Molecular Phylogeny of Section Parrya of Pinus (Pinaceae) Based on Chloroplast matk Gene Sequence Data

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1 Acta Botanica Sinica 2004, 46 (2): Molecular Phylogeny of Section Parrya of Pinus (Pinaceae) Based on Chloroplast matk Gene Sequence Data ZHANG Zhi-Yong 1,2, LI De-Zhu 1* (1. Laboratory of Plant Biodiversity and Biogeography, Kunming Institute of Botany, The Chinese Academy of Sciences, Kunming , China; 2. Agricultural College, Jiangxi Agricultural University, Nanchang , China) Abstract: The molecular phylogenetics of sect. Parrya Myre of Pinus L. was analyzed based on chloroplast matk gene sequence data. The section was resolved as paraphyletic because members of the sect. Strobus were nested within a clade composed by the Asian members of the section, including the Vietnamese P. krempfii Lecomte, which was strongly supported with a bootstrap value of 92%. In this topology, the three sampled species of sect. Strobus formed a strongly supported monophyletic group, while their relationships of Asian species of sect. Parrya were not clear. P. krempfii was grouped with P. gerardiana Wall. ex D. Don with low bootstrap support. The relationships among the Asian members of the sect. Parrya, i.e. P. bungeana Zucc. ex Loud., P. gerardiana and the recently described endangered pine, P. squamata X. W. Li, was not resolved, although the monophyly of the three pines was strongly supported in the combined analysis of four cpdna sequences. The topology of the neighbor joining tree revealed an assemblage of the American members of the section, which also appeared in the majority rule tree with weak bootstrap support. However, this assemblage was not resolved in the consensus tree of the parsimonious analysis. The American subsect. Balfourianae Engelm. formed a weakly supported group including P. aristata Engelm., while the relationships among and within the other two American subsections, Cembroides Englem. and Rzedowskianae Carv., were not resolved, as the members of them formed a polytomy in the consensus tree of the parsimonious analysis. The biogeographical implications of the results are also discussed in this paper. Key words: sect. Parrya ; Pinus ; molecular phylogeny; matk gene Pinus L. (Pinaceae) is the largest genus of conifers and a widespread genus of trees (sometimes shrubs) in the Northern Hemisphere. Because of their diversity, in addition to their ecological importance as a major component of many temperate forests and economical importance as a source of timber, pines received much attention to their taxonomy and phylogeny (Shaw, 1914; Pilger, 1926; Mirov, 1967; Little and Critchfield, 1969; van der Burgh, 1973; Farjon, 1984; 1996; Schirone et al., 1991; Piovesan, 1993; Price et al., 1998; Liston et al., 1999; Wang et al., 1999). However, most studies were concentrated on subgen. Pinus, or the hard pines (Li, 1997) and/or the sect. Strobus of the subgen. Strobus with a few samples in sect. Parrya (Wang et al., 1999). In contrast, sect. Parrya Mayr received less attention. Bailey (1970) and Malusa (1992) studied two American subsections of it, respectively. Zhang et al. (2003) studied systematic position of the Chinese endemic species P. squamata. As a very heterogeneous section, Parrya comprises several subsections in different taxonomic treatments of Pinus (Shaw, 1914; Pilger,1926; Little and Critchfield, 1969; van der Burgh, 1973; Farjon, 1984; 1996; Price et al., 1998). The common characters (maybe a symplesiomorphy) among species of sect. Parrya are only found in the wood anatomy: ray tracheids with smooth walls and ray cells with small pits (Shaw, 1914; Mirov, 1967; Farjon, 1984). In Shaw s (1914) classification, species of sect. Parrya were recognized to be included in the subsection of Paracembra, which comprised three groups, Group Gerardianae, Group Balfourianae and Group Cembroides. In comparison with Shaw s system, Pilger s classification shows some changes in the arrangement of sect. Parrya (named as sect. Paracembra in original literature). P. krempfii, which is one of the most morphologically unique species in Pinus, was added and placed close to P. balfouriana and P. aristata. Little and Critchfield (1969) arranged these pines in their classification of Pinus in a way very similar to that of Shaw (1914), while they placed P. krempfii in a independent subgenus, subgen. Ducampopinus (A. Cheval) de Ferrérather than in the subgen. Strobus. The classification of van der Burgh (1973) Received 17 Jun Accepted 16 Aug Supported by the Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX B). * Author for correspondence. <dezhuli@hotmail.com>.

2 differed significantly in structure from that of Little and Critchfield (1969). In his system, the sect. Parrya was divided into three subsections: Balfourianae (including P. krempfii), Gerardianae and Cembroides. This treatment of sect. Parrya was followed by Farjon (1984) with some amendments. Farjon (1984) pointed out that sect. Parrya was very heterogeneous, which was divided into six subsections, i.e. Krempfianae, Balfourianae, Aristatae, Gerardianae, Nelsoniae and Cembroides. This classification was later revised (Farjon, 1996), with a distinct change which placed P. rzedowskii as a monotypic subsection in sect. Parrya. Price et al. (1998) evaluated all data available to them and proposed a comprehensive system of pines in which sect. Parrya was treated to be comprised of subsections of Balfourianae, Krempfianae, Cembroides, Rzedowskianae and Gerardianae. Little data was available for the newly discovered Chinese species P. squamata X. W. Li at that time, which was tentatively placed in subsect. Gerardianae with an annotation of probability of change to be a new subsection. Increasing studies especially evidence from molecular systematics demonstrated that sect. Parrya was paraphyletic (Strauss and Doerksen, 1990; Govindaraju et al., 1992; Wang and Szmidt, 1993; Price et al., 1998; Liston et al., 1999; Wang et al., 1999). However, the subsectional relationships were not well resolved because of insufficient samples (Strauss and Doerksen, 1990; Govindaraju et al., 1992; Wang and Szmidt, 1993; Wang et al., 1999) or for the high levels of divergence of sequences (Liston et al., 1999). The chloroplast matk gene sequence has been found to be more variable than other coding cpdna sequences tested (Wolfe, 1991). Wang et al. (1999) used matk combined with other three cpdna sequences to reconstruct phylogenetic relationships of Eurasian pines, but only five species of two subsections in Parrya were sampled in their analysis. In this study, we sampled most species of sect. Parrya representing all subsections described by previous authors and some little known species such as P. rzedowskii, P. nelsonii and P. squamata. Our main objectives in this study were: (1) to provide additional information for the assessment of relationships among subsections and species in sect. Parrya using matk gene sequence data; and (2) to study the phylogenetic relationships of several little known taxa in the light of molecular phylogenetics based matk gene divergence. 1 Materials and Methods 1.1 Species sampled Twenty-one species were included in this study (Table 1), including three species of sect. Strobus, 15 species of sect. Parrya representing all subsections and three species of subgen. Pinus as outgroups. Because section Strobus was a well monophyly in previous studies (Strauss and Doerksen, 1990; Liston et al., 1999; Wang et al., 1999), only three representatives were sampled in this study. Vouchers are deposited in herbarium of Kunming Institute of Botany, The Chinese Academy of Sciences except for those whose DNA samples were provided by Dr. A. LISTON of the Oregon State University, USA. The matk sequences of 11 species were downloaded from GenBank, others were sequenced during this study. 1.2 DNA isolation, PCR amplification and sequencing Silica-gel-dried leaves or leaves from herbarium specimens were used for genomic DNA isolation. Part of DNA samples were from Dr. Aaron Liston. Genomic DNA was isolated from needles using a modified CTAB procedure of Doyle and Doyle (1987). The primers for matk were the same as those used by Wang et al. (1999). The PCR reaction volumes (20 µl) contained 1.5 U of AmpliTaq DNA polymerase (Perkin-Elmer), Replitherm TM buffer, 1.5 mmol/l MgCl 2, 1 mmol/l dntp, 0.15 µmol/l primer and ng of sample DNA. PCR reactions were performed in a GeneAmp 9600 (Perkin-Elmer, Applied Biosystems). PCR amplification was carried out at 94 for initial denaturation, followed by 30 cycles of denaturation at 94 for 45 s, primer annealing at 58 for 50 s, an extension at 72 for 80 s, and a termination at 72 for 5 min. PCR products were purified by Watson s purification kit prior to being sequenced. Double-stranded purified PCR products were sequenced by using the Dideoxy Chain Termination method with an ABI PRISM TM Bigdye Terminator Cycle Sequencing Ready Reaction Kit and AmpliTaq DNA polymerase FS (Perkin- Elmer, Norfolk, Connecticut). Reactions and programs were chosen according to recommendations of the manufacturers, with slight modification in some cases. Samples were electrophoresed on an ABI 310 Genetic Analyzer (Applied Biosystems Inc.) Phylogenetic analyses DNA sequences were edited and aligned using SeqMan and Megalign (DNASTAR), and adjusted manually where necessary. In all phylogenetic analyses, characters were weighted equally. Maximum parsimony (MP) analysis was performed using PAUP 4.0b (Swofford 2001) treating gaps as the fifth character. We used the heuristic search options with random replications of stepwise data addition and TBR branch-swapping. Tree fit measures from the MP analysis were calculated using consistency (CI) and retention (RI)

3 ZHANG Zhi-Yong et al.: Molecular Phylogeny of Section Parrya of Pinus (Pinaceae) Based on Chloroplast matk Gene Sequence Data Table 1 List of species sampled, system of classification followed to Price et al. (1998) Subgen. Sect. and subsect. Species GenBank accession No. Voucher information Strobus Lemmon Sect. Strobus Subsect. Cembrae Lordon Pinus koraiensis Siebold et Zuccarini ABO19834 Subsect. Strobi P. armandii Franchet ABO19841 P. wallichiana Jackson ABO19838 Sect. Parrya Mayr Subsect. Balfourianae Engelmann P. aristata Engelmann ABO19842 P. balfouriana Balfour ABO19843 P. longaeva Bailey AY DSG Subsect. Gerardianae Loudon P. bungeana Zuccarini ex Loudon ABO19845 P. gerardiana Wallich ex D. Don ABO19844 P. squamata X. W. Li AF Zhang 001 Subsect. Cembroides Engelm. P. cembroides Zuccarini AY Zhang 003 P. quadrifolia Parlatore AY Zhang 004 P. maximartinezii Rzedoski AY Zhang 005 P. remota (Little) Bailey AY Zhang 002 P. monophylla Torrey et Frémont AY DSG P. pinceana Gordon AY DSG P. nelsonii Shaw AY DSG Subsect. Krempfianae P. krempfii Lecomte ABO19831 Little et Critchfield Subsect. Rzedowskianae Carvajal P. rzedowskii Madrigal et Caballero AY EAB-94 Pinus (outgroups) P. massoniana Lambert ABO19852 P. tabulaeformis Carrière ABO19853 P. yunnanensis Franchet ABO19847 *, downloaded from GenBank. indices. A genetic distance tree was also constructed by using the neighbor joining (NJ) method (Swofford, 2001). 2 Results 2.1 Sequences analysis The overall characteristics of pines matk gene were discussed by Wang et al. (1999). The results showed that the matk sequences analyzed in their study had a distinctly higher evolutionary rate than rbcl sequence within subgen. Pinus. In addition, the variation of matk in Pinus was even higher than that in some noncoding regions of cpdna. However, the matk diverged at a rate very similar to rbcl within their samples of subgen. Strobus. In our study the average divergence for matk within sect. Parrya was (Juke-Cantor distance), a little higher than the average divergence within subgen. Strobus. No alignment gaps were found among the taxa of sect. Parrya. The aligned sequences have 937 characters, among them 63 are variable and 43 are parsimony-informative characters. 2.2 Phylogenetic analyses A strict consensus tree of four most parsimonious trees resulted from the unweighted parsimony analysis is shown in Fig.1. The monophyly of subgen. Strobus was strongly supported. However, within the sect. Parrya, only the American subsect. Balfourianae, including P. aristata, formed a weakly supported clade with a bootstrap value of 54%. The sampled species of sect. Strobus nested in a well supported clade comprise Asian members of sect. Parrya (92%, bootstrap value). Within this topology, P. krempfii was unexpectedly grouped with P. gerardiana, but with low bootstrap support (61%). The remaining species of sect. Parrya all from North America, formed a polytomy with little resolution. The topology of the majority rule tree (Fig. 2) was slightly different from the strict consensus tree. The monophyly of subgen. Strobus, and a clade comprised Asian members of sect. Parrya and sect. Strobus were resolved with strong bootstrap support. All American species of sect. Parrya formed a poorly resolved clade with low bootstrap support (only 53%). Again, the three sampled species of sect. Strobus was strongly supported as monophyletic but nested within a clade comprised Asian members of Parrya. The monophyly of subsect. Balfourianae was weakly supported with a bootstrap value of 54%. P. rzedowskii was grouped with P. monophylla with low bootstrap support (less than 50%). The relationships among members of subsect. Cembroides were still unclear on the majority rule tree. Again, because sect. Strobus was nested a clade

4 Fig.1. Strict consensus tree of four most parsimonious trees based on the matk sequences of 21 Pinus species. CI=0.929; RI=0.959; Tree length is 70 steps. Bootstrap values > 50% are indicated above branches. The species of sect. Parrya are indicated in broadened lines. comprised Asian members of Parrya, the section became paraphyletic. The topology of the neighbor joining tree (Fig.3) was somewhat similar to that of the majority rule tree but with slightly better resolution. The monophyly of Asian members of Parrya together with the three sampled species of the subgen. Strobus formed a monophyletic clade with a bootstrap support of 96%. However, the American members of the section formed a clade with bootstrap support below 50%. Within this topology, P. monophylla and P. rzedowskii grouped together with low bootstrap support (60%). Except for P. monophylla and P. cembroides, all other members of subsect. Cembroides formed an assemblage with bootstrap support below 50%. The subsect. Balfourianae was also weakly supported with a bootstrap value of 51%. 3 Discussion 3.1 Systematic implications As pointed out by earlier authors (Strauss and Doerksen, 1990; Wang and Szmidt,1993; Wang et al., 1999; Liston et al., 1999), sect. Parrya was a paraphyletic group basal in subgen. Strobus. Based on our majority rule tree and NJ tree, American species of sect. Parrya formed a branch and may be sister to the clade consisted of Asian members of sect. Parrya and sect. Strobus. Although the former branch was weakly supported, the later was strongly supported with high bootstrap value. Similar topology was also suggested by Wang et al. (1999) but without the samples of subsect. Cembroides and subsect. Rzedowskianae. On

5 ZHANG Zhi-Yong et al.: Molecular Phylogeny of Section Parrya of Pinus (Pinaceae) Based on Chloroplast matk Gene Sequence Data Fig.2. Majority rule tree of four most parsimonious trees of 70 steps based on matk sequences. Bootstrap values > 50% are indicated above branches. The species of sect. Parrya are indicated in broadened lines. Strauss and Doerksen s (1990) trees, the representative of Asian species of sect. Parrya, P. gerardiana, was also clustered with members of sect. Strobus. All these studies implied that Asian members of sect. Parrya and sect. Strobus had close affinities, which may be one of early clades along with the clade of American species of sect. Parrya after the genus Pinus was evolved into two lineages of subgenera Strobus and Pinus. In the lineage of Asian members of sect. Parrya and sect. Strobus, the relationships of Asian species of sect. Parrya were still poorly resolved. P. krempfii, the morphologically unusual species (with two peculiar, flat, sometimes hypostomatic leaves) endemic to south central Vietnam (Dallat Plateau), was grouped with P. gerardiana. This is beyond our expectation, because P. krempfii is so morphologically special that Chevalier (1944) elevated this species to an independent monospecific genus in Pinaceae and named it Ducampopinus krempfii. Some authors created a separate subgenus, Ducampopinus, in the genus Pinus to accommodate it (de Ferr, 1953; Gaussen, 1960; Little and Critchfield, 1969). However, more and more studies showed that it has some linkages with subsect. Gerardianae in respect of its cone morphology (Mirov, 1967), wood chemistry (Erdtman et al., 1966), cpdna PCR- RFLP variation (Wang et al., 2000) and cpdna sequences variation (Wang et al., 1999). Our results may reflect the essence of their relationships. It is clear that P. krempfii is a member of the subgen. Strobus but its relationships with

6 Fig.3. Neighbor joining tree of sect. Parrya based on the matk sequences of 21 Pinus species. The distances are marked above each branch, bootstrap values above branches or denoted by arrows (in boldface). other members of the subgenus need to be further investigated. P. squamata X. W. Li is an extremely endangered species of pine recently described from the northeastern corner of Yunnan Province, China (Li, 1992). Because of its peculiarity, e.g. five needles per bundle and long-winged seed, a new series, Squamatae X. W. Li et Hsueh (subgen. Strobus sect. Parrya), was established to accommodate this species. On the basis of an analysis of chemical constituents of seed oils of P. bungeana and seven other related pines, Li and Zhu (1993) suggested that P. squamata should be a member of the central American subsect. Balfourianae of their new proposed subgen. Parrya. Our previous study based on four cpdna sequences and ITS sequence data revealed that it was a member of the subsect. Gerardianae (Zhang et al., 2003a). In the present study, P. squamata was a stable member of the clade comprising Asian members of the sect. Parrya and members of sect. Strobus. But its relationships with P. bungeana and P. gerardiana were not resolved in this study, which maybe attribute to the low divergence rate of matk gene in Pinus. As for the assemblage of American members of sect. Parrya, only P. aristata, P. balfouriana and P. longaeva formed a monophyletic group on different trees with low bootstrap support, which corresponding to subsect. Balfourianae. These pines thought to be a natural group and closely related tertiary relics confined to high elevations in certain mountainous areas of southwestern states in America. P. aristata was a member of subsect. Balfourianae and the subsect. Aristatae (van der Burgh,

7 ZHANG Zhi-Yong et al.: Molecular Phylogeny of Section Parrya of Pinus (Pinaceae) Based on Chloroplast matk Gene Sequence Data 1973) seemed to be unnecessary. The relationships within subsect. Cembroides and P. rzedowskii were not resolved on the NJ tree. However, P. rzedowskii and P. monophylla clustered together, except for P. cembroides, other members of subsect. Cembroides formed an assemblage. The result implied that subsect. Cembroides might not be a monophyletic group. Malusa (1992) suggested that some members of subsect. Cembroides were more closely related to taxa not in subsect. Cembroides, and the pinyon pines (subsect. Cembroides sens. lato.) were a paraphyletic group according to his cladistic analysis. The results based on the molecular systematic analysis of ITS (Liston et al., 1999) also showed that subsect. Cembroides was not monophyletic. P. rzedowskii, the poorly known pine, had been added to sect. Parrya (Klaus, 1989) for its similar feature of wood anatomy, but there was no evidence for this relationship (Malusa, 1992). Taxonomically, Pinus rzedowskii is one of the most interesting species in the genus. Its foliar morphology and anatomy places it close to P. cembroides and P. maximartinezii. Preliminary results from the analysis of cpdna of a limited number of Mexican pine species place it and P. cembroides as unresolved clades away from P. maximartinezii and other species in subgen. Strobus (Pérez de la Rosa et al., 1995). The elongation of shoots prior to leaf development is shared with P. maximartinezii. The bark of P. rzedowskii is unlike any other in Strobus and at least externally resembles that of species in subgen. Pinus. The seed cones and seeds are also morphologically more similar to some species in subgen. Pinus. The seeds are likewise small, with well-developed, articulate wings three to four times larger than the seeds. In this respect there is no resemblance to either P. cembroides or P. maximartinezii, nor to any other pinyon pine, in which the seeds are large and the wings vestigial. The present analysis suggested that P. rzedowskii might have a close relationship with P. monophylla, in contrast with previous results, but its position is not consistent on different trees and may collapse. The phylogenetic relationships of this peculiar pine need more studies. P. nelsonii was described by Shaw (1914) based on E. W. Nelson s collection in June 1898 on a mountain above Miquihuana, Tamaulipas, near the border with Nuevo León. The very distinct connate leaves and the long pedunculate, curious cones uniquely distinguished this pine from all others that Shaw (1914) had known. Its connate leaves, held together by persistent fascicle sheaths, are morphologically and anatomically distinct from any of the other pinyon pines. So van der Burgh (1973) elevated P. nelsonii to a separate monotypic subsect. Nelsoniae, and Farjon (1996) had also accepted the subsection but as a ditypic group together with P. pinceana. However, other author still placed P. nelsonii in subsect. Cembroides (Price et al., 1998). Pérez de la Rosa et al. (1995) examined restriction site variation in PCR-amplified fragments representing 12 Mexican pine species and a single Picea species, revealing that P. nelsonii had a isolated position. The results of ITS sequences analysis showed that P. nelsonii was basal to all haploxylon pines. Our result preferred to support that P. nelsonii was a real pinyon pines and should be included in subsect. Cembroides. But because of the low bootstrap support, the phylogenetic relationships of P. nelsonii merit more investigation. 3.2 Biogeographical implications In consideration of the paraphyly of sect. Parrya, we should therefore be careful about any previous biogeographical studies on it. Strauss and Doerksen (1990) suggested that the high diversity among species within sect. Parrya was a reflection of the section s ancientness. Many of its current species and taxa may be relicts with long evolutionary histories. Its biogeography was complex, suggesting the possibility of more than one intercontinental migration. Strauss and Doerksen (1990) also suggested that the ancestral species in Parrya maybe originated from North America, their progenitors appear to have given rise to a currently Asian group of species, subsect. Gerardianae, which then gave rise to the widespread sect. Strobus, which occurs over substantial parts of both continents. In the present analysis we are not sure if the species of sect. Parrya in North America are more ancestral than Asian members of the section. The members of American sect. Parrya are sister group to Asian sect. Parrya and sect. Strobus as shown in Fig.2 and Fig.3, which implied that the progenitors of subgen. Strobus might give rise to two or more clades of sect. Parrya very early and the ancestors of subsect. Gerardianae may then give rise to species of sect. Strobus subsequently. Some descendants of sect. Strobus migrated into America through land bridges. Most members of sect. Parrya disappeared from many middle-latitude areas during early Tertiary and were replaced by the diverse angiosperms of boreotropical flora, which were adapted to the equable, tropical climate (Millar, 1993). A few species of sect. Parrya shifted to some refuges during the warm period of early Tertiary. Subsect. Cembroides seems to have been limited to western North America and Central American refuges, the small subsect. Balfourianae appears to have been entirely concentrated in middle-latitude Rocky Mountain refuge and a Tethys refuge area of

8 pines in Southeast Asia also have fossil evidence and evidence from extant pines such as P. krempfii, P. gerardiana, P. bungeana and the recently-described P. squamata. Nevertheless, the destinies of species in different refuges are different. Within subsect. Cembroides, extensive radiation and speciation were triggered by the active mountainbuilding, cooling and drying of the climate afterward. For Asia refuges, the climates were relatively stable and fit for the development of angiosperms, so the species still restrict in refuges and have little chance to radiation and speciation. For certain species such as P. squamata, the living conditions are still threatened by broad-leave trees (Zhang et al., 2003b). As revealed in the majority rule tree (Fig.2) which was supported by earlier authors (Liston et al., 1999; Wang et al., 1999) and our combined analysis of five cpdna sequences (Zhang, 2003), there were two major evolutionary lineages in Pinus, i.e. subgen. Pinus and subgen. Strobus. The Strobus lineage was separated into two groups, the American members of sect. Parrya as one, and a clade consisted of sect. Strobus and Asian members of the sect. Parrya as the other. This distribution pattern was similar to some angiosperm taxa (Wen, 1999) such as bamboos (Clark et al., 1995). Acknowledgements: We are very indebted to Dr. Aaron Liston for his kindly providing DNA samples and to Dr. Wang Xiao-Ru for her kindly providing data matrix. Hearty thanks are due to Dr. Zsolt Debreczy for his presenting many experimental materials. We thank Miss Tian Xin for her reading an earlier version of the manuscript. References: Bailey D K Phytogeography and taxonomy of Pinus subsect. Balfourianae. Ann Mo Bot Gard 57: Chevalier A Notes sur les Coniféres de l lndochine. Rev Bot Appl Agric Tropicale, 24:7 34. Clark L G, Zhang W, Wendel J F A phylogeny of glass family (Poaceae) based on ndhf sequence data. Syst Bot, 20: Doyle J J, Doyle J L A rapid DNA isolation procedure for small quantities of fresh leaf material. Phytochem Bull 19: Erdtman H, Kimland B, Norin T Wood constituents of Ducampopinus krempfii (Lecomte) Chevalier (Pinus krempfii Lecomte). Phytochemistry, 5: Farjon A Pines: drawings and Descriptions of the Genus Pinus. Leiden: E. J. Brill & Dr W. Baxkhuys. Farjon A Biodiversity of Pinus (Pinaceae) in Mexico: speciation and palaeo-endemism. Bot J Linn Soc, 121: de Ferr Division de genre Pinus en quatres sous-genres. Compt Rend Hebd Séances Acad Sci (Paris), 236: Gaussen H Les Gymnosperms Actuelles et Fossils. Fascicle VI. Les Coniferales. Chap. XI. G n ralit s, Genre Pinus. Toulouse: Travaux du Laboratorie Forestier de Toulouse Govindaraju D, Lewis P, Cullis C Phylogenetic analysis of pines using ribosomal DNA restriction fragment length polymorphisms. Plant Syst Evol, 179: Klaus W Mediterranean pines and their history. Plant Syst Evol, 162: Li D Z A reassessment of Pinus subgen. Pinus in China. Edinb J Bot, 54: Li X-P, Zhu Z-D Studies on constituents of fatty acids from seed oils of Pinus bungeana and its taxonomic position. J Nanjing Forest Univ, 17: (in Chinese with English abstract) Li X-W A new series and a new species of Pinus from Yunnan. Acta Bot Yunnan, 14: (in Chinese with English abstract) Liston A, Robinson W A, Pinero W D, Alvarez-buylla E R Phylogenetics of Pinus (Pinaceae) based on nuclear ribosomal DNA internal transcribed spacer region sequences. Mol Phylogenet Evol, 11: Little E L Jr, Critchfield W B Subdivisions of the Genus Pinus. Washington DC: USDA Forest Service Miscellaneous Publication Malusa J Phylogeny and biogeography of the pinyon pines (Pinus subsect. Cembroides). Syst Bot, 17: Millar C I Impact of the Eocene on the evolution of Pinus L. Ann Mo Bot Gard, 80: Mirov N T The Genus Pinus. New York: Ronald Press Company. Pérez de la Rosa, Harris S A, Farjon A Noncoding chloroplast DNA variation in Mexican pines. Theor Appl Genet, 91: Pilger R Pinus. Engler A, Prantl K. Die Naturlichen Pflanzenfamilien. Vol. Leipzig: Wilhelm Engelmann Piovesan G, Pelosi C, Schirone A, Schirone B Taxonomic evaluations of the genus Pinus (Pinaceae) based on electrophoretic data of salt soluble and in soluble seed storage proteins. Plant Syst Evol, 186: Price R A, Liston A, Strauss S H Phylogeny and systematics of Pinus. Richardson D M. Ecology and Biogeography of Pinus. Cambridge, UK: Cambridge University Press Schirone B, Piovesan G, Bellarosa R, Pelosi C A taxonomic analysis of seed proteins in Pinus spp. (Pinaceae). Plant Syst Evol, 178: Shaw G R The Genus Pinus. Massachusetts: The Murray

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