DOI: 10.5061/dryad.95x69p8hf
title: Random forests for predicting species identity of forensically important blow flies (Diptera: Calliphoridae) and flesh flies (Sarcophagidae) using geometric morphometric data: proof of concept

description: Wing shape variation has been shown to be useful for delineating
 forensically important fly species in two Diptera families: Calliphoridae
 and Sarcophagidae. Compared to DNA-based identification, the cost of
 geometric morphometric data acquisition and analysis is relatively much
 lower because the tools required are basic, and stable softwares are
 available. However, to date, an explicit demonstration of using wing
 geometric morphometric data for species identity prediction in these two
 families remains lacking. Here, geometric morphometric data from 19
 homologous landmarks on the left wing of males from seven species of
 Calliphoridae (n=55), and eight species of Sarcophagidae (n=40) were
 obtained and processed using Generalized Procrustes Analysis. Allometric
 effect was removed by regressing centroid size (in log10) against the
 Procrustes coordinates. Subsequently, principal component analysis of the
 allometry-adjusted Procrustes variables was done, with the first 15
 principal components used to train a random forests model for species
 prediction. Using a real test sample consisting of 33 male fly specimens
 collected around a human corpse at a crime scene, the estimated percentage
 of concordance between species identities predicted using the random
 forests model and those inferred using DNA-based identification was about
 80.6% (approximate 95% confidence interval = [68.9%, 92.2%]). In contrast,
 baseline concordance using naive majority class prediction was 36.4%. The
 results provide proof of concept that geometric morphometric data has good
 potential to complement morphological and DNA-based identification of blow
 flies and flesh flies in forensic work. 
description: The fly training set samples were taken from archived fly
 collection at the Molecular Genetics Laboratory MP2, University of
 Malaya. The fly test samples were collected from a
 murder crime scene in 2016 at the state of Selangor, Malaysia, where the
 victim was estimated to have been dead for more than 24 hours. The
 collected flies were anesthetised in ethyl acetate in a covered
 bottle. The left wing of each fly specimen was detached
 after overnight relaxation, mounted onto a glass slide using euparal as
 the mounting medium, and covered with a coverslip. The slides were left
 overnight at 56°C to clear out bubbles. Wing images were captured using a
 digital camera (20X magnification) attached to a binocular microscope
 (Motic Microscope 2.0, China). For each specimen, coordinate data from 19
 homologous landmarks on the left wing image were recorded by a single
 person (NADMP), using the tpsDig 2.0 (Version 2.17) software and saved in
 tps file format. Sequence data from the COII gene were
 obtained from eight Sarcophagidae test samples, and five out of 26 of the
 Calliphoridae test samples. DNA was extracted from two legs of a specimen
 using QIAamp® DNA and Blood Mini Kit (Qiagen, USA). For detailed molecular
 protocols, see the associated publication. For
 processing and analysing geometric morphometric data, we used R Version
 3.2.1. General Procrustes Analysis (GPA) (geomorph R package, Version
 3.0.3) was applied to two separate data sets: one containing only the
 training samples (for inspection of patterns of shape variation within and
 among species), and another containing both the training and the test
 samples (for prediction using random forests).  Linear
 regression was applied on the resultant Procrustes coordinate variable
 against the logarithm (base 10) of the centroid size was done to remove
 potential effects of wing shape allometry. The data were subsequently
 transformed to uncorrelated principal component scores using principal
 component analysis (PCA). 
description: The data set can be used as it is. See the README.txt file for
 descriptions of file contents.
description: # Random forests for predicting species identity of forensically important
 blow flies (Diptera: Calliphoridae) and flesh flies (Sarcophagidae) using
 geometric morphometric data: proof of concept
 Manuscript title: Random
 Forests for Predicting Species Identity of Forensically Important Blow
 Flies (Diptera: Calliphoridae) and Flesh Flies (Diptera: Sarcophagidae)
 using Geometric Morphometric Data: Proof of Concept Authors: T.F.Khang,
 N.A.D. Mohd Puaad, S.H.Teh, Z. Mohamed. Email:
 tfkhang[at]um[dot]edu[dot]my or zulq[at]um[dot]edu[dot]my Journal: Journal
 of Forensic Sciences Date: 17 October 2020 ## Description of the data and
 file structure 1. Image_data This folder contains three subfolders. From
 these images, landmark coordinate data were taken using the tpsDIG2
 software. The folder Calliphoridae contains JPEG images of the left wing
 taken from seven species. The folder Sarcophagidae contains JPEG images of
 the left wing taken from eight species. The folder Test samples contains
 JPEG images of the left wing taken from fly samples collected at a murder
 crime scene. 2. tpsdata This folder contains 16 subfolders. One folder
 ("test_samples") contains the tpsdata for the test samples from
 the folder "Test samples" in Image_data. The remaining
 subfolders are organised according to species, and they contain text files
 in .TPS extension generated from the JPEG images in Image_data folder. 3.
 DNA_data This folder contains four files: (i) IQtree_result.NWK - The
 inferred maximum likelihood phylogenetic tree (Fig.2) using IQ-TREE, in
 newick file format. (ii)translatorX_result.FAS - A fasta file containing
 the multiple sequence alignment result obtained using the translatorX
 server. (iii) trimmed_MSA_data.FAS - A fasta file containing the multiple
 sequence alignment of the sequences used in Fig. 2 that has been manually
 trimmed at the right end. (iv) sequence_data_partition.PART - a text file
 containing instructions for IQ-TREE to apply different DNA-substitution
 models for different codon positions of the multiple sequence alignment
 file "trimmed_MSA_data.FAS". In principle, one should be able to
 reproduce the tree in IQtree_result.NWK using the files (ii)-(iv) using
 the Stable Release Version 1.6.12 (August, 2019) of IQ-TREE. Slight
 variations are possible if the job is submitted to the webserver at
 http://iqtree.cibiv.univie.ac.at/ since it may run on some later version.
 4. GUIDE_files This folder contains files associated with the GUIDE
 program. (i) fly_gpa_pca_allo.csv - A csv file containing the training and
 the test samples and 15 principal component scores based on the residuals
 from regressing Procrustes coordinates against log10 of centroid size.
 (ii) fly_gpa_pca_allo.DSC - The GUIDE description file which specifies how
 the variables in fly_gpa_pca_allo.csv should be used by GUIDE. (iii)
 flywings_rf.IN - A text file containing the input parameters of the GUIDE
 program used for the analysis. 5. mainscript.R This file is the R script
 used to process the raw data and run the analyses described in the
 manuscript.