Phylogeography of the freshwater red alga Sirodotia (Batrachospermales,Rhodophyta) in Brazil

Considering the lack of knowledge on genetic variation on members of the freshwater red algal of the order Batrachospermales in tropical regions, phylogeographic patterns in Sirodotia populations were investigated using two mitochondrial regions: the cox2‐3 spacer and partial cox1 gene (barcode). Individuals identified as Sirodotia delicatula were analyzed from 14 stream segments across its distribution in Brazil. Phylogenetic analyses based on the ribulose‐1,5‐bisphosphate carboxylase/oxygenase large sub‐unit gene showed three clades, one representing S. delicatula, from all locations in southeastern Brazil and other regions from Brazil. The remaining samples formed two clades, which were highly divergent and distantly positioned from those of S. delicatula: 2.5–2.7% and 3.4–3.7%. This level of variation would warrant the species split of these taxa from mid‐western Brazil. A total of eight cox2‐3 spacer and nine cox1 haplotypes were observed among the 122 individuals studied. One location had two cox2‐3 haplotypes and three locations had two cox1 haplotypes; all others had a single dominant haplotype each. The existence of high intraspecific genetic variation among individuals of distinct locations (several haplotypes), but little variation within a location seems to be a pattern for the Batrachospermales. Haplotype networks showed low variation among the haplotypes from southeastern Brazil (10 locations with divergence of 0.3–1.1% for cox2‐3, 0.1–0.3% for cox1) and high variation among the haplotypes from the mid‐west region (four locations, 4.0–9.3% for cox2‐3, 6.2–8.4% for cox1). Thus, the present data clearly suggest the existence of cryptic species in Sirodotia in Brazil.     

Phylogeographic analysis of the red seaweed Palmaria palmata reveals a Pleistocene marine glacial refugium in the English Channel

Phylogeography has provided a new approach to the analysis of the postglacial history of a wide range of taxa but, to date, little is known about the effect of glacial periods on the marine biota of Europe. We have utilized a combination of nuclear, plastid and mitochondrial genetic markers to study the biogeographic history of the red seaweed Palmaria palmata in the North Atlantic. Analysis of the nuclear rDNA operon (ITS1-5.8S-ITS2), the plastid 16S-trnI-trnA-23S-5S, rbcL-rbcS and rpl12-rps31-rpl9 regions and the mitochondrial cox2-3 spacer has revealed the existence of a previously unidentified marine refugium in the English Channel, along with possible secondary refugia off the southwest coast of Ireland and in northeast North America and/or Iceland. Coalescent and mismatch analyses date the expansion of European populations from approximately 128,000 BP and suggest a continued period of exponential growth since then. Consequently, we postulate that the penultimate (Saale) glacial maximum was the main event in shaping the biogeographic history of European P. palmata populations which persisted throughout the last (Weichselian) glacial maximum (c. 20,000 BP) in the Hurd Deep, an enigmatic trench in the English Channel.     

Gracilaria species (Gracilariaceae, Rhodophyta) from southeastern Australia, including a new species, Gracilaria perplexa sp. nov.: Morphology, molecular relationships and agar content

Select species of the agarophyte Gracilaria were studied from southeastern Australia. The morphology and anatomy of species is described and molecular relations are inferred based on plastid and mitochon‐drial DNA sequence data. Agar yields and qualities are determined for each species. Gracilaria chilensis, found in Tasmania and Victoria, is morphologically and molecularly similar to G. chilensis from New Zealand and Chile and has low agar yields of 11–16%. Gracilaria cliftonii from Victoria, has high crude agar yield (52%) and is molecularly uniform. Gracilaria perplexa sp. nov., known only from Botany Bay, New South Wales, has an agar yield of 39%. The agar of G. perplexa is unusual in requiring the addition of 0.1 mol L^?1 NaCl for alcohol precipitation and is cold‐water (25°C) soluble because of the very high sulfate ester content. Molecular phylogeny shows that G. perplexa is closely related to Gracilaria preissiana from western Australia, but differs from the latter in its reduced branching and narrower more terete axes.     

In vivo therapeutic potentiality of red seaweed,Asparagopsis (Bonnemaisoniales,Rhodophyta) in the treatment of Vibriosis in Penaeus monodon Fabricius

The crude extract of the red seaweed, Asparagopsis sp. was evaluated for in vivo antibacterial activity against the shrimp vibrio pathogens. The algal extract was rationalized with commercial shrimp feed and orally administered for different duration of time followed by the artificial bacterial challenge experiment. In dose titration experiments, the oral administration of Asparagopsis sp. at a dosage of 850 mg kg^–1 of biomass was highly efficacious in the treatment of natural infestations of Vibriosis in Penaeus monodon. The results of the confirmatory dose experiment revealed that the prophylactic treatment with moderate dose of 850 mg kg^–1 of biomass day^–1 for four weeks followed by 14 days of post infection therapy was highly effective in controlling Vibrio infection in shrimps. Moreover, results of the percent survival index and microbiological analysis clearly show that Asparagopsis extract incorporated medicated feed had broad therapeutic potential for managing shrimp Vibriosis. In addition, in vivo trials and results obtained in this work are based on the crude organic extract sourced from an unidentified Asparagopsis cryptic lineage, therefore further molecular analysis to identify the species will be required.     

Is the tetrasporophyte of Asparagopsis armata (Bonnemaisoniales) limited by inorganic carbon in integrated aquaculture?1

Seaweeds cultivated in traditional land‐based tank systems usually grow under carbon‐limited conditions and consequently have low production rates, if no costly artificial source of inorganic carbon is supplied. In integrated aquaculture, the fish effluents provide an extra source of dissolved inorganic carbon (DIC) to seaweeds due to fish respiration. To evaluate if the tetrasporophyte of Asparagopsis armata (Harv.) F. Schmitz (the Falkenbergia stage) is carbon limited when cultivated with effluents of a fish (Sparus aurata) farm in southern Portugal, we characterized the DIC forms in the water, assessed the species photosynthetic response to the different DIC concentrations and pH values, and inferred for the presence of a carbonic anhydrase (CA)–mediated mechanism. Results showed that A. armata relies mainly on CO₂ to meet photosynthetic needs. Nevertheless, from pH 7.5 upward, the CO₂ supply to RUBISCO seems to derive also from the external dehydration of HCO₃^– mediated by CA. The contribution of this mechanism was essential for A. armata to attain fully saturated O₂‐evolution rates at the natural seawater DIC concentration (2–2.2 mM) and pH values (～8.0). We revealed in this study that seaweeds cultivated in fish‐farm effluents benefit not only from a rich source of ammonia but also from an important and free source of DIC for their photosynthesis. If supplied at relatively high turnover rates (～100 vol · d^?1), fish‐farm effluents provide enough carbon to maximize the photosynthesis and growth even for species with low affinity for HCO₃^–, avoiding the artificial and costly supply of inorganic carbon to seaweed cultures.     

Phylogeography of a subterranean amphipod reveals cryptic diversity and dynamic evolution in extreme environments

Extreme conditions in subsurface are suspected to be responsible for morphological convergences, and so to bias biodiversity assessment. Subterranean organisms are also considered as having poor dispersal abilities that in turn generate a large number of endemic species when habitat is fragmented. Here we test these general hypotheses using the subterranean amphipod Niphargus virei. All our phylogenetic analyses (Bayesian, maximum likelihood and distance), based on two independent genes (28S and COI), revealed the same tripartite structure. N. virei populations from Benelux, Jura region and the rest of France appeared as independent evolutionary units. Molecular rates estimated via global or Bayesian relaxed clock suggest that this split is at least 13 million years old and accredit the cryptic diversity hypothesis. Moreover, the geographical distribution of these lineages showed some evidence of recent dispersal through apparent vicariant barrier. In consequence, we argue that future analyses of evolution and biogeography in subsurface, or more generally in extreme environments, should consider dispersal ability as an evolving trait and morphology as a potentially biased marker.     

Microsatellite markers in an invasive strain of Asparagopsis taxiformis (Bonnemaisoniales, Rhodophyta): insights in ploidy level and sexual reproduction

Eight polymorphic nuclear microsatellite loci were identified from the invasive Indo-Pacific Mediterranean strain of Asparagopsis taxiformis. Microsatellite markers were tested against a panel of specimens collected along the Italian (Elba, Naples) and Californian (Catalina Island) coasts, all belonging to the same mitochondrial lineage. In addition, we used Hawaiian specimens, belonging to a closely related mitochondrial lineage. The markers amplified in all of the specimens but failed consistently in thalli of two more distantly related mitochondrial lineages of A. taxiformis as well as in specimens belonging to the sister species Asparagopsis armata. Since haploid female individuals among the Mediterranean specimens contained cystocarps, genotyping was performed on supposedly haploid female specimens and supposedly diploid cystocarps separately. As expected, external allelic contribution was detected in the cystocarps. However, even after removal of these reproductive structures, gametophyte thalli exhibited patterns consisting of up to three alleles in all of the tested populations indicating polyploidy. An elevated number of distinct genotypes (up to 85%) were found per population, suggesting high intra-population variation. Results showed high genetic similarity between the two Mediterranean populations screened and lower similarity between these two and the Californian one within the same mitochondrial lineage. Lowest similarity was found between these three and the Hawaiian population belonging to the other related mitochondrial lineage 1.     

Genetic and morphological variation in an ecosystem engineer,Lithophyllum byssoides (Corallinales,Rhodophyta)

Lithophyllum byssoides is a common coralline alga in the intertidal zone of Mediterranean coasts, where it produces biogenic concretions housing a high algal and invertebrate biodiversity. This species is an ecosystem engineer and is considered a target for conservation efforts, but designing effective conservation strategies currently is impossible due to lack of information about its population structure. The morphological and molecular variation of L. byssoides was investigated using morphoanatomy and DNA sequences (psbA and cox2,3) obtained from populations at 15 localities on the Italian and Croatian coasts. Lithophyllum byssoides exhibited a high number of haplotypes (31 psbA haplotypes and 24 cox2,3 haplotypes) in the central Mediterranean. The psbA and cox2,3 phylogenies were congruent and showed seven lineages. For most of these clades, the distribution was limited to one or a few localities, but one of them (clade 7) was widespread across the central Mediterranean, spanning the main biogeographic boundaries recognized in this area. The central Mediterranean populations formed a lineage separate from Atlantic samples; psbA pair‐wise divergences suggested that recognition of Atlantic and Mediterranean L. byssoides as different species may be appropriate. The central Mediterranean haplotype patterns of L. byssoides were interpreted as resulting from past climatic events in the hydrogeological history of the Mediterranean Sea. The high haplotype diversity and the restricted spatial distribution of the seven lineages suggest that individual populations should be managed as independent units.     

Phylogeography of the common ragworm Hediste diversicolor (Polychaeta: Nereididae) reveals cryptic diversity and multiple colonization events across its distribution

Previous studies on the common ragworm Hediste diversicolor (Polychaeta: Nereididae) revealed a marked genetic fragmentation across its distribution and the occurrence of sibling taxa in the Baltic Sea. These results suggested that the phylogeographic patterns of H. diversicolor could reflect interactions between cryptic differentiation and multiple colonization events. This study aims to describe the large-scale genetic structuring of H. diversicolor and to trace the phylogeographic origins of the genetic types described in the Baltic Sea. Samples of H. diversicolor (2 <  n  < 28) were collected at 16 locations across the NE Atlantic coasts of Europe and Morocco and in the Mediterranean, Black and Caspian Seas and sequenced at two mitochondrial gene fragments (COI and cyt b , 345 and 290 bp, respectively). Bayesian analyses revealed deep phylogeographic splits yielding three main clades corresponding to populations (i) from the NE Atlantic coasts (from Germany to Morocco) and from part of the Western Mediterranean, (ii) from the Mediterranean Sea, and (iii) from the Black and Caspian Seas. These clades are further divided in well-supported subclades including populations from different regions of NE Atlantic and Mediterranean (i.e. Portugal/Morocco, Western Mediterranean, Adriatic Sea). The Baltic Sea comprises three sympatric lineages sharing a common evolutionary history with populations from NE Atlantic, Western Mediterranean and Black/Caspian Seas, respectively. Hence, the current patterns of genetic structuring of H. diversicolor appear as the result of allopatric isolation, multiple colonization events and possible adaptation to local environmental conditions.     

Reproductive ecology of the freshwater red alga Batrachospermum delicatulum (Batrachospermales, Rhodophyta) in three tropical streams

Batrachospermum delicatulum specimens from three stream segments were analyzed from a tropical region in south‐ eastern Brazil (20°18′– 20°49′S, 49°13′– 49°46′W). Physical and chemical parameters and the spatial placement of thalli were investigated along with the reproductive characteristics of the gametophytic phase. Sequence data of the cox 2‐ 3 spacer region was also utilized to evaluate genetic variation in individuals within and among stream segments. Gametophyte occurred under relatively diverse environmental conditions, whereas thalli abundance was weakly or not correlated to environmental variables within the stream segments. All specimens examined were dioecious. The ratio of male/female plants was relatively low (0.5 to 1.3) and male plants tended to occur as clumps (two or three plants together). High reproductive success was observed, as indicated by the occurrence of 100% fertilized (carposporophytic) female plants. This is similar to previous reports for this and other dioecious species, which is remarkable considering the relatively low proportion of male/female plants. Results support the two hypotheses to explain the high reproductive success in dioecious species. The occurrence of male plants in clumps was evidence for a strict spatial relationship (i.e. male plants located in upstream position of female plants in order to release spermatia, which would be carried by eddies through female plants). In contrast, the occurrence of male and female plants adjacent to each other allowed outcrossing among neighboring plants with intermingled male and female branches, which seemed more applicable to some situations (low turbulence habitats). The cox 2‐ 3 spacer region from the 18 individuals sequenced was 376 bp and the DNA sequence was identical with no base pair substitutions. Likewise, a previous study of another Batrachospermum species showed that the same haplotypes were present in all stream segments from the same drainage basin, even though the stream segments were a considerable distance apart. Short distance dispersal either by small birds or waterway connectivity might explain these findings.     

Global sampling reveals low genetic diversity within Compsopogon (Compsopogonales,Rhodophyta)

Twenty-five specimens of the freshwater red alga Compsopogon were collected from locations in North America, South America, Europe, Asia, Australasia and Oceania, and from an aquarium, with the goal of determining genetic diversity among specimens and ascertaining the number of phylogenetic species. Specimens were morphologically identified as having either the ‘caeruleus’ morphology, with regular polyhedral cortical cells, or the ‘leptoclados’ morphology, with irregular cortical cells with rhizoidal outgrowths. The ‘leptoclados’ morphology has been used by some researchers to distinguish the genus Compsopogonopsis from Compsopogon, or at least to distinguish C. leptoclados from other Compsopogon species. Sequence data for the rbcL gene and cox1 barcoding region were obtained for most specimens. In addition, SSU and partial LSU (barcode) rDNA were explored for a few specimens, but all sequences were identical. For the 25 newly generated and eight previously published rbcL gene data, there were seven unique haplotypes, but the sequence divergence was very low (≤7 bp, ≤ 0.7%). One haplotype was widespread, represented by 21 specimens from diverse locations in all regions sampled. Likewise, the 22 new and one previously published cox1 barcode region sequences yielded seven unique haplotypes with little sequence divergence (≤13 bp, ≤ 2.0%). One haplotype was widespread, being shared among 16 specimens from all regions. The combined molecular and morphological data showed no genetic differentiation between the ‘caeruleus’ and ‘leptoclados’ morphologies. The ubiquitous distribution of Compsopogon in tropical/subtropical regions and its low genetic variation are probably facilitated by the alga's ability to tolerate a wide range of stream conditions and its propagation via asexual spores. Given the findings of previous culture-based studies, morphometric research and field observations, coupled with the results of our study, we conclude there is only a single monospecific genus worldwide and that the species is correctly called C. caeruleus, since this is the oldest validly published name; all other previously described species of Compsopogon and Compsopogonopsis are synonyms.     

Phylogeography of Padina boryana (Dictyotales,Phaeophyceae) around the Thai-Malay Peninsula

The Indo-Pacific Ocean is a biodiversity hotspot for marine organisms. In this area, most of the research has focused on marine animals, such as reef fish, molluscs and other associated coral fauna, but very little has been done on macroalgae. The Thai-Malay Peninsula is an important north–south barrier in this area, which faces two different oceans – the Indian Ocean and the Pacific Ocean. This study aims to investigate genetic distribution patterns of Padina boryana Thivy around the Thai-Malay Peninsula, where it is common. Three DNA marker regions, the mitochondrion-encoded cytochrome c oxidase subunit 3 gene (cox3); the plastid rbcL, and the nuclear internal transcribed spacer 2 (ITS2) were used to evaluate genetic diversity and the relationships within and between populations. Samples were collected from both the Andaman Sea and Gulf of Thailand sides of the peninsula. Parsimony networks and maximum likelihood and Bayesian analyses showed clearly that there are two separated P. boryana lineages, one restricted to the Gulf of Thailand and the other to the Andaman Sea and other areas of the Indo-Pacific. The effect of different ocean currents along the Andaman Sea and Gulf of Thailand may have shaped these populations of P. boryana. This phylogeographic separation, based on persistent currents in the area, may affect other marine organisms along the Thai peninsula.     

Circumscription of species in the genus Sirodotia (Batrachospermales,Rhodophyta) based on molecular and morphological data

Species level taxonomy and phylogeographical distribution patterns in the freshwater rhodophyte Sirodotia are resolved through phylogenetic inferences based on rbcL and cox2–3 sequence data. Previous studies focused on the taxonomy of specific Sirodotia species or the distributions across a limited geographical region. Our molecular phylogenies included samples attributable to five recognized Sirodotia species and include collections from Australia, Brazil, Costa Rica, Canada, Finland, Mexico, New Zealand, South Africa and the United States. Both rbcL and cox2–3 phylogenies inferred S. suecica, S. tenuissima and S. goebelii as a monophyletic group with little sequence divergence. This result supports the synonymy of S. tenuissima and S. goebelii with S. suecica (the species name with priority). Within this clade, samples collected from Australia and New Zealand formed a monophyletic group with no other discernible phylogeographical patterns within S. suecica. This result seems to be somewhat unusual in the Batrachospermales, as other species have shown greater genetic variation among geographically distant locations. As in previous studies, S. huillensis and S. delicatula were inferred as a separate species based on the rbcL phylogeny, supporting the current taxonomy. A specimen of S. aff. huillensis from South Africa, may represent a new species but further research is necessary before it can be designated as such.     

Assessing global range expansion in a cryptic species complex: insights from the red seaweed genus Asparagopsis (Florideophyceae)

The mitochondrial genetic diversity, distribution and invasive potential of multiple cryptic operational taxonomic units (OTUs) of the red invasive seaweed Asparagopsis were assessed by studying introduced Mediterranean and Hawaiian populations. Invasive behavior of each Asparagopsis OTU was inferred from phylogeographic reconstructions, past historical demographic dynamics, recent range expansion assessments and future distributional predictions obtained from demographic models. Genealogical networks resolved Asparagopsis gametophytes and tetrasporophytes into four A. taxiformis and one A. armata cryptic OTUs. Falkenbergia isolates of A. taxiformis L3 were recovered for the first time in the western Mediterranean Sea and represent a new introduction for this area. Neutrality statistics supported past range expansion for A. taxiformis L1 and L2 in Hawaii. On the other hand, extreme geographic expansion and an increase in effective population size were found only for A. taxiformis L2 in the western Mediterranean Sea. Distribution models predicted shifts of the climatically suitable areas and population expansion for A. armata L1 and A. taxiformis L1 and L2. Our integrated study confirms a high invasive risk for A. taxiformis L1 and L2 in temperate and tropical areas. Despite the differences in predictions among modelling approaches, a number of regions were identified as zones with high invasion risk for A. taxiformis L2. Since range shifts are likely climate‐driven phenomena, future invasive behavior cannot be excluded for the rest of the lineages.     

Phylogeography of Asparagopsis taxiformis revisited: Combined mtDNA data provide novel insights into population structure in Japan

Six intraspecific lineages (Lineages 1–6) of Asparagopsis taxiformis have been previously established based on mitochondrial cox2‐cox3 intergenic spacer and a partial cox1 sequences. ‘Lineage 2’ (L2) was suggested to be a recent introduction to the Mediterranean Sea, but its source population has not yet been identified. In order to clarify the nature of northwestern Pacific populations, we performed extensive sampling in Japan (60 individuals from 16 locations) and molecular phylogenetic analyses based on mitochondrial sequences. Sixteen additional individuals, collected from eight locations in the Indo‐Pacific, Caribbean, and Mediterranean regions, were also analyzed. Combined sequence analyses revealed that the Japanese populations only consisted of L2. Out of 19 combined haplotypes identified within L2, two are shared between Japan and the Mediterranean Sea and the Hawaiian Islands, and 12 were identified as endemic to Japan. Genetic analyses of population differentiation suggested that Japanese populations are genetically isolated from the Mediterranean and the Hawaiian populations. A genetic disjunction appears to separate two subpopulations within Japan: one between Toi and Kagoshima and the other between Ojikajima Island and Kagoshima in the Kyushu area.     

Three gene phylogeny of the Thoreales (Rhodophyta) reveals high species diversity

The freshwater red algal order Thoreales has triphasic life history composed of a diminutive diploid “Chantransia” stage, a distinctive macroscopic gametophyte with multi‐axial growth and carposporophytes that develop on the gametophyte thallus. This order is comprised of two genera, Thorea and Nemalionopsis. Thorea has been widely reported with numerous species, whereas Nemalionopsis has been more rarely observed with only a few species described. DNA sequences from three loci (rbcL, cox1, and LSU) were used to examine the phylogenetic affinity of specimens collected from geographically distant locations including North America, South America, Europe, Pacific Islands, Southeast Asia, China, and India. Sixteen species of Thorea and two species of Nemalionopsis were recognized. Morphological observations confirmed the distinctness of the two genera and also provided some characters to distinguish species. However, many of the collections were in “Chantransia” stage rather than gametophyte stage, meaning that key diagnostic morphological characters were unavailable. Three new species are proposed primarily based on the DNA sequence data generated in this study, Thorea kokosinga‐pueschelii, T. mauitukitukii, and T. quisqueyana. In addition to these newly described species, one DNA sequence from GenBank was not closely associated with other Thorea clades and may represent further diversity in the genus. Two species in Nemalionopsis are recognized, N. shawii and N. parkeri nom. et stat. nov. Thorea harbors more diversity than had been recognized by morphological data alone. Distribution data indicated that Nemalionopsis is common in the Pacific region, whereas Thorea is more globally distributed. Most species of Thorea have a regional distribution, but Thorea hispida appears to be cosmopolitan.