Polymorphic microsatellite loci for species of Australian freshwater cod, <Emphasis Type="Italic">Maccullochella</Emphasis>

The Australian freshwater cod genus, Maccullochella is represented by three species: Murray cod, M. peelii peelii, eastern freshwater cod, M. ikei, and trout cod, M.macquariensis. Seven novel microsatellite loci from M. ikei and six previously published loci from M. peelii peelii were tested on wild populations of Murray, eastern and trout cod. Levels of polymorphism varied between species with 13 loci polymorphic in Murray cod, 9 in trout cod and 7 in eastern cod. Observed heterozygosities ranged from 0.053 to 0.842. This suite of microsatellite loci will facilitate future studies of the genetic status of wild and hatchery bred populations of Maccullochella.     

Isolation and characterization of 102 new microsatellite loci in Murray cod,Maccullochella peelii peelii (Percichthyidae), and assessment of cross‐amplification in 13 Australian native and six introduced freshwater species

We have isolated 102 polymorphic microsatellite loci from an enriched Murray cod DNA library and also assessed their amplification success in 13 native and six introduced freshwater fish species. The loci will serve the dual purpose of assessing wild population genetic structure for future conservation efforts, and for identifying markers for key quantitative trait loci important for aquaculture.     

Migratory behaviour shapes spatial genetic structure of cyprinid fishes within the Lake Malawi catchment

• Genetic differences among freshwater fish populations are dependent on life‐history characteristics of the species, including the range of adult dispersal and the extent of homing to natal breeding grounds. However, the effects of variation in such characteristics on population genetic connectivity are rarely studied comparatively among closely related species.
• We studied population genetic structure within three congeneric cyprinid species from the Lake Malawi catchment that differ substantially in life‐history traits and conservation status, using a combination of microsatellite and mitochondrial DNA markers. Mpasa (Opsaridium microlepis) is a large (70 cm total length) migratory species that spawns in rivers, but as an adult is exclusively known from the main lake body. Sanjika (Opsaridium microcephalum), is a medium size (30 cm total length) species that exists in lake breeding, river‐lake migratory and apparently landlocked populations. Dwarf sanjika (Opsaridium tweddleorum) is a small non‐migratory species (15 cm total length) that persists in small tributaries surrounding the main lake and adjoining rivers.
• The results revealed striking differences among the three species in spatial genetic structuring. The river‐lake migratory mpasa showed only weak yet significant population genetic structure within the main Lake Malawi catchment, suggesting that there is no strong natal homing. The habitat‐generalist sanjika showed only weak spatial genetic differentiation at microsatellite loci within the Lake Malawi catchment, but moderate structure in mitochondrial DNA, potentially reflecting male‐biased dispersal. The river‐restricted dwarf sanjika showed strong genetic structure in both microsatellite and mitochondrial DNA, suggesting strictly limited dispersal at both adult and juvenile stages.
• We conclude that contrasting migration life histories have resulted in dramatically different patterns of population genetic structure among these congeneric species. The observed patterns demonstrate how divergent life‐history evolution may strongly influence broader patterns of population genetic connectivity in freshwater fish, with consequences for management and conservation. Specifically the results suggesting gene flow among Lake Malawi populations of mpasa, an IUCN red‐listed ‘Endangered’ species endemic to the lake catchment, imply that conservation initiatives operating at both local and catchment scales are needed to reverse local population decline.

     

The development of 10 novel polymorphic microsatellite markers through next generation sequencing and a preliminary population genetic analysis for the endangered Glenelg spiny crayfish, Euastacus bispinosus

The Glenelg spiny crayfish, Euastacus bispinosus, is an iconic freshwater invertebrate of south eastern Australia and listed as ‘endangered’ under the Environment Protection and Biodiversity Conservation Act 1999, and ‘vulnerable’ under the International Union for Conservation of Nature’s Red List. The species has suffered major population declines as a result of over-fishing, low environmental flows, the introduction of invasive fish species and habitat degradation. In order to develop an effective conservation strategy, patterns of gene flow, genetic structure and genetic diversity across the species distribution need to be clearly understood. In this study we develop a suite of polymorphic microsatellite markers by next generation sequencing. A total of 15 polymorphic loci were identified and 10 characterized using 22 individuals from the lower Glenelg River. We observed low to moderate genetic variation across most loci (mean number of alleles per locus = 2.80; mean expected heterozygosity = 0.36) with no evidence of individual loci deviating significantly from Hardy–Weinberg equilibrium. Marker independence was confirmed with tests for linkage disequilibrium, and analyses indicated no evidence of null alleles across loci. Individuals from two additional sites (Crawford River, Victoria; Ewens Ponds Conservation Park, South Australia) were genotyped at all 10 loci and a preliminary investigation of genetic diversity and population structure was undertaken. Analyses indicate high levels of genetic differentiation among sample locations (F _ST  = 0.49), while the Ewens Ponds population is genetically homogeneous, indicating a likely small founder group and ongoing inbreeding. Management actions will be needed to restore genetic diversity in this and possibly other at risk populations. These markers will provide a valuable resource for future population genetic assessments so that an effective framework can be developed for implementing conservation strategies for E. bispinosus.     

Population genetics of wild Siniperca knerii Garman, 1912 in China as evaluated by microsatellites

The Chinese perch Siniperca knerii Garman, 1912 is an endemic perciform freshwater fish species mainly distributed in the Yangtze River and the Pearl River of China. To evaluate their genetic diversity in these rivers, microsatellite markers were used to assess the genetic structure and genetic variability among nine wild populations of S. knerii. In this study, the 12 loci used were polymorphic, with observed heterozygosity (Ho) ranging from 0.4079 to 0.8886 and polymorphism information content (PIC) ranging from 0.4511 to 0.7146. Results showed that six populations from the Yangtze River had a higher genetic diversity than those from the three Pearl River populations. The Bayesian STRUCTURE analysis recovered genetic clusters that matched localities perfectly or the kind of ecology environment with a significant Fst between them. The findings of this research should provide useful information for management and conservation of S. knerii stocks.     

A multigene molecular assessment of cryptic biodiversity in the iconic freshwater blackfishes (Teleostei: Percichthyidae: Gadopsis) of south‐eastern Australia

Freshwater biodiversity is under ever increasing threat from human activities, and its conservation and management require a sound knowledge of species‐level taxonomy. Cryptic biodiversity is a common feature for aquatic systems, particularly in Australia, where recent genetic assessments suggest that the actual number of freshwater fish species may be considerably higher than currently listed. The freshwater blackfishes (genus Gadopsis) are an iconic group in south‐eastern Australia and, in combination with their broad, naturally divided distribution and biological attributes that might limit dispersal, as well as ongoing taxonomic uncertainty, they comprise an ideal study group for assessing cryptic biodiversity. We used a multigene molecular assessment including both nuclear (51 allozyme loci; two S7 introns) and matrilineal markers (cytb) to assess species boundaries and broad genetic substructure within freshwater blackfishes. Range‐wide examination demonstrates the presence of at least six candidate species across two nominal taxa, Gadopsis marmoratus and Gadopsis bispinosus. Phylogeographical patterns often aligned to purported biogeographical provinces but occasionally reflected more restricted and unexpected relationships. We highlight key issues with taxonomy, conservation, and management for a species group in a highly modified region. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 521–540.     

Isolation and characterization of twelve novel microsatellites in yellow catfish, Pelteobagrus fulvidraco

The yellow catfish Pelteobagrus fulvidraco is a freshwater fish species. Due to overfishing and pollution of freshwater ecosystems, the wild stocks of this fish reduced substantially. We isolated and characterized 12 polymorphic microsatellites of this species. The number of alleles at the 12 microsatellite loci ranged from four to eight, with an average of 6.6/locus. The average observed heterozygosity was 0.72, whereas the expected heterozygosity ranged from 0.60 to 0.86 (average: 0.80). All 12 microsatellites conformed to Hardy–Weinberg Equilibrium and were in linkage equilibrium. These 12 novel microsatellites could facilitate studies of genetic diversity and population structure of the yellow catfish to supply necessary information of conservation of the yellow catfish.     

Isolation of polymorphic microsatellite loci in three phantom midge species of the genus Chaoborus (Diptera: Chaoboridae)

Because of its widespread distribution in lakes and ponds Chaoborus is of great interest to many freshwater ecologists. Interestingly some species are restricted to small fish‐less water bodies, whereas other species live mostly in large lakes. To eventually test the genetic and evolutionary implications of these different lifestyles we identified microsatellite loci in three species in this preliminary study: C. obscuripes, C. crystallinus and C. flavicans. Using a biotin/streptavidin capture technique of repetitive sequences in a 96 well format, we obtained microsatellite‐enriched genomic libraries for all three species and identified six polymorphic microsatellite markers for each species.     

Genetic variation and phylogenetic relationship between two species of yellow catfish, <Emphasis Type="Italic">Horabagrus brachysoma</Emphasis> and <Emphasis Type="Italic">H. nigricollaris</Emphasis> (Teleostei: Horabagridae) based on RAPD and microsatellite markers

The two species of yellow catfish, Horabagrus brachysoma and H. nigricollaris are categorized as ‘endangered’ and ‘critically endangered’ respectively in their wild habitat. Proper knowledge of genetic structure and variability of these endangered species are highly essential for the management, conservation and improvement of fish stocks. Therefore, genetic variation and phylogenetic relationships between these species of yellow catfish sampled from Chalakkudy River in the hot spot of biodiversity-Western Ghats region, Kerala, India were analyzed by using Random amplified polymorphic DNA (RAPD) and microsatellite markers. 85 RAPD and five microsatellites loci were detected to analyze the genetic variation and phylogenetic relationships among these species. Out of 85 RAPD loci produced only 52.94% were polymorphic whereas in microsatellite, all 5 loci were polymorphic (100%). Species-specific RAPD bands were found in both species studied. In microsatellite, the number of alleles across the five loci ranged from 1 to 8. The observed heterozygosities in H. brachysoma and H. nigricollaris were 0.463 and 0.443, respectively. Here, both RAPD and microsatellite methods reported a low degree of gene diversity and lack of genetic heterogeneity in both species of Horabagrus which strongly emphasize the need of fishery management, conservation and rehabilitation of these species.     

Phylogeography of the Australian freshwater turtle Chelodina expansa reveals complex relationships among inland and coastal bioregions

We examined range‐wide mitochondrial phylogeographical structure in the riverine freshwater turtle Chelodina expansa to determine whether this species exhibits deep genetic divergence between coastal and inland hydrological provinces, as seen in co‐distributed freshwater taxa. We sequenced two mitochondrial loci, genealogical relationships were assessed using a network approach, and relationships among biogeographical regions were tested using analyses of molecular variance. Population history was evaluated using neutrality tests, indices of demographic expansion, and mismatch analyses. Twenty‐one haplotypes were recovered across two mitochondrial haplogroups separated by approximately 4% nucleotide divergence. The haplogroups have discrete geographical boundaries but only partially support a hypothesis of deep divergence between coastal and inland bioregions. The first haplogroup comprises populations from the inland Murray‐Darling Basin and from coastal catchments south of the Mary River in south‐east Queensland. The second haplogroup comprises populations from coastal catchments north of the Mary River. Cryptic phylogeographical barriers separating adjacent coastal populations are congruent with those demonstrated for other freshwater taxa and may result from the combined influences of the Conondale Range and alluvial deposits at the mouth of the Mary River. The findings of the present study demonstrate that freshwater taxa commonly display genetic differentiation within a biogeographical region where no boundaries have been recognized, highlighting the need to uncover cryptic microbiogeographical regions to aid conservation of freshwater biota. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 789–805.     

Isolation and characterization of microsatellite loci in the endangered freshwater fish <Emphasis Type="Italic">Pararasbora moltrechti</Emphasis> (Cyprinidae) using PCR-based isolation of microsatellite arrays (PIMA)

Pararasbora moltrechti (Cyprinidae) is an endangered freshwater fish with a scattered distribution in the central Taiwan. In order to undertake a conservation program for this species in Taiwan, it is essential to evaluate its genetic diversity and population structure. For this purpose, we described the development of eight microsatellite loci in P. moltrechti for genetic studies. These new molecular markers were tested in 36 individuals of the rare species. The number of alleles per locus ranged from 8 to 14. As the expected heterozygosity varied from 0.770 to 0.932, all microsatellite loci, however, were significantly deviated from Hardy–Weinberg expectations due to the heterozygote deficiency. Hung-Du Lin and Teh-Wang Lee contributed equally.     

Daily age determination and growth rates of freshwater fish throughout a regulated lotic system of the Murray‐Darling Basin Australia

To facilitate future research in freshwater fish recruitment response to environmental flow delivery, size‐at‐age and growth models are presented for eight fish species occurring in south‐eastern Australia; three small‐bodied species and the juvenile 0+ age classes of five large‐bodied species. Otolith increments were used to estimate the daily age of golden perch Macquaria ambigua, bony bream Nematalosa erebi, common carp Cyprinus carpio; Murray cod Maccullochella peelii, freshwater eel‐tailed catfish Tandanus tandanus, Australian smelt Retropinna semoni, un‐specked hardyhead Craterocephalus stercusmuscarum fulvus and Murray–Darling rainbowfish Melanotaenia fluviatilis. Linear growth models provided the best fit for length‐at‐age data of juvenile 0+ age large‐bodied species; whereas von Bertalanffy growth functions provided the best fit to length‐at‐age data of small‐bodied species. The results provide novel baseline data for future research in this area.     

Microsatellites from the burbot (Lota lota), a freshwater gadoid fish (Teleostei)

We developed 21 polymorphic dinucleotide microsatellite loci, (CA)_n and (CT)_n, for the Holarctic freshwater fish, Lota lota, using an enriched genomic library protocol. The species has an interesting life history because winter‐spawning adults migrate over long distances to form spawning aggregations, a behaviour which should maintain genetic homogeneity across large spatial scales. Availability of the reported microsatellites will facilitate the investigation of population genetic structure with regard to postglacial colonization history and conservation strategies. The primers were screened on 30 individuals from a natural population (Lake Constance, southern Germany), revealing three to 24 alleles per locus with expected heterozygosities ranging from 0.48 to 0.93.     

Microsatellite markers and mtDNA data indicate two distinct groups in dwarf galaxias, Galaxiella pusilla (Mack) (Pisces: Galaxiidae), a threatened freshwater fish from south-eastern Australia

Galaxiella pusilla is a small, non-migratory freshwater fish, endemic to south-eastern Australia and considered nationally threatened. To assist in the conservation of the species, microsatellite markers were developed and used to characterize genetic variation in 20 geographically distinct populations across its range. Substantial genetic differentiation was found between an eastern (Victoria east of the Otway Ranges and Tasmania) and western (South Australia and Victoria west of, and including, the Otway Ranges) region. This major separation was also observed in data from a mitochondrial gene and supports a previously proposed split. Populations from the eastern region had overall lower genetic diversity for both the microsatellite and mtDNA markers. There was substantial genetic differentiation between populations within the two regions, suggesting that gene flow is limited by the isolation of freshwater streams. Genetic structure, consistent with an isolation-by-distance model, was also evident in both regions. Patterns of genetic variation in this threatened species are compared to those obtained for other taxa across the same region. The need to consider separate conservation strategies for the two sets of populations is emphasized.     

Development of polymorphic microsatellite loci for the design of management and conservation strategies of the critically endangered Barrens topminnow (Fundulus julisia,Williams & Etnier, 1982)

The decline of the Barrens topminnow (Fundulus julisia), a small schooling fish endemic to the Barrens Plateau region of Middle Tennessee, is one of the most dramatic cases of species imperilment in eastern North America. Loss of undisturbed habitat, coupled with the introduction of the invasive Western Mosquitofish (Gambusia affinis) has led to the extirpation of 12 out of 14 natural populations since the 1980s. Identified is a set of 14 microsatellite loci to improve conservation and management strategies for the species. Four loci were cross‐amplified using primers designed for congeneric taxa and 10 loci were developed from whole genome Illumina sequencing. Initial surveys in two populations suggest significant structuring of genetic variation and differing levels of heterozygosity among populations. These markers will contribute valuable information to ongoing conservation and management efforts.     

Incongruence between mtDNA and nuclear data in the freshwater mussel genus Cyprogenia (Bivalvia: Unionidae) and its impact on species delineation

Accurately identifying species is a crucial step for developing conservation strategies for freshwater mussels, one of the most imperiled faunas in North America. This study uses genetic data to re‐examine species delineation in the genus Cyprogenia. Historically, Cyprogenia found west of the Mississippi River have been ascribed to Cyprogenia aberti (Conrad 1850 ), and those east of the Mississippi River were classified as Cyprogenia stegaria (Rafinesque 1820). Previous studies using mitochondrial DNA sequences indicated that C. aberti and C. stegaria were not reciprocally monophyletic groups, suggesting the need for systematic revision. We generated a novel dataset consisting of 10 microsatellite loci and combined it with sequence data from the mitochondrial ND1 gene for 223 Cyprogenia specimens. Bayesian analysis of the ND1 nucleotide sequences identified two divergent clades that differ by 15.9%. Members of these two clades occur sympatrically across most sampling locations. In contrast, microsatellite genotypes support recognition of three allopatric clusters defined by major hydrologic basins. The divergent mitochondrial lineages are highly correlated with the color of the conglutinate lures used by mussels to attract and infest host fishes, and tests for selection at the ND1 locus were positive. We infer that the incongruence between mtDNA and microsatellite data in Cyprogenia may be the result of a combination of incomplete lineage sorting and balancing selection on lure color. Our results provide further evidence that mitochondrial markers are not always neutral with respect to selection, and highlight the potential problems of relying on a single‐locus‐marker for delineating species.     

Movements and Habitat Use by the Endangered Australian Freshwater Mary River cod, Maccullochella peelii mariensis

The decline of the endangered Mary River cod, Maccullochella peelii mariensis, of Queensland, Australia, has been attributed to anthropomorphic habitat alterations, however, the relationships between this subspecies and its physical environment are poorly understood. We used radiotelemetry to investigate the movements and use of habitats by nine Mary River cod (total length 420–760mm; weight 1.3–5.5kg) in the Mary River system over a 20 month period. The mean distance moved each month was positively correlated with monthly stream discharge, but the direction of movements was unpredictable. Patterns of movement varied considerably among individuals and appeared to be unrelated to size. Cod established home ranges of between 70 and 820m in length. Five of the tagged cod returned to a previous home range after moving more than 10km, whereas three did not move more than 2km from their capture location for the duration of the study. Cod used large woody debris complexes more than any other type of habitat, and rarely used areas of open water. Managers should give high priority to maintenance of fish passage and protection/rehabilitation of large woody debris habitats if cod populations are to recover.     

Polymorphic microsatellite markers in an Australian freshwater fish,southern pygmy perch Nannoperca australis

Five microsatellite loci were isolated and characterized for the southern pygmy perch, Nannoperca australis, a regionally vulnerable Australian freshwater fish. Screening of 22 individuals from southeastern Australia revealed four to 15 alleles per locus, and observed levels of heterozygosity ranged from 0.09 to 0.45. All loci conformed to Hardy–Weinberg proportions and were in linkage equilibrium.     

Characterization of microsatellite markers in the endangered Sinojackia xylocarpa (Styracaceae) and cross‐species amplification in closely related taxa

Twenty‐four polymorphic microsatellite loci were isolated and characterized from an AAG‐enriched genomic library of Sinojackia xylocarpa. The average allele number of these microsatellites was 3.3 per locus, ranging from two to seven. The observed and expected heterozygosities at population level were 0.10–0.83 and 0.10–1.00, respectively. In addition, successful cross‐species amplification of this set of microsatellites in three other species of Sinojackia and a closely related taxon, Changiostyrax dolichocarpa, suggested that this set of microsatellite markers should provide a useful tool for genetic and conservation studies of Sinojackia species and other closely related taxa in the Styracaceae.     

Contemporary genetic structure reflects historical drainage isolation in an Australian snapping turtle,Elseya albagula

Effective spatial classification of freshwater biodiversity remains a worldwide conservation challenge. The isolating nature of catchment boundaries over evolutionary timescales makes them potentially important in defining natural units for biodiversity management. We sought to clarify biogeographical relationships amongst drainages within Australia's biodiverse mid‐eastern coastal region (Fitzroy, Burnett, and Mary Catchments) where freshwater communities face considerable urban pressure, using a locally endemic riverine specialist, the white‐throated snapping turtle, Elseya albagula. Mitochondrial and nuclear microsatellite data sets were employed to investigate past and present influences on population connectivity and to identify units for management. Populations within catchments were largely well connected genetically. However, the Fitzroy Catchment contained a distinct genetic lineage, deeply divergent from a second lineage present across the Burnett and Mary Catchments. The two lineages can be considered evolutionarily significant units that reflect historical isolation of the Fitzroy and recent coalescence of the Burnett‐Mary Catchments during lowered Pleistocene sea levels. Congruence with geological evidence and patterns reported for fish and macroinvertebrates supports a shared biogeographical history of a diverse regional biota. This work highlights the need for better spatial classification of freshwater biodiversity at local as well as regional scales, including recognition of potentially cryptic diversity amongst individual river drainages. © 2013 The Linnean Society of London