Computational protocol: Chaetopteryx bucari sp. n., a new species from the Chaetopteryx rugulosa group from Croatia (Insecta, Trichoptera, Limnephilidae) with molecular, taxonomic and ecological notes on the group

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[…] Fieldwork. We collected specimens of Chaetopteryx including Chaetopteryx rugulosa group species in the continental (central Croatia, Banovina, Hrvatsko zagorje, Kordun, Slavonia), mountain (Gorski kotar, Lika regions) and Mediterranean (Istria and Dalmatia) regions of Croatia. Collecting methods included the use of entomological nets and handpicking specimens from walls of small buildings or wells, or from the riparian vegetation near springs and headwater streams. In one spring (Pecki spring, Banovina region) () we installed 5 pyramid-type emergence traps in 2010 and 2011 to investigate the emergence dynamics of caddisflies (). This investigation is part of a multi-year study on emergence dynamics of aquatic insects in springs and other aquatic habitats in Croatia and the Dinaric karst of the Balkan Peninsula (Bosnia and Herzegovina) (, , , , , M. Kučinić unpublished data). The emergence trapping methodology was presented in detail by and .In pyramid-type emergence traps caddisflies were collected in 1% formaldehyde and thereafter stored in 80% alcohol. All other collected specimens were stored directly in 80% or 96% alcohol. All specimens were deposited in the collections of the first and second authors. The holotype is deposited in the Croatian Natural History Museum in Zagreb.Laboratory work. For the phylogenetic analysis we compiled mtCOI DNA sequence data for 103 specimens from the Chaetopteryx rugulosa group (). We also sequenced several outgroup taxa of varying putative phylogenetic depths including congeneric species (e.g., Chaetopteryx gessneri McLachlan, 1876, Chaetopteryx fusca Brauer, 1857, Chaetopteryx major McLachalan, 1876, Chaetopteryx villosa (Fabricius, 1798)), other members of the tribe Chaetopterygini (Chaetopterygopsis maclachlani (Stein, 1874)), other members of the subfamily Limnephilinae (Limnephilus centralis Curtis, 1834), and members of a different subfamily of Limnephilidae (e.g. Metanoea rhaetica Schmid, 1955, Drusus alpinus (Meyer-Dür, 1875), Drusus rectus McLachlan, 1868).Systematic presentation follows . The terminology and morphological assessment of the Chaetopteryx rugulosa group follows , , , , , , and . Comparative assessments of morphological features of Chaetopteryx bucari were based on the other specimens collected in Croatia (Chaetopteryx rugulosa rugulosa, Chaetopteryx marinkovicae) or based on literature (e.g., Chaetopteryx rugulosa schmidi, Chaetopteryx rugulosa mecsekensis, , ). Morphological features of genitalia of Chaetopteryx bucari were analysed from 84 specimens (40 males and 44 females).The mitochondrial COI barcodes were generated at the Canadian Centre for DNA Barcoding, University of Guelph, Canada. Standard barcoding protocols for DNA extraction (), PCR amplification and COI sequencing (, ) were used. Full-length COI-5P DNA barcodes were amplified using C_LepFolF/C_LepFolR (, ) and LCO1490/HCO2198 () primer sets. COI barcodes and detailed specimen information can be found in the Barcode of Life Data Systems (BOLD; http://www.boldsystems.org/) () within the project “Chaetopteryx of Europe.” Unpublished COI barcodes of additional Chaetopteryx outgroups were provided by Karl Kjer, Rutgers University, USA (). The sequence of Limnephilus centralis Curtis, 1834 was taken from ().Phylogenetic analysis. Sequences were edited manually and aligned using the program Geneious 5.4 (). The final alignment was 617 base pairs (bp) long. Bayesian phylogenetic analyses were performed using the Markov chain Monte Carlo method (B/MCMC) using MrBayes 3.2 (, ). We selected the best-fitting models of DNA substitution using Akaike information criterion (AIC) implemented in jModelTest 0.1.1 (, ). jModelTest indicated a general time reversible model () with a significant proportion of invariant sites (I=0.607) and with gamma-distributed rate heterogeneity (α=1.049) (GTR+I+G). We conducted Bayesian tree construction with 6 chains, 2 independent runs and 8 million generations. Trees were sampled every 1000th generation. The first 9000 generations were discarded as burn-in. We plotted the log-likelihood scores of sample points against generation time using Tracer 1.5 () to ensure that stationary was achieved after the first 9000 generations by checking whether the log-likelihood values of the sample points reached a stable equilibrium plateau. We used the remaining trees with average branch lengths to create a 50% majority-rule consensus tree with the sumt option of MrBayes. Posterior probabilities (pp) were obtained for each clade, whereby pp≥0.95 indicated significant support for clades. Finally, we also calculated the uncorrected pairwise distances between individuals based on mtCOI sequences using MEGA 5.1 ().Microphotography and measuring. Microphotographic images of genitalia and forewing measurements were taken using a Leica Wild MZ8 stereomicroscope and Olympus SP-500 UZ digital camera. The photographs were processed with the Olympus Quick Photo Camera 2.2. software package. Geographic coordinates and altitudes of sampling localities were recorded with a Garmin ‘Oregon 450' GPS device. […]

Pipeline specifications

Software tools BOLD, Geneious, MrBayes, jModelTest, MEGA
Applications Phylogenetics, WGS analysis