Gábor Sramkó

Molecular phylogeny and evolutionary history of the Eurasiatic orchid genus Himantoglossum s.l. (Orchidaceae).

BACKGROUND AND AIMS Lizard orchids of the genus Himantoglossum include many of Eurasias most spectacular orchids, producing substantial spikes of showy flowers. However, until recently the genus had received only limited, and entirely traditional, systematic study. The aim of the current work was to provide a more robust molecular phylogeny in order to better understand the evolutionary relationships among species of particular conservation concern. METHODS All putative species of Himantoglossum s.l. were sampled across its geographical range. A large subsample of the 153 populations studied contributed to an initial survey of nuclear ribosomal internal transcribed spacer (nrITS) ribotypes. Smaller subsets were then sequenced for four plastid regions and the first intron of the low-copy-number nuclear gene LEAFY. Rooted using Steveniella as outgroup, phylogenetic trees were generated using parsimony and Bayesian methods from each of the three datasets, supplemented with a ribotype network. KEY RESULTS The resulting trees collectively determined the order of branching of the early divergent taxa as Himantoglossum comperianum > H. robertianum group > H. formosum, events that also involved significant morphological divergence. Relaxed molecular clock dating suggested that these divergences preceded the Pleistocene glaciations (the origin of the H. robertianum group may have coincided with the Messinian salinity crisis) and occurred in Asia Minor and/or the Caucasus. Among more controversial taxa of the H. hircinum-jankae clade, which are only subtly morphologically divergent, topological resolution was poorer and topological incongruence between datasets was consequently greater. CONCLUSIONS Plastid sequence divergence is broadly consistent with prior, morphologically circumscribed taxa and indicates a division between H. hircinum-adriaticum to the west of the Carpathians and H. jankae-caprinum (plus local endemics) to the east, a distinction also suggested by nrITS ribotypes. LEAFY phylogenies are less congruent with prior taxonomic arrangements and include one likely example of paralogy. Himantoglossum metlesicsianum fully merits its IUCN Endangered status. Potentially significant genetic variation was detected within Steveniella satyrioides, H. robertianum and H. hircinum. However, confident circumscription of the more derived species of Himantoglossum s.s., including local endemics of hybrid origin, must await future morphometric and population-genetic analyses.

Appearance of Planktothrix rubescens Bloom with [D-Asp3, Mdha7]MC–RR in Gravel Pit Pond of a Shallow Lake-Dominated Area

Blooms of toxic cyanobacteria are well-known phenomena in many regions of the world. Microcystin (MC), the most frequent cyanobacterial toxin, is produced by entirely different cyanobacteria, including unicellular, multicellular filamentous, heterocytic, and non-heterocytic bloom-forming species. Planktothrix is one of the most important MC-producing genera in temperate lakes. The reddish color of cyanobacterial blooms viewed in a gravel pit pond with the appearance of a dense 3 cm thick layer (biovolume: 28.4 mm3 L−1) was an unexpected observation in the shallow lake-dominated alluvial region of the Carpathian Basin. [d-Asp3, Mdha7]MC–RR was identified from the blooms sample by MALDI-TOF and NMR. Concentrations of [d-Asp3, Mdha7]MC–RR were measured by capillary electrophoresis to compare the microcystin content of the field samples and the isolated, laboratory-maintained P. rubescens strain. In analyzing the MC gene cluster of the isolated P. rubescens strain, a deletion in the spacer region between mcyE and mcyG and an insertion were located in the spacer region between mcyT and mcyD. The insertion elements were sequenced and partly identified. Although some invasive tropical cyanobacterial species have been given a great deal of attention in many recent studies, our results draw attention to the spread of the alpine organism P. rubescens as a MC-producing, bloom-forming species.

Genetic diversity and population structure of the rare and endangered plant species Pulsatilla patens (L.) Mill in East Central Europe

Pulsatilla patens s.s. is a one of the most endangered plant species in Europe. The present range of this species in Europe is highly fragmented and the size of the populations has been dramatically reduced in the past 50 years. The rapid disappearance of P. patens localities in Europe has prompted the European Commission to initiate active protection of this critically endangered species. The aim of this study was to estimate the degree and distribution of genetic diversity within European populations of this endangered species. We screened 29 populations of P. patens using a set of six microsatellite primers. The results of our study indicate that the analyzed populations are characterized by low levels of genetic diversity (Ho = 0.005) and very high levels of inbreeding (FIS = 0.90). These results suggest that genetic erosion could be partially responsible for the lower fitness in smaller populations of this species. Private allelic richness was very low, being as low as 0.00 for most populations. Average genetic diversity over loci and mean number of alleles in P. patens populations were significantly correlated with population size, suggesting severe genetic drift. The results of AMOVA point to higher levels of variation within populations than between populations.The results of Structure and PCoA analyses suggest that the genetic structure of the studied P. patens populations fall into three clusters corresponding to geographical regions. The most isolated populations (mostly from Romania) formed a separate group with a homogeneous gene pool located at the southern, steppic part of the distribution range. Baltic, mostly Polish, populations fall into two genetic groups which were not fully compatible with their geographic distribution.Our results indicate the serious genetic depauperation of P. patens in the western part of its range, even hinting at an ongoing extinction vortex. Therefore, special conservation attention is required to maintain the populations of this highly endangered species of European Community interest.

The orchid flora of Turkish graveyards: a comprehensive field survey

Abstract Löki V., Tökölyi J., Süveges K., Lovas-Kiss Á, Hürkan K., Sramkó G. & Molnár V. A.: The orchid flora of Turkish graveyards: a comprehensive field survey. — Willdenowia 45: 231–243. 2015. — Version of record first published online on 17 July 2015 ahead of inclusion in August 2015 issue; ISSN 1868-6397;

Higher seed number compensates for lower fruit set in deceptive orchids

1.Floral deception is widespread in orchids, with more than one third of the species being pollinated this way. The evolutionary success of deceptive orchids is puzzling, as species employing this strategy are thought to have low reproductive success (less flowers yielding fruits) because of low pollination rates. However, direct measurements of total seed production in orchids – which is a better measure of reproductive success – are scarce due to the extremely small size of their seeds. 2.Here, we quantified seed numbers in 1,015 fruits belonging to 48 orchid species from the Pannonian ecoregion (central Europe) and obtained fruit-set and thousand-seed weight data for these species from the literature. We used phylogenetic comparative methods to test the hypothesis that deceptive species should compensate for their lower fruit-set by having either more flowers, larger seeds or more seeds in a fruit. 3.Similarly to previous studies, we found that deceptive orchids have substantially lower fruits-set than nectar-rewarding ones. Also, we found that deceptive species have more seeds in a fruit but not more flowers or larger seeds compared to nectar-rewarding ones. Based on our results, deceptive species compensate for their lower fruit-set by having higher seed numbers per fruit. As a consequence, their seed numbers per shoot do not differ from that of nectar-rewarding ones. 4.Together with other benefits of deceptive pollination (e.g. lower energy expenditure due to the lack of nectar production and higher genetic variability due to decreased probability of geitonogamous pollination), our results can explain why deceptive strategies are so widespread in the orchid family. 5.Synthesis. Our results indicate that deceptive orchids can compensate for their lower fruit-set by having more (but not larger) seeds in a fruit than rewarding species. These findings highlight possible ways in which plants can increase their reproductive success in face of pollinator limitation. We emphasize that fruit-set in itself is an inappropriate measure of the reproductive success of orchids – the total number of seeds per shoot is a much better approximation.

Flood induced phenotypic plasticity in amphibious genus Elatine (Elatinaceae)

Vegetative characters are widely used in the taxonomy of the amphibious genus Elatine L. However, these usually show great variation not just between species but between their aquatic and terrestrial forms. In the present study we examine the variation of seed and vegetative characters in nine Elatine species (E. brachysperma, E. californica, E. gussonei, E. hexandra, E. hungarica, E. hydropiper, E. macropoda, E. orthosperma and E. triandra) to reveal the extension of plasticity induced by the amphibious environment, and to test character reliability for species identification. Cultivated plant clones were kept under controlled conditions exposed to either aquatic or terrestrial environmental conditions. Six vegetative characters (length of stem, length of internodium, length of lamina, width of lamina, length of petioles, length of pedicel) and four seed characters (curvature, number of pits / lateral row, 1st and 2nd dimension) were measured on 50 fruiting stems of the aquatic and on 50 stems of the terrestrial form of the same clone. MDA, NPMANOVA Random Forest classification and cluster analysis were used to unravel the morphological differences between aquatic and terrestrial forms. The results of MDA cross-validated and Random Forest classification clearly indicated that only seed traits are stable within species (i.e., different forms of the same species keep similar morphology). Consequently, only seed morphology is valuable for taxonomic purposes since vegetative traits are highly influenced by environmental factors.

Patterns of plastid DNA differentiation in Erythronium (Liliaceae) are consistent with allopatric lineage divergence in Europe across longitude and latitude

Little attention has been paid so far to the genetic legacy of the oceanic-continental gradient across Europe. Due to this gradient, steppe regions become more extensive and mesic environments become more scattered towards the East. A well-suited system to study the impact of this gradient on lineage differentiation is the temperate mesophilic plant Erythronium dens-canis (Liliaceae), which is widespread in southern Europe with a distribution gap in the Pannonian Plain. Moreover, the large disjunction between E. dens-canis and its sister species E. caucasicum coincides with the Pontic steppe region. By applying range-wide sampling of E. dens-canis and limited sampling of E. caucasicum, we explored their phylogeography using the plastid regions rpl32-trnL and rps15-ycf1. Three major phylogroups were identified: a Caucasian lineage, a highly structured and narrowly distributed Transylvanian lineage, and a more homogenous and widely distributed ‘non-Transylvanian’ lineage. Apparently, both physiographic (mountain) and climatic (steppe) barriers have caused allopatric differentiation in European Erythronium. The Southern Carpathians constitute a latitudinal barrier and the Pannonian Plain a longitudinal barrier between the Transylvanian and ‘non-Transylvanian’ lineages of E. dens-canis. The eastern Carpathian Basin likely functioned as a combination of cryptic eastern (mesic) and cryptic northern refugia for E. dens-canis during glacial periods. The Eastern Carpathians and particularly the Pontic steppe regions acted as a longitudinal barrier between E. dens-canis and E. caucasicum. Steppe-dominated gaps in the distribution range of Erythronium are mirrored by genetic discontinuities along longitudes; this highlights the important role of the oceanic-continental gradient throughout Europe for lineage differentiation.

Floral miniaturisation and autogamy in boreal-arctic plants are epitomised by Iceland’s most frequent orchid, Platanthera hyperborea

Background and Aims. This paper concludes our series of publications comparing island and mainland speciation in European butterfly-orchids, by studying the morphology, phylogenetics and reproductive biology of the controversial circum-arctic species Platanthera (Limnorchis) hyperborea—the most frequent of seven Icelandic orchids. We draw particular attention to its phylogenetic placement, remarkable reproductive biology and morphological convergence on other Platanthera lineages through floral miniaturisation. Methods. Five populations of P. hyperborea in southwest Iceland were measured for 33 morphological characters and subjected to detailed multivariate and univariate analyses, supported by light and scanning electron microscopy of selected flowers. Representative samples from six populations were sequenced for nrITS and placed in a taxonomically broader phylogenetic matrix derived from previous studies. Key Results . Section Limnorchis consists of three distinct ITS-delimited clades based on P. stricta, P. sparsifolia–limosa–aquilonis and P. dilatata–hyperborea. Within the latter group, supposed species boundaries overlap; instead, the data indicate a crude stepwise series of ribotypic transitions extending eastward from North America to Iceland. Morphometric data failed to identify any taxonomically meaningful partitions among Icelandic P. hyperborea populations, despite the presence of a distinct and apparently plesiomorphic ribotype at the most glacially influenced habitat sampled. Microscopic study of the flowers revealed several distinguishing features (some not previously reported), including resupinate lateral sepals, toothed bract margins, club-shaped papillae shared by both the interior of the labellar spur and the stigmatic surface, and an exceptionally adhesive stigma that is reliably covered in disaggregated pollen masses prior to anthesis; auricles are absent. Conclusions. Ribotypes suggest that Icelandic P. hyperborea represents the terminus of a migration route that may have begun in East Asia before passing through North America and presumably Greenland. The incohesive pollinia, rapidly desiccating anther locules, weakly developed rostellum, exceptionally adhesive stigma and the close juxtaposition of compact male and female reproductive organs together conspire to cause routine autogamy and frequent cleistogamy, despite the continued production of substantial nectar reservoirs in the spur and consequent ongoing attraction to the flowers of insects, including mosquitoes. When considered in combination with independently derived lineages of Platanthera on the Azorean and Hawaiian archipelagos also bearing small green flowers, our observations show allometric and paedomorphic reductions in flower size as the primary evolutionary driver, but also indicate strong developmental and functional constraints.

Relationships within the Melitaea phoebe species group (Lepidoptera: Nymphalidae): new insights from molecular and morphometric information

The genus Melitaea consists of about 80 species, divided into ten species groups, which are all restricted to the Palaearctic region. The Melitaea phoebe group was defined by Higgins based on morphological characters such as wing pattern and genital structures. According to his interpretation, the M. phoebe group included seven species: M. phoebe, M. sibina, M. scotosia, M. aetherie, M. collina, M. consulis and M. turkmanica. The taxonomy of the phoebe species group has been poorly resolved and recent results on the species composition within the group suggest the need for a re‐evaluation. In this study molecular sequences (5985 bp) including one mitochondrial (COI) and up to six nuclear (CAD, EF‐1α, GAPDH, MDH, RpS5 and wingless) gene regions from 38 specimens of the Melitaea phoebe species group sensu Higgins and some closely related taxa from the Palaearctic region were analysed. The possible evolution of the processus posterior of the male genitalia was also reconstructed based on a shape mapping technique. The analysis of the combined data shows a very clear pattern and almost all relationships are highly supported. Based on the combined Bayesian tree and the shape of the processus posterior of the male genitalia, four main groups are recognised: (i) collina group, (ii) arduinna group, (iii) aetherie group and (iv) phoebe group. The status of M. ornata, M. zagrosi and M. scotosia as species is confirmed, and the results also indicate that M. telona (s.s.) from Israel is a separate species.

Rediscovery of the Hungarian birch mouse (Sicista subtilis trizona) in Transylvania (Romania) with molecular characterisation of its phylogenetic affinities

Abstract The Southern birch mouse (Sicista subtilis) is a small-sized rodent species characteristic of the Palearctic steppes with westernmost occurrences in central Europe. The species was considered to be extinct in Transylvania (central Romania), but in our field survey we captured three living individuals near the city of Cluj-Napoca. On the basis of nuclear interphotoreceptor retinoid-binding protein and mitochondrial cytochrome c oxidase subunit 1 sequences, we assessed the taxonomic status of the newly found S. subtilis population by comparing them to available sequences, including the sequences of its subspecies. The Transylvanian samples were found to be genetically closest to the Hungarian samples of S. subtilis trizona. These new records extend the known geographic range of this rediscovered species and provide additional information on its habitat preference and external morphological features. Moreover, our phylogenetic tree reconstruction for seven Sicista taxa provides a basic insight into the phylogenetic relationships of the genus, placing the northern Eurasian taxa (S. betulina and S. subtilis) at the crown of the tree and the central Asian taxa at the base of the tree. The Transylvanian occurrence of S. subtilis trizona, which is endemic to the Carpathian Basin, is of high faunistic value as a result of an increase in the number of known populations of this subspecies – one of the most endangered rodents of Europe – from one to two.