Pure Appl. Biol., 8(4): 2213-2227, December, 2019 http://dx.doi.org/10.19045/bspab.2019.80167 Research Article Effect of outgroup on phylogeny reconstruction: a case study of family Solanaceae Ishrat Jamil*, Syeda Qamarunnisa and Abid Azhar The Karachi Institute of Biotechnology and Genetic Engineering (KIBGE), University of Karachi, Karachi-Pakistan *Corresponding author’s email: [email protected] Citation Ishrat Jamil, Syeda Qamarunnisa and Abid Azhar. Effect of outgroup on phylogeny reconstruction: a case study of family Solanaceae. Pure and Applied Biology. Vol. 8, Issue 4, pp2213-2227. http://dx.doi.org/10.19045/bspab.2019.80167 Received: 06/02/2019 Revised: 10/07/2019 Accepted: 15/07/2019 Online First: 23/07/2019 Abstract In phylogenetic analysis, direction of ingroup evolution depends on outgroup, therefore, the determination of correct outgroup is very critical. In the current study, effect of different outgroups on tree topology within Solanaceae was examined using Bayesian analysis. Different outgroups were selected from closely related families of Solanaceae (Convolvulaceae, Hydrolaceae and Montiniaceae), distantly related families (Acantaceae, Scrophularaceae and Boraginaceae) and more distantly related group (Gymnospermic families; Cycadaceae and Pinaceae). Single taxa from each family, multiple taxa from a single genus and multiple taxa from different genera of the same sister family were selected to evaluate their resolving power. The single taxa of a genus belong to Convolvulaceae produced more consistent result as compared to the multiple taxa of the single genus. In addition, single taxa produced similar tree topology as produced by multiple taxa of different genera. Among single taxa, Evolvulus pilosus, observed to be the best outgroup because it resolved the associations between genus, tribes, and subfamilies within Solanaceae followed by Humbertiama dagascariensis. The lineages established by the successful outgroups suggest the evolutionary pattern within Solanaceae is Cestroideae to Petunoideae to Solanoideae. Distantly related outgroup was unable to resolved the tree topology. It may be suggested that for lower level taxonomical relationships of Solanaceae, Evolvulus pilosus could be used as outgroup to infer true associations. Keywords: Ingroup; Outgroup; Phylogenetics; rbcL; Solanaceae Introduction topology of the phylogenetic tree. However, there Molecular phylogenetic infers the hypothesis of is no extensive research presented on its use in the evolutionary relationships in the organisms or reconstruction of the phylogenetic tree. It usually group of organisms by means of phylogenetic been selected randomly or on the basis of vague tree. The topology of the tree, which is based on relationships between in group and outgroup taxa hypothesis of relationships, is effected by several [1, 3, 5-8]. An out group is used to root the assumptions and factors including sample size, unrooted cluster. It infers the theoretical selection of coding or noncoding region of the ancestral origin and provides the array of time of genome, sequence length of the selected region, all subsequently evolutionary events led to alignment methodology, optimality criterion, diverging the sequence, because the root of a tree treatment of data, parameter values and is first or deepest split [1, 5-9]. An out group is assumptions of each phylogenetic inference necessary for phylogenetic analysis to understand methods [1-4]. Selection of outgroup is among the evolution of the group under study (ingroup). those explicit and implicit factors that affects the As the direction of ingroup evolution depends on Published by Bolan Society for Pure and Applied Biology 2213 Jamil et al. the out group, therefore, the selection of correct Boraginaceae were also tested. More distantly outgroup is very critical. An incorrect outgroup related taxa belong to Gymnosperm; Cycas may mislead the inference of hypothetical revoluta (Cycadaceae) and Pinus gerardiana taxonomic relationships and character evolution. (Pinaceae) were also checked for their resolution Therefore, in any phylogenetic analysis the power in Solanaceae phylogenetic tree. Multiple determination of correct root location with taxa that belong to same and different genera accuracy is very important. Incorrectly rotted tree were also taken into consideration to reconstruct may provide erroneous in phylogenetic signals the phylogeny of Solanaceae. A list of selected that may lead to the “random outgroup effect” outgroup species with their classification and and Long Branch Attraction (LBA). To avoid accession numbers is given in (Table 2) while such errors, outgroup should be from outside the their sequence length and GC content is ingroup but closely related to the group of study. mentioned in (Table 3). Usually sister group of the ingroup is preferably DNA extraction and PCR chosen as outgroup. Moreover, outgroup contains A modified Cetyl trimethyl ammonium bromide multiple taxa has been given more consideration (CTAB) method [17] was used to extract the total because of better inference [2, 3, 6-13]. However, genomic DNA from the fresh leave samples. By it is not always necessary that the closely related using primer3 software version 0.4.0 [18] family sister taxa provide correct hypothesis of specific primers were designed for the coding phylogenetic signals especially in case of the region (rbcL) of chloroplast genome. Polymerase relatively high evolutionary rate or if the only chain reaction (PCR) was performed in thermal single taxon, as outgroup, is present in the sister cycler to amplify the rbcL gene. Amplification of group [1, 5, 8]. the rbcL gene was carried out in 20 µL reaction In the present work, effort was made to study the volume containing 1X PCR buffer, 2 mM MgCl2, possible effects of different outgroups to infer the 0.2 mM dNTPs, 0.3 µM of each primer (Forward, subfamilial relationships in Solanaceae. 5'-TTCCGATTCACCGGATCTTA-3' and Materials and methods reverse, 5’-ACTCATTGTTATAGCTGGAT-3'), Ingroup plant material 1.25 units of DNA Taq Polymerase Plant samples for ingroup were collected from (MOLEQULE-ON, New Zealand), 100 ng DNA different areas of Karachi during their flowering template and an appropriate amount of Milli-Q season and identified with the help of key water. The PCR conditions were set as: initial characters present in flora of Pakistan [14] and denaturation of double stranded DNA at 94°C for other floras [15, 16]. Herbarium sheets of the 4 minute, 35 cycles of 94°C for 30 second, primer identified species were prepared and submitted to annealing at 54°C for 1 minute, extension at 72°C Karachi University Herbarium. Their names, for 1.5 minutes and after 35 cycles final extension locations, voucher number and GenBank at 72°C for 7 minutes. Amplified products were accession numbers are given in (Table 1). observed on 1% agarose gel and purified using Outgroup selection column based PCR purification kit (MOLQULE- Most of the outgroup species sequences were ON, NZ). Purified products were sent for direct retrieved from GenBank. Outgroup was selected sequencing at MOLEQULE-ON (NZ). as closely related, distantly related and more Sequence Editing and Alignment distantly related members with respect to Basic Local Alignment Search Tool, Blastn [19] Solanaceae. An outgroup Schizantus pinnatus was performed to analyze each nucleotide was also selected randomly from the ingroup sequence. Open reading frame, ORF [20] was (Solanaceae, subfamily Schizanthoideae). performed to observe the putative conserved Different members from sister families of domain of the rbcL gene. The discrepancy Solanaceae (Convolvulaceae, Hydrolaceae, present in nucleotide sequence was resolved by Montineaceae) were chosen as outgroup. aligning forward and reverse primer sequences Different species of same genera were also using Multalin software [21]. Nucleotide checked for their potency to be selected as sequences were translated into protein using outgroup. Taxa from distantly related families online available ExPASy translate tool [22]. The like Acantaceae, Scrophularaceae and refined sequences were submitted to NCBI 2214 Pure Appl. Biol., 8(4): 2213-2227, December, 2019 http://dx.doi.org/10.19045/bspab.2019.80167 (National Center for Biotechnology Information) species of Cuscuta, one species of Convolvulus, Genbank to get their specific accession numbers. two species of Evolvulus and one species of Phylogenetic tree reconstruction Humbertia were able to resolve the true topology Phylogenetic tree was reconstructed by using of Solanaceae tree. One ingroup species each chosen outgroup. The model based Bayesian Schizanthus pinnatus also resolved associations inference method was used to infer the within Solanaceae. All potential outgroup species relationship within Solanaceae. The best showed different branches of three subfamilies substitution model for almost all the (Solanoideae, Petunoideae, Cestroideae), which combinations was found be Hasegawa, Kishino further sub-divided into seven tribes (Solaneae, and Yano (HKY) as determined by JModelTest Capsiceae, Physleae, Datureae, Lycieae, 2.1.3 software [23]. The program BEAUTi [24] Hyoscymeae, Cestreae). As Petunia is was executed to generate the xml. file which represented by only one member therefore a further executed in BEAST. MCMC chain length separate branch protruding tribe Petuneae can was set as 800,000 generations. Tree was also be seen. Evolutionary lineage of subfamilies screened at every 10,000 generations.