Genetic variation within and between Iliamna corei and I. remota (Malvaceae): implications for species delimitation

Iliamna corei and I. remota are classified as endangered species; however, their designation as separate species has been questioned. In order to address this problem, intersimple sequence repeat (ISSR) data were generated to examine patterns of genetic differentiation within and between these two taxa. ISSRs were used to screen individuals for genetic diversity of I. corei from the single known natural population and two garden populations, and individuals of I. remota from the six natural populations and four garden populations. Using ten primers, 140 informative markers were generated. Ninety-four percent of loci detected revealed polymorphisms. Cluster analysis (neighbour-joining, NJ) revealed that the two species are genetically distinct and that the Illinois populations of I. remota were genetically distinct from the Virginia populations of I. remota . Ordination by Principal Coordinate Analysis (PCoA) supported the findings of the NJ, with a separation of I. corei and I. remota . Analysis of Molecular Variance (AMOVA) revealed that the majority of variation detected was within populations, which is consistent with other self-incompatible plants. The results indicate a correlation between the geographical distributions of the species and gene flow.  © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society , 2006, 151 , 345–354.     

Population genetics of Dioon edule Lindl. (Zamiaceae, Cycadales): biogeographical and evolutionary implications

Dioon edule Lindl. (Zamiaceae) is a cycad endemic to Mexico, that occurs as one species D. edule and the geographical variety D. edule var. angustifolium (Miq.) Miq. Dioon edule has a north to south distribution in eastern Mexico. In this study, we analysed 14 allozymic loci in eight populations of D. edule from its total distribution range by sampling all known populations. Patterns of diversity and genetic variability, within and among populations, were obtained. The mean number of alleles per locus ( A ) was 1.44 and the percentage of polymorphic loci was relatively high ( P  = 54.78). The mean observed ( H _O ) and expected heterozygosity ( H _E ) were 0.27 and 0.24, respectively. F -statistics revealed an excess of heterozygous genotypes, locally and globally ( F  = −0.17 and f  = −0.27, respectively). The genetic variation explained by differences among populations was only 7.5%. We also detected a negative relationship between genetic diversity and latitude. On average, the gene flow between population pairs was relatively high ( Nm  = 2.98); furthermore, gene flow between population pairs was significantly correlated with geographical distances ( r  = −0.38, P  = 0.025). Therefore, patterns of genetic diversity in D. edule appear to be associated with the post-Pleistocene spread of the species, from its southerly (origin) to its northerly range (derived populations, including its central distribution). The biogeographical and evolutionary aspects of the results of this study are discussed. We recognize Dioon angustifolium Miq. for the northernmost disjunct populations.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 80 , 457–467     

Parnassia palustris: a genetically diverse species in Scandinavia

Patterns of variation at nine enzyme loci were examined in 528 plants representing diploid and tetraploid populations of Parnassia palustris s. l. in Europe to assess genetic variation patterns and migration history. Half of the plants showed a unique multilocus phenotype and 75% of all phenotypes occurred only in Scandinavia. Diploid populations showed similar levels of genetic diversity as other widespread outbreeding species with animal-mediated pollination and F -statistics indicated excessive heterozygosity and low rates of gene flow among them. In spite of dramatic population histories caused by the ice ages, diploid populations have maintained the same genetic diversity in Scandinavia as in central and southern Europe. Northern populations have apparently been established through the gradual advance of genetically variable populations and patterns of variation at individual loci indicate different migration routes, from the south-south-west and the east-north-east, respectively. The data strongly support a repeated autoploid origin of the tetraploid cytotype which has been much more successful than the diploid progenitors in colonizing new land since the last ice age. High genetic diversity in Scandinavia has apparently been obtained by a combination of immigration of plants from different source areas and recurrent formation of autotetraploids from diploid progenitors.  © 2003 The Linnean Society of London, Botanical Journal of the Linnean Society , 2003, 142 , 347−372.     

Low population differentiation and high genetic diversity in the invasive species Carduus acanthoides L. (Asteraceae) within its native range in the Czech Republic

Colonizing species are predicted to suffer from reductions in genetic diversity during founding events. Although there is no unique mode of reproduction that is characteristic of successful plant colonizers, many of them are predominantly self-fertilizing or apomictic species, and almost all outcrossing colonizers are self-compatible. Carduus acanthoides comprises a species of disturbed habitats with wind-dispersed seeds that colonizes open spaces of various sizes. Population genetic diversity was expressed by assessing patterns of variation at nine putatively neutral allozyme loci within and among 20 natural populations in its native distribution range in the Czech Republic. Overall, C. acanthoides displayed high levels of genetic diversity compared to other herbaceous plants. The percentage of polymorphic loci was 84.5, with values of 2.37, 0.330, and 0.364 for the mean number of alleles per polymorphic locus ( A ), observed heterozygosity ( H _o), and expected heterozygosity ( H _e), respectively. There was only weak evidence of inbreeding within populations ( f  = 0.097) and very low genetic differentiation among populations ( θ  = 0.085). Analyses of the data provide strong evidence for isolation-by-distance for the whole study area. Even the colonizing species, C. acanthoides , currently supports a substantial amount of allozyme variation at both the species and population levels.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 98 , 596–607.     

Little genetic differentiation across Europe between early-flowering and late-flowering populations of the rapidly declining orchid Neotinea ustulata

Amplified fragment length polymorphism (AFLP) was used to characterize genetic diversity of the endangered Burnt Orchid, Neotinea (formerly Orchis ) ustulata . Fingerprinting of Estonian and British populations revealed surprisingly little genetic differentiation between populations but larger amounts of diversity within populations, especially in Britain. The resulting mean F _st value of 0.51 is unusually high for an orchid species. Much of the variation follows a west–east cline across Europe, whereas the much-discussed early- and late-flowering taxa of N. ustulata are considered insufficiently distinct to be viewed as separate subspecies. The later flowering N. ustulata var. aestivalis probably evolved independently on two or three occasions, each time diverging from the earlier flowering nominate race. The identity of the genes underpinning phenology in the species, and the potential selective advantages of phenological divergence, merit further study. Overall genetic diversity within populations is sufficiently high to render impoverishment an unlikely cause of their recent, precipitous decline.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 87 , 13–25.     

Genetic and environmental components of phenotypic variation in body shape among populations of Atlantic cod (Gadus morhua L.)

A common-garden experiment was conducted on larvae to test for genetic differences in body shape among populations of Atlantic cod ( Gadus morhua ). Offspring from four north-west Atlantic regions were reared from hatching to postmetamorphosis at two temperatures (7 ± 1 °C and 11 ± 1 °C) and two food levels (1500 and 4500 prey L⁻¹). Body shape differed between populations and treatments. Population differences were greatest between south-west Scotian Shelf cod and those further north; the former were characterized by a deeper body, larger head, and longer caudal peduncle than cod from the other populations. Significant differences were also observed between two putative populations on the south-west Scotian Shelf, suggesting genetic divergence between spawning aggregations at small spatial scales (< 100 km). Temperature and food supply also influenced body shape, with the effect of the former being more pronounced. Individuals reared at the higher temperature or food level had a deeper body and a larger head than those reared at the lower temperature or food supply. Phenotypic responses to changes in the rearing environment also differed among populations, indicating genetic differences in phenotypic plasticity. Differences between populations in morphology and in phenotypic plasticity suggest genetic divergence at both large (> 1000 km) and small (< 100 km) spatial scales. The genetic differences at large spatial scales counteracted the expected effects of temperature differences in the wild, suggesting countergradient variation in morphology among these populations.  © 2006 Her Majesty the Queen in Right of Canada. Journal compilation © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 88 , 351–365.     

The genetic structure of the steppe plant Iris aphylla L. at the northern limit of its geographical range

Genetic diversity of Iris aphylla was examined in seven Polish populations located at the northern limit of the geographical range of the species. Using RAPD analysis, 491 DNA fragments were amplified, all of which were polymorphic. The peripheral populations were characterized by a low number of unique bands (one to three per population) and moderate values of genetic diversity parameters (percent of polymorphic loci = 30.6%, Shannon's index = 0.149 and Nei's gene diversity = 0.097, on average), which can be explained as being due to the recent expansion of the European biota and the extremely low frequency or absence of seed set and of juveniles, as well as limited pollen and seed dispersal. There was also no significant correlation between ecological properties (population size; number of flowering ramets and fruits) and genetic diversity parameters. Despite this fact, more fixed loci were noted in the smallest populations than were in the largest ones. All populations were significantly differentiated and geographically structured. Analysis of molecular variance between regions showed a low and nonsignificant value, but relatively high molecular variation was partitioned among and within populations. Only one group of south-eastern populations was structured according to isolation by distance pattern and neighbour-joining cluster analysis. © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society , 2006, 152 , 245–255.     

Cryptic speciation, genetic diversity and gene flow in the California turret spider Atypoides riversi (Araneae: Antrodiaetidae)

Populations of the turret spider Atypoides riversi from eight central Californian sites were compared based on variation at ten allozyme loci. Multidimensional scaling of interpopulation genetic distances defined four population units (Coast Range, Sierran, Valley, Jenness Camp), corroborating the distinctness of Coast Range and Sierran populations indicated by a prior study. While the species status of these units has yet to be determined, Jenness Camp is the most likely to represent a new species, given its clear genetic uniqueness (two fixed allelic differences). Populations in all units were generally in Hardy–Weinberg equilibrium with no evidence of inbreeding, though variability was minimal (mean H _o = 2.8%, mean P  = 15.4%). Reduced variability in these populations may be the result of repeated bottlenecks, environmental homogeneity, and/or directional selection. Interpopulation differentiation within units was significant in the absence of intervening forest habitat and was substantially less in its presence, indicating that gene flow is likely only when forest corridors exist. To foster preservation of the existing gene pool and enhance participation in it, management of the units of At. riversi should focus on maintaining as many populations in situ as possible and facilitating connections between them, while also creating or restoring habitat for potential colonization.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 82 , 27–37.     

Microsatellite DNA analysis of northern pike (Esox lucius L.) populations: insights into the genetic structure and demographic history of a genetically depauperate species

The northern pike Esox lucius L. is a freshwater fish exhibiting pronounced population subdivision and low genetic variability. However, there is limited knowledge on phylogeographical patterns within the species, and it is not known whether the low genetic variability reflects primarily current low effective population sizes or historical bottlenecks. We analysed six microsatellite loci in ten populations from Europe and North America. Genetic variation was low, with the average number of alleles within populations ranging from 2.3 to 4.0 per locus. Genetic differentiation among populations was high (overall θ_ST = 0.51; overall ρ_ST = 0.50). Multidimensional scaling analysis of genetic distances between populations and spatial analysis of molecular variance suggested a single phylogeographical race within the sampled populations from northern Europe, whereas North American and southern European populations were highly distinct. A population from Ireland was monomorphic at all loci, presumably reflecting founder events associated with introduction of the species to the island in the sixteenth century. Bayesian analysis of demographic parameters showed differences in θ (a product of effective population size and mutation rate) among populations from large and small water bodies, but the relative differences in θ were smaller than expected, which could reflect population subdivision within the larger water bodies. Finally, the analyses showed drastic population declines on a time scale of several thousand years within European populations, which we ascribe to either glacial bottlenecks or postglacial founder events.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 84 , 91–101.     

Population genetics and the conservation status of the threatened Iberian steppe grass Puccinellia pungens (Pau) Paunero (Poaceae)

Puccinellia pungens (Pau) Paunero is a narrowly endemic grass found in two continental saline lagoons of north-eastern Spain. This rare plant has been classified as 'at risk of extinction' in several national and European catalogues of endangered species. Recent demographic studies indicate that population sizes greatly exceed several million individuals, challenging that threat category. Our genetic analysis, based on allozymes, has shown that in spite of the large population sizes, very low levels of genetic variation were found in P. pungens . Genetic variation was similar in most populations, but the largest, Gallocanta lagoon as a whole, had less variation (35% polymorphic loci, 1.4 alleles/locus, H _T = 0.038) than the more restricted Royuela range (45% polymorphic loci, 1.5 alleles/locus, H _T = 0.056), suggesting a recent population expansion of the Gallocanta populations from few founder lines. The low genetic distances among populations also suggest a recent divergence. The low genetic variation observed cannot be explained fully by eventual clonal spread and rare seedling establishment in the hypersaline environment. This low variation seems to result from extreme recent population bottlenecks as a consequence of habitat conversion to agricultural fields. In the light of our data, it seems unlikely that reinforcement of populations could increase the genetic diversity of the populations. Hence, conservation efforts should focus on avoiding further habitat loss of this endangered steppe grass species.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 154 , 269–281.     

Heterozygote excess in ancient populations of the critically endangered Dioon caputoi (Zamiaceae,Cycadales) from central Mexico

Dioon caputoi is a long‐lived cycad known from only four populations that range in size from 50 to 120, mostly adult individuals. Dioon caputoi has the most narrow geographical range of all Dioon spp. (less than 10 km), existing completely within the boundaries of the Tehuacán–Cuicatlán Biosphere Reserve, Mexico. Negative inbreeding values were found in all four populations (F_IT = ?0.242) and within subpopulations (F_IS = ?0.379). Only c. 10% of the total genetic variation was partitioned among populations (F_ST = 0.099). We also found that most mean values of genetic variation (A = 1.91 ± 0.12; P = 78.9 ± 10.2; H_E = 0.35 ± 0.01) are within the range reported for other Dioon species with larger populations and with wider geographical ranges. These results support recent findings that rare plant species maintain high levels of genetic diversity. The heterozygote excess found at all loci is discussed in detail from a neutral evolutionary perspective, leaving arguments as working hypotheses for further research. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158 , 436–447.     

Extremely low levels of genetic diversity in the terrestrial orchid Epipactis thunbergii (Orchidaceae) in South Korea: implications for conservation

Levels of allozyme variation, population genetic structure, and fine-scale genetic structure (FSGS) of the rare, both sexually and clonally reproducing terrestrial orchid Epipactis thunbergii were examined for eight ( N  = 734) populations in a 20 × 20-km area in South Korea. Twenty-three putative allozyme loci resolved from 15 enzyme systems were used. Extremely low levels of allozyme variation were found within populations: the mean frequency of polymorphic loci was 3.8% [isocitrate dehydrogenase ( Idh-2 ) with two alleles was polymorphic across populations], the mean number of alleles per locus was 1.04, and the mean expected heterozygosity was 0.013. The overall fixation index was not significantly different from zero ( F _IS = 0.069), although the species is self-compatible. However, a significantly high degree of population differentiation was found between populations at Idh-2 ( F _ST = 0.388) in the studied area. Furthermore, spatial autocorrelation analyses revealed a significant FSGS (up to 3 m) within populations. These observations suggest that the main explanatory factors for the extremely low levels of genetic diversity and the shaping of the population genetic structure of E. thunbergii are genetic drift as a result of a small effective population size, a restricted gene flow, and the isolation of populations. Considering the current genetic structure of E. thunbergii , three guidelines are suggested for the development of conservation strategies for the species in South Korea: (1) protection of habitats of standing populations; (2) prohibition by law of any collection of E. thunbergii ; and (3) protection of nearby pollinator populations, given the fact that fruit set in natural habitats is very low.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 155 , 161–169.     

Implications of contrasting patterns of genetic variability in two vespertilionid bats from the Indonesian archipelago

Wallacean island populations of two Vespertilioninae bats, Myotis muricola and Scotophilus kuhlii , which have similar geographical ranges, showed marked contrast in the amount and pattern of genetic variation. Within islands, genetic variation was on average much higher in M. muricola but declined from west to east, whereas all populations of S. kuhlii had uniformly low levels of genetic variation by mammalian standards. S. kuhlii showed little genetic differentiation between islands and estimates of gene flow were substantial whereas island populations of M. muricola differed markedly and there was a strong isolation-by-distance effect associated with the extent of the sea crossing between islands. Furthermore, the lower mean heterozygosity and small genetic distances between eastern island populations of M. muricola is evidence that there has been a bottleneck associated with the colonization of this area. The attenuation of genetic diversity to the east is also seen in some other mammalian species and may indicate limits to dispersal and have implications for species management. The patterns of variability in S. kuhlii may be a consequence of its strong dispersal capacity and close association with human activity, which, together with other factors, suggest a panmictic population.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 83 , 421–431.     

High levels of genetic diversity in populations of Iris aphylla L. (Iridaceae), an endangered species in Poland

We used RAPDs (Random Amplified Polymorphic DNAs) to investigate genetic diversity and its partition within and between three populations of Iris aphylla in Poland. Analysis of Molecular Variance (AMOVA) of 84 distinct RAPD multiband genotypes revealed higher variation within populations (77.2%) than genetic differentiation between them (22.8%, P  < 0.002). Values of genetic diversity indices ( H ) were similar in all three sites (0.21–0.24). The differentiation of the populations corresponded to low average gene flow ( Nm  = 0.81). Our results indicated that genetic diversity was independent of population size. We concluded that although sexual reproduction and gene flow between populations of I. aphylla were very limited, they preserved high levels of genetic diversity. Relatively large number of seeds, which migrated in the past to populations, as well as patterns of reproduction and life history of I. aphylla may explain this situation.  © 2003 The Linnean Society of London, Botanical Journal of the Linnean Society , 2003, 142 , 65–72.     

Two approaches for the selection of Relevant Genetic Units for Conservation in the narrow European endemic steppe plant Boleum asperum (Brassicaceae)

Several methods based on population biology, biogeography, ecology, and genetics have been traditionally used for the identification of units for conservation below the species level. We use a combination of two methods based on population genetic structure estimators and on probabilities of loss of rare alleles to identify the Relevant Genetic Units for Conservation (RGUCs). The aims were to assess the genetic diversity and population structure of the endemic steppe plant Boleum asperum (Brassicaceae), and to determine how many and which populations significantly represent the total genetic diversity and the rarest allelic variation. Despite the high amplified fragment length polymorphism genetic diversity values detected in B. asperum ( h _T = 0.744), caused probably by its hexaploidy and allogamy, moderate spatial genetic differentiation was detected among populations (< 20%) and geographical ranges (> 13%), suggesting the existence of an ancestral continuous distribution range that was fragmented into separate 'islands' in more recent historical times. Five RGUCs, accounting for the 85.10% of the total genetic variation and representative of the entire geographical distribution of the species, were selected for in situ conservation. Ex situ conservation is proposed to complement the preservation of B. asperum . This method of objective selection of populations may be applied to other candidate taxa for conservation with prior adjustment of the threshold values of diversity required for effective protection of each particular species.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 94 , 341–354.     

Arbuscular mycorrhizal fungi: genetics of multigenomic, clonal networks and its ecological consequences

Arbuscular mycorrhizal fungi are thought to have remained asexual for 400 million years, although recent studies have suggested that considerable genetic and phenotypic variation could potentially exist in populations. A brief discussion of these multigenomic organisms is presented.  © 2003 The Linnean Society of London, Biological Journal of the Linnean Society , 2003, 79 , 59–60.     

Volatile leaf oil diversity in the narrow range endemic Eucalyptus argutifolia (Myrtaceae) and its widespread congener Eucalyptus obtusiflora

Variation in the qualitative composition of volatile leaf oil was examined in the rare Eucalyptus argutifolia and its widespread congener Eucalyptus obtusiflora . The results revealed that, consistent with the pattern seen with allozymes, E. argutifolia had less variation within populations than E. obtusiflora . Total leaf oil diversity was also significantly lower in the rare species. As found with allozymes, most leaf oil diversity was within populations, but there was also a significant proportion of the variation between populations (25.2% and 27.3% for E. argutifolia and E. obtusiflora , respectively). There were significant associations between phenotypic distance based on leaf oils and geographical distance and between phenotypic and genetic distance across all populations, but these associations were not evident within species. Factors leading to reduced variation in E. argutifolia appear to affect all types of variation, but the relationships between different types of variation within the species are less apparent.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 96 , 738–745.     

Genetic differentiation among the Maculinea species (Lepidoptera: Lycaenidae) in eastern Central Europe

The present study aimed to analyse the level of genetic variation in the eastern Central European (Slovenia, Hungary, and Romania/Transylvania) populations of the Large Blues ( Maculinea ) to analyse the pattern of differentiation both between and within the species. One objective was to compare the level of differentiation between the two disputed species ( Maculinea alcon and Maculinea rebeli ) with that among the other species. Imagos were collected from 23 localities in eastern Central Europe in 2002. Enzyme polymorphism was analysed using polyacrylamide gel electrophoresis. Fourteen enzyme loci were studied in all samples. In the analysis of the data, F -statistics and Nei's genetic distances were calculated and a dendrogram (unweighted pair group method with arithmetic mean) was constructed on the basis of the distance matrix. A multivariate analysis of variance was performed to study the pattern of genetic differentiation among the samples. Principal component analysis analysis was also carried out using the allele frequency data of the samples. Our results indicated that the large blues are generally less polymorphic than other European lycaenid butterflies studied. At the same time, the level of genetic differentiation was high, even among local populations within the species. A low level of genetic variation within the populations coupled with strong differentiation among them implies the effect of genetic drift. Strong genetic differentiation of four Maculinea species ( M. alcon , Maculinea teleius, Maculinea nausithous , and Maculinea arion ) was confirmed. Significant differentiation was not found between M. alcon and M. rebeli .  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 91 , 11–21.     

Historical diversification of floodplain forest specialist species in the Amazon: a case study with two species of the avian genus Xiphorhynchus (Aves: Dendrocolaptidae)

Phylogeographical and population genetics methods are used to reconstruct the diversification history of two species of the genus Xiphorhynchus (Aves: Dendrocolaptidae) associated with seasonally flooded forest types in Amazonia. Sequences of the mitochondrial gene cytochrome b were assessed for 21 and 30 individuals, belonging to eight and ten populations, of Xiphorhynchus kienerii and Xiphorhynchus obsoletus , respectively. Uncorrected genetic distances among unique haplotypes recovered ranged only from 0.01% to 0.4% for both species. Over 90% of the genetic variation detected in both species was partitioned within populations, and therefore was not structured geographically. Mismatch distributions and values of Tajima's D -tests indicate that both X. kienerii and X. obsoletus have had small evolutionary effective population sizes, but experienced a recent demographic expansion. These demographic expansions are tentatively dated as occurring over the last 18 000 years BP, a time frame which coincides with the establishment of the early and mid-Holocene age floodplain forest in most of central and eastern Amazonia, following a period of increased river stages throughout the basin. Based on phylogenetic, phylogeographical, and populations genetics data obtained for X. kienerii and X. obsoletus , an evolutionary scenario is proposed to account for the historical diversification of floodplain specialist species in Amazonia.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 89 , 383–395.     

Immigration history and gene dispersal: allozyme variation in Nordic populations of the red campion, Silene dioica (Caryophyllaceae)

Most of the Nordic region was ice-covered during the last (Weichselian) glaciation. During the postglacial period, plant and animal species recolonized the region from several directions and the geographic structuring of genetic variation within Nordic species may still contain a historic component that reflects patterns of postglacial immigration. The present investigation of 69 populations of Silene dioica represents the first large-scale allozyme study of a widespread herbaceous plant in the Nordic region. Although the frequencies of individual alleles showed a range of different geographic patterns, mapping of the axis scores from an ordination of variation at eight polymorphic loci revealed a division into two main geographic groups of populations. The broadly south-western and north-eastern distributions of these two groups of populations suggest that immigration into the region may have involved both eastern and southern geographic sources. However, the geographic boundaries between the two groups of populations are diffuse, and the relatively low between-population component of genetic diversity (G_ST = 16.4%) suggests a history of extensive gene dispersal by pollen.  © 2002 The Linnean Society of London, Biological Journal of the Linnean Society , 2002, 77 , 23–34.