Tracking biological invasions in space and time: elucidating the invasive history of the green alga Codium fragile using old DNA

With the advent of 'ancient DNA' studies on preserved material of extant and extinct species, museums and herbaria now represent an important although still underutilized resource in molecular ecology. The ability to obtain sequence data from archived specimens can reveal the recent history of cryptic species and introductions. We have analysed extant and herbarium samples of the highly invasive green alga Codium fragile , many over 100 years old, to identify cryptic accessions of the invasive strain known as C. fragile ssp. tomentosoides , which can be identified by a unique haplotype. Molecular characterization of specimens previously identified as native in various regions shows that the invasive tomentosoides strain has been colonizing new habitats across the world for longer than records indicate, in some cases nearly 100 years before it was noticed. It can now be found in the ranges of all the other native haplotypes detected, several of which correspond to recognized subspecies. Within regions in the southern hemisphere there was a greater diversity of haplotypes than in the northern hemisphere, probably as a result of dispersal by the Antarctic Circumpolar Current. The findings of this study highlight the importance of herbaria in preserving contemporaneous records of invasions as they occur, especially when invasive taxa are cryptic.     

Conspecificity of the model organism Ulva mutabilis and Ulva compressa (Ulvophyceae,Chlorophyta)

As one of the most abundant and ubiquitous representatives of marine and brackish coastal macrophytobenthos communities, the genus Ulva is not only an important primary producer but also of ecological and morphogenetic interest to many scientists. Ulva mutabilis became an important model organism to study morphogenesis and mutualistic interactions of macroalgae and microorganisms. Here, we report that our collections of Ulva compressa Linnaeus (1753) from Germany are conspecific with the type strains of the model organism U. mutabilis Føyn (1958), which were originally collected at Olhão on the south coast of Portugal and have from that time on been maintained in culture as gametophytic and parthenogenetic lab strains. Different approaches were used to test conspecificity: (i) comparisons of vegetative and reproductive features of cultured material of U. mutabilis and German U. compressa demonstrated a shared morphological pattern; (ii) gametes of U. compressa and U. mutabilis successfully mated and developed into fertile sporophytic first‐generation offspring; (iii) molecular phylogenetics and species delimitation analyses based on the Generalized Mixed Yule‐Coalescent method showed that U. mutabilis isolates (sl‐G[mt+]) and (wt‐G[mt‐]) and U. compressa belong to a unique Molecular Operational Taxonomic Unit. According to these findings, there is sufficient evidence that U. mutabilis and U. compressa should be regarded as conspecific.     

Putting incertae sedis taxa in their place: a proposal for ten new families and three new genera in Sphaeropleales (Chlorophyceae,Chlorophyta)

Best known for aquatic colonial algae such as Hydrodictyon, Pediastrum, or Scenedesmus, the order Sphaeropleales also contains numerous coccoid taxa from aquatic and terrestrial habitats. Recent findings indicate that coccoid lineages in this order are very diverse genetically and may be the prevalent form, although their diversity is often hidden morphologically. This study characterizes coccoid algae recently discovered from desert soil crusts that share morphological and ecological features with the genera Bracteacoccus, Pseudomuriella, and Chromochloris. Analyses of a multi‐gene data set that includes members from all sphaeroplealean families are used to examine the monophyly of these morphologically similar taxa, which are shown instead to be phylogenetically distinct and very divergent. We propose new generic names for these lineages: Bracteamorpha, Rotundella, and Tumidella. In addition, we propose an updated family‐level taxonomy within Sphaeropleales that includes ten new families of coccoid algae to accommodate the newly presented genera and many incertae sedis taxa in the order: Bracteamorphaceae, Chromochloridaceae, Dictyococcaceae, Dictyochloridaceae, Mychonastaceae, Pseudomuriellaceae, Rotundellaceae, Schizochlamydaceae, Schroederiaceae, and Tumidellaceae.     

Ulva (Chlorophyta,Ulvales) Biodiversity in the North Adriatic Sea (Mediterranean,Italy): Cryptic Species and New Introductions

Ulva Linnaeus (Ulvophyceae, Ulvales) is a genus of green algae widespread in different aquatic environments. Members of this genus show a very simple morphology and a certain degree of phenotypic plasticity, heavily influenced by environmental conditions, making difficult the delineation of species by morphological features alone. Most studies dealing with Ulva biodiversity in Mediterranean waters have been based only on morphological characters and a modern taxonomic revision of this genus in the Mediterranean is not available. We report here the results of an investigation on the diversity of Ulva in the North Adriatic Sea based on molecular analyses. Collections from three areas, two of which subject to intense shipping traffic, were examined, as well as historical collections of Ulva stored in the Herbarium Patavinum of the University of Padova, Italy. Molecular analyses based on partial sequences of the rbcL and tufA genes revealed the presence of six different species, often with overlapping morphologies: U. californica Wille, U. flexuosa Wulfen, U. rigida C. Agardh, U. compressa Linnaeus, U. pertusa Kjellman, and one probable new taxon. U. californica is a new record for the Mediterranean and U. pertusa is a new record for the Adriatic. Partial sequences obtained from historical collections show that most of the old specimens are referable to U. rigida. No specimens referable to the two alien species were found among the old herbarium specimens. The results indicate that the number of introduced seaweed species and their impact on Mediterranean communities have been underestimated, due to the difficulties in species identification of morphologically simple taxa as Ulva.     

Species delimitation and phylogeographic analyses in the Ectocarpus subgroup siliculosi (Ectocarpales,Phaeophyceae)

The genus Ectocarpus (Ectocarpales, Phaeophyceae) contains filamentous algae widely distributed in marine and estuarine habitats of temperate regions in both hemispheres. While E. siliculosus has become a model organism for genomics and genetics of the brown macroalgae, accurate species delineation, distribution patterns and diversity for the genus Ectocarpus remain problematic. In this study, we used three independent species delimitation approaches to generate a robust species hypothesis for 729 Ectocarpus specimens collected mainly along the European and Chilean coasts. These approaches comprised phylogenetic reconstructions and two bioinformatics tools developed to objectively define species boundaries (General Mixed Yule Coalescence Method and Automatic Barcode Gap Discovery). Our analyses were based on DNA sequences of two loci: the mitochondrial cytochrome oxidase subunit 1 and the nuclear internal transcribed spacer 1 of the ribosomal DNA. Our analyses showed the presence of at least 15 cryptic species and suggest the existence of incomplete lineage sorting or introgression between five of them. These results suggested the possible existence of different levels of reproductive barriers within this species complex. We also detected differences among species in their phylogeographic patterns, range and depth distributions, which may suggest different biogeographic histories (e.g., endemic species or recent introductions).     

Surveying native egg parasitoids and predators of the invasive Halyomorpha halys in Northern Italy

The invasive Halyomorpha halys (Heteroptera: Pentatomidae) is a key pest of fruits in the Emilia‐Romagna region of Italy. For the development of a sustainable management programme, knowledge of its native natural enemy community and its efficacy is essential. A three‐year field survey was conducted exposing H. halys egg masses in different types of habitats to investigate the efficacy of native natural enemies in reducing the H. halys populations in the Emilia‐Romagna region, where the stinkbug was first detected in 2012. Over the first year of the study, sentinel eggs from laboratory cultures were stapled to the underside of leaves in various host trees, whereas in following years H. halys adults were directly caged on branches in sleeve cages to allow natural oviposition. Over the examined years, low rates of parasitism (1%–3%) and predation (2%–5%) were observed. Parasitism was caused exclusively by the generalist parasitoid Anastatus bifasciatus.     

A combined morphological,ultrastructural, molecular,and biochemical study of the peculiar family Gomontiellaceae (Oscillatoriales) reveals a new cylindrospermopsin‐producing clade of cyanobacteria

Members of the morphologically unusual cyanobacterial family Gomontiellaceae were studied using a polyphasic approach. Cultured strains of Hormoscilla pringsheimii, Starria zimbabweënsis, Crinalium magnum, and Crinalium epipsammum were thoroughly examined, and the type specimen of the family, Gomontiella subtubulosa, was investigated. The results of morphological observations using both light microscopy and transmission electron microscopy were consistent with previous reports and provided evidence for the unique morphological and ultrastructural traits of this family. Analysis of the 16S rRNA gene confirmed the monophyletic origin of non‐marine repre‐sentatives of genera traditionally classified into this family. The family was phylogenetically placed among other groups of filamentous cyanobacterial taxa. The presence of cellulose in the cell wall was analyzed and confirmed in all cultured Gomontiellaceae members using Fourier transform infrared spectroscopy and fluorescence microscopy. Evaluation of toxins produced by the studied strains revealed the hepatotoxin cylindrospermopsin (CYN) in available strains of the genus Hormoscilla. Production of this compound in both Hormoscilla strains was detected using high‐performance liquid chromatography in tandem with high resolution mass spectrometry and confirmed by positive PCR amplification of the cyrJ gene from the CYN biosynthetic cluster. To our knowledge, this is the first report of CYN production by soil cyanobacteria, establishing a previously unreported CYN‐producing lineage. This study indicates that cyanobacteria of the family Gomontiellaceae form a separate but coherent cluster defined by numerous intriguing morphological, ultrastructural, and biochemical features, and exhibiting a toxic potential worthy of further investigation.     

Mitochondrial divergence suggests unexpected high species diversity in the opsariichthine fishes (Teleostei: Cyprinidae) and the revalidation of Opsariichthys macrolepis

Opsariichthine (sensu Oceanologi Et Limnologia Sinica, 1982, 13, 293–298) is a cyprinid group consisting of five genera and endemic to East Asia. Previous studies suggested that there may be many possible cryptic species in this group, but this has not been confirmed. In this study, using mitochondrial cyt b sequences on 1,388 samples and 739 haplotypes, we showed very high species diversity within this group. The results showed that phylogenetic relationships of the opsariichthine group were as ([Nipponocypris‐Parazacco‐Candidia] + [Zacco + Opsariichthys]), and there were multiple deep lineages within several species, flagging putative cryptic species. When a 3% genetic distance was used as a threshold for species delimitation, 35 haplogroups were found, nine haplogroups in Candidia‐Parazacco‐Nipponocypris group, six haplogroups in the Zacco group, and 20 haplogroups in the Opsariichthys group. We consider all of them to be putative until determination of distinct species based on the tree topology, geographic distributions, or a combination of both. In addition, two kinds of species delimitation tools, ABGD and PTP, were applied to construct molecular operational taxonomic units (MOTUs). The ABGD method revealed nine MOTUs in Candidia‐Parazacco‐Nipponocypris group, two MOTUs in the Zacco group, and 17 MOTUs in the Opsariichthys group. And the PTP method revealed 10 MOTUs in Candidia‐Parazacco‐Nipponocypris group, 10 MOTUs in the Zacco group, and 29 MOTUs in the Opsariichthys group. Therefore, there should be more species in the opsariichthine group than presently described. Based on the molecular data and morphological characteristics, we proposed Opsariichthys macrolepis as a valid species and described its morphological diagnostic characters.     

Genetic diversity and haplotype distribution of Pachymeniopsis gargiuli sp. nov. and P. lanceolata (Halymeniales,Rhodophyta) in Korea,with notes on their non‐native distributions

The red alga Pachymeniopsis lanceolata, formerly known as Grateloupia lanceolata, is a component of the native algal flora of northeast Asia and has been introduced to European and North American waters. It has been confused with a cryptic species collected from Korea and Italy. Our analyses of rbcL, cox3 and ITS from P. lanceolata and this cryptic species has revealed two distinct entities, forming a clade, which were clearly separated from its congeners and positioned with other Asian species. Here, we describe the cryptic species as P. gargiuli sp. nov., a species that differs from others by molecular sequence and subtle anatomical characters. We hypothesize that P. gargiuli may have been recently dispersed by anthropogenic vectors, possibly at or near the same time as was P. lanceolata. Our cox3 data set revealed that one haplotype of P. gargiuli, shared between Korea and Italy, and two haplotypes of P. lanceolata, commonly occurring in Korea and USA, are invasive haplotypes. This is the first report of the utility of the mitochondrial coding cox3 sequences in red algae.     

Morphological and molecular differentiation of invasive freshwater species of the genus Corbicula (Bivalvia, corbiculidea) suggest the presence of three taxa in French rivers

Asiatic Clams are common in brackish and fresh water in Asia, and they were introduced into North America in 1924 and have now spread throughout the continent. During the last two decades they have been reported in Europe, but the number of species here is uncertain. Populations of Corbicula from France and the Netherlands were analysed morphologically and genetically to quantify the degree of species and/or population differentiation. The morphological and genetic data, based on allozymes and mitochondrial sequences, were in full agreement. They indicate that there are two distinct species, identified as C. fluminalis and C. fluminea, in the two countries. Analyses of the mitochondrial COI gene revealed an unexpected divergent population of Corbicula in the Rhône. All these individuals were morphologically identified as C. fluminea, but had a COI sequence different from the two previous species. This population may, therefore, be a more ancient population, or a distinct species introduced via a different colonization route.     

Biodiversity and Molecular Phylogeny of Australian Clevelandella Species (Class Armophorea,Order Clevelandellida,Family Clevelandellidae), Intestinal Endosymbiotic Ciliates in the Wood‐Feeding Roach Panesthia cribrata Saussure, 1864

There are over 100 species in the Order Clevelandellida distributed in many hosts. The majority is assigned to one of the five families, the Nyctotheridae. Our knowledge of clevelandellid genetic diversity is limited to species of Nyctotherus and Nyctotheroides. To increase our understanding of clevelandellid genetic diversity, species were isolated from intestines of the Australian wood‐feeding roach Panesthia cribrata Saussure, 1864 from August to October, 2008. Four morphospecies, similar to those reported in Java and Japan by Kidder [Parasitologica, 29 :163–205], were identified: Clevelandella constricta, Clevelandella nipponensis, Clevelandella parapanesthiae, and Clevelandella panesthiae. Small subunit rRNA gene sequences assigned all species to a “family” clade that was sister to the clade of species assigned to the Family Nyctotheridae in the Order Clevelandellida. Genetics and morphology were consistent for the first three Clevelandella species, but isolates assigned to C. panesthiae were assignable to three different genotypes, suggesting that this may be a cryptic species complex.     

Five Cyanophora (Cyanophorales,Glaucophyta) species delineated based on morphological and molecular data

Cyanophora is an important glaucophyte genus of unicellular biflagellates that may have retained ancestral features of photosynthetic eukaryotes. The nuclear genome of Cyanophora was recently sequenced, but taxonomic studies of more than two strains are lacking for this genus. Furthermore, no study has used molecular methods to taxonomically delineate Cyanophora species. Here, we delimited the species of Cyanophora using light and electron microscopy, combined with molecular data from several globally distributed strains, including one newly established. Using a light microscope, we identified two distinct morphological groups: one with ovoid to ellipsoidal vegetative cells and another with dorsoventrally flattened or broad, bean‐shaped vegetative cells containing duplicated plastids. Our light and scanning electron microscopy clearly distinguished three species with ovoid to ellipsoidal cells (C. paradoxa Korshikov, C. cuspidata Tos.Takah. & Nozaki sp. nov., and C. kugrensii Tos.Takah. & Nozaki sp. nov.) and two species with broad, bean‐shaped cells (C. biloba Kugrens, B.L.Clay, C.J.Mey. & R.E.Lee and C. sudae Tos.Takah. & Nozaki sp. nov.) based on differences in cell shape and surface ornamentations of the vegetative cells under the field‐emission scanning electron microscope. Molecular phylogenetic analyses of P700 chl a apoprotein A2 (psaB) genes and internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (rDNA), as well as a comparison of secondary structures of nuclear rDNA ITS‐2 and genetic distances of psaB genes, supported the delineation of five morphological species of Cyanophora.     

Euchlorocystis gen. nov. and Densicystis gen. nov., Two New Genera of Oocystaceae Algae from High‐altitude Semi‐saline Habitat (Trebouxiophyceae,Chlorophyta)

The Oocystaceae family is generally considered to contain common freshwater eukaryotic microalgae, and few are reported living in semi‐saline habitats. Our latest ecological survey in Qinghai Lake and Angzicuo Lake, both large, closed, high‐altitude, semi‐saline lakes located on the Qinghai‐Tibet plateau in China, revealed Oocystaceae species as a dominant group among plankton. Since limited knowledge exists about semi‐saline species in the Oocystaceae family, a taxonomical study was carried out using morphological and phylogenetic methods. Using this approach, four new strains of Oocystaceae were identified and successfully cultured in the lab. Molecular results correlated with morphological characters and resolved these species into at least three genera. A new genus, Euchlorocystis, with type species Euchlorocystis subsalina, is described here as having the distinctive morphology of multiple pyrenoids per chloroplast among Oocystaceae, and an independent phylogenetic position at the base of the Oocystaceae. Similarly, the genus Densicystis, with type species Densicystis glomerata, is newly proposed here as having a unique colony morphology of dozens or hundreds of little cells tightly embedded in ellipsoid to round mucilage masses. Oocystis marina, originally described from the Baltic Sea, was also identified in Qinghai Lake and Angzicuo Lake and phylogenetically positioned in the semi‐saline clade of the Oocystaceae. The result that a marine species was detected in the closed inland lakes implies a further need to reevaluate the origins of these species.