Comparative Efficacy of Abscisic Acid and Methyl Jasmonate for Indirect Somatic Embryogenesis in Medicago sativa L.

The effects of methyl jasmonate (MeJA) in relation to abscisic acid (ABA) on different phases of somatic embryogenesis were studied in Medicago sativa L. Different concentrations of both the growth inhibitors (0.0, 0.5, 5.0, 50.0 and 500.0 μM) were tested in five distinct phases of somatic embryogenesis, viz., induction, proliferation, differentiation, maturation and regeneration. Like ABA, MeJA also inhibited callus induction, callus growth, proliferation of embryogenic suspension as well as germination and conversion of somatic embryos. However, its inhibitory effects on various phases of somatic embryogenesis were less pronounced as compared to that due to ABA. In contrast to ABA, MeJA did not have any significant influence on the development of somatic embryos when applied in the differentiation phase. The study showed that ABA used routinely as an inducer of somatic embryo maturation in M. sativa could not be replaced by MeJA.     

Effect of low temperature on dormancy breaking and growth after planting in lily bulblets regenerated in vitro

Lilies regenerating on scale segments may develop dormancy in vitro depending on the culture conditions. The dormancy is broken by storage for several weeks at a low temperature (5 °C). The effect of the low temperature on sprouting, time of leaf emergence and further bulb growth was studied. Dormant and non-dormant bulblets were regenerated in vitro on bulb scale segments cultured at 20 °C or 15 °C, respectively. The low temperature not only affected the number of sprouted bulblets but also the time of emergence. The longer the cold storage, the faster and more uniform leaf emergence occurred. Both dormant and non-dormant bulblets grew faster after a low temperature treatment of six weeks. Thus, during dormancy breaking the tissue is prepared not only for sprouting but also for subsequent bulb growth. These processes are rather independent as low temperature stimulates growth in non-dormant bulblets whereas these bulblets sprout also without treatment at low temperature. Moreover, the hormone gibberellin induces rapid sprouting but has no influence on further bulb growth. Good growth in bulblets exposed to the low temperature coincided with production of an increased leaf weight. However, the relationship is not absolute as bulblets that were cold-treated for six weeks grew larger than bulblets cold-treated for four weeks but the formation of leaf biomass was similar. During storage at low temperature starch was hydrolyzed in the bulb scales and sugars accumulated. This indicates that during this period, preparation for later bulb growth involves mobilization of carbohydrate reserves which play a role in leaf growth and development of the photosynthetic apparatus. Starch hydrolysis proceeded in the outer scales after planting. Approximately six weeks later, the switch from source to sink took place in the bulblet, which became visible as a deposition of starch in the middle scales.     

外源钙和茉莉酸甲酯诱导番茄植株抗灰霉病研究

以‘辽园多丽’番茄幼苗为材料,研究了经钙(Ca)、钙螯合剂(EGTA)和茉莉酸甲酯(MeJA)处理后接种番茄灰霉病幼苗叶片的病情指数、活性氧(H2O2、O2.-)含量和过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、过氧化物酶(POD)活性的变化。结果显示:(1)Ca、MeJA、MeJA+Ca处理番茄幼苗的灰霉病发病率分别比对照显著降低32.5%、38.0%和54.5%,而MeJA+Ca处理又显著低于Ca、MeJA处理32.6%和15.3%;MeJA+EGTA处理高于MeJA处理30.3%,但低于EGTA处理13.1%;Ca处理低于EGTA处理34.2%。(2)Ca、MeJA及MeJA+Ca处理番茄幼苗叶片中活性氧积累量高于对照,MeJA+Ca处理又高于Ca、MeJA处理;但MeJA+EGTA处理活性氧积累量低于MeJA处理,而高于EGTA处理;Ca处理的活性氧含量高于EGTA处理。(3)Ca、MeJA及MeJA+Ca处理幼苗叶片的SOD、CAT、POD的活性均比对照提高,且以MeJA+Ca处理最高;而MeJA+EGTA处理抗氧化酶活性低于MeJA处理,但高于EGTA处理;Ca处理抗氧化酶活性高于EGTA处理。研究表明,钙在茉莉酸甲酯诱导番茄抗灰霉病过程中具有重要调节作用,这种作用与钙促进茉莉酸甲酯诱导番茄活性氧积累和抗氧化酶活性有关。     

茉莉酸甲酯对烟草愈伤组织一些活性氧生成和相关酶活性的影响

烟草愈伤组织用茉莉酸甲酯(MJ, 1mgml-1)处理;同时将愈伤组织在含AIP(0.1mmol/L,水杨酸合成抑制剂)和/或AOA(1mmol/L,乙烯合成抑制剂)的MS培养基上进行暗培养,测定在活性氧产生和脂质过氧化中起作用的相关酶活性及一些代谢物的含量。结果表明,茉莉酸甲酯能激活超氧阴离子的产生,提高脂氧合酶同工酶1(LOX1)的活性从而启动脂质过氧化,对脂氧合酶同工酶3(LOX3)没有明显影响;降低过氧化氢(H2O2)的含量、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)及抗坏血酸过氧化物酶(APX)等保护酶的活性,减少了膜脂过氧化中有毒物质丙二醛(MDA)的含量。AIP和AOA都对MJ的作用有不同程度的影响。MJ调节超氧阴离子和过氧化氢生成以及相关酶活性是通过不同的信号转导途径,MJ调节超氧阴离子和MDA生成、SOD和APX活性很可能是通过乙烯起作用,且对MDA生成和APX活性的调节可能通过水杨酸起作用;MJ直接调节LOX1活性,但对LOX3活性没有明显作用。     

Identification of jasmonic acid and its methyl ester as gum-inducing factors in tulips

The purpose of this study was to identify endogenous factors that induce gummosis and to show their role in gummosis in tulip (Tulipa gesneriana L. cv. Apeldoorn) stems. Using procedures to detect endogenous factors that induce gum in the stem of tulips, jasmonic acid (JA) and methyl jasmonate (JA-Me) were successfully identified using gas–liquid chromatography–mass spectrometry. Total amounts of JA and JA-Me designated as jasmonates in tulip stems were also estimated at about 70–80 ng/g fresh weight, using deuterium-labeled jasmonates as internal standards. The application of JA and JA-Me as lanolin pastes substantially induced gums in tulip stems with ethylene production. The application of ethephon, an ethylene-generating compound, however, induced no gummosis although it slightly affected jasmonate content in tulip stems. These results strongly suggest that JA and JA-Me are endogenous factors that induce gummosis in tulip stems.     

茉莉酸甲酯对花生幼苗生长和抗旱性的影响

经过茉莉酸甲酯处理的花生幼苗,在形态、解剖和生理上都发生变化,其中以１２５ｍｇ／Ｌ处理最显著。处理后的植株幼苗矮化,叶小而厚,叶片贮水细胞大、蒸腾减弱、内源脱落酸和脯氨酸含量增多、过氧化物酶活性加大。由于水分的丧失减少,叶片水分的贮存增加,从而提高幼苗的抗旱性。     

Methyl jasmonate-induced rosmarinic acid biosynthesis in Lithospermum erythrorhizon cell suspension cultures

Summary A dramatic increase in rosmarinic acid (RA) content in cultured cells of Lithospermum erythrorhizon was observed after their exposure to methyl jasmonate (MJ). Preceding the induced RA accumulation, phenylalanine ammonia-lyase (PAL) and 4-hydroxyphenylpyruvate reductase (HPR) activities increased rapidly and transiently, whereas tyrosine aminotransferase (TAT) activity showed only a slight increase. The elicitation activity of MJ was much higher than that of yeast extract (YE) in terms of the induction of PAL and HPR activities, RA accumulation and incorporation of both ¹⁴C-phenylalanine and ¹⁴C-tyrosine into RA. However, the response of the cultured cells to MJ-treatment was slower than that to YE-treatment.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - LS Linsmaier and Skoog - HPR 4-hydroxyphenylpyruvate reductase - PAL phenylalanine ammonia-lyase - TAT tyrosine aminotransferase - MJ methyl jasmonate - YE yeast extract     

低温对东方百合试管鳞茎解除休眠及生长的影响

以东方百合‘索邦’和‘西伯利亚’鳞片为外植体得到的一代试管小鳞茎为试材,研究0℃冷藏不同时间对试管鳞茎生理指标变化,以及试管鳞茎解除休眠和移栽后生长发育的影响。结果表明,冷藏0--28dN,随着冷藏时间的延长,试管鳞茎出苗率逐渐增加,冷藏处理28d达到最高,之后逐渐降低。冷藏期间,试管鳞茎中淀粉含量持续降低,而可溶性总糖和还原性糖含量表现出先升高后下降的趋势,冷藏处理28d的含量最高。同时,随着冷藏时间的延长,IAA和ZR含量逐渐升高,ABA含量逐渐下降,而GA。含量表现出先上升后下降的趋势,并且也在冷藏处理28dN-含量达到峰值。另外,采用隶属函数值对低温处理的试管苗栽培1年后所收获种球的数量、质量等指标进行评价,结果显示,均以低温处理28d时达到最大值。由此得出,0℃低温处理试管小鳞茎28d为解除休眠及促进生长发育的最适处理时间。     

Purification and partial amino acid sequences of an esterase from tomato

Screening of 18 suspension plant cell cultures of taxonomically distant species revealed that a methyl jasmonate hydrolysing enzyme activity (0.21-5.67 pkat/mg) occurs in all species so far analysed. The methyl jasmonate hydrolysing esterase was purified from cell cultures of Lycopersicon esculentum using a five-step procedure including anion-exchange chromatography, gel-filtration and chromatography on hydroxylapatite. The esterase was purified 767-fold to give an almost homogenous protein in a yield of 2.2%. The native enzyme exhibited a M(r) of 26 kDa (gel-filtration chromatography), which was similar to the M(r) determined by SDS-PAGE and MALDI-TOF analysis (M(r) of 28547 kDa). Enzyme kinetics revealed a K(m) value of 15 microM and a V(max) value of 7.97 nkat/mg, an pH optimum of 9.0 and a temperature optimum of 40 degrees C. The enzyme also efficiently hydrolyzed methyl esters of abscisic acid, indole-3-acetic acid, and fatty acids. In contrast, methyl esters of salicylic acid, benzoic acid and cinnamic acid were only poor substrates for the enzyme. N-Methylmaleimide, iodacetamide, bestatin and pepstatin (inhibitors of thiol-, metal- and carboxyproteases, respectively) did not inactivate the enzyme while a serine protease inhibitor, phenylmethylsulfonyl fluoride, at a concentration of 5 mM led to irreversible and complete inhibition of enzyme activity. Proteolysis of the pure enzyme with endoproteinase LysC revealed three peptide fragments with 11-14 amino acids. N-Terminal sequencing yielded an additional peptide fragment with 10 amino acids. Sequence alignment of these fragments showed high homologies to certain plant esterases and hydroxynitrile lyases that belong to the alpha/beta hydrolase fold protein superfamily.     

咖啡酸和氯化钴对茉莉酸甲酯诱导抗病相关酶活性的影响

烟草愈伤组织在含咖啡酸(5mmol/L)和／或CoCl2(10mmol/L,乙烯合成抑制剂)的MS培养基上暗培养,同时用莱莉酸甲酯(1mg／d,简称MJ)处理愈伤组织。处理后测定乙烯、水杨酸和病程相关蛋白(PR)含量及一些抗病相关酶活性。MJ明显促进乙烯产生、增加水杨酸和PR蛋白含量,提高苯丙氨酸解氨酶(PAL)、β1,3—葡聚糖苷酶和几丁酶的活性。咖啡酸降低MJ对乙烯和水杨酸诱导,CoCl2明显降低MJ对乙烯的诱导,但没有明显影响MJ对水杨酸的诱导,两者都促进MJ诱导PAL活性而抑制MJ诱导β1,3—葡聚糖苷酶活性。咖啡酸明显影响MJ诱导内切几丁酶,几乎完全抑制对外切几丁酶的诱导;CoCl2对MJ诱导内切几丁酶没有影响,促进对外切几丁酶的诱导。实验结果表明,不同的抗病相关酶活性诱导有不同的信号传递途径,在所测几种酶的诱导中,水杨酸起主要作用,乙烯作用较小,MJ的诱导作用主要是由水杨酸所转导。     

Methyl jasmonate effect on diterpenoid accumulation in Salvia sclarea hairy root culture in shake flasks and sprinkle bioreactor

Hairy root cultures of Salvia sclarea were grown in shake flasks and 10 L nutrient sprinkle bioreactor, running for 30 days and the effects of methyl jasmonate (MJ) on their growth and capacity to accumulate diterpenoids were measured. We found that MJ concentration and exposure time to the elicitor were factors that strongly affected the diterpenoid production. The highest diterpenoid accumulation (67.5 ± 7.1 mg g⁻¹ dry weight, calculated as a sum of ferruginol, salvipisone, aethiopinone and 1-oxoaethiopinone) without reduction of biomass, was achieved when the 23-day-old hairy roots in bioreactor culture were exposed to 125 μM MJ for 7 days. The roots produced 9 and 3.8 times as much aethiopinone (40 ± 5.9 mg g⁻¹ dry weight) and salvipisone (12.6 ± 0.4 mg g⁻¹ dry weight), respectively, as roots cultured in shake flasks. Our results imply that cultivation of S. sclarea hairy roots in sprinkle bioreactor after elicitation with MJ may be valuable to enhance production of the bioactive diterpenoids.     

Jasmonates Inhibit Flowering in Short-Day Plant Pharbitis nil

The role of jasmonates in the photoperiodic flower induction of short-day plant Pharbitis nil was investigated. The plants were grown in a special cycle: 72 h of darkness, 24 h of white light with lowered intensity, 24-h long inductive night, 14 days of continuous light. At 4 h of inductive night the cotyledons of non-induced plants contained about two times the amount of endogenous jasmonates (JA/JA-Me) compared to those induced. A 15-min long pulse of far red light (FR) applied at the end of a 24-h long white light phase inhibited flowering of P. nil. The concentration of jasmonates at 2 and 4 h of inductive night in the cotyledons of the plants treated with FR was similar. Red light (R) could reverse the effect of FR. R light applied after FR light decreased the content of jasmonates by about 50%. Methyl jasmonate (JA-Me) applied to cotyledons, shoot apices and cotyledon petioles of P. nil inhibited the formation of flower buds during the first half of a 24-h long inductive or 14-h long subinductive night. Application of JA-Me to the cotyledons was the most effective. None of the plants treated with JA-Me on the cotyledons in the middle of the inductive night formed terminal flower buds. The aspirin, ibuprofen and phenidone, jasmonates biosynthesis inhibitors partially reversed the effect of FR, stimulating the formation of axillary and terminal flower buds. Thus, the results obtained suggests that phytochrome system control both the photoperiodic flower induction and jasmonates metabolism. Jasmonates inhibit flowering in P. nil.     

水杨酸和茉莉酸甲酯对丹参幼苗叶片显微结构、光合及非结构糖积累的影响

研究了水杨酸(SA)和茉莉酸甲酯(MeJA)处理对丹参(Salvia miltiorrhiza Bunge)幼苗叶片显微结构、叶片光合能力及幼苗中非结构糖积累的影响.结果显示:SA处理增加了丹参幼苗叶片气孔密度;叶肉细胞排列紧密、体积减小,叶肉细胞内叶绿体数目减少,但叶绿体体积增大,叶绿体基粒片层结构的数目增加;叶片中叶绿素a、b含量、叶气孔导度、蒸腾速率以及净光合速率均增加;同时,幼苗根中和叶片中酸性转化酶活性降低,幼苗地上部分蔗糖含量及可溶性糖总量显著高于对照.MeJA处理减少了叶片气孔密度,气孔发育畸形;叶肉细胞间隙增大,栅栏细胞层数减少,叶肉细胞内叶绿体数目减少,叶绿体体积减小,叶绿体基粒片层结构被破坏;叶片中叶绿素a及类胡萝卜素含量、叶片的净光合速率低于对照,叶气孔导度、蒸腾速率增强;同时,幼苗根中及叶中酸性转化酶活性增加,幼苗根中蔗糖含量及可溶性糖总量显著低于对照.可见,SA处理能促进植物叶片显微结构发育,增强叶片光合能力,抑制蔗糖降解并促进蔗糖积累;而MeJA处理则破坏了植物叶片显微结构,降低了叶片光合能力,促进了蔗糖降解并减少蔗糖积累.     

外源茉莉酸甲酯对盐胁迫下小桐子幼苗渗透调节和脯氨酸代谢的影响

以小桐子幼苗为材料,采用人工气候箱内水培试验,设置不同浓度(0、20、40、60、80、100μmol·L^(-1))茉莉酸甲酯(MeJA)和150 mmol·L^(-1)NaCl胁迫处理,分析不同处理条件下小桐子幼苗叶片的组织活力、MDA含量、水势、含水量和叶片渗透调节物质脯氨酸、甜菜碱和可溶性糖的含量,以及脯氨酸代谢关键酶P5CS、OAT和甜菜碱合成关键酶BADH活性和相关基因表达水平,探讨外源茉莉酸甲酯对盐胁迫下小桐子幼苗渗透调节能力的影响及其机制。结果表明:(1)外源MeJA处理可提高盐胁迫下小桐子幼苗叶片的组织活力和叶片含水量,降低小桐子幼苗叶片的MDA含量和水势,且60μmol·L^(-1)浓度处理效果最佳。(2)外源MeJA处理可提高盐胁迫下小桐子幼苗叶片的脯氨酸、甜菜碱和可溶性糖的含量,且60μmol·L^(-1)MeJA处理显著提高了盐胁迫下小桐子幼苗内源茉莉酸、脯氨酸和甜菜碱的含量。(3)60μmol·L^(-1)MeJA也提高了盐胁迫下小桐子BADH、P5CS和OAT的活性,并上调了JcBADH、JcP5CS、JcOAT基因的表达水平,但MeJA降低了脯氨酸降解酶ProDH的活性,下调了JcProDH基因的表达。研究发现,在盐胁迫条件下,外源MeJA通过活化脯氨酸生物合成的谷氨酸和鸟氨酸途径,尤其是鸟氨酸途径,以及抑制脯氨酸降解途径来促进小桐子幼苗脯氨酸的积累,同时MeJA也激活了幼苗体内甜菜碱的生物合成过程,从而强化了盐胁迫下幼苗的渗透调节作用和耐盐性,表明MeJA诱导的渗透调节在小桐子耐盐性形成过程中发挥着重要作用。     

茉莉酸甲酯对木豆不定根染料木素含量及抗氧化系统影响

为探讨茉莉酸甲酯(Methyl Jasmonate,MJ)对木豆不定根影响,研究了茉莉酸甲酯的不同浓度及处理时间对木豆不定根中染料木素的含量及抗氧化系统的影响。研究结果表明,诱导子的浓度和处理时间对不定根中染料木素的含量有重要影响。在MJ浓度为100μmol·L^-1,处理时间为48 h时,染料木素的含量最高,为1.38 mg·g^-1,是对照组的4.03倍。在此基础上,研究了100μmol·L^-1的MJ处理对木豆不定根抗氧化系统的影响,结果表明,MJ处理的木豆不定根明显发生了氧化胁迫,O_2^-速率、H_2O_2和MDA含量明显升高,同时不定根中SOD和POD活性明显升高,但是抗氧化酶活性的提高并未消除不定根的氧化胁迫,这种氧化胁迫导致了木豆不定根次生代谢产物合成能力提高。     

The Involvement of Intracellular Glutathione in Methyl Jasmonate Signaling in Arabidopsis Guard Cells

We examined the involvement of intracellular glutathione (GSH) in methyl jasmonate (MeJA) signaling. The chlorina1-1 (ch1-1) mutation decreased GSH in guard cells and narrowed the stomatal aperture. GSH monoethyl ester increased intracellular GSH, diminishing this phenotype. GSH did not affect MeJA-induced reactive oxygen species production or cytosolic Ca²⁺ oscillation, suggesting that GSH modulates MeJA signaling downstream of production and oscillation.     

The Effect of Abscisic Acid and Methyl Jasmonate on Carbonic Anhydrase Activity in Pea

Short-term (2 h) treatment with 10 μM abscisic acid decreased stomatal conductance and net photosynthetic rate, and increased carbonic anhydrase activity in pea seedlings. The treatment with 10 μM methyl jasmonate did not significantly affect these parameters. This revised version was published online in June 2006 with corrections to the Cover Date.     

氨基茚磷酸(AIP)和氨基氧乙酸(AOA)对茉莉酸甲酯诱导的烟草一些酶活性的影响

用茉莉酸甲酯(MJ,1mg ml^-1)处理培养在含0．1mmol／L AIP(水杨酸合成抑制剂)和／或1mmol／L AOA(乙烯合成抑制剂)的MS培养基上的烟草愈伤组织,测定某些酶的活性。结果表明：MJ明显提高过氧化物酶(POD)、β1,3-葡聚糖苷酶和几丁质外切酶的活性,略微促进苯丙氨酸解氨酶(PAL)、多酚氧化酶(PPO)的活性,抑制几丁内切酶的活性,而AOA和AIP则明显抑制MJ对POD和β1,3-葡聚糖苷酶活性的诱导作用,但对MJ诱导的PAL和PPO的活性影响很小,AOA和AIP可能作为逆境因子促进PAL的活性。AOA能部分解除MJ对几丁内切酶的抑制作用,但对MJ诱导的几丁外切酶的影响较小,而AIP抑制几丁内切酶的活性,也抑制MJ对此酶的诱导作用。因此我们认为：MJ对POD、PPO和几丁内切酶的影响可能是通过乙烯途径,对β1,3-葡聚糖苷酶和几丁外切酶的影响可能是通过水杨酸(SA)途径,而对PAL的影响可能是通过其它途径。     

Effect of elicitors on the production of gossypol and methylated gossypol in cotton hairy roots

The effect of two chemical elicitors, salicylic acid and methyl jasmonate, on the production of gossypol, 6-methoxygossypol, and 6,6′-dimethoxygossypol in Gossypium barbadense hairy roots was examined. Methyl jasmonate, but not salicylic acid, was found to increase the production of gossypol and its methylated forms, but with a concomitant reduction in culture growth. The optimal methyl jasmonate dose was between 100 and 300 μM for hairy roots harvested 7 days after elicitation. After 20 d of induction with 100 μM methyl jasmonate, an eightfold increase in the level of gossypol was observed in elicited cultures compared with control cultures, double the highest gossypol levels previously reported for any cotton tissue. A two to threefold increase in the level of 6-methoxygossypol and a slight increase in the levels of 6,6′-dimethoxygossypol were also observed. Although methyl jasmonate stimulated the production of both optical forms of gossypol, the distribution of the enantiomers was different between elicited and control cultures.     

Effect of Temperatures on Dormancy and Germination in Three Species in the Lamiaceae Occurring in Northern Wetlands

In the temperate region temperature is the main factor influencing the germination period of plant species. The purpose of this study was to examine effects of constant and fluctuating temperatures on dormancy and germination under laboratory and field conditions in the three wetland species Lycopus europaeus, Mentha aquatica and Stachys palustris. The results should give indications if the temperature-dependent regulation of dormancy and germination is phylogenetically constrained. Tests for germination requirements showed a minimum temperature for germination of 9 °C in Mentha and 12 °C in Lycopus and Stachys, and a maximum temperature of 33 °C for Lycopus and 36 °C for Mentha and Stachys. Fluctuating temperatures promoted germination in all three species but the amplitude required for high germination (>50%) differed: it was 8 °C in Mentha, 10 °C in Stachys and 14 °C in Lycopus (mean temperature 22 °C). The effect of temperatures on the level of dormancy was examined in the laboratory by imbibing seeds at temperatures between 3 °C and 18 °C for periods between 2 and 28 weeks, as well as by a 30-month burial period, followed by germination tests at various temperatures, in light and darkness. In the laboratory only low temperatures (≤12 °C) relieved primary dormancy in seeds of Lycopus, while in Mentha and Stachys also higher temperatures lead to an increase of germination. Dormancy was only induced in Lycopus seeds after prolonged imbibition at 12 °C in the laboratory. Buried seeds of all species exhibited annual dormancy cycles with lower germination in summer and higher germination from autumn to spring. Exhumed seeds, however, showed considerable differences in periods of germination success. Dormancy was relieved when ambient temperatures were below 12 °C. Ambient temperatures that caused an induction of dormancy varied depending on species and test condition, but even low temperatures (8 °C) were effective. At high test temperatures (25 °C) in light, exhumed seeds of all three species showed high germination throughout the year. The three species showed various differences in the effects of temperatures on dormancy and germination. Similarities in dormancy and germination found among the species are in common with other spring-germinating species occurring in wetlands, so it seems that the temperature dependent regulation of dormancy and germination are related to habitat and not to phylogenetic relatedness.