Symbiotic effectiveness of streptomycin-resistant Rhizobium spp. mutants of chickpea (Cicer arietinum L.)

Summary Antibiotic-resistant Rhizobium spp. strains have been used in ecological studies of legumerhizobia symbiosis. It has been suggested that in the course of acquiring resistance against high doses of antibiotics, rhizobia might lose their symbiotic effectiveness. Evidence both for and against this argument has been presented (Kremer and Peterson 1982; Materon and Hagedron 1983). This communication reports our experience with streptomycin-resistant (Str⁺) mutants of chickpea Rhizobium spp. strains. Parent strains were used as controls.Research paper No. 5233 from the Experiment Station, G.B.P.U.A.&T. Pantnagar, Nainital     

Effect of inoculum rate on the performance of chickpea (Cicer arietinum L.) Rhizobium strains in the field

Summary The influence of three inoculum rates on the performance of three chickpea (Cicer arietinum L.) Rhizobium strains was examined in the field on a Mollisol soil. Increasing amounts of inoculum improved the performance of the strains. A normal dose (10⁴ cells per seed) applied at different intervals gave non-significant increases in nodulation, nitrogenase activity (acetylene reduction assay), nitrogen uptake and grain yield. A ten-fold increase in inoculum increased nodule number, shoot dry weight, nitrogenase activity (ARA) and grain yield, but increases over the control were significant only for nodule dry weight and nitrogen uptake by shoot and grain. The highest level of inoculum (100 × normal) significantly increased nodule dry weight, grain yield, total nitrogenase activity (ARA) and nitrogen uptake by shoot and grain. Strain TAL 620 was more effective than the other two. Combined nitrogen (60 kg N ha^–1) suppressed nodulation and nitrogenase activity (ARA).Research paper No. 4345 from the Experiment Station, G. B. P. U. A. & T., Pantnagar, Nainital, U. P.     

Displacement of native rhizobia nodulating chickpea (Cicer arietinum L.) by an inoculant strain through soil solarization

Summary Soil solarization greatly reduced the native chickpea Rhizobium population. With inoculation, it was possible to increase the population of the Rhizobium in solarized plots. In the 1st year, 47% nodulation was obtained with chickpea inoculant strain IC 59 when introduced with a cereal crop 2 weeks after the soil solarization and having a native Rhizobium count of <10 g^-1 soil, and only 13% when introduced 16 weeks after solarization at the time the chickpeas were sown, with 2.0×10² native rhizobia g^-1 soil. In the non-solarized plots inoculated with 5.6×10³ native rhizobia g^-1 soil, only 6% nodulation was obtained with the inoculant. In the succeeding year, non-inoculated chickpea was grown on the same plots without any solarization or Rhizobium inoculation. The treatment that showed good establishment of the inoculant strain in year 1 formed 68% inoculant nodules. Other treatments indicated a further reduction in inoculant success, from 1%–13% to 1%–9%. Soil solarization thus allowed an inoculant strain to successfully displace the high native population in the field and can serve as a research tool to compare strains in the field, irrespective of competitive ability. In year 1, Rhizobium inoculation of chickpea gave increased nodulation and increased plant growth 20 and 51 days after sowing, and increased dry matter, grain yield, and grain protein yield at maturity. These beneficial effects of inoculation on plant growth and yield were not measured in the 2nd year.Submitted as Journal Article No. JA 945 by the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru, Andhra Pradesh 502 324, India     

Differential responses of chickpea (Cicer arietinum L.) — Rhizobium combinations to saline soil conditions

Summary Chickpea cultivars (Cicer arietinum L.) and their symbiosis with specific strains of Rhizobium spp. were examined under salt stress. The growth of rhizobia declined with NaCl concentrations increasing from 0.01 to 2% (w : v). Two Rhizobium spp. strains (F-75 and KG 31) tolerated 1.5% NaCl. Of the 10 chickpea cultivars examined, only three (Pusa 312, Pusa 212, and Pusa 240) germinated at 1.5% NaCl. The chickpea — Rhizobium spp. symbiosis was examined in the field, with soil varying in salinity from electrical conductivity (EC) 4.5 to EC 5.2 dSm^-1, to identify combinations giving satisfactory yields. Significant interactions between strains and cultivars caused differential yields of nodules, dry matter, and grain. Four chickpea — Rhizobium spp. combinations, Pusa 240 and F-75 (660 kg ha^-1), Pusa 240 and IC 76 (440 kg ha^-1), Pusa 240 and KG 31 (390 kg ha^-1), and Pusa 312 and KG 31 (380 kg ha^-1), produced significantly higher grain yields in saline soil.     

Tolerance of chickpea (Cicer arietinum L.) lines to root-knot nematode, Meloidogyne javanica (Treub) Chitwood

The root-knot nematode, Meloidogyne javanica (Treub) Chitwood is an important parasite of chickpea (Cicer arietinum L.). Four chickpea genotypes were evaluated for tolerance to M. javanica in naturally infested fields at three locations. Each genotype was evaluated for number of galls, gall size, root area covered with galls and number of egg masses produced. All the cultivars were susceptible or highly susceptible. Seed yield, weight of 100 undamaged seeds, total dry matter and plant height were compared with checks. Chickpea cultivar Annigeri and a local check were used as nematode susceptible checks in all locations. The four promising nematode tolerant genotypes produced significantly greater yield and total dry matter than the checks in fields naturally infested with M. javanica at three locations. These M. javanica tolerant lines represent new germplasm and they are available in the chickpea genebank at the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) bearing the identification numbers ICC 8932, ICC 11152, ICCV 90043 and ICCC 42.     

RAPD and seed coat morphology variation in annual and perennial species of the genus Cicer L.

Molecular and morphological variation of six perennial and five annual species including domesticated chickpea, C. arietinum, were inferred on the basis of RAPD and S.E.M. seed coat features using three outgroup taxa (Lens ervoides, Lathyrus japonica and Pisum sativum). Of the 66 decamer arbitrary primers tested, eight primers revealed 87 informative fragments. Neighbor-joining cluster analysis using Jaccard's coefficient of similarity on the basis of polymorphic fragments indicated a narrow variation in C. arietinum and recognized two major clusters in the genus Cicer. The first one included four species of sect. Monocicer: C. echinospermum, C. arietinum, C. reticulatum and Iranian material of C. bijugum. The second cluster contained annual and perennial species belonging to sections Chamaecicer, Polycicer and Acanthocicer. The character state of morphological and ecological traits on the RAPD phenogram indicated no monophyletic incision. Our results suggested that the high genetic difference between annual and perennial species might be regarded as a rapid speciation of section Monocicer. Reconsideration of traditional classification of sections Polycicer and Acanthocicer is necessary. The Desi and Kabuli types of C. arietinum could not be separately grouped at the DNA level, and the low level of genetic variation of C. arietinum may result from a bottleneck during the domestication process.     

Evaluation of annual wild <Emphasis Type="Italic">Cicer</Emphasis> species for drought and heat resistance under field conditions

About 90% of chickpea (Cicer arietinum L.) in the world is grown under rainfed conditions where it is subjected to drought and heat stress. Unlike the cultivated chickpea, annual wild Cicer species possess sources of resistance to multiple stress; annual wild Cicer species were therefore evaluated for resistance to drought and heat stress. Eight annual wild Cicer species (Cicer bijugum, C. chorassanicum, C. cuneatum, C. echinospermum, C. judaicum, C. pinnatifidum, C. reticulatum, and C. yamashitae) were compared with special checks, the cvs ICC 4958 and FLIP 87-59C (drought resistant) and ICCV 96029 (very early double-podded). ILC 3279 and 8617 as drought susceptible checks were sown after every 10 test lines. Yield losses due to drought and heat stress in some accessions and susceptible checks (ILC 3279 and ILC 8617) reached 100%. Accessions were evaluated for drought and heat resistance on a 1 (free from drought and heat damage)−9 (100% plant killed from drought and heat) visual scale. Four accessions of C. reticulatum and one accession of C. pinnatifidum were found to be as resistant to drought and heat stress (up to 41.8°C) as the best checks. C. reticulatum should be taken account in short term breeding programs since it can be crossed with the cultivated chickpea.     

Evaluation of 15N-isotope dilution for measurement of nitrogen fixation in chickpea (Cicer arietinum L.)

Summary N accumulation, nodulation, and acetylene reduction activity were measured at frequent intervals during the growth of two chickpea genotypes, and N₂ fixation was estimated by an isotope-dilution method, using safflower as a non-N₂-fixing reference. Safflower was more efficient at N uptake than both the chickpea genotypes for at least the first 50 days and thus could not be used as an accurate reference control. We recommend that further work should employ non-nodulatiog genotypes of chickpea as reference plants and use slow-release forms of 15N fertilizer. Direct genotype comparison by isotope dilution estimated that genotype K 850 fixed 16–18 kg ha^–1 more N than G 130, and this difference was supported by the greater nodule mass and acetylene reduction activity in the K 850 cultivar. Inoculation with an ineffective chickpea Rhizobium sp. led to 69% nodulation on cultivar K 850 but only 33% on G 130. While nodule weight, N uptake, and acetylene reduction activity decreased with inoculation in K 850, the isotope dilutions were similar for both inoculation treatments. The lack of a significant effect on N₂ fixation was ascribed to the partial success of inoculant establishment.Published as Journal Article No. JA 692 of the International Crops Research Institute for the Semi Arid Tropics, Patancheru, A.P. 502324, India     

RAPD and ISSR fingerprinting in cultivated chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) and its wild progenitor <Emphasis Type="Italic">Cicer reticulatum</Emphasis> Ladizinsky

Detection of genetic relationships between 19 chickpea cultivars and five accessions of its wild progenitor Cicer reticulatum Ladizinsky were investigated by using RAPD and ISSR markers. On an average, six bands per primer were observed in RAPD analysis and 11 bands per primer in ISSR analysis. In RAPD, the wild accessions shared 77.8% polymorphic bands with chickpea cultivars, whereas they shared 79.6% polymorphic bands in ISSR analysis. In RAPD analysis 51.7% and 50.5% polymorphic bands were observed among wild accessions and chickpea cultivars, respectively. Similarly, 65.63% and 56.25% polymorphic bands were found in ISSR analysis. The dendrogram developed by pooling the data of RAPD and ISSR analysis revealed that the wild accessions and the ICCV lines showed similar pattern with the dendrogram of RAPD analysis. The ISSR analysis clearly indicated that even with six polymorphic primers, reliable estimation of genetic diversity could be obtained, while nearly 30 primers are required for RAPD. Moreover, RAPD can cause genotyping errors due to competition in the amplification of all RAPD fragments. The markers generated by ISSR and RAPD assays can provide practical information for the management of genetic resources. For the selection of good parental material in breeding programs the genetic data produced through ISSR can be used to correlate with the relationship measures based on pedigree data and morphological traits to minimize the individual inaccuracies in chickpea.     

Comparison of some endogenous hormone levels in different parts of chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.)

The objective of this study was to detect plant hormone levels in different plant parts of chickpea, and to compare cultivars with different characteristics such as simple leaf vs. normal leaves, simple leaf vs. bipinnate leaf, kabuli type vs. desi types, double-podded vs. single-podded. It was found that there was a great variation in basic plant hormone levels among genotypes, and plant hormone concentrations varied according to leaf type, pod and seed characteristics in these genotypes. IAA level in leaves was the highest in ICC 6119 (bipinnate leaf) and followed by ICC 552 (double-pod) and Kusmen 99 (simple leaf), while it was highest level in pods of ICC 552 (double-pod). Zeatin concentration in leaves and pods was the highest level in ICC 6119 (bipinnate leaf). GA₃ concentration was the highest in leaves of ICC 6119 (bipinnate leaf). ICC 552 (double-pod) has the highest GA₃ in leaves. It was postulated that leaf type and pod characteristics may be related to hormones induced growth and development.     

Sources of resistance to cyst nematode in cultivated and wild Cicer species

Summary Among the nematodes infesting chickpea (Cicer arietinum L.) plants in Syria, cyst nematode (Heterodera ciceri Vovlas, Greco et Di Vito) is the most important. It is uneconomical to grow chickpea in fields infested with cyst nematode and to control this nematode with nematicide. Therefore, investigations were conducted at ICARDA, Syria from 1987 to 1991 to identify sources of resistance to cyst nematode in 7258 lines of C. arietinum and 102 lines of eight annual Cicer species including C. bijugum K.R. Rech. (13 lines), C. chorassanicum (Bge) M. Pop. (3 lines), C. cuneatum Hochst. ex Rich. (3 lines), C. echinospermum P.H. Davis (8 lines), C. judaicum Boiss. (18 lines), C. pinnatifidum Jaub. & Sp. (18 lines), C. reticulatum Ladiz. (36 lines), and C. yamashitae Kitamura (3 lines). All lines were grown in a greenhouse at 15–25°C in pots containing soil infested with 20 eggs of the nematode g^-1 soil. Nematode infestation was evaluated on a 0 to 5 scale based on number of females and cysts on roots. Resistance was found in one line of C. bijugum, six lines of C. pinnatifidum, and one line of C. reticulatum. No lines of C. arietinum, C. chorassanicum, C. cuneatum, C. echinospermum, C. judaicum, or C. yamashitae was resistant to cyst nematode. Plants with resistance have been recovered in the F₃ generation from crosses between the cultigen and C. reticulatum, indicating the possibility of transfer of gene(s) for resistance to cyst nematode from wild to cultivated Cicer species.Joint contribution from Istituto di Nematologia Agraria, ICARDA and ICRISAT (International Crops Research Institute for the Semi-Arid Tropics), Patancheru P.O., A.P. 502 324, India.     

Identification of duplicates and fingerprinting of primary and secondary wild annual <Emphasis Type="Italic">Cicer</Emphasis> gene pools using AFLP markers

Wild annual Cicer gene pools contain valuable germplasm for chickpea improvement programs. Previous research showed that duplication might exist in accessions collected from these gene pools, which would hinder chickpea breeding and related research. AFLP (amplified fragment length polymorphism) markers were used to fingerprint the world collections of the primary and secondary gene pools including C. reticulatum Lad., C. bijugum K.H. Rech., C. judaicum Boiss. and C. pinnatifidum Jaub. et Sp. Duplicates were detected in a total of 24 accessions in both the gene pools, highlighting the necessity to fingerprint the germplasm. Genotypic difference was detected as gene pool specific, species specific and accession specific AFLP markers. These were developed into fingerprinting keys for accession identification between and within species and gene pools. Use of AFLP markers to detect duplicates and to identify accessions is a reliable method which will assist in the characterisation and use of wild annual Cicer germplasm in chickpea improvement programs. We recommend the procedure presented in this paper as a standard approach for the precise genetic identification and characterisation of future world collections of wild Cicer, to keep germplasm integrity and to benefit chickpea breeding and related research programs.     

Preliminary screening and selection for cold tolerance in annual wild <Emphasis Type="Italic">Cicer</Emphasis> species

The study was aimed to select cold tolerant accessions of annual wild Cicer species in the highlands in the west Mediterranean area of Turkey. A total of 43 accessions of eight annual wild Cicer species was screened for cold tolerance and compared with the best cold tolerant cultivars, ILC 8262 and FLIP 93-53C. After the sensitive check, ILC 533, was killed, accessions were evaluated using a 1–9 scale. Three accessions of Cicer bijugum, 2 accessions of Cicer reticulatum and 2 accessions of Cicer echinospermum were highly cold tolerant, while 3 accessions of C. bijugum, 9 accessions of C. reticulatum, 2 accessions of C. echinospermum and 1 accession of Cicer pinnatifidum were cold tolerant. All accessions of Cicer judaicum were sensitive to cold. Whereas Cicer chorassanicum and Cicer cuneatum were killed, Cicer yamashitae was recorded as highly sensitive to cold. The cold tolerant accessions of annual wild Cicer species were superior to the best cultivar as far as hardiness is concerned.     

Potential of wild species for genetic enhancement of some semi-arid food crops

Discovery and incorporation of genes from wild species provide means to sustain crop improvement, particularly when levels of resistance in the cultigens are low and virulent strains of pests and pathogens overcome the host plant resistance. The extent of utilization and the potential of the wild genepool for genetic enhancement were reviewed in five important food crops viz. sorghum, pearl millet, chickpea, pigeonpea and groundnut grown in the semi-arid tropics. Introgression from compatible wild germplasm in the primary gene pool resulted in transfer of new cytoplasmic male sterility systems in pearl millet and pigeonpea, development of high protein, cleistogamous flower and dwarf pigeonpea lines and foliar disease resistant groundnut cultivars. Utilization of wild species in secondary and tertiary gene pools has been generally limited due to sterility, restricted recombination or cross incompatibility. Nevertheless, these species are extremely important as they contain high levels of resistance to several important biotic and abiotic stresses. Several of them, like those belonging to the Parasorghum section and the rhizomatous Arachis species are sources of multiple resistances and hold great promise to sustain crop productivity.     

Diversity of seed globulins in Lathyrus sativus L. and some related species

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of reduced seed globulins covered 141 accessions of Lathyrus sativus and the following related species: L. amphicarpos, L. blepharicarpus, L. cicera, L. gorgoni, L. marmoratus, L. pseudocicera and L. stenophyllus. In the total examined material 71 globulin polypeptide bands were distinguished. The number of polypeptide bands detected in particular species varied from 14 in L. amphicarpos to 29 in L. sativus; all the species under study showed intraspecific variation due to individual variation and/or differences among accessions. Electrophoretic data were subjected to statistical analysis using hierarchical UPGMA grouping. Frequency distribution of polypeptide bands in L. sativus showed some correlations with geographical origin and certain seed characteristics (seed coat colour, seed weight) of the studied accessions. As regards interspecific relationships, the studied species showed to be distantly related taxa except for the rather closely allied L. cicera and L. marmoratus. The obtained results are compared with the electrophoretic seed albumin data reported earlier for the same taxa.     

Genetic relationships among Arachis hypogaea L. (AABB) and diploid Arachis species with AA and BB genomes

The cultivated peanut (Arachis hypogaea L.) is an allotetraploid, with two types of genomes, classified as AA and BB, according to cytogenetic characters. Similar genomes to those of A. hypogaea are found in the wild diploid species of section Arachis, which is one of the nine Arachis sections. The wild species have resistances to pests and diseases that affect the cultivated peanut and are a potential source of genes to increase the resistance levels in peanut. The aim of this study was to analyze the genetic variability within AA and BB genome species and to evaluate how they are related to each other and to A. hypogaea, using RAPD markers. Eighty-seven polymorphic bands amplified by ten 10-mer primers were analyzed. The species were divided into two major groups, and the AA and the BB genome species were, in general, separated from each other. The results showed that high variation is available within species that have genomes similar to the AA and the BB genomes of A. hypogaea.     

Identification and overcoming barriers between Brassica rapa L. em. Metzg. and B. nigra (L.) Koch crosses for the resynthesis of B. juncea (L.) Czern.

Brassica juncea (2n = 36, AABB) is an amphidiploid derived from its diploid progenitor species B. rapa (2n = 20, AA) and B. nigra (2n = 16, BB). Resynthesis of B. juncea by exploiting the wider gene pool of the present day diploids offers a powerful tool for obtaining novel genetic variation. Hybridization between the two species is difficult because of the presence of crossability barriers. Barriers to hybridization were identified using Aniline Blue Fluorescence (ABF) method. Crosses were classified as having pre- or post-fertilization barriers based on whether less than or more than one per cent ovules showed pollen tube entry. Of the 23 crosses studied, seven crosses all with B. rapa as the female parent showed pre-fertilization barriers. Only 0 to 0.3 per cent ovules were fertilized in these crosses. The remaining 16 crosses, four with B. rapa as the female parent and 12 with B. nigra as the female parent, showed post-fertilization barriers. In crosses with pre-fertilization barriers use of bud pollination and stump pollination ensured or increased fertilization. These two simple techniques were used in combination with ovary-ovule culture to ensure recovery of hybrids. In crosses with post-fertilization barriers, ovary-ovule culture alone helped to obtain hybrids. Hybrids were obtained at higher frequency with the use of direct ovule culture compared to ovary-ovule culture. The F₁ plants obtained in vitro were multiplied on MS medium + 2 mg/l Kn + 0.2 mg/l IAA. These F₁ plants were confirmed as true hybrids through morphology, leaf isozymes and cytology. In all 17 hybrids were obtained of which three were amphidiploids.     

Dual origin of the cultivated rice based on molecular markers of newly collected annual and perennial strains of wild rice species, Oryza nivara and O. rufipogon

The direct ancestor of rice (Oryza sativa L.) is believed to be AA genome wild relatives of rice in Asia. However, the AA genome wild relatives involve both annual and perennial forms. The distribution of the retrotransposon p-SINE1-r2, a short interspersed nuclear element (SINE) at the waxy locus was analyzed in diverse accessions of the AA genome wild relatives of rice (O. rufipogon sensu lato). Most annual wild rice accessions had this retrotransposon, while most perennial types lacked this element, contradicting results to the previous studies. Results presented here suggest that O. sativa has dual origin that lead to indica-japonica differentiation. Results suggest the indica line of rice varieties evolved from the annual genepool of AA genome and the japonica varieties from the perennial genepool of AA genome wild rice.     

Diversity and genetic resources of wild <Emphasis Type="Italic">Vigna</Emphasis> species in India

Diversity in morphological characters of 206 accessions of 14 wild Vigna species from India was assessed. Of these, 12 species belonged to Asian Vigna in the subgenus Ceratotropis and two were V. vexillata and V. pilosa belonging to subgenus Plectotropis and Dolichovigna, respectively. Data on 71 morphological traits, both qualitative and quantitative, were recorded. Data on 45 qualitative and quantitative traits exhibiting higher variation were subjected to multivariate analysis for establishing species relationships and assessing the pattern of intraspecific variation. Of the three easily distinguishable groups in the subgenus Ceratotropis, all the species in mungo-radiata group, except V. khandalensis, viz. V. radiata var. sublobata, V. radiata var. setulosa, V. mungo var. silvestris and V. hainiana showed greater homology in vegetative morphology and growth habit. The species, however, differed in other plant, flower, pod and seed characteristics. Within species variation was higher in V. mungo var. silvestris populations and three distinct clusters could be identified in multivariate analysis. V. umbellata showed more similarity to V. dalzelliana than V. bourneae and V. minima in the angularis-umbellata (azuki bean) group. Within species variations was higher in V. umbellata than other species in the group. In the aconitifolia-trilobata (mothbean), V. trilobata populations, were more diverse than V. aconitifolia. The cultigens of the conspecific wild species were more robust in growth, with large vegetative parts and often of erect growth with three- to five-fold increase in seed size and seed weight, except V. aconitifolia, which has still retained the wild type morphology to a greater extent. More intensive collection, characterisation and conservation of species diversity and intraspecific variations, particularly of the close wild relatives of Asian Vigna with valuable characters such as resistance to biotic/abiotic stresses, more number of pod bearing clusters per plant etc. assumes great priority in crop improvement programmes.     

Assessment of genetic diversity, and phylogenetic relationships based on ribosomal DNA repeat unit length variation and Internal Transcribed Spacer (ITS) sequences in chickpea (Cicer arietinum) cultivars and its wild species

Repeat unit length variation and internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA were used to assess genetic diversity, and phylogenetic relationships in chickpea (C. arietinum) cultivars, and its related wild species. Total genomic DNAs of 76 accessions of 10 Cicer species, belonging to three sections of the genus, were restricted with seven enzymes and the restriction fragments were hybridized to heterologous ribosomal clones of wheat pTa71 and Vicia faba probes Ver 6-5 and Ver18-6. A single repeat unit length class of 11.4 kb or 10.5 kb was recognized across Cicer accessions with pTa71. The intraspecific variation was negligible in those species where more than one accession was studied, except the four C. judaicum accessions, which were different from the rest. EcoRI and DraI digests gave two and one-two fragments, respectively. All the accessions produced three and three-five bands with BamHI and SacI, respectively. Both the accessions of C. yamashitae differed in their rDNA repeat unit length as well as restriction site variation. Maximum likelihood tree with rDNA RFLP recognized five clades which were more or less congruent with the previous data. Length of ITS-1 region was more variable (235–239 bp) than the ITS-2 region (212–213 bp). Cladistic analysis of ITS data revealed two major clades, clade I consisting of C. arietinum, C. reticulatum and C. echinospermum, and clade II comprised of C. judaicum, C. chorassanicum, C. bijugum and C. cuneatum. C. microphyllum grouped with the above four species. C. pinnatifidum was present as a separate branch. C. yamashitae emerged as the most distinct species.