Integrative taxonomy of the Rhinolophus macrotis complex (Chiroptera,Rhinolophidae) in Vietnam and nearby regions

The taxonomic status of Rhinolophus macrotis sensu lato (s.l.) in Vietnam and adjacent territories remains problematic. To address this issue, we performed an integrated study of morphological, acoustic, and genetic characters of R. macrotis s.l. specimens and compared these with sympatric species within the philippinensis group (R. marshalli, R. paradoxolophus, and R. rex). Our results reveal that in addition to a cryptic species of R. macrotis previously found in Jiangxi and Jingmen, China, R. macrotis s.l. in continental Asia includes three further species, namely R. cf. siamensis, R. cf. macrotis, and R. cf. macrotis “Phia Oac.” These four taxa are distinguished from genuine R. macrotis in Nepal and R. siamensis in Thailand by their morphological and/or genetic features. Further taxonomic evaluation of the subspecies of R. macrotis s.l. is needed to determine their affinities with recently recognized cryptic species and to possibly describe new taxa. Our results also show that interspecific divergences in mitochondrial DNA sequences (Cytb and COI genes) among taxa within the philippinensis group (particularly between R. cf. siamensis/R. cf. macrotis and R. rex/R. paradoxolophus) are significantly lower than those of other morphological groups in the genus. These phylogenetic patterns might be explained by recent allopatric speciation or ancient introgression events among ancestors of the taxa during the Pleistocene. However, further investigations including genetic analyses of nuclear genes are needed to test the latter hypothesis.     

Deep‐sea anglerfishes (Lophiiformes: Ceratioidei) from the western North Atlantic: Testing the efficacy of DNA barcodes

The taxonomy of ceratioid fishes is challenging and frequently based on a small number of female specimens described for each species. Twenty ceratioid specimens caught on Flemish Cap and Grand Bank (western North Atlantic), representing 12 species and six families: Ceratias holboelli and Cryptopsaras couesii (Ceratiidae); Himantolophus albinares (Himantolophidae); Melanocetus johnsonii (Melanocetidae); Lophodolos acanthognathus, Oneirodes eschrichtii, Dolopichthys karsteni, and Leptacanthichthys gracilispinis (Oneirodidae); Caulophryne polynema (Caulophrynidae); and Haplophryne mollis, Linophryne brevibarbata, and L. bicornis (Linophrynidae) were identified by examination of morphological characters. DNA barcode sequences, from the 5′ end of the COI mitochondrial gene, were developed for 18 specimens and compared with all ceratioid barcode sequences available in public repositories. The analyses extended the ranges of some quantitative traits for certain species, highlighted the possible existence of cryptic species in C. couesii with distinct ranges in the Atlantic and Pacific oceans, and indicated a close relationship between Bertella and Dolopichthys meriting further attention. The authoritative identification of the 18 voucher specimens made possible detection of erroneous identifications of some sequences extracted from the repositories and highlighting of taxonomic conflicts that should be the subject of future studies.     

The Halomonhystera disjuncta population is homogeneous across the Håkon Mosby mud volcano (Barents Sea) but is genetically differentiated from its shallow‐water relatives

The deep sea has a high biodiversity and a characteristic bathyal fauna. Earlier evidence suggested that at least some shallow‐water species invaded the ecosystem followed by radiation leading to endemic deep‐sea lineages with a genetic and/or morphological similarity to their shallow‐water counterparts. The nematode Halomonhystera disjuncta has been reported from shallow‐water habitats and the deep sea [Håkon Mosby mud volcano (HMMV)], but the morphological features and the phylogenetic relationships between deep‐sea and shallow‐water representatives remain largely unknown. Furthermore, nothing is known about the genetic structure of the H. disjuncta population within the HMMV. This study is the first integrative approach in which the morphological and phylogenetic relationships between a deep‐sea and shallow‐water free‐living nematode species are investigated. To elucidate the phylogenetic relationships, we analysed the mitochondrial gene Cytochrome oxidase c subunit I (COI) and three nuclear ribosomal genes (Internal Transcribed Spacer region, 18S and the D2D3 region of 28S). Our results show that deep‐sea nematodes comprise an endemic lineage compared to the shallow‐water representatives with different morphometric features. COI genetic divergence between the deep‐sea and shallow‐water specimens ranges between 19.1% and 25.2%. Taking these findings into account, we conclude that the deep‐sea form is a new species. amova revealed no genetic structure across the HMMV, suggesting that nematodes are able to disperse efficiently in the mud volcano.     

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.     

Cryptic speciation in the field vole: a multilocus approach confirms three highly divergent lineages in Eurasia

Species are generally described from morphological features, but there is growing recognition of sister forms that show substantial genetic differentiation without obvious morphological variation and may therefore be considered ‘cryptic species’. Here, we investigate the field vole (Microtus agrestis), a Eurasian mammal with little apparent morphological differentiation but which, on the basis of previous sex‐linked nuclear and mitochondrial DNA (mtDNA) analyses, is subdivided into a Northern and a Southern lineage, sufficiently divergent that they may represent two cryptic species. These earlier studies also provided limited evidence for two major mtDNA lineages within Iberia. In our present study, we extend these findings through a multilocus approach. We sampled 163 individuals from 46 localities, mainly in Iberia, and sequenced seven loci, maternally, paternally and biparentally inherited. Our results show that the mtDNA lineage identified in Portugal is indeed a distinct third lineage on the basis of other markers as well. In fact, multilocus coalescent‐based methods clearly support three separate evolutionary units that may represent cryptic species: Northern, Southern and Portuguese. Divergence among these units was inferred to have occurred during the last glacial period; the Portuguese lineage split occurred first (estimated at c. 70 000 bp ), and the Northern and Southern lineages separated at around the last glacial maximum (estimated at c. 18 500 bp ). Such recent formation of evolutionary units that might be considered species has repercussions in terms of understanding evolutionary processes and the diversity of small mammals in a European context.     

Eco-physiological characteristic of some common caprellid species in the Possjet Bay (the Japan Sea)

Parameters of equations relating wet and dry body weight to length have been calculated for four species of caprellids from the Japan Sea (Possjet Bay), specifically Caprella cristibrachium, C. kroyeri, C. penantis, and C. bispinosa. A common equation was obtained for oxygen consumption rate in relation to body weight. It was shown that within the Order Amphipoda the metabolic rate of caprellids is 1.5 times lower than that of gammarids. The differences in the fecundity of gravid females of four species and females of one species in the spring and summer, respectively, are shown. At the same time the relationship between number of eggs per female and wet body weight of female is approximated by one equation. The equation obtained shows that caprellids represent an ecologically homogeneous group.     

Phylogeny of Gracilariaceae (Rhodophyta): evidence from plastid and mitochondrial nucleotide sequences

Gracilariaceae are mostly pantropical red algae and include ~230 species in seven genera. Infrafamilial classification of the group has long been based on reproductive characters, but previous phylogenies have shown that traditionally circumscribed groups are not monophyletic. We performed phylogenetic analyses using two plastid (universal plastid amplicon and rbcL) and one mitochondrial (cox1) loci from a greatly expanded number of taxa to better assess generic relationships and understand patterns of character distributions. Our analyses produce the most well‐supported phylogeny of the family to date, and indicate that key characteristics of spermatangia and cystocarp type do not delineate genera as commonly suggested. Our results further indicate that Hydropuntia is not monophyletic. Given their morphological overlap with closely related members of Gracilaria, we propose that Hydropuntia be synonymized with the former. Our results additionally expand the known ranges of several Gracilariaceae species to include Brazil. Lastly, we demonstrate that the recently described Gracilaria yoneshigueana should be synonymized as G. domingensis based on morphological and molecular characters. These results demonstrate the utility of DNA barcoding for understanding poorly known and fragmentary materials of cryptic red algae.     

Congruent population genetic structure but differing depths of divergence for three alpine stoneflies with similar ecology and geographic distributions

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DNA barcoding of genus Toxoptera Koch (Hemiptera: Aphididae): Identification and molecular phylogeny inferred from mitochondrial COI sequences

Abstract Identification of aphid species is always difficult due to the shortage of easily distinguishable morphological characters. Aphid genus Toxoptera consists of species with similar morphology and similar to Aphis in most morphological characters except the stridulatory apparatus. DNA barcodes with 1 145 bp sequences of partial mitochondrial cytochrome‐coxidase I (COI) genes were used for accurate identification of Toxoptera. Results indicated mean intraspecific sequence divergences were 1.33%, whereas mean interspecific divergences were greater at 8.29% (0.13% and 7.79% if T. aurantii 3 and T. aurantii 4 are cryptic species). Sixteen samples were distinguished to four species correctly by COI barcodes, which implied that DNA barcoding was successful in discrimination of aphid species with similar morphology. Phylogenetic relationships among species of this genus were tested based on this portion of COI sequences. Four species of Toxoptera assembled a clade with low support in maximum‐parsimony (MP) analysis, maximum‐likelihood (ML) analysis and Bayesian phylogenetic trees, the genus Toxoptera was not monophyletic, and there were two sister groups, such as T. citricidus and T. victoriae, and two clades of T. aurantii which probably presented cryptic species in the genus.     

Nuclear genes (but not mitochondrial DNA barcodes) reveal real species: Evidence from the Brenthis fritillary butterflies (Lepidoptera,Nymphalidae)

Discordance between entities revealed by nuclear versus mitochondrial genes is a common phenomenon in evolutionary and taxonomic studies. However, little attention has been paid to analysis of how such discordant entities correspond to traditional species detected through investigation of their morphology, ecology, and distribution. Here, we used one mitochondrial (COI, DNA barcode fragment) and four nuclear (CAD, Ca‐ATPase, arginine kinase, wg) genes to analyze the genetic structure of the taxonomically well‐studied butterfly genus Brenthis (Lepidoptera, Nymphalidae). Analysis of COI revealed multiple diverged allopatric and sympatric mitochondrial lineages within the known Brenthis species hinting at possible presence of unrecognized cryptic species. However, these multiple‐species hypotheses were not supported by further studies of nuclear genes and phenotypic traits. The discovered mitochondrial lineages did not correspond to the clusters revealed by nuclear genes. Simultaneously, we found a complete congruence between (a) traditional species boundaries, (b) clusters recognized by nuclear genes, and (c) clusters identified via cladistic analysis of phenotypic traits (genitalia and wing pattern characters, ecological preferences, and chromosome numbers). We conclude that in case of the genus Brenthis, nuclear genes rather than mtDNA barcodes reveal real species boundaries. Additionally, we suggest to support each DNA barcode‐based taxonomic conclusion by analysis of phased alleles of nuclear genes, avoiding widely used practice of nuclear and mitochondrial genes concatenation without any examination of interaction of these different types of data.     

Cryptic or pseudocryptic: can morphological methods inform copepod taxonomy? An analysis of publications and a case study of the Eurytemora affinis species complex

Interest in cryptic species has increased significantly with current progress in genetic methods. The large number of cryptic species suggests that the resolution of traditional morphological techniques may be insufficient for taxonomical research. However, some species now considered to be cryptic may, in fact, be designated pseudocryptic after close morphological examination. Thus the “cryptic or pseudocryptic” dilemma speaks to the resolution of morphological analysis and its utility for identifying species. We address this dilemma first by systematically reviewing data published from 1980 to 2013 on cryptic species of Copepoda and then by performing an in‐depth morphological study of the former Eurytemora affinis complex of cryptic species. Analyzing the published data showed that, in 5 of 24 revisions eligible for systematic review, cryptic species assignment was based solely on the genetic variation of forms without detailed morphological analysis to confirm the assignment. Therefore, some newly described cryptic species might be designated pseudocryptic under more detailed morphological analysis as happened with Eurytemora affinis complex. Recent genetic analyses of the complex found high levels of heterogeneity without morphological differences; it is argued to be cryptic. However, next detailed morphological analyses allowed to describe a number of valid species. Our study, using deep statistical analyses usually not applied for new species describing, of this species complex confirmed considerable differences between former cryptic species. In particular, fluctuating asymmetry (FA), the random variation of left and right structures, was significantly different between forms and provided independent information about their status. Our work showed that multivariate statistical approaches, such as principal component analysis, can be powerful techniques for the morphological discrimination of cryptic taxons. Despite increasing cryptic species designations, morphological techniques have great potential in determining copepod taxonomy.     

Morphological evidence that the molecularly determined Ciona intestinalis type A and type B are different species: Ciona robusta and Ciona intestinalis

Ciona intestinalis is considered a widespread and easily recognizable tunicate, the sister group of vertebrates. In recent years, molecular studies suggested that C. intestinalis includes at least two cryptic species, named ‘type A’ and ‘type B’, morphologically indistinguishable. It is dramatic to certify that two different species may be hidden under the name of a species widely used as a model species in biological researches. This raised the problem of identifying diagnostic morphological characters capable of distinguishing these types. We compared the morphology of specimens belonging to the two types and found that only type A specimens possess tunic tubercular prominences, allowing unambiguous discrimination. Remarkably, these structures were already described as distinctive of the Japanese species Ciona robusta, Hoshino and Tokioka, 1967; later synonymized under C. intestinalis (sensu Millar, 1953). In this study, we have confirmed that C. intestinalis type A corresponds to C. robusta. Based on the geographic distribution of C. intestinalis type B, and considering that the original C. intestinalis species was described from North European waters, we determined that C. intestinalis type B corresponds to C. intestinalis as described by Millar in 1953 and possibly to Linnaeus' Ascidia intestinalis L., 1767 for which we have deposited a neotype (from Roscoff, France) and for which we retain the name Ciona intestinalis (Linnaeus, 1767).     

A Molecular Phylogenetic Study of the Tribe Corallineae (Corallinales,Rhodophyta) with an Assessment of Genus‐Level Taxonomic Features and Descriptions of Novel Genera

A multigene phylogeny using COI‐5P (mitochondrial cytochrome c oxidase subunit 1), psbA (PSII reaction center protein D1), and EF2 (elongation factor 2) sequence data for members of the tribe Corallineae was constructed to assess generic boundaries. We determined that traditional reliance on conceptacle position as an indicator of generic affinities in the Corallineae is not supported and taxonomic changes are required. We found that species currently assigned to Pseudolithophyllum muricatum resolved within the Corallineae in all analyses. This is the first record of crustose members in the subfamily Corallinoideae. Further‐more, the genus Serraticardia was polyphyletic; we propose to synonomize Serraticardia with Corallina, transfer the type species S. maxima to Corallina (C. maxima (Yendo) comb. nov.), and describe the new genus Johansenia for S. macmillanii (J. macmillanii (Yendo) comb. nov.). Our molecular data also indicate that species in the genus Marginisporum have evolutionary affinities among species of Corallina and these genera should also be synonymized. This necessitates the combinations C. aberrans (Yendo) comb. nov. for M. aberrans (Yendo) Johansen & Chihara, C. crassissima (Yendo) comb. nov. for M. crassissimum (Yendo) Ganesan, and C. declinata (Yendo) comb. nov. for M. declinata (Yendo) Ganesan. Corallina elongata was divergent from all other members of Corallina and is transferred to a new genus, Ellisolandia (E. elongata (J. Ellis & Solander) comb. nov). In addition, COI‐5P and internal transcribed spacer (ITS) data combined with morphological characters were used to establish that rather than the four Corallina species recognized in Canada, there are nine.     

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.     

Classification of crucigenoid algae: phylogenetic position of the reinstated genus Lemmermannia,Tetrastrum spp. Crucigenia tetrapedia,and C. lauterbornii (Trebouxiophyceae,Chlorophyta)1

The subfamily Crucigenioideae was traditionally classified within the well‐characterized family Scenedesmaceae (Chlorophyceae). Several morpho‐logical revisions and questionable taxonomic changes hampered the correct classification of crucigenoid species resulting in a high number of synonymous genera. We used a molecular approach to determine the phylogenetic position of several Tetrastrum and Crucigenia species. The molecular results were correlated with morphological and ontogenetic characters. Phylogenetic analyses of the SSU rDNA gene resolved the position of Tetrastrum heteracanthum and T. staurogeniaeforme as a new lineage within the Oocystis clade of the Trebouxiophyceae. Crucigenia tetrapedia, T. triangulare, T. punctatum, and T. komarekii were shown to be closely related to Botryococcus (Trebouxiophyceae) and were transferred to Lemmer‐mannia. Crucigenia lauterbornii was not closely related to the other Crucigenia strains, but was recovered within the Chlorella clade of the Trebouxiophyceae.     

Systematics,genetics, and biogeography of intertidal mites (Acari,Oribatida) from the Andaman Sea and Strait of Malacca

This study demonstrates for the first time the presence of marine‐associated mites in the Andaman Sea and Strait of Malacca and reveals a relatively high diversity of these taxa with six species from two different families: Selenoribatidae and Fortuyniidae. Indopacifica, a new genus of Selenoribatidae, is described from Thailand and Malaysia, with two new species, Indopacifica pantai n. sp. and Indopacifica parva n. sp. The genus is characterized by the unique combination of following characters: lacking lamellar ridges, incomplete dorsosejugal suture, fourteen pairs of notogastral setae, and presence of epimeral foveae. A phylogenetic reconstruction based on 18S ribosomal RNA sequences clearly confirms the distinctness of the new genus Indopacifica and places it close to the genus Rhizophobates. The lack of molecular genetic data of possible relatives impedes a clear assessment, and hence, we emphasize the need for further combined approaches using morphological and molecular genetic sequence data. All species show wide distribution areas within this geographic region suggesting that these taxa are good dispersers despite their minute size and wingless body. Molecular genetic data demonstrate recent gene flow between far distant populations of I. pantai n. sp. from the coasts of Thailand and two islands of Malaysia and hence confirm this assumption. The seasonally changing surface currents within this geographic area may favor hydrochorous dispersal and hence genetic exchange. Nevertheless, morphometric data show a slight trend to morphological divergence among the studied populations, whereas this variation is suggested to be a result of genetic drift but also of habitat differences in one population of Alismobates pseudoreticulatus.     

Pseudoparamoeba garorimi n. sp., with Notes on Species Distinctions within the Genus

A new marine species of naked lobose amoebae Pseudoparamoeba garorimi n. sp. (Amoebozoa, Dactylopodida) isolated from intertidal marine sediments of Garorim Bay, Korea was studied with light and transmission electron microscopy. This species has a typical set of morphological characters for a genus including the shape of the locomotive form, type of subpseudopodia and the tendency to form the single long waving pseudopodium in locomotion. Furthermore, it has the same cell surface structures as were described for the type species, Pseudoparamoeba pagei: blister‐like glycostyles with hexagonal base and dome‐shaped apex; besides, cell surface bears hair‐like outgrowths. The new species described here lacks clear morphological distinctions from the two other Pseudoparamoeba species, but has considerable differences in the 18S rDNA and COX1 gene sequences. Phylogenetic analysis based on 18S rDNA placed P. garorimi n. sp. at the base of the Pseudoparamoeba clade with high PP/BS support. The level of COX1 sequence divergence was 22% between P. garorimi n. sp. and P. pagei and 25% between P. garorimi n. sp. and P. microlepis. Pseudoparamoeba species are hardly distinguishable by morphology alone, but display clear differences in 18S rDNA and COX1 gene sequences.     

Genetic and morphological discrimination of three species of ninespined stickleback Pungitius spp. (Teleostei,Gasterosteidae) in France with the revalidation of Pungitius vulgaris (Mauduyt, 1848)

The taxonomy of French ninespined sticklebacks (Pungitius spp.) has long been controversial. To clarify the taxonomy in this group, we use mitochondrial (COI) and nuclear (RNF213) sequence markers, as well as morphological data. In France, both genetic markers discriminate three evolutionary lineages. Morphological analysis on fresh and type specimens supports the different lineages and the existence of three species in France. Pungitius pungitius, occurring in the North of France and Rhone basin, is characterized by specimens longer than 35 mm SL, by a flat head with a straight or slightly concave snout, typically 9–10 dorsal spines, 10–11 dorsal soft rays, 9–10 anal soft rays, 0–12 scutes on the caudal peduncle with a keel reaching the last anal‐fin ray, longer pelvic fin, post‐dorsal and caudal peduncle lengths, and a slim caudal peduncle (caudal peduncle depth/length 11.8%–21.9%). Pungitius laevis, occurring in France, in the English Channel basins and Loire drainage, differs from the other species by a head rounded with concave snout in specimens longer than 35 mm SL, accentuating the impression of fleshy lips, 0–4 scutes on the caudal peduncle and a higher caudal peduncle depth/length ratio (15.7%–34.5%). Finally, Pungitius vulgaris, endemic to the Vienne River and rivers of south‐western France as far north as the Garonne estuary, is differentiated by a rounded head with a straight or slightly convex snout, the absence of scutes on the caudal peduncle and by having 11 pectoral‐fin rays. Our data confirm the existence of a hybridization zone in the North of France between P. pungitius and P. laevis. As a result, Pungitius lotharingus is invalid, as it was described based on hybrid specimens. A lectotype for P. laevis was designated because the syntypes included hybrids. This revision provides new perspectives for evolutionary biology studies and will have consequences for Pungitius conservation in France.     

Genetic and morphological differentiation of Porphyra and Pyropia species (Bangiales,Rhodophyta) coexisting in a rocky intertidal in Central Chile

A recent molecular taxonomic study along the Chilean coast (18° S–53° S) described 18 candidate species of bladed Bangiales of which only two were formally described. Few studies focused on local genetic and morphological diversity of bladed Bangiales and attempted to determine their intertidal distribution in contrasting habitats, and none were performed in Chile. To delimit intertidal distributions of genetic species, 66 samples of bladed Bangiales were collected at Maitencillo (32° S) in four zones: a rocky platform, a rocky wall, and two boulders zones surrounded by sandy and rocky bottoms, respectively. These samples were identified based on sequences of the mitochondrial COI and chloroplast rbcL markers. We also collected 87 specimens for morphological characterization of the most common species, rapidly assessing their putative species identity using newly developed species‐diagnostic (PCR‐RFLP) markers. Eight microscopic and two macroscopic morphological traits were measured. We described and named three of four species that predominate in Maitencillo (including Pyropia orbicularis): Pyropia variabilis Zapata, Meynard, Ramírez, Contreras‐Porcia, sp. nov., Porphyra luchea Meynard, Ramírez, Contreras‐Porcia sp. nov., and Porphyra longissima Meynard, Ramírez, Contreras‐Porcia, sp. nov. With the exception of Po. longissima restricted to boulders surrounded by sandy bottom, and a morphotype of Py. variabilis restricted to rocky walls, the other species/morphotypes have overlapping intertidal distributions. Except for Po. longissima, which is clearly differentiated morphologically (longest and thinnest blades), we conclude that morphology is not sufficient to differentiate bladed Bangiales. Our findings underscore the importance of refining our knowledge of intrinsic and environmental determinants on the distribution of bladed Bangiales.