Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 77(4): 13-23, 2024 Firenze University Press https://riviste.fupress.net/index.php/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-3052 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Gu, S.-R., Li, H.-Y., Huang, X.-X., Yang, H., Peng, X., Song, Z.-Q., Duan, L., Shi, M.-M., Wang, X.-P., Zhao, Z.-T., Li, S.-J., Tu, T.-Y. & Zhang, D.-X. (2024). Cytogenetics of Cheniella (Leguminosae: Cercidoideae) from China and Vietnam. Caryologia 77(4): 13-23. doi: 10.36253/caryologia-3052 Received: October 23, 2024 Accepted: April 16, 2025 Published: July 15, 2025 © 2024 Author(s). This is an open access, peer-reviewed article pub- lished by Firenze University Press (https://www.fupress.com) and distrib- uted, except where otherwise noted, under the terms of the CC BY 4.0 License for content and CC0 1.0 Uni- versal for metadata. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. ORCID S-RG: 0000-0003-4504-0500 M-MS: 0000-0002-3285-4065 X-PW: 0000-0002-0645-6370 Z-TZ: 0000-0002-7733-9542 S-JL: 0000-0002-6596-5124 T-YT: 0000-0001-7385-008X D-XZ: 0000-0001-6549-8872 Cytogenetics of Cheniella (Leguminosae: Cercidoideae) from China and Vietnam Shi-Ran Gu1,2, Hong-Yan Li1,2, Xiang-Xu Huang1,2, Hong Yang1,2,3, Xia Peng1,2,3, Zhu-Qiu Song1,2, Lei Duan1,2, Miao-Miao Shi1,2, Xiang-Ping Wang1,2, Zhong-Tao Zhao1,2, Shi-Jin Li1,2, Tie-Yao Tu1,2,*, Dian-Xiang Zhang1,2, j 1 State Key Laboratory of Plant Diversity and Specialty Crops, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, 510650, China 2 South China National Botanical Garden, Guangzhou, 510650, China 3 University of Chinese Academy of Sciences, Beijing, 100049, China *Corresponding author. E-mail: tutieyao@scbg.ac.cn Abstract. The cytogenetics of Cheniella, a recently segregated genus of the early- diverging subfamily Cercidoideae of Leguminosae, remain understudied, hindering our understanding of the cytological evolution and the utilization of this important plant group. Here we conducted comparative cytogenetic studies on 11 species and one subspecies of Cheniella and one species of its sister genus Phanera. Unlike ear- lier reports which recovered 2n=28 for two Cheniella species, we consistently observed chromosome counts of 2n=26 for all 11 species of Cheniella, supporting the segrega- tion of Cheniella from Phanera, which consistently exhibited 2n=28 in this and previ- ous studies. Our analyses, along with previous cytogenetic data, indicates that 2n=14, 2n=26 and 2n=28 are the predominant chromosome numbers in the basal-most genus Cercis, Cheniella and the remainder genera, respectively. The ancestor of the subfamily is most probably a diploid with 2n=14, with subsequent polyploidization followed by chromosome reduction events leading to 2n=28 and 2n=26 in the other lineages. Our results provide new insight into the cytotaxonomy and chromosome evolution of Cer- cidoideae, also lay the foundation for future genomics research. Keywords: Bauhinia s.l., chromosome counts, cytology, Fabaceae, Phanera, Southeast Asia. INTRODUCTION The plant family Leguminosae Juss. (or Fabaceae Lindl.) is currently recognised by the Legume Phylogeny Working Group to consist of six sub- families (LPWG, 2017), of which the Cercidoideae LPWG contains about 14 genera and 340 species distributed pantropically and in some subtropi- cal regions. Various species of Cercidoideae are used for food, timber, dyes, ropes and medicine, and widely cultivated as ornamental trees in many areas of the world (Clark et al., 2017; Gu et al., 2024). The flowers of many Cerci- https://riviste.fupress.net/index.php/caryologia https://doi.org/10.36253/caryologia-3052 https://doi.org/10.36253/caryologia-3052 https://www.fupress.com https://creativecommons.org/licenses/by/4.0/legalcode https://creativecommons.org/publicdomain/zero/1.0/legalcode https://orcid.org/0000-0003-4504-0500 https://orcid.org/0000-0002-3285-4065 https://orcid.org/0000-0002-0645-6370 https://orcid.org/0000-0002-7733-9542 https://orcid.org/0000-0002-6596-5124 https://orcid.org/0000-0001-7385-008X https://orcid.org/0000-0001-6549-8872 mailto:tutieyao@scbg.ac.cn 14 Shi-Ran Gu et al. doideae species are highly attractive and fragrant, with great value or potential as garden ornamental plants. The subfamily Cercidoideae currently contains 14 genera including Adenolobus (Harv. ex Benth. & Hook.f.) Torre. & Hillc., Barklya F.Muell., Bauhinia L., Brenierea Hum- bert, Cercis L., Cheniella R.Clark & Mackinder, Gigasi- phon Drake, Griffonia Baill, Lysiphyllum (Benth.) de Wit, Phanera Lour., Piliostigma Hochst., Schnella Raddi; Tournaya A.Schmitz, and Tylosema (Schweinf.) Torre & Hillc. (Wunderlin, 1976; Lewis & Forest, 2005; LPWG, 2017; Clark et al., 2017; Sinou et al., 2020). The initially diverged lineage of Cercidoideae, Cer- cis, exhibits a somatic chromosome number of 2n=14, whereas most other lineages in this subfamily were con- sistently reported to have a somatic chromosomal count of 2n=28, with a few exceptions of 2n=24, 2n=26, or even 2n=42, 2n=56 found in several species of Barklya, Bauhinia, Gigasiphon, Lysiphyllum, and Piliostigma (Table 1) (Sharma & Raju, 1968; Goldblatt, 1981; Yeh et al., 1986; Kumari & Bir, 1989). Intraspecific chromosom- al variations are also observed. For example, Bauhinia monandra Kurz exhibits counts of 2n=24, 2n=28, and 2n=42 (Sharma & Raju, 1968; Gill & Husaini, 1982; Darlington & Wylie, 1955), Bauhinia acuminata L. has 2n=26 and 2n=28, and Lysiphyllum hookeri (F.Muell.) Pedley shows both 2n=26 and 2n=28 (Sharma & Raju, 1968; Singhal et al., 1980b; Goldblatt, 1981; Sarkar et al., 1982; Basumatari & Das, 2017). Cheniella R.Clark & Mackinder, a recently segre- gated genus from Bauhinia s.l., contains 16 species and three subspecies, and is closely related to Phanera (Clark et al., 2017; Gu et al., 2024; Peng et al., 2024). The cen- tre of diversity of Cheniella is in southern China, and its full distribution range extends westward to India and southeast through Indochina into Malesia (Clark et al., 2017). The genus is characterised as being tendrilled lia- nas with a deeply to slightly bilobed or emarginate leaf blade, elongate hypanthia, a fleshy disc on which the sta- minodes are mounted, glabrous or densly hirsute, oblong and compressed, indehiscent or tardily dehiscent pods with numerous seeds (Fig. 1). The chromosome numbers of two species in Cheniella have been previously report- ed, C. corymbosa (Roxb.) R.Clark & Mackinder and C. quinnanensis (Benth.) R.Clark & Mackinder, both with 2n=28 chromosomes (Sharma & Raju, 1968; Singhal et al., 1980a). It must be noted that the initial identifica- tions of C. corymbosa and C. quinnanensis by Sharma & Raju (1968) and Singhal et al. (1980a) were Bauhinia corymbosa and (probably) Bauhinia glauca respectively, of which the former name was synonymised to C. corym- bosa and the latter was probably erroneously identified, the correct name being C. quinnanensis. Beside the misi- dentification, the accuracy and reliability of the chromo- some numbers in previous studies needed to be tested especially for those groups that were poorly studied or for those that have various chromosome counts reported. To test the cytogenetics of Cheniella, we counted the chromosome numbers of 11 species and one subspe- cies of Cheniella, as well as one species of Phanera. By combining evidence from cytology and morphology, this study aims to provide the chromosomal data and cyto- taxonomy of Cheniella and to compare these with other members of subfamily Cercidoideae. MATERIALS AND METHODS All seeds or transplanted living plants studied were collected in the field of southern and southwestern China and adjacent regions except for one sample was collected from Vietnam. Detailed collection informa- tion is shown in Table 1. The vouchers of all collections and permanent slides are deposited in the herbarium of South China Botanical Garden, Chinese Academy of Sci- ences (IBSC). All cytological observations were made from root tip cells obtained either from seeds or from transplant- ed living individuals. All root tips were obtained from germinating seeds, mature and dry seeds were cut the seed coat and placed in petri dishes lined with moist filter paper and cultured at room temperature until 1–2 cm root sprouted. Root tips were pretreated in a satu- rated 1,4-dichlorobenzene solution for 150 min, then fixed with Carnoy’s fluid (absolute alcohol: glacial ace- tic acid, 3:1, v/v) at 4 °C for at least 30 min. The fixed roots were hydrolysed in 1 N HCl solution at 60 °C for 4 min, stained with modified phenol magenta stain for 2 h and squashed for cytological observation. The best metaphase plates were photographed using a Nikon DS- Fi2 digital camera attached to the BX41 Olympus micro- scope. Permanent slides were made using the standard liquid nitrogen method. RESULTS AND DISCUSSION The interphase nuclei of 11 species from Cheniella and one species from Phanera studied in this paper show the similar shape and distribution pattern of chromatin, which are dispersed evenly throughout the nuclei (Fig. 1, A). According to Tanaka (1971, 1977), they can be categorised as the complex chromocentre type, which is characterised by darkly stained chromocentres of irreg- ular shape and lightly stained chromatin threads. The 15Cytogenetics of Cheniella (Leguminosae: Cercidoideae) from China and Vietnam Ta bl e 1. S ta tis tic s o n ch ro m os om e nu m be rs in C er ci do id ea e. G en us Sp ec ie s C hr om os om e nu m be r ( 2n ) Lo ca lit y Vo uc he r Re fe re nc es N ot es Ad en ol ob us A . p ec hu eli i ( K un tz e) K or cz . & H ill c. 28 W al vi s B ay , N am ib ia Se el y s.n . G ol db la tt (1 98 1) Ba rk ly a B. sy ri ng ifo lia F .M ue ll. 26 C ul t. in A us tr al ia Pe dl ey A 17 72 ( M O ) G ol db la tt (1 98 1) Ba uh in ia B. a cu m in at a L. 28 - - Sh ar m a & R aj u (1 96 8) 26 U tta r P ra de sh , I nd ia Si ng ha l 2 30 01 Si ng ha l e t a l. (1 98 0b ) * 28 H ow ra h, In di a C BL H 4 51 2 Sa rk ar e t a l. (1 98 2) 28 - - K um ar i & B ir (1 98 9) 28 - - Si nh a & S in gh (2 01 3b ) 28 G uw ah at i, As sa m - Ba su m at ar i & D as (2 01 7) B. c or ni cu la ta B en th . 28 - - Sh ar m a & R aj u (1 96 8) B. d iv ar ica ta L . 28 - - Sa nd hu & M an n (1 98 8) B. fo rfi ca ta L in k 28 - - G ill & H us ai ni (1 98 6) 28 Pa ra ná , B ra zi l E. B io nd o 30 3 (I C N ) Bi on do e t a l. (2 00 5) B. lu na ri oi de s A .G ra y ex S .W at so n 28 - Th ar p 44 15 9 (T EX ) Tu rn er (1 95 6) B. g al pi ni i N .E .B r. 28 - - Sh ar m a & R aj u (1 96 8) 28 - - Pa iv a & L ei ta o (1 98 9) 28 U tta r P ra de sh , I nd ia Si ng ha l 2 35 02 Si ng ha l e t a l. (1 99 0) B. m on an dr a K ur z 28 - - Sh ar m a & R aj u (1 96 8) 24 - - G ill & H us ai ni (1 98 2) * 42 - - D ar lin gt on & W yl ie (1 95 5) * B. p et er sia na B ol le 28 - - Sh ar m a & R aj u (1 96 8) B. p ur pu re a L. 28 - - Sh ar m a & R aj u (1 96 8) 28 - - Ye h et a l. (1 98 6) 28 - - K um ar i & B ir (1 98 9) 28 - - Si nh a & S in gh (2 01 3a ) 28 - - Si nh a & S in gh (2 01 3b ) B. ra ce m os a La m . 28 - - Sh ar m a & R aj u (1 96 8) 28 - - K um ar i & B ir (1 98 9) 28 - - Si nh a & S in gh (2 01 3b ) B. r uf es ce ns L am . 28 - - Sh ar m a & R aj u (1 96 8) 56 - - Sh ar m a & R aj u (1 96 8) * B. to m en to sa L . 28 - - Sh ar m a & R aj u (1 96 8) 28 - - G ill & H us ai ni (1 98 2) 28 - - K um ar i & B ir (1 98 9) B. u ng ul at a L. 28 Be lé m , B ra zi l So uz a 14 So uz a & B en ko -I se pp on (2 00 4) (C on tin ue d) http://N.E.Br 16 Shi-Ran Gu et al. G en us Sp ec ie s C hr om os om e nu m be r ( 2n ) Lo ca lit y Vo uc he r Re fe re nc es N ot es B. v ar ie ga ta L . 28 - - At ch iso n (1 95 1) 28 - - Sh ar m a & R aj u (1 96 8) 28 - - Bi r & K um ar i ( 19 79 ) 28 - - Si nh a & S in gh (2 01 3a ) 28 - - Si nh a & S in gh (2 01 3b ) 28 Si rm au r, In di a 56 68 8 (P U N ) R an i e t a l. (2 01 3) 28 K an gr a, In di a 56 27 6 (P U N ) R an i e t a l. (2 01 3) 28 N ag ao n, A ss am - Ba su m at ar i a nd D as (2 01 7) 28 G ua ng zh ou , C hi na - Zh on g et a l. (2 02 2) B. × bl ak ea na D un n 28 - - Sh ar m a & R aj u (1 96 8) 28 - - Si nh a & S in gh (2 01 3a ) Ce rc is C . c an ad en sis L . 14 C ul t. in U SA C ur tis 1 01 ( M O ) C ur tis (1 97 6) 14 - - H ill (1 98 9) 14 - - Bl ac kw el l ( 19 90 ) C . c hi ne ns is Bu ng e 14 - - Ye h et a l. (1 98 6) 14 - - C he n et a l. (2 00 3) 14 G ua ng xi , C hi na - Li e t a l. (2 02 3) C . c hi ng ii C hu n 14 Jia ng su , C hi na - C he n et a l. (1 99 1) C . s ili qu as tr um L . 14 - - Fe rn an de s e t a l. (1 97 5) 14 Pr eb al ka n, B ul ga ria BK 7 31 92 K uz m an ov (1 97 5) Ch en ie lla C . c le m en sio ru m ( M er r.) R .C la rk & M ac ki nd er 26 D a H an g Pr o, V ie tn am LB o7 79 ( IB SC ) Th is stu dy C . c or ym bo sa ( Ro xb .) R .C la rk & M ac ki nd er 26 H ai na n, C hi na ZQ B5 9 (I BS C ) Th is stu dy 28 - - Sh ar m a & R aj u (1 96 8) * C . d id ym a (H .Y. C he n) R .C la rk & M ac ki nd er 26 G ua ng do ng , C hi na Tu T Y4 69 1 (I BS C ) Th is stu dy C . h up eh an a co m b. n ov . i ne d. 26 H ub ei , C hi na ZQ B6 8 (I BS C ) Th is stu dy C . l on gi pe s ( H os ok .) S. R .G u, T .Y. Tu & D .X .Z ha ng 26 H ai na n, C hi na ZQ B5 6 (I BS C ) Th is stu dy C . l on gi sta m in ea S .R .G u, T .Y. Tu & D .X .Z ha ng 26 G ua ng xi , C hi na Ze ng Q B1 98 ( IB SC ) Th is stu dy C . o va tif ol ia ( T. C .C he n) R .C la rk & M ac ki nd er 26 G ua ng xi , C hi na Tu T Y4 79 9 (I BS C ) Th is stu dy C . p ar ag la uc a sp . n ov . n om . i ne d. 26 G ua ng do ng , C hi na G uS R1 25 ( IB SC ) Th is stu dy C . q ui nn an en sis ( T. C .C he n) R .C la rk & M ac ki nd er su bs p. qu in na ne ns is 26 G ua ng xi , C hi na Tu T Y4 81 6 (I BS C ) Th is stu dy 28 U tta r P ra de sh , I nd ia Si ng ha l 2 28 12 Si ng ha l e t a l. (1 98 0a ) * C . q ui nn an en sis su bs p. v ill os a R .C la rk & M ac ki nd er 26 G ua ng do ng , C hi na Tu T Y4 84 8 (I BS C ) Th is stu dy C . t en ui flo ra ( W at t e x C .B .C la rk e) R .C la rk & M ac ki nd er 26 Yu nn an , C hi na G uS R0 21 ( IB SC ) Th is stu dy C . t ia nl in en sis × o va tif ol ia 26 G ua ng xi , C hi na ZQ B3 2 (I BS C ) Th is stu dy C . t ou ra ne ns is (G ag ne p. ) R .C la rk & M ac ki nd er 26 G ua ng xi , C hi na Tu T Y4 79 5 (I BS C ) Th is stu dy (C on tin ue d) Ta bl e 1. (C on tin ue d) . http://S.R.Gu http://T.Y.Tu http://S.R.Gu http://T.Y.Tu 17Cytogenetics of Cheniella (Leguminosae: Cercidoideae) from China and Vietnam G en us Sp ec ie s C hr om os om e nu m be r ( 2n ) Lo ca lit y Vo uc he r Re fe re nc es N ot es G ig as ip ho n G . m ac ro sip ho n (H ar m s) B re na n 26 M ua h ill s, K en ya G ac ha th i s .n . G ol db la tt (1 98 1) Ly sip hy llu m L. d ip hy llu m ( Ba nk s) d e W it 28 - - Sh ar m a & R aj u (1 96 8) 28 - - Bi r & K um ar i ( 19 79 ) 28 - - K um ar i & B ir (1 98 9) L. h oo ke ri ( F. M ue ll. ) P ed le y 28 - - Sh ar m a & R aj u (1 96 8) 26 - - Sh ar m a & R aj u (1 96 8) 26 C ul t. in A us tr al ia Pe dl ey A 77 71 ( M O ) G ol db la tt (1 98 1) Ph an er a P. ch am pi on ii Be nt h. 28 Ta iw an , C hi na 47 28 Pe ng e t a l. (1 98 6) 28 G ua ng xi , C hi na - Lu e t a l. (2 02 4) P. in te gr ifo lia ( Ro xb .) Be nt h. 28 - - Sh ar m a & R aj u (1 96 8) P. ro xb ur gh ia na ( Vo ig t) B an dy op ., An an d K um ar & C ha kr ab . 28 - - Sh ar m a & R aj u (1 96 8) 28 U tta ra kh an d, In di a Si ng ha l 2 32 81 Si ng ha l e t a l. (1 99 0) P. se m ib ifi da ( Ro xb .) Be nt h. 28 - - Sh ar m a & R aj u (1 96 8) P. v ah lii ( W ig ht & A rn .) Be nt h. 28 - - Sa nd hu & M an n (1 98 8) 28 Si rm au r, In di a 56 00 4 (P U N ) R an i e t a l. (2 01 3) P. y un na ne ns is (F ra nc h. ) W un de rli n 28 Yu nn an , C hi na G uS R0 97 ( IB SC ) Th is stu dy Pi lio sti gm a P. m al ab ar icu m ( Ro xb .) Be nt h. 28 - - Sh ar m a & R aj u (1 96 8) 28 - - K um ar i & B ir (1 98 9) P. th on ni ng ii (S ch um ac h. ) M iln e- Re dh . 26 - - Ye h et a l. (1 98 6) * in di ca te s t he c hr om os om e nu m be rs sh ou ld b e in te rp re te d w ith c au tio n be ca us e th ey a re d iff er en t f ro m o th er st ud ie s o r f ro m th e us ua l p er ce pt io n. Ta bl e 1. (C on tin ue d) . 18 Shi-Ran Gu et al. similar pattern is consistent with the other reported Cer- cidoideae species. Heterochromatin and euchromatin segments are clearly seen at mitotic prophase in all samples. The heterochromatin segments are located in the proximal regions that are deeply stained, indicating early con- densation, while the euchromatin segments in the distal regions of chromosomes are lightly stained and extend- ed, indicating late condensation (Fig. 1, B–C). According to Tanaka (1971, 1977), the prophase chromosomes of all species in this study are of the proximal type. The prochromosomes in the pro-metaphase are curly and gradually arranged on the equator of the spin- dle with indistinct edges (Fig. 1, D). Paired sister chro- matids are clearly visible during late metaphase stage (Fig. 1, E). Successful separation of daughter chromo- somes is visible in late anaphase stage, moving from the equatorial plate to the poles of the spindle, but new nuclear membranes have not yet formed (Fig. 1, F). There was little difference in size between the chro- mosomes in each species of Cheniella and Phanera (Fig. 1, G–T). Chromosomes in all species of Cheniella were rod-shaped or oblong in mitotic metaphase nuclei, where- as they were round or punctate in Phanera yunnanensis (Franch.) Wunderlin. The cell size and mitotic metaphase nuclei chromosome size of P. yunnanensis were smaller in comparison with Cheniella. Chromosomes in species of both Cheniella and Phanera are so small at mitotic met- aphase nuclei that karyotypes cannot be clearly distin- guished, but the number can be clearly counted. All stud- ied Cheniella species have the same chromosome number 2n=26 (Fig. 2, G–R), while the chromosome number of P. yunnanensis is 2n=28 (Fig. 2, S–T). These results demon- strate the differences between Cheniella and P. yunnan- ensis in cytological characters. The chromosome count of Cheniella species is here determined to be 2n=26, sug- gesting that the previous reported chromosome number of 2n=28 (Singhal et al., 1980a; Sharma & Raju, 1968) might be erroneous. Consistency in chromosome num- bers between different species within the genus indicates that speciation within Cheniella is not driven by poly- ploidy or chromosomal number variation. The seeds of the artificial hybrid Cheniella tianlinen- sis × ovatifolia were harvested from the field, hand-pol- linated and bagged, the mature legumes were collected for cytological analysis, revealing a chromosome number of 2n=26 (Fig. 2, R). The maternal parent of the hybrid was Cheniella tianlinensis (T.C.Chen & D.X.Zhang) S.R.Gu, T.Y.Tu & D.X.Zhang and the paternal parent was Cheniella ovatifolia (T.C.Chen) R.Clark & Mackinder. Although chromosome counts for C. tianlinensis were not obtained, the chromosome number of C. ovatifolia was 2n=26. Given the successful production of hybrid seeds with 2n=26, it is reasonable to infer that C. tian- linensis also has a chromosome number of 2n=26. These findings support the inclusion of C. tianlinensis within Cheniella, and are consistent with Gu et al. (2024). Taxonomy of Cheniella and Phanera Based on derived floral characters, palynology and previous molecular evidence, Clark et al. (2017) estab- lished the genus Cheniella to include 10 species and three subspecies. This was supported by the prior study of Hao et al. (2003) which presented a phylogenetic anal- ysis of the nuclear ITS region, recovering a clade of five species later reassigned to Cheniella. However, in a phy- logenetic study by Sinou et al. (2020) which sequenced Legcyc1, Legcyc2, matK and trnL-F for 17 liana species from Asia, a polytomy resulted, including Cheniella and Phanera. Cheniella appeared non-monophyletic, with sampled species dispersed in two clades, raising ques- tions about the validity of the genus. In contrast, Gu et al. (2024) analysed the concat- enated sequences of 77 CDS, 103 IGS, 19 introns, and 4 rRNA genes, recovering two distinct clades for Chen- iella and Phanera, and presenting a sister relationship between them. Unlike Sinou et al. (2020), P. yunnanensis grouped with other Phanera species rather than Cheniel- la corymbosa. Moreover, P. yunnanensis differs morpho- logically from Cheniella in characters that are informa- tive at the generic level, having a raceme or simple cyme of two flowers, staminodes not joined at the base on a fleshy disc (Fig. 1, O–P), and a coriaceous legume that dehisces along both sutures. In the treatment of Clark et al. (2017), P. tianlinen- sis was not included in Cheniella due to its pubescent legumes and rarity in herbaria. Gu et al. (2024) found that the fruit traits and flower structures of P. tianlin- ensis align with Cheniella. Additionally, P. tianlinensis also cluster with the Cheniella clade phylogenetically. Intergrating evidence of the morphological and molecu- lar studies, Gu et al. (2024) concluded that Cheniella is a natural group that includes P. tianlinensis. In the present study, all Cheniella species exhibited rod-shaped or oblong chromosomes in mitotic metaphase nuclei, unlike Phanera yunnanensis, which displayed round or punctate chromosomes. Additionally, cell size and mitotic metaphase chromosome size in P. yunnanen- sis were smaller in comparison with Cheniella. All exam- ined Cheniella species possessed a chromosome number of 2n=26, whereas P. yunnanensis had a chromosome number of 2n=28, which consistent with numbers reported from other studies of Phanera (Sharma & Raju, 1968; Peng et http://S.R.Gu http://T.Y.Tu 19Cytogenetics of Cheniella (Leguminosae: Cercidoideae) from China and Vietnam al., 1986; Singhal et al., 1990; Lu et al., 2024). Cheniella tianlinensis has a chromosome number of 2n=26, as it can hybridize with C. ovatifolia (2n=26), producing offspring with chromosome number of 2n=26. These findings high- light the differences between Cheniella and Phanera, and confirm that C. tianlinensis belongs to Cheniella. Chromosome number evolution within Cercidoideae The subfamily Cercidoideae of Leguminosae con- tains 14 genera and a diverse array of species, many of which exhibit significant intraspecific or interspecific variability in chromosome numbers, 2n=14, 24, 26, 28 Figure 1. Morphological diversity in Cheniella and comparison with Phanera. A: Cheniella didyma; B: C. corymbosa; C: C. quinnanensis subsp. villosa; D: C. quinnanensis subsp. quinnanensis; E–F: C. longistaminea; G–H: C. longipes; I: C. ovatifolia; J: C. tenuiflora; K: C. par- aglauca sp. nov. nom. ined.; L: C. hupehana comb. nov. ined.; M: C. touranensis; N: C. clemensiorum; O–P: Phanera yunnanensis. Photos: A, G–H & O–P, Qiu-Biao Zeng; B–F & I, Tie-Yao Tu; J & K, Shi-Ran Gu; L, Yi-Chen Zhang; M, Kai-Wen Jiang; N, Bo Li. 20 Shi-Ran Gu et al. Figure 2. Comparative cytological features between Cheniella and Phanera. Scale bars=2 μm. A: Mitotic interphase of Cheniella didyma. B: Early prophase of C. ovatifolia. C: Late prophase of C. corymbosa. D: Pro-metaphase of C. quinnanensis subsp. villosa. E: Late metaphase of C. longistaminea. F: Mitotic anaphase of Phanera yunnanensis. G–T: Mitotic metaphases, G: C. longipes, 2n=26; H: C. touranensis, 2n=26; I: C. hupehana comb. nov. ined., 2n=26; J: C. clemensiorum, 2n=26; K: C. longistaminea, 2n=26; L: C. corymbosa, 2n=26; M: C. paraglauca nom. ined., 2n=26; N: C. quinnanensis subsp. villosa, 2n=26; O: C. ovatifolia, 2n=26; P: C. quinnanensis, 2n=26; Q: C. tenuiflora, 2n=26; R: Cheniella tianlinensis × ovatifolia, 2n=26; S–T: P. yunnanensis, 2n=28. 21Cytogenetics of Cheniella (Leguminosae: Cercidoideae) from China and Vietnam (42, 56) (Doyle, 2012; Steven et al., 2015; Roberts & Wer- ner, 2016; LPWG, 2017). The earliest diverging lineage within Cercidoideae, Cercis, has a somatic chromosome number of 2n=14, whilst most other lineages in this sub- family share the chromosome number 2n=28, including Adenolobus, Griffonia, Phanera, Piliostigma, and most species of Bauhinia (Table1). Our study has confirmed that Cheniella possesses a somatic chromosome num- ber of 2n=26, which is the same as several species of Barklya, Bauhinia, Gigasiphon, Lysiphyllum, and Pili- ostigma (Table1). Exceptions to the predominant chro- mosome numbers have been observed occasionally in B. monandra (2n=24 and 2n=42) and B. rufescens (2n=56) (Darlington & Wylie, 1955; Sharma & Raju, 1968; Gill & Husaini, 1982). Given the basal-most phylogenetic position of Cer- cis within Cercidoideae (Hao et al., 2003; LPWG, 2017; Gu et al., 2019; Sinou et al., 2020; Gu et al., 2024), it is reasonable to infer that the ancestral state of chromo- some number for this subfamily was likely a diploid with 2n=14. Cercis retains the characteristics of the dip- loid ancestors, whereas the ancestor of the sister clade of Cercis, which comprises all the remaining genera expe- rienced a whole genome duplication event, resulting in the chromosome number of 2n=28, with probably a few undergoing further duplications to achieve higher chro- mosome numbers. This was followed by at least three independent aneuploidy chromosomal variation events, reducing the chromosome numbers to 2n=26. Reported chromosome counts of 2n=24, 2n=42 and 2n=56 in cer- tain genera or species should be interpreted with cau- tion. Understanding chromosomal evolution within this group is crucial for elucidating the broader evolutionary patterns that shape its biodiversity. ACKNOWLEDGEMENTS This work was financially supported by the Nation- al Natural Science Foundation of China (31270222) and the Guangdong Provincial Special Fund for Natural Resource Affairs on Ecology and Forestry Construc- tion (GDZZDC20228704). 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