© 2016 The Japan Mendel Society Cytologia 81(4): 409–421

A Comparative Analysis of Multi-Probe Fluorescence In Situ Hybridisation (FISH) Karyotypes

in 26 Pinus Species (Pinaceae)

Fukashi Shibata*, Yukari Matsusaki and Masahiro Hizume

Faculty of Education, Ehime University, Matsuyama, Ehime 790–8577, Japan

Received March 24, 2016; accepted July 12, 2016

Summary Every species has a unique karyotype, but certain genera have common karyptypes among species. The markers (chromosome length and centromere position) used in traditional karyotyping do not distinguish all chromosome pairs in the genus Pinus. However, the application of multi-probe fluorescence in situ hybridisation (FISH) procedures allowed exact karyotyping of 26 Pinus congeners. We used these new data to examine species relationships. The 35S rDNA and 5S rDNA, Arabidopsis-type telomere repeat sequences, and the proximal CMA band-specific repeat (PCSR) of P. densiflora were used as FISH probes for our analysis of chromosomes in 26 Pinus species. Each species had a unique FISH karyotype and most homologous chromosome pairs were identi-fied. The FISH karyotypes were used to compare corresponding or homologous chromosomes among the species. Common or similar FISH signal patterns appeared in closely related species. Species that had inherited common FISH signal patterns were classified into four karyotype groups. We used cluster analyses to compare quantitative differences in FISH signals within these groups. The results of these analyses were consistent with recent system-atic interpretations and resolved differences among existing taxonomic systems based on diverse methodologies. Our results indicate that FISH signal patterns reflect the history of species differentiation and that comparative FISH karyotyping has potential as an important tool for studying the taxonomy or phylogeny of Pinus.

Key words Chromosome, Comparative karyotyping, FISH, Pinaceae, Pinus, Phylogeny, Taxonomy.

Pinus, the largest genus in the Pinaceae, comprises ca. 100 species. Most of the congeners occur in the Northern Hemisphere (Little and Critchfield 1969, Far-jon 2005). The species are divided into three subgenera (Pinus, Strobus, and Ducampopinus) based on morpho-logical features. Subdivisions of the subgenera into sec-tions and subsections vary among different taxonomic systems. Taxonomic decisions based on morphological and molecular phylogenetic studies are not fully congru-ent, particularly at the subsection level; large differences among taxonomic systems exist at this level (Price et al. 1998, Gernandt et al. 2005).

Pinus species have a common chromosome number (2n=24) and a symmetrical karyotype. The identifica-tion of all homologous chromosome pairs is difficult because of similarities in morphology (Saylor 1972, 1983, Hizume 1988, Mehes-Smith et al. 2011). Fluo-rescent banding procedures using chromomycin A3 (CMA) and 4′,6-diamidino-2-phenylindole (DAPI) have been helpful in distinguishing some of the chromo-some pairs. Stained chromosomes of Pinus species have many fluorescent bands. Data on banding patterns can be used for comparative karyotyping in some species, and they detect inter- and intraspecific variation patterns

(Hizume et al. 1983, 1989, 1990); however, fluorescent banding does not provide sufficient information for the discrimination of all homologous chromosome pairs. Not all homologous pairs in Pinus species were identi-fied by probe signals in studies of karyotypes by FISH that used 35S rDNA and 5S rDNA probes (Doudrick et al. 1995, Liu et al. 2003, Cai et al. 2006, Bogunić et al. 2011), telomere sequences (Fuchs et al. 1995, Shibata et al. 2005, Islam-Faridi et al. 2007), or simple sequence repeats (SSR) (Pavia et al. 2014). Combined fluorescent banding and FISH investigations have identi-fied interstitial and proximal CMA bands that are consis-tent with 35S rDNA and/or the short repetitive sequence proximal CMA band-specific repeat (PCSR) loci, and interstitial DAPI bands that are consistent with signals of repetitive sequences containing the Arabidopsis-type telomere sequence (Hizume et al. 2001, Shibata et al. 2005). A FISH probe combining 35S rDNA, 5S rDNA, the Arabidopsis-type telomere sequence, and the PCSR distinguished all homologous pairs in four Pinus spe-cies, thereby allowing karyotype comparisons among these species (Hizume et al. 2002). The relationships among the four species that emerged from the FISH pat-terns were similar to the results of a phylogenetic analy-sis. Comparative FISH karyotype analysis is among the most important tools available for exploring the relation-ships of species with conserved karyotypes, such as the

* Corresponding author, e-mail: shibatafukashi@hotmail.comDOI: 10.1508/cytologia.81.409

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Pinaceae genera. A previous phylogenetic analysis of FISH signal patterns in the genus Picea based on three probes (Shibata and Hizume 2008) revealed relation-ships among the species.

In this study, we applied multi-probe FISH karyotyp-ing to the chromosomes of 26 Pinus species to explore species relationships and resolve taxonomic and phylo-genetic discrepancies between taxonomies and phylog-enies based on morphological vs. molecular data.

Materials and methods

Plant materials and chromosome preparationSources and/or localities of the seeds of 26 Pinus spe-

cies used in this study are listed in Table 1. The seeds were sown on sterilised filter papers held in petri dishes. After 10–14 days, the primary root tips were excised and treated with 0.05% colchicine for 12 h at 20°C, and fixed in a chilled solution of ethanol : chloroform : acetic acid (2 : 1 : 1) overnight. Fixed root tips were macer-ated in an enzyme mixture containing 2 or 3% cellulase Onozuka RS (Yakult), 0.5% pectolyase Y23 (Seishin), and 5 mM EDTA in 2× SSC buffer (pH 4.5) at 37°C for 30–240 min. The meristematic cells were squashed under coverslips on glass slides. The coverslips were re-moved by the dry-ice method; the glass slides were then air dried and stored in a freezer.

Fluorescence in situ hybridisationThe Arabidopsis-type telomere sequence repeats

(TTT AGG G)n were amplified using the primers (TTT AGG G)5 and (CCC TAA A)5 (Ijdo et al. 1991, Cox et al. 1993) and labelled with biotin using the BioNick Label-ing System (Invitrogen). We used EcoRI-digested plas-mid with inserted wheat 35S rDNA (pTa71; Gerlach and Bedbrook 1979) and 5S rDNA amplified by PCR using the primers 5Sl1 (CCA TCA GAA CTC CGC AGT TA) and 5Sl2 (CGG TGC ATT AAT GCT GGT AT) (Hizume 1993) from Rumex acetosa genomic DNA. EcoRI-digested wheat 35S rDNA is generally identified as 45S rDNA, but identified here as 35S rDNA following the de-scription of Garcia and Kovarik (2013). We labelled the 35S and 5S rDNAs with digoxigenin (DIG) using DIG-High Prime (Roche). The PCSR was amplified from the plasmid DNA of PDCD501 (accession no. AB051860, Hizume et al. 2001) using a short repeat-specific primer (PD501RP1, GAA ACC CCA AAT TTT) and the M13 reverse primer (M13-1, AGC GGA TAA CAA TTT CAC ACA GGA), and then labelled with reactive isothio-cyanate fluorescein (FITC) using FITC-High Prime (Roche). Our FISH procedure followed the methodology of Hizume et al. (2002). Biotin- or DIG-labelled probes were visualised with Streptavidin-Cy5 (Invitrogen) and anti-digoxigenin rhodamine conjugate (Roche). The slides were counterstained with 0.1 µg mL-1 DAPI. We recorded the FISH signals with a cooled CCD camera (Sensys 1400, Photometrics), and pseudocolour images were generated using IPLab (Scanalytics). The 5S rDNA loci were identified by the reproving method (Heslop-Harrison et al. 1992). First round FISH procedures were

Table 1. Sources and/or provenances of pine materials included in this study.

Subgenus Species Source or locality

Pinus P. canariensis Commercial source (Clyde Robin Seed Co. Inc., CA, U.S.A.)P. clausa U.S.A. (Sheffield’s Seed Co. Inc., NY, U.S.A.)P. densiflora Iyo, Ehime Prefecture, JapanP. echinata Commercial source (Clyde Robin Seed Co. Inc., CA, U.S.A.)P. elliottii var. elliottii Louisiana, U.S.A. (Sheffield’s Seed Co. Inc., NY, U.S.A.)P. halepensis Slovenia (Sheffield’s Seed Co. Inc., NY, U.S.A.)P. khasya China (Nanjing Forestry University, Nanjing, China)P. luchuensis Nago, Okinawa Prefecture, JapanP. massoniana China (Sheffield’s Seed Co. Inc., NY, U.S.A.)P. mugo SwitzerlandP. muricata California, U.S.A. (Sheffield’s Seed Co. Inc., NY, U.S.A.)P. nigra Oravsky Biely Potok, West Highland Tatras, Slovak RepublicP. pinaster Italy (Sheffield’s Seed Co. Inc., NY, U.S.A.)P. ponderosa Macdoel, CA, U.S.A. (Sheffield’s Seed Co. Inc., NY, U.S.A.)P. radiata Auckland, New Zealand (School of Biological Science, Auckland University, Auckland New Zealand)P. resinosa New Mexico, U.S.A. (Sheffield’s Seed Co. Inc., NY, U.S.A.)P. rigida Hardy, WV, U.S.A. (United States Forest Tree Seed Center, Forest Service, GA, U.S.A.)P. roxburghii Dehr Dun, IndiaP. sylvestris Tribec-Drazovce, Slovak RepublicP. tabulaeformis China (Nanjing Forestry University, Nanjing, China)P. taeda Auckland, New Zealand (School of Biological Science, Auckland University, Auckland, New Zealand)P. thunbergii Masaki, Ehime Prefecture, JapanP. virginiana York, SC, U.S.A. (United States Forest Tree Seed Center, Forest Service, GA, U.S.A.)P. yunnanensis China (Nanjing Forestry University, Nanjing, China)

Strobus P. aristata Commercial source (Clyde Robin Seed Co. Inc., CA, U.S.A.)P. peuce Slovenia (Sheffield’s Seed Co. Inc., NY, U.S.A.)

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performed using only 5S rDNA as a probe. After detec-tion and data recording, the specimens were subjected to second round FISH probing using 35S rDNA, 5S rDNA, the PCSR, and the Arabidopsis-type telomere repeats de-scribed above. The first and second round images were compared and 5S rDNA loci were identified in multico-lour FISH images. We did not observe the colocalisation between 35S rDNA and 5S rDNA reported for some co-nifers (Garcia and Kovarik 2013).

Cluster analysisWe performed a cluster analysis of dissimilarities in

FISH signal data among 50 sites in karyotype group A (Supplementary Data Table S1) and among 60 sites in karyotype group D (Supplementary Data Table S2) using Ward’s method. We constructed a dendrogram using the Mulcel add-in package in Excel software (Yanai 2005). We performed a cluster analysis using a single probe but the output was unsatisfactory because the distances between pairs of species were often distorted (either too distant or too close) due to the low level of variation in the signals. Better FISH karyotyping performance was obtained by using combinations of probes.

Results

All of the 26 Pinus species investigated in this study had a diploid chromosome number of 24, as in previ-

ous counts. The chromosome features of the subgenera Pinus and Strobus differed significantly, as reported by Saylor (1972, 1983) and Hizume (1988). We therefore treated them separately.

Karyotyping of subgenus PinusUsing multicolour FISH with probes of 35S rDNA,

5S rDNA, Arabidopsis-type telomere sequences (TTT AGG G)n, and the PCSR, we examined 24 spe-cies in the subgenus Pinus and identified homologous chromosome pairs in each by the signal patterns. To compare FISH karyotypes of corresponding chromo-somes among species, we assigned chromosomes with similar characteristics to one of the chromosome groups (I–XII) identified by Hizume et al. (2002). Within each species, the chromosome members of a single group may have evolved from the same ancestral chromosome, i.e., chromosomes in the same group were homologous. We assigned the multi-FISH karyotype of each of the 24 species in the subgenus Pinus to one of four groups (karyotype groups A, B, C, and D) by the FISH sig-nal patterns. Karyotype group A contained P. clausa, P. echinata, P. elliottii var. elliottii, P. muricata, P. ponderosa, P. radiata, P. rigida, P. taeda, and P. virginiana. Karyotype group B contained P. canariensis and P. roxburghii. Karyotype group C contained P. halepensis. Karyotype group D contained P. densiflora, P. khasya, P. luchuensis, P. massoniana,

Table 2. Subgeneric classification of the pine species included in this study by Gernandt et al. (2005) and Price et al. (1998) using different taxo-nomic procedures.

Gernandt et al. (2005)Species Karyotype group

Price et al. (1998)

Section Subsection Subsection Section

Trifoliae Contortae P. clausa Karyotype group A Contortae New World Diploxylon PinesP. virginiana

Australes P. echinata AustralesP. elliottii var. elliottiiP. rigidaP. taedaP. muricata AttenuataeP. radiata

Ponderosae P. ponderosa PonderosaePinus Pinaster P. canariensis Karyotype group B Canarienses Pinus

P. roxburghiiP. halepensis Karyotype group C HalepensesP. pinaster Karyotype group D Pinus

Pinus P. densifloraP. khasyaP. luchuensisP. massonianaP. mugoP. nigraP. resinosaP. sylvestrisP. tabulaeformisP. thunbergiiP. yunnanensis

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P. mugo, P. nigra, P. pinaster, P. resinosa, P. sylvestris, P. tabulaeformis, P. thunbergii, and P. yunnanensis (Table 2).

We identified 12 signal types among the 50 signal sites in karyotype group A (Table 3); the positions of

the signals on the chromosomes are shown in Figs. 1, 4, and Supplementary Figs. S1–S9. We detected polymor-phisms at several signal sites; these are indicated by blue arrowheads in Supplementary Figs. S1–S9.

We identified five types of signals among 47 signal

Table 3. FISH signals of species in karyotype group A. Telomere, telomere repeat (TTT AGG G)n signal; PCSR, proximal CMA band-specific repeat (PCSR) signal; 35S rDNA, 35S rDNA signal; 35S+PCSR, 35S rDNA signal with a small PCSR signal; PCSR/35S, mixed bal-anced signals of 35S rDNA and the PCSR; 5S rDNA, 5S rDNA signal; Telo+PCSR, telomere repeat signal with a small PCSR sig-nal; PCSR+Telo, PCSR signal with a small telomere repeat signal; Telo/PCSR, mixed balanced signal of telomere repeats and the PCSR; Telo/35S+PCSR, strong telomere repeat and 35S rDNA signals with a PCSR signal adjacent to the telomere repeat signal; PCSR+Telo+35S, PCSR signal with small telomere repeat and 35S rDNA signals; Telo+35S rDNA, telomere repeat signal with a small 35S rDNA signal.

Chromo-some group

Signal position

P. clausa P. echinataP. elliottii var.

elliottiiP. muricata P. ponderosa P. radiata P. rigida P. taeda P. virginiana

I 1a 5S rDNA 5S rDNA 5S rDNA 5S rDNA 5S rDNA 5S rDNA 5S rDNA 5S rDNA 5S rDNA1b Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere1c CEN Telo+PCSR Telomere Telomere Telo/PCSR Telomere Telo+PCSR Telo/35S+PCSR PCSR+Telo+35S Telo+PCSR1e Telomere Telomere Telomere Telomere Telomere Telomere Telomere

II 2a 35S rDNA 35S+PCSR 35S rDNA PCSR/35S 35S rDNA 35S rDNA 35S rDNA 35S rDNA2b Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere2c CEN Telo+PCSR Telomere Telomere Telo+PCSR Telomere Telo/PCSR Telo+PCSR Telomere Telo+PCSR2d Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere2e Telomere2f 5S rDNA 5S rDNA 5S rDNA 5S rDNA 5S rDNA 5S rDNA 5S rDNA

III 3a 35S rDNA 35S rDNA 35S rDNA 35S rDNA PCSR/35S 35S rDNA 35S rDNA 35S rDNA 35S rDNA3b Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere3c CEN PCSR+Telo PCSR+Telo Telomere Telo/PCSR Telomere Telo/PCSR PCSR+Telo Telomere PCSR+Telo3d Telomere Telomere

IV 4a Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere4b CEN Telo+PCSR Telo+PCSR Telomere Telomere Telo/35S+PCSR Telo+PCSR Telo/35S+PCSR Telomere Telo+PCSR4c Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere4d Telomere Telomere Telomere Telomere Telomere Telomere Telomere

V 5a Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere5b CEN Telo+PCSR Telo/35S+PCSR Telo/35S+PCSR Telo+PCSR Telo/35S+PCSR Telo+PCSR Telo/35S+PCSR Telo/35S+PCSR Telo/35S+PCSR5c Telomere Telomere Telomere Telomere Telomere Telomere Telomere

VI 6a Telomere Telomere Telomere6b CEN Telo+PCSR Telo+PCSR PCSR Telomere Telomere Telo+PCSR PCSR+Telo+35S PCSR+Telo+35S Telo/35S+PCSR6c Telomere Telomere Telomere Telomere Telomere Telomere6d 35S rDNA 35S rDNA 35S rDNA 35S rDNA PCSR/35S 35S rDNA 35S rDNA 35S rDNA 35S rDNA

VII 7a 35S rDNA 35S+PCSR 35S rDNA 35S rDNA 35S rDNA 35S rDNA 35S rDNA 35S rDNA7b Telomere Telomere Telomere Telomere Telomere Telomere Telomere7c CEN PCSR PCSR Telomere Telo+PCSR PCSR/35S Telo/35S+PCSR Telo+PCSR Telo+PCSR7d Telomere Telomere Telomere Telomere

VIII 8a 35S rDNA 35S rDNA 35S rDNA 35S rDNA PCSR/35S 35S rDNA 35S rDNA 35S rDNA 35S rDNA8b Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere8c CEN PCSR+Telo PCSR+Telo+35S PCSR PCSR Telo+35S rDNA PCSR PCSR+Telo8d Telomere Telomere Telomere Telomere Telomere8e Telomere

IX 9a 35S rDNA 35S+PCSR 35S rDNA 35S rDNA PCSR/35S 35S rDNA 35S rDNA 35S rDNA 35S rDNA9b Telomere Telomere Telomere Telomere Telomere Telomere Telomere9c CEN Telo+PCSR Telo+PCSR Telo+PCSR Telomere Telomere Telo+PCSR PCSR+Telo+35S PCSR+Telo Telo+PCSR9d Telomere Telomere9e 5S rDNA 5S rDNA 5S rDNA 5S rDNA 5S rDNA

X 10a 35S rDNA 35S+PCSR 35S rDNA 35S rDNA PCSR/35S 35S rDNA 35S rDNA 35S rDNA10b Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere10c CEN Telo+PCSR Telo/35S+PCSR Telo/PCSR PCSR Telomere Telo/PCSR 35S+PCSR PCSR+Telo+35S Telo+PCSR10d Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere10e Telomere Telomere

XI 11a Telomere Telomere Telomere Telomere Telomere Telomere Telomere11b CEN Telo+PCSR Telo+PCSR Telo+PCSR Telomere Telo/35S+PCSR Telo+PCSR Telomere Telo+PCSR Telo+PCSR11c Telomere Telomere Telomere Telomere Telomere Telomere

XII 12a CEN PCSR+Telo Telo+35S rDNA Telomere Telomere Telo/35S+PCSR Telomere Telomere Telomere Telomere12b Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere

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sites in karyotype group B (Table 4); the positions of the signals on the chromosomes are shown in Figs. 2, 4, and Supplementary Figs. S10 and S11.

Four signal types were identified among 38 signal sites in karyotype group C (Table 5); the positions of the signals on the chromosomes are shown in Figs. 2, 4, and Supplementary Fig. S12.

We observed 10 signal types among 60 signal sites in karyotype group D (Table 6); the positions of the

signals on the chromosomes are shown in Figs. 3, 4, and Supplementary Figs. S13–S24. Polymorphisms occurred at several signal sites, which are indicated by yellow ar-rowheads in the supplementary figures.

Hizume et al. (2001, 2002) did not report weak PCSR signals on the secondary constrictions in P. densiflora, P. sylvestris, or P. thunbergii; however, we detected weak PCSR signals on most secondary constrictions, probably because of the enhanced sensitivity of the FISH

Fig. 1. FISH karyotype of species in karyotype group A. Red signals, 35S rDNA and 5S rDNA; red arrowheads indicate 5S rDNA sites; green signals, telomere repeat (TTT AGG G)n sequences; magenta signals, proximal CMA band-specific re-peat (PCSR). Scale bar=5 µm.

414 F. Shibata et al. Cytologia 81(4)

detection system that we used. In karyotype group A, the patterns of mixed sympatric or very closely-located signals were much more diverse than patterns in other karyotype groups (Table 3, Supplementary Figs. S1–S9). In karyotype groups B and C, all of the signals were simpler than those in karyotype groups A and D, but the PCSR signals of groups B and C were weaker than those of the other two groups (Supplementary Figs. S10–S12). Karyotype groups A and D shared no similarities with one another or any other karyotype group. Seven chro-mosome pairs had similar FISH signal patterns between karyotype groups B and C (Fig. 2). Nine species of karyotype group A were previously assigned to section Trifoliae (Gernandt et al. 2005) or to the New World Diploxylon Pines group (Price et al. 1998). Species of karyotype groups B, C, and D were previously assigned to section Pinus (Price et al. 1998, Gernandt et al. 2005). Following the taxonomy of Price et al. (1998), species in karyotype group B were consistent with those in subsection Canarienses, species in karyotype group C were consistent with those in subsection Halepenses, and species in karyotype group D were consistent with those in subsection Pinus (Table 2). In the taxonomy of Gernandt et al. (2005), P. canariensis (karyotype group B), P. roxburghii (karyotype group B), P. halepensis (karyotype group C), and P. pinaster (karyotype group D) were assigned to subsection Pinaster (Table 2).

Cluster analysis of the FISH signals of species in FISH karyotype groups A and D

We used cluster analysis to compare the FISH signal patterns in karyotype groups A and D. Cluster analysis groups objects by their similarity. Thus, we used Ward’s method to cluster the FISH signals for the species by their similarities (Figs. 5, 6). The lengths of the branches

from the bifurcations in Figs. 5 and 6 represent the distances between species. The PCSR signal strengths at the secondary constrictions (indicated by the 35S rDNA+PCSR signals in Tables 3 and 6) were very weak and may have been at the lower limit of FISH detection. Some chromosome signals were not detected, even on the same slides. Thus, for our cluster analyses we pooled values for 35S rDNA and 35S rDNA+PCSR in the same signal. Similarly, the weak values for 35S rDNA in the PCSR+35S rDNA combination (Tables 3, 6) were at the limit of FISH detection, and we accordingly pooled values for the PCSR and PCSR+35S rDNA in the same signal.

The cluster analysis of nine species in karyotype group A identified two relatively close clusters, one containing P. clausa and P. virginiana and one con-taining P. rigida and P. taeda (Fig. 6). The other five species had low similarities with other taxa, especially P. ponderosa, which had unique chromosome features. The closely-clustered species P. clausa and P. virginiana were assigned to the subsection Contortae; P. rigida and P. taeda were assigned to subsection Australes. Accord-ing to previous molecular phylogenetic reports, these two groups of species are closely related (Gernandt et al. 2005, Eckert and Hall 2006). Only P. ponderosa was as-signed to subsection Ponderosae, i.e., karyotype group A in this study.

The cluster analysis of 12 species in karyotype group D detected three clusters (Fig. 7). One cluster contained P. densiflora, P. sylvestris, P. mugo, and P. pinaster. Among the species included in this study, P. densiflora and P. sylvestris had the most similar chromosomal FISH signals. The two species grouped in the same clade with P. mugo in previous molecular phylogenetic analy-ses (Gernandt et al. 2005, Eckert and Hall 2006); among

Fig. 2. FISH karyotype of species in karyotype group B and C. Red signals, 35S rDNA and 5S rDNA; red arrowheads indicate 5S rDNA sites; green signals, telomere repeat (TTT AGG G)n sequences; magenta signals, proximal CMA band-specific repeat (PCSR). Yellow lines between pairs of karyotypes indicate closely-related chromosome groups. Scale bar=5 µm.

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the three species in this clade, P. mugo was a phyloge-netic outlier. Our karyotype relationship analyses of species were congruent with the molecular phylogenetic

analyses. P. pinaster is considered to be taxonomically distant from the other species based on morphology and molecular data used by Price et al. (1998) to assign the species to subsection Pinus. However, Gernandt et al. (2005) used a molecular phylogenetic analysis to assign P. pinaster to subsection Pinaster.

Our cluster analysis detected another group contain-ing P. luchuensis, P. thunberghii, P. yunnanensis, P. khasya, and P. tabulaeformis, which were included in the same clade in earlier molecular phylogenetic inves-tigations (Gernandt et al. 2005, Eckert and Hall 2006). P. massoniana, P. nigra, and P. resinosa constituted a

Table 4. FISH signals of species in karyotype group B. 35S rDNA, 35S rDNA signal; 35S+PCSR, 35S rDNA signal with a small proximal CMA band-specific repeat (PCSR) signal; 5S rDNA, 5S rDNA signal; Telomere, telomere repeat (TTT AGG G)n signal; 35S+Telo, 35S rDNA signal with a small telomere repeat signal.

Chromosome group Signal position P. canariensis P. roxburghii

I 1a Telomere Telomere1b CEN 35S rDNA 35S rDNA1c Telomere Telomere

II 2a 5S rDNA 5S rDNA2b Telomere Telomere2c CEN Telomere Telomere2d Telomere Telomere

III 3a 35S+PCSR 35S+PCSR3b Telomere Telomere3c CEN Telomere3d Telomere Telomere3e 5S rDNA

IV 4a 35S+PCSR4b Telomere Telomere4c CEN Telomere Telomere4d Telomere Telomere

V 5a 35S+PCSR 35S+PCSR5b Telomere Telomere5c CEN 35S rDNA 35S rDNA5d Telomere Telomere

VI 6a 35S+PCSR 35S+PCSR6b Telomere Telomere6c CEN 35S rDNA 35S rDNA6d Telomere

VII 7a 5S rDNA 5S rDNA7b Telomere Telomere7c CEN 35S rDNA Telomere7d Telomere Telomere7e 35S+PCSR 35S+PCSR

VIII 8a 35S+PCSR 35S+PCSR8b Telomere Telomere8c CEN8d Telomere Telomere8f 35S+PCSR

IX 9a Telomere Telomere9b CEN 35S rDNA 35S rDNA9c Telomere Telomere

X 10a 35S+PCSR 35S+PCSR10b Telomere Telomere10c CEN 35S rDNA 35S+Telo10d Telomere Telomere

XI 11a 35S+PCSR 35S+PCSR11b Telomere Telomere11c CEN 35S rDNA 35S rDNA11d Telomere Telomere

XII 12a CEN 35S rDNA 35S rDNA12b Telomere Telomere

Table 5. FISH signals of species in karyotype group C. 35S rDNA, 35S rDNA signal; 35S+PCSR, 35S rDNA signal with a small proximal CMA band-specific repeat (PCSR) signal; 5S rDNA, 5S rDNA signal; Telomere, telomere repeat (TTT AGG G)n signal.

Chromosome group Signal position P. halepensis

I 1a 5S rDNA1c CEN Telomere1d Telomere

II 2a 35S rDNA2b Telomere2c CEN 35S rDNA2d 5S rDNA2e 5S rDNA

III 3a 35S rDNA3b Telomere3c CEN Telomere3d Telomere

IV 4a 35S rDNA4b Telomere4c CEN 35S rDNA4d Telomere

V 5a Telomere5b Telomere5c 35S rDNA

VI 6a 35S rDNA6b Telomere6c Telomere

VII 7a Telomere7b 35S rDNA

VIII 8a Telomere8b Telomere

IX 9a 35S rDNA9b Telomere9c Telomere9d 35S rDNA

X 10a 35S rDNA10b Telomere10c Telomere

XI 11a CEN 35S rDNA11b Telomere11c 35S+PCSR

XII 12a CEN 35S rDNA12b Telomere

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Table 6. FISH signals of species in karyotype group D. 35S rDNA, 35S rDNA signal; 35S+PCSR, 35S rDNA signal with a small proximal CMA band-specific repeat (PCSR) signal; PCSR, PCSR signal; PCSR+35S, PCSR signal with a small 35S rDNA signal; PCSR/35S, PCSR/35S, mixed balanced signals of 35S rDNA and PCSR; 5S rDNA, 5S rDNA signal; Telo+PCSR, Telo/PCSR, mixed balanced sig-nal of telomere repeats and PCSR; P/T/35, mixed balanced signals of PCSR, telomere repeats and 35S rDNA; Telomere; telomere repeat (TTT AGG G)n signal; Telomere+35S, mixed signals of telomere repeats and 35S rDNA.

Chromo-some group

Signal position

P. densiflora

P. khasya

P. luchuensis

P. massoniana

P. mugo

P. nigra

P. pinaster

P. resinosa

P. sylvestris

P. tabulaeformis

P. thunbergii

P. yunnanensis

I 1a PCSR/35S PCSR/35S1b Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere1c Telomere Telomere Telomere Telomere Telomere Telomere Telomere1d CEN PCSR+35S PCSR PCSR/35S Telomere PCSR Telomere Telomere PCSR+35S PCSR+35S PCSR PCSR+35S1e Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere1f 35S+PCSR 35S+PCSR PCSR/35S 35S+PCSR 35S+PCSR 35S rDNA PCSR/35S 35S+PCSR PCSR/35S 35S rDNA PCSR

II 2a 35S+PCSR 35S+PCSR 35S+PCSR PCSR/35S 35S+PCSR PCSR/35S 35S rDNA PCSR/35S 35S rDNA 35S+PCSR 35S+PCSR 35S+PCSR2b 5S rDNA 5S rDNA 5S rDNA 5S rDNA 5S rDNA 5S rDNA 5S rDNA 5S rDNA 5S rDNA 5S rDNA 5S rDNA 5S rDNA2c Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere2d CEN PCSR+35S PCSR+35S PCSR+35S Telomere Telomere PCSR+35S PCSR PCSR PCSR+35S PCSR2e Telomere Telomere Telomere Telomere Telomere Telomere Telomere P/T/35 Telomere Telomere Telomere Telomere2f Telomere

III 3b PCSR/35S3c Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere3d CEN PCSR+35S Telomere Telomere Telomere Telomere Telomere PCSR Telomere Telomere Telomere3e Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere3f PCSR/35S

IV 4a Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere4b 35S+PCSR 35S+PCSR 35S+PCSR PCSR/35S 35S+PCSR PCSR/35S 35S rDNA PCSR/35S 35S+PCSR 35S+PCSR 35S+PCSR 35S+PCSR4c Telomere Telomere Telomere Telomere Telomere Telomere Telomere4d Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere4e CEN PCSR+35S PCSR+35S PCSR PCSR+35S PCSR PCSR PCSR+35S PCSR4f Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere

V 5a Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere5b CEN PCSR+35S PCSR PCSR+35S PCSR PCSR PCSR PCSR PCSR5c Telomere Telomere Telomere Telomere+35S Telomere Telomere Telomere5d Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere5e Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telo+PCSR Telomere Telomere Telomere Telomere

VI 6a Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere6b CEN PCSR+35S PCSR PCSR+35S 35S+PCSR PCSR PCSR+35S PCSR PCSR/35S PCSR PCSR PCSR+35S PCSR6c Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere6d PCSR/35S

VII 7a Telomere7b Telomere7c PCSR PCSR7d CEN Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere7e Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere7f 35S+PCSR 35S rDNA 35S+PCSR PCSR/35S 35S+PCSR PCSR/35S 35S rDNA PCSR/35S 35S+PCSR 35S+PCSR 35S+PCSR 35S rDNA

VIII 8a 35S+PCSR 35S rDNA 35S+PCSR PCSR/35S 35S+PCSR PCSR/35S 35S rDNA PCSR/35S 35S+PCSR 35S rDNA 35S+PCSR 35S rDNA8b Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere8c PCSR+35S PCSR+35S PCSR/35S PCSR/35S PCSR PCSR+35S PCSR PCSR+35S PCSR+35S8d PCSR8e Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere8f Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere8g Telomere8h 5S rDNA 5S rDNA 5S rDNA 5S rDNA 5S rDNA 5S rDNA 5S rDNA 5S rDNA 5S rDNA 5S rDNA 5S rDNA 5S rDNA

IX 9a 35S+PCSR 35S+PCSR 35S+PCSR PCSR/35S PCSR/35S 35S+PCSR PCSR/35S 35S rDNA 35S rDNA 35S rDNA PCSR/35S9b Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere9c CEN Telomere Telomere PCSR+35S Telomere PCSR Telomere Telomere Telomere PCSR or

TelomerePCSR PCSR

9d Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere

X 10a 35S+PCSR 35S+PCSR 35S+PCSR PCSR/35S 35S+PCSR PCSR/35S PCSR/35S 35S+PCSR 35S rDNA 35S+PCSR 35S+PCSR10b Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere10c CEN PCSR+35S Telomere Telomere Telomere PCSR Telomere PCSR+35S PCSR Telomere Telomere Telomere10d Telomere Telomere Telomere

XI 11a Telomere11b CEN PCSR+35S PCSR PCSR PCSR+35S PCSR PCSR PCSR PCSR11c CEN Telomere Telomere Telomere Telomere Telomere Telomere11d Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere Telomere

XII 12a CEN PCSR+35S PCSR/35S PCSR/35S PCSR/35S PCSR PCSR/35S Telomere PCSR/35S PCSR PCSR/35S PCSR/35S PCSR+35S12b Telomere

2016 Multicolour FISH Karyotyping in Pinus 417

single cluster in our analysis. However, P. resinosa was relatively distant from the other two species, although all three had common FISH signal traits on the second-ary constrictions, where 35S rDNA signals were similar in strength to those of PCSR signals. Thus, these three species may have originated from a single common an-cestor.

FISH karyotyping of the subgenus StrobusWe performed similar FISH analyses on two species

in the subgenus Strobus; we were unable to identify the locus of 5S rDNA in these species. We did not observe PCSR signals or interstitial signals of the Arabidopsis-type telomere sequence in P. aristata or P. peuce. The signal patterns of the 35S and 5S rDNA probes were ex-tremely different between P. aristata and P. peuce; thus we were unable to identify a chromosome group (Fig. 7, Supplementary Figs. S25, S26).

Fig. 3. FISH karyotype of species of karyotype group D. Red signals, 35S rDNA and 5S rDNA; red arrowheads indicate 5S rDNA sites; green signals, telomere repeat (TTT AGG G)n sequences; magenta signals, proximal CMA band-specific re-peat (PCSR). Scale bar=5 µm.

418 F. Shibata et al. Cytologia 81(4)

Discussion

The descendants in each karyotype group may gain unique chromosome features at the time of differentia-tion from the ancestral species. Each species in a single karyotype group inherits FISH signal patterns from the karyotype group ancestor. We classified 24 Pinus spe-cies into four karyotype groups (A, B, C, and D) by their FISH karyotypes. Each group had unique FISH karyo-type features.

PCSR and species differentiationThe PCSR is a tandemly arranged repetitive sequence

that was cloned from the centromeric chromosomal regions of P. densiflora by microdissection (Hizume et al. 2001). A similar DNA sequence was found in the DNA database for P. taeda (PT_7Ba2900I02: accession number AC241294). The sequence homologies were in the range of 79–94%. The PCSR was a major repeti-tive sequence in karyotype groups A and D, but not in

karyotype groups B or C, in which minor signals were detected. Furthermore, we did not find any PCSR FISH signals in P. aristata or P. peuce (subgenus Strobus). The phylogenetic study of Gernandt et al. (2008) indi-cated that the subgenera Pinus and Strobus differenti-ated 72 or 87 Ma, suggesting that the ancestor of the sub-genus Strobus lacked the PCSR, which first emerged or was amplified in the ancestor of the subgenus Pinus. In the subgenus Pinus, the ancestor of karyotype group A and the common ancestor of karyotype groups B, C, and D differentiated 25 or 31 Ma; the ancestor of karyotype group D and the common ancestor of karyotype groups B and C separated 13 or 16 Ma. The amount of PCSR in the common ancestor of karyotype groups B and C may have decreased before the separation of karyotype groups B and C.

Fig. 4. Ideogram and FISH signal sites for each karyotype group in Pinus.

Fig. 5. Cluster analysis of karyotype group A.Fig. 6. Cluster analysis of karyotype group D.

2016 Multicolour FISH Karyotyping in Pinus 419

Interstitial FISH signals for (TTT AGG G)n during spe-cies differentiation

Interstitial telomere signals were important chromo-some markers in the subgenus Pinus. These interstitial signals contain Arabidopsis-type telomere sequences and/or a degenerate Arabidopsis-type telomere sequence mixture (Shibata et al. 2005). In the subgenus Strobus, neither P. aristata nor P. peuce had any trace of an in-terstitial telomere signal. These two species have been assigned to different subsections (P. aristata to subsec-tion Balfourianae, and P. peuce to subsection Strobus). The separation of these two subsections formed the first phylogenetic branch in the subgenus Strobus (Gernandt et al. 2005), suggesting that the ancestor of the subgenus Strobus had no interstitial telomere repeats.

Colocalisation of 35S rDNA and the PCSR during spe-cies differentiation

Colocalisation of 35S rDNA and the PCSR was de-tected by FISH analyses in P. densiflora (Hizume et al. 2001), P. thunbergii, and P. nigra (Hizume et al. 2002). In our study we observed colocalisation of 35S rDNA and the PCSR in 22 species assigned to the subgenus Pinus; the proportions of 35S rDNA and the PCSR dif-fered among species and signal sites. The proportions of the PCSR were especially elevated at the secondary constriction in P. massoniana, P. nigra, P. resinosa, and P. ponderosa and at the secondary constriction of chro-mosome group X in P. thunbergii. This increased ratio probably occurred at least three times independently: (i) in the ancestor of P. massoniana, P. nigra, and P. resinosa, (ii) in the ancestor of P. ponderosa, and (iii) in the secondary constriction of chromosome group X in P. thunbergii.

Comparisons between taxonomic assignments and FISH karyotyping in karyotype group A

Although karyotype group A contained species as-signed to three or four different subsections (Table 2), the chromosome groupings were congruent with spe-cies assignations within each of the subsections, which was not the case for karyotype groups B, C, or D. This congruence is indicative of close relationships among species in karyotype group A. It is possible that (i) the

section Trifoliae in the taxonomy erected by Gernandt et al. (2005) and (ii) the New World Diploxylon Pines in the taxonomy erected by Price et al. (1998) may be rec-ognised as members of the same subsection.

The groups identified by our cluster analysis of karyo-type group A did not align well with previous taxonomic and molecular phylogenetic studies. The complexity of FISH signal characteristics partially accounts for the discrepancy among studies. At some of the sites, sev-eral types of signals were mixed in variable proportions, which added a measure of complexity and increased the difficulties in producing a satisfactory cluster analysis. FISH may not be an appropriate taxonomic procedure for karyotype group A. Accumulation of FISH karyo-types for other members of karyotype group A is a re-quirement for robust phylogenetic analysis.

Comparisons between taxonomic assignments and FISH karyotyping in karyotype groups B, C, and D

Karyotype groups B, C, and D were congruent with the section Pinus in the taxonomic systems of both Price et al. (1998) and Gernandt et al. (2005). These systems differ at the subsection level within the ge-nus. The taxonomy of Gernandt et al. (2005) is based on chloroplast DNA and that of Price et al. (1998) is based on morphology and molecular data. According to Gernandt et al. (2005), the section Pinus is divided into subsections Pinus and Pinaster. Price et al. (1998) divided section Pinus into four subsections: Pinus, Canarienses, Halepenses, and Pinea. P. pinaster was assigned to the subsection Pinaster by Gernandt et al. (2005), and to subsection Pinus by Price et al. (1998). The assignments of P. pinaster, P. canariensis, P. roxburghii, and P. halepensis differ among taxonomies (Little and Critchfield 1969, Van der Burgh 1973, Price et al. 1998, Farjon 2005, Gernandt et al. 2005). Different molecular phylogenetic analyses have produced different taxonomic assignments (Liston et al. 1999, Wang et al. 1999, Gernandt et al. 2005); however, P. pinaster is not included in subsection Pinus in any of these studies.

Our classification of the section Pinus based on FISH karyotyping was congruent with that of Price et al. (1998). P. pinaster had a karyotype similar to those of other species in karyotype group D or subsection Pinus.

Fig. 7. FISH karyotype of species in subgenus Strobus. Red signals, 35S rDNA and 5S rDNA; green signals, telomere repeat (TTT AGG G)n sequences. Scale bar=5 µm.

420 F. Shibata et al. Cytologia 81(4)

The disparity between the molecular phylogenetic analy-sis and FISH karyotyping suggests the possibility of introgression from the ancestor of subsection Pinaster to the ancestor of P. pinaster; introgression has been reported for some congeners of Pinus (Senjo et al. 1999, Chen et al. 2004, Stewart et al. 2012). The chromosome characteristics of karyotype group D (subsection Pinus; Price et al. 1998) were not similar to those of karyotype group B (subsection Canarienses; Price et al. 1998) or karyotype group C (subsection Halepenses; Price et al. 1998). These findings are in agreement with the classifi-cation of section Pinus by Price et al. (1998). Karyotype groups B and C had common signal sites on seven chro-mosomes, indicating that these groups have close rela-tionships; species in these karyotype groups fell within the same clade in earlier molecular phylogenetic analy-ses based on chloroplast DNA (Gernandt et al. 2005, Eckert and Hall 2006). The difference in chromosomal characteristics between karyotype groups B and C may have developed more rapidly than the differentiation of nucleotide sequences.

Our analyses showed that differences in FISH karyo-types are indicative of distances among species. Thus, FISH karyotyping will become an important tool for taxonomic decision making. New probes that provide more information on chromosomal differentiation are required; they will contribute to a better understanding of the phylogenetic history of the genus Pinus.

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