Tracing ancestry with chromosomal sequences

Most of the large Drosophila species of Hawaii are single-island endemics. Chromosomal sequences show that species at the new end of the archipelago have been derived stepwise from ancestral populations on older islands. The oldest high island has an endemic species with sequences that match some in the Nearctic-Palearctic robusta species group. Colonization from a continent by long-distance dispersal seems a likely origin for the Hawaiian drosophilids. Telmatogeton, a worldwide genus of marine midges, has five Hawaiian species inhabiting freshwater streams. Chromosomal sequences of a marine species in Hawaiian waters match the freshwater forms, indicating colonization from the ocean.     

Molecular insights into the phylogenetic structure of the spider genus Theridion (Araneae, Theridiidae) and the origin of the Hawaiian Theridion-like fauna

The Hawaiian happy face spider ( Theridion grallator Simon, 1900), named for a remarkable abdominal colour pattern resembling a smiling face, has served as a model organism for understanding the generation of genetic diversity. Theridion grallator is one of 11 endemic Hawaiian species of the genus reported to date. Asserting the origin of island endemics informs on the evolutionary context of diversification, and how diversity has arisen on the islands. Studies on the genus Theridion in Hawaii, as elsewhere, have long been hampered by its large size (> 600 species) and poor definition. Here we report results of phylogenetic analyses based on DNA sequences of five genes conducted on five diverse species of Hawaiian Theridion , along with the most intensive sampling of Theridiinae analysed to date. Results indicate that the Hawaiian Islands were colonised by two independent Theridiinae lineages, one of which originated in the Americas. Both lineages have undergone local diversification in the archipelago and have convergently evolved similar bizarre morphs. Our findings confirm para- or polyphyletic status of the largest Theridiinae genera: Theridion , Achaearanea and Chrysso . Convergent simplification of the palpal organ has occurred in the Hawaiian Islands and in two continental lineages. The results confirm the convergent evolution of social behaviour and web structure, both already documented within the Theridiidae. Greater understanding of phylogenetic relationships within the Theridiinae is key to understanding of behavioural and morphological evolution in this highly diverse group.     

Evolution of insular Pacific Pittosporum (Pittosporaceae): origin of the Hawaiian radiation.

We investigated the origin of Hawaiian Pittosporum and their relationship to other South Pacific Pittosporum species using internal transcribed spacer sequences of nuclear ribosomal DNA. We performed both maximum-parsimony and maximum-likelihood analyses, which produced congruent results. Sequence divergence was 0.0% between Hawaiian members of Pittosporum. These taxa formed a strongly supported clade, suggesting a single colonization event followed by phyletic radiation. Sister to the Hawaiian clade were two South Pacific species, P. yunckeri from Tonga and P. rhytidocarpum from Fiji. This result presents convincing evidence for a South Pacific origin of Hawaiian Pittosporum. Our results also identify a monophyletic group comprising three species representing the Fijian Province and East Polynesia, two introductions onto New Caledonia, and at least one (but possibly two) introduction(s) onto New Zealand. Whether the New Zealand taxa form a monophyletic group is unclear from these data. Previous morphologically based hypotheses, however, suggest the presence of four different lineages occupying New Zealand. The nonmonophyly of the New Caledonian species was not surprising based on the extent of their morphological diversity. Although this latter result is not strongly supported, these species are morphologically complex and are currently the subject of taxonomic revision and molecular systematic analyses.     

Origin and evolution of North American polyploid Silene (Caryophyllaceae)

Nuclear DNA sequences from introns of the low-copy nuclear gene family encoding the second largest subunit of RNA polymerases and the ribosomal internal transcribed spacer (ITS) regions, combined with the psbE-petL spacer and the rps16 intron from the chloroplast genome were used to infer origins and phylogenetic relationships of North American polyploid Silene species and their closest relatives. Although the vast majority of North American Silene species are polyploid, which contrasts to the diploid condition dominating in other parts of the world, the phylogenetic analyses rejected a single origin of the North American polyploids. One lineage consists of tetraploid Silene menziesii and its diploid allies. A second lineage, Physolychnis s.l., consists of Arctic, European, Asian, and South American taxa in addition to the majority of the North American polyploids. The hexaploid S. hookeri is derived from an allopolyploidization between these two lineages. The tetraploid S. nivea does not belong to any of these lineages, but is closely related to the European diploid S. baccifera. The poor resolution within Physolychnis s.l. may be attributed to rapid radiation, recombination among homoeologues, homoplasy, or any combination of these factors. No extant diploid donors could be identified in Physolychnis s.l.     

A molecular phylogeny of four endangered Madagascar tortoises based on MtDNA sequences

Four of the five tortoise species in Madagascar, Pyxis arachnoides, P. planicauda, Geochelone radiata, and G. yniphora, are endemic and on the verge of extinction. Their phylogenetic relationships remain controversial and unresolved. Here we address the phylogeny of this group using DNA sequences for the 12S and 16S rDNA and cyt b genes in mitochondrial DNA. As outgroups we used two species of Geochelone, pardalis (mainland Africa) and nigra (Galápagos), as well as a more distant North American tortoise, Gopherus polyphemus. We conclude that the two Pyxis species are sister taxa and are imbedded in the genus Geochelone, rendering this latter genus paraphyletic. There is moderate support for the sister status of the two Madagascar Geochelone and for the monophyletic origin of all four endemics, suggesting a single colonization of the island. The separation of Madagascar from other land masses (90-165 mya) predates the origin of the endemic tortoises (estimated to be 14-22 mya). This suggests founding by rafting, a process known to have occurred with other tortoises. The derived morphological divergence of the Pyxis species in a relatively short period of time (13-20 my) stands in contrast to the notoriously slow rate of morphological evolution in most lineages of Chelonia.     

Origin of the Hawaiian endemic mints within North American Stachys (Lamiaceae)

The Hawaiian endemic mints constitute a major island radiation, displaying a remarkable diversity of floral, fruit, and vegetative features. Haplostachys and Phyllostegia have flowers associated with insect pollination, whereas Stenogyne has flowers typical of bird pollination. The three genera had been thought to be closely related to East Asian members of Lamioideae tribe Prasieae because of the fleshy nutlets borne by Phyllostegia and Stenogyne. We evaluated the origins of the Hawaiian mints using phylogenetic analyses of DNA sequence data from the plastid rbcL and trnL intron loci and the nuclear ribosomal 5S nontranscribed spacer. The Hawaiian genera were found to be monophyletic but deeply nested inside another lamioid genus, Stachys. In particular, they were found to be most closely related to a group of temperate North American Stachys from the Pacific coast, suggesting that the Hawaiian mints derived from a single colonization event from western North America to the Hawaiian Islands. Furthermore, Stachys, which contains amphiatlantic and transberingian clades, was found to be polyphyletic, with some species more closely related to Gomphostemma, Phlomidoschema, Prasium, and Sideritis than to other species of Stachys. Based on chromosomal evidence and our phylogenetic analyses, we hypothesize that the Hawaiian mints may be polyploid hybrids whose reticulate genomes predate the Hawaiian dispersal event and are derived from Stachys lineages with flowers exhibiting insect- vs. bird-pollination characteristics. Thus, the Hawaiian endemic mints may provide yet another insular system for the combined study of polyploidy, hybrid cladogenesis, and adaptive radiation.     

Phylogenetic relationships of Hemidactylus geckos from the Gulf of Guinea islands: patterns of natural colonizations and anthropogenic introductions estimated from mitochondrial and nuclear DNA sequences

Mitochondrial DNA (12S rRNA, 16S rRNA, and cytochrome b) sequences and nuclear sequences (C-mos and alpha-Enolase) were analyzed within all known Hemidactylus species from all three volcanic islands in the Gulf of Guinea that have never been connected to the continent. These comprise both endemic and widespread species. Our aim was to determine if the widespread species was introduced anthropogenically, to determine the number of distinct genetic lineages within the islands, and to determine if the endemic forms constituted a monophyletic group. Our results suggest that a previously undescribed species on S?o Tomé is the sister taxon to Hemidactylus newtoni, endemic to Annobon. Genetic variation between populations of Hemidactylus greefii from S?o Tomé and Principe is very high based on mtDNA sequences, but the forms cannot be distinguished using the nuclear DNA sequences. Hemidactylus mabouia appears to have been anthropogenically introduced to all three islands. The island endemics do not form a monophyletic group, suggesting multiple independent colonizations of the islands.     

Inferring the history of the polyploid Silene aegaea (Caryophyllaceae) using plastid and homoeologous nuclear DNA sequences

The origin of the rare allotetraploid Silene aegaea was inferred from plastid rps16 intron sequences, homoeologous copies of nuclear ribosomal internal transcribed spacer (ITS) sequences, and an intron from the nuclear gene coding for the second largest subunit of RNA polymerase II (RPB2). The nuclear DNA regions support the S. sedoides and S. pentelica lineages as most closely related to the two S. aegaea paralogues. A few recombinant ITS sequences were found, but as PCR recombination could be demonstrated, no true recombination could be demonstrated. No recombination was found in the RPB2 sequences. Plastid rps16 intron sequences strongly support S. pentelica as the maternal lineage. The strength of the approach of using homoeologous sequences of several loci is demonstrated, and its usefulness for the study of phylogenies of groups including polyploids is emphasized.     

Molecular phylogeny of fire ants of the Solenopsis saevissima species-group based on mtDNA sequences

The systematics of South American fire ants (Solenopsis saevissima species-group) has been plagued by difficulties in recognizing species and their relationships on the basis of morphological characters. We surveyed mtDNA sequences from 623 individuals representing 13 described and undescribed species within the species-group and 18 individuals representing other major Solenopsis lineages to generate a phylogeny of the mitochondrial genome. Our analyses support the monophyly of the S. saevissima species-group, consistent with a single Neotropical origin and radiation of this important group of ants, as well as the monophyly of the socially polymorphic species within the group, consistent with a single origin of polygyny (multiple queens per colony) as a derived form of social organization. The mtDNA sequences of the inquiline social parasite S. daguerrei form a clade that appears to be distantly related to sequences from the several host species, consistent with the view that advanced social parasitism did not evolve via sympatric speciation of intraspecific parasites. An important general finding is that species-level polyphyly of the mtDNA appears to be the rule in this group of ants. The existence of multiple divergent mtDNA lineages within several nominal species (including the pest S. invicta) suggests that the pattern of widespread polyphyly often stems from morphological delimitation that overcircumscribes species. However, in two cases the mtDNA polyphyly likely results from recent interspecific hybridization. While resolving species boundaries and relationships is important for understanding general patterns of diversification of South American fire ants, these issues are of added importance because invasive fire ants are emerging as global pests and becoming important model organisms for evolutionary research.     

Allozyme diversity and the evolution ofSymplocos (Symplocaceae) on the Bonin (Ogasawara) Islands

To study the origin and speciation of plants in oceanic islands, electrophoretic analyses have been done on three endemic species ofSymplocos in the Bonin Islands as well as on three other species;S. kuroki, S. nakaharae andS. tanakae which are considered to be closely related to the Bonin endemics. There occur three species:S. kawakamii, S. pergracilis andS. boninensis in Bonin. The genusSymplocos is one which is considered to be diversified in the Bonin Islands. Seven enzyme systems presumed to be encoded by 18 loci were examined. The genetic diversity was low in the island species, as reported in some oceanic island plants of Hawaii and the Bonin Islands. The three endemics share high genetic identities and they clustered together in the tree drawn by the UPGMA method, suggesting that they are a monophyletic group, that is, they result from a single introduction.     

Molecular biogeography and diversification of the endemic terrestrial fauna of the Hawaiian Islands

Oceanic islands have played a central role in biogeography and evolutionary biology. Here, we review molecular studies of the endemic terrestrial fauna of the Hawaiian archipelago. For some groups, monophyly and presumed single origin of the Hawaiian radiations have been confirmed (achatinelline tree snails, drepanidine honeycreepers, drosophilid flies, Havaika spiders, Hylaeus bees, Laupala crickets). Other radiations are derived from multiple colonizations (Tetragnatha and Theridion spiders, succineid snails, possibly Dicranomyia crane flies, Porzana rails). The geographic origins of many invertebrate groups remain obscure, largely because of inadequate sampling of possible source regions. Those of vertebrates are better known, probably because few lineages have radiated, diversity is far lower and morphological taxonomy permits identification of probable source regions. Most birds, and the bat, have New World origins. Within the archipelago, most radiations follow, to some degree, a progression rule pattern, speciating as they colonize newer from older islands sequentially, although speciation often also occurs within islands. Most invertebrates are single-island endemics. However, among multi-island species studied, complex patterns of diversification are exhibited, reflecting heightened dispersal potential (succineids, Dicranomyia). Instances of Hawaiian taxa colonizing other regions are being discovered (Scaptomyza flies, succineids). Taxonomy has also been elucidated by molecular studies (Achatinella snails, drosophilids). While molecular studies on Hawaiian fauna have burgeoned since the mid-1990s, much remains unknown. Yet the Hawaiian fauna is in peril: more than 70 per cent of the birds and possibly 90 per cent of the snails are extinct. Conservation is imperative if this unique fauna is to continue shedding light on profound evolutionary and biogeographic questions.     

Phylogeny and age of diversification of the planitibia species group of the Hawaiian Drosophila

The Hawaiian Drosophila offer a unique opportunity to examine evolutionary questions because of the known ages of the Hawaiian Islands and the large number of species endemic to this archipelago. One of the more well studied groups of Hawaiian Drosophila is the planitibia species group, a long-standing population genetic model system. Here we present a molecular phylogenetic hypothesis of all 17 taxa in the planitibia group based on nucleotide sequences from two mitochondrial (16S and COII) and four nuclear (Adh, Gpdh, Yp1, and Yp2) loci, accounting for over 4kb of sequence per taxon. We use these data to estimate major divergence times within this group. Our results suggest that the basal diversification within this group, calculated at 6.1 +/- 0.47 MY, predates the oldest high island of Kauai. The older diversifications in this group took place on Kauai, with subsequent colonization and speciation events occurring as new islands became available to Drosophila. Understanding of the phylogenetic relationships of this important group will place the existing population genetic work in a macroevolutionary context and stimulate additional work, particularly on those taxa endemic to the Maui Nui complex of islands.     

Hawaiian angiosperm radiations of North American origin

Background

Putative phytogeographical links between America (especially North America) and the Hawaiian Islands have figured prominently in disagreement and debate about the origin of Pacific floras and the efficacy of long-distance (oversea) plant dispersal, given the obstacles to explaining such major disjunctions by vicariance.

Scope

Review of past efforts, and of progress over the last 20 years, toward understanding relationships of Hawaiian angiosperms allows for a historically informed re-evaluation of the American (New World) contribution to Hawaiian diversity and evolutionary activity of American lineages in an insular setting.

Conclusions

Temperate and boreal North America is a much more important source of Hawaiian flora than suggested by most 20th century authorities on Pacific plant life, such as Fosberg and Skottsberg. Early views of evolution as too slow to account for divergence of highly distinctive endemics within the Hawaiian geological time frame evidently impeded biogeographical understanding, as did lack of appreciation for the importance of rare, often biotically mediated dispersal events and ecological opportunity in island ecosystems. Molecular phylogenetic evidence for North American ancestry of Hawaiian plant radiations, such as the silversword alliance, mints, sanicles, violets, schiedeas and spurges, underlines the potential of long-distance dispersal to shape floras, in accordance with hypotheses championed by Carlquist. Characteristics important to colonization of the islands, such as dispersibility by birds and ancestral hybridization or polyploidy, and ecological opportunities associated with ‘sky islands’ of temperate or boreal climate in the tropical Hawaiian archipelago may have been key to extensive diversification of endemic lineages of North American origin that are among the most species-rich clades of Hawaiian plants. Evident youth of flowering-plant lineages from North America is highly consistent with recent geological evidence for lack of high-elevation settings in the Hawaiian chain immediately prior to formation of the oldest, modern high-elevation island, Kaua‘i.     

Origin and diversification of the endemic Hawaiian tree snails (Achatinellidae: Achatinellinae) based on molecular evidence

Tree snails of the endemic subfamily Achatinellinae comprise a diverse and important component of the Hawaiian fauna. In recent decades anthropogenic impacts have resulted in devastating extinction rates in Hawaiian tree snails. To address long-standing biogeographic, systematic, and evolutionary questions we used cytochrome c oxidase subunit I (COI) gene sequences to reconstruct the phylogeny of 23 extant species spanning the range of the subfamily from five Hawaiian Islands. To investigate family-level relationships, data were analyzed from 11 terrestrial pulmonate families. Although nodal support for monophyly of the endemic Pacific family Achatinellidae and endemic Hawaiian subfamily Achatinellinae was strong, bifurcation order among deeper ingroup nodes was not well-supported by bootstrap resampling. We hypothesize that lineage extinction and rapidity of lineage formation may have rendered evolutionary reconstruction difficult using a standard phylogenetic approach. Use of an optimized evolutionary model, however, improved resolution and recovered three main clades. The diversification pattern inferred contradicts the traditional biogeographic hypothesis of a Maui origin of the achatinelline lineage. Taxa comprising the basal ingroup clade (Achatinella spp.) and seeding lineages for subsequent clades originated on O'ahu. Therefore it appears that the ancestral colonizing species of achatinellines arrived first on O'ahu from an unknown source, and that O'ahu is the Hawaiian origin of the subfamily. Species previously defined by morphological criteria were generally found to be phylogenetically distinct, and the overall colonization pattern follows the island-age progression rule with several instances of generic polyphyly and back-colonization.     

Inferring the History of the Polyploid Silene aegaea (Caryophyllaceae) Using Plastid and Homoeologous Nuclear DNA Sequences

The origin of the rare allotetraploid Silene aegaea was inferred from plastid rps16 intron sequences, homoeologous copies of nuclear ribosomal internal transcribed spacer (ITS) sequences, and an intron from the nuclear gene coding for the second largest subunit of RNA polymerase II (RPB2). The nuclear DNA regions support the S. sedoides and S. pentelica lineages as most closely related to the two S. aegaea paralogues. A few recombinant ITS sequences were found, but as PCR recombination could be demonstrated, no true recombination could be demonstrated. No recombination was found in the RPB2 sequences. Plastid rps16 intron sequences strongly support S. pentelica as the maternal lineage. The strength of the approach of using homoeologous sequences of several loci is demonstrated, and its usefulness for the study of phylogenies of groups including polyploids is emphasized.     

Single origin and subsequent diversification of central Andean endemic Umbilicaria species

We studied an Andean endemic group of species of the lichen-forming fungal genus Umbilicaria from the subalpine and low-alpine zone, with their biogeographic center in Bolivia and Peru. A number of species and varieties have been described from this element, but apparent instability in several morphological traits has made it difficult to precisely delimit taxa. Based on DNA sequences of nuclear ITS, LSU and mitochondrial SSU from extensive collections from Argentina, Bolivia, Chile, Colombia, Ecuador and Peru, we present here a molecular phylogenetic analysis of this Andean endemic element within genus Umbilicaria. All analyses (MP, ML and Bayesian) support a single origin for the element and a division into two major groups characterized by different apothecium types: the Umbilicaria dichroa group and U. calvescens group. Taxa U. krempelhuberi, U. peruviana and U. subcalvescens are nested withinn U. calvescens and are treated as conspecific with the latter species. The endemic element shares a most recent common ancestor with the Umbilicaria vellea group, which has a worldwide distribution and contains several asexually reproducing (sorediate) species. Independent reversals to sexual reproduction might explain the evolution of two types of apothecia in this monophyletic endemic lineage. A number of cosmopolitan, mostly high-alpine, species of Umbilicaria also present in the central Andes are related only remotely to the endemic element and do not exhibit speciation into endemics. Because the An-dean element dominates the Umbilicaria habitats of the low- and subalpine zones we propose that the founder colonized the Andes at a time when the mountains had not yet reached their current elevation while the high-alpine species arrived more recently.     

Molecular phylogeny of Lysimachia (Myrsinaceae) based on chloroplast trnL-F and nuclear ribosomal ITS sequences

Both nuclear ribosomal ITS and chloroplast trnL-F sequences were acquired for 57 species (accessions) of Lysimachia and its close relatives, and were analyzed together with sequences retrieved from databases. The results of phylogenetic analyses based on these data (separately or combined) show that Lysimachia is paraphyletic, with the monotypic genus Glaux nested deeply inside. Previous suggestions that Anagallis and Trientalis could be ingroups of Lysimachia were not corroborated by our results. The molecular phylogenies do not support the current infrageneric divisions of Lysimachia. Subgenus Lysimachia contains at least five independent lineages. The Hawaii endemic subgenus Lysimachiopsis was shown to group with subgenera Palladia and Heterostylandra, instead of subgenus Idiophyton as previously suggested. The two North American representatives of Lysimachia, subgenus Seleucia and section Verticillatae of subgenus Lysimachia are group together as the most basal clade of the genus. Parallel and independent evolutions were inferred for morphological characters that were previously used as diagnostic criteria. Molecular phylogenies do not offer clear inferences on the overall historical biogeography of Lysimachia, but Southeast Asia origins of several clades, including the Hawaiian endemic clade and the Iberian Lysimachia ephemerum are strongly supported.     

Origin of Hawaiian Polystichum (Dryopteridaceae) in the context of a world phylogeny

A genus-wide molecular phylogeny for Polystichum and allied genera (Dryopteridaceae) was reconstructed to address the biogeographic origin and evolution of the three Hawaiian Polystichum species, all endemic there. The analysis was based on the cpDNA sequences rbcL and the trnL-F spacer from a taxonomically and geographically diverse sample. Parsimony and Bayesian phylogenetic analyses of the combined data support a monophyletic Polystichum and corroborate recent hypotheses as to membership and sequence of origin of the major groups within the genus. The Hawaiian Polystichum species are polyphyletic; two separate lineages appear to have arrived independently from the Old World. The provenance of the diploid Polystichum hillebrandii is continental eastern Asia, while the source of the polyploid lineage comprising tetraploid P. haleakalense and octoploid P. bonseyi is likely continental Asia. From our results, the origin of the Hawaiian species of Polystichum, like many Hawaiian fern genera with several species, is the result of multiple migrations to the islands, rather than single migrations yielding nearly all the local diversity as in the angiosperms. This emerging pattern provides a modern test of the premise that propagule vagility has a central role in determining pattern of evolution.     

Phylogenetic relationships of xenodontine snakes inferred from 12S and 16S ribosomal RNA sequences

The phylogenetic relationships of xenodontine snakes are inferred from sequence analyses of portions of two mitochondrial genes (12S and 16S ribosomal RNA) in 85 species. Although support values for most of the basal nodes are low, the general pattern of cladogenesis observed is congruent with many independent molecular, morphological, and geographical data. The monophyly of xenodontines and the basal position of North American xenodontines in comparison with Neotropical xenodontines are favored, suggesting an Asian-North American origin of xenodontines. West Indian xenodontines (including endemic genera and members of the genus Alsophis) appear to form a monophyletic group belonging to the South American clade. Their mid-Cenozoic origin by dispersal using ocean currents is supported. Within South American mainland xenodontines, the tribes Hydropsini, Pseudoboini, and Xenodontini are monophyletic. Finally, our results suggest that some morphological and ecological traits concerning maxillary dentition, macrohabitat use, and foraging strategy have appeared multiple times during the evolution of xenodontine snakes.     

Multiple origins of Hawaiian drosophilids: Phylogeography of Scaptomyza Hardy (Diptera: Drosophilidae)

Scaptomyza is a highly diversified genus in the family Drosophilidae, having undergone an explosive radiation, along with the Hawaiian‐endemic genus Idiomyia in the Hawaiian Islands: about 60% of 269 Scaptomyza species so far described are endemic to the Hawaiian Islands. Two hypotheses have been proposed for the origin and diversification of Hawaiian drosophilids. One is the “single Hawaiian origin” hypothesis: Scaptomyza and Idiomyia diverged from a single common ancestor that had once colonized the Hawaiian Islands, and then non‐Hawaiian Scaptomyza migrated back to continents. The other is the “multiple origins” hypothesis: Hawaiian Scaptomyza and Idiomyia derived from different ancestors that independently colonized the Hawaiian Islands. A key issue for testing these two hypotheses is to clarify the phylogenetic relationships between Hawaiian and non‐Hawaiian species in Scaptomyza. Toward this goal, we sampled additional non‐Hawaiian Scaptomyza species, particularly in the Old World, and determined the nucleotide sequences of four mitochondrial and seven nuclear genes for these species. Combining these sequence data with published data for 79 species, we reconstructed the phylogeny and estimated ancestral distributions and divergence times. In the resulting phylogenetic trees, non‐Hawaiian Scaptomyza species were interspersed in two Hawaiian clades. From a reconstruction of ancestral biogeography, we inferred that Idiomyia and Scaptomyza diverged outside the Hawaiian Islands and then independently colonized the Hawaiian Islands, twice in Scaptomyza, thus supporting the “multiple origins” hypothesis.