Peripatric speciation in an endemic Macaronesian plant after recent divergence from a widespread relative.

The Macaronesian Scrophularia lowei is hypothesized to have arisen from the widespread S. arguta on the basis of several phylogenetic studies of the genus, but sampling has been limited. Although these two annual species are morphologically distinct, the origin of S. lowei is unclear because genetic studies focused on this Macaronesian species are lacking. We studied 5 S. lowei and 25 S. arguta populations to determine the relationship of both species and to infer the geographical origin of S. lowei. The timing of S. lowei divergence and differentiation was inferred by dating analysis of the ITS region. A phylogenetic analysis of two nuclear (ITS and ETS) and two chloroplast (psbJ-petA and psbA-trnH) DNA regions was performed to study the relationship between the two species, and genetic differentiation was analysed by AMOVA. Haplotype network construction and Bayesian phylogeographic analysis were conducted using chloroplast DNA regions and a spatial clustering analysis was carried out on a combined dataset of all studied regions. Our results indicate that both species constitute a well-supported clade that diverged in the Miocene and differentiated in the Late Miocene-Pleistocene. Although S. lowei constitutes a well-supported clade according to nDNA, cpDNA revealed a close relationship between S. lowei and western Canarian S. arguta, a finding supported by the spatial clustering analysis. Both species have strong population structure, with most genetic variability explained by inter-population differences. Our study therefore supports a recent peripatric speciation of S. lowei-a taxon that differs morphologically and genetically at the nDNA level from its closest relative, S. arguta, but not according to cpDNA, from the closest Macaronesian populations of that species. In addition, a recent dispersal of S. arguta to Madeira from Canary Islands or Selvagens Islands and a rapid morphological differentiation after the colonization to generate S. lowei is the most likely hypothesis to explain the origin of the last taxon.

Scrophularia arguta, a widespread annual plant in the Canary Islands: a single recent colonization event or a more complex phylogeographic pattern?

Many studies have addressed evolution and phylogeography of plant taxa in oceanic islands, but have primarily focused on endemics because of the assumption that in widespread taxa the absence of morphological differentiation between island and mainland populations is due to recent colonization. In this paper, we studied the phylogeography of Scrophularia arguta, a widespread annual species, in an attempt to determine the number and spatiotemporal origins of dispersal events to Canary Islands. Four different regions, ITS and ETS from nDNA and psbA-trnH and psbJ-petA from cpDNA, were used to date divergence events within S. arguta lineages and determine the phylogenetic relationships among populations. A haplotype network was obtained to elucidate the phylogenetic relationships among haplotypes. Our results support an ancient origin of S. arguta (Miocene) with expansion and genetic differentiation in the Pliocene coinciding with the aridification of northern Africa and the formation of the Mediterranean climate. Indeed, results indicate for Canary Islands three different events of colonization, including two ancient events that probably happened in the Pliocene and have originated the genetically most divergent populations into this species and, interestingly, a recent third event of colonization of Gran Canaria from mainland instead from the closest islands (Tenerife or Fuerteventura). In spite of the great genetic divergence among populations, it has not implied any morphological variation. Our work highlights the importance of nonendemic species to the genetic richness and conservation of island flora and the significance of the island populations of widespread taxa in the global biodiversity.

Evolution of the staminode in a representative sample of Scrophularia and its role as nectar safeguard in three widespread species.

Approximately 30% of the genera of Scrophulariaceae s.str. have a staminode, which is the remnant of a sterile stamen. However, there are no studies of the functionality or evolutionary pattern of staminodes in that family. This paper investigates three Scrophularia species with different staminode sizes to determine if the staminode safeguards nectar from dilution by rainwater and if it influences pollinator behavior. We also study staminode evolution and ancestral state reconstruction onto a phylogeny containing 71 species and subspecies with four different staminode developmental stages: tiny, large, enormous, and absent. The results showed that large staminodes did not hinder nectar collection or modify pollinator-visiting time but acted as a barrier to reduce rainwater entry. The latter reduced the dilution of nectar, which did not occur with tiny staminodes. The phylogenetic study revealed that the ancestral state in the genus corresponds with the presence of a large staminode vs. the tiny and enormous staminodes that are considered as derived. The complete disappearance of the staminode has occurred independently at least twice. Events occurred that increased or reduced the staminode size in one of the clades (Clade II), which includes species of sect. Caninae; most of these events occurred during the Pleistocene (0.6-2.7 Ma).

The role of birds and insects in pollination shifts of Scrophularia (Scrophulariaceae).

The mixed vertebrate-insect pollination system is rare in Holarctic plants. Phylogenetic relationships of 116 Scrophularia taxa were investigated based on two plastid (ndhF and trnL-trnF) and one nuclear (ITS) DNA regions. A wider time-calibrated analysis of ndhF sequences of the Lamiales revealed that Scrophularia diverged as early as in the Miocene (<22 Ma). Results of maximum-likelihood optimizations supported wasp pollination as the ancestral pollination system from which other systems derived (hoverfly, mixed vertebrate-insect and bird systems). Four origins for a mixed vertebrate-insect (MVI) pollination system were inferred, in which two western Mediterranean species (S. sambucifolia and S. grandiflora) and two island species (the Tirrenian S. trifoliata and the Canarian S. calliantha) were involved. S. calliantha is the only species in which a more complex MVI system, including pollination by the lizard Gallotia stehlini, has evolved. In addition, bird (hummingbird) floral traits found in the New Mexican S. macrantha appear to have been independently acquired. In contrast, we failed to find evidence for an ancient role of hummingbirds in the evolution of European Scrophularia. Indeed, paleontological data revealed that extinction of European hummingbirds (30-32 Ma) occurred earlier than the divergence of European MVI lineages of Scrophularia. In conclusion, our results showed that a role of birds in pollination of Scrophularia may not have been effective in the Miocene-Pliocene, but bird pollination that shows its origin in the Pliocene-Pleistocene is still operating independently in different islands and continents.

Insects, birds and lizards as pollinators of the largest-flowered Scrophularia of Europe and Macaronesia.

BACKGROUND AND AIMS: It has traditionally been considered that the flowers of Scrophularia are mainly pollinated by wasps. We studied the pollination system of four species which stand out for their large and showy flowers: S. sambucifolia and S. grandiflora (endemics of the western Mediterranean region), S. trifoliata (an endemic of the Tyrrhenian islands) and S. calliantha (an endemic of the Canary Islands). Our principal aim was to test whether these species were pollinated by birds or showed a mixed pollination system between insects and birds. METHODS: Censuses and captures of insects and birds were performed to obtain pollen load transported and deposited on the stigmas. Also, a qualitative and quantitative analysis of the flowers and inflorescences was carried out. KEY RESULTS: Flowers were visited by Hymenoptera and by passerine birds. The Canarian species was the most visited by birds, especially by Phylloscopus canariensis, and its flowers were also accessed by juveniles of the lizard Gallotia stehlini. The most important birds in the other three species were Sylvia melanocephala and S. atricapilla. The most important insect-functional groups in the mixed pollination system were: honey-bees and wasps in S. sambucifolia; bumble-bees and wasps in S. grandiflora; wasps in S. trifoliata; and a small bee in S. calliantha. CONCLUSIONS: The species studied show a mixed pollination system between insects and passerine birds. In S. calliantha there is, in addition, a third agent (juveniles of Gallotia stehlini). The participation of birds in this mixed pollination system presents varying degrees of importance because, while in S. calliantha they are the main pollinators, in the other species they interact to complement the insects which are the main pollinators. A review of different florae showed that the large showy floral morphotypes of Scrophularia are concentrated in the western and central Mediterranean region, Macaronesia and USA (New Mexico).

Self-sterility in two Cytisus species (Leguminosae, Papilionoideae) due to early-acting inbreeding depression.

In most angiosperms, the endosperm develops before the embryo, but with harmony between the two structures until final seed formation. In an embryological study, we show that inbreeding depression causes disharmony in development of the two structures in two Leguminosae shrubs, Cytisus multiflorus and C. striatus. Our main objective was to test the causes of self-sterility in the two species by comparing the embryological development of the self seeds with that of cross seeds. In developing selfed seeds of C. multiflorus, the embryo reaches at most the globular stage and never forms mature seeds, while in C. striatus a few mature selfed seeds are formed. In both species, the main cause of abortion of developing selfed seeds is diminished endosperm development (low values of the ratio of endosperm to embryo), which triggers collapse of the endosperm and embryo. The results indicate that self-sterility in C. striatus is postzygotic because of strong, early inbreeding depression, while in C. multiflorus there exists a mixed pre- and postzygotic mechanism; the prezygotic mechanism causes rejection of some self-pollen tubes in the style/ovary, and the early inbreeding depression triggers abortion of fertilized ovules that escaped that action.

Pollen-stigma interference in two gynodioecious species of Lamiaceae with intermediate individuals.

BACKGROUND AND AIMS: Intermediate individuals (perfect flowers with very high degree of pollen abortion) in a gynodioecious plant species are very rare. A study is made of male-female relationships in each flower type and how floral characters can enhance the avoidance of 'pollen discounting' and 'self-pollination' in two gynodioecious species, Teucrium capitatum and Origanum syriacum. METHODS: The relationship between stigma receptivity and pollen viability was studied in two gynodioecious protandrous species of Lamiaceae, in addition to measuring some floral morphological characters over the life span of the flowers. KEY RESULTS: Three plant types in each species were found: plants bearing hermaphrodite (or male fertile) flowers (MF), female (or male sterile) flowers (MS) and intermediate flowers (INT). Plant types differed in flower size, with MS types being shorter than the other two types. There was no difference in style length among plant types in T. capitatum. Stigma receptivity decayed with floral age and was negative and significantly correlated with pollen viability in the two species, and positive and significantly correlated with style length in O. syriacum but only in MS flowers of T. capitatum. CONCLUSIONS: Reduction in size of floral characters is associated with male sterility, except style length in T. capitatum. MF flowers have two successive reproductive impediments: self-pollination and pollen-stigma interference. In both species, self-pollination is avoided by dichogamy (negative correlation between stigma receptivity and pollen viability), and pollen-stigma interference shows two different patterns: (1) style elongation in O. syriacum is characterized by a significant length increase, final MF dimensions are greater than those of MS dimensions, and style length is positively and significantly correlated with stigma receptivity; and (2) style movement in T. capitatum is characterized by a non-significant increase in style length, final MF floral dimensions are similar to those of MS dimensions, and there is no correlation between style length and stigma receptivity.

Megasporogenesis, megagametogenesis and ontogeny of the aril in Cytisus striatus and C. multiflorus (Leguminosae: Papilionoideae).

BACKGROUND AND AIMS: There are few embryological reports on wild legumes and even fewer on their seminal appendages. There are no existing studies on the complete ontogeny of these appendages in Cytiseae, a very important Papilionoideae tribe in Mediterranean ecosystems. In this work megasporogenesis, megagametogenesis and aril ontogeny were studied in Cytisus multiflorus and C. striatus, endemics from the western Mediterranean region. METHODS: Ovaries and ovules from flower buds, flowers at anthesis and hand cross-pollinated flowers were sectioned with a rotary microtome and studied under light and fluorescence microscopy. KEY RESULTS: A monosporic Polygonum-type of megagametogenesis is observed in both species but with megasporogenesis characterized by formation of a triad of cells after incomplete meiosis. The original cell wall of the megaspore mother cell and triad, including the transverse walls between the latter, are surrounded by a callose layer that isolates them from the surrounding diploid tissue; this callose layer gradually disappears during embryo sac formation. There are no antipodals in the mature embryo sac. Aril ontogeny starts in pre-anthesis with the formation of the aril primordium, and its normal development will occur only after fertilization, more specifically after endosperm initiation. After fertilization, a reactivation of meristem capacity takes place in the aril cells resulting in slow and sparse growth. Later, this type of development gradually decreases but the aril cells continue to grow by cell expansion, which in the last period of seed development is the only type of growth of the aril. In the mature seed, the seminal appendage acquires an irregular U-shape in transverse section, showing vacuolated cells with a large central vacuole that stores lipids and some proteins. CONCLUSIONS: Meiotic triad formation is due to a failure in meiosis II of the chalazal cell of the dyad. In Cytisus seeds the aril has a funicular origin with predominantly post-fertilization development, but a normal growth of the endosperm is needed for proper aril development.