Evolutionary history of the polyploid complex of Cardamine amara (Brassicaceae): isozyme evidence

 In samples from 56 populations of Cardamine amara, representing four diploid subspecies (subspp. amara, opicii, balcanica, and pyrenaea) and two tetraploid subspecies (subspp. austriaca and olotensis) from different parts of the European distribution area, four enzyme systems with 23 alleles were studied. These data, together with previous morphological and karyological data, suggest that the distribution and variation pattern within the species was strongly influenced by the last glacial period and postglacial migrations. Cardamine amara subsp. pyrenaea is monomorphic for a unique allele, and subsp. balcanica has a unique allele, too which, however, is not fixed in all populations of the taxon. Both taxa seem to be relic ones, although otherwise subsp. balcanica in respect of allelic spectrum much resembles subsp. amara. The other two diploid subspecies, subsp. amara and subsp. opicii, are not characterised by presence of unique alleles but differ in allele frequencies. The two tetraploid subspecies have different evolutionary histories. C. amara subsp. austriaca seems to be an autopolyploid derivative of subsp. amara which colonised open space offered by retreating glaciers in the Eastern Alps. C. amara subsp. olotensis from the Iberian Peninsula represents most probably a polyploid of preglacial time. Received June 22, 2001; accepted May 17, 2002 Published online: September 13, 2002     

Evolution of novel morphological and reproductive traits in a clade containing Antirrhinum majus (Scrophulariaceae)

Phylogenetic analysis of DNA sequences of the chloroplast genes rcbL and ndhf revealed a highly supported clade composed of the families Plantaginaceae, Callitrichaceae, and Hippuridaceae in close association with the model organism Antirrhinum majus and other members of family Scrophulariaceae. Plantago has miniature actinomorphic wind-pollinated flowers that have evolved from zygomorphic animal-pollinated precursors. The aquatic Hippuridaceae have reduced windpollinated flowers with one reproductive organ per whorl, and three, rather than four, whorls. In monoecious aquatic Callitrichaceae, further reduction has occurred such that there is only one whorl per flower containing a single stamen or carpel. Optimization of character states showed that these families descended from an ancestor similar to Antirrhinum majus. Recent studies of plant developmental genetics have focused on distantly related species. Differences in the molecular mechanisms controlling floral development between model organisms are difficult to interpret due to phylogenetic distance. In order to understand evolutionary changes in floral morphology in terms of their underlying genetic processes, closely related species exhibiting morphological Variation should be examined. Studies of genes that regulate morphogenesis in the clade described here could aid in the elucidation of a general model tot such fundamental issues as how changes in floral symmetry, organ number, and whorl number are achieved, as well as providing insight on the evolution of dicliny and associated changes in pollination syndrome.     

Phylogeny and biogeography in the evolution of migration: shorebirds of the Charadrius complex

Aim We explore whether molecular phylogeny and biogeography can complement evolutionary ecology in developing a method to address a long-standing issue in the evolution of migration: have migrations between breeding and non-breeding grounds, which may be on different continents, evolved through origins in the breeding grounds with successive shifts of the non-breeding distribution or vice versa? Methods To accommodate the biology of migration, we treated breeding and non-breeding distributions as characters to be mapped onto a phylogeny derived from mitochondrial DNA sequence data and so examined the ancestral home issue as a study in the direction of character evolution. Results Our main findings from applying this approach to a subset of the Charadrius complex of shorebirds (Aves: Charadriinae) are that a case can be made for shifts of breeding distributions having occurred in the ancestries of C. alexandrinus and C. veredus as those species evolved their present migration patterns. Our results also argue for a southern hemisphere origin (specifically South America) for the Charadrius complex as a whole. A South American origin implies other shifts in breeding distributions having occurred in the evolution of the species C. semipalmatus and C. vociferus. On applying the methods we developed for dealing with phylogenetic uncertainty, these results are reinforced and the merit of testing them further is suggested. Conclusions By way of a new approach to the evolution of migration, our study adds to a consensus emerging from the evolutionary ecology of migrant birds, arguing that shifts of breeding distributions are commonly, though not necessarily exclusively, involved in the evolution of migration.     

Marine shelf habitat: biogeography and evolution

We synthesize the evolutionary implications of recent advances in the fields of phylogeography, biogeography and palaeogeography for shallow‐water marine species, focusing on marine speciation and the relationships among the biogeographic regions and provinces of the world. A recent revision of biogeographic provinces has resulted in the recognition of several new provinces and a re‐evaluation of provincial relationships. These changes, and the information that led to them, make possible a clarification of distributional dynamics and evolutionary consequences. Most of the new conclusions pertain to biodiversity hotspots in the tropical Atlantic, tropical Indo‐West Pacific, cold‐temperate North Pacific, and the cold Southern Ocean. The emphasis is on the fish fauna, although comparative information on invertebrates is utilized when possible. Although marine biogeographic provinces are characterized by endemism and thus demonstrate evolutionary innovation, dominant species appear to arise within smaller centres of high species diversity and maximum interspecies competition. Species continually disperse from such centres of origin and are readily accommodated in less diverse areas. Thus, the diversity centres increase or maintain species diversity within their areas of influence, and are part of a global system responsible for the maintenance of biodiversity over much of the marine world.     

Molecular evidence for naturalness of genera in the tribe Antirrhineae (Scrophulariaceae) and three independent evolutionary lineages from the New World and the Old

The tribe Antirrhineae consists of 29 genera distributed in the New World and the Old. Phylogenetic analyses of ITS and ndhF sequences served to recognize six main lineages: Anarrhinum group (Anarrhinum, Kickxia); Linaria group (Linaria); Maurandya group (Cymbalaria, Asarina, Maurandella, Rhodochiton, Lophospermum); Schweinfurthia group (Pseudorontium, Schweinfurthia); Antirrhinum group (Antirrhinum, Pseudomisopates, Misopates, Acanthorrhinum, Howeliella, Neogarrhinum, Sairocarpus, Mohavea, Galvezia); Chaenorrhinum group (Chaenorrhinum, Albraunia, Holzneria). Parsimony (cladistics), distance-based (Neighbor-Joining), and Bayesian inference reveal that: (1) the tribe is a natural group; (2) genera such as Linaria, Schweinfurthia, Kickxia, and Antirrhinum also form natural groups; (3) three Antirrhineae lineages containing genera from the New and Old World are the result of three intercontinental disjunctions displaying similar levels of ITS-sequence divergence and differentiation times (Oligocene-Miocene); (4) evolution of flower shapes is not congruent with primitiveness of personate flowers; (5) both polyploidy and dysploidy appear to be responsible for most variation in chromosome number in the six main lineages. Nuclear and chloroplast evidence also supports the split of American and Mediterranean species of Antirrhinum into different genera, a result that should be contemplated in the interest of a more natural (monophyletic) taxonomy. Nucleotide additivity causes poor resolution in the ITS analysis of 22 species of Mediterranean Antirrhinum and lead us to interpret extensive hybridization in the Iberian Peninsula.This research was supported by the Spanish Dirección General de Investigación Científica y Técnica (DGICYT) through the project Flora Iberica (PS91-0070-C03-01). We thank Jorge Martínez and Emilio Cano for his assistance in the lab, Rafael Rubio and Omar Fiz for helping on the Bayesian inferences, the curators/collection managers of BCF, E, JACA, MA, SALA, SALAF, SEV, TARI, VAL for loans and access to specimens, and two anonymous reviewers for improving the quality of this publication.     

Phylogeny, biogeography and evolution of clutch size in South American lizards of the genus Kentropyx (Squamata: Teiidae)

The lizard genus Kentropyx (Squamata: Teiidae) comprises nine species, which have been placed in three species groups (calcarata group, associated to forests ecosystems; paulensis and striata groups, associated to open ecosystems). We reconstructed phylogenetic relationships of Kentropyx based on morphology (pholidosis and coloration) and mitochondrial DNA data (12S and 16S), using maximum parsimony and Bayesian methods, and evaluated biogeographic scenarios based on ancestral areas analyses and molecular dating by Bayesian methods. Additionally, we tested the life‐history hypothesis that species of Kentropyx inhabiting open ecosystems (under seasonal environments) produce larger clutches with smaller eggs and that species inhabiting forest ecosystems (under aseasonal conditions) produce clutches with fewer and larger eggs, using Stearns’ phylogenetic‐subtraction method and canonical phylogenetic ordination to take in to account the effects of phylogeny. Our results showed that Kentropyx comprises three monophyletic groups, with K. striata occupying a basal position in opposition to previous suggestions of relationships. Additionally, Bayesian analysis of divergence time showed that Kentropyx may have originated at the Tertiary (Eocene/Oligocene) and the ‘Pleistocene Refuge Hypothesis’ may not explain the species diversification. Based on ancestral reconstruction and molecular dating, we argued that a savanna ancestor is more likely and that historical events during the Tertiary of South America promoted the differentiation of the genus, coupled with recent Quaternary events that were important as dispersion routes and for the diversification at populational levels. Clutch size and egg volume were not significantly different between major clades and ecosystems of occurrence, even accounting for the phylogenetic effects. Finally, we argue that phylogenetic constraints and phylogenetic inertia might be playing essential roles in life history evolution of Kentropyx.     

Polyploid speciation inHedera (Araliaceae): Phylogenetic and biogeographic insights based on chromosome counts and ITS sequences

Variation in chromosome number and internal transcribed sequences (ITS) of nrDNA is used to infer phylogenetic relationships of a wide range ofHedera species. Polyploidy was found to be frequent inHedera, with diploid, tetraploid, hexaploid and octoploid populations being detected. Nucleotide additivity occurs in the ITS sequences of one tetraploid (H. hibernica) and two hexaploid species (H. maderensis, H. pastuchovii), suggesting that all three species originated by allopolyploidisation. ITS sequence polymorphism and nucleotide characters may indicate the presence of an ancient genome persistent only in some allopolyploid species. Phylogenetic analyses of ITS sequence data reveal two lineages ofHedera: one containing all sequences belonging to extant diploids plus the tetraploidH. algeriensis, and a second that includes this ancient ITS type and others exclusive to several polyploid species. The origin of the polyploids is evaluated on the basis of morphology, chromosome counts, ITS sequence polymorphism, and phylogenetic analyses. Reconstruction of reticulate evolution inHedera agrees with two allopolyploid areas on both sides of the Mediterranean basin. Morphological, molecular and cytological evidence also suggests an active dispersal ofHedera populations that may account for three independent introductions in Macaronesia.     

The shape of evolution: systematic tree topology

Three hypotheses that predict probabilities associated with various tree shapes, or topologies, are compared with observed topology frequencies for a large number of 4, 5, 6 and 7-member trees. The united data on these n-member trees demonstrate that both the equiprobable and proportional-to-distinguishable-types hypotheses poorly predict tree topologies, while all observed topology frequencies are similar to predictions of a simple Markovian dichotomous branching hypothesis. Differences in topology frequencies between phenetic and non-phenetic trees are observed, but their statistical significance is uncertain. Relative frequencies of highly asymmetrical topologies are larger, and those of symmetrical topologies are smaller, in phenetic than in non-phenetic trees. The fact that a simple Markovian branching process, which assumes that each species has an equal probability of speciating in each time period, can predict tree topologies offers promise. Refinement of Markovian branching hypotheses to include the possibility of multiple furcations, differential speciation and extinction rates for different groups of organisms as well as for a single group through geological time, hybrid speciation, introgression, and lineage fusion will be necessary to produce realistic models of lineage diversification.     

Phylogeny of the tribe Antirrhineae (Scrophulariaceae) based on morphological andndhF sequence data

Phylogenetic relationships within the tribe Antirrhineae (Scrophulariaceae) are analysed and discussed on the basis of parsimony analyses of morphological andndhF gene sequence data. The results indicate that the tribe Antirrhineae consists of four major groups of genera, theAnarrhinum clade, theGambelia clade, theMaurandya clade, and theAntirrhinum clade. TheAnarrhinum clade, consisting of the Old World bee-pollinated generaAnarrhinum andKickxia, is sister to the rest of the tribe. TheGambelia clade consists of the New World generaGambelia andGalvezia, which are very closely related and pollinated by hummingbirds. TheMaurandya clade consists of one subclade includingMaurandya and a number of related bee- or hummingbird-pollinated New World genera and another subclade with the Old World bee-pollinated generaAsarina andCymbalaria. TheAntirrhinum clade consists mainly of bee-pollinated Old World genera, such asAntirrhinum, Linaria, Chaenorhinum, and their segregates, but also includes the New World generaMohavea andHowelliella, of which the latter is known to be partly pollinated by hummingbirds. It is concluded that hummingbirdpollination has evolved independently within Antirrhineae at least three times from bee-pollinated ancestors.     

Stigma behavior in Mimulus aurantiacus (Scrophulariaceae)

The bilobed stigma of many species in the order Scrophulariales closes in response to touch by an animal pollinator. In hummingbird-pollinated bush monkey flower, Mimulus aurantiacus (Scrophulariaceae), closure is rapid, occurring within seconds of tactile stimulus. We investigated the proximate causes of stigma closure and subsequent reopening in M. aurantiacus, as well as potential costs and benefits of stigma closure for female fitness. Stigma closure is elicited by both touch and pollen, but closure in response to pollen is much slower, requiring 0.5–1.5 h. Stigmata reopen within 2.5–4.5 h if touch, but no pollen, is applied. Upon receipt of pollen, most stigmata remain closed for the remaining lifetime of the flower, even if less pollen is received than is needed for full seed set. Those stigmata that do reopen after pollination generally require between 20 and 28 h to do so, much longer than for unpollinated stigmata. Reopening after pollination appears to be a response to low seed set rather than to low pollen load. Natural pollination of stigmata manipulated to prevent closure shows that closure does not increase capture of pollen or seed set. In fact, closure reduces the average pollen load received by flowers. Despite this, there is no evidence that stigma closure has any negative effect on female fitness in terms of seed set or germinability. Hypotheses for the adaptive significance of stigma closure are discussed. Understanding proximate causes of stigma closure and reopening is essential in the evaluation of these hypotheses.     

Chloroplast DNA phylogeny, reticulate evolution, and biogeography of Paeonia (Paeoniaceae)

The coding region of the mat K gene and two intergenic spacers, psb A-trn H and trn L(UAA)-trn F(GAA), of cpDNA were sequenced to study phylogenetic relationships of 32 Paeonia species. In the psb A-trn H intergenic spacer, short sequences bordered by long inverted repeats have undergone inversions that are often homoplasious mutations. Insertions/deletions found in the two intergenic spacers, mostly resulting from slipped-strand mispairing, provided relatively reliable phylogenetic information. The mat K coding region, evolving more rapidly than the trnL-trn F spacer and more slowly than the psb A-trn H spacer, produced the best resolved phylogenetic tree. The mat K phylogeny was compared with the phylogeny obtained from sequences of internal transcribed spacers (ITS) of nuclear ribosomal DNA. A refined hypothesis of species phylogeny of section Paeonia was proposed by considering the discordance between the nuclear and cpDNA phylogenies to be results of hybrid speciation followed by inheritance of cpDNA of one parent and fixation of ITS sequences of another parent. The Eurasian and western North American disjunct distribution of the genus may have resulted from interrruption of the continuous distribution of ancestral populations of extant peony species across the Bering land bridge during the Miocene. Pleistocene glaciation may have played an important role in triggering extensive reticulate evolution within section Paeonia and shifting distributional ranges of both parental and hybrid species.     

The old wasp and the tree: fossils, phylogeny and biogeography in the Orussidae (Insecta, Hymenoptera)

The phylogenetic relationships of the fossil orussid taxa Mesorussus taimyrensis and Minyorussus luzzi are examined by analysing them together with a large data set compiled previously for the extant Orussidae. The fossils are placed in an unresolved trichotomy with the extant Orussidae. The phylogeny is used for evaluating the hypothesis that the ancestors of the family had reduced body size; the results of this analysis are inconclusive. The biogeographical history of the Orussidae is explored. The common ancestor of the family was probably widespread, the initial splitting events taking place prior to or coinciding with the separation of Laurasia from Gondwana. Later putative vicariance events can be correlated with the gradual breakup of Gondwana. However, the biogeographical history of the Orussidae is dominated by speciation within regions and dispersal. The minimum age of the common ancestor of the Orussidae is >180 Myr when estimated from the biogeographical pattern, >95 Myr when estimated from the phylogenetic position of the fossils; the earlier date is considered to be the most likely.  © 2004 The Linnean Society of London , Biological Journal of the Linnean Society , 2004, 82 , 139–160.     

The evolution of Spartina anglica C.E. Hubbard (Gramineae): origin and genetic variability

An extensive survey of isozyme phenotypes in British populations of the amphidiploid salt marsh grass Spartina anglica and its putative parents has confirmed that the species arose by chromosome doubling in S. × townsendii , a sterile hybrid between S. maritima and S. alterniflora. Isozyme phenotypes and seed protein profiles indicate that S. anglica is almost totally lacking in genetic variation. Isozyme evidence also indicates that the parental species are characterized by low levels of genetic variation. The lack of variation in S. anglica is proposed as being due to a narrow genetic base resulting from a single origin, or a multiple origin from uniform parents; the fact that many populations are derived from very small founder populations; and because preferential pairing between identical homologous chromosomes prevents recombination between the divergent component genomes of the species. The low levels of isozyme variation that occur appear to be due to chromosome loss.
The consequences for the future evolution of S. anglica , given its lack of genetic variation, are discussed.     

A few varietal combinations in Californian Keckiella and Penstemon (Scrophulariaceae)

Combinations are made forKeckiella antirrhinoides var.microphylla, K. rothrockii var.jacintensis, K. ternata var.septentrionalis, Penstemon fruticiformis var.amargosae, P. grinnellii var.scrophularioides, P. heterodoxus var.cephalophorus, P. heterodoxus var.shastensis, andP. newberryi var.berryi.     

Phylogenetic relationships and biogeography of midwife toads (Discoglossidae: Alytes)

Aim To devise a robust phylogenetic hypothesis for midwife toads (Alytes: Anura: Discoglossidae) and to discuss its implications for the reconstruction of the biogeographical history of the group. Location Western Palearctic. Methods Analysis of sequences of mitochondrial DNA (cytochrome b and 16S RNA, 861 bp) and 29 characters of cranial osteology of all species and subspecies within Alytes. Results Phylogenetic analyses support a sister group relationship between Alytes dickhilleni and A. muletensis, and of this clade with A. maurus. The monophyly of A. obstetricans is controversial; in particular, the phylogenetic position of A. obstetricans almogavarii is uncertain. The estimated dates for the cladogenetic events within Alytes are congruent with those derived from independent analyses (allozymes), except for the differentiation of A. o. almogavarii and the split between A. dickhilleni and A. muletensis. Main conclusions The phylogeny based on the analysis of morphological and mtDNA data differs from previous hypotheses in the positions of A. o. almogavarii and A. maurus. Events associated with the radiation of Alytes are the formation of large inland saline lakes in Iberia c. 16 Ma and the 11 ° C dramatic decrease in average annual temperature during the Middle–Late Badenian transition c. 14–13.5 Ma (A.cisternasii vs. the ancestor of the remaining clades), the structuring of the Neo‐Pyrenees and the reopening of the Betic Strait c. 10–8 Ma(A. o. almogavarii vs. A. obstetricans and vs. the subgenus Baleaphryne), the opening of the Gibraltar Strait at the end of the Messinian Salinity Crisis at c. 5.3 Ma (A. maurus vs. the ancestor of the A. dickhilleni A. muletensis clade) and a transmarine colonization event at c. 3 Ma (the ancestor of A. muletensis vs. A. dickhilleni). Following the new hypothesis, A. maurus, previously considered a subspecies of A. obstetricans, deserves species status. Second, A. o. almogavarii is a well‐differentiated lineage that was isolated from other A. obstetricans more than c. 5 Ma, but later lost its genetic and specific identity following secondary contact, hybridization and introgression with the main stock. The presence of a marked morphological and genetic diversity within A. obstetricans renders reconstruction of the evolutionary history of the genus more complicated than previously appreciated.     

Historical biogeography of Androcymbium Willd. (Colchicaceae) in Africa: evidence from cpDNA RFLPs

The cpDNA restriction variation in 39 populations representing a geographical sampling of 18 species of Androcymbium in southwestern and northern Africa was examined to assess the historical biogeography of the genus. The cpDNA phylogeny indicates that the disjunction between South and North Africa is best explained by the dispersal of southern African ancestors into North Africa. Divergence time estimates suggest that the geographic range of the genus may have extended considerably north (perhaps to Tanzania and Kenya) prior to the global desiccation of Africa in the Miocene. Further expansion of the genus northward was probably stalled until climatic changes in the late Miocene brought about the gradual replacement of a subtropical woodland savanna with the arid landscape that gave rise to the Sahara. Aridification of the northern quarter of the continent provided the ecological conditions for fostering the expansion of Androcymbium along the Mediterranean fringe (probably east to west) and its introduction into the Canary Islands. Unlike their South African congeners, the northern species have experienced expansions, fragmentations, and local extinctions in response to the severe climatic shifts in this area during the Pliocene-Pleistocene. According to our divergence time estimates, the arid track may have already existed as a continuous area connecting southern and northern Africa in the late Miocene.     

A new section of Linaria (Scrophulariaceae: Antirrhineae)

An investigation of species of the Antirrhineae (Scrophulariaceae), primarily from the point of view of seed morphology, has indicated that the inclusion of Linaria chalepensis and L. armeniaca in section Versicolores is unsatisfactory. Several unusual features of both the seeds and other parts of the plants provide a basis for recognizing a distinct section to include these two species. An overlooked infrageneric taxon of Linaria has been reinstated as section Macrocentrum (C. Koch) D. Sutton. All taxa included in this section are described and illustrated. The significance of the unusual seed morphology is discussed in relation to the affinities of the genus.     

Floral ontogeny inMazus pumilus (Scrophulariaceae)

InMazus pumilus, all the floral appendages are initiated in acropetal sequence in the second cell layer (except stamens) of the floral primordium by periclinal divisions. The actinomorphic calyx tube is formed due to zonal growth. The zygomorphy in corolla is evident from the inception of petal primordia which arise sequentially as independent units in order of one anterior, a pair of anterio-lateral followed by a pair of posterio-lateral. Later these primordia exhibit differential growth because of which zygomorphy becomes more pronounced. The upper corolla tube is formed by interprimordial growth and lower corolla tube by zonal growth. Stamens are initiated in the third layer of the floral apex. Unlike sepals and petals, in the development of stamens (4) underlying cells of corpus also contribute. Posterior stamen is absent. The stamens become epipetalous because of interprimordial and zonal growth in the common region below the bases of petals as well as stamens. The two carpel primordia arise as crescent shaped structures which become continuous due to interprimordial growth. The ovary is formed by a ring of zonal meristem. The style develops later between stigma and ovary because of intercalary growth. The residual apex grows vertically along with the ovary and forms the septum of the ovary. All the floral appendages exhibit similar pattern of histogenesis and early growth suggesting thereby the appendicular nature of these appendages.     

Atlantic reef fish biogeography and evolution

Aim To understand why and when areas of endemism (provinces) of the tropical Atlantic Ocean were formed, how they relate to each other, and what processes have contributed to faunal enrichment. Location Atlantic Ocean. Methods The distributions of 2605 species of reef fishes were compiled for 25 areas of the Atlantic and southern Africa. Maximum‐parsimony and distance analyses were employed to investigate biogeographical relationships among those areas. A collection of 26 phylogenies of various Atlantic reef fish taxa was used to assess patterns of origin and diversification relative to evolutionary scenarios based on spatio‐temporal sequences of species splitting produced by geological and palaeoceanographic events. We present data on faunal (species and genera) richness, endemism patterns, diversity buildup (i.e. speciation processes), and evaluate the operation of the main biogeographical barriers and/or filters. Results Phylogenetic (proportion of sister species) and distributional (number of shared species) patterns are generally concordant with recognized biogeographical provinces in the Atlantic. The highly uneven distribution of species in certain genera appears to be related to their origin, with highest species richness in areas with the greatest phylogenetic depth. Diversity buildup in Atlantic reef fishes involved (1) diversification within each province, (2) isolation as a result of biogeographical barriers, and (3) stochastic accretion by means of dispersal between provinces. The timing of divergence events is not concordant among taxonomic groups. The three soft (non‐terrestrial) inter‐regional barriers (mid‐Atlantic, Amazon, and Benguela) clearly act as ‘filters’ by restricting dispersal but at the same time allowing occasional crossings that apparently lead to the establishment of new populations and species. Fluctuations in the effectiveness of the filters, combined with ecological differences among provinces, apparently provide a mechanism for much of the recent diversification of reef fishes in the Atlantic. Main conclusions Our data set indicates that both historical events (e.g. Tethys closure) and relatively recent dispersal (with or without further speciation) have had a strong influence on Atlantic tropical marine biodiversity and have contributed to the biogeographical patterns we observe today; however, examples of the latter process outnumber those of the former.