Speciation in sexually deceptive orchids: pollinator-driven selection maintains discrete odour phenotypes in hybridizing species

Ophrys orchids mimic the female sex pheromones of their pollinator species to attract males for pollination. Reproductive isolation in Ophrys is based on the selective attraction of only a single pollinator species. A change of floral odour can result in the attraction of a new pollinator species that acts as an isolation barrier towards other sympatrically occurring Ophrys species. Ophrys lupercalis, Ophrys bilunulata, and Ophrys fabrella grow sympatrically and bloom consecutively on Majorca and are pollinated by three species of Andrena. We investigated variation of phenotypic and genotypic flower traits, aiming to study the role of the floral odour for reproductive isolation and speciation. Using chemical and electrophysiology (gas chromatography coupled with an electroantennographic detector) methods, we show that the three Ophrys species use the same odour compounds for pollinator attraction, but in different proportions. A comparison of the floral odour bouquets in a multivariate analysis revealed a clear grouping of plants from the same species, although with an overlap between species. A comparison of the same plants using molecular markers gave a contrasting result. Although O. lupercalis and O. fabrella were genetically well separated, plants of O. bilunulata did not form a distinct group but were similar to either O. lupercalis or O. fabrella. Our data indicate gene flow and hybridization to occur between O. bilunulata and O. lupercalis as well as between O. bilunulata and O. fabrella. All plants of O. bilunulata, despite having different genotypes, showed a very similar floral odour. This reflects a strong selective pressure by the pollinating males. The overlap of genotypes of O. bilunulata and O. fabrella supports our hypothesis that O. fabrella diverged from O. bilunulata by scent variation and the attraction of a new pollinator species, Andrena fabrella. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98 , 439–451.     

Morphological phylogenetic analysis of Ophrys (Orchidaceae): insights from morpho‐anatomical floral traits into the interspecific relationships in an unresolved clade

Reconstructing the phylogeny of the sexually deceptive orchid genus Ophrys is crucial to our understanding of the evolution of its complex floral morphology. Molecular phylogenetic analyses showed that section Pseudophrys forms a well supported clade with Ophrys bombyliflora, O. tenthredinifera and O. speculum, but were unable to elucidate the relationships between these four groups of taxa. Here we conduct a morphological phylogenetic analysis of this unresolved clade of Ophrys based on a data matrix of 45 macro‐ and micromorphological and anatomical floral characters, using maximum parsimony and Bayesian inference. Our cladistic analysis yielded a single most parsimonious tree and a Bayesian 50% majority‐rule consensus tree which differed in their overall topology but agreed that O. tenthredinifera and O. bombyliflora are not sister groups. The phylogenetic placement of O. tenthredinifera was ambiguous since it shares six valid synapomorphies each with the cluster of O. speculum–O. bombyliflora and with section Pseudophrys. In contrast, O. bombyliflora is most likely the sister group to O. speculum, a finding that rejects an earlier morphological phylogenetic hypothesis and favours the existing molecular trees based on nuclear ITS rather than plastid data. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179 , 454–476.     

The value of leaf cuticle characteristics in the identification and classification of Iberian Mediterranean members of the genus Pinus

This study reports the value of leaf cuticle characteristics in the identification and classification of Iberian Mediterranean species of the genus Pinus (P. nigra subsp. salzmannii, P. pinaster, P. pinea and P. halepensis), with the aim of using these characters to identify isolated cuticles and stomata in palynology slides. Preparations were made of the cuticles of pine needles belonging to one natural Iberian population of each of the above species. A number of epidermal morphological characteristics were then recorded with the aim of distinguishing these species from one another. The structure of the stomatal complex (the shape and arrangement of the subsidiary cells) was different in each species. The aperture of the epistomatal chamber was significantly smaller in P. pinea than in the other species examined, and the variables recorded for the thickening of the guard cells provided relationships that clearly distinguished all four taxa. The width and length of the stomata and the upper woody lamellae, the central distance between the external limits of the medial lamellae borders and the length of the stem were the most useful variables in this respect. The present results contribute to the ongoing discussion regarding the taxonomic classification of the members of Pinus, and provide valuable clues for the identification of Iberian Mediterranean pine species from small pine needle fragments or isolated stomata. After validation of the present results for multiple populations, these results could also be used to help identify fossil leaf macroremains and the scattered/isolated stomata commonly observed in palaeopalynological samples. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 161 , 436–448.     

A study of Sarcocornia A.J. Scott (Chenopodiaceae) from Western Mediterranean Europe

The latest publications on Sarcocornia taxonomy and phylogeny recognize six taxa in this genus on the Iberian Peninsula: S. perennis, S. fruticosa, S. alpini, S. alpini subsp. carinata, S. hispanica, and S. pruinosa. The present study represents a comprehensive revision of the different taxa in the Sarcocornia genus present in Western Mediterranean Europe by means of morphological, micromorphological and phylogenetic internal transcribed spacer (ITS) analysis. Morphological and micromorphological data were studied from Sarcocornia samples from 113 populations in coastal salt marshes and inland salt pans in Portugal, France, Spain and Italy. Sixteen new ITS sequences were obtained from Mediterranean Sarcocornia species and analysed together with previous reported data. Published karyological, ecological and biogeographical data from Western Mediterranean Europe were also reviewed. The results indicate the presence of a new species, S. lagascae, found growing in coastal Mediterranean areas of the Iberian Peninsula. The species S. fruticosa was found to be absent from the Iberian territories.     

Reassessing the Odontites purpureus group (Orobanchaceae) from south‐east Spain and north‐west Africa

We describe a species of Odontites, O. bolligeri E.Rico, L.Delgado & Herrero, endemic to the south‐eastern Iberian Peninsula and north Africa, from Morocco to Tunisia. This species belongs to the O. purpureus group and corresponds to the taxon that the monographer Markus Bolliger called O. squarrosus subsp. squarrosus. However, according to the International Code of Botanical Nomenclature (ICBN), this is an invalid name, and hence we propose a new name for these plants here. Drawings of the new species are also supplied. In addition, we compare and discuss the other species of the O. purpureus group, propose a key for them and include a karyological study of the two Iberian species of the group. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158 , 701–708.     

Dispersal,fragmentation, and isolation shape the phylogeography of the European lineages of Polyommatus (Agrodiaetus) ripartii (Lepidoptera: Lycaenidae)

Polyommatus ripartii is a biogeographically and taxonomically poorly understood species of butterfly with a scattered distribution in Europe. Recently, it has been shown that this species includes several European endemic and localized taxa (galloi, exuberans, agenjoi) that were previously considered species and even protected, a result that poses further questions about the processes that led to its current distribution. We analysed mitochondrial DNA and the morphology of P. ripartii specimens to study the phylogeography of European populations. Three genetically differentiated but apparently synmorphic lineages occur in Europe that could be considered evolutionarily significant units for conservation. Their strongly fragmented and counterintuitive distribution seems to be the result of multiple range expansions and contractions along Pleistocene climatic oscillations. Remarkably, based on the 79 specimens studied, these genetic lineages do not seem to extensively coexist in the distributional mosaic, a phenomenon most evident in the Iberian Peninsula. One of the important gaps in the European distribution of P. ripartii is reduced by the discovery of new Croatian populations, which also facilitate a better understanding of the biogeography of the species. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 817‐829.     

New cytological data in Ophrys sect. Pseudophrys Godfery and comparative karyomorphological studies in Ophrys L. (Orchidaceae)

Eight species of Ophrys sect. Pseudophrys (Orchidaceae) were cytogenetically studied. The analysed species possess the most symmetrical karyotypes of the genus (M_CA ranged from 10.21 to 15.87 and CV_CL from 19.61 to 23.93) with 2n = 2x = 36, being composed of mainly metacentric chromosomes. The karyotype formulae were: 36m for Ophrys archimedea, 32m + 4sm for O. flammeola, 32m + 4sm for O. funerea, 36m for O. laurensis, 36m for O. lojaconoi, 34m + 2sm for O. lucifera, 34m + 2sm for O. obaesa and 36m for O. pallida. Karyotype morphometric characters were evaluated by calculating M_CA and CV_CL for the assessment of karyotype asymmetry, and CV_CI for the evaluation of heterogeneity in the position of the centromeres. The relationships between species were thus finally elucidated. The species characterised by wide distribution show greater karyomorphological distance than those with restricted distribution. The possible evolutionary role of chromosomal rearrangements as well as gene mutations in the speciation of Ophrys is discussed.     

A multivariate morphometric study of the iberian representatives of<Emphasis Type="Italic">Potentialla</Emphasis> sect.<Emphasis Type="Italic">Recta</Emphasis> (<Emphasis Type="Italic">Rosaceae</Emphasis>)

A multivariate analysis has been performed in order to study the polymorphisms of the three species ofPotentilla sect.Recta (Th. Wolf)Juzepchuk represented in the Iberian Peninsula:P. asturica Rothm.,P. hirta L. andP. recta L. The variability of 20 characters (quantitative, binary and qualitative) has been evaluated in 76 collections mostly from the Iberian Peninsula and from other European territories, but also from North America, and North Africa. The analyses performed provide support for the recognition of the three species. The most discriminant among the quantitative characters is the width of the long trichomes on the stem, and then the width of the long trichomes on the leaves and the maximum length of the long trichomes. The interval of number of carpels, presence or absence of short eglandular trichomes and the abundance of long eglandular trichomes (>3 mm) are the most discriminant ones among the qualitative ones. The taxonomical position ofP. asturica, which has been previously subordinated as a subspecies within the other two taxa, is discussed. Our results support a good delimitation of this taxon which, on the basis of the morphological characters studied by us, seems to be morphologically more close toP. hirta than toP. recta, but differs significantly from both. A key to the three species is provided.     

Combining traditional morphometrics and molecular markers in cryptic taxa: towards an updated integrative taxonomic treatment for Veronica subgenus Pentasepalae (Plantaginaceae sensu APG II) in the western Mediterranean

The aim of this study was to quantify the phenotypic differences amongst the taxa included in Veronica subgenus Pentasepalae, represented in the western Mediterranean area. In combination with previously obtained data on genotypic differences, this enabled the recognition of eight taxonomic units. The delimitation of the boundaries among species and subspecies was mainly based on molecular data, whereas the best characters to discriminate taxa were selected after an accurate study of herbarium material and living populations. Such a selection was also supported by a detailed morphometric study of 30 quantitative leaf traits in 88 populations. Several morphological characters, including the length of the trichomes, length of the petiole and leaf division, were found to be robust, and could be useful in the identification of the taxa involved. As a conclusion, a key to the taxa is proposed, as well as a complete nomenclatural treatment for the group, which includes the typification of several names. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 159 , 68–87.     

Karyological and taxonomic notes on Ophrys (Orchidoideae, Orchidaceae) from the Iberian Peninsula

Karyological information on Iberian Ophrys species is very limited. This paper provides the haploid and diploid chromosome numbers of 11 taxa of sect. Pseudophrys and sect. Ophrys , both of which are well represented in the Iberian Peninsula, and two taxa from Tunisia. The first data on chromosome numbers for O. vasconica (2 n  = 72, 74), O. ficalhoana (2 n  = 36), O. picta (2 n  = 36), O. sphegifera ( n  = 18, 2 n  = 36, 38) and O. passionis (2 n  = 36) are also presented, confirming the stability of the chromosome number in Ophrys . In addition, populations of the group O. omegaifera ( O. dyris and O. vasconica ), together with tetraploidy, pentaploidy and the existence of aneuploid phenomena, are reported for the first time in Iberia. The basic diploid number is always 2 n  = 36. The karyotypes of several species were analysed. Evolutionary trends in Ophrys chromosomes are discussed. Taxonomic and phytogeographical details are provided on several species or groups of species from Iberia.  © 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003 , 142 , 395−406.     

Unravelling the stylar polymorphism in Melochia (Malvaceae): reciprocity and ancillary characters

Heterostyly is a genetic polymorphism in which plant populations possess two (distyly) or three (tristyly) morphs with flowers differing reciprocally in stigma and anther height. Sex organ deployment has been described as being highly variable among and within species of several distylous taxa belonging to different taxonomic groups. However, the number of studies considering within‐species disparities is still limited. For a better overview of the existing amount of variation that can occur within and between heterostylous species, we sampled 46 populations of six Melochia spp., a style‐polymorphic genus in Cuba. We characterized the floral morphology in all populations and described a set of ancillary characters per species. All of these Melochia spp. are distylous, except for the monomorphic M. nodiflora. The S‐morph produces fewer, larger pollen than L‐morphs, and has verrucose ornamentation. The L‐morph produces reticulate pollen and has larger stigmatic papillae than the S‐morph. The monomorphic M. nodiflora shows ancillary characteristics that are similar to the L‐morph individuals in the related species. As expected, there are differences in ancillary characters among species and also dissimilarities in reciprocity among and within species of Melochia. Our results highlight the importance of considering intraspecific variation in the morphometric characterization of heterostylous taxa. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176 , 147–158.     

Unmasking cryptic species: morphometric and phylogenetic analyses of the Ibero‐North African Linaria incarnata complex

Linaria incarnata has been treated as a widely distributed Ibero‐North African species in the latest taxonomic reviews. Morphological and phylogenetic disparity between populations of this species has been previously reported. Here we present new morphological and phylogenetic evidence for the disintegration of L. incarnata into three distinct species: L. incarnata from the western Iberian Peninsula; L . mamorensis sp. nov. from north‐western Morocco; and L. onubensis from south‐western Spain. The relatively poor morphological differentiation between these taxa (which can be regarded as cryptic species) and their distinct phylogenetic positions indicate that characters of the L. incarnata morphotype have been acquired multiple times in the evolution of Linaria section Versicolores. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 00, 000–000.     

Revision of Chaetodipus arenarius (Rodentia: Heteromyidae)

Chaetodipus arenarius is an endemic species of the Baja California Peninsula. It has undergone taxonomic changes in the last few years. Today, two species, C. arenarius and Chaetodipus dalquesti, are formally recognized with an unknown range for each. However, genetic analysis showed the possible presence of a third species. The objectives of this study were to use molecular data (cytochrome b and cytochrome c oxidase subunit III genes) to determine how many genetic groups are present in C. arenarius in the Baja California Peninsula; if these groups have morphological characteristics that could be used to distinguish them; if the genetic groups have limited distributions; identify taxonomic implications; and establish if there are any associations amongst genetic groups and different habitats. Bayesian inference, maximum parsimony, maximum likelihood, distance optimality criteria, and minimum spanning network indicated three monophyletic groups with high genetic differences (5.54–13.59%). These groups are morphologically diagnosable based on pelage and morphometric characters and geographical range. The three genetic groups correspond to three taxa previously described as subspecies or species. Based on our results, these should be considered to be different species: C. arenarius occupies the most extreme habitats with hot summers; C. dalquesti occupies the moistest coastal areas in the southern part of the peninsula, and Chaetodipus siccus is restricted by mountain ranges to the isolated Los Planes Basin and Cerralvo Island. © 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010.     

Relationship of Hypochaeris salzmanniana (Asteraceae, Lactuceae), an endangered species of the Iberian Peninsula, to H. radicata and H. glabra and biogeographical implications

Hypochaeris salzmanniana DC. (Asteraceae, Lactuceae) is an endangered species on the Iberian Peninsula, known from only eight coastal populations. Most authors have treated it as a variety, subspecies or simply as a synonym of H. glabra L. On the basis of morphological and cytological characters, Talavera recently separated H. salzmanniana (2n = 8) from H. glabra (2n = 10). Material of H. salzmanniana, H. glabra and H. radicata was collected from Spain, Italy, Sicily and Tunisia in order to assess taxonomic status and population relationships. Amplified Fragment Length Polymorphism (AFLP) analysis revealed three well-differentiated species. A close relationship between H. salzmanniana and H. radicata is also confirmed by AFLP analysis and chromosome number (2n = 8), morphology, and rDNA localization (FISH, fluorescence in situ hybridization). Hypochaeris salzmanniana and H. radicata share three fixed diagnostic AFLP fragments out of 348 fragments scored. The population structure of H. salzmanniana reveals distinct groups in southern Spain that are separated geographically. High differentiation among a western (Conil to Zahara), an intermediate (Punta Paloma and Los Algarbes) and an eastern (Algeciras and La Línea) group may reflect ancient separation. Population sizes and genetic compatibility differ greatly among populations and can be used to explain levels of within-population genetic diversity, together with recent documented loss of habitats resulting from tourist developments. Population structures of H. radicata and H. glabra show a similar geographical patterning: strongly differentiated populations from the Betic Cordillera and from the Iberian Massif, which are separated at present by the Guadalquivir river. Geological events at the end of the Tertiary (Tortonian–Messinian Miocene) might help explain patterns of differentiation in these three species of sect. Hypochaeris. © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society, 2004, 146 , 79–95.     

Morphometric study of the complex Moehringia sect. Pseudomoehringia McNeill from the western Mediterranean

Moehringia. sect. Pseudomoehringia McNeill is endemic to the western Mediterranean, having its origin and diversification centre in the Iberian Peninsula and mountains of northern Morocco. Both the relationship with the genus Arenaria as well as the taxonomy within the section have been largely controversial. To disentangle these issues, we conducted a morphometric study using herbarium material. We measured 12 relevant morphological traits from 148 selected herbarium sheets. Data were analyzed using different statistical methods: general linear models, multiple factorial analysis and linear discriminant analysis. We found significant differences between Arenaria balearica and the rest of the genus Moehringia taxa. Within this genus, we identified three well-discriminated species: Moehringia fontqueri, M. glochidisperma and M. intricata. Within M. intricata complex, we discerned virtually no differences amongst most of the subspecies (intricata, giennensis and tejedensis) with the exception of subsp. castellana. We propose: (i) to maintain A. balearica separate from Moehringia sect. Pseudomoehringia; (ii) to consider three species in the section Pseudomoehringia: M. fontqueri, M. glochidisperma and M. intricata; (iii) to recognize only two subspecies within M. intricata complex: subsp. intricata and subsp. castellana. To clarify the taxonomy of this threatened group is of great interest because it might help to prioritize conservation measures.     

A review of the Iberian Sphingonotini with description of two novel species (Orthoptera: Acrididae: Oedipodinae)

The genus Sphingonotus Fieber, 1853 is one of the most species‐rich grasshopper genera in the world. We studied the morphology of c. 1000 individuals from the Iberian Peninsula to review the taxonomy of the genus and its relatives. Moreover, we inferred a molecular phylogeny of the Iberian Sphingonotini based on two mitochondrial genes. The Iberian and north‐west African Sphingonotini comprise two recent radiations, within which the genetic relationships are not fully resolved. A multivariate morphometric analysis showed that S. azurescens (Rambur, 1838) and S. morini (Defaut, 2005) can be clearly discriminated, supporting their species status. Based upon the combined data, the genus Granada Koçak & Kemal, 2008 is synonymized with Sphingonotus Fieber, 1853 and its type species re‐assigned to Sphingonotus imitans Brunner von Wattenwyl, 1882 comb. rev . The data also supported species rank for Sphingonotus lusitanicus Ebner, 1941 comb. rev. Oedipoda callosa Fieber, 1853 is considered as nomen dubium. Two novel species are described: Sphingonotus ( Neosphingonotus ) almeriense Llucià‐Pomares sp. nov . and Sphingonotus (Neosphingonotus) nodulosus Llucia‐Pomares sp. nov. from the southern part of the Iberian Peninsula. The new species are compared with other Iberian Sphingonotini and a key to the species is provided. © 2013 The Linnean Society of London     

High mortality and female‐biased operational sex ratio result in low encounter rates and moderate polyandry in a spider

The taxonomy of the Old World bat genus Otomops (Chiroptera: Molossidae) has been the subject of considerable debate. The failure of classical morphological studies to provide consistent patterns regarding interspecific relationships within Otomops has limited any understanding of the evolutionary history of the genus. We used traditional and geometric morphometric approaches to establish the species limits of taxa from sub‐Saharan Africa, the Arabian Peninsula, and Madagascar. Morphometric data supported the recent recognition of three distinct Afrotropical taxa: Otomops madagascariensis from Madagascar; Otomops martiensseni s.s. from southern, eastern, central, and western Africa; and an undescribed taxon from north‐east Africa and the Arabian Peninsula. Analyses of craniodental measurements and landmark‐based data showed significant cranial size and shape divergence between the three taxa. Cranial size and shape variation within Afro‐Arabian Otomops were strongly influenced by altitude, seasonality of precipitation, and precipitation in the driest month. Based on morphometric patterns and molecular divergence estimates, we suggest that morphological evolution within Afro‐Arabian Otomops occurred in response to the fluctuating climate during the Pleistocene on the one hand, and the increasing aridity and seasonality over north‐eastern Africa on the other. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, •• , ••–••.     

Mitochondrial DNA diversity and taxa delineation in the land snails of the Iberus gualtieranus (Pulmonata,Helicidae) complex

Iberus gualtieranus is a species complex of land snails that is endemic to the Iberian Peninsula. The species taxonomy of the group is based merely on the basis of shell morphology, but validity of the existing taxonomy is uncertain. Using mitochondrial DNA (mtDNA) data (cytochrome oxidase I and 16S rRNA sequences) we were able to validate the observed phylogenetic taxa within the I. gualtieranus s.l. complex by means of the analysis of specimens of the different morphospecies, together with the study of topotypes. Strong incongruences were obtained between morphology and molecular data. The Iberus alonensis morphospecies comprised several genetically divergent but morphologically cryptic lineages. Considering (1) the allopatric distribution of the operational taxonomic units (OTUs), (2) the morphological differentiation, (3) the possible occurrence of hybridization among the different lineages, and (4) the strong differentiation of the mtDNA phylogroups, we suggest the main lineages obtained, for the time being, may be treated as evolutionary species. The robust phylogenetic reconstruction obtained allows us to consider I. alonensis s.s., Iberus campesinus, Iberus carthaginiensis, and Iberus gualtieranus s.s. as valid species. Two additional unnominated taxa of the alonensis shell type have also been identified. Further subdivisions are also considered, including Iberus gualtieranus mariae and Iberus gualtieranus ornatissimus. © 2008 The Linnean Society of London, Zoological Journal of the Linnean Society, 2008, 154 , 722–737.