Morphological evolution and genetic differentiation in Daphnia species complexes

Despite many ecological and evolutionary studies, the history of several species complexes within the freshwater crustacean genus Daphnia (Branchiopoda, Anomopoda) is poorly understood. In particular, the Daphnia longispina group, comprising several large-lake species, is characterized by pronounced phenotypic plasticity, many hybridizing species and backcrossing. We studied clonal assemblages from lakes and ponds comprising daphnids from several species complexes. In order to reveal patterns of reticulate evolution and introgression among species, we analysed three data sets and compared nuclear, mtDNA and morphological divergence using animals from 158 newly established clonal cultures. By examining 15 nuclear and 11 mitochondrial (12S/16S rDNA) genetic characters (allozymes/restriction enzymes), and 48 morphological traits, we found high clonal diversity and discontinuities in genotypic and morphological space which allowed us to group clones by cytonuclear differentiation into seven units (outgroup D. pulex). In contrast to six groups emerging from nuclear divergence (related to three traditional species, D. cucullata, D. galeata, D. hyalina and three pairwise intermediate hybrids), a seventh group of clones was clearly resolved by morphological divergence: distinct mtDNA haplotypes within one nuclear defined cluster, ‘D. hyalina’, resembled traditional D. hyalina and D. rosea phenotypes, respectively. In other nuclear defined clusters, association between mtDNA haplotype and morphology was low, despite hybridization being bidirectional (reciprocal crosses). Morphological divergence was greatest between young sister species which are separated on the lake/pond level, suggesting a significant role for divergent selection during speciation along with habitat shifts. Phylogenetic analyses were restricted to four cytonuclear groups of clones related to species. mtDNA and nuclear phylogenies were consistent in low genetic divergence and monophyly of D. hyalina and D. rosea. Incongruent patterns of phylogenies and different levels of genetic differentiation between traditional species suggest reticulate evolutionary processes.     

Evolution of Dystaenia takesimana (Apiaceae), endemic to Ullung Island, Korea

Dystaenia (Apiaceae) consists of two species, one distributed in Japan (D. ibukiensis), and the other endemic to Ullung Island, Korea (D. takesimana). In comparison with representative outgroup taxa in Ligusticum, Seseli, Angelica, and Osmorhiza, Dystaenia is shown to be monophyletic based on sequences from chloroplast trnL-F intron and spacer regions confirming previously published results using ITS sequences. Loss of one large part of trn L-F in D. takesimana strongly suggests that this species evolved from D. ibukiensis rather than the reverse. AFLP analysis within and among twelve populations (six from each species; total 126 individuals) using three primer combinations reveals 130 reliable fragments. Neighbour-joining analysis shows the two species to be distinct populational systems. Levels of overall genetic variation as measured by Shannon Diversity are significantly higher in D. takesimana. Geographic structuring of genetic variation occurs within D. ibukiensis but not within D. takesimana, suggesting that the Ullung species exists as a single population. It is hypothesised that after a founder-effect reduction of genetic variation, anagenetic speciation may have occurred in D. takesimana by gradual morphological divergence accompanied by accumulation of genetic variation through mutation, recombination and drift.     

An ITS-based phylogenetic analysis of theEuryptera species group inLomatium (Apiaceae)

Lomatium, the largest genus of Apiaceae in western North America, includes many narrow endemics whose relationships are uncertain. Although no infrageneric classification exists forLomatium, several informal groups have been recognized. TheEuryptera group comprises seven narrowly endemic species distributed primarily in California. We conducted parsimony and maximum likelihood (ML) analyses using sequences of the internal transcribed spacers (ITS) of the nuclear ribosomal DNA from species of theEuryptera group and several other species ofLomatium. When considered with distribution, morphological, and cpDNA data, the ITS analyses are consistent with the monophyly of theEuryptera group and suggests that speciation in this group has occurred through geographical divergence. Inferences from ITS data also identify putative progenitors of the polyploidEuryptera species.     

Characterization of an Indonesian Isolate of Paecilomyces reniformis

An entomopathogenic fungus (IndGH 96), identified as Paecilomyces reniformis, was isolated from long-horned grasshoppers (Orthoptera: Tettigoniidae) in Sulawesi, Indonesia. The phenotypic and molecular data identified the IndGH 96 as a P. reniformis. We present the first comprehensive characterization of this species using morphological features, sequencing of the ITS1-5.8s-ITS2 region, D1/D2 region of 28S of rDNA, and a portion of the tubulin gene, and laboratory bioassays. Distinguishing features include a hyphal body stage during vegetative growth and the production of distinctly curved, light-green conidia. High dosage bioassays showed that IndGH 96 was infectious to both long-horned and short-horned grasshoppers but not to the house cricket, Acheta domestica, or to the lepidopterans velvetbean caterpillar, Anticarsia gemmatalis or fall armyworm, Spodoptera frugiperda. Phenotypic and genetic analyses suggest that IndGH 96 and other isolates of P. reniformis are more closely related to Nomuraea rileyi than to other species of Paecilomyces.     

A study of wing morphology and fluctuating asymmetry in interspecific hybrids between Drosophila buzzatii and D. koepferae

In this work we investigate the effect of interspecific hybridization on wing morphology using geometric morphometrics in the cactophilic sibling species D. buzzatii and D. koepferae. Wing morphology in F₁ hybrids exhibited an important degree of phenotypic plasticity and differs significantly from both parental species. However, the pattern of morphological variation between hybrids and the parental strains varied between wing size and wing shape, across rearing media, sexes, and crosses, suggesting a complex genetic architecture underlying divergence in wing morphology. Even though there was significant fluctuating asymmetry for both, wing size and shape in F₁ hybrids and both parental species, there was no evidence of an increased degree of fluctuating asymmetry in hybrids as compared to parental species. These results are interpreted in terms of developmental stability as a function of a balance between levels of heterozygosity and the disruption of coadaptation as an indirect consequence of genomic divergence.     

Morphological and phylogenetic analyses of <Emphasis Type="Italic">Uromyces appendiculatus</Emphasis> and <Emphasis Type="Italic">U. vignae</Emphasis> on legumes in Japan

Uromyces appendiculatus, inclusive of three varieties, is distinguished from U. vignae primarily by the position of urediniospore germ pores and putative host specificity. However, opinions concerning these morphological and physiological features as taxonomic characters have varied greatly, and distinction of these species has often been confused. To clarify the taxonomy of these two species, morphological features of urediniospores and teliospores of 225 rust fungus specimens on species of Phaseolus, Vigna, Apios, Lablab, and Dunbaria were examined by light microscopy and scanning electron microscopy. Forty-five specimens were subjected to molecular phylogenetic analyses. As a result, the position of germ pores in urediniospores and the teliospore-wall thickness were considered as good characters to separate three morphological groups. In molecular analyses, the specimens fell into two and three clades based on the nucleotide sequence at D1/D2 domain of LSU rDNA and ITS regions, respectively. One of the D1/D2 clades corresponded to one morphological group whereas another D1/D2 clade included two other morphological groups. In contrast, each of the three ITS clades corresponded to a separate morphological group. Neither morphological groups nor molecular clades were host limited. It is suggested that the three morphological groups that corresponded to three distinct ITS clades constitute distinct species.Contribution no. 186 from the Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

Parallel and non‐parallel morphological divergence among foraging specialists in European whitefish (Coregonus lavaretus)

Parallel phenotypic evolution occurs when independent populations evolve similar traits in response to similar selective regimes. However, populations inhabiting similar environments also frequently show some phenotypic differences that result from non‐parallel evolution. In this study, we quantified the relative importance of parallel evolution to similar foraging regimes and non‐parallel lake‐specific effects on morphological variation in European whitefish (Coregonus lavaretus). We found evidence for both lake‐specific morphological characteristics and parallel morphological divergence between whitefish specializing in feeding on profundal and littoral resources in three separate lakes. Foraging specialists expressed similar phenotypes in different lakes in both overall body shape and selected measured morphological traits. The morphology of the two whitefish specialists resembled that predicted from other fish species, supporting the conclusion of an adaptive significance of the observed morphological characteristics. Our results indicate that divergent natural selection resulting from foraging specialization is driving and/or maintaining the observed parallel morphological divergence. Whitefish in this study may represent an early stage of divergence towards the evolution of specialized morphs.     

Systematic analysis of Dendrobium Swartz section Dendrocoryne in the Australian region

Dendrobium sect. Dendrocoryne is a difficult taxonomic group of allied species and species complexes, occurring on the eastern Australian coastline, Lord Howe Island and New Caledonia. Significant morphological characters were assessed from all key areas of the distribution range. Patterns of variation between species, varieties and hybrids were investigated by principal coordinate analysis. The analyses affirm 12 species in the sect. Dendrocoryne described by Schlechter (1912, 1982) and Dockrill (1969, 1992), including a cluster of small statured D. gracilicaule, D. adae, D. fleckeri, and D. finniganense, and three variable species complexes – D. speciosum, D. kingianum and D. tetragonum. Recently described D. finniganense and D. callitrophilum are interpreted as members of the section. Phylogenetic relationships are presented based on parsimony analysis of 31 morphological characters. Analyses do not support proposals to recognize new genera for D. tetragonum, D. callitrophilum and D. aemulum, or to create new species within D. tetragonum, D. jonesii, D. speciosum and D. kingianum. The phylogeny indicates that rainforest taxa are earlier lineages and xerophytic taxa are more derived.     

Geographical diversity and genetic relationships among Cedrus species estimated by AFLP

Genetic diversity was described in 17 cedar populations covering the geographical range of the four species of the genus Cedrus. The study was conducted using amplified fragment length polymorphism (AFLP) on haploid tissues (megagametophytes). Eleven selective AFLP primer pairs generated a total of 107 polymorphic amplification products. Correspondence and genetic distance analyses indicated that Cedrus deodara constitutes a separate gene pool from the Mediterranean cedars. Within Mediterranean cedars, we distinguished two groups: the first one is made of Cedrus atlantica, while the second one is made of Cedrus libani and Cedrus brevifolia, these latter two species being genetically similar despite important divergence previously observed for morphological and physiological traits. The lowest intrapopulation variability was found in the two C. deodara populations analyzed. Surprisingly, C. brevifolia, the endemic taxon from the island of Cyprus that is found in small and fragmented populations, showed one of the highest levels of diversity. This unexpected pattern of diversity and differentiation observed for C. brevifolia suggests a recent divergence rather than a relictual, declining population. Patterns of diversity within- and among-populations were used to test divergence and fragmentation hypotheses and to draw conclusions for the conservation of Cedrus gene pools.     

The systematics of Australian Daphnia (Cladocera: Daphniidae). Multivariate morphometrics

Forty nine characters were measured in each of 76 male and 200 female Daphnia including specimens from all eleven taxa described from the genus in Australia, except D. jollyi. Separate multivariate analyses (cluster, principal components and discriminant) of males and females each revealed only five morphological groups in the genus corresponding to the species D. occidentalis, D. lumholtzi, D. cephalata, D. nivalis, and a conglomerate of all other taxa described from the D. carinata complex, D. carinata (sensu lato). Interpretation of the male results was straightforward, but that for females was not because of the occurrence of an environmentally induced seasonal change in morphology (cyclomorphosis) in females. Log transformations of the female data successfully isolated the effects of cyclomorphosis. The seasonal morphs formed two groups which were further differentiated into taxonomic groups.     

Behavioural Reproductive Isolation in a Rotifer Hybrid Zone

A hybrid zone between two Brachionus plicatilis rotifer mitochondrial DNA (mtDNA) lineages was recently described in the Iberian Peninsula between a pond (Santed 2) and a lake (Gallocanta). The patterns of mitochondrial and nuclear genetic variation observed suggested that gene flow is mainly male-mediated from the lake to the pond. Here we test two hypotheses: (a) that male-mediated gene flow occurs through assortative mating between individuals from these ponds, (b) that behavioural isolation occurs between the two mtDNA lineages. We isolated, reared and genotyped rotifer clones from resting eggs collected in the sediments of these and two other distant ponds. We devised a quick, inexpensive RFLP method to discriminate between B. plicatilis and its sibling species B. ‘Manjavaeas’ and between both mtDNA B. plicatilis lineages. Behavioural no-choice tests using new-born, virgin males and females were performed between five clones. B. ‘Manjavacas’ and B. plicatilis were reproductively isolated. B. plicatilis clones did not show evidence of reproductive isolation, regardless of their mtDNA lineage, except Santed 2 males, which discriminated strongly against Gallocanta females. These results could help to explain the discrepancies between mitochondrial and nuclear genetic variation reported in the two populations.     

Taxonomy and species boundaries in the coral genus Favia Milne Edwards and Haime, 1857 (Cnidaria: Scleractinia) from Thailand revealed by morphological and genetic data

While Faviidae is a widely and uniformly distributed coral family throughout the Indo-Pacific, the extensive phenotypic plasticity of colony surface and corallite features often confounds the use of macromorphological characters in species identification, and contributes to conflict between traditional classification and molecular analyses of the group. Recent advances in morphological and molecular techniques now provide a suite of methods to re-address coral taxonomy in complex groups, such as that represented by the Faviidae. This study combines morphologic measurements including “3D coordinates landmarks” data with phylogenetic assessments of nuclear (ITS) and mitochondrial (COI-trnM) DNA to assess species boundaries in nine species of Faviidae with para-septothecal walls from Thailand. Strong concordance was found between morphological features and a priori groupings based on both morphospecies and genetically defined groups (ITS and COI-trnM). Favia truncatus was the most well-defined species based on morphological analyses, and it was also shown to be monophyletic using phylogenetic analyses. Besides F. truncatus, the only other species that was found to be monophyletic in analyses of both genes was F. cf. helianthoides, but its skeletal morphology overlapped with the F. favus species complex (comprised of F. favus, F. speciosa, F. matthaii and F. rotumana). Although not genetically monophyletic, the F. favus species complex and F. pallida were fairly well delineated morphologically. Morphospecies within the F. favus species complex are therefore possibly a result of genetic drift and/or stable polymorphisms driven by divergent selection. These results represent a first step toward a taxonomic revision of the Indo-Pacific Favia, which will integrate morphological methods with the study of type material, genetic information, reproductive data, and tests of phenotypic plasticity—given that multiple lines of evidence are needed to resolve ambiguous species and assign species names.     

Long‐term acclimation to reciprocal light conditions suggests depth‐related selection in the marine foundation species Posidonia oceanica

Phenotypic differences among populations of the same species reflect selective responses to ecological gradients produced by variations in abiotic and biotic factors. Moreover, they can also originate from genetic differences among populations, due to a reduced gene flow. In this study, we examined the extent of differences in photo‐acclimative traits of Posidonia oceanica (L.) Delile clones collected above and below the summer thermocline (i.e., ?5 and ?25 m) in a continuous population extending along the water depth gradient. During a reciprocal light exposure and subsequent recovery in mesocosms, we assessed degree of phenotypic plasticity and local adaptation of plants collected at different depths, by measuring changes in several traits, such as gene expression of target genes, photo‐physiological features, and other fitness‐related traits (i.e., plant morphology, growth, and mortality rates). Samples were also genotyped, using microsatellite markers, in order to evaluate the genetic divergence among plants of the two depths. Measures collected during the study have shown a various degree of phenotypic changes among traits and experimental groups, the amount of phenotypic changes observed was also dependent on the type of light environments considered. Overall plants collected at different depths seem to be able to acclimate to reciprocal light conditions in the experimental time frame, through morphological changes and phenotypic buffering, supported by the plastic regulation of a reduced number of genes. Multivariate analyses indicated that plants cluster better on the base of their depth origin rather than the experimental light conditions applied. The two groups were genetically distinct, but the patterns of phenotypic divergence observed during the experiment support the hypothesis that ecological selection can play a role in the adaptive divergence of P. oceanica clones along the depth gradient.     

Species boundaries,phylogenetic relationships,and ecological differentiation inAnthriscus (Apiaceae)

Species boundaries and phylogenetic relationships of 17 taxa ofAnthriscus (Apiaceae), with special emphasis on the critical sect.Cacosciadium, were explored using morphological data with principal component analysis, phenetics, and phylogenetics. The analyses did not provide satisfactory resolution of taxa from sect.Cacosciadium and only four species were retained. The total number of species was reduced to nine. Sect.Cacosciadium is distinguished by only two synapomorphies while sects.Anthriscus andCaroides are better supported. Present geographic and ecological variation suggests that the radiation ofAnthriscus occurred through divergence of peripheral isolated populations adapting to different habitats: high montane meadows and screes, shady climax forests, and seasonally dry habitats at lower altitudes. The adaptive significance of particular morphological traits is discussed.     

Chromosome studies in Alouatta belzebul

The chromosome constitution of Alouatta belzebul was studied with G-, C-, and silver staining. In ten specimens identified as Alouatta belzebul, the chromosome constitution of males (2n = 49) differed from that of females (2n = 50) owing to a Y-autosome translocation. In another single female specimen, probably Alouatta belzebul nigerrima, the diploid chromosome number was also 50, though its karyotype was drastically different from that of Alouatta belzebul belzebul. Chromosome studies, taken together with phenotypic and field observations, suggest that Alouatta belzebul belzebul is phenotypically variable in respect to pelage coloration. This attribute is therefore unreliable for the precise identification of Alouatta belzebul subspecies. Conversely, relatively minor phenotypic differences, allowing for the characterization of subspecies within a same species, coexist with unparalleled, drastic karyotypic divergence. These findings clearly question gross morphological attributes as discriminative characteristics of Alouatta belzebul subspecies.     

Morphological and molecular phylogenetic analysis of <Emphasis Type="Italic">Melampsora</Emphasis> species on poplars in China

Many species of Melampsora on Populus have been reported in China, based on morphological characteristics of both uredial and telial states, and on host species, but their morphology and taxonomy are still poorly defined. In this study, 196 specimens representing Melampsora species on poplars and collected from various areas of China were used for morphological observations. The morphological characteristics of urediniospores and teliospores were examined with light and scanning electron microscopy. The specimens could be classified into five groups based on their morphology. For the sequencing of the nuclear large subunit rDNA (D1/D2), 5.8S rDNA and their internal transcribed spacers, ITS1 and ITS2 region, 54 specimens were selected from the specimens used in morphological observations. These specimens were separated into six clades by phylogenetic analyses of the D1/D2 and ITS regions. Correlations among morphological groups and phylogenetic clades based on these results suggest a revision of these species. In particular, no evidence to discriminate specimens of M. acedioides, M. magnusiana, and M. rostrupii was found from either morphological characteristics or sequence analysis.Contribution no. 185 Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

Intragenomic diversity and phylogenetic systematics of wild rosemaries (Rosmarinus officinalis L. s.l., Lamiaceae) assessed by nuclear ribosomal DNA sequences (ITS)

Nuclear ribosomal sequences (ITS) were used to study species boundaries and to infer phylogenetic patterns in wild rosemaries (Rosmarinus officinalis, R. eriocalyx, R. tomentosus). Intragenomic polymorphisms (overlapping peaks and in some cases unreadable sequences) were found throughout the sequencing electrophoretograms of most Rosmarinus accessions. Sequencing the cloned ITS products from representative individuals resulted in 25 ribotypes differing at 59 variable sites. Average sequence divergence among clones was 1.75%, and the most divergent sequences differed by 3.48%. No single ribotype was shared between any two-paired species. The highest values of intragenomic divergence were similar in R. officinalis (1.63%) and R. eriocalyx (1.14%–2.12%), and contrast with those shown by R. tomentosus (0.97%). Sequence data suggest that most divergent rDNA sequences within individuals belong to paralogous loci that apparently are not pseudogenes. A detailed inspection of direct and cloned sequences does not show evidence that the intragenomic polymorphism found is due to interspecific hybridization. Phylogenetic analyses of cloned sequences suggested that both R. officinalis and R. tomentosus were monophyletic, whereas R. tomentosus clones were nested within a paraphyletic R. eriocalyx.     

Formation of morphological defences in response to YOY perch and invertebrate predation in two <Emphasis Type="Italic">Daphnia</Emphasis> species coexisting in a mesotrophic lake

This study examined the formation of morphological defences by two coexisting Daphnia species, the large-sized D. pulicaria (2 mm) and the small-sized D. mendotae (1.4 mm), in response to the presence of young-of-the-year (YOY) yellow perch (Perca flavescens) and invertebrate predators (Chaoborus, Leptodora) during summer in a mesotrophic lake. We hypothesized that due to differential size-selective predation risk by YOY fish and invertebrates, the large-sized and the small-sized Daphnia species would show different morphological responses to predation threats. We followed changes in two morphological traits (relative length of the tail spine in D. pulicaria and of the helmet in D. mendotae) among different periods during summer according to YOY fish and invertebrate predation. We defined four YOY fish predation periods based on the presence of YOY perch in the pelagic zone of the lake and the relative abundance of Daphnia preys in their gut contents, and two invertebrate predation periods based on exclusive or mutual occurrence of the invertebrate predators. The large-sized (D. pulicaria) and the small-sized (D. mendotae) species showed different morphological responses to YOY fish and invertebrate predators, respectively. The tail spine ratio of the juveniles and adults of D. pulicaria did not change in response to YOY fish predation or to invertebrate predation. A gradual increase in the helmet ratio was observed in the small-sized D. mendotae over the summer period. This change was related to the co-occurrence of the invertebrate predators (Chaoborus and Leptodora) and to YOY fish predation. The warmer temperature cannot be accounted for helmet elongation since it was constant across depths, and not related with the co-occurrence of D. mendotae and YOY perch. Guest editor: Piet Spaak Cladocera: Proceedings of the 7th International Symposium on Cladocera     

Evolutionary Acceleration and Divergence in <Emphasis Type="Italic">Procolobus kirkii</Emphasis>

We investigated the role of geographical insularity in divergence and speciation of Procolobus kirkii by examining cranial morphology. The sample (n = 369) included museum specimens of Procolobus spp. and recently deceased individuals of P. kirkii from the main island of Zanzibar and 2 smaller islands in the archipelago. Geometric morphometrics evinced pronounced divergence of Procolobus kirkii from mainland Procolobus, including members of P. badius ssp., P. pennantii ssp., P. rufomitratus, P. gordonorum and also representatives of the assemblage of red colobus populations from Central Equatorial Africa. Procolobus kirkii has a small cranium, consistent with the island rule for large mammals, reduced sexual dimorphism consistent with Rensch’s rule, and a distinct cranial form. Analyses of phenotypic variance of Procolobus kirkii gave no evidence for population bottlenecks in the history of the species, but there is a clear indication that the species has experienced accelerated morphological evolution of size, probably as a result of insularity. Their highly distinctive morphology lends weight to the argument that they are a unique insular endemic species in need of active conservation. An erratum to this article can be found at     

Phylogenetic and morphometric analyses reveal ecophenotypic plasticity in freshwater mussels Obovaria jacksoniana and Villosa arkansasensis (Bivalvia: Unionidae)

Freshwater mollusk shell morphology exhibits clinal variation along a stream continuum that has been termed the Law of Stream Distribution. We analyzed phylogenetic relationships and morphological similarity of two freshwater mussels (Bivalvia: Unionidae), Obovaria jacksoniana and Villosa arkansasensis, throughout their ranges. The objectives were to investigate phylogenetic structure and evolutionary divergence of O. jacksoniana and V. arkansasensis and morphological similarity between the two species. Our analyses were the first explicit tests of phenotypic plasticity in shell morphologies using a combination of genetics and morphometrics. We conducted phylogenetic analyses of mitochondrial DNA (1416 bp; two genes) and morphometric analyses for 135 individuals of O. jacksoniana and V. arkansasensis from 12 streams. We examined correlations among genetic, morphological, and spatial distances using Mantel tests. Molecular phylogenetic analyses revealed a monophyletic relationship between O. jacksoniana and V. arkansasensis. Within this O. jacksoniana/V. arkansasensis complex, five distinct clades corresponding to drainage patterns showed high genetic divergence. Morphometric analysis revealed relative differences in shell morphologies between the two currently recognized species. We conclude that morphological differences between the two species are caused by ecophenotypic plasticity. A series of Mantel tests showed regional and local genetic isolation by distance. We observed clear positive correlations between morphological and geographic distances within a single drainage. We did not observe correlations between genetic and morphological distances. Phylogenetic analyses suggest O. jacksoniana and V. arkansasensis are synonomous and most closely related to a clade composed of O. retusa, O. subrotunda, and O. unicolor. Therefore, the synonomous O. jacksoniana and V. arkansasensis should be recognized as Obovaria arkansasensis (Lea 1862) n. comb. Phylogenetic analyses also showed relative genetic isolation among drainages, suggesting no current gene flow. Further investigation of in‐progress speciation and/or cryptic species within O. arkansasensis is warranted followed by appropriate revision of conservation management designations.