Study species
Cnidoscolus aconitifolius (Euphorbiaceae) is a shrub native to Mexico and Central America24,39. It has been suggested that this plant was domesticated on the Yucatan Peninsula by the Maya where it is called “chaya”40,41. Leaves of the cultivar are edible, and while it is mainly grown in Mesoamerica, it has recently expanded beyond its native distribution range42. Across all cultivation areas, chaya is clonally propagated from stem cuttings25,40,42. In its domestication centre, chaya is still cultivated by the Maya in traditional agroecosystems, particularly home gardens where it coexists with its wild relatives25,41. Despite their co-occurrence, there is almost complete reproductive isolation between wild and domesticated forms, mainly due to poor pollen production and reduced fertility of the latter12. Clonal propagation of the cultivar is, therefore, obligate. The male flowers of wild plants produce abundant pollen, and female flowers set dry fruit with up to three seeds43. Clonal propagation is also possible in the wild relatives but unlikely without human assistance25,43. Domestication syndrome includes the increased production of bigger leaves with significantly fewer trichomes and more succulent stems25,44. Traditional growers usually select the thicker stems of secondary branches from apparently healthy plants for propagation25.
This study complied with relevant institutional, national, and international guidelines and legislation. Cnidosculus aconitifolius is not at risk of extinction or under the protection of international or local authorities. Additionally, plant material (stem cuttings) used in this study was collected in private lands and therefore no permission from local government was required. The identity of the specimens was confirmed by the curator (Dr. J Tun-Garrido) of the herbarium Alfredo Barrera Marín where a specimen was deposited (Voucher: UADY-23474).
Resistance to desiccation
In autumn 2020, I took 60 stem cuttings (35 cm long) from the secondary branches of 60 different plants (30 wild and 30 domesticated) from an experimental orchard established as a part of a bigger project in 2017 in the municipality of Merida in central Yucatan (see reference 25 for details). I used this orchard as source of cuttings to control for environmental and mother effects and thus, isolate the effect of domestication per se. All donor plants were exposed to full sunlight, watered evenly once a week; are the same age and similar in height (2-2.5 m tall). Once in the laboratory, the basal diameter of the cuttings was measured, and a 5 cm segment was cut from the 35-cm-long segment. Both segments (30 cm & 5 cm) were weighed for all cuttings and the small ones were oven-dried at 75 °C for 72 h to estimate water content. The 30 cm segments were placed in a controlled environment chamber (Binder Inc., KBW 240, Tuttlingen, Germany) at a constant temperature of 26 °C with a photoperiod of 12 h light/dark, light was provided by high-pressure sodium lamps (PFD = 46.89 µmol· m2· s-1). The only source of water for the cuttings was environmental humidity (≈ 60%) which was homogeneous throughout the chamber. All cuttings were weighed twice a week for a month to estimate the rate of water loss (measured as weight loss). The temperature and photoperiod used resembled the average values observed during the summer on the Yucatan Peninsula.
Resource storage
Using the same source plants and selection criteria outlined above; one month later, I cut another stem section of ca. 10 cm in length to measure soluble sugars and assess the presence of starch. To identify the presence and distribution of starch, I cut a fine slice (3-4 mm thick) from the end opposite the apex of each stem section and immediately added approximately 1 ml of Lugo’s solution (5g I2 + 10g KI + 85ml H2O) to all the slices simultaneously. After 3 minutes, the slices were washed in distilled water to remove the excess solution. The presence and distribution of starch was easily recognized as it stains dark blue. I indirectly measured total soluble sugars in the remaining portion of the stems by using the method outlined by Okamura and colleagues45, which consists of measuring the sugar content of the sap, obtained by squeezing the stem sections, and using a digital refractometer with automatic temperature compensation (HI96801, Hanna Instruments Inc., Rhode Island, USA). Although the refractometer gives the sugar content in Brix units, it is a reliable proxy for % total soluble sugars (r=0.96, P < 0.0145). I was able to obtain enough sap to perform the measurements on the stems of 19 wild and 21 domesticated plants; the remaining stems were too hard and/or too dry to obtain enough sap to test.
Shoot production and cutting longevity
Twice a week over four months (120 days), I counted the new shoots sprouting from the 30-cm-long cuttings in the controlled environmental chamber described above in Resistance to desiccation. The presence of leaves emerging from the shoots was also recorded. During the experiment, all new shoots were labeled to avoid underestimating the total number of shoots because some of them withered and fell off during the experiment. In addition to shoot emergence time, I recorded the time to cutting death, defined as the time when a cutting showed clear signs of wilting as well as shoot abortion and leaf abscission (when these had developed). I discarded dead cuttings to prevent the proliferation of fungi and bacteria in the chamber, and the potential contamination of the remaining cuttings.
Rooting
In January 2021, I took 57 stem cuttings, 30 cm in length, from 57 plants (26 wild and 31 domesticated) following the same procedure and criteria described above in Resistance to desiccation. Two days after collection, the cuttings were planted in 2L plastic pots using a mix of gravel and soil (70:30) as the substrate. Before planting the cuttings, I removed all of the leaves with pruning shears. I left the pots with these cuttings in a plant nursery located next to the experimental orchard mentioned before. All cuttings were exposed to the same light environment (full light exposure) and watered to field capacity once or twice a week during the experiment. After four weeks, I gently removed the cuttings by turning the pot upside down, without pulling on the cutting in order to avoid any damaging the roots. Once the cutting had been removed I washed the part that had been buried to eliminate all traces of soil. I carefully examined the cuttings with a magnifying glass in search of roots. I recorded the presence/absence of roots, the number of main roots (i.e. roots that emerged directly from the cuttings) and measured the length of the longest root, if present. Additionally, the presence/absence of any leaves on the aerial part of all cuttings was recorded.
Clone survival
To assess the long-term survivorship of clonally propagated plants (clones), in June 2019 160 stem cuttings (30 cm long) from 40 different mother plants (20 wild and 20 domesticated, four cuttings per plant) were collected using the donors, procedure and selection criteria described above in Resistance to desiccation. The cuttings were planted in 20L pots using the same substrate and procedure described in the Rooting section. The plants were left in the plant nursery described above, under full light exposure and were watered to field capacity once a week. I checked clone survivorship monthly for nine months (270 days) starting after the fifth month. I did this because survivorship is difficult to assess during the first months of life. For example, cuttings may have no leaves, but may have roots, or may even already have died with no clear signs of wilting because watering keeps the aerial part of the cuttings turgid. A clone was considered dead when it had lost its leaves and the stem presented clear signs of wilting.
Statistical analyses
Resistance to desiccation. I estimated water content by subtracting the final weight of oven-dried cuttings from their initial weight. Water content, expressed as a proportion of total weight, was compared between the stems of wild and domesticated plants (i.e. domestication factor) with an ANCOVA, including the diameter of fresh cuttings as a covariable. The domestication x diameter interaction was also included in the model. To improve the normality of the data, the proportion of water was arcsine square root transformed. The rate of water loss was assessed with a mixed-linear model, with weight as the response variable and domestication factor, time and their interaction as fixed effects. Time nested in cuttings was also included in the random part of the model to account for repeated measures.
Resource storage. I assessed differences in total soluble sugar between the stems of wild and domesticated plants with an ANCOVA test, including stem diameter and its interaction with domestication factor in the model.
Shoot production and cutting longevity. The effect of domestication on the incidence and the total number of shoots was assessed using generalized linear models with a binomial (shoot sprouting incidence) and Poisson (number of shoots) error distribution, respectively. In both models, the initial diameter and weight of the cuttings were included as covariables. The effect of domestication on the time when the first shoot was recorded and cutting survival were assessed with time to an event (survival) analyses assuming a Weibull and exponential distribution, respectively. In both models, the initial diameter and weight of cuttings were included as covariables.
Rooting. Rooting incidence, the number of roots and the length of the largest root were compared between cuttings of wild and domesticated plants using generalized lineal models (3 models in total) with binomial, Poisson and Gaussian error distributions, respectively. In all models, the initial diameter of the cuttings and its interaction with domestication were included as explanatory variables. The proportion of cuttings that developed leaves was compared between wild and domesticated plants with a proportion test. I assessed whether the number of main roots and the length of the largest root predicted the development of leaves (presence vs. absence of leaves) on cuttings from wild plants using a generalized linear model with a binomial error distribution. I did not include the data for the cuttings of domesticated plants in this analysis because all except one developed at least one leaf.
Clone survival. The survivorship of clones propagated from the cuttings of wild and domesticated plants were compared with survival models assuming a constant (exponential) hazard.
All data analyses were run in R .6.2.46
Data availability
The raw data is included as online supplementary material.