Evolution of seed characters and of dispersal modes in Aizoaceae.

The family Aizoaceae includes ~1880 species and is one of the more diverse groups within Caryophyllales, particularly in arid areas in the western part of southern Africa. Most species are dwarf succulent-leaf shrubs. In response to the harsh climatic conditions prevalent where they occur, many representatives have evolved special reproductive adaptations. These include hygrochastic capsules (mostly found in Mesembryanthemoideae and Ruschioideae), burr-like indehiscent and one-seeded, winged diaspores, and fast germination of seeds after rain. We focused on anatomical features, evolutionary trends, and the ecological significance of various morpho-anatomical structures found in the seeds. The seeds of 132 species from 61 genera were studied, and 18 diagnostic characters were discovered. All studied characters were compared with those of other families from core Caryophyllales. The seed notch and embryo shape were added to the list of characteristics distinguishing major clades within the family. In addition, the presence of longitudinal ridges and a keel on the seed are additional characters of Aizooideae and combined Ruschioideae-Apatesieae, respectively. Puzzle-like borders of testa cells are a common trait in Ruschioideae and Mesembryanthemoideae. Most taxa in Aizoaceae have a thin seed coat, which is the ancestral state within the family. This may facilitate fast germination. We observed several shifts to a medium-thick or thick seed coat in members of Ruschioideae and Acrosanthoideae. These inhabit fire-prone environments (in vegetation types known as fynbos and renosterveld), where the thickened seed coat may protect against damage by fire. Multi-seeded fruits are the ancestral state within Aizoaceae, with several shifts to one-(two-)seeded xerochastic fruits. The latter are dispersed via autochory, zoochory, or anemochory. This trait has evolved mainly in less succulent subfamilies Acrosanthoideae, Aizooideae, and Sesuvioideae. In highly succulent subfamilies Ruschioideae and Mesembryanthemoideae, fruits are almost exclusively multi-seeded and hygrochastic with ombrohydrochoric dispersal. A reduction in the number of seeds within a dispersal unit is rare. Within Apatesieae and Ruschieae, there are also a few unusual genera whose fruits fall apart into one- to two-seeded mericarps (that are mainly dispersed by wind).

Selecting the best candidates for resurrecting extinct-in-the-wild plants from herbaria.

Resurrecting extinct species is a fascinating and challenging idea for scientists and the general public. Whereas some theoretical progress has been made for animals, the resurrection of extinct plants (de-extinction sensu lato) is a relatively recently discussed topic. In this context, the term 'de-extinction' is used sensu lato to refer to the resurrection of 'extinct in the wild' species from seeds or tissues preserved in herbaria, as we acknowledge the current impossibility of knowing a priori whether a herbarium seed is alive and can germinate. In plants, this could be achieved by germinating or in vitro tissue-culturing old diaspores such as seeds or spores available in herbarium specimens. This paper reports the first list of plant de-extinction candidates based on the actual availability of seeds in herbarium specimens of globally extinct plants. We reviewed globally extinct seed plants using online resources and additional literature on national red lists, resulting in a list of 361 extinct taxa. We then proposed a method of prioritizing candidates for seed-plant de-extinction from diaspores found in herbarium specimens and complemented this with a phylogenetic approach to identify species that may maximize evolutionarily distinct features. Finally, combining data on seed storage behaviour and longevity, as well as specimen age in the novel 'best de-extinction candidate' score (DEXSCO), we identified 556 herbarium specimens belonging to 161 extinct species with available seeds. We expect that this list of de-extinction candidates and the novel approach to rank them will boost research efforts towards the first-ever plant de-extinction.

Hidden Diversity-A New Speciose Gall Midge Genus (Diptera: Cecidomyiidae) Associated with Succulent Aizoaceae in South Africa.

Aizoaceae (Caryophyllales) constitute one of the major floral components of the unique Greater Cape Floristic Region (GCFR), with more than 1700 species and 70% endemism. Within succulent Aizoaceae, the subfamily Ruschioideae is the most speciose and rapidly diversifying clade, offering potential niches for the diversification of specialized herbivorous insects. Nevertheless, insect diversity on these plants has not been studied to date, and knowledge of gall-inducing insects in the Afrotropics is generally scarce. Our recent observations indicate that succulent Aizoaceae in the GCFR support a rich and largely unstudied community of gall midges (Diptera: Cecidomyiidae). Here, we provide a first report of their diversity with a description of a new genus, Ruschiola Dorchin, and ten new species, based on morphological and molecular analyses of material collected during a three-year targeted survey across major GCFR vegetation types. A high degree of morphological uniformity in Ruschiola suggests recent diversification and necessitated the use of molecular data and laboratory rearing from host plants to verify species boundaries and host ranges.

Diagnostics, taxonomy, nomenclature and distribution of perennial Sesuvium (Aizoaceae) in Africa.

The taxonomy of perennial Sesuvium species in Africa has been poorly investigated until now. Previously five perennial species of Sesuvium were recognised in Africa (S. congense, S. crithmoides, S. mesembryanthemoides, S. portulacastrum, and S. sesuvioides). Based on the differing number of stamens, S. ayresii is accepted here as being distinct from S. portulacastrum. Field observations in Angola also led the authors to conclude that S. crystallinum and S. mesembryanthemoides are conspecific with S. crithmoides. A new subspecies, Sesuvium portulacastrum subsp. persoonii, is described from West Africa (Cape Verde, Gambia, Guinea-Bissau, Mauritania, Senegal). The molecular phylogeny indicates the position of S. portulacastrum subsp. persoonii within the "American lineage" as a part of the Sesuvium portulacastrum complex which needs further studies. A diagnostic key and taxonomic notes are provided for the six perennial species of Sesuvium found in Africa and recognised by the authors (S. ayresii, S. congense, S. crithmoides, S. portulacastrum subsp. portulacastrum, S. portulacastrum subsp. persoonii, S. verrucosum and the facultatively short-lived S. sesuvioides). The distribution of S. crithmoides, previously considered to be endemic to Angola, is now confirmed for the seashores of Republic of Congo and DR Congo. The American species S. verrucosum is reported for the first time for Africa (the Macaronesian islands: Cape Verde and the Canaries). It is locally naturalised in Gran Canaria, being a potentially invasive species. These findings as well as new records of S. verrucosum from Asia and the Pacific Islands confirm its proneness to transcontinental introduction. Lectotypes of S. brevifolium, S. crithmoides, S. crystallinum and S. mesembryanthemoides are selected. The seed micromorphology and anatomy of the perennial African species is studied. Compared to the seeds of some annual African Sesuvium investigated earlier, those of perennial species are smooth or slightly alveolate. The aril is one-layered and parenchymatous in all species and usually tightly covers the seed. The aril detachments from the seed coat that form a white stripe near the cotyledon area easily distinguish S. verrucosum from other species under study.

Out of southern Africa: Origin, biogeography and age of the Aizooideae (Aizoaceae).

The Aizooideae is an early-diverging lineage within the Aizoaceae. It is most diverse in southern Africa, but also has endemic species in Australasia, Eurasia and South America. We derived a phylogenetic hypothesis from Bayesian and Maximum Likelihood analyses of plastid DNA-sequences. We find that one of the seven genera, the fynbos-endemic Acrosanthes, does not belong to the Aizooideae, but is an ancient sister-lineage to the subfamilies Mesembryanthemoideae & Ruschioideae. Galenia and Plinthus are embedded inside Aizoon and Aizoanthemum is polyphyletic. The Namibian endemic Tetragonia schenckii is sister to Tribulocarpus of the Sesuvioideae. For the Aizooideae, we explored their possible age by means of relaxed Bayesian dating and used Bayesian Binary MCMC reconstruction of ancestral areas to investigate their area of origin. Early diversification occurred in southern Africa in the Eocene-Oligocene, with a split into a mainly African lineage and an Eurasian-Australasian-African-South American lineage. These subsequently radiated in the early Miocene. For Tetragonia, colonisation of Australasia via long-distance dispersal from Eurasia gave rise to the Australasian lineage from which there were subsequent dispersals to South America and Southern Africa. Despite the relatively old age of the Aizooideae, more than half the species have radiated since the Pleiocene, coinciding with the large and rapid diversification of the Ruschioideae. The lineage made up of Tetragonia schenckii &Tribulocarpus split from the remainder of the Sesuvioideae already in the mid Oligocene and its disjunct distribution between Namibia and north-east Africa may be the result of a previously wider distribution within an early Arid African flora. Our reconstruction of ancestral character-states indicates that the expanding keels giving rise to hygrochastic fruits originated only once, i.e. after the split of the Sesuvioideae from the remainder of the Aizoaceae and that they were subsequently lost many times. Variously winged and spiky fruits, adapted to dispersal by wind and animals, have evolved independently in the Aizooideae and the Sesuvioideae. There is then a greater diversity of dispersal systems in the earlier lineages than in the Mesembryanthemoideae and Ruschioideae, where dispersal is mainly achieved by rain.

How succulent leaves of Aizoaceae avoid mesophyll conductance limitations of photosynthesis and survive drought.

In several taxa, increasing leaf succulence has been associated with decreasing mesophyll conductance (g M) and an increasing dependence on Crassulacean acid metabolism (CAM). However, in succulent Aizoaceae, the photosynthetic tissues are adjacent to the leaf surfaces with an internal achlorophyllous hydrenchyma. It was hypothesized that this arrangement increases g M, obviating a strong dependence on CAM, while the hydrenchyma stores water and nutrients, both of which would only be sporadically available in highly episodic environments. These predictions were tested with species from the Aizoaceae with a 5-fold variation in leaf succulence. It was shown that g M values, derived from the response of photosynthesis to intercellular CO2 concentration (A:C i), were independent of succulence, and that foliar photosynthate δ(13)C values were typical of C3, but not CAM photosynthesis. Under water stress, the degree of leaf succulence was positively correlated with an increasing ability to buffer photosynthetic capacity over several hours and to maintain light reaction integrity over several days. This was associated with decreased rates of water loss, rather than tolerance of lower leaf water contents. Additionally, the hydrenchyma contained ~26% of the leaf nitrogen content, possibly providing a nutrient reservoir. Thus the intermittent use of C3 photosynthesis interspersed with periods of no positive carbon assimilation is an alternative strategy to CAM for succulent taxa (Crassulaceae and Aizoaceae) which occur sympatrically in the Cape Floristic Region of South Africa.

A phylogenetic hypothesis for the recently diversified Ruschieae (Aizoaceae) in southern Africa.

The Ruschieae is a large tribe of about 1600 species of succulent perennials. They form a major component of the arid parts of the Greater Cape Floristic Region, both in numbers of species and in their density of coverage. So far phylogenetic relationships within the tribe have been unresolved, largely through the paucity of variable molecular characters and this is ascribed to the tribe's recent and rapid radiation. Our phylogeny is based on 10 chloroplast gene regions and represents a nearly complete sampling of the 100 currently recognised genera of the Ruschieae. These chloroplast regions yielded relatively few phylogenetically informative characters, consequently providing only limited resolution in and poor support for many parts of the phylogeny. Nevertheless, for the first time, we provide well-supported evidence that taxa with mostly mesomorphic, often ephemeral leaves and weakly persistent fruits form a basal grade of lineages in the Ruschieae. These lineages subtend a large polytomy of taxa with almost exclusively xeromorphic, persistent leaves and strongly persisting fruits. Among the basal grade of lineages, those occurring within the winter-rainfall region typically shed their leaves or form (at least partly) a protective, dry sheath around the apical bud during the dry summer months, as a means of escaping the summer drought. This contrasts with taxa of the basal grade from outside the winter-rainfall region, in which the leaves persist. Our results show that, in both strongly and weakly persistent fruits, specialised characteristics of the fruit evolved repeatedly and so these structures are highly homoplasious. Perhaps as a consequence of repeated changes towards increased persistence and specialisation of leaves and fruits, several clades show little morphological cohesion. However, as in other groups in the Cape Flora, most clades in the Ruschieae represent regional groupings. Our analysis of sequences of the nuclear gene 'chloroplast-expressed glutamine synthetase' (ncpGS) revealed extensive paralogy within the Ruschieae, but found an intact reading frame in all its members. More data on the cytology of the Ruschieae is needed to evaluate whether the paralogy observed is due to gene duplication or polyploidy.

Duplication of the Asymmetric Leaves1/Rough Sheath 2/Phantastica (ARP) gene precedes the explosive radiation of the Ruschioideae.

The Mesembryanthemoideae and Ruschioideae subfamilies are a major component of the Greater Cape Floristic Region in southern Africa. The Ruschioideae show an astonishing diversity of leaf shape and growth forms. Although 1,585 species are recognised within the morphologically diverse Ruschioideae, these species show minimal variation in plastid DNA sequence. We have investigated whether changes in selected leaf development transcription factors underpin the recent, rapid diversification of this large group of succulent plants. Degenerate primers designed to conserved regions of Asymmetric Leaves1/Rough Sheath 2/Phantastica (ARP) and the Class III HD-ZIP family of genes, were used to amplify sequences corresponding to these genes from several species within the Mesembryanthemoideae and Ruschioideae subfamilies. Two members of the Class III HD-ZIP family were identified in both the Mesembryanthemoideae and Ruschioideae, and were derived from an ancient gene duplication event that preceded the divergence of gymnosperms and angiosperms. While a single ARP orthologue was identified in the Mesembryanthemoideae, two paralogues, ARPa and ARPb, were identified in the Ruschioideae subfamily. ARPa was present in all species of Ruschioideae analysed in this study. ARPb has been lost from the Apatesieae and Dorotheantheae tribes, which form an early evolutionary branch from the Ruschieae tribe, as well as from selected species within the Ruschieae. The recent duplication and subsequent selected gene loss of the ARP transcription factor correlates with the rapid diversification of plant forms in the Ruschioideae.

A phylogenetic hypothesis for the Aizoaceae (Caryophyllales) based on four plastid DNA regions.

The Aizoaceae is the largest family of leaf succulent plants, and most of its species are endemic to southern Africa. To evaluate subfamilial, generic, and tribal relationships, we produced two plastid DNA data sets for 91 species of Aizoaceae and four outgroups: rps16 intron and the trnL-F gene region (both the trnL intron and the trnL-F intergenic spacer). In addition, we generated two further plastid data sets for 56 taxa restricted to members of the Ruschioideae using the atpB-rbcL and the psbA-trnH intergenic spacers. In the combined tree of the rps16 intron and trnL-F gene region, three of the currently recognized subfamilies (Sesuvioideae, Mesembryanthemoideae, and Ruschioideae) are each strongly supported monophyletic groups. The subfamily Tetragonioideae is polyphyletic, with Tribulocarpus as sister to the Sesuvioideae and Tetragonia embedded in the Aizooideae. Our study showed that the group consisting of the Sesuvioideae, Aizooideae, and Tetragonioideae does not form a monophyletic entity. Therefore, it cannot be recognized as a separate family in order to accommodate the frequently used concept of the Mesembryanthemaceae or "Mesembryanthema," in which the subfamilies Mesembryanthemoideae and Ruschioideae are included. We also found that several genera within the Mesembryanthemoideae (Mesembryanthemum, Phyllobolus) are not monophyletic. Within the Ruschioideae, our study retrieved four major clades. However, even in the combined analysis of all four plastid gene regions, relationships within the largest of these four clades remain unresolved. The few nucleotide substitutions that exist among taxa of this clade point to a rapid and recent diversification within the arid winter rainfall area of southern Africa. We propose a revised classification for the Aizoaceae.