The significance of the radial nerve cords in asteroids and echinoids

Summary The radial nerve cords of a number of different species of echinoids and asteroids have been examined with the electron microscope. They have been shown to consist of tracts of small naked axons with discrete regions of neuropile. Synapses within these areas of neuropile have been described and their significance discussed. A careful examination has failed to reveal any connections between the ectoneural nerve and the mesodermal nerves and muscles. Synapses across the connective tissue sheet separating these regions have been proposed to account for known functional connection. A hypothetical model of the nervous system has been proposed to account for known structure and function.The author wishes to acknowledge the help of Prof. M. S. Laverack during this study. Part of this work was carried out while the author was in receipt of a Queen Elizabeth Fellowship from the Commonwealth Government of Australia.     

Morphogenesis and organogenesis in the regenerating planktotrophic larvae of asteroids and echinoids

In a previous study, we described complete body regeneration (with organogenesis) following surgical bisection in the planktotrophic larvae of the asteroids Luidia foliolata and Pisaster ochraceus. Here we present further detailed observations of these unique regenerative processes not presented in the previous paper. Furthermore, we describe for the first time complete regeneration following surgical bisection of planktotrophic larvae of the regular echinoid Lytechinus variegatus and the irregular echinoid Dendraster excentricus. Larvae of both asteroids and echinoids displayed a capacity for rapid regeneration regardless of their developmental stage. Within 48 h after bisection, aggregations of mesenchyme cells with pseudopodia were observed at the site of surgical bisection. These cellular aggregations were similar in appearance to the mesenchymal blastemas that form in adult echinoderms prior to their arm regeneration, and to those described in other deuterostomes that undergo regeneration. When asteroid larvae were surgically bisected in the early stages of their development, clusters of mesenchyme cells developed into completely new pairs of coelomic pouches located anterior to the newly regenerated digestive tract. This indicates that cell fate in regenerating asteroid larvae remains indeterminate during early development. In the larvae of P. ochraceus, regardless of the developmental stage at the time of bisection, both the anterior and posterior portions regenerated all their missing organs and tissues. However, the larvae of L. foliolata displayed differential regenerative capacity in bisected larval halves at the late bipinnaria stage. The differences observed may be due to differences in larval development (L. foliolata has no brachiolaria stage), and may have evolutionary implications. In the regular echinoid L. variegatus, both larval portions regenerated into morphologically and functionally normal larvae that were indistinguishable from non-bisected control larvae. The regenerative processes were similar to those we observed in planktotrophic asteroid larvae. Regenerating larvae readily metamorphosed into normal juveniles. In the irregular echinoid D. excentricus, posterior portions of larvae completed regeneration and metamorphosis, but anterior portions regenerated only partially during the 2-week study. Our observations confirm that asteroid and echinoid larvae provide excellent models for studies of regeneration in deuterostomes.     

Prefeeding larval development time is not correlated with egg size in regular echinoids (Strongylocentrotus species)

Summary

We compared prefeeding development times, from fertilized egg to prism larva, for Strongylocentrotus embryos from four clutches of eggs (each from a different species) differing in size. Development times did not vary consistently with egg diameter, and trends among eggs of different sizes varied with stage of development. In some cases, development times for eggs of intermediate diameter (S. franciscanus) were longer than those for larger or smaller eggs. Although mean egg diameters in clutches ranged from 84 μm (S. purpuratus) to 162 μm (S. pallidus), differences in development time to the last embryonic stage (prism) were very small. We conclude that the inverse relationship between parental investment in offspring and premetamorphic development time in echinoids depends only on the functional consequences of reduced size of feeding larval stages: effects of egg size on prefeeding development time are not evident.     

Divergent decapentaplegic expression patterns in compound eye development and the evolution of insect metamorphosis

In the fruit fly Drosophila, the patterning genes decapentaplegic and wingless contribute to the spatial control of retina development in an antagonistic manner. We examined the expression patterns of these genes in the developing visual system of the hemimetabolous grasshopper Schistocerca americana and the primitive holometabolous beetle species Tribolium castaneum. The pattern of wingless expression was strongly conserved as a pair of lateral domains at the anterior margins of both the developing retina and the developing optic lobes. The expression of decapentaplegic, on the other hand, is different. Unlike in Drosophila, no decapentaplegic expression was detected before the onset of photoreceptor differentiation in the retinal precursor tissue of either grasshopper or beetle. Moreover, the subsequent expression of decapentaplegic in the latter species was not concentrated in the moving front of retina differentiation, as in Drosophila, but observed in anterior and posterior regions. Our results indicate that Drosophila eye development contains elements of both ancestral and derived regulatory gene functions. The requirement for decapentaplegic as an antagonist of wingless during the early development of the Drosophila retina might have originated during the evolution of insect metamorphosis.     

Metacommunity patterns in larval odonates

The growth of metacommunity ecology as a subdiscipline has increased interest in how processes at different spatial scales structure communities. However, there is still a significant knowledge gap with respect to relating the action of niche- and dispersal-assembly mechanisms to observed species distributions across gradients. Surveys of the larval dragonfly community (Odonata: Anisoptera) in 57 lakes and ponds in southeast Michigan were used to evaluate hypotheses about the processes regulating community structure in this system. We considered the roles of both niche- and dispersal-assembly processes in determining patterns of species richness and composition across a habitat gradient involving changes in the extent of habitat permanence, canopy cover, area, and top predator type. We compared observed richness patterns and species distributions in this system to patterns predicted by four general community models: species sorting related to adaptive trade-offs, a developmental constraints hypothesis, dispersal assembly, and a neutral community assemblage. Our results supported neither the developmental constraints nor the neutral-assemblage models. Observed patterns of richness and species distributions were consistent with patterns expected when adaptive tradeoffs and dispersal-assembly mechanisms affect community structure. Adaptive trade-offs appeared to be important in limiting the distributions of species which segregate across the habitat gradient. However, dispersal was important in shaping the distributions of species that utilize habitats with a broad range of hydroperiods and alternative top predator types. Our results also suggest that the relative importance of these mechanisms may change across this habitat gradient and that a metacommunity perspective which incorporates both niche- and dispersal-assembly processes is necessary to understand how communities are organized. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Divergent patterns of breakpoint reuse in Muroid rodents

Multiple Genome Rearrangement (MGR) analysis was used to define the trajectory and pattern of chromosome rearrangement within muroid rodents. MGR was applied using 107 chromosome homologies between Mus, Rattus, Peromyscus, the muroid sister taxon Cricetulus griseus, and Sciurus carolinensis as a non-Muroidea outgroup, with specific attention paid to breakpoint reuse and centromere evolution. This analysis revealed a high level of chromosome breakpoint conservation between Rattus and Peromyscus and indicated that the chromosomes of Mus are highly derived. This analysis identified several conserved evolutionary breakpoints that have been reused multiple times during karyotypic evolution in rodents. Our data demonstrate a high level of reuse of breakpoints among muroid rodents, further supporting the “Fragile Breakage Model” of chromosome evolution. We provide the first analysis of rodent centromeres with respect to evolutionary breakpoints. By analyzing closely related rodent species we were able to clarify muroid rodent karyotypic evolution. We were also able to derive several high-resolution ancestral karyotypes and identify rearrangements specific to various stages of Muroidea evolution. These data were useful in further characterizing lineage-specific modes of chromosome evolution.     

Genetic variance and covariance patterns of larval development in the small white butterflyPieris rapae crucivora Boisduval

Quantitative genetic theory indicates that genetic covariance patterns among life history characters should have played an important role as genetic constraint in life history evolution. Highly positve (and negative) genetic correlations between larval development time (or larval growth rate) and adult size characters were detected by means of sib analysis for the small white butterfly Pieris rapae crucivora. The genetic associations suggested that evolution of developmental characteristics and adult phenotypic traits were constrained by pleiotropy. The positive genetic correlations between development time and adult body size may be compatible with the trade-off between them, but the negative genetic correlations between larval growth rate and adult body size are not predicted from theories of optimal energy allocation. That phenotypic correlations drastically differed from the genetic correlations indicates limitations of evolutionary inferences based only on phenotypic variation.     

Stage- and tissue-specific patterns of cell division in embryonic and larval tissues of amphioxus during normal development

The distribution of dividing cells is described for embryos and larvae of amphioxus (Branchiostoma floridae) pulse labeled with bromodeoxyuridine. Because cell division is assessed for all of the developing tissues, this is the first comprehensive study of developmental cell proliferation for an animal lacking a stereotyped cell lineage. In amphioxus, cell divisions are virtually synchronous during cleavage, but become asynchronous at the blastula stage. Starting at the neurula stage, after the origin of the mesoderm, the proportion of dividing cells progressively declines in the somitic mesoderm and notochord. Other tissues, however, deviate from this pattern. For example, in the mid-neurula, there is a brief, intense burst of mitosis at the anterior end of the neural plate. Also, from the neurula through the early larval stage, all of the ectoderm cells cease dividing and develop cilia that propel the animal through the water; subsequently, in the epidermis of later larvae, mitosis resumes and the proportion of ciliated cells declines as muscular undulation gradually replaces ciliation for swimming. Finally, in the early larvae, there is a terminal arrest of cell division in three cell types that differentiate early to participate in feeding as soon as the mouth opens-namely the ciliated pharyngeal cells that produce the feeding current and the secretory cells of the club-shaped gland and endostyle that export food-trapping mucus into the pharynx. In sum, these stage- and tissue-specific changes in cell proliferation intensity illustrate how the requirements of embryonic and larval natural history can shape developmental programs.     

Enzyme patterns in mitochondria of eggs, liver, and skeletal muscle during larval development of Xenopus

The amebo-flagellate Naegleria gruberi contains three major RNA polymerase activities separable from whole cell extracts by DEAE-Sephadex column chromatography. One is resistant to the durg α-amanitin and the other two are sensitive to it. These separated activities were characterized and then examined during the differentiation of ameba to flagellate, which is dependent on RNA synthesis and which exhibits gross changes in RNA metabolism under the conditions employed. It was found that no detectable changes occurred qualitatively or quantitatively in these polymerases during differentiation. Care was taken to account for total enzyme activity in every fraction of the extraction procedure. It is concluded from the above observation that gross changes in RNA synthesis as well as differential gene activity can occur with absolutely no major fluctuations in the DNA-dependent RNA polymerases.     

Life styles and patterns of development of gills and muscles in larval cyprinids (Cyprinidae; Teleostei)

A morphometric study of gill structures and of the body musculature during the first weeks after hatching was carried out on larvae of six cyprinid species: Leuciscus cephalus, L. leuciscus, Rutilus rutilus, Alburnus albumus, Chondrostoma nasus and Abramis brama . In all species a unicellular layer of red muscle fibres covers the central muscle mass; this layer is of greatest extent shortly after hatching but diminishes gradually in mass by contracting towards the lateral region of the body until it merges with (or gives rise to) the adult red muscle fibres proper. There is a close relationship between the rate of differentiation of gill structures and the rate at which the larval red muscle layer disappears, the pattern of this relationship reflecting the life style of the species. The longer the larvae delay the start of their free-swimming existence after hatching (which in A. alburnus may be as long as 10 days) the longer does the red layer of muscle fibres serve as the organ of gas exchange and the longer is gill development suppressed. It appears that the metabolism of the swimming muscles is almost entirely aerobic so long as gas exchange takes place across the whole body surface, the glycolytic capacity of the central muscle mass developing only slowly in conjunction with the switch from red layer to gills as the major respiratory organ.     

Connexin 43 expression reflects neural crest patterns during cardiovascular development

We used transgenic mice in which the promoter sequence for connexin 43 linked to a lacZ reporter was expressed in neural crest but not myocardial cells to document the pattern of cardiac neural crest cells in the caudal pharyngeal arches and cardiac outflow tract. Expression of lacZ was strikingly similar to that of cardiac neural crest cells in quail-chick chimeras. By using this transgenic mouse line to compare cardiac neural crest involvement in cardiac outflow septation and aortic arch artery development in mouse and chick, we were able to note differences and similarities in their cardiovascular development. Similar to neural crest cells in the chick, lacZ-positive cells formed a sheath around the persisting aortic arch arteries, comprised the aorticopulmonary septation complex, were located at the site of final fusion of the conal cushions, and populated the cardiac ganglia. In quail-chick chimeras generated for this study, neural crest cells entered the outflow tract by two pathways, submyocardially and subendocardially. In the mouse only the subendocardial population of lacZ-positive cells could be seen as the cells entered the outflow tract. In addition lacZ-positive cells completely surrounded the aortic sac prior to septation, while in the chick, neural crest cells were scattered around the aortic sac with the bulk of cells distributed in the bridging portion of the aorticopulmonary septation complex. In the chick, submyocardial populations of neural crest cells assembled on opposite sides of the aortic sac and entered the conotruncal ridges. Even though the aortic sac in the mouse was initially surrounded by lacZ-positive cells, the two outflow vessels that resulted from its septation showed differential lacZ expression. The ascending aorta was invested by lacZ-positive cells while the pulmonary trunk was devoid of lacZ staining. In the chick, both of these vessels were invested by neural crest cells, but the cells arrived secondarily by displacement from the aortic arch arteries during vessel elongation. This may indicate a difference in derivation of the pulmonary trunk in the mouse or a difference in distribution of cardiac neural crest cells. An independent mouse neural crest marker is needed to confirm whether the differences are indeed due to species differences in cardiovascular and/or neural crest development. Nevertheless, with the differences noted, we believe that this mouse model faithfully represents the location of cardiac neural crest cells. The similarities in location of lacZ-expressing cells in the mouse to that of cardiac neural crest cells in the chick suggest that this mouse is a good model for studying mammalian cardiac neural crest and that the mammalian cardiac neural crest performs functions similar to those shown for chick.     

Coordinated shifts to non-planktotrophic development in spatangoid echinoids during the Late Cretaceous

Despite widespread interest in the interplay between evolutionary and developmental processes, we still know relatively little about the evolutionary history of larval development. Many clades exhibit multiple shifts from planktotrophic (feeding) to non-planktotrophic (non-feeding) larval development. An important question is whether these switches are scattered randomly through geological history or are concentrated in particular intervals of time. This issue is addressed using the Cretaceous spatangoid sea urchins, which are unusual in that larval strategy can be determined unambiguously from abundantly fossilized adult tests. Using a genus-level phylogeny, we identify five clades of non-planktotrophic taxa, each of which first appears in the fossil record in the Campanian or Maastrichtian (the final two Cretaceous stages). No examples of non-planktotrophy have been identified in any of the earlier stages of the Cretaceous. This strongly suggests that shifts to non-planktotrophic development are clustered in certain episodes of geological history, and this, in turn, implies that extrinsic factors operating at these times are responsible for driving shifts in developmental strategy.     

Major muscle systems in the larval caenogastropod,Ilyanassa obsoleta,display different patterns of development

This study describes the anatomical and developmental aspects of muscular development from the early embryo to competent larval stage in the gastropod Ilyanassa obsoleta. Staining of F‐actin revealed differential spatial and temporal patterns of several muscles. In particular, two major muscles, the larval retractor and pedal retractor muscles originate independently and display distinct developmental patterns similar to observations in other gastropod species. Additionally, together with the larval retractor muscle, the accessory larval muscle developed in the embryo at the trochophore stage. Therefore, both these muscles develop prior to ontogenetic torsion. The pedal retractor muscle marked the most abundant growth in the mid veliger stage. Also during the middle stage, the metapodial retractor muscle and opercular retractor muscle grew concurrently with development of the foot. We show evidence that juvenile muscles, such as the buccal mass muscle and siphon muscle develop initially during the late veliger stage. Collectively, these findings substantiate that larval myogenesis involves a complex sequence of events that appear evolutionary conserved within the gastropods, and set the stage for future studies using this model species to address issues concerning the evolution and eventual fates of larval musculature in molluscs. J. Morphol., 2009. © 2009 Wiley‐Liss, Inc.     

Divergent timing and patterns of species accumulation in lowland and highland neotropical birds

Late Pliocene and Pleistocene climatic instability has been invoked to explain the buildup of Neotropical biodiversity, although other theories date Neotropical diversification to earlier periods. If these climatic fluctuations drove Neotropical diversification, then a large proportion of species should date to this period and faunas should exhibit accelerated rates of speciation. However, the unique role of recent climatic fluctuations in promoting diversification could be rejected if late Pliocene and Pleistocene rates declined. To test these temporal predictions, dateable molecular phylogenies for 27 avian taxa were used to contrast the timing and rates of diversification in lowland and highland Neotropical faunas. Trends in diversification rates were analyzed in two ways. First, rates within taxa were analyzed for increasing or decreasing speciation rates through time. There was a significant trend within lowland taxa towards decreasing speciation rates, but no significant trend was observed within most highland taxa. Second, fauna wide diversification rates through time were estimated during one-million-year intervals by combining rates across taxa. In the lowlands, rates were highest during the late Miocene and then decreased towards the present. The decline in rates observed both within taxa and for the fauna as a whole probably resulted from density dependent cladogenesis. In the highlands, faunawide rates did not vary greatly before the Pleistocene but did increase significantly during the last one million years of the Pleistocene following the onset of severe glacial cycles in the Andes. These contrasting patterns of species accumulation suggest that lowland and highland regions were affected differently by recent climatic fluctuations. Evidently, habitat alterations associated with global climate change were not enough to promote an increase in the rate of diversification in lowland faunas. In contrast, direct fragmentation of habitats by glaciers and severe altitudinal migration of montane vegetation zones during climatic cycles may have resulted in the late Pleistocene increase in highland diversification rates. This increase resulted in a fauna with one third of its species dating to the last one million years.     

Divergent patterns of age-dependence in ornamental and reproductive traits in the collared flycatcher

Sexual ornaments are predicted to honestly signal individual condition. We might therefore expect ornament expression to show a senescent decline, in parallel with late-life deterioration of other characters. Conversely, life-history theory predicts the reduced residual reproductive value of older individuals will favor increased investment in sexually attractive traits. Using a 25-year dataset of more than 5000 records of breeding collared flycatchers (Ficedula albicollis) of known age, we quantify cross-sectional patterns of age-dependence in ornamental plumage traits and report long-term declines in expression that mask highly significant positive age-dependency. We partition this population-level age-dependency into its between- and within-individual components and show expression of ornamental white plumage patches exhibits within-individual increases with age in both sexes, consistent with life-history theory. For males, ornament expression also covaries with life span, such that, within a cohort, ornamentation indicates survival. Finally, we compared longitudinal age-dependency of reproductive traits and ornamental traits in both sexes, to assess whether these two trait types exhibit similar age-dependency. These analyses revealed contrasting patterns: reproductive traits showed within-individual declines in late-life females consistent with senescence; ornamental traits showed the opposite pattern in both males and females. Hence, our results for both sexes suggest that age-dependent ornament expression is consistent with life-history models of optimal signaling and, unlike reproductive traits, proof against senescence.     

施氏鲟仔鱼发育圾异速生长模型

施氏鲟仔鱼的生长发育可分为两个时期:卵黄囊期(或称为自由胚期),即从刚出膜(0日龄,10.17±0.63 mm)到初次开口(9日龄,18.93±0.74 mm);晚期,从开口摄食至器官发育基本完全(38日龄,41.89±5.09 mm).卵黄囊期仔鱼的感觉、摄食、呼吸、游泳等器官快速分化;晚期仔鱼各骨板分化并发育,在形态上逐渐完成向成鱼的转变.对施氏鲟仔鱼异速生长进行的研究表明,仔鱼许多关键器官均存在异速生长现象,如眼径、口宽、尾鳍长、胸鳍长分别在2日龄、8~9日龄、10日龄、11日龄出现生长拐点,拐点之前器官快速生长,拐点之后生长速度减慢甚至近似等速生长.施氏鲟仔鱼各器官呈现出协调和快速发育的特征,随着重要的感觉、摄食、呼吸、游泳等器官的发育和完善,仔鱼快速地具备了躲避敌害和摄食的能力,其生存能力大大提高.     

施氏鲟仔鱼发育及异速生长模型

施氏鲟仔鱼的生长发育可分为两个时期:卵黄囊期(或称为自由胚期),即从刚出膜(0日龄,10.17±0.63 mm)到初次开口(9日龄,18.93±0.74 mm);晚期,从开口摄食至器官发育基本完全(38日龄,41.89±5.09 mm).卵黄囊期仔鱼的感觉、摄食、呼吸、游泳等器官快速分化;晚期仔鱼各骨板分化并发育,在形态上逐渐完成向成鱼的转变.对施氏鲟仔鱼异速生长进行的研究表明,仔鱼许多关键器官均存在异速生长现象,如眼径、口宽、尾鳍长、胸鳍长分别在2日龄、8～9日龄、10日龄、11日龄出现生长拐点,拐点之前器官快速生长,拐点之后生长速度减慢甚至近似等速生长.施氏鲟仔鱼各器官呈现出协调和快速发育的特征,随着重要的感觉、摄食、呼吸、游泳等器官的发育和完善,仔鱼快速地具备了躲避敌害和摄食的能力,其生存能力大大提高.     

Divergent patterns of telomere shortening in tropical compared to temperate stonechats

Telomeres have emerged as important biomarkers of health and senescence as they predict chances of survival in various species. Tropical birds live in more benign environments with lower extrinsic mortality and higher juvenile and adult survival than temperate birds. Therefore, telomere biology may play a more important role in tropical compared to temperate birds. We measured mean telomere length of male stonechats (Saxicola spp.) at four age classes from tropical African and temperate European breeding regions. Tropical and temperate stonechats had similarly long telomeres as nestlings. However, while in tropical stonechats pre‐breeding first‐years had longer telomeres than nestlings, in temperate stonechats pre‐breeding first‐years had shorter telomeres than nestlings. During their first breeding season, telomere length was again similar between tropical and temperate stonechats. These patterns may indicate differential survival of high‐quality juveniles in tropical environments. Alternatively, more favorable environmental conditions, that is, extended parental care, may enable tropical juveniles to minimize telomere shortening. As suggested by previous studies, our results imply that variation in life history and life span may be reflected in different patterns of telomere shortening rather than telomere length. Our data provide first evidence that distinct selective pressures in tropical and temperate environments may be reflected in diverging patterns of telomere loss in birds.     

Arrested larval development in cattle nematodes

Most economically important cattle nematodes are able to arrest their larval development within the host - entering a period of dormancy or hypobiosis. Arrested larvae have a low death rate, and large numbers can accumulate in infected cattle during the grazing season. Because of this, outbreaks of disease caused by such nematodes can occur at times when recent infection with the parasites could not have occurred, for example during winter in temperature northern climates when cattle are normally housed. The capacity to arrest is a heritable trait. It is seen as an adaptation by the parasite to avoid further development to its free-living stages during times when the climate is unsuitable for free-living survival. But levels of arrestment can vary markedly in different regions, in different cattle, and under different management regimes. Climatic factors, previous conditioning, host immune status, and farm management all seem to affect arrestment levels. In this article, James Armour and Mary Duncan review the biological basis of the phenomenon, and discuss the apparently conflicting views on how it is controlled.     

Ocean-scale patterns of 'biodiversity' of Atlantic asteroids determined from taxonomic distinctness and other measures

We examine patterns of ‘gamma’ (within-rcgion) and ‘beta’ (between-region) diversity from analysis of a presence/absence dataset for species of asteroids encompassing the whole Atlantic Ocean partitioned into 26 regions. Absolute species numbers (a poor measure of biodiversity) in shallow coastal areas and the deep sea are the same, although species richness per area for two well-sampled regions suggests, qualitatively, that coastal areas may be more speciose. Taxonomic distinctness (A*), an index which is markedly less sample-size dependent than other common diversity measures, shows no significant association with geographic area and no clear pattern with depth, suggesting an absence of latitudinal and coastal/deep trends. Cluster analysis shows that distinctive faunal assemblages are most evident in shelf/ shallow waters, where six groupings separate recognizably acccording to geographical location. Three of these arc the southernmost regions of the Atlantic (southeast of S. America, S. Angola/S. Africa & Tristan da Cunha/Gough Island) and arc characterized by their isolation and high levels of endemism. As depth increases so does the amount of faunal similarity between regions. This indicates that beta diversity is highest in shelf regions and lowest in lower bathyal/abyssal regions. Our results may support the contention which questions the emerging paradigm that the deep sea has exceptionally high diversity. It is evident, however, that comparisons (e.g. between coasts and the deep sea) are problematic and can depend very much on the element(s) of biodiversity measured, sampling methods and the spatial scales (e.g. alpha, beta or gamma diversity) over which assessment is made. Any wider conclusions should therefore be drawn cautiously, particularly since assessment is made of only one faunal group. Other findings include significant correlation between the depth range of asteroids and their geographical range. The utility of low-resolution datasets is reviewed. It is concluded that within limitations they can be of value for determining broad (e.g. regional, ocean-scale and global-scale) patterns of diversity and community structure, especially when analysed using measures relatively uninfluenced by sample size.