Phylogeography of the marine macroalga Sargassum hemiphyllum (Phaeophyceae,Heterokontophyta) in northwestern Pacific

Sargassum hemiphyllum is commonly found in Japan and Korea, with a variety, var. chinense, that is found distributed in the southern Chinese coast. We previously reported distinct genetic differentiation between the two taxa based on the PCR‐RFLP data of plastid RubiscoL‐S spacer. The present study aims at elucidating the phylogeographic pattern of S. hemiphyllum based on more markers in the nuclear and extranuclear genomes, with a view to reveal the occurrence of hybridization. The two allopatrically distributed taxa were found to be genetically distinct in nuclear ITS2, plastidial Rubisco (Rbc) and mitochondrial TrnW_I (Trn) spacers. Their divergence was postulated to be attributable to the vicariant event which resulted from the isolation of the Sea of Japan during the late Miocene (6.58–11.25 Mya). Divergence within both S. hemiphyllum and the chinense variety was observed based on Trn spacer, while the divergence in S. hemiphyllum was further confirmed in Rbc spacer. This divergence appears to correspond to the separation of the Japanese populations between the Sea of Japan and the Pacific that occurred around 0.92–2.88 Mya (the early Pleistocene). The presence of an ITS2 clone resembling var. chinense sequences in a Japanese population of S. hemiphyllum (JpNS) raises the possibility of the introgression of var. chinense individuals into S. hemiphyllum population. Compared to that between S. hemiphyllum and the chinense variety, hybridization among the Japanese and Korean populations of S. hemiphyllum is highly probable as all these individuals share a pool of nuclear ITS2 sequences, possibly attributable to incomplete concerted evolution of ITS2.     

GENETIC STRUCTURE AND EVOLUTION OF SPECIES IN THE MANGROVE GENUS AVICENNIA (AVICENNIACEAE) IN THE INDO-WEST PACIFIC

Allozyme variation in species of the mangrove genus Avicennia was screened in 25 populations collected from 22 locations in the Indo-West Pacific and eastern North America using 11 loci. Several fixed gene differences supported the specific status of Avicennia alba, A. integra, A. marina, and A. rumphiana from the Indo-West Pacific, and A. germinans from the Atlantic-East Pacific. The three varieties of A. marina, var. marina, var. eucalyptifolia, and van australasica, had higher genetic similarities (Nei's I) and no fixed gene differences, confirming their conspecific status. Strong genetic structuring was observed in A. marina, with sharp changes in gene frequencies at the geographical margins of varietal distributions. The occurrence of alleles found otherwise in only one variety, in only immediately adjacent populations of another variety, provided evidence of introgession between varieties. The varieties appear to have diverged recently in the Pleistocene and are apparently not of ancient Cretaceous origin, as suggested earlier. Despite evidence of high degrees of outcrossing, gene flow among populations was relatively low (N_em < 1–2), except where populations were geographically continuous, questioning assumptions that these widespread mangrove species achieve high levels of long-distance dispersal.     

Phylogeography of the fungal pathogen Histoplasma capsulatum

Until recently, Histoplasma capsulatum was believed to harbour three varieties, var. capsulatum (chiefly a New World human pathogen), var. duboisii (an African human pathogen) and var. farciminosum (an Old World horse pathogen), which varied in clinical manifestations and geographical distribution. We analysed the phylogenetic relationships of 137 individuals representing the three varieties from six continents using DNA sequence variation in four independent protein‐coding genes. At least eight clades were idengified: (i) North American class 1 clade; (ii) North American class 2 clade; (iii) Latin American group A clade; (iv) Latin American group B clade; (v) Australian clade; (vi) Netherlands (Indonesian?) clade; (vii) Eurasian clade and (viii) African clade. Seven of eight clades represented genetically isolated groups that may be recognized as phylogenetic species. The sole exception was the Eurasian clade which originated from within the Latin American group A clade. The phylogenetic relationships among the clades made a star phylogeny. Histoplasma capsulatum var. capsulatum individuals were found in all eight clades. The African clade included all of the H. capsulatum var. duboisii individuals as well as individuals of the other two varieties. The 13 individuals of var. farciminosum were distributed among three phylogenetic species. These findings suggest that the three varieties of Histoplasma are phylogenetically meaningless. Instead we have to recognize the existence of genetically distinct geographical populations or phylogenetic species. Combining DNA substitution rates of protein‐coding genes with the phylogeny suggests that the radiation of Histoplasma started between 3 and 13 million years ago in Latin America.     

An introduced population of Chorda asiatica (Chordaceae,Laminariales) in Puget Sound,Pacific coast of North America

Northeastern Pacific Ocean and northwestern Atlantic Ocean populations of Chorda species, which have not been examined in previous phylogenetic studies, were investigated. All specimens that were collected in Hood Canal, Puget Sound, WA, USA, Pacific coast of North America, showed identical ITS‐5.8S rDNA sequences, and they were included in the clade of Japanese Chorda asiatica. With morphological data added to the molecular data, they were identified as C. asiatica and were concluded to be non‐indigenous populations, most likely introduced with oyster spat together with Sargassum muticum. Specimens collected in New York, NY, USA, Atlantic coast of North America, were genetically closest to C. filum from Newfoundland and were identified as C. filum. The genetic divergence of the North Atlantic populations of C. filum was relatively small compared to that of Japanese C. asiatica considering their broader distributional ranges on both sides of the Atlantic.     

MOLECULAR PHYLOGENY OF GRACILARIA SPECIES INFERRED FROM MOLECULAR MARKERS BELONGING TO THREE DIFFERENT GENOMES1

The nucleotide sequence data of molecular markers 18S rRNA, RUBISCO spacer, and cox2‐3 intergenic spacer were integrated to infer the phylogeny of Gracilaria species, collected from the western coast of India, reducing the possibility of misidentification and providing greater phylogenetic resolution. A phylogenetic tree was constructed using cox2‐3 and RUBISCO spacer sequences, exhibiting the same clustering but differing slightly from that of the rRNA‐based phylogenetic tree. The phylogeny inferred from the combined data set confers an analogous pattern of clustering, compared with those of trees constructed from individual data sets. The combined data set resulted in a phylogeny with better resolution, which supported the clade with higher consistency index, retention index, and bootstrap values. It was observed that Gracilaria foliifera (Forssk.) Børgesen is closer to G. corticata (J. Agardh) J. Agardh varieties, while G. salicornia (C. Agardh) E. Y. Dawson and G. fergusonii J. Agardh both originated from the same clade. The position of G. textorii (Suringar) De Toni faltered and toppled between G. salicornia and G. dura (C. Agardh) J. Agardh; however, G. gracilis (Stackh.) M. Steentoft, L. M. Irvine et W. F. Farnham was evidently distant from the rest of the species.     

Biosystematic studies on the genus Heloniopsis (Melanthiaceae) I. Phylogeny inferred from plastid DNA sequences and taxonomic implications

Based on a combined dataset of plastid DNA sequences (atpB‐rbcL, trnG, trnL‐trnL‐trnF, trnK 5' intron and matK) from 60 individuals, we conducted parsimony and likelihood analyses to clarify the phylogenetic relationships among the six species and three varieties that are commonly recognised in Heloniopsis, in addition to the related genera Ypsilandra and Helonias, using Chamaelirium and Chionographis as an outgroup. According to the single most parsimonious tree, which was identical to the maximum‐likelihood tree in topology, Helonias, Ypsilandra and Heloniopsis are all monophyletic with 100% bootstrap support (BS). In Heloniopsis, there are two highly supported clades (BS 94–97%): a clade of Korean species and a clade of Japanese and Taiwanese species. The latter clade comprised the following four subclades (BS 99–100%): 1) H. orientalis var. orientalis, 2) H. orientalis var. breviscapa and var. flavida, 3) H. kawanoi and 4) H. leucantha and H. umbellata. Because subclades 1 and 2 did not form a monophyletic group, and do show clear morphological differences – including nectary position, nectary‐sac structure and leaf margin undulation – they should be distinguished at the species level: H. orientalis for subclade 1 and H. breviscapa for subclade 2. In subclade 2, neither var. breviscapa nor var. flavida was monophyletic; instead, var. breviscapa plus var. flavida (thick‐leaved entity) was monophyletic (BS 62–63%) and var. flavida (thin‐leaved entity) was monophyletic (BS 86–87%). As var. breviscapa and var. flavida (thick‐leaved entity) share basally ± pinkish wide tepals and dark‐coloured thick leaves, in contrast to var. flavida (thin‐leaved entity), which has completely white narrow tepals and light‐coloured thin leaves, the two varieties should may be kept distinct after the merge of var. flavida (thick‐leaved entity) with var. breviscapa.     

DEFINING TAXON BOUNDARIES IN MEMBERS OF THE MORPHOLOGICALLY AND GENETICALLY PLASTIC GENES CAULERPA (CAULERPALES,CHLOROPHYTA)1

Numerous attempts to capture the morphological variability of the genus Caulerpa have resulted in an unstable classification of numerous varieties and formae. In the present study we attempted to test taxon boundaries by investigating morphological and genetic variation within and between seven taxa of Caulerpa, supposedly belonging to four species, sampled at different sites in a Philippine reef system. Using both field and culture observations, we described the relation between the variability of a set of morphological characters and ecological parameters, such as wave exposure, light intensity, and substrate type. Statistical analyses showed that the limits between two (out of three) ecads of the C. racemosa (Forsskål) J. Agardh complex were obscured by the presence of morphological plasticity. Other studied taxa of Caulerpa (i.e. C. cupressoides [Vahl] C. Agardh, C. serrulata [Forsskål] J. Agardh, and two formae of C. sertularioides [S. Gmelin] Howe) could be grouped based on morphology despite the presence of morphological plasticity. Our results indicated a strong association between light intensity and several quantitative morphological variables. Genetic diversity of these taxa was assessed by partial sequencing chloroplast rbcL and tufA genes and the ycf10‐chlB chloroplast spacer. In all phylogenetic analyses, C. serrulata, C. cupressoides, C. sertularioides, and the three ecads of C. racemosa emerged as distinct genetic units. Despite the presence of morphological plasticity and morphological convergence, a subset of morphological characters traditionally used in taxonomic delimitation still had sufficient discriminative power to recognize the terminal phylogenetic clades.     

GLOBAL SYSTEMATIC AND PHYLOGENETIC ANALYSIS OF SARGASSUM IN THE GULF OF MEXICO,CARIBBEAN AND PACIFIC BASIN

Sargassum is one of the most species‐rich genera in the brown algae with over 400 described species worldwide. The bulk of these species occurs in Pacific‐Indian ocean waters with only a small portion found on the Atlantic side of the Isthmus of Panama. Sargassum also has one of the most subdivided and complex taxonomic systems used within the algae. Systematic distinctions within the genus are further complicated by high rates of phenotypic variability in several key morphological characters. Molecular analyses in such systems should allow testing of systematic concepts while providing insights into speciation and evolutionary patterns. Global molecular phylogenetic analyses using both conserved and variable regions of the Rubisco operon (rbcL and rbcL‐IGS‐rbcS) were performed with species from the Gulf of Mexico, Caribbean, and Pacific basin. Results confirm earlier analyses based on rbcL‐IGS‐rbcS from Pacific species at the subgeneric and sectional level while providing additional insights into the systematics and phylogenetics on a global scale. For example, species east of the Isthmus of Panama form a distinct well‐resolved clade within the tropical subgenus. This result in sharp contrast to traditional systematic treatments but provides a window into the evolutionary history of this genus in the Pacific and Atlantic Ocean basins and a possible means to time speciation events.     

AN EXAMINATION OF PACHYMENIA AND AEODES (HALYMENIACEAE,RHODOPHYTA) IN NEW ZEALAND AND THE TRANSFER OF TWO SPECIES OF AEODES IN SOUTH AFRICA TO PACHYMENIA1

A phylogenetic study was conducted of species of Halymeniaceae from New Zealand presently placed in Aeodes or Pachymenia, based on maximum‐likelihood (ML), maximum‐parsimony (MP), and Bayesian analyses of rbcL and nuclear internal transcribed spacer (ITS) rDNA sequences. We used molecular and morphological data in combination with exhaustive sampling of herbarium collections to clarify the taxonomy and distributions of New Zealand members of Pachymenia and Aeodes. Our study confirms the presence of three erect species of Pachymenia on the New Zealand mainland, and we resurrect the name Pachymenia dichotoma J. Agardh for the widely distributed, southernmost species. Species of Aeodes from South Africa are shown to be closely related to Pachymenia carnosa (J. Agardh) J. Agardh, the type species of Pachymenia, and are accordingly transferred to Pachymenia.     

DNA SEQUENCE DATA DEMONSTRATE THE POLYPHYLY OF THE GENUS CYSTOSEIRA AND OTHER SARGASSACEAE GENERA (PHAEOPHYCEAE)1

Phylogenetic relationships in the Sargassaceae were explored using three DNA markers, and the monophyly of its genera was challenged. Nineteen out of 24 currently recognized genera were sampled, representing 63 species. The variable mt23S‐tRNA Val intergenic spacer could only be aligned within genera and could not be used to infer intergeneric relationships. The partial mt23S was also useful to delineate genera and was alignable at the family level but provided few informative characters. Analysis of mt23S DNA sequences together with chloroplast‐encoded psbA sequences resulted in a better resolved phylogeny. Hormophysa was the first genus to branch off within the Sargassaceae, followed by Myriodesma; then the three genera Caulocystis, Carpoglossum, and Scaberia in unresolved order; and then Acrocarpia. The other taxa studied here were divided over three major clades, but there was no branch support for the monophyly of two of these. The genera Bifurcaria, Cystoseira, Halidrys, and Sargassum appeared polyphyletic. The following taxonomic changes are proposed: a new genus Brassicophycus for Bifurcaria brassicaeformis (Kützing) E. S. Barton; reinstatement of the genus Sargassopsis for Sargassum decurrens (R. Brown ex Turner) C. Agardh; reinstatement of the genus Sirophysalis for Indo‐Pacific Cystoseira trinodis (Forsskål) C. Agardh; reinstatement of the genus Polycladia for the western Indian Ocean species Cystoseira indica (Thivy et Doshi) Mairh, Cystoseira myrica (S. G. Gmelin) C. Agardh, and Acystis heinii Schiffner; and reinstatement of the genus Stephanocystis for the North Pacific Cystoseira species and Halidrys dioica N. L. Gardner. The European Cystoseira species should be split into three genera, but no name changes are proposed yet, because diagnostic characters were found only for the clade including the type species. Some evolutionary trends could be discerned from the mt23S + psbA phylogeny.     

POLYMORPHISM IN THE DESMID MICRASTERIAS PINNATIFIDA AND ITS TAXONOMICAL IMPLICATIONS1

A case of polymorphism in the desmid Micrasterias pinnatifida (Kütz.) Ralfs is described. It is based on material collected from the overflow channel of a dam in Caeté, Minas Gerais State. After careful examination of nearly 500 specimens, 26 different morphological expressions of the alga were found which led the authors to make the following deductions: (1) for delimitation of species and infraspecific taxa in desmids it is absolutely necessary to analyze sample populations; (2) the morphological characteristics presently used, for delimination of some varieties of M. Pinnatifida (Kütz.) Ralfs seem to have no taxonomical value if only isolated individuals are examined; (3) the varieties granulata, inflata, and tridentata must be considered with some caution, until further studies are carried out, because they can be merely distinct morphological expressions of M. pinnatifida (Kütz.) Ralfs var. pinnatifida.     

Morphology and molecular phylogeny of coccoid green algae Coelastrella sensu lato (Scenedesmaceae,Sphaeropeales), including the description of three new species and two new varieties

The genus Coelastrella was established by Chodat (Bull. Soc. Bot. Geneve, 13 [1922] 66), and was characterized as being unicellular or in few‐celled aggregations with many longitudinal ribs on the cell wall. Many species of this genus showed strong ability to accumulate carotenoids and oils, so they have recently attracted much attention from researchers due to its potential applicability in the energy and food industries. In this study, a total of 23 strains of Coelastrella were sampled from China, and three new species and two new varieties were described: C. thermophila sp. nov., C. yingshanensis sp. nov., C. tenuitheca sp. nov., C. thermophila var. globulina var. nov., C. rubescens var. oocystiformis var. nov. Besides 18S rDNA and ITS2 sequences, we have newly sequenced the tufA gene marker for this taxon. Phylogenetic analysis combined with morphological studies revealed four morphotypes within the Coelastrella sensu lato clade, which contained the morphotype of original Coelastrella, original Scotiellopsis, Asterarcys, and morphotype of C. vacuolata and C. tenuitheca sp. nov. The relationships between morphological differences and phylogenic diversity based on different markers were discussed. Our results support that 18S rDNA was too conserved to be used a species‐specific or even a genus‐specific marker in this clade. The topology of tufA gene‐based phylogenetic tree had a better match with the morphological findings.     

ESSENTIAL OILS AS TAXONOMIC AIDS IN THE CLASSIFICATION OF DICHANTHIUM PARVIFLORUM

Essential oil components and gross morphological characters are closely correlated in Dichanthium parviflorum (R. Br.) de Wet et Harlan (Gramineae) and related species. Different species, varieties, and geographical races, as well as hybrids between them, can be identified on the basis of absence or presence and quantity of essential oil components. The morphologically variable D. parviflorum was subdivided into four varieties: var. parviflorum, var. capilliflorum (Steud.) de Wet et Harlan comb. nov., var. mutispiculum (Ohwi) de Wet et Harlan comb. nov., and var. spicigerum (S. T. Blake) de Wet et Harlan comb. nov. These varieties differ from each other morphologically in having respectively racemes with 1-4 and awned, 3-5 and awned, 1-2 and awnlass, and 4-10 and awned spikelet pairs per raceme.     

PHYLOGEOGRAPHIC STUDIES IN THE TROPICAL SEAWEED CLADOPHOROPSIS MEMBRANACEA (CHLOROPHYTA,ULVOPHYCEAE) REVEAL A CRYPTIC SPECIES COMPLEX1

The seaweed Cladophoropsis membranacea (Hofman Bang ex. C. Agardh) Børgesen is a widely distributed species on coral reefs and along rocky coastlines throughout the tropics and subtropics. In a recent population‐level survey openface>1600 individuals with eight microsatellite loci, a number of isolates from biogeographically disjunct locations could not be amplified for any of the loci. Nonamplifiable and amplifiable isolates co‐occurred within the Canary Islands, Cape Verde Islands, and in the Caribbean. These unexpected results led to question whether or not C. membranacea is a single species. Phylogenetic relationships were evaluated using rDNA ITS1 and ITS2 sequence comparisons from 42 isolates sampled from a subset of 30 of the 66 locations. Four well‐supported clades were identified. Sequence divergence within clades was <1%, whereas between‐clade divergence was 2%–3%. Intraindividual variation was extremely low with no effects on the analysis. A strong, but imperfect, correspondence was found between ITS clades and amplifiable microsatellite loci. It is concluded that C. membranacea consists of three cryptic species. Using Pacific isolates as an outgroup, the most basal clade included the Central Canary Islands, Cape Verde, and Bonaire (Caribbean) isolates and thus spanned the widest latitude. Two derived sister clades consisted of a southern transtropical group stretching across the SE Caribbean to the Cape Verde Islands and African coast (but not the Canary Islands) and a NE‐Canary Island‐Mediterranean clade that also included the Red Sea. The detection of overlapping biogeographic distributions highlights the importance of ecotypic differentiation with respect to temperature and the importance of shifting sea surface isotherms that have driven periodic extinctions and recolonizations of the Canary Islands—a crossroads of marine floral exchange—since the last glacial maximum.     

Genomic signatures of evolution in Nautilus—An endangered living fossil

Living fossils are survivors of previously more diverse lineages that originated millions of years ago and persisted with little morphological change. Therefore, living fossils are model organisms to study both long‐term and ongoing adaptation and speciation processes. However, many aspects of living fossil evolution and their persistence in the modern world remain unclear. Here, we investigate three major aspects of the evolutionary history of living fossils: cryptic speciation, population genetics and effective population sizes, using members of the genera Nautilus and Allonautilus as classic examples of true living fossils. For this, we analysed genomewide ddRAD‐Seq data for all six currently recognized nautiloid species throughout their distribution range. Our analyses identified three major allopatric Nautilus clades: a South Pacific clade, subdivided into three subclades with no signs of admixture between them; a Coral Sea clade, consisting of two genetically distinct populations with significant admixture; and a widespread Indo‐Pacific clade, devoid of significant genetic substructure. Within these major clades, we detected five Nautilus groups, which likely correspond to five distinct species. With the exception of Nautilus macromphalus, all previously described species are at odds with genomewide data, testifying to the prevalence of cryptic species among living fossils. Detailed F_ST analyses further revealed significant genome‐wide and locus‐specific signatures of selection between species and differentiated populations, which is demonstrated here for the first time in a living fossil. Finally, approximate Bayesian computation (ABC) simulations suggest large effective population sizes, which may explain the low levels of population differentiation commonly observed in living fossils.     

PHYLOGENY OF AULACOSEIRA (BACILLARIOPHYTA) BASED ON MOLECULES AND MORPHOLOGY1

The phylogeny of 67 populations representing 45 species of Aulacoseira Thwaites was estimated by maximum parsimony methods using a combination of nucleotide sequence data and qualitative and quantitative morphological characteristics of the silica cell wall gathered primarily from original observation by LM and SEM. A new type of character using continuous quantitative variables that describe the ontogenetic‐allometric trajectories of cell wall characteristics over the life cycle (size range) of diatoms is introduced. In addition to the 45 Aulacoseira species, the phylogeny also incorporated one Miosira Krammer, Lange‐Bertalot, and Schiller species and two outgroup species (Melosira varians Agardh and Stephanopyxis nipponica Gran & Yendo). Fifteen species, represented by 24 populations, also contained molecular data from the nuclear genome (18S rDNA), and 11 of these species (18 populations) contained data from the chloroplast genome (rbcL) as well, which were sequenced or downloaded from GenBank. The phylogeny of Aulacoseira is composed of five major clades: 1) an A. crenulata (Ehrenburg) Thwaites and A. italica (Ehrenburg) Simonsen clade, which is the most basal; 2) an A. granulata (Ehrenburg) Simonsen complex clade; 3) an A. ambigua (Grunow) Simonsen clade; 4) an A. subarctica (O. Müller) Haworth and A. distans (Ehrenburg) Simonsen clade; and 5) an A. islandica (O. Müller) Simonsen clade that also contained endemic species from Lake Baikal, Siberia and many extinct Aulacoseira taxa. Monophyly of Aulacoseira can only be achieved if Miosira is no longer given separate generic status.     

Origin of the pantropical and nutriceutical Morinda citrifolia L. (Rubiaceae): comments on its distribution range and circumscription

Aim Morinda citrifolia L., commercially known as noni or the Indian mulberry plant, is morphologically variable and the only widely distributed member of the pantropical genus Morinda sensu stricto (Rubiaceae). This large distribution has been attributed partly to the ability of the seeds of the large‐fruited M. citrifolia L. var. citrifolia L. to be transported by oceanic drifting. This form of M. citrifolia var. citrifolia has been predicted to be the progenitor colonizer of the island endemic Morinda species. Using a phylogenetic approach and large sampling of the widespread, large‐fruited M. citrifolia var. citrifolia, we assessed the potential area of origin of M. citrifolia and tested the hypothesis that the large‐fruited M. citrifolia var. citrifolia is an ancestral colonizer. Location Tropics. Methods We performed Bayesian analyses of 22 species of the tribe Morindeae (including 11 individuals of the three currently recognized varieties of M. citrifolia) based on combined nrETS, nrITS, rps16 and trnT–F sequence data. Geographic origins of the studied taxa were mapped onto the Bayesian majority rule consensus tree. Results Nine sequenced individuals of M. citrifolia from diverse geographic locations formed a highly supported clade, which was sister to the Australo‐Micronesian clade that included M. bracteata var. celebica and M. latibracteata. These sister clades are part of the broader Asian, arborescent Morinda clade. We found no support for the current varietal classification of M. citrifolia. Main conclusions Our analyses suggest a Micronesian origin of M. citrifolia. This implies that the large‐fruited M. citrifolia var. citrifolia might well have been present in the Pacific before the arrival of the Micronesian and Polynesian ancestors from Southeast Asia. The wide distribution of this form of M. citrifolia var. citrifolia is attributed partly to the trans‐oceanic dispersal of its buoyant seeds, self‐pollination and its ability to produce flowers and fruits year‐round. The hypothesis that the widespread, large‐fruited M. citrifolia var. citrifolia is the progenitor colonizer of the island endemic Morinda species is inconsistent with its derived position within the Asian, arborescent Morinda clade and with the fact that the nine sampled individuals of M. citrifolia form a clade.