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The phylogenetic position of a freshwater green alga, Aegagropila linnaei (Cladophorales, Ulvophyceae), was investigated using nuclear 18S rRNA gene sequences. This alga has usually been called Cladophora aegagropila (L.) Rabenhorst or Cladophora sauteri (Nees ex Kütz.) Kütz. Based on morphology, it was formerly classified into the section Aegagropila or into the subgenus Aegagropila, together with several marine species of the genus Cladophora. This classification is not supported by the present phylogenetic analyses in which two very distinct Cladophorales clades are recognized. Aegagropila linnaei groups together in a well‐supported clade with Cladophora sp., Pithophora sp., Chaetomorpha okamurae, Arnoldiella conchophila, Wittrockiella lyallii, and Cladophora conchopheria. Aegagropila linnaei and its closely related species share some ultrastructural and biochemical characteristics, like pyrenoid structure, carotenoid composition, and cell wall composition. Freshwater species, included in the analysis, were located in two distantly related lineages, indicating that adaptation from a marine to a freshwater habitat has happened at least twice independently in the Cladophorales.

Photosynthetic pigments were analyzed by HPLC for 27 samples of the Cladophorales (Ulvophyceae, Chlorophyta). The carotenoid compositions of the examined algae were classified into three types based on the final compound of biosynthesis of the a-carotene branch: lutein type, characterized by containing lutein as a major carotenoid and lacking loroxanthin and siphonaxanthin; loroxanthin type, characterized by containing loroxanthin and lacking siphonaxanthin; and siphonaxanthin type, characterized by containing siphonaxanthin. We constructed molecular phylogenetic tree of the species examined in the present study using 18S rRNA gene sequences and mapped the carotenoid types of the species onto the tree. The molecular phylogenetic analysis divided the Cladophorales into two major clades, clade 1 and Aegagropila-clade (clade 2), and divided clade 1 into subclade 1-1 and subclade 1-2. All the examined species positioned in the Aegagropila-clade and those of the subclade 1-1 belonged to the loroxanthin type, whereas both lutein type and siphonaxanthin type appeared only in the subclade 1-2. The clades delineated by molecular phylogenetic analysis were congruent with distribution of the carotenoid types, indicating that the carotenoid types are of taxonomic significance in the Cladophorales.Considering the distribution pattern of these carotenoid types and minimum state changes in the Cladophorales, we concluded that the loroxanthin type was the primitive (plesiomorphic) state and the siphonaxanthin type and lutein type appeared in the subclade 1-2 as advanced (apomorphic) state within this order and suggested that the cladophoralean siphonaxanthin type would have been secondarily acquired.

Summer distributions of the invasive signal crayfish (Pacifastacus leniusculus) were investigated in relation to physicochemistry in a Kusiro Moor marsh and its inflows and outflows in northern Japan. Maximum crayfish abundance and biomass were 1.04 individuals/m 2 and 3.56 g dry mass (DM)/m 2 in littoral marsh habitats, and 5.84 individuals/m 2 and 13.48 g DM/m 2 in stream habitats. Classification tree analysis was used to predict crayfish occurrence at 102 sites from all habitats (i.e. littoral marsh, pelagic marsh and stream) while regression tree analyses were used to predict crayfish abundance at littoral marsh and stream sites separately. The classification tree showed that crayfish occurrence was primarily determined by undercut bank volume regardless of habitat identity. When undercut bank volume was <0.0054 m 3 , crayfish were predicted to be absent at marsh sites, but expected to occur at stream sites where pH and water temperature exceeded 6.5 and 14.3°C, respectively. The regression tree using only littoral marsh sites showed that undercut bank volume, followed by dissolved oxygen level, determined the splits of the tree. Crayfish abundance was highest when undercut bank volume was >0.61 m 3 , and moderately high when dissolved oxygen was >9.09 mg/l and undercut bank volume was <0.61 m 3 . On the other hand, the regression tree using only stream sites showed that water temperature was the major predictor that determined the splits. We discuss the roles of physicochemical factors as limiting factors of the distribution pattern of the invasive crayfish.

Aim Aegagropila linnaei is a freshwater macroalga that is generally regarded as a rare species. It is apparently absent from large but seemingly suitable areas of the Northern Hemisphere, implying a limited dispersal potential and an imprint of Pleistocene glaciations in its biogeography. However, despite the popularity of its enigmatic lake ball-form, detailed biogeographical studies of A. linnaei have never been conducted. The main means of reproduction of A. linnaei is fragmentation and akinetes are not formed, supporting the assumption of limited dispersal capacity. The aim of this study was to reconstruct the biogeography of A. linnaei, and to identify possible refugia during glaciations, as well as to evaluate dispersal potential by quantitative desiccation experiments.Location Palaearctic.Methods The current distribution of A. linnaei was inferred from herbarium specimens, literature data and recent field observations. All herbarium specimens were morphologically re-examined. Desiccation experiments were performed with vegetative filaments of three isolates of A. linnaei, as no specialized resistant stages are known. For comparison, the widespread freshwater algae Cladophora glomerata and Rhizoclonium sp. were included. Internal transcribed spacer (ITS) ribosomal DNA sequences were generated and a ribotype network was constructed.Results Aegagropila linnaei was recorded from 283 locations in freshwater and brackish environments. The majority of locations were in central and northern Europe in previously glaciated areas. Desiccation experiments showed that A. linnaei is very susceptible to desiccation. Based on ITS sequences of 34 samples, five different ribotypes were identified. Four of these ribotypes had a restricted distribution. Aegagropila linnaei represents a single species with little genetic variation (0.1-0.5%).Main conclusions This is the most comprehensive study of this species so far, reporting many new locations and tackling several taxonomic problems. Few additional finds were made from North America, and the origin of A. linnaei is inferred to be in Asia. The highest density of its present-day locations is in previously glaciated areas in Europe, where glacial ice-dammed lakes might have functioned as refugia. Low effective long-distance dispersal capacity is inferred, based on high susceptibility to desiccation and its modes of dispersal.