Maternal Lineages of Pinus Densata, a Diploid Hybrid

Molecular Ecology (2002) 11, 1057–1063

BlackwellMaternal Science, Ltd lineages of Pinus densata, a diploid hybrid

BAO-HUA SONG,* XIAO-QUAN WANG,* XIAO-RU WANG,*† LAN-JU SUN,* DE-YUAN HONG* and PEI-HAO PENG‡ *Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China, †National Institute for Working Life, S-907 13 Umeå, Sweden, ‡Sichuan Academy of Forestry, Chengdu 610081, China

Abstract Previous morphological, allozyme and chloroplast DNA data have suggested that Pinus densata originated through hybridization between P. tabuliformis and P. yunnanensis. In the present study, sequence and restriction site analyses of maternally inherited mitochondrial nad1 intron were used to detect variation patterns in 19 populations of P. tabuliformis, P. yunnanensis and P. densata. A total of three mitotypes (A, B, C) were detected. All but one of the populations of P. yunnanensis possessed mitotype B while all populations of P. tabuliformis had mitotype A. Pinus densata populations, on the other hand, harboured both mitotypes A and B, which are characteristic of P. tabuliformis and P. yunnanensis, respectively. This result gives strong additional evidence supporting the hybrid origin of this diploid pine. The distribution of mitotypes indicated very different mating compositions and evolutionary history among P. densata populations. It seems that local founder populations and backcrosses may have played important roles in the early establishment of P. densata populations. The uplift of the Tibetan Plateau had a significant impact on the distribution of maternal lineages of P. densata populations.

Keywords: hybridization, mitochondrial DNA, mitotype, nad1 intron, Pinus, speciation Received 22 November 2001; revision received 18 February 2002; accepted 22 February 2002

is morphologically and anatomically intermediate between Introduction P. tabuliformis and P. yunnanensis, its two putative parents Hybridization among species may have several evolutionary (Wu 1956; Mirov 1967; Guan 1981; Wang & Szmidt 1994). consequences, including increased intraspecific genetic The three species have very different ecological require- diversity, the transfer of genetic adaptations, the origin of ments and do not form broad overlaps (Mirov 1967; Cheng new ecotypes or species, and the reinforcement or breakdown 1983; Li & Liu 1984; Wang & Szmidt 1994). In addition, of reproductive barriers (Anderson 1948; Stebbins 1950; P. densata is mainly distributed at high mountain eleva- Grant 1981; Ellstrand & Elam 1993; Whitham et al. 1994; tions from 2700 m to 4200 m, where neither of the potential Rieseberg & Gerber 1995; Levin et al. 1996; Rieseberg 1997). parents can normally grow (Guan 1981; Li & Liu 1984). Pre- Although reticulate evolution played a very important role vious allozyme and chloroplast (cp) DNA analyses have in the establishment of species diversity in plants (Arnold provided much new evidence supporting the hybrid origin 1997; Rieseberg 1997), homoploid hybrid speciation is not of P. densata (Wang et al. 1990, 2001; Wang & Szmidt 1990, as common as polyploid speciation due to a combination of 1994; Yu et al. 2000). It was further suggested that the spe- factors such as hybrid sterility, hybrid breakdown and ciation of P. densata was related to the uplift of the Tibetan difficulties in evolving reproductive isolation in sympatry Plateau and that a third unknown or extinct taxon, apart (Grant 1981). from P. tabuliformis and P. yunnanensis, might have also Pinus densata represents an example of homoploid been involved in its origin (Wang & Szmidt 1994; Wang hybrid speciation in gymnosperms. It has a huge distri- et al. 2001). However, these studies could not fully reveal bution in western Sichuan, the northwestern corner of the hybridization history at the population level, nor the Yunnan, and southeastern Tibet (Fu et al. 1999). The species stabilization mechanisms of this advanced hybrid, due to insufficient sampling of the hybrid populations in the Correspondence: Dr Xiao-Quan Wang. Fax: 86–10–62590843; allozyme analysis and lack of maternal information in the E-MAIL: [email protected] cpDNA analysis.

Pines transmit their mitochondria (mt) and chloroplast investigate the variation patterns of the nad1 intron in 19 genomes through opposite sex gametes, mtDNA being populations of P. densata, P. yunnanensis and P. tabulae- inherited maternally and cpDNA being inherited pater- formis and ask the following questions. Do the new mtDNA nally (Neale & Sederoff 1989; Mogensen 1996). The dif- data support the hybrid hypothesis of P. densata? Do differ- ferent uniparental inheritance of mtDNA and cpDNA ent P. densata populations vary in their maternal lineages? provides an exceptional opportunity for studying paternal What are the possible pathways of the hybrid population and maternal genetic lineages within a single species establishment? (Palmer 1992) and for discriminating female (seed) and male (pollen) components of gene flow across hybridizing Materials and methods species and populations (Ennos 1994; Watano et al. 1996). Although uniparental inheritance of organellar genomes Population sampling results in molecular lineages being established in populations, which in turn may be interpreted in terms of A total of 295 trees from 19 natural populations of Pinus historical relationships, the phylogenetic value of pollen- tabulaeformis, P. densata and P. yunnanensis were sampled in transmitted cpDNA markers decreases when consider- southwestern China (Fig. 1). Each population was repres- able genetic exchange takes place among the populations ented by 10–23 trees that were at least 50 m apart. The (Provan et al. 1998). In conifers, gene migration via pollen designation, location, elevation and sample size of each can be extensive, and thus the historical differentiation population are shown in Table 1. Among them, one among populations is homogenized (Ennos 1994). In con- population (ZD) of P. densata was from the sympatric trast, the maternally transmitted mitochondrial markers region with P. yunnanensis. The remaining populations of can be regarded as almost ideal tools in evolutionary individual species did not overlap. Needles were collected studies because the geographical distributions of seed- from each individual tree and preserved in silica gel. dispersed mtDNA haplotypes mirror the former gene flow through migration and are suitable for describing DNA analysis pathways of colonization and population differentiation (Gugerli et al. 2001a). Genomic DNA was extracted from silica-gel-dried Recently, variants in an intron between the exons B and needles using the modified CTAB method of Rogers & C of the mitochondrial gene encoding subunit 1 of NADH Bendich (1988) and used as template in polymerase dehydrogenase (nad1) were successfully used in studies of chain reaction (PCR). We amplified a segment of the population differentiation and introgression of pines and nad1 intron between exons B and C using primers nad1F1 ′ ′ ′ Norway spruce (Watano et al. 1996; Senjo et al. 1999; Isoda (5 -GATCGGCCATAAATGTACTCC-3 ) and nad1R1 (5- et al. 2000; Mitton et al. 2000; Soranzo et al. 2000; Gugerli CCCCATATATTCCCGGAGC-3′). These two primers et al. 2001a, 2001b; Sperisen et al. 2001). In this study, we covered a region of 1492 base pairs (bp) relative to the P.

Fig. 1 The distribution of mtDNA haplotypes in populations of Pinus tabuliformis, P. densata and P. yunnanensis sampled in this study. Open circles represent haplotype A; filled circles represents haplotype B; partially filled circle demonstrates the coexistence of haplotypes A and B; and the star indicates haplotype C. The area framed with dash line shows the presumed historical hybridization zone. The prefixes T-, D-, Y- represent P. tabuliformis, P. densata and P. yunnanensis, respectively.

Table 1 Populations of Pinus species analysed in this study and their mitotype composition

Population Sample Latitude/ Elevation Mitotype Species Province code size Longitude (m) scored Chlorotype scored*

P. densata TibetBS 10 30 °00′/94°00′ 3450 A (Pt)† BY 23 29°36′/94°12′ 3200 A (Pt) LZ 18 29°24′/94°40′ 3100 A (Pt) Sichuan LXA 13 31°24′/103°06′ 2000 B (Py) Pd1−2: 52% Pt 32% Py 16% 3rd type LXB 11 31°24′/103°06′ 2000 B (Py) LXC 16 31°24′/103°06′ 2450−2500 B (Py) KD 15 30°00′/101°54′ 3050−3150 A (Pt) Pd3−6: 31−69% Py, no Pt, 31−69% 3rd type DB 20 30°48′/101°54′ 2750−3300 B (Py) SLK 19 29°52′/102°06′ 3600 A (Pt) Yunnan ZD 16 27°42′/99°42′ 3400 38% A (Pt); 62% B (Py) P. tabuliformis Beijing SS 16 40°48′/115°54′ 1100−1500 A Shaanxi FP 23 33°30′/107°54′ 1350−2000 A Sichuan GY 16 32°24′/105°48′ 1300−1450 A P. yunnanensis Sichuan BX 12 30°18′/102°48′ 1600 C ELS 15 29°48′/102°12′ 1650 B Yunnan LJ 15 26°48′/100°12′ 2900 B CX 10 25°00′/101°30′ 2400 B KM 13 25°00′/102°42′ 2200 B GJ 14 23°18′/103°06′ 1450 B Total 19 295

*Data from Wang & Szmidt (1994). †Pt represents P. tabulaeformis; Py represents P. yunnanensis.

sylvestris genome from position 472 to 1963 (Soranzo et al. ware clustal W (Thompson et al. 1994). The obtained 2000). The PCR amplification was carried out in a volume sequence variation pattern was used to search for restric- of 25 µL, containing 5–50 ng of genomic DNA, 6.25 pmol tion site polymorphisms. One restriction enzyme HaeIII of each primer, 0.2 mm of each dNTP, 2 mm MgCl2 and had a recognition site on the identified polymorphic 0.75 Units of Taq DNA polymerase. Amplification was sequence region (Fig. 2) and created two different restric- conducted in a GeneAmp PCR System 9600 (Perkin-Elmer) tion fragment length polymorphism (RFLP) patterns that programmed as follows: 4 min for initial incubation at differentiate two mitotypes. Thus, this enzyme was 70 °C, followed by four cycles of 2 min at 94 °C, 20 s at selected to analyse the nad1 intron segment of the remain- 50 °C, 2 min at 72 °C, and then 36 cycles of 20 s at 94 °C, 20 s ing individuals except those of BX. The digestions of the at 50 °C, and 2 min at 72 °C, with a final extension step of nad1 intron were implemented by 1-h incubation at 37 °C 6 min at 72 °C. The PCR products were purified using in the presence of 1 unit HaeIII and 1× restriction buffer GFX™ PCR DNA and a Gel Band Purification Kit (Takaya). The cleaved products were resolved on 2.5% (Pharmacia). Sequencing reactions were performed with agarose gel and visualized under UV light. the two primers listed above and an internal primer nad1R3 (5′-CGAGGTACTATAAGAGGCAC-3′) to cover the Results whole PCR segment using ABI Prism Bigdye™ Terminator Cycle Sequencing Ready Reaction Kit on GeneAmp PCR To obtain information about the degree of sequence System 9600 (Perkin Elmer). The sequencing reaction variation among individuals and populations of the three products were purified through precipitation with 95% pine species, we first sequenced four to eight individuals ethanol and 3 m sodium acetate (pH 5.2) and then applied from each of the 19 populations. Relative to the Pinus to the ABI 377 automatic sequencer. sylvestris nad1 intron, which is 2376 bp long (Soranzo et al. A total of 102 individual trees (four to eight per popula- 2000), our sequenced region covered ≈ 63% of the intron. In tion) were initially sequenced for the whole PCR amplified general, sequence variation in this region was very low segment. Sequence alignment was made using the soft- among our samples. The aligned 102 individual sequences

Fig. 2 Positions of the primers used in the present study and the three different mitotypes detected from nad1 intron of P. densata, P. tabuliformis and P. yunnanensis. A HaeIII recognition site is shown.

were 1183 bp in length. The variants were mainly in the region from position 345 to 371, starting from the primer nad1F1 site (Fig. 2). No polymorphism was detected in the whole region covered by primers nad1R1 and nad1R3. A total of three mtDNA sequence types, designated as mitotypes A, B and C, respectively (Fig. 2), were detected among the 102 individual nad1 intron sequences. Haplotype A (1172 bp) differed from haplotype B (1179 bp) in both length and sequence. In mitotype B, there were two variants (B1, B2) differing by a single nucleotide substitution (Fig. 2), but they were treated as the same mitotype. Haplotypes B and C (1183 bp) differ by single nucleotide substitution and four 1-bp insertions in mitotype C. These insertions were single nucleotide additions to four mono- Fig. 3 Minimum spanning tree generated by ntsys (version 2.02a) for mitotypes of nad1 intron. Numbers at nodes indicate the nucleotide short repeats, compared to mitotype B. Thus, numbers of variable sites. mitotypes B and C are closely related. A minimum spanning tree (Fig. 3), showing the relationship of the four mitotypes, was constructed using ntsys version 2.02a the sequence and RFLP data are given in Table 1 and their (Rohlf 1970) based on the Tamura–Nei distance (Tamura & geographical distribution is illustrated in Fig. 1. Five of the Nei 1993). The four mitotypes were divided into two six P. yunnanensis populations possessed mitotype B, while groups: one consisted of mitotype A and the other of B (B1 all 12 individuals of the northernmost population (BX) had and B2) and C. Analysis of the 102 individual sequences mitotype C. All three populations of P. tabuliformis (SS, FP, revealed that mitotype C was restricted to population BX GY) had mitotype A. In P. densata, both mitotypes A of P. yunnanensis, while the other five populations of P. and B were detected. Among the 10 populations of P. yunnanensis had mitotype B. It is therefore likely that densata, five populations (LZ, BY, BS, KD and SLK) had mitotype C is a subtype derived from mitotype B. The mitotype A and four (DB, LXA, LXB, LXC) had mito- sequences of the three mitotypes A, B and C were type B (Table 1). Interestingly, population ZD from the registered in GenBank with accession numbers AF440388, southern margin of the distribution of P. densata, which AF440385/AF440386 and AF440387, respectively. overlaps with that of P. yunnanensis (Fig. 1), had both mito- RFLP analysis with HaeIII showed two different banding types A and B. Of the 16 individuals analysed six (38%) patterns corresponding to mitotypes A and B. Mitotype B were of mitotype A and 10 (62%) were of mitotype B. had eight HaeIII restriction sites in the nad1 intron segment amplified. Due to the sequence variation in the region indi- Discussion cated in Fig. 2, one HaeIII site was absent in mitotype A. This difference created a mitotype A RFLP pattern with a In genetic analysis of hybrid populations, the markers 382-bp fragment, while the corresponding fragment in should show stable qualitative differences between parental mitotype B was 339 bp + 50 bp. This RFLP marker can be species and little intraspecific variation (Rieseberg et al. easily scored by agarose gel electrophoresis. Mitotype C, 1988, 1990; Szmidt 1990; Wang & Szmidt 1990; Rieseberg & however, cannot be differentiated from mitotype B by Ellstrand 1993). Recently, mtDNA has been frequently RFLP analysis. Thus, the remaining six individuals of used as a species-specific marker (Sutton et al. 1991; Sinclair population BX were also analysed by sequencing. The et al. 1999; Mitton et al. 2000; Sperisen et al. 2001). The RFLP analysis was conducted on the remaining 187 indi- present study showed that the markers we employed are viduals from 18 populations of the three species. stable and can be regarded as species specific. All The mitotype compositions of each population based on populations of Pinus tabuliformis were fixed for mitotype

A. In P. yunnanensis, five out of six populations were fixed Pd-6 derived from hybridization events in the opposite for mitotype B. Mitotype C (a derivative of type B) was direction. restricted to the northernmost population (BX) of the Some studies have shown abundant mitotype poly- species. We can thus regard mitotype B as typical of morphisms within hybrid zones or populations (Watano et al. P. yunnanensis. 1996; Senjo et al. 1999). The distribution of polymorphisms In P. densata both mitotypes (A and B) characteristic of in the advanced hybrid populations can be used to retrieve the putative parents were detected (Table 1). Five of the 10 the history of hybrid population establishment. Among the P. densata populations had mitotype A, another four had 10 P. densata populations, five had P. tabuliformis as their mitotype B, and one had both. This result lends strong maternal lineage and four P. yunnanensis, while one had additional support to the hybrid nature of P. densata and both. Several processes could contribute to this variation in the involvement of P. tabuliformis and P. yunnanensis in its maternal lineages among populations. Firstly, the hybrid origin. In addition, due to the maternal nature of mtDNA populations might have experienced drastic founder effects in pines, our results also indicate that both P. tabuliformis by which the mitotype was purified. This phenomenon and P. yunnanensis have acted as mothers in the hybridiza- was observed during postglacial re-colonization of Norway tion events. spruce in the western Alps (Gugerli et al. 2001b). Secondly, Populations LXA to LXC of P. densata were from the low- due to the maternal nature of mtDNA markers, the mito- est altitude of the species’ distribution. Morphologically type of a hybrid population can be easily ‘captured’ and they were very similar to P. tabuliformis. However, these maintained in the following generations through random three populations possessed mitotype B, which is typical of mating among hybrid individuals, or through either direc- P. yunnanensis. Perron & Bousquet (1997) also reported a tion of back crossing (Rieseberg & Soltis 1991). In contrast lack of correspondence between morphological and mole- to other populations, population ZD of P. densata possessed cular classifications in a study on natural hybridization both mitotypes A and B. This population was from the between Picea mariana and Picea rubens, and considered sympatric region with P. yunnanensis and may represent an that molecular markers were more effective in identifying introgression zone. Yu et al. (2000) sampled nine popula- hybrids/introgressants. The same phenomenon was also tions of P. densata from the same region ZD in an allozyme observed in the hybrids of Abies veitchii and Abies homolepis analysis and found relatively high gene diversity as com- (Isoda et al. 2000). pared to other gymnosperms. This was attributed to gene Comparison of the distribution pattern of mitotypes exchange between P. densata and P. yunnanensis in that with cpDNA haplotypes, which have opposite uniparental region (Yu et al. 2000). inheritance in pines, is very informative for inferring the Anderson (1948) emphasized the importance of habitat direction of gene flow and historical components of hybrid disturbance for facilitating breakdown of premating re- populations. In a hybrid zone, Watano et al. (1996) revealed productive barriers between previously isolated parental that cpDNA introgression had occurred uni-directionally species and for providing suitable habitat for hybrid from Pinus parviflora var. pentaphylla to Pinus pumila while segregation, often causing ecological divergence of the the mtDNA introgression had occurred in the opposite hybrids from both parents (Rieseberg 1997). The uplift of direction, i.e. from P. pumila to P. parviflora var. pentaphylla. the Tibetan Plateau in the past 40 million years has caused The situation in P. densata is more complex. The localities of the regional climate to cool down markedly and changed the the P. densata populations KD, LXA, LXB and LXC, inves- regional topography. This has led to sharp changes in the tigated in the present study, are close to those of popu- vegetation (Chang 1983; Hou 1983; Ruddiman & Kutzbach lations Pd-1 to Pd-6 that were analysed for cpDNA 1991). Wang & Szmidt (1994) suggested that the drastic composition by Wang & Szmidt (1994). Populations Pd-1, geographical and climatic changes in that period could Pd-2 and LXA to LXC are close to each other, while popu- have either brought distant species together or separ- lation KD is close to Pd-3 to Pd-6. According to the present ated sympatric species. The uplift of the plateau clearly analysis and the results of Wang & Szmidt (1994), all indi- created a new territory and an opportunity for hybrids to viduals of LXA to LXC had the P. yunnanensis mitotype develop. Our combined analysis of mtDNA and cpDNA while ≈ 50% individuals of Pd-1 and Pd-2 had the cpDNA haplotypes indicates that populations sampled from haplotype of P. tabuliformis and > 30% individuals had southwest Sichuan and northwest Yunnan, i.e. the east that of P. yunnanensis. In contrast, all individuals of popu- margin of the Tibetan Plateau, are very heterogeneous, lation KD had the mitotype of P. tabulaeformis while Pd-3 to with both P. tabuliformis and P. yunnanensis maternal Pd-6 had no P. tabuliformis cpDNA component (Wang & lineages present (Fig. 1). Since mtDNA markers carry Szmidt 1994; Wang et al. 2001). These data suggest that strong historical footprints, this region was most likely populations LXA to LXC and Pd-1 and Pd-2 have derived the ancient zone of contact and hybridization between the two from one or more P. tabuliformis × P. yunnanensis hybrid- parental species in the early stage of the plateau uplift ization events, while the populations KD and Pd-3 to (Fig. 1).

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