Genetic evidence for depth- and spatially separated stocks of the deep-water spikey oreo in Australasian waters

Samples of Neocyttus rhomboidalis from eight areas off southern Australia and eastern New Zealand were examined to assess the stock structure of the species. No spatial heterogeneity was detected for mitochondrial DNA variation after restriction analysis by two enzymes detecting polymorphic cut sites. Twenty mtDNA haplotypes were recorded, with an overall haplotype diversity of 0·683. Twenty-eight allozyme loci were examined; the mean heterozygosity per locus was 13·0%. Significant sample heterogeneity was detected at three of twelve polymorphic loci: MPI*, PGM-1 * and, most strikingly (G_ST=0·43), sSOD *. Most of the sSOD * differentiation was associated with depth rather than geographical separation; sSOD * allele frequencies of shallow and deep samples being very different. It is suggested that the correlation between the sSOD * allele frequencies and depth is more likely to reflect natural selection than reproductive isolation. The spatial patterning of the MPI * and PGM-1 * data suggest there are at least three geographic stocks of N. rhomboidalis in Australasian waters: (1) New Zealand, (2) Western Australia and South Australia, (3) west Tasmania to New South Wales and Lord Howe Rise.     

Mitochondrial DNA sequence divergence between two cryptic species of Lethenteron, with reference to an improved identification technique

Genetic divergences and population structures were examined in the cryptic Lethenteron sp. N and sp. S, based on mitochondrial DNA (mtDNA) cytochrome oxidase subunit I (CO I) region sequences. An improved method of discrimination between L . sp. N and sp. S was found using PCR, with diagnostic primers for each species-specific sequence in the mtDNA CO I region. Identification of 50 individuals of each species by this analysis was consistent with that by allozyme analysis of nuclear DNA. L . sp. N and sp. S, identified on the basis of diagnostic alleles at five allozymic loci, were independently grouped in a neighbour-joining (NJ) tree, with a large sequence difference (mean ±  s . d . = 9·10 ± 0·36%) between them. Within each species, the values of sequence divergence among localities were significantly higher in L . sp. S (1·61 ± 0·44%) than in L . sp. N (1·10 ± 0·48%). On the tree and nested clade analyses, several phylogenetic groups comprising geographically close localities were detected in the former, although scarcely detected in the latter, probably resulting from dispersal pattern differences between them.     

Evidence for independent origin of two spring‐spawning ciscoes (Salmoniformes: Coregonidae) in Germany

Combined analyses of mitochondrial DNA (mtDNA) and microsatellite loci were performed to assess the genetic differentiation of two spring‐spawning ciscoes from each other and from sympatric Coregonus albula in two German lakes. Polymorphism was screened at six microsatellite loci and mtDNA for a total of 247 and 94 ciscoes, respectively. Microsatellite data showed a weak differentiation between spring‐spawning Coregonus fontanae and sympatric C. albula in Lake Stechlin ( F _ST = 0–0·008), whereas a significant differentiation was observed between spring‐spawning Coregonus lucinensis and sympatric C. albula in Lake Breiter Luzin ( F _ST = 0·013–0·039). A more pronounced genetic difference was observed between both spring‐spawning species ( F _ST = 0·05–0·128). Shared mtDNA haplotypes among sympatric species within both Lake Stechlin and Lake Breiter Luzin were observed, whereas no haplotype was shared between C. fontanae and C. lucinensis . These results suggest an independent origin for spring‐spawning ciscoes in each lake. Evidence is also provided for mtDNA introgression of Coregonus sardinella into C. lucinensis and C. albula in Lake Breiter Luzin. Postglacially, this species or at least a population which showed mtDNA introgression has colonized the Baltic Sea basin up to the glacial margin that was located between Lakes Stechlin and Breiter Luzin.     

Population structure and phylogeography of Aphyocypris kikuchii (Oshima) based on mitochondrial DNA variation

Aphyocypris kikuchii is a cyprinid species endemic to northern and eastern Taiwan and is the only primary freshwater fish native east of the Coastal Mountain Range. In total, 92 individuals of A. kikuchii from seven populations in three regions of the island were surveyed for mitochondrial DNA (mtDNA) variation. High haplotype diversity ( h = 0·989) and low nucleotide diversity ( π = 0·009) of mtDNA were detected. Negative values of Tajima's D and unimodal mismatch distributions probably reflect a history of recent demographic expansions from small populations. Three major haplotype clusters displayed geographically non-overlapping distributions, indicating a long-term isolation between regions. Hierarchical analysis of molecular variance showed significant genetic structuring among populations ( Φ _ST= 0·66). Significant haplotype heterogeneity was also detected among populations within regions ( Φ _SC= 0·41, P < 0·001) and among regions ( Φ _CT= 0·43, P < 0·05). Molecular clock estimates of coalescence in the three major mtDNA lineages indicated coalescence in the most recent common ancestor c. 0·11–0·39 million years ago. Haplotypes of cluster B nested as interior nodes in the haplotype network, indicating that migrations from Shueilian (SL) populations to the northern region (cluster A) and to the eastern region (cluster C) may have occurred independently. Lineages A and B + C should be managed as two distinct evolutionarily significant units, while the northern, SL and southern groups should be managed as separate management units.     

Natural hybridization between endangered and introduced species of Pseudorasbora, with their genetic relationships and characteristics inferred from allozyme analyses

From 25 populations of Pseudorasbora in Japan, polymorphism at 22 allozyme loci indicated that the level of genetic differentiation between Pseudorasbora parva and Pseudorasbora pumila ( D  = 0·421–0·517) was greater than that between the two subspecies of P. pumila , P. pumila pumila and P. pumila subsp. ( D  = 0·164), consistent with morphological differences. While P. parva displayed genetic variation ( H  = 0·003–0·100) similar to other freshwater fishes, P. pumila pumila and P. pumila subsp. populations showed no genetic variation. In five of the 15 populations collected from the contact zone between P. parva and P. pumila pumila , hybrids were detected by allozyme analyses. All hybrids were presumed to represent the F₁ generation, because they were heterozygous at all 12 loci diagnostic between P. parva and P. pumila pumila . Although four populations were characterized by high frequencies of F₁ hybrids ( c . 40%), only one of the parental species was observed in each case. The results indicated that the two species have hybridized easily under natural conditions, but cannot coexist in the long-term. It is suggested that continued invasion of P. parva would hasten the extinction of P. pumila pumila .     

Morphological and mitochondrial DNA divergence validates blackmouth, Galeus melastomus, and Atlantic sawtail catsharks, Galeus atlanticus, as separate species

A total of 60 morphometric traits and nucleotide sequences of the entire mtDNA NADH dehydrogenase subunit 2 (ND2) gene [1047 base pair (bp)] in 23 individuals of blackmouth, Galeus melastomus , and 13 individuals of sawtail catsharks, Galeus atlanticus , caught in Southern Portugal, were examined to test the validity of these two taxa. These sharks closely resemble each other, have overlapping geographical ranges and are difficult to identify by morphological characters. Non-metric multidimensional scaling of morphometric variables indicates a clear separation between the two species, with 10 characters each contributing 2·12–2·45% of the total variability between species. Maximum likelihood, parsimony and neighbour-joining trees revealed two major mtDNA haplotype clades, corresponding to the two species, with an average corrected sequence divergence between them of 3·39 ± 0·56%. Within species divergences between haplotypes averaged 0·27 ± 0·18% in G. melastomus and 0·12 ± 0·08% in G. atlanticus . A total of 35 diagnostic nucleotide site differences and four restriction fragment length polymorphism recognition sites in the ND2 gene can be used to distinguish the two species.     

Effects of temperature on prey consumption and growth in mass of juvenile trahira Hoplias aff. malabaricus (Bloch, 1794)

The influence of temperature on prey consumption and growth in mass of juvenile trahira Hoplias aff. malabaricus were investigated. Consumption of small-sized lambari Astyanax altiparanae (mean standard length, L _S, 5·43 cm) varied from zero to 65 over a period of 30 days. Temperatures ranged from 14 to 34° C and the size of trahiras ranged from 17·5 to 24·7 cm L _S. Prey consumption differed significantly among temperatures. Trahiras at 18° C consumed significantly less than those at 30° C. A linear multiple regression model including temperature, prey consumption and L _S explained 89·4% of the variability in growth in mass. Some caution is suggested when inferring the impact of H. aff. malabaricus piscivory on assemblage structures in systems that, despite their location in tropical regions, are subjected to seasonal thermal variations.     

Stock discrimination and movements of narrow‐barred Spanish mackerel across northern Australia as indicated by parasites

The parasite fauna of Spanish mackerel Scomberomorus commerson from 10 sites across northern Australia and one site in Indonesia, was examined to evaluate the degree of movement and subsequent stock structure of the fish. Kupang fish (Indonesia) had very few Terranova spp., Grillotia branchi , Otobothrium cysticum or Pterobothrium sp. compared to Australian fish, indicating that no Australian fish enter the Kupang fishery. Univariate and discriminant function analysis of four 'temporary' parasite species, the copepod Pseudocycnoides armatus and the monogeneans Gotocotyla bivaginalis , Pricea multae and Pseudothoracocotyla ovalis , demonstrated little similarity between areas of northern Australia, indicating minimal short‐term exchange between neighbouring groups of S. commerson . Analyses of five 'permanent' parasite species, the larval helminths G. branchi , O. cysticum , Pterobothrium sp., Callitetrarhynchus gracilis and Paranybelinia balli , also revealed large differences between areas thus indicating long‐term separation. There are at least six parasitological stocks across northern Australia: Fog Bay/Bathurst Island, Cape Wessel, Groote/Sir Edward Pellew, Mornington Island, Weipa, and the Torres Strait. The occurrence of a few irregular fish in the samples suggested that up to 5% of fish moved between stocks during their lifetime. The similarity of within‐school variability to that between schools showed that the fish do not form long‐term school associations.     

Genetic variation and stock structure of school mackerel and spotted mackerel in northern Australian waters

The total mean sample heterozygosity calculated from eight polymorphic loci was 0·172 (0·047 S.E.) (F_st 0·025) for school mackerel Scomberomorus queenslandicus , and for spotted mackerel S. munroi was 0·110 (0·074 S.E.) (F_st 0·038). There was no evidence of temporal variation for either species as significant genetic differences were not detected between months or year classes within areas. Spatially, school mackerel have a complex stock structure, with stocks being associated with large embayments. In contrast, spotted mackerel appear to comprise a single stock in Australian east coast waters. Both species showed a significant pattern of stock structure between Australian east coast and northern (Arafura Sea) samples.     

Some aspects of the biology of the red bass, Lutjanus bohar (Forsskal), from the Tigak Islands, Papua New Guinea

The growth rate, food, reproduction and mortality rates of the red bass, Lutjanus bohar , are described for fish associated with coral reefs in the Tigak Islands of Papua New Guinea. Spawning intensity by month was investigated by examination of gonad maturation stages. The length–at–maturity of female L. bohar was estimated from the relationship between changes in gonad index with increasing size. Female fish matured at about 45–0 cm fork length. The Petersen method was used to obtain age–at–length data from length frequency data of L. bohar captured by hook–and–line fishing. The growth of L. bohar was described by a von Bertalanffy growth curve with the parameters AT=0.27 and L_∞ = 81.7 cm. There was a positive correlation between water depth (D) and the length of L. bohar (L) that was described by L = 318–8+ 3.050 D . The mortality rates ( Z ) of L. bohar from shallow (≤30m) and deep (≤80m) water were estimated from length converted catch curves and were 1–181 and 0–64 respectively. The high Z value for the shallow water population of L. bohar was ascribed to the migration of larger specimens into deeper water. The deep water population of L. bohar remains unfished and the estimate of Z was believed to be equivalent to the natural mortality rate ( M ). An empirical method of calculating M gave an estimate of 0.59. Lutjanus bohar is a carnivorous piscivore that feeds throughout the water column.     

Genetic differentiation of charrs in the Russian north and far east

Genetic differences and relationships within charrs Salvelinus alpinus , northern and southern S. malma, S. taranetzi, S. levanidovi and S. leucomaenis , were studied by examination of 31–35 allozyme loci and PCR-RFLP analysis of mtDNA segment (about 2600 bp) including the cytochrome b gene and D-loop. Data for mtDNA were also obtained for samples of S. drjagini, S. albus and for European samples of S. alpinus. Generally allozyme and mtDNA segment analyses produced similar pictures of genetic divergence and relationships. The southern S. malma exhibited the greatest genetic variability (allozyme heterozygosity and mtDNA haplotype diversity). S. alpinus, S. malma, S. levanidovi and S. leucomaenis had fixed differences from one another at various allozyme loci, and differed in fixed (but polymorphic in S. malma ) individual mtDNA haplotypes produced with MspI restrictase. Such genetic individuality is typical of distinct taxa. S. taranetzi is closest genetically to allopatric S. alpinus , but exhibited fixed differences from the northern S. malma in sympatry. In all samples of S. alpinus the only mtDNA haplotype was shared with S. drjagini. S. albus shared its single haplotype with the northern S. malma from the same locality. S. leucomaenis is differentiated from the other species to the greatest extent, possibly reflecting an early evolutionary divergence. Some evolutionary and taxonomic implications of the genetic relationships of charrs studied are discussed.     

Low mtDNA diversity among widespread Australian diamondback moth Plutella xylostella （L,） suggests isolation and a founder effect

Populations of Australian diamondback moth （DBM） Plutella xylostella （L.）, a serious pest of cruciferous crops, display extremely low levels of genetic differentiation across Australia and New Zealand sample locations, as determined previously using microsatellite markers. These data suggest high levels of contemporary gene flow that is consistent with Australian DBM being a vagile species. Here we examine Australian DBM samples for haplotype variation using the mitochondrial DNA sequences of a 257 bp fragment of the CO1 gene. We compare this variation to equivalent mtDNA sequence variation in samples from New Zealand, Kenya and Korea. Using 42 moths collected throughout Australia we show that Australian DBM have both low mtDNA haplotype and nucleotide diversities. The three Australian haplotypes detected are closely related and they cluster with the common haplotype group from Indonesia. In addition the Australian haplotype frequency distribution resembled more that from Indonesia than that from Kenya or Korea. These data are consistent with an original strong Australian/New Zealand founder effect, from a south-eastern Asian source, with subsequent continued isolation. In a single season, the frequency of PXMt01, the most common Australian haplotype, was estimated at 15 locations spread across southern Australia and New Zealand using a polymerase chain reaction BiPASA method. The PXMt01 haplotype frequency variation was heterogenous, suggesting a small degree of population isolation that was not detected using microsatellites. Differentiation was not a function of geographical distance. These data suggest transient and sporadic local colonisation events by small numbers of founding females.     

Genetic verification of native brown trout from the Persian Gulf (Catak Cay River, Tigris basin)

Analysis of the mtDNA control region in six individual brown trout Salmo trutta from the Catak Cay River (Tigris basin) revealed a single, new, haplotype, 1·0 to 1·5% divergent from five other Da lineage haplotypes analysed, that groups with the Danubian clade with low bootstrap support. This highly divergent haplotype combined with unique phenotypic characteristics underscores the novelty and native status of this population, which has probably been isolated from other brown trout lineages for at least several hundred thousand years.     

Markedly discordant mitochondrial DNA and allozyme phylogenies of tube-nosed fruit bats, Nyctimene, at the Australian–Oriental biogeographical interface

Australo-Papuan tube-nosed fruit bats of the genus Nyctimene reach their most westerly distribution on the island archipelagos of eastern Indonesia. A recent morphological examination indicates three species occur on Moluccan islands. Both allozyme electrophoresis and mitochondrial DNA (mtDNA) sequencing reveal there are only two species present, Nyctimene cephalotes and Nyctimene albiventer , but there is considerable disparity between the results obtained from the two genetic data sets. Allozyme data indicate N. albiventer occurs on Wokam, which sits on the Australian continental shelf and was joined to Australo-Papua during the last glacial maximum, and N. cephalotes on the other Moluccan islands, all of which are off the continental shelf. Divergence of these two species is dated at approximately 1.5 Mya. By contrast, the mtDNA gene tree shows two deep clades, one containing all specimens from Wokam and Yamdena, and the second all the specimens from the other islands. This especially marked incongruence between the two genetic data sets is ascribed to either a single introgression event of N. albiventer mtDNA into N. cephalotes on Yamdena at around 100 000 ya or lineage sorting of very old mtDNA lineages that coalesce a rather long time before the speciation event. These results highlight that caution should be exercised when relying on mtDNA as barcodes in species taxonomy. The continental-associated N. albiventer on Wokam has higher levels of allozyme heterozygosity and mtDNA nucleotide diversity than the N. cephalotes populations occurring on the more remote islands, indicating that this colonizing species has experienced bottlenecks and/or a low effective population size since speciation.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 93 , 589–602.     

Hepatocytes primary culture from the Neotropical fish, trahira Hoplias malabaricus (Bloch)

Hepatocytes were isolated and cultured from Hoplias malabaricus using a non-enzymatic protocol. From 6·9 to 9·4 × 10⁷ hepatocytes g⁻¹ of liver were isolated and cell viability was 65–85%. Fish hepatocytes attached well to untreated polystyrene flasks (Corning) as well as on treated surfaces with extracellular matrix proteins, i.e. fibronectin, matrigel or type I collagen coatings. Cells rapidly started migrating and reorganizing in cord-like groups after seeding, with spaces similar to bile canaliculi in vivo . Prominent nucleolus and euchromatin-rich rounded nucleus, abundant mitochondria, well-developed rough endoplasmic reticulum, polysomes and Golgi apparatus were seen under ultrastructural analysis. Cells remained functional and metabolically active after 6 days in culture. The protocol established in the current work provides the basis for further studies of native fishes for accurate in vitro toxicological studies.     

Molecular and morphological divergence in the butterfly genus Lycaeides (Lepidoptera: Lycaenidae) in North America: evidence of recent speciation

Male genital morphology, allozyme allele frequencies and mtDNA sequence variation were surveyed in the butterfly species Lycaeides idas and L. melissa from across much of their range in North America. Despite clear differences in male genital morphology, wing colour patterns and habitat characteristics, genetic variation was not taxonomically or geographically structured and the species were not identifiable by either genetic data set. Genetic distances (Nei's D=0.002–0.078, calculated from allozyme data) between all populations of both species were within the range commonly observed for conspecific populations of other butterflies. The most frequent mtDNA haplotype was present in individuals of both species in populations from southern California to Wisconsin. We conclude that speciation has probably happened recently and the lack of genetic differentiation between the species is the product of either (1) recent or ongoing gene flow at neutral loci, and/or (2) an insufficiency of time for lineage sorting. The evolution of male genital morphology, wing colour patterns and ecological characteristics has proceeded more rapidly than allozyme or mtDNA evolution.     

Extreme mtDNA divergences in a terrestrial slug (Gastropoda, Pulmonata, Arionidae): accelerated evolution, allopatric divergence and secondary contact

Extremely high levels of intraspecific mtDNA differences in pulmonate gastropods have been reported repeatedly and several hypotheses to explain them have been postulated. We studied the phylogeny and phylogeography of 51 populations (n = 843) of the highly polymorphic terrestrial slug Arion subfuscus (Draparnaud, 1805) across its native distribution range in Western Europe. By combining the analysis of single stranded conformation polymorphisms (SSCP) and nucleotide sequencing, we obtained individual sequence data for a fragment of the mitochondrial 16S rDNA and a fragment of the nuclear ITS1. Additionally, five polymorphic allozyme loci were scored. Based on the 16S rDNA phylogeny, five monophyletic haplotype groups with sequence divergences of 9-21% were found. Despite this deep mitochondrial divergence, the haplotype groups were not monophyletic for the nuclear ITS1 fragment and haplotype group-specific allozyme alleles were not found. Although there is evidence for an accelerated mtDNA clock, the divergence among the haplotype groups is older than the Pleistocene and their current allopatric ranges probably reflect allopatric divergence and glacial survival in separate refugia from which different post-glacial colonization routes were established. A range-overlap of two mtDNA groups (S1 and S2, 21% sequence divergence) stretched from Central France and Belgium up to the North of the British Isles. The nuclear data suggest that this secondary contact resulted in hybridization between the allopatrically diverged groups. Therefore, it seems that, at least for two of the groups, the deep mtDNA divergence was only partially accompanied by the formation of reproductive isolation.     

Genetic consequences of introducing allopatric lineages of Bluestriped Snapper (Lutjanus kasmira) to Hawaii

A half century ago the State of Hawaii began a remarkable, if unintentional, experiment on the population genetics of introduced species, by releasing 2431 Bluestriped Snappers (Lutjanus kasmira) from the Marquesas Islands in 1958 and 728 conspecifics from the Society Islands in 1961. By 1992 L. kasmira had spread across the entire archipelago, including locations 2000 km from the release site. Genetic surveys of the source populations reveal diagnostic differences in the mtDNA control region (d = 3.8%; φ_ST = 0.734, P < 0.001) and significant allele frequency differences at nuclear DNA loci (F_ST = 0.49; P < 0.001). These findings, which indicate that source populations have been isolated for approximately half a million years, set the stage for a survey of the Hawaiian Archipelago (N = 385) to determine the success of these introductions in terms of genetic diversity and breeding behaviour. Both Marquesas and Society mtDNA lineages were detected at each survey site across the Hawaiian Archipelago, at about the same proportion or slightly less than the original 3.4:1 introduction ratio. Nuclear allele frequencies and parentage tests demonstrate that the two source populations are freely interbreeding. The introduction of 2431 Marquesan founders produced only a slight reduction in mtDNA diversity (17%), while the 728 Society founders produced a greater reduction in haplotype diversity (41%). We find no evidence of genetic bottlenecks between islands of the Hawaiian Archipelago, as expected under a stepping‐stone model of colonization, from the initial introduction site. This species rapidly colonized across 2000 km without loss of genetic diversity, illustrating the consequences of introducing highly dispersive marine species.     

Post mortem shrinkage of four species of temperate and tropical marine fishes, without freezing or preservation

The present study indicated significant post mortem decreases in length for four Australian teleost species from temperate to tropical habitats: black bream Acanthopagrus butcheri , King George whiting Sillaginodes punctata , summer whiting Sillago ciliata and redthroat emperor Lethrinus miniatus . Shrinkage averaged 5·0 mm after 24 h (range 0–10 mm) for A. butcheri , 4 mm (0–8 mm) for S. punctata , 2·2 mm (0·7–3·7 mm) for S. ciliata and 5·4 mm (−2·5–15·0 mm) for L. miniatus . For A. butcheri held under three different post mortem treatments, the mean length of fish in all treatments decreased but there was no significant effect of treatment type on the extent of shrinkage. The rate of shrinkage of S. ciliata varied with treatment, but the ultimate extent did not. For A. butcheri shrinkage was most rapid (2·5 mm h⁻¹) between 1 and 2 h postcapture. The results from these studies confirm that a post mortem decrease in length is a common phenomenon, even in fishes that are not frozen or preserved. Such shrinkage has implications for the enforcement of minimum legal length legislation, and may cause bias in biological investigations, including growth estimates from tag and recapture studies.     

Shared Stocks of Snappers (Lutjanidae) in Australia and Indonesia: Integrating Biology, Population Dynamics and Socio-Economics to Examine Management Scenarios

Joint Australia–Indonesia scientific workshops on the fisheries of the Arafura Sea, held in 1992 and 1994, concluded that the two countries might share stocks of the red snappers Lutjanus malabaricus and L. erythropterus and the gold-band snapper Pristipomoides multidens. At that time, no information concerning stock structure, distribution and movements of these species was available. Moreover, data on the population biology and on commercial catches were inadequate. Such data are crucial for stock assessment and for managing the stocks. Clearly, if the stocks being fished were shared, joint management would be appropriate. In order to answer the questions related to managing shared stocks, a collaborative research project was initiated by Australia (CSIRO as the lead agency) and Indonesia in 1999. The objectives were firstly, to describe the population dynamics, stock structure and biology of snappers relevant to the management of stocks shared between Australian and Indonesian fisheries; secondly, to characterize the social and financial structures of the Indonesian fishery so they could be taken into account in the development of management strategies; and thirdly, to explore ways of developing complementary management for the long term sustainability of the snapper fisheries. This project finished in 2003 and in this paper we bring together the results of the biological, genetic, population dynamics and socioeconomic research in relation to managing shared stocks in the context of managed versus unmanaged fisheries, small scale and industrial fisheries, and in both developed and developing country regulatory environments. Severe data limitations necessitated an innovative approach making use of comparative analyses, often data-poor values, and the drawing together of fishery dependent and independent data to evaluate the status of the stocks.