Biodiversity of leaf beetles (Coleoptera: Chrysomelidae) in a tropical montane rainforest ecosystem assessed with DNA barcoding

The aim of the present study was the assessment of an unknown tropical insect fauna without traditional taxonomy. For this purpose, the diversity of leaf beetles (Coleoptera: Chrysomelidae) in the montane rainforest of the Reserva Biológica San Francisco (RBSF) and parts of the Podocarpus National Park in southern Ecuador was investigated. Beetles were sampled at three different elevations, ’1000 m’ (Bombuscaro; 1020–1075 m a.s.l.), ’2000 m’ (Estación Científica San Francisco – ECSF; 1913–2089 m a.s.l.), and ’3000 m’ (Cajanuma; 2805–2891 m a.s.l.) with a set of different collection methods. Beetles were mainly sampled with sweep netting, beating, and hand-collection from the lower understorey vegetation of 36 sampling plots (12 per elevation, six of them in the valleys, six on the ridges) following a standardized sampling protocol. A total of 4286 leaf beetles have been collected, 1775 of these (usually one of each morphospecies per sample) were sorted into 515 different morphospecies, DNA barcoded, and assigned to molecular operational taxonomic units (MOTUs). The study covers aspects of community structure and its changes with increasing elevation. Methodological aspects of rapid biodiversity assessment are evaluated: Different collection methods and morphological and sequence-based methods for species delimitation are compared. General leaf beetle diversity patterns in an Andean mountain forest Leaf beetle assemblages showed patterns typical for tropical arthropods: They were species-rich, with few common species but a high percentage of rare species. 1583 specimens were sorted into 473 morphospecies, and for 1334 of them a DNA barcode could be obtained. They belong to 416 morphospecies and were grouped into 459 MOTUs. Species accumulation curves showed no saturation indicating a further increase in species numbers with additional sampling. Species number estimates ranged up to 916 morphospecies (chao2) for the 1583 analysed individuals, and 705 morphospecies, respectively 805 MOTUs for the 1334 barcoded individuals. The higher MOTU number compared to morphospecies number suggests a high level of potential cryptic diversity that was not recognized by the morphospecies approach alone. The leaf beetle community showed an uneven distribution of incidence and abundance with very few common morphospecies (5% found in more than ten samples, 10% represented by more than ten individuals) and a high percentage of uniques (morphospecies found in one single sample; 50% of all morphospecies), respectively singletons (one single individual found; 45% of all morphospecies). The singleton curve did not reach saturation. Most morphospecies were restricted to one single elevational level (91%), indicating a high turnover of communities with elevation. This pattern was even more apparent for MOTUs (96%) and haplotypes (99%). More than half of the morphospecies belonged to Alticinae (53%), 21% were Galerucinae, 14% Eumolpinae, 5% Hispinae, and 4% Cassidinae. Criocerinae, Chrysomelinae, Lamprosomatinae, and Cryptocephalinae together accounted for 3% of all morpho- species. Rank order remained the same when number of individuals was considered. Composition of the subgroups changed slightly with elevation. Diversity patterns along an elevational gradient inferred with DNA barcode data Leaf beetle assemblages from the 36 study plots were sampled and differences between the three elevations and the two microhabitats (forest on ridges and in valleys) were analysed based on DNA barcode data. The importance of small-scale topography for elevational diversity patterns was evaluated: It was tested whether elevational diversity differs between ridge and valley forests and if the species turnover between and within habitats varies with elevation and changes patterns of elevational diversity when scaling up from the local (sampling plot) to the regional (elevational belt) level. MOTUs were determined using PTP modelling and data was analysed using permutational MANOVA analysis and ordinary linear models. When study sites of both habitats were pooled, local leaf beetle diversity showed a clear mid- elevational peak pattern. However, only leaf beetle diversity in ridge forests peaked at mid- elevations, while the diversity in valley forests was similarly high at 1000 and 2000 m a.s.l. and declined at highest elevations. When scaling up to the regional scale, levels of diversity were generally similar at the two lower elevations and declined at 3000 m a.s.l. The scale-dependent shift in diversity patterns was caused by a higher turnover of species communities between and within habitats at lower than at mid-elevations, suggesting more specialized herbivore communities in the more productive lower elevations. The study underscores the importance of topography and spatial scale for the inference of diversity patterns. Changes in ecosystem productivity but also area and temperature with elevation might also influence the genetic diversity within species, however, levels of genetic diversity (haplotype diversity per MOTU) did not differ among elevational levels. Biodiversity patterns along the elevational gradient were revealed by MOTUs and morphospecies in the same way. Comparison of morphospecies sorting and DNA barcoding 1475 barcoded individuals were assigned to MOTUs and the results were compared with the morphospecies sorting. The barcode approach estimated 10% higher species numbers (448 morphospecies, 493 MOTUs). This was caused by a higher number of splittings than lumpings of morphospecies. The similar numbers of morphospecies and MOTUs arose partly due to the fact that splittings and lumpings compensated one another. However, the number of perfect matches was comparatively low: 63% of all morphospecies corresponded exactly with one MOTU. Most lumpings united individuals of two morphospecies in one MOTU (76%), in some cases, individuals of up to five morphospecies (4%) were lumped. Similarly, most splittings divided a morphospecies in two networks (69%), only once a morphospecies was split into six MOTUs (1%). The subgroups most challenging for morphospecies sorting were Galerucinae and especially Alticinae. Difficulties most probably arose due to the large number of specimens and species. DNA barcoding showed to be a valuable tool in cases were morphospecies sorting is exacerbated by pronounced intraspecific variation in colour, shape, or size, and may reveal cryptic diversity. Especially in species that are small and/or lack conspicuous external characters barcoding is a useful tool to complement morphospecies sorting. Particularly in large, specimen- and species-rich data sets DNA barcoding can facilitate morphospecies sorting and can result into a more accurate species delimitation. Influence of different species delimitation methods on species richness estimates For a subset of 674 barcoded specimens, a set of four different DNA-based species delimitation methods and their influence on species richness estimates were compared. Distance-based clustering, statistical parsimony analysis, GMYC-, and PTP modelling led to highly similar results. The reason probably lies within the structure of the underlying data set: It is geographically restricted and undersampled with a high proportion of singletons what turns it insensitive against differences in species delimitation methods. Several cases of splittings and lumpings led to discrepancies between morphospecies and MOTU assignment and generally MOTU numbers were ~8% higher than morphospecies numbers. Morphospecies sorting and DNA barcoding allow similar conclusions on leaf beetle diversity: The leaf beetle fauna is species-rich with a strong turnover among elevations. Most morphospecies where found only at a single elevational level, also when singletons and doubletons have been excluded. This pattern was even more visible for MOTUs and haplotypes. The high turnover between leaf beetle communities at the different elevations is also visible in the species accumulation curves: If to the specimens of one elevation the specimens of a second elevation where added, the curves showed once more a further increase. Comparison of sampling methods Within the present study a total of 1174 samples were taken. They varied considerably in size and effort as different sampling methods were used. The focus was on standardized sampling with sweep netting, beating, and hand-collection on the sampling plots. Malaise trapping, light trapping, and additional hand-collection completed the sampling. In sweep netting-, beating-, hand-collection-, and light trap samples on average only few individuals and morphospecies were caught per single sample (less than five). In contrast, the Malaise traps were highly efficient on a per sample basis: They yielded a mean of 31 individuals and 15 morphospecies per sample. Collection efficiency for certain subgroups slightly differed between the different methods. Even after 298.5 sampling hours the species accumulation curve of the standardized plot samples showed no saturation indicating that a further increase of morphospecies number is expected with further sampling

Parametric and non-parametric masking of randomness in sequence alignments can be improved and leads to better resolved trees

<p>Abstract</p> <p>Background</p> <p>Methods of alignment masking, which refers to the technique of excluding alignment blocks prior to tree reconstructions, have been successful in improving the signal-to-noise ratio in sequence alignments. However, the lack of formally well defined methods to identify randomness in sequence alignments has prevented a routine application of alignment masking. In this study, we compared the effects on tree reconstructions of the most commonly used profiling method (GBLOCKS) which uses a predefined set of rules in combination with alignment masking, with a new profiling approach (ALISCORE) based on Monte Carlo resampling within a sliding window, using different data sets and alignment methods. While the GBLOCKS approach excludes variable sections above a certain threshold which choice is left arbitrary, the ALISCORE algorithm is free of <it>a priori </it>rating of parameter space and therefore more objective.</p> <p>Results</p> <p>ALISCORE was successfully extended to amino acids using a proportional model and empirical substitution matrices to score randomness in multiple sequence alignments. A complex bootstrap resampling leads to an even distribution of scores of randomly similar sequences to assess randomness of the observed sequence similarity. Testing performance on real data, both masking methods, GBLOCKS and ALISCORE, helped to improve tree resolution. The sliding window approach was less sensitive to different alignments of identical data sets and performed equally well on all data sets. Concurrently, ALISCORE is capable of dealing with different substitution patterns and heterogeneous base composition. ALISCORE and the most relaxed GBLOCKS gap parameter setting performed best on all data sets. Correspondingly, Neighbor-Net analyses showed the most decrease in conflict.</p> <p>Conclusions</p> <p>Alignment masking improves signal-to-noise ratio in multiple sequence alignments prior to phylogenetic reconstruction. Given the robust performance of alignment profiling, alignment masking should routinely be used to improve tree reconstructions. Parametric methods of alignment profiling can be easily extended to more complex likelihood based models of sequence evolution which opens the possibility of further improvements.</p

Testing a Short Nuclear Marker for Inferring Staphylinid Beetle Diversity in an African Tropical Rain Forest

The use of DNA based methods for assessing biodiversity has become increasingly common during the last years. Especially in speciose biomes as tropical rain forests and/or in hyperdiverse or understudied taxa they may efficiently complement morphological approaches. The most successful molecular approach in this field is DNA barcoding based on cytochrome c oxidase I (COI) marker, but other markers are used as well. Whereas most studies aim at identifying or describing species, there are only few attempts to use DNA markers for inventorying all animal species found in environmental samples to describe variations of biodiversity patterns.In this study, an analysis of the nuclear D3 region of the 28S rRNA gene to delimit species-like units is compared to results based on distinction of morphospecies. Data derived from both approaches are used to assess diversity and composition of staphylinid beetle communities of a Guineo-Congolian rain forest in Kenya. Beetles were collected with a standardized sampling design across six transects in primary and secondary forests using pitfall traps. Sequences could be obtained of 99% of all individuals. In total, 76 molecular operational taxonomic units (MOTUs) were found in contrast to 70 discernible morphospecies. Despite this difference both approaches revealed highly similar biodiversity patterns, with species richness being equal in primary and secondary forests, but with divergent species communities in different habitats. The D3-MOTU approach proved to be an efficient tool for biodiversity analyses.Our data illustrate that the use of MOTUs as a proxy for species can provide an alternative to morphospecies identification for the analysis of changes in community structure of hyperdiverse insect taxa. The efficient amplification of the D3-marker and the ability of the D3-MOTUs to reveal similar biodiversity patterns as analyses of morphospecies recommend its use in future molecular studies on biodiversity

Youth in international nature conservation: The example of youth participation in the Convention on Biological Diversity (CBD)

Jungen Menschen bieten sich heute viele Möglichkeiten, ihr Engagement im Naturschutz auch international zu gestalten. Insbesondere in der globalen Umweltpolitik und in multilateralen Umweltabkommen wird die Jugend zunehmend als wichtiger Interessenvertreter und Akteur wahrgenommen. In der Literatur zur Rolle nichtstaatlicher Akteure in internationalen Umweltabkommen wurde die Beteiligung der Jugend allerdings bisher kaum betrachtet. Der Artikel analysiert erstmals Umfang und Qualität der Jugendpartizipation innerhalb des Übereinkommens über die biologische Vielfalt (Convention on Biological Diversity, CBD) und identifiziert Grenzen und Erfolgsfaktoren für Jugendpartizipation. Zur Bewertung des Umfangs der Partizipation werden Vergleiche mit vier weiteren multilateralen Umweltabkommen gezogen (UNFCCC, UNCCD, Ramsar und CITES). In der CBD ermöglichen die Motivation der Jugend selbst und ein hoher Grad an Selbstorganisation in einem starken Netzwerk in Kombination mit einer positiven Wahrnehmung von Seiten anderer Akteure eine gut ausgeprägte Partizipation mit viel inhaltlicher Mitarbeit. Zuletzt werden Vorschläge für eine weitere Stärkung der Jugendpartizipation abgeleitet

Tetramorium boehmei sp. n. - a new ant (Hymenoptera: Formicidae) species from the Kakamega Forest, Western Kenya

Volume: 57Start Page: 359End Page: 36

Exploring the Leaf Beetle Fauna (Coleoptera: Chrysomelidae) of an Ecuadorian Mountain Forest Using DNA Barcoding

Background Tropical mountain forests are hotspots of biodiversity hosting a huge but little known diversity of insects that is endangered by habitat destruction and climate change. Therefore, rapid assessment approaches of insect diversity are urgently needed to complement slower traditional taxonomic approaches. We empirically compare different DNA-based species delimitation approaches for a rapid biodiversity assessment of hyperdiverse leaf beetle assemblages along an elevational gradient in southern Ecuador and explore their effect on species richness estimates. Methodology/Principal Findings Based on a COI barcode data set of 674 leaf beetle specimens (Coleoptera: Chrysomelidae) of 266 morphospecies from three sample sites in the Podocarpus National Park, we employed statistical parsimony analysis, distance-based clustering, GMYC- and PTP-modelling to delimit species-like units and compared them to morphology-based (parataxonomic) species identifications. The four different approaches for DNA-based species delimitation revealed highly similar numbers of molecular operational taxonomic units (MOTUs) (n = 284–289). Estimated total species richness was considerably higher than the sampled amount, 414 for morphospecies (Chao2) and 469–481 for the different MOTU types. Assemblages at different elevational levels (1000 vs. 2000 m) had similar species numbers but a very distinct species composition for all delimitation methods. Most species were found only at one elevation while this turnover pattern was even more pronounced for DNA-based delimitation. Conclusions/Significance Given the high congruence of DNA-based delimitation results, probably due to the sampling structure, our study suggests that when applied to species communities on a regionally limited level with high amount of rare species (i.e. ~50% singletons), the choice of species delimitation method can be of minor relevance for assessing species numbers and turnover in tropical insect communities. Therefore, DNA-based species delimitation is confirmed as a valuable tool for evaluating biodiversity of hyperdiverse insect communities, especially when exact taxonomic identifications are missing

Continuation of Fig 2.

<p>Continuation of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148268#pone.0148268.g002" target="_blank">Fig 2</a>.</p

Overview of splittings and lumpings.

<p>Overview of splittings and lumpings.</p