388 Phytologia (December2012) 94(3) TAXONOMY OF THE TURBINATE SHAPED SEED CONE TAXA OFJUNIPERUS, SECTION SABINA: SEQUENCE ANALYSIS OF nrDNA AND FOURcpDNA REGIONS Robert P. Adams Biology Department, Baylor University, Box 97388, Waco, TX 76798, USA email [email protected] and Andrea E. Schwarzbach Department ofBiomedicine, University ofTexas at Brownsville, Brownsville, TX 78520, USA. ABSTRACT An analysis of the turbinate shaped seed cone taxa of Jimiperus sect. Sabina is presented, based on nrDNA and four cpDNA regions. These DNA data revealed several previously unknown affinities. Plants collected asJ. przewalskiifpendula were found to be J. convallium (J. convallium forma pendula, comb. nov.). Several infraspecific taxa were found to be so distinct to warrant recognition at the specific level: J.fargesii (=J. squamata var.fargesii ); J. uncinata, & Stat. comb. nov. (=J. recurva var. uncinata);J. carimta (=J. pingii var. carinata); J. rushfortltiana, stat. nov. (= J. indica var. nishforthiana). J. squamata f wilsonii is best treated as /. squamata var. wilsonii^ comb. nov. Phytologia 94(3): 388-403 (December 1, 2012). KEY WORDS: Taxonomy, Juniperus, section Sabina, turbinate seed cones, nrDNA, petN-psbM, tmS-tmG, tmD-tmT, tmL-tmF, J. convallium f pendula, comb, nov., J. uncinata, stat. & comb, nov., J. squamatavar. wilsonii, com. nov.,J. rushforthiana, stat. nov. The turbinate shaped seed cone junipers of section Sabina comprise a distinct clade (Mao et al., 2010), having seed cones with elongated, pointed fips (Fig. 1). The cone elongation is more Phytologia (December2012) 94(3) 389 apparent in immature cones. Although most of the taxa have only scale-like leaves in the adult foliage (Fig. 1), several taxa have only decurrent type leaves in the adult foliage (Fig. 2). Decurrent leaves (juvenile) have a sheath that clasps the stem and a free blade that may be appressed (J. squamata var. squamata. Fig. 2), free at about 30 - 45° (J. sq. f wilsomi\ j convdilium free at about 60°- 90° {J. sq. var. fargesii), elongated and slightly free Figure 1. J. convallium leaves (10°- 20°) inJ. morrisonicola, to and turbinate shaped seed cones, very appressed as inJ. recurva and J. coxii (Fig. 2). Because the seed cones are similar, decurrent leaf shape has been used to lump J. squamata, J. sq. f. wilsonii and J. sq. var.fargesiiandJ. morrisonicola (J. sq. var. morrisonicola (Hayata) H- & L. Li H. Keng). Juniperus squamata has, historically, included several taxa with decurrent leaves that don't fit into other turbinatejunipers. Part of the confusion seems to have arisen by the nature ofthe type specimen from a sheet that has three specimens from at leasttwo and likely, three taxa (Fig. 3); Farjon (2005, p. 382) designated this sheet as the lectotype for J. squamata, but he failed to indicate which one of the specimens. As the sheet contains three plants, the lectotype is clarified W here as: W. S. Webb 6043C (lectotype K-W, right-most specimen on W sheet 6043C). The original description of Buch.-Ham. [Lambert, Descr. Pinus 2: 17 (1824)] reads "leaves 3, appressed, imbricate, ovate- oblong, acute, acuminate; withered persistent; inflexed when young with apex somewhat obtuse, fruits ovate and umbilicate at the top, branches and branchlets crowded, terete, stem prostrate." Clearly, the right-most specimen best fitsthe description ofJ. squamata. A comparison of leaves from the type specimens of J. squamata, f. wilsonii and V2ix.fargesii (Fig. 4) reveals the nature ofthe appressed leafblades inJ. squamata and f. wilsonii, and the free, very 390 Phytologia (December2012) 94(3) ^Q^^^^^^ J. squamata var. squamata wHsonii f. J. recun/a ^- coxii Figure 2. Comparison ofthe decurrent leaves ofsix turbinate seed cone taxa. Phytologia (December2012) 94(3) 391 W Figure 3. Photo of type ofJ. squamata Buch.-Ham., W. S. Webb, 6043C at K (K000395285). Left-most: cf J. indica, center: cf J. squamata vav.fargesii, right-most: J. squamata. 392 Phytologia (December2012) 94(3) divergent leafblades in J. squamata var.fargesii. The seed cones ofJ. squamata appear largerthan those ofJ. s. f wilsonii(Fig. 4). J. squamata,typeW6043c, K tJ.ypsequWialmsaotna9f85w,ilEsonn. tJ.ypsequFaarmgaetsa1v5ar3.,fAargesii, Figure 4. Comparison of leaves from the type specimens of J. squamata, f wilsonii and vav.fargesii. Although Mao et al. (2010) analyzed several of the turbinate junipers (J. convaliium, J. coxii, J. indica, J. komarovii, J. pingii, J. przewalskii, J. pseudosabina, J. saltuaria, J. squamata, J. tibetica), they did not includeJ. indica var. caespitosa, J. i. var. rushforthiana, J. microsperma, J. morrisonicola, J. pingii var. carinata, J. przewalskii f pendula, J. recun^a var. recurva, J. r. var. uncinata, J. squamata var. fargesiiandJ. s. f wilsonii. The purpose ofthe current study is to present analysis ofall the turbinate seed cone taxa of section Sabina using the most informative nuclear (nrDNA - ITS) and cpDNA regions (petN-psbM, trnS-trnG, trnD-trnT, trnL-trnF). Phytologia (December2012) 94(3) 393 Table 1. Comparison of Adams and Farjon taxonomic treatments of taxa in this study. Taxa with DNA sequencing support for a modified taxonomic status are in bold. Adams(20in Farion (2005, 2010) Supported, this study J. convallium J. convallium J. convallium J. coxii J. recurvav. coxii J. coxii J. indica J. indica J. indica J. i. var. caespitosa J. i. var. caespitosa J. recurva var. caespitosa? J. ivar. rushforthiana J. indica J. rushforthiana J. komarovii J. komarovii J. convallium var. komaroviil J. microsperma J. convallium var. J. microsperma microsperma J. morrisonicola J. squamata J. morrisonicola J. pingii J. pingii J. pingii J. p. var. carinata J. p. var. wilsonii J. carinata J. przewalskii J. przewalskii J. przewalskii J. p.f.pendula J. przewalskii J. convalliumf. pendula J. pseudosabina J. pseudosabina J. pseudosabina J. recurva J. recurva J. recurva J. r. var. uncinata J. recuj^a? J. uncinata J. saltuaria J. saltuaria J. convallium var. saltuarial J. squamata J. squamata J. squamata J. s. var.fargesii J. squamata J.fargesii J. s. f. wilsonii J. pingii f wilsonii J. s, var. wilsonii J. tibetica J. tibetica J. tibetica 394 Phytologia (December2012) 94(3) MATERIALS AND METHODS Specimens used in this study: J. convallium, Adams 6781-83, 17 km e Tewo, Gansu, China, J. communis L. var. communis, Adams 7846, 7847, Stockholm, Sweden (outgroup), J. coxii, Adams 8137-38, clone from Type tree, Abbotsmarsh Arboretum, UK, ex Burma, Chimili valley,J. indica, Adams 8775-77, Dumpa, Jomson,Nepal,J. indica var. caespitosa, Adams 7625-27, near Yangjin Gompa, Nepal, J. indica var. rushforthiana, Adams 8140-41, Abbotsmarsh Arboretum, UK, ex Lingshi, Bhutan, J. komarovii, Adams 8518-20, near Zhe Gu Mtn., Maerkan County, Sichuan, China, J. microsperma, Adams 8522-24, near Zhe Gu Mtn., Maerkan County, Sichuan, China, J. morrisonicola, Adams 8681-2, Younger Bot. Gard., Scotland, ex Taiwan, J. pingii, Adams 8506-7, near White Horse Mtn., Deqin County, Yunnan, China, J. pingii var. carinata, Adams 8497-99, near White Horse Mtn., Deqin County, Yunnan, China, J. pseudosabina, Adams 7808-10, 30 km n Jarkent (Paniflor), Kazakhstan, J. przewalskii, Adams 6775-77, 25 km w Jone, Gansu, China, J. przewalskii f. pendula, Adams 6779, Langmusi, Gansu, China, J. recw-va, Adams 7215, 7217, 7219, Cholan Pati lodge, Nepal, J. recurva var. uncinata, Adams 7212-14, Lauri Binayak, Nepal, J. saltuaria, Adams 6789-91, on Duoer River, 23 km e Forestry Station, Gansu, China, J. squamata, Adams 6795-96, Xian Bot. Garden, Shaanxi, China, J. s. var. fargesii, Adams 8491-93, near White Horse Mtn., Zhongdian County, Yunnan, China, J. s. f wilsonii, Adams 5521, Accession 1010-64A, cultivated from seeds from E. H. Wilson 985 (Holotype) collection, Arnold Arboretum, USA, ex. China, J. tibetica, Adams 8512-16, on Maerkan River, near Zhe Gu Mtn., Maerkan County, Sichuan, China. Voucher specimens are deposited in the herbarium, BAYLU, Baylor University. DNA extraction, PCR amplification, sequencing and data analyses - see Adams and Schwarzbach (2012a). RESULTS AND DISCUSSION The turbinate seed conejunipers are relatively uniform with a few distinct groups (Fig. 5). As previously reported (Adams 201 1), J. recurva and J. indica taxa are in a clade, but well resolved from J. Phytologia (December2012) 94(3) 395 Turbinate Seed Cone Junipers indicav. 'rushforthiana Bayesian Tree L—rnecurvav. recurva based on 4292 bp 100 from nrDNA, petN-psbM, 100 indicav. caespitosa trnS-G, trnD-T, trnL-F indicav. indica recurvav. uncinata coxii 87 — squamata v. fargesii f—przewalskiif.pendula r- 87 l<omarovii 99 L.convallium 96 pingii\j. carinata 94 ^67 p//?g//V. p/ng// 53 saltuaria microsperma przewalskii 100 morrisonicola 100 100 squamataf. wilsonii 199 tibetica 100 squamatav. squamata pseudosabina J. communis, outgroup Figure 5. Bayesian tree for the turbinate seed cone taxa, sect. Sabina. Numbers atthe branch points are posteriorprobabilities (as percent). indica var. rushforthiana (Fig. 5). Juniperus recurva is a small tree with lax foliage and tightly appressed decurrent leaves (Fig. 2), and J. indica has generally erect foliage with typical scale-like leaves of(cf. Fig. 1), otherwise, the taxa are similar in morphology (Adams 201 1). Juniperus recurva var. uncinata is in a clade with J. coxii and J. squamata var.fargesii. 396 Phytologia (December2012) 94(3) Juniperus comallium, J. komarovii and the putative J. przewalskii f.pendula are in an unresolved clade (Fig. 5). J. pingii and J. p. var. carinata are in a clade, but are well differentiated. J. microsperma and J. saltuaria are associated with the J. comallium - komarovii clade (Fig. 5). Finally, it is noteworthy thatJ. przewalskii, J morrisonicola, J. squamata f. wilsonii, J. s. var. squamata and J. tibetica form a distinct clade (Fig. 5). J. pseudosabina is well supported as an outlierto the otherturbinatejunipers (Fig. 5). Taxonomic Considerations DNA In addition to illuminating phylogeny, sequence data are useful in validating and elucidating taxonomy. Coding all nucleotide substitutions and indels (as single mutations) resulted in 225 mutational events (MEs). These 225 MEs were used to construct a minimum spanning network (Fig. 6). Two patterns are immediately visible: the central nature ofJ. convallium among the taxa, and relative uniformly large differences between taxa (even taxa in the same species, such as J. indica, J. i. var. caespitosa, J. i. var. rushforthiana. Fig. 6). The most obvious taxonomic anomaly is the difference ofonly 2 MEs (out of225) between J. convallium and putativeJ. przewalskiif. pendula (Fig. 6). Clearly, this pendulous form is a part of J. convallium and should be recognized as: Juniperus convallium formapendula (Cheng & L-K. Fu) R. P. Adams, comb. nov. Basionym: Sabinaprzewalskii(Kom.) W-C, Cheng & L-K. Fu f.pendula Cheng & L-K. Fu, Acta Phytotax. Sin. 13(4): 86. 1975. Type: PE. Juniperusprzewalskii i. pendula R. P. Adams and G-L. Chu, J. Essential Oil Res. 6:17. 1994. TheJ. convallium - komarovii- saltuaria complex is separated by only 6 - 8 MEs (Fig. 6). Adams and Schwarzbach (2012a, 2012b) found varieties in Juniperus sections Juniperus and Sabina to differ by 6-8 MEs, whereas generally accepted species differed by 10 - 12 to 20 MEs (using the same set of gene regions). Both Adams (201 1) and Farjon (2005, 2010) recognize these three as species in their monographic treatments (Table 1); it seems wise to continue such , Phytologia (December2012) 94(3) 397 recognition although there is only marginal support for such treatment DNA, in the data set. Turbinate Cone Junipers ^ communis(outgroup) Minimum Spanning p/ng//V. Network 96 carinata 225 MEs , microsperma 15, 12 15 squamata saltuad^ V. fargesii cbnvallium pingiiV. p/ng// recurvav. 13 'ppreznewdaullsakii f. 21 uncinata 17/ 17 13 przewal?l<ii squamata coxn f. wilson,t indicav, rushforthiaiia\ 31 ' morrisonicola indica dicaV. tibkfica indica caespitosa pseudosabina Figure 6. Minimum spanning network based on 225 MEs from nrDNA, and cpDNA (petN-psbM, trnS-G, trnD-T, trnL-F). Numbers next to the lines are the number ofMEs separating taxa. Dashed lines arethe second nearest link. Juniperus pingii is a tall tree, with long, sharp-pointed decurrent leaves and J. pingii var. carinata is a small shrub with short, scale-like leaves (Fig. 7). Juniperus pingii differs by only 10 MEs