MPMI Vol. 28, No. 12, 2015, pp. 1338–1352. http://dx.doi.org/10.1094/MPMI-06-15-0141-R
Rhizobial Diversity and Nodulation Characteristics of the Extremely Promiscuous Legume Sophora flavescens Yin Shan Jiao,1 Yuan Hui Liu,1 Hui Yan,1 En Tao Wang,1,2 Chang Fu Tian,1 Wen Xin Chen,1 Bao Lin Guo,3 and Wen Feng Chen1 1
State Key Laboratory of Agrobiotechnology, Beijing 100193, China; College of Biological Sciences and Rhizobia Research Center, China Agricultural University, Beijing 100193, China; 2Departamento de Microbiolog´ıa, Escuela Nacional de Ciencias 3 Biologicas, ´ Instituto Politecnico ´ Nacional, Mexico ´ D. F. 11340, Mexico; ´ Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China Submitted 15 June 2015. Accepted 13 September 2015.
In present study, we report our extensive survey on the diversity and biogeography of rhizobia associated with Sophora flavescens, a sophocarpidine (matrine)-containing medicinal legume. We additionally investigated the cross nodulation, infection pattern, light and electron microscopies of root nodule sections of S. flavescens infected by various rhizobia. Seventeen genospecies of rhizobia belonging to five genera with seven types of symbiotic nodC genes were found to nodulate S. flavescens in natural soils. In the cross-nodulation tests, most representative rhizobia in class a-Proteobacteria, whose host plants belong to different cross-nodulation groups, form effective indeterminate nodules, while representative rhizobia in class b-Proteobacteria form ineffective nodules on S. flavescens. Highly host-specific biovars of Rhizobium leguminosarum (bv. trifolii and bv. viciae) and Rhizobium etli bv. phaseoli could establish symbioses with S. flavescens, providing further evidence that S. flavescens is an extremely promiscuous legume and it does not have strict selectivity on either the symbiotic genes or the species-determining housekeeping genes of rhizobia. Root-hair infection is found as the pattern that rhizobia have gained entry into the curled root hairs. Electron microscopies of ultra-thin sections of S. flavescens root nodules formed by different rhizobia show that the bacteroids are regular or irregular rod shape and nonswollen types. Some bacteroids contain poly-b-hydroxybutyrate (PHB), while others do not, indicating the synthesis of PHB in bacteroids is rhizobia-dependent. The extremely promiscuous symbiosis between S. flavescens and different rhizobia provide us a basis for future studies aimed at understanding the molecular interactions of rhizobia and legumes.
The association between leguminous plants and symbiotic nitrogen-fixing rhizobia is one of the most important symbioses and has attracted extensive studies. In rhizobia, the nodulation (nod) genes encoding for Nod factors are believed to be the determinants of rhizobial host specificity (D´enari´e et al. 1996; Ferguson 2013). Based upon the differences in their nod genes, some rhizobial strains can only nodulate limited plant species, such as Mesorhizobium muleiense CCBAU 83963T for Cicer Corresponding author: Wen Feng Chen; Telephone: +86 10 6273 4009; E-mail:
[email protected] *The e-Xtra logo stands for “electronic extra” and indicates that seven supplementary figures and one supplementary table are published online. © 2015 The American Phytopathological Society
1338 / Molecular Plant-Microbe Interactions
arietinum (Zhang et al. 2012), while other strains, such as Sinorhizobium sp. strain NGR234, have a broad host range and can nodulate with many different legumes, even the nonlegume Parasponia andersonii (Pueppke and Broughton 1999). Similarly, some legumes, like Astragalus sinicus (Wang et al. 2014), Medicago truncatula (Barker et al. 1990), and Cicer arietinum (Zhang et al. 2012) are hosts specific for a limited number of rhizobial species or even strains. In contrast, other legumes, such as common bean (Phaseolus vulgaris) (Michiels et al. 1998), Acacia tortilis (Ba et al. 2002), Siratro (Macroptilium atropurpureum) (Angus et al. 2013; Moulin et al. 2001), Sophora alopecuroides (Zhao et al. 2010), Sarothamnus scoparius (Sajnaga et al. 2001), and the nonlegume Parasponia andersonii (Op den Camp et al. 2012) are known as promiscuous hosts. The legume genus Sophora contains about 50 species, in which the nodulation of about 20 species is documented (Sprent 2001), and fewer of them have been studied about their rhizobia in detail (Sprent 2001; Zhao et al. 2010). Previously, various rhizobia belonging to 11 genospecies in six genera harboring five nodA gene types have been isolated from the root nodules of Sophora alopecuroides (Han et al. 2008; Zhao et al. 2010). Most recently, Tan et al. (2015) studied 48 rhizobial isolates from the New Zealand native Sophora spp. and found that all isolates were classified into genus Mesorhizobium. These results demonstrate that Sophora spp. are a promiscuous host for diverse rhizobia. Sophora flavescens, another species in genus Sophora, is a perennial medicinal plant native to northeastern Asia (Han et al. 2015). It is increasingly cultured in north China, since the nitrogen-containing sophocarpidine (matrine) in its roots acts as a traditional insecticide and an anticarcinogen (Han et al. 2015). Crow et al. (1981) reported that S. flavescens was a distinctive species and that the rhizobia isolated from S. angustifolia could nodulate S. flavescens, while others suggested that these two species were synonymous (Sprent 2001; 2009). Despite the nomenclature argument, the rhizobia associated with S. flavescens and their nodulation procedures have not yet been studied extensively. Three novel species, Rhizobium sophorae, Rhizobium sophoriradicis, and Phyllobacterium sophorae, were just recently recorded by our research group (Jiao et al. 2015a and b). Because of the wider distribution of S. flavescens than S. alopecuroides, it is possible that the genetic diversity of rhizobia is greater in the former than the latter. In this study, we performed the first systematic analysis of the genetic diversity and distribution of rhizobia isolated from root nodules of S. flavescens grown in different ecoregions. Based on these results, we then carried out extensive
cross-nodulation tests under laboratory conditions to explore the symbiotic promiscuity of S. flavescens. In addition, morphologies of the root nodule and nodule section, the infection process, and the presence of the bacteroids in the nodule cells were examined. Our studies revealed that S. flavescens is an extremely promiscuous host for various rhizobia and it can serve as a good candidate for future studies on the symbiotic relationship between rhizobia and legumes. RESULTS Isolation of root-nodule bacteria and soil characterization. In total, 269 pure rhizobial isolates were obtained from Sophora flavescens nodules collected from three ecoregions (Supplementary Table S1). Except for three isolates (CCBAU 03429, CCBAU 03419, and CCBAU 71316), the remaining 266 isolates formed effective nodules on S. flavescens, as evidenced by the bright green leaves and the red color of the nodule section (Supplementary Fig. S3). The results of soil characteristics showed that the three ecoregions differed mainly in soil pH values. Ecoregion I in Shanxi province had a slightly alkaline soil (7.26 to 7.95), ecoregion II in Shaanxi province had an acidic soil (5.44 to 6.68), and ecoregion III in Liaoning province had an alkaline soil (8.36 to 8.59) (Table 1). In addition, ecoregion I had a much higher concentration of available potassium (AK) than the other two ecoregions. Ecoregion III had total nitrogen content, organic matter, available nitrogen, available phosphorus, available potassium, and total salt levels lower than those of the other two ecoregions (Table 1). Genetic diversity and distribution of rhizobia associated with S. flavescens. Based on analysis of the recA gene sequence screening, 17 genotypes were identified and 35 representative isolates were further characterized with a multilocus sequence analysis (MLSA) of three housekeeping genes (atpD, glnII, and recA). In the maximum likelihood phylogenetic tree reconstructed with the MLSA data, these 35 representative isolates were
classified into 17 genospecies (Fig. 1; Table 2), belonging to genera Bradyrhizobium, Sinorhizobium, Mesorhizobium, Rhizobium, and Phyllobacterium. Nine genospecies corresponding to Sinorhizobium fredii, Mesorhizobium septentrionale, Bradyrhizobium elkanii, Rhizobium yanglingense, Mesorhizobium sp. strain I, R. sophorae, R. sophoriradicis, Mesorhizobium huakuii, and Rhizobium sp. strain I were the major groups containing 12 to 46 isolates (Table 2). Sinorhizobium fredii was the most widely spread major genospecies occurring on eight sites (S3, S4, S7 to S11, and S18). Eight genospecies, including Phyllobacterium sophorae, Bradyrhizobium sp. strain I, Mesorhizobium sp. strain II, R. mongolense, R. lusitanum, Rhizobium spp. II, III, and V were minor groups containing 1 to 4 isolates (Table 2), which were found in one to three sampling sites. The highest diversity index of Shannon-Weiner (H9) was found on site S18 (1.68), followed by that on site S5 (1.58) and site S17 (1.22) (Table 2), and seven genospecies were found on each of these three sites. The lowest H9 values (0) were found on sites S2, S9, S10, S14, and S15, with only one genospecies isolated on those sites. The other sampling sites had H9 values ranging from 0.97 to 0.30. The values of Simpson’s index (D) varied between 0.78 and 0 on 18 sampling sites and were well consistent with the H9 values. Evenness index Pielou (J) varied from 0.97, in the case of sites S1 and S7, to 0.27, in the case of site S16. These results demonstrated that the diversity and genospecies composition of the rhizobial community associated with S. flavescens varies on different sampling sites. Soil factors influencing the geographic distribution of rhizobia. According to the length of the arrows (the longer the arrow, the more highly related that this variable is to the genospecies distribution) and the angles between the arrow and the genospecies (the smaller, the more closely related) shown in Figure 2, the soil pH, total salt, and available potassium had an obviously positive correlation with the distribution of genospecies isolated from ecoregion I, but a negative correlation with the distribution of genospecies isolated from ecoregion II and III. Soil pH is one of the most important soil factors related to the distribution of
Table 1. Geographic information, climate, and soil characteristics of the 18 sampling sites Ecoregion,a geographic origin, and sampling sites Ecoregion I: Shanxi province S1: Zhenxin town, Changzhi S2: Zhenxin town, Changzhi S3: Zhenxin town, Changzhi S4: Wugu Mt., Changzhi S5: Niusi town, Qinxian S6: Niusi town, Qinxian S7: Hanbei town, Wuxiang S8: Hanbei town, Wuxiang S9: Hanbei town, Wuxiang S10: Hanbei town, Wuxiang S11: Hanbei town, Wuxiang Ecoregion II: Shaanxi province S12: Maping town, Luonan S13: Maping town, Luonan S14: Maping town, Luonan S15: Youfang town, Luonan Ecoregion III: Liaoning province S16: Heishui, Jianping S17: Changlong, Jianping S18: Yangshuling, Jianping a b c
GPS
Soil characteristicsc
Altitude (m)
Longitude
Latitude
Rainfall (mm)b
1,224 1,224 1,224 1,041 993 993 1,225 1,225 1,225 1,225 1,225
113°03ʹ08ʺE 113°03ʹ10ʺE 113°03ʹ09ʺE 113°03ʹ02ʺE 112°03ʹ30ʺE 112°03ʹ36ʺE 113°11ʹ56ʺE 113°11ʹ55ʺE 113°11ʹ57ʺE 113°11ʹ49ʺE 113°11ʹ50ʺE
36°02ʹ24ʺN 36°02ʹ27ʺN 36°02ʹ30ʺN 36°02ʹ24ʺN 36°54ʹ36ʺN 36°54ʹ35ʺN 36°45ʹ40ʺN 36°45ʹ25ʺN 36°45ʹ32ʺN 36°45ʹ35ʺN 36°45ʹ31ʺN
411 411 411 411 606 606 560 560 560 560 560
1.18 1.06 0.82 1.43 0.89 0.94 1.01 1.02 0.90 1.19 1.06
16.7 15.4 16.3 30.4 11.4 10.3 23.0 28.4 17.7 23.8 20.9
70.0 53.4 42.0 44.3 58.5 44.5 103.0 90.4 50.9 52.2 57.3
5.9 56.7 19.9 25.8 14.8 25.4 33.4 10.9 20.8 7.3 16.7
436 138 122 170 273 390 398 322 470 316 562
0.75 0.70 0.83 1.27 0.85 0.86 0.91 0.90 0.73 0.67 0.80
7.95 7.52 7.76 7.26 7.75 7.78 7.76 7.81 7.91 7.84 7.91
1,066 1,066 1,050 1,109
110°04ʹ10ʺE 110°04ʹ03ʺE 110°03ʹ59ʺE 110°02ʹ44ʺE
34°12ʹ06ʺN 34°12ʹ11ʺN 34°12ʹ06ʺN 34°13ʹ14ʺN
700 700 700 700
1.27 1.33 1.43 1.06
18.4 18.0 20.2 13.0
52.7 123.0 89.3 83.5
23.1 34.8 25.5 18.9
93 80 110 65
0.41 0.43 0.45 0.53
5.80 5.44 5.57 6.68
554 526 690
119°29ʹ48ʺE 119°23ʹ16ʺE 119°45ʹ58ʺE
42°03ʹ33ʺN 41°58ʹ41ʺN 41°50ʹ36ʺN
500 500 500
0.74 0.47 0.73
49.8 36.6 49.2
9.0 1.6 7.3
86 40 81
0.47 0.64 0.55
8.36 8.59 8.46
TN
OM
AN
AP
AK
TS
pH
8.18 5.6 9.29
Ecoregion I: clay loam, semihumid, alkaline soil; ecoregion II: sandy loam, moist, acid soil; and ecoregion III: sandy loam, semiarid land, highly alkaline soil. Mean annual rainfall (mm). TN, total nitrogen content (g/kg); OM, organic matter (g/kg); AN, available nitrogen (mg/kg); AP, available phosphorus (mg/kg); AK, available potassium (K) (mg/kg); TS, total salt (g/kg); and pH, soil pH. Vol. 28, No. 12, 2015 / 1339
Fig. 1. Maximum likelihood phylogenetic tree based on the three concatenated genes (atpD-glnII-recA). The model GTR+G+I was used to construct the tree. Only bootstrap values greater than 50% are shown at branch nodes. Bar, 5% nucleotide substitution per site. Boldfaced strains were isolated from Sophora flavescens. R., M., B., S., and P. = Rhizobium, Mesorhizobium, Bradyrhizobium, Sinorhizobium, and Phyllobacterium. T = type strain. 1340 / Molecular Plant-Microbe Interactions
these rhizobia (P = 0.0020) (Fig. 2; Table 1). R. yanglingense, R. mongolense, and Rhizobium sp. strain IV were solely found in alkaline soil at ecoregion III, while R. lusitanum, Mesorhizobium sp. strains I and II, M. huakuii, Bradyrhizobium elkanii were only found in acidic soil at ecoregion II.
were identical to the reference strains in the same species that originated from soybean. Type IV was defined among the isolates of R. sophorae, R. sophoriradicis and Rhizobium sp. strain I from ecoregion I, whose symbiotic genes identical to those of some common bean (Phaseolus vulgaris)-nodulating rhizobia (bv. phaseoli). The nodC types V and VII were from the isolates of R. yanglingense, unique for ecoregion III, in which type V was similar to reference strains in the same species from Gueldenstaedtia multiflora/Medicago ruthenica while the type VII was a separated lineage. Type VI was specific for the isolates of B. elkanii from ecoregion II and their nodC and nifH genes were very similar to those of the soybean (Glycine max)nodulating strain (B. elkanii USDA 76T) in the same species. In the cross nodulation tests, 54 strains representing different species of the rhizobia in the classes of alpha-Proteobacteria (a-rhizobia) and beta-Proteobacteria (b-rhizobia) were selected to inoculate S. flavescens (Fig. 3 and Table 3). Most test strains, including the highly host-specific Rhizobium leguminosarum bv. viciae and R. etli bv. phaseoli, could effectively form nodules on S. flavescens. Another highly host-selective strain, R. leguminosarum bv. trifolii LMG 8820T, could nodulate S. flavescens too, but the symbiosis were inefficient. The following 11 strains, Bradyrhizobium japonicum USDA 6T, B. diazoefficiens USDA 110T, B. yuanmingense CCBAU 10071T, Bradyrhizobium sp. strain ORS278, B. oligotrophicum LMG 10732T, Ochrobactrum lupini LMG 22726T, M. muleiense CCBAU 83963T, M. amorphae
Diversity of symbiotic genes and cross nodulation. Excepting 3 isolates CCBAU 03429, CCBAU 03419 and CCBAU 71316 that failed to nodulate S. flavescens, the symbiotic genes nodC and nifH were amplified successfully from 266 isolates. As shown by the phylogenetic trees of nodC (Fig. 3) and nifH genes (Supplementary Fig. S2), the grouping results of S. flavescens isolates were similar and 7 symbiotic gene types (I ; VII) were distinguishable. The nodC type I was found in all of the three ecoregions, including all of the isolates in genospecies M. septentrionale. Several isolates defined as R. mongolense, M. huakuii, Mesorhizobium sp. strain I and sp. strain II, had nodC genes identical or very similar to those of the reference strain M. septentrionale CCBAU 03074 that originated from Astragalus membranaceus. Type II included the isolates of Sinorhizobium fredii, M. septentrionale and P. sophorae from ecoregion I, which were identical or very similar to those of reference strains for Mesorhizobium species associated with Astragalus adsurgens, Glycyrrhiza pallidiflora and Caragana intermedia. Type III was composed of isolates belonging to Sinorhizobium fredii from ecoregion I, and they
Table 2. Representative genospecies identified based on multilocus sequence analysis (MLSA) similarities and their distribution in each sampling site and ecoregion Number of isolates in different ecoregions and sitesc Ecoregion I (n = 105) CCBAU no.a 03470 03386 03360 03429 03419 11560 71316 11559 11536 03373 71358 71303 71320 03405 71325 03416 03422 Diversity indexesd
a b c d
Genospeciesb R. sophoriradicis (100.0%) R. sophorae (100.0%) Rhizobium sp. strain I (95.8%) Rhizobium sp. strain II (95.5%) Rhizobium sp. strain III (94.8%) Rhizobium sp. strain IV (94.5%) R. lusitanum (99.5%) R. mongolense (96.0%) R. yanglingense (97.8%) S. fredii (99.2%) Mesorhizobium sp. strain I (95.4%) Mesorhizobium sp. strain II (94.5%) M. huakuii (96.4%) M. septentrionale (97.1%) B. elkanii (96.5%) Bradyrhizobium sp. strain I (94.5%) P. sophorae (100%) Total Shannon-Wiener index (H9) Simpson index (D) Pielou index (J)
S1 2
S2
S3
2
S4
S5
S6
S7
Ecoregion III (n = 83)
Ecoregion II (n = 81) S8
S9
S10
S11
S12
S13
S14
S15
S16
S17
12 3
3
2
2
2
11 2
1
1
5
S18
Total
4
20
3
25
2
12
1
1
1
2 1
3 1
2
2
5
20
3
6
2
8
2 1
2
1
4
27
1
1
29
1
46 29
1 1
28
2 11 1
9
2 8
1
24 6
6
23
29 1
1 2 2 5 2 6 34 19 3 5 7 2 8 14 14 16 23 28 29 37 17 0.67 0.00 0.45 0.85 1.58 0.64 0.67 0.41 0.00 0.00 0.66 0.66 0.97 0.00 0.00 0.30 1.22 1.68 0.48 0.00 0.28 0.97 Null 0.65
0.53 0.77
0.72 0.44 0.48 0.24 0.00 0.00 0.81 0.92 0.97 0.59 Null Null
0.36 0.60
0.36 0.60
0.59 0.89
0.00 Null
19 41
0.00 Null
0.13 0.27
0.55 0.63
0.78 0.86
4 269 / / /
CCBAU no.) Three isolates (03429 [from site S5], 03419 [from site S5], 71316 [from site S13]) could not nodulate Sophora flavescens in the nodulation tests in laboratory conditions and the nodC gene could not be amplified. MLSA similarity to known species. n = 17. R., M., B., S., and P., = Rhizobium, Mesorhizobium, Bradyrhizobium, Sinorhizobium, and Phyllobacterium. Nodules collected in S1 to S11 were from Shanxi province; S12 to 15 were from Shaanxi province; and S16 to S18 were from Liaoning province. The Shannon-Wiener index (H9) is the diversity considering the species richness in a community; the Simpson index (D) shows the species dominance in a community; the Pielou index (J) shows the species evenness in a community. / = no data.
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ATCC 19665T, R. galegae HAMBI 1174T, Burkholderia nodosa LMG 23741T and Burkholderia phymatum STM 815T could not infect S. flavescens (Fig. 3; Table 3). Sinorhizobium meliloti USDA 1002T, CCBAU 01199, and 1021 could occasionally nodulate, but they fix nitrogen effectively on S. flavescens (Fig. 3; Table 3). The b-rhizobia Burkholderia sp. strain ICMP 19430 and ICMP 19869, Burkholderia tuberum LMG 21444T, and Cupriavidus taiwanensis LMG 19424T could nodulate S. flavescens, but the nitrogen fixation was inefficient and the nodules were white, small, and spherical, indicative of deficient function (Supplementary Figs. S4 and S7). The isolates that originated from S. flavescens belonging to nodC types VI (B. elkanii CCBAU 71325 and CCBAU 71331), type IV (R. sophorae CCBAU 03468, CCBAU 03386 and R. sophoriradicis CCBAU 03470), type II (M. septentrionale CCBAU 03399), and V (R. yanglingense CCBAU 11536) could nodulate effectively on Glycine max, Phaseolus vulgaris, Arachis hypogaca, Astragalus membranaceus, and Caragana intermedia, respectively (Table 3). While the isolates in types I (Mesorhizobium sp. strains I CCBAU 71358 and CCBAU 71303 and M. septentrionale CCBAU 03405) and II (P. sophorae CCBAU 03422) could not nodulate either Astragalus sinicus or A. membranaceus (Table 3).
Infection pattern, morphology, and efficiency of nodules. Cellular curl, branch, or swell could be visible on the tips of root hairs of S. flavescens inoculated with the representative rhizobium (Sinorhizobium fredii CCBAU 45436 or R. yanglingense CCBAU 01603) under light-field microscopy (Fig. 4B through E). Aggregation on the bifurcating site, formation of the infection pocket or thread were observed clearly on and in the root hairs infected by lacZ-labeled rhizobia (strains CCBAU 45436 or CCBAU 01603) (Fig. 4F through H). Nodule primordium was found after 15 days of inoculation (Fig. 4I through J). Most nodules were found on lateral roots and only a few on the taproots. Most reference strains and isolates (like M. septentrionale CCBAU 03399) formed effective nitrogenfixation nodules with red color in the nodule section, while reference strain M. huakuii 7653R formed low-efficiency nodules on the plants that showed yellowish leaves. Significant differences (P < 0.05) for chlorophyll content were observed between the plants inoculated with strains CCBAU 03399 and 7653R. Sphere-like and deficient nodules were formed by b-rhizobia of Burkholderia tuberum LMG 21444T, Burkholderia spp. ICMP 19869 and ICMP 19430 (not shown), and Cupriavidus taiwanensis LMG 19424T on S. flavescens, while most a-rhizobia formed effectively indeterminate
Fig. 2. Biplot showing relationships between the 17 genospecies and soil factors in three ecoregions, drawn using CANOCO software. AN = available nitrogen, AP = available phosphorus, AK = available potassium, OM = organic materials, TS = total salt. The arrows and triangles show soil factors and genospecies, respectively. The circles (s), squares (N), and diamonds (♢) show sampling sites in the three ecoregions I, II, and III. Triangles (4) show the distribution of genospecies. The longer the arrow is, the greater the influence the soil factor has on the distribution of the genospecies; the smaller the angle between the arrow and the line (not drawn) linking the triangle and original point (O), the closer relationship the soil factor and the specific rhizobial genospecies have. 1342 / Molecular Plant-Microbe Interactions
(rod-shape or bifurcated) nodules on S. flavescens. Different parts showing the growth of the infection, nitrogen-fixation, and senescence zones were observed clearly in the section of effective nodules induced by a-rhizobium M. septentrionale CCBAU 03399 (Supplementary Fig. S5). The root nodules formed by b-rhizobia were much smaller (about 2 mm in diameter) than those formed by a-rhizobia. No more than half of the plant cells within a nodule were infected by b-rhizobium (e.g., Burkholderia tuberum LMG 21444T), while most of the plant cells within a nodule were full of a-rhizobium (e.g., M. septentrionale CCBAU 03399). Yellowish leaves of the plants inoculated with b-rhizobia were visualized and chlorophyll content of the leaves was only 23 to 25 µg/cm2 (SPAD 502), lower than in the a-rhizobia-inoculated plants (approximately 30 µg/cm2). Bacteroids in the nodules. Observation of mature nodules (35 days old) through electron microscopy indicated that the bacteroids in nodule cells of S. flavescens were regular or irregular rods with lengths
