Extra-pair paternity and intraspecific brood parasitism in wild zebra finches Taeniopygia guttata,revealed by DNA fingerprinting

Summary The frequency of extra-pair parentage in a wild population of zebra finches Taeniopygia guttata was examined by DNA fingerprinting. A total of 25 families, comprising 16 pairs of parents and 92 offspring (in broods of 1 to 6) were examined. Ten cases of extra-pair parentage, presumed to constitute intraspecific brood parasitism, were detected (10.9% of offspring or 36% of broods), including one possible instance of quasi-parasitism (parasitism by a female fertilized by the male nest owner). The average number of parasitic eggs per clutch detected by fingerprinting was 1.10±0.32 SD, very similar to the one egg difference in average clutch size between parasitised (6.0±0.82) and unparasitised nests (5.0±0.95). Two cases of extra-pair paternity (EPP) were detected among 82 offspring whose maternity was confirmed: 2.4% of offspring, or 8% of broods. In both cases EPP accounted for only a single offspring within a brood. Behavioural observations show that EPP occurs through extra-pair copulation rather than rapid mate switching. The results are discussed in the light of what is known about the fertile period and sperm precedence patterns in this species. Offprint requests to: T.R. Birkhead     

Genetic mating system and timing of extra-pair fertilizations in the Kentish plover

It is still unclear why females in many bird species pursue extra-pair copulations. Current hypotheses focus mainly on indirect benefits such as obtaining particular good genes for their offspring or maximizing genetic compatibility between themselves and the father of their offspring. Supporting the latter, a recent study of shorebirds suggests that extra-pair matings may function to avoid the negative effects of genetic similarity between mates. Here, we further investigate genetic parentage in the Kentish plover, Charadrius alexandrinus, a shorebird with a highly variable social mating system. DNA fingerprinting revealed that most pairs were genetically monogamous: 7.9% of the broods (7/89) contained extra-pair young, comprising 3.9% of all chicks (9/229). These cases represented, however, three alternative reproductive behaviors: extra-pair paternity, quasi-parasitism (extra-pair maternity) and intraspecific brood parasitism. This is the first study showing the occurrence of all three behaviors in one shorebird species. We also found that extra-pair fertilizations (extra-pair paternity and quasi-parasitism) were more frequent later in the breeding season. There was no consistent relationship between genetic similarity of mates and laying date; the pattern, as well as the degree of genetic similarity, differed among breeding sites within the study population.Communicated by M. Webster     

Co-operative breeding by the Australian Bell Miner Manorina melanophrys Latham: A test of kin selection theory

Summary A population of Bell Miners Manorina melanophrys cosisted of two social units (clans), each consisting of one or more monogamous paris and numerous helpers. Individuals of a breeding pair occupied one home range, while non-breeding individuals occupied partially overlapping home ranges. Attendants (i.e. parents and helpers) showed clear preferences as to whom they aided (fed) and how much aid they provided. A significant correlation was found between the amount of aid given by an attendant and the genetic relationship between the attendant and the recipient. This provides strong quantitative evidence for the theory of kin selection. Only 14% of eggs laid produced juveniles which lived longer than 3 months. It is suggested that the extremely high juvenile mortality favours individuals which help relatives, even while such individuals have dependent offspring of their own.     

Temporal variation and distribution of trace metals in freshwater and fish from Calabar River, S.E. Nigeria

An investigation on the abundance and distribution of trace metals (Fe, Cu, Zn, Mn, Cr, Cd and Pb) in water, and nine species of fish samples from Calabar river was carried out in 1992. The concentrations of iron (6000–7240gl^–1), zinc (4910–7230gl^–1), and cadmium (3–7gl^–1) showed moderate pollution while those of copper (420–630gl^–1), manganese (23–48gl^–1), chromium (<10–20gl^–1) and lead (<1–10gl^–1) in water were well below WHO permissible levels. Significant seasonal changes (0.001p0.25) were obtained for iron, copper, zinc, manganese and cadmium in water. Furthermore, iron, zinc and cadmium showed statistically significant spatial changes (0.005p0.10). Of the nine fish species studied, no statistically significant relationship between body weight and the concentrations of the metals was observed. The concentrations of the metals per mean total body weight apparently decreases in the order Fe>Zn>Cu>Mn>Pb>Cd=Cr and were within the limits that were safe for consumption.     

Effect of parentage and relatedness on the individual contribution to cooperative chick care in carrion crows Corvus corone corone

Carrion crows Corvus corone corone in northern Spain form complex cooperative groups that contain non-dispersing offspring and immigrants. Reproduction is often shared among group members, with polyandry prevalent over joint egg laying. However, due to incest avoidance or sexual immaturity, reproduction is potentially available only to a fraction of group members (potential breeders), while others do not breed (non-breeders). We combined molecular data with video-recorded observations at nests in order to investigate whether potential breeders and non-breeders adjust their individual effort in nestling feeding respectively to their level of parentage and the relatedness with the chicks. Overall, parents of at least one chick in the brood showed the highest feeding rates. Among potential breeders, genetic mothers and fathers fed chicks at significantly higher rates than individuals with no parentage, but they did not adjust their effort according to the proportion of offspring generated. Current direct fitness benefits are therefore important in determining a high provisioning effort, but crows may lack a mechanism to finely assess their share of parentage. Among non-breeders, males contributed more than females to chick feeding, but we found no significant correlation between feeding rate and relatedness to the nestling. We discuss how the latter result can be reconciled with the fact that kin selection has been shown to be important in shaping the crow cooperative society.Communicated by C. Brown     

Replacement of breeders and within-group conflict in the cooperatively breeding acorn woodpecker

Summary Breeding males or females were removed from groups of the cooperatively breeding acorn woodpecker to study (1) competition among individuals from different groups to fill the breeding vancancies, (2) conflict between parents and offspring within groups over replacement of the removed bird, and (3) conflict between parents and offspring for breeding status. Intense fights (power struggles) among nonbreeders from other groups to fill the breeding vacancies developed after the removals. In groups containing only helpers of the opposite sex as the removed bird, replacement was rapid, but in groups containing helpers of the same sex as the removed bird replacement was delayed substantially. In these latter groups parent-offspring conflict developed: helpers could not breed because their parent of the opposite sex was present, and they attacked potential replacements, while their remaining parent advertised for a new mate by calling. Once replacement had occurred, dominance interactions between parents and offspring and among siblings appeared to prevent some individuals from breeding. We conclude that the potential for competition among close kin is high in cooperative species because breeding opportunities are limited.     

Levels of intracellular free amino acids used for salinity tolerance by oysters (Crassostrea virginica) are altered by protozoan (Perkinsus marinus) parasitism

Free amino acid (FAA) levels were measured from May through October 1991 in gill tissues of two groups of juvenile oysters (Crassostrea virginica Gmelin), one transferred from a low salinity field site (8) to a field site of high salinity (20) and high Perkinsus marinus (Mackin, Owen, and Collier) prevalence, the other kept at the low salinity field site. Within 24 h, glycine levels in the oysters transferred to high salinity increased 8-fold, taurine concentrations doubled and the total FAA pool rose from 150 mol g^–1 dry wt to 400 mol g^–1 dry wt. Taurine levels reached a plateau within 20 d after transfer to high salinity and remained at that level until P. marinus infections were detected 85 d after transfer. Taurine and glycine levels declined by 40% in the high salinity population as infection intensity increased between 70 and 105 d. Total FAA declined by approximately 33% over this period. The oysters kept at low salinity were not infected and continued to grow while the infected high salinity oysters showed no increase in shell length after Day 85. FAA levels in the low salinity group remained relatively constant throughout the experiment except for an initial rise triggered by an increase in ambient salinity from 8 to 12. The results suggest that salinity tolerance mechanisms in C. virginica may be impaired by P. marinus infection.     

Female choice on male quantitative traits in lizards — why is it so rare?

Female choice on the basis of male traits has been described in an array of taxa but has rarely been demonstrated in reptiles. In the sand lizard (Lacerta agilis), and possibly in other non-territorial reptiles, a male's contribution to a female's fitness is restricted to his genes. In order to choose males of high genetic quality, females have to trade the fitness gain against the costs of active choice. In a Swedish population of sand lizards, long-lived males sired offspring with higher embryonic survival compared to offspring sired by short-lived males. In spite of this female sand lizards did not mate selectively with older and/or larger males. There appeared to be mo reliable cues to male longevity; age-specific male body size was highly variable. Furthermore, estimates of male nuptial coloration did not covary with ectoparasite load and, hence, females cannot use male coloration as a cue to heritable resistance to pathogenic parasite effects. When cues to male genetic quality are poor, or inaccurate, and males make no parental investment, we predict that female choice will be rare. Sand lizard females mating with many partners lay clutches with higher hatching success. Thus, females may obtain good genes for their young by multiple mating, thereby avoiding costs associated with mate choice.     

Lead Contamination of Topsoil and Vegetation in the Vicinity of a Battery Factory in Nigeria

Levels of Pb, Zn, Cu, Cr, Ni and Cd were determined in topsoil and vegetation in the vicinity of a factory manufacturing lead- batteries in Ibadan, Nigeria. The samples were collected along five transects in different directions, and varying distances up to 1000m from the factory. Soil lead levels were found to be elevated around the factory, with average levels of about 2000mgkg^–1 close to the fence that declined gradually to about 50mgkg^–1 some 750m away. Soil-lead level around a primary school located about 500m from the factory was as high as 1450mgkg^–1. Lead levels were equally elevated in the vegetation, though average levels in vegetation were slightly lower than in the soil. Cadmium concentrations in soil and vegetation, though low, were more positively correlated with lead levels than any of the other metals are with lead.     

Low frequency of extra-pair paternity in pied flycatchers revealed by DNA fingerprinting

Summary Genetic parentage of 135 nestlings from 27 broods of polygynous and monogamous pied flycatchers Ficedula hypoleuca was analyzed by means of multilocus DNA fingerprinting. The minisatellite probe alpha-globin 3HVR detected approximately 12 scorable bands per fingerprint, and the proportion of bands shared between presumably unrelated adults averaged 0.22+0.08 SD. The fingerprints of 125 of the 135 nestlings made a complete match to those of their putative parents. In 4 nestlings a single mismatched band occurred, but since band sharing with both putative parents was high, the single mismatches were assumed to be caused by mutation. The 6 remaining nestlings had 5 or more mismatched bands each, low band-sharing proportions with their putative father and high band-sharing proportions with their putative mother. We thus conclude that they were all sired through extra-pair copulations (EPCs). Hence, only 4% of nestlings were sired through EPCs, and none resulted from intraspecific brood parasitism. One of the cuckolding males was identified, explaining all 5 mismatched bands in the nestling's fingerprint. Three of the illegitimate nestlings were from primary nests of polygynous males; 3 were from nests of monogamous males. The fact that many males in this study started to advertise for a second female in a distant territory several days before their first mate began egglaying, and still managed to secure almost exclusive paternity in their first brood, suggests that male polyterritoriality is not costly in terms of lost paternity. Common anti-cuckoddry tactics performed by male birds, like high rate of within-pair copulation and continuous mate-guarding thoughout the female's fertilizable period, do not seem to be important in pied flycatchers.Offprint requests to: J.T. Lifjeld     

Laboratory spawning and rearing of a marine fish,the silverside Menidia menidia menidia

Adult silversides, Menidia menidia menidia (Linnaeus), were collected in early March, 1974 and maintained in 3 recirculating seawater tanks in the laboratory. Respective groups were fed Moore-Clark Fry Fine at 3, 7 and 10% of their body weight per day. The photoperiod (light intensity approximately 2000 lux) was increased in increments of 10 min/day from 12 h light to 14 h light. The water temperature was increased by 1C°/day from the ambient collection temperature, 14°C, to 22°C. Twenty-four days after beginning laboratory conditioning, fish in each tank were stripped. There was a significant increase (², =0.05) in the number of ripe males at all three feeding levels, compared to an initial field-collected group that was checked at the beginning of the conditioning period. Females also showed significant increases in ripeness at the 7 and 10% but not at the 3% feeding level. The gonadal indices (gonad weight expressed as percentage of body weight) of both sexes were significantly greater than those measured for the initial field-collected group, but did not differ from those of adults collected from the field at the time laboratory conditioning was terminated. Techniques for maintaining eggs from field-ripened adults in the laboratory have been developed, and the effect of salinity on the percentage emergence of larvae determined. The highest emergence rate of larvae was 61% when eggs were maintained at 30 S. Emergence was 56% at 20 S and 47% at 10 S. The effect of delayed feeding on survival and growth of larvae was determined at 20 and 30 S and 25°C. Survival and growth was best for larvae fed Artemia sp. nauplii immediately after emergence at 30 S.Contribution No. 252, Gulf Breeze Environmental Research Laboratory.Associate Laboratory of the National Environmental Research Center, Corvallis, Oregon, USA.     

Genetic analysis of parentage within experimental populations of a male dimorphic beetle,<Emphasis Type="Italic"> Onthophagus taurus</Emphasis>, using amplified fragment length polymorphism

We used the multilocus DNA fingerprinting technique, amplified fragment length polymorphism, to examine parentage of 902 offspring from eight experimental populations of the dung beetle, Onthophagus taurus. The males of this species exhibit a suit of morphological and behavioural traits that characterise alternative mating tactics. Hornless minor males sneak copulations with females that are guarded by horned major males. Our aims were to provide a prospective assessment of the potential role of frequency dependence in the maintenance of alternative mating tactics, to assess the levels of polyandry, and to determine the patterns of sperm usage by multiply mated females. The average proportion of offspring sired by major and minor males did not co-vary with the relative frequency of each morph present in experimental populations. However, there was some indication that the effective mating frequency (number of females producing offspring sired by a given male) of major and minor males may exhibit frequency dependence. Mating success of both male types declined with increasing numbers of major males. Paternity was positively associated with effective mating frequency. Females produced offspring sired from between one and eight males and, on average, paternity was distributed equally amongst a females mates, regardless of the number of males mated. Differences in fertilisation success among males were not associated with alternative male phenotypes. Neither did a males fertilisation success depend on his genetic dissimilarity with the female. These results are discussed in the context of the evolution of alternative mating strategies, and mechanisms of postcopulatory sexual selection.Communicated by N. Wedell     

Genetic parentage in the indigo bunting: a study using DNA fingerprinting

Summary Parentage of nestlings in a North Carolina population of indigo buntings (Passerina cyanea) was analyzed using DNA fingerprinting. Three minisatellite DNA probes (wild type M13, Jeffreys' 33.15 and 33.6) were used to analyze nuclear DNA isolated from muscle biopsies of 63 nestlings, their parents, and other local adults. Each probe detected approximately 15 scorable fragments per individual, with 18%–39% overlap between probes. The proportion of bands shared (using all fragments over all three probes) between presumably unrelated adults averaged 0.23. Of the 63 offspring analyzed, 35 had at least one fragment not present in either putative parent. The distribution of offspring with novel fragments was distinctly bimodal. The lower mode (offspring with 0, 1, or 2 novel fragments, N=41) fit a Poisson distribution, a pattern expected if mutation (estimated rate per fragment= 0.01) were the source of the novel fragments. The remaining 22 offspring had more novel fragments than could be explained by mutation alone (minimum of four independent fragments across all three probes, =8.2). A low band-sharing proportion with the resident male ( =0.24) and high band-sharing with the resident female ( =0.60) implicated extra-pair fertilizations as the source of all 22. Thus in this sample, 35% of all nestlings came from extra-pair fertilizations and none from intra-specific brood parasitism. Of 25 broods sampled, 12 (48%) had at least one excluded offspring. In 3 broods all of the offspring excluded the resident male. Band-sharing proportions between excluded nestlings within a brood could not distinguish between single and multiple extra-pair paternity. Although young males tended to be excluded less often than older males, wing length and weight were not associated with the frequency of exclusion. Weight and wing length of females also were not associated with involvement in EPCs. Six of the 22 excluded offspring (in 3 broods) shared a large proportion of bands and had fewer than four novel fragments when compared to the fingerprints of a neighboring territorial male, implicating those males as actual fathers. The parentage of the remaining 16 offspring (in 9 broods) could not be clearly assigned because (1) one or more neighbors were not sampled or (2) difficulties in scoring across gels prevented confident alignment of fingerprint bands, and insufficient DNA was obtained from muscle samples to allow reanalysis of potential actual fathers on the same gel as excluded nestlings.     

Effect of temperature and salinity on larval development of southern king crab (Lithodes antarcticus)

Larvae of Lithodes antarcticus Jacquinot were reared in October, 1981 from hatching to the glaucothoe stage at 16 temperature/salinity combinations (5.5°; 7.5°; 9.5° and 13.5°C; 26, 29, 32 and 35 S) to determine optimal environmental conditions for larval development. The highest survival percentage was obtained in the culture at 7.5°C and diminished according to temperature increase or decrease. High temperature cultures significantly shorten the larval life duration, but produce large mortalities. At 5.5°C mortality occurred almost exclusively during the moult to glaucothoe stage. Higher survival percentages were obtained as salinity was increased. In the lowest salinity culture (26 S) no zoea reached the post-larvae stage at culture temperatures. The best T/S combination was obtained at 7.5°C and 35 S, with a survival percentage of 29%. The shortest zoeal developments were obtained at 32 S in all culture temperatures. Salinity also affects larvae coloration: there is a pigment concentration on erythrophores, which causes a color decrease.     

Spatial mesoscale patterns of West Pacific picophytoplankton as analyzed by flow cytometry: their contribution to subsurface chlorophyll maxima

Vertical distributions of picophytoplankton (ppp) (<2 m) were studied by ship-board flow cytometry during two cruises in Western Pacific waters to Palau and to Australia in 1990. Weak red-fluorescing small ppp, supposed to be free-living prochlorophytes (Chisholm et al. 1988), were abundant in the area surveyed. These ppp, designated the prochlorophytes, were abundant in the surface waters (>10⁴ cells ml^-1) at the northern region (27°03N; 7°11N) in November, whereas in December at the southern tropical stations (0°23.54S; 9°20.30S; 13°50.6S), they formed subsurface maximum layers (>10⁵ cells ml^-1) on a nitracline at a depth of 3.5 to 5.4% surface irradiation. Their fluorescence intensity increased with depth below 10% surface irradiation. The prochlorophytes at a depth of 1% surface irradiation had ten times higher fluorescence than those at the surface layer. The total fluorescence intensity of the prochlorophytes accounted for 32 to 63% of the sum of the total fluorescence intensity of all fluorescing phytoplankton detected at subsurface chlorophyll maxima in the tropical area. These results suggest that distribution of the prochlorophytes is greatly affected by nitracline and by light intensity and that their chlorophyll is a major contributor to the subsurface chlorophyll maximum in the pelagic West Pacific Ocean.     

Salinity tolerance of benthic estuarine diatoms as tested with a rapid polarographic measurement of photosynthesis

Measurements of net photosynthesis of benthic estuarine diatoms were made by polarographic registration of oxygen saturation. A measuring cell was constructed in which media with salinities of 2.0 to 100.7 were pumped over the algae between measurements. Diatoms from unialgal cultures and mixed populations from intertidal flats appeared to be highly tolerant of extreme salinities. During short-term exposures (20 min) the net photosynthesis of the algae did not drop below 70% of the initial values, within the salinity range 4.0 to 60.0. Prolonged exposure (up to 6 h) gave essentially the same results. Populations of benthic diatoms, sampled from field stations with mean salinities of about 30, 12, and below 5, showed only gradual differences in their tolerance of salinities between 2.0 and 33.7. Two species, Navicula arenaria and Nitzschia sigma, were cultured in media ranging in salinity from 8.0 to 45.0 and from 2.0 to 45, respectively. The tolerance to changing salinity was only slightly affected by the salinity of the medium in the preculture. The role of salinity in the production and distribution of intertidal diatoms is discussed.     

Feeding,growth and bioluminescence of the heterotrophic dinoflagellate Protoperidinium huberi

Feeding, growth and bioluminescence of the thecate heterotrophic dinoflagellate Protoperidinium huberi were measured as a function of food concentration for laboratory cultures grown on the diatom Ditylum brightwellii. Ingestion of food increased with food concentration. Maximum ingestion rates were measured at food concentrations of 600 g C l^-1 and were 0.7 g C individual^-1 h^-1 (1.8 D. brightwelli cells individual^-1 h^-1). Clearance rates decreased asymptotically with increasing food concentration. Maximum clearance rates at low food concentration were ca. 23 l ind^-1 h^-1, which corresponds to a volume-specific clearance rate of 5.9x10⁵ h^-1. Cell size of P huberi was highly variable, with a mean diameter of 42 m, but no clear relationship between cell size and food concentration was evident. Specific growth rates increased with food concentration until maximum growth rates of 0.7 d^-1 were reached at a food concentration of 400 g C l^-1 (1000 cells ml^-1). Food concentrations as low as 10 g C l^-1 of D. brightwellii (25 cells ml^-1) were able to support growth of P. huberi. The bioluminescence of P. huberi varied with its nutritional condition and growth rate. Cells held without food lost their bioluminescence capacity in a matter of days. P. huberi raised at different food concentrations showed increased bioluminescence capacity, up to food concentration that supported maximum growth rates. The bioluminescence of P. huberi varied over a diel cycle, and these rhythmic changes persisted during 48 h of continuous darkness, indicating that the rhythm was under endogenous control.     

Male quality,reproduction, and survival in the great tit (Parus major)

Summary Three possible measures of male quality (social dominance, song, and size), reproduction, and survival were studied in a single population of great tits. Winter dominance position on a feeder was related to strophe length (number of phrases per strophe), inversely related to positive drift (decrease of the singing rate of the phrases in a strophe), but not related to song repertoire size. Neither winter dominance position nor song were related to size (wing length, tarso-metatarsus length, weight).Singing capacity was not correlated with individual reproductive success in a single breeding season, using a rather limited data set. However, better singers (males which sing longer strophes, show less positive drift, and have larger song repertoires) survived better and had a higher individual lifetime reproductive success (on the basis of a male's recruited offspring of all breeding seasons). Our results show that there exist measurable differences whereby birds that are dominant in winter sing better, survive longer, and produce more surviving offspring during their life time. We suggest that differences in male quality are the common cause (direct and indirect) of all these effects.     

Dynamics of social nesting in overwintering meadow voles (Microtus pennsylvanicus): possible consequences for population cycling

Summary A small population of meadow voles (Microtus pennsylvanicus) in a field enclosure was studied from August to February in Apalachin, New York, USA. Radiotelemetry provided direct measures of intraspecific spacing and social nesting through the fall-winter transition. Data on weather and predation were collected concurrently. A total of 32 voles were radiotracked during 6 tracking sessions, with an average of 17.3 voles (11 to 25 range) tracked per session (Figs. 1, 2a). Discrete social nesting constellations first occurred during October, primarily as a result of the formation of extended maternal families (Figs. 1, 2e). Recruitment of adult males and offspring into these early nesting groups was male biased. The average number of voles in these groupings varied from 3.2 (Jan; 3–4 range) to 7.0 (Oct; 4–10 range); but the average number of voles that slept together at any given time, the nesting cluster, remained steady at 2.4 (2–5 range) (Fig. 2e). During late December and early January under the protection of snow, many voles shifted their home areas and nesting affiliations with the result that non-lineage nesting constellations formed (Fig. 1, 3). The thermoregulatory benefits of huddling and the threat of predation appear to be important governors of movement, group formation and dispersion. The existence of an optimum group size produces and Allee Effect that may contribute importantly to population lows and multi-annual cycles.     

Effect of ionizing radiation on haemoglobin of marine lamellibranchs

The effect of ionizing radiation on the iron-linked protein (haemoglobin) of the marine lamellibranchs Anadara granosa (Linn.) and Cardita antiquata (Lam.) from Bombay waters, India is discussed. Purified haemoglobin solutions were exposed to a ⁶⁰Cobalt source delivering a dose of about 4,600 rad/min, at the sample irradiation point. Radiation damage or degradation was measured spectrophometrically by studying changes in the absorption spectra following irradiation in the presence and absence of oxygen. Exposure to ionizing radiation in general caused a decrease in absorption of both haemoglobins, irrespective of location, viz extra-versus intracellular, at Soret (412 m), (540 to 42 m) and (574 to 76 m) peaks, and an increase in absorption at 510 and 630 m. Upon exposure to a higher dose, O₂Hb of C. antiquata showed an increase in absorption at the protein peak (280 m); exposure to a lower dose, however, resulted in decreased absorption. Furthermore, the changes following irradiation were dependent upon the initial state of the pigment. Oxyhaemoglobin, when exposed to radiation, oxidized to hemiglobin, and hemiglobin reduced to oxyhaemoglobin. The extracellular haemoglobin of high molecular weight (3x10⁶) of the false cockle C. antiquata was found to be extremely radio-resistant, whereas intracellular haemoglobin of low molecular weight (74,000) of the arcid clam A. granosa was highly radiosensitive, since it could not be exposed to doses exceeding 18,000 r.