Species delimitation under the general lineage concept: an empirical example using wild North American hops (Cannabaceae: Humulus lupulus)

There is an emerging consensus that the intent of most species concepts is to identify evolutionarily distinct lineages. However, the criteria used to identify lineages differ among concepts depending on the perceived importance of various attributes of evolving populations. We have examined five different species criteria to ask whether the three taxonomic varieties of Humulus lupulus (hops) native to North America are distinct lineages. Three criteria (monophyly, absence of genetic intermediates, and diagnosability) focus on evolutionary patterns and two (intrinsic reproductive isolation and niche specialization) consider evolutionary processes. Phylogenetic analysis of amplified fragment length polymorphism (AFLP) data under a relaxed molecular clock, a stochastic Dollo substitution model, and parsimony identified all varieties as monophyletic, thus they satisfy the monophyly criterion for species delimitation. Principal coordinate analysis and a Bayesian assignment procedure revealed deep genetic subdivisions and little admixture between varieties, indicating an absence of genetic intermediates and compliance with the genotypic cluster species criterion. Diagnostic morphological and AFLP characters were found for all varieties, thus they meet the diagnosability criterion. Natural history information suggests that reproductive isolating barriers may have evolved in var. pubescens, potentially qualifying it as a species under a criterion of intrinsic reproductive isolation. Environmental niche modeling showed that the preferred habitat of var. neomexicanus is climatically unique, suggesting niche specialization and thus compliance with an ecological species criterion. Isolation by distance coupled with imperfect sampling can lead to erroneous lineage identification using some species criteria. Compliance with complementary pattern- and process-oriented criteria provides powerful corroboration for a species hypothesis and mitigates the necessity for comprehensive sampling of the entire species range, a practical impossibility in many systems. We hypothesize that var. pubescens maintains its genetic identity, despite substantial niche overlap with var. lupuloides, via the evolution of partial reproductive isolating mechanisms. Variety neomexicanus, conversely, will likely persist as a distinct lineage, regardless of limited gene flow with vars. lupuloides and pubescens because of ecological isolation--adaptation to the unique conditions of the Rocky Mountain cordillera. Thus, we support recognition of vars. neomexicanus and pubescens as species, but delay making a recommendation for var. lupuloides until sampling of genetic variation is complete or a stable biological process can be identified to explain its observed genetic divergence.     

Pollinator effectiveness and fruit set in common ivy, Hedera helix (Araliaceae)

Flowers of common ivy (Hedera helix L.) provide late season pollen and nectar for several insect groups, and its fruits are a winter and spring food source for frugivorous birds. Ivy benefits from insect pollination in order to set fruit, but it is unknown which flower-visiting insects are the most effective pollinators. Our observations suggest that Vespula wasps are potentially the most effective pollinators since they were frequent visitors, had relatively fast foraging rates, carried large numbers of pollen grains on their bodies and had the highest ‘Pollination potential PP index’ score (a measure of pollinator effectiveness) of all the insect groups examined. There was also a positive linear relationship between the proportion of ivy flowers that set fruit and wasp foraging activity in 0.5 m² quadrats. Visits by Vespula wasps may therefore be important for ensuring a supply of ivy fruits for birds.     

Polyploidy and invasion of English ivy (Hedera spp., Araliaceae) in North American forests

Polyploidy is a common feature of agricultural weeds and natural area invaders. There are few studies comparing related diploid and polyploid exotics, however, and it is unclear what ecological and genetic factors favor the establishment of weedy polyploids. This research characterizes the geographic distribution and phenotypic characteristics of diploid Hedera helix and tetraploid Hedera hibernica, European species that are invading North American forests. To confirm the taxonomic affinity of invasive plants, we sequenced five non-coding cpDNA regions for 108 individuals (105 populations) as well as reference samples representing all species in the genus Hedera. Because diploid H. helix and tetraploid H. hibernica are poorly distinguished by morphology and DNA sequence, we used flow cytometry to determine their distribution (585 individuals). More than 90 % of sampled plants had cpDNA sequences identical or similar to H. helix sensu lato and H. hibernica. Diploid H. helix was dominant on the U.S. east coast (78.5 % of sampled plants) while tetraploid H. hibernica was dominant on the U.S. west coast (72.2 % of sampled plants), mirroring the species’ occurrence in maritime versus continental climates of Europe. Moreover, for sympatric occurrences in the Pacific Northwest, H. hibernica was larger and more frequently reproductive than H. helix. In a 2-year garden experiment, tetraploid H. hibernica had substantial architectural differences compared to diploid H. helix, including larger (but less numerous) leaves and thicker (but less branched) stems. Field experiments are needed to evaluate “pre-adaptation” (directional ecological filtering) and other factors mediating the invasion of H. helix and H. hibernica.     

Chloroplast and nuclear evidence for multiple origins of polyploids and diploids of Hedera (Araliaceae) in the Mediterranean basin

Chloroplast (trnT-L) and nuclear rDNA (ITS) sequence analyses of the Araliaceae provide strong molecular evidence for the monophyly of the genus Hedera. Phylogenetic reconstructions suggest multiple origins and an active polyploidization process not only in the formation of tetraploids (2n = 96), hexaploids (2n = 144), and octoploids (2n = 192), but also of diploids (2n = 48). A high basic chromosome number of x = 24, extensive polyphyly in widespread diploids, and terminal placement of Hedera in phylogenies of the Araliaceae reveal that extant diploid taxa may be, in fact, assemblages of ancestral polyploids from plants of n = 12. Four major lineages containing four types of chloroplast (chlorotypes I, II, III, and IV), which are defined by different trnT-L nucleotide substitutions and two large insertions (50- and 30-bp), provide evidence for evolutionary processes and historical biogeography in Hedera. We propose a scenario where an initial colonization in the Mediterranean basin by Asian ancestors (carrying the ancestral Araliaceae chlorotype I) is followed by differentiation into the four chlorotypes of the Mediterranean region, and then recolonization of Asia and northern Europe only by chlorotype III. The Macaronesian taxa (Hedera azorica, Hedera maderensis ssp. maderensis, and Hedera canariensis) appear to have originated from a single-colonization event to each archipelago with no further contact either with continental or insular species.     

Phylogenetic relationships ofPseudopanax species (Araliaceae) inferred from parsimony analysis of rDNA sequence data and morphology

Sequence data from internal transcribed spacer (ITS) regions of rDNA and data from morphology, cytology and wood anatomy are used to study phylogenetic relationships inPseudopanax. The molecular and non-molecular data are analysed as independent data sets and in combination using parsimony. Results supported the conclusion that the genusPseudopanax is polyphyletic.Pseudopanax species emerge in two major monophyletic groups. The Anomalus group containsPseudopanax anomalus, P. edgerleyi, andP. simplex; these species share a common ancestor withCheirodendron trigynum and more distantly withPseudopanax gunnii. The second major group consists of two smaller groups: the Arboreus group, includingPseudopanax arboreus, P. colensoi, P. kermadecensis, P. laetus, andP. macintyrei, and the Crassifolius/Discolor group, includingP. chathamicus, P. crassifolius, P. discolor, P. ferox, P. gilliesii, P. lessonii, andP. linearis. Meryta species are close relatives of thePseudopanax Arboreus and Crassifolius/Discolor groups.     

Comparative palynology and seed morphology in annual pansies (Viola sect. Melanium,Violaceae): implications for species delimitation

Abstract

This integrated study provides new insights into pollen and seed morphology and pollen heteromorphism of four closely related annual taxa of Viola sect. Melanium. The plant material, both fresh and dried, was collected in Italy and studied using light and scanning electron microscope. Palynological data for V. hymettia together with a detailed comparative analysis of seed morphology and micromorphology of the four species are reported for the first time. Results of this work highlight some pollen and seed features as useful diagnostic characters. The pollen size proves to be of diagnostic value to easily separate V. kitaibeliana, having the smallest pollen grains, from the others, especially from V. arvensis with the largest ones. Exine ornamentation is microreticulate, showing no relevant differences among species. We can partially confirm the diagnostic value of the prevailing pollen morph as it can be useful only for V. arvensis (five-aperturate) versus V. tricolor and V. hymettia (four-aperturate). The macro- and micromorphology of seeds provide additional useful distinguishing characters. Particularly, seed size was found to be a good delimitating character, especially to distinguish V. kitaibeliana (with the smallest seeds) from V. arvensis, and easy to be measured with no need of particular equipment.     

Multidisciplinary approaches for species delimitation in Sisyrinchium (Iridaceae)

A remarkable morphological similarity in natural populations of different groups of Sisyrinchium spp. (Iridaceae) makes classification of some species difficult. The present paper focused on two morphological categories of S. sellowianum (MC‐I and MC‐II) with distinct morphological traits. Morphological, cytogenetic, reproductive biology and genetic studies were performed, aiming to elucidate and better characterize such variation. The basic chromosome number established for the species was x = 9 and diploid and tetraploid populations were observed for MC‐I, whereas MC‐II showed only diploid populations. Different pollen morphologies were recognized in each morphological category. Based on pollination experiments, MC‐I is out‐crossing, whereas MC‐II is selfing. The populations were highly differentiated (F_ST = 0.46, θ^B = 0.62). In MC‐I, observed variation was greater within populations (69%) than among populations (31%), whereas in MC‐II, 61% of the variation was among populations and 39% within populations. This study evidenced that the two morphological categories correspond to distinct and reproductively isolated species and that floral traits reflect their breeding system, with allogamous plants in MC‐I and autogamous in MC‐II. Furthermore, MC‐II is unveiled as S. platycaule, a neglected taxon of Sisyrinchium in Brazil. The tools applied in this study were substantially able to check the identity of the two morphological categories as two different species and present the variation encountered in the taxa.     

Utility of microsatellites and mitochondrial DNA for species delimitation in the spruce budworm (Choristoneura fumiferana) species complex (Lepidoptera: Tortricidae)

Species identifications have been historically difficult in the Choristoneura fumiferana group, an important insect pest complex. We examined the utility of simple sequence repeats (SSRs, also referred to as microsatellites) and mitochondrial DNA (mtDNA) for delimiting and identifying eight currently recognized species sampled across North America. Four of these species formed discrete clusters using SSRs, while only two species were delimited with mtDNA. There was evidence for hybridization or incomplete lineage sorting between several species pairs. An integrative approach, using both phenotypic traits and molecular markers, allowed for the discrimination of more biologically relevant species units than did the use of molecular markers alone. As species are currently identified using putatively adaptive phenotypic traits, the differences observed between recognized species and neutral SSRs or mtDNA suggests that these species (or evolutionary significant units) have diverged via natural selection in spite of some gene flow.     

Isozyme variation in Calliandra calothyrsus (Leguminosae): its implications for species delimitation and conservation

Patterns of genetic diversity within and among populations of Calliandra calothyrsus, an important multipurpose tree species, were examined using isozyme analysis. C. calothyrsus is a widespread species distributed throughout Central America and southern Mexico, across a variety of environments. Morphologically and ecologically distinct populations can be identified within this range, but they are currently considered to represent a single species. C. calothyrsus has been introduced to many parts of the tropics, where it is cultivated as a source of fuelwood, animal fodder, green manure, and shade by rural communities. Some of these introductions are known to have originated from Guatemala, but very little is known about the genetic diversity of either the native or naturalized populations. Isozyme electrophoresis of 23 loci across 17 populations of C. calothyrsus indicated that the majority of genetic diversity was partitioned between populations (FST = 0.802) and that within-population heterozygosity was low (mean Ho = 0.057). Naturalized populations had lower than expected heterozygosities and were most similar to material from Santa Maria de Jesus, a natural population in southern Guatemala. Four distinct groups of populations were identified on the basis of Nei's genetic distances and Population Aggregation Analysis (PAA), and correlate with the morphological and ecological differences that can be observed within the species. The results are discussed in relation to species delimitation and conservation.     

Systematics and phylogenetic species delimitation within Polinices s.l. (Caenogastropoda: Naticidae) based on molecular data and shell morphology

Here, we present the first phylogenetic analysis of a group of species taxonomically assigned to Polinices sensu latu (Naticidae, Gastropoda) based on molecular data sets. Polinices s.l. represents a speciose group of the infaunal gastropod family Naticidae, including species that have often been assigned to subgenera of Polinices [e.g. P. (Neverita), P. (Euspira), P. (Conuber) and P. (Mammilla)] based on conchological data. The results of our molecular phylogenetic analysis confirm the validity of five genera, Conuber, Polinices, Mammilla, Euspira and Neverita, including four that have been used previously mainly as subgenera of Polinices s.l. Our results furthermore indicate a close relationship of members of the Polinicinae to Sinum??a genus traditionally placed in the naticid subfamily Sininae. We furthermore present conchological analyses to determine the validity of shell characters used traditionally in species designation in the genus Polinices. Our data reveal several characters (e.g. protoconch, operculum colour, parietal callus) to be informative, while many characters show a high degree of homoplasy (e.g. umbilicus, shell form). Among the species arranged in the genus Polinices s.s., four conchologically very similar taxa often subsumed under the common Indo-Pacific species P. mammilla are separated distinctly in phylogenetic analyses. Despite their striking conchological similarities, none of these four taxa are related directly to each other. Additional conchological analyses of available name-bearing type specimens and type figures reveal the four ??mammilla??-like white Polinices species to include true P. mammilla and three additional species, which could be assigned to P. constanti (replacement name for P. dubius), P. jukesii and possibly P. tawhitirahia, based on protoconch and operculum characteristics.     

DNA barcoding and species delimitation of butterflies (Lepidoptera) from Nigeria

Molecular Biology Reports - Accurate identification of species is a prerequisite for successful biodiversity management and further genetic studies. Species identification techniques often require...     

SpedeSTEM: a rapid and accurate method for species delimitation

We describe a software package (SpedeSTEM) that allows researchers to conduct a species delimitation analysis using intraspecific genetic data. Our method operates under the assumption that a priori information regarding group membership is available, for example that samples are drawn from some number of described subspecies, races or distinct morphotypes. SpedeSTEM proceeds by calculating the maximum likelihood species tree from all hierarchical arrangements of the sampled alleles and uses information theory to quantify the model probability of each permutation. SpedeSTEM is tested here against empirical and simulated data; results indicate that evolutionary lineages that diverged as few as 0.5N generations in the past can be validated as distinct using sequence data from little as five loci. This work enables speciation investigations to identify lineages that are evolutionarily distinct and thus have the potential to form new species before these lineages acquire secondary characteristics such as reproductive isolation or morphological differentiation that are commonly used to define species.     

Phylogenetic relationships,population demography,and species delimitation of the Alouatta belzebul species complex (Atelidae: Alouattinae)

Primates - Howler monkeys (genus Alouatta) exhibit the most extensive distribution among platyrrhines, comprising Mesoamerican and South American species groups, with the South American group...     

(New) Species concepts,species delimitation and the inherent limitations of taxonomy

The species problem, despite decades of heated debates, has not been resolved yet. Recently, two new species concepts have been published, the mitonuclear compatibility species concept and the inclusive species concept. I briefly discuss them, together with a recent attempt at standardizing taxonomic decisions, in the broader framework of what I believe is an inherent limitation of taxonomy—imposing a discrete system on a continuous process (evolution) that leads to fuzzy boundaries in nature. In the light of this, taxonomists, biologists in general and conservationists alike will have to accept the fact that completely nonarbitrary species delimitation is impossible. This has serious ramifications in all disciplines that rely on species, and particularly species counts, as a basic currency for quantitative analyses (ecology, evolutionary biology) and practical decision-making (conservation and environmental policy).     

Molecular taxonomy and species delimitation in Andean Schistocerca (Orthoptera: Acrididae)

The acridian genus Schistocerca comprises about 50 species which are endemic to the New World, except the Old World locust S. gregaria. Their morphological identification is rendered difficult by phase polyphenism, geographical overlap due to migrations or swarming, the difficulty to easily differentiate genitalia and the occurrence of interspecific hybrids. The three species reported from Peru include the swarming species S. interrita, a pest that can be recognized only by taxonomists. We show that it can be unambiguously identified using a mitochondrial DNA fragment known to have barcoding properties in this genus. We used several methods to delimitate Peruvian species. While S. interrita and S. pallens were well characterized, S. piceifrons peruviana was split into several taxa by a phylogeny-based method, whereas a combination of population genetics methods led one to identify only the three nominal species. A tentative reconstruction of the species history shows that several populations of S. piceifrons peruviana have recently increased in number, while exchanging some migrants, whereas an isolated population at the northern margin of the species range is substantially differentiated while exchanging no migrants with the others. This complex history has resulted in an atypical lineage pattern that appears to have confounded the standard assumptions underlying available species delimitation methods. Because of its behavioral property which tends to keep it panmictic, the identification of the swarming S. interrita remained unaffected.     

Morphometric studies and taxonomic delimitation in Menonvillea scapigera and related species (Cremolobeae: Brassicaceae)

Previous systematic treatments of Menonvillea recognized a group of six morphologically related species, the ‘M. scapigera group,' distinguished by having pinnately lobed leaves and united filaments of median staminal pairs. The number of recognized species ranged from six to two, one with three subspecies. In order to clarify the taxonomy of this group, multivariate and univariate analyses of qualitative and quantitative characters from herbarium specimens were performed. Additionally, to test the morphological results, the phylogenetic relationships and degree of exclusive ancestry were studied using ITS sequences and the genealogical sorting index. Our results support the recognition of three species (M. famatinensis, M. hirsuta, M. scapigera) delimited by a unique combination of qualitative characters. Quantitative characters support the division of the latter species into two subspecies: scapigera and longipes. The molecular data are congruent with the morphology and support the inclusion of M. hookeri within M. scapigera. A key to taxa of the M. scapigera group is presented.     

A toolbox for integrative species delimitation in Machilis jumping bristletails (Microcoryphia: Machilidae)

Accurate species delimitation is fundamental for many fields of biology. Although a wide range of evolutionary-biological patterns are known to promote failure in species delimitation when any single information source is considered, the majority of species characterisations are still based on single disciplines. Using Alpine jumping bristletails we here showcase the establishment of morphological and molecular tools for multi-source species delimitation in a taxonomically problematic arthropod group and stress its advantages over single-discipline approaches. Once sound species delimitations have been achieved, Alpine jumping bristletails will be available as a prime model system for addressing questions related to the emergence of endemism and parthenogenesis.     

Essential oils from the leaves of three New Zealand species of Pseudopanax (Araliaceae)

Essential oils from three of the eleven endemic New Zealand species of Pseudopanax, P. arboreus, P. discolor and P. lessonii, were found to have a fairly uniform composition which was different from that of the oils of Raukaua species that were formerly classified in the Pseudopanax genus. Oils of the three Pseudopanax species all contained significant proportions of viridiflorol and a closely related unidentified hydroazulene alcohol in common. In addition, the oil of P. arboreus contained bicyclogermacrene, linalool and long chain hydrocarbons. The oil of P. discolor contained nerolidol in abundance (36.3%) together with linalool and epi-alpha-muurolol. The oil of P. lessonii contained a complex mixture of sesquiterpene alcohols including epi-alpha-muurolol and a mixture of long chain hydrocarbons. Nerolidol and linalool provided the oil of P. discolor with a pleasant floral aroma, but the yield of oil was very low (0.01%).