Computational protocol: Four New Bat Species (Rhinolophus hildebrandtii Complex) Reflect Plio Pleistocene Divergence of Dwarfs and Giants across an Afromontane Archipelago

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Protocol publication

[…] Chromatograms were visualized and aligned using BioEdit v7.0.1 . The resultant datasets comprised: 1) control region - 33 taxa and 525 bp (101 bp parsimony-informative); 2)12S - 27 taxa and 368 bp (23 bp parsimony-informative); 3) cytochrome b - 20 taxa and 535 bp; and 4) Chd1 - 20 taxa and 778 bp (15 bp parsimony-informative). Sequences were deposited in GenBank under the following accession numbers: JQ929202–JQ929301. Not all individuals would amplify for all gene fragments resulting in many missing sequences. Thus the datasets were not combined, but analysed separately. Phylogenetic hypotheses were estimated using both parsimony and Bayesian analyses for the control region, 12S and Chd1 data sets. Parsimony analysis was done using the heuristic search option, with all site changes weighted equally in PAUP* 4.0b10 . Tree-bisection-reconnection (TBR) branch swapping was used and starting trees were obtained via 100 random stepwise additions. Bootstrap support was calculated using 1000 replicates. Calculation of uncorrected (not based on a particular model of evolution) pairwise genetic distances for each gene was conducted using PAUP* 4.0b10.The most appropriate model of molecular evolution was determined for each gene fragment using the Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC) as implemented in jModelTest 0.1.1 . The model parameters for each dataset were used in subsequent analyses.Bayesian analyses (BA) were conducted using Mr Bayes 3.1.2 . Four incrementally heated chains were run for three million generations, with parameters sampled every 1000 generations. Convergence of the MCMC chains was assessed by inspecting whether the standard deviation of split frequencies approached zero and the potential scale reduction factor (PSRF) reached 1.0 for all parameters. We also investigated the convergence using Tracer v 1.4.1 . A 25% burnin was used and the 50% majority rule consensus tree was constructed from the remaining tree data. [...] To estimate divergence dates, the cytochrome b data set was analysed in BEAST 1.4 . We included additional R. hildebrandtii sequences from Pafuri and Tanzania (EU436676, ) as well as other African Rhinolophus species downloaded from GenBank: R. darlingi (EU436675); R. eloquens (EU436677); R. fumigatus (FJ457614); R. landeri (EU436668, FJ457612) and R. ruwenzorii (EU436679 and FJ185203). In order to be able to use fossil calibration points we incorporated sequences downloaded from GenBank for six species in the family Hipposideridae, sister to the Rhinolophidae: H. armiger (DQ865345), H. caffer (EU934461), H. cyclops (EU934466), H. gigas (EU934469), H. pratti (EF544427) and H. ruber (EU934485). jModelTest was used to determine the most appropriate model of evolution. BEAUTi was used to set model parameters and the monophyly of the ingroup (Rhinolophus) was constrained during analysis. We used the HKY model with empirical base frequencies and the substitution rate was not fixed. The Yule speciation process was used as the tree prior and a relaxed uncorrelated lognormal molecular clock model was selected. A normal distribution for the tree prior for the node delimiting time to the most recent common ancestor was selected. As calibration points we followed Teeling et al. (2003) and Eick et al. (2005) and used a minimum of 37 Mya and maximum of 55Mya for the split between the Rhinolophidae and the Hipposideridae , . The MCMC chain was run for 20 million generations, with parameters logged every 1000 generations.Results were evaluated using Tracer v1.4.1 . The Effective Sample Size (ESS) values were >200 for all parameters, suggesting the MCMC run was sufficient and independent samples were incorporated to obtain valid parameter estimates . Trees were collated using TreeAnnotator 1.6 where mean heights and a burnin of 10% were selected. [...] In additional to morphometric analysis of continuous characters, we scored the following qualititative, craniodental characters: the presence, position (external or within toothrow) and relative size (small or “tiny”) of the small anterior upper premolar and the relative height of the anterior nasal swelling and sagittal crest in lateral view.For morphometric analysis, adult rhinolophids were selected based on degree of tooth wear, and extent of ossification of epiphyses in the finger bones. We used two morphometric approaches: analysis of traditional linear measurements as well as landmarks placed on dorsal and lateral images of crania.The following 12 cranial measurements were taken to the nearest 0.01 mm using Mitutoyo digital callipers with accuracy of 0.01 mm: greatest length of skull measured dorsally from occiput to anterior point of skull (GSL); condylo-incisive length from occipital condyles to front of incisors (CIL); condylocanine length from occipital condyles to front of canines (CCL); zygomatic width, the greatest distance across the zygoma (ZW); mastoid width, the greatest distance across the lateral projections of the mastoid processes (MW); width of maxilla between outer edges of M3 (M3M3); braincase width measured at dorsal surface of posterior root of zygomatic arches (BCW); least interorbital width between orbits (IOW); upper toothrow length from anterior surface of C to posterior surface of M3 (CM3); greatest width across anterior lateral nasal inflations (NW); length from occipital condyles to front of nasal inflations (NL); and height of nasal inflation directly above the anterior cingulum of M2 (NH) , . Principal component analysis (PCA) and canonical variates analysis (CVA) was carried out on log-transformed variables using the programme XLSTAT version 2008.2.03 .A Sony Cybershot DSC-H2 digital camera (6 megapixel; 12× optical zoom and ×2 converter; macro function), mounted on a tripod at a fixed distance of 20 cm from the skull (which was always mounted on graph paper), was used to take dorsal and lateral images for 22 skulls from the same sample used for linear measurements.Landmark placement and further analyses were performed using the thin plate spline (TPS) series of programmes. The programme tpsDig version 2.1 was used to capture landmarks in two dimensions for dorsal (13 landmarks) and lateral (12 landmarks) views (see and for position of landmarks on lateral and dorsal images respectively). The programme tpsRelw version 1.42 was used to conduct a Generalised Procrustes Analysis or GPA (Generalised Least Squares, GLS, ), which serves to translate, rotate and scale the landmark configurations, and produces a consensus configuration for the entire suite of specimens in the analysis via a series of iterations. GPA residuals are further decomposed into both non-uniform (non-affine), and uniform (affine) shape components. Non-affine shape expresses localized shape changes, and is represented by the weights matrix, W, of partial warp scores. Affine shape expresses shape changes that affect the entire configuration (i.e. dilation or sheer), and this component is represented by two vectors, U1 and U2. Together, U+W represent total shape. Relative warps analysis performs a PCA of the covariance matrix of the total shape matrix (U+W).For introductions to geometric morphometrics and its application to mammalian systematics see , , , , . […]

Pipeline specifications

Software tools BioEdit, PAUP*, jModelTest, BEAST
Application Phylogenetics
Diseases Dwarfism, Gigantism