SVEN O. KULLANDER
Department of Zoology, Swedish Museum of Natural History, PO Box 50007, SE-104 05 Stockholm, Sweden
MD. MIZANUR RAHMAN
Department of Zoology, University of Dhaka, Dhaka-1000, Bangladesh:
MICHAEL NORÉN
Department of Zoology, Swedish Museum of Natural History, PO Box 50007, SE-104 05 Stockholm, Sweden
ABDUR ROB MOLLAH
Department of Zoology, University of Dhaka, Dhaka-1000, Bangladesh:
DNA barcode, Freshwater, Morphometrics, Phylogeny, Taxonomy
Zoology Department, University of Dhaka, Dhaka
Conservation and Biodiversity
Specimens are kept in the following collections: NRM, Swedish Museum of Natural History, Stockholm; DU, Zoology Department, University of Dhaka, Dhaka. Some comparative material is kept in the Natural History Museum, London (BMNH). Measurements were taken with digital callipers to a precision of 0.1 mm. Counts and measurements were made according to Fang (1997) and Kullander (2015). Colour pattern terminology follows Fang (1998) with modifications for special markings as explained by Kullander (2015). Horizontal stripes are identified by alphanumeric annotations. Fin-ray and vertebral counts were taken from X-radiographs made with a Philips MG-105 low voltage X-ray unit and Kodak EDR2 plates. Abdominal vertebrae counts include the Weberian apparatus (assumed to contain four centra). Statistics were calculated using SYSTAT v. 13 (Systat Software, 2009), except that the principal component analysis (PCA) of measurements was made using a separate procedure for component shearing, partially out multivariate-size residues from the second and further components as described by Humphries et al. (1981). The PCA was made with log-transformed measurement data to a tenth of a millimetre in a covariance matrix, and without rotation. An asterisk (*) marks counts from the holotype.Species delimitation was tested using the standard mitochondrial 5’ cytochrome oxidase subunit I (COI, also known as mt-CO1 or COX1) DNA barcoding fragment, as suggested by the Barcode of Life consortium (Steinke & Hanner, 2011).DNA was extracted using a Thermo Scientific™ KingFisher™ Duo (Thermo Fisher Scientific) fully automated liquid-handling instrument, with the Thermo Scientific KingFisher Cell and Tissue DNA Kit (Thermo Fisher Scientific) and recommended protocol. The COI fragment was amplified using the fish barcoding primers Fish-F1 and Fish-R1 (Ward et al. 2005). PCR was performed with the puReTaq Ready-To-Go PCR kit (Amersham Biosciences AB, Uppsala, Sweden). PCR cycling: 94°C 4min; 35 * (94°C 30s; 52°C 30s; 72°C 30s); 72°C 8min). PCR products were checked on agarose gel and purified by adding 5μL of a mix consisting of 20% Exonuclease I (EXO) and 80% FastAP Thermosensitive Alkaline Phosphatase (Fermentas/Thermo Fischer Scientific, Gothenburg, Sweden) to each 25 μL PCR reaction, incubated at 37°C for 30 minutes, then heated to 80°C for 15 minutes.Sequencing of both strands of all fragments was carried out by Macrogen Europe (Amstelveen, Holland). All sequences were proofread and assembled using the software Geneious R8 (Kearse et al., 2012).651 base pairs from the 5’ end of the COI gene of three individuals of Danio annulosus, including the holotype, and all other available species of chain danios were sequenced. Additional sequences were obtained from GenBank and the supplementary dataset S1 in Collins et al. (2012). A total of 40 sequences representing 14 species were aligned using the software Geneious with the MUSCLE (Edgar, 2004) plug-in, and edges were manually trimmed to a total alignment length of 651 base pairs. DNA sequences and vouchers are listed in Table 1.Sequences prefixed with RC were taken from Collins et al. (2012). They submitted their COI sequences to the Barcode of Life Database (BOLD). However, it turns out that they, probably by mistake have uploaded their Rhodopsin data (labelled as Rhodopsin) instead of the COI data to BOLD. As a result, Collins et al. (2012) COI sequences are available neither from BOLD nor GenBank, and they have no GenBank accession number. Although the COI sequences in Collins et al. (2012) are not published in GenBank, are based on aquarium specimens, and presently are only available as supplementary information with Collins et al. (2012: their Dataset S1, doi:10.1371/journal.pone.0028381.s004), we elected to include them, as they correspond to those identified from wild material. Species identifications based on photos in BOLD had been made previously by Kullander (2015). Phylogenetic analysis was performed using the software MrBayes v3.3 (Huelsenbeck & Ronquist, 2001; Ronquist et al., 2014). Data were partitioned according to codon position (first, second, third) and parameters estimated separately for each partition. Devario was designated outgroup in all analyses. The GTR + Γ + I model was used as suggested by ModelTest (Posada & Crandall, 1998). Samples were taken every 1000 generations, and the first 25% of samples were discarded as ‘burn-in’. The analysis was run for ten million generations. Convergence was confirmed with MrBayes (average standard deviation of split frequencies <0.004), and the software Tracer v1.6 (Rambaut et al., 2014) (effective sample size = 8612). Unless otherwise stated all distances are uncorrected pairwise p-distances, as recommended by Srivathsan & Meier (2011). The software Geneious with the plug-in Species Delimitation (Masters et al., 2011) was used to obtain “corrected” estimates of pairwise p-distance and for calculating the probability of reciprocal monophyly under a model of random coalescence.
Zootaxa 3994 (1): 053–068
Journal