Baraminology of Cucurbitaceae Based on Chloroplast Genome Analysis

Introduction

Baraminology is the study of the created kinds, mentioned in the Genesis creation account. A kind, or holobaramin, is a reproductive community consisting of species that are continuous with one another and discontinuous with all other species. The goal of a baraminology study is to use various types of data (the Bible, hybridization data, DNA sequences, morphological, ecological, and behavioral data) to classify species into their appropriate kinds.

The gourd family (Cucurbitaceae) is an agriculturally important group of food crops that include cucumbers, gourds, squashes, melons, pumpkins, zucchinis, and luffas. The secondary metabolites found in gourds called glycoalkaloids are used in the pharmaceutical industry. The family is quite diverse and can be classified into 15 tribes, 95 genera, and around 1000 species (Chomicki, Schaefer, and Renner 2020).

Hybridization data exists for 131 unique species pairs of cucurbits, including data from experimental cross-pollination events, natural hybridization, and embryo/ovule cultures. Previous baraminology research based on hybridization found eight monobaramins: Cucurbita, Citrullus, Cucumis, Lagenaria, Coccinia + Diplocyclos, Trichosanthe s+ Momordica, Luffa, and Bryonia (Brophy, Gregory, and Townsend 2023). This study was further augmented by the sequence analysis of ITS regions of nuclear ribosomal genes for 21 cucurbits taken from a study by Jobst, King, and Hemleben (1998). The ITS region is a segment of the DNA situated between the small-subunit rRNA and the large-subunit rRNA and is used widely in phylogenetic studies.

However, the two ITS regions combined amount to approximately 450 bp. Besides the ITS region, the whole chloroplast genome may be used to get more precise baraminic relationships, as it is much longer, approximately 155–160 Kbp. Its sequence is also very conserved, easy to isolate, abundant in cells, and has low mutation rates (Kousar and Park 2023). More closely related species are more likely to have similar genome sizes and characteristics. Therefore, it makes sense to put two plant species into the same baramin based on a high chloroplast genome sequence similarity (Xiao-Ming et al. 2017).

This study would be the second of its kind, after the analysis of the chloroplast genomes of 163 species from the order Liliales (Cserhati 2023). The results from the present study can be compared with hybridization data from Brophy , Gregory, and Townsend (2023) and the previous results from the analysis of Liliales.

Materials and Methods

The chloroplast genome of 28 species from Cucurbitaceae as well as three species from the genus Fagus and three from the genus Lycium, as outgroups, were downloaded from the Chloroplast Genome Database (CpGDB) at https://www.gndu.ac.in/CpGDB/index.aspx (Singh et al. 2020). Two chloroplast genomes for Gomphogyne cissiformis var. cissiformis and var. villosa were downloaded from the NCBI database at https://www.ncbi.nlm.nih.gov/nucleotide. These 36 sequences were then aligned using the MAFFT software (Katoh, Rozewicki, and Yamada 2019; Kuraku et al. 2013) using the --retree 2 and --reorder parameters.

The ‘dist.alignment’ command from the ‘seqinr’ package in R was used to create the sequence identity matrix using the ‘identity’ flag parameter. The dist.alignment command uses Fitch matrixes, which contain the square-root of the pairwise distances (Fitch 1966). The ‘heatmap’ function in R was used to create the heatmap of the 34 species seen in fig. 1. Species clustering was performed using the ‘ward.D’ method, which implements Ward’s agglomerative clustering method (Ward 1963).

Fig. 1. Baraminic heatmap of 30 species from Cucurbitaceae, three species of Fagus, and three species of Lycium. Darker, greener colors represent species with high chloroplast genome sequence similarity. These species likely belong to the same monobaramin. Lighter, yellower colors represent species with lower sequence similarity, and which may belong to different baramins. Eight putative monobaramins were found: orange: Sicyoaea + Bryonieae + Momordiceae, light green: Cucurbita, blue: Cucumis, cyan: Citrullus, green: Gynostemma, red: Gomphogyne+Hemsleya, magenta: Fagus, purple: Lycium.

The ‘upgma’ method was used to create a UPGMA-based baraminic tree for fig. 2. Bootstrapping was done using the ‘boot.phylo’ method in R for 100 bootstrap replicates. The ward.D2 clustering method was used. See Cserhati (2023) for a more detailed description of methods utilized in this study.

Fig. 2. Baraminic tree of the four Cucumis species analyzed in this study. Cucumis melo may be an earlier form in this monobaramin than the other species. The tree was constructed using the WPGMA algorithm.

Version 4.3.1 of R was used. The New King James Version of the Bible was used in the biblical analysis. All supplementary files and figures are available on Zenodo at https://zenodo.org/records/10372288.

Biblical Analysis

Mentions of cucurbits in the Bible are scant. Isaiah 1:8 mentions cucumbers: “So the daughter of Zion is left as a booth in a vineyard, as a hut in a garden of cucumbers, as a besieged city.” 2 Kings 4:39 mentions gourds: “So one went out into the field to gather herbs, and found a wild vine, and gathered from it a lapful of wild gourds, and came and sliced them into the pot of stew, though they did not know what they were.” Jonah 4:6–10 mentions a plant called קִּיקָיוֹן (kikayon), which some allege to be either a gourd, or the castor-oil plant, but which is disputed. The word for gourd in 2 Kings 4:39 is פַּקֻּעָה (paquah), which is different from the word in Jonah 4.

Lastly, Numbers 11:5 mentions two members of the family Cucurbitaceae: “We remember the fish which we ate freely in Egypt, the cucumbers, the melons, the leeks, the onions, and the garlic.” Since cucumbers and melons are listed separately, this might be an indication that they possibly belong to separate baramins, but this is not decisive. Cucumbers and many melons belong to the genus Cucumis. However, ‘melon’ is a common term and may cover other species outside Cucumis. Since the genus is generally held to be the lower bound of the baramin, some researchers believe that cucumbers and many of the melons are members of the same baramin (Brophy et al. 2023).

Results and Discussion

The 36 species of Cucurbitaceae, Fagus, and Lycium were clustered into eight groups, all but one with significant p-values (fig. 1). The elbow plot in Supplementary fig. 1 (available on Zenodo at https://zenodo.org/records/10372288) appears to show a drastic drop at seven clusters. However, the silhouette plot is inconsistent and appears to show that the optimal number of clusters is two (Supplementary fig. 2). The Hopkins statistic has a value of 0.881 which indicates very good clustering. The two outgroups, Fagus and Lycopis form groups distinct from the cucurbit species. The sequence identity matrix, the putative clusters, and the descriptive statistics for each of the eight putative groups found in this study are available in Supplementary File 1.

The first cluster is made up of nine species, from the genus Gynostemma. The mean sequence similarity within this group is 0.829. This group was not analyzed by Brophy, Gregory, and Townsend (2023), so they may indeed be a separate holobaramin within Cucurbitaceae.

Hemsleya lijiangensis is very similar to H. zhejiangensis, which both cluster closely to the genus Gynostemma in a chloroplast genome tree in Li et al. (2019). However, in another chloroplast study including two subspecies of Gomphogyne cissiformis (var. cissiformis and var. villosa), H. lijiangensis clustered together with the two Gomphogyne species (Zhang et al. 2018). The question then is, does Hemsleya + Gomphogyne form a monobaramin within Gynostemma, or are they their own holobaramin?

Based on the heatmap, Hemsleya + Gomphogyne form a separate small putative holobaramin, with a mean sequence similarity of 0.85. The tribe Gomphogyneae consists of mainly dioecious perennial lianas that grow in tree canopies but also annual creepers (Schafer and Renner 2011). However, the p-value for this group is 0.107, so further analysis is needed to confirm the baraminic status of these two genera.

Three putative holobaramins were also discovered corresponding to the genera Cucurbita (Cucurbita pepo, Cucurbita moschata, and Cucurbita maxima), Cucumis (Cucumis x hytivus, Cucumis hystrix, Cucumis melo, and Cucumis sativus), and Citrullus (Citrullus rehmii, Citrullus amarus, Citrullus colocynthis, Citrullus mucosospermus, Citrullus lanatus). These three groups were also identified via hybridization data by Brophy et al. (2023).

Zhou et al. (2023) found that the largest chloroplast genome sequence divergence calculated for seven species of Citrullus was 0.00641 (between C. naudinianus and C. lanatus subsp. vulgaris). The chloroplast genome size for these seven species is 157,005±102 bp, suggests that the genomes of the species in this baramin have remained fairly stable throughout their history.

The four species of Cucumis were also found to form a holobaramin, the baraminic tree of which can be seen in fig. 2. The genome size ranges from 154,673 to 155,670 bp in several species of Cucumis studied by Zhai et al. (2021), which is less than 1 Kbp. Exons and inverted repeat (IR) regions were also found to be conserved in the chloroplast genome of Cucumis x hytivus (Zhai et al. 2021). It may be that C. melo is an earlier form in the putative Cucumis holobaramin. The mean chloroplast genome sequence similarity value between C. melo and C. x hytivus, C. hystrix, and C. sativus is only 82.8±0.8%, whereas among the latter three Cucumis species it is 89.7±2.9%. Analyses of chromosomal numbers and ITS regions indicate that C. melo (2n = 24) is ancestral to other species (Sebastian et al., 2010; Garcia-Mas, Monforte, and Arús 2004), (which have a chromosome configuration of 2n = 14). Therefore C. melo seems to be an earlier form of the Cucumis.

Interestingly, although 66 members of the genus Cucumis have a monoecious (hermaphrodite) mating system, and annual lifestyle, whereas other species have a dioecious mating system (male and female reproductive organs on separate plants) and perennial habit (Sebastian et al. 2010). Apparently, there may be a more or less simple genetic switch that lets species alternate between monoecious and dioecious mating systems within a baramin. Similarly, Wood and Cavanaugh (2001) found that various species of the subtribe Flaveriinae use the C₃, C₃-C₄ and C₄ photosynthetic pathways. Both monoecious and dioecious mating systems and the C₃, C₃-C₄ and C₄ photosynthetic pathways appear to be specially designed morphological and molecular structures that allow plants to plastically adapt to new environments.

The last group is made up of six species from various genera: Coccinia grandis, Hodgsonia macrocarpa, Lagenaria siceraria, Momordica charantia, Siraitia grosvernorii, and Trichosanthes kirilowii. This group has a mean sequence similarity of 0.847, which is the lowest of all eight groups, but has a p-value of 5.59E-20. Brophy, Gregory, and Townsend (2023) separate Lagenaria and Coccinia + Diplocyclos into their own groups, although both groups are represented by only two species in their study. However, Brophy, Gregory, and Townsend (2023) place Trichosanthes, Luffa, Echinopepon, Cyclanthera, Sicyos, Bryonia, Ecballium, and Momordica into one single putative monobaramin. They did this based on the genetic distances between 21 cucurbit species using the ITS region analyzed by Jobst, King, and Hemleben (1998).

For example, based on Brophy, Gregory, and Townsend’s (2023) hybridization data, Momordica charantia and Trichosanthes anguina hybridize directly and M. charantia hybridizes with the same third species as T. cucumerina and T. lobata, indicating that they all belong to the same holobaramin. The genetic distance between M. charantia and T. cucumerina based on ITS regions is 0.149 (Jobst, King, and Hemleben 1998; see also Supplementary File 2). By inference, any species pair with distances less than 0.142 may possibly also be able to hybridize. This means that the 20 species in the previously mentioned eight genera might be united into one single holobaramin. This would also support the six species in the last group being assigned to their own holobaramin.

The chloroplast genome map for 26 cucurbits and the outlier species Lycium ruthenicum can be seen in fig. 3. The gene order appears largely conserved between all 26 species, with only minor differences. This is evidence that all cucurbits could be united into a single holobaramin (Brophy, Gregory, and Townsend 2023). However, this is not decisive. The gene order in the mitochondrial genome is also almost the same among the primates, yet we know that humans belong to a separate baramin compared to apes. The chloroplast gene order also seems to be conserved in non-cucurbit plant species as well, such as Nicotiana, Arabidopsis, Panax, and Oenothera (Plader et al. 2007). Each element in the chloroplast genome on the genome map is listed in Supplementary File 3.

Fig. 3. Chloroplast genome map of several species of the six putative Cucurbitaceae holobaramins and one species from Lycium as a reference.

Summary and Conclusion

This is the second chloroplast genome-based baraminology study performed to date after the one on Liliales (Cserhati 2023). The present study benefited from the extra hybridization data analysis done by Brophy, Gregory, and Townsend (2023). The present study confirmed the holobaraminic status of Cucurbita, Cucumis, Citrullus, and Coccinia grandis, Hodgsonia macrocarpa, Lagenaria siceraria, Momordica charantia, Siraitia grosvernorii, and Trichosanthes kirilowii. Two further putative holobaramins were predicted, namely Gynostemma and Hemsleya + Gomphogyne, although the latter needs further analysis.

Brophy, Gregory, and Townsend (2023) consider Cucurbitaceae to be a single holobaramin. Their finding is based on the inclusion of species considered to possibly be in the same baramin based only on genetic similarity of hybridizing species. This genetic distance was determined by the sequence similarity of the ITS region. However, according to Tippery and Les (2008), genetic distances mean the reliability of such data deteriorates above the genus level. As such, the holobaraminic status of Cucurbitaceae is only tentative. While this may be a valid assessment on the part of Brophy, Gregory, and Townsend, the Silhouette value analysis shows that the maximum Silhouette value is 0.6 for eight clusters as opposed to 0.52 for four clusters (Cucurbitaceae, Gynostemma, Fagus, Lycium). The Silhouette values can be seen in Supplementary File 2. This consideration indicates that eight holobaramins may be a better division of the data as opposed to four.

This study demonstrated how chloroplast genomes can be used to classify species into holobaramins. Since chloroplast genomes are very easy to determine, many more chloroplast genome-based baraminology studies can be carried out in the future. Results from mitochondrial and chloroplast genome studies can be compared as supportive evidence for baraminic classification alongside hybridization and other datasets.

This might prove to be more difficult than may be anticipated. This is because mitochondrial genomes range from 200 to 2,400 Kbp in size (rivaling the genome size of some bacteria) in plants (Ward, Anderson, and Bendich 1981). Some of these plant mitochondrial genomes are also multichromosomal. For example, the mitochondrial genome size of Citrullus lunatus (watermelon) is 379 Kbp, that of Cucurbita pepo (field pumpkin) is 983 Kbp, and that of Cucumis melo (musk melon) is 2,740 Kbp, the longest mitochondrial genome in existence (Rodríguez-Moreno et al. 2011). The determination of baraminic status of species in the cucumber family may be complicated due to different inheritance patterns between the nucleus, the chloroplast and the mitochondrion. In Cucumis species, for example, mitochondrial DNA is paternally inherited, whereas chloroplast DNA is maternally inherited (Shen et al. 2015; Zhai et al. 2021).

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Supplementary Materials

All supplementary files and figures are available on Zenodo at https://zenodo.org/records/10372288.