First molecular phylogeny of Paralucilia Brauer & Bergenstamm, 1891 (Insecta, Diptera, Calliphoridae): A preliminary approach

Accepted Manuscript Title: First molecular phylogeny of Paralucilia Brauer and Bergenstamm, 1891 (Insecta, Diptera, Calliphoridae): a preliminary approach Authors: Ta´ıs Madeira-Ott, Marco A.T. Marinho, Juliana Cordeiro, Patricia J. Thyssen PII: DOI: Article Number:

S0001-706X(19)30559-5 https://doi.org/10.1016/j.actatropica.2019.105096 105096

Reference:

ACTROP 105096

To appear in:

Acta Tropica

Received date: Revised date: Accepted date:

24 April 2019 15 July 2019 15 July 2019

Please cite this article as: Madeira-Ott T, Marinho MAT, Cordeiro J, Thyssen PJ, First molecular phylogeny of Paralucilia Brauer and Bergenstamm, 1891 (Insecta, Diptera, Calliphoridae): a preliminary approach, Acta Tropica (2019), https://doi.org/10.1016/j.actatropica.2019.105096 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1 First molecular phylogeny of Paralucilia Brauer & Bergenstamm, 1891 (Insecta, Diptera, Calliphoridae): a preliminary approach

Taís Madeira-Otta, Marco A. T. Marinhob, Juliana Cordeirob, Patricia J. Thyssenc

Department of Microbiology and Parasitology, Federal University of Pelotas – UFPel, PC 96010-

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900, Pelotas, Rio Grande do Sul, Brazil

Department of Ecology, Zoology and Genetics, Federal University of Pelotas – UFPel, PC 96010-

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b

900, Pelotas, Rio Grande do Sul, Brazil c

Department of Animal Biology, University of Campinas – UNICAMP, PC 13083-862,

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Campinas, São Paulo, Brazil

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Corresponding author:

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Taís Madeira-Ott

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Department of Microbiology and Parasitology, IB, Federal University of Pelotas (UFPel), Campus Universitário Capão do Leão, Capão do Leão, State of Rio Grande do Sul, 96160-000, Brazil.

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e-mail: [email protected]

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Graphical abstract

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2

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Highlights

The first phylogeny of Paralucilia is presented.

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At least, three out of five Paralucilia species currently valid (sensu Mello (1996)) are well

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Here we record two polymorphic morphotypes of Paralucilia fulvinota: P. fulvinota

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defined in this study: P. fulvinota, P. pseudolyrcea and P. paraensis.

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"Amazonia" and P. fulvinota "Atlantic Forest".

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Abstract. Paralucilia Brauer & Bergenstamm, 1891 (Diptera, Oestroidea, Calliphoridae) is a small genus of blowflies restricted to the Neotropical region, which is commonly reported on decaying corpses and vertebrate carcasses. The number of species currently assigned to this genus

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and their denominations are contentious, with either three or five species recognized by different authors. This taxonomic instability results in a lack of consensus in species determination, making it impossible to compare results from different studies as well as to elaborate confident taxonomic keys. In order to solve some of the most commonly reported taxonomic conflicts within this genus, to the best of our knowledge, this study presents the first phylogenetic hypothesis for the relationships among Paralucilia species, based on molecular analysis of the COI, ITS2, 28S, and 16S genes. Maximum parsimony, maximum likelihood, and Bayesian inference analyses were

3 used for phylogenetic reconstruction and divergence time estimation analyses. Intra- and interspecific genetic distances were calculated among species using the COI dataset. The results showed that at least three of the five currently accepted species are well defined: P. fulvinota, P. pseudolyrcea, and P. paraensis, however, a significant level of intraspecific variation was observed in P. fulvinota. These findings will assist future revisions of the description, classification, and distribution of species of Paralucilia, as well as in the elaboration of taxonomic keys. Additionally, we show that it is possible to clarify the evolutionary history of this Neotropical

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genus using supplementary evidence such as morphology and molecular data.

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Key words. Blow flies; Oestroidea; Taxonomy impediment; Necrophagous; Diversification.

1. Introduction

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Calliphoridae are dipterans (Arthropoda, Insecta) with worldwide distribution (Shewell,

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1987). There are currently more than 1,500 known species (Yeates et al., 2007; Pape et al., 2011),

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of which approximately 130 have been recorded in the Neotropical region (Amorim et al., 2002;

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Whitworth, 2010, 2012, 2014). They present a wide variety of feeding habits and behaviors,

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including saprophagy and parasitism (Ferrar, 1987). Many species are observed in anthropized environments (Greenberg, 1971), and in addition to annoyance they may cause damage to the

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health of humans and other animals by acting as vectors of numerous pathogens, or by causing obligatory or facultative myiasis (Guimarães and Papavero, 1999). In this context, a significant

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amount of resources is spent annually in an effort to eliminate or control populations of synanthropic species (Grisi et al., 2014).

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Necrophagous blowflies play an important role in the forensic field as they are among the

first insects to colonize carrion, and usually the most abundant flies found feeding on carrion (Marcondes and Thyssen, 2017). Consequently, taking into account the biology, ecology, and geographical distribution of several species of Calliphoridae (Linhares and Thyssen, 2012), in particular of the genera Calliphora Robineau-Desvoidy, 1810, Chrysomya Robineau-Desvoidy,

4 1830, Cochliomyia Townsend, 1915; Hemilucilia Brauer, 1895, Lucilia Robineau-Desvoidy, 1830,

and Paralucilia Brauer & Bergenstamm, 1891, it is possible to estimate the post-mortem interval, to identify and associate suspects with a crime, to indicate cases of negligence against incapacitated persons, and to infer cause of death (e.g., Carvalho et al., 2000; Pujol-Luz et al., 2006; Kosmann et al., 2011; Thyssen et al., 2012; Souza et al., 2013; Thyssen et al., 2018).

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Paralucilia (Diptera, Calliphoridae, Chrysomyinae) is a small genus of blowflies restricted to the Neotropical region (Mariluis et al., 1994; Dear, 1985; Kosmann et al., 2013). Discrepancies,

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with respect to the number of valid species, have been reported since the first comprehensive review of the genus conducted by Mello (1969). Although the current revision (Mello, 1996) recognizes five valid species – Paralucilia borgmeieri (Mello, 1969), P. fulvinota (Bigot, 1877),

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P. nigrofacialis (Mello, 1969), P. paraensis (Mello, 1969), P. pseudolyrcea (Mello, 1969) – it is

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intriguing to note that several studies associated with Paralucilia since then seem to be unaware

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of the discussions conducted by Mariluis et al. (1994) on the mistakes made by Mello (1969) and

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Dear (1985), which were also observed by Mello (1996). In addition, polymorphisms within P.

and the other “darker.”

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fulvinota have been reported by Mariluis et al. (1994), who recognized two forms, one “lighter”

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Reliable taxonomic information is necessary for consensus and stability in nomenclature classification systems. In particular, in the designation of Paralucilia species it has been observed

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recurrent problems considering the in situ and ex situ species surveys (e.g., Pape et al., 2004; Bermudéz, 2007; Amat, 2009; Barbosa et al., 2014; Aguirre & Barragán. 2015), biodiversity and

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population fluctuation studies (e.g., Esposito et al., 2010; Ferraz et al., 2010; Batista-da-Silva et al., 2011; Amat et al., 2016), synanthropy (e.g., Baumgartner and Greenberg, 1985; Vianna et al., 1998; Olea et al. 2012), bionomy and description of immature stages (e.g., Greenberg and Szyska, 1984; Barros-Souza et al., 2012; Silva et al., 2018), forensic entomology (e.g., Pujol-Luz et al.,

5 2006; Pastrana and Echeverry, 2011; Armani et al., 2015; Ramos-Pastrana et al. 2018), and also in the dichotomic keys available (e.g., Dear, 1985; Mello, 2003; Kosmann et al., 2013). In order to shed a light in some of the most common taxonomic conflicts reported in the literature, this study proposes the first phylogenetic hypothesis for species of Paralucilia, based on molecular evidence, using sequences of the COI, ITS2, 28S, and 16S genes. Estimates of

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genetic distances and time of divergence are also discussed.

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2. Material and Methods

2.1 Taxonomic sampling

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Specimens of Paralucilia were sourced from surveys conducted in various locations in

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Brazil and Colombia (Table 1). Hand held entomological nets and baited traps (Shannon and Van

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Someren-Rydon) were used for the collection of the adult Diptera, using fish, beef liver, or chicken

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gizzard as bait. In the laboratory, identification of individuals was conducted using taxonomic keys

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and species descriptions (Mello, 1969; Dear, 1985; Mariluis et al., 1994; Mello, 1996). The holotype of the species P. paraensis, P. pseudolyrcea, P. nigrofacialis and P. borgmeieri was

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analyzed to confirm the identification of the species. Sampled specimens were stored at -20°C in absolute ethanol.

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Six morphotypes of Paralucilia were selected for this study: (i) P. nigrofacialis; (ii) P.

paraensis; (iii) P. pseudolyrcea – all sensu Mello (1996); (iv) P. pseudolyrcea d.p. (determined a

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posteriori, sensu Mello 1996 – the sample had previously been identified as P. xanthogeneiates sensu Dear (1985), see Table 1); (v) and (vi) P. fulvinota "darker morphotype" and "lighter morphotype," respectively, sensu Mariluis et al. (1994).

2.2 Molecular techniques and sequence editing

6 Genomic DNA was extracted from one to three legs of each individual using the DNeasy Blood and Tissue Kit™ (Qiagen, Valencia, CA, USA) following the manufacturer's protocol. Voucher specimens were deposited at the ZUEC Museum (Museum of Zoology ‘Adão José Cardoso’, University of Campinas, São Paulo, Brazil). Four molecular markers were amplified and subsequently sequenced: (i) the whole region

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of the Internal Transcribed Spacer 2 (ITS2); (ii) the 5′ region of the nuclear large ribosomal DNA subunit (28S rDNA), both from the nuclear ribosomal DNA cluster, (iii) the 5′ region of the

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cytochrome c oxidase subunit I (COI), and (iv) the 3′ portion of the mitochondrial large ribosomal DNA subunit (16S rDNA), both from the mitochondrial genome.

PCR amplification reactions were conducted with 1x PCR buffer, 0.6 μL (0.2 mM) of

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dNTPs, 1.25 μL (3.0 mM) of MgCl2, 1.0 μL (0.4 pmol/μL) of each forward and reverse primer,

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1.25 U of Taq DNA polymerase, 15.6 μL of water and 2 μL (25 ng) of extracted DNA for a 25 μL

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reaction. DNA was quantified using a NanoVue™ spectrophotometer (GE Health Care).

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Corresponding primers and annealing temperatures are shown in Supplementary Material A. PCR

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products were visualized by electrophoresis at 80 V in 1x TAE 1% agarose gels stained with GelRed™ (Biotium Inc., CA, USA). The amplified fragments were sized by comparison with Low

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DNA Ladder (Life Technologies, CA, USA). Amplicons were purified using the QIAquick™ PCR Purification (Qiagen, Valencia, CA,

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USA) kit following the manufacturer's protocol. Sanger sequencing was carried out by an outsourced company. Nucleotide sequences were edited using the Staden Package Gap4™

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software (Staden, 1996).

2.3 Phylogenetic analysis Sequences of Neotropical Chrysomyinae species obtained from GenBank were included in the analyses: Chloroprocta idioidea Robineau-Desvoidy, 1830, Cochliomyia macellaria

7 (Fabricius,

1775),

Compsomyiops

fulvicrura

(Robineau-Desvoidy,

1830),

Hemilucilia

semidiaphana (Rondani, 1850), and Chrysomya megacephala (Fabricius, 1794). Chrysomya megacephala was selected as an out-group based on previous Chrysomyinae phylogenetic studies conducted by Singh and Wells (2011). GenBank accession numbers are listed in Table 1. Maximum parsimony (MP) analysis was performed in TNT™ v1.5 (Goloboff and

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Catalano, 2016) using the ‘new technology search’ option with the following parameters: search at level = 15, initial add seqs = 10, find minimum tree length = 10 times. Two analyses were

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performed taking into account the treatment of gaps (a) as missing data (MD), and (b) as a fifth character state (5C). Node supports were evaluated by bootstrap resampling (BS = 500 replicates). Subsequently, maximum likelihood (ML) and Bayesian inference (BI) analyses were

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conducted using GARLI™ v2.01 (Zwickl, 2006) and MrBayes™ v3.2.6 (Ronquist et al., 2012),

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respectively.

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For ML and BI analyses, nucleotide model selection and definition of the best partition

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scheme was performed with the PartitionFinder 2.1 software (Lanfear et al., 2016). A priori, each

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molecular marker was treated as a partition with distinct evolutionary rates, and the COI region was then further partitioned according to codon position (1st, 2nd and 3rd positions). The partitioning

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scheme suggested by the software was as follows: GTR+I+G (COI 1st+2nd), GTR+G (COI 3rd), GTR+G (ITS2) and HKY+I (28S+16S). For the phylogenetic analyses, models applied to each

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partition were considered unlinked, with parameters inferred from different distributions [GARLI: linkmodels = 0, subsetspecificrates = 1; MrBayes: prset applyto = (all) ratepr = variable, unlink

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statefreq = (all) revmat = (all) shape = (partitions 1,2,3) pinvar = (partitions 1,4)]. Partitioned ML analysis was performed as follows: 10 independent replicates, 100,000,000

generations, 10 individuals per generation, standard parameters for automated shutdown. Phyutility (Smith and Dunn, 2008) was used to generate the consensus tree and measure the stability of the branches/nodes by BS (500 replicates).

8 BI analysis was performed for 10,000,000 generations (nchains = 6; sample frequency = 1000) and burn-in adjusted to 25% after convergence verification. Node supports were analyzed by their posterior probabilities (PP) in the 50% extended majority-rule consensus tree, in which clades with less than 0.5 of PP are added to the final tree if they do not contradict previously established clades (option “sumt contype = allcompat” in MrBayes).

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Divergence times were estimated using BEAST™ v2.4.4 (Bouckaert et al., 2014), following the same configurations used for the BI analysis conducted in MrBayes. Additional

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priors were set as follows: a coalescent Yule model for the tree prior, with a uniform distribution prior for relative birth rates, gamma shape parameters with exponential priors, and all rates with priors following a gamma distribution using values provided by the software.

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A relaxed log-normal clock model was used to estimate the divergence times, with an

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exponentially distributed mean (ucldMean.c; mean = 10) and a gamma distributed standard

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deviation. Two secondary calibrations were used on two nodes in the tree, obtained from the

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Cerretti et al. (2017) study, and in each case a normal distribution prior was used to estimate the

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Time to Most Recent Common Ancestor (TMRCA), assuming that all groups are monophyletic. These calibrations comprised: (i) a node common to all Chrysomyinae, modeled with a normal

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distribution with a mean of 17.5 and σ = 1.8 [17.5 ± 4.5 million years ago (mya)] and (ii) a clade comprised [Paralucilia + Cochliomyia + Compsomyiops], modeled with a normal distribution

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with a mean of 9.0 and σ = 1.1 (9.0 ± 3.0 mya). TreeAnnotator v2.4.0 (included in the BEAST package v2.4.4) was used to calculate the maximum clade credibility tree and posterior

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probabilities of nodes based on the 10,000 trees sampled by the MCMC analysis.

2.4 Genetic distances analyses The tree obtained by BI was used to define groups of species for the analysis of intra- and interspecific genetic distances, based on the DNA Barcoding method (Hebert et al., 2003). COI

9 sequences were analyzed in the MEGA6 software (Tamura et al., 2013) with the Neighbor-Joining method (NJ) using the K2-p substitution model, similar to the HKY model proposed by jModelTest 2 (Darriba et al., 2012). In order to confirm the number of hypothetical specific clusters in the data set, we used the Automatic Barcode Gap Discovery (ABGD) tool (Puillandre et al.,

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2012).

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3. Results

3.1 Molecular phylogeny

Concatenated data for each of the 14 Paralucilia specimens resulted in a 2,889 bp sequence

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(700 bp for COI, 568 bp for ITS2, 646 bp for 16S and 985 bp for 28S). The phylogenetic

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trees are shown in Supplementary Material – B.

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reconstruction trees resulting from the BI, ML and MP analyses are shown in Fig. 1. Alternative

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The monophyly of Paralucilia is highly supported in the ML (BS = 91.4) and BI (PP = 1)

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analyses, but only moderately supported in the MP analysis (BS-MD = 69; BS-5C = 68). There was low support for the relationship between Paralucilia and the other Chrysomyinae in all the

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different analyses (MP-5C-BS = 3; MP-MD-BS = 4; ML-BS = 36.6; IB-PP = 0.45). Nevertheless, most of the analyses recovered C. fulvicrura as a sister taxon of Paralucilia, as well as Co.

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macellaria as sister taxon of this group. Four highly supported clades for the species of Paralucilia were recovered in all analyses:

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[P. fulvinota "darker morphotype" + P. nigrofacialis], [P. fulvinota "lighter morphotype"], [P. pseudolyrcea + P. pseudolyrcea d.p.], and [P. paraensis]. Paralucilia fulvinota "lighter morphotype", P. fulvinota "darker morphotype" and P. nigrofacialis were grouped in the same clade, supporting the hypothesis that they comprise the same species. Likewise, P. pseudolyrcea

10 was grouped with P. pseudolyrcea d.p.", suggesting that these groups also belong to the same species. In general, [P. pseudolyrcea + P. pseudolyrcea d.p.] was recovered as a sister group of P. paraensis. However, the relationships between P. paraensis and the other groups of the genus remain unclear, with only moderate to low support across the various analyses (ML-BS = 57.8;

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IB-PP = 0.61). This topology changes among the clusters in MP analysis, where P. pseudolyrcea

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was recovered as a sister group of P. fulvinota (MP-MD-BS = 68; MP-5C-BS = 69).

3.2 Divergence time estimation

The BI tree, shown in Fig. 1 A, indicates the Paralucilia species divergence time. The

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divergence between the present species analyzed is dated to approximately 4.37 mya. The

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divergence between [P. fulvinota "darker morphotype" + P. nigrofacialis + P. fulvinota "lighter

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morphotype"] and [P. pseudolyrcea + P. pseudolyrcea d.p.] occurred approximately 3.77 mya.

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The estimated divergence time within each clade is approximately 1.61 mya within [P. fulvinota

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"darker morphotype" + P. nigrofacialis + P. fulvinota "lighter morphotype"], 0.69 mya within [P. pseudolyrcea + P. pseudolyrcea d.p.], and 0.63 mya within [P. paraensis]. The estimated

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divergence time between [P. fulvinota "darker morphotype" + P. nigrofacialis] and [P. fulvinota

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"lighter morphotype"] is approximately 0.75 mya.

3.3 Intra- and interspecific genetic distances

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The lowest interspecific values here found were 0.1% between P. fulvinota “darker

morphotype” and P. nigrofacialis and 0.3% between P. pseudolyrcea and P. pseudolyrcea d.p.. The highest intraspecific value found was 1.3% for P. fulvinota “lighter morphotype” (more results in Supplementary Material – C).

11 The intra- and interspecific genetic distances of each potential species are presented taking into account four hypotheses of species arrangements (Fig. 2): - hypothesis 1: which includes six potential species: P. fulvinota “darker morphotype”, P. fulvinota “lighter morphotype”, P. nigrofacialis, P. pseudolyrcea, P. pseudolyrcea d.p. and P. paraensis;

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- hypothesis 2: which includes five potential species: P. fulvinota "darker morphotype" + P. fulvinota "lighter morphotype", P. nigrofacialis, P. pseudolyrcea, P. pseudolyrcea d.p. and P.

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paraensis;

- hypothesis 3: which includes four potential species: P. fulvinota “darker morphotype” + P. nigrofacialis, P. fulvinota “lighter morphotype”, P. pseudolyrcea + P. pseudolyrcea d.p. and P.

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paraensis.

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- hypothesis 4: which includes three potential species: P. fulvinota “darker morphotype” +

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P. fulvinota “lighter morphotype” + P. nigrofacialis, P. pseudolyrcea + P. pseudolyrcea d.p. and

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P. paraensis;

The ABGD analysis recovered four potential Paralucilia species in our dataset with values

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of maximum intraspecific genetic distances (P) of 0.00278 [(P. fulvinota "darker morphotype" + P. nigrofacialis), (P. fulvinota "lighter morphotype”), (P. pseudolyrcea + P. pseudolyrcea d.p.)

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and (P. paraensis)], and three potential species for values of 0.00464 [(P. fulvinota "darker morphotype" + P. nigrofacialis + P. fulvinota "lighter morphotype"), (P. pseudolyrcea + P.

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pseudolyrcea d.p.) and (P. paraensis)]. Additionally, the DNA barcoding gap value of 1.8% was detected only in hypotheses 3 and 4 (Fig. 4).

4. Discussion

12 The results of this study endorse the monophyly of Paralucilia as published by Singh and Wells (2011), who also compared the relationships of this genus with other genera of Chrysomyinae. In agreement with these authors, the grouping of Paralucilia with the genus Compsomyiops and Cochliomyia is the stronger phylogenetic hypothesis. For the first time, genetic divergence times of Paralucilia species were estimated using

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secondary calibration data obtained from the study of Cerretti et al. (2017). The difficulty of obtaining preserved fossils of insects limits our understanding of the evolutionary past of these

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species and other Calliphoridae species. In the absence of fossil representatives for the family, we used the calibrations of older relationships, and therefore the accuracy of the divergence time between the taxa analyzed may be limited.

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Despite the divergence of this genus from the other Chrysomyinae occurring approximately

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9 mya, the lineages leading to the extant species began to diverge less than 5 mya. Given these

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species are currently found in forest fragments and are apparently sensitive to anthropic

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disturbances (e.g. Baumgartner and Greenberg, 1985; Mariluis et al., 1990; Ururahy-Rodrigues et

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al., 2013; Barbosa et al., 2014; Gadelha et al., 2015; Amat et al., 2016), it is possible that these species evolved exclusively in a forest environment. This recent divergence time, as well as the

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coexistence of these species in certain localities, may be reflected in the low interspecific divergence here observed.

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Although the DNA barcode method proposes an interspecific divergence threshold value

of 3% for identify species (Hebert et al., 2003), studies conducted by Meiklejohn et al. (2012),

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Madeira et al. (2016) and Buenaventura et al. (2018) show that it is possible to distinguish species within the same genus or family with very low genetic variations, i.e., below 3%. Likewise, as also observed by the same authors, it seems plausible to take into account the discrepancies between the highest intraspecific value and the lowest interspecific value (= barcoding gap or reciprocal monophyly) to delimit species. In this way, our results indicate that hypotheses 3 and 4 emerge as

13 the most favorable to represent the number of Paralucilia species here analyzed, even considering the non-evident barcoding gap value. We can assume that these data is reflecting the need for increasing the number of Paralucilia species analyzed. Certainly, the identification of distinct Paralucilia species would improve with a larger species data set combined to different data type, molecular and morphological for example.

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The four highly supported clades of Paralucilia observed in this study (Fig. 1 A) corroborate the morphology-based proposal of Mariluis et al. (1994), including the polymorphisms

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within P. fulvinota. Dear (1985) proposed the existence of only three species, but mistakenly described two species previously described by Mello (1969) and ignored other. From our analyses, we can highlight the mistake of Dear (1985) regarding P. pseudolyrcea, improperly treated in his

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work as P. xanthogeneiates (denominated here as P. pseudolyrcea d.p.). Mello (1996) has correctly

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synonymized P. xanthogeneiates/P. pseudolyrcea d.p. in P. pseudolyrcea, and our data

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corroborates this synonymization, showing that the genetic divergence between them is very low

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Unfortunately, several studies still use the incorrect denomination of P. xanthogeneiates sensu

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Dear (1985), instead of P. pseudolyrcea, which we reinforce to be the only correct denomination, according to the criteria of priority established by the International Code of Zoological

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Nomenclature.

Mello (1996) recognized five valid species within genus Paralucilia, but our results show

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that P. nigrofacialis is not genetically distinct from P. fulvinota "darker morphotype". Although we cannot state about P. nigrofacialis synonymization, due to the low number of specimens

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analyzed in this study, the type-material examined by us does not differ morphologically from P. fulvinota "darker morphotype" (data not shown – specimen not included in the molecular analyses). All this taxonomic issue will only be elucidated with an in-depth morphological examination of the Paralucilia genus type-species and also studies addressing both morphological and molecular aspects of Paralucilia species and populations. There is an ongoing study aimed at

14 this subject and the proper taxonomic treatment, with nomenclatural changes, will only be possible after its conclusion. Nevertheless, this preliminary study provides evidences to which hypothesis should be further pursued and investigated. Paralucilia fulvinota exhibited greater intraspecific distances in comparison to the other species of the genus. The ABGD analysis differentiated P. fulvinota "darker morphotype" and P.

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fulvinota "lighter morphotype" into two distinct species. Remarkably, these two morphotypes are restricted to distinct biogeographical regions comprising the tropical forests of the Chacoan

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(Atlantic Forest) and Brazilian (Amazon Rainforest) subregions respectively, which are separated by the Chacoan dominion (Morrone, 2014). This distribution can be explained by historical connections between these forests, as demonstrated in various biogeographic studies of South

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American species (e.g., Costa, 2003; Batalha-Filho et al., 2013; Peres et al., 2017; Machado et al.,

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2018). The different selective pressures that P. fulvinota has been exposed into distinct

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environments might have led to the morphological and molecular changes observed today. The

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increase in the number of Paralucilia species and specimens sample, representing the distinct

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biogeographic domains in a phylogeographyc study would be of great importance to clarify the genetic diversity, as well as the evolution of the genus Paralucilia.

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Our data clarifies some of the most common taxonomic conflicts within Paralucilia genus reported in the literature and proposes the first phylogenetic hypothesis for species within this

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genus. Our data show that the association of morphological and molecular information in integrative taxonomy studies are crucial to enlighten taxonomic problems (e.g., Grella et al., 2015)

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and phylogenetic relationships (e.g., Heethoff et al., 2011). We hope that the information provided here will aid future studies dealing with revision,

classification, taxonomic keys, and distribution of the species of Paralucilia. Additionally, we show it is possible to clarify the evolutionary history of this Neotropical genus using supplementary evidence such as morphology and molecular.

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4. Conclusions The results obtained in this study showed that at least three out of the five currently valid species of Paralucilia (sensu Mello (1996)) are well defined: P. fulvinota, P. pseudolyrcea and P. paraensis.

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Our analyses regarding P. fulvinota are in agreement with the observations of polymorphic specimens made by Mariluis et al. (1994), which were named by the authors as "lighter" and

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"darker” morphotypes. Here, two morphotypes were evidenced: P. fulvinota Amazonia Rainforest and P. fulvinota Atlantic Forest, corresponding to the "lighter" and "darker” morphotypes sensu Mariluis et al. (1994), respectively. Further studies are needed to investigate the distribution and

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existence of other polymorphisms associated with this species.

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Acknowledgments

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The authors would like to thank Dr. Eduardo Amat (INPA, Brazil) and Luz Miryam

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Gómez-Piñerez (Grupo de Investigación Ciencias Forenses y Salud, Tecnológico de Antioquia, Colombia) for providing specimens for analysis from Santa Marta, Colombia. This study was

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financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001 to the first author. Access registre at Sistema Nacional de Gestão do

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Patrimônio Genético e do Conhecimento Tradicional Associado (SisGen) - #A2CA844. All collections were authorized by the Brazilian Ministry of the Environment (MMA), through the

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Biodiversity Authorization and Information System (SISBIO), processes numbers 54049-1 and 5947-1.

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Zwickl, D.J., 2006. Genetic algorithm approaches for the phylogenetic analysis of large biological sequence datasets under the maximum likelihood criterion. Ph.D. dissertation, The University of Texas at Austin.

22 Table 1. Information on specimens examined and GenBank accession number for all molecular markers analyzed in this study (“-” indicates no information; “MR” indicates museum registration number-catalog; "underline" indicates sequences obtained in this study).

GenBank accession number COI

ITS2

16S

28S

Chloroprocta idioidea

-

JQ246658

EF560180

JQ246708

JQ246603

Cochliomyia macellaria

-

JQ246666

EF560182

JQ246715

JQ246611

Compsomyiops fulvicrura

-

FJ025607

-

FJ025428

FJ025504

Hemilucilia semidiaphana

-

JQ246668

JQ246572

JQ246717

JQ246613

Chrysomya megacephala

-

JQ246662

EF560175

HM016572

JQ246607

P. fulvinota “darker morphotype” ind.1

1 ♂: [Paralucilia fulvinota]; [Extrema, MG, Brazil]; [MR: ZUEC DIP 3363]

MH688062

MH699084

P. fulvinota “darker morphotype” ind.2

1 ♂: [Paralucilia fulvinota]; [Extrema, MG, Brazil]; [MR: ZUEC DIP 3364]

MH716212

P. fulvinota “darker morphotype” ind.3

1 ♀: [Paralucilia fulvinota]; [Cabreúva, SP, Brazil]; [MR: ZUEC DIP 3365]

P. fulvinota “lighter morphotype” ind.1

A

P. nigrofacialis

P. pseudolyrcea d.p. ind.1

SC R

U

MH699111

MH703517

MH699112

A

N

MH699083

MH703525

MH699085

MH703518

MH699118

1 ♀: [Paralucilia fulvinota]; [Novo Airão, AM, Brazil]; [MR: ZUEC DIP 3367]

MH791333

MH699090

MH703520

-

1 ♀: [Paralucilia fulvinota]; [Novo Airão, AM, Brazil]; [MR: ZUEC DIP 3368]

MH987763

MH699088

MH703521

-

1 ♂: [Paralucilia nigrofacialis]; [Cabreúva, SP, MH987764 Brazil]; MR: ZUEC DIP 3366

MH699086

MH703522

MH699114

1 ♀: [Paralucilia xanthogeneiates]; [Extrema, MG, Brazil]; [MR: ZUEC DIP 3371]

MH699113

MH703523

MH699123

ED

M

MH751442

PT

CC E

P. fulvinota “lighter morphotype” ind.2

IP T

Information from material examined

Species

MH987767

23

MH987768

MH699116

MH703526

MH699379

P. pseudolyrcea d.p. ind.3

1 ♀: [Paralucilia xanthogeneiates]; [Extrema, MG, Brazil]; [MR: ZUEC DIP 3373]

MH987769

MH699120

MH703528

MH699380

P. pseudolyrcea ind.1

1 ♀: [Paralucilia pseudolyrcea]; [Extrema, MG, Brazil]; [MR: ZUEC DIP 3369]

MH987765

MH699087

MH703519

P. pseudolyrcea ind.2

1 ♀: [Paralucilia pseudolyrcea]; [Extrema, MG, Brazil]; [MR: ZUEC DIP 3370]

MH987766

MH699089

P. paraensis ind.1

1 ♂: [Paralucilia paraensis]; [Novo Airão, AM, Brazil]; [MR: ZUEC DIP 3374]

MH987770

P. paraensis ind.2

1 ♀: [Paralucilia paraensis]; [Santa Marta, Colômbia]

1 ♂: [Paralucilia paraensis]; [Santa Marta, Colômbia]

A

CC E

SC R

MH699121

MH699122

MH699119

MH703529

-

MH987771

MH699117

MH703530

-

MH987772

MH699115

MH703531

-

A

N

U

MH703524

M

ED

PT

P. paraensis ind.3

IP T

P. pseudolyrcea d.p. ind.2

1 ♂: [Paralucilia xanthogeneiates]; [Extrema, MG, Brazil]; [MR: ZUEC DIP 3372]

24 Fig. 1. Phylogenetic reconstruction trees of Paralucilia genus species using concatenated dataset (COI, ITS2, 16S, 28S). Chrysomya megacephala was used as an out-group. (A) Bayesian inferred time-calibrated phylogeny. The blue bars length of the nodes indicates 95% confidence interval whithin node age. Numbers indicate the posterior probabilities. Scale time unit: 2.5 million years. Highly supported clades of Paralucilia are highlighted: P. fulvinota (cluster 1), P. pseudolyrcea (cluster 2) e P. paraensis (cluster 3). (B) Maximum parsimony phylogenetic reconstruction tree. The strict consensus tree was constructed from 2 (MP-5C) and 12 (MP-MD) equally parsimonious

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trees. Bootstrap support values are shown in the respective nodes. (C) Maximum likelihood and Bayesian inference phylogenetic reconstruction tree. Posterior probability (PP) and bootstrap (BS)

CC E

PT

ED

M

A

N

U

SC R

values are shown next to the respective nodes (PP/BS).

Fig. 2. Frequency distribution of intraspecific and interspecific genetic distances in Paralucilia. The following species hypotheses are presented: A) Hypothesis 1 (6 species) - P. fulvinota "darker

A

morphotype", P. fulvinota "lighter morphotype", P. nigrofacialis, P. peseudolyrcea, P. pseudolyrcea d.p., P. paraensis; B) Hypothesis 2 (5 species) - P. fulvinota, P. nigrofacialis, P. pseudolyrcea, P. pseudolyrcea d.p., P. paraensis; C) Hypothesis 3 (4 species) - P. fulvinota, P. pseudolyrcea, P. paraensis; D) Hypothesis 4 (3 species) P. fulvinota, P. fulvinota "lighter morphotype", P. pseudolyrcea, P. paraensis.

A ED

PT

CC E

IP T

SC R

U

N

A

M

25

I N U SC R

Supplementary material - A

26

A

Table A.1 Primer and PCR conditions for amplification of COI, 28S, ITS2 and 16S molecular markers of Paralucilia specimens.

M

Molecular markers / Primers LCO HCO

ED

COI (Folmer, 1994)

(GGTCAACAAATCATAAAGATATTGG)

Initial denaturation step 3 min/ 95°C

PT

3 min/ 95°C

CC E

3 min/ 95°C

A

(CCATTGCACTAATCTGCC)

95°C/ 45 sec

52°C/ 45 sec

72°C/ 45 sec

72°C/5 min

52°C/ 45 sec

72°C/ 45 sec

72°C/5 min

52°C/ 1 min

72°C/ 1 min

72°C/5 min

52°C/ 1 min

72°C/ 1 min + 30 sec 72°C/5 min

95°C/ 45 sec 95°C/ 2 min 35 cycles

(GTTAGTTTCTTTTCCTCCCCT)

LR-N-13398 16S (CGCCTGTTTAACAAAAACAT) (Marinho et al., L1R 2012)

Extension

35 ciclos

(CTGTTTCGGTCTTCCATCAGGG)

5.8S ITS2 (ATCACTCGGCTCGTGGGATTCGAT) (Marinho et al., 28S 2012)

Annealing

35 cycles

(TAAACTTCAGGGTGACCAAAAAATCA)

28S-F1 28S (GGGAGGAAAAGAAACTAACAAGG) (Marinho et al., 28S-R1 2012)

Final Extension

Denaturation

3 min/ 95°C

95°C/ 1 min 35 cycles

27

Supplementary material – B

Fig. B.1 Maximum Parsimony (MP) tree generated in the software TNT v. 1.5 with bootstrap support assigning Chrysomya megacephala as outgroup and gaps treated as 5th character. The

M

A

N

U

SC R

IP T

strict consensus tree was constructed from 2 equally parsimonious trees.

ED

Fig. B.2 Maximum Parsimony (MP) tree generated in the software TNT v. 1.5 with bootstrap support assigning Chrysomya megacephala as outgroup and gaps treated as missing data. The

A

CC E

PT

strict consensus tree was constructed from 12 equally parsimonious trees.

28

Fig. B.3 Maximum Parsimony (MP) consensus tree generated in the software TNT v. 1.5 assigning Chrysomya megacephala as outgroup and gaps treated as 5th character. The strict

M

A

N

U

SC R

IP T

consensus tree was constructed from 2 equally parsimonious trees.

Fig. B.4 Maximum Parsimony (MP) consensus tree generated in the software TNT v. 1.5

ED

assigning Chrysomya megacephala as outgroup and gaps treated as missing data. The strict

A

CC E

PT

consensus tree was constructed from 12 equally parsimonious trees.

29

Fig. B.5 Maximum Likelihood (ML) consensus tree generated in the software GARLI v.2.01

N

U

SC R

IP T

assigning Chrysomya megacephala as outgroup.

A

Fig. B.6 Bayesiana Inference (BI) tree generated in the software MrBayes v.3.1.6 with posterior

A

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PT

ED

M

probabilities assigning Chrysomya megacephala as outgroup.

I N U SC R

Supplementary material - C

30

A

Table C.1 Intra-specific (bold) and interspecific genetic distances (%) for P. pseudolyrcea, P. paraensis, P. nigrofacialis, P. fulvinota "lighter morphotype," P. fulvinota "darker morphotype" P. pseudolyrcea - d.p., and for the hypothetical groups of species (P. fulvinota “darker morphotype” + P. nigrofacialis), (P. fulvinota “darker morphotype” + P. fulvinota “lighter morphotype”), (P. fulvinota “darker morphotype” + P. nigrofacialis + P. fulvinota “lighter morphotype”) and (P. pseudolyrcea + P. pseudolyrcea - d.p.). (*): only one specimen was analyzed. (-): nterspecific genetic distances not analyzed.

M

Species

ED

1. P. pseudolyrcea (n = 2)

2

3

4

5

6

7

8

9

2.8

0.2

3. P. nigrofacialis (n = 1)

2.4

1.9

*

4. P. fulvinota “lighter morphotype” (n = 2)

2.8

3.2

1.7 1.3

5. P. fulvinota “darker morphotype” (n = 3)

2.3

2.0

0.1 1.7

0.2

6. P. pseudolyrcea - d.p. (n = 3)

0.3

2.7

2.3 2.9

2.3

0.3

7. P. fulvinota “darker morphotype” + P. nigrofacialis (n = 4)

-

2.0

-

-

-

0.1

8. P. fulvinota “darker morphotype” + P. fulvinota “lighter 2.5 morphotype” (n = 5)

2.5

0.7 -

-

2.5

-

1.2

9. P. fulvinota “darker morphotype” + P. nigrofacialis + P. fulvinota “lighter morphotype” (n = 6)

2.4

-

-

-

-

-

-

1.0

10. P. pseudolyrcea + P. pseudolyrcea - d.p. (n = 5)

2.7

-

2.8

-

-

2.3

-

2.5

PT

10

0,0

2. P. paraensis (n = 3)

CC E A

1

-

1.7

0.3