BACKGROUND
eight main islands and  <dig> small islands, atolls and shoals comprise the hawaiian archipelago, which encompasses  <dig>  km <dig> and represents the largest oceanic archipelago in the world  <cit> , <cit> .
the islands are volcanically-derived, and form successively as the pacific plate moves in a northwestern direction over a fixed “hot spot”, such that the eight current high islands  at the southeastern end of the chain represent only the last  <dig>  million years  of a 75– <dig> my history of volcanic activity  <cit> .
the main hawaiian islands are characterized by steep gradients of elevation , rainfall  and island age , which have generated a remarkable range of ecological conditions  <cit> , <cit> .
the hawaiian islands are well known as a uniquely isolated and time-calibrated environment in which to study evolution; a place where numerous lineages of plants and animals exemplify the phenomena of endemism and adaptive radiation  <cit> - <cit> .
the geographical isolation of the main hawaiian islands  is a strong force influencing the biogeography of hawaii, and initial colonization of the islands was accomplished through the arrival and survival of dispersed individuals  <cit> .
subsequent colonization likely occurred through a combination of long-distance dispersal, and, increasingly commonly, dispersal from adjacent islands  <cit> .
a number of studies on the freshwater algae of the hawaiian islands have appeared in the literature  <cit> - <cit> .
eight hundred taxonomic records were compiled from the literature in a bibliographic checklist spanning the years 1876– <dig>  <cit> , which included representatives of almost all major freshwater algal lineages.
based on these literature records , the overall level of endemism for the hawaiian non-marine algae was estimated to be 5%, or a total of  <dig> taxa.
this endemism level was noted as being very low in comparison to other organisms such as marine red algae , marine invertebrates , ferns and lycophytes , flowering plants , and insects   <cit> , <cit> , <cit> .
it was subsequently noted that the stream algal flora of hawaii was “suspiciously cosmopolitan” in composition  <cit> , drawing attention to the large proportion of hawaiian taxonomic records in common with very different biogeographic regions of the world.
others have noted that freshwater algae around the world have often been considered to be cosmopolitan, that this notion is unlikely to hold true when tested with genetic data, and that endemism is most likely obscured by the “force-fitting” of european names  <cit> .
competing explanations exist for the biogeographic distributions of freshwater algae.
distributions of microbial algae can be examined in the context of “everything is everywhere, but, the environment selects”  <cit> , which predicts that microorganisms are globally distributed as a result of their vast population sizes and unlimited dispersal, and whether or not a species becomes established is determined solely by local conditions  <cit> , <cit> .
in contrast, a number of researchers have reported evidence of biogeographic patterning of microorganisms by using larger sample sizes, detailed observations, and/or molecular assessments of diversity  <cit> - <cit> , and still others invoke a combination of ubiquity and endemism to explain cryptic molecular lineages of widespread morphospecies  <cit> , <cit> .
whether hawaiian freshwater algae are truly cosmopolitan when molecular comparisons are taken into account, or whether they display levels of endemism that parallel other elements of the flora and fauna of the archipelago, is currently unknown.
given current estimates of biodiversity loss, it is critical to understand the scope of endemism versus cosmopolitanism in the poorly characterized freshwater flora of a region already recognized as a biodiversity hotspot for other lineages of organisms.
loss of biodiversity is proceeding faster in freshwaters than in any other major biome  <cit> - <cit> .
the hawaiian freshwater algal biodiversity survey  was carried out to establish baseline data to further assess human impacts on freshwater hawaiian ecosystems and monitor change in the context of biodiversity conservation.
the goals of the survey were to collect, document, and characterize as many non-marine algal taxa as possible from the main hawaiian islands, and to use the resultant data to examine distribution patterns and develop biogeographic hypotheses to explain these patterns.
the first author  held responsibility for the characterization of the macroalgal samples resulting from the survey, and as such the focus of the present report is on these taxa.
expeditionary collections were made on the islands of kauai, oahu, molokai, lanai, maui and hawaii, and macroalgal specimens were characterized morphologically and, where possible, using molecular methods.
the distributional patterns and biogeographic affinities of taxa based on both literature records and dna sequence analyses were examined in the context of distributions characterized by cosmopolitanism  versus endemism .
with very little information currently available for biogeographic patterns of specific freshwater algae based on molecular data trends, no predictions of biogeographic affinity were made for the hawaiian non-marine algae.
however, an increase in the number of recognized endemic taxa was predicted, given the frequency with which freshwater algae are demonstrated to harbor cryptic  diversity or pseudocryptic  diversity when molecular tools are employed.
RESULTS
summary of collections
a total of  <dig>  distinct sites were sampled  .
collection sites were concentrated on the wetter, windward sides of the islands, and on the larger islands that had a greater number of perennial streams and associated moist habitats.
sampling sites were not chosen randomly, but were selected on the basis of accessibility and likelihood of harboring interesting algal diversity; thus, the following numbers should be interpreted as a summary of the characteristics of the collections rather than an indication of available habitats in the hawaiian islands.
areas not sampled lacked suitable habitats or were not accessible.
streams comprised  <dig> % of all sites, followed by terrestrial sites  and wet walls , with smaller proportions of the remaining habitat categories .
a total of  <dig>  isolate accessions  were characterized, of which  <dig>  were macroalgae.
slightly more than  <dig> % of these isolate accessions were green algae, while diatoms , red algae , cyanobacteria , xanthophytes  and euglenoids  comprised the remainder .
additional file  <dig> summarizes the taxa identified, along with their islands of distribution and the habitat types from which they were collected and identified.
the most widely collected taxa included the green algae spirogyra spp.
, mougeotia spp.
, oedogonium spp.
, cladophora glomerata , microspora spp.
, cloniophora spicata , and rhizoclonium spp.
, the red algal species compsopogon caeruleus  and the xanthophyte vaucheria spp.
.
dna barcoding
a total of  <dig> dna sequences were generated as part of the survey, representing eight different markers  .
the upa marker, which was employed as an initial screen for all samples, accounted for more sequences than any other marker , while ssu , rbcl , lsu , coi  and 16s rrna  comprised the majority of the remainder.
sequence data for the 16s rrna, coi, lsu, ssu, rbcl, and upa markers were employed to investigate the molecular diversity of specific lineages of the hawaiian non-marine algal flora.
sequence diversity for each sampled taxon is represented in neighbor-joining trees based on uncorrected p-distances .
rather than attempting to reconstruct phylogenetic relationships for each taxon, we aimed to use these data for an assessment of patterns of molecular diversity within the hawaiian flora.
thus, higher order relationships are not inferred from these analyses; comparisons should be restricted to closely-related taxa.
more in-depth phylogenetic analyses have been published or are in progress for a number of taxa, including green and red algae  <cit> , <cit> , cyanobacteria  <cit>  and diatoms  <cit> .
rbcl
sequences of the hawaiian non-marine charophycean green algae.
sequence diversity based on the rbcl marker for the hawaiian non-marine charophycean green algae.
the neighbor-joining tree is based on uncorrected p-distances nucleotide model in mega  <dig> .
scale bar = substitutions per site.
rbcl
sequences of the hawaiian non-marine red algae.
sequence diversity based on the rbcl marker for the hawaiian non-marine red algae.
the neighbor-joining tree is based on uncorrected p-distances nucleotide model in mega  <dig> .
scale bar = substitutions per site.
summary taxon labels on each tree reflect the lowest taxonomic level to which a confident assignment can be made for a cluster of identical or near-identical sequences .
the vast majority of samples were not reproductive at the time of collection, which limited the number of taxa that could be identified to the species level.
nonetheless, clustering patterns of closely related sequences can be used as an indication of the number of entities represented in the hawaiian flora for a given genus .
for example, the upa analysis of cyanobacteria suggests eight species of the genus nostoc were collected and characterized during the surveys , two of which were included in the 16s rrna analysis .
four species of the red algal form genus “chantransia” were identified from the upa analysis, representing members of the batrachospermales and thoreales   <cit> , and these species-level groupings were consistent with groups recovered in the coi , rbcl  and ssu  analyses.
the most species-rich genus of freshwater macroalgae studied in this survey was the charophycean green alga spirogyra, which was demonstrated to consist of  <dig> clusters of sequences for the upa marker ,  <dig> for rbcl , and nine for ssu ; an in-depth comparison of these sequences to accessions worldwide and a comparative analysis of thallus morphology is underway .
categories of distribution
the best-studied and best-represented taxa from the survey were examined for distributional trends: eight red algae,  <dig> green algae,  <dig> cyanobacteria, four diatoms and one xanthophyte, for a total of  <dig> taxa .
twelve taxa were determined to be in the category of broadest distribution ,  <dig> were in the category of second most broadly distributed ,  <dig> were members of the third category , and five were members of the putatively endemic category .
it should be noted, however, that sampling effort has not been consistent among these regions of the world, with some having received many times the effort  than others  over the course of the past several centuries.
for each of the taxa in each of the categories above, thallus size and dispersal unit size ranges  were plotted to assess patterns associated with dispersal category.
thallus sizes were found to range from microscopic to macroscopic for representatives of all dispersal categories  .
in contrast, dispersal unit size ranges were found to be uniformly ≤1 mm, and much smaller for both endemic and cosmopolitan taxa  .
discussion
a comparative overview of the hawaiian non-marine macroalgal flora
the  <dig>  macroalgal samples collected during the hawaiian freshwater algal biodiversity survey represent, by far, the most exhaustive survey of non-marine algae for a tropical oceanic island archipelago yet reported.
previous studies in the tropical pacific have yielded between seven and  <dig> samples, and have resulted in algal checklists of 12– <dig> taxa  <cit> , <cit> - <cit> .
algal studies of the islands of yap, in the caroline islands, revealed a diverse flora of cyanobacteria, green algae, red algae, euglenoids and dinoflagellates totaling  <dig> taxa  <cit> , <cit> , while, in contrast, a study of stream macroalgae of the islands of fiji suggested an impoverished stream algal diversity of  <dig> taxa representing cyanobacteria, green algae and red algae  <cit> .
similar to the flora from fiji, a rapid survey of fresh waters from the austral islands of french polynesia yielded  <dig> species, representing cyanobacteria, green algae and xanthophytes  <cit> .
a number of previous studies of hawaiian stream algae have been conducted, which yielded from  <dig> taxa  <cit>  to  <dig> taxa  <cit>  to  <dig> taxa  <cit> ; albeit all identifications from these past studies were morphologically based, and thus are not completely comparable to the current research, and were also more limited in collection intensity.
for example, many of the common taxa previously reported from the hawaiian islands have been subsequently demonstrated through molecular analyses to represent different, but recognized species , endemic hawaiian taxa , or members of different genera .
the present study is the first to integrate taxonomic collections from a multitude of non-marine algal habitats, including streams, wet walls, high elevation bogs, taro fields, ditches and flumes, cave walls and terrestrial areas for a tropical island algal flora.
two of these habitats were sufficiently well represented in the present survey collections to warrant individual treatment, and these have been published elsewhere as reports on the non-marine algae of hawaiian taro fields  <cit>   and high elevation bogs  <cit>  .
molecular markers and example within-lineage patterns
a total of eight different molecular markers were employed in this study to characterize the non-marine algal diversity of the hawaiian islands.
due to the large scope of phylogenetic diversity encompassed in the collections , we aimed to establish a multi-step molecular assessment that allowed initial broad-based characterization of all collections , with subsequent use of one or more lineage-specific markers  for comparisons among closely related samples.
our previous biodiversity survey of the hawaiian rhodophyta  as well as other algal investigations  <cit> - <cit>  have provided support for the use of the upa marker as a near-universal region of the plastid genome that enables construction of a molecular biodiversity framework for almost all algae, and we advocate for the use of this marker in multi-step molecular characterizations or analyses of environmental dna samples where characterization of both cyanobacterial and eukaryotic algal diversity is desirable.
our second most widely applied marker, which spans the v <dig> region of nuclear ssu  <cit> , <cit> , also resulted in generation of a large number of sequences, albeit fewer than upa and of a more conserved nature than that marker .
a comparison of the use of both markers for the characterization of environmental dna from stream periphyton is underway .
a number of taxa were well-represented in the survey, and the hawaiian biodiversity represented by these lineages was characterized using a combination of the molecular approaches described above and microscopic analysis.
for example, the charophycean genus spirogyra has been noted as one of the most common freshwater macroalgae across the hawaiian islands in numerous publications dating back to  <dig>  <cit> - <cit> , <cit> , and our survey collections of this genus spanned  <dig> locations on six islands, including diverse habitats such as streams, ditches, taro fields, bogs and ponds.
the red algal “form genus” chantransia was previously thought to be represented in the hawaiian islands by two lineages, sheathia arcuata and nemalionopsis shawii <cit> ; however, two additional taxa were added as a result of the survey collections , doubling the known diversity of these freshwater red algal sporophytes in hawaii  <cit> .
some non-marine representatives of the cladophorales , which can be difficult or impossible to identify using only morphological characters were demonstrated in some cases to be molecularly homogeneous, as demonstrated by others, despite substantial morphological variation , and in other cases to harbor genus-level molecular diversity masked by a simple morphology .
thus, one of the most valuable outcomes of the biodiversity survey approach of collecting and characterizing all non-marine macroalgae from a geographical region is that it allows multi-taxon large scale comparisons that can be compared to more accurately assess taxonomy and infer broader distributional patterns.
biogeographic patterns of the hawaiian non-marine algae
baas becking’s ideas of microorganisms being globally distributed apply to organisms too small to see with the unaided eye, yet may be applicable to the study of freshwater macroalgae given that the dispersal forms of these organisms  fall into the <1 mm size range .
as a consequence, organism size may not predict distribution patterns well.
dispersal mechanisms of non-marine algae have been the subject of much speculation but little empirical research.
a number of means of transport have been proposed, including air currents, vector-assisted transport , or rafting  <cit> - <cit> .
although the initial colonization of the volcanically produced hawaiian islands by non-marine algae must have occurred by long-distance transport  <cit> , dispersal among the islands likely played an increasingly important role in shaping the flora of the archipelago.
the non-marine algal flora of the hawaiian islands has almost certainly been impacted by humans since their first arrival in the islands .
polynesian introductions of numerous plant species for food and other uses may account for the first anthropogenic changes to the flora, but an acceleration of human impact over the past several hundred years  has probably had an even greater influence its composition, with spores and fragments being transported to and among the islands from activities such as the aquarium and ornamental plant trades, hiking, transport of building materials and heavy machinery, and farming.
any attempts to interpret algal biogeographic patterns must take these influences into consideration.
what is evident from the survey collection analyses is that no consistent biogeographic pattern emerges to explain the origins of the non-marine algal flora of the hawaiian islands.
some remarkably widespread taxa were characterized that are also known from many locations worldwide, including the clonally propogating red alga compsopogon caeruleus and the green alga cladophora glomerata; distribution of the former to hawaii may be a result of the aquarium trade  <cit> , while ubiquity in the latter case may be a result of either anthropogenic or non-anthropogenic dispersal.
a few taxa were demonstrated to consist of multiple, cryptic lineages that likely represent intraspecific taxonomic variation, including sheathia arcuata and some spirogyra spp.
a number of other taxa were found to have affinities with samples from a more limited number of other regions, but with little obvious biogeographical connection, for example, cloniophora spicata, nemalionopsis shawii, pithophora roettleri, schizomeris leiblienii and thorea hispida .
in the absence of well-populated reference frameworks of collections from numerous locations it seems likely that these distributions are a product of under-sampling rather than being representative of true biogeographic patterns.
conversely, several taxa were identified that are currently considered endemic to the hawaiian islands , and it is possible that expanded distributions of these taxa may be recognized in the future with increased sampling and characterization of the non-marine algal floras of other tropical pacific islands.
it should also be noted that a number of microalgal taxa have also been recently described from the hawaiian islands as a result of this biodiversity survey, which serves to further emphasize the rich flora of this archipelago that is being illuminated through this opportunity for focused taxonomic attention  <cit> , <cit> - <cit> .
accurate taxonomic identification of algae is not straightforward, and much depends on the species concept employed.
many of the taxa reported in this survey were described decades, if not centuries ago, and the formal descriptions of these taxa are anchored in morphological characters.
while a great deal of information can be gleaned from macroscopic and microscopic observation, over the past two decades numerous examples of cryptic and pseudo-cryptic variation in algae have been highlighted through the analysis of molecular data  <cit> , <cit> , and a definitive application of a pre-existing taxonomic name can only be made in light of demonstrated molecular affinity with type material; an exhausting and impossible prospect for the plethora of collections resulting from a biodiversity survey.
thus, it is expected that the taxonomic names applied to hawaiian non-marine algae will be re-visited and re-examined as more comparative data become available.
finally, several practical difficulties played into a tradeoff between the number of samples that could be characterized and the number for which species-level taxonomic names could be applied.
most non-marine algal collections were relatively small in size  and were often heavily epiphytized; obtaining sufficient amounts of clean material for dna extraction and pcr amplification was challenging.
additionally, many of the common taxa are typically assigned taxonomic names based heavily on characters pertaining to sexual reproduction , which was very infrequently observed in the field-collected samples.
attempts to induce sexual reproduction in culture in the laboratory were frequently unsuccessful; these observations suggest that sexual reproduction in hawaiian non-marine algae may naturally occur much less frequently than for those in other geographical locations.
CONCLUSIONS
the hawaiian freshwater algal biodiversity survey yielded the largest sequence data compilation yet for tropical non-marine algae, which will serve as a baseline for comparison of new collections within the hawaiian islands and to other tropical regions.
key features of the biodiversity survey include the following: 1) the hawaiian freshwater algal database, which includes data for both collection sites and individual algal samples, which was designed and implemented as a key organizational component of the study, and serves as a web-accessible project portal to the scientific and broader communities.
these kinds of digital resources are becoming increasingly recognized as critical data sources to those extracting and compiling broader scale patterns  <cit> .
2) analysis of the  <dig>  collecting sites and resultant  <dig>  macroalgal samples, which informed the construction of the first comprehensive checklists for non-marine algae from unusual habitats in the hawaiian islands , and an updated taxonomic checklist of hawaiian non-marine macroalgae .
3) in-depth assessment of common hawaiian taxa, including representatives of the green algae , red algae  and several cyanobacteria .
future research characterizing other tropical non-marine algal floras is needed to be able to draw comparisons between the flora of the hawaiian islands and elsewhere, to foster an understanding of the level of endemism in these isolated systems, and to illuminate the biodiversity of these understudied components of tropical island floras.

 eight main islands and small islands, atolls and shoals comprise the hawaiian archipelago, which encompasses  km and represents the largest oceanic archipelago in the world,. the islands are volcanically-derived, and form successively as the pacific plate moves in a northwestern direction over a fixed “hot spot”, such that the eight current high islands at the southeastern end of the chain represent only the last million years of a 75– my history of volcanic activity. the main hawaiian islands are characterized by steep gradients of elevation, rainfall and island age, which have generated a remarkable range of ecological conditions,. the hawaiian islands are well known as a uniquely isolated and time-calibrated environment in which to study evolution; a place where numerous lineages of plants and animals exemplify the phenomena of endemism and adaptive radiation -. the geographical isolation of the main hawaiian islands is a strong force influencing the biogeography of hawaii, and initial colonization of the islands was accomplished through the arrival and survival of dispersed individuals. subsequent colonization likely occurred through a combination of long-distance dispersal, and, increasingly commonly, dispersal from adjacent islands. a number of studies on the freshwater algae of the hawaiian islands have appeared in the literature -. eight hundred taxonomic records were compiled from the literature in a bibliographic checklist spanning the years 1876–, which included representatives of almost all major freshwater algal lineages. based on these literature records, the overall level of endemism for the hawaiian non-marine algae was estimated to be 5%, or a total of taxa. this endemism level was noted as being very low in comparison to other organisms such as marine red algae, marine invertebrates, ferns and lycophytes, flowering plants, and insects,,. it was subsequently noted that the stream algal flora of hawaii was “suspiciously cosmopolitan” in composition, drawing attention to the large proportion of hawaiian taxonomic records in common with very different biogeographic regions of the world. others have noted that freshwater algae around the world have often been considered to be cosmopolitan, that this notion is unlikely to hold true when tested with genetic data, and that endemism is most likely obscured by the “force-fitting” of european names. competing explanations exist for the biogeographic distributions of freshwater algae. distributions of microbial algae can be examined in the context of “everything is everywhere, but, the environment selects”, which predicts that microorganisms are globally distributed as a result of their vast population sizes and unlimited dispersal, and whether or not a species becomes established is determined solely by local conditions,. in contrast, a number of researchers have reported evidence of biogeographic patterning of microorganisms by using larger sample sizes, detailed observations, and/or molecular assessments of diversity -, and still others invoke a combination of ubiquity and endemism to explain cryptic molecular lineages of widespread morphospecies,. whether hawaiian freshwater algae are truly cosmopolitan when molecular comparisons are taken into account, or whether they display levels of endemism that parallel other elements of the flora and fauna of the archipelago, is currently unknown. given current estimates of biodiversity loss, it is critical to understand the scope of endemism versus cosmopolitanism in the poorly characterized freshwater flora of a region already recognized as a biodiversity hotspot for other lineages of organisms. loss of biodiversity is proceeding faster in freshwaters than in any other major biome -. the hawaiian freshwater algal biodiversity survey was carried out to establish baseline data to further assess human impacts on freshwater hawaiian ecosystems and monitor change in the context of biodiversity conservation. the goals of the survey were to collect, document, and characterize as many non-marine algal taxa as possible from the main hawaiian islands, and to use the resultant data to examine distribution patterns and develop biogeographic hypotheses to explain these patterns. the first author held responsibility for the characterization of the macroalgal samples resulting from the survey, and as such the focus of the present report is on these taxa. expeditionary collections were made on the islands of kauai, oahu, molokai, lanai, maui and hawaii, and macroalgal specimens were characterized morphologically and, where possible, using molecular methods. the distributional patterns and biogeographic affinities of taxa based on both literature records and dna sequence analyses were examined in the context of distributions characterized by cosmopolitanism versus endemism. with very little information currently available for biogeographic patterns of specific freshwater algae based on molecular data trends, no predictions of biogeographic affinity were made for the hawaiian non-marine algae. however, an increase in the number of recognized endemic taxa was predicted, given the frequency with which freshwater algae are demonstrated to harbor cryptic diversity or pseudocryptic diversity when molecular tools are employed.  summary of collections a total of distinct sites were sampled. collection sites were concentrated on the wetter, windward sides of the islands, and on the larger islands that had a greater number of perennial streams and associated moist habitats. sampling sites were not chosen randomly, but were selected on the basis of accessibility and likelihood of harboring interesting algal diversity; thus, the following numbers should be interpreted as a summary of the characteristics of the collections rather than an indication of available habitats in the hawaiian islands. areas not sampled lacked suitable habitats or were not accessible. streams comprised % of all sites, followed by terrestrial sites and wet walls, with smaller proportions of the remaining habitat categories. a total of isolate accessions were characterized, of which were macroalgae. slightly more than % of these isolate accessions were green algae, while diatoms, red algae, cyanobacteria, xanthophytes and euglenoids comprised the remainder. additional file summarizes the taxa identified, along with their islands of distribution and the habitat types from which they were collected and identified. the most widely collected taxa included the green algae spirogyra spp., mougeotia spp., oedogonium spp., cladophora glomerata, microspora spp., cloniophora spicata, and rhizoclonium spp., the red algal species compsopogon caeruleus and the xanthophyte vaucheria spp.. dna barcoding a total of dna sequences were generated as part of the survey, representing eight different markers. the upa marker, which was employed as an initial screen for all samples, accounted for more sequences than any other marker, while ssu, rbcl, lsu, coi and 16s rrna comprised the majority of the remainder. sequence data for the 16s rrna, coi, lsu, ssu, rbcl, and upa markers were employed to investigate the molecular diversity of specific lineages of the hawaiian non-marine algal flora. sequence diversity for each sampled taxon is represented in neighbor-joining trees based on uncorrected p-distances. rather than attempting to reconstruct phylogenetic relationships for each taxon, we aimed to use these data for an assessment of patterns of molecular diversity within the hawaiian flora. thus, higher order relationships are not inferred from these analyses; comparisons should be restricted to closely-related taxa. more in-depth phylogenetic analyses have been published or are in progress for a number of taxa, including green and red algae,, cyanobacteria and diatoms. rbcl sequences of the hawaiian non-marine charophycean green algae. sequence diversity based on the rbcl marker for the hawaiian non-marine charophycean green algae. the neighbor-joining tree is based on uncorrected p-distances nucleotide model in mega. scale bar = substitutions per site. rbcl sequences of the hawaiian non-marine red algae. sequence diversity based on the rbcl marker for the hawaiian non-marine red algae. the neighbor-joining tree is based on uncorrected p-distances nucleotide model in mega. scale bar = substitutions per site. summary taxon labels on each tree reflect the lowest taxonomic level to which a confident assignment can be made for a cluster of identical or near-identical sequences. the vast majority of samples were not reproductive at the time of collection, which limited the number of taxa that could be identified to the species level. nonetheless, clustering patterns of closely related sequences can be used as an indication of the number of entities represented in the hawaiian flora for a given genus. for example, the upa analysis of cyanobacteria suggests eight species of the genus nostoc were collected and characterized during the surveys, two of which were included in the 16s rrna analysis. four species of the red algal form genus “chantransia” were identified from the upa analysis, representing members of the batrachospermales and thoreales, and these species-level groupings were consistent with groups recovered in the coi, rbcl and ssu analyses. the most species-rich genus of freshwater macroalgae studied in this survey was the charophycean green alga spirogyra, which was demonstrated to consist of clusters of sequences for the upa marker, for rbcl, and nine for ssu; an in-depth comparison of these sequences to accessions worldwide and a comparative analysis of thallus morphology is underway. categories of distribution the best-studied and best-represented taxa from the survey were examined for distributional trends: eight red algae, green algae, cyanobacteria, four diatoms and one xanthophyte, for a total of taxa. twelve taxa were determined to be in the category of broadest distribution, were in the category of second most broadly distributed, were members of the third category, and five were members of the putatively endemic category. it should be noted, however, that sampling effort has not been consistent among these regions of the world, with some having received many times the effort than others over the course of the past several centuries. for each of the taxa in each of the categories above, thallus size and dispersal unit size ranges were plotted to assess patterns associated with dispersal category. thallus sizes were found to range from microscopic to macroscopic for representatives of all dispersal categories. in contrast, dispersal unit size ranges were found to be uniformly ≤1 mm, and much smaller for both endemic and cosmopolitan taxa. discussion a comparative overview of the hawaiian non-marine macroalgal flora the macroalgal samples collected during the hawaiian freshwater algal biodiversity survey represent, by far, the most exhaustive survey of non-marine algae for a tropical oceanic island archipelago yet reported. previous studies in the tropical pacific have yielded between seven and samples, and have resulted in algal checklists of 12– taxa, -. algal studies of the islands of yap, in the caroline islands, revealed a diverse flora of cyanobacteria, green algae, red algae, euglenoids and dinoflagellates totaling taxa,, while, in contrast, a study of stream macroalgae of the islands of fiji suggested an impoverished stream algal diversity of taxa representing cyanobacteria, green algae and red algae. similar to the flora from fiji, a rapid survey of fresh waters from the austral islands of french polynesia yielded species, representing cyanobacteria, green algae and xanthophytes. a number of previous studies of hawaiian stream algae have been conducted, which yielded from taxa to taxa to taxa; albeit all identifications from these past studies were morphologically based, and thus are not completely comparable to the current research, and were also more limited in collection intensity. for example, many of the common taxa previously reported from the hawaiian islands have been subsequently demonstrated through molecular analyses to represent different, but recognized species, endemic hawaiian taxa, or members of different genera. the present study is the first to integrate taxonomic collections from a multitude of non-marine algal habitats, including streams, wet walls, high elevation bogs, taro fields, ditches and flumes, cave walls and terrestrial areas for a tropical island algal flora. two of these habitats were sufficiently well represented in the present survey collections to warrant individual treatment, and these have been published elsewhere as reports on the non-marine algae of hawaiian taro fields and high elevation bogs. molecular markers and example within-lineage patterns a total of eight different molecular markers were employed in this study to characterize the non-marine algal diversity of the hawaiian islands. due to the large scope of phylogenetic diversity encompassed in the collections, we aimed to establish a multi-step molecular assessment that allowed initial broad-based characterization of all collections, with subsequent use of one or more lineage-specific markers for comparisons among closely related samples. our previous biodiversity survey of the hawaiian rhodophyta as well as other algal investigations - have provided support for the use of the upa marker as a near-universal region of the plastid genome that enables construction of a molecular biodiversity framework for almost all algae, and we advocate for the use of this marker in multi-step molecular characterizations or analyses of environmental dna samples where characterization of both cyanobacterial and eukaryotic algal diversity is desirable. our second most widely applied marker, which spans the v region of nuclear ssu,, also resulted in generation of a large number of sequences, albeit fewer than upa and of a more conserved nature than that marker. a comparison of the use of both markers for the characterization of environmental dna from stream periphyton is underway. a number of taxa were well-represented in the survey, and the hawaiian biodiversity represented by these lineages was characterized using a combination of the molecular approaches described above and microscopic analysis. for example, the charophycean genus spirogyra has been noted as one of the most common freshwater macroalgae across the hawaiian islands in numerous publications dating back to -,, and our survey collections of this genus spanned locations on six islands, including diverse habitats such as streams, ditches, taro fields, bogs and ponds. the red algal “form genus” chantransia was previously thought to be represented in the hawaiian islands by two lineages, sheathia arcuata and nemalionopsis shawii; however, two additional taxa were added as a result of the survey collections, doubling the known diversity of these freshwater red algal sporophytes in hawaii. some non-marine representatives of the cladophorales, which can be difficult or impossible to identify using only morphological characters were demonstrated in some cases to be molecularly homogeneous, as demonstrated by others, despite substantial morphological variation, and in other cases to harbor genus-level molecular diversity masked by a simple morphology. thus, one of the most valuable outcomes of the biodiversity survey approach of collecting and characterizing all non-marine macroalgae from a geographical region is that it allows multi-taxon large scale comparisons that can be compared to more accurately assess taxonomy and infer broader distributional patterns. biogeographic patterns of the hawaiian non-marine algae baas becking’s ideas of microorganisms being globally distributed apply to organisms too small to see with the unaided eye, yet may be applicable to the study of freshwater macroalgae given that the dispersal forms of these organisms fall into the <1 mm size range. as a consequence, organism size may not predict distribution patterns well. dispersal mechanisms of non-marine algae have been the subject of much speculation but little empirical research. a number of means of transport have been proposed, including air currents, vector-assisted transport, or rafting -. although the initial colonization of the volcanically produced hawaiian islands by non-marine algae must have occurred by long-distance transport, dispersal among the islands likely played an increasingly important role in shaping the flora of the archipelago. the non-marine algal flora of the hawaiian islands has almost certainly been impacted by humans since their first arrival in the islands. polynesian introductions of numerous plant species for food and other uses may account for the first anthropogenic changes to the flora, but an acceleration of human impact over the past several hundred years has probably had an even greater influence its composition, with spores and fragments being transported to and among the islands from activities such as the aquarium and ornamental plant trades, hiking, transport of building materials and heavy machinery, and farming. any attempts to interpret algal biogeographic patterns must take these influences into consideration. what is evident from the survey collection analyses is that no consistent biogeographic pattern emerges to explain the origins of the non-marine algal flora of the hawaiian islands. some remarkably widespread taxa were characterized that are also known from many locations worldwide, including the clonally propogating red alga compsopogon caeruleus and the green alga cladophora glomerata; distribution of the former to hawaii may be a result of the aquarium trade, while ubiquity in the latter case may be a result of either anthropogenic or non-anthropogenic dispersal. a few taxa were demonstrated to consist of multiple, cryptic lineages that likely represent intraspecific taxonomic variation, including sheathia arcuata and some spirogyra spp. a number of other taxa were found to have affinities with samples from a more limited number of other regions, but with little obvious biogeographical connection, for example, cloniophora spicata, nemalionopsis shawii, pithophora roettleri, schizomeris leiblienii and thorea hispida. in the absence of well-populated reference frameworks of collections from numerous locations it seems likely that these distributions are a product of under-sampling rather than being representative of true biogeographic patterns. conversely, several taxa were identified that are currently considered endemic to the hawaiian islands, and it is possible that expanded distributions of these taxa may be recognized in the future with increased sampling and characterization of the non-marine algal floras of other tropical pacific islands. it should also be noted that a number of microalgal taxa have also been recently described from the hawaiian islands as a result of this biodiversity survey, which serves to further emphasize the rich flora of this archipelago that is being illuminated through this opportunity for focused taxonomic attention, -. accurate taxonomic identification of algae is not straightforward, and much depends on the species concept employed. many of the taxa reported in this survey were described decades, if not centuries ago, and the formal descriptions of these taxa are anchored in morphological characters. while a great deal of information can be gleaned from macroscopic and microscopic observation, over the past two decades numerous examples of cryptic and pseudo-cryptic variation in algae have been highlighted through the analysis of molecular data,, and a definitive application of a pre-existing taxonomic name can only be made in light of demonstrated molecular affinity with type material; an exhausting and impossible prospect for the plethora of collections resulting from a biodiversity survey. thus, it is expected that the taxonomic names applied to hawaiian non-marine algae will be re-visited and re-examined as more comparative data become available. finally, several practical difficulties played into a tradeoff between the number of samples that could be characterized and the number for which species-level taxonomic names could be applied. most non-marine algal collections were relatively small in size and were often heavily epiphytized; obtaining sufficient amounts of clean material for dna extraction and pcr amplification was challenging. additionally, many of the common taxa are typically assigned taxonomic names based heavily on characters pertaining to sexual reproduction, which was very infrequently observed in the field-collected samples. attempts to induce sexual reproduction in culture in the laboratory were frequently unsuccessful; these observations suggest that sexual reproduction in hawaiian non-marine algae may naturally occur much less frequently than for those in other geographical locations.  the hawaiian freshwater algal biodiversity survey yielded the largest sequence data compilation yet for tropical non-marine algae, which will serve as a baseline for comparison of new collections within the hawaiian islands and to other tropical regions. key features of the biodiversity survey include the following: 1) the hawaiian freshwater algal database, which includes data for both collection sites and individual algal samples, which was designed and implemented as a key organizational component of the study, and serves as a web-accessible project portal to the scientific and broader communities. these kinds of digital resources are becoming increasingly recognized as critical data sources to those extracting and compiling broader scale patterns. 2) analysis of the collecting sites and resultant macroalgal samples, which informed the construction of the first comprehensive checklists for non-marine algae from unusual habitats in the hawaiian islands, and an updated taxonomic checklist of hawaiian non-marine macroalgae. 3) in-depth assessment of common hawaiian taxa, including representatives of the green algae, red algae and several cyanobacteria. future research characterizing other tropical non-marine algal floras is needed to be able to draw comparisons between the flora of the hawaiian islands and elsewhere, to foster an understanding of the level of endemism in these isolated systems, and to illuminate the biodiversity of these understudied components of tropical island floras. 
the hawaiian freshwater algal biodiversity survey represents the first comprehensive effort to characterize the non-marine algae of a tropical region in the world using both morphological and molecular tools. dna sequences were generated representing eight different markers, which enabled an assessment of the number of taxonomic entities for genera collected as part of the survey. aquatic and terrestrial habitats and distinct collections of non-marine macroalgae were collected from the islands of kauai, oahu, molokai, maui, lanai and hawaii from the years 2009– targeted habitats included streams, wet walls, high elevation bogs, taro fields, ditches and flumes, lakes/reservoirs, cave walls and terrestrial areas. we conducted a biodiversity survey of the non-marine macroalgae of the six largest main hawaiian islands using molecular and microscopic assessment techniques. algal distributionbiodiversity surveybiogeographycyanobacteriadispersalfreshwater algaehawaiian islandsmolecular characterizationtaxonomyupa approximately 50% of the identifications were of green algae, with lesser proportions of diatoms, red algae, cyanobacteria, xanthophytes and euglenoids. a remarkable range of environmental conditions is present in the hawaiian islands due to their gradients of elevation, rainfall and island age. despite being well known as a location for the study of evolutionary processes and island biogeography, little is known about the composition of the non-marine algal flora of the archipelago, its degree of endemism, or affinities with other floras. we aimed to evaluate whether endemism or cosmopolitanism better explain freshwater algal distribution patterns, and provide a baseline data set for monitoring future biodiversity changes in the hawaiian islands. these analyses yielded an updated checklist of the non-marine macroalgae of the hawaiian islands, and revealed varied biogeographic affinities of the flora that are likely a product of both natural and anthropogenic dispersal. this analysis revealed no clear biogeographic affinities of the flora, with % characterized as “cosmopolitan”, % “endemic”, and % as intermediate. survey data were entered in the hawaiian freshwater algal database, which serves as a digital repository of photographs and micrographs, georeferenced localities and dna sequence data. sites that lacked freshwater macroalgae were typically terrestrial or wet wall habitats that were sampled for diatoms and other microalgae.
 a remarkable range of environmental conditions is present in the hawaiian islands due to their gradients of elevation, rainfall and island age. despite being well known as a location for the study of evolutionary processes and island biogeography, little is known about the composition of the non-marine algal flora of the archipelago, its degree of endemism, or affinities with other floras. we conducted a biodiversity survey of the non-marine macroalgae of the six largest main hawaiian islands using molecular and microscopic assessment techniques. we aimed to evaluate whether endemism or cosmopolitanism better explain freshwater algal distribution patterns, and provide a baseline data set for monitoring future biodiversity changes in the hawaiian islands.  aquatic and terrestrial habitats and distinct collections of non-marine macroalgae were collected from the islands of kauai, oahu, molokai, maui, lanai and hawaii from the years 2009– targeted habitats included streams, wet walls, high elevation bogs, taro fields, ditches and flumes, lakes/reservoirs, cave walls and terrestrial areas. sites that lacked freshwater macroalgae were typically terrestrial or wet wall habitats that were sampled for diatoms and other microalgae. approximately 50% of the identifications were of green algae, with lesser proportions of diatoms, red algae, cyanobacteria, xanthophytes and euglenoids. dna sequences were generated representing eight different markers, which enabled an assessment of the number of taxonomic entities for genera collected as part of the survey. forty-four well-characterized taxa were assessed for global distribution patterns. this analysis revealed no clear biogeographic affinities of the flora, with % characterized as “cosmopolitan”, % “endemic”, and % as intermediate.  the hawaiian freshwater algal biodiversity survey represents the first comprehensive effort to characterize the non-marine algae of a tropical region in the world using both morphological and molecular tools. survey data were entered in the hawaiian freshwater algal database, which serves as a digital repository of photographs and micrographs, georeferenced localities and dna sequence data. these analyses yielded an updated checklist of the non-marine macroalgae of the hawaiian islands, and revealed varied biogeographic affinities of the flora that are likely a product of both natural and anthropogenic dispersal. algal distributionbiodiversity surveybiogeographycyanobacteriadispersalfreshwater algaehawaiian islandsmolecular characterizationtaxonomyupa 