Evolution and Classification of Musaceae Based on Male Floral Morphology

Classification of the banana family (Musaceae) into three genera, Musa, Ensete and Musella, and infrageneric ranking are still ambiguous. Within the genus Musa, five formerly separated sections were recently merged into sections Musa and Callimusa based on seed morphology, molecular data and chromosome numbers. Nevertheless, other key morphological characters of the genera, sections, and species have not been clearly defined. This research aims to investigate male floral morphology, classify members of the banana family based on overall similarity of morphological traits using 59 banana accessions of 21 taxa and make inferences of the evolutionary relationships of 57 taxa based on ITS, trnL-F, rps16 and atpB-rbcL sequences from 67 Genbank and 10 newly collected banana accessions. Fifteen quantitative characters were examined using principal component analysis and canonical discriminant analysis and 22 qualitative characters were analyzed by the Unweighted Pair Group Method with an Arithmetic Mean (UPGMA). The results showed that fused tepal morphology, median inner tepal shape and length of style supported the three clades of Musa, Ensete and Musella, while shapes of median inner tepal and stigma classified the two Musa sections. In conclusion, a combination of morphological characters of male flowers and molecular phylogenetics well support the taxonomic arrangement within the banana family and the Musa genus and assist in selection of characters to construct an identification key of Musaceae.


Introduction
Musa L., Ensete Bruce ex Horan. and Musella (Franch.) C.Y. Wu ex H.W.Li belong to the banana family [1]. In 1947, Cheesman separated Ensete from Musa based on a monocarpic habit and divided the genus Musa into four sections mainly on inflorescence orientation, seed shapes and chromosome numbers, including sect. Musa (inflorescence pendant, seed sub-globose, compressed or irregular angulate and x = 11), sect. Rhodochlamys (inflorescence erect, seed sub-globose, compressed or irregular angulate, x = 11), sect. Australimusa (inflorescence pendant, seed sub-globose, x = 10) and sect. Callimusa (inflorescence erect, seed cylindrical, barrel-shaped, or top-shaped, x = 10) [2]. Later, Argent placed M. ingens into a new sect. Ingentimusa (inflorescence erect, seed sub-globose to irregular angulate, x = 7) [3]. Based on molecular phylogenetic analyses and chromosome numbers, Häkkinen later proposed a new sectional classification of the genus Musa by merging the five sections into two: Musa sect. Musa (n = x = 11) and Musa sect. Callimusa (n = x = 10, 9, 7) [4], nevertheless, did not refer to the morphologically discriminating characters between the two sections. In 2015, Swangpol and her team reported a new species of Musa, M. nanensis with remarkable staminal features and a unique arrangement of tepals [5]. The species The scatter plot of the first two principal components from 15 floral morphological measurements indicated that all accessions clustered into two separated groups of Musaceae, i.e., Ensete taxa separated from those of Musa and Musella ( Figure 1). However, PCA based on quantitative analysis of male flowers cannot distinguish Musella from Musa. The first and second principal components strongly distinguished Ensete from Musa and Musella, and the first principal component weakly separated Musella from Musa (Table 2).  Canonical Discriminant Analysis (CDA) provided 100% of the variation between the three genera in the first two dimensions (Table 3, Figure 2). The results of the CDA indicated that the characters most significantly separated flowers of Ensete from Musa and Musella were, i.e., length from style base to stigma head, width of ovary, length of median inner tepal, length of filament and length of ovary (Table 4-Function 1). The characters that separated Musella from Ensete and Musa includes length of compound tepal, length of anther, width of lateral outer compound tepal lobe, width of compound tepal, length of inner compound tepal lobe, width of central outer compound tepal lobe, width of inner compound tepal lobe, length of central outer compound tepal lobe and width of median inner tepal (Table 4-Function 2).

Morphological Analysis
The cluster analysis was computed based on UPGMA of 22 characters (Figures 3-5) from 59 samples of the Musaceae male flowers and presented in Figure 6. Ensete is grouped with Musella and separated from Musa. The four species of Ensete are clearly separated from Musella. There are two groups in the Musa cluster, the first includes Musa (incl. Rhodochlamys) and the second is Callimusa (incl. Australimusa & Ingentimusa). 16 Ratio of median inner tepal length/fused tepal length (0) 1/3-1/4 (1) more than 20 mm

Character Evolution and Analysis
Using fresh flowers, dried specimens or pictures from original publication, five qualitative characters of male flowers including perianth symmetry (bilateral, radial and asymmetric), lateral inner tepal shape (acicular and ovate), median inner tepal shape (tricuspidate, obovate, oval-lanceolate, oblanceolate, oblong, elliptic, ovate and fused with perianth tube), stigma shape (clavate, spatulate and capitate) and style length (shorter than 18 mm and longer than 18 mm) were investigated (Table S1). The data matrix was coded into binary or multistate variables with unordered states. The species of which specimens cannot be observed were treated as a missing data. Evolution of the floral characters   Figure 8; character no. 9-15 in Figure 9; character no. 17-21 in Figure 10).

Discussion
Previous study on overall morphology and characters of perianths by Simmonds [30] have paved way towards the clarification of the classification of Musaceae, however, could not solve generic status clearly. Recently, works on stem, inflorescence, seed morphology and molecular biology of the family independently created debates on taxonomic arrange-  Figure 3; character no. 9-15 in Figure 4; character no. 17-21 in Figure 5; Table 5). The dissimilarities of the male floral morphological characters between the banana species were used in constructing a key to species of the two sections within Musa.

Morphological Characters in the Classification of Musaceae
The phylogeny shows that bilateral perianth with five-fused compound tepals and one free median inner tepal is commonly founded within Musaceae, while radial perianths with six-fused tepals is the autapomorphic character found in M. nanensis ( Figure 3B(b), Figure 4B(e) and Figure 7). In Zingiberales, bilateral perianths are mostly found excepted the asymmetric flowers of Heliconiaceae ( Figure 7). The zygomorphic flower is ancestral state in this order.   The phylogenetic relationship included the families within Zingiberales (Musaceae, Strelitziaceae, Lowiaceae, Heliconiaceae, Costaceae, Cannaceae, Marantaceae and Zingiberaceae). The tree separated banana clades including Musella (blue), Ensete (red), species of sect. Musa (green) and species of sect. Callimusa (purple). Circles on nodes show character changes between the taxa, red pie represent node absent. Morphological characters from other publications were labeled with reference number [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30]. Based on the shape of the outer tepal-lobe apex and inner tepals, there are two types of Musaceae flowers ( Figure 3C). Shared character is the flowers with the acute outer tepal-lobe apex and ovate inner tepals, which were found in Musa and Musella. On the other hand, shifting to the flower with round outer tepal-lobe apex and long acicular inner tepals were only founded in Ensete ( Figure 8A).   All Musaceae members, except for M. nanensis, have free median inner tepal ( Figure 8B). Although the shape of this tepal is various, especially in Musa, it is always tricuspidate in Ensete. The obovate median inner tepal found in Musella is also found in Musa textilis. In the section Callimusa, oblong median tepal is ancestral state, shifting to lanceolate twice and from lanceolate to obovate once. In the section Musa (including Rhodochlamys) the elliptic median tepal shape is the ancestral state, shifted to ovate three times, and then changed to the median tepal that fused with other tepals (Figures 4A and 8B). The ancestor of median inner tepal shape cannot be inferred.
Three stigma shapes were found in the male flowers of Musaceae ( Figure 5B). Clavate stigma, found in the section Musa and in Ensete species, is a plesiomorphic character of the family. A spatulate stigma positioned on a slender style is a synapomorphy of Callimusa (incl. Australimusa). A capitate stigma is an autapomorphic character in Musella ( Figure 9A).
Our results indicate that a clavate stigma is the ancestral state within Musaceae.
The style length is various among the different banana groups ( Figure 5D). The style and stigma of Ensete are reduced in male flowers with a style length shorter than 18 mm, being shorter than the length of the filament and a stigma length that is less than 0.5 mm. The style of Musella and Musa are as long as the length of the compound tepal except for some cultivated M. ornata where it is very short ( Figure 9B and Figure S2.13). A long style with a stigma that is positioned at the same height as the compound tepal is the ancestral state within Musaceae.
Differences observed along male floral morphological characters in relation to the phylogenetic hypothesis (Figures 7-9) were used in to construct a key for the classification at inter-and intraspecific level (see all keys in Section 3)

Discussion
Previous study on overall morphology and characters of perianths by Simmonds [30] have paved way towards the clarification of the classification of Musaceae, however, could not solve generic status clearly. Recently, works on stem, inflorescence, seed morphology and molecular biology of the family independently created debates on taxonomic arrangement of the three genera members [6,7,31,32]. Our investigation on the male floral morphology concurrently with molecular study, however, initiate a set of discriminated morphological characters based on phylogenetic inference and able to resolve the infrafamilial evolutionary relationship within Musaceae.
Analyses of the male floral morphology accompanied by the molecular data in our study confirmed that the characters can be used to classify Musaceae at generic level into Musa, Ensete, and Musella ( Figure S1) as well as to construct an identification key (Figures 7-9).
Ensete possesses three outer tepals which fuse only at the base and adnate at the margin upwardly. The genus is separated from the other genera by round outer tepal apex lobes with long acicular inner tepals and tricuspidate median inner tepal. These characters were also used in a general key to describe this genus (Figures 3-5). Meanwhile, a monocarpic habit is not a suitable character to discriminate at genus level, since Ensete plants can produce suckers in cultivation or under abiotic stress [31].
Musella is the sister genus of Ensete based on molecular phylogenetic evidence [6,7], in contrast to morphological indications including rhizomatous habit and perianth is similar to Musa [26,30]. Meanwhile, similarities in inflorescences were used to group Musella with Ensete [2]. However, our investigation fortified that they are separated genera by their genetics, which is in contrast to the conclusions of Simmond [6] and Bakker [7], but supports the results of Christelova et al. [32] and Janssens et al. [8]. Moreover, the molecular phylogeny indicated that Ensete and Musella share their ancestor.
Our results agreed with work of Christelova [32] that the capitate stigma ( Figure 5A) of Musella separates it from the other genera, therefore, this character has a high discriminative power and can be easily used in an identification key. The generic classification of Musella remains unclear if only morphological characters were used, while the combination of morphology and molecular phylogenetics can clearly resolve the conflict of these classifications. Musella was placed under Musa based on a polycarpic habit and the perianth structures [26,30]. However, several characters of Musella, i.e., dwarf, congested pseudostems, compact rosette inflorescences and embryological characters, placed Musella as a separated genus [33][34][35][36]. The molecular data suggested that Musella is closely related to Ensete [6,7]; the two genera possess quite similar inflorescences [2,37]. However, the male floral characters of Ensete and Musella are obviously different. The two genera are grouped in the same cluster.
In spite of these evidences, it was found that, judging from molecular phylogenetics combined with multivariate analyses of the male flowers and the degree of perianth fusion, Musella is more similar to Musa than to Ensete. The fact indicated that the perianth fused at base of Ensete are apomorphic in Musaceae. Furthermore, the shared characters of having a perianth fused until the base of the apex in Musella and Musa suggests that this character is more primitive than a perianth fused at base. Moreover, the long acicular lateral inner tepal, tricuspidate free tepal and reduced style and stigma in male flowers can satisfactorily be used to separate Ensete from Musa and Musella. The tricuspidate shape of the free tepal was often found in Ensete [2], the obovate shape in Musella while the elliptic, ovate, oblong, lanceolate and obovate shapes in Musa. Musa nanensis is the only taxon which has a median tepal that is fused with the perianth tube [5].
The multivariate analysis of the 15 male floral morphological characters did not separate the genus Musa at sectional level, whereas the molecular phylogeny clearly divided the genus into two sections: Musa + Rhodochlamys and Callimusa + Australimusa [6,7,30].
Discriminating characters that delimit the genera of Musaceae are shapes of median inner tepals and stigmas. The median inner tepals of Musa species which varies in shapes and length and apical shapes and length clearly separated the genus into two groups. These variations in floral morphology, especially in perianth and androecium dissimilarities, which also occur in the other Zingiberales taxa, may have caused by differences in pollination syndrome [4,9,10]. Moreover, pollinator syndromes in Musaceae are assumed to have switched several times between bats and birds or other animals, except for Musella, which is always pollinated by insects [38,39]. Banana species with erect buds, excluding Musella, are suitable to be pollinated by sunbirds, whereas those with pendent buds are suitable to be pollination by bats [38,40]. Watery and gelatinous nectar types attract different pollinators and were suggested to be related to bud and floral positions. Watery nectar is found in erect inflorescences, whereas gelatinous nectar is found in horizontal (later pendent) inflorescences [41]. In the genus Musa, the median inner tepal changes in length and shape and long free median inner tepals and perianth tubes were found in erect inflorescences, except for M. rubra and M. siamensis. Conversely, short free median inner tepals were found in pendent inflorescences. Additionally, bats can use their tongues to lick up the sticky nectar from pendent inflorescence, whereas birds can take nectar from both erect and pendent inflorescences [41]. Therefore, fluidity of the nectar depends likely corroborate floral and bud orientations.
Our results indicated that, in bananas, shapes of the median inner tepal opposing nectary gland is different in erect versus pendent inflorescences are different. The elliptic median inner tepal shape with boat-shaped curve easing logging of sticky nectar is found in Musa and Ensete with hanging inflorescences and pollinated by long-tongued fruit bats. On the other hand, elliptic shape is found in some species of the section Musa and the former section Rhodochlamys, i.e., M. rubra and M. siamensis, with erect inflorescences pollinated by birds [40,42]. The difference between the pollinators may also be related to changing length of free median inner tepal, floral direction and viscosity of nectar in the section Musa. Moreover, some species of Rhodochlamys, i.e., M. ornata, M. velutina and Callimusa + Australimusa, do not possess boat-shaped curve of median inner tepals, the tepal length is as long as that of the fused tepal and form perianth tube which contains higher volume of watery nectar [43]. Though both Rhodochlamys and Callimusa + Australimusa were pollinated by birds and have long median inner tepal, the median inner tepal shape is different. The increasing of floral size of bananas in erect inflorescences ( Figure S2.10) suggested adaptation to produce large amount of nectar for bird pollinators [43].
The pistil size of the banana flowers in the genus Musa is reduced in male stage [44]. Two shapes of stigma, clavate and spatulate, were found, in the section Musa and in Callimusa + Australimusa, respectively. The spatulate stigma can be distinguished from the clavate one by its slender style and clearly separation of stigma head at connective zone. The stigma shapes of the two sections are unrelated to the position of the inflorescences [6,7,30,[45][46][47].
Curiously, Musa nanensis found recently by Swangpol and her team in Thailand is unique in floral symmetry and its placement within the Musa section have been in question [5]. The molecular phylogenetics of combined ITS, trnL-F, rps16 and atpB-rbcL sequences did not distinguish M. nanensis from Musaceae, on the other hand, placed it closely to the section Musa + Rhodochlamys with the erect inflorescence, the tubular flower and the long free tepal. The clavate stigma of M. nanensis is also a key character to classify it within the Musa section. Finally, within the section, though M. nanensis is similar to M. rubra (often called previously by its synonym, M. laterita [48]) based on vegetative features [5], its reproductive morphology and present molecular phylogenetic analysis placed it as sister to M. ornata and M. velutina.
The six tepals fused as a perianth tube and actinomorphic flower of M. nanensis is a reverse evolution in Musaceae. Moreover, among angiosperm lineages, bilateral floral symmetry has evolved multiple times from the radial symmetric ancestors in response to natural selection associates with adaptations to pollinators [49][50][51].

Identification of Musa Species Using Floral Morphological Characters
In our present banana phylogenetic trees, M. acuminata, M. serpentina, M. rubra (=M. laterita), M. siamensis and M. rosea are not separated. However, these species are different from each other based on morphological features, i.e., short elliptic free tepal with long apex wing found in M. serpentina, M. rubra (=M. laterita) and M. siamensis and long ovate free tepal found in M. rosea as seen in Figure 8B.
Musa rubra, M. laterita and M. siamensis are three ambiguous species with similar morphology including itinerant rhizomes and smooth surface on subglobose seeds [52][53][54][55]. While M. laterita was recently reduced into a synonym of M. rubra [49], M. siamensis was reduced to a variety of M. rubra [56,57]. The cases were supported by the resemblance of the male flowers in all accessions of the three taxa ( Figure 6).
The flowers of different subspecies of M. acuminata are similar, i.e., with short elliptic free tepal and short apex wing (Figures 6-9). The result agrees with the Musaceae phylogenetic tree based on nrDNA and plastid combined data ( Figure S1). Molecular phylogenetic study revealed that M. acuminata subspecific classification is paraphyletic [7] and the raise of each subspecies into species is not supported.

Taxon Sampling
Fifty-nine banana accessions in 21 taxonomic categories (Table S1)  For morphological studies, freshly collected mature flowers were kept in 70% ethanol and dried specimens were soaked in hot water and kept in 70% ethanol before being observed.

Sequence Alignment and Phylogeny Reconstruction
The sequences were aligned in MAFFT [65] and edited manually using Bioedit v7.0.5 [66]. Substitution rates for Bayesian analyses were selected under the Akaike information criterion (AIC) using Jmodeltest2 on XSEDE (2.1.6) [67] and the Bayesian inference (BI) models performed was conducted using MrBayes 3.2.7a [68], both on the CIPRES web portal [69]. Substitution model was selected as GTR+I+G for ITS and GTR+G for rps16, trnL-F and atpB-rbcL with number of substitutions as six. The Markov Chain Monte Carlo (MCMC) was performed using independent runs with four chains, 10,000 print results, saving ten every 1000 generations, for a total of five million of generations.
Maximum Likelihood was reconstructed using RAxML [70] on CIPRES portal with GTRGAMMAI model and four chains to parallel search the tree. A total of 1000 bootstrap replicates were generated by random sequence addition. To generated a starting tree for the parsimony inference, 12,345 was a random number used.

Multivariate Analysis
Fifteen quantitative characters ( Figure 10) of the floral structures including tepals, stamens and pistils of ten male flowers (three flowers from each herbarium specimen, except for a scarce specimen which only one flower was used) from either fresh or dried accessions were measured using ruler (Table S1). The range of variation between taxa on each quantitative character was visualized asbox-plots and transferred into decimal logarithms. Correlations between variations of quantitative data were determined by PCA. To estimate homogeneity of the morphology within groups of Musaceae, CDA was applied. Stepwise discriminant analysis, unstandardized coefficients and Maholanobis distance were used to determine characters that separate groups. Principal component and discriminant scores were constructed into scatter plots to identify groups within Musaceae. PCA and CDA were computed using PASW Statistics 18 (SPSS, Inc.). To determine similarities between samples, Gower similarity index was computed and the distributions of quantitative variables were visualized as box-plot and violin-plot in Past 4.05 [71].
Stepwise discriminant analysis, unstandardized coefficients and Maholanobis distance were used to determine characters that separate groups. Principal component and discriminant scores were constructed into scatter plots to identify groups within Musaceae. PCA and CDA were computed using PASW Statistics 18 (SPSS, Inc.). To determine similarities between samples, Gower similarity index was computed and the distributions of quantitative variables were visualized as box-plot and violin-plot in Past 4.05 [72].

Morphological Analyses
Ten male flowers (one to three flowers from each herbarium specimens) were observed under dissecting microscope (Olympus SZ40 light microscope, Tokyo, Japan). Twenty-two qualitative characters of the floral structure including two whorls of tepal, which composed of three outer and three inner tepals, stamen and pistil were coded into binary or multistate variables. To determine similarities between samples, Gower similarity index was perform using Past 4.05 [72]. The UPGMA performed on 59 OTUs was based

Morphological Analyses
Ten male flowers (one to three flowers from each herbarium specimens) were observed under dissecting microscope (Olympus SZ40 light microscope, Tokyo, Japan). Twenty-two qualitative characters of the floral structure including two whorls of tepal, which composed of three outer and three inner tepals, stamen and pistil were coded into binary or multistate variables. To determine similarities between samples, Gower similarity index was perform using Past 4.05 [71]. The UPGMA performed on 59 OTUs was based on 22 floral morphological characters, of which states were coded as in Table 5. These characters were used to construct an identification key to the banana species in this present study.

Character Evolution and Analysis
Using fresh flowers, dried specimens or pictures from original publication, five qualitative characters of male flowers including perianth symmetry (bilateral, radial and asymmetric), lateral inner tepal shape (acicular and ovate), median inner tepal shape (tricuspidate, obovate, oval-lanceolate, oblanceolate, oblong, elliptic, ovate and fused with perianth tube), stigma shape (clavate, spatulate and capitate) and style length (shorter than 18 mm and longer than 18 mm) were investigated (Table S1). The data matrix was coded into binary or multistate variables with unordered states. The species of which specimens cannot be observed were treated as a missing data. Evolution of the floral characters was traced over the phylogenetic trees using Mesquite version 3.6 [72]. The selected characters were constructed into identification keys to genera and Musa sections. We used Fitch parsimony [73] as a criterion for character optimization. To account for phylogenetic uncertainty, we traced character histories on 5001 post burn-in trees from the Bayesian analysis using the 'Trace Character Over Trees' command in Mesquite 3.6 [71].

Conclusions
The morphological characters of the male flowers within the banana family (Musaceae) show several distinguishable characters for the classification. These characters were used to construct a key to genera and sections in the genus Musa. The cluster analysis using PCA and CDA indicated that the three genera, Ensete, Musa and Musella, are different with supports by degrees of perianth fusion, median inner tepal shapes, lengths of styles and stigma shapes. Molecular phylogenetic analysis of ITS, trnL-F, rps16 and atpB-rbcL indicated that Musella is closer related to Ensete than to Musa. Based on capitate stigma, supporting by anther vegetative and reproductive morphological characters, Musella should be treated as a distinct genus, and not as a member of the genus Ensete. The elliptic and ovate shape of the median inner tepal and capitate stigma can clearly be used to separate the section Musa (including Rhodochlamys) from the section Callimusa (including Australimusa and Ingentimusa) that have obovate, oval-lanceolate, oblanceolate and oblong shapes combine with spatulate stigma. The species with undetermined section, M. nanensis with its uniquely different flowers including radial symmetry, six stamens and their fused filaments, should be placed in the section Musa based on its molecular phylogeny and the occurrence of a clavate stigma. The results and analyses from this study provide significant information on male floral characters as key characters to classify genera and section within Musaceae.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/plants12081602/s1, Table S1: List of banana accessions used in the phenetic analysis; Table S2: List of sequences of Zingiberales-Musaceae used in the phylogenetic analysis; Figure