Tree Genetics & Genomes (2018) 14:6 https://doi.org/10.1007/s11295-017-1223-z ORIGINAL ARTICLE Phylogenetic relationships of Aurantioideae (Rutaceae) based on RAD-Seq Yukio Nagano1 & Takashi Mimura2 & Nobuhiro Kotoda2 & Ryoji Matsumoto2 & Atsushi J. Nagano3,4,5 & Mie N. Honjo3 & Hiroshi Kudoh3 & Masashi Yamamoto6 Received: 31 July 2017 /Revised: 27 November 2017 /Accepted: 25 December 2017 # Springer-Verlag GmbH Germany, part of Springer Nature 2018 Abstract The economically and nutritionally important genus Citrus belongs to the subfamily Aurantioideae in the family Rutaceae. Here, we analyzed the phylogenetic relationships of the subfamily Aurantioideae based on RAD-Seq. The RAD-Seq data produced phylogenetic trees with high support values, clear discriminations based on branch length, and elucidations of early branching events. Our genetic classification corresponded well with the classical morphological classification system and supported the subdivision of Citreae, one of two tribes of the Aurantioideae, into three subtribes—Triphasiinae, Citrinae, and Balsamocitrinae. Additionally, it was largely consistent with the subdivision of Clauseneae, the other tribe of the Aurantioideae, into three subtribes—Micromelinae, Clauseninae, and Merrillinae; the exception was Murraya paniculata. With the exception of members of primitive citrus fruit trees, namely, Severinia buxifolia and Hesperethusa crenulata, lower-level morphological groupings under subtribes based on genetic and morphological classifications corresponded well. The phylogenetic relationship between Asian Btrue citrus fruit trees^ (genera Citrus, Poncirus,and Fortunella) and Australian/New Guinean citrus fruit trees (genera Microcitrus, Eremocitrus,andClymenia) was incon- sistent between present classification based mainly on the nuclear genome and the previous classification based on the chloroplast genome. This inconsistency may be explained by chloroplast capture. Our findings provide a valuable insight into the genetic relationships of the subfamily Aurantioideae in the family Rutaceae. Keywords Citrus . Aurantioideae . Rutaceae . RAD-Seq . Phylogeny . Genetic classification Communicated by W.-W. Guo Introduction Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11295-017-1223-z) contains supplementary material, which is available to authorized users. Citrus is an economically and nutritionally important genus and a member of the subfamily Aurantioideae in the family * Yukio Nagano Rutaceae. In addition to Citrus species, some other members [email protected] of Aurantioideae are used for various purposes such as foods, spices, medicinal chemicals, cosmetics, and garden trees. 1 Analytical Research Center for Experimental Sciences, Saga Therefore, it is important to understand the phylogenetic rela- University, 1 Honjo-machi, Saga 840-8502, Japan tionships of the subfamily Aurantioideae. 2 Faculty of Agriculture, Saga University, 1 Honjo-machi, Classical morphology divided the subfamily Saga 840-8502, Japan Aurantioideae into two tribes—Clauseneae and Citreae 3 Center for Ecological Research, Kyoto University, 509-3 2-chome, (Swingle and Reece 1967). Recent molecular classifica- Hirano, Otsu, Shiga 520-2113, Japan tions, using small numbers of DNA sequences, also clear- 4 JST PRESTO, 4-1-8, Honcho, Kawaguchi, Saitama 332-0012, Japan ly discriminated Citreae from Clauseneae (Bayer et al. 5 Faculty of Agriculture, Ryukoku University, 1-5 Yokotani, Seta 2009;Penjoretal.2013;Schwartzetal.2015). Oe-cho, Otsu, Shiga 520-2194, Japan Morphological classification subdivided each tribe into 6 Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, three subtribes. The tribe Clauseneae consists of Kagoshima 890-0065, Japan Micromelinae, Clauseninae, and Merrillinae, and the tribe 6 Page 2 of 11 Tree Genetics & Genomes (2018) 14:6 Citreae consists of Triphasiinae, Citrinae, and RAD-Seq analysis Balsamocitrinae (Swingle and Reece 1967). However, re- cent molecular studies did not clearly reveal this level of The DNA purification procedure was identical to that described classification (Bayer et al. 2009;Penjoretal.2013; by Penjor et al. (2014). The method used to create the library Schwartz et al. 2015), probably because they did not have for double-digest RAD-Seq was identical to that described by the capability to determine earlier branching events. Sakaguchi et al. (2015), which is a modification of the original The data size used in previous molecular analyses was double-digest RAD-Seq (Peterson et al. 2012)asfollows:BglII small, i.e., small numbers of markers or short DNA se- was used as the first restriction site adjacent to the binding site quences. Therefore, these previous studies did not dis- of the primer to read a single-end sequence, and EcoRI was criminate the earlier branching events that separated major used as the second restriction site adjacent to the binding site to taxa. In addition, it was difficult to reveal genetic relation- read an index sequence. The library was sequenced with 49 bp ships among closely related species. Next-generation se- single-end reads in one lane of an Illumina HiSeq2000 quencing produces a large amount of data; hence, it is (Illumina, San Diego, CA, USA) by BGI Hong Kong. At worthwhile using it to reveal the genetic relationships BGI, the raw data were modified using the following two steps: within the subfamily Aurantioideae. In comparison with (1) reads that were polluted by adapter sequences were deleted, whole genome sequencing, restriction site-associated and (2) reads that contained > 50% low-quality bases (quality DNA sequencing (RAD-Seq) can analyze many samples value ≤ 5) or > 10% N bases were removed. inexpensively and simultaneously, and it is suitable for studying the relationships between genetically similar in- Phylogenetic analysis based on the pyRAD program dividuals (Baird et al. 2008). The original report of RAD- Seq(Bairdetal.2008) has more than 1000 Google Reads were further cleaned using the process_shortreads pro- Scholar citations, and the technique has been applied to gram of the Stacks package (version 1.46) with -c (clean data, many types of model and non-model organisms (Andrews remove any read with an uncalled base) and -q (discard reads et al. 2016). In our case, we used this method to analyze with low-quality scores) options (Catchen et al. 2011, 2013). Citrus species (Penjor et al. 2014, 2016), which are ge- For the construction of phylogenetic trees, multiple alignments netically similar because of their cross-compatibilities. were created using the pyRAD program (version 3.0.66) The relationships between genetically distant individuals (Eaton 2014). In multiple alignments c80m12, c85m12, and at the genus and/or family level have been analyzed using c90m12, Wclust (clustering threshold as a decimal) was set to RAD-Seq data with the new software PyRAD (Eaton 0.80, 0.85, and 0.90, respectively, with Mindepth (min cover- 2014). The clustering method of PyRAD does not use age for a cluster) = 6, NQual (max # sites with quality < 20) = the reference genome and allows for lower similarity 4, MinCov (min samples in a final locus) = 12, and MaxSH thresholds and inclusion of indels. Therefore, it is possible (max inds with shared hetero site) = 3. In multiple alignments to analyze relationships within the subfamily c80m4, c85m4, and c90m4, Wclust was set to 0.80, 0.85, and Aurantioideae using RAD-Seq. The RAD-Seq method 0.90, respectively, with Mindepth = 6, NQual = 4, MinCov = and the programs required to analyze the resulting data 4, and MaxSH = 3. In these calculations, the default parameter were reviewed by Andrews et al. (2016). of maximum depth filtering was used. For all six multiple In the present study, we analyzed the genetic relationships alignments, phylogenetic trees based on maximum likelihood within the subfamily Aurantioideae based on RAD-Seq. The were constructed using the RAxML program (version 8.2.10) obtained phylogenetic trees elucidated branching order well. (Stamatakis 2014) (-f = a, -x = 12,345, -p = 12,345, -N (boot- Herein, we describe new findings and confirm previous find- strap value) = 1000, and -m = GTRGAMMA). For multiple ings. Our molecular classification is consistent with the clas- alignments c80m12, c85m12, and c90m12, phylogenetic trees sical morphological classification (Swingle and Reece 1967). based on Bayesian inference were constructed using the MrBayes program (version 3.2.2) (Ronquist and Huelsenbeck 2003) (lset nst = 6, rates = invgamma, mcmc Materials and methods ngen = 100,000, samplefreq = 1000, nchains = 4, and savebrlens = yes). In each analysis, the midpoint was used as Plant materials a root. The number of phylogenetically informative sites was calculated using MEGA version 7 (Kumar et al. 2016). Thirty-five species from 22 genera of the subfamily Aurantioideae were used in this study (Table 1). The ma- Phylogenetic analysis based on the Stacks program terials have been preserved at the Faculty of Agriculture, Saga University, and the Faculty of Agriculture, For the de novo analysis, the cleaned reads were analyzed Kagoshima University. using the denovo_map.pl script of the Stacks package Tree Genetics & Genomes (2018) 14:6 Page 3 of 11 6 Table 1 Species used to analyze the genetic relationships within Aurantioideae Tribe Subtribe Group Latin name (common name/accession name) Source Accession no. Clauseneae Micromelinae Micromelum minutum (Forst.) Wt. & Arn. Saga Univ. 8650 Clauseninae Clausena