphological data. We also used this phylogeny to assess the extent to which the evolution of Schizopygopsis has been driven by paleo‐drainage changes linked to uplifting of the Qinghai‐Tibetan Plateau. Results indicated that all Schizopygopsis taxa formed a monophyletic group comprising five major clades, which were inconsistent with the taxonomic relationships based on morphology for this group. Our results also strongly supported the validity of S. anteroventris and S. microcephalus as distinct species within Schizopygopsis. Molecular calibrations showed that species within the middle Yangtze species diverged earlier (~4.5 Mya) than species within the Indus River (~3.0 Mya), the Mekong River (~2.8 Mya) and the Tsangpo + Salween rivers (~2.5 Mya). The most recent evolutionary splits occurred among species from the upper and lower Yangtze River, the Yellow River and the Qiadam Basin at about 1.8 to 0.3 Mya. Our molecular evidence and use of the molecular clock calibration have allowed us to associate speciation events within the genus Schizopygopsis to the formation and separation of paleo‐drainage connections caused by tectonic events during the uplifting of the Qinghai‐Tibetan Plateau (~4.5 Mya). This work underlines the dominant role of vicariance in shaping the evolutionary history of the genus Schizopygopsis. Further research using multiple loci and more extensive sampling will reveal a more complete picture of the phylogenetic relationships and biogeography of Schizopygopsis fishes.     

DNA barcoding of Bradysia (Diptera: Sciaridae) for detection of the immature stages on agricultural crops

We investigated the usefulness of mitochondrial cytochrome c oxidase (COI) DNA barcoding of the genus Bradysia for the detection of immature stages and cryptic species complex. Although the larvae of some species in this genus are agricultural pests, immature stages are rarely identified due to the lack of key morphological characteristics. We constructed partial sequences of the COI gene for 25 species of Bradysia as a first step towards a DNA barcode. Using these data, Bradysia impatiens, B. procera and B. peraffinis were identified from larval specimens collected, respectively, from paprika, ginseng and oak sawdust beds used for cultivating shiitake. Our findings reveal a complex of three species within the B. tilicola group. These species were all identified as important pest B. ocellaris based on the morphology of male genital structures; however, the interspecific genetic divergence of the COI region was significantly greater (16.1–19.4%) than the intraspecific variation in each species. Therefore, B. ocellaris may consist of at least three species. The results demonstrate that COI DNA barcodes are useful for Bradysia species identification.     

A review of the Iberian Sphingonotini with description of two novel species (Orthoptera: Acrididae: Oedipodinae)

The genus Sphingonotus Fieber, 1853 is one of the most species‐rich grasshopper genera in the world. We studied the morphology of c. 1000 individuals from the Iberian Peninsula to review the taxonomy of the genus and its relatives. Moreover, we inferred a molecular phylogeny of the Iberian Sphingonotini based on two mitochondrial genes. The Iberian and north‐west African Sphingonotini comprise two recent radiations, within which the genetic relationships are not fully resolved. A multivariate morphometric analysis showed that S. azurescens (Rambur, 1838) and S. morini (Defaut, 2005) can be clearly discriminated, supporting their species status. Based upon the combined data, the genus Granada Koçak & Kemal, 2008 is synonymized with Sphingonotus Fieber, 1853 and its type species re‐assigned to Sphingonotus imitans Brunner von Wattenwyl, 1882 comb. rev . The data also supported species rank for Sphingonotus lusitanicus Ebner, 1941 comb. rev. Oedipoda callosa Fieber, 1853 is considered as nomen dubium. Two novel species are described: Sphingonotus ( Neosphingonotus ) almeriense Llucià‐Pomares sp. nov . and Sphingonotus (Neosphingonotus) nodulosus Llucia‐Pomares sp. nov. from the southern part of the Iberian Peninsula. The new species are compared with other Iberian Sphingonotini and a key to the species is provided. © 2013 The Linnean Society of London     

Aphis (Hemiptera: Aphididae) species groups found in the Midwestern United States and their contribution to the phylogenetic knowledge of the genus

A phylogeny of the genus Aphis Linnaeus, 1 758 was built primarily from specimens collected in the Midwest of the United States. A data matrix was constructed with 68 species and 41 morphological characters with respective character states of alate and apterous viviparous females. Dendrogram topologies of analyses performed using UPGMA (Unweighted Pair Group Method with Arithmetic Mean), Maximum Parsimony and Bayesian analysis of Cytochrome Oxidase I, Elongation Factor 1‐α and primary endosymbiont Buchnera aphidicola 16S sequences were not congruent. Bayesian analysis strongly supported most terminal nodes of the phylogenetic trees. The phylogeny was strongly supported by EF1‐α, and analysis of COI and EF1‐α molecular data combined with morphological characters. It was not supported by single analysis of COI or Buchnera aphidicola 16S. Results from the Bayesian phylogeny show 4 main species groups: asclepiadis, fabae, gossypii, and middletonii. Results place Aphis and species of the genera Protaphis Börner, 1952, Toxoptera Koch, 1856 and Xerobion Nevsky, 1928 in a monophyletic clade. Morphological characters support this monophyly as well. The phylogeny shows that the monophyletic clade of the North American middletonii species group belong to the genus Protaphis: P. debilicornis (Gillette & Palmer, 1929 ), comb. nov., P. echinaceae (Lagos and Voegtlin, 2009 ), comb. nov., and P. middletonii (Thomas, 1879 ). The genus Toxoptera should be considered a subgenus of Aphis (stat. nov.). The analysis also indicates that the current genus Iowana Frison, 1954 should be considered a subgenus of Aphis (stat. nov.).     

Phylogeny and biogeography of Muusoctopus (Cephalopoda: Enteroctopodidae)

Deep‐sea octopuses of the genus Muusoctopus are thought to have originated in the Pacific Northern Hemisphere and then diversified throughout the Pacific and into the rest of the World Ocean. However, this hypothesis was inferred only from molecular divergence times. Here, the ancestral distribution and dispersal routes are estimated by Bayesian analysis based on a new phylogeny including 38 specimens from the south‐eastern Pacific Ocean. Morphological data and molecular sequences of three mitochondrial genes (16S rRNA, COI and COIII) are presented. The morphological data confirm that specimens newly acquired from off the coast of Chile comprise two species: Muusoctopus longibrachus and the poorly described species, Muusoctopus eicomar. The latter is here redescribed and is clearly distinguished from M. longibrachus and other closely related species in the region. A gene tree was built using Bayesian analysis to infer the phylogenetic position of these species within the species group, revealing that a large genetic distance separates the two sympatric Chilean species. M. longibrachus is confirmed as the sister species of Muusooctopus eureka from the Falkland Islands; while M. eicomar is a sister species of Muusoctopus yaquinae from the North Pacific, most closely related to the amphi‐Atlantic species Muusoctopus januarii. Molecular divergence times and ancestral distribution analyses suggest that genus Muusoctopus may have originated in the North Atlantic: one lineage dispersed directly southward to the Magellan region and another dispersed southward along the Eastern Pacific to the Southern Ocean and Antarctica. The Muusoctopus species in the Southern Hemisphere have different phylogenetic origins and represent independent invasions of this region.