Past and present patterns of connectivity among populations of four cryptic species of freshwater mussels Velesunio spp. (Hyriidae) in central Australia

We examined genetic structure and levels of connectivity among subpopulations within each of four cryptic species belonging to the freshwater mussel genus Velesunio. We used allozymes and a fragment of the mitochondrial cytochrome c oxidase I gene to examine genetic variation in populations from isolated waterholes, belonging to four major inland drainages in eastern Australia. Based on evidence from other invertebrates in the region we predicted that, in each species, we would find evidence of historical connectivity among populations from different drainages. This was clearly not the case, as for the two species that occurred in more than one drainage there was evidence of both current and past restriction to gene flow. Moreover, given the potential for extensive dispersal of these mussels through the river systems during flood times via their fish hosts, we predicted low levels of genetic variation among populations from waterholes in the same drainage. Contrary to our expectations, all four species showed some evidence of restricted gene flow among waterholes within drainages. This suggests that either (a) mussel larvae are not produced during flood times, when their fish hosts would be free to move between waterholes, or (b) mussel larvae are attached to their hosts at these times, but the fish movement is limited between waterholes.     

Dispersal ability and genetic structure in aquatic invertebrates: a comparative study in southern California streams and reservoirs

1. The natural seasonal drying and flooding of southern California streams have been altered over the past century by activities related to agriculture, flood control, and reservoir construction. The genetic structure and diversity of aquatic invertebrates inhabiting these environments is largely unexplored, and may be important for conservation. 2. We sampled two species of aquatic invertebrates with different dispersal abilities to assess genetic structure and diversity, and make inferences about the evolutionary processes that underlie these genetic patterns. The mayfly Fallceon quilleri, which has a winged terrestrial stage, was sampled from perennial and intermittent streams from three catchments across San Diego County. The amphipod Hyalella azteca was sampled from streams (perennial and intermittent) and reservoirs in a single catchment (San Dieguito). Because it is obligately aquatic throughout its life‐cycle, H. azteca was assumed to disperse less than F. quilleri. 3. Intrapopulation and overall genetic diversity was higher in F. quilleri than in H. azteca. In F. quilleri there was very little genetic divergence among populations, and most of the genetic differentiation that was observed could be attributed to a single population. In H. azteca, populations were markedly differentiated between the upper and lower segments of the San Dieguito basin, which are separated by a c. 10 km section of stream that rarely has surface flow. Within both segments, genetic divergence between sites connected by reservoirs and perennial streams was not significantly different. 4. Our results suggest that F. quilleri disperses widely and thus avoids genetic bottlenecks and marked levels of population differentiation that may be expected from frequent extinctions and recolonizations. In contrast, restricted dispersal in H. azteca is associated with relatively low genetic diversity and high genetic divergence across a portion of the catchment in which surface flow is rare.     

Distribution of aquatic vascular plants in lowland rivers: separating the effects of local environmental conditions, longitudinal connectivity and river basin isolation

1. Changes in species distributions along rivers have rarely been observed independently of changes in environmental conditions and meaningful comparison between different catchments is made difficult by the limited geographical distribution of species. This study presents a new approach to quantify the effect of the spatial structure of lowland river networks on aquatic plant distribution and to explore the potential underlying processes using species life‐history characteristics. 2. Twenty‐five species of aquatic vascular plants recorded in 62 sites across five calcareous river basins were used to investigate (i) the temporal turnover of plant species, (ii) the habitat utilisation of species, (iii) the trade‐offs between different plant life‐history characteristics and (iv) the relationship between species life‐history characteristics and habitat utilisation. 3. The annual plant turnover within a 3‐year period was, although significant, extremely low. It suggests that results from spatial surveys conducted over 3 years should not be undermined by temporal changes. 4. Spatial connectivity along and between rivers was more important than in‐channel physical characteristics in shaping species assemblages. Neither chemical factors (ammonium, phosphate) nor extrinsic biotic competitors (filamentous green algae) significantly influenced plant distribution. 5. The most common combinations of life‐history characteristics were neither related to environmental conditions nor to spatial isolation. Instead, they could reflect natural selection processes associated with larger scales than those considered in this study. 6. Plant distribution was most strongly related to the dispersal and regeneration abilities of the plants, supporting the hypotheses relating to longitudinal connectivity. The hypothesis that different growth forms would be associated with different in‐channel physical features was not verified. As expected, there were no substantial differences in plant life‐history characteristics between river basins.     

Local habitat restoration in streams: Constraints on the effectiveness of restoration for stream biota

Summary   The restoration of physical habitat has emerged as a key activity for managers charged with reversing the damage done by humans to streams and rivers, and there has been a great expenditure of time, money and other resources on habitat restoration projects. Most restoration projects appear to assume that the creation of habitat is the key to restoring the biota ('the field of dreams hypothesis'). However, in many streams where new habitat is clearly required if populations and communities are to be restored, there may be numerous other factors that cause the expected link between habitat and biotic restoration to break down. We discuss five issues that are likely to have a direct bearing on the success, or perceived success of local habitat restoration projects in streams: (i) barriers to colonization, (ii) temporal shifts in habitat use, (iii) introduced species, (iv) long-term and large-scale processes, and (v) inappropriate scales of restoration. The purpose of the study was primarily to alert ecologists and managers involved in stream habitat restoration to the potential impacts of these issues on restoration success. Furthermore, the study highlights the opportunities provided by habitat restoration for learning how the factors we discuss affect populations, communities and ecosystems.     

湿地生物连通性影响因素及测度方法研究进展

湿地是地球上最多样化和最具生产力的生态系统之一,但近年来由于人类活动的频繁干扰,自然环境以前所未有的速度变化,两者的协同作用导致湿地呈现破碎化趋势。湿地的退化、转化使得湿地生物连通性减弱。良好的湿地生物连通性有利于湿地生态系统服务功能的维持、生物多样性的保护以及生物对气候变化适应水平的提高。因此理解、评估和保护生物连通性对于湿地生态系统而言至关重要。综述了湿地生物连通性的定义、总结了生物连通发生与变化的四个相互作用因子,从结构连通性、功能连通性两个测定角度,论述动物、植物和微生物三类研究对象生物连通性的测度方法,最后从湿地生物连通的时空尺度,连通与物质循环的耦合关系,湿地生物连通性评估分析模型改进角度提出了未来的研究方向,从而确保湿地生物连通网络的完整有效。可为湿地生物多样性、湿地保护和管理提供新的思路。     

Restoring habitat connectivity over the road: vegetation on a fauna land‐bridge in south‐east Queensland

The Compton Road overpass in southern Brisbane is known to be allowing passage by numerous birds, reptiles and amphibians; but what type of vegetation is providing habitat suitable for these movements? An evaluation of the reconstructed vegetation on the overpass shows that 45 species of plants were present after 4 years of growth. Most of these were in the original planting list but over 40% have almost certainly come from salvaged native topsoil used in the bypass construction. The resulting habitat, 4 years later, now appears similar to a typical coastal eucalypt forest secondary succession, although not yet identical to nearby forest. The current presence of a dense mid‐layer of small trees and shrubs may be an important factor in allowing even the edge‐avoiding species to cross the road. On‐going investigation of successional changes and the changing responses of the birds and other taxa will contribute to understanding the functionality of such crossing structures.     

Dissolved oxygen decline in ice-covered rivers of northern Alberta and its effects on aquatic biota

To determine whether existing dissolved oxygen (DO) regulationswere adequate to protect riverine biota, the Northern River Basins Studyrequired a research and assessment program to establish the effect ofpulp mill and municipal sewage discharges on under-ice DO concentrationsand aquatic biota in the Athabasca, Wapiti and Smoky rivers of northernAlberta, Canada. Analysis of monitoring data collected over >30 yearsshowed that pulp mill and municipal effluents have caused sags in DObelow their discharges and contributed to an increased rate of declinein DO over 10s of kilometers in the Wapiti-Smoky rivers or 100s ofkilometers in the Athabasca River. To permit forecasting of DO declinesin response to changes in river discharge or effluent biochemical oxygendemand (BOD), a one-dimensional steady-state river water quality modelwas implemented that successfully (r² =0.74–0.92) predicted DO concentrations over an 820 km reach of theAthabasca River for the 1990–1994 winters. Experiments on nativefish and benthic macroinvertebrate species showed that exposure to lowDO and low temperatures (2–3°C) caused delays in hatch ofmountain whitefish eggs (at 6.5 mg/L DO), reduced mass of bull troutalevins post-hatch (at 5.0 mg/L DO), extended the spawning period ofburbot (at 6.0 mg/L DO), and depressed feeding rates and loweredsurvival of the mayfly Baetis tricaudatus (at 5 mg/L DO).As a result of recommendations from this integrated program of rivermonitoring, modelling and experimentation, effluent BOD loading has beenreduced to the Athabasca and Wapiti rivers and the provincial DOguideline for protection of aquatic life has been increased from 5.0 to6.5 mg/L.     

Barriers to the recovery of aquatic insect communities in urban streams

1. Worldwide, increasing numbers of stream restoration projects are being initiated to rehabilitate waterways modified by urbanisation. However, many of these projects have limited success in restoring stream communities. Prompted by this, we investigated previously unrecognised barriers to aquatic insect colonisation in urban streams. 2. To investigate whether the availability of suitable substrata for oviposition limited the longitudinal distribution of caddisflies, large boulders were added to the upstream reaches of one stream. Prior to the addition, more egg masses were observed downstream and this longitudinal pattern persisted subsequently. 3. Malaise trapping revealed that adult caddisfly diversity and abundance was greater downstream than upstream. Furthermore, in a previous study the authors found larval caddisflies reflected the longitudinal distribution of adults. 4. The only obvious potential obstructions between reaches were roads beneath which the stream flowed through culverted crossings. Malaise trapping was used to examine the effect of road culverts and bridges on caddisfly dispersal. Numbers of caddisflies caught declined upstream and about 2.5 × more individuals were taken in traps immediately below than above five culverts. 4. Bridges, which had a more open structure than culverts, had no significant effect on the size of catches made above and below them. 5. Road culverts could act as partial barriers to upstream flight, with consequences for larval recruitment in urban streams. We recommend that urban planners and designers of restoration projects consider possible synergistic effects of poor oviposition habitat and barriers to aquatic insect dispersal, which may be critical for the colonisation of urban headwater streams and for the maintenance of stream insect populations.     

Efforts to restore habitat connectivity for an upland tropical rainforest fauna: A trial of underpasses below roads

Summary Four large underpasses, specifically designed for movements by fauna, form part of a major road upgrade project on the Atherton Tablelands in northeast Queensland, Australia. We describe the design and rationale of a project to test their effectiveness in restoring habitat continuity for tropical rainforest fauna. The large blocks of upland rainforest divided by the road are recognized for their high faunal conservation significance, forming habitat for many rare or threatened species. Ecologists, road engineers and the Atherton Tablelands conservation community have united with a common conservation goal: to design the 'furniture' within the underpasses and accomplish rainforest revegetation to provide protective cover and attract fauna to underpass entrances. Prior to construction, small mammals were trapped weekly for several months in habitats close to the road upgrade. The small mammal community comprised grassland species in abandoned pasture and differed significantly from the rainforest specialists found in three closed canopy habitats: rainforest edge, rainforest interior and Lantana shrubland. Rainforest restoration works designed to restore connectivity for rainforest fauna across this abandoned pastureland (via the underpasses to major rainforest blocks to the north and south of the road) are currently in progress. Monitoring of the effectiveness of the underpasses in allowing faunal movements will involve the use of infra-red-triggered cameras within the underpasses and near underpass entrances, and a survey of road-killed fauna both prior to and postconstruction. Further examination of small mammal community structure and movements, with respect to both the new road and the underpasses, will be undertaken once rainforest plantings have become established. This evaluation should provide insights for further road-associated restoration projects in rainforest regions.     

Mitochondrial DNA and allozymes reveal high dispersal abilities and historical movement across drainage boundaries in two species of freshwater fishes from inland rivers in Queensland, Australia

This study used allozymes and mtDNA variation to test that: 1) populations of two fish species, Nematolosa erebi and Retropinna semoni , in lowland rivers in central Australia were highly connected within drainages, 2) populations in different drainages were highly differentiated and 3) there was evidence of historical connections between two major lowland drainages in inland Australia. Levels of genetic differentiation among populations within drainages were low, but still statistically significant, indicating that populations were not as highly connected as had been predicted. Populations from the Murray–Darling and the Lake Eyre drainages were highly differentiated, indicating no contemporary dispersal across drainage boundaries. Both species showed evidence of historical connections between the two drainage basins, although estimates of the time that these last occurred differed between the two species. Nematolosa erebi populations from the two drainages were estimated to have been separated c . 150 000 years ago, whereas populations of R. semoni , were estimated to have been separated c . 1.5 million years ago.     

Long-term recovery of macroinvertebrate biota in grossly polluted streams: Re-colonisation as a constraint to ecological quality

To meet targets imposed by the European Water Framework Directive (2000/60/EC) it is vital that measures to improve the status of rivers are both effective and economically viable. Achievement of such aims needs robust understanding of biological responses to changes in water quality vis-à-vis mechanisms of and constraints to the colonization of previously polluted sites. This study therefore examined the long-term chemical and biological changes in historically polluted rivers to elucidate the responses of macroinvertebrate biota to improvements in chemical water quality. For three historically polluted sites in the English Midlands, data from surveys over a period of ca. 50 years were analysed. Ammonia (NH₃) and 5-day biochemical oxygen demand (BOD₅) were used as chemical water quality indicators. Variations in the ecological recovery of the study sites were assessed using an average pollution sensitivity score (Average Score Per Taxon) and the number of taxa present (usually to family level) present in hand-net samples. Ecological recovery varied widely and was influenced by the intensity and spatial extent of the pollution and the proximity of available sources of potential colonisers. At the site most isolated from potential sources of colonizing taxa, no clean-water macroinvertebrate taxa were recorded 30 years after the major sources of pollution ceased. Where clean-water colonisers were more readily available, significant improvements in ecological quality followed within 2–5 years of the improvements in chemical quality. Macroinvertebrate communities and hence monitoring data may thus be indicative of long past conditions or of biological isolation rather than contemporaneous chemical conditions. Combined chemical and biological data were used to explore a generic model for predicting recovery rates and success. Neither BOD₅ nor NH₃ were found to provide a consistent and meaningful prediction of either average pollution tolerance of macroinvertebrate taxa or of the number of taxa present. Long-term relationships between macroinvertebrate variables and chemical water quality variables, however, were non-linear, suggesting that water quality thresholds may have to be exceeded before biological recovery can occur. Even when chemical water quality has been improved substantially, the apparent ecological status of macroinvertebrate communities may not reflect reduced pollution levels attained until adequate time to allow for re-colonisation (possibly decades) has elapsed.     

Towards routine DNA metabarcoding of macroinvertebrates using bulk samples for freshwater bioassessment: Effects of debris and storage conditions on the recovery of target taxa

1. Macroinvertebrates are commonly sampled for bioassessment of freshwater ecosystems. However, current bioassessment protocols involve laborious sorting of the animals from the debris (sample matrix) and morphological identification, where species level identifications are often difficult. DNA metabarcoding has the potential to improve bioassessment by reducing the time taken to process samples and improve the accuracy and speed of macroinvertebrate species identification.
2. In this study, we evaluated DNA metabarcoding of macroinvertebrate samples, which include macroinvertebrates and the debris collected in the sample nets, to test if bulk, unsorted samples can be used to assess macroinvertebrate diversity. First, we tested if the sample matrix prevented the detection of six target macroinvertebrate taxa when DNA metabarcoding. Second, we tested if sample storage influenced the detection of the same six target macroinvertebrates. We also explored different levels of replication at the sample, sub-sample, and polymerase chain reaction levels and compared the overall macroinvertebrate families detected using DNA metabarcoding to those identified morphologically.
3. We found that the presence of the sample matrix did not interfere with or inhibit the detection of the six target macroinvertebrate taxa. Furthermore, we found that the various sample storage methods did not affect target macroinvertebrate detection. The reliability of detection of the target macroinvertebrates improved as hierarchical levels of replication were combined. We found strong overlap between the detection of overall macroinvertebrate family diversity when comparing DNA metabarcoding to morphological identification.
4. Extracting DNA from the bulk macroinvertebrate samples that included the sample matrix and using this for DNA metabarcoding could improve bioassessment by removing the need for laborious sorting of samples. Furthermore, DNA metabarcoding detection of the six target taxa was not dependent on sample storage of up to 1 year in 95% ethanol, at room temperature or after heating. DNA metabarcoding had the advantage of identifying macroinvertebrate species, but good DNA barcode libraries are needed for widespread species identifications. Further investigation should focus on including multiple samples with different macroinvertebrate composition and densities to refine and standardise bulk sample processing protocols, and on building comprehensive DNA barcode libraries for aquatic macroinvertebrates.

     

Restoring freshwater ecosystems in riverine landscapes: the roles of connectivity and recovery processes

1. This paper introduces key messages from a number of papers emanating from the Second International Symposium on Riverine Landscapes held in August 2004 in Sweden, focusing on river restoration. Together these papers provide an overview of the science of river restoration, and point out future research needs. 2. Restoration tests the feasibility of recreating complex ecosystems from more simple and degraded states, thereby presenting a major challenge to ecological science. Therefore, close cooperation between practitioners and scientists would be beneficial, but most river restoration projects are currently performed with little or no scientific involvement. 3. Key messages emanating from this series of papers are: The scope, i.e. the maximum and minimum spatial extent and temporal duration of habitat use, of species targeted for restoration should be acknowledged, so that all relevant stages in their life cycles are considered. Species that have been lost from a stream cannot be assumed to recolonise spontaneously, calling for strategies to ensure the return of target species to be integrated into projects. Possible effects of invasive exotic species also need to be incorporated into project plans, either to minimise the impact of exotics, or to modify the expected outcome of restoration in cases where extirpation of exotics is impractical. 4. Restoration of important ecological processes often implies improving connectivity of the stream. For example, longitudinal and lateral connectivity can be enhanced by restoring fluvial dynamics on flood‐suppressed rivers and by increasing water availability in rivers subject to water diversion or withdrawal, thereby increasing habitat and species diversity. Restoring links between surface and ground water flow enhances vertical connectivity and communities associated with the hyporheic zone. 5. Future restoration schemes should consider where in the catchment to locate projects to make restoration most effective, consider the cumulative effects of many small projects, and evaluate the potential to restore ecosystem processes under highly constrained conditions such as in urban areas. Moreover, restoration projects should be properly monitored to assess whether restoration has been successful, thus enabling adaptive management and learning for the future from both successful and unsuccessful restorations.     

Effects of land use on water quality and aquatic biota of three North Carolina Piedmont streams

Three streams in the Piedmont ecoregion of North Carolina were studied to evaluate the effect of land use (forested, agricultural, urban) on water quality and aquatic biota. In comparison with the forested stream, there were few changes in water quality at the agricultural and urban streams. Suspended-sediment yield was greatest for the urban catchment and least at the forested catchment. Suspended-sediment concentrations during storm events followed this same pattern, but at low-moderate flows suspended-sediment concentrations were greatest at the agricultural site. Most nutrient concentrations were highest at the agricultural site, and the amount of available dissolved nitrogen was elevated at both the urban and agricultural sites. High concentrations of metals (totals) in the water column were sometimes observed at all sites, but maximum average concentrations were recorded at the urban site (especially Cr, Cu, and Pb). Maximum sediment metal concentrations, however, were not found at the urban site, but were usually recorded at the forested site. Only minor differences were noted between fish communities of the forested and agricultural sites, although both abundance and average size of some species increased at the agricultural site. The fish community at the urban site was characterized by low species richness, low biomass, and the absence of intolerant species.Invertebrate taxa richness, a biotic index, and the number of unique invertebrate species (found at only one site) indicated moderate stress (Fair water quality) at the agricultural site and severe stress (Poor water quality) at the urban site. At the agricultural site, declines in taxa richness within intolerant groups were partially offset by increases within tolerant groups. The agricultural stream had the highest abundance values, indicating enrichment. The urban site, however, was characterized by low species richness for most groups and very low abundance values. Analysis of seasonal patterns suggested detritus was the most important food source for invertebrates in the forested stream, while periphyton was of greater importance in the agricultural stream. Dominant macroinvertebrate groups shifted from Ephemeroptera at the forested site, to Chironomidae at the agricultural site, and Oligochaeta at the urban site. There was little between-site overlap in dominant species (8–7%), indicating that land use strongly influenced the invertebrate community. Chemical and physical parameters measured at the three sites did not seem sufficient to account for all of the observed differences in the invertebrate communities, suggesting some unmeasured toxicity. Biological measurements, especially macroinvertebrates community structure, consistently indicated strong between-site differences in water and habitat quality.     

Small-scale patterns of genetic variation in the mayfly Bungona narilla (Ephemeroptera: Baetidae) in rainforest streams, south-east Queensland

SUMMARY 1. Genetic structure of the mayfly Bungona narilla was examined using allozymes and a section of the cytochrome oxidase I gene.
2. The study had two major aims. The first was to determine whether patterns of genetic variation in mitochondrial DNA were similar to those found previously for allozymes, i.e. that more variation was evident among pools within a single stream than among streams. The mitochondrial DNA results were similar to those reported previously for allozymes, supporting the idea that larvae within any particular pool were unrepresentative of the total population and may result from a few matings.
3. The second aim was to test the hypothesis that the variation among pools within a stream was greater after dry periods than after wet periods. This was because after wet periods, larvae would have greater opportunity for mixing because of movement among pools. This hypothesis was partly supported by the mitochondrial DNA data but not by the allozyme data, in which variation among pools was extremely low on both sampling occasions. The reasons for this difference are unclear.     

Biodiversity in extreme aquatic environments: Lakes, ponds and streams of the Ross Sea sector, Antarctica

The Ross Sea Sector (RSS) of Antarcticallies between the lines of longitude 150°E and 150°W and contains diverse landscapes with a variety of lakes, ponds and streams. Neither insects nor crustacean species have been recorded in these ecosystems but most contain planktonic and/or benthic communities that are composed exclusively of microscopic organisms. Microbial brodiversity is low with a small number of species (e.g. filamentous cyanobacteria of the family Oscillatoriaceae) occurring under a broad range of environmental conditions throughout the region. There is no evidence to date of microbial endemism in the RSS; however, there is a need to apply molecular and cellular techniques to compare biodiversity and genetic characteristics with assemblages elsewhere in Antarctica and with comparable communities in the north polar zone. A series of hypotheses are advanced to help guide further work. These derive from the conclusion that environmental extremes plus biogeographical isolation control the biodiversity of RSS communities, and that biological interactions (competition, grazing, predation, parasitism) are weak and play a minor role by comparison with temperate latitude ecosystems.     

The application of molecular markers to the study and conservation of fish populations, with special reference to Salmo

The main molecular techniques which can be used to generate genetic markers, and the applications of these markers to studies of fish populations are outlined. Published and ongoing studies, in the authors' laboratories, on brown trout and Atlantic salmon are used to compare the resolution and applicability of allozyme, mitochondrial DNA and minisatellite (variable number of tandem repeats) markers for studies on population structuring, genetic variation within populations, and the impact of the accidental and deliberate introduction of non-native salmonids on the genetic make-up of natural populations.     

Comparative study of monitoring South-Finnish rivers and streams using macroinvertebrate and benthic diatom community structure

In southern Finland, most of the rivers are turbid and suffer from eutrophication and leaching of suspended solids from diffuse sources. We first related benthic diatom and macroinvertebrate structure to environmental factors using direct ordination. Second, benthic diatoms and macroinvertebrates were simultaneously sampled in several South-Finnish rivers and streams to compare two monitoring methods. The study sites constituted of some large, moderately nutrient rich rivers and some smaller, less eutrophic streams situated on the south coast of Finland. Diatom species distribution was most affected by conductivity, total P and latitude. Species distribution of macroinvertebrates was mostly related to channel width, conductivity and pH. For diatoms, separation of community structure between sampling stations was clear, but corresponding macroinvertebrate communities were more similar to each other. Correlation between diatom and macroinvertebrate pollution indices was rather low and insignificant (r = 0.28). As a whole, variation of macroinvertebrate index values (CV = 4.7%) among replicate samples was slightly lower than for diatom index (CV = 6.0%). On the contrary, community similarity between the replicate samples was slightly lower among macroinvertebrates (r = 0.770) due probably to their larger local scale spatial variation, sampling of more habitats and lower density compared to diatoms (r = 0.874). In conclusion, multiple pressures affecting the river ecosystems at different spatial and temporal scales should lead to choosing more than one biological monitoring method with clearly identifiable responses.     

Population structure and biogeography of migratory freshwater fishes (Prochilodus: Characiformes) in major South American rivers

Mitochondrial DNA (mtDNA) sequences were used to infer the phylogenetic relationships of Prochilodus species in the Paraná, Amazonas, Orinoco, and Magdalena basins. Sequences of ATPase subunits 6 and 8 (total 840 bp) were obtained for 21 Prochilodus specimens from the four river systems. Using Semaprochilodus as an outgroup, phylogenetic analyses showed that: (i) each river basin contains a monophyletic group of mtDNA lineages; and (ii) the branching order places Magdalena in a basal position with subsequent branching of Orinoco, Amazon and Paraná. The mitochondrial control region was sequenced for 26 P. lineatus (from the Paraná basin) and six other Prochilodus specimens from the Magdalena, Orinoco and Amazon. All 26 control region haplotypes were unique with sequence divergence ranging from 0.3 to 3.6%. The control region phylogeny is well resolved but phylogenetic structure is not associated with geography. For example, mtDNA haplotypes from the upper Paraná (Mogui Guassú) and the upper Bermejo, separated by at least 2600 km, have close genealogical ties. Phylogeographic analyses, including nested clade analysis, suggest high levels of gene flow within this basin.