褶皱臂尾轮虫同胞物种复合类群分类的研究进展

褶皱臂尾轮虫包含复杂的同胞物种,对其分类研究经过了一个长期发展的过程。综述了轮虫分类手段的发展进程,介绍和分析了轮虫特别是褶皱臂尾轮虫同胞物种复合类群分类的研究进展。褶皱臂尾轮虫的L-型和S-型属于B.plicatilis和B.rotundiformis两个物种。这个复合类群中还包括更多的种类,如B.plicatilis sensu stricto、B.ibericus和B.rotundiformis,其中B.plicatilis sensu stricto属于L型,B.ibericus属于SM-型,B.rotundiformis属于SS-型。此类研究丰富了轮虫系统分类学,提高了水域生态调查的准确性。     

The association between the caprellid Pariambus typicus Krøyer (Crustacea,Amphipoda) and ophiuroids

The work of Kazutsugu Hirayama over the past 25 yearspromoted the wide use of the marine rotifer Brachionusplicatilis as an experimental model in zooplanktonecology. His reports about the nature of geneticvariation in the B. plicatilis complex stimulated usto investigate how mate recognition maintains speciesboundaries. For the past several years, we haveexamined chemical communication between female andmale B. plicatilis. Here we report on the comparativebinding of polyclonal antibody against the materecognition pheromone (MRP) to three B. plicatilisstrains and three B. rotundiformis strains.Quantification of anti-MRP binding permitsinvestigation of how the female mating signal differsamong closely related Brachionus species and strains.Antibody binding reflects differentiation independentof the male receptor which has been describedelsewhere. Anti-MRP bound to females of all sixstrains and was localized in the corona. Antibodybinding greatly reduced mating in all three B. plicatilis strains. However, antibody bindingsignificantly reduced mating in only one of the B. rotundiformis strains. The MRP of both species has asimilar molecular weight, but the differential bindingsuggests that the mate recognition pheromone onfemales has differentiated in B. plicatilis and B. rotundiformis.     

Interference with the quorum sensing systems in a Vibrio harveyi strain alters the growth rate of gnotobiotically cultured rotifer Brachionus plicatilis

AIMS: To evaluate the effect of Vibrio harveyi strains on the growth rate of the gnotobiotically cultured rotifer Brachionus plicatilis, and to establish whether quorum sensing is involved in the observed phenomena. METHODS AND RESULTS: Gnotobiotic B. plicatilis sensu strictu, obtained by hatching glutaraldehyde-treated amictic eggs, were used as test organisms. Challenge tests were performed with 11 V. harveyi strains and different quorum sensing mutants derived from the V. harveyi BB120 strain. Brominated furanone [(5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone] as a quorum sensing inhibitor was tested in Brachionus challenge tests. Some V. harveyi strains, such as strain BB120, had a significantly negative effect on the Brachionus growth rate. In the challenge test with MM77, an isogenic strain of BB120 in which the two autoinducers (HAI-1 and AI-2) are both inactivated, no negative effect was observed. The effect of single mutants was the same as that observed in the BB120 strain. This indicates that both systems are responsible for the growth-retarding (GR) effect of the BB120 strain towards Brachionus. Moreover, the addition of an exogenous source of HAI-1 or AI-2 could restore the GR effect in the HAI-1 and AI-2 nonproducing mutant MM77. The addition of brominated furanone at a concentration of 2.5 mg l(-1) could neutralize the GR effect of some strains such as BB120 and VH-014. CONCLUSIONS: Two quorum sensing systems in V. harveyi strain BB120 (namely HAI-1 and AI-2-mediated) are necessary for its GR effect on B. plicatilis. With some other V. harveyi strains, however, growth inhibition towards Brachionus does not seem to be related to quorum sensing. SIGNIFICANCE AND IMPACT OF THE STUDY: Interference with the quorum sensing system might help to counteract the GR effect of some V. harveyi strains on Brachionus. However, further studies are needed to demonstrate the positive effect of halogenated furanone in nongnotobiotic Brachionus cultures and eventually, in other segments of the aquaculture industry.     

Interspecific interactions in the marine rotifer microcosm

Copepods and protozoans often co-exist in marinerotifer mass cultures. Interspecific interactionbetween the rotifer Brachionus rotundiformisTschugunoff and eight other zooplankton species,namely Brachionus plicatilis O. F. Müller (rotifer),Diaphanosoma celebensis Stingelin (cladoceran),Tigriopus japonicus Mori, Acartia sp. (copepod),Euplotes sp., Vorticella sp., an unidentifiedprotozoan species (P1 strain) (protozoan) and Artemiasp. (anostracan) at two developmental stages (nauplii –0.95 mm, 0 days old; adults – 3.3 mm, 19 days old) wereinvestigated in the laboratory.There was no contaminating species that contributedto an increase in rotifer population growth during theexperiments. Four types of interspecific interactionswere seen between B. rotundiformis and otherco-existing zooplankton species. These include effectson population growth: (1) both species declined, (2) onespecies is promoted while the other is not influenced,(3) one species is declined while the other is notinfluenced and (4) one species is promoted while theother declined. The first type was exhibited by B. rotundiformis vs B. plicatilis, B. rotundiformis vsD. celebensis and B. rotundiformis vs Artemia sp. The second type was exhibited by B. rotundiformis vsVorticella sp. and the third type by B. rotundiformisvs Euplotes sp. and B. rotundiformis vs T. japonicus. The fourth type was exhibited by B. rotundiformis vs Acartia sp. and B. rotundiformis vs P1 strain.     

On the taxonomy of three sympatric sibling species of the Brachionus plicatilis (Rotifera) complex from Spain, with the description of B. ibericus n. sp.

The accumulation of evidence during the last decade led to thesplitting of the Brachionus plicatilis complex (Rotifera) intotwo morphologically recognizable species: B. plicatilis Müllerand B. rotundiformis Tschugunoff, previously referred to asL- and S-type B. plicatilis (s.l.), respectively. However, recentpopulation genetics and molecular studies have revealed thateach of these taxa concern cryptic species complexes. In particular,in Torreblanca Marsh, a wetland on the Mediterranean coast ofSpain, three genetically distinct groups in this rotifer complexhave been shown to co-occur. Differences in genetic markers,ecological preferences, mixis responses, mating behaviour andno evidence of gene flow between them have led to the conclusionthat they must be considered as three different biological species.In this study we present a detailed comparative analysis usinglight and scanning electron microscopy of laboratory strainsenabling a morphological characterization of the three species.Fine morphology and morphometry revealed taxonomic charactersconstant enough to recognize three well-defined morphologies,which always correspond with the respective biological speciesidentified. Since no type material of both B. plicatilis andB. rotundiformis is available, we designed our Spanish clonesas neotypes, which would allow further comparative work. Furthermore,B. ibericus n. sp. is described. This study sets the first stepfor a complete characterization of the biological diversitycontained within this economically important species complex.     

Two circular chromosomes of unequal copy number make up the mitochondrial genome of the rotifer Brachionus plicatilis

The monogonont rotifer Brachionus plicatilis is an emerging model system for a diverse array of questions in limnological ecosystem dynamics, the evolution of sexual recombination, cryptic speciation, and the phylogeny of basal metazoans. We sequenced the complete mitochondrial genome of B. plicatilis sensu strictu NH1L and found that it is composed of 2 circular chromosomes, designated mtDNA-I (11,153 bp) and mtDNA-II (12,672 bp). Hybridization to DNA isolated from mitochondria demonstrated that mtDNA-I is present at 4 times the copy number of mtDNA-II. The only nucleotide similarity between the 2 chromosomes is a 4.9-kbp region of 99.5% identity including a transfer RNA (tRNA) gene and an extensive noncoding region that contains putative D-loop and control sequence. The mtDNA-I chromosome encodes 4 proteins (ATP6, COB, NAD1, and NAD2), 13 tRNAs, and the large and small subunit ribosomal RNAs; mtDNA-II encodes 8 proteins (COX1-3, NAD3-6, and NAD4L) and 9 tRNAs. Gene order is not conserved between B. plicatilis and its closest relative with a sequenced mitochondrial genome, the acanthocephalan Leptorhynchoides thecatus, or other sequenced mitochondrial genomes. Polymerase chain reaction assays and Southern hybridization to DNA from 18 strains of Brachionus suggest that the 2-chromosome structure has been stable for millions of years. The novel organization of the B. plicatilis mitochondrial genome into 2 nearly equal chromosomes of 4-fold different copy number may provide insight into the evolution of metazoan mitochondria and the phylogenetics of rotifers and other basal animal phyla.     

Application of unicellular algae Chlorella vulgaris for the mass-culture of marine rotifer Brachionus

Condensed suspension of Chlorellavulgaris was used for the food of the rotifer Brachionus plicatilis and B. rotundiformis inplace of Nannochloropsis oculata. Thisreport describes the characteristics of C. vulgaris as arotifer food in comparison with N. oculata and thepresent status of this field.The cell components of C. vulgarissuch as protein content, amino acids, minerals andvitamins are generally similar to those of N. oculata. However, the taxonomic status of thesealgal species are different. Based on thesimilarity of cell components, the dietary value ofC. vulgaris is equal in value to that of N. oculata for rotifer growth. Dietary value ofC. vulgaris can be improved by addition ofvitamin B₁₂. This improved C. vulgaris is currently widely used as an indispensable food organism for rotifer culture. Recent investigationshave shown that the use of the condensed suspensionof C. vulgaris makes it possible tosignificantly increase the rotifer density atharvest. Application of condensed C. vulgaris has made rotifer culture quite easy because theculture of N. oculata is no longer required,and intensive rotifer production in aquaculture cannow be realized.     

Effect of unionized ammonia,viscosity and protozoan contamination on reproduction and enzyme activity of the rotifer Brachionus rotundiformis

We determined the effect of environmental stressors on the physiological condition of Brachionus rotundiformis. For two morphologically distinct B. rotundiformis strains: Hawaii (average lorica length = 222 m) and Langkawi strains (average lorica length 180 m), neonates hatched from resting eggs were exposed to different levels of unionized ammonia (0.7–9.8 mg l^–1), viscosity (relative viscosity against natural seawater = 1–1.17) and Euplotes sp. (protozoan) contamination (1–40 cells ml^–1). Increasing stress decreased fecundity and lifespan of both rotifer strains. Glucosidase and phospholipase activities were correlated with reproductive responses of both the strains exposed to unionized ammonia. When culture water viscosity was changed, the activity of esterase and phospholipase was correlated with reproductive responses of the Hawaiian strain, and glucosidase activity was correlated with those of Langkawi strain.With the protozoan contamination, esterase and glucosidase activities were correlated only with reproductive responses of the Hawaiian strain, while activity of all three enzymes was correlated to those of the Langkawi strain. Glucosidase activity proved to be a reliable indicator of stress for cultured B. rotundiformis.     

营养强化时褶皱臂尾轮虫对饵料微藻的摄食

选取适宜浓度利用单种微藻和混合微藻对轮虫进行营养强化,采用实验生态学方法研究了轮虫滤水率和摄食率的动态变化.结果表明:微藻浓度、微藻种类和培养时间均对轮虫的滤水率和摄食率有显著影响;轮虫对几种单种微藻的滤水率和摄食率均随培养时间的延长而下降,在实验条件下,6h内轮虫对3种微藻的滤水率大小顺序为小球藻＞球等鞭金藻＞牟氏角毛藻,12h内轮虫对3种和,微藻的滤水率大小顺序则为球等鞭金藻＞小球藻＞牟氏角毛藻;轮虫在混合微藻中的选择顺序为球等鞭金藻＞小球藻＞牟氏角毛藻.     

Predatory interactions between a cyclopoid copepod and three sibling rotifer species

SUMMARY 1. Cyclopoid copepod predation on rotifers affects the dynamics and structure of zooplankton communities. We address the differential vulnerability of three sympatric rotifer sibling species belonging to the Brachionus plicatilis species complex. These co-occur with their cyclopoid predator, Diacyclops bicuspidatus odessanus .
2. Using video recording and tracking, we analysed the steps in predation including attack distance, attack angle, and rotifer species swimming in the presence and absence of the predator. Our results show the greater vulnerability of B. rotundiformis (the smallest species) to D. b. odessanus predation, which is associated with a high percentage of attacks after contact. Brachionus plicatilis (the biggest species) is the less vulnerable prey, with low percentage of attacks after contact and captures after attacks. Branchionus ibericus , the intermediate sized species, had also intermediate vulnerability.
3. The differential vulnerability provides insight into the coexistence and seasonal succession of these competing rotifer species. Our results show that the competitive superiority of B. rotundiformis may be balanced by its greater vulnerability to copepod predation.     

Rhodomonas salina (Cryptophyta) pastes as feed for Brachionus plicatilis (Rotifera)

Rotifers are an important live feed for first feeding larvae of many fish species. The use of concentrated algae cells in the mass culture of the rotifer Brachionus plicatilis (Brachionidae) has opened new horizons for research on this organism. Pastes of Rhodomonas salina (Pyrenomonadaceae) obtained either by centrifugation or flocculation with chitosan were preserved, with or without vitamin C, at -20 degrees C for four weeks and were evaluated biochemically (proteins, lipids, pigments and fatty acids contents) and subsequently, were used to feed the rotifer Brachionus plicatilis at a ratio of 25 mg/L/day. Four different microalgae pastes were prepared: (1) centrifuged and preserved with vitamin C (CV), (2) centrifuged and preserved without vitamin C (C), (3) flocculated and with vitamin C (FV) and (4) flocculated without vitamin C (F). All treatments showed similar contents of proteins and total lipids with respect to control culture (a fresh culture of R. salina), with mean values of 40.0 +/- 2.32% and 12.0 +/- 1.45%, respectively. The pheophytin a/chlorophyll a ratio, a general indicator of the chemical status of microalgal concentrates, was similar (0.09-0.11) between centrifuged pastes and control culture, but was found to be higher in flocculated pastes (1.28-1.48). The fatty acid profile varied with respect to the control culture, mainly in the proportion of the essential polyunsaturated fatty acids (PUFAs): eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Total PUFAs, EPA and DHA contents were statistically similar between centrifuged pastes and control culture (PUFAs: 47%, EPA: 4% and DHA: 4.7%), whereas values obtained for flocculated pastes were significantly lower. The rotifers grew equally well when fed with centrifuged pastes or control culture (maximum density: 320 rotifers/mL; instantaneous growth rate: 0.23 rotifers/day, fecundity: 1.49 eggs/female and productivity: 43 x 10(3) rotifers/L/day. No significant effect of vitamin C was found when used as a paste preservative. We concluded that centrifugation is an effective harvesting method, and that freezing to -20 degrees C for four weeks (no vitamin added), may help maintain the nutritional quality of R. salina paste, similar to fresh microalgae and can be offered to Brachionus plicatilis.     

蒙古裸腹溞与褶皱臂尾轮虫的种间关系

在实验室内将蒙古裸腹溞按0．06、0．10、0．30、0．60个·ml^-1的密度与褶皱臂尾轮虫(密度为0．3个·ml。)进行}昆合培养,同时按上述密度分别单种培养了蒙古裸腹溞和褶皱臂尾轮虫作为对照．结果表明,蒙古裸腹溞与褶皱臂尾轮虫之间存在种间竞争,当二者共存时,褶皱臂尾轮虫对蒙古裸腹溞种群产生压制作用,使蒙古裸腹溞在混合培养体系中以很低的密度存在,而褶皱臂尾轮虫种群受蒙古裸腹溞影响不大．通过进一步的饥饿实验发现90％的褶皱臂尾轮虫经过144h饥饿后仍然存活,而经过120h饥饿后的蒙古裸腹溞100％死亡,褶皱臂尾轮虫耐饥饿能力较强是其在与蒙古裸腹溞竞争中获胜的原因之一。     

Inheritance of mitochondrial DNA in the rotifer <Emphasis Type="Italic">Brachionus plicatilis</Emphasis>

By crossing Brachionus plicatilis s.s. NH1L strain and German strain, we obtained two types of hybrids, NH1L female × German male designated as NXG and German female × NH1L male designated as GXN. To confirm the crossing of the two hybrid strains at the genetic level, random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis using 10 kinds of primers (10 and 12 mers) was carried out. Some amplified DNA fragments from RAPD of the hybrid strain showed mixed patterns of both parental strains, thus confirming that both hybrids were crossbreeds of the NH1L and German strains. Using these hybrids, we investigated the mode of mitochondrial inheritance in B. plicatilis. Full-length mtDNA of the four strains was amplified by PCR, and digested with restriction enzymes to obtain restriction fragment length polymorphism (RFLP) patterns. Both hybrid strains had the same RFLP patterns as their female parents. This result shows that mitochondrial inheritance in rotifers is maternal. Guest editors: S. S. S. Sarma, R. D. Gulati, R. L. Wallace, S. Nandini, H. J. Dumont and R. Rico-Martínez Advances in Rotifer Research     

Genetic variation among marine Brachionus strains and function of mate recognition pheromone (MRP)

Recent studies on morphology and genetics of marinerotifer populations have demonstrated the existence ofconsiderable variation. Differences in theglycoprotein structure of a mate recognition pheromone(MRP) probably have a primary role in maintainingspecies boundaries among Brachionus species.This study examined factors involved in mating toclarify their relation with genetic variability. Threeexperiments were performed using 3 Brachionusplicatilis strains (Russia, Germany and Tokyo) and 4B. rotundiformis strains (Hamana, Fiji, Thai andSpain).Selfing and cross mating of 7 rotifer strains wasconducted in the first experiment. Russian malesmated with B. plicatilis females in thefollowing increasing order, based on mating attempts:German, Tokyo and Russia. There was little matingattempts with females of B. rotundiformisstrains. In a second set of experiments, the binding of anantibody (anti-MRP) derived from the MRP of Russianstrain was tested. A fluorescent label was attached toanti-MRP and the antibody reacted with the MRP onfemales of seven strains. The fluorescence intensity,indicating the degree of antibody binding, wasmeasured with epifluorescence microscopy and imageanalysis. The binding intensity of the anti-MRP to theMRP was in the following increasing order: Hamana,Fiji, Spain, German, Tokyo, Thai and Russia.It was expected that when the anti-MRP binds tothe MRP of a female, the males recognition of thefemale would be inhibited. In the third experiment,male mating with females exposed to the anti-MRP wascompared with unexposed females. Russia Tokyo, Thaiand Spain females exposed to anti-MRP, elicited fewermale mating attempts than unexposed females. Matingfrequency and anti-MRP binding significantlycorrelated with genetic distance obtained from isozymeanalysis.     

Characterization of Satellite DNA Sequences from the Commercially Important Marine Rotifers Brachionus rotundiformis and Brachionus plicatilis

This study was initiated to search for species-specific and strain-specific satellite DNA sequences for which oligonucleotide primers could be designed to differentiate between various commercially important strains of the marine monogonont rotifers Brachionus rotundiformis and Brachionus plicatilis. Two unrelated, highly reiterated satellite sequences were cloned and characterized. The eight sequenced monomers from B. rotundiformis and six from B. plicatilis had low intrarepeat variability and were similar in their overall lengths, A + T compositions, and high degrees of repeated motif substructure. However, hybridizations to 19 representative strains, sequence characterizations, and GenBank searches indicated that these two satellites are morphotype-specific and population-specific, respectively, and share little homology to each other or to other characterized sequences in the database. Primer pairs designed for the B. rotundiformis satellite confirmed hybridization specificities on polymerase chain reaction and could serve as a useful molecular diagnostic tool to identify strains belonging to the SS morphotype, which are gaining widespread usage as first feeds for marine fish in commercial production. Received April 12, 1999; accepted July 29, 1999.     

Collection and culture of live foods for aquaculture in Taiwan

Collection and culture of live foods to be used instudies of feeds for rearing finfish and shellfishlarvae in Taiwan began in 1982. Today, 31 species (49strains) of microalgae, three species (nine strains)of rotifers, one cladoceran and one copepod are holdas start culture. Microalgae are collected from localwaters and obtained from foreign collection centers.The most common genera are Chlorella,Nannochloropsis, Tetraselmis, Chaetoceros, Skeletonema, Isochrysis, and Pavlova. Someinteresting genera such as Ellipsoidion,Nannochloris, Synechococcus, and Alexandriumare also included. Three types of rotifers, i.e. L, S,and SS-type, which are classified as Brachionusplicatilis, B. rotundiformis, and Brachionus sp. are found in Taiwan waters. Among therotifers, six strains have been isolated and cultured.Another L-type strain and two SS-type strains wereobtained from foreign sources. The cladoceran Diaphanosoma aspinosum and copepod Apocyclopsroyi are the most common species used in aquaculture.Studies of live foods including their morphology,culture techniques, fatty acid composition andnutritional value as feeds have been undertaken.     

长江口疏浚弃土悬沙对褶皱臂尾轮虫的影响

采用克隆培养法 (编制生命表 )和群体累积培养法 ,研究了长江口疏浚弃土悬沙对褶皱臂尾轮虫(Brachionusplicatilis)的影响 .实验设 0 (CK)、1、3、5、7、9mg·ml-16个悬沙梯度和 2 0、12× 10 6cells·ml-1两个饵料密度 .结果表明 ,克隆培养时 ,悬沙可使该轮虫种群的内禀增长率 (rm)下降 2 9.6 %～ 6 4 .1% ,最大降幅为 130 .0 % .悬沙对轮虫种群的存活率 (lx)有影响 ,而对种群的繁殖率 (mx)无显著影响 .群体累积培养时 ,悬沙对轮虫种群的雌体密度、雄体密度、孤雌生殖卵和休眠卵的密度以及种群的动态变化规律无显著影响 .可见 ,长江口疏浚工程会对轮虫动物有一定的负面影响     

赤潮藻塔玛亚历山大藻对褶皱臂尾轮虫生活史特征的影响

采用生命表方法,研究了不同浓度塔玛亚历山大藻对褶皱臂尾轮虫生命周期中各发育阶段历时以及种群增长参数的影响．结果表明：塔玛亚历山大藻对褶皱臂尾轮虫的生长发育有明显影响,使轮虫的繁殖前期和世代时间延长,繁殖期、繁殖后期和平均寿命缩短,产卵量和繁殖率降低．轮虫的净生殖率和内禀增长率均低于对照．不同藻浓度下轮虫能够维持一定的种群增长．     

Effect of juvenile hormone and serotonin (5-HT) on mixis induction of the rotifer Brachionus plicatilis Muller

Juvenile hormone (JH) and serotonin (5-HT) were previously shown to enhance mictic (sexual) female production of the rotifer Brachionus plicatilis in batch cultures. To explore the basis of these effects, experiments were conducted on isolated individuals. JH treatment of maternal rotifers with 5 and 50 μgml(-1) (18.8 and 187.7 μM) resulted in significantly higher (P<0.05) mictic female production in the second (F(2)) and third (F(3)) generations. JH treatment was effective even at a lower food concentration of 7x10(5) cellsml(-1), but it was not effective when free ammonia was added at 2.4 and 3.1 μgml(-1). Mictic female production was not increased with exposure to 5-HT up to 50 μgml(-1) (129.1 μM) concentrations. When food level was reduced to 7x10(5) cellsml(-1), however, 5-HT-treated rotifers produced significantly (P<0.05) more mictic females than the control, particularly in F(3) generation. Mictic female production of 5-HT-treated rotifers did not differ from that of the control with or without free ammonia, but the intrinsic rate of natural increase (r) of 5-HT-treated rotifers at 3.1 μgml(-1) free ammonia was significantly higher than the control. These results show that juvenile hormone increases mictic female production under optimum and sub-optimum food levels, whereas 5-HT increases both mictic female production at low food level and population growth rate at high free ammonia concentrations. These compounds could be used to manage rotifer cultures and probe the mechanisms controlling the rotifer life cycle as it switches to mictic reproduction.     

Persistent genetic signatures of colonization in Brachionus manjavacas rotifers in the Iberian Peninsula

Recent phylogeographical assessments have consistently shown that continental zooplankton display high levels of population subdivision, despite the high dispersal capacity of their diapausing propagules. As such, there is an apparent paradox between observed cosmopolitanism in the zooplankton that is associated with long-distance dispersal, and strong phylogeographical structures at a regional scale. Such population dynamics, far from migration-drift equilibrium, have been shown in the rotifer species complex Brachionus plicatilis, a group of over a dozen species inhabiting salt lakes and coastal lagoons worldwide. Here we present the mitochondrial DNA phylogeography of one of these species, Brachionus manjavacas, in the Iberian Peninsula, where it often co-occurs with the morphologically similar species B. plicatilis sensu stricto. We obtained sequences from 233 individuals from diapausing eggs and clonal cultures from 16 lakes in the Iberian Peninsula, and a Tunisian lake. Two strongly supported deep mitochondrial DNA clades were found (A and B). Phylogenetic and nested clade analysis showed that clade A has a strong phylogeographical structure, with a strong similarity of phylogeographical patterns between B. manjavacas clade A and B. plicatilis s.s. These include (i) signatures of allopatric fragmentation between central and southern populations, and (ii) range expansions in the Iberian Peninsula, both likely to have occurred during the Pleistocene. We find evidence for a glacial refugium in the Guadiana basin. Clades A and B co-occurred in several of these lakes because of range expansion and secondary contact between both clades. The co-occurrence between B. plicatilis s.s. and B. manjavacas is not recent, and both species might have experienced similar environmental challenges during the Pleistocene. The strong correlation of genetic and geographical distance found suggests that historical events can lead to such correlation, mirroring the effects of 'isolation by distance' in equilibrium populations.