Bot. Bull. Acad. Sin. (2002) 43: 31-35

Yang et al. — Systematics of the Genus Machilus

Application of cecidomyiid galls to the systematics of the genus Machilus (Lauraceae) in Taiwan

Sue-Yen Yang^1, *, Ming-Yih Chen¹, and Jeng-Tze Yang²

¹Department of Botany, National Chung-Hsing University, Taichung 402, Taiwan

²Department of Entomology, National Chung-Hsing University, Taichung 402, Taiwan

(Received October 12, 2000; Accepted September 10, 2001)

Abstract. The species of the genus Machilus (Lauraceae) in Taiwan sustain diverse cecidomyiid galls induced by the insects of the genus Daphnephila (Cecidomyiidae). This work examines the feasibility of applying cecidomyiid galls to the systematics of the genus Machilus. Amplified fragment length polymorphism (AFLP) was used to analyze the 38 gall-bearing trees of four Machilus taxa including Machilus kusanoi, M. thunbergii, M. zuihoensis var. zuihoensis, and M. zuihoensis var. mushaensis. The UPGMA cluster analysis of the AFLP data revealed three distinct clusters, including M. kusanoi, M. thunbergii, and M. zuihoensis variety complex. Machilus zuihoensis var. zuihoensis and M. zuihoensis var. mushaensis were indistinguishable from the three primer combinations. These two varieties could be considered the same taxon. PCR and DNA sequencing methods were used to analyze the nucleotide sequences of the mitochondrial 16S rDNA gene of the twenty gall midges from three types of galls from four Machilus taxa. The phylogenetic tree from the partial 16S rDNA sequence by UPGMA method of proportion distance revealed that the gall midges can be divided into three groups according to gall types. The phylogenetic tree cannot separate the two varieties of M. zuihoensis within each group. Machilus zuihoensis var. zuihoensis and M. zuihoensis var. mushaensis cannot be distinguished according to the AFLP or DNA sequencing methods, and they are more closely related to M. thunbergii than to M. kusanoi. The systematic relationships among the Machilus from the data of host plants are congruent with the data from the gall inducers. Results in this study imply that the gall inducers of genus Daphnephila provide information for resolving the plant systematic relationships based on molecular techniques.

Keywords: AFLP; Cecidomyiidae; DNA sequence; Insect gall; Machilus; Systematics.

Introduction

The genus Machilus of Lauraceae is distributed in the tropical and subtropical areas of Asia (Liu et al., 1994). There are six taxa of Machilus in Taiwan: Machilus kusanoi, M. obovatifolia, M. zuihoensis var. zuihoensis, M. zuihoensis var. mushaensis, M. thunbergii and M. japonica. All except M. thunbergii and M. japonica are endemic species in Taiwan. Currently, plant taxonomists differ in their systematic arrangement of the Machilus taxa in Taiwan.

A gall is an abnormal growth on some part of a plant as a result of the activity of another organism (virus, nematode, arthropod etc.) and the gall inducer uses this structure as a shelter and nutrition source (Csóka, 1997). The genus Machilus plants in Taiwan have a high diversity of cecidomyiid galls induced by the insects of the genus Daphnephila (Cecidomyiidae). Harris (1994) demonstrated that the family Cecidomyiidae is one of the major groups of gall inducers and that most gall midges are highly host specific. The galls of phytophagous arthropods are traits associated with plants and may be

useful for separating plant taxa (Floate et al., 1996). Abrahamson et al. (1998) indicated that cynipid occurrences offer helpful information concerning some aspects of oak systematics. Our work examines the feasibility of applying cecidomyiid galls to the systematics of the genus Machilus (Lauraceae) in Taiwan. Amplified fragment length polymorphism (AFLP) was used to analyze the phylogeny of Machilus taxa. PCR and DNA sequencing methods were used to examine the phylogeny of gall inducers gathered from galled plants.

Materials and Methods

Gall-Bearing Plants

Plant materials. Thirty eight gall-bearing trees of four Machilus taxa including Machilus kusanoi (code K), M. thunbergii (T), M. zuihoensis var. zuihoensis (Z) and M. zuihoensis var. mushaensis (M) were sampled throughout Taiwan (Figure 1). Leaf samples from each plant were packed in a paper bag and stored in a box of silica gel desiccant. The dried leaves were used for DNA extraction. Only four Machilus taxa were analyzed in the preliminary study because there were not enough M. obovatifolia samples that are only distributed on the Hengchun Peninsula, and M. japonica was difficult to identify.

*Corresponding author. E-mail: syyang160@yahoo.com.tw

Botanical Bulletin of Academia Sinica, Vol. 43, 2002

DNA extraction. The leaves of the Machilus contain a large amount of polysaccharide that inhibits DNA extraction. DNA was extracted from dried leaf material according to the Kobayashi et al. method (1998) so that polysaccharides and polyphenolics could be removed from the DNA extraction protocol and high quality DNA could be extracted.

AFLP analysis. The AFLP technique, as reported by Vos et al. (1995), consists of three major steps: (1) restriction endonuclease digestion of the DNA and ligation of the adapters, (2) amplification of the restricted fragments, (3) gel analysis of the amplified fragments. The AFLP procedure was performed using the AFLP analysis system I (Life Technologies, Inc.) according to the manufacturer's instructions. The total genomic DNA was restricted with Eco RI and Mse I. The DNA fragments were ligated to Eco RI and Mse I adapters provided in the kit. Selective PCR was performed using three primer pairs: Eco RI+AAC and Mse I+CAA, Eco RI+ACC and Mse I+CTC, and Eco RI+ACG and Mse I+CAA.

Data analysis. The presence/absence of each scorable fragment was recorded in a binary data matrix. Data from the three primer combinations were combined, and the pairwise similarities between samples were calculated using a simple matching coefficient. The resultant similarity matrix was input into both a UPGMA cluster analysis and a principal coordinate analysis using NTSYS-PC, Version 2.0 (Rohlf, 1993).

Gall-Inducing Insects

Insect materials. Twenty specimens were gathered from three types of midge galls, including urn-shaped, mouse-like, and coniform galls obtained from four Machilus taxa

Figure 1. The Machilus collection sites in Taiwan.

Yang et al. — Systematics of the Genus Machilus

(Table 1). The specimens were preserved in 70% alcohol at room temperature.

DNA extraction. The entire insect body was homogenized by a glass homogenizer in 500 µl digestion buffer that contained 100 mM Tris-Cl (pH 8.0), 10 mM EDTA, 100 mM NaCl, 0.5% SDS, 50 mM dithiothreitol, and 0.5 mg/l proteinase K. The mixture was shaken horizontally (180 rpm) at 50°C overnight. The DNA template was generated using a phenol-chloroform extraction of total nucleic acids (Yeh and Yang, 1997). The extracted crude DNA was dissolved in 50 µl distilled H₂O.

PCR and DNA sequencing. A polymerase chain reaction (PCR) was employed to amplify part of the 3´ end sequence of the mitochondrial 16S rDNA gene. The primers used to amplify the region were 5´-GCCTGTTTA TCAAAAACAT-3´ and 5´-CCGGTCTGAACTCAGATCA-3´. PCR amplification was performed in a 100 µl reaction mix that contained 100 mM Tris-Cl (pH 9.0), 50 mM KCl, 1.5 mM MgCl₂, 0.01% gelatin, 0.1% Trutib-X100, 2U SuperTag polymerase (HT Biotechnology, LTD), 0.2 mM of each dNTP, 20 pmoles of each primer, and 2 µl DNA template. A Perkin-Elmer 9600 thermal cycler was employed with the following temperature profile: 95°C for 2 min; 39 cycles of 95°C for 40 s, 48°C for 1 min, and 72°C for 30 s; 72°C for 10 min; and 4°C at the end. The target DNA was recovered from the gel by a Bio101 kit after electrophoresis, and the DNA products were sequenced directly using the AmpliCycle Sequencing kit (Perkin Elmer) for 29 cycles with the following temperature profiles: 95°C for 30 s, 55°C for 30 s, and 72°C for 20 s.

Data analysis. The partial 16S rDNA gene sequences were aligned using the GCG (Genetic Computer Group, Version 7.0) Pileup program (Devereux et al., 1991) and then visually checked. Phylogentic analysis of the aligned nucleotide sequences was performed by the UPGMA method of proportion distance using the MEGA program (Kumar et al., 1993). A bootstrap analysis was performed for 1000 replications in the clustering method.

Figure 3. The phylogenetic tree inferred from the partial 16S rDNA sequence by the UPGMA method of proportion distance. The values (percentages) on the branches were the result of 1000 bootstrap replications.

Results

The three primer combinations employed for the AFLP of gall-bearing plants revealed 114 polymorphic bands. The number of polymorphic bands differed depending upon the primer combinations. The UPGMA cluster analysis revealed three distinct groups (Figure 2) and the cophenetic correlation coefficient was 0.858. The three distinct groups were categorized into Machilus kusanoi, M. thunbergii, and the M. zuihoensis variety complex. Machilus zuihoensis var. zuihoensis and M. zuihoensis var. mushaensis were indistinguishable, and they appeared to be the same taxon. Taxonomists disagree about the classification of the genus Machilus in Taiwan. The interference with the clustering may result from the difficulty of specimen identification.

GenBank accession numbers of the partial 16S rDNA gene sequence data of all twenty individual gall inducers are AF334186—AF334205. The average nucleotide base compositions of guanine, adenine, thymine and cytosine were 10.1%, 40.1%, 44.4% and 5.4%, respectively. The phylogenetic tree constructed by the UPGMA method using proportion distance could be divided into three groups according to the gall types: urn-shaped, mouse-like, and coniform galls (Figure 3). The bootstrap and distance analysis is illustrated in the tree (Figure 3). There is over 90% support for monophyly of each gall type gathered from the various Machilus taxa. The phylogenetic tree of each group cannot separate the two varieties of M. zuihoensis. The synthetic systematic relationships of the four Machilus taxa from DNA data of gall inducers imply that the two varieties of M. zuihoensis could be considered the same taxon, and they are more closely related to M. thunbergii than to M. kusanoi.

Discussion

Knowledge of the systematics of genus Machilus in Taiwan is limited because the morphological characteris

Figure 2. The dendrogram of the gall-bearing plants based on polymorphic AFLP bands.

Botanical Bulletin of Academia Sinica, Vol. 43, 2002

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tics are often confused. Two troublesome Machilus taxa are M. zuihoensis var. mushaensis and M. japonica. The two varieties of Machilus zuihoensis were considered two species by Yang et al. (1999), whereas Liu et al. (1994) categorized them as two varieties. Machilus japonica was considered as M. pseudolongifolia by Yang et al. (1999) and was often confused with M. kusanoi. Taxonomists differ in the systematics of the Machilus taxa using morphological characteristics such as wood anatomy (Chang, 1994), pollen morphology (Huang, 1972), ultra-structure of leaf epidermis (Ou, 1989), and biochemistry (Ou, 1975; Kuo, 1985) alone. We used the molecular technique AFLP to examine the systematics and found that Machilus zuihoensis var. zuihoensis and M. zuihoensis var. mushaensis may be the same taxa and they are more closely related to M. thunbergii than to M. kusanoi. Analysis results imply a coevolution between the Machilus plants and the Daphnephila gall inducers.

The Cecidomyiidae probably began plant feeding during the flowering plant radiation period in the late Cretaceous (Roskam, 1992). Cecidomyiidae presumably were pre-adapted for plant feeding and gall-inducing in an earlier period of angiosperm radiation (Roskam, 1992). The Cecidomyiidae family of insects is a major group of gall inducers, and most gall midges are highly host specific (Harris, 1994). The genus Daphnephila of Cecidomyiidae has only been recorded in India (Gagné, 1973) and Japan (Yukawa and Masuda, 1996). The gall inducer specimens gathered in Taiwan are difficult to identify into species level.

Phytophagous insects are a natural bioassay that may be used to segregate closely related plant taxa (Floate and Whitham, 1995). The distributions of gall inducers may serve to distinguish hybrid plants (Aguilar and Boecklen, 1992; Fritz et al., 1994; Floate and Whitham, 1995) and even intrapopulational categories of plant genotypes (Floate et al., 1996). The systematic relationships of four Machilus taxa gathered from the data of gall inducers herein are congruent with the data gathered from gall-bearing plants. These results suggest that gall midges Daphnephila can distinguish the characteristics among Machilus taxa and can help resolve plant systematic problems. These cecidomyiid galls on Machilus may have already taken a long time to adapt to the host plants in this region. Future studies will collect more specimens of gall-bearing plants and gall-inducing insects to clarify the intact systematic relationships of genus Machilus and genus Daphnephila.

Acknowledgements. The authors would like to thank the Council of Agriculture of the Republic of China for financially supporting this research under the project 89NCF-07(07).

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