1 Plastome Structure and Phylogenetic Relationships of Styracaceae 2 (Ericales) 3 4 Xiu-lian Caia, Jacob B. Landisb , Hong-Xin Wang a, Jian-Hua Wang a, Zhi-Xin Zhu a, 5 Hua-Feng Wang a,* 6 7 aHainan Key Laboratory for Sustainable Utilization of Tropical Bioresources, School 8 of Life and Pharmaceutical Sciences, Hainan University, Haikou 570228, China 9 10 bSchool of Integrative Plant Science, Section of Plant Biology and the L.H. Bailey 11 Hortorium, Cornell University, Ithaca, NY 14850, USA 12 13 14 15 16 *Authors for correspondence: 17 Hua-Feng Wang, E-mail: [email protected]; 18 19 20 21 1 22 ABSTRACT 23 Background: The Styracaceae are a woody, dicotyledonous family containing 12 24 genera and an estimated 160 species. Recent studies have shown that Styrax is 25 monophyletic, Alniphyllum and Bruinsmia cluster into a clade with an approximately 26 20-kb inversion in the LSC. Halesia and Pterostyrax are not supported as 27 monophyletic, while Melliodendron and Changiostyrax always from a clade sister to 28 the rest of the family. However, the phylogenetic relationship of Styracaceae at the 29 level of genera remains ambiguous. 30 Results: We collected 28 complete plastomes of Styracaceae, including 12 sequences 31 newly reported here and 16 publicly available complete plastome sequences, 32 comprising 11 of the 12 genera of Styracaceae. All species possessed the typical 33 quadripartite structure of angiosperm plastomes, and the sequence difference is small, 34 except for the large 20-kb (14 genes) inversion region found in Alniphyllum and 35 Bruinsmia. Seven coding sequences (rps4, rpl23, accD, rpoC1, psaA, rpoA and ndhH) 36 were identified to possess positively selected sites. Phylogenetic reconstructions based 37 on seven data sets (i.e., LSC, SSC, IR, Coding, Non-coding, combination of 38 LSC+SSC and concatenation of LSC+SSC+one IR) produced similar topologies and 39 most relationships are consistent with previous findings. In our study, Pterostyrax was 40 strongly supported as monophyletic; Melliodendron and Changiostyrax as 41 successively sister to the rest of the family. 42 Conclusion: Our results clearly indicate that Pterostyrax is monophyletic, and the 43 establishment of Perkinsiodendron and Changiostyrax are supported. A 20-kb reverse 44 sequence was found in the newly published sequence of Alniphyllum fortunei, which 45 confirmed the existence of large inversion sequence in Alniphyllum and Bruinsmia. 46 47 Keywords: Styracaceae; Plastome; Genome structure; Phylogeny; positive selection 48 49 2 50 Background 51 The Styracaceae DC. & spreng (Ericales) comprise an angiosperm clade of 12 52 genera and over 160 species, mainly distributed in regions of Asia, as well as tropical 53 and temperate America, and the Mediterranean [1]. The family consists of shrubs or 54 trees with stellate pubescent or epidermal scales, simple leaves, inflorescence of 55 raceme, cyme or panicle, and actinomorphic flowers with varying degrees of 56 synsepaly and sympetaly [2]. The fruit of Styracaceae is a drupe or capsule, with 57 persistent calyx, surrounding or united with the fruit. The Styracaceae have been 58 included in a number of morphological studies, including leaf anatomy [3], wood 59 anatomy [4], pollen morphology [5] and floral morphology and anatomy [2], but 60 distinguishing between genera in the family primarily involves variation in fruit 61 morphological characters (e.g. hypanthium at maturity). On one hand the ovary is 62 inferior with a persistent hypanthium combined with the fruit at maturity (i.e., 63 Changiostyrax C.T. Chen, Halesia J. Ellis ex L , Melliodendron Hand.-Mazz, 64 Parastyrax Siebold & Zucc., Perkinsiodendron P. W. Fritsch, Pterostyrax W.W. Sm., 65 Rehderodendron Hu, and Sinojackia Hu), while on the other hand the ovary is 66 superior and a persistent hypanthium forms only at the base of the fruit at maturity 67 (Alniphyllum Matsum., Bruinsmia Boerl. & Koord, Styrax L.,)(Fig.1). Moreover, the 68 ovary of Huodendron Rehder is semisuperior with a persistent hypanthium extending 69 from the base to about two-thirds of the fruit length [1, 2], a feature considered to be 70 transitional. 71 The systematic position of Styracaceae has been revised numerous times. Early 72 researchers thought Styracaceae was positioned in the order Ebenales, along with the 73 well-known Sapotaceae, Ebenaceae, and Symplocaceae, and the small family 74 Lissocarpaceae [6, 7, 8, 9]. However, Cronquist [6] showed that there were some 75 original characteristics and some new evolutionary characters in each family of 76 persimmons, which may have arisen via parallel evolution. Based on embryological 77 and anatomical studies, Herbert [10] suggested that the family Styracaceae and 78 Theaceae, which have many common characteristics, may have originated from the 3 79 ancestors of Theaceae. According to molecular systematic studies, Styracaceae has 80 been recognized as part of the order Ericales sensu lato [11]. Within the family, 81 phylogenetic resolution generally remains poor. At most 17 genera have been included 82 in Styracaceae, with Symplocos L, Diclidanthera Mart, Afrostyrax Perk et Gil, 83 Foveolaria Ruiz et pav., Pamphilia Mart. ex A. DC, Huapierre et De Wil, and 84 Lissocarpa Benth having been placed in the Styracaceae by various authors [12]. 85 Symplocos, Diclidanthera and Lissocarpa were excluded from Styracaceae by Perkins 86 [13], after which they were treated as independent families (Symplocos) or combined 87 into Polygalaceae (Diclidanthera) and Ebenaceae (Lissocarpa), Diclidanthera is 88 placed in Polygalaceae [6, 7, 14], and Lissocarpa is the closest relative of Ebenaceae 89 [15]. Afrostyrax was once included in the genus Styrax [16], but was later reclassified 90 into Huaceae [6, 7, 14,17]. According to taxonomic revisions, Pamphilia was 91 classified into Styrax [18]. Fritsch [19] combined Foveolaria into Styrax by 92 implementing morphological phylogenetic analyses. In addition, two new genera have 93 been established in the family Styracaceae: (1) Chen [20] segregated Sinojackia 94 dolichocarpa as a new monotypic genus Changiostyrax, and (2) according to 95 morphological and DNA sequences, Halesia macgregorii was removed from Halesia 96 to become a new genus, Perkinsiodendron P.W. Fritsch [21]. 97 Although the phylogenetic placement of the family has been resolved, there are 98 few phylogenetic studies above the genus level, with the phylogenetic relationships 99 between genera remaining ambiguous. In the phylogeny of Ericales based on the 100 single chloroplast gene rbcL [22], the results showed that Styrax and Clethra 101 Gronov.ex L. (Clethraceae) were clustered in a clade, while Halesia, Rehderodendron, 102 and Sinojackia formed a clade that was sister to Diapensia L. and Galax Rafin. 103 (Diapensiaceae). Therefore, Styracaceae was considered to be polyphyletic. However, 104 this conclusion does not always hold true. Olmstead et al. [23] inferred the phylogeny 105 of Asteridae based on multiple genes, including Styrax and Halesia, which formed a 106 strongly supported sister-group relationship. Albach et al. [24] came to the same 107 conclusion as Olmstead et al. [23] when analyzing molecular evolution of four genes 108 within the Asterids. In addition, the phylogeny of Styracaceae based on morphology 4 109 plus three DNA sequences (chloroplast trnL intron/trnL-trnF spacer and rbcL with the 110 nuclear ribosomal DNA region ITS) recovered a monophyletic relationship of 111 Styracaceae [1]. Early molecular phylogenetic studies of Styracaceae based on 112 morphological characters and three DNA loci showed that Pterostyrax and Halesia 113 were not supported as monophyletic, Styrax and Huodendron formed a clade that was 114 sister to a clade of Alniphyllum and Bruinsmia, and a sister relationship was found 115 between Halesia macgregorii and Rehderodendron macrocarpum [1]. Based on ITS, 116 the plastid psbA-trnH intergenic spacer, and microsatellite data, Yao et al. [25] 117 recovered Sinojackia as monophyletic and reported a similar topology as Fritsch et al. 118 [1] with weak support for six genera within Styracaceae. Yan et al. [26] conducted 119 phylogenetic analyses of the Styracaceae based on 19 chloroplast genomes. The 120 results showed that Styrax was monophyletic, while Huodendron, Alniphyllum and 121 Bruinsmia clustered in a clade with an approximate 20-kb inversion in the Large 122 Single-Copy (LSC) region. The tree species Pterostyrax were not supported as 123 monophyletic, however, Halesia carolina L and Pterostyrax hispidus Siebold & Zucc 124 formed a clade that was sister to the remainder of the family. Additionally, the 125 systematic positions of Halesia and Pterostyrax have not yet been fully resolved. 126 The chloroplast genomes of most angiosperms are of matrilineal inheritance. The 127 rate of evolution of genes in the chloroplast is relatively slow, but the rates of 128 evolution across different regions of the genome have observed differences, which 129 can be applied to phylogenentic studies of various taxonomic scales. Due to a 130 conserved structure, small effective population size, and lack of recombination, 131 chloroplast genomes have been extensively used to infer phylogenetic relationships 132 and histories [27, 28, 29]. With the advent of next-generation sequencing (NGS) 133 technologies, whole-plastome sequencing has become cheaper and faster than ever 134 before. As a result, whole-plastome sequence data have recently been employed to 135 generate highly resolved